Protective effect of a human C5a receptor antagonist against hepatic ischaemia-reperfusion injury in rats

BACKGROUND/AIMS: Complement activation is induced by ischaemia-reperfusion (I/R) and the complement factor C5a plays an important role in organ specific I/R injuries. This study investigated the efficacy of a small molecule C5a receptor (C5aR) antagonist against hepatic I/R injury. METHODS: Total hepatic ischaemia or partial hepatic ischaemia were induced in rats, followed by a period of reperfusion. The C5aR antagonist, AcF-[OPdChaWR], was administered at 1 mg/kg i.v. or 10 mg/kg p.o. or s.c. before induction of ischaemia. Total hepatic I/R-induced mortality was measured and partial hepatic ischaemia injury was assessed by measuring the serum levels of liver enzymes, tissue or serum TNFalpha, liver and lung myeloperoxidase activity, the number of infiltrating neutrophils, neutrophilia and liver histopathology. RESULTS: C5aR antagonist treatment reduced total hepatic I/R-induced mortality. In partial hepatic I/R rats, treatment with the C5aR antagonist significantly attenuated the increases in liver enzymes, serum and tissue TNFalpha, myeloperoxidase activity, infiltrating neutrophils, neutrophilia, and also reduced liver histopathology. CONCLUSIONS: This study shows that an orally active, small molecule C5aR antagonist is effective in reducing the markers of tissue damage caused by I/R in the rat, suggesting an important role for C5a in I/R injuries in the liver.     

C5a-C5aR在脓毒症中的作用

脓毒症中补体系统的过度激活导致大量C5a的产生,并诱导其受体(C5aR)在细胞上的表达异常,C5a-C5aR相互作用调节炎症介质的表达、干扰凝血通路和诱导中性粒细胞功能障碍,最终导致器官功能损伤.     

补体5a受体在阿霉素致急性心力衰竭心肌损伤的早期研究

目的:探讨补体系统补体5a受体(complement 5a receptor,C5aR)在急性心力衰竭(心衰)早期,对心脏功能影响以及对心肌损伤的作用。方法:选择12 w龄C57BL/6J野生型及C5aR敲除小鼠各12只,分别随机分为野生小鼠对照组、野生小鼠心衰组、C5aR敲除小鼠对照组和C5aR敲除小鼠心衰组等共4组,每组6只。采用单次腹腔注射阿霉素20 mg/kg建立小鼠急性心衰模型,对照组采用同计量0.9%氯化钠液注射;阿霉素注射3d后显微超声检测小鼠心室射血分数和短轴缩短率,处死后测量体质量以及心脏质量;运用半定量PCR、蛋白印迹技术Western Blotting、免疫荧光等实验方法观察C5aR在野生小鼠心脏中的表达。C5aR敲除小鼠心脏组织采用HE染色观察心肌形态、Masson染色观察心肌纤维化程度,WGA染色观察心肌横截面大小,免疫组化染色观察转化生长因子-β(TGF-β)及α-平滑肌肌动蛋白(α-SMA)在心脏中表达。结果:与野生小鼠对照组相比,野生小鼠心衰组中C5aR的mRNA及蛋白表达均显著上调(P<0.05);与野生小鼠心衰组相比,C5aR敲除小鼠心衰组体质量及血压均显著升高(均为P<0.05),心室射血分数和短轴缩短率均显著升高(均为P<0.05),C5aR敲除小鼠心衰组胶原沉积及α-SMA表达均显著降低(均为P<0.05),TGF-β表达显著降低(P<0.01)。结论:C5aR在阿霉素诱导心衰模型中表达上调、加重了心肌损伤且促进了心脏纤维化,而C5aR敲除保护小鼠心功能并抑制纤维化,提示C5aR在小鼠急性心衰早期具有促进心脏纤维化作用。     

Differential contributions of C3, C5, and decay-accelerating factor to ethanol-induced fatty liver in mice

