Alteration of L-selectin by mAb, cytoskeletal association, and fatty acid membrane content
by Jeffrey Gordon Leid
A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of
Philosophy in Veterinary Molecular Biology
Montana State University
© Copyright by Jeffrey Gordon Leid (2000)
Abstract:
L-selectin is an adhesion protein expressed on most leukocytes and is responsible for the initial event in
the multistep process of transendothelial cell migration, tethering and rolling along the vessel wall.
Leukocytes are captured from the blood stream by engagement of carbohydrate moieties, expressed on
large molecular weight glycoproteins, by a calcium-dependent lectin domain expressed on L-selectin.
L-selectin is expressed at the tips of the microvilli of leukocytes in humans and mice. Here, we
extended this observation to bovine lymphocytes. Also, bovine &gama;δ T cells were smaller in size
and contain greater than twofold the number of microvilli than αβ T cells. Engagementof L-selectin by
its carbohydrate ligands may have functional consequences in the cell, such as calcium mobilization,
tyrosine phosphorylation, and may also cause a conformational change in L-selectin that leads to
stronger tethering and rolling interactions. L-selectin binding by an anti-L-selectin mAb, LAM1-116,
caused a conformational change in L-selectin that was recognized by another anti-L-selectin mAb,
EL-246. Both mAbs bind to distinct, functional epitopes on L-selectin. Furthermore, the
conformational change induced in L-selectin by LAM1-116 predisposed L-selectin to cytoskeletal
association, which was triggered by engagement of the EL-246 epitope by EL-246 mAb. Electron
microscopy was employed to further characterize the nature of the lymphocyte cytoskeleton. The
bovine lymphocyte cytoskeleton was dramatically different than an intact, resting cell. Compared to
normal lymphocytes, mild detergent treatment of the lymphocyte generated a smaller, smooth-surfaced
cell remnant that contained little cytoplasm and the cell nucleus. We also surveyed 28 human and
bovine cell surface proteins and compared their patterns of cytoskeletal association to that of
L-selectin. Our studies demonstrated that L-selectin fits a pattern of cytoskeletal association similar to
that of other adhesion proteins involved in tethering and rolling. Finally, we have shown that
enrichment of the lymphocyte cell membrane with polyunsaturated fatty acids inhibits the ability of
L-selectin to associate with the cytoskeleton, and may reduce L-selectin-mediated adhesive events in
vitro and in vivo. ALTERATION OF L-SELECTIN BY MAB, CYTOSKELETAL
ASSOCIATION, AND FATTY ACID MEMBRANE CONTENT
■ by
Jeffrey Gordon Leid
A dissertation submitted in partial fulfillment of
the requirements for the degree
Doctor of Philosophy
Veterinary Molecular Biology
MONTANA STATE UNIVERSITY
Bozeman, Montana
April, 2000
©COPYRIGHT
by
Jeffrey Gordon Leid
2000
All Rights Reserved
APPROVAL
of a dissertation submitted by
Jeffrey Gordon Leid
This dissertation has been read by each member of the dissertation committee and
has been found to be satisfactory regarding content, English usage, format, citations,
bibliographic style, and consistency, and is ready for submission to the College of
Gradutate Studies.
Approved for the Department of Veterinary Molecular Biology
Mark A. Jutila, Ph.D.
Department Head
Date
Approved for the College of Graduate Studies
Bruce R. McLeod, Ph.D.
Graduate Dean
Date
STATEMENT OF PERMISSION TO USE
In presenting this dissertation in partial fulfillment of the requirements for a
doctoral degree at Montana State University, I agree that the Library shall make it
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Signature
Date
This dissertation is dedicated to my parents, Katherine M. Leid and R. Wes Leid,
who have been both loving and wonderful inspirations to me throughout my life. Mom, I
love you dearly and cherish every day I get to enjoy your company. Remember, you are
the next in line! And Pops, what else can I say except that I have grown up just like you
and of all the accomplishments in my life, I can think of none more fulfilling. I love you,
thank you both.
ACKNOWLEDGEMENTS
I would like to thank first and foremost my advisor. Dr. Mark Jutila. He has been
an excellent mentor and I look forward to working with him throughout my career as a
scientist. Secondly, I would like to thank my dissertation committee members, Dr. CA.
Speer, Dr. David Pascual, Dr. Jim Cutler, Dr. Martin Teintze, Dr. Stephen Perkins, and
Dr. Don Boyd. Thank you all for your critical input. I would also like to acknowledge
Dr, Thomas Tedder and Dr. Douglas Steeber who helped complete this dissertation by
providing reagents and performing experiments. Finally, I would like to thank the faculty
of the Department of Veterinary Molecular Biology for all of their support.
Many graduate students were also a great help and I thank you all, both past and
present. Specifically, I would like to thank a most dear friend, Karen Sipes, who without
her support and knowledge, I would not have finished in such a timely manner, nor would
I have enjoyed my stay to such a great extent. And to Jessica Borgquist, thank you for
everything and good luck On reaching your own personal goals as a scientist. You are a
bright shinning star in our department and a wonderful human being.
I would also like to thank my dear friends, Kemal Aydintug, Don Frye, Bill Frye
and the entire Frye family. You all have been vital to my success as a scientist and more
importantly, as a human being. Finally, to Miss Julie Oakason, who has brightened my
life in ways that are not accurately reflected in my words here, I love you dearly and look
forward to enjoying your company for the rest of my life. You are, and always will be,
my Sweet Pea
vi .
TABLE OF CONTENTS
Page
1. INTRODUCTION................................................................................................
I
Leukocyte Trafficking............................................................................................
Historical Perspective........................................................................................
Constitutive Lymphocyte Homing...................................................................
Tissue-selective Trafficking.................................................................
Lymphocyte Subset-specific Homing....... ................... ;.................................
Inflammation-induced Homing..........................................................................
Leukocyte-endothelial Cell Interactions..............................................
Multistep Model of Leukocyte Trafficking......................................................
Adhesion Molecules Involved in Rolling.............................................
L-selectin..................................................................................
History of L-selectin.....................................................................
Structure of L-selectin.......................................................................................
Expression of L-selectin.............................................................:....................
L-selectin Ligands............................................................................................
L-selectin, Adhesion, and Rolling....................................................................
L-selectin Regulation.........................
L-selectin Signaling......................................................................
L-selectin and the Cytoskeleton......................................................................
Summary................................................................................................................
Brief Overview of Chapter Contents................................................................
References Cited....................................................................................................
I
I
2
11
12
13
17
18
21
22
25
26
28
2. COMPARATIVE ULTRASTRUCTURE AND EXPRESSION OF
L-SELECTIN ON BOVINE ap AND yS T CELLS.............................................
43
2
4
4
5
6
8
9
Introduction..............................................................................
Materials and Methods.......................................................................................... 45
Animals.............................................................................................................. 45
Monoclonal Antibodies..................................................................................... 46
Cell Lines........................................................................................................... 46
Isolation of y5 and a(3 T Cells........................................................................... 46
Scanning Electron Microscopy.......................................................................... 47
4
VU
TABLE OF CONTENTS - CONTINUED
Page
Transmission Electron Microscopy............ ...................................................
Flow Cytometry.............................................................................................
Results....................................... ................... ;............................ .......................
Scanning Electron Microscopy of Bovine a|3 and y6 T Cells .....................
Transmission Electron Microscopy of Bovine ap and yS T Cells.................
Flow Cytometric Analysis of L-selectin on ocP versus y5 T Cells.................
Discussion...............................................
References Cited.........................................................................
48
48
49
49
49
52
53
55
3. ANTIBODY BINDING TO A CONFORMATION-DEPENDENT
EPITOPE INDUCES L-SELECTIN LINKAGE TO
THE CYTOSKELETON.....................................................................................
58
Introduction.................................................................................
Materials and Methods........................................................................................
Animals............................................................................................
Cell Preparations.............................................................................................
Monoclonal Antibodies...................................................................................
Cell Lines............................................................................................
Flow Cytometry of Non-detergent Treated Cells...........................................
Flow Cytometry of Detergent-solubilized Cells.....................
Generation of LAMl-116 and DREG 56 Fab.................................................
Phase Contrast Fluorescence Microscopy......................................................
Immunoprecipitation of L-selectin and Densiometric
Analysis of Band Intensities...........................................................................
Results..................................................
MAb LAMl-116 Enhances Leukocyte Staining of L-selectin
by mAb EL-246...............................................................................................
LAM1-116 Fab also Induced an Increase in Expression of
the EL-246 Epitope....................................................
The Tyrosine Kinase Inhibitors Herbimyein A and Genistein
do not Block LAMl-116 Upregulation of the EL-246 Epitope........... ..........
Expression of the EL-246 Epitope on Recombinant
Selectins Generated by Swapping Different Domains of
L-, E-, and P-selectin..................................................................
58
61
61
61
62
62
63
63
64
65
65
66
66
71
72
73
vm
TABLE OF CONTENTS - CONTINUED
Page
Treatment of Detergent Lysate Preparations with
LAMl-116 mAh Specifically Enhances L-selectin
Immunoprecipitation by EL-246 mAh
Covalently Linked to CNBr Beads................ ..................................... ...........
EL-246 mAh Can Induce L-selectin Cytoskeletal
Association in the Presence of LAM1-116.....................................................
Staining of L-selectin on Wild Type and LACyto
Transfectants Lacking an Intact Cytoplasmic Tail..........................................
Discussion............................................................................................................
References Cited..............................................................................
4. COMPARISON OF L-SELECTIN CYTOSKELETAL
ASSOCIATION TO THAT OF HUMAN AND BOVINE
LYMPHOCYTE SURFACE ANTIGENS.........................................................
Introduction.........................................................................................................
Materials and Methods........................................
Animals.....................................................
Monoclonal Antibodies...................................................................................
Cell Preparations.............................................................................................
Scanning and Transmission Electron Microscopy..........................................
Flow Cytometric Analysis of Non-detergent Treated Cells...........................
Flow Cytometric Analysis of Detergent Treated Cells..............
SDS-PAGE Analysis of Detergent-soluble and
Detergent-insoluble Membrane Fractions......................................................
PUFA Enrichment of Bovine T Cells.......... ............................. :.....................
Results............ ............................................ :......................................................
Detergent Treatment of Human and Bovine
Lymphocytes Generated a Homogeneous
Population of Actin Cytoskeletal Particles..................................................,..
Electron Microscopy Studies on the Actin Cytoskeleton..............................
Flow Cytometric Analysis of Human and
Bovine Lymphocyte Cytoskeletal Association
of Cell Surface Proteins......................
SDS-PAGE Analysis of Detergent-soluble and
Detergent-insoluble Membrane Fractions.......................................................
Characterization of Other Human and Bovine Surface Proteins.....................
74
74
80
83
89
95
95
99
99
99
101
101
102
103
104
106
106
106
108
111
115
118
ix
TABLE OF CONTENTS - CONTINUED
Page
PUFA Treatment Specifically Reduced L-selectin
Expression and Inhibits L-selectin Cytoskeletal Association......................... 119
Discussion......................
125
References Cited.................................................................................................. 134
5. SUMMARY OF WORK DESCRIBED IN CHAPTERS 2-4 AND
OTHER COLLABORATIVE,EFFORTS NOT
PRE SENTED IN THIS DISSERTATION........................................................ 143
Summary.............................................................................................................. 143
Collaborative Work During My Graduate Program...................
148
References Cited......................................................................
149
X
LIST OF TABLES
Table
Page
LI. Tissues Involved in Tissue-specific Homing....:..............................................
3
1.2. Receptor/ligand Pairs Involved in Rolling............................................................
8
1.3. L-selectin Ligands...............................................................................................
13
3.1. Expression of the EL-246 Epitope on Leukocytes
Under Various Conditions...........:............................. .......... ;............................. 70
3.2. EL-246 Staining of Chimeric Recombinant Selectin Transfectants..................
74
4.1. Human Lymphocyte Cytoskeletal
Association of Various Proteins...........................................................................
119
4.2. Bovine Lymphocyte Cytoskeletal
Association of Various Proteins...........................................................................
120
xi
LIST OF FIGURES
Figure
- Page
LI. The Multi-step Model of Leukocyte Migration..............................................
7
1.2. Diagram of the Selectin Family.......................................................................
11
2.1. SEM of Bovine yh T Cell.................................................................................
50
2.2. SEM of Bovine aP T Cell................................................................................
50
2.3. TEM of a Bovine y5 T Cell Showing L-selectin Localization by
MAb DREG 56 and Colloidal Gold....................................................... ............
51
2.4. High Magnification TEM of L-selectin Expressed at the
Tips of the Microvilli of Two Adjacent y5 T Cells.............................................
51
2.5. Staining of L-selectin by Four Different Anti-L-selectin mAbs;......................
52
3.1. LAM1-116 Enhancement of the EL-246 Epitope............................................
67
3.2. Other anti-L-selectin mAbs, as well as mAbs
Directed Against Distinct Surface Antigens
do not Enhance the Expression of the EL-246 Epitope.......................................
68
3.3. LAMl-116 Fab Enhancement of the EL-246 Epitope.....................................
71
3.4. LAM l-116 Treatment of Detergent-solubilized L-selectin
Increases EL-246-mediated Immunoprecipitation of L-selectin..........................
76
3.5. In the Presence of LAMl-116, EL-246 Triggers
L-selectin Cytoskeletal Association by Flow Cytometry...................................
78
3.6. In the Presence of LAM l-116, EL-246 Triggers
L-selectin Cytoskeletal Association by Fluorescence Microscopy....................
79
3.7. EL-246 Epitope Expression is Reduced on L-selectin
Transfectants Lacking an Intact Cytoplasmic Tail............................. ................
81
xii
LIST OF FIGURES - CONTINUED
Figure
Page
3.8. LAMI-116 Treatment Enhances EL-246 Staining of
L-selectin on Wild Type and Cytoplasmic Tail Deletion
Transfectants.........................................................................................................
82
4.1. Low Dose Detergent Treatment of Human and
Bovine Lymphocytes Generates Homogeneous Cytoskeletal Particles.............. 107
4.2. Scanning Electron Microscopy of
Bovine Lymphocyte Cytoskeleton....................................................................... 109
4.3. Transmission Electron Microscopy of
Bovine Lymphocyte Cytoskeleton........................................................
HO
4.4. Analysis of Human Lymphocyte Cell
Surface Protein Cytoskeletal Association...... ....................................................... 112
4.5. Analysis of Bovine Lymphocyte Cell
Surface Protein Cytoskeletal Association.................................................
114
4.6. SDS-PAGE Analysis of Human Cell
Surface. Protein Association with the Detergent-soluble
and Detergent-insoluble Membrane Fraction......................................................... 116
4.7. SDS-PAGE Analysis of Bovine Cell
Surface Protein Association with the Detergent-soluble
and Detergent-insoluble Membrane Fraction......................................................... 118
4.8. EPA-treatment Reduces L-selectin Expression.................................................. 121
4.9. LAMI-116 Enhances Expression of EL-246 Epitope
Lost on EPA-treated Bovine Lymphocytes................................................ ;......
4.10. EPA-treatment of Bovine Lymphocytes Blocks
L-selectin Cytoskeletal Association.....................................................................
1,23
124
4.11. Bovine yS TCR and WCl Antigens Associate
with the Cytoskeleton on EPA-treated Cells...................................................... 125
xiii
Abstract
L-selectin is an adhesion protein expressed on most leukocytes and is responsible
for the initial event in the multistep process of transendothelial cell migration, tethering
and rolling along the vessel wall. Leukocytes are captured from the blood stream by
engagement of carbohydrate moieties, expressed on large molecular weight glycoproteins,
by a calcium-dependent lectin domain expressed on L-selectin. L-selectin is expressed at
the tips of the microvilli of leukocytes in humans and mice. Here, we extended this
observation to bovine lymphocytes. Also, bovine y§ T cells were smaller in size and
contain greater than twofold the number of microvilli than a(3 T cells. Engagementof Lselectin by its carbohydrate ligands may have functional consequences in the cell, such as
calcium mobilization, tyrosine phosphorylation, and may also cause a conformational
change in L-selectin that leads to stronger tethering and rolling interactions. L-selectin
binding by an anti-L-selectin mAb, LAMl-116, caused a conformational change in Lselectin that was recognized by another anti-L-selectin mAb, EL-246. Both mAbs bind to
distinct, functional epitopes on L-selectin. Furthermore, the conformational change
induced in L-selectin by LAMI-116 predisposed L-selectin to cytoskeletal association,
which was triggered by engagement of the EL-246 epitope by EL-246 mAb. Electron
microscopy was employed to further characterize the nature of the lymphocyte
cytoskeleton. The bovine lymphocyte cytoskeleton was dramatically different than an
intact, resting cell. Compared to normal lymphocytes, mild detergent treatment of the
lymphocyte generated a smaller, smooth-surfaced cell remnant that contained little
cytoplasm and the cell nucleus. We also surveyed 28 human and bovine cell surface
proteins and compared their patterns of cytoskeletal association to that of L-selectin.
Our studies demonstrated that L-selectin fits a pattern of cytoskeletal association similar
to that of other adhesion proteins involved in tethering and rolling. Finally, we have
shown that enrichment of the lymphocyte cell membrane with polyunsaturated fatty
acids inhibits the ability of L-selectin to associate with the cytoskeleton, and may reduce
L-selectin-mediated adhesive events in vitro and in vivo.
I
CHAPTER I
INTRODUCTION
Leukocyte Trafficking
Historical Perspective
Cohnheim (1889) first described leukocyte rolling along venule walls almost a
century before Gallatin and his colleagues (1983) suggested a function for L-selectin
(1889). After his observation in 1889, others began to study the process of lymphocyte
recirculation. At the turn of the 20th century, Davis and Carlson (1909), and later Sjovall
(1936), hypothesized that lymphocytes exit the blood through the endothelium and enter
the lymphatics.
In 1972, Atherton and Bom reported the adhesion of circulating
polymorphonuclear leukocytes to blood vessel walls (1972). Exquisite studies by James
Gowans and others in the late 1950s and early 1960s led to the foundation of what we
now understand to be a multistep, highly regulated process of lymphocyte, recirculation
from the blood, into the lymph, and back again into the blood. Specifically, Gowans
reported on the effects of continuous re-infusion of lymphocytes on thoracic duct
lymphocyte output in a rat model (1957).
Two years later, he reported on the
recirculation of lymphocytes from the blood to the lymph in the rat (Gowans, 1959).
Further studies conducted by McGregor and Gowans demonstrated that if the
recirculating pool of lymphocytes is eliminated, a primary immune response against
2
sheep red blood cells or skin grafts does not develop (1963). Reconstituting the animal
with competent lymphocytes, however, results in restoration of the immune response.
Thus, not only is the presence of lymphocytes important for immune system function,
but the trafficking of lymphocytes is also vital to the generation of an immune response.
Constitutive Lymphocyte Homing
Naive lymphocytes traffic to secondary lymphoid sites in the body (Butcher et
al., 1999).
This type of homing is called constitutive homing, or lymphocyte
recirculation, because it occurs continuously and in the absence of inflammation. Naive
lymphocytes enter secondary lymphoid organs, such as peripheral lymph nodes (PLNs),
Peyeris patches (PPs), and tonsils, and if they do not encounter foreign antigen, exit
through the efferent lymphatics, reenter the blood stream, and traffic to another
secondary site in the body (Mackay et al., 1990).
Constitutive homing allows
lymphocytes to continually recirculate through secondary lymphoid organs, where
foreign antigens are presented, and enhances the chance that antigen-specific B or T cells
recognize these antigens and respond accordingly (Butcher et al., 1999).
Tissue-selective Trafficking
Once a naive cell encounters antigen and proliferates, it becomes an
effector/memory cell. Memory cells exhibit a different homing pattern than naive cells
and often recirculate back to the place they originally encountered antigen (Mackay et al.,
1992, Bradley and Watson, 1996, and Butcher et al., 1999). In general, memory cells
3
home preferentially to peripheral tissues, such as the skin, as well as other areas, such as
the gut mucosa (Mackay, 1992). Moreover, CD4+ memoiy T cells do not migrate into
PLN in the absence of antigen (Bradley et al., 1999). Lymphocytes isolated from the
efferent lymph of sheep mesenteric lymph nodes (MLN) preferentially home to sheep
mucosal tissues upon reinjection into that animal. Similarly, lymphocytes isolated from
the efferent lymph of peripheral lymph nodes migrate preferentially back to the PLN
(Reynolds et al., 1982, Scollay et al., 1976, and Cahill et al., 1977). In addition, sheep
lymphocytes collected from gut-draining lymphatics, having been exposed to foreign
antigens, labeled with FITC, and injected into syngeneic animals, home to the gut versus
the peripheral lymphatics (Mackay et al., 1992). The cells were re-isolated, phenotyped,
and found to consist primarily of memory lymphocytes (Mackay et al., 1992). Thus,
these studies, and others, suggest that lymphocytes traffic in a preferential manner to
specific sites in the body and their patterns of trafficking can be influenced by the tissue
of origin. A variety of tissues/organs are involved in tissue-specific homing, as illustrated
in Table 1.1 below.
Table 1.1. Tissues Involved in Tissue-specific Homing
Tissue Sites__________________________ References
1) PLN
2) Gut-associated lymphoid tissue
3) Lung-associated lymphoid tissue
_____ ;________________ _
(Reynolds et al., 1982, and Butcher, 1986)
(Cahill et al., 1977, and Butcher, 1999)
(Butcher, 1986 and Yednock and Rosen,
1989)
4) Nasal-associated lymphoid tissue
(Csensits et al., 1999)
5) Skin
(Picker et al., 1993)
61 Gutlam inapropia________ '_________ !Rose et al.. 1978. and Butcher. 19991_____
4
Lymphocyte Subset-specific Hominp
Besides the naive and memory subsets of lymphocytes described above, other
lymphocyte subsets exist that exhibit specific homing properties. Gamma delta T cells, a
recently defined subset of T cells, preferentially home to extralymphoid, epithelial sites in
the body, such as skin and mucosal tissues, and homing correlates with specific TCR
usage (Cahill et ah, 1996). For example, high numbers of Vy4/VSl y§ T cells are found in
the reproductive tract of mice (Mincheva-Nilsson et al., 1997, and Rakasz et al., 1998).
Bovine yS T cells also predominate in the spleen (Holder et al., 1999, and Wilson et al.,
1999).
