Central And Peripheral Pathways Mediating Modulation Of Immune System Function By Cerebellar Fastigial Nuclei

Objectives:Our previous work has shown that the cerebellar fastigial nucleus (FN) participates inthe modulation of lymphocyte functions. Since there are no direct connections in structurebetween the cerebellum and the immune system, it appears especially important to explorethe potential pathways and mechanisms underlying the cerebellar immunomodulation. Thehypothalamus is an important immunoregulatory center in the central nervous system.Herein, we on the one hand further revealed the pathways and properties of the FNprojections to the hypothalamus and on the other hand investigated the effects of FNγ-aminobutyric acid (GABA)-ergic and glutamatergic projections to the hypothalamus onlymphocyte functions so as to explore the central and peripheral pathways involved in thecerebellar FN immunomodulation and obtain more comprehension and knowledge oncerebellar functions.Methods:1. Texas red dextran amine (TRDA), an anterograde tracer, was injected into unilateralcerebellar FN to trace the traveling courses and ending locations of the neuronalprojections from the cerebellar FN to the hypothalamus. Further, retrograde tracing byinjection of Fluoro-Ruby (FR) into unilateral lateral hypothalamic area (LHA) combinedwith GABA or glutamate fluorescence immunohistochemistry were performed to identifythe properties of FN neurons projecting to the hypothalamus.2. Vigabatrin (VGB), an inhibitor of GABA-transaminase (GABA-T) that degradesGABA, or3-mercaptopropionic acid (3-MP), an antagonist of glutamic acid decarboxylase (GAD) that synthesizes GABA, was microinjected into bilateral cerebellar FN. Rats withintact or saline-infused FN were used as controls. On the third day after the injections,carboxyl fluorescein succinimidyl ester (CFSE) and anti-CD3antibody double-labelingflow cytometry was used to detect concanavalin A (Con A)-induced T lymphocyteproliferation. Enzyme-linked immunosorbent assay (ELISA）was employed to measureanti-sheep red blood cell (SRBC) IgM antibody level in the serum. On day three followingthe second injection of VGB or3-MP, NK cell cytotoxicity to YAC-1target cells wasevaluated by means of CAM/EH-1double-labeling flow cytometry. Simultaneously, thenumber of GABA-immunoreactive neurons in FN-LHA projections and GABA content inthe hypothalamus were measured by FR retrograde tracing combined with GABAfluorescence immunohistochemistry and high performance liquid chromatography (HPLC),respectively.3. Rats were immunized with bovine serum albumin (BSA). On the third day after theimmunization,6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase forglutamate synthesis, was microinjected in bilateral FN and D,L-threo-β-hydroxyasparticacid (THA), an inhibitor of glutamate transporters on plasma membrane, wasmicroinjected in both sides of LHA or ventrolateral thalamic nuclei (VL). On day threeafter the injections, flow cytometric assay with anti-CD3, CD4, CD8, CD45RA andNKR-P1A antibody labeling was employed to examine the percentage of T lymphocytesand its subsets, B lymphocytes and NK cells in the peripheral blood. CD3/CD25andCD3/CFSE double-labeling flow cytometry were used to detect Con A-induced Tlymphocyte activation and proliferation, respectively. Real-time PCR was used to measuremRNA expressions of IL-2, IFN-γ, IL-4, IL-5, IL-10, TGF-β, IL-22and IL-17in Tlymphocytes of the mesenteric lymph nodes and ELISA was used to detect theconcentration of IL-2, IFN-γ, IL-4, IL-10, TGF-β, IL-22and IL-17in the cell culturesupernatant. In addition, levels of anti-BSA IgM and IgG antibodies in the serum andsplenic NK cell cytotoxicity were measured by ELISA and flow cytometry, respectively.Similarily, when the lymphocyte functions were assayed, the number of glutamate-immunoreactive neurons in FN–LHA projections and glutamate content in thehypothalamus and thalamus were measured by FR retrograde tracing combined withglutamate fluorescence immunohistochemistry and HPLC, respectively.4. On the third day after treatment with DON and THA, the levels ofadrenocorticotropic hormone (ACTH), cortisol, thyroid stimulating hormone (TSH),triiodothyronine (T3) and thyroxin (T4) in the serum and norepinephrine (NE) content inthe spleen and lymph nodes were detected by ELISA and HPLC, respectively.Results:1. TRDA-labeled fibers from the FN traveled