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.     

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.     

Interferon-gamma mediates antigen trafficking to MHC class II-positive late endosomes of enterocytes

MHC class II-positive late endosomes of enterocytes are thought to be involved in antigen presentation to CD4(+) T cells. In contrast to enterocytes of BALB/c mice, severe combined immunodeficiency (SCID) enterocytes lack MHC class II expression and fail to transport internalized ovalbumin (OVA) into late endosomes. IFN-gamma is known to induce MHC class II in enterocytes and antigen targeting to late endosomes in macrophages. In this study, we investigated the influence of IFN-gamma and MHC class II on the processes of antigen traffic in enterocytes. Subcellular targeting of OVA and MHC class II expression within enterocytes were examined in SCID, IFN-gamma-treated SCID, BALB/c and C57BL/6 MHC class II knockout (KO) mice after a single feed with OVA. Sorting of OVA into late endosomes was found in enterocytes from BALB/c, C57BL/6 KO and IFN-gamma-stimulated SCID mice, but not from untreated SCID mice. MHC class II expression was restricted to enterocytes of IFN-gamma-treated SCID and BALB/c mice, present at basolateral membranes and within endosomal compartments. These enterocytes further revealed colocalization of class II antigens and OVA in endosomes. We suggest that antigen trafficking into late endosomes of enterocytes is mediated by IFN-gamma and occurs in the absence of MHC class II.     

MHC antigen expression in human oral squamous carcinoma cell lines.

This study quantified the constitutive and interferon-gamma (IFN-gamma) stimulated expression of MHC class I (HLA-ABC and beta 2 microglobulin) and class II antigens (HLA-DR, -DP, -DQ) on normal and malignant oral keratinocytes using radioimmunoassay and immunocytochemical techniques. Normal keratinocytes and three of four malignant cell lines (H103, H157, H314) expressed MHC class I antigens constitutively; IFN-gamma increased MHC class I expression with significant changes in normals, H157 and H314. Normal keratinocytes expressed significantly more constitutive MHC class I antigens than H103 and H157 and significantly more IFN-gamma stimulated MHC class I antigens than H103, H157 and H314. MHC class II antigens predominantly were not expressed constitutively on normals, H103 and H157 but, in H314, HLA-DR, -DP and -DQ antigens were demonstrated on 35, 11 and 5 per cent of cells, respectively, and resulted in a non-coordinated pattern of expression (HLA-DR greater than -DP = -DQ). IFN-gamma induced HLA-DR on normals, H103 and H157, whilst HLA-DP and -DQ remained undetectable. In H314, IFN-gamma enhanced HLA-DR, -DP and -DQ (significant increase of HLA-DQ) but the interrelationship between these antigens was maintained (HLA-DR greater than -DP = -DQ). Normal keratinocytes expressed significantly more IFN-gamma stimulated HLA-DR than H103 and H157 but significantly less HLA-DR than H314 under similar experimental conditions. One oral malignant cell line (H191) did not express MHC class I and MHC class II antigens either constitutively or in response to IFN-gamma. The results demonstrate aberrant patterns of MHC expression (absence, enhanced, diminished) in the different malignant oral keratinocyte cell lines.     

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.     

Expression of MHC class I and II molecules by cadaver retinal pigment epithelium cells: optimization of post-mortem HLA typing

The objective of this study was to investigate the expression of MHC antigens by retinal pigment epithelium cells (RPE) after stimulation with interferon-gamma (IFN-gamma) and to improve the currently practised technique of cadaver HLA typing. A concentration of 100 U/ml IFN-gamma induced expression of class I molecules up to greater than 90% 3 days after stimulation, whereas 50 U/ml were required for the expression of HLA-DR to greater than 90%. A concentration of 750 U/ml induced 35-45% expression of HLA-DP and less than 25% HLA-DQ after 3 days. Cells were serologically typed using the standard lymphocytotoxicity assay 3 days after stimulation with 250 U/ml IFN-gamma. Typing of class I specificities was complemented by one-dimensional isoelectric focusing (1D-IEF). We observed high concordance between the results of the RPE typing and the lymphocytotoxicity test on the same donors. Our results show complete typing of class I and II antigens post-mortem, which, in particular, enables graft matching and improvement of graft survival in recipients of organs removed many hours after death such as the cornea.     

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.     

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.     

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.     

