Regulation of cytokine production in the human thymus: epidermal growth factor and transforming growth factor alpha regulate mRNA levels of interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6 in human thymic epithelial cells at a post-transcriptional level.

Human thymic epithelial (TE) cells produce interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6, cytokines that are important for thymocyte proliferation. The mRNAs for these cytokines are short-lived and are inducible by multiple stimuli. Thus, the steady-state levels for IL-1 and IL-6 mRNAs are critical in establishing the final cytokine protein levels. In this study we have evaluated the effect of epidermal growth factor (EGF), a growth factor for TE cells, and its homologue transforming growth factor alpha (TGF-alpha), on primary cultures of normal human TE cells for the levels of IL-1 alpha, IL-1 beta, IL-6, and TGF-alpha mRNA. We showed that TE cells expressed EGF receptors (EGF-R) in vitro and in vivo, and that treatment of TE cells with EGF or TGF-alpha increased IL-1 and IL-6 biological activity and mRNA levels for IL-1 alpha, IL-1 beta, and IL-6. Neither EGF nor TGF-alpha increased transcription rates of IL-1 alpha, IL-1 beta, and IL-6 genes, but rather both EGF and TGF-alpha increased cytokine mRNA stability. By indirect immunofluorescence assay, TGF-alpha was localized in medullary TE cells and thymic Hassall's bodies while EGF-R was localized to TE cells throughout the thymus. Thus, TGF-alpha and EGF are critical regulatory molecules for production of TE cell-derived cytokines within the thymus and may function as key modulators of human T cell development in vivo.

Adhesion of T lymphoblasts to epidermal keratinocytes is regulated by interferon gamma and is mediated by intercellular adhesion molecule 1 (ICAM-1).

The cell surface expression and function of the LFA-1 ligand, intercellular adhesion molecule 1 (ICAM-1), on epidermal keratinocytes (EK) was studied. ICAM-1 expression on the surface of cultured EK was either absent or weak, but was induced by treating EK with rIFN-gamma or TNF for 4-48 h. IFN-gamma and TNF were synergistic. IFN-gamma treatment increased T lymphoblast adhesion from less than 2% to 20-40%, with a concentration dependence similar to that seen for ICAM-1 induction. All of the adhesion to EK was inhibited by LFA-1 and ICAM-1 mAbs, but not by HLA-DR, CD2, or LFA-3 mAbs. There was no difference in the level of T lymphoblast adhesion to IFN-gamma-treated allogeneic or autologous EK. ICAM-1 purified from the HeLa epithelioid cell line and reconstituted into planar membranes also supported efficient adhesion of T lymphoblasts that was blocked by LFA-1 mAb bound to the T lymphoblasts or ICAM-1 mAb bound to the planar membranes. T lymphoblasts adherent to EK or ICAM-1 planar membranes were isolated by panning, and surface markers were analyzed by immunofluorescence flow cytometry. The adherent T cells were a phenotypically skewed subpopulation. They were enriched for CD8+ cells and expressed 1.5-2.5-fold higher LFA-1 and CD2 compared with the unseparated population.

Anti-cell surface pemphigus autoantibody stimulates plasminogen activator activity of human epidermal cells. A mechanism for the loss of epidermal cohesion and blister formation.

Binding of anti-cell surface pemphigus autoantibodies to cultured human epidermal cells stimulates synthesis and secretion of plasminogen activator (PA). Increases in PA activity were detected within 6 h of the addition of IgG and stimulation was dependent upon IgG concentration. Stimulation of PA activity was inhibited by cycloheximide, which indicates that synthesis of protein was necessary. Pharmacological doses of dexamethasone also prevented IgG-induced stimulation of PA. Electrophoretic profiles of PA secreted by cultured human epidermal cells in the presence or absence of pemphigus IgG were similar. The majority of the PA activity comigrated with the higher-molecular-weight species of human urokinase (approximately 55,000). Explants of normal human skin incubated with pemphigus vulgaris IgG displayed loss of epidermal cohesion similar to that observed in patient biopsies. The histologic changes were potentiated by the inclusion of human plasminogen. Loss of epidermal cohesion in normal skin explants incubated with pemphigus foliaceous IgG was dependent upon the addition of plasminogen and was inhibited by aprotinin or lima bean trypsin inhibitor, which indicated that plasmin is the active enzyme in producing acantholysis. These data support the hypothesis that stimulation of PA by the anti-cell surface autoantibodies of pemphigus results in a localized increase in plasmin, which through proteolysis produces the loss of epidermal cohesion characteristic of pemphigus.

