Beulah Holmes

In Vitro Bactericidal Capacity of Human Polymorphonuclear Leukocytes: Diminished Activity in Chronic Granulomatous Disease of Childhood *

Diminished bactericidal capacity was found to be characteristic of polymorphonuclear leukocytes (PMN) from five children with the clinical syndrome of granulomatous disease of childhood. The PMN from these children demonstrated nearly normal phagocytic capacity, and the majority of viable bacteria, after 2 hours of incubation in the phagocytosis system, were found associated with leukocytes. The morphology of the unstimulated polymorphonuclear leukocytes from patients with chronic granulomatous disease was similar to those from normal persons of similar ages by light and electron microscopy. In addition, the total lysozyme and phagocytin activity of leukocyte extracts from these patients was similar to those from equal numbers of leukocytes from controls.A striking difference in the cytoplasmic response after phagocytosis characterized the PMN of the patients with granulomatous disease. Whereas degranulation, vacuole formation, and rapid bacterial digestion were the rule in the PMN from controls, little degranulation and persistence of intact bacteria in the cytoplasm characterized disease. The deficiency of bactericidal capacity and the minimal degranulation after active phagocytosis by the PMN of these children with an inherited syndrome suggest that separate metabolic processes are involved in phagocytosis and in intracellular digestion. Continuing study of the metabolic function of leukocytes from these children should provide an opportunity for increased understanding of the metabolic basis for degranulation and intracellular digestion in phagocytic cells.

Specific degranulation of human polymorphonuclear leukocytes

THE human polymorphonuclear leukocyte (PMN) contains two types of cytoplasmic granules, specific or heterophil granules and azurophil granules1. These granules contain enzymes and various non-enzymatic proteins. The specific granule contains alkaline phosphatase and most of the lysozyme of the cell while the azurophil granule contains the acid hydrolases and other enzymes which identify it as a lysosome1. Recent work2 has shown that in the degranulation accompanying phagocytosis the specific or heterophil granules combine with the phagosome first, followed by the azurophil granules. This implies that degranulation may be a complex event with more than one signal involved. If this were true then one might expect that the events should be separable, that is, that one type of granule might be stimulated and the other little affected. Experiments in our laboratories have indicated that only one type of PMN granule responds to phorbol myristate acetate (PMA). Five minutes after application of nanogramme quantities of the agent the cell begins to show large intracellular vacuoles. Granules which demonstrate an alkaline phosphatase reaction disappear in 30 min while those staining for myeloperoxidase remain3. Here we report that PMA causes both a dose and a time dependent release of enzyme activity associated with specific granules while those associated with azurophil granules or lysosomes remain associated with the cell as did a cytoplasmic marker enzyme glucose-6-phosphate dehydro-genase.

The pattern of genetic transmission of the leukocyte defect in fatal granulomatous disease of childhood

The leukocyte-phagocytic function test which was found to be abnormal in boys with fatal granulomatous disease of childhood has been found to be abnormal to an intermediate extent in their mothers. Nine of nine mothers were shown to be abnormal, whereas none of eight fathers and none of five healthy brothers exhibited a defect. 10 of 16 female siblings were abnormal to the same degree as their mothers, as were all three maternal grandmothers available for study. Assuming that this intermediate functional defect represents the heterozygous state, the nine family pedigrees are entirely compatible with the concept that the trait is transmitted on the X-chromosome.A tetrazolium dye-phagocytosis histochemical test was also abnormal in the carrier females and provided independent confirmation of the selection of the female siblings suspected of being carriers for the trait. In addition, this procedure gives indirect evidence that the gene in question is subject to the random inactivation that appears to affect many X-linked genes in mammalian females. The family members were also studied with two of the metabolic assays that have been shown to be abnormal in the cells of affected boys. One assay, the oxidation of the first carbon of glucose-1-(14)C by the isolated leukocytes, was significantly abnormal in the cells of carrier females. The other assay, the oxidation of formate-(14)C by leukocytes of heterozygotes was not significantly different from control values. The practical problem of diagnosing patients would appear to be best solved with a tetrazolium dye procedure, whereas the more subtle abnormality in carrier females is best detected with the leukocyte function test. Improved methods for the function test are being developed.

Effects of Phorbol Myristate Acetate on the Metabolism and Ultrastructure of Neutrophils in Chronic Granulomatous Disease

Previous investigations have demonstrated that phorbol myristate acetate (PMA), the active principle of croton oil, stimulates alterations in normal polymorphonuclear leukocytes (PMN) that resemble closely the changes that develop in the cells after phagocytosis of bacteria. The present study has compared the effects of PMA and heat-killed bacteria on the oxygen uptake, glucose oxidation, nitroblue tetrazolium (NBT) reduction, and ultrastructure of normal neutrophils and PMN from six patients with chronic granulomatous disease (CGD). PMA stimulated oxygen consumption, hexose monophosphate shunt activity, and NBT reduction in normal cells but failed to produce similar effects in CGD neutrophils. However, PMA did induce formation of cytoplasmic vacuoles in the CGD cells similar to those observed in normal neutrophils. The results indicate that PMA is a useful nonparticulate agent for distinguishing between normal and CGD neutrophils and for studying basic mechanisms of phagocytosis in normal and abnormal PMN.

