Canine Granulocytopathy Syndrome an Inherited Disorder Ofleukocyte Function

Canine Granulocytopathy Syndrome An Inherited Disorder ofLeukocyte Function

Harland W. Renshaw, DVM, PhD, and William C. Davis, PhD

A disease closely resembling the human neutrophil dysfunction syndromes has been identified in a colony of dogs. The syndrome, referred to as the canine granulocytopathy syndrome, is characterized by recurrent life-threatening bacterial infections and a greatly shortened life span. The disease is genetically determined, being transmitted as an autosomal recessive trait. The increased susceptibility to pyogenic infections and shortened life span is related to an impairment of leukocvte function at the cellular level. Preparations of neutrophils from affected animals exhibit impaired in ritro killing of Escherichia coli. The defect in bactericidal activity is associated with reduced glucose oxidation by the hexose monophosphate shunt and an increased capacity to reduce nitroblue tetrazolium dye. The data obtained thus far indicate the canine granulocytopathy syndrome will be of considerable value as a model for the study of granulocytopathy syndromes in man. (Am J Pathol 95:731-744, 1979)

STUDIES OF LEUKOCY-TE structure and function in host defense failure syndromes of man and animals have provided dramatic evidence for the necessitv of adequate neutrophil function for normal host resistance.`6 A group of human diseases, characterized clinically bv enhanced susceptibilitv to pyogenic infections, has been related to impaired killing of microorganisms by neutrophils and monocytes.7-12 A direct correlation has been demonstrated between defective intraneutrophilic microbicidal capacitv and enhanced susceptibilitv to infections in Chediak-Higashi syndrome (C-HS) of humans,12 chronic granulomatous disease (CGD) of childhood,13-15 mveloperoxidase (MPO) deficiencv,'-'8 glucose-6-phosphate dehydrogenase (G-6-PD) deficiencv,"" congenital abnormalities of specific granules,22 and lysozvme and lactoferrin deficiency with congenital absence of specific granules.2 Evidence from detailed clinical and laboratory studies of the affected patients and their relatives supports the hypothesis that several, if not all, of these diseases are genetically determined clinical svndromes.5,1324,25

From the Department of Veterinary Microbiology and Parasitology. College of Veterinary Medi- cine. Texas A&M Uni-ersity. College Station. Texas. and the Department of Veterinary Microbiology and Pathology. College of Veterinan Medicine. Washington State University. Pullman. Washington Supported in part by US Public Health Service Grants AI-15952-01 and AI-12826402 from the National Institute of Allergy and Infectious Diseases. Accepted for publication January 222 1979. Address reprint requests to Harland WV Renshaw. DVM. PhD, Department of Veterinarv Micro- biology and Parasitology. College of Veterinary Medicine. Texas A&MN University. College Station. TX 77843 0002-9440/79/0607-0731 $01.00 731 © 1979 American Association of Pathologists 732 RENSHAW AND DAVIS American Journal of Pathology Recently, we observed a spontaneously occurring disease in a male Irish setter dog, which had clinical features closely resembling those observed in human neutrophil dysfunction syndromes.26 Increased susceptibility to infection in the affected animal was associated with defective bactericidal capacity of neutrophil-rich leukocyte preparations. Affected canine neutrophils appear normal ultrastructurally with no morphologically identi- fiable alteration in lysosome formation.27 Results of preliminary studies of this condition, tentatively designated the canine granulocytopathy syndrome (CGS), suggest that it is similar to, but uniquely different from, any neutrophil dysfunction syndrome described thus far in man or animals. Because this new disease could have considerable value in comparative biology studies, controlled breeding experiments were conducted to determine whether the CGS is a genetically determined metabolic disorder. In this report, we present the results of the breeding experiments and describe studies that indicate that the increased disease susceptibility of affected dogs is associated with impaired in vitro killing of bacteria and a metabolic anomaly in hexose monophosphate shunt (HMPS) activity by their neutrophils.

