INTERNATIONAL JOURNAL OF LEPROSY^ Volume 54, Number 2

Printed in the U.S.A.

Mechanism of Phagocytosis of Mycobacteria bySchwann Cells and Their Comparison with Macrophages'

Abdul Hamid Band, Shobha D. Chitamber, Alok Bhattacharya,and Gursaran P. Talwar2

Schwann cells, the predominant neuraltarget cells of Mycobacterium leprae, lack amechanism for the specific uptake of AI.leprae ( 2). Rat sciatic nerve-derived Schwanncells as well as the rat Schwannoma cells(33B cell line) phagocytosed all the myco-bacteria tested, as well as inert latex parti-cles. In this property they resemble mac-rophages which are professional phagocyticcells. However, it is not known whether themechanism of phagocytosis by Schwanncells is similar to that of macrophages. Thisreport describes the influence of a numberof factors on the phagocytosis of mycobac-teria by these two cell types.

MATERIALS AND METHODSThe 33B rat Schwannoma cell culture, the

harvesting and culture of rat peritonealmacrophages, and the culture and enumer-ation of mycobacteria were as described ( 2).

Radiolabeling of "Mycobacterium w" with' 4C-acetate. "Mycobacterium iv- was incu-bated in Middlebrook 7H9 medium (DifcoLaboratories, Detroit, Michigan, U.S.A.)containing 10 pCi/m1 of (U-' 4C)-sodiumacetate (56.7 mCi/mM; Bhabha Atomic Re-search Centre, Bombay, India) for 4 hr at37°C. The bacteria were washed 3 times andcentrifuged at 2500 rpm for 30 min at 4°Cin Dulbecco's modified Eagle's medium(DME) without antibiotics. The pellet wasresuspended in DME equivalent to the orig-inal volume and an aliquot (5-10 pl) was

' Received for publication on 2 January 1985; ac-cepted for publication in revised form on 16 December1985.

A. H. Band, M.B.B.S, M.D., Senior Research Of-ficer; S. D. Chitamber, Ph.D., Research Officer; G. P.Talwar, D.Sc., F.I.C.A., F.A.Sc., F.N.A., Director, Na-tional Institute of Immunology, P.O. Box 4922, NewDelhi 110029, India. A. Bhattacharya, Ph.D., Scientist,Tata Research Development and Design Center, Pune,India.

Reprint requests to Dr. A. H. Band, Division ofImmunogenetics, Dana-Farber Cancer Institute, 44Binney Street, Boston, Massachusetts 02115, U.S.A.

counted in a liquid scintillation counter. La-beled bacteria were stored frozen (-20°C)in aliquots until used.

Radiometric phagocytosis assay using' 4C-labeled "Mycobacterium w." For thephagocytosis assay, 5 X 104 33B cells or 2 x10' peritoneal exudate cells were plated in0.5 ml medium (DME + 10% fetal calf se-rum) in wells of 24-well plates. After 24 hr,nonadherent cells were removed by gentlewashing; 20,000 to 40,000 cpm of ' 4 C-ace-tate-labeled "Mycobacterium w," equiva-lent to about 2.5 x 10 6 bacilli, were thenadded per well and the cells were incubatedat 37°C. At the end of the incubation period,the medium containing the unphagocytosedbacteria was removed. The cells were washed3 to 4 times with warm (37°C) DME, andthen 0.5 ml of distilled water was added tolyse the cells. After 15 min, the lysates weretransferred to scintillation vials. The wellswere further washed twice with 0.5 ml vol-umes of distilled water and the washes werealso added to the vials. The vials were thendried in an oven at 110°C, and the radio-activity was counted in a Packard model3320 liquid scintillation counter. The back-ground counts, obtained by incubating thesame amount ' 4C-labeled "Mycobacteriumw" in wells without cells, were subtractedfrom the experimental values.

