Identification of novel genes for secreted and membrane-anchored proteins in human keratinocytes.

BACKGROUND: Both intercellular and intracellular signals are transduced primarily by interactions of secreted and/or membrane-anchored polypeptides, and they play a pivotal role in regulating proliferation, differentiation and apoptosis of keratinocytes within the epidermis. Despite recent identification of these polypeptides, it is likely that several important molecules remain undisclosed. OBJECTIVES: To identify novel genes encoding secreted or membrane-anchored polypeptides expressed by human keratinocytes. METHODS: We employed a signal sequence (SS) trap of a 5'-end-enriched cDNA library prepared from primary cultured human keratinocytes. Gene expression analysis was performed using Northern blotting. R Screening of 4018 cDNA clones yielded 82 positive clones (57 independent genes), most of which encoded SSs in their N-termini. Most of the positive clones were known genes registered in the GenBank database. Seven genes were identified in the EST database, four of which encoded novel membrane-anchored polypeptides with features of type I transmembrane proteins; the other three genes encoded novel non-type I transmembrane polypeptides. These EST genes were expressed differentially by keratinocytes subjected to low vs. high calcium concentrations and by basal vs. squamous cell carcinomas. CONCLUSIONS: Using the SS trap, we isolated many genes known to be involved in constituting epidermal structures and others that had not previously been associated with keratinocytes. In addition, we identified novel genes (EST genes) that differ in kinetics of gene expression in keratinocyte differentiation. Our results validate the effective use of this SS trap method for identifying secreted and membrane-anchored polypeptides expressed by human keratinocytes. The identification will better illuminate the molecular mechanisms responsible for co-ordinated regulation of epidermal homeostasis.

Expression of NF-kappaB in epidermis and the relationship between NF-kappaB activation and inhibition of keratinocyte growth.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a transcription factor involved in a number of signalling pathways in many cell types. NF-kappaB in mice has been implicated as an important regulator of keratinocyte proliferation and differentiation. OBJECTIVES: To evaluate the role of NF-kappaB in keratinocyte growth in human beings, we examined its expression in keratinocytes both in culture and in situ, and studied the relationship between NF-kappaB activation and the inhibition of keratinocyte proliferation induced by known modulators of keratinocyte growth. METHODS: The expression of subunits of the NF-kappaB family was examined in human skin, primary cultured keratinocytes and an immortalized keratinocyte line by immunohistochemistry and reverse transcriptase-polymerase chain reaction analysis. NF-kB activation was examined in keratinocytes treated with various modulating agents by electrophoretic mobility shift assay (for DNA-binding activity) and by immunocytochemistry (nuclear translocation). The proliferative capacity of treated keratinocytes was also examined by 3H-thymidine incorporation, cell cycle analysis, and expression of Ki-67, a nuclear marker for cell proliferation. The involvement of NF-kappaB was assessed using sodium salicylate, which inhibits NF-kappaB activation. RESULTS: The NF-kappaB subunits, p50, p65, RelB, and c-Rel (but not p52), were detected in keratinocytes and in normal epidermis at mRNA and protein levels. The four subunits were expressed in a cytoplasmic (rather than a nuclear) pattern in both basal and suprabasal keratinocytes. Phorbol myristate acetate (PMA), tumour necrosis factor alpha, and interferon gamma each activated NF-kappaB and inhibited keratinocyte proliferation. Lipopolysaccharide and dexamethasone did not activate NF-kappaB and had the least effect on proliferation. Finally, a high concentration of calcium (Ca2+) and retinoic acid each failed to activate NF-kappaB, but were potent inhibitors of keratinocyte proliferation, respectively. PMA-induced cell cycle arrest of keratinocytes was blocked by pretreatment with sodium salicylate. CONCLUSIONS: NF-kappaB is constitutively expressed in a resting state in both human cultured keratinocytes and the epidermis. Activation of NF-kappaB is required for PMA-induced keratinocyte growth arrest.

Activation of HIV in human skin by ultraviolet B radiation and its inhibition by NFkappaB blocking agents.

