Complement peptide C3a receptor 1 promotes optic nerve degeneration in DBA/2J mice.

BACKGROUND: The risk of glaucoma increases significantly with age and exposure to elevated intraocular pressure, two factors linked with neuroinflammation. The complement cascade is a complex immune process with many bioactive end-products, including mediators of inflammation. Complement cascade activation has been shown in glaucoma patients and models of glaucoma. However, the function of complement-mediated inflammation in glaucoma is largely untested. Here, the complement peptide C3a receptor 1 was genetically disrupted in DBA/2J mice, an ocular hypertensive model of glaucoma, to test its contribution to neurodegeneration. METHODS: A null allele of C3ar1 was backcrossed into DBA/2J mice. Development of iris disease, ocular hypertension, optic nerve degeneration, retinal ganglion cell activity, loss of RGCs, and myeloid cell infiltration in C3ar1-deficient and sufficient DBA/2J mice were compared across multiple ages. RNA sequencing was performed on microglia from primary culture to determine global effects of C3ar1 on microglia gene expression. RESULTS: Deficiency in C3ar1 lowered the risk of degeneration in ocular hypertensive mice without affecting intraocular pressure elevation at 10.5 months of age. Differences were found in the percentage of mice affected, but not in individual characteristics of disease progression. The protective effect of C3ar1 deficiency was then overcome by additional aging and ocular hypertensive injury. Microglia and other myeloid-derived cells were the primary cells identified that express C3ar1. In the absence of C3ar1, microglial expression of genes associated with neuroinflammation and other immune functions were differentially expressed compared to WT. A network analysis of these data suggested that the IL10 signaling pathway is a major interaction partner of C3AR1 signaling in microglia. CONCLUSIONS: C3AR1 was identified as a damaging neuroinflammatory factor. These data help suggest complement activation causes glaucomatous neurodegeneration through multiple mechanisms, including inflammation. Microglia and infiltrating myeloid cells expressed high levels of C3ar1 and are the primary candidates to mediate its effects. C3AR1 appeared to be a major regulator of microglia reactivity and neuroinflammatory function due to its interaction with IL10 signaling and other immune related pathways. Targeting myeloid-derived cells and C3AR1 signaling with therapies is expected to add to or improve neuroprotective therapeutic strategies.

The Significance of VSIG4 Expression in Ovarian Cancer.

OBJECTIVES: The protein V-set and Ig domain-containing 4 (VSIG4), a novel B7 family-related macrophage protein with the capacity to inhibit T-cell activation, has a potential role in cancer. Here we suggest its possibility as a therapeutic target and prognostic biomarker of ovarian cancer. METHODS: Between January 2011 and June 2015, tumor tissues and peripheral blood samples were obtained during surgery from 10 patients with benign ovarian tumors and 22 patients with ovarian cancers. Messenger RNA and protein expression levels of VSIG4 in benign tumor and cancer tissues were examined by the reverse transcription polymerase chain reaction and Western blot, respectively. Soluble VSIG4 concentrations were measured by an enzyme-linked immunosorbent assay. The correlation between VSIG4 expression and the prognosis of ovarian cancer was analyzed according to the patients' clinicopathologic characteristics. RESULTS: VSIG4 messenger RNA and protein expression levels in ovarian cancer tissues were higher than those in benign ovarian tumors (P = 0.0013 and 0.0001, respectively). Soluble VSIG4 concentrations were increased in patients with ovarian cancer compared with that in patients with benign ovarian tumors (P = 0.0452). Moreover, soluble VSIG4 levels were significantly increased in advanced-stage and recurrent ovarian cancer (P = 0.0244 and 0.0288, respectively). High VSIG4 expression of cancer tissue and low VSIG4 expression of plasma (soluble VSIG4) were associated with a longer disease-free interval (P = 0.0246 and 0.0398, respectively). CONCLUSIONS: VSIG4 is overexpressed in ovarian cancers compared with that in benign tumors. This finding supports VSIG4 being used as a potential therapeutic target for ovarian cancer. Furthermore, soluble VSIG4 levels are associated with the progression and recurrence of ovarian cancer, indicating that soluble VSIG4 may be used as a potential biomarker for predicting tumor prognosis.

