C3-dependent effector functions of complement.

C3 is the central effector molecule of the complement system, mediating its multiple functions through different binding sites and their corresponding receptors. We will introduce the C3 forms (native C3, C3 [H2 O], and intracellular C3), the C3 fragments C3a, C3b, iC3b, and C3dg/C3d, and the C3 expression sites. To highlight the important role that C3 plays in human biological processes, we will give an overview of the diseases linked to C3 deficiency and to uncontrolled C3 activation. Next, we will present a structural description of C3 activation and of the C3 fragments generated by complement regulation. We will proceed by describing the C3a interaction with the anaphylatoxin receptor, followed by the interactions of opsonins (C3b, iC3b, and C3dg/C3d) with complement receptors, divided into two groups: receptors bearing complement regulatory functions and the effector receptors without complement regulatory activity. We outline the molecular architecture of the receptors, their binding sites on the C3 activation fragments, the cells expressing them, the diversity of their functions, and recent advances. With this review, we aim to give an up-to-date analysis of the processes triggered by C3 activation fragments on different cell types in health and disease contexts.

Microglial V-set and immunoglobulin domain-containing 4 protects against ischemic stroke in mice by suppressing TLR4-regulated inflammatory response.

Ischemic stroke is a leading cause of death among human in the world, and a critical cause for long-term disability. Accumulating studies have indicated that inflammatory response regulated by microglia contributes a lot to neuronal death, but the molecular mechanism still remains unclear. V-set and immunoglobulin domain-containing 4 (Vsig4), a complement receptor of the immunoglobulin superfamily (CRIg) that specifically expresses in resting tissue-resident macrophages, plays a critical role in regulating various inflammatory diseases via multiple signaling pathways. However, the effects of Vsig4 on ischemic stroke have not been investigated. In this study, we identified that Vsig4 expression was decreased after cerebral ischemic injury induced by middle cerebral artery occlusion (MCAO). Immunofluorescence staining showed that Vsig4 was co-localized with Iba1 in microglial cells from the infarct region of MCAO-operated mice. After over-expressing Vsig4 in mice, MCAO-induced infarction area and neurological deficits score were markedly attenuated. In addition, neurological dysfunction due to MCAO surgery was improved by Vsig4 over-expression. Microglial M1 polarization was detected in mice with MCAO surgery, which was markedly inhibited by Vsig4 over-expression, as evidenced by the markedly reduced expression of CD16, CD11b, inducible nitric oxide synthase (iNOS) and interleukin 6 (IL-6); however, the expression of M2-like phenotype hallmarks such as arginase 1 (Arg1), CD206, IL-10 and Ym-1 was significantly up-regulated. Mechanistically, the anti-inflammatory role of Vsig4 was mainly through the blockage of toll-like receptor 4/nuclear factor kappa B (TLR4/NF-κB) signaling via the in vivo and in vitro experiments. Also, we found that microglial TLR4 expression in the cerebral infarct area of MCAO mice was highly suppressed by Vsig4 over-expression. In vitro, the neuron-glial mixed culture by fluorescent staining showed that oxygen glucose deprivation (OGD) treatment led to significant cell death, while being attenuated by Vsig4 over-expression in primary microglial cells. Finally, we showed that Vsig4 could interact with TLR4 and repress its expression, subsequently alleviating ischemic stroke. Collectively, our findings demonstrated that microglial Vsig4 protected against post-stroke neuro-inflammation mainly through interacting with TLR4.

C5L2 and C5aR interaction in adipocytes and macrophages: insights into adipoimmunology.

