Abnormal Bactericidal, Metabolic, and Lysosomal Functions of ...

Abnormal Bactericidal, Metabolic, and Lysosomal

Functions of Chediak-Higashi Syndrome Leukocytes

RMcicu K. RooT, ALANS. ROSENTHAL,and DomnIc J. BALESIA

From the Laboratory of Clinical Investigation, National Institute ofAllergy and Infectious Diseases, National Institutes of Health,Bethesda, Maryland 20014

A B S T R A C T Phagocytic, antimicrobial, and metabolicfunctions were studied in leukocytes obtained from threepatients with the Chediak-Higashi syndrome (CHS)and compared to normals, individuals, heterozygous forChediak-Higashi syndrome, and two subjects withchronic granulomatous disease of childhood (CGD).Chediak-Higashi syndrome leukocytes showed normalingestion of a variety of bacteria, Candida albicans, andpolystyrene latex particles. Intracellular destruction wassignificantly impaired for Staphylococcus aureus, GroupD streptococci, and a rough strain of Type II pneumo-cocci over a 2 hr incubation. Killing of Serattia marces-ccns was consistently delayed at 1 hr whereas that ofEscherichia coli and C. albicans appeared normal, unlessthe incubations were shortened to 20 min. Examinationof the rates of killing indicated that the greatest defectoccurred in the first 20 min of contact between Chediak-Higashi syndrome cells and bacteria. Separation ofChediak-Higashi syndrome granulocytes from monocytesrevealed that the former were most defective in bacteri-cidal activity. After phagocytosis, Chediak-Higashi syn-drome granulocytes displayed a normal burst in oxygenconsumption and oxidation of glucose-l-'4C and glucose-6-uC and formate-14C. Oxidation of glucose-12T4C by non-phagocytizing Chediak-Higashi syndrome granulocytesand monocytes averaged 2-3 times normal, whereasglucose-6-14C and formate-12C oxidation were not sig-nificantly increased by resting cells. Iodination of intra-cellular protein by Chediak-Higashi syndrome leukocytes

Dr. Root's present address is Infectious Disease Section,Department of Medicine, University of Pennsylvania, 552Johnson Pavilion, Philadelphia, Pa. 19104.

An abstract of this report has been published in Clin. Res.,April 1971 and portions were reported at the annual meet-ing of the American Federation for Clinical Research on1 May 1971.

Received for publication 26 March 1971 and in revisedform 28 July 1971.

was significantly increased above normal in both theresting and phagocytizing state. Electron microscopichistochemistry revealed that almost all peroxidase ac-tivity was localized to the giant granules in Chediak-Higashi granulocytes, and after bacterial ingestionthere was a failure of delivery of peroxidase to manyphagosomes. Upon longer incubation more phagosomesacquired peroxidase activity, presumably through afusion process, although many giant granules remainedintact. The contrasting patterns and kinetics of the kill-ing defects and the differing metabolic properties ofChediak-Higashi syndrome and chronic granulomatousdisease leukocytes emphasize the pleiomorphic nature ofinherited disorders of leukocyte function.

INTRODUCTIONThe Chediak-Higashi syndrome (CHS)1 is a rare auto-somal recessive disorder characterized by partial oculo-cutaneous albinism and the presence of giant abnormallysosomes in most granule-containing cells (1). A simi-lar disorder has been described in mink and cattle (1),and more recently in mice (2). A major clinical mani-festation of this disease in affected patients is the occur-rence of multiple bacterial infections, principally withGram-positive organisms and, in particular, Staphylo-coccus aureus (3). Such infections are a major causeof death in this disease. Until recently (4), extensivestudies in mink and cattle (5) failed to determine themechanisms responsible for this decreased resistance toinfection and the few reported investigations of patients

1Abbreviations used in this paper: BSA, bovine serumalbumin; CGD, chronic granulomatous disease; CHS, Che-diak-Higashi syndrome; HBSS, Hanks's balanced salt solu-tion; HMS, hexose monophosphate shunt; MHS, modifiedHanks's solution; NBT, nitroblue tetrazolium; PBS, phos-phate-buffered saline; PMN, polymorphonuclear leukocytes;PSB, polystyrene balls; WBC, white blood cells.

The Journal of Clinical Investigation Volume 51 1972 649

(1, 6-8) including leukocyte bactericidal assays in oneindividual (8) were likewise unrevealing.

Four patients with the CHS from three differentfamilies have been available for study at the NationalInstitutes of Health since 1967. Previous investigations(9) have confirmed normal immunoglobulin levels (savefor patients in the advanced stage of illness), normalantibody formation, delayed hypersensitivity, and reticu-loendothelial clearance functions previously reported forother similarly affected patients (6-8). Studies of leuko-cyte functions have documented the occurrence of neu-tropenia and decreased marrow granulocyte responses(10), diminished granulocyte responses to infection(9), impaired skin window migration and chemotacticresponsiveness of their granulocytes (11), all of whichmight be incriminated in the pathogenesis of decreasedhost resistance. The present report deals with the phago-cytic, bactericidal, and fungicidal properties of leuko-cytes obtained from three of these patients. In addition,data is presented on metabolic functions important toantimicrobial activity, as well as histochemical electronmicroscopic studies of lysosomal degranulation. Thefindings indicate that such leukocytes have defectivebactericidal capabilities for a variety of bacteria whichare most pronounced for S. aureus, streptococci, andpneumococci and can be characterized primarily as adelay in intracellular killing. Comparative studies withleukocytes obtained from two children with chronicgranulomatous disease of childhood (CGD) suggestthat this diminished antibacterial capacity is not due tometabolic deficiencies related to the generation of hy-drogen peroxide but rather to abnormal lysosomaldegranulation.

METHODSCHSpatients. Two male siblings and one female formed

the CHS study group. Some clinical features of all havebeen previously published by others (12, 13). The female,presently 8 yr of age, is in the accelerated or lymphomatousphase of the disease (1). This phase is characterized byvarying degrees of granulocytopenia, anemia, thrombocyto-penia, and mononuclear cell infiltration of organs with hepato-splenomegaly and lymphadenopathy (1). Intermittent therapywith corticosteroids and vincristine has been required forcontrol of the manifestations of hypersplenism and organinvasion. Whenever possible, studies were performed whenshe was infection-free and not receiving steroids, and at least3 wk after her last dose of vincristine. Because of the com-plicating factor of this type of chemotherapy, the majorityof investigations were performed on the two male siblings(La. R., aged 18, and Le R., 19), both of whom are hema-tologically normal save for mild granulocytopenia and clini-cally well with the exception of recurrent sinopulmonaryand skin infections. Neither patient has required corticosteroidand vincristine therapy and all studies were performed whenthey were free of infection.

CGDpatients. Two males (S. E., aged 9, and B. P., 5),with typical sex-linked CGD, including histories of recurrentseptic and granulomatous pulmonary lymph node and hepatic

infections, and defective leukocyte bactericidal and respira-tory capacities, were used for comparison. Some features ofthese patients have been reported (14, 15).' Studies wereperformed on these patients both when they were infectedand clinically well. No significant alterations of the data pre-sented were seen during infection.

Controls. Normal individuals of both sexes aged 10-35,parents of the male CHS patients, and a 65 yr old femalewith bronchiectasis served as controls. All normal subjectswere in good health at the time of study and none had ahistory of increased susceptibility to infection.

