Effects of ultraviolet B irradiation on cutaneous leishmaniasis

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Effects of Ultraviolet B Irradiation on Cutaneous Leishmaniasis S.H. G i a n n i n i

Protection against many infectious diseases is mediated by cellular immunity in the competent host. Ultraviolet (UV) radiation, a component of sunlight, is a potent suppressor of ceU-mediated immune responses. Suzanne Holmes Giannini discusses the possible relevance of ambient levels of UVB to pathogenesis and immunity in infectious diseases, with special reference to cutaneous leishmaniasis. Ultraviolet radiation, with wavelengths between 180 and 400nm, lies in the electromagnetic spectrum between visible light and X-rays. Most of the UV radiation reaching the earth's surface in sunlight is UVA (wavelengths of 320-400 nm). The shorter-wavelength UVB (290-320 nm), also called the 'sunburn spectrum', is the more damaging biologically; its intensity varies greatly, depending on latitude, season and thickness of the ozone layer, which absorbs these wavelengths 1. At present, UVC (180-290nm) is completely blocked by the atmosphere. The recent discoveries of a 'hole' in the ozone layer over the Antarctic 2 and depletion of the stratospheric ozone layer fuel concerns that levels of terrestrial UVB will increase, not only in the tropics but also in the temperate zones. UVB in tropical sunlight is not only more intense than in the temperate zones, but it is also qualitatively different in that it contains a higher proportion of the biologically damaging shorter wavelengths. As the wavelength of UVB decreases, it is more injurious to cells, for example, as measured by the number of single-strand breaks induced in DNA (broken curve in Fig. 1). The spectral power distribution of solar UVB is illustrated for two areas endemic for cutaneous leishmaniasis: Tumaco, Colombia, at I°N of the equator, and Bukhara, Uzbekistan, at 39°N (solid curves in Fig. 1). It is significant that the highly biologically active UVB near 290 nm is present in equatorial sunlight at up to approximately 2500-fold the intensity found in temperate zone sunlight, although the exposures to total UVB differ by less than fivefold. In the laboratory, UVB causes an impressive array of immunological effects, including depression of cell-mediated immune responses in contact hypersensitivity and delayed-type hyperSuzanne Holmes Giannini is at the University of Maryland School of Medicine, Department of Microbiology and Immunology, 655 West Baltimore Street, Baltimore, MD 21201, USA.

sensitivity, altered rates of secretion of cytokines, alteration in lymphocyte homing patterns and the induction of T-suppressor cells (for Reviews, see Refs 3-5). UVB at 150-300J m -2 damages Langerhans cells (the antigen-presenting cells in the epidermis)6; at 22-34kJ m -2 it alters lymphocyte homing patterns 7, at 4 9 k J m -2 it causes systemic suppression of contact hypersensitivity 8 and at 8 6 k J m -2 it causes immunological unresponsiveness to UV-induced tumors 9. For comparison, the minimum erythemal dose of UVB needed to cause sunburn in skin type I humans (fair-skinned humans who always burn, never tan) is approximately 200J m -2. On clear days in the latitudes between 25°N and 25°S, approximately 1 - 4 J m -2 of UVB reach ground level every second during midday 1, so that over a 10-h period, humans can be readily exposed to even the highest experimental level of biologically effective UVB, ie. 8 6 k J m -2. Systemic immunosuppressive effects depend on the cumulative dose of UVB and not the rate of exposure 1°, so that doses that are fractionated and delivered over

6-

[,.

A 4-

r~

1

2

2'

0' ~-''-~-A

-2" -4

i

z

--i

~

' -2

280

300

4oo W A V E L E N G T H (NM)

Fig. I. Spectral intensity and biological activity of UV in temperate (Bukhara) and tropical (Tumaco) sunlight When temperate and tropical sunlight are compared, the greatest spectral differences in wavelength intensity occur for the most biologically damaging wavelengths. The graph shows the relative intensities in J m-2 (solid curves) and the biological activities (broken curve, with filled triangles, representing differences in the number of DNA strand breaks caused by the same intensity of UV) for the UVB (290-320nm) and UVA (320-400nm) spectra. Terrestrial UVC (<290 nm) does not reach detectable levels. Loglo breaks per I O7 daltons of DNA per J m-2 were calculated flora published data4°. Total daily UV exposures at Bukhara (open squares) and Tumaco (solid diamonds) were modeled from Nimbus 7 Satellite measurements (H. Pitcher, pers. commun.). ~ ) 1992, Elsevier Science Publishers Ltd, (UK)