Interactions between epithelial cells and T lymphocytes: role of adhesion molecules.

Cell-cell interaction is critical for normal T cell development and function. A number of adhesion molecules important in T cell interactions with other cell types have been defined. This paper reviews the role of two adhesion pathways, CD2/LFA-3 and LFA-1/ICAM-1, in T cell interactions with epithelial cells of the thymus and skin. While thymic epithelium-T cell interactions are mediated by both the LFA-1/ICAM-1 pathway and the CD2/LFA-3 pathway, epidermal-T cell interactions are mediated primarily by the LFA-1/ICAM-1 pathway. Although ICAM-1 is not expressed in vivo on epidermal keratinocytes in normal skin, ICAM-1 is expressed by epidermal keratinocytes at the site of T cell infiltration in inflammatory dermatitis. ICAM-1 is expressed in vivo on thymic epithelium. These antigen independent adhesion molecules play an important role in the cell-cell interactions associated with T cell differentiation and function.

Autodegradation of 125I-labeled human epidermal cell surface proteins.

Triton X-100 extracts of cultured human epidermal cells exhibited proteolytic activity as measured by the hydrolysis of [3H]-casein at neutral pH. The majority of endogenous proteolytic activity was inhibited by parahydroxy mercuribenzoate and by mersalyl acid, indicating the enzyme(s) was a thiol class proteinase(s). Crude Triton X-100 extracts were prepared from epidermal cells following labeling of proteins with 125I. Autodegradation of labeled proteins at 37 degrees C was detected as early as 1 hr and reached a plateau level by 4 hr. Degradation was inhibited by thiol class proteinase inhibitors. Among the detergent-solubilized radiolabeled proteins a polypeptide chain of Mr 155,000 was particularly sensitive to degradation by endogenous thiol proteinase(s).

Epidermal keratinocytes express the adhesion molecule intercellular adhesion molecule-1 in inflammatory dermatoses.

Using indirect immunofluorescence assays on frozen tissue sections of skin from healthy subjects and subjects with inflammatory skin diseases, we found that intercellular adhesion molecule-1 (ICAM-1) was expressed in a cell surface pattern on epidermal keratinocytes at the site of lymphoid infiltration in cutaneous dermatoses. ICAM-1 was not expressed on epidermal keratinocytes in noninflamed skin. Its expression was not related solely to epidermal hyperproliferation, as hyperproliferative, tape-stripped epidermis did not express ICAM-1. We have reported previously that ICAM-1 expression on epidermal keratinocytes was upregulated by treatment with interferon gamma and that activated T lymphocytes bound to cultured epidermal keratinocytes in vitro by lymphocyte function associated-1 (LFA-1) molecules on T cells and ICAM-1 on epidermal keratinocytes. Taken together, these data suggest that upregulation of expression of ICAM-1 is an important feature of cutaneous inflammation.

Proteinase activation: a mechanism for cellular dyshesion in pemphigus.

An in vitro model system using cultured newborn epidermal cells was employed to investigate the binding of pemphigus autoantibody and subsequent loss of adhesion between epidermal cells. Pemphigus antibodies bound to both mouse and human cultured epidermal cells. Incubation of cultured newborn mouse epidermal cells with pemphigus antibody followed by gentle agitation induced loss of adhesion between the epidermal cells and the plastic culture dish. Release of viable epidermal cells from the dish was inhibited by the proteinase inhibitors, soybean trypsin inhibitor and alpha 2-macroglobulin. These observations suggest that pemphigus antibody induces viable epidermal cells to activate cellular proteinases which then degrade the glycocalyx and cause cellular dyshesion and acantholysis.

The role of adhesion molecules in epithelial-T-cell interactions in thymus and skin.

