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.     

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.     

Macrophage inhibitor factor (MIF) in cutaneous lymphoproliferative diseases

Macrophage migration inhibitor factor (MIF) activity in the sera of patients with mycosis fungoides, Sézary syndrome, and cutaneous lymphoma was observed in the sera of eight of the ten patients with stage II (infiltrative) mycosis fungoides, but in only one of the eight patients with stage I and in neither of the two patients with stage III mycosis fungoides. Two of the three patients with Sézary syndrome had MIF in the serum. No MIF was observed in cutaneous lymphoma. These data support the concept that Sézary syndrome and mycosis fungoides are T-cell diseases, and transitional, prelymphomatous diseases.     

Epidermal cell growth-dependent arylhydrocarbon-hydroxylase (AHH) activity in vitro

Summary Cytochrome P-450-dependent arylhydrocarbon-hydroxylase (AHH) activity and inducibility by benzanthracene (BA) was measured in cultured guinea pig and human epidermal cells. Basal AHH-activity (AHH_b) in guinea pig epidermal cells was much higher than in human epidermal cells. AHH_b in guinea pig epidermal cells was directly related to the labeling index and decreased to the original level between the 5th and 7th day of cell culturing. On the other hand, the induction-ratio of AHH reached its maximum level when the number of cells began to rise (proliferation phase) and remained high at day 7 of the cell culture. These results suggest a cell growth dependent activity and inducibility of carcinogen-metabolizing enzymes, such as AHH, in isolated epidermal cells.Dedicated to Prof. Dr. Dr. h.c. Otto Braun-Falco on the occasion of his 65th birthday     

Drug-induced lymphocyte proliferation in the experimental sulbenicillin (SBPC) rash of guinea pigs and the enhancement effects of interleukin 2 (IL2)

The effects of interleukin 2 (IL2) on drug-specific lymphocyte proliferative responses and their suppression by elicitation of sulbenicillin (SBPC) rash and by induction of SBPC tolerance were studied. GR-LC and TO-LC, the draining lymph node cells taken from animals with SBPC generalized rash (GR) and SBPC tolerance (TO), respectively, were incubated with IL2 rich medium (IL2RM) overnight, then washed, and tested for antigen-specific response in a standard 3-day proliferation assay. The GR-LC and TO-LC showed low responsiveness or unresponsiveness to specific drug before overnight incubation with IL2LM, but recovered a distinct responsiveness after overnight incubation with IL2LM. This result is encouraging for the establishment of the drug-specific lymphocyte proliferation test for determination of the causative drug in patients with delayed type hypersensitivity drug eruptions.     

Epidermal growth factor receptor (EGFR) expression in periocular and extraocular sebaceous carcinoma

Sebaceous carcinoma has a predominant periocular origin but can also be extraocular. These two groups have distinct clinical courses. Insight into the molecular determinants of tumorigenesis and metastasis is limited. There is no effective treatment for metastatic sebaceous carcinoma. Epidermal growth factor receptor (EGFR) is implicated in tumorigenesis and can be a therapeutic target in certain settings. We evaluated EGFR levels by immunohistochemistry (IHC), comparing its expression between periocular and extraocular tumors and assessed EGFR mutation status. IHC was performed in 36 cases: 19 periocular and 17 extraocular (10 associated with Muir‐Torre syndrome—MTS). EGFR IHC was scored for percentage of positive cells (< 5%, 5–25%, 26–50%, > 50%) and intensity (+1 = low , +2 = moderate , +3 = high ). Extraocular carcinomas showed markedly increased levels of EGFR when compared to periocular carcinoma cases, both in terms of distribution (88% were > 25% of tumor cells vs. 16%) and intensity (77% were 2+ or 3+ vs. 21%) (p < 0.001). Among extraocular cases, there was significantly lower EGFR expression in MTS‐related cases (p < 0.05). No EGFR mutations were identified. Our results underscore the divergent mechanisms underlying the tumorigenesis of periocular and extraocular sebaceous carcinoma and suggest an association between aggressive behavior and increased EGFR expression in extraocular sebaceous carcinoma. Ivan D, Prieto VG, Esmaeli B, Wistuba II, Tang X, Lazar AJF. Epidermal growth factor receptor (EGFR) expression in periocular and extraocular sebaceous carcinomas.     

