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*
Department of Medical Biochemistry and
Pathology (Neuropathology Laboratory), University of Wales College of Medicine, Cardiff, Wales, United Kingdom; and
INSERM Unit 78, Institut Fèdèratif de Recherches Multidisciplinaires sur les Peptides, Chemin de la Bretéque, Bois-Guillaume, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells respond to anaphylatoxins via specific receptors. The receptor for C5a (C5aR) was cloned in 1990 (9), and the generation of specific Abs using synthetic peptides enabled the tissue distribution of C5aR to be examined (10, 11). The C5aR was expressed on both myeloid cells (monocytes/macrophages, neutrophils, and eosinophils) and nonmyeloid cells (hepatocytes, epithelia, endothelia, mast cells, vascular smooth muscle, and glial cells) (3, 4, 5, 6, 7, 10, 11, 12, 13, 14, 15, 16). Much less is known about the cellular distribution of the C3aR. C3a binding experiments and assessment of C3a functional effects indicated that a receptor for C3a was present on monocyte/macrophage cells and cell lines, platelets, polymorphonuclear leukocytes, mast cells, and adipocytes (3, 12, 17, 18, 19, 20, 21, 22, 23). The human C3aR cDNA has recently been cloned from the HL60 cell line (24), LPS-activated neutrophils (25), and the PMA-differentiated U937 monocyte cell line (26). Northern blot analysis revealed that the 2.2-kb C3aR mRNA was expressed in all tissues, particularly in lung, placenta, heart, spleen, and brain (25). However, little is known about the expression of C3aR at the protein level and on nonmyeloid cells in these tissues.
We and others are interested in the role of complement in the brain, and we have shown that a full complement system can be expressed by resident cells in the brain, particularly after cytokine stimulation (for review, see 27 . The brain is an immunoprivileged organ isolated from the peripheral immune system, and we have proposed that complement expressed locally in response to infection or inflammation plays an important role as an antipathogen (27). Human and mouse glial cells respond to and are activated by anaphylatoxins (14, 28, 29), and we have recently shown that the C5aR was expressed by astrocytes, the most abundant glial cell type (14). Immunohistochemistry demonstrated that expression of C5aR was low in normal brain but abundant in inflamed brain, suggesting that the anaphylatoxin receptor was involved in the inflammatory process (16).
We here describe the expression of the C3aR by myeloid cells and brain cells in vitro and in brain tissue. C3aR expression was demonstrated in cells and tissue at the protein and mRNA levels, and immunohistochemistry was conducted on normal, multiple sclerosis (MS), and bacterial meningitis (BM) brain tissue sections. In myeloid and glial cells the expressed C3aR was a protein of 65 kDa; expression of the receptor was highly elevated on myeloid cells, astrocytes, microglia, and smooth muscle cells (pericytes) in inflamed CNS tissue. The results further emphasize the important role played by complement activation in CNS inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A multiple array, 30-amino acid, C3aR peptide
PSGFPIEDHETSPLDNSDAFLSTHLKLFPS (MAP-C3aR peptide) corresponding to
amino acid residues 270 to 300 of the C3aR loop (see Fig. 1
) was synthesized on an Applied
Biosystems Synergy Synthesizer (Applied Biosystems, Warrington, U.K.).
Anti-peptide antisera were raised in New Zealand White rabbits by
repeated s.c. immunization (total of five) with peptide (100
µg/animal/immunization) in CFA using standard protocols. The animals
were test-bled at intervals, and the titer of the anti-peptide
response was measured. When the antipeptide response had plateaued, the
animals were sacrificed by exsanguination, and the antiserum was stored
at -20°C.
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For FACS analysis, affinity-purified anti-C3aR (1.5 mg) was also biotinylated with 1 mg of biotinamidocaproate N-hydroxysuccimide (Sigma, Poole, U.K.) and dialyzed to eliminate free biotin. All preparations of Abs were stored at 4°C in PBS/0.1% NaN3. The specificity and titer of the anti-peptide Ab were assessed in an ELISA using 96-well plates coated with the MAP-C3aR peptide or nonspecific peptides (C3a peptide, WWGKKYRASKLGLAR; MAP-C5aR peptide, MNSSFEINYDHYGTMDPNIPADGIHLPKRQ) produced in our laboratory (14). In some experiments, the anti-C3aR was blocked by preincubation with various concentrations of the MAP-C3aR peptide before application to the 96-well plate. Specificity was also assessed by flow cytometry on C3aR+ (THP1) and C3aR- (Ramos) cell lines and by Western blot analysis of cell lysates and a C3aR loop-glutathione-S-transferase (GST) fusion protein.
To detect C3aR extracted from cells, lysates were immunoprecipitated using anti-C3aR Sepharose. For this purpose, 5 mg of affinity-purified anti-C3aR was coupled to 1.5 g (5–7 ml) of cyanogen bromide-activated Sepharose 4B according to the manufacturer’s protocol (Pharmacia, Piscataway, NJ).
Chemicals, cytokines, and Abs
Recombinant human IFN-
, IL-1ß, and TNF-
were gifts from
Hoffmann-La Roche (Nutley, NJ). PMA, protein A-Sepharose, pepstatin,
and leupeptin were obtained from Sigma. Mouse and rabbit Abs against
the N-terminal extracellular sequence of the human C5aR have been
characterized previously (11, 14). Abs against CD Ags (CD4, CD14, CD16,
CD18, CD19, CD35, and CD68) were purchased from Dako (High Wycombe,
U.K.). mAbs against CD44 (clones BRIC222 and BRIC235) and CD59
(BRIC229) were obtained from IBGRL (Elstree, U.K.). Other Abs used for
immunocytochemistry of adherent cells on coverslips and for
immunohistochemistry of tissue sections were mouse anti-glial
fibrillary acidic protein (anti-GFAP; clone GA5; 1/1000; Sigma),
mouse anti-galactocerebroside (1/1000) (16), mouse
anti-neuron-specific enolase (clone BBS/NC/VI-H14; 1/2000; Dako),
mouse anti-CD11b (clone 2LPM19c; 1/50; Dako), mouse anti-CD68
(1/50; clones KP1, PG-M1, and EBM11; Dako), and mouse anti-HLA
class II (1/50; clone LN3; Biotest, Solihull, U.K.).