Thus, a variety of lymphocyte subsets exist that exhibit specific homing
characteristics.
Inflammation-induced Homing
In addition to the homing patterns described above, there exists a second general
type of tissue-specific homing mediated by inflammation.
Inflammation-induced
trafficking is a response to insult and injury in the animal and the process preferentially
recruits cells with specific effector functions. Neutrophils, leukocytes that normally do
not home to any tissues, are recruited to sites of inflammation (Jutila et al., 1989a).
Another type of leukocyte, called a monocyte, is also recruited from the blood to tissue
sites of inflammation (Jutila et al., 1989b, and Spertini et al., 1992). Natural killer (NK)
lymphocytes, so-called because they do not need previous exposure to antigen to
function and are effective anti-tumor killing cells, also show a preference for mucosal
5
tissue in response to IL-2 activation (Uksila et al., 1997).
In addition, lymphocytes
isolated from sites of inflammation migrate back to the site where the inflammatory event
originally occurred, and also home more effectively to other sites of tissue inflammation
(Chin and Hay, 1980, Issekutz and Hay, 1980, Picker and Butcher, 1992). Therefore, not
only are lymphocytes recruited to inflammatory sites, but other leukocytes are as well,
including granulocytes, monocytes, and NK cells (Butcher et al., 1999).
Leukocyte-endothelial Cell Interactions
In order for leukocytes in the blood to traffic to a specific site in the body, they
must first interact with the endothelium. Constitutive homing interactions primarily take
place in postcapillary venules (PCV) of the various secondary lymphoid organs on
specialized endothelial cells, termed high endothelial venules (HEV) (Stamper and
Woodruff, 1976, Picker and Butcher, 1992, Dianzani and Malavasi, 1995, Stoolman et al.,
1984, and Stoolman, 1989). HEV are plump, cuboidal endothelial cells that, because of
their large surface area, are well-suited for lymphocyte binding.
The regions of
postcapillary venules expressing HEV look physiologically different than the regular,
flattened appearance of the rest of the endothelial cells lining the PCV. In humans and
mice, each of the secondary lymphoid organs, with the exception of the spleen, have areas
on the endothelium where HEV are present.
Memory cells, like naive cells, also
preferentially traffic through PCV, into the tissues where they first encountered antigen
(Mackay et al., 1992).
6
As one can imagine from the above descriptions of homing, leukocyte-endothelial
interactions exhibit substantial specificity. Mouse lymphocytes incubated on thin frozen
sections of lymph nodes and PP, as described originally by Stamper and Woodruff,
specifically adhere to HEV (1976, and Rosen et al., 1985).
Rodent and human
lymphocytes also display similar characteristics (Stoolman et al., 1987).
Monocyte
adhesion is also mediated by HEV binding (Grober et al., 1993). However, monocyte
attachment to the endothelium also occurs in the absence of HEV (Grober et al., 1993).
Collectively, the process of leukocyte trafficking from the blood into secondary lymphoid
or other tissues, is a highly regulated and specific process', mediated in part by leukocyteendothelial interactions.
Multistep Model of Leukocyte Trafficking
Leukocytes travel through the blood stream at -4000 pm/sec (Bargatze et al.,
1995). In order for tight adhesion to take place, the leukocyte must slow down. The
adhesion proteins expressed on leukocytes and the endothelium are the molecular braking
mechanisms, slowing the cells down 100-fold to -40 pm/sec (Bargatze et al., 1995, and
Figure 1.1A). This first step in the multistep process is called tethering and rolling.
Tethering is the formation of labile adhesion on the vessel walls which allows for the
subsequent rolling of leukocytes in the direction of blood flow (Fabbri et al., 1999).
The
second event occurs with activation of the leukocyte leading to tight adhesion (Butcher et
al., 1999).
Through G-protein-linked signaling receptors, the leukocyte undergoes
7
functional alterations, adheres tightly and flattens on the endothelium, usually within 20
seconds (Bargatze and Butcher, 1993, and Butcher et ah, 1999, Figure LlB and C). If
further G-protein-mediated signals are present, the leukocytes eventually transmigrate
through the endothelium (Butcher et ah, 1999, and Figure LID).
Importantly, each step
is a reversible process and if specific stimuli do not exist, the cells release back into the
blood stream.
Figure LI. The multi step model of leukocyte transmigration showing rolling (A),
activation (B) and tight adhesion (C), and transendothelial migration (D).
8
Adhesion Molecules Involved in Rnllinp
A variety of adhesion molecules are involved in rolling as described in the previous
model (Figure 1.1).
In addition, a number of receptor/ligand pairs contribute to the
distinct tissue-selective homing patterns. Table 1.2 lists some of the adhesion molecules
which have been shown to be involved in rolling.
Table 1.2. Receptor/ligand Pairs Involved in Rolling
Homing Adhesion Receptor_______ Vascular Addressin________ Tissue of Interaction
«4(37
a4 p i
CLA
PSGL-I
L-selectin
MAdCAM-I
VCAM-I
E-selectin
P-selectin
PNAd
GALT
Inflammation
Skin
Acute Inflammation
PLN
Three major families of adhesion molecules exist, the selectins, the integrins, and
the immunoglobulin-like superfamily. The selectin family of adhesion proteins mediates
the initial interaction of the leukocyte with the endothelium, an interaction which, as
mentioned above, is termed rolling. Integrins mediate the tight adherence of leukocytes to
the endothelium as well as participate in the process of transmigration.
The
immunoglobulin superfamily of adhesion proteins are expressed on the endothelium and
act as counterreceptors for the integrins. As my dissertation focuses on L-selectin, the
remaining portion of this discussion will provide a detailed overview of this adhesion
protein.
9
L-selectin
History of L-selectin
Since the early 1960’s, lymphocytes have been known to associate with
specialized vessels, termed postcapillary high endothelial venules. Indeed, in 1964, James
Gowans and E.J. Knight (1964) noted this interaction and postulated potential homing
mechanisms. However, it was not until 1983, that scientists began to understand the
molecular process of homing in more detail with the discovery of L-selectin (Gallatin et
al., 1983). In their study, Gallatin and colleagues blocked lymphocyte binding to HEV in
the Stamper and Woodruff ex vivo adhesion assay with a monoclonal antibody, MEL-14
(1983, and Stamper and Woodruff, 1976).
Additionally, injection Of MEL-14 into
recipient animals blocked lymphocyte homing to PLN in vivo. These studies led to the
original characterization of L-selectin as the PLN homing receptor.
As further research was performed, it became clear that the peripheral homing
receptor, which was recognized by the MEL-14 mAh, interacted with the endothelium
through a carbohydrate binding activity. In 1984, Stoolman and colleagues demonstrated
that phosphomannosyl carbohydrates, such as phosphomarman (PPME), inhibited
lymphocyte binding to HEV (Stoolman et al., 1984). Similarly, PPME-coated beads bind
to lymphocytes in a calcium-dependent manner, and this interaction is blocked by MEL14 mAb treatment (Yednock et al., 1987a and b). Further studies corroborated the lectin­
like activity of the MEL-14 antigen (Geoffrey and Rosen, 1989, and Imai et al., 1990).
10
Together, these data suggested that the peripheral homing receptor contained a
carbohydrate binding domain that is responsible for the interaction between lymphocytes
and endothelium.
Extensive cloning work done in the late 1980s led to the molecular characterization
of L-selectin. In 1989, five different papers were published describing the cloning of
cDNA encoding L-selectin in the mouse and human (Lasky et al., 1989, Siegehnan et al.,
1989, Siegelman and Weissman, 1989, Bowen et al., 1989, and Tedder et al., 1989) and
subsequent work identified this protein in the cow (Walcheck et al., 1992). The cloning
work confirmed the functional studies mentioned above and established that L-selectin
contains a lectin domain that binds carbohydrate. During this same time period, two
other adhesion proteins were cloned, E-selectin and P-selectin, which shared homology to
L-selectin (Bevilacqua et al., 1989, and Johnston et al., 1989). E-selectin is an inducible
adhesion molecule that is present on endothelial cells and is an active participant in the
process of leukocyte rolling (Bevilacqua et al., 1987, and Jutila et al., 1994). P-selectin,
also an inducible adhesion molecule with similar functions to L- and E-selectin, is found in
alpha granules of platelets and Weible-Palade bodies in endothelial cells (Bevilacqua et al.,
1989, Jutila et al., 1994, and McEver et al., 1989). In 1991, scientists agreed upon a
common name, "selectin,” for these three newly described cell adhesion molecules
(Bevilacqua et al., 1991).
11
Structure o f L-selectin
All three selecting contain five different domains: a Ca2+-dependent C-type lectin
domain; an epidermal growth factor-like domain (EOF); short consensus repeat domains
(SCR) that have homology to complement binding proteins; a transmembrane domain, and
a small cytoplasmic tail. L-selectin differs from E- and P-selectin in the number of SCR
domains. In the mouse, L-selectin has two SCRs, whereas E-selectin has five and Pselectin has eight. In humans, E-selectin has six SCR domains and P-selectin has nine.
The number of SCR’s in L-selectin does not vary from species to species.
L-selectin
E-selectin
P-selectin
Transmembrane
Domain
plasmic
Tail
Figure 1.2. Diagram of the selectin family of adhesion molecules showing their common
domains; Lectin, EGF, SCR, Transmembrane, and Cytoplasmic tail domains.
12
The C-type lectin domain serves as the calcium-dependent carbohydrate binding
domain for all three selectins (Stoolman et al., 1984, and reviewed in Varki, 1994, Jutila
1996, and Kansas, 1996). Treatment of any of the selectins with EDTA, a divalent
chealtor of calcium, diminishes the ability of each to bind carbohydrates (Yednock et al.,
1987b). The EGF and SCR domains are important to the structure of the molecule,
although some mAbs directed against these domains block the function of L-selectin under
flow, suggesting that these domains also contribute to ligand binding (Siegelman et al.,
1990, Watson et al., 1991, and Kansas et al., 1991). The cytoplasmic tail has also been
shown to be important, especially in L-selectin function (Kansas et al., 1993, Evans et al.,
1999, and Kahn et al., 1998). Of all the domains, homology is highest between the lectin
domains of the selectins, which accounts for their ability to recognize overlapping ligands
(Mebius and Watson, 1993, Tu et al., 1999, and Kansas, 1996).
Expression of L-selectin
Besides its expression on lymphocytes, L-selectin is also found on other
leukocytes including monocytes, eosinophils, and neutrophils (Lewinsohn et al., 1987,
and Jutila et al., 1989b). As with the studies on lymphocytes, bone marrow neutrophils
bind to HEV in frozen sections and their binding is blocked by MEL-14 (Lewinsohn et
al., 1987, and Jutila et al., 1989a). Recently, L-selectin expression has also been reported
on dendritic cells, and may account for the ability of dendritic cells to home to PLN in
vivo (Munro et al., 1996, and Vremec and Shortman, 1997).
13
Different cells express varying amounts of L-selectin. Walcheck and colleagues
(1994) have shown that bovine y5 T cells express 2-5 times the amount of L-selectin than
do af3 T cells. At the time, it was not clear whether this was due simply to enhanced Lselectin syndissertation or a physical difference in yS versus a (3 T cells. The functional
nature of varying levels of L-selectin on a|3 versus y§ T cells has not been well defined,
but our lab has shown that yS T cells exhibit highly efficient rolling interactions by in
vitro assays done under physiological shear (Jutila et al., 1994, and Jutila and Kurk,
1996).
L-selectin is located at the tips of the microvilli of leukocytes (Picker et al., 1991,
and Bruehl et al., 1996). Other adhesion proteins involved in rolling also are expressed at
the tips of leukocyte microvilli including E-selectin ligand-1 and P-selectin glycoprotein
ligand-1 (Steegmaier et al, 1997, and Moore et al., 1995). Thus, these adhesion proteins
involved in the first step of the multistep pathway are well positioned for their role in
leukocyte/endothelial rolling interactions.
L-selectin Ligands
A variety of ligands have been defined for L-selectin (Table 1.3).
14
Table 1.3. L-selectin Ligands
Ligand
Expression
Peripheral Node Addressin
Mucosal Addressin Cell Adhesion Molecule I
E-selectin
P-selectin Glycoprotein Ligand I__________
Endothelium
Endothelium
Endothelium
Leukocyte
Peripheral node addressin (PNAd) was originally defined as a group of 5 different
molecular weight proteins that are precipitated by the MECA-79 mAh (Streeter et al.,
1988). Four of these have been well characterized and include Glycam-1, CD34, Sgp-200,
and a recently defined protein called podocalyxin (Lasky et al., 1992, Baumhueter et al.,
1993, Berg et al., 1991, Hemmerich et al., 1994, and Sassetti et al., 1998). Most are
present on HEV and can mediate rolling interactions with L-selectin. However, Glycam-I
is a soluble protein and CD34 knockout mice do not show a decrease in the rolling arid
homing capacities of lymphocytes (Lasky et al., 1992, and Suzuki et al., 1996). Thus,
these two carbohydrate ligands may not play a vital role in L-seleCtin-mediated
constitutive homing. Podocalyxin is currently under investigation and has not yet been
shown to be important in vivo (Sassetti et al., 1998). Sgp-200 can be present in both
membrane-bound and soluble forms and can mediate L-selectin binding (Hemmerich et al.,
1994). However, its interactions with L-selectin are still under investigation and the
molecule has yet to be cloned. Therefore, it is not clear whether one or all of the MECA79 antigens are the principal L-selectin ligands on HEV. Together though, these antigens
are important in L-selectin-mediated constitutive homing as MECA-79 blocks homing of
15
lymphocytes in vivo and blocks HEV binding by lymphocytes in vitro (Streeter et al,
1988).
Mucosal addressin cell adhesion molecule-1, or MAdCAM-1, is the vascular
addressin that was originally defined to bind a4|37, but also binds L-selectin (Briskin et
al., 1993, and Berg et al., 1993). It is a 58-66 kD glycoprotein that has the unique feature
of containing both immunoglobulin-like domains as well as mucin-like domains in its
structure (Briskin et al., 1993). Mucins are glycoproteins rich in serine and threonine
residues and tend to be heavily O-glycosylated at these sites. This point is important as
most of the defined L-selectin ligands are mucins that contain clustered serine/threonine
residues which serve as linkage points to the carbohydrate moieties that L-selectin
recognizes (Varki, 1994, and Kansas, 1996). MAdCAM-I is expressed in PP, MLN, and
lamina propia, and MECA-367, a mAh that specifically binds to MAdCAM-1, blocks
lymphocyte recognition of intestinal postcapillary venules in vitro and in vivo (Streeter et
al., 1988 and Nakache et al., 1989). L-selectin binds to MAdCAM-I when the PNAd
carbohydrates are expressed on its surface (Berg et al., 1993) and through this interaction,
L-selectin plays a role in homing to mucosal sites as well as peripheral sites. Rolling of
lymphocytes on PPs is initiated by the L-selectin/MAdCAM-1 interaction but is
sustained by the binding and subsequent rolling of a4|37 on MAdCAM-I (Bargatze et al.,
1995).
16
Picker and colleagues originally reported that neutrophil L-selectin presents
carbohydrates to E- and P-selectin and suggested that selectin-selectin binding could
mediate rolling interactions (Picker et al., 1991). Kishimoto and colleagues expanded on
this and reported that L-selectin and E-selectin mediate common adhesion pathways in
vitro (Kishimoto et al., 1991).
Other studies have implicated L-selectin-E-selectin
interactions as well (Zoller et al., 1997, and Jones et al., 1997). Zoller and colleagues
(1997) demonstrated that human neutrophil L-selectin, but not mouse, binds to E-selectin
in vitro. Jones and colleagues (1997) confirmed these observations by demonstrating that
an E-selectin chimera expressing the E-selectin extracellular domain fused to an
immunoglobulin G constant region, specifically precipitated L-selectin from neutrophil
membrane lysates.
Thus, it is clear that E-selectin can be a ligand for L-selectin,
depending upon the type of leukocyte expressing L-selectin. However, this interaction
has yet to be shown to be important in vivo.
In addition to mediating leukocyte-endothelial interactions, L-selectin also
mediates leukocyte-leukocyte interactions (Bargatze et al., 1994, and Jutila and Kurk,
1996). One counterreceptor expressed on the leukocyte cell surface that binds to Lselectin is P-selectin glycoprotein ligand-1 (PSGL-I) (Guyer et al., 1996, Spertini et al.,
1996, Tu et al., 1996, and Walcheck et al., 1996). PSGL-I is a 230 kD homodimer
expressed on most leukocytes and was originally defined as the primary ligand for Pselectin (Moore et al., 1992, and Sako et al., 1993). MAbs against PSGL-I that block Pselectin-mediated rolling interactions do not block L-selectin-mediated interactions to the
17
same extent, suggesting the presence of another important ligand in L-selectin-mediated
leukocyte-leukocyte interactions (Walcheck et ah, 1996). Further characterization found
that E-selectin can also bind PSGL-I, yet the importance of these interactions are not
clear (Asa et ah, 1995, Goetz et ah, 1997, and Borges et ah, 1997). PSGL-I knockout
mice show a dramatic reduction in P-selectin-mediated lymphocyte rolling and capture
whereas PSGL-I is not needed for E-selectin mediated rolling events in vivo (Yang et ah,
1999). Future investigation may clarify the issue as other ligands for L- and E-selectin are
discovered.
L-selectin. Adhesion, and Rolling
As mentioned earlier, Gallatin and colleagues (1983) first demonstrated a direct
interaction between the lymphocyte and endothelium using the mAh, MEL-14.
Lewinsohn and Jutila (1987 and 1989b, respectively) broadened this observation to Lselectin on other leukocytes. Lewinsohn (1987) provided the first evidence of a common
molecular mechanism shared by all leukocytes in their recognition of host endothelium.
Jutila (1989b), by MEL-14 mAb binding to L-selectin, ascertained that pretreated
neutrophils do not home to inflamed tissues and neutrophils deficient in L-selectin lacked
trafficking capabilities. With new intravital microscopy techniques, scientists began to
biochemically characterize leukocyte rolling interactions on endothelium in the animal,
von Andrian demonstrated that neutrophils use L-selectin to roll on activated endothelium
in rabbits (1991). Ley and colleagues confirmed these results showing that L-selectin
18
mediates leukocyte rolling in mesenteric venules in rats in vivo (1991).
Recently, L-
selectin was shown to be important in HEV-mediated rolling in vivo, substantiating the
early ex vivo HEV binding assays (von Andrian, 1996).
L-selectin also mediates rolling interactions on other immobilized leukocytes
(Bargatze et al, 1994). Bargatze and colleagues, using an in vitro closed loop circulation
assay, showed that neutrophils roll on other adherent neutrophils under shear forces
similar to those found physiologically, and that rolling is dependent upon the presence of
L-selectin on the rolling cells, as determined by mAh blocking studies (1994). Gamma
delta T cells, a subset of lymphocytes found in particularly high numbers in ruminants,
also exhibit L-selectin-dependent rolling interactions on other immobilized yd T cells
(Jutila and Kurk, 1996).
It has been proposed that leukocyte-leukocyte rolling
interactions serve to augment leukocyte-endothelial interactions (Bargatze et al., 1994,
and Alon et al., 1996).
L-selectin Regulation
Neutrophils isolated from sites of inflammation, which no longer have the
capacity to home to other tissues, lack L-selectin, suggesting a regulatory role for. Lselectin cleavage (Jutila et al., 1989a). Following activation of either lymphocytes or
myeloid cells with chemotactic factors or phorbol esters, there is a rapid increase in
functional avidity which, under most circumstances, is followed by shedding of the cell
surface molecule (Spertini et al., 1991, Kishimoto, et al., 1989, Griffin, et al., 1990 and
19
Smith, et ah, 1991). There is an inverse relationship between the downregulation of Lselectin and the activation of the p2 integrin, Mac-1 (Jutila et ah, 1989a, and Kishimoto et
ah, 1989).
L-selectin is shed from the cell surface of neutrophils while Mac-1 is
upregulated (Jutila et ah, 1989a, and Kishimoto et ah, 1989). The basis for the increased
functional avidity in L-selectin. immediately following activation of the leukocyte is
poorly understood, but it may be due to phosphorylation of L-selectin, clustering of the
J
protein, cytoskeletal association of L-selectin through its cytosplasmic tail, or
conformational changes induced in L-selectin (Haribabu, et ah, 1997, Li et ah, 1998, and
Evans et ah, 1999). Hyperthermic treatment of lymphocytes also increases L-selectin
function in vitro and in vivo (Wang et ah 1998). In their study, Wang and colleagues
demonstrated that hyperthermic-treated lymphocytes bind better to HEV in ex vivo
assays and home more efficiently to PLN in vivo (1998).
L-selectin shedding is caused by proteolysis, which can be blocked by protease
inhibitors that target a unique membrane-bound metalloproteinase (Kishimoto et ah, 1989,
Jutila et ah, 1991, Kishimoto et ah, 1995, Feehan et ah, 1996, Walcheck et ah, 1996, and
Bennet et ah, 1996). Endoproteolytic release of L-selectin from the surface of leukocytes
is regulated by certain structural requirements of L-selectin and mutational analysis of the
cleavage site demonstrates that conformation is the most important aspect in L-selectin
shedding (Chen et ah, 1995, and Migaki et ah, 1995). A recent report by Kahn and
colleagues suggests calmodulin, a calcium regulatory protein that specifically co­
20
precipitates with L-selectin through a direct association with the cytoplasmic tail, also
regulates L-selectin shedding (1998). Calmodulin inhibitors disrupt L-selectin-dependent
adhesion by inducing proteolytic release of L-selectin from the cell surface (Kahn et al,
1998).
The putative metalloprotease that cleaves L-selectin has been recently defined.
Tumor necrosis factor alpha converting enzyme (TACE), an enzyme responsible for
cleaving TNF-a, has been implicated in L-selectin cleavage from the cell surface (Peschon
et al., 1998). Peschon and colleagues generated mice deficient in the active form of TACE
by mutating the Zn2+ binding domain, thus inactivating its metalloproteinase activity
(1998). However, this mutation is lethal so embryonic cells were used to characterize
TACE activity on L-selectin. Embryonic thymocytes isolated from the TACE mutants
did not downregulate L-selectin from the cell surface when treated with PMA in
comparison to wild type embryonic thymocytes (Peschon et al., 1998). Also, TACE
cleaves a peptide that matches the proteolytic site on L-selectin (Peschon et al., 1998).
