Complement fragment C3d is colocalized within the lipid rafts of T cells and promotes cytokine production.

UNLABELLED: The complement system plays an important role in tissue inflammation and damage in SLE patients. High levels of C3d were detected on the surface of erythrocytes and lymphocytes of SLE patients. The objective of this study was to assess the functional consequences of C3d fragments deposited on the surface membrane of SLE T cells. METHODS: 46 SLE patients, 43 patients with other autoimmune diseases (OAD) and 33 healthy individuals (N) were enrolled in this study. T cells were isolated from peripheral blood and flow cytometry studies were conducted to assess the levels of C3d fragments, Ca++ influx responses and cytokine production. Confocal microscopy was used to study co-localized molecules. Student's t-test was performed to determine statistical significance among study groups. RESULTS: A significant percentage of the SLE T cells were found to be positive for C3d (13.58 ± 3.92%) when compared with normal T cells (4.52 ± 2.92%) (p < 0.0000547) and T cells from patients with other autoimmune diseases (6.31 ± 4.57%) (p < 0.00513). Peak Ca++ influx responses were significantly higher in C3d- SLE T cells compared with C3d+ SLE T cells (p < 0.011). C3d+ T cells produced significantly more IL-2, IFN-gamma, IL-4 and IL-17. In contrast to the increased production of IL-2 by the C3d+ T cells, the overall SLE T cell population produced less IL-2 when compared with T cells from normal individuals or patients with other autoimmune disease. The C3d fragments were found to be localized within the lipid rafts. CONCLUSION: C3d fragments are localized in the lipid rafts of SLE T cells and contribute to abnormal T cell function by modulating Ca++ influx responses and increased cytokine production.

Prospective assessment of C4d deposits on circulating cells and renal tissues in lupus nephritis: a pilot study.

Complement activation plays a key role in the pathogenesis of lupus nephritis (LN), a severe complication of systemic lupus erythematosus (SLE). We prospectively evaluated 15 LN subjects and two control groups: 13 non-SLE renal subjects (control A) and 239 SLE subjects without LN (control B). All had C4d levels on circulating erythrocytes (E-C4d), reticulocytes (R-C4d) and platelets (P-C4d) measured by flow cytometry, while C4d deposition in renal tissue was semiquantitatively assessed in LN subjects and control A using immunoperoxidase staining. Compared with control A, LN biopsies had higher glomerular-C4d scores (p = 0.003), which were associated with more frequent granular glomerular immunofluorescence staining and electron dense deposits (p < 0.001). Compared with control A and B groups, LN subjects had higher E-C4d (p = 0.002 and p = 0.005) and R-C4d levels (p = 0.002 and p = 0.008), respectively. LN subjects were more likely to have P-C4d compared with control A (p = 0.016). In LN, only E-C4d correlated with National Institutes of Health (NIH) activity index (r = 0.55, p = 0.04). In conclusion, LN biopsies showed frequent glomerular-C4d staining associated with immune complex deposits. LN subjects had higher E-C4d and R-C4d levels compared with both control groups. E-C4d levels also correlated with NIH activity index. These findings suggest a potential role of C4d on circulating cells as a biomarker for lupus nephritis.

Experimental system for ex vivo measurement of murine aortic stiffness.

While vascular stiffness is universally studied using pulse wave velocity, this method overestimates the stiffness of small calibre blood vessels. We have developed and rigorously validated an ex vivo system for measuring stiffness of the mouse aorta. The system consists of a temperature-controlled tissue bath, a pressurization loop and a helium-neon laser micrometer. We harvested thoracic aortas from 8 (n = 56), 11 (n = 6) and 14 (n = 6) week male C57BL/6J mice, mounted them within a tissue chamber and applied an intraluminal pressure waveform while measuring mid vessel outer diameter. Vessel stiffness (E(p), mmHg) was calculated from the pressure-diameter response. Vessels were then stained for endothelial cells, smooth muscle cells, elastin fibres and collagen. The data indicate highly reproducible stiffness measurements in 8 week mice (E(p) = 602.4 +/- 160.2; p = 0.934), age-related stiffening between 11 and 14 week mice (11 week E(p) = 646.9 +/- 62.4, 14 week E(p) = 795.4 +/- 87.5, p = 0.008), and a morphologically intact vessel wall. These results represent the first ex vivo measurements of murine aortic stiffness and illustrate that our methods are feasible and reliable. Since we demonstrate that the system is sensitive to age-related stiffening and does not damage the vessel, this approach is useful for investigating the pathophysiology of vascular disease from biomechanical and histological perspectives.

New insights into complement: a mediator of injury and marker of disease activity in systemic lupus erythematosus.

