INFECTION AND IMMUNITY, Sept. 1975, p. 687-693Copyright i 1975 American Society for Microbiology

Vol. 12, No. 3Printed in U.S.A.

Characterization of Monkey Peripheral Neutrophil GranulesDuring Infection

P. GREGORY RAUSCH* AND PETER G. CANONICO

United States Army Medical Research Institute of Infectious Diseases, Frederick, Maryland 21701

Received for publication 23 May 1975

Rhesus monkey (Macaca mulatta) neutrophils were shown to contain theazurophilic granule marker enzymes myeloperoxidase and ,B-glucuronidase butwere deficient in the specific granule markers alkaline phosphatase (AKP) andlysozyme. Isopycnic centrifugation of leukocyte homogenates on linear sucrosegradients resulted in cosedimentation of myeloperoxidase and ,-glucuronidasewith an equilibrium density of 1.18. After an intravenous inoculation of monkeyswith Salmonella typhimurium AKP activity became marked, whereas that of,-glucuronidase decreased and myeloperoxidase remained unchanged. Lysozymewas undetected throughout the course of the experiment, but was present inoil-induced peritoneal macrophages and peripheral mononuclear cells. Theinduced AKP exhibited partial latency and had an equilibrium density of 1.15. Itis unclear, however, whether the induced AKP is associated with specificgranules or cytoplasmic membranes. Hence, while these data are consistent withthe presence of azurophilic granules in polymorphonuclear neutrophils frominfected monkeys, the presence of specific granules in polymorphonuclearneutrophils of both uninfected and infected monkeys remains moot.

Ingestion of microorganisms by polymor-phonuclear neutrophils (PMN) results in rapiddegranulation of the phagocyte as cytoplasmicgranules fuse with the phagocytic vacuoles (4).Many studies evaluating bactericidal and diges-tive mechanisms of PMN have therefore cen-tered on the biochemistry and morphology ofthese granules (1). Studies of rabbit neutrophilsindicate the presence of two major types ofgranules (5). The larger, electron-dense, azuro-philic (primary) granules are formed during thepromyelocyte stage of cellular maturation (5).These granules contain myeloperoxidase(MPO) (EC 1.11.1.7), lysozyme (LZM) (EC3.2.1.17), and acid hydrolases, which classifiesthem as true lysosomes (3). The smaller, lessdense, specific (secondary) granules appear dur-ing the later myelocyte stage of cellular matura-tion (5, 6) and contain LZM, alkaline phospha-tase (AKP) (EC 3.1.3.1) (3), lactoferrin (2, 21),and collagenase (EC 3.4.4.-) (30).While the enzymatic complement of PMN

granules in other species is generally similar tothat of rabbit, a number of notable exceptionshave been reported. For example, in humangranulocytes, AKP is localized to cytoplasmicmembranes (32). Chicken leukocytes have beenreported to lack MPO (9), whereas those ofcattle are deficient in LZM (25). The histo-chemical absence ofAKP has also been reported

in leukocytes of cats, dogs (18), mice, andrhesus monkeys (15).We now report that rhesus monkey PMN are

also deficient in LZM. Alterations in enzymecontent and distribution of monkey PMN gran-ules after an experimentally induced infectionare also reported. Results indicate that AKP,but not LZM, is markedly induced after infec-tion.

MATERIALS AND METHODSAnimals. Eight young adult rhesus monkeys

housed in individual cages under controlled tempera-ture and light were fed water and Purina monkeychow ad libitum. All monkeys were evaluated fordisease and three blood samples were obtained forbase-line determinations before an intravenous inocu-lation of 109 Salmonella typhimurium, a dose previ-ously shown (29) to cause fever but not death inrhesus monkeys. Five 25-ml blood samples wereobtained from each monkey on days 1, 3, 7, 10, and 14postinoculation. Rectal temperatures were monitoredtwice daily.

Leukocyte isolation. Leukocytes were harvestedfrom ethylene-diaminetetraacetic acid (EDTA)-anti-coagulated blood by differential sedimentation at4 C in dextran, molecular weight 500,000 (PharmaciaFine Chemicals), as described by Skoog and Beck(31). After contaminating erythrocytes were lysed byexposure to 3 volumes of 0.87% NH4Cl for 10 min,leukocytes were washed three times with Hanksbalanced salts solution (Microbiological Associates)

687

on April 4, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

688 RAUSCH AND CANONICO

and adjusted to a final concentration of 5 x 106 PMN/ml. One- to 3-ml suspensions of leukocytes consist-ing of 60 to 75% PMN were routinely obtained. Hu-man and rabbit peripheral leukocytes were also har-vested by the above procedure.

