IL-6 receptor blockage inhibits the onset of autoimmune kidney disease in NZB/W F1 mice

In the present study, we examined the preventive effect of anti-mouse IL-6 receptor (IL-6R) antibody, MR16-1, on the development of autoimmune kidney disease in female NZB/W F₁ (BWF₁) mice. Immunological tolerance to MR16-1 or isotype-matched control antibody, KH-5, was induced by the simultaneous administration of anti-CD4 MoAb in mice. Thereafter, mice were intraperitoneally given 0.5 mg of MR16-1, 0.5 mg of KH-5 or saline once a week from 13 to 64 weeks of age. MR16-1 treatment dramatically suppressed proteinuria and prolonged the survival time of BWF₁ mice. Only one out of 10 mice died with high levels of proteinuria throughout the experiment. MR16-1 almost completely suppressed the production of IgG forms of anti-DNA and anti-TNP antibodies, but not the IgM forms of these antibodies. In particular, all IgG subclasses (IgG1, IgG2a, IgG2b and IgG3) of anti-DNA antibody production were significantly suppressed. Moreover, serum IgG1, IgG2a and IgG3 levels in MR16-1-treated mice were lower than those in saline- and KH-5-treated mice, whereas serum IgM and IgA levels were not influenced. In conclusion, MR16-1 potently suppressed the development of autoimmune disease in BWF₁ mice, and this was attributed to its effect of specific suppression of IgG class antibody production.     

抗鼠IL－1α、IL－6单抗对狼疮倾向NZB／W F1小鼠的免疫调节研究

对狼疮倾向ＮＺＢ／ＷＦ１小鼠在４月龄时腹腔注射抗鼠ＩＬ－１α、抗ＩＬ－６单抗，每日１次１０μｇ×３ｄ，后每月１次×３次，１１月龄活杀，发现这样的剂量不能完全防治狼疮性肾炎，但可减轻蛋白尿，明显降低血清ＩＬ－１α水平，抑制腹腔巨噬细胞分泌ＩＬ－１α。对ＩＬ－６和ＴＮＦ－α水平无明显影响。     

NZB/W狼疮鼠体外IFN-α信号调控网络研究

通过IFN-α刺激基因(ISG)表达谱勾画SLE发病机制中可能的IFN-α异常调控通路。采用基因芯片技术,比较分析发病前新西兰黑/白(NZB/W)F1代狼疮鼠和年龄性别相匹配的对照组BALB/c小鼠脾脏单个核细胞3 h干扰素刺激基因表达谱,并运用信号通路分析软件(CRSD)解析IFN-α信号调控网络。结果:IFN-α体外刺激诱导上调了BALB/c小鼠而非NZB/W狼疮鼠脾脏单个核细胞p53通路相关基因转录产物的表达;IFN-α共同诱导了NZB/W狼疮鼠和BALB/c小鼠Th1型细胞因子和趋化因子网络,相对于BALB/c鼠,IFN-α刺激后IL15、IP10、XCL1、CCL3、CCL7及CCL12的转录产物在NZB/W狼疮鼠中高表达(P值均<0.01)。NZB/W狼疮鼠对IFN-α抑制细胞增殖作用和促炎作用的异常反应,参与并加重了NZB/W狼疮鼠自身免疫性疾病的进展。     

Immunologic abnormality in NZB/W F1 mice. Thymus-independent expansion of B cells responding to interleukin-6.

We have previously reported that B cell abnormality in NZB/W F1 mice developed independently of thymus. Here we examined further whether B cells from NZB/W F1 mice respond to interleukin-6 (IL-6), a factor for terminal differentiation of B cells. When freshly isolated splenic B cells were incubated for 5 days in the presence of human IL-6, an increased production of both IgM and IgG, including anti-DNA antibody, was evident in NZB/W F1 mice; there was no increase in BALB/c mice. A magnitude of augmentation in IgG but not IgM production by IL-6 became more apparent in older NZB/W F1 mice. The increased immunoglobulin production seen with IL-6 was neutralized by treatment with rabbit anti-recombinant human IL-6 antibody. As B cells from athymic NZB/W F1 nude mice also responded to IL-6, it was suggested that B cells in NZB/W F1 mice differentiated into the IL-6-responding state in a thymus-independent manner. This B cell abnormality may be associated with the pathogenesis of autoimmune disease in NZB/W F1 mice.     

