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Department of Microbiology, Division of Clinical Immunology and Rheumatology, University of Alabama, Birmingham, AL 35294; and
Department of Molecular Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 nM) and a molecular mass ranging from 40 to 65
kDa depending upon the cell type expressing the receptor (6). Like most
chemoattractant receptors, C5a-induced chemotaxis requires C5aR
coupling to the pertussis toxin-sensitive 
subunit of Gi proteins
(Gi) (18). The trafficking of T cells under physiological or pathological conditions involves numerous chemoattractant receptors, the majority of which have been cloned only recently (19). However, until now there was no direct proof of C5aR expression in human T cells. In this report, we provide the first evidence that the C5aR is expressed by human T cells at the protein and mRNA level. Moreover, we demonstrate that human rC5a is a chemotactic factor for unstimulated and PHA-stimulated T cells, and that C5aR+ T cells are present in abundance in the central nervous system (CNS) of rats with experimental allergic encephalomyelitis (EAE). These data suggest that C5aR may play an important role in the trafficking of disease-causing T cells into the CNS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rC5a was prepared as described previously (20). Mouse anti-human CD3, CD4, and CD8 Abs were purchased from Serotec (Washington, DC). FITC-labeled goat anti-rabbit IgG, phycoerythrin-labeled goat anti-mouse IgG, and unlabeled mouse control IgG were supplied by Southern Biotechnology Associates (Birmingham, AL). Unlabeled rabbit control IgG was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Preparation and characterization of rabbit anti-human C5aR Ab
Rabbit anti-human C5aR Ab was prepared by immunizing animals with a peptide corresponding to amino acids 7–24 (TTPDYGHYDDKDTLDLNT) of the extracellular amino-terminus of C5aR, using the multiple antigenic peptide resin technology (Research Genetics, Huntsville, AL). The antisera were characterized by ELISA using the immunizing peptide and by flow cytometry Ab staining in which the Ab bound to the rat basophilic leukemia cell line RBL-2H3 (American Type Culture Collection, Manassas, VA) stably transfected with human C5aR cDNA but not with cells transfected with vector alone.
Cells, cell lines, and cell culture conditions
RBL-2H3 cells stably transfected with either human C3a receptor (C3aR) cDNA or human C5aR cDNA were grown as described previously (21). Jurkat T cells (kindly provided by Dr. Ed Blalock, Department of Physiology and Biophysics, University of Alabama at Birmingham) were maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. In most experiments, human lymphocytes were isolated by elutriation from PBLs of healthy donors. Otherwise, mononuclear cells were isolated by density gradient centrifugation on LymphoH (Atlanta Biologicals, Norcross, GA) and T cell enrichment was performed using nylon wool columns. Both of these procedures yielded lymphocytes of 85–95% purity as assessed by flow cytometry using anti-CD3 Ab staining. When needed for the assessment of C5aR mRNA by RT-PCR, highly purified T cell populations were obtained by cell sorting CD3+ cells on a cell sorter (FACScalibur, Becton Dickinson, Heidelberg, Germany) under sterile conditions. T cells maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 50 µM 2-ME, 100 IU/ml penicillin, and 100 µg/ml streptomycin were cultivated with or without 2.5 or 10 µg/ml PHA (Sigma) for 1–60 h. In some experiments, T cells were treated with cycloheximide (CHX) (10 µg/ml) (Sigma, St. Louis, MO) without PHA stimulation or starting at 2 h before PHA stimulation.
Flow cytometric analysis
In each experiment, 0.5 x 106 to 1 x 106 cells of the enriched T cell preparation were incubated on ice for 30 min with polyclonal rabbit anti-C5aR IgG alone or in combination with anti-CD3, anti-CD4, or anti-CD8 mAbs. Single staining experiments with the CD markers mentioned above were performed in parallel. As a control, cells were incubated with the same concentration of nonimmune polyclonal rabbit IgG or unlabeled control mouse IgG. After two washes, cells were stained with FITC-conjugated anti-rabbit IgG and/or phycoerythrin-conjugated anti-mouse IgG or PBS containing 2% FCS to evaluate nonspecific binding of the secondary Abs. All Abs were used at saturating concentrations. Next, cells were washed twice in PBS, assessed in a FACScan flow cytometer, and analyzed by CellQuest software (Becton Dickinson).
