MHC class II antigen expression in human vascular smooth muscle cells is induced by interferon-gamma and modulated by tumour necrosis factor and lymphotoxin

Arterial smooth muscle cells (SMC) express major histocompatibility complex (MHC) class II antigens in experimental vasculitis and in the human atherosclerotic plaque. We have therefore studied the regulation of expression of MHC antigens in cultured human arterial SMC, using immunofluorescence, radioimmunoprecipitation and a quantitative cell-surface immunoradiometric assay. SMC expressed class I, but not class II, antigens on their cell surfaces under basal conditions. Treatment of SMC with recombinant or natural interferon-gamma (IFN-gamma) induced expression of class II antigens in the following order of intensity, DR greater than DP greater than DQ. HLA-DR protein in SMC showed the same MW as that synthesized by B-lymphoblastoid cells. Antibodies to IFN-gamma blocked all HLA-DR-inducing activity in mixed leucocyte reaction (MLR) supernatants and PHA-stimulated peripheral blood mononuclear cell (PBMC)-conditioned media, indicating that IFN-gamma is the only lymphokine secreted under these conditions that is capable of de novo induction of HLA-DR expression in SMC. Treatment of SMC with recombinant human tumour necrosis factor-alpha (TNF) or lymphotoxin (LT) did not per se induce class II antigen expression. However, both TNF and LT substantially enhanced IFN-gamma-induced expression of HLA-DQ while decreasing that of HLA-DP. TNF, but not LT, increased HLA-DR expression. Also, in dermal fibroblasts, IFN-gamma-induced HLA-DP expression was significantly inhibited in the presence of TNF. These data demonstrate that TNF and LT differentially modulate IFN-gamma-induced MHC antigen expression in mesenchymal cells. The fact that SMC can express MHC class II antigens suggests that this cell type may serve as an accessory cell in the initiation of the immune response.     

Up-regulation of the dendritic cell marker CD83 on polymorphonuclear neutrophils (PMN): divergent expression in acute bacterial infections and chronic inflammatory disease

Upon cultivation with interferon-gamma (IFN-gamma ) and granulocyte/macrophage-colony stimulating factor (GM-CSF) polymorphonuclear neutrophils (PMN) acquire characteristics of dendritic cells, including expression of major histocompatibility complex (MHC) class II antigens, of the co-stimulatory antigens CD80, CD86 and of CD83, the latter considered to be specific for dendritic cells. Dendritic-like PMN were also able to present to T cells antigens in a MHC class II-restricted manner. To assess whether dendritic-like PMN are also generated in vivo, cells of patients with acute bacterial infections and of patients with chronic inflammatory diseases (primary vasculitis) were tested. During acute infection up to 80% of PMN acquired CD83, but remained negative for MHC class II, CD80 or CD86. PMN of patients with primary vasculitis expressed MHC class II antigens, CD80 and CD86, but not CD83, indicating that up-regulation of MHC class II and of CD83 are not necessarily linked to each other. Indeed, parallel studies with PMN of healthy donors showed that while IFN-gamma and granulocyte/macrophage colony stimulating factor (GM-CSF) induced both, MHC class II and CD83, tumour necrosis factor (TNF)-alpha selectively induced de novo synthesis of CD83. The function of CD83 on PMN is still elusive. A participation in the MHC class II-restricted antigen presentation could be ruled out, consistent with the segregation of MHC class II and CD83 expression. Regardless, however, of its function, CD83 expression could serve as a marker to differentiate between acute and chronic inflammation.     

Induction of class II major histocompatibility complex antigens in murine placenta by 5-azacytidine and interferon-gamma involves different cell populations

