Expression of HLA-DR antigens by thyroid cells: the effect of Graves' IgG

We have investigated factors which influence HLA-DR expression on thyroid cells. While bTSH (100 mU/ml) did not enhance HLA-DR expression, it increased when brought about by IFN-gamma. Graves' IgG showed a dose-dependent (0.1-2 mg/ml) increase in DR expression and at a concentration of 2 mg/ml prolonged the time for which DR was expressed. The pathway of DR induction by Graves' IgG apparently differs from that by IFN-gamma. The humoral response in Graves' disease, by inducing DR expression, may be instrumental in propagating thyroid specific autoimmunity.     

Induction of a polarized micro-environment by human T cells and interferon-gamma in three-dimensional spheroid cultures of human endometrial epithelial cells

Expression of human leukocyte antigen (HLA)-DR molecules andproliferation of epithelium in human endometrium are polarized.We have suggested that the induction of such a polarized micro-environmentis T cell and interferon (IFN)-gamma dependent. The presentstudy was designed to demonstrate the induction of such a micro-environmentaround T cells and around the source of IFN-gamma. Spheroidsreminiscent of endometrial glands were formed by allowing three-dimensionalaggregation of endometrial epithelial cells of a cloned HLA-DRnegative endometrial carcinoma cell line (ECC1) over agarose.Both HLA-DR expression and inhibition of proliferation werefound to be directly dependent on the dose of IFN-gamma thatwas allowed to diffuse in the agarose beneath the spheroids.To show that the interaction of the epithelial cells with activatedT cells also induces HLA-DR molecules in a paracrine fashionin the epithelial cells, ECC1 spheroids were co-cultured withincreasing numbers of allogeneic peripheral blood T cells forvarious time-intervals. T cells bound to the ECC1 cells, andbecame activated as indicated by the expression of interleukin(IL)-2 receptor and HLA-DR molecules. A focal HLA-DR expressionbecame apparent in the ECC1 cells adjacent to the T cells. Asthe number of T cells added to spheroid cultures was increased,a concomitant increase in the number of HLA-DR positive ECC1cells occurred and HLA-DR immunoreactivity was enhanced in eachcell. There was a corresponding decrease in the proliferationof the ECC1 cells in T cell-ECC1 spheroid co-cultures. Basedon these data, we suggest that activation of T cells is associatedwith the induction of HLA-DR expression and inhibition of proliferationin a paracrine fashion in the epithelial cells and may be responsiblefor the creation of a polarized micro-environment in vivo.     

Effect of cytokines on HLA-DR and IL-1 production by a monocytic tumour, THP-1.

The monocytic tumour, THP-1, expresses many of the properties of monocytes, both by cell surface staining and its capacity to produce monokines. It was used as a source of homogenous monocytic cells as a model to determine whether a variety of highly purified or recombinant cytokines could induce HLA-DR expression and the production of interleukin-1 (IL-1). Interferon-gamma (IFN-gamma) alone induced HLA-DR. Tumour necrosis factor (TNF), lymphotoxin (LT) and granulocyte-macrophage colony-stimulating factor (GM-CSF) alone were able to induce IL-1 but not HLA-DR. When IFN-gamma was combined with TNF, induction of HLA-DR and IL-1 was enhanced in a synergistic manner. These effects were detectable at a pretranslational level as synergistic effects were observed on DR alpha mRNA and IL-1 beta mRNA levels. The results demonstrate the specificity of IFN-gamma as the inductive stimulus for HLA-DR expression by THP-1 cells. As IFN-gamma and TNF are products of activated T cells, the synergistic role for these molecules in macrophage activation is discussed.     

Cow's milk protein induced changes in the expression of HLA-DR antigens on colonic epithelial cells.