BACKGROUND AND AIMS: The complement pathway is an important component of the innate and adaptive immune response. Here we tested the hypothesis that activation of complement is required for development of ethanol-induced fatty liver. METHODS: Wild-type mice and mice lacking the third (C3) or fifth (C5) components of the complement activation pathway, as well as mice lacking decay-accelerating factor (CD55/DAF), a complement regulatory protein, were fed Lieber-DeCarli ethanol-containing diets for 6 weeks or pair-fed control diets. RESULTS: Ethanol feeding to wild-type mice increased C3a in plasma. Wild-type and C5-/- mice fed the ethanol diet developed hepatic steatosis characterized by microvesicular and macrovesicular lipid accumulation and increased triglyceride content. C3-/- mice did not develop steatosis, while CD55/DAF-/- mice accumulated even more hepatic triglyceride after ethanol feeding than wild-type mice. Levels of serum alanine aminotransferase and hepatic tumor necrosis factor alpha, indicators of hepatocyte injury and inflammation, respectively, were increased in wild-type and CD55/DAF-/- mice but not in C5-/- mice after ethanol feeding. In contrast to the protective effect of C3-/- against ethanol-induced steatosis, levels of both alanine aminotransferase and tumor necrosis factor alpha were increased in C3-/- mice after ethanol feeding. CONCLUSIONS: Here we have identified several elements of the complement system as important contributors to ethanol-induced fatty liver. C3 contributed primarily to the accumulation of triglyceride in the liver, whereas C5 was involved in inflammation and injury to hepatocytes. Further, the absence of CD55/DAF exacerbated these responses, suggesting that CD55/DAF serves as a barrier to ethanol-induced fatty liver.     

Targeting the minor pocket of C5aR for the rational design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief

Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. Pharmacological therapies currently available for certain types of pain are only partially effective and may cause severe adverse side effects. The C5a anaphylatoxin acting on its cognate G protein-coupled receptor (GPCR), C5aR, is a potent pronociceptive mediator in several models of inflammatory and neuropathic pain. Although there has long been interest in the identification of C5aR inhibitors, their development has been complicated, as for many peptidomimetic drugs, mostly by poor drug-like properties. Herein, we report the de novo design of a potent and selective C5aR noncompetitive allosteric inhibitor, DF2593A, guided by the hypothesis that an allosteric site, the “minor pocket,” previously characterized in CXC chemokine receptors-1 and -2, is functionally conserved in the GPCR class. In vitro, DF2593A potently inhibited C5a-induced migration of human and rodent neutrophils. In vivo, oral administration of DF2593A effectively reduced mechanical hyperalgesia in several models of acute and chronic inflammatory and neuropathic pain, without any apparent side effects. Mechanical hyperalgesia after spared nerve injury was also reduced in C5aR^−/− mice compared with WT mice. Furthermore, treatment of C5aR^−/− mice with DF2593A did not produce any further antinociceptive effect compared with C5aR^−/− mice treated with vehicle. The successful medicinal chemistry strategy confirms that a conserved minor pocket is amenable for the rational design of selective inhibitors and the pharmacological results support that the allosteric blockade of the C5aR represents a highly promising therapeutic approach to control chronic inflammatory and neuropathic pain.Inflammatory and neuropathic pain are the most prevalent types of pathological pain and represent important health problems. Whereas inflammatory pain is one of the classic symptoms of the inflammatory process, neuropathic pain arises from any of multiple nerve lesions or diseases, with symptoms including hyperalgesia or allodynia (1, 2). Some of the most powerful painkillers, including opioids and nonsteroidal anti-inflammatory drugs, are only partially effective and prolonged exposure can cause unwanted effects (3, 4). As a result, there is continuous effort to identify novel therapeutics for pain control with alternative biological mechanisms and that elicit fewer side effects.Inflammatory mediators, including cytokines/chemokines, play a critical role in the pathogenesis of inflammatory and neuropathic pain (5, 6). Emerging evidences suggest that C5a, the anaphylatoxin produced by complement activation, has potent nociceptive activity in several models of inflammatory and neuropathic pain by interacting with its selective receptor C5aR (7, 8). C5aR belongs to the class A subfamily of the seven-transmembrane (TM) G protein-coupled receptors (GPCR) (9) and is widely expressed in immune cells, including neutrophils (polymorphonuclear cells, PMN), monocytes, microglia, and in nonimmune cells, including neurons in the CNS and dorsal root ganglia (10, 11).Evidence for a role of C5a in nociception sensitization has been obtained in several models of inflammatory pain. For example, C5a was produced at the inflammatory sites and elicited mechanical hyperalgesia by activating the C5aR on infiltrated PMN (7). Direct intraplantar injection of C5a in mice elicited both heat and mechanical hyperalgesia by sensitizing primary afferent C-nociceptors (12, 13). Local activation of C5aR has been also implicated in the pathogenesis of postsurgical pain, a model of postoperative pain (13). Finally, local administration of PMX-53, a C5aR antagonist, attenuated mechanical hyperalgesia induced by carrageenan, zymosan, or lipopolysaccharide (7). In addition to the peripheral role of C5a/C5aR in inflammatory pain, up-regulated levels of C5 and C5aR have been found in spinal cord microglia in animals subjected to spared nerve injury (SNI), a model of neuropathic pain (8). Indeed, C5-null mice or the infusion of PMX-53 into the intrathecal space reduced neuropathic pain hypersensitivity in the SNI model (8). Collectively, these data suggest that a neuroimmune interaction in the periphery and spinal cord through activation of the complement cascade and the production of C5a contributes to the genesis of both inflammatory and neuropathic pain.As for other peptidergic GPCRs, the efforts to identify small molecular weight C5aR antagonists have led to a limited number of molecules, mostly lacking adequate potency and selectivity (14). The most promising candidate so far described, PMX-53, is a cyclic peptidomimetic antagonist designed to mimic the C-terminal portion of C5a (15). Despite the encouraging results obtained in preclinical studies, as for many peptide drugs, the development of PMX-53 has been limited by its short half-life and unfavorable bioavailability (16). In the present study, we report the successful design of a nonpeptidic C5a allosteric small molecular weight inhibitor driven by the structural information on a minor pocket spanning between TM1, -2, -3, -6, and -7 that is highly conserved across the GPCR family and that has been recently proposed as a key motif for the intracellular activation process. Reparixin was previously reported as a neutral allosteric inhibitor of CXCR1 and CXCR2 that binds the TM in a region that overlaps the minor pocket (17, 18). Combining the information from independent sources on structural and functional features of allosteric sites in homologous chemokine receptors, this paper intends to provide what is, to our knowledge, the first example of de novo design of a new class of allosteric small molecular weight inhibitors of a GPCR not belonging to the chemokine receptor family, C5aR. The preclinical candidate, DF2593A, is a potent and orally active C5a noncompetitive allosteric inhibitor with significant antinociceptive effects in a wide range of inflammatory and neuropathic pain models.     