Obesity is associated with inflammation characterized by increased infiltration of macrophages into adipose tissue. C5aR-like receptor 2 (C5L2) has been identified as a receptor for acylation-stimulating protein (ASP) and the inflammatory factor C5a, which also binds C5aR. The present study examines the effects of ligands ASP and C5a on interactions between the receptors C5L2 and C5aR in 3T3-L1 adipocytes and J774 macrophages. BRET experiments indicate that C5L2 and C5aR form homo- and heterodimers in transfected HEK 293 cells, which were stable in the presence of ligand. Cell surface receptor levels of C5L2 and C5aR increased during 3T3-L1 adipocyte differentiation; both receptors are also highly expressed in J774 macrophages. Using confocal microscopy to evaluate endogenous receptors in adipocytes following stimulation with ASP or C5a, C5L2 is internalized with increasing perinuclear colocalization with C5aR. There is little C5a-dependent colocalization in macrophages. While adipocyte-conditioned medium (ACM) increased C5L2-C5aR colocalization in macrophages, this was blocked by C5a. ASP stimulation increased Akt (Ser(473)) phosphorylation in both cell types; C5a induced slight Akt phosphorylation in adipocytes with less effect in macrophages. ASP, but not C5a, increased fatty acid uptake/esterification in adipocytes. C5L2-C5aR homodimerization versus heterodimerization may thus contribute to differential responses obtained following ASP vs C5a stimulation of adipocytes and macrophages, providing new insights into the complex interaction between these two cell types within adipose tissue. Studying the mechanisms involved in the differential responses of C5L2-C5aR activation based on cell type will further our understanding of inflammatory processes in obesity.

Evaluation of renal protective effects of inhibiting TGF-ß type I receptor in a cisplatin-induced nephrotoxicity model.

PURPOSE: The use of cisplatin, the first of the platinum-containing anti-cancer drugs, is limited by the development of a myriad of adverse reactions, including nephrotoxicity. We conducted this study therefore to find out whether SB-431542, potent and specific inhibitor of type I transforming growth factor-beta receptor (TGF-ßR1), could prevent or attenuate kidney damage in rats, and to elucidate its possible mechanism of action. METHODS: Fifty rats were treated with cisplatin (10 mg/kg) in the presence (1 and 3 mg/kg) or absence of SB-431542. Morphological changes were assessed in kidney sections stained with H/E. Oxidative stress was evaluated in kidney homogenates by measuring malondialdehyde (MDA) and superoxide dismutase (SOD). Kidney samples were used for measurements of TGF-ßR1, TGF-ß1 and sCD93 by ELISA. Kidney tissue apoptosis was assessed by measuring caspase-3 activity. RESULTS: The renal protective effect of SB-431542 was confirmed by the normal appearance of renal tissue and the relatively unaffected serum creatinine and urea levels. With SB-431542, there was significantly lower renal MDA and increased SOD compared with the cisplatin group. Furthermore, in the SB-431542 group, renal TGF-ßR1, TGF-ß1, sCD93 and caspase-3 levels were significantly lower. CONCLUSIONS: Inhibition of TGF-ßR1 provides protective effects against cisplatin-induced nephrotoxicity through several mechanisms, including attenuation of oxidative stress, inhibition of pro-inflammatory cytokines, blocking of renal fibrosis markers, and anti-apoptotic effects.

Excretion of complement proteins and its activation marker C5b-9 in IgA nephropathy in relation to renal function.

BACKGROUND: Glomerular damage in IgA nephropathy (IgAN) is mediated by complement activation via the alternative and lectin pathways. Therefore, we focused on molecules stabilizing and regulating the alternative pathway C3 convertase in urine which might be associated with IgAN pathogenesis. METHODS: Membrane attack complex (MAC), properdin (P), factor H (fH) and Complement receptor type 1 (CR1) were quantified in urine samples from 71 patients with IgAN and 72 healthy controls. Glomerular deposition of C5, fH and P was assessed using an immunofluorescence technique and correlated with histological severity of IgAN and clinical parameters. Fibrotic changes and glomerular sclerosis were evaluated in renal biopsy specimens. RESULTS: Immunofluorescence studies revealed glomerular depositions of C5, fH and P in patients with IgAN. Urinary MAC, fH and P levels in IgAN patients were significantly higher than those in healthy controls (p < 0.001), but CR1 was significantly lower than that in healthy controls (p < 0.001). Urinary MAC and fH levels were positively correlated with serum creatinine (sCr), urinary N-acetyl-ß-D-glucosaminidase (u-NAG), urinary ß2 microglobulin (u-Bm), urinary protein (p < 0.001), interstitial fibrosis (MAC: p < 0.01, fH: p < 0.05) and the percentage of global glomerular sclerosis (p < 0.01). Urinary P was positively correlated with u-NAG, u-Bm, and urinary protein (p < 0.01). CONCLUSIONS: Complement activation occurs in the urinary space in IgAN and the measurement of levels of MAC and fH in the urine could be a useful indicator of renal injury in patients with IgAN.