Cell preparation and culture
Primary cultures of human fetal astrocytes were grown in our laboratory from tissue supplied by the Medical Research Council Fetal Tissue Bank (London, U.K.). All cultures were established from fetal brain (6- to 10-wk-old fetus) as described previously (14). Primary cultures (passage 0, days 1–7) contained clusters of neurons (demonstrated by immunostaining with an anti-70K neurofilament mAb) that survived 7 to 10 days, lying on a monocellular layer. Some 60 to 80% of the cells in this monocellular layer expressed the astrocytic marker, GFAP; 5 to 7% were microglia, and 15 to 40% were fibroblasts. Microglia were removed by shaking (350 rpm, 37°C, for 1.5 h followed by a second shaking at 250 rpm and 37°C overnight). Adherent cells after these two sequential shakings were subcultured in DMEM containing 10% FCS (Life Technologies, Paisley, U.K.), 1% L-glutamine (Life Technologies), and 1% penicillin/streptomycin (Life Technologies). Experiments were conducted from passages 2 to 5, when cultures contained >95% GFAP +ve cells and <5% neurons, fibroblasts, and microglia.
Normal adult temporal lobe tissue was obtained fresh from biopsies of patients undergoing therapeutic resection for intractable epilepsy. The tissue was used for culture of adult astrocytes as previously described (16, 30). The use of human tissue was in accordance with procedures and regulations established by the local ethical committee of the University of Wales College of Medicine.
The two human astrocyte cell lines, T98G and CB193, were cultured and characterized as described previously (14). Neutrophils (PMN) and mononuclear cells (MNC) were isolated from heparinized blood obtained by venepuncture of healthy volunteers. Briefly, leukocytes were separated from erythrocytes by dextran sedimentation using 0.6% (w/v) dextran (Fisons, Loughborough, U.K.). The leukocyte-rich upper layer was then fractionated by layering on Histopaque (Sigma) followed by centrifugation at 220 x g for 25 min at room temperature. MNC were collected from the interface and washed in PBS/BSA. Residual erythrocytes in the PMN-rich cell pellet were removed by hypotonic lysis, and PMN were washed in PBS-BSA. Cytocentrifuge preparations of cells were stained with Wright’s stain, and >90% of cells were PMN. FACS analysis of PMN preparations revealed that 90 to 95% of cells were CD11b+++, CD18+++, CD16++, CD14+, CD19-, CD35+. Lymphocytes and monocytes constituted >90% of the MNC preparation, and no contaminating PMN were identified. In the MNC preparation, 10 to 15% of cells analyzed by FACS were strongly CR3+. Human monocyte-derived cell lines, THP1 and U937, and neuroblastoma cell lines, IMR32 and SKNSH, were obtained from American Type Culture Collection (Rockville, MD). The human endothelial cell line ECV 304, the human erythroleukemia cell line K562, the human B lymphocyte cell line Ramos, and the human T lymphocyte cell line Molt 4 were obtained from European Cell Culture Collection (ECACC) (Salisbury, U.K.). A subclone of the B lymphocyte cell line Raji (Raji+3) expressing high levels of CD59 (31) was routinely grown in our laboratory and used for RT-PCR analysis. The human hepatoma cell line HepG2 was obtained from Dr. M. Daveau (INSERM Unit 78, Rouen, France). The human NK cell line (YT) was a gift from Dr. G. Griffiths (London, U.K.) and was cultured in the presence of IL-2.
Cell phenotype was confirmed using immunocytochemistry or FACS
analysis, as described previously (14). Astrocytes and the two
astrocyte cell lines presented the phenotype (GFAP+,
CD11b-, HLAII-). For some experiments, cells
were stimulated with cytokines (200 IU/ml of IFN- or IL-1ß and
1000 IU of TNF-) or with PMA (10 ng/ml) for 24 h. To induce
differentiation of the two monocyte cell lines, THP1 and U937, cells
were treated with 10 ng/ml of PMA over a period of 3 days, replacing
medium with fresh PMA-containing medium every day. Under these
conditions, PMA was not toxic to the cells, which by day 2 had adhered
strongly to the plastic.
Monocyte cell lines THP1 and U937 were characterized as the monocyte/macrophage (the latter after PMA (10 ng/ml) treatment for 3 days) cell phenotype with, respectively, for monocyte/macrophage: GFAP-/-, CD11b+/+++, CD68+/+++, CD35+/++, HLAII+/++, C5aR+/+++, as previously described (14).
Flow cytometry and testing specificity using competitive MAP-C3aR peptide
Astrocytes or cell lines were harvested from culture by incubation in FACS buffer (PBS containing 2% BSA and 0.1% NaN3) supplemented with 10 mM EDTA. Cells were washed and resuspended at 106 cells/ml in the same buffer without EDTA, incubated with the appropriate primary Ab (0.5–5 µg/ml unless stated otherwise) for 30 min on ice, washed three times in cold FACS buffer, incubated with the appropriate phycoerythrin (PE)-labeled secondary Ab for 30 min on ice, and washed an additional three times before analysis on a Becton Dickinson FACScan (San Jose, CA). PE-conjugated rabbit anti-mouse IgG (Dako) and PE-conjugated goat anti-rabbit IgG (Sigma) were used at a 1/100 dilution. When cells were incubated with the biotin-labeled affinity-purified anti-C3aR, the staining was visualized after incubation with FITC-labeled avidin (Sigma) (1/100) or with R-PE-labeled streptavidin (Jackson ImmunoResearch, Stratech, Luton, U.K.). To confirm the specificity of the anti-C3aR, dilutions of Abs (0.4–3.25 µg/ml) were preincubated with a fixed concentration of competing MAP-C3aR peptide (5 µg/ml) at 4°C for 30 min to 1 h before applying them to the cell suspension. The remainder of the FACS protocol was identical with that described above.
