Rina Ulmansky

Resistance to Adjuvant Arthritis Is Due to Protective Antibodies Against Heat Shock Protein Surface Epitopes and the Induction of IL-10 Secretion

Adjuvant arthritis (AA) is an experimental model of autoimmune arthritis that can be induced in susceptible strains of rats such as inbred Lewis upon immunization with CFA. AA cannot be induced in resistant strains like Brown-Norway or in Lewis rats after recovery from arthritis. We have previously shown that resistance to AA is due to the presence of natural as well as acquired anti-heat shock protein (HSP) Abs. In this work we have studied the fine specificity of the protective anti-HSP Abs by analysis of their interaction with a panel of overlapping peptides covering the whole HSP molecule. We found that arthritis-susceptible rats lack Abs to a small number of defined epitopes of the mycobacterial HSP65. These Abs are found naturally in resistant strains and are acquired by Lewis rats after recovery from the disease. Active vaccination of Lewis rats with the protective epitopes as well as passive vaccination with these Abs induced suppression of arthritis. Incubation of murine and human mononuclear cells with the protective Abs induced secretion of IL-10. Analysis of the primary and tertiary structure of the whole Mycobacterium tuberculosis HSP65 molecule indicated that the protective epitopes are B cell epitopes with nonconserved amino acid sequences found on the outer surface of the molecule. We conclude that HSP, the Ag that contains the pathogenic T cell epitopes in AA, also contains protective B cell epitopes exposed on its surface, and that natural and acquired resistance to AA is associated with the ability to respond to these epitopes.

Treatment with a Laminin-Derived Peptide Suppresses Lupus Nephritis

The role of DNA as the target for pathogenic lupus autoantibodies in systemic lupus erythematosus is equivocal and renal damage may be due to cross-reactivity of lupus Abs with glomerular components. We have previously shown that lupus autoantibodies bind to the laminin component of the extracellular matrix. In the present work, we have analyzed the fine specificity of the interaction of pathogenic murine lupus autoantibodies with this molecule and the effect of inhibiting their binding to laminin during the course of the disease. We have found that pathogenic murine lupus autoantibodies react with a 21-mer peptide located in the globular part of the α-chain of laminin. Immunization of young lupus-prone mice with this peptide accelerated renal disease. Analysis of transgenic, congenic, and RAG-1−/− mice confirmed the importance of this epitope in the pathogenesis of lupus renal disease. We have synthesized a panel of peptides that cross-react with the anti-laminin Abs and have found that the binding of lupus autoantibodies to the extracellular matrix could be inhibited in vitro by some of these competitive peptides. Treatment of MRL/lpr/lpr mice with these peptides prevented Ab deposition in the kidneys, ameliorated renal disease, and prolonged survival of the peptide-treated mice. We suggest that laminin components can serve as the target for lupus Abs. The interaction with these Ags can explain both the tissue distribution and the immunopathological findings in lupus. Moreover, inhibition of autoantibody binding to the extracellular matrix can lead to suppression of disease.

Anti-DNA antibodies cross-reacting with laminin inhibit trophoblast attachment and migration: implications for recurrent pregnancy loss in SLE patients.

PROBLEM: Systemic lupus erythematosus (SLE), an autoimmune disease, is associated with reduced fetal survival, recurrent abortions, and other pregnancy complications. Some of the autoantibodies found in SLE bind to laminins (LNs), which play an important role in the implantation of the fertilized ovum in humans.
 METHOD OF STUDY: To elucidate the role of these specific autoantibodies, chorionic villous explants from 6–7‐week‐old human placentas were established as organ cultures on laminin‐1 (LN‐1), collagen IV (CN‐IV) or uncoated culture dishes. The cultures were then exposed to a mouse monoclonal anti‐DNA/anti‐LN‐1 antibody, to human polyclonal lupus antibodies cross‐reacting with LN‐1, a function‐blocking polyclonal antibody to LN‐1, polyclonal antibodies to CN‐IV, or IgG control.
 RESULTS: The explants attached to LN‐1 and CN‐IV, but not to uncoated culture dishes. LN‐1 promoted migration of trophoblast, whereas CN‐IV promoted migration of fibroblast‐like cells. Trophoblast attachment and migration were abolished in a dose‐dependent manner by all three antibodies to LN‐1, but not by antibodies to CN‐IV or IgG control. Furthermore, the effect of anti‐LN antibodies was abolished by preincubating them with LN‐1.
 CONCLUSIONS: These studies suggest that anti‐DNA antibodies cross‐reacting with LNs may play a role in early pregnancy failure in SLE patients by interfering with placental implantation.

