INFECTION AND IMMUNrrY, Oct. 1974, p. 928-937Copyright i) 1974 American Society for Microbiology

Vol. 10, No. 4Printed in U.S.A.

Leukocyte Dysfunction in the Bovine Homologue of theChediak-Higashi Syndrome of Humans

HARLAND W. RENSHAW, WILLIAM C. DAVIS, H. HUGH FUDENBERG, AND G. A. PADGETTDepartments of Veterinary Microbiology and Veterinary Pathology, Washington State University, andPioneering Research Laboratory Western Region, Agricultural Research Service, U.S. Department of

Agriculture, Pullman, Washington 99163; and Department of Medicine, University of California School ofMedicine, San Francisco, California 94122

Received for publication 9 August 1974

The increased susceptibility to pyogenic infections noted in cattle with theChediak-Higashi syndrome trait has been related to an impairment of leukocytefunction at the cellular level. Investigations of the relationship of abnormalgranule formation to increased susceptibility to infection, conducted with cellsuspensions containing high concentrations of polymorphonuclear leukocytes,revealed a bactericidal defect that was associated with abnormal intracellularkilling and not due to defective particle ingestion. The in vitro bactericidal defectwas associated with a metabolic anomaly in the hexose monophosphate shunt,but not with an alteration in the capacity to reduce nitroblue tetrazolium dye.Ultrastructural histochemical studies of phagocytosis and phagolysosome forma-tion in polymorphonuclear leukocytes suggest that the impairment in bacteri-cidal capacity is correlated also with either a delay or failure of primary granulesto degranulate.

The Chediak-Higashi syndrome (C-HS) is arare autosomal recessive disease in man whichis characterized by increased susceptibility tobacterial infections, partial oculocutaneous al-binism, and the presence of large abnormalgranules (C-HS granules) in a number of differ-ent cell types (2, 6, 10, 30, 39). Over the past fewyears, homologues of this disease have beenidentified in four phylogenetically disparatespecies: mink (22, 31, 38), cattle (31), mice (4,23), and killer whales (R. F. Taylor and R. K.Farrell, Fed. Proc., abstr. 3403, vol. 32, 1973).Although the exact site of the primary gene de-fect has not been established, comparative ul-trastructural studies have revealed that C-HSgranules occur both as abnormal enlarged pri-mary (virgin) lysosomes or related granules, andas enlarged phagolysosomes, depending uponthe type of cell and its function (11, 12). Theorigin of these granules has been best char-acterized in leukocytes. In neutrophils, theC-HS granules arise from the aberrent forma-tion and fusion of the primary (azurophil) gran-ules (11). The defect is selective in that only onepopulation of granules is affected, i.e., sec-ondary (specific) granules are unaffected (11).The development of C-HS granules in basophilsand eosinophils follows the same pattern (12).

Information on the relation of abnormal gran-ulogenesis to the debility of affected humansand animals is limited; however, recent studies

92t

indicate that increased susceptibility to infec-tion may be primarily associated with neutro-phil dysfunction (8, 9, 15, 34). In vitro studies ofphagocytosis by phase microscopy have shownthat the large pleomorphic primary granules failto degranulate after ingestion of bacteria (28,34). Root et al. (34) have demonstrated that inhumans this alteration in functionality is ac-companied by an impaired capacity to killcertain gram-negative and gram-positive bacte-ria.The purpose of the present report is to de-

scribe our recent findings on the bovine homo-logue of the C-HS, which show that abnormaldegranulation in neutrophils is associated witha marked impairment in bactericidal capacityand a decrease in hexose monophosphate shunt(HMPS) activity.

MATERIALS AND METHODSAnimals. The normal cattle and cattle affected

with the C-HS trait used in these studies weremaintained on a nutritionally adequate diet at theexperimental facilities at Washington State Univer-sity.

