AN ABSTRACT OF THE THESIS OF
DAN V. MOURICH FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN
MICROBIOLOGY PRESENTED ON MARCH 20. 1996. T1TLE: ANALYSIS OF
IMMUNE MODULATORS IN RAINBOW TROUT (Oncorkvnchus mykiss
Redacted for Privacy
Abstract approved:
The immune systems of various teleost fish have been studied in some detail for
the past several decades. One aspect of fish immunity, that of endogenously produced
modulating factors, has recently received a great deal of attention. Understanding the
functions and roles of endogenous factors that regulate fish immunity is paramount to
expanding the fields of fish immunology and vaccinology. It is know that several
lymphoid cell derived factors are detectable in in vitro cell culture systems and exhibit
immune modulating effects similar to well studied proteins in mammals. However in
comparison, few genes or gene products involved in the modulation of the trout immune
responses have been isolated, cloned and characterized.
The studies described herein were designed to isolate specific genes from rainbow
trout (0 ncorhynchus mykiss ) and characterize their involvement in the modulation and
regulation of the trout immune system. Two distinct genes were isolated cloned and
sequenced. The first, non-specific cytotoxic cell enhancement factor (NCEF) gene is
closely related to a human gene termed "natural killer enhancement factor" (NKEF) which
is important in the modulation of human natural killer cell activity. The second gene is
closely related to a group of recently characterized mammalian genes involved in the signal
transduction of cytokines termed "STATs". The role of these genes and their respective
protein products will be examined and discussed.
The antigenic structures of the fish proteins (NCEF and STAT5) were examined
by western blot and immunohistochemistry. Monoclonal antibodies derived against the
respective human proteins were found to cross react with the corresponding trout proteins,
demonstrating antigenic relatedness. The monoclonal regents were also used to analyze
the expression of these proteins in fish cells of lymphoid and non lymphoid origin.
In vitro cell culture analysis was used to determine the effects and roles of NCEF
and STAT5 gene products in the trout immune system. The cytolytic and apoptotic killing
activities of spleen, head kidney and peripheral blood leukocytes were found to be
enhanced by NCEF. Mitogenic stimulation of peripheral blood lymphoid cells resulted in
the trout STAT 5 protein binding to a know sequences contained with in the promoters of
genes transcriptionally activated in response to cytokine exposure.
ANALYSIS
OF IMMUNE MODULATORS IN RAINBOW
TROUT (Oncorhynchus mykiss )
by
Dan V. Mourich
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed March 20, 1996
Commencement June 1996
© Copyright by Dan V. Mourich
March 20, 1996
All Rights Reserved
Doctor of Philosophy thesis of Dan V. Mourich presented on March 20, 1996
APPROVED:
Redacted for Privacy
or Professor, representing
crobiology
Redacted for Privacy
air of Department of Microbiol
Redacted for Privacy
Dean of Gradate School
I understand that my thesis will become part of the permanent collection of Oregon State
University libraries. My signature below authorizes release of my thesis to any reader
upon request.
Redacted for Privacy
Dan V. Mourich, Author
ACKNOWLEDGMENTS
The one person most instrumental in making this thesis possible was Dr. Jo-Ann
Leong. Without her constant support, guidance and confidence in my abilities and this
work these studies would have not been possible. I thank her specifically for allowing me
the opportunity to work for her as both a technician and a graduate student over the past
years. It has been a privilege and honor to learn from her in both capacities.
I also thank my various lab mates, colleagues, fellow students, friends and
teachers; Jon Piganelli for his friendship and unending interest in immunology, Marc
Johnson for his steadfast work ethic, Grant Trobridge for his expertise and jokes across
the bench, Eric Anderson for the 6 am mountain bike rides and late night guitar sessions,
Jon Hansen for his friendship and brewing excellence, Peter Chiou for his friendship and
cooking skills, Dr. Linda Boot land and Harriet Lorz for their constant help, Dr. Carol
Kim for her expertise and friendship, Don Stevens for his help and eye for detail, all of
the Oregon Department of Fish and Wildlife people for their help and morning coffee, Jon
Pampush for fishing, hunting, rafting, basketball and all the things that got me out of the
lab and kept me sane, and Dr. Steven Kaattari for making immunology the first science I
thought was cool.
CONTRIBUTION OF AUTHORS
Dr. Jo-Ann Leong was involved in all aspects of the work reported within this
thesis including experimental design, data analysis, interpretation, writing criticism and
financial support. Dr. Jon Hansen was involved in supplying both the library and
molecular screening expertise for the isolation of the NCEF clone described in Chapter 3.
Marc Johnson was involved in all aspects of cloning, sequencing and biological
characterization of the STATS gene described in Chapter 5. Dr. Carol Kim provided
expertise and work crucial to the immunohistochemical confocal microscopic analysis of
the STATS protein in chapter 5.
TABLE OF CONTENTS
Chapter
Page
1. THESIS INTRODUCTION
2
Objectives
Approach
Isolation of Genes
Assessment of Biological Activity
Introduction to Thesis Chapters
2.
1
LITERATURE REVIEW
Perspective
Nomenclature History
Defining Fish Cytokines
Molecular Evidence of Cytokines in Fishes
Cytokine Signal Transduction
3. NATURAL KILLER CELL ENHANCEMENT FACTOR-LIKE GENE
IN RAINBOW TROUT (Oncorhynchus mykiss)
Introduction
Acknowledgments
References
3
3
4
5
7
7
7
9
20
28
31
32
36
36
4. CHARClERIZATION OF A NON-SPECIFIC CYTOTOXIC CELL
ENHANCEMENT GENE PRODUCT (NCEF) IN RAINBOW TROUT
(Oncorhynchus mykiss
)
Abstract
Introduction
Methods and Materials
Results and Discussion
Acknowledgments
References
38
39
40
41
51
65
65
5. CLONING AND SEQUENCE OF A STAT 5-LIKE GENE IN RAINBOW
TROUT (Oncorhynchus mykiss ): DISTRIBUTION IN LYMPHOID
CELLS
Abstract
Introduction
Materials and Methods
Results and Discussion
Acknowledgments
68
69
69
72
78
98
TABLE OF CONTENTS (Continued)
Chapter
References
Pu
98
6. Thesis Summary
Immune Modulatory Genes in Rainbow Trout
Biological Characterization of Gene Products
101
101
101
BIBLIOGRAPHY
103
LIST OF FIGURES
Figure
Page
3.1 Complete nucleotide sequence of trout NKEF related clone.
34
Autoradiogram of 35S-methionine labeled in vitro
transcription product derived from NCEF gene insert..
53
4.2
Western blots with anti -NKEF monoclonal antibodies
55
4.3
Immunohistochemical analysis of CHSE-214 cells transfected with
CMV-NCEF or CMV-Pgal .
55
4.4
Results of chromium release assay with head kidney leukocyte effectors.
58
4.5
Results of chromium release assay with spleen leukocyte effectors
58
4.6
Results of chromium release assay with peripheral blood
leukocyte effectors
60
Results of DNA fragmentation assay for peripheral blood
leukocyte effectors
63
Results of DNA fragmentation assay for headkidney leukocyte effectors.
63
4.1
4.7
4.8
5.1a Sequence of STAT 5-like gene segment (nucleotides 1 to 420) from
rainbow trout (0. mykiss).
79
5.1b Sequence of STAT 5-like gene segment (nucleotides 421 to 840) from
rainbow trout (0. mykiss).
81
5.1c Sequence of STAT 5-like gene segment (nucleotides 900 to 960) from
rainbow trout (0. mykiss).
83
5.2
Predicted amino acid sequence of trout (0. mykiss) STAT 5-like
protein and its relationship related to other STAT5 proteins from
human, sheep and mouse
85
5.3
Detection of STAT 5 proteins fish cell lines
88
5.4
Distribution of STAT 5 protein in trout tissues.
90
5.5
Confocal immunohistochemical analysis of trout cells with
STAT 5 specific monoclonal antibody.
93
Electromobility shift and super shift assay with PBL nuclear extracts
harvested 1 hour after exposure to Con A.
96
5.6
LIST OF TABLES
Table
Page
2.1
The pleiotropic and redundant activities cytokines
5.1
PCR primers pairs and gene segments along the human STAT 5 gene
11
74
DEDICATION
I dedicate this thesis to my family,
who supported me with their love and faith
over the many years.
ANALYSIS
OF IMMUNE MODULATORS IN RAINBOW
TROUT (Oncorhynchus mykiss )
CHAPTER 1
THESIS INTRODUCTION
In the past decade much has been learned about the various proteins which control
and regulate the mammalian immune system. These proteins are functional components in
various immune mechanisms such as cell to cell communication, antigen recognition or
signal transduction. Since all of these mechanisms result in the modulation of cells of the
immune system, the proteins involved can be described in broad terms as "immune
modulators". The genes of many of these immune modulators have been isolated, cloned
and characterized. Examples of these are cytokines, cytokine receptors, T cell receptors
and transcription factors. This had led to a detailed understanding of the mammalian
immune system and made possible the manipulation of immune responses for the benefit of
combating disease.
There is biological evidence for the existence of immune modulators in fish.
However, little is known about the specific proteins involved in fish immune responses or
the genes which code them. Many fish cell culture derived factors with immune
modulatory activities similar to various mammalian cytokines have been described
(Secombes, 1994). Yet, few genes coding for these factors have been cloned and
characterized in fish. There are many possible reasons why isolation of such genes from
fish has been elusive. Some of the difficulty likely resides in the techniques used and the
assumptions that must be made when using these techniques. For example, most
researchers including this laboratory have attempted to isolate cytokine gene sequences by
employing the polymerase chain reaction (PCR). The initial assumption that designing
2
PCR primers to homologous regions found by comparisons of published cytokine gene
sequences may be erroneous. By and large cytokine gene sequences available for
homology comparisons are mammalian. Very little cytokine sequence data is available for
species bridging the phylogenetic gap between mammals and fish. It is likely that
insufficient homology between fish and mammalian genes, particularly in these primer
regions, could account for negative results. A more successful approach in isolating the
genes involved in the fish immune response might be the use of PCR to amplify genes
whose sequences were conserved throughout a phylogenically diverse range of species.
Other proteins involved in immune modulation besides cytokines might be better
candidates for gene isolation from fish by PCR. Genes that code for proteins that are
dependent on structural features intrinsic to their function, such as DNA binding motifs,
nuclear translocation signals or protein modification sites, may be more conserved
throughout evolution.
Using these two strategies for designing PCR primers, i.e. 1) conserved gene
sequence information from various taxa and 2) conserved sequences based on the required
structure for the functional features of proteins, may be more suitable for isolating genes of
immune modulators from fish. Isolation and characterization of genes and their respective
proteins involved in fish immune modulation ,specifically rainbow trout, would not only
help in understanding the salmonid immune system more fully but would also be valuable
in designing drug or vaccine strategies to combat fish disease.
Objectives
The objectives of these studies were to 1) isolate genes which code for proteins
involved in the modulation of rainbow trout immune responses 2) compare the relative
homologies of these trout genes to know gene sequences from other species 3) analyze the
3
functions of the protein products from these genes in trout immune modulation and 4)
compare the functions of these trout proteins to their mammalian counterparts.
Approach
To study the immune modulating molecules of rainbow trout (Oncorhynchus
mykiss ), complementary DNA clones (cDNAs) of the genes of interest had to be isolated,
cloned and sequenced. Once sequence information was obtained then sequence homology
comparisons of the specific trout gene to the known gene sequences could be made by
computer analysis. If sufficient homology existed between an isolated fish gene and
known gene sequences then antibodies to the known protein might be used to determine
structural relatedness. Monoclonal antibodies to known mammalian immune modulating
proteins were obtained to determine if the trout proteins were structurally similar to the
mammalian proteins. In vitro studies were then designed and carried out to examine the
biological activities of these trout proteins and determine if there were functional similarities
to the mammalian proteins. Two genes were isolated from rainbow trout and examined in
these studies. The first is a nonspecific cytotoxic cell enhancement factor (NCEF) which is
related to a human natural killer cell enhancement factor (NKEF). The second is related to
group of proteins involved the signal transduction of cytokines termed signal transduction
and activator of transcription (STAT). Specifically the STAT5 gene for rainbow trout was
isolated which is related to the group of STAT proteins involved in interleuldn-2 (IL-2)
signal transduction.
Isolation of Genes
PCR amplification technique was used to isolate immune modulator genes from
trout. Synthetic primers were designed to well conserved regions of a known gene
4
sequence found in humans termed natural killer enhancement factor gene (NKEF) that
shares homology with related genes found in a phylogenetically diverse range of
organisms. Primers were also designed to well conserved regions found in the STAT5
gene which encodes a protein highly conserved among mammalian species and sequences
based upon functional structures in the protein. In both cloning attempts, primers were
designed to regions coding for amino acids with low codon degeneracy.
The template source for the PCR reactions was a cDNA pool was made from
messenger RNA (mRNA) derived from peripheral blood lymphocytes (PBLs). Products
derived from the PCR reactions were isolated, cloned and sequenced. Then, computer
aided analysis was used to determine their relative similarity with known gene sequences.
After confirming that a trout derived gene sequence was significantly related to its
mammalian counterpart, the cloned gene segment in the case of the trout-NKEF was used
as a probe to screen a rainbow trout cDNA library. In the case of the STAT5 gene,
sequence information obtained form the initial PCR amplification was used to synthesize
new primers for subsequent screening of the cDNA pool for larger portions of the gene.
Assessment of Biological Activity
In the case of the nonspecific cytotoxic cell enhancing factor gene (NCEF) or trout-
NKEF, a recombinant form of the protein was produced in a salmonid cell line, CHSE­
214, by transfection of a eukaryotic expression plasmid containing the NCEF cDNA insert.
The transfected cell lines expressing the factor served as a target cell source to assess the
effect of NCEF protein on trout leukocytes activity placed in the transfected cultures. The
enhancement effect of NCEF was examined by measuring the increased cytotoxicity
(necrosis and apoptosis) of target cells expressing the NCEF upon exposure to trout
spleen, head kidney, and peripheral blood derived leukocytes. Target cells transfected with
DNA for the expression of 13-galactosidase (n-gal) served as controls.
5
The binding of the trout STAT5 protein to DNA was examined in electromobility
shift and super-shift assays (EMSA). Lymphocytes were exposed to mitogen stimulus in
culture for 1 hour and then the nuclear proteins were extracted. The presence of STAT5
like proteins was assessed by their ability to bind to a specific double-stranded 32P labeled
synthetic primer. The primer sequence was derived from sequence elements found in the
promoters of genes, whose expression is regulated by cytokines through the binding of
STAT5 proteins. Binding reaction mixtures were fractionated on non-denaturing
polyacrylamide gels and protein-primer binding was detected by autoradiography. To
determine if the positive binding reaction was due to a specific STAT5 protein-primer
interaction, the mixture was pre-incubated with an anti-STAT5 monoclonal antibody.
STAT5 binding was confirmed by a super shift in the banding pattern for the monoclonal
antibody treated material as compared to the untreated material.
Introduction to Thesis Chapters
Chapter 3 is a published gene sequence report of the complete coding sequence of a
trout derived gene, and its similarity to two human genes coding for natural killer
enhancement factors (NKEF A and B). Initially, primers were designed to conserved
regions found in the two human genes. These primers were used in a PCR reaction to
amplify a segment of the gene from a trout blood cell cDNA pool. This PCR product was
cloned, sequenced and confirmed to be related to the NKEF genes. The cloned product
was then used to screen a rainbow trout cDNA library from which a full length trout cDNA
was obtained. This is the first report of a trout gene with significant homology to a known
human gene involved in the enhancement of an immunological response.
Chapter 4 is a manuscript reporting the biological and antigenic relatedness of the
trout NKEF -like protein termed, nonspecific cytotoxic cell enhancement factor (NCEF), to
the two human NKEF proteins. The trout protein was found to be similar in molecular
6
weight to NKEF proteins and shared epitopes with NKEF A or B. This was demonstrated
in western blots with monoclonal antibodies which differentiate between the two human
NKEF proteins. The trout NCEF protein was also found to enhance the necrotic killing
activity of trout leukocytes similar to the effects of NKEF A and B on human natural killer
cells. Also, apoptosis which has not yet been demonstrated for the human NKEF proteins
was enhanced by the trout NCEF protein
.
Chapter 5 is a manuscript reporting the existence of a trout gene whose encoded
protein, is structurally and functionally related to the mammalian gene STAT5, a protein
known to be involved in the transduction of cytokine signals. Signal transduction and
activator of transcription (STAT) proteins are necessary components in the signaling
process of many cytokines. These proteins, in response to the binding of specific
cytokines to their cognate receptors are phosphorylated and translocated to the nucleus.
Once in the nucleus they bind to target sequences found in the promoter elements of
cytokine regulated genes and activate transcription of these genes. The finding of these
proteins in salmonid cells will be valuable in defining trout cytokines and aid in the
isolation of these genes.
7
CHAPTER 2
LITERATURE REVEIW
Perspective
The predominant portion of the literature describing the activities, gene sequence
and signal transduction mechanisms of cytokines has been based primarily on studies in
mammals. Over the years, the results of these studies have formed the basis of a number
of different definitions and attempts to categorize cytokines. The study of cytokines in
more primitive animals, namely fish, is at the stage where mammalian cytoldnes were three
decades ago. There are many studies describing fish cell derived "factors" exhibiting
activities that parallel mammalian cytokines; but there have been far fewer studies dealing
with cloned genes homologous to mammalian cytokines. The purpose of this chapter is not
to describe in detail the vast amount of information available on each cytokine, but to
summarize the information available on cytokines in fish. The information on cytokine -like
factors found in fish will be examined for functional and molecular similarities with their
mammalian counterparts.
Nomenclature History
Mediators of cellular immunity defined as non-antibody, lymphocyte-derived,
proteins were first termed "lymphokines" by Dumonde et al. (1969). The term "cytokine ",
defined as "soluble proteins that mediate communication between cells", was next proposed
by Cohen et al. (1974) to distinguish mediators that were only produced by lymphocytes.
The defmition of "interleukin" was coined soon after to recognize cytokines that function
exclusively as communication signals between leukocytes (Aarden, 1979). However,
these terms are inadequate because many interleuldns were found to be produced by and
8
have an effect on a variety of cell types. Throughout the past three decades cytokines have
undergone several changes in classification as additional information has been gained about
their particular function, source, structure and molecular basis for activity. Ever since the
presence of these factors was first hypothesized by Bloom and Bennett in 1966, the field of
immunology has grown. The isolation, characterization and use of cytokines have not only
aided in understanding the intricacies of the immune system, but have also made possible
the manipulation of immune responses for therapeutic benefit.
The first two decades of cytokine research dealt with classifying the biological
activities of "factors" found in preparations of crude or partially purified culture conditioned
media supernatant (CMS) fluids. Derived from lymphoid cells induced with various
mitogens or antigens, these CMS factors were added to cultures of "indicator cells" and the
ensuing cytological phenomena would be observed and measured. For example,
preparations able to activate macrophages were said to contain macrophage activation factor
or MAF (Nathen et al., 1983). The number of acronyms grew as new factors were found
to exhibit chemoattraction, inhibition, maturation, growth, transformation, differentiation,
necrotizing and stimulation activities on various cell types (as reviewed by Paul, 1989;
Vilcek and Lee, 1994). Factors were then characterized by source cell type; for example, if
the source was a cell from monocyte/macrophage lineage, the factor was categorized as a
"monokine". A wide range of activities were observed from different cell types under
different induction conditions and it was hypothesized that a single substance could be
assigned to each identified activity (as reviewed by Kishimoto et al., 1994).
It was not until the third decade of cytokine research and the advent of recombinant
DNA technology that substantial progress was made in characterizing the properties of an
individual cytokines. Recombinant DNA technology enabled scientists to produce large
quantities of individual cytokines free from contaminating cellular proteins. A purified
cytokine separated from the mixture of substances found in a given CMS made it possible
to test the hypothesis that a single immunological function could be assigned to each
9
cytokine. These studies showed that purified cytokines often exhibited multiple functions
and overlapping activities. The ability of a cytokine to exert more than one action on
multiple cell types has been termed " cytokine pleiotropy". The term "cytokine redundancy"
describes shared activities by different cytokines (Paul et al., 1989). Even though each
cytokine is different by gene sequence and protein product, their roles in immune responses
are more complicated than previously thought. Therefore categorizing cytokines based on a
single activity is not possible.
Recombinant technology has also made possible the cloning and sequencing of the
cognate receptors for the various cytokines. This lead to another scheme for categorizing
cytokines, based upon conserved receptor structures. For example the receptors for
interleukins 2-6,7,9 and 12, granulocyte macrophage stimulatory factor (GMCSF),
prolactin and growth hormone all share a common structure and have been placed in a
group known as the "hematopoietins" (Paul et al., 1994). The elucidation of shared
receptor structures helped in understanding the pleiotropic and redundant nature of different
cytokines. If receptor structures for different cytokines are similar, then the secondary
messenger molecules that interact with the receptors may also be similar. In fact, recent
information regarding signal transduction has led to a new categorization scheme for
cytokines based upon the families of secondary messengers used in the transduction of
signals (Lin et al., 1995)
Defining Fish Cytokines
There are several technical obstacles that make it difficult to classify as specific
cytokines the different biologically active factors found in primitive animals. The first is
that cytokines are pleiotropic and redundant. This makes it difficult to assign a factor
exhibiting a single activity to one specific cytokine without the complete characterization of
that factor. For example, the activation of macrophages can be induced by interferon
10
gamma; however, other cytokines can activate macrophages as well, such as granulocytemacrophage colony stimulation factor (GMCSF) (Gasson et al., 1991) and interleukin-3
(IL-3) (Schrader et al., 1988). Neutrophil activation is another example of redundant
cytokine activities; induced by interleuldn-1 (IL-1) this activity is also shared by
interleukin-8 (IL-8) (Luger et al., 1983). The pleiotropic nature of cytokines can be seen in
the example of interleukin-2 (IL-2) activity. IL-2 can effect various cell types in different
ways such as the growth of T cells (Paul and Ohara, 1987) or the activation of NK cells
(Trinchieri et al., 1984). The pleiotropic and redundant nature of the specific cytokines
interferon, IL-1 and IL-2 can be seen in Table 2.1 which shows examples of alternate
cytokines exhibiting these activities. It becomes evident from these examples that assigning
a factor found in a CMS to a specific cytokine is difficult unless all of the possible activities
of that factor are determined.
The cytokines interferon, IL-1 and IL-2 were chosen for this comparison because
the majority of non-mammalian literature describing cytokine-like factors assign activity to
these particular cytokines. The experiments in fish systems which describe the presence of
these cytokine activities will be discussed in the following sections. Determining other
possible activities is the second obstacle to defining fish derived factors as specific
cytokines. This is mainly due to the lack of assay systems to study the fish immune
response. In most cases the highly specific and sensitive assay systems used for the
detection of mammalian cytokines are not available for fish. For example, cytokine­
dependent cell culture lines that require specific cytokines to sustain growth do not exist for
analysis of fish cytokine -like factors. The first such cell line to be described is the murine
CTLL line which requires IL-2 or IL -4 for growth (Gillis, 1978). This cell line is sensitive
to cytokine concentrations between 50 pg/mL - 50 ng/ mL of cytokine (Gearing and
Thorpe, 1988). Although long term cultures of fish lymphocytes are possible, the
11
NAME
ACTIVITY
ALTERNATE
REFERENCE
Interferons
antiviral activity
Lymphotoxin
Hamblin et al., 1993
Weissenbach et al, 1980
Mestan et al., 1986
Interleukin-6
TNF
macrophage
activation
Interleukin-3
Interleukin-4
Interleukin-8
GMCSF
Lymphotoxin
Interleukin-1
(endogenous
pyrogen)
fever induction,
shock
neutrophil
activation
acute phase
reaction
Interleukin-2
(T Cell Growth
Factor)
Schrader et al., 1988
Paul et al., 1991
Luger et al., 1983
Gasson et al., 1991
Vilcek et al., 1991
GMCSF
Okusawa et al., 1988
Vilcek et al., 1991
Gasson et al., 1991
Interleukin-8
Interleukin-3
Interleukin-6
Luger et al., 1983
Schrader et al., 1988
Sehgal et al, 1995
Lymphotoxin
Dinarello et al, 1991
TNF
Lymphotoxin
induces growth and Interleukin-3
proliferation of T Interleukin-4
Cells
Interleukin-7
Interleukin-9
Interleukin-12
Interleukin-15
Londei et al., 1989
Paul & Ohara,1987
Noguchi et al., 1993
Moeller et al., 1990
Zeh et al., 1994
Thomson et al., 1994
increased NK
TNF
activity
Interferon a
Ostensen et al., 1987
Lucero et al., 1981
Perussia et al., 1980
Weigent et al., 1982
Trinchieri et al., 1984
Nagler et al., 1988
Edington et al., 1994
Naume et al., 1992
Shau & Golub, 1993
Interferon
13
Interferon y
Interleukin-2
Interleukin-4
Interleukin-7
Interleukin-12
NKEF
Table 2.1 The pleiotropic and redundant activities of cytokines.
