Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for immune-related molecules by central nervous system mixed glial cell cultures.

Cytokines secreted within the central nervous system (CNS) are important in the development of multiple sclerosis (MS) lesions. The balance between Th1, monocyte/macrophage (M/M) and Th2 cytokines in the CNS may be pivotal in determining the outcome of lesion development. We examined the effects of mixtures of cytokines on gene expression by CNS glial cells, as mixtures of cytokines are present in MS lesions, which in turn contain mixtures of glial cells. In this initial analysis by gene array, we examined changes at 6 hours to identify early changes in gene expression that represent primary responses to the cytokines. Rat glial cells were incubated with mixtures of Th1, M/M and Th2 cytokines for 6 hours and examined for changes in early gene expression employing microarray gene chip technology. A minimum of 814 genes were differentially regulated by one or more of the cytokine mixtures in comparison to controls, including changes in expression in a large number of genes for immune system-related proteins. Expression of the proteins for these genes likely influences development and inhibition of MS lesions as well as protective and regenerative processes. Analysing gene expression for the effects of various combinations of exogenous cytokines on glial cells in the absence of the confounding effects of inflammatory cells themselves should increase our understanding of cytokine-induced pathways in the CNS.

Interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta inhibit cyclic AMP-induced Schwann cell differentiation.

Schwann cells differentiate in vivo in response to contact with axons, and cAMP simulates some of these aspects of differentiation in vitro, particularly morphologic changes and expression of certain phenotypic molecules. Unfractionated inflammatory cytokines inhibit cAMP-induced Schwann cell expression of galactolipids (Gal). We sought to identify which cytokines were responsible for this inhibition and to determine whether other phenotypic indicators of Schwann cell differentiation were also affected. Neonatal rat Schwann cells were incubated in vitro with 1 mM 8 Bromo cAMP (8 Br cAMP) with or without the addition of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-6, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or transforming growth factor-beta (TGF-beta). Cells were then examined for morphologic changes and for expression of surface Gal and low-affinity nerve growth factor receptor (NGFRp75), employing indirect immunofluorescence. 8 Br cAMP induced Schwann cell upregulation of Gal, downregulation of NGFRp75, and the cells became enlarged and somewhat amorphous and irregular in appearance. Cells treated with IFN-gamma or TNF-alpha alone were more bipolar and more evenly distributed on coverslips than were control cells, whereas TGF-beta alone induced elongated cells often in a swirling pattern. None of the cytokines alone induced upregulation of Gal or downregulation of NGFRp75. TNF-alpha, IFN-gamma, and TGF-beta inhibited the 8 Br cAMP-induced morphologic changes, as well as the upregulation of Gal and downregulation of NGFRp75. The other cytokines had no effects on Gal or NGFRp75 expression. Thus, these three cytokines, which are present in inflammatory lesions in the peripheral nervous system, are capable of inhibiting Schwann cell differentiation.

Cell-surface expression of lymphocyte activation markers in myasthenia gravis.

An analysis of the cell-surface expression of activation markers on B- and T-cells was done to compare patients with myasthenia gravis (MG) and healthy non-myasthenic controls. Marker expression was determined by immunostaining of peripheral blood mononuclear cells (PBMC) isolated from MG patients and from controls. The percentage of B-cells in PBMC that expressed CD71, a transferrin receptor, was significantly greater in patients compared to controls, particularly, in patients who were seropositive for acetylcholine receptor-specific antibodies. When subgroups of MG patients were studied, our data showed that within the first year after disease onset, patients had a significantly higher percentage of T-cells in PBMC that were CD25+ (interleukin-2 receptor alpha) and CD26+ (dipeptidyl peptidase IV ectoenzyme) in comparison to patients with disease symptoms for longer than one year and to healthy controls. Our data also showed that patients with generalized MG had significantly lower percentages of gamma/delta T-cells in peripheral blood compared to healthy controls. The results of this study demonstrate important differences in the cell-surface expression of lymphocyte markers between MG patients and healthy non-myasthenic controls. In addition, differences between subgroups of patients demonstrate that patients with MG are heterogeneous in clinical presentation and in immunological parameters.

Inflammatory cytokines inhibit upregulation of glycolipid expression by Schwann cells in vitro.

OBJECTIVE: To determine whether products of inflammatory cells can inhibit differentiation and synthesis of myelin glycolipids by Schwann cells. BACKGROUND: Infiltration of the peripheral nervous system by inflammatory cells is a feature of acquired demyelinating neuropathies. It is not clear what role these cells have in causing demyelination or inhibiting myelin synthesis. METHODS: Nonmyelinating rat Schwann cells were incubated with 1) different concentrations of activated supernatants (AS) from mitogen-activated inflammatory cells; 2) 8-bromo cyclic adenosine monophosphate (8Br cAMP), known to induce Schwann cell differentiation and synthesis of glycolipids; 3) 8Br cAMP and varying concentrations of AS; 4) 8Br cAMP and cytosine arabinoside (Ara C), which inhibits Schwann cell proliferation; 5) 8Br cAMP, AS, and Ara C; or 6) additional medium. RESULTS: AS inhibits the capacity of cAMP to induce Schwann cell expression of myelin-associated glycolipids. Inhibition of glycolipid expression was independent of the capacity of these AS to induce Schwann cell proliferation. CONCLUSIONS: These data suggest that inflammatory mediators are capable of inhibition of Schwann cell differentiation and synthesis of myelin.