To determine whether ultraviolet B (UVB) irradiation leads to activation of HIV in human skin, we conducted prospective and controlled studies in two academic medical centers in Texas from July 1995 to April 1999. HIV-positive patients with UV-treatable skin diseases were enrolled at each center, 18 subjects at one and 16 at the other. In one center, specimens from lesional and nonlesional skin biopsies were taken before and after sham- or UVB-irradiation administered in vivo or in vitro. In the other center, UVB phototherapy was administered three times weekly and specimens from skin biopsies were taken before and after 2 weeks (six treatments). Cutaneous HIV load was assessed using reverse transcriptase-polymerase chain reaction and reverse transcriptase-polymerase chain reaction in situ hybridization. UVB irradiation led to a 6-10-fold increase in the number of HIV in skin. To ascertain a role for nuclear factor kappa B (NFkappaB) in UVB-inducible HIV activation, two types of blockers, NFkappaB oligonucleotide decoy and sodium salicylate, were tested; each inhibited UVB-inducible HIV activation in skin partially. We conclude that UVB irradiation leads to increased numbers of HIV in human skin via processes that include release of cytoplasmic NFkappaB.

FRAP DNA-dependent protein kinase mediates a late signal transduced from ultraviolet-induced DNA damage.

Ultraviolet radiation induces signal transduction at both early (<6 h) and late (>6 h) times after exposure. The inflammatory and immunosuppressive cytokine tumor necrosis factor alpha is induced at late times, and is induced by ultraviolet-induced DNA damage, as defects in DNA repair increase, and enhanced photoproduct repair reduces, tumor necrosis factor alpha expression. Here we show that late tumor necrosis factor alpha gene expression is sensitive to rapamycin, implicating FKBP12-rapamycin-associated protein, a member of the DNA protein kinase family, as a signal transducer of ultraviolet-induced DNA damage. FKBP12-rapamycin-associated protein was localized in the nucleus of keratinocytes and its level was increased following ultraviolet irradiation. Immuno- precipitated FKBP12-rapamycin-associated protein was stimulated by ultraviolet-irradiated DNA to phosphorylate p53 in vitro, and in vivo rapamycin reduced ultraviolet induction of p53 by 20%. Rapamycin further inhibited the ultraviolet-induced phosphorylation of the FKBP12-rapamycin-associated protein downstream target kinase p70S6K. In mice, topical application of rapamycin before ultraviolet exposure protected against suppression of the contact hypersensitivity that is a hallmark of ultraviolet-induced cytokine gene expression. These results demonstrate that the FKBP12-rapamycin-associated DNA protein kinase transduces the signal of ultraviolet-induced DNA damage into production of immunosuppressive cytokines at late times after ultraviolet irradiation.

Molecular identification of metastatic cancer to the skin using laser capture microdissection: a case report.

BACKGROUND: In the current study the authors report a 57-year-old woman with a scalp tumor and cervical lymphadenopathy who had a previously resected duodenal carcinoid. Histologic and immunophenotypic characteristics of the duodenal carcinoid differed from those of the scalp and cervical lymph node tumors, prompting the use of molecular methodologies to make the diagnosis. METHODS: Paraffin embedded tissues from the duodenal carcinoid, scalp, and lymph node tumors were dissected using microscopic visualization and laser capture microdissection. DNA was extracted and polymerase chain reaction (PCR) was performed to evaluate loss of heterozygosity and microsatellite alterations using primers flanking 22 polymorphic microsatellite markers from 9 chromosomal regions, including genes associated with MEN-1 (11q), CDKN2 (9p), p53 (17p), and bronchial carcinoid (3p). Microdissected lymphocytes from the three tissues were used as source of constitutional DNA (controls). RESULTS: Fourteen of the 22 markers were informative (heterozygous in control lymphocytes). A marker on 3p12 showed loss of the same parental allele in the three tumors. A different marker on 3p14.2 showed an identical shifted band in the three tumors indicative of a common microsatellite alteration. CONCLUSIONS: The shared molecular abnormalities among the three tumors indicated a common clonal origin, leading to a diagnosis of primary duodenal carcinoid with clear cell metastases to the scalp and cervical lymph nodes. These findings led to radiation therapy and immunotherapy rather than chemotherapy. This case illustrates the novel application of laser capture microdissection combined with PCR-based analyses of genomic markers for the identification of the origin of metastatic disease.