Separation of leukocytes. Erythrocyteswere removedfromheparinized (10 U/ml) venous blood by dextran sedimenta-tion followed by hypotonic lysis (16). The remaining leuko-cytes were collected by low speed centrifugation (150 g for7 min) and washed twice in modified Hanks's solution(MHS) (17) with or without 10% heat-inactivated fetalcalf serum. Total and differential leukocyte counts were per-formed by hemocytometer or electronic particle counter(Coulter Electronics, Inc., Hialeah, Fla.) and examinationof Wright's stained smears, respectively. To obtain leuko-cyte preparations containing a high percentage of polymor-phonuclear cells (92-96%o ), a minor modification of theFicoll-Hypaque (Pharmacia, Uppsala, Sweden) density gra-dient centrifugation technique of Boyum (18) followed bydextran sedimentation was employed. Heparinized blood wasdiluted 2: 1 with isotonic saline, layered in 15-ml volumeson 3 ml of the Ficoll-Hypaque mixture, and centrifuged at400 g for 40 min at 200C. After removal of the mononuclearcell layer 1 ml of homologous or AB plasma was added tothe erythrocyte-granulocyte pellet and the mixtures pooled.An equivalent volume of 3%o dextran was added and aftersedimentation of the erythrocytes, the granulocytes were ob-tained as noted above. Mononuclear cells obtained from thegradient were washed twice in MHS-10% fetal calf serumand the percentage of monocytes determined by Wright'sstaining and ingestion of 0.01% neutral red dye. Before use,cells were suspended in Hanks's balanced salt solution(HBSS) at a concentration of 10 X 10' phagocytes (PMNplus monocytes)/ml. Viability as determined by 0.1% trypanblue dye exclusion was > 99% in all preparations examined.

Microorganisms. Representative Gram-positive organismsincluded a Staphylococcus aureus (phage type 29), a roughType II pneumococcus, a Group D streptococcus, and aBacillus subtilis. Gram-negative organisms included Esche-richia coli and Serattia marcescens. The fungus employedwas a Candida albicans type A.' All bacteria were grownovernight in trypticase soy or Todd-Hewitt broth (pneumo-cocci), centrifuged for 20 min at 2000 g, washed once inMHS, and adjusted to an approximate concentration of1 X 10'/ml turbidometrically with a Coleman (Coleman In-

'Ward and Schlegel (14) felt that patient B. P. did nothave typical CGDbecause of the absence of a killing defectfor Staphylococcus aureus. This finding was not confirmedin the present study and additional data to be noted belowtogether with intermediate abnormalities found for the motherindicate that this patient is indeed a typical CGDchild.

'Micro-organisms were kindly provided as follows: S.aurcus, Group D streptococci, E. coli, and S. marcescens byDr. James MacLowery, National Institutes of Health, Clini-cal Center; the pneumococcus by Dr. W. Barry Wood, Jr.,Johns Hopkins University, School of Medicine; the B. sub-tilis by Dr. William Davis, Washington State University,School of Veterinary Medicine; and the C. albicans by Dr.Martin Cline, University of California at San Francisco,School of Medicine.

650 R. K. Root, A. S. Rosenthal, and D. J. Balestra

struments, Maywood, Ill.) Junior spectrophotometer set at650 mu. C. albicans was grown in Sabouraud's broth at 330Covernight with agitation and allowed to stand several daysat room temperature before use. The number of candida permilliliter was determined by counting in a hemocytometer.A portion was spun down at 2000 g for 10 min and broughtup in HBSS at a concentration of 1 X 10'/ml.

Measurement of phagocytosis. Phagocytosis of microor-ganisms was determined in two ways. First, mixtures con-sisting of 5 X 106 phagocytes/ml in 10% AB serum-HBSSwere incubated with viable and heat-killed organisms at370C with tumbling (Fisher Roto-Rack, Fisher ScientificCompany, Pittsburgh, Pa.) for 20 min and Wright's stainedsmears were made. The number of cells which had ingestedone or more bacteria or candida were noted in counts of 100-200 phagocytes. Second, the kinetics of the phagocytic processby the entire cell population was determined by measuringthe uptake of radiolabeled bacteria. S. aureus were labeledwith "C by growing bacteria in trypticase soy broth towhich either alanine-"C or mixed universally labeled-'Camino acids (New England Nuclear Corp., Boston, Mass.)were added in a concentration of 10 MuCi/ml. "C-labeled E.coli were similarly obtained by growing bacteria with thymi-dine-"C in trypticase soy broth at a concentration of 10tCi/ml. At the end of an overnight incubation at 37'C thebacteria were killed by placement in a boiling water bathfor 20 min, washed three times in saline by centrifugationat 2000 g for 20 min and adjusted to a final concentrationof 5 X 10'/ml in saline. "C-labeled S. aureus obtained inthis way conntained approximately 250,000-350,000 cpm/mland E. coli 60,000-80,000 cpm/ml. They were stored at 40Cuntil used, which was always within 3 wk after preparation.

Phagocytosis of the "C-labeled bacteria by cells in sus-pension was determined by tumbling at 37'C 5 X 10. phago-cytes in 1 ml of 10%o AB serum-HBSS in 12 X 75 mmplastic tubes (Falcon Plastics, Div. of B-D Laboratories,Inc., Los Angeles, Calif.). At successive time intervals in-gestion was halted by the addition of 102 M sodium fluorideand placing the tubes in ice. Noningested bacteria were sepa-rated from the leukocytes by centrifugation at 4VC at 100 gfor 10 min followed by two washes and repeated centrifuga-tion using 4 ml of 10%o fetal calf serum in MHScontaining10-2 M NaF. The resulting leukocyte pellets were allowedto dry overnight and digested the following day with 0.5 mlof 0.2 N NaOHfor at least 2 hr at 56'C. The solutions wereneutralized at the end of the digestion with 0.2 ml of 3%glacial acetic acid, 0.5 ml of distilled water was added, and1 ml portions counted in 10 ml of Aquasol (New EnglandNuclear Corp., Boston, Mass.) in a scintillation counter(Packard Tri-Carb, Packard Instrument Co., Inc., DownersGrove, Ill.).

To measure phagocytosis by monolayers, preparations ofgranulocytes or monocytes (4-5 X 10' cells in 2 ml of 10%ofetal calf or AB serum-HBSS) were placed in 35 mmplastic Petri dishes (Falcon Plastics, Los Angeles, Calif.),and allowed to adhere at 370C for 90 min. At the end of thetime nonadherent cells were removed by four gentle washeswith warm HBSS and final incubation mixtures preparedusing 2-ml volumes of 10%o AB serum-HBSS. 'C-labeledbacteria were added and the monolayers incubated at 370C.At different times phagocytosis was stopped by pouring offthe supernates and gently washing four times with warmHBSS. The monolayers were allowed to dry and then di-gested with NaOH, neutralized, and counted as above. Insome experiments, portions were saved for the determinationof protein content by the Lowry technique (19). The mono-

layers could also be examined microscopically after stainingwith Wright's stain.

Antimicrobial assays. For bactericidal studies, a modifica-tion of the technique of Hirsch and Strauss (20) was em-ployed. Duplicate suspensions containing 5-10 X 10' phago-cytes in 10% AB or autologous serum and HBSS wereestablished in siliconized 13 X 100 mmglass or 12 X 75 mmplastic tubes and incubated at 370C for 5-10 min before theaddition of 0.1 ml of the bacterial suspension. The tubeswere then rotated end over end at 370C. Portions (0.01 or0.001 ml) were removed at intervals using a calibratedplatinum loop and placed in distilled water containing 0.01%human serum albumin to rupture the cells for total viablebacterial counts. For extracellular counts the majority(> 95%) of the leukocytes was deposited by centrifuga-tion at 150 g for 5 min at 40C and similar samples of thecell-free supernates removed and placed in isotonic saline.Serial 10-fold dilutions were made and appropriate dilutionsplated on trypticase soy agar. When pneumococci were used,1.0 ml of rabbit blood was added to each plate to facilitategrowth. The plates were incubated at 370C and the numberof colonies counted the following day to determine the num-ber of viable bacteria present at each time interval. Zerotime counts were obtained by taking the mean of three sus-pensions which did not contain cells and which had beenhandled in a similar manner.

