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In this study, the temporal and spatial expression of the chemotactic factor monocyte chemotactic protein-1 (MCP-1) was examined in the rabbit retina after challenge with the proinflammatory cytokine interleukin-1beta (IL-1). In these tissues, IL-1 induces an acute inflammatory response of the epiretinal vessels that peaks approximately 24 h postintraocular injection (pi) with the cytokine. At 2 h after challenge with IL-1, MCP-1 mRNA was expressed by perivascular microglial cells and astrocytes that form the glial limitans. Protein analysis at 3 h pi with IL-1 confirmed these sites of MCP-1 expression. The intensity of the mRNA and protein signals increased at 6 h and at 24 h. At these time points, MCP-1 message and protein also were detected in infiltrating macrophages and, at the latest time point, in endothelial cells as well. These data support the conclusion that IL-1 provides a strong stimulus for the rapid expression of MCP-1 mRNA and protein in retinal tissues, and they further support the role of endogenous glial cells as important sources of mediators involved in the regulation of inflammation occurring within the nervous system., (Copyright 2000 Wiley-Liss, Inc.)

Chemokines are secreted proteins that function as chemoattractants, mediating the recruitment of specific subsets of leukocytes to sites of tissue damage and immunological reactions. Chemokines may also function as antiviral agents, since viruses such as human immunodeficiency virus type 1 (HIV-1) use chemokine receptors as co-receptors for viral entry. This study examines whether virus-induced interferon, IFNbeta, or immune-related interferon, IFNgamma, affects the production of beta-chemokines by CNS microglia and peripheral monocytes. When IFNbeta was used as the stimulus, induction of MIP-1alpha, MIP-1beta, MCP-1, and RANTES mRNA and protein was observed within 12 h of stimulation in microglia. By contrast, when IFNgamma was used as the stimulus, only MCP-1 was induced. IFNbeta stimulation of blood monocytes resulted in upregulation of MIP-1alpha, MIP-1beta, and MCP-1. Thus, type I and II interferons differentially regulate beta-chemokines in human fetal microglia and peripheral blood monocytes. These observations may have relevance for the therapeutic activity of IFNbeta in multiple sclerosis and for the antiviral effects of IFNbeta for HIV-1 infection of monocytes and microglia., (Copyright 2000 Wiley-Liss, Inc.)

Nitric oxide generated from the inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of multiple sclerosis. Because significant species- and cell-specific differences exist in the expression of iNOS, we used primary human glial cell cultures to screen for an inhibitor of iNOS expression. Remarkably, among numerous soluble factors tested, interferon-beta (IFN-beta) alone showed a selective and potent inhibition of interleukin-1beta/interferon-gamma (IL-1beta/IFN-gamma)-induced iNOS expression in astrocytes. Inhibition of iNOS may provide a mechanism by which IFN-beta can ameliorate inflammation and cytotoxicity in the central nervous system of patients with multiple sclerosis.

In this study we have shown that an antibody to CD18 identified a population of cells in the rabbit retina that resembled the perivascular macrophage found in other regions of the central nervous system. In the normal retina these cells possessed a ramified morphology and presented in an ordered array on the vitreal surface in association with the epiretinal vessels. Approximately 50% of the perivascular macrophages constitutively expressed MHC class II. In response to interleukin-1 beta (IL-1 beta)-induced inflammation, these cells became activated, as evidenced by a change from a ramified to an ameboid morphology and increased expression of MHC class II, and migrated away from the vessels. These changes were first detected around 3 h post-intraocular challenge coincident with the onset of inflammation. At the peak of the inflammatory response (approximately 24 h post-challenge), many activated perivascular macrophages were no longer associated with the vessels and formed long "cord" of MHC class II+ cells associated with underlying deposits of fibrin. In eyes challenged with heat-inactivated IL-1, no change in the morphology or distribution of the perivascular macrophage was noted. At 3 weeks post-challenge with IL-1, the number and distribution of the perivascular macrophages were restored to baseline values, although with a reduced cell size. Since these changes closely resemble those that occur in non-lymphoid dendritic cells in the skin, heart, and/or kidney following activation with cytokines or bacterial products, the results suggest that the perivascular macrophage represents the dendritic cell of the retina and may thus play an important role in immune surveillance in the eye and maintenance of the blood-retina barrier.