The treatment of systemic lupus erythematosus (SLE) in NZB/W F1 hybrid mice; studies with recombinant murine DNase and with dexamethasone

The effects of recombinant mouse DNase on a well established murine model of spontaneous SLE have been evaluated. Daily intraperitoneal injections of DNase were given to female NZB/NZW F₁ mice during the period of disease development from 4 to 7 months of age or at the height of disease activity from the age of 7 months for 3 weeks. This treatment was compared with the injections of diluent and with an immunosuppressive dose of dexamethasone. The effects of treatment were evaluated using the immunological parameters of disease activity (antinucleoprotein antibody, immune complexes, serum immunoglobulins, anti-cardiolipin antibodies), proteinuria, serum creatinine and renal histopathology (light microscopy, immunofluorescence and electron microscopy). The dose of dexamethasone used (1 mg/kg per day from the age of 4 months) was sufficient to suppress the development of lupus entirely. Treatment with DNase starting at the age of 4 months postponed the development of the disease by about 1 month and extended the period from the onset of disease to death by about 30%. Mice treated for 3 weeks during the most active phase of the disease at 7 months of age showed more dramatic effects. Proteinuria and serum creatinine were significantly reduced and renal histopathology was strikingly less severe than in the control group. Immune complexes involving DNA-containing antigens are believed to play a crucial role in the pathogenesis of SLE. DNA-nucleoprotein, even in immune complexes, can be destroyed by DNase. This enzyme therefore provides a rational way to interfere with the disease process. The results reported here encourage a trial of recombinant human DNase in human SLE and lupus nephritis.     

Attenuation of immune complex nephritis in NZB/WF1 mice by a prostacyclin analogue.

Although prostaglandins have been shown to inhibit the evolution of the nephritis in NZB/W mice, the mechanisms of this effect are unknown. To characterize such inhibition, we injected the prostacyclin (PGI2) analogue, beraprost, into NZB/W mice, using 0.5 mg, 1.0 mg or 5.0 mg beraprost/kg body weight of test animals three times in 1 week when the mice were 2 months old. Evaluation included measurement of urine albumin excretion, serological parameters and splenic T cell subset, as well as examination of renal histology by light and fluorescence microscopy. Mice given beraprost showed a marked decrease in urine albumin excretion and in glomerular hypercellularity compared with untreated controls. Maximal beneficial effects occurred when the dose was 5.0 mg/kg of beraprost. These effects correlated with a reduction of immune complex deposition in glomeruli. In addition, beraprost reduced serum levels of immunoglobulins and anti-double-stranded DNA antibodies, and decreased the number of helper (L3T4+) T cells in splenocytes. These results indicate that beraprost attenuates the nephritis of NZB/W mice, and that the source of this effect is the reduced production of autoantibodies and deposition of immune complexes in glomeruli.     

Soluble human CD83 ameliorates lupus in NZB/W F1 mice

In the present study we explored the immunomodulatory potential of prokaryotically expressed soluble CD83 in the treatment of murine lupus using the NZB/W F1 mouse model. Therefore female NZB/W F1 lupus mice were treated either with sCD83 or PBS for 4 weeks. sCD83 treated mice showed a significantly delayed onset of anti-dsDNA autoantibody production when compared with the control group. Importantly, during the treatment period with sCD83 none of the mice showed elevated levels of anti-dsDNA autoantibodies. In addition, NZB/W F1 mice which received sCD83 displayed lower concentrations of anti-histone IgG autoantibodies. Furthermore, there was no difference in total IgG antibodies, indicating a modulatory role for sCD83 in the production of self-reactive antibodies without decreasing total IgG. These results indicate that administration of sCD83 has profound immune-modulatory effects on the induction of autoantibodies in NZB/W F1 lupus mice and may thus be a promising approach to interfere with autoimmunity in SLE and other autoantibody-driven diseases.     

