Vol. 51, No. 3INFECTION AND IMMUNITY, Mar. 1986, p. 838-8430019-9567/86/030838-06$02.00/0Copyright © 1986, American Society for Microbiology

Suppression of Pathogenesis in Cutaneous Leishmaniasis byUV IrradiationM. S. H. GIANNINI

Division of Tropical Medicine, Columbia University, College of Physicians & Surgeons, New York, New York 10032

Received 19 August 1985/Accepted 30 November 1985

The effects of suberythematous levels of UV-B radiation on the development of cutaneous leishmaniasis werestudied in B10.129(10M) mice. Doses of 15 mJ cm2 UV-B applied locally to the injection site suppressed thedevelopment of skin lesions after the inoculation of Leishmania major promastigotes. The primary targets ofUV-B radiation appeared to be host cells and not leishmanial parasites, because UV-B irradiation of parasitescultured in vitro did not affect their viability, but did kill host cells. Furthermore, the same numbers ofparasites were recovered from skin at the in,ection site in both irradiated and control mice. UV-B irradiationabrogated the induction of contact hypersensitivity to dinitrofluorobenzene and also abrogated the induction ofdelayed-type hypersensitivity responses to leishmanial antigens. These results suggest that local perturbationsin the functions of the skin-associated lymphoid tissue during the initial phases of leishmanial infection canprofoundly influence immunological response and the subsequent development of clinical disease.

Cutaneous leishmaniasis results from infection by orga-nisms of the Leishmania species, obligate intracellular pro-tozoan parasites of macrophages, monocytes, and histi-ocytes. Leishmania species are transmitted by phlebotomineflies (sandflies). Depending on the species of Leishmania,infection may result in a broad spectrum of cutaneousdiseases, encompassing asymptomatic infection, transientinduration, a self-limiting skin ulcer (Oriental sore), nonheal-ing skin lesion (leishmaniasis recidivans), metastasis to distalskin sites (incurable diffuse cutaneous leishmaniasis), ormetastasis to the mucocutaneous junctions (nonhealingespundia or mucocutaneous leishmaniasis).

Little is known about the development of pathology duringthe cutaneous phase of leishmaniasis. Certainly the im-munogenetic background of the host is a major factor in theresponse to cutaneous leishmanial infection. Certain ethnicgroups of humans are more susceptible than others tomutilating lesions of mucocutaneous leishmaniasis (31) andto leishmaniasis recidivans (14). In mice, the sequelae toleishmanial infections are characteristic for each inbredstrain (3, 5, 7, 8, 15, 16). Yet not all infected individuals ingroups at high risk for these sequelae develop severe dis-ease. Thus, other factors are probably involved.Cutaneous leishmaniasis in humans generally occurs on

parts of the body that are naturally exposed both to the bitesof sandflies and to solar radiation. UV light in the sunburnportion of the spectrum, 280 to 320 nm (herein referred to asUV-B), is known to have profound effects on the immunesystem (reviewed in references 4, 9, 17, 20, and 27). Thepurpose of this study was to determine whether local per-turbations in the function of the skin-associated lymphoidtissue (28) during the initial phases of leishmanial infectionwould influence the development of cutaneous disease.

MATERIALS AND METHODS

Parasites. Leishmania major (Leishmania tropica major)strain WR300 promastigotes were used throughout. Thehistory of the strain and methods of maintenance, enumer-

ation, cultivation, and harvest have been described else-where (lla).

Hosts. Male and female BALB/cJ mice and maleB10.129(10M) ScSn mice (25) (Jackson Laboratory, BarHarbor, Maine) were sex and age matched and used forexperiments between 1 and 4 months of age. TheB10.129(10M) strain is chronically susceptible to visceralleishmaniasis (8), unlike most other inbred strains, andrelatively resistant to cutaneous leishmaniasis (7). Femalesare much more susceptible than males, however (S. H.Giannini, Parasite Immunol., in press). BALB/c mice arehighly susceptible to cutaneous leishmaniasis, developingexpanding skin ulcers with fatal metastases to distal skinsites and internal lymphoid organs (16).J774A.1 cell line. J774A.1, a macrophagelike cell line of

BALB/c origin (23), was obtained from the American TypeCulture Collection (Rockville, Md.) and maintained at 35°Cas described by Chang (6).UV-B irradiation source. Mice or cultures were irradiated

with a Waldmann UV800 UV-B source (Schwenningen,Federal Republic of Germany) equipped with six Phillips TL20W/12 UV-B bulbs. Output was monitored with an IL-782spectroradiometer (International Light, Newburyport,Mass.). The peak output of the light source was at 300 nm,with 79% of the UV output occurring between 280 and 320nm (the remaining output occurred at wavelengths >320nm). The flux was 1.33 mW cm-2, and the time to deliver 15mJ cm-2 was 12 s.UV irradiation of mice. Mice were restrained in plastic

holders, and their tails were immobilized with tape at theirtips. The dorsal surfaces of their tails were irradiated,whereas their bodies were shielded with aluminum foil. Tocontrol for heat and stress induced by handling, unirradiatedcontrol mice were similarly restrained, but both their bodiesand tails were shielded from the UV light source.

