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This study relates to a novel mediator signaling between the nervous system and the spleen following an immune challenge. Using enzyme-linked immunospot and cell proliferation assays, we found that supernatants of cultured splenocytes prepared from subcutaneously trypanosome-inoculated rats and mice spleens obtained immediately after inoculation and added to naive cells significantly stimulate interferon-gamma production and cell proliferation compared to phosphate-buffered saline-inoculated animals. This action was abrogated by surgical denervation of the spleen. Using the fluorescent differential display technology, the gene involved in this process was identified and further cloned and its sequence was mapped to chromosome 14 (GenBank accession number: EU552928). Protein expression revealed approximately 15 kDa molecule with biological activities similar to the cultured supernatants of splenocytes obtained directly from parasite-inoculated animals. Antibodies raised against the protein blocked the activities of both the protein and the supernatant and also recognized a band in the active supernatant with the same molecular mass as the protein. Furthermore, the protein was able to reactivate experimentally immunosuppressed cells by regaining their ability to proliferate, suggesting that such a nervous system-induced immune system-released activating agent (ISRAA) may have a potential therapeutic benefit in immunocompromised situations and in further understanding the mechanism for innate immunity commencement and action.

Critical interactions between the nervous system and the immune system during experimental autoimmune myasthenia gravis (EAMG) were examined in an animal model for human MG after immunization of adult female Lewis rats with Torpedo acetylcholine receptor (AChR) and complete Freund's adjuvant. Immunized rats depicted marked clinical severity of the disease. Using enzyme-linked immunospot (ELISPOT) assay and in situ hybridization techniques, immune responses in these animals were examined and showed elevated numbers of anti-AChR IgG secreting B cells and AChR reactive interferon (IFN)-gamma-secreting cells, enhanced mRNA expression of the proinflammatory cytokines IFN-gamma and tumour necrosis factor (TNF)-alpha as Th1 subset and the anti-inflammatory cytokines interleukin (IL)-4 and IL-10 as a Th2 subset, and transforming growth factor (TGF)-beta as a Th3 cytokine. Corticosterone and prostaglandin E(2) (PGE(2)) levels were measured by radioimmunoassay and illustrated increased production after immunization. Surgical denervation of the spleen reduced significantly the clinical severity of the disease, suppressed the numbers of IgG and IFN-gamma-secreting cells, down-regulated the mRNA expression for cytokines and reduced corticosterone and PGE(2) production. As controls, sham-operated rats were used and showed results as the EAMG non-denervated control rats. The data present herein, and for the first time, substantial effects of the nervous system on immune responses that may influence the outcome of EAMG. These effects were not dependent on cytokine inhibitory mediators such as prostaglandins or stress hormones. IL-10 and TGF-beta, the two potent immunosuppressive cytokines, were also suppressed, indicating a general suppression by splenic denervation. More investigations are initiated at our laboratories to understand the evident neural control over the immune system during challenges leading to the break of tolerance and development of autoimmunity, which may assist in innovative therapeutic approaches.

Objective: To test the hypothesis that the nervous system participates in modulating the immune response during experimental African trypanosomiasis caused by Trypanosoma brucei brucei., Methods and Results: Using in situ hybridization and immunochemistry, we studied the effects of splenic sympathectomy on mRNA gene expression and protein production of IL-1beta and IL-6 in splenic and peritoneal macrophages (PMPhi) of Sprague-Dawley rats infected with T. brucei brucei and non-infected rats. The enhancements of mRNA gene expression and production of IL-1beta and IL-6 by peritoneal macrophages were significantly suppressed by the splenic sympathectomy in both infected and non-infected rats., Conclusions: Our data indicate a probably stimulatory role of the sympathetic nervous system during the host immune response in both normal and T. brucei brucei-infected rats., (Copyright 2004 S. Karger AG, Basel)