Experimental therapy of systemic lupus erythematosus: the treatment of NZB/W mice with mouse soluble interferon-γ receptor inhibits the onset of glomerulonephritis

Female NZB/W F1 mice develop an autoimmune disease similar to human systemic lupus erythematosus (SLE), and ultimately die of glomerulonephritis. Starting at the age of 16 weeks NZB/W F1 mice were treated for a period of 19 weeks with soluble interferon-γ receptor (sIFN-γR), anti-IFN-γ monoclonal antibody (mAb) or IFN-γ. All mice treated with sIFN-γR or anti-IFN-γ mAb were alive 4 weeks after the treatment was discontinued, whereas 50% of mice died in the placebo groups and 78% of the mice died in the IFN-γ-treated group. Histologically, there was severe membrano-proliferative glomerulonephritis in IFN-γ- and placebo-treated mice, and minimal or no mesangioproliferative disease in mice receiving sIFN-γR or anti-IFN-γ mAb. The renal mononuclear infiltrate (T lymphocytes and monocytes), expression of major histocompatibility complex class II antigen and glomerular immunoglobulin and complement deposition were reduced in those mice. These data suggest that an IFN-γ inhibitor, such as the soluble IFN-γR, can be used for SLE therapy in the early stages of the disease.     

Tamoxifen decreases renal inflammation and alleviates disease severity in autoimmune NZB/W F1 mice

It has been documented that sex hormone may play a role in the pathogenesis of murine lupus. To determine the effect of tamoxifen (TAM) on NZB/W F1 female mice, a total dose of 800 microg (22 mg/kg body weight) of TAM was administered subcutaneously every 2 weeks. The control mice were injected with peanut oil only. After treatment with TAM for 5 months, the mice were killed and immunological parameters were evaluated. The results suggest that NZB/W F1 mice treated with TAM had less severe proteinuria and increased survival rate compared to controls. Flow cytometric analysis of splenocytes revealed a significantly lower percentage of B cells and CD5+ B cells in the TAM-treated group. There was a significantly lower serum level of soluble tumour necrosis factor (TNF) receptor I and II molecules in the TAM-treated mice. Immunohistological study showed that control mice had severe immune complex deposition in the kidney. In contrast, TAM-treated mice had much less pathological change. In summary, this study demonstrated that TAM treatment might be able to alleviate the symptoms of lupus nephritis, influence B-cell count, modulate the expression of cytokine receptors and thereby subsequently affect immune function. Further studies to determine the cellular mechanisms in lupus nephritis may increase our understanding of this complex disease and provide additional targets for therapeutic intervention.     

Beneficial effect of polyclonal immunoglobulins from malaria-infected BALB/c mice on the lupus-like syndrome of (NZB × NZW)F1 mice

We previously reported that infection of BALB/c mice with the parasite Plasmodium chabaudi induces high production of natural autoantibodies. Here we demonstrate that such an infection of lupus-prone (NZB × NZW)F1 (B/W) mice retards the development of their autoimmune disease. Survival and disease hallmarks (high-grade proteinuria and IgG anti-DNA antibodies) were delayed for 6 months when parasite inoculation was given at either 3 or 7 months of age, i.e. before or after the onset of the clinical symptoms. Similar beneficial effects, although less pronounced, were obtained when mice were treated with a total of 800 m?g of IgG (P-IgG) or IgM (P-IgM) or 300 m?g of cryoglobulin preparations isolated from P. chabaudi-infected BALB/c mice while similarly prepared fractions from uninfected mice had little effect. Compared to these fractions, P-IgG and P-IgM contained higher levels of natural antibodies bearing the D23 idiotype characteristic of polyreactive natural autoantibodies with enhanced activity against Fab and Fc fragments of IgG. In surviving mice, the level of anti-DNA antibodies, particularly those of IgG1 isotype, were significantly decreased. Flow cytometric analysis of various T cell subsets showed that the number of cells expressing γδ T cell receptor (TcR) antigens which did not vary with age was not modified after P-IgG or P-IgM treatment. In contrast, the number of T cells expressing Vβ8.1,2, Vβ10 and Vβ14 TcR antigens, which increased with age, were significantly reduced. Taken together, these results indicate that parasite infection of mice induces the synthesis of populations of IgM and IgG natural autoantibodies with immunoregulatory properties and that these antibodies attempt, at least transitorily, to rescue a natural autoantibody network that is deficient in B/W mice.     

