MOLECULAR AND FUNCTIONAL CHARACTERIZATION
OF BOVINE C5a RECEPTOR
By
Sailasree Nemali
A Thesis Submitted in Partial Fulfillment
of The Requirements of The Degree
of
Master of Science
In
Veterinary Molecular Biology
MONTANA STATE UNIVERSITY
Bozeman, Montana
April 2006
© COPYRIGHT
By
Sailasree Nemali
2006
All Rights Reserved
ii
APPROVAL
of a thesis submitted by
Sailasree Nemali
This thesis has been read by each member of the thesis 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 Division of Graduate Education
Mark T. Quinn
Approved for the Department of Veterinary Molecular Biology
Mark T. Quinn
Approved for the Division of Graduate Education
Joseph J. Fedock
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a master degree
at Montana State university-Bozeman, I agree that the Library shall make it available to
borrowers under rules of the Library. I further agree that copying of this thesis is
allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S.
Copyright Law. Requests for extensive copying or reproduction of this thesis should be
referred to ProQuest Information and Learning, 300 North Zeeb Road, Ann Arbor,
Mochigan, 48106, to whom I have granted “exclusive right to reproduce and distribute
my thesis in and from microfilm along with the non-exclusive right to reproduce and
distribute my abstract in any format in whole or in part”
Sailasree Nemali
April, 2006.
iv
ACKNOWLEDGEMENT
I would like to thank my supervisor Dr. Mark T. Quinn for his support during my stay
in his laboratory. I also like to thank my advisory committee members, especially Dr.
Mark Jutila, for the scientific collaboration.
I also would like to thank my husband, Dr. R. Krishna Mohan for his support, sense
of humor, and friendship. I will always cherish the support I have received at VTMB, and
the friendships I have made.
v
TABLE OF CONTENTS
1. MOLECULAR AND FUNCTIONAL CHARACTERRIZATION
OF BOVINE C5A RECEPTOR .............................................................................1
Introduction ..........................................................................................................1
C5a-Anaphylatoxin and C5a Receptor (C5aR) .................................................6
Signal Cascades and Neutrophil Recruitment
to The Site of Infection ...................................................................................9
Heterologous Desensitization of C5a Receptor
and IL-8 Receptor ............................................................................................12
The C5a Receptor Expression on Bovine Peripheral
Blood Leukocytes ............................................................................................16
Effect of C5a on The Function of Epithelial Cells-A Possible
Functional Interaction between Toll-Like Receptor (TLR)
and C5a Receptor.............................................................................................17
Methods ................................................................................................................21
Animals.............................................................................................................21
Cloning and Expression of Bovine C5a Receptor ............................................21
Cloning .......................................................................................................21
Generation of Bovine C5a Receptor cDNA ...............................................22
Plasmid Construction..................................................................................23
Generation of CHO-K1-pCDNA 3.1-bovine
C5aR c-Myc/His Stable Cell Line ...........................................................24
DNA Sequencing ........................................................................................25
Clustal W Alignment of VertebrateC5a Receptor
Protein Sequence and Phylogentic Tree Construction.............................25
Analysis of 5’-end of Bovine Genomic DNA Sequence
Obtained from ENSMBL..........................................................................25
Analysis of C5a Receptor Mediated Signaling in
Bovine Neutrophils ........................................................................................26
Bovine Neutrophil Isolation........................................................................26
Preparation of Bovine Peripheral Blood Leukocytes .................................26
FACS Analysis of Bovine Leukocytes .......................................................26
Intracellular Calcium Flux ..........................................................................27
Cloning and Expression of Bovine C5a ...........................................................28
C5a PCR with Degenerate Primers.............................................................28
Cloning and Expression of Bovine C5a......................................................29
Monoclonal Antibody Generation ..................................................................30
Immunogen Synthesis.................................................................................30
Monoclonal Antibody Generation ..............................................................30
Characterization of Mouse Anti-bovine C5R ...................................................31
Isotype Determination of 3D5 ...................................................................31
Protein G Purification of 3D5....................................................................31
vi
TABLE OF CONTENTS-CONTINUED
Preparation of Peripheral Blood
Mononuclear Cells (PBMC) from Bovine Blood .......................................32
Indirect Enzyme-linked Immunosorbant Assay .........................................32
FACS Analysis ..........................................................................................32
Immunocytochemistry ...............................................................................33
C5a Receptor Expression Analysis...................................................................34
C5a Receptor Expression Analysis on Mac-T cells..........................................34
RT-PCR Analysis of The Bovine C5a Receptor ........................................34
FACS Analysis ...........................................................................................35
Results...............................................................................................................35
Cloning and Analysis of Bovine C5a Receptor ................................................35
Cloning and Sequence Homology Analysis ...............................................35
In vitro and in vivo Expression of Bovine C5a Receptor ...........................37
Analysis of Bovine C5a Receptor-Clustal W Alignment ...........................38
Analysis of Vertebrate C5a Receptors........................................................40
5’UTR Sequence Analysis of Bovine C5a Receptor
Genomic Sequence ....................................................................................43
Activation of Bovine Neutrophils by C5a and C5a des arg........................44
Activation of Bovine Neutrophils by C5a and
C5a des arg ......................................................................................................44
C5a Receptor Stimulated L-selectin Shedding in
Bovine Neutrophils ........................................................................................46
Cloning of bovine C5a cDNA ...........................................................................48
C5a Receptor Desensitization...........................................................................50
Homologous Desensitization of C5a Receptor...........................................50
Heterologous Desensitization of C5a Receptor ..........................................51
Production and Characterization of Monoclonal
Antibody to Bovine C5a Receptor Peptide...............................................54
Production of a Bovine C5a Receptor Specific
Monoclonal Antibody ...............................................................................53
Cross-reactivity of Bovine C5a Receptor-specific
Antibody with Human Neutrophils...........................................................54
Characterization of Monoclonal Antibody (3D5) Directed
Against the Predicted N-Terminal Amino Acid Sequence
Of Bovine C5a Receptor........................................................................55
Isotype Determination of 3d5 .....................................................................55
Indirect Enzyme Linked Immunosorbant Assay ........................................56
Binding of 3D5 to Bovine C5a Receptor Expressed
in Cos-7 Cells ...........................................................................................56
3D5 Recognizes Surface C5a Receptor Expression
on Bovine Neutrophils ..............................................................................57
vii
TABLE OF CONTENTS-CONTINUED
Surface Expression Analysis of Bovine C5a Receptor on
Peripheral Blood Leukocytes (PBMC) .....................................................59
Expression Analysis of Bovine C5a Receptor in
Mac-T cells.....................................................................................................61
RT-PCR Analysis of Bovine C5a Receptor Expression
in Mac-T Cells ...........................................................................................60
FACS Analysis of Bovine C5a Receptor Expression
in Mac-T Cells .........................................................................................61
Discussion...............................................................................................................62
Summary.................................................................................................................72
Future Studies .........................................................................................................76
REFERENCE LIST ......................................................................................................79
viii
LIST OF TABLES
Table
Page
1. Vertebrate C5a Receptor cDNA Sequence Comparison ...............................................42
2. Vertebrate C5a Receptor protein Sequence Comparison...............................................42
ix
LIST OF FIGURES
Figure
Page
1. The Complement Pathway Activation in Peripheral Blood ..............................................8
2. Cellular Signaling Mechanisms Mediated by TheC5a ReceptorA Schematic Representation of G-protein Mediated Signaling......................................9
3. The Signaling During Neutrophil Activation and Migration.............................................15
4a.The cDNA Sequence of The Bovine C5a Receptor..........................................................36
4b.Snake Plot of The Bovine C5aR .......................................................................................37
5. The Expression Bovine C5a Receptor in CHO-K1 Cells ..................................................38
6. The Phylogenetic Tree Analysis of Vertebrate C5a Receptors .........................................40
7. Clustal W Alignment of Bovine C5a Receptor with Vertebrate
C5a Receptors .................................................................................................................41
8. 5’-UTR Sequence Analysis of The Bovine C5a Receptor ...............................................44
9. Intracellular Calcium Flux Induced by C5a and C5a des arg
In Bovine Neutrophils......................................................................................................45
10. Effect of Pertussis toxin (ptx) on C5a Receptor Mediated
Intracellular Calcium flux in Bovine Granulocytes .......................................................46
11. The Analysis of L-Selectin Shedding on Bovine Neutrophils
Due to Chemotactic Factors...........................................................................................47
12a.The Effect of Human C5a on The Bovine C5a Receptor................................................49
12b.The Cloning and Expression of Bovine C5a...................................................................49
13. Homologous Desensitization of The Bovine C5a Receptor ............................................51
14. Heterologous Desensitization of The Bovine C5a Receptor ...........................................52
15. Hydropathy Plot of Predicted the Bovine C5a Receptor .................................................54
16. Bovine C5a Receptor Specific Antibody Interaction With
Human Neutrophils........................................................................................................55
17. Indirect Immunosorbant Assay for The Bovine C5a Receptor Peptide...........................56
18. 3D5 Binds Specifically to The C5a Receptor Expressed in vivo.....................................57
19a.3D5 Recognizes Neutrophils in Bovine Peripheral Blood Leukocytes ..........................58
19b FACS Analysis of Bovine Peripheral Blood Leukocytes with 3D5 ...............................58
20a.The Bovine C5a Receptor Expression on Peripheral
Blood Leukocytes .........................................................................................................59
20b.The analysis of plastic-adherent peripheral bovine blood leukocytes
for The C5a receptor expression ..................................................................................60
21. PCR Analysis of The C5a Receptor Gene in Mac-T Cells..............................................61
22. FACS Analysis of Bovine C5a Receptor on Mac-T Cells...............................................62
x
ABSTRACT
Anaphylatoxin C5a is an important chemotactic factor for bovine neutrophils, and is
the earliest inflammatory agent formed during bovine mastitis. Bovine neutrophils
respond to C5a and its truncated form C5a des arg with similar affinities unlike human
neutrophils. Therefore, to test the hypothesis that the bovine C5a receptor structure and
signaling differ from that of the human C5a receptor, we cloned and analyzed the bovine
C5a receptor. In the present investigation, the bovine C5a receptor encoding cDNA from
bovine bone marrow was cloned and the recombinant C5a receptor protein was expressed
in CHO-K1 cells. Analysis of predicted C5a receptor amino acid sequence demonstrated
69.1%, 71.3%, 59.4%, 61.6%, and 33.2% identity with that of human, dog, mouse, rat,
and trout respectively. The bovine C5a receptor mediated signaling was via pertussis
toxin insensitive Gα-protein, and mediated L-selectin shedding on the activated
neutrophils. The homologous-, and heterologous desensitization experiments provided
further evidence that C5a and C5a des arg could desensitize C5a receptor signaling as
well as IL-8 receptor mediated signaling. We further analyzed the expression profiles of
the C5a receptor on bovine peripheral blood leukocytes with the receptor-specific
antibody and FITC labeled C5a des arg. The monoclonal antibody as well as C5a des arg
FITC failed to detect C5a receptor on peripheral blood mononuclear cells. Furthermore,
we probed for the expression of C5a receptor gene and protein expression in Mac-T cell
line--an immortalized bovine mammary epithelial cell line--, to test the hypothesis that
bovine epithelial cells express C5a receptor protein. The results of RT-PCR analysis and
FACS analysis for the expression of C5a receptor demonstrated the presence of the C5a
receptor in these cells. This investigation led to the elucidation of the structure and
function of bovine C5a receptor in neutrophils. The analysis of the C5a receptor
expression in peripheral blood leukocytes demonstrated that it was constitutively
expressed in neutrophils only. Mac-T, a mammary epithelial cell line expresses C5a
receptor. Future studies understanding the function of C5a receptor in these cells will
contribute to the knowledge of bovine inflammation.
1
MOLECULAR AND FUNCTIONAL CHARACTERIZATION
OF BOVINE C5a RECEPTOR
Introduction
The innate immune response to invading pathogens is the first line of defense, and is
required for the elimination of infection. The primary goal of inflammation is to restrain
pathogens by mounting a global non-specific response, which will limit both the
proliferation and spreading of pathogens, and is achieved by the recruitment of
neutrophils, monocytes, and macrophages to the site of infection. The elimination of
infection by neutrophils is via 1) activation, and trans-endothelial migration of
neutrophils to the site of infection and 2) phagocytosis of the pathogen, and it’s
elimination by the generation of anti-microbicidal products (1). One such inflammatory
disease of economic importance in the dairy industry is clinical mastitis. Mastitis is a
major cause of economic losses in dairy industry, costing approximately $2 billion
annually in the United States alone (National Mastitis Council 1999), by increasing the
somatic cell count to unacceptable levels in bovine milk. Although the increase in
somatic cell count confers protection in clinical mastitis (2;3), accumulation, and
activation of neutrophils (leading to release of reactive oxygen species) along with
generation of proinflammatory cytokines leads to tissue damage resulting in scarred
tissue and decreased milk production. Furthermore, in humans, mastitis can mediate
transfer of human immunodeficiency virus (4;5) and bacteria (6) from an infected mother
to the child. A link between C5a receptor and HIV transmission in monocyte-derived
macrophages has been demonstrated in vitro, and shown to be related via C5a receptor
2
stimulated secretion of tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) (7;8).
In addition, C5a receptor down regulation in advanced AIDS leads to impaired
interleukin 8 (IL-8) secretion during cryptococcal infection (9). A complete
understanding of complex inflammatory process on epithelial cell surfaces, such as
mammary gland infection that can be applied to human mastitis is lacking. An
understanding of interactions between inflammatory agents such as C5a and IL-8 in a
biological paradigm that does not respond to f-MLF (10) can shed some light in human
mastitis, since the dominant effect of f-MLF receptor on the signaling cascades mediated
by C5aR and IL-8R is completely absent (11).
The infection due to extracellular pathogens leads to neutrophilic inflammation,
mobilizing and recruiting neutrophils in large numbers, leading to a non-specific immune
response to eliminate the pathogens. Mature neutrophils circulating in blood contain a
polymorphic, multi-lobed nucleus and different classes of cytoplasmic granules. These
granules contain receptors, including CD11/CD18, chemotactic receptors, TNF-α
receptor; bactericidal peptides such as defensins, azurocidin, lysozyme, myeloperoxidase,
cathepsin G, bactericidal permeability increasing protein; peroxidases and alkaline
phosphatase. These granules fuse with the neutrophil plasma membrane during their
priming and activation by exocytosis, leading to upregulation of receptors for neutrophil
migration, phagocytosis, and reactive oxygen species production. Bovine neutrophils are
unique in containing large peroxidase-negative granules with unknown function. The
lactoferrin released from novel and secondary granules during bovine inflammatory
condition chelates iron that is required for the growth of bacteria. The change in
morphology of the neutrophil is regulated by proinflammatory cytokines, such as PAF
3
and TNF-α, in order to control the movement of neutrophils to the site of infection. The
recruitment of neutrophils to mammary gland in bovine mastitis is largely dependent
upon the strength of a chemotactic agent to recruit the neutrophils to the site of infection
[for review see (12)].
Strategies to develop resistance to mastitis-causing pathogens by either conventional
breeding procedures or transgenic technology do not address mass amelioration of
treatments to mastitis. Conventional breeding of cows to develop resistance to mastitisinducing pathogens is hampered due to low heritability in cows for this trait [for review
see (13;14)]. In addition, it is often time consuming and costly. Another alternative
strategy taken was aimed at the generation of transgenic cows expressing lysostaphin, an
enzyme that degrades Staphylococcus aurus (S. aureus) cell walls leading to the
pathogen’s death (15). However, these cows lack immunity to acute coliform bacterial
infections as the enzyme expressed is specific to S. aureus, and the technology is still too
primitive to be used on a large scale. During mastitis, the milk is rendered commercially
unviable due to increased somatic cell count, which includes neutrophils, mononuclear
leukocytes, and epithelial cells. Thus, chemotactic factors play an important role in the
activation and recruitment of neutrophils and leukocytes to the site of infection.
Therefore, there is a need for a comprehensive understanding of the influences exerted by
different chemotactic factors on neutrophil activation and the identification of whether
dominant chemotactic factor that is obligatory to recruit neutrophils to the site of
bacterial infections.
Efficient migration of neutrophils to the site of infection, mediated by
proinflammatory agents, plays an important role in the resolution of Escherichia coli (E.
4
coli) infection in bovine mastitis (16). Recent studies on the presence of
proinflammatory agents during inflammation in cows--due to experimental mastitis -indicate that C5a is the first phlogistic response to be formed, followed closely by IL-8.
E. coli infected cow milk whey contains about 20ng/ml of C5a or C5a des arg, while IL-8
concentration in this milk whey is about 1.2ng/ml. The kinetics of appearance of C5a
coincides with an increased bacterial growth in the mastitis models induced by gramnegative bacteria. These results suggest that C5a formation is via the alternate
complement pathway on the pathogen surface and is dependent on the nature of the
pathogen (infection due to S. aureus did not elicit formation of C5a to same extent).
Coincidentally, the formation of proinflammatory cytokines also followed the same trend,
and ΤΝF-α and interleukin-1β (IL-1β) appeared later than C5a and IL-8. The
accumulation of somatic cells and appearance of serum albumin, an indication of
epithelial cell barrier disruption, followed the appearance of C5a in milk whey from the
infected bovine mammary gland (17-20) .
Although studies conducted by Persson and Sandgren demonstrated that endotoxin,
IL-1β, leukotriene Β4 (LΤΒ4), and C5a are potent chemotactic factors in the order
mentioned, formation of C5a is followed by IL-8 in all mastitis models (21).
Nonetheless, the implication of this phenomenon is not well understood. The conclusions
from these studies lead to the hypotheses that the potency of endotoxin in vivo may be
due to: 1) the endotoxin mediated formation of C5a, which is an end target chemotactic
factor and modulator of epithelial cell function via C5a receptor in the presence of
endotoxin; or 2) epithelial cell stimulation leading to the secretion of IL-8, a potent
5
intermediary chemotactic factor, which induces trans-endothelial and epithelial migration
of leukocytes (22).
Bovine neutrophils are equally responsive to anaphylatoxin C5a, and its truncated
form-C5a des arg (23). In contrast, bovine neutrophils are non-responsive to formylated
peptides (10), and a functional formylated peptide receptor has not been identified in
cattle. Therefore, a conclusive study to determine the importance of the C5a receptor
during mastitis infection is warranted in bovine species, in order to understand the
function of C5a in bovine inflammation. In addition, currently there are no antagonists
that can completely inhibit C5a-mediated responses in sheep (24), these compounds have
not yet been tested in bovine inflammatory condition such as mastitis. In other words,
due to the lack of sequence information of bovine C5a receptor, there are no antagonists
specific to bovine C5a receptor. A close inspection of the literature in the field of bovine
mastitis, a model for bovine inflammation, indicates that there is a correlation between
the appearance of C5a and neutrophil accumulation at the site of infection. Moreover,
formation of IL-8 temporally follows C5a formation with a sustained peak for 42 hours in
milk whey of a cow with E. coli induced mastitis. Although IL-8 is a potent chemotactic
factor, mutation in CXCR2 gene in cows leads to impaired expression of adhesion
molecules and chemotaxis not only to IL-8, but also to zymosan-activated serum (25).
Although the studies conducted on the ability of bovine IL-8 to recruit neutrophils
showed that IL-8 was an important chemotactic factor, they did not rule out the
possibility of other chemotactic factors influencing neutrophil recruitment (26).
However, the mutual influence of C5a and IL-8 on the signal transduction pathways
mediated by their receptors, and the consequence of such mutual influence is not obvious
6
in bovine inflammation. Thus, the objectives of this study are to provide a partial
characterization of structure and function of C5a receptor, and investigate the influence
of IL-8 on C5a receptor mediated signaling.
C5a- Anaphylatoxin and C5a Receptor (C5aR)
The anaphylatoxin C5a is a major by product of complement pathway. C5a is formed
by the activation of classical-, lectin-, and alternate complement activation pathways
(figure 1). C5a is a small cationic peptide of 74 amino acids and is a proteolytic product
of C5, formed by the action of C5 convertase complex. C5a, or its truncated form-C5a
des arg, mediate signaling via their cognate receptor, which belongs to a G-protein
coupled receptor (GPCR) of the rhodopsin subfamily and involves heterotrimeric Gproteins (27;28). The protein-coding region of the gene has two exons separated by an
intron (9Kb in length). C5a receptor-mediated signaling through pertussis toxin-sensitive
G-proteins by the activation of both p38 mitogen activated protein kinase pathway
(MAPK), as well as extracellular signal regulated protein kinases (ERK) (29), leading to
down stream effects that include intracellular calcium flux, chemotaxis due to
cytoskeletal re-arrangements, reactive oxygen species production, and gene regulation via
transcription (1;22;29-33) (fig 2).
The cognate receptor for C5a is a heptahelical transmembrane glycoprotein with Nlinked glycosylation via asperagine at N-terminus. The protein also contains six cystine
residues, which form three intra-molecular disulphide bonds that are required for the
stabilization of C5a structure. Human C5a receptor protein has 34% sequence homology
with that of the human formyl peptide receptor (f-MLF receptor). The highest sequence
7
homology exists between transmembrane domains, with diversity between sequences
encoding extracellular surfaces of the receptor. The proposed model for C5a binding is a
two-site binding model, involving the charge-charge interaction, with the N-terminus of
the cationic peptide region of C5a tethered to the negatively charged N-terminus (due to
aspartic acid residues) of the C5a receptor (C5aR), thus stabilizing the binding of the
ligand to its receptor (34;35). The C-terminus of C5a then interacts with the
transmembrane domain, along with arginine 206 and glutamate 199 in the third
extracellular loop to mediate C5a C-terminal binding and transduction of intracellular
signaling via G-protein dissociation into Gα and Gβγ (36). The second intracellular loop
contains a signature DRF motif, which is important for GPCR function. The DRF motif
may have a functional importance in the desensitization and internalization of GPCRs as
demonstrated by the studies in the marmoset gonadotropin releasing hormone(GnRH)
receptor (37). Human C5a receptor has high binding affinity for C5a, but low binding
affinity for C5a des arg (38), and the N-terminal glycosylation at asparagine is one of the
contributing factors to ligand binding affinities (39). C5a and C5a des arg elicit similar
physiological responses in bovine neutrophils, although the bovine C5a molecule is not
glycosylated unlike that of human C5a. The C5aR antagonists exhibit species-specific
inhibition of receptor activity as well (24). Therefore, investigation to understand these
differences requires structural information about the bovine C5a receptor. There is lack of
information with regard to the expression profile of the C5a receptor, although several
studies in different species indicated that both myeloid cells and non-myeloid cells
express C5a receptor (22;40). In addition, monoclonal antibody generated against the Nterminus of human C5a receptor does not interact with bovine neutrophils (unpublished
8
observation). These results indicate that bovine C5a receptor may be different from that
of human, warrants cloning of the bovine C5a receptor, and generation of specific
detection reagents such as monoclonal antibodies to detect the surface expression of this
receptor. Thus, we pursued cloning of bovine C5a receptor and its ligand C5a, and
generation of a mouse monoclonal antibody to N-terminus of bovine C5a receptor to
probe for the expression of C5a receptor in neutrophils and monocytes as the first
objective of this study.