Immunocytochemistry
For CD44 and C3aR staining, adult astrocytes, fetal astrocytes, and astrocyte cell lines were cultured on sterile glass coverslips for 5 to 8 days, and after washing with PBS, cells were fixed with 1% formaldehyde for 20 min. Cells were then washed intensively in PBS/0.2 M glycine to block aldehyde groups. For GFAP immunostaining, cells were fixed and permeabilized with a mixture of 95% ethanol/5% acetic acid for 5 min at -20°C. Abs were used at optimal dilution (1 µg/ml in PBS/1% BSA) and incubated with fixed cells overnight at 4°C in a humid chamber. Affinity-purified rabbit anti-C3aR was tested over the concentration range 7.5 to 0.81 µg/ml in the presence or the absence of competing peptide. After washing, coverslips were incubated for 30 min at 37°C with either FITC-labeled secondary Ab (F(ab')2 rabbit anti-mouse IgG (1/100; Dako) or goat anti-rabbit IgG (Sera-Lab, Sussex, U.K.)) or peroxidase-labeled secondary Ab (rabbit anti-mouse IgG (1/100; Bio-Rad, Richmond, CA) or goat anti-rabbit IgG (1/100; Bio-Rad)). After intensive washing, coverslips for fluorescence microscopy were mounted in Citifluor (Citifluor, London, U.K.) and sealed. For peroxidase immunostaining, coverslips were incubated in a solution of DAB/H2O2 (Sigma) before hematoxylin counterstaining and mounting. Fluorescence was imaged by confocal laser scanning microscopy on a Leica TCS microscope (Leica, Heidelberg, Germany). Twelve optical sections were collected per field at 0.3- to 0.5-µm intervals from the bottom to the top of the cell. Sections were then assembled as extended focus views, or individual sections were viewed as a gallery. DAB immunostaining (brown positive staining) was photographed on a Leica DMLB microscope with brightfield at two magnifications (x500 and x1250).
Double staining of the CB193 astrocyte cell line for CD44 and C3aR was performed in an identical manner, except that both Abs were applied to the same coverslip and were detected using a rhodamine-conjugated goat anti-mouse IgG (Sigma) and a FITC-conjugated goat anti-rabbit IgG (Seralab), respectively. Fluorescence was imaged by confocal microscopy using specific cut-off filters for FITC and rhodamine.
Sources of tissues, processing, and immunohistochemistry
Brain tissue was obtained locally at autopsy or from specialist tissue collections. Tissue was collected from individuals with a variety of brain disorders (demyelination and CNS infection) and from normal surgical controls. Tissue from cases of MS (three acute plaques and one chronic plaques) were obtained from Dr. Jia Newcombe (MS Society Laboratory, London, U.K.) and locally (J. W. Neal, Neuropathology Laboratory, Cardiff, U.K.). Tissues from four cases of BM were obtained locally, all of which were characterized by a marked infiltration of neutrophils in the meninges. Normal control brain tissues was obtained at autopsy or brain surgery from individuals with no evidence of neurodegenerative disease, ischemia, or gliosis. Autopsy samples were obtained at a similar postmortem interval (maximum of 30 h) as the disease samples. Brains were cut coronally, and individual blocks from areas of the brain containing macroscopic evidence of pathology were dissected. Tissue was either snap-frozen and kept at -40°C or fixed in 10% formalin before processing for cryosections or paraffin wax embedding and sectioning, respectively.
Rehydrated paraffin sections were counterstained with hematoxylin/eosin to display morphology, and Luxol fast blue stain was used to identify demyelinating plaque areas in MS tissue (16). Rehydrated paraffin wax sections and cryosections (8 µm) from normal and diseased brains were immunostained with different dilutions of the affinity-purified anti-C3aR (7.5 to 0.81 µg/ml) and anti-cell markers diluted in PBS/BSA using an indirect immuno-horseradish peroxidase/DAB method as described previously (32). Peroxidase-labeled swine anti-rabbit IgG and rabbit anti-mouse IgG (Dako; 1/100 dilution) were used as secondary Abs.
For double immunofluorescence (IF), tissue sections were incubated simultaneously with the rabbit anti-C3aR and mouse anti-brain cell markers (clone GA5 for GFAP and clone LN3 for HLA class II) followed by FITC-labeled goat anti-rabbit (Sera-Lab) and rhodamine-labeled goat anti-mouse (Sigma) Abs. Fluorescence was imaged on a Leica DMLB epifluorescence microscope using specific filters.
Cell lysates, Western blotting, and immunoprecipitation
Western blotting was performed on cells (astrocytes and cell lines) solubilized in PBS containing 2% Nonidet P-40 together with enzyme inhibitors as previously described (14). To enrich for C3aR before Western blotting, cell lysates were immunoprecipitated. Freshly made cell lysate (1 ml; 2 x 107 cells/ml) was incubated with mixing overnight at 4°C with 50 µl of affinity-purified rabbit anti-C3aR Sepharose. After five washings with 0.5% Nonidet P-40 in PBS, C3aR retained on the Ab-Sepharose was eluted by mixing in an Eppendorf microfuge with 50 µl of 0.1 M glycine, pH 2.5, for 30 min at room temperature. Sepharose was removed by centrifugation at 10,000 x g for 10 min, and the eluted protein was diluted in an equal volume of SDS-PAGE sample buffer. The incorporation of the glycine elution step prevented the release of rabbit anti-C3aR, simplifying interpretation of the Western blots. THP1 and Ramos cell lysates were tested as positive and negative controls for C3aR expression to validate the immunoprecipitation protocol. Samples were fractionated on 15% or 7.5% SDS-PAGE, electroblotted onto nitrocellulose and stained overnight at 4°C with either the affinity-purified anti-C3aR (0.75 µg/ml) or rabbit anti-decay-accelerating factor (anti-DAF; CD55) also produced in our laboratory (0.25 µg/ml), essentially as previously described (14). After washing and incubation with peroxidase-labeled goat anti-rabbit IgG (Bio-Rad; 1/4000), blots were developed using the enhanced chemiluminescence system (ECL, Pierce, Chester, U.K.). Prestained broad range protein markers from New England Biolabs (Beverly, MA) were used as m.w. standards. Human THP1, unstimulated or differentiated with PMA (10 ng/ml, 3 days), were used as the C3aR-positive control.