High levels of complement C3a receptor in the glomeruli in lupus nephritis.

BACKGROUND: Taking into consideration the key role of the complement system in renal diseases, we investigated molecular and cellular properties of the human complement C3a receptor (C3aR) in vitro and in situ, looking at its expression in several human renal pathological states. METHODS: Several antibodies were generated and used for immunohistochemistry and Western blot analyses to address C3aR expression and its regulation in vitro on cell lines of myeloid cells and nonmyeloid cell lineages. Furthermore, C3aR distribution was investigated in control nephrectomized kidneys and 116 biopsy specimens from patients with renal diseases, including lupus nephritis (lupus-N). RESULTS: C3aR is a highly N-glycosylated protein with an apparent molecular mass of 65 to 95 kd expressed by myeloid and endothelial cells. C3aR is particularly upregulated in response to interferon gamma treatment, but was unaffected by the other inflammatory cytokines, such as tumor necrosis factor alpha and transforming growth factor beta. In normal human kidney, C3aR staining was not observed. However, glomerular C3aR staining was detected in 42.9% of lupus-N specimens in association with immunoglobulin G immune-complex depositions. Staining intensity correlated with disease severity. C3aR was found in the endothelial area of 81.3% of samples classified as World Health Organization class IV with active lesions. Conversely, C3aR was not detected by means of immunohistochemistry in kidneys from patients with other renal diseases. CONCLUSION: Our data indicate that C3aR expression is tightly regulated and altered in certain disease conditions. C3aR may be used as a unique biomarker of diagnosis and disease activity in patients with lupus-N.

Cleavage fragments of the third complement component (C3) enhance stromal derived factor-1 (SDF-1)-mediated platelet production during reactive postbleeding thrombocytosis.

We hypothesized that the third complement component (C3) cleavage fragments (C3a and (des-Arg)C3a) are involved in stress/inflammation-related thrombocytosis, and investigated their potential role in reactive thrombocytosis induced by bleeding. We found that platelet counts are lower in C3-deficient mice in response to excessive bleeding as compared to normal littermates and that C3a and (des-Arg)C3a enhance stromal-derived factor-1 (SDF-1)-dependent megakaryocyte (Megs) migration, adhesion and platelet shedding. At the molecular level, C3a stimulates in Megs MAPKp42/44 phosphorylation, and enhances incorporation of CXCR4 into membrane lipid rafts increasing the responsiveness of Megs to SDF-1. We found that perturbation of lipid raft formation by statins decreases SDF-1/C3a-dependent platelet production in vitro and in an in vivo model statins ameliorated post-bleeding thrombocytosis. Thus, inhibition of lipid raft formation could find potential clinical application as a means of ameliorating some forms of thrombocytosis.

Expression of the anaphylatoxin receptors C3aR and C5aR is increased in fatal asthma.

BACKGROUND: The mechanisms leading to death from asthma are not completely understood. Recent studies suggest the involvement of the anaphylatoxins C3a and C5a, generated during complement activation, and their receptors C3aR and C5aR in the pathogenesis of asthma. OBJECTIVE: The aim of our study was to investigate the expression of C3aR and C5aR in fatal asthma. METHODS: We analyzed lung tissue from 14 subjects who died of asthma (fatal asthma; FA) and 14 subjects who died of nonpulmonary causes (controls) and bronchial biopsy specimens from 16 subjects with mild intermittent asthma (MIA). C3aR and C5aR expression was evaluated by immunohistochemistry, and a semiquantitative analysis of the intensity of staining was performed according to a visual analogue scale (score, 0-3). RESULTS: C3aR was expressed on airway epithelium, smooth muscle, submucosal, and parenchymal vessels. C5aR was expressed on myeloid cells infiltrating the submucosa and on airway epithelium. Statistical analysis demonstrated higher expression of C3aR on submucosal vessels in FA compared with controls and MIA (median [minimum-maximum], controls, 0.24 [0-1.48]; MIA, 0.0 [0-1.00]; FA, 1.56 [0.13-3]; P = .002). C3aR was also increased on parenchymal vessels in FA (controls, 0.56 [0-2.00]; FA, 1.81 [0.5-3]; P = .0004). C5aR expression on airway epithelium was increased in FA compared with controls and MIA (controls, 1.25 [0.25-3]; MIA, 1.00 [0-2.00]; FA, 3.00 [1.13-3.00]; P = .001). CONCLUSION: The results of our study suggest a role of complement in FA.