Classical
Lectin
(IgM/IgGbound to antigen) (Repetitive microbial surface patterns)
C1q
C1r
MBL
C1r'
C1s
Alternate pathway
(c3b amplification
on foreign surface)
C1s'
C2
C2a, C2b
MSP2 MSP-1
B
D
H2O
iC3
C4 C4a, C4b
C3
C4b+C2a
C3b, C3a
C5b+C6+C7+C8+(C9)n
iC3+Bb+P
C3b+Bb+P
B
D
C5
C5a C5b
Membrane attack complex
Opsonisation
Figure 1: The complement pathway activation in peripheral blood: Three main pathwaysclassical, lectin, and alternate pathways can activate complement formation. In the
classical pathway, conformational change on the IgM or IgG molecules leads to the
recruitment of C1q molecule-a complex of C1r, C1s, thus activating serine protease
activation cascade, and generation of C4b, C3b and finally C5b. C5b forms a membrane
attack complex along with C6, C7, C8 and C9. All the pathways converge at the cleavage
step of C5 to C5a and C5b.
9
α
P P
Des
ous
olog
Hom eptor n
Rec itizatio
ens
P
P1-3
kinase-γ
PI (4,5)P
PI
GAP
GEF
βγ
PL C
PI (,4,5) di
phosphate
PI + DAG
Intracellular Calcium
Flux
PI (3,4,5) P3
PI(3)P
Actin Polymerization
Chemotaxis
Respiratory Burst
PKC Activation
MAPK Activation
L-Selectin Shedding
Heterologous Receptor
Desensitization
Figure 2: Cellular signaling mechanisms mediated by C5a receptor-A schematic representation
of G-protein mediated signaling: The G-protein coupled receptor, for example C5a receptor,
upon binding to its ligand, undergoes conformational changes leading to its interaction with Gprotein. The G-protein is made up of three cytosolic subunits Gαβγ . Gα subunit is a guanine
nucleotide binding protein, and recycles between GDP bound inactive form and GTP bound
active form. The ligand-bound GPCR induces exchange of GDP to GTP on Gα, leading to its
dissociation and release of the membrane-bound βγ subunits. These βγ subunits, once released,
activate intracellular calcium flux via phospholiase C, thus initiating a cascade of physiological
responses. In case of neutrophils, this leads to intracellular calcium flux, loss of L-selectin
surface expression, chemotaxis, NADPH oxidase activation, and phagocytosis. The termination
of receptor signal is by homologous receptor desensitization and endocytosis.
Signal Cascades and Neutrophil Recruitment to The Site of Infection
The activation of neutrophils and their egress to the site of infection from blood has
been proposed to occur by a multi-step paradigm involving: 1) the formation of an
10
inflammation synapse between neutrophils and activated endothelium [activated by
lipopolysaccharide (LPS), IL-1β and TNF-α], 2) transendothelial migration, and 3)
chemotaxis through concentration gradients mediated by end-target chemotactic factors
such as f-MLF or C5a. According to the multi-step paradigm, which was proposed by
Butcher and Springer (figure 3), the neutrophils under shear flow in blood exhibit
constitutive expression of L-selectin (CD62L) and platelet sialoglycoprotein ligand-1
(PSGL-1) on their surface (41-46). During inflammation, LPS or proinflammatory
cytokines can upregulate ligands for these two glycoproteins on endothelial surface.
TNF-α and C5a is a potent inducer of E-selectin (ligand for L-selectin) expression on
human umbilical vein endothelial cells (47;48). Once L-selectin and PSGL-1 sense their
ligands, they tether the cell to endothelium, and the cell rolls on the surface of the
activated endothelium and samples the surface. During this process, the neutrophil senses
chemokines (e.g., IL-8) or LTB4. Chemokines signaling along with engagement of Lselectin induce clustering and changes in the affinity of β2-integrins on the neutrophil
surface. Ultimately, this process leads to arrest and firm adhesion of neutrophils under
shear flow. This phenomenon of β2-integrin affinity and avidity change is an obligatory
step in neutrophil extravasation and requires synergistic action of L-selectin and PSGL-1
binding to their ligands, IL-8 receptor activation leading to activation of pathways
involving MAPK, PI(3) kinase, and small G-protein (Rap-1 and Rho-1). These events in
turn bring changes in β2-integrin distribution and clustering on neutrophils (49). The
above findings lead to hypothesis that transient chemokine signaling leads to PI(3) kinase
activation and calcium influx induced calpain activation, which leads to leukocyte
11
functional antigen-1 (LFA-1) localization to lamellipodia and induction of transendothelial migration. This signaling leads to down regulation of L-selectin upon endtarget chemotactic factor encounter (50;51). The corroborative evidence for this
hypothesis is provided by studies conducted in CD18 knockout mice as well as combined
E-selectin and P-selectin double knockout mice, which exhibit defects in neutrophil
recruitment to the site of infection (52). Mac-1, which is also known as CD11b, is a β2
integrin subunit that is expressed predominantly on neutrophils. It forms a heterodimeric
complex with CD18. However, Mac-1deficient mice do not exhibit such severe
deficiencies (53;54) in neutrophil recruitment to the site of inflammation.
Studies conducted by Diez-Fraille (55) on the expression of L-selectin and Mac-1
in E. coli-induced mastitis after infection demonstrated the loss of L-selectin occurred
only after peak inflammatory reaction due to E. coli, but not during leukocyte infiltration
to the site of infection. This peak inflammatory response coincides with the maximum
C5a formation in the infected mammary glad. Hence, it suggests that the C5a induces
shedding of CD62L and increases expression of CD11b via neutrophil activation. Blake,
et al. (56) further demonstrated that the trans-epithelial migration induced by f-MLF is
dependent on Mac-1 expression. C5a induced trans-epithelial migration is independent
of Mac-1 in vitro. In addition, investigations in differentiated HL-60 cells (into a
neutrophilic phenotype) devoid of Mac-1 show that these cells migrate across an
epithelial cell barrier in response to C5a (57). Therefore, transepithelial migration of HL60 cells induced by C5a does not require Mac-1. Thus, L-selectin shedding appears to be
one of the earliest steps of neutrophil activation due to C5a. The ability of C5a to induce
12
CD62L shedding is yet to be shown in bovine neutrophils, although this was
demonstrated in human neutrophils (58;59).
The ability to induce intracellular calcium flux by both C5a and C5a des arg and the
effect of pertussis toxin on their signal transduction machinery has not been not fully
addressed in bovine neutrophils. Studies conducted by Gennaro (1;60) indicate that both
of these ligands elicit similar chemotactic responses in bovine neutrophils, and bovine
C5a induces a calcium response in human neutrophils. In human neutrophils, the C5amediated signaling occurs via a pertussis toxin sensitive GPCR (61;62). However, there
is no evidence to demonstrate that the bovine C5a receptor couples with a pertussis toxin
sensitive G-protein in bovine neutrophils. Therefore, we addressed the effect of C5a, C5a
des arg, and IL-8 on the activation of neutrophils, which is measured by the loss of Lselectin, and addressed the role of pertussis toxin in anaphylatoxin-mediated intracellular
calcium flux.
Heterologous Desensitization of C5a Receptor and IL-8 Receptor
The studies conducted in bovine inflammation models implicate C5a and C5a des
arg as important chemoattractants for bovine neutrophils (63). The lack of bovine
neutrophil functional response to f-MLF leads to the hypothesis that C5a is a dominant
end target chemotactic factor over IL-8 in activating neutrophils and plays an
important role in epithelial cell function via its cognate receptor expression.
C5a forms at the site of infection and, therefore, is classified as an end-target
chemotactic factor; a category that includes the formylated peptides. These chemotactic
factors mediate directed migration of neutrophils through a chemotactic gradient to reach
13
the site of infection, while the initial transendothelial migration of neutrophils requires
intermediary chemotactic factors, such as IL-8 and LTB4. Intermediary chemotactic
factors are by definition endogenously synthesized and secreted by endothelial cells,
epithelial cells, macrophages, and monocytes (30;64;65). C5a acts as an end-target to
attract neutrophils to the site of infection, in addition to stimulation and secretion of
intermediary targets such as IL-8 from endothelial cells (66), monocytes (31), and
epithelial cells (30). Leukocytes under shear flow recognize intermediary chemotactic
factors, which are synthesized in response to proinflammatory stimulus at endothelial and
epithelial surface and are presented tethered to the surface of the endothelium by
transendocytosis (67), mediating their activation (68). Thus, one can envision a scenario
where the intermediary factors mediate trans-endothelial and trans-epithelial migration,
priming of neutrophils to upregulate chemotactic receptors (69), and neutrophil migration
along the concentration gradient of end-target chemotactic factors.
Neutrophils that migrate from the blood to the site of infection encounter a plethora
of proinflammatory agents, including chemotactic agents. They have to navigate through
complex chemotactic gradients of migratory and inhibitory signals. Therefore, there
should be an established hierarchy between the signaling cascades mediated by
intermediary chemotactic factors and end target factors, leading to target specific
recruitment of neutrophils to the site of infection. Studies conducted on the mutual
influence of multiple chemotactic factors in human neutrophils led to the concept of
heterologous desensitization. These studies demonstrated that formylated peptide
receptor-mediated signaling can uni-directionally desensitize signaling cascades mediated
by C5a and IL-8 receptors (11;70-73) upon sequential exposure in vitro (in calcium
14
assays and in under agarose chemotactic migration assays). Functional studies performed
by Heit et al. (69) further indicated a functional crosstalk between signaling cascades
induced by chemoattractants. Their studies show that C5a and f-MLF employ the p38
MAPK pathway, while IL-8 preferentially activates downstream events via PI3K
activation. Moreover, their studies also show that during chemotaxis, neutrophils migrate
efficiently along the chemotactic gradient of the f-MLF, in spite of experiencing optimal
and opposing gradient of IL-8. Neutrophils experiencing higher local concentrations of
IL-8 still migrated toward end-target chemoattractants. These results, along with unique
nature of bovine neutrophils to respond to formylated peptides, indicate that the signaling
hierarchy between end-target and intermediary chemotactic factors in bovine neutrophils
will be distinct from that in human neutrophils. Hence, we explored the ability of IL-8
and C5a to desensitize their receptors reciprocally.
15
A
Neutrophil activation
Tethering Rolling Adhesion
Arrest
Diapedsis
Shear flow
Endothelium
Epithelium
End Target
Concentration
Gradient
TLR Stimulation
Endotoxins
On Epithelial cells
C5a+ C5a des arg
Pathogen
C5 from Serum
N
C
C
C
N
Β
C
N
N
IL-8, PAF, LTB4
C
N
LFA-1
IL-8, PAF, LTB4
TNF-α, LPS, IL-1β
TNF-α, LPS, IL-1β,C5a
N
C
Neutrophil
LFA-1
Mac-1
Intermediary
chemotactic
factor
Chemotactic receptor
PSGL-1
Selectin
ICAM
Figure 3: Signaling during neutrophil activation and migration (A&B): A schematic
representation of C5a-induced inflammation during Gram-negative bacterial infection: C5a
formed on the surface of bacteria by the alternate complement pathway mediates recruitment of
neutrophils (Figure A) by activating endothelium, stimulating cytokine production and mediating
chemotaxis of neutrophils along its concentration gradient (74). Figure B represents the
interaction between neutrophil and activated endothelium at the inflammatory synapse, which
leads to trans-endothelial migration.
16
The C5a Receptor Expression on Bovine Peripheral Blood Leukocytes:
In the present study, we tested whether C5a receptor is expressed on bovine nonadherent as well as plastic-adherent bovine peripheral blood leukocytes cultured for 72
hours in vitro using both C5a des arg FITC and anti-C5a receptor antibody 3D5. These
studies were performed because the human C5a receptor is constitutively expressed on
monocytes, and neutrophils (75). Moreover, it mediates the recruitment of monocytes
and their differentiation indirectly through the induction of TNF-α and prostaglandin E2
(76). It also induces IL-8 gene expression in human peripheral blood mononuclear cells
via the NF-κB pathway (31). In addition, recent experiments in mouse macrophage cells
demonstrate that C5a negatively regulates the Toll-like receptor pathway to regulate the
inflammation mediated by bacterial cell wall components, such as LPS, through C5a
receptor expression (77;78). These studies demonstrate C5a receptor’s function in the
myloid cells. Human T lymphocytes (5.8%) constitutively express C5a receptor, and
phytohaemaglutinin stimulation led to an increased number of T cells that exhibited C5a
receptor expression (79). Studies conducted by Kim et al. (80) further demonstrated the
importance of C5a receptor in the generation of anti-viral CD8+ T cells in vivo,
presumably via activation of antigen-presenting cells and T cells through an unknown
mechanism. Thus, investigation into the expression profiles of the bovine C5a receptor
will shed some light on its function in bovine immunity, since the spatial and temporal
C5a receptor expression profiles on cells that participate in innate immune response will
determine the outcome of C5a mediated inflammatory response. It will also determine
whether the functions of C5a receptor are species-specific.
17
The mutational analysis of human C5a receptor demonstrated that N-terminus
aspartic acid residues (at positions 10, 15, and 16, respectively) are required for the
recognition of C5a by its receptor, with the C5a binding residues residing between amino
acids 21-30. Further studies demonstrated that when aspartic acid residues 21 and 27
were mutated in the human C5a receptor, the extent of phosphorylation at the C-terminus
of the receptor decreased compared to that on the wild-type receptor (81-83). Recent
studies on S. aureus protein interaction with human C5a receptor (84) and studies on
human C5a receptor by Siciliano et al. (85) suggested the N-terminus of the C5a receptor
is important for tethering to its ligand, which serves as the primary site for C5a receptorligand interaction. Taking these data into account, the generation of a monoclonal
antibody to this region is not only important to investigate the C5a receptor cellular
expression, but also to produce an antibody that may interfere with the binding of C5a to
its cognate receptor. Therefore, generation of a monoclonal antibody to the predicted
hydrophilic N-terminus of the bovine C5a receptor and expression analysis of the C5a
receptor on bovine peripheral blood leukocytes and epithelial cells is important to
understand the pleiotropic effects of the C5a receptor in bovine inflammation.
Effect of C5a on The Function of Epithelial Cells-A Possible
Functional Interaction between Toll-Like Receptor (TLR) and C5a Receptor
In an inflammatory condition like bovine mastitis, the first cell to sense pathogens is
the epithelial cell. Hence, the epithelial cell surface acts as a first line of defense and
mediates the secretion of proinflammatory cytokines, such as IL-8, upon sensing
pathogens (86). The earliest sensors of pathogen-associated molecular patterns such as
LPS (present in the cell wall of Gram-negative bacteria), and lipoteichoic acid (a cell wall
18
component of Gram-positive bacteria) are Toll-like receptors (TLR). These receptors
belong to the interleukin-1 receptor superfamily, type-one transmembrane proteins and
mediate signaling cascades by engaging myloid differentiation factor-88 (an intracellular
adaptor protein), which in turn activates NF-κB via interleukin receptor-associated kinase
(for review see (87)). TLRs are differentially expressed on the epithelial cell surface, and
these cells play an important role in microbial pathogenesis as a first line of defense by
secreting proinflammatory cytokines. The expression of TLR-4 and -2 increases in
bovine mammary glands during mastitis (88), implicating TLRs not only in microbial
pathogenesis of the mammary gland, but also in mediating protection against tissue injury
during inflammation by maintaining the integrity of epithelial cells by endogenous
ligands (89). In addition, in vitro studies on the effect of LPS or lipoteichoic acid, the
ligands for TLR-4 and 2, demonstrated their ability to induce proinflammatory cytokines
in bovine mammary epithelial cells, as well as in Mac-T cells (a cell line that serves as a
cellular model for bovine epithelial cells). Bovine mammary epithelial cells, as well as
Mac-T cells upregulate proinflammatory cytokine genes, such as IL-8, IL-1β, TNF-α,
and NF-κB in response to LPS or lipoteichoic acid (90;91), which is mediated via the
TLR pathway. Recent data in rat alveolar epithelial cells indicate that C5a acts
synergistically with LPS to induce proinflammatory cytokine gene production (22).
Moreover, human bronchial epithelial cells constitutively express C5a receptor. Upon
activation, C5a receptor mediates IL-8 secretion (92). Hence, one can hypothesize that
C5a mediates IL-8 secretion via C5a receptor in mammary epithelial cells in the presence
of LPS, similar to that in human mononuclear cells (93).
19
Studies conducted by Liu et al. (94) on transepithelial migration of neutrophils
through IL-1β−treated epithelial monolayers demonstrated that IL-8 is the major
chemotactic factor secreted by epithelial cells leading to transepithelial migration of
neutrophils with from the basolateral to apical surface of epithelial cells. However, C5a
may also act as a gatekeeper for TLR signaling when there is continuous exposure of
epithelial cells to bacteria, controlling the production of proinflammatory cytokines. For
example, recent studies in mouse macrophages on the ability of C5a to interfere with
TLR-mediated signaling cascades demonstrated its potency to negatively regulate TLRmediated secretion of IL-12 (95).
Investigation into bovine mastitis demonstrated that there is upregulation of
proinflammatory cytokines, such as IL-8, IL-1β, TNF-α, and IL-6. Epithelial cells, a
component of innate immunity, readily respond to bacterial cell wall components and
initiate inflammation (91;96;97). Investigations on immortalized squamous mammary
epithelial cells, Mac-T cells, demonstrated that these cells are capable of secreting large
amounts of IL-8 (98-100), and upregulate proinflammatory cytokine gene expression
upon stimulation with LPS or lipoteichoic acid (101). Mac-T cells secrete IL-8 and TNFα, bind to lactoferrin (102), and upregulate CXCL5, caspase activators, IL-6, and
RANTES gene transcription in response to LPS. However, the role of C5a in modulating
the function of epithelial cells has not been addressed. Studies in other vertebrate models
of inflammation (22) demonstrated that C5a can act synergistically with LPS to elicit a
proinflammatory response by the synthesis of IL-1β, IL-6, and TNF-α. The expression
of C5a receptor is in turn regulated by these proinflammatory cytokines (103-106). This
20
study also demonstrated the autocrine effect of C5a on its gene expression in rat alveolar
epithelial cells. Based on these studies, we hypothesized that bovine mammary epithelial
cells express C5a receptor. The detection of the bovine C5a receptor will be a first step
towards the exploration of its function in epithelial cells, as this will lead to investigations
testing the ability of C5a to interfere with LP-induced secretion of IL-8 in Mac-T cells.
C5a may act either synergistically or antagonistically with the TLR-mediated signaling
cascade to regulate the inflammatory process and regulate the extent of pathogenmediated infection.
The expression of the C5a receptor on epithelial cells may regulate epithelial cell
function in inflammation, such as LPS induced IL-8 secretion, which has been shown to
be an important chemokine in bovine inflammatory models. Therefore, we used antibovine C5a receptor-specific monoclonal antibodies and FITC-labeled C5a des arg to
probe for receptor expression on Mac-T cells (107). RT-PCR analysis demonstrated gene
transcription, and FACS analysis showed the presence of surface C5a receptor
expression.
Overall, this study will address the structure of the C5a receptor, the reciprocal effect
of IL-8 and C5a on induction of intracellular calcium flux, the expression of the C5a
receptor in bovine peripheral blood leukocytes, and the expression of the C5a receptor in
mammary epithelial cells.
21
Methods
TRIzol reagent was from Invitrogen. DNA-free was from Ambion. Pfu turbo DNA
polymerase was from Stratagene. pGEM-Teasy, pCDNA 3.1 and pCDNA 3.1 cmyc/His, pertussis toxin, human recombinant IL-8, and goat anti-mouse IgG (H+L) Fab
fragment-fluorescently labeled (FITC or PE) were obtained from Promega, Invitrogen,
Calbiochem (San Diego, CA), and Jackson Laboratories respectively. All the
oligonucleotide PCR primers were obtained from Integrated DNA technologies (IDT).
Anti-L-selectin antibody- DREG56 was gift from Dr. Mark Jutila, Montana State
University, Bozeman, MT.
Animals
Holstein calves were maintained according to MSU animal care and use.
Cloning and Expression of Bovine C5a Receptor
Cloning: Bovine C5a receptor cDNA was cloned by 5’-rapid amplification of cDNA
ends (5’-RACE). Cells from bovine bone marrow, liver, and kidney were extracted after
hypotonic lysis of red blood cells (RBC) for 40 seconds. Total RNA was synthesized
from these cells using TRIzol reagent, according to the manufacturer’s instructions.
Genomic DNA contamination was eliminated by treating total RNA with DNA-free
treatment, per manufacturer’s instructions prior to cDNA synthesis. A partial bovine
C5aR cDNA was generated using the following primers:
Forward primer 5’-GACCGCTGTATTTGTGTCCTG-3’ and
Reverse primer 5’-GGACGACTGGACTTAATCAAG-3’.
22
Based on the bovine C5a receptor sequence information obtained, the following
primers were designed to clone the entire coding sequence of bovine C5a receptor by the
3’-RACE technique. The following 5’-RACE primer was used to generate 5’ ends of the
bovine C5a receptor: 5’-GAGTAGGGCAACCAGAAGATAAAGAAGCT-3’.
The cDNA synthesis and rapid amplification of cDNA ends were performed
according to the manufacturer’s instructions. The cycling parameters for 5’-RACE were
5 cycles of 94oC for 5 seconds followed by 72oC, 5 cycles of 94oC for 5 seconds followed
by 70oC for 10 seconds and 72oC for 3 minutes, and 26 cycles of 94oC for 5 seconds
followed by 69oC for 10 seconds and 72oC for 3 minutes. The PCR reaction was diluted
50-fold in Tricine EDTA buffer (1mM Tricine-KOH, pH 8.5, with 1mM EDTA), and
was subjected to nested PCR. The PCR fragment obtained was TA cloned into pGEMTeasy after gel purification and sequenced. The gene-specific 3’-RACE primer was
designed from the 5’-RACE product, and PCR was performed according to the
manufacturer’s instruction.
The following 3’-RACE primer was used to generate the 3’ end of the bovine C5a
receptor: 5’-CGATGTGTGTCGTCGCATACGGTAA-3’.
