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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.)

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.)

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.)

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.

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.

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.

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.

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.)

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.

The primary age-related loss in B cell progenitors is thought to be at the pro- to pre-B cell transition. However, we show that the frequencies and absolute numbers of all progenitor populations for the B cell lineage, including B-lineage-committed pro-B cells and multipotent B-lymphoid progenitors, decline in aged C57BL/6 mice. Moreover, when derived from aged mice, lymphoid progenitors within every population examined exhibited suboptimal IL-7 responsiveness, demonstrating that age-associated suboptimal IL-7R signaling is a general property of all early B-lineage precursors. Collectively, these data indicate that aging results in a previously unappreciated decline in the earliest stages of B cell development.

It has been suggested that the human C1qRp is a receptor for the complement component C1q; however, there is no direct evidence for an interaction between C1q and C1qRp. In this study, we demonstrate that C1q does not show enhanced binding to C1qRp-transfected cells compared with control cells. Furthermore, a soluble recombinant C1qRp-Fc chimera failed to interact with immobilized C1q. The proposed role of C1qRp in the phagocytic response in vivo is also unsupported in that we demonstrate that this molecule is not expressed by macrophages in a variety of human tissues and the predominant site of expression is on endothelial cells. Studies on the rodent homolog of C1qRp, known as AA4, have suggested that this molecule may function as an intercellular adhesion molecule. Here we show that C1qRp is the Ag recognized by several previously described mAbs, mNI-11 and two anti-CD93 Abs (clones X2 and VIMD2b). Interestingly, mNI-11 (Fab') has been shown to promote monocyte-monocyte and monocyte-endothelial cell adhesive interactions. We produced a recombinant C1qRp-Fc chimera containing the C-type lectin-like domain of C1qRp and found specific binding to vascular endothelial cells in sections of inflamed human tonsil, indicating the presence of a C1qRp ligand at this site. This interaction was Ca(2+) independent and was not blocked by our anti-C1qRp mAb BIIG-4, but was blocked by the proadhesive mAb mNI-11. Collectively, these data indicate that C1qRp is not a receptor for C1q, and they support the emerging role of C1qRp (here renamed CD93) in functions relevant to intercellular adhesion.

B cells and dendritic cells (DCs) each develop from poorly described progenitor cells in the bone marrow (BM). Although a subset of DCs has been proposed to arise from lymphoid progenitors, a common developmental pathway for B cells and BM-derived DCs has not been clearly identified. To address this possibility, we performed a comprehensive analysis of DC differentiative potential among lymphoid and B lymphoid progenitor populations in adult mouse BM. We found that both the common lymphoid progenitors (CLPs), shown here and elsewhere to give rise exclusively to lymphocytes, and a down-stream early B-lineage precursor population devoid of T and NK cell precursor potential each give rise to DCs when exposed to the appropriate cytokines. This result contrasts with more mature B-lineage precursors, all of which failed to give rise to detectable numbers of DCs. Significantly, both CLP and early B-lineage-derived DCs acquired several surface markers associated with functional DCs, and CLP-derived DCs readily induced proliferation of allogeneic CD4(+) T cells. Surprisingly, however, DC differentiation from both lymphoid-restricted progenitors was accompanied by up-regulation of CD11b expression, a cell surface molecule normally restricted to myeloid lineage cells including putative myeloid DCs. Together, these data demonstrate that loss of DC developmental potential is the final step in B-lineage commitment and thus reveals a previously unrecognized link between early B cell and DC ontogeny.

A lymphoid-committed progenitor population was isolated from mouse bone marrow based on the cell surface phenotype Thy-1.1(neg)Sca-1(pos)c-Kit(low)Lin(neg). These cells were CD43(pos)CD24(pos) on isolation and proliferated in response to the cytokine combination of steel factor, IL-7, and Flt3 ligand. Lymphoid-committed progenitors could be segregated into more primitive and more differentiated subsets based on expression of AA4.1. The more differentiated subset generated only B lymphoid cells in 92% of total colonies assayed, lacked T lineage potential, and expressed Pax5. These studies have therefore defined and isolated a B lymphoid-committed progenitor population at a developmental stage corresponding to the initial expression of CD45R.