Interferon-gamma in vivo. Induction and loss of class II MHC antigens and immature myelomonocytic cells in rat organs

The effects of recombinant rat interferon-gamma on class II major histocompatibility complex antigen expression in vivo were studied by immunohistology in LEW rats after continuous intravenous infusion for three days. Interferon-gamma administration led to a systemic induction of class II molecules in previously negative parenchymal and stromal cells. The induction patterns observed were highly reproducible, but not closely dose dependent within a 25-fold dose difference tested. However, the effect of interferon infusion differed profoundly in individual cell types, and appeared to be related to the differentiation stage of each cell population. Thus, epithelial cells like duct epithelia, urothelium or basal ear skin keratinocytes as well as endothelia in big vessels were strongly and easily induced for class II antigen expression. Parenchymal cells like cardiomyocytes and hepatocytes showed intermediate reactivity, while capillary endothelia, neurons in the brain, straight proximal kidney tubules or endocrine pancreatic islet cells did not express class II antigens. The induced expression was rapidly lost from most cells within one or two days after interferon withdrawal; the only exception occurred in keratinocytes. Long-term alterations were, however, still found 14 days after infusion. Interstitial class II-positive dendritic-shaped cells were increased in the organs and hepatic Kupffer cells carried class II antigens. On conventional histology all organs appeared perfectly normal at this date. After three days of interferon, cells of an immature myelomonocytic phenotype occluded medium-sized and small veins in all organs and occurred in granuloma-like lesions in the liver. Although these cells quickly disappeared after interferon withdrawal they might have been at least partially responsible for single deaths on day three. Our study provides a basis for testing the immunological in vivo function of parenchymal class II antigen expression and its differentiation-specific regulation.     

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.     

Histological changes of bile duct in experimental graft-versus-disease across minor histocompatibility barriers. III. Immunoelectron microscopic observations

Graft-versus-host disease (GVHD) was produced by injecting lymphoid cells from B10.D2 mice into BALB/C mice. Both are H-2-identical but differ only at multiple minor H loci. The expression and localization of MHC class II antigens on the bile duct epithelium was examined using an immunoelectron microscopical method. All GVHD mice developed bile duct lesions of chronic nonsuppurative cholangitis and expressed MHC class II antigens on their bile duct epithelium, while none of the control mice, which injected with the same number of syngeneic lymphocytes, developed bile duct lesions or expressed the antigens. The antigenic expression was characteristically localized on the basolateral surface of the bile duct epithelium but not on the apical surface. Furthermore, the expression varied markedly in its intensity and distribution within the same liver and even within a single bile duct. The infiltration of Lyt-1-positive helper/inducer lymphocytes in the duct epithelial layer apparent by electron microscopy was predominant to a much degree than that of Lyt-2-positive cytotoxic/suppressor lymphocytes and non-lymphocytic cells. The immunological mechanisms involving helper/inducer T cells in association with MHC class II antigens on bile duct epithelium may be important in the induction and progression of the bile duct lesions apparent in the present GVHD model.     

Class II MHC molecules and monocytes/macrophages in the respiratory system of conventional, germ-free and interferon-gamma-treated rats.

The localization of I-A-like class II major histocompatibility complex (MHC) molecules and cells of the monocyte/macrophage lineage was studied immunohistologically in the trachea and lungs of conventional, specified pathogen-free (SPF) and germ-free rats. In the three groups of animals I-A-like class II MHC molecules occurred in epithelia of the bronchus-associated lymphatic tissue (BALT), in B lymphocytes, in dendritic-shaped and elongated interstitial cells and in type II pneumocytes. Conventional and SPF rats were distinguished from germ-free animals only by the larger number of class II MHC-positive respiratory epithelial cells in the lower trachea and main bronchi. The distribution of monocytes/macrophages (ED1-positive cells) did not differ between the groups. After systemic treatment of SPF rats with interferon-gamma class II MHC molecules were newly induced in all respiratory epithelia and in the endothelium of large vessels. In addition, interferon-gamma sometimes led to pulmonary infiltration and caused class II-positive activated monocytes to accumulate in medium-sized pulmonary vessels and in alveolar capillaries. It is concluded that the microbial status does not qualitatively alter the distribution of class II MHC molecules and monocytes/macrophages in rat respiratory organs. Interferon-gamma can, however, provoke profound changes.     

