Lifa Sciaaces Vol . 21, pp . 1575-1584 Priatad in thn U.S .A .

Pargamon Preae

SiJPERO%IDE PRODUCTION BY RABBIT PUI~NARY ALVEOLAR MACROPHAOEB D. B. Lowrie and V . R . Aber bIItC Unit for laboratory studies of tuberculosis, Royal Postgraduate ~éedical School, Du Cane Road, London, W12 OHS (Received is final form October 10, 1977) SvmmarY : Nitroblue tetrazolium (NBT) reduction by alveolar '^°^ rophages from normal and BCG-granulomatous rabbit lunge was inhibited by superoaide dismutase (SOD) . Superoxide ( .0 2 ) might therefore be involved, either directly or indirectly, in the bactericidal activities of such cello . Cells from BCGgranulomatous rabbits did not, however, reduce significantly more NBT per cell than cells from normal rabbits . Superoxide radical ( .0 2 ) plays a prominent part is the antimicrobial activities of polymorphonuclear leucocytes, either by functioning ae a final tout effector agent itself, or, perhaps more importantly, as as intermediate through which H202 . and further free radicals are evolved (1,2,3) . In contrast, little is known of the role of .0 2 is the microbicidal powers of macrophages . Staphylococcal killing by human peripheral blood monocytea has been ahown_to involve .0 2 (4) and, using cytochrome c reduction as an indicator, .02 production by mouse and guinea pig macrophages has been demonstrated (5), other workers have, hovev~er, failed to find .0 2 production by rabbit macrophages with this indicator (6) . Reduction of nitroblue tetrazolium (NBT) to a dark blue insoluble formazan can be used as an alternative to cytochrome c as an indicator of .O2 evolution. Thus the intra-leucocytec reduction of NBT by phagocytosing polymorphonuclear cells can be inhibited by the addition o_f superozide diamutase (SOD), an enzyme vhich specifically removes .0 (1,T) " NBTreduction tests have been widely used to indicate the atâte of activation of human leucocyte antimicrobial functions (8,9) sad the occurrence of formazan in peripheral blood monocytes and derived macrophages has been described (8,10) . However, an effect of SOD on NBT reduction in either monocytes ôr macrophages has not previously been reported . In this study we ezamiaed the ability of rabbit pulmonary alveolar Cells macrophages to reduce NBT via intermediary 02 from the lungs of normal animals were compared with cells from chronic granulomatous lungs of BCGrvaccinated animals, since the latter have been shown to have greater bactericidal properties (11,12) which, we hypothesised, might be mediated by .0 2 .

