Search

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.

Aim: The aim of this study is to analyse the prognostic value of complement anaphylatoxin receptors in patients with non-ischaemic cardiomyopathy undergoing endomyocardial biopsy., Methods and Results: In 102 patients (72.5% male patients, median age 54 years) with non-ischaemic cardiomyopathy, myocardial expression of C3aR was assessed among other parameters. The primary study endpoint was a composite of death, heart transplantation, heart failure-related re-hospitalization, and deterioration of left ventricular ejection fraction within a mean follow-up of 11.9 months. The number of cells, which stained positive for C3aR, was significantly increased in patients with inflammatory compared with non-inflammatory cardiomyopathy (1.75 ± 0.31 cells in inflammatory cardiomyopathy vs. 0.94 ± 0.26 in non-inflammatory cardiomyopathy, P = 0.049). Subsequently, positive expression for C3aR was judged based on a semi-quantitative scoring system. Significantly, more patients with positive MHCII and CD68 expression showed an increased number of C3aR-positive cells. C3aR expression based on this score was more pronounced in patients with human herpesvirus 6 viral genome detection. Kaplan-Meier curves illustrate that the C3aR-negative group reached the primary endpoint significantly more often (mean follow-up 11.9 months, log rank 5.963, P = 0.015). Lack of C3aR expression was a strong independent predictor for the primary endpoint in Cox regression analysis [hazard ratio 0.46 (0.26-0.82, P = 0.009)]., Conclusions: C3aR-positive cells are found more often in patients with inflammatory cardiomyopathy. The relevance of C3aR-positive cells in patients with non-ischaemic cardiomyopathy should be further evaluated as potential predictors or modulators of adverse cardiac remodelling, the substrate of progressive heart failure., (© 2018 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of the European Society of Cardiology.)

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.

CD93 belongs to the group XIV C-type lectin like domain (CTLD) and is closely related to thrombomodulin (CD141). Although CD93 is known to be involved in the regulation of cell adhesion and phagocytosis, its role in innate immunity remains to be fully investigated. Critically, published data about CD141 suggest that CD93 CTLD could be involved in the control of inflammation. In order to address further functional and structural analyses, we expressed human CD93 CTLD with several disulfide bonds in an E. coli expression system. As the E. coli cytoplasm is a reducing compartment, production of disulfide-bond proteins remains a challenge. Hence, we decided to over express CD93 CTLD in commercially available strains of E. coli and co-expressed a sulfhydryl oxidase (Erv1p) and a disulfide isomerase (DsbC). This strategy led to high yield expression of a native form of CD93 CTLD. NMR studies revealed that Ca 2+ was not able to bind to CD93 CTLD. We also showed that the recombinant protein could alter LPS pro-inflammatory activity on THP1. This work provides new tool for further functional and structural studies to decipher the functions associated to the CTLD of CD93. This approach may also be used for others members of the group XIV C-type lectin like domain (CD141, CD248 and CLec14A)., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Epithelial-mesenchymal transition (EMT) and stem-like glioma cells display hallmark therapeutic resistance. Understanding of the mechanisms underlying these properties will be vital for the development of effective therapies. In this study, we found that VSIG4 protein is upregulated in glioblastoma. Overexpressing VSIG4 induced EMT and significantly promoted invasion and migration in glioblastoma U-87MG cells. Moreover, we showed that its overexpression promoted formation of glioma stem cell phenotypes in U-87MG cells. P4HB, VAMP8 and Connexin 43 (CX43) can promote temozolomide (TMZ) resistance in human glioma cells. We showed that P4HB, VAMP8 and CX43 protein were upregulated by VSIG4 in U-87MG cells, implying its upregulation might be a cause for temozolomide resistance. We found that let-7g-5p can inhibit VSIG4 protein expression, but it cannot degrade VSIG4 mRNA in U-87MG cells. Contrary to VSIG4, we demonstrated that overexpressing let-7g-5p promoted mesenchymal-epithelial transition (MET) and significantly inhibited invasion and migration consistent with the reduction of glioblastoma stem cell phenotypes in U-87MG cells. Thus, we concluded that let-7g-5p inhibits epithelial-mesenchymal transition (EMT) consistent with reduction of glioma stem cell (GSC) phenotypes by targeting VSIG4 in glioblastoma.

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., (© 2016 Authors; published by Portland Press Limited.)

Leukemia stem cells (LSCs) are thought to share several properties with hematopoietic stem cells (HSCs), including cell-cycle quiescence and a capacity for self-renewal. These features are hypothesized to underlie leukemic initiation, progression, and relapse, and they also complicate efforts to eradicate leukemia through therapeutic targeting of LSCs without adverse effects on HSCs. Here, we show that acute myeloid leukemias (AMLs) with genomic rearrangements of the MLL gene contain a non-quiescent LSC population. Although human CD34(+)CD38(-) LSCs are generally highly quiescent, the C-type lectin CD93 is expressed on a subset of actively cycling, non-quiescent AML cells enriched for LSC activity. CD93 expression is functionally required for engraftment of primary human AML LSCs and leukemogenesis, and it regulates LSC self-renewal predominantly by silencing CDKN2B, a major tumor suppressor in AML. Thus, CD93 expression identifies a predominantly cycling, non-quiescent leukemia-initiating cell population in MLL-rearranged AML, providing opportunities for selective targeting and eradication of LSCs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Integration of cellular and humoral arms of the innate immune response is fundamental to the development of powerful effector functions in host defence as well as aberrant immune responses. Here, we provide evidence in support of the relationship between complement activation and NK cell functional modulation. We demonstrate that human NK cells and both CD56(bright)CD16(-) and CD56(dim)CD16(+) populations express receptors known to detect the biologically active peptides C3a and C5a (i.e. C3aR, C5aR, C5L2) and the covalently-bound fragments C3b and metabolites iC3b and C3d which serve in immune adhesion (e.g. CR3, CR4). We also show that several pathogen- or tumour/inflammation-related stimuli differentially regulated those complement receptor expression. Furthermore, our results suggest that C3 fragments (C3a, iC3b) have a negative regulatory effect on IFN-γ production in NK cells. This work provides extensive information of human complement receptors relevant to the integrated actions of complement and NK cells which has been suggested by animal studies. The observations may act as a resource that allows further understanding and exploitation of role of complement in human health and immune mediated diseases., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