Purpose: To explore the role of the complement system and complement regulatory proteins in an immune-privileged organ, the eye., Methods: Eyes of normal Lewis rats were analyzed for the expression of complement regulatory proteins, membrane cofactor protein (MCP), decay-acceleration factor (DAF), membrane inhibitor of reactive lysis (MIRL, CD59), and cell surface regulator of complement (Crry), using immunohistochemistry, Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR). Zymosan, a known activator of the alternative pathway of complement system was injected into the anterior chamber of the eye of Lewis rats. Animals were also injected intracamerally with 5 microl (25 microg) of neutralizing monoclonal antibody (mAb) against rat Crry (5I2) or CD59 (6D1) in an attempt to develop antibody induced anterior uveitis; control animals received 5 microl of sterile phosphate-buffered saline (PBS), OX-18 (25 microg), G-16-510E3 (25 microg), or MOPC-21 (25 microg). The role of complement system in antibody-induced uveitis was explored by intraperitoneal injection of 35 U cobra venom factor (CVF), 24 hours before antibody injection. Immunohistochemical staining and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Western blot analysis were used to detect the presence of membrane attack complex (MAC) and C3 activation products, respectively, in normal and antibody-injected rat eyes., Results: Complement activation product MAC was present in the normal rat eye, and intraocular injection of zymosan induced severe anterior uveitis. The complement regulatory proteins, MCP, DAF, CD59, and Crry, were identified in the normal rat eye. Soluble forms of Crry and CD59 were also detected in normal rat aqueous humor. Severe anterior uveitis developed in Lewis rats injected with a neutralizing mAb against Crry, with increased formation of C3 split products. Systemic complement depletion by CVF prevented the induction of anterior uveitis by anti-Crry mAb. Intracameral injection of anti-rat CD59 (6D1), anti-rat MHC class I antigen (OX-18), anti-rat Ig (G-16-510E3), or MOPC-21 caused no inflammatory reaction., Conclusions: The results suggest that the complement system is continuously active at a low level in the normal eye and is tightly regulated by intraocular complement regulatory proteins.

gC1q receptor, a protein originally described as the cell surface receptor for the globular heads of complement factor C1q, has been found to bind human H-kininogen with high affinity and specificity. Therefore, gC1qR has been considered candidate kininogen docking site on the surfaces of platelets, neutrophils and endothelial cells. Recent work demonstrating that gC1qR is an intracellular protein that is tightly associated with mitochondria rather than targeted to the cell surface has challenged this view. To further probe cellular trafficking routes of gC1qR, we overexpressed human gC1qR in a mammalian cell and monitored cell surface exposure of recombinant gC1qR by virtue of its capacity to bind labeled H-kininogen. Transient transfection of COS1 cells with the full-length cDNA of human gC1qR resulted in a high level of recombinant protein that matched the pool of endogenous gC1qR present in these tells. Overexpression of gC1qR did not significantly increase the number of H-kininogen binding sites exposed by the transfected cells thus denying the possibility that alternative routing of gC1qR to the surface of COS1 cells occurs at significant levels. Hence gC1qR has the capacity to tightly bind H-kininogen, but because gC1qR is routed to mitochondria it cannot fulfill the postulated functions as a cell docking site for kininogens and complement factors.

