Frank Alderuccio

Autoantibodies in neuropsychiatric lupus.

The American College of Rheumatology presented a consensus document in 1999 proposing the classification of 19 different syndromes defined by neurological and psychiatric manifestations of systemic lupus erythematosus (SLE). The detection of autoantibodies in patients serum or cerebrospinal fluid has not been used as diagnostic markers for the proposed neuropsychiatric lupus classifications as their disease associations remain highly contentious. Autoantibodies detected in the serum and/or cerebrospinal fluid, that have been reported to segregate with patients presenting with neuropsychiatric lupus include: (1) anti-neuronal antibodies, (2) brain-lymphocyte cross-reactive antibodies, (3) anti-ribosomal P antibodies, (4) anti-phospholipid antibodies and (5) anti-ganglioside antibodies. Tests for anti-neuronal, anti-brain-lymphocyte cross-reactive and anti-ganglioside antibodies remain highly specialized whereas tests for ribosomal P antibodies and for antiphospholipid antibodies are currently routinely available in most diagnostic laboratories. Anti-ribosomal antibodies segregate with SLE. Antiphospholipid P antibodies are markers for the antiphospholipid syndrome. This syndrome may be associated with another disease, commonly SLE. In this setting, neuropsychiatric manifestations in SLE may arise as a consequence of thrombotic episodes involving the cerebral vasculature. There is a pressing need for antibodies to ribosomal P and to phospholipids to be standardized for routine diagnostic application. We conclude that the search for specific antibody marker(s) that can be applied for the routine laboratory diagnosis for neuropsychiatric lupus remains elusive.

Local Transgenic Expression of Granulocyte Macrophage-Colony Stimulating Factor Initiates Autoimmunity

Mechanisms leading to breakdown of immunological tolerance and initiation of autoimmunity are poorly understood. Experimental autoimmune gastritis is a paradigm of organ-specific autoimmunity arising from a pathogenic autoimmune response to gastric H/K ATPase. The gastritis is accompanied by autoantibodies to the gastric H/K ATPase. The best characterized model of experimental autoimmune gastritis requires neonatal thymectomy. This procedure disrupts the immune repertoire, limiting its usefulness in understanding how autoimmunity arises in animals with intact immune systems. Here we tested whether local production of GM-CSF, a pro-inflammatory cytokine, is sufficient to break tolerance and initiate autoimmunity. We generated transgenic mice expressing GM-CSF in the stomach. These transgenic mice spontaneously developed gastritis with an incidence of about 80% after six backcrosses to gastritis-susceptible BALBc/CrSlc mice. The gastritis is accompanied by mucosal hypertrophy, enlargement of draining lymph nodes and autoantibodies to gastric H/K ATPase. An infiltrate of dendritic cells and macrophages preceded CD4 T cells into the gastric mucosa. T cells from draining lymph nodes specifically proliferated to the gastric H/K ATPase. CD4 but not CD8 T cells transferred gastritis to nude mouse recipients. CD4+ CD25+ T cells from the spleen retained anergic suppressive properties that were reversed by IL-2. We conclude that local expression of GM-CSF is sufficient to break tolerance and initiate autoimmunity mediated by CD4 T cells. This new mouse model should be useful for studies of organ-specific autoimmunity.

The causative H+/K+ ATPase antigen in the pathogenesis of autoimmune gastritis

The gastric H+/K+ ATPase is the causative autoantigen recognized by CD4+ T cells that mediate autoimmune gastritis. Pathogenic CD4+ T cells are regulated by CD25+CD4+ T cells. Here, it is proposed that waves of activation and migration of antigen presenting cells and CD4+ T cells to and from the target organ and draining lymph node result in tissue damage.

Cutting Edge Issues in Autoimmune Gastritis

Autoimmune gastritis is the outcome of a pathological CD4 T cell-mediated autoimmune response directed against the gastric H/K-ATPase. Silent initially, the gastric lesion becomes manifest in humans by the development of megaloblastic pernicious anemia arising from vitamin B12 deficiency. Cutting edge issues in this disease relate to its epidemiology, immunogenetics, a role for Helicobacter pylori as an infective trigger through molecular mimicry, its immunopathogenesis, associated organ-specific autoimmune diseases, laboratory diagnosis, and approaches to curative therapy.

Expression of the Gastric H/K-ATPase α-Subunit in the Thymus may Explain the Dominant Role of the β-Subunit in the Pathogenesis of Autoimmune Gastritis

The two subunits of the gastric H/K ATPase, namely the catalytic α-subunit and the glycoprotein β-subunit, are the major targets of parietal cell autoantibodies associated with human and murine autoimmune gastritis. The murine disease induced by neonatal thymectomy is T cell-mediated. We have previously shown that transgenic expression of the H/K ATPase β-subunit gene in the thymus prevented the development of autoimmune gastritis induced by thymectomy. However, little is known of the contribution of the H/K ATPase α-subunit in disease development. Here, we show that (1) in contrast to the gastric H/K ATPase β-subunit, the α-subunit gene is expressed in normal BALB/c thymus, (2) transgenic expression of the gastric H/K ATPase α-subunit gene in the thymus failed to prevent the development of autoimmune gastritis and (3) normal BALB/c and transgenic mice expressing the α-subunit in the thymus develop autoimmune gastritis following immunisation with purified murine gastric H/K ATPase, whereas transgenic mice...

