In this paper we report that macrophages can be stimulated to express detectable levels of IFN-gamma-specific mRNA. Macrophages from lipopolysaccharide (LPS)-responsive, C3H/OuJ mice are induced by LPS to increase steady-state levels of IFN-gamma-specific mRNA, while those from LPS-hyporesponsive C3H/HeJ mice are not. This interstrain variation is apparently the result of LPS-specific signal differences since macrophages derived from both Lpsn and Lpsd mouse strains are able to produce comparable levels of IFN-gamma-specific mRNA following stimulation with polyinosinic-polycytidylic acid. The identity of the cell type responsible for this IFN-gamma message appears to be the macrophage as IFN-gamma-specific mRNA was also detectable following T and natural killer cell depletion, in the LPS-stimulated RAW 264.7 cell line, and in a homogeneous population of mature macrophages propagated in vitro by stimulation of bone marrow progenitors with recombinant colony stimulating factor-1. Immunofluorescent staining of fixed and permeabilized LPS-stimulated macrophages confirmed the presence of immunoreactive IFN-gamma protein. The possible significance of IFN-gamma production by macrophages is discussed in the context of normal macrophage differentiation as well as the inflammatory immune response.

Venezuelan equine encephalitis virus (VEE) produces an acute infection in humans and induces a well-characterized cytopathic effect in neurons of the central nervous system (CNS). However, little is known about the role of glial cells in response to VEE infection of the CNS. Our results demonstrate that VEE is capable of a productive infection in primary astrocyte cultures and that this infection is cytotoxic. Further, there were signi®cant differences in the growth kinetics comparing virulent and attenuated strains of VEE. Additionally, VEE infection of astrocyte cultures induced gene expression of two neuro-immune modulators, tumor necrosis factor-alpha (TNF-a) and inducible nitric oxide synthase (iNOS). Assays for TNF-a protein and nitric oxide (NO) demonstrated high levels of TNF-a protein and low levels of NO in response to VEE infection of astrocytes. These observations suggest an important role of astrocytes in this virus-induced encephalitis, and that interactions between astrocytes, other glial cells, and neurons may be important in VEE pathogenesis. Such interactions, which could impact neuronal survival, may include loss of functional changes in astrocytes or, alternatively, their production of neurotoxic molecules.

Macrophages secrete interferon (IFN), as well as other cytokines, following lipopolysaccharide (LPS) stimulation. The interferon regulatory factors (IRFs) comprise a family of DNA-binding proteins that have been implicated in the transcriptional regulation of IFN and certain IFN-inducible genes. We therefore characterized basal and LPS-inducible levels of IRF-1, IRF-2, and interferon consensus sequence binding protein (ICSBP) mRNA in LPS-responsive macrophages and compared the expression of these genes in macrophages that typify two murine models of LPS hyporesponsiveness. In the first model, the LPS-hyporesponsive phenotype of the C3H/HeJ mouse is genetically determined and maps to the Lps locus on mouse chromosome 4. In the second model, normally LPS-responsive macrophages acquire a transient LPS-hyporesponsive phenotype following a prior exposure to LPS, a phenomenon referred to as ''endotoxin tolerance.'' Using reverse transcription PCR, we detected basal levels of IRF-1 mRNA in LPS-responsive (Lps n) macrophages that were approximately 15 times higher than those found in LPS-hyporesponsive (Lps d) macrophages. Conversely, Lps d macrophages expressed basal levels of IRF-2 mRNA that were approximately 18 times higher than those expressed in Lps n macrophages. LPS stimulation resulted in a dose-and time-dependent accumulation of IRF-1, IRF-2, and ICSBP mRNA only in Lps n macrophages. Cycloheximide inhibited the accumulation of LPS-stimulated IRF-2 and ICSBP mRNA, but not IRF-1 mRNA, thus designating IRF-1 an immediate-early, LPS-inducible gene. Finally, macrophages rendered tolerant to endotoxin expressed elevated but nonmaximal mRNA levels for all three transcription factors that are not reinduced upon secondary challenge with LPS. Thus, the IRFs may represent yet an additional molecular pathway in the complex response to LPS.

A large number of studies have indicated that the activation of T helper cells for both antibody (1-6) and cytolytic T lymphocyte (CTL) 1 (7-11) 2 responses is restricted by products of the I region of the major histocompatibility complex (MHC), while the activation of CTL themselves is restricted by products of the K/D regions of the MHC (12-14). However, it is less clear how and when during their developmental pathway T ceils acquire their MHC-restricted self-recognition specificity. Studies with chimeras (12, 15, 16) and thymus-grafted nude mice (17, 18) have indicated that the MHC phenotype of the thymus dictates the particular MHC determinants that T cells recognize as self-recognition elements. In addition, the MHC phenotype of the extra-thymic environment has been implicated in the process that determines the restriction specificities of T cells (11,14,17,19,20). In vivo manipulation of the expression of MHC products may yield a better understanding of how the host environment (thymic or extra-thymic) determines restriction specificities of T ceils. One approach to achieving such manipulation is by in vivo administration of antibodies (Ab) to MHC products. A few reports describe the effect of in vivo anti-Ia Ab (5, 21-26) but no data on actual expression of I region products in the recipients are so far available. A recent report described suppression of B lymphocyte development in mice injected from birth with monoclonal anti-I-A Ab (27), i.e., surface Ia +, IgM ÷, and IgD ÷ B ceils did not develop in such mice.The purpose of the present study was to determine whether such chronic administration of anti-Ia Ab into mice during their first weeks of life might influence their T cell self-recognition repertoire. We show here that chronic injection of anti-I-A k Ab, starting in neonatal H-2 k mice, abrogates their ability to generate alloreactive and trinitrophenyl (TNP)-specific splenic CTL responses. Different mechanisms for this

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