68, 1^ Correspondence

Phagocytic Myeloperoxidase in Leprosy Pathogenesis

To THE EDITOR:

ft is well known that a causative agent ofleprosy, Mycobacterium leprae, survivesand multiplics in cells of the mononuclearphagocyte line (MPL). MPL cells act as ini-tiators and regulators of the immtme re-sponse and they are involved in nonspecificrcsistance and antimicrobicidal activities. Ina view of improvement and development ofnew methods for the prevcntion and therapyof leprosy, a detailed study of the mecha-nisms ofM. leprae persistencc in MPL cellsseems to bc urgem.

Today a lot of researchers devote them-selves to studyine. myeloperoxidase (MP)of phagocyting cells, especially in chronicinfectious and granulomatous diseases,since it is known that the acquired or hered-itary deficit of MP activity might favor thedevelopment of chronic infectious diseases,infectious complications of chronic infec-tious diseases or granulomatoses.

According to prescnt-day conceptions,acid hydrolases destroy only those bacteriain phagolysosomes which have been killcdby the MP system, nonenzymatic cationicproteins, lysozyme and lactoferrin ()).

Decrcased MP activity of neutrophils ofperipheral blood has been observed in pa-tients with chronic myeloleukosis and sub-leukemic myelosis. In this group there wasa high percent of the cases with infectiouscomplications (2). With strongly insufficientactivity of MP, MPL cells are not capable ofkilling candida, stztphylococci and variousgram-neg,ative microorganisms (). Lowindices o—f MP activity of peripheral bloodphagocytes are found in patients with infil-trated dermatophytoses of the scalp C) andpatients with chronic tonsillitis ('). The dataobtained suggest that the levei of MP activ-ity of MPL edis might be a reliable crite-rion for the assessment of their phagocyticability.

In M. leprae-infected armadillos Al. lep-toe were absent in macrophages with highactivity of MP, and, contrary, macrophageswith low MP activity contained numerousM. lepme in their cytoplasm (8)•

We carried out electron cytochemical in-vestigations of skin granulomas from lepro-matous leprosy patients and found thatmacrophage cytoplasm with low MP activ-ity contained clusters of intact M. lepraeand, contrary, intensive lysis of Al. lepraeWaS observed in macrophages with highMP activity e). Our long-term (over 14years) obscrvations on the possible relation-ship bctween the terms of appearance andstability of clinicai improvement and activ-ity of phagocytic MP could establish astrong association between the rate of MPactivity in inacrophages of skin lepromas(as obscrved at the patient's admittance)and the time of appearance of the first signsof clinicai improvement and stability ofreg,ress. A high levei of MP activity in gran-uma macrophages strongly correlatedwith the fast elimination of Al. leprae andLhe highest peak of effectiveness of therapyadministered. Low activity of macrophageMP was correlated with the slow regrcss ofthe disease and risk of relapse (`').

Further, the goal was sought to find acorrelation between the intensity of phago-cytic responses and MP activity in experi-ments on mice subject to ai] induced decreasein MP activity in MPL cells, as well as toelucidate whether it is possible to simulate aprocess of persistence of pathogenic my-cobacteria (Al. leprae and M. tubetrulosis)in MPL cens under the above conditions.

With this atm in mind, the followingtasks should be solved: to evolve a methodof decreasing the activity of phagocytic MPand to define optimal agent concentrationthat would not affect cell ultrastructure.

ft is well known that one of the mainfunctions of MPL is to bind hydrogen per-oxide preventing its cellular accumulation;the enzyme therewith loses its activity. Alithings considered, wc attempted to reduceMP activity in MPL cens by saturatingthem with hydrogen peroxide solution.

In order to obtain peritoneal macro-phages (PM), mice were stimulateci bymeans of intraperitoneal (i.p.) injection of2% peptone casem n solution. Two hr afterstimulation and 1-2 hr before Al. leprae in-

72^ International Journal af Leprosy^ 2000

Fio. 1. Electron micrographs of peritoneal macro-phages (PM) of test (a) and control (h) mice 6 clays ai-ter AI. leprae (ML) inoculation; ps = pseudopodia, v =vacuoles (uranil acetate and lead citrate stain; bar = 0.5mm).

oculation the mice were injected i.p. withtest solutions of hydrogen peroxide (0.3%,0.6% and 1.0%) at a dose of 2 ml. Micewere inoculated i.p. with 1 x 10' organisms

tuberculosis obtained from patientswith pulmonary tuberculosis and M. lepmepassed on mice were suspended in 1 ml ofsalt solution). The mice were sacrificed at 2hr, 1, 2, 3, 4, 5, and 6 days after inoculation.PM activity was assayed electron micro-scopically and cytochemically as well as inan absorption test.