The scintillation mixture consisted of 5 gPPO (2,4-diphenyloxazole; Sigma Chemi-cal Co., St. Louis, Missouri, U.S.A.) and 0.1g of POPOP (1,4-bis(2-(5-phenyloxazoly1)benzene); Sigma) per liter of sulfur-free tol-uene (Sarabhai Chemicals Ltd., India). Theefficiency of counting was 89%.

The sodium azide, colchicine, cytochala-sin B, and dibutyryl cyclic AMP used in ourstudy were obtained from Sigma. Cyto-chalasin B was dissolved at 2.5 mg/ml indimethyl sulfoxide (DMSO); other modu-lators were made in DME. The controls re-ceived the solvent only.

294

54, 2^ Band, et al.: Phagocytosis of Mycobacteria^295

4

3

2

1

00 4 8 12 16 20 24

Time(Hr)FIG. 1. Time course of the uptake of "C-labeled

"Mycobacterium w" by 33B cells; 20,000 cpm of la-beled mycobacteria were added per well. Back-ground = counts in wells without 3313 cells. Each pointis the mean ± S.D. of triplicates.

The statistical significance of the varioustreatments was determined by Student'st test. All values have been presented asmean ± standard deviation (S.D.) of trip-licates.

RESULTSIt was necessary to utilize a rapid and

simple assay for measuring quantitativelythe uptake of mycobacteria. It was also im-portant to avoid the fallacies in morpho-logical counting of mycobacteria due to lossof acidfastness by some mycobacteria, asencountered earlier with Al. smegmatis ( 2 ).A radiometric phagocytosis assay was,therefore, set up.

"Mycobacterium w" (8, 2°) was used as amodel mycobacterium for these experi-ments because it is nonpathogenic ( 20) butantigenically crossreactive with M. leprae( 15- 17) and one of the candidates for an an-tileprosy vaccine ( 23). Moreover, it grows asan evenly dispersed suspension in Middle-brook medium and is easy to label with"C-acetate (3).

Phagocytosis of 14C-labeled "Mycobac-terium w" by 33B cells and macrophages.

TABLE 1. Uptake of "C-labeled "Myco-bacterium w" by 33B cells and macro-phages.

Uptake (mean cpm ± S.D.).

Background^Test3313 cells 211 ± 38 2541 ± 213Macrophages 198 ± 33 1811 ± 165

" Cells were incubated with labeled mycobacteria(20,000 cpm/well) for 6 hr. Background representscounts in control wells without cells. Each value ismean ± S.D. of triplicates.

A time course study showed that the amountof labeled "Mycobacterium w" phagocy-tosed by 33B cells increased with time (Fig.1). The background counts remained lowthroughout the time course. The assay coulddetect significant phagocytosis as early as 4to 6 hr. A similar time-related increase inthe uptake of labeled bacteria by peritonealmacrophages was observed, as expected(data not shown). Phagocytosis at 6 hr wasused for subsequent experiments. At thistime point, considerable uptake of labeledmycobacteria with very low backgroundswas observed in both 33B cells and mac-rophages (Table 1).

Effect of various modulators on phago-cytosis by 33B cells and macrophages. Tostudy the effect of various modulators, thecells were preincubated with respectiveagents for 1 hr at 37°C (except for the effectof incubation at low temperature) beforeadding labeled mycobacteria. Where stocksolutions were made in DMSO, the controlswere treated with the highest concentrationof DMSO used in the experiment. In theconcentrations used, DMSO by itself didnot significantly affect phagocytosis.

Uptake of ' 4C-labeled "Mycobacteriumw" by 33B cells as well as macrophages wassignificantly decreased by incubation at 4°C,by treatment with sodium azide, and by For-malin-fixation of the host cells (Table 2).These experiments indicate that the phago-cytosis of mycobacteria by both of these celltypes requires live and actively metaboliz-ing host cells.

