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Urocanic Acid Binds to GABA but not to Histamine (H1, H2, or H3) Receptors
LETTERS TO THE EDITOR
Urocanic Acid Binds to GABA but not to Histamine (H1, H2, or H3) Receptors (Fig 1). Their S-shaped competition curves showed IC50 values of 51 and 38 µM for cis-UCA and trans-UCA, respectively (CI from 21 to 126 µM and from 3 to 532 µM, respectively). Thus, it is possible that the UCA compounds displace GABA from subtypes of GABA receptors. The binding data must be evaluated against the physiologic concentrations of UCA in the skin. The mean concentrations of UCA in the unirradiated skin of a Caucasian population ranged between 2.3 and 62 nmol per cm2 (Jansen et al, 1991; Snellman et al, 1992, 1997; Gilmour et al, 1993b; Kavanagh et al, 1995), corresponding to 0.3–8.9 mM within the µ70 µm thick epidermis (Bruls et al, 1984). When cis-UCA can be raised to 50% of the total epidermal UCA concentrations with suberythemal doses of solar-simulated irradiation (Snellman et al, 1997), the levels of cis-UCA in vivo are comparable with the effective concentrations for GABA receptor binding in vitro. Because these data did not show affinity of the UCA isomers to the three histamine receptors, they suggest that the effects of UCA on histaminergic functions must be indirect. Binding of trans-UCA (cisUCA not studied) to rat cortical membrane GABA receptors has been reported, but it was much weaker than that of GABA (Matheson et al, 1987). Our data also show slight displacement of bound GABA by trans-UCA, but a clearly stronger effect by cis-UCA. This is, to our knowledge, the first report of interaction between cis-UCA and GABA receptors. Nerve fibers containing calcitonin gene-related peptide, a vasodilating neurotransmitter, are intimately associated with epidermal Langerhans cell bodies in mice (Hosoi et al, 1993). In other murine systems, neurogenic mediators (e.g., calcitonin gene-related peptide,
To the Editor: Urocanic acid (UCA) is a known epidermal component and a major chromophore for ultraviolet (UV) light. Photoisomerization from transto cis-UCA upon UV irradiation is thought to be an important event initiating changes in immune functions. Recently, the UV-induced suppression of local and systemic immune functions and generation of antigen-specific tolerance have been attributed both to UCA photoisomerization (reviewed by Norval, 1996) and to DNA damage (reviewed by Vink et al, 1996). Although several experimental designs have revealed stereospecific effects of the two isomers on intracellular signaling, cytokine networks, and systemic immunity, a specific receptor for UCA has not yet been discovered. The conception that cis-UCA might act through histamine receptors rests largely on indirect evidence obtained from experiments with histamine receptor antagonists/agonists (Norval et al, 1990; Gilmour et al, 1993a); however, the action of UCA through histaminergic receptors in keratinocytes (Mitra et al, 1993), monocytes (Hart et al, 1993), and Langerhans cells (Beissert et al, 1997) has been rebutted. Because of its structural relationship to γ-amino butyric acid (GABA), UCA has also been included in screening of in vivo biologic activity mediated by the GABA receptors, found typically in the central nervous system, and substantial effectiveness was observed (Matheson et al, 1986, 1987). Direct competition binding studies with UCA have been reported only on α2- and imidazol(in)e receptors1 (no binding), and GABA (weak binding with trans-UCA; cis-isomer not studied) (Tunnicliff et al, 1985; Matheson et al, 1987). In this study, we analyzed the binding of UCA isomers to the histamine H1, H2, and H3 receptors, and to the GABA receptors in radioligand competition assays in vitro. Cis-UCA was prepared from trans-UCA (Aldrich) according to the method of Morrison et al (1980), and purified by anion exchange column chromatography. The purity of the isomers was checked with high performance liquid chromatography (Pasanen et al, 1990) and [1H]NMR using DMSO-d6 as the solvent. Experiments (n 5 16) with different batches of rat cortex membranes were done, and the efficiency of UCA to displace bound radioligands from H1- (n 5 3; [3H]pyrilamine), H2- (n 5 6; [3H]tiotidine), and H3-(n 5 2; [3H]R-α-methylhistamine) histamine receptors as well as from GABA (n 5 5; [3H]GABA) receptors was studied (methods reviewed by Enna et al, 1977; Hill, 1990). The specific receptor binding was transformed into percentage of control (100% when no competitor was present). The two UCA stereoisomers (ø1 mM of UCA tested) did not show consistent displacement of H1, H2, or H3 binding. Thus no reliable IC50 values could be calculated for histamine receptor binding. [3H]GABA binding was clearly displaced by nonlabeled GABA with an IC50 value of 58 nM and 95% confidence interval (CI) from 46 to 72 nM. On the other hand, cis-UCA and trans-UCA partially displaced [3H]GABA by 48% and 19%, respectively
Figure 1. Competition binding curves of cis-UCA, trans-UCA, and GABA for [3H]GABA in the rat cortical membranes. Each curve includes combined data from five separate binding experiments. The final [3H]GABA concentration was 8 nM and the mean protein concentration was 1.13 mg per ml. The IC50 values were determined by fitting the specific binding data to a sigmoidal curve using GraphPad Prism 2.01 for Windows 95 (GraphPad Software, San Diego, CA). The symbols (s, trans-UCA, n, cis-UCA, u, GABA) and error bars represent the mean 6 SEM (n 5 3–10 for other data except for controls where n 5 40).
