Kenichi Sekita

Dermatopathological studies on skin lesions of MRL mice

SummaryThe MRL-lpr/lpr(MRL/l) mouse is a new animal model for human systemic lupus erythematosus (SLE) and skin lesions with hair loss and scab formation are one of the characteristic manifestations in this mouse. We investigated the histopathology of the skin lesions in MRL/l mice and studied the related autoimmune phenomenon. Light microscopical observations revealed hyperkeratosis, acanthosis, hypergranulosis, liquefaction, vasodilation in the dermis and T-cell infiltration into the dermis at the age of 5 months (mo). Immunohistological studies showed the presence of immunoglobulins and/or complement depositions at the dermal-epidermal junction (DEJ). In some mice there was deposition of immunoglobulin at the DEJ at 2 mo and in 90%–100% of MRL/l mice at over 5 mo. Temporal relationship was present among cutaneous immunoglobulin depositions, the occurrence of anti-DNA antibodies and proteinuria. These findings suggest that MRL/l mice might provide a new aid for studying the biological mechanisms of the development of skin lesions in human SLE.

Genetic regulation of the class conversion of dsDNA-specific antibodies in (NZB×NZW) F1 hybrid

To investigate the possible effects of NZW genes on the class conversion of dsDNA-specific antibodies in NZB × NZW (B/W) F1 hybrids, we measured IgM, IgGl, and IgG2 dsDNA-specific antibodies, using the Crithidia luciliae kinetoplast immunofluorescence test, in NZB, NZW, B/W F1 hybrid, B/W F1 × NZB backcross, and B/W F1 × NZW backcross mice at 4, 7, and 10 months of age. The highest serum levels of IgM dsDNA-specific antibodies were observed in NZB mice at the ages tested; however, the amounts of IgG1 and IgG2 antibodies were scanty. In contrast, a large amount of both IgG1 and IgG2 dsDNA-specific antibodies was produced in B/W F1 hybrids, in which the serum IgM antibodies were lower than those observed in NZB mice. NZW mice were virtually negative for these antibodies. Progeny testing suggested that a combined effect of two unlinked dominant genes of the NZB strain determines the production of dsDNA-specific antibodies and that these genes only act to produce IgM antibodies. These traits are to a great degree modified by the NZW loci in B/W F1 hybrids, and a combined effect of two unlinked dominant genes leads to conversion of the class of the antibodies from IgM to IgG, which, in turn, increases the serum levels of dsDNA-specific antibodies. The F1 hybrid of C57BL/6 and NZW strains produced no dsDNA-specific antibodies, indicating that the relevant NZB predisposing genes are required for the NZW gene action. Linkage studies showed that one of such NZW genes is to some extent linked to the H-2 complex on chromosome 17, but not to Mup-1 (chromosome 4) or a coat color locus (chromosome 2). The appearance of IgG dsDNA-specific antibodies correlated well with the incidence of renal disease in B/W F1 × NZB backcross mice.

Relationship of Intraglomerular Coagulation and Platelet Aggregation to Glomerular Sclerosis

In order to investigate the relationship between intraglomerular coagulation and glomerular sclerosis, the distribution of fibrin-related antigen (FRA) in glomeruli without extracapillary lesions was examined by immunoperoxidase microscopy in 80 patients with IgA nephropathy (IgA-N). A total of 302 glomeruli were examined, including 20 with global sclerosis, 31 with segmental sclerosis (SS glomeruli), and 251 nonsclerosed glomeruli. In the nonsclerotic areas of SS glomeruli, the deposition of FRA was significantly greater than in the nonsclerosed glomeruli. In the nonsclerosed glomeruli FRA was mainly found in the mesangium, while in the nonsclerotic areas of SS glomeruli FRA was not only present in the mesangium but also in the endothelium of the glomerular capillary loops. FRA-positive microclots were also often observed attached to the endothelium of the capillaries of the nonsclerotic areas of SS glomeruli. Cross-linked FRA was also observed in the endothelium of the same capillaries using the monoclonal antibody DD3B6/22. Deposition of von Willebrand factor (vWF) was greater in the endothelium than in the mesangium in the same areas. Aggregated platelets adhering to the glomerular capillary walls in these areas were frequently detected using the monoclonal antibody P2. Such distribution of platelets and vWF showed that the endothelium of the nonsclerotic areas of SS glomeruli was more severely damaged than that of nonsclerosed glomeruli. These findings suggest that endothelial cell damage might activate the intraglomerular coagulation, which might be one of the factors in the development of global glomerular sclerosis.

Circulating Immune Complexes of IgG, IgA, and IgM Classes in Various Glomerular Diseases

In order to examine the correlation between immunoglobulin (Ig) classes of immunoglobulins in circulating immune complexes (CIC) and immunoglobulins in glomerular deposits, we have measured CIC of IgG, IgA and IgM classes in various glomerular diseases. Serum CIC were measured using a modified conglutinin binding assay (K assay) using 125I-labeled anti-gamma, anti-alpha and anti-mu antisera. IgA class CIC were detected in more patients with IgA nephropathy than patients with non-IgA nephropathy. There was an association between the presence of IgG deposits in kidneys and the presence of IgG in CIC. Patients with IgA deposits in their kidneys also had IgA class CIC. There was also an association between the presence of IgM deposits in kidneys and the presence of IgM in CIC. These results suggest that the Ig class in CIC and the Ig class in glomerular deposits were correlated, and that in IgA nephropathy the mesangial IgA deposits may be derived from IgA class CIC.

