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.

Determination of the structural basis for selective binding of Epstein-Barr virus to human complement receptor type 2.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus that selectively infects and immortalizes human B lymphocytes. One determinant of this narrow tropism is human CR2, the only viral receptor within the superfamily of proteins that contain short consensus repeats (SCRs). Human CR2 serves as a receptor for both C3dg and the gp350/220 glycoprotein of EBV, and binds the monoclonal antibody (mAb) OKB7, which blocks binding of both ligands to the receptor. In contrast, although murine CR2 is capable of binding human C3dg and this interaction can be blocked with the mAb 7G6, it does not bind OKB7 or EBV. We have determined the structural basis for absolute specificity of EBV for human CR2 through characterization of a panel of 24 human-murine chimeric receptors, all of which bind human C3dg. The results indicate that preferential binding of EBV to human CR2 is not due to unique amino acids that are capable of binding the virus, but reflects a distinct receptor conformation that can be achieved in murine CR2 with single amino acid substitutions in two discontinuous regions of the primary structure: replacement of proline at position 15 with the corresponding serine from human CR2, and elimination of a potential N-linked glycosylation site between SCR-1 and SCR-2. Furthermore, species-specific binding of EBV, OKB7, and 7G6 can all be manipulated through substitutions among residues 8-15, suggesting that this octapeptide is part of a structural determinant that is critical for binding of both viral and natural ligands to CR2.

Molecular interactions of complement receptors on B lymphocytes: a CR1/CR2 complex distinct from the CR2/CD19 complex.

The complement system augments the humoral immune response to low concentrations of antigen. This effect may be partly mediated by complement receptors on the surface of B lymphocytes that bind immunogenic complexes bearing fragments of C3 and C4. We have shown by immunoprecipitation analysis that the two complement receptors expressed by B lymphocytes, complement receptor 1 (CR1) and CR2, form a detergent-sensitive complex on the surface of tonsillar B lymphocytes and on K562 erythroleukemia cells that were co-transfected with cDNAs encoding CR1 and CR2. The CR1/CR2 complex is distinct from the CR2/CD19 complex and may assist B cell activation by efficiently capturing C3b-containing immunogens and maintaining such immunogens on the B cell after CR1 and factor I-mediated cleavage to iC3b and C3dg. The complement activating immunogen may then trigger signal transduction by the CR1/CR2 complex, the CR2/CD19 complex, or membrane immunoglobulin.

Functional dissection of the CD21/CD19/TAPA-1/Leu-13 complex of B lymphocytes.

The CD21/CD19/TAPA-1 complex of B lymphocytes amplifies signal transduction through membrane immunoglobulin (mIg), recruits phosphatidylinositol 3-kinase (PI3-kinase), and induces homotypic cellular aggregation. The complex is unique among known membrane protein complexes of the immune system because its components represent different protein families, and can be expressed individually. By constructing chimeric molecules replacing the extracellular, transmembrane, and cytoplasmic regions of CD19 and CD21 with those of HLA-A2 and CD4, we have determined that CD19 and TAPA-1 interact through their extracellular domains, CD19 and CD21 through their extracellular and transmembrane domains, and, in a separate complex, CD21 and CD35 through their extracellular domains. A chimeric form of CD19 that does not interact with CD21 or TAPA-1 was expressed in Daudi B lymphoblastoid cells and was shown to replicate two functions of wild-type CD19 contained within the complex: synergistic interaction with mIgM to increase intracellular free calcium and tyrosine phosphorylation and association with the p85 subunit of PI3-kinase after ligation of mIgM. The chimeric CD19 lacked the capacity of the wild-type CD19 to induce homotypic cellular aggregation, a function of the complex that can be ascribed to the TAPA-1 component. The CD21/CD19/TAPA-1 complex brings together independently functioning subunits to enable the B cell to respond to low concentrations of antigen.

Mapping of the C3b-binding site of CR1 and construction of a (CR1)2-F(ab')2 chimeric complement inhibitor.

CR1/CR2 chimeric receptors in which various short consensus repeats (SCRs) of CR1 were attached to CR2 were transiently expressed on COS cells, and assessed for the binding of polymerized C3b (pC3b) and anti-CR2 by immunofluorescence. Of COS cells expressing chimeras containing SCR 1-4, 1-3, 2-4, 1-2, and 2-3 of the long homologous repeats (LHRs) -B or -C, 96%, 66%, 23%, 0%, and 0%, respectively, bound pC3b. K562 cells were stably transfected with wild-type CR1, deletion mutants of CR1, and the CR1/CR2 chimeras, respectively, and assayed for binding of 125I-pC3b. The dissociation constants (Kd) for pC3b of wild-type CR1 and the LHR-BD and -CD constructs were in the range of 1.0-2.7 nM, and of the CR1/CR2 chimeras containing SCRs 1-4, 1-3, and 2-4 of LHR-B or -C were 1.8-2.4, 6-9, and 22-36 nM, respectively. The factor I-cofactor function of the CR1/CR2 chimeras paralleled the C3b-binding function of the constructs. A CR1/immunoglobulin (Ig) chimeric protein was prepared by fusing SCRs 1-4 of LHR-B to the heavy chains of a murine F(ab')2 anti-nitrophenacetyl (NP) monoclonal antibody. The (CR1)2-F(ab')2 chimera, which retained its specificity for NP, was as effective as soluble, full-length CR1 in binding pC3b, serving as a cofactor for factor I-mediated cleavage of C3b, and inhibiting activation of the alternative pathway, indicating that the bivalent expression of these SCRs reconstitutes the alternative pathway inhibitory function of CR1. The feasibility of creating CR1/Ig chimeras makes possible a new strategy of targeting complement inhibition by the use of Ig fusion partners having particular antigenic specificities.

