The role of apoptosis in the ameliorating effects of a CDR1-based peptide on lupus manifestations in a mouse model

Experimental systemic lupus erythematosus (SLE) can be induced in mice following immunization with an anti-DNA mAb expressing a major Id, 16/6Id. Treatment with a peptide, designated human CDR1 (hCDR1; Edratide), that is based on the sequence of CDR1 of the 16/6Id ameliorated disease manifestations. In the present study, we investigated the roles of apoptosis and related molecules in BALB/c mice with induced experimental SLE following treatment with hCDR1. A higher state of activation and increased rate of apoptosis were found in lymphocytes of SLE-afflicted mice as compared with healthy controls. The latter effects were associated with up-regulated caspase-8 and caspase-3, and down-regulated Bcl-x(L). The ameliorative effects of hCDR1 were associated with down-regulation of caspase-8 and caspase-3, up-regulation of Bcl-x(L), and a reduced rate of apoptosis. Treatment of diseased mice with an apoptosis-reducing compound that inhibited caspases down-regulated the secretion of the pathogenic cytokine IFN-gamma and lowered the intensity of glomerular immune complex deposits and the levels of proteinuria. Furthermore, coincubation of Bcl-x(L) inhibitors with hCDR1-treated cells abrogated the ability of hCDR1 to reduce the activation state of lymphocytes and to down-regulate the secretion of IL-10 and IFN-gamma. Moreover, the Bcl-x(L)-expressing CD4(+)CD25(+) cells from hCDR1-treated mice induced the expression of Bcl-x(L) in CFSE-labeled CD4(+)CD25(-) cells of the SLE-afflicted mice. Thus, the reduction of apoptosis and the up-regulation of Bcl-x(L), which plays an apparent role in tolerance induction, contribute to at least part of the beneficial effects of hCDR1 on lupus manifestations.     

Down-regulation of stromal cell-derived factor-1alpha-induced T cell chemotaxis by a peptide based on the complementarity-determining region 1 of an anti-DNA autoantibody via up-regulation of TGF-beta secretion

Systemic lupus erythematosus (SLE) can be induced in mice by immunizing them with a monoclonal human anti-DNA Ab that expresses a major Id, designated 16/6Id. In addition, a peptide based on the sequence of the CDR 1 (hCDR1) of the 16/6Id ameliorated the clinical manifestations of SLE in experimental models. In this study we examined the effects of treating mice with human complementary-determining region 1 (hCDR1) on the subsequent chemotaxis of T cells derived from 16/6Id-primed mice. First we demonstrated elevated levels of stromal cell-derived factor-1alpha (SDF-1alpha) in the sera of SLE-afflicted mice and in the sera and lymphoid tissues of 16/6Id-immunized BALB/c mice shortly after the immunization. We then found that administration of hCDR1 to 16/6Id-immunized mice specifically down-regulated SDF1alpha-induced T cell chemotaxis through fibronectin and collagen type I. This was accompanied by diminished SDF1-alpha-induced T cell adhesion and ERK phosphorylation. Treatment with hCDR1 up-regulated TGF-beta secretion, which, in turn, inhibited the murine T cell adhesion to and chemotaxis through fibronectin as well as their ERK phosphorylation. Thus, the secretion of TGF-beta after treatment of 16/6Id-immunized mice with hCDR1 plays an important role in the down-regulation of SDF-1alpha-mediated T cell activation and the interactions with extracellular matrix moieties observed in the present study.     

The inhibition of autoreactive T cell functions by a peptide based on the CDR1 of an anti-DNA autoantibody is via TGF-beta-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-beta secretion 10 days later. Addition of TGF-beta suppressed the 16/6Id-stimulated T cell proliferation similarly to hCDR1. In addition, we provide evidence that one possible mechanism underlying the hCDR1- and TGFbeta-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 (alphaLbeta2) and CD44, which operate as accessory molecules in mediating APC-T cell interactions. Indeed, T cells of mice treated with hCDR1 showed a TGF-beta-induced suppression of adhesion to the LFA-1 and CD44 ligands, hyaluronic acid and ICAM-1, respectively, induced by stromal cell-derived factor-1alpha 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-beta on the expression and function of T cell adhesion receptors and, consequently, on T cell stimulation, adhesion, and proliferation.     