Materials and Methods Subjects The subjects used in this study were obtained from two sources: 1) a series of controlled breeding experiments involving matings of the propositus, his clinically normal dam, an unrelated clinically normal female, and breedings of some of the resultant clinically normal offspring (Text-figure 1); and 2) a population of clinically normal unrelated mongrel dogs. Healthy animals were maintained under conventional conditions in ken- nels. A combination of clinical and laboratory evidence was used to arrive at a diagnosis of CGS. Affected animals were treated with antibiotics as soon as signs of the disease were evident. Representative Case Report Data Prominent clinicopathologic features of the CGS are omphalophlebitis after birth, suppurative skin lesions, gingivitis, lymphadenopathy, pododermatitis, and, in most ad- vanced cases, osteomyelitis. Hematologic studies, even in very young affected puppies, reveal a persistent leukocytosis with a regenerative left shift. Normal dogs usually have a total leukocyte count of 8000 to 14,000/cu mm with 55 to 75% neutrophils. In affected dogs that have been stabilized with antibiotic therapy total leukocyte counts are usually in the range of 25,000 to 65,000/cu mm with 75 to 90% neutrophils. During exacerbations the counts will rise to 120,000 to 540,000 with a corresponding increase in immature forms. Mature neutrophils contain nuclei that are often hypersegmented and both peroxidase- positive lysosomes and specific granules. Neither immature nor atypical lymphocytes or undifferentiated nongranular neutrophil forms are observed in Wright's-stained blood smears. Most affected animals have a moderate normocytic, normochromic anemia. The syndrome is often associated with hypergammaglobulinemia. The major classes of canine immunoglobulins are present. Histopathologic examination of necropsy or biopsy material Vol. 95, No. 3 CANINE GRANULOCYTOPATHY- - - . . . - - . - .. SYNUHUME 733 May 1979

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...,, ,,,., .... , S,:.,.''',, ,j_..,|,...... '...- t,,- ,*.,j _;-;:S,j,,~~~~~~~~~~~~~~~~~~~~~~~~~~~~.,.,...... W.,,....,v 734 RENSHAW AND DAVIS American Journal of Pathology reveals granular brown-staining pigment (lipofuscin) in reticuloendothelial cells through- out the lymphoid tissues with an absence of granulomatous lesions. Radiographic examination of head, thorax, abdomen, and limbs reveals no visceral or skeletal abnormalities except those associated with cases of osteomyelitis. Serum glutamic transaminase and alkaline phosphatase activities are generally normal, as are results of blood urea nitrogen determinations, urinalysis, and the direct Coomb's test. Between episodes of severe infections, the animals eat and drink well and are alert. However, during exacerbations of the disease, anorexia, weight loss, dehydration, and severe depression accompany periods of pyrexia and extreme neutrophilia. By 1 month of age, the affected animals are usually only slightly greater than half the size and weight of their clinically normal littermates. Continuous therapy with a broad-spectrum antibiotic appears to be necessary to keep affected individuals alive for more than a few weeks. Even with antibiotic therapy and extensive supportive therapy, the affected dogs have a greatly shortened life span. Leukocyte Preparation Heparinized blood (15 U/ml) specimens were collected from affected and clinically normal dogs of the same ages. Mixed cell preparations of peripheral blood leukocytes were isolated from the heparinized blood by lysing the erythrocytes in three volumes of 0.87% NH4C1.28 The leukocytes were pelleted by low-speed centrifugation at 200g for 5 minutes at 22 C and suspended in sterile saline for counting. Preparations containing an enriched population of polymorphonuclear neutrophils (PMN) were obtained using a modification of the IsopaqueFicoll density gradient centrifugation technique of B6yum.29 The heparinized blood was mixed with an equal volume of heparinized (15 U/ml) Eagle's minimal essential medium (MEM). The heparinized blood- MEM mixture (30 ml) was layered on 20 ml of Isopaque-Ficoll separation fluid (density 1.077 g/ml), and centrifugated at 450g for 40 minutes at 22 C. The mononuclear cells banded at the interface region were removed and discarded. The bottom fraction containing erythrocytes and leukocytes was resuspended and the erythrocytes lysed in 3 volumes of 0.87% NH,C1. Leukocytes were collected by centrifugation at 200g for 5 minutes at 22 C and suspended in sterile saline for counting. Cell viability, determined by 0.1% trypan blue dye exclusion, was > 95% for all mixed cell preparations and > 90% in all PMN preparations. Total and differential leukocyte counts were performed with a hemacytometer and by examination of Wrights's-stained smears, respectively. Standardized leukocyte suspensions, both mixed cells and PMN, containing 10 X 108 neutrophils/mI were prepared in heparinized MEM with 10% fetal calf serum. Leukocyte Bactericidal Assay The bactericidal activity of canine leukocytes was assessed using Escherichia coli (078:K80) as the test organism. Bacteria were grown at 37 C for 4 hours in brain-heart infusion broth (Difco Laboratories, Inc., Detroit, Michigan), centrifuged at 2000g for 20 minutes at 22 C, washed three times in sterile phosphate-buffered saline solution (pH 7.4), and adjusted to an optical density of 0.6 with a Bausch and Lomb Spectronic 20 at 620 nm. The leukocyte bactericidal assay 6.27.30 was performed in 12 X 75 mm plastic snap cap tubes. The reaction mixture contained 0.2 ml of standardized viable E coli suspension, 0.2 ml of freshly collected canine serum, 0.6 ml MEM, and 1.0 ml of PMN suspension (containing 10 X 106 neutrophils/ml). The bacteria were added after the remainder of the mixture had been incubated at 37 C for 15 minutes on a rotator at 20 revolutions per minute. The bacterial suspension was added to achieve a bacteria-to-neutrophil ratio in the range of 0.2: 1 to 1: 1. At given time intervals, fractions of the leukocyte-bacteria mixture were removed and mixed thoroughly in sterile distilled water to lyse the leukocytes. Serial tenfold dilutions were made and appropriate dilutions plated on nutrient agar Vol. 95, No. 3 CANINE GRANULOCYTOPATHY SYNDROME 735 June 1979