Membrane-bound complement regulatory proteins inhibit complement activation by an immunotherapeutic mAb in a syngeneic rat colorectal cancer model.

MAb-mediated immunotherapy offers a potential tool for destroying metastasizing colorectal tumor cells. Promising results have been obtained by using xenograft models. However, overexpression of membrane-bound complement regulatory proteins (mCRP) impedes complement-mediated destruction of tumor cells in vitro. mCRP operate in a species selective manner. Therefore a syngeneic animal model is needed to investigate the contribution of mCRP in mAb-mediated immunotherapy. Here we present a syngeneic rat colorectal carcinoma model, which fulfills the conditions necessary to investigate the effect of mCRP expression on mAb-mediated immunotherapy of metastases of solid tumors.CC531 rat colorectal cancer cells were injected subcapsularly into the liver of syngeneic WAG/Rij rats. Four mAb (MG1(IgG2a), MG2(IgG2a), MG3(IgG3) and MG4(2a)(IgG2a)) directed against CC531 cells, were tested for their complement activating abilities in vitro and tumor homing capacities in vivo. Only MG4(2a) was found to activate complement in vitro and home to the tumor cells in vivo. This mAb induced C3-deposition and C-mediated lysis of CC531 cells in vitro when the effects of the C-inhibitors Crry/p65 and CD59 were neutralized. This implies an important role for these mCRP in restricting the effector functions of tumor-associated mAb on these cells. Although C activation could be induced by MG4(2a) in situ on tumor tissue sections, no deposition of C3 could be found on the tumor cells positive for MG4(2a) in vivo. This suggests that complement activation in vivo was inhibited by mCRP. The results indicate the suitability of this syngeneic animal model for studying the effects of mAb immunotherapy. However, the effect of mCRP on tumor cells need to be overcome, e.g. by the use of mAb against tumor antigens and mCRP.

[Changes of ECR1 genomic density polymorphism, quantitative expression and the activity of ECR1 natural adhesion in patients with chronic hepatitis].

OBJECTIVE: To study the changes of genomic density polymorphism, quantitative expression and the adhesion activity of complement receptor type 1 (ECR1) on erythrocytes in patients with chronic hepatitis. METHODS: Polymerase chain reaction (PCR) and Hind restriction enzyme digestion, the quantitative assay of ECR1 and the activity of erythrocytes immune adhesion test were applied. RESULTS: The spot mutation rate (25.0%-30.3%) of ECR1 density gene in patients with chronic hepatitis was not significantly different from that of healthy individuals (28.0%). The amount of ECR1 in patients with chronic hepatitis, except for the diseases with normal liver function, was significantly lower than that of healthy individuals (t=9.87,P<0.000 1). The quantitative expression of ECR1 in decompensated cirrhosis was obviously lower than that of compensated cirrhosis (t=2.21,P<0.05). CONCLUSIONS: Defective expression of ECR1 in chronic hepatitis B may be acquired through central and/or peripheral mechanisms. It is very important to study the quantitative expression in the patients with chronic hepatitis.

Overexpression of complement receptors and related antigens on the surface of bone marrow mast cells in patients with systemic mastocytosis.