Pathogenic idiotypes of autoantibodies in autoimmunity: lessons from new experimental models of SLE

Systemic lupus erythematosus (SLE) is considered a classical autoimmune disease that involves many biological systems. Similar to other autoimmune conditions, its etiology is multifactorial entailing genetic, environmental, hormonal, and immunologic factors. In this review we demonstrate that by using a pathogenic idiotype of anti-DNA autoantibodies, it is possible to explain some of the pathogenesis and diversity of clinical and serological manifestations reported by SLE patients. The 16/6 idiotype (Id) is a representative pathogenic idiotype of anti-DNA autoantibodies. The serum titers of 16/6 Id in SLE patients correlate with clinical activity of the disease, and are deposited in afflicted tissues in SLE patients. SLE was experimentally induced in naive mice after immunization with 1 microgram of the Id. The disease is characterized clinically (proteinuria), serologically (e.g., anti-dsDNA, anti-Sm antibodies), and by pathological findings (e.g., deposition of 16/6 Id in the kidneys). The condition can be induced by other human and mouse antibodies carrying the 16/6 Id, as well as by mouse antimonoclonal-16/6 Id and by T cell lines and clones specifically reactive with 16/6 Id. There are strain-dependent differences in susceptibility to the induction of systemic lupus erythematosus (SLE). Induction of SLE is directly correlated with the ability to respond to the 16/6 idiotype (or 16/6 Id)2 by anti-Id antibody production. It is easier to induce the disease in females, and it can be modulated by manipulation of sex hormones. Being able to identify the pathogenic idiotype allowed us to generate T suppressor (Ts) cells specific for the 16/6 Id. Treatment of mice with these T cells abrogated the disease. Our studies point to the importance of pathogenic idiotypes of autoantibodies in autoimmunity, which suggests that SLE may represent a dysregulation of a functional network of idiotypes-anti-idiotypes interactions among autoreactive B cells, T helper cells, and T suppressor cells.     

The Tolerogenic Peptide,hCDR1, Down-Regulates the Expression of Interferon-α in Murine and Human Systemic Lupus Erythematosus

Background

The tolerogenic peptide, hCDR1, ameliorated manifestations of systemic lupus erythematosus (SLE) via the immunomodulation of pro-inflammatory and immunosuppressive cytokines and the induction of regulatory T cells. Because type I interferon (IFN-α) has been implicated to play a role in SLE pathogenesis, we investigated the effects of hCDR1 on IFN-α in a murine model of SLE and in human lupus.

Methodology/Principal Findings

(NZBxNZW)F1 mice with established SLE were treated with hCDR1 (10 weekly injections). Splenocytes were obtained for gene expression studies by real-time RT-PCR. hCDR1 down-regulated significantly IFN-α gene expression (73% inhibition compared to vehicle treated mice, p = 0.002) in association with diminished clinical manifestations. Further, hCDR1 reduced, in vitro, IFN-α gene expression in peripheral blood mononuclear cells (PBMC) of 10 lupus patients (74% inhibition compared to medium, p = 0.002) but had no significant effects on the expression levels of IFN-α in PBMC of primary anti-phospholipid syndrome patients or of healthy controls. Lupus patients were treated for 24 weeks with hCDR1 (5) or placebo (4) by weekly subcutaneous injections. Blood samples collected, before and after treatment, were frozen until mRNA isolation. A significant reduction in IFN-α was determined in hCDR1 treated patients (64.4% inhibition compared to pretreatment expression levels, p = 0.015). No inhibition was observed in the placebo treated patients. In agreement, treatment with hCDR1 resulted in a significant decrease of disease activity. IFN-α appears to play a role in the mechanism of action of hCDR1 since recombinant IFN-α diminished the immunomodulating effects of hCDR1 on IL-1β, TGFβ and FoxP3 gene expression.