Interaction of T lymphocytes with other cell types is important for normal T-cell development and function. Recently, a number of adhesion molecules important in T-cell interactions with other cell types have been defined. In this paper we review the role of two adhesion pathways, CD2/LFA-3 and LFA-1/ICAM-1, in T-cell interactions with epithelial cells of the thymus and skin. While thymic epithelium-T-cell interactions were mediated by both the LFA-1/ICAM-1 pathway and the CD2/LFA-3 pathway, epidermal-T-cell interactions were mediated primarily by the LFA-1/ICAM-1 pathway. Although ICAM-1 was not expressed in vivo on epidermal keratinocytes in normal skin, ICAM-1 was expressed by epidermal keratinocytes at the site of T-cell infiltration in inflammatory dermatitis. ICAM-1 was expressed in vivo on thymic epithelium. Both LFA-3 and ICAM-1 were expressed on epithelial cells of thymus and skin early on in fetal ontogeny. These antigen-independent adhesion molecules play an important role in the cell-cell interactions associated with T-cell differentiation and function.

Expression of antigens by cultured epithelial cells: comparison of epidermis and thymic epithelium.

We have established long term in vitro cultures of human thymic epithelium and human epidermis free of contaminating fibroblasts. The cultured cells were examined using a panel of monoclonal antibodies which were raised against human thymic stroma and recognize tissue specific differentiation antigens of human epidermis and thymic epithelium. A subset of cultured epidermal cells (50%) and thymic epithelial cells (18%) expressed the TE-4 antigen characteristic of basal keratinocytes in skin and endocrine epithelium found in the subcapsular cortex and medulla of the thymus. Subpopulations of the cultured cells expressed the antigens detected by antibodies TE-8 and TE-15. In tissue sections antibodies TE-8 and TE-15 bound to the stratum granulosum and stratum corneum of skin and to the Hassall's bodies of thymus, and therefore recognize antigens characteristic of late stages of keratinized epithelial differentiation. In addition, a subset of thymic epithelial cells expressed the antigen detected by antibody TE-3 which is expressed by nonendocrine thymic epithelium found in the thymic cortex. Thus, in vitro cultures of both epidermal and thymic epithelial cells expressed the entire array of differentiation antigens detected by our panel of monoclonal antibodies. This approach can be used to evaluate the role of components of the thymic microenvironment at various stages of differentiation on developing T lymphocytes. In addition, the cultured epidermal cells can be used to evaluate epidermis as a site of extrathymic T cell maturation.

Plasminogen activator in cultured human epidermal cells.

Primary cultures of human epidermal cells produce plasminogen activator (PA) as demonstrated by the ability of conditioned medium or cell lysates to hydrolyze fibrin in the presence of plasminogen, and to cleave [125I]plasminogen to characteristic fragments. The major molecular species of PA in human epidermal cells was inhibited by diisopropylfluorophosphate and comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the high molecular weight band of human urokinase (Mr 55,000). Production of PA by human epidermal cells was inhibited by cycloheximide, stimulated by colchicine, and not affected by cytochalasin B or the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate. Both cholera toxin and epidermal growth factor stimulated PA activity in human epidermal cells, and PA activity was maximal at concentrations that best support in vitro growth of human epidermal cells. Examination of individual cells indicated that at least 15% of cells within a culture produced detectable amounts of PA.

Removal of fibroblasts from human epithelial cell cultures with use of a complement fixing monoclonal antibody reactive with human fibroblasts and monocytes/macrophages.

A complement fixing IgM monoclonal antibody (1B10) that reacts with surface membrane molecules of human fibroblasts, tissue macrophages, and peripheral monocytes was produced. In Western blot analysis of detergent extracts of cultured human foreskin fibroblasts, antibody 1B10 detected protein bands of Mr 43,000 and 72-80,000. We used the 1B10 antibody with complement to eliminate most 1B10 positive nonepithelial cells from thymic epithelial (TE) cell cultures, thereby allowing us to grow highly enriched populations of human TE cells.

Epithelial-thymocyte interactions in human thymus.