A protein of 33 kDa (p33) that tightly binds to the globular domains of the first complement component, C1q, is thought to serve as the major C1q receptor (gC1qR) on B cells, neutrophils, and mast cells. However, the cellular routing and the subcellular localization of p33/gC1qR are unknown. We have performed confocal laser-scanning microscopy and found that p33/gC1qR is present in intracellular compartments, where it colocalizes with the mitochondrial marker protein, pyruvate dehydrogenase. No surface staining for p33/gC1qR on endothelial EA.hy926 cells was observed. A fusion protein of the p33/gC1qR presequence with green fluorescent protein translocated to the mitochondria of transfected COS-7 cells. Concomitantly, a 6-kDa portion of the fusion protein was proteolytically removed. The 33 amino-terminal residues of the presequence proved sufficient to direct reporter constructs to mitochondria. Association of p33/gC1qR with mitoplasts indicated that the mature protein of 209 residues resides in the matrix and/or the inner membrane of mitochondria. Immunocytochemistry of fetal mice tissues revealed a ubiquitous expression of p33/gC1qR, most prominently in tissues that are rich in mitochondria. Thus, the candidate complement receptor p33/gC1qR of intact cells cannot interact with plasma C1q due to mutually exclusive localizations of the components. The functional role of p33/gC1qR needs to be reconsidered.

The complement component C1q can interact with a variety of different cells, resulting in multiple functional consequences depending on the cell type. mAbs R3 and R139, which recognize a 126,000 Mr (reduced) cell surface protein, are able to abrogate the C1q-mediated enhancement of monocyte phagocytosis. The cDNA encoding this C1q receptor that modulates phagocytosis, C1qRP, has recently been cloned. Using a DNA probe based on the coding region of the receptor, Northern blot and RT-PCR analysis of RNA isolated from different cell types showed C1qRP expression in cells of myeloid origin and in endothelial cells, but not in cells of lymphoid origin nor in the HeLa epithelial-like cell line or iliac artery smooth muscle cells. FACS analysis of cell surface expression of C1qRP, as detected by mAb R139 and R3, corresponded in all cases to the mRNA levels detected. Using the anti-C1qRP mAb, the 126,000 Mr receptor was also detected in lysates of human platelets. Interestingly, C1qRP is not expressed by the promyelocytic leukemia cell line HL-60, and differentiation of these cells with various chemical compounds did not induce C1qRP expression. It has been reported that C1q can induce specific receptor-mediated responses in fibroblasts. However, RNA and cell surface expression analysis for C1qRP indicate that this particular C1q receptor is not expressed by either human gingival or human skin fibroblasts. These data demonstrate selective expression of C1qRP in specific cell types and support the hypothesis that there is more than one C1q receptor mediating the diverse responses triggered by C1q.

gC1qBP is a 33 kDa glycoprotein that binds to the globular 'heads' of C1q. We have cloned cDNAs encoding the rat and mouse homologues of gC1qBP. Comparison of the cDNA-derived amino acid sequences of gC1qBP reveals that either of the rodent sequences is 89.9% identical to the reported human sequence. Recombinant rat gC1qBP binds avidly to human C1q. gC1qBP mRNA is abundantly expressed in every rat and mouse tissue analysed. Rat mesangial cells synthesise gC1qBP, but do not express gC1qBP on the cell surface. In rat serum, gC1qBP is present at low levels.

The Epstein-Barr virus (EBV) EBNA-1 protein has a central role in the maintenance of a latent EBV infection and is the only virus-encoded protein expressed in all EBV-associated tumors. EBNA-1 is required for replication of the episomal form of the latent viral genome and transactivates the latency C and LMP-1 promoters. The mechanisms by which EBNA-1 performs these functions are not known. Here we describe the cloning, expression, and characterization of a cellular protein, P32/TAP, which strongly interacts with EBNA-1. We show that P32/TAP is expressed at high levels in Raji cells and is synthesized as a proprotein of 282 amino acids (aa) that is posttranslationally processed by a two-step cleavage process to yield a mature protein of 209 aa. It has been previously reported that P32/TAP is expressed on the cell surface. Our transient expression assays detected full-length P32/TAP (1-282 aa) in the cytoplasm while mature P32/TAP protein localized to the nucleus. Three lines of evidence support P32/TAP interaction with EBNA-1. First, in the yeast two-hybrid system we mapped two interactive N-terminal regions of EBNA-1, aa 40-60 and aa 325-376, each of which contains arginine-glycine repeats. These regions interact with the C-terminal half of P32/TAP. Second, the full-length cytoplasmic P32/TAP protein can translocate nuclear EBNA-1 into the cytoplasm. Third, P32/TAP co-immunoprecipitated with EBNA-1. We have confirmed that a Gal4 fusion protein containing the C-terminal region of P32/TAP (aa 244-282) transactivates expression from a reporter containing upstream Gal4-binding sites. Deletion of the P32/TAP interactive regions of EBNA-1 severely diminished EBNA-1 transactivation of FRTKCAT in transient expression assays. Our data suggest that interaction with P32/TAP may contribute to EBNA-1-mediated transactivation., (Copyright 1997 Academic Press.)