Changes in the cytokine profile of lupus-prone mice (NZB/NZW)F1 induced by Plasmodium chabaudi and their implications in the reversal of clinical symptoms

We have previously observed that aged lupus-prone (NZB/NZW)Fl (BWF1) mice when infected with Plasmodium chabaudi show an improvement in their clinical lupus-like symptoms. In order to study the mechanisms involved in the long-lasting protective effect of the P. chabaudi infection in lupus-prone mice we analysed specific aspects of the cellular response, namely the profiles of cytokine mRNA expression and cytokine secretion levels in old BWF1 mice, in comparison with uninfected age-matched BWF1 mice and infected or uninfected BALB/c mice. Two months after infection, cells from BWF1 mice were stimulated with concanavalin A (Con A) and demonstrated a recovery of T cell responsiveness that reached the levels obtained with BALB/c cells. Old BWF1 mice showed high levels of interferon-gamma (IFN-gamma) and IL-5 production and correspondingly low levels of IL-2 and IL-4 secretion before infection with P. chabaudi. Infection did not modify the IFN-gamma levels of BWF1 T cells, whereas it considerably increased the secretion of the Th2-related cytokines IL-4, IL-5 and IL-10. In addition, only BWF1 T cells showed increased mRNA expression of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta). This counter-regulatory cytokine network of infected BWF1 mice may be involved in the improvement of their lupus symptoms. The results of our investigations using the complex model of P. chabaudi infection can be extended and, by using more restricted approaches, it may be possible to explain the multiple regulatory defects of lupus-prone mice.     

A study of mast cells in autoimmune NZB/W F1 mice: possible relationship between mast cells and increased vascular permeability in the thymus of NZB/W F1 mice.

We examined the possible relationship between thymic mast cells and increased vascular permeability in the thymus of autoimmune NZB/W F1 mice. Light-microscopic observation of tissue sections showed that non-autoimmune BDF1 mast cells increased with age. In contrast, autoimmune NZB/W F1 mast cells did not increase in the thymic parenchyma at the age of 9 weeks. However, NZB/W F1 mast cells resumed the age-associated increase from the age of 12 weeks and exceeded the number of BDF1 mast cells at the age of 30 weeks. Blood histamine levels of 9-week-old NZB/W F1 mice were higher than those of BDF1 mice of comparable age. Furthermore, peritoneal mast cells of NZB/W F1 mice were more sensitive to compound 48/80 than those of BDF1 mice. Increased blood histamine levels of NZB/W F1 mice seem to be due to the enhanced histamine release from mast cells. These results suggest a possible correlation between the high histamine levels by degranulation of mast cells and increased vascular permeability in the thymus of NZB/W F1 mice.     

Suppression of autoimmune disease in New Zealand mice associated with infection in malaria. II. NZB mice

The onset of a positive Coombs test was significantly delayed in NZB mice infected with Plasmodium berghei at the age of 1 month. At the age of 12 months, twelve out of fourteen malaria-infected NZB mice had become Coombs positive but their reticulocyte counts and mean spleen weight were significantly lower than those of control NZB mice of the same age.     

Acceleration of autoimmunity in NZB/NZW F1 mice by graft-versus-host disease.

