KRN/I-Ag7 mouse arthritis is independent of complement C3.

BACKGROUND: KRN/I-A(g7) (KxB/N) is a mouse model of inflammatory arthritis, which resembles human rheumatoid arthritis. Arthritis in these animals is caused by autoreactivity to a ubiquitously expressed autoantigen, glucose-6 phosphate isomerase. Tolerance is broken at both the T cell and B cell level. The sera from KRN/I-A(g7) mice can induce mouse arthritis in healthy mice. Complement components of the alternative complement pathway, including C3, have been shown to be required in induction of mouse arthritis by serum transfer. METHODS: We have bred KRN/I-A(g7) mice onto a C3-deficient background and followed cohorts for the spontaneous appearance of arthritis. We have also transferred KxB/N serum to B6.I-A ( g7 ) recipients. RESULTS: C3-deficient KRN/I-A(g7) mice spontaneously developed severe, destructive arthritis, comparable to that seen in C3-intact KRN/I-A(g7) mice. However, serum transfer experiments confirmed the strong requirement for C3 in the passive model. CONCLUSION: The pathogenesis of spontaneous KRN/I-A(g7) arthritis can largely proceed by complement-independent pathways and must have pathology effector mechanisms in addition to those seen in the passive serum transfer model.

In vivo, multimodal imaging of B cell distribution and response to antibody immunotherapy in mice.

BACKGROUND: B cell depletion immunotherapy has been successfully employed to treat non-Hodgkin's lymphoma. In recent years, increasing attention has been directed towards also using B-cell depletion therapy as a treatment option in autoimmune disorders. However, it appears that the further development of these approaches will depend on a methodology to determine the relation of B-cell depletion to clinical response and how individual patients should be dosed. Thus far, patients have generally been followed by quantification of peripheral blood B cells, but it is not apparent that this measurement accurately reflects systemic B cell dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Cellular imaging of the targeted population in vivo may provide significant insight towards effective therapy and a greater understanding of underlying disease mechanics. Superparamagnetic iron oxide (SPIO) nanoparticles in concert with near infrared (NIR) fluorescent dyes were used to label and track primary C57BL/6 B cells. Following antibody mediated B cell depletion (anti-CD79), NIR-only labeled cells were expeditiously cleared from the circulation and spleen. Interestingly, B cells labeled with both SPIO and NIR were not depleted in the spleen. CONCLUSIONS/SIGNIFICANCE: Whole body fluorescent tracking of B cells enabled noninvasive, longitudinal imaging of both the distribution and subsequent depletion of B lymphocytes in the spleen. Quantification of depletion revealed a greater than 40% decrease in splenic fluorescent signal-to-background ratio in antibody treated versus control mice. These data suggest that in vivo imaging can be used to follow B cell dynamics, but that the labeling method will need to be carefully chosen. SPIO labeling for tracking purposes, generally thought to be benign, appears to interfere with B cell functions and requires further examination.

Immunomodulatory effect of DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) on complement system and macrophages.

DDT (bis[4-chlorophenyl]-1,1,1-trichloroethane) is responsible for many immuno-dysregulatory functions in exposed animals, but data particularly on complement system and macrophages are limited. In this study we have shown that DDT activates the complement system through the alternative pathway in the absence of any pathogen. A significant (p<0.05) increase in C3b, C3d and C3a generation, and decline in complement hemolytic activity was observed in insecticide exposed sera. The uncontrolled complement consumption reduces the lytic activity of the complement, which enhances the susceptibility to pyogenic infection if the exposure to DDT remains unabated. Further, DDT induced the significant (p<0.05) production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in macrophages and thus contributes inflammatory reactions, cytokine imbalance and immune-dysregulation. These molecular changes in macrophages lead to structural aberrations like heterochromatin condensation, loss of pseudopodia, cytoplasmic vacuolization, DNA fragmentation and hypodiploid nuclei as seen in our study, suggesting apoptosis. However, in presence lipopolysaccharide, DDT induced significant (p<0.05) suppression of TNF-alpha and NO generation, suggestive of impairment of macrophage microbiocidal effects. This study concludes that the functional and structural derangements of macrophages in association with uncontrolled and excessive complement consumption by DDT are perhaps one of the major mechanisms contributing to the immunosuppressive effects of insecticide.

Modulation of CR1 transcript in systemic lupus erythematosus (SLE) by IFN-gamma and immune complex.

Reduced expression of Erythrocyte Complement Receptor 1 (E-CR1) is envisaged to contribute significantly to the pathophysiology of systemic lupus erythematosus (SLE). We determined the levels of CR1 transcript in the neutrophils from 25 untreated patients with active SLE and 25 normal healthy individuals and, studied the effect of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and immune complexes (IC) on the same. The study revealed a marked decline in the levels of neutrophil CR1 (N-CR1) transcript in the patients with SLE, and differential pattern of IFN-gamma and IL-4 expression in the neutrophils from normals and patients. Opsonized immune complexes down regulated CR1 transcript in patients and IFN-gamma up regulated the same both in normals and patients. Immune complexes suppressed this effect of IFN-gamma. IL-4 also suppressed the effect of IFN-gamma but effect confined only to the normals. This is the first real-time RT-PCR data comparing the neutrophil CR1 expression in normals and patients with SLE and its modulation by IFN-gamma, IL-4 and immune complexes. IFN-gamma and immune complexes, respectively, emerged as the positive and negative modulators of neutrophil CR1 transcript in SLE.

Association of leukocyte CR1 gene transcription with the disease severity and renal involvement in systemic lupus erythematosus.

