The mycotoxin deoxynivalenol inhibits the cell surface expression of activation markers in human macrophages.

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. It exhibits several toxic effects including impaired growth and immune dysregulation. Macrophages play pivotal role in the host defense; upon activation, they express several specific cell surface receptors that are important in adhesion and cell signaling. Several studies have demonstrated that DON can affect macrophages, however, very few data are available concerning the effect of DON on human macrophages, and the effect on macrophage cell surface receptors is unknown. In the present study, human blood monocytes, differentiated in vitro into macrophages, were activated with IFN-gamma, in the presence or absence of low concentrations of DON. The expression of CD11c, CD13, CD14, CD18, CD33, CD35, CD54, CD119 and HLA-DP/DQ/DR was analyzed by flow cytometry. As expected, macrophage activation by IFN-gamma upregulated the expression of CD54, CD14, CD119 and HLA-DP/DQ/DR. Incubation with DON decrease the cell surface expression of these activation markers in a dose-dependent manner. When cells were treated with 5muM DON, the mean fluorescence intensity measured for the expression of these receptors was the same as that observed in non-activated macrophages. This inhibitory effect of DON was only observed when the mycotoxin was applied before the activation signal. Taken together, our results suggest that low concentration of DON alter macrophage activation as measured by the expression of cell surface markers. This may have implications for human health when consuming DON contaminated feed.

Comparison of the transcriptional regulation of classical and non-classical MHC class II genes.

The class II transactivator (CIITA) regulates expression of the classical and non-classical MHC class II genes, HLA-DR, -DP, -DQ and -DM, but not the B cell-specific HLA-DO (DO). Here we show that only HLA-DR expression is completely dependent on CIITA, since residual expression of HLA-DM, -DP and the beta chain of DQ was observed in CIITA-deficient RJ2.2.5 cells. Although DO shows a unique expression pattern compared to other MHC class II genes, prolonged IFN-gamma treatment of HeLa cells induced DOB expression. Similar to all MHC class II promoters, the DOB promoter contains the highly conserved W, X1, and Y boxes in addition to a putative OCT box. Mutational analysis of the DOB promoter demonstrated that the X1, Y and OCT boxes are necessary for maximum promoter activity.Furthermore, our results demonstrate that CREB-1, RFXANK and Oct-2 occupy the DOB promoter in vivo, However, CIITA and Bob-1 were only minimally recruited. Finally, fusion of Bjab, a DOB-negative B cell line, with.174 B cells that lack the complete MHC class II region (including the DO genes), lead to DO expression. These data indicate that the expression of DO is regulated by an unidentified factor in B cells.

Glucocorticoids enhance interleukin-4 production to neo-antigen (hyaluronidase) in children immunocompromised with cytostatic drugs.

Immunoglobulin E (IgE)-mediated immediate-type allergic reactions to hyaluronidase have been observed in children with central nervous system (CNS) tumors. Glucocorticoids, used as therapy for brain edema, are discussed controversially as T helper 2 (Th2) stimulatory factors. In this study we investigated the role of glucocorticoids on a Th2 cytokine-promoting effect in children with CNS tumors. Peripheral blood mononuclear cells (PBMCs) from: 29 children suffering from malignant brain tumors, of whom 23 received short-term glucocorticoid treatment (for 3-4 days) during the course of chemotherapy; 18 children with nephrotic syndrome or renal transplantation receiving long-term glucocorticoid treatment; and 13 healthy children, were incubated with phytohemagglutinin (PHA) and/or anti-CD28 monoclonal antibody (mAb) and, in a second approach, with hyaluronidase. The concentrations of Th cell-mediated cytokines - interleukin (IL)-4, IL-10, and interferon-gamma (IFN-gamma) - were measured in supernatants. The IL-4 production of PBMCs incubated with PHA/anti-CD28 mAb from children with repeated co-administration of glucocorticoids, hyaluronidase, and cytostatic drugs (median: 249.9 pg/ml; range: 234.4-261.7) was significantly higher (p < 0.0001) than IL-4 production of PBMC from children of all the other groups (median: 86.18; range: 16.0-212.5). There was no significant difference in the levels of IL-10 and IFN-gamma within the groups. PBMCs stimulated only with hyaluronidase failed to produce detectable levels of cytokines. The results of this study indicate that repeated co-administration of glucocorticoids and hyaluronidase (a neo-antigen) enhance IL-4 production in vitro and thus may induce the production of specific IgE antibodies in children immunocompromised with cytostatic drugs. Hyaluronidase itself does not stimulate in vitro IL-4 synthesis in PBMCs of children receiving cytostatic drugs.

