"Mechanism of renal damage in systemic autoimmune disorders." Journal of Chemotherapy, 10(2), pp. 167–168

OBJECTIVE:To study the specificity of antibodies reactive with renal antigens in mixed cryoglobulinemia.METHODS:Sera from mixed cryoglobulinemia (MC) patients were tested on human kidney extracts by immunoblot. A partially purified renal antigen was subjected to N-terminal sequencing.RESULTS:Antibodies reactive with a renal antigen of 48 kD were detected in 7 out of 11 patients with MC and renal involvement. N-terminal sequencing of this antigen showed that it was identical with alpha-enolase. This result was confirmed by the reactivity of the renal antigen with a rabbit anti-serum specific for alpha-enolase.CONCLUSIONS:The results indicate that antibodies specific for alpha-enolase are frequently produced by mixed cryoglobulinemia patients with renal involvement.

Kidney involvement in immuno-mediated diseases is a life threatening complication to be early detected. Glomerulo-tubular functional indices, kidney-released enzymes and metabolic profiles were assessed in 21 patients with systemic lupus erythematosus, progressive systemic sclerosis and mixed cryoglobulinaemia, without overt nephropathy at a current laboratory examination, and in 31 age-sex-matched healthy controls. All patients had a urinary total protein excretion rate higher than controls (353.6±182.4 vs 243.0±108.2 mg/24 h, p<0.01); 12 of them resulted albuminuric (775.5±1192.4 mg/24 h), while 9 were normoalbuminuric (16.6±7.6 mg/24 h). Urinary enzyme excretion rates (GGT and NAG) were significantly heightened compared to healthy subjects, both in albuminuric and in normoalbuminuric patients. Serum albumin resulted significantly lower in all patients, independent of their urinary albumin leakage. Finally, all subjects with connective tissue diseases had significantly higher triglycerides, lower HDL cholesterol and double serum fasting insulin than normals.

The frequency and specificity of antibodies to native and denatured collagens were evaluated in systemic sclerosis (SSc) and in primary Raynaud's phenomenon (PRP) by direct and competitive ELISA. Antibodies reactive with denatured collagen type I (CI) were found in 43% of the SSc sera, and anti-CIV and anti-CV in 31%. In PRP, anti-CI, anti-CIV and anti-CV antibodies were detected in 8% of patient sera. Anti-CI, anti-CIV and anti-CV antibodies reacted with determinants expressed on the native as well as on the denatured molecule. Anti-CI and anti-CIV were cross-reactive; a reactivity with CII and a lower one with CV were detected. Anti-CV antibodies also reacted with CI and CII and, in a smaller proportion of cases, with CIV. Anti-collagen antibodies, affinity-purified from blotted collagen IV and V and cyanogen bromide (CBr)-digested CI, displayed the cross-reactivities shown by inhibition studies on sera. Moreover, antibodies eluted from a CBr fragment of CI reacted with the other CBr fragments as well. These data show that one-third of SSc sera contain antibodies that react with epitopes expressed on native as well as on heat-denatured CI, CII, CIV and CV, and therefore have the potential to bind collagens in vivo.

Recent studies suggest that anti-DNA antibodies may arise from the immune response to a complex of DNA and a DNA-binding protein. One of the protein targets frequently recognized by anti-DNA antibodies is the enzyme DNAase I. To investigate the possible role of DNAase I in the induction of anti-DNA antibodies, we immunized mice with a complex of DNA and DNAase I. Mammalian double strand DNA was crosslinked with DNAase I by ultraviolet light (UV) treatment and emulsified in complete Freund's adjuvant. BALB/c mice were immunized at the base of the tail with the DNA-DNAase complex, boosted after 2 weeks with the immunogen in incomplete adjuvant and bled one week after the boost. Control mice received UV treated DNA in adjuvant. In one-third of the mice immunized with the DNA-DNAase complex, IgG anti-DNA antibodies were detectable in serum; the antibodies reacted with single and double strand DNA. No anti-DNA response was elicited by immunization with DNA alone. These data show that immunization with a DNA-DNAase complex can induce anti-DNA antibodies in non-autoimmune mice strains and suggest that DNA-binding proteins may act as carriers in the immune response that leads to anti-DNA antibody production.

