FORMATION OF SPHEROPLASTS OF MYCOBACTERIUM TUBERCULOSIS BY LYSOZYME IN COMBINATION WITH

CERTAIN ENZYMES OF RABBIT PERITONEAL MONOCYTES

HILDA POPE WILLETT AND HARSI-IAD TI-IACORE Department of n/Iicrobiology, Duke University Medical Center, Durham, North Carolina

Received July 5, 1966

Spheroplasts of ~Mycobacteriz~m tr~bercl~losis were formed by culture of the organism in a nutritionally complete medium to which Iysozyme and an extract obtained from rabbit peritoneal monocytes ulere added. The potentiating elfect of the factor, or factors, present in the monocyte extract was lost upon pro- longed storage a t -20 O C or heat a t 90 OC for 5 minutes, but was unaflected by dialysis. It \ \as inactive in the presence of fluoride or oxalate. Enzymatic analysis of the monocyte extract sho\ved the presence of lysozyme, acid phos- phatase, phospholipase, and trypsin. When acid phosphatase or phospholipase were substituted for the monocyte extract in the lysozyme test system, sphero- plast formation was obtained.

Introduction In tuberculosis, the mononuclear phagocyte determines the fate of the

invading organis~n follo\ving infection. One of the enzymes which has been shown to be present in these cells and which probably plays an important role in resistance to tuberculosis is lysozyme (2, 12).

The complex nature of the cell wall of Mycobacteriurn tuberculosis makes these organisms relatively resistant in vitro to the action of lysozyme. When ethylenediaminetetraacetic acid (EDTA), however, is included in the growth medium with lysozyme, osmotically fragile spheroplasts are formed (17). Since lysozyme is important in the body's natural defense mechanisms, the question arises whether there are, within the phagocyte cells of the body, other factors which will play a similar role to the EDTA used in the in vitro system, and allolv complete expression of the lysozyme effect. The existence of such factors was indicated in previous studies by the ability to initiate spheroplast formation in a monocyte tissue culture system by exposing the organism to lysozylne a t the time of infection (18).

The following is a report of our findings on spheroplast formation in an in vitro system utilizing an extract from rabbit monocytes in combination with lysozyme. The finding by other worlters ( 1 , 4 , 5 , 13) of hydrolytic enzymes in the monocyte extract prompted our examining the extract for various enzymes that might alter the cell surface of the tubercle bacillus in such a way that the mucopeptide substrate would be made more readily accessible to lysozyme.

Materials and Methods Cultures

The tubercle bacillus used in this investigation was the H37 Ra strain of M. tuberculosis maintained in stock culture by surface growth on Prosltauer- Beck synthetic medium and adapted to subsurface growth for these experi- ments by weeltly transfer and daily intermittent shaking in a Tween 80 medium without albumin (20). Cells were harvested after 3 days' growth and Canadian Journal of Microbiology. Volume 13 (1967)

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482 CAKADIAX JOURNAL OF MICROBIOLOGY. VOL. 13. 1967

washed once with Tween 80 - all~umin medium (6). They were resuspended in a small volume of this medium to give a final optical density reading of 0.03 a t 450 mp when included in the final reaction system.

Test System for the Fornzation of Spheroplasts For the formation of spheroplasts in liquid growth mediunl, a modified

Tween 80 - albumin ~nediu~ii was used. The components of the test system were prepared as follows.

Solution I contained Bacto-asparagine, 2.0 g ; I<1HJ'O4, 1.0 g ; Na2HP0.2. 121H20, 6.3 g ; Casamino acids, 1.0 g; ferric a m m o n i ~ ~ m citrate, 0.05 g ; i\4gS0,1. 7H20, 1 ml of 1% solution; CaCIz, 1 ml of 0.05% solution; ZnSO.,, 1 1111 of 0.01% solution; CuSO.2, 1 ml of 0.01% solution; Tween SO certified, 0.5 g. The pH was adjusted so that there \vould be a final pH of 7.1 after autoclaving of tlie solution a t 15 Ib pressure for 15 ininutes. Distilled water n7as added to a volume of 800 ml and tlie medium mas distributed in 28-1111 portions in 125-1111 flasks.