LL-37-induced host cell cytotoxicity depends on cellular expression of the globular C1q receptor (p33).

The human host-defence peptide (HDP) LL-37 not only displays anti-microbial activity but also immune-modulating properties that trigger intracellular signalling events in host cells. Since the cytolytic activity of high LL-37 concentrations affects cell viability, the function of LL-37 requires tight regulation. Eukaryotic cells therefore benefit from protective measures to prevent harmful effects of LL-37. p33, also known as globular C1q receptor (gC1qR), is reported to act as an LL-37 antagonist by binding the peptide, thereby reducing its cytotoxic activity. In the present report, we show that high levels of endogenous p33 correlate with an increased viability in human cells treated with LL-37. Sub-cellular localization analysis showed p33 distribution at the mitochondria, the plasma membrane and in the cytosol. Strikingly, cytosolic overexpression of p33 significantly antagonized detrimental effects of LL-37 on cell fitness, whereas the reverse effect was observed by siRNA-induced down-regulation of p33. However, modulation of p33 expression had no effect on LL-37-induced plasma membrane pore forming capacity pointing to an intracellular mechanism. A scavenging function of intracellular p33 is further supported by co-immunoprecipitation experiments, showing a direct interaction between intracellular p33 and LL-37. Thus, our findings support an important role of intracellular p33 in maintaining cell viability by counteracting LL-37-induced cytotoxicity.

Role of complement activation in obliterative bronchiolitis post-lung transplantation.

Obliterative bronchiolitis (OB) post-lung transplantation involves IL-17-regulated autoimmunity to type V collagen and alloimmunity, which could be enhanced by complement activation. However, the specific role of complement activation in lung allograft pathology, IL-17 production, and OB is unknown. The current study examines the role of complement activation in OB. Complement-regulatory protein (CRP) (CD55, CD46, complement receptor 1-related protein y/CD46) expression was downregulated in human and murine OB; and C3a, a marker of complement activation, was upregulated locally. IL-17 differentially suppressed complement receptor 1-related protein y expression in airway epithelial cells in vitro. Neutralizing IL-17 recovered CRP expression in murine lung allografts and decreased local C3a production. Exogenous C3a enhanced IL-17 production from alloantigen- or autoantigen (type V collagen)-reactive lymphocytes. Systemically neutralizing C5 abrogated the development of OB, reduced acute rejection severity, lowered systemic and local levels of C3a and C5a, recovered CRP expression, and diminished systemic IL-17 and IL-6 levels. These data indicated that OB induction is in part complement dependent due to IL-17-mediated downregulation of CRPs on airway epithelium. C3a and IL-17 are part of a feed-forward loop that may enhance CRP downregulation, suggesting that complement blockade could be a therapeutic strategy for OB.

VSIG4 expression on macrophages facilitates lung cancer development.

Tumor-associated macrophages are a prominent component of lung cancer stroma and contribute to tumor progression. The protein V-set and Ig domain-containing 4 (VSIG4), a novel B7 family-related macrophage protein that has the capacity to inhibit T-cell activation, has a potential role in the development of lung cancer. In this study, 10 human non-small-cell lung cancer specimens were collected and immunohistochemically analyzed for VSIG4 expression. Results showed massive VSIG4(+) cell infiltration throughout the samples. Immunofluorescent double staining showed that VSIG4 was present on CD68(+) macrophages, but absent from CD3(+) T cells, CD31(+) endothelial cells, and CK-18(+) epithelial cells. Moreover, VSIG4 was coexpressed on B7-H1(+) and B7-H3(+) cells in these tumor specimens. Transfection of the VSIG4 gene into 293FT cells demonstrated that the VSIG4 signal could inhibit cocultured CD4(+) and CD8(+) T-cell proliferation and cytokine (IL-2 and IFN-γ) production in vitro. Interestingly, in a murine tumor model induced by Lewis lung carcinoma cell line, we found that tumors grown in VSIG4-deficient (VSIG4(-/-)) mice were significantly smaller than those found in wild-type littermates. All of these results demonstrate that macrophage-associated VSIG4 is an activator that facilitates lung carcinoma development. Specific targeting of VSIG4 may prove to be a novel, efficacious strategy for the treatment of this carcinoma.