Conclusions/Significance

We reported previously that hCDR1 affected various cell types and immune pathways in correlation to disease amelioration. The present studies demonstrate that hCDR1 is also capable of down-regulating significantly (and specifically to lupus) IFN-α gene expression. Thus, hCDR1 has a potential role as a novel, disease specific treatment for lupus.     

A conserved anti-DNA antibody idiotype associated with nephritis in murine and human systemic lupus erythematosus

In order to identify unique structural features of pathogenic autoantibodies to DNA in SLE, a murine anti-anti-DNA (anti-Id) mAb (mAb 1C7) was produced in response to immunization of lupus mice with a syngeneic anti-DNA mAb (mAb 3E10). Immunization of lupus mice with mAb 3E10 inhibited production of native anti-DNA antibodies, suppressed development of lupus kidney disease (nephritis), and induced production of anti-anti-DNA (anti-Id) antibodies. mAb 1C7 bound F(ab')2 fragments of mAb 3E10, and it bound other murine anti-DNA mAb, but not murine mAb or polyclonal serum antibodies unreactive with DNA. Moreover, binding of mAb 1C7 anti-Id to mAb 3E10 was inhibited by DNA, suggesting anti-Id binding within or near the binding site for DNA. Furthermore, mAb 1C7 bound serum IgG immunoglobulins from 9/12 patients with lupus nephritis and serum anti-DNA antibodies compared to only 3/12 SLE patients with comparable serum levels of anti-DNA antibodies, but without nephritis (p = 0.04), and only 1/53 SLE patients without serum anti-DNA antibodies, 0/49 patients with rheumatoid arthritis, and 1/47 healthy subjects (p less than 0.001). These results provide evidence that mAb 1C7 identifies a conserved Id associated with anti-DNA antibodies in murine and human SLE and may be useful as a structural probe to characterize pathogenic anti-DNA antibodies in SLE.     

Blockade of programmed death-1 in young (New Zealand black x New Zealand white)F1 mice promotes the activity of suppressive CD8+ T cells that protect from lupus-like disease

The programmed death-1 (PD-1)/programmed death-1 ligand 1 (PD-L1) pathway regulates both stimulatory and inhibitory signals. In some conditions, PD-1/PD-L1 inhibits T and B cell activation, induces anergy, and reduces cytotoxicity in CD8(+) T cells. In other conditions, PD-l/PD-L1 has costimulatory effects on T cells. We recently showed that induction of suppressive CD8(+)Foxp3(+) T cells by immune tolerance of lupus-prone (New Zealand black × New Zealand white)F(1) (BWF(1)) mice with the anti-DNA Ig-based peptide pConsensus (pCons) is associated with significantly reduced PD-1 expression on those cells. In this study, we tested directly the role of PD-1 by administering in vivo neutralizing Ab to PD-1 to premorbid BWF(1) and healthy control mice. Anti-PD-1-treated mice were protected from the onset of lupus nephritis for 10 wk, with significantly improved survival. Although the numbers of T cells declined in aging control mice, they were maintained in anti-PD-1-treated mice, including CD8(+)Foxp3(+) T cells that suppressed syngeneic CD4(+)CD25(-) T cell proliferation and IFN-γ production, reduced production of IgG and anti-dsDNA IgG, induced apoptosis in syngeneic B cells, and increased IL-2 and TGF-β production. The administration of anti-PD-1 Ab to BWF(1) mice after induction of tolerance with pCons abrogated tolerance; mice developed autoantibodies and nephritis at the same time as control mice, being unable to induce CD8(+)Foxp3(+) T suppressor cells. These data suggest that tightly regulated PD-1 expression is essential for the maintenance of immune tolerance mediated by those CD8(+)Foxp3(+) T cells that suppress both T(h) cells and pathogenic B cells. PD-1 regulation could represent a target to preserve tolerance and prevent autoimmunity.     