pour plates. Plates were incubated at 37 C for 18 hours, and the number of colonies present on the plates was counted with a Biotran colony counter (New Brunswick Scientific Co. Inc.. New Brunswick, NJ) to determine the number of viable organisms at each time period. Zero time counts were obtained by making serial dilutions from the leukocyte- bacteria suspensions immediately after adding bacteria. Results were expressed as the number of viable bacteria remaining per milliliter of the reaction mixture per unit time and plotted as the logarithm of the mean number of viable bacteria ± 1 standard deviation for affected and normal dogs.

Nitroblue Tetrazolium Test Neutrophils from normal and affected dogs were tested for their ability to reduce nitroblue tetrazolium (NBT) dye (Grade 3, Sigma Chemical Co., St. Louis, Mo) to blue formazan after phagocytosis of 0.81-gm latex spheres (Difco Laboratories, Inc.. Detroit. Mich). The previouslv described modification of the spectrophotometric method of Baehner and Nathan ii was used to assess NBT dve reduction by mixed cell preparations." Results were recorded as the optical density of reduced NBT in pyridine ex- tracts from resting and phagocytizing cells. From these values the difference in N'BT dye reduction between resting and phagocytizing cells (A difference) for affected and normal canine leukocvtes was determined. Glucose Oxidation Oxidation of [1-14C] glucose (New England Nuclear, Boston. Mass) by mixed cell preparations from the propositus and normal dogs w-as determnined as previouslI described.612 The release of C402 from [1-14C1 glucose was used as an indicator of HM PS activity and values measured as counts per minute (cpm) were recorded for both resting and phago- cvtizing cells.