Secondary structure composition and pH-dependent conformational changes of soluble recombinant HLA-DM.

HLA-DM catalyzes the release of invariant chain fragments from newly synthesized major histocompatibility complex (MHC) class II molecules, stabilizes empty class II molecules, and edits class II-associated peptides by preferentially releasing those that are loosely bound. The ability of HLA-DM to carry out these functions in vitro is pH dependent, with an optimum at pH 4.5-5.5 and poor activity at pH 7. The structural basis for these properties of HLA-DM is unknown. Sequence homology suggests that HLA-DM resembles classical, peptide-binding MHC class II molecules. In this study, we examined whether HLA-DM has a secondary structure composition consistent with an MHC fold and whether HLA-DM changes conformation between pH 5 and pH 7. Far-UV circular dichroism (CD) spectra of recombinant soluble HLA-DM (sDM) indicate that HLA-DM belongs to the alpha/beta class of proteins and structurally resembles both MHC class I and class II molecules. The CD peak around 198 nm increases upon going from neutral to endosomal pH and drops sharply upon denaturation below pH 3.5, distinguishing at least three states of sDM: the denatured state and two highly similar folded states. Fluorescence emission spectra show a slight blue-shift and a approximately 20% drop in intensity at pH 5 compared with pH 7. Unfolding experiments using guanidinium chloride show that the stability of sDM is somewhat reduced but not lost at pH 5. These results indicate that sDM undergoes a pH-dependent conformational change between neutral and endosomal pH. The change seems to involve both hydrogen bonding patterns and the hydrophobic core of sDM and may contribute to the pH dependence of DM activity.

HLA class II expression in uninducible hepatocarcinoma cells after transfection of AIR-1 gene product CIITA: acquisition of antigen processing and presentation capacity.

The AIR-1-encoded CIITA transcriptional activator is crucial for both constitutive and IFN-gamma-induced MHC class II gene transcription. We show here that the MHC class II negative phenotype of the human hepatocarcinoma cell lines Alexander and HepG2 remains unmodified after treatment with IFN-gamma, although MHC class I expression is up-modulated. This correlates with absence of CIITA mature transcripts. Transfection of an expressible CIITA cDNA in Alexander cells resulted in a very high cell surface expression of all three human class II subsets, HLA-DR, -DP and -DQ, indicating that normally observed induction of CIITA expression by IFN-gamma is probably blocked, in the hepatocarcinoma cell lines, at the level of CIITA transcription and not at the level of IFN-gamma receptor binding and signal transduction mechanisms. To assess whether MHC class II expression on CIITA-transfected Alexander cells could have functional relevance, we tested their capacity to present antigenic peptides to an HLA-DR-restricted T cell line specific for a peptide of Mycobacterium tuberculosis Ag85 protein. It was found that the transfected cells could not only present the exogenously supplemented peptide but also process Ag85 protein to generate the specific epitope recognized by the HLA-DR-restricted T cell line. Similar results were obtained with CIITA-transfected CFPAC-1 pancreatic adenocarcinoma cells, which differed from Alexander cells in that they were inducible by IFN-gamma. These results suggest new strategies to act on CIITA for increasing the potential of a tumor cell to present putative tumor Ags to the immune system.

Binding of copolymer 1 and myelin basic protein leads to clustering of class II MHC molecules on antigen-presenting cells.