To eliminate the possibility of cross-reactivity of affinity-purified anti-C3aR Ab with C5aR, PMA-differentiated THP1 cell lysate (1 ml; 2 x 107 cells) was immunoprecipitated with W17 mouse monoclonal anti-C5aR preadsorbed on protein A-Sepharose (Sigma; 20 µg of Ab for 6.25 mg of PAS). After elution in Laemmli buffer, samples were tested by Western blot using the affinity-purified anti-C3aR.
RNA extraction and RT-PCR for C3aR mRNA
Total RNA from unstimulated and stimulated cultures of human astrocytes and cell lines was prepared using the Ultraspec RNA isolation system (Biotecx, Houston, TX) according to the manufacturer’s instructions. RNA integrity was confirmed on agarose gels, and concentrations were determined from absorbance at 260 nm. Before reverse transcription, total RNA (50 µg) was treated for 20 min at 37°C with 10 U of RQ1 RNase-free DNase (Promega, Madison, WI) in 100 µl of buffer (40 mM Tris/HCl (pH 8), 10 mM NaCl, 6 mM MgCl2, and 10 mM CaCl2) and 200 U of RNAsin (Promega) to remove all trace of DNA. The mixture was then phenol and chloroform extracted. The aqueous phase was ethanol precipitated, and the pellet after centrifugation was resuspended in diethylpyrocarbonate-treated water.
The reverse transcription was conducted at 37°C for 120 min in 30 µl (final volume) with 2 µg of total RNA (DNA-free), 60 U of RNAsin (Promega), 1 mM dNTPs (Bioline, London, U.K.), 250 pmol of random hexamer primers (pdN6 from Pharmacia), and 400 U of Moloney murine leukemia virus-RT (Life Technologies, Paisley, U.K.) in the reaction buffer (10 mM Tris/HCl, 15 mM KCl, 0.6 mM MgCl2, and 5 mM DTT). The absence of contaminants was routinely checked by RT-PCR assays of negative control samples, in which the RNA samples were replaced with sterile water, or Moloney murine leukemia virus-RT was not added.
PCR was conducted with 3 µl of reverse transcribed RNA mixture in a 50-µl final reaction volume with 100 pmol of each C3aR primer (generated in-house on a Beckman oligo synthesizer, Beckman, Palo Alto, CA) in 10x buffer (Promega) containing 1.5 mM MgCl2, 200 µM dNTP, and 1.25 U of Taq DNA polymerase (Promega). The PCR protocol used was: denaturation step at 94°C for 4 min; five cycles of 94°C for 30 s, annealing 60°C for 1 min, and extension at 72°C for 2 min; 25 cycles of 94°C for 10 s, 60°C for 30 s, and 72°C for 45 s; and elongation at 72°C for 15 min. PCR was performed in a Hybaid (Teddington, U.K.) Omnigene thermocycler.
Seven oligonucleotides were selected from the published C3aR cDNA sequence (23, 24, 25). Their sequences from 5' to 3' and their positions in parentheses are given according to the sequence published by Roglic et al. (EMBL access no. U28488) (24): GCT CAT CCC CTC CAT CAT TG (sense, 369), GGG TGG TGG CTT TTG TGA TG (sense, 521), GTC CCC ACT GTC TTC CAA CC (sense, 754), GGT TGG AAG ACA GTG GGG AC (antisense, 754), CAG CAG GAA ACC CAC TA (antisense, 1122), AGG GCA TAA AGG AAG GGA TT (antisense, 1391), and TGA ATG GAC TGC CTT GCT TT (antisense, 1433). All samples were subjected to RT-PCR for ß-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a positive control and as an internal standard not affected by any cytokine or PMA treatment. The sequences of human ß-actin (EMBL M10278) primers were: actin-1, CTA CAA TGA GCT GCG TGT GG (311); and actin-2, CTC ATT GCC AAT GGT GAT GA (800). The sequences of GAPDH (EMBL M33197) primers were: GAPDH-Up, GAA CGG GAA GCT TGT CAT CA; and GAPDH-Do, TGA CCT TGC CCA CAG CCT TG.
RT-PCR analysis for complement receptor type 2 (CR2), C5aR, and C4 mRNAs was also conducted on all RNA samples to confirm the specificity of the RT-PCR protocol (20, 33). The sequences of human CR2 primers (EMBL J03564) were CCC ATT GCT GTT GGT ACC GT (181) and ACA CAG GTT GGT AGT CGT (503). The sequences of human C5aR primers (EMBL no. M62505/JO5327) were GAC CAG AAC ATG AAC TCC TT (16) and TGT CGC CTA CAC TGC CTG (1083). The sequences of human C4 primers (EMBL no. K02403) were CGG GTC TTT GCW CTG GAT CA (515) and CTT CAC CTC RAA GTT GGG AA (773). Samples of RT-PCR products were loaded onto a 1% agarose gel in 1x TBE buffer, separated by electrophoresis at 50 to 100 mA, and transferred onto nylon membranes (Hybond N+, Amersham, Aylesbury, U.K.) for Southern blotting. Ladders (123 bp and 1 kb; Life Technologies) were used as DNA size markers. All C3aR cDNAs obtained after RT-PCR of the astrocyte cell line CB193 were purified (Quiaquick PCR purification column, Qiagen, Dorking, U.K.) and sequenced using the ABI PRISM dye terminator cycle sequencing kit from Perkin-Elmer (Warrington, U.K.) and the 373A automatic sequencer from Applied Biosystems (Foster City, CA). Sequences were compared with the published human C3aR using the DNAstar software package (DNA Star, London, U.K.).