Results Genetic Stdies The pedigree of matings used to investigate the possibilitv of genetic transmission of the CGS is presented in Text-figure 1. Three of 11 littermates of the originally recognized animal with CGS died soon after birth, and there was no history of illness present in siblings that survived the first week. None of the 7 siblings was available for study. The mother and father of the propositus were clinically normal. Mating of the mother with a different clinicallv normal male resulted in the birth of 8 clinically normal puppies (3 females and 3 males-data not shown). NMating of the propositus to his mother, however, resulted in the birth of 3 affected daughters with shortened life spans and 2 clinically normal offspring. Mating of the propositus to an unrelated clinically normal female resulted in the birth of 4 clinicallv normal females and a male that died of suffocation at 2 davs of age from being lain upon. Three of these females survived to breeding age and were mated with the clinicallv normal male obtained from the mating of the propositus and his mother; this clinically normal male was also bred to his mother. From these four matings a total 736 RENSHAW AND DAVIS American Journal of Pathology of 40 offspring were obtained. Since 5 puppies in one litter were lost due to suffocation during the first 3 days of life, only 35 survived long enough for a diagnosis to be made. Puppies affected with the CGS were obtained from each of the four matings. There was a total of 10 affected puppies (6 females and 4 males) in the 35 puppies (19 females and 16 males) available for study. These genetic studies indicated that the CGS has an autosomal recessive pattern of transmission. Leukocyte Bactericidal Assay The Isopaque-Ficoll technique yielded cell populations containing high percentages of neutrophils from CGS and normal individuals. The CGS cell preparations contained approximately 96% neutrophils (96.3 ± 4.4%; range 86 to 100%) vs approximately 87% (87.2 ± 5.8%; range 79 to 98%) for the normals. The majority of the remaining cells were mononuclear cells and eosinophils. Mean lymphocyte and monocyte contamination of the final cell suspensions was in all cases less than 9.0% for the different experiments. Cells from both normal and affected animals were cultured with freshly collected serum from normal animals. When PMN suspensions from normal dogs were cultured with E coli for 2 hours, there was nearly complete killing of the microorganisms. However, as shown in Text-figure 2, PMN suspensions from animals with CGS exhibited a pronounced defect in bactericidal capacity. The difference in the rate of killing between the different cell-serum mixtures was attributable to cellular and not serum factors. Since neutrophils were the principal effector cells in the leukocyte bactericidal assays, the defect is, in large part, associated with dysfunction of neutrophils. No information was obtained on whether monocytes are similarly affected. Nitroblue Tetrazolium Test In other granulocytopathies, such as CGD of childhood, defective intraneutrophilic microbicidal capacity is associated with impaired NBT dye reduction by phagocytizing cells.31 Efforts to define the site of the primary gene defect in the CGS have shown that the abnormality is not correlated with decreased NBT dye reduction (Text-figure 3). Mixed cell preparations from normal (.035 ± .008 OD,15nm) and affected (.036 ± 027 OD515nm) dogs reduced comparable amounts of NBT dye to blue formazan in the resting state. However, more (P < 0.001) NBT dye was reduced following phagocytosis of polystyrene latex spheres by the cells with defective bactericidal capacity (.180 ± .064 OD515nm) than by the cells with normal bactericidal capacity (.116 ± .043 OD515nm). Further- Vol. 95, No. 3 CANINE GRANULOCYTOPATIHY YNUDUME 737-7 7 June 1979 10s

49 TExr-FIGtuRE 2-In vitro bacteri- X cidal capacity of peripheral blood . leukocyte suspensions containing high concentrations of neutrophils from normal (circles) and X 16: CGS-affected (triangles) dogs. > Study of the kIlling of opsonized U. Escherichia coli. The logarithm of ° the number of viable bacteria is . plotted as a function of time. a Geometric means of the results of z eight studies and one standard 10\ deviation are shown.

5 x to6 PMN/mi

0 30 60 120 MINUTES more, the difference in NBT dye reduction between resting and phagocytizing cells (,A difference) from affected animals (.144 ± .061 OD515nm) was higher (P < 0.001) than for the normals (.081 ± .043 OD5l5nm). acsoOAN A series of metabolic events referred to as the "respiratory burst" underlies all oxygen-dependent killing by neutrophils. Stimulation of the normal neutrophil causes an increase in 02 uptake, 02- production, H202 production, and HMPS activity.32'" Oxidation of [1-14C] glucose by affected and normal cells was studied to determine whether any alteration could be detected in glucose metabolism by the HMPS pathway. As shown in Text-figure 4 glucose oxidation by the HMPS for resting cells from normal (266 ± 67 cpm) and affected (222 ± 91 cpm) cell preparations was similar. However, following ingestion of latex spheres, glucose oxidation by affected cells (2741 ± 792 cpm) was significantly less (P < 0.01) than for normal cells (4726 ± 1979 cpm). This interesting observa- tion, as will be discussed later, establishes the CGS as a host defense 738 RENSHAW AND DAVIS American Journal of Pathology

0.300 RESTING o PHAGOCYTIZING MMADIFFERENCE o.0.250 TEXT-FIGURE 3-Quan- 10.200 titative NBT reduction o _ _ by neutrophils from nor- o Mm m mal (24 tests, 24 ani- 0.5 ~...mals) and CGS-affected (24 tests, 7 animals) E _ _ _ ;:; ^ *@ Z ;@ @ dogs. Geometric means * t .t tX and one standard devia-