In some experiments a modification of the technique ofHolmes, Quie, Windhorst, and Good (21) was used to deter-mine the number of intracellular bacteria present. After a20 min period of incubation to permit the completion ofphagocytosis, streptomycin (50 ,ug/ml) and penicillin (100/Ag/ml) were added to the medium after a portion was re-moved for the determination of total and leukocyte-associatedbacteria. At suitable time intervals thereafter, additional0.5-ml portions were taken from the 2.0 ml mixture, cen-trifuged at 150 g at 40C and washed twice with 4 ml 10%fetal calf serum-MHS. 5 ml of distilled water was thenadded to lyse the leukocytes and pour plates were made todetermine the remaining viable bacteria. The staphylococcusemployed in these studies was penicillin-sensitive.

To evaluate candidacidal activity of cells the methyleneblue vital staining technique of Lehrer and Cline (22) wasemployed without modification.

Oxygen consumption. Oxygen consumption was measuredin triplicate or quadruplicate suspensions of 14-15 X 106 pha-gocytes using a Gilson (Gilson Medical Electronics, Inc.,Middleton, Wis.) differential respirometer. All suspensions(2.0 ml total volume) were prepared in 15-ml Warburgflasks to which were added leukocytes in HBSS, AB serum(25% final concentration), and 1.0 / polystyrene balls (PSB)(Dow Chemical Co., Midland, Mich.) diluted 1: 5 withHBSS (approximately 2 X 10' total particles). All suspen-sions were incubated for at least 30 min before tipping inthe PSB from the side arm to compare resting with phago-cytizing responses.

Glucose oxidation. The oxidation of glucose labeled inthe first or sixth position with "C was measured by apreviously published technique (17). Suspensions of 5-10 X106 polymorphonuclear leukocytes in 2 ml of 25% ABserum-HBSS (11.2 ,moles and 0.5 MCi glucose) with andwithout PSB (approximately 2 X 10') were prepared. CO2was trapped in 0.2 ml of a 10% solution of KOH sus-pended from the rubber stopper in a plastic center well(Kontes Glass Co., Vineland, N. J.) after incubations ofthe suspensions for differing times and the addition of 0.5ml of 1.0 N HCl. The well containing the KOHwas then

Defective Bacterial Activity of Chediak-Higashi Syndrome Leukocytes 651

TABLE IDifferential Counts of Dextran-Separated Peripheral Leukocytes* from Normal

and Chediak-Higashi Syndrome (CHS) Subjects

Percentage of total WBC(mean ASE)Number of

Subject preparations PMN Monocytes Eosinophils Lymphocytes

L. R. 33 56.4 ±1.7 10.8 40.9 4.5 ±1.0 28.6 41.5P value4 <0.001 <0.001 >0.20 <0.001

La. R. 22 59.7 ±2.9 9.7 ±1.1 4.1 ±1.0 26.0 ±2.7P value4 <0.001 <0.001 >0.20 <0.001

T. H. 5 37.8 45.4 11.2 ±2.2 0.8 40.6 50.2 ±5.3P value4 <0.001 <0.001 >0.10 <0.001

<0.01§ >0.20 >0.10 <0.001Normal 57 81.8 ±1.3 3.7 ±0.6 4.2 ±0.7 9.9 40.9

* For details of separation procedure see text.$ Two sample t test compared with normal.§ Comparisons with other CHSsubjects.

placed in 20 ml of a scintillation solution (17) and countedfor sufficient time to insure a counting error of 1.5% orless.

Formate oxidation. Hydrogen peroxide production by leu-kocytes was measured by the oxidation of formate-"C (23)in a manner similar to glucose oxidation with the followingexceptions: 10 X 106 granulocytes were used routinely and250 nmoles of cold sodium formate (0.5 juCi Na-formate-14C)were added to the flasks in place of glucose-'C. Incubationswere for 60 min at 370C with trapping of C02 and countingas described above. Cell-free controls were always preparedand the values for spontaneous formate oxidation subtractedfrom the preparations containing leukocytes.

Iodination of protein by leukocytes. The ability of leuko-cytes to iodinate intracellular protein precipitated by 10%trichloroacetic acid (TCA) in the resting state and withphagocytosis of heat-killed S. aureus was measured using aminor modification of the method of Klebanoff (24). Finalsuspensions in 12 X 75 mmtubes contained 5 X 10' PMN,10% AB serum, 1 X 108 heat-killed S. aureus, 20 nmoles and0.2 aCi of Na-'I in HBSS (total volume 0.6 ml). Thesewere incubated at 370C with shaking for varying time in-tervals. The iodination reaction was halted by the additionof 0.01 M sodium thiosulfate and the tubes were placed onice. 1 ml of cold 10% TCA was added and the tubes cen-trifuged at 2000 g for 5 min. The TCA precipitates werewashed and centrifuged three times with cold 10% TCAand the "2I in the pellet determined by placing each tubeinside an 18 X 150 mmtube and counting in a sodium iodidecrystal well scintillation counter (Nuclear-Chicago Corpora-tion, Des Plaines, Ill., model No. 4223). Appropriate cell-freesuspensions were prepared with the TCA-precipitable countsin these added to background and subtracted from the restingand phagocytizing cell counts. In some experiments sodiumazide (0.01 mmfinal concentration) was added to the reac-tion mixtures to inhibit the peroxidase-mediated iodinationreaction (25).

Nitroblue tetrazolium dye reduction. Quantitative mea-surement of the reduction of the colorless dye nitrobluetetrazolium to blue formazan was carried out using thetechnique of Baehner and Nathan (26).

Electron microscopic cytochemistry. 10 X 106 granuloctyeswere incubated with a 100:1 ratio of heat-killed S. aureus

in 1 ml volumes of 10% AB serum-HBSS at 370C as above.At various times phagocytosis was halted and the cells fixedin suspension by the addition of 1 ml of 2% gluteraldehydein Tyrode's solution. The cells were washed in phosphate-buffered saline pH 7.4 (PBS) and the cytochemical reactionproduct developed with 3,3'-diaminobenzidine for 30 min atroom temperature (27). The cell suspension was then washedtwice with PBS containing 10 mmpotassium cyanide, sus-pended in 10% bovine serum albumin (BSA) and sedimentedby centrifugation. After removal of the excess BSA, thecell pellet was gelled by the addition of 25 ul of 8% gluteral-dehyde and the pellet minced into 1 mmcubes. The cubeswere then suspended in 1% osmic acid, dehydrated withethanol, passed through propylene oxide and embedded inMaraglas (The Marblette Co., Div. Allied Products Corp.,Long Island City, N. Y.). Thick sections were stained withtoluidine blue and examined under the light microscope.Thin sections were stained with lead citrate and examinedin a Philips (Philips Electronic Instruments, Mount Vernon,N. Y.) 300 electron microscope.

RESULTSDifferential counts of leukocyte preparations. In Ta-

ble I differential counts of leukocytes prepared by thedextran sedimentation procedure are presented from the3 CHS patients and 28 control subjects. All three pa-tients were significantly neutropenic when compared tonormal and had significantly higher mean percentages ofmnonocytes and lymphocytes than the controls. The de-gree of the neutropenia of the two male siblings wasmild compared to the female in the advanced stage ofthe CHSwho had mean granulocyte percentages signifi-cantly below and lymphocyte percentages higher thanboth males. Eosinophil counts of all three CHS patientswere not significantly different from normal. When cellpreparations were equalized for phagocyte content in dif-ferent studies, the mean percentage of granulocytes inthe CHS phagocytic cell populations averaged from alow of 76.3% to a high of 81.3% in the bactericidal and


TABLE IIPhagocytosis of Different Strains of Bacteria by Normal and Chediak-Higashi Syndrome (CHS) Leukocytes*

PhagocytosislNumber Ratio Phagocytic¶

Organism Subjectst experiments bacteria/cellsi Total >5 index

S. aureus CHS (3) 7 0.7-4.0/1 71.5 ±6.2 25.0 :1=12.0 3.0 4:0.3Normal (5) 6 0.7-4.0/1 66.7 :1:4.0 14.0 ±4.0 2.6 40.3CHS-heterozygote (2)** 1 3/1 63.0 A1.0 15.5 42.5