through the superior cerebellar peduncle(SCP), crossed in decussation of SCP (XSCP), entered the hypothalamus, and primarilyterminated in the LHA. By injecting FR in the LHA, the FR-stained fibers retrogradelypassed through the XSCP and reached the FN. Combined with fluorescenceimmunohistochemistry, we observed that among these FR-positive neurons in the FN,there were GABA and glutamate immunoreactive cells.2. After VGB injection into the bilateral FN, the Con A-induced T lymphocyteproliferation, the level of anti-SRBC IgM antibody in the serum and the NK cellcytotoxicity to YAC-1cells were all decreased in comparison with control rats with intactor saline-infused FN. However,3-MP injection in the bilateral FN resulted in the contrarychanges. Meanwhile, VGB treatment notably raised but3-MP remarkably reduced thenumber of GABAergic neurons in the FN-LHA projections and GABA content in thehypothalamus.3. After DON injection into bilateral FN, the number of CD3+and CD3+CD4+Tlymphocytes as well as CD4/CD8ratio in the peripheral blood were all less than those ofthe intact and saline-treated control rats, but the number of CD3+CD8+T lymphocytes hadno significant change. The Con A-induced T lymphocyte activation and proliferationdecreased following DON treatment in comparison with the two control rats. The mRNAexpressions of proinflammatory cytokines of IL-2, IFN-γ, IL-17, IL-22in T lymphocytesand their concentration in the cell culture supernatant were all significantly reduced following DON treatment when compared with the intact and saline-infused rats, while themRNA expressions of IL-4, IL-10, IL-5, TGF-β and concentration of IL-4, IL-10, TGF-βin the cell culture supernatant had no remarkable change. In addition, DON treatment inthe FN notably attenuated B lymphocyte number in the peripheral blood and anti-BSA IgMand IgG levels in the serum. Injection of DON in the bilateral FN also diminished thenumber of NK cells in the peripheral blood and NK cell cytotoxicity in the spleen.Simultaneously, the number of glutamatergic neurons in the FN-LHA projections andglutamate content in the hypothalamus were also reduced after DON treatment in the FN.Combined treatment with DON in the FN and with THA in the LHA resulted in theenhancement in the number of CD3+, CD3+CD4+T lymphocytes and CD4/CD8ratio inthe peripheral blood, T lymphocyte activation and proliferation, and mRNA expressionsand concentration in the supernatant of proinflammatory cytokines when compared withDON injection alone. In addition, B lymphocyte number in peripheral blood and levels ofanti-BSA antibodies in the serum as well as the NK cell number in peripheral blood andNK cytotoxicity in the spleen were also raised relative to DON treatment alone. Combinedtreatment with DON and THA increased glutamate content in the hypothalamus incomparison with DON injection alone. There were direct glutamatergic projections fromthe FN to the VL. However, co-treatment with DON in the FN and with THA in the VL didnot significantly alter DON-dependent changes in the number and functions of T, B andNK cells, although the co-treatment altered DON-dependent glutamate content in thethalamus.4. DON injection into bilateral FN increased NE content in the spleen and lymphnodes relative to the intact and saline-treated rats, co-treatment with DON in the FN andTHA in the LHA reduced the NE content compared with DON treatment alone. Afterinjection with DON and THA, the levels of ACTH, cortisol, TSH, T3and T4in the serumdid not significantly alter. Conclusions:1. There are direct GABAergic and glutamatergic projections from the cerebellar FNto the LHA.2. The cerebellar FN GABAergic neurons regulate the functions of T, B and NK cells,this effect is mediated by the GABAergic neuronal projections from the cerebellar FN tothe hypothalamus.3. The cerebellar FN glutamatergic neurons modulate the number and functions of T,B and NK cells, this effect is mediated by the FN glutamatergic projections to thehypothalamus, but not the thalamus.4. The sympathetic nerve but not the adrenal cortical hormone or thyroid hormonepathways mediate the immunomodulatory effect of the cerebellar FN glutamatergicprojections to the hypothalamus.5. In conclusion, the central and peripheral pathways involved in mediating thecerebellar FN immunomodulation may be the FN GABAergic and glutamatergicneurons—hypothalamus—sympathetic nerves—lymphocytes.