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.

Purpose: Müller cells, a major type of glial cell found in the eye, are postulated to play an important role in many retinal diseases, including diabetic retinopathy (DR). Complement is an integral part of innate immunity, and the activation of complement has been associated with retinal diseases. However, the role of complement in the regulation of Müller cell function remains unclear. We were trying to address these issues in this study., Methods: Using primary human Müller cells and a spontaneously immortalized human Müller cell line, we examined the expression of complement receptor C5aR both at mRNA and protein levels. Regulation of C5aR expression on Müller cells by prostaglandin E2 and by hyperglycemia, both of which are integrally involved in DR, were studied. Significance of C5aR on Müller cells was also investigated by examining relevant cytokine productions and their impacts on retinal endothelial cell proliferation/permeability after ligating the receptor using its ligand, C5a., Results: C5aR is constitutively expressed in human Müller cells. Prostaglandin E2 and hyperglycemia individually and synergistically upregulate C5aR expression in Müller cells. Signaling through C5aR on Müller cells upregulates production of IL-6 and VEGF, which promotes the proliferation of human retinal endothelial cells and increases their permeability., Conclusions: These results indicate that complement can regulate Müller cells through C5aR, which may contribute to the pathogenesis of retinal diseases, including DR.

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.

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., (Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Objectives: The aim of this study was to determine the expression of the anaphylatoxin receptors complement C3a receptor (C3aR) and C5a receptor (C5aR) in the placentas of pregnancies complicated by severe early onset preeclampsia., Study Design: We recruited women with pregnancies complicated by severe early-onset preeclampsia (n=19, 11 of which were further complicated with IUGR) and women with preterm pregnancies not affected by preeclampsia (n=8). Gene and protein expression of C3aR and C5aR was analysed by quantitative RT-PCR and Western blotting, respectively., Results: C3aR was detected in the Hofbauer cells in the villous stroma of the placenta. C5aR staining was detected in the syncytiotrophoblast and endothelial cells. We found significantly decreased expression of C3aR mRNA and protein expression in placentas with preeclampsia compared to controls. However, C5aR expression was not significantly different between preeclamptic and control placentas at either the mRNA or protein level., Conclusions: Decreased C3aR expression indicates a dysregulation of the complement system in the placentas of preeclamptic women. Further studies would elucidate the exact mechanisms that complement has in preeclampsia., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

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.

Introduction: The complement system is crucial for the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). In particular, C5a plays a central role. In this study, plasma and urinary levels of C5a as well as renal C5a receptors (CD88 and C5L2) expression were investigated in patients with AAV., Methods: Twenty-four patients with AAV in the active phase, 19 patients with AAV in the remission phase, and 20 patients with lupus nephritis (LN) were included. Plasma and urinary levels of C5a were measured with enzyme-linked immunosorbent assay (ELISA). The staining of CD88 and C5L2 in renal specimens was detected with immunohistochemistry., Results: The level of plasma C5a was significantly higher in patients with AAV in the active phase than that in patients in remission, that in patients with LN, and that in normal controls. The urinary C5a level was significantly higher in patients with AAV in the active phase than that in patients in remission and that in normal controls, but not significantly different between patients with active AAV and patients with LN. The mean optical density of CD88 staining in the tubulointerstitium was significantly lower in AAV patients than that in normal controls (0.0052 ± 0.0011 versus 0.029 ± 0.0042; P = 0.005). The mean optical density of C5L2 in glomeruli was significantly higher in AAV patients than that in normal controls (0.013 ± 0.0027 versus 0.0032 ± 0.0006; P < 0.001). The mean optical density of CD88 staining closely correlated with the initial eGFR (r = 0.835; P < 0.001) in AAV patients. Double-labeling immunofluorescence assay suggested that CD88 did not express on neutrophils, monocytes, or macrophages, but C5L2 expressed on neutrophils (or monocytes) and macrophages., Conclusion: The elevated plasma and urinary C5a levels indicated complement activation in human AAV. The level of renal CD88 expression could reflect the disease severity of ANCA-associated glomerulonephritis. CD88 expression was downregulated, and C5L2 was upregulated in ANCA-associated glomerulonephritis.

While it has been known for some time that CD93 regulates several processes involved in innate immunity and inflammation including phagocytosis and adhesion, the function of CD93 in disease progression is only now being elucidated. Recent in vivo studies in mice, and genome wide studies in mice and humans, have provided clues about its molecular function. Following a comprehensive review of CD93 expression patterns, this review will focus on recent findings over the last three years that address the putative function of CD93 in inflammation and innate immunity.