12
characterization of the particular cell type and specific cytokine response characteristics of
this cell type have yet to be determined (Lin et al., 1992).
A third obstacle to fish cytokine study is that some immune phenomena dependent
on specific cytokines are not present in fish, such as antibody class switching. Interleukin­
4 or interleuldn-13 must be available to differentiating B-cells in vivo in order to switch to
class E (IgE) or IgG4 immunoglobulin synthesis (Pene et al., 1988; Punnonen et at,
1993).
The last obstacle to studying fish cytokines is the paucity of cloned cytokine fish
genes available for characterization or comparison. Cytokine sequence information from
one fish species would be valuable in isolating these genes from another species. Cloned
cytokine genes from fish would also help in the characterization of these cytokines.
Purified cytokine proteins could be made by recombinant techniques and used in assay
systems to determine which cell types are affected and the different activities induced.
Attempts to purify these factors from fish by conventional methods such as
chromatography have been done but this has often resulted in different molecular weight
fractions containing the same activities. Because of these various obstacles, literature
citations for fish and lower animals generally refer to cytokine activities with the
qualification hyphenated "like" to suggest that the factor(s) being described exhibits some
but not all the qualities of a particular cytokine.
Interferons
The hallmark experiment by Isaacs and Lindemann in 1957 first described
interferon (INF) as a "non-hemagglutinating, heat stable, macromolecule that was released
from chick embryos following incubation with inactivated influenza virus and that
interfered with the growth of live virus ". Interferons have since been found to be
produced by several cell types from a variety of species (as reviewed by Secombes, 1994).
13
Three classes are described in mammals, INF-a, INF-0 and INF-y, and these in turn are
classified into different group types on the basis of their biological and biochemical
properties (Stewart, 1980). Type I interferons include INF-a and INF-ii Both are stable
to pH 2.0 and are produced by many cell types including fibroblastic, epithelial and
leukocytic cells. Though very similar biologically, they share only 30 % amino acid
homology, 45 % nucleotide homology and are thought to have diverged from a common
ancestral gene hundreds of millions of years ago (Taniguchi et al., 1980).
There is abundant literature demonstrating evidence of type I interferon-like factors
in various species of fish including salmonids, carp, flat fish, fathead minnow and goldfish
(as reviewed by Secombes, 1994). The majority of these reports deal with in vitro studies
measuring the production of interferon-like activity from fibroblastic and epithelial cell lines
in response to viral infection (Gravel and Marlsberg, 1965; De Sena and Rio, 1975).
Interferon activity was also demonstrated to be elicited in vivo in response to pathogenic
viruses VHS or Egtved (Dorson et al., 1975; de Kinkelin et al., 1982). In these studies,
serum samples were assayed for the capacity to induce non-specific protection to virus
challenge. This was done by means of tissue culture assay or passive transfer to live
animals after removal of virus specific antibodies. The interferon-like activity was shown
to be non-specific since it induced protection against an unrelated pathogenic virus,
infectious pancreatic necrosis virus (IPNV). Biochemical evidence showed that these
serum or culture-derived factors responsible for the induction of virus protection were
similar to type I interferons. Interferon-like factors from both sources, in vivo and in
vitro, are acid- and heat-stable. Surprisingly, they differ widely in molecular weights, 26 k
Da and 94 k Da, respectively (Dorson et al., 1975; De Sena and Rio, 1975). There is little
molecular evidence that fish interferons are related to mammalian type I interferons
(reviewed under the section Molecular Evidence of Cytokines in Fish in this chapter).
However, the cell type source, induction method and chemical stability suggest they are
similar.
14
Type II interferon, INF-y, appears unrelated to both type I interferons since the
proteins not only share less than 10 % amino acid homology , but are also biochemically
and functionally distinct (Epstein, 1984). Wheelock reported in 1965 a substance that
inhibited virus growth similar to previously described interferons, yet it was not acid or
heat stable. Also this substance was alternatively produced by leukocytes induced with a
lectin, phytohemagglutinin, and not by the classical viral induction methods. Later it was
determined that T cells were the primary cell source for the newly designated interferon,
INF-y and it was thus categorized as a lymphokine (Kiener and Spitalny, 1987).
Additional to the biochemical, inducing agent and cell source differences, INF-y is a
multifunctional cytokine mediating a wide range of activities besides inhibition of virus
growth. These include activation and differentiation of hematopoietic cells and regulation
of expression of other cytokines and cytokine receptors (Hamblin, 1994). A well
characterized example of cell activation induced by INF-y is macrophage activation.
Macrophage activating factor (MAF) was the designation given to culture supernatants
containing an activity that enhanced the oxygen-dependent and independent antimicrobial
and anti-tumor mechanisms of macrophages (Cohn, 1978; North, 1978).
Although many cytokines exhibit MAF activity, it was first shown in the human and
murine systems that a lymphokine biochemically identical to INF-y could specifically
increase the oxidative metabolism and respiratory burst of macrophages measured by the
reduction of ferricytochrome C (Nathen et al., 1983; Fukazawa et al., 1984 ).
Activation of rainbow trout (Oncorhynchus mykiss) macrophages in culture has
been measured by both increased respiratory burst and bacterial killing. Two by-products
of macrophage respiratory burst have been measured by an alternative method using the
colorimetric indicators nitroblue tetrazolium (NBT) to indicate superoxide anion and phenol
red (dependent upon the oxidation of horseradish peroxidase) to indicate hydrogen
peroxide production. In the first such study phorbol myristate acetate (PMA) and
concanavalin A (Con A), known inducing agents of INF-y in mammals (Yip et al., 1981),
15
were added to cultures of rainbow trout head kidney and blood leukocytes in vitro
(Graham and Secombes, 1988). After three hours the media were removed and the
cultures were washed several times to remove residual inducing agent. New medium was
placed on the cultures and allowed to incubate for an additional 48-72 hours. The CMS
supernatants were then used to treat a partially purified culture of head kidney
macrophages. It was found that the levels of reactive oxygen species and bacterial killing
were increased significantly. This was one of the first studies to suggest that a
lymphocyte-derived factor or a lymphokine existed in fish for which a specific immune
response mechanism could be measured, namely macrophage bactericidal activity. In a
follow up study, panning experiments were performed on the leukocytes with a
monoclonal antibody specific for teleost immunoglobulin (Deluca et al., 1983) and it was
found that leukocytes negative for surface immunoglobulin were responsible for the
production of macrophage activating factor (Graham and Secombes, 1990). This study not
only determined the type of cell which produced MAF, but also provided further evidence
that trout lymphocyte populations, like mammalian lymphocytes, can be separated into T
and B-like cells based on immunoglobulin and cytokine expression . Of course to confirm
the T cell hypothesis, the trout derived MAF would have to fit some of the defining
characteristics of INF-y. The necessary chemical and biological experiments were
performed in a third study that supported this hypothesis (Graham and Secombes, 1990).
First, CMS fluids containing the factor were shown to confer viral resistance on a trout
epithelial cell line, a finding that characterized the factor as an interferon. Second, both the
MAF and interferon activities co-eluted from HPLC size exclusion chromatography which
demonstrated that a single factor with an apparent molecular weight of 19 k Da was
responsible for both activities. Lastly, the factor displayed sensitivities to acid and
temperature resembling those of mammalian INF -'y.
16
Interleuldn-1
Interleukin-1 (IL-1) is one of the most widely described interleukins. It is
produced by a variety of cell types, but mainly those from the monocyte and macrophage
lineage (Di-Giovine and Duff, 1990). It is also the most pleiotropic of the cytokines
including the interferons. The range of biological activities includes fever, shock, acute
phase response and cytokine synthesis induction, activation of neutrophils and endothelial
cells as well as induction of mitogenic responses in T, B and fibroblastic cells (Hamblin,
1993). With such a variety of sources and actions, it is not surprising that IL-1 like factors
have been described for a number of species from across the phylogenetic spectrum.
Factors exhibiting lL-1 activity have been found in echinoderms, tunicates, annelids,
gastropods, amphibians, birds and teleost (Beck et al., 1989; Beck and Habicht, 1986;
Paemen et al., 1992; Granath et al., 1994; Watkins et al., 1987; Hayari et al., 1982 and
Verburg-van Kemenade et al., 1995).
The earliest evidence for the necessity of an IL-1 like factor in fish came from
studies of channel catfish. Paralleling the murine experiments that determined the
requirement for accessory cells (monocytes/macrophages) in order for Con A induced T
cell mitogenesis to occur (Lee et al., 1976), the same need was demonstrated for fish
leukocyte cultures (Clem et al., 1985). This requirement for accessory cells in the murine
system was attributed to IL-1, which was provided by monocytes or macrophages and was
a necessary T cell co-mitogen with the Con A. This was confirmed by eliminating the need
for accessory cells when the culture media were supplemented with interleuidn-1 (Chu et
al., 1985). Again, a similar experiment was done with fish (El laser, 1989). Macrophagedepleted channel catfish lymphocyte cell cultures supplemented with lipopolysaccharide
(LPS) conditioned culture media were found to respond as well to Con A as did
macrophage intact cultures. The same paper goes on to show that human or murine IL-1
can substitute for accessory cells in catfish and that antibodies against human IL-1 can
17
block this activity
.
Purification of the catfish IL-1-like factor from monocyte CMS
revealed two forms with molecular weights of 15 and 70 k Da. The 15 k Da protein was
active on the murine T lymphoma line LBRMSS-1AS used for the detection of human and
murine interleukin -1 (Klaus, 1987). The 70 k Da protein was active only on catfish cells.
Western blot analysis also confirmed that the isolated catfish proteins with IL-1-like activity
cross reacted with antibody to human IL -1.
Recently, IL-1-like factors have been characterized for carp (Cyprinus carpio)
(Verburg-van Kemenade et al., 1995). Macrophage CMS were similar to the channel
catfish material in that they contained specified proteins with IL -i activity
.
The
supernatants tested positive for IL-1 bioactivity in both fish cells and the IL-1 dependent
murine cell line D10(N4) bioassay system. Sheep-derived polyclonal antisera to human
IL-1 was able to nullify the activity in the supernatants in both assay systems. Western blot
analysis results were similar to the catfish study in that two proteins were revealed, with
respective molecular weights of 15 k Da and 22 k Da.
Accessory cell replacement factors like IL-1 have been demonstrated for rainbow
trout (Ortega, 1993). In contrast to the catfish and carp studies, it was shown that purified
human and not murine IL-1 affected trout lymphocytes as measured by in vitro antibody
response to defined antigens after glucocorticosteroid suppression (Tripp, 1987). Two
studies cite the specific presence of a rainbow trout IL-1-like protein antigenically similar to
human IL-1. Both use commercially available ELISA based kits for the detection of human
cytokines in serum or tissue culture samples. The first such study used supernatants from
mitogen (Con A or LPS) stimulated rainbow trout lymphocyte cultures and found elevated
levels in stimulated cultures (Ahne, 1994). The second study analyzed serum levels of IL­
1 after in vivo infection with virus and detected elevated IL-1 levels relative to those of
non-infected fish (Ahne, 1994).
18
Interleuldn-2
Interleukin-2 (IL-2) was first defined as a growth-promoting factor for bone
marrow-derived T lymphocytes secreted from leukocyte cultures stimulated with T-cell
mitogens (Morgan et al.
1976).
It has since been recognized as a pleiotropic cytokine
exhibiting various effects on a multitude of cell types. These include not only the growth
and differentiation of T cells but also B cells, natural killer (NK) cells, monocytes,
macrophages, lymphokine activated killer (LAK) cells and oligodendrocytes (Goldsmith
and Greene, 1994).
Unlike the situation with interleukin-1 or interferon, there are few reports of IL -2­
like activity in fish. The primary obstacle has been the definitive characterization of T cells
in fish lymphocyte populations. Monoclonal antibodies which recognize unique T cell
determinants such as those found on mammalian T cells have not yet been developed.
This would be necessary for positive identification and isolation of T cells from
heterogeneous populations of fish lymphocytes. Thus, most fish studies investigating IL2-like factors have been confined to the definition of factors produced by classical T cell
mitogens on heterogeneous lymphocyte populations or surface immunoglobulin negative
enriched populations. To add to this difficulty, the reports of the few studies that
characterize fish derived IL-2-like activities are sometimes contradictory.
The first two reports of IL -2 -like activity in fish used phytohemagglutinin-M
(PHA), phorbol myristate acetate (PMA) and alloantigenic stimulation of carp head kidney
or peripheral blood lymphocytes to generate factor containing CMS (Capsi and Avatalion,
1984;
Grondel and Hamersen,
1984).
Both studies found that these types of CMS were
mitogenic on putative T cell lymphoblasts while only weakly mitogenic for resting cells.
This activity is similar to mammalian T cells in that activated cells respond to IL-2 to a
greater extent than do resting cells (Gillis,
1978).
Murine derived IL-2 from the EL-4 cell
line did not affect fish lymphoblast growth, suggesting that IL-2 does not have the same
19
cross species activity of IL-1. In the catfish system, a similar finding for incompatibility of
murine IL-2 was shown (Clem et al., 1990). In this case, culture supernatants from catfish
putative T-cell lines did not effect the mouse CTLL cell line used to assay for mouse IL-2.
When recombinant mouse IL-2 was tested on catfish cells, it also had no mitogenic effect
on the fish cells. In contrast, it was reported by Caspi and Avtalion, 1984 that rat, human
and monkey cell line derived IL -2 containing supernatants did induce a mitogenic response
in carp T lymphoblasts. Yet, in the same study when partially purified human IL-2 was
used on carp cells no effect was seen.
It becomes more confusing when the data from the study done by Ahne, 1994 is
considered. In this study IL-1 and IL-2 were detected in trout sera using anti-human
interleukin monoclonal antibody based ELISA kits. IL-1 and IL -2 were detected in sera
from virus infected rainbow trout and in trout cell derived CMS. It is difficult to
understand why IL-2 containing CMS from rat, monkey and human would affect fish cells
but not purified mouse or human IL-2 as demonstrated by Caspi and Avtalion, 1984. This
appears to be in contradiction with Ahne' s findings that trout IL-2 is detected by anti­
human IL-2 monoclonals. A probable explanation lies in the composition of the various
CMS from rat, human and monkey. Since they are crude supernatants known to contain
IL-2, it is also likely that they contain some of the many interleukins which support T cell
growth (refer to Table 2.1). These may be the interleukins detected in Caspi and Avtalion
in vitro assay system and not IL-2. It is also possible that the CMS contained small
amounts of IL-2 which acted in a synergistic fashion with other T cell growth promoting
cytokines present in the mixture. The synergistic affect of different cytokines has been
demonstrated in mice (Vilcek and Le, 1994). It is possible that the IL-2 from human or
mouse though antigenically similar, demonstrated by Ahne, are insufficiently cross reactive
to effect fish cells without the presence of complimenting cytokines, which would be
present in the rat, monkey or human cell CMS mixtures tested.
20
Molecular Evidence of Cytokines in Fishes
There are only two reports of fish cytokine genes which have been cloned by
designing primers to homologous regions of mammalian cytokine gene sequences and
employing PCR amplification to obtain a cDNA clone. One possible reason that it has been
so difficult to clone these genes is that such fish genes may not be sufficiently similar to the
mammalian genes, and the primers designed for PCR amplification do not attain the
necessary binding required for the detection of the particular cytokine gene (Secombes,
1994). It has also been suggested that an alternative method for isolating cytokine genes
from fish could be more successful (Secombes, 1994). In vitro analysis of gene products
screened from cDNA libraries has often been successfully employed in mammals to obtain
different cytokines (Yokota et al., 1985) and the same method might prove valuable for fish
systems. Actually, both methods have been used successfully to isolate IL-2, INF, NKEF
and STAT like genes from two species of fish, flatfish (Paralichthys olivaceus) (Tamai et
al., 1992 and 1993) and rainbow trout (Oncorhynchus mykiss) (Mourich et al., 1995).
Interferons
Human INF cDNAs have been used as probes in Southern blot analysis to detect if
cross hybridization occurs with genomic DNAs isolated from a number of vertebrates
including fish (Wilson et al., 1983). Both INF-a and INF-13 probes were used to
distinguish what gene families might be conserved in the various animals. Complex
multigene families were detected in all mammals tested for INF-a but no such genes were
detected in fish. However, under low stringency hybridization condition, gene sequences
related to INF -13 could be detected in perch, minnow, dace and the stone loach. This
molecular evidence, along with a number of biological studies (to be discussed later),
21
strongly suggests that INF genes are present in fish, but share limited sequence homology
mammalian gene sequences.
An INF-like gene from flatfish has been cloned and expressed by the method of
cDNA library screening (Tamai et al., 1993). A cDNA library was constructed from
mRNA isolated from a flatfish leukocyte line immortalized by the transfection of human c­
fos and c-ras oncogenes (Tamai et al., ESACT proceedings in press). Gene products were
expressed in an in vitro translation system and assayed for INF bioactivity on EPC cells
against Hirame rhabdovirus. The INF gene was isolated, sequenced and inserted into a
mammalian expression vector for production in a transformed BHK-21 cell line. The
complete nucleotide sequence for the flatfish INF cDNA contained a single open reading
frame coding for a 138 amino acid polypeptide which was determined to have a molecular
weight of 16 k Da by SDS-polyacrylamide gel electrophoresis.
Comparison of the flatfish INF amino acid sequence to various mammalian alpha
and beta INFs showed little conservation. The highest degree of homology 24 % was with
the human INF-a gene. Conservation with human INF-13 protein was much less, with
only 14 % identity. This is contradictory to the findings of Wilson and coworkers, 1993
that human alpha INF families are not detectable in fishes. However, flatfish genomic
DNA was not used in their analysis.
The flatfish INF-like protein shared little sequence homology with mammalian
INFs and appeared antigenically and chemically dissimilar. Antiserum to human INF-a or
INF-13 did not cross-react with the flatfish protein. Furthermore, the flatfish protein was
not stable to pH 2.0, at which pH type I INFs are stable.
It may be that the flatfish protein described by Tamai and co-workers, is not a type I
but a type II INF. The protein has more of the attributes of a type II interferon since it is
derived from leukocytes is acid labile and yet still retains anti-viral activity. Although this
may be possible, no such sequence or bioactivity comparisons were made in this report.
22
Interleukin-2
Interleukin-2 cDNAs have been isolated, cloned and sequenced from a number of
species including human, mouse, rat, cow, gibbon, sheep and pig (Taniguchi et al., 1983;
Kashima et al., 1985; McKnight et al., 1989; Chen et al., 1985; Reeves et al., 1986; Seow
et al., 1990 and Goodall et al., 1991). A significant degree of amino acid homology (44
%) exists among all seven species, including the positional conservation of three cysteine
residues (Goldsmith and Greene, 1994). The primary polypeptide has an average length of
153 residues with the exception of the mouse IL-2 which has an insertion of 14 residues
beginning at residue 27 (Kashima et al., 1985). The primary polypeptide undergoes
several postranslational processing events which are essential for bioactivity. There is a
cleavage of the first 20 residues which acts as a signal peptide. There is an addition of
carbohydrate to the threonine residue at position 3 and the formation of a disulfide bond
between the cysteines at positions 58 and 105 (Robb et al., 1983). The final 15.5 k Da
protein is almost exclusively produced by activated T cells, but some reports suggest that
activated B cells also have a limited ability to produce IL-2 (Taira et al., 1987; Walker et
al., 1988).
Only one report of a fish derived IL-2-like cDNA has been made to date. This gene
was cloned from the genomic DNA of the Japanese flat fish by PCR amplification, using
primers designed to well conserved regions of the gibbon, mouse, human and bovine IL-2
sequences (Tamai et al., 1992). The homologous regions included conserved intervening
sequences, introns, found in the genomic sequences of the various interleukin-2 genes.
Three PCR product were produced from three different primer pairs. The PCR products
were independently cloned into pUC119 and the inserted sequences were determined. The
sequences were joined through a process that was not well described to generate the full
length cDNA. The final sequence was inserted into a mammalian expression plasmid
vector and the recombinant plasmid was used to transform COS-1 cells for expression into
23
culture supernatants. The final cDNA encoded a polypeptide consisting of 102 residues
with a predicted molecular weight of 10.28 k Da that was bioactive on flatfish PL51
lymphocyte putative T cell line.
A comparison of the predicted amino acid sequence of the flatfish to mouse, gibbon
and human demonstrated a relatively significant degree of homology of 42 %. Although
the homology was significant, the flatfish IL-2 gene was missing some of the essential
amino acid residues that have been found to be necessary for mammalian IL -2 biological
activity. Only two cysteines are encoded in the flatfish IL-2, not three as in mammals,
which are not in the correct position to form the internal disulfide bond necessary for IL -2
protein structure. Also although there was an amino terminal glycosylation site the position
of the threonine residue was not conserved with mammalian IL-2s.
The flatfish IL-2 gene appears to be related to mammalian interleukins to a limited
degree. However, most of the literature citing IL-2 activity in fish and cross reactivity with
mammalian interleukins suggests that fish IL -2 would not be closely related to mammals
.
Enhancing Factors of Natural Killer Cells
Natural killer (NK) cells have been well defined in mammals (as reviewed by
Trinchieri, 1989). The presence of these cells was first observed in studies dealing with
cytotoxicity of normal human lymphocytes on both allogeneic and syngeneic tumor cells
(West et al., 1975). It was found that in the absence of pre-sensitization of the responding
effector population to the target tumor line, spontaneous cytotoxicity was rapid and
unrestricted by MHC recognition. This non-adaptive, non-MHC-restricted cell mediated
cytotoxicity was defined as "natural" cytotoxicity and the effector cell responsible for this
activity was defined as the natural killer (NK) cell. Although the lineage of these cells still
remains unclear, they have been characterized, along with their functional activity, as large
granular lymphocytes that are CD3, CD4 negative and CD16, CD56 positive (Trinchieri,
24
1994). The central role of NK cells is immune surveillance to eliminate neoplastic tumors,
virus infected cells and various microbial organisms (Ritz et al., 1988; Trinchieri, 1989).
Also, NK cells are involved in immune development by the regulation of hematopoiesis
(Janeway, 1989).
NK cell are less well defined in fish, since there are no distinguishing surface
markers available for fish lymphocytes. However, similar physiological and biological
attributes have been described for a fish lymphocyte population termed "nonspecific
cytotoxic" (NC) cell. First demonstrated in various warm water fish species and soon after
in studies with channel catfish, leukocytes were able to lyse tumor cell lines in a non-MHC­
restricted manner (Hinuma et al., 1980; Graves et al., 1984 and Evans et al., 1984). Some
biophysical aspects of this cell type have demonstrated similarities to NK cells such as cell
density, non-adherency, and a lack of phagocytic activity (Evans et al., 1984; Evans et al.,
1987). Metabolic requirements for the cytolytic activity and the effect of antibody on NC
cells suggests that these cells are the piscine counterpart to the mammalian NK cell (Carlson
et al., 1985). The NC cell of channel catfish has also been shown to eliminate parasitic
infections in a similar fashion to mammalian NK cells (Graves et al., 1985).
