Human keratinocytes are a source for tumor necrosis factor alpha: evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light.

Tumor necrosis factor alpha (TNF-alpha), in addition to being cytotoxic for certain tumor cells, has turned out as a multifunctional cytokine that is involved in the regulation of immunity and inflammation. Since human keratinocytes have been demonstrated to be a potent source of various cytokines, it was investigated whether epidermal cells synthesize and release TNF-alpha. Supernatants derived from normal human keratinocytes (HNK) and human epidermoid carcinoma cell lines (KB, A431) were tested both in a TNF-alpha-specific ELISA and a bioassay. In supernatants of untreated epidermal cells, no or minimal TNF-alpha activity was found, while after stimulation with lipopolysaccharide (LPS) or ultraviolet (UV) light, significant amounts were detected. Western blot analysis using an antibody directed against human TNF-alpha revealed a molecular mass of 17 kD for keratinocyte-derived TNF-alpha. These biological and biochemical data were also confirmed by Northern blot analysis revealing mRNA specific for TNF-alpha in LPS- or ultraviolet B (UVB)-treated HNK and KB cells. In addition, increased TNF-alpha levels were detected in the serum obtained from human volunteers 12 and 24 h after a single total body UVB exposure, which caused a severe sunburn reaction. These findings indicate that keratinocytes upon stimulation are able to synthesize and release TNF-alpha, which may gain access to the circulation. Thus, TNF-alpha in concert with other epidermal cell-derived cytokines may mediate local and systemic inflammatory reactions during host defense against injurious events caused by microbial agents or UV irradiation.

Novel production of interleukin-1 receptor antagonist peptides in normal human cornea.

Interleukin-1 receptor antagonist (IL-1ra) is an important modulator of IL-1 activity in a variety of tissues. IL-1ra is differentially produced by different cell types as a 22-26-kD secreted peptide (sIL-1ra) and/or a smaller 16- or 18-kD intracellular peptide (icIL-1ra). This study was undertaken to evaluate the production of IL-1ra in the human cornea. IL-1ra mRNA can be detected in early passage human corneal epithelial cells and corneal stromal fibroblasts and is significantly enhanced by IL-1. Corneal endothelial cells do not express IL-1ra mRNA. Immunohistochemical studies of cultured corneal cells and whole human cornea demonstrate IL-1ra protein production by both the epithelial and stromal cells but not the endothelial cells. Reverse transcriptase polymerase chain reaction, ELISA, and immunoprecipitation studies indicate that corneal epithelial cells are capable of producing both icIL-1ra and sIL-1ra forms of IL-1ra whereas the corneal stromal cells produce only icIL-1ra. In addition to the larger 18-kD icIL-1ra, both corneal epithelial and stromal cells are also capable of producing a smaller recently described 16-kD icIL-1ra. Thus, the differential production of IL-1ra in the human cornea is unique; whereas both epithelial and stromal cells produce icIL-1ra (type 1 and type 2), the epithelial cells appear to also produce sIL-1ra. It is proposed that these IL-1ra proteins may play an important role in regulating IL-1-induced corneal inflammation.

Human keratinocytes are a major source of cutaneous platelet-derived growth factor.

PDGF has been implicated as one of the principal mitogens involved in cutaneous wound healing. While it has been previously reported that both platelets and monocytes are a source of PDGF in human dermal wound repair, the production of PDGF by human keratinocytes has not yet been described. In this manuscript, we report the production of PDGF by cultured human keratinocytes. Both PDGF A and B chain mRNA can be detected in cultured cells. While only PDGF-AA polypeptide is found in significant levels in keratinocyte-conditioned culture media, all three PDGF isoforms (AA, AB, and BB) are present in detergent-solubilized cell extracts. No evidence of PDGF receptor expression was observed in cultured keratinocytes when analyzed for either mRNA levels or polypeptide expression, suggesting that PDGF does not play an autocrine role in keratinocyte growth. Analysis of cryosections of human cutaneous wounds by immunostaining for PDGF showed that both PDGF A and B chain is constitutively expressed in normal epidermis, as well as in newly reconstituted wound epidermis. No evidence for PDGF receptor polypeptide expression in the epidermis was detected by immunostaining of cryosections.