Human CD93, an epidermal growth factor (EGF)-like domain containing transmembrane protein, is predominantly expressed in the vascular endothelium. Studies have shown that AA4, the homolog of CD93 in mice, may mediate cell migration and angiogenesis in endothelial cells. Soluble CD93 has been detected in the plasma of healthy individuals. However, the role of soluble CD93 in the endothelium remains unclear. Recombinant soluble CD93 proteins with EGF-like domains (rCD93D123, with domains 1, 2, and 3; and rCD93D23, with domains 2 and 3) were generated to determine their functions in angiogenesis. We found that rCD93D23 was more potent than rCD93D123 in stimulating the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Production of matrix-metalloproteinase 2 increased after the HUVECs were treated with rCD93D23. Further, in a tube formation assay, rCD93D23 induced cell differentiation of HUVECs through phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase and extracellular signal-regulated kinases-1/2 signaling. Moreover, rCD93D23 promoted blood vessel formation in a Matrigel-plug assay and an oxygen-induced retinopathy model in vivo. Our findings suggest that the soluble EGF-like domain containing CD93 protein is a novel angiogenic factor acting on the endothelium.

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-β1 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., (© 2010 John Wiley & Sons A/S.)

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.

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.

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.

Growing evidence shows that C1q modulates the growth and function of cells committed to the monocyte-derived dendritic cell (DC) lineage. Because C1q regulates both innate and acquired immune responses, we postulated that C1q modulates the transition from monocytes to DCs, i.e. the interface between innate and acquired immunity. Human peripheral blood monocytes cultured with soluble C1q and DC growth factors (granulocyte-macrophage colony-stimulating factor + Interleukin-4) failed to down-regulate monocyte-associated (CD14, CD16) and up-regulate DC-associated (CD83, CD86) markers. Impaired DC differentiation was not due to apoptosis; further analysis revealed the development of CD14(hi)CD11c(hi)CD16 (+/-) cells that have previously been associated with both innate and acquired immunity. Monocyte-DC precursors expressed gC1qR, the receptor for globular heads of C1q, from the outset, while cC1qR, the receptor for the collagen tails of C1q, was expressed at low levels. Notably, the binding pattern of monoclonal antibodies specific to the globular heads of C1q indicated that C1q is bound to monocytes via globular heads, presumably through gC1qR. Moreover, gC1qR levels decreased, while cC1qR levels were dramatically amplified as monocytes differentiated into immature DC. Thus, specific C1q/C1q receptor (R) interactions may control the transition from the monocyte state (innate immunity) toward the professional antigen-presenting cell state (adaptive immunity).

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.

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.

Self cells depend on surface complement regulators to protect them from autologous complement mediated attack. CD4(+)CD25(+)foxp3(+) T regulatory (Treg) cells are critical in maintaining immune homeostasis, however, which complement regulators are expressed on them and how they are protected from autologous complement attack remains unknown. We report here that mouse Treg cells express virtually no DAF or CR1. Instead, all of them express Crry and approximately half of them express CD59. Both Crry(-/-) and CD59(-/-) Treg cells exhibit greater complement mediated injury than WT Treg cells. These results clarify the status of cell surface complement regulators on mouse Treg cells and indicate that both Crry and CD59 are required to protect Treg cells from autologous complement mediated injury. Additionally, these data also argue that different from previous assumption, at least in mice, CD4(+)CD25(+)foxp3(+) Treg cells are not homogenous and could be further divided into subgroups based on CD59 expression.

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.

Background: Mast cells (MC) are key effector cells of allergic diseases and resistance to helminthic parasites and induce or amplify diverse innate and adaptive immune responses. The signals controlling MC mobilization during inflammation are not fully understood., Results: Since anaphylatoxins are attractive candidates as MC chemoattractants, we investigated expression and function of anaphylatoxin receptors in murine MC. Precursor cell-derived MC cultured with IL-3 in the presence or absence of SCF did not express significant amounts of surface C5a receptor (C5aR) or C3a receptor (C3aR). MC required approximately 4 h of stimulation with Ag (DNP-albumin, following preincubation with IgE anti-DNP), ionomycin, or PMA to enable a strong chemotactic response towards C5a, paralleled by a distinct C5aR upregulation. Likewise, C5a induced intracellular calcium fluxes solely in activated MC. In contrast, C3a proved to be a weak MC chemotaxin and unable to increase intracellular calcium. Primary peritoneal MC did not express detectable amounts of anaphylatoxin receptors, however, similar to precursor cell-derived MC, stimulation with Ag or ionomycin for 4 h induced a prominent surface expression of C5aR whereas C3aR remained undetectable., Conclusion: Collectively, our results suggest that Ag-dependent as well as -independent activation induces an inflammatory MC phenotype which is distinguished by neoexpression of a functional C5aR as a novel effector mechanism in MC-mediated pathogenesis.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by B cell hyper-reactivity, autoantibody production, immune complex (IC) deposition, and multiple organ damage. The contribution of IC and B cell-mediated changes in the pathogenesis of SLE is well established, however, the exact role of IC-binding receptors expressed on B cells, Fcgamma receptors, and complement receptors CR1 and CR2 in these pathological processes is unclear. Development of lupus-like symptoms in mice defective for the inhibitory Fc-gammaRIIb and genetic association of certain FcgammaR genes with SLE demonstrate a significant role for these receptors but reports indicating alterations of Fcgamma or complement receptor-mediated B cell functions in human SLE are relatively few. The present review highlights a selected set of data including our own discussing the significance of animal models, genetics, and functional alterations of these IC-binding receptors in the etiopathogenesis of SLE.

