C5a Receptor Expression Research Articles

Expression of complement factors and functional C5a receptors in human embryonic stem cells, and induced pluripotent stem cells

Expression of complement factors and functional C5a receptors in human embryonic stem cells, and induced pluripotent stem cells

Novel insights in localization and expression levels of C5aR and C5L2 under native and post-transplant conditions in the kidney

AimsThe complement system, and especially C5a, plays an important role in the pathophysiology of renal diseases and post-transplant renal injury. The two receptors for C5a are C5a receptor (C5aR) and C5a-like-receptor-2 (C5L2). Only renal C5aR expression has been reported, although exact localization and alterations in expression after transplantation are unknown. Materials and resultsRenal C5aR and C5L2 expression and localization were analyzed immunohistochemically. C5aR and C5L2 expression was analyzed in human kidney biopsies obtained from living donors and patients suffering from acute tubular necrosis, acute cellular and vascular rejection or IF/TA.C5aR was expressed in the thick ascending limb of Henle's loop and first part of the distal convoluted tubule (DCT). Under inflammatory conditions, C5aR was de novo expressed in proximal tubuli. C5L2 was expressed in the kidney and localized to DCT1, DCT2 and connecting tubule. Persistent distal tubular expression of both receptors was demonstrated after renal transplantation. ConclusionsThis study shows distinct renal expression patterns for C5aR and C5L2. Our findings suggest a functional role for renal C5L2 rather than being a C5a decoy receptor. Future studies focusing on renal C5a–C5aR interaction should take differential C5aR and C5L2 expression into account, alongside abundant C5aR expression on infiltrating cells.

Decreased expression of complement 3a receptor (C3aR) in human placentas from severe preeclamptic pregnancies

The aim of this study was to determine the expression of the anaphylatoxin receptors complement C3a receptor (C3aR) and C5a receptor (C5aR) in the placentas of pregnancies complicated by severe early onset preeclampsia. We recruited women with pregnancies complicated by severe early-onset preeclampsia (n=19, 11 of which were further complicated with IUGR) and women with preterm pregnancies not affected by preeclampsia (n=8). Gene and protein expression of C3aR and C5aR was analysed by quantitative RT-PCR and Western blotting, respectively. C3aR was detected in the Hofbauer cells in the villous stroma of the placenta. C5aR staining was detected in the syncytiotrophoblast and endothelial cells. We found significantly decreased expression of C3aR mRNA and protein expression in placentas with preeclampsia compared to controls. However, C5aR expression was not significantly different between preeclamptic and control placentas at either the mRNA or protein level. Decreased C3aR expression indicates a dysregulation of the complement system in the placentas of preeclamptic women. Further studies would elucidate the exact mechanisms that complement has in preeclampsia.

Enhancement of human cancer cell migration and invasion by anaphylatoxin C5a viaaberrantly expressed C5a-receptor.

e21084 Background: Accumulated evidence has revealed implications of chemokines and their receptors in the pathophysiology of cancer. Anaphylatoxin C5a, a fragment of the complement 5th component (C5), is a potent chemokine for leukocytes that express the C5a receptor (C5aR). Recent reports indicate production of C5a by proteases of various origins, besides a by-product of activation of the complement system, and expression of C5aR in non-myeloid cells, suggesting an involvement of C5a in these cell biology. However, little is known about roles of C5aR in human cancer. Aim of our study was to investigate the expression and the role of C5aR on cancer cells in proliferation and invasion. Methods: Surgically resected cancer tissues and cancer cell lines were investigated C5aR expression. The effect of C5a on cancer cell proliferation, cell shape and invasion activity were examined in vitro and in vivo. Results: By immunohistochemical analysis, C5aR was strongly expressed in human cholangiocarcinoma samples. Using RT-PCR, immunoblotting and flow cytometric analysis human cholangiocarcinoma cell line MEC but not HuCCT1 were found to express C5aR. Incubation of both cancer cells with C5a did not increase these cells, indicating no mitogenic effect of C5a on these cells. Interestingly, C5a stimulated invasion of MEC and C5aR-transgenic HuCCT1 when examined by Matrigel invasion assay, which was suppressed by anti C5aR antibody. F-actin staining and human MMPs array revealed that C5a induced actin polymerization and MMPs production, respectively, in MEC and C5aR-transgenic HuCCT1. In contrast, these C5a effects were not seen in HuCCT1. Conclusions: These results illustrate a novel activity of C5a-C5aR axis that promotes cancer cell invasion through cytoskeletal rearrangement and MMP release. Thus, targeting this signaling pathway may provide a useful therapeutic option for cancer treatment.