Cutting edge: lupus susceptibility interval Sle3/5 confers responsiveness to prolactin in C57BL/6 mice

Prolactin is of interest in the pathogenesis of systemic lupus erythematosus (SLE) because almost 25% of SLE patients display hyperprolactinemia, and serum prolactin correlates with disease activity in some patients. Furthermore, hyperprolactinemia causes early mortality in lupus-prone mice and induces a lupus-like phenotype in nonspontaneously autoimmune mice. We show here that the immunomodulatory effects of prolactin are genetically determined; hyperprolactinemia breaks B cell tolerance and causes a lupus-like serology in BALB/c mice expressing a transgene encoding the H chain of an anti-DNA Ab but not in C57BL/6 transgenic mice. In C57BL/6 mice that express both the H chain transgene and the lupus susceptibility interval Sle3/5, prolactin induces increased serum titers of anti-DNA Ab and glomerular Ig depositions. The increase in costimulation due to prolactin-mediated up-regulation of both CD40 on B cells and CD40L on T cells would appear to play a central role in lupus induction in this model.     

Aberrant genetic control of invariant TCR-bearing NKT cell function in New Zealand mouse strains: possible involvement in systemic lupus erythematosus pathogenesis

Both suppressive and promoting roles of NKT cells have been reported in the pathogenesis of systemic lupus erythematosus (SLE). Herein, we found that although New Zealand mice have normal frequencies of NKT cells, their in vitro potential to produce IL-4 and IFN-gamma in response to alpha-galactosylceramide was remarkably impaired in New Zealand Black (NZB) mice prone to mild SLE, while production was highly up-regulated in nonautoimmune New Zealand White (NZW) mice and at intermediate levels in (NZB x NZW)F(1) mice, which are prone to severe SLE. Because this aberration is evident in young mice before disease onset, genetic mechanisms are thought to be involved. Genome-wide quantitative trait locus analysis and association studies revealed that a locus linked to D11Mit14 on chromosome 11 may be involved in the difference in cytokine-producing potential between NZB and NZW NKT cells. Additionally, (NZB x NZW)F(1) x NZB backcross progeny with the NZW genotype for D11Mit14 showed significantly increased frequencies of age-associated SLE phenotypes, such as high serum levels of IgG, IgG anti-DNA Abs, and lupus nephritis. In coculture studies, alpha-galactosylceramide-stimulated NKT cells from NZW and (NZB x NZW)F(1) mice, but not from NZB mice, showed significantly enhanced Ig synthesis by B cells. These findings suggest that the D11Mit14-linked NZW locus may contribute to the development of SLE in (NZB x NZW)F(1) mice through a mechanism that up-regulates NKT cell function. Thus, this NZW allele may be a candidate of the NZW modifiers that act to promote (NZB x NZW)F(1) disease.     

The role of the idiotypic network in the induction of experimental systemic lupus erythematosus

Systemic lupus erythematosus (SLE) has been induced in C3H.SW mice by their immunization with a human monoclonal anti-DNA antibody that bears a common idiotype-16/6 Id. Following immunization, high levels of murine anti-16/6 and anti-anti-16/6 antibodies were detected in the sera of the immunized mice. Elevated titers of autoantibodies reacting with ssDNA, dsDNA, poly(I), poly(G), RNP, Ro, and La were also observed. The serological findings were associated with significant proteinuria, leukopenia, and elevated erythrocyte sedimentation rate. Immune complex deposition in the glomerular mesangium and sclerosis of the glomeruli were demonstrated. To study whether or not anti-idiotype antibodies are involved in the induction of the disease, a murine monoclonal antibody against the 16/6 Id was prepared and injected into C3H.SW mice. The anti-16/6 Id antibody induced experimental SLE similarly to the 16/6 Id with an accelerated kidney pathology. A study performed on different mouse strains indicated that the susceptibility to the induction of SLE by the 16/6 Id is strain dependent and directly correlates to their ability to produce anti-16/6 Id specific antibodies.     