Solution I1 was prepared fresh before use and consisted of 28.0 1111 of solu- tion I to which tile follon~ing were added: 4.0 ml bovine (V-fraction) albumin (2.19y0 in 0.85% saline), 4.0 ml glucose solution (21.93% in 1120), 8.5 1111

sucrose solution (8.7 g dissolved in 8.5 ml solution I ) , 0.5 1111 3'IgSO., . 71-120 (17.54y0 in solution I ) . This solution was sterilized l ~ y Seitz filtration.

The complete test system for ol~taining spheroplast forlnation was con- tained in a total of 10.0 1111 of tlie follonring composition: 0.5 1111 suspeilsio~i of cells, 6.0 ml solution 11, and 3.5 ml of solution I containing the factor or factors under stucly. 'The tubes were incubated a t 37 "C and shalcen gently daily to resuspend the cells, and optical density readings talcen a t 450 Inp. Cultures were examined a t 24-hour intervals with dark phase contrast optics for change in inorphology of the cells.

Monocyte Extracts Extracts were prepared from the cells collected from peritoneal esudates

of rabbits according to the method of Fong et c~ l . (8). Rabbits injected intra- peritoneally with 45-50 In1 of heavy mineral oil (Nujol brand from Plough, Inc.. New York) were sacrificed on the fifth day and the peritoneal cavity washed with 200-250 1711 of chilled Tyrode's solution. Cells were collected by centrifuging the exudate a t 146 X g for 7 minutes and the cells nrashed twice with chilled Tyrode's solution. Total and differential cell counts were inade on cell suspensions. i lpprosi~n;~tely 90% of the cells obtained from the peri- toneal exudate were mononuclear cells. They were resuspended in chilled solution I of tlie Tween 80 culture nledium used in setting up the test systein. and vibrated for 15 minutes in a 9-lic I i a y t h e o ~ ~ sonic oscillator. The chamber containing the inonocyte suspension n7as chilled during this period by cir- culating ice water around the vibrating rod. The monocyte suspension, com- pletely disrupted by vibration, was then centrifuged a t 20,000 X g for 1 hour in a refrigerated Sorvall centrifuge.

The extract was standardized before use by a phosphate determination by Fiske and SubbaRow's procedure (7) . One milliliter of the standardized ex- tract contained 1.4 pmole phosphate, corrected for the phosphate in the

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\\"ILLETT AXD THACORE: SPHEROPLAST FORMATION BY LYSOZYME 483

mediu111 in which the monocytes were suspended. The extract, which was used immediately, was sterilized by passage through a HA 0.45 p millipore filter. Varying concentrations of standardized nlonocyte extract (0.02-2.0 ml) were used, alone and in colnbination with three different co~~centrat ions of lysozy~lle (50, 80, and 100 pg/ml). To test the effect of dialysis on activity of the extract, a portion of it was dialyzed for 24 hours a t 4 "C: against four changes of the suspending culture mediu111.

Enzyme Deternzinations The following methods were used in testing for the presence of certain

enzymes in the monocyte extract. Lysozyme.-'The lysozyme activity of the monocyte extract was determined

by a method suggested by Sh~igar (15). The substrate consisted of \Vorthingto11 dried il4icrococcz~s lysodeikticus cells, 0.3 nlg/nlI in 0.1 64 phosphate buffer, pH 7.0. To 2.9 1111 of this substrate, 0.1 ml of the standardized monocyte extract was added and the optical density read a t 15-second intervals a t 450 nip.

l'rypsin.-This was determined by Sch\vert and Taltenaka's (14) method, in which use is made of a difference in absorbancy a t 253 mp between N- benzoyl-1-arginine and its ester. The substrate consisted of 0.00025 Ad benzoyl- 1-arginine ethyl ester in 0.0667 Ad phosphate buffer, pH 7.0. The substrate (2.9 ml) was incubated with 0.2 nll of standardized ~nonocyte extract for 21 hours a t 37 "C.