Studies performed during the past several decades have demonstrated a role for the complement system in both the etiology and pathogenesis of systemic lupus erythematosus (SLE). However the specifically defective molecular and cellular pathways responsible for the disease and its complications have generally not been identified. In this report, we describe two recent advances in complement pathobiology that highlight future directions for promising investigation toward enhancing our capacity to diagnose SLE, to monitor activity of the disease, and to identify molecular and cellular defects in SLE that can be targeted by therapeutic inhibitors of complement activation. In the first example, we describe recently developed assays to detect erythrocyte C4d and complement receptor 1 for diagnosis and monitoring of disease activity in SLE. In the second example, we describe a recently discovered role for complement in mediating fetal loss in antiphospholipid syndrome and discuss the potential for this observation to facilitate identification and development of complement based biomarkers to predict poor fetal outcome in pregnant patients with SLE. These two examples are meant to underscore the importance of complement in the etiology and pathogenesis of SLE and its complications, and to stress the need for further investigation focused on the link between the complement system and SLE.

Apoptosis of skeletal muscle cells and the pathogenesis of myositis: a perspective.

Apoptosis is a genetically controlled form of cell death that occurs in many biologic processes including embryogenesis, immune cell development, and maintenance of peripheral immune tolerance. Recent studies have yielded evidence suggesting that apoptosis of parenchymal cells may play a role in providing self-antigens to initiate autoimmune reactions. Skeletal muscle cells are fully differentiated and multinucleated. Apoptosis has been described in developing myoblasts and, recently, in mature myotubes. However, the involvement of apoptosis in skeletal muscle pathologies is unclear. This article reviews the available data concerning the occurrence of skeletal muscle cell apoptosis in selected muscle diseases. It also discusses the potential role of muscle cell apoptosis in the development of autoimmune diseases such as idiopathic inflammatory myopathies.

Apoptosis, clearance mechanisms, and the development of systemic lupus erythematosus.

Cell death by apoptosis is an integral part of many biologic processes, including embryonic development, T- and B-cell selection, the elimination of potentially autoreactive lymphocytes in the periphery, and maintenance of lymphocyte homeostasis through activation-induced cell death. There is also increasing evidence that apoptosis may maintain immune tolerance and that it may be the process that generates the self antigens responsible for the initial development of autoimmunity. This review discusses some of the biochemical steps involved in the apoptotic process, how potentially immunogenic self antigens are generated during apoptosis, and the mechanisms by which the products of apoptosis are cleared and processed to avoid breaking immune tolerance.

Spontaneous conception in the presence of stage IIIC endometrioid ovarian cancer.

OBJECTIVE: To describe a rare case of spontaneous conception in a patient with a preexisting metastatic ovarian cancer. DESIGN: Case report. SETTING: University hospital. PATIENT(S): A 39-year-old Asian woman who conceived while undergoing an evaluation for primary infertility and newly detected bilateral adnexal masses. INTERVENTION(S): Staging laparotomy and total abdominal hysterectomy and bilateral salpingo-oophorectomy. MAIN OUTCOME MEASURE(S): Anatomic pathology diagnosis. RESULT(S): Blighted ovum and stage IIIC endometrioid adenocarcinoma of ovary. CONCLUSION(S): Metastatic ovarian cancer does not prevent either spontaneous ovulation or spontaneous conception.

The globular heads of C1q specifically recognize surface blebs of apoptotic vascular endothelial cells.

Complement protein C1q is required to maintain immune tolerance. The molecular mechanism responsible for this link has not been determined. We have previously demonstrated that C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes, suggesting that it may participate in clearance of self Ags generated during programmed cell death. Here, we demonstrate that C1q also binds directly to apoptotic blebs of vascular endothelial cells and PBMC. These apoptotic cells are recognized by the globular heads of C1q, which bind specifically to the surface blebs, and deposition increases as the blebs mature on the cell surface. These observations suggest that C1q may participate in the clearance of apoptotic cells from the circulation and from the walls of the vascular lumen. The interaction of surface blebs with the globular heads of C1q suggests that surface blebs may be capable of directly activating the classical pathway of complement under certain circumstances, generating C4- and C3-derived ligands for receptors such as CR1, CR2, CR3, and CR4. Appropriate recognition of apoptotic cells by C1q and targeted clearance of the molecular contents of surface blebs to complement receptors may be critical for the maintenance of immune tolerance.

Apoptosis and autoimmunity: complement deficiency and systemic lupus erythematosus revisited.

Apoptosis may have a dual role in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus. First, this process may be integral in the clonal deletion of self-reactive lymphocytes and maintenance of peripheral tolerance. Second, apoptosis generates altered self-antigens with the potential for breaking self-tolerance. This review will discuss these two aspects of apoptosis and autoimmunity, and explore the potential role of the classical complement pathway in this context.

