UVB Radiation-Mediated Expression of Inducible Nitric Oxide Synthase Activity and the Augmenting Role of Co-Induced TNF-α in Human Skin Endothelial Cells
2004; Elsevier BV; Volume: 123; Issue: 5 Linguagem: Inglês
10.1111/j.0022-202x.2004.23422.x
ISSN1523-1747
AutoresChristoph V. Suschek, Csaba Mahotka, Oliver Schnorr, Victoria Kolb-Bachofen,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoNitric oxide (NO) plays a pivotal role in ultraviolet radiation-induced inflammation in human skin. We had earlier reported on the inducible nitric oxide synthase (iNOS) inducing activity of UVA radiation. We now demonstrate that UVB-exposure induces expression of the iNOS in vessel endothelia of normal human skin and in cultured human dermal endothelial cells (HUDEC), although by a molecular mechanism different from UVA-mediated induction. With HUDEC, UVB induces iNOS expression and leads to significant enzyme activities, although at app. 5-fold lower levels than can be achieved with proinflammatory cytokines. In contrast to our earlier observation with UVA, cytokine-challenge combined with simultaneous UVB-exposure had no additive effects on iNOS expression nor activity. Interestingly, a time-delay between UVB-irradiation and cytokine-challenge enhances endothelial iNOS enzyme activity 2.5-fold over cytokines activation only. This time-dependent effect strongly correlates with UVB-induced endothelial TNF-α expression. In HUDEC addition of TNF-α results in enhanced expression of the inducible arginine transporter system CAT-2 essential for substrate supply and thus iNOS activity. In summary, UVB induces iNOS mRNA and enzyme activity in HUDEC. Moreover, UVB augments CAT-2 expression through a TNF-α- dependent mechanism which essentially contributes to increased iNOS activity. Nitric oxide (NO) plays a pivotal role in ultraviolet radiation-induced inflammation in human skin. We had earlier reported on the inducible nitric oxide synthase (iNOS) inducing activity of UVA radiation. We now demonstrate that UVB-exposure induces expression of the iNOS in vessel endothelia of normal human skin and in cultured human dermal endothelial cells (HUDEC), although by a molecular mechanism different from UVA-mediated induction. With HUDEC, UVB induces iNOS expression and leads to significant enzyme activities, although at app. 5-fold lower levels than can be achieved with proinflammatory cytokines. In contrast to our earlier observation with UVA, cytokine-challenge combined with simultaneous UVB-exposure had no additive effects on iNOS expression nor activity. Interestingly, a time-delay between UVB-irradiation and cytokine-challenge enhances endothelial iNOS enzyme activity 2.5-fold over cytokines activation only. This time-dependent effect strongly correlates with UVB-induced endothelial TNF-α expression. In HUDEC addition of TNF-α results in enhanced expression of the inducible arginine transporter system CAT-2 essential for substrate supply and thus iNOS activity. In summary, UVB induces iNOS mRNA and enzyme activity in HUDEC. Moreover, UVB augments CAT-2 expression through a TNF-α- dependent mechanism which essentially contributes to increased iNOS activity. cationic amino acid transporters human dermal endothelial cells inducible nitric oxide synthase tumor necrosis factor α ultraviolet A/B/C Prolonged exposure of the human skin to ultraviolet radiation (UVR) can result in inflammation including erythema and edema formation, and also in premature aging, immune suppression and skin cancer (Jagger, 1985Jagger J. Solar-UV Action on Living Cells. Praeger, New York1985Google Scholar;Gange, 1987Gange R.W. Acute effects of ultraviolet radiation in the skin.in: Fitzpatrick T.B. Eisen A.Z. Wolff K. Dermatology in General Medicine. McGraw-Hill, New York1987: 1451-1457Google Scholar). Erythema formation is the result of local increases in blood flow in both the superficial and deep vascular plexus of the dermis (Greaves, 1986Greaves M.W. Ultraviolet erythema: Causes and consequences.Curr Probl Dermatol. 