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Materials and Methods Preparation of pulmonary alveolar leucocytes . Female NZW rabbits weighing 2-3 kg were sacrificed using sodium pentobarbitone anaesthesia and alveolar cells collected by a lung lavage technique (13) . The lungs were fully distended with ice-cold 0 .25 M sucrose containing 1 mM vexsene (pH î .0), the cells were collected by centrifugation at 150 ~ at 4 °C for 15 min . and washed twice in the same buffer and once in Hanks' balanced salts solution by repeated suspension and centrifugation . BCCr-induced alveolar cells were obtained from vaccinated rabbits 2-3 weeks after injection of 100 ug of BCG (Glaxo) in 0.3 ml of Freund's incomplete adjuvant (Difco) into a marginal ear vein . Phase-contrast haemocytometer counts shoved that about 5 x 10~ leucocytes were recovered from a normal rabbit and about 5 x 10 8 from a vaccinated rabbit . The cells were finally suspended in a 2 :1 by volume mixture of Hanks' balanced salts solution sad rabbit serum (GibcoBiocult) to a concentration of about 3 x 10 6 cells per ml . Preliminary tests shoved that addition of 33~ serum did not alter NBT reduction but increased cell retention on nylonwrool columns in NBT reduction tests . Pulmonary granuloma indices were determined according to the method of Moors and IQyrvik (14) and defined as (lung weight x 250)/body weight . Differential cell counts were done by microscopial examination of Giemsa-stained smears made from the final cell suspension . Over 95x of the leucocytes from the lungs of normal rabbits were classed ss macrophages, the remainder being predominantly epithelial cells and small lymphocytes ; large lymphocytes were not distinguished from macrophages . The leucocytes from the chronic-granulomatous lungs of vaccinated rabbits were classed as 80-90x macrophages and up to lOK polymorphonuclear leucocytes . Ia cells from normal rabbits, the number of type A macrophages (blur cytoplasm, blue/violet nucleus ; reference 15) approximately equalled the number of type B macrophages (pink cytoplasm, red/violet nucleus) ; after vaccination type A exceeded type B at a ratio of about 2 .5 :1 . Incubation of cells with NBT and other materials . To a aeries of duplicate po]ypropyleae tubes 2 ml-cryostorage screw-top ampoules, Sterilin) were added 1 .5 ml of freshly-prepared cell suspension is 33,E serum and 0 .2 ml of 0 .15 M saline or appropriate concentrations of test materials in saline . The tubes were warmed to 3i C and then vas added 0 .1 ml of a heparin-NBT complex suspension (H-NBT) which contained 4 mM NBT (Sigma) and 60 units of heparin (Evens Pharmaceutical) per ml in 430 mM sucrose. Final concentrations of test materials were : SOD (Sigma), 11 ug protein (39 activity unite) per ml ; heat-inactivated SOD, prepared as described by Babior et al . (16), 11 ug per ml ; saline-dialysed zymosan, 100 particles per cell ; endotoxin ( E . coli 012Z :B8, Difco), 5 .5 ug per ml . In each experiment, control tubes were included which contained &ell suspension and saline only (no H-NHT) . All tubes were incubated at 3T C for 25 min . titation of reduced NBT formazan extraction . The nylon column-NBT teat of Segel and Peters 17) vas used to estimate the amount of NBT reduced by the cells . The incubated cell suapénsioas, lean about 50 ul, were transferred onto 100-mg nylon wool columns and the cells were washed on the columns with saline, water and HC1 . Formazan vas immediately extracted from the trapped cells with 2 .5 ml diozane . After centrifugation of the eztracts their optical density vas determined at 520 nm using a Perkia E1mer spectrophotometer with a diozane blank . Formazan standards were

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Formazan yield per prepared as described by Segal and Peters (1T) . leucocyte vas calculated for each column using S5~ = 29 .g5 and haemocytometer estimates of the number of cells applied tb the column and the number found in the saline slants . Eluted cells ware concentrated before counting by centrifugation at 150 g for 15 min and resuspenaion in 0.5 ml of Hanks' balanced salts solution . Appropriate correction was made in the calculation of formazan yield per leucocyte according to the optical densities of control "no H-NBT" column extracts . An air-dried smear on a Microscopical survey of 1~BT-reducing cells . microscope slide vas made Pram the residue of each incubated suspension which remained after the bulk of the suspension had been applied to a ~rloa column . Sunears were fined in methanol and stained with O .lx eafranin in 10Z glycerol for 5 min" Random fields of each smear were exa= ; aed microscopically using s x 100 oil-immersion objective until between 500 and 2000 leucocytes had been encountered . Each cell was scored according to three criteria : size (large or small, i .e . greater than or lees than about 11 um) ; presence or absence of distinct cellassociated formazan deposits ; whether it was a single cell or closely associated with another cell (i .e . paired) . In the latter circumstance the size and formazan status of the associated cell were also recorded . Whey more than two cells appeared closely associated or when cells were not sufficiently widely dispersed in a microscope field to disti~~ ; sh single fra~m paired they were scored as single . Ao attempt was made to distinguish between macrophages and lymphocytes in safranin-stained smears . However, polymorphonuclear cells were essentially absent from the "large" cell category . Results The effect of SOD and phaAOCytic stimuli on the amount of NBT reduced per cell in cells from normal and granulamatous rabbit lungs . The results of duplicate quantitative ABT tests on cells from four normal and four vaccinated (granulomatous) rabbits are s~~~+ aed in Table 1 . SOD consistently inhibited ABT reduction when cells were incubated with H-ABT, either alone or together with phagocytosable zymosan particles . 7ymoean itself usually inhibited ABT reduction . Analysis of variance of the log-transformed data (eadotozin data ezcluded) shoved that the effect of SOD and the effect of zymoean were significant (P < 0 .01, normal rabbits, P < 0 .001, vaccinated rabbits) and independent of each other . Analysis which included the endotozin data showed that the small increase in the average amount of ABT reduced by cells from vaccinated animals compared to cells from normal animals was not significant (P > 0 .05) ; the variation Pram animal to animal within each group was highly significant (P < 0 .001) . There was no significant interaction between animals and treatments (P > 0 .05) " The effect of SOD vas shown to be due to enzymatic activity . Quadruplicate tests oa cells from a normal rabbit detected no effect of heat-inactivated 80D on NBT reduction, whereas active SOD significantly decreased formazan production (P < 0 .01; analysis of variance) . The proportion and type of almsolar cells which reduced ABT . The results of differential survey oP safranin-stained smears of cells which had been incubated with ABT or with ABT + endoto~.n are summarised is Table 2 . Since exposure of the cells to endotoxin did not affect the results the