E. coli CHS (3) 3 2.0/1 58.0 ±8.9 1.1 40.2Normal 3 2.0/1 47.5 ±2.5 - 1.0 ±0.1

S. marcescens CHS (3) 3 1.0-10.0/1 74.0 ±18.0 49.0 ±23.5Normal (2) 2 1.0-10.0/1 62.5 427.5 39.0 438.0

Group D streptococci CHS (2) 3 1.8-5.0/1 55.5 ±3.0 12.0 ±3.8Normal (3) 3 1.8-5.0/1 51.5 ±5.2 8.0 ±3.6CHS-heterozygote (2) 1 5/1 45.5 ±12.5 3.5 ±1.5

Group D pneumoniae CHS (2) 4 20.0/1 95.0 42.0 88.8 ±5.6(R 1136) Normal (2) 2 20.0/1 95.5 40.5 91.0

B. subtilis CHS (2) 4 1.0-3.0/1 62.3 413.5 24.5 ±13.4Normal (2) 2 1.0-3.0/1 63.0 419.0 24.5 ±21.5

S. albus CHS (3) 8 5.0-10.0/1 89.0 43.8 76.0 ±2.3Normal 3 5.0-10.0/1 92.7 ±5.4 78.7 ±6.8CHS-heterozygote (5) 5 5.0-10.0/1 90.6 ±1.6 73.4 ±3.3

* Dextran-sedimented, washed leukocytes suspended in 10% AB or homologous (S. albus) serum-HBSS at a concentration of5 X 101 phagocytes per ml and incubated with tumbling at 370C for 20 min with heat-killed or viable bacteria. For differentialcounts see text.t Numbers in parentheses refer to the number of subjects studied.§ Number of bacteria per phagocyte in initial inoculum, given as range in different experiments.11 Per cent of granulocytes and monocytes containing one or more (total) and greater than five bacteria (100-200 cells counted).Results given as mean and standard error with principal variability due to wide range of inoculums employed.¶ Mean number of intracellular bacteria per phagocyte. Results given as mean and standard error.** Parents of CHSsubjects.

phagocytic assays and from 76.0 to 86.1% in the meta-bolic studies. Similar values for normals ranged from87.3 to 96.2%. Monocytes as a percentage of total phago-cytes in the CHS cell preparations varied from a low of11.7 to 21.2% in the bactericidal assays and from 5.2to 23.2% in the metabolic investigations vs. 0.2 to 7.5%for the normals. Despite these significant mean differ-ences among both groups, granulocyte and monocytepercentages were occasionally the same for CHS andnormal cells in some experiments.

Differential counts obtained from dextran-sedimentedCGDleukocytes were in all cases similar to normal.

In contrast to these findings with dextran-sedimentedcells, the Ficoll-Hypaque technique yielded equivalentpopulations of granulocytes from CHSand normal indi-viduals with mean percentages ranging from 89.5 to96.0% in different groups of experiments. Eosinophilsmade up the majority of the remaining cells. Meanmonocyte and lymphocyte contamination was in allcases less than 2.5% and usually less than 2% for dif-

ferent groups of experiments. Mononuclear cell prepa-rations from the Ficoll-Hypaque gradients were like-wise similar in both normal and CHS subjects withthe percentage of monocytes ranging from a mean of32.4 to 38%, with lymphocytes making up the remain-der of the cells. Granulocyte contamination of the mono-

nuclear cell layer averaged less than 1% and usuallywas zero.

Phagocytosis. As indicated in Table II, phagocytosisof a variety of microorganisms by mixed leukocytes ob-tained from all three CHS subjects was similar to nor-

mal when evaluated microscopically regardless if theserum in the medium was homologous (experimentswith Staphylococcus albus) or obtained from a common

AB pool to minimize variability due to differing op-sonins. In all experiments in which a wide range ofbacteria to cell ratios was utilized both the per cent ofCHS phagocytes containing bacteria and the numberof organisms per cell (phagocytic index) did not differsignificantly from normal.


201

2 15 - 8

EI10v

0 Normal (5'0 CHS(2)

5 -

0 6 12 18 24TIME (min)

FIGURE 1 Phagocytosis of "C-labeled S. aureus by mixedleukocytes from five normal and two CHS subjects. Theline denotes the mean and the bars the standard error ofthe mean of the radioactivity (cpm) associated with thenormal leukocyte pellets at each time interval. For leukocytedifferential counts see text.

Phagocytosis by CHS monocytes appeared to beessentially similar to normal monocytes and was lessthan PMNwhen examined microscopically.

Data obtained on the kinetics of the ingestion processby measuring the uptake of "C-labeled bacteria by cellsin suspension or monolayers confirmed the microscopicobservations of normal phagocytosis by the CHS leu-kocytes. In preliminary experiments it was determinedthat the number of counts associated with the leukocytepellet was directly proportional to the number of intra-cellular organisms at each time interval. As shown inFig. 1, which depicts the ingestion of "C-labeled S.aureus by mixed populations of CHS and normal cells,the uptake of radioactivity by leukocytes from the twomales was equivalent to the mean of five normal con-trols. Similar information was obtained when "C-labeledE. coli were used and when granulocytes and mononu-clear cells from one subject (Le. R.) separated by theFicoll-Hypaque technique were studied in suspension.In fact, at early times phagocytosis by the CHS gran-ulocytes appeared to be faster than normal (68% forCHS vs. 57% uptake for normal cells at 5 min). Up-take of "C-labeled S. aureus by leukocytes in suspensionfrom one CGDsubject (B. P.) was equivalent to nor-mal (data not shown).

A better examination of the phagocytic capabilitiesof monocytes was obtained by measuring the ingestionof "C-labeled S. aureus in monolayers which had beenwashed free of most of the contaminating lymphocytes.As shown in Fig. 2 the mean ingestion rate of "C-

labeled S. aureus by monocytes in two experiments fromone patient (Le. R.) was greater than the mean ofthree normal controls; however, the differences werenot statistically significant. It is noteworthy that thekinetics of the ingestion process was different by cellsadherent to a plastic surface compared to those in sus-pension. The density of the cells on the xnonolayers ap-peared similar when examined microscopically and thevalues for cellular protein determined in one experimentwere similar (mean lg protein/plate = 0.311 for normalvs. 0.227 for CHS).

Phagocytosis of "C-labeled S. aureus by PMN insimilarly prepared monolayers was suggestive of en-hanced activity by CHS cells when compared to nor-mal; however, the results obtained were quite variabledue to variations in adherence of these cells to theplastic Petri dishes and therefore are inconclusive.

Bactericidal activity. The bactericidal activity ofmixed CHS, CGD, and normal leukocytes against fivedifferent species of bacteria are shown in Table III.Since there were no significant differences among indi-vidual patients the data from each disorder has beenpooled in comparing the patient groups against normalsubjects. As expected, from previous studies (28)CGD leukocytes consistently showed impaired killingof the catalase-positive organisms, S. aureus, E. coli,and S. marcescens, as well as C. albicans (Fig. 3). Kill-ing of the catalase-negative Group D streptococcus wasnormal at the 60- and 120-min time intervals studied. Incontrast, CHS leukocytes displayed bactericidal defectsfor both catalase-negative and catalase-positive bacteriadepending upon the time of incubation. For instance, kill-

CY

x

EaL

25

20

15/

0-- 0 CHS_-. Normal

0 5 10

TIME (min)15 20 25

FIGURE 2 Phagocytosis of '4C-labeled S. aureus by mono-cytes adherent to plastic Petri dishes as a monolayer fromthree normal and one CHS subject (two experiments). Thebars denote the standard error of the mean radioactivity(cpm) at each time interval as denoted by the open orclosed circles.

