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.     

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.     

Preparation of human epidermal tissue for functional immune studies

A trypsinization technique was optimized for preparation of human epidermal single-cell suspensions from suction blisters. The conditions which provided the highest epidermal cell yield and viability was Sigma trypsin 0.25% (Type II, crude; cat. no. T8128). The incubation time should be 45 min at 37 degrees C with continuous shaking of the epidermal tissue. The use of suction blisters was evaluated for in vivo determination of epidermal thymocyte activating factor (ETAF/Il-1). Trypsinization of epidermis diminished ETAF/Il-1 activity. Dialysis of epidermal homogenates improves determination of ETAF/Il-1-like activity due to removal of low-weight inhibitors. Determination of ETAF/Il-1-like activity in homogenates of suction blisters constitutes a reliable model with acceptable reproducibility.     

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.     

Production of interleukin 1 alpha by a trichilemmoma cell line

In order to obtain a good source of human epidermal cell derived thymocyte activating factor (ETAF) for biological study, several cell lines of epidermal origin were cultured. One of the trichilemmoma cell lines, KTL-1, was found to produce ETAF satisfactorily in its culture supernatant. The culture supernatant was collected and analyzed to determine the nature of the ETAF activity. The ETAF activity was completely inhibited by anti-IL 1 alpha antibody, but not by anti-IL 1 beta antibody. Messenger RNA expression by KTL-1 cells was analyzed by using cDNA of IL 1 alpha as a probe. Messenger RNA was not detected in KTL-1 cells by either dot blot analysis or northern blot analysis. However, it was expressed weakly in the cytoplasm by in situ hybridization technique. Messenger RNA expression was easily detected in KTL-1 cells stimulated by lipopolysaccharide (LPS). Messenger RNA expression reached a peak at 3 hours after stimulation of LPS. A trichilemmoma cell line, KTL-1, can be a good source of human ETAF for biological study.     

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.     

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.     

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.     

Interleukin 1

The last decade has witnessed a remarkable expansion in the understanding of the skin as an immunologic organ. A prime example of this is the discovery that keratinocytes can synthesize and secrete immunoregulatory and proinflammatory glycoproteins termed cytokines. Initial studies focused on interleukin 1-like molecules produced by keratinocytes, initially termed epidermal cell-derived thymocyte activating factor (ETAF). It is now clear that the functional activities described by the term ETAF are composed mainly of interleukin 1 alpha and interleukin 1 beta, although other epidermal cytokines may contribute to the biologic activity. The biologic properties of interleukin 1 are the basis of this review.     

Effect of X-irradiation on epidermal immune function: decreased density and alloantigen-presenting capacity of Ia+ Langerhans cells and impaired production of epidermal cell-derived thymocyte activating factor (ETAF)

The mechanisms involved in the modulation of cutaneous immune responses by UV radiation have been extensively investigated; by contrast, few studies have addressed the effects of x-irradiation on epidermal immune function. We therefore investigated the effect of x-irradiation of mice on: (a) the density of epidermal Ia+ Langerhans cells (LC) in immunofluorescence studies, (b) epidermal cell (EC) allostimulatory capacity in the allogeneic EC-lymphocyte reaction (ELR), and (c) production of epidermal cell-derived thymocyte activating factor (ETAF). C3H/He and BALB/c mice were irradiated with 900, 1,800, 2,700, or 3,600 rad from a 137Cs source, and sacrificed 10 h or 3 days later. X-irradiation of mice 10 h previously only slightly decreased the density of epidermal Ia+ LC and did not affect the capacity of their EC to stimulate allogeneic responder lymphocytes in the ELR. X-irradiation of mice 3 days previously, however, resulted in a dose-dependent decrease in the density of Ia+ LC. This decrease was accompanied by a substantial reduction in EC allostimulatory capacity in the ELR at all doses of x-irradiation. ETAF production by cultured EC from mice x-irradiated 3 days previously was also found to be diminished at all doses of x-irradiation. Trypan blue exclusion studies demonstrated that the observed decreases in EC allostimulatory capacity and ETAF production were not the result of a generalized lethal effect of x-irradiation on EC. The reduction in EC allostimulatory capacity following in vivo x-irradiation could not be reversed by addition of exogenous ETAF or interleukin-1 in the ELR. Taken together, these results indicate that x-irradiation decreases the density of Ia+ LC, impairs LC alloantigen-presenting function, and reduces ETAF production. Thus cutaneous x-irradiation may affect inflammatory and neoplastic processes not only by its antimitotic activity, but also by a direct effect on EC which subserve immunologic functions.     

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.     

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.     

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.     