Autocrine production of IFN-gamma by macrophages controls their recruitment to kidney and the development of glomerulonephritis in MRL/lpr mice

Anti-DNA autoantibody production is a key factor in lupus erythematosus development; nonetheless, the link between glomerular anti-DNA autoantibody deposition and glomerulonephritis development is not understood. To study the inflammatory and destructive processes in kidney, we used IFN-gamma(+/-) MRL/lpr mice which produce high anti-DNA Ab levels but are protected from kidney disease. The results showed that defective macrophage recruitment to IFN-gamma(+/-) mouse kidney was not caused by decreased levels of monocyte chemoattractant protein-1, a chemokine that controls macrophage migration to MRL/lpr mouse kidney. To determine which IFN-gamma-producing cell type orchestrates the inflammation pathway in kidney, we transferred IFN-gamma(+/+) monocyte/macrophages or T cells to IFN-gamma(-/-) mice, which do not develop anti-DNA autoantibodies. The data demonstrate that IFN-gamma production by infiltrating macrophages, and not by T cells, is responsible for adhesion molecule up-regulation, macrophage accumulation, and inflammation in kidney, even in the absence of autoantibody deposits. Therefore, in addition to monocyte chemoattractant protein-1, macrophage-produced IFN-gamma controls macrophage migration to kidney; the degree of IFN-gamma production by macrophages also regulates glomerulonephritis development. Our findings establish the level of IFN-gamma secretion by macrophages as a link between anti-DNA autoantibody deposition and glomerulonephritis development, outline the pathway of the inflammatory process, and suggest potential treatment for disease even after autoantibody development.     

CD8+ T cell-mediated suppression of autoimmunity in a murine lupus model of peptide-induced immune tolerance depends on Foxp3 expression

Systemic lupus erythematosus is an autoimmune disease caused by autoantibodies, including IgG anti-DNA. New Zealand Black/New Zealand White F(1) female mice, a model of spontaneous polygenic systemic lupus erythematosus, tolerized with an artificial peptide (pConsensus) based on anti-DNA IgG sequences containing MHC class I and class II T cell determinants, develop regulatory CD4+CD25+ T cells and CD8+ inhibitory T cells (CD8+ Ti), both of which suppress autoantibody production. CD8+ Ti inhibit primarily via secretion of TGF-beta. In the present study, we show that the inhibitory function of CD8+ T cells from tolerized mice is sustained for up to 8 wk and at all times depends on expression of Foxp3. Both CD28-positive and CD28-negative CD8+ T cells contain inhibitory cells, but the expression of mRNA for Foxp3 and for TGF-beta is higher and lasts longer in the CD28- subset. In vitro addition of TGF-beta (in the presence of IL-2) induces Foxp3 expression in a dose-response manner. Gene inhibition or blockade with small interfering RNA of Foxp3 abrogates the ability of the CD8+ Ti to inhibit anti-DNA production and the proliferation of CD4+ Th cells. Moreover, a significant correlation between expression of Foxp3 and ability of CD8+ Ti to secrete TGF-beta is observed. Therefore, CD8+ Ti in this system of tolerance are similar to CD4+CD25+ regulatory T cells in their dependence on expression of Foxp3, and there may be a bidirectional Foxp3/TGF-beta autocrine loop that determines the ability of the CD8+ T cells to control autoimmunity.     

Mesenchymal stem cell transplantation inhibits abnormal activation of Akt/GSK3β signaling pathway in T cells from systemic lupus erythematosus mice

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by activation and proliferation of autoreactive T cells and B cells. We examined changes in cell cycle progression of T cells from MRL/lpr mice with or without allogenic bone marrow mesenchymal stem cells (BMMSCs) treatment and analyzed the expression of cell cycle associated proteins. In addition, the Akt/GSK3β protein kinase cascade was studied. We demonstrated that high-dose MSCs transplantation effectively ameliorated disease activity in MRL/lpr mice. BMMSCs treatment inhibited G1/S transition of the abnormal lupus T lymphocytes. Moreover, it increased the expression of p21(WAF1/CIP1) and p27(Kip1) and decreased the expression of CDK2. Furthermore, high-dose MSCs inhibited abnormal activation of the Akt/GSK3β signaling pathway of T cells from MRL/lpr mice. Our results suggest that high-dose BMMSCs transplantation successfully treated MRL/lpr lupus mice by inhibiting abnormal activation of Akt/GSK3β signaling pathway of T cells.     