Manuscript received October 8, 1997; revised April 22, 1998; accepted for publication June 10, 1998. Abbreviations: GABA, γ-amino butyric acid; UCA, urocanic acid. 1Savola J-M, Laihia JK, Jansen CT. Urocanic acid stereoisomers do not bind to adrenergic α2-receptors or nonadrenergic imidazol(in)e receptors. J Invest Dermatol 98:652, 1992 (abstr.)
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0022-202X/98/$10.50 Copyright © 1998 by The Society for Investigative Dermatology, Inc.
705
706
LETTERS TO THE EDITOR
substance P, and nitric oxide) have also been shown to contribute to UVB-induced erythema (Benrath et al, 1995), immunosuppression (Gillardon et al, 1995), and production of cytokines like TNF-α in mast cells (Ansel et al, 1993; Niizeki et al, 1997). Because GABA is an inhibitory neurotransmitter, it might be possible that cis-UCA binds as an antagonist to possible cutaneous GABA receptors. Such function could disinhibit the secretion of cutaneous neuropeptides modulating local immune reactions. Because no reports of GABA receptors in the skin have been found, our next goal will be to study whether GABA receptors exist in the skin. Jarmo K. Laihia, Martti Attila,†‡ Kari Neuvonen,* Paavo Pasanen,* Leena Tuomisto,‡ and Christer T. Jansen, Departments of Dermatology and *Chemistry, University of Turku, Turku, Finland †Pharmacology and Toxicology, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland ‡Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland REFERENCES Ansel J, Brown J, Payan D, Brown M: Substance P selectively activates TNF-α gene expression in murine mast cells. J Immunol 150:1–8, 1993 Beissert S, Mohammad T, Torri H, Lonati A, Yan Z, Morrison H, Granstein RD: Regulation of tumor antigen presentation by urocanic acid. J Immunol 159: 92–96, 1997 Benrath J, Eschenfelder C, Zimmerman M, Gillardon F: Calcitonin gene-related peptide, substance P and nitric oxide are involved in cutaneous inflammation following ultraviolet irradiation. Eur J Pharmacol 293:87–96, 1995 Bruls WAG, Slaper H, van der Leun JC, Berrens L: Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem Photobiol 40:485–494, 1984 Enna SJ, Wood JH, Snyder SH: Gamma-aminobutyric acid (GABA) in human cerebrospinal fluid: radioreceptor assay. J Neurochem 28:1121–1124, 1977 Gillardon F, Moll I, Michel S, Benrath J, Weihe E, Zimmermann M: Calcitonin generelated peptide and nitric oxide are involved in ultraviolet radiation-induced immunosuppression. Eur J Pharmacol 293:395–400, 1995 Gilmour JW, Norval M, Simpson TJ, Neuvonen K, Pasanen P: The role of histamine-
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
like receptors in immunosuppression of delayed hypersensitivity induced by cisurocanic acid. Photodermatol Photoimmunol Photomed 9:250–254, 1993a Gilmour JW, Vestey JP, Norval M: The effect of UV therapy on immune function in patients with psoriasis. Br J Dermatol 129:28–38, 1993b Hart PH, Jones CA, Jones KL, Watson CJ, Santucci I, Spencer LK, Finlay-Jones JJ: Cis-urocanic acid stimulates human peripheral blood monocyte prostaglandin E2 production and suppresses indirectly tumor necrosis factor-α levels. J Immunol 150:4514–4523, 1993 Hill SJ: Distribution, properties, and functional characteristics of three classes of histamine receptor. Pharmacol Rev 42:45–83, 1990 Hosoi J, Murphy GF, Egan CL, Lerner EA, Grabbe S, Asahina A, Granstein RD: Regulation of Langerhans cell function by nerves containing calcitonin gene-related peptide. Nature 363:159–162, 1993 ¨ yra¨s P: A Jansen CT, Lammintausta K, Pasanen P, Neuvonen K, Varjonen E, Kalimo K, A non-invasive chamber sampling technique for HPLC analysis of human epidermal urocanic acid isomers. Acta Derm Venereol (Stockh) 71:143–145, 1991 Kavanagh G, Crosby J, Norval M: Urocanic acid isomers in human skin: analysis of site variation. Br J Dermatol 133:728–731, 1995 Matheson GK, Freed E, Tunnicliff G: Novel GABA analogues as hypotensive agents. Neuropharmacol 25:1191–1195, 1986 Matheson GK, Freed E, Tunnicliff G: Central receptor binding and cardiovascular effects of GABA analogues in the cat. Gen Pharmacol 18:269–273, 1987 Mitra RS, Shimizu Y, Nickoloff BJ: Histamine and cis-urocanic acid augment tumor necrosis factor-α mediated induction of keratinocyte intercellular adhesion molecule1 expression. J Cell Physiol 156:348–357, 1993 Morrison H, Avnir D, Zarella T: Analysis of Z and E isomers of urocanic acid by highperformance liquid chromatography. J Chromatogr 183:83–86, 1980 Niizeki H, Alard P, Streilein JW: Calcitonin gene-related peptide is necessary for ultraviolet B-impaired induction of contact hypersensitivity. J Immunol 159:5183–5186, 1997 Norval M: Chromophore for UV-induced immunesuppression: urocanic acid. Photochem Photobiol 63:386–390, 1996 Norval M, Gilmour JW, Simpson TJ: The effect of histamine receptor antagonists on immunosuppression induced by the cis-isomer of urocanic acid. Photodermatol Photoimmunol Photomed 7:243–248, 1990 ¨ yra¨s P: Urocanic acid Pasanen P, Reunala T, Jansen CT, Ra¨sa¨nen L, Neuvonen K, A isomers in epidermal samples and suction blister fluid of nonirradiated and UVBirradiated human skin. Photodermatol Photoimmunol Photomed 7:40–42, 1990 ¨ yra¨s P, Jansen CT: Snellman E, Koulu L, Pasanen P, Lammintausta K, Neuvonen K, A Effect of psoriasis heliotherapy on epidermal urocanic acid isomer concentrations. Acta Derm Venereol (Stockh) 72:231–233, 1992 Snellman E, Jansen CT, Laihia JK, Mila´n T, Koulu L, Leszczynski K, Pasanen P: Urocanic acid concentration and photoisomerization in Caucasian skin phototypes. Photochem Photobiol 65:862–865, 1997 Tunnicliff G, Welborn KL, Ngo TT: Identification of potential GABA-mimetics by their actions on brain GABA recognition sites. Gen Pharmacol 16:25–29, 1985 Vink AA, Yarosh DB, Kripke ML: Chromophore for UV-induced immunesuppression: DNA. Photochem Photobiol 63:383–386, 1996
UV Immunosuppression and Skin Cancer To the Editor: We read with considerable interest the paper by Yamawaki et al ‘‘Genetic variation in low-dose UV-induced suppression of contact hypersensitivity and in the skin photocarcinogenesis response,’’ published in the Journal of Investigative Dermatology (109:716, 1997). The data in Figs 4 and 5 of this paper compare tumor incidence and tumor yield in C3H/HeN and C3H/HeJ mice treated with either UV alone (Fig 4) or a single dose of UV followed by 12-O-tetradecanoylphorbol-13-acetate promotion (Fig 5). These two mouse strains differ in the Lps gene that is defective in C3H/HeJ mice and controls a variety of B lymphocyte and macrophage responses. These mice have also been reported to differ in susceptibility to the ‘‘local’’ immunosuppressive effects of UV radiation, proposed to be an important factor in UV carcinogenesis. We have some comments on this paper. Statistical analysis It is stated (p. 719) that there are significant differences in tumor incidence and yield between the two strains in Fig 5, i.e., after treatment with UV and 12-O-tetradecanoyl-phorbol13-acetate, but not in Fig 4, i.e., after treatment with UV alone. By visual inspection this would appear to be the case, but we were unable to find any mention of any statistical test for significance. It is essential to see the results of the application to this data, e.g., survival analysis with censoring, which accounts for any tumor-free deaths during the experiment, and appropriate statistical analysis, e.g., Kaplan–Meier
