Phagocytic Myeloperoxidase in Leprosy Pathogenesis

68, 1^ CorrespondencePhagocytic Myeloperoxidase in Leprosy PathogenesisTo 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 initiatorsand regulators of the immtme responseand 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 mechanismsofM. leprae persistencc in MPL cellsseems to bc urgem.Today a lot of researchers devote themselvesto studyine. myeloperoxidase (MP)of phagocyting cells, especially in chronicinfectious and granulomatous diseases,since it is known that the acquired or hereditarydeficit of MP activity might favor thedevelopment of chronic infectious diseases,infectious complications of chronic infectiousdiseases 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 patientswith chronic myeloleukosis and subleukemicmyelosis. 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 infiltrateddermatophytoses of the scalp C) andpatients with chronic tonsillitis ('). The dataobtained suggest that the levei of MP activityof MPL edis might be a reliable criterionfor the assessment of their phagocyticability.In M. leprae-infected armadillos Al. leptoewere 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 investigationsof skin granulomas from lepromatousleprosy patients and found thatmacrophage cytoplasm with low MP activitycontained 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 relationshipbctween the terms of appearance andstability of clinicai improvement and activityof 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 granumamacrophages 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 phagocyticresponses and MP activity in experimentson 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 mycobacteria(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 peroxidepreventing 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 macrophages(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^ 2000TABLE 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. afterM. /eprae cotim per mouse foot pael x 10''inoculation^Test mice^Control lince3 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 ± 1.211 58.1 + 6.2" 10.0 ±- 1.6Mice received til. leprae from a lepromatous leprosypatient which had been passed three times inmice. Data are given as mean + SEM.: N = 6.Statistical analysis = Student's t test, p = 0.05.Fio. 1. Electron micrographs of peritoneal macrophages(PM) of test (a) and control (h) mice 6 clays aiterAI. 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' organismstuberculosis 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 microscopicallyand 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 reductionin phagocytic activity. Electron microscopyof H,0,-treated PM at 6 claysshowed numerOus-mycobacterial Mis withintact ultrastructure (Fig. la). In controlsamples, mycobacteria were completelylysecl in 2-3 days (Fig. lb). Initial inclicesof PM absorption capability (phagocytic indexand phagocytic cotim) markedly increased,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 accompanieclby a significant clecrease in thenumber o{ PM pseudopodia and their Ilattening(macrophages became round) as well asdisturbed lysosome-phagosome fusionThus, 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 solutionof hydrogen peroxide at a dose of0.3-0.5 ml per foot pad. A single injectionof hydrog,en peroxide resulted in a more intensivemultiplication of leprosy bacilli inLhe mouse foot pads as compared with standardmethods 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^ 73TABLE 2. AFB iii print smears of internalorgans of mire inoculated with 1 x 10'M. leprae in the foot path"Internai^Mouseorgans^groupLiver^TestControlKidney TestPresence of^lepra('3^5^7^9^11mos. mos. mos. mos. mos.Control^-^-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 importantrole of the MP system of phagocytesin 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 inflammatorydiseases, offering strong possibilitiesfor scarches and screening immunocorrectiveagents of selective effect on bactericidalphagocytic system to compensatefor MPL insufficiency.—Alexander K. Maslov, M.D.Leprosy Research InstituteOstrovsky pas.3Astrakan 414057, RussiaREFERENCES1. DRA(;()NERErsKy, V. D. and BAzitoRA, Y. 1. Assessmentof bactericida' system of polymorphonuclearleucocytes in patients with chronic tonsillitis.Laboratornoye Delo. 11 (1986) 649-652.GUSEVA, S. A., TislictIENK(), L. M. andGAIDUKOVA, S. N. Myeloperoxidase activity andinfectious complications: a relationship in myeloproliferativedisorders. Archiv Patologii 1 (1988)52-55.3. HAMAZINA, O. Sil. Assessment of myeloperoxidaseactivity in patients with dermatophytosesagainst the background of immune therapy. Abstractsof 6th All-Russian Congress of Dermatologistsand Venerologists M. (1989) 356-357.4. MAsi.ov, A. K. A method of simulation of macrophagedefect: Patent No 2105352. Discoveries Inventions5(1988).5. MAsi.ov, A. K. and JuscENK0, A. A. Assessmentof functional activity of leprous macrophages.Archiv. Patologii. 11 (1988) 51-54.6. MAsi.ov, A. K. and JuscENK0, A. A. A method forprediction of leprosy relapse: Author's CertilicateNo 1636717 (USSR). Discoveries Inventions 11(1991) 122.7. MASI.OV, A. K. and KM.YANINA, O. V. A methodof simulation of leprosy infection. Patent No.102482. Discoveries Inventions 2(1998).8. McKEENER, P. E., WALs0, G. P., SToRRs, E. E. andBALENTINE, J. D. Electron microscope of peroxidaseand acid phosphatase in leprous and uninfectedarmadillo macrophages: a macrophage subpopulationcontains peroxisomes and lacks bacilli.Aro. J. Trop. Med. Hyg. 27 (1978) 1019-1929.9. PIGAREVSKY, V. E. The new trend in the teachingon phagocytosis and nonspecilic resistance.Archiv Patologii 2 (1977) 84-94.10. UCHITEI., I. YA. Macrophages in immunity. M.Medicine. 1978,199 pp.