Leukocytes. Peripheral blood leukocytes were iso-lated from heparinized (10 U/ml) venous blood bylysing the erythrocytes in 3 volumes of 0.87% ammo-nium chloride (NH4Cl; 20). The leukocytes werepelleted by low speed centrifugation (150 x g for 10min) and washed twice in Eagle minimal essentialmedium (MEM) with 10% heat-inactivated fetal calf

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serum. Viability was determined by 0.1% trypan bluedye exclusion and was >96% in all preparationsexamined. Total and differential leukocyte countswere performed by heomcytometer and by examina-tion of Wright stained smears, respectively. To obtainleukocyte preparations containing a high percentageof polymorphonuclear leukocytes (PMN), and toavoid a peculiar irreversible agglutination which oc-curs with bovine leukocytes, a modification of theFicoll (Pharmacia, Uppsala, Sweden)-Hypaque (Win-throp Laboratories, N.Y.) density gradient centrifu-gation technique of Boyum was employed (7). Hepa-rinized blood was centrifuged at 300 x g for 15 min at20 C. The buffy coat was harvested and diluted 2:1with MEM, layered in 30-ml volumes on 15 ml of theFicoll-Hypaque mixture, and centrifuged at 400 x gfor 40 min at 20 C. After removal of the mononuclearcell layer, the erythrocyte-granulocyte pellet wasresuspended in 5 ml of homologous plasma. Erythro-cytes were removed by lysis with 3 volumes of 0.87%NH4Cl, and leukocytes were collected by centrifuga-tion at 150 x g for 10 min. Then the cells were washedtwice with MEM and prepared for immediate use.

Peritoneal exudate cells were collected in heparin-ized MEM (10 U/ml) 12 to 14 h after intraperitonealinfusion of 4 to 5 liters of 0.1% glycogen (NutritionalBiochemicals Corporation, Cleveland, Ohio) in saline.After removal of contaminating erythrocytes by lysiswith 0.87% NH4Cl, the leukocytes were pelleted andwashed twice in MEM.

Microorganisms. Field isolates of Bacillus sub-tilis, Staphylococcus aureus, Streptococcus pyogenes,Sarcina lutea, Escherichia coli, and Salmonellapullorum were used as test organisms to assessbacterial activity of bovine leukocytes. All bacteriawere grown overnight in brain heart infusion broth(Difco, Detroit, Mich.), centrifuged for 20 min at2,000 x g, and washed three times in sterile phos-phate-buffered saline, pH 7.4. The samples wereadjusted to an optical density of 0.6 with a Bauschand Lomb Spectronic 20 set at 620 nm. The bacterialconcentrations were determined by appropriate stan-dard curves.

Bactericidal assays. A modification of the methodof Hirsch and Strauss (19) was employed to determinethe in vitro bactericidal capacity of normal andaffected cattle leukocytes. Suspensions containing 5to 10 x 106 PMN/ml in 10% autologous serum andMEM were placed in plastic tubes (12 by 75 mm) andincubated at 37 C for 15 min. Either 0.1 or 0.2 ml ofthe bacterial suspension was added to produce abacteria to PMN ratio in the range of 1:1 to 10:1. Thetubes were rotated end over end (20 revolutions permin) at 37 C. Portions of the cell bacteria mixturewere removed at intervals, lysed in sterile distilledwater, diluted serially by 10-fold dilutions, and platedon standard nutrient agar pour plates. For noncell-associated counts, the majority (>95%) of the leuko-cytes were pelleted by centrifugation (150 x g for 10min at 4 C). Samples of the cell-free supernatantswere placed in sterile distilled water. Serial dilutionswere then dispensed in agar pour plates and incu-bated at 37 C. The number of colonies growing on theplates were counted the following day to determinethe number of viable bacteria present for each time

period. Zero time counts were obtained by taking themean of two bacterial suspensions that did notcontain leukocytes. Results were expressed as thenumber of viable bacteria remaining per milliliter ofthe reaction mixture per unit time, or as the percent-age of the original bacteria in the inoculum survivingper unit time.

