Properties of murine and human epidermal cell-derived thymocyte-activating factor

Keratinocytes, the predominant cell within the epidermis, perform some macrophage-like functions, such as endocytosis and phagocytosis. We therefore investigated whether keratinocytes may also exert some nonspecific immunoregulatory functions through the secretion of mediators. Tissue cultures of freshly isolated murine and human keratinocytes as well as keratinocyte cell lines secrete a cytokine, epidermal cell-derived thymocyte-activating factor (ETAF), which augments in vitro lymphoproliferative responses. Keratinocyte cell line cells produce increased levels of ETAF activity after exposure to a variety of cell-damaging agents such as silica, endotoxin, phorbol myristate acetate, hydroxyurea, and mechanical disruption. Biochemical studies showed that murine and human ETAF, like interleukin 1 (IL 1), had a molecular weight between 12,000 and 20,000, interacted with hydrophobic phenyl-Sepharose, and was eluted from anion but not cation exchangers. Like IL 1, murine ETAF had a single isoelectric point whereas human ETAF eluted as three peaks of activity (pI 7.2, 5.8, and 5.0). Partially purified ETAF of either species also had the same biological properties of IL 1. That is, ETAF enhanced IL 2 production by T cells in culture, was chemotactic for polymorphonuclear leukocytes, and was directly mitogenic for fibroblasts. When injected into C3H/HeJ mice ETAF induced hepatocyte production of serum amyloid A, an acute phase protein. Furthermore, ETAF, like IL 1, may act as an endogenous pyrogen and induce fever when injected into rabbits. These findings indicate that production of IL 1-like molecules is not confined to cells of the immune system and that ETAF production by keratinocytes may have important implications in would healing, as well in the pathogenesis of inflammatory and neoplastic diseases.     

Differential effects of epidermal growth factor, transforming growth factor-alpha, and vaccinia virus growth factor in the positive regulation of IFN-gamma production

We have recently shown that epidermal growth factor (EGF) is capable of positive regulation of IFN-gamma production, thus establishing a functional relationship between nonhemopoietic growth factors and the immune system. In order to study this relationship further, EGF and the EGF-related growth factors transforming growth factor-alpha (TGF-alpha) and vaccinia virus growth factor (VGF), which stimulate cellular proliferation via binding to the EGF receptor, were studied for their functional and physicochemical effects on IFN-gamma production. In contrast to the positive signal of purified murine EGF and recombinant human EGF (both at 1 nM), neither synthetic TGF alpha nor recombinant VGF were capable of restoring competence for IFN-gamma production by Th cell-depleted spleen cell cultures. TGF-alpha and VGF, in molar excess, also failed to block the helper signal of EGF for IFN-gamma production. Thus TGF-alpha and VGF failed to functionally compete for the EGF receptor in the murine spleen cell system. Both TGF-alpha and VGF stimulated murine 3T3 cell proliferation at concentrations similar to those of EGF, and thus their failure to provide help for IFN-gamma production was not due to a general lack of biologic activity. Binding studies with 125I-EGF suggest that the EGF receptor on murine lymphocytes is not constitutively expressed, but inducible by the T cell mitogen staphylococcal enterotoxin A. TGF-alpha did not compete with 125I-EGF for the induced receptor. The data suggest that lymphocytes express a novel inducible EGF receptor that differs from that expressed on cells such as 3T3 fibroblasts.     

A new role for epidermal cell-derived thymocyte activating factor/IL-1 as an antagonist for distinct epidermal cell function

