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Side population (SP) cells are identified as cells capable of excluding the fluorescent Hoechst dye and anticancer drugs, and it represents hematopoietic stem cells and chemoresistant cells from several solid tumors. In this study, we confirmed the presence of SP cells in tumors from melanoma patients. Melanoma SP cells overexpressed ATP-binding-cassette (ABC) transporters, ABCB1 and ABCB5. We generated a direct in vivo xenograft model, and demonstrated that SP cells were resistant to paclitaxel, a substrate of ABCB1, both in vitro and in vivo. However, melanoma SP cells were also resistant to temozolomide, which is not a substrate for ABC transporters, through IL-8 upregulation. In addition, gene profiling studies identified three signaling pathways (NF-κB, α6-β4-integrin, and IL-1) as differentially upregulated in melanoma SP cells, and there was a significant increase of PCDHB11 and decrease of FUK and TBX2 in these cells. Therefore, we provide evidence that SP is an enriched source of chemoresistant cells in human melanomas, and suggest that the selected genes and signaling pathways of SP cells may be a potential target for effective melanoma therapies. To our knowledge, this is a previously unreported study to isolate SP cells from melanoma patients and to investigate the gene expression profiling of these cells.

Human adipose tissue-derived mesenchymal stem cells (ASCs) stimulate regeneration of injured tissues by secretion of various cytokines and chemokines. Wound healing is mediated by multiple steps including inflammation, epithelialization, neoangiogenesis, and proliferation. To explore the paracrine functions of ASCs on regeneration of injured tissues, cells were treated with tumor necrosis factor-α (TNF-α), a key inflammatory cytokine, and the effects of TNF-α-conditioned medium (CM) on tissue regeneration were determined using a rat excisional wound model. We demonstrated that TNF-α CM accelerated wound closure, angiogenesis, proliferation, and infiltration of immune cells into the cutaneous wound in vivo. To assess the role of proinflammatory cytokines IL-6 and IL-8, which are included in TNF-α CM, IL-6 and IL-8 were depleted from TNF-α CM using immunoprecipitation. Depletion of IL-6 or IL-8 largely attenuated TNF-α CM-stimulated wound closure, angiogenesis, proliferation, and infiltration of immune cells. These results suggest that TNF-α-activated ASCs accelerate cutaneous wound healing through paracrine mechanisms involving IL-6 and IL-8.

Xeroderma pigmentosum is a rare, autosomal recessive disease in which patients develop excessive solar damage at an early age and have a 1000-fold increased risk of developing cutaneous neoplasms. Xeroderma pigmentosum can be classified into seven complementation groups (A-G) with defects in different DNA nucleotide excision repair genes. Xeroderma pigmentosum patients also have impaired immune function including reduced natural killer cell activity and impaired induction of interferon-gamma. We hypothesized that altered cytokine induction may contribute to the immune defect in xeroderma pigmentosum patients. We examined cytokine mRNA expression after ultraviolet B irradiation using reverse transcriptase polymerase chain reaction in fibroblasts derived from five xeroderma pigmentosum patients in complementation groups A, C, and D and in complemented XP-A and XP-D cells. Cytokines interleukin-1beta and interleukin-6 displayed impaired ultraviolet B induction whereas interleukin-8 had normal induction in the xeroderma pigmentosum fibroblasts. Stable complementation of XP-A and XP-D cell lines increased ultraviolet-B-induced interleukin-1beta and interleukin-6 expression. These results demonstrate a deficient response of xeroderma pigmentosum fibroblasts to ultraviolet B in terms of cytokine interleukin-1beta and interleukin-6 induction but normal interleukin-8 induction and exhibit a role for DNA repair in cytokine induction.