Hence, TACE mediates L-selectin cleavage from the cell surface.
L-selectin shedding may allow leukocytes to break their tight bonds with native
ligands and proceed with tight adhesion and transmigration (Kishimoto et al., 1991). A
recent report suggests that in vivo, a hydroxamic acid-based metalloproteinase inhibitor
(KD-IX-73-4) that blocks L-selectin cleavage, actually slows the rolling velocity of
leukocytes compared to untreated cells (Hafezi and Ley, 1999).
Another important
21
measure of L-selectin function is the number of rolling cells per unit time of observation
(rolling flux) and KD-IX-73-4 treatment increases the rolling flux of leukocytes observed
in vivo (Hafezi and Ley, 1999). Additionally, the number of firmly adhering leukocytes,
as well as the number of transmigrating cells, increased (Hafezi and Ley, 1999). These
data suggest that L-selectin shedding significantly impacts leukocyte recruitment and
provides a physiological role for L-selectin shedding in vivo, expanding on Jutila and
colleagues’ original observation that leukocytes isolated from inflammatory sites lack Lselectin (Jutila et ah, 1989a).
L-selectin Signaling
L-selectin has been implicated in cellular signaling events which regulate the
function of other adhesion molecules on the cell surface and the activation state of the
leukocyte. Engagement of L-selectin on neutrophils by mAb leads to activation and tight
adhesion, potentiates the oxidative burst, and enhances tyrosine phosphorylation as well
as activating MAP kinase (Waddell et ah, 1994, Crocket-Torabi et ah, 1995, Simon et ah,
1995, and Waddell et ah, 1995).
In these studies, crosslinking of L-selectin by a
secondary reagent specific to the anti-L-selectin mAb, or by ligand binding (Glycam-1), is
requisite for the signaling events. Other examples exist where engagement of L-selectin by
primary antibody alone, directly activates the cell.
Binding of L-selectin by a mAb
directed against a highly conserved region of L-selectin alone, causes activation of
neutrophils and their subsequent tight adherence (Steeber et ah, 1996). Engagement of
22
neutrophil L-selectin by sulfatides, carbohydrate moieties, increases cytosolic free Ca+2,
increases expression of TNF-a and IL-8 mRNA, and also transiently induces oxygen
radicals (Laudanna et ah, 1994, and Bengtsson et al, 1996). More recently it has been
reported that neutrophils arrest on ICAM-I when activated independently through Lselectin under physiological shear force (Gopalan et al., 1997).
In addition, human
neutrophil L-selectin signaling causes alterations in the cytoskeleton as well as co­
localization with CD 18 (Simon et al., 1999).
This observation, and those above,
demonstrate a correlation between L-selectin engagement,
albeit some with artificial
ligands, and downstream activation events.
L-selectin and the Cytoskeleton
The actin-based cytoskeleton is made up of three primary components,
microtubules, microfilaments, and intermediate filaments, and is vital in maintaining the
integrity of various immune cells in the body, including endothelial cells and leukocytes
(Kim et al., 1989, and Wechezak et al., 1989, and reviewed in Hall, 1998). Recently,
much work has focused on the actin cytoskeleton and lymphocyte function (Penninger
and Crabtree, 1999). Numerous studies have shown that the functional capacity of
lymphocytes, as well as proteins contained internally and externally, depends on a
competent actin cytoskeleton.
Cytoskeletal defects are important in various disease
states including cancer (Saunders et al., 2000), Wiskott-Aldrich Syndrome (Gallego et al.,
1997, Wu et al., 1998, Zicha et al., 1998, Snapper and Rosen, 1999, and Trasher and
23
Bums, 1999), B-cell function (Cheng et al., 1999), HIV pathogenesis (Pearce-Pratt et al.,
1994, Soli .and Kennedy, 1994, Soil, 1997, Fackler et al., 1999, and Wang et al., 2000) and
other immune cell function deficiencies (Zhang et al., 1999, and Roberts et al., 1999).
Thus, the ability of a protein to associate with the cytoskeleton plays a vital role in its
function.
Cytoskeletal association of adhesion molecules has been investigated for some
time. Most studies have focused on integrin function as it relates to the cytoskeleton
controlling the avidity of PI and (32 integrals in leukocytes (Shaw et al., 1990, Pavalko
and LaRoche, 1993, Otey et al., 1993, Pardi et al., 1992, Pavalko and Otey, 1994, and
Sampath, et al., 1998). . These reports suggest that integrals associate with detergentresistant membranes (DRMs) through direct binding of the integrin cytosplasmic tail to
the actin cytoskeleton. A variety of other proteins have been studied as well, including
CD2, CD4, CDS, CD44, CD45, and class I MHC (Geppert and Lipsky, 1991, and Gur et
al., 1997). Depending upon the treatment of proteins by either primary antibody alone or
crosslinking, they can be induced to associate with the cytoskeleton (Geppert and
Lipsky, 1991, and Evans et al., 1999). However, some molecules are linked directly to
the cytoskeleton immediately after syndissertation and transport from the Golgi (Brown
and Rose, 1992, Varma and Mayor, 1998, and Melkonian et al., 1999).
Numerous signaling molecules constitutively associate with the cytoskeleton
through GPI-anchored interactions that form what some refer to as “lipid rafts”, which
24
play a role in T cell activation (Moran and Miceli, 1998). The concept of lipid raft was
first proposed by Brown and Rose (1992) and has since received much attention. In
addition to lipid rafts, supramolecular activation clusters (SMAC) exist as organized
contact sites at the physical interface between T cells and antigen presenting cells and the
proteins involved at such sites are linked to the cytoskeleton (Monks et al., 1997, Shaw
and Dustin, 1997, and Monks et al., 1998). Recent studies have also elucidated the
importance of the Ras superfamily of small guanosine triphosphates (GTPases),
especially the Rho family, and the actin cytoskeleton in immune cell function (Hall, 1998,
and Magee and Marshall, 1999). Indeed, Rho family deficiencies, in which cytoskeletal
interactions are affected, show abnormalities in neutrophil function and host defense,
decreases in integrin-mediated cell adhesion, decreases in cell motility in neutrophils, and
abnormalities in growth factor induced chemotaxis in neutrophils and macrophages
(Roberts et al., 1999, Laudanna et al., 1996, Stasia et al., 1991, Allen et al., 1997, Allen et
al., 1998, and Zicha et al., 1998).
Rho GTPases also induce T cell adhesion to
MAdCAM-1, and control migration and polarization of adhesion molecules and ERM
components in T lymphocytes, and defects in these GTPases affect T cell trafficking
(Zhang, et al., 1999, and del Pozo et al., 1999).
Linkage to the cytoskeleton is also important to the function of L-selectin. The
cytoplasmic tail of L-selectin is required for leukocyte rolling and adhesion (Kansas et al,
1993). Deletion mutants lacking the carboxyl terminal 11 amino acids of the cytoplasmic
tail of L-selectin do not bind to HEV nor establish rolling interactions in vivo (Kansas et
25
al., 1993).
The cytoplasmic tail of L-selectin binds to the cytoskeletal-associated
proteins a-actinin and vinculin (Pavalko et al, 1995). Evans and colleagues determined by
antibody crosslinking, hyperthermic treatment, and ligand binding assays, that the
cytoplasmic tail of L-selectin is important to L-selectin function by regulating linkage to
the actin cytoskeleton through specific binding interation (1999).
Presumably, this
interaction is mediated by a-actinin binding to actin and the cytoplasmic tail of L-selectin.
Specific concentrations of cytochalasin B, which inhibit actin polymerization, block Lselectin-mediated rolling, although it is not clear what mechanisms are involved in the
inhibition of rolling interactions (Kansas et al., 1993).
These data suggest a causal
relationship between the ability of L-selectin to associate with the cytoskeleton and its
ability to form strong. tethers and thus high avidity rolling interactions with its
carbohydrate ligands. However, the study by Evans and colleagues does not directly
address this question.
Summary
In this dissertation, I have explored the nature of L-selectin regulation by a variety
of techniques in an effort to test the following hypothesis:
Expression and conformation of L-selectin, as well as fatty acid composition of the
cell membrane, impact its linkage to the cytoskeleton.
26
In regards to this hypothesis, the following issues are addressed.
1) Ultrastructure of L-selectin expression on bovine lymphocytes.
2) Characterization of a mAb-induced conformational change in L-selectin and
impact of this conformational change on L-selectin cytoskeletal linkage.
3) Comparison of L-selectin linkage to the cytoskeleton with the linkage of other
important cell surface molecules.
4) Alteration of the linkage of L-selectin to the cytoskeleton.
Brief Overview of Chapter Contents
The second chapter deals with the ultrastructure and expression of L-selectin on
a(3 versus yd T cells and was published in the Journal of Leukocyte Biology as detailed
below:
Leid, J.G., CA. Speer, and M.A. Jutila.
1998.
Comparative ultrastructure and
expression of L-selectin on bovine ccP and yd T cells. J Leukoc. Biol 64:104-107.
Chapter 3 focuses on a conformational change in L-selectin that predisposes the
protein to cytoskeletal association and some of the work was done in collaboration with
Drs. Thomas Tedder and Douglas Steeber in the Department of Immunology at Duke
University Medical Center. Chapter 4 reports on the comparison of the cytoskeletal
association of L-selectin to that of other functionally characterized proteins as well as
characterizes the role of PUFA treatment on L-selectin cytoskeletal linkage. Chapter 5 is
27 .
a summary of the work presented in chapters 2-4 as well as other collaborations I have
worked on during my graduate program.
28
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cytoskeletal-associatedprotein. JBiolChem. 273:5765-5770.
Yang, J., T. Hirata, K. Croce, G.M. Skoloff, B. Tchemychev, E. Williams, R.
Flaumenhaft, B.C. Furie, and B. Furie. Targeted gene disruption demonstrates
that P-selectin glycoprotein-1 (PSGL-I) is required for P-selectin-mediated but
not E-selectin-mediated neutrophil rolling and migration. J Exp Med. 190:17691782.
Yednock, TA., and S.D. Rosen. 1989. Lymphocyte homing. Adv Immunol. 93:81-94.
Yednock, TA ., E.C. Butcher, L.M. Stoolman, and S.D. Rosen. 1987a. Receptors
involved in lymphocyte homing: relationship between the carbohydrate-binding
receptor and the MEL-14 antigen. J Cell Biol. 104:725-731.
Yednock, TA., Stoolman, L.M., and S.D. Rosen. 1987b. Phosphomannosyl-derivatized
beads detect a receptor involved in lymphocyte homing. J Cell Biol. 104:713723.
42
Zhang, W.V., Y. Yang, R.W. Berg, E. Leung, and G.W. Krissansen. 1999. The small
OTP-binding proteins Rho and Rac induce T cell adhesion to the mucosal
addressin MAdCAM-1 in a hierarchical fashion. Eur J Immunol. 29:2875-2885. ■'
■
■
•'
-
Zicha, D., W.E. Allen, P.M. Brickell, C. Kinnon, GA. Dunn, arid G.E. Joties. 1998.
Chemotaxis of macrophages is abolished in the Wiskott-Aldrich Syndrome. Brit J
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Zoller, O., M.C. Renter, J.E. Blanks, E. Borges, M. Steegmaier, H.G. Zerwes, and D.
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directly to E-selectin. J Cell Biol. 136:707-716.
43
CHAPTER 2
COMPARATIVE ULTRASTRUCTURE AND EXPRESSION OF L-SELECTIN
ON BOVINE ap AND y5 T CELLS
Introduction
Although y8 T cells were discovered over a decade ago, their functions are still not
clearly defined. Some suggest that these cells play a role in the initial host response
against a number of different bacterial, viral, and parasitic agents, contributing to what is
referred to as “the first line of defense” (Libero, 1997). Gamma delta T cells have many
characteristics that distinguish them from other T cells, including molecularly unique
receptors for antigens (y8 TCRs) (Raulet, 1989, and Hein and Mackay, 1991), lack of
either CD4 or CDS expression (Mackay and Hein, 1989) though some y8 T cells express
a low level of CDS, and unique trafficking behaviors in the animal (Libero, 1997, Mackay
and Hein, 1989, Jutila, 1996, and Jutila 1998). In the context of trafficking, y8 T cells
have been shown in many animals to exclude secondary lymphoid tissue during normal
circulation throughout the body and instead' preferentially recirculate through
extralymphoid sites, such as the skin, gut mucosa, reproductive tract, and sites of
inflammation (Jutila, 1998).
Only recently have studies been done to compare the
molecular mechanisms used by y8 T cells and other lymphocytes to migrate throughout
the body and begin to address the basis for their unique trafficking behavior.
44
The process of trafficking involves recognition of the vascular lining of vessels in
tissues by the circulating leukocyte, resulting in a sequential multistep process
characterized by I) rolling of the leukocyte along the vessel wall, 2) tight adhesion and
arrest, and 3) transendothelial cell migration (Springer, 1994). Once leukocytes tightly
adhere to the vessel wall, they can serve as adhesive substrates for continued recruitment
of new leukocytes (Bargatze et al, 1994, Walcheck et al., 1996, Tu et al, 1996, and Alon
et al., 1996).
Most leukocyte-on-endothelial cell and leukocyte-on-leukocyte rolling
interactions are mediated by a family.of adhesion proteins called selectins (Springer, 1994,
Tedder et al., 1995, and Jutila and Kurk, 1996). Three selectins have been defined: Lselectin on most leukocytes, E-selectin on inflamed endothelium, and P-selectin on
activated platelets and inflamed endothelium (Tedder et al., 1995, and Jutila and Kurk,
1996). L-selectin mediates leukocyte rolling on endothelial cell monolayers as well as
leukocyte-on-leukocyte rolling (Tedder et al., 1995).
E- and P-selectin also support
rolling interactions, but only at the level of the endothelium or immobilized platelets
(Tedder et al., 1995, and Jutila.and Kurk, 1996).
A number of years ago, we began a comparison of the trafficking mechanisms used
by yS T cells and other lymphocytes with an emphasis on selectin-mediated rolling (Jutila
and Kurk, 1996, Jutila et al., 1994, and Jutila et al., 1997). We have used the bovine
model for these studies because newborn calves, which provide homogenous lymphocyte
populations with respect to their state of activation and memory cell status, can be
45
utilized. Also, for reasons undefined, yS T cells are the predominant population of
circulating T cells in newborn calves. Therefore, large numbers of cells are easily isolated
for study. We have found that bovine y§ T cells exhibit a much greater capacity to roll
on monolayers of endothelial cells, platelets, or recombinant E-selectin than other
lymphocytes in assays done under physiologic flow (Jutila and Kurk, 1996, Jutila et al.,
1994, and Jutila et al., 1997). Furthermore, we have shown that y5 T cells avidly roll on
other immobilized yS T cells, arid rolling is completely blocked by anti-L-selectin mAbs
(Jutila and Kurk, 1996). Finally, we found that bovine yS T cells express 2-5 times the
level of L-selectin as other lymphocytes; whereas, the expression of other adhesion
molecules, such as the CD 18 integrins, does not differ (Walcheck and Jutila, 1994). Here,
we have used electron microscopy (EM) to further compare bovine y8 and a [3 T cells to
determine whether the increased capacity of yS T cells to interact with endothelial cells
and other T cells correlates with possible differences in the physical properties of these
cells.
Materials and Methods
Animals
One-week to three-month-old Holstein calves, housed in the Montana State
University large animal facility, were used as sources of peripheral blood.
46
Monoclonal Antibodies
The anti-L-selectin mAbs DREG 56, DREG 200, EL-246, and GD 4.22 have been
described elsewhere (Kishimoto et al., 1990, Jutila et al, 1992, and Jutila and Kurk,
1996). Anti-WCl mAb IL-A29 has also been described elsewhere (American Tissue
Type Culture Collection, Manassas, VA). FITC-conjugated goat anti-mouse antibodies
were used to visualize primary antibody staining of L-selectin in the flow cytometry
studies (Jackson ImmunoResearch, West Grove, PA).
Cell Lines
Human E-selectin transfectants were made by transfecting mouse L-cells with
human ELAM (E-selectin) cDNA as previously described (Kishimoto et al., 1991), and
were grown to confluency in T-175 flasks for yS T cell isolation, as described (Walcheck
e ta l, 1993).
Isolation of yS and aft T Cells
Gamma delta T cells were isolated, as described (Walcheck et al., 1993). Briefly,
peripheral blood was collected from Holstein calves into heparin vacutainer tubes by
jugular venipuncture. The blood was diluted 1:1 with Hank’s Balanced Salt Solution
(HBSS, Sigma, St. Louis, MO), underlayed with 15 mL of Histopaque™, and centrifuged
for 30 min at 700g. The buffer coat layer of PBMC’s was collected and incubated on
plastic in Dulbecco’s Modified Eagle Medium (DMEM, Sigma) containing 0.7% BSA
47
(Sigma) for 30 min at 37°C. Non-adherent lymphocytes were collected and incubated on
confluent layers of E-selectin transfectants for 30 min as described (Walcheck et al.,
1993). Gamma delta T cells specifically bind to E-selectin in a calcium-dependent manner
whereas a (3 T cells do not (Walcheck et al., 1993). Non-E-selectin adherent cells were
poured off the monolayers washed, and the yS T cells isolated by incubation with 2 mM
EDTA. This procedure yielded >95% yS T cells, as analyzed by their expression of yS
TCR (Walcheck et al., 1993). The non-E-selectin adherent cells contained > 85% a (3 T
cells as determined by flow cytometry (data not shown). Both lymphocyte subsets were
collected and prepared for electron microscopy.
Scanning Electron Microscopy
Bovine lymphocytes were prepared as described above and scanning electron
microscopy performed as described (Speer et al., 1979).
Briefly, lymphocytes were
adhered to 12 mm glass cover slips (Ted Pella, Redding CA) coated with CellTak® (100
pg/mL, Collaborative Biomedical Products, Bedford, MA). Adherent cells were fixed in
2.5% (v/v) glutaraldehyde in Millonig’s phosphate buffer overnight, dehydrated, critical
point dried, sputter coated with gold-palladium, and examined with a JEOL I OOCX
scanning/transmission electron microscope (Speer et al., 1979).
48
Transmission Electron Microscopy
Transmission electron microscopy (TEM) studies were done as described (Speer
et al., 1997).
Briefly, bovine lymphocytes were isolated and fixed in modified
Kamovsky’s fixative overnight. Samples were partially dehydrated in ethanol, prestained
with 1% (w/v) phosphotungstic acid (Ted Pella) and 1% (w/v) uranyl acetate (Ted Pella)
in 70% ethanol, completely dehydrated and embedded in Spurrs1 (Ted Pella) medium.
Ultrathin sections were stained with lead citrate and examined by TEM.
Flow Cvtometrv
Flow cytometry was performed, as previously described (Walcheck et al., 1993a).
Briefly, lymphocytes were collected, suspended at IxlO7 cells/mL, and 100. JiL aliquots
transferred into FACS tubes (Fisher Scientific Co., Pittsburgh, PA) for analysis. The
mAbs described above were added to the samples at a concentration of 50 pg/mL and
incubated on ice for 30 min, washed in PBS containing horse serum and sodium azide
(FACS buffer). FITC-conjugated goat anti-mouse second stage was added and the cells
incubated for an additional 30 min on ice.
The cells were washed a final time,
resuspended in FACS buffer, and analyzed on either a FACScan or FASCalibur (Becton
Dickenson). 10-20,000 events were collected for each sample.
49
Results
Scanning Electron Microscopy
of Bovine a P and y5 T Cells
By scanning electron microscopy (SEM)5 we found that yS T cells have more
microvilli (i.e., folds or projections from the plasmalemma) on a per cell basis than a (3 T
cells (Figure 2.1 versus Figure 2.2). Gamma delta T cells had 59+1.3 (n=19) microvilli/cell
whereas aP T cells had 28+1.8 (n=18) microvilli/cell. A blind count was employed to
confirm our observation. Surprisingly, yS T cells were ~32 % smaller than cc|3 T cells
(3.9+0.01 mm versus 5.7+0.01 mm; n=33 and n-26 respectively; Figure 2.1, Panel B
versus Figure 2.2, Panel B). All data are representative of 3 different preparations from 3
different Holstein calves.
Transmission Electron Microscopy
of Bovine aft and yS T Cells
We also examined the expression of L-selectin on bovine lymphocytes to
determine if, as shown with human leukocytes (Picker et ah, 1991, and Bruehl et ah,
1996), L-selectin is localized to the tips of microvilli. As found for human neutrophils
and lymphocytes, L-selectin was localized at the tips of microvilli of bovine lymphocytes
(Figure 2.3, Panel A). Little or no L-selectin was detected on the rest of the plasmalemma
of yS T cells, concurrent with the data previously reported for human leukocytes (Picker
et ah, 1991, and Bruehl et ah, 1996). In contrast, WC1, a y8 T cell subset marker,
50
Figure 2.1. (A) Low-magnification SEM showing the uniform size of several yfi T
cells; bar = 5 gm; original magnification X6,000. (B) Higher magnification SEM
of a y5 T cell showing numerous microvilli (Mv); bar = I jam; orig. mag. X20,000.
Figure 2.2. (A) Low-magnification SEM showing several a(3 T cells with a
few platelets (Pt); bar = 5 pm; orig. mag. X6,000. (B) Higher magnification
SEM of an aP T cell showing microvilli (Mv); bar = I pm; orig. mag.
X20,000.
51
Figure 2.3. (A) TEM of a y5 T cell treated with DREG 56 (anti-L-selectin)
plus colloidal gold-conjugated antibody; note gold label at tips of microvilli
(arrows); bar = I pm, orig. mag. X20,000. (B) TEM of WCl (anti-y8 T
cell) labeled with colloidal gold on the plasmalemma (Pl) of a y5 T cell;
Mv, microvilli; bar = 0.5 pm; orig. mag. X70,000.
Figure 2.4. (A) TEM showing localization of L-selectin on two adjacent bovine
y5 T cells by DREG 56 and colloidal gold-conjugated antibody; note that Lselectin is localized primarily at the tips of the microvilli of adjacent cells; bar =
0.5 pm; Nu, nuclei; *, extracellular space; orig. mag. X70,000. (B) Highmagnification TEM of the tips of microvilli of two y5 T cells in close proximity
showing colloidal gold-labeled L-selectin; bar = 0.1 pm; orig. mag. Xl 50,000.