Electron microscopy cytochemistry. A modifica-tion of the method of Graham and Karnovsky forstaining of peroxidase (18) was used as a cytochemicaltechnique for detecting myeloperoxidase (MPO) ac-tivity in ultrastructural studies of the PMN, and forexamining the sequence of events leading to fusion ofprimary granules with the phagosomes after ingestionof bacteria. The leukocyte suspensions in MEM with10% autologous serum were adjusted to a concentra-tion of 10 x 106 PMN/ml and incubated at 37 C on arotator for 30 min with B. subtilis at a bacteria-cellratio of 10:1 or 40:1. Samples of cells were collected at5, 15, and 30 min after the addition of bacteria to thecell suspension. The cells were centrifuged, fixed in2% potassium phosphate-buffered (0.1 M, pH 7.3)glutaraldehyde, with 1% added sucrose, for 1 h, rinsedin a potassium phosphate-buffered (0.1 M, pH 7.3)solution of 1% sucrose, and then incubated for 9 minat room temperature in a saturated solution of3,3'-diaminobenzidine in 0.05 M Tris-hydrochloridebuffer (pH 7.6) containing 0.01% hydrogen peroxide(H202). The cells were rinsed in distilled water andthen postfixed in potassium phosphate-buffered 1%osmium tetroxide (0.1 M, pH 7.3) for 30 min. Thespecimens were stained en bloc with an aqueoussolution of 1% uranyl acetate, dehydrated through agraded series of ethanol, and embedded in Epon-aral-dite (26). Thin sections were collected on uncoated200-mesh grids, stained with lead or uranyl salts (33,37), and examined in a Philips 200 electron micro-scope.

Nitroblue tetrazolium test. Neutrophils from nor-mal cattle and cattle heterozygous and homozygousfor the C-HS trait were examined for their functionalcapacity to reduce nitroblue tetrazolium (NBT; gradeIII, Sigma Chemical Company, St. Louis, Mo) afterphagocytosis of 0.81-gim latex spheres (Difco, Detroit,Mich.). The ability of neutrophils to reduce NBT wastested by a modification of the spectrophotometricmethod of Baehner and Nathan (1). A suspension ofleukocytes (0.1 ml at a concentration of 25 x 106PMN/ml) was reacted with polystyrene latex spheres(0.05 ml of 0.81-um latex spheres dialyzed againstphosphate-buffered saline, pH 7.4), potassium cya-nide (0.1 ml of a 0.01-M solution of KCN), NBT (0.4ml of 0.1% solution), and phosphate-buffered saline(0.35 ml in phagocytizing tubes with spheres, and0.40 ml in control resting tubes without spheres) ina total volume of 1.0 ml. The cell mixtures were in-cubated for 15 min at 37 C; then 5 ml of 0.05 M HClwas added to stop the reaction. The pelleted cellswere washed, boiled for 15 min in 4 ml of pyridine(reagent grade), and then centrifuged (1,000 x g for10 min) to remove the sediment. The optical densityof reduced NBT in the extract was read on a Bauschand Lomb Spectronic 20 spectrophotometer at 515nm against a pyridine blank.

Glucose oxidation. Glucose oxidation was deter-

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mined with [1-_4C]glucose and [6-"4C]glucose by amodification of the method of Mickenberg, Root, andWolff (25). Suspensions of 5 x 106 PMN, in 2 ml ofMEM containing 25% autologous serum (11.2 ,umoland 0.5 uCi of glucose), with and without 0.81-Mmpolystyrene latex spheres (approximately 2 x 109),were placed in 25-ml Erlenmeyer flasks. The flaskswere covered with a rubber stopper with an attachedpolyethylene centerwell (Kontes Glass Co., Vineland,N.J.). After the stoppers were in place, 0.2 ml of afreshly prepared 10% solution of KOH was added tothe center wells to collect the CO2. The flasks wereincubated for 1 h at 37 C in a shaking water bath (60oscillations per min). The reaction was stopped byinjecting 0.5 ml of 1.0 N HCl through the cap into themixture. After an additional 15 min of incubation, thecontents of the polyethylene center well containingthe labeled CO2 were placed in a counting vial con-taining 20 ml ofaqueous phosphor (42 ml of Liquifluor,New England Nuclear, Boston Mass.; 660 ml of tolu-ene; and 300 ml of methanol). The samples werecounted in a Packard Tri-Carb liquid scintillationspectrometer with at least a 50% counting efficiencyand at a standard deviation of less than 2.5%. Thecounts per minute were converted to disintegrationsper minute from an appropriate quench curve.