Class II MHC antigen expression is required for recognition of an alloantigen and generation of immune response. In rodents as well as in humans primary trophoblasts do not express class II MHC antigens. In this study we focused our interest on the mechanism(s) of class II antigen suppression on murine trophoblasts. First, we examined the possibility of gene inactivation by methylation and second the possibility of lymphokine regulation of the class II genes. The first possibility was tested by treatment of placental cells with 5-azacytidine (5-AzaC), a cytidine analog which upon incorporation into the DNA inhibits further methylation, thus leading to gene activation. In order to test the second possibility we treated placental cells with interferon-gamma (IFN-gamma) or interleukin 4 (IL4) which are known to induce class II antigen expression in many systems. We showed that treatment with 5-AzaC or IFN-gamma but not IL4 significantly increased class II expression on cytokeratin-positive and vimentin-negative adherent placental cells. Following placental cell fractionation we distinguished three cell subsets with different responsiveness to 5-AzaC and IFN-gamma. The first, characterized as placental macrophages, were induced to express class II MHC antigens only after IFN-gamma treatment. The other two subsets, characterized as trophoblasts, were isolated from the labyrinthine- and spongio-trophoblast layer of the placenta and showed class II inducibility to 5-AzaC and IFN-gamma, respectively. The results show that depending on the anatomical localization of trophoblasts within the placenta, various regulatory elements control gene expression, so that the placental barrier provides fetal protection at different levels.     

Schwann cells co-cultured with stimulated T cells and antigen express major histocompatibility complex (MHC) class II determinants without interferon-gamma pretreatment: synergistic effects of interferon-gamma and tumor necrosis factor on MHC class II induction

Schwann cells (SC) do not express major histocompatibility complex (MHC) class II antigens under normal culture conditions. SC can, however, be induced in vitro to express MHC class II molecules by exposure to high concentrations of interferon-gamma (IFN-gamma) and can present antigens to antigen-specific T cell lines. In the present study immunohistochemical labeling showed that most SC (greater than 90%) prepared from rat neonatal sciatic nerves expressed MHC class II molecules when cultured together with mycobacterial antigen and T cells, and as a consequence were able to function as antigen-presenting cells in lymphoproliferation assays, without requiring pretreatment with IFN-gamma. Antigen or T cells alone were ineffective in stimulating MHC class II expression and induction of class II molecules was MHC restricted, requiring the presence of syngeneic T cells. Addition of monoclonal antibody DB1, directed against IFN-gamma to co-cultures of SC and T lymphocytes stimulated with antigen, prevented the induction of MHC class II antigen on SC. When SC were incubated with recombinant (r)IFN-gamma alone, up to 50% of SC showed positive labeling for MHC class II antigen. This level of expression was enhanced to greater than 80% when recombinant tumor necrosis factor (rTNF) was also added. rTNF alone had no effect, and addition of DBI antibody inhibited the synergistic effects of rTNF on MHC class II expression. The effects of rIL 4 were also investigated but neither rIL 4 alone nor rIL 4 in combination with rIFN-gamma induced MHC class II expression by SC. These results show that in the presence of sensitized T lymphocytes and antigen, SC do not require pretreatment with exogenous rIFN-gamma to express MHC class II antigens and function as antigen-presenting cells. T cell-derived TNF and IFN-gamma appear to act as mediators of the T cell-induced expression of MHC class II by SC.     

MHC class II antigen expression is not induced on murine epidermal keratinocytes by interferon-gamma alone or in combination with tumour necrosis factor-alpha

Epidermal keratinocytes are induced to express MHC class II molecules in a variety of disease states associated with immune activity. To investigate the mechanism of this process we have exposed murine and rat keratinocytes to a variety of lymphokines and monitored changes in their MHC molecule expression. Murine cultured keratinocytes were treated with recombinant interferon-gamma (IFN-gamma), and MHC antigen expression quantified by flow cytometry. IFN pretreatment resulted in the up-regulation of class I molecule expression, but no class II expression was detected. In addition, cultured murine keratinocytes exposed to a combination of recombinant tumour necrosis factor (TNF)-alpha and IFN-gamma, or crude lymphocyte supernatants, failed to show positive membrane staining for class II molecules. However, rat keratinocytes cultured under conditions identical to murine cells were induced to express class II molecules after IFN-gamma pretreatment. The inability of IFN to induce class II expression on murine keratinocytes appears not to result from cell culture, as subcutaneous injection of IFN fails to induce epidermal class II antigen expression. However, class II expression can be induced on rat epidermis in vivo. Thus, the response of epidermal keratinocytes to IFN-gamma appears to show species variation.     

Reduced stimulation of helper T cells by Ki-ras transformed cells.