Non-specific cytotoxic cell activity has also been demonstrated with lymphoid cells
in other fish species. For example, damselfish lymphocytes have been shown to have lytic
activity against neurofibromas (Mckinney and Schmale 1994). Also, various species of
salmonids have lymphoid cells with NC activity. In rainbow trout, NC cells exhibit
cytolytic mechanisms of apoptosis, necrosis and MHC-unrestricted recognition of human,
mouse and teleost target cells (Greenlee et al., 1991).
The role of NC cells in virus resistance was demonstrated by enhanced killing of
infected allogenic target cells by rainbow trout, Atlantic salmon (Salmo salar ) as well as
golden shiner (Notemigonous crysoleucas) leukocytes (Moody et al., 1985). Because of
these functional and physical similarities, it has been suggested that fish NC cells are
phylogenetic progenitors of the mammalian NK cell (Evans et al., 1992). However, a
25
number of distinguishing properties warrant the separate name designation. The NC cells
of channel catfish are different from NK cells of mammals in killing kinetics, radiation
sensitivity, temperature optima and a lack of cytoplasmic granules (Evans et al., 1984;
Carlson et al., 1985).
Various studies with teleost have used lectin stimulation to enhance the activity of
nonspecific cytotoxic cells. Lectin-dependent cell mediated cytotoxicity (LDCC)
enhancement attributed to NK cell activity has been demonstrated in mammals (Bevan et
al., 1975; Bonavida and Bradley, 1976; Rubens and Henny, 1977 and Davignon and
Laux, 1978). As well, enhanced activity of rainbow trout (Oncorhynchus mykiss) and
brook trout (Salvelinus fontenalus) NC cells has been demonstrated by the addition of
phytohemagglutinin (PHA) to culture media conditions (Hayden and Laux, 1985). PHA
and concanavalin-A (Con A) have been shown to have similar effects on carp and channel
catfish NCC activity (Evans et al., 1992; LeMorvo-Rocher et al., 1994).
Besides lectins, infection of target cells virus (Hergerman et al., 1977) or certain
microorganisms (Wolfe et al., 1976) and exposure to a number of different cytokines (as
reviewed by Trinchieri, 1989) can also affect the in vitro or in vivo activity of NK cells.
Several cytokines manifest these effects on NK cells but those that have been observed to
have a "direct effect on increased killing capacity" are type I and II interferons (Lucero et
al., 1981; Perussia et al., 1980 and Weigent et al., 1982), tumor necrosis factor (Ostensen
et al., 1987), interleukin-2 (Trinchieri et al., 1984), interleukin-4 (Nagler et al., 1988),
interleukin-7 (Edington, 1994), interleukin-12 (Naume, et al., 1992) and a newly cloned
and characterized factor termed natural killer enhancement factor (NKEF) (Shau et al.,
1994).
The presence of an uncharacterized natural killer enhancement factor was first
postulated when it was observed that red blood cells (RBCs) played a role in the
modulation of NK mediated lysis (Shau and Golub, 1988) and the growth and activity of
IL -2 activated killer (LAK) cells (Yannelli et al., 1988). Shau and Golub carried out
26
specific in vitro experiments to observe the effects of RBCs on NK activity. They
determined that the cytotoxicity of NK cells was enhanced in a dose dependent fashion
based upon the number of RBCs present in cytolytic assays, whether from autochthonous,
allogeneic or xenogeneic sources. Enhanced killing was seen against two NK sensitive
tumor cell lines K562 and U937 in three different assay systems, chromium release, lactate
dehydrogenase release and inhibition of thymidine incorporation. Yannelli and co-workers
(1988) made a similar observation during clinical studies of cancer patients receiving IL-2
leukophoesis therapy. This type of therapy involved the removal and purification of
peripheral blood lymphocytes from 40 different patients followed by in vitro exposure to
IL-2. It was found that the number and cytolytic potential of LAK cells decreased as more
autologous RBCs were removed during the culturing period.
Shau and co-workers went on to identify and characterize the factor in RBCs
influencing the growth and activity of natural killer cells (Shau et al., 1993). Red blood
cell proteins were precipitated by ammonium sulfate and fractionated by gel filtration high
performance liquid chromatography (HPLC). The individual fractions were tested for NK
enhancement and it was determined that the factor was a trypsin sensitive cytosolic protein
with a native molecular weight between 300 and 400 kDa. Under SDS-PAGE denaturing
conditions the protein gave an apparent molecular weight between 48 and 24 kDa. The
purified proteins were used to immunize rabbits to produce polyclonal antiserum. This
antiserum was then used to determine if the activity induced by the protein could be
specifically inhibited by pre-incubation. Immune anti-serum was found to inhibit the
enhancement activity while control rabbit serum had no effect. The serum to the newly
termed "natural killer enhancing factor" (NKEF) was also used to detect the presence of
NKEF in the cytosol of the NK-sensitive erythroleukemic cell line K562 by western blot
analysis. Both K562 and red blood cells produced a reactive band at 24 kDa. Tryptic
digests of NKEF were then made and three peptides were isolated and sequenced.
27
Computer searches of protein data bases revealed that one peptide was related to a known
protein of a murine erthroleukemia-related gene MER5 (Yamamoto et al., 1989) (Swiss
Protein accession number P20108).
Using the anti-NKEF serum in a immunoblot screening technique, Shau's group
probed a lambda gtl l cDNA expression library derived from the K562 cell line. Two
closely related genes, sharing 88 % amino acid and 71 % nucleotide identity, were isolated
and termed NKEF A and B (Shau et al., 1994). Analysis of the predicted amino acid
sequences indicated several phosphorylation sites and no glycosylation sites. Computer
analysis of gene sequences revealed several homologous proteins from a variety of
organisms ranging from prokaryotes to mammals. The previously examined MER5 protein
shared 61 % and 64 % identity with NKEF A and B respectively. One protein, mouse
macrophage stress related protein (MSP23) (Ishii et al., 1993) (GenBank accession number
D16142), exhibited a noteworthy degree of identity, 93 % and 76 %, with the respective
human NKEF A and B proteins. The function of this 23 k Da protein is yet to be fully
characterized but it has been found to be expressed by murine macrophages exposed to
hydrogen peroxide (Sato et al., 1993). Two other genes, related to the NKEF gene
sequence, appear to also be involved in responses to oxidative stress conditions in the cell.
The thiol-specific antioxidant gene protein product (TSA) (Chae et al., 1993) (GenBank
accession number S64263) of Saccharomyces cerevisiae is 57 % identical to NKEF A and
66 % identical NKEF B and has been shown to be oxygen-inducible with anti-oxidant
activity (Kim et al., 1989). Also, the alkyl hydroperoxide reductase C22 subunit gene
(AHPC) of Salmonella typhimurium shares 34 % and 30 % amino acid identity with
NKEF A and B proteins respectively. The expression of this gene has been shown to be
under the control of the regulatory element OxyR which up regulates the expression nine
other genes in S. typhimurium responsible for controlling oxidative stress (Tartaglia et al.,
1990).
28
Although no other function has been identified for the human NKEF proteins
besides the enhancement of NK activity, it has been postulated by Shau that since related
proteins are so well conserved throughout evolution, NKEF A and B may also be involved
in coping with oxidative stress in RBC and other cells. A newly reported gene with
significant homology to human NKEF has been found in rainbow trout (Mourich et al.,
1995). This gene sequence shares 79 % and 72 % nucleotide sequence identity and 75.5 %
and 71 % amino acid identity with human NKEF A and B, respectively. The trout gene
protein product (NCEF), produced by in vitro translation has a molecular weight similar to
that of the human proteins, 25 k Da by SDS-PAGE. Western blot analysis with
monoclonals specific for either the A or B NKEF demonstrates that the trout protein is
related antigenically to both human proteins. To determine if the trout protein was
functionally related to human NKEF, effects on NCC activity were tested by radio-labeled
chromium (51Cr) release and DNA fragmentation of target cells. The NCEF gene was sub
cloned into a eukaryotic expression plasmid under the control of a CMV promoter and
transfected into a salmonid cell line CHSE-214 by lipid-DNA complex. The expression of
NCEF in the transfected cell cultures was confirmed by immunohistochemistry with a cross
reactive monoclonal that recognizes both NKEF A and B in native form. Enhanced
cytolysis of the 51Cr labeled target cells expressing NCEF was seen for lymphocytes from
trout pronephros (head kidney), spleen and peripheral blood. Also, a similar increase in
apoptotic killing measured by DNA fragmentation was seen in target cultures expressing
NCEF.
Cytokine Signal Transduction
Cell to cell communication is mediated in part through membrane bound receptors.
Some receptors are specific for soluble factors, for example cytokines, produced by cells of
the immune or hematopoietic systems (Hamblin, 1993). The binding of a cytokine to its
29
cognate receptor induces receptor dimerization and the subsequent triggering of intercellular
signaling molecules, "secondary messengers" (He ldin, 1995). The resulting effect is the
rapid and selective transcriptional activation of target genes regulated by specific cytokine
exposure. The protein products derived from these genes control cellular functions such as
growth, differentiation or effector states (Sabath et al., 1990). The step in this process
between cytokine-receptor binding and gene activation or "signal transduction" has recently
become a focus of study in immune regulation.
It has long been known that the phosphorylation state of various cytoplasmic
proteins are involved in signal transduction. However, the specific protein kinases and
substrates mediating this process remained unknown in earlier studies (Hatakeyama et al.,
1991). Since the intracellular domains of cytokine receptors lack cytoplasmic catalytic
domains, other receptor associated protein were thought to be "recruited" to transmit
secondary signaling processes (Torigoe et al., 1992; Venkitaraman and Cowling, 1992). A
study examining the signaling properties of cytokine receptors demonstrated the presence
of two important cytoplasmic domains which interact with catalytic proteins. The
membrane distal domain was shown to be involved in the activation of the Ras pathway,
while the membrane-proximal domain was shown to activate tyrosine protein kinases (Ih le
and Kerr, 1995). The tyrosine protein kinases that associate with cytokine receptors and
function in these pathways were identified in earlier studies as kinases belonging to the
Janus family (Jak) (Argetsinger et al., 1993; Russell et al., 1994).
Cytokine receptor binding activates Jak proteins which in turn catalyzes the
phosphorylation of a class of transcriptional factors termed "signal transducers and
activators of transcription" (STAT) proteins. Phosphorylation of STAT proteins results in
their rapid dimerization, nuclear translocation and association with secondary
transcriptional factors. These protein complexes will then bind to specific sequences found
in the promoters various target genes which results in active gene transcription. (Darnell et
al., 1994).
30
To date six different STAT proteins (STAT 1-6) have been characterized and
identified. These have been shown to be involved in the signal transduction of several
different cytokines including; INF a, INF 13, IL-2, IL-4, IL-6, IL -7, IL-10, IL-12, IL-13,
IL-15 (as reviewed by Ivashkiv, 1995) and growth factors EGF and prolactin (RuffJamison et al., 1995; Gilmor and Reich, 1994).
At this time little information on signal transduction of cytokines and cytokine-like
factors in teleost fishes is available. In chapter 5 of this thesis we report on a partial gene
sequence derived from rainbow trout (Oncorhynchus mykiss) that closely resembles the
signal transduction and transcription activating factor STAT5 found in various species of
mammals.
STAT5 is involved in the signal transduction process of interleukins 2, 7 and 15
(Lin et al., 1995). Cells expressing the corresponding receptors will respond to these
cytokines by Jak driven phosphorylation of Src homologous-2 (SH2) domains in STAT5
proteins (Frank et al., 1995). Trans location to the nucleus and binding to promoter
sequences containing the consensus inverted repeat G/TAA structure called the y-INF­
activation site (GAS) results in the rapid transcriptional activation of these genes (Gilmor
and Reich, 1994). Interleukin-2 as well as mitogenic stimulation have been shown to
rapidly induce nuclear translocation and DNA binding activity of STAT5 in freshly isolated
PBLs. This binding activity appears to be transient since it drops off dramatically only
hours after stimulation (Hou et al., 1995).
The tissue distribution of STAT5 has also been determined (Lin et al., 1995).
Northern blot analysis demonstrates active transcription of STAT5 gene in heart, placenta,
lung, muscle, spleen, thymus , prostate, testis, ovary, intestine, colon and peripheral blood
lymphocytes. However, transcription of STAT5 is undetected in the brain, liver, kidney
and pancreas.
31
CHAPTER 3
NATURAL KILLER CELL ENHANCEMENT FACTOR-LIKE GENE IN
RAINBOW TROUT (Oncorhynchus mykiss)
Dan V. Mourich, John Hansen and Jo Ann Leong*
Department of Microbiology and the Center for Salmon Disease Research,
Oregon State University, Corvallis, Oregon 97331, U.S.A.
*Correspondence should be addressed to this author
Published September 1995
Immunogenetics
42 (5):438-439
32
Introduction
Two closely related human genes termed natural killer enhancing factor A and B
(NKEF) encode cytosolic proteins expressed in both human red blood cells and the natural
killer sensitive erythroleukemic cell line K562 (Shau et al. 1993). These proteins are found
to augment the cytolytic effects of human natural killer (NK) cells (Shau and Golub 1988)
and increase yields of lymphokine activated killer (LAK) cells (Yannelli et al. 1988).
When the deduced amino acid sequences of NKEF A and B were compared to deposited
sequences, significantly related regions were found in genes of other unrelated species such
as bacteria, amoebae, yeast and mice (Shau et al. 1994). This high degree of sequence
conservation through a broad evolutionary range of species suggests that it may be possible
to amplify related gene segments from trout cDNA using primers designed to these
conserved regions.
Primers were constructed from two homologous protein motifs found within the
various sequences. These primers were then used in the polymerase chain reaction (PCR)
to amplify a product from a trout derived peripheral blood cell cDNA pool. The 5' primer
ME 244 was constructed using the coding sequence for the motif FVCPTE which is 100
% homologous in all species compared. The 3' primer ME 245 is complementary to the
coding sequence for the motif WKPGSDT. This primer is 100 % homologous to the
human NKEF genes and the mouse gene MSP23 (D16142), but is less well conserved in
the other related genes. A PCR product of the predicted size [405 base pairs (bp)] was
visible in an ethidium bromide stained agarose gel. The band was excised and ligated into
the TA cloning vector (Invitrogen). Plasmid clones were screened for insert and prepared
for T7 and SP6 primer sequencing.
33
Comparison of the nucleotide and predicted amino acid sequence of the trout
derived clone with the human NKEF sequences demonstrated a high degree of
conservation for the region amplified. The putative trout NKEF gene segment was then
labeled with 32 P-dCTP and used as a probe to screen a cDNA library derived from trout
peripheral blood cells. A full length clone of 1127 by was isolated and sequenced. The
nucleotide and amino acid sequence shown in figure 1 confirms that the trout gene shares a
high degree of identity with both human NKEF A and NKEF B deduced coding
sequences. Specifically, the trout NKEF shares 79 % and 72 % nucleotide identity and
75.5 % and 71 % amino acid identity with human NKEF A and B, respectively.
Mammalian Natural Killer (NK) cells are defined as large granular lymphocytes that
are CD3, CD4 and CD16 negative, whose central role is immune surveillance (Ritz et al.
1988). Their functions include MHC unrestricted recognition and elimination of neoplastic
tumors, virus infected cells and various microbial organisms, through cytolytic mechanism
of apoptosis and necrosis (Trinchieri 1989). NK cells are less well defined in fish due of
the lack of surface marker reagents. However, similar biological activities of cytolysis
have been described for a nonspecific cytotoxic cell (NCC) found in various teleost, such
as channel catfish (Evans et al. 1984) and salmonids (Moody et al. 1985). Also NC cells
of channel catfish, like mammalian NK cells, function in the elimination of some
microorganisms (Graves et al. 1985). Tumor recognition of Damsel fish NC cells has been
demonstrated by cytotoxicity toward neurofibromas (McKinney and Schmale 1994). In the
specific case of rainbow trout, NC cells have exhibited cytolytic mechanisms of apoptosis,
necrosis and MHC unrestricted recognition of human, murine and teleost target cells
(Greenlee et al. 1991). From this functional related evidence, it has been postulated that the
NC cells of fish are phylogenetic progenitors of the mammalian NK cell counterpart (Evans
et al. 1992). These cytolytic cell types from two phylogenetically different taxa not only
share common biological mechanisms of cytolysis and surveillance but it is now evident
that a specific gene involved in the regulation of these functions is also closely related.
34
Figure 3.1
Complete nucleotide sequence of trout NKEF-related clone. Alignment of
deduced amino acid sequences for trout (Gen Bank accession number
U27125), Human A (L19284) and Human B (L19185) natural killer cell
enhancing factors. The primers used for amplification of the initial trout
derived probe are shown in upper case. (-) indicates identity with the
human sequences. (*) indicate deletions.
35
44
Trout 5'gagcaagctgccgcagtgggaccgaagtagaggcaaaagtgaag
atggctgcaggtaaagcacgaatcgggcatctggcccctggcttcacggccaaagcagtgatgccagacggccagttcaaagacatcagc
t--t
t
t
c- -t t
c-ct
caa
aaHumanA ---t--t- -a--t--taa -t
**t- t- -gcc
c- -g- g
a
a
as -g ca
99 9 ag
Human8
c
g -c
ct-c
Trout
TroutMAAGKARIGHLAPGFTAKAVMPDGQFKDIS
HumanA HumanB
S
S
-
K
N
S
N
K
T
D
K
T
V
A
*
-
E
V
TroutM5DYRGKYVVEFFIPLDFTFVCPTEITAFS
Trout
HumanA
HumanB
Trout
HumanA
HumanB
A
A
E
E
R
F
R
K
I
G
K
-
L
N
C
E V
Q
G
A
S
V
D
S
H
F
C
H
L
A
W
-L -V --
L
D
I
T
N
V
T
cctcgtaagcacggtggtctgggtgccatgaagattcctctggtagccgacacaatgcgttccatctccacggactatggggtgtttgaa
c
a*­
ca
tg-tca- t
c
t
t-a- -c-g-a
at
HumanA ---aag--a -a--a- a
c--c--c---c-t-gt---gtg-cca-acg-t-g--tga---t--c--c---c-*---
HumanB --c--q--ag-g- a at- -gc c
TroutPRKHGGLGAMKIPLVADTMRSISTDYGVFE
-5-PK-T-AQ-LG-VT-RL-EK
Q
P
-
N
E
P
L
N
-
L
K
L
K
ggagggatgagggcatcgcctacagggggcctttttatcattgacgacaagggcgtcttgaggcagatcaccatcaacgatcttccggtg
Trout
*
tg a--t--c-c---cLgt
to -tc-tc
t -t
t g t
HumanA -ct *- -a
tg-t--t---t-g--t---
c -t -g
t- c-tc-c
c
t
*
HumanB aaca .
Trout
GGMRASPTGGLFIIDDKGVLRQITINDLPV
HumanAADEGI-FR- *
V
V
I
HumanEITCKGIAYR- *
G
3.ACCTTTGOA
ggacggtgcgttgacgagatcctqcgtctggtgcaggcctttcagttcactgacaaacacggagaagtctgtcctgccggctggaaacca
t
g c--a -t
t
g
c
ctt -a a a t
HumanA t-c--c-ct--g--t
t
c--t
g
t
c- -a- -a- -g-g- t g
g
c
gct
c-c- -g t
HumanB
R
C
V
D
I
L
R
L
V
Q
A
F
Q
F
T
D
K
H
G
E
-
C
P
A
G
W
120
494
150
K
P
584
180
A
CCGTCACTATGG 5.ME 245
ggaagtgataccatcaagcccgacgtgcagaagagcaaagacttcttctccaagcagcagtaa­
Trout
a
g
-c
t- t -c-ca
a-at
HumanA -c
a -ca-t-g­
g tg-c
g
a-at
a
c--g--t
HumanB -c
TroutC5DTIKPDVQKSKDFFSKQQ*
-K.
P -T-EYHumanA
HumanB
V
404
T
5
5
E
90
C
P
Trout
G
Trout
HumanA C
HumanB
314
I
Trout
HumanA
HumanB
60
L
R
N
224
L
K
K
gacgctgccgaggagtttaggaagatcggttgtgaggtcattggtgcctctgtcgattcccacttctgccatcttgcttggaccaacaca
t
a--a---gt---t---
t
t
g
tagg a -a a- -a- -ac- aac--cc a g
t
c
-aa- c g
a -gc-g--c-t -g g- c t g
c
cc-c -c-g--c
-a
a-- cg
D
30
K
5.TTTGTGTGCCCCACGGAG 3'ME244
atgtctgattacagagggaagtatgtggtgttcttcttctacccgctggacttcacctttgtgtgccccactgagatcatcgccttcagt
Trout
t -t
g
t
t
t
t
ca
-a--a
HumanA c
t
c
g
c
t
c
cc- -t
a
g
c
HumanB cHumanA L
HumanB L
134
N -DD- -
E
Y
-
647
200
-HN.
gacgacacatgtctgaagcctgctgtaggacagctagaattacagtacatttcaagagaacaatggtgaagagcagtagcctacctctgtgcctgag
actgtagaacaacatcttcagcatccccttatctcatgtgtttgggtagagtggaaattattatgaccaaactatgaagattatattaacgtatagc
ttggagatgttttatattgtagtcatttagcagacactcttatcttagtgtcattgatgtcggcctattttattcttctgtatcatttttgtaacat
ctgaggtggacattcgtttttgcctttaggccacagatgggatagtctgaaatgaggaacagaagctttacaggacaaaacttgtggcgaaaactcc
1127
agttagctttttgtctctttatttacaactcaatgtcattcatcttctaaataaacactttacaaaggcaaaaaaaaaaaaaaaaaaaa
Figure 3.1
36
Acknowledgments
This study was supported by the United States Department of Agriculture to
the Western Regional Aquaculture Consortium under the grant 92-38500-7195, project no.
92080441; an Oregon Sea Grant with funds from the National Oceanic and Atmospheric
Administration, Office of Sea Grant, Department of Commerce, under grant NA89AA-D­
SG108, project R/FSD-16; grant NA36RG451, projects R/FSD-23 and Amend. No. 2; a
grant from the National Institutes of Allergy and Infectious Disease, T32A107379; and a
grant from the National Oceanic and Atmospheric Administration (Salton-Kennedy funds),
NA46FD0490. Oregon Agricultural Experiment Station Technical Paper No. 10,761.
References
Evans, D. L., Harris, D. T., and Jaso-Freidmann, L. (1992). Function associated
molecules on nonspecific cytotoxic cells: Role in calcium signaling, redirected
lysis, and modulation of cytotoxicity. Dev. Comp. Immunol., 16: 383-394,
Evans, D. L., Graves, S., Cobb, D., and Dawe, D. L. (1984). Nonspecific cytotoxic
cells in fish (Ictalurus punctatus), II. Parameters of target cell lysis and
specificity. Dev. Comp. Immunol., 8: 303-312.
Graves, S. S., Evans D. L., and Dawe D. L. (1985). Mobilization and activation of
nonspecific cytotoxic cells in the channel catfish infected with Ichthyopthirius
multifiliis.. Dev. Comp. Immunol., 8: 43-51.
Greenlee, A. R., Brown, R. A., and Ristow, S. S. (1991). Nonspecific cytotoxic cells
of rainbow trout (Oncorhynchus mykiss) kill YAC-1 target cells by both necrotic
and apoptotic mechanisms. Dev. Comp. Immunol., 15: 153-164.
McKinney, E. C., and Schmale, M. C. (1994). Damselfish with neurofibromatosis
exhibit cytotoxicity toward targets. Dev. Comp. Immunol., 18: 305-313.
Moody, C. E., Serreze, D. V., and Reno P. W. (1985) Nonspecific cytotoxicity activity
of teleost leukocytes. Dev. Comp. Immunol., 9: 51-64.
Ritz, J., Schmidt, R.E., Michon, J., Hercend, T., and Schlossman, S. F. (1988).
Characterization of functional surface structures on human natural killer cells.
Adv. Immunol., 42: 181-211.
37
Shau, H., Gupta, R. K., and Golub, S. H. (1993). Identification of a natural killer factor
(NKEF) from human erythroid cells. Cell. Immunol., 147: 1-11.