Proopiomelanocortin-derived peptides are synthesized and released by human keratinocytes.

Proopiomelanocortin (POMC), the precursor for melanotropic, corticotropic, and opioid peptides such as alpha-melanocyte-stimulating hormone (alpha MSH), ACTH, and other related peptides, was originally identified as a product of the pituitary gland. However, recent evidence shows that POMC products can also be produced by nonpituitary tissues. Because keratinocytes, the major constituent of the epidermis exhibit the capacity to release a variety of proinflammatory and immunomodulatory mediators, the present study was performed to investigate whether human keratinocytes are able to produce POMC-derived peptides. Supernatants of human normal keratinocytes and an epidermal carcinoma cell line (A431) contained significant levels of immunoreactive alpha MSH and ACTH. Upon immuneprecipitation and size-exclusion chromatography, keratinocyte-derived alpha MSH exhibited a molecular mass of approximately 1 kD and was biologically active as demonstrated in a tyrosinase bioassay. Northern blot analysis revealed the expression of POMC-specific transcripts (1.3 kb) in both normal keratinocytes and A431 cells. The production of alpha MSH and ACTH could be significantly upregulated both at the protein and mRNA level upon treatment with phorbol myristate acetate, ultraviolet light, or interleukin 1. These data provide first evidence that human keratinocytes produce POMC-derived peptides such as alpha MSH and ACTH. Because POMC-derived peptides recently have been recognized as potent immunomodulatory mediators, their presence in the epidermis may have a major impact on the skin immune system.

Expression and release of interleukin-1 by different human melanoma cell lines.

The influence of immunologic parameters on the clinical course of malignant melanoma is increasingly evident. However, it is not known which factors contribute to the immunologic host reaction against malignant melanoma. Because epidermal cells and, in particular, normal as well as transformed keratinocytes recently have been demonstrated to release various immunomodulating cytokines, the capacity of melanoma cells to produce interleukin-1 (IL-1) was examined. Accordingly, supernatants derived from different melanoma cell lines contained significant levels of IL-1 activity. Upon high-performance liquid chromatography (HPLC) gel filtration, melanoma cell-derived IL-1 (MEL-IL-1) exhibited molecular weight heterogeneity, and HPLC chromatofocusing revealed major activity at pH 5.0 and minor activity at pH 7.0. A monoclonal antibody directed against monocyte-derived IL-1 blocked MEL-IL-1 activity significantly and was able to precipitate four species of biosynthetically radiolabeled MEL-IL-1 (25, 17, 6, and 4 kilodaltons), suggesting that MEL-IL-1 is identical to monocyte-derived IL-1. This was also confirmed by Northern blot analysis detecting IL-1 alpha specific mRNA in melanoma cells by hybridization with a cDNA fragment encoding for IL-1 alpha. Thus, melanoma cells, like other epidermal cells, exhibit the capacity to release the immunomodulating cytokine IL-1 and, therefore, probably have the potency to influence host defense mechanisms directed against malignant melanoma.

Antitumor effects of granulocyte-macrophage colony-stimulating factor production by melanoma cells.

