Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins

Abstract

Background: In multiple sclerosis, inflammatory cells are found in both active and chronic lesions, and it is increasingly clear that cytokines are involved directly and indirectly in both formation and inhibition of lesions. We propose that cytokine mixtures typical of Th1 or Th2 lymphocytes, or monocyte/macrophages each induce unique molecular changes in glial cells.

Methods: To examine changes in gene expression that might occur in glial cells exposed to the secreted products of immune cells, we have used gene array analysis to assess the early effects of different cytokine mixtures on mixed CNS glia in culture. We compared the effects of cytokines typical of Th1 and Th2 lymphocytes and monocyte/macrophages (M/M) on CNS glia after 6 hours of treatment.

Results: In this paper we focus on changes with potential relevance for neuroprotection and axon/glial interactions. Each mixture of cytokines induced a unique pattern of changes in genes for neurotrophins, growth and maturation factors and related receptors; most notably an alternatively spliced form of trkC was markedly downregulated by Th1 and M/M cytokines, while Th2 cytokines upregulated BDNF. Genes for molecules of potential importance in axon/glial interactions, including cell adhesion molecules, connexins, and some molecules traditionally associated with neurons showed significant changes, while no genes for myelin-associated genes were regulated at this early time point. Unexpectedly, changes occurred in several genes for proteins initially associated with retina, cancer or bone development, and not previously reported in glial cells.

Conclusion: Each of the three cytokine mixtures induced specific changes in gene expression that could be altered by pharmacologic strategies to promote protection of the central nervous system.

Figure 1

Effect of Th1 cytokines on microglia. CNS mixed glial cultures were incubated with Th1 cytokines or additional culture medium for 4 days. Cultures were fixed with 4% paraformaldehyde for 10 minutes, washed and then incubated with mouse anti-ED-1 (IgM) and mouse anti-rat MHC class II (IgG) followed by Alexa 488-conjugated goat anti-mouse IgM and Cy 3-conjugated donkey anti-mouse IgG. Cultures were examined for indirect immunofluoresence employing a Leitz Orthoplan 2 fluorescent microscope. The Th1 treated microglia (ED-1+ cells) have a different appearance when compared to those incubated with additional medium (control). Control microglia do not express MHC class II whereas Th1 treated microglia strongly express MHC class II molecules.

Figure 2

Effect of Th1 cytokines on oligodendrocyte precursors. CNS mixed CNS glial cultures were incubated with Th1 cytokines or additional medium and fixed as in Figure 1. Cultures were then incubated with mouse A2B5 (IgM) and mouse anti rat MHC class II (IgG) followed by Alexa 488-conjugated goat anti-mouse IgM and Cy 3-conjugated donkey anti-mouse IgG. Cultures were examined by indirect immunofluoresence as above. Compared to control, the Th1 treated A2B5+ oligodendrocyte precursors have a much more mature appearance including more extensive process formation although the cells still express A2B5, but do not express MHC class II.