1986; 15: 18-24Crossref PubMed Google Scholar). UVB-induced erythema were shown to be significantly reduced by inhibitors of nitric oxide synthases (NOS) indicating the involvement of nitric oxide (NO) in regulation of local vascular tone and blood flow in normal human skin (Goldsmith et al., 1996Goldsmith P.C. Leslie T.A. Hayes N.A. Levell N.J. Dowd P.A. Foreman J.C. Inhibitors of nitric oxide synthase in human skin.J Invest Dermatol. 1996; 106: 113-118https://doi.org/10.1111/1523-1747.ep12328204Crossref PubMed Scopus (87) Google Scholar). Endothelial-derived NO plays a physiological role in blood pressure regulation and in inhibition of platelet aggregation and platelet adhesion and is also held responsible for erythema and edema formation through vascular leakage as a marker for local inflammation but also modulates leukocyte adhesion (Langrehr et al., 1993Langrehr J.M. Hoffman R.A. Lancester Jr, R.R. Simmons R.L. Nitric oxide. A new endogenous immunomodulator.Transplantation. 1993; 55: 1205-1212Crossref PubMed Scopus (164) Google Scholar). During inflammatory processes, cytokines are known modulators of endothelial cell functions (Pober and Cotran, 1990Pober J.S. Cotran R.S. Cytokines and endothelial cell biology.Physiol Rev. 1990; 70: 427-451Crossref PubMed Scopus (1099) Google Scholar). One of the prominent effects that cytokines can exert on endothelial cells is the induction of the expression of the inducible isoform of the NOS-family, the iNOS, ensuing high-output NO synthesis (Suschek et al., 1993Suschek C. Rothe H. Fehsel K. Enczmann J. Kolb-Bachofen V. Induction of a macrophage-like nitric oxide synthase in cultured rat aortic endothelial cells.J Immunol. 1993; 151: 3283-3291PubMed Google Scholar;Suschek et al., 1994Suschek C. Fehsel K. Kröncke K-D Sommer A. Kolb-Bachofen V. Primary cultures of rat capillary endothelial cells: Constitutive and cytokine-inducible macrophage-like nitric oxide synthases are expressed and activities regulated by glucose concentration.Am J Pathol. 1994; 145: 485-695Google Scholar;Hoffmann et al., 1999Hoffmann G. Schobersberger W. Rieder J. et al.Human dermal microvascular endothelial cells express inducible nitric oxide synthase in vitro.J Invest Dermatol. 1999; 112: 387-390https://doi.org/10.1046/j.1523-1747.1999.00505.xCrossref PubMed Scopus (17) Google Scholar). High-output NO production may serve for local defense against viruses, bacteria, protozoa, and helminths (Liew and Cox, 1991Liew F.Y. Cox F.E.G. Nonspecific defense mechanism: The role of nitric oxide.Immunol Today. 1991; 12: A17-A21https://doi.org/10.1016/0167-5699(91)90107-5Abstract Full Text PDF PubMed Scopus (497) Google Scholar), but is also involved in toxic effects on neighboring cells (Mills, 1991Mills C.D. 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Type 1 (insulin-dependent) diabetes mellitus and nitric oxide.Diabetologia. 