Mononuclear (MN) phagocytes were elicited fromanother group of normal monkeys by intraperitonealinjection of 50 ml of sterile Bayol F. After 4 days,animals were killed and cells were harvested byperitoneal lavage and washed as above.

Highly purified mononuclear cell suspensions wereprepared from normal monkey peripheral blood usingBoyum's technique (7).

Histochemistry. EDTA-anticoagulated bloodsmears were fixed for 30 s in ice-cold 10% formalin inmethanol, air dried, and stained for AKP by the azodye technique; 100 cells were counted and scored (19).Leukocytes were also stained for MPO by the methodof Kaplow (20). Normal human peripheral bloodsmears served as stain controls.

Electron microscopy. Purified monkey peripheralblood leukocytes were fixed in suspension in cacody-late-buffered glutaraldehyde, stained, and processedaccording to the method of Farquhar et al. (16).Enzyme determinations. For cellular enzyme de-

terminations, isolated leukocyte suspensions weresonicated in an ice bath at 80 W (Bronson Sonifier)with three 30-s periods of exposure. Triton X-100 wasthen added to a final concentration of 0.1%. Proteinwas determined by the Lowry method, using bovineserum albumin as standard (22). AKP was deter-mined in 0.62 M 2-amino-2-methyl-1-propanol buffer(pH 10.2) with 15.2 mM p-nitrophenylphosphate and0.1 mM MgCl2 at 37 C for 30 min. The reaction wasstopped with 1.25 M NaOH and absorbance was readin a Beckman DU recording spectrophotometer at 420nm (11). MPO was assayed with 3.3 mM o-anisidinein 0.1 M phosphate buffer (pH 6.8) with 10 mMhydrogen peroxide. Change in absorbance was moni-tored continuously at 460 nm and results were ex-pressed as change in optical density per min (26).,B-Glucuronidase (,GU) (EC 3.2.1.31) activity wasdetermined with 1.0 mM phenolphthalein glucuro-nide in 0.1 M acetate buffer (pH 5.0) after the methodof Canonico and Bird (10). LZM was determined bythe lysoplate method of Osserman and Lawlor (24).

Fractionation of leukocyte granules. Peripheralleukocyte granule suspensions have been characteris-tically difficult to prepare because peripheral leuko-cytes, in contrast to peritoneal exudate cells, cannotbe ruptured readily without simultaneous lysis ofintracellular granules. Satisfactory granule prepara-tions, however, were obtained by swelling peripheralcells in hypotonic media before homogenization.

Leukocytes from 75 ml of pooled EDTA-anticoagulated blood were isolated as described aboveand suspended in a small volume of ice-cold 0.34 Msucrose in 5 mM NaHCOs, pH 8.2; 4 volumes of anice-cold 4 mM NaHCOs solution containing 1 mMethylene-glycoltetraacetic acid were added. The cellswere allowed to swell for 2 min at 4 C and thenhomogenized for three 1-min periods in a motor-driven glass-Teflon Porter Duall homogenizer with aclearance of 0.2 mm. Each 1-min period consisted of

eight up and down cycles with the pestle rotating at1,000 rpm. Isotonicity was restored with 0.1 volume of9.0% NaCl, and a postnuclear supernatant fractionwas prepared by centrifugation at 400 x g for 10 min.In some experiments, acridine orange (50 ,g/ml) wasadded to leukocyte suspensions before homogeniza-tion (14) to assess the extent of cellular lysis. Fluores-cence microscopy of the homogenate revealed thatover 95% of the PMN were disrupted. Granularorange-red fluorescence in the postnuclear superna-tant confirmed the presence of intact granules,whereas enzyme assays of the supernatant resultingfrom a high-speed centrifugation (40,000 x g for 20min) of the homogenate demonstrated an 18 to 30%release of the granular enzymes.