Copolymer 1 (Cop 1), a synthetic copolymer of amino acids, effective in suppression of experimental allergic encephalomyelitis (EAE) and myelin basic protein (MBP), was shown to bind extensively and promiscuously to the class II MHC molecules on antigen-presenting cells (APC) without prior processing. In the case of human APC, binding has earlier been demonstrated to DR but not DQ or class I molecules. In the present study, we examined whether binding of Cop 1 and MBP affects MHC class II expression on the cell membrane. Biotinylated derivatives of these antigens were used to monitor their direct binding to MHC molecules on living APC by flow cytometry using phycoerythrin-streptavidin, while the levels of MHC surface expression were monitored by staining with FITC-conjugated anti-class I- and class II-specific antibodies. When Cop 1 or MBP were incubated with the APC, intensity of cell staining with anti-DR, but not with anti-DQ or anti-class I antibodies, was significantly increased, compared to the staining of control APC not reacted with these antigens. In contrast, staining intensity was unaffected when p84-102, a human immunodominant epitope of MBP, or ovalbumin (OVA), a protein which undergoes proteolytic degradation prior to binding, were incubated with the APC. Cycloheximide, a protein synthesis inhibitor, had no effect on the enhanced staining intensity with anti-DR antibody of cells treated with Cop 1 or MBP, whereas it inhibited the enhanced staining of both DR and DQ molecules caused by the respective antibodies, in the absence of these antigens. Brefeldin A, a protein transport inhibitor, lowered the levels of staining intensity with anti-DR and anti-DQ antibodies in both cases, with and without antigen added to the APC. Fluorescence microscopic analysis revealed that cells incubated with Cop 1 or MBP, but not with p84-102 or OVA, exhibit both bright staining of the cell membrane and clusters produced by the aggregation of DR molecules with these antigens. Taken together, these observations indicate that Cop 1 and MBP, due to their polyvalent character, lead to increased fluorescence intensity of their complexes with HLA-DR, possibly due to recruitment and clustering of previously synthesized DR molecules. This can explain the efficient binding of these antigens to the MHC class II molecules.

Leukocyte infiltration in synovial tissue from the shoulder of patients with polymyalgia rheumatica. Quantitative analysis and influence of corticosteroid treatment.

OBJECTIVE: To investigate the immunologic features of synovitis in patients with polymyalgia rheumatica (PMR) and to assess the modifications induced by corticosteroids. METHODS: Arthroscopic biopsies of shoulder synovium were obtained from 12 patients with untreated PMR and from 7 patients with PMR that had been treated. Immunohistochemistry was performed on frozen sections utilizing a panel of monoclonal antibodies and computerized image analysis. RESULTS: Synovitis was present in 10 of 12 (83%) untreated patients and in only 2 of 7 (29%) treated patients. The synovitis was characterized by vascular proliferation and leukocyte infiltration. Infiltrating cells consisted predominantly of macrophages and T Lymphocytes. Almost all T lymphocytes were CD45RO positive. A few neutrophils, but no B cells, natural killer cells, or gamma/delta T cells were found. Intense expression of HLA class II antigens (DR moreso than DP moreso than DQ) was found in the lining layer cells as well as in macrophages and lymphocytes. DR, but not DP or DQ, was expressed by the endothelium of a few vessels. Class II antigen expression correlated with the number of macrophages and lymphocytes. Macrophage infiltration of arteriole walls was observed in 1 untreated patient without giant cell arteritis (GCA). In untreated patients, there was a positive correlation between the percentage of infiltrating T cells and the duration of disease. Steroid therapy was associated with a significant reduction in the number of blood vessels and of HLA class II expression. One treated patient who no longer had symptoms of PMR still had active synovitis: a relapse occurred 4 months after the biopsy. CONCLUSION: Our findings support the hypothesis that synovitis is a major cause of the musculoskeletal symptoms of PMR. There are immunologic similarities with the vascular inflammation observed in GCA. Corticosteroids act on both the vascular and cellular components of synovitis.

Immunomodulation: an important concept in modern anaesthesia.

This review summarises evidence for immunomodulatory effect of drugs administered peri-operatively. The clinical significance of the balance of pro- and anti-inflammatory cytokines may be seen in certain disease states, for example, meningococcal meningitis and Lyme arthritis. This balance may be altered peri-operatively. Traditionally, these changes are considered to be due to the stress response of surgery, the response to cardiopulmonary bypass, or endotoxaemia. This review presents in vitro evidence suggesting that drugs modulating this cytokine balance include non-steroidal anti-inflammatory agents, phosphodiesterase inhibitors and opioids, acting through effects on intracellular cyclic nucleotide messenger systems. An important consequence of the pro-inflammatory cytokine activity is increased adhesion of neutrophils. Aspects of this process may be inhibited by avoiding low blood flow states, by reducing adhesion molecule expression (for example by use of pentoxifylline), or by use of negatively charged anions such as heparin. Neutrophil activity is generally depressed by intravenous anaesthetic induction agents, but is enhanced by opioids. Natural killer cell activity, which is involved in immunity against tumour cells and virally infected cells is transiently depressed by volatile anaesthetic agents and opioids. In contrast catecholamines enhance natural killer cell activity. Whereas decrease in immunoglobulin levels occur peri-operatively, this is not thought to be as a result of drugs at clinically used concentrations but rather due to haemodilution.

