Jin Seino

Circulating anti-DNA immune complexes in active lupus nephritis

PURPOSE The role that circulating anti-DNA immune complexes play in autoimmunity has not yet been elucidated in humans. The aim of this study was to relate circulating anti-DNA immune complexes to a variety of renal histologic features and to immunoglobulin deposits in active lupus nephritis. PATIENTS AND METHODS The study population consisted of 47 patients with active lupus nephritis, 28 with active systemic lupus erythematosus (SLE) in the absence of renal lesions, and 40 with other categories of the disease. All patients were examined for anti-DNA circulating immune complexes (CIC) and their anti-DNA idiotype expression by an isoelectrofocusing analysis. Patients with renal lesions were also examined for renal histologic and immunofluorescent findings in renal biopsy specimens. RESULTS Anti-DNA CIC expressing an anti-DNA idiotype termed 0-81 Id occurred in patients with active lupus nephritis but not in acute episodes lacking renal involvement or in remission. Positive test results for anti-DNA CIC were associated with the incidence of diffuse proliferative glomerulonephritis (DPGN). Patients with anti-DNA CIC were also found to have a statistically significant increase in the prevalence of immunoglobulin immune deposits in the subendothelial area of the renal glomeruli. CONCLUSION The findings suggest that anti-DNA CIC preferentially occurred in lupus patients with DPGN. Examination for anti-DNA CIC may be a useful predictor of renal lesions, and therefore may contribute to the management of SLE. The results also indicate that anti-DNA CIC may be associated with immunoglobulin deposition in the subendothelial area of the renal glomeruli.

A novel ELISA assay for the detection of C3 nephritic factoro

We have developed an ELISA procedure for the detection of C3 nephritic factor (C3NeF), in which wells are coated with a fixed concentration of 2 micrograms C3b per well, and subsequently reacted with B and D. The presence of increasing concentrations of NiCl2 showed a NiCl2 concentration-dependent generation of C3bBb and very little solid-phase bound C3bBb was generated with MgCl2. The formation of solid-phase C3bBb in the presence of an optimal concentration of 1 mM NiCl2, was time-dependent and plateau values were reached after 30 min at 37 degrees C. IgG purified from the serum of a patient with membranoproliferative glomerulonephritis (MPGN) type II containing C3NeF stabilizing activity was bound to the C3bBb generated on microwells in a dose-dependent manner whereas normal IgG exhibited only minor reactivity. C3NeF activity was measured using the ELISA method in patients with MPGN type II (n = 15) and other diseases (n = 17) and in normal controls (n = 15). Most of the patients with MPGN type II exhibited positive C3NeF at various levels, while two of the disease controls showed only slight reactivities. C3NeF titers measured by this new ELISA procedure correlated well with previously described hemolytic assays (r = 0.617, p < 0.01).

Quantitation of C3 nephritic factor of alternative complement pathway by an enzyme-linked immunosorbent assay.

We have developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of C3 nephritic factor of the alternative pathway of complement (NeFA). Incubation of the NeFA-positive serum (patient KS serum) with normal human serum (NHS) in Mg-EGTA resulted in the formation of C3-B-IgG complex. No complex was formed in EDTA. At first this was detected as three types of complexes: C3-IgG, B-IgG and B-C3, by the combination of antibodies. The reaction mixture in Mg-EGTA was filtered through an ACA 22 column, from which the complexes were eluted in the same part as the first protein peak. When IgG purified from KS serum was incubated with NHS in Mg-EGTA, B-C3 complex increased in proportion to the dose of IgG. These results indicated that only one kind of complex consisting of IgG, C3 and B (IgG-C3-B) was generated by the addition of NeFA to NHS. Serum NeFA could be quantified as the titer of B-C3 complex formed after its incubation with NHS in Mg-EGTA. Using the ELISA method, NeFA was positive in five out of six patients with membranoproliferative glomerulonephritis (MPGN) type II and in only one of 17 with MPGN type I. Titers obtained by the new method were in good accordance with those by C3 conversion and C3bBb stabilization assays for NeFA, and the new method was more exact and simple than the conventional methods.

Participation of CR1 (CD35), CR3 (CD11b/CD18) and CR4 (CD11c/CD18) in membranoproliferative glomerulonephritis type I

Intraglomerular expression of complement receptors (CR) was investigated chronologically in 22 repeatedly biopsied patients with membranoproliferative glomerulonephritis (MPGN) type I by indirect immunoperoxidase staining using MoAbs. Patients were divided into two groups based on whether intraglomerular C3c deposition was decreased at the second biopsy (2nd Bx) (group A, n= 12), or not (group B, n= 10). At the first biopsy (1st Bx), the severity of glomerular injury and the degree of glomerular C3c deposition were compatible between the two groups. Four patterns of CR1 (CD35) expression on podocytes were recognized: normal; generally decreased; focally/ segmentally lost; and completely lost. The numbers of CR3 (CD11b/CD18)‐ and CR4 (CD11c/ CD18)‐positive cells per glomerular cross‐section were counted. At the 1st Bx, no significant difference was found in the number of CR3+ or CR4+ cells between the two groups. At the 2nd Bx. the numbers of both the CR3+ and CR4+ cells were significantly decreased only in group A (P <0·01). The numbers of CR3+ and CR4+ cells were significantly higher in cases with moderate or marked C3c deposits than in those with no or mild C3c deposits. The intensity of CR1 expression in group B was less than that in group A at both the 1st and 2nd Bx (1 st, P <0·05; 2nd, P < 0·01), and chronological improvement of CR1 expression was observed only in group A. The severity of glomerular injury was increased only in group B (P <0·01), and was associated with persistent massive proteinuria and hypocomplementaemia. Our results suggest that, in cases with an adverse outcome, a more severe defect of CR1 initially exists and the expression of CR1 is not recoverable chronologically. This irreversible decrease or loss of CR1 may partly contribute to the continuous C3c deposition and intraglomerular infiltration of CR3+ and CR4+ cells.