Shau, H., and Golub, S. H. (1988). Modulation of natural killer-mediated lysis by red
blood cells. Cell. Immunol., 116: 60-72
Shau, H., Butterfeild, L. H., Chiu, R., and Kim, A. (1994). Cloning and sequence
analysis of candidate human natural killer-enhancing factor genes.
Immunogenetics., 40: 129-134.
Trinchieri, G. (1989). Biology of natural killer cells. Adv. Immunol., 47: 187-376.
Yannelli, J. R., Thurman, G.B., Mrowca-Bastin, A., Pennington, C. S., West, W. H.,
and Oldman R.K. (1988). Enhancement of human lymphokine- activated killer
cell cytolysis and a method for increasing lymphokine-activated killer cell yields to
cancer patients. Cancer Res., 48: 5696-5700.
38
CHAPTER 4
CHARCTERIZATION OF A NON-SPECIFIC CYTOTOXIC CELL
ENHANCEMENT GENE PRODUCT (NCEF) IN RAINBOW TROUT
(Oncorhynchus mykiss )
Dan V. Mourich and Jo Ann Leong*
Department of Microbiology and the Center for Salmon Disease Research,
Oregon State University, Corvallis, Oregon 97331, U.S.A.
*Correspondence should be addressed to this author
(for submission to Cellular Immunology)
This Paper reports a portion of the work encompassed by a thesis submitted to Oregon
State University, Department of Microbiology, in partial fulfillment of that required for the
degree of Ph. D., for Dan V. Mourich
39
Abstract
The nonspecific cytotoxic cell (NCC) of teleost fish is functionally related to the
natural killer cells of mammals. A gene with significant homology to human natural killer
enhancement factor (NKEF) genes was isolated from a cDNA library derived from rainbow
trout head kidney cells. The sequence of this gene shares 79 % and 72 % nucleotide
identity and 75.5 % and 71 % amino acid identity with human NKEF A and B,
respectively. Here we demonstrate the structural and functional similarities of these two
related gene products from phylogenically distant animals. A protein product derived from
the trout cDNA (NCEF) by in vitro transcription and translation has a molecular weight
(25 k Da by SDS-PAGE) similar to those of the two human proteins. Western blot
analysis with three different monoclonal antibodies specific for the NKEF A or B proteins
demonstrate that the trout protein is antigenically related to both human proteins. The
NCEF gene was sub cloned into a eukaryotic expression plasmid under the control of a
CMV promoter and subsequently transfected into the salmonid CHSE-214 cell line by
lipid-DNA complex. Expression of NCEF in the transfected cell cultures was confirmed
by immunohistochemistry with a cross reactive monoclonal that recognizes both NKEF A
and B. The trout protein was determined to be functionally related to human NKEFs by
effects on NCC activity tested by radio-labeled chromium (51Cr) release and thymidine­
labeled DNA fragmentation of target cells. Enhanced cytolysis of the 51Cr labeled,
NCEF-expressing target cells was seen for trout derived lymphocytes from pronephros
(head kidney), spleen and peripheral blood. Also, a similar increase in DNA fragmentation
was seen in target cultures expressing NCEF.
40
Introduction
Nonspecific cytotoxic cells (NCC) of various fishes have been postulated to be the
teleost equivalent of mammalian natural killers cells (NK) (Lester et al., 1994). These
cytolytic cell types from two phylogenetically different taxa share similar attributes. Among
these are; the ability to lyse tumor cell lines in a non-MHC-restricted manner (Hinuma et
al., 1980; Graves et al., 1984; Evans et al., 1984; Mckinney and Schmale 1994), the
metabolic requirements for and antibody influence on cytolysis (Carlson et al. 1985), the
elimination of parasitic infections (Graves et al., 1985), role in virus resistance (Moody et
al., 1985) and cell destruction by apoptosis (Greenlee et al., 1991). Some biophysical
aspects of NCC cells are similar to NK cells such as cell density, non-adherency, lack of
phagocytic activity and surface molecules (Evans et al., 1984, 1987 and 1992). Although
studies demonstrating lectin-induced enhanced activity of mammalian NK cells are similar
for NC cells of fish (Hayden and Laux, 1985; LeMorvo-Rocher et al., 1994), no
endogenously produced teleost factors have been shown to enhance NCC activity.
Many substances besides lectins can effect the in vitro or in vivo activity of NK
cells including infection with viruses and certain microorganisms (Hergerman et al., 1977
and Wolfe et al., 1976), and a number of cytokines (as reviewed by Trinchieri, 1989).
Recently, a cytosolic protein expressed in both human red blood cells (RBC) and the
natural killer sensitive erythroleukemic cell line K562 has been shown to augment the
cytolytic effects of human natural killer (NK) cells (Shau et al., 1993) and increase yields
of lymphokine activated killer (LAK) cells (Yannelli et al., 1988). High performance
liquid chromatography (HPLC) purified RBC cytosolic protein preparations exhibiting this
activity were used to produce polyclonal immune antisera (Shau et al., 1993). This antisera
was used to screen a cDNA expression library derived from the K562 cell line. Two
distinct genes, similar in nucleotide sequence were isolated and termed NKEF A and B.
41
Recombinant forms of these proteins added to in vitro assay conditions were shown to
induce a significant increase in the killing activity of human NK cells (Shau et al. 1994).
Previously we reported the sequence of a cDNA clone derived from a trout head
kidney library that exhibited significant homology with both human NKEF gene sequences
(Mourich et al., 1995). The isolation methodology is detailed in this report. In this present
study we examined if the trout-derived NKEF-like gene product, termed NCEF, has
antigenic and biological properties similar to the human NKEF proteins. Specifically, we
expressed the trout gene in both prokaryotic and eukaryotic systems and tested for cross
reactivity with monoclonals raised against the human isomers of NKEF. Also,
enhancement of rainbow trout NCC activity was examined by measuring the necrotic and
apoptotic destruction of cell cultures expressing either the NCEF or beta-galactosidase as a
control protein.
Methods and Materials
Animals and Cell Lines
Rainbow trout (Oncorhynchus mykiss ), Shasta strain, were obtained from the
Food Toxicology Research Center at Oregon State University and maintained at the Center
for Salmon Disease Research Laboratory in Corvallis, Oregon. The facility supplies
pathogen-free water at a constant temperature of 120 C. Fish were fed Oregon Moist Pellet
(OMP) commercial salmon food daily. Blood and tissues (anterior kidney and spleen)
were obtained by aseptic sampling or dissection after lethal overdose anesthesia in
benzocaine (ethyl p-amino-benzoate: Sigma). The Chinook salmon embryo derived cell
line CHSE-214 (Lannan et al., 1984) was used as a target cell. The cells were maintained
in RPMI-1640 media (Sigma) supplemented with 10 % heat inactivated fetal bovine serum
(Intergen), antibiotics and L-glutamine (penicillin, 100 IU/mL; streptomycin, 100 ilg/mL;
42
and L-glutamine, 2 mM) (Gibco BRL). The cells and cellular assays were maintained at
17° C in an incubator culture chamber (C.B.S. Scientific) perfused with a blood gas
mixture composed of 9.9% mol/mol CO2, 10.2% mol/mol 02 and 79.95 mol/mol N2.
Leukocyte Isolation
To obtain effector cells, leukocytes were isolated as a source of effector cells from
peripheral blood, spleen and anterior kidney. Separate single cell suspensions of spleen or
anterior kidney were prepared by mincing the tissues in a petri dish containing 10 mL
RPMI-1640 and subsequently passed through a 100-mesh sieve. The cells were
centrifuged and washed twice in 20 mL of media and suspended in final 12 mL of RPMI­
1640. Whole blood collected in a heparinized vacuum tube was centrifuged to remove
plasma. The packed blood cells were then washed twice with media and suspended to a
final volume of 12 mL in RPMI-1640. All cell suspensions were underlayed with an
equivalent amount of Histopaque-ficol 1077 (Sigma). After centrifugation, the white cells
contained at the interface were removed and washed twice with supplemented media.
Viability and number were determined by trypan blue exclusion. Cell number was adjusted
by addition of supplemented media or by centrifugation followed suspension in new
supplemented media.
Generation of Rainbow Trout NKEF-like Probe
Primers were constructed from two homologous protein motifs found within the
human NKEF gene sequences. All primers were synthesized at the Oregon State Center
for Gene Research and Biotechnology Central Services facility. The 5' primer ME244 = 5'
ITTGTGTGCCCCACGGAG-3' was constructed using the coding sequence for the motif
43
FVCPTE. The 3' primer ME245 = 3'-ACCTTTGGACCGTCACTATGG-5' is
complementary to the coding sequence for the motif WKPGSDT. These primers were
used in the polymerase chain reaction (PCR) to amplify a product from a trout derived
peripheral blood cell cDNA pool. Total RNA using the RNAso1 method ( Tel-Test, INC.)
was isolated from a cell pack taken from 1 mL of trout peripheral blood. To produce a
cDNA pool, a reverse transcription reaction was carried out on 2 pig of the isolated RNA in
a 20 µL mixture containing ; 0.51.1g dT17-adapter primer [ ME102 = 5'
GACTCGAGTCGACATCGA(T)17 3' ], 300 units MMLV RT, 10 units RNasin , 0.02
mM dNTPs and 1 X MMLV RT buffer (Promega). The reaction mixture was incubated at
37° C for 1 hour followed by 950 C for 5 minutes. The polymerase chain reaction was
carried out in a 100 'IL reaction mixture containing; 2µL of the cDNA pool, 0.2 mM
dNTPs, 511M primers ME244 and ME245, 1.5 mM MgC12, 50 mM KC1, 10 mM Tris­
HC1 (pH 9.0), 0.1 % Triton X-100 and 1 unit Taq polymerase (Promega). The mixture
was overlaid with mineral oil and placed into a Themiolyne thermocycler (Barstead) for
amplification. The reaction was heated to 950 C for 2 minutes after which 5 cycles of a
low stringency initiation profile of [95° C for 1 min., 47° C for 1 min. and 72° C for 1
min. ], followed by 30 cycles of a high stringency amplification profile of [ 95° C for 1
min., 53° C for 1 min. and 72° C for 1 min.] were run. This was immediately followed
by an over-hang extension incubation of 720 C for 10 minutes. Ten µL of the reaction
mixture was analyzed by agarose gel electrophoresis. A PCR product of the predicted size
[405 base pairs (bp)] was visible in an ethidium bromide stained gel. The band was
excised and ligated into the TA cloning vector and subsequently transformed into E. coli
according to manufacture's protocols (Invitrogen).
Plasmid clones were isolated and screened for insert size by restriction enzyme
digest analysis with EcoR I. Clones containing inserts of predicted size were then prepared
for 77 and SP6 primer sequencing to confirm sequence homology to the human NKEF
gene sequences. A single clone was selected, based on NKEF similarity, and used as a
44
homologous probe for screening a rainbow trout cDNA library. The rainbow trout
pNCEF1 clone was digested with Eco R1, separated by agarose gel electrophoresis and
the insert isolated in an agarose gel slice. This was subsequently purified (Geneclean; Bio
101), and again separated on an agarose gel, followed by a secondary purification. The
twice purified insert was randomly labeled to an approximate specific activity of 1.5 x 109
cprns/pg with a 32P dCTP (ICN) using a commercially available kit (BRL, Gaithersberg,
MD). Removal of non-incorporated nucleotides was done using a G-50 Quick-Spin
column (BMB) prior to using the labeled probe for hybridization analysis or screening.
Library Screening
A rainbow trout Uni-ZAPXRII cDNA library derived from trout pronephros cells
was used to isolate a full length cDNA clone encoding the gene related to human NKEF.
The phage library was titered and plated on 150 x 25 mm petri dishes (Corning) so that
each plate contained approximately 5 x 104 PFUs. A total of 1 x 106 PFUs (20 plates)
were plaque-lifted onto BA-85-supported nitrocellulose (Schleicher and Schuell) per
manufacture's protocol. The filters were pre-hybridized in a Techne Hybridiser HB-1D
(Techne, Princeton, NJ) for 6 h at 580 C in a solution containing 5 x standard sodium
citrate (SSC), 5 x Denhardt's solution , and 0.5% sodium dodecyl sulfate [(SDS) (w/v)].
Hybridization conditions were the same as prehybridization except for the addition to the a
[32-P] dCTP labeled probe. Hybridization was allowed to proceed for 20 hr after which
the filters were washed for 15 min. and again for 5 min. at 580 C in a solution containing 3
x SSC and 0.5 % SDS. All heated washes were carried out in a model 2564 heated
shaking water bath (Forma Scientific). The filters were removed to a room temperature
wash containing 0.2 x SSC and 0.2 % SDS for an additional 15 minutes. Autoradiographs
of the hybridizing plaques were made with Kodak XAR-5 film. Positive plaques were
located, selected and titered. Titers were adjusted to 50 and 500 PFUs for each positive
45
plaque selected and plated onto 100 x 20 mm petri dishes. Duplicate filter lifts were made
for each plate as before and were pre-hybridized for 1 hr and hybridized in the presents of
the labeled probe for 20 hr at 65° C in 3 x SSC and 0.5
SDS. Duplicate filters were
split and washed under either a low stringency (42° C, 3 x SSC / 0.5% SDS) or a high
stringency (65° C , 0.2 SSC/0.2% SDS) condition for 15 min. followed by a 15 min.
room temperature wash in 0.2 x SSC and 0.2 % SDS. Autoradiographs were again made
and the duplicates were compare for position of positive plaques. Twelve positive plaques
found in both duplicates were selected and used for preparation of in vivo excision
phagemid clones.
Sub-cloning into Eucaryotic Expression Plasmid
Twelve separate phagemid clones were isolated from the positive plaques by the
method of in vivo excision (Hay and Short, 1992) using the ExAssist TM helper phage
system (Stratagene). The pBluescript phagemid containing bacterial clones generated by
the excision process were cultured in 5 mL of LB media plus ampicillin (120 pg /mL) in
overnight shaking cultures at 370 C. Phagemid DNA was isolated with mini-prep columns
(Qiagen) and digested with Eco R1 and Xho 1 restriction enzymes to determine insert sizes.
The inserts were isolated, purified and sub cloned into the eukaryotic expression plasmid
pCDNA3 (Invitrogen). This plasmid contains a CMV promoter upstream of the multiple
cloning site for expression of cloned inserts in transfected eukaryotic cells. The subcloned
plasmids were also screened for insert size and then prepared for sequencing with T7 and
SP6 promoter primers.
46
Sequencing and Analysis
The twelve clones derived from the library screening, in vivo excision and
subcloning processes were sequenced in part by both manual and automated DNA
sequencing methods based upon dideoxy chain termination chemistry. The Sequenase 2.0
system (US Biochemicals) was used for manual sequencing while automated sequencing
was performed at the Center for Gene Research and Biotechnology (Oregon State
University) with an ABI 373A automated sequence machine. After confirmation of
sequence similarity to NKEF, additional primers were synthesized [ME266 =
5'GGCAGATCACCATCAACGATCTT 3' & ME267 =
CCATCTGTGGCCTAAAGGCAAA] and used to sequence the full length of the cDNA by
automated DNA sequencing.
Analysis of Predicted Protein Sequence by in vitro Translation
The CMV promoter plasmid containing the full length coding sequence for the
rainbow trout gene related to human NKEF (pNCEF) was digested with Xho I. This was
isolated in an agarose gel slice after electrophoresis and purified (Gene Clean, BIO 101).
This was repeated again to twice purify the linear plasmid. The plasmid DNA was ethanol
precipitated washed twice with 75 % ethanol and Speed Vac (SAVANT) dried with heat for
15 min. The DNA was then suspended in nuclease free water to approximately 2 lig / RL.
Two micrograms served as template in a 50 !IL T7 RNA polymerase directed in vitro
transcription reaction containing; 4 mM DTT, 20 units RNasin (Promega), 0.125 mM
rNTPs, 10 units T7 RNA polymerase, 40 mM Tris-HCL pH 7.9, 6 mM MgC12, 2 mM
spermindine and 10 mM NaCl. The reaction was incubated at 370 C for 1 hr followed by
ethanol precipitation and two 75 % ethanol washes. The pellet was again vacuum dried and
suspended in 5 !IL of nuclease free water. The transcription reaction product was heated to
47
650 C for 2 min. prior to addition to a 50 µL in vitro translation reaction mixture
containing; 10 units RNasin, 25 pL rabbit reticulocyte lysate (Promega), 2011M amino
acid mix minus methionine (Promega) and 60 ACi 35S methionine (ICN). The mixture
was incubated at 300 C for 1 hr and then stored at -700 C. Aliquots were taken and
added to either denaturing or non-denaturing sample buffer. Samples to be analyzed under
denaturing conditions were heated to 950 C for 5 min. and cooled on ice prior to loading 15
of each to separate wells of a 15 % SDS-PAGE gel. SDS-PAGE pre-stained broad
range molecular weight standards (BIO-RAD) were loaded in an adjoining reference well.
The gel was run at 165 V constant for 4 hr, then dried on to a piece of blotting paper with a
vacuum dryer (Hoeffer). This was exposed to autoradiograph film (HYPER-film, Kodak)
for 18 hours.
Western Blot Analysis
The in vitro translation product was fractionated on a 15 % SDS-PAGE gel as
before, also included was a concentrated sample of rabbit reticulocyte lysate mixture. The
proteins were transferred to nitrocellulose blotting paper (Schleicher and Schuell) in a
electrobloting chamber (BIO-RAD). The blot was blocked for 1 hr in 5 % non-fat dry milk
(SACO), 1 % BSA (Fraction V, Sigma) and cut into 3 strips. The strips were placed into
separate containers and incubated with one of the three anti-NKEF monoclonal antibodies
diluted 1:1500 in 100 mM Tris-HC1 pH 7.2, 0.5 M NaCl, 0.05 % Tween-20 and 1% BSA
113SA) for 1 hr. The strips were washed 3 times in ITBSA and then incubated with
either, anti-mouse IgG gamma chain specific or anti-mouse IgM IA chain specific, alkaline
phosphatase conjugate (BIO-RAD), diluted 1:1000 in TTBSA, depending on isotype of the
monoclonal. The strips were washed 3 times in TTBSA, equilibrated in 100 mM Tris pH
9.0 and developed with BCIP/NBT one component substrate (Kirkegaard & Peny Labs).
48
Transfection of Target Cell Line
Transfected CHSE-214 cells served as both producers of the NCEF protein and as
a target cell source. Cells were trypsinized and seeded onto 6 well tissues culture plates
(Coming). The cultures were grown to 80-90 % confluence (approximately 2 x 106 cells /
well) and then washed 3 times in 2 mL of Opti-MEM (Gibco BRL). The washed cells
were kept at 170 C with 1 mL of Opti-MEM / well while DNA-lipid complexes were made.
One i.tg of either pNCEF or pCMVBga1 with 200 .tL of OptiMem in a 12.5 mL tube
(Falcon) was added to a separate tube containing 9 III, of LipofectAMINE reagent (Gibco
BRL) with 200 gL of OptiMem to form the DNA-lipid complexes. The contents mixed and
incubated at room temperature for 1 hour, after which 600 pL of OptiMem was added to
the mixture. The media was removed from the cells and immediately overlaid with the
mixture and incubated 18 hr at 170 C. The wells were washed 3 times with OptiMem,
overlaid with RPMI-1640 media (Sigma) supplemented with 10 % heat inactivated fetal
bovine serum (Intergen), antibiotics and L-glutamine (penicillin, 100 IU/mL;
streptomycin, 100 µg/mL; and L-glutamine, 2 mM) (Gibco BRL) and returned to 170 C for
48-72 hr until needed for assays or analysis.
f3-gal Activity and Immunohistochemistry
Transfection efficiency of the pCMVItal transfected cells produced DNA-lipid
complex incubation was determined by colorimetric enzymatic conversion of X-gal
substrate by the method of Lim and Chae (1989). A sub-sample of the cell culture was
plated and fixed for 10 min. with 1 % gluteraldehyde in 0.1M PBS pH 7.0. The wells
were washed 3 times with PBS pH 7.0 and overlaid with substrate solution containing
0.2% X-gal, 10 mM sodium phosphate buffer (pH 7.0), 150 mM NaC1, 1 mM MgC12,
49
3.3 mM K4Fe(CN)6H20 and 3.3 mM K3Fe(CN)6 for 4 hr at 370 C. The cells were
examined microscopically and blue staining cells were scored as positives.
Immunohistochemical staining was used to determine the transfection efficiency of
the pNCEF transfected cultures. A sub-sample of the culture was plated, fixed and rinsed
in the same manner described for the Pgal transfected cultures. The wells were blocked
with 5 % non-fat dry milk at 4° C overnight, rinsed, and probed with anti-human NKEF
monoclonals diluted 1:100 in PBS pH 7.2 for 1 hr at 37° C. The wells were washed 5
times with PBS and incubated with avidin labeled anti-mouse (Vector).
DNA Fragmentation Assay
Target cell DNA fragmentation levels were determined using a modification of the
method of Duke et al. (1986). Transfected CHSE-214 cell monolayers (approx. 1.0 x 106
cells) were labeled by the addition of 501.tCi methyl -3H- thymidine in 1 mL of
supplemented RPMI for 24 hr at 17° C. After radiolabel incubation, the cells were washed
3 times with 2 mL RPMI, overlaid with 2 mL supplemented media and allowed to incubate
for an additional 24 hr. The cell were trypsinized to a produce single cell suspension and
adjusted to 1 x 105 cells / mL in supplemented media. One hundred microliters of the cell
suspension (1x104 cells) was dispensed into sterile 2.0 mL conical bottom microcentrifuge
tubes (ICN) and allowed to incubate for 10 hours. One hundred microliters of effector
cells (head kidney or peripheral blood leukocytes) were added to quadruplicate tubes at cell
concentrations of either 1, 0.5, or 0.25 x 106 cells / mL. An additional 2001..LL of media
was added to each tube, after which the tubes were centrifuged for 1 second at 14,000 g to
initiate cell to cell contact and incubated at 17° C for 6 hours. After the incubation period
the tubes were centrifuged for 15 sec at 14,000 x g and the supernates (supemate A) were
transferred to 7 mL scintillation vials (VWR). Cell pellets were then lysed by freeze thaw
and the addition of 500 µL lysis buffer (10 mM Tris, 1 mM EDTA, 0.2% Triton X-100,
50
pH 7.5) to each tube. To separate fragmented DNA from intact chromatin the lysates were
centrifuged for 20 min. at 14,000 x g at 4° C. Supernates (supernate B) containing the
fragmented DNA were transferred to new scintillation vials. The lysed cell pellets (Pellet)
were suspended in 500 µL of lysis buffer and transferred to separate scintillation vials.
Radioactivity in the samples was measured by adding 5.5 mL of Cytoscint scintillation
cocktail (ICN) and counting the vials containing the separate pellets and supernates in a
model LS1800 (Beckman) liquid scintillation spectrometer. The percentage of DNA
fragmentation was calculated using the formula [ % fragmentation = 100 x (( supernate A
cpms + supernate B cpms) / ( Total supernate cpms + Pellet cpms)) J. The percentage of
specific DNA fragmentation was calculated using the results of the above equation in the
formula [ % specific DNA fragmentation = 100 x (( % experimental % spontaneous) /
( 100 - % spontaneous))].
51Chromium Release Assay:
Cytolytic activity on a fish target cell line was determined using a modification of
the method of Moody et al. (1985). Transfected CHSE-214 cells were plated into 96 well
microtiter plates (Coming) at 1 x 104 cells/ well in 100 µL media. An additional 100 µL of
media containing 1 t.tCi of 51Cr (sodium chromate, ICN) was added to each well followed
by a 20 hr incubation at 17° C. The wells were washed three times with 200 !IL of RPMI
and leukocyte suspensions were added to triplicate wells at E:T ratios of 200:1, 100:1, 50:1
or 25:1 in 200 !IL of media. Culture medium alone was added to triplicate wells used for
determination of spontaneous release. Total release was determined by adding 200 !IL of
1% Triton X-100 in PBS pH 7.2 to triplicate wells. The plates were incubated for 18 hr at
17° C in a culture chamber perfused with blood gas mixture. One hundred microliters was
removed from each well and transferred to separate 6 mL Wheaton, Omni-vials (VWR).