IFN-gamma-stimulated enhancement of MHC class II antigen expression by the human mast cell line HMC-1.

The expression of MHC class II molecules by human mast cells has been reported in immunohistochemical surveys of inflammatory conditions, such as in tuberculin hypersensitivity. While these data suggest that human mast cells may act as antigen-presenting cells under inflammatory conditions, the induction of class II antigens on human mast cells has not been examined. In this study, we determined the effects of the inflammatory cytokines IFN-gamma and IL-4 on the expression of class II antigens HLA-DR, -DP, and -DQ by the human mast cell line HMC-1. HMC-1 cells were incubated with or without 1000 U/ml recombinant human IFN-gamma (rhIFN-gamma) and IL-4 (rhIL-4) for 72 hr and analyzed for expression of MHC class II antigens by direct immunofluorescence and flow cytometry. HMC-1 cells expressed significant levels of HLA-DR and moderate levels of HLA-DP and -DQ at baseline and when cultured without exogenous cytokines. Stimulation by rhIFN-gamma for 72 hr significantly increased the levels of HLA-DR and -DP expression but did not affect levels of HLA-DQ. Stimulation by rhIL-4 for 72 hr had minimal effect on expression of class II molecules, but induced a significant difference in levels of ICAM-1 (CD54) expression, indicating that this cytokine is involved instead in the control of certain accessory molecules. Our data showing constitutive expression of MHC class II molecules on HMC-1 cells and upregulation of that expression by rhIFN-gamma suggest that human mast cells function as antigen-presenting cells at sites where inflammatory cytokines are present.

Modulation of MHC class II expression in human cells by dexamethasone.

The effect of dexamethasone on human MHC class II expression was examined on various cell types including lymphocytes, monocytes, and epithelial cells. Dexamethasone decreased the surface expression of HLA-DR and -DP, but not HLA-DQ, on lymphocytic cell lines that constitutively express these molecules. In addition, dexamethasome down-regulated the mRNA levels of HLA-DRA, but not of HLA-DQB, in Jijoye cells, a human lymphoblastic cell line. Similarly, dexamethasone decreased HLA-DR expression on epithelial and monocytic cell lines that express HLA-DR upon IFN-gamma treatment. In total, these results suggest that dexamethasone inhibits both constitutive and IFN-gamma-inducible MHC class II expression in several cell types. Moreover, these results indicate that the inhibitory effect of dexamethasone on MHC class II expression is selective for HLA-DR and -DP but not HLA-DQ. Possible mechanisms of dexamethasone-mediated regulation of MHC class II expression are discussed.

Isotype-specific regulation of MHC class II gene expression in human monocytes by exogenous and endogenous tumor necrosis factor.

The control of expression of MHC class II molecules on antigen-presenting cells is important for the induction of immunity, while aberrant expression of these molecules plays a role in the immunopathology of autoimmune diseases. This study explored the role of tumor necrosis factor alpha (TNF) in controlling the level of HLA class II mRNA in human monocytes. Exposure of monocytes to exogenous recombinant TNF (rTNF) selectively up-regulated DR alpha-mRNA but not DP or DQ alpha-mRNA. Inhibitors of TNF synthesis, pentoxifylline (PTX) and thalidomide, inhibited TNF mRNA accumulation in LPS-activated monocytes and down-regulated DR mRNA but not DP or DQ mRNA. The inhibitory effect of anti-TNF monoclonal antibody (MAb) indicated that endogenously generated TNF acted extracellularly. Anti-p75 TNF-R2 receptor and to a lesser extent anti-p55 TNF-R1 MAbs inhibited TNF-mediated up-regulation of DR mRNA and TNF mRNA. Taken together, this implies that endogenously generated TNF plays a role in controlling isotype-specific MHC class II gene expression in human monocytes/macrophages. These results may have some implications for anti-tumor response and autoimmunity.

Interleukin-6 exacerbates glomerulonephritis in (NZB x NZW)F1 mice.

