calpain and Autoimmune-Diseases

A variant of the PTPN22-encoded Lyp phosphatase (Lyp620W) confers risk for autoimmune disease, but the mechanisms underlying this association remain unclear. We show here that mice expressing the Lyp variant homolog Pep619W manifest thymic and splenic enlargement accompanied by increases in T-cell number, activation and positive selection and in dendritic- and B-cell activation. Although Ptpn22 (Pep) transcript levels were comparable in Pep619W and wild-type Pep619R mice, Pep protein levels were dramatically reduced in the mutant mice, with Pep619W protein being more rapidly degraded and showing greater association with and in vitro cleavage by calpain 1 than Pep619R. Similarly, levels of the Lyp620W variant were decreased in human T and B cells, and its calpain binding and cleavage were increased relative to wild-type Lyp620R. Thus, calpain-mediated degradation with consequently reduced Lyp/Pep expression and lymphocyte and dendritic cell hyperresponsiveness represents a mechanism whereby Lyp620W may increase risk for autoimmune disease.

Topics: Animals; Autoimmune Diseases; B-Lymphocytes; Calpain; Dendritic Cells; Isoenzymes; Lymphocyte Activation; Male; Mice; Mice, Mutant Strains; Organ Size; Protein Tyrosine Phosphatase, Non-Receptor Type 22; Spleen; T-Lymphocytes; Thymus Gland

Since calcium-activated neutral proteinase (calpain) activity and expression are significantly increased in activated glial/inflammatory cells in the central nervous system of animals with autoimmune demyelinating diseases, this enzyme may also play a role in peripheral organ systems in these diseases. In this study, the activity and expression of calpain and the endogenous inhibitor, calpastatin, were evaluated at transcriptional and translational levels in spleens of Lewis rats with acute experimental allergic encephalomyelitis (EAE) prior to the onset of clinical symptoms. Calpain activity and translational expression were increased by 475.5% and 44.3% respectively, on day 4 post-induction in adjuvant controls and animals with EAE. These levels remained elevated compared to normal controls on days 8 and 12. Calpastatin translational expression was similarly increased at these time points although transcriptional expression was not significantly altered at any time following induction of EAE. Likewise, transcriptional expression of mu-calpain was unchanged following induction, while small increases in m-calpain transcriptional expression were observed on days 2 and 8. Most calpain expression was observed in activated splenic macrophages at day 8 post-induction even though activated T cells were also calpain positive. In spinal cords of animals with EAE, calpain expression was significantly increased in rats with severe disease compared to those exhibiting only mild symptoms at day 12 post-induction. Thus, prior to symptomatic EAE, increased calpain activity and expression in peripheral lymphoid organs may play an important role in T cell migration and subsequent disease progression.

Topics: Adjuvants, Immunologic; Animals; Autoimmune Diseases; Calcium-Binding Proteins; Calpain; Encephalomyelitis, Autoimmune, Experimental; Enzyme Induction; Lymphocyte Activation; Lymphocytes; Macrophage Activation; Macrophages; Male; Protein Biosynthesis; Rats; Rats, Inbred Lew; Spinal Cord; Spleen; Time Factors; Transcription, Genetic

Since myelin proteins are degraded in autoimmune demyelinating diseases such as optic neuritis, proteinases are believed to participate in myelinolysis. Calpain (calcium activated neutral proteinase) degrades myelin proteins at physiological pH and is found in glial and inflammatory cells involved in demyelination. To examine the putative role of calpain in myelinolysis, the activity and expression (translational and transcriptional) of this enzyme and endogenous inhibitor, calpastatin were examined in optic nerves of Lewis rats with experimental allergic encephalomyelitis (EAE), an animal model of optic neuritis. Calpain activity was examined via Western blotting by measuring the extent of myelin protein degradation and calpain-specific fodrin proteolysis in optic nerves from controls versus rats with experimental optic neuritis. RT-PCR studies demonstrated no significant change in millicalpain, microcalpain, or calpastatin expression at the mRNA level in optic nerves from animals with experimental optic neuritis compared to controls. However, myelin associated glycoprotein (MAG) levels were decreased by 25.5% while calpain translational expression and calpain-autolyzed fodrin levels were increased by 72.1% and 462.8% respectively, in experimental optic neuritis compared to controls. Translational expression of calpastatin isoforms (80, 68 and 55 KD) was not significantly different in rats with experimental optic neuritis compared to controls. Thus, increased activity and translational expression of calpain in experimental optic neuritis suggests this proteinase may participate in the degradation of myelin and cytoskeletal proteins in demyelinating diseases such as optic neuritis.

Topics: Animals; Autoimmune Diseases; Blotting, Western; Calcium-Binding Proteins; Calpain; Cysteine Proteinase Inhibitors; Cytoskeletal Proteins; Encephalomyelitis, Autoimmune, Experimental; Male; Myelin Proteins; Optic Neuritis; Protein Biosynthesis; Rats; Rats, Inbred Lew; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic

The Wiskott-Aldrich syndrome (WAS) is an inherited platelet/T-lymphocyte disease characterized by small platelets, thrombocytopenia and immunodeficiency. Because degradative events have a significant role, we directly examined calpain (Ca(2+)-dependent neutral protease), a prominent protease in the affected cells, by functional and antigenic quantitation. Calpain activity in platelets of seven WAS patients was decreased to 59 +/- 3.7% (P < 0.01) relative to platelets of 11 normals. Platelets of two patients with immune thrombocytopenia had normal calpain activity. By immunoblotting, mu-procalpain, the mu-calpain species in resting (unstimulated) blood cells, was decreased in platelets of nine WAS patients to 58 +/- 14.6% (P < 0.01) relative to paired normals. In contrast, mu-procalpain levels in lymphocytes of seven WAS patients did not differ from normal lymphocytes. Normal platelets and lymphocytes have different mechanisms for Ca(2+)-dependent mu-procalpain activation. On addition of ionophore and Ca2+ to stirred platelets, 80kD mu-procalpain was rapidly (0.5 min) and quantitatively converted to 76 kD active mu-calpain; this process was the same in WAS platelets. In lymphocytes, mu-procalpain activation was slow, only partially complete (40 min), and the active species was 78 kD. The marked depletion of calpain in WAS platelets demonstrated in this study may result from inappropriate stimulation of platelets and be related to the severe thrombocytopenia that characterizes this disease.

Topics: Autoimmune Diseases; Blood Platelets; Calcimycin; Calcium; Calpain; Enzyme Precursors; Humans; Immunoblotting; Lymphocytes; Male; Thrombocytopenia; Wiskott-Aldrich Syndrome