logrank test, before it can be concluded that significant differences do in fact exist. Strain differences in UV immunosuppression There is not universal agreement that C3H/HeN and C3H/HeJ strains do differ in susceptibility to the immunosuppressive effects of UV. The UV dose– responses for suppression of contact hypersensitivity in these two strains are in fact identical if the ‘‘systemic’’ model is used (Noonan and De Fabo, 1990) and would have predicted the findings in Fig 4. ‘‘Low-dose’’ versus ‘‘high-dose’’ immunosuppression It is simply incorrect to say, as is stated in the Introduction (p. 716), that ‘‘A relatively low dose of UV radiation is all that is required to produce immunosuppression if the antigen is applied directly to the UV-exposed skin site (local or low-dose immune suppression). On the other hand, when a greater UV dose is administered, immunosuppression results even if the antigen is applied to a non-UV exposed skin site (systemic or high-dose immune suppression).’’ In fact, a direct comparison between UV-induced ‘‘local’’ and ‘‘systemic’’ immunosuppression of contact hypersensitivity showed that the UV dose–responses for these effects are the same (Noonan and De Fabo, 1990). The UV dose–responses differ, however, between mouse strains (Noonan and De Fabo, 1990; Noonan and Hoffman, 1994) and the kinetics of ‘‘local’’ and ‘‘systemic’’ suppression differ. A time lag of 2–3 d after UV before antigen application has long been known to be necessary for the detection of systemic suppression (Noonan et al,
Urocanic Acid Binds to GABA but not to Histamine (H1, H2, or H3) Receptors (Fig 1). Their S-shaped competition curves showed IC50 values of 51 and 38 µM for cis-UCA and trans-UCA, respectively (CI from 21 to 126 µM and from 3 to 532 µM, respectively). Thus, it is possible that the UCA compounds displace GABA from subtypes of GABA receptors. The binding data must be evaluated against the physiologic concentrations of UCA in the skin. The mean concentrations of UCA in the unirradiated skin of a Caucasian population ranged between 2.3 and 62 nmol per cm2 (Jansen et al, 1991; Snellman et al, 1992, 1997; Gilmour et al, 1993b; Kavanagh et al, 1995), corresponding to 0.3–8.9 mM within the µ70 µm thick epidermis (Bruls et al, 1984). When cis-UCA can be raised to 50% of the total epidermal UCA concentrations with suberythemal doses of solar-simulated irradiation (Snellman et al, 1997), the levels of cis-UCA in vivo are comparable with the effective concentrations for GABA receptor binding in vitro. Because these data did not show affinity of the UCA isomers to the three histamine receptors, they suggest that the effects of UCA on histaminergic functions must be indirect. Binding of trans-UCA (cisUCA not studied) to rat cortical membrane GABA receptors has been reported, but it was much weaker than that of GABA (Matheson et al, 1987). Our data also show slight displacement of bound GABA by trans-UCA, but a clearly stronger effect by cis-UCA. This is, to our knowledge, the first report of interaction between cis-UCA and GABA receptors. Nerve fibers containing calcitonin gene-related peptide, a vasodilating neurotransmitter, are intimately associated with epidermal Langerhans cell bodies in mice (Hosoi et al, 1993). In other murine systems, neurogenic mediators (e.g., calcitonin gene-related peptide,