The structural basis for complement receptor type 2 (CR2, CD21)-mediated alternative pathway activation of complement: studies with CR2 deletion mutants and vaccinia virus complement-control protein-CR2 chimeras.

The role of complement receptor 2 (CR2) short consensus repeats (SCR) in binding of hydrolyzed C3 (iC3) to form an alternative pathway (AP) convertase, and promoting C3 fragment deposition following AP activation, was examined. We used (1) K562 cells transfected with CR2 constructs, where the C3d-binding site of CR2 (SCR1+2) was replaced with the four-SCR vaccinia virus complement control protein (VCP), or truncation mutants thereof, and (2) COS cells transfected with wild-type (wt) CR2, or deletion mutants thereof. AP activation required iC3 binding in both systems. Thus, the VCP-CR2 chimera had an iC3 binding efficiency of 11.4 %, compared to wtCR2, and a relative AP activity of 5.5 %, the truncation mutants being inactive. Of the CR2 mutants, only EK (DeltaSCR10 - 11) had AP activity similar to wtCR2. NN (DeltaSCR6 - 8) and NOP (DeltaSCR6-mid14) had reduced AP activity, but near normal iC3 binding. XB (DeltaSCR3 - 6) and PP (DeltaSCR3-mid14) were inactive in both assays. We conclude that, whilst iC3 binding to CR2 via SCR1 - 4 is essential for AP activation, the efficiency of C3 deposition also depends on the midportion of CR2.

Functional characterization of soluble and membrane-bound forms of vaccinia virus complement control protein (VCP).

Vaccinia virus secretes a 35 kD protein, vaccinia virus complement control protein (VCP), that inhibits the classical and alternative pathways of complement at several points, indicating that it may be a viral analogue of human complement receptor type 1 (CR1; CD35). Structurally, however, CR1 is composed of 30 short consensus repeats (SCRs), whereas VCP consists entirely of four SCRs. We have begun a structure-function analysis of VCP to define the minimum number of SCRs necessary for function, the functional differences between VCP and CR1, and the potential therapeutic roles for VCP. We addressed these questions by creating and characterizing recombinant soluble and membrane-bound forms of VCP. We have determined that (1) VCP requires all four SCRs to bind C3b, (2) whereas CR1 binds C3b and iC3b, VCP binds C3b but not iC3b, and (3) although normally secreted, if expressed on the membrane of mammalian cells, VCP effectively protects the cells from complement-mediated lysis. Thus, VCP appears to be a compact and unique complement regulatory protein with the ability to inhibit both arms of the complement cascade, but lacking affinity for iC3b. By releasing rather than capturing iC3b-bearing complexes following inactivation of C3b, VCP may 'recycle' its active site locally among infected cells, and thereby enable the virus to evade more efficiently host immune and inflammatory responses. The unique function, compact structure, and capacity of VCP to protect mammalian cells from complement-mediated attack, suggests that it could be used both to better understand the structure-function relationship of complement regulatory proteins, in general, and also to rationally design and develop novel therapeutic agents.

Systemic lupus erythematosus and complement deficiency: clues to a novel role for the classical complement pathway in the maintenance of immune tolerance.

Complete deficiency of C1q, the first component of the classical pathway of complement activation, is almost invariably associated with the development of systemic lupus erythematosus. Understanding why complement deficiency results in the specific autoimmune phenotype of SLE may provide valuable clues to the role of complement in the maintenance of immune tolerance. The following review will focus on the characteristics of complement-deficient SLE and the experimental evidence in support of our hypothesis that C1q may critically influence the immune response to self-antigen contained within surface blebs generated by apoptotic cells.

Viral complement regulatory proteins.

The inactivation of complement provides cells and tissues critical protection from complement-mediated attack and decreases the associated recruitment of other inflammatory mediators. In an attempt to evade the host immune response, viruses have evolved two mechanisms to acquire complement regulatory proteins. They can directly seize the host cell complement regulators onto their outer envelope and/or they can produce their own proteins which are either secreted into the neighboring intercellular space or expressed as membrane-bound proteins on the infected host cell. The following review will concentrate on the viral homologues of the mammalian complement regulatory proteins, specifically those containing complement control protein (CCP) repeats.

C1q binds directly and specifically to surface blebs of apoptotic human keratinocytes: complement deficiency and systemic lupus erythematosus revisited.