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.

The evolutionary origin of allorecognition in vertebrates is unknown. Urochordates, being the closest living relatives of vertebrates [Delsuc F, Brinkmann H, Chourrout D, Philippe H]. Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 2006; 439: 965-8], have efficient mechanisms to prevent both allogeneic fusion and self fertilization. To shed light on allorecognition in urochordates and on the molecules involved in preventing self fertilization, we compared gonadal cDNAs of three genetically unrelated Ciona intestinalis individuals by suppression subtractive hybridisation (SSH). Here, we report the discovery and characterization of a highly polymorphic gene coding for a transmembrane protein with several short consensus repeat domains (SCR/CCP). The protein, termed variable complement receptor-like 1 (vCRL1), is structurally similar to vertebrate complement receptors. However, in contrast to vertebrate complement receptors, vCRL1 shows an unprecedented high degree of amino acid variations among Ciona individuals and is expressed in follicle cells as well as in hemocytes. Based on our data we propose that in the absence of MHC Ciona uses variable components of the complement system as individuality markers.

The in vitro macrophage response to zymosan has been attributed to Toll-like receptor 2 (TLR2). Whether TLR2 is obligatory for the zymosan-induced in vivo response has not been assessed. The importance of this question is underscored by the fact that zymosan activates complement in a cell-independent manner. We have investigated whether the in vitro observation of TLR2 as the dominant zymosan receptor on macrophages would translate to an experimental peritonitis model in vivo. We have treated mice with zymosan, resulting in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. Zymosan-mediated leukocyte recruitment was TLR2 independent, but was predominantly dependent on the complement components, C3 and C5a with a minor contribution from LTB4. Peritoneal neutrophilia was 50% mast cell dependent and this defect was reproduced using C5a receptor (C5aR)-deficient mast cells in mast cell-deficient mice, suggesting that C5aR is responsible for mast cell activation following zymosan challenge. By 24 h, the response to zymosan involved primarily monocyte recruitment and was C3 and C5aR independent. Taken together, these studies indicate that the in vivo inflammatory response to zymosan does not necessarily mimic the TLR2 dependence observed in vitro, and that complement plays a dominant role in early, but not late, zymosan-mediated peritonitis.

Complement and dendritic cells (DCs) are essential components of innate immunity. Both participate in local inflammation and moreover have roles in the initiation of the acquired immunity response and in the maintenance of tolerance. Recent studies have demonstrated the ability of DCs to synthesize C1q, C3, Factor I, Factor B and complement receptors 3 and 4. In this study, we demonstrate that human DCs are a source of other soluble complement proteins including C1q, C4b binding protein (C4BP), C7 and C8. Complement receptors (CR)1 and the CD18 chain (common for CR3 and CR4) were also present on DCs while CR2 was not detected.

Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various cells, particularly in granulocytes and monocytes . We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances : an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (-) cells and Annexin V positive (+)/7AAD positive (+) cells, respectively, in U937 cells after 6 hr of treatment. The modulation of CD93 expression on U937 cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (-) cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated cell sorter (FACS). The mean fluorescence intensity (MFI) of the cells that stained positive for CD93 mAb (mNI-11) among the treated U937 cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 beta; LFA-1beta) and CD54 (intercellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 cells identified as Annexin V positive (+)/7AAD negative (-) cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 cells. Importantly, however , the U937 cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 cells with apoptotic properties, and that the decrease in CD93 expression on U937 cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid cells.

Z39Ig is a transmembrane protein containing two Ig homology domains with unknown functions. Immunohistochemical analyses of human carotid atherosclerotic plaques detected Z39Ig staining in areas rich in foamy macrophages. Z39Ig staining was also observed in macrophages in the lining layers and sublining areas of rheumatoid arthritis synovium. Z39Ig staining in the osteoarthritis synovium was restricted to macrophages in the lining layers. To identify the role(s) of Z39Ig in the function of macrophages, we used human monocytic cell lines TF-1A (Z39Ig-negative) and THP-1 (Z39Ig-positive). The expression of Z39Ig was induced in TF-1A cells ,when they were differentiated into macrophages by treatment with PMA. The stimulation of PMA-treated TF-1A or THP-1 cells with immobilized anti-Z39Ig mAb induced the secretion of IL-8 and matrix metalloproteinase (MMP)-9, which was dependent on NF-kappaB activation. These data indicate that the macrophage Z39Ig is involved in the pathogenesis of inflammatory diseases through chemokine induction, which will promote the migration of inflammatory cells into the lesion area, and MMP-9 induction, which will contribute to cartilage destruction or extracellular matrix degradation.

In mammals, the bioactive fragment C3a, released from C3 during complement activation, is a potent mediator of inflammatory reactions and exerts its functional activity through the specific binding to cell surface G protein-coupled seven-transmembrane receptors. Recently, we demonstrated a Ciona intestinalis C3a (CiC3a)-mediated chemotaxis of hemocytes in the deuterostome invertebrate Ciona intestinalis and suggested an important role for this molecule in inflammatory processes. In the present work, we have cloned and characterized the receptor molecule involved in the CiC3a-mediated chemotaxis and studied its expression profile. The sequence, encoding a 95,394 Da seven-transmembrane domain protein, shows the highest sequence homology with mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is expressed abundantly in the heart and neural complex and to a lesser extent in the ovaries, hemocytes, and larvae. Three polyclonal Abs raised in rabbits against peptides corresponding to CiC3aR regions of the first and second extracellular loop and of the third intracellular loop react specifically in Western blotting with a single band of 98-102 kDa in hemocyte protein extracts. Immunostaining performed on circulating hemocytes with the three specific Abs revealed that CiC3aR is constitutively expressed only in hyaline and granular amoebocytes. In chemotaxis experiments, the Abs against the first and second extracellular loop inhibited directional migration of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal sequence, thus providing the compelling evidence that C. intestinalis expresses a functional C3aR homologous to the mammalian receptor. These findings further elucidate the evolutionary origin of the vertebrate complement-mediated proinflammatory process.