Human preimplantation embryos, gametes and cumulus cells were studied for expression of the complement-binding proteins CD46 (membrane cofactor protein), CD55 (decay accelerating factor) and CD59 (membrane attack complex inhibitory factor) as well as complement receptors type 1 (CRI), type 2 (CR2) and type 3 (CR3). Both the CD55 and CD59 glycosyl phosphatidylinositol (GPI)-anchored proteins were expressed by the plasma membrane and zona pellucida of oocytes, early embryos and expanded preimplantation blastocysts; in contrast, CD46 was expressed only on the plasma membrane. Cumulus cells consistently expressed CD46 and, most strongly, CD59 whereas CD55 expression was variable. Monoclonal antibodies (mAbs) to CRI, CR2 and CR3 epitopes gave only occasional reactivity on oocytes and were unreactive with blastocysts, spermatozoa and cumulus cells. CD46 is expressed only on the spermatozoal inner acrosomal membrane, and CD59 on the plasma membrane; CD55 expression was confirmed on the plasma membrane as well as the inner acrosomal membrane. Control mAbs specific for factor H were usually unreactive with gametes, blastocysts and cumulus cells. These data support the concept that gametes and early embryonic cells are protected from complement-mediated attack by expression of CD46, CD55 and CD59, although these complement-binding proteins may have additional roles in reproductive events.

Although previous studies have shown that different cells and cell lines of murine origin bind human C1q, suggesting that they display cell surface receptors for C1q, no information is available to indicate whether mouse or human mast cells express C1q receptors. This paper presents the first evidence to show that murine mast cells express specific receptors for C1q. Western blot analysis of cell membrane proteins prepared from a bone marrow-derived mouse cell line using two monospecific polyclonal Abs, one directed against the 60-kDa C1q receptor (C1q-R) that binds to the collagen-like stalk of C1q (cC1q-R) and the other directed against the 33-kDa molecule that binds to the globular "heads" of C1q (gC1q-R), show that both of these receptors are present on these cells. In addition, C1q can induce mast cell migration in a specific and dose-dependent manner. Interestingly, the C1q-induced migratory response was found to be biphasic; the first response peaked at a C1q concentration of 0.1 nM, whereas the second phase peaked at approximately 40 nM. Checkerboard analysis of the mast cell migratory response to C1q showed that the first phase was primarily due to chemotaxis and the second phase was attributable to chemokinesis. Preincubation of C1q with Abs specific for the collagen-like tail of the molecule abolished both its chemotactic and chemokinetic response, whereas heat inactivation of C1q (56 degrees C, 1 h) resulted in 85% abrogation of the chemotactic phase and 42% reduction in the chemokinetic phase. The observed mast cell migratory responses were mediated by cell surface C1q-R(s), as inclusion of a mixture of anti-C1q-R and anti-gC1q-R Abs with the cells inhibited their migratory response toward C1q. However, incubation of cells with various doses of C1q did not result in histamine release. Furthermore, engagement of mast cell C1q-Rs by the ligand C1q induced an antiproliferative response, as coculturing of mast cells with C1q resulted in a specific and dose-dependent decrease in DNA synthesis. These data suggest that C1q-Rs may play a significant role in mast cell function and regulation by providing an important signal through which mast cells can be recruited to inflammatory sites of increased C1q concentration.