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several days are as effective as when they are given all at once. Furthermore, the effects of UVB persist for months once suppression of immunity is induced 9'11. Thus, studies of the immunological effects of even kiloJoule exposures of UVB may be physiologically relevant. A growing body of epidemiological and clinical evidence suggests that UVB irradiation can affect both pathogenesis and immunity in infectious diseases involving skin. The effect of UV irradiation on the progression of skin disease has been the subject of study for almost a century (for Review, see Ref. 12). The negative effects of exposure to UVB have progressively emerged, despite decades of 'good press' starting with Niels Finsen, who was awarded the Nobel Prize in 1903 for successfully treating lupus vulgaris, or tuberculosis of the skin, with UV irradiation (which he called 'chemical rays'). Until the advent of sulfa drugs, actinic therapy (the application of UV radiation) was the only effective treatment for erysipelas (for Review, see Ref. 12). In a more recent study, the skin lesions of herpes zoster were ameliorated by irradiation with erythemal doses of UVB 13. Note that, despite the seemingly beneficial cosmetic effect of UV on the appearance of infected skin, no long-term follow-up was done in these studies with respect to numbers of viable causative agents in irradiated skin, or later incidence of systemic disease. Yet UV can cause adverse effects in other infectious diseases. Finsen showed that smallpox lesions are worsened by exposure to sunlight ~4. The lesions of herpes simplex viruses type I (HSV-I) and II (HSVII) are re-activated by exposure to UVB 15-17. Epidemiological evidence suggests that exposure of immunosuppressed patients to sunlight leads to increased incidence of viral warts caused by papillomavirus ls49, presumably due to the UVB component. Long-term exposure to UVB is now associated with increased incidence of skin cancer and clearly contributes to the aging of skin. Considerable evidence in several animal models indicates that exposure to UVB during the first

encounter with viral, bacterial, fungal and protozoan infectious disease agents or their antigens modulates the immune response2°. UVB irradiation altered primary lesion development, reactivated healed lesions, increased numbers of organisms and/or abrogated delayed-type hypersensitivity responses in models for HSV-I, HSV-II, Moraxella

bovis, Mycobacterium bovis, Candida albicans, Plasmodium yoelii and P. chabaudi. UVB-irradiated mice, infected with agents that are usually nonlethal, developed high mortality rates in some experiments, compared with infected, nonirradiated controls. It is evident from these observations that UVB profoundly affects the ability of the host to mount an immune response against invading micro-organisms. This is well illustrated by a mouse model system for leishmaniasis. Effect o f U V B on cutaneous leishmaniasis in mice

Sandfly vectors deposit Leishmania in the upper layer of the dermis or the epidermis, which is also most likely to be exposed to UVB in sunlight. Disease progresses in two phases: (1) replication of organisms in the skin, where they may or may not induce an ulcer (Oriental sore, the most common form of leishmaniasis) and (2) in some individuals, dissemination of the organisms beyond the primary skin site and draining lymph nodes to distal skin (to cause incurable diffuse cutaneous leishmaniasis), mucocutaneous membranes (mutilating mucocutaneous leishmaniasis, or espundia) or visceral lymphoid organs (systemic, often fatal, visceral leishmaniasis or kala-azar). Known risk factors include the species of parasite 21 and the genetic background of the host (for Review, see Ref. 22). We have developed a mouse model to investigate the effects of UVB on pathogenesis and immunity in cutaneous leishmaniasis. The model applies to two strains of mice: B10.129(10M)Sn z3'24 and the more readily available C3H/HeJ. Both of these strains develop persistent, but non-fatal, skin lesions after infection with L. major. In both strains, low doses of UVB irradiation (<1 kJ m -2) suppress

Table I. Effect of U V B irracliation of the primary injection site on lesion severity and numbers of Leishmania in skin and lymph nodes of C 3 H / HeJ mice

Experiment number a

T i m e post infection

Treatment

Pathology index b of primary lesion

I

4 weeks

2

7 weeks

3

3 months

4

6 months

Control UVB Control UVB Control UVB Control UVB

2.0; area 24 mm z 2.0; area d 17 mm z 2.0; area 30 mm 2 2.0; area d 8.5 mm 2 2.0 1.0d 3.0 1.5c

Log Leishmania c Skin 2.0 2.0 2.0 4.0 ne e ne 3.0 2.5

L y m p h nodes 2.3 2.8 3.0 5.5 4.0 4.0 3.0 3.5

=Mice in groups of five were UVB-irradiated (600Jm -z) on the tails, 48 and 24h before infection. They were injected intradermally with I × J0 6 L. major stationary phase promastigotes and then irradiated every 2-3 days for one month. b Pathology index of I = no lesion or depigmentation only; 2 = nodule; 3 = ulcer ~<5 mm in diameter; 4 = ulcer >5 mm in diameter; 5 = metastasis to distal skin; 6 = death. c Equal amounts of tissue were triturated in I ml medium; 10 !~1 of triturate was assayed by limiting dilution culture. d Differences significantat P<0.05, by the Wilcoxon Rank Sum Test. e Ne, parasites present but no~cenumerated.