RESULTS

Differential counts on leukocytepreparations. There were no statistically sig-nificant differences in the differential counts ofleukocyte cell suspensions obtained from nor-

mal cattle and cattle with the C-HS trait (Table1). This contrasts with the neutropenia noted inhuman subjects with the C-HS (5), but agrees

with the observations that neutropenia does notexist in the animal homologues (4, 9). Differen-tial counts of leukocyte cell suspensions ob-tained by NH4Cl lysis of heparinized wholeblood were within normal limits for cattle (3).There was a threefold enrichment of the PMNcontent of the cell suspensions by Ficoll-Hypaque density gradient centrifugation ofbuffy coat cells. The degree of monocyte andlymphocyte contamination in these prepara-

tions were similar in normal and affected cattle.Bactericidal assays. A comparison of the in

vitro capacity of peripheral blood leukocytesfrom normal and affected cattle revealed a

marked difference in their capacity to kill both

gram-positive and gram-negative bacteria: B.subtilis, S. aureus, S. pyogenes, S. lutea, E.coli, and S. pullorum. As shown for B. subtilis,impaired function was evident at 1 h andpronounced at 2 and 4 h (Fig. 1). Although lessextensive, studies with peritoneal exudate cellsyielded similar results (Fig. 2).

Further studies with cells isolated with Ficoll-Hypaque demonstrated that reduction in bacte-ricidal activity was associated with an intracel-lular defect in PMN. Cell preparations, contain-ing 70 to 80% PMN and less than 3% monocytes,reacted with four different species of bacteriaexhibiting a prominent impairment in bacteri-cidal capacity. This defect was very similar tothat obtained with unseparated cells (Table 2).The difference in the rate of killing was not

attributable to a variation in the uptake ofbacteria (Table 2). PMN from affected animalswere as effective as normal cells in clearingbacteria from the leukocyte-bacteria cell sus-pension.Electron microscopy cytochemistry. In pre-

vious studies by light microscopy, C-HS gran-ules have been observed in affected neutrophilsfor prolonged periods of time after phagocytosisof bacteria, with the granules persisting aroundthe phagocytic vacuole and in some cases

apparently localized within phagosomes (28,34). Therefore, studies were conducted to verifythis point and to determine the extent to whichimpaired postphagocytic degranulation mightcontribute to the reduction in bactericidal ca-

pacity. A cytochemical stain for MPO was usedto facilitate the identification of primary gran-

ules. Both qualitative and quantitative differ-ences in granule content could be distinguished(Fig. 3 and 6). The primary granules (C-HSgranules) in cells from affected animals were

pleomorphic and fewer in number than innormal cells (Fig. 3 and 5).Two differences were noted in PMN after in-

gestion of bacteria. Whereas degranulation was

readily detected in normal cells at 15 and 30min (Fig. 3 and 4), few changes were evident inC-HS cells even after 30 min (Fig. 5 and 6). Thelarge C-HS granules were seen generally intactin the cytoplasm (Fig. 6) or, on occasion,

TABLE 1. Differential counts ofperipheral blood leukocytes from normal cattle and cattle with the C-HS trait

Percentage of total leukocytes (mean standard error)Cell origin No. of prepn Method of prepn

PMN Lymphocytes Monocytes Eosinophils Basophils

Normal 8 0.87% NH4Cl lysis 22.7 4.2 68.1 ± 9.4 5.8 4 3.1 2.9 ± 0.7 0.5 0.2C-HS 10 0.87% NH4Cl lysis 24.8 ± 3.7 65.9 ± 8.1 6.2 + 2.6 2.6 ± 1.3 0.6 ± 0.3Normal 5 Ficoll-Hypaque 74.3 ± 6.1 16.4 + 4.5 2.1 + 0.9 7.2 ± 1.9 0.2 + 0.1C-HS 4 Ficoll-Hypaque 75.5 ± 5.5 15.8 + 5.1 1.8 ± 1.1 6.4 + 2.4 0.5 + 0.2