Complete deficiency of C1q is almost invariably associated with the development of systemic lupus erythematosus. It has been suggested that this association may result from a generalized failure to clear Ag-Ab complexes. However, it has not been demonstrated how such a broad impairment results in this specific and consistent autoimmune phenotype, in which photosensitive skin disease is the most prominent manifestation. We believe there is another role for the classical pathway in maintaining immune tolerance. Surface blebs of apoptotic keratinocytes are concentrated sources of autoantigens, and these packages may define a novel immune context and challenge self-tolerance if not properly cleared and processed. We demonstrate here that when human keratinocytes are rendered apoptotic, they also develop the capacity to specifically and directly bind to C1q in the absence of Ab. C1q may mediate Ab-independent clearance of apoptotic keratinocytes, and prevent immunization with autoantigens of cutaneous origin.

Mechanism of suppression of cell-mediated immunity by measles virus.

The mechanisms underlying the profound suppression of cell-mediated immunity (CMI) accompanying measles are unclear. Interleukin-12 (IL-12), derived principally from monocytes and macrophages, is critical for the generation of CMI. Measles virus (MV) infection of primary human monocytes specifically down-regulated IL-12 production. Cross-linking of CD46, a complement regulatory protein that is the cellular receptor for MV, with antibody or with the complement activation product C3b similarly inhibited monocyte IL-12 production, providing a plausible mechanism for MV-induced immunosuppression. CD46 provides a regulatory link between the complement system and cellular immune responses.

Antibody response to a T-dependent antigen requires B cell expression of complement receptors.

Several lines of evidence indicate that antibody responses to T-dependent antigens require complement receptors expressed on either B lymphocytes or follicular dendritic cells. We have used RAG-2 deficient blastocyst complementation to create mice specifically lacking B cell complement receptors. Despite normal expression of complement receptor 1 (CR1[CD35]) and CR2 (CD21) on follicular dendritic cells, these mice have a profound defect in their capacity to mount a T-dependent antibody response. This is the first direct demonstration in vivo that B cell expression of complement receptors is required for a humoral immune response. This is the first direct demonstration in vivo that B cell expression of complement receptors is required for a humoral immune response. This suggests that CD21 and/or CD35 on B lymphocytes may be required for cellular activation, adsorptive endocytosis of antigen, recruitment to germinal centers, and/or protection from apoptosis during the humoral response to T-dependent antigens.

Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B cell response to T-dependent antigen.

Covalent attachment of activated products of the third component of complement to antigen enhances its immunogenicity, but the mechanism is not clear. This effect is mediated by specific receptors, mCR1 (CD35) and mCR2 (CD21), expressed primarily on B cells and follicular dendritic cells in mice. To dissect the role of mCR1 and mCR2 in the humoral response, we have disrupted the Cr2 locus to generate mice deficient in both receptors. The deficient mice (Cr2-/-) were found to have a reduction in the CD5+ population of peritoneal B-1 cells, although their serum IgM levels were within the range of normal mice. Moreover, Cr2-/- mice had a severe defect in their humoral response to T-dependent antigens that was characterized by a reduction in serum antibody titers and in the number and size of germinal centers within splenic follicles. Reconstitution of the deficient mice with bone marrow from MHC-matched Cr2+/+ donors corrected the defect, demonstrating that the defect was due to B cells themselves. These results indicate an obligatory role of B cell complement receptors in responses of the B cells to protein antigens.

Novel packages of viral and self-antigens are generated during apoptosis.

Immune context is an essential determinant of the host response to potential autoantigens. The clustering of the autoantigens targeted in systemic lupus erythematosus within surface blebs of apoptotic cells generates high concentrations of autoantigen within discrete subcellular packages. We demonstrate here that when apoptosis is induced by Sindbis virus infection, viral antigens and autoantigens cocluster exclusively in small surface blebs of apoptotic cells. The surface of these blebs is rich in viral glycoproteins, and virions can be seen blebbing from their surface. We propose that these blebs of mixed foreign and self-origin define a novel immune context that may challenge self-tolerance.

Constitutional trisomy 8 mosaicism and gestational trophoblastic disease.

Concurrence of congenital trisomy 8 mosaicism and gestational trophoblastic disease in a 42-year-old gravida IV, para IV female is described in the present report. In contrast to other cases described in the literature, our patient had no known additional confounding chromosomal abnormalities other than trisomy 8. The finding of trisomy 8 mosaicism in yet another type of cancer provides further support for the hypothesis of an increased predisposition to cancer in tissues with constitutional genomic imbalance, which can manifest itself as numerical chromosomal abnormalities (e.g., trisomies) or structural chromosomal abnormalities (e.g., translocations). To the best of our knowledge, this is the only report in the English literature of constitutional trisomy 8 mosaicism associated with gestational trophoblastic disease, a rare gynecologic disease entity in itself.