654 R. K. Root, A. S. Rosenthal, and D. J. B3alestra

ing of S. aureus, rough pneumococci, and Group D strep-tococci was significantly less than normal through 2 hrof incubation, whereas that of S. marcescens was lessthan normal at 1 hr but not at 2 hr. Killing of E. coliwas only significantly less than normal at the 20 minincubation time. Killing of a strain of B. subtilis forwhich leukocytes from CHS cattle have a bactericidaldefect (4) was also less than normal in five out of eightexperiments (data not shown) through 2 hr incubation;however, because of variability the mean differences forall the studies were not statistically significant. Killingof C. albicans (Fig. 3) was less than normal at 60 minof incubation on one occasion only, with one of two CHSsubjects. Consistent with the observations on phago-cytosis noted previously, clearance of bacteria from thecell-free medium at 2 hr was similar to normal in bothpatient groups. The majority of surviving bacteria forwhich defective killing was documented were thereforeassociated with the leukocyte pellets.

The differences in the kinetics of the killing processin the two types of patients compared to normal areshown in Fig. 4 which graphically depicts the survivalof S. aureus over a 2 hr incubation. Killing of the totalinoculum by both CHSand CGDcells was similar overthe first 20 min and significantly less than normal. Afterthis time, killing by CHS leukocytes continued whereasthat by the CGD cells halted after the initial 20 minincubation. Organisms within CHS leukocytes ("intra-

50

401-

I

xwCz

-J4C'34C

zU

301

20i-

A

S

0

Aa

a0

Ioh a

a

0NORMAL CHS CGD CGD

- CARRIER

FIGURE 3 Comparative candidacidal activity of normal (0),CHS (A, A), CGD (-, E1), and ? CGD carrier (E)subjects. The candidacidal index refers to the per cent ofcandida stained with methylene blue, 1 hr after mixing cells(2.5 X 106) with fungi (2.5 X 10). All values represent themean result of duplicate determinations (300 candida counted).

cellular") as determined after the addition of antibioticswere in fact killed at a rate equivalent to normal, al-though significantly more bacteria were present through

TABLE IIIBactericidal Activity of Leukocytes* from Normal, Chediak-Higashi Syndrome (CHS), and

Chronic Granulomatous Disease (CGD) Subjects

Per cent survivalsNumber Inoculumt Cell-associated, 11 Per cent¶

Organism Subject experiments X 106 20 min 60 min 120 min 120 min cleared

S. aureus Normal (15)** 18 12.8 +1.4 25.4 44.6 12.4 +3.5 6.1 +1.6 5.6 4:1.8 98.3 +0.8CHS (3) 13 13.0 41.5 57.1 t5.9It 45.3 t7.Oft 25.2 :1:6.1tt 23.6 hS.11 98.4 A-1.1

CGD(2) 5 15.4 +5.7 51.5 +6.7tt 54.7 1417.5tt 49.1 :1:16.5f 45.5 +14.6tt 95.9 +1.8

Group D streptococci Normal (7) 9 18.8 +2.1 - 1.2 40.2 0.30 +0.18 - -

CHS (2) 5 19.3 +3.5 - 9.7 +3.2tt 2.9 +1.6f - -

CGD(1) 2 16.9 +0.2 - 1.6 +0.6 0.11 +0.04 - -

Group D pneumoniae Normal (2) 3 1.2 +0.8 - 3.6 +0.6 0.45 40.10 0.26 +0.09 99.8 +0.1

(RII 36) CHS (2) 4 1.1 :10.5 - 21.6 19.0tt 6.9 41.614 5.8 -11.5$t 98.9 +0.3

E. coli Normal (11) 9 9.6 +0.5 21.4 44.1 3.2 +1.1 1.7 +0.5 1.8 +0.3 99.7 40.1

CHS (3) 13 9.8 +0.5 54.5 +12.5tt 8.0 +1.8 2.1 +0.7 1.3 40.4 98.2 40.7CGD(2) 5 11.6 +0.8 - 17.9 +5.Ott 18.3 +6.1tt 15.1 45.2tt 96.6 +1.3

S. marcescens Normal (5) 12 19.7 43.7 - 8.0 41.9 3.2 +1.2 2.2 +0.9 99.0 +0.5CHS (3) 12 24.2 +4.1 - 20.8 +4.01t 4.0 +1.1 2.2 +0.9 98.2 +0.6CGD(2) 4 23.1 +9.3 - 29.9 +9.61t 15.7 +5.6tt 10.7 +2.8ft 95.0 42.8tt

* Cells separated by dextran sedimentation (for differential counts see text) and incubated with bacteria in 10% AB serum-HBSS in a concentration of5 X 106 phagocytes (PMN and monocytes)/milliliter.t Number of viable organisms at zero time as determined by pour plate counts (mean ASE).5 Per cent survival (mean ASE) of bacterial inoculum at time intervals noted.

Per cent (mean +SE) of original inoculum associated with leukocyte pellet after centrifugation at 150 g.¶ Per cent (mean =SE) bacteria removed from cell-free supernate (150 g centrifugation) by adherence to or ingestion by leukocytes at 120 min.* Number of different subjects studied.1t Difference from normal statistically significant (P < 0.05 or less two sample t test).

Defective Bacterial Activity of Chediak-Higashi Syndrome Leukocytes

-- I

655

z5

cc1

:2

011

0 20 60 120 0 20 80 140TIME (min)

FIGURE 4 Comparative killing of staphylococci by mixednormal, CGD, and CHS leukocytes expressed as the per centsurviving viable bacteria with time. The number of experi-ments with 3 CHS, 2 CGD, and 7 ("intracellular") to 15("total") normal subjects are shown in parentheses. Thebars denote the standard error of the mean at each timeinterval. "Total" bacteria were determined by sampling por-tions of the entire leukocyte suspension. "Intracellular" bac-teria refer to those organisms which remained after theaddition of penicillin and streptomycin to the medium andwashing the leukocyte pellets several times before sampling.Stars denote differences which are significant by two samplet testing (P <0.05 or greater).

80 min due to the early lag in killing. By 140 min, over-lapping values for killing of intracellular S. aureus wereseen in some experiments and when the incubations wereprolonged through 4 hr (data not shown), killing byCHS leukocytes in two experiments became equivalentto normal.

Comparison of antistaphylococcal action of CHSgranulocytes and monocytes. Since, as noted above,the CHS-mixed phagocyte populations differed fromnormal in their greater content of monocytes it becamedesirable to examine separately the bactericidal prop-erties of both monocytes and granulocytes. Fig. 5demonstrates the antistaphylococcal activity of Ficoll-Hypaque-separated granulocytes and mononuclear cells,and indicates that killing is slightly but not significantlyless for normal PMNobtained by this procedure whencompared to dextran-separated cells. Killing by CHSgranulocytes was found to be similar to the mixed cellpopulation (5-6: 1 PMNto monocytes) and was sig-nificantly less than normal through 2 hr of incubation.In addition, killing by CHS monocytes also was de-layed at 1 hr, becoming normal by 2 hr.

Comparative oxidative metabolism of CHS, CGD, andnormal leukocytes. Oxygen consumption by mixedCHS leukocytes was greater than normal at rest andshowed a marked increase with phagocytosis (Fig. 6).This contrasted sharply with similar studies with CGDcells, which as expected (29) showed no postphagocyticrespiratory burst (data not shown).

Similar to these observations the oxidation of glucose-1-14C by mixed CHS phagocytes was greater than nor-mal in the nonphagocytic state at both 30- and 60-minincubation times (Table IV), although the differenceswere not statistically significant with the small numberof experiments done. Postphagocytic activity was higherat 30 min in one experiment but equivalent to normalby 60 min. CGDleukocytes demonstrated the previouslyobserved (29) deficiencies in glucose oxidation via the

TIME (min)

FIGURE 5 Antistaphylococcal activity of granulocytes (>95%) and mononuclear cells (30-35% monocytes) separatedby Ficoll-Hypaque gradient centrifugation as noted in thetext. Numbers in parentheses refer to the number of experi-ments with two CHS and four normal subjects. The barsdenote the standard error of the mean at each time interval.Symbols indicate differences that are significantly different(P < 0.05) by two sample (*) or paired sample (+) t tests.

hexose monophosphate shunt (HMS) both at rest andwith phagocytosis.