Chimpanzee is a unique animal model for HCV infection, in which about 50% of infections resolve spontaneously. It has been reported that the magnitude of T cell responses to HCV core in recovered chimpanzees is greater than that in chronically infected ones. However, the mechanism(s) by which the chimpanzees with resolved infection overcome core-mediated immunosuppression remains unknown. In this study, we examined the effect of HCV core on T cell responsiveness in chimpanzees with resolved and chronic HCV infection. We found that core protein strongly inhibited T cell activation and proliferation in chimpanzees with chronic infection, while this inhibition was limited in chimpanzees with resolved infection. Notably, the level of gC1qR, as well as the binding of core protein, on the surface of T cells was lower in recovered chimpanzees when compared to chimpanzees with chronic HCV infection. Intriguingly, the observed differences in gC1qR expression levels and susceptibility to core-induced suppression amongst HCV-chronically infected and recovered chimpanzees were observed prior to HCV challenge, suggesting a possible genetic determination of the outcome of infection. These findings suggest that gC1qR expression on the surface of T cells is crucial for HCV core-mediated T cell suppression and viral clearance, and that represents a novel mechanism by which a virus usurps host machinery for persistence.

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.

Signaling of the C3a anaphylatoxin through its G protein-coupled receptor, C3aR, is relevant in a variety of inflammatory diseases, but its role in lupus nephritis is undefined. In this study, we show that expression of C3aR was significantly increased in prediseased and diseased kidneys of MRL/lpr lupus mice compared with MRL/+ controls. To investigate the role of C3aR in experimental lupus, a small molecule antagonist of C3aR (C3aRa) was administered continuously to MRL/lpr mice from 13 to 19 wk of age. All 13 C3aRa-treated mice survived during the 6-wk treatment compared with 9 of 14 (64.3%) control animals given vehicle (p = 0.019). Relative to controls, C3aRa-treated animals were protected from renal disease as measured by albuminuria (p = 0.040) and blood urea nitrogen (p = 0.021). In addition, there were fewer neutrophils, monocytes, and apoptotic cells in the kidneys of C3aRa-treated mice. C3aRa treatment also led to reduced renal IL-1beta and RANTES mRNA and phosphorylated phosphatase and tensin homologue deleted on chromosome 10 protein, whereas the mass of phosphorylated protein kinase B/Akt was increased by C3aRa. Thus, C3aR antagonism significantly reduces renal disease in MRL/lpr mice, which further translates into prolonged survival. These data illustrate that C3aR is relevant in experimental lupus nephritis and may be a target for therapeutic intervention in the human disease.

CD93 is a highly glycosylated transmembrane protein expressed on monocytes, neutrophils, endothelial cells, and stem cells. Antibodies directed at CD93 modulate phagocytosis, and CD93-deficient mice are defective in the clearance of apoptotic cells from the inflamed peritoneum. In this study we observe that CD93, expressed on human monocytes and neutrophils, is susceptible to phorbol dibutyrate-induced protein ectodomain shedding in a time- and dose-dependent manner. The soluble fragment found in culture supernatant retains the N-terminal carbohydrate recognition domain and the epidermal growth factor repeats after ectodomain cleavage. Importantly, a soluble form of the CD93 ectodomain was detected in human plasma, demonstrating that shedding is a physiologically relevant process. Inhibition of metalloproteinases with 1,10-phenanthroline inhibited shedding, but shedding was independent of TNF-alpha-converting enzyme (a disintegrin and metalloproteinase 17). Phorbol dibutyrate-induced CD93 shedding on monocytes was accompanied by decreased surface expression, whereas neutrophils displayed an increase in surface expression, suggesting that CD93 shed from the neutrophil surface was rapidly replaced by CD93 from intracellular stores. Cross-linking CD93 on human monocytes with immobilized anti-CD93 mAbs triggered shedding, as demonstrated by a decrease in cell-associated, full-length CD93 concomitant with an increase in CD93 intracellular domain-containing cleavage products. In addition, the inflammatory mediators, TNF-alpha and LPS, stimulated ectodomain cleavage of CD93 from monocytes. These data demonstrate that CD93 is susceptible to ectodomain shedding, identify multiple stimuli that trigger shedding, and identify both a soluble form of CD93 in human plasma and intracellular domain containing cleavage products within cells that may contribute to the physiologic role of CD93.