Standard and semiquantitative RT-PCR analyses
Total RNA was isolated from unstimulated and PHA-stimulated enriched T cell preparations, unstimulated and PHA-stimulated cell-sorted T cells, Jurkat T cells, C5aR-transfected RBL cells (RBL-C5aR), and C3aR-transfected RBL cells (RBL-C3aR). RNA was extracted using RNA-STAT (Tel-Test B, Friendswood, TX) according to the manufacturer’s instructions. RNA (1 µg) was reverse transcribed as described previously (15). The reverse-transcribed products were amplified using primers for C5aR (based on sequences 400–421 and 919–940) (obtained from Stratagene, La Jolla, CA) and primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (based on sequences 363–386 and 731–755) (kindly provided by Meg Mosteller-Barnum, Department of Gastroenterology, University of Alabama at Birmingham). Samples were amplified for 35 cycles for C5aR and 25 cycles for GAPDH. In semiquantitative RT-PCR experiments, various numbers of PCR cycles were performed followed by extension for 10 min as described previously (22). In all cases, products were separated on 1.5% agarose gels containing ethidium bromide for visualization and transferred to Nylon filter paper (Micron Separations, Westborough, MA) for Southern blot analysis as described previously using 32P-labeled C5aR and GAPDH cDNA fragments labeled by the random prime method with a kit from Boehringer Mannheim (Mannheim, Germany). Blots were washed at high stringency (final wash with 2 x SSC containing 0.1% SDS at 65°C for 30 min) and exposed to Fuji film at -70°C for 15–30 min. For semiquantitative RT-PCR, the amount of hybridized probe was quantified by analysis on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The intensity of the specific hybridization signal was plotted against the number of PCR cycles to ensure that comparisons were conducted in the linear range. The ratio of C5aR to GAPDH amplification products was determined for each different PCR cycle.
In situ hybridization (ISH) and immunohistochemistry
Female Lewis rats (8–9 wk old) obtained from Charles River France (Cléon, France) were immunized for EAE as described previously (23). EAE rats were killed by CO2 inhalation at different timepoints postimmunization, and central nervous tissue was processed for ISH and immunohistochemistry as reported previously (16). ISH was performed on 8-µm-thick transverse sections using digoxigenin-labeled cRNA and detection by alkaline phosphatase color reaction as described previously (16). C5aR riboprobes were prepared by linearizing a murine C5aR cDNA clone prepared in our laboratory (16), using XbaI and ClaI endonucleases (Promega, Madison, WI) for the generation of antisense and sense riboprobes, respectively. In vitro transcription was performed using an RNA transcription kit (Promega), and probes were labeled with digoxigenin-UTP (Boehringer Mannheim). To identify T cells expressing C5aR mRNA, ISH was combined with immunohistochemistry using anti-TCR mAb (Serotec) specific for T cells. Infiltrating macrophages or activated microglia were identified using the ED1 mAb (Serotec), which binds to a specific moiety expressed on these cells. Immunohistochemistry was initially performed according to an immunoenzymatic staining method supplied by the manufacturer (Vectastain ABC kit, Vector, Burlingame, CA) and as described elsewhere (24). The use of RNase free reagents including diethyl pyrocarbonate-treated phosphate buffer allowed good preservation of the mRNA before ISH was performed. As a control, and to verify that immunohistochemistry procedures did not interfere with ISH, an isotype-matched mouse mAb was used for immunohistochemistry; ISH was performed using both a sense and an antisense C5aR probe.
Migration assay
The ability of human rC5a to chemoattract T cells was evaluated using transwell plates (Costar, Cambridge, MA) with polycarbonate inserts with 8-µm pores (Costar 3422) as described previously (25). Wells with inserts were loaded with 200 µl of migration media (MM) consisting of RPMI 1640 and 0.5% BSA (fraction V; Sigma) and were incubated for 1 h at 37°C, 5% CO2. Next, lower wells were aspirated and loaded with 200 µl of MM alone or containing rC5a at concentrations of 1 or 10 nM. For each condition, experiments were conducted in triplicate. To avoid any possibility of T cell stimulation through the cross-linking of CD3, we purified T cells by negative isolation. Unstimulated T cell preparations were stained with an anti-CD11b Ab (Boehringer Mannheim) recognizing monocyte/macrophages and with an anti-CD19 Ab (Becton Dickinson) recognizing B cells. Cells negative for both markers were sorted (FACScalibur, Becton Dickinson) under sterile conditions. The purity of the T cell population isolated by this procedure was analyzed by FACS analysis and was 98–99% CD3+. Equal numbers of T cells were loaded in each well and incubated for 5 h at 37°C, 5% CO2. The cells in the lower chamber were collected, fixed in 1% paraformaldehyde, transferred to 12 x 75-mm test tubes, and counted using a FACScan for 30 s with a constant flow rate of 140 µl/min as described previously (25). The results of three independent experiments performed on freshly isolated, unstimulated T cells were pooled. In one experiment, T cells were stimulated for 1 h with 10 µg/ml PHA, isolated by negative selection, and submitted to a chemotactic assay as described above.