The ability of interleukin-6 (IL-6) to modulate immune parameters and mesangial cell function suggests a role for this cytokine in the development of autoimmune glomerulonephritis. This hypothesis was tested in 6-month-old female (NZB x NZW)F1 mice that were administered recombinant human IL-6 (rhIL-6) (50 and 250 micrograms/kg s.c.) for 12 weeks, resulting in an accelerated and severe form of membranoproliferative glomerulonephritis associated with marked upregulation of mesangial major histocompatibility complex class II antigen and glomerular ICAM-1 expression. To distinguish direct effects of rhIL-6 on the renal mesangium from those mediated through the immune system, (NZB x NZW)F1 mice were immunosuppressed with cyclosporin. Immunosuppression by cyclosporin inhibited the development of glomerulonephritis, decreased class II antigen expression, and abrogated IL-6-mediated effects. Administration of neutralizing anti-IL-6 antibody had no effect on the spontaneous development of glomerulonephritis in (NZB x NZW)F1 mice. This finding, together with undetectable IL-6 serum levels, makes a pathogenetic role of endogenously produced IL-6 in this disease model unlikely. In contrast to (NZB x NZW)F1 mice, parental NZW or BALB/c mice given high doses of rhIL-6 (500 micrograms/kg) or recombinant murine IL-6 (100 micrograms/kg) daily for 4 weeks failed to develop morphological or biochemical evidence of glomerulonephritis. Induction of acute phase proteins, anemia, thrombocytosis, and induction of renal class II antigen confirmed the biological activity of IL-6 in these mice. In conclusion, while non-nephritogenic in normal mice, IL-6 accelerates the development of the genetically determined glomerulonephritis of (NZB x NZW)F1 mice through effects mediated by a modulated immune system. Since neutralizing IL-6 antibody treatment did not prevent the development of glomerulonephritis, it is unlikely that increased IL-6 production plays a role in the pathogenesis of lupus nephritis.

Immunosuppressive effects of 1,25-dihydroxyvitamin D3 and its analogue calcipotriol on epidermal cells.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3; calcitriol) is the biologically active form of vitamin D. This hormone is a potent immunoregulatory agent. Calcipotriol is a synthetic analogue of 1,25(OH)2D3, with similar receptor binding, and comparable effects on cell proliferation and differentiation, but less potent effects on calcium metabolism. As a step towards understanding the mechanisms by which vitamin D compounds affect T-cell activation by epidermal cells (EC), we assessed the effects of 1,25(OH)2D3 and calcipotriol on the human allogeneic mixed epidermal cell-lymphocyte reaction. All experiments were performed both with 1,25(OH)2D3, and calcipotriol, with similar results. Both compounds had potent immunoinhibitory properties on this model, and enhanced the immunosuppressive effects of cyclosporin A. Using preincubation experiments, we found that pretreatment of EC with 1,25(OH)2D3 resulted in a more pronounced inhibition than preincubation of lymphoid cells. The epidermal targets of this inhibitory effect have been further investigated, using cultures with freshly isolated Langerhans cells (LC) or LC-depleted keratinocytes, separated by an immunomagnetic particle technique. Pretreatment of LC induced a 30% decrease of proliferation, compared with vehicle-treated LC. These calcitriol-pulsed LC did not decrease the proliferation induced by unmodified autologous EC. As expected, LC-depleted keratinocytes failed to stimulate allogenic lymphocytes. When added to autologous unmodified EC, however, calcitriol-pulsed keratinocytes induced an 85% decrease of proliferation, compared with vehicle-treated keratinocytes. The phenotypic expression of HLA-DR, -DQ, and -DP antigens on EC, assessed by immunoalkaline phosphatase staining, was not modified after a 2-h or 24-h pulse with 1,25(OH)2D3 or calcipotriol.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethanol influences class I and class II MHC antigen expression on human fetal islet-like cell clusters.

Several autoimmune diseases have been linked to an aberrant expression of major histocompatibility complex (MHC) products Ethanol enhances Class I and Class II products on a variety of cell types, and there is evidence for an autoimmune etiology in numerous pathologies associated with alcoholism. We examined whether ethanol alters the expression of Class I and Class II MHC products on human fetal islet-like cell clusters. Incubation of islet-like clusters for 48 hr in ethanol at a starting concentration of 1.5% increased the percentage of single cells expressing Class I. The percentage of cells expressing Class II did not change, but their relative mean fluorescence increased significantly. These findings suggest that alcohol ingestion could alter MHC expression on pancreatic islet cells in vivo perhaps affecting the development of diabetes in genetically predisposed individuals. These findings also support the hypothesis that the rising incidence of type 1 diabetes seen in areas of the world where the per capita consumption of alcohol is also increasing may be a consequence of the immunological effects of alcohol intake.