Mapping of the Epstein-Barr virus and C3dg binding sites to a common domain on complement receptor type 2.

Complement receptor type 2 (CR2;CD21), a member of the superfamily of proteins containing short consensus repeats (SCRs), is the B cell receptor for both the gp350/220 envelope protein of Epstein-Barr virus (EBV), and for the C3dg protein of complement. By analysis of CR2 deletion mutants and chimeras formed with CR1 (CD35) we determined that of the 15 SCRs in CR2, the NH2-terminal two SCRs are necessary and sufficient to bind both gp350/220 and C3dg with affinities equivalent to those of the wild-type receptor. The epitope for OKB-7, a mAb that blocks binding of both EBV and C3dg and shares with these ligands B cell-activating capabilities, also requires both SCR-1 and SCR-2, whereas mAbs lacking these functions bind to other SCRs. Thus, EBV, a polyclonal activator of B cells, has selected a site that is proximate or identical to the natural ligand binding site in CR2, perhaps reflecting the relative immutability of that site as well as its signal transducing function.

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.

Suppression of the immune response by a soluble complement receptor of B lymphocytes.

The CD19-CR2 complex of B lymphocytes contains proteins that participate in two host-defense systems, the immune and complement systems. The ligand for the subunit of the immune system, CD19, is not known, but the complement receptor subunit, CR2 (CD21), binds activation fragments of the C3 component of the complement system and may mediate immunopotentiating effects of complement. A recombinant, soluble CR2 was prepared by fusing the C3-binding region of the receptor to immunoglobulin G1 (IgG1). The (CR2)2-IgG1 chimera competed with cellular CR2 for C3 binding and suppressed the antibody response to a T cell-dependent antigen when administered to mice at the time of immunization. This inhibitory effect of (CR2)2-IgG1 demonstrates the B cell-activating function of the CD19-CR2 complex and suggests a new method for humoral immunosuppression.

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.

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.

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: a review of clinico-laboratory features and immunogenetic markers in 150 patients with emphasis on demographic subsets.

Clinical and laboratory features as well as immunogenetic markers were analyzed in 150 patients with SLE to determine if demographic factors--age at diagnosis, sex and race--influenced the expression of disease. The overall series included 103 white females, 35 black females, 10 white males and 2 black males; the mean age at diagnosis was 32.5 years. Males had a significantly older mean age at diagnosis than females (40.4 versus 31.8 years) and a significantly higher frequency of peripheral neuropathy (50% versus 18.8%). No other differences in clinical or laboratory features or HLA-DR or DQ phenotype frequencies were noted. Blacks had a significant younger mean age at diagnosis than whites (26.9 versus 33.4 years) as well as significantly higher frequencies of nephritis, hypertension, acute lupus pneumonitis, discoid rash, hyperglobulinemia and hypocomplementemia. There were no differences in autoantibody frequencies between race-specific subgroups. HLA-DR2, DRw52 and DQ1 were significantly associated with SLE in whites compared to controls; no HLA-DR or DQ associations were found with SLE in blacks. In whites, HLA-DR2 was associated with the presence of anti-Ro(SS-A) antibody while HLA-DR3 was associated with the presence of both anti-Ro(SS-A) and anti-La(SS-B) antibody. In blacks, HLA-DR2 was associated with the presence of anti-nDNA antibody. In whites, patients with late-onset SLE (age at diagnosis greater than or equal to 50 years) had significantly lower frequencies of nephritis and mesenteric vasculitis but, on the other hand, a higher frequency of secondary Sjögren syndrome than patients with age at diagnosis less than or equal to 22 years. Similar findings were noted when blacks aged 35 and above were compared to those aged 17 and below at diagnosis. In whites, the frequency of both anti-Ro(SS-A) and La(SS-B) antibodies increased with increasing age as did that of HLA-DR3; HLA-DR2, however, was more frequent in those with younger age at diagnosis. These data suggest the existence of two serologic-genetic subsets of SLE with different age at diagnosis.

Epidemiology and genetics of ankylosing spondylitis.

The spondyloarthropathies represent a group of disorders characterized by the presence of inflammatory peripheral and axial arthritis with sacroiliac involvement and a tendency to familial aggregation; prototype clinical syndromes are ankylosing spondylitis (AS) and Reiter's syndrome. The descriptive epidemiology of AS, including course and prognosis in patients cohorts, is reviewed. Particular attention is focused on the genetic epidemiology of AS, especially the evidence supporting causal association of disease with the class I histocompatibility antigen HLA-B27. In addition, analytical studies that support associations of infectious agents are reviewed. The spondyloarthropathies, especially AS and Reiter's syndrome, clearly illustrate the epidemiologic concept of host-environment interaction in the development and expression of disease.

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.

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.

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.

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.