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Results |
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Using a rabbit polyclonal anti-C5aR Ab, we observed that a
subset of PBL-derived unstimulated T cells were labeled by FACS
analysis (Figs. 1
and
2; Table I). The percentage of C5aR and CD3
double-positive cells in fresh preparations of T cells varied between
individuals, ranging from 1 to 8.6%, and could be up-regulated after
24 h of stimulation with 2.5 µg PHA (Fig. 1). After 60 h of
stimulation with either 2.5 or 10 µg/ml of PHA, T cells consistently
demonstrated a striking up-regulation of C5aR expression on both CD4
and CD8 subsets compared with cells cultivated for 60 h without
PHA (Table I). Moreover, in kinetic studies, PHA-induced C5aR
up-regulation was clearly time-dependent and could be observed as early
as 1 h after PHA stimulation and continued to increase for the
remaining timepoints (Fig. 2). A PHA-induced up-regulation of C5aR
appears to be, in large part, independent of protein synthesis, as
treatment with CHX inhibited increased C5aR expression by only 16% and
29% after 4 h and 12 h of exposure, respectively (Fig. 3). Moreover, T cells treated with CHX
for 2 h and then stimulated with PHA for 1 h showed a
striking increase in C5aR expression that was comparable with the level
observed in the absence of CHX (Fig. 3). It is also worth noting that
CHX treatment for 
24 h did not alter the constitutive expression of
C5aR on T cells (data not shown). To confirm C5aR expression on the T
cell lineage, we also examined the Jurkat T cell line and found a low
constitutive level of C5aR expression by FACS analysis (data not
shown). However, in contrast to PBL-derived T cells, C5aR expression on
Jurkat cells was not reproducibly up-regulated upon PHA stimulation
(data not shown).
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Using Northern blot analysis, we were not able to readily detect
C5aR mRNA in enriched preparations of human T cells. Therefore, to
further document C5aR expression on T cells, C5aR mRNA expression was
assessed by RT-PCR analysis of total RNA extracted from unstimulated
and PHA-stimulated T cell preparations as well as Jurkat T cells. Total
RNA from RBL-C5aR and RBL-C3aR was also analyzed as a positive and a
negative control, respectively. In all samples except RBL-C3aR, an
appropriately sized band of 550 bp was amplified (Fig. 4A). In parallel, samples were
amplified using the GAPDH primers as an internal control (data not
shown). To ensure that the amplified C5aR mRNA was actually derived
from T cells, RT-PCR experiments were also performed on cell-sorted
CD3+ cells. As shown in Fig. 4B, the same
results were obtained, thus formally demonstrating that C5aR mRNA is
expressed in human T cells, although at a low level.
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To assess C5aR mRNA regulation in PHA-stimulated T cells,
semiquantitative RT-PCR experiments were performed using GAPDH
amplification products as a measure of input RNA. Based on our flow
cytometry data, we chose to analyze C5aR mRNA expression in T cells
stimulated with 10 µg of PHA for 60 h, a timepoint for which
C5aR expression is maximal. In two independent experiments, enriched T
cell preparations were cultured for 60 h with or without PHA,
sorted for CD3 expression, and analyzed for C5aR expression by
semiquantitative RT-PCR. Surprisingly, we found that C5aR mRNA
decreased by >50% in PHA-stimulated T cells compared with
unstimulated T cells (Fig. 4
C).