Thymidine dinucleotides inhibit contact hypersensitivity and activate the gene for tumor necrosis factor alpha1.

DNA is a target for ultraviolet-B-induced inhibition of contact hypersensitivity, and small DNA fragments such as thymidine dinucleotides (pTpT) can simulate several ultraviolet-induced effects. To determine whether pTpT mimics the suppressive influence of ultraviolet-B on contact hypersensitivity, we compared the effects of topical application of pTpT with those of ultraviolet-B irradiation on C57BL/6 mice sensitized to dinitrofluorobenzene. Mice pretreated with pTpT or ultraviolet-B irradiation showed markedly suppressed ear swelling responses to dinitrofluorobenzene challenge. Because tumor necrosis factor alpha mediates ultraviolet-B-induced suppression of contact hypersensitivity, and because pTpT exerts many ultraviolet-mimetic effects by augmenting mRNA and protein levels of effector molecules, we asked if pTpT mimics ultraviolet-B's upregulatory influence on tumor necrosis factor alpha expression. Using transgenic mice carrying a chloramphenicol acetyl transferase reporter linked to the tumor necrosis factor alpha promoter, we examined effects of ultraviolet-B irradiation versus intradermal injection of pTpT on tumor necrosis factor alpha gene transcription. Both treatments induced cutaneous chloramphenicol acetyl transferase activity. Ultra- violet-B or pTpT treatment of cultured dermal fibroblasts from these mice also stimulated chloramphenicol acetyl transferase activity. To determine whether human cells responded similarly, a well- differentiated ultraviolet-responsive human squamous cell carcinoma line was treated with pTpT. pTpT increased tumor necrosis factor alpha mRNA expression and protein secretion in a dose-dependent manner. Our findings expand the spectrum of ultraviolet effects mimicked by pTpT to include inhibition of contact hypersensitivity and activation of the tumor necrosis factor alpha gene. These results support the hypothesis that DNA photoproducts and/or their repair intermediates trigger many of the biologic consequences of ultraviolet irradiation.

HIV-related eosinophilic folliculitis: a panel discussion.

Eosinophilic folliculitis is a common cause of morbidity in patients infected with the human immunodeficiency virus (HIV) and a marker of the acquired immunodeficiency syndrome (AIDS). No causative organism has yet been identified, although an aberrant Th2-type immune response to a follicular antigen appears relevant to pathogenesis. A variety of treatments have been reported to minimize the inflammatory component, relieve the concomitant pruritus, and/or favorably alter the cutaneous microenvironment.

Ultraviolet therapy of HIV-infected individuals: a panel discussion.

Patients infected with the human immunodeficiency virus (HIV) frequently develop skin diseases that are responsive to ultraviolet (UV) radiation. Studies on the effects of UV on HIV and on the immune system in vitro and in transgenic animals have raised questions regarding the safety of UV exposure in these patients. In this article, invited experts address issues concerning the safety of ultraviolet therapy in HIV-infected patients by discussing their clinical and/or research experience.

Adenovirus-mediated blockade of lymphotoxin-beta inhibits the induction of contact sensitivity in mice.

Lymphotoxin-beta is a newly recognized member of the tumor necrosis factor ligand family. Recent studies have suggested a role for this cytokine in delayed-type hypersensitivity responses. To determine whether lymphotoxin-beta contributes to the development of contact sensitivity, we utilized an inhibitor protein that can effectively block binding of lymphotoxin-beta to its receptor. An adenoviral vector was created that encodes for a lymphotoxin-beta inhibitor protein consisting of the extracellular domain of the lymphotoxin-beta receptor fused to IgG heavy chain. Intravenous injection of the recombinant virus into BALB/c mice yielded plasma levels of inhibitor protein > 500 micrograms that persisted for 1 week. Mice treated in this manner were compared with control animals injected with adenovirus encoding beta-galactosidase, with respect to their ability to mount contact sensitivity responses to epicutaneously applied dinitro-fluorobenzene. Mice transduced with the lymphotoxin-beta inhibitor prior to the induction of contact sensitivity showed significantly suppressed ear swelling responses. By contrast, mice treated with the lymphotoxin-beta inhibitor prior to the elicitation of contact sensitivity showed no change in ear swelling responses in comparison to controls. These findings indicate that lymphotoxin-beta plays an important role in the afferent phase of the contact sensitivity response.