T lymphocytes bearing the gamma delta T-cell receptor have been found in the central nervous system of patients with multiple sclerosis in association with demyelinated lesions. Although the biological function of these cells remains to be established, it has been proposed that they are involved in the response to highly conserved antigens, such as heat shock proteins (hsp), expressed during tissue damage and thus may contribute to the development of an autoimmune response. Using polymerase chain reaction, we probed for the presence of T-cell receptor gamma delta cells in fresh-frozen early autopsy brain tissue from patients with multiple sclerosis and patients with non-multiple sclerosis conditions. The results demonstrated the presence of two major V-J combinations of the T-cell receptor delta chain--V delta 2-J delta 3, V delta 2-J delta 1--and we used a direct sequencing technique to determine whether this gamma delta T-cell population was clonal or diverse. In chronic-active plaques from 9 patients with multiple sclerosis, we found a striking predominant gene rearrangement within the V delta 2-J delta 3 T-cell receptor population that was not present in central nervous system tissue from patients with other neurological diseases. In contrast, within the V delta 2-J delta 1 T-cell receptor population, a predominant rearrangement pattern was detected in only 1 of the multiple sclerosis patients. The sequence of the predominant V delta 2-J delta 3 gene rearrangement was confirmed by cloning and sequencing the gene products from 1 multiple sclerosis patient.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferation of microglia/macrophages is a common finding in many central nervous system diseases. To identify mitogenic signals for human microglia, we examined primary cultures of human fetal and adult microglia after stimulation with cytokines, colony stimulating factors (CSFs), or LPS, using proliferating cell nuclear antigen (PCNA) expression as an index of cell proliferation. The results showed that both M-CSF and GM-CSF induced microglial proliferation in fetal and adult human cultures, but that GM-CSF provided a much stronger stimulus. At 96 h post-stimulation, the mean PCNA labeling index was 2.4 for M-CSF and 13.3 for GM-CSF in fetal microglia; in adult microglia, the PCNA labeling index was 4.7 for M-CSF and 9.0 for GM-CSF. The effect of GM-CSF on fetal microglia was dose dependent and synergistic with M-CSF. LPS abolished the basal level of PCNA labeling in adult microglia, but in fetal microglia, caused a slight increase in PCNA labeling (1.9) at 96 h and consistently enhanced microglial cell survival and differentiation into highly branched cells. The production of GM-CSF in purified human fetal astrocyte and microglial cultures was examined after stimulation with LPS, TNF-alpha, or IL-1 beta. Unlike M-CSF, neither cell type produced GM-CSF in unstimulated cultures; however, when stimulated with IL-1 beta, astrocytes expressed GM-CSF mRNA and protein, which accumulated in the culture through 72 h. In microglia, LPS was the only effective inducing agent. An immunocytochemical study performed to identify in vivo sources of GM-CSF revealed selective labeling of reactive astrocytes in active lesions of multiple sclerosis and senile plaques of Alzheimer's disease. Our data demonstrate that both fetal and adult human microglia are capable of proliferation in response to CSFs, GM-CSF being the more effective stimulus.

Microglia are associated with central nervous system (CNS) pathology of both Alzheimer's disease (AD) and the acquired immunodeficiency syndrome (AIDS). In AD, microglia, especially those associated with amyloid deposits, have a phenotype that is consistent with a state of activation, including immunoreactivity with antibodies to class II major histocompatibility antigens and to inflammatory cytokines (interleukin-1-beta and tumor necrosis factor-alpha). Evidence from other studies in rodents indicate that microglia can be activated by neuronal degeneration. These results suggest that microglial activation in AD may be secondary to neurodegeneration and that, once activated, microglia may participate in a local inflammatory cascade that promotes tissue damage and contributes to amyloid formation. In AIDS, microglia are the primary target of retroviral infection. Both ramified and ameboid microglia, in addition to multinucleated giant cells, are infected by the human immunodeficiency virus (HIV-1). The mechanism of microglial infection is not known since microglia lack CD4, the HIV-1 receptor. Microglia display high affinity receptors for immunoglobulins, which makes antibody-mediated viral uptake a possible mechanism of infection. In AIDS, the extent of active viral infection and cytokine production may be critically dependent upon other factors, such as the presence of coinfecting agents. In the latter circumstance, very severe CNS pathology may emerge, including necrotizing lesions. In other circumstances, HIV infection of microglia probably leads to CNS pathology by indirect mechanisms, including release of viral proteins (gp120) and toxic cytokines. Such a mechanism is the best hypothesis for the pathogenesis of vacuolar myelopathy in adults and the diffuse gliosis that characterizes pediatric AIDS, in which very little viral antigen can be detected.

The effect of gangliosides on the clinical course of experimental allergic neuritis was tested in Lewis rats sensitized with bovine intradural root myelin in complete Freund's adjuvant. A mixture of bovine brain gangliosides (GM1, GD1a, GD1b, GT1b) was injected intramuscularly at a daily dose of 20 mg per kilogram of body weight, beginning 6 days after inoculation. The results from seven different experiments show that in most cases, the administered gangliosides were partially protective. Particularly striking was the reduction in mortality rate to half or less of saline-injected controls. Cumulative clinical index scores were also significantly lower with ganglioside treatment in five of the seven experiments. The cause of the wide variability is not known, but it was noted that better results were obtained when the animals were sensitized with freshly isolated myelin.

Our studies have focused on the mechanisms involved in blood-brain barrier and blood-nerve barrier alterations in inflammatory demyelinating diseases. The results support the conclusion that these diseases are initiated by delayed hypersensitivity reactions. They further demonstrate a role for mast cell degranulation and vasoactive amines in the peripheral nerve. In the central nervous system, our studies have documented both active and passive mechanisms of edema formation and support the conclusion that cytokines may be involved in altered blood-brain barrier permeability and inflammation. We conclude that several mechanisms of barrier breakdown are activated in the inflammatory demyelinating diseases and that alteration in endothelial cell function is a major component of disease induction.

Spinal cord sections from rats sensitized to develop experimental allergic encephalomyelitis (EAE) were immunostained with antibodies against glial fibrillary acidic protein (GFAP), carbonic anhydrase, and vimentin, to see whether the latter two antigens could be detected in GFAP-positive reactive astrocytes. Sixteen days after sensitization (16 dpi) there was intense carbonic anhydrase immunostaining in GFAP-positive cells in the spinal cords of EAE rats, particularly in the white matter. At 13 and 20 dpi carbonic anhydrase immunostaining in astrocytes was less intense, and in the spinal cord white matter of control animals carbonic anhydrase was not detected in the few GFAP-positive cells. In the spinal cords of EAE rats vimentin immunostaining was observed in inflammatory cells and astrocytes. In the latter, GFAP and carbonic anhydrase were colocalized with vimentin. The data suggest that carbonic anhydrase expression in astrocytes is an acute response to injury and that vimentin can be detected in astrocytes, as well as inflammatory cells, as early as 16 dpi.