Human T cells are chemotactic to C5a
To determine whether the C5aR on T cells was functionally active,
we decided to evaluate the ability of C5a to chemoattract purified T
cells. Three independent experiments were performed using fresh
PBL-derived T cells that had been isolated by negative selection as
described in Materials and Methods. T cells were then
examined for their ability to migrate in response to human rC5a. The
results presented in Fig. 5 demonstrate
that C5a at concentrations as low as 1 nM reproducibly and
significantly induced chemotaxis of unstimulated T cells. In one set of
experiments, fresh T cells were stimulated with PHA (10 µg/ml) for
1 h, cell-sorted, and used in a chemotactic assay. As shown in
Fig. 5, the results of these experiments demonstrate a dramatic
increase in the number of T cells that migrated in response to C5a when
compared with MM alone. Interestingly, with knowledge of the total
number of loaded cells and the total number of migrated cells, we were
able to evaluate the percentage of loaded cells that migrated in
response to C5a. We found that 7% of loaded cells migrated
in the experiments using freshly isolated T cells, whereas 15% of
loaded cells migrated in the experiments using PHA-stimulated T cells.
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Recently, we analyzed the kinetics of C5aR expression during EAE
in the Lewis rat (24). Using ISH and immunohistochemistry, we observed
that numerous blood-derived infiltrating cells expressed C5aR mRNA but
did not belong to the monocyte/macrophage lineage based on staining
experiments using the ED1 Ab (24). To verify whether some of the
C5aR+ cells were T cells, we performed a combination of
immunohistochemistry using an anti-TCR Ab and ISH for the C5aR on
spinal cord sections from animals sacrificed at the clinical peak of
the disease (Fig. 6). Staining with the
anti-TCR Ab alone identified a number of infiltrating T cells in
the spinal cords of animals with EAE (Fig. 6A). An
isotype-matched control Ab did not show any immunoreactivity (Fig. 6B). The combination of immunohistochemistry and ISH
demonstrated that a large proportion of the cells recognized by the
anti-TCR Ab strongly expressed the C5aR mRNA (Fig. 6C).
These cells were located in the parenchyma in or around blood vessels
as well as in the meninges. Using a C5aR sense probe, we did not
observe any double-staining, although T cells could be easily
recognized by immunohistochemistry (Fig. 6D). In parallel
experiments, we were also able to demonstrate C5aR mRNA by ISH on
cytospin preparations of PBL-derived human T cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). These data suggest that the increased C5aR expression induced by
PHA is not due, at least at early timepoints, to de novo synthesis. In
addition, these data are consistent with the hypothesis that T cells
have significant intracellular pools of the C5aR. Experiments are
currently in progress to investigate the intracellular trafficking of
C5aR in T cells and to determine whether PHA stimulation alters the
turnover of C5aR from the cell surface to intracellular pools of
preformed C5aR proteins. Using RT-PCR, we were able to detect C5aR mRNA in both human T cells and Jurkat T cells. Indeed, other cell types such as astrocytes and endothelial cells also express low amounts of C5aR mRNA despite readily detectable expression of C5aR protein at the cell surface (15, 28). Surprisingly, in semiquantitative RT-PCR experiments, we found that PHA-stimulated T cells express a lower amount of C5aR mRNA compared with unstimulated T cells. This result further supports the hypothesis that C5aR up-regulation on the cell surface is regulated posttranscriptionally in PHA-stimulated T cells.
To investigate the function of C5aR on human T cells, we performed
chemotaxis assays and found that human rC5a was able to chemoattract
unstimulated and PHA-stimulated T cells. This result is in agreement
with previous work showing chemotaxis of enriched preparations of human
T cells toward C5a (29). It is worth noting that the percentage of
migrating cells was evaluated at 7% of loaded cells, which is in the
range of unstimulated T cells expressing C5aR (between 1 and 8.6%).
Interestingly, the percentage of migrating cells reached 15% of
loaded cells when T cells were stimulated for 1 h with 10 µg/ml
PHA before the chemotactic assay. Thus, in both unstimulated and
PHA-stimulated T cells, the ability of C5a to chemoattract human T
cells appears to correlate with the number of C5aR-expressing T cells.
This finding, along with the fact that C5a chemoattracts T cells at
nanomolar concentrations, argues for a C5aR-mediated mechanism.