Roles of promoter and 3′ untranslated motifs in expression of the human C5a receptor

The C5a receptor (C5aR) is a 7 transmembrane G-protein coupled receptor (GPCR) that mediates the powerful pro-inflammatory effect of the complement activation product C5a. Excess C5a generated under pathological conditions has been implicated in a variety of conditions including sepsis, asthma and rheumatoid arthritis, but very little is known about the regulation of expression of the C5aR. The 5′ promoter region and 3′ untranslated region (UTR) of the C5aR mRNA were cloned, generating enhanced green fluorescent protein (EGFP)-reporter plasmids, which were transfected into the monocytic cell line U937. Most of the cloned 2kb 5′ region was dispensable for the expression of the reporter constructs and the majority of regulatory sequences are in the first 200bp. Three motifs, a NFκB, a CCAAT and a NFAT site, were identified to be of importance by site directed mutagenesis for basal expression. Analysis of the 3′UTR of the C5aR mRNA showed that it contained two AU-rich elements (AREs), however site directed mutagenesis showed that these had no effect on basal expression. While the phorbol ester PMA and dibutyryl cAMP increased C5aR protein expression, these agents had no effect on the regulation of expression via the promoter or the 3′UTR. This is the first study to investigate the role of both the promoter and 3′UTR in regulating C5aR expression and our results show that regulation of the human C5aR is similar but not identical to that of the mouse C5aR.

444 ABERRANT EXPRESSION OF C5AR IN METASTATIC RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2012444 ABERRANT EXPRESSION OF C5AR IN METASTATIC RENAL CELL CARCINOMA Yoshihiro Maeda, Yoshihiro Wada, Yoshiaki Kawano, Ken Kikuchi, Wataru Takahashi, Jiro Honda, Juro Nakanishi, Takahisa Imamura, and Masatoshi Eto Yoshihiro MaedaYoshihiro Maeda Kumamoto, Japan More articles by this author , Yoshihiro WadaYoshihiro Wada Kumamoto, Japan More articles by this author , Yoshiaki KawanoYoshiaki Kawano Kumamoto, Japan More articles by this author , Ken KikuchiKen Kikuchi Kumamoto, Japan More articles by this author , Wataru TakahashiWataru Takahashi Kumamoto, Japan More articles by this author , Jiro HondaJiro Honda Kumamoto, Japan More articles by this author , Juro NakanishiJuro Nakanishi Kumamoto, Japan More articles by this author , Takahisa ImamuraTakahisa Imamura Kumamoto, Japan More articles by this author , and Masatoshi EtoMasatoshi Eto Kumamoto, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.511AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Anaphylatoxin C5a is a-fragment of complement 5th component, which is a potent chemoattractant. Recent studies showed C5a receptor (C5aR) is aberrantly expressed in human cancers, which plays a crucial role in cancer invasion via enhancing cancer cell motility. In this study, we analysed expression of C5aR in metastatic and non-metastatic renal cell carcinoma (RCC). The possible role of C5aR in renal cancer cells was also investigated by in vitro analysis. METHODS We retrospectively analysed data from 127 Renal Cell Carcinoma patients who had received radical or partial nephrectomy or biopsy between 2002 and 2011. C5aR expression in renal cell carcinoma samples were analysed by immunohistochemistry using Formalin-Fixed Paraffin-Embedded tissue samples, and correlation of C5aR staining and clinicopathological parameters was analysed. For in vitro analysis, 293 cells, which are derived from human renal epithelial cells, were transiently transfected with C5aR expression plasmid then cell lysate was subjected to immunoblotting with anti-total or phospho ERK antibodies. RESULTS This study used data from 127 RCC patients (86 men and 41 women). The median (range) age of the patients was 62 (22-87) years. 97 patients (66 men and 31 women) had non-metastatic RCC, whose median (range) age was 63 (22-85) years, and 30 patients (20 men and 10 women) had metastatic RCC (mRCC), whose median (range) age was 59 (30-87) years. Immunohistochemical analysis showed that 96.7% of mRCC was C5aR positive, whereas 50.5% of non-metastatic RCC expressed C5aR (Fisher&undefined;fs Exact Test, p<0.001). There is no correlation between C5aR expression and other clinicopathological parameters (e.g. stage, Fuhrman grade, histological subtypes). In vitro experiment revealed that overexpression of C5aR triggers activation of ERK in renal epithelium-derived cells, which is a crucial molecular event for renal cell carcinoma progression. CONCLUSIONS This study suggests that aberrant expression of C5aR may be a pivotal step for cancer cell metastasis. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e182 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yoshihiro Maeda Kumamoto, Japan More articles by this author Yoshihiro Wada Kumamoto, Japan More articles by this author Yoshiaki Kawano Kumamoto, Japan More articles by this author Ken Kikuchi Kumamoto, Japan More articles by this author Wataru Takahashi Kumamoto, Japan More articles by this author Jiro Honda Kumamoto, Japan More articles by this author Juro Nakanishi Kumamoto, Japan More articles by this author Takahisa Imamura Kumamoto, Japan More articles by this author Masatoshi Eto Kumamoto, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Expression of Receptors for Anaphylatoxins C3a and C5a on Rat Islet Preparations