The use of immunomodulating gene therapy in the treatment of malignant disease is under intensive investigation. In this study, we examined the potential of melanoma-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) to inhibit melanoma progression in a murine model. The HGH18 murine melanoma cell line was transfected with the murine GM-CSF gene in a SV40 expression vector that resulted in melanoma clones that produced varying amounts of GM-CSF. Syngeneic mice inoculated s.c. with HFH18 parental melanoma cells or HFH18 cells transfected with the GM-CSF gene n the noncoding 3'-5' orientation [HFH18/GM-CSF(-) cells] develop large tumors that reach a mean tumor volume of 3300 mm3 by day 30. In contrast, animals inoculated with two melanoma clones producing high levels of GM-CSF [HFH18/GM-CSF(++) and HFH18/GM-CSF(+ + +)] either completely reject the tumor cells or develop tumors with a mean volume of only 40 mm3. In comparison, animals inoculated with a melanoma clone producing low levels of GM-CSF [HFH18/GM-CSF(+)] develop large tumors averaging 2000 mm3, thus demonstrating a dose-response effect of tumor inhibition by melanoma-derived GM-CSF. Additionally, vaccination with irradiated GM-CSF-producing melanoma cells conferred optimal immunogenicity against a subsequent challenge with HFH18 cells. Tissue sections from excised GM-CSF-producing tumor cell inoculation sites but not from HFH18 parental or HFH18/GM-CSF(-) inoculation sites demonstrate a dense inflammatory infiltrate composed of neutrophils, tissue macrophages, and numerous CD4- and CD8-positive lymphocytes but few melanoma cells. Large numbers of dendritic cells and cells expressing the B7-2 costimulatory molecule are detected only within HFH18/GM-CSF(+ + +) melanoma inoculation sites. Our results lend further support to clinical trials of GM-CSF gene therapy in the treatment of advanced malignant melanoma, possibly by the recruitment of dendritic antigen-presenting cells.

Fever and increased serum IL-1 activity as a systemic manifestation of acute phototoxicity in New Zealand White rabbits.

Ultraviolet (UV) radiation is a significant environmental hazard for humans and animals. Although the clinical effect of an acute UV exposure such as cutaneous inflammation, malaise, somnolence, chills, and fever have been appreciated for many years, the underlying mechanisms mediating these effects are poorly understood. Since chills and fever are the most dramatic systemic sequelae after a prolonged exposure to UV, we specifically examined the effect of whole-body UV irradiation on core body temperature and serum endogenous pyrogen activity of New Zealand White rabbits, correlating this with serum interleukin 1 (IL-1) activity and alterations of serum divalent cation levels. We found that an acute dose of UV irradiation (Westinghouse FS-40 lamps, 0.2 mJ/cm2/s X 8 h) resulted in a significant increase in the core body temperature 2 h post UV (0.8 degree C), peaking 5 h post UV (1.8 degree C), and returning to normal 24 h post UV. Likewise, the sera from the UV-irradiated rabbits had significant endogenous pyrogen activity when transferred into naive recipient animals, causing an increase in core body temperature within 45 min (0.65 +/- 0.12 degree C), decreasing over the next 2 h, and returning to normal 6 h post injection. No endotoxin contamination was detected in any serum samples. This post-UV febrile response was accompanied by a prolonged increase in serum IL-1 activity (5-10 X) and a significant alteration in serum divalent cation levels, with the rabbits becoming euthermic even as the serum IL-1 levels remained elevated. These findings provide new information concerning the pathogenesis and kinetics of these systemic effects after an acute dose of UV irradiation.

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.

Melanoma-derived interleukin 6 inhibits in vivo melanoma growth.

