Heidy Zinger

Cellular Sensitivity to Collagen in Thromboangiitis Obliterans

We studied 39 patients with thromboangiitis obliterans to determine their cellular and humoral immune responses to native human collagen Type I and Type III, which are constituents of blood vessels. Cell-mediated sensitivity to these collagens was measured by an antigen-sensitive thymidine-incorporation assay. The mean stimulation index--the ratio of thymidine incorporation in the presence of antigen to that in its absence--with both Type I and Type III collagens used as antigens was significantly higher in patients with thromboangiitis obliterans than in patients with arteriosclerosis obliterans or in healthy male controls. Lymphocytes from 77 per cent of the patients with thromboangiitis obliterans exhibited cellular sensitivity to human Type I or Type III collagens (or both). Furthermore, in 17 of 39 serum samples from the patients with thromboangiitis obliterans a low but significant level of anticollagen antibody activity was detected, whereas there was no antibody activity in serum samples from controls. These results suggest that there is a distinct etiologic factor in this disease and also raise the possibility of differentiating between thromboangiitis obliterans and arteriosclerosis obliterans by immunologic means.

Beneficial effects of the anti-oestrogen tamoxifen on systemic lupus erythematosus of (NZBxNZW)F1 female mice are associated with specific reduction of IgG3 autoantibodies.

Background: Sex hormones have been shown to influence the immune system and to modify the course of autoimmune disorders. Objective: To examine the effects of the oestrogen antagonist tamoxifen on the course of systemic lupus erythematosus (SLE) in (NZB×NZW)F1 mice. Methods: Groups of 8 week old (NZB×NZW)F1 female mice were treated with tamoxifen (800 μg/mouse; twice a week) or with double distilled water for four months. Mice were evaluated monthly for the presence of autoantibodies directed against DNA and nuclear extract (NE) by enzyme linked immunosorbent assay (ELISA). White blood cells and thrombocytes were quantified by a cell counter and proteinuria by combistix kit. At 6 months of age, all mice that did not die spontaneously were killed and evaluated for the presence of glomerular immune deposits by indirect immunofluorescence assay. IgG isotypes of autoantibodies in the mouse sera and glomeruli were determined by γ chain specific antibodies. Results: Tamoxifen treatment significantly reduced autoantibody production directed against either NE or DNA. The latter reduction was mainly in autoantibodies of the IgG3 isotype. Furthermore, tamoxifen had significant beneficial effects on the course of SLE in (NZB×NZW)F1 mice. At 6 months of age, 40% of the untreated mice died spontaneously, whereas all the tamoxifen treated mice were still alive. All untreated mice showed severe thrombocytopenia and persistent proteinuria, with diffuse glomerular immune deposits of IgG2a and IgG3 isotypes in their kidneys. In contrast, the tamoxifen treated mice had a normal number of thrombocytes and only minimal proteinuria. Moreover, glomerular immune deposits were detected in <40% of the tamoxifen treated mice. The latter were mainly of the IgG2a but not of the IgG3 isotype. Conclusion: The results clearly show the remarkable therapeutic effects of tamoxifen on SLE of (NZB×NZW)F1 female mice and suggest that these beneficial effects are related to the specific reduction of IgG3 autoantibodies.

Suppression of experimental systemic lupus erythematosus (SLE) in mice via TNF inhibition by an anti-TNFα monoclonal antibody and by pentoxiphylline

We have previously shown that the clinical manifestations of experimental systemic lupus erythematosus (SLE) correlate with an early increased secretion of TNFα and IL-1. In the present study, we examined the efficacy of two therapeutic modalities which lower TNFα production or activity, on the clinical manifestations of the disease. Experimental SLE was induced in naive C3H.SW mice by injection of the human anti-DNA monoclonal antibody (mAb) bearing the common idiotype, 16/6 Id. Two weeks after booster injections, treatment with either an anti-TNFα mAb, or pentoxiphylline (PTX) was started, for a period of 6 weeks. Production of TNFα (by splenocytes) and IL-1 (by peritoneal macrophages) was determined 3 and 7 months after disease induction. The experimental mice were also followed for disease manifestations. Both treatment protocols, with anti-TNFα mAb and with PTX, reduced the production of the two pro-inflammatory cytokines, TNFα and IL-1, in mice with experimental SLE. Anti-DNA antibodies were significantly lower in the mice treated with either protocol. In addition, a significantly lower rate of leukopenia, proteinuria and immune complex deposition was observed in treated mice. Abrogation of TNFα and IL-1 production in the early stages of experimental SLE by an anti-TNFα mAb or by PTX improves the clinical status of mice affiicted with this autoimmune disease.

A peptide based on the complementarity determining region 1 of a human monoclonal autoantibody ameliorates spontaneous and induced lupus manifestations in correlation with cytokine immunomodulation.

