Joseph M. Ahearn

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.

Cardiovascular disease in autoimmune rheumatic diseases

Various autoimmune rheumatic diseases (ARDs), including rheumatoid arthritis, spondyloarthritis, vasculitis and systemic lupus erythematosus, are associated with premature atherosclerosis. However, premature atherosclerosis has not been uniformly observed in systemic sclerosis. Furthermore, although experimental models of atherosclerosis support the role of antiphospholipid antibodies in atherosclerosis, there is no clear evidence of premature atherosclerosis in antiphospholipid syndrome (APA). Ischemic events in APA are more likely to be caused by pro-thrombotic state than by enhanced atherosclerosis. Cardiovascular disease (CVD) in ARDs is caused by traditional and non-traditional risk factors. Besides other factors, inflammation and immunologic abnormalities, the quantity and quality of lipoproteins, hypertension, insulin resistance/hyperglycemia, obesity and underweight, presence of platelets bearing complement protein C4d, reduced number and function of endothelial progenitor cells, apoptosis of endothelial cells, epigenetic mechanisms, renal disease, periodontal disease, depression, hyperuricemia, hypothyroidism, sleep apnea and vitamin D deficiency may contribute to the premature CVD. Although most research has focused on systemic inflammation, vascular inflammation may play a crucial role in the premature CVD in ARDs. It may be involved in the development and destabilization of both atherosclerotic lesions and of aortic aneurysms (a known complication of ARDs). Inflammation in subintimal vascular and perivascular layers appears to frequently occur in CVD, with a higher frequency in ARD than in non-ARD patients. It is possible that this inflammation is caused by infections and/or autoimmunity, which might have consequences for treatment. Importantly, drugs targeting immunologic factors participating in the subintimal inflammation (e.g., T- and B-cells) might have a protective effect on CVD. Interestingly, vasa vasorum and cardiovascular adipose tissue may play an important role in atherogenesis. Inflammation and complement depositions in the vessel wall are likely to contribute to vascular stiffness. Based on biopsy findings, also inflammation in the myocardium and small vessels may contribute to premature CVD in ARDs (cardiac ischemia and heart failure). There is an enormous need for an improved CVD prevention in ARDs. Studies examining the effect of DMARDs/biologics on vascular inflammation and CV risk are warranted.

Biomarkers for systemic lupus erythematosus

The urgent need for lupus biomarkers was demonstrated in September 2011 during a Workshop sponsored by the Food and Drug Administration: Potential Biomarkers Predictive of Disease Flare. After 2 days of discussion and more than 2 dozen presentations from thought leaders in both industry and academia, it became apparent that highly sought biomarkers to predict lupus flare have not yet been identified. Even short of the elusive biomarker of flare, few biomarkers for systemic lupus erythematosus (SLE) diagnosis, monitoring, and stratification have been validated and employed for making clinical decisions. This lack of reliable, specific biomarkers for SLE hampers proper clinical management of patients with SLE and impedes development of new lupus therapeutics. As such, the intensity of investigation to identify lupus biomarkers is climbing a steep trajectory, lending cautious optimism that a validated panel of biomarkers for lupus diagnosis, monitoring, stratification, and prediction of flare may soon be in hand.

Biomarkers for systemic lupus erythematosus: a review and perspective.

Purpose of reviewDespite decades of extensive work in the understanding of the etiopathogenesis of systemic lupus erythematosus, few biomarkers have been validated and widely accepted for this disease. The lack of reliable, specific biomarkers not only hampers clinical management of systemic lupus erythematosus but also impedes development of new therapeutic agents. This paper reviews briefly the historical aspects of systemic lupus erythematosus biomarkers and summarizes recent studies on candidate biomarkers. Recent findingsRecognizing the urgent need for lupus biomarkers, a Lupus Biomarker Working Group has recently been initiated to facilitate collaborative efforts aimed at identifying and validating biomarkers for systemic lupus erythematosus. Based on available data, several laboratory markers have shown promise as biomarkers for susceptibility, diagnosis, and disease activity. These include Fc receptor genes (disease susceptibility), complement C4d-bound erythrocytes (diagnosis or disease activity), CD27high plasma cells (disease activity), ‘interferon signature’ (disease activity), and anti-C1q antibodies (disease activity and organ involvement). SummaryThere is a longstanding and recently rejuvenated enthusiasm for biomarkers that precisely and specifically reflect the pathophysiologic and clinical changes in systemic lupus erythematosus. Promising candidate biomarkers have been identified but must still be validated through rigorous, large-scale multicenter studies.

Erythrocyte C3d and C4d for monitoring disease activity in systemic lupus erythematosus

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 E-C4d 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-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.

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.

Biomarkers in systemic lupus erythematosus: challenges and prospects for the future

The search for lupus biomarkers to diagnose, monitor, stratify, and predict individual response to therapy is currently more intense than ever before. This effort is essential for several reasons. First, epidemic overdiagnosis and underdiagnosis of lupus, even by certified rheumatologists, leads to errors in therapy with concomitant side effects which may be more serious than the disease itself. Second, identification of lupus flares remains as much an art as it is a science. Third, the capacity to stratify patients so as to predict those who will develop specific patterns of organ involvement is not currently possible but would potentially lead to preventive therapeutic strategies. Fourth, only one new drug for the treatment of lupus has been approved by the US Food and Drug Administration in over 50 years. A major obstacle in this pipeline is the dearth of biomarkers available to prove a patient has responded to an experimental therapeutic intervention. This review will summarize the challenges faced in the discovery and validation of lupus biomarkers, the most promising lupus biomarkers identified to date, and the promise of future directions.