Malignant melanomas are capable of producing a wide range of cytokines with multiple biologic functions, including interleukin 6 (IL-6). We have observed an inverse relationship between IL-6 production of three B16-derived murine melanoma cell lines (NP133, HFH18, and HFH(M)) and the tumorigenicity of these melanoma cells in syngeneic mice. To further test the effect of IL-6 on melanoma growth, a non-IL-6-producing murine B16-derived melanoma cell line (HFH18) was transfected with a murine IL-6 expression vector, resulting in stable transfectants (HFH18/IL-6(+)) that expressed significant amounts of IL-6 mRNA and secreted high levels of bioactive IL-6. Syngeneic C57BL/6 mice inoculated subcutaneously with HFH18/IL-6(+) cells developed tumors that reached a final mean diameter of less than half the size of tumors that developed in mice inoculated with either HFH18 parental or HFH18 cells transfected with the IL-6 cDNA in the non-coding 3'-5' orientation (HFH18/IL-6(-) cells). In addition, mice bearing IL-6-producing HFH18/IL-6(+) tumors survived twice as long as mice bearing HFH18 parental or HFH18/IL-6(-) tumors. The specificity of melanoma growth inhibition by IL-6 was confirmed by the reversal of the slow-growing phenotype of HFH18/IL-6(+) cells by local peritumoral administration of neutralizing alpha-murine IL-6 antibody. IL-6-producing melanoma cells exerted a growth-inhibitory effect on distant parental tumors in a dose-dependent manner. The growth of HFH18/IL-6(+) melanomas was also decreased in nude mice, suggesting that melanoma-derived IL-6 may mediate this anti-tumor effect independently of a normal host B- and T-cell immune response. Thus, melanoma-derived IL-6 exerts a significant inhibitory effect on cutaneous melanoma growth and progression. These results indicate that melanoma cytokines may have a profound effect on tumor pathogenesis.

Increased interleukin 6 production by T cells derived from patients with atopic dermatitis.

Many immunologic aspects of atopic dermatitis have been studied, but basic pathobiologic mechanisms of this disease remain unknown. In this study, we measured the production of interleukin-6 (IL-6) by peripheral blood T cells and monocytes from patients with atopic dermatitis in comparison to normal control subjects and patients with chronic psoriasis. We found that peripheral blood T cells isolated from patients with atopic dermatitis produced significantly higher levels of IL-6 (36.1 +/- 5.1 units/ml, n = 22) than T cells derived from either normal subjects (12.6 +/- 1.9 units/ml, n = 22) or patients with chronic psoriasis (26.7 +/- 4.1 units/ml, n = 7). T-cell activation was also measured in the patients with atopic dermatitis by soluble serum IL-2 receptor levels and were found to be significantly higher (623.7 +/- 8.1 units/ml, n = 8) than normal subjects (357.2 +/- 26.0 units/ml, n = 8). In contrast to the increased production of IL-6 by T cells in atopic dermatitis, there was no significant difference in the IL-6 production by peripheral blood monocytes derived from patients with atopic dermatitis compared to normal subjects. Thus, peripheral blood T cells derived from patients with AD spontaneously produce increased amounts of IL-6 compared to T cells from normal subjects, which may reflect the increased activation state of T cells in atopic dermatitis. These data support the concept that activated T cells or subsets of T cells may be important effector cells in mediating inflammatory activity in atopic disease.

Protein gene product 9.5 is expressed by fibroblasts in human cutaneous wounds.

In a study initially designed to evaluate reinnervation of human cutaneous wounds using an antibody to the neuroneal marker protein gene product (PGP) 9.5, we observed marked immunostaining of cells with morphologic features of fibroblasts in the wounds. PGP 9.5 has recently been shown to be an important enzyme in the highly conserved ubiquitin system of proteolysis. Because the ubiquitin system is known to play an important role in regulating the cell cycle, the presence of PGP 9.5 in cells at a wound site was of considerable interest. Our objectives were to clarify the time frame for the appearance of PGP 9.5 and ubiquitin in wounds, to verify that PGP 9.5 is produced by wound fibroblasts, and to evaluate a potential role for these proteins in the tissue repair process. Standard incisional human wounds were stained with antibodies specific for PGP 9.5 and ubiquitin. At 7 d, stellate cells with morphologic features of fibroblasts stained for PGP 9.5, whereas earlier wounds were generally negative. In 14 and 21 d incised wounds and in chronic granulation tissue from nonhealing ulcers there was strong cellular staining for PGP 9.5 and for ubiquitin. These stellate cells also showed expression of mRNA for PGP 9.5 by reverse transcriptase-polymerase chain reaction in situ hybridization. PGP 9.5 was detected in cultured fibroblasts both by reverse transcriptase-polymerase chain reaction and by northern blot analysis. Confocal microscopy showed colocalization of antibodies to PGP 9.5 and prolyl-4-hydroxylase (a fibroblast marker) as well as colocalization of PGP 9.5 and the platelet derived growth factor beta receptor. We conclude that ubiquitin and PGP 9.5 were expressed by fibroblasts during the granulation tissue and remodeling phases wound healing. The mRNA for PGP 9.5 was demonstrated in stellate cells in chronic wounds and in fibroblasts in culture. The appearance of these degradative proteins in later wounds suggests a downregulation function in the wound healing response.

