Effects of ultraviolet radiation on the pathogenesis of Mycobacterium lepraemurium infection in mice Jeevat-i A. Gilliat-n K, Heard H. Kripke M L. Effects of ultraviolet radiation on the pathogenesis of Mycohacterium lepraemurium infection in mice. Exp Dermatol 1992: 1: 152 160. Abstract: The purpose of this study was to determine whether exposing mice to ultraviolet radiation (UVR) would alter the pathogenesis of infection with Mycobacleriwn lepraemurium (MLM), which causes a chronic, progt-essive, lethal disease in susceptible mouse strains. BALB/c mice were irradiated on dorsal skin with various doses of UVR from FS40 sunlamps 3 days befot-e infection with MLM in the hind footpad. The cour.se of disease was followed by assessing the nut-nber of acid-fast bacteria in the footpad, regional lymph node and spleen, and measuring the size of the lesion at the site of MLM infection at various times after infection. Mice were also tested periodically for a delayed-type hypersensitivity (DTH) t-esponse by it-ijecting MLM antigen into the uninfected footpad and t-i-ieasuring footpad swellit-ig 24 hours later. Mice treated with a single high dose of UVR (45 kJ/m") had significantly more bacteria in the infected footpad, lymph node and spleen than unirradiated control anit-nals. They also had larger lesions at the site of MLM infection and exhibited significant suppression of the DTH response at 3 and 6 months after infectiot-i. It-ijection of mice s.c. in the footpad with MLM 3 d after 45 k.l/t-n- UVR t-educed the t-nedian survival tit-|-ie from 391 to 305 d and after j.v. infection from 171 to 139 d. Dose-response studies indicated that exposing mice to 2.3 kJ/m- of UVR, which is approximately 1 minimal et-ythemal dose for this strain, suppressed the DTH t-esponse by 50% at 3 n-ionths after infection. Significant increases in the number of bacteria in the footpad, spleen and lyt-i-tph node were detected with doses of UVR>5.6 kJ/n-)-. Mice exposed chronically to UV radiation also showed impaired responses to MLM. Thus, exposing mice to a single or multiple doses of UVR before infection increased the severity of a chronic mycobacterial infection and decreased the it-nmune response to mycobacterial antigej-is.

Introduction Mycobacterial diseases, such as leprosy and ftiberculosis, are major public bealtli problems in developing countries. It is estimated that there are 15 million cases of leprosy in the world today and 10 tiiillion new cases of tuberculosis annually (1, 2). In the United States, mycobacterial infections have assumed increasing importance because of their rising incidence and because of atypical t-i-iycobacterial infections in persons with AIDS (3-5). Mycohacterium lepraemurium (MLM) is an obligate intraeellular pathogen that causes a slow, progressive disease in susceptible mouse strains (6). Although the chronic disease process caused by 152

Amminikutty Jeevan, Kelly Gilliam, Hillary Heard and Margaret L. Kripke Depaitment of Immunology, The Univeisity ot Texas M. D. Anderson Cancer Center, Houston, Texas, tJ.S.A.

Key woids: UVR, ultraviolet radiation; MLM, Mycobacterium lepraemurium: DTH, delayed-type hypersensitivity: AFB, acid-fast bacteria Amminikutty Jeevan, Ph.D., Department of Immunology, Box 178, The University ot Texas M. D. Anderson Cancer Centei, 1515 Holcombe Boulevard, Houston, TX 77030, U.S.A. Accepted for publication 14 September 1992

MLM resetnbles that of human leprosy in sotne respects (6, 7), infection of mice with MLM is generally used as a rnodel of chronic mycobacterial infection (8). Some inbred strains of mice develop a progressive, lethal disease from MLM infection; others develop a self-limiting infection. The outcorne of the disease is also influenced by the dose of bacteria injected and route of injection, in addition to genetic factors (9-11). Resistance to this and many other intracellular pathogens is thought to be tnediated by cellular, rather than hutnoral. immune responses (6, 7), and the delayed-type hypersensitivity (DTH) response has been shown to be an important protective mechanism in controlling these infections (12-15).

Effect of UVR on MLM infection Ultraviolet radiation (UVR) was once used to treat patients with rnyeobaeterial infections (reviewecl in 16), but in recent years it has been shown to impair cellular itntnune responses, including DTH (17-21). The demonstration that UV irradiation can impair cellular itnmune responses to antigens introduced locally, within the UV-irradiated site (19, 20), and at distant, unirradiated sites (17, 18, 21) has raised the question of whether UV irradiation might also interfere with the development of imtnune responses to tnicrobial pathogens and thereby increase the severity of infectious diseases. This question has been addressed in studies of tnice infected with herpes simplex virus types 1 and 2 (22-25), Leishmania major (26), Candida albieans (27, 28), Sehistosoma numsoni (29), and Myeobacteriuin bovis BCG (29-31). However, none of these studies addressed the ability of UV irradiation to influence the course of a chronic, lethal disease process. Therefore, we have exatnined the effects of UV radiation on the pathogenesis of a chronic, progressive infection in mice caused by MLM. Material and methods