A number of viral genes and cellular oncogenes inhibit major histocompatibility complex (MHC) antigen expression at the cell surface. In the case of inhibition of class I MHC antigens by viral genes this results in a reduced recognition by antigen-specific cytotoxic T cells. The activated Ki-ras cellular oncogene carried by the Ki-murine sarcoma virus (Ki-MuSV) in contrast inhibits class II MHC (or Ia) antigen expression on transformed cells. We have studied how transformation with Ki-ras affects recognition by alloreactive helper T cells. We found that the Ki-ras inhibition of class II MHC antigen expression led to greatly reduced stimulation of alloreactive T cells to proliferate and to secrete interferon-gamma (IFN-gamma). These findings support our hypothesis that the ability of an oncogene to reduce class II MHC antigen expression is crucial to its ability to produce tumour cells.     

Systemic anti-IFN-gamma treatment and role of macrophage subsets in the foreign body reaction to dermal sheep collagen in rats

The application of a biomaterial induces a foreign body reaction. By controlling this reaction, biocompatibility could be improved. We previously demonstrated that impregnation of a biodegradable biomaterial with antibodies against interferon-gamma (IFN-gamma) inhibits the foreign body reaction. In this study we investigate whether systemic administration of the antibody can induce similar reactions. Several parameters are compared between control and anti-IFN-gamma-treated rats: cellular ingrowth; degradation of the biomaterial; ingrowth of macrophage (MO) subsets, T cells, B cells, NK cells, and granulocytes; and expression of the major histocompatibility complex class II (MHC class II) molecule on antigen presenting cells. Treatment with anti-IFN-gamma results in increased cellular ingrowth and biomaterial degradation and a decreased expression of MHC class II. Overall, systemic treatment with anti-IFN-gamma is insufficient to modulate the foreign body reaction. This suggests an alternative mechanism for MO activation besides IFN-gamma. The role of T cells and MO subsets in the foreign body reaction is discussed.     

Role of interferon-gamma in T-cell responses to Semliki Forest virus-infected murine brain cells

Primary brain cell cultures prepared from newborn C3H mice were infected with Semliki Forest virus (SFV) or treated with a beta-propiolactone-inactivated preparation of SFV (BPL-SFV). The effects of recombinant interferon-gamma (IFN-gamma) treatment on SFV replication, SFV antigen display, major histocompatibility complex (MHC) class I and class II antigen expression, susceptibility to lysis by SFV-specific cytotoxic T lymphocytes (CTL) and the ability to stimulate SFV-specific T lymphocytes to release IFN-gamma were determined. The IFN-gamma treatment prevented replication of SFV, as determined by incorporation of [3H]uridine into SFV-RNA, and reduced expression of SFV antigens on the cell surface, as determined by lysis with antibody and complement or indirect immunofluorescence. BPL-SFV-treated brain cells expressed no SFV antigen detectable by lysis with antibody and complement or indirect immunofluorescence. IFN-gamma increased expression of MHC class I and class II antigens, measured by indirect immunofluorescence, susceptibility to killing by alloreactive T-cell lines and ability to stimulate an allogeneic mixed lymphocyte reaction (MLR). Brain cells infected with SFV or treated with BPL-SFV were susceptible to killing by the CTL. The killing was MHC restricted and neither uninfected nor untreated cells were killed. IFN-gamma treatment prior to SFV infection or BPL-SFV treatment resulted in an augmentation of lysis by the CTL, indicating that even where SFV antigen expression is reduced or present at very low levels, in the context of enhanced MHC class I expression cells remain susceptible to CTL killing. Brain cells treated with BPL-SFV stimulated SFV-specific T cells to release IFN-gamma. Pretreatment of brain cells with IFN-alpha beta or IFN-gamma prior to BPL-SFV treatment markedly increased the ability of the cells to stimulate the SFV-specific T cells to release IFN-gamma. Release of IFN-gamma was MHC restricted and brain cells untreated with BPL-SFV did not stimulate IFN-gamma release. IFN-gamma released by T cells stimulated with BPL-SFV-treated brain cells increased class II MHC expression by brain cells as assessed by indirect immunofluorescence.     