These observations were extended to purified prepa-rations of PMNand mononuclear cells and a largernumber of experiments were performed. The patternof greater activity by nonphagocytizing CHS leuko-cytes was found to exist both for PMNat 30- and 60-min incubation times and mononuclear cells at 60 minwhether in suspension or adherent to glass with con-taminating lymphocytes removed (Table V). The dif-ferences were statistically significant. Postphagocyticoxidation of glucose-l-4C, on the other hand, was simi-lar to normal by CHS PMNat both 30 and 60 min,whereas that by monocytes was greater than normal atthe later incubation time. It is noteworthy that post-phagocytic glucose-1-"C oxidation was significantly lessfor normal PMNobtained by the longer Ficoll-Hypaqueseparation procedure than by dextran sedimentation(Table IV) at the 60 min incubation time (P <0.02,


t test). Similar differences were found for the CHSleukocytes, but due to variability were not statisticallysignificant.

The oxidation of glucose-6-'4C and formate-"4C byresting CHS leukocytes also averaged higher than nor-mal but the differences were not statistically significant.Phagocytizing CHSgranulocytes and mixed phagocyteswere similar to normal in both these functions (TableIV). Again CGD leukocytes demonstrated the pre-viously documented (29) deficiencies in formate oxida-tion.

Iodination of intracelular protein. In parallel withthe findings on glucose oxidation by CHS leukocytes,intracellular iodination of protein, a ;myeloperoxidase-mediated reaction (24), was greater than normal byresting cells (Table VI). In addition, higher post-phagocytic values were observed, using heat-killed S.aureus as the phagocytic particle. The addition of 1 mMazide to the reaction mixtures resulted in equivalentsuppression of both normal (97.8%) and CHS (94.9%)activities and placed them in the range of those foundfor the CGD leukocytes (30). Measurement of iodina-

I 80

N0

20

10 20 30 40TIME (min)

50 60 70

FIGuRE 6 Oxygen consumption before and after the addi-tion of polystyrene latex particles (PSB) to suspensions ofmixed CHS (subjects Le. R. and La. R.) and normal leuko-cytes. Phagocytes numbered 14 X 106 in the mixtures andthe lines represent the mean of triplicate determinations. Themean number of each cell type which made up the CHSphagocytic population was 76% PMN, 23.2% monocytes,and 0.8% eosinophils vs. the normal values of 92.2% PMN,0.2% monocytes, and 2.5% eosinophils.

tion at 10- and 30-min time intervals gave results simi-lar to the above with CHS cells always exhibitinggreater activity than normal (data not shown).

Phagocytic reduction of NBT dye. As shown inFig. 7, CHS-mixed leukocytes were capable of nor-mally reducing NBT due to blue formazan duringphagocytosis of polystyrene latex particles. This repre-

sented another differentiating feature of these cellswhen compared to CGDleukocytes.

Cytochemtical analysis of degranulation by electronmicroscopy. Since the metabolic studies suggested thatthe observed bactericidal defects in CHS phagocytescould not be explained by a failure of peroxide forma-tion or tnyeloperoxidase-mediated peroxidation, atten-tion was turned to the postphagocytic degranulationprocess. In preliminary studies the microscopic obser-vation, previously noted for CHS mink and cattle leu-kocytes (5), that the large granules persisted seeminglyintact within CHS PMNfor prolonged periods of timeafter phagocytosis of bacteria was confirmed. Evalua-tion with phase microscopy disclosed the presence ofmany of these granules around phagocytic vacuoles afteringestion of S. aureus, and in some cases apparent lo-calization within phagosomes. Better morphologicaldocumentation of the degranulation process was ob-tained through the use of a cytochemical assay for per-oxidase activity in conjunction with electron micros-copy. In nonphagocytic CHS granulocytes almost alldetectable peroxidase activity was localized to the giantgranules (Fig. 8 B), which are few in number whencompared to the large peroxidase-positive primarygranule population of normal PMN (Fig. 8A). 15min after mixing with staphylococci, the differences inthe appearance between intracellular bacteria in thetwo cell types was striking. Within normal granulocytesalmost all phagocytic vacuoles were seen to contain per-oxidase reaction product and the bacteria appeared tobe undergoing degeneration (Figs. 8C and D). In con-trast, in CHS PMNmany phagosomes contained noperoxidase activity and the bacteria within these re-tained their defining structural characteristics (Figs.

0.500

0400

0.300 F- ]

0.200

0.1001

NORMAL CHS CGD

FIGURE 7 Quantitative reduction of nitroblue tetrazoliumdye by 2.5 X 106 mixed phagocytes from seven normal, threeCHS, and two CGD subjects after phagocytosis of poly-styrene latex particles. Values given as the mean of all spec-trophotometric determinations at 515 m~uwith the bars de-noting the standard error.


TABLE IVOxidation of Glucose-1-54C, Glucose-6-14C, and Formate-'4C by Normal and CHSLeukocytes*

Cell Number* IncubationSubstrate Subject: preparations experiments time Resting: Phagocytizing¶

min nmoles substrate oxidized (mean dsE)Glucose-1-54C Normal (2) Mixed 3 30 62.6 ± 10.0 169 ±28

CHS(2) Mixed 3 30 131 424 240P value** <0.10 >0.20

Normal (2) Mixed 2 60 58.2 ±7.3 385 412.8CHS (2) Mixed 2 60 125 433 391 4105

P value <0.20 >0.20CGD(2) Mixed 3 60 15.9 ±2.5 18.2 ±2.2

P value <0.01 <0.001

Glucose-6-14C Normal (4) PMN 4 60 1.95 40.37 6.23 ±2.20CHS (2) PMN 4 60 3.12 ±0.56 5.40 40.75

P value >0.10 >0.20

Formate-54C Normal (8) Mixed 12 60 0.17 ±0.04 0.53 40.10CHS (3) Mixed 7 60 0.28 40.09 0.72 40.19

P value >0.20 >0.20CGD(2) Mixed 7 60 0.046 ±0.01 0.081 40.01

P value <0.05 <0.01

* For studies of glucose-54C oxidation reaction mixtures (2 ml) contained 5 X 108 phagocytes in 25%AB serum in HBSSwith0.5 ,Ci and 11.2 pmoles of glucose. Formate-54C oxidation was measured in mixtures (2.5 ml) containing 10 X 106 phagocytes,20%AB serum in HBSSwith 0.5 1Ci, and 250 nmoles of sodium formate. Phagocytosis was initiated by the addition of polysty-rene latex particles and the mixtures were incubated for the times noted with trapping of 14C-O2 and counting as per text.$ Numbers in parentheses refer to different subjects studied.§ Mixed cells refer to those obtained by dextran sedimentation (for differential counts see text). PMNwere obtained by centri-fugation on Ficoll-Hypaque.

jj Amount of substrate oxidized to 14C-02 by nonphagocytizing cells.¶ Amount of substrate oxidized to 14C-02 by cells ingesting polystyrene latex particles.** Two sample t test.

9A and B). In some areas an occasional giant lysosomecould be found apparently extruding its contents intoa phagocytic vacuole, with corresponding alteration inthe appearance of the bacteria. By 60 min of incubationthese differences were less distinct. In normal cellsperoxidase-positive granules were rarely seen in thecytoplasm and bacteria were collected within one ortwo vacuoles which contained peroxidase activity ineach section examined (Fig. 9C). In CHSgranulocytesgiant lysosomes were frequently found which did notappear to have any connection with phagocytic vacuoles(Fig. 9D); however, in most sections a greater num-ber of vacuoles contained peroxidase activity.