For isopycnic centrifugation, the postnuclear su-pernatant was layered on a 28 to 60% linear sucrosegradient and centrifuged at 25,000 rpm for 1 h (W2t =1,880 x 107 rad2/s) in a SW27 rotor. One- to 2-mlfractions were collected with a Buchler autofractiona-tor. The sucrose concentration of each fraction wasassessed with an Abbe 3-L refractometer (Bausch &Lomb).

Three subcellular fractions were isolated by differ-ential centrifugation of the homogenate. A nuclearpellet was separated at 2,000 x g for 6 min. Theresulting supernatant was centrifuged at 25,000 x gfor 10 min to obtain a granule and a postgranularfraction. Enzyme latency in tissue fractions wasdetermined by measurement of enzyme activitiesbefore and after addition of Triton X-100 (0.1%), andresults were expressed as relative specific activities aspreviously described (13).

RESULTSMonkey, rabbit, and human PMN differed

markedly in content of MPO, BGU, AKP, andLZM (Table 1). Human PMN contained thehighest levels ofMPO and LZM, whereas rabbitPMN had higher levels of #GU and AKP.Monkey PMN contained moderate levels ofMPO and ,BGU, but AKP and LZM activities, ifpresent, were lower than the sensitivity of theenzyme assays used.

Histochemical activity of MPO and AKP ineach species closely approximated the biochem-ical activity. MPO reaction product was visual-ized as fine cytoplasmic particles in monkeyand human PMN; larger, though fewer, grainswere observed in rabbit PMN. Positive reac-tions were occasionally seen in monocytes of allspecies but not in lymphocytes. AKP reactionproduct appeared as very small cytoplasmicgrains in rabbit and human PMN, but monkeyneutrophils and other leukocytes and plateletsfrom all species were uniformly negative.An electron micrograph of an isolated periph-

eral PMN from a normal monkey is shown inFig. 1. Two morphologically distinct granulepopulations are apparent. The predominant

INFECT. IMMUN.

on April 4, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

VOL. 12, 1975 MONKEY PMN GRANULES IN INFECTION 689

TABLE 1. Comparison of PMN granular enzyme levels between species

Enzyme activity/5 x 106 PMN/minaSpecies No. __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

MPO ,GU AKP LZM

Human 3 67.7 + 5.2 2.47 4 0.41 8.5 ± 1.60 101.8 22.6Rabbit 3 24.0 + 4.0 2.56 + 0.76 106.3 + 30.99 47.8 + 8.9Rhesus monkey 5 66.0 ± 3.0 1.90 ± 0.35 < 0.5 < 1.0

aValues are means + standard errors. MPO,substrate hydrolyzed; LZM, micrograms.

1

I,.;,It. IZ .ma -.~._ .w,0.vI

FIG. 1. Electron micrograph of normal monkeyPMN (x8,000) showing two granule populations. (1)Large, spherical granules, 0.2 to 0.4 jum in diameter;(2) smaller rod-shaped granules, 0.1 x 0.4 um; Lm,lymphocyte.

form is represented by relatively larger, roundgranules measuring 0.2 to 0.4 Mm in diameter. Inaddition, smaller elongated bodies (0.1 by 0.4um) are occasionally seen. Both forms appearequally electron dense.

Isopycnic fractionation of leukocyte granulesfrom normal monkeys is shown in Fig. 2. MPOand ,BGU cosedimented in a single band with anequilibrium density of 1.18. AKP activity wasnot detected throughout the gradient.

After inoculation with S. typhimurium allmonkeys rapidly developed lassitude and ano-rexia. Fever (> 102.5 F [Ca. 39 C]) occurred inall monkeys within 12 h of bacterial challengeand persisted for 5 days (Fig. 3). Neutropeniadeveloped rapidly with the nadir occurring onday 3; thereafter lymphocytosis ensued whilethe granulocyte count returned to normal. Byday 14 all animals were afebrile but lym-phocytosis persisted. All survived and nonerequired antibiotic treatment.Enzyme activities for the preinoculation con-

trol period, the peak febrile period (days 1 and3), and the postfebrile period (days 7, 10, and14) are listed in Table 2. MPO activity re-