Anti-DNA antibody idiotypes in primary biliary cirrhosis

A significant increase in 16/6 Id--a major cross-reactive idiotype of anti-DNA antibodies (Ab) derived from a patient with systemic lupus erythematosus (SLE) and hitherto identified in SLE patients and their relatives, was found in 16/17 patients with primary biliary cirrhosis (PBC). The increased serum level of Ab with the 16/6 idiotype (16/6 Id) in PBC patients (median 50 ng/ml) was not found in 6/7 of the patients' spouses nor among 27/28 healthy controls or most patients with other types of cirrhosis. The quantity of 16/6 Id was not correlated to either the stage of disease or the presence of antimitochondrial, antinuclear, or anti-dsDNA antibodies. However, 16/6 Id could be shown to be associated with anti-ssDNA antibodies. The high frequency of the lupus-derived 16/6 Id in PBC may accompany the polyclonal B-cell activation seen in that disease. Of 14 healthy first-degree relatives of the PBC patients, 4 (29%) also had elevated serum 16/6 Id (20-25 ng/ml) and the cluster of 3 of them in a single family may indicate a genetic predisposition to develop PBC.     

Tumor cell lysate-pulsed dendritic cells are more effective than TCR Id protein vaccines for active immunotherapy of T cell lymphoma

TCR Id protein conjugated to keyhole limpet hemocyanin (KLH) (TCR Id:KLH) and injected with a chemical adjuvant (QS-21) induces a protective, Id-specific immune response against the murine T cell lymphoma, C6VL. However, Id-based immunotherapy of C6VL has not demonstrated therapeutic efficacy in tumor-bearing mice. We report here that C6VL lysate-pulsed dendritic cells (C6VL-DC) vaccines display enhanced efficacy in both the prevention and the therapy of T cell lymphoma compared with TCR Id:KLH with QS-21 vaccines. C6VL-DC vaccines stimulated potent tumor-specific immunity that protected mice against lethal challenge with C6VL and significantly enhanced the survival of tumor-bearing mice. Tumor-specific proliferation and secretion of IFN-gamma indicative of a Th1-type immune response were observed upon ex vivo stimulation of vaccine-primed lymph node cells. Adoptive transfer of immune T cell-enriched lymphocytes was sufficient to protect naive recipients from lethal tumor challenge. Furthermore, CD8(+) T cells were absolutely required for tumor protection. Although C6VL-DC and control vaccines stimulated low levels of tumor-specific Ab production in mice, Ab levels did not correlate with the protective ability of the vaccine. Thus, tumor cell lysate-pulsed DC vaccines appear to be an effective approach to generate potent T cell-mediated immune responses against T cell malignancies without requiring identification of tumor-specific Ags or patient-specific Id protein expression.     

Spontaneous shift of anti-DNA antibody idiotypes in systemic lupus erythematosus

Spontaneous shift in Id expression of polyclonal anti-DNA antibodies in a patient, BS, with SLE was investigated. BS had active lupus nephritis in 1982 and developed central nervous system lupus in 1986 without evidence of active nephritis. Two rabbit polyclonal anti-Id (BS-82 and BS-86 R-anti-Id) were raised against affinity-purified anti-DNA antibodies prepared from 1982 serum (BS-82) and 1986 serum (BS-86), respectively. In addition, murine monoclonal anti-Id was prepared against BS-82 Id. Direct binding assays showed that all three anti-Id had preferential binding to the immunizing anti-DNA antibodies (the homologous Id) and poor binding to anti-DNA antibodies prepared from the different dated sample of BS. This was confirmed by inhibition assays of binding of anti-Id to the homologous Id by various Id. Moreover, inhibition assays of binding of various Id to DNA by the R-anti-Id showed that the R-anti-Id was the most effective inhibitor for the homologous Id. Testing for Id expression in serial (1982 to 1986) serum samples of BS with the R-anti-Id as probes showed that BS-82 Id declined and was undetectable after October, 1984, whereas BS-86 Id was first detectable in July, 1985, and increased by June, 1986. These results clearly demonstrate spontaneous shifts in Id expression of human anti-DNA antibodies. The phenomenon of Id shift should be considered in any future strategy for the diagnosis and therapy of human autoimmune disease by anti-Id.     