Evidence that the extracellular N-terminal domain of C5aR contains amino-acid residues crucial for C5a binding

Abstract: The human C5a anaphylatoxin is a cationic 74 amino-acid long glycopeptide which derives from proteolysis of the fifth component of complement. It interacts with high affinity with a receptor that belongs to the G protein-coupled receptor superfamily. Several studies have previously suggested that multiple contact points between C5a and the receptor are required to achieve high-affinity interaction. However, at the receptor level little is known about the sites of interaction with C5a. We have investigated by in vitro mutagenesis whether the N-terminal extracellular sequence of the C5a receptor, which is rich in aspartic acid residues, could play some role in C5a binding. Conversion of Asp¹⁰ into asparagine did not impair the level of expression at the plasma membrane, nor did it alter the affinity for C5a. However, we consistently observed a discrepancy between an apparent high level of surface expression and a weak capacity to bind C5a with high affinity, suggesting that many receptor molecules, although present on the cell surface, might be misfolded and unable to bind C5a. Replacement of Pro⁹ by an isoleucine had little effect, if any, on either the affinity or the C5a-binding capacity, whereas the conversion of Pro³⁶ into leucine dramatically reduced the expression of high-affinity receptor at the cell surface. N-glycosylation of human C5a receptor was found to be dispensable for the function of the receptor.     