Generation of Bovine C5a Receptor cDNA The bovine C5a receptor was cloned
using a gene-specific sense primer (based on the sequence information obtained from 5’-,
and 3’-RACE studies) comprising of start site 5’-GATGGACTCCATGGCTCTCA-3’.
The anti-sense primer used in the 3’-UTR region to amplify the complete coding
sequence was, 5’-GAAGCAGGACAAGTCCGAGAA-3’. PCR was performed using Pfu
turbo DNA polymerase, according to the manufacturer’s instructions. The PCR
23
parameters were: initial denaturation for 5 minutes at 94oC followed by 29 cycles of
denaturation at 94oC for 1 minute, annealing at 52oC for 30 seconds and extension at
72oC for 1 minute followed by final extension at 72oC for 10 minutes. The PCR product
was gel-purified and incubated with Taq DNA polymerase in the presence of magnesium
chloride and dNTP mix, and TA cloned into the pGEM-Teasy vector. At least five clones
from three independent PCR experiments were sequenced using ABI sequencing
chemistry and an ABI 377 DNA sequencer.
Plasmid Construction The bovine C5aR PCR fragment obtained was inserted into the
EcoR1 site of pcDNA 3.1, transformed into TOP 10 F’ E. coli, and clones of E. coli
expressing the plasmid were selected for their resistance to ampicillin on LB-agar plates
containing 100 μg/mL ampicillin). The isolated plasmid DNA clones were sequenced
using a vector-specific primer for the T7 promoter sequence. A clone with the bovine
C5aR insert in the right reading frame and orientation was chosen for further analysis.
To generate C5a receptor tagged to a c-Myc epitope at its C-terminus, the receptor
was amplified with a forward primer including a BamH1 site and reverse primer with an
EcoR1 site, as follows:
Forward primer: 5’-CGGATCCAAGATGGACTCCATGGCTCTCA-3’;
Reverse primer: 5’-CAGAATTCCCACTGCCGTGCTCTTCAC-3’
The resulting PCR fragment was cloned into pCDNA3.1/Myc-His (+). The bovine
C5a receptor sequence containing its entire reading frame (devoid of stop codon) was
cloned upstream of the human c-Myc epitope sequences, and the presence of bovine C5a
receptor in the plasmid was confirmed by restriction enzyme digestion analysis with
24
EcoR1 and BamH1. The clones containing insert (approximate size of 1.2Kb) were
chosen and sequenced with a primer for the T7 promoter to confirm the sequence. In
addition, bovine C5aR gene specific primer was used as a sequencing primer to check the
splice site between C5a receptor and the tags, which contained DNA sequence coding for
12 amino acids. The clones containing the C5a receptor sequence in-frame with the cMyc epitope tag were selected for further studies.
Generation of CHO-K1-pCDNA 3.1bovine C5aR
c-Myc/His Stable Cell Line Based on previous studies (108), we chose the CHO-K1
cell line for expression analysis of the bovine C5a receptor. CHO-K1 cells grown to log
phase and at 80% confluence were transfected with 1.5μg pCDNA3.1bovineC5aR-cmyc/His using Lipofectamine. The cells were split 24 hours after transfection and
selected with G418 (500μg/mL) in F12 medium with 10% FBS, 2mM L-glutamine, and
100 units penicillin for 14 days prior to screening stable clones for protein expression by
fluorescent immunocytochemistry as well as by FACS for c-Myc (using mouse antihuman c-myc antibody clone MYC 1-9E 10.2). The stable clones were plated in 8-well
Labtech chamber slides, grown until they were partially confluent, and fixed with freshly
made 4% paraformaldehyde in phosphate buffered saline (PBS), pH 7.4, for 15 minutes
at room temperature. The cells were washed extensively with PBS to remove any residual
paraformaldehyde and blocked with PBS containing 10% normal goat serum (NGS) and
0.01% Triton-X 100 for 1 hr at room temperature (RT). The cells were incubated with
anti c-Myc antibody (1μg/mL) diluted in Tris buffered saline containing 10% NGS and
0.01% Triton X-100, for 1 hr at RT on a rocker. The cells were then washed 5 times with
PBS containing 1.0% NGS and 0.001% Triton-X100. The cells were further incubated
25
with 1:1000 diluted goat anti-mouse IgG1 (H+L) and 1 μM nuclear dye TO-PRO3 for 1
hr at RT before repeating the washing step. The slide was washed with PBS and mounted
using Prolong Anti-fade reagent (Molecular Probes).
DNA Sequencing The plasmid DNA was sequenced using ABI big dye sequencing
chemistry and analyzed on an ABI 377 DNA sequencer.
Clustal W Alignment of Vertebrate C5a Receptor Protein
Sequences and Phylogentic Tree Construction The protein coding sequences of all
known C5a receptors were aligned using the MEGA 3.1 software program. The option of
obtaining Clustal W alignment displaying non-conserved amino acids was chosen. The
phylogenetic tree was constructed using the unweighted pair group method with
arithmetic mean (UPGMA). We assumed that the rate of evolution of C5a receptor is
constant among different lineages. Options for the test were: bootstrap (10000 replicates;
seed=67866), gaps/missing data (complete deletion), model applied (Dayhoff Matrix
Model), all substitutions were included, pattern among lineages were assumed to be the
same (homogeneous), rates among sites were assumed to be uniform rates, number of
sites included in the analysis were 170, and number of bootstrap reps = 10000. If the
bootstrap values were >95%, then the calculation was considered correct.
Analysis of 5’-end of Bovine Genomic DNA Sequence The genomic sequence of
bovine C5a receptor was obtained using its cDNA sequence as query for the bovine
genome database. The sequence was confirmed to contain C5a receptor gene sequence
and analyzed for the gene structure using promoter scan to search for the TATA box
(109). The result obtained was also cross-checked by using the TRANSFAC program
26
(110-112), and by manual inspection. The results presented were obtained after
conservative analysis of 500 base pairs upstream of the initiating codon in the 5’-UTR of
the bovine C5a receptor.
Analysis of C5a Receptor-Mediated Signaling in Bovine Neutrophils
Bovine Neutrophil Isolation Blood from Holstein calves (6-18 months of age) was
collected into tubes containing 5 mM 2, 2-ethylenediaminotetra acetic acid (EDTA).
Neutrophils were isolated by hypotonic lysis of RBC and separation from mononuclear
cells on a two-step Histopaque gradient, as described previously (113). Cells purified by
this technique were 95% pure by flow cytometric analysis and ≥ 98% viable, as
determined by Trypan blue exclusion.
Preparation of Bovine Peripheral Blood Leukocytes Peripheral blood leukocytes
were prepared by incubating 5mL of blood with 30mL of ACK lysis buffer (0.15M
NH4Cl, 1mM KHCO3, and 0.1mM Na2-EDTA at pH 7.4) for 10 minutes at RT prior to
the addition of 15mL of HBSS. The leukocytes were pelleted by centrifugation at 1500
rpm for 10 minutes at 4oC, and resuspended in HBSS at a concentration of 107 cells/mL
for FACS analysis.
FACS Analysis of Bovine Leukocytes Bovine leukocytes (or neutrophils) were
blocked with 10% normal goat serum for 10 minutes at 4°C (106 cells/100μl) prior to the
addition of 100μl of hybridoma supernatant of a relevant antibody, or 100μl of Protein G
purified relevant antibody at a final concentration of 1 μg/mL. The cells were incubated
with primary antibody for 30 minutes at 4°C. As an isotype control for primary antibody,
27
cells were incubated with an equal concentration of mouse anti-human c-Myc antibody
from clone MYC 1-9E 10.2. The primary antibody was washed from the cells with 5mL
of wash buffer (Hank’s balanced salt solution without calcium and magnesium containing
2% horse serum). Cells labeled with primary antibody were incubated with goat antimouse IgG (H+L) Fab fragment conjugated to fluorophores (FITC or PE) at a dilution of
1: 250 of stock solution for 30 minutes at 4°C, and the washing step was repeated prior to
cell suspension in 300μl of wash buffer. The cells were analyzed using a FACSCaliber,
and data was collected and analyzed using CellQuest software.
Intracellular Calcium Flux Neutrophils were loaded with 2μM Fluo-4/108 cells for
30 minutes at RT in calcium free Hanks balanced salt solution. The cells were washed
with excess volume of the same buffer and resuspended in Hanks balanced salt solution
with 10mM HEPES. Cells were resuspended at a concentration of 108/mL, and 14.4μl of
cell suspension was used in a volume of 144μl. The stimulants were added to achieve the
required concentration in a volume of 160μl. The response was measured at λ540 nm
(F1), after exciting at λ488 nm, and compared with baseline (Fo). In order to obtain
percent inhibition, the ratio between F1 and Fo were calculated. The percent response
was calculated using the following formula :[(ratio F1/Fo of untreated cells- ratio F1/Fo
of treated cells) x100 / ratio F1/Fo of untreated cells].
The cells were also loaded with the ratiometric dye Fura-2AM, and intracellular
calcium flux was measured using a Perkin-Elmer LS-50B fluorimeter. The ratio between
λ 340 and 380 nm was calculated.
28
Whenever the desensitization due to one ligand over the other was tested, the
response due to the first ligand was measured until the response returned to baseline,
followed by the addition of another ligand. The above-mentioned formula was used since
the measurements were made in intact cells. Untreated cells were stimulated once with
ligand, and not subjected to further sequential stimulation. This formula was used to
measure the percent reduction in stimulation when cells were sequentially stimulated, in
relation to the cells stimulated once.
Cloning and Expression of Bovine C5a
C5a PCR with Degenerate Primers The C5a sequence (Swiss protein accession
number being P12082) from NCBI was and reverse-translated into cDNA sequence using
the back-translation Tool (available from Markus Fischer distributed over the World
Wide Web by the author, Entelechon GmbH, Regensburg, Germany). The cDNA
sequence obtained was optimized for eukyaryotic gene codon usage, and primers were
designed based on the cDNA sequence (114). Inosine residues were introduced where
amino acid degeneracy was high. PCR was performed using the following
oligonucleotide sequences:
Forward primer: 5’-GCCATGCTNAARAARAARATYGARGA-3'
Reverse primer: 5’-YCTIGGITCYTGCAGRTTYTTRTGRT-3’.
PCR was performed using Taq polymerase on bovine liver cDNA with following
parameters: initial strand dissociation for 3 minutes at 94oC, followed by 3 cycles of 94oC
for strand-dissociation for one minute, annealing at 50oC for 30 seconds, and extension of
strand at 72oC for one minute. This was followed by 30 PCR cycles, where all of the
29
above-mentioned parameters were kept constant, but annealing temperature was changed
to 52oC for 30 seconds. The PCR product with the expected length was gel purified,
ligated into pGEMTeasy vector, and sequenced using ABI sequencing chemistry and ABI
377 DNA sequencer. This sequence was translated, using the Expasy proteomic tools,
and confirmed to be the C5a peptide sequence. This sequence was used as a query to
search for the cDNA sequence in an expression sequence tag database to obtain the
sequence for C5a that confirmed the cloned C5a sequence.
Cloning and Expression of Bovine C5a Bovine C5a was cloned into the pET 30
vector using Nco1 and EcoR1 sites. The splice sites were checked by sequencing to
confirm that C5a was in-frame with the His-tag, and chemically transformed into the BL21 strain of E. coli. The kanamycin resistant bacterial colonies were selected, cultured in
LB medium containing 50μg/mL kanamycin, and protein was induced by the addition of
1mM IPTG at the end of the log phase of the bacterial growth for 6 hours. The cells were
disrupted by sonication, in the presence of the recommended concentrations of Sigma
protease inhibitor cocktail, and total protein was analyzed on 5-15% SDS-PAGE gradient
gels.
Monoclonal Antibody Generation
Immunogen Synthesis A synthetic peptide sequence corresponding to the N-terminus
of predicted the bovine C5ar receptor (NH2-TPDYSDYDKWTSNPDVLVDC-COOH)
was coupled to KLH in order to generate monoclonal antibody against the bovine C5a
receptor (115;116). C5a receptor peptide was synthesized with cysteine at the C-
30
terminus of the peptide to facilitate conjugation of the peptide to keyhole limpet
haemocyanin (KLH) and was linked to KLH through a spacer (sulfo-SMCC). This was
synthesized at Macromolecular Resources (Colorado State University, CO). The
manufacturer also coupled of hapten to KLH and confirmed its identity by mass
spectrometry.
Monoclonal Antibody Generation Mice were immunized with intra-peritoneal
injections of immunogen (100μg) using TiterMax Classic as an adjuvant. The mice were
boosted once by intra-peritoneal injection of immunogen plus TiterMax. ELISA tested
immunoreactivity to the immunogen on day 8 after the boost, either for the peptide or by
the recognition of neutrophils in FACS analysis. In ELISA, both KLH linked peptide as
well as the peptide alone were tested. Once the mouse antiserum was tested positive, the
mouse was boosted with 100μg of immunogen diluted in sterile PBS intravenously. The
mouse splenocytes were extracted from the spleen 4 days after the final injection and
fused with the SP2/0 cell line- a mouse myeloma cell line (animal dissection was
performed according to approved animal care and use by MSU, Bozeman, MT).
The splenocytes were fused to the mouse SP2/0 cell line (mouse myeloma cell line
that does not secrete IgG heavy and light chain and generated from Balb/c) at a ratio of
1:2. The standard hybridoma fusion protocol using polyethylene glycol was used.
The clones resistant to hypoxathine, thymidine, and aminopterin were selected and
screened for their ability to secrete IgG (14 days after fusion), which recognized bovine
neutrophils by FACS analysis. The positive clones were subcloned, and the hybridoma
subclones (derived from a single cell) that were tested positive in FACS analysis were
31
chosen. The desired clone was subcloned once again to establish a stable IgG-secreting
hybridoma clone (designated as 3D5) and was further characterized. The monoclonal
antibody specificity to bovine C5a receptor was confirmed by immunocytochemistry on
bovine C5aR expressing Cos-7 cells.
Characterization of Mouse Anti-bovine C5aR Antibody
Isotype Determination of 3D5 The Isostrip Mouse Monoclonal Antibody Isotyping
Kit from Roche Diagnostics was used to determine the isotype of 3D5, according to the
manufacturer's instructions
Protein G Purification of 3D5 Protein G Sepharose 4 fast flow was used to purify
3D5 from either hybridoma supernatant, or ammonium sulfate precipitates of 3D5.
Briefly, the beads were equilibrated in antibody-binding buffer (20mM phosphate buffer,
pH 7.0 at RT). The antibody in binding buffer was incubated with the beads overnight at
4oC, washed with excess volume of binding buffer until no protein was detected by the
Bradford protein detection method (Biorad), and eluted with glycine buffer (0.1M, pH
3.0). The eluted protein was collected in 1mL aliquots, and equilibrated to pH7.4
immediately with 1M Tris.Cl, pH 9.0. The protein was estimated by the Biorad Bradford
assay method, and fractions with highest protein content were pooled, dialyzed against
PBS (pH 7.4) containing 0.02% sodium azide, and stored at 4oC.
Preparation of Peripheral Blood Mononuclear Cells (PBMC) Blood was collected
from healthy Holstein calves. The peripheral blood leukocytes were obtained after water
lysis and separation on Histopaque 1044 gradients. The mononuclear cells at the
32
interphase of Histopaque and buffer were collected, resuspended in RPMI 1640 medium
(containing 2mM L-glutamine, non-essential amino acids, 100U/μg
penicillin/streptomycin, 10% fetal calf serum), and cultured for 72 hours prior to FACS
analysis of adherent cells. Approximately 1-2x106 cells were cultured per well in 6-well
costar tissue culture plates.
Indirect Enzyme-linked Immunosorbant Assay Immulon plates (Costar) were coated
with 10μg/ml bovine C5a receptor peptide or KLH-C5a receptor peptide in PBS
overnight at 4oC. The ELISA was performed according to standard procedures, and
visualized using goat anti-mouse IgG (H+L)-HRP. The substrate used to detect HRPlinked goat anti-mouse antibody was 2, 2’-azinobis (3-ethylbenzthiazoline sulfonic acid).
The reaction was monitored using a SpectraMax microtiter plate reader at λ 405 nm.
FACS analysis For FACS analysis, the cells (either bovine neutrophils or peripheral
blood leukocytes) were suspended in PBS (Dulbecco’s phosphate buffered saline, pH 7.4
at RT) containing 2% fetal bovine serum at 2x107 cells/ml. 50μl of the cells were
incubated with an equal volume of either hybridoma supernatant or purified 3D5
(1μg/ml) or an irrelevant antibody for one hour at 0oC. 3D5 pre-incubated with the
bovine C5a receptor peptide for 30 minutes at RT was also used to determine the
specificity of the antibody to the epitope. To confirm the hybridomas were secreting
antibody specific to bovine C5aR, the antibody was incubated with different
concentrations of bovine C5aR peptide NH2-TPDYSDYDKWTSNPDVLVD-COOH
ranging from 10μg to 0.01ng/reaction. The primary antibodies were detected using goat
anti-mouse IgG (H+L)-phycoerythrin
33
Immunocytochemistry Cos-7 cells (harvested in log phase) were plated on lab Tek 8well chamber slides to achieve 80% confluence after 24 hour in vitro. They were
transiently transfected with pCDNA 3.1 c-myc/His and pCDNA 3.1 bovine C5aR cmyc/His using Mirus Transit-LT1. The cells were fixed 36 hours after transfection with
fresh 4% paraformaldehyde made in phosphate buffered saline (PBS, pH 7.4) for 15
minutes at RT. The cells were washed extensively with PBS to remove any residual
paraformaldehyde and blocked with PBS containing 10% normal goat serum (NGS) and
0.01% Triton-X100 for 1 hr at RT. The cells were incubated with anti c-Myc antibody
(1μg/ml) in TBS containing 10% NGS and 0.01% Triton X-100 for 1hr at RT on a
rocker. The primary antibody was washed off the cells with PBS containing 1.0% NGS
and 0.001% Triton-X100 before incubating with goat anti-mouse IgG1 conjugated to Cy2 (1:500 dilution). Cells were stained with the nuclear dye TO-PRO3 (1μM) for 1hr at
RT before repeating the washing step. The slide was finally washed with PBS, and
mounted using Prolong Anti-fade reagent (Molecular Probes). The images of stained cells
were collected using a Zeiss LSM510 META confocal microscope with Ar laser at λexc
488 nm and He-Ne laser at λexc 633 .nm using LSM-Meta software.  The images were
compiled as 3-D images using LSM-Meta software and presented using Adobe
Photoshop. The primary antibodies used were 3D5, MYC1.9E10.2, and IgG1κ (54.1) as
an isotype control.
C5a receptor Expression Analysis
The 3D5 antibody and C5a des arg-FITC were used at 1μg/ml and 100nM,
respectively. Isotype-matched control irrelevant antibody was used at a concentration of
34
1μg/ml as well. Cells were blocked for 10 minutes with PBS containing 2% NGS,
incubated with either primary antibody or C5a des arg-FITC for 1 hr at 0oC prior to
washing the cells with PBS containing 2% NGS. The Cells were then incubated with goat
anti-mouse IgG (H+L)-phycoerythrin to visualize anti-C5a receptor (recognized by 3D5)
or for the bovine monocyte-macrophage cell marker (BN 1-80). The cells were analyzed
for the expression of C5a receptor by FACS analysis, and the data were analyzed using
CellQuest software (Becton Dickinson).
C5a Receptor Expression Analysis on Mac-T cells
Cell Culture Mac-T cells were cultured in Dulbecco’s modified eagles’s medium
(DMEM) supplemented with 10% fetal bovine serum, penicillin (100μ/ml), streptomycin
(100μg/ml), and bovine insulin (10μg/ml) at 37oC in a humidified atmosphere of 5%
CO2.
RT-PCR Analysis of the Bovine C5a Receptor The full-length bovine C5a receptor
was cloned using gene-specific sense primer (based on the sequence information obtained
from 5’- and 3’- RACE studies) comprised of the following sequence:
5’-GACCGCTTCCTGCTGGTGTTCAAT-3’;
5’-CTGGTAGGGCAGCCAGAAGACAA-3’.
The PCR was performed using Taq DNA polymerase (Promega) according to the
manufacturer’s instructions. The PCR parameters were: initial denaturation for 5 minutes
at 94oC followed by 30 cycles of denaturation at 94oC for 1minute, annealing at 60oC for
30 seconds, extension at 72oC for 1 minute, followed by final extension step at 72oC for
35
10 minutes. The PCR product was gel purified and TA cloned into pGEM-Teasy vector.
At least three clones were sequenced using ABI sequencing chemistry and an ABI 377
DNA sequencer.
FACS Analysis The cells were removed from the culture substratum by incubating
with 2mM EDTA for 15 minutes at 37oC after washing away the medium from cells with
PBS lacking calcium and magnesium. The 3D5 antibody and C5a des arg-FITC were
used at 1μg/ml and 50nM, respectively. Cells were blocked for 10 minutes with PBS
containing 10% normal goat serum (NGS) on ice and incubated with either primary
antibodies or C5a des arg-FITC for 1 hr at 0oC prior to washing the cells off with PBS
containing 2% NGS. Cells were then incubated with goat anti-mouse IgG (H+L)phycoerythrin to visualize anti C5a receptor (recognized by 3D5). Cells were analyzed
for the expression of C5a receptor by FACS analysis, and the data collected by
FACSCalibur was analyzed using CellQuest software (Becton Dickinson).