Acid phosphnluse.-?'he activity \vas determined by the method described by Valentine and Beclc (19). The substrate, sodium-P-glyceropl~ospl~ate, was incubated for 1 hour a t 37 "C with 0.2 ml of standardized monocyte extract in 0.028 44 veronal buffer, pH 4.5. The reaction mixture was stopped by precipitating the protein with 10% trichloracetic acid. After centrifugation a t 1475 X g for 10 minutes, the supernatant was used for phosphate deter- mination, by the method of Fislce and Subbarow (7).

Phospholipase C.-A modification of the method of ;\iIacI;arlane and Icnight (11) was used. The substrate (2% homogenate of egg in water) was incubated with standardized monocyte extract in a 0.1 M Tris-maleate buffer system, pH 7.3, a t 37 "C for 24 hours. After precipitation of unchanged lecithin the liberated phosphoryl choline \\:as digested and measured as inorganic pllos- phorus.

Other enzymes tested for but \\rhich were not detected in the volume of monocyte extract used with lysozylne in the spheroplast induction system included allcaline phosphatase, alpha-amylase, carboxypeptidase A, carboxy- peptidase B, chymotrypsin. beta-glucuronidase, lipase, pepsin, veilom phos- phodiesterase, and spleen phosphodiesterase.

Results and Observations The Effect of Monocyte Extract on the Formation of Spheroplasts

In testing the effect of the standardized monocyte extract, all optillla1 system was found to be one containing 80 pg/ml lysozyme and 0.2 ml of the monocyte extract. At the end of 7 days a 10% inhibition of growth was obtained in the system containing 0.2 ml of the extract alone (Fig. 1). During

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484 CANADIAN JOURNAL O F MICROBIOLOGY. VOL. 13, 1967

I ! 4 I , I I ! I I I 1

0 1 2 3 4 5 6 7 8 9 1 0

DAYS

FIG. 1. The effect of 80 pg/ml lysozyme and of 0.2 ml of a standardized monocyte ex- tract (M.E.) alone and in combination on the growth of IM. tz~berculosis in a Tween 80 - albumin medium containing- 0.34 M sucrose and 1.2 mg/ml of ME++.

the first 3 days no change in the morphology of the cells was observed. During the next 4 days, although a very small percentage of cells showed a slight degree of swelling, most of them resembled the control cells with the absence of demonstrable swelling (Fig. 2B).

The inhibition of growth by SO pg/inl of lysozynle is shown in Fig. 1, and is approximately 50% by the end of 1 week. No obvious change was observed in the morphology of the organism during the first 4 days of exposure to lysozyme. During the next 2 days some cells appeared slightly swollen; many seemed to be transparent and undergoing lysis (Fig. 2C). The surviving cells of this system, however, showed no obvious changes and continued to multiply.

In a system containing lysozy~ne and monocyte extract, more inhibition was obtained than when either was used singly (Fig. 1). An increase in amount of either the lysozyme or monocyte extract used resulted in an increased degree of lysis. A ma?tiinum number of spheroplasts was obtained in a system containing 80 ,ug/ml lysozyrne and 0.2 ml monocyte extract. This coinbina- tion of lysozyme and extract produced approximately 60y0 inhibition of growth as opposed to SOYo with lysozyme alone. A steady rise in the optical density was observed during the experiment, indicating that all cells were not equally susceptible to the conlbined action of lysozyme and monocyte extract. This is in contrast to observations on the action of lysozyme and EDTA under similar growth conditions in which the optical density leveled off after the third day (17).

The first indication of morphological changes in the cells of the lysozyme- monocyte extract system appeared on the third day when the cells were observed to be swollen and appeared transparent (Fig. 2D). During the next 2 days there was an increase in the number and degree of swelling of the cells, and from the sixth day on, an increasing number of spheroplasts were observed in the system (Fig. ZE, F). Not all cells of the system were equally susceptible to lysozyme action and many remained unchanged; others were optically less dense, with or without swelling.