Psoriasis is a chronic inflammatory skin disease in which epidermal hyperplasia results from the release of cytokines by infiltrating type 1 T cells. Up- regulation of endogenous interleukin-10 controls type 1 skin responses in animal models; however, interleukin-10 production is low in psoriatic lesions. Consistent with an important role of interleukin-10 in psoriasis, we and colleagues have recently demonstrated clinical efficacy of subcutaneous administration of recombinant interleukin-10 to affected patients. Here, we studied the effects of interleukin-10 on disease-related inflammatory pathways. Patients were treated with recombinant interleukin-10 over 6 wk in an open-label phase II clinical trial. Tissue was obtained before and after therapy and examined by histology/immunohistochemistry, in situ hybridization, and quantitative real-time reverse transcription-polymerase chain reaction. Ten of 14 patients showed a marked reduction of the clinical disease activity. The clinical response was associated with a significant decrease of cutaneous T cell infiltration and the lesional expression of type 1 cytokines interferon-gamma and tumor necrosis factor-alpha. Interleukin-10 inhibited the epidermal interleukin-8 pathway by downregulating the expression of interleukin-8, its receptor CXCR2, and its inducer interleukin-17, and partially reversed the aberrant keratinocyte maturation defining psoriatic epidermal pathology. Remarkably, there was evidence that genetic factors are involved in the response to interleukin-10 as individual variations in the downregulation of tumor necrosis factor-alpha were related to the presence of polymorphisms in the tumor necrosis factor-alpha promoter. These data suggest that excessive production of type 1 cytokines in human skin disease can be counter-regulated by the administration of recombinant interleukin-10. Genotypic analysis may help to identify patients that will preferentially respond to interleukin-10 therapy.

The in vivo response to ultraviolet B (UVB) radiation in skin is characterized by the accumulation of both mononuclear and polymorphonuclear cells within the dermis and an induction of vascular endothelial adhesion molecules. Epidermal production of cytokines (IL-8 and TNF-alpha) has been strongly implicated in the development of UVB-induced inflammation. In the current study, we examined the time course of IL-8 and TNF-alpha mRNA and protein expression in the epidermis over a 24-h period after in vivo UVB irradiation. Also, the induction of adhesion molecule expression and the accumulation of neutrophils within the dermis were followed. We found constitutive expression of both cytokines (mRNA and protein) in the epidermis of unirradiated skin. IL-8 was rapidly upregulated after irradiation and mRNA and protein increased at 4 h, reaching a maximum between 8 and 24 h. TNF-alpha mRNA and protein was minimally increased by 8 h after UVB irradiation and reached a maximum by 24 h. No significant alteration in ICAM-1 or VCAM-1 expression was observed. E-selectin expression, which was absent from control samples, was increased from 4 h onward and also reached a maximum at 24 h, coinciding with peak neutrophil accumulation. A strong correlation (r = 0.96) was found between number of E-selectin-positive vessels and numbers of infiltrating neutrophils at this time. Moreover, because E-selectin expression was increased before any apparent increase in TNF-alpha protein (4 h), TNF-alpha does not appear to be involved in the early induction of the adhesion molecule, but cytokines such as TNF-alpha and IL-8 may act subsequently to augment the inflammatory response.

In response to exogenous stimuli such as phorbol-12-myristate 13-acetate, ultraviolet B radiation, and lipopolysaccharide, human keratinocytes produce soluble mediators that are important in primary contact irritancy including cytokines that are associated with proinflammatory properties (interleukin-1 alpha [IL-1 alpha], tumor necrosis factor alpha), chemotaxis (IL-8), and growth activation (granulocyte/macrophage colony stimulating factor, IL-6, transforming growth factor alpha). We examined qualitative and quantitative changes in selected intracellular and secreted cytokines in human keratinocyte cultures in response to non-sensitizing contact irritants (croton oil, sodium lauryl sulfate, methyl salicylate, ethyl phenylpropiolate), sensitizing irritants (oxazolone, dinitrofluorobenzene), and ulcerative agents (phenol, benzalkonium chloride, chromium trioxide). The chemicals were also applied to mouse skin to assess whether the chemical-specific pattern of inflammation correlated with the in vitro production of keratinocyte-derived cytokines. Although all agents elicited neutrophils to the site of chemical application, time dependent and chemical-specific patterns of inflammation could be detected. Sodium lauryl sulfate, phenol, and croton oil induced increases in IL-8 production at non-cytotoxic concentrations in semi-confluent human keratinocyte cultures. Phenol and croton oil stimulated tumor necrosis factor alpha production, whereas croton oil was the only agent found to induce granulocyte/macrophage colony-stimulating factor production. Croton oil, phenol, benzalkonium chloride, and dinitrofluorobenzene induced the intracellular production of IL-1 alpha without a concomitant release into the medium. The release of cytokines occurred in parallel with a relative increase in cytokine-specific mRNA transcripts. Studies using neutralizing antibodies to tumor necrosis factor alpha and IL-1 alpha demonstrated that IL-8 induction by croton oil and phenol occurred directly rather than through autocrine circuits. These data suggest that a given pattern of cytokine production is chemical-specific and may predict the contribution of keratinocytes to skin inflammation.

