Chediak-Higashi Syndrome Neutrophils Are Characterized by the A bsence of Both Normal Azurophilic Granules BURION C. WEST, MD From the Section of Infectious Diseases, Department of Medicine, Louisiana State University School of Medicine, Shreveport, Louisiana; and the Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland Neutrophils from two Chediak-Higashi syndrome identified was eosinophil granule peroxidase at Q = 1.24 brothers were isolated, suspended in heparinized sucrose, g/ml (band E). Alkaline phosphatase, not a granule lysed, and filtered. The granule-rich filtrate was cen- marker, was twice normal at the normal density, Q = trifuged on a sucrose gradient (Q = 1.287-1.10 g/ml) at 1.14-1.15 g/ml, consistent with an increase in unidentified a mean force of95,000g for 4 hours. The gradients con- membranes. A lysate gradient suggested that the giant tained one band at Q = 1.18 g/ml (band C) which was azurophilic granules were Q = 1.25-1.27 g/ml. These neu- broader than normal and lacked normal bands A, Q = trophils contain blue-grey or slate-grey giant granules, 1.22 g/ml, and B, Q = 1.20 g/ml. Gradient fractions were which are not truly azurophilic or basophilic, but should assayed for enzyme activities and protein. No marker en- continue to be identified as azurophilic to conform to the zymes identified densities normally occupied by bands convention making "azurophilic" and "peroxidase- A and B, and of the enzymes measured, only lysozyme positive" synonymous. The eosinophils contain normal showed peak activity with band C. Thus, only normal eosinophil granules as well as giant inclusion granules. specific granules were present. Two azurophil gran- In contrast, neutrophils are deficient in both normal ules, normally present and separable, were absent. Also azurophilic granules. (Am J Pathol 1986, 122:177-189) THE CHEDIAK-HIGASHI SYNDROME, described ules, which appear to form from fusion of azurophil in 1943,1 is a rare autosomal recessive disorder, fre- and specific granules.2 8 12 Moreover, no studies address quently complicated by infections, occurring in man the number and character of granules present in the pe- and animals; it is regarded as a lysosomal disease be- ripheral blood neutrophil of human beings with the cause of the presence of giant granules with lysosomal Chediak-Higashi syndrome. The studies presented here properties in leukocytes and other cells.2-4 Accessible address this question. They provide no evidence for the neutrophils from human beings with the Chediak- presence of normal azurophil granules of any kind in Higashi syndrome have been extensively studied,5-18 al- the Chediak-Higashi syndrome neutrophil from periph- though few studies have examined isolated neutrophil eral human blood. Furthermore, they support the con- granules.7'13-18 Interest in the formation of the giant cept that all azurophil granules in this cell are in fact granules in neutrophils has been histologic and cyto- abnormal. They show that specific granules are pres- chemical.2' 1015 Even the deficiency of natural killer cells ent at the density in sucrose gradients at which normal in the Chediak-Higashi syndrome appears to be related specific granules are known to be at equilibrium to reduced numbers and defective functioning of large density25; this previous report of granule separation granular lymphocytes19 where poorly characterized large using normal human neutrophils constitutes concur- abnormal granules might constitute the major defect. rently performed controls for this report.25 The method Concurrent interest has focused upon neutrophil mem- of granule separation has been confirmed.26-31 branes, particularly the membrane lipids and micro- tubular interconnections of membranes.20-24 Membrane Accepted for publication August 16, 1985. and microtubule defects are present, and although not Address reprint requests and correspondence to Burton C. completely characterized, have been considered impor- West, MD, LSU Medical Center, P.O. Box 33932, Shreveport, tant to understanding the formation of the giant gran- LA 71130-3932. 177 178 WEST AJP * January 1986 Materials and Methods ameter, often exceeding 2 .. 