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Abstract-Hyperphosphorylation of the cardiac Ca 2ϩ release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) at serine-2808 has been proposed to be a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Here we used various approaches to investigate the phosphorylation of RyR2 by PKA. Mutating serine-2808, which was thought to be the only PKA phosphorylation site in RyR2, did not abolish the phosphorylation of RyR2 by PKA. Two-dimensional phosphopeptide mapping revealed two major PKA phosphopeptides, one of which corresponded to the known serine-2808 site. Another, novel, PKA phosphorylation site, serine 2030, was identified by Edman sequencing. Using phospho-specific antibodies, we showed that the novel serine-2030 site was phosphorylated in rat cardiac myocytes stimulated with isoproterenol, but not in unstimulated cells, whereas serine-2808 was considerably phosphorylated before and after isoproterenol treatment. We further showed that serine-2030 was stoichiometrically phosphorylated by PKA, but not by CaMKII, and that mutations of serine-2030 altered neither the FKBP12.6-RyR2 interaction nor the Ca 2ϩ dependence of [ 3 H]ryanodine binding. Moreover, the levels of phosphorylation of RyR2 at serine-2030 and serine-2808 in both failing and non-failing canine hearts were similar. Together, our data indicate that serine-2030 is a major PKA phosphorylation site in RyR2 responding to acute ␤-adrenergic stimulation, and that

Antineutrophil cytoplasmic autoantibodies (ANCAs) are identified in the circulation of approximately 80% of patients with pauci-immune necrotizing and crescentic glomerulonephritis and systemic small vessel vasculitis, such as microscopic polyangiitis and Wegener granulomatosis. The most common antigen target for ANCAs is myeloperoxidase (MPO), which is found in neutrophils and monocytes. We report definitive experimental animal evidence that ANCAs are pathogenic. MPO knockout (Mpo–/–) mice were immunized with mouse MPO. Splenocytes from these mice or from control mice were injected intravenously into recombinase-activating gene-2–deficient (Rag2–/–) mice, which lack functioning B lymphocytes and T lymphocytes. All mice that received splenocytes developed mild to moderate glomerular immune deposits, but only mice that received 1 × 108 or 5 × 107 anti-MPO splenocytes developed severe necrotizing and crescentic glomerulonephritis, granulomatous inflammation, and systemic necrotizing vasculitis, including necrotizing arteritis and hemorrhagic pulmonary capillaritis. To test the pathogenic potential of antibodies alone, purified anti-MPO IgG or control IgG was injected intravenously into Rag2–/– mice and wild-type mice. Mice that received anti-MPO IgG but not mice that received control IgG developed focal necrotizing and crescentic glomerulonephritis with a paucity of glomerular Ig deposition. Thus, anti-MPO IgG alone was able to cause pauci-immune glomerular necrosis and crescent formation in the absence of functional T or B lymphocytes in Rag2–/– mice and in the presence of an intact immune system in wild-type C57BL/6J mice. This animal model offers strong support for a direct pathogenic role for ANCA IgG in human glomerulonephritis and vasculitis

Background: The histopathology of multiple sclerosis (MS) is characterized by a loss of myelin and oligodendrocytes, relative preservation of axons, and a modest inflammatory response. The reasons for this selective oligodendrocyte death and demyelination are unknown. Materials and Methods: In light of the T lymphocyte and macrophage infiltrates in MS lesions and the numerous cytokines these cells secrete, the direct influence of cytokines on survival of cultured oligodendrocytes and sensory neurons was investigated. Expression of cytokines in vivo was determined by immunolabeling cryostat sections of snap-frozen tissue containing chronic active lesions from four different patients. The samples were also analyzed for the presence of apoptotic nuclei by in situ labeling of 3'-OH ends of degraded nuclear DNA.Results: The results showed: (i) interferon-y (IFNy) to be a potent inducer of apoptosis among oligodendrocytes in vitro and that this effect can be reversed by leukemia inhibitory factor (LIF); (ii) IFN-y has a minimal effect on the survival of cultured neurons; (iii) IFNy at the margins of active MS plaques but not in unaffected white matter; (iv) evidence for apoptosis of oligodendrocytes at the advancing margins of chronic active MS plaques. Conclusions: Injury to a substantial number of oligodendrocytes in MS is the result of programmed cell death rather than necrotic cell death mechanisms. We postulate that IFNy plays a role in the pathogenesis of MS by activating apoptosis in oligodendrocytes.

Numerous mechanisms of action have been proposed for intravenous Ig (IVIG). In this study, we used IgG passive transfer murine models of bullous pemphigoid (BP), pemphigus foliaceus (PF), and pemphigus vulgaris (PV) to test the hypothesis that the effect of IVIG in autoantibody-mediated cutaneous bullous diseases is to accelerate the degradation of pathogenic IgG by saturation of the MHC-like Fc receptor neonatal Fc receptor (FcRn). BP, PF, and PV are organ-specific antibody-mediated diseases in which autoantibodies target the hemidesmosomal antigen BP180 and desmosomal antigens Dsg1 and Dsg3, respectively. Antibodies against BP180, Dsg1, and Dsg3, when injected into neonatal mice, induce the BP, PF, and PV disease phenotypes, respectively. We found that FcRn-deficient mice were resistant to experimental BP, PF, and PV. Circulating levels of pathogenic IgG in FcRndeficient mice were significantly reduced compared with those in WT mice. Administration of high-dose human IgG (HDIG) to WT mice also drastically reduced circulating pathogenic IgG levels and prevented blistering. In FcRn-deficient mice, no additional protective effect with HDIG was realized. These data demonstrate that the therapeutic efficacy of HDIG treatment in the pemphigus and pemphigoid models is dependent on FcRn. Thus, FcRn is a promising therapeutic target for treating such IgG-mediated autoimmune diseases.

