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Mar 5, 1989 - Gillis, S., G. R. Crabtree, and K. A. Smith. 1979. ... McCune, R. M., F. M. Feldmann, H. P. Lambert, and W. ... Robb, and P. C. Nowell. 1986.
Vol. 57, No. 6

INFECTION AND IMMUNITY, June 1989, p. 1719-1724

0019-9567/89/061719-06$02.00/0 Copyright © 1989, American Society for Microbiology

Mechanisms of Recrudescence of Mycobacterium bovis BCG Infection in Mice JOSEPHINE H. COX,t* BRUCE C. KNIGHT, AND JURAJ IVANYI

MRC Tuberculosis and Related Infections Unit, Hammersmith Hospital, DuCane Road, London W12 OHS, England Received 29 November 1988/Accepted 5 March 1989

The capacity of various immunosuppressive agents to cause a recrudescence of the replication of Mycobacterium bovis BCG in the spleens of chronically infected mice was investigated. The actions of three corticosteroid preparations, cyclosporin A, and anti-T-cell subset monoclonal antibodies were compared. Treatment of mice with hydrocortisone acetate, which depressed the number of splenic lymphocytes and suppressed T-cell responses, most effectively exacerbated the stationary BCG counts, at 4 to 6 months after infection. The magnitude of reactivation was more pronounced in innately resistant CBA/Ca mice than in the susceptible C57BL/6 strain of mice. Splenic bacterial counts were also amplified by anti-L3T4 antibody when the antibody was injected at the chronic phase, whereas cyclosporin A had an effect only during the initial 6 weeks after BCG infection. Cultures of spleen cells from chronically infected mice showed a significant increase in the numbers of viable BCG recovered after 7 days of incubation in the presence of dexamethasone but not with cyclosporin A. The observed differences between the tested immunosuppressive agents indicate that the stationary bacterial counts during chronic BCG infection are maintained by discrete T-cell actions on the infected macrophages.

The murine host response to infection with mycobacteria and other intracellular bacteria essentially consists of three phases. Initially, bacterial replication is restrained by nonimmune, macrophage-mediated innate resistance (23). In the second phase, the effector mechanisms of acquired protective immunity have been attributed to CD4 T-cell-derived lymphokines, which activate bactericidal macrophages as part of a granulomatous reaction, and possibly also to CD8 cells, which may cause cytolysis of infected cells (8). However, T-cell-mediated immunity usually turns a progressive murine infection into a stable chronic one instead of sterilizing the convalescent host. Thus, the third protracted phase of the host response is represented either by the persistence of metabolically dormant bacilli or by a dynamic equilibrium between the replication and death rates of organisms (18). The need for a better understanding of mechanisms involved in this chronic phase of infection is apparent from the fact that in humans, a breakdown in host resistance and the subsequent recrudescence of bacterial replication represent the pathogenesis of the most common clinical form, i.e., adult pulmonary tuberculosis. Although there are few clues to the nature of impairment of host immune resistance in adults with typical pulmonary disease, the higher incidence of tuberculosis as well as of nontuberculous mycobacterioses has been associated with general immunosuppression resulting from human immunodeficiency virus infection (24). Reactivation rather than primary infection is the likely pathogenesis, particularly in populations with high endemic occurrence of the infection. Corticosteroid treatment was only rarely reported to enhance known or unrecognized tuberculosis in humans but can aggravate the natural course and the postchemotherapy relapse of tuberculous infection of several laboratory animal species (10, 11, 18, 19). In view of the pleiotropic suppres-

sive effects of hydrocortisone on lymphocyte function and distribution, as well as on macrophage function and granuloma formation (13), the mechanisms of action on mycobacterial infection have not yet been explained. The purpose of this study was to compare the hydrocortisone-induced enhancement of chronic Mycobacterium bovis BCG infection in innately resistant (CBA/Ca) and sensitive (C57BL/6) strains of mice and to compare the efficacy and time course of other potent immunosuppressive agents, such as cyclosporin A (CsA) and anti-T-cell antibodies. Moreover, the demonstration of reactivation of chronic infection by using an attenuated strain such as BCG per se is surprising and offers a convenient model for detailed study of phenomena which so far have been associated only with pathogenic human-type tubercle bacilli. MATERIALS AND METHODS Animals. All mice used were females, 6 to 8 weeks old at the start of the experiment. Inbred strains C57BL/6 (H-2b) and CBA/Ca (H-2") were purchased from Olac, Bicester, Oxford, United Kingdom. Infection. M. bovis (BCG Glaxo) was grown for 3 weeks in Middlebrook 7H9 medium (Difco Laboratories, Detroit, Mich.) before being dispensed and stored at 107 viable organisms per vial in liquid nitrogen. Freshly thawed suspensions were diluted in phosphate-buffered saline, and mice were injected intravenously (i.v.) with approximately 104 viable units. CFU. Spleens were harvested from the mice at various times after BCG infection and homogenized in 5 ml of sterile distilled water in a stomacher (Seward Medical, London, United Kingdom). Serial 10-fold dilutions of the homogenate were plated on Middlebrook 7H11 agar (Difco). The plates were incubated for 21 days. The limit of detection in this assay system is 100 mycobacteria per spleen. Drug treatment. CsA (Sandimmun; Sandoz, Basel, Switzerland) was given daily by subcutaneous (s.c.) injections of 100 mg/kg of body weight in olive oil for a period of 14 days. Hydrocortisone acetate (HCA; Boots, Nottingham, United