Effects of UV radiation on autoimmune strains of mice: increased mortality and accelerated autoimmunity in BXSB male mice.

Systemic lupus erythematosus is an immunologically mediated autoimmune disease which has been reported to be aggravated by certain environmental agents such as ultraviolet irradiation (UV). To further investigate this interaction, we examined the consequences of UV exposure on the autoimmune process of several strains of autoimmune mice. Strains of age- and sex-matched, 3- to 4-month old, autoimmune (MRL-lpr/lpr, (NZB X NZW)F1, BXSB) and nonautoimmune (BALB/c, B10.A) mice were shaved and exposed to an acute (2 h daily X 7 days) and a chronic (3 h/weekly X 4 weeks) dose of UV (FS40 lamps, 2 mJ/cm2/s). UV-induced changes in survival, autoantibody production, splenic B-cell activity, and target organ pathology were examined. After an acute UV exposure there were (10/15) deaths in the UV BXSB males and (4/15) in the UV BXSB females, compared to (1/15) in the non-UV BXSB male group and (0/15) in the non-UV BXSB females. No deaths occurred in the other UV autoimmune or nonautoimmune groups. Likewise, chronic UV resulted in increased UV BXSB male mortality (13/15) compared to UV BXSB females (2/15) and non-UV BXSB males and females. No deaths occurred in the other autoimmune (MRL-lpr/lpr, (NZB X NZW)F1) or nonautoimmune (BALB/c, B10.A) strains, after chronic UV exposure. Equivalent doses of Mylar-filtered FS40 UV (UVB deleted) resulted in no deaths in the UV BXSB male group. Acute and chronic UV also resulted in a significant increase in serum single-stranded DNA antibody production, splenic polyclonal B-cell activity, and renal glomerular inflammatory changes in the UV BXSB male mice. Thus, UV resulted in premature death and accelerated autoimmunity in UV BXSB males and may serve as a useful model for phototoxicity in autoimmunity.

Regulation of epidermal cell interleukin-6 production by UV light and corticosteroids.

Epidermal cells (EC) are well known as a source of cytokines, including interleukin (IL)-6. In the present study, we investigated whether ultraviolet (UV) light and corticosteroids (CS) affect IL-6 production by normal (HNK) or malignant (KB) human keratinocytes. Supernatants derived from UVB (100 J/m2)- but not from UVA (100-1500 kJ/m2)-exposed EC (HNK and KB) contained significantly increased levels of IL-6 activity. This was also confirmed by Western blot analysis, resulting in specific bands at 23 kD and 27 kD. Northern blot analysis revealed an enhanced IL-6 mRNA expression after UVB exposure. Addition of hydrocortisone, prednisolone, or dexamethasone immediately after UVB irradiation significantly blocked UVB or IL-1-induced IL-6 mRNA expression and production by EC. The suppressive effect was observed at doses in the physiologic (10(-7)-10(-9) M) as well as pharmacologic (10(-5)-10(-7) M) range. In contrast, the nonactive steroid prednisone did not affect EC IL-6 mRNA expression. These findings indicate that increased IL-6 production by EC after UVB irradiation may mediate local and systemic inflammatory reactions following extensive sun exposure. Thus, the therapeutic effect of corticosteroids observed in various inflammatory diseases may be partly due to their downregulating capacity of IL-6 production.