Effect of UV-B on the induction of contact hypersensitivity.Mice were shielded or irradiated on their tails with 60 or 15mJ of UV-B per cm2 (described above), sensitized 24 h afterirradiation, and challenged after 5 days with dinitrofluoro-benzene (DNFB) as described previously (29). These doses

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have been shown selectively to damage Langerhans cells,which function as antigen-presenting cells in the skin (1).Mice were then coded, and ear thicknesses were measuredwith micrometer calipers (Fisher Scientific Co., Springfield,N.J.) at 4, 24, and 48 h. Results are expressed as thedifference between the thicknesses of the ears (DNFBpainted minus solvent painted).

Irradiation schedule and infection of mice. At 24 h afterirradiation, mice were anaesthetized with ketamine hydro-chloride (Bristol Laboratories, Syracuse, N.Y.) and injectedintradermally in the dorsal surface of the tail with 106promastigotes in a 10-pu volume. At 24 h after infection, andsubsequently every 48 to 72 h for 1 month, the UV-Birradiation treatment was repeated to maintain immunosup-pression. This was necessary because the number of cellsbearing surface markers characteristic of antigen-presentingcells reaches a minimum 48 h after the skin is irradiated andthen begins to return to normal levels (1, 29).

Assessment of lesion pathology. The extent of lesion devel-opment was recorded weekly for infected mice. As asemiquantitative measure of pathology, each category ofcutaneous lesion appearance was assigned a numericalvalue. The indices of pathology and the lesions they repre-

sented were as follows: 1, no lesion or healed lesion; 2,nodule or swelling (5-mm diameter); 3, small ulcer, thediameter of the nodule; 4, expanding ulcer (larger than thenodule, diameter >5 mm); 5, tail loss or metastatic skinlesion; 6, death.

In some mice, a small depigmented macule 1 to 5 mm indiameter was observed at the injection site. Such depigmen-tation in the absence of induration was also scored as 1,because depigmented areas on the tail were present inuninfected B10.129(10M) mice.UV irradiation of J774A. 1 cells and quantitation of infection

in vitro. J774A.1 cells were plated overnight in 35- by 10-mmpetri dishes (Falcon no. 1008; Becton Dickinson Labware,Oxnard, Calif.) at a density of 2 x 105 cells per cm2. Dosesof irradiation are given in Table 2. Cultures were irradiatedor shielded and infected 24 h later with L. major promasti-gotes in a ratio of five promastigotes per host cell. Cells wereirradiated again at 24 and at 72 h after infection; controlswere shielded. Cells were harvested 48 h after the lastirradiation.

Petri dishes containing J774A.1 cells were gently washedthree times to remove nonadherent cells and free parasites.The host cells per 1-cm2 area of the petri dish were countedby using an inverted microscope fitted with a calibratedocular grid. Four representative fields per plate were

counted at x256 magnification. Cells were then suspendedwith a rubber policeman in 4 ml of medium. Cytospinpreparations were made (Shandon Southern Instruments,Inc., Sewickley, Pa.), fixed in absolute methanol, andGiemsa stained. The ratio of parasites per host cell was thendetermined microscopically at x 1,000 magnification.

Determination of the viability of leishmanias after UV-Birradiation. For in vitro experiments, infected irradiatedJ774A.1 cells and infected unirradiated controls were sus-

pended as described above.For in vivo experiments, tail skin surrounding the injec-

tion site was removed (-1.5-cm length) and triturated in 0.5ml of Schneider's medium (GIBCO Laboratories, GrandIsland, N.Y.) supplemented with 20% (vol/vol) fetal bovineserum (Hyclone; Sterile Systems) and 1% (vol/vol) of a 100xantibiotic-antimycotic solution (GIBCO) (complete me-

dium).Parasite viability was determined by culturing serial 10-

fold dilutions of triturated tail skin or suspended cells inflat-bottomed 96-well microtiter plates (Linbro; Flow Labo-ratories, Inc., McLean, Va.) as previously described (lla).A culture well seeded with one or more L. major amastigoteswill become positive within 2 to 10 days (lla ).