1992; 35: 796-797PubMed Google Scholar) and in the pathogenesis of septic shock and cytokine-induced hypotension (Kilbourn and Belloni, 1990Kilbourn R.G. Belloni P. Endothelial cell production of nitrogen oxides in response to interferon-gamma, with tumor necrosis factor, interleukin-l, or endotoxin.J Natl Cancer Inst. 1990; 82: 772-776Crossref PubMed Scopus (433) Google Scholar). Although numerous cytotoxic effects on a variety of mammalian cells were described to be associated with iNOS-derived high-output NO formation, more recently, many publications point to a powerful protective activity of iNOS-generated NO during oxidative stress resulting from exposure to reactive oxygen species (ROS) such as superoxide, hydrogen peroxide or alkyl peroxides (Wink et al., 1995Wink D.A. Cook J.A. Pacelli R. Liebmann J. Krishna M.C. Mitchell J.B. Nitric oxide (NO) protects against cellular damage by reactive oxygen species.Toxicol Lett. 1995; 82/83: 221-226Crossref Scopus (143) Google Scholar;Wink et al., 1996Wink D.A. Hanbauer I. Grisham M.B. et al.Chemical biology of nitric oxide: Regulation and protective and toxic mechanisms.Curr Top Cell Reg. 1996; 34: 159-187Crossref PubMed Scopus (246) Google Scholar) all of which are known intracellular mediators of UVR-induced cytotoxicity (Danpure and Tyrrell, 1976Danpure H.J. Tyrrell R.M. Oxygen dependence of near-UV (365 nm) lethality and the interaction of near-UV and X-rays in two mammalian cell lines.Photochem Photobiol. 1976; 23: 171-177Crossref PubMed Scopus (90) Google Scholar;Black, 1987Black H.S. Potential involvement of free radical reactions in ultraviolet light-mediated cutaneous damage.Photochem Photobiol. 1987; 46: 213-221Crossref PubMed Scopus (293) Google Scholar). Indeed, we could show recently that endogenously produced as well as exogenously applied NO effectively protects from UVA-induced and ROS-mediated damage and apoptosis of endothelial cells (Suschek et al., 1999Suschek C.V. Krischel V. Bruch-Gerharz D. Berendji D. Krutmann J. Kröncke K-D Kolb-Bachofen V. Nitric oxide fully protects against UVA-induced apoptosis in tight correlation with Bcl-2 up-regulation.J Biol Chem. 1999; 274: 6130-6137https://doi.org/10.1074/jbc.274.10.6130Crossref PubMed Scopus (150) Google Scholar,Suschek et al., 2001aSuschek C.V. Briviba K. Bruch-Gerharz D. Sies H. Kröncke K.D. Kolb-Bachofen V. Even after UVA-exposure will nitric oxide protect cells from reactive oxygen intermediate-mediated apoptosis and necrosis.Cell Death Differ. 2001; 8: 515-527https://doi.org/10.1038/sj.cdd.4400839Crossref PubMed Scopus (52) Google Scholar). We had previously shown that UVA irradiation, capable of penetrating far into the dermal layers of human skin, will induce human dermal endothelial iNOS expression in situ in the absence of proinflammatory cytokines. Moreover, UVA irradiation significantly augments iNOS expression and activity induced by the addition of Th-1-type cytokines (Suschek et al., 2001bSuschek C.V. Bruch-Gerharz D. Kleinert H. Förstermann U. Kolb-Bachofen V. Ultraviolet A1 radiation induces nitric oxide synthase-2 expression in human skin endothelial cells in the absence of proinflammatory cytokines.J Invest Dermatol. 2001; 117: 1200-1205https://doi.org/10.1046/j.0022-202x.2001.01502.xCrossref PubMed Google Scholar). Here, we now examine the effects of UVB radiation on the expression of the iNOS in specimens of normal human skin maintained in organ culture and on iNOS expression and activity in cultures of human dermal endothelial cells. We here show that UVB radiation induces iNOS mRNA expression similar to UVA-mediated effects. In contrast to UVA, however, UVB irradiation modulates endothelial iNOS activity by a mechanism involving endogenous TNF-α production and also increases the expression of the L-arginine transport system CAT (cationic amino acid transporters)-2. UVB irradiation (50 mJ per cm2) of normal human skin specimens maintained in organ culture results in de novo iNOS protein expression in small vessel-lining endothelia within 24 h post irradiation. As positive controls, specimens were