To the Editor: Urocanic acid (UCA) is a known epidermal component and a major chromophore for ultraviolet (UV) light. Photoisomerization from transto cis-UCA upon UV irradiation is thought to be an important event initiating changes in immune functions. Recently, the UV-induced suppression of local and systemic immune functions and generation of antigen-specific tolerance have been attributed both to UCA photoisomerization (reviewed by Norval, 1996) and to DNA damage (reviewed by Vink et al, 1996). Although several experimental designs have revealed stereospecific effects of the two isomers on intracellular signaling, cytokine networks, and systemic immunity, a specific receptor for UCA has not yet been discovered. The conception that cis-UCA might act through histamine receptors rests largely on indirect evidence obtained from experiments with histamine receptor antagonists/agonists (Norval et al, 1990; Gilmour et al, 1993a); however, the action of UCA through histaminergic receptors in keratinocytes (Mitra et al, 1993), monocytes (Hart et al, 1993), and Langerhans cells (Beissert et al, 1997) has been rebutted. Because of its structural relationship to γ-amino butyric acid (GABA), UCA has also been included in screening of in vivo biologic activity mediated by the GABA receptors, found typically in the central nervous system, and substantial effectiveness was observed (Matheson et al, 1986, 1987). Direct competition binding studies with UCA have been reported only on α2- and imidazol(in)e receptors1 (no binding), and GABA (weak binding with trans-UCA; cis-isomer not studied) (Tunnicliff et al, 1985; Matheson et al, 1987). In this study, we analyzed the binding of UCA isomers to the histamine H1, H2, and H3 receptors, and to the GABA receptors in radioligand competition assays in vitro. Cis-UCA was prepared from trans-UCA (Aldrich) according to the method of Morrison et al (1980), and purified by anion exchange column chromatography. The purity of the isomers was checked with high performance liquid chromatography (Pasanen et al, 1990) and [1H]NMR using DMSO-d6 as the solvent. Experiments (n 5 16) with different batches of rat cortex membranes were done, and the efficiency of UCA to displace bound radioligands from H1- (n 5 3; [3H]pyrilamine), H2- (n 5 6; [3H]tiotidine), and H3-(n 5 2; [3H]R-α-methylhistamine) histamine receptors as well as from GABA (n 5 5; [3H]GABA) receptors was studied (methods reviewed by Enna et al, 1977; Hill, 1990). The specific receptor binding was transformed into percentage of control (100% when no competitor was present). The two UCA stereoisomers (ø1 mM of UCA tested) did not show consistent displacement of H1, H2, or H3 binding. Thus no reliable IC50 values could be calculated for histamine receptor binding. [3H]GABA binding was clearly displaced by nonlabeled GABA with an IC50 value of 58 nM and 95% confidence interval (CI) from 46 to 72 nM. On the other hand, cis-UCA and trans-UCA partially displaced [3H]GABA by 48% and 19%, respectively
Figure 1. Competition binding curves of cis-UCA, trans-UCA, and GABA for [3H]GABA in the rat cortical membranes. Each curve includes combined data from five separate binding experiments. The final [3H]GABA concentration was 8 nM and the mean protein concentration was 1.13 mg per ml. The IC50 values were determined by fitting the specific binding data to a sigmoidal curve using GraphPad Prism 2.01 for Windows 95 (GraphPad Software, San Diego, CA). The symbols (s, trans-UCA, n, cis-UCA, u, GABA) and error bars represent the mean 6 SEM (n 5 3–10 for other data except for controls where n 5 40).
Manuscript received October 8, 1997; revised April 22, 1998; accepted for publication June 10, 1998. Abbreviations: GABA, γ-amino butyric acid; UCA, urocanic acid. 1Savola J-M, Laihia JK, Jansen CT. Urocanic acid stereoisomers do not bind to adrenergic α2-receptors or nonadrenergic imidazol(in)e receptors. J Invest Dermatol 98:652, 1992 (abstr.)
·
0022-202X/98/$10.50 Copyright © 1998 by The Society for Investigative Dermatology, Inc.