A optical density (10- 3); ,GU, AKP: nanomoles of

3-

I-

0

C.0at

-50

-40wen00

-30 Dae

-20

5 10 15 20 25

FRACTION NUMBER

FIG. 2. Isopycnic fractionation of postnuclearPMN homogenates from normal monkeys. Percent oftotal activity in each fraction is shown for ,3GU andMPO; AKP was undetected. Total enzymatic recover-ies were: ,3GU, 92%; MPO, 85%.

mained constant throughout the experimentalperiod. 3GU activity declined to one-half of thecontrol value of 1.21 nmol/5 x 106 PMN/minduring the peak febrile period and returned tocontrol values during the postfebrile period.AKP, undetected during the control period,showed marked activity (27.6 nmol/5 x 106PMN/min) during the peak febrile period andremained at 40% of maximal activity throughday 14. No correlation was observed betweenAKP activity in the cell lysates and the totalwhite count or absolute PMN count (r = 0.201and 0.198, respectively). LZM activity was notdetected in peripheral leukocyte homogenatesfrom the experimental group, but was present inperitoneal macrophages and peripheral mono-cytes isolated from control monkeys.

Histochemical evidence of AKP activity wasnoted as early as 12 h after inoculation. Granu-lar reaction product was observed within theperinuclear region of PMN. A consistent rela-tionship was observed between the enzymaticactivities of cell lyastes and the histochemicalreactivity of AKP and MPO.

on April 4, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

690 RAUSCH AND CANONICO

106-, 105

1 04-103-

010

oEE0z

TEMPERATURE1(102.5)

14DAYS

FIG. 3. Clinical responses of M. mulatta to S. typhimurium infection. Temperature, total leucocytes andtotal PMN are shown as means; vertical lines represent standard errors of the mean (n = 8).

Isopycnic fractionation of leukocytes ob-tained from monkeys 3 days after inoculationdemonstrated that AKP activity was presentand sedimented at an equilibrium density of1.15 (Fig. 4). The sedimentable portion of #GUactivity shifted to an equilibrium density of1.20.The distribution of AKP and MPO in differ-

entially fractionated homogenates of peripheralleukocytes from infected monkeys (Fig. 5) dem-onstrated maximal concentration in the granulefraction. The enzymatic distribution and par-tial structure-linked latency appear consistentwith a granular localization. However, the pos-sibility of an association of AKP with cytoplas-mic membranes cannot be excluded at thistime, since this enzyme, in contrast to MPO,also showed partial latency in the postgranularfraction.

DISCUSSIONPrevious studies of the enzymatic content of

PMN granules have been limited primarily tothose of rabbit and human; however, since therhesus monkey is frequently used as a labora-tory model for a variety of infectious diseases, itis important to define differences between rhe-sus and human PMN. Initial comparisons ofenzymatic activities in human, monkey, andrabbit PMN emphasized the marked interspe-cies variations that occur and illustrate thedifficulties of extrapolation of data betweenspecies. The demonstration that monkey PMN

were deficient in AKP and LZM, but not fGUor MPO, prompted the studies reported here.MPO is restricted to azurophilic granules in

rabbit (34) and human (32) PMN. These gran-ules also contain acid hydrolases such as fGU(32). LZM is a component of both azurophilicand specific granules in many species (2, 8),whereas AKP is a specific granule marker inrabbit (2) but not human PMN (32). The factthat rhesus monkey PMN lack AKP and LZMsuggests either a multiple deficiency of specificgranule enzymes or an absolute absence ofspecific granules. Since electron micrographs ofmonkey PMN demonstrated marked morpho-logical heterogeneity of cytoplasmic granules,separation of the various granules was at-tempted by isopycnic centrifugation.The azurophilic markers MPO and flGU were

associated with a sedimentable particle havinga mean equilibrium density of 1.18. Structure-linked latency of MPO was also demonstrated.These characteristics are similar to those ofMPO, 8GU, and other azurophilic constituentsin rabbit (34) and human (17) PMN. Accord-ingly, it is reasonable to suggest that MPO and,GU are associated with, and are reliable mark-ers for, monkey azurophilic granules. However,since specific granule markers were undetected,we were unable to determine if a second class ofgranules was separated in the gradients. Hence,we attempted to induce a detectable level ofthese enzymes by utilizing the phenomenon ofenzyme activation after bacterial infection (1).