It is known that many immunologic responses to IL-1 are antagonized by the neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH). This led us to investigate the possible reciprocal effects of IL-1 and the functionally related epidermal cytokines, epidermal cell-derived thymocyte activating factor (ETAF) and IL-6, on the melanogenic effect of alpha-MSH on murine Cloudman melanoma cells. When these cells were treated with ETAF in combination with alpha-MSH or its potent analog [Nle4,D-Phe7]-alpha-MSH, the melanotropin induced increase in tyrosinase activity, and thus melanin synthesis, was abrogated. This inhibitory effect of ETAF was not mediated by competitive binding to the melanotropin receptor, because ETAF also blocked the melanogenic response of melanoma cells to isobutyl methylxanthine (IBMX) and to PGE1 and PGE2. ETAF had no effect on cellular proliferation. Inhibition of the stimulated tyrosinase activity by ETAF was not due to diminished cAMP synthesis or increased cAMP degradation. Cells treated concomitantly with ETAF and alpha-MSH, IBMX, or PGE1 had the same cAMP levels as cells treated with alpha-MSH, IBMX, or PGE1 alone. In contrast to ETAF, human rIL-1 alpha or IL-1 beta alone or in combination did not have an inhibitory effect on melanogenesis. IL-6 significantly inhibited the basal level of tyrosinase and partially abrogated the alpha-MSH-induced tyrosinase activity. IL-6 also stimulated cellular proliferation when added alone or in combination with alpha-MSH. Granulocyte-macrophage colony stimulating factor (GM-CSF) did not alter either the tyrosinase activity or cellular replication at the concentrations tested. IL-1 alpha, GM-CSF, and IL-6 or IL-1 alpha and GM-CSF added together did not significantly affect the MSH-induced tyrosinase activity. These results ascribe a new potential function for ETAF and IL-6 as modulators of the melanogenic response of pigment cells.     

Epidermal cells synthesize a cytokine with interleukin 3-like properties

Interleukin 3 (IL 3) is produced by T lymphocytes and T cell lines (EL 4), as well as by a monomyelocytic cell line (WEHI 3), and it activates lymphocytes as well as mast cells. Recently we have demonstrated that epidermal cells (EC) perform monocyte/macrophage-like functions through the release of an interleukin 1-like immunomodulating mediator (EC-derived thymocyte activating factor; ETAF. Because mast cells predominantly are located in the skin, in the present study we investigated whether EC in addition to ETAF may produce IL 3. Normal as well as transformed keratinocytes were able to secrete an IL 3-like mediator (EC IL 3) that induces the proliferation of IL 3-dependent cell lines. Furthermore, both EC IL 3 and WEHI IL 3 have a similar m.w. of 30,000. In addition, an antibody against IL 3 also blocked EC IL 3 activity, suggesting that these molecules appear to be very similar. EC IL 3 production was greatly enhanced by the addition of concanavalin A, phorbol myristate acetate, lipopolysaccharide, and silica. Factor production was completely blocked by inhibiting protein synthesis. These findings demonstrate that keratinocytes synthesize an additional cytokine with the biologic and biochemical properties of IL 3, but distinct from ETAF. Thus, through the production of EC IL 3, EC may participate in the activation of mast cells and thereby mediate inflammatory as well as hypersensitivity reactions.     

Protein tyrosine kinase Syk modulates EGFR signalling in human mammary epithelial cells

Signalling through protein tyrosine kinases (PTKs) is critical in the regulation of important cellular processes and its deregulation is associated with pathophysiological disorders such as cancer. We investigated the function of the PTK spleen tyrosine kinase (Syk) in the regulation of growth factor signalling pathways in human mammary epithelial cells. Our results show that downregulation of endogenous Syk expression enhances the ligand-induced activity of the epidermal growth factor receptor (EGFR) but not that of the closely related human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 3 (HER3) receptors. Moreover, Syk function interfered with EGFR-mediated cell responses such as proliferation and survival of mammary epithelial cells. A mechanistic link between Syk and EGFR is further supported by the colocalisation of the two PTKs in membrane fractions as well as the regulatory feedback effects of the EGFR kinase on Syk activity. Our findings demonstrate that Syk acts a negative control element of EGFR signalling.     

Phorbol myristic acetate enhances the production of Interleukin 2

Interleukin 2 is an antigen-nonspecific factor produced by Concanavalin A (Con A)-activated mouse spleen cells that has a number of biologic activities including the capacity to stimulate thymocyte proliferation, support the growth of continuous T cell lines, augment the antibody response of nude mouse spleen cells, and support the generation of antigen-specific cytotoxic T cells. In order to obtain increased amounts of Interleukin 2 for further purification and biologic studies, we have examined the use of Phorbol Myristic Acetate (PMA) as a costimulant. In this report we demonstrate that the addition of PMA to Con A-induced mouse spleen cell cultures results in a 5- to 20-fold increase in the production of Interleukin 2 activity under serum-containing and serum-free culture conditions.     