Our data demonstrate that the epithelial component of the human thymic microenvironment is not an inert cell type, but rather is capable of being directly involved in the promotion of both early and late stages of T-cell maturation. Data from our laboratory [54,69], together with the work of Plunkett et al. [61] and Shaw et al. [70] suggest that an endogenous ligand for the CD2 molecule in humans is the LFA-3 molecule. Using an SV40 transformed human thymic epithelial cell line of subcapsular cortical origin, Mizutani et al. have confirmed that thymic epithelial cells bind thymocytes via a CD2/LFA-3 interaction [78]. The data reviewed in this paper suggest that within the thymus one endogenous ligand for the alternative pathway of thymocyte activation via the CD2 molecule is the LFA-3 molecule on TE cells. Following thymocyte binding to TE cells, immature thymocytes are directly activated to proliferate, and their response to both IL1 and IL2 is augmented. Also, following TE-thymocyte binding, TE-IL1 secretion is augmented and TE cell MHC class II antigen expression is induced. Moreover, while undergoing activation, thymocytes appear to be able to modulate their microenvironment milieu of MHC antigens and IL1. Further analysis of the sequelae of TE-thymocyte interactions using phenotypic characterization of thymocytes with anti-T-cell MoAbs, coupled with molecular analysis of thymocyte T-cell receptor genes, should allow for the determination of the precise sequential stages that immature T cells undergo enroute to functional maturity. Understanding these steps in T-cell maturation will be critical to our understanding of the events that transpire in the genesis of autoimmune, lymphoproliferative, and immunodeficiency diseases.

In vitro growth and phenotypic characterization of mesodermal-derived and epithelial components of normal and abnormal human thymus.

Long-term in vitro cultures of human thymic tissue were established and phenotypically characterized using monoclonal reagents that define distinct components of the human thymic microenvironment. The epithelial component of the thymus, defined by monoclonal antibodies TE-3, TE-4, BBTECS, and AE1 (anti-keratin) was isolated from the mesodermal component, defined by antibody TE-7, and maintained separately in long-term culture. The epithelial cells were subcultured repeatedly and recovered from storage in liquid nitrogen. The in vitro phenotype of the cultured cells was compared to that of cultured human epidermal cells. A subpopulation of cultured thymic epithelial cells along with a subpopulation of cultured epidermal cells expressed antigens (TE-8, TE-15) characteristic of late stages of keratinized epithelial cell differentiation. Thus, we have established a system whereby components of the human thymic microenvironment can be cultivated in vitro while maintaining the capacity to differentiate. This approach can be used to evaluate the role of components of the thymic microenvironment at various stages of differentiation on developing T lymphocytes. In addition, keratin-containing thymic epithelial cells were successfully cultured from thymuses obtained from patients with myasthenia gravis and thymoma. Cultivation of abnormal thymic epithelium will provide insight into aberrant T lymphocyte-thymic epithelial interaction.

Human thymic epithelial cells: adhesion molecules and cytokine production.

The ability to culture human thymic epithelial cells has greatly facilitated studies of direct cell-cell interaction between thymic epithelial cells and T lymphocytes in vitro, as well as cytokine production and regulation of cytokine production. In vitro, human thymic epithelial cells bind to T lymphocytes via two adhesion pathways: CD2-lymphocyte function-associated antigen-3 and lymphocyte function-associated antigen-1-intercellular adhesion molecule-1. Cultured human thymic epithelial cells produce interleukins-1 alpha, -1 beta, -3, -6 and -8, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, leukemia inhibitory factor and transforming growth factor-alpha. Production of thymic epithelial cell-derived cytokines is regulated by both adhesion molecules (lymphocyte function-associated antigen-3) and soluble factors via both autocrine (interleukin-1 alpha, transforming growth factor-alpha) and paracrine (interleukin-4, interferon-gamma) pathways. Transforming growth factor-alpha and epidermal growth factor regulate various cytokine mRNA at a post-transcriptional level by increasing cytokine mRNA stability.

Characterization of an antigen shared by human thymic epithelium and human T cell leukemia virus p19 Gag protein.