Delayed-type hypersensitivity (DTH) response to leishma-nial antigens. For leishmanin skin testing, antigen was pre-pared by freezing and thawing 2 x 108 L. major promasti-gotes per ml in 0.01 M phosphate-buffered saline, pH 7.2.Mice were injected intradermally in the right rear footpadwith 10 Ll of antigen and in the left rear footpad with 10 [LI ofphosphate-buffered saline. Footpad thicknesses were mea-sured with micrometer calipers (Fisher) at 24 and 48 h.

Statistical analysis. The statistical significance of resultswas determined by the Wilcoxon rank sum test (32), whichdoes not require a normal distribution of data.

RESULTS

Effects of UV-B on development of contact hypersensitivityand cutaneous leishmaniasis. Doses of 60 and 15 mJ of UV-Bradiation per cm2 applied to the site of sensitization abro-gated the induction of contact hypersensitivity (Table 1).The lower dose was subsequently used to assess the effect ofUV-B irradiation on cutaneous leishmaniasis. It is importantto realize that this amount of radiation, 15 mJ cm-2, is wellbelow the minimum erythematous dose of 20 to 40 mJ cm-2(20) and is less than the amount of UV-B radiation in 1 h ofexposure to sunlight on a bright day at noon (11).With the lower dose of UV-B which suppressed sensitiza-

tion of mice to DNFB, the development of cutaneousleishmaniasis in a relatively resistant host, the B10.129(10M)male, was indeed affected (Fig. 1). Surprisingly, lesiondevelopment was suppressed in irradiated mice. As early as2 months postinfection, most of the irradiated mice had nolesions, whereas control mice were well on their way toulceration (Fig. 1) (P < 0.01).Lack of effect of UV-B on parasite viability. To determine

whether UV-B directly damaged the parasites, the J774A.1cell line, infected with L. major promastigotes, was irradi-ated on a schedule similar to that used for the mice.Radiation did not reduce the numbers of leishmania per hostcell in infected cultures. However, the number of host cellsin cultures irradiated with 15 mJ cm-2 was reduced, com-pared with unirradiated or untreated controls (Table 2).The lack of a parasiticidal effect of UV-B was further

TABLE 1. Suppression of contact sensitivity to DNFB by UV-Birradiation in mice

Difference in ear thickness"(mm x 10-2)

TreatmentB10.129 BALB/c

(1OM) mice mice

Unirradiated, sensitized, 12.5 ± 6.4 4.2 ± 2.3challenged

Unirradiated, not sensitized, 5.7 ± 2.7" 0 ± 1.3"challenged

60 mJ cm-2 UV-B, sensitized, 5.7 ± 6.2" 2.4 ± 1.6challenged

15 mJ cm-2 UV-B, sensitized, 3.8 ± 5.2" 2.8 ± 2.3challenged

" Difference between thicknesses of DNFB-challenged ear and ear paintedwith solvent. Values given are means + standard deviations; four to six miceper group.

bSignificantly different from unirradiated sensitized controls (P < 0.05).

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FIG. 1. Effect of local UV-B irradiation on the development ofcutaneous lesions in mice. Groups of nine mice were infectedintradermally in the tail with 106 L. major promastigotes. Irradiatedmice (=II) received 15 mJ of UV-B per cm2 on the tails at 24 hbefore and 24 h after infection and every 48 or 72 h thereafter for 4weeks. Control mice (_) were shielded. Lesion development was

significantly different at 2 months postinfection (P < 0.01).

confirmed in vivo. In two experiments, the numbers ofparasites recovered from irradiated injection sites were

not significantly different than from shielded controls (Table3).Taken together, these results suggest that the primary

effect of UV irradiation is on the host cell.Long-term effect of UV-B irradiation at inoculation site. In

a second in vivo experiment, the development of lesionpathology was followed for 5 months after the final UVirradiation, for a total of 6 months. A progressive dichotomyin the severity of lesions in the control and irradiated micewas clearly present (Fig. 2), evidence of a long-lasting effectof early immunological events on subsequent developmentof pathology.