Mast cell anaphylatoxin receptor expression can enhance IgE-dependent skin inflammation in mice.

BACKGROUND: Mast cells express receptors for complement anaphylatoxins C3a and C5a (ie, C3a receptor [C3aR] and C5a receptor [C5aR]), and C3a and C5a are generated during various IgE-dependent immediate hypersensitivity reactions in vivo. However, it is not clear to what extent mast cell expression of C3aR or C5aR influences C3a- or C5a-induced cutaneous responses or IgE-dependent mast cell activation and passive cutaneous anaphylaxis (PCA) in vivo. OBJECTIVE: We sought to assess whether mouse skin mast cell expression of C3aR or C5aR influences (1) the cells' responsiveness to intradermal injections of C3a or C5a or (2) the extent of IgE-dependent mast cell degranulation and PCA in vivo. METHODS: We measured the magnitude of cutaneous responses to intradermal injections of C3a or C5a and the extent of IgE-dependent mast cell degranulation and PCA responses in mice containing mast cells that did or did not express C3aR or C5aR. RESULTS: The majority of the skin swelling induced by means of intradermal injection of C3a or C5a required that mast cells at the site expressed C3aR or C5aR, respectively, and the extent of IgE-dependent degranulation of skin mast cells and IgE-dependent PCA was significantly reduced when mast cells lacked either C3aR or C5aR. IgE-dependent PCA responses associated with local increases in C3a levels occurred in antibody-deficient mice but not in mice deficient in FcɛRIγ. CONCLUSION: Expression of C3aR and C5aR by skin mast cells contributes importantly to the ability of C3a and C5a to induce skin swelling and can enhance mast cell degranulation and inflammation during IgE-dependent PCA in vivo.

Complement activation fragment C5a receptors, CD88 and C5L2, are associated with neurofibrillary pathology.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative dementia characterized by the decline of cognition and the presence of neuropathological changes including neuronal loss, neurofibrillary pathology and extracellular senile plaques. A neuroinflammatory process is also triggered and complement activation has been hypothesized to have a relevant role in this local inflammatory response. C5a, a proinflammatory anaphylatoxin generated after complement activation, exerts its chemotactic and inflammatory functions through the CD88 receptor while the more recently discovered C5L2 receptor has been postulated to have an anti-inflammatory role. Previously, we reported that a CD88 specific antagonist (PMX205) decreased the pathology and improved cognition in transgenic models of AD suggesting that C5a/C5aR interaction has an important role in the progression of the disease. METHODS: The present study characterizes the expression of the two receptors for C5a in human brain with confirmed post mortem diagnosis of vascular dementia (VD) or AD as well as age matched controls by immunohistochemistry and Western blot analysis using several antibodies against different epitopes of the human receptors. RESULTS: The CD88 and C5L2 antibodies revealed increased expression of both receptors in AD samples as compared to age-matched controls or VD brain tissue by Western blot and immunohistochemistry, using multiple antibodies and distinct cohorts of brain tissue. Immunostaining showed that both the C5L2 and CD88 antibodies similarly labeled abundant neurofibrillary tangles, neuropil threads and dystrophic neurites associated with plaques in the hippocampus and frontal cortex of AD cases. In contrast, little or no neuronal staining, tangles or dystrophic neurites associated with plaques were observed in control or VD brains. CD88 and C5L2 receptors are associated with both early (AT8) and mature (PHF1) neurofibrillary tangles and can be found either independently or colocalized with each other. CONCLUSIONS: The observed association of CD88 and C5L2 with neurofibrillary pathology suggests a common altered pathway of degradation.

Anaphylatoxin receptors and complement regulatory proteins in human articular and non-articular chondrocytes: interrelation with cytokines.