52
localized on the plasmalemma covering the y8 T cell body but was absent on microvilli
(Figure 2.3, Panel B). A representative micrograph of two 76 T cells in close proximity is
also shown in Figure 2.4, Panel A. In the same figure, Panel B shows a high magnification
(XI 50,000) of two microvilli in contact and a cluster of L-selectin localized at the tips of
the microvilli.
Flow Cytometric Analysis of
L-selectin on a p Versus y§ T Cells
Finally, we have previously shown by flow cytometry that y8 T cells have more
L-selectin on their surface than a (3 T cells by DREG 56 mAb staining (Walcheck and
Jutila, 1993a). This finding was reconfirmed here.
Four mAbs, which stain distinct
epitopes on L-selectin, all stained brighter on y5 versus a (3 T cells (Figure 2.5). Thus,
there clearly is more L-selectin on bovine yS T cells, which we now hypothesize is due to
an increased number of microvilli on these cells.
DREG 56
EL-246
E3
DREG 200
o rfj
r t-e ii*
§ cells
GD 4.22
X l i x l e I l i K i r e s e e B ie e s l s i i r i i i i ^
of
cjt|i 11n et
rF c e l l s
Figure 2.5. Mode fluorescence anti-L-selectin staining of a (3 versus y8 T cells
by four distinct anti-L-selectin mAbs. All four mAbs stained y8 T cells
brighter than otp T cells.
53
Discussion
Our in vitro studies have shown that y§ T cells are far more efficient at initiating
adhesive rolling interactions on both endothelial and adherent leukocyte monolayers under
shear forces comparable to those found in blood flow in vivo (Jutila and Kurk3 1996,
Jutila et ah, 1994, and Jutila et al., 1997). This may be due, in part, to differences in
expression levels of adhesion molecules on y5 T cells, but the observations shown here
provide the basis for an alternative explanation. Increased numbers of microvilli, where
some adhesion molecules are located, would effectively increase the number of contact
sites, thus enhancing the opportunity for adhesion to occur. The smaller size of the y5 T
cell may also be a benefit. Due to its lower profile, the y8 T cell would not experience the
same shear forces in blood flow as aP T cells. Thus, three differences have been defined
which likely contribute to the increased efficiency of bovine yS T cell/endothelial cell
interactions:
these cells have been shown to I) have increased expression of some
adhesion molecules, 2) are smaller in size, and 3) have considerably more surface
microvilli to initiate cell/cell contact.
In summary, these results further define the differences between bovine y5 and a p
T cells. Previously, these cell types could be distinguished by their T cell receptors for
antigen (TCRs), expression of CD4/CD8 on the a|3 T cell and other lineage-specific
antigens, such as WCI, on the y8 T cell.
Differences in the size and surface topography
54
of j8 and a(3 T cells may be important in the interaction of these cells with other host
cells, such as endothelial cells, or targets they intend to kill, such as tumor cells or
infectious agents.
55
References Cited
Alon5 R., R.C. Fuhlbirgge5E.B. Finger5 and T.A. Springer. 1996. Interactions through Lselectin between leukocytes and adherent leukocytes nucleate rolling adhesions on
selectins and VCAM-I in shear flow. J Cell Biol. 135:849-865.
Bargatze5 R.F., S. Kurk5 E.C. Butcher, and M.A. Jutila. 1994. Neutrophils roll on
adherent neutrophils bound to cytokine-stimulated endothelium via L-selectin on
the rolling cells. JExp Med. 180:1785-1792.
Bruehl5 R E., T.A. Springer5 and D.F. Bainton. 1996. Quantitation of L-selectin
distribution on human leukocyte microvilli by immunogold labeling and electron
microscopy. JHistochem Cytochem. 44:835-844.
Hein5W.R., and C.R. Mackay. 1991. Prominence of gamma/delta T cells in the ruminant
immune system. Immunol Today 12:30-34.
Jutila5 M.A., G. Watts5 B. Walcheck5 and G.S. Kansas. 1992. Characterization of a
functionally important evolutionarily conserved epitope mapped to the short
consensus repeats of E-selectin and L-selectin. JExp Med. 175:1565-1573.
Jutila5 M.A., R.F. Bargatze5 S. Kurk5 RA. Wamock5 N. Ehsani5 S. Watson5 and B.
Walcheck. 1994. Cell surface P- and E-selectin support shear-dependent rolling of
bovine yS T cells. J Immunol. 153:3917-3928.
Jutila5 M.A. 1996. Gamma/delta T cell/endothelial cell interactions. Vet Immunol
lmmunopathol. 54:105-110. '
Jutila5 M.A., and S. Kurk. 1996. Analysis of bovine yS T cell interactions with E-, P-,
and L-selectin: characterization of lymphocyte on lymphocyte rolling and the
effects of O-glycoprotease. J Immunol. 156:289-296.
Jutila5M.A., E. Wilson, and S. Kurk. 1997. Characterization of an adhesion molecule that
mediates leukocyte rolling on 24 hr cytokine- or lipopolysaccharide-stimulated
bovine endothelial cells under flow conditions. JExp Med. 186:1701-1711.
Jutila5 M.A. 1998. Recruitment of y5 T cells and other T cell subsets to sites of
inflammation. C. Serhan and P. Ward, eds. In Molecular and Cellular Basis o f
Inflammation. Humana Press Inc., Totowa5New Jersey.
56
Kishimoto5 T.K., M.A. Jutila5 and E.C. Butcher. 1990. Identification of a human
peripheral lymph node homing receptor: a rapidly down-regulated adhesion
molecule. ProcNatlAcadSci., USA 87:2244-2248.
Kishimoto5T.K., R.A. Wamock5M.A. Jutila5E.C. Butcher, C. Lane5 D.C. Anderson5 and
C.W. Smith. 1991. Antibodies against human neutrophil LECAM-I (LAMl/Leu-8/DREG 56 antigen) and endothelial cell ELAM-I inhibit a common CD 18independent adhesion pathway in vitro. Blood 78:805-811.
Libero5G. 1997. Sentinel function of broadly reactive human y5 T cells. Immunol Today
18:22- 26 .
Mackay5C.R., W.R. Hein. 1989. A large proportion of bovine T cells express the y5 T
cell receptor and show a distinct tissue distribution and surface phenotype. Int
Immunology 1:540-545.
Picker, L.J., A. Wamock5C. Bums5E. Doerschuk5 E. Berg5 and E.C. Butcher. 1991. The
neutrophil selectin LECAM-I presents carbohydrate ligands to the vascular
selectins ELAM-I and GMP-140. Cell 66:921-933.
Raulet5D.H. 1989. The structure, function, and molecular genetics of the gamma delta T
cell receptor. Ann Rev Immunol. 7:175-201.
Speer5 CA., A.A. Marchiondo5 B. Mueller, and D.W. Duszynski. 1979. Scanning and
transmission electron microscopy of the oocyst wall of Isospora lacazei.
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Speer5CA., J.P. Dubey5JA . Blixt5and K. Prokop. 1997. Time lapse video microscopy
and ultrastructure of penetrating sporozoites, types I and 2 parasitophorous
vacuoles, and transformation of sporozoites to tachyzoites of the VEG strain of
Toxoplasma gondii. J Parasitology 83:565-574.
Springer5 TA . 1994. Traffic signals for lymphocyte recirculation and leukocyte
emigration: The multistep paradigm. Cell 76:301-314.
Tedder5T.F., DA. Steeber5A. Chen5and P. Engel. 1995. The selectins: vascular adhesion
molecules. FASEB J. 9:866-873.
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Tedder. 1996. L-selectin binds to P-selectin glycoprotein ligand-1 on leukocytes:
57
interactions between the lectin, epidermal growth factor, and consensus repeat
domains of the selectins determine ligand binding specificity. J Immunol.
157:3995-4004.
Walcheck, B., G. Watts, and M.A. Jutila. 1993. Bovine y5 T cells bind E-selectin via a
novel glycoprotein receptor: First characterization of a lymphocyte/E-selectin
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Walcheck, B., and M.A. Jutila. 1994. Bovine yS T cells express high levels of functional
peripheral lymph node homing receptor (L-selectin). Int Immunol. 6:81-91.
Walcheck, B., K.L. Moore, R.P. McEver, and T.K. Kishimoto. 1996. Neutrophilneutrophil interactions under hydrodynamic shear stress involve L-selectin and
PSGL-I. A mechanism that amplifies initial leukocyte accumulation on P-selectin
in vitro. J Clin Invest. 98:1081-1087.
58
CHAPTER 3
ANTIBODY BINDING TO A CONFORMATION-DEPENDENT EPITOPE
INDUCES L-SELECTIN LINKAGE TO THE CYTOSKELETON
Introduction
Recruitment of leukocytes from the blood into tissues is controlled by a variety of
adhesion molecules on the surface of the endothelium and circulating leukocytes, one of
which is L-selectin (Butcher et ah, 1999). L-selectin is important in the normal migration
and circulation of both memory and naive lymphocytes (Gallatin et al., 1983 and Steeber
et al., 1996a, and 1996b) and is central to leukocyte-endothelial cell interactions, including
neutrophil rolling along the vessel wall (von Andrian et al., 1991). We, and others, have
also shown that L-selectin and corresponding ligands are important in leukocyte-onleukocyte rolling interactions that may amplify further recruitment of leukocytes from the
blood into tissues (Bargatze et al., 1994a, Jutila and Kurk, 1996, Fuhlbrigge et al., 1996,
Alon et al., 1996 and Walcheck et al., 1996).
A common feature of all adhesive
interactions mediated by L-selectin is that they occur under flow in the bloodstream.
Indeed, threshold levels of shear promote adhesion through L-selectin (Finger et al., 1996,
and Lawrence, et al, 1997).
L-selectin is constitutively expressed on most circulating leukocytes, but is
uniquely regulated when the cell becomes activated. Following activation of lymphocytes
or myeloid cells with chemotactic factors or phorbol esters, there is a rapid increase in
59
functional avidity (Spertini et al., 1991) which, under most circumstances, is followed by
proteolytic cleavage of the protein from the cell surface .(Kishimoto, et al., 1989, Griffin,
et al., 1990 and Smith, et al., 1991). The basis for the increased functional activity in Lselectin immediately following activation of the leukocyte is poorly understood. It may
be due to phosphorylation of L-selectin or other proteins, dimerization, hyperthermic
conditions, cytoskeletal association of L-selectin through its cytosplasmic tail, or
conformational changes in the protein (Haribabu, et al., 1997, Li et al., 1998, Wang et al.,
1998 and Evans et al., 1999). Endoproteolytic release of L-selectin from the surface of
leukocytes is regulated by structural features of the L-selectin protein (Chen, et al., 1995,
Migaki et al., 1995). Calmodulin, an intracellular calcium regulatory protein, specifically
co-precipitates with L-selectin through a direct association with the cytoplasmic tail and
calmodulin inhibitors disrupt L-selectin-dependent adhesion by inducing proteolytic
release of L-selectin from the cell surface (Kahn et al., 1998).
Many reports suggest that L-selectin can also function as a signal transduction
molecule. Crosslinking of human L-selectin with mAbs leads to neutrophil activation as
measured by Ca2+ flux, superoxide generation, increased adhesiveness, and activation of
intracellular protein pathways, such as tyrosine phosphorylation and MAP kinase
production (Po et al., 1995, Waddell et al., 1995, Crockett-Torabi et al., 1995, Simon et
al., 1995, Sikorski et al., 1996, and Gopalan et al, 1997). Crosslinking of L-selectin also
potentiates the response of neutrophils to formyl peptides (Waddell et al, 1994). In
most studies, crosslinking of L-selectin using primary anti-L-selectin mAh followed by a
60
secondary reagent is requisite for signaling, though important exceptions exist.
For
example, signaling through L-selectin can also be induced by sulfatides, which bind
directly to L-selectin (Laudana et al, 1994). Further, a mAh directed against a highly
conserved region of L-selectin can signal and cause increased adhesion of lymphoid cells
transfected with human L-selectin, neutrophils, and lymphocytes, in the absence of a
crosslinking secondary antibody (Steeber et al., 1997).
Therefore, it is clear that L-
selectin can act as a signaling molecule under certain conditions.
The cytoplasmic tail of L-selectin is vital to its function during leukocyte rolling
and adhesion (Kansas et al., 1993). Deletion mutants lacking the carboxyl terminal 11
amino acids of the cytoplasmic tail of L-selectin do not bind to HEV and do not establish
rolling interactions in vivo (Kansas et al., 1993). Subsequent work demonstrated a direct
link between the cytoplasmic tail of L-selectin and the cytoskeletal proteins a-actinin and
vinculin (Pavalko et al., 1995) and recently, L-selectin was shown to dynamically
associate with the cytoskeleton (Evans et al., 1999).
Antibody crosslinking,
hyperthermic treatment, and ligand binding studies demonstrate that the cytoplasmic tail
is important to the function of L-selectin by regulating linkage to the actin cytoskeleton
through direct binding of the cytoplasmic tail (Evans et al., 1999). However, the nature of
the linkage has not been clearly shown and it is not known what predisposes L-selectin to
link to the cytoskeleton.
61
Here, we have examined the effect of treating leukocytes with an anti-L-selectin
mAh that recognizes a highly conserved and functionally important epitope on L-selectin
expression. We show that one mAh (LAM1-116), which binds an epitope in the lectin
domain (Steeber et al., 1997), causes a structural change in human, bovine, and ovine Lselectin in the absence of cellular activation that is detected by increased staining of a
second anti-L-selectin mAb, EL-246. The induced conformation predisposes L-selectin
to link to the cytoskeleton. As both mAbs bind functional epitopes on L-selectin, this
type of structural regulation may be important in L-selectin function.
Materials and Methods
Animals
One-week to three-month-old Holstein calves, and eighteen-month-old sheep,
housed in the Montana State University large animal facility, and healthy human donors
were used as sources of peripheral blood.
Cell Preparations
Peripheral blood was collected by venipuncture into citrate or heparin
anticoagulant tubes as previously described (Jutila et al., 1992). Total leukocytes were
harvested using a hypotonic solution for 10 sec followed by rapid dilution in either HBSS
or PBS (Sigma, St. Louis, MO) and centrifuged at 200g for 5 min. The process was
repeated as necessary to rid preparations of red blood cells.
62
Monoclonal Antibodies
The following mAbs were used.
Anti-L-selectin mAbs included DREG 55,
DREG 56, DREG HO, DREG 152, and DREG 200 (Kishimoto et ah, 1990); L A M l-I,
LAM1-5, LAMI-101, LAM1-102, LAMl-104, LAMl-108, LAM l-110, LAM l-115,
LAM l-116, LAMI-118, LAMl-119, LAM1-120, LAMI-126 (Steeber et ah, 1997), EL246 and GD 4.22 (Jutila, 1992 and 1996), and Leu-8 (Becton Dickenson, Mountain View,
CA). Anti-CD 18 mAbs included Rl 5.7 (gift from R. Rothlein), IB4 (ATCC, Manassas,
VA: ATCC #HB-10164), and MHM-23 (Dako, Carpinteria, CA). Anti-CDllb mAh
Leu-15 (Becton Dickenson) was also used. Other mAbs included HECA 452 [anti-CLA
(Picker et ah, 1990)], Hermes-3 [anti-CD44 (Picker et ah, 1989)], GD 3.5 [anti-yS T cell
(unknown yS T cell specific marker, Jones et ah, 1996)], GD 3.8 [anti-y8 TCR (Wilson et
al, 1998)], and EL-112 [anti-E-selectin (Bagatze et ah, 1994b)].
Cell Lines
The mouse pre-B 300.19 cells transfected with either full length human L-selectin
cDNA (300.19/L-selectin) or a deletion mutant lacking the carboxyl-terminal 11 amino
acid residues of the cytoplasmic domain (300.19/LAcyto) have been previously described
(Kansas et ah, 1993). The selectin tranfectants used in the antibody mapping studies
were described elsewhere (Tu et ah, 1996).
63
Flow Cytometry of Non-detergent-treated Cells
Isolation of leukocytes and flow cytometric analysis were as described (Jutila et
al., 1992, and Bargatze et al., 1994b). Briefly, IxlO6 leukocytes were incubated with I jag
of LAMl-116 or other mAbs at 37°C or 4°C, 10-100 mM sodium azide (Sigma Co.), 1100 mM Herbimycin A (Calbiochem), Genistein (Calbiochem), or calpeptin (Calbiochem)
for 15 min, or buffer alone. After incubation with mAh, the cells were placed on ice and
FITC-conjugated (Molecular Probes, Eugene, OR) EL-246, biotin-conjugated (Pierce,
Rockford IL) EL-246, or other FITC- or PE-conjugated mAbs were added. Cells were
incubated with mAbs for 30 min on ice, washed in PBS with horse serum (FACS buffer),
and staining measured on either a FACScan or FACSCalibur (Becton Dickenson,
Mountain View, CA). Data were collected from 10,000 cells and mode fluorescence
staining values reported in table form or as representative histograms. L-selectin levels
were also measured by an indirect stain on the 300.19 L-selectin transfectants, as
described (Jutila et al., 1992). LAMl-116 and DREG 56 Fab treatment of leukocytes
was performed as above.
. Flow Cytometry of Detergent-solubilized Cells
Flow cytometric analysis of L-selectin association with the detergent-insoluble
cytoskeleton was as described (Geppert and Lipsky, 1991, and Nebe et al., 1997), with
minor exceptions. Specifically, leukocytes were harvested and isolated as described above
and incubated with LAMl-116 or other anti-L-selectin mAbs at 37°C for 15 min. The
64
cells were washed in FACS buffer and either treated with another anti-L-selectm mAh for
15 min at 37°C, or treated directly with 0.5% NP-40 lysis buffer (150 mM NaCl5 2 mM
CaCl2, 2 mM MgCl2, 10 mM HEPES, 2% Goat Serum, 0.5% NP-40) for 15 min at room
temperature in the absence of antibody crosslinking, as described (Nebe et al., 1997).
Mock buffer without NP-40 was used as a control. The cells were washed in FACS
buffer and incubated with PE-labeled goat anti-mouse F(ab’)2 (Jackson ImmunoResearch
Labs, Inc., West Grove PA) for 30 min on ice. Staining was measured by gating on the
detergent-insoluble cytoskeletal fraction using forward and side scatter. The gated
population was confirmed by phalloidin-FITC (Sigma), which specifically stains the
detergent-insoluble cytoskeleton (Nebe et al., 1997).
Generation of LAMl-116 and DREG 56 Fab
Monovalent Fab fragments were generated from whole IgG molecules with papain
by the ImmunoPure® Fab Preparation Kit from Pierce, per manufacturer’s instructions.
To confirm Fab production, 8% SDS-PAGE gels were run under non-reducing and
reducing conditions and molecular weight determined by Coomassie stain. In some cases,
the Fab preparation was also incubated with Protein G beads (Boehringer Mannheim,
Germany) to reduce the levels of contaminating Fe fragments.
65
Phase Contrast Fluorescence Microscopy
Bovine lymphocytes were isolated as described (Jones et ah, 1996), treated with
mAbs, incubated with 0.5% NP-40 lysis buffer for 10 min at room temperature, and
stained with PE-labeled goat anti-mouse F(ab’)2 (Jackson ImmunoReSeareh) secondary
antibody on ice for 30 min. Cells were washed in FACS buffer and incubated with
phalloidin-FITC (Sigma) for 20 min on ice. Cells were washed a final time in FACS
buffer and placed in 16-well, LabTek glass chamber slides (Nunc, Naperville, IL) for
microscopic examination. Fluorescent microscopy was performed using a super high
pressure mercury lamp power supply (Nikon, Melville, NY) model HB-1013AF, linked
to a Nikon inverted microscope (Eclipse TE300), ,and digital data captured using a Spot
digital imaging system (Diagnostic Instruments, Inc., Sterling Heights, MI). Results are
visualized at 400X magnification. The fluorescence micrographs are overlays of red and
green images to show double positive regions of cells (orange).
Immunoprecipitation of L-selectin and
Densiometfic Analysis of Band Intensities
MAb EL-246 was covalently linked to activated cyanogen-bromide (CNBr)
Sepharose 4B beads (Pharmacia LKB, Uppsala, Sweden), which were then blocked with
IM glycine at 25°C for 3 hrs (Walcheck et ah, 1993). Bovine lymphocytes were surface
labeled with biotin (Pierce), as described (Jones et ah, 1996), and detergent lysates
prepared from these cells. The preparations were incubated with CNBr beads for 2 hrs at
66
25°C as a pre-clearing step (Jones et al., 1996). The lysates were drained from the CNBr
columns and incubated without mAh, LAMl-116, other. anti-L-selectin mAbs, or
irrelevant mAbs at 37°C for 15 min. After incubation, an equal quantity of EL-246labeled CNBr beads were added to each lysate and L-selectin was immimoprecipitated
either at 4°C overnight or at 37°C for 2.5 hrs. The beads were washed 3x with wash
buffer (Walcheck et al., 1993), mixed with reducing or non-reducing buffer, boiled for 3
min, and loaded directly onto an 8% polyacrylamide gel. Gels were electrophoresed and
the proteins were transferred to PVDF membrane (Bio-Rad, Hercules, CA) overnight at
4°C. Proteins were visualized using a streptavadin horseradish peroxidase (Amersham,
Buckinghamshire, England) reaction and ECL detection system (Amersham) and
developed on X-OMAT (Kodak) film. Densiometric analysis was performed on the
intensity of the immunoprecipitated L-selectin bands using I-D MULTI on an Alpha
Innotech Corp. IS-IOOO Digital Imaging System.
Protein G (Boehringer Mannheim,
Germany) immunoprecipitation using anti-L-selectin mAh GD 4.22 was used as a
positive control for L-selectin immunoprecipitation, as described (Jones et al., 1996).
Results
MAb LAM1-116 Enhances Leukocyte
Staining of L-selectin bv mAh EL-246
Pretreatment of human neutrophils or bovine lymphocytes with mAh LAM l-116
at 37°C for 15 minutes enhanced their subsequent staining of L-selectin by FITC-Iabeled
67
A
Log 10 Fluorescence
B
■ No Treatment
Human
Neutrophils
68 LAM1-116 Treatment
*
Bovine
Lymphocytes
*
0
50
100
150
200
250
300
EL-246 Staining (Mode Fluorescence Intensity)
Figure 3.1. LAMl-116 treatment of human or bovine leukocytes at 37°C increases
the presentation of a functionally conserved epitope on L-selectin as measured by
another directly FITC-Iabeled mAb, EL-246. Representative overlay histograms are
shown in Panel A: EL-246 alone (dotted line), EL-246 following pretreatment with
LAMl-116 (thick solid line). Panel B represents staining of human neutrophils and
bovine lymphocytes with and without LAMl-116 treatment.