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C0

D 103

Ez

0(2

0 60 120 180 240

MINUTES

FIG. 1-2. In vitro study of the killing of opsonizedB. subtilis by leukocytes from normal cattle andcattle with the C-HS trait. The logarithm of thenumber of viable intracellular bacteria is plotted as a

function of time. Cell suspensions were prepared bythe 0.87% NH4CI lysis method.

FIG. 1. In vitro bactericidal capacity of peripheralblood leukocytes from normal cattle and cattle withthe C-HS trait. (Note the difference in the rate ofkilling of ingested bacteria.)

apparently located in phagocytic or autophagicvacuoles (Fig. 6, insert). In addition, thereappeared to be a difference in the morphology ofingested organisms. Some of the bacteria ob-served in normal cells that had been incubatedfor 30 min appeared partially degraded (Fig. 4).Those in C-HS cells were much more distinctand similar to uningested bacteria (Fig. 6).Nitroblue tetrazolium test. Studies with the

NBT test were unrevealing, although this testhas been used effectively to show alterations inmetabolic activity in other granulocytopathies,such as chronic granulomatous disease of child-hood (1). Leukocytes from cattle homozygousand heterozygous for the C-HS trait were as

capable as those obtained from normal cattle inreducing NBT dye to blue formazan after thephagocytosis of polystyrene latex spheres(Table 3). The differences between resting andphagocytizing cells from the different groups ofanimals were not significant. As will be dis-cussed later, this is a rather interesting observa-tion in light of the correlation between bacteri-cidal capacity and dye reduction found in othergranulocytopathies.Glucose oxidation. Since the NBT test did

not reveal any alteration in the metabolic activ-

ity of affected cells, further studies were con-ducted to determine whether any alterationcould be detected in glucose metabolism by theHMPS or Krebs cycle pathway. Contrary to anearlier report (29), a significant difference wasdemonstrated in the activity of affected andnormal leukocytes. This was most pronouncedin the oxidation of [1-14C ]glucose. HMPS activ-ity in mixed and Ficoll-Hypaque-separatedC-HS leukocytes was significantly reduced inboth resting and phagocytizing cells (Table 4).Statistically significant but less striking differ-ences were noted in oxidation of [6-14C]glucoseand here only with Ficoll-Hypaque-separatedcells (Table 4).

DISCUSSIONAlthough the biochemical defect(s) in the

C-HS has not been definitively elucidated, thedemonstration that C-HS granules are the re-sult of an abnormality in a biosynthetic path-way common to granule-forming cells has pro-vided new insight into the genetic basis of thisinteresting disease. Furthermore, it has allowedfor a more meaningful analysis of how C-HSgranules contribute to the general debility andincreased susceptibility to infection characteris-tic of this disease (10, 11, 12). C-HS granulesrepresent aberrant forms of normal constituentsof cells, not tertiary granules or accumulationsof cell products as in the lipidosis (17). In PMN,C-HS granules represent pleomorphic forms ofprimary granules (11). Thus, dysfunction of this

0 60 120 180 240MINUTES

FIG. 2. In vitro bactericidal capacity of peritonealexudate cells from normal cattle and cattle with theC-HS trait.

