Treatment of established relapsing experimental autoimmune encephalomyelitis with the proteasome inhibitor PS-519.

PLP139-151-induced relapsing experimental autoimmune encephalomyelitis (R-EAE) in SJL mice is a Th1-mediated autoimmune demyelinating disease model for multiple sclerosis (MS) in which the primary disease relapse is mediated by T cells specific for the endogenous PLP178-191 epitope. This complex inflammatory process requires the co-ordinated expression of a wide variety of immune-related genes active at a variety of stages of the autoimmune process which are regulated, in part, by the transcription factor nuclear factor (NF)-kappaB which is activated via the ubiquitin-proteasome pathway. We asked if in vivo administration of a selective inhibitor of the ubiquitin-proteasome pathway, PS-519, which downregulates activation of NF-kappaB, could downregulate ongoing R-EAE. Administration of PS-519 during the remission phase, following acute clinical disease was effective in significantly reducing the incidence of clinical relapses, CNS histopathology, and T cell responses to both the initiating and relapse-associated PLP epitopes. The inhibition of clinical disease was dependent upon continuous administration of PS-519 in that recovery of T cell function and onset of disease relapses developed within 10-14 days of drug withdrawal. The data suggest that targeting the ubiquitin proteasome pathway, in particular NF-kappaB, may offer a novel and efficacious approach for the treatment of progressive autoimmune diseases, including MS.

Differential expression of inflammatory cytokines parallels progression of central nervous system pathology in two clinically distinct models of multiple sclerosis.

Multiple sclerosis is an immune-mediated demyelinating disease of unknown etiology that presents with either a chronic-progressive or relapsing-remitting clinical course. Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) and relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE) in the SJL/J mouse are both relevant murine CD4+ T cell-mediated demyelinating models that recapitulate the multiple sclerosis disease phenotypes. To determine the cellular and molecular basis for these observed differences in clinical course, we quantitatively analyzed the temporal expression of pro- and antiinflammatory cytokine mRNA expression in the central nervous system (CNS) and the phenotype of the inflammatory mononuclear infiltrates. TMEV-infected SJL/J mice expressed IFN-gamma, TNF-alpha, IL-10, and IL-4 mRNA during the preclinical phase, and their levels continued to increase throughout the duration of the chronic-progressive disease course. These data correlated with the continued presence of both CD4+ T cells and F4/80+ macrophages within the CNS infiltrates. In contrast, SJL/J mice with PLP(139-151)-induced R-EAE displayed a biphasic pattern of CNS expression for the proinflammatory cytokines, IFN-gamma and TNF-alpha, with expression peaking at the height of the acute phase and relapse(s). This pattern correlated with dynamic changes in the CD4+ T cell and F4/80+ macrophage populations during relapsing-remitting disease progression. Interestingly, IL-4 message was undetectable until disease remission(s), demonstrating its potential role in the intrinsic regulation of ongoing disease, whereas IL-10 was continuously expressed, arguing against a regulatory role in either disease. These data suggest that the kinetics of cytokine expression together with the nature of the persistent inflammatory infiltrates are major contributors to the differences in clinical course between TMEV-IDD and R-EAE.

Characterization of and functional antigen presentation by central nervous system mononuclear cells from mice infected with Theiler's murine encephalomyelitis virus.

We examined the phenotype and function of cells infiltrating the central nervous system (CNS) of mice persistently infected with Theiler's murine encephalomyelitis virus (TMEV) for evidence that viral antigens are presented to T cells within the CNS. Expression of major histocompatibility complex (MHC) class II in the spinal cords of mice infected with TMEV was found predominantly on macrophages in demyelinating lesions. The distribution of I-As staining overlapped that of the macrophage marker sialoadhesin in frozen sections and coincided with that of another macrophage/microglial cell marker, F4/80, by flow cytometry. In contrast, astrocytes, identified by staining with glial fibrillary acidic protein, rarely expressed detectable MHC class II, although fibrillary gliosis associated with the CNS damage was clearly seen. The costimulatory molecules B7-1 and B7-2 were expressed on the surface of most MHC class II-positive cells in the CNS, at levels exceeding those found in the spleens of the infected mice. Immunohistochemistry revealed that B7-1 and B7-2 colocalized on large F4/80(+) macrophages/microglia in the spinal cord lesions. In contrast, CD4(+) T cells in the lesions expressed mainly B7-2, which was found primarily on blastoid CD4(+) T cells located toward the periphery of the lesions. Most interestingly, plastic-adherent cells freshly isolated from the spinal cords of TMEV-infected mice were able to process and present TMEV and horse myoglobin to antigen-specific T-cell lines. Furthermore, these cells were able to activate a TMEV epitope-specific T-cell line in the absence of added antigen, providing conclusive evidence for the endogenous processing and presentation of virus epitopes within the CNS of persistently infected SJL/J mice.