Critical role of C5a in sickle cell disease

Innate immune complement activation may contribute to sickle cell disease (SCD) pathogenesis. Ischemia-reperfusion physiology is a key component of the inflammatory and vaso-occlusive milieu in SCD and is associated with complement activation. C5a is an anaphylatoxin, a potent pro-inflammatory mediator that can activate leukocytes, platelets, and endothelial cells, all of which play a role in vaso-occlusion. We hypothesize that hypoxia-reoxygenation (H/R) in SCD mice activates complement, promoting inflammation and vaso-occlusion. At baseline and after H/R, sickle Townes-SS mice had increased C3 activation fragments and C5b-9 deposition in kidneys, livers and lungs and alternative pathway Bb fragments in plasma compared to control AA-mice. Activated complement promoted vaso-occlusion (microvascular stasis) in SS-mice; infusion of zymosan-activated, but not heat-inactivated serum, induced substantial vaso-occlusion in the skin venules of SS-mice. Infusion of recombinant C5a induced stasis in SS, but not AA-mice that was blocked by anti-C5a receptor (C5aR) IgG. C5a-mediated stasis was accompanied by inflammatory responses in SS-mice including NF-κB activation and increased expression of TLR4 and adhesion molecules VCAM-1, ICAM-1, and E-selectin in the liver. Anti-C5aR IgG blocked these inflammatory responses. Also, C5a rapidly up-regulated Weibel-Palade body P-selectin and von Willebrand factor on the surface of human umbilical vein endothelial cells in vitro and on vascular endothelium in vivo. In SS-mice, a blocking antibody to P-selectin inhibited C5a-induced stasis. Similarly, an antibody to C5 that blocks murine C5 cleavage or an antibody that blocks C5aR inhibited H/R-induced stasis in SS-mice. These results suggest that inhibition of C5a may be beneficial in SCD.     

Binding of fluorescein-labeled anaphylatoxin C5a to human peripheral blood, spleen, and bone marrow leukocytes.

The expression of C5a receptors (C5aR) on human leukocytes was evaluated by flow cytometry using fluorescein-labeled human C5a (C5a-F). Granulocytes and CD14+ mononuclear cells (MNL) but not CD3+, CD20+, CD16+, CD56+, or CD11b+ lymphocytes in peripheral blood and spleen bound C5a-F. C5a-F binding was saturable and inhibitable by anti-C5a monoclonal antibody (MoAb) C17/5 or unlabeled C5a. During hemodialysis, which led to the generation of C5a, only granulocytes and monocytes increased their expression of the adhesion molecule CD11b (CR3). In vitro, C5a induced an increase of CR3 and p 150/95 (CD11c/CR4) only on myeloid cells. However, treatment of leukocytes with phorbol 12-myristate 13 acetate increased CR3 and CR4 expression on both myeloid cells and a lymphocyte subpopulation. Stimulation of MNL in mixed lymphocyte cultures or by treatment with conditioned medium or with IFN-gamma did not induce binding sites for C5aR on lymphocytes and reduced the binding of C5a-F to monocytes. The expression of C5aR on low-density bone marrow cells was analyzed by setting appropriate gates during flow cytometry. Cells that bound C5a-F were found in all populations that contained granulocyte and monocyte precursors, but not in lymphocyte precursor populations. All C5aR+ bone marrow cells were CD34 and expressed high levels of CR3, which suggests a late appearance of C5aR during myeloid cell maturation. Our results indicate that C5aR is exclusively expressed on myeloid cells within the hematopoetic cell population.     

Detection of activated complement complex C5b-9 and complement receptor C5a in skin biopsies of patients with systemic sclerosis (scleroderma)

OBJECTIVE: Upregulated matrix synthesis is a hallmark of systemic sclerosis (SSc). There are indications that growth factors such as platelet derived growth factor (PDGF) are involved in proliferative pathways in SSc lesions. As activated complement releases PDGF from endothelial cells, we searched for activated complement and the complement receptor for C5a (C5aR) in skin biopsies of patients with SSc. METHODS: Snap frozen sections of 8 patients with early SSc and 5 patients with longterm SSc were examined. Using monoclonal antibodies against activated complement complex C5b-9 and the C5aR, skin biopsies derived from both clinically involved and non-involved skin were examined by APAAP immunohistochemistry. RESULTS: A pattern of activated complement C5b-9 and the CSaR could be detected in SSc microvasculature. Eleven of the 13 patients (7/8 patients with early SSc) showed positive staining for C5b-9. The CSaR was detected in 6 of the 8 patients with early SSc. In 3 patients with longterm disease, C5aR expression could also be detected in non-involved skin. CONCLUSION: Activated complement and complement receptors could be detected in early and late stages of SSc skin lesions. The presence of complement receptors in non-involved skin may indicate preclinical activation of pathways resulting in growth factor dependent matrix synthesis.     