incubated with proinflammatory cytokines resulting in the identical positive signal for iNOS protein (Figure 1a). With human dermal endothelial cell (HUDEC) cultures, UVB irradiation in the absence of cytokines, concentration dependently (1.25–10 mJ per cm2), leads to the induction of expression of iNOS mRNA (Figure 1c) as well as protein (Figure 1b and c). Again, cytokine incubation of HUDEC (interleukin (IL)-1β+interferon-gamma (IFN-γ), each 500 U per mL) was used as a positive control and led to iNOS mRNA and iNOS protein expression as previously shown (Suschek et al., 2001bSuschek C.V. Bruch-Gerharz D. Kleinert H. Förstermann U. Kolb-Bachofen V. Ultraviolet A1 radiation induces nitric oxide synthase-2 expression in human skin endothelial cells in the absence of proinflammatory cytokines.J Invest Dermatol. 2001; 117: 1200-1205https://doi.org/10.1046/j.0022-202x.2001.01502.xCrossref PubMed Google Scholar). The expression of iNOS mRNA parallels iNOS enzyme activity (Figure 2a and Table I). In contrast to our observation with UVA (Suschek et al., 2001bSuschek C.V. Bruch-Gerharz D. Kleinert H. Förstermann U. Kolb-Bachofen V. Ultraviolet A1 radiation induces nitric oxide synthase-2 expression in human skin endothelial cells in the absence of proinflammatory cytokines.J Invest Dermatol. 2001; 117: 1200-1205https://doi.org/10.1046/j.0022-202x.2001.01502.xCrossref PubMed Google Scholar), the combination of UVB irradiation and subsequent cytokine addition do not augment iNOS expression or activity above cytokine treatment only. Interestingly, a delayed addition of cytokines 18 h after UVB irradiation leads to a strong and significant increment in iNOS expression and activity as compared to cytokines only.Table IImpact of cationic amino acid transport and TNF-α on endothelial iNOS enzyme activityNitrite (nM)TreatmentNo additives+lys/ornARG depr.+L-NIO+anti-TNF-α+thal+thal +TNF-α+thal+IL-6+thal+IL-8Resting111±1493±4576±3726±8121±788±2488±22110±3291±12UVB250±30*p<0.001 as compared to the resting controls.77±1881±2636±25247±22236±7191±21231±31201±18βγ612±90**p<0.001 as compared to resting or UVB-irradiated only cell cultures.94±988±1620±12**p<0.001 as compared to resting or UVB-irradiated only cell cultures.590±32660±67521±62563±22624±27UVB+βγ769±82**p<0.001 as compared to resting or UVB-irradiated only cell cultures.86±1068±2211±9544±41602±109623±44547±21603±41UVB→βγ1361±139***p<0.001 as compared to all other cell cultures.85±3175±2314±8517±33****p<0.001 as compared to respective cell cultures cultivated in the absence of any additives.794±51****p<0.001 as compared to respective cell cultures cultivated in the absence of any additives.1221±119*****p<0.001 as compared to the respective cultures treated with thalidomide only.644±34635±38In resting or cytokine activated (βγ, IL-1β+IFN-γ, each 500 U per mL) and/or UVB-challenged (10 mJ per cm2) human dermal endothelial cells (HUDEC) nitrite formation and its dependence on exogenous arginine or on CAT-mediated arginine-transport or on TNF-α function was determined. Values represent the mean±SD of three to six individual experiments.iNOS, inducible nitric oxide synrhase; UVB, ultraviolet B; lys/orn, 10 mM lysine+ornithine; ARG depr., arginine deprivation of the growth medium; L-NIO, NOS inhibitor L-NIO (0.25 mM); anti-TNF-α, anti-human TNF-α antibodies; thal, thalidomide (20 μg per mL) alone or simultaneously with IL-6 or IL-8. UVB+βγ, UVB-irradiation and cytokine-challenge were performed simultaneously; UVB→βγ, HUDEC were irradiated by UVB and 18 h later activated by the respective cytokines.* p<0.001 as compared to the resting controls.** p<0.001 as compared to resting or UVB-irradiated only cell cultures.