51Cr release was determined by counting the vials for 1 min. in a model 5500 autogamma
51
counter (Beckman). The percent of specific release was calculated using the formula [ %
specific release = 100 x ((experimental cpms- spontaneous cpms) / (total cpms
spontaneous cpms))].
Results and Discussion
Molecular Size and Antigenic Similarities of NCEF and NKEF
To determine if the predicted open reading frame and deduced amino sequence for
the trout derived NKEF- related clone were correct in vitro 35S methionine radio-labeling
of proteins was done during the translation reaction. The reaction was fractionated through
a 10 % SDS-PAGE gel and exposed to X-ray film. Figure 4.1 shows the autoradiogram
and molecular weight marker indications. Two bands are apparent with corresponding
molecular weights of approximately 25 and 50 k Da. This is similar to what was seen for
the initial studies on human NKEF proteins isolated and purified from red blood cells
(Shau et al., 1993). Under non reducing conditions the human protein appears to dimerize
exhibiting an apparent molecular weight of 48 k Da, while under reducing conditions a
single band at 24 k Da is seen. This may account for the two bands seen in the
autoradiogram of the labeled trout protein. The faint upper band probably represents
dimerized pairs not fully reduced under the conditions used.
The in vitro translation proteins were again fractionated on SDS-PAGE and then
transferred to nitrocellulose for western blotting analysis. Three separate blot were
prepared all containing pre-stained molecular weight markers, in vitro translation reaction
and control rabbit reticulate lysate concentrate. Each blot was probed with a separate
monoclonal antibody preparation that is specific for either NKEF A, NKEF B or both
forms. A band at approximately 25 k Da was detected in all three blots for the lanes
52
corresponding to the in vitro translation product (figure 4.2) suggesting that the trout
derived protein is antigenically related to both human NKEF proteins.
Analysis of twelve separate NCEF clones isolated from the trout derived cDNA
library showed that the DNA sequences were nearly identical. This information suggests
that there may be only one form of this protein in trout instead of two, as seen in humans.
The western blot data suggest that the NCEF protein is related to both the A and B NKEF
forms. The protein derived from in vitro translation of the trout NCEF gene reacts with
two different monoclonals that are specific for the different human proteins. It is possible
that the trout gene represents an ancestral form of the gene. It has been suggested that the
two human forms arose from a primordial gene that was duplicated over the course of
evolution (Shau et al., 1994).
It is not known if NKEF A and B function in vivo as a strict heterodimer, two
separate homodimers or a combination of both dimeric forms. However, some type or
dimer is detected in portion extracts of human red blood cells by western blot (Shau et al.,
1993). If dimerization is necessary to the function of these proteins it appears that the trout
NCEF protein also has the capacity to form homodimers.
Detection of NCEF in Transfected CHSE-214 Cell Cultures
Separate cell cultures were transfected with CMV promoter driven eukaryotic
expression plasmids containing either inserts coding for NCEF or beta-galactosidase.
Immunohistochemistry was used to detect if the NCEF protein was being expressed in
transfected cell cultures. X-gal substrate incubation was used to detect expression of beta
galactosidase in the pCMVBga1 transfected cell cultures. Only the monoclonal antibody
able to recognize both forms of the human NKEF proteins was able to detect the expression
of NCEF in the transfected cultures. Beta galactosidase expressing cell cultures were
53
Figure 4.1
Autoradiogram of 35S-methionine labeled in vitro translation products
derived from NCEF plasmid gene insert Position and size of molecular
weight markers are indicated. Arrows indicate 25 k Da and 50 k Da
products. Lane 1: Pelleted proteins from translation reaction. Lane 2:
Supernatant proteins from translation reaction.
54
1
2
208
112
80
45
36
27
18
Figure 4.1
55
Figure 4.2 Western blots with anti-NKEF monoclonal antibodies. NCEF in vitro
translation products (pelleted proteins and supernatant) and control (rabbit
reticulocyte lysate) were probed with anti-human NKEF monoclonals.
Molecular weight marker sizes are indicated. Panel A: Lane 1: control,
Lane 2: pellet and 3: supernatant vs. anti-A NKEF. Panel B: Lane 1
supernatant, Lane 2: pellet and Lane 3: control vs. anti-B NKEF.
Panel C: Lane 1 supernatant, Lane 2: pellet and Lane 3: control vs. anti-A/B
NKEF. Arrow indicates the position of the 25 k Da product.
Figure 4.3 Immunohistochemical analysis of CHSE-214 cells transfected with CMV­
NCEF (panel A) or CMV-rigal (panel B). Fixed cell monolayers were
probed with anti-AB NKEF followed by anti-mouse alkaline phosphatase
conjugate and substrate indicator. Cells expressing NCEF are indicated
with arrows.
56
A
1
2
C
B
3
1
2
3
1
208
112
80
45
36
27
18
7.1
Figure 4.2
A
B
Figure 4.3
2
3
57
negative when tested with all anti-NKEF monoclonal antibodies. The staining of NCEF
indicates that the expression appears to be cytoplasmic with little surface staining (figure
4.3).
Enhanced Necrotic Attack in Cultures Expressing NCEF Measured by Cell Lysis
Necrosis of target cells is one result of mammalian NK or teleost NC cell mediated
cellular destruction (Russell et al., 1980; Greenlee et al., 1991). Necrotic lesions produced
in target cells under cytolytic attack are exhibited by membrane pore formation, cytoplasmic
swelling and finally cell death by lysis (Dennert and Podack, 1983). Target cells
radiolabeled with 51chromium can be used to measure the extent of cell lysis in both
mammal and fish culture systems (Duke et al, 1986; Moody et al. 1985). Employing this
assay system and using plasmid transfected target cells we have demonstrated that NCC
activity of fish can be enhanced by NCEF present in assay culture conditions. Specifically,
target cell cultures expressing NCEF were found to be lysed to a greater degree by spleen,
head kidney and peripheral blood lymphocytes than control target cell cultures expressing
13gal. The effector lymphocytes were added to labeled target cell cultures in ratios of
(effector:target) 200, 100, 50 and 25:1 and incubated for 20 hours. The percent specific
51chromium release was calculated (see methods) for each ratio and for the two different
transfected target cell conditions. An increase of cytolytic activity followed the increase in
effector to target cell ratio culture conditions. Head kidney effector cells gave the largest
average increase in activity with 38 % +1- 14.7 % followed by spleen cells 21 % +1- 12.66
% and peripheral blood lymphocytes with 18.7 % +/- 11.6 %. These increases are slightly
higher than the average of 13 % found by Shau's group when non purified human red
blood cell cytosol extracts were used (Shau et al., 1993).
58
Figure 4.4
Results of chromium release assay with head kidney leukocytes
effectors. Head kidney leukocyte effectors were incubated for 20 hours
with chromium labeled CHSE-214 targets transfected with either CMVPgal (E) or CMV-NCEF (J). Results are expressed as an average percent
specific release of the mean of triplicate wells for separate fish leukocyte
preparations (n= 3 fish). Error bars indicate the standard deviation of the
mean for the three fish.
Figure 4.5
Results of chromium release assay with spleen leukocyte effectors.
Effectors were incubated for 20 hours with chromium labeled CHSE-214
targets transfected with either CMV-13gal (E) or CMV-NCEF (J). Results
are expressed as a percent specific release of label from targets for the
mean of triplicate wells from separate fish leukocyte preparations (n=3
fish). Error bars indicate the standard deviation of the mean average
specific release for the three fish.
Percent Specific Realease from 1 x104 Target Cells
1
Percent Specific Release from l x104 Target Cells
60
Figure 4.6
Results of chromium release assay for peripheral blood leukocyte effectors.
Cells (E=25, 50, 100 or 200 x 104 cells) were incubated for 20 hours with
chromium labeled CHSE-214 targets (T=1 x 104 cells) transfected with
either CMV-kal (E) or CMV-NCEF (3). Results are expressed as an
average percent specific lysis of the mean of triplicate wells for separate
fish leukocyte preparations (n=3 fish). Error bars indicate the standard
deviation of the mean average specific lysis for the three fish.
Percent Specific Release from lx104 Target Cells
62
Yet when the wide range of variation is accounted for in the fish assays the increased
activity due to NCEF is near that produced in the human assays by NKEF. Therefore the
cytolytic data suggests that these two closely related genes from trout and humans produced
similar effects on the activity of innate killing cells.
Enhancement of Apoptosis in Cell Cultures Expressing NCEF Measured by DNA
Fragmentation
Apoptosis is another measurable result of NK or NCC activity on target cells (Duke
and Cohen, 1986; Greenlee et al., 1991). Apoptotic lesions on target cells are characterized
by chromatin margination and condensation, cytoplasmic blebbing, membrane swelling and
fragmentation of cellular DNA into nucleosomal subunits (Wyllie, 1980). The extent of
this type of target cell destruction can be measured by the isolation and quantification of
labeled fragmented DNA from assay culture (Duke et al., 1983). Transfected target cells,
labeled with 3H-thymidine prior to 6 hour assay incubation with effector cells from head
kidney or peripheral blood lymphocytes. Effector to target ratios of 25, 50 and 100:1 were
tested with target cell cultures transfected with either CMV-NCEF or CMV-I3gal. An
increase in target cell DNA fragmentation was observed in the CMV-NCEF transfected cell
cultures over the CMV-13gal transfected cell cultures for both head kidney and peripheral
blood lymphocyte effectors. Specifically, the head kidney effectors and the peripheral
blood lymphocyte effectors showed respective average increases of 9.4 % +/- 1.4 % and
8.5 % +/- 1.9 % specific DNA fragmentation of NCEF targets over Pgal targets. Although
this type of cellular destruction activity has not been tested in the human system in respect
to NKEF effect on NK cells, it appears that cell killing mechanism of apoptosis is enhanced
by NCEF in fish NC cells.
63
Figure 4.7
Results of DNA fragmentation assay for peripheral blood leukocyte
effectors. Cells (E=25, 50, 100 x 104 cells) were incubated for 6 hours
with 3H-thymidine labeled CHSE-214 targets (T=1 x 104 cells) transfected
with either CMV-Pgal (E) or CMV-NCEF (J). Results are expressed as an
average percent of the mean of quadruplicate wells for separate fish
leukocyte preparations (n=6 fish). Error bars indicate the standard deviation
of the mean average specific lysis for the six fish.
Figure 4.8
Results of DNA fragmentation assay for head kidney leukocyte
effectors. Cells (E=25, 50, 100 x 104 cells) were incubated for 6 hours
with 3H-thymidine labeled CHSE-214 targets (T=1 x 104 cells) transfected
with either CMV-Pgal (E) or CMV-NCEF (J). Results are expressed as an
average percent of the mean of quadruplicate wells for separate fish
leukocyte preparations (n=6 fish). Error bars indicate the standard deviation
of the mean average specific lysis for the six fish.
Percent Specific Fragmentation of Target Cell DNA
Percent Specific Fragmentation of Target Cell DNA
Z
65
Acknowledgments
This study was supported by the United States Department of Agriculture to the
Western Regional Aquaculture Consortium under the grant 92-38500-7195 project no.
92080441; an Oregon Sea Grant with funds from the National Oceanic and Atmospheric
Administration, Office of Sea Grant, Department of Commerce, under grant NA89AA-D­
SG108, project R/FSD-16; grant NA36RG451, projects R/FSD-23 and Amend. No. 2; a
grant from the National Institutes of Allergy and Infectious Disease, T32A107379; and a
grant from the National Oceanic and Atmospheric Administration (Salton-Kennedy funds),
NA46FD0490. Monoclonal antibodies were provided as a kind gift from Dr. Hungyia
Shau at UCLA medical school, Department of Surgery and Dr. Charles Shi, Pharmacia
labs.
References
Carlson, R. L., Evans, D. L., and Graves, S. S. (1985). Nonspecific cytotoxic cell in
fish (Ictalurus punctatus). V. Metabolic requirements of lysis. Dev. Comp.
Immunol., 9: 271-280.
Dennert, G. and Podack, E. R. (1983). Assembly of tubular complexes on target cell
membranes. J. Immunol ., 157: 1483-1495.
Duke, R. C. and Cohen J. J. (1986). Differences in target cell DNA fragmentation
induced by mouse cytotoxic T lymphocytes and nonspecific killer cells. J.
Immunol., 137: 1442-1447.
Duke, R.C., Chervenak, R. and Cohen J. J. (1983). Endogenous endonuclease-induced
DNA fragmentation. An early event in cell mediated cytolysis. Proc. Natl.
Acad. Sci. USA., 80: 6361-6356.
Evans, D. L., Graves, S., Cobb, D., and Dawe, D. L. (1984). Nonspecific cytotoxic
cells in fish ( Ictalurus punctatus), H. Parameters of target cell lysis and
specificity. Dev. Comp. Immunol., 8: 303-312.
66
Evans, D. L., Harris, D. T., and Jaso-Freidmann, L. (1992) Function associated
molecules on nonspecific cytotoxic cells: Role in calcium signaling, redirected
lysis, and modulation of cytotoxicity. Dev. Comp. Immunol., 16: 383-394.
Graves, S. S., Evans D. L., and Dawe D. L. (1985). Mobilization and activation of
nonspecific cytotoxic cells in the channel catfish infected with Ichthyopthirius
multifiliis. Dev. Comp. Immunol., 8: 43-51.
Greenlee, A. R., Brown, R. A., and Ristow, S. S. (1991). Nonspecific cytotoxic cells of
rainbow trout (Oncorhynchus mykiss) kill YAC-1 target cells by both necrotic and
apoptotic mechanisms. Dev. Comp. Immunol., 15: 153-164.
Hayden, B. J., and Laux, D. C. (1985). Cell-mediated lysis of murine target cells by
nonimmune salmonid lymphoid preparations. Dev. Comp. Immunol., 9: 627­
639.
Herberman, R. B., Nunn, M. E., Holden, H. T., Staal, S., and Djeu, J. Y. (1977).
Augmentation of natural cytotoxic reactivity of mouse lymphoid cells against
syngeneic and allogeneic target cells. Int. J. Cancer , 19: 555-562.
Himuma, S., Abo, T., Kumagai, K., and Hata, M. (1980). The potent activity of fresh
water fish kidney cells in cell killing. I. Characterization and species -distribution
of cytotoxicity. Dev. Comp. Immunol., 4: 653.
LeMorvan-Rocher, C., Troutland, D., and Deschaux, P. (1995). Effects of temperature
on carp leukocyte mitogen- induced proliferation and nonspecific cytotoxic activity.
Dev. Comp. Immunol., 19: 87-95.
Lester III, J. P., Evans, D. L., Leary III, J. H., Fowler, S. C., Jaso-Friedmann, L.
(1994). Identification of a target cell antigen recognized by nonspecific cytotoxic
cells using a anti-idiotype monoclonal antibody. Dev. Comp. Immunol., 18: 219­
229.
McKinney, E. C., and Schmale, M. C. (1994). Damselfish with neurofibromatosis
exhibit cytotoxicity towards targets. Dev. Comp. lmmunol., 18: 305-313.
Moody, C. E., Serreze, D. V., and Reno P. W. (1985) Nonspecific cytotoxicity activity
of teleost leukocytes. Dev. Comp. Immunol., 9: 51-64.
Mourich, D. V., Hansen, J., and Leong, J. C. (1995). Natural killer cell enhancement
factor-like gene in rainbow trout (Oncorhynchus mykiss). Immunogenetics, 42:
438-439.
Ritz, J., Schmidt, R.E., Michon, J., Hercend, T., and Schlossman, S. F. (1988).
Characterization of functional surface structures on human natural killer cells.
Adv. Immunol., 42: 181-211.
Russell, J. H., Masakowski. V. R., and Dobos, C. B. (1980). Mechanisms of immune
lysis. I. Physiological distinctions between target cell death mediated by cytotoxic
T lymphocytes and antibody plus complement. J. Immunol., 124: 1100-105.
Shau, H., and Golub, S. H. Modulation of natural killer-mediated lysis by red blood
cells. (1988). Cell. Immunol., 116: 60-72.
67
Shau, H., Butterfeild, L. H., Chiu, R., and Kim, A. (1994) Cloning and sequence
analysis of candidate human natural killer-enhancing factor genes.
Immunogenetics., 40: 129-134.
Shau, H., Gupta, R. K., and Golub, S. H. (1993). Identification of a natural killer
enhancement factor (NKEF) from human erythroid cells. Cell. Immunol.,
147: 1-11.
Trinchieri, G. (1989). Biology of natural killer cells. Adv. Immunol., 47: 182-376.
Wolfe, S.A. Tracey, D.E., and Henney, C. S. (1977). Induction of natural killer cells by
BCG. Nature, 262: 584-587.
Wylie, A. H. (1980). Glucocorticoid-induced thymocyte apoptosis with endogenous
endonuclease activation. Nature, 284: 555-556.
Yannelli, J. R., Thurman, G.B., Mrowca-Bastin, A., Pennington, C. S., West, W. H.,
and Oldman R.K. (1988). Enhancement of human lymphokine- activated killer
cell cytolysis and a method for increasing lymphokine-activated killer cell yields to
cancer patients. Cancer Res., 48: 5696-5700.
68
CHAPTER 5
CLONING AND SEQUENCE OF A STAT5-LIKE GENE IN RAINBOW
TROUT (Oncorhynchus mykiss): DISTRIBUTIONAL EXPRESSION IN
LYMPHOID CELLS.
Dan V. Mourich, Marc C. Johnson, Carol Kim and Jo Ann Leong*
Department of Microbiology and the Center for Salmon Disease Research
Oregon State University, Corvallis, Oregon 97331, U.S.A.
*Correspondence should be addressed to this author
(for submission to Immunogenetics)
This Paper reports a portion of the work encompassed by a thesis submitted to Oregon
State University, Department of Microbiology, in partial fulfillment of that required for the
degree of Ph. D., for Dan V. Mourich
69
Abstract
We report here a 960 nucleotide rainbow trout gene sequence derived from two
separate polymerase chain reactions (PCR) with primers designed to conserved regions of
the STAT5 gene found in sheep, humans and mice. Rainbow trout (Oncorhynchus
mykiss) cDNA from peripheral blood lymphocytes served as template. The consensus
sequence from the two PCR products shares 79.6 % over all nucleotide identity and 81.4
% apparent amino acid homology with the STAT5 sequence from human, mouse and
sheep. Also, the presence of a STAT5-like protein was detected with a monoclonal
antibody specific for STAT5 in various rainbow trout tissues, corresponding to a similar
tissue distribution found in humans. This was confirmed in both western blot and
immunohistochemical analysis. Cell lines from chinook salmon, rainbow trout and carp
were also positive for STAT5 by western blot. Lastly, we demonstrated that mitogenic
stimulation of trout lymphocytes with Con A results in the rapid translocation of a STAT5
like protein to the nucleus where it binds to a DNA sequence containing the GAS motif.
Introduction
Cytoldnes are soluble secreted proteins that regulate and coordinate communication
between cells of the immune and hematopoietic systems (Kishimoto et al., 1994). This cell
to cell communication is mediated through surface bound receptors specific for each
cytokine (Hamblin, 1993). The binding of a cytokine to its cognate receptor results in
receptor dimerization and the subsequent triggering of intercellular signals (Heldin, 1995).
The resulting effect is the rapid transcriptional activation of target genes which may control
production of soluble proteins such as antibodies or cytokines or the growth and
differentiation of a responding cell (Sabath et al., 1990). It is the processes between
70
receptor binding and gene activation that have recently become a focus of study in immune
regulation. It has long been known that phosphorylation states of proteins are involved,
however, early on the identification and characterization of the responsible kinases and
substrates remained unknown (Hatakeyama et al., 1991). Since intracellular domains of
cytokine receptors lack intrinsic kinase activity, other receptor associated protein kinases
must be recruited to transmit secondary signaling processes (Torigoe et al., 1992;
Venkitaraman and Cowling, 1992; Kishimoto et al., 1994). The kinases now known to
associate with cytokine receptors and function in these pathways belong to the Janus kinase
(Jak) family (Russell et al., 1994). Receptor activated Jak proteins in turn catalyze the
phosphorylation of a class of transcriptional factors termed signal transducers and
activators of transcription (STAT) proteins. Phosphorylation of STAT proteins results in
their dimerization and nuclear translocation. Once in the nucleus STATs alone or in
association with other transcriptional factors bind to response element sequences for
transcriptional activation of target genes (Darnell et al., 1994). To date six different STAT
proteins (STAT 1-6) have been characterized and identified to be involved in the signal
transduction of several cytokines including INF a, INF 13, IL-2, IL-4, IL -6, IL-7. IL-10,
IL-12, IL-13, IL -15 ( as reviewed by Ivashkiv, 1995) and growth factors EGF and
prolactin (Ruff -Jamison et al., 1995; Gilmor and Reich, 1994).
Although there is evidence that cytokine -like factors exist in phylogenetically
primitive animals including fishes, most of this remains as characterizations of biologically
active culture supernatant proteins (as reviewed by Secombes, 1994). To date only two
fish derived genes resembling cytokines have been cloned (Tamai et al., 1992 and 1993).
Yet, little is known about signal transduction of cytokines and cytokine -like factors in
teleost fishes. Here we report on a partial gene sequence derived from rainbow trout
(Oncorhynchus mykiss) that closely resembles the signal transduction and transcriptional
factor STAT5 found in various mammals. The cell type distribution of the STAT5-like
protein was examined by western blot and confocal immunohistochemical analysis. Also
71
conditions for the activation, nuclear translocation and DNA element binding were
examined.
STAT5 is involved in the signal transduction processes of interleukins 2, 7 and 15
(Lin et al., 1995). Cell expressing the corresponding receptors will respond to these
cytokines by Jak driven phosphorylation of Src homologous-2 (SH2) domains in STAT5
proteins (Frank et al., 1995). Trans location to the nucleus and binding to promoter
sequences containing the consensus inverted repeat G/TAA of structure called the y-INF­
activation site (GAS) results in the rapid transcriptional induction of some genes (Gilmor
and Reich, 1994). Interleukin -2 as well as mitogenic stimulation have been shown to
rapidly induce nuclear binding activity of STAT5 in freshly isolated PBLs. This binding
activity appears to be transient since it drops off dramatically only hours after stimulation
(Hou et al., 1995).
The tissue distribution of STAT5 expression has also been determined (Lin et al.,
1995). Northern blot analysis demonstrates active transcription of STAT5 gene in heart,
placenta, lung, muscle, spleen, thymus , prostate, testis, ovary, intestine, colon and
peripheral blood lymphocytes. While in the brain, liver, kidney and pancreas transcription
is dramatically reduced or undetectable.
We report here a partial gene sequence derived from a series of polymerase chain
reactions (PCR) with primers designed to conserved regions of published STAT5 genes
and rainbow trout cDNA as template. Also, the presence of a STAT5-like protein is
detected in various cell types and cell lines from fish with a monoclonal specific for
STAT5. Lastly, we show that mitogenic stimulation of trout lymphocytes with Con A
results in the rapid translocation of a STAT5 like protein to the nucleus which binds to a
DNA sequence containing the GAS motif.
72
Materials and Methods
Animals and Tissues
Rainbow trout (Oncorhynchus mykiss ), Shasta strain, were obtained from the
Food Toxicology Research Center at Oregon State University and maintained at the Center
for Salmon Disease Research Laboratory in Corvallis, Oregon. The facility supplies
pathogen-free water at a constant temperature of 120 C. Fish were fed Oregon Moist Pellet
(OMP) commercial salmon food daily. Blood and tissues (anterior kidney, liver and
spleen) were obtained by aseptic sampling or dissection after lethal overdose anesthesia in
benzocaine [(ethyl p-amino-benzoate: Sigma) (Kaattari and Irwin, 1985)]. The fish
derived cell lines CHSE-214, EPC and RTG-2 were maintained in RPMI-1640 media
(Sigma) supplemented with 10 % heat inactivated fetal bovine serum (Intergen), antibiotics
and L-glutamine (penicillin, 100 IU/mL; streptomycin, 100 ug/mL; and L-glutamine, 2
mM) (Gibco BRL). The cells were maintained and cellular assays were done at 17° C in
an incubator culture chamber (C.B.S. Scientific) perfused with a blood gas mixture
composed of 9.9% mol/mol CO2, 10.2% mol/mol 02 and 79.95 mol/mol N2.