Immunopathological correlation between mesangial C3d-deposition and C3d-fixing circulating immune complexes in lupus nephritis

By a direct immunofluorescent technique, glomerular C3d deposition was examined in a total of 50 renal biopsy specimens from patients with lupus nephritis. C3d deposition was then compared with disease activity, glomerular IgG and C3c deposition, and the levels of circulating immune complexes (CIC) measured by a solid-phase anti-C3d assay. There was a good correlation between disease activity and the positivity of glomerular C3d deposits (P less than 0.001), as well as C3c deposits (P less than 0.001). Even in clinically inactive patients, a relatively high percentage (59%) of C3d deposits were positive compared with C3c deposits (17%). Mesangial C3d deposition correlated with clinical disease activity more significantly (P less than 0.005) than capillary wall C3d deposition (P less than 0.025). C3d deposits were detected in all of the 30 cases with positive C3c deposits, and moreover, in 15 of the 20 (75%) cases with negative C3c deposits. Glomerular IgG deposits were almost always associated with C3d deposits, both in mesangial areas and along capillary walls, with statistical significance (P less than 0.005, P less than 0.001, respectively). The serum levels of C3d-fixing immune complexes (IC) were significantly correlated with the positivity and intensity of mesangial C3d deposits. This study demonstrates glomerular deposition of C3d in patients with lupus nephritis and reveals a significant correlation between mesangial C3d deposition and disease activity.

Immunoelectron Microscopic Localization of Fibrin-Related Antigen in Human Glomerular Diseases

The distribution of fibrin-related antigen (FRA) in glomeruli was examined by immunoelectron microscopy in 9 patients with idiopathic membranous nephropathy (MN), 8 patients with minimal-change nephrotic syndrome, and 10 patients with IgA nephropathy (IgA-N), using antisera against human gamma--chain, alpha-chain, mu-chain, and fibrinogen. Electron-dense reaction products of FRA were observed in the endothelium, subendothelium, and/or in electron-dense deposits (EDD). Among the three glomerular diseases, the amount of electron-dense reaction products of FRA in the endothelium was highest in MN. This suggests that coagulation occurs on the endothelium in MN. Although the mesangial EDD of IgA-N were intensely stained with reaction products of FRA, the staining was weak in the subepithelial EDD of MN. This suggests that FRA hardly penetrates into the subepithelial EDD in MN.

Evaluation of circulating immune complexes and antinuclear antibodies in Japanese patients with leprosy

SummaryIn 79 patients with leprosy a significant increase of anti-extractable nuclear antigen (ENA) antibodies and circulating immune complexes (CIC) was found. No correlation between CIC and anti-ENA antibodies was demonstrable. Since such a correlation is known from antinuclear antibodies and CIC in patients with systemic lupus erythematosus, it appears likely that anti-ENA antibodies do not play a causative role in CIC-mediated pathogenesis of leprosy.

A Comparative Immunologic Study of IgA Nephropathy

A conglutinin binding assay has been used to detect circulating immune complexes (CIC) containing IgA, IgG, or IgM in sera from patients with IgA nephropathy. IgA class CIC were detected in 40.7% of patient. IgG class CIC were detected only in patients with glomercular IgG deposits. IgM class CIC were detected more often in patients with glomerular IgM deposits than in patients without glomerular IgM deposits. These results demonstrate an association between the immunoglobulin in CIC and those in glomerular deposits. CIC were not detected in sera from most patients with IgA nephropathy by a Clq binding assay, however, since this assay does not detect IgA class CIC. Immunoelectronmicroscopic studies of IgA nephropathy have shown that C3 deposits are localized to the same areas as IgA deposits. In conclusion, we suggest that mesangial IgA deposits are composed of immune complexes and may be derived from CIC.

Ultrastructural Distribution of von Willebrand Factor in Human Glomerular Diseases

The distribution of the von Willebrand factor (vWF) as the factor-VIII-related antigen in glomeruli was examined by immunoelectron microscopy in 10 patients with idiopathic membranous nephropathy (MN), 8 patients with minimal-change nephrotic syndrome (MCNS), and 11 patients with IgA nephropathy (IgA-N). Electron-dense reaction products of vWF were observed in the endothelium and mesangium in all specimens examined. However, they were not detected in subepithelial electron-dense deposits of MN. The amount of electron-dense reaction products of vWF in the endothelium was significantly higher in MN than that in MCNS or IgA-N. This finding suggests that the glomerular endothelium in MN is the site of the local activation of coagulation and platelet aggregation system in glomerular capillary wall lesions.

Effects of single-stranded DNA on circulating immune complexes in SLE sera detected by C1q solid-phase assay

Circulating immune complexes (CIC) and anti-DNA antibodies are frequently detected in sera of patients with systemic lupus erythematosus (SLE). In 1973, Harbeck et al. [10] suggested the presence of circulating DNA/anti-DNA complexes in SLE sera by using the DNase digestion method. However, Izui et al. [12] have reported that these DNA complexes are not a major component of CIC. Recent evidence appears to support the concept that DNA/anti-DNA complexes are present in sera of patients with SLE [2, 5, 17, 18]. In order to clarify the role of DNA in CIC, we designed this inhibition study of C lq-binding activity by single-stranded (ss)DNA. Thirty serum samples were collected from 20 patients with SLE who visited the Department of Dermatology, Kyoto University Hospital. Pair sera were obtained from 10 patients. These patients satisfied the diagnostic criteria of the American Rheumatism Association for SLE [4], and their age ranged from 20 to 45 years. Activity of SLE was judged by the presence of at least two of the following items: (1) facial erythema, (2) arthralgia, (3) Raynauds phenomenon, (4) unexplained fever, (5) central nerve abnormalities, (6) serositis, (7) onset of edema, (8) hypertension, (9) LE cells, (10) leukopenia, and (11) proteinuria. For a comparative study, sera from 12 patients meeting the criteria for rheumatoid arthritis (RA) [16], 8 patients with pemphigus vulgaris, and 8 patients with