Langerhans' cell histiocytosis in adults.

Three cases of Langerhans' cell histiocytosis with unusual clinical and histopathologic features are described. The first two cases illustrate diagnostic pitfalls that underscore the importance of considering Langerhans' cell histiocytosis in the differential diagnosis of purpuric papular eruptions of the scalp and intertriginous areas, particularly in association with hypothalamic, pituitary, or liver disease. The third case is the first report of Langerhans' cell histiocytosis presenting as a vesicular eruption.

Is phototherapy safe for HIV-infected individuals?

Patients infected with human immunodeficiency virus (HIV) have a high prevalence of UV radiation-responsive skin diseases including psoriasis, pruritus, eosinophilic folliculitis and eczemas. On the other hand, UV has been shown to suppress T cell-mediated immune responses and to induce activation and replication of HIV. These developments have prompted clinicians and investigators to question whether phototherapy is safe for HIV-infected individuals. We have reviewed these issues and hereby provide a summary and critique of relevant laboratory and clinical evidence.

Basic science answers to questions in clinical contact dermatitis.

Since the first clear demonstration in the 1970s of an immunologic linkage between UV-light exposure of laboratory mice and the development of skin cancers in these animals, much progress has been achieved in our understanding of the effects of UV light on the immune system. At the cellular level, two overlapping mechanisms have been expounded to explain UVB-induced immunosuppression: (1) altered cutaneous antigen presentation due to perturbations in Langerhans' cells and/or the recruitment of macrophage-like cells into skin, and (2) the initiated or up-regulated secretion by skin cells (particularly but not limited to keratinocytes) of soluble mediators of immunosuppression (alpha-melanocyte-stimulating hormone, interleukin 10, tumor necrosis factor alpha). An end result common to both mechanisms is inhibition of Th1-type immunity in favor of preserved or heightened Th2-type immunity. At the molecular level, two targets of UVB radiation are the subject of intense investigation. Circumstantial evidence has accumulated to implicate a direct effect of UVB on DNA (in antigen-presenting cells and/or cytokine-producing cells) as the triggering cause of UVB-evoked immunosuppression. On the other hand, UVB-triggered changes in cell membrane molecules (epidermal growth factor receptor, intercellular adhesion molecule 1, B7) have been correlated with temporally distal UV effects, including the activation of gene transcription pathways and the down-regulation of antigen presentation independent of DNA damage.

Interleukin-15 expression by human endothelial cells: up-regulation by ultraviolet B and psoralen plus ultraviolet A treatment.

The purposes of the present study were to determine whether endothelial cells express IL-15 and to evaluate effects of ultraviolet B (UVB) and 8-methoxypsoralens plus UVA (PUVA) on such expression. Cultured human endothelial cells derived from dermis or umbilical veins were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analyses for the detection of IL-15 mRNA and protein, respectively. Both dermal and umbilical vein endothelial cells were shown to express IL-15 mRNA and protein, and these markers were upregulated following UVB or PUVA treatment (but not by UVA or 8-methoxypsoralens alone). Also using RT-PCR, dermal and umbilical vein endothelial cells were shown to express IL-2R gamma c mRNA. These results expand the sources of IL-15 in skin to include keratinocytes, dermal fibroblasts, and now endothelial cells. That IL-15 from all three skin cells can be upregulated by UV treatment suggests a role for this cytokine in photosensitive disorders. Finally, the possibility of an autocrine effect of IL-15 on endothelial cells is raised by the expression of IL-2R gamma c in these cells.