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the induction of contact hypersensitivity to dinitrofluorobenzene (DNFB) applied to irradiated skin. In both strains, similarly low doses of UVB irradiation convert these moderately susceptible mice into seemingly resistant hosts that have cosmetically less severe skin lesions. But the actual parasite load is not reduced in the UVB-irradiated, lesion-free mice, which have the same or larger numbers of parasites in skin at the injection site and in the draining lymph nodes, compared with the nonirradiated, obviously diseased mice (Table 1 and Refs 23-25). The lack of effect of low doses of UVB on parasite viability and infectivity, in vivo and in vitro, indicates that the target of UVB is the host skin immune system rather than Leishmania 23. The critical role of the immune system is illustrated by the data in Table 2, which show that UVB irradiation modulates primary lesion development only in strains of mice, such as the C3H/HeJ and B10.129(10M)Sn, that are UVB-sensitive, ie. in which low doses of UVB suppress the induction of contact hypersensitivity to a sensitizer, such as DNFB, applied to irradiated skin. In contrast, the BALB/c mouse is UVB resistant, and neither its response to a contact sensitizer nor the development of primary lesions of cutaneous leishmaniasis are affected by low doses of UVB (data not shown). The ulcer of cutaneous leishmaniasis is largely caused by the immune response, so in retrospect it is not surprising that immune depression can ameliorate the dermatologic symptoms without reducing parasite numbers. This is a particularly crucial point for the evaluation of the efficacy of vaccines and other therapies, where reduction in lesion severity is often taken as evidence of protection. Although this interpretation is probably valid in most cases, our results indicate that treatments that may induce tolerance to the parasites can abrogate lesion development although the total parasite load in the tissues is unchanged, or increased.

In addition to affecting pathology at the primary injection site in UVB-sensitive mice, broadband UVB abrogates protective immunity against a subsequent re-infection at a distal skin site. Infection with L. major generally confers immunity to subsequent infections, so that skin lesions resulting from a later challenge infection are either reduced in size or do not develop. UVB-irradiated or nonirradiated B 10.129(10M) Sn mice were infected with L. major and then challenged two weeks later in a nonirradiated distal skin site. Throughout a fourmonth observation period, protective immunity was evident in the nonirradiated mice, in which lesions developing at the challenge site were significantly smaller than in the nonimmune controls; in contrast, the challenge lesions of the UVB-irradiated mice were indistinguishable from those of the nonimmune controls 25. In other experiments with C3H/HeJ mice, UVB irradiation of the primary injection site abrogated the development of protective immunity to a challenge infection at a distal skin site. Upon re-infection, UVB-irradiated mice were compared with nonirradiated, infected controis and were found to have higher numbers of Leishmania, detected by limiting dilution culture assay, in skin at the challenge site (lOglo = 1.5 versus none detected per 10~tl triturated skin, respectively; P < 0.05), which were accompanied by significantly more severe skin lesions (nodules versus no detectable lesions at the injection site, respectively; P < 0.01). UVB affects immunological memory as early as two weeks post infection with L. major 25. From this it is clear that resistance or susceptibility to leishmanial disease is promoted by early immunological events occurring in the skin and draining lymph nodes, events that are significantly perturbed by UVB irradiation. The doses of UVB effective in the mouse model (150-600Jm -2, depending on the mouse strain) were chosen to cause minimal modulation of the immune response, namely, local suppression of

Table 2. I m m u n o m o d u l a t i o n by U V B irradiation in UVB-sensitive mice

Treatment Dose of UVB (J m -z)

0 0 150 600 0 Effective dose f

Antigen applied DNFB ¢ DNFB DNFB L. m a j o r e L major

Effect of UVB irradiation of sensitization site BI0.129 (10M)Sn C3H/HeJ Ear thickness a % Suppression Pathology Ear thickness % Suppression index b (mm) (mm) 0+_0.3 0.6 _+ 0.3 1.0 ___0.5 0 1.3 _+ 0.6 0 0.4 -t- 0.5 100 d 0.8 + 0.2 20 0.4 -I- 0.2 60 d 0.6 _+ 0.6 100 d 3.0 I .S d -