Complement and FcgammaR effector pathways are central triggers of immune inflammation; however, the exact mechanisms for their cooperation with effector cells and their nature remain elusive. In this study we show that in the lung Arthus reaction, the initial contact between immune complexes and alveolar macrophages (AM) results in plasma complement-independent C5a production that causes decreased levels of inhibitory FcgammaRIIB, increased levels of activating FcgammaRIII, and highly induced FcgammaR-mediated TNF-alpha and CXCR2 ligand production. Blockade of C5aR completely reversed such changes. Strikingly, studies of pertussis toxin inhibition show the essential role of G(i)-type G protein signaling in C5aR-mediated control of the regulatory FcgammaR system in vitro, and analysis of the various C5aR-, FcgammaR-, and G(i)-deficient mice verifies the importance of Galpha(i2)-associated C5aR and the FcgammaRIII-FcgammaRIIB receptor pair in lung inflammation in vivo. Moreover, adoptive transfer experiments of C5aR- and FcgammaRIII-positive cells into C5aR- and FcgammaRIII-deficient mice establish AM as responsible effector cells. AM lacking either C5aR or FcgammaRIII do not possess any such inducibility of immune complex disease, whereas reconstitution with FcgammaRIIB-negative AM results in an enhanced pathology. These data suggest that AM function as a cellular link of C5a production and C5aR activation that uses a Galpha(i2)-dependent signal for modulating the two opposing FcgammaR, FcgammaRIIB and FcgammaRIII, in the initiation of the inflammatory cascade in the lung Arthus reaction.

Sepsis is associated with extensive complement activation, compromising innate immune defenses, especially in neutrophils (PMN). Recently, a second C5a receptor (C5L2) was detected on PMN without evidence of intracellular signaling. The current study was designed to determine changes in C5L2 in blood PMN during sepsis. In vitro exposure of PMN to C5a, but not to fMLP, led to reduced content of C5L2. Following cecal ligation and puncture-induced sepsis in rats, PMN demonstrated a time-dependent decrease in C5L2. In vivo blockade of C5a during experimental sepsis resulted in preservation of C5L2. Similarly, PMN from patients with progressive sepsis showed significantly reduced C5L2 expression (n = 26), which was virtually abolished in patients who developed multiorgan failure (n = 10). In contrast, sepsis survivors exhibited retention of C5L2 (n = 12/13). The data suggest that C5L2 on PMN diminishes during sepsis due to systemic generation of C5a, which is associated with a poor prognosis.

Recombinant soluble human complement receptor type 1 (sCR1) is a highly glycosylated glycoprotein intended for use as a drug to treat ischemia-reperfusion injury and other complement-mediated diseases and injuries. sCR1-sLe(x) produced in the FT-VI-expressing mutant CHO cell line LEC11 exists as a heterogeneous mixture of glycoforms, a fraction of which include structures with one or more antennae terminated by the sialyl Lewis X (sLe(x)) [Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc]) epitope. Such multivalent presentation of sLe(x) was shown previously to effectively target sCR1 to activated endothelial cells expressing E-selectin. Here, we describe the use of the soluble, recombinant alpha2-3 sialyltransferase ST3Gal-III and the alpha1-3 fucosyltransferase FT-VI in vitro to introduce sLe(x) moieties onto the N-glycan chains of sCR1 overexpressed in standard CHO cell lines. The product (sCR1-S/F) of these in vitro enzymatic glycan remodeling reactions performed at the 10-g scale has approximately 14 N-glycan chains per sCR1 molecule, comprised of biantennary (90%), triantennary (8.5%), and tetraantennary (1.5%) structures, nearly all of whose antennae terminate with sLe(x) moieties. sCR1-S/F retained complement inhibitory activity and, in comparison with sCR1-sLe(x) produced in the LEC11 cell line, contained twice the number of sLe(x) moieties per mole glycoprotein, exhibited a twofold increase in area under the intravenous clearance curve in a rat pharmacokinetic model, and exhibited a 10-fold increase in affinity for E-selectin in an in vitro binding assay. These results demonstrate that in vitro glycosylation of the sCR1 drug product reduces heterogeneity of the glycan profile, improves pharmacokinetics, and enhances carbohydrate-mediated binding to E-selectin.

The C3aR is expressed throughout the CNS and is increased in expression on glial cells during CNS inflammation. However, the role that C3a and the C3aR play in chronic inflammation, such as in the demyelinating disease experimental autoimmune encephalomyelitis (EAE), remains unclear. We show in this study that deletion of the C3aR is protective in myelin oligodendrocyte glycoprotein-induced EAE in C57BL/6 mice. C3aR-deficient (C3aR(-/-)) mice had a significantly attenuated course of EAE compared with control mice during the chronic phase of the disease. Immunohistochemical analysis demonstrated modestly reduced macrophage and T cell infiltration in the spinal cords of C3aR(-/-) mice. To examine the role of C3a in EAE, we developed a transgenic mouse that expresses C3a exclusively in the CNS using the glial fibrillary acidic protein (GFAP) promoter. We observed that C3a/GFAP mice had exacerbated EAE during the chronic phase of the disease, with significant mortality compared with nontransgenic littermates. C3a/GFAP mice had massive meningeal and perivascular infiltration of macrophages and CD4(+) T cells. These studies indicate that C3a may contribute to the pathogenesis of demyelinating disease by directly or indirectly chemoattracting encephalitogenic cells to the CNS.

Although complement activation and deposition have been associated with a variety of glomerulopathies, the pathogenic mechanisms by which complement directly mediates renal injury remain to be fully elucidated. Renal parenchymal tissues express a limited repertoire of receptors that directly bind activated complement proteins. We report the renal expression of the receptor for the C3 cleavage product C3a, a member of the anaphylatoxin family. C3aR is highly expressed in normal human and murine kidney, as demonstrated by immunohistochemistry and in situ hybridization. Its distribution is limited to epithelial cells only, as glomerular endothelial and mesangial cells showed no evidence of C3aR expression. The C3aR is also expressed by primary renal proximal tubular epithelial cells in vitro as demonstrated by FACS, Western blot, and RT-PCR. In vitro C3aR is functional in terms of its capacity to bind 125I-labeled C3a and generate inositol triphosphate. Finally, using microarray analysis, four novel genes were identified and confirmed as transcriptionally regulated by C3aR activation in proximal tubular cells. These studies define a new pathway by which complement activation may directly modulate the renal response to immunologic injury., (Copyright 2004 The American Association of Immunologists, Inc.)