Locally produced C5a binds to T cell-expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis

Our recent studies have shown that immune cell-produced complement provides costimulatory and survival signals to naive CD4(+) T cells. Whether these signals are similarly required during effector cell expansion and what molecular pathways link locally produced complement to T-cell survival were not clarified. To address this, we stimulated monoclonal and polyclonal T cells in vitro and in vivo with antigen-presenting cells (APCs) deficient in the complement regulatory protein, decay accelerating factor (DAF), and/or the complement component C3. We found that T-cell expansion induced by DAF-deficient APCs was augmented with diminished T-cell apoptosis, whereas T-cell expansion induced by C3(-/-) APCs was reduced because of enhanced T-cell apoptosis. These effects were traced to locally produced C5a, which through binding to T cell-expressed C5aR, enhanced expression of Bcl-2 and prevented Fas up-regulation. The results show that C5aR signal transduction in T cells is important to allow optimal T-cell expansion, as well as to maintain naive cell viability, and does so by suppressing programmed cell death.     

Blocking the receptor for C5a in patients with rheumatoid arthritis does not reduce synovial inflammation

OBJECTIVES: All complement pathways lead to the formation of C5a, which is believed to contribute to the influx and activation of C5a-receptor (C5aR) bearing cells into the joints of patients with rheumatoid arthritis (RA). Studies in animal models of RA have suggested therapeutic potential of C5aR blockade. In this study, we examined the effects of the C5aR blockade on synovial inflammation in RA patients. METHODS: We performed a double-blind, placebo-controlled study using an orally administered C5aR-antagonist. Twenty-one patients with active RA were randomized 2:1 to treatment with a C5aR-antagonist AcF- (OpdChaWR) (PMX53) vs placebo for 28 days. Serum concentrations of PMX53 were determined. Synovial tissue was obtained at baseline and after 28 days of treatment for pharmacodynamic analysis using immunohistochemistry and digital image analysis. RESULTS: All patients completed the study. Areas under the curve (AUCs) of PMX53 in patients' blood samples showed a mean of 40.8 nmol h/l. There was neither decrease in cell infiltration, nor changes in key biomarkers associated with clinical efficacy after active treatment. In addition, there was no trend towards clinical improvement in the C5aR-antagonist-treated group compared with placebo nor was there a correlation between the AUC and clinical response. CONCLUSIONS: Treatment with PMX53 did not result in a reduction of synovial inflammation despite reaching serum levels of PMX53 that block C5aR-mediated cell activation in vitro. The data suggest that C5aR blockade does not result in reduced synovial inflammation in RA patients.     

Activation of Akt through 5-HT2A receptor ameliorates serotonin-induced degradation of insulin receptor substrate-1 in adipocytes

Serotonin (5-hydroxytryptamine, 5-HT) was found to be elevated in the serum of diabetic patients. In this study, we investigate the mechanism of insulin desensitization caused by 5-HT. In 3T3-L1 adipocytes, 5-HT treatment induced the translocation of insulin receptor substrate-1 (IRS-1) from low density microsome (LDM), the important intracellular compartment for its functions, to cytosol, inducing IRS-1 ubiquitination and degradation. Moreover, inhibition of 5-HT-stimulated Akt activation by either ketanserin (a specific 5-HT2A receptor antagonist) or knocking-down the expression of 5-HT2A receptor promoted 5-HT-stimulated IRS-1 dissociation from 14-3-3β in LDM, leading to drastic ubiquitination. Interestingly, sarpogrelate, another antagonist of 5-HT2A receptor, protected IRS-1 from degradation through activation of Akt. This implicates the importance of Akt activation in extending IRS-1 life span through maintaining their optimal sub-location into adipocytes. Taken together, this study suggest that activation of Akt may be able to compensate the adverse effects of 5-HT by stabilizing IRS-1 in LDM.     

C5a receptor enables participation of mast cells in immune complex arthritis independently of Fcγ receptor modulation

Objective

Mast cells are tissue‐resident immune sentinels that are implicated in the pathogenesis of inflammatory joint disease. The aim of this study was to test our hypothesis that complement fragments could be key activators of synovial mast cells in autoimmune arthritis.

Methods

In vivo studies used the murine K/BxN arthritis model, a distal symmetric polyarthritis mediated by IgG immune complexes. Expression of C5aR on synovial mast cells was determined by immunohistochemical and functional studies. C5aR^−/− and control mast cells were engrafted into mast cell–deficient WBB6 F1‐Kit^w/Kit^W‐v (W/Wv) mice to examine the requirement for this receptor in arthritis. C5aR‐dependent activation of mast cells was investigated in C5aR^−/− animals and in murine and human mast cell cultures.