Tissue trauma induces an inflammatory response associated with a cytokine release that may engage complement pathways. Cytokine-mediated complement expression may contribute to cartilage degradation. Hence, we analysed the complement expression profile in primary articular and non-articular chondrocytes and its interrelation with cytokines. The expression of the anaphylatoxin receptors (C3aR and C5aR) and the complement regulatory proteins (CPRs) CD35, CD46, CD55 and CD59 was studied in cultured articular, auricular and nasoseptal chondrocytes using RTD-PCR and immunofluorescence labelling. The complement profile of peripheral blood mononuclear cells (PBMCs) was opposed to the expression in articular chondrocytes. The time-dependent regulation (6 and 24 h) of these complement factors was assessed in articular chondrocytes in response to the cytokines TNFα, IL-10 or TNFα combined with IL-10 (each 10 ng/mL). C3aR, C5aR, CD46, CD55 and CD59 but almost no CD35 mRNA was expressed in any of chondrocyte types studied. The anaphylatoxin receptor expression was lower and that of the CRPs was higher in chondrocytes when compared with PBMCs. The majority of the studied complement factors were expressed at a significantly lower level in non-articular chondrocytes compared with the articular chondrocytes. TNFα significantly increased the C3aR expression in chondrocytes after 6 and 24 h. TNFα + IL-10 significantly downregulated C5aR and IL-10 significantly inhibited the CD46 and CD55 gene expression after 24 h. C5aR and CD55 could be localised in cartilage in situ. Anaphylatoxin receptors and CRPs are regulated differentially by TNFα and IL-10. Whether cytokine-induced complement activation occurs in response to cartilage trauma has to be further identified.

The complement inhibitors Crry and factor H are critical for preventing autologous complement activation on renal tubular epithelial cells.

Congenital and acquired deficiencies of complement regulatory proteins are associated with pathologic complement activation in several renal diseases. To elucidate the mechanisms by which renal tubular epithelial cells (TECs) control the complement system, we examined the expression of complement regulatory proteins by the cells. We found that Crry is the only membrane-bound complement regulator expressed by murine TECs, and its expression is concentrated on the basolateral surface. Consistent with the polarized localization of Crry, less complement activation was observed when the basolateral surface of TECs was exposed to serum than when the apical surface was exposed. Furthermore, greater complement activation occurred when the basolateral surface of TECs from Crry(-/-)fB(-/-) mice was exposed to normal serum compared with TECs from wild-type mice. Complement activation on the apical and basolateral surfaces was also greater when factor H, an alternative pathway regulatory protein found in serum, was blocked from interacting with the cells. Finally, we injected Crry(-/-)fB(-/-) and Crry(+/+)fB(-/-) mice with purified factor B (an essential protein of the alternative pathway). Spontaneous complement activation was seen on the tubules of Crry(-/-)fB(-/-) mice after injection with factor B, and the mice developed acute tubular injury. These studies indicate that factor H and Crry regulate complement activation on the basolateral surface of TECs and that factor H regulates complement activation on the apical surface. However, congenital deficiency of Crry or reduced expression of the protein on the basolateral surface of injured cells permits spontaneous complement activation and tubular injury.

CD93/AA4.1: a novel regulator of inflammation in murine focal cerebral ischemia.

The stem-cell marker CD93 (AA4.1/C1qRp) has been described as a potential complement C1q-receptor. Its exact molecular function, however, remains unknown. By using global expression profiling we showed that CD93-mRNA is highly induced after transient focal cerebral ischemia. CD93 protein is upregulated in endothelial cells, but also in selected macrophages and microglia. To elucidate the potential functional role of CD93 in postischemic brain damage, we used mice with a targeted deletion of the CD93 gene. After 30 min of occlusion of the middle cerebral artery and 3 d of reperfusion these mice displayed increased leukocyte infiltration into the brain, increased edema, and significantly larger infarct volumes (60.8 +/- 52.2 versus 23.9 +/- 16.6 mm(3)) when compared with wild-type (WT) mice. When the MCA was occluded for 60 min, after 2 d of reperfusion the CD93 knockout mice still showed more leukocytes in the brain, but the infarct volumes were not different from those seen in WT animals. To further explore CD93-dependent signaling pathways, we determined global transcription profiles and compared CD93-deficient and WT mice at various time points after induction of focal cerebral ischemia. We found a highly significant upregulation of the chemokine CCL21/Exodus-2 in untreated and treated CD93-deficient mice at all time points. Induction of CCL21 mRNA and protein was confirmed by PCR and immunohistochemistry. CCL21, which was formerly shown to be released by damaged neurons and to activate microglia, contributes to neurodegeneration. Thus, we speculate that CD93-neuroprotection is mediated via suppression of the neuroinflammatory response through downregulation of CCL21.