Tumorigenicity and metastatic behavior in nude mice of two human squamous cell carcinoma lines that differ in production of the cytokine ETAF/IL-1

The purpose of these studies was to determine whether the production of the cytokine epidermal cell thymocyte-activating factor (ETAF) by human squamous cell carcinoma (SCC) cells correlated with their tumorigenicity and metastatic potential in athymic nude mice. Cells of the human SCC line A431 produced rapidly growing subcutaneous tumors, few experimental lung metastases, and low levels of ETAF activity in vitro. In contrast, cells of the SCC Colo-16 line produced slower growing subcutaneous tumors, high numbers of experimental lung metastases, and a high level of ETAF activity in culture supernatants. The apparent relationship between production of ETAF and experimental metastasis formation was not consistent. Clonal populations of the SCC A431 and Colo-16 were isolated in vitro. The clones of Colo-16 varied in their ability to produce experimental metastases and in production of ETAF in vitro. However, the levels of ETAF production did not correlate with the propensity of the SCC cells to produce experimental metastases. We conclude that while the growth and metastasis of human SCC in nude mice may benefit from production of the cytokine ETAF, the ETAF production per se is not invariably linked with the capability of the SCC cells to metastasize.     

De novo synthesis of lysozyme by human epidermal cells

The presence of lysozyme in human epidermis was determined in extracts from the surface of human skin and in sonicates of human epidermal cell preparations with the use of a bacteriolytic assay employing Micrococcus lysodeikticus cell walls as substrate. Preincubation of the extracts with the Fab portion of the IgG fraction of an antiserum to human lysozyme abolished the lytic activity of the extracts showing the specificity of the assay. De novo synthesis of lysozyme by human epidermal cells was demonstrated by radiolabeling studies. Human epidermal cells cultured in serum-free medium and pulsed with [3H]leucine for 24 h were sonicated and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A major band at Mr 14,500 exhibited both immunoreactivity with a rabbit antihuman lysozyme antibody and radioactivity. Gel filtration of the cell sonicate revealed bacteriolytic activity in the fractions containing radioactive and immunoreactive proteins. These findings suggest that lysozyme is newly synthesized by human epidermal cells.     

Production of paf-acether by human epidermal cells

The production of the inflammatory mediator paf-acether (paf) from human epidermal cells was investigated in vitro. Human epidermal cells, freshly isolated from normal skin or in culture, were incubated in Tyrode's buffer containing 0.25% lipid-free bovine serum albumin in the presence of 2 microM calcium ionophore A23187, at 37 degrees C, for 1 to 60 min. Paf production slightly began at the first min of stimulation, was significant after 10 min, reached a maximum at 20 min (251 +/- 25 pg/l X 10(6) cells, mean +/- 1 SD), and decreased thereafter. About 50% of the paf amount produced by epidermal cells was recovered in supernatants. Addition of the non-acetylated paf precursor 1-O-octadecyl-sn-glycero-3-phosphocholine, i.e., lyso-paf, at 0.1 microM to epidermal cells during A23187-stimulation did not alter this production. In contrast, addition of acetyl-coenzyme A at 0.1 mM enhanced paf production by 5 times. The material produced by epidermal cells was identical to synthetic paf because: 1) the aggregation of aspirin-treated and ADP-insensitive washed rabbit platelets it induced was inhibited by BN 52021, an antagonist of the paf putative receptor; 2) the factor was inactivated by phospholipase A2 but was insensitive to lipase from Rhizopus arrhizus; 3) it exhibited the same retention time as synthetic paf during standard and reverse-phase (RP) high-pressure liquid chromatography (HPLC) elution. The paf precursors, i.e., lyso-paf and 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine, were also detected in epidermal cells, stimulated with A23187 or not. As determined by RP-HPLC analysis and confirmed by gas chromatography analysis, these precursors and the paf produced by epidermal cells exhibited more than 90% of a hexadecyl chain at the sn-1 position of the molecule. The present results demonstrate the synthesis and release of paf by normal human epidermal cells. Paf production within the epidermis might account for the development of cutaneous inflammation and the pathogenesis of many skin disorders.     

热处理对体外培养正常人黑素细胞活性的影响

目的 探讨热处理对体外培养的正常人黑素细胞的增殖活性、黑素合成及其酪氨酸酶活性的影响。方法 无菌操作获取正常人包皮环切术后的包皮,按照皮肤表皮细胞培养法获取黑素细胞,利用碱性成纤维细胞生长因子（bFGF）为主要有丝分裂原建立正常人表皮黑素细胞培养系,采用Masson-Fontana染色法对培养细胞进行鉴定,确定细胞种系,噻唑蓝（MTT）比色法测定不同温度条件（39、41、42、43、45 ℃）处理对黑素细胞活力的影响并选择最佳的温度条件,以左旋多巴为底物测定酪氨酸酶活性,比色法测定黑素含量,并将实验组与对照组结果进行比较。结果 体外培养的正常人黑素细胞在5个不同的温度条件作用后,以42 ℃作用后的细胞增殖活性最佳。酪氨酸酶活性和黑素含量测定结果显示,42 ℃每天1 h连续干预3 d后,体外培养的正常人黑素细胞的酪氨酸酶活性增长率为36.4%,黑素合成增长率为78%,均明显高于对照组（P值均 < 0.01）。结论 热处理可以增强体外培养的人黑素细胞的增殖活性,促进其黑素合成,并且可提高其酪氨酸酶的活性。