Results

Murine synovial mast cells express functional C5aR. Unlike their wild‐type counterparts, C5aR^−/− mast cells adoptively transferred into W/Wv mice were not competent to restore arthritis, despite equivalent synovial engraftment. Activation of C5aR^−/− mast cells by K/BxN serum in vivo remained intact, indicating that C5aR is dispensable for normal IgG‐mediated triggering. Consistent with this result, cultured mast cells treated with C5a failed to modulate the expression of Fcγ receptors (FcγR) or to otherwise alter the activation threshold. In human mast cells, C5a promoted the production of the neutrophil chemotaxin interleukin‐8, and recruitment of neutrophils at 24 hours after serum administration was impaired in C5aR^−/− mice, suggesting that enhanced neutrophil chemoattractant production underlies the requirement for C5aR on mast cells in arthritis.

Conclusion

Stimulation via C5aR is required to unleash the proinflammatory activity of synovial mast cells in immune complex arthritis, albeit via a mechanism that is distinct from C5a‐modulated expression of FcγR.

     

蛋白激酶C与钙敏感受体在心肌缺血预适应中的保护作用

目的 探讨蛋白激酶C(PKC)与钙敏感受体(CaR),在心肌缺血预适应(IPC)中的保护作用.方法 采用细胞培养方法 ,体外培养大鼠乳鼠心肌细胞,模拟缺血预适应模型,实验分为7组:①正常对照组(C),②缺血再灌注组(1/R),③IPC组,④IPC+PKC抑制剂组(IPC+PKCI),⑤IPC+PKCI+CaR激动剂组(IPC+PKCI+CARS),⑥IPC+CaRS组,⑦IPC+CaR抑制剂组(IPC+CaRI).分别用TUNEL,Hoechst 33342染色法检测细胞凋亡,四甲基偶氮唑比色法(MTT)观察细胞存活率,Western Blot法检测胞浆内caspase-12,CaR,钙蛋白酶(calpain)表达.结果 光镜下,I/R组细胞核缩小,呈强蓝色荧光,染色质浓缩,出现凋亡小体,其他各实验组有不同程度的荧光增强,尤以IPC+PKCI+CaRS组多见强蓝色荧光细胞核.心肌细胞存活率和凋亡率,I/R组[(62.99±0.65)%,(19.13±0.87)%],IPC组[(78.67±0.37)%,(14.21±0.74)%],IPC+PKCI组[(71.09±0.52)%.(20.46±0.81)%],IPC+PKCI+CaRS组[(66.10±0.75)%,(24.89±1.43)%],IPC+CaRS组[(69.56±0.44)%,(21.64±0.77)%],IPC+CaRI组[(85.81±0.60)%,(13.12±0.69)%]明显低于或高于对照组[(100.00)%,(6.02±0.31)%],除IPC+CaRI组心肌细胞存活率外,其他各组与对照组比较差异有统计学意义(P＜0.05或＜0.01).Western Blot测定,胞浆白CaR蛋白在IPC+PKCI,IPC+CaRS,IPC+PKCI+CaRS组表达较IPC组高,IPC+CaRI组较IPC组低,caspase-12蛋白在I/R,IPC+CaRS,IPC+PKCI+CaRS 组,相对分子质量(胁)为60×103的活性片段表达均较高,calpain在I/R,IPC,IPC+PKCI,IPC+PKCI+CaRS,IPC+CaRS组表达均高于对照组和IPC+CaRI组,其中I/R组最高,对照组最低,IPC+CaRI组次之.结论 PKC与CaR受体之间的相互作用在IPC中可以通过减少肌浆网钙释放而起到对心肌的保护作用.Protective mechanism of the interaction between protein kinase C and calcium sensing receptor in jschemiapreconditioning DU Li-juan,WANG Yah-li,SUN Zhi-rui,ZHAO Ya-jun,LI Quan-feng,WANG Li-na,ZHANG Wei-hua Departmem of Pothophysioloty,Harbin Medical University,Harbin 150081,China     

Induction of anaphylatoxin C5a receptors in rat hepatocytes by lipopolysaccharide in vivo: mediation by interleukin-6 from Kupffer cells