Depending on their stage of maturation and other factors, mast cell (MC) subsets differ from each other in terms of the expression of complement-associated antigens. This study analysed the expression of various complement-related cell surface antigens (CD11b/CR3, CD11c/CR4, CD35/CR1, CD55/DAF, CD59/MIRL, CD88/C5aR) on bone marrow mast cells (BMMC) in patients suffering from systemic mastocytosis (SM), other haematological diseases and non-haematological disorders (control groups). Expression of complement-associated cell surface antigens was analysed by flow cytometry. There were clear immunophenotypic differences between BMMC obtained from patients with SM and those from the control subjects: the percentage of patients expressing surface CD11c, CD35 and CD88 was significantly higher in patients with SM (76%, 100%, 54%) than in the control subjects (58%, 11%, 18%) (P < 0.05). In addition, the levels of CD11c, CD35 and CD88 expressed per MC (sites per cell) were significantly higher (P < 0.05) in SM than in the control group. Expression of the complement regulatory molecules CD55 and CD59 was detected in BMMC in all patients analysed. However, the levels of CD59 per BMMC were higher in patients with SM as compared with the control subjects, which could help to explain the formation of BMMC aggregates in the former group of individuals. Together, our results showed that BMMC in systemic mastocytosis overexpressed the cell surface membrane receptors involved in binding of complement components and complement-mediated cell activation. Whether this pathological expression of complement receptors is of pathophysiological significance remains to be determined.

Essential role for the C5a receptor in regulating the effector phase of synovial infiltration and joint destruction in experimental arthritis.

A characteristic feature of rheumatoid arthritis is the abundance of inflammatory cells in the diseased joint. Two major components of this infiltrate are neutrophils in the synovial fluid and macrophages in the synovial tissue. These cells produce cytokines including tumor necrosis factor alpha and other proinflammatory mediators that likely drive the disease through its effector phases. To investigate what mechanisms underlie the recruitment of these cells into the synovial fluid and tissue, we performed expression analyses of chemoattractant receptors in a related family that includes the anaphylatoxin receptors and the formyl-MetLeuPhe receptor. We then examined the effect of targeted disruption of two abundantly expressed chemoattractant receptors, the receptors for C3a and C5a, on arthritogenesis in a mouse model of disease. We report that genetic ablation of C5a receptor expression completely protects mice from arthritis.

6-Sulfo LacNAc, a novel carbohydrate modification of PSGL-1, defines an inflammatory type of human dendritic cells.

The monoclonal antibody M-DC8 defines a major subset of human blood dendritic cells (DCs). Here we identify the M-DC8 structure as 6-sulfo LacNAc, a novel carbohydrate modification of the P selectin glycoprotein ligand 1 (PSGL-1). In contrast to previously described blood DCs, M-DC8+ DCs lack the cutaneous lymphocyte antigen (CLA) on PSGL-1 and fail to bind P and E selectin. Yet they express anaphylatoxin receptors (C5aR and C3aR) and the Fcgamma receptor III (CD16), which recruit cells to inflammatory sites. While sharing with DC1 the expression of myeloid markers and a potent capacity to prime T cells in vitro, M-DC8+ DCs produce far more TNF-alpha in response to the bacterial endotoxin lipopolysaccharide (LPS). Thus, 6-sulfo LacNAc-expressing DCs appear as a novel proinflammatory DC subset.

Phagocytosis by emigrated, intra-abdominal neutrophils is depressed during human secondary peritonitis.

The phagocytic function of neutrophils is a crucial element in host defense against invading microorganisms. Patients with diffuse peritonitis depend on adequate reactivity of neutrophils, in particular locally in the peritoneal cavity as well as in the circulation. This study examined phagocytosis as well as numerical expression of Fcgamma I-III (CD16, CD32, CD64) and complement receptors (CD18, CD35) of emigrated, intra-abdominal and circulating neutrophils during human secondary peritonitis using fluorescence-activated cell analysis. Optimally opsonized E. coli bacteria were used independently of the well-known low level of opsonic molecules during peritonitis. Compared with controls (abdominal surgery without peritonitis), the percentage of emigrated neutrophils which engulfed E. coli bacteria was significantly depressed until 48 h after diagnosis of, and surgery for, peritonitis. When patients with complicated peritonitis (septic shock, multiple organ failure) were compared with patients without complications, phagocytosis was even more depressed in patients with complications. Numerical expression of CD64 (Fcgamma RI) and CD35 (CR1) increased significantly on emigrated polymorphonuclear leukocytes (PMNs) during peritonitis when compared to controls. There was no difference in CD18 and CD32 (Fcgamma RII) expression between the two groups. Numerical expression of CD16 (Fcgamma RIII) on emigrated PMNs decreased significantly in peritonitis. This was more pronounced in patients with complicated peritonitis. We conclude that there is a long-lasting depression of phagocytosis by emigrated PMNs during peritonitis, independent of the opsonic activity. Our data suggest that decreased phagocytosis might be correlated to the profound drop in CD16 on these cells.