705
706
LETTERS TO THE EDITOR
substance P, and nitric oxide) have also been shown to contribute to UVB-induced erythema (Benrath et al, 1995), immunosuppression (Gillardon et al, 1995), and production of cytokines like TNF-α in mast cells (Ansel et al, 1993; Niizeki et al, 1997). Because GABA is an inhibitory neurotransmitter, it might be possible that cis-UCA binds as an antagonist to possible cutaneous GABA receptors. Such function could disinhibit the secretion of cutaneous neuropeptides modulating local immune reactions. Because no reports of GABA receptors in the skin have been found, our next goal will be to study whether GABA receptors exist in the skin. Jarmo K. Laihia, Martti Attila,†‡ Kari Neuvonen,* Paavo Pasanen,* Leena Tuomisto,‡ and Christer T. Jansen, Departments of Dermatology and *Chemistry, University of Turku, Turku, Finland †Pharmacology and Toxicology, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland ‡Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland REFERENCES Ansel J, Brown J, Payan D, Brown M: Substance P selectively activates TNF-α gene expression in murine mast cells. J Immunol 150:1–8, 1993 Beissert S, Mohammad T, Torri H, Lonati A, Yan Z, Morrison H, Granstein RD: Regulation of tumor antigen presentation by urocanic acid. J Immunol 159: 92–96, 1997 Benrath J, Eschenfelder C, Zimmerman M, Gillardon F: Calcitonin gene-related peptide, substance P and nitric oxide are involved in cutaneous inflammation following ultraviolet irradiation. Eur J Pharmacol 293:87–96, 1995 Bruls WAG, Slaper H, van der Leun JC, Berrens L: Transmission of human epidermis and stratum corneum as a function of thickness in the ultraviolet and visible wavelengths. Photochem Photobiol 40:485–494, 1984 Enna SJ, Wood JH, Snyder SH: Gamma-aminobutyric acid (GABA) in human cerebrospinal fluid: radioreceptor assay. J Neurochem 28:1121–1124, 1977 Gillardon F, Moll I, Michel S, Benrath J, Weihe E, Zimmermann M: Calcitonin generelated peptide and nitric oxide are involved in ultraviolet radiation-induced immunosuppression. Eur J Pharmacol 293:395–400, 1995 Gilmour JW, Norval M, Simpson TJ, Neuvonen K, Pasanen P: The role of histamine-
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
like receptors in immunosuppression of delayed hypersensitivity induced by cisurocanic acid. Photodermatol Photoimmunol Photomed 9:250–254, 1993a Gilmour JW, Vestey JP, Norval M: The effect of UV therapy on immune function in patients with psoriasis. Br J Dermatol 129:28–38, 1993b Hart PH, Jones CA, Jones KL, Watson CJ, Santucci I, Spencer LK, Finlay-Jones JJ: Cis-urocanic acid stimulates human peripheral blood monocyte prostaglandin E2 production and suppresses indirectly tumor necrosis factor-α levels. J Immunol 150:4514–4523, 1993 Hill SJ: Distribution, properties, and functional characteristics of three classes of histamine receptor. Pharmacol Rev 42:45–83, 1990 Hosoi J, Murphy GF, Egan CL, Lerner EA, Grabbe S, Asahina A, Granstein RD: Regulation of Langerhans cell function by nerves containing calcitonin gene-related peptide. Nature 363:159–162, 1993 ¨ yra¨s P: A Jansen CT, Lammintausta K, Pasanen P, Neuvonen K, Varjonen E, Kalimo K, A non-invasive chamber sampling technique for HPLC analysis of human epidermal urocanic acid isomers. Acta Derm Venereol (Stockh) 71:143–145, 1991 Kavanagh G, Crosby J, Norval M: Urocanic acid isomers in human skin: analysis of site variation. Br J Dermatol 133:728–731, 1995 Matheson GK, Freed E, Tunnicliff G: Novel GABA analogues as hypotensive agents. Neuropharmacol 25:1191–1195, 1986 Matheson GK, Freed E, Tunnicliff G: Central receptor binding and cardiovascular effects of GABA analogues in the cat. Gen Pharmacol 18:269–273, 1987 Mitra RS, Shimizu Y, Nickoloff BJ: Histamine and cis-urocanic acid augment tumor necrosis factor-α mediated induction of keratinocyte intercellular adhesion molecule1 expression. J Cell Physiol 156:348–357, 1993 Morrison H, Avnir D, Zarella T: Analysis of Z and E isomers of urocanic acid by highperformance liquid chromatography. J Chromatogr 183:83–86, 1980 Niizeki H, Alard P, Streilein JW: Calcitonin gene-related peptide is necessary for ultraviolet B-impaired induction of contact hypersensitivity. J Immunol 159:5183–5186, 1997 Norval M: Chromophore for UV-induced