Platelet C4d Is Associated With Acute Ischemic Stroke and Stroke Severity

Background and Purpose— Platelets bearing complement C4d were recently reported to be 99% specific for a diagnosis of systemic lupus erythematosus (SLE) and associated with neuropsychiatric lupus. We compared the prevalence of platelet C4d and investigated the clinical associations of platelet C4d in patients with acute ischemic stroke. Methods— We recruited 80 patients hospitalized for acute ischemic stroke. Stroke severity was measured by the National Institutes of Health Stroke Scale (NIH-SS). Infarct volume was determined by MRI. Platelet C4d was measured by flow cytometry. Results— Mean age was 57.9 years (range: 24.6 to 86.8 years), 58% were male, and 91% were white. Eight patients (10%) with acute ischemic stroke were platelet C4d-positive, which was significantly higher in prevalence compared to healthy controls (0%, P<0.0001) and non-SLE patients with immune/inflammatory disease (2%, P=0.004). The median NIH-SS score and infarct volume for acute stroke patients were 6 (interquartile range [IQR]: 2 to 13) and 3.4 cc (IQR: 1.1 to 16.6), respectively. Platelet C4d-positive patients were more likely to have a severe stroke compared to those with negative platelet C4d (NIH-SS median: 17.5 versus 5, P=0.003). Positive platelet C4d was independently associated with stroke severity (P=0.03) after controlling for age, anticardiolipin antibody (aCL) status, and total anterior circulation of stroke involvement, and also with infarct volume (P=0.005) after controlling for age, aCL status, and old stroke by MRI. Conclusions— Platelet C4d is associated with severe acute ischemic stroke. Platelet C4d may be a biomarker as well as pathogenic clue that links cerebrovascular inflammation and thrombosis.

Lymphocyte‐Bound Complement Activation Products as Biomarkers for Diagnosis of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is frequently misdiagnosed due to the lack of definitive diagnostic tests. The purpose of this study was to determine specifically whether complement activation products (CAP) are deposited on lymphocytes of SLE patients and whether lymphocyte‐bound CAP (LB‐CAP) may serve as novel biomarkers for the diagnosis of SLE. We conducted a cross‐sectional study of 224 patients with SLE, 179 patients with other diseases, and 114 healthy controls. LB‐CAP on peripheral blood lymphocytes was measured by flow cytometry. Diagnostic utility of LB‐CAP was determined by receiver operating characteristic (ROC) analysis. Significantly elevated levels of C4d and C3d were detected specifically on T and B lymphocytes (designated T‐C4d, T‐C3d, B‐C4d, and B‐C3d) of SLE patients. As diagnostic tools, T‐C4d and B‐C4d, respectively, were 56% sensitive/80% specific and 60% sensitive/82% specific in differentiating SLE from other diseases. Moreover, compared with measurement of anti‐dsDNA, serum C3, or serum C4, measurement of T‐C4d/B‐C4d was significantly more sensitive in identifying SLE patients during a single clinic visit. This is the first investigation of lymphocytes bearing complement activation products in human disease. T‐C4d and B‐C4d have high diagnostic sensitivity and specificity for SLE and may have added value to current laboratory tests for SLE diagnosis.

Cell-bound complement activation products in systemic lupus erythematosus: comparison with anti-double-stranded DNA and standard complement measurements

Objective To compare the performance characteristics of cell-bound complement (C4d) activation products (CBCAPS) on erythrocyte (EC4d) and B cells (BC4d) with antibodies to double-stranded DNA (anti-dsDNA) and complement C3 and C4 in systemic lupus erythematosus (SLE). Methods The study enrolled 794 subjects consisting of 304 SLE and a control group consisting of 285 patients with other rheumatic diseases and 205 normal individuals. Anti-dsDNA and other autoantibodies were measured using solid-phase immunoassays while EC4d and BC4d were determined using flow cytometry. Complement proteins were determined using immunoturbidimetry. Disease activity in SLE was determined using a non-serological Systemic Lupus Erythematosus Disease Activity Index SELENA Modification. A two-tiered methodology combining CBCAPS with autoantibodies to cellular and citrullinated antigens was also developed. Statistical analyses used area under receiver operating characteristic curves and calculations of area under the curve (AUC), sensitivity and specificity. Results AUC for EC4d (0.82±0.02) and BC4d (0.84±0.02) was higher than those yielded by C3 (0.73±0.02) and C4 (0.72±0.02) (p<0.01). AUC for CBCAPS was also higher than the AUC yielded by anti-dsDNA (0.79±0.02), but significance was only achieved for BC4d (p<0.01). The combination of EC4d and BC4d in multivariate testing methodology with anti-dsDNA and autoantibodies to cellular and citrullinated antigens yielded 80% sensitivity for SLE and specificity ranging from 70% (Sjogrens syndrome) to 92% (rheumatoid arthritis) (98% vs. normal). A higher proportion of patients with SLE with higher levels of disease activity tested positive for elevated CBCAPS, reduced complement and anti-dsDNA (p<0.03). Conclusions CBCAPS have higher sensitivity than standard complement and anti-dsDNA measurements, and may help with the differential diagnosis of SLE in combination with other autoantibodies.