Results
Cloning and Analysis of Bovine C5a Receptor
Cloning and Sequence Homology Analysis Bovine C5a receptor cloned using 5’RACE technique has protein sequence homology 69%, 59.4%, 92.5%, 61.6%, and 3.2%
with that of human, mouse, sheep, rat, and trout, respectively. The cloned bovine C5a
receptor sequence has Kozak sequence of AAG ATG G, instead of AAC ATG G/A. The
predicted theoretical pI/Mw for the protein sequence of bovine C5a receptor obtained
from cDNA was 9.06/38983.95 kDa. Moreover, the protein was produced by this coding
36
region when the entire coding frame was cloned into pCDNA 3.1 and in vitro translated
(figure 4).
atggactccatggctctcagcacccctgactactctgattatgataaatggacctcgaac
M D S M A L S T P D Y S D Y D K W T S N
cccgatgtcttggtggatgcctcttcccccacccacagactgcgtgtcccagatgtgatc
P D V L V D A S S P T H R L R V P D V I
gccctggtgatcttcgcggccgtcttcctgatcggggtgcctggcaacgccatggtggtc
A L V I F A A V F L I G V P G N A M V V
tgggtgacagggtccgaggtcaagcggaccgtcaacgccatctggtttctcaacctagcg
W V T G S E V K R T V N A I W F L N L A
gtggccgacttcctctcctgcctggcactgcccatcttgtttacatccatcatccagcac
V A D F L S C L A L P I L F T S I I Q H
aaccactggtcctttggtgatgctgcatgtcgaatcctgccctctctcatccttctcaac
N H W S F G D A A C R I L P S L I L L N
atgtacgccagcatcttgctcctggccaccatcagcgccgaccgcttcctgctggtgttc
M Y A S I L L L A T I S A D R F L L V F
aatcccgtctggtgccagaactaccgaggggcccgcgtggcctgggtggcctgtgccgtg
N P V W C Q N Y R G A R V A W V A C A V
gcctggggcttggctctgctgctgaccataccgtcctttgtgttccgtcaggtccacata
A W G L A L L L T I P S F V F R Q V H I
gaccacttcccacccaagacgatgtgtgtcgtcgcatacggtaaggaccgtgaatatgcg
D H F P P K T M C V V A Y G K D R E Y A
gagaaggccgtggccatcgtccggctggccatgggcttcctggggccgctggtcacgctt
E K A V A I V R L A M G F L G P L V T L
acaatctgttacaccttcctcctgcttcgggcatggagccgcactgccacgcgctccgcc
T I C Y T F L L L R A W S R T A T R S A
aagacgctcaaggtggtggtggccgtggtgaccagcttctttgtcttctggctgccctac
K T L K V V V A V V T S F F V F W L P Y
caggtgacggggatgatgctggcctttttcaatgtgcacggggacagcttcaaactcgtg
Q V T G M M L A F F N V H G D S F K L V
tccagtctagatgccctgtgcgtctccctagcttatatcaactgctgcattaaccccatc
S S L D A L C V S L A Y I N C C I N P I
atttacgtgttcgccgcccagggctttcacgcccggttcctgaagaccctccccgccagg
I Y V F A A Q G F H A R F L K T L P A R
ctccggggcgtgttgactgaagagtcggtgcgcagggagagcaagtcccagacgctctcc
L R G V L T E E S V R R E S K S Q T L S
acggcggataccccggccgtgaagagccaggcagtgtaaagcaaggcctctctgagcgcc
T A D T P A V K S Q A V -
Figure 4: The cDNA sequence of bovine C5a receptor: The cDNA sequence was
obtained using RACE studies. It was translated using Expasy proteomic tools (117), and
found to contain all amino acid that were shown to be functionally important in the
human C5a receptor (4a). Snake plot of bovine C5a receptor was generated using the
RbDg program. The white circles indicate the deletions made by the program, in order to
avoid long extracellular domains. This confirms that the translated amino acid sequence
from cloned bovine C5a receptor is indeed a G-protein coupled receptor (4b).
37
Figure 4-Continued
In vitro and in vivo Expression of Bovine C5a Receptor The bovine C5a receptor
cDNA sequence was cloned into the mammalian expression vector pCDNA 3.1, was
translated in vitro in the presence of radioactive S35-methionine, and subjected to
autoradiography (Figure 5A). C5a receptor was tagged to human c-Myc epitope down
steam of the 3’ coding sequence in order to provide evidence that it was a full-length
cDNA, which can be translated into membrane protein. The bovine C5a receptor with
its native Kozak sequence was cloned into pCDNA 3.1 c-Myc tagged vector devoid of
either Kozak sequence or the initiation codon ATG. A stable CHO-K1 cell line
expressing this chimeric protein successfully generated as shown by
38
immunocytochemistry to human c-Myc epitope tag (figures 5B, 5C, and 5D). These
results demonstrate successful cloning of bovine C5a receptor protein coding cDNA by
RACE, expression of protein in vitro and in vivo, and analysis of protein structure.
This also provided protein sequence information to generate a monoclonal antibody
that can recognize the N-terminal sequence of the bovine C5a receptor, a powerful tool
to detect cell-surface expression of bovine C5a receptor on cells involved in the innate
immune system (Figure 5).
A
B
C
60x
D
60x
100x
Figure 5 The expression bovine C5a receptor in CHO-K1 cell line: In vitro translation of
bovine C5a receptor cloned into mammalian expression vector pCDNA 3.1, using TNT
quick-coupled transcription and translation kit containing rabbit reticulocyte lysates
(Promega), demonstrates that the cloned sequence was indeed translated (A). The antihuman c-Myc mouse monoclonal antibody did not recognize CHO-K1 cells (B), while it
recognized c-Myc tagged C5a receptor expressed in stable CHO-K1 cell line (C and D).
Analysis of Bovine C5a Receptor-Clustal W Alignment A comparison between
bovine C5a receptor and other vertebrate C5a receptor was made, using MEGA 3.1
genetic analysis software (118), in order to understand the relationship between the
bovine C5a receptor and other vertebrate C5a receptors. The clustal W alignment of
bovine C5a receptor amino acid sequence (Figure 6) revealed interesting details, which
39
may have contributed to the unique behavior of bovine C5a receptor in responding to
C5a and C5a des arg. In addition to that, human C5a receptor-specific antibody did not
recognize bovine neutrophils in FACS analysis (data not shown), although mouse antihuman C5a receptor antibody is directed against the N-terminus of human C5a receptor.
The translated sequence has heptahelical G-protein coupled receptor configuration
and unique features, which are different from that of the human, in lacking an N-linked
glycosylation site at the N-terminus of bovine C5a receptor. The amino acid sequence
alignment (Figure 7) showed that the N-terminus of bovine C5aR had an alanine at the
fifth position, unlike that of human, and lacked a putative N-linked glycosylation site. It
differed from that of the human sequence significantly in amino acid composition at the
N-terminus. However, the positions of aspartic acid residues that were shown to be
important for tethering of C5a to the receptor (10 and 15) were present. There was an
insertion of two basic amino acids (histidine followed by arginine) just before the first
transmembrane region. Bovine C5a receptor, however, exhibits variations in amino acids
at positions 42, 43, 61, 67, 68, 65, 121, 192, 240, and 249 that were otherwise highlyconserved in mammalian C5a receptor. Bovine C5a receptor had histidine and arginine
at positions 42 and 43, respectively, and alanine and methionine at positions 57 and 58,
while other vertebrate species had phenylalanine and leucine respectively in those
positions. At position 74, glycine replaced the highly-conserved alanine in bovine C5a
receptor. The highly-conserved threonine (amino acid 231) in the third intracellular
domain, which had been implicated as required region for activating G-protein and
phospholipase (119),was replaced by alanine, and this substitution was seen in ovine
C5aR and trout C5aR only. Another replacement of threonine with alanine in the third
40
intracellular loop was seen at amino acid 240. In transmembrane region VII, bovine C5a
receptor had alanine instead of serine, which was a consensus amino acid present at that
position in other vertebrate species.
Phylogenetic Analysis of Vertebrate C5a Receptors Phylogenetic analysis of
vertebrate C5a receptors (Figure 6, Table 1 and 2) showed that C5a receptors of cow,
sheep, and pig were closely related, when compared to primate C5a receptors and rodent
C5a receptors. This clade was closer than rodent C5a receptors to primate C5a receptors.
98 Human
100
Pan troglodytes
Macaca mulatta
85
99 Pongo pygmaeus
44
Oryctolagus cuniculus
Sus scrofa
59
Bos taurus
98
100
94
Ovis aries
Canis familiaris
Meriones unguiculatus
100
Mus musculus
78
68
Rattus norvegicus
Cavia porcellus
Gallus gallus
Oncorynchus mykiss
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Figure 6: Phylogenetic tree analysis of vertebrate C5a receptors: An UPGMA
phylogenetic tree for C5a receptors generated shows that cow, sheep, and pig C5a
receptors form a clade (120).
41
#Bos_taurus
#Ovis_aries
#Meriones_unguiculatus
#Oryctolagus_cuniculus
#Canis_familiaris
#Sus_scrofa
#Human
#Macaca_mulatta
#Pan_troglodytes
#Pongo_pygmaeus
#Mus_musculus
#Rattus_norvegicus
#Cavia_porcellus
#Gallus_gallus
#Oncorynchus_mykiss
MDSMALSTPD
-------------MDLID.
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LM.SPVPWHT
-MDDMC.ILT
YSDYDKWTSN
---------PTYDYEN.TL
DYTNYDSLGT
.P..GTA.LD
---------DYGHYDDKDT
DYGHYDDKDT
DYGHYDDKDT
DYEHYDDNDM
EIN..HYGTM
ITYDYSDGTP
V.YDYDYN.T
PTPFPDYSDL
EEELSLYNIT
PDVLVDASSP
---------NYYDPVDGP.
L.PSTPVDNT
.NIF..E.LN
---------L.LNTPVDKT
L.ANTPVDKT
L.LNTPVDKT
L.ANTPVDKT
DPNIPADGIH
NPDMPADGVY
FLPDGFVDNY
NN.TFNDIGG
DCEF.KPGGL
THRLRVPDVI
---------IPWMPPG.IV
VR...PTTIV
.PK.S...M.
---------SNT.....IL
SNT.....IL
SNT.....IL
SNT.....IL
LPKRQPG..A
IPKMEPG.IA
VE..SFG.LV
H..IPWTHRV
GPV.GPRHLS
ALVIFAAVFL
---------..I.YS....
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60]
60]
60]
60]
60]
60]
60]
60]
60]
60]
#Bos_taurus
#
#Ovis_aries
#Meriones_unguiculatus
#Oryctolagus_cuniculus
#Canis_familiaris
#Sus_scrofa
#Human
#Macaca_mulatta
#Pan_troglodytes
#Pongo_pygmaeus
#Mus_musculus
#Rattus_norvegicus
#Cavia_porcellus
#Gallus_gallus
#Oncorynchus_mykiss
#Bos_taurus
#Ovis_aries
#Meriones_unguiculatus
#Oryctolagus_cuniculus
#Canis_familiaris
#Sus_scrofa
#Human
#Macaca_mulatta
#Pan_troglodytes
#Pongo_pygmaeus
#Mus_musculus
#Rattus_norvegicus
#Cavia_porcellus
#Gallus_gallus
#Oncorynchus_mykiss
WVTGSEVKRT
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[120]
[[112200]]
[120]
[120]
[120]
[120]
[120]
[120]
[120]
[120]
[120]
[120]
[120]
[120]
[120]
[180]
[180]
[180]
[180]
[180]
[180]
[180]
[180]
[180]
[180]
[180]
[180]
[180]
[180]
[180]
#Bos_taurus
#Ovis_aries
#Meriones_unguiculatus
#Oryctolagus_cuniculus
#Canis_familiaris
#Sus_scrofa
#Human
#Macaca_mulatta
#Pan_troglodytes
#Pongo_pygmaeus
#Mus_musculus
#Rattus_norvegicus
#Cavia_porcellus
#Gallus_gallus
#Oncorynchus_mykiss
DHFPPKTMCV
E.Y.......
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EY....VL.G
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.PYSDSIL.N
E..SF.VY..
.P.ADAE...
QLSTS.SE.L
VAY---GKDR
...---.RGH
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.D.---.HEG
.D.---SGVG
.D.---.R.G
.D.---SH..DH---.H..D.---SH..DH---.H.IN.---.GGS
ID.---S.GP
TD.---.R.I
.D.GAARQH.
GL.---TVAS
EYAEKAVAIV
..........
IKK.R...VL
VR..R.....
VLV.RG...L
F.I.RV..LI
KRR.R.....
KRR.R....A
KRR.R.....
KRR.R.....
FPK......L
FFI...I..L
SKER-...L.
RF..AFT.AL
AW.N---TTA
RLAMGFLGPL
...V......
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LLRAWSRT-........-..KT...K-...T...N-.I.T...K-.I.T...S-...T...R-...T...R-...T...R-...T...R-...T...K-.I.T...K-...T...K-.A.LHA.H-YS..RRGSGV
[240]
[240]
[240]
[240]
[240]
[240]
[240]
[240]
[240]
[240]
[240]
[240]
[240]
[240]
[240]
#Bos_taurus
#Ovis_aries
#Meriones_unguiculatus
#Oryctolagus_cuniculus
#Canis_familiaris
#Sus_scrofa
#Human
#Macaca_mulatta
#Pan_troglodytes
#Pongo_pygmaeus
#Mus_musculus
#Rattus_norvegicus
#Cavia_porcellus
#Gallus_gallus
#Oncorynchus_mykiss
#Bos_taurus
#Ovis_aries
#Meriones_unguiculatus
#Oryctolagus_cuniculus
#Canis_familiaris
#Sus_scrofa
#Human
#Macaca_mulatta
#Pan_troglodytes
#Pongo_pygmaeus
#Mus_musculus
#Rattus_norvegicus
#Cavia_porcellus
#Gallus_gallus
#Oncorynchus_mykiss
#
------ATRS
------....
------....
------G...
------....
------....
------....
------....
------....
------....
------....
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------....
------F..T
GPGRVSEA..
LAYINCCINP
.--------.......V..
...V......
V.........
I--------F.........
F.........
F.........
F.........
.......V..
.......V..
F.........
...A.S.A..
...C.S.L..
AKTLKVVVAV
..........
T..V...A..
T.........
T.........
T...E..A..
T.........
T.........
T.........
T.........
T......M..
T......M..
...V......
RRAAGT.L..
RR..R.I...
IIYVFAAQG---------....M.GH.....V.GK.....L....---------....V.G......V.G......V.G......V.G......M.G......M.G......V.GH.AVRSDDEGSP
LL..CLGR.-
VTSFFVFWLP
..........
.SC.......
.V...I....
.V....L...
.G...I....
.A...I....
.A...I....
.A...I....
.A...I....
.IC..I....
..C.......
.S........
IVT...C...
SL...LC.F.
---------------------------------------------------------------------------------------------------------------------PRSGWDWSCP
----------
YQVTGMMLAF
..........
.....V.M.W
...M..I..L
.......M.L
.....V....
.....I.MS.
.......MS.
.....I.MS.
.......MS.
.....V.I.W
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.H.V.LI..A
LHILDFLVLS
---------------------------------------------------------------------------------------------------------------------RVRRRVEEQH
----------
FNVHGDSFKL
.QPQW.....
LPSASPT..K
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LEPSSPT.L.
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LPPSSPTL.R
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TAPGAALYQG
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---------------------------------------------------------------------------------------------------------------------FGPGGLLPCS
----------
VSSLDALCVS
..........
.KR..S....
AIR..P..IA
..R..S...A
..........
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LKK..S..I.
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LKK..S..I.
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.ER.NS....
TKA...V..A
AMAA.PVLTG
IQLAHT.ALC
---------F
------------------.
---------.
---------.
------------------.
---------.
---------.
---------.
---------.
---------.
---------.
PEDIHNFWA.
---------.
[300]
[300]
[300]
[300]
[300]
[300]
[300]
[300]
[300]
[300]
[300]
[300]
[300]
[300]
[300]
[360]
[360]
[360]
[360]
[360]
[360]
[360]
[360]
[360]
[360]
[360]
[360]
[360]
[360]
[360]
[403]
Figure 7: Clustal W alignment of bovine C5a receptor with vertebrate C5a receptors:
The bovine C5a receptor amino acid sequence was aligned by Clustal W method, after
translating cDNA sequences of the bovine C5a receptor to amino acid sequence.
Alignment shows high sequence homology between transmembrane domains, followed
by intracellular domains and extra cellular domains. Bovine C5a receptor (121).
42
Figure 7-Continued
#
#
#
#
#
#
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#
#
#
#
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.
C
.
T
T
T
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.
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R
-
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.
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E
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-
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I
-
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
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]
]
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]
]
Table 1:
[ 1]
[ 2]
[ 3]
[ 4]
[ 5]
[ 6]
[ 7]
[ 8]
[ 9]
[10]
[11]
[12]
#Bos_taurus
0.03
#Ovis_aries
0.14
#Sus_scrofa
0.18
#Canis_familiaris
0.21
#Homo_sapiens
0.22
#PREDICTED:_Pan
#Oryctolagus_cuniculus 0.24
0.27
#Cavia_porcellus
0.30
#Rattus_norvegicus
0.30
#Mus_musculus
0.74
#PREDICTED:_Gallus
#Oncorhynchus_mykiss 0.79
0.15
0.18
0.21
0.22
0.23
0.28
0.29
0.29
0.74
0.80
0.20
0.20
0.22
0.24
0.28
0.30
0.29
0.74
0.81
0.24
0.24
0.23
0.28
0.32
0.31
0.76
0.78
0.02
0.20
0.29
0.27
0.25
0.69
0.80
0.21
0.29
0.29
0.26
0.70
0.82
0.27
0.27
0.27
0.63
0.73
0.33
0.32 0.12
0.70 0.79 0.79
0.76 0.79 0.79 0.80
Table2:
[ 1]
[ 2]
[ 3]
[ 4]
[ 5]
[ 6]
[ 7]
[ 8]
[ 9]
[10]
[11]
[12]
[13]
[14]
[15]
#Bos_taurus
#Ovis_aries
#Meriones_unguiculatus
#Oryctolagus_cuniculus
#Canis_familiaris
#Sus_scrofa
#Human
#Macaca_mulatta
#Pan_troglodytes
#Pongo_pygmaeus
#Mus_musculus_1
#Rattus_norvegicus
#Cavia_porcellus
#Gallus_gallus
#Oncorynchus_mykiss
0.08
0.41 0.44
0.39 0.37 0.41
0.36 0.38 0.48 0.41
0.25 0.26 0.43 0.41 0.40
0.35 0.38 0.36 0.31 0.41 0.36
0.35 0.38 0.35 0.31 0.42 0.36 0.05
0.35 0.38 0.36 0.31 0.40 0.36 0.01 0.07
0.33 0.36 0.34 0.30 0.41 0.35 0.04 0.01 0.06
0.54 0.52 0.33 0.48 0.56 0.53 0.46 0.46 0.46 0.45
0.44 0.45 0.33 0.42 0.46 0.40 0.39 0.41 0.40 0.40 0.29
0.51 0.53 0.49 0.53 0.61 0.51 0.51 0.51 0.50 0.50 0.62 0.53
1.11 1.12 1.32 1.09 1.25 1.14 1.20 1.21 1.21 1.21 1.41 1.12 1.22
1.39 1.34 1.64 1.44 1.52 1.50 1.48 1.48 1.48 1.49 1.66 1.37 1.55 1.52
Table 1 and 2: A comparison of vertebrate C5a receptors: Table 1 indicates Tamuara-Nei
distances for vertebrate C5a receptor cDNA. Table 2 shows Dayhoff Matrix Model
distance comparison between vertebrate C5a receptor amino acid sequences. The table
shows that bovine C5a receptor is closely related to that of sheep and pig, and distantly
related to that of chicken and trout.
43
5’UTR Sequence Analysis of Bovine C5a Receptor Genomic Sequence The
genomic sequence was obtained using the bovine C5a receptor cDNA as a query
sequence in the bovine genome database. Genomic sequence analysis of the bovine C5a
receptor showed a two-exon structure separated by a single intron, similar to that of
human and mouse C5a receptor genomic sequences. However, the bovine C5a receptor
putative promoter sequence does not contain a TATA box, unlike its human and mouse
counterparts. The first 500 base pairs of the 5’-untranslated region (5’-UTR) sequence of
the bovine C5aR was obtained from the bovine genome (at Ensembl, the search
alignment score was 31 and the E-value was 1.7x10-6). The percentage identity was 100,
which means that the sequence matched to the query is the genomic sequence of bovine
C5a receptor. The first part of the gene is located to the genomic location SCAFFOLD
130237:8331-8361, and the second part of the gene is located to the SCAFFOLD
130237:20216-21475 of the genomic location, AFC01396709 (Figure 8). Analysis of
the 5’-UTR sequence for putative transcription binding sites indicated the presence of
core binding region, PU.1 site, and NF-κB sites and an IL-6 responsive element binding
site juxtaposed with the NF-κB binding site. The sequence also contains SP-1, TF2D,
and UBP-1 (upstream binding protein) sites.
44
Figure 8: 5’-UTR sequence analysis of bovine C5a receptor gene: The genomic sequence
was obtained from a bovine genome database using the C5a receptor cDNA sequence
cloned from bovine bone marrow cDNA.The genomic sequence upstream of the initiation
codon and Kozak sequence lacks a TATA box, has a core binding region, and has sites
for myeloid transcription factors NF-κB, PU.1, and IL-6 responsive binding element
protein.
Activation of Bovine Neutrophils by C5a and C5a des arg
Concentration-dependent activation of bovine neutrophil calcium flux by C5a and
C5a des arg was analyzed. As shown in Figure 9, both agonists induced a similar
response, which is consistent with both C5a and C5a des arg being equally effective at
activation of the bovine receptor. This is is contrast to the human C5a receptor, where
C5a des arg is 100-fold less active as an agonist. Bovine netrophils were also treated with
45
different concentrations of pertussis toxin, an inhibitor of the Gα subunit of G protein.
The results indicate that the C5a receptor induced intra-cellular calcium flux was
marginally inhibited when bovine neutrophils were treated with pertussis toxin for 90
minutes at 37oC (Figure 10). The inhibition achieved by 1μg/mL of pertussis toxin was
nmoles of calcium
released
5% ± 2.5, 7% ± 2.5, and 2.5% ± 1.5 for C5a, C5a des arg and PAF, respectively.
110
100
90
80
70
60
50
40
30
20
10
0
-10
-13 -12 -11 -10
C5a
C5a des arg
EC50
-9
-8
-7
C5a des arg
C5a
1.089e-009 9.887e-010
-6
-5
concentration of stimulant in M
(Log M)
Figure 9: Intracellular calcium flux induced by C5a and C5a des arg in bovine
neutrophils: Bovine neutrophils stimulated with different concentrations of C5a and C5a
des arg induce similar dose response, indicating that these ligands are equally effective in
their ability to stimulate the bovine C5a receptor.
46
10.0
% Inhibition in intracellular
calcium flux
C5a 10nM
C5a des arg 10nM
PAF 100nM
7.5
5.0
2.5
0
0.1
0.5
1.0
0.0
Concentration of pertussis toxin in μg/ml
Figure 10: Effect of pertussis toxin (ptx) on C5a receptor-mediated intracellular calcium
flux in bovine granulocytes: Bovine neutrophils were analyzed for the ability of C5a
receptor to induce intracellular calcium flux. PAF was used as a control. The change in
the absorbance of Fluo-4, AM was measured at λ 538 nm. The ratio between baseline and
response was used to calculate percent inhibition due to pertussis toxin (mean ± S.D).
Pertussis toxin had a marginal effect on C5a receptor induced intracellular calcium flux,
implicating pertussis toxin insensitive Gα subunits (n=2).