cDNA probes, nucleotide sequencing, and Southern blot
The human C3aR cDNA probe was obtained from PMA-differentiated U937 RNA by RT-PCR using two C3aR oligonucleotides (754 and 1391). The RT-PCR product was purified by the PCR DNA purification system (Hybaid) and subcloned in the PGEM-T plasmid (Promega, Southampton, U.K.). Positive bacterial clones were identified by blue/white screening and were selected. Inserts were PCR amplified using primers SP6 and T7 (developed in-house) derived from vector sequence flanking the insert site and then sequenced as described above. The probe isolated by this procedure was 100% homologous with the reported C3aR cDNA (24) between positions 754 and 1391 bp.
For Southern blotting the C3aR probe was labeled by the random oligo
procedure with [-32P]dCTP (3000 Ci/mmol; Redivue,
Amersham). Blots were prehybridized (2 h) and hybridized (16–20 h) in
a hybridization oven at high stringency (50% deionized formamide, 5x
SSPE, 1% SDS, 5x Denhardt’s reagent, 5% dextran sulfate, and 100
µg/ml denatured and fragmented herring sperm DNA). Posthybridization
washings were performed in (2x SSPE/0.1% SDS, twice at room
temperature; 2x SSPE/0.1% SDS for 1 h at 68°C; 1x SSPE/0.1%
SDS for 1 h at 68°C; 0.1x SSPE/0.1% SDS for 1 h at
68°C). Blots were then exposed to Kodak 4 film (Eastman Kodak,
Rochester, NY) for 5 to 10 min at room temperature.
GST-C3aR loop fusion protein
To further confirm the specificity of the anti-C3aR peptide we generated a fusion protein by cloning the cDNA encoding the second C3aR extracellular loop (between TM4 and TM5 domains, amino acids 161–332) in a GST fusion protein system (Pharmacia). The C3aR loop cDNA was produced by RT-PCR from PMA-differentiated THP1 RNA using two specific oligonucleotides (F-C3aR-s (562, CGG GAT CCC GGG AAA TCT TCA CTA CAG AC) and R-C3aR-s (1077, CGG GAT CC TCA G GGT GTT GGC ACT TGA TCG TC) and Vent DNA polymerase (New England Biolabs, Hertfordshire, U.K.) to minimize PCR errors. The sequence TCA (double underlined) was introduced in the downstream primer to create a stop codon TGA. A BamHI site (underlined) was introduced in both primers and was used to clone the C3aR cDNA into the pGex-2T expression vector. After transfection in Escherichia coli (strain BL21, Pharmacia), plasmid from individual bacterial colonies was tested to confirm the cDNA insert orientation and the fidelity of the C3aR cDNA sequence. The expression of the fusion protein was induced by the addition of 1 mM isopropyl-ß-thiogalactopyranoside (Promega) in 0.5 l bacterial culture (OD600 nm = 0.8) for 2 to 3 h. Bacteria were pelleted at 5000 x g for 30 min, and lysates were prepared using 1% Nonidet P-40/PBS. Crude lysate was analyzed by Western blot, and the expressed fusion protein was affinity purified on glutathione-Sepharose 4B according to the manufacturer’s instructions. The predicted size of the fusion protein was 50 kDa (30 kDa for GST and 19.4 kDa for the C3aR loop).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 30-amino acid sequence from the second extracellular loop (amino
acids 270–300; inset in Fig. 1
) was chosen for Ab
production based upon predictions of hydrophilicity/antigenicity. This
peptide had no significant homology with any other cloned protein
reported in the BLAST databank and had no homology with the human C5aR.
The C3aR peptide was synthesized as a multiple array on a lysine core
and was used for immunization of rabbits. The antiserum was purified by
affinity chromatography on peptide-Sepharose. The yield of
affinity-purified anti-C3aR Abs was approximately 10% of the
applied IgG. Affinity-purified anti-C3aR IgG was used throughout
the study. The specificity of the anti-C3aR was tested first by
staining the THP1 monocyte line and the Ramos B cell line followed by
FACS analysis (Fig. 1). THP1 was strongly stained even when cells were
incubated with as little as 0.81 µg/ml of the Ab. Ramos was
consistently negative for C3aR. Preincubation of anti-C3aR with a
100-fold molar excess of the C3aR peptide completely abolished staining
of THP1 (not shown). The titer and specificity of the anti-C3aR
were further tested by ELISA (Fig. 2).
The anti-C3aR gave a strong positive signal even at high dilutions
of C3aR peptide, and preincubation with peptide prevented binding of
Ab; no binding of anti-C3aR occurred on plates coated with C3a or
C5aR peptide (latter not shown). In all subsequent experiments
anti-C3aR was used at 0.4 µg/ml unless stated otherwise.
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We have previously described the expression of C5aR by leukocytes
(primary cells and cell lines); the same approach was used to
characterize the expression of C3aR using FACS analysis. THP1,
undifferentiated or differentiated with PMA (3 days), were stained for
C3aR; PMA-differentiated cells had a mean fluorescence double that of
undifferentiated cells (Table I). We have
previously shown that PMA differentiation increased the expression of
C5aR on THP1 by a factor of 10- to 20-fold, and these results were here
confirmed in parallel with the measurements of C3aR expression (Table I). To characterize in more detail the regulation of C3aR expression by
monocyte cell lines, U937 were cultured for 48 h in the presence
of three different concentrations of recombinant cytokines (IFN-,
IL-1ß, and TNF-) or phorbol ester (PMA). C3aR expression on the
cell membrane was analyzed by FACS, and the results are presented in
Table II. PMA increased the expression of
C3aR, but only by a factor of 1.5 at 48 h poststimulation; the
most dramatic effect was obtained after stimulation of U937 with
IFN-. Even at a very low concentration (10 IU/ml) IFN- increased
C3aR expression by 2-fold, and at 1000 IU/ml, C3aR expression was
elevated by 6-fold. TNF- and IL-1ß had no effect on C3aR
expression by U937 (Table II). IFN- also up-regulated C3aR
expression by THP1. Neutrophils and monocytes both expressed C3aR,
whereas the various lymphocyte subsets were all negative (Table I).