Background Complement activation has been implicated in the development of the instant blood-mediated inflammatory reaction (IBMIR). In particular, anaphylatoxins C3a and C5a elicit a broad range of proinflammatory effects, including chemotaxis of inflammatory cells and cytokine release. We have previously shown that 2 types of receptors for C5a are expressed on isolated islets. In the present study, we investigated this component in detail. Methods C3aR, C5aR, and C5L2, together with CD11b and CD31, on freshly isolated islets (fresh group) and islets cultured with (cytokine group) or without (culture group) TNF-α, IL-1β, and IFN-γ for ∼12 hours were analyzed by flow cytometry. In addition, these 3 kinds of receptors were analyzed on nonendocrine cells. Results C5aR and C5L2 were expressed on the isolated islets (C5aR: 7.91 ± 2.83%; C5L2: 2.45 ± 1.34%) and the expression of both C5a receptors was markedly attenuated by culture for 12 hours (C5aR: P < .005; C5L2: P < .05). Compared with the culture group, the expression was significantly up-regulated in the cytokine group (C5aR: P < .05; C5L2: P = .05). C5aR-positive cells expressed CD11b but not CD31. In contrast to islets, nonendocrine cells expressed C5L2 predominantly. C3aR was scarcely expressed on isolated islets or nonendocrine cells. Conclusions These data suggest that C5aR and C5L2 are expressed on CD11b-positive leukocytes in islet preparations. Depletion of C5a receptors by culturing appropriately could be an attractive therapeutic strategy in clinical islet transplantation.

Complement and the Alternative Pathway Play an Important Role in LPS/D-GalN-Induced Fulminant Hepatic Failure

Fulminant hepatic failure (FHF) is a clinically severe type of liver injury with an extremely high mortality rate. Although the pathological mechanisms of FHF are not well understood, evidence suggests that the complement system is involved in the pathogenesis of a variety of liver disorders. In the present study, to investigate the role of complement in FHF, we examined groups of mice following intraperitoneal injection of LPS/D-GalN: wild-type C57BL/6 mice, wild-type mice treated with a C3aR antagonist, C5aR monoclonal antibody (C5aRmAb) or CR2-Factor H (CR2-fH, an inhibitor of the alternative pathway), and C3 deficient mice (C3−/− mice). The animals were euthanized and samples analyzed at specific times after LPS/D-GalN injection. The results show that intraperitoneal administration of LPS/D-GalN activated the complement pathway, as evidenced by the hepatic deposition of C3 and C5b-9 and elevated serum levels of the complement activation product C3a, the level of which was associated with the severity of the liver damage. C3a receptor (C3aR) and C5a receptor (C5aR) expression was also upregulated. Compared with wild-type mice, C3−/− mice survived significantly longer and displayed reduced liver inflammation and attenuated pathological damage following LPS/D-GalN injection. Similar levels of protection were seen in mice treated with C3aR antagonist,C5aRmAb or CR2-fH. These data indicate an important role for the C3a and C5a generated by the alternative pathway in LPS/D-GalN-induced FHF. The data further suggest that complement inhibition may be an effective strategy for the adjunctive treatment of fulminant hepatic failure.