Treatment with increasing concentra-tions of colchicine caused a dose-related in-hibition of the phagocytosis by both 33Bcells and macrophages (Fig. 2). The inhi-bition of phagocytosis by 33B cells was sig-

Cell type

3.0

2.5

2.0

1.5

1.0

0.5

0

296^ International Journal of Leprosy^ 1986

0 10 -7^10 -5^10-3

Colchicine(Molar)FIG. 2. Effect of colchicine on the uptake of labeled

"Mycobacterium w" by 33B cells and macrophages(MO). Each point is the mean ± S.D. of triplicates.

nificant at concentrations of 10 -3 M (p <0.025), and that by macrophages was sig-nificant at 10 -4 M (p < 0.005) and 10 -3 M(p < 0.025) concentrations. However, theinhibition was less than 100%, even at 10 -3M concentration. Higher concentrationswere avoided due to cytotoxicity.

Cytochalasin B produced a concentra-tion-related inhibition of the phagocytosis

by 33B cells as well as by macrophages (Fig.3). The effect on both of these cell types wassignificant at 5 µg/ml and at 25 µg/m1 con-centrations (p < 0.005). Inhibition wascomplete at the higher concentrations.

Dibutyryl cyclic AMP had a small inhib-itory effect on the phagocytosis by both 33Bcells and macrophages at 10 -3 M concen-trations (Fig. 4). The effect on 33B cells wassignificant (p < 0.05).

The above experiments suggest a depen-dence of phagocytosis on microtubule andmicrofilament systems. Moreover, these ex-periments demonstrate a close parallel be-tween the phagocytic mechanisms of 33Bcells and macrophages regarding the uptakeof mycobacteria.

DISCUSSIONIn order to study the mechanism of my-

cobacteria-Schwann cell interaction, 33Bcells were compared with macrophages re-garding their phagocytosis of ' 4C-labeled"Mycobacterium w" under different con-ditions. Phagocytosis requires live and ac-tively metabolizing host cells, as demon-strated by a drastic inhibition of the uptakeof mycobacteria by incubation at 4°C or withsodium azide, and fixation of host cells withFormalin. This is consonant with the re-ported inhibition of macrophage phagocy-tosis with such treatments (4). Uptake oflatex particles by 33B Schwannoma cells was

TABLE 2. Modulation of uptake of "C-labeled "Mycobacterium w."

Cell type Treatment° Uptake (mean cpm ± S.D.)h p value

33B 37°C 3721 ± 4684°C 715 ± 223

54, 2^Band, et al.: phagocytosis of Mycobacteria^297

5

4

O 3

0 0.2 1^5^25

Cytochalasin B(pg/mI)Fin. 3. Effect of cytochalasin B on the uptake of

labeled "Mycobacterium iv- by 33B cells and macro-phages (MO). Each point is the mean ± S.D. of trip-licates.

5

400

32

2

1

00 10-7^10-5^10-3

Dibutyryl cAMP(Molar)Fin. 4. Effect of dibutyryl cyclic AMP on the uptake

of labeled "Mycobacterium w" by 33B cells and mac-rophages (MO). Each point is the mean ± S.D. oftriplicates.

earlier shown to be similarly affected by theabove treatments (Band, Bhattacharya andTalwar, unpublished observations), indi-cating a similar requirement for energy pro-duction for both mycobacteria and latexphagocytosis in these cells.

The mechanism of phagocytosis is thoughtto involve elements of the cytoskeleton, acomplex intracellular network comprised ofthree major interacting fiber systems, i.e.,the microtubules, microfilaments, and in-termediate filaments. The microfilamentsprovide the motive force for the engulfmentof particles, while the microtubules arethought to mediate the intracellular trans-port of phagosomes and lysosomes(9, 13, 18.21).) Therefore, inhibitors of the con-tractile system were used to further char-acterize the mechanism of phagocytosis.Colchicine is known to depolymerize themicrotubules of the cell and thus reducephagocytosis by macrophages ( 10). Inhibi-tion of phagocytosis by 33B cells and mac-rophages with colchicine, therefore, indi-cates the involvement of microtubules inthe phagocytic process. On the other hand,as observed earlier, latex phagocytosis by33B cells is relatively refractory to colchi-

cine (unpublished observations). A similardifference in susceptibility to the colchicineeffect has been observed in macrophages de-pending on the size and, possibly, physicalproperties of the particles being phagocy-tosed (' 9).