*** p<0.001 as compared to all other cell cultures.**** p<0.001 as compared to respective cell cultures cultivated in the absence of any additives.***** p<0.001 as compared to the respective cultures treated with thalidomide only. Open table in a new tab In resting or cytokine activated (βγ, IL-1β+IFN-γ, each 500 U per mL) and/or UVB-challenged (10 mJ per cm2) human dermal endothelial cells (HUDEC) nitrite formation and its dependence on exogenous arginine or on CAT-mediated arginine-transport or on TNF-α function was determined. Values represent the mean±SD of three to six individual experiments.iNOS, inducible nitric oxide synrhase; UVB, ultraviolet B; lys/orn, 10 mM lysine+ornithine; ARG depr., arginine deprivation of the growth medium; L-NIO, NOS inhibitor L-NIO (0.25 mM); anti-TNF-α, anti-human TNF-α antibodies; thal, thalidomide (20 μg per mL) alone or simultaneously with IL-6 or IL-8. UVB+βγ, UVB-irradiation and cytokine-challenge were performed simultaneously; UVB→βγ, HUDEC were irradiated by UVB and 18 h later activated by the respective cytokines. Since iNOS activity depends on the availability of its substrate L-arginine transported via the CAT (Closs, 2002Closs E.I. Expression, regulation and function of carrier proteins for cationic amino acids.Curr Opin Nephrol Hypertens. 2002; 11: 99-107Crossref PubMed Scopus (87) Google Scholar) we also searched for an impact of UVB on CAT-2 expression. As shown in Figure 2b, HUDEC constitutively express CAT-2. UVB irradiation (10 mJ per cm2) alone does not influence the CAT-2-mRNA expression levels, whereas activation with proinflammatory cytokines IL-1β+IFN-γ (each 500 U per mL) strongly augments CAT-2 mRNA expression as expected. Delayed (18 h) cytokine challenge post-UVB irradiation leads to a significant additional enhancement of CAT-2 mRNA expression over the increment by cytokines alone. Again, UVB irradiation immediately followed by cytokine challenge does not increase CAT-2 expression. Thus, the pattern of CAT-2 mRNA expression completely parallels that of iNOS expression and activity (Table I). Competition for arginine transport by excess lysine+ornithine (10 mM each) or lack of exogenous arginine both completely inhibit the cytokine and/or UVB-induced iNOS enzyme activity to the same degree as does incubation with the NOS inhibitor L-NIO (L-N5-(1-Iminoethyl)-ornithin) (0.25 mM) (Table I). Examination of CAT-1 mRNA expression reveals a constitutive expression unchanged by UVB or cytokine treatments (data not shown). TNF-α is a potent costimulatory factor for iNOS expression and activity (Suschek et al., 1993Suschek C. Rothe H. Fehsel K. Enczmann J. Kolb-Bachofen V. Induction of a macrophage-like nitric oxide synthase in cultured rat aortic endothelial cells.J Immunol. 1993; 151: 3283-3291PubMed Google Scholar). As UVB radiation is known to induce TNF-α expression in dermal cells (de Kossodo et al., 1995de Kossodo S. Cruz Jr, P.D. Dougherty I. Thompson P. Silva-Valdez M. Beutler B. Expression of the tumor necrosis factor gene by dermal fibroblasts in response to ultraviolet irradiation or lipopolysaccharide.J Invest Dermatol. 1995; 104: 318-322https://doi.org/10.1111/1523-1747.ep12665361Crossref PubMed Scopus (38) Google Scholar;Strickland et al., 1997Strickland I. Rhodes L.E. Flanagan B.F. Friedmann P.S. TNF-alpha and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: Correlation with neutrophil accumulation and E-selectin expression.J Invest Dermatol. 