4,12,15,34,35 In two experi- Patients ments 0.1-ml aliquots of lysates and 5-,u filtrates, and in one of these experiments, 0.1 ml of the 5-,A and 2-,p Two brothers with the Chediak-Higashi syndrome2.32 filtrates, were diluted to 2.0 ml with 0.34 M sucrose con- were the source of blood neutrophils. At the time of taining 0.25 mM Na2H2EDTA and assayed for enzyme these experiments they were 22 and 21 years old and activities and protein (Table 1). In another, a lysate and were free of infection. Concurrent parallel control ex- a 5-si filtrate were separately subjected to density gra- periments using venous blood from 14 normal adult dient centrifugation. donors as the source of neutrophils previously reported provided the description of normal neutrophil gran- ules.25'26 These data are not repeated here. Permission Sucrose Density Gradients after informed consent for venipuncture was obtained sucrose gradients were made as previ- with guidelines. Continuous in keeping institutional ously described with the use of a 3-ml cushion of su- crose, Q = 1.287 g/ml, 15 ml of the same dense sucrose, Materials and 15 ml of light sucrose, Q = 1.10 g/ml. Volumes were to allow room for a 2.5-5.0-ml sample at the chemicals were used throughout. Wa- adjusted Reagent grade top.25 ter was triply distilled and autoclaved. For centrifugation we used a Model L2-65B and an SW 27 rotor with cups which held 38.5 ml cellulose ni- Blood Neutrophils trate tubes (Beckman Instruments Inc., Palo Alto, Calif). Centrifugations were conducted at 4 C for 4 Peripheral venous blood was drawn in 5 mM hours at 27,000 rpm (mean force, 95,000g). Gradients Na2H2EDTA, pH 7.3, in a volume of 100 ml twice from were photographed and collected from below into 41-46 La. R. and 200 ml once from Le. R. Neutrophils were fractions and the pellet fraction, which was scraped promptly isolated by Hypaque-Ficoll gradients and dex- from the bottom and resuspended in one ml 0.34 M tran sedimentation and clarified of erythrocytes.25 33 The sucrose.25 Fractions were frozen at -20 C and assayed leukocytes, greatly enriched in neutrophils, were cen- promptly, all within 10 weeks. trifuged at 150g and washed in 30 ml of 0.34 M sucrose with 0.25 mM Na2H2EDTA; then the cell suspension was centrifuged at 150g and adjusted to 50 x 106 leu- Biochemical Determinations kocytes/4 ml of 0.34 M sucrose with 0.25 mM Na2H2EDTA. A Gilford Model 240 spectrophotometer (Gilford In- strument Laboratories, Inc., Oberlin, Ohio) was used throughout. Because of fraction volume, all assays could Preparation of Neutrophil Granules not be performed on every gradient. The number of To each 4-ml cell suspension was added sodium hepa- gradients on which an assay was performed is indicated. rin, 0.5 ml, containing 5000 units (The Upjohn Com- Assay descriptions and conditions are identical to those pany, Kalamazoo, Mich). Cell disruption, aided by previously reported.25 Protein, utilizing egg white lyso- heparin, was accomplished by 11-15 cycles of suction- zyme as standard, is expressed as Mg/ml.36 Because of ing the suspension through a 9-cm 18-gauge needle at- interference of hydrolyzed sucrose in the accuracy of tached to a 6-ml polypropylene syringe.2526 Because of the Lowry method, the values reported are considered concern about the destruction of the giant azurophilic estimates.37 Myeloperoxidase (EC 1.11.1.7) is expressed granules by the lysis procedure, an attempt was made as microgram equivalents of horseradish peroxidase per to minimize resuctioning. Lysis was monitored by phase milliliter.25 38 Lysozyme (EC 3.2.1.17) is expressed as microscopy. micrograms of egg white lysozyme equivalent per mil- Lysates were filtered through a 5-,. (pore size) poly- liliter.39 (-Glucuronidase (EC 3.2.1.31) is expressed as carbonate filter, 25 mm in diameter (Nuclepore, General micrograms phenolphthalein released per hour per mil- Electric Corp., Pleasanton, Calif), followed by filtra- liliter.40'41 Acid phosphatase (EC 3.1.3.2) was determined tion of the 5-,A filtrate through a 2-,u pore size filter.25 by two methods and is expressed either as micromoles Each 5-. and 2-, filtrate was applied to a sucrose gra- of p-nitrophenylphosphate released per hour per mil- dient. liliter or as nanograms inorganic phosphorous liber- A limited effort was made to assess the presumably ated (from sodium P-glycerophosphate) per minute per deleterious effects of filtration of Chediak-Higashi syn- milliliter.43'44 Alkaline phosphatase (EC 3.1.3.1) is ex- drome giant granules, which range from 1 to 4 IA in di- pressed as micromoles of p-nitrophenylphosphate Vol. 122 * No. 1 CHEDIAK-HIGASHI SYNDROME NEUTROPHILS 179 Table 1-The Distribution of Protein Concentration and Enzyme Activities in Lysates, 5-p (Pore Size) Filtrates, and 5-J and 2-M (Pore Size) Filtrates of Normal Human Neutrophils and Ch6diak-Higashi Syndrome Neutrophils, the Latter as Estimated From Two Experiments Normals* P Ch6diak-Higashi syndrome Protein (mg/ml) Lysate 0.85 ± 0.03 (22)t <0.01 0.45, 0.52 5-f filtrate 0.62 ± 0.04 (21) NS 0.45, 0.59 5-2-1j filtrate 0.62 ± 0.02 (22) NS 0.63, 0.48 Enzyme activityt Myeloperoxidase Lysate 0.94 + 0.05 (23) NS 0.85 ± 0.16 (4) 5-r filtrate 0.77 ± 0.04 (23) <0.001 0.33 ± 0.04 (4) 5-2-MA filtrate 0.76 ± 0.04 (22) <0.001 0.23, 0.24 P-Glucuronidase Lysate 74.6 ± 5.6 (11) NS 55 5-p filtrate 55.6 ± 6.5 (10) <0.05 15 5-2-IA filtrate 63.2 ± 5.4 (10) - - Lysozyme Lysate 72.1 + 4.5 (18) <0.001 137 ± 40 (3) 5-,.