S. typhimurium inoculation caused a marked

INFECT. IMMUN.

on April 4, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

MONKEY PMN GRANULES IN INFECTION

TABLE 2. Effects of infection on granular enzymes in M. mulatta

Enzyme activity/5 x 106 PMN/minaTime

MPO ,GU AKP

Preinfection 63.8 + 5.1 1.21 + 0.11

692 RAUSCH AND CANONICO

X 6 MPOa.CD 5

11J4 N

2

0

0 20 40 60 100

% TOTAL PROTEIN

FIG. 5. Differential centrifugation of leukocyte ho-

mogenates from infected monkeys. Ordinate: relative

specific activity (percentage of total recovered activi-

ty/percentage of total recovered protein) for AKP and

MPO. Abscissa: fractions represented by relative

protein content in the order in which they were

isolated. N, nuclear; G, granular; S, postgranular.

Height of each bar expresses relative specific activity

in presence of 0.1% Triton X-100; hatched regions are

enzyme activities determined in absence of Triton

x-ioo.

and sheep (P. G. Rausch and T. G. Moore, Fed.

Proc. 34:861, 1975). In these instances, there do

not appear to be any changes in the bactericidalcapacities of the cells. Similarly, monkey PMNwith their multiple deficiencies of specific gran-ule constituents do not appear to have alteredbactericidal capacity since PMN isolated fromnormal monkeys effectively kill Escherichia coliin vitro (28). There are no data suggesting thatthis species has an increased susceptibility tobacterial infection in vivo. Hence, the relation-ship of LZM content to the overall bactericidalcapacity of the PMN remains to be determined.

ACKNOWLEDGMENT

We thank John D. White for preparation of electronmicrographs.

LITERATURE CITED

1. Baggiolini, M. 1972. The enzymes of the granules of

polymorphonuclear leukocytes and their functions.Enzyme 13:132-160.

2. Baggiolini, M., C. de Duve, P. L. Masson, and J. F.Heremans. 1970. Association of lactoferrin with specificgranules in rabbit heterophil leukocytes. J. Exp. Med.131:559-570.

10.

INFECT. IMMUN.

Baggiolini, M., J. G. Hirsch, and C. de Duve. 1970.Further biochemical and morphological studies ofgranule fractions from rabbit heterophil leukocytes. J.Cell Biol. 45:586-597.

Bainton, D. F. 1973. Sequential degranulation of the twotypes of polymorphonuclear leukocyte granules duringphagocytosis of microorganisms. J. Cell Biol.58:249-264.

Bainton, D. F., and M. G. Farquhar. 1966. Origin ofgranules in polymorphonuclear leukocytes. J. Cell Biol.28:277-301.

Bainton, D. F., J. L. Ullyot, and M. G. Farquhar. 1971.The development of neutrophilic polymorphonuclearleukocytes in human bone marrow. J. Exp. Med.134:907-934.

Boyum, A. 1968. Isolation of mononuclear cells andgranulocytes from human blood. Scand. J. Clin. Lab.Invest. 21(Suppl. 97):77-89.

Bretz, U., and M. Baggiolini. 1974. Biochemical andmorphological characterization of azurophil and spe-cific granules of human neutrophilic polymorphonu-clear leukocytes. J. Cell Biol. 63:251-269.

Brune, K., and J. K. Spitznagel. 1973. Peroxidaselesschicken leukocytes: isolation and characterization ofantibacterial granules. J. Infect. Dis. 127:84-94.

Canonico, P. G., and J. W. C. Bird. 1970. Lysosomes inskeletal muscle tissue. Zonal centrifugation evidencefor multiple cellular sources. J. Cell Biol. 45:321-333.

DeChatelet, L. R., and M. R. Cooper. 1970. A modifiedprocedure for the determination of leukocyte alkalinephosphatase. Biochem. Med. 4:61-68.

DeChatelet, L. R., J. V. Volk, C. E. McCall, and M. R.Cooper. 1971. Studies on leukocyte phosphatases. II.Inhibition of leukocyte alkaline phosphatase by aminoacids and its reversal by zinc. Clin. Chem. 17:210-213.

de Duve, C., B. C. Pressman, R. Gianetto, R. Wattiaux,and F. Appelmans. 1955. Tissue fractionation studies.6. Intracellular distribution patterns of enzymes inrat-liver tissue. Biochem. J. 60:604-617.

