Constitutive Expression Of Class Research Articles

Cytoprotection of human umbilical vein endothelial cells against apoptosis and CTL-mediated lysis provided by caspase-resistant Bcl-2 without alterations in growth or activation responses.

Graft endothelial cells are primary targets of host CTL-mediated injury in acute allograft rejection. As an in vitro trial of gene therapy to reduce CTL-mediated endothelial injury, we stably transduced early passage HUVEC with a caspase-resistant mutant form (D34A) of the anti-apoptotic gene Bcl-2. Bcl-2 transductants were compared with HUVEC transduced in parallel with an enhanced green fluorescent protein (EGFP) gene. Both transduced HUVEC have equivalent growth rates in complete medium and both show contact inhibition of growth. However, compared with EGFP-transduced HUVEC, the Bcl-2-transduced cells are resistant to the apoptotic effects of serum and growth factor withdrawal and are also resistant to the induction of apoptosis by staurosporine or by ceramide, with or without TNF. Transduced Bcl-2 did not reduce TNF-mediated NF-kappaB activation or constitutive expression of class I MHC molecules. HUVEC expressing D34A Bcl-2 were significantly more resistant to lysis by either class I-restricted alloreactive or PHA-redirected CTL than were HUVEC expressing EGFP. We conclude that transduction of graft endothelial cells with D34A Bcl-2 is a possible approach for reducing allograft rejection.

Constitutive and visna virus induced expression of class I and II major histocompatibility complex antigens in the central nervous system of sheep and their role in the pathogenesis of visna lesions.

Expression of major histocompatibility complex (MHC) antigens was studied in the brains of 10 healthy sheep 2 months to 5 years old and 13 sheep infected with visna virus by intracerebral inoculation and killed one and 6 months post infection (p.i.). In healthy sheep there was prominent expression of class I, mainly on endothelial cells but also detected on ependyma, choroid plexus and in the leptomeninges. Class II expression was sparse. It was observed on perivascular cells, in choroid plexus, leptomeninges and on microglial cells in the white matter. No definite increase with age in the constitutive expression of class I and II was observed, confirming that we are dealing with a true constitutive expression. In visna-infected sheep a considerable induction of MHC antigens on microglia was observed, which correlated with severity of lesions and was mainly found in or adjacent to inflammatory infiltrates of the white matter. Increase in class II antigen expression was detected in all sheep but class I only in sheep with the most severe lesions 6 months p.i., an indication of a higher threshold for induction of class I than class II antigens on microglia. Few cells expressed viral antigens, indicating that direct immune-mediated destruction of infected cells plays a minor role in evolution of lesions. Since the preferential induction of MHC antigens on microglia in the white matter correlated with the lesion pattern, activated microglia may play a considerable role in the pathogenesis of lesions.

Constitutive Expression of Class 3 Aldehyde Dehydrogenase in Cultured Rat Corneal Epithelium

Mammalian Class 3 aldehyde dehydrogenase (ALDH) is normally associated with neoplastic transformation or xenobiotic induction by aromatic hydrocarbons in liver. However, Class 3 ALDH is constitutively expressed at it's highest specific activity in corneal epithelium. Tissue-specific, differential gene expression is often controlled by alternative, independent molecular pathways. We report here the development of an in vitro corneal epithelium culture system that retains constitutive high expression of the ALDH3 gene. This model system was used to establish, by enzymatic assays, Western and Northern analyses, histochemical and immunocytochemical staining, and 5'3' RACE methodologies that constitutive and xenobiotic induction of Class 3 ALDHs occurs from a single gene. Our results also provide a plausible explanation for the very high Class 3 ALDH activity in mammalian cornea, as the primary mechanism of oxidation of lipid peroxidation-derived aldehydes. Further studies with corneal epithelium suggest the presence of additional mechanisms, other than Ah-receptor-mediated, by which the ALDH3 gene can be differentially regulated in a tissue-specific manner.

Macrophage synthesis of nitric oxide in the mouse mixed leucocyte reaction

The production of nitric oxide (•NO) in the splenocyte mixed leucocyte reaction (MLR) results in inhibition of allospecific lymphocyte effector function. In order to more clearly define the circumstances which promote •NO synthesis in the MLR, responder accessory cell depleted spleen cells (ACDSC) were co-cultured with allogeneic macrophage cell lines or peritoneal macrophages. •NO synthesis and C57BL/6 (H-2 b) ACDSC proliferation were concurrently monitored in cultures comparing RAW 264.7 (H-2 d, a high •NO producer), P388D1 (H-2 d, a low •NO producer) and BALB/c (H-2 d) peritoneal macrophages as allogeneic antigen presenting cells (APC). A concentration-dependent increase in lymphocyte proliferation was observed in the presence of 1×10 4 to 1×10 5 P388D1. In contrast, addition of N G-monomethyl- L-arginine (NMA), a competitive inhibitor of •NO synthase, was necessary in order to observe lymphocyte proliferation in the presence of increasing numbers of RAW 264.7 and BALB/c peritoneal macrophages. The addition of both anti-IL-2 and anti-IFNγ (interferon-gamma) monoclonal antibodies inhibited •NO synthesis in alloantigen-stimulated cultures. The IFNγ induced expression of class II antigen, as well as the constitutive expression of class I antigen, on RAW 264.7 was similar in the presence or absence of NMA, indicating that induction of •NO synthesis by IFNγ does not inhibit H-2 antigen expression. Thus, cytokines produced as a result of alloimmune interaction initiate macrophage •NO synthesis. However, allogeneic APC function, as assessed by H-2 antigen expression and subsequent stimulatory capacity in MLR, is not affected by initiation of the •NO pathway.