Class II MHC antigens in the rat digestive system. Normal distribution and induced expression after interferon-gamma treatment in vivo

The normal and interferon-gamma induced expression of class II MHC antigens was investigated immunohistologically in the digestive system of LEW rats. In the normal state class II molecules were present in interstitial dendritic cells, B lymphocytes and epithelial cells. Epithelial class II expression was restricted to enterocytes in certain portions of the small intestine and to some duct epithelia in salivary glands. After continuous intravenous infusion of interferon-gamma (IFN-gamma) for 3 days, class II MHC antigens were induced in large vessel endothelium and in the surface epithelia of the tongue, oesophagus and proventricle. In the gastric glands class II molecules appeared in mucous neck cells and in parietal cells, while adjacent mucous surface cells and chief cells did not acquire class II reactivity. All enterocytes, including the previously negative colonic epithelium, were induced to express class II antigens. In the salivary glands class II antigens appeared in all duct epithelia. Serous acinar cells were induced in the parotids, but in the submandibular glands and in the pancreas the serous gland epithelium stayed negative. Our study thus shows that the effects of IFN-gamma on class II MHC antigen expression in vivo depend on the differentiation pathway of the individual cell. The normal distribution in rats suggest that class II MHC antigens may play a role in peptide transport across specialized epithelia. It remains to be determined whether such a function is enhanced after IFN treatment.     

Influence of cytokines and immunosuppressive drugs on major histocompatibility complex class I/II expression by human cardiac myocytes in vitro

Human cardiac myocytes do not express detectable levels of major histocompatibility complex (MHC) class II antigens and express low levels, if any, of MHC class I antigens. During rejection episodes, cardiac biopsies show massive increases of MHC antigens, which are thought to be induced by cytokines released by donor-sensitized recipient mononuclear cells. In efforts to determine the nature of the cytokines that induce MHC expression on cardiac myocytes, human fetal cardiac myocyte cultures were established. Interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), interleukin (IL)-1, IL-2, IL-3, IL-4, and tumor necrosis factor (TNF)-alpha were added to these cultures and dose/kinetics of MHC class I/II induction quantitated. Data show that IFN-gamma induces both MHC class I and II expression, and all the other cytokines (except IL-2) induce only MHC class I but not class II. Cytokines used in combination showed that IFN-alpha with TNF-alpha was the only combination that induced MHC class II expression. Addition of immunosuppressive drugs such as cytoxan, azathioprine, cyclosporine-A, and FK-506, even when added at the initiation of the cultures, did not appreciably affect the ability of the appropriate cytokines to induce MHC expression by the myocytes in vitro.     

Luminal bacterial antigen-specific CD4+ T-cell responses in HLA-B27 transgenic rats with chronic colitis are mediated by both major histocompatibility class II and HLA-B27 molecules

Rats transgenic (TG) for the human major histocompatibility complex (MHC) class I HLA-B27 and beta2-microglobulin genes develop chronic colitis under specific pathogen-free (SPF) but not sterile (germ-free, GF) conditions. We investigated the role of antigen-presenting molecules involved in generating immune responses by CD4+ mesenteric lymph node (MLN) cells from colitic HLA-B27 TG rats to commensal enteric micro-organisms. All TG MLN cells expressed HLA-B27. A higher level of MHC class II was expressed on cells from TG rats, both SPF and GF, compared to non-TG littermates. In contrast, rat MHC class I expression was lower on TG than non-TG cells. Both TG and non-TG antigen presenting cells (APC) pulsed with caecal bacterial antigens induced a marked interferon-gamma (IFN-gamma) response in TG CD4+ T lymphocytes but failed to stimulate non-TG cells. Blocking MHC class II on both TG and non-TG APC dramatically inhibited their ability to induce TG CD4+ T cells to produce IFN-gamma. Blocking HLA-B27 on TG APC similarly inhibited IFN-gamma responses. When the antibodies against MHC class II and HLA-B27 were combined, no APC-dependent IFN-gamma response was detected. These data implicate both native rat MHC class II and TG HLA-B27 in CD4+ MLN T-cell IFN-gamma responses to commensal enteric microflora in this colitis model.     

Susceptibility to Theiler's virus-induced demyelinating disease correlates with astrocyte class II induction and antigen presentation.