Neutral endopeptidase expression and distribution in human skin and wounds.

Cutaneous sensory nerves mediate inflammation and wound healing by the release of neuropeptides such as substance P. Neutral endopeptidase is a cell surface enzyme that degrades substance P and thereby terminates its biologic actions. The distribution of neutral endopeptidase in normal skin and wounded human skin, however, has not been examined. The objectives of this study were to evaluate neutral endopeptidase expression in wounded and unwounded skin as well as in cells derived from human skin. Neutral endopeptidase was strikingly localized in normal skin by immunohistochemistry to keratinocytes of the epidermal basal layer, to hair follicles, eccrine and sebaceous glands as well as to endothelium of blood vessels and to large nerves. Standard incisional human wounds were studied at several time points between 1 h and 28 d after wounding. Staining for neutral endopeptidase was noted in the wound bed 6 h after wounding. In contrast to normal skin, staining of all the epidermal cell layers was noted in the migrating tongue of epithelium in l d wounds. Similar full-thickness staining was noted in 3 d and 7 d wounds in all layers of the new wound epithelium and in a "transition epithelium" near the wound edge. By 28 d post wounding neutral endopeptidase staining again was detected only in the basal layer of the epidermis. Neutral endopeptidase mRNA was detected in normal skin and wounds as well as cultured keratinocytes, fibroblasts and endothelial cells. Neutral endopeptidase enzymatic bioactivity was demonstrated in cultured keratinocytes. While it is known that several metalloproteinases important to tissue repair are produced by keratinocytes, this is the first evidence that keratinocytes produce neutral endopeptidase. Neutral endopeptidase may terminate the proinflammatory and mitogenic actions of neuropeptides in normal skin and wounds.

Human keratinocytes constitutively express interleukin-18 and secrete biologically active interleukin-18 after treatment with pro-inflammatory mediators and dinitrochlorobenzene.

Interleukin-18 is a potent inducer of interferon-gamma by activated T cells, macrophages, and monocytes and is synthesized as an inactive precursor. Pro-interleukin-18 must be cleaved by interleukin-1-beta-converting enzyme for secretion of the biologically active form. We report that among selected non-bone marrow derived skin cells, interleukin-18 mRNA is constitutively expressed by human keratinocytes and not by dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes. Interleukin-18 mRNA and intracellular protein levels are neither changed in human keratinocytes nor induced in human dermal microvascular endothelial cells, dermal fibroblasts, or melanocytes by exposure to pro-inflammatory stimuli. Exposure of human keratinocytes to phorbol 12-myrisate 13-acetate, lipopolysaccharides or the contact sensitizer DNCB results in the secretion of immunoprecipitable interleukin-18 protein. Human keratinocyte-secreted interleukin-18 is biologically active, in that conditioned media from phorbol 12-myrisate 13-acetate, lipopolysaccharide and DNCB-treated human keratinocytes induce interferon-gamma expression by peripheral blood mononuclear cells. This bioactivity is neutralized by anti-interleukin-18, but not anti-interleukin-12 antibodies. By immunohistochemistry, interleukin-18 protein is detected in basal keratinocytes of normal human skin, but its expression is markedly upregulated in suprabasal keratinocytes in psoriasis. These findings indicate that human keratinocytes are a source of biologically functional interleukin-18 and thus are capable of playing an initiating part in the local interferon-gamma-dependent inflammatory processes through expression, activation, and secretion of interleukin-18.

Expression of proopiomelanocortin peptides in human dermal microvascular endothelial cells: evidence for a regulation by ultraviolet light and interleukin-1.