C3aR was not detected on the membranes of the undifferentiated Raji+3 B
cell line, the K562 erythroleukemia cell line, the Molt 4 T lymphocyte
cell line, or the YT NK cell line (data not presented).
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Two astrocyte cell lines were tested by FACS for C3aR expression.
The well-differentiated astrocytoma cell line CB193 expressed twice as
much C3aR as the undifferentiated glioblastoma cell line T98G (Table I). Both lines express the astrocyte-specific marker GFAP and have been
extensively used as a model of the human astrocyte (33). Indirect IF
and confocal microscopy confirmed that C3aR was expressed abundantly on
the membrane of the CB193 astrocyte cell line (Fig. 3, b and c).
No staining was detected in the nucleus or the cell cytoplasm. C3aR
staining was patchy, a pattern identical with that described for
expression of C5aR on astrocyte lines (14). The C3aR staining intensity
and pattern were reproducible (n > 10),
specific, and blocked by the competitive peptide. To exclude the
possibility that the patchy C3aR staining was an artifact of the
immunochemistry procedure, we simultaneously stained CB193 for
membrane-associated CD44 and C3aR. Figure 3, d and
e, clearly shows that CD44 membrane staining was distributed
homogeneously, whereas C3aR staining on the same cells was patchy and
clustered.
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). Adult astrocytes are adherent cells
with long, ramified processes and stain strongly for GFAP (Fig. 4a). The C3aR staining on adult astrocytes was again
patchy and confirmed our results with the CB193 astrocyte cell line
(Fig. 4d and Fig. 3, b, c, and
e). Neurons and oligodendrocytes in fetal brain
cultures did not express C3aR at a detectable level (not shown),
whereas microglia in fetal and adult brain cultures were stained for
C3aR (Fig. 4b). Adult and fetal brain cultures and
CB193 were stimulated for 24 h with various cytokines (IFN-,
IL-1ß, and TNF-) or with PMA. No change in C3aR expression was
detected on any cell type in the cultures (data not shown).
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Although the GST-C3aR fusion protein was readily detected by
immunoblotting (Fig. 5A) and DAF was easily
detected in cell lysates (Fig. 5B), no positive
results were obtained for C3aR from cell lysates. To improve
sensitivity, cell lysates were first immunoprecipitated on
Sepharose-bound anti-C3aR. Western blotting of immunoprecipitates
revealed a protein with an apparent molecular mass of 65 kDa in lysates
from unstimulated CB193, undifferentiated THP1, and PMA-differentiated
THP1 (Fig. 5C). The intensity of the 65-kDa band was
greater in the PMA-stimulated THP1 lane compared with that of
undifferentiated THP1. No protein was detected when Ramos or K562 cell
lysates were immunoprecipitated under the same conditions. When
THP1/PMA cell lysate was immunoprecipitated with either Sepharose-bound
polyclonal anti-C5aR or monoclonal anti-C5aR (W17/1) adsorbed
on protein A-Sepharose to enrich the preparation for C5aR, no protein
was detected upon Western blotting with anti-C3aR (not shown). This
simple experiment eliminated the possibility that the anti-C3aR
might cross-react with the C5aR.
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RT-PCR analysis was used to characterize the expression of C3aR in
isolated cells from human brain. The cDNAs so obtained were cloned,
sequenced, and compared with the published sequence. Seven
oligonucleotides were designed and used for RT-PCR analysis. The human
C3aR intron/exon organization has not yet been reported, so we were not
able to choose primers spanning an intron sequence. Thus, all RNA
samples were treated with Rq1 DNase to eliminate contamination with
genomic DNA, and it was confirmed that no PCR fragment was obtained
when the RT step was omitted, when RNA was substituted with water, or
when no primer was added to the PCR mix. The different combinations of
C3aR primers were used in RT-PCR with total RNA extracted from control
U937 (Fig. 6A) and
PMA-differentiated U937 (Fig. 6B). The same strategy
was used for RT-PCR with total RNA from the CB193 astrocyte cell line
and fetal astrocytes (data not shown). C3aR mRNA was detected in U937,
particularly after PMA differentiation, and also in CB193 and fetal
astrocytes. We were not able to obtain adult astrocytes at sufficient
purity in culture for analysis of the expression of C3aR mRNA. The
astrocytoma (CB193) C3aR cDNAs were sequenced and were 100% identical
between positions 369 and 1433 bp with the C3aR cDNA cloned from
HL60 (24).
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, TNF-, or PMA
for 24 h followed by RT-PCR analysis had no effect on C3aR mRNA
expression, whereas IL-1 at 200 IU/ml decreased expression of C3aR mRNA
(Fig. 6D).
The activities of all cytokines and PMA treatment were checked by
examining the effects on expression of C5aR by U937 (Fig. 6C) and of C4 by CB193 (data not shown). After
24 h, C5aR mRNA expression by U937 was slightly (IFN- effect)
or highly (PMA effect) up-regulated, in agreement with previous reports
(34, 35). C4 mRNA expression by CB193 was up-regulated by IFN- and
TNF-, but was not affected by the other treatments as reported
previously (36).