The reduced level of complement receptor 1 (CR1) on erythrocytes is speculated as a key mechanism contributing to immune complex (IC) overload and exaggerated complement (C) activation in systemic lupus erythematosus (SLE). Comparatively, fewer studies documented lower levels of CR1 on leukocytes and glomerular podocytes in this disease. The decline in E-CR1 is largely believed as an acquired phenomenon caused due to the proteolytic cleavage of CR1 from erythrocyte membrane. The mechanism underlying reduced CR1 expression on nucleated cells is under constant investigation. Recently, reduced leukocytes CR1 gene transcription had been demonstrated in SLE and was suggested as the main cause of decline in leukocyte CR1 (L-CR1). The relationship of L-CR1 gene transcription with severity and pathophysiology of disease needs to be elucidated. We determined the levels of L-CR1 in 30 active SLE patients and compared with normal healthy controls (n = 30). Patients were categorized into two groups i.e., with nephritis (n = 14) or without nephritis (n = 16). The expression of L-CR1 at transcriptional level was correlated with the levels of serum CIC, C3 and anti dsDNA antibodies. The levels of L-CR1 transcription were significantly reduced in all SLE patients as compared to controls (P < 0.001). This decline in L-CR1 however, was more marked in patients with nephritis than those without nephritis. In addition, the serum levels of CIC, anti dsDNA antibodies were higher and the levels of serum C3 were lower than the normal range in the patients. The difference was much more marked in SLE patients with nephritis than those without nephritis. The levels of L-CR1 transcription correlated negatively with the levels of CIC and anti dsDNA antibodies and positively with serum C3 levels. Thus, between SLE patients with and without nephritis, we found significant difference in the levels of L-CR1 transcription (P < 0.01), CIC (P < 0.05), anti dsDNA antibodies (P < 0.01) and C3 (P < 0.01). Our findings suggest that L-CR1 is drastically reduced in patients with severe form of SLE, i.e., lupus nephritis. Determination of L-CR1 expression at transcriptional level in addition to disease hallmarks like C3, CIC and anti-dsDNA antibodies may facilitate the assessment of severity of SLE and discrimination between patients with or without renal involvement.

Reduced complement receptor 1 (CR1, CD35) transcription in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by the production of a broad spectrum of autoantibodies against nuclear, cytoplasmic and cell surface antigens and immune complex overload. Complement receptor 1 (CR1, CD 35), a transmembrane glycoprotein found on the surface of erythrocytes, leukocytes and glomerular podocytes plays a key role in the clearance of immune complexes and regulation of complement cascade. A drastic decline in the level of cell surface CR1 appears to be an important event in pathology of SLE. However, the etiology of lower than normal expression of cell surface CR1 in this disease is poorly understood. We studied the level of leukocyte CR1 transcription in 30 patients with active SLE and 30 controls by reverse transcriptase-polymerase chain reaction (RT-PCR) and related the same with the level of CR1 protein expression monitored by Western blotting. For RT-PCR, ratio of CR1/beta-actin was considered for semiquantitation of the level of CR1 transcription. Despite individual variation at the level of transcription, 70% (21 out of 30) of the patients expressed CR1 transcript at the lowest range of 0-15% as compared to the controls wherein only 30% (9 out of 30 individuals) demonstrated CR1 transcript in this range. Majority of the controls (70%) expressed CR1 transcript at the level above 15%. Mean level of CR1 transcript in patients (mean +/- S.D. = 21.09 +/- 14.3) was significantly lower than the controls (mean +/- S.D. = 48.91 +/- 26.34) (P < 0.001). The level of CR1 transcription correlated inversely with circulating immune complexes (CIC) (r = 0.52, P < 0.01). This may suggest that although erythrocyte CR1 is the chief vehicle for CIC clearance, drastic decline in leukocyte CR1 expression may impair the phagocyte mediated immune complex clearance and contribute to increased complement consumption in SLE. Total leukocyte CR1 protein expression was also significantly reduced in patients (P < 0.001) as compared to controls. This decline at the protein level gave a very significant positive correlation with CR1 transcript (r = 0.67, P < 0.01). A marginal increase in soluble CR1 (sCR1) was observed in the plasma (ELISA) of SLE patients compared to the controls but was insignificant. This paper for the first time brings evidence to suggest that reduced synthesis of CR1 contributes substantially to the low cell surface CR1 expression in SLE. Our findings also suggest increased proteolytic cleavage of leukocyte cell surface CR1 in these patients. However, evidence for the latter is indirect.

Association of complement receptor 1 (CR1, CD35, C3b/C4b receptor) density polymorphism with glomerulonephritis in Indian subjects.

Complement receptor 1 (CR1, CD35, C3b/C4b receptor), a polymorphic membrane bound glycoprotein is important both as a complement regulatory protein, and as a vehicle for immune complex clearance. It is differentially expressed on erythrocytes, eosinophils, monocytes, B and T-lymphocytes, dendritic cells and kidney podocytes. It also occurs in the plasma as soluble CR1 (sCR1) and in urine as urinary CR1 (uCR1). Different population studies have either suggested or refuted the functional and physiological significance of genomic (HH, high erythrocyte CR1 expression; HL, intermediate and LL, low expression) polymorphism of CR1 in health and disease. Prevalence of autoimmune disorders like RA, GN and SLE is higher in Asian-Indians compared to the western world. Although several studies from India emphasize the modulation of E-CR1 levels as a key factor in the pathophysiology of glomerulonephritis (GN), none of them, however, provide much information on the role of CR1 gene variance in this context. We, therefore, carried out the study of CR1 polymorphism in 117 normal Indian subjects and 65 patients suffering from glomerulonephritis in order to study its possible association with the disease and E-CR1 levels. This is the first study of its kind in the Indian population, in which, the direct effect of a particular genotype on the E-CR1 levels and its possible association with the disease has been studied simultaneously.