Mucosal type mast cells express complement receptor type 2 (CD21).

Fragments of complement component C3 generated upon activation of the cascade play an important role in the induction and regulation of immune responses. Receptors interacting with various fragments of this versatile complement protein are expressed on a wide variety of cell types, including lymphocytes, macrophages, dendritic cells, follicular dendritic cells, granulocytes, erythrocytes and consequently, C3-products may influence several biological functions at different sites of the body, where complement activation occurs. Regarding the expression of various C3-receptors on mast cells, mainly rodent serosal type mastocytes have been investigated so far. It has been known for a long time that C3a triggers the release of mediators of immediate type hypersensitivity via binding to serosal-type cells. Complement receptor type 1 (CR1/CD35) and type 2 (CR2/CD21) interacting with the larger activation products, such as C3b and C3d, have so far been shown on serosal type mast cells only. In this study, the expression of CR1/2 on mucosal type mast cells is demonstrated. Using mouse CR1/2 specific single chain antibodies and the natural ligand C3d in cytofluorimetric measurements, we show that the rat mucosal mast cell line RBL-2H3 and mouse bone marrow-derived mast cells (BMMC) express CD21. RT-PCR experiments carried out with mouse CR1 and CR2 specific primers show CD21, but not CD35 specific products in BMMC. It is also demonstrated that, in contrast to serosal type mast cells, mucosal mastocytes do not express CD19. In an attempt to reveal the possible function of CR2 on mucosal type mast cells, the effect of receptor-clustering was tested regarding degranulation, Ca-response and IL-6 production, but no CR2-mediated change was detected in any of these processes.

C1qR(P), a myeloid cell receptor in blood, is predominantly expressed on endothelial cells in human tissue.

C1qR(P) is a type I cell surface glycoprotein that has been shown to enhance ingestion of suboptimally opsonized targets by phagocytes in vitro. In this study, we developed and characterized polyclonal antibodies to study the tissue distribution of this receptor targeted to either the N- or C-terminal portion of the molecule. C1qR(P) was detected in vascular endothelial cells and in a subset of pyramidal neurons in the brain, as well as neutrophils, but it was absent in most tissue macrophages. Analysis of in vitro differentiation of blood monocytes to dendritic cells demonstrated a down-regulation of the receptor as monocytes differentiate to dendritic cells, providing a possible explanation for the lack of reactivity of these cells in tissue. The predominant presence of C1qR(P) in endothelial cells, while compatible with a phagocytic role in host defense and/or clearance of cellular material, suggests other possible novel roles for this receptor.

Assays for membrane complement receptors.

Leukocytes express several types of receptors for activated products of the complement (C) system. This unit describes assays for a subset of these receptors: those specific for fixed fragments of C3 (CR1, CR2, CR3, and CR4). Although the assays have been designed for tests of human C receptors, they can often be carried out with rodent (e.g., mouse, rat, or guinea pig) cells by substitution of rodent-specific reagents. Two basic protocols are presented for rosette assay of CR1, CR2, CR3, and CR4 on adherent and nonadherent cells. Isolated leukocytes may be tested for rosette formation in suspension or adherent to a surface. Nonadherent cells such as lymphocytes, NK cells, or cells from an undifferentiated leukocyte cell line must be tested in suspension. The unit also contains three support protocols for formation and quantitation of sheep erythrocyte-C3 complexes for use in the two basic protocols.