NORIAL CELLS CGS CELLS failure syndrome characterized by an aberration of respiratory burst activitv in neutrophils. Discussion Data obtained from genetic, clinical, and laboratory studies of the CGS indicate that the condition is a genetically determined defect in host resistance to infection that is inherited as an autosomal recessive trait. Several of the genetic observations are consistent with this pattern of inheritance. Thus, backcrossing the affected propositus with his dam yielded a litter with both affected and phenotypically normal offspring, whereas all offspring from a mating with a clinically normal unrelated female were phenotypically normal (Text-figure 1). Subsequent breedings of these first generation offspring yielded litters with both male and female affected puppies. As would be expected if the CGS is inherited as an autosomal recessive trait, the observed ratio of affected to phenotypically normal offspring was close to 1 :3 (actually 1: 2.5) when presumed heterozygous parents were mated. Thus, the mode of inheritance of the CGS is the same as the autosomal recessive form of CGD of childhood,34 but differs from the sex-linked form. CGS to of The can, therefore, be addedhs,temdtheoflist genetically determinedc defectsheterozygousoparentsin host resistancewesrdermtdsto infection thatcmlenmtaoinherhave been describedreitain manlofhand animals during the last several decades. Enhanced susceptibility to infection can result from immunoglobulin deficiencies,t36i3a defective cellular femalewerephenotypicalynormalsof (Text-figuen1). Subseque breedings .- -- ...... - .11, 7 n Vol. 95, No. 3 CANINE GRANULOCYTOPATHY SYNDHUME '3i June 1979

TEXT-FICGRE 4-Glucose oxidation via the HMPS by peripheral O0 blood leukocvtes from normal (16 x _ tests, 16 animals) and CGS-af- _ fected (9 tests. propositus only) * dogs. The release of "4CO2 from [1-`C] glucose measured as counts per minute was used as an indicator of HM PS activit. Geo- metric means and one standard deviation are show n.

m CELLS Cm Cs endotheliosis,'3'" and functional defects in phagocvtic cells.25" The familial nature of several host defense failure svndromes has been clearlv documented bv the analvsis of extensive pedigrees. The genetic evidence suggests that most of these svndromes have either an autosomal recessive or sex-linked pattern of inheritance. The CGS is the first granulocvtopathv svndrome described in a sub- human species that is not associated with a morphologically identi- fiable aberration in Iysosome formation. Mature CGS neutrophils contain morphologically normal-appearing primary (peroxidase-positive; azurophil) and secondarv granules.27 This is in contrast to the situation in humans and animals with the C-HS where large abnormal granules (C- HS granules) in neutrophils arise from aberrant formation and fusion of the primarv (azurophil) granules."'5 Increased susceptibilitv to infection, noted in humans and several animal homologues, of this disease has been related to an impairment of leukocvte function.'6,',-' Genetic studies conducted in several of the affected species indicate the C-HS, like the CGS, is inherited as an autosomal recessive trait.4 The results of previous clinical and laboratorv studies had suggested that the increased susceptibility to pyogenic infections and shortened life span noted in CGS-affected animals were related to an impairment of 740 RENSHAW AND DAVIS American Journal of Pathology leukocyte function at the cellular level.26'27 Mixed leukocyte cell preparations from normal animals and the propositus had similar ability to clear bacteria, by either adherence or ingestion, from leukocyte-bacteria cell suspensions. 27 However, as compared to normals, the leukocytes from the propositus had significantly impaired bactericidal capacity. As noted in the data presented here, the leukocyte defect in the CGS is certainly associated with the neutrophil. Leukocyte preparations containing high concentrations of neutrophils exhibit impaired killing of E coli, as do mixed cell preparations.27 Further studies are needed to determine if monocytes as well as neutrophils are defective in bactericidal capacity. In CGD both cell types are defective.813-15 The studies of the CGS that have been conducted thus far revealed that this interesting disease has certain similarities and dissimilarities with the granulocytopathy syndromes recognized in man. Leukocytes taken from animals affected with the CGS, while apparently being phagocytically competent, are defective in intracellular bactericidal capacity. A similar situation exists in a number of human neutrophil dysfunction syndromes. Unlike the situation in severe G-6-PD deficiency 19-21 and CGD,13-15,49 in CGS the capacity to kill bacteria does not depend on whether the micro- organism is catalase positive or negative. The main correlate that has been found is that the defect in killing is associated with impaired functioning of the HMPS. In comparison with normal dogs, there is a lower rate of glucose utilization via the HMPS by phagocytizing cells from the affected animals. Interestingly, the alteration in HMPS activity is not reflected in the NBT test. In fact, our studies indicate that in comparison with cells from clinically normal dogs there is an elevation in the cellular activity that leads to the reduction of NBT dye by phagocytizing cells from affected dogs. The reason for this remains obscure. In CGD the fundamental abnormality is the inability of the phagocyte to undergo a respiratory burst.50" Such cells fail to show increases in 02 uptake, 02- production, H202 production, and HMPS activity following exposure to a phagocytic stimulus. Phagocytes from patients with CGD 31 and severe G-6-PD deficiency 19 do not reduce NBT, apparently because of the absence of the respiratory burst. NBT reduction by phagocytes is thought to result from a chemical reaction between the dye and the 2- generated during the respiratory burst.55 When viewed in the context of present knowledge about the relationship of phagocyte function and metabolism, the results from the present study are very intriguing. Para- doxically, while depressed HMPS activity by affected canine phagocytes suggests reduced respiratory-burst activity, the increased capacity to reduce NBT dye to blue formazan implies that 2- production is increased Vol. 95, No. 3 CANINE GRANULOCYTOPATHY SYNDROME 741 June 1979 as would be observed were respiratory-burst activity increased. Further detailed biochemical studies will be necessarv to clarifv the significance of these findings. Evidence from this and previous studies suggests that, although similar, the CGS is probablyr not homologous to anv of the granulocvtopathy syndromes described in humans and animals. It would appear that one of the potential values of this recently discovered host defense failure svndrome would be the unique opportunitv it provides for careful analvsis of defects leading to impaired metabolic and functional activitv of phagocytes. Because the normal neutrophil possesses multiple nonoxidative and oxidative bactericidal mechanisms, a detailed analvsis w%ill be necessary to identifv the precise biochemical defect(s) in the CGS. It is apparent that other aspects of the functionalitv of CGS neutrophils, such as phago- cvtosis, chemotaxis, and degranulation, should be thoroughly evaluated. Such studies mav document other abnormalities besides the bactericidal defect. Since the CGS is a genetically determined defect in host resistance, it will be possible to breed research animals and studv manifestations of the disease in both carrier and affected animals.