Each panel is
representative of over 10 separate experiments.
*A paired students t test was performed on the untreated vs. LAMI-116 treated cells
for both human neutrophils and bovine lymphocytes, p<0.005.
68
EL-246
DREG 56
DREG 200
GD 4.22
Leu-8
so
ioo
mAh
iso
200
Staining
B
L A M l-116
0 EL-246
R15.7
HECA 452
■ mAh + EL-246
Hermes-3
GD 3.8
GD 3.5
0
50
100
150
200
EL-246 Staining
Figure 3.2. LAMI-116 specifically increases the presentation of the EL-246
epitope. Other isotype-matched and mismatched anti-L-selectin mAbs directed
against distinct epitopes are not enhanced by LAMI-116 pretreatment (Panel A).
MAbs directed against other cell surface antigens do not enhance the presentation
of the EL-246 epitope (Panel B). Data is representative of 3 separate experiments
and the S E M. shown.
69
EL-246 mAb, as measured by flow cytometric analysis (Figure 3.IA5 and B). The same
effect was seen with all leukocyte cell types tested from humans, cattle, and sheep (data
not shown).
The increased staining by EL-246 after leukocyte treatment with the
LAMl-116 mAb was dose-dependent with I pg/ml of LAMl-116 mAb giving optimal
results (data not shown). Importantly, the increase in EL-246 staining was not due to
increased expression of L-selectin protein, since the staining of four other FITC- or PElabeled anti-L-selectin mAbs was unaffected (Figure 3.2A). This effect was also detected
by EL-246 labeled with biotin (Table 3.1). Moving human or bovine leukocytes from
4°C to 37°C for 15 minutes greatly reduced the staining of L-selectin by EL-246 in the
absence of L-selectin shedding (Table 3.1, and data not shown). In contrast, staining by
other anti-L-selectin mAbs was not altered by this treatment (data not shown).
To determine the specificity of the LAMl-116 mAb-induced upregulation of the
EL-246 epitope, human and bovine leukocytes were pretreated with 19 other anti-Lselectin mAbs, including at least 6 reactive with the same domain as LAMl-116, as well
as mAbs directed to other Surface antigens, including R15.7 (anti-CD18), HECA 452
(anti-CLA), Hermes-3 (anti-CD44), GD 3.5 (unknown ligand on yS T cells) and GD 3.8
-
(anti-yd TCR). None of the other anti-Lrselectin mAbs or other mAbs listed above
induced increased EL-246 staining [Figures 3.2A (example of 4 other anti-L-selectin
mAbs) and 3.2B and data not shown]. In similar experiments, LAM l-116 pretreatment
had no effect on the expression of other surface antigens, such as CD 18 and TCR (data
70
not shown). To be confident staining of L-selectin by FITC-Iabeled EL-246 was specific,
excess (IOx) unlabeled EL-246 or excess isotype-matched (IOx) GD 3.8 (anti-y5 TCR)
antibodies were added along with FITC-Iabeled EL-246, as above. The addition of excess
unlabeled EL-246 mAb effectively blocked the staining of FITC-Iabeled EL-246 (Table
3.1). However, treatment with excess GD 3.8 mAb did not inhibit staining of FITClabeled EL-246 to an appreciable extent (Table 3.1).
Table 3.1. Expression of the EL-246 Epitope on Leukocytes Under Various Conditions
Cell Treatment
EL-246 Expression
4°C
100%
37°C
50% + 7%
LAM l-116
270% ± 20% *
LAM1-116**
250% ± 6%**
LAMI-116 with excess unlabeled EL-246
4% ± 4%
LAMl-116 with excess unlabeled GD 3.8
240% ± 9%
LAMl-116 with 50 mM sodium azide
220% ± 5%
LAMl-116 with 50 |iM Herbimycin A
225% ± 7%
LAMl-116 with 50 |LM Genistein
23594 ±994
Level of EL-246 expression, measured by EL-246 staining, was normalized to 100% for
each experiment and the mean ± S.E.M. listed.
All treatments were done at 37°C unless otherwise stated. At least 3 separate
experiments were done for all the treatments listed.
*The amount of EL-246 epitope enhancement varied depending upon the amount of yS T
cells present in the preparations as y§ T cells express 2-5 fold more L-selectin than a(3 T
cells.
**The prior readout was EL-246-FITC and these numbers are for EL-246-Biotin.
71
LAMl-116 Fab also Induced an Increase
in Expression of the EL-246 Epitope
To determine whether intact LAMl-116 was requisite for enhanced EL-246
reactivity, a Fab fragment of LAMl-116 was generated by papain digestion, as described
in Materials and Methods. The Fab fragment of LAMl-116 was able to enhance EL-246
epitope expression, although to a lesser extent than the whole IgG antibody (Figure 3.3).
This data suggested that LAMl-116 did not induce crosslinking of adjacent L-selectin
molecules as monovalent Fab fragments do not have a second binding site needed for
effective crosslinking. The larger increase in EL-246 staining seen with the IgG was likely
due to the ability of dual binding sites to act upon L-selectin and enhance the EL-246
epitope more effectively. To ensure the specificity of the LAMl-116 Fab interaction,
Fab fragments of DREG 56 mAb were generated, and although the antibody preparations
stained L-selectin, they did not increase EL-246 reactivity in parallel assays (data not
shown).
EL-246
LAMl-116 F(ab’)2
LAMl-116 IgG
\Y ////////////////////////////////////////////s
0
50
100
150
200
250
300
350
400
450
EL-246 Staining
Figure 3.3. LAMl-116 monovalent Fab enhances the presentation of the EL-246
epitope on bovine lymphocytes. Leukocytes from human and bovine preparations
were enhanced by LAMl-116 Fab treatment (data not shown). Data are representative
of 3 separate experiments and the S.E.M. shown.
72
The Tyrosine Kinase Inhibitors Herbimycin A
and Genistein do not Block LAMl-116
Upreeulation of the EL-246 Epitope
Tyrosine kinase activity may play a role in the function of L-selectin (Waddell ,et
al, 1995). To address whether tyrosine kinases are involved in upregulation of the EL246 epitope, we tested the effects of two tyrosine kinase inhibitors, Herbimycin A and
Genistein, to see if they could block the increased presentation of the EL-246 epitope in
the presence of the LAMl-116 mAh. As shown in Table 3.1, neither Herbimycin A or
Genistein blocked the LAMl-116 enhancement of the EL-246 epitope. No inhibition of
EL-246 epitope enhancement was seen over a range of 1-100 mM of each inhibitor. Thus,
tyrosine kinase activity seems to have no role in the conformational change induced in Lselectin by the LAMI-116 mAh. To further investigate whether other signaling events in
the cell were responsible for the upregulation of the EL-246 epitope, studies were
undertaken at 4°C, and in the presence of sodium azide, which blocks electron transport
and eventual ATP production. Both treatments had no inhibitory effect on the LAM l116 mAh's ability to induce the conformation that increases the expression of the EL-246
epitope (Table 3.1). Therefore, enhancement of the EL-246 epitope is not dependent
upon common signaling events.
73
Expression of the EL-246 Epitope on Recombinant
Selectins Generated by Swapping Different
Domains of L-. E-. and P-selectin
EL-246 mAh recognizes a conserved epitope on both E- and L-selectin and
original mapping studies using recombinant chimeric selectins comprised of L- and Pselectin extracellular domains demonstrated that the SCR domain of L-selectin was
important for optimal EL-246 binding (Jutila et al., 1992). Additional chimeric selectin
molecules were generated, expressed and analyzed for EL-246 staining to gain further
insight into the nature of the EL-246 epitope.
As expected, EL-246 recognized
recombinant L and E-selectin (Table 3.2, LLL and EEE), but not P-selectin (data not
shown).
Staining of multiple chimeric molecules, generated by swapping different
domains of each selectin, failed to provide a precise location of the EL-246 epitope. The
EL-246 epitope did not reside in the SCR domain of L-selectin, since EL-246 failed to
stain a construct containing the lectin domain of P-selectin, and EGF and SCR domains of
L-selectin (Table 3.2, PLL). Weak reactivity was seen with a construct containing the
lectin domain of L-selectin, and EGF and SCR domains of P-selectiri (LPP). Replacing
the SCR domain in this latter construct with an SCR from L-selectin increased staining
slightly, but not nearly as much as seen in our earlier study (Jutila et al., 1992), suggesting
variability in expression of the EL-246 epitope on the same transfectants analyzed at
different times. The lectin domain of L-selectin did not confer EL-246 reactivity, even if
combined with EGF or SCR domains of E-selectin (Table 3.2, LEE and LLE). The
greatest amount of EL-246 staining was seen with a chimera expressing the lectin and
74
EGF domains of E-selectin and SCR domain of L-selectin (Table 3.2, EEL). From these
stains we conclude that the EL-246 epitope is not encoded by a specific domain of Lselectin, but whose expression is dependent upon a proper conformation of the molecule
that can be altered by changes in one or more of its domains.
Chimeric
Selectin
None
LLL
LEE
ELL
EEE
EEL
LLE
LLP
LPP
PLL
LPL
EL-246
3.2
18.4
3.2
60.8
21.3
142
3.9
2.8
6.2
2.8
7.2
LAMl-14 fSCR)
3.0
67.4
3.0
26.4
2.6
57.3
16.4
2.6
2.9
19.7
33
LAMI -3 ILectinI
3.4
161
20
2.9
2.8
2.8
34
11.3
74
2.5
60
EL-246 staining of L-, E-, and P-selectin chimeras was analyzed by flow cytometry.
LAM1-14 and LAMl-3 mAb staining is also shown as a comparison to EL-246. Mean
fluorescence intensity is reported. Staining of the parent cell lines is reported in the row
titled None. Data was generated by Dr. Doug Steeber at Duke University.
Treatment of Detergent Lysate Preparations with
LAMI-116 mAb Specifically Enhances L-selectin
Immunoprecipitation by EL-246 mAb
Covalently Linked to CNBr Beads
To test whether or not the intact leukocyte was needed for a conformational
change to take place in the L-selectin protein, bovine lymphocytes were surface labeled
with biotin and detergent lysates prepared. The biotin-labeled lysates were divided into
75
fractions, which were treated with the LAMl-116 mAh, a negative control antibody, or
no antibody at all, for 15 minutes at 37°C.
The treated lysates were then
immunoprecipitated with EL-246 covalently attached to Sepharose 4B beads either at
4°C or 37°C. As shown in Figure 3.4A, lane 2, EL-246 weakly immunoprecipitated Lselectin from control lysates.
By contrast, treatment of lysates with LAM l-116
increased the amount of L-selectin subsequently immunoprecipitated by EL-246 when
compared to the negative control, an irrelevant, isotype-matched mAh, EL-112 (Figure
3.4A, lanes 3 and 4). Other anti-L-selectin mAbs, including DREG 56, were tested, and
only LAM l-116 caused increased immunoprecipitation of L-selectin (data not shown).
Protein G bead immunoprecipitation of L-selectin with anti-L-selectin mAh GD 4.22 was
used as a control for L-selectin precipitation (Figure 3.4A, lane I). Densiometric analysis
was employed to quantify the differences in band intensity.
Analysis of the bands
showed that LAMl-116 mAh treatment of the detergent lysates increased the amount of
L-selectin immunoprecipitated by EL-246 mAb four - fivefold compared to non-treated
or other mAb treated fractions (Figure 3.4B, lanes 2-4).
EL-246 mAb Can Induce L-selectin Cytoskeletal
Association in the Presence of LAMl-116
Recently, L-selectin was shown to dynamically associate with the actin
cytoskeleton under a variety of conditions (Evans et al., 1999). To investigate L-selectin
cytoskeletal interactions, cells were treated with primary anti-L-selectin mAbs as
76
B
Figure 3.4. LAMl-116 treatment increases the amount of L-selectin immunoprecipitated
from bovine lysates by EL-246 covalently linked to activated CNBr beads. Panel A
represents a SDS-PAGE gel with L-selectin immunoprecipitation. Lane I, protein-G
control immunoprecipitation of L-selectin with anti-L-selectin mAb, GD 4.22. Lane 2,
untreated lysate immunoprecipitated with EL-246-labeled CNBr beads. Lane 3, LAMI116 treated lysate immunoprecipitated with EL-246 beads. Lane 4, EL-112 treated lysate
immunoprecipitated with EL-246 beads (irrelevant, isotype-matched anti-E-selectin mAb).
Panel B, densiometric analysis of lanes 2-4 in Panel A. LAMl-116-treated lysate was
normalized to 100% L-selectin immunoprecipitation. Data are representative of 3 separate
experiments and the S E M shown.
described in Materials and Methods, subjected to 0.5% NP-40 buffer, and L-selectin
staining o f the detergent-insoluble cytoskeleton visualized by goat F(ab’)2 anti-mouse
antibody labeled with phycoerythrin. Thus, if cytoskeletal association o f L-selectin was
detected, it was due to a direct effect o f the mAbs on L-selectin and not crosslinking since
the cells were treated with detergent prior to the addition o f the secondary antibody.
This is in contrast to the recent report by Evans and colleagues (1999) in which
crosslinking by second stage antibody
cytoskeletal association of L-selectin.
was
a prerequisite
to
antibody-induced
77
Treatment of human and bovine lymphocytes with the LAM l-116 or the EL-246
mAbs alone did not induce association of L-selectin with the cytoskeleton (Figure 3.5A).
However, in the presence of LAMl-116, EL-246 induced L-selectin association with the
cytoskeleton without subsequent crosslinking of the surface protein (Figure 3.5B). Other
anti-L-selectin mAbs tested did not induce L-selectin association in tandem with LAM l116 mAh treatment, suggesting that EL-246 mAh cytoskeletal induction was specific to
the LAMl-116/EL-246 interaction with L-selectin (data not shown).
Crosslinking
studies parallel to those done by Evans et al. (1999) did show L-selectin cytoskeletal
association but the association was no more dramatic than LAMl-116/EL-246 antibody
treatment alone (data not shown).
Cytoskeletal association of L-selectin was also visualized by two-color
fluorescent microscopy with PE-labeled secondary antibody and phalloidin-FITC, which
specifically stains the actin cytoskeleton (Nebe et al., 1997).
Parallel to our flow
cytometry results, engagement of the EL-246 epitope in the presence of the LAM l-116
mAh caused L-selectin to associate with the cytoskeleton (Figure 3.6B).
The
micrographs are overlays of a red (L-selectin) and a green (phalloidin-FITC, actin
cytoskeleton) digital image. A phase contrast micrograph of detergent-treated cells,
matching the micrograph in 3.6B, is shown in 3.6A.
Phalloidin-FITC staining was
specific as it did not stain viable, non-detergent-treated cells (data not shown). Also, nohdetergent-treated lymphocytes exhibited a normal, punctate L-selectin staining pattern
(data not shown). As seen with our flow cytometry data (Figure 3.5), neither the LAMl-
78
A
B
Figure 3.5. In the presence of LAMl-116, EL-246 can induce association of Lselectin with the actin cytoskeleton. Panel A, LAMl-116 or EL-246 alone do not
induce L-selectin cytoskeletal association [solid line, EL-246, dotted line, LAMl-116,
stippled line, second stage goat anti-mouse F(ab’)2 PE]. Panel B, treatment of bovine
lymphocytes with LAMI-116 predisposed L-selectin to cytoskeletal association,
which in turn was triggered by EL-246 epitope engagement [solid line, EL-246
treatment in the presence of LAMl-116, dotted line, second stage goat anti-mouse
F(ab’)2 PE alone]. Data are representative of 4 separate experiments.
79
Figure 3.6. Phase contrast fluorescent microscopy of bovine lymphocytes. Panel
A, phase contrast micrograph of the fluorescent overlay in Panel B. Panel B,
overlay of green (phalloidin-FITC) and red (anti-L-selectin mAbs LAMI-116
and EL-246 visualized with goat anti-mouse F(ab’)2 PE) stains showing
cytoskeletal association of L-selectin (red and orange). Arrows represent
examples of cytoskeletal association (red and orange) of L-selectin in the
micrograph. Panel C, overlay of green (phalloidin-FITC) and red (LAMI-116).
Notice the lack of red and/or orange staining demonstrating no cytoskeletal
association of L-selectin with LAMl-116 alone. Panel D, overlay of green
(phalloidin-FITC) and red (EL-246). Notice lack of red or orange staining with
EL-246 alone. Data are representative of 4 separate experiments. Micrographs
were taken at 400X magnification, except for Panels C and D; 200X, bar = 10
pm.
80
116 mAb nor the EL-246 mAh alone induced L-selectin association with the cytoskeleton
(Figures 3.6C and D). These data suggest that the conformational change in L-selectin
induced by the LAMl-116 mAb predisposes L-selectin to cytoskeletal association.
Staining of L-selectin on Wild Type and LACyto
Transfectants Lacking an Intact Cytoplasmic Tail
Since the EL-246 epitope appears to require a unique conformation of L-selectin,
we tested whether an intact cytoplasmic tail domain, which can link to the cytoskeleton,
was required for optimal staining. L-selectin transfectants that lack the carboxyl terminal
11 amino acids of the cytoplasmic tail domain (LACyto) of L-selectin do not bind to HEV
in vitro nor do they exhibit rolling interactions in vivo in exteriorized rat mesenteric
venules, suggesting that the cytoplasmic domain of L-selectin regulates leukocyte
adhesion by controlling cytoskeletal interactions and/or receptor avidity (Kansas et al.,
1993). Staining of the EL-246 epitope on L-selectin in the LACyto versus the wild type
transfectants was reduced (Figure 3.7A). Indeed, EL-246 staining of the majority of the
LACyto mutant cells fell below the upper threshold of background staining. In
comparison, LAMl-116 staining of the LACyto mutants remained high (Fig. 3.7B). To
test whether or not LAMl-116 could enhance the expression of the EL-246 epitope on
the cytoplasmic tail mutants, the mutants were pretreated with LAMl-116 parallel to the
studies described above. Interestingly, EL-246 epitope expression could be enhanced by
LAMl-116 pretreatment on both the wild type and the LACyto mutants (Figure 3.8A
81
A
EL-246 +
Wild type
GAM FITC
____ ago-<,AM „ . * * * o » *
EL-246 +
GAM FITC
LACyto
B
aoo-iAM
LAMl-116 +
GAM FITC
Wild type
LACyto
I
LAMl-116 +
GAM FITC
Figure 3.7. Staining of L-selectin with EL-246 and LAMl-116 on wild type and
LACyto transfectants. Panel A, EL-246 staining of wild type and LACyto
transfectants.
Panel B, LAMl-116 staining of wild type and LACyto
transfectants. Histograms are representative of 2 separate experiments. Data was
generated by Dr. Doug Steeber at Duke University.
82
A. Wild-type L-selectln
EL-246-F1TC
B. L-selectin cytoplasmic tail deletion
Figure 3.8. LAMI-116 treatment of wild type and cytoplasmic tail deletion
transfectants upregulates the EL-246 epitope. Panel A, EL-246 staining on wild type
transfectants (thin line), EL-246 staining with LAMI-116 treatment (thick line), and
second stage goat anti-mouse FITC alone (dotted line). L-selectin staining by LAMI116 is shown to the immediate left. Panel B, EL-246 staining on cytoplasmic tail
deletion transfectants (thin line), EL-246 staining with LAMI-116 treatment (thick
line), and second stage goat anti-mouse FITC alone (dotted line). L-selectin staining
by LAMI-116 is shown to the immediate left. Histograms are representative of 2
separate experiments and were performed by Dr. Doug Steeber at Duke University
Medical Center.
83
and B). There was a dramatic upregulation of the EL-246 epitope on the cytoplasmic tail
mutants as the EL-246 epitope expression increased well beyond the minimal staining
levels seen in Figure 3.7. These data suggest that an intact cytoplasmic tail of L-selectin
is not need for the LAMl-116 induction of the conformational change that increases EL246 epitope expression.
Discussion
In this report, we demonstrate a conformation-induced change in L-selectin
mediated by a mAh that recognizes a functionally conserved epitope on L-selectin. The
mAh, LAM l-116, generated in L-selectin deficient mice, specifically caused a
conformational change in L-selectin as measured by an increase in FITC-Iabeled EL-246
staining of L-selectin (Figure 3.1).
This effect was specific to mAh LAM l-116.
Nineteen other anti-L-selectin mAbs did not induce the EL-246 epitope, nor did other
antibodies against cell surface markers, such as the CD 18 integrals and CD44 (Figure
3.2A and data not shown). Also, LAMl-116 induction of the EL-246 epitope was
specific to the EL-246 mAh. Other mAbs against L-selectin, as well as mAbs directed
against other cell surface markers, were not enhanced by treatment with LAMl-116 (data
not shown and Figure 3.2B). These data relating to the specificity of this interaction are
important since others have shown that L-selectin engagement by some mAbs can
activate cellular processes and cause integrin-dependent adhesion of neutrophils under
flow (Steeber et al., 1997, and Gopalan et ah, 1997).
84
As stated previously, EL-246 recognizes a conserved epitope on both L-selectin
and E-selectin (Jutila et ah, 1992).
Past mapping studies using L-selectin/P-selectin
chimeras showed that the EL-246 epitope requires the SCR domains, as well as the lectin
domain, for optimal antibody binding (Jutila et ah, 1992). At that time, it was proposed
that the EL-246 epitope was contained within the L-selectin SCR domains. These studies
were extended here and we now show that none of the domains of L-selectin can, by
themselves, confer the EL-246 epitope. Staining of a number of recombinant selectins
containing different domains of L- E-, and P-selectin show that optimal presentation of
the EL-246 epitope in the absence of LAMl-116 requires both the lectin and SCR
domains from E- or L-selectin and a complete cytoplasmic tail (Figure 3.7 and Table 3.2).
Recently, studies have been .done which analyze L-selectin function inside the cell.