Suppression of lesion development despite presence of par-asites in skin. To determine whether the reduced lesiondevelopment in the UV-B-irradiated groups might resultfrom the killing of potential host cells and hence reflect anabsence of parasites, leishmanias were sought in skin eycised from the injection site. Despite highly significantreductions in lesion pathology (P < 0.01), the skin wasinfected in 100% of animals, even those with no discernible

TABLE 3. Effect of UV-B irradiation in vivo" on numbers ofviable L. major organismsb recovered from skin at injection sites

of B10.129(10M) mice

Expt Time post- Log amastigotes per 15 1.l of triturated skinExpt infection (mo) 15 mJ cm-2 Shielded

UV-B controls

1 3.5 3.3 ± 3.2 1.3 ± 1.5 NS'2 9 8.5 ± 2.6 8.0 ± 3.1 NS

"Groups of four to eight mice were irradiated with 15 mJ of UV-B per cm2on the tails 24 h before and 24 h after infection with 104 (experiment 1) or 106(experiment 2) L. major promastigotes and subsequently irradiated every 48or 72 h for 4 weeks. Male mice (experiment 1) or female mice (experiment 2)were used.bDetermined by culturing serial 10-fold dilutions as described previously

(la).c NS, Not significant.

lesions (Table 4). These results show that the suppression oflesion development induced by UV-B irradiation does notindicate an absence of leishmanias.Absence of DTH response to leishmanial antigens in UV-B

irradiated mice. Because UV-B irradiation of the site ofapplication of antigen was able to suppress the developmentof contact hypersensitivity to DNFB, the effect of UV-Birradiation on the animals' subsequent ability to mount aDTH response to parasite antigens was tested in a third invivo experiment. At 2 and 6 weeks after infection, irradiatedmice challenged intradermally with leishmanial antigensshowed minimal DTH responses, compared with the unirra-diated controls (Table 5).

DISCUSSION

This study was designed to investigate whether low dosesof UV-B at the inoculation site at the time of infection couldinfluence the subsequent development of clinical disease incutaneous leishmaniasis. It is important to note that theeffective dose of radiation used here, 15 mJ cm-2, is wellbelow the minimum erythematous dose of 40 mJ cm2 (20)and is the minimal amount of radiation shown by othersselectively to damage Langerhans cells (1). This dose isconsiderably lower than those used in earlier studies ofsuppression of contact hypersensitivity by UV-B irradiation(4, 9, 10, 27, 29). Low levels of UV-B irradiation, similar tothose used in the present study, would likely be encountered

TABLE 2. Growth and viability of L. major in UV-B-irradiated J774A.1 cells in vitro

Expt UV-B Leishmanias per Host cells per Leishmanias Log1o viableExpt (mJ cm-2) 100 host cells" 0.06 mm2" per 0.06 mm2" parasites"1 15 64 + 9 39± 14" 25 Not done

15" Not infected 45 ± 20' 0 Not doneNone 19 + 4 83 31 16 Not doneNoned Not infected 91 ± 15 0 Not done

2 15 360 40 17 10' 61 4.5 15 330 11 50 21 164 5.2 11.5 330 60 45 16 150 4.3 10.5 300±50 61±16 183 5.2±1None 350 ± 10 43 ± 16 150 5.0 1

"Means + standard deviations of four cultures.Calculated by multiplying the mean number of leishmanias per host cell times the mean number of host cells per 0.06 mm2.

'*Significantly lower than unirradiated control (P < 0.01).dControl for the effect of L. major parasites on growth of host cells; cultures were not infected.

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FIG. 2. Effect of local UV-B irradiation on the long-term devel-opment of cutaneous lesions in mice. Groups of eight or nine micewere infected intradermally in the tail with 106 L. major promasti-gotes. Irradiated mice (=II) received 15 mJ of UV-B per cm2 as

described in the legend to Fig. 1. Control mice (_) were shielded.Lesion development was significantly different at 6 months postin-fection (P < 0.01).

by populations at risk for leishmaniasis, especially ruralpopulations.A single dose of 15 mJ cm2 abrogated the development of

contact hypersensitivity in B10.129(10M) mice, but not inBALB/c mice (Table 1). That BALB/c mice (as well as some

other strains) are radiation resistant has been shown byothers (4). Consistent with this, UV-B irradiation does notaffect lesion development in BALB/c mice (data not shown).Contrary to what might be expected, radiation resistance,which varies among strains of mice, is not correlated withskin pigmentation (4). We might then predict that any effectsof UV-B on the functioning of the skin-associated lymphoidtissue in humans need not be race specific, but rather mightreflect the workings of some other genetic polymorphism.