M cells expressing the complement C5a receptor are efficient targets for mucosal vaccine delivery

In the mucosal immune system, M cells are known as specialized epithelial cells that take up luminal antigens, although the receptors on M cells and the mechanism of antigen uptake into M cells are not well-understood. Here, we report the expression of the complement C5a receptor (C5aR) on the apical surface of M cells. C5ar mRNA expression in co-cultured Caco-2 human M-like cells was six-fold higher than in mono-cultured cells. C5aR expression was detected together with glycoprotein 2, an M-cell-specific protein, on the apical surface of M-like cells and mouse Peyer's patch M cells. Interestingly, after oral administration of Yersinia enterocolitica which expresses outer membrane protein H (OmpH) that is homologous to the Skp α1 domain of Escherichia coli, a ligand of C5aR, dense clustering and phosphorylation of C5aR were detected in M cells. Finally, targeted antigen delivery to M cells using C5aR as a receptor was achieved using the OmpH α1 of Y. enterocolitica such that the induction of ligand-conjugated antigen-specific immune responses was confirmed in mice after oral immunization of the OmpH β1α1-conjugated antigen. Collectively, we identified C5aR expression on M cells and suggest that C5aR could be used as a target receptor for mucosal antigen delivery.

In this issue

In this issue

C5a contributes to intraocular inflammation by affecting retinal pigment epithelial cells and immune cells

BackgroundThe complement activation molecule C5a has been found in the eye and is implicated in the pathogenesis of ocular inflammatory diseases. In this study, the authors sought to investigate C5a's...

Increased renal C5a receptor expression and systemic complement activation in brain dead donors

Increased renal C5a receptor expression and systemic complement activation in brain dead donors

Complement C5a inhibition reduces atherosclerosis in ApoE –/– mice

The complement C5a receptor, CD88, is present on many of the cells found within human atherosclerotic plaques, but little is known about the role of C5a in atherogenesis. Using real-time PCR, we determined that ApoE(-/-) mice fed a normal diet express more aortic CD88 mRNA compared with controls, and this increase coincides with atherosclerotic lesion development (P<0.001 for 3- vs. 25-wk-old animals). Conversely, mRNA expression of the alternative C5a receptor, C5L2, in aortas of ApoE(-/-) mice, was lower than controls at all time points. Using immunohistochemistry, we confirmed the presence of CD88 on macrophages, smooth muscle cells, and activated endothelial cells in plaques from brachiocephalic arteries. Treatment of ApoE(-/-) mice with a CD88 antagonist (PMX53; 3 mg/kg s.c. 3 ×/wk plus 1 mg/kg/d p.o.) for 25 wk reduced lesion size and lipid content in the plaque by ∼ 40% (P<0.05). Our study provides evidence for a proatherogenic role for C5a and identifies the CD88 antagonist PMX53 as a potential antiatherosclerotic drug.

Blockade of complement activation product C5a activity using specific antibody attenuates intestinal damage in trinitrobenzene sulfonic acid induced model of colitis

Complement represents a chief component of innate immunity in host defense. However, excessive complement activation has been involved in the pathogenesis of inflammatory diseases. In this study, we investigated the contribution of complement to intestinal pathology of patients and rodents with inflammatory bowel disease. The expression of complement effectors (C3a and C3) was increased remarkably in inflamed colons of IBD patients compared with those of normal counterparts. In accordance with this, the sustained activation of complement in serum and colon (including elevated C3a and C5a levels, enhanced hemolytic activity, downregulated expression of C5a receptors) was observed, following the establishment of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, which peaked at 24 h. Mice pretreated with neutralizing anti-C5a antibodies (−2, 0, and 2 days after TNBS instillation) had significantly reduced weight loss and improved macroscopic/microscopic scores, comparable to the efficacy of prednisolone treatment. Strikingly, treatment with anti-C5a at 24 h after TNBS instillation showed remarkable therapeutic effects, whereas prednisolone did not. The efficacy of anti-C5a administration was associated with decreased release of proinflammatory chemokines and cytokines, inhibition of infiltration of neutrophils into colons, and enhanced Th2 response. These findings suggest a disease-promoting role of complement, particular C5a, in the pathology of TNBS-induced colitis in mice, indicating possible therapeutic potentials for C5a-specific antibody in IBD.

