As a result we performed a preliminary PCR screen on RNA isolated from the CNS of WT and TNFR1-deficient hosts on day 10 after transfer of WT Th1 cells

As a result we performed a preliminary PCR screen on RNA isolated from the CNS of WT and TNFR1-deficient hosts on day 10 after transfer of WT Th1 cells. endothelial cells produced CXCL2 in the early stages of inflammation. Therefore, productive inflammation results from a conversation, or mutually responding signals, between the initiating T cells and cells in the parenchyma of the spinal cord. Experimental autoimmune encephalomyelitis (EAE) is an autoimmune demyelinating disease of the central nervous system (CNS). Because of similarities in clinical symptoms and CNS histology,1C5 EAE serves as a model for multiple sclerosis and can be induced in mice by either immunization with myelin protein or peptides6 or by adoptive transfer of myelin-specific Th1 CD4+ T cells.7,8 As a result, inflammatory cells consisting mostly of CD4+ T lymphocytes and macrophages infiltrate the CNS leading to demyelination and neuronal damage.9 Tumor necrosis factor receptor 1 (TNFR1), also known as the p55 kd TNF receptor, binds to two ligands, TNF and lymphotoxin-.10 The dominant signaling pathway for TNFR1 promotes inflammation by up-regulating inflammatory cytokines, chemokines, and adhesion molecules11C17 and also suppresses apoptosis by the induction of IAPs (inhibitors of apoptosis) through nuclear factor (NF)-B-dependent pathways.18 TNFR1 also has the ability to induce apoptosis through a death domain in its cytoplasmic region that initiates the FADD-dependent extrinsic apoptotic pathway.19 Our previous experiments have demonstrated that TNFR1 contributes to the pathogenesis of EAE mainly by promoting CNS inflammation,20 and thereby, in the absence of TNFR1 expression, clinical disease is limited. Through our adoptive transfer experiments we have found a dramatic difference in the location PGK1 of encephalitogenic T cells within the CNS of wild-type (WT) versus TNFR1-null mice. Although we observed equivalent numbers of T cells in the CNS of TNFR1-deficient mice compared to WT mice, we found that the T cells in the TNFR1-null animals were confined to the leptomeninges and perivascular spaces of the spinal cord, whereas in the WT animals an abundant number of inflammatory cells were found within the CNS parenchyma. A critical role for macrophages in EAE has been supported by a number of studies. Monocytes along with T cells make up the majority of the inflammatory infiltrate within CNS lesions, and histological evidence of lipid-laden macrophages within multiple sclerosis and EAE lesions has been described.4,5 In addition, it has been demonstrated that depletion of peripheral macrophages using dichloromethylene diphosphonate prevents EAE and PEG3-O-CH2COOH demyelination in rats and mice.21,22 In this report, we show that TNFR1 plays an important role in the production of the chemokines CXCL2 and CCL2, which are associated with inflammation and disease. Bone marrow chimera experiments demonstrate that the critical TNFR1-expressing cell(s) for induction of the inflammatory response resides in host tissue, most likely cells PEG3-O-CH2COOH within the CNS. This TNFR1-dependent production of PEG3-O-CH2COOH chemokines by cells in the CNS appears to play an important role in the initiation of early inflammation. Materials and Methods Animals C57BL/6, congenic Thy1.1 mice (B6.PL-Thy1a/Cy), and congenic CD45.1 mice (B6.SJL-for 10 minutes. Pelleted material was resuspended in 70% Percoll (Pharmacia Biotech, Piscataway, NJ) and additional 37% and 30% layers were added above the cells. Density gradients were centrifuged for 30 minutes at 1200 The debris layer (30% Percoll) was removed and discarded. The remaining gradient PEG3-O-CH2COOH was resuspended in 50 ml of HBSS and centrifuged at 500 for 10 minutes. Pellets were resuspended in 1 ml of HBSS supplemented with 0.2% bovine serum albumin (Sigma), 0.1% sodium azide (Sigma), and 15 mmol/L HEPES before antibody staining and flow cytometry. The following antibodies were used: anti-CD11b-FITC, anti-CD11b-APC, anti-CD45.1-PE, and anti-CD45.2-FITC (BD PharMingen, San Diego, CA). All analyses were conducted on a FACScan using CellQuest software (BD Biosciences, San Jose, CA). FACS Analysis of Splenocytes Animals were sacrificed and the spleen was removed and passed through sterile nylon cell strainers to form single cell suspensions. Cells were resuspended in 1 ml of HBSS supplemented PEG3-O-CH2COOH with 0.2% bovine albumin serum, 0.1% sodium azide, and 15 mmol/L HEPES before antibody staining and flow cytometry using anti-CD11b-APC, anti-CD45.1-PE, and anti-CD45.2-FITC (BD PharMingen). All analyses were conducted on a FACScan using CellQuest software (BD Biosciences). Immunohistochemistry CNS tissues removed from mice perfused with saline were embedded and frozen in OCT. Ten-m sections from the lumbar-sacral spinal cord were placed on Superfrost Plus slides (Fisher Scientific Co., Pittsburgh, PA), fixed in acetone for 5 minutes, immunostained for either Thy1.1, CD11b, or.