Several conclusions may be drawn from this study. Ofparticular significance is the finding that the absence of a skinlesion need not necessarily indicate an absence of leishma-nial parasites. This is a particularly crucial point for theevaluation of the efficacy of vaccines, where reduction inlesion development upon challenge is considered evidence ofprotection. Although this interpretation is probably valid inmost cases, results presented here indicate that treatmentswhich may induce tolerance to the parasites can suppress

TABLE 4. Reduction of lesion pathology despite presence ofparasites at injection sites of mice irradiated with UV-B and

infected with L. major

Duration of Lesion pathology indexainfection (mo) Controls Irradiatedb P

2 2.5 ± 0.8 1.1 ± 0.4 <0.016 4.2 ± 0.4 2.1 ± 1.4 <0.01

a Lesion pathology indices: 1, negative; 2, nodule; 3, small ulcer (<5-mmdiameter); 4, large ulcer (>5-mm diameter); 5, tail loss or metastatic skinlesions. Results are means ± standard deviations for groups of seven to ninemice.

b Mice were irradiated on the tails with 15 mJ of UV-B per cm2 and infected24 h later. They were irradiated 24 h postinfection and subsequently every 48to 72 h for 4 weeks. The Log viable amastigotes per 15 pJ of triturated skin was>6.0 ± 0 for all mice in both groups (not significant).

lesion development without reducing parasite numbers in thetissues.

It is also evident that low doses of UV-B, such as wouldlikely be encountered by persons at risk for leishmaniasis,can suppress the development of cutaneous lesions withoutkilling leishmanias in the skin. The main targets of UV-Birradiation are host cells, and one of the effects of UV-Birradiation in vivo is the abrogation of DTH responses toleishmanial antigens in hosts irradiated at the injection siteduring the early stages of infection. The types of lymphoidcells known to be sensitive to UV-B include Langerhanscells (1, 29), primed T cells (19), and keratinocytes (2, 24),whereas T suppressor cells are relatively resistant and mayeven be induced by UV-B irradiation in vivo (10, 12, 13). Theidentity of the particular host cell type(s) affected remains tobe elucidated by further studies in vitro, now in progress.

Protective immunity in leishmaniasis is cell mediated (21,22, 26), and a DTH response to leishmanial antigens isgenerally considered indicative of the onset of healing.However, it has recently been shown that murine cutaneousleishmaniasis is exacerbated by adoptive transfer of helper Tcells which mediate Leishmania-specific DTH (30), whereasprotection against murine leishmaniasis is transferred byLyt-1+2- cells that do not mediate a cutaneous DTH (18).Certainly in human infections, both mucocutaneous leishma-

TABLE 5. DTH responses of UV-B-irradiated micea infectedwith L. major after challenge with leishmanial antigensb

Difference in footpadMice

Time post- thickness' (10-2 mm)infection (wk)

4 h 24 h 48 h

Uninfected controls 0 8 0

Unirradiated 2 8d 25d 12dUV-B irradiated 2 0 11 0

Unirradiated 6 12d 1l 18dUV-B irradiated 6 0 0 0

a Four to five B10.129(10M) mice per group were UV-B irradiated with 15mJ cm-2 on the tails 24 h before and 24 after infection with 106 L. majorpromastigotes and subsequently every 48 or 72 h for 4 weeks.

I Mice were challenged in the left rear footpad with 10 p.l of PBS containing2 x 106 promastigotes, solubilized by freezing and thawing in PBS. The rightrear footpad was injected with 10 p.l of PBS.

' Thicknesses of left footpads minus those of right footpads. Median valuesare given because lesion pathology (and therefore the responses of the mice)are not normally distributed (Fig. 1 and 2).

d Significantly different from the primary response of uninfected controls (P< 0.05).

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niasis and leishmaniasis recidivans elicit strongly positiveantiparasite DTH responses accompanied by extensive le-sions with no tendency to self-heal. It may be that thecell-mediated immunity in leishmaniasis consists of twocomponents, one affording protection and the other mediat-ing tissue destruction. Identification of specific parasiteantigens eliciting each type of immune response could pro-vide the basis for rational development of vaccines.

Finally, we can conclude that local perturbations in thefunctions of the skin-associated lymphoid tissue during theinitial phase of leishmanial infection can profoundly influ-ence the immunological response to the parasites and thesubsequent development of clinical disease.

ACKNOWLEDGMENTS

Benvenuto Pernis, Jeanette Thorbecke, Vincent DeLeo, FrancisGasparro, Leonard Harber, and Philip D'Alesandro made manyhelpful comments and suggestions. Expert technical assistance wasprovided by Susie Azon and Allan Ho. The manuscript was greatlyimproved by the critical evaluation of Edmond Goidl, who alsocontributed helpful advice during the course of the work.

This study was supported by Public Health Service grant Al 18937from the National Institutes of Health.

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