Anaerobic growth of gonococci does not alter their Opa-mediated interactions with human neutrophils.

Gonococci grown anaerobically (anaerobic gonococci) in the presence of nitrite induce the expression of at least three novel outer membrane proteins (PANs 1 to 3). Although PAN 1 is expressed by gonococci during gonorrhea, the function of the PAN proteins remains unknown. In the absence of serum, gonococci possessing opacity-associated (Opa, formerly PII) outer membrane proteins adhere to, stimulate, and are phagocytically killed by human neutrophils. Gonococci lacking Opa proteins demonstrate none of these activities. We investigated whether the PAN proteins, or any other characteristics of anaerobic gonococci, altered the ability of nonpiliated, Opa+ or Opa- gonococci to adhere to, stimulate, or be phagocytically killed by neutrophils. The expression of Opa4 by strain F62, as determined by its relative mobility on sodium dodecyl sulfate-polyacrylamide gels, appeared to be unaltered by anaerobic growth, as seen previously (V. L. Clark, L. A. Campbell, D. A. Palermo, T. M. Evans, and K. W. Klimpel, Infect Immun. 55:1359-1364, 1987). Anaerobic and aerobic Opa+ gonococci adhered to and stimulated neutrophils to the same extent. Similarly, anaerobic and aerobic Opa- gonococci adhered to and stimulated neutrophils equally poorly. Finally, anaerobic and aerobic Opa+ gonococci were equally sensitive to phagocytic killing by neutrophils, while anaerobic and aerobic Opa- gonococci were equally resistant to killing. Thus, the role of Opa proteins in mediating the interactions of gonococci with human neutrophils appears unaltered by anaerobic growth, and the PAN proteins, or other cryptic properties of anaerobic gonococci, do not seem to modulate or mediate these phenomena.     

Growth of Neisseria gonorrhoeae in CMP-N-acetylneuraminic acid inhibits nonopsonic (opacity-associated outer membrane protein-mediated) interactions with human neutrophils.

Gonococci possessing certain opacity-associated (Opa) outer membrane proteins adhere to and are phagocytosed by human neutrophils in the absence of serum. Recently, it has been shown that serum-sensitive strains of Neisseria gonorrhoeae possessing the appropriate lipooligosaccharide phenotype become serum resistant when grown in the presence of CMP-N-acetylneuraminic acid (CMP-NANA) because of sialylation of their lipooligosaccharide. We investigated whether such sialylation affects nonopsonic (antibody- and complement-independent) interactions of gonococci with human neutrophils in vitro. We grew Opa+ gonococci in the presence of up to 50 micrograms of CMP-NANA per ml, incubated them with neutrophils in vitro, and measured their abilities to adhere to neutrophils, stimulate neutrophil luminol-dependent chemiluminescence (LDCL), and be phagocytically killed by neutrophils. Growth in CMP-NANA dramatically inhibited (in a dose-dependent manner) the ability of Opa+ gonococci to adhere to neutrophils and stimulate neutrophil LDCL. Growth of Opa+ gonococci in 50 micrograms of CMP-NANA per ml appeared to delay, but did not inhibit, their killing by neutrophils. Sialidase treatment of sialylated Opa+ gonococci, i.e., gonococci grown with CMP-NANA, totally restored their abilities to adhere to neutrophils and stimulate neutrophil LDCL. Opa- gonococci grown in the presence of 50 micrograms of CMP-NANA per ml and opsonized with fresh human serum bound to neutrophils only about 30% less efficiently than did Opa- gonococci grown without CMP-NANA and opsonized. The results of our studies show that sialylated Opa+ gonococci