Background: Phagocytosis of IgG- or complement-opsonized bacteria and antibody production by lymphocytes are regulated by cell surface receptors for IgG (FcgammaRI, FcgammaRII and FcgammaRIII) and complement (CR1 and CR3). We measured the effect of uraemia and dialysis treatment on FcgammaR and CR expression on leukocytes in blood., Methods: Blood samples were obtained from children: 40 treated with peritoneal dialysis (PD), 23 with haemodialysis (HD), 46 not yet dialysed (CRF) and 33 healthy (HC). White blood cells, isolated from EDTA-blood by centrifugation after cell fixation with paraformaldehyde, were labelled with FITC-conjugated CD16 (FcgammaRIII), CD32 (FcgammaRII), CD64 (FcgammaRI), CD11b (CR3) and CD35 (CR1) monoclonal antibodies and analysed by flow cytometry., Results: In PD, HD, CRF and HC, monocytes and neutrophils were all positive for FcgammaR and CR, except for CD16 on monocytes (20% positive). Lymphocytes expressed CD16 and CD32 but not CD64. PD, HD and CRF children had lower percentages of CD16(+) and CD32(+) lymphocytes compared with HC. The percentage of CD11b(+) lymphocytes was lower only in PD and the percentage of CD35(+) lymphocytes was lower in HD and CRF compared with HC. The median CD32 mean fluorescense intensity (MFI) on lymphocytes, monocytes and neutrophils was lower in PD, HD and CRF compared with HC. On the other hand, CD11b MFI on lymphocytes, monocytes and neutrophils was higher in PD, HD and CRF children compared with HC. CD16 and CD64 MFI were not different among the groups and CD35 MFI was only lower on lymphocytes from PD, HD and CRF compared with HC., Conclusions: In children with chronic renal failure, whether dialysed or not, FcgammaRII expression on lymphocytes, monocytes and neutrophils was reduced and CR3 expression was increased. Furthermore, CR1 expression on lymphocytes, important for the humoral response, was lower in children with renal failure. Age and uraemia are associated with these abnormalities and might contribute to impaired immune function in children with chronic renal failure.

Combinatorial Ig libraries with phage display allow in vitro generation of human Ig fragments without the need to maintain hybridomas in ongoing cell culture or to select circulating Ig from human serum. Identifying tumor-associated antigens on the surface of intact tumor cells, as opposed to purified proteins, presents a challenge due to the difficulty of preserving complex 3-D epitopic sites on the cell surface, the variable expression of antigens on different malignant cell types and the stereotactic interference of closely associated proteins on the intact membrane surface limiting accessibility to antigenic sites. A combinatorial Ig library of 10(10) clones was generated from the cDNA of PBMCs derived from patients with breast adenocarcinoma. Following subtractive panning, the library was enriched for Ig (Fab fragment) binding to intact adenocarcinoma cells and the resultant Fabs were screened against a cDNA expression library, itself generated from breast cancer cells. Using this approach, we isolated clones from the cDNA library expressing gC1q-R, a glycoprotein comprising the major structure of C1, the first component of the complement system. gC1q-R is a 33 kDa glycoprotein expressed not only on the cell surface but also intracellularly, with motifs that target it to mitochondria and complete homology with HABP and human HeLa cell protein p32, which is copurified with pre-mRNA SF2. Sequencing of the gene encoding tumor-associated gC1q-R did not reveal any consistent tumor-specific mutations. However, histochemical staining with anti-gC1q-R MAb demonstrated marked differential expression of gC1q-R in thyroid, colon, pancreatic, gastric, esophageal and lung adenocarcinomas compared to their nonmalignant histologic counterparts. In contrast, differential expression was not seen in endometrial, renal and prostate carcinomas. Despite high expression in breast carcinoma, gC1q-R was also expressed in nonmalignant breast tissue. Although the precise relation of gC1q-R to carcinogenesis remains unclear, our finding of tumor overexpression and the known multivalent binding of gC1q-R to not only C1q itself but also a variety of circulating plasma proteins as well as its involvement in cell-to-cell interactions suggest that gC1q-R may have a role in tumor metastases and potentially serve in molecule-specific targeting of malignant cells., (Copyright 2004 Wiley-Liss, Inc.)

A growing body of evidence supports the hypothesis that atherosclerosis has an inflammatory component, and that immune mechanisms, including complement activation, are likely to be involved. gC1q-R/p33 (gC1q-R) is a multifunctional and multicompartmental cellular protein, which is postulated to play a role in inflammation and thrombosis by interacting with C1q and high molecular weight kininogen (HK). To examine the expression of gC1q-R and its major ligands, C1q and HK, in human atherosclerotic lesions, sections of carotid arteries removed during endarterectomy and coronary arteries obtained at autopsy were stained with specific polyclonal or monoclonal antibodies. Control sections were stained with irrelevant rabbit IgG or isotype matched murine monoclonal antibody (MOPC), respectively. Tissue sections were counterstained with hematoxylin and examined by light microscopy. Specific staining for gC1q-R, C1q, and HK was observed in and around atherosclerotic lesions. In contrast to control antibodies, antibodies directed against gC1q-R reacted with endothelial cells, foam cells, smooth muscle cells, and inflammatory cells present in the intima and media of atherosclerotic lesions. In addition, the necrotic central core of advanced lesions with calcifications, fibrin, and lipids, stained intensely for gC1q-R, and negligibly with control antibodies. HK demonstrated a similar staining pattern, whereas C1q was most heavily expressed in the fibrous cap and necrotic core of atherosclerotic lesions. The localization of gC1q-R and its ligands C1q and HK in atherosclerotic lesions, and the previously described ability of gC1q-R to modulate complement, kinin, and coagulation cascades, suggest that gC1q-R may play an important role in promoting inflammation and thrombosis in atherosclerotic lesions., (Copyright 2004 Elsevier Ltd.)