Ultraviolet B radiation up-regulates the expression of IL-15 in human skin.

Ultraviolet B (UVB) radiation is a potent modulator of skin-related immune responses, particularly those involving the synthesis and the secretion of cytokines. The discovery of a new T cell mitogen, IL-15, prompted use to investigate its expression in skin and to examine the effects of UVB radiation on such expression. RNA from unirradiated and UVB-irradiated epidermal and dermal sheets derived from human foreskin as well as from unirradiated and UVB-irradiated skin cell populations were assayed for IL-15 expression by semiquantitative RT-PCR. Constitutive levels of IL-15 mRNA were detected in dermal sheets, but not in epidermal sheets. Following UVB treatment, IL-15 mRNA was induced in epidermal sheets and enhanced in dermal sheets. UVB-inducible epidermal expression of IL-15 mRNA was traced to HLA-DR- cells (presumably keratinocytes) and not to HLA-DR+ cells (Langerhans cells). Cultured keratinocytes and dermal fibroblasts displayed basal levels of IL-15 mRNA that were also up-regulated following UVB exposure. Immunoblot analysis revealed secretion of IL-15 protein by keratinocytes that was enhanced following UVB treatment. These results constitute the first report of IL-15 mRNA expression and protein production in human skin. In addition to expanding the known influence of UVB radiation on the capacity of keratinocytes and dermal fibroblasts to express immunomodulatory cytokines, these findings suggest a new mechanism by which UVB can promote Ag-independent T cell responses via elaboration of IL-15.

Expression of the tumor necrosis factor gene by dermal fibroblasts in response to ultraviolet irradiation or lipopolysaccharide.

To examine the effects of different wavelengths of ultraviolet (UV) radiation on tumor necrosis factor (TNF) production, we took advantage of mice carrying a chloramphenicol acetyl transferase (CAT) reporter transgene bearing the entire TNF promoter and 3'-untranslated region. Aside from constitutive expression in the thymus, CAT activity was detected only in locally UVB- or UVC-irradiated skin. After UVB irradiation, markedly greater amounts of CAT activity were traced to the dermis rather than the epidermis; by contrast, almost all CAT activity was localized to the epidermis after UVC irradiation. Fibroblasts have not been shown previously to express the TNF gene, i.e., the TNF gene is highly methylated and inaccessible to exogenous modulation in 3T3 fibroblasts. However, the present report reveals that cultured dermal fibroblasts are capable of producing both CAT and TNF in response to treatment in vitro with either UVB irradiation, UVC irradiation, or lipopolysaccharide. These findings indicate that dermal fibroblasts may serve not only as a target for but also as a source of TNF.

A comparison of the efficacy and safety of Jessner's solution and 35% trichloroacetic acid vs 5% fluorouracil in the treatment of widespread facial actinic keratoses.

BACKGROUND AND DESIGN: We compared the efficacy and safety of a medium-depth chemical peel with those of the standard regimen of topical fluorouracil in the treatment of widespread facial actinic keratoses (AK). Fifteen patients with severe facial actinic damage and similar numbers of AK on both sides of the face were treated on the left side with a single application of Jessner's solution and 35% trichloroacetic acid and on the right side with twice daily applications of 5% fluorouracil cream for 3 weeks. Evaluations were conducted before treatment and at 1, 6, and 12 months after treatment. Visible AK were counted, random skin biopsies performed, adverse effects monitored, and patients questioned about preference and perception of efficacy. RESULTS: Both treatments reduced the number of visible AK by 75% and produced equivalent reductions in keratinocyte atypia, hyperkeratosis, parakeratosis, and inflammation, with no significant alteration of preexisting solar elastosis and telangiectasia. Except for erythema that lasted 3 months in one patient, no untoward side effects were observed with the chemical peel. The majority of patients preferred the peel over fluorouracil because of the single application and less morbidity. CONCLUSION: The medium-depth peel induced by Jessner's solution and 35% trichloroacetic acid is a useful alternative therapeutic option for widespread facial AK, particularly for poorly compliant patients, because it equals fluorouracil in efficacy while being superior in terms of the convenience of a single application with little associated morbidity.