Modulation of JE/MCP-1 expression in dermal wound repair.

The tissue macrophage plays a prominent role in wound repair, yet the parameters that influence macrophage migration into the wound bed are not well understood. To better understand the process of macrophage recruitment, the production of JE, the murine homologue of monocyte chemoattractant protein 1(JE/MCP-1), was examined in a murine model of dermal wound repair. High levels of JE/MCP-1 mRNA were found in dermal punch wounds at 12 hours and 1 day (24 hours) after wounding; mRNA levels slowly decreased to undetectable by day 21. In situ hybridization analysis of wounds revealed that JE/MCP-1 was predominantly expressed by monocytic and macrophage-like cells, as well as by occasional fibroblasts and other interstitial cells. To correlate JE/MCP-1 production with macrophage migration, macrophage infiltration into the wound bed was quantitated. The number of macrophages within the wound increased to a maximum at day 3 (11.3 +/- 4.5 macrophages per high power field), began to decrease at day 5 (4.8 +/- 1.9 macrophages per high power field), and reached near base line at day 10 (3.0 +/- 1.1 macrophages per high power field). The results demonstrate that JE/MCP-1 production within wounds is closely linked to the time course and distribution of macrophage infiltration, with maximal JE/MCP-1 mRNA levels occurring 1 to 2 days before maximal macrophage infiltration. The results support a role for JE/MCP-1 in the recruitment of wound macrophages and suggest that macrophages, through the production of JE/MCP-1, may sustain the recruitment of additional monocytes and macrophages into sites of injury.

Cytokine production and heterogeneity of primary stromal cells that support B lymphopoiesis.

Many reports document that bone marrow stromal cells or their cytokine products can influence the formation of B cells in vitro. Most of this data comes from studies using lines or clones of stromal cells after multiple passage in culture, which could alter gene expression. Our aim in the present study was to determine which cytokines are produced by normal stromal cells under conditions that promote B lymphopoiesis. Primary cultured stromal cells were isolated on FACS from active Whitlock cultures. These cells proved to be relatively homogeneous in expression of cell surface antigens (CD44, VCAM-1, MECA10, and a molecule marked by hamster anti-mouse 8.28 monoclonal antibody). RNA from unselected Whitlock cultured adherent cells and sorted stromal cells from the same cultures were subjected to reverse transcriptase polymerase chain reaction to assess constitutive expression of several cytokine genes. Transcripts for interleukin-1 beta (IL-1 beta), IL-7, macrophage (M)-colony-stimulating factor (CSF), stem cell growth factor (SCGF), insulin-like growth factor 1 (IGF-1) and occasionally leukemia inhibitory factor were detected in RNA from intact cultures. Messages for IL-7, M-CSF, and SCGF were selectively contained within the isolated stromal cell fraction; whereas, IL-1 beta was found solely within the non-stromal cell fraction. IGF-1 was transcribed by both stromal cells and macrophages in Whitlock cultures. No evidence was found for constitutive expression of IL-1 alpha, IL-4, IL-6, or granulocyte-macrophage-CSF. This is in contrast to some reported stromal cell lines and clones. To determine if all primary stromal cells from active lymphopoietic cultures produced IL-7, the isolated cells were stained to reveal cytoplasmic IL-7 protein. A majority of the cells produced IL-7, but about 20% had no detectable IL-7 protein. Taken together, our results suggest that the primary stromal cells are a distinguishable cell type but functional subsets may exist. In regard to the differences in IL-7 production, the primary cell phenotype appears to mirror at least one division noted among the stromal cell lines.