1578

Superoxide and Alveolar Macrophages

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Superozidn and Alveolar Macrophages

Vol . 21, No . 11, 1977

counts from cells incubated with and without endotoxin were pooled and, for clarity, only the pooled results are shown . Formazan deposits were observed almost exclusively in the large cells which constituted the bulk of the cells harvested Pram the lungs ; between T and 21z of the large cells reduced NBT (Table 2, col . 8) . Scores for the "small" cell category are not displayed but calculation shows that only 0 .2-5z of formazan positive cells were small . Examination of duplicate smears which heal been Giemsa-stained indicated that large formazaa~ositive cells were predominantly type A and occasio nul,s ~y type H macrophages but fewer formazan~positive cells could be detected with this counter-staining technique . During microscopical survey it was noted that formazan seemed often to be segregated in a region of the cell in close contact with another cell (Fig . 1) . Since thin suggested that NBT reduction might be associated with cell :cell interaction, the relationship between celh pairing and formazaa deposition was also investigated (Table 2) . Analysis of variance showed that large cells had a greater tendency than small cells to form pairs (P < 0 .001 ; Table 2, col . 12 c .f . col. q) and that amongst paired cells, large cells were more often formazan positive than were large cells in the population as a whole (P < 0.001 ; col . 13 c .f . col . 8) . Thus formazan deposition and the formation of cell pairs tended to occur together in large cells .

FIG . 1 Formazan deposition at the region of contact between a pair of apparently closely-adherent macrophages . Safranin counter stain, x 1500 .

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Large variation between animals is apparent in the results in Table 2 and ana]ysis failed to shox any consistent effect of vaccination . The proportion of formazan~ositive cells in individual smears (results not shorn) did not correlate significantly (P > 0 .05) with the mean amount of formazan per cell found on the respective nylon columns . Discussion Inhibition of NBT reduction by S_OD constitutes evidence that rabbit alveolar macrophages can generate 02 Therefore, at least part of the H 0 2 shown by others to be produced by " these cells (18,19,20) might arise ye a intermediary

.02.

Free NBT has been shown to be tout for human leucocytes suspended in a protein-free solution (21) but it seems unlikely that macrophage .02 Morphological production ras e pathological response to NBT tozicity . damage was not apparent by light microscopy and others have shown that phagocytosis of H-NBT complex did not cause ultrastructural damage is âuman leucocytes (22) . The rate of formazen production per cell (up to 1 flnol/25 min .) xas about one-tenth of the rate observed by Segal and Peters (lq) in human leucocytes (up to 8 fmol/20 min .) . Nevertheless, just as in human blood only about lOz of leucocytes reduced NBT (9), so between 4 and 20z of rabbit pulmonary alveolar cells reduced the dye . Thus the active rabbit cells had about a tenth of the activity of the active human leucocytea . Polymorphonuclear cells did not contribute substantially to the total dye reduction . Since formazan vas essentially confined to type A macrophages, a functional distinction is apparent between type A and type B macrophages . NBT reduction was particularly associated with those type A macrophages which shored a tendency to pair . A similar association of NBT reduction with pairing cells has been observed in studies of human polymorphonuclear cells (23) but the cause is unknown : close cell :cell membrane contact might potentiate NBT reduction ; H-NBT complez might form stable bridges between cells ; both NBT reduction and cell :cell adherence might be independent indications of phagocyte maturity . H 02 evolution, and the closely-linàed activity of the hexose monophospha~e shunt (HMPS), are increased during phagocytosis in alveolar macrophages (18,19,2+) and it vas anticipated that .02 production might also be increased during phagocytosis as it i_s in polymorphonuclear leucocytes (3,25,26) . Indeed, increased 0 production has been found with phagocytosing mouse and guinea-pig peritoneal macrophages (5,27), mouse alveolar macrophages (5) and human monocytes (28) but not trith guineapig alveolar macrophages (5) " Other xorkere have found ao significant increase in NBT reduction in rabbit alveolar macrophages during phagocytosis of latex spheres(29) and in the present study NBT reduction was significantly depressed by zymosan phagocytosis . Gee and Khandvala (30) have recently cited unpublished observations of a depression of release from rabbit alveolar macrophages caused by phagocytosis .