Cytochalasin B is a microfilament depo-lymerizing agent and is known to inhibitmacrophage phagocytosis ('• 5). Dose-relat-ed and complete inhibition of phagocytosisof mycobacteria by both 33B cells and mac-rophages on treatment with cytochalasin B,therefore, indicated the crucial dependenceof the phagocytic process on intact micro-filaments. In this, characteristic phagocy-tosis of mycobacteria by 33B cells is similarto that of latex, which is also completelyinhibited by cytochalasin B (unpublishedobservations).

Cyclic AMP and agents which stimulateits intracellular accumulation have beenshown to either decrease (6.7. 12.22) or in-crease (10,14) phagocytosis by macrophagesand macrophage-like cell lines. This com-pound had a small inhibitory effect on thephagocytosis by both of the cell types. CyclicAMP, on the other hand, had no effect onthe phagocytosis of latex particles by 33B

298^ International Journal of Leprosy^ 1986

cells at comparable concentrations (unpub-lished observations).

The foregoing results demonstrate a closeparallel between Schwannoma cell andmacrophage phagocytosis. However, an in-hibitory effect of rabbit antimycobacterialantiserum on the phagocytosis of "Myco-bacterium w" by Schwannoma cells but notby macrophages (data not shown) suggestsmarked differences between the two celltypes regarding the recognition of myco-bacteria.

SUMMARYFactors influencing the phagocytosis of

mycobacteria by 33B rat Schwannoma cellsand rat peritoneal macrophages were stud-ied. Uptake of ' 4C-acetate-labeled Myco-bacterium w by these cells was used to setup a radiometric phagocytosis assay. Incu-bation at 4°C and treatment with sodiumazide (0.2% to 1%), cochicine (10 -7 to 10 -3M), cytochalasin B (0.2 µg/ml to 25 pig/m1),and dibutyryl cyclic AMP (10 -7 to 10 -3 M)inhibited the phagocytosis by both cell typesin a similar manner. These experimentsdemonstrate similarities in the mechanismof phagocytosis of mycobacteria by Schwanncells and macrophages.

RESUMENSc estudiaron los factores que influyen en fa fago-

citosis de micobacterias por las celulas del Schwan-noma 33B y por los macrOfagos peritoneales de la rata.Se estableciO un ensayo radiometrico para medir elconsumo de Mycobacterium w marcado con acetatoC 14 .

La incubaciem a 4°C y el tratamiento con azida desodio (0.2 al 1%), colchicina (10 -7 a 10 - ' M), citoca-lasina B (0.2 a 0.25 pg/m1), y dibutiril AMP ciclico(10 -7 a 10 -3 M) inhibicron, todos, la fagocitosis porambos tipos celulares de manera similar. Estos expe-rimentos demuestran analogias en los mecanismos defagocitosis de micobacterias por las cêlulas de Schwanny por los macrOfagos peritoneales de la rata.

RÉSUMÉ

On a etudie les facteurs influencant la phagocytosedes mycobacteries par les cellules de schwannome derats 33B et par les macrophages peritoneaux du rat. Lacapture de Mycobacterium w marque au 14 C-acetatepar ces cellules a servi pour mettre au point une epreuveradiomêtrique d'êtude de la phagocytose. La phago-cytose par l'une et l'autre espece de cellules a etc inhibeede la merne maniere par une incubation a 4°C et parle traitement par l'azide de sodium (0,2% a 1%), la

colchicine (10 -7 a 10 -1 M), la cytochalasine B (0,2 pg/ml a 25 pg/m1), et I'AMP cyclique dibutyryle (10 - ' a10 -1 M). Ces experiences demontrent des similitudesdans le mecanisme de phagocytose des mycobacteriespar les cellules de Schwann et par les macrophages.

Acknowledgments. We thank Professor Martin C.Ralf for a gift of 3311 cell line and M/s Sunder Singh,P. Mannickam and A. Robin for technical assistance.

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