0UVtNE PERIPEtRAL BLOOD LEUKOCYT IEI~ Bocillus subtilis

5xiOr Cells

0-0 C - HS

0C- Normal

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TABLE 2. Bactericidal activity of leukocytes from normal cattle and cattle with the C-HS traita

Viable bacteria (%)c Cell-associated ClearedeOrganism Cell origin" 120 mind (%)

20 min 60 min 120 min (%)

S. aureus Normal (4) 15.3 8.6 6.3 ± 3.0 3.9 2.3 3.4 ± 2.1 98.4 ± 0.8C-HS (4) 38.9 7.1' 26.8 6.9' 24.2 7.6' 23.0 5.4' 98.9 0.3

S.pyogenes Normal (4) 18.6 ± 6.1 5.3 ± 3.4 3.8 ± 1.6 2.9 ± 2.4 99.3 ± 0.2C-HS (4) 36.7 ± 9.0' 22.9 ± 4.8' 15.9 ± 3.6t 14.3 ± 2.9' 99.1 ± 0.5

B. subtilis Normal (3) 12.4 ± 4.5 3.2 ± 1.6 0.7 ± 0.5 0.5 ± 0.08 98.5 ± 1.0C-HS (3) 47.5 ± 12.2' 28.9 ± 4.7' 25.4 ± 8.6' 23.6 i 7.3t 99.0 ± 0.6

E. coli Normal (3) 18.3 ± 4.2 3.7 ± 2.7 1.8 ± 1.5 1.1 ± 0.7 98.2 ± 0.7C-HS (3) 26.9 i 5.7 18.9 ± 4.1' 12.6 ± 2.2' 11.7 ± 2.0' 98.9 ± 0.4

a Cells separated by the Ficoll-Hypaque density gradient centrifugation of buffy coat cells. They were

incubated with bacteria in 10% autologous serum in a concentration of 5 x 106 polymorphonuclear leukocytesper ml.

b Number in parentheses is amount of experiments conducted, each on a different subject.c Percent survival (mean standard error of the mean) of original bacterial inoculum at the time intervals

indicated.d Percentage (mean ± standard error of the mean) of original inoculum associated with leukocyte pellet after

centrifugation at 150 x g for 10 min.ePercentage (mean X standard error of the mean) of bacteria cleared from cell-free supernate (150 x g for 10

min) by adherence to or ingestion by leukocytes at 120 min.'Difference from normal is statistically significant (P < 0.05, two-sample t test).

lpi

- pd-w

p~~~~~~~~

FIG. 3-6. Electron photomicrograph ofPMN from cattle after in vitro incubation (with or without bacteria)for 30 min. The cells were histochemically stained for myeloperoxidase present in the primary granules with3,3'-diaminobenzidine, and the cell suspensions were prepared by the 0.87% NH4CI lysis method.

FIG. 3. Normal PMN incubated without bacteria. Cell contains primary (P) and secondary (S) granulescharacteristic ofa mature PMN. x 18,000.

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¢_ .- 4 .f s + .; >>1

S j F s

t s . _ gEs t.* X ' t; S;t - -,,l a ^- ;, f^Y, -

t 4 .: 4 e v t .;;;r- . -

,.e *,. ,)

FIG. 4. Normal PMN incubated in the presence of B. subtilis. Phagocytic vacuole formation and degranula-tion of most primary granules has occurred. One primary granule in the process of degranulation (D) is evi-dent. Some of the bacteria (arrow) present within the phagocytic vacuoles appear partially degraded. x30,OOO.

cell type should correlate with the abnormalfunctioning of this organelle. In retrospect, theinitial observations of phagocytosis did revealan alteration in degranulation after ingestion ofbacteria, as evidenced by the persistence ofC-HS granules, apparently intact, for prolongedperiods of time after phagocytosis (28). Theimportance of this observation has been ampli-fied by the recent findings in humans (34) andnow cattle, which have demonstrated that thisanomaly is accompanied by abnormal bacteri-cidal and metabolic functions. Even thoughingestion of bacteria is normal (28, 34), the rateof killing is diminished, being reflected as analteration in the rate and not the absence ofkilling. Thus, a serial sample assay methodreveals the defect, whereas a single-point assaymethod yields variable results. This may, inpart, account for the results obtained in anearlier report which indicated no bactericidaldefect in affected cattle phagocytes (28).Although the exact significance has not been

determined, depression of glucose oxidation

accompanies the abnormal bactericidal andlysosomal functions noted in affected cattleleukocytes. In contrast to the human cells (43),a depression of glucose oxidation by bothHMPS and Krebs cycle pathways was observedin resting and phagocytizing cattle cells. Theresults of this study are seemingly at variancewith previous observations on glucose oxidationby normal and affected cattle cells (29). How-ever, the decreased HMPS activity noted in thecurrent report may have been obscured by theuse of peritoneal exudate cells, where a majorityof the cell population is macrophages, and byrecording the data as the ratios of [1-14C ]glucoseand [6-14C]glucose oxidation. We made directmeasurements of [1-14C ]glucose oxidation bycell populations derived from the peripheralblood which contained a high concentration ofPMN.

