The effect of in vitro and in vivo UV irradiation on the production of ETAF activity by human and murine keratinocytes

Cultured epidermal cells and keratinocytes produce a potent hormone-like factor called epidermal cell-derived thymocyte-activating factor (ETAF). ETAF appears to be similar if not identical to a monocyte-derived lymphokine, known as interleukin 1 (IL-1). These two cytokines are able to amplify a diverse number of proliferative and inflammatory processes. Several recent investigations have suggested that UV-induced immunosuppression may be due in part to the inhibition of IL-1/ETAF production by monocytes and keratinocytes, respectively. We therefore decided to directly study the effects of various doses of in vitro and in vivo UV radiation (UVR) on the production of ETAF by normal murine epidermal cells and a murine (Pam 212) and a human (SCC) keratinocyte cell line. Our results surprisingly demonstrated an increase in both the extracellular and the intracellular ETAF activity of the murine epidermal, Pam 212, and SCC after sublethal amounts of in vitro UVR. Likewise, increased ETAF activity of murine epidermal cells was detected after sublethal doses of in vivo UVR. The UV-induced ETAF activity was cycloheximide-sensitive, suggesting that de novo synthesis of ETAF rather than cell membrane leakage was responsible for the increased ETAF activity. The fact that UV irradiation can increase ETAF activity by keratinocytes could have important local and systemic consequences for the host and may provide an efficient, contaminant-free method for generating ETAF activity for further biochemical and immunologic studies.     

Epidermal cell-derived thymocyte activating factor (ETAF) is a potent T-cell chemoattractant

The epidermis, in particular epidermal cytokines, have been shown to modulate a number of inflammatory and cellular immune responses. In this study we have demonstrated that the partially purified epidermal cytokine, epidermal thymocyte activating factor (ETAF), a polypeptide released by keratinocytes, is a potent T-cell chemoattractant. Compared to its ability to augment lectin-stimulated thymocyte proliferation, ETAF is much more active as a T-cell chemoattractant. The results of this study give further support to the role of local epidermal factors in immune reactivity. This finding may have particular relevance to pathologic states characterized by T-cell infiltration in the skin, such as cutaneous T-cell lymphoma.     

Epidermal interleukin-1 is increased in cutaneous T-cell lymphoma

Interleukin-1 (IL-1) is a monocyte-derived polypeptide with immunoregulatory and proinflammatory functions. Although monocytes are the principle source of IL-1, other cells, such as keratinocytes, endothelial cells, renal mesangial cells, and neutrophils, produce a factor with IL-1 activity. The IL-1-like polypeptide produced by keratinocytes, epidermal-derived thymocyte-activating factor (ETAF), is similar on biological, biochemical, and molecular levels to monocyte-derived IL-1. Studies of IL-1 or ETAF have for the most part been undertaken using cell culture supernatants or cell lysates, and in situ localization of ETAF has not been demonstrated. Previous studies have suggested that ETAF is involved in the pathogenesis of cutaneous T-cell lymphoma (CTCL). To gain insights into the role of these cytokines in disease states, we investigated whether IL-1 could be localized in tissue sections using a direct immunofluorescence technique with a monoclonal antibody directed against IL-1. This monoclonal antibody partially inhibited ETAF activity and totally inhibited IL-1 activity in the co-stimulator assay and so could be used to detect IL-1 or ETAF. We studied skin biopsies from 10 healthy individuals, 10 patients with CTCL, and 11 patients with various inflammatory dermatoses. Intense epidermal fluorescence was demonstrated in all cases of CTCL, whereas minimal reactivity was visible in normal biopsies and the inflammatory dermatoses. Most patients with CTCL showed an intercellular pattern, while none of the normal controls or those with inflammatory dermatoses showed this pattern. An irrelevant IgM monoclonal antibody, used as a negative control, did not demonstrate epidermal staining. To further demonstrate specificity, we incubated the anti-IL-1 antibody with recombinant beta IL-1:Epidermal reactivity was completely blocked. In a separate experiment, COLO 16 cells, a squamous cell carcinoma cell line that constitutively produces ETAF, stained positively for the IL-1 antibody. We conclude that epidermal IL-1 is elevated in CTCL.     

