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.

Objective. C4-derived activation fragments are the only complement ligands present on the surfaces of normal erythrocytes. The significance of this observation is unknown, and the role of erythrocyte-bound C4 (E-C4) in human disease has not been explored. More than any other human disease, the pathogenesis of systemic lupus erythematosus (SLE) has been characterized by defects in clearance of complement-bearing immune complexes via erythrocytes expressing complement receptor 1 (CR1). This study was undertaken to determine whether these functional defects might be reflected by abnormal patterns of E-C4 and E-CR1 expression on erythrocytes of patients with SLE.Methods. We conducted a cross-sectional study of 100 patients with SLE, 133 patients with other diseases, and 84 healthy controls. Erythrocytes were characterized by indirect immunofluorescence and by flow cytometry for determination of levels of C4d and CR1.Results. Patients with SLE had higher levels of E-C4d and lower levels of E-CR1 than did patients with other diseases (P < 0.001) or healthy controls (P < 0.001). The test was 81% sensitive and 91% specific for SLE versus healthy controls and 72% sensitive and 79% specific for SLE versus other diseases, and it had an overall negative predictive value of 92%.Conclusion. This is the first report of abnormal levels of E-C4d in human disease. We found that abnormally high levels of E-C4d and low levels of E-CR1 are characteristic of SLE, and combined measurement of the 2 molecules has high diagnostic sensitivity and specificity for lupus. Determination of E-C4d/E-CR1 levels may be a useful addition to current tests and criteria for SLE diagnosis.It has been known for decades that proteolytic fragments of complement component C4, generated during activation of the classical pathway, are present on surfaces of normal erythrocytes. C4 is a class III HLA molecule, and its polymorphism is responsible for the Rogers and Chido blood group antigens; however, the physiologic significance of erythrocyte-bound C4 (E-C4) is entirely unknown. Although there has been extensive study of serum C4 levels in human disease, abnormalities of E-C4 and the potential role of E-C4 molecules during inflammatory and immune responses have not been investigated.A large body of evidence accumulated over several decades has demonstrated that abnormalities in complement activation and clearance of immune complexes by erythrocytes are fundamental to the pathogenesis of systemic lupus erythematosus (SLE) (1,2). These include reduced levels of CR1 on erythrocytes, deficiencies in components of the classical pathway including C4, and saturation of CR1 by preexisting immune complexes (3-12). These observations led to our hypothesis that abnormalities in complement activation specific to SLE may be reflected by molecular events involving both receptors and ligands on erythrocyte surfaces and that

Objective. Complement-activation product C4d is deposited on normal erythrocytes, while abnormal levels have been observed on the surface of erythrocytes of patients with systemic lupus erythematosus (SLE). This study examines whether C4d also deposits on human platelet surfaces, and whether platelet-bound C4d may provide a biomarker for SLE.Methods. We conducted a cross-sectional study of 105 patients with SLE, 115 patients with other diseases, and 100 healthy controls. Levels of C4d on the surface of platelets were examined by flow cytometry and scanning confocal microscopy. Statistical analyses were performed to determine the clinical variables associated with platelet C4d.Results. Abnormal levels of platelet C4d were found to be highly specific for SLE. Platelet C4d was detected in 18% of patients with SLE, being 100% specific for a diagnosis of SLE compared with healthy controls and 98% specific for SLE compared with patients with other diseases (P < 0.0001). In addition, platelet C4d was significantly associated with positivity for lupus anticoagulant (P < 0.0001) and anticardiolipin antibodies of the IgG (P ‫؍‬ 0.035) or the IgM (P ‫؍‬ 0.016) isotype. Platelet C4d was also significantly associated with SLE disease activity according to the SLE Disease Activity Index (P ‫؍‬ 0.039), low serum C4 (P ‫؍‬ 0.046), an elevated erythrocyte sedimentation rate (P ‫؍‬ 0.006), and abnormal levels of C4d on erythrocytes (P < 0.0001).Conclusion. This observation suggests that platelet-bound C4d may be a useful biomarker for SLE and may be a clue to the pathogenic mechanisms responsible for the myriad thrombotic and vascular complications of lupus associated with antiphospholipid antibodies.

Objective-Disease activity in systemic lupus erythematosus (SLE) is typically monitored by measuring serum C3 and C4. However, these proteins have limited utility as lupus biomarkers, because they are substrates rather than products of complement activation. The aim of this study was to evaluate the utility of measuring the erythrocyte-bound complement activation products, erythrocyte-bound C3d (E-C3d) and E-C4d, compared with that of serum C3 and C4 for monitoring disease activity in patients with SLE.Methods-The levels of E-C3d and E-C4d were measured by flow cytometry in 157 patients with SLE, 290 patients with other diseases, and 256 healthy individuals. The patients with SLE were followed up longitudinally. Disease activity was measured at each visit, using the validated Systemic Lupus Activity Measure (SLAM) and the Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA) version of the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI).Results-At baseline, patients with SLE had higher median levels of E-C3d and E-C4d (P < 0.0001) in addition to higher within-patient and between-patient variability in both E-C3d and EC4d when compared with the 2 non-SLE groups. In a longitudinal analysis of patients with SLE, E-C3d, E-C4d, serum C3, and anti-double-stranded DNA (anti-dsDNA) antibodies were each significantly associated with the SLAM and SELENA-SLEDAI. In a multivariable analysis, E-© 2010, American College of Rheumatology Address correspondence and reprint requests to Amy H. Kao, MD, MPH, S721 Biomedical Science Tower, 3500 Terrace Street, Pittsburgh, PA 15261. ahk7@pitt.edu. Ms Navratil has received royalties (less than $10,000) for the cell-bound C4d/C3d assay licensed to Cypress Bioscience, Inc. Dr. Hawkins has received consulting fees from Cellatope, Inc. (more than $10,000). Dr. Ahearn has received consulting fees, speaking fees, and/or honoraria from Cellatope, Inc. (more than $10,000) and has received licensing fees from Cypress Bioscience, Inc. Dr. Manzi has received consulting fees, speaking fees, and/or honoraria from Cellatope, Inc. (more than $10,000) and has received licensing fees from Cypress Bioscience, Inc. AUTHOR CONTRIBUTIONSAll authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Kao had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. C4d remained significantly associated with these SLE activity measures after adjusting for serum C3, C4, and anti-dsDNA antibodies; however, E-C3d was associated with the SLAM but not with the SELENA-SLEDAI.Conclusion-Determining the levels of the erythrocyte-bound complement activation products, especially E-C4d, is an informative measure of SLE disease activity as compared with assessing serum C4 levels and should be considered for monitoring disease activity in patients with SLE. In lieu of more useful lupus biomar...