* Corresponding author. t Present address: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, 12441 Parklawn Drive, Rockville, MD, 20852.

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Kingdom), hydrocortisone sodium succinate (The Upjohn Co., Kalamazoo, Mich.), and dexamethasone (DEX; Merck Sharp & Dohme, West Point, Pa.) were given s.c. by the following standard regimen or as indicated in the text; 2.5 mg on day 1 of treatment followed by 3 injections of 1.25 mg each s.c. on days 6, 8, and 10. Spleens were harvested for bacterial enumeration 1 month after the start of the treatments or as indicated in Results. Depletion of T-cell subsets in vivo. Rat immunoglobulin G 2b monoclonal antibodies (MAbs) used for in vivo depletion were directed against Lyt-2 marker on T-cytotoxic cells (YTS.169) and L3T4 marker on T helper cells (YTS.191). These were produced as ascitic fluid by growing the hybridomas (1) (kind gift from H. Waldmann, Department of Pathology, Cambridge, University, United Kingdom) in (Lou x DA)F1 rats. Ascitic fluid at approximately 10 mg/ml was stored at -20°C and centrifuged at 1,000 x g for 10 min before use. Mice were injected with 200 ,u1 of ascitic fluid i.v. on day 1 of treatment and given intraperitoneal injections on days 2 and 3. Spleen cell culture. Single-cell suspensions from spleens of BCG-infected mice made up in RPMI 1640 medium (GIBCO, Paisley, Scotland) supplemented with 5% fetal calf serum and glutamine (without antibiotics) were dispensed into 24-well plates. After incubation with 10-5 or 10-7 M DEX or soluble CsA (Sandimmun) at 1 or 10 p,g/ml for 7 days at 37°C, the supernatants were removed and the cells were lysed with distilled water. Samples were then plated onto 7H11 agar for enumeration of BCG as described above. DTH. For sensitization, 100 ,ll of a 0.01% sheep erythrocyte (Tissue Culture Services, Slough, United Kingdom) suspension was injected i.v. into mice. Four days later, the delayed-type hypersensitivity (DTH) response was elicited by injecting each footpad with 40 of a 20% sheep erythrocyte suspension. Footpad thickness before and 24 h after injection was measured with a dial caliper gauge (Pocotest, Kroplin, Surrey, United Kingdom). Fluorescence-activated cell sorter analysis. Spleen cells were incubated with anti-Lyt-2 (YTS.169), anti-L3T4 (YTS.191) or anti-Thy-1 (YTS.154, Sera lab, Sussex, United Kingdom) for 40 min at 4°C. The cells were washed by centrifugation in phosphate buffered saline containing 1% fetal calf serum and then incubated for 40 min at 4°C with fluorescein isothiocyanate-conjugated rabbit anti-rat F(ab')2 (Serotec, Kidlington, Oxford, United Kingdom) diluted 1 in 50 in 10% normal mouse serum. Cells were fixed after being stained in 1% paraformaldehyde and were stored at 4°C before analysis by fluorescence-activated cell sorter by using an Epics profile (Coulter Electronics, Inc., Hialeah, Fla.). Cell proliferation assay. Spleen cells were cultured in flat-bottomed 96-well microdilution plates at 5 x 106 cells per ml in RPMI 1640 medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U of penicillin per ml, and 100 p.g of streptomycin per ml with 5 p.g of concanavalin A per ml or medium alone in a 200-,ul volume. In some experiments, concanavalin A at 2 ,ug/ml was added along with a final concentration of 10-5 or 10' M DEX or soluble CsA at 1 or 10 ,ug/ml. After incubation for 4 days at 37°C, the cultures were pulsed for 8 to 16 h with 1 ,uCi of tritiated methyl thymidine (Amersham Corp., Arlington Heights, Ill.) and harvested onto glass fiber filters. Radioactive counts were measured by liquid scintillation spectroscopy. Results are expressed as mean counts per minute of triplicate cultures.