Edna Mozes

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.

In vitro proliferative responses and antibody titers specific to human acetylcholine receptor synthetic peptides in patients with myasthenia gravis and relation to HLA class II genes.

To investigate which parts of the acetylcholine receptor are involved in the initiation and development of myasthenia gravis (MG), peptides representing different sequences of the human acetylcholine receptor alpha-subunit were synthesized. These peptides were tested for their ability to stimulate T cells of myasthenic patients and healthy control patients in proliferation assays and to bind to sera antibodies. Three of eight peptides discriminated significantly between the two groups in the proliferation assay, as well as in their ability to bind to serum antibodies. HLA-DR3 and DR5 were associated with proliferative responses to specific AChR peptides in the group of myasthenics. Acetylcholine receptor epitopes that might play a specific role in myasthenia gravis thus were demonstrated.

The beneficial effects of treatment with tamoxifen and anti-oestradiol antibody on experimental systemic lupus erythematosus are associated with cytokine modulations.

In an attempt to elucidate the role of oestrogens in systemic lupus erythematosus (SLE) we investigated the effects of treatment with an oestrogen antagonist – tamoxifen and a monoclonal anti‐oestradiol (anti‐E2) antibody on mice in which experimental systemic lupus erythematosus (SLE) was induced by a human monoclonal anti‐DNA antibody bearing the 16/6 idiotype (16/6 Id). Thus, groups of BALB/c female mice were immunized with the 16/6 Id and 3 weeks following the booster injection, when antibody titres were elevated in the injected mice, treatment protocols with anti‐oestradiol or tamoxifen were initiated. Control groups that were not immunized with the 16/6 Id but were similarly treated with the above agents were included in the study. The treatment with the above agents had no effect on the total autoantibody titres; however, a decrease in the immunoglobulin G (IgG)2a/IgG1 ratio of the anti‐DNA antibodies was determined in the 16/6 Id immunized and treated mice. Further, both the anti‐oestradiol and tamoxifen had beneficial effects on the clinical manifestations (white blood cell counts, levels of protein in the urine and immune complex deposits in the kidneys) of the 16/6 Id immunized and treated mice. We have previously observed a significant elevation in interleukin‐1 (IL‐1) and tumour necrosis factor‐α (TNF‐α) secretion in mice with experimental SLE and a reduction in IL‐2, IL‐4 and interferon‐γ (INF‐γ) levels as compared with the levels detected in healthy controls. Treatment with either the anti‐oestradiol antibody or with tamoxifen restored the levels of all the above cytokines to the normal levels observed in the control mice.These findings suggest that cytokine modulation may be the basis for the therapeutic effects of both anti‐oestrogens in experimental SLE.

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.

Activity of matrix metalloproteinase-9 is elevated in sera of patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and by systemic clinical manifestations and damage to multiple organs. The aim of the present study was to analyse matrix metalloproteinase (MMP)‐9 activity in sera of patients with active and inactive SLE in order to evaluate its role in the pathogenesis and course of the disease, as well as its diagnostic value. We measured activity levels of MMP‐9 and MMP‐2, using both gel zymography and activity assay kits, in sera of 40 SLE patients and of 25 healthy controls. We found that MMP‐9 activity, but not MMP‐2 activity, is significantly elevated in the sera of SLE patients compared with sera samples of healthy controls. High activity levels of MMP‐9 were determined in sera of 68% of the SLE patients. Elevated levels of MMP‐9 were correlated with the presence of discoid rash, Raynaud phenomenon, pneumonitis, mucosal ulcers and anti‐phospholipid antibodies. Changes in activity levels of MMP‐9, but not of MMP‐2, were observed in sera of the same patient at different periods of the disease course. High levels of MMP‐9 did not correlate with disease activity index (SLEDAI, BILAG) in female patients, but correlated with SLE activity in the group of male patients. The results of the present study suggest that MMP‐9 plays a role in the pathogenesis of SLE.

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.

The importance of the pathogenic 16/6 idiotype in the induction of SLE in naive mice.