Pathology index 4.2 2. I d

"Mice in groups of five were sensitized with DNFB five days before challenge, except for the first group, which received solvent alone. Mean +_ so are shown for differences in thickness between ear challenged with DNFB and ear painted with solvent alone, measured 48 h after challenge. b Pathology index of primary skin lesion at six months post infection: see footnote to Table I. DNFB, dinitrofluorobenzene, d Significantly different from nonirradiated control; P<0.05. e Mice were infected in the tail with I x 106 promastigotes. The injection site was irradiated at days - 2 and - I before infection, then three times per week for the next four weeks. f Effective doses to suppress induction of contact hypersensitivity to DNFB: 150 J m -2 for B I0.129(10M)Sn and 600 J m-z for C3H/HeJ.

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contact hypersensitivity. Such low exposures improved the cosmetic appearance of cutaneous lesions. However, over several days' exposure to equatorial midday sun, a dose of UVB can be acquired that is sufficient in mice to suppress the immune response to UV-induced tumors (calculated from Ref. 1). Such doses could easily be absorbed by people exposed to sunlight in the tropics and semitropics, where infectious diseases are endemic. The effects of UVB irradiation on pathogenesis in B10.129(10M)Sn mice show a pronounced wavelength dependence 24 in that 320 nm UVB significantly improves the cosmetic appearance of infected skin, while 290 nm does not. In experiments with mouse macrop]hage cell lines, the uptake of Leishmania promastigotes, in vitro, was not affected by sublethal doses of broad band UVB ranging from 5 to 6 0 0 J m -2 (Ref. 23). Nor did sublethal exposures to narrow band UV (half-bandwidth 10 nm) from 280 to 320 nm significantly affect the uptake of Leishmania (S.H. Giannini and A.M. Harris, unpublished; data not shown) for parasite:macrophage ratios of 1:1, 10:1 or 30:1, irradiated with doses of UVB that left 90% survivors or with doses that left 75% viable, macrophages. Therefore we conclude that UVB perturbs steps subsequent to phagocytic uptake, ie. antigen processing, antigen display, cytokine se,cretion and macrophage activation. Other investigators have shown that UVB irradiation of mice alters the secretion of the cytokines interleukin 1, interleukin 2, gamma-interferon 26, and tumor necrosis factor-alpha/cachectin 27, believed to modulate immunity in murine cutaneous leishmaniasis. Experiments are in progress to monitor the effects of UVB on cytokine profiles and pathogenesis to identify cellular mechanisms modulating the effects of UVB on immunity in leishmaniasis. In both B10.129(10M)Sn and C3H/HeJ mice, suberythematous leve.ls of UVB, which suppress the induction of contact ihypersensitivity, also alter the development of primary skin lesions, affect the development of delayed hypersensitivity reactions to leishmanial antigens and depress protective immune responses. In both strains the improved cosmetic appearance of primary skin lesions after exposure to low levels of UVB is accompanied by three negative factors: (1) a diagnostic indicator that infection has occurred is lost; (2) despite the absence of lesions, UVB-exposed, infected individuals remain heavily infected and (3) UVBexposed mice fail to develop protective immunity to re-infection. At a later time, immunosuppression caused by environmental or other factors could reactivate certain species of Leishmania to cause systemic disease. It is significant that the levels of UVB effective in our experimental model are considerably lower than those attainable over several hours of exposure to tropical sunlight,

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which might cause even more extensive immunosuppression.