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Flc;. 2. C l i a~~gcs i l l the ~liorpliol(~g>. o[ :If. tflberclllc~.si.s $ro\\.ll i l l a T\\.ee~i 80 - albumill 111ccliu111 r o ~ ~ t a i ~ i i ~ i ~ O..% .I! S [ I C ~ O S ~ :~llcl 1.1 11ig/1ii1 hlg+-'- i l l 11 s).ste111 colitaitii~~g 80 pg/1111 Iysox).~iic ;llicl 0 . 2 1111 01 sta~i(larclixccl 111011c)cyte cxtri~ct, illolie alicl ill c o ~ i i b i ~ ~ a t i o ~ i : (.\) control, 6 cl;~!.s; (I!! cxtr;lrt alone, 6 clays; ( C j Iysozynle a l o ~ ~ c , 6 cla\.s; ( I> ) Iysozy~~ic ancl extract, 3 cla:.~; (I:) I>-soz).lnc ant1 extract, 6 (lays; (F) lysozyn~e a ~ l d extract, 9 clays. X 5238.

Can. J. Microbial.-Willett and Thacorc

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FIG. 3. Formation of spheroplasts of A$. tllbercrtlosis ill 'Tween 80 - a l l )~~min rnctl;urn containing 0.31 dd sucrose alitl 1.1 111g/rnl R~lg~'~+ i l l the presence of 100 pg/ml 1)-sozynic a~i t l 3.5 pg/lnl acid phosphatasc or 2.5 pg/nil ~)hospholipase C : (:\) co~ltl.ol, 6 tla!.s; (B) acid phosphatase olily, 6 days; ( C ) phos~)holipase C only, 6 tin)-s; ( I ) ) I).soz).~nc ol~ly, 6 days; ( E ) lysozyme aiicl acid phosphat:~sc, 3 clays; ( F ) Iysoz!-me a ~ ~ c l acid phosphat;~sc, 6 clays; (G) lysozyme and phospholipase C, -I days; ( H ) lysozyme ant1 phospholipasc C, 6 days. X 5238.

Can. J. Microbio1.-\\'illett and Tllacore

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WILLETT AND THACORE: SPHEROPLAST FORM.ATION B1' LYSOZYME 485

N a t ~ ~ r e of the "Active Substance" in Monocyte Extracts iVIonocyte extracts lost inost of their activity when stored a t -20 O C for 2

months, as shown by the observation that very few spl~eroplasts were formed when used in coinbination with SO pg/inl of lysozyme. Activity was coin- pletely lost when the monocyte extract was heated a t 90 O C for 5 minutes.

Dialyzed monocyte extract alone had no effect on the n~orphology of the tubercle bacillus, but when 0.2 in1 of the dialyzed extract was used in com- bination with lysozyine (80 pg/ml) spl~eroplasts were observed on the sixth day, as with the undialyzed extract-lysozyine combination. The active component of the monocyte extract which worlts in con~bination with lysozyme is thus a heat-lal~ile substance of high inolecular weight.

The e'fect of the inonocyte extract on the morphology of the organisin is completely eliminated when either oxalate or fluoride is included in the inonocyte-lysozyme system in a concentration which alone does not inhibit growth or affect the morphology of the organism. Since oxalate and fluoride are inhibitors of acid phosphatase, the findings suggest that acid phosphatase is one of the enzymes present in monocyte extracts responsible for making the inucopeptide layer of the cell wall available to the lytic enzyme, lysozyme.

Enzymatic Analysis of Monocyte Extract Of the 14 enzymes tested for, only 4 were found in the extract in a sufficient

amount to be detected under the conditions used for testing. Lysozyme was found in a concentration of 15 pg/inl and trypsin in a concentration of 6 pg/ml when based on the activity of twice crystallized Worthington enzymes. In the standardized lnoilocyte extract an activity of 17.5 pg of acid phospha- tase and 6.5 pg of phospholipase C were detected per milliliter of the extract. Failure to find the other enzyines tested for does not preclude their presence, but simply indicates that they could not be detected by the procedures used, when tested for in the monocvte extract in an amount 10 tiines that which was sufficient to produce spl~eroplasts.