Expression of C3aR mRNA in astrocyte cell lines and a range of other
cell lines of myeloid and nonmyeloid lineage was compared by RT-PCR
(Fig. 7). C3aR mRNA was expressed by the
THP1 monocyte cell line and the ECV 304 endothelial cell line, but not
by B and T lymphocyte cell lines, the YT NK cell line, or the HepG2
hepatoma cell line (Fig. 7A). Southern blot analysis
using a specific C3aR cDNA probe cloned from U937 confirmed the
identity of the cDNA and revealed that C3aR mRNA was present at a very
low level in Molt4 T lymphocyte, YT NK cell, and HepG2 cell lines (Fig. 7B). Multiple controls for false positive and false
negative results were performed. Figure 7, C and
D, shows RT-PCR analysis for CR2 cDNA and GAPDH cDNA on the
same samples (same RT) as those used for C3aR RT-PCR. GAPDH cDNA was
present in all RNA samples and at the same level; CR2 cDNA was detected
only in Raji, Molt4, and CB193 astrocyte cell lines as previously
described (37). The expression of CR2 mRNA by the ECV endothelial cell
line demonstrated here is a new finding and merits further study.
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By RT-PCR analysis the expression of C3aR mRNA was detected in the
temporal and frontal lobes, the cerebellum, and the caudate nucleus of
normal control human brain (data not shown). To localize the cellular
distribution of the C3aR, anti-C3aR Ab was applied to sections of
normal and diseased brain. The Ab was highly specific for C3aR and was
positive on frozen or formalin-fixed tissue. In all sections from
normal brain, C3aR staining was almost undetectable except for a very
faint, but reproducible, staining on some astrocytes and a stronger
staining on peripheral macrophages. However, sections of MS (Fig. 8, a–d) and acute BM
(Fig. 8, e–h) brain gave a strong and specific C3aR
staining. Reactive astrocytes (Fig. 8, b and
e), ameboid microglia (Fig. 8c),
and ramified microglia (Fig. 8d) were consistently
and strongly stained for C3aR in both conditions. In MS, but not in BM,
cells in the brain blood vessel wall were also strongly stained for
C3aR (Fig. 8b), and these cells were identified as
smooth muscle cells (pericytes). In acute MS, C3aR was expressed at
high level on infiltrating perivascular macrophages, but not on
infiltrating lymphocytes (data not shown). In the lumen of the vessels,
erythrocytes were always C3aR negative (see Fig. 8b,inset). Infiltrating PMN in BM were also stained for C3aR
(Fig. 8, f and h). At high magnification
it was noted that C3aR staining on neutrophils was variable in
distribution, i.e., patchy on some cells and homogeneously distributed
on others (Fig. 8, f and h). Occasional
perivascular macrophages present in BM sections were also stained for
C3aR (Fig. 8g). Double IF staining of MS tissue
sections (Fig. 9) confirmed that all
cells positive for C3aR were also either GFAP positive
(astroglia) or HLA class II positive (macrophage/microglia).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of a C3aR on monocyte cell lines was first demonstrated by
showing a specific and saturable binding of C3a to U937 cells (18, 34).
Differentiation of U937 using PMA only slightly increased C3a binding,
whereas differentiation with dibutyryl cAMP increased binding 7-fold.
We here show, using the specific anti-C3aR Ab, that monocyte lines
THP1 and U937 express C3aR and that PMA differentiation caused an
up-regulation of C3aR levels. Up-regulation of C3aR was detectable only
after prolonged incubation with PMA; in contrast, CR3 was up-regulated
within 1 h of PMA exposure. The rapid up-regulation of CR3 is a
consequence of mobilization of an intracellular pool, and the slow
response of C3aR suggests than no PMA-responsive pool is present.
Up-regulation of C5aR by PMA followed kinetics similar to those of
C3aR, but the increase was much greater, up to 10-fold. IFN- in our
hands was the most powerful regulator of C3aR expression by two
different monocyte cell lines. Even at 10 IU/ml, IFN- caused a
2-fold increase in C3aR expression on U937. The effect of IFN- was
time and dose dependent. Our data confirm previous reports that showed
that [125I]C3a binding to U937 was elevated when
cells were cultured for 3 days with IFN- (34). Neutrophils isolated
from blood expressed C3aR and at higher levels than did
undifferentiated THP1 or blood-derived monocytes. Lymphocytes were
consistently negative for C3aR. We have yet to examine whether C3aR can
be up-regulated on monocytes and neutrophils or expressed de novo by
lymphocytes when cells are primed with cytokines or chemokines.
We have previously reported the expression of C5aR on glial cells in vitro and the remarkable correlation between expression by glia in vivo and brain inflammation (14, 15, 16). We have proposed that expression of C5aR in the inflamed brain is important in perpetuation of the inflammatory response. The primary aim in generating reagents for detection of C3aR was to ascertain whether it follows a similar pattern of expression in brain cells and tissue. In the original descriptions of cloning, the 2.2-kb C3aR mRNA was detected by Northern analysis in human brain tissue (24, 25, 26); in the present report we confirm this finding. However, no data were available on the cell types expressing C3aR in brain. Staining with the specific anti-C3aR Ab demonstrated that astrocyte cell lines and primary fetal and adult astrocytes expressed C3aR in vitro, with the more differentiated of the cell lines and adult astrocytes expressing more than the undifferentiated cell line and fetal astrocytes. To further confirm that the receptor expressed by astrocytes was identical with that cloned from leukocyte cell lines we used a PCR strategy to clone and sequence astrocyte C3aR cDNA. The majority of the astrocyte C3aR cDNA sequence (residues 369–1433) was obtained in this manner and was 100% identical with the published sequence obtained from HL60 cells (24).
Although all cells in astrocyte cultures expressed C3aR, the
distribution on individual cells was not homogeneous; C3aR was present
in dense patches on the membrane, particularly near the ends of
cellular processes (Fig. 3). The distribution is very reminiscent of
that reported for C5aR on astrocytes (14); double-staining studies will
be necessary to demonstrate whether these two proteins are colocalized.