References 1. Davis WC, Douglas SD: Defective granule formation and function in the Chediak- Higashi syndrome in man and animals. Semin Hematol 9:431-450, 1972 2. Klebanoff SJ: Intraleukocytic microbicidal defects. Annu Rev Med 22:39-62, 1971 3. Lund JE, Padgett GA, Ott RL: Cyclic neutropenia in grey collie dogs. Blood 29:432-461, 1967 4. Padgett GA, Holland JM, Davis WC, Henson JB: The Chediak-Higashi syndrome: A comparative review. Curr Top Pathol 31:175-194, 1970 3. Quie PG: Pathology of bactericidal power of neutrophils. Semin Hematol 12:143- 160, 1973 6. Renshaw HWN, Davis WVC, Fudenberg HH, Padgett GA: Leukocyte dysfunction in the bovine homologue of the Chediak-Higashi syndrome of humans. Infect Immun 10:928-937, 1974 7. Baehner RL: Disorders of leukocy-tes leading to recurrent infection. Pediatr Clin North Am 19:935-956, 1972 8. Davis WC, Huber H, Douglas SD, Fudenberg HH: A defect in circulating mononuclear phagocytes in chronic granulomatous disease of childhood. J Immunol 101:1093-1093, 1968 9. Nathan DG, Baehner RL: Disorders of phagocvtic cell function. Prog Hematol 7:235-234, 1971 10. Quie PG: Infections due to neutrophil malfunction. Medicine 32:411-417, 1973 11. Quie PG: Infection associated with phagocytic cell deficiencies, Infection and the Compromised Host. Edited by JC Allen. Baltimore, Williams & Wilkins Company, 1976, pp 81-101 12. Root RK, Rosenthal AS, Balestra DJ: Abnormal bactericidal, metabolic, and lvso- somal functions of Chediak-Higashi syndrome leukocvtes. J Clin Invest 51:649-665, 1972 742 RENSHAW AND DAVIS American Journal of Pathology