C5a Receptor Stimulated L-selectin Shedding in Bovine Neutrophils
Neutrophils prepared from bovine peripheral blood were stimulated with different
chemotactic factors at 100 nM, including C5a, C5a des arg, platelet activating factor
(PAF), and IL-8, as well as a neutrophil activator, phorbol 12-myristate 13-acetate
(PMA). Stimulated neutrophils shed L-selectin when stimulated for 30 minutes at 37oC;
however, complete loss of L-selectin was not seen, which does occur human neutrophils
due to either PMA or C5a (Figure 11). The percent loss of surface L-selectin on the the
neutrophil surface was 56%, 53.64%, and 44.9 due to C5a, C5a des arg and PAF,
respectively, which is significant when compared to unstimulated cells (p=0.01 between
47
control and treatments). PMA induced 45% of L-selectin loss when compared to that on
unstimulated cells held at RT. Interestingly, 100 nM IL-8 induced marginal loss of Lselectin (insignificant loss of 17.8%) and did not synergized with C5a to induce Lselectin shedding, although the combined action of 100 nM IL-8 and 100 nM C5a
induced 50% loss of L-selectin, which was significant (p=0.05). The transfer of bovine
neutrophils from ambient temperature to physiological temperature also caused loss of Lselectin; however, it was not significant (22% loss), when compared to chemotactic
factors that mediate signaling via classical GPCRs, such as C5a, C5a des arg, and PAF.
Figure 11: Analysis of L-selectin shedding on bovine neutrophils due to chemotactic
factors: Neutrophils were stimulated with different chemotactic factors for 30 minutes in
a volume of 100 μl and analyzed for the cell surface expression of L-selectin. Cell
preparations from two different calves were analyzed. Statistical analysis was performed
using one-way ANOVA. The comparison between and treatments were made by
Dunnet’s multiple comparison.
48
Cloning of Bovine C5a cDNA
Gennaro et al. (122) purified, characterized bovine C5a peptide and provided with
amino acid sequence information. Nonetheless, there is a lack of information on its
cDNA sequence that is needed to produce recombinant bovine anaphylatoxin C5a and its
truncated form C5a des arg. Hence, cloning of cDNA encoding bovine C5a was achieved
using degenerate polymerase chain reaction (Figure 12b). In addition, the effect of
human C5a on bovine C5a receptor mediated intracellular calcium flux warranted the
need for purified bovine C5a recombinant protein (Figure 12a). Previous studies have
purified bovine C5a or C5a des arg from yeast-activated bovine serum; however, we
cannot completely rule out the possibility of traces of C5a des arg contamination. Hence,
we cloned the cDNA sequence of bovine C5a using degenerate PCR technique with the
primer set:
Forward primer: 5’-GCCATGCTNAARAARAARATYGARGA-3'
Reverse primer: 5’-YCTIGGITCYTGCAGRTTYTTRTGRT-3'
The bovine C5a cDNA obtained was cloned into an expression vector pET 30 (a),
and its expression was induced in E. coli (Figure 12b). However, its activity is yet to be
tested.
49
Response of Neutrophils to hC5a
Bovine PMN+10nMC5a
+10μM ATP+1mM
1μMPAF
PAF
Human PMN
+10nMC5a+10μM ATP
1.12 1.68 1.76
1.3 1.25 1.37
1.03
ATP
C5a
PAF
C5a
ATP
Time in Seconds
Figure 12a: Effect of human C5a on bovine neutrophils: The effect of human C5a, which
is a glycopeptide unlike bovine C5a, was analyzed on bovine neutrophil intracellular
calcium flux. It differed significantly when compared to its ability to activate human
neutrophils. PAF was used as a positive control for bovine neutrophils.
Mr
UI
I
Figure 12b: The cDNA sequence and expression of bovine C5a: Bovine C5a cDNA was
cloned by degenerate PCR from bovine liver cDNA. Expression of the protein was
induced with 1 mM IPTG for six hours (Lane I), resolved on a gradient gel (5-15%), and
the total bacterial proteins visualized demonstrated the expression of bovine C5a.
50
C5a Receptor Desensitization
Homologous Desensitization of C5a Receptor Sequential stimulations of the bovine
C5a receptor with C5a and C5a des arg led to the desensitization of the receptor when the
ligand concentration was either equal or ten times greater than that of secondary
stimulant (Figure 13). This indicates that at saturating concentrations of bovine C5a
receptor ligands, receptor desensitization may be achieved.Whenever secondary stimulant
concentration was greater than that of primary stimulant, agonist elicited a response,
although at a reduced level. This may be due to intracellular calcium response by naive
C5a receptors. It has been demonstrated that a few C5a receptors are sufficient to elicit
detectable intracellular calcium flux due to GPCRs in neutrophils (123). When bovine
neutrophils were stimulated with 100nM of ligands, followed by stimulation with 10 nM
of the same ligand, the response was reduced to about 50%. This result can be
extrapolated to the behavior of bovine neutrophils at the site of infection, where the C5a
concentration is high. Both C5a and C5a des arg elicited a similar response, which may
lead to termination of the C5a receptor chemotactic signal at the site of infection.
51
% Reduction in intracellular
calcium flux
0 nM
* ≤ 0.01
125
10 nM
10 nM + 100 nM
100
75
50
100 nM
100 nM + 10 nM
*
10 nM + 10 nM
*
*
25
10 nM + 1 nM
*
1 nM
1nM + 10 nM
1nM + 1 nM
0
C5a
C5a des arg
Concentration (nM)
Figure 13: Homologous desensitization of bovine C5a receptor: Neutrophils stimulated
sequentially with either C5a or C5a des arg had reduced intracellular calcium response
compared to those neutrophils exposed once to identical concentration of these ligands.
The response obtained due to primary stimulation was adjusted to 0% reduced response,
and the response due to subsequent stimulation is expressed as percent reduced
stimulation in relation to uninhibited response. The results represent experiments
repeated at least three times, using neutrophils from two calves, and are expressed as
mean ± S.D. Statistical analysis was performed using one-way ANOVA. The comparison
between and treatments were made by Dunnet’s multiple comparison test.
Heterologous Desensitization of C5a Receptor The mutual reciprocal effect of C5a
receptor stimulation and IL-8 receptor stimulation was addressed by the intracellular
calcium flux assay. IL-8 (10 nM) elicited reduced intracellular flux (15%±2.5) when
cells were exposed to 10nM C5a prior to stimulation with IL-8 (10nM) (Figure 14). C5a
des arg elicited 22% reduction in IL-8 mediated intracellular calcium flux. The reduction
in IL-8-mediated intracellular calcium flux was more pronounced, when cells were
exposed to either C5a or C5a des arg at 10nM, followed by 1nM IL-8 (Figure 14). Here,
the sequential stimulation was performed soon after first ligand stimulation in order to
52
mimic an inflammatory condition where neutrophils experience more than one
chemoattractant at any given time. The results show that there is reciprocal heterologous
desensitization when either C5a receptor or IL-8 receptors are stimulated. The results
indicate that IL-8 receptor stimulation had a pronounced effect on C5a-mediated receptor
stimulation. However, the functional implication of this phenomenon (heterologous
desensitization) has to be tested as a part of future studies. The results deviate
significantly from that of the human C5a receptor (70;124-126). The caveat in the
experiments presented here is the use of human recombinant IL-8. Therefore, these
results should be corroborated with ae functional assay ,such as chemotaxis in a milieu of
IL-8 and C5a, and the significance of heterologous desensitization has to be deciphered.
λ 540
C5a 10 nM+IL8 10 nM
C5a des 10 nM+IL8 10 nM
0+10nM IL-8
Response in seconds
Figure 14: Heterologous desensitization of bovine C5a receptor: Neutrophils stimulated
sequentially with either C5a or C5a des arg and IL-8 elicit reduced intracellular calcium
response compared to those neutrophils exposed once to identical concentration of these
ligands. The response obtained due to primary stimulation was adjusted to 0% reduced
response, and the response due to subsequent stimulation is expressed as percent reduced
stimulation in relation to uninhibited response. The results represent experiments
repeated at least three times, using neutrophils from two calves, and were expressed as
mean ± SEM. Statistical analysis was performed using one-way ANOVA. The
comparison between and treatments were made by Dunnet’s multiple comparison test.
53
25
C5a
C5a des arg
20
15
10
5
nM
-8
+
10
IL
-8
IL
10
nM
+
10
nM
10
nM
IL
1n
M
IL
-8
-8
1n
M
IL
nM
10
+
10
nM
-8
0
10
% Reduction in intracellualr
calcium flux
Figure 14-Continued
Treatment
Production and Characterization of Monoclonal Antibody
to Bovine C5a Receptor Peptide (8-26 amino acids)
Production of a Bovine C5a Receptor Specific Monoclonal Antibody The bovine
C5a receptor deduced amino acid sequence (containing 351 amino acids) demonstrated
that it indeed represents the bovine C5a receptor full-length sequence, belongs to the
rhodopsin subfamily of GPCRs, and hydropathy analysis (Figure 15) shows that the Nterminus contains a hydrophilic region. Based on the information obtained from multiple
sequence alignments presented above, as well as information available on the structural
analysis of human C5a receptor, we chose the N-terminal region of bovine C5a receptor
encompassing amino acids 8-26 to raise a monoclonal antibody to this receptor
54
(35;127;128). Murine polyclonal sera were tested for the presence of antibodies to
recognize bovine neutrophils prior to fusion. After fusion, the antibody producing
hybridoma supernatants were screened for their ability to recognize bovine neutrophils.
The monoclonal antibody thus selected was named 3D5, and it was further characterized.
I
#Bovine_C5aR
#Human_c5aR
#Rat_C5aR
#Dog_C5aR
#Rabbit_C5aR
#Mouse_C5aR
#Trout_C5aR
II
III
IV
MDSMALSTPD
.N.FNYT...
..PISNDSSE
.A..NF.P.E
-AP.EN..Y.
----MN.SFE
--MDDMCSIL
V
VI
YSDYD-KWTS
.GH..D.D.L
ITYDYSDG.P
.P..G-TA.L
.TN..SLG.L
INYDH-YG.M
TEEELSLYNI
VII
NPDVLVDASS
DLNTP..KT.
...MPA.GVY
D.NIF..E.L
D.STP..NTV
D.NIPA.GIH
TDCEF.KPGG
Figure 15: Hydropathy plot of predicted bovine C5a receptor: The predicted amino acid
sequence of the bovine C5a receptor shows a heptahelical tansmembrane structure, with
hydrophilic N-terminus and C-terminus. The Kyte-Doolittle algorithm (129) was used to
generate the hydropathy plot. The region encompassing amino acid sequence used as a
synthetic peptide is depicted in the plot (dark line), and a multiple C5a receptor sequence
alignment of that region is presented.
Cross-reactivity of Bovine C5a Receptor-specific
Antibody with Human Neutrophils FACS analysis of human neutrophils showed
these cells were not recognized by bovine C5a receptor-specific antibody (Figure 16),
despite the presence of consensus aspartic acid residues in the N-terminal sequence,
55
similar to that of human C5a receptor. These data are in agreement with the results we
have obtained with anti-human C5a receptor mouse monoclonal antibody (Pharmingen),
which does not recognize the bovine neutrophil surface in FACS analysis (data not
shown).
Characterization of Monoclonal Antibody (3D5) Directed
Against the Predicted N-Terminal Amino Acid Sequence Of Bovine C5a Receptor
Isotype Determination of 3d5 As determined by isotype analysis, 3D5 belongs to
class IgG1 and contains κ light chain.
Figure 16: Bovine C5a receptor-specific antibody interaction with human neutrophils:
The monoclonal antibody generated against the amino acid sequence of bovine C5a
receptor 8-26 was tested for its cross-reactivity with human neutrophils. As a positive
control, human neutrophils were stained with anti-human mouse monoclonal antibody
56
that recognizes N-terminus of human C5a receptor. The results show that 3D5 does not
cross react with the human C5a receptor.
Indirect enzyme linked Immunosorbant Assay The results show that protein-G
purified 3D5 recognized plate-bound bovine C5a receptor peptide (8-26) with an ED50
Peptide concentration in μg/ml
titer of 0.3 μg/mL. In contrast, 54.1, and antibody of same isotype, failed to bind to the
peptide above the background levels (Figure 17).
Figure 17: Indirect immunosorbant assay for bovine C5a receptor peptide: ELISA was
performed according to standard procedures. Peptide was plated at a concentration of 10
μg/mL, and the concentration of primary antibody was varied across the x-axis. The
ED50 titer of the antibody is 0.3 μg/mL, when peptide was plated at a concentration of 10
μg/mL. As background control, irrelevant antibody of an identical isotype (54.1) was
used to determine the nonspecific binding due to primary antibody. Phosphate-buffered
saline (PBS) alone served as a negative control to estimate the background caused by
secondary antibody.
Binding of 3D5 to bBovine C5a Receptor Expressed in Cos-7 Cells Cos-7 cells
transiently transfected with bovine C5a receptor cloned into pCDNA3.1c-myc/his were
probed with 3D5, mouse anti-c-Myc antibody, and an isotype control (irrelevant mouse
IgG1). Results demonstrated that 3D5 recognizes C5a receptor, and staining was
57
inhibited in the presence of bovine C5a receptor N-terminal peptide (8-26). The staining
pattern of 3D5 was identical to anti-c-Myc antibody staining, which was located at the Cterminus of bovine C5a receptor (Figure 18).
A
B
C
A: Bovine C5a receptor + 3D5
B: Bovine C5a receptor + anti-c-Myc
C: Vector control + anti-c-Myc
D: Bovine C5a receptor + 3D5+C5aR
N-terminus peptide
E: Bovine C5a receptor + Isotype
D
E
control
Figure18: Antibody 3D5 binds specifically to C5a receptor expressed in vivo: Cos-7 cells
transfected with either bovine C5ar-pCDNA3.1 c-myc/His (panels A-D) or pCDNA 3.1
(panel E) c-myc/His were probed with a) 3D5, b) mouse anti-c-myc antibody, c) 3D5
preincubated with 10 μg/mL bovine C5a receptor peptide, d) irrelevant isotype antibody
and e) anti-c-Myc antibody. The monoclonal antibody is specific to bovine C5aR.
3D5 Recognizes Surface C5a Receptor Expression on Bovine Neutrophils Bovine
neutrophils constitutively express C5a receptor, as demonstrated by their physiological
response to C5a or C5a des arg. We further confirmed the presence of C5a receptor on
neutrophils by staining with C5a des arg-FITC (data not presented).
Immunocytochemistry, as well as FACS analysis of bovine neutrophils stained with 3D5
demonstrated the presence of bovine C5a receptor on the neutrophil surface. This
58
interaction could be inhibited by incubating the antibody with N-terminal peptide of the
bovine C5a receptor (Figure 19).
Figure 19a: 3D5 recognize neutrophils in bovine peripheral blood leukocytes: Cytospin
preparations of bovine peripheral blood leukocytes were fixed and processed for
immunocytochemistry with 3D5. Normal mouse serum (A) shows background stain,
while 3D5 (B) stains neutrophils. TOPRO-3 was used to visualize the nucleus.
3D5 + 200μg/ml
bovineC5aR peptide
3D5 + 20μg/ml
bovineC5aR peptide 3D5 + 200μg/ml
random peptide
C5a receptor expression
Figure 19b: FACS analysis of bovine peripheral blood leukocytes with 3D5: Bovine C5a
receptor-specific antibody 3D5 recognizes bovine neutrophils in peripheral blood
leukocytes, and it was competitively inhibited by bovine C5aR peptide (8-26).
59
Surface Expression Analysis of Bovine C5a Receptor on
Peripheral Blood Leukocytes (PBMC) Bovine C5a receptor expression on freshly
isolated bovine mononuclear cells, and cultured plastic-adherent bovine peripheral blood
leukocytes cultured for 72 hours in vitro was probed using 3D5 antibody. The results
show that 3D5 does not recognize bovine mononuclear cells (Figure 20A) or plastic
adherent bovine peripheral blood leukocytes cultured for 72 hours (Figure 20B). This
result was corroborated by lack of binding of C5 des arg-FITC. BN 1-80 antibody was
used to identify monocytes, which recognizes bovine monocytes and macrophages.
Bovine peripheral blood mononuclear cells, whether fresh or cultured, were not
recognized by either 3D5 or bovine C5a des arg-FITC (100nM).
A
A
3D5
15.38%
N
L
B
BN 1-80
20.61%
C
M
Figure 20A: Analysis of bovine C5a receptor expression on peripheral blood leukocytes:
Bovine peripheral blood leukocytes stained with 3D5 and BN1-80 respectively (panels B
and C) show that C5a receptor specific 3D5 does not recognize bovine mononuclear
cells. N= neutrophils, L= lymphocytes, and M= monocytes.
60
60
50
50
40
40
30
30
20
20
10
10
0
0
Is
o
BN
ry
A
b
on
ce
lls
se
co
nd
a
180
-4
4
ty
p
ec
fM
on
LP
tro
C5
-F
l
IT
ad
C
es
10
ar
0n
g
M
FI
TC
10
0n
M
60
3D
5
70
ly
70
% of total events
Mean Fluorescence intensity
mean fluorescence Intensity
Total number of Events
Figure 20B: Analysis of plastic-adherent peripheral bovine blood leukocytes for C5a
receptor expression: Bovine mononuclear cells were cultured in vitro for 72 hours and
analyzed for the expression of C5a receptor and monocyte-mcrophage marker by
antibodies 3D5 and BN-1-80, respectively. C5a des arg FITC was used as a positive
control for C5a receptor expression at saturating concentrations (100 nM), and f-MLFFITC was used as a negative control for fluorescent peptide labeling.
Expression Analysis of Bovine C5a Receptor in Mac-T Cells:
RT-PCR analysis of bovine C5a receptor expression in Mac-T cells C5a receptor
expression analysis by RT-PCR, probed with gene-specific primers spanning intracellular
loop 2 and transmembrane 6, demonstrated that C5a receptor is indeed expressed in MacT cell line (Figure 21). H2O (lane 1) was used as a control for background, which did not
generate specific PCR bands. C5a receptor full-length cDNA cloned into pCDNA 3.1
(lane 2) was used as a positive control (50-100 ng/reaction). Mac-T cDNA was in lane 3
and demonstrated a PCR fragment of the correct size. The PCR fragment was cloned into
61
pGEM-Teasy and sequenced to ensure that the amplified PCR fragment was indeed the
desired C5a receptor fragment.
1
2
3
Figure 21: PCR analysis of the C5a receptor gene in Mac-T cell line. PCR analysis (30
cycles) of Mac-T cells (lane 3) demonstrated the presence of the C5a receptor gene
expression. Positive control of bovine C5a receptor cDNA is shown in lane 2, and
negative control of H2O is shown in lane 1.
FACS Analysis of Bovine C5a Receptor Expression in Mac-T Cells In order for C5a
receptor to modulate epithelial cell function, C5a receptor should be expressed on the
surface of the cells. Mac-T cells cultured for 24 hours were analyzed for their ability to
bind to bovine C5a receptor-specific antibody 3D5 and C5a des arg-FITC (50nM), in
order to determine the surface expression of C5a receptor their surface. The results
indicate that C5a des arg-FITC (50 nM) and 3D5 recognized the same population of cells,
confirming the presence of C5a receptor on the Mac-T cells (Figure 22). Since the MacT cell line is a transformed cell line, all of the cells may not exhibit the same phenotype
in vitro. In addition, the low fluorescence intensity due to 3D5 binding indicates low
expression of C5a receptor, as compared to that in neutrophils (Figure 22).
3D5
62
C5a des arg FITC (50nM)
Figure 22: FACS analysis of bovine C5a receptor on Mac-T cells: Mac-T cells were
stained with 3D5 hybridoma supernatant (upper right) and C5a des arg FITC (50nM)
(lower left) alone, as well as with both reagents simultaneously (lower right). The results
indicate C5a receptor-pecific staining on Mac-T cells. The results represent at least three
replicates.
Discussion
C5a is formed during bovine mastitis, which represents inflammation on the
epithelial cell surface, and its formation is dependent on the nature of the pathogen.
However, the role of bovine C5a-mediated cellular responses to infection has not been
characterized in a complex milieu of inflammatory agents. The evidence gleaned from
mastitis models, nevertheless, provides correlative evidence for a C5a-mediated
inflammatory response. Thus, in order to decipher the importance of C5a-mediated
cellular responses during inflammation, we isolated and expressed cDNA encoding
bovine C5a receptor, along with its 5’-untranslated sequences (Figures 4 and 5).
63
Comparison of gene coding cDNA sequences and protein sequences indicated that bovine
C5a receptor is closely related to the corresponding receptor in sheep and pig, and
distantly related to that in trout and chicken. However, it is phylogenetically closer to
both primate and rodent C5a receptors.
The translated sequence of bovine C5a receptor did not contain a putative N-linked
glycosylation site at the N-terminus, unlike that of its human counterpart. Moreover,
bovine C5a receptor has a histidine at position 42 and an arginine at position 43.
Functionally, histidine can be a proton donor at neutral pH and prefers to form an αhelix, while arginine is a positively charged amino acid at neutral pH with no significant
preference to any of the protein secondary structures. These residues are absent in a
majority of vertebrate C5a receptors, while canine C5a receptor has proline (which aids
in turning of the polypeptide chain) and lysine (positively charged at neural pH and
favors α-helix formation) instead of histidine and arginine. Rat and mouse C5a receptors
have a single amino acid insertion of lysine. This could be of some functional importance
due to positive charges imparted by both histidine and arginine at physiological pH, as
the bovine and chicken C5a receptors have histidine at position 42, while pig has arginine
at position 43. The bovine C5a receptor has alanine and methionine at positions 67 and
68, while other vertebrate species’ receptors have leucine and phenylalanine, respectively
in those positions. The bovine C5a receptor also has glycine, which aids in free rotation
of the polypeptide, at position 64 instead of highly conserved alanine. The highly
conserved threonine (amino acid 241) in the third intracellular domain is replaced by
alanine (position 241 in the bovine C5a receptor), and this substitution is seen in ovine
and trout C5a receptors only. Mutations in this region have been shown to be important
64
for C5a receptor-mediated phospholipase activity (130). Another feature unique to cow,
sheep and pig C5a receptors is that they have alanine instead of serine in the
transmembrane VI region.
Bovine C5a receptor has high sequence divergence with those of other vertebrate
C5a receptors at the N-terminus, third extracellular loop, C-terminus, first intracellular
loop, and second intracellular loop, with decreasing divergence in the order mentioned.