Expression and Function of C5a Receptor in a Fatal Anaphylaxis After Honey Bee Sting*

The mechanisms leading to death from anaphylaxis after insect sting involve antigen cross-linkage of antibody molecules that activate immunoglobulin receptors on inflammatory cells. The aim of our study was to investigate the pathomorphology and the expression of C5aR in fatal anaphylaxis in a patient after a fatal insect sting. A 38-year-old women was stung by a honeybee. C5R1 expression was detected in many dilated capillaries in the lungs. Pulmonary epithelial cells did not bind the monoclonal antibody for C5R1; however, intensive cytoplasmic staining was detected in endothelial cells. The findings of this case provide evidence for an active role of complement in fatal anaphylaxis elicited by bee venom. C5aR detection could be useful in the identification of sudden death cases because of unwitnessed fatal insect sting cases. Authors can recommend this immunohistochemical analysis on all sudden unexpected deaths outdoors where a possible bee sting might occur.

Anaphylatoxin C5a-induced toll-like receptor 4 signaling in bovine neutrophils

It is well known that signaling in neutrophils through both the complement component 5a (C5a) and C5a receptor (C5aR) and the toll-like receptor 4 (TLR4) pathways plays an essential role in innate defense. Neutrophil dysfunction, as seen during sepsis in severe mastitis during the periparturient period, is correlated with elevated concentrations of anaphylatoxin C5a. The aim of the current study was to elucidate the effect of C5a on TLR4 signaling in bovine neutrophils. Neutrophils were incubated with a high (but physiological) dose of purified C5a, and mRNA was extracted from neutrophils at different time points postincubation (PI). The incubation with C5a resulted in a biphasic C5aR expression profile, a phenomenon that might be explained by internalization (at 10min PI) with subsequent reconstitution (starting at 40min PI) of this receptor. The expression of TLR4, as well as its coreceptor, CD14, showed a similar biphasic change as observed with C5aR. In addition, changes in the mRNA expression levels of several genes belonging to the TLR4 pathway, such as TICAM-1, IKKα, and MAP3K7 were noted. The maximal expression of TLR4, CD14, and C5aR mRNA at 80min PI was accompanied by a peak in IL8 mRNA, indicating that C5a is able to induce IL-8 production in neutrophils in vitro without the need of a costimulatory factor such as lipopolysaccharide. Moreover, a relatively constant expression of RELA was accompanied by increased expression of ATF3, an endogenous inhibitor of nuclear factor-κB mediated transcription, implying that C5a regulates TLR4 signaling and IL-8 synthesis independently. A significant time-dependent correlation was found between C5aR and TLR4, with the majority of the selected TLR4-dependent genes showing a significant correlation with C5aR at 80min PI, when C5aR and TLR4 mRNA expression reached its maximum, suggesting crosstalk between both receptors. Taken together, this study showed that C5a is able to (1) alter the expression of genes belonging to the TLR4 pathway and (2) induce IL8 gene expression in bovine neutrophils. In addition, indications for cross-talk between complement activation and TLR4 signaling were found in the present study.

Increased expression of C5a receptor (CD88) mRNA in canine mammary tumors

Mammary tumors are among the most common neoplastic conditions in dogs, and there is evidence that inflammation plays a role in the development of some tumor types in dogs. The complement system is a major participant in the inflammatory process and the complement activation component, C5a, is a potent inflammatory peptide. This study investigated the mRNA expression of the major receptor for C5a (C5aR; CD88) in histopathological samples of canine mammary tumors by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) using canine-specific primers for CD88. A total of seven canine mammary tumors (four malignant carcinomas, two benign mixed mammary tumors, and one myoepithelioma) and eight normal mammary glands were analysed. All the tumor samples expressed low levels of CD88 mRNA, while none of the normal mammary tissues showed any detectable expression. These preliminary results suggest that C5a–CD88 interaction may play a contributory role in the inflammatory response associated with mammary tumor development in dogs. Further studies investigating the mechanisms behind complement activation and C5a receptor expression in canine mammary tumors are warranted.