Transplantation of Bone Marrow Transduced to Express Self-Antigen Establishes Deletional Tolerance and Permanently Remits Autoimmune Disease

Autoimmune diseases are incurable. We have hypothesized that these diseases can be cured by the transplantation of bone marrow (BM) stem cells that have been genetically engineered to express self-Ag. Here we have tested this hypothesis in experimental autoimmune encephalomyelitis (EAE) induced by the self-Ag myelin oligodendrocyte glycoprotein (MOG). We show that, in mice, transplantation of BM genetically modified to express MOG prevented the induction and progression of EAE, and combined with antecedent corticosteroid treatment, induced long-term remission of established disease. Mice remained resistant to EAE development upon subsequent rechallenge with MOG. Transfer of BM from these mice rendered recipients resistant to EAE. Splenocytes from these mice failed to proliferate or produce IL-17, IFN-γ, and GM-CSF in response to MOG35–55 peptide stimulation and they failed to produce MOG autoantibody. Mechanistically, we demonstrated in vivo reduction in development of CD4+ MOG35–55-specific thymocytes, indicative of clonal deletion with no evidence for selection of Ag-specific regulatory T cells. These findings validate our hypothesis that transplantation of genetically modified BM expressing disease-causative self-Ag provides a curative approach by clonal deletion of disease-causative self-reactive T cells.

α and β Subunits of the Gastric H/K -ATPase Are Concordantly Targeted by Parietal Cell Autoantibodies Associated with Autoimmune Gastritis

We have previously shown that parietal cell autoantibodies predominantly react with a 60-90 kDa gastric autoantigen1,2, subsequently identified as the (3 subunit of the gastric H+/K+-ATPase (EC 3.6.1.3) (proton pump)3-5 whereas Karlsson et al6 showed that these autoantibodies primarily target the 95 kDa α subunit of the pump. In view of these discordant results, we have reassessed the reactivity of parietal cell autoantibodies with the two subunits of the gastric H+/K+-ATPase. We show here that all 26 parietal cell autoantibody-positive sera immunoblot both subunits under appropriate, but mutually exclusive, conditions. Thus, reactivity of anti- parietal cell autoantibodies with the 95 kDa α subunit is optimal when the SDS-PAGE is carried out with samples which are reduced but not boiled. Whereas reactivity with the 60-90 kDa β subunit is optimal with samples which are boiled but not reduced. Autoantibody reactivity with the β subunit is critically dependent on the presence of a full complement of N-linke...

A Novel Method for Isolating Mononuclear Cells from the Stomachs of Mice with Experimental Autoimmune Gastritis

Autoimmune gastritis induced in BALB/c mice by neonatal thymectomy is a CD4+ T cell-mediated disease. The disease is characterised by mononuclear cell infiltrates in the gastric mucosa, loss of gastric parietal and chief cells and autoantibodies to the gastric H/K ATPase. Here we describe a simple non-enzymatic method for isolating cellular infiltrates from stomachs of gastric mice by injection of medium directly into stomach walls, causing swelling and rupture. Using this method, large numbers of viable lymphocytes were released from stomachs for analysis by flow cytometry. An 8.3 fold increase in the total number of lymphocytes from diseased stomachs compared to normal controls was observed. Total cell numbers of CD4+ and B cells were increased 4.8 fold and 39.5 fold respectively, in diseased stomachs compared with controls. No change was observed in the CD8+ T cell population. This method will allow detailed quantitative analysis of cellular infiltrates during the development of the gastric lesion and enrichment of pathogenic T cells for analysis and cloning. This procedure may have general application for the isolation of cellular infiltrates from lesion sites of other organs.

Transplantation of bone marrow genetically engineered to express proinsulin II protects against autoimmune insulitis in NOD mice.

Type 1 diabetes (T1D) is a T‐cell‐dependent autoimmune disease resulting from destructive inflammation (insulitis) of the insulin‐producing pancreatic β‐cells. Transgenic expression of proinsulin II by a MHC class II promoter or transfer of bone marrow from these transgenic mice protects NOD mice from insulitis and diabetes. We assessed the feasibility of gene therapy in the NOD mouse as an approach to treat T1D by ex vivo genetic manipulation of normal hematopoietic stem cells (HSCs) with proinsulin II followed by transfer to recipient mice.

Methylprednisolone induces reversible clinical and pathological remission and loss of lymphocyte reactivity to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of human multiple sclerosis (MS). EAE, induced by immunisation with myelin-associated autoantigens, is characterised by an inflammatory infiltrate in the central nervous system (CNS) associated with axonal degeneration, demyelination and damage. We have recently shown in an experimental mouse model of autoimmune gastritis that methylprednisolone treatment induces a reversible remission of gastritis with regeneration of the gastric mucosa. Here, we examined the effect of oral methylprednisolone on the mouse EAE model of human MS induced by immunisation with myelin oligodendrocyte glycoprotein peptide (MOG35–55). We examined the clinical scores, CNS pathology and lymphocyte reactivity to MOG35–55 following treatment and withdrawal of the steroid. Methylprednisolone remitted the clinical signs of EAE and the inflammatory infiltrate in the CNS, accompanied by loss of lymphocyte reactivity to MOG35–55 peptide. Methylprednisolone withdrawal initiated relapse of the clinical features, a return of the CNS inflammatory infiltrate and lymphocyte reactivity to MOG35–55 peptide. This is the first study to show that methylprednisolone induced a reversible remission in the clinical and pathological features of EAE in mice accompanied by loss of lymphocyte reactivity to the encephalitogen. This model will be useful for studies directed at a better understanding of mechanisms associated with steroid-induced disease remission, relapse and remyelination and also as an essential adjunct to an overall curative strategy.