TNF-alpha and TGF-beta act synergistically to kill Schwann cells.

Interactions between cytokines and Schwann cells (SC) are important in development, repair, and disorders of the peripheral nervous system (PNS). Tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) are two prominent cytokines which may be involved in these processes and their gene products are upregulated in some experimental neuropathies. This study focuses on the in vitro effects of these cytokines, both singly and in combination, on cultured SC. Expression of both Type I and Type II TNF-alpha receptors was demonstrated on the SC surface by immunocytochemistry. Treatment of SC with a combination of TNF-alpha plus TGF-beta causes significant detachment and cell death while treatment with each cytokine alone is not significantly cytotoxic. When compared with control cultures, SC treated with the combination of cytokines exhibit an increase in the number of cells with condensed nuclei and evidence of DNA fragmentation, characteristics consistent with cells undergoing programmed cell death. Thus, TNF-alpha plus TGF-beta induce SC loss of adhesion which is predominantly due to cell death. Apoptotic mechanisms are likely to contribute to some extent to this cell death. These findings provide in vitro evidence to support the hypothesis that cytokines can directly damage SC in PNS disorders.

The role of cytokines in Schwann cell damage, protection, and repair.

Cytokines, proteins that are secreted by many cells, including inflammatory and glial cells, mediate interactions between cells, generally through paracrine and autocrine networks. Their effects are highly pleiotropic, with overlap of some activities. The pathogenesis of Guillain-Barré syndrome (GBS), especially the classic inflammatory demyelinating polyneuropathy form, seems to involve lymphocytes and macrophages, which are rich sources of cytokines. Macrophages likely have a role in the pathogenesis of the primarily axonal, less inflammatory forms of GBS. Cytokines appear to be involved in damage to Schwann cells, myelin, and axons, although the exact roles of the different cytokines is uncertain. There is increasing evidence that cytokines, including some proinflammatory cytokines that ordinarily cause damage, may also protect the cells of the peripheral nervous system and aid in its repair. The evolution of inflammatory and demyelinating disorders, including the degree of recovery, is probably dependent on the interactions of the different cytokines.

Induced upregulation of IL-1, IL-1RA and IL-1R type I gene expression by Schwann cells.

We analyzed two distinct phenotypes of Schwann cells (SC), non-differentiated and differentiated, for their ability to produce IL-1alpha and IL-1beta, and to express message for IL-1 receptor antagonist (IL-IRA) and IL-1R type I, in vitro. SC were stimulated with: lipopolysaccharide (LPS), products of activated splenocytes (ASP), products from LPS stimulated SC (SCP), rat recombinant IL-1beta (rrIL-1beta) or dexamethasone. IL-1alpha, IL- 1beta and IL-1RA mRNA levels were highly upregulated after stimulation with LPS, ASP, SCP or rrIL-1beta. SC constitutively expressed low levels of message for IL-1alpha and IL-1beta but not IL-1RA. Specific mRNAs for both IL-1 isotypes were highly upregulated 2 to 4 h after LPS stimulation and then decreased and were undetectable by 24 h. IL-1RA mRNA was detectable after 6 h of LPS stimulation and was maximally upregulated at 24 h. IL-1 gene expression was inducible in both SC phenotypes. IL-1beta could be detected by immunofluorescence in SC, one to three days after LPS stimulation. At the same time IL-1 bioactivity was maximal in SC supernatants. Treatment with either SCP, rrIL-1beta or dexamethasone induced upregulation of IL-1R type I mRNA with maximal expression between 2 to 4 h, in SC. Inducible expression of genes for IL-1alpha, IL-1beta, IL-1RA and IL-1R type I in both differentiated and non-differentiated SC suggest autocrine and paracrine regulation of IL-1 by SC.

Upregulation of intercellular adhesion molecule-1 (ICAM-1) on rat Schwann cells in vitro: comparison of interferon-gamma, tumor necrosis factor-alpha and interleukin-1.

We investigated the expression and upregulation of intercellular adhesion molecule-1 (ICAM-1) in cultures of neonatal rat Schwann cells. Unstimulated cells expressed little or no ICAM-1 as determined by binding of monoclonal antibody to rat ICAM-1 using qualitative and quantitative immunofluorescence. ICAM-1 positive cells when present represented a very small minority (<4%) of Schwann cells. Incubation with recombinant rat interferon-gamma, recombinant rat IL-1beta, recombinant rat tumor necrosis factor-alpha, unfractionated rat cytokines but not purified porcine transforming growth factor-beta or recombinant rat IL-2, resulted in upregulation of ICAM-1 on Schwann cells. Different kinetics were noted for ICAM-1 upregulation by the different recombinant and purified cytokines. The upregulation of ICAM-1 on Schwann cells may be important in inflammatory diseases of the peripheral nervous system as well as in myelination and remyelination.