This is in agreement with previous studies on molecular characterization of mouse and
canine C5a receptors (131;132). Bovine C5a receptor has aspertate at amino acid
position 181, which is an exception to other primate C5a receptors, and similar to rodent
and chicken C5a receptors. The studies conducted by Klco et al. (133) demonstrated that
combined mutation of Y181D, F182L, and P184A led to constitutive activation of human
C5a receptor in yeast. In the bovine C5a receptor, histidine is present at position 181,
and aspartic acid is present at 180. However, proline at position 184 is conserved in
ruminants, pig, and primates only. Hence one can hypothesize that this region in the
extracellular loop 2 (EC2) along with structural diversity in EC2 towards transmembrane
V leads to lack of a differential response to C5a and C5a des arg. Anaphylatoxin C5a and
C5a des arg interact with bovine C5a receptor with similar dissociation constants (134)
(Figure 9).
Although bovine C5a receptor has amino acid sequence homology of 69% to that of
human and conserved consensus amino acids that are functionally important, the above
stated differences might have been acquired by the bovine C5a receptor in order to
respond equally well to C5a and its truncated version C5a des arg. This may prolong the
effect of C5a during infection in bovine inflammatory conditions, unlike that in some
65
other vertebrates (27;135), where proteolytic cleavage of C5a to C5a des arg decreases its
affinity to C5a receptor and dampens the effect of C5a receptor-mediated signaling
during inflammation (136-140). Pathogens such as group B streptococci encode for
carboxy peptidases that convert C5a into C5a des arg as part of immune evasion strategy
(141-143). However, studies have not addressed such inactivation of C5a in bovine
mastitis, in spite of group B streptococcal infections mediating mastitis (144;145). Future
studies exploring the functional differences due to these amino acids will elucidate their
specific role in the C5a response during bovine innate immunity (Figures 5 and 6).
Furthermore, analysis of the putative genomic sequence of bovine C5a receptor
obtained from bovine genomic sequence from ENSMBL shows that it has analogous
gene structure to that of human and mouse C5a genes, with two exons separated by one
intron and 30 bases of 5’-UTR sequence. The putative sequence distal to the first intron
encodes 351 amino acids before a termination codon TAA. Analysis 5’-UTR sequence
for promoter elements showed lack of a TATA box in the putative promoter region of
C5a receptor. In addition, the 5’-UTR sequences isolated by 5’-RACE do not have
identical sequence length. Moreover, TF2D, CAAT box, and SP-1 sites are present in the
5’-UTR and can bind to RNA polymerase II. Hence, we hypothesize that bovine C5a
receptor contains a TATA-less promoter, and is under transcriptional regulation of
myloid transcriptions factors such as PU.1, NF-κB, and IL-6. However, this hypothesis
has to be tested in vivo.
Bovine C5a receptor induced intracellular calcium flux via a pertussis toxininsensitive Gα pathway, since pertussis toxin did not abolish intracellular calcium flux
due to C5a and PAF. The studies conducted on trout C5a receptor demonstrated it
66
mediated chemotaxis that was partially inhibited by pertussis toxin (146), while that of
human was coupled to Gα(i) in polymorphonuclear cells, and C5a signaling was
inhibited by pertussis toxin in differentiated HL60 and U937 (61;147;148). Nonetheless,
C5a receptor has been shown to couple to promiscuous pertussis toxin-resistant Gα16,
but not to Gα11 Gαq (62). These results indicate that the C5a receptor can mediate
signaling via G-proteins that are both pertussis toxin-sensitive and insensitive. Thus, the
C5a receptor coupling to different classes of G-proteins may be species-specific, and the
results we obtained show that the bovine C5a receptor may be signaling via pertussis
toxin-insensitive G-proteins. However, a comparison with human neutrophils will rule
out possible experimental artifacts. This result should be supported with an in vitro
biochemical measurement of bovine C5a receptor association with the Gα subunit that is
insensitive to pertussis toxin.
Neutrophils shed L-selectin, a constitutively expressed adhesion molecule on the cell
surface, when stimulated with C5a, PAF, and f-MLF or during inflammation, thus acting
as an early marker for neutrophil activation (149-151). Ivestigation into L-selectin
shedding mediated by C5a, C5a des arg, PAF, and IL-8 on bovine neutrophils indicated
that the classical chemoattractants, which mediate signaling via G-protein coupled
receptors, caused significant L-selectin shedding at a concentration of 100 nM, although
IL-8 did not cause a significant loss of L-selectin. This is contrary to a report that IL-8
can cause L-selectin shedding at 40 ng/ml (152). The result also suggested that IL-8 had
an antagonistic effect on C5a mediated L-selectin shedding, since neutrophils stimulated
with IL-8 and C5a had slightly higher expression of L-selectin when compared to those
67
stimulated with C5a or C5a des arg. The concentration of hrIL-8 used was 100 nM. The
dose-response (1-100 nM) in intracellular calcium flux showed that 1 nM of hrIL-8 was
sufficient to induce intracellular calcium flux. The same concentration was sufficient to
induce chemotaxis of bovine neutrophils. Therefore, in spite of its ability to induce
physiological responses at low concentrations, hr IL-8 did not mediate L-selectin
shedding at 100 nM. PAF-induced L-selectin shedding served as a positive control for
this study, although there are contradictory results on the effect of PAF-mediated bovine
L-selectin shedding on neutrophils (153;154). Analysis of Mac-1 expression only
showed a marginal increase over its constitutive expression (data not presented).
Bovine C5a has been purified from activated serum; however, recombinant protein
required for complete analysis of bovine C5a receptor is not available. Therefore, as a
part this study, we cloned bovine C5a cDNA and expressed it as a recombinant protein
that contained a His tag at its N-terminus. The advantage of cloning bovine C5a is that it
can be expressed in bacteria, since it is not a glycoprotein, without any amino acid
additions to the native sequence.
Therefore, in the present study, molecular and functional analysis bovine C5a
receptor was conducted. We present evidence to support the hypothesis that bovine C5a
receptor is structurally and functionally different from that of human C5a receptor,
stimulates bovine neutrophils via a pertussis toxin-insensitive pathway, and mediates
partial loss of L-selectin when stimulated with 100 nM C5a or C5a des arg. C5a does not
act synergistically with IL-8 in mediating L-selectin shedding, indicating that IL-8mediated signal transduction via CXC receptor may differ from C5a receptor-mediated
stimulation in neutrophils to cause L-selectin shedding. This may have functional
68
significance in the recruitment of bovine neutrophils to the site of inflammation and
reflect in vivo conditions (155). Furthermore, homologous desensitization of bovine C5a
receptor by either C5a or C5a des arg occurs when C5a receptor is sequentially
stimulated with a saturating concentration of C5a. These results indicate that under
persistent high concentrations of C5a or C5a des arg, similar to E. coli-induced bovine
mastitis, the neutrophils will show no further response to high concentrations of C5a or
C5a des arg, and other proinflammatory cytokines such as TNF-α will stimulate
phagocytosis and bacterial killing by reactive oxygen species generation (156-158).
Moreover, the investigation into reciprocal heterologous desensitization mediated by C5a
and IL-8 revealed that under in vitro conditions C5a would be the major neutrophil
activator (Figure 14), since IL-8 and C5a can reciprocally desensitize each other only at
equimolar concentrations. Considering nearly a 100-fold difference in the concentration
of IL-8 and C5a present in the inflamed mammary glad (17) and the ability of C5a or C5a
des arg to desensitize IL-8-mediated intracellular calcium flux at a 10-fold difference
leads to the conclusion that C5a receptor-mediated signaling may act as dominant signal
for neutrophils to reach the site of infection once they egress into mammary gland.
Future studies investigating the effects of IL-8 and C5a on neutrophil chemotaxis in vitro
will further define the role of C5a in neutrophil migration to the mammary gland
(69;159). Finally, the C5a cDNA cloned during this investigation should facilitate
detailed molecular studies on bovine C5a receptor’s ability to interact with both C5a and
C5a des arg.
69
The molecular cloning of bovine C5a receptor provides sequence information to
detect the transcription of bovine C5a receptor in different tissues, such as lungs, liver,
and bone marrow. However, it does not address protein expression. Thus, we sought to
produce a monoclonal antibody, which will facilitate investigation into modulation of
receptor expression during inflammation. Results of this study demonstrate successful
generation of a monoclonal antibody, specific for synthetic peptide based on predicted
the bovine C5a receptor sequence, which can indeed bind to the bovine C5a receptor. It
recognizes transiently expressed the bovine C5a receptor in Cos-7 cells and binds to solid
phase bound peptide in indirect ELISA. The flow cytometric analysis of bovine
neutrophils indicates that these cells bind to anti-C5a receptor (8-26) in a specific
manner, and this binding is inhibited in the presence of synthetic peptide.
Immunocytochemical analysis of bovine neutrophils also corroborates with the above
results. This antibody, however, did not cross-react with human neutrophils, which is in
agreement with lack of reactivity of bovine neutrophils with mouse anti-human C5a
receptor (data not shown). These results suggest that this site-directed monoclonal
antibody specifically interacts with bovine C5a receptor and can be used for the
expression analysis of C5a receptor.
The approach taken in this study was based on the successful generation of
monoclonal antibody to a synthetic peptide of human C5a receptor predicted amino acid
(9-29). Since mutational analysis of human C5a receptor demonstrated that N-terminus
aspartic acid residues are required for C5a binding to its receptor, it was proposed to be
the tethering region for its ligand (160), which forms the recognition site along with the
70
third extracellular loop of the C5a receptor, in a two-site binding model proposed for
C5a receptor (128). By competitive inhibition of this antibody binding to bovine
neutrophils using synthetic peptide, we demonstrate that this antibody epitope lies in the
C5a recognition region of bovine C5a receptor. Lack of interspecies cross-reactivity of
both human and bovine C5a receptor antibodies, despite the presence of consensus
aspartic acid residues at the N-terminus, leads to the conclusion that the epitopes of these
two antibodies are highly species-specific and differ from each other. Further epitope
mapping and interference with C5a mediated function will delineate whether this
antibody can serve as blocking antibody to study C5a-mediated signal transduction. This
will further our understanding of C5aR structural contribution to its unique ability to
interact with both C5a and C5a des arg.
Anaphylatoxin receptor for C5a is expressed on both myeloid and non-myeloid cells
in vertebrates. C5a is a major chemotactic factor in pathogen-induced neutrophilic
inflammation and mediates signaling via its cognate receptor-C5a receptor. Apart from
being a chemotactic factor, it acts as a proinflammatory cytokine in monocytes,
macrophages, and epithelial cells (22;30;161-166). Unless C5a receptor protein
expression is regulated on bovine innate immune cells, there could be enhanced
inflammation due the unique ability of C5a receptor to interact with C5a as well as C5a
des arg. Due to the lack of sequence information, the cellular expression of bovine C5a
receptor has not been addressed so far, in spite of the presence of C5a in bovine mastitis.
The results of the present investigation show that bovine C5a receptor is neither
constitutively expressed on bovine peripheral blood mononuclear cells, nor it is up
regulated after culturing them in vitro. This was further confirmed by stimulating bovine
71
mononuclear cells with Convalin A, IL-2, and phytohaeglutanin for 24 hours in vitro
prior to FACS analysis with 3D5 antibody (data not shown). This is in contrast to the
previous studies performed in human and mice. Future studies probing C5a receptor gene
expression, and upregulation in the presence of proinflammatory cytokines such as IL1β and interferon-γ may elucidate the role of the bovine C5a receptor in immune cells.
Bovine mastitis serves as a model for bovine inflammation, and mammary gland
infection serves as a paradigm to study pathogen-mediated inflammation on the epithelial
surface. Thus, epithelial cells are one of the key players in modulating inflammation
during economically devastating bovine mastitis, via the secretion of proinflammatory
cytokines. The recent studies in other inflammatory models indicate that C5a receptor
modulates epithelial cell function and has the effect of a double-edged sword in
controlling LPS-induced effects (29;167). Since bovine C5a receptor responds to both
C5a and C5a des arg, C5a is the major inflammatory agent formed in mammary gland
soon after infection, and mammary epithelial cells secrete proinflammatory cytokines in
response to bacterial endotoxins, we probed for the expression of C5a receptor in Mac-T
cell line, an in vitro cellular model for bovine squamous mammary epithelial cell line.
RT-PCR analysis of bovine C5a receptor gene expression demonstrated the expression of
C5a receptor in Mac-T cells. Furthermore, FACS analysis of these cells demonstrated
C5a receptor protein expression, which was supported by binding of 50 nM C5a des argFITC to the same C5a des arg-binding populations of cells (Figure 22). However, only a
subpopulation of cells recognized the receptor-specific antibody as well as C5a des argFITC. Nonetheless, this binding was specific, as f-MLF-FITC (50nM) or isotypematched irrelevant antibody did not bind to the cells (data not shown). Thus, we speculate
72
that Mac-T cells, although a good model to represent bovine mammary epithelial cells
may have lost the true nature of bovine mammary epithelial cells due to the
immortalization process and duration in culture. Mac-T cells exhibit significant
differences, both in the propensity to form mammospheres and to respond to endotoxins,
when compared to that of bovine primary mammary epithelial cell cultures (168).
Nevertheless, this is the first report of bovine C5a receptor expression in a squamous
epithelial cell line.
Future studies directed towards understanding the role of C5a receptor on bovine
epithelial cells will shed light on C5a receptor-mediated modulation of inflammation.
Summary
Bovine mastitis is a major pathogen-induced neutrophilic inflammatory disease in
cows and leads to economic losses in dairy industry ($2 billion in the United States alone,
according to the National Mastitis Council) by rendering milk unviable for commercial
use. Acute mastitis caused by Gram-negative bacterial inflammation leads to generation
of proinflammatory factors, such as C5a, IL-8, TNF-α, and IL-1β, and accumulation of
neutrophils at the site of infection. However, the importance of C5a is not obvious due to
lack of the bovine C5a receptor knowledge.
The present study on bovine C5a receptor was conducted to address the gaps that
hinder the understanding of the role of C5a in bovine inflammation. In this study, we
asked whether bovine C5a receptor is significantly different from human C5a receptor,
addressed mutual effects of IL-8 and C5a on their respective receptors, and analyzed
expression of C5a receptor on bovine mononuclear and epithelial cells.
73
The present thesis on bovine C5a receptor demonstrated that it is structurally and
functionally different from that of humans; however, it is more closely related to the
human C5a receptor than that of rodents. The bovine immune system is unique in the lack
of a functional response to formylated peptides, and knowledge about C5a receptor
provides ways to study and understand the role of anaphylatoxin in infectious diseases in
general.
The structural information obtained by cloning and sequencing led to the
development of bovine C5a receptor-specific antibodies as a part of this study. The
sequence analysis demonstrated that bovine C5a receptor retained all the amino acid
absolutely required for its structure and ligand binding; however, it is different from that
of human C5a receptor at the N-terminus, second extra-cellular domain, and lacks an Nlinked glycosylation site. One of the interesting observations was that the monoclonal
antibodies to the N-terminus of the C5a receptor are species-specific, indicating the
difference in structure of the receptor at its N-terminus. Further analysis with anti-bovine
C5a receptor specific monoclonal antibody demonstrated that it does not recognize
human C5a receptor, despite the presence of consensus aspartic acid residues at the Nterminus. C5a receptor-mediated signaling is insensitive to pertussis toxin, similar that of
trout, and is in contrast to human C5a receptor. Furthermore, activation of neutrophils, as
measured by the shedding of L-selectin, indicated that C5a receptor mediates L-selectin
shedding, while IL-8 could not at 100 nM concentration. Taking the in vivo
concentrations of C5a and IL-8 into consideration, one can deduce that C5a and PAF
mediate arrest of leukocyte rolling and not IL-8. The heterologous desensitization of IL-8
by C5a occurs when C5a is 10-fold greater in concentration. Considering the 300-fold
74
difference in mammary gland C5a concentration during E. coli-induced mastitis, one can
conclude that when C5a and IL-8 are generated at the site of infection, C5a will be the
dominant chemoattractant. However, when C5a receptor was stimulated with 100 nM
C5a or C5a des arg, the response to 10 nM C5a was reduced, thus leads to the speculation
that neutrophils arrive at the site of infection along the C5a chemotactic gradient,
experience receptor desensitization, and concomitant stimulation with other chemokines
or proinflammatory agents will prime neutrophils to mediate phagocytosis and reactive
oxygen species generation. This needs to be tested in vitro as a part of future studies.
Results on expression analysis of bovine C5a receptor on mononuclear cells
demonstrated that bovine peripheral blood mononuclear cells do not have constitutive
expression of C5a receptor. In vivo mastitis models suggest recruitment of macrophages
to engulf apoptotic neutrophils formed after clearing of infection (169); nonetheless, the
major chemotactic factor to recruit monocytes and macrophages is yet to be discovered in
bovine inflammation. Future studies on C5a receptor gene expression and regulation in
bovine monocytes can shed some light on the enigma surrounding the lack of constitutive
expression of C5a receptor in monocytes.
We also analyzed C5a receptor expression on Mac-T cells, which indicates that C5a
receptor is expressed and may have some functional role in epithelial cells. Using bovine
mastitis as a paradigm to study an inflammation that occurs on epithelial surface,
deciphering the role of C5a receptor in bovine mammary epithelial cells may lead to the
containment of inflammation that is deleterious to the mammary gland. This is the first
report of C5a receptor expression in bovine epithelial cells, and further analysis of C5a
75
receptor function in these cells may lead to better understanding of role of C5a in Gramnegative bacteria-induced acute mastitis.
Future Studies
The predicted amino acid sequence of bovine C5a receptor is different from that of
human C5a receptor and may lead to understanding of its unique ability to respond to
C5a and C5a des arg. Although it has all the motifs and important consensus amino acid
residues, additional mutational analysis of unique substitutions in the bovine C5a receptor
may shed some light on its ability to respond to C5a and C5a des arg. Furthermore, C5a
receptor-mediated signaling induces L-selectin shedding; however, the pathway leading
to L-selectin shedding is yet to be deciphered. C5a receptor-mediated signaling induces
pertussis toxin-insensitive intracellular calcium flux in neutrophils, which is contrary to
studies performed in human neutrophils and similar to the behavior of trout C5a receptor.
Desensitization of C5a receptor due to its ligand is in agreement with that of human C5a
receptor, but requires saturating concentrations of C5a or C5a des arg. However, this
evidence points out that both C5a and C5a des arg can mediate identical cellular
response, i.e., desensitizing the neutrophil response to anaphylatoxin at the site of
infection. Future studies addressing the functional relevance of homo- and heterologous
desensitization by IL-8 and C5a receptors are required. The phagocytic function of
neutrophils may require additional signals along with C5a receptor-mediated signals
(170;171), and a review of literature on bovine mastitis supports this implication
(17;19;20;172;173). Heterologous desensitization experiments to understand the mutual
influence of C5a and IL-8 on their receptor signals indicate that C5a can suppress the
76
effect on IL-8 when cells were exposed to a ten-fold higher concentration of either C5a or
C5a des arg prior to IL-8 exposure. However, sequential stimulation of neutrophils with
equal concentrations of these two chemotactic factors elicited insignificant suppression of
the IL-8 signal. In order to understand the translation of these subtle differences in
calcium signaling into chemotaxis, future studies addressing the mutual influences of
these chemotactic factors in chemotaxis will have to be performed (69;159;174-176). All
the studies performed on human neutrophils have invariably addressed the dominant
effect of f-MLF, which has been proposed to be dominant chemotactic factor over C5a
and IL-8. However, a study performed on the mutual effect of f-MLF and IL-8
demonstrated that IL-8 induced a stop signal during neutrophil migration via
sequestration of cyclic adenosine mono-phosphate (cAMP) and reduced f-MLF-mediated
adhesion of neutrophils on collagen. Therefore, this extends the model proposed by
Foxman et al. (177;178), which proposes cellular memory of a neutrophils of previous
environment as one of the key factors in migrating across multiple chemotactic gradient
and shows that IL-8 provides a stop signal for neutrophils traveling along an f-MLF
gradient (177;178). Studies in bovine mastitis indicated the presence of C5a as well as
IL-8 in mammary gland. Thus, the significance of this finding can only be understood by
investigating the mutual effect of C5a and IL-8 in chemotaxis.
The expression profile of bovine C5a receptor indicates constitutive expression on
neutrophils; nonetheless, it was not expressed on mononuclear cells, unlike that of human
and mouse C5a receptors. Thus, the expression of C5a receptor on monocytes, which can
link the innate and adaptive immune responses, may be under tight regulation of the
proinflammatory response (78). Future studies to understand this lack of constitutive C5a
77
receptor expression on bovine monocytes may shed some light on the role of C5a in the
recruitment of monocytes to the site of infection. Moreover, functional analysis of C5a
receptor expression on mononuclear cells, such as chemotaxis due to C5a and C5a des
arg, will delineate the role of C5a in bovine the inflammatory response (179). In the
absence of a functional response, future studies focusing on C5a receptor message
expression in monocytes and the influence of inflammatory cytokines will further our
understanding of bovine C5a receptor biology.
Recent studies exploring the expression and function of C5a receptor on nonmyeloid cells, e.g., epithelial cells, demonstrated that C5a can act in conjunction with
TLRs and enhance the proinflammatory response. However, in macrophages, it
negatively regulates TLR-mediated inflammation. In the present study, we demonstrated
C5a receptor gene as well as protein expression in a bovine mammary epithelial cell line.
Future studies on the function of C5a receptor in Mac-T cells and its effect on LPSinduced IL-8 expression would shed light on modulation of epithelial cell function by
C5a.
Thus, this thesis forms the basis for the understanding of the bovine C5a receptor,
which may eventually lead to development of low cost pharmacological agents to contain
the damage due to bovine mastitis.
78
REFERENCE LIST
1. Gennaro,R., Pozzan,T., and Romeo,D. 1984. Monitoring of cytosolic free Ca2+ in
C5a-stimulated neutrophils: loss of receptor-modulated Ca2+ stores and Ca2+
uptake in granule-free cytoplasts. Proc. Natl. Acad. Sci. U. S. A 81:1416-1420.
2. Paape,M.J., Schultze,W.D., Corlett,N.J., and Weinland,B.T. 1988. Effect of
abraded intramammary device on outcome in lactating cows after challenge
exposure with Streptococcus uberis. Am. J. Vet. Res. 49:790-792.
3. Paape,M.J., and Corlett,N.J. 1984. Intensification of milk somatic cell response to
intramammary device. Am. J. Vet. Res. 45:1572-1575.
4. Semba,R.D. 2000. Mastitis and transmission of human immunodeficiency virus
through breast milk. Ann. N. Y. Acad. Sci. 918:156-162.
5. Semba,R.D., Kumwenda,N., Taha,T.E., Hoover,D.R., Quinn,T.C., Lan,Y.,
Mtimavalye,L., Broadhead,R., Miotti,P.G., van der,H.L. et al 1999. Mastitis and
immunological factors in breast milk of human immunodeficiency virus-infected
women. J. Hum. Lact. 15:301-306.