Objective: To examine critical interactions between the nervous system and the immune system during experimental African trypanosomiasis., Methods and Results: Inoculation of Trypanosoma brucei brucei resulted in early interferon (IFN)-gamma production, elevated corticosterone and prostaglandin E(2) (PGE(2)) levels and increased splenocyte proliferation, as measured by enzyme-linked immunospot assay, radioimmunoassay and thymidine incorporation assay, respectively. Splenic denervation suppressed IFN-gamma, corticosterone and PGE(2) production, enhanced splenocyte proliferation, and significantly reduced parasitemia and prolonged rat survival., Conclusions: Our data show substantial effects of the nervous system on early immune responses that may influence the outcome of this disease. These effects were not dependent on cytokine inhibitory mediators such as prostaglandins or stress hormones. More investigations are required to understand the evident neural control over the immune system during infectious challenges, which may assist in novel therapeutic approaches., (Copyright 2000 S. Karger AG, Basel)

The cross talk between the central nervous system (CNS) and the immune system includes among others, the modulation of immune responses by the autonomic nervous system. Here, we investigated the effects of a splenic denervation on cytokine induction in early experimental African trypanosomiasis. Profiles of the cytokine mRNA expression for interleukin (IL)-4, interleukin (IL)-6, interleukin (IL)-10, interleukin (IL)-12, tumour necrosis factor (TNF)-alpha, tumour necrosis factor (TNF)-beta, transforming growth factor (TGF)-beta and interferon (IFN)-gamma were examined at 4 h, 8 h and 12 h postinfection (p.i.), and in noninfected controls. Only IFN-gamma and IL-12 were significantly expressed over noninfected controls. Already at 4 h p.i. both cytokines were expressed and showed more increased levels at 12 h. Sympathetic denervation of the spleen markedly reduced the mRNA expression for both IFN-gamma and IL-12. Con A was used as a positive control and showed an enhanced mRNA expression, which was also suppressed by a splenic denervation. To demonstrate that the mRNA expression had resulted in a cytokine production, we looked for the protein level of IFN-gamma at 4 h p.i. by immunohistochemistry and found increased levels of IFN-gamma, which was also inhibited by the denervation. Sham-operated animals exhibited similar responses as the nondenervated controls. Our data present for the first time very early kinetics for a cytokine gene expression during an experimental African trypanosomiasis. Furthermore, the data suggest a regulatory role for the autonomic nervous system on cytokine responses at both the mRNA and the protein levels.

Cytokines are important signalling proteins, which have been shown to contribute to immunopathogenesis of several inflammatory and infectious diseases such as African trypanosomiasis. The present study was conducted in order to evaluate the early induction of five potential cytokines in the central nervous system (CNS) and spleens from Trypanosoma brucei brucei (T. b. brucei)-inoculated and uninfected control Sprague-Dawley rats. In brain, choroid plexus and spleen, cytokine levels were examined by in situ hybridization and immunohistochemistry, while ELISA was used to measure cytokine levels in cerebrospinal fluid (CSF). Our results showed that interferon (IFN)-gamma and transforming growth factor (TGF)-beta were highly expressed in all compartments, but low interleukin (IL)-4, IL-10 and tumour necrosis factor (TNF)-alpha mRNA levels were registered. The pattern of these cytokines is in context with the severity of the disease because (i) IFN-gamma was previously demonstrated to promote parasite growth (ii) TNF-alpha was previously demonstrated to kill the parasites and (iii) IL-4 was previously demonstrated to promote antibody production necessary for elimination of the infection. These data support the hypothesis that cytokines may have a role in developing the disease either by enhancing the parasite growth or by suppressing the immune response.

We examined the induction of 4 chemokines during early experimental African trypanosomiasis using in situ hybridization and immunocytochemistry. mRNA expression and protein production of Rantes, MCP-1, MIP-1a and MIP-2 were studied in splenocytes obtained at 0 h, 4 h and 12 h post-infection. Splenic denervation was performed to study the role of the central nervous system in early infection. The mRNA for Rantes increased at 4 h and declined at 12 h, but the protein level was high at both time-points. MCP-1 and MIP-la had elevated mRNA and protein levels at 12 h post-infection. MIP-2 mRNA was high at both 4 h and 12 h, but the protein level was only increased at 12 h. Splenic denervation, but not sham operation, suppressed these responses. The upregulation of these chemokines during very early infection suggests a chemokine role in the developing immunopathology The sympathetic nervous system may, however, participate in modulation of such early immune responses.