Theiler's murine encephalomyelitis virus (TMEV) is a picornavirus which induces a chronic demyelinating disease of the central nervous system (CNS) in certain susceptible mouse strains. Demyelination has been shown to result from immunopathological responses mediated by CD4+, major histocompatibility complex (MHC) class II-restricted T cells. As little or no class II is expressed in the normal mouse CNS, the ability of astrocytes to express these proteins and present antigen to T cells from TMEV-infected mice was investigated here. It is shown that astrocytes are capable of presenting TMEV to virus-specific T cells in vitro, and that this ability is dependent on prior induction of MHC class II by interferon-gamma (IFN-gamma) treatment. Unlike other viruses such as murine hepatitis virus-JHM (a coronavirus) and measles, TMEV is not capable of inducing class II on astrocytes directly. There is a correlation between the ease of class II induction on astrocytes from different mouse strains by IFN-gamma and mouse strain susceptibility to TMEV-induced demyelinating disease. These results suggest that following viral infection and initial T-cell infiltration into the CNS, class II induction on astrocytes is a key step allowing local antigen presentation and amplification of immunopathological responses within the CNS and hence the development of demyelinating disease.     

Effects of tumor necrosis factor on the interferon-gamma-induced major histocompatibility complex class II antigen expression by human endothelial cells

Effects of tumor necrosis factor-alpha (TNF) and interferon-beta (IFN-beta) on the IFN-gamma-induced major histocompatibility complex (MHC) class II expression on human umbilical vein endothelial (HUVE) cells are reported. TNF inhibited the induction of MHC class II expression by IFN-gamma markedly, when added before or simultaneously with IFN-gamma. However, TNF added to the cells 24 h after IFN-gamma enhanced the expression of MHC class II antigens. IFN-beta inhibited the MHC class II expression irrespective of the time at which it was added to the cells. Addition of IFN-beta, TNF, IFN-gamma, and the combination of IFN-beta and IFN-gamma or TNF and IFN-gamma, resulted in all cases in an enhanced MHC class I antigen expression. Antibodies directed against IFN-beta reversed the inhibition of MHC class II expression by both TNF and IFN-beta. The enhancing effect of TNF could not be inhibited by anti-IFN-beta indicating that TNF mediates enhancement of IFN-gamma-induced MHC class II expression via a pathway other than IFN-beta. The role of TNF in the up-regulation as well as in the down-regulation of MHC class II expression in inflammatory processes is discussed.     

Polymorphonuclear neutrophils as accessory cells for T-cell activation: major histocompatibility complex class II restricted antigen-dependent induction of T-cell proliferation

Polymophonuclear cells (PMN) of healthy donors do not express major histocompatibility complex (MHC) class II antigens or the T-cell costimulatory molecules CD80 or CD86. Expression of these receptors, however, is seen in patients with chronic inflammatory diseases. We now report that, by culturing PMN of healthy donors with autologous serum, interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF), de novo synthesis of MHC class II, CD80 and CD86 could be induced. MHC class II-positive PMN acquired the capacity to present staphylococcus enterotoxin to peripheral T cells, apparent as induction of interleukin-2 (IL-2) synthesis and proliferation of the T cells. Moreover, the PMN also processed tetanus toxoid (TT) and induced proliferation of TT-specific T cells in a MHC class II-restricted manner. Taken together, these data indicate that PMN can be activated to function as accessory cells for T-cell activation.     

Activation by interferon-gamma of expression of ICAM-1 and MHC class II antigens in tumour cells from colorectal carcinomas

Aims-To determine whether lack of MHC class II antigen and intercellular adhesion molecule-1 (ICAM-1) expression in some tumours is due to the inability of the tumour cells to respond to the cytokine interferon-gamma (IFN-gamma), an important activator of these surface molecules.Methods-Cells from 40 colorectal tumours which did not constitutively express class II MHC antigens or ICAM-1 were kept in short term culture after disaggregation for a few days to two weeks without significant loss of viability. These were treated with IFN-gamma. Expression of class II MHC antigens and ICAM-1 was determined using immunohistological techniques.Results-There was clear induction in vitro of both MHC class II antigens and ICAM-1 in cells from eight of the tumours, with between 50 and 80% of the tumour cells in the cultures staining positively. The staining was apparent within 24 hours, appeared maximal at about three days, and declined thereafter. There were no obvious differences in cell morphology or viability between the cultures which were inducible and those which were not, nor were there obvious differences between the tumours from which they were derived.Conclusions-Expression of MHC class II antigens and ICAM-1 may be induced by IFN-gamma in a small proportion of colorectal tumours which do not constitutively express these antigens, showing that only a minority of tumours are capable of responding to this cytokine.