Expression of AA4.1 marks lymphohematopoietic progenitors in early mouse development.

The hematopoietic system of mice is established during the early to midgestational stage of development. However, the earliest lymphohematopoietic progenitors that appear during mouse development have been less well characterized compared with the hematopoietic stem cell compartment of fetal liver and bone marrow. We isolated the earliest lymphohematopoietic progenitors by using embryonic stem (ES) cell culture in vitro. Cells with the c-Kit(+)Lin(-) cell surface phenotype were present abundantly in ES cells cocultured with stromal cell lines. We further separated the cells into two distinct cell subsets based on AA4.1 expression. Although AA4.1(+) and AA4.1(-) cells had equivalent potency to generate myeloid cell lineages, the lymphoid potential in ES-cell-derived cells was largely restricted to the cells expressing AA4.1. The same cell type was present abundantly in the early yolk sac and in fewer numbers (approximately 5% of that in the yolk sac) in the caudal half of the developing embryos. These data suggest that AA4.1 is a cell surface marker that can identify the earliest lymphohematopoietic progenitors in mouse development.

Complement-dependent T-cell lymphopenia caused by thymocyte deletion of the membrane complement regulator Crry.

Although complement lysis is frequently used for the purification of lymphocyte subpopulations in vitro, how lymphocytes escape complement attack in vivo has not been clearly delineated. Here, we show that conditional gene targeting of a murine membrane complement regulator Crry on thymocytes led to complement-dependent peripheral T-cell lymphopenia. Notably, despite evidence of hypersensitivity to complement attack, Crry-deficient T cells escaped complement injury and developed normally in the thymus, because of low intrathymic complement activity. Crry-deficient T cells were eliminated in the periphery by a C3- and macrophage-mediated but C5-independent mechanism. Thus, Crry is essential for mature T-cell survival in the periphery but not for lymphogenesis in the thymus. The observation that the thymus is a complement-privileged site may have implications for complement-based antitumor therapies.

Peri-implantitis fibroblasts respond to host immune factor C1q.

BACKGROUND AND OBJECTIVE: Current therapies for peri-implantitis apply the same clinical protocols as those used for the treatment of periodontitis; however, outcomes remain unpredictable. We hypothesized that resident fibroblasts of the peri-implantitis stroma and periodontitis stroma differ in their phenotype and response to host immune factors. Fibroblasts are highly heterogeneous and comprise discrete subtypes with the potential of modulating inflammatory activities. The aim of the present study was to characterize the expression of receptors for complement C1q of innate immunity on human peri-implantitis fibroblasts and investigate effects of C1q on the proinflammatory properties of the cells. MATERIAL AND METHODS: Fibroblasts were cultured from gingival tissues exhibiting peri-implantitis and periodontitis, and from healthy gingivae as a control. Expression of C1q receptors for the collagen (cC1qR) and globular domains (gC1qR) of the protein was determined by flow cytofluorometric analysis (FITC) of specific antibodies bound to the surface of the cells. Secretion of C1q-inducible proinflammatory mediators was quantified after 24 h incubation using array-based ELISAs. RESULTS: The percentage of fibroblasts FITC-positive for cC1qR was 67, 75 and 12% in peri-implantitis, healthy and periodontitis cultures, respectively, whereas the percentage of gC1qR FITC-positive fibroblasts was 5, 3 and 59%, respectively. The C1q interactions with peri-implantitis and healthy fibroblasts increased secretion of the chemokines interleukin-6 and interleukin-8 twofold, and monocyte chemoattractant protein-1 fourfold over baseline values, whereas periodontitis fibroblasts were unresponsive. Complement C1q increased levels of vascular endothelial growth factor sevenfold and transforming growth factor-ß1 12-fold over baseline values in peri-implantitis cultures, only. CONCLUSIONS: Peri-implantitis fibroblasts differ from periodontitis fibroblasts in phenotypic expression of cC1qR and function, and from healthy fibroblasts in proinflammatory, angiogenic and fibrogenic function. Peri-implantitis fibroblasts may represent a novel subtype.