Phagocytosis of either bacteria or latexspheres in normal PMN is accompanied by adramatic burst of metabolic activity, whichincludes increased oxygen consumption, glycol-

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S

SS

.s0- I'tA'4 e ..

As. *S.

FIG. 5. C-HS PMN incubated without bacteria.characteristic of mature C-HS neutrophil. x18,000.

ysis, and oxidation of glucose through theHMPS (27, 35). It has been suggested that theincreased HMPS activity may serve to supplyH202 to the PMN, by reoxidation of reducednicotinamide adenine dinucleotide phosphateby its oxidase in the presence of molecularoxygen (21). Although HMPS activity doesappear to be associated with 02 uptake andH202 production, the relationship is not com-pletely understood (36). Metabolic reactionsthat result in the reduction of molecular oxygento H202 in the PMN are critical to host defensebecause of the importance of peroxide formationfor the MPO-H202-halide microbicidal system.The bactericidal activity generated by theMPO-H202-halide microbicidal system is effec-tive against both gram-negative and gram-posi-tive organisms (24). Thus, there may be arelationship between bactericidal impairmentnoted in leukocytes from cattle with the C-HStrait and reduced HMPS pathway activity inthe affected cells. It could be postulated thatthe reduced HMPS activity results in reducedH202 production and impaired functioning ofthe MPO-H202-halide microbicidal system.

Cell contains C-HS (P) and secondary (S) granules

The finding of altered postphagocytic de-granulation in affected human and cattle cellsextends the microscopic observation that thegiant MPO-positive lysosomes of the granulo-cytes persist, apparently intact, for hours afterthe ingestion of bacteria (28, 34). These obser-vations suggest that abnormal delivery of lyso-somal contents to the phagocytic vacuole inaffected cattle and human cells may be relatedto diminished intracellular bactericidal capac-ity. Collectively, the reduced HMPS pathwayactivity and altered postphagocytic degranula-tion may explain why affected cattle cells ex-hibit a prominent bactericidal defect to abroader spectrum of bacteria than do humanC-HS cells (34). Additional studies will beneeded to determine the relationship betweenthe microbicidal defect and the abnormal meta-bolic and lysosomal activities noted in thisstudy.

Studies conducted with affected human (34)and cattle cells indicate that there is not adirect association of NBT dye reduction withHMPS activity. Root et al. (34) found anincrease in HMPS activity in resting cells and

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normal activity in phagocytic human C-HSneutrophils. However, we found a decrease inHMPS activity in both resting and phagocyticneutrophils from cattle with the C-HS trait.

Yet, the data from the human cells (34) and our

data revealed normal NBT dye reduction byC-HS cells. Although NBT dye reduction byneutrophils is a poorly understood biochemical

.4

FIG. 6. C-HSPMN incubated in the presence of B. subtilis. Phagocytic vacuole formation has occurred withan intact, apparently undigested bacterium (arrow) without the vacuole. However, C-HS granules remaining inthe cytoplasm after a 30-min incubation indicate that the degranulation of primary granules (P) was delayed.x30,000. Insert shows phagocytic or autophagic vacuole apparently containing an intact C-HS granule.x30,000.