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.     

Epidermal interleukin 1 alpha functional activity and interleukin 8 immunoreactivity are increased in patients with cutaneous T-cell lymphoma

Previous studies have suggested that epidermal-derived interleukin-1 is involved in the pathogenesis of cutaneous T-cell lymphoma (CTCL); however, the findings are conflicting and studies that combine immunohistochemistry and functional activity have not been performed. We investigated the interleukin-1 level in epidermis of patients with cutaneous T-cell lymphoma using both immunohistochemistry, enzyme-linked immunosorbent assays, and the thymocyte co-stimulation assay. Using supernatants obtained from epidermal cell cultures, we found a significant but small increase of interleukin 1 alpha protein release from involved CTCL epidermis compared to normal epidermis from healthy individuals. Both keratinocytes and leukocytes could release interleukin-1 alpha, but the majority was derived from the keratinocytes. Interleukin-1 beta protein was not detectable. In the thymocyte assay, interleukin-1 alpha was found to be biologically active. When lymphokines derived from a T-cell clone obtained from involved CTCL skin were co-cultured with epidermal cells, an enhanced release of epidermal interleukin-1 alpha could be demonstrated. Because interleukin 1 alpha was increased, we investigated the presence of interleukin 1-inducible keratinocyte-derived interleukin 8 and found it increased in CTCL epidermis compared to normal epidermis from healthy individuals. This study demonstrated an elevated epidermal IL-1 alpha level and IL-8 immunoreactivity in CTCL epidermis, which suggests that this elevated level is induced by lymphokines released from activated T cells.     

Trimethylmethoxyphenyl-retinoic acid (Ro 10-1670) and lymphocyte DNA synthesis activity in vitro

Trimethylmethoxyphenyl-retinoic acid (TMMPRA), the main active therapeutic form of etretinate, was used for in vitro studies on human lymphocytes. Lymphocyte DNA synthesis remained unchanged when TMMPRA was added to the cultures at a concentration range of 1.25-25 micrograms/ml culture medium. No modulatory activity of the drug on DNA synthesis was seen on cultured lymphocytes stimulated by lectins (phytohemagglutinin, pokeweed mitogen, concanavalin A) or by a mitogen selective for a T lymphocyte subpopulation, phorbol myristate acetate. Concanavalin A induced T suppressor cell activity tested toward the proliferative response to allogeneic stimuli was lowered by TMMPRA. The results indicated that TMMPRA alone or in the presence of lectins or phorbol myristate acetate did not inhibit or stimulate DNA synthesis activity in vitro but that the drug could lower T suppressor cell activity.     

Epidermal tight junctions: ZO-1 and occludin are expressed in mature, developing, and affected skin and in vitro differentiating keratinocytes.

This study demonstrates the presence of tight junction antigens in adult and developing human epidermis. Indirect immunofluorescence labeling and immunoelectron microscopy with antibodies to ZO-1 and occludin localized tight junction components ZO-1 and occludin to a narrow zone of the granular cells of adult epidermis. Double immunolabeling for tight junction components with adherens junction or desmosome proteins suggested that occludin is more specific for tight junctions than ZO-1, which may also be associated with adherens junctions. In developing skin, tight junctions interconnected the peridermal cells, and after the fetal stratification localized to the granular cell layer. Immunolabeling of psoriasis, lichen planus, and ichthyosis vulgaris, representing aberrant differentiation of the epidermis, showed that these conditions were associated with relocation of ZO-1 and occludin to the spinous cells. Cultures of epidermal keratinocytes, which offer a useful model for the formation of cellular contacts, revealed that tight junction components, ZO-1 and occludin, displayed a marked degree of colocalization relatively late during the process when the fusion zone had assumed a linear appearance. This suggests that the formation of adherens junctions and desmosomes precedes that of tight junctions. We speculate that the epidermal barrier, isolating the human body from the external environment, is in part formed by tight junctions of stratum granulosum.     

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.