Different effects of TPA on two skin-derived cell lines: murine (HEL-30) and human (NCTC) epidermal cells

12-O-tetradecanoylphorbol-13-acetate (TPA) caused a rapid activation of protein kinase C in a murine (HEL-30) and in a human (NCTC) epidermal cell line. In HEL-30 cells, protein kinase C activation is followed by ornithine decarboxylase stimulation and cell proliferation, events inhibited by H-7, a specific inhibitor of protein kinase C. TPA in NCTC cells inhibited the basal ornithine decarboxylase activity and cell growth, whereas H-7 did not modify TPA effect. The response of NCTC cells was not due to direct toxicity of TPA. These data confirm that in murine epidermal cells, the proliferation induced by TPA is mediated by protein kinase C, whereas in a human skin-derived cell line these events are not or inversely associated.     

Transforming growth factors (TGFs): Properties and possible mechanisms of action

Transforming growth factors (TGFs) are growth-promoting polypeptides that cause phenotypic transformation and anchorage-independent growth of normal cells. They have been isolated from several human and animal carcinoma and sarcoma cells. One TGF is sarcoma growth factor (SGF) which is released hy murine sarcoma virus-transformed cells. The TGFs interact with epidermal growth factor (EGF) cell membrane receptors. TGFs are not detectable in culture fluids from cells which contain high numbers of free EGF cell membrane receptors. SGF acts as a tumor promoter in cell culture systems and its effect on the transformed phenotype is blocked by retinoids (vitamin A and synthetic analogs). The production of TGFs by transformed cells and the responses of normal cells to the addition of TGFs to the culture medium raise the possibility that cells “autostimulate” their own growth by releasing factors that rebind at the cell surface. The term “autocrine secretion” has been proposed for this type of situation where a cell secretes a hormone-like substance for which it has external cell membrane receptors. The autocrine concept may provide a partial explanation for some aspects of tumor cell progression.     

Interleukin 2 regulation of mitogen induction of immune interferon (IFN gamma) in spleen cells and thymocytes

Interleukin 2 (IL 2) or T-cell growth factor induced the production of immune interferon (IFN_γ) in C57B1/6 mouse spleen cell cultures, and enhanced mitogen-induced IFN_γ production in both spleen cells and thymocytes. Staphylococcal enterotoxin A, but not phytohemagglutinin P (PHA-P), induced IFN_γ production in thymocytes. IL 2 enhanced this production by almost 12-fold, while having no effect on the negative response to PHA-P. The IFN activity was shown to be IFN_γ by neutralization with specific antiserum. A strict correlation between IL 2 induction or enhancement of IFN_γ production and cell proliferation was not observed, probably indicating that non-IFN-producing cells also proliferated in the presence of IL 2. The data indicate that IL 2 can both induce IFN_γ and modulate mitogen induction of IFN_γ.     

Interleukin 1 enhances synovial cell hyaluronate synthesis

Interleukin 1 enhances proliferation of murine thymocytes in the presence of lectins, and is also known to stimulate the release of prostaglandins and neutral proteases from a variety of cell types. We have previously shown that a factor isolated from the culture media of disaggregated lining cells of the human synovial membrane was indistinguishable from monocyte-derived interleukin 1. We report here that interleukin 1 from either source stimulates hyaluronate synthesis by synovial membrane cells. Upon gel filtration or isoelectric focusing of synovial cell supernatants, the hyaluronate-stimulatory activity co-fractionates with the interleukin 1 activity. Enhanced cell secretion of hyaluronate is a newly described metabolic effect of interleukin 1.     