A peptide based on the sequence of the complementarity determining region (CDR) 1 of a human monoclonal anti-DNA autoantibody that bears the 16/6 idiotype (16/6Id) was synthesized as a potential candidate for the treatment of SLE patients. The peptide, designated hCDR1, did not induce experimental SLE upon active immunization of mice. The ability of the peptide to treat an already established lupus that was either induced in BALB/c mice or developed spontaneously in (NZB × NZW)F1 mice was tested. Ten weekly injections of hCDR1 (200, 50 μg/mouse) given subcutaneously mitigated disease manifestations (e.g., leukopenia, proteinuria and kidney damage) and resulted in a prominent reduction in the dsDNA specific antibody titers. Furthermore, treatment with hCDR1 resulted in reduced secretion and expression of the “pathogenic” cytokines [i.e., INFγ, IL-1β, TNFα (in the induced model) and IL-10], whereas the immunosuppressive cytokine TGFβ was up-regulated. Thus, the significant ameliorating effects of hCDR1 are manifested at least partially via the immunomodulation of the cytokine profile. These results suggest that hCDR1 is a potential candidate for a novel treatment of SLE patients.

Treatment of lupus patients with a tolerogenic peptide, hCDR1 (Edratide): immunomodulation of gene expression.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by dysregulation of cytokines, apoptosis, and B- and T-cell functions. The tolerogenic peptide, hCDR1 (Edratide), ameliorated the clinical manifestations of murine lupus via down-regulation of pro-inflammatory cytokines and apoptosis, up-regulation of the immunosuppressive cytokine TGF-beta, and the induction of regulatory T-cells. In the present study, gene expression was determined in peripheral blood mononuclear cells of 9 lupus patients that were treated for 26 weeks with either hCDR1 (five patients), or placebo (four patients). Disease activity was assessed by SLEDAI-2K and the BILAG scores. Treatment with hCDR1 significantly down-regulated the mRNA expression of the pathogenic cytokines IL-1beta, TNF-alpha, IFN-gamma, and IL-10, of BLyS (B-lymphocyte stimulator) and of the pro-apoptotic molecules caspase-3 and caspase-8. In contrast, the treatment up-regulated in vivo gene expression of both TGF-beta and FoxP3. Furthermore, hCDR1 treatment resulted in a significant decrease in SLEDAI-2K (from 8.0+/-2.45 to 4.4+/-1.67; P=0.02) and BILAG (from 8.2+/-2.7 to 3.6+/-2.9; P=0.03) scores. Thus, the tolerogenic peptide hCDR1, immunomodulates, in vivo, the expression of genes that play a role in SLE, consequently restoring the global immune dysregulation of lupus patients. Hence, hCDR1 has a potential role as a novel disease-specific treatment for lupus patients.

Prevention of Systemic Lupus Erythematosus-Like Disease in (NZBxNZW)F1 Mice by Treating with CDR1- and CDR3-Based Peptides of a Pathogenic Autoantibody

Two peptides based on the complementarity-determining regions (CDR) of a pathogenic murine anti-DNA antibody were employed in an attempt to prevent the spontaneous systemic lupus erythematosus (SLE)-like disease of (NZBxNZW)F1 mice. Female mice, at the age of 2 months, were injected with either the CDR1- or the CDR3-based peptides (pCDR1, pCDR3) subcutaneously or intravenously in aqueous solution for a total of 8–10 treatments. A reduction was observed in the total and pathogenic IgG2a and IgG3 anti-DNA antibody titers in the CDR-treated groups. Treatment reduced the number of mice that developed proteinuria and immune complex deposits in their kidneys. The severity of renal pathology was significantly reduced in the pCDR3 (P < 0.02) and pCDR1 (P ≤ 0.05) treated mice. Thus, both CDR-based peptides administered in aqueous solution were capable of preventing the SLE-like disease in (NZBxNZW)F1 mice, although the beneficial effects of pCDR3 appeared to be more pronounced than those of pCDR1 in the treated mice.

Methotrexate treatment in murine experimental systemic lupus erythematosus (SLE); clinical benefits associated with cytokine manipulation.

The objective of this study was to determine the effects of Methotrexate (MTX) on the development and the course of experimental murine SLE, as well as on the cytokine profile involved in the disease. SLE was induced in naive BALB/c female mice by injection of the human anti‐DNA MoAb bearing a common idiotype (16/6 Id). Six weeks following immunization, when high levels of autoantibodies were demonstrated, the mice were treated with MTX (2 mg/kg once a week) for a period of 10 months. MTX treatment had no effect on 16/6 Id‐induced autoantibody production. However, MTX treatment had beneficial effects on the clinical manifestations of the experimental disease (i.e. leucocyte counts, levels of protein in the urine and immune complex deposits in the kidneys). Thus, only 20% of 16/6 Id‐immunized BALB/c mice that were treated with MTX had immune complex deposits in their kidneys compared with 100% of SLE‐afflicted BALB/c mice that were not treated. We have observed a significant elevation in IL‐1, tumour necrosis factor (TNF) and IL‐10 secretion in BALB/c mice afflicted with experimental SLE. IL‐2, IL‐4, IL‐6 and interferon‐gamma (INF‐γ) levels were decreased in these mice compared with the levels detected in healthy controls. Treatment with MTX reversed the levels of all the above cytokines to normal levels observed in control mice. These studies demonstrate therapeutic effects of MTX on murine experimental SLE. The normal cytokine profile observed following treatment with MTX is suggested to play a role in the amelioration of the clinical manifestations of experimental SLE.