TABLE 3. Quantitative reduction of NBT by leukocytes from normal cattle and cattle heterozygous andhomozygous for the C-HS traita

No. of Optical densityCell origin No. of tests animals

tested Resting Phagocytizing A Difference

Normal 10 7 0.104 4 0.071b 0.202 ± 0.110 0.098 ± 0.0440.023 to 0.220c 0.093 to 0.355 0.042 to 0.175

C-HS (heterozygous) 10 10 0.105 + 0.069 0.208 i 0.084 0.103 + 0.0570.035 to 0.235 0.095 to 0.340 0.042 to 0.255

C-HS (homozygous) 10 3 0.103 i 0.069 0.182 + 0.071 0.079 + 0.0420.020 to 0.230 0.085 to 0.285 0.020 to 0.145

a Results are expressed as optical density at 515 nm against a pyridine blank and are given for 2.5 x 106neutrophils per 15 min. Cell suspensions were prepared by the 0.87% NH4Cl lysis method.

b Mean ± standard error of the mean.c Range of values.

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TABLE 4. Glucose oxidation by peripheral blood leukocytes from normal cattle and cattle with the C-HS trait inthe presence and absence of polystyrene latex spheres

Substrate Cell origin No. of animals tested Cell prepna Restinge Phagocytizing

[1-'4C]Glucose Normal 8 Mixed 42.3 + 6.2 (8) 133.8 + 31.0 (8)C-HS 5 Mixed 22.9 + 7.5 (9) 49.3 + 20.7 (10)

P value 0.001 0.001

Normal 5 PMN 42.3 + 5.3 (5) 131.3 + 32.1(5)C-HS 4 PMN 27.0 ± 5.7 (4) 45.3 + 6.0 (4)

Pvalue 0.02 0.001

[6-"4C]Glucose Normal 5 PMN 1.30 + 0.02 (5) 2.57 + 0.62 (5)C-HS 4 PMN 1.08 0.09 (4) 1.354+ 0.09 (4)

Pvalue 0.01 0.01

aMixed cells refer to those obtained by 0.87% NH4Cl lysis; PMN were obtained by centrifugation onFicoll-Hypaque (for differential counts see the text). P value from two sample t tests.

b Values are expressed as nanomoles of glucose oxidized per 5 x 101 polymorphonuclear leukocytes per hour:mean + standard error of the mean. Each experiment was conducted with triplicate or quadruplicate suspen-sions. Number of experiments is shown in parentheses.

event, the test has been used as a marker ofenzymatic activation, which in normal PMNcorresponds to the activity of intracellular kill-ing of bacteria. In children with chronic granu-lomatous disease, phagocytosis is quantitativelynormal, but there is an in vitro defect inbactericidal capacity (16). In this hereditarydefect, the inability of the PMN to kill certainbacteria corresponds to the defect in NBT dyereduction (1). NBT dye reduction has been usedas a clinical test for the diagnosis of chronicgranulomatous disease and is almost as sensi-tive as bactericidal assays in detecting individu-als homozygous and heterozygous for the trait(1, 13). However, in humans with the C-HS andin affected cattle, there is no correlation be-tween defective bactericidal activity of PMNand defective NBT dye reduction.

It is perhaps worth emphasizing the unique-ness of the bovine homologue of the C-HS andthe potential it offers for the study of the C-HSand the other granulocytopathies described inman (14). It is the only animal model, at thepresent time, with well documented abnormali-ties in leukocyte function that are associatedwith increased susceptibility to infection (30,32). Thus, C-HS cattle show the closest resem-blance to the disease occurring in man and, assuch, may serve as the best model for analyzingthe genetic and biochemical basis of the disease.Additionally, the presence of defects both in themetabolic and lysosomal activity of neutrophilsprovides an opportunity to compare the relativerole of each in effecting the destruction ofbacteria.

ACKNOWLEDGMENTSWe thank Donald Gayman and Susan Renshaw for excel-

lent technical assistance, Nancy Carter and Lynda Pitkin for

help in preparing this manuscript, and Harold Conners forphotography.

This investigation was supported by Public Health Servicegrants GM-00691-12 from the National Institute of GeneralMedical Sciences, AI-06591 and AI-09145 from the NationalInstitute of Allergy and Infectious Diseases, and HD-05894from the National Institute of Child Health and HumanDevelopment, and by General Research Support FR 5465.

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