The partial characterization and expression of the C1q receptor (C1q-R) in relation to other complement receptors present on the surface of neutrophils has been examined, as well as the effects of free C1q on cell function. A polyclonal anti-C1q-R antibody recognizes a 68-kD neutrophil surface protein. C1q-R expression was not upregulated upon warming, priming, or exposure to FMLP, but decreased after exposure to phorbol myristate acetate (PMA), because of shedding of the receptor into the extracellular medium, as detected by enzyme-linked immunosorbent assay. CR3 and CR1 expression was upregulated from intracellular pools after cell stimulation by PMA. No evidence of intracellular pools of C1q-R was found, as assessed by immunoblotting of subcellular fractions. But C1q-R appeared to be expressed early in cell differentiation, was detected on undifferentiated HL-60 cells, and like CR3 expression, increased upon 5 days differentiation towards a neutrophil lineage. However, C1q-R expression decreased upon additional culture, whereas CR3 expression continued to increase. A large variation in the percentage of peripheral cells expressing C1q receptors in donors was observed, ranging from 13% to 100%, contrasting with CR3 receptors that exhibited less variability. Interactions between free monomeric C1q and neutrophils were also studied. Incubation of stimulated neutrophils with 10 to 100 micrograms/mL C1q resulted in a further increase in CR3 expression and adherence to albumin-coated surfaces. Staphylococci opsonized with low quantities of C1q (0.1 to 1 microgram/mL) mediated a moderate and sustained respiratory burst in neutrophils, whereas a burst of similar magnitude was generated only with free C1q at concentrations 10- to 100-fold higher. Stimulation was only partially inhibited if cells were first treated with anti-C1q-R antibody, suggesting other C1q binding proteins may be present on the cell surface. In summary, neutrophil C1q receptor is approximately 68-kD, exhibits varying expression on different subjects, and is not upregulated from intracellular stores on exposure to soluble stimuli. Stimulated, but not resting, neutrophils selectively respond to raised levels of free C1q, resulting in altered cell function and enhanced CR3 receptor expression. These studies thus suggest complex roles for C1q in neutrophil function.

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)

C1q receptor (C1qR) expression was determined by immunofluorescence flow cytometry on neutrophils from paired peripheral blood and synovial fluid samples from 21 patients with rheumatoid arthritis (RA) and 13 patients with other articular diseases (OAD). In both patient groups the levels of C1qR on circulating neutrophils were similar to that observed for normal control subjects, whereas on synovial fluid neutrophils significantly higher levels of receptor expression were observed. The mean percentage increases observed were: RA patients 47%, OAD patients 72%. C1q-bearing immune complexes were most prevalent in patients with RA, with the highest concentrations being found in synovial fluid samples. No correlation between immune complex levels and neutrophil C1qR expression was noted. Upregulation of C1qR expression is a feature of activated neutrophils from inflammatory joint fluids.

In injury and inflammation, interactions of complement C1q with C1q receptors may provide attachment sites for cell localization and tissue regeneration. Cultured smooth muscle cells (58%), epithelial cells (26%), and endothelial cells (25%) attach to C1q-coated surfaces, while only 6% of cultured B cells (Raji) attach. Endothelial and Raji cells express C1q receptors, but C1q receptors (C1qR) on smooth muscle cells and epithelial cells have not previously been demonstrated. Evidence is provided that smooth muscle cells express an average of 1.5 x 10(6) C1qR/cell (K alpha = 10(8) M-1) and that epithelial cells express an average of 0.7 x 10(6) C1qR/cell (K alpha = 1.4 x 10(8) M-1). Binding properties of C1qR, and immunoreactivity to anti-C1qR antibodies, are characterized. The antibodies specifically recognize a 67-kDa component of smooth muscle cell lysates and inhibit cell attachment to C1q substrates. We conclude that distribution of C1qR may be ubiquitous; binding properties, size, and antigenicity of various C1qR may be related, but adhesive function may be tissue specific.