1997; 108: 763-768https://doi.org/10.1111/1523-1747.ep12292156Crossref PubMed Scopus (160) Google Scholar), we next examined whether the UVB-induced effects or parts of it are under the influence of endogenous endothelial TNF-α production. Indeed, UVB irradiation induces TNF-α mRNA expression after 30 min (Figure 3a) and after 4 h significant amounts of TNF-α protein (Figure 3b) are detected in culture supernatants. When cell cultures were grown in the presence of thalidomide (20 μg per mL), TNF-α synthesis was effectively blocked (Figure 3b). Furthermore, in UVB-irradiated cell cultures the time delay for cytokine-mediated increases in iNOS activity can be explained by the UVB-induced endogenous TNF-α expression and the time needed for protein secretion (Figure 3b vs c) and indeed neutralizing anti-TNF-α-antibodies completely block the increment in NO-synthesis. Neither UVA irradiated nor non-irradiated HUDEC produce TNF-α (not shown), demonstrating the UVB specificity of this response. Next, we examined the role of endogenous TNF-α production on iNOS and CAT-2 mRNA expression or iNOS activity, respectively, and find indeed that TNF-α production is a crucial factor, since inhibition of endogenous TNF-α formation by the presence of thalidomide (20 μg per mL) effectively blocks the cytokine-mediated increments of both, iNOS and CAT-2 mRNA expressions (Figure 4) and also of iNOS activity (Table I). Furthermore, addition of recombinant human TNF-α completely reverses these thalidomide effects (Figure 4 and Table I). Neither human IL-6 nor IL-8, which represent two proinflammatory endothelial cytokines that may also be affected by thalidomide were able to reverse the thalidomide-mediated suppression (Table I). Thalidomide, however, does not block the UVB-mediated de novo iNOS expression in resting cells nor does it interfere with IL-1β/IFN-γ-induced iNOS or CAT-2 activity or expression, respectively. These data give strong evidence for UVB-induced endogenous endothelial TNF-α production as an important enhancing factor in UVB-induced iNOS expression and activity. Recently, we have demonstrated that UVA irradiation induces a de novo expression of the iNOS in human dermal endothelia in the absence of cytokines and significantly augments endothelial iNOS activity in the presence of proinflammatory stimuli (Suschek et al., 2001bSuschek C.V. Bruch-Gerharz D. Kleinert H. Förstermann U. Kolb-Bachofen V. Ultraviolet A1 radiation induces nitric oxide synthase-2 expression in human skin endothelial cells in the absence of proinflammatory cytokines.J Invest Dermatol. 2001; 117: 1200-1205https://doi.org/10.1046/j.0022-202x.2001.01502.xCrossref PubMed Google Scholar). The timing of the de novo expression of this NO-synthase isotype as induced by UVA or as demonstrated here by UVB strongly correlates with the appearance of erythema formation which peaks 24 h after UV-irradiation (Greaves, 1986Greaves M.W. Ultraviolet erythema: Causes and consequences.Curr Probl Dermatol. 1986; 15: 18-24Crossref PubMed Google Scholar). Thus, it appears that in addition to the neuronal NOS activity in dermal keratinocytes, as hypothesized earlier (Deliconstantinos et al., 1995Deliconstantinos G. Villiotu V. Stravides J.C. Release by ultraviolet B (u.v.B.) radiation of nitric oxide (NO) from human keratinocytes: A potential role for nitric oxide in erythema production.Br J Pharmacol. 1995; 114: 1257-1265Crossref PubMed Scopus (115) Google Scholar), iNOS expression in endothelia might contribute to erythema formation. Although both, UVA (Suschek et al., 2001bSuschek C.V. Bruch-Gerharz D. Kleinert H. Förstermann U. Kolb-Bachofen V. Ultraviolet A1 radiation induces nitric oxide synthase-2 expression in human skin endothelial cells in the absence of proinflammatory cytokines.J Invest Dermatol. 