13. Good RA, Quie PG, Windhorst DB, Page AR, Rodey GE, White J, Wolfson JJ, Holmes BH: Fatal (chronic) granulomatous disease of childhood: A hereditary defect of leukocyte function. Semin Hematol 5:215-254, 1968 14. Kaplan EL, Laxdal T, Quie PG: Studies of polymorphonuclear leukocytes from patients with chronic granulomatous disease of childhood: Bactericidal capacity for streptococci. Pediatrics 41:591-599, 1968 15. Quie PG, White JG, Holmes B, Good RA: In vitro bactericidal capacity of human polymorphonuclear leukocytes: Diminished activity in chronic granulomatous disease of childhood. J Clin Invest 46:668-679, 1967 16. Lehrer RI: Measurement of candidacidal activity of specific leukocyte types in mixed cell populations. I. Normal, myeloperoxidase-deficient, and chronic granulomatous disease neutrophils. Infect Immun 2:42-47, 1970 17. Lehrer RI, Cline MJ: Leukocyte myeloperoxidase deficiency and disseminated candidiasis: The role of myeloperoxidase in resistance to candida infection. J Clin Invest 48:1478-1488, 1969 18. Lehrer RI, Hanifin J, Cline MJ: Defective bactericidal activity in myeloperoxidase- deficient human neutrophils. Nature (London) 223:78-79, 1969 19. Baehner RL, Johnston RB Jr, Nathan DG: Comparative study of the metabolic and bactericidal characteristics of severely glucose-6-phosphate dehydrogenase-deficient polymorphonuclear leukocytes and leukocytes from children with chronic granulomatous disease. J Reticulendothel Soc 12:150-169, 1972 20. Cooper MR, DeChatelet LR, McCall CE, LaVia MF, Spurr CL, Baehner RI: Complete deficiency of leukocyte glucose-6-phosphate dehydrogenase with defective bactericidal activity. J Clin Invest 51:769-778, 1972 21. Gray GR, Stamatoyannopoulos G, Naiman SC, Kliman MR, Klebanoff SJ, Austin T, Yoshida A, Robinson GCF: Neutrophil dysfunction, chronic granulomatous disease, and non-spherocytic haemolytic anaemia caused by complete deficiency of glucose-6-phosphate dehydrogenase. Lancet 2:530-534, 1973 22. Strauss RG, Bove KE, Jones JF, Mauer AM, Fulginiti VA: An anomaly of neutrophil morphology with impaired function. N Engl J Med 290:478-484, 1974 23. Spitznagel JK, Cooper MR, McCall AE, DeChatelet LR, Welsh IRH: Selective deficiency of granules associated with lysozyme and lactoferrin in human poly- morphs (PMN) with reduced microbicidal capacity. J Clin Invest 51:93a, 1972 24. Douglas SD: Analytic review: Disorders of phagocyte function. Blood 35:851-866, 1970 25. Stossel TP: Phagocytosis. N Engl J Med 290:717-723, 774-780, 833-839, 1974 26. Renshaw HW, Chatburn C, Bryan GM, Bartsch RC, Davis WC: Canine granulocytopathy syndrome: Neutrophil dysfunction in a dog with recurrent infections. J Am Vet Med Assoc 166:443-447, 1975 27. Renshaw HW, Davis WC, Renshaw SJ: Canine granulocytopathy syndrome: De- fective bactericidal capacity of neutrophils from a dog with recurrent infections. Clin Immunol Immunopathol 8:385-395, 1977 28. Hummeler K, Harris TN, Tomassini N, Hechtel M, Farber MB: Electron micro- scopic observations on antibody-producing cells in lymph and blood. J Exp Med 124:255-262, 1966 29. B1oyum A: Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by com- bining centrifugation and sedimentation at lg. Scand J Clin Lab Invest 21, Suppl 97:77-89, 1968 30. Hirsch JG, Strauss B: Studies on heat-labile opsonin in rabbit serum. J Immunol 92:145-154, 1964 31. Baehner RL, Nathan DG: Quantitative nitroblue tetrazolium test in chronic granulomatous disease. N Engl J Med 278:971-976, 1968 Vol. 95, No. 3 CANINE GRANULOCYTOPATHY SYNDROME 743 June 1979