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by the production of a broad spectrum of autoantibodies against nuclear, cytoplasmic and cell surface antigens and immune complex overload. Complement receptor 1 (CR1, CD 35), a transmembrane glycoprotein found on the surface of erythrocytes, leukocytes and glomerular podocytes plays a key role in the clearance of immune complexes and regulation of complement cascade. A drastic decline in the level of cell surface CR1 appears to be an important event in pathology of SLE. However, the etiology of lower than normal expression of cell surface CR1 in this disease is poorly understood. We studied the level of leukocyte CR1 transcription in 30 patients with active SLE and 30 controls by reverse transcriptase-polymerase chain reaction (RT-PCR) and related the same with the level of CR1 protein expression monitored by Western blotting. For RT-PCR, ratio of CR1/beta-actin was considered for semiquantitation of the level of CR1 transcription. Despite individual variation at the level of transcription, 70% (21 out of 30) of the patients expressed CR1 transcript at the lowest range of 0-15% as compared to the controls wherein only 30% (9 out of 30 individuals) demonstrated CR1 transcript in this range. Majority of the controls (70%) expressed CR1 transcript at the level above 15%. Mean level of CR1 transcript in patients (mean +/- S.D. = 21.09 +/- 14.3) was significantly lower than the controls (mean +/- S.D. = 48.91 +/- 26.34) (P < 0.001). The level of CR1 transcription correlated inversely with circulating immune complexes (CIC) (r = 0.52, P < 0.01). This may suggest that although erythrocyte CR1 is the chief vehicle for CIC clearance, drastic decline in leukocyte CR1 expression may impair the phagocyte mediated immune complex clearance and contribute to increased complement consumption in SLE. Total leukocyte CR1 protein expression was also significantly reduced in patients (P < 0.001) as compared to controls. This decline at the protein level gave a very significant positive correlation with CR1 transcript (r = 0.67, P < 0.01). A marginal increase in soluble CR1 (sCR1) was observed in the plasma (ELISA) of SLE patients compared to the controls but was insignificant. This paper for the first time brings evidence to suggest that reduced synthesis of CR1 contributes substantially to the low cell surface CR1 expression in SLE. Our findings also suggest increased proteolytic cleavage of leukocyte cell surface CR1 in these patients. However, evidence for the latter is indirect.

Human CD93 (known as C1qRp) has been shown to be a phagocytic receptor involved in the in vitro C1q-dependent enhancement of phagocytosis. However, binding of CD93 to C1q and its function remain controversial. In this study, we have generated CD93-deficient mice (CD93(-/-)) to investigate its biological role(s). The CD93(-/-) mice were viable and showed no gross abnormalities in their development. Thioglycolate-elicited peritoneal macrophages deficient in CD93 showed a similar enhancement in complement- and FcgammaR-dependent uptake of RBC to the wild-type macrophages when plated on C1q-coated surfaces suggesting that the lack of this receptor had no effect on these C1q-mediated events. There was no impairment in either complement- or FcgammaR-dependent phagocytic assays in vivo. By contrast, the CD93(-/-) mice had a significant phagocytic defect in the clearance of apoptotic cells in vivo (human Jurkat T cells and murine thymocytes: p=0.0006 and p=0.0079, respectively) compared with strain-matched controls. However, in vitro, the CD93(-/-) macrophages showed similar engulfment of apoptotic cells to wild-type macrophages. Furthermore, no supporting evidence for a role of CD93 as an adhesion molecule was found using intravital microscopy or analyzing peritoneal cell recruitment in response to three different inflammatory stimuli (thioglycolate, zymosan A, and IL-1beta). Thus, our findings indicate that murine CD93 is expressed on the peritoneal macrophage, especially on thioglycolate-elicited cells, but does not appear to play a key role in C1q-mediated enhancement of phagocytosis or in the intercellular adhesion events tested. However, our results suggest that it may contribute to the in vivo clearance of dying cells.

By-products of complement activation and complement regulatory proteins are increasingly recognized to play an important pathogenic role in a variety of vascular diseases including atherosclerosis, ischemia and reperfusion injury, hyperacute graft rejection, vasculitis, and the vascular complications of human diabetes. "Self" damage by autologous complement is mediated by activation products of the complement cascades or by direct insertion of the membrane attack complex (MAC) into cell membranes. Specifically, insertion of MAC complexes into endothelial cells results in the release of an array of growth factors and cytokines that induces proliferation, inflammation and thrombosis in the vascular wall. This paper reviews complement and complement regulatory proteins with specific focus on the vasculature and vascular diseases; it highlights complement and its regulators as potential targets for the rational design of mechanism-specific drugs for the treatment of some of the most prevalent human diseases.