Antibodies against small nuclear ribonucleoproteins (snRNP) are frequently produced in systemic autoimmune disorders and different mechanisms have been proposed to account for their production. Molecular mimicry is a well-established phenomenon which can involve exogenous antigens as well as autoantigens. An example of an exogenous antigen, i.e. a viral protein, was reported by Guldner et al. (1990). A 5-amino acid sequence of the p68 protein, a component of U1 snRNP, is shared with the matrix protein of the influenza B virus. A subset of patients with autoimmune disorders, whose sera contain anti-p68 antibodies, react with this shared sequence and with the entire matrix protein. The same p68 protein bears in another region a strong sequence-homology with the third complementarity-determining region (CDR3) of an immunoglobulin light chain. Immunization of normal animals with the light chain induces the production of anti-p68 antibodies (Puccetti et al, 1990). In this case, the sequence mimicking the ribonucleoprotein is contained in the variable region of the immunoglobulin, where the idiotope is located.

Mixed cryoglobulinaemia (MC) is a disorder characterized by the presence of large amounts of cryoprecipitating IgM-IgG complexes. An immune complex glomerulonephritis develops in one third of all patients, but its occurrence does not seem related to the amount of cryoglobulins in the sera, nor to their complement-fixing ability. In this study we investigated the presence of IgG antibodies reactive with kidney antigens in 33 MC patients (11 with glomerulonephritis, 22 without renal involvement). A total glomerular extract was run on a 10% acrylamide gel, blotted to nitrocellulose and probed with the patients' sera. Sera from half of the patients without renal involvement reacted with several glomerular antigens whose molecular weight ranged between 200 and 29 kD. In the group with renal involvement, sera from 7/11 patients reacted with an antigen of 50 kD, which is also expressed in thymus, but not in the heart or liver. In a follow-up study of four patients with renal involvement, the amount of serum antibody specific for the 50-kD antigen fluctuated, either spontaneously or in response to therapy. These results show that antibodies specific for glomerular antigens are detectable in MC sera. The immune response against a 50-kD antigen expressed in the kidney and thymus seems to be restricted to a subset of MC patients with renal involvement. Circulating autoantibodies specific for glomerular antigens might contribute to the induction of glomerulonephritis in MC forming immune complexes in situ.

Epstein-Barr virus (EBV) infection is associated with production of autoantibodies. The N-terminal 35-58 sequence of EBNA I, one of the nuclear antigens encoded by EBV, is highly homologous to the C-terminal 95-119 region of the ribonucleoprotein SmD. Autoantibodies specific for SmD are present only in systemic lupus (SLE) sera and are therefore considered a serological marker of SLE. We measured antibodies to the EBNA I 35-58 sequence in EBV-related diseases and in autoimmune disorders. Antibodies to the EBNA I 35-58 peptide were present in 30% of normal sera, 12% Burkitt lymphoma, 22% infectious mononucleosis, 25% rheumatoid arthritis, 38% SLE and 33% Sjogren's syndrome. Antibodies to the SmD 95-119 peptide were detectable in 32% of SLE sera, 17% infectious mononucleosis and 12% Burkitt lymphoma. The specificity of anti-EBNA I 35-58 antibodies affinity-purified from nine sera was analysed by means of an inhibition assay. Only anti-EBNA I 35-58 antibodies affinity-purified from SLE sera have a similar affinity for the viral peptide and the SmD C-terminal one; they also bind the recombinant SmD in western blot. The results indicate that antibodies to EBNA I 35-58 are produced in normals, in EBV-related diseases and in autoimmune disorder, but only SLE sera contain anti-viral antibodies cross-reactive with an autoantigen.

Autoantibodies against the ribonucleoproteins B, B' and D are a serological marker of systemic lupus erythematosus (SLE). We mapped the epitopes recognized by autoantibodies on the SmD molecule by means of 7 synthetic peptides corresponding to the entire length of the protein. By ELISA assay, 25% of the lupus sera contained IgG antibodies specific for the C-terminal SmD sequence 95-119. This reactivity was confirmed by synthesizing the sequence as a multiple antigen peptide (MAP): antibodies reactive with the MAP 95-119 were present only in SLE and not in other connective tissue disorders. Sera containing high titers of anti-MAP 95-119 antibodies reacted in immunoblot with the SmD protein. These results indicate the presence of a dominant epitope in the C-terminal region of SmD, which is highly homologous to the Epstein-Barr virus induced nuclear protein EBNA I.