DISCUSSIONThe most significant abnormality in function uncoveredin the present investigations for phagocytes obtainedfrom three patients with the CHS was an alteration inthe rate of killing of several different classes of bac-teria. Comparison with leukocytes obtained from twomales with sex-linked CGDindicated that in contrast tothe bactericidal defect for catalase-positive organisms

found in these subjects (28), both catalase-positive (S.aureus, S. marscescens, and E. coli) and catalase-nega-tive (Group D streptococcus and pneumococci) bacteriawere inactivated at abnormal rates by CHS cells. Thisdefect in killing was most pronounced in the first 20 minof contact between CHS leukocytes and bacteria; there-after bactericidal rates increased, a finding which fur-ther distinguished the CHS from the CGDleukocytes.The magnitude and duration of the defects observedfor the CHS cells varied with the organism studied andwere most marked for S. aureus and least so for E. coli.This pattern corresponds to the bacteria most frequentlyimplicated in infections in these patients, with staphylo-cocci having accounted for 65.7% of the 51 infectiousepisodes observed over a 3J yr observation period inour subjects (9).

Previous data available on the bactericidal capabilit-ies of CHS cells has been limited and seemingly atvariance with our findings. For instance, employing atechnique in which antibiotics are added to the medium15 min after mixing cells and bacteria with sampling ofthe suspensions for surviving bacteria at 1 and 3 hr,


TABLE VOxidation of Glucose-1-'4C by Normal and CHSLeukocytes Obtained

by Centrifugation on Ficoll-Hypaque*

Number IncubationSubject: Cell prep§ experiments time Resting!1 Phagocytizing¶

min nmoles substrate oxidized (mean 4sE)

Normal (6) PMN 6 30 58.1 ±8.0 168 ±48.2CHS (2) PMN 6 30 117 ±11.3 135 431.4

P value** <0.01 >0.20Normal (6) PMN 6 60 58.9 ±14.4 206 ±26.2CHS (2) PMN 4 60 196 ±25.7 250 ±53.2

P value <0.001 >0.20Normal (5) Monos (suspension) 5 60 27.2 ±6.4 161 ±25.6CHS (2) Monos (suspension) 2 60 93.9 ±20.2 187

P value <0.01 >0.20Normal (4) Monos (adherent) 4 60 11.5 ±3.0 130 ±4.4CHS (2) Monos (adherent) 2 60 31.9 ±2.4 180 ±6.0

P value <0.02 <0.01

* For details of the separation procedure see text. All incubation mixtures consisted of 5 X 101 phago-cytes in 2 ml HBSS-25%AB serum with 0.5 ,Ci and 11.2 jmoles of glucose. Phagocytosis was initiatedby the addition of 2 X 109 polystyrene latex particles and 14C-O2 trapped and counted as noted in text.t Numbers in parentheses refer to different subjects studied.§ PMNwere in suspension at a concentration of 5 X 106/ml. "Monos (suspension)" were mixtures ofmonocytes and lymphocytes (approximately 30% and 70%, respectively) maintained in suspensionsimilar to PMNat a concentration of 5 X 106 monocytes/ml. "Monos (adherent)" were obtained bylayering mixtures of monocytes (5 X 106) and lymphocytes in the 25-ml Erlenmeyer reaction flaskfor 90 min, and then washing off the nonadherent lymphocytes and platelets with HBSSbefore measure-ment of glucose-1-'4C oxidation.

Nonphagocytizing cells.¶ Cells which were incubated with polystyrene latex particles.** Two sample t test.

Windhorst et al. were unable to find an abnormality of sampling times might have missed the delay in intra-killing of staphylococci in two experiments on one pa- cellular killing, the most consistent feature of our re-tient (8). As noted in Fig. 4, however, the use of such sults. In another unpublished investigation using tech-

TABLE VIfodination of TCA-Precipitable Protein by CHS, CGD, and Normal Leukocytes*

No. PhagocytizingSubject: Addition experiments Resting cells cells

nmoles nmoles

Normal (5) None 5 0.139 ±0.034§ 0.534 ±0.084CHS (2) None 3 0.791 40.274 1.499 ±0.267

P valuejf <0.01 <0.01Normal (3) 1 mMazide 3 0.018 ±0.009 0.012 40.001CHS (2) 1 mMazide 2 0.018 0.076 ±0.036CGD(2) None 2 0.038 ±0.018 0.072 ±0.035

P value <0.05 <0.01

* Reaction mixtures consisted of 5 X 106 PMN, 20 nmoles and 0.2 ,Ci Na-1251 withor without 1 X 108 heat-killed S. aureus and 1 mMazide in 10% AB serum-HBSSincubated for 60 min at 370C. Leukocytes were obtained by dextran sedimentation(for differential counts see text).t Numbers in parentheses refer to number of different individuals studied.§ Mean ±SE of nmoles iodide fixed to protein.1I Two sample t test.



A..Aab..-

vr7

niques similar to ours, Quie found an impairment inkilling of S. aureus by leukocytes from one CHS pa-tient in the range reported here; however, the experi-ments could not be repeated because of the patient'ssevere neutropenia and early demise.4 A third study onanother patient has reported impairments in killing ofS. aureus and S. marcesceng similar to the findings re-ported here (31). Recently, Davis reported that leuko-cytes from CHS cattle, but not mink, had defective kill-ing of S. aureus and B. subtilis (4).

Because of the kinetics of the abnormal killing shownby the CHS cells, it became important to establish thatthe delay was not due to diminished phagocytic rates.Microscopic studies indicated that at the end of a 20min incubation period CHSgranulocytes and monocytesparticipated in the phagocytic process in numbers simi-lar to normal, and the content of intracellular organismsappeared the same. A better measure of the rate of bac-teria ingestion was afforded by the use of radiolabeledorganisms in which the phagocytic activity by the en-tire cell population could be frequently sampled. Inpreliminary experiments it was determined that theamount of radioactivity associated with the cell pelletcorrelated closely with the number of intracellular bac-teria. It was then demonstrated that the uptakes by bothmale patients were similar to normal controls, leadingto the conclusion that the marked delay in killing ob-served over the first 20 min could only be due to ab-normal intracellular inactivation. Similar data on in-gestion rates was obtained on one CGD subjectconfirming previous observations of their normal phago-cytic capabilities (21).

Due to the neutropenia of the CHS patients themixed cell populations obtained by dextran sedimenta-tion differed from normal not only in their diminishednumber of granulocytes but also in their larger per-centage of monocytes and lymphocytes. The numbers ofeosinophils, on the other hand, were similar in bothpatient and normal groups. The question then arises asto whether the larger number of monocytes in the CHSphagocytic suspensions could cause an apparent impair-ment of bactericidal activity because of their reducedphagocytic capabilities compared to granulocytes (32).Several points argue strongly against this conclusion.First, although the percentage of monocytes was greaterthan normal, the number of granulocytes in the CHScell preparations outweighed monocytes by at least fiveto one in all studies. Second, the kinetics of phagocy-

4Quie, P. G. Personal communication.

tosis by the mixed cell populations was found to benormal as noted above. Third, the data obtained fromthe CHS female, who was significantly more neutro-penic than both males, was identical to the males. Fi-nally, when CHS granulocytes and monocytes were ob-tained separately from Ficoll-Hypaque gradients andmatched in percentages identical to normal cells, theabnormalities observed for the mixed cell populationpersisted. Granulocytes were found to ingest "C-labeledS. aureus at rates equivalent to normal, yet had a di-minished rate of killing which persisted over 2 hr.MIonocytes were likewise found to have normal ratesof bacterial ingestion whether maintained in suspensionwith lymphocytes or allowed to adhere to a plastic sur-face and washed free of the latter cells. In contrast togranulocytes, however, kill rates for S. aureus wereimpaired over the 1st hr of incubation only.