Mice Inbred, female BALB/cAnNCr mice were obtained from the NCI Frederick Cancer Research Facility Anitnal Production Area (Frederick, MD). Mice were 8-12 wk old at the beginning of each experiment. The animals wet e housed in a specific-pathogen-free facility, accredited by the American Association for Accreditation of Laboratory Anitnal Care, for at least 2 wk before use in experiments. Mice were given free access to National Institutes of Health fortnula 31 tnouse fbod and sterilized water; ambient lighting was autotnatically controlled to provide 12-h light - 12-h dark cycles. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee. Source of MLM The stock cultures of MLM (Douglas strain) bacilli were obtained frotn Dr. G. L. Asherson, Division of Imtnunological Medicine, MRC Clinical Research Centet; Harrow, UK. A large batch of MLM was grown in C3H tnice infected i.v. 14-18 wk previously with 10'' MLM (32). The bacilli were harvested frotn the infected liver and spleen of mice by the method described previously (32-34). Briefly, tissues were hotnogenized in 4 tnl of Trissaline medium (0.15 M NaCl, 2 M Tris base) and centrifuged at lOOOOx g for 10 min at 4 X . The sediment was washed twice in Tween-saline (0.15 M NaCl, pH 7.2; 1 M MgSO4 and 0.1% Tween

80). The pellet was then suspended in buffered I M Tween-saline (0.1% Tween 80), MES (2-Nmorpholinoethane sulfonic acid, pH 6.8), and Percoll (Sigma Fine Chemicals) was added to give a final concentration of 30%. This suspension was centrifuged at 27 000 x g for 40 min at 4 C. The band of bacteria at the top layer was collected, washed once in Tween-saline, and resuspended in 0.15 M NaCl. The acid-fast bacteria were counted by the standard smear technique (35).

UV irradiation of miee The technique of UV irradiation has been described before (30). Briefly, tbe dorsal hair of mice was retnoved using electric clippers, and the miee were exposed to single or multiple doses of UV radiation from 6 FS40 sunlamps (Westinghouse, Blootnfield, NJ). Approximately 65% of the radiation emitted frotn these lamps is within the UVB (280-320 nm) range. The average irradiance of the UV souree was 8 W/m- as tneasured with a calibrated IL700 radiometer (hiternational Light, Inc., Newburyport, MA) with an SEE240 detector fitted with an SEE280 filter and a W2372 quartz diffuser. During irradiation, mice were housed in individual compartments under a wire mesh lid, which reduced the irradiance by 40%. The dose used for chronic UV irradiation was the amount that induced a barely perceptible erythetnatous reaction (minimal erythema dose, MEL)) on BALB/c skin. This was previously found to be 2.25 k,I/tn- (31). For chronic UV radiation, mice were shaved once a week and exposed to 1 MED 3 times a week for various periods of time.

MLM infeetion and DTH Mice were itijected s.c. with 50 \.\.\ of 5-10 x 10' live MLM bacilli in the left footpad 3 d after a single treatment with UV irradiatioti or 3 d after the last UV exposure in the case of chronic UV treatment. At regular intervals after MLM infection, groups of infected and uninfected mice were tested for a DTH response by injecting 50 |.tl of MLM antigen (equivalent to 1 x fO'"* MLM bacilli) into the contralateral footpad. The MLM antigen was prepared by autoclaving live MLM bacilli for 15 tnin at 15 lb/tn- at 120 C. The footpad swelling was measured with a spring-loaded tnicrometer (Mitutoyo, Tokyo, Japan) just before challenging the mice with MLM antigen and 24 h later. The degree of specific swelling was calculated by subtracting the swelling of the footpads of uninfected mice frotn those of the infected mice. 153

Jeevan et al. Course of MLM infeetion Since MLM cannot be grown in vitro, the course of infection was followed by assessing the number of acid-fast bacilli from tissue smears of the infected footpad, popliteal lymph node, and spleen at regular intervals, as described previously (30). The tissues from 5 mice were individually homogenized in 4% NaOH containing 0.2% potassium alum, incubated at 37"C for 30 min, washed once in phosphate buffer (0.067 M, pH 6.7) by centrifuging at 3000 rpm, and suspended in a known volume of 0.1% albumin in saline. Appropriate dilutions were made, and a standard volume was smeared onto each of four circles of multitest slides (Flow

Laboratories, Inc., McLean, VA). The slides were stained by the Ziehl-Neelsen method and bacteria counted by the standard smear technique (35). The number of acid-fast bacteria in 20 to 60 fields (vertical and horizontal) from each of the three circles was counted under oil immersion. The mean counts from three different circles were assessed, and the total number of bacilli per organ was calculated. MLM infection was also assessed by measuring the local inflammatory reaction to MLM bacilli at the site of infection in a group of 10-20 tnice. Tbe thickness of the infected footpads was measured at regular intervals after infeetion and subtracted from that of the uninfected footpad. Effeet of UV on lethality from MLM

A, FOOTPAD

To detertnine whether prior exposure of mice to a single dose of UV radiation would affect the course of a lethal infection with MLM, miee were exposed to 45 kJ/nr UVB radiation and infected either i,v. or via the footpad. The mice were observed for signs of systemic infection, and the mortality was recorded at regular intervals.

MLM UV MLM

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Effects of ultraviolet radiation on the pathogenesis of Mycobacterium lepraemurium infection in mice.

The purpose of this study was to determine whether exposing mice to ultraviolet radiation (UVR) would alter the pathogenesis of infection with Mycobac...
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