BACKGROUND: Normal colonic epithelial cells do not express HLA-DR antigens unless they become inflamed. It is possible that colonic epithelial cells may function as antigen-presenting cells once HLA-DR is induced by infection or inflammation. OBJECTIVE: The aim of this study was to investigate whether cow's milk protein (CMP) is capable of inducing changes in HLA-DR expression on colonic epithelial cells, providing indirect evidence that CMP may activate cell-mediated immune mechanisms within intestinal mucosa. METHODS: HLA-DR expression was evaluated by flow cytometry on cultured human colonic epithelial cell line (HT-29) before and after treatment with bacterial lipopolysaccharide (LPS) or recombinant gamma interferon (IFN-gamma). Untreated and LPS- or IFN-gamma-treated HT-29 cells were then cultured in the presence of CMP. The changes in epithelial HLA-DR expression induced by CMP on untreated and LPS- or IFN-gamma-treated HT-29 cells were examined. RESULTS: Untreated HT-29 cells expressed very little HLA-DR molecule. Bacterial LPS or IFN-gamma induced a significant HLA-DR expression on HT-29 cells. When untreated HT-29 cells were cultured in the presence of CMP, there was little induction of HLA-DR expression. Culture of LPS- or IFN-gamma-treated HT-29 cells in the presence of CMP induced a significant increase in HLA-DR expression, which was much greater than on HT-29 cells treated with bacterial LPS or IFN-gamma only. CONCLUSIONS: Our results suggest that CMP initiates an immune response in the intestinal mucosa and may be responsible for the activation of cell-mediated immunity after enteric infection or inflammation.     

Expression of MHC antigens by intestinal epithelial cells. Effect of transforming growth factor-beta 2 (TGF-beta 2).

The effect of interferon-gamma (IFN-gamma) and TGF-beta 2 on expression of MHC antigens by the human intestinal epithelial cell line HT-29 was examined by immunohistochemistry and flow cytometry. Untreated HT-29 cells constitutively expressed HLA-ABC but little HLA-DR. Expression of both molecules was increased by IFN-gamma (100 U/ml, 24 h). TGF-beta 2 at concentrations > or = 0.5 ng/ml given before or simultaneously with IFN-gamma, inhibited the IFN-induced expression of HLA-DR. Small increases in HLA-ABC expression by IFN-gamma were further increased by pretreatment with TGF-beta 2, while a strong induction of HLA-ABC was inhibited by the TGF-beta 2 pretreatment. Our results suggest that the inhibitory action of the TGF-beta 2 on both HLA-ABC and HLA-DR correlates with the degree of induction following IFN-gamma treatment. Since TGF-beta 2 is present in milk and is produced by gut epithelial cells, one of its possible functions may be to regulate expression of HLA antigens in the neonatal intestine and/or diseased intestine.     

Modulation of expression of class II histocompatibility antigens by secretion of a cellular inhibitor in K562 leukemic cells

In this report we show that it is possible to induce the expression of HLA-DR antigens on K562 cells, previously reported to be unresponsive to interferon-gamma (IFN-gamma). However, only low cell concentrations and a high dose of IFN-gamma allowed the induction of HLA-DR antigens. Furthermore, the recombinant glycosylated IFN-gamma is 100-fold more efficient than the unglycosylated form. This induction of HLA-DR antigens on K562 was not related to a stage of differentiation or to the presence of cells subsets specifically sensitive to IFN-gamma, since repeated sorting of K562 HLA-DR-positive and negative cells did not lead to the selection of a cell subset with a different potential of induction for HLA-DR. The difficulty in obtaining induction is due to the production of a soluble endogenous inhibitor of proteic nature, whose action is not restricted to the K562 cell line since it operates also on both epithelial and fibroblastic cells. Treatment of normal human epithelial and fibroblastic cells with conditioned medium from K562 cultures caused a marked decrease in the expression of HLA class II antigens (DR and DP) induced by IFN-gamma (10,000 U/ml), but had no effect on cell growth; however, it also affected expression of HLA class I antigens. This inhibition is not mediated by prostaglandin or an IFN-alpha or IFN-beta-dependent mechanism. Production of this inhibitor by pluripotent human leukemic cells could cause an unbalance in the complex control exerted by the immunological system during hematopoietic differentiation or leukemic progression.     