6. Bingen,E., Denamur,E., Lambert-Zechovsky,N., Aujard,Y., Brahimi,N.,
Geslin,P., and Elion,J. 1992. Analysis of DNA restriction fragment length
polymorphism extends the evidence for breast milk transmission in Streptococcus
agalactiae late-onset neonatal infection. J. Infect. Dis. 165:569-573.
7. Doepper,S., Kacani,L., Falkensammer,B., Dierich,M.P., and Stoiber,H. 2002.
Complement receptors in HIV infection. Curr. Mol. Med. 2:703-711.
8. Kacani,L., Banki,Z., Zwirner,J., Schennach,H., Bajtay,Z., Erdei,A., Stoiber,H.,
and Dierich,M.P. 2001. C5a and C5a(desArg) enhance the susceptibility of
monocyte-derived macrophages to HIV infection. J. Immunol. 166:3410-3415.
9. Meddows-Taylor,S., Pendle,S., and Tiemessen,C.T. 2001. Altered expression of
CD88 and associated impairment of complement 5a-induced neutrophil responses
in human immunodeficiency virus type 1-infected patients with and without
pulmonary tuberculosis. J. Infect. Dis. 183:662-665.
10. Brown,G.B., and Roth,J.A. 1991. Comparison of the response of bovine and
human neutrophils to various stimuli. Vet. Immunol. Immunopathol. 28:201-218.
11. Ali,H., Richardson,R.M., Tomhave,E.D., Didsbury,J.R., and Snyderman,R. 1993.
Differences in phosphorylation of formylpeptide and C5a chemoattractant
receptors correlate with differences in desensitization. J. Biol. Chem. 268:2424724254.
79
12. Paape,M., Mehrzad,J., Zhao,X., Detilleux,J., and Burvenich,C. 2002. Defense of
the bovine mammary gland by polymorphonuclear neutrophil leukocytes. J.
Mammary. Gland. Biol. Neoplasia. 7:109-121.
13. Rupp,R., and Boichard,D. 2003. Genetics of resistance to mastitis in dairy cattle.
Vet. Res. 34:671-688.
14. Rupp,R., and Boichard,D. 1999. Genetic parameters for clinical mastitis, somatic
cell score, production, udder type traits, and milking ease in first lactation
Holsteins. J. Dairy Sci. 82:2198-2204.
15. Wall,R.J., Powell,A.M., Paape,M.J., Kerr,D.E., Bannerman,D.D., Pursel,V.G.,
Wells,K.D., Talbot,N., and Hawk,H.W. 2005. Genetically enhanced cows resist
intramammary Staphylococcus aureus infection. Nat. Biotechnol. 23:445-451.
16. Hill,A.W. 1981. Factors influencing the outcome of Escherichia coli mastitis in
the dairy cow. Res. Vet. Sci. 31:107-112.
17. Bannerman,D.D., Paape,M.J., Lee,J.W., Zhao,X., Hope,J.C., and Rainard,P. 2004.
Escherichia coli and Staphylococcus aureus elicit differential innate immune
responses following intramammary infection. Clin. Diagn. Lab Immunol. 11:463472.
18. Riollet,C., Rainard,P., and Poutrel,B. 2000. Kinetics of cells and cytokines during
immune-mediated inflammation in the mammary gland of cows systemically
immunized with Staphylococcus aureus alpha-toxin. Inflamm. Res. 49:486-496.
19. Riollet,C., Rainard,P., and Poutrel,B. 2000. Differential induction of complement
fragment C5a and inflammatory cytokines during intramammary infections with
Escherichia coli and Staphylococcus aureus. Clin. Diagn. Lab Immunol. 7:161167.
20. Shuster,D.E., Kehrli,M.E., Jr., Rainard,P., and Paape,M. 1997. Complement
fragment C5a and inflammatory cytokines in neutrophil recruitment during
intramammary infection with Escherichia coli. Infect. Immun. 65:3286-3292.
21. Persson,K., Larsson,I., and Hallen,S.C. 1993. Effects of certain inflammatory
mediators on bovine neutrophil migration in vivo and in vitro. Vet. Immunol.
Immunopathol. 37:99-112.
22. Riedemann,N.C., Guo,R.F., Sarma,V.J., Laudes,I.J., Huber-Lang,M.,
Warner,R.L., Albrecht,E.A., Speyer,C.L., and Ward,P.A. 2002. Expression and
function of the C5a receptor in rat alveolar epithelial cells. J. Immunol. 168:19191925.
80
23. Gennaro,R., Simonic,T., Negri,A., Mottola,C., Secchi,C., Ronchi,S., and
Romeo,D. 1986. C5a fragment of bovine complement. Purification, bioassays,
amino-acid sequence and other structural studies. Eur. J. Biochem. 155:77-86.
24. Woodruff,T.M., Strachan,A.J., Sanderson,S.D., Monk,P.N., Wong,A.K.,
Fairlie,D.P., and Taylor,S.M. 2001. Species dependence for binding of small
molecule agonist and antagonists to the C5a receptor on polymorphonuclear
leukocytes. Inflammation 25:171-177.
25. Rambeaud,M., and Pighetti,G.M. 2005. Impaired neutrophil migration associated
with specific bovine CXCR2 genotypes. Infect. Immun. 73:4955-4959.
26. Caswell,J.L., Middleton,D.M., and Gordon,J.R. 1999. Production and functional
characterization of recombinant bovine interleukin-8 as a specific neutrophil
activator and chemoattractant. Vet. Immunol. Immunopathol. 67:327-340.
27. Gerard,N.P., and Gerard,C. 1991. The chemotactic receptor for human C5a
anaphylatoxin. Nature 349:614-617.
28. Boulay,F., Mery,L., Tardif,M., Brouchon,L., and Vignais,P. 1991. Expression
cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells.
Biochemistry 30:2993-2999.
29. Riedemann,N.C., Guo,R.F., Hollmann,T.J., Gao,H., Neff,T.A., Reuben,J.S.,
Speyer,C.L., Sarma,J.V., Wetsel,R.A., Zetoune,F.S. et al 2004. Regulatory role of
C5a in LPS-induced IL-6 production by neutrophils during sepsis. FASEB J.
18:370-372.
30. Fukuoka,Y., and Medof,E.M. 2001. C5a receptor-mediated production of IL-8 by
the human retinal pigment epithelial cell line, ARPE-19. Curr. Eye Res. 23:320325.
31. Hsu,M.H., Wang,M., Browning,D.D., Mukaida,N., and Ye,R.D. 1999. NFkappaB activation is required for C5a-induced interleukin-8 gene expression in
mononuclear cells. Blood 93:3241-3249.
32. Albrecht,E.A., Chinnaiyan,A.M., Varambally,S., Kumar-Sinha,C., Barrette,T.R.,
Sarma,J.V., and Ward,P.A. 2004. C5a-induced gene expression in human
umbilical vein endothelial cells. Am. J. Pathol. 164:849-859.
33. Riedemann,N.C., Guo,R.F., Gao,H., Sun,L., Hoesel,M., Hollmann,T.J.,
Wetsel,R.A., Zetoune,F.S., and Ward,P.A. 2004. Regulatory role of C5a on
macrophage migration inhibitory factor release from neutrophils. J. Immunol.
173:1355-1359.
81
34. Wetsel,R.A. 1995. Structure, function and cellular expression of complement
anaphylatoxin receptors. Curr. Opin. Immunol. 7:48-53.
35. Mery,L., and Boulay,F. 1993. Evidence that the extracellular N-terminal domain
of C5aR contains amino-acid residues crucial for C5a binding. Eur. J. Haematol.
51:282-287.
36. Huber-Lang,M.S., Sarma,J.V., McGuire,S.R., Lu,K.T., Padgaonkar,V.A.,
Younkin,E.M., Guo,R.F., Weber,C.H., Zuiderweg,E.R., Zetoune,F.S. et al 2003.
Structure-function relationships of human C5a and C5aR. J. Immunol. 170:61156124.
37. Byrne,B., McGregor,A., Taylor,P.L., Sellar,R., Rodger,F.E., Fraser,H.M., and
Eidne,K.A. 1999. Isolation and characterisation of the marmoset gonadotrophin
releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe. J.
Endocrinol. 163:447-456.
38. Bokisch,V.A., and Muller-Eberhard,H.J. 1970. Anaphylatoxin inactivator of
human plasma: its isolation and characterization as a carboxypeptidase. J. Clin.
Invest 49:2427-2436.
39. Pease,J.E., and Barker,M.D. 1993. N-linked glycosylation of the C5a receptor.
Biochem. Mol. Biol. Int. 31:719-726.
40. Laudes,I.J., Chu,J.C., Huber-Lang,M., Guo,R.F., Riedemann,N.C., Sarma,J.V.,
Mahdi,F., Murphy,H.S., Speyer,C., Lu,K.T. et al 2002. Expression and function
of C5a receptor in mouse microvascular endothelial cells. J. Immunol. 169:59625970.
41. Butcher,E.C. 1992. Leukocyte-endothelial cell adhesion as an active, multi-step
process: a combinatorial mechanism for specificity and diversity in leukocyte
targeting. Adv. Exp. Med. Biol. 323:181-194.
42. Butcher,E.C. 1991. Leukocyte-endothelial cell recognition: three (or more) steps
to specificity and diversity. Cell 67:1033-1036.
43. Alon,R., Chen,S., Puri,K.D., Finger,E.B., and Springer,T.A. 1997. The kinetics of
L-selectin tethers and the mechanics of selectin-mediated rolling. J. Cell Biol.
138:1169-1180.
44. Springer,T.A., and Anderson,D.C. 1986. The importance of the Mac-1, LFA-1
glycoprotein family in monocyte and granulocyte adherence, chemotaxis, and
migration into inflammatory sites: insights from an experiment of nature. Ciba
Found. Symp. 118:102-126.
82
45. Springer,T.A. 1995. Traffic signals on endothelium for lymphocyte recirculation
and leukocyte emigration. Annu. Rev. Physiol 57:827-872.
46. Springer,T.A. 1994. Traffic signals for lymphocyte recirculation and leukocyte
emigration: the multistep paradigm. Cell 76:301-314.
47. Albrecht,E.A., Chinnaiyan,A.M., Varambally,S., Kumar-Sinha,C., Barrette,T.R.,
Sarma,J.V., and Ward,P.A. 2004. C5a-induced gene expression in human
umbilical vein endothelial cells. Am. J. Pathol. 164:849-859.
48. Thorp,K.M., Southern,C., Bird,I.N., and Matthews,N. 1992. Tumour necrosis
factor induction of ELAM-1 and ICAM-1 on human umbilical vein endothelial
cells--analysis of tumour necrosis factor-receptor interactions. Cytokine 4:313319.
49. Rot,A., Hub,E., Middleton,J., Pons,F., Rabeck,C., Thierer,K., Wintle,J., Wolff,B.,
Zsak,M., and Dukor,P. 1996. Some aspects of IL-8 pathophysiology. III:
Chemokine interaction with endothelial cells. J. Leukoc. Biol. 59:39-44.
50. Jutila,M.A., Kishimoto,T.K., and Butcher,E.C. 1990. Regulation and lectin
activity of the human neutrophil peripheral lymph node homing receptor. Blood
76:178-183.
51. Simon,S.I., and Green,C.E. 2005. Molecular mechanics and dynamics of
leukocyte recruitment during inflammation. Annu. Rev. Biomed. Eng 7:151-185.
52. Lawson,J.A., Burns,A.R., Farhood,A., Lynn,B.M., Collins,R.G., Smith,C.W., and
Jaeschke,H. 2000. Pathophysiologic importance of E- and L-selectin for
neutrophil-induced liver injury during endotoxemia in mice. Hepatology 32:990998.
53. Ding,Z.M., Babensee,J.E., Simon,S.I., Lu,H., Perrard,J.L., Bullard,D.C.,
Dai,X.Y., Bromley,S.K., Dustin,M.L., Entman,M.L. et al 1999. Relative
contribution of LFA-1 and Mac-1 to neutrophil adhesion and migration. J.
Immunol. 163:5029-5038.
54. Lu,H., Smith,C.W., Perrard,J., Bullard,D., Tang,L., Shappell,S.B., Entman,M.L.,
Beaudet,A.L., and Ballantyne,C.M. 1997. LFA-1 is sufficient in mediating
neutrophil emigration in Mac-1-deficient mice. J. Clin. Invest 99:1340-1350.
55. ez-Fraille,A., Mehrzad,J., Meyer,E., Duchateau,L., and Burvenich,C. 2004.
Comparison of L-selectin and Mac-1 expression on blood and milk neutrophils
during experimental Escherichia coli-induced mastitis in cows. Am. J. Vet. Res.
65:1164-1171.
83
56. Blake,K.M., Carrigan,S.O., Issekutz,A.C., and Stadnyk,A.W. 2004. Neutrophils
migrate across intestinal epithelium using beta2 integrin (CD11b/CD18)independent mechanisms. Clin. Exp. Immunol. 136:262-268.
57. Carrigan,S.O., Weppler,A.L., Issekutz,A.C., and Stadnyk,A.W. 2005. Neutrophil
differentiated HL-60 cells model Mac-1 (CD11b/CD18)-independent neutrophil
transepithelial migration. Immunology 115:108-117.
58. Jutila,M.A., Kishimoto,T.K., and Butcher,E.C. 1990. Regulation and lectin
activity of the human neutrophil peripheral lymph node homing receptor. Blood
76:178-183.
59. Kishimoto,T.K., Jutila,M.A., Berg,E.L., and Butcher,E.C. 1989. Neutrophil Mac1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors.
Science 245:1238-1241.
60. Gennaro,R., Simonic,T., Negri,A., Mottola,C., Secchi,C., Ronchi,S., and
Romeo,D. 1986. C5a fragment of bovine complement. Purification, bioassays,
amino-acid sequence and other structural studies. Eur. J. Biochem. 155:77-86.
61. Rollins,T.E., Siciliano,S., Kobayashi,S., Cianciarulo,D.N., Bonilla-Argudo,V.,
Collier,K., and Springer,M.S. 1991. Purification of the active C5a receptor from
human polymorphonuclear leukocytes as a receptor-Gi complex. Proc. Natl.
Acad. Sci. U. S. A 88:971-975.
62. Amatruda,T.T., III, Gerard,N.P., Gerard,C., and Simon,M.I. 1993. Specific
interactions of chemoattractant factor receptors with G-proteins. J. Biol. Chem.
268:10139-10144.
63. Gennaro,R., Simonic,T., Negri,A., Mottola,C., Secchi,C., Ronchi,S., and
Romeo,D. 1986. C5a fragment of bovine complement. Purification, bioassays,
amino-acid sequence and other structural studies. Eur. J. Biochem. 155:77-86.
64. Monsinjon,T., Gasque,P., Chan,P., Ischenko,A., Brady,J.J., and Fontaine,M.C.
2003. Regulation by complement C3a and C5a anaphylatoxins of cytokine
production in human umbilical vein endothelial cells. FASEB J. 17:1003-1014.
65. Ember,J.A., Sanderson,S.D., Hugli,T.E., and Morgan,E.L. 1994. Induction of
interleukin-8 synthesis from monocytes by human C5a anaphylatoxin. Am. J.
Pathol. 144:393-403.
66. Monsinjon,T., Gasque,P., Chan,P., Ischenko,A., Brady,J.J., and Fontaine,M.C.
2003. Regulation by complement C3a and C5a anaphylatoxins of cytokine
production in human umbilical vein endothelial cells. FASEB J. 17:1003-1014.
84
67. Middleton,J., Neil,S., Wintle,J., Clark-Lewis,I., Moore,H., Lam,C., Auer,M.,
Hub,E., and Rot,A. 1997. Transcytosis and surface presentation of IL-8 by
venular endothelial cells. Cell 91:385-395.
68. Cinamon,G., Shinder,V., and Alon,R. 2001. Shear forces promote lymphocyte
migration across vascular endothelium bearing apical chemokines. Nat. Immunol.
2:515-522.
69. Heit,B., Tavener,S., Raharjo,E., and Kubes,P. 2002. An intracellular signaling
hierarchy determines direction of migration in opposing chemotactic gradients. J.
Cell Biol. 159:91-102.
70. Richardson,R.M., Ali,H., Tomhave,E.D., Haribabu,B., and Snyderman,R. 1995.
Cross-desensitization of chemoattractant receptors occurs at multiple levels.
Evidence for a role for inhibition of phospholipase C activity. J. Biol. Chem.
270:27829-27833.
71. Campbell,J.J., Foxman,E.F., and Butcher,E.C. 1997. Chemoattractant receptor
cross talk as a regulatory mechanism in leukocyte adhesion and migration. Eur. J.
Immunol. 27:2571-2578.
72. Foxman,E.F., Kunkel,E.J., and Butcher,E.C. 1999. Integrating conflicting
chemotactic signals. The role of memory in leukocyte navigation. J. Cell Biol.
147:577-588.
73. Foxman,E.F., Campbell,J.J., and Butcher,E.C. 1997. Multistep navigation and the
combinatorial control of leukocyte chemotaxis. J. Cell Biol. 139:1349-1360.
74. Simon,S.I., and Green,C.E. 2004. Molecular Mechanics and Dynamics of
Leukocyte Recruitment During Inflammation. Annu. Rev. Biomed. Eng.
75. Van Epps,D.E., and Chenoweth,D.E. 1984. Analysis of the binding of fluorescent
C5a and C3a to human peripheral blood leukocytes. J. Immunol. 132:2862-2867.
76. Soruri,A., Riggert,J., Schlott,T., Kiafard,Z., Dettmer,C., and Zwirner,J. 2003.
Anaphylatoxin C5a induces monocyte recruitment and differentiation into
dendritic cells by TNF-alpha and prostaglandin E2-dependent mechanisms. J.
Immunol. 171:2631-2636.
77. Soruri,A., Kim,S., Kiafard,Z., and Zwirner,J. 2003. Characterization of C5aR
expression on murine myeloid and lymphoid cells by the use of a novel
monoclonal antibody. Immunol. Lett. 88:47-52.
78. Hawlisch,H., Belkaid,Y., Baelder,R., Hildeman,D., Gerard,C., and Kohl,J. 2005.
C5a negatively regulates toll-like receptor 4-induced immune responses.
Immunity. 22:415-426.
85
79. Nataf,S., Davoust,N., Ames,R.S., and Barnum,S.R. 1999. Human T cells express
the C5a receptor and are chemoattracted to C5a. J. Immunol. 162:4018-4023.
80. Abe,M., Hama,H., Shirakusa,T., Iwasaki,A., Ono,N., Kimura,N., Hugli,T.E.,
Okada,N., Katsuragi,T., and Okada,H. 2005. Contribution of anaphylatoxins to
allergic inflammation in human lungs. Microbiol. Immunol. 49:981-986.
81. Chen,Z., Zhang,X., Gonnella,N.C., Pellas,T.C., Boyar,W.C., and Ni,F. 1998.
Residues 21-30 within the extracellular N-terminal region of the C5a receptor
represent a binding domain for the C5a anaphylatoxin. J. Biol. Chem. 273:1041110419.
82. Mery,L., and Boulay,F. 1994. The NH2-terminal region of C5aR but not that of
FPR is critical for both protein transport and ligand binding. J. Biol. Chem.
269:3457-3463.
83. Mery,L., and Boulay,F. 1993. Evidence that the extracellular N-terminal domain
of C5aR contains amino-acid residues crucial for C5a binding. Eur. J. Haematol.
51:282-287.
84. Postma,B., Kleibeuker,W., Poppelier,M.J., Boonstra,M., van Kessel,K.P., van
Strijp,J.A., and de Haas,C.J. 2005. Residues 10-18 within the C5a receptor N
terminus compose a binding domain for chemotaxis inhibitory protein of
Staphylococcus aureus. J. Biol. Chem. 280:2020-2027.
85. Siciliano,S.J., Rollins,T.E., DeMartino,J., Konteatis,Z., Malkowitz,L., Van,R.G.,
Bondy,S., Rosen,H., and Springer,M.S. 1994. Two-site binding of C5a by its
receptor: an alternative binding paradigm for G protein-coupled receptors. Proc.
Natl. Acad. Sci. U. S. A 91:1214-1218.
86. McClenahan,D.J., Sotos,J.P., and Czuprynski,C.J. 2005. Cytokine response of
bovine mammary gland epithelial cells to Escherichia coli, coliform culture
filtrate, or lipopolysaccharide. Am. J. Vet. Res. 66:1590-1597.
87. Hopkins,P.A., and Sriskandan,S. 2005. Mammalian Toll-like receptors: to
immunity and beyond. Clin. Exp. Immunol. 140:395-407.
88. Goldammer,T., Zerbe,H., Molenaar,A., Schuberth,H.J., Brunner,R.M., Kata,S.R.,
and Seyfert,H.M. 2004. Mastitis increases mammary mRNA abundance of betadefensin 5, toll-like-receptor 2 (TLR2), and TLR4 but not TLR9 in cattle. Clin.
Diagn. Lab Immunol. 11:174-185.
89. Jiang,D., Liang,J., Fan,J., Yu,S., Chen,S., Luo,Y., Prestwich,G.D.,
Mascarenhas,M.M., Garg,H.G., Quinn,D.A. et al 2005. Regulation of lung injury
and repair by Toll-like receptors and hyaluronan. Nat. Med. 11:1173-1179.
86
90. Goldammer,T., Zerbe,H., Molenaar,A., Schuberth,H.J., Brunner,R.M., Kata,S.R.,
and Seyfert,H.M. 2004. Mastitis increases mammary mRNA abundance of betadefensin 5, toll-like-receptor 2 (TLR2), and TLR4 but not TLR9 in cattle. Clin.
Diagn. Lab Immunol. 11:174-185.
91. Pareek,R., Wellnitz,O., Van,D.R., Burton,J., and Kerr,D. 2005. Immunorelevant
gene expression in LPS-challenged bovine mammary epithelial cells. J. Appl.
Genet. 46:171-177.
92. Floreani,A.A., Heires,A.J., Welniak,L.A., Miller-Lindholm,A., Clark-Pierce,L.,
Rennard,S.I., Morgan,E.L., and Sanderson,S.D. 1998. Expression of receptors for
C5a anaphylatoxin (CD88) on human bronchial epithelial cells: enhancement of
C5a-mediated release of IL-8 upon exposure to cigarette smoke. J. Immunol.
160:5073-5081.
93. Hsu,M.H., Wang,M., Browning,D.D., Mukaida,N., and Ye,R.D. 1999. NFkappaB activation is required for C5a-induced interleukin-8 gene expression in
mononuclear cells. Blood 93:3241-3249.