Studies done by Pavalko et al., (1995) first described a linkage between the cytoplasmic
tail of L-selectin and cytoskeletal proteins, and this observation was shown to be
functionally important in intact cells by Evans and Colleagues (1999). The latter was the
first report of dynamic cytoskeletal association of L-selectin with the actin cytoskeleton,
presumably by a linkage of a-actinin with the cytoplasmic tail of L-selectin and the actin
cytoskeleton.
Our studies show that the conformation-induced change in L-selectin,
caused by LAMl-116, predisposes the protein to cytoskeletal association, which is
triggered by EL-246 (Figures 3.5 and 3.6). Other mAbs directed against functionally
distinct epitopes failed to induce cytoskeletal association of L-selectin (data not shown).
85
Thus, we have expanded on the data presented by Evans and colleagues (1999) and
demonstrate that a specific conformation of L-selectin predisposes the protein to
cytoskeletal association in the absence of crosslinking (Figures 3.5 and 3.6). We propose
that this conformational change regulates L-selectin such that a high affinity binding
epitope, the EL-246 epitope, is increased in expression and when this epitope is engaged,
dynamic cytoskeletal association takes place.
This data parallels other reports of
conformation playing a role in L-selectin function. Studies on L-selectin shedding have
shown that the cleavage site recognized by the membrane-bound metalloprotease has
relaxed sequence specificity suggesting that the conformation of L-selectin is the most
important factor in protease recognition and this conformation may be regulated by the
presence of calmodulin binding to the cytoplasmic tail (Chen et al., 1995, Migaki et al.,
1995, and Kahn et al., 1998).
As stated earlier, others have shown that L-selectin can act as a signal transduction
molecule (Po et al., 1995, Waddell et al., 1995, Crockett-Torabi et al., 1995, Simon et al.,
1995, Sikorski et al., 1996, Gopalan et al., 1997, and Steeber et al., 1997). However, a
signaling event through L-selectin is not responsible for the increased binding of EL-246
to L-selectin. The event occurs at 4°C and in the presence of sodium azide and tyrosine
kinase inhibitors (Table 3.1). Furthermore, LAMl-116 induces the EL-246 epitope on Lselectin in detergent lysates. This latter finding is important, since EL-246 does not
recognize shed, soluble L-selectin very well and is, generally, a poor immunoprecipitating
86
antibody (M.A. Jutila, unpublished observation). A previous report by Schleitfenbaum
and colleagues (1992) has shown that soluble L-selectin is conformationally distinct from
cell surface L-selectin which may be the reason for poor L-selectin immunoprecipitation
by EL-246, although EL-246 binding was not tested in their study.
LAMl-116 likely
alters this distinct conformation of soluble L-selectin. These observations clearly show
that the cell is not needed for the enhancement of EL-246 epitope and a cellular signal is
not required for increased presentation of the EL-246 epitope. Thus, if our hypothesis is
correct, overt cellular signaling is not needed for mcreased functional activity of L-selectin.
The cytoplasmic tail of L-selectin is important for adhesive events including
leukocyte rolling, receptor avidity, and cytoskeletal association of L-selectin (Kansas et
al, 1993, and Evans et al., 1999). Thus, we tested whether or not the expression of
specific epitopes on L-selectin are regulated by the cytoplasmic tail. In comparison to
wild type L-selectin transfectant staining, the EL-246 staining of LACyto L-selectin was
reduced to minimal levels (Figure 3.7A). In contrast, staining of the LAMl-116 epitope
-
on the LACyto transfectants remained high (Figure 3.7B). However, in the presence of
LAM l-116, the EL-246 epitope can be enhanced on the LACyto transfectants (Figure
3.8) suggesting that an intact cytoplasmic tail is not required for the conformational
change induced in L-selectin and the subsequent increase in the El-246 epitope expression.
Based on our data, we propose that initial engagement with ligand induces a
conformational change in L-selectin, leading to exposure of a high avidity binding site (EL-
87
246 epitope) for the original ligand, or possibly a second ligand, to bind and induce
stronger tethers and slower rolling. In effect, our data also suggests that LAM l-116
binding to L-selectin may make it more “E-selectin-like.” E-selectin is an inducible
member of the selectin family of adhesion molecules and is expressed on the cell surface at
its highest levels 4-6 hours after an activation signal, such as TNF-cc. It is synthesized de
novo after activation and is expressed in a functional form that allows immediate capture
of leukocytes from the blood stream.
After the primary activation signal on the
endothelium, no further activation is needed to mediate E-selectin capture of leukocytes.
E-selectin mediates a more avid interaction than L-selectin, since leukocyte rolling is
slower on the former in assays done under similar shear forces (Alon et al, 1997). In
vivo, L- and E-selectin can recognize the same naturally occurring ligands on HEV
(Mebius and Watson, 1993, and Tu et al., 1999).
Past studies in our lab have
demonstrated that moving E-selectin-expressing cells to 37°C does not decrease EL-246
staining.
In addition, EL-246 preferentially binds to E-selectin versus L-selectin in
competitive binding assays (Bargatze et al., 1994b). If this model is correct, it could
explain why EL-246 if effective as a therapeutic agent during ischemia and reperfusion
injury, as well as other shear dependent events (Ma et al., 1993, Steinberg et al., 1994,
Pizcueta and Luscinskas, 1994, Ramamoorthy et al., 1996, Seekamp et al., 1997, and
Stotland and Kerrigan, 1997).
Presently, We are not able to functionally test the
hypothesis that LAMl-116 induces higher ligand binding avidity, because both mAbs
88
(LAM1-116. and EL-246) and native ligand, block the function of L-selectin. However,
studies are under way comparing binding affinities of EL-246 for E-selectin and LAM l116-bound L-selectin.
Here, we report that a conformational change in L-selectin induced by an artificial
ligand may play an important regulatory role in the molecular interaction of L-selectin
with its ligands,, and predisposes the protein to cytoskeletal association upon engagement
of a second functionally important epitope.
The functional importance of the
conformational change described is currently under investigation. Our report provides
additional insight into the structural features of L-selectin and may lead to a better
understanding of L-selectin/ligand interactions.
89
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95
CHAPTER 4
COMPARISON OF L-SELECTIN CYTOSKELETAL ASSOCIATION TO THAT OF
OTHER HUMAN AND BOVINE LYMPHOCYTE SURFACE ANTIGENS
Introduction
Lymphocyte trafficking and recirculation is essential for maintaining a healthy
immune system.
Various adhesion molecules on the leukocyte surface, including L-
selectin, are involved in trafficking and mediate interactions with the vascular endothelium
as well as with other immobilized leukocytes (Butcher, 1999, Bargatze et al., 1994, and
Jutila and Kurk, 1996).
L-selectin is constitutively expressed on leukocytes and is
involved in tethering and rolling as it engages appropriate carbohydrate ligands expressed
on the endothelium or other leukocytes. The interactions of L-selectin and its ligands
mediate specific trafficking events in vivo. For example, L-selectin binds to the peripheral
node addressin, a group of glycoproteins collectively termed PNAd, and this interaction
controls lymphocyte trafficking into peripheral lymph nodes (Gallatin et al., 1983,
Streeter et al., 1988, Lasky et al., 1992, Berg et al., 1991, Hemmerich et al., 1994, and
Sassetti et al., 1998, Kansas, 1996, von Andrian et al., 1996, and Butcher, 1999). When
PNAd is expressed on the mucosal addressin, MAdCAM-1, L-selectin can also mediate
lymphocyte traffic into intestinal sites (Berg et al., 1993, and Butcher, 1999). Leukocyteleukocyte rolling, controlled by an L-selectin/P-selectin glycoprotein ligand -I interaction,
also plays a role in trafficking by augmenting endothelial capture and rolling of other
96
leukocytes (Walcheck et al., 1996). In addition, cells that do not express L-selectin do not
home efficiently to sites of inflammation (Jutila et ah, 1989).
Recently, it has been shown that L-selectin dynamically associates with the actin
cytoskeleton by mAh crosslinking, hyperthermic treatment, or ligand binding (Evans et
al., 1999). We have demonstrated that upon induction of a conformational change in Lselectin and subsequent engagement of a conserved epitope on L-selectin, cytoskeletal
association takes place in the absence of antibody crosslinking (Chapter 3). Presumably,
L-selectin associates with the actin cytoskeleton by linkage of a-actinin to the
cytoplasmic tail of L-selectin and the actin cytoskeleton (Pavalko et al., 1995 and Evans
et al., 1999).
Previous work has demonstrated that the cytoplasmic tail of L-selectin is
important to L-selectin function as mutants lacking the 11 terminal amino acids of the
cytoplasmic tail do not exhibit rolling interactions in vitro and in vivo (Kansas et al.,
1993). These same cells are also incapable of L-selectin cytoskeletal linkage under a
variety of conditions (Evans et al., 1999).
Cytoskeletal association of adhesion molecules has .been investigated for some
time. Most studies have focused on integral function as it relates to the cytoskeleton
controlling the avidity of (31 and (32 integrals in leukocytes (Shaw et al., 1990, Pavalko
and LaRoche, 1993, Otey et al., 1993, Pardi et al., 1992, Pavalko and Otey, 1994, and
Sampath et al., 1998).
These reports suggest that integrals associate with detergent
resistant membranes (DRMs) by direct binding of the integral cytoplasmic tail to the
97
actin cytoskeleton. A variety of other proteins have been studied as well, including CD2,
CD3, CD4, CD5, CDS, CD9, CD28, CD44, CD45, and class I MHC (Geppert and
Lipsky, 1991 Gur et ah, 1997, and Yashiro-Ohtani et ah, 2000). Depending upon the
treatment of specific proteins by either primary antibody alone or by antibody
crosslinking, these proteins can be induced to associate with the cytoskeleton.
In
addition, numerous signaling molecules are thought to constitutively associate with the
cytoskeleton through GPI-anchored interactions that form what some refer to as “lipid
rafts” and play a role in T cell activation (Brown and Rose, 1992, Moran and Miceli,
1998, and Penninger and Crabtree, 1999). However, the term lipid rafts has since been
broadened to other interactions such as the TCR and BCR (Janes et ah, 1999, and Cheng
et ah, 1999).
Further studies have shown that supramolecular activation clusters
(SMACs) exist as organized contact sites at the physical interface between T cells and
antigen presenting cells and the proteins involved at such sites are linked to the
cytoskeleton (Monks et ah, 1997, Shaw and Dustin, 1997, and Monks et ah, 1998).
Recent studies have also elucidated the importance of the Ras superfamily of small
guanosine triphosphates
(GTPases), especially the Rho family, and the actin
cytoskeleton, in immune cell function (reviewed in Hall, 1998, and Magee and Marshall,
1999). Indeed, Rho family deficiencies, in which cytoskeletal association is impaired,
result in abnormalities in neutrophil function and host defense, decreased integrinmediated cell adhesion in a lymphoid cell line, decreased cell motility in neutrophils and
98
growth factor-induced chemotaxis in neutrophils and macrophages (Roberts et al., 1999,
Laudanna et al., 1996, Stasia et al., 1991, Allen et al., 1997, and Allen et al., 1998).
Cell membranes are composed of a variety of compounds, including saturated
fatty acids, and their alteration can disrupt membrane linkage to the cytoskeleton. For
example, polyunsaturated fatty acids (PUFAs) can replace the saturated fatty acids in the
cell membrane when taken through diet or incubated directly with cultured cells (Li and
Steiner, 1991, Yosefy et al., 1996, Khalfoun et al., 1997, and Stulnig et al., 1998). PUFA
treatment of immune cells has a wide range of effects that are both immunoregulatory and
anti-inflammatory (Calder, 1998). The mechanism of PUFA action is not clear but is
hypothesized to act via effects on cell adheion molecules, the cytoskeleton, PKC,
eicosanoid receptors, prostaglandin synthases, and/or lipoxygenases (Chen et al., 1992,
and Johanning and Lin, 1995). However, a recent report demonstrates that enrichment of
the T cell membrane with PUFAs disrupts the ability of T cells to signal via the TCR by
disrupting the cytoskeletal association of Lck and Fyn kinases with the cytoskeleton
(Stulnig et al., 1998).
To further elucidate the nature of L-selectin cytoskeletal association, we have
examined a variety of cell surface proteins on human and bovine lymphocytes and
compared their patterns of association to that of L-selectin.
Our data provides a
comprehensive analysis of the lymphocyte cytoskeleton at the ultrastructural level and
describes three cytoskeletal linkage patterns of various proteins in relation to L-selectin.
99
We also analyze the impact of PUFA treatment on the cytoskeletal association of Lselectin. ■
Materials and Methods
Animals
One-week-to-three-month-old Holstein calves, housed in the Montana State
University large animal facility, and healthy human donors were used as sources of
peripheral blood.
Monoclonal Antibodies
The following mAbs were used. Anti-L-selectin included DREG 55, DREG 56,
DREG 200 (Kishimoto et al., 1990), LAMl-116 (Steeber et ah, 1997), GD 4.22 (Jutila
and Kurk, 1996), and EL-246 (Jutila et al., 1992). Anti-human a [3 and y§ T cell receptor
included WT-31 andy/81, respectively (Becton Dickenson, Mountain View, CA). Antibovine y5 T cell receptor included GD 3.8, GD 3.1, and GD 197 (Wilson et ah, 1999), and
anti-WCI included IL-A29 (American Tissue Type Culture Collection, Manassas, VA)
and GD 6.22a (deBoer and Jutila, unpublished). Anti-CD2 included CC42 for bovine
studies (Naessens, et al., 1997) and Tl I-Fluoroscein Isothiocyanate (FITC) on human
cells (Coulter Immunology, Hialeah, FL). Anti-CD3 included M M lA on bovine cells
(VMRD, Pullman, WA) and CD3-FITC for human studies (Becton Dickenson). AntiCD4 included CC30 on bovine cells (Naessens et ah, 1997) and LeU-3-FITC on human
100
cells (Becton Dickenson), and anti-CD5 included CC17 on bovine cells (Naessens et al,
1997). Anti-CDS included CC58 on bovine cells (Naessens et al., 1997) and Leu-2aPhycoerythrin (PE) on human cells (Becton Dickenson). Anti-CDlS included IB4 and
MHM-23 for bovine studies (ATCC #10164, and Dako, Carpinteria, CA, respectively)
and MHM-23 and RDI 68-5A5 for human studies (Dako and Research Diagnostics Inc.,
Flanders NI, respectively) and anti-CD44 included Hermes-3 (Picker et al., 1989). AntiCD45 RA included GS5A on bovine cells (VMRD) and Leu-16 on human cells (Becton
Dickenson). Anti-CD45 RO included GD 6.10a on bovine cells (Walcheck, de Boer and
Jutila, unpublished) and CD45 RO for human studies (Becton Dickenson). Anti-B-cell
included CD 19 on human cells (Becton Dickenson), and anti-E-selectin ligand(s) included
P11.4 chimera/human IgG (Tsang, et al., 1995, and Jones et al., 1997). Anti-GD 3.5, an
unknown antigen on bovine y5 T cells, included IhAb GD 3.5 (Jones et al., 1996), and
B N l-80, an unknown antigen on bovine monocytes and a small fraction of neutrophils,
included B N l-80 (Soltys et al., 1999).
FITC- or PE-conjugated goat anti-mouse
secondary antibody was added to samples for visualization by flow cytometry where
appropriate (Jackson ImmunoResearch, West Grove, PA). For visualization of the Pl 1.4
chimera, R-PErconjugated F(ab’)2 goat anti-human IgG, Fcy specific, was added
(Jackson).
101
Cell Preparations
Peripheral blood was collected by venipuncture into citrate or heparin
anticoagulant tubes as described previously (Jones et al, 1997). Whole blood was diluted
1:1 with Hanks' balanced salt solution (HESS, Gibco, BRL, Grand Island, NY),
underlayed with Histopaque 1077 (Sigma, St. Louis, MO) and centrifuged at 700 g for 30
minutes. The huffy coat containing peripheral blood mononuclear cells was collected,
washed with HESS, and if red blood cells were present, they were lysed in a hypotonic
solution for 10 seconds followed by rapid dilution in HESS and centrifuged. In some
cases, monocytes were depleted from the sample by incubation on plastic in Dulbecco’s
Modified Eagle Medium (DMEM, Sigma) containing 0.7% bovine serum albumin (ESA,
Sigma) for 30 mins at 37°C.
Purified lymphocytes were collected, centrifuged, and
suspended in either HESS or phosphate buffered saline (PBS, Sigma).
Scanning and Transmission Electron Microscopy
Bovine yS T cells were collected and prepared for scanning electron microscopy,
as described (Leid et al., 1998).
Briefly, y§ T cells were treated with Nonidet-40
detergent lysis buffer (150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM Hepes, 2%
goat serum, and 0.5% NP-40) (cytoskeletal buffer) as described (Nebe et al., 1997) for 15
min at room temperature (RT), or not, washed in PBS containing 0.7% horse serum, and
adhered to 12 mm glass cover slips (Ted Pella, Redding CA) coated with CellTak® (100
pg/mL, Collaborative Biomedical Products).
Adherent cells were fixed in 2.5% (v/v)
102
glutaraldehyde in Millonig’s phosphate buffer overnight, dehydrated, critical point dried,
sputter
coated
with
gold-palladium,
and
examined with
a
JEOL
I OOCX
scanning/transmission electron microscope (Speer et ah, 1979).
Transmission electron microscopy (TEM) studies were done as described (Leid et
ah, 1998). Briefly, bovine lymphocytes were isolated as above, treated with cytoskeletal
buffer for 15 min at RT, or not, washed, and fixed in modified Karnovsky’s fixative
overnight.
Samples were partially dehydrated in ethanol, prestained with 1% (w/v)
phosphotungstic acid (Ted Pella) and 1% (w/v) uranyl acetate (Ted Pella) in 70% ethanol,
completely dehydrated and embedded in Spurr’s (Ted Pella) medium. Ultrathin sections
were stained with lead citrate and examined by TEM.
Flow Cytometric Analysis of
Non-detergent-treated Cells
Flow cytometry was performed as previously described (Leid et ah, 1998).
Briefly, lymphocytes were collected, suspended at IxlO7 cells/mL in HBSS, arid 100 (iL
aliquots transferred into FACS tubes (Fisher Scientific Co., Pittsburgh, PA) for staining.
The mAbs described above were added to the cells, incubated on ice for 30 min, washed in
PBS containing horse serum and sodium azide (FACS buffer), and, if appropriate,
fluorochrome-labeled second stage was added and the cells incubated for an additional 30
min on ice.
The cells were washed a final time, resuspended in FACS buffer, and
analyzed on either a FACScan or FASCaliber (Becton Dickenson)..
Second stage
103
fluorochrome alone, was added as a control for background staining. For each sample,
10,000-20,000 events were collected and representative histograms are shown.
Flow Cytometric Analysis
of Detergent-Treated Cells
Cells membrane protein linkage to the cytoskeleton was assayed by flow
cytometry as described with some exceptions (Geppert and Lipsky, 1991 and Nebe et al.,
1997). In order to more thoroughly study protein linkage to the cytoskeleton, three
separate detergent treatments were developed and we have classified each type of
association as either constitutive, inductive, or mAh crosslink-induced.
Constitutive:
lymphocytes were treated first with 100 pL of cytoskeletal buffer for 15 min at RT,
washed with FACS buffer, and primary mAb added and incubated on ice for 30 min.
These samples were then washed again and, if appropriate, second stage FITC or PEconjugated goat anti-mouse antibodies added, incubated for 30 min on ice, washed a final
time, and analyzed by flow cytometry. Staining only occurred for those proteins linked
to the cytoskeleton prior to detergent treatment. Inductive: lymphocytes were treated
with primary mAb for 15 min at 37°C, washed, and 100 (iL of cytoskeletal buffer added
to each sample for 15 min at RT. Samples were washed, second stage fluorochromelabeled antibodies added if necessary for 30 min on ice, washed a final time, and analyzed
by flow cytometry.
This treatment procedure allowed for visualization of mAb or
chimera/Ig induced association to the cytoskeleton in the absence of secondary mAb-
104
induced crosslmking.
MAb Crosslink-induced:
lymphocytes were treated with
primary mAh for 15 min at 37°C, washed, and either PE- or FITC-Iabeled second stage
antibodies added to the appropriate samples for 30 min on ice to induce crosslinking. In
the event that directly Guorochrome-Iabeled antibody was utilized in this treatment
procedure, unlabeled goat anti-mouse IgG was added as a crosslinking agent, as described
(Geppert and Lipsky, 1991).
The cells were then washed, treated with 100 JiL of
cytoskeletal buffer for 15 min at RT, washed again, and analyzed by Gow cytometry.
Mock cytoskeletal buffer without NP-40 was used as a control for all procedures and did
not affect protein expression (data not shown). Second stage FITC- or PE-labeled goat
anti-mouse and anti-human antibody alone was added as a control for background staining
in all three treatments. Staining was measured by gating on the detergent insoluble
cytoskeletal fraction (see Figure 4.1) by forward and side scatter. The gated population
was confirmed to be the cytoskeleton by phalloidin-FITC (Sigma), which speciGcally
stains the detergent insoluble cytoskeleton (Nebe et al, 1997).
For each experiment,
10,000-20,000 events were collected.
SDS-PAGE Analysis of Detergent-soluble
and Detergent-insoluble Membrane Fractions
Human and bovine lymphocytes were surfaced labeled with biotin, as described
(Jones et al., 1997), and low dose detergent soluble and insoluble membrane fractions
prepared according to the following procedure.
Cell preparations were treated with
105
cytoskeletal buffer containing 0.5% NP-40 for 15 min at RT, centrifuged for 5 min at
200 g, and the supernatant fluid collected. This aliquot was labeled the detergent-soluble
membrane fraction. The detergent-insoluble pellet was further treated with 2% NP-40
buffer for I hr on ice with constant vortexing, centrifuged at 200g, and the supernatant
fluid collected. This aliquot was labeled the detergent-insoluble membrane fraction. Each
preparation was then incubated with mouse serum and protein G beads (Boehringer
Mannheim, Germany) overnight at 4°C under constant rotation to reduce non-specific
immunoprecipitation as described (Jones et al, 1996). The samples were centrifuged, the
supernatant fluid collected, and the beads discarded. Samples were aliquoted into 120 JiL
fractions and 10 Jig of mAb added at RT for I hour, and proteins immunoprecipitated
overnight with protein G at 4°C under constant rotation. The beads were washed 3X
with wash buffer, mixed with non-reducing buffer, boiled for 3 min, centrifuged, and
loaded directly onto an 8% polyacrylamide gel.