T h e E f e c t of Various Enzymes on the Growth of il~ycobacterizlnz Tz~berculosis The folloxving enzymes were tested in concentrations of 10 pg to 80 pg/ml

for their effect on tubercle bacilli, alone and in combination with lysozyme (100 pglinl) in the Tween-albumin system containing 0.34 M sucrose and a high Mg++ concentration (1.2 ing/ml supplied as i\?gS04.7H20): acid phos- phatase, alkaline phosphatase, alpha-amylase, carboxypeptidase A, carboxy- peptidase B, chyinotrypsin, beta-glucuronidase, lipase, pepsin, phosphodies- terase (venom), phosphodiesterase (spleen), phospholipase C, and trypsin. When tested alone, only three of these enzyines exerted an inhibitory growth effect on the organism. A 20% inhibition of growth was produced by 10 pg/ml of beta-glucuronidase and spleen phosphodiesterase; a 15% inhibitory effect was shown with the same concentration of phospholipase C. When phospholi- pase C was used in combination with 100 pg/ml of lysozyine the inhibitory effect a t varying concentrations appeared to be additive. With the other two enzyines, however, no greater inhibition was produced than by lysozyme alone. None of the 13 enzymes when used alone had any effect on the mor- phology of the organisms during the 2-week period over which the experi- ment was followed.

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486 CANADIAN JOIJKNAL OF MICROBIOLOGY. VOL. 13. 1967

Although trypsin, chymotrypsin, pepsin, and lipase were without effect on the tubercle bacillus when used alone, a greater degree of gro\vth inhibition was observed when each of these enzymes was used in conlbination \vith lysozyn~e. This effect was 111ost pronounced with trypsin where 10 pgjml together with 100 pgjml of lysozyme produced a 95% inhibition of growth.

1Iicroscopic examination for changes in the morphologic appearance of the organisms revealed changes only \vhen acid phosphatase or phospholipase C was used in combination \vith lysozyme.

1;ormut~on of Spl~eropltrsts i n n Lysozynze - ,lcid Plzosphutase Systenz No inhibition of growth or any change in the morphologic appearance of

the organism was ol~served with 2.5 pgjml of acid phosphatase (Fig. 3B). X 60% inhil~ition of gro\vth was produced hy lysozyme alone, but the mor- phological changes produced were minimal ancl were late in appearing (Fig. 3D) as reported previously (17).

A system containing 2.5 pgjml of acid phosphatase and 100 pgjml of lysozyme provided an optimal system for observing the changes produced. In this s).ste~n the cells were swollen, either on the ends or in the middle of the rod, on the third day (Fig. 3E). During the next 24 hours, more s\vollen cells wcre ol~scrved, and l ~ y the sisth day spherical spheroplasts were seen (Fig. 3F). After this time cell lysis was evident, and there was a decrease in the number of spheroplasts. ,\lthough higher concentrations of acid phos- phatase (5-80 pgjml) did not affect the rate of multiplication of the organisms, an increased a~lloiint of Iysis was observed \vhen the higher concentrations were used in coml,ination wit11 lysozyme. The lysoz~me - acid phosphatase system resembled more closely thc lysozyme - rnonocyte extract system than the one with lysozyme and ED'I'X (17). This \vas true not only in respect to the time of appearance of the spheroplasts but also the type of gro\vth curve obtained.

Fornzation 01 Splzeropl(zsts i n n Lysozyme - Plzospl~olipuse C System Similar results \\rere obtained with phospholipase C as with acid phospha-

tase. Spheroplasts \\rere obtained in a system containing 2.5-5.0 pg/ml phos- pholipase C and 100 p g / ~ n l lysozyme. Figure 3G shou-s intermediate stages in the formation of spheroplasts, whereas Fig 3H she\\-s the final stage on the sixth day. Fewer spheroplasts, ho\vever, were formed in this system as compared to the lysozyme - acid phosphatase system.

Discussion In tuberculosis the most significant factor in the n~echanisms involved in

both immunity and natural resistance is the capacity of the mononuclear phagocyte to destroy or inhibit the gro\vth of the invading organism. Studies during recent years have demonstrated that in chronic infections such as tuberculosis functional and morphologic alterations occur. Lurie's early observations (10) of an increased inhibitory effect of monocytes on phago- cytized tubercle bacilli stimulated interest in the enzymatic components of these phagocytic cells in normal and in infected aninlals (1, 4, 5, 13).