Chronic graft-versus-host (GVH) disease was induced in NZB/NZW F1 (B/W) hybrid female mice by the weekly injection of parental NZB spleen cells. Control mice received injections of syngeneic spleen cells only. The mice were assayed for antibodies to [3H]DNA and [3H]polyadenylic-polyuridylic acid by a cellulose ester filter radioimmunoassay, and for antibody to thymocytes by a cytotoxicity method. GVH disease accelerated the development of all three antibodies in B/W mice. In addition, sucrose density gradient ultracentrifugation of pooled sera suggested that an accelerated switch from 19S to 7S anti-DNA production may be an early effect of GVH. The mechanism of acceleration is discussed in terms of immunological and viral factors generated by the GVH reaction.     

Perforin mRNA expression in the inflamed tissues of NZB/W F1 lupus mice decreases with methylprednisolone treatment.

Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this study, the authors examined perforin mRNA levels in the kidney, spleen, liver, lung, heart, and brain of NZB/W F1 lupus mice and NZW mice. Perforin mRNA levels in the kidney, spleen, liver, and lung of NZB/W F1 mice increased significantly with age, whereas those in the heart and brain of NZB/W F1 mice showed little change between 2 and 10 months of age. In all tissues examined in NZW, control mice perforin mRNA levels showed little change during the experimental period. In addition, the authors examined the effect of methylprednisolone (MPSL) on perforin gene expression in the tissues of NZB/W F1 mice. MPSL ameliorated the increase in perforin mRNA levels in the kidney, spleen, liver, and lung of NZB/W F1 mice. These findings suggest that perforin may contribute to tissue injuries in autoimmune lupus mice and that MPSL may be effective in lupus partly by decreasing perforin expression.     

Beneficial effect of Salmonella typhimurium infection and of immunoglobulins from S. typhimurium-infected mice on the autoimmune disease of (NZB × NZW)F1 mice

Various infections can precede or aggravate autoimmune diseases. Yet a beneficial effect of infection has also been described and various mechanisms have been postulated to explain this effect. The aim of this study was to examine the hypothesis that infection can have an immunoregulatory effect on the autoimmune process via the increased production of natural polyreactive antibodies. The effect of Salmonella typhimurium infection on the lupus-like disease of (NZB × NZW)F₁ (B/W) mice was therefore studied. The effect of IgM and IgG preparations isolated from the serum of S. typhimurium-infected C57Bl/6 and CBA mice on the autoimmune disease of B/W mice was also tested. C57Bl/6 and CBA mice were chosen because they are respectively genetically susceptible and resistant to S. typhimurium infection and they differ in their antibody response during the early phase of infection. CBA mice can mount a specific anti-bacterium antibody response, whereas C57Bl/6 mice present increased production of polyreactive antibodies. The infection effect was evaluated on several disease parameters, i.e. survival, incidence of high grade proteinuria and serum IgM and IgG antibody activity directed against a panel of autoantigens. Our main findings were: (i) infection of B/W mice with an attenuated strain of S. typhimurium delayed the course of the autoimmune disease when performed before the appearance of autoimmune symptoms; and (ii) IgM and IgG preparations from S. typhimurium-infected C57Bl/6 mice had a similar effect, whereas the IgM and IgG preparations from infected CBA mice, as well as from normal C57Bl/6 and CBA mice, were ineffective. These results suggest that S. typhimurium infection can beneficially influence the development of the autoimmune disease of B/W mice. The immunoregulatory effect of the infection seems to be related, at least partially, to the increase of a particular population of antibodies, the polyreactive antibodies.     

The effects of CP 17193, an immunosuppressive pyrazaloquinoline, on the development of spontaneous lupus disease in NZBW F1 hybrid mice.

The effects of the immunosuppressive agent CP 17193 on the development of spontaneous lupus disease in female NZBW F1 hybrid mice were investigated. Long term dosing with CP 17193 markedly delayed the onset of mortality but did not extend the long term survival of the mice. CP 17193 significantly inhibited immune complex deposition in the glomeruli of 30- and 35-week-old mice and also reduced the levels of proteinuria in the 35-week-old mice. There was a slight reduction in the levels of circulating antinuclear antibody to ds DNA in CP 17193-treated mice but this was not statistically significant. Studies on immune cell function of 35-week-old mice dosed with CP 17193 showed significant reduction in the total numbers of spontaneous polyclonal antibody producing cells. Analysis of the results revealed these effects to result from a marked reduction in total spleen cell numbers in CP 17193-treated mice. When results were expressed as activity per cell unit the differences between drug-treated and control mice were small. Spleen cells from mice given a shorter dosing schedule of 7 weeks with CP 17193 showed an augmentation of IL-2 production and responsiveness. These results show CP 17193 having interesting selective immunomodulating activity on the immunopathogenesis of spontaneous murine lupus disease. Furthermore, compounds with this profile of activity may have a potential role in the treatment of some autoimmune diseases.     