The antigenic heterogeneity of the bile duct epithelium in alcoholic liver disease. VA Cooperative Study Group 275

The chronic alcoholic patient is usually immunosuppressed, but the significance of this phenomenon in terms of bile duct injury is unclear. The immunoreactivity of the bile duct cells was examined in a series of 69 frozen liver biopsy specimens obtained from patients with alcoholic liver disease, comprising 29 cases of cirrhosis, 26 of alcoholic hepatitis, 10 cases of alcoholic fatty liver, and 4 specimens from normal livers. Liver diseases such as primary biliary cirrhosis and human hepatic allograft rejection, known to have an autoimmune basis, share the characteristic feature of damage to the bile duct epithelial cells. In both instances the damage seems to be immune mediated, but the nature of the antigens involved is not established. We used the avidin-biotin-peroxidase complex method to test in alcoholic liver disease for the expression of a battery of surface antigen markers that have been incriminated in tissue injury and are usually present in lymphoid cells but also expressed by epithelium. In this study we investigated the expression of the following molecules: HLA class I (ABC) and class II (HLA-DR, HLA-DP, HLA-DQ), CD29, CD45RA, CD45RO, CD56, interleukin 1 (IL-I), IL-2, IL-4, interferon (IFN-gamma), tumor necrosis factor beta, and transforming growth factor beta1 (TGF-beta1). The bile duct epithelial cells strongly expressed HLA-ABC in all cases, CD56 in 47 of 55, IL-4 in 15 of 41, TGF-beta1 in 14 of 25, and CD29 in 4 of 25 cases. The other markers including IFN-gamma, HLA-DR, HLA-DP, and HLA-DQ were not expressed by bile duct cells. The expression of HLA class I agrees with previous observations while the absence of class II expression does not. The expression by the bile duct epithelium of CD56 confirms our own previous report. A new observation is the finding of molecules such as IL-4, TGF-beta1, and CD29 strongly expressed in the bile ducts cells. The presence of these molecules, taken together with the lack of IFN-gamma expression, contradicts previous speculations that attributed to IFN-gamma a role in the induction of major histocompatibility antigens and adhesion molecules in immune-mediated alcoholic liver disease.     

Suppression of HLA class II expression on thyrocytes by interferon-alpha 1

Inappropriate expression of HLA class II molecules by human thyroid epithelial cells (thyrocytes) is commonly associated with autoimmune thyroid disease. HLA class II expression can be modulated in thyrocytes in vitro by a variety of substances: in particular, it is readily induced by interferon-gamma (IFN-gamma). Here we show that recombinant IFN-alpha 1 (rIFN-alpha 1) does not induce HLA class II expression by thyrocytes, but rather it suppresses the induction of such expression by rIFN-gamma. Similar effects were observed with IFN-alpha derived from a lymphoblastoid cell line. The effect of rIFN-alpha 1 on thyrocytes differs from its effect on human monocytes, reported by others, in which it was found to enhance the expression of HLA class II. Thus, rIFN-alpha 1 appears to have a differential effect on HLA class II expression, depending on the cell type involved.     

Differential Expression of HLA-DR,HLA-DP,and HLA-DQ Antigenic Determinants of the Major Histocompatibility Complex in Human Endometrium

ABSTRACT: We reported on the expression of HLA-DR molecules of the major histocompatibility complex in human endometrium. We now report on the expression of two other class II molecules, the HLA-DP and HLA-DQ determinants. These molecules were localized by monoclonal antibodies in 11 proliferative and 12 secretory endometria by avidin-biotin-complex (ABC) procedure. The expression of all three molecules was invariable and consistent throughout the entire menstrual cycle in endothelial and lymphoid cells. HLA-DR molecules were expressed in endometrial epithelium, particularly in the basalis in the mid to late proliferative phases of the cycle. In contrast, through the entire menstrual cycle, the expression of HLA-DP and HLA-DQ antigenic determinants was absent, and only occasionally a focal expression of these molecules was seen in endometrial epithelium. The in vitro induction of expression of the class II molecules in human endometrial epithelial cell cultures (HEE) by IFN-gamma was dose-dependent. After treatment with low doses of IFN-gamma, these cells primarily expressed HLA-DR molecules in vitro. The differential expression of the molecules of the major histocompatibility complex in human endometrial epithelium in vivo may be due to the differential sensitivity of the epithelium in response to cytokine(s).     