In vitro bactericidal capacity of Blaberus craniifer hemocytes

Abstract Blaberus craniifer hemocytes, maintained in short-term culture, are capable of phagocytosing and destroying Staphylococcus aureus, Staphylococcus albus, Streptococcus faecalis, Serratia marcescens, and Proteus mirabilis. The observed bactericidal activity of the hemocyte suspensions was entirely a function of the phagocytes; the medium, the hemolymph, and cell products elaborated during incubations were not bactericidal. No humoral opsonic factors were required for, or facilitated, bacterial phagocytosis in vitro. Washed hemocyte monolayers bathed by hemolymph-free medium were capable of phagocytosing bacteria. The addition of hemolymph concentrated by ultrafiltration did not increase the bactericidal capacity of the hemocytes. Bacteria opsonized with concentrated hemolymph were not killed more efficiently than were untreated bacteria. A partial blockage of bactericidal capacity was induced by prior exposure of the hemocytes to bacteria or to latex particles. The functional blockade was more complete with bacteria than with latex particles. Pseudomonas aeruginosa, Escherichia coli, Salmonella typhosa, and Diplococcus pneumoniae were phagocytosed but not killed by the hemocytes. This lack of bactericidal activity suggests that roaches may encounter difficulty in eliminating these organisms from the hemocoel. However, deficient bactericidal capacity probably does not entirely correlate with pathogenicity since the known insect pathogens, Staphylococcus albus, Serratia marcescens, and Proteus mirabilis, are killed by the hemocytes. Pathogenicity seems to depend on a complex of factors including bacterial strain, dose received, and intracellular survival of ingested bacteria. A possible connection between the lack of hemocytic bactericidal capacity and the role of roaches as potential disease vectors warrants further investigation.

Metabolic activities in leukocytes of newborn infants

Leukocytes of the newborn infant manifest an activated metabolic state similar in some ways to that associated with phagocytosis. Oxygen consumption, hexose monophosphate pathway activity, and nitroblue tetrazolium (NBT) reduction by newborn leukocytes were significantly increased in the resting state. These cells of the newly born infant showed normal metabolic response to phagocytosis in vitro and normal staphylocidal capacity in the presence of pooled normal human serum, indicating a functional maturity of these leukocytes as phagocytes.

Acute infection of mice with lactate dehydrogenase-elevating virus enhances Fc and complement receptor activity of peritoneal macrophages.

Peritoneal macrophages isolated from Balb/c mice 1 day after infection with lactate dehydrogenase-elevating virus (LDV) exhibited a 5- to 10-fold enhancement of attachment and ingestion of sheep red blood cells coated with immunoglobulin (EAIgG) or immunoglobulin plus complement (EAIgMC). Macrophages isolated from mice 7 days after LDV infection or macrophages infected with LDV in culture were also slightly more active than macrophages from uninfected mice, but the differences were not significant. The results indicate that a specific increase in the number of Fc and C3 receptors on macrophages occurs during the acute phase of infection. This increase correlates with the transient appearance of interferon in acutely infected mice. We postulate that during the acute phase the productive infection of a subpopulation of macrophages that is permissive for LDV results in the synthesis of sufficient interferon to cause activation of the remaining non-permissive macrophages in the animal.

Spectrum of function of neutrophils from carriers of sex-linked chronic granulomatous disease

Bactericidal and metabolic activities were compared for polymorphonuclear leukocytes from normal controls, patients with sex-linked chronic granulomatous disease, five obligate carriers, and six potential carriers of CGD. Bacteria surviving at one hour were quantitated in a standardized assay which employed 1.25 Staphylococcus aureus per neutrophil. Heat-killed bacteria or a chemical agent, phorbol myristate acetate, were used to stimulate increases in utilization of oxygen, oxidation of [1--14C] glucose, and reduction of neotetrazolium chloride by PMN. The results demonstrate that PMN from the individual obligate carriers of CGD have a broad spectrum of functional capabilities. Neutrophils from one obligate carrier performed in the above in vitro tests and others on a par with normal control cells, whereas the PMN of others displayed deficiencies nearly as profound as those of the affected CGD patients. The observations parallel the broad range of phenotypic expression observed in heterozygotic carriers of other sex-linked recessive disorders as a result of random inactivation of the X chromosome. Although predictable from the current concept of random X inactivation, the spectrum has not been previously demonstrated for carriers of sex-linked recessive CGD and thus has important implications for the detection and counseling of carriers of CGD.

Comparative biochemistry of phagocytizing insect hemocytes

Abstract 1. 1. The respiratory increment associated with phagocytosis by mammalian leukocytes is absent in Blaberus craniifer hemocytes. 2. 2. The glycolytic pathway provides energy for particle engulfment in both mammalian leukocytes and B. craniifer phagocytes. 3. 3. Hexose monophosphate pathway activity of roach hemocytes, unlike mammalian cells, is not stimulated by phagocytosis. 4. 4. Nitroblue tetrazolium reduction, characteristic of mammalian phagocytes, is absent in B. craniifer cells. 5. 5. B. craniifer hemocytes utilize antimicrobial systems other than the myeloperoxidase-H 2 O 2 -halide system of mammals.

Chronic granulomatous disease in an adult male: A proposed X-linked defect

A 25-year old patient with chronic granulomatous disease of somewhat unusual history is described. The diagnosis of CGD was based on increased susceptibility to infection, granulomatous appearance of tissues, and diminished bactericidal and metabolic response of leukocytes during phagocytosis: the clinical and cellular features considered phenotypic of CGD. A 16-year-old female sibling had bactericidal and metabolic abnormalities of leukocyte function similar to those of the patients leukocytes. Leukocytes from another sister, 26 years of age, were intermediate in bactericidal capacity. Two populations of leukocytes were identified by a histochemical test of NBT reduction. Both normal and abnormal polymorphonuclear leukocytes were present in the leukocyte population of the two sisters. Leukocytes from the patients mother and maternal grandmother were normal by all methods tested. These findings are taken as evidence of a germ-line mutation in the chromosomal gene causing CGD, with transmission of the genetic defect from the mother to the son.