Dingle, J. T., and A. J. Barrett. 1969. Uptake of biologi-cally active substances by lysosomes. Proc. R. Soc.London Ser. B. 173:85-93.

Eng, L.-I. L. 1964. Alkaline phosphatase activity of theleukocytes in animals. Nature (London) 204:191-192.

Farquhar, M. G., D. F. Bainton, M. Baggiolini, and C. deDuve. 1972. Cytochemical localization of acid phospha-tase activity in granule fractions from rabbit polymor-phonuclear leukocytes. J. Cell Biol. 54:141-156.

Folds, J. D., I. R. H. Welsh, and J. K. Spitznagel. 1972.Neutral proteases confined to one class of lysosomes ofhuman polymorphonuclear leukocytes. Proc. Soc. Exp.Biol. Med. 139:461-463.

Jain, N. C. 1968. Alkaline phosphatase activity in leuko-cytes of some animal species. Acta Haematol. (Basel)39:51-59.

Kaplow, L. S. 1955. A histochemical procedure forlocalizing and evaluating leukocyte alkaline phospha-tase activity in smears of blood and marrow. Blood10:1023-1029.

Kaplow, L. S. 1965. Simplified myeloperoxidase stainusing benzidine dihydrochloride. Blood 26:215-219.

Leffel, M. S., and J. K. Spitznagel. 1972. Association oflactoferrin with lysozyme in granules of human poly-morphonuclear leukocytes. Infect. Immun. 6:761-765.

Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

McCall, C. E., I. Katayama, R. S. Cotran, and M.Finland. 1969. Lysosomal and ultrastructural changesin human "toxic" neutrophils during bacterial infec-tion. J. Exp. Med. 129:267-293.

Osserman, E. F., and D. P. Lawlor. 1966. Serum andurinary lysozyme (muramidase) in monocytic and

on April 4, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

MONKEY PMN GRANULES IN INFECTION 693

monomyelocytic leukemia. J. Exp. Med. 124:921-952.25. Padgett, G. A., and J. G. Hirsch. 1967. Lysozyme: its

absence in tears and leukocytes of cattle. Aust. J. Exp.Biol. Med. Sci. 45:569-570.

26. Paul, B. B., R. R. Strauss, A. A. Jacobs, and A. J. Sbarra.1970. Function of H202, myeloperoxidase and hexosemonophosphate shunt enzymes in phagocytizing cellsfrom different species. Infect. Immun. 1:338-344.

27. Prieur, D. J., H. M. Olson, and D. M. Young. 1974.Lysozyme deficiency-an inherited disorder of rabbits.Am. J. Pathol. 77:283-298.

28. Proctor, R. A., J. D. White, E. Ayala, and P. G. Canonico.1975. Phagocytosis of Francisella tularensis by rhesusmonkey peripheral leukocytes. Infect. Immun.11:146-151.

29. Rayfield, E. J., D. T. George, and W. R. Beisel. 1974.Altered growth hormone hemeostasis during acutebacterial sepsis in the rhesus monkey. J. Clin. Endo-crinol. Metab. 38:746-754.

30. Robertson, P. B., R. B. Ryel, R. E. Taylor, K. W. Shyu,and H. M. Fullmer. 1972. Collagenase: localization inpolymorphonuclear leukocyte granules in the rabbit.Science 177:64-65.

31. Skoog, W. A., and W. S. Beck. 1956. Studies on thefibrinogen, dextran and phytohemagglutinin methodsof isolating leukocytes. Blood 11:436-454.

32. Spitznagel, J. K., F. G. Dalldorf, M. S. Leffell, J. D.Folds, I. R. H. Welsh, M. H. Cooney, and L. E. Martin.1974. Character of azurophil and specific granulespurified from human polymorphonuclear leukocytes.Lab. Invest. 30:774-785.

33. Trubowitz, S., E. Moschides, and D. Feldman. 1961.Alkaline phosphatase activity of the polymorphonu-clear leukocyte in rapidly induced leukopenia andleukocytosis. J. Lab. Clin. Med. 57:747-754.

34. Zeya, H. I., and J. K. Spitznagel. 1971. Isolation ofpolymorphonuclear leukocyte granules from rabbitbone marrow. Lab. Invest. 24:237-245.

VOL. 12, 1975

on April 4, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/