Cross-linking HLA-DR molecules on Th1 cells induces anergy in association with increased level of cyclin-dependent kinase inhibitor p27(Kip1)

HLA class II molecules play pivotal roles in antigen presentation to CD4+ T cells. We investigated signaling via HLA-DR molecules expressed on CD4+ T cells. When HLA-DR or CD3 molecules on cloned CD4+ T cells were cross-linked by solid-phase mAbs, T cells proliferated, and this resulted in anergy. Whereas cross-linking of HLA-DR and CD3 resulted in secretion of the same levels of IFN-gamma and IL-8, secretion of IL-10 induced by cross-linking of HLA-DR was less than that induced by cross-linking of CD3 on CD4+ T cells. Interestingly, expression of p27(Kip1) but not p21(Cip1) increased after stimulation by either anti-HLA-DR or anti-CD3 mAb. This was indeed the case, when T cells were rendered anergic using a soluble form of antigenic peptide. In contrast, T cells stimulated by peptide-pulsed PBMC expressed little p27(Kip1). We propose that signaling via HLA-DR molecules on CD4+ T cells at least in part contributes to the induction of T cell anergy, through the upregulated expression of the p27(Kip1). The implication of our finding is that HLA-DR molecules play a role in human T cell anergy induced by a soluble form of antigenic peptide.     

Modulation of HLA-DR antigen and ICAM-1 molecule expression on airway epithelial cells by sodium nedocromil.

OBJECTIVE: To test in vitro and in vivo the hypothesis that sodium nedocromil could modulate the expression of surface molecules on airway epithelial cells. METHODS: Human bronchial epithelial cells, obtained from surgically resected bronchi, were cultured and stimulated with recombinant IFN-gamma in the presence of sodium nedocromil. The intensity of the expression of surface molecules HLA-DR and ICAM-1 molecules on bronchial epithelial cells in vitro, was quantified by specific antibody staining and flow-cytometry analysis. Furthermore, we studied the effect of the drug on airway inflammation in vivo and on allergic rhinitis patients sensitized to house dust mites. Nasal epithelial cells were collected by brushing, at baseline and 2 to 3 weeks after treatment with sodium nedocromil. The expression of HLA-DR and ICAM-1 molecules was measured by flow-cytometry, and the proportions of neutrophils and eosinophils "contaminating" the epithelial cells evaluated by light microscopy examination of nasal brushings. RESULTS: The enhanced HLA-DR and ICAM-1 expression, induced by IFN-gamma, was effectively downregulated, in a dose-dependent manner, by sodium nedocromil. At all the concentrations tested (10(-9) to 10(-4) M), the inhibitory activity of the drug was stronger on HLA-DR than on ICAM-1 expression (P<.05, all comparisons). As compared with healthy subjects, patients with allergic rhinitis had a higher expression of HLA-DR (P<.05) but not of ICAM-1 molecules (P>.05) on nasal epithelial cells, and higher proportions of nasal eosinophils (P<.05). Treatment with sodium nedocromil downregulated the expression of HLA-DR (P<.05), but not of ICAM-1 (P>.05), and induced a mild, but not statistically significant, decrease of nasal eosinophilia (P>.05). CONCLUSION: These data demonstrate that the antiinflammatory activity of sodium nedocromil may include modulation of surface molecule expression on airway epithelial cells.     

Bordetella pertussis infection of primary human monocytes alters HLA-DR expression

Bordetella pertussis is the causative agent of whooping cough, a potentially lethal respiratory disease in children. In immunocompetent individuals, B. pertussis infection elicits an effective adaptive immune response driven by activated CD4(+) T cells. However, live B. pertussis persists in the host for 3 to 4 weeks prior to clearance. Thus, B. pertussis appears to have evolved short-term mechanisms for immune system evasion. We investigated the effects of B. pertussis wild-type strain BP338 on antigen presentation in primary human monocytes. BP338 infection reduced cell surface expression of HLA-DR and CD86 but not that of major histocompatibility complex class I proteins. This change in cell surface HLA-DR expression reflected intracellular redistribution of HLA-DR. The proportion of peptide-loaded molecules was unchanged in infected cells, suggesting that intracellular retention occurred after peptide loading. Although B. pertussis infection of monocytes induced rapid and robust expression of interleukin-10 (IL-10), HLA-DR redistribution did not appear to be explained by increased IL-10 levels. BP338-infected monocytes exhibited reduced synthesis of HLA-DR dimers. Interestingly, those HLA-DR proteins that were generated appeared to be longer-lived than HLA-DR in uninfected monocytes. BP338 infection also prevented gamma interferon (IFN-gamma) induction of HLA-DR protein synthesis. Using mutant strains of B. pertussis, we found that reduction in HLA-DR surface expression was due in part to the presence of pertussis toxin whereas the inhibition of IFN-gamma induction of HLA-DR could not be linked to any of the virulence factors tested. These data demonstrate that B. pertussis utilizes several mechanisms to modulate HLA-DR expression.     