BACKGROUND & AIMS: In normal rat liver, anaphylatoxin C5a induces glucose output from hepatocytes indirectly via prostanoids released from Kupffer cells. Correspondingly, it was found that hepatocytes, in contrast to Kupffer cells, did not express C5a receptors. Lipopolysaccharide (LPS) has been reported to enhance C5a receptor expression in murine livers. This might be the result of de novo expression in hepatocytes. METHODS: C5a receptor expression was investigated in hepatocytes after in vivo treatment of rats with LPS and in vitro stimulation of isolated cells with LPS and proinflammatory cytokines on messenger RNA (mRNA) and protein level, and functionally in isolated hepatocytes and perfused liver. RESULTS: In vivo treatment of rats with LPS induced C5a receptor mRNA and protein in hepatocytes with a maximum after 8-10 hours. At this time-point, C5a directly activated glycogen phosphorylase in isolated hepatocytes and enhanced glucose output in perfused livers without the involvement of prostanoids. LPS failed to induce C5a receptors in cultured hepatocytes in vitro, whereas interleukin (IL) 6 and IL-1beta, which are known to be released from Kupffer cells on stimulation with LPS, did so. In cocultures of hepatocytes with Kupffer cells, LPS induced C5a receptors in hepatocytes in an IL-6-dependent manner. CONCLUSIONS: Thus, IL-6 from Kupffer cells appears to be the main mediator of LPS-induced de novo expression of C5a receptors in hepatocytes.     

Contribution of C5-mediated mechanisms to host defence against Echinococcus granulosus hydatid infection

The aim of this work was to investigate the contribution of complement C5-mediated mechanisms, with an emphasis on inflammation, to host defences against Echinococcus granulosus hydatid disease. Thus, we compared the systemic and local inflammatory responses induced by the parasite, and the outcome of infection, between congenic C5-sufficient (B10.D2 n/SnJ) and C5-deficient (B10.D2 o/SnJ) mice challenged with protoscoleces. Indirect evidence of in-vivo complement activation during the establishment phase was obtained; infection induced serum amyloid P and eosinophil responses which were dependent on C5. Early recruitment of polymorphonuclear cells was not dependent on the presence of C5. The higher capacity of C5-sufficient mice to recruit eosinophils was also observed during the cystic phase of infection, and mice recruiting more eosinophils developed lower parasite masses. Analysis of the outcome of infection after 8 months showed that C5-sufficient mice were more resistant to infection than C5-deficient mice in terms of individuals with no cysts; this trend was not statistically significant. In addition, C5-deficient mice developed higher numbers of large (> 5 mm in diameter) cysts and higher cyst weights than C5-sufficient mice indicating that C5-mediated mechanisms are detrimental for parasite growth. Taken together, our results suggest that complement, through C5-mediated effectors, contributes to host defences by both restricting the establishment of infection and controlling the growth of established cysts. This contribution may, at least partially, be associated with the ability of C5a to promote eosinophil infiltration.     

Biological Effect of Anaphylatoxin C5a on the Generation of Anti‐inflammatory Substances in Leukocyte Adsorption

Anaphylatoxins, which are involved in both pro‐inflammatory processes and a variety of anti‐inflammatory effects, are produced during granulocyte and monocyte adsorptive apheresis. We noticed the anti‐inflammatory effects of C5a, the strongest anaphylatoxin, in granulocyte and monocyte adsorptive apheresis. The aim of this study was to investigate the effect of C5a on interleukin‐1 receptor antagonist (IL‐1ra) and hepatocyte growth factor (HGF) generation in granulocyte and monocyte adsorption. Peripheral blood containing nafamostat mesilate as an endogenous complement activation inhibitor was divided into four groups: (1) no recombinant C5a added, no contact with cellulose acetate (CA) beads (control group); (2) no C5a added, contact with CA beads; (3) C5a added, no contact with CA beads; and (4) C5a added, contact with CA beads. After incubation, IL‐1ra and HGF in plasma were measured. IL‐1ra was significantly higher in group 3, in which only C5a was added in the absence of CA beads, compared to groups 2 (P < 0.01) and 4 (P < 0.05). HGF was significantly higher only in group 4, in which C5a was added in the presence of CA beads (P < 0.05), but did not increase in the absence of CA beads. C5a can directly induce IL‐1ra generation without the granulocyte and monocyte adsorption stimuli to CA beads, but can synergistically induce HGF generation with the adsorption stimuli, indicating C5a has different effects on IL‐1ra and HGF generation.