The Inhibition of Autoreactive T Cell Functions by a Peptide Based on the CDR1 of an Anti-DNA Autoantibody Is via TGF-β-Mediated Suppression of LFA-1 and CD44 Expression and Function

Systemic lupus erythematosus (SLE), which is characterized by the increased production of autoantibodies and defective T cell responses, can be induced in mice by immunization with a human anti-DNA mAb that expresses a major Id, designated 16/6Id. A peptide based on the sequence of the CDR1 of the 16/6Id (human CDR1 (hCDR1)) ameliorated the clinical manifestations of SLE and down-regulated, ex vivo, the 16/6Id-induced T cell proliferation. In this study, we examined the mechanism responsible for the hCDR1-induced modulation of T cell functions related to the pathogenesis of SLE. We found that injection of hCDR1 into BALB/c mice concomitant with their immunization with 16/6Id resulted in a marked elevation of TGF-β secretion 10 days later. Addition of TGF-β suppressed the 16/6Id-stimulated T cell proliferation similarly to hCDR1. In addition, we provide evidence that one possible mechanism underlying the hCDR1- and TGFβ-induced inhibition of T cell proliferation is by down-regulating the expression, and therefore the functions, of a pair of key cell adhesion receptors, LFA-1 (αLβ2) and CD44, which operate as accessory molecules in mediating APC-T cell interactions. Indeed, T cells of mice treated with hCDR1 showed a TGF-β-induced suppression of adhesion to the LFA-1 and CD44 ligands, hyaluronic acid and ICAM-1, respectively, induced by stromal cell-derived factor-1α and PMA. The latter suppression is through the inhibition of ERK phosphorylation. Thus, the down-regulation of SLE-associated responses by hCDR1 treatment may be due to the effect of the up-regulated TGF-β on the expression and function of T cell adhesion receptors and, consequently, on T cell stimulation, adhesion, and proliferation.

Amelioration of lupus manifestations by a peptide based on the complementarity determining region 1 of an autoantibody in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes of systemic lupus erythematosus (SLE) patients

A peptide based on the complementarity determining region (CDR)1 of a human monoclonal anti‐DNA autoantibody (hCDR1) was shown to either prevent or treat an already established murine lupus in systemic lupus erythematosus (SLE)‐prone mice or in mice with induced experimental SLE. The present study was undertaken to determine the therapeutic potential of hCDR1 in a model of lupus in severe combined immunodeficient (SCID) mice engrafted with peripheral blood lymphocytes (PBL) of patients with SLE. To this end, PBL obtained from lupus patients were injected intraperitoneally into two equal groups of SCID mice that were treated either with the hCDR1 (50 µg/mouse) once a week for 8 weeks, or with a control peptide. Mice were tested for human IgG levels, anti‐dsDNA autoantibodies, anti‐tetanus toxoid antibodies and proteinuria. At sacrifice, the kidneys of the successfully engrafted mice were assessed for human IgG and murine complement C3 deposits. Of the 58 mice transplanted with PBL of SLE patients, 38 (66%) were engrafted successfully. The mice that were treated with the control peptide developed human dsDNA‐specific antibodies. Treatment with hCDR1 down‐regulated the latter significantly. No significant effect of the treatment on the levels of anti‐tetanus toxoid antibodies could be observed. Treatment with hCDR1 resulted in a significant amelioration of the clinical features manifested by proteinuria, human IgG complex deposits as well as deposits of murine complement C3. Thus, the hCDR1 peptide is a potential candidate for a novel specific treatment of SLE patients.

MHC class I expression regulates susceptibility to spontaneous autoimmune disease in (NZBxNZW)F1 mice

(NZBxNZW)F1 mice spontaneously develop with age an autoimmune disease that resembles the human disease, systemic lupus erythematosus (SLE). Previous studies have demonstrated that susceptibility to experimentally induced SLE depended on the expression of MHC class I molecules: mice deficient in β2-microglobulin did not express cell surface class I and were resistant to the induction of experimental SLE. Furthermore, the spontaneous SLE-like disease of (NZBxNZW)F1 mice was ameliorated by treatment with an agent that reduces MHC class I expression, methimazole (MMI). In the present study, the role of MHC class I has been examined in (NZBxNZW)F1 mice deficient in β2-microglobulin expression. Homozygous (NZBxNZW)F1b2m-/- mice do not express class I or develop CD8+ T cells. Surprisingly, they show an increased susceptibility to disease. In sharp contrast, heterozygous (NZBxNZW)F1β2m+/- express class I, albeit at reduced levels, develop normal levels of CD8+ T cells and are less susceptible to autoimmune disease, relative to their wild-type litter mates. Taken together, these findings suggest that class I expression regulates the development of disease, both positively and negatively. We speculate that MHC class I expression itself confers susceptibility to disease through presentation of self-peptides, while also selecting for a CD8+ suppressor T cell population that mitigates disease.