Scrapie pathogenesis: the role of complement C1q in scrapie agent uptake by conventional dendritic cells.

Mice lacking complement components show delayed development of prion disease following peripheral inoculation. The delay could relate to reduced scrapie prion protein (PrP(Sc)) accumulation on follicular dendritic cells (DCs). However conventional DCs (cDCs) play a crucial role in the early pathogenesis of prion diseases and complement deficiency could result in decreased PrP(Sc) uptake by cDCs in the periphery. To explore this possibility, we cultured murine splenic or gut-associated lymph node cDCs with scrapie-infected whole brain homogenate in the presence or absence of complement. Uptake decreased significantly if the serum in the cultures was heat-inactivated. Because heat inactivation primarily denatures C1q, we used serum from C1q(-/-) mice and showed that PrP(Sc) uptake was markedly decreased. PrP(Sc) internalization was saturable and temperature-dependent, suggesting receptor-mediated uptake. Furthermore, uptake characteristics differed from fluid-phase endocytosis. Immunofluorescence showed colocalization of C1q and PrP(Sc), suggesting interaction between these molecules. We evaluated the expression of several complement receptors on cDCs and confirmed that cDCs that take up PrP(Sc) express one of the C1q receptors, calreticulin. Our results show that C1q participates in PrP(Sc) uptake by cDCs, revealing a critical role for cDCs in initial prion capture, an event that takes place before the PrP(Sc) accumulation within the follicular DC network.

C3a and C5a are chemotactic factors for human mesenchymal stem cells, which cause prolonged ERK1/2 phosphorylation.

Mesenchymal stem cells (MSCs) have a great potential for tissue repair, especially if they can be delivered efficiently to sites of tissue injury. Since complement activation occurs whenever there is tissue damage, the effects of the complement activation products C3a and C5a on MSCs were examined. Both C3a and C5a were chemoattractants for human bone marrow-derived MSCs, which expressed both the C3a receptor (C3aR) and the C5a receptor (C5aR; CD88) on the cell surface. Specific C3aR and C5aR inhibitors blocked the chemotactic response, as did pertussis toxin, indicating that the response was mediated by the known anaphylatoxin receptors in a G(i) activation-dependent fashion. While C5a causes strong and prolonged activation of various signaling pathways in many different cell types, the response observed with C3a is generally transient and weak. However, we show herein that in MSCs both C3a and C5a caused prolonged and robust ERK1/2 and Akt phosphorylation. Phospho-ERK1/2 was translocated to the nucleus in both C3a and C5a-stimulated MSCs, which was associated with subsequent phosphorylation of the transcription factor Elk, which could not be detected in other cell types stimulated with C3a. More surprisingly, the C3aR itself was translocated to the nucleus in C3a-stimulated MSCs, especially at low cell densities. Since nuclear activation/translocation of G protein-coupled receptors has been shown to induce long-term effects, this novel observation implies that C3a exerts far-reaching consequences on MSC biology. These results suggest that the anaphylatoxins C3a and C5a present in injured tissues contribute to the recruitment of MSCs and regulation of their behavior.

Role of the receptor for the globular domain of C1q protein in the pathogenesis of hepatitis C virus-related cryoglobulin vascular damage.