Experimental versus clinical effects of UVB How relevant to clinical cutaneous leishmaniasis is our experimental model? Mice and humans appear similar in several respects in their responses to leishmanial infection and to UVB irradiation. By judiciously selecting a panel of inbred mouse strains, one can obtain, in laboratory mice, the full spectrum of leishmanial diseases and immune dysfunctions occurring in human infections. Together with epidemiological data, these experiments suggest a strong immunogenetic component to disease resistance in both species (for Review, see Ref. 28). UVB irradiation of mouse skin causes immunological and fine structural alterations 6 also seen in human skin, such as changes in Langerhans cell morphology 6'29. The density of Langerhans cells in human skin is diminished by exposure to sunlight and this effect is independent of melanocyte density or tanning response 3°. Limited data suggest that mice and humans may be immunologically sensitive to similar levels of UVB, which cause equivalent structural damage to skin in both species: selective damage to Langerhans cells (<150Jm-2), and virtual depletion of ATPase surface markers of these cells, along with damage to other epidermal cells (800 J m -z) (Ref. 6). For ethical reasons, only one type of photoimmunological effect has been experimentally induced in humans, namely, the suppression of local contact hypersensitivity (CHS) (although the minimum immunosuppressive dose of UVB is uncertain). Here, too, the relative sensitivities of the two species are similar: four daily exposures totaling 2400-2640Jm -2 for mice 31'3z or 5760Jm -2 for humans 33'34 almost completely abrogate the induction of CHS when the sensitizing dose of a contact allergen is applied within 24h to UVBirradiated skin. Chronically sun-damaged human skin shows a diminished ability to mount cell-mediated immune responses to contact sensitizers and to commonly encountered antigens 3°. Sensitivity to UVBinduced modulation of contact hypersensitivity is governed in mice by two or three independent genetic loci and is unrelated to pigmentation 31. Just as different strains of mouse may be UVB susceptible or UVB resistant, so is the response to UVB polymorphic in the human population, with approximately 40% of humans in the USA being susceptible to local immunosuppression by low doses of UVB. UVB susceptibility in humans also shows no relationship to skin pigmentation 33. The immunological effects of lifelong UVB exposure are poorly understood, but chronic UVB irradiation leads to premature aging of skin 35 and shortened life span 36. Acute exposure of susceptible individuals to UVB radiation, particularly at the

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time of initial infection, could influence the outcome of the infection process and the subsequent development of clinical disease. UVB-induced immunosuppression could thus be one of the significant factors in the persistence, severity and recrudescence of leishmanial infections in tropical populations. By extrapolation from the murine experimental model, the effects of UVB on clinical leishmaniasis could occur at several levels. In sub-tropical and tropical regions, high UVB exposures might abrogate or decrease the size of the lesion in UVBsensitive individuals, thereby eliminating this diagnostic indicator of infection. However, these individuals with minimal or no skin lesions would nevertheless remain infected. Subsequent to environmental or other immunosuppression at a later date, they could then develop disseminated leishmaniasis. Such reactivation is well documented in patients who are cryptically infected with certain species of Leishmania, who later develop acquired immunodeficiency syndrome and then full-blown visceral or diffuse cutaneous leishmaniasis 37'38. Furthermore, individuals exposed to UVB during a primary infection might fail to develop protective immunity, leaving them susceptible to recurrent infections with the same Leishmania species. In equatorial Tumaco, a focus of recurrent cutaneous leishmaniasis has been described in which reinfection with closely related but genotypically distinctive strains of L. braziliensis panamensis occurs within months or years of recovery from infection 39. Perhaps in this focus, UVB exposure plays a role in the failure of an effective immune response to develop. Conclusions Sunlight in the tropics and semitropics currently contains levels of UVB that are immunosuppressive in several experimental immunological systems, with effects ranging from local suppression of contact hypersensitivity to systemic suppression of tumor immunity. In these systems, the effects do not depend on the rate of exposure but rather on the total dose; the effects are cumulative. With ozone layer depletion, UVB levels will likely increase in temperate and arctic zones and even further in the tropics and semitropics. Epidemiological studies in humans suggest that exposure to sunlight (presumably the UVB component) may alter pathology and susceptibility to some infectious diseases. Exposures to levels of UVB, already attainable in the tropics and semitropics, affect pathogenesis and immunity in animal models for several infectious diseases, including cutaneous leishmaniasis. Further research is needed to determine, for UVB, the effective exposures, action spectra and cellular targets modifying pathogenesis and protective immunity in models for cutaneous leishmaniasis and other infectious diseases.