Immunotoxicology of cadmium and mercury on B-lymphocytes — I. Effects on lymphocyte function

Heavy metals have been shown to exert immunotoxic effects on humoral immunity. To ascertain the mechanisms by which these immunotoxic effects are exerted, the effects of CdCl 2 and HgCl 2 on the biology of murine B-lymphocytes were studied. It was shown that CdCl 2 aand HgCl 2 inhibited B-cell RNA and DNA synthesis. The IC 50 (the concentration required to inhibit a specific B-cell function by 50%) for CdCl 2 was 30 μM for RNA synthesis and DNA synthesis. The IC 50 for HgCl 2 was 50 and 120 nM for RNA and DNA synthesis, respectively. Cell cycle analysis revealed that B-cells were arrested throughout the cell cycle with CdCl 2 and HgCl 2. The inhibitory effects exerted by CdCl 2 were rapid, inhibiting RNA synthesis within 2 h of activation. Differentiation to Ig secretion was inhibited by CdCl 2 and HgCl 2 in culture and there appeared to be selective effects on specific Ig isotypes. IgG 3 production was most sensitive to inhibition by CdCl 2 and HgCl 2 followed by IgG 1 and IgG 2b and then IgM and IgG 2a. Changes in the expression of B-cell surface antigens induced by LPS were also influenced by CdCl 2. LPS-induced increases in class II MHC expression was inhibited by CdCl 2, as was the constitutive expression of class I MHC antigen. A summary of the IC 50 for CdCl 2 are presented. In summary, both CdCl 2 and HgCl 2 exert early, inhibitory effects on B-cell activation. This is manifested by the inhibition of RNA, DNA and antibody synthesis. However, selective effects on the production of specific Ig isotypes by these metals may influence the ability of B-cells to mount effective immune responses to pathogens.

The function of vacuolar beta-1,3-glucanase investigated by antisense transformation. Susceptibility of transgenic Nicotiana sylvestris plants to Cercospora nicotianae infection.

Vacuolar class I beta-1,3-glucanases (EC 3.2.1.39) are believed to be important in the induced defense reaction of plants to fungal infection. We used antisense transformation to test this hypothesis and to identify other possible physiological functions of this enzyme. Nicotiana sylvestris plants were transformed with antisense constructions containing the region from position 27 to 608 of the coding sequence of the basic, vacuolar beta-1,3-glucanase gene GLA of tobacco regulated by cauliflower mosaic virus 35S RNA expression signals. Plants homozygous for this transgene showed a marked, ca. 20-fold reduction in the constitutive expression of class I beta-1,3-glucanase antigen in their leaves. RNA blot analysis indicated that the antisense plants expressed low levels of the sense transcript of the host beta-1,3-glucanase gene and the antisense transcript of the transgene. Immune blot analysis of plant extracts indicated that only expression of the N. sylvestris homologue of class I tobacco beta-1,3-glucanase and not the acidic, class II isoforms of the enzyme was blocked in the antisense plants. Class I isoforms of beta-1,3-glucanase and chitinase were coordinately induced in leaves of untransformed and empty-vector-transformed N. sylvestris plants treated with ethylene or infected with the fungal leaf pathogen Cercospora nicotianae. In antisense plants, chitinase but not beta-1,3-glucanase was induced under these conditions indicating that antisense transformation effectively blocks constitutive as well as induced expression of class I beta-1,3-glucanase. Under greenhouse conditions, antisense plants developed normally and were fertile. The plants did not exhibit increased susceptibility to C. nicotianae infection. These results suggest that expression of the beta-1,3-glucanase isoform blocked by antisense transformation is not necessary for 'housekeeping' functions of N. sylvestris nor defense against the fungal pathogen tested.

Expression of major histocompatibility complex antigens on inflammatory peripheral nerve lesions

The expression of major histocompatibility complex (MHC) antigens by cells of the rat peripheral nervous system (PNS) was studied using a model of peripheral nerve transplantation. Monoclonal antibodies to polymorphic determinants of MHC class I and class II (Ia) molecules were used to determine donor or recipient origin of MHC antigen-bearing cells in nerve allografts. The expression of class I and class II antigens by PNS parenchymal cells was modified during varying alloimmune conditions. Baseline, constitutive expression of class I antigens on endothelial and perivascular cells and class II antigens on interstitial cells were identified. Decreased MHC antigen expression was noted following in vitro culture of nerve allografts prior to implantation. After transplantation, enhanced donor-derived MHC antigen expression was demonstrated by both cultured and untreated allograft endothelial, perivascular and interstitial cells in a pattern which was distinct from isografts. This data supports a concept of perivascular monocytic and/or parenchymal cell (Schwann cell or resident macrophage-like cell) activity as the resident antigen-presenting cell for PNS immune processes.