A sequence motif, GXRXGGGXGD, located in the putative channel-forming domain, is conserved in all known ryanodine receptors and inositol 1,4,5-trisphosphate receptors. The functional significance of this conserved region was investigated by using site-directed mutagenesis together with functional assays consisting of Ca 2؉ Ryanodine receptors (RyRs) 1 are members of a superfamily of intracellular Ca 2ϩ channels that include the inositol 1,4,5-trisphosphate receptors (IP 3 Rs). These channels play an essential role in intracellular Ca 2ϩ signaling by virtue of releasing Ca 2ϩ from the lumen of sarco(endo)plasmic reticulum to the cytosol of muscle and non-muscle cells (1, 2).RyR is a homotetrameric structure composed of four identical subunits, each having ϳ5000 amino acids. Sequence analysis reveals that one-fifth of the COOH terminus of the molecule is likely to form the channel conducting pore. The remaining ϳ4000 amino acid residues apparently constitute the cytoplasmic "foot" domain (3-7). A truncated RyR in which the foot domain has been deleted has been shown to function as a Ca 2ϩ release channel. The truncated RyR channel was still regulated by Ca 2ϩ , was modified by ryanodine, and exhibited a single channel conductance similar to that of the full-length RyR (8). These studies indicate that the sites for Ca 2ϩ activation and ryanodine binding, and the ion conduction pathway are located within the COOH-terminal ϳ1000 amino acid residues. A glutamate residue located in the putative transmembrane sequence M2 has recently been identified as the Ca 2ϩ sensor of RyR (9). The locations of the ryanodine binding site and the pore-forming segment of RyR, however, have yet to be defined.RyRs and IP 3 Rs share some sequence homology, in particular in the COOH-terminal channel-forming domain (10). Considering their sequence homology and similar conduction properties (11-13), RyRs and IP 3 Rs are likely to share similar structural features in the channel pore. A hydrophobic region between the M5 and M6 transmembrane sequences of the mouse type 1 IP 3 R has been proposed to be the pore-forming region (14). The equivalent region in RyR, corresponding to the M9 transmembrane sequence proposed by Zorzato et al. (15), is also hydrophobic. Sequence alignment of these regions reveals a GXRXGGGXGD motif that can be found in all known RyRs and IP 3 Rs (Fig. 1). To investigate its role in RyR function, we have introduced point mutations into this highly conserved region and examined the functional consequences of these point mutations. Our data indicate that this region is critical for ryanodine binding and ion conduction and probably constitutes the pore-forming segment of RyRs. EXPERIMENTAL PROCEDURES Materials-Ryanodine was obtained from Calbiochem. [3 H]Ryanodine was from NEN Life Science Products. Monoclonal antibody 34C was a generous gift from Dr. John L. Sutko (16).Cloning of the Mouse Cardiac RyR cDNA-Total RNA from mouse heart tissue, isolated by the method of Chomczynski and Sacchi (17), was used to generate firs...

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IntroductionBullous pemphigoid (BP) is an acquired autoimmune skin disease characterized by autoantibodies against two hemidesmosomal antigens, BP230 (BPAG1) and BP180 (BPAG2), and subepidermal blisters (1). These antihemidesmosomal autoantibodies can be detected, along with complement proteins, bound to the dermal-epidermal junction (DEJ) of perilesional skin. In the skin lesions of these patients, basal keratinocytes detach from the underlying dermis at the level of the lamina lucida, leading to subepidermal blistering (1, 2). Eosinophils (3, 4), neutrophils (5), lymphocytes (6), monocyte/macrophages (7,8), and mast cells (MCs; 7, 9) are present in the upper dermis of lesional areas in patients with BP. Interestingly, MC degranulation is a feature of BP (7, 9). Chemoattractants from MCs, including eosinophilic/neutrophilic chemotactic factors and histamine, are present at high concentrations in BP blister fluids (10, 11). Similar skin lesions are observed in the pregnancy-associated nonviral disorder herpes gestationis (12). These observations imply that MCs may play a role in blister formation.We have used an experimental model of BP that involves the passive transfer of anti-mBP180 antibodies into neonatal BALB/c mice to reproduce the key immunopathological features of this human autoimmune disease: IgG and complement deposition at the DEJ, inflammatory infiltration of the upper dermis, and subepidermal blistering (13). The pathogenicity of anti-mBP180 antibodies depends on complement activation (14) and polymorphonuclear leukocyte (PMN) recruitment (15). In the present study, we have investigated the role of MCs in experimental BP using mice genetically deficient in MCs. Preparation of pathogenic rabbit anti-mouse IgG. The preparation of recombinant mBP180 and the immunization of rabbits were performed as described previ- Methods Laboratory animals. Breeding pairs of MC-deficient WCB6F1-Mgf