Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-α undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-α) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

SUMMARY Colony stimulating factor-1 (Csf-1) receptor and its ligand Csf-1 control macrophage development, maintenance, and function. The development of both Langerhans cells (LCs) and microglia is highly dependent on Csf-1 receptor signaling but independent of Csf-1. Here we show that in both mice and humans, interleukin-34 (IL-34), an alternative ligand for Csf-1 receptor, is produced by keratinocytes in the epidermis and by neurons in the brain. Mice lacking IL-34 displayed a marked reduction of LCs and a decrease of microglia, whereas monocytes, dermal, and lymphoid tissue macrophages and DCs were unaffected. We identified IL-34 as a nonredundant cytokine for the development of LCs during embryo-genesis as well as for their homeostasis in the adult skin. Whereas inflammation-induced repopulation of LCs appears to be dependent on Csf-1, once inflammation is resolved, LC survival is again IL-34-dependent. In contrast, microglia and their yolk sac precursors develop independently of IL-34 but rely on it for their maintenance in the adult brain.

Fas/APO-1 is a transmembrane protein of the nerve growth factor/TNFa receptor family which signals apoptotic cell death in susceptible target cells. We have investigated the susceptibility of seven human malignant glioma cell lines to Fas/APO-1-dependent apoptosis. Sensitivity to Fas/APO-1 antibody-mediated cell killing correlated with cell surface expression of Fas/APO-1. Expression of Fas/APO-1 as well as Fas/APO-1-dependent cytotoxicity were augmented by preexposure of human malignant glioma cells to IFNy and TNFa. Further, pretreatment with TGFp2, IL1 and IL8 enhanced Fas/APO-1 antibody-induced glioma cell apoptosis whereas other cytokines including TNFfi, IL6, macrophage colony-stimulating factor, IL10 and EL13 had no such effect. None of the human malignant glioma cell lines was susceptible to TNFa-induced cytotoxicity. Fas/ APO-1 antibody-sensitive glioma cell lines (n = 5), but not Fas/APO-1 antibody-resistant glioma cell lines (n = 2), became sensitive to TNFa when co-treated with inhibitors of RNA and protein synthesis. Resistance of human glioma cells to Fas/APO-1 antibody-mediated apoptosis was mainly related to low level expression of Fas/APO-1 and appeared not to be linked to overexpression of the antiapoptotic protooncogene, bcl-2. Given the resistance of human malignant glioma to surgery, irradiation, chemotherapy and immunotherapy, we propose that Fas/APO-1 may be a promising target for a novel locoregionary approach to human malignant glioma. This strategy gains support from the demonstration of Fas/APO-1 expression in ex vivo human malignant glioma specimens and from the absence of Fas/APO-1 in normal human brain parenchyma. (J. Clin. Invest. 1994. 94:954-964.)

In this study microglial cells isolated from brain cell cultures of newborn mice were characterized and investigated for morphology, their responses to growth factors and their functional properties. The microglial cells were phagocytic, contained nonspecific esterase activity and expressed Fc (IgG1/2b) and type-3 complement receptors. Scanning electron microscopy revealed that in analogy to brain tissue two types of microglial cells are present in the cultures: the ameboid and the ramified type which both display similar appearance by transmission electron microscopy. Interleukin 3 and the granulocyte-macrophage colony-stimulating factor were potent growth factors for the cultured microglial cells. The cells were negative for class II antigens (Ia) of the major histocompatibility antigen complex. However, upon treatment with interferon-gamma (IFN-gamma) microglial cells became Ia+ and functioned as antigen-presenting cells when tested on ovalbumin-specific Ia-restricted helper T cells. Furthermore, microglial cells exposed to IFN-gamma and endotoxin developed tumor cell cytotoxicity and produced tumor necrosis factor alpha. Taken together, microglial cells share the characteristics of cells of the macrophage lineage.

Interleukin 6 (IL6) was found to be produced in the central nervous system (CNS) of ICR+/+ mice infected with lymphocytic choriomeningitis virus (LCMV) or with vesicular stomatitis virus (VSV). When infecting athymic ICR nu/nu mice which cannot develop T cell-mediated meningitis after LCMV infection, no significant synthesis of IL6 was detected in the CNS. IL6 was found, however, to be produced intrathecally in ICR nu/nu mice infected with VSV, which causes a T cell-independent acute encephalitis. This suggested that IL6 may also originate from cells not belonging to the T cell compartment. Indeed, in vitro assays showed that both virus-infected microglial cells and astrocytes secreted IL6. In astrocytes, the infection resulted in the induction of the 1.3-kb messenger RNA IL6. Besides its effect on the development of B cell immunity in the brain, IL6 may be involved in repair mechanisms initiated in the course of viral-induced tissue damage. As shown here, IL6 induced an increase of the secretion of a neurotrophic factor, nerve growth factor by astrocytes. Thus, the intrathecal synthesis of IL6 may be part of the host response to infection favoring immune-mediated elimination of the infectious agent as well as trophic support for neurons.