Glucocorticoids in nano-liposomes administered intravenously and subcutaneously to adjuvant arthritis rats are superior to the free drugs in suppressing arthritis and inflammatory cytokines

We have previously shown that intravenous (i.v.) treatment with sterically stabilized nano-liposomes (NSSL) actively remote-loaded with the glucocorticoid (GC) methylprednisolone hemisuccinate (NSSL-MPS) or betamethasone hemisuccinate (NSSL-BMS) significantly decreased severity of adjuvant arthritis in Lewis rats (a model of human rheumatoid arthritis) throughout all disease stages. Here, we compared i.v. or subcutaneous (s.c.) weekly treatment with each of the two NSSL-GC to weekly or daily treatment with the free drugs or with the TNF-α antagonists Infliximab and Etanercept. Therapeutic efficacy and effects on the profile of pro-inflammatory (IL-6, TNF-α, and INF-γ) and anti-inflammatory (IL-10 and TGF-β) cytokines in rat sera and splenocyte tissue culture supernatants were compared to those of the liposomal and free drugs. Both s.c. and i.v. NSSL-GC suppressed arthritis significantly, compared to higher doses of the free drugs or to TNF-α antagonists. NSSL-GC also suppressed the secretion of pro-inflammatory cytokines, but did not change the levels of TGF- β. The highly efficacious anti-inflammatory therapeutic feature of these nano-drugs makes them candidates for treatment of human rheumatoid arthritis.

Prevalence and Clinical Significance of Anticardiolipin, Anti-β2-Glycoprotein-1, and Anti-Heat Shock Protein-70 Autoantibodies in Sudden Sensorineural Hearing Loss

Sudden sensorineural hearing loss (SSNHL) is frequently classified as ‘idiopathic’ since the causative factor responsible for its onset is not identified in most cases. In the present study, we determined whether SSNHL is clinically associated with serum anti-heat shock protein-70 (anti-HSP70) and antiphospholipids (anti-PLs) autoantibodies and whether these autoantibodies have an impact on the prognosis of SSNHL. Sera from 63 patients with SSNHL were screened prospectively for the presence of anti-HSP70 and anti-PLs autoantibodies by an enzyme-linked immunosorbent assay test. Anti-PLs antibodies in this study consisted of anticardiolipin, and anti-β2-glycoprotein-1 antibodies. Serum was assayed for anti-HSP70 IgG antibodies using recombinant human HSP70. Demographic, clinical, and audiometric variables were analyzed to find the possible role of serum autoantibodies in SSNHL patients. Sixteen patients (25.4%) had demonstrable anti-HSP70 antibodies in serum. Twenty-one patients (33.3%) showed a positive result for at least one isotype (IgM or IgG) of anti-PLs. In 19% of the patients, anti-HSP70 and anti-PLs antibodies were positive in two combinations. A statistically significant association was found between anti-HSP70 antibodies and the Siegel recovery grade subgroup. SSNHL patients who were positive for anti-HSP70 antibodies showed a significantly higher rate of complete recovery and incomplete but partial recovery than SSNHL patients without anti-HSP70 antibodies (p = 0.0496). Statistically significant association was found between total anticardiolipin, total anti-β2-glycoprotein-1, total anti-PLs, and anti-PLs in combination with anti-HSP70 antibodies and age (p = 0.0229). The detection of autoantibodies to HSP70 and PLs offers a pliable explanation for the immune-mediated mechanism of SSNHL. The present study confirms and supports previous studies regarding the association between anti-HSP70 and anti-PLs antibodies with SSNHL, and is the first to identify a positive association between anti-HSP70 antibodies and a positive outcome of SSNHL. Further studies are necessary in order to identify and further clarify the immunologic role of the presence of autoantibodies and their impact on the prognosis of SSNHL.

