Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), is usually mediated by myelin-specific autoreactive T cells that cause inflammation and demyelination in the central nervous system (CNS), with significant contributions from activated microglia and macrophages. inflammatory plaques in the brain and spinal cord (1). Experimental autoimmune encephalomyelitis (EAE) shares both neuropathological and clinical Mouse monoclonal to CD59(PE) features of MS (2). Studies of MS and EAE provide evidence that T lymphocytes specific for myelin antigens contribute to disease pathogenesis (3). Inflammation in EAE is usually mediated EPO906 by major histocompatibility complex (MHC) class IICrestricted, Th1-type CD4+ myelin reactive, and Th17-type T cells (4C6). Autore-active T cells activate in the periphery, mix the blood-brain hurdle to enter the central nervous system (CNS) and serve as important disease initiators, affecting both the local cytokine milieu and the recruitment and activation of numerous effector cells (7C9). Microglia and macrophages also contribute to EAE; they produce cytokines that promote inflammation during induction, but also phagocytose and obvious apoptotic cell body, debris and inhibitory substances that limit remyelination and axon regeneration (10,11). The molecular mechanisms that control growth, activation and CNS trafficking of myelin-specific autoreactive T cells and the complex functions of microglia and macrophages in EAE are incompletely comprehended. Allograft inflammatory factor-1 (Aif-1) (also known as ionized Ca2+ binding adapter-1 [Iba-1]) is usually a 17-kDa, interferon (IFN)-Cinducible, EF hand motif protein encoded within the class III EPO906 region of the MHC (human chromosome 6p21.3, mouse chromosome 17B1) in an area densely clustered with inflammatory response genes (12,13). Largely comparable gene products arising from the same locus have been named Iba1, microglial response factor-1 (MRF1) and daintain; Iba1 in particular is usually a well-known histologic marker of microglia and of their activation in pathological CNS conditions. Aif-1 is usually differentially expressed in numerous mouse and human tissues (14,15) and in multiple leukocyte types including macrophages and T cells at basal levels (16C18). In inflammatory disease models, upregulated Aif-1 manifestation has been reported in microglia, macrophages, EPO906 T cells, synoviocytes, pancreatic -cells and adipocytes under numerous pathological conditions representing encephalomyelitis, uveitis, neuritis, arteriopathies, arthritis and diabetes, respectively (19). The significance of increased Aif-1 manifestation in neuroinflammatory diseases such as EAE (20,21) has not been characterized. Overexpression of Aif-1 in MOLT-4 T cells increases proliferation, migration and activation (17) and in macrophage cell lines enhances production of interleukin (IL)-6, IL-12 and IL-10 (22). On the other hand, impaired Aif-1 function decreases microglial phagocytosis (23). Extrapolation from these findings suggests that Aif-1 deficiency might ameliorate EAE by limiting T cell and macrophage inflammatory activity, but could also allow cellular debris to accumulate, secondarily exacerbating EPO906 inflammation and neurotoxicity and impairing regenerative processes. We recently developed an Aif-1Cdeficient mouse collection (24) that can be used to determine the net effect of loss of Aif-1 in disease models. MATERIALS AND METHODS Animals Aif-1Cdeficient mice were generated through a homologous recombination gene targeting strategy (24). The targeted allele was backcrossed onto the C57BT/6 strain for eight decades, and the corresponding knockout ((25). Induction of EAE and Evaluation of Clinical Disease EAE was induced in mice as previously explained (26). Briefly, 10- to 12-wk-old male mice were immunized subcutaneously in the lower dorsum with 300 g myelin oligodendrocyte glycoprotein (MOG35C55) peptide (MEVGWYRSPFSRVVHLYRNGK; Celtek Bioscience, Nashville, TN, USA) in a 200 T emulsion of incomplete Freund adjuvant (IFA) made up of 5 mg/mL H37RA (Difco Laboratories, Detroit, MI, USA). Subsequent to immunization, the mice received intraperitoneal injections of pertussis toxin (500 ng; List Biological Laboratories, Campbell, CA, USA) on the first day of sensitization and again after 2 deb. We considered the day after MOG immunization as deb 1. The EAE disease activity was scored as follows: 0, no symptoms; 1, floppy tail; 2, hindlimb weakness; 3, hindlimb paralysis; 4, forelimb and hindlimb paralysis; and 5, death. Histologic and Immunofluorescence Analysis of Spinal Cords For pathological analysis, EAE mice were anesthetized at the time points indicated and perfused with phosphate-buffered saline (PBS) via cardiac puncture. The spinal cord was flushed by hydrostatic pressure by using PBS. The lumbar spinal cord was postfixed overnight with 4% paraformaldehyde, and the tissues were paraffin embedded. To assess.