Effects of interferons alpha and gamma on cytokine production and phenotypic pattern of human bronchial epithelial cells

Human bronchial epithelial cells are involved in airway immune mechanisms through secretion of cytokines and through cell-cell contacts with immunocompetent cells. The aim of our study was to assess the ability of interferon (IFN) alpha and gamma alone and in combination to modulate human bronchial epithelial cell (HBECs) release of the inflammatory cytokines IL-8 and IL-6 and fibronectin and to induce the surface expression of HLA-DR and ICAM-1 molecules involved in immune interactions with other cells. HBECs spontaneously secreted a limited amount of IL-8, which was significantly increased by IFN gamma. IFN alpha inhibited IFN gamma stimulated IL-8 secretion in a concentration-dependent manner. Further, IFN gamma induced IL-6 and fibronectin secretion, and this was also inhibited by IFN alpha. The expression of HLA-DR antigens was significantly increased by IFN gamma and partially inhibited by co-stimulation with IFN alpha. In contrast, IFN gamma also induced ICAM-1 expression by HBECs but co-stimulation with IFN alpha had no significant effect on the expression of this surface antigen. IFN alpha modulation of HBEC functions does not seem to be restricted to IFN gamma stimulation since either stimulatory or inhibitory effects of INF alpha on IL-8 production have been found in pilot experiments using IL-1 beta, TNF alpha, and TGF beta as stimuli. In summary, IFN-gamma induces a number of responses in HBECs including increased secretion of IL-6, IL-8 and fibronectin and increased expression of HLA-DR and ICAM-1. IFN alpha can inhibit all these except expression of ICAM-1 which is unaffected. IFN alpha can also interact with other inflammatory cytokines, but whether the effects are inhibitory or augmentive depends on the cytokines.     

Effect of Pseudomonas aeruginosa exotoxin A on IFN-gamma synthesis: expression of costimulatory molecules on monocytes and activity of NK cells.

The aim of the study was (1) to evaluate the effect of Pseudomonas aeruginosa Exotoxin A (P-ExA) on the production of IFN-gamma in anti-CD3 induced human peripheral blood mononuclear cells (PBMC) and (2) to establish the effect of P-ExA on the IFN-gamma dependent cellular activities such as the expression of costimulatory molecules on monocytes and cytotoxicity of NK cells. The toxin in a high dose (100 ng/ml) inhibited IFN-gamma synthesis. Inhibitory effect of P-ExA was abolished by IL-1alpha which in a combination with P-ExA exerted a strong synergistic effect on IFN-gamma synthesis. Other monokines such as IL-1beta, IL-6, TNF-alpha neither reversed the inhibitory effect of P-ExA nor induced production of IFN-gamma. P-ExA also inhibited IFN-gamma-induced cellular events: (1) expression of costimulatory molecules on monocytes (CD80, CD86, ICAM-1, HLA-DR); (2) cytotoxic activity of NK cells. Inhibition of NK cells activity by P-ExA was not reversed by cytokines such as IL-2, IFN-alpha and TNF-alpha, which are known to enhance effector functions of NK cells. From these results we conclude that: (1) inhibition of IFN-gamma synthesis, as well as IFN-gamma-induced expression of costimulatory molecules and NK-cell effector functions may lead to suppression of specific and non-specific defense mechanisms, respectively, which are necessary for elimination of PA bacteria; (2) enhancement of IFN-gamma synthesis induced by P-ExA in a combination with IL-1alpha may cause harmful, Th1 cells dependent, inflammatory reactions of the host (septic shock, tissue damage) during infection with Pseudomonas aeruginosa.     

