Isolated necrotizing myopathy associated with ANTI-PL12 antibody.

INTRODUCTION: Immune-mediated myopathies are a heterogeneous group of chronic autoimmune disorders. Autoantibodies associated with this disease complex are classified into myositis-associated and myositis-specific. Anti-tRNA synthetase antibodies are the most well known of the myositis-specific antibodies. Previous reports have revealed an association of tRNA synthetase autoantibodies with systemic connective tissue disorders. METHODS: Our case report involved a 49-year-old man who developed difficulty walking and climbing stairs 5 months prior to his initial visit. No rash or skin changes were observed. RESULTS: Laboratory testing was positive for anti-PL12 autoantibody with a negative evaluation for connective tissue disorder (CTD). The patient was found to have necrotizing myopathy associated with anti-PL12 antibodies in the absence of inflammatory changes on biopsy, significant derangement of muscle enzymes, or findings characteristic of a typical CTD. CONCLUSION: A high index of suspicion must be maintained for immune-mediated necrotizing myopathy despite the absence of an identifiable CTD and milder symptoms.

Altered regulation of IL-2 production in systemic lupus erythematosus: an evolving paradigm.

In systemic lupus erythematosus (SLE), IL-2 production by T lymphocytes in vitro is impaired. Deficient IL-2 production may be an outcome of a primary SLE T cell disorder that is due to impaired signal transduction. In this issue of the JCI, evidence is presented that an anti-TCR/CD3 complex autoantibody present in SLE sera can bind to T cells and activate the Ca(2+)-calmodulin kinase IV (CaMKIV) signaling cascade, resulting in downregulation of IL-2 transcription and IL-2 production. Because IL-2 may contribute to the maintenance of T cell tolerance, deficient IL-2 production could promote a breach of T cell tolerance that results in autoantibody production in SLE.

Protein kinase A and signal transduction in T lymphocytes: biochemical and molecular methods.

Abnormal T-cell effector functions in systemic lupus erythematosus (SLE) are present and may be associated with disease immunopathogenesis. Our work has led to the characterization of a signaling defect, involving protein kinase A (PKA), leading to abnormal T-cell effector functions in SLE. PKA is a component of the adenylyl cyclase/cyclic adenosine monophosphate/PKA (AC/cAMP/PKA) pathway, a principal signal transduction system in T cells. The aim of this chapter is to provide a comprehensive, technical, step-by-step approach to studying PKA function in T cells. The methods detailed here are (a) chromatographic fractionation of PKA-I and PKA-II isozymes and PKA phosphotransferase activity in purified T cell populations, (b) Western immunoblotting to identify the presence of regulatory (R)-subunit proteins of PKA, and (c) isolation of RNA, and quantification of PKA R subunit-specific transcripts by competitive polymerase chain reaction. Although our emphasis in the chapter is T cells, these methods may be useful for investigation of signaling via PKA in other cell types as well.

Down-regulation of IL-2 production in T lymphocytes by phosphorylated protein kinase A-RIIbeta.

The beta isoform of the type II regulatory subunit (RIIbeta) of protein kinase A suppresses CREB transcriptional activity and c-Fos production in T cells following activation via the TCR. Because CREB is an integral nuclear transcription factor for IL-2 production by T cells, we tested the hypothesis that RIIbeta down-regulates IL-2 expression and IL-2 production in T cells. Stable transfection of RIIbeta in Jurkat T cells led to an approximately 90% reduction in IL-2 mRNA and IL-2 protein following T cell activation. The inhibition of IL-2 production was associated with phosphorylation of the RIIbeta subunit at serine 114 (pRIIbeta) and localization of pRIIbeta in intranuclear clusters. A serine 114 phosphorylation-defective mutant, RIIbeta(S114A), did not form these intranuclear clusters as well as wild-type RIIbeta, and did not inhibit IL-2 mRNA and protein synthesis, indicating that serine 114 phosphorylation is required for both nuclear localization and down-regulation of IL-2 production by RIIbeta. In contrast to its effect on IL-2, RIIbeta induced constitutive up-regulation of CD154 mRNA and cell surface expression. Thus, pRIIbeta differentially regulates gene expression following T cell activation. Unexpectedly, we also found that stable overexpression of another protein kinase A regulatory subunit, RIalpha, had the opposite effect on IL-2 expression, causing a 3- to 4-fold increase in IL-2 production following stimulation. In summary, our data demonstrate a novel mechanism by which serine 114 phosphorylation and nuclear localization of RIIbeta controls the regulation of gene expression in T cells.