.02

(31) .

Human leucocytes exposed to endotoxin show enhanced NBT reduction Eadotoxin apparently disturbs the cell membrane and stimulates NBT

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reduction in a manner analogous to a m~cimat phagocytic stimulus (32) . Thus the lack of a stimulatory effect of endotoxin on dye reduction in rabbit macrophages might imply that the cells were already max~mßi ly stimulated in the basic test situation, perhaps because H-NBT itself constitutes a phagocytic stimulus (22) . Conversely, endotoxin might be an inappropriate stimulus for these cells . Selectivity in metabolic responsiveness to other membrane-active substances has been observed in rabbit alveolar macrophages (33) . However, even exposure to phospholipase c, which stimulates the HMPS (33), failed to enhance NBT reduction in preliminary experiments . A pre-existing state of maximum stimulation therefore seems likely ; this would also account for an absence of stimulation with zymosan . The observed depression with zymosan could then b_e due to competition between zymosan and H-NBT particles for the sites of 0 evolution since the likelihood of the unstable .02 reaching H-NBT would 2 thereby be decreased. Alveolar macrophages from BCG-vaccinated rabbits are not only more efficiently bactericidal than those from normal rabbits, they also exhibit greater HMPS activation during phagocytosis (12) which implies greater H 0 2 2 production . Similar observations have been reported in comparisons of peritoneal macrophages from normal and BCGrvaccinated mice (27) . However, these differences were not reflected in a greater capacity to produce .02 " in macrophages Pram vaccinated mice (27) or in the present observations, in macrophages from vaccinated rabbits . The increased H2O production capacity of macrophages _from vaccinated animals presumably reflec~s the increased activity of .0 2 -independent H202generating reactions . Acknowled~ eme nt

The technical assistance of Miss M. E. W. Carrol is gratefully acknowledged . References 1. 2. 3" 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 "

R . B . JOHHSTON, B. B. KEELS, H . P . MISRA, J . E . r, :v~ , L. S . WEBB, R . L . BAEHNER and K. V . RAJAGOPALAN, J. clin . Invest . ~, 1357-1372 (1975) " S . J. KLEBAFOFF, Semin. Hematol. 12, ll7-142 (1975) . R . S. WEENIAG, R. WEVER and D. ROOS, J . Lab . clin . Med . 8~, 245-252 (1975) " A . L. SAGONE, G. W. KING and E . N . METZ, J . clin . Invest . ~, 1352-1353 (1976) . D . B. DRATH and M. L . KAIiNOVSKY, J : exp . Med. 141, 257-262 (1975) . L. R. DeCHATELET, D. MULLTxrN and D . E . McCALL, JJ . infect . Dis . 1~1, 443-446 (1975) . R. L. BAEHNSR, 8 . K. MURRMANN, J . DAMS and R . B . JOHNSTON, J. clin . Invest . ~, 571-576 (1975) . J . K. LACE, J . S . TAN and C. WATANAKfmiAKORN, Amer . J . Med . ~, 685-694 (1975) " A. W. SEGAL, Lancet ü , 1248-1252 (1974) " H. W. won HEMDEN and D . won HEMDEN, Blut ~, 37- 42 (1974) . D. G . EVANS and Q . N . MYRVIK, J . Reticulcendothel . Soc . _4, 428-429 (1967) . Q . N . MYRVIK, J. Reticuloendothel . Soc . 11, 459-468 (1972) " Q . &. MYRV2K, E. S . LEAKS and B. FARISS,J. Immunol. 86, 128-132 (1961) . V . L . MJORE and Q. N. MYRVIK, Infect . Immun . 2, 810-814 (1970) . D . ROMDO, R. CRAMER, T . MARZI, M. R. SORANZO, G . ZABUCCHI and F . ROSSI, J . Reticuloendothel . Soc . 1~, 399-409 (1973) .