Lymphokines: their role in lymphocyte responses. Properties of interleukin 1

Interleukin 1, or IL 1, otherwise known as lymphocyte-activating factor, is a macrophage-derived 12,000- to 15,000-dalton polypeptide. Isoelectric focusing of human IL 1 reveals three peaks at pI's of 5.2, 6.0 and 6.9 respectively. IL 1 can be depleted of lymphocyte-derived IL 2 by SP-Sephadex chromatography. IL 1 augments the mitogenic response of PNA- Lyt 1+ thymocytes, and promotes thymocyte helper functions and B cell antibody production. IL 1 induces stable E rosette formation and the production of lymphokines such as T cell growth factor (IL 2) by peripheral T lymphocytes. Others have shown that IL 1 or closely related factors also stimulate hypothalamic cells to induce fever; induce in vitro fibroblast growth, prostaglandin, and collagenase production; and stimulate hepatocytes to produce acute phase proteins such as serum amyloid A. Murine epidermal cells also produce a 15,000-dalton factor that is mitogenic for thymocytes and may be similar to IL 1. We have recently hybridized spleen cells from mice sensitized with partially purified human IL 1 with a myeloma cell line. Clones have been isolated that produce supernatants that partially inhibit the thymocyte proliferative response to IL 1 but not the T cell growth factor activity of IL 2. Should these hybridoma products prove to be monoclonal anti-IL 1 antibodies, they will facilitate the further purification and characterization of IL 1.     

Immunity to herpes simplex virus type 2: viral antigen-presenting capacity of epidermal cells and its impairment by ultraviolet irradiation

Ia+ epidermal cells (EC) had accessory cell function for herpes simplex virus type 2 (HSV-2)-induced T cell proliferation of immune lymph node cells (LNC). The EC-mediated virus-induced proliferative response of immune LNC was inhibited by ultraviolet B (UVB) irradiation. When EC were pulsed with viral antigen before UVB irradiation, the response was partially restored by addition of epidermal cell-derived thymocyte-activating factor (ETAF). Although normal EC secreted prostaglandin E2, the levels secreted by UVB-irradiated EC were significantly reduced, and the UVB-induced suppression of the proliferative response of immune LNC was not corrected by indomethacin. Soluble factor(s) that suppresses proliferation was generated in supernatants from cultures containing UVB-irradiated but not nonirradiated EC. Sephadex chromatography revealed the presence of factors differentially modulating the proliferative response of HSV-stimulated immune LNC and concanavalin A-stimulated normal lymphoid cells.     

Effect(s) of lipopolysaccharide on lectin-induced T-cell activation

The present study focuses on the effect of lipopolysaccharide (LPS) on the cellular events leading to T-cell activation by concanavalin A (Con A). Interleukin 2 (Il-2) production is much reduced in Con A-stimulated cultures of spleen cells derived from LPS-treated mice. This depressed Il-2 synthesis is not related to the eventual activity of LPS-activated suppressive B cells. Rather, it reflects an ineffective collaboration between adherent cells and T lymphocytes. The low level of Il-2 produced by LPS-sensitized spleen cells is sufficient for lectin-induced T-cell proliferation. Moreover, acquisition of responsiveness to Il-2 is unaltered by LPS. No strict correlation was found between the deficiency in Il-2 production and the inability of LPS-sensitized spleen cells to generate a thymus-dependent response. Less time (5 hr) is needed for LPS to exert its inhibitory effect on an anti-sheep red blood cell response than on Il-2 synthesis (at least 24 hr). Results are discussed in terms of cellular interactions implicated in a polyclonal T-cell response and with regard to the contribution of Il-2 to the LPS-induced immune unresponsiveness.     

Synergistic hematopoietic growth factors

A number of growth factors acting on hematopoietic stem cells have now been purified and characterized. These include erythropoietin, granulocyte-macrophage colony-stimulating activity (GM-CSA), granulocyte colony-stimulating activity and colony-stimulating factor-1 (CSF-1). Factors which act in concert with these defined factors and appear to act relatively early in the hematopoietic stem cell lineage are currently under study. Interleukin 3 appears to have both the characteristics of a differentiating hormone and the ability to generate proliferation of relatively early stem cells. Interleukin 3 acts in concert with at least CSF-1 and erythropoietin to enhance their effect on stem cell proliferation and differentiation. A new class of hematopoietic growth factor activities termed synergizing activities also exist. These activities appear to have no intrinsic capacity to stimulate hematopoietic colony formation by themselves but enhance the effects of other differentiating hormones such as GM-CSA and CSF-1. Activities which appear to represent synergizing activities have now been found to evolve from a human bladder carcinoma line, a cell line derived from murine marrow adherent cells and normal murine marrow and thymic cells. These activities may act on very primitive hematopoietic progenitors to allow them to express receptors to various differentiating hormones or alternatively they may act as commitment factors in a commitment-progression model of stem cell regulation.     

Immunoregulatory effects of transforming growth factor-beta in a prolonged period of culture.

It is well known that transforming growth factor-beta (TGF-beta) strikingly inhibits numerous immune functions in short-term cultures. In this study we investigated the effects of TGF-beta on the immune responses of murine spleen cells in a prolonged period of culture. The addition of exogenous TGF-beta (0.1 ng/ml) inhibited the proliferation of Con A- or LPS-stimulated spleen cells, polyclonal IgM and IgG antibody production, and NK cell activity during 4 days of the initial culture and subsequently enhanced their responses on Day 10. The augmented polyclonal IgM and IgG responses in murine spleen cells induced by LPS and TGF-beta on Day 10 were suppressed by the secondary addition of TGF-beta on Day 6. These results suggest that TGF-beta acts as an immunoregulator in prolonged period responses by immunoactivators.     

Human epidermal cells and squamous carcinoma cells synthesize a cytokine that augments natural killer cell activity

Normal as well as transformed epidermal cells (EC) have recently been reported to produce a cytokine--EC-derived thymocyte-activating factor (ETAF), which according to its biologic as well as biochemical properties is indistinguishable from macrophage-derived interleukin 1 (IL 1). In the present study, the effect of supernatants (SN) derived from normal EC and a human squamous carcinoma cell (SCC) line were tested for their effects on natural killer (NK) cell activity. EC- as well as SCC-derived SN were able to augment in vitro NK cell activity of peripheral blood lymphocytes against K 562 cells. In contrast, adherent cell-derived, IL 1-containing SN did not affect NK cell activity. Upon high-pressure liquid chromatography (HPLC) gel filtration, ETAF and the EC-derived NK cell activity-augmenting factor (ENKAF) exhibited a similar m.w. However, by using reverse-phase HPLC, ETAF and ENKAF eluted as distinct peaks of activity, indicating that SCC cell-derived ENKAF is different from ETAF. Furthermore, ENKAF does not contain interleukin 2 (IL 2) or interferon (IFN) activity. The enhancement of NK cell activity was dose dependent and evident after 20 hr of preincubation of effector cells. Pretreatment of target cells with ENKAF did not affect the susceptibility of the target cells. The NK activity of large granular lymphocytes (LGL) purified by discontinuous Percoll gradient centrifugation and further depleted of high-affinity sheep erythrocyte rosetting cells was enhanced by ENKAF. In contrast, no NK cell activity was expressed by LGL-depleted T cell populations before or after treatment with ENKAF. In a single cell cytotoxicity assay in agarose, the number of lymphocyte binding to K 562 was not affected by ENKAF, but the frequency of dead conjugated target cells and presumably of active killer cells was increased by pretreatment with ENKAF. Additional incubation of LGL with ETAF did not further increase ENKAF-mediated augmentation of NK activity. In contrast to ETAF, ENKAF was not chemotactic for polymorphonuclear leukocytes. These results indicate that normal as well as transformed EC release a unique cytokine--ENKAF--which augments NK cell activity of LGL but is distinct from ETAF, IL 2, and IFN.     

Biologic properties of chromatographically separated murine thymoma-derived Interleukin 2 and colony-stimulating factor

Two growth factors, interleukin 2 (T cell growth factor) and colony-stimulating factor, are produced concomitantly by a murine EL-4 thymoma cell line after stimulation by phorbol myristate acetate. As shown elsewhere, these thymoma-derived factors appear to be biochemically and functionally indistinguishable from the interleukin 2 and colony-stimulating factor produced by mitogen-stimulated mouse spleen cells. Both factors co-elute during gel filtration with apparent m.w. in the range of 30,000, and both exhibit overlapping isoelectric point profiles between pH 4 and pH 5. Because we were unable to separate these 2 factors by methods based on either m.w. or charge, we have used phenyl-Sepharose chromatography, a method based on hydrophobic interactions, to completely separate murine interleukin 2 and colony-stimulating factor. In contrast with published reports, each of the separated factors exhibits unique biologic activities on lymphocytes and macrophages. Interleukin 2 provides help for antibody synthesis in the nude mouse, but neither enhances interferon production by macrophages nor stimulates macrophage growth. Colony-stimulating factor does not enhance antibody synthesis in the nude mouse but does enhance interferon production by macrophages and stimulate macrophage growth.     

Serotonin derivative, N-(p-Coumaroyl)serotonin, isolated from safflower (Carthamus tinctorius L.) oil cake augments the proliferation of normal human and mouse fibroblasts in synergy with basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF)

N-(p-Coumaroyl)serotonin (CS) with antioxidative activity is present in safflower oil. We have reported that CS inhibits proinflammatory cytokine generation from human monocytes in vitro. As reactive oxygen species (ROS) affect cell proliferation, in this study the effect of CS on the proliferation of various cell types was examined. CS augments the proliferation of normal human and mouse fibroblast cells. The cells continue to proliferate in the presence of CS and form a transformed cell-like focus without transformation. CS, however, does not augment the proliferation of other cell types, either normal or tumor cells. CS augments the proliferation of fibroblasts in synergy with basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF), but not with acidic FGF(aFGF) or platelet-derived growth factor (PDGF). This study using synthesized derivatives of CS reveals that the growth-promoting activity is not due to antioxidative activity. These findings indicate that CS is a natural compound with unique growth-promoting activity for fibroblasts.     

Characterization of a long-term T-helper-cell line which produces IL-2 and induces immunoglobulin secretion

Six OKT4+ human T-cell lines that require continuing PHA stimulation and TCGF for continuous growth were established. The cells from all six of these T-cell lines became smaller in size and lost the cell surface Ia antigen when they were grown in phytohemagglutinin (PHA)-depleted growth factor. These cells were unable to survive in the absence of PHA even if exogenous factor was present in great abundance. One of the cell lines (FL) was capable of providing helper functions. In the presence of PHA and phorbol myristate acetate, FL cells produced a growth factor, tentatively identified as Interleukin 2 (IL-2) by its ability to promote the proliferation of an IL-2-dependent murine T-cell line. Moreover, when FL cells were cocultured with B cells, pokeweed mitogen-induced immunoglobulin production was enhanced.     

Effect of glucocorticosteroids on epidermal cell-induced immune responses

Recent reports indicate that pharmacologic doses of glucocorticosteroids induce structural alterations in epidermal Langerhans cells. In this study we hoped to determine whether steroid-induced changes in Langerhans cell surface characteristics are paralleled by alterations in Langerhans cell-dependent immunologic functions of epidermal cells. We found that both topically and systemically administered steroids led to a dose-dependent reduction in the number of Ia-bearing epidermal cells. This numerical decrease was paralleled by a substantial impairment of Langerhans cell-dependent immunologic functions of epidermal cells in that their capacity to induce antigen-specific, syngeneic, and allogeneic proliferation of T cells from non-steroid-treated animals was substantially reduced. The capacity of epidermal cells to generate ETAF activity, however, was not adversely affected by the steroid treatment. After cessation of treatment, Langerhans cell numbers and Langerhans cell-dependent in vitro functions slowly and gradually returned to normal values. We propose that the ability of glucocorticosteroids to interfere with the generation of T cell-dependent immune responses may be due, at least in part, to their interference with antigen-presenting cell function.