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Acknowledgements This work was supported in part by grants from the Medical •Biotechnology Center of the Maryland Biotechnology Institute. Edmond A. Goidl (University of Maryland School of Medicine, Baltimore, MD), Robert Luebke (US Environmental Protection Agency, Research Triangle Park, NC) and Janice Longstreth (Battelle Pacific Northwest Laboratories, Washington, DC) provided helpful comments and discussion. I am grateful to Hugh Pitcher (US Environmental Protection Agency, Washington DC, now at Pacific Northwest Laboratories), for providing the estimates of ambient UVB used in Fig. I. References 1 Frederick, J.E. (1986) in Effects of Changes in Stratospheric Ozone and Global Climate, Vol. I (Titus, J.G., ed.), pp 121-128, US Environmental Protection Agency 2 Rowland, F.S. (1991) Environ. Sci. Technol. 25,622-628 3 Kripke, M.L. (1984) Immunol. Rev. 80, 87-102 4 Krutmann, J. and Elmets, C.M. (1988) Photochem. Photobiol. 48, 787-798 5 De Fabo, E.C. and Noonan, F.P. (1990) in Laboratory Methods in Immunology, Vol. 2 (Zola, H., ed.), pp 77-95, CRC Press 6 Aberer, W. et al. (1981)J. Invest. Dermatol. 76, 202-210 7 Spangrude, G.J. et al. (1983)J. Immunol. 130, 2974-2981 8 Kripke, M.L. and Morison, W.L. (1986) Photodermatology 3, 4-14 9 De Fabo, E.C. and Kripke, M.L. (1979) Photochem. Photobiol. 30, 385-390 10 De Fabo, E.C. and Kripke, M.L. (1980) Photochem. Photobiol. 32, 183-188 11 Howie, S., Norval. M. and Malngay, J. (1986) J . Invest. Dermatol. 86, 125-128 12 Licht, S. (1983) in Therapeutic Electricity and Ultraviolet Radiation (Stillwell. G.K., ed.), pp 174-193, Williams & Wilkins 13 Szigeti, B., Chapiro, J. and Salnt-Girons, J.M. (1976) J. Radial. Electrol. 57, 549-551 14 Finsen, N.R. (1901) Phototherapy, Edward Arnold 15 Wheeler, C.E., Jr (1975)J. Invest. Dermatol. 65,341-346 16 Spruance, S.L. (1985)J. Clin. Microbial. 22, 366-368 17 Klein, K.L. and Linnemann, C.C., Jr (1986) Lancet i, 796-797 18 Boyle, J. et al. (1984) Lancet i, 702-705 19 DyaU-Smith, D. and Varigos, G. (1985) Aust. J . Dermatol. 26, 102-107 20 Giannini, S.H. (1990) in Global Atmospheric Change and Public Health (White, J.C., ed.), pp 33-45, Elsevier Science Publishers 21 World Health Organization (1984) WHO Tech. Rep. Ser. No. 701 22 Bradley, D.J. (1987) in The Leishmaniases, Vol. 2 (Peters, W. and Killick-Kendrick, R., eds), pp 551-581, Academic Press 23 Giannini, M.S.H. (1986) Infect. Immun. 51,838-843 24 Giannini, S.H. and De Fabo, E.C. (1989) in Leishmaniasis: The Current Status and New Strategies for Control (Hart, D.T., ed.), pp 677-684, Plenum Press 25 Giannini, S.H. (1986) in Effects of Changes in Stratospheric Ozone and Global Climate, Vol 2 (Titus, J.G., ed.), pp 101-112, US Environmental Protection Agency 26 Araneo, B.A. et al. (1989)J. Immunol. 143, 1737-1744 27 Vermeer, M. and Streilein, J.W. Photodermatol. Photoimmun. Photomed. (in press) 28 Howard, J.G. (1985) in Human Parasitic Diseases, Vol. I (Chang, K-P. and Bray, R.S., eds), pp 140-162, Elsevier Science Publishers 29 Scheibner, A. et al. (1986) Photodermatology 3, 15-25 30 O'Dell, B.L. et al. (1980)Arch. Dermatol. 116, 55%561 31 Streilein, J.W. and Bergstresser, P.R. (1988) Immunogenetics 27, 252-258 32 Cruz, P.D., Jr et al. (1988) Photodermatology 5, 126-132 33 Vermeer, M. et al. J . Invest. Dermatol. (in press) 34 Yoshikawa, T. et al. (1990)J. Invest. Dermatol. 95, 530-536 35 Johnson, B.E. (1984) Physiol. Pathophysiol. Skin 8, 2413-2492 36 Davies, R.E. and Forbes, P.D. (1986) in Effects of Changes in Stratospheric Ozone and Global Climate, Vol. 2 (Titus, J.G., ed.), pp 23-25, US Environmental Protection Agency 37 Fernandez-Guerrero, M.L. et al. (1987) Am. J. Med. 83, 1098-1102 38 Clauvel, J.P. et al. (1986) Tram. R. Sac. Trap. Med. Hyg. 80, 1011-1012 39 Saravia, N.G. et al. (1990) Lancet 336, 398--402 40 Rosenstein, B.S. and Ducore, J.M. (1983) Photochem. Photobiol. 38, 51-55