Mixed cryoglobulinemia (MC) is a lymphoproliferative disorder observed in approximately 10 to 15% of hepatitis C virus (HCV)-infected patients. Circulating, nonenveloped HCV core protein, which has been detected in cryoprecipitable immune complexes, interacts with immunocytes through the receptor for the globular domain of C1q protein (gC1q-R). In this study, we have evaluated circulating gC1q-R levels in chronically HCV-infected patients, with and without MC. These levels were significantly higher in MC patients than in those without MC and in healthy controls and paralleled specific mRNA expression in PBL. Soluble gC1q-R circulates as a complexed form containing both C1q and HCV core proteins. Higher serum gC1q-R levels negatively correlated with circulating concentrations of the C4d fragment. The presence of sequestered C4d in the vascular bed of skin biopsies from MC patients was indicative of in situ complement activation. In vitro studies showed that release of soluble gC1q-R is regulated by HCV core-mediated inhibition of cell proliferation. Our results indicate that up-regulation of gC1q-R expression is a distinctive feature of MC, and that dysregulated shedding of C1q-R molecules contributes to vascular cryoglobulin-induced damage via the classic complement-mediated pathway.

Complement receptor 1 expression on mouse erythrocytes mediates clearance of Streptococcus pneumoniae by immune adherence.

Complement-containing immune complexes can be presented to phagocytes by human erythrocytes bearing complement receptor 1 (CR1). Although this has long been assumed to be a mechanism by which humans are able to protect themselves from "extracellular" bacteria such as pneumococci, there is little direct evidence. In these studies we have investigated this question by comparing results for erythrocytes from transgenic mice expressing human CR1 on their erythrocytes to the results for wild-type mouse erythrocytes that do not express CR1. We demonstrate that human CR1 expression on murine erythrocytes allows immune adherence to beads opsonized with either mouse or human serum as a source of complement. The role of CR1 in immune adherence was supported by studies showing that it was blocked by the addition of antibody to human CR1. Furthermore, human CR1 expression enhances the immune adherence of opsonized pneumococci to erythrocytes in vitro, and the pneumococci attached to erythrocytes via CR1 can be transferred in vitro to live macrophages. Even more importantly, we observed that if complement-opsonized pneumococci are injected intravenously with CR1(+) mouse erythrocytes into wild-type mice (after a short in vitro incubation), they are cleared faster than opsonized pneumococci similarly injected with wild-type mouse erythrocytes. Finally, we have shown that the intravenous (i.v.) injection of pneumococci into CR1(+) mice also results in more rapid blood clearance than in wild-type mice. These data support that immune adherence via CR1 on erythrocytes likely plays an important role in the clearance of opsonized bacteria from human blood.

Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" of C1q.

This work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein. This protein designated gC1q-R, was first isolated from Raji cells and was found to bind to the globular "heads" of C1q molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum. The NH2-terminal amino acid sequence of the first 24 residues of the C1q-binding protein was determined and this information allowed the synthesis of two degenerate polymerase chain reaction primers for use in the preparation of a probe in the screening of a B cell cDNA library. The cDNA isolated, using this probe, was found to encode a pre-pro protein of 282 residues. The NH2 terminus of the protein isolated from Raji cells started at residue 74 of the predicted pre-pro sequence. The cDNA sequence shows that the purified protein has three potential N-glycosylation residues and is a highly charged, acidic molecule. Hence, its binding to C1q may be primarily but not exclusively due to ionic interactions. The "mature" protein, corresponding to amino acid residues 74-282 of the predicted pre-pro sequence, was overexpressed in Escherichia coli and was purified to homogeneity. This recombinant protein was also able to inhibit the complement-mediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gel filtration in nondissociating conditions. Northern blot and RT-PCR studies showed that the C1q-binding protein is expressed at high levels in Raji and Daudi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 cell line. However, it is not expressed in the K562 cell line. Comparison of gC1q-R NH2-terminal sequence with that of the receptor for the collagen-like domain of C1q (cC1q-R) showed no similarity. Furthermore, antibodies to gC1q-R or an 18-amino acid residue-long NH2-terminal synthetic gC1q-R peptide did not cross-react with antibodies to cC1q-R. Anti-gC1q-R immunoblotted a 33-kD Raji cell membrane protein, whereas anti cC1q-R recognized a molecule of approximately 60 kD. The NH2-terminal sequence of gC1g-R appears to be displayed extracellularly since anti-gC1g-R peptide reacted with surface molecules on lymphocytes, polymorphonuclear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses.(ABSTRACT TRUNCATED AT 400 WORDS)

N-formylpeptide and complement C5a receptors are expressed in liver cells and mediate hepatic acute phase gene regulation.