One of the enzymes \vhich has been shown to be present in the mononuclear phagocyte is lysozyme. This enzyme is present in higher concentrations in

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U'ILLETT A N D TIIACORE: SPI-IEROPLAST FORMATIOX BY LYSOZYM'IE 487

alveolar macrophages than in peritoneal ~nacropl~ages, and is elevated in ~nacrophages derived from tuberculous animals (2, 12). Lysozyme destroys bacteria by hydrolyzing the basal nlucopeptide component of the cell wall. Tubercle bacilli are relatively resistant to the action of lysoz~.me altl~ough when included with EDTA in a system suitable for gronrth, oslnotically fragile spheroplasts are formed (17).

Our studies with the monocyte extracts obtained from peritoneal exudates of normal rabbits emphasize the importance of factors other than lysozyme within these phagocytic cells. The active substance in the monocyte extract was found to be heat-labile, non-dialyzable, and inactive in the presence of fluoride or oxalate. The properties suggested the enzyme acid phosphatase as an important component of the lysozyme - monocyte extract system. T h e substitution of acid phosphatase for the monocyte extract in this in vitro system induced spheroplast production.

The role of acid phosphatase in the host's resistance to tubercle bacilli has been emphasized before (3, 16). Like lysozyme, it is present in higher concentrations in alveolar ~nonocvtes than in oil-induced ~eri toneal ~nonocvtes (9). Infection with tubercle bacilli also induces an increase of acid phosphatase levels (13).

Of the other enzymes tested, only pl~ospl~olipase C in coml~ination \\it11 lysozyn~e was capable ol initiating spl~eroplast formation. As observed with acid phosphatase, no alteration in the structure of the organisms was obtained unless lysozy111e was included in the s j stem. Since an enzynle with phos- pholipase activity was detected in the monocyte extract, as was acicl phos- phatasc and lysozyme, it probably also plays an important role in limiting intracellular growth of tuhercle bacilli.

Altl~ougl~ the ~llucopeptide substrate of Iysozyme is present in tubercle bacilli, it constitutes a snlaller percentage of the cell wall material than it does in the gram-positive organisms. The presence of large amounts of lipo- protein and lipopolysaccl~aride exterior to the mucopeptide layer necessitates the presence of a second accessory agent for ~nalting the mucopeptide sub- strate available. Once the wall structures are altered other substrates of the bacterial cell are then vulneral~le to attaclc by various lytic enzymes nrithin the host cell.

The finding that certain enzj7mes nor~nally present in the monocyte are capable of inducing spheroplast formation when used in combination with lysozy~ne is of fundamental ilnportance because of their possible role in the induction of an L-type growth of -11. tztberczilosis in their intracellular locale within the monocytes of the infected animal. Our observations also enlphasize the need for a reevaluation of techniclues employed for the study of the rate of tubercle bacilli after infection, if one is to take into consideration this possible induction of an L-for111 of growth \vhose co~nponents are not demon- strable by the standard techniques of culture and microscopy.

Acknowledgment This study was supported in part by USPHS training grant 5 T I GhI 1019

from the National Institute of General hdedicine, by Public Health Service Research Grant A1 03988-03 from the National Institute of Allergy and

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488 CANADIAN JOURXAL OF MICROBIOLOGY. VOL. 13. 1967

Infectious Diseases, and by a research grant from the American Thoracic S o c i e t y , i\/Iedical Section of the N a t i o n a l Tuberculosis Association.

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6. Dunos, R. J . and DAVIS, B. D. 1946. Factors affecting the growth of tubercle bacilli in liauicl medium. 1. E s ~ t l . hled. 83. 409-423.

7. F I S K E , ' ~ . H. and S;BBAI<OW, Y. 1925: The colorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400.

8. FONG, J., SCIIMEIDER, F., and ELBERG, S. S. 1956. Studies on tubercle bacillus - monocyte relationship. I. Qr~antitative analysis of effect of serum of animals vaccinated with BCG upon bacterium - monocyte system. J. Exptl. RlIed. 104, 455-465.

9. LEAICE, E. S., GOSZALEZ-OJEDA, D., and MYRVIIC, Q. x. 1964. Enzymatic differences between normal alveolar macrophages and oil-iriduced peritoneal macrophages obtained from rabbits. Exptl. Cell Res. 33, 553-561.

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