The experimental data showed that inmice injected i.p. with the experimentallychosen 0.6% concentration of hydrogenperoxide at a dose of 2 ml, MP activity inMPL cells was significantly decreasedwithin 6 clays, with a simultaneous reduc-tion in phagocytic activity. Electron mi-croscopy of H,0,-treated PM at 6 claysshowed numerOus-mycobacterial Mis withintact ultrastructure (Fig. la). In control

TABLE 1. Coutas qf M. leprae permonse fool pad in animais receiving0.03-0.05 of 0.6% kvdrogen pero.videinto the foot pad 1-2 lir befin-e inoculationof 1 x M.leprae."

Mos. after M. /eprae cotim per mouse foot pael x 10''inoculation^Test mice^Control lince

3 27.19 ± 2.3" 9.48 ± 1.75 101.6 ± 5.8" 3.71 ± 0.367 378.0 ±- 9.8" 38.17 ± 4.19 109.0 -± 7.8" 12.1 ± 1.2

11 58.1 + 6.2" 10.0 ±- 1.6

Mice received til. leprae from a lepromatous lep-rosy patient which had been passed three times inmice. Data are given as mean + SEM.: N = 6.

Statistical analysis = Student's t test, p = 0.05.

samples, mycobacteria were completelylysecl in 2-3 days (Fig. lb). Initial inclicesof PM absorption capability (phagocytic in-dex and phagocytic cotim) markedly in-creased, but 3 clays afterward they leveledoff and approached control inclices.

A study of the ultrathin structure of PMshowed that a drop in activity of MP 2-3clays after the mice were inoculated was ac-companiecl by a significant clecrease in thenumber o{ PM pseudopodia and their Ilatten-ing (macrophages became round) as well asdisturbed lysosome-phagosome fusion

Thus, a single saturation of MPL censwith a 0.6% solution of hydrogen peroxidecauses reduced MP activity in phagocytes;hence, resulting in long-term persistence ofpathogenic organisms (Al. tuberculosis andM. leprae) in their cytoplasm.

Based on the above investigations andconsidering the actuality of developing anoptimal leprosy model and identification offactors favoring mycobacterial persistencein host cells, we tried to improve Shepard'stechnique by selectively acting on the MPsystem of phagocytes. For this purpose, 1-2hr before M. leprae inoculation the micewere given a 0.6% freshly prepared solu-tion of hydrogen peroxide at a dose of0.3-0.5 ml per foot pad. A single injectionof hydrog,en peroxide resulted in a more in-tensive multiplication of leprosy bacilli inLhe mouse foot pads as compared with stan-dard methods of inoculation (Table 1) and ageneralization of the infection (M. lepraewere seen in print smears of lung andspleen tissue of mice) (Table 2). Electron

68, 1^ Correspondence^ 73

TABLE 2. AFB iii print smears of inter-nal organs of mire inoculated with 1 x 10'M. leprae in the foot path"

Presence of^lepra('

3^5^7^9^11mos. mos. mos. mos. mos.

Liver^TestControl

Kidney TestControl^-^-

Lung^Test^+++^+

Control^-^-

Spleen 'fest^ ++^+++ ++Control

"Mycohacteria in the internai organs were estimatedas follows: = AFB/lield; + = 1-20 AF13/lield, ++ =20-50 AFB/lield, +++ = numerous AFB/lield.

cytochemistry demonstrated low activity ofMP in phagocytic cells, suggesting an im-portant role of the MP system of phago-cytes in the mycobactcrial killing andpathogenesis of leprosy (').

Our studics provide an opportunity for adirected altcration of phagocytic activity ofMPL cells. A proposed approach to inducephagocytic insufficiency is similar to themechanism of human immunodeficienciesin a =bei- of chronic infectious and in-flammatory diseases, offering strong possi-bilities for scarches and screening immuno-corrective agents of selective effect on bac-tericidal phagocytic system to compensatefor MPL insufficiency.

—Alexander K. Maslov, M.D.Leprosy Research InstituteOstrovsky pas.3Astrakan 414057, Russia

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8. McKEENER, P. E., WALs0, G. P., SToRRs, E. E. andBALENTINE, J. D. Electron microscope of peroxi-dase and acid phosphatase in leprous and unin-fected armadillo macrophages: a macrophage sub-population contains peroxisomes and lacks bacilli.Aro. J. Trop. Med. Hyg. 27 (1978) 1019-1929.

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Internai^Mouseorgans^group