Interleukin (IL)-8 and gro peptides are members of the intercrine-alpha family of chemotaxins known to be present in biologically active form in psoriasis lesions. However, the relative contribution of the three different gro genes to the expression of this material is unknown, as is the stimulus for gro overexpression in psoriatic lesions. To address these questions, Northern blot and semiquantitative polymerase chain reaction analysis were performed on RNA extracted from keratome biopsies of normal skin, untreated plaques of psoriasis, or plaques treated for 1 week with low-dose cyclosporin A (CsA). Northern blot analysis revealed a significant correlation between gro and IL-8 mRNA levels in psoriasis lesions from 26 different individuals (r = 0.61, p = 0.0009), and overexpression of gro was markedly reduced by CsA prior to detectable clinical improvement (79.3%, p = 0.01, n = 22). To determine which form(s) of gro were overexpressed in psoriatic lesions, total keratome RNA (1 microgram) was analyzed by semiquantitative reverse transcription-polymerase chain reaction (SQRT-PCR). In five patients known to markedly overexpress gro and IL-8 mRNAs by Northern blotting, gro-alpha was approximately six times more abundant than gro-beta, and 25 times more abundant than gro-gamma. In cultured human keratinocytes, all three forms of gro mRNA were increased by IL-1 alpha or by interferon (IFN)-gamma plus tumor necrosis factor (TNF)-alpha. However, in contrast to the situation in vivo, CsA had no inhibitory effect on cytokine-stimulated gro expression in cultured keratinocytes. Taken together, these results demonstrate that the gro-alpha gene is selectively overexpressed in psoriatic lesions and strongly suggest that overexpression of gro is a keratinocyte response to activated T cells in psoriasis.

In order to better understand the factors regulating disease promotion and activity in psoriasis (PS), we searched for the in situ expression of mRNA for various cytokines in long-standing PS skin lesions. Specific hybridization with a NAP-1/IL-8 anti-sense RNA probe was keratinocyte associated and yielded strong and specific signals exclusively in the upper layers of the lesional epidermis, but not in uninvolved skin from psoriatic patients or normal skin from non-psoriatics. Interestingly, NAP-1/IL-8 transcripts were focally clustered in a spotty pattern predominantly between the tips of elongated papillae, but were absent in the lower epidermal region and the dermal compartment. We consistently failed to detect appreciable numbers of TNF-alpha and/or IL-6 mRNA-containing cells in psoriatic lesions. These results support the notion that IL-8, rather than IL-6, is an important disease-promoting cytokine in PS. In view of the known in vitro and in vivo effects of IL-8, it is conceivable that this substance greatly contributes to the major pathologic changes seen in psoriatic skin, i.e., keratinocyte hyperproliferation and leucocyte infiltration. In this case, local pharmacologic down-regulation of NAP-1/IL-8 activity could be a promising therapeutic strategy in PS.

Cytokines are (glyco)proteins that are synthesized and secreted by various cells, which bind to specific receptors on target cells and which regulate activation, proliferation, and differentiation of immune as well as non-immune cells. Keratinocytes upon injury release interleukin (IL)-1, IL-6, IL-8, colony-stimulating factors, and tumor-necrosis factor, as well as growth and suppressor factors. There is also strong evidence for a network of interacting cytokines, which has been only partially characterized so far, maintaining a proper balance. However, excessive or insufficient production of these mediators may contribute to certain disease states, particularly those with infectious and autoimmune genesis. Therefore the understanding of cytokine interactions may be helpful in elucidating the pathomechanisms of such diseases. Moreover, certain cytokines, as well as their analogues and antagonists, may prove to be of therapeutic value.

Cytokines (hormone-like polypeptide mediators) play a major role in inflammatory and immunoregulatory responses. Skin, and particularly keratinocytes in the skin, represent a potent source for many cytokines, including interleukins 1, 6, 8, and the hemopoietic colony stimulating factors. Cytokines initiate their biologic action by interacting with target cells bearing cytokine receptors and then initiating a cascade of cellular interactions. Certain inflammatory skin diseases have been associated with overproduction of cytokines, alteration in cytokine receptors, or dysregulation of cytokines. While data is still quite preliminary, it is likely that cytokines contribute to the pathogenesis of many inflammatory skin diseases.