However, we cannot exclude the possibility that C5a binds to another
receptor that is not recognized by our Ab and/or that C5a induces the
synthesis of soluble mediators that, in turn, exert chemotactic
activities. Previous studies have described different C5aR subtypes
with different biochemical characteristics and various affinities for
their ligands (6, 17). Similarly, the cell-specific effects of C5a may
occur through a differential regulation of C5aR protein G
coupling. Thus, it is conceivable that C5a might elicit different types
of responses from activated T cells depending upon the input stimuli,
the T cell subtype (CD4 vs CD8), and/or the profile of cytokine
secretion (Th1 vs Th2).
Interestingly, using a combination of ISH and immunohistochemistry, we were able to detect C5aR mRNA in T cells infiltrating the CNS of rats with EAE. This is of particular interest because C5aR-expressing cells invade the CNS during EAE far before the onset of clinical symptoms (24). This finding raises the possibility that at the earliest stages of the disease, activated T cells could be chemoattracted in the CNS through an interaction between the C5aR and its ligand.
The experiments reported in the present study clearly demonstrate that human T cells express the C5aR at the protein and mRNA level and are chemotactic toward human rC5a. These results are in accordance with recent findings showing that C5a plays a major role in vivo in the recruitment of delayed-type hypersensitivity effector CD4+ T cells during the elicitation of contact sensitivity (11, 12). Similarly, anti-C5 mAbs were shown to inhibit collagen-induced arthritis, a T cell mediated autoimmune disorder, when administered before or during the efferent phase of the disease (30). Additional studies are needed to determine whether an Ag-driven stimulation can up-regulate C5aR expression on human T cells. In addition to chemotaxis, C5a elicits a broad range of activities on cells of the myeloid lineage and may exert similar effects on T cells, including cytokine synthesis and an augmentation of adhesion to endothelial cells and/or the extracellular matrix (4, 28). The demonstration of C5aR-expressing T cells in the CNS of EAE rats also suggests that C5a could be involved in T cell trafficking in the CNS. This hypothesis is currently being tested using C5aR-deficient mice.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Scott Barnum, Department of Microbiology, University of Alabama, 701 19th Street South, LHR/141, Birmingham, AL 35294. E-mail address:
3 Abbreviations used in this paper: C5aR, C5a receptor; C3aR, C3a receptor; CNS, central nervous system; EAE, experimental allergic encephalomyelitis; ISH, in situ hybridization; RBL-C5aR cells, C5aR-transfected rat basophilic leukemia cells; RBL-C3aR cells, C3aR-transfected rat basophilic leukemia cells; CHX, cycloheximide; MM, migration media; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received for publication August 12, 1998. Accepted for publication January 12, 1999.
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: a possible initiating role of B cells. J. Exp. Med. 186:1015.-mediated expression of the receptor for anaphylatoxin C5a (C5aR) on neurons in experimental Listeria meningocephalitis. J. Immunol. 159:861.[Abstract]
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R. F. Tsuji, I. Kawikova, R. Ramabhadran, M. Akahira-Azuma, D. Taub, T. E. Hugli, C. Gerard, and P. W. Askenase Early Local Generation of C5a Initiates the Elicitation of Contact Sensitivity by Leading to Early T Cell Recruitment J. Immunol., August 1, 2000; 165(3): 1588 - 1598. [Abstract] [Full Text] [PDF]
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A. J. Szalai, F. W. van Ginkel, Y. Wang, J. R. McGhee, and J. E. Volanakis Complement-Dependent Acute-Phase Expression of C-Reactive Protein and Serum Amyloid P-Component J. Immunol., July 15, 2000; 165(2): 1030 - 1035. [Abstract] [Full Text] [PDF]
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Y. Wang, J. Kristan, L. Hao, C. S. Lenkoski, Y. Shen, and L. A. Matis A Role for Complement in Antibody-Mediated Inflammation: C5-Deficient DBA/1 Mice Are Resistant to Collagen-Induced Arthritis J. Immunol., April 15, 2000; 164(8): 4340 - 4347. [Abstract] [Full Text] [PDF]
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N. Davoust, S. Nataf, R. Reiman, M. V. Holers, I. L. Campbell, and S. R. Barnum Central Nervous System-Targeted Expression of the Complement Inhibitor sCrry Prevents Experimental Allergic Encephalomyelitis J. Immunol., December 15, 1999; 163(12): 6551 - 6556. [Abstract] [Full Text] [PDF]
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