Mechanisms of Pain and Immune Processes

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Overview: Chronic neuropathic pain can affect any part of the body, including the oral cavity and facial nerves. Neuropathic pain can occur due to a variety of insults, infections, autoimmune disorders such as Sjogrens Syndrome, or metabolic disorders such as diabetes (diabetic neuropathy). We are testing the hypothesis that, in some patients, chronic pain is maintained by immunopathological processes related to autoantibodies generated against proteins in peripheral nerve. Autoantibodies are known culprits in certain large fiber peripheral neuropathies. Where pain is a component, we hypothesize the presence of autoantibodies to proteins found in nerve endings arising from small diameter, pain-sensing (nociceptive) C-fiber or A-delta nerve fibers. In support of this idea, it has been reported that approximately 30% of Sjogrens syndrome (SjS) patients exhibit a small fiber neuropathy that produces painful paresthesias in the upper and lower extremities. Similar neuropathic pain occurs prominently in Type II diabetes and in cancer patients treated with certain chemotherapeutic agents. To test the hypothesis that painful neuropathic conditions have an autoimmune component we established a sensitive, quantitative, liquid phase luminescence assay that uses recombinant antigen tracers expressed in mammalian cells. This assay called luciferase immunoprecipitation systems (LIPS) can measure antibodies in serum, plasma, cerebrospinal fluid saliva and other bodily fluids. This translational research program addresses molecular and pathophysiological processes of nociceptive transmission and new ways to investigate chronic pain conditions in human patients. Our goals are to understand (1) the molecular and cell biological mechanisms underlying human chronic pain disorders. In addition, the assay methodology we established has great versatility due to its sensitivity, modularity, and use of recombinant DNA methodology to generate protein antigen tracers. Thus, numerous conditions have been examined as we evaluate the range of diseases and disorders that antibodies play a role in. These include various infectious diseases, with and without nervous system involvement, and various autoimmune disorders, all of which have nervous system symptomology in subsets of patients. Over the past year we have extended the SjS study to include a comparison of salivary antibody levels to those in serum. Using only 5 microliters, the LIPS assay readily detected the major SjS autoantigens in saliva, yielding the same sensitivity and specificity as in serum. These results highlight the feasibility of establishing non-invasive, saliva based assays for many types of human diseases and for monitoring of vaccine immune status for large populations of people. The major Sjogren's antigens have a peculiar feature in that even normal people exhibit a high titer of antibodies to these proteins. One of these proteins Ro52 is an interferon-inducible member of the tripartite motif family a bearing RING domain functioning as an E3 ubiquitin ligase that ubquitinates interferon regulatory factor 8 and other proteins. Analysis of Ro52 and 20 other established antigens revealed that Ro52 had the highest antibody titer adn most likely represents the most immunogenic human protein. While the antibody titers in many of the SjS patients were significantly and substantially higher than controls, all healthy individuals also had antiRo52 autoantibodies. Recombinant expression and LIPS testing of various Ro52 fragments showed that the C-terminal region, containing the B30.2/SPRY protein interaction domain yielded higher antibody titers in the SjS patients compared to controls and that this region was responsible for the high level of Ro52 immunoreactivity in healthy individuals, too. We will begini to confirm these observations using serum from the SICCA Sjogern's registry maintained at the School of Dentistry, UCSF. In many neural autoimmune disorders the major autoantigens are frequently plasma membrane receptors or ion channels. This year we reported on a receptor-based autoimmune disorder, Myasthenia Gravis. In Myasthenia, a neurological autoimmune disorder against a membrane-bound, ligand-gated ion channel, patients display antibodies to the muscle nicotinic receptor (AChR). LIPS detected autoantibodies that reacted with specific truncation mutants suggesting antigenic presentation of the extracellular domain may depend on intracellular folding of the second intracellular loop. These data provide a heuristic template for further studies of the AChR and similar membrane bound receptors and ion channels. We are currently working on several additional inter-institute and inter-institutional collaborations to obtain well-characterized patients with Complex Regional Pain Syndrome (CRPS, a neuropathic pain disorder), other neuropathies, and other CNS and PNS disorders and infectious diseases that have neurological manifestations, especially pain, such as shingles and post-herpetic neuralgia. We have begun the analysis of neuropathic pain patients with CRPS using samples obtained from Rush University. We also are using this assay to explore neuro-glial autoantibodies in HIV-induced painful peripheral neuropathies. One of the most compelling aspects of this project is the progressive layering and evolution of the data set. As we increase the number of test antigens and assay across conditions and diseases, we assemble comprehensive evaluations of immune and autoimmune responses. This is accomplished by determination of (a) the extent and specificity of immune response to orthologous proteins and protein fragments, (b) overlap in antigen profiles indicative of a common denominator or general mechanism, and (c) antigenicity within entire signaling pathways involved in inter- or intracellular communication. One example is the antigenicity of the TRIM family of proteins in Sjogren's Syndrome. As time progresses, full multiple antigen profiling can be implemented to obtain a new level of understanding of many complex human disease states. In order to meet the increased demands of such broad-spectrum autoimmunome profiling and take advantage of the layering, we are in the process of scaling up the throughput of the assay from 96 well plates to 384 well plates and eventually to 1536 well plates. This will conserve serum and allow us to examine individuals in much greater depth than what is presently possible.