Proopiomelanocortin peptides such as alpha-melanocyte-stimulating hormone and adrenocorticotropin are expressed in the epidermal and dermal compartment of the skin after noxious stimuli and are recognized as modulators of immune and inflammatory reactions. Human dermal microvascular endothelial cells mediate leukocyte-endothelial interactions during cutaneous inflammation by the expression of cellular adhesion molecules and cytokines such as interleukin-1. This study addresses the hypothesis that human dermal microvascular endothelial cells express both proopiomelanocortin and prohormone convertases, which are required to generate proopiomelanocortin peptides. Semiquantitative reverse transcriptase polymerase chain reaction and northern blot studies revealed a constitutive expression of proopiomelanocortin mRNA by human dermal microvascular endothelial cells in vitro that was time- and concentration-dependently upregulated by interleukin-1 beta. Furthermore, irradiation of human dermal microvascular endothelial cells with ultraviolet A1 (30J per cm(2)) or ultraviolet B (12.5 mJ per cm(2)) enhanced proopiomelanocortin expression as well as the production and release of the proopiomelanocortin peptides adrenocorticotropin and alpha-melanocyte-stimulating hormone. In addition to proopiomelanocortin, prohormone convertase 1 mRNA expression was detected by reverse transcriptase polymerase chain reaction in unstimulated human dermal microvascular endothelial cells and was augmented after exposure to alpha-melanocyte- stimulating hormone, interleukin-1 beta, or irradiation with ultraviolet. These findings demonstrate that human dermal microvascular endothelial cells express proopiomelanocortin and prohormone convertase 1 required for the generation of adrenocorticotropin. Additionally, human dermal microvascular endothelial cells express mRNA for the prohormone convertase 2 binding protein 7B2. Taken together these findings indicate that human dermal microvascular endothelial cells upon stimulation express both proopiomelanocortin and prohormone convertases required for the generation of alpha-melanocyte-stimulating hormone. As proopiomelanocortin peptides were found to regulate the production of human dermal microvascular endothelial cell cytokines and adhesion molecules and to have a variety of anti-inflammatory properties these peptides may significantly contribute to the modulation of skin inflammation. J Invest Dermatol 115:1021-1028 2000

The neurosensory tachykinins substance P and neurokinin A directly induce keratinocyte nerve growth factor.

Nerve growth factor is an essential neurotrophic factor required for the growth and maintenance of cutaneous sensory nerves. In the skin, keratinocytes are a significant source of nerve growth factor; however, the regulation of cutaneous nerve growth factor production still remains to be fully understood. In this study we tested the hypothesis that neuropeptides released by cutaneous sensory nerves have the capacity to modulate directly the expression of keratinocyte nerve growth factor, which would have important implications for the maintenance and repair of nerves in the skin. In order to address this question experimentally we examined the effect of the neuropeptides, substance P and neurokinin A, on nerve growth factor expression in human keratinocytes and the murine keratinocyte PAM 212 cell line by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and the PC-12 nerve growth factor bioassay. The results of these studies indicated that substance P and neurokinin A can directly induce nerve growth factor mRNA expression and the secretion of bioactive nerve growth factor protein in both human and murine keratinocytes. The specificity of these responses was demonstrated using neuropeptide receptor antagonists and nerve growth factor blocking antibodies. Additional studies also demonstrated a significant in vivo upregulation of keratinocyte nerve growth factor expression in murine epidermis after the topical application of the neuropeptide releasing agent capsaicin. This is the first report demonstrating the induction of cutaneous nerve growth factor by sensory nerve-derived neuropeptides such as substance P and neurokinin A. This direct effect of the neurosensory system on keratinocyte nerve growth factor production may have important consequences for the maintenance and regeneration of cutaneous nerves in normal skin and during inflammation and wound healing.

Alpha-melanocyte stimulating hormone prevents lipopolysaccharide-induced vasculitis by down-regulating endothelial cell adhesion molecule expression.