Intraglomerular Immune Cell Infiltration and Complement 3 Deposits in Membranoproliferative Glomerulonephritis Type I: A Serial-Biopsy Study of 25 Cases

We examined chronological changes in intraglomerular immune cell infiltration in comparison to the changes in glomerular complement 3 (C3) deposits (C3-D) and serum complement levels in 25 patients with membranoproliferative glomerulonephritis (MPGN) type I. These patients were divided into the following two groups: group A (n = 13), cases in which there were fewer intraglomerular C3-D at the second biopsy (2nd-Bx) than at the first biopsy (1st-Bx); and group B (n = 12), those in which the amount of C3-D at the 2nd-Bx was greater than or equal to that at the 1st-Bx. At the 1st-Bx, monocytes (Mo)/macrophages (M phi) and total leukocytes (TLC) were the predominant cell types in both groups, whereas T cells were less marked. At the 2nd-Bx, only group A showed a significant decrease in the number of either Mo/M phi (P < 0.01), TLC (P < 0.01), pan-T cells (P < 0.01), or intraglomerular nuclei per glomerular cross-section ([NIN] P < 0.01). In group B, there was a positive correlation between the number of intraglomerular pan-T cells (CD3-positive cells) and M phi (CD68-positive cells, P < 0.05 at the 1st-Bx and P < 0.01 at the 2nd-Bx), but not in group A. An improvement in light-microscopic findings and a significant decrease of urinary protein excretion (P < 0.05) at the 2nd-Bx was observed only in group A. Hypocomplementemia (hypo-C) was found in 12 of 13 cases of group A and in eight of 12 cases of group B at the 1st-Bx. Hypo-C in group A was not found at the 2nd-Bx. On the other hand, in group B, hypo-C was still observed in the eight cases and was found in one additional case at the 2nd-Bx.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of C4 nephritic factor by an enzyme-linked immunosorbent assay

We have developed an enzyme-linked immunosorbent assay (ELISA) for the quantitation of C4 nephritic factor (C4NeF). Incubation of the C4NeF-positive serum from patient M.I. with normal human serum (NHS) in the presence of human aggregated IgG (AHG) resulted in the formation of stable C4-C2 complex. No complex was formed in EDTA or under the condition free of AHG. The reaction mixture was filtered through an ACA 22 column, from which the C4-C2 complex was eluted at the first protein peak. When IgG purified from M.I. serum was incubated with NHS and AHG, C4-C2 complex also increased in proportion to dose of the purified M.I. IgG. These results show that C4NeF in M.I. serum stabilizes C4b2a convertase of the classical complement pathway, and is quantified by the ELISA. C4NeF activity was measured, using the ELISA method, in patients with various glomerular diseases, and found elevated in three of 24 patients with membranoproliferative glomerulonephritis (MPGN) type I and slightly but distinctly positive in seven of 24. No C4NeF was detected in two C3 nephritic factor-positive patients with MPGN type II and six with active systemic lupus erythematosus. The new method was more simple and quantitative than C4b2a stabilization assay for C4NeF.

Alteration of C3 Nephritic Factor in a Patient with Membrano- proliferative Glomerulonephritis Type II

J. Seino, 2nd Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-Cho, Aoba-Ku, Sendai 980 (Japan) Dear Sir, C3-nephritic factor (C3NeF) is often found in patients with membranoprolifera-tive glomerulonephritis (MPGN) type II [1]. In plasma, low levels of C3, caused by activation of the alternative pathway of complement, are accompanied by relatively normal levels of Cl, C4, and C2 [2]. Performing immu-nohistological studies, C3 deposition is also frequently observed in the glomeruli. However, the relationship between C3NeF, hypo-complementemia, and immunological renal lesions is still controversial. We observed a case with MPGN type II who developed chronic renal failure and whose C3NeF level decreased and disappeared with progression of chronic renal failure and reappeared after starting hemodialy-sis. The relation between CH50/C3 and C3NeF levels was completely reciprocal during the clinical course of this patient. A 7-year-old girl presented with facial edema and oliguria 2 weeks after catching a common cold in 1975. Heavy proteinuria and slight microhematuria were pointed out by her doctor. There was no family history of any medical diseases. In 1977, a renal biopsy specimen was obtained because of a persistent proteinuria. She was diagnosed as having MPGN by light microscopy, showing lobular formation with mesangial and endocapillary proliferation in all glomeruli. However, the study by electron microscopy demonstrated no dense deposits in the glomerular basement membrane, but only lamellation of the lamina densa. In 1982, a second renal biopsy specimen was obtained because heavy proteinuria and nephrotic syndrome had continued. Electron microscopy revealed typical dense deposits of MPGN type II on the lamina densa in all glomerular basement membranes [3]. A detailed complement profile of this patient was first studied in 1983. Then renal function tests revealed values within normal ranges: serum creatinine 0.6 mg/dl, blood urea nitrogen 11 mg/dl, and creatinine clearance (C„) 96 ml/min. Complement assays showed extremely reduced CH5Π (15 U/ml) and C3 (10 mg/dl) levels (fig. 1), a high titer of C3d, and a normal C4 titer (34 mg/dl).