Down-regulation of surface antigens recognized by systemic lupus erythematosus antibodies on embryonal cells following differentiation and exposure to corticosteroids

We have previously suggested that anti‐DNA antibodies present in systemic lupus erythematosus patients can bind directly to tissues as a result of cross‐reactivity with embryonal tissue‐based antigens. Here we have analyzed the interaction between polyclonal and monoclonal mouse and human lupus autoantibodies and an embryonal cell line. We report that a murine embryonal stem cell line (ES) expresses a surface antigen which is recognized by mouse and human lupus autoantibodies. This surface antigen is down‐regulated following maturation of the cells or incubation with corticosteroids. Adhesion molecules may serve as the target membrane antigen in ES cells since preincubation with these antibodies decreases the ability of ES cells to adhere to the plate.

A Humanized Monoclonal Antibody against Heat Shock Protein 60 Suppresses Murine Arthritis and Colitis and Skews the Cytokine Balance toward an Anti-Inflammatory Response

We have previously shown that naturally occurring as well as acquired Abs against the Mycobacterium tuberculosis heat shock protein (HSP)65 protect against the induction of murine autoimmune inflammatory arthritis. In the present work, we have studied the anti-inflammatory effect of prozumab, a humanized anti-HSP mAb in murine inflammatory arthritis and colitis, and its effects on cytokine secretion. Prozumab was shown to bind to HSP60, the highly conserved mammalian homolog of the bacterial protein, and it was found to be effective in protecting and suppressing autoimmune arthritis in the models of adjuvant arthritis and collagen-induced arthritis in rats and mice, respectively, as well as in acute hapten-mediated colitis and chronic, spontaneous colitis models. Mechanistically, prozumab induces IL-10 secretion from naive human PBMCs and suppresses the secretion of IFN-γ and IL-6 from anti-CD3–activated human PBMCs. These findings make prozumab a promising potential drug for treating human rheumatoid arthritis and inflammatory bowel disease, as well as a wide range of autoimmune inflammatory diseases.

Susceptibility and resistance to experimental adjuvant arthritis.

Autoimmunity is the result of an abnormal immune response against constituents of body tissues. For many years, the study of animal models of human diseases was aimed at defining the factors participating in the autoimmune process. During the past two decades, much of the attention was diverted to another intriguing aspect of animal models: the mechanisms rendering some animal strains autoimmune-susceptible and others resistant. In this report, we focus on one experimental model, adjuvant arthritis (AA) which is inducible in the Lewis rat following stimulation of the immune system by heat-killed mycobacterium and its 65kDa heat shock protein. We describe genetic loci regulating the severity of this disease as well as the contribution of microbial flora and endocrine activity to susceptibility and resistance. In our opinion, a better understanding of the processes underlying susceptibility and resistance to AA is an important step towards the development of new therapeutic approaches to autoimmunity.

Chapter 84 – Anticollagen Antibodies

Collagen is a triple-helical molecule, which functions as the major scaffold protein in tissues and participates in cell–matrix interactions. Its deposition in the extracellular compartment renders it an immunogen and a target for attack by autoantibodies in susceptible individuals. Several types of collagens have been described as the target of autoantibodies in autoimmune diseases, and the clinical features of the ensuing disease reflect the organ location of the specific collagen. Collagens that are implicated in autoimmunity include types I, II, III, V (e.g., adult and juvenile rheumatoid arthritis (RA)), type IV (Goodpasture syndrome), types VII and XVII (immunobullous diseases), and type XIII (Graves’ disease). In some cases, the causative role of antibodies has been clearly established in animal models. In everyday clinical practice, identification of anticollagen antibodies in the appropriate setting may help the clinician in establishing an accurate diagnosis and in predicting the prognosis.

ANTIBODIES TO LAMININ

Abstract Laminins are major components of the basement membrane, forming essential contacts with cells and other components of the basal lamina, thereby inducing dynamic interactions in the tissue through intracellular signaling, promoting/regulating cell adhesion, spreading, and differentiation. Autoantibodies to laminin have been found in autoimmune disorders such as pemphigoids, rheumatic fever, systemic lupus erythematosus (SLE), and cutaneous lupus erythematosus as well as in Chagas disease and in women with recurrent abortions and/or infertility. The antibodies bind mainly the LG domain of the α chain of laminin, and in some cases they seem to appear as a consequence of antigenic mimicry. Their pathogenic role and diagnostic value are currently being investigated.