In vivo evidence that ultraviolet B-induced suppression of allergic contact sensitivity is associated with functional inactivation of Th1 cells.

Having previously shown in vitro that ultraviolet B (UVB)-treated Langerhans cells (LC) can induce antigen-specific proliferative unresponsiveness and tolerance in Th1 (but not Th2) cells, we wanted to determine whether cutaneous exposure to UVB radiation prior to hapten-painting would produce similar differential effects in hapten-reactive Th1 and Th2 T cells in vivo. C3H/HeN mice were exposed to UVB (200 J/m2/day) through abdominal skin on days -4 through -1, followed by painting dinitrofluorobenzene (DNFB) on the irradiated skin on days -1 and 0. Induction of allergic contact sensitivity (CS) was assayed by ear swelling responses to DNFB and by the proliferative responses of draining lymph node cells (LNC) to DNBS. UVB-irradiated and hapten-painted mice (in comparison to a control panel of unirradiated and DNFB-painted mice) displayed suppressed ear swelling responses to DNFB and suppressed LNC proliferation to DNBS. However, LNC from either panel of mice proliferated well in response to exogenous interleukin 2 (IL-2). To examine effects on Th1 and Th2 cells, lymphokines were assayed from supernatants of DNBS-stimulated LNC. The Th1-associated lymphokines, interferon-gamma and IL-2, were the predominant cytokines detected in samples taken from unirradiated and DNFB-painted mice. Both of these cytokines were reduced markedly in samples from UVB-treated and DNFB-painted mice. Except for miniscule amounts of IL-10, no Th2-associated lymphokines were detected in LNC supernatants from either panel of mice. These results suggest that UVB-induced suppression of CS in vivo is associated with functional inactivation of hapten-reactive Th1 cells.

A spectrum of inflammatory metastasis to skin via lymphatics: three cases of carcinoma erysipeloides.

We report a case in which carcinoma erysipeloides was the first sign of the primary malignancy in a patient with a rare form of prostate carcinoma (mixed adenosquamous type) and two cases in which carcinoma erysipeloides was a marker of tumor recurrence in two patients with breast carcinoma. The value of recognizing the distinctive inflammatory manifestation of carcinoma erysipeloides and the significance of dermal lymphatic involvement in this form of skin metastasis are discussed.

Profiles of cytokine mRNA expressed by dendritic epidermal T cells in mice.

The epidermis of mice contains, in addition to Langerhans cells, a second dendritic population that is Thy-1+/CD3+/CD4-/CD8-/T-cell receptor-V gamma 3/V delta 1+. These dendritic epidermal T cells (DETC) are now thought to comprise one element in the family of epithelial tissue-resident gamma delta T cells. In the present study, DETCs were examined for their expression of mRNA for cytokines, using a reverse transcription-polymerase chain reaction. Freshly isolated Thy-1+ epidermal cells constitutively expressed mRNA for gamma-interferon, but not IL-2. Within 24 h after stimulation with Con A, these cells then expressed mRNA for gamma-interferon and IL-2, but not IL-4. The rapid onset of expression of mRNA for IL-2 occurred exclusively within the Thy-1+ population, and in a Con A-dependent fashion. When freshly isolated epidermal cells were first stimulated with Con A and then expanded in bulk with rIL-2 for 20-24 d, cells expressing IL-4 mRNA then emerged, upon secondary stimulation with Con A. These "short-term" DETC lines also expressed mRNA for IL-2, interferon-gamma, IL-1 alpha, IL-3, IL-6, IL-7, tumor necrosis factor alpha and beta, and granulocyte macrophage-colony stimulating factor. Interestingly, mRNA for IL-4 and IL-6 was no longer detected in long-term (> 1 year) DETC lines 7-17 and 12-12. In addition, one line (7-17) maintained IL-3 mRNA expression, whereas another (12-12) had lost this capacity. These results emphasize the concept that, as resident cells in epidermis, DETCs exhibit several different immunorelevant activities, and the heterogeneity in cytokine mRNA profiles suggests that DETCs may divide into functional subsets.