2001; 117: 1200-1205https://doi.org/10.1046/j.0022-202x.2001.01502.xCrossref PubMed Google Scholar) and UVB radiation are potent inducers of endothelial iNOS expression and activity, the molecular basis of this response is not identical. Simultaneous challenge of endothelial cells with UVA+cytokines significantly increases iNOS expression and activity relative to cells treated with cytokines only. In contrast with UVB, such increases are not observed, rather here only a time-delay between UVB irradiation and cytokine challenge increase strongly iNOS-mRNA expression as well as enzyme activity. This indicates the involvement of an additional UVB-inducible event serving as modulator or enhancer of iNOS expression and activity. One obvious candidate for such a factor is TNF-α, since this gene is responsive to UVB and UVC but not to UVA (Bazzoni et al., 1994Bazzoni F. Kruys V. Shakhov A. Jongeneel C.V. Beutler B. Analysis of tumor necrosis factor promoter responses to ultraviolet light.J Clin Invest. 1994; 93: 56-62Crossref PubMed Scopus (40) Google Scholar;de Kossodo et al., 1995de Kossodo S. Cruz Jr, P.D. Dougherty I. Thompson P. Silva-Valdez M. Beutler B. Expression of the tumor necrosis factor gene by dermal fibroblasts in response to ultraviolet irradiation or lipopolysaccharide.J Invest Dermatol. 1995; 104: 318-322https://doi.org/10.1111/1523-1747.ep12665361Crossref PubMed Scopus (38) Google Scholar). UVB-induced TNF-α expression in known to occur in keratinocytes (Köck et al., 1990Köck A. Schwarz T. Kirnbauer R. Urbanski A. Perry P. Ansel J.C. Luger T.A. Human keratinocytes are a source of tumor necrosis factor alpha: Evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light.J Exp Med. 1990; 172: 1609-1614https://doi.org/10.1084/jem.172.6.1609Crossref PubMed Scopus (611) Google Scholar) and dermal fibroblasts (de Kossodo et al., 1995de Kossodo S. Cruz Jr, P.D. Dougherty I. Thompson P. Silva-Valdez M. Beutler B. Expression of the tumor necrosis factor gene by dermal fibroblasts in response to ultraviolet irradiation or lipopolysaccharide.J Invest Dermatol. 1995; 104: 318-322https://doi.org/10.1111/1523-1747.ep12665361Crossref PubMed Scopus (38) Google Scholar). For endothelial iNOS expression and activity, TNF-α is not sufficient for de novo induction (Suschek et al., 1993Suschek C. Rothe H. Fehsel K. Enczmann J. Kolb-Bachofen V. Induction of a macrophage-like nitric oxide synthase in cultured rat aortic endothelial cells.J Immunol. 1993; 151: 3283-3291PubMed Google Scholar), but represents a strong costimulator, a finding confirmed by the present data. We here show that UVB induces endothelial TNF-α formation, but that this event is not involved in UVB-induced de novo iNOS expression. On the other hand, endothelial TNF-α protein production reaches a maximum 18–24 h after UVB challenge and serves as a potent enhancer of iNOS expression and enzyme activity. This effect becomes apparent when UVB-irradiated cells are subsequently challenged with proinflammatory cytokines where the necessary the time-delay matches the time needed for maximal endogenous TNF-α formation. In addition, TNF-α has also been shown to represent a key signal for the induction of the “inducible” cationic amino acid transporter CAT-2 responsible for the influx of higher L-arginine concentration (Closs et al., 2000Closs E.I. Scheld J.S. Sharafi M. Forstermann U. Substrate supply for nitric-oxide synthase in macrophages and endothelial cells: Role of cationic amino acid transporters.Mol Pharmacol. 2000; 57: 68-74PubMed Google Scholar;Closs, 2002Closs E.I. Expression, regulation and function of carrier proteins for cationic amino acids.Curr Opin Nephrol Hypertens. 