Interleukin-1 alpha, but not interleukin-1 beta, is a co-mitogen for neonatal rat Schwann cells in vitro and acts via interleukin-1 receptors.

The culture of neonatal rat Schwann cells (SC) with unfractionated cytokines induces an increase in SC proliferation. Previous studies demonstrated that while incubation of SC with interleukin-1(IL-1) does not result in enhanced SC mitogenesis, a mixture of antibodies to IL-1 alpha plus IL-1 beta inhibits cytokine-induced proliferation. We undertook the current studies to: (i) confirm that neither isoform of IL-1 directly causes SC proliferation; (ii) determine if there is a difference in the effect of antibodies to IL-1 alpha versus IL-1 beta; and (iii) determine if IL-1 contribution to cytokine-induced proliferation of SC is mediated via IL-1 receptors. IL-1 alpha or IL-1 beta from several sources, over a wide range of concentrations, failed to induce SC proliferation. Polyclonal antibodies to IL-1 alpha from several suppliers and a monoclonal antibody to IL-1 alpha inhibited SC proliferation, whereas similar antibodies to IL-1 beta had no effect on cytokine-induced SC proliferation. Addition of excess IL-1 alpha to an incubation mixture of unfractionated cytokines plus anti-IL-1 alpha abolished the inhibitory effect of the antibodies. Addition of IL-1 receptor antagonist (IL-1 Ra) to unfractionated cytokines inhibited SC proliferation. Therefore, while neither IL-1 alpha nor IL-1 beta is a solitary mitogen for neonatal rat SC, IL-1 alpha but not IL-1 beta acts as a co-mitogen. Moreover, IL-1 alpha seems to exert its co-mitogenic effect via receptors for IL-1.

A cost-benefit analysis of four hormonal contraceptive methods.

This paper presents the results of a cost-benefit analysis conducted for pregnancy prevention treatment with four hormonal methods of contraception using a managed-care viewpoint. The therapies analyzed are medroxy-progesterone acetate injection (Depo-Provera), levonorgestrel subdermal implants (Norplant), progestogenonly oral tablets (Nor-QD), and combination progestogen/estrogen oral tablets (Ortho-Novum 7/7/7). Cost and benefits associated with the use of therapies are identified and analyzed based on the cost per patient-day of effective pregnancy prevention. The analysis demonstrates that all four methods have a positive net benefit, with Depo-Provera having the highest net benefit. This information can provide decision makers within a pharmacy and therapeutics committee of a managed-care organization the framework on which to base formulary decisions.

Antibodies to interleukin-6 inhibit Schwann cell proliferation induced by unfractionated cytokines.

We have previously demonstrated that unfractionated cytokines induce proliferation of neonatal rat Schwann cells in vitro. Incubation of Schwann cells with natural or recombinant human interleukin-1 (IL-1) does not induce Schwann cell proliferation, but antibodies to IL-1 inhibit Schwann cell proliferation induced by unfractionated cytokines of human origin. In this study we investigated the effect of interleukin-6 (IL-6), another monokine, on proliferation of rat Schwann cells. Incubation of Schwann cells with recombinant (r) mouse IL-6 and two types of human rIL-6 did not induce proliferation of Schwann cells or of endoneurial fibroblasts. However, antibodies to rhIL-6 inhibited Schwann cell proliferation induced by unfractionated cytokines but had no effect on fibroblast proliferation. Addition of excess rhIL-6 to a mixture of unfractionated cytokines plus anti-rhIL-6 abrogated the inhibitory effect of the anti-rhIL-6. Therefore, IL-6 is a co-mitogen for neonatal Schwann cells in vitro.

Differences in the capacity of gamma-interferons from different species to induce class I and II major histocompatibility complex antigens on neonatal rat Schwann cells in vitro.

We compared the capacity of unfractionated cytokines and recombinant gamma-interferons (gamma-IFN) to induce major histocompatibility (MHC) antigens on neonatal rat Schwann cells and endoneurial fibroblasts in vitro. Rat and mouse gamma-IFN were capable of induction of both class I and class II MHC antigens on both cell types although rat gamma-IFN was more potent than that of mouse. Human gamma-IFN failed to induce MHC class I or class II on either cell type. Unfractionated cytokines from the different species also differed in their capacity to induce MHC antigens. Differences in species of origin of cytokine mixtures as well as in individual cytokines, such as gamma-IFN need to be considered in studies of MHC induction. The presence of MHC class I and class II antigens on Schwann cells could render them susceptible to cytotoxic reactions and allow for the presentation of antigen, respectively.

Antibodies to interleukin-1 inhibit cytokine-induced proliferation of neonatal rat Schwann cells in vitro.

Unfractionated cytokines have been shown to induce in vitro proliferation of neonatal rat Schwann cells but the nature of the mitogen(s) is not known. A mixture of rabbit antibodies specific for recombinant interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta (IL-1 beta) inhibited Schwann cell proliferation induced by unfractionated human cytokines whereas antibodies to interleukin-2 (IL-2) and control IgG did not. However, purified human IL-1 and recombinant human IL-1 alpha or beta did not induce Schwann cell proliferation on their own.