Isolation of Rainbow Trout STAT5 Gene Segments
Synthetic primer were made to conserved regions found in the published STAT5
gene sequences of human, sheep and mouse. All primers were synthesized at the Oregon
State Center for Gene Research and Biotechnology Central Services facility. These primers
were then used in the polymerase chain reaction (PCR) to amplify a product from a cDNA
pool derived from trout peripheral blood cells. Using the RNAsol method (Tel-Test, INC.)
total RNA was isolated from a cell pack taken from 1 mL of trout peripheral blood. To
73
produce a cDNA pool, a reverse transcription reaction was carried out on 2 ug of the
isolated RNA in a 20 gL mixture containing ; .5 pig dT17-adapter primer [ ME102 = 5'
GACTCGAGTCGACATCGA(T)17 3' ], 300 units Moloney Murine Leukemia Virus
Reverse Transcriptase (MMLV RT), 10 units RNasin, 0.02 mM dNTPs and 1 X MMLV
RT buffer ( Promega). The reaction mixture was incubated at 37° C for 1 hour followed by
950 C for 5 minutes. The polymerase chain reaction was carried out in a 100 µL reaction
mixture containing; 11.th of the cDNA reaction mixture was diluted 1:1000 in nuclease free
water, 0.2 mM dNTPs, 51.IM primers, 1.5 mM MgC12, 50 mM KC1, 10 mM Tris-HC1
(pH 9.0), 0.1 % Triton X-100 and 1 unit Taq polymerase (Promega). The mixture was
overlaid with mineral oil and placed into a Thermolyne thermocycler (Barstead) for
amplification. The reaction was heated to 950 C for 2 minutes after which 5 cycles of a
low stringency initiation profile of [95° C for 1 min., 450 C for 1 min. and 72° C for 1
mini, followed by 30 cycles of a high stringency amplification profile of [95° C for 1
min., 58° C for 1 min. and 72° C for 1 mini were run. This was immediately followed
by an over-hang extension incubation of 720 C for 10 minutes. Ten FAL of the reaction
was analyzed on an ethidium bromide stained agarose gel. PCR products of the predicted
size were excised and either directly sequenced or ligated into the TA cloning vector and
subsequently transformed into E. coli according to manufacture's protocols (Invitrogen).
Plasmid clones were isolated and screened for insert size by restriction enzyme digest
analysis with EcoR I. Clones containing inserts of predicted size were then prepared for
17 and SP6 primer sequencing to confirm sequence homology to STAT5 gene sequences.
Refer to Table 5.1 for primer sequences and map position along human STAT5 gene.
Sequencing
The Sequenase 2.0 system (US Biochemicals) was used for manual sequencing,
following manufacture's suggested protocol.. Automated sequencing was performed at the
74
Center for Gene Research and Biotechnology (Oregon State University) with an ABI 373A
automated sequencing machine.
Primer Name
Sequence 5'-3'
Position
ME 318
gacgacgagctgatccagtg
750-769
ME 319
aacaactgctgcgtgatgg
1195-1215
ME 320
cccgtcgaaccactgcca
1716-1736
ME 326
gcacctccgccttgtacttc
1542-1562
HUMAN STAT 5 LIANA 2400 bases
ME 318
41
llob
ME 319
Table 5.1
ME 326
ME 320
PCR primer pairs and gene segments positions along the human STAT5
gene.
Isolation of Cells
Leukocytes were isolated from peripheral blood, spleen and anterior kidney.
Separate single cell suspensions of spleen, liver or anterior kidney were prepared by
mincing the tissues in a petri dish containing 10 mL RPMI-1640 and subsequently passed
through a 100-mesh tissue sieve. Cells were centrifuged and washed twice in 20 mL of
media and finally suspended in 12 mL of unsupplemented RPMI-1640. Whole blood
collected in a heparinized vacuum tubes was centrifuged. After removal plasma, the packed
blood cells were washed twice with media and suspended to a final volume of 12 mL in
75
RPMI-1640. All cell suspensions except the hepatocytes were underlayed with an
equivalent amount of Histopaque-ficol 1077 (Sigma). After centrifugation, the white cells
contained at the interface were removed and washed twice with supplemented media.
Viability and number were determined by trypan blue exclusion for all of the cell
preparations.. Cell number was adjusted to 1 x 106 cells per mL in complete media and
plated into 6 well culture plates (Corning) at 1 mL per well.
Western Blot Analysis
Samples were taken from cell culture, tissues or lymphocyte preparations and
adjusted to 1 x 10 6 total cells in 100 !IL Phosphate buffered saline (PBS). An equal
volume of 2 X SDS-PAGE sample buffer was added to each sample which was then placed
in a boiling water bath for 5 minutes. The samples were fractionated on a 12 % SDS­
PAGE gel and then transferred to nitrocellulose paper (Schleicher and Schuell) in a
electrobloting chamber. The blot paper then was blocked for 1 hr in 5 % non-fat dry milk
(SACO), 1 % BSA (Fraction V, Sigma) after which it was transferred to a dish containing
anti -STATS monoclonal antibody (Transduction Labs, Lexington, Kentucky) diluted
1: 250 in PBS and incubated for 2 hours at room temperature. This was followed by three
separate 10 minute shaking wash steps in PBS containing 0.05 % Tween-20 and 1% BSA.
An anti-mouse IgG specific biotinylated secondary antibody (BIO-RAD), diluted 1:1000 in
PBS, was added for 2 hours at room temperature. The blot was again washed 3 times as
before. At this time an avidin-HRP conjugate diluted 1:500 in PBS (Sigma) was added for
a 1 hour incubation. Five washes followed in PBS containing 0.05 % Tween-20 and two
in PBS alone. The blot was then removed to a dish containing pre-mixed Cl -HRP
SuperSignal luminol enhancer solution (Pierce) for 20 min. The excess liquid was drained
off and the blot placed between two plastic cover sheets. This was exposed to hyper film
X-ray film (Kodak) for 10 minutes. The film was immediately developed and dried.
76
Nuclear Protein Extraction
Nuclear proteins were obtained from the peripheral blood lymphocyte cultures after
a 1 hour pulse with 5 ng per mL concanavalin A (Con A) (Sigma) by the method of
Schreiber et al. (1989). Pulsed cells, approximately 1 x 106 cells per preparation, were
harvested and washed with 10 mL of ice cold Tris buffered saline (TBS) by pelleting at
1500 x g for 5 min. in a 40 C centrifuge. The pellet was suspended in 1 mL of cold TBS
and transferred to a 1.5 mL eppendorf tube and pelleted again at 1000 x g for 15 sec in a
microfuge. The TBS was removed and the cell pellet suspended in 400 µL of ice cold
buffer A (10 mM Hepes pH 7.9, 10 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM
DTI' and 0.5 mM PMSF) by gentle pipetting. The tube containing the suspended cells was
placed on ice for 15 min., after which 25 [IL of a 10 % solution of NP-40 was added. The
tube was vortexed for 10 sec and then immediately centrifuged for 30 sec at 2000 x g. The
supernatant was removed and the remaining nuclear pellet was suspended in 501.1L of ice
cold buffer C (20 mM Hepes pH 7.9, 0.4 mM NaC1, 1 mM EDTA, 1 mM EGTA, 1 mM
DTT and 1 mM PMSF). The tube was shaken vigorously for 15 min. at 40 C on a shaking
platform. After a 5 min. 40 C centrifugation at 5000 x g the supernatant containing the
nuclear extract was removed to a new tube and kept frozen at 700 C.
Electromobility Shift and Super Shift Assay
Two !IL of nuclear extract material containing approximately 4 i.tg of total protein
was used in the binding and super shift assays. The super shift assay involved a pre
incubation step of 1 hr with 21AL (0.25 mg per mL) of the anti -STATS monoclonal
antibody with nuclear extract at room temperature. The nuclear extract alone or pre
incubated with the anti-STAT5 monoclonal were added to the binding reaction mixture
77
for 1 hr at room temperature. The reaction mixture consisted of 5 mM Tris-HC1 pH 7.5,
25 mM NaC1, 0.5 mM EDTA, 2.5 % glycerol, 0.5 mM DTT, 3 lig Poly I:C and 2
picomoles of double stranded y 32P end labeled primer containing the GAS motif (5'
GCCTGATTTCCCCGAAATGATGA 3') in a total volume of 30 p.L. After incubation,
51AL of loading buffer (reaction buffer with a trace of bromophenol blue dye and 5 %
glycerol) was added and the samples loaded into individual wells of a 5 % acrylamide (30:1
acrylamide:bisacrylamide) 0.5 M Tris-Borate EDTA (TBE) gel. The sample were
electrophoresed for 1.5 hr. at 100 volts. The gel was dried and exposed to hyper film Xray film (Kodak) for 72 hours. The film was developed in an Xomat (Kodak) automatic
film developer.
Immunohistochemical Confocal Microscopic Analysis of STAT5 in Trout Cells
Spleen, peripheral blood and head kidney lymphocytes isolated by ficol gradient
centrifugation as well as mixed population of trout hepatocytes were used in this analysis.
The cells were washed twice with PBS by lightly spinning the cells to the bottom of the
microfuge tube at low rpm. Cells were fixed by suspending the cell pellet in 1 mL of 3.7%
formaldehyde (Fluka) in PBS. To permeabilize, the cells were incubated with 0.5%
Triton-X 100 in PBS for 5 minutes at room temperature. The cells were washed and then
allowed to adhere to Poly L-lysine coated cover slips for 20 minutes. After the allotted
time, the cover slips were washed in PBS to clear away any non-adherent cells. A solution
of 1% BSA with 0.1% Tween-20 in PBS was used as a blocking agent and incubated with
the cells for 30 minutes at room temperature. At this point, the blocking solution was used
as the diluent for the primary and secondary antibody preparations. The samples were
incubated with a 1:100 dilution of mouse anti-STAT-5 for one hour. The cells were
washed and subsequently incubated with goat anti-mouse IgG conjugated with Texas Red
(10 1.1g/mL, Molecular Probes, Inc.) for one hour. After washing the samples, fluorescein
78
conjugated Concanavalin A (10 ug/mL, Molecular Probes, Inc.) and Hoescht dye was to
added to a total concentration of 10011M and allowed to incubate for 5 minutes. The cells
were washed, allowed to air dry, and then mounded in Cytoseal (Stephens Scientific). The
samples were then analyzed with a Leica TCD4 Confocal Microscope using a 100 X oil
immersion lens (NA 1.3).
Results and Discussion
Sequence Comparison:
The combined data from the isolated gene sequences obtained from rainbow trout
(Oncorhynchus mykiss) show a high degree of identity to known STAT5 gene sequences .
When this 906 nucleotide sequence (figures 5.1 a, b and c) was compared to published
sequences from human (GenBank accession # L4114, Hou et al., 1995), sheep (GenBank
accession # X78428, Wakao et al., 1994) and mouse STAT5a and STAT5b clones
(GenBank accession #'s U21103, U21110, Liu et al., 1995; 248538, 248539, Mui et al.,
1995) shared identities of 79.5 %, 77.6 % , 80.3 % and 81.2 % respectively, were
observed. The predicted amino acid sequence for the trout gene segment (figure 5.2)
shares 81.1 %, 79.3 %, 82% and 83 % identity with human, sheep, mouse STAT5 A and
mouse STAT5 B, respectively.
The significant level of conservation of the STAT5 genes seen throughout these
different species, suggest that the function of STAT5 may be the same in trout.
Specifically, STAT5's function in mammals is the transduction of cytokine signals in
response to IL-2, 7 and 15. Although, these cytokines have yet to be fully characterized in
fish, the trout STAT5 gene sequence data implies that one of the necessary components for
their signal transduction is present in fish.
79
Figure 5.1a
Sequence of STAT5-like gene segment (nucleotides 1 to 420) from
rainbow trout (0. mykiss). Alignment with other published STAT5
cDNA sequences. A consensus of the cloned sequences for trout is
shown. (.) indicates identical nucleotides to the human STAT5A
reference sequence. (-) indicates gaps.
80
HUMSTAT5A
OAMGF
MMU21103
MMT21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
AGCGGCGGCA GCAGCTGGCC GGGAACGGCG GGCCCCCCGA GGGCAGCCTG GACGTGCTAC
G
CG.T A CG A..AGC-... ..T .......
AA
G
T
G
AA
G
T
G
AA
G
T
G
AA
G
T
G
..A.A..A.. T ................. A. .T..T..A.. ...GG.G... ...A.C..G.60
AGTCCTGGTG TGAGAAGTTG GCCGAGATCA TCTGGCAGAA CCGGCAGCAG ATCCGCAGGG
G
A
MMU21103
MMT21110
MMSTAT5A
MMSTAT5B
TROUT
A
AG A 120
A
CG.
HUMSTAT5A
CTGAGCACCT CTGCCAGCAG CTGCCCATCC CCGGCCCAGT GGAGGAGATG CTGGCCGAGG
OAMGF
MMU21103
.0
MMT21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
MMU21103
MMT21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
MMU21103
MMT21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
MMU21103
MMT21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
MMU21103
MMT21110
MMSTAT5A
MMSTAT5B
TROUT
.A..A
c
G
A
C
T G
A
C
G
G
A
A
C
GA.A A
T
C
CA. C
T
T
T
C A
T
T
AA .CC180
TCAACGCCAC CATCACGGAC ATTATCTCAG CCCTGGTGAC CAGCACATTC ATCATTGAGA
C
C
C
T
C
G
C
C
C
G
C
C
T
C
G
C
C
C
G
C
AGT
A
C
A
C
C T ..... A..
240
AGCAGCCTCC TCAGGTCCTG AAGACCCAGA CCAAGTTDGC AGCCACCGTA CGCCTGCTGG
C
C
G
G
G
G
G
G
G
G
T G
C
G
T T C
A TG ..T..C..A.300
TGGGCGGGAA GCTGAACGTG CACAaAATC CCCCCCAGGT GAAGGCCACC ATCATCAGTG
C
G
C
....G..A.. ...... T... ........ C. ....G ........... G... ........ C.
G
T
C
G
G
C
....G..A.. ...... T... ........ C. ....G ........... G... ........ C.
G
T
C
G
G
C
....T ..... ...C..T... ........ C. ....G ..... C....G.... ........ T.360
AGCAGCAGGC CAAGTCTCTG
................ C...
................ C...
................ C...
................ C...
................ C...
.A...G.... G...G.CT..
CTIAAAAATG
..C..G..C.
..C..G....
..C..G....
..C..G....
..C..G....
T.C..G..T.
Figure 5.1a
AGAACACCCG CAACGAGTGC
.......... ...T ......
.......... ...T ......
.......... ...T..T.A.
.......... ...T ......
.......... ...T..T.A.
........ A. G..T..CA..
AGTGGTGAGA
..C..G....
..C..C....
..C..C....
..C..C....
..C..C....
..G..G....420
81
Figure 5.1b
Sequence of STAT5-like gene segment (nucleotides 421 to 840) from
rainbow trout (0. mykiss). Alignment with other published STAT5
cDNA sequences. A consensus of the cloned sequences for trout is
shown. (.) indicates identical nucleotides to the human STAT5A
reference sequence. (-) indicates gaps.
82
HUMSTAT5A
OAMGF
MMU21103
b4W21110
MMSTAT5A
MMSTAT5B
TROUT
TCCTGAACAA CTGCTGCGTG ATGGAGTACC ACCAAGCCAC GGGCACCCTC AGTGCCCACT
T
GCG
A
C
T
C
T
G
G
C
T
C
G
T
A
C
T
C
G
T
G
C
T
C
G
T
G
C
...CT
AA
T.
G .
A A
G ..T..T....480
OAMGF..........
HUMSTAT5A
NINIU21103
NINTT21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
MNIU21103
NINIT21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
A4NDU21103
MMT21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
NENAU21103
MNIT21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
MNIU21103
MNIT21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
MNIU21103
Mh4T21110
MMSTAT5A
MMSTAT5B
TROUT
TCAGGAACAT GTCACPGAAG AGGATCAAGC GTGCTGACCG GCGGGGTGCA GAGTCCGTGA
...C..C... .......... .A ........ ...A..C... ..... T....
A
A
A
A
C. C.
A .
....A ..... ...0 ..... A C.A
C. C.
A .
....A ..... ...0 ..... A C.A
C
C
.A. ...T ........... G....
A .GT
A .AT
T .GT.G
C..T..G... .G...A..A.
A
...T ........... G....
C T
A A
T. C. G.
. G. C.540
CAGAGGAGAA GTTCACAGTC CTGTITGAGT CTCAGTTCAG TGTTGGCAGC AATGAGCTTG
A
G
A
.G
C
C
G
.G
A
GA
C
A
C. C. .TG .A
.0 .
G.
G
C
C
G
.G
A
GA
C
A
C. C. .TG .A
.0 .
G.
T T A
T .T
C. G. .GG.. . .0 .
G.600
TGTTCCAGGT GAAGACTCTG TCCCTACCTG TGGTTGTCAT CGTCCACGGC AGCCAGGACC
C
T C
T
C
C
C T
T
.C..T..A.. C
CT..
G..0 .G
.G .G
T T
A
C
C
C T
T
.C..T .A
C
CT..
.G .0 .G
.G .G
T T
A
.0
C
C
AT.A ..T.NT..C. .C..G..G.. A..T ..... T ..T..A...A660
ACAATGCCAC GGCTACTGTG CTGTGGGACA ATGCCTTTGC TGAGCCGGGC AGGGTGCCAT
G
T
T C C
C
C
C
A
T
T
T C C
C
A
T
....0
...A..G.TC .T T
T T
A
720
T CC
AC
AC
CC
7TGCCGTGCC TGACAAAGTG CTGTGGCCGC AGCTGTGTGA GGCGCTCAAC ATGAAATTCA
C
G
C
T
G
A
G
A
T
G
A
G
A
.C.T. .T
T
G
C
T
A. .C. .AG
780
AGGCCGAAGT GCAGAGCAAC CGGGGCCTGA CCAAGGAGAA CCTCGTGTTC CTGGCGCAGA
G
GT.
.T
.T ..... A
T
A
A
A
T
. T
A
A
T
A
.T ..... A
T
A
....A..GA. ...AG.TGC
TT TTG.TCGC.. ...G..C... ..... C....840
.T .....
Figure 5.1b
83
Figure 5.1c
Sequence of STAT5-like gene segment (nucleotides 900 to 960) from
rainbow trout (0. mykiss). Alignment with other published STAT5
cDNA sequences. A consensus of the cloned sequences for trout is
shown. (.) indicates identical nucleotides to the human STAT5A
reference sequence. (-) indicates gaps.
84
HUMSTAT5A
OAMGF
NINVU21103
h4t2T21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
h4W21103
hOTT21110
MMSTAT5A
WASTAMB
TROUT
AACTGTICAA CAACAGCAGC AGCCACCTGG AGGACTACAG
.G
C
A
T
A
C
A
T
A
C
A
T
A
C
A
T
A
C
A
.GGCA....G ..G.GC.... .T.A...CA. .A
. C
TGGCCTGTCC GTGTCCTGGT900
C
A .
. T
CA A.
.T
CA A
CA
A.
.T
CA A
CA
A. A.
A
G
CCCAGTTCAA CAGGGAGAAC TI'GCCGGGCT GGAACTACAC CTTCTGGCAG TGGTTTGACGG960
C
C
C
C
C
C
T
A AC ....T..... T
T
C
C
C
C
T
A AC ....T..... T
T
A
C
G
A GA
T
T
A CA
Figure 5.1c
85
Figure 5.2
Predicted amino acid sequence of trout (Oncorhynchus mykiss) STAT5­
Ike protein and its relationship related to other STAT5 proteins from
human, sheep and mouse. (.) indicate identity to the human protein
sequence. (-) indicates deletions.
86
HUMSTAT5A
OAMGF
QWKRRQQLAG NGGPPEGSLD VLQSWCEKLA EIIWQNRQQI RRAERLCWL PIPGPVEEML
HDWR MEA
R­
h4M21103
MMU21110
MMSTAT5A
MMSTAT5B
TROUT
A-Q .H
HUMSTAT5A
OAMGF
AEVNATITDI ISALVTSTFI IEKQPPQVLK TQTKFAATVR LLVGGKLNVH MNPPQVKATI
.......G.. I
T
R
I
L
MMU21103
MMU21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
NDL.S
V
ISEQQAKSLL KNENTRNECS GEILNNCCVM EYHQATGTLS AHFRNMSLKR IKRADRRGAE
MMU21103
MMU21110
DY.
MMSTAT5A
MMSTAT5B
S
G
DY.
TROUT
....E..A.F
HUMSTAT5A
OAMGF
SVTEEKFTVL FESQFSVGSN ELVFQVKTLS LPVVVIVHGS QDHNATATVL WDNAFAEPGR
MMU21103
MMU21110
MMSTAT5A
MMSTAT5B
TROUT
HUMSTAT5A
OAMGF
MMU21103
MMU21110
DSR
N
P
D
I
D
I
M
G
H
HUMSTAT5A
OAMGF
X
VPFAVPDKVL WPQLCEALNM KFKAEVQSNR GLTKENLVFL AQKLFNNSSS HLEDYSGLSV
L
N M
I
I
MMSTAT5A
MMSTAT5B
TROUT
WS
I
...v ..............
I
N
N
N
N
D. .Y...M.GA. ..FDR ..... ...A.SSA.I NP
SWSQFNRENL PGWNYTFWQW FDG*
MMU21103
MMU21110
R
MMSTAT5A
MMSTAT5B
TROUT
..A.L...S. ..R.F....F NGS.
R
Figure 5.2
NSM..
NSM..
NSM..
NSM
RSMTM
87
Antigenic Analysis and Tissue Distribution of STAT5 in Rainbow Trout
To determine if STAT5-like proteins were present in cells of fish, western blot
analysis was used. A monoclonal antibody specific for STAT5 (Transduction labs),
shown to cross react with STAT5 proteins from human, mouse and sheep, was used to
probe cell extracts from cell lines from chinook salmon (CHSE-214), rainbow trout (RTG­
2) and carp (EPC). Cell samples were boiled in SDS-PAGE sample buffer and fractionated
on a 10 % SDS-PAGE gel and transferred to nitrocellulose paper. A control sample of
mouse cell extract supplied by the manufacture was included as a positive control for
STAT5. Figure 5.3 shows a common protein band at approximately 91 k Da detected by
the STAT5 monoclonal antibody for all cell lines tested. This result, together with the
sequence information for rainbow STAT5-like gene confirms that a protein related to
mammalian STAT5 is present in fish cells.
The distribution of STAT5 in mammalian cells has been demonstrated by northern
blot analysis (Lin et al., 1995). The constitutive expression of STAT5 mRNA is seen in
heart, placenta, lung, muscle, spleen, thymus , testis, ovary, intestine, colon and peripheral
blood lymphocytes. Transcription levels are much lower in the brain, liver, kidney and
pancreas and in most cases beyond the level detection for northern blot analysis. A similar
tissue expression profile was seen in rainbow trout by western blot analysis. Figure 5.4
shows the STAT5 monoclonal reacting with a 91 k Da protein in cell samples taken from
rainbow trout. Specifically, a high level of protein is seen in both peripheral blood and
spleen derived lymphocytes. STAT5 protein was also detected in head kidney leukocyte
samples, but at considerably lowered levels when compared to peripheral and spleen
lymphocytes. No STAT5 protein was detected in sample preparations from liver or red
blood cells.
88
Figure 5.3
Detection of STAT5 proteins in fish cell lines. Western blot results of
cell proteins probed with a STAT5 specific monoclonal antibody. Lane 1:
positive control mouse RSV-3T3 fibroblastic cell line sample supplied by
manufacture. Lane 2: CHSE-214 cells. Lane 3: EPC cells. Lane 4:
RTG-2 cells. Molecular weight marker positions and sizes are indicated.