immunesuppression: urocanic acid. Photochem Photobiol 63:386–390, 1996 Norval M, Gilmour JW, Simpson TJ: The effect of histamine receptor antagonists on immunosuppression induced by the cis-isomer of urocanic acid. Photodermatol Photoimmunol Photomed 7:243–248, 1990 ¨ yra¨s P: Urocanic acid Pasanen P, Reunala T, Jansen CT, Ra¨sa¨nen L, Neuvonen K, A isomers in epidermal samples and suction blister fluid of nonirradiated and UVBirradiated human skin. Photodermatol Photoimmunol Photomed 7:40–42, 1990 ¨ yra¨s P, Jansen CT: Snellman E, Koulu L, Pasanen P, Lammintausta K, Neuvonen K, A Effect of psoriasis heliotherapy on epidermal urocanic acid isomer concentrations. Acta Derm Venereol (Stockh) 72:231–233, 1992 Snellman E, Jansen CT, Laihia JK, Mila´n T, Koulu L, Leszczynski K, Pasanen P: Urocanic acid concentration and photoisomerization in Caucasian skin phototypes. Photochem Photobiol 65:862–865, 1997 Tunnicliff G, Welborn KL, Ngo TT: Identification of potential GABA-mimetics by their actions on brain GABA recognition sites. Gen Pharmacol 16:25–29, 1985 Vink AA, Yarosh DB, Kripke ML: Chromophore for UV-induced immunesuppression: DNA. Photochem Photobiol 63:383–386, 1996
UV Immunosuppression and Skin Cancer To the Editor: We read with considerable interest the paper by Yamawaki et al ‘‘Genetic variation in low-dose UV-induced suppression of contact hypersensitivity and in the skin photocarcinogenesis response,’’ published in the Journal of Investigative Dermatology (109:716, 1997). The data in Figs 4 and 5 of this paper compare tumor incidence and tumor yield in C3H/HeN and C3H/HeJ mice treated with either UV alone (Fig 4) or a single dose of UV followed by 12-O-tetradecanoylphorbol-13-acetate promotion (Fig 5). These two mouse strains differ in the Lps gene that is defective in C3H/HeJ mice and controls a variety of B lymphocyte and macrophage responses. These mice have also been reported to differ in susceptibility to the ‘‘local’’ immunosuppressive effects of UV radiation, proposed to be an important factor in UV carcinogenesis. We have some comments on this paper. Statistical analysis It is stated (p. 719) that there are significant differences in tumor incidence and yield between the two strains in Fig 5, i.e., after treatment with UV and 12-O-tetradecanoyl-phorbol13-acetate, but not in Fig 4, i.e., after treatment with UV alone. By visual inspection this would appear to be the case, but we were unable to find any mention of any statistical test for significance. It is essential to see the results of the application to this data, e.g., survival analysis with censoring, which accounts for any tumor-free deaths during the experiment, and appropriate statistical analysis, e.g., Kaplan–Meier
logrank test, before it can be concluded that significant differences do in fact exist. Strain differences in UV immunosuppression There is not universal agreement that C3H/HeN and C3H/HeJ strains do differ in susceptibility to the immunosuppressive effects of UV. The UV dose– responses for suppression of contact hypersensitivity in these two strains are in fact identical if the ‘‘systemic’’ model is used (Noonan and De Fabo, 1990) and would have predicted the findings in Fig 4. ‘‘Low-dose’’ versus ‘‘high-dose’’ immunosuppression It is simply incorrect to say, as is stated in the Introduction (p. 716), that ‘‘A relatively low dose of UV radiation is all that is required to produce immunosuppression if the antigen is applied directly to the UV-exposed skin site (local or low-dose immune suppression). On the other hand, when a greater UV dose is administered, immunosuppression results even if the antigen is applied to a non-UV exposed skin site (systemic or high-dose immune suppression).’’ In fact, a direct comparison between UV-induced ‘‘local’’ and ‘‘systemic’’ immunosuppression of contact hypersensitivity showed that the UV dose–responses for these effects are the same (Noonan and De Fabo, 1990). The UV dose–responses differ, however, between mouse strains (Noonan and De Fabo, 1990; Noonan and Hoffman, 1994) and the kinetics of ‘‘local’’ and ‘‘systemic’’ suppression differ. A time lag of 2–3 d after UV before antigen application has long been known to be necessary for the detection of systemic suppression (Noonan et al,