Several other membrane proteins stained using an identical protocol
gave a homogeneous distribution pattern on astrocytes, eliminating the
possibility that this unusual distribution pattern of C3aR and C5aR is
an artifact of the staining procedure. We suggest that the enrichment
of the receptor at specific areas on the cell membrane is of functional
significance, perhaps enhancing the capacity of the cells to respond to
the anaphylatoxins. It will be interesting to examine whether the
distribution of the anaphylatoxin receptors alters when cells are
stimulated with C3a or C5a and when cells are migrating along an
anaphylatoxin gradient.
Microglia derived from fetal and adult brain were also strongly stained
for C3aR (Fig. 4b). This finding was anticipated
because microglia are derived from the monocyte/macrophage lineage (38)
and strongly express C5aR (16). The demonstration of C3aR expression by
human microglia also supports the recent report that C3a induces
calcium fluxes in cultured murine microglia (29). Oligodendrocytes and
neurons in culture were always negative for C3aR.
The molecular mass of expressed C3aR has not previously been
determined. The cDNA sequence predicts a mature protein of 482 amino
acids and a molecular mass of 54 kDa; however, the sequence contains
two putative N-glycosylation sites. Immunoprecipitation of
C3aR from leukocyte lines or from astrocyte lines followed by Western
blotting demonstrated that the protein from both sources had a
molecular mass of 65 kDa, implying that the C3aR is heavily
glycosylated in both cell types (Fig. 5C). Although
all immunoprecipitates were from whole cells, the predicted 54-kDa
unglycosylated C3aR precursor was never detected. We are currently
confirming the above data by growing cells in tunicamycin to inhibit
glycosylation before immunoprecipitation. Experiments are also underway
to define the molecular mass of C3aR from eosinophils, neutrophils,
monocytes, and primary astrocytes. The principal difficulty in these
experiments is to obtain sufficient numbers of cells in sufficient
purity for use in the immunoprecipitation protocol.
Expression of C3aR was confirmed for the various cell lines and primary
cells at the mRNA level by RT-PCR. All cells positive by IF were also
positive by RT-PCR (Fig. 7). The endothelial cell line ECV304, untested
in IF, was also positive, whereas the hepatoma cell line HepG2, also
untested in IF, was negative. The absence of detectable message for
C3aR in HepG2 cells is interesting in that this cell line is reported
to express the C5aR (5, 13); it may thus represent the unusual
occurrence of expression of one anaphylatoxin receptor without the
other. To confirm specificity and increase sensitivity, a
combination of RT-PCR and Southern blotting was used. With this
protocol, C3aR message was detected at very low levels in the various
lymphocyte cell lines and in HepG2 (Fig. 7).
Given the above data on glial cell expression of C3aR and the presence
of C3aR message in brain tissue (24, 25, 26), it was expected that C3aR
would be detected by immunohistochemistry in brain. However, normal
brain tissue was almost completely negative for C3aR expression,
although the Ab readily detected C3aR in other normal tissues (lung,
liver, and adrenal gland) subjected to a similar processing protocol
(data not included). In marked contrast, inflamed brain tissue (MS or
meningitis) was strongly positive for C3aR (Figs. 8 and 9). This
finding is, again, very similar to that obtained for C5aR, which was
barely detectable in normal brain but was highly expressed in
inflammation (16). The distribution of C3aR expression differs between
the two conditions chosen for study. In both, astrocytes and microglia
in the areas of pathology and infiltrating phagocytes (macrophages in
MS, neutrophils in meningitis) express C3aR. In MS, a strong
perivascular staining was observed, which was not present in meningitis
tissue, that appeared to be associated with pericytes. The functional
significance of expression of C3aR by pericytes in the vessel wall is
uncertain, but it does suggest that the anaphylatoxins might influence
vessel tone or permeability in MS brain. Elevated expression of
anaphylatoxin receptors in CNS tissue appears to be a hallmark of
inflammatory processes and is probably associated with intrathecal
complement activation and local generation of the anaphylatoxins (27, 39). The expressed receptors may then contribute to an autocrine
pathway, activating glia and recruiting myeloid and nonmyeloid cells
into the brain tissue (38). This scenario is supported by the in vitro
observation that C3a and C5a stimulate and also induce chemotaxis of
astrocytes and microglia (14, 28, 29, 40). Monocyte/macrophage
expression of cytokines, chemokines, and other immune molecules is
regulated by C3a and C5a (3, 12, 19, 41); we are now testing whether
C3a and/or C5a have similar effects on astrocytes and microglia in
culture.
To address the roles of the anphylatoxins and their receptors in CNS inflammation it will be necessary to use models in which the receptors and their ligands can be manipulated. We have established rodent models of demyelination and ischemia (both conditions characterized by a severe brain inflammation with complement activation and genesis of anaphylatoxins), and we plan to block specifically the effects of both anaphylatoxins using specific antagonist peptides and neutralizing Abs for ligands and receptors. Mouse and rat C5aR have been cloned, and recently, mouse C3aR has been cloned from a brain cDNA library (42, 43, 44). The reagents necessary for receptor blockade in rodents are currently being generated.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Philippe Gasque, Department of Medical Biochemistry, Brain Inflammation and Immunity Group, University of Wales College of Medicine, Cardiff, Wales CF44XN. E-mail address:
3 Abbreviations used in this paper: PMN, polymorphonuclear cells; C5aR, receptor for C5a; C3aR, receptor for C3a; MS, multiple sclerosis; BM, bacterial meningitis; CNS, central nervous system; MAP, multiple array peptide; GST, glutathione-S-transferase; GFAP, glial fibrillary acidic protein; MNC, mononuclear cells; PE, phycoerythrin; DAB, diaminobenzidene; IF, immunofluorescence; DAF, decay-accelerating factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Received for publication August 21, 1997. Accepted for publication December 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß synthesis. J. Immunol. 156:3455.[Abstract]
and PMA in U937 cells and related myeloblastic cell lines. J. Immunol. 157:5574.[Abstract]
upregulates the human C5a receptor (CD88) in myeloblastic U937 cells and related cell lines. J. Immunol. 155:4419.[Abstract]
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