Ultraviolet radiation inhibits alloantigen presentation by epidermal cells: partial reversal by the soluble epidermal cell product, epidermal cell-derived thymocyte-activating factor (ETAF)

It has been postulated that ultraviolet radiation (UVR) alters antigen presentation by macrophages. This is thought to be due, in part, to inhibition of macrophage-derived interleukin 1 (IL-1), which is a hormone-like factor with immunoregulatory functions. Conventional stimulator cells for antigen presentation are macrophages; however, other cell types such as epidermal Langerhans cells are capable of antigen presentation. Keratinocytes also play a role in the immune system by providing a factor with IL-1-like activity, termed Epidermal cell-derived Thymocyte-Activating Factor (ETAF). The purpose of this study was to determine whether UVR affects alloantigen presentation by epidermal cells and if so, whether the UV-induced change is due to UVR alteration in ETAF activity. Epidermal cells from UV-treated BALB/c mice (UV-EC) or from non-UV-treated mice (EC) were x-irradiated and then cocultured for 5 days with allogeneic T cells from C57Bl/6 mice. UV-EC caused less T-cell stimulation than did EC from non-UV-treated animals. When chromatography purified fractions of ETAF were added to cultured UV-EC, partial restoration of T-cell stimulation was seen. These results suggest that this UV-induced defect in alloantigen presentation is due, in part, to decreased ETAF activity.     

Characteristics of an epidermal cell thymocyte-activating factor (ETAF) produced by human epidermal cells and a human squamous cell carcinoma cell line

In this study we show that both cultured normal human epidermal cells (EC) and a human squamous cell carcinoma (SCC) cell line produce a thymocyte-activating factor (ETAF). EC-ETAF and SCC-ETAF both have a Mr of 15,000 and were eluted from chromatofocusing at the same isoelectric points of 7.2, 5.8, and 5.0. Both activities were maintained at alkaline pH and were destroyed at temperatures above 60 degrees C. In addition to stimulating thymocyte proliferation, human ETAF exhibited a variety of other pertinent biologic activities. Although EC-ETAF or SCC-ETAF by themselves exhibited no T-cell growth factor activity, both ETAF preparations enhanced Interleukin 2 production by cultured human peripheral blood lymphocytes when stimulated with polyclonal T-cells stimulants (Concanavalin A and phorbol myristate acetate). Human ETAF also was chemotactic for rabbit polymorphonuclear leukocytes and was directly mitogenic for cultured human dermal fibroblasts. Injection of human ETAF into C3H/HeJ mice, resulted in inducing serum amyloid A (SAA) production by murine hepatocytes. The thymocyte growth-enhancing activity, the fibroblast-stimulating activity, and the SAA-inducing capacity of ETAF all coeluted off AcA54 gel. These biologic as well as biochemical properties of human keratinocyte-derived ETAF are identical with those of human macrophage-derived Interleukin 1. The ability of keratinocytes to release an immunomodulating factor with such diverse consequences may play an important role in normal wound healing and in diseases involving epithelial tissues.     

Epidermal cell-derived lymphocyte differentiating factor (ELDIF) inhibits in vitro lymphoproliferative responses and interleukin 2 production

We have examined the biologic characteristics and immunologic properties of epidermal cell-derived lymphocyte differentiating factor (ELDIF), a lymphocyte differentiating factor produced by cultured human keratinocytes. The ELDIF was semipurified by a gel filtration procedure. This factor, which is distinct from prostaglandins, epidermal cell-derived thymocyte activating factor (ETAF), and the well-known thymic hormones (thymulin, thymopoietin, and thymosin alpha 1) did not exhibit any interleukin (IL)-1, IL-2, or IL-3 activity. It strongly inhibited in vitro lymphoproliferative responses of normal mouse spleen cells to phytohemagglutinin, concanavalin A, and lipopolysaccharide. This dose-dependent phenomenon was associated with a suppression of IL-2 production rather than any toxic effect. It can be concluded that ELDIF, a product of human epidermal cells, which displays in vitro T-cell differentiation and regulatory activities, could be of major importance in vivo in the control of cutaneous inflammatory reactions.     

Hydrocortisone reduces both constitutive and UV-elicited release of epidermal thymocyte activating factor (ETAF) by cultured keratinocytes

Epidermal thymocyte activating factor (ETAF) is spontaneously released into the media by PAM 212 and A 431 cell lines and cultured normal human keratinocytes. ETAF from all 3 cell types can substitute for interleukin 1 (IL-1) in the augmentation of proliferation of a helper T-cell clone (D10.G4.1) induced by mitogen. Hydrocortisone (HC) substantially reduces the release of ETAF by these keratinocytes and, further, appears to induce the release of an inhibitor of lymphocyte activating factor activity of IL-1. Irradiation with UVC causes increased ETAF release into the media. Hydrocortisone abrogates this effect. Thus HC reduces both constitutive and elicited release of ETAF. ETAF plays a major role in inflammation; the ability of HC to block ETAF release by keratinocytes may account for the anti-inflammatory effect of glucocorticosteroids on the skin.     