We have previously demonstrated the pathogenicity of the common anti‐DNA idiotype designated 16/6 Id. Immunization of naive mice with the 16/6 Id induced SLE‐like disease characterized by serological (e.g. anti‐dsDNA and anti‐Sm auto‐antibodies), clinical (increased ESR, leucopenia and proteinuria), and pathological (16/6 Id deposition in kidneys) parameters. To elucidate further the role of the 16/6 Id in SLE induction the following studies were carried out: BALB/c mice were immunized with SA‐1, a human anti‐DNA monoclonal antibody carrying the 16/6 Id; TB‐68, a mouse monoclonal anti‐tuberculosis (TB) glycolipid, which binds dsDNA and carries the 16/6 Id; TB‐72, a mouse monoclonal anti‐TB glycolipid that binds DNA and does not harbour the 16/6 Id; and 4B4, a human anti‐Sm antibody that carries the 16/6 Id. SLE was induced in BALB/c mice only when immunized with SA‐l, TB‐68, and 4B4, namely antibodies with diverse binding capacities albeit having the 16/6 Id. Our studies further support previous evidence on the pathogenic role attributed to the 16/6 Id in SLE, and suggest that SLE is most probably an idiotype‐induced disease.

Effect of aging on cytokine production in normal and experimental systemic lupus erythematosus afflicted mice

Aging mice of strains susceptible to the induction of experimental systemic lupus erythematosus (SLE) develop a milder disease than young animals. To find out whether the decrease in susceptibility to disease is due to age-associated changes in cytokine profile, we first examined the secretion of cytokines by healthy mice aged 2-15 months. A gradual age-related decline in the levels of interleukin (IL)-2 and interferon (IFN) gamma, and an increase in IL-4, IL-10, IL-1, and tumor necrosis factor (TNF) alpha were observed. Experimental SLE was induced in 2- and 10-month-old mice by immunization with the monoclonal anti-DNA antibody bearing the 16/6 Id. Early increased production of pro-inflammatory cytokines (TNFalpha and IL-1), followed by a peak of the Th1-type cytokines (IL-2, IFNgamma) were observed in young mice. The Th2-type cytokines (IL-4, IL-10) peaked later. In contrast, only a mild increase in all of the above cytokines was determined in 10-month immunized mice. It thus appears that the decline in susceptibility to SLE induction in older mice may be related to changes in the capacity to produce cytokines.

The Suppression of Murine Lupus by a Tolerogenic Peptide Involves Foxp3-Expressing CD8 Cells That Are Required for the Optimal Induction and Function of Foxp3-Expressing CD4 Cells

A peptide, designated human CDR1 (hCDR1), that is based on the CDR1 of an anti-DNA Ab ameliorates systemic lupus erythematosus (SLE) in murine models via the induction of CD4+CD25+ regulatory T cells (Tregs). In the present study, the involvement of CD8 Tregs in the mode of action of hCDR1 was investigated in SLE-afflicted (NZB × NZW)F1 mice and in SJL mice following immunization with the lupus-inducing anti-DNA mAb that bears a common Id, 16/6Id. Treatment with hCDR1 up-regulated Foxp3-expressing CD8+CD28− Tregs in association with clinical amelioration of lupus manifestations. Furthermore, the in vivo depletion of the latter cells diminished the clinical improvement and the inhibitory effects of hCDR1 on the secretion of IFN-γ and resulted in the up-regulation of IL-10. However, the stimulatory effect of hCDR1 on the secretion of TGF-β was not affected by the CD8 Tregs. In the absence of CD8 Tregs, CD4+CD25+ Tregs were unable to expand in the hCDR1-treated mice, and the expression of Foxp3 was reduced, thereby interfering further with the suppressive function of CD4+CD25+ Tregs as determined in the in vitro assays. However, CD8 cells from hCDR1-treated mice that were adoptively transferred into SLE-afflicted mice led to up-regulation of CD4+CD25+ cells with intensified Foxp3 expression in the recipient mice. Thus, a functional link between two subsets of Tregs is demonstrated in which CD8+CD28− Tregs are required for both the optimal expansion and function of lupus ameliorating hCDR1-induced CD4+CD25+ Tregs.