The specific signals mediating the activation of microglia and astrocytes as a prelude to, or consequence of, CNS inflammation continue to be defined. We investigated TLRs as novel receptors mediating innate immune responses in human glial cells. We find that microglia express mRNA for TLRs 1–9, whereas astrocytes express robust TLR3, low-level TLR 1, 4, 5, and 9, and rare-to-undetectable TLR 2, 6, 7, 8, and 10 mRNA (quantitative real-time PCR). We focused on TLRs 3 and 4, which can signal through both the MyD88-dependent and -independent pathways, and on the MyD88-restricted TLR2. By flow cytometry, we established that microglia strongly express cell surface TLR2; TLR3 is expressed at higher levels intracellularly. Astrocytes express both cell surface and intracellular TLR3. All three TLRs trigger microglial activation upon ligation. TLR3 signaling induces the strongest proinflammatory polarizing response, characterized by secretion of high levels of IL-12, TNF-α, IL-6, CXCL-10, and IL-10, and the expression of IFN-β. CXCL-10 and IL-10 secretion following TLR4 ligation are comparable to that of TLR3; however, other responses were lower or absent. TLR2-mediated responses are dominated by IL-6 and IL-10 secretion. Astrocytes respond to TLR3 ligation, producing IL-6, CXCL-10, and IFN-β, implicating these cells as contributors to proinflammatory responses. Initial TLR-mediated glial activation also regulates consequent TLR expression; while TLR2 and TLR3 are subject to positive feedback, TLR4 is down-regulated in microglia. Astrocytes up-regulate all three TLRs following TLR3 ligation. Our data indicate that activation of innate immune responses in the CNS is not homogeneous but rather tailored according to cell type and environmental signal.

We examined whether oligodendrocytes, neurons, and astroglia derived from the human central nervous system differ in susceptibility to injury mediated by tumor necrosis factor (TNF)-alpha and by activated CD4+ T cells acting via a TNF-independent mechanism. Injury was assessed either as cell membrane-directed (lysis), measured by 51chromium (Cr) or lactate dehydrogenase (LDH) release, or nucleus-directed (apoptosis), measured by morphologic features based on propidium iodide (PI) staining and by DNA fragmentation measured by a terminal transferase (TdT)-mediated dUTP biotin nick end labeling technique (TUNEL). TNF did not induce 51Cr or LDH release in any cell targets, but did induce nuclear (66 +/- 2% of cells) and DNA (68 +/- 2% of cells) fragmentation selectively in the oligodendrocytes over 96 hr. At this time, there was no significant loss of oligodendrocyte cell number. Nuclear injury could be induced in neurons by serum deprivation and in malignant astrocytes by the combination of TNF and low serum. CD4+ T cells activated with phytohemagglutin (pha) or anti-CD3 plus interleukin-2 induced significant 51Cr and LDH release in all target cells tested; only pha-activated CD4+ T-cell cocultures showed reduced target cell numbers. Significant nuclear fragmentation was observed only for glioma cells (22 +/- 1% of cells). Differences in susceptibility to different immune effector mechanisms and in the nature of the injury response to the same effector mediator among human CNS-derived neural cells will need to be considered in design of therapeutic strategies aimed at protecting or limiting target cell injury consequent to disease or trauma.

The mechanisms whereby immune cells infiltrating the CNS in multiple sclerosis patients contribute to tissue injury remain to be defined. CD4 T cells are key players of this inflammatory response. Myelin-specific CD4 T cells expressing CD56, a surrogate marker of NK cells, were shown to be cytotoxic to human oligodendrocytes. Our aim was to identify NK-associated molecules expressed by human CD4 T cells that confer this oligodendrocyte-directed cytotoxicity. We observed that myelin-reactive CD4 T cell lines, as well as short-term PHA-activated CD4 T cells, can express NKG2C, the activating receptor interacting with HLA-E, a nonclassical MHC class I molecule. These cells coexpress CD56 and NKG2D, have elevated levels of cytotoxic molecules FasL, granzyme B, and perforin compared with their NKG2C-negative counterparts, and mediate significant in vitro cytotoxicity toward human oligodendrocytes, which upregulated HLA-E upon inflammatory cytokine treatment. A significantly elevated proportion of ex vivo peripheral blood CD4 T cells, but not CD8 T cells or NK cells, from multiple sclerosis patients express NKG2C compared with controls. In addition, immunohistochemical analyses showed that multiple sclerosis brain tissues display HLA-E+ oligodendrocytes and NKG2C+ CD4 T cells. Our results implicate a novel mechanism through which infiltrating CD4 T cells contribute to tissue injury in multiple sclerosis.