Monoclonal antibodies (MAbs) to the capsular polysaccharide of the pathogenic fungus Cryptococcus neoformans can prolong survival and decrease organ fungal burden in experimental murine cryptococcosis. To investigate the mechanism of antibody-mediated protection, the interaction of C. neoformans and murine macrophage-like J774.16 cells was studied in the presence and absence of MAbs differing in isotype. Immunoglobulin G2a (IgG2a) and IgG2b isotype switch variants were isolated from an IgM hybridoma to complete the IgG subclass set. IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA MAbs were studied for their ability to promote phagocytosis and reduce the number of CFU in C. neoformans and J774.16 cell cocultures. The MAbs in this set had similar if not identical fine specificities and were derived from a single B cell. All isotypes promoted phagocytosis; however, the IgG subclasses were more effective opsonins than IgM or IgA. All isotypes enhanced J774.16 anti-C. neoformans activity in vitro, as measured by a reduction in the number of CFU. The IgG1 MAbs were consistently more active in promoting opsonization and reducing the number of CFU. Addition of IgG1 MAb to C. neoformans and J774.16 cocultures resulted in rapid reduction in the number of CFU, which is consistent with fungal killing. Electron microscopy revealed that MAb-opsonized C. neoformans cells were internalized and appeared damaged. Administration of IgM, IgG1, IgG2a, and IgG2b isotype switch variant MAbs revealed that the IgG2a and IgG2b subclasses were the most and least effective isotypes, respectively, in prolonging survival in an intraperitoneal murine infection model. The results indicate that murine antibody subclasses differ in their ability to enhance macrophage anti-C. neoformans activity and suggest that antibody enhancement of macrophage function is a mechanism by which antibodies modify infection in vivo.

Microglia, the tissue macrophages of the central nervous system (CNS), intimately interact with neurons physically and through soluble factors that can affect microglial activation state and neuronal survival and physiology. We report here a new mechanism of interaction between these cells, provided by the formation of gap junctions composed of connexin (Cx) 36. Among eight Cxs tested, expression of Cx36 mRNA and protein was found in microglial cultures prepared from human and mouse, and Cx45 mRNA was found in mouse microglial cultures. Electrophysiological measurements found coupling between one-third of human or mouse microglial pairs that averaged below 30 pico-Siemens and displayed electrical properties consistent with Cx36 gap junctions. Importantly, similar frequency of low-strength electrical coupling was also obtained between microglia and neurons in cocultures prepared from neocortical or hippocampal rodent tissue. Lucifer yellow dye coupling between neurons and microglia was observed in 4% of pairs tested, consistent with the low strength and incidence of electrical coupling. Cx36 expression level and/or the degree of coupling between microglia did not significantly change in the presence of activating agents, including lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, interferon-gamma, and tumor necrosis factor-alpha, except for some reduction of Cx36 protein when exposed to the latter two agents. Our findings that intercellular coupling occurs between neuronal and microglial populations through Cx36 gap junctions have potentially important implications for normal neural physiology and microglial responses in neuronopathology in the mammalian CNS.

IL-16 is a natural ligand for the CD4 molecule and is known for its chemotactic and anti-HIV-1 activities. We determined IL-16 expression in human brain tissue with HIV-1 encephalitis by specific immunocytochemistry and showed that infiltrating lymphocytes and activated microglia express IL-16. IL-16 immunoreactivity was particularly pronounced in microglial nodules. In vitro, human foetal microglia and not astrocytes produce IL-16, and HIV-1 infection up-regulates microglial IL-16 release in a Nef-dependent manner. These results support the notion that, in the brain, IL-16 is a macrophage-lineage specific modulator of the inflammatory response and HIV-1 expression. Recruitment of IL-16+ T cells and microglia/macrophages may represent an innate response to HIV-1 infection in the central nervous system that counterbalances viral stimulatory factors.

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As part of a study on the role of cytokines in central nervous system development and dysfunction, we determined the pattern of cytokine production in highly purified cultures of microglia and astrocytes isolated from second-trimester human fetal brains. Levels of TNF-alpha, IL-1 beta, and IL-6 mRNA and protein were determined by Northern blot analysis and ELISA before and after stimulation with LPS, TNF-alpha, or IL-1 beta. In microglia, LPS induced mRNA for all three cytokines. High protein levels of IL-6 and TNF-alpha were also found in the medium, whereas IL-1 beta protein was mostly cell associated. IL-1 beta also induced message for all three cytokines, in the rank order of IL-1 beta > IL-6 > TNF-alpha. TNF-alpha induced mRNA and protein for IL-1 beta but not for TNF-alpha or IL-6. In contrast, LPS failed to stimulate either mRNA or protein expression for any of the three cytokines in astrocytes. On the other hand, IL-1 beta provided a strong stimulus for astrocytes. IL-1 beta induced mRNA and protein for both TNF-alpha and IL-6, but the kinetics of the response differed for the two cytokines. TNF-alpha mRNA and protein levels peaked early (at 4 h and 16 h, respectively) and were undetectable by 72 h, whereas IL-6 mRNA peaked later (at 16 h) and protein levels continued to accumulate in the medium through 72 h. IL-1 beta did not induce IL-1 beta mRNA or protein in astrocytes. TNF-alpha did not induce expression of any of the cytokines in astrocytes. In conclusion, our results demonstrate that cytokine production can be induced in human fetal microglia and astrocytes but that the stimuli for induction differed significantly for the two cell types. Whereas LPS was a potent stimulus for microglia, astrocytes primarily responded to IL-1 beta. The data further suggest that microglia may be key regulators of astrocyte response, working primarily through the expression of cell-associated IL-1 beta.