Mechanisms of deficient type I protein kinase A activity in lupus T lymphocytes.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which the immune response to antigen results in exaggerated CD4(+) T helper and diminished CD8(+) T cytotoxic responses. To determine the mechanisms underlying impaired T cell effector functions, we have investigated the cAMP/protein kinase A (cAMP/PKA) signaling pathway. The results demonstrate that diminished PKA-catalyzed protein phosphorylation is the result of deficient type I (PKA-I) and type II (PKA-II) isozyme-specific activities. The prevalence of deficient PKA-I and PKA-II activities in SLE T cells is approximately 80% and 40%, respectively. Diminished PKA-I activities are not associated with disease activity and appear to be stable over time. Two disparate mechanisms account for these low PKA-I and PKA-II isozyme activities. Moreover, novel transcript mutations of the RI alpha gene have been identified that are characterized by deletions, transitions, and transversions. Most mutations are clustered adjacent to GAGAG motifs and CT repeats. In conclusion, aberrant signaling via the cAMP/PKA pathway occurs in SLE T cells, and this is proposed to contribute to abnormal T cell effector functions.

Protein kinase A regulatory subunit type II beta directly interacts with and suppresses CREB transcriptional activity in activated T cells.

Levels of the type IIbeta regulatory subunit (RIIbeta) of protein kinase A are abnormally high in the nuclei of T cells of some subjects with the autoimmune disorder systemic lupus erythematosus (SLE). However, the role of nuclear RIIbeta in the regulation of T cell function is unknown. Based on previous studies demonstrating that nuclear protein kinase A-RII subunits can modify cAMP response element (CRE)-dependent transcription, we tested the hypothesis that nuclear RIIbeta can alter CRE-directed gene expression in T cells through interaction with the nuclear transcription factor CRE-binding protein CREB. To test this hypothesis, we used the RIIbeta-deficient S49 and the Jurkat T cell lines. In both cell lines, transient transfection of RIIbeta resulted in nuclear localization of a portion of the ectopically expressed RIIbeta. In vitro and in vivo analyses revealed a novel, specific interaction between RIIbeta and CREB that mapped to the N-terminal 135 aa of RIIbeta. In functional studies, RIIbeta inhibited the transcriptional activity of a GAL4-CREB fusion protein by 67% in Jurkat T cells following activation with anti-CD3 and anti-CD28 mAbs. Importantly, deletion of the CREB-binding region of RIIbeta completely abrogated inhibition. Additionally, RIIbeta suppressed CRE-directed reporter gene expression and substantially reduced induction of promoter activity and endogenous protein levels of the CREB-dependent gene, c-fos, in activated T cells. We conclude that nuclear RIIbeta can act as a repressor of CREB transcriptional activity in T cells, providing a potential functional significance for aberrant levels of nuclear RIIbeta in systemic lupus erythematosus T cells.

Treatment of primary Sjögren's syndrome with low-dose human interferon alfa administered by the oromucosal route: combined phase III results.

OBJECTIVE: This study tested the safety and efficacy of human interferon (IFN) alfa for treatment of salivary hypofunction and dry mouth symptoms in primary Sjögren's syndrome patients. METHODS: Combined results are reported from 2 phase III clinical trials in which a total of 497 subjects with primary Sjögren's syndrome received 150 international units of human IFN alfa or matching placebo 3 times per day for 24 weeks by the oromucosal route. RESULTS: Subjects given IFN alfa had a significantly (P = 0.01) greater mean increase in unstimulated whole saliva (UWS) flow, compared with subjects given placebo. In IFN alfa patients, increases in UWS correlated positively and significantly with improvements noted in 7 of 8 symptoms associated with oral and ocular dryness. The coprimary endpoints of stimulated whole saliva flow and oral dryness were not significantly improved in the IFN alfa group relative to placebo. No significant differences were found between the groups with respect to overall adverse event incidence or severity. CONCLUSION: IFN alfa given at low dosage by the oromucosal route can significantly increase UWS flow in patients with primary Sjögren's syndrome, without causing significant adverse events.

Deficient protein kinase a in systemic lupus erythematosus: a disorder of T lymphocyte signal transduction.

Systemic lupus erythematosus (SLE) is an idiopathic autoimmune disease characterized by impaired T lymphocyte immune effector functions. We have identified a disorder of signal transduction in SLE T cells involving the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Cyclic AMP-stimulated PKA-catalyzed protein phosphorylation is markedly diminished owing to profound deficiencies of both type I (PKA-I) and type II (PKA-II) isozyme activities. Deficient PKA-I isozyme is characterized by a significant reduction in the amount of type I regulatory beta subunit (RI beta) steady state mRNA by competitive polymerase chain reaction. This is associated with a 30% decrease in RI alpha protein and a 65% reduction in RI beta protein. Indeed, T cells from approximately 25% of SLE subjects have no detectable RI beta protein. Transient transfection of T cells not expressing RI beta protein with autologous SLE RI beta cDNA bypassed the block in translation, reconstituting PKA activity and augmenting IL-2 production. Of importance was the initial identification of novel RI alpha mRNA mutations characterized by heterogeneous transcript mutations, including deletions, transitions, and transversions. Most mutations are clustered adjacent to GAGAG motifs and CT repeats. By contrast, deficient PKA-II activity is the result of spontaneous dissociation of the cytosolic RII beta(2)C(2) holoenzyme, aberrant RII beta translocation to the nucleus from the cytosol, and retention of RII beta in the nucleus. In conclusion, distinct mechanisms account for deficient PKA-I and PKA-II isozyme activities in SLE T cells.