Gels were electrophoresed and the
proteins were transferred to PVDF (Bio-Rad, Hercules, CA) overnight at 4°C. Proteins
were
visualized
using
a
streptavadin
horseradish
peroxidase
(Amersham,
Buckinghamshire, England) reaction and ECL (Amersham) detection system and
developed on X-OMAT (Kodak) film.
106
PUFA Enrichment of Bovine T Cells
Fatty acid composition of T cell membranes was modified as described (Stulnig et
al., 1998). Briefly, bovine lymphocytes were incubated with 25-50 pM of PUFAs
(Cayman Chemical Co., MI) for 2 days in DMEM containing penicillin/streptomycin (50
U/mL and 50 fig/mL, respectively, Gibco BRL, Gaithersburg, MD), 2 mM glutamine
(Gibco BRL), 0.4% (w/v) BSA (fraction V, containing less than 3 pM total fatty acids,
Sigma), I mg/L transferrin, and 8.1 mg/L a-monothioglycerol at 37°C in humidified
atmosphere in the presence of 10% CQ2. Control cells were incubated in DMEM
containing 10% BSA, and glutamine, and antibiotics as above. Cell viability after culture
was >90% as determined by trypan blue exclusion. PUFA-cultured and control cultured
cells were prepared for FACS and analyzed as described above.
/
Results
Detergent Treatment of Human and Bovine
Lymphocytes Generated a Homogeneous
Population of Actin Cvtoskeleton Particles
-
Previous reports have demonstrated that protein association with the actin
cytoskeleton can be studied by flow cytometry (Nebe et al., 1997, Gur, et al., 1997, and
Capote and Maccioni, 1998). One characteristic of the actin cytoskeletal network is that
phalloidin, a phallotoxin, specifically stains F-actin and can be used to positively identify
107
A
B
phall 14 d ec-99.006
9
________ phall 14dec-99.01Q
r.' =
200
400
600
800
1000
FSCH
FLI-H
Log 10 Fluorescence
C
►
D
phall 14-deg-99.00l
200
400
600
FSOH
800
1000
Log 10 Fluorescence
Figure 4.1.
Phalloidin-FITC specifically stains detergent-treated cytoskeletal
particles versus intact, non-detergent treated cells. Panel A, scatter profile of
cytoskeletal particles showing uniform, small size. Panel B, staining of the particles
in Panel A with phalloidin-FITC. Panel C, scatter profile of non-detergent-treated
bovine peripheral blood mononuclear cells (PBMCs) with a selective gate around the
lymphocyte population. Panel D, staining of the gated population of PBMCs with
phalloidin-FITC. Note the lack of fluorescence of non-detergent treated cells by
phalloidin-FITC staining. Data are representative of 3 separate experiments.
108
cytoskeletal particles by flow cytometry. Low dose detergent treatment of human or
bovine lymphocytes generated a homogeneous population of cytoskeletal particles
relatively small in size which were stained brightly by phalloidin-FITC (Figure 4.1, A and
B). In contrast, non-detergent-treated cells maintained their normal size and were not
stained by phalloidin-FITC (Figure 4.1, C and D).
Electron Microscopy Studies
on the Actin Cvtoskeleton
In an effort to further understand the nature of the lymphocyte cytoskeleton and
the particles analyzed in Figure 4.1, scanning and transmission electron microscopy
techniques were employed. By scanning electron microscopy (SEM), the surface of the
actin cytoskeleton was smooth, with little or no surface topology compared to that of an
intact, non-detergent-treated cell (Figure 4.2A, intact bovine yS T cell, and B, bovine y5 T
cell cytoskeleton). Note the numerous microvilli on the non-detergent-treated, intact y§ T
cell versus the detergent-treated y5 T cell, and the smaller size of the detergent-treated y8
T cell compared to a non-detergent-treated yS T cell (3 |im versus 4-8 pm for bovine and
human lymphocytes, Leid et ah, 1998, and Picker et ah, 1991).
Transmission electron microscopy was utilized to investigate the internal makeup
of the cytoskeletal sphere seen in Figure 4.2. Strikingly, detergent treatment of bovine y5
T cells left highly uniform cellular ‘ghosts’ that consisted primarily of the cell nucleus,
109
Figure 4.2. Scanning electron micrographs o f an untreated and a detergent-treated bovine
y d T cell. Panel A , untreated y d T cell show ing numerous m icrovilli (M v). Original
m agnification, X 15,000. Panel B, cellular remnant o f a low dose detergent-treated bovine
y5 T cell show ing a sm ooth surface with little or no surface topography. Original
m agnification, X 20.000.
no
Figure 4.3. Transm ission electron micrographs o f untreated and detergent-treated bovine
lym phocytes. Panel A , untreated y§ T cell show ing numerous m icrovilli (M v), euchromatin
(Eu), G olgi com plex (G o), heterochromatin (H e), mitochondria (M i), nuclear envelope (N e),
and plasm alem m a (PI). Original m agnification. X 25.000. Panel B, bovine lym phocyte treated
with low dose detergent show ing incom plete plasm alem m a (PI), indistinct nuclear envelope
(N e), and lack o f cytoplasm ic organelles; euchromatin (Eu); heterochromatin (He); and
nucleolus (N o). Original m agnification, X 48,000.
Ill
and had little cytoplasm and few or no cellular organelles (Figure 4.3B). The TEM of the
detergent-treated lymphocytes showed that the smooth surface seen by SEM was
actually the plasmalemma of the cell. By TEM5 the plasmalemma was found to have
collapsed upon and surrounded the nucleus. This was in contrast to a non-detergenttreated bovine lymphocyte, which had an intact plasmalemma, nuclear membrane,
cytoplasm and typical cytoplasmic organelles (Figure 4.3A).
Flow Cytometric Analysis of Human
and Bovine Lymphocyte Cytoskeletal
Association of Cell Surface Proteins
L-selectin has previously been shown to dynamically associate with the actin
cytoskeleton (Evans et al., 1999). Therefore, we wanted to compare the association of
numerous proteins on the surface of human and bovine lymphocytes in an effort to relate
patterns of association to their known functions. Human lymphocytes were treated as
described in Materials and Methods and analyzed by flow cytometry. As previously
described (Geppert and Lipsky, 1991), CD44 was found to constitutively associate with
the actin cytoskeleton following detergent treatment, and exhibited bright staining
patterns under all three treatment conditions (Figure 4.4A).
CD3, a co-stimulatory
molecule, could be induced to associate with the cytoskeleton by primary mAb binding
but was absent if lymphocytes were treated with detergent prior to mAb binding alone
(Figure 4.4B). Similar results were seen with a FITC conjugated anti-CD3 mAb (data not
shown). In contrast, the a (3 TCR on human lymphocytes was only linked after
112
Hermes (CD44)
C ytoskeletal
Hermes (CD44)
C ontrol
A
FL IH
CD3 (CD3)
F L I-H
CD3 (CD3)
B
F L l-H
W T -3 1 (<xp TcR )
FLl H
W T 31 ( o 3 TcR)
C
Cell
Number
FLl H
FLI H
Log IO Fluorescence
Figure 4.4. Flow cytometric analysis of the cytoskeletal association of human lymphoycte
surface proteins. Panel A, CD44 is constitutively associated with the cytoskeleton (thick
line, constitutive*; thin line, inductive*; dotted line, mAh crosslink*). Panel B, CD3 is
induced to associate with the cytoskeleton by mAb binding alone (constitutive*,
inductive*, mAb crosslink*). Panel C, ap TCR links to the cytoskeleton only when
crosslinked by secondary mAb (constitutive*, inductive*, mAb crosslink*). Control stains
of non-detergent-treated cells are shown to the right of each cytoskeletal histogram (thick
line, mAb staining; dotted line, second stage goat anti-mouse F(ab’)2 labeled
fluorochrome. Data are representative of 3 separate experiments.
*See Materials and Methods for details on treatments.
113
crosslmking with a secondary antibody, similar to L-selectin (Figures 4.4C, and Evans et
al., 1999). Control staining of non-detergent-treated protein expression is shown to the
immediate right of each histogram (Figure 4).
WCI, a lineage specific antigen on the surface of bovine yS T cells, was found to
constitutiyely associate with the actin cytoskeleton (Figure 4.5A).
This result was
similar to that seen for CD44 and other proteins examined (Figure 4.4, and data not
shown), although treatment with primary mAh alone, as well as crosslinking, increased
the amount of WCl associated with the cytoskeleton compared to CD44. Another mAh
that recognizes WC1, GD 6.22a, produced similar results (data not shown). Bovine y5
TCR, like human CD3 above, could be induced to associate with the cytoskeleton by
primary mAh binding alone (Figure 4.5B). Directly FITC-conjugated mAh gave similar
results (data not shown). This result was in contrast to the human a (3 TCR seen above.
Three unique bovine anti-y8 TCR mAbs all induced cytoskeletal association, suggesting
the effect was not due to engagement of a specific epitope on the yS TCR (data not
shown and Figure 4.7). Finally, as previously shown, L-selectin did not link to the
cytoskeleton until crosslinked by secondary antibodies (Figure 4.5C). Control staining of
' non-detergent-treated protein expression is shown immediately to the right of each
histogram.
114
I1.-A 29 ( W C l )
C ytoskeletal
A ssociation
c;i> 3 .8 (y5 T c R )
D R E C 5 6 ( I .-s e le c tin )
1C -A 29 ( W C l )
C ontrol
C D 3 .8 (y8 T c R )
D R E G 5 6 < L -selectin )
S 1--------------------- -----------@4
Cell
Number
FL2-H
Log IO Fluorescence
>
Figure 4.5.
Flow cytometric analysis of the cytoskeletal association of bovine
lymphocyte surface proteins. Panel A, WCl is constitutively associated with the
cytoskeleton (thick line, constitutive*; thin line, inductive*; dotted line, crosslink*).
Panel B, y5 TCR is induced to associate with the cytoskeleton by mAb binding alone
(constitutive*, inductive*, mAb crosslink*). Panel C, L-selectin links to the cytoskeleton
only when crosslinked by secondary mAb (constitutive*, inductive*, mAb crosslink*).
Control stains of non-detergent-treated cells are shown to the right of each cytoskeletal
histogram (thick line, mAb staining; dotted line, second stage goat anti-mouse F(ab’)2
labeled fluorochrome. Data are representative of 3 separate experiments.
*See Materials and Methods for details on treatments.
115
SDS-PAGE Analysis of Detergent-soluble
and Detergent-insoluble Membrane Fractions
In order to confirm our flow cytometry results at the protein level, SDS-PAGE
analysis of immunoprecipitated proteins was performed.
Membrane fractions were
prepared as in Materials and Methods and antigens immunoprecipitated with mAbs.
CD44 was found in the detergent-insoluble membrane fraction, parallel to the results seen
with flow cytometric studies (Figure 4.6, lane 5). CD44 was also found in the detergentsoluble fraction, indicating that either some CD44 was not constitutively linked to the
cytoskeleton or that upon mild detergent treatment, some CD44 was lost from the surface
(Figure 4.6, lane 6). As mentioned above, CD 18 was consistently found to be weakly
associated with the detergent-insoluble membrane fraction (Figure 4.6, lane 7). However,
it was readily precipitated from the detergent-soluble fraction (Figure 4.6, lanes 7). In
contrast, L-selectin and E-selectin ligand(s) were not seen in the insoluble fraction, in
support of the flow data (Figure 4.6, lane I versus 2, and 3 versus 4, respectively). The
arrow points to a specific E-selectin ligand band at 130 kD.
SDS-PAGE analysis of bovine lymphocyte surface antigens also confirmed the
flow cytometry data. WCI, a lineage specific y8 T cell marker thought to function in
various capacities as a signal transduction protein, and CD5, an important protein
involved in T and B cell differentiation and function, both were immunoprecipitated in the
detergent-insoluble membrane fraction (Figure 4.7, lanes I and 3, respectively). Similar to
the human studies with CD44, both proteins were also present in the detergent-soluble
116
L-selectin
Pl 1.4
CD44
CD 18
220 kD
97 kD
66 kD
Figure 4.6. SDS-PAGE analysis of human lymphocyte surface proteins in detergentsoluble (S) and detergent-insoluble (IN) membrane fractions. Lanes I and 2, L-selectin;
lanes 3 and 4, Pl 1.4 chimera; lanes 5 and 6, CD44; and lanes 7 and 8, CD18. Single
arrow denotes specific E-selectin ligand immunoprecipitation band -130 kD. Double
arrows denote specific CD 18 immunoprecipitation bands. Note weak presence in the
detergent-insoluble membrane fraction.
Data are representative of 3 separate
experiments.
fraction (Figure 4.7, lanes 2 and 4, respectively). CD4, which was not seen constitutively
associated with the cytoskeleton by FACS, was weakly associated by SDS-PAGE
analysis (Figure 4.7, lane 5). However, most CD4 was seen in the detergent-soluble
membrane fraction (Figure 4.7, lane 6). Bovine CD3 and CD2 were found only in the
detergent-soluble membrane fraction (Figure 4.7, lanes 8 and 10 respectively). As seen in
the human studies above, L-selectin was predominantly found in the detergent-soluble
117
membrane fraction (Figure 4.7, lane 12), though a small amount was precipitated from the
detergent-insoluble membrane fraction (Figure 4.7, lane 11).
Previous reports have
suggested that a small amount of L-selectin may be constitutively associated with the
cytoskeleton (Pavalko et al., 1995, and Evans et al., 1999), thus explaining our
immunoprecipitation results. However, it is clear, as these papers suggest, that most Lselectin is not constitutively associated with the actin-based cytoskeleton (Figure 4.7,
lane 11 versus 12). E-selectin ligand(s) were not precipitated from the detergent-insoluble
membrane fraction in the absence of E-selectin chimera (Figure 7, lanes 13 versus 14).
The arrow denotes a specific band that has been shown previously to be an E-selectin
ligand on bovine lymphocytes (Jones et al., 1997). As mentioned above, the bovine yS
TCR was not constitutively linked to the cytoskeleton, but three distinct bovine anti-y8
TCR mAbs alone could induce the TCR to associate with the cytoskeleton (Figure 4.5).
Accordingly, bovine yS TCR was specifically immunoprecipitated from the detergentsoluble membrane fraction by anti-y8 TCR mAbs GD 197, and GD 3.1 (Figure 4.7, lanes
16 and 18, respectively). Thus, in addition to mAh GD 3.8 (Figure 4.5B, and data not
shown), GD 197 and GD 3.1 also demonstrate that bovine yS TCR is not constitutively
associated with the cytoskeleton.
118
Figure 4.7. SDS-PAGE analysis of bovine lymphocyte surface proteins in detergentinsoluble (IN) and detergent-soluble (S) membrane fractions. Lanes I (IN) and 2 (S), WCI,
lanes 3 and 4, CD5, lanes 5 and 6, CD4, lanes 7 and 8, CD3, lanes 9 and 10, CD2, lanes 11
and 12, L-selectin, lanes 13 and 14, P U .4 chimera, lanes 15 and 16, y5 TCR (mAh GD 197),
and lanes 17 and 18, y6 TCR (mAh GD 3.1). Note the presence of the WCl and CD5
antigens in the insoluble membrane fraction (lanes I and 3, respectively). Also, note the
weak presence of bovine CD4 in the insoluble membrane fraction. Small arrow denotes
specific CD2 band. Medium arrow denotes y5 TCR Large arrow represents PU 4 (Eselectin ligands) bands. The data are from 2 separate gels run on the same day and are
representative of 3 separate experiments.
Characterization of Other Human
and Bovine Surface Proteins
Further characterization of additional human and bovine cell membrane proteins
was undertaken as well, and those results are summarized in Tables 4.1 and 4.2. Based
upon data generated by flow cytometry, all the proteins were grouped into 3 patterns of
cytoskeletal association: constitutive, inductive, and mAb crosslink-induced.
119
Table 4.1. Human Lymphocyte Cytoskeletal Association of Various Proteins
Antigens_________________Constitutive________Inductive__________ Crosslink
CD2
No
No
Yes
CD3
No
Yes
Yes
CD4
Yes
Yes
Yes
CD 8
Yes
Yes
Yes
CD18
Weak
Yes
Yes
CD44
Yes
Yes
Yes
CD45RA
No
Yes
Yes
CD45RO
No
Weak
Yes
Ctp TCR
No
No
Yes
ySTCR
No
■No
No
E-selectin Ligand(s)
No ■
Yes
Yes
L-selectin*
No
No
Yes
*Five different anti-L-selectin mAbs were employed for these studies and all gave similar
results (See Materials and Methods).
All the data in the table were generated from flow cytometric analysis with at least 3
separate experiments for each antigen. In some cases, SDS-PAGE was employed to
confirm the flow cytometry data (data not shown).
PUFA Treatment Specifically Reduced
L-selectin Expression and Inhibits
L-selectin Cytoskeletal Association
PUFAs have previously been shown to decrease the expression level of some
proteins, including E-selectin, as well as disrupt the ability of some proteins, such as Lck
and Fyn, to associate with the cytoskeleton (Hoover et ah, 1981, Stulnig et ah, 1998, and
De Caterina and Libby, 1996). We tested the effects of PUFA treatment on the linkage of
representative surface antigens from each pattern of cytoskeletal association, which were
L-selectin, TCR, and WC1, as presented in Tables 4.1 and 4.2. Enrichment of bovine
lymphocyte cell membranes with Eicosapentaenoic Acid (EPA) reduced the expression
120
Table 4.2. Bovine Lymphocyte Cytoskeletal Association of Various Proteins
Antisens
CD2
CD3
CD4
CD 8
CD 18
CD45RA
CD45RO
WCl (IL-A29)
WCl (GD 6.22a)
GD 3.5
yb TCR (GD 3.8)
ySTCR (GD 3.1)
yb TCR (GD 197)
B N l-80
E-selectin Ligand(s)
L-selectin**
Constitutive
No
No
No*
Yes
■Weak
No
Yes
Yes
Yes
No
No
No
No
Yes
No
No
Inductive
Yes
Yes
Yes
Yes
Yes •
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Crosslink
Yes
Yes
Yes
Yes .
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
*There was a weak constitutive association of CD4 with the detergent-insoluble
membrane fraction by SDS-PAGE gel electrophoresis. However, this result was not seen
on a consistent basis.
**Five different anti-L-selectin mAbs were employed for these studies and all gave
similar results.
All the data in the table were generated from flow cytometric analysis with at least 3
separate experiments for each antigen. In some cases, SDS-PAGE was employed to
confirm the flow cytometry data (data not shown).
level of L-selectin by anti-L-selectin mAb DREG 56 staining compared to control (Figure
4.8A, mode fluorescence intensity average (MFI) of 98 ± 9 S.E.M. vs 187 ± 17 S.E.M.
for 3 separate experiments, p<0.01). Docosapentaenoic Acid (DPA) or Docosahexaneoic
acid (DHA) treatment gave similar results (data not shown). Interestingly, the expression
of a conserved epitope on L-selectin, recognized by mAb EL-246, was dramatically
121
Figure 4.8. Treatment of bovine lymphocytes with EPA reduced the expression of Lselectin but did not alter the expression of y5 TCR or WCI. Panel A, staining of Lselectin by mAb DREG 56 on control cultured (c.c.) cells (thick line) versus EPA-treated
cells (thin line). Panel B, staining of L-selectin by mAb EL-246 on c.c. (thick line)
versus EPA-treated cells (thin line). Panel C, staining of y8 TCR by mAb GD 3.8 on c.c
(thick line) versus EPA-treated cells (thin line). Panel D, staining of WCl by mAb ILA29 on c.c. (thick line) versus EPA-treated cells (thin line). Data are representative of 3
separate experiments.
122
reduced compared to its expression on control cultured cells (Figure 4. SB, MFI of 22 + 8
S.E.M. vs 107 ± 10 S.E.M. for 3 separate experiments, p< 0.01). This result was not due
to the absence of L-selectin as DREG 56 mAh stained EPA treated cells. In contrast to
L-selectin, EPA treatment of bovine lymphocytes did not change the surface expression
of y§ TCR or WCl (Figure 4.8 C and D, respectively).
We have previously, characterized a mAb-induced (LAM1-116) conformational
change in L-selectin that increased the expression of another conserved epitope (EL-246)
and predisposed L-selectin to cytoskeletal association (Chapter 3).
Thus, we tested
whether or not LAMl-116 could restore the loss of the EL-246 epitope expression on
EPA-treated bovine lymphocytes. Enhancement of the EL-246 epitope occurred on Lselectin for both the control cultured and EPA-treated lymphocytes (Figure 4.9A and B,
respectively).
L-selectin on untreated, resting cells can be induced to associate with the
cytoskeleton by the mAb-induced conformational change described above as well as by
antibody crosslinking.
However, L-selectin on EPA-treated lymphocytes did not
associate with the cytoskeleton by treatment with LAMl-116 and EL-246 (Figure
4.1GA), in spite of the fact that LAMl-116 binding to L-selectin on EPA-treated cells did
enhance the expression of the EL-246 epitope. This result was not due to the lack of
surface expression of L-selectin, as detected by DREG 56 staining (Figure 4.1GA). In
contrast, L-selectin on bovine lymphocytes cultured without EPA was able to link to the
123
J i
«3
--
FrL f - I I
B
s if: -
Cell
Number
Vo 1
'
Log IO Fluorescence
"
»=l 1 -M
Figure 4.9. Enhancement of the EL-246 epitope by mAh LAMl-116 occurred on control
cultured as well as EPA-treated cells as measured by EL-246-FITC staining of L-selectin.
Panel A, LAMl-116 treatment of control cultured cells enhanced the expression of the
EL-246 epitope (LAM l-ll 6-treated, thick line, EL-246 alone, thin line). Panel B,
LAMl-116 treatment of EPA cultured cells enhanced the expression of the EL-246
epitope (LAM l - l l 6-treated, thick line, EL-246 alone, thin line). Data are representative
of 3 separate experiments.
cytoskeleton by LAMl-ll6/EL-246 mAh engagement (Figure 4.10A).