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16 .

B . M . BABIOR, J . T . CURNUT7.'E and R . S . KIPNES, J . Lab . clin . Med . ~,

17 . 18 .

A. J. J. E.

19 . 20 . 21 . 22 . 23 . 24 .

235-244 (1975) .

W . 3EGAL and T . J . PETERS, Clin . Sci . mol . Med . ~, 591-596 (1975) " B . L . GEE, C . L . VA3ALL0, P . BFr "r" , J . KASKIN,R . E . BASFORD and B . FIELD, J . clin . Invest . 4Q, 1280-1287 (1970) . OUCHI, R . J . SELVARAJ and A . J . 3BARRA, Exp . Cell Res . 40, 456-468

(1965) .

B . B . PAUL, R . R . STRAUSS, A . A . JACOBS and A . J . SSARRA, Infect . Immun . 1, 338-344 (1970) . A . W . SEGAL and A . J . LEVI, Clin . ezp . Immunol . 1~, X9 - 318 (1975) " B . M . C ~eRx~!r^~xr , D . H . COWAN and R . W . SELCHER, Amer . J . clin . Path .

64, 34-40 (1975) "

C . KOCH, Acta path . microbiol . stead . Sect . C,B~, 139-143 (1975) . S . J . KL~e1~FF and C . B . HAè~N, in 'Mononuclear Ph c es in Immunity . Infection and Patholo~r ' (R . VAN FÜRTH ed . p . 507, Blackwell Scientific Press, Oxford, London, Edinburgh, Melbourne

(1975) "

25 . 26 .

J . T . CURNUR'PE and B . M . BASIOR, J . clin . Invest . ,, 1662-1672 (1974) . R . B . JOHNSTON, B . B . KEELE, H . P . MISRA, L . S . WEBB, J . E . i .FH~1F'YF.R and K . V . RAJAGOPALAN, in 'The c is Cell and Host Resistance' (J . A . Brrs.AxmI and D . H . DAYTON eds . p . 1, Raven Press, Nev York

27 .

M . L . KARNOVSKY, J . LAZDINS, D . DRATH and A . HARPER, Ann . N .Y . Aced .

28 .

R . B . JOHNSTON, J . E . i.FÜMF!YTi!R and L . A . GU'I'HRIE, J . exp . Med . lam,

29 .

W . D . BIGGAR, S . BURON and B . HOLMES, Infect . Immun . 14, 6-10 (1976) . J . B . L . GEE and A . S . KHANDWALA, J . Reticulcendothel . Soc . 1~,

30 . 31 . 32 . 33 .

(1975) .

Sci . 2~, 266-274 (1975) . 1551-1556 (1976) .

229-236 (19T6) .

B . H . PARK and R . A . GOOD, Lancet ü, 616 (1970) . A . W . SEGAL and A . J . LEVI, Clin . Sci . mol . Med . 48, 201-212 (1975) " F . ROSSI, G . ZABUCCHI and D . ROMEO, in 'Mononuclear Pha o es in Immunity, Infection and Patholop.Y ' (R . VAN . FÜRTH ed . , p . 1, B1ackFrell Scientific Press, Oxford, London, Edinburgh, Melbourne

(1975) .

Superoxide production by rabbit pulmonary alveolar macrophages.

Lifa Sciaaces Vol . 21, pp . 1575-1584 Priatad in thn U.S .A . Pargamon Preae SiJPERO%IDE PRODUCTION BY RABBIT PUI~NARY ALVEOLAR MACROPHAOEB D. B. L...
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