SummaryExperimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system that can be transferred to naive mice via CD4 + T cells isolated from appropriately immunized mice. We have evaluated the effects of recombinant murine interleukin 12 (rmi1:12), a potent inducer of interferon 3/(IFN-3') and promoter ofThl T cell development, on the course of adoptively transferred EAE. The transfer of lymph node cells (LNC) isolated from proteolipid protein (PLP)-primed animals and stimulated in vitro with PLP to naive mice resulted in a progressive paralytic disease culminating in complete hind limb paralysis in the majority of the recipients. When mice were injected with LNC that had been stimulated in vitro with PLP in the presence of rmi1:12, the subsequent course of disease was more severe and prolonged. The addition of rmlL-12 during the in vitro stimulation with PLP resulted in a 10-fold increase in IFN-'y and a 2-fold increase in tumor necrosis factor (TNF) ct in the supernatants, relative to LNC stimulated with PLP alone. However, neutralization of IFN-3, or TNF-c~ in vitro with specific antibodies did not abrogate the ability of rmlL-12 to exacerbate the subsequent disease. Similarly, mice treated with rmi1:12 in vivo after the transfer of antigen-stimulated LNC developed a more severe and prolonged course of disease compared with vehicle-treated control animals. In contrast, treatment of mice with an antibody to murine I1:12 after cell transfer completely prevented paralysis, with only 40% of the mice developing mild disease. These results demonstrate that in vitro stimulation of antigen primed LNC with PLP and rmlb12 enhances their subsequent encephalitogenicity. Furthermore, inhibition of endogenous IL-12 in vivo after LNC transfer prevented paralysis, suggesting that endogenous 11:12 plays a pivotal role in the pathogenesis of this model of autoimmune disease.

The cysteinyl leukotrienes (CysLTs) are important mediators of human asthma. Pharmacologic and clinical studies show that the CysLTs exert most of their bronchoconstrictive and proinflammatory effects through activation of a putative, 7-transmembrane domain, G-protein-coupled receptor, the CysLT1 receptor. The initial molecular characterization of the CysLT1 receptor showed by in situ hybridization, the presence of CysLT1 receptor messenger RNA (mRNA) in human lung smooth-muscle cells and lung macrophages. We confirmed the results of these in situ hybridization analyses for the CysLT1 receptor, and produced the first immunohistochemical characterization of the CysLT1 receptor protein in human lung. The identification of the CysLT1 receptor in the lung is consistent with the antibronchoconstrictive and antiinflammatory actions of CysLT1 receptor antagonists. We also report the expression of CysLT1 receptor mRNA and protein in most peripheral blood eosinophils and pregranulocytic CD34+ cells, and in subsets of monocytes and B lymphocytes.

IP10 and MIG are two members of the CXC branch of the chemokine superfamily whose expression is dramatically up-regulated by interferon (IFN)-gamma. The proteins act largely on natural killer (NK)-cells and activated T-cells and have been implicated in mediating some of the effects of IFN-gamma and lipopolysaccharides (LPSs), as well as T-cell-dependent anti-tumor responses. Recently both chemokines have been shown to be functional agonists of the same G-protein-coupled receptor, CXCR3. We now report the pharmacological characterization of CXCR3 and find that, when heterologously expressed, CXCR3 binds IP10 and MIG with Ki values of 0.14 and 4.9 nM, respectively. The receptor has very modest affinity for SDF-1alpha and little or no affinity for other CXC-chemokines. The properties of the endogenous receptor expressed on activated T-cells are similar. Surprisingly, several CC-chemokines, particularly eotaxin and MCP-4, also compete with moderate affinity for the binding of IP10 to CXCR3. Eotaxin does not activate CXCR3 but, in CXCR3-transfected cells, can block IP10-mediated receptor activation. Eotaxin, therefore, may be a natural CXCR3 antagonist.

We have evaluated the effects of rmIL-12 on the course of adoptively transferred EAE. When mice were injected with LNC that had been stimulated in vitro with PLP in the presence of rmIL-12, the subsequent course of disease was more severe and prolonged than controls. In vitro stimulation with PLP in the presence of IL-12 was associated with an increase in IFN-gamma and decrease in IL-4-producing cells, indicating a preferential expansion of Th1 effector cells. At peak disease, no notable differences in either the cellular composition or cytokine expression within CNS lesions was seen between groups. However, the frequency of macrophages that stained positively for inducible nitric oxide synthase (iNOS) was increased in animals challenged with rmIL-12 treated LNC. These data suggest that in addition to promoting the preferential expansion of IFN-gamma-producing cells by rmIL-12 treatment in vitro, in vivo effects leading to macrophage activation and iNOS expression may contribute to the severe, protracted course of CNS inflammation in this model. In contrast, treatment of mice with an antibody to murine IL-12 following cell transfer completely prevented paralysis with only 40% of the mice developing mild disease. These data suggest that endogenous IL-12 plays a pivotal role in the pathogenesis of this model of autoimmune disease.