89
1
2
3
Figure 5.3
4
90
Figure 5.4
Distribution of STAT5 protein in trout tissues. Western blot analysis of
proteins derived from various cell types from rainbow trout probed with
STAT5 specific monoclonal antibody. Molecular weight marker positions
are indicated. Lane 1 Pre-stained molecular weight markers. Lane 2:
Peripheral blood lymphocytes. Lane 3: Liver cells. Lane 4: Head kidney
lymphocytes. Lane 5: Spleen derived lymphocytes. Lane 6: Red blood
cells.
91
2
1
3
a
204
121
84
50
Figure 5.4
4
5
6
92
The distribution of STAT5 expression was also examined by immunohistochemical
confocal microscopy. Figure 5.5 shows the same reaction profile for STAT5 expression as
was seen in western blot analysis for peripheral blood, head kidney and spleen derived
lymphocytes. STAT5 protein was not detected in liver cells by this method, confirming the
results of the western blot of STAT5 tissue distribution . The tissue distribution and
relative level of STAT5 expression may be due to the profiles of cytokines and growth
factors to which these types of cells respond. STAT5 regulates the signal transduction of
various lymphokines such as IL-2, 7 and 15 (Lin et al., 1995) which are produced by and
elicit responses in T cells.
Though antibody regents to T cell specific surface makers, such as CD4 or CD 8,
have yet to be developed for teleost fishes, there is biological evidence suggesting the
existence fish T cells (Secombes et al., 1994). Most of this evidence centers around
reports of fish lymphocytes responding to classic T cell mitogens. The predominant
responses to lectin based T cell mitogenic stimulus are demonstrated fish cell cultures of
spleen, head kidney and peripheral blood lymphocytes, indicating that T-like cells might be
present in these tissues (Caspi and Avatlion, 1984; Grondel and Hamersen, 1984).
Presumably, since STAT5 is a signal transducer protein for such cytokines as IL-2,
a T cell autocrine growth factor, the presence of this protein could be used as an indicator
of T like-cells in fish tissues. However, in mammals expression of STAT5 mRNA appears
to be constitutive in many tissue types including PBLs and spleen. STAT5 expression is
absent in the human kidney, but we observed protein expression in the head kidney cells of
rainbow trout, albeit at low levels This may be due in part to the anatomical differences
between fishes and humans. The pronephros or head kidney, unlike the human kidney, is
also a hematopoietic organ containing a number of leukocytes.
93
Figure 5.5
Confocal immunohistochemical analysis of rainbow trout cells with
STAT5 specific monoclonal antibody. Red indicates STAT5 specific
staining. Green counter stain is Con A FITC. Panel A: Spleen derived
lymphocytes. Panel B: Peripheral blood lymphocytes. Panel C: Liver cells
and Panel D: Head kidney leukocytes.
94
B
D
Figure 5.5
95
Mitogen Signal Transduction and STATS in PBLs of Rainbow Trout
To determine if the STATS -like protein detected in PBLs was functionally similar to
the STATS proteins of mammals, we used the electromobility shift and super shift assay
systems. It has been shown that, upon mitogenic or cytokine simulation of lymphocytes,
STATS is rapidly phosphorylated and translocated to the nucleus where it binds to target
gene sequence containing the GAS DNA sequence motif (Gilmor and Reich, 1994).
Peripheral blood lymphocytes were exposed to Con A for 1 hr in culture, and nuclear
extracts were harvested soon after. The extracts were incubated with a radio-labeled GAS
primer and then fractionated over a polyacrylamide gel. Figure 5.6 demonstrates a protein
binding the labeled GAS primer in lane 3. This suggests that DNA binding proteins,
specific for the sequence motif bound by mammalian STAT5s during signal transduction of
cytokine or T cell mitogen response, are present in fish PBLs.
To determine if the protein binding the GAS primer was antigenically related to
STATS we used a monoclonal antibody specific for STATS to super shift the band seen in
the shift assay. Again, nuclear extracts were isolated from trout PBLs after Con A mitogen
stimulation. An additional step of pre-incubating the nuclear extract proteins with the
STATS specific monoclonal antibody was done before adding the labeled GAS primer.
After GAS primer was added, the reaction mixture was fractionated over a polyacrylamide
gel. The resulting antibody-STATS complex caused a super shift of the band due to the
increased molecular weight of the protein complex. This super shifted band (lane 4 of
figure 5.6) confirms that a STATS -like protein in rainbow trout is functionally similar to
mammalian STATS. The result also indicates that the fish STATS protein is functionally
similar to mammalian since it is also translocated to the nuclease with in 1 hour after
mitogenic stimulus.
96
Figure 5.6
Electromobility shift and super shift assay with PBL nuclear extracts
harvested 1 hour after exposure to Con A. Lane 1: 7 32P labeled 1 k by
ladder DNA size markers. Lane 2: control reaction lacking nuclear extract.
Lane 3: electromobility shift binding assay with nuclear extract and Lane
4: electromobility Super shift binding assay nuclear extracts pre incubated
with anti-STAT5 monoclonal antibody. Arrows indicate shift and super
shifted bands.
97
1
2
3
Figure 5.6
4
98
Acknowledgments
This study was supported by the United States Department of Agriculture to the Western
Regional Aquaculture Consortium under the grant 92-38500-7195 project no. 92080441;
an Oregon Sea Grant with funds from the National Oceanic and Atmospheric
Administration, Office of Sea Grant, Department of Commerce, under grant NA89AA-D­
SG108, project R/FSD-16; grant NA36RG451, projects R/FSD-23 and Amend. No. 2; a
grant from the National Institutes of Allergy and Infectious Disease, T32A107379; and a
grant from the National Oceanic and Atmospheric Administration (Salton-Kennedy funds),
NA46FD0490. Oregon Agricultural Experiment Station Technical Paper No. 10,761.
References
Caspi, R. R., and Avatlion, R. R. (1984). Evidence for the existence of an IL-2 like
lymphocyte growth promoting factor in bony fish, Cyprinus carpio. Dev. Comp.
Immunol., 8: 51-60.
Darnell, J. E., Kerr, I. M., and Stark, G. R. (1994). Jak-STAT pathways and
transcriptional activation in response to INFs and other extracellular signaling
proteins. Science, 264: 1415-1421.
Frank, D. A., Robertson, M. J., Bonni, A., Ritz, J., and Greenburg, M. E. (1995).
Interleukin 2 signaling involves the phosphorylation of STAT proteins. Proc.
Natl. Acad. Sci. USA, 92: 7779-7783.
Gilmour, K. C., and Reich N. C. (1994). Receptor to nucleus signaling by prolactin and
interleukin-2 via activation of latent DNA-binding factors. Proc. Natl. Acad. Sci.
USA, 91: 6850-6854.
Grondel, J. L., and Hamersen, E. G. (1984). Phylogeny of interleukins: growth factors
produced by leukocytes of cyprinid fish, Cyprinus carpio. Immunology, 52, 477­
482.
Hamblin, A. S. (1993). Cytokines one by one. Cytokines and Cytokine Receptors., D.
Male and D. Rickwood, eds., IRL Oxford University Press, New York, 21-43.
99
Hatakeyama, M., Kono, T., Kobayashi, N., Jawawhara, A., Levin, S.D., Perlmutter,
R.M., Taniguchi, T. (1991). Interaction of the IL-2 receptor with the src-family
kinase p56". Identification of novel intermolecular association. Science , 252:
1523-1528.
Heldin, C. H. (1995). Dimerization of cell surface receptors in signal transduction. Cell,
80: 213-223.
Hou, J., Schindler, U., Henzel, W.J., Wong, S. C., and McKnight S. L. (1995).
Identification of human STAT proteins activated in response to interleukin-2.
Immunity, 2: 321-329.
Ivashkiv, L. B. (1995). Cytoidnes and STATs: How can signals achieve specificity ?
Immunity, 3: 1-4.
Lin, J., Mignone, T., Tsang, M., Fiedmann, M., Weatherbee, J. A., Yamauchi, L. A.,
Bloom, E. T., Mietz, J., John, S., and Leonard, W. J. (1995). The role of
shared receptor motifs and common STAT proteins in the generation of cytokine
pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13 and IL-15. Immunity, 2:
331-339.
Liu, X., Robinson, G.W., Gouilleux, F., Groner. B., and Hennighausen, L. (1995).
Cloning and expression of StatS and an additional homologue (Stat5b) involved in
prolactin signal transduction in mouse mammary tissue. Proc. Natl. Acad.
Sci. USA, 92: 8831-8835.
Kishimoto, T., Taga, T., and Akira, S. (1994). Cytokine signal transduction. Cell, 76:
253-262.
Mui, A.L., Wakao, H., O'Farrell, A. M., Harada, N., and Miyajima, A. (1995).
Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5
transduce signals through two STAT5 homologs. EMBO J. , 14: 1166-1175.
Ruff-Jamison, S., Chen, K., and Cohen, S. (1995). Epidermal growth factor induces
the tyrosine phosphorylation and nuclear translocation of STAT5 in mouse liver.
Proc. Natl. Acad. Sci. USA, 92: 4215-4218.
Russel, S. M., Johston, J. A., Noguchi, M., Kawamura, M., Bacon, C. M., Friedman,
M., Berg, M., McVicar, D. W., Witthuhn, B. A., Silvennoinen, 0., Goldman,
A. S., Schmalstieg F. C., Ihle J. N., O'Shea, J. J., and Leonard W. J. (1994).
Interaction of IL -2R(3 and y chains with Jaki and Jak3: Implications for XSCID
and XCID. Science, 266: 1042-1047.
Sabath, D. E., Podolins, P. L., Comber, P. G., and Prystowsky, M. B. (1990). cDNA
Cloning and characterization of interleukin-2 induced genes in a cloned T helper
lymphocyte. J. Biolog. Chem., 265: 12671-12678.
Schreiber, E., Matthias, P., Muller, M. M., and Schaffner, W. (1989). Rapid detection
of octamer binding proteins with "mini-extracts", prepared from a small number of
cells. Nuc. Acid. Res., 17: 6419.
100
Secombes, C. J. (1994). The Phylogeny of Cytokines. The Cytokine Handbook, 2nd
ed. The Cytokine Handbook, A. W. Thomson, ed., Academic Press, New York,
567-594.
Tamai, T., Sato, N., Kimura, S., Shirahata, S., and Murakami, H. (1992). Cloning and
expression of flatfish interleuldn 2 gene. Animal Cell Technology: Basic &
Applied Aspects., H. Murakami, ed., Kloewer Academic Publishers,
Netherlands, 509-513.
Tamai, T., Shirahata, S., Noguchi, T., Sato, N., Kimura, S., and Murakami, H. (1993).
Cloning and expression of flatfish (Paralichthys olivaceus) interferon cDNA.
Biochim Biophys Acta, 1174(2): 182-6.
Torigoe, T., Saragovil, H. U., and Reed, J. C. (1992). Interleukin -2 regulates the
activity of the lyn protein-tyrosine kinase in a B-cell line. Proc. Natl. Acad. Sci.
USA, 89: 2674-2678.
Venkitaraman, A. R., and Cowling, R. J. (1992). Interleukin-7 receptor functions by
recruiting the tyrosine kinase p59 through a segment of its cytoplasmic tail.
Proc. Natl. Acad Sci. USA, 89: 12083-12087.
Wakao, H., Gouilleux, F. and Groner, B. (1994) Mammary gland factor (MGF) is a
novel member of the cytokine regulated transcription factor gene family and
confers the prolactin response. EMBO J., 13: 2182-2191.
101
CHAPTER 6
THESIS SUMMARY
Immune Modulatory Genes in Rainbow Trout
Two distinct genes sequences were isolated from rainbow trout. Chapter 3
describes the isolation of a gene from trout peripheral blood cells that is significantly related
to two human genes termed natural killer enhancement factor (NKEF) (Shau et al., 1994).
The protein products from these human genes have been show to enhance the cytolytic
killing activity of NK cells (Shau et al., 1993). Chapter 4 describes the isolation of a gene
sequence that is also significantly related to a group of mammalian genes termed signal
transduction and activators of translation (STAT). Specifically, the STAT gene involved in
the transduction of lymphokines IL-2, IL -7 and IL-15, is closely related to the rainbow
trout gene. To date only two genes involved in immune modulation have been isolated
from fish (Tamai et al 1992 and 1993). These findings help us achieve an understand of
the evolutionary aspects of immune regulation in phylogenetically distant species.
Biological Characterization of Gene Products
By examining the biological functions of the products of these two genes we
demonstrated their roles in immune modulation of rainbow trout, and relatedness to their
mammalian counterparts. Chapter 4 showed that the protein product from the nonspecific
cytotoxic cell enhancement factor (NCEF) gene was antigenically and functionally related
to the human NKEF protein. NC cell cytolytic activities of rainbow trout lymphocytes
were enhanced significantly when exposed to target cells expressing the NCEF protein.
Also, apoptotic killing activity was shown to be enhanced by this protein. NC cells are
important to the resistance of fish to viral and parasitic infections (Moody et al., 1985;
Graves et al., 1985). Enhancement of the activity of this cell type may be useful in
102
combating diseases of fishes. This factor could be employed as either a prophylactic or
therapeutic means for managing fish health. Specifically, fish rearing facilities could
administer this factor to fish prior to situation of stress or potential encounters with viral
pathogens. Enhance immune surveillance mediated by NC cells would aid in combating
the initial stages of virus infection and increase the survivability of infected fish.
Chapter 5 demonstrates the existence of STAT5-like protein in trout leukocytes.
This protein like its mammalian counterpart may be involved in the intracellular
transduction of mitogenic signals. The electromobility shift and super shift assays show
that trout STAT5 protein is rapidly translocated to the nucleus and binds to the GAS motif
DNA sequence after mitogenic stimulus. This not only demonstrates the functional
similarity of these proteins from two phylogenetically distant taxa, but also implies that
promoter element sequences for cytokine regulated genes are similar. This is important
since few genes in fish have been characterized as cytoldne inducible genes and such
sequence data may aid in their isolation. The electromobility shift assay could potentially
be used determine the type of cytokines to which fish cells can respond. Assay systems to
date have relied on relatively crude culture systems to assay for certain cytokine activities.
Fish-derived proteins suspected to have cytokine activity could be categorized, as they are
in mammals, based on what type of STAT proteins are activated.
103
BIBLIOGRAPHY
Aarden, L. A. (1979). Revised nomenclature for antigen-nonspecific T cell proliferation
and helper factors. J. Immunol., 123: 2928-2929.
Ahne, W. (1993). Presence of interleukins (IL-1,IL-3,M-6) and the tumor necrosis
factor (TNF alpha) in fish sera. Bull. Eur. Ass. Fish Pathol., 13: 105-107.
Ahne, W. (1994). Evidence for the early appearance of interleukins and tumor necrosis
factor in the phylogenesis of vertebrates. Immunol. Today, 15: 137.
Argetsinger, L.S., Campbell, G.S., Yang, X., Witthuhn, B.A., Silvennoinen, 0., Ihle,
J.N., and Carter-Su, C. (1993). Identification of JAK2 as a growth hormone
receptor-associated tyrosine kinase. Cell , 74: 237-244.
Beck, G., and Habicht, G. S. (1986). Isolation and characterization of a primitive
interleukin -1 -like protein from an invertebrate, Asterias forbesi. Proc. Natl. Acad.
Sci. USA, 83: 7429-7433.
Beck, G., Vasta, G. R., Marchalonis, J. J., and Habicht, G. S. (1989). Characterization
of interleukin-1 activity in tunicates. Comp. Biochem. Physiol., 92B: 93-98.
Bloom, B., and Bennett, B. (1966). Mechanism of a reaction in vitro associated with
delayed-type hypersensitivity. Science, 153: 80-82.
Bowcock, A. M., Ray, A., Erlich, H. A., and Sehgal, P. B. (1989). The molecular
genetics of beta-2 interferon/interleukin-6 (IFN beta 211L6) alpha. Ann. N. Y.
Acad. Sci. USA, 557: 345-52.
Carlson, R. L., Evans, D. L., and Graves, S. S. (1985). Nonspecific cytotoxic cell in
fish (Ictalurus punctatus). V. Metabolic requirements of lysis. Dev. Comp.
Immunol., 9: 271-280.
Caspi, R. R., and Avtalion, R. R. (1984). Evidence for the existence of an IL-2 like
lymphocyte growth promoting factor in bony fish, Cyprinus carpio. Dev. Comp.
Immunol., 8: 51-60.
Chae, H. Z., Kim, I. H., Kim, K., and Rhee, S. G. (1993). Cloning, sequencing and
mutation of thiol-specific antioxidant gene of Saccharomyces cerevisiae. J. Biol.
Chem., 268: 16815-16821.
Chen, S. J., Holbrook, N. J., Mithchell, K. F., Vallone, C. A., Greengard, J. S.,
Crabtree, G. R., and Lin, Y. (1985). A viral long terminal repeat in the
interleukin 2 gene of a cell line that constitutively produces interleukin 2. Proc.
Natl. Acad. Sci. USA, 82: 7284-7288.
104
Chu, E. T., Lareua, M., Rosenwasser, L. J., Dinerello, C. A., and Geha, R. S. (1985).
Antigen presentation by EBV-B cells to resting and activated T cells: role of
interleukin-1. J. Immunol., 134: 1676-1679.
Clem, L. W., Miller, M. W., and Bly, J. E. (1990). Evolution of lymphocyte
subpopulation. their interactions and temperature sensitivities. Phylogenesis of
Immune Function., G. W. Warr and N. Cohen, eds., CRC Press, Boca Raton,
191-213.
Clem, L. W., Sizemore, R. C., and Ellsaesser, C. F. (1985). Monocytes as accessory
cells in fish immune responses. Dev. Comp. Immunol., 9: 803-809.
Cohen, R. L., Bigazzi, P. E., and Yoshida, T. (1974). Cell Immunol., 12: 150-159.
Cohn, Z. A. (1978). The activation of mononuclear phagocytes: Facts, fancy, and
future. J. Immunol., 121: 813-816.
Darnell, J. E., Kerr, I. M., and Stark, G. R. (1994). Jak -STAT pathways and
transcriptional activation in response to INFs and other extraceLlular signaling
proteins. Science, 264: 1415-1421.
deKinkelin, P., Dorson, M., and Hattenberger-Baubouy, A. M. (1982). Interferon
synthesis in trout and carp after viral infection. Dev. Comp. Immun., Suppl. 2:
167-174.
Deluca, D., Wilson, M., and Warr, G. W. (1983). Lymphocyte heterogeneity in the
trout, Salmo gairdneri, defined with monoclonal antibodies to IgM. Eur. J.
Immunol., 13: 546-551.
Demetri, G. D., and Griffin, J. D. (1991). Granulocyte colony-stimulating factor and its
receptor. Blood, 78: 2791-808.
Dennert, G. and Podack, E. R. (1983). Assembly of tubular complexes on target cell
membranes. J. Immunol., 157: 1483-1495.
De Sena, J., and Rio, G. J. (1975). Partial purification and characterization of RTG-2
fish cell interferon. Infect. and Immunity, 11: 815-822.
Di-Giovine, F. S., and Duff, G. W. (1990). IL-1: the first interleukin. Immunol.
Today, 11: 13-20.
Dorson, M., Barde, A., and deKinkelin, P. (1975). Egtved virus induces rainbow trout
serum interferon: some physiochemical properties. Ann. Microbiol., 126: 485­
489.
Duke, R. C. and Cohen J. J. (1986). Differences in target cell DNA fragmentation
induced by mouse cytotoxic T lymphocytes and nonspecific killer cells. J.
Immunol., 137: 1442-1447.
Duke, R.C., Chervenak, R. and Cohen J. J. (1983). Endogenous endonuclease-induced
DNA fragmentation. An early event in cell mediated cytolysis. Proc. Natl.
Acad. Sci. USA, 80: 6361-6356.
105
Dumonde, D. C., Wolstencroft, R. A., Panayi, G. S., Matthew, M., Morley, J., and
Howson, W. T. (1969). Lympholcines : Non-antibody mediators of cellular
immunity generated by lymphocyte activation. Nature, 224: 38-42.
Edington, H., and Lotze, M. T. (1994). Interleukin-7. The Cytokine Handbook., A. W.
Thomson, ed., Academic Press, New York, 169-185.
Ellsaesser, C. F. M. (1989). Identification and Characterization of a High and Low
Molecular Weight Species of Channel Catfish Interleukin-1 with Differential
activities on Fish and Mouse Lymphocytes., Doctoral dissertation, University of
Mississippi, Jackson.
Epstein, L. B. (1984). The special significance of interferon-gamma. Interferon, J.
Vilcek and E. DeMaeyer, eds., Elsevier Science Publishers, Amsterdam, 195­
220.
Evans, D. L., Carlson, R. L., Graves, S. S., and Hogan, K. T. (1984a). Nonspecific
cytotoxic cell in fish (Ictalurus punctatus). 1 V. Target cell binding and recycling
capacity. Dev. Comp. Immunol., 8: 823-833.
Evans, D. L., Carlson, R. L., Graves, S. S., and Hogan, K. T. (1984b). Nonspecific
cytotoxic cell in fish (Ictalurus punctatus). II. Parameters of target cell lysis and
specificity. Dev. Comp. Immunol., 8: 303-312.
Evans, D. L., Harris, D. T., and Jaso-Freidman, L. (1992). Functional associated
molecules on nonspecific cytotoxic cells: Role in calcium signaling, redirect lysis,
and modulation of cytotoxicity. Dev. Comp. Immunol, 16: 383-394.
Evans, D. L., Hogan, K. T., and Graves, S. S. (1984c). Nonspecific cytotoxic cells in
fish (Ictalurus punctatus). III. Biophysical and biochemical properties affecting
cytolysis. Dev. Comp. Immunol., 8: 599-610.
Evans, D. L., Smith, E. E., and Brown, F. E. (1987). Nonspecific cytotoxic cell in fish
(Ictalurus punctatus). VI. Flow cytometric analysis. Dev. Comp. Immunol., 11:
95-104.
Frank, D. A., Robertson, M. J., Bonni, A., Ritz, J., and Greenburg, M. E. (1995).
Interleukin 2 signaling involves the phosphorylation of Stat proteins. Proc. Natl.
Acad. Sci. USA, 92: 7779-7783.
Fukazawa, Y., Kagaya, K., Miura, H., Shinoda, T., Natori, K., and Yamazaki, S.
(1984). Biological and biochemical characterization of macrophage activating
factor (MAF) in murine lymphocytes: physiochemical similarity of MAF to
gamma interferon (INF-y). Microbiol. Immunol., 28, 691-702.
Gasson, J. (1991). Molecular physiology of Granulocyte-Macrophage ColonyStimulating Factor. Blood, 77: 1131-1145.
Gearing, A. J. H., Cartwright, J. E., and Wadhwa, M. (1994). Biological and
immunological assays for cytokines. The Cytokine Handbook, A. W. Thomson,
ed., Academic Press, New York, 507-522.
106
Gillis, S., Baker, P. E., Ruscetti, F. W., and Smith, K. A. (1978a). Long-term culture
of human antigen-specific cytotoxic T-cell lines. J. Exp. Med., 148(4): 1093­
1098.
Gillis, S., Ferm, M. M., Ou, W., and Smith, W. A. (1978b). T cell growth factor:
parameters of production and a quantitative micro assay for activity. J. Immunol.,
120: 2027-2032.
Gilmour, K. C., and Reich N. C. (1994). Receptor to nucleus signaling by prolactin and
interleukin -2 via activation of latent DNA-binding factors. Proc. Natl. Acad. Sci.
USA, 91: 6850-6854.
Gold Smith, M. A., and Greene, W. C. (1994). Interleukin -2 and the interleukin -2
receptor. The Cytokine Hanbook, A. W. Thomson, ed., Academic Press, New
York, 57-80.
Goodall, J. C., Emery. D. C., Bailey, M., English, L. S., and Hall, L. (1991). cDNA
cloning of porcine interleukin 2 by polymerase chain reaction. Biochim. Biophys.