Effects of antigen and internal environment on anti-phosphorylcholine immune responses of autoimmune aged NZB/W F1 mice.

The idiotypic profile of anti-phosphorylcholine plaque-forming cell responses and their evolution with ageing were studied in (NZB X NZW) F1 mice. Our results showed that the anti-phosphorylcholine plaque-forming cell response induced by phosphorylcholine coupled to keyhole limpet haemocyanin and, paralleling, the T15 idiotype clonal dominance declined with ageing. This loss of immune competence was also observed with another thymus-dependent (phosphorylcholine coupled to egg globulin) as well as thymus-independent (capsular polysaccharide of Streptococcus pneumoniae strain R36a) antigens. In contrast, old mice challenged with an antigenic preparation of Neisseria meningitidis showed an immune response not significantly different from that elicited by the same antigen in young mice. The hapten-augmentable plaque-forming cells were assayed to determine whether a putative auto-antiidiotypic regulation underlies this loss of immune competence. Only minimal numbers and non-significant differences between young and old mice immunized with any antigen could be detected. Further studies using an adoptive transfer system demonstrated that cells from aged mice were able to support a normal anti-phosphorylcholine response when transferred into lethally irradiated young recipients. Our results suggest that no permanent cellular defects, but rather internal environment or/and radioresistant suppressor cells, are involved in this loss of immune competence. The role played by these factors and their effect on distinct subpopulations of B cells are discussed.     

Immune enhancement by novel vaccine adjuvants in autoimmune-prone NZB/W F1 mice: relative efficacy and safety

Background

Vaccines have profoundly impacted global health although concerns persist about their potential role in autoimmune or other adverse reactions. To address these concerns, vaccine components like immunogens and adjuvants require critical evaluation not only in healthy subjects but also in those genetically averse to vaccine constituents. Evaluation in autoimmune-prone animal models of adjuvants is therefore important in vaccine development. The objective here was to assess the effectiveness of experimental adjuvants: two phytol-derived immunostimulants PHIS-01 (phytanol) and PHIS-03 (phytanyl mannose), and a new commercial adjuvant from porcine small intestinal submucosa (SIS-H), relative to a standard adjuvant alum. Phytol derivatives are hydrophobic, oil-in water diterpenoids, while alum is hydrophilic, and SIS is essentially a biodegradable and collagenous protein cocktail derived from extracellular matrices.

Results

We studied phthalate -specific and cross-reactive anti-DNA antibody responses, and parameters associated with the onset of autoimmune disorders. We determined antibody isotype and cytokine/chemokine milieu induced by the above experimental adjuvants relative to alum. Our results indicated that the phytol-derived adjuvant PHIS-01 exceeded alum in enhancing anti-phthalate antibody without much cross reactivity with ds-DNA. Relatively, SIS and PHIS-03 proved less robust, but they were also less inflammatory. Interestingly, these adjuvants facilitated isotype switching of anti-hapten, but not of anti-DNA response. The current study reaffirms our earlier reports on adjuvanticity of phytol compounds and SIS-H in non autoimmune-prone BALB/c and C57BL/6 mice. These adjuvants are as effective as alum also in autoimmune-prone NZB/WF1 mice, and they have little deleterious effects.

Conclusion

Although all adjuvants tested impacted cytokine/chemokine milieu in favor of Th1/Th2 balance, the phytol compounds fared better in reducing the onset of autoimmune syndromes. However, SIS is least inflammatory among the adjuvants evaluated.