Although the classical chemotactic receptor for complement anaphylatoxin C5a has been associated with polymorphonuclear and mononuclear phagocytes, several recent studies have indicated that this receptor is expressed on nonmyeloid cells including human endothelial cells, vascular smooth muscle cells, bronchial and alveolar epithelial cells, hepatocytes, and in the human hepatoma cell line HepG2. In this study, we examined the possibility that other members of the chemotactic receptor family are expressed in HepG2 cells and human liver, and the possibility that such receptors mediate changes in acute phase gene expression in HepG2 cells. Using polymerase chain reaction (PCR) amplification of HepG2 mRNA with primers based on highly conserved regions of the chemotactic subgroup of the G protein-coupled receptor family, we identified a PCR fragment from the formyl-methionyl-leucyl-phenylalanine (FMLP) receptor, as well as one from the C5a receptor. Immunostaining with antipeptide antisera to FMLPR confirmed the presence of this receptor in HepG2 cells. Receptor binding studies showed specific saturable binding of a radioiodinated FMLP analogue to HepG2 cells (Kd approximately 2.47 nM; R approximately 6 x 10(3) plasma membrane receptors per cell). In situ hybridization analysis showed the presence of FMLPR mRNA in parenchymal cells of the human liver in vivo. Both C5a and FMLP mediated concentration- and time-dependent changes in synthesis of acute phase proteins in HepG2 cells including increases in complement C3, factor B, and alpha 1-antichymotrypsin, as well as concomitant decreases in albumin and transferrin synthesis. The effects of C5a and FMLP on the synthesis of these acute phase proteins was evident at concentrations as low as 1 nM, and they were specifically blocked by antipeptide antisera for the corresponding receptor. In contrast to the effect of other mediators of hepatic acute phase gene regulation, such as interleukin 6, the effects of C5a and FMLP were reversed by increased concentrations well above the saturation point of the respective receptor. These results suggest that acute phase gene regulation by C5a and FMLP is desensitized at high concentrations, a property that is unique among the several known mechanisms for hepatic acute phase gene regulation.

Interleukin-33 Amplifies Human Mast Cell Activities Induced by Complement Anaphylatoxins.

Both, aberrant mast cell responses and complement activation contribute to allergic diseases. Since mast cells are highly responsive to C3a and C5a, while Interleukin-33 (IL-33) is a potent mast cell activator, we hypothesized that IL-33 critically regulates mast cell responses to complement anaphylatoxins. We sought to understand whether C3a and C5a differentially activate primary human mast cells, and probe whether IL-33 regulates C3a/C5a-induced mast cell activities. Primary human mast cells were generated from peripheral blood precursors or isolated from healthy human lung tissue, and mast cell complement receptor expression, degranulation, mediator release, phosphorylation patterns, and calcium flux were assessed. Human mast cells of distinct origin express constitutively higher levels of C3aR1 than C5aR1, and both receptors are downregulated by anaphylatoxins. While C3a is a potent mast cell degranulation inducer, C5a is a weaker secretagogue with more delayed effects. Importantly, IL-33 potently enhances the human mast cell reactivity to C3a and C5a (degranulation, cytokine and chemokine release), independent of changes in C3a or C5a receptor expression or the level of Ca2+ influx. Instead, this reflects differential dynamics of intracellular signaling such as ERK1/2 phosphorylation. Since primary human mast cells respond differentially to anaphylatoxin stimulation, and that IL-33 is a key regulator of mast cell responses to complement anaphylatoxins, this is likely to aggravate Th2 immune responses. This newly identified cross-regulation may be important for controlling exacerbated complement- and mast cell-dependent Th2 responses and thus provides an additional rationale for targeting anti-IL33 therapeutically in allergic diseases.