Acta., 1089: 257-259.
Graham, S., and Secombes, C. J. (1988). The production of a macrophage-activating
factor from rainbow trout Salmo gaidneri leukocytes. Immunology, 65: 293­
297.
Graham, S., and Secombes, C. J. (1990a). Cellular requirements for lymphokine
secretion by rainbow trout Salmo gairdneri leukocytes. Dev. Comp. Immunol.,
14: 59-68.
Graham, S., and Secombes, C. J. (1990b). Do fish lymphocytes secrete interferon -y?
J. Fish Biol., 36: 563-573.
Granath, W. 0., Connors, V. A., and Tarleton, R. L. (1994). Interleuldn-1 activity in
heamolymph from strains of the snail Biophalaria glabrata varying in susceptibility
to the human blood fluke, Schistosoma mansoni : Presence, differential
expression and biological function. Cytokine, 6: 21-27.
Gravell, M., and Marlsberg, R. G. (1965). A permanent cell line from flathead minnow
(Pimephales promelas). Ann. N.Y. Acad. Sci., 126: 555-565.
Graves, S. S., Evans, D. L., Cobb, D., and Dawe, D. L. (1984). Nonspecific cytotoxic
cells in fish (Ictalurus punctatus), I. Optimum requirements for target cell lysis.
Dev. Comp. Immunol., 8: 293-302.
Graves, S. S., Evans, D. L., and Dawe, D. L. (1985). Mobilization and activation of
nonspecific cytotoxic cells in the channel catfish infected with Ichthyopthirus
multifilis. Dev. Comp. Immunol., 8: 43-51.
Greenlee, A. R., Brown, R. A., and Ristow, S. S. (1991). Nonspecific cytotoxic cells
of rainbow trout (Oncorhynchus mykiss) kill YAC-1 target cells by both necrotic
and apoptotic mechanisms. Dev. Comp. Immunol., 15: 43-51.
107
Grondel, J. L., and Hamersen, E. G. (1984). Phylogeny of interleukins: growth factors
produced by leukocytes of cyprinid fish, Cyprinus carpio. Immunology, 52, 477­
482.
Hamblin, A. S. (1993). Cytokines one by one. Cytokines and Cytokine Receptors., D.
Male and D. Rickwood, eds., IRL Oxford University Press, New York, 21-43.
Hatakeyama, M., Kono, T., Kobayashi, N., Jawawhara, A., Levin, S.D., Perlmutter,
R.M., Taniguchi, T. (1991). Interaction of the IL-2 receptor with the src-family
kinase p561ck . Identification of novel intermolecular association. Science , 252:
1523-1528.
Hayari, Y., Schauenstein, K., and Globerson, A. (1982). Avian lymphokines, II:
Interleukin-1 activity in supernatants of stimulated adherent splenocytes of
chickens. Dev. Comp. Immunol., 5: 785-789.
Hayden, B. J., and Laux, D. C. (1985). Cell-mediated lysis of murine target cells by
nonimmune salmonid lymphoid preparations. Dev. Comp. Immunol., 9: 627­
639.
Heldin, C. H. (1995). Dimerization of cell surface receptors in signal transduction.
Cell , 80: 213-223.
Herberman, R. B., Nunn, M. E., Holden, H. T., Staal, S., and Djeu, J. Y. (1977).
Augmentation of natural cytotoxic reactivity of mouse lymphoid cells against
syngeneic and allogeneic target cells. Int. J. Cancer, 19: 555-562.
Himuma, S., Abo, T., Kumagai, K., and Hata, M. (1980). The potent activity of fresh
water fish kidney cells in cell killing. I. Characterization and species -distribution
of cytotoxicity. Dev. Comp. Immunol., 4: 653.
Hou, J., Schindler, U., Henzel, W.J., Wong, S. C., and McKnight S. L. (1995).
Identification of human STAT proteins activated in response to interleukin-2.
Immunity, 2: 321-329.
Isaacs, A., and Lindermann, J. The interferon. Virus interference., London, 258-267.
Ishii, T., Yamada, M., Sato, H., Matsue, M., Taketani, S., Nakayama, K., Sugita, Y.,
and Bantu, S. (1993). Cloning and characterization of a 23 k Da stress-induced
mouse peritoneal macrophage protein. J. Biol. Chem., 268: 18633-18636.
Ivashkiv, L. B. (1995). Cytokines and STATs: How can signals achieve specificity ?
Immunity, 3: 1-4.
Janeway, C. A. (1989). A primitive immune system. Nature, 341: 108-111.
Kashima, N., Nishi-Takaoka, C., Fujita, T., Taki, S., Yamada, G., Hamuro, J., and
Taniguchi, T. (1985). Unique structure of murine interleukin-2 as deduced from
cDNAs. Nature, 313: 402-408.
Kiener, P. A., and Spitalny, G. L. (1987). Induction, production and purification of
murine gamma interferon. Lymphokines and Interferons., M. J. Clemens, A. G.
Morris, and A. J. H. Gearings, eds., IRL Press, Oxford, 15-28.
108
Kim, I. H., Kim, K., and Rhee, S. G. (1989). Induction of an antioxidant protein of
Saccharomyces cerevisiae 02, Fe3+ or 2-mercaptoethanol. Proc. Natl. Acad.
Sci. USA, 86: 6018-6022.
Kishimoto, T., Taga, T., and Akira, S. (1994). Cytokine signal transduction. Cell, 76:
253-262.
Lee, K. C., Shiozawa, C., Shaw, A., and Diener, E. (1976). Requirements for
accessory cell in antibody response to T cell antigens in vitro. Eur. J. Immunol.,
6: 63-69.
LeMorvan-Rocher, C., Trout land, D., and Deschaux, P. (1995). Effects of temperature
on carp leukocyte mitogen-induced proliferation and nonspecific cytotoxic activity.
Dev. Comp. Immunol., 19: 87-95.
Lester III, J. P., Evans, D. L., Leary III, J. H., Fowler, S. C., Jaso-Friedmann, L.
(1994). Identification of a target cell antigen recognized by nonspecific cytotoxic
cells using a anti-idiotype monoclonal antibody. Dev. Comp. Immunol., 18: 219­
229
Lin, G. L., Ellsaesser, C. F., Clem, L. W., and Miller, N. W. (1992). Phorbol
ester/calcium ionophore activate fish leukocytes and induce long term cultures.
Dev. Comp. Immunol., 16: 153-163.
Lin, J., Mignone, T., Tsang, M., Fiedmann, M., Weatherbee, J. A., Yamauchi, L. A.,
Bloom, E. T., Mietz, J., John, S., and Leonard, W. J. (1995). The role of
shared receptor motifs and common STAT proteins in the generation of cytokine
pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-13 and IL-15. Immunity, 2:
331-339.
Londei, M., Verhoef, A., Berardinis, P. D., Kissonerghis, M., Grubeck-Loebenstein,
B., and Feldman, M. (1989). Definition of a population of CD4-8- T cells that
express the ar3 T-cell receptor and respond to interleuldns 2, 3, and 4. Proc.
Natl. Acad. Sci. USA, 86: 8502-8506.
Lucero, M. A., Fridman, W. H., Provost, M. A., Billardon, C., Pouillart, P., Dumont,
J., and Falcoff, E. (1981). Effects of various interferons on the spontaneous
cytotoxicity exerted by lymphocytes from normal and tumor bearing patients.
Cancer Res., 41: 294-297.
Luger, T. A., Charon, J. A., Co lot, M., Micksche, M., and Oppenheim, J. J. (1983).
Chemotatic properties of partially purified human epidermal cell-derived
thymocyte-activating factor (ETAF) for polymorphonuclear and mononuclear
cells. J. Immunol., 131: 816-820.
May, L. T., Santhanam, U., Tatter, S. B., Ghrayeb, J., and Sehgal, P. B. (1989).
Multiple forms of human interleukin -6. Phosphoglycoproteins secreted by many
different tissues. Ann. N. Y. Acad. Sci, 557: 114-121.
McKinney, E. C., and Schmale, M. C. (1994). Damselfish with neurofibromatosis
exhibit cytotoxicity towards targets. Dev. Comp. Immunol., 18: 305-313.
109
McKnight. A. J., Mason D. W., and Barclay A. N. (1989). Sequence of rat interleukin 2
and anomalous binding of a mouse interleukin 2 cDNA probe to rat MHC class IIassociates invariant chain mRNA. Immunogenetics , 30: 145-147.
Mestan, J., Digel, W., Mittnacht, S., Hillen, H., Blohm, D., Moeller, A., Jacobsen, H.,
and Kirchner, H. (1986). Antiviral effects of tumor necrosis factor in vitro.
Nature, 323: 816-819.
Moeller, J., Hultner, L., Schmitt, E., Breuer, M., and Dormer, P. (1990). Purification
of MEA, a mast cell growth enhancing activity, to apparent homogeneity and its
partial amino acid sequencing. J. Immunol., 144: 4231-4234.
Moody, E. C., Serreze, D. V., and Reno, P. W. (1985). Nonspecific cytotoxic cell
activity of teleost fish. Dev. Comp. Immunol., 9: 51-64.
Morgan, D. A., Ruscetti, F. W., and Gallo, R. (1976). Selective in vitro growth of T
lymphocytes from normal bone marrow. Science, 1193: 1007-1008.
Mourich, D. V., Hansen, J., and Leong, J. C. (1995). Natural killer cell enhancement
factor-like gene in rainbow trout (Oncorhynchus mykiss). Immunogenetics, 42:
438-439.
Nagler, A., Lanier, L. L., and Philips, J. H. (1988). The effects of IL-4 on human
natural killer cells. A potent regulator of IL-2 activation and proliferation. J.
Immunol., 141: 2349-2344.
Nathen, C. F., Murray, H. W., Wiebe, M. E., and Rubin, B. Y. (1983). Identification
of an interferon as a lymphokine that activates human macrophage oxidative
metabolism and antimicrobial activity. J. Exp. Med., 158: 670-689.
Naume, B., Gately, M., and Espevik, T. (1992). A comparative study of IL-12
(cytotoxic lymphocyte maturation factor), IL-2, and IL-7 induced effects on
immunomagnetically purified CD56+ NK cells. J. Immunol., 148: 2429-2436.
Noguchi, M., Nakamura, Y., Russell, S. M., Ziegler, S. F., Tsang, M., Cao, X., and
Leonard, W. J. (1993). Interleukin-2 receptor gamma chain: a functional
component of the interleukin-7 receptor. Science, 262: 1877-1880.
North, R. J. (1978). The concept of the activated macrophage. J. Immunol., 121: 806­
809.
Okusawa, S., Gelfand, J. A., Ikejima, T., Connolly, R. J., and Dinarello, C. A. (1988).
Interleukin 1 induces a shock-like state in rabbits. Synergism with Tumor
Necrosis Factor and the effects of cyclooxygenase. Jr. Clin. Invest., 81: 1162­
1 172.
Ortega, H. (1993). Mechanisms of accessory cell function in rainbow trout
(Oncorhynchus mykiss). Masters of Science, Oregon State University, Corvallis.
Ostensen, M. E., Thiele, D. L., and Lipsky, P. E. (1987). Tumor necrosis factor-alpha
enhances cytolytic activity of human natural killer cells. J. Immunol., 138: 4185­
4188.
110
Paemen, L. P., Porchet-Hennere, E., Mason, M., Leung, M. K., Hugues, T. K., and
Stefano, G. B. (1992). Glial localization of interleukin-la in invertebrate ganglia.
Cell. Mol. Neurobiol., 12: 463-472.
Paul, W. E. (1991). Interleukin-4: A Prototypic Immunoregulatory Lymphokine. Blood,
77: 1859-1870.
Paul, W. E., and Ohara, J. (1987). B-Cell Stimulatory Factor-1/ Interleukin 4. Ann.
Rev. Immunol, 5: 429-459.
Paul, W. E., and Sader, R. A. (1994). Lymphocyte responses and cytokines. Cell, 76:
241-251.
Pene, J., Rousset, F., Briere, F., Chretien, I., Bonnefoy, J. Y., Spits, H., Yokota, T.,
Arai, N., Arai, K., Banchereau, J., and Vreis, J. E. (1988). IgE production by
normal human lymphocytes is induced by interleukin 4 and suppressed by
interferons gamma and alpha and prostaglandin E2. Proc. Natl. Acad. Sci. USA,
85: 6880-6884.
Perussia, B., Santo li, D., and Trinchieri, G. (1980). Interferon modulation of natural
killer cell activity. Ann. N.Y. Acad. Sci., 350: 55-72.
Punnonen, J., Aversa, G., Cocks, B. G., McKenzie, A. N. J., Menon, S., Zurawski,
G., deWaal-Malefyt, R., and Vreis, J. E. (1993). Interleukin-13 induces
interleukin-4-independent IgG4 and IgE synthesis and CD23 expression by
human B cells. Proc. Natl. Acad. Sci. USA, 90: 3730-3734.
Ray, A., Tatter, S. B., Santhanam, U., Helfgott, D. C., May, L. T., and Sehgal, P. B.
(1989). Regulation of expression of interleukin-6. Molecular and clinical studies.
Ann. N. Y. Acad. Sci, 557: 353-362.
Reeves, R., Speis, A. G., Nissen, M. S., Buck, C. D., Weinberg, A. D., Barr, P. J.,
Magnuson, N. S., and Magnuson, J. A. (1986). Molecular cloning of a
functional bovine interleukin-2 cDNA. Proc. Natl. Acad. Sci. USA, 83: 3223­
3229.
Ritz, J., Schmidt, R. E., Michon, J., Hercend, T., and Schlossman, S. F. (1988).
Characterization of functional surface structures on human natural killer cells.
Adv. Immunol., 42: 181-211.
Robb, R.J. and Greene, W. C. (1983). Direct demonstration of the identity of T cell
growth factor binding protein and the TAC antigen. J. Exp. Med., 158: 1332­
1337.
Rook, G. A. W., Steele, J., Umar, S., and Dockrell, H. M. (1985). A simple method
for the solubilization of reduced NBT, and its use as a colorimetric assay for
activation of human macrophages by y-interferon. J. Immunol. Methods, 82:
161-167.
Ruff-Jamison, S., Chen, K., and Cohen, S. (1995). Epidermal growth factor induces
the tyrosine phosphorylation and nuclear translocation of StatS in mouse liver.
Proc. Natl. Acad. Sci. USA, 92: 4215-4218.
111
Russel, S. M., Johston, J. A., Noguchi, M., Kawamura, M., Bacon, C. M., Friedman,
M., Berg, M., Mc Vicar, D. W., Witthuhn, B. A., Silvennoinen, 0., Goldman,
A. S., Schmalstieg F. C., lh le J. N., O'Shea, J. J., and Leonard W. J. (1994).
Interaction of IL -2R[ and y chains with Jakl and Jak3: Implications for XSCID
and XCID. Science, 266: 1042-1047
Russell, J. H., Masakowski. V. R., Dobos, C. B. (1980). Mechanisms of immune
lysis. I. Physiological distinctions between target cell death mediated by cytotoxic
T lymphocytes and antibody plus complement. J. Immunol., 124: 1100-105.
Sabath, D. E., Podolins, P. L., Comber, P. G., and Prystowsky, M. B. (1990). cDNA
Cloning and characterization of interleukin-2 induced genes in a cloned T helper
lymphocyte. J. Biolog. Chem., 265: 12671-12678.
Sato, H., Ishii, T., Suguta, Y., Tateishi, N., and Bannai, S. (1993). Induction of a 223
k Da stress protein by oxidative and sulfhydryl-reactive agents in mouse peritoneal
macrophages. Biochem. Biophys. Acta., 1148: 127-132.
Schrader, J. W., Clark-Lewis, I., Crapper, R. M., Leslie, K. B., Schrader, S., Varigios,
G., and Zilterner, H. J. (1988). Interleukin 3: The panspecific hemopoietin.
Lymphokines 15, J. W. Schrader, ed., Academic Press, San Diego, 281-311.
Secombes, C. J. (1994). The Phylogeny of Cytokines. The Cytokine Handbook, 2nd
ed. The Cytokine Handbook, A. W. Thomson, ed., Academic Press, New York,
567-594.
Sehgal, P. B., Wang, L., Rayanade, R., Pan, H., and Margulies, L. (1995).
Interleukin-6-type cytokines. Ann. N. Y. Acad. Sci, 762: 1-14.
Seow, H. F., Rothel J. S., Radford, A. J., and Wood, P. R. (1990). The molecular
cloning of ovine interleukin 2 gene by the polymerase chain reaction. Nuc. Acids
Res., 18: 7175.
Shau, H., Butterfield, L. H., Chiu, R., and Kim, A. (1994). Cloning and sequence
analysis of a candidate human natural kill-enhancing factor gene.
Immunogenetics, 40: 129-134.
Shau, H., and Golub, S. H. (1988). Modulation of natural killer-mediated lysis by red
blood cells. Cell. Immunol., 116: 60-72.
Shau, H., Gupta, R. K., and Golub, S. H. (1993b). Identification of a natural killer
enhancing factor (NKEF) from human erythroid cells. Cell. Immunol., 147: 1­
11.
Stewart, W. E. (1980). Interferon nomenclature. Nature, 286: 110.
Tamai, T., Sato, N., Kimura, S., Shirahata, S., and Murakami, H. (1992). Cloning and
expression of flatfish interleukin 2 gene. Animal Cell Technology: Basic &
Applied Aspects., H. Murakami, ed., Kloewer Academic Publishers,
Netherlands, 509-513.
112
Tamai, T., Shirahata, S., Noguchi, T., Sato, N., Kimura, S., and Murakami, H.
(1993a). Cloning and expression of flatfish (Paralichthys olivaceus) interferon
cDNA. Biochim Biophys Acta, 1174(2): 182-6.
Tamai, T., Shirahata, S., Sato, N., Kimura, S., Nonaka, M., and Murakami, H.
(1993b). Purification and characterization of interferon-like antiviral protein
derived from flatfish (Paralichthys olivaceus) lymphocytes immortalized by
oncogenes. Cytotechnology, 11(2): 121-31.
Taniguchi, T., Mantei, N., Schwarzstein, M., Nagata, S., Muramatsu, M., and
Weissman, C. (1980). Human leukocyte and fibroblastic interferons are
structurally related. Nature, 285: 547-549.
Taniguchi, T., Matsui, H., Fujita, T., Takaoka, C., Kashima, N., Yoshimoto, R., and
Hamura, J. (1983). Structure and expression of a cloned cDNA for human
interleukin-2. Nature, 302: 305-310.
Taria, S., Matsui, M., Hayakawa, K., Yokoyama, T., and Nariuchi, H. (1987).
Interleukin 2 secretion by B cell lines and spleen derived B cells stimulated with
calcium ionophore and phorbol ester. J. Immunol., 139: 2957-2964.
Tartaglia, L. A., Storz, G., Brodsky, M. H., Lai, A., and Ames, B. N. (1990). Alkyl
hydroperoxide reductase from Salmonella typhimurium . Sequence and homology
to thioredoxin reductase and other flavoprotein disulfide oxidoreductases. J. Biol.
Chem., 265: 10535-10540.
Thomson, A. W., and Lotze, M. T. (1994). Interleukins 13, 14 and 15. The Cytokine
Handbook, A. W. Thomson, ed., Academic Press, New York, 261-262.
Torigoe, T., Saragovil, H. U., and Reed, J. C. (1992). Interleukin-2 regulates the
activity of the lyn protein-tyrosine kinase in a B-cell line. Proc. Natl. Acad. Sci.
USA, 89: 2674-2678.
Trinchieri, G. (1994). Definition and biology of natural killer cells. Molecular Biology
Intelligence Unit: MHC Antigens and NK Cells, J. Pena and R. Solana, eds., R.
G. Landers company, CRC press, Austin, 1-15.
Trinchieri, G. (1989). Biology of natural killer cells. Adv. Immunol., 47: 182-376.
Trinchieri, G., Matsumoto-Kobayashi, M., Clark, S. C., Sheehra, J., London, L., and
Perussia, B. (1984). Response of resting human peripheral blood natural killer
cells. J. Exp. Med., 160: 1147-1153.
Tripp, R. A. (1988). Glucocorticoid Regulation of Salmonid B Lymphocytes., Doctoral
Dissertation, Oregon State University, Corvallis.
Venkitaraman, A. R., and Cowling, R. J. (1992). Interleukin-7 receptor functions by
recruiting the tyrosine kinase p59fYn through a segment of its cytoplasmic tail.
Proc. Natl. Acad. Sci. USA, 89: 12083-12087.
Verburg-van Kemenade, B. M. L., Weyts, F. A. A., Debets, R., and Flik, G. (1995).
Carp macrophages and neutrophilic granulocyte secrete an interleuldn-l-like
factor. Dev. Comp. Immunol., 19: 59-70.
113
Vilcek, J., and Lee, J. (1994). Immunology of Cytokines: An introduction. The
Cytokine Handbook, A. W. Thomson, ed., Academic Press, New York, 6-7.
Vilcek, J., and Lee, T. H. (1991). Tumor Necrosis Factor: New Insights Into The
Molecular Mechanisms of its Multiple Actions. J. Biol. Chem., 266: 7313-7316.
Walker, E., Leemhuis, T., and Roeder, W. (1988). Murine B lymphoma cell lines
release functionally active interleukin 2 after stimulation with S. aureus . J.
Immunol. , 140: 859-865.
Watkins, D., Parsons, S. C., and Cohen, N. (1987). A factor with interleukin -1 -like
activity is produced by peritoneal cells from the frog Xenopus laevis.
Immunology, 62: 669-673.
Weigent, D. A., Langford, M. P., Fleishmann, W. R., and Stanton, G. J. (1982).
Enhancement of natural killing activity by different types of interferons. Human
Lymphokines, A. Khan and N. 0. Hills, eds., Academic Press, New York,
539.
Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U.,
Wallach, D., Perricaudet, M., Tiollais, P., and Revel, M. (1980). Two interferon
mRNAs in human fibroblasts: In vitro translation and Escherichia coli cloning
studies. Proc. Natl. Acad. Sci. USA, 77: 7152-7156.
West, W. H., Cannon, G. B., Kay, H. D., Bonnard, G. D., and Heberman, R. B.
(1977). Natural cytotoxic reactivity of human lymphocytes against a myeloid cell
line: Characterization of the effector cell. J. Immunol., 118: 355-367.
Wheelock, E. F. (1965). Interferon-like virus-inhibitor in induced in human leukocytes
by phytohemagglutinin. Science, 149: 310-311.
Wolfe, S.A. Tracey, D.E., and Henney, C. S. (1977). Induction of natural killer cells by
BCG. Nature, 262: 584-587.
Wylie, A. H. (1980). Glucocorticoid-induced thymocyte apoptosis with endogenous
endonuclease activation. Nature, 284: 555-556
Yamamoto, T., Matsui, Y., Natori, S., and Obinata, M. (1989). Cloning of a
housekeeping-type gene (MER5) preferentially expressed in murine
erthroleukemia cells. Gene, 80: 337-343.
Yannelli, J. R., Thurman, G. B., Mrowca-Bastin, A., Pennington, C. S., West, W. H.,
and Oldman, R. K. (1988). Enhancement of human lymphokine-activated killer
cell yields to cancer patients. Cancer Res., 48: 5696-5700.
Yip, Y. K., Pang, R. H. L., Urban, C., and Vilcek, J. (1981). Partial purification and
characterization of human-g (immune) interferon. Proc. Natl. Acad. Sci. U.S.A.,
78: 1601-1608.
Yokota, R., Arai, N., Lee, R., Rennick, D., Mosmann, T., and Arai, K. I. (1985). Use
of a cDNA expression vector for isolation of mouse interleukin-2 clones:
expression of T-cell growth factor activity after transfection of monkey cells.
Proc. Natl. Acad. Sci. USA, 82: 68-74.
114
Zeh, H. J., Tahara, H., and Lotze, M. T. (1994). Interleukin -12. The Cytokine
Handbook, A. W. Thomson, ed., Academic Press, New York, 245-256.