The molecular basis for cross-reactive antibody binding to human T cell leukemia virus type I (HTLV-I) p19 core protein and human thymic epithelium has been defined with two monoclonal antibodies (mAbs), 12/1-2 and 13B12, raised to HTLV-I p19. The mAb 12/1-2 has previously been shown to react with HTLV-I p19, HTLV-II p22, and antigens of normal human thymic epithelium, placenta, and foreskin, whereas mAb 13B12 binds only to the carboxyl terminus of HTLV-I p19. In the present study, mAb 12/1-2 bound to a subset of Triton X-100-insoluble intermediate filaments in human thymic epithelium also recognized by antikeratin antibodies AE1 and AE3. The mAb 12/1-2 also reacted in Western blot assays with proteins of 54, 46, and 40 kDa present in extracts of human thymic epithelium and with hexameric peptides containing overlapping sequences of HTLV-I p19 with the amino acids IPP (amino acids 117-119). In contrast, the HTLV-I-specific mAb 13B12 did not bind to human thymic epithelium and reacted with a single hexameric peptide containing the carboxy-terminal HTLV-I p19 sequence IPPPYV (amino acids 117-122). Binding of mAb 12/1-2 to thymic epithelium could be inhibited by adsorption with peptide SP-79 containing a C-terminal sequence (amino acids 112-125) of p19. The crossreactive IPP site is within a region of p19 that has been previously shown to be highly immunogenic in HTLV-I-infected individuals and that is also encoded by genes or mRNA of human cytokeratin 17, keratin 4, epidermal cytokeratin 2, and 50-kDa type I epidermal keratin. Thus, our studies define the sequence of a cross-reactive antigen on HTLV-I p19 that is also associated with keratin intermediate filaments from human thymic epithelium and other normal human tissues and that could serve as a focus of an autoimmune response during HTLV-I infection.

CD44 antibody against In(Lu)-related p80, lymphocyte-homing receptor molecule inhibits the binding of human erythrocytes to T cells.

The cluster of differentiation 44 (CD44) Ag system, originally described in brain and mature T cells, has been subsequently shown to be identical with the human lymphocyte homing-receptor defined by the Hermes-1 antibody and to be involved in T cell/endothelial cell interactions in synovium, mucosa, and lymph node. CD44 is also present on human E. On E, CD44 has been shown to be regulated by the In(Lu) dominant inhibitor gene and to express the Ina and Inb blood group Ag. Because human E have been shown to interact with human T cells via CD2 on T cells and LFA-3 on human E, we have studied the ability of human E and T lymphocyte CD44 Ag to participate in CD2/LFA-3 interactions between human E and T cells. In this study, we demonstrate that a mAb (A3D8) against the CD44, In(Lu)-related p80, lymphocyte homing-receptor molecule inhibited the binding of human E to human T cells. Whereas whole CD44 antibody molecules inhibited human E binding to T cells, saturating amounts of CD44 Fab fragments did not inhibit human E to T cell binding. Our data demonstrated that anti-CD44 antibody A3D8 acted at the level of the E to inhibit CD2/LFA-3 interactions between human E and T cells.

Molecular size of secreted and cell-associated plasminogen activators from cultured epidermal cells.

The molecular sizes of secreted and cell-associated plasminogen activators from four cultured cell types were determined using an SDS-PAGE technique in which plasminogen and casein were included during polymerization of the polyacrylamide gel. The major bands of plasminogen activators secreted by human neonatal epidermal cells, human adult epidermal cells and transformed human squamous cells migrated the same distance as the high molecular weight band of authentic urokinase, indicating that the apparent molecular weight of these plasminogen activators was approximately 55,000 daltons. Plasminogen activator extracted from normal adult human epidermis also migrated with this major band of plasminogen activator, and a minor higher molecular weight band was also detected. In contrast, plasminogen activators secreted by transformed mouse squamous cells migrated between the high molecular weight band (approximately 55K) and the low molecular weight band of urokinase (approximately 32K), indicating that plasminogen activators of mouse epidermal cells differ from those of human epidermal cells. The mobility of the major bands of plasminogen activators detected in cell lysates of the four cell types was identical to that of secreted plasminogen activators.

Interaction of cytotoxic T lymphocytes with target cells. I. Specific inhibition by detergent-solubilized, partially purified mouse histocompatibility antigens.

H-2 antigens from EL4 (H-2b) and RLmale 1 (H-2d) leukemias were solubilized with deoxycholate and partially purified based on their adherence to a Lens culinaris hemagglutinin column. In double reciprocal experiments we have shown that these preparations specifically inhibit conjugate formation between target cells and alloimmune peritoneal lymphocytes, a preparation rich in cytotoxic T lymphocytes.