Lupus serum and normal human serum contain anti-DNA antibodies with the same idiotypic marker

We have previously shown that serum from patients with active SLE contain high levels of Id-16/6 and anti-DNA antibodies. In this study we investigated whether serum Id 16/6 is related to anti-DNA antibodies. Sera from 12 patients with active SLE were absorbed individually with poly(dT) cellulose (to purify anti-DNA antibodies) and rabbit (R) anti-Id-16/6 Sepharose (to purify Id 16/6 Ig). Removal of all anti-DNA activity removed most of the Id-16/6. Conversely, removal of all Id 16/6 removed most of the anti-DNA activity. Although there was no measurable anti-DNA antibody activity in normal serum, such antibodies were isolated by absorption with poly(dT) cellulose. The eluted immunoglobulins also had Id 16/6 activity. Similarly, Id 16/6 with anti-DNA activity were isolated from normal serum by absorption with R anti-Id 16/6 Sepharose. We conclude that a large fraction of anti-DNA antibodies in SLE serum are Id-16/6+, and that most Id 16/6 immunoglobulins in lupus serum have anti-DNA activity. Our observations suggest that lupus anti-DNA antibodies result from an overproduction of autoantibodies that are present in normal people.     

Tolerogenic treatment of lupus mice with consensus peptide induces Foxp3-expressing, apoptosis-resistant, TGFbeta-secreting CD8+ T cell suppressors

Lupus-prone (NZB x NZW)F1 mice spontaneously develop elevated titers of anti-DNA Abs that contain T cell determinants in their V(H) regions. We have previously shown that tolerization with an artificial peptide based on these T cell determinants (pConsensus (pCons)) can block production of anti-DNA Abs and prolong survival of the mice. In this study, we show that this protection depends in part on the generation of peripheral TGFbeta- and Foxp3-expressing inhibitory CD8+ (Ti) cells. These CD8+ Ti cells suppress anti-DNA IgG production both in vitro and in vivo and require up-regulated expression of both Foxp3 and TGFbeta to exert their suppressive function, as indicated by microarray analyses, small interfering RNA inhibition studies, and blocking experiments. Additionally, CD8+ Ti cells from pCons-tolerized mice were longer-lived suppressors that up-regulated expression of Bcl-2 and were more resistant to apoptosis than similar cells from naive mice. These data indicate that clinical suppression of autoimmunity after administration of pCons depends in part on the generation of CD8+ Ti cells that suppress secretion of anti-DNA Ig using mechanisms that include Foxp3, TGFbeta, and resistance to apoptosis.     

IL-1 beta-deficient mice are resistant to induction of experimental SLE

IL-1 is one of the most pleiotropic pro-inflammatory and immunostimulatory cytokines. Overproduction of IL-1 has been shown to be involved in the pathogenicity of various autoimmune inflammatory diseases, including systemic lupus erythematosus (SLE). However, the different contributions that the IL-1 agonistic molecules make in their in vivo native milieu, IL-1beta which is mainly secreted against IL-1alpha which is mainly cell-associated, have not been established. Experimental SLE can be induced in mice by injection with monoclonal anti-DNA antibodies bearing a major idiotype designated, 16/6Id. In the present study, experimental SLE was induced in mice deficient in specific IL-1 molecules, i.e. IL-1alpha(-/-), IL-1beta(-/-), IL-1alpha/beta(-/-) (double KO) and in control BALB/c mice. Mice deficient in IL-1beta , i.e. IL-1beta(-/-) and IL-1alpha/beta(-/-) mice, developed lower levels of anti-dsDNA antibodies after immunization with 16/6Id, as compared to IL-1alpha(-/-) or control BALB/c mice. Disease manifestations were milder in mice deficient in IL-1beta expression. The representative cytokine cascade that is characteristic of overt experimental SLE was also shown to be reduced in groups of mice that lacked IL-1beta as compared to mice deficient in IL-1alpha, which is mainly cell-associated. Altogether, our results point to the importance of secretable IL-1beta, rather than cell-associated IL-1alpha, in the immunostimulatory and inflammatory phenomena that mediate the pathogenesis of experimental SLE.