Human keratinocytes and epidermoid carcinoma cell lines produce a cytokine with interleukin 3-like activity

Keratinocytes are capable of releasing distinct immunomodulating cytokines such as epidermal cell-derived thymocyte activating factor (ETAF) and an epidermal cell-derived natural killer cell augmenting factor (ENKAF). The present study was performed to determine whether human keratinocytes also may secrete an interleukin 3 (IL-3)-like mediator and thereby participate in the regulation of mast cell activity in the skin. Supernatants of freshly isolated human epidermal cells (EC) and malignant keratinocyte cell lines (A 431, SCC) were tested for their capacity to induce the proliferation of IL-3-dependent cell lines 32 DCL and FDCP. Human epidermal cell interleukin 3 (EC IL-3) is spontaneously released by freshly isolated EC, A 431, and squamous cell carcinoma (SCC) cells. However, both normal EC and A 431 cells produced increased levels of EC IL-3 activity when cultured in the presence of different stimulants, such as phorbol myristate acetate and lipopolysaccharide. The EC IL-3 activity was not inhibited when treated with a monoclonal anti-IL-1 or anti-IL-2-antibody. Biochemical characterization showed that human EC IL-3 has a molecular weight of 17K, elutes of DEAE-ion exchange high-performance liquid chromatography (HPLC) as one major peak at 0.36 M NaCl, and upon HPLC-chromatofocusing exhibits 3 isoelectric points of 7.8, 7.5, and 5.6. Upon reversed-phase HPLC, EC IL-3 activity eluted at about 100% acetonitrile. When highly purified EC IL-3 was labeled with 125I and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a single homogeneous band exhibiting a molecular weight of 17K was seen, which correlated with the IL-3 activity and was free of ETAF/IL-1, IL-2, and interferon activity. These data indicate that human EC synthesize an IL-3-like cytokine which is distinct from ETAF/IL-1, IL-2, and interferon and thereby may participate in the regulation of mast cell activity during inflammatory and fibrotic, as well as hypersensitivity reactions.     

Autocrine growth stimulation of human keratinocytes by epidermal cell-derived thymocyte-activating factor: implications for skin aging

Summary The monocyte-derived cytokine interleukin-1 (IL-1) has growth-promoting activity for a variety of cell types, including lymphocytes and fibroblasts. We have previously shown that the epidermal cell-derived thymocyte-activating factor (ETAF) strongly resembles IL-1 in terms of biological, biochemical, and molecular biological properties. Because some lymphokines are known ot alter epidermal cell growth and differentiation and because cultured epidermal keratinocytes are capable of autocrine growth stimulation in vitro through conditioning of their culture medium, we sought to evaluate the effect of ETAF on keratinocyte growth. While there was marked donor variability in the responsiveness of keratinocytes to ETAF, partially purified preparations of ETAF showed substantial ability to stimulate the growth of keratinocytes, particularly those of newborn donors. In addition, in conditioned media there appeared to be activities distinct from ETAF that also promoted keratinocyte growth. Keratinocytes in serum-free medium secreted large amounts of ETAF, as reported previously, and keratinocyte cultures derived from newborn donors secreted significantly more than did those derived from adult donors. These results are consistent with an autocrine growth regulatory role of ETAF in human epidermis and with an age-associated loss of this phenomenon.     

Stimulation of muscle protein degradation by murine and human epidermal cytokines: relationship to thermal injury

Accelerated muscle proteolysis is a characteristic of systemic reaction following trauma, sepsis, or extensive thermal injury. The factors involved in this accelerated muscle breakdown have not been fully described. However, recently leukocytic pyrogen or interleukin 1 (IL-1) have been implicated in the induction of muscle protein degradation in septicemia or trauma. The epidermal cytokine epidermal cell-derived thymocyte activating factor (ETAF) is biochemically and functionally similar to IL-1. Injury to skin can augment ETAF activity. Using a murine model, we found that thermal injury can significantly enhance ETAF/IL-1 activity in a dose-dependent fashion. In addition, ETAF can cause net muscle protein breakdown in vitro. Thus, increased amounts of ETAF produced by thermally injured skin may contribute to the accelerated muscle breakdown in extensive thermal injury.     