C5a-Inhibitory Peptide Combined with Gabexate Mesilate Prevents the Instant Blood-Mediated Inflammatory Reaction in a Rat Model of Islet Transplantation

Background The instant blood-mediated inflammatory reaction (IBMIR), in which the activation of both the coagulation and the complement cascades plays a key role, is one of the main obstacles to successful islet transplantation. At present, however, no useful protocol is clinically available. Therefore the aim of this study was to examine whether complementary peptides against an active region of C5a were safe to suppress IBMIR, owing to their extremely low molecular mass, when combined with a clinically available anticoagulant. Methods Complement receptors on pancreatic tissues and isolated islets were analyzed by immunohistochemical staining and flow cytometry. Two-and-a-half islet equivalents per gram of syngeneic rat islet grafts were transplanted intraportally into 4 groups of 10–13 animals each after streptozotocin induction of diabetes: control, gabexate (Gab), C5a-inhibitory peptide (C5aINH), and C5aINH plus Gab. Recipients injected with equivalent amounts of saline solution served as control subjects. Plasma samples were collected at 0, 0.5, 1, 3, 6, and 24 h after transplantation for analysis. We also evaluated the curative rate, intravenous glucose tolerance test, and insulin amounts in the liver of the recipients. Results C3a receptor (C3aR) was scarcely expressed on the isolated islets with relatively strong expression of C5a receptor (C5aR): C3aR: 0.44 ± 0.38%; C5aR: 7.91 ± 2.83%). However, C5aR was not expressed on pancreatic tissues before the isolation procedures. Thrombin-antithrombin complex was significantly suppressed in the 3 treated groups ( P = .0015). The curative rate was also significantly improved (0% vs 33% vs 67% vs 100%, respectively; P = .03). Glucose tolerance was significantly improved among the 3 treated groups ( P < .0005). Insulin amounts in the liver were considerably higher among treated versus control hosts. Notably, the treatment did not affect the increased body weight of the recipient. Conclusions This study suggested that C5a-inhibitory peptide combined with gabexate mesilate may be a useful approach to control the IBMIR induced in clinical islet transplantation and one that is free of side effects.

Role of G-protein Coupled Receptor Kinases GRK2 and GRK3 in C3a Receptor Signaling in Human Mast Cells (133.12)

Abstract Studies with animal models have shown that complement components C3a and C5a play an important role in the effector phase of allergic asthma. We have previously shown that C3a activates human CD34+-derived mast cells to induce degranulation. To determine the role of GRKs on C3a receptor (C3aR) signaling in human mast cells, we used shRNA to knockdown GRK2 and GRK3 a human mast cell line, HMC-1 and CD34+-derived cultures of primary mast cells. We found that GRK2 and GKR3 knockdown inhibited C3aR desensitization as measured by enhanced C3a-induced Ca2+ mobilization in HMC-1 cells and greater degranulation in CD34+ mast cells but had no effect on receptor internalization. In addition, GRK3-knockdown in HMC-1 cells showed increased expression of cell surface C3aR and this was correlated with enhanced extracellular-signal-regulated kinase (ERK) activation and a four-fold increase in C3a-induced chemotaxis. Surprisingly, there was no enhancement of these responses in GRK2 knockdown mast cells. These findings suggest that while GRK2 and GRK3 are required for C3aR sensitization, presumably by promoting receptor phosphorylation, they play distinct roles in modulating downstream signaling such as ERK phosphorylation and chemotaxis.

Complement C3a Regulates Late Asthmatic Response and Airway Hyperresponsiveness in Mice

Allergic asthma is a chronic inflammatory disorder of the airways characterized by biphasic airway obstruction and airway hyperresponsiveness. In this study, we attempted to elucidate the contribution of the complement C3a to these asthmatic symptoms. BALB/c mice sensitized by i.p. injections of OVA plus alum were challenged with OVA intratracheally four times. The fourth challenge caused a biphasic asthmatic response peaking at 10 min and 3-4 h, as well as airway hyperresponsiveness to methacholine. Histological examination revealed increased expression of C3a receptors in the lung on the fourth challenge. Additionally, the C3 level in serum 4 h after the fourth challenge was significantly reduced compared with that before the challenge. When a C3a receptor antagonist, SB290157, was administered i.p. 30 min before the fourth challenge, the late-phase asthmatic response and airway hyperresponsivness induced by the fourth challenge were significantly inhibited, although the early-phase response was not influenced. In bronchoalveolar lavage fluid, neutrophil infiltration 24 h after the fourth challenge was reduced by the treatment. On the other hand, SB290157 suppressed the increased expression of IL-1beta in the lung in this model, and the intratracheal administration of IL-1beta induced airway obstruction, airway hyperresponsiveness, and neutrophil infiltration in normal mice. These results illustrate that C3a is involved in the development of the late asthmatic response and airway hyperresponsiveness. The mechanism leading to the development of these symptoms may correlate with the recruitment of neutrophils and/or the production of IL-1beta induced by C3a.