Exogenous type-1 cytokines modulate mercury-induced hyper-IgE in the rat

Suppression of IgE responses is a major goal for immunotherapy, especially in the field of allergy. The Th2 subset of helper T cells plays a vital role in class switching of B cells to IgE production by releasing IL-4. In susceptible rat strains, mercuric chloride (HgCl2) induces activation of Th2 cells, with enhanced expression of IL-4, polyclonal B cell activation and very high levels of circulating IgE. We have previously shown that spontaneous regulation of this response coincides with enhanced expression of Th1/type-1 cytokines, including interferon-gamma (IFN-gamma) and IL-12. We now report the effects of administration of exogenous type-1 cytokines on HgCl2-induced Th2 responses. At high doses, recombinant rat IFN-gamma markedly reduced serum IgE levels. Recombinant mouse IL-12 was less effective at suppressing the IgE response following HgCl2, although it caused marked up-regulation of IFN-gamma gene expression in the spleen. In Lewis rats, which are resistant to HgCl2-induced autoimmunity, a rise in serum IFN-gamma was observed after HgCl2, but administration of polyclonal anti-IFN-gamma antibodies did not render them susceptible to induction of a Th2 response by HgCl2. Our data show that individual type-1 cytokines are capable of suppressing the dramatic Th2 response induced by HgCl2 in the rat, even when they are not given until after starting HgCl2 administration. IFN-gamma is a pivotal cytokine in ameliorating the Th2 response and measures aimed at selective up-regulation of this cytokine may be of therapeutic value in suppression of unwanted IgE responses.     

De novo HLA class II and enhanced HLA class I molecule expression in SV40 transfected human thyroid epithelial cells.

Human thyroid follicular cells normally synthesize and express HLA Class I molecules but in glands affected by autoimmune or neoplastic diseases they express Class II molecules. We have investigated the effect of SV40 virus transformation on the expression of MHC molecules in human thyrocytes. Primary cultures of human thyroid from two different glands were transfected with the plasmid pX-8, containing the 'early' region of SV40 virus, and two continuous lines of thyrocytes were obtained. The cell lines maintained features of parental thyroid epithelial cells showing the characteristic cytokeratin filament network, microvillar protrusion and tight junctions. In addition, the SV40-transfected cells responded to graded doses of TSH with increased c-AMP production. Thyrocytes from both cell lines hyperexpressed Class I molecules and a significant proportion of them also acquired constitutive Class II expression, as determined by indirect immunofluorescence (IFL), flow cytometry and Northern blotting hybridization using a DR beta probe. These cells were found to be normally up-regulated by interferon (IFN)-gamma. Indirect IFL and flow cytometry analysis were used to detect and quantify the expression of HLA-DR, DP and DQ subregions. A co-ordinated expression (DR greater than DP much greater than DQ), reminiscent of the inappropriate HLA expression found in thyroid autoimmune disease in vivo and of the in vitro regulation in normal thyrocytes, was observed. Clones derived from these cell lines differed in their level of constitutive Class II expression and in their sensitivity to Class II induction by IFN-gamma. In conclusion, these thyroid cell lines could provide a useful tool for further investigation of HLA gene regulation in thyroid cells and for elucidating on the mechanism involved in the inappropriate HLA expression described in autoimmune and neoplastic diseases of the thyroid.     

Synergistic effects of recombinant tumour necrosis factor and interferon-gamma on rat thyroid cell growth and Ia antigen expression

In this study, we have investigated the effects of tumour necrosis factor (TNF), interferon-gamma (IFN-gamma) and interleukin-1 (IL-1) on rat thyroid cells, using the continuously growing, differentiated FRTL5 cell line. No effect on cell growth was found with any cytokine alone, but the combination of TNF and IFN-gamma was cytostatic. These cells were not killed in a 18-hr assay for cytotoxicity, but prolonged incubation (4 days) with TNF and IFN-gamma produced cell detachment and death. Normal rat thyroid cells in primary culture were also susceptible to the combination of TNF and IFN-gamma for 4 days, but not 18 hr. In addition, IFN-gamma treatment induced class II (Ia) antigens on thyroid cells, and this was enhanced by TNF. TNF alone did not cause Ia expression on thyroid cells but did increase the constitutive expression of class I antigens. These results suggest that IFN-gamma and TNF, which are likely to be released by the lymphocytes and macrophages infiltrating the gland in thyroid autoimmunity, may synergize to impair cell growth and produce aberrant Ia antigen expression in thyroiditis. This could be an important mechanism for disease perpetuation.