L-selectin on
freshly isolated, as well as control cultured, bovine lymphocytes linked to the
cytoskeleton by DREG 56 mAh crosslinking (Figure 4.1OB). However, DREG 56 mAh
crosslinking of L-selectin did not induce cytoskeletal association of L-selectin on EPA-
124
A
B
s J
sU
a S J
Cell
Number
Log 10 Fluorescence
Figure 4.10. EPA-treatment inhibits L-selectin cytoskeletal association. Panel A, LAMl116/EL-246-induced L-selectin cytoskeletal association on control cultured (c.c.) and EPAtreated cells (thick line, c.c. cells, thin line, EPA-treated cells, dashed line, second stage
goat anti-mouse control, and dotted line, DREG 56 staining of EPA-treated cells. Panel B,
DREG 56 mAb crosslink-induced cytoskeletal association (dotted line, freshly isolated
cells, thin line, c.c cells, thick line, EPA-treated cells, dashed line, DREG 56 mAb staining
of L-selectin on EPA-treated cells. Data are representative of 3 separate experiments.
treated cells (Figure 4.1OB). The lack of DREG 56 mAb crosslinking-induced L-selectin
cytoskeletal association was not due to the absence of L-selectin on EPA-treated cells
(Figure I OB). Furthermore, bovine y5 TCR and WCl antigen were associated with the
cytoskeleton on EPA-treated cells suggesting that the cytoskeletal-linkage machinery for
these proteins was still intact (Figure 4.1 IA and B, respectively). Thus, PUFA treatment
appears to have a unique effect on the linkage of only some cell surface proteins.
125
A
Cell
Number
Log 10 Fluorescence
Figure 4.11. Cytoskeletal association of bovine yd TCR and WCl is not altered by EPA
treatment. Panel A, bovine y5 TCR was associated with the cytoskeleton after treatment
with the mAb GD 3.8 (thin line, control cultured, thick line, EPA-treated, and dotted
line, second stage goat anti-mouse control. Panel B, WCI, a lineage specific marker on
bovine yd T cells, associated with the cytoskeleton after treatment with EPA (thin line,
control cultured, thick line, EPA-treated, and dotted line, second stage goat anti-mouse
control). Data are representative of 2 separate experiments.
Discussion
This report expands upon previous knowledge of the lymphocyte cytoskeleton
and provides additional insight into the structure and nature of the cytoskeleton as well as
furthers the knowledge of the relationship between protein function and cytoskeletal
linkage. For example, ultrastructural studies of the lymphocyte cytoskeleton revealed
126
that following detergent treatment, the surface topography of the lymphocyte was
remarkably smooth and lacked microvilli compared to that of an intact yd T cell, which
consisted of numerous microlli (compare Figures 4.2A and B). Furthermore, detergenttreated cells were smaller in size.
Previous measurements of bovine yd T cells
demonstrated a uniform size of approximately 4 pm (Leid et al., 1998), whereas
detergent-treated yd T cells were 3 pm in the studies described here. TEM provided
additional information regarding the nature of the lymphocyte cytoskeleton.
As
previously reported, bovine yd T cells had numerous microvilli, an intact plasmalemma
and nuclear envelope, and various organelles including mitochondria and Golgi complex
(Leid et al., 1998). By TEM, detergent-treated yd T cells consisted of a nucleus, little or
no cytoplasm, and a plasmalemma that had collasped upon the nucleus. The detergenttreated yd T cell lacked microvilli, while the plasmalemma and nuclear envelope remained
partially intact. Thus, the detergent-treated lymphocyte consisted primarily of a nucleus
surrounded by the plasmalemma.
In an attempt to better understand the functional consequences of L-selectin
protein association with the cytoskeleton, its linkage was compared to the linkage of 28.
other human and bovine surface antigens.
From our flow cytometry and gel
electrophoresis studies, we collated the nature of cytoskeletal association into three
distinct patterns; I) constitutive, 2) inductive, and 3) antibody crosslink-induced
association. Human lymphocyte CD44 and bovine lymphocyte WCl antigens, both
127
reported to act as signal transduction molecules, associate with the cytoskeleton prior to
detergent treatment. This result is similar to previous reports that CD44 associates with
lipid rafts and generates cellular activation signals utilizing the signaling machinery of the
plasma membrane microdomains (Ilangumaran et al., 1999). CD44 binds hyaluronate and
mediates the binding of lymphocytes to specialized high endothelial venules (Aruffo et
al, 1990). Antibodies against CD44 induce homotypic cell aggregation as well as augment
binding of human leukocytes to fibronectin (Cao et al., 1995 and Cao et al., 1996-7).
Also, anti-CD44 mAbs or native CD44 ligands activate several signaling pathways that
culminate in cell proliferation, cytokine secretion, chemokine gene expression and
cytolytic effector functions (reviewed in Ilangumaran et al., 1999). Thus, it is clear that
engagement of CD44 can lead directly to activation of lymphocytes (Jackson et al., 1992
and Aruffo et al., 1990).
WCl is a lineage specific protein on bovine yS T cells that can be expressed as 3
different isoforms, depending on specific gene rearrangements that take place (Davis et al.,
1987, Clevers et al., 1990, and Winjgaard et al., 1992). The extracellular domain of the
protein contains 11 homologous regions that have strong identity to the scavenger
receptor cysteine-rich (SRCR) family (Winjgaard, et al., 1994). Scavenger receptors
react with host serum proteins and may participate in host defense against bacterial
infection (Chiang et al., 1995). Recently, WCl has been suggested to control reversible yS
T cell growth arrest in an IL-2 dependent manner (Takamatsu et al., 1997, Kirkham, et al.,
128
1997 and Kirkham et al., 1998). Steroids can also affect the expression of WCl on the
surface of the 78 T cell.
In vivo treatment with dexamethasone causes WCl
downregulation from the cell surface (Burton and Kehrli5 1996). Thus5 it is clear that
WCl has the potential to act directly as a signal transduction molecule without co­
stimulation, although studies are still under way to elucidate more about how this protein
functions.
Other proteins found to be constitutively linked to the cytoskeleton include
human CD4 and CDS, bovine CD4 (to a small extent by SDS-PAGE), CDS, CDS, CD 18
(to a small extent by SDS-PAGE), CD45RO, and a yet undefined antigen on bovine
monocytes recognized by mAb B N l-80 (Tables 4.1 and 4.2). Most of these proteins
share the similar trait of signal transduction molecules, that upon engagement, cause
activation of the lymphocyte. Although there is a difference between molecules such as
CD44 and WCI, which have not yet been shown to require accessory molecule
engagement for lymphocyte activation, and CD4, CDS, and CDS, it is apparent that all
can transduce signals. Thus, our studies of the various proteins above suggest cell surface
signal transduction proteins are constitutively linked to the actin cytoskeleton after
syndissertation and transport, and linkage to the cytoskeleton provides a scaffolding for
requisite protein function. In addition to these proteins, other proteins can transduce
signals as well, but their linkage to the cytoskeleton may be regulated by ligand
engagement.
129
The T cell receptor for antigen is important in recognition of foreign antigens and
an intact cytoskeleton is vital for lymphocyte activation and function (Penninger and
Crabtree, 1999). Upon activation, the TCR undergoes cytoskeletal rearrangement and
associates closely with other important antigen recognition proteins such as CD2, CDS,
and CDS, forming supramolecular activation clusters (SMACs) that aid in directing the
cellular immune response (Monks et al, 1997, Monks et ah, 1998, and Shaw and Dustin,
1997). Therefore, we investigated the cytoskeletal association of some of these proteins
to further investigate the patterns of association as it related to their defined functions.
For example, we have studied CDS, a co-stimulatory molecule that associates with the a (3
and y§ TCR, and the TCR itself.
These lymphocyte cell surface markers are not
constitutively linked to the cytoskeleton but can be linked by mAb engagement alone. It
is possible that some primary mAbs may cause crosslinking alone, however, this seems
unlikely because three different anti-yb TCR mAbs produced similar results.
Recent
work has shown a role for lipid rafts in B cell antigen receptor (BCR) signaling and
antigen targeting and aggregation of lipid rafts has been shown to accompany signaling via
the TCR (Cheng et ah, 1999, and Janes et ah, 1999). Both of these results were generated
by crosslinking, however, and our data suggests that the bovine y8 TCR does not need to
be crosslinked to associate with the cytoskeleton, as opposed to the human a|3 TCR, or
the BCR. This may be due to different reagents used in the studies that may bind more
functionally important epitopes on the yS TCR versus the a (3 TCR. However, in many
130
cases, different mAbs directed against the same surface antigen were used and gave similar
results suggesting that this was likely not the case. A more reasonable explanation is the
fundamental difference of antigen recognition between an a(3 T cell and a 76 T cell.
Numerous studies have demonstrated that y8 T cells can recognize antigens without the
aid of classical accessory molecules, such as MHC. Thus, engagement of the y§ TCR
with mAbs may mimic ligand engagement in vitro and in vivo. Future studies will help
define the uniqueness of primary antibody cytoskeletal induction of the y8 TCR as well
as further characterize the features of yS TCR association with lipid rafts, and y§ T cell
function in general.
CD 19 and CD45RA in humans, and CD2 and GD 3.5, a bovine y8 T cell specific
antigen, also can be induced to link to the cytoskeleton (Tables 4.1 and 4.2).
The
distinction between the bovine yS T cell specific antigens WCl and GD 3.5 may provide
clues to the function of GD 3.5 antigen, and is important as their respective cytoskeletal
association patterns further distinguish these molecules of similar molecular weight. Also,
E-selectin ligand(s) were induced to associate with the cytoskeleton by engagement with
recombinant E-selectin/Ig chimera (Tables 4.1 and 4.2).
Some differences were seen
between the cytoskeletal association of human and bovine CD2 and CD45RA.
For
example, CD2 could be induced to associate with the cytoskeleton by mAb engagement
alone on bovine lymphocytes whereas it had to be crosslinked on human lymphocytes.
Also, bovine CD45RA was not constitutively linked to the cytoskeleton, in contrast to
131
CD45RA in humans. These differences may be a result of the developmental stage of the
lymphocytes from the animal tested.
Young calves are immunologically immature,
compared to adult human donors whose circulating lymphocytes are functionally mature.
Therefore some of the proteins on human lymphocytes may have reached a
developmentally mature stage which is characteristic of CD45RA constitutive
cytoskeletal linkage. The CD2 result may also be explained in a similar manner.
A previous report demonstrated that L-selectin dynamically associates with the
cytoskeleton upon mAh crosslinking, hyperthermic treatment, and Glycam-I binding
(Evans et ah, 1999). Here, as with Evans and colleagues, we also show that L-selectin is
linked to the cytoskeleton by mAb crosslinking. We have previously characterized a
conformational change in L-selectin, caused by an anti-L-selectin mAb, that predisposes
L-selectin to cytoskeletal association when a functionally distinct epitope is engaged by a
separate anti-L-selectin mAb (Chapter 3).
We believe that induction through a
conformational change may more accurately represent a physiological process similar to
native ligand binding. Indeed, as mentioned above, Glycam-1, a defined ligand for Lselectin, can induce L-selectin association directly (Evans et al., 1999). Therefore, ligand
binding, which can be mimicked by mAb binding, may cause a conformational change in
L-selectin, exposing a separate conserved epitope to a greater extent, and predisposing Lselectin to associate with the cytoskeleton. In turn, cytoskeletal association allows for
tighter tethering and rolling interactions, slowing the lymphocyte rolling velocity, and
allowing the L-selectin captured cell to more closely sample the microenvironment.
132
Hence, without the ability to associate with the cytoskeleton, strong L-selectin-mediated
leukocyte capture can not take place. However, this possibility cannot be effectively
studied at this time as both mAh and ligand binding block the function of L-selectin under
flow. Nevertheless, L-selectin mutants that lack the 11 terminal amino acids of the
cytoplasmic tail, do not associate with the cytoskeleton, and do not establish efficient
rolling interactions in vivo and in vitro, suggesting a causal role between L-selectin
cytoskeletal linkage and function (Kansas et al., 1993, Evans et ah, 1999).
Similar to the genetic manipulation of the cytoplasmic tail of L-selectin,
polyunsaturated fatty acids also inhibit the ability of L-selectin to associate with the
cytoskeleton, while two other patterns of cytoskeletal association, constitutive and
inductive, are not altered.
Importantly, PUFA treatment also inhibited cytoskeletal
association of tandem mAb treatment which may mimic direct ligand binding.
This
suggests that the machinery that mediates L-selectin cytoskeletal association is different
than the other two types of proteins.
Importantly though, PUFAs disrupt the cell
membrane while keeping L-selectin intact and this system may provide a more
physiologically relevant tool in understanding L-selectin cytoskeletal association in vivo.
Indeed, studies have shown that diets supplemented with PUFAs have anti-inflammatory
effects which may be explained by a decrease in L-selectin, as well as E-selectin ligandmediated selective recruitment of lymphocytes to sites of inflammation (Li and Steiner,
1991, and Calder, 1998). These issues are currently under investigation. The mechanism
of PUFA action on L-selectin expression and cytoskeletal association remains unclear but
133
is similar to other published reports which demonstrate a reduction in adhesion molecule
expression upon PUFA treatment as well as a reduction in protein association with the
cytoskeleton (Sethi et al., 1996, Stulnig et ah, 1998, and Hughes and Finder, 2000). For
L-selectin, PUFA treatment most likely blocks the interaction of a-actinin binding to
actin by altering the spatial arrangement of these two proteins in the cell. This could
explain the differences in the effects seen with varying degrees of unsaturation of fatty
acids as eladic acid, a PUFA with a single degree of unsaturation, did not exhibit as
dramatic an effect than other PUFAs with higher degrees of unsaturation (data not
shown).
We have investigated a variety of surface proteins on human and bovine
lymphocytes in an effort to provide more clues as to the function of L-selectin
cytoskeletal association. Among other things, we have further described the nature of the
lymphocyte cytoskeleton and compared its appearance and internal makeup to that of
intact, non-detergent-treated cells.
Also, these data suggest that adhesion molecules
involved in rolling, i.e. L-selectin and E-selectin ligand(s), are induced to associate with the
actin-based cytoskeleton upon mAh or ligand engagement and this may play a role in their
ability to induce strong tethers. As the mechanisms underlying cytoskeletal association
are further elucidated, more patterns of protein association with the actin-based
cytoskeletal network, as well as the consequences of disrupting this linkage, will emerge,
and provide additional information linking the cytoskeleton to overall cell function.
134'
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CHAPTER 5
SUMMARY OF WORK DESCRIBED IN CHAPTERS 2-4 AND OTHER
COLLABORATIVE EFFORTS NOT PRESENTED IN THIS DISSERTATION
Summary
This dissertation describes the novel regulation of L-selectin by mAb, cytoskeletal
association, and fatty acid membrane content. L-selectin is expressed on the tips of the
microvilli on human leukocytes (Picker et ah, 1991 and Bruehl et al., 1996). Here, we
extend these observations to bovine lymphocytes (Leid et al., 1998).
Transmission
electron microscopy demonstrated that L-selectin is located at the tips of the microvilli of
y8 T cells as visualized by anti-L-selectin mAb DREG 56 and colloidal-gold conjugated
goat anti-mouse second stage staining. Scanning electron microscopy demonstrated that
bovine yS T cells contain greater than twofold the number of microvilli than ccp T cells
and that bovine yS T cells are smaller in size (4 pm versus 6 pm). It has previously been
shown that bovine yS T cells express 2-5 times the amount of L-selectin versus a(3 T cells
(Walcheck et al., 1994). We confirmed this finding here and suggest that because of the
location of L-selectin at the tips of the microvilli, yS T cells express more L-selectin
simply because these cells contain more microvilli than a(3 T cells. Differences in the size
and surface topography of y8 and a [3 T cells may be important in the interaction of these
144
cells with other host cells, such as endothelial cells, or targets they intend to kill, such as
tumor cells or infectious agents.
A recent report demonstrates that L-selectin associates with the cytoskeleton by
mAh crosslinking, hyperthermic treatment, and Glycam-I binding (Evans et' al., 1999).
We have characterized a mAb-induced (LAM1-116) conformational change in L-selectin
that predisposed the protein to cytoskeletal association. In the presence of this structural
change in L-selectin, a second functional epitope on L-selectin, recognized by mAb EL246, is upregulated, and when it is engaged by EL-246, cytoskeletal association occurs.
The conformational change and ultimate cytoskeletal association of L-selectin was
specific to these two antibodies as other anti-L-selectin mAbs, as well as other cell
surface antigens, did not trigger cytoskeletal association. It is interesting to note that both
mAbs recognize distinct functional epitopes on L-selectin and only these two epitopes
were involved in non-mAb-crosslinked-induced L-selectin cytoskeletal association. The
functional importance of the conformational change and eventual cytoskeletal association
described is currently under investigation. Unfortunately, both mAb and native ligand,
which all induce cytoskeletal association, also block the function of L-selectin. However,
other work done in different systems have suggested a causal link between L-selectin
cytoskeletal association and function.
L-selectin presumably associates with the cytoskeleton through binding of aactinin to the cytoplasmic tail of L-selectin and actin. Previous work demonstrates that
145
the cytoplasmic tail is important in the function and regulation of L-selectin (Kansas et
al., 1993 and Kahn et al, 1998) and transfectants lacking the 11 terminal amino acid
residues of L-selectin do not roll in vitro and in vivo (Kansas et al., 1993). A separate
study further concluded that L-selectin on these same transfectants does not associate
with the cytoskeleton under any conditions (Evans et al., 1999). Therefore, it is implied
that cytoskeletal association of L-selectin is important in mediating L-selectin rolling
events.
The cytoskeleton is vital to lymphocyte activation and function (Penninger and
Crabtree, 1999). To further investigate the lymphocyte cytoskeleton, as well as Lselectin cytoskeletal association and its relation to protein function, we compared the
cytoskeletal association of L-selectin to that of 28 other cell surface proteins on human
and bovine lymphocytes. Interestingly, scanning electron microscopy demonstrates that
low dose detergent treatment leaves a highly uniform lymphocyte cytoskeleton with a
remarkably smooth surface compared to an intact, non-detergent-treated yS T cell.
Detergent treament of bovine lymphocytes strips the cell of its microvilli, or surface
projections from the plasmalemma. Also, the lymphocyte cytoskeleton is smaller in size
than an intact y§ T cell. A previous report has shown that bovine yS T cells are
approximately 4 (im in size. Here, we show that detergent treatment reduces the size of
the bovine yS T cell by -25%, resulting in a detergent-treated lymphocyte of
approximately 3 pm.
Upon additional investigation by transmission electron
146
microscopy, detergent treatment of bovine lymphocytes demonstrates that the
plasmalemma collaspes around the nucleus, exhibiting little or no cytoplasm. In addition,
detergent treatment leaves the cell without any obvious organelles or microvilli extending
from the plasmalemma. As with a previous report, an intact, non-detergent lymphocyte
retained these characteristics, including the presence of mitochondria and Golgi in the
cytoplasm.
Three different types of protein cytoskeletal association are described herein,
constitutive, inductive, and mAh crosslink-induced. Each characterization depends upon
the timing of low dose detergent treatment to the cells compared to mAh treatment.
Proteins constitutively associated with the cytoskeleton are visualized by mAb staining
after detergent treatment. Some of these proteins included CD44 in the human and WCl
and CDS in the cow. All of the proteins discovered to be constitutivley linked to the
cytoskeleton in these studies can act directly as signaling molecules, thus suggesting that
constitutive cytoskeletal linkage is important in a cells ability to transduce effective
signals. Indeed, when the ability of some proteins to associate with the cytoskeleton is
inhibited, efficient signaling does not take place (Stulnig et ah, 1998). Other proteins are
induced to associate with the cytoskeleton by primary mAb binding alone.
These
included molecules such as.CDS, E-selectin ligand(s), and bovine y§ TCR. Under certain
conditions, L-selectin cytoskeletal association mirrors that of E-selectin' ligands, in that
both are induced to associate with the cytoskeleton by direct ligand and/or
147
counterreceptor binding. L-selectin can also be induced to associate with the cytoskeleton
by mAb crosslinking. Thus, for L-selectin, the ability to associate with the cytoskeleton
depends upon a specific conformation of L-selectin.
L-selectin can be induced to
associate with the cytoskeleton by direct ligand binding or by dual anti-L-selectin mAb
treatment that causes a conformational change in L-selectin, predisposing the protein to
associate with the cytoskeleton when a distinct, conserved epitope recognized by mAb
EL-246, is engaged.
Taken together, these data suggest that cytoskeletal association
provides a mechanism of regulation between various proteins and their receptors. Indeed,
when these interactions are disrupted by incorporation of PUFAs into the cell membrane,
the adhesive quality of L-selectin is dramatically reduced.
In summary, this work describes the novel alteration of L-selectin by mAb,
cytoskeletal association, and fatty acid membrane content. A ligand mimicking mAb,
LAMl-116, induces a conformational change in L-selectin, increasing the expression of a
conserved epitope recognized by a distinct mAb, EL-246, and predisposes L-selectin to
cytoskeletal association when the EL-246 epitope is engaged. Cytoskeletal association of
L-selectin is important in its function as an adhesion molecule.
By describing the
regulation of an important epitope on L-selectin (EL-246), it may help design a better
theraputic agent during ischemia and reperfusion injury, as well as other shear dependent
events. In the past, EL-246 has been shown to be an effective reagent against these
injuries and now, with a better understanding of its interaction with L-selectin, may lead
148
to the next generation of reagents suited to fight both acute and chronic inflammatory
events.
Collaborative Work During Mv Graduate Program
Alexander, S.R., EU. Gilmore, C. Wang, J.G. Leid, and B. Walcheck. (2000). Detection
of a cell surface molecule on human hematopoietic cell lines by particular anti-E-selectin
monoclonal antibodies. Submitted.
This paper describes the unusual expression of an E-selectin-like molecule on
human hematopoietic cell lines by the staining of a variety of functionally important antiE-selectin mAbs.
It also demonstrates that the E-selectin-like protein can be
immunoprecipitated from the cell membrane lysates of these human hematopoietic cell
lines.
Speer, CA., K. Prokop, G. Watts, J.A. Blixt, M.A. Jutila, and J.G. Leid. (2000).
Monoclonal antibody kills equine protozoal myeloencephalitis parasite. In preparation.
This paper describes the novel killing mechanism of an antibody that recognizes a
cell surface protein on an equine protozoal myeloencephalitis parasite. Co-culture of the
parasite with the mAh results in parasite death within 4 hours and total killing within 48
hours.
149
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MONTANA STATE UNIVERSITY - BOZEMAN