A CIITA-independent pathway that promotes expression of endogenous rather than exogenous peptides in immune-privileged sites

A CIITA-independent pathway of MHC class II expression has been found in the eye and the brain, both immune-privileged sites. Although corneal endothelial cells were unable to express MHC class II in response to IFN-gamma alone, these cells readily expressed MHC class II molecules via a CIITA-independent pathway when triggered by simultaneous exposure to IFN-gamma and TNF-alpha. CIITA-independent expression of MHCclass II molecules enabled corneal endothelial cells to present cytosolic, but not endosomal, ovalbumin (OVA) to OVA-primed T cells. To determine whether CIITA-independent expression of MHC class II is relevant in vivo, minor H-only-incompatible corneal allografts prepared from CIITA knockout (KO) mice, MHC class II KO mice or wild-type donors were placed in eyes of normal mice. Cornea allografts from wild-type and CIITA KO mice suffered similar rejection fates, whereas far fewer class II-deficient corneas were rejected. In addition, MHC class II-bearing macrophages were observed in cuprizone-induced inflammatory and demyelinating brain lesions of CIITA KO mice. We conclude that class II expression via the CIITA-independent pathway enhances the vulnerability to rejection of corneal grafts expressing minor antigens. The potential relevance of CIITA-independent MHC class II expression at immune-privileged sites is discussed in relation to tolerance to strong autoantigens.     

Expression of HLA-D-locus (DP, DQ, DR)-coded antigens, beta 2-microglobulin, and the interleukin 2 receptor in Sj?gren's syndrome

The expression of beta 2-microglobulin (beta 2m), class II major histocompatibility complex (MHC) antigens (HLA-DP, -DQ, -DR) and the interleukin 2 receptor (IL-2R) on resident and infiltrating cells in labial salivary glands of patients with Sj?gren's syndrome (SS) was studied using an immunoperoxidase technique based on staining with monoclonal antibodies. The progression of the inflammatory process was accompanied by increasing numbers of immunocompetent cells as well as glandular epithelial cells expressing beta 2m and class II MHC antigens. Up to 60% of the infiltrating lymphocytes were DQ- and DR-positive while fewer cells stained for DP antigen. Glandular epithelium (acinar and ductal cells) stained for products of all three HLA-D subregions but with varying degrees of expression following the pattern DR greater than DP greater than DQ related to the severity of inflammation. A minor portion of the infiltrating lymphocytes expressed IL-2R. These findings emphasize the potential importance of epithelial expression of class II antigens in the local activation of T lymphocytes in salivary gland lesions of SS patients. They also indicate that differences may exist in this respect between T-cell reactions restricted by different class II MHC-encoded gene products.     

Expression of MHC class II antigens on bile duct epithelium in experimental graft versus host disease

Recently we observed the expression of MHC class II antigens on bile duct epithelium in human liver transplantation rejection. In this report we present evidence for the expression of MHC class II antigens on bile duct epithelium in experimental GVHD. These findings support the idea, that the target tissue elements in liver transplantation rejection and GVHD are the bile ducts, on the other hand this is a new experimental model for the analysis of MHC class II antigen expression on epithelial tissue.     

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.     

HISTOLOGICAL CHANGES OF BILE DUCT IN EXPERIMENTAL GRAFT-VERSUS-HOST DISSEASE ACROSS MINOR HISTOCOMPATIBILITY BARRIERS III. IMMUNOELECTRON MICROSCOPIC OBSERVATIONS

Graft-vesus-host disease (GVHD) was produced by injecting lymphoid cells from B10·D2 mice into BALB/C mice. Both are H-2-identical but differ only at multiple minor H loci. The expressin and localizaton of MHC class II antigens on the bile duct epithelium was examined using an immunoelectron microscopical method. All GVHD mice developed bile duct lesions of chronic nonsuppurative cholangitis and expressed MHC class II antigens on their bile duct epithelium, while none of the control mice, which injected with the same number of syngeneic lymphocytes, developed bile duct lesions or expressed the antigens. The antigenic expression was characteristically localized on the basolateral surface of the bile duct epithelium but not on the apical surface. Furthermore, the expression varied markedly in its intensity and distribution within the same liver and even within a single bile duct. The infiltration of Lyt-1-positive helper/inducer lymphocytes in the duct epithelial layer apparent by electron microscopy was predominant to a much greater degree than that of Lyt-2-positive cytotoxic/suppressor lymphocytes and non-lymphocytic cells. The immunological mechanisms involving helper/inducer T cells in association with MHC class II antigens on bile duct epithelium may be important in the induction and progression of the bile duct lesions apparent in the present GVHD model.     

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.