The neuroendocrine hormone alpha-melanocyte stimulating hormone (MSH) has profound antiinflammatory and immunomodulating properties. Here we have examined the possibility that alpha-MSH may interfere with the expression and function of cell adhesion molecules (CAMs) expressed by human dermal microvascular endothelial cells (HDMECs) in response to lipopolysaccharide (LPS) or TNFalpha in vitro and in vivo. In HDMEC, alpha-MSH (10(-8)/10(-12) M) profoundly reduced the mRNA and protein expression of E-selectin, vascular CAM (VCAM)-1, and intercellular CAM (ICAM)-1 induced by LPS or TNFalpha as determined by semiquantitative RT-PCR, ELISA, and fluorescence-activated cell sorter analysis. In addition, alpha-MSH significantly impaired the LPS-induced ICAM-1 and VCAM-1-mediated adhesion of lymphocytes to HDMEC monolayer in a functional adhesion assay. Likewise, alpha-MSH effectively inhibited the transcription factor nuclear factor-kappaB activation in HDMEC, which is required for CAM gene expression. Importantly in vivo, in murine LPS-induced cutaneous vasculitis (local Shwartzman reaction), a single ip injection of alpha-MSH significantly suppressed the deleterious vascular damage and hemorrhage by inhibiting the sustained expression of vascular E-selectin and VCAM-1. This persistent expression has been implicated in the dysregulation of diapedesis and activation of leukocytes, which subsequently leads to hemorrhagic vascular damage. Our findings indicate that alpha-MSH may have an important therapeutical potential for the treatment of vasculitis, sepsis, and inflammatory diseases.

Retinal pigment epithelial cells secrete interleukin-6 in response to interleukin-1.

Interleukin-6 (IL-6) is a peptide whose properties include the ability to activate T-lymphocytes, stimulate the secretion of immunoglobulin, induce neuronal differentiation, and trigger the release of acute phase proteins. We have detected IL-6-like activity in conditioned medium from cultured human retinal pigment epithelial (RPE) cells with a bioassay based on the ability of IL-6 to induce the proliferation of murine B-9 plasmacytoma cells. Biologic activity increased approximately 90-fold when the cells were cultured in the presence of IL-1 alpha (30 units/ml). Western blot analysis confirmed that conditioned medium from IL-1 alpha-stimulated RPE cells contained peptides with molecular weights ranging between 19,000 and 30,000 and reactive with antibody to IL-6. Finally, Northern blot analysis indicated that cells cultured in the presence of interleukin-1 contained a 1.2 kilobase transcript that hybridized to a cDNA probe specific for IL-6 messenger RNA. IL-6 peptide on Western blots and mRNA on Northern blots were undetectable unless cells were cultured in the presence of IL-1 alpha. Although IL-6 is synthesized by a variety of cell types, this report is the first to detect its synthesis by an eye-specific cell type. Furthermore, these observations indicate that retinal pigment epithelial cells respond to IL-1, a cytokine that previously has been implicated in ocular inflammation.

Detection of mRNA for the cytokines, interleukin-1 alpha and interleukin-8, in corneas from patients with pseudophakic bullous keratopathy.

PURPOSE: Cytokines have been implicated in corneal inflammatory diseases. The authors sought to determine if corneal diseases with different patterns of inflammation are accompanied by characteristic patterns of cytokine expression. METHODS: The authors used reverse transcription-polymerase chain reaction to detect mRNA for 12 specific cytokines from patients with Fuch's dystrophy (n = 7), pseudophakic bullous keratopathy (n = 12), or normal corneas (n = 4). RESULTS: Using Wilcoxon rank sum analysis, mRNA for interleukin (IL)-1 alpha or IL-8 was significantly more abundant in corneas from patients with pseudophakic bullous keratopathy relative to either comparison group. mRNA for the T-cell marker, CD4, and for T-cell derived cytokines, IL-2, IL-4, and interferon gamma, could not be detected in any corneal sample. Message for IL-1 receptor antagonist and transforming growth factor-beta 1 or -beta 2 was readily detectable in most corneas regardless of diagnosis. CONCLUSION: The findings indicate that the pattern of cytokine mRNA expression reflects differences in the pathogenesis of these corneal diseases.