94. Liu,L., Mul,F.P., Lutter,R., Roos,D., and Knol,E.F. 1996. Transmigration of
human neutrophils across airway epithelial cell monolayers is preferentially in the
physiologic basolateral-to-apical direction. Am. J. Respir. Cell Mol. Biol. 15:771780.
95. Hawlisch,H., Belkaid,Y., Baelder,R., Hildeman,D., Gerard,C., and Kohl,J. 2005.
C5a negatively regulates toll-like receptor 4-induced immune responses.
Immunity. 22:415-426.
96. Boudjellab,N., Chan-Tang,H.S., and Zhao,X. 2000. Bovine interleukin-1
expression by cultured mammary epithelial cells (MAC-T) and its involvement in
the release of MAC-T derived interleukin-8. Comp Biochem. Physiol A Mol.
Integr. Physiol 127:191-199.
97. Boudjellab,N., Chan-Tang,H.S., Li,X., and Zhao,X. 1998. Interleukin 8 response
by bovine mammary epithelial cells to lipopolysaccharide stimulation. Am. J. Vet.
Res. 59:1563-1567.
98. Huynh,H.T., Robitaille,G., and Turner,J.D. 1991. Establishment of bovine
mammary epithelial cells (MAC-T): an in vitro model for bovine lactation. Exp.
Cell Res. 197:191-199.
99. Zavizion,B., Gorewit,R.C., and Politis,I. 1995. Subcloning the MAC-T bovine
mammary epithelial cell line: morphology, growth properties, and cytogenetic
analysis of clonal cells. J. Dairy Sci. 78:515-527.
87
100. Boudjellab,N., Chan-Tang,H.S., Li,X., and Zhao,X. 1998. Interleukin 8 response
by bovine mammary epithelial cells to lipopolysaccharide stimulation. Am. J. Vet.
Res. 59:1563-1567.
101. Strandberg,Y., Gray,C., Vuocolo,T., Donaldson,L., Broadway,M., and Tellam,R.
2005. Lipopolysaccharide and lipoteichoic acid induce different innate immune
responses in bovine mammary epithelial cells. Cytokine 31:72-86.
102. Rejman,J.J., Turner,J.D., and Oliver,S.P. 1994. Characterization of lactoferrin
binding to the MAC-T bovine mammary epithelial cell line using a biotin-avidin
technique. Int. J. Biochem. 26:201-206.
103. Gasque,P., Singhrao,S.K., Neal,J.W., Gotze,O., and Morgan,B.P. 1997.
Expression of the receptor for complement C5a (CD88) is up-regulated on
reactive astrocytes, microglia, and endothelial cells in the inflamed human central
nervous system. Am. J. Pathol. 150:31-41.
104. Riedemann,N.C., Guo,R.F., Neff,T.A., Laudes,I.J., Keller,K.A., Sarma,V.J.,
Markiewski,M.M., Mastellos,D., Strey,C.W., Pierson,C.L. et al 2002. Increased
C5a receptor expression in sepsis. J. Clin. Invest 110:101-108.
105. Burg,M., Martin,U., Rheinheimer,C., Kohl,J., Bautsch,W., Bottger,E.C., and
Klos,A. 1995. IFN-gamma up-regulates the human C5a receptor (CD88) in
myeloblastic U937 cells and related cell lines. J. Immunol. 155:4419-4426.
106. Laudes,I.J., Chu,J.C., Huber-Lang,M., Guo,R.F., Riedemann,N.C., Sarma,J.V.,
Mahdi,F., Murphy,H.S., Speyer,C., Lu,K.T. et al 2002. Expression and function
of C5a receptor in mouse microvascular endothelial cells. J. Immunol. 169:59625970.
107. Aarum,J., Sandberg,K., Haeberlein,S.L., and Persson,M.A. 2003. Migration and
differentiation of neural precursor cells can be directed by microglia. Proc. Natl.
Acad. Sci. U. S. A 100:15983-15988.
108. Pease,J.E., Burton,D.R., and Barker,M.D. 1993. The generation of stable CHO
cell lines expressing very high levels of complement C5A receptors and
subsequent modulation of binding affinity for C5A. Biochem. Mol. Biol. Int.
29:339-347.
109. Prestridge,D.S. 1995. Predicting Pol II promoter sequences using transcription
factor binding sites. J. Mol. Biol. 249:923-932.
110. Wingender,E. 2004. TRANSFAC, TRANSPATH and CYTOMER as starting
points for an ontology of regulatory networks. In Silico. Biol. 4:55-61.
88
111. Wingender,E., Chen,X., Fricke,E., Geffers,R., Hehl,R., Liebich,I., Krull,M.,
Matys,V., Michael,H., Ohnhauser,R. et al 2001. The TRANSFAC system on gene
expression regulation. Nucleic Acids Res. 29:281-283.
112. Wingender,E., Chen,X., Hehl,R., Karas,H., Liebich,I., Matys,V., Meinhardt,T.,
Pruss,M., Reuter,I., and Schacherer,F. 2000. TRANSFAC: an integrated system
for gene expression regulation. Nucleic Acids Res. 28:316-319.
113. Davis,A.R., Clements,M.K., Bunger,P.L., Siemsen,D.W., and Quinn,M.T. 2000.
Cloning and characterization of bovine low molecular weight GTPases (Rac1 and
Rac2) and rho GDP-dissociation inhibitor 2 (D4-GDI). Vet. Immunol.
Immunopathol. 74:285-301.
114. Lathe,R. 1985. Synthetic oligonucleotide probes deduced from amino acid
sequence data. Theoretical and practical considerations. J. Mol. Biol. 183:1-12.
115. Wilkins,M.R., Gasteiger,E., Bairoch,A., Sanchez,J.C., Williams,K.L.,
Appel,R.D., and Hochstrasser,D.F. 1999. Protein identification and analysis tools
in the ExPASy server. Methods Mol. Biol. 112:531-552.
116. Appel,R.D., Bairoch,A., and Hochstrasser,D.F. 1994. A new generation of
information retrieval tools for biologists: the example of the ExPASy WWW
server. Trends Biochem. Sci. 19:258-260.
117. Gasteiger,E., Gattiker,A., Hoogland,C., Ivanyi,I., Appel,R.D., and Bairoch,A.
2003. ExPASy: The proteomics server for in-depth protein knowledge and
analysis. Nucleic Acids Res. 31:3784-3788.
118. Kumar,S., Tamura,K., and Nei,M. 2004. MEGA3: Integrated software for
Molecular Evolutionary Genetics Analysis and sequence alignment. Brief.
Bioinform. 5:150-163.
119. Kolakowski,L.F., Jr., Lu,B., Gerard,C., and Gerard,N.P. 1995. Probing the
"message:address" sites for chemoattractant binding to the C5a receptor.
Mutagenesis of hydrophilic and proline residues within the transmembrane
segments. J. Biol. Chem. 270:18077-18082.
120. Kumar,S., Tamura,K., and Nei,M. 2004. MEGA3: Integrated software for
Molecular Evolutionary Genetics Analysis and sequence alignment. Brief.
Bioinform. 5:150-163.
121. Kumar,S., Tamura,K., and Nei,M. 2004. MEGA3: Integrated software for
Molecular Evolutionary Genetics Analysis and sequence alignment. Brief.
Bioinform. 5:150-163.
89
122. Gennaro,R., Simonic,T., Negri,A., Mottola,C., Secchi,C., Ronchi,S., and
Romeo,D. 1986. C5a fragment of bovine complement. Purification, bioassays,
amino-acid sequence and other structural studies. Eur. J. Biochem. 155:77-86.
123. Sklar,L.A., Hyslop,P.A., Oades,Z.G., Omann,G.M., Jesaitis,A.J., Painter,R.G.,
and Cochrane,C.G. 1985. Signal transduction and ligand-receptor dynamics in the
human neutrophil. Transient responses and occupancy-response relations at the
formyl peptide receptor. J. Biol. Chem. 260:11461-11467.
124. Richardson,R.M., Ali,H., Tomhave,E.D., Haribabu,B., and Snyderman,R. 1995.
Cross-desensitization of chemoattractant receptors occurs at multiple levels.
Evidence for a role for inhibition of phospholipase C activity. J. Biol. Chem.
270:27829-27833.
125. Tomhave,E.D., Richardson,R.M., Didsbury,J.R., Menard,L., Snyderman,R., and
Ali,H. 1994. Cross-desensitization of receptors for peptide chemoattractants.
Characterization of a new form of leukocyte regulation. J. Immunol. 153:32673275.
126. Didsbury,J.R., Uhing,R.J., Tomhave,E., Gerard,C., Gerard,N., and Snyderman,R.
1991. Receptor class desensitization of leukocyte chemoattractant receptors. Proc.
Natl. Acad. Sci. U. S. A 88:11564-11568.
127. Morgan,E.L., Ember,J.A., Sanderson,S.D., Scholz,W., Buchner,R., Ye,R.D., and
Hugli,T.E. 1993. Anti-C5a receptor antibodies. Characterization of neutralizing
antibodies specific for a peptide, C5aR-(9-29), derived from the predicted aminoterminal sequence of the human C5a receptor. J. Immunol. 151:377-388.
128. Siciliano,S.J., Rollins,T.E., DeMartino,J., Konteatis,Z., Malkowitz,L., Van,R.G.,
Bondy,S., Rosen,H., and Springer,M.S. 1994. Two-site binding of C5a by its
receptor: an alternative binding paradigm for G protein-coupled receptors. Proc.
Natl. Acad. Sci. U. S. A 91:1214-1218.
129. Kyte,J., and Doolittle,R.F. 1982. A simple method for displaying the hydropathic
character of a protein. J. Mol. Biol. 157:105-132.
130. Kolakowski,L.F., Jr., Lu,B., Gerard,C., and Gerard,N.P. 1995. Probing the
"message:address" sites for chemoattractant binding to the C5a receptor.
Mutagenesis of hydrophilic and proline residues within the transmembrane
segments. J. Biol. Chem. 270:18077-18082.
131. Gerard,C., Bao,L., Orozco,O., Pearson,M., Kunz,D., and Gerard,N.P. 1992.
Structural diversity in the extracellular faces of peptidergic G-protein-coupled
receptors. Molecular cloning of the mouse C5a anaphylatoxin receptor. J.
Immunol. 149:2600-2606.
90
132. Perret,J.J., Raspe,E., Vassart,G., and Parmentier,M. 1992. Cloning and functional
expression of the canine anaphylatoxin C5a receptor. Evidence for high
interspecies variability. Biochem. J. 288 ( Pt 3):911-917.
133. Klco,J.M., Wiegand,C.B., Narzinski,K., and Baranski,T.J. 2005. Essential role for
the second extracellular loop in C5a receptor activation. Nat. Struct. Mol. Biol.
12:320-326.
134. Gennaro,R., Simonic,T., Negri,A., Mottola,C., Secchi,C., Ronchi,S., and
Romeo,D. 1986. C5a fragment of bovine complement. Purification, bioassays,
amino-acid sequence and other structural studies. Eur. J. Biochem. 155:77-86.
135. Gerard,N.P., Lu,B., Liu,P., Craig,S., Fujiwara,Y., Okinaga,S., and Gerard,C.
2005. An anti-inflammatory function for the complement anaphylatoxin C5abinding protein, C5L2. J. Biol. Chem. 280:39677-39680.
136. Asai,S., Sato,T., Tada,T., Miyamoto,T., Kimbara,N., Motoyama,N., Okada,H.,
and Okada,N. 2004. Absence of procarboxypeptidase R induces complementmediated lethal inflammation in lipopolysaccharide-primed mice. J. Immunol.
173:4669-4674.
137. Campbell,W.D., Lazoura,E., Okada,N., and Okada,H. 2002. Inactivation of C3a
and C5a octapeptides by carboxypeptidase R and carboxypeptidase N. Microbiol.
Immunol. 46:131-134.
138. Campbell,W., Okada,N., and Okada,H. 2001. Carboxypeptidase R is an
inactivator of complement-derived inflammatory peptides and an inhibitor of
fibrinolysis. Immunol. Rev. 180:162-167.
139. Ogasawara,W., Abe,N., Hagio,T., Okada,H., and Morikawa,Y. 1997. Purification
and characterization of a dipeptidyl carboxypeptidase from Pseudomonas sp.
WO24. Biosci. Biotechnol. Biochem. 61:858-863.
140. Ikai,M., Itoh,M., Joh,T., Yokoyama,Y., Okada,N., and Okada,H. 1996.
Complement plays an essential role in shock following intestinal ischaemia in
rats. Clin. Exp. Immunol. 106:156-159.
141. Bohnsack,J.F., Zhou,X.N., Williams,P.A., Cleary,P.P., Parker,C.J., and Hill,H.R.
1991. Purification of the proteinase from group B streptococci that inactivates
human C5a. Biochim. Biophys. Acta 1079:222-228.
142. Hill,H.R., Gonzales,L.A., Knappe,W.A., Fischer,G.W., Kelsey,D.K., and
Raff,H.V. 1991. Comparative protective activity of human monoclonal and
hyperimmune polyclonal antibody against group B streptococci. J. Infect. Dis.
163:792-798.
91
143. Hill,H.R., Bohnsack,J.F., Morris,E.Z., Augustine,N.H., Parker,C.J., Cleary,P.P.,
and Wu,J.T. 1988. Group B streptococci inhibit the chemotactic activity of the
fifth component of complement. J. Immunol. 141:3551-3556.
144. Onile,B.A. 1985. Review of group B streptococci and their infections. Afr. J.
Med. Med. Sci. 14:131-143.
145. McDonald,J.S., McDonald,T.J., and Anderson,A.J. 1975. Characterization of and
bovine intramammary infection by group B Streptococcus agalactiae of human
origin. Proc. Annu. Meet. U. S. Anim Health Assoc.150-156.
146. Holland,M.C., and Lambris,J.D. 2004. A functional C5a anaphylatoxin receptor
in a teleost species. J. Immunol. 172:349-355.
147. Shum,J.K., Allen,R.A., and Wong,Y.H. 1995. The human chemoattractant
complement C5a receptor inhibits cyclic AMP accumulation through Gi and Gz
proteins. Biochem. Biophys. Res. Commun. 208:223-229.
148. Vanek,M., Hawkins,L.D., and Gusovsky,F. 1994. Coupling of the C5a receptor to
Gi in U-937 cells and in cells transfected with C5a receptor cDNA. Mol.
Pharmacol. 46:832-839.
149. Jutila,M.A., Kishimoto,T.K., and Butcher,E.C. 1990. Regulation and lectin
activity of the human neutrophil peripheral lymph node homing receptor. Blood
76:178-183.
150. Jutila,M.A., Berg,E.L., Kishimoto,T.K., Picker,L.J., Bargatze,R.F., Bishop,D.K.,
Orosz,C.G., Wu,N.W., and Butcher,E.C. 1989. Inflammation-induced endothelial
cell adhesion to lymphocytes, neutrophils, and monocytes. Role of homing
receptors and other adhesion molecules. Transplantation 48:727-731.
151. ez-Fraile,A., Meyer,E., Paape,M.J., and Burvenich,C. 2003. Analysis of selective
mobilization of L-selectin and Mac-1 reservoirs in bovine neutrophils and
eosinophils. Vet. Res. 34:57-70.
152. Videm,V., and Strand,E. 2004. Changes in neutrophil surface-receptor expression
after stimulation with FMLP, endotoxin, interleukin-8 and activated complement
compared to degranulation. Scand. J. Immunol. 59:25-33.
153. ez-Fraile,A., Meyer,E., Duchateau,L., and Burvenich,C. 2004. Effect of
proinflammatory mediators and glucocorticoids on L-selectin expression in
peripheral blood neutrophils from dairy cows in various stages of lactation. Am. J.
Vet. Res. 65:1421-1426.
154. Swain,S.D., Bunger,P.L., Sipes,K.M., Nelson,L.K., Jutila,K.L., Boylan,S.M., and
Quinn,M.T. 1998. Platelet-activating factor induces a concentration-dependent
92
spectrum of functional responses in bovine neutrophils. J. Leukoc. Biol. 64:817827.
155. ez-Fraille,A., Mehrzad,J., Meyer,E., Duchateau,L., and Burvenich,C. 2004.
Comparison of L-selectin and Mac-1 expression on blood and milk neutrophils
during experimental Escherichia coli-induced mastitis in cows. Am. J. Vet. Res.
65:1164-1171.
156. Chiang,Y.W., Murata,H., and Roth,J.A. 1991. Activation of bovine neutrophils by
recombinant bovine tumor necrosis factor-alpha. Vet. Immunol. Immunopathol.
29:329-338.
157. Rainard,P., Riollet,C., Poutrel,B., and Paape,M.J. 2000. Phagocytosis and killing
of Staphylococcus aureus by bovine neutrophils after priming by tumor necrosis
factor-alpha and the des-arginine derivative of C5a. Am. J. Vet. Res. 61:951-959.
158. Sample,A.K., and Czuprynski,C.J. 1990. Recombinant bovine interferon-gamma,
but not interferon-alpha, potentiates bovine neutrophil oxidative responses in
vitro. Vet. Immunol. Immunopathol. 25:23-35.
159. Heit,B., and Kubes,P. 2003. Measuring chemotaxis and chemokinesis: the underagarose cell migration assay. Sci. STKE. 2003:L5.
160. Carney,D.F., and Hugli,T.E. 1993. Site-specific mutations in the N-terminal
region of human C5a that affect interactions of C5a with the neutrophil C5a
receptor. Protein Sci. 2:1391-1399.
161. Braunwalder,A.F., Musmanno,D., Galakatos,N., Garlick,R.H., Haston,W.O.,
Rediske,J.J., Wennogle,L., Seligmann,B., and Sills,M.A. 1992. Characterization
of the binding of Bolton-Hunter labeled [125I]C5a to human neutrophil,
monocyte and U-937 cell membranes. Mol. Immunol. 29:1319-1324.
162. Burg,M., Martin,U., Rheinheimer,C., Kohl,J., Bautsch,W., Bottger,E.C., and
Klos,A. 1995. IFN-gamma up-regulates the human C5a receptor (CD88) in
myeloblastic U937 cells and related cell lines. J. Immunol. 155:4419-4426.
163. Ember,J.A., Sanderson,S.D., Hugli,T.E., and Morgan,E.L. 1994. Induction of
interleukin-8 synthesis from monocytes by human C5a anaphylatoxin. Am. J.
Pathol. 144:393-403.
164. Fayyazi,A., Sandau,R., Duong,L.Q., Gotze,O., Radzun,H.J., Schweyer,S.,
Soruri,A., and Zwirner,J. 1999. C5a receptor and interleukin-6 are expressed in
tissue macrophages and stimulated keratinocytes but not in pulmonary and
intestinal epithelial cells. Am. J. Pathol. 154:495-501.
93
165. Furebring,M., Hakansson,L.D., Venge,P., Nilsson,B., and Sjolin,J. 2002.
Expression of the C5a receptor (CD88) on granulocytes and monocytes in patients
with severe sepsis. Crit Care 6:363-370.
166. Haviland,D.L., McCoy,R.L., Whitehead,W.T., Akama,H., Molmenti,E.P.,
Brown,A., Haviland,J.C., Parks,W.C., Perlmutter,D.H., and Wetsel,R.A. 1995.
Cellular expression of the C5a anaphylatoxin receptor (C5aR): demonstration of
C5aR on nonmyeloid cells of the liver and lung. J. Immunol. 154:1861-1869.
167. Hawlisch,H., Belkaid,Y., Baelder,R., Hildeman,D., Gerard,C., and Kohl,J. 2005.
C5a negatively regulates toll-like receptor 4-induced immune responses.
Immunity. 22:415-426.
168. Strandberg,Y., Gray,C., Vuocolo,T., Donaldson,L., Broadway,M., and Tellam,R.
2005. Lipopolysaccharide and lipoteichoic acid induce different innate immune
responses in bovine mammary epithelial cells. Cytokine 31:72-86.
169. Sladek,Z., and Rysanek,D. 2001. Neutrophil apoptosis during the resolution of
bovine mammary gland injury. Res. Vet. Sci. 70:41-46.
170. Smits,E., Burvenich,C., Guidry,A.J., Heyneman,R., and Massart-Leen,A. 1999.
Diapedesis across mammary epithelium reduces phagocytic and oxidative burst of
bovine neutrophils. Vet. Immunol. Immunopathol. 68:169-176.
171. Rainard,P., Riollet,C., Poutrel,B., and Paape,M.J. 2000. Phagocytosis and killing
of Staphylococcus aureus by bovine neutrophils after priming by tumor necrosis
factor-alpha and the des-arginine derivative of C5a. Am. J. Vet. Res. 61:951-959.
172. Rainard,P., Poutrel,B., and Caffin,J.P. 1984. Assessment of hemolytic and
bactericidal complement activities in normal and mastitic bovine milk. J. Dairy
Sci. 67:614-619.
173. Riollet,C., Rainard,P., and Poutrel,B. 2001. Cell subpopulations and cytokine
expression in cow milk in response to chronic Staphylococcus aureus infection. J.
Dairy Sci. 84:1077-1084.
174. Foxman,E.F., Kunkel,E.J., and Butcher,E.C. 1999. Integrating conflicting
chemotactic signals. The role of memory in leukocyte navigation. J. Cell Biol.
147:577-588.
175. Foxman,E.F., Campbell,J.J., and Butcher,E.C. 1997. Multistep navigation and the
combinatorial control of leukocyte chemotaxis. J. Cell Biol. 139:1349-1360.
176. Campbell,J.J., Foxman,E.F., and Butcher,E.C. 1997. Chemoattractant receptor
cross talk as a regulatory mechanism in leukocyte adhesion and migration. Eur. J.
Immunol. 27:2571-2578.
94
177. Blackwood,R.A., Hartiala,K.T., Kwoh,E.E., Transue,A.T., and Brower,R.C. 1996.
Unidirectional heterologous receptor desensitization between both the fMLP and
C5a receptor and the IL-8 receptor. J. Leukoc. Biol. 60:88-93.
178. Kitayama,J., Carr,M.W., Roth,S.J., Buccola,J., and Springer,T.A. 1997.
Contrasting responses to multiple chemotactic stimuli in transendothelial
migration: heterologous desensitization in neutrophils and augmentation of
migration in eosinophils. J. Immunol. 158:2340-2349.
179. Wilson,E., Hedges,J.F., Butcher,E.C., Briskin,M., and Jutila,M.A. 2002. Bovine
gamma delta T cell subsets express distinct patterns of chemokine responsiveness
and adhesion molecules: a mechanism for tissue-specific gamma delta T cell
subset accumulation. J. Immunol. 169:4970-4975.