Having demonstrated impaired bactericidal activityby CHS leukocytes in a pattern that differed from theCGDcells it became of interest to further compare theactivities of these cells in metabolic studies importantto the bactericidal process. In contrast to the CGDleukocytes (21) and similar to studies previously re-ported for mink and cattle with the CHS (5), it wasquickly determined that CHS phagocytes were capableof a respiratory "burst" after phagocytosis (33). Mea-surements of the oxidation of glucose-12-4C, glucose-6-`4C and formate-"C were also similar to normal afterphagocytosis at two incubation times, indicative ofnormally functioning pentose shunt and Krebs' cyclepathways and normal production of hydrogen peroxide(23). As expected, glucose-1-"4C and formate oxidationby the CGD cells was significantly diminished fromnormal (21). CHS leukocytes were also capable ofutilizing peroxide and myeloperoxidase to enzymaticallyiodinate intracellular protein (24) with activities thatexceeded similar numbers of normal granulocytes inboth the resting and the phagocytizing state. The addi-tion of sodium azide to the incubation mixtures resultedin equivalent suppression of normal and CHS iodina-tion to the range seen with untreated CGDcells (25).

In addition to the increased iodination, glucose-1-14Coxidation by nonphagocytizing CHS leukocytes wasalso significantly greater than normal. When this wasinvestigated further by comparing glucose-1-'4C oxida-tion activities of cells separated by the Ficoll-Hypaquetechnique, both granulocytes and monocytes exhibitedthis abnormality. In addition, monolayers of CHSmonocytes had significantly higher than normal post-phagocytic glucose-1-14C oxidation that could not be

Defective Bacterial Activity of Chediak-Higashi Syndrome Leukocytes

FIGuRE 8 Electron micrographs of granulocytes histochemically stained for peroxidase activity.Panel A is a normal and panel B a CHS granulocyte without ingested particles. Arrows pointto peroxidase-positive lysosomes. Panels C and D are representative sections of normal granulo-cytes 15 min after mixing with S. aureus. Note the dark staining peroxidase activity in phago-somes surrounding bacteria and evidence of active degranulation (arrow, panel D).

661

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explained by different cell numbers. These findings areparalleled by the earlier observation of increased sphin-golipid turnover in peripheral leukocytes from the CHSpatients (34). What the significance of these findingsis in relation to membrane turnover or inherent phago-cytic capabilities (35) of these cells is indeterminate.It is of interest that in another series of studies (36)CHS leukocytes were capable of ingesting greateramounts of a paraffin oil emulsion than normal cells.Both the increased iodination of protein and theexaggerated oxidation of glucose-1-"4C by nonphago-cytizing CHS leukocytes could be explained by elevatedperoxide production (24, 37). In keeping with this, theoxidation of formate-14C by these cells was also higheron the average than normal; however the differencebetween the means was not statistically significant dueto experimental variability. Similarly, the oxidation ofglucose-6-"C was increased in resting CHS leukocytesin some but not all experiments and the means were notsignificantly different.

As initially suggested by the finding of abnormalkilling of both catalase-positive and catalase-negativebacteria, these observations indicate that metabolic ab-normalities related to the generation of hydrogen per-oxide are not involved in the pathogenesis of thedefect seen in the CHS cells. Previous data obtainedin our laboratory have indicated that CHSgranulocytescontain reduced activities of the primary lysosomal en-zymes myeloperoxidase and fi-glucuronidase (38); how-ever, the degree of reduction of peroxidase is clearlynot enough to impair intracellular iodination as notedabove. Therefore, it appears unlikely that peroxidasedeficiency, per se, is responsible for the observed find-ings.

Attention was then directed to the manner in whichthe abnormal lysosomes participate in the normal post-phagocytic chain of events. Similar to previously re-ported findings for mink and cattle (5), the giant lyso-somes were noted by light and phase microscopy toremain apparently intact for prolonged periods of timeafter phagocytosis. A cytochemical technique in con-junction with electron microscopy was employed to bet-ter observe the manner in which peroxidase is deliveredinto phagocytic vacuoles in CHS cells. As noted pre-viously (39), almost all peroxidase activity withinCHS granulocytes was localized to the giant lysosomes.

Furthermore, after bacterial ingestion the delivery ofperoxidase activity appeared spotty and inefficient whencompared to normal, with many phagosomes devoid ofthis activity 15 min after the start of ingestion. As acorollary, the appearance of most of the bacteria withinthe CHS granulocytes was strikingly different fromnormal in that many staphylococci retained details ofmorphology similar to noningested organisms. In somesections giant lysosomes were seen discharging theircontents into scattered phagosomes and in these bac-terial morphology was altered similar to that seen innormal cells. By 60 min, more vacuoles contained per-oxidase activity, but in many sections giant granuleswere observed to remain apparently intact and withoutrelationship to phagocytic vesicles. These observationssuggest that the increased delivery of peroxidase intomore phagocytic vacuoles with time either through de-layed degranulation or fusion of peroxidase-negativewith peroxidase-positive vacuoles might be responsiblefor the increase in bactericidal rates observed in theCHS cells. In this regard, it is of interest that thekinetics of the bactericidal process in CHS leukocytesbears a resemblance to that seen in leukocytes froman individual with myeloperoxidase deficiency (40).Whether the delay in killing in the first 20 min isdue to a lack of peroxidase or some other bactericidalsubstance in the majority of phagosomes within CHScells can only be speculated, however. Biochemical sup-port for these morphological observations by measure-ment of intraphagosomal activity of two enzymes asso-ciated with primary lysosomal granules (peroxidaseand P-glucuronidase) (41) has been obtained employinga technique to isolate phagocytic vesicles (42) and isthe subject of another report (36).

These studies provide evidence that in addition toimpaired chemotactic responses and neutropenia, de-layed inactivation of intracellular bacteria, in particularstaphylococci, might play a role in the pathogenesis ofrecurrent skin and sinopulmonary infections character-istic of patients with the CHS. Furthermore, the con-trasting patterns and kinetics of the bactericidal defectsexhibited by the CHS and CGD leukocytes as well astheir differing metabolic properties illustrate the multi-plicity of factors involved in leukocyte antimicrobialmechanisms and the pleiomorphic nature of inheriteddefects of leukocyte function.

FIGURE 9 Electron micrographs of granulocytes which have phagocytized S. aureus and arehistochemically stained for peroxidase. Panels A and B depict sections of CHS granulocytestaken 15 min after mixing with staphylococci. Note the lack of peroxidase activity around mostphagosomes with the exception of those into which giant lysosomes (arrows) appear to be dis-charging their contents. Panel C is a normal granulocyte 60 min after mixing with staphylo-cocci showing phagosomal fusion with peroxidase activity in the phagosomes and a lack ofperoxidase-positive granules in the cytoplasm. Panel D is a CHS granulocyte at the same timeinterval which depicts the persistence of structurally intact peroxidase-positive giant lysosomes(arrows). Peroxidase activity can be seen in some, but not all, phagosomes.


ACKNOWLEDGMENTSThe authors are indebted to Dr. David Alling for help withstatistical analyses and to Dr. Sheldon M. Wolff for sup-port, encouragement, and critical evaluation of this manuscript.

REFERENCES1. Padgett, G. A. 1968. The Chediak-Higashi syndrome.

Advan. Vet. Sci. 12: 239.2. Lutzner, M. A., C. T. Lowrie, and H. W. Jordan. 1967.

Giant granules in leukocytes of the beige mouse. J.Hered. 58: 299.

3. Kritzler, R. A., J. Y. Terner, J. Lindenbaum, J. Magid-son, R. Williams, R. Preisig, and G. B. Philips. 1964.Chediak-Higashi syndrome. Cytologic and serum lipidobservations in a case and family. Amer. J. Med. 36:583.

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