2002; 11: 99-107Crossref PubMed Scopus (87) Google Scholar) as an essential prerequisite for maximal iNOS activity (Schnorr et al., 2003Schnorr O. Suschek C.V. Kolb-Bachofen V. The importance of cationic amino acid transporter expression in human skin.J Invest Dermatol. 2003; 120: 1016-1022https://doi.org/10.1046/j.1523-1747.2003.12139.xAbstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar;Suschek et al., 2003Suschek C.V. Schnorr O. Hemmrich K. Aust O. Klotz L.O. Sies H. Kolb-Bachofen V. Critical role of L-arginine in endothelial cell survival during oxidative stress.Circulation. 2003; 107: 2607-2614PubMed Google Scholar). Indeed, the pivotal role of TNF-α on CAT-2 expression and the impact of CAT-2 on iNOS enzyme activity is highlighted by our findings that iNOS-mediated NO formation after UVB challenge parallels TNF-α-dependent CAT-2 expression, and that blocking TNF-α formation or activity abolishes increases in CAT-2 expression and in iNOS activity. In our experiment, we intend to block TNF-α expression by addition of thalidomide which is not TNF-specific but also unspecifically affects the expression of a variety of other cytokines (Matthews and McCoy, 2003Matthews S.J. McCoy C. Thalidomide: A review of approved and investigational uses.Clin Ther. 2003; 25: 342-395https://doi.org/10.1016/S0149-2918(03)80085-1Abstract Full Text PDF PubMed Scopus (168) Google Scholar). Here, the inhibitory effects of thalidomide on iNOS or CAT mRNA expression can be completely restored by addition of TNF-α but not IL-6 and IL-8, two other cytokines known to be affected by thalidomide. Furthermore, regarding iNOS activity we used TNF-α specific monoclonal antibodies and could completely block the mentioned UVB-induced increases in endothelial nitrite formation. Again, this effect was completely restored by addition of recombinant TNF-α but not IL-6 or IL-8. Therefore, although thalidomide is not a specific TNF-α inhibitor, the experiments presented here give a strong evidence for endogenous TNF-α as mediator of UVB action on CAT-2 expression and thus NO formation in human dermal endothelial cells. In conclusion, we here demonstrate that UVB radiation induces iNOS mRNA, protein, and function in HUDEC. Further, cytokines strongly augment UVB-induced iNOS and CAT-2 expression in a delayed pattern and through a TNF-α-dependent mechanism, and also iNOS activity and NO generation. Thus, TNF-α is required for maximal increases in iNOS mRNA expression and activity following UVB exposure under inflammatory conditions, although it is not sufficient in itself for iNOS induction. This effect may also contribute to the known time-delay in UV-induced erythema formation. The study was approved by the ethical committee of the Heinrich-Heine-University of Düsseldorf and conducted according to Declaration of Helsinki Principles. Patients gave written informed consent. Recombinant human IL-1β was purchased from HBT (Leiden, Netherlands), recombinant human IFN-γ, and recombinant human TNF-α from Genzyme (Cambridge, Massachusetts). The lipopolysaccharide (LPS) content in this cytokine batches never exceeded 0.1 ng per μg protein. The polyclonal anti-human-TNF-α-antiserum, monoclonal anti-human-TNF-α-antibody, the human TNF-α ELISA kit, IL-6 and IL-8 were purchased from R&D Systems (Wiesbaden, Germany), Neutral Red (3% solution), the oligo dT16-primer, and anti-α-tubulin antibody from Sigma (Deisenhofen, Germany), the monoclonal anti-iNOS and anti-eNOS antibodies from Transduction Laboratories (Lexington, KT, USA), peroxidase-conjugated goat anti-mouse IgG from Zymed Laboratories (San Francisco, CA, USA), trypsin, EDT
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