32. Babior BM: Oxygen-dependent microbial killing by phagocytes. N Engl J Med 298:659-668, 721-725, 1978 33. Quie PG, Mills EL, Holmes B: Molecular events during phagocytosis by human neutrophils. Prog Hematol 10:193-210, 1977 34. Azimi PH, Bodenbender JG, Hintz RL, Kontras SB: Chronic granulomatous disease in three female siblings. JAMA 206:2865-2870, 1968 35. Windhorst DB, Page AR, Holmes B, Quie PG, Good RA: The pattern of genetic transmission of the leukocyte defect in fatal granulomatous disease of childhood. J Clin Invest 47:1026-1034, 1968 36. Biggar WD: Analysis and treatment of immunodeficiency diseases, Infection and the Compromised Host. Edited by JC Allen. Baltimore, Williams & Wilkins Com- pany, 1976, pp 1-27 37. Douglas SD, Fudenberg HH: Genetically determined defects in host resistance to infection: Cellular immunologic aspects. Med Clin North Am 53:903-922, 1969 38. Fudenberg H, Good RA, Goodman HC, Hitzig WH, Kunkel HG, Roitt IM, Rosen FS, Rowe DS, Seligmann M, Soothill JR: Primary immuno-deficiencies: Report of a WHO committee. Pediatrics 47:927-946, 1971 39. Good RA: Recent studies on the immunodeficiencies of man. Am J Pathol 69:484- 490, 1972 40. Provost T: Infections associated with T-cell dysfunction, Infection and the Com- promised Host. Edited by JC Allen, Baltimore, Williams & Wilkins Company, 1976, pp 49-79 41. Provost T, Allen JC: Susceptibility to infection related to deficiencies in the complement system, Infection and the Compromised Host. Edited by JC Alleii. Williams & Wilkins Company, 1976, pp 133-149 42. Ruddy S, Gigli I, Austen KF: The complement system of man. N Engl J Med 287:642-646, 1972 43. Miller DR: Familial reticuloendotheliosis: Concurrence of disease in five siblings. Pediatrics 38:986-995, 1966 44. Schoech VW, Peterson RDA, Good RA: Familial occurrence of Letterer-Siwe disease. Pediatrics 32:1055-1063, 1963 45. Davis WC, Spicer SS, Greene WB, Padgett GA: Ultrastructure of bone marrow granulocytes in normal mink and mink with the homolog of the Chediak-Higashi trait of humans. I. Origin of the abnormal granules present in the neutrophils of mink with the C-HS trait. Lab Invest 24:303-17, 1971 46. Clark RA, Kimball HR, Padgett GA: Granulocyte chemotaxis in the Chediak- Higashi syndrome of mink. Blood 39:644-649, 1972 47. Gallin JI, Bujak JS, Patten E, Wolff SM: Granulocyte function in the Chediak- Higashi syndrome of mice. Blood 43:201-206, 1974 48. Wolff SM, Dale DC, Clark RA, Root RK, Kimball HR: The Chediak-Higashi syndrome: Studies of host defenses. Ann Intern Med 76:293-S06, 1972 49. Mandell GL, Hook EW: Leukocyte bactericidal activity in chronic granulomatous disease: Correlation of bacterial hydrogen peroxide production and susceptibility to intracellular killing. J Bacteriol 100:531-532, 1969 50. Baehner RL, Nathan DG: Leukocyte oxidase: Defective activity in chronic granulomatous disease. Science 155:835-S6, 1967 51. Curnutte JT, Whitten DM, Babior BM: Defective superoxide production by granulocytes from patients with chronic granulomatous disease. N Engl J Med 290:593- 597, 1974 52. Holmes B, Page AR, Good RA: Studies of the metabolic activity of leukocytes from patients with a genetic abnormality of phagocytic function. J Clin Invest 46:1422- 1432, 1967 53. Johnston RB Jr, Keele BB Jr, Misra HP, Lehmeyer JE, Webb LS, Baehner RL, 744 RENSHAW AND DAVIS American Journal of Pathology

Rajagopalan KV: The role of superoxide anion generation in phagocytic bactericidal activity: Studies with normal and chronic granulomatous disease leukocytes. J Clin Invest 55:1357-1372, 1975 54. Mandell GL, Hook EW: Leukocyte function in chronic granulomatous disease of childhood: Studies on a seventeen year old boy. Am J Med 47:473-486, 1969 55. Baehner RL, Boxer LA, Davis J: The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease polymorphonuclear leukocytes. Blood 48:309-313, 1976

Acknowledgment We wish to thank Ms. Susan J. Renshaw for excellent technical assistance and graphics work.