Characterization of endogenous Chinese hamster ovary cell surface molecules that mediate T cell costimulation.

Chinese hamster ovary (CHO) cells are commonly used in the generation of transfectants for use in in vitro costimulation assays. However, we have noted that nontransfected CHO cells can themselves provide a low-level B7/CD28 independent costimulatory signal for CD3-mediated murine T cell activation and IL-2 production. This study set out to identify those molecules that contribute to this CHO-dependent costimulatory activity. We describe a CHO subline capable of delivering potent CD28-independent costimulation to murine T cells and the generation of monoclonal antibodies against these CHO cells that inhibited this costimulatory activity. These blocking antibodies do not affect CHO cell-independent costimulation or bind mouse cells, suggesting an effect mediated by their target molecules on the costimulatory competent CHO cells. Immunoprecipitation and expression cloning revealed that these antibodies bound the hamster homologues of Crry (CD21/35), CD44, CD54 (ICAM-1), CD63, CD87, CD147, and an 80- to 90-kDa protein which could not be cloned. Expression of these hamster genes on COS cells demonstrated that hamster CD54 was able to costimulate both CD3-mediated IL-2 secretion and T cell proliferation by naive murine T cells independent of the other molecules identified.

Characterization and molecular cloning of rat C1qRp, a receptor on NK cells.

Here we report the generation of monoclonal antibodies (mAb) LOV3 and LOV8 to a 110-130-kDa membrane glycoprotein expressed by rat NK cells. This NK surface molecule was identified by eucaryotic expression cloning as the structural orthologue of the phagocytosis-stimulating receptor for complement factor C1q and mannose-binding lectin on human macrophages, C1qRp. Rat C1qRp is a monomeric type I integral membrane protein consisting of 643 amino acids with an N-terminal lectin-like domain, five epidermal growth factor-like domains, a transmembrane domain and a 45-residue cytoplasmic domain. It is encoded by a single gene on rat chromosome 3q41-q42 and is 67% and 87.5% identical at the amino acid level to human and mouse C1qRp, respectively. Rat C1qRp is expressed by resting and by activated NK cells, on subpopulations of NKR-P1(+) T cells (NK/T cells), dendritic cells, macrophages and granulocytes, but not by B cells or NKR-P1(-) T cells. Expression of this innate immune receptor is therefore not restricted to hematopoietic cells of the myeloid lineage, but is also expressed on subsets of cells of lymphoid origin. The mAb did not affect the cytotoxic function of NK cells, and C1qRp on NK cells may have functions not related to NK killing.

Localization of P32 protein (gC1q-R) in mitochondria and at specific extramitochondrial locations in normal tissues.

P32 protein, also known as the gC1q receptor for complement component C1q, is a binding protein for nuclear pre-mRNA splicing factor SF2/ASF and numerous other nuclear and cell surface proteins, yet is targeted to the mitochondrial matrix compartment where these proteins are not present. In the present study, we use immunogold electron microscopy to evaluate the subcellular distribution of P32 protein (gC1q-R) in cultured cell lines and in rat tissues embedded in the acrylic resin LR Gold. Immunogold labeling of Raji lymphoma, CHO, human fibroblasts, HeLa and B-SC-1 cells shows reactivity primarily within mitochondria. Highly specific labeling of mitochondria is also obtained in rat tissues, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen, testes and thyroid. However, strong P32 (gClq-R) reactivity is also present in (i) zymogen granules, condensing vacuoles, endoplasmic reticulum, and on the cell surface of pancreatic acinar cells, (ii) on the cell surface of microvascular endothelial cells in pancreas and kidney, (iii) on the cell surface and in nuclei of splenic lymphocytes, and (iv) in the acrosome of developing spermatids in testes. Western immunoblots show that the polyclonal antibody to P32 (gC1q-R) used in this study reacts specifically with a 32-kDa protein in both purified pancreatic zymogen granules and in mitochondria, and no other proteins are reactive. These results provide evidence that P32 (gC1q-R) is a mitochondrial protein that also localizes outside mitochondria in certain cells and tissues under normal physiological conditions.