Immunomodulation using an epidermal cytokine (ETAF)

Tissue cultures of freshly isolated murine and human keratinocytes as well as transformed keratinocyte cell lines secret a cytokine, epidermal cell derived thymocyte activating factor (ETAF), which augments in vitro lymphoproliferative responses. Keratinocytes produce increased levels of ETAF activity after exposure to a variety of cell damaging agents such as silica, endotoxin, phorbol esters, hydroxyurea, mechanical disruption and UV-irradiation. Biochemical studies showed that ETAF is a heat- and pH-stable low molecular weight (15K) protein which is produced in low amounts and active at low (10(-10)-10(-15) M) concentration. The most important biological property of ETAF is an antigen non specific stimulation of the immunological system. ETAF in conjunction with the antigen presenting function of Langerhans cells results in an activation of T-lymphocytes and increased production of lymphokines such as Interleukin 2, which is responsible for the activation of T- and B-lymphocytes. In addition ETAF is chemotactic for polymorphonuclear leukocytes, monocytes and natural killer cells and is directly mitogenic for fibroblasts. When injected into mice ETAF induces production of acute phase proteins such as Serum Amyloid A. Furthermore ETAF may act as an endogenous pyrogen and induce fever. According to its biochemical characteristics and biological properties ETAF can not be separated from the macrophage derived Interleukin 1(IL 1), suggesting that both ETAF and IL 1 are identical. These findings indicate that production of IL 1-like molecules is not confined to cells of the immunological system and ETAF production by keratinocytes may have important implications in the pathogenesis of inflammatory as well as neoplastic skin diseases.     

Interleukin-1 decreases the number of Ia+ epidermal dendritic cells but increases their expression of Ia antigen

It is generally accepted that ETAF/IL-1 is produced in epidermis by both keratinocytes and Langerhans' cells. We have studied the density and morphology of Ia+ epidermal dendritic cells in mice after systemic or intracutaneous injection of recombinant IL-1 beta. We found that rIL-1 beta decreased the density of Ia+ dendritic cells in the time period 2-7 days after rIL-1 beta administration. However, the remaining dendritic cells were enlarged and more arborized with increased expression of Ia antigen 1-4 days after injection of rIL-1 beta. The implication of the results is that ETAF/IL-1 modulates the function of Langerhans' cells through autocrine and paracrine regulation.     

Interleukin 1 alpha, tumor necrosis factor alpha, and interferon gamma in psoriasis

Although recent studies have suggested that a variety of cytokines released by keratinocytes and inflammatory leukocytes could contribute to induction or persistence of the inflammatory processes in psoriasis, it remains unclear how production of these cytokines is regulated in psoriatic patients. To elucidate the biologic relevance of these cytokines to the pathogenesis of psoriasis, we investigated serum levels of interleukin 1 alpha, tumor necrosis factor alpha, and interferon gamma in 21 patients with psoriasis vulgaris, together with 21 healthy controls. The mean serum levels of interleukin 1 alpha and tumor necrosis factor alpha were not significantly different from those in controls, while those of interferon gamma were significantly elevated in the patients with psoriasis. Serum levels of interleukin 1 alpha correlated negatively with clinical disease severity expressed as psoriasis area and severity index score and with duration of psoriasis. In contrast, interferon gamma levels were related, although not significantly, to disease severity. In addition, an inverse correlation was noted between the interleukin 1 alpha levels and interferon gamma levels. These results indicate that interleukin 1 alpha and interferon gamma may be relevant to the induction and perpetuation, respectively, of the inflammatory responses in psoriasis, and that these cytokines, which have similar biologic properties, may strictly regulate one another's production in vivo.     

Substance P induction of murine keratinocyte PAM 212 interleukin 1 production is mediated by the neurokinin 2 receptor (NK-2R)

The neurological system plays an important role in modulating some inflammatory skin diseases. Neuro-cutaneous interactions may be mediated by the release of neuropeptides such as substance P (SP) which activate immunocompetent cells in the skin by binding to high affinity neurokinin receptors (NKR). Since epidermal keratinocytes produce a variety of cytokines and are intimately associated with cutaneous sensory fibers, we tested the ability of these cells to participate in the cutaneous neuroimmune system by the secretion of potent cytokines such as interleukin 1 (IL-1) in response to released SP. RT-PCR studies demonstrated that cultured PAM 212 murine keratinocytes expressed mRNA for NK-2R but not NK-1R. Correspondingly, the addition of SP to these cells resulted in a rapid increase in intracellular Ca2+ levels that could be specifically blocked by an NK-2R antagonist. NK-2R was also shown in normal mouse epidermis by immunohistochemistry. SP augmented the expression of PAM 212 keratinocyte IL-1alpha mRNA in a dose and time dependent manner and this induction was inhibited by an NK-2R antagonist. Secretion of bioactive IL-1alpha by the PAM 212 keratinocytes was likewise stimulated by SP in a dose dependent manner. These data support the hypothesis that SP released from cutaneous sensory nerves contributes to neuroimmune inflammatory responses in the skin by modulating the expression and release of cytokines from epidermal keratinocytes.     

Evaluation of the potential role of cytokines in toxic epidermal necrolysis

Toxic epidermal necrolysis is a rare disease observed as a consequence of adverse reactions to drugs. It results in the widespread apoptosis of epidermal cells and has a high mortality rate. The mechanisms leading to this apoptosis are not yet elucidated. We investigated whether the cytokines present in the blister fluid, which accumulates under necrotic epidermis, originated from T lymphocytes and may play a role in the propagation of keratinocyte apoptosis. Interferon gamma (IFN-gamma), soluble tumor necrosis factor alpha (TNF-alpha), soluble Fas ligand (sFas-L) were present in much higher concentration in the blister fluids of 13 toxic epidermal necrolysis (TEN) patients than in control fluids from burns. The results of RT-PCR studies, however, indicated that only IFN-gamma and to a lesser extent interleukin (IL)-18 were produced by mononuclear cells present in the fluid. That suggests that the other cytokines also present (TNF-alpha, sFas-L, IL-10) rather originated from activated keratinocytes. Fas-L was indeed overexpressed on the membranes of keratinocytes in lesional skin in situ. The Th1 profile of T lymphocyte activation found in the blister fluid of patients with TEN is consistent with a key role for drug-specific cytotoxic T lymphocytes (CTL) as previously reported, the activation of keratinocytes by IFN-gamma making them sensitive to cell-mediated cytolysis. We propose the hypothesis that the production of Fas-L, TNF-alpha, and IL-10 by keratinocytes could be a defense mechanism against CTL rather than a way of propagating apoptosis among epidermal cells.     

Cyclosporin increases granulocyte/macrophage colony-stimulating factor (GM-CSF) activity and gene expression in murine keratinocytes.

Keratinocytes produce multiple cytokines in response to a variety of stimuli. The release of interleukin 1 (IL-1) from keratinocytes may be significant in initiation of cutaneous inflammation, and the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) is thought to be important in the regulation of antigen-presenting function by epidermal Langerhans cells. Because cyclosporin inhibits interleukin 2 release from T cells, it has been suggested that cyclosporin may function as an anti-inflammatory agent within the epidermis through inhibition of keratinocyte cytokine release. This investigation examined the direct effect of cyclosporin on the production of GM-CSF by murine keratinocytes and the keratinocyte cell line PAM 212. GM-CSF bioactivity increased in cell supernatants from keratinocytes exposed in vitro to 1 microgram/ml cyclosporin for up to 24 h. GM-CSF and IL-1 mRNA levels in keratinocytes cultured under similar conditions or in the presence of lipopolysaccharide also increased. The lack of inhibition of GM-CSF expression following cyclosporin treatment is consistent with recent observations in T cells and is opposite to the effect of cyclosporin on interleukin 2.     

Lidocaine-induced inhibition of the production of interleukin 1-like epidermal cell-derived thymocyte activating factor

For the purpose of investigating the influence of the cationic anesthetic, lidocaine, on the production of epidermal cell-derived thymocyte activating factor (ETAF), murine epidermal cells were incubated for 1 h with 0.02-6 mg/ml lidocaine, washed, and incubated for an additional 23 h. The ETAF activity was assayed as the co-mitogenic activity of the crude epidermal cell supernatants on phytohemagglutinin-stimulated murine thymocytes. Lidocaine reduced the ETAF activity significantly, most markedly at a concentration of 2 mg/ml. The reduction was not caused by cytotoxicity, by co-production of inhibitory factors, or by modification of the ETAF molecule. Although the murine thymocyte assay was highly sensitive to lidocaine, the reduction of ETAF activity was not the result of carryover of lidocaine to the thymocyte assay. Our results indicate that lidocaine inhibits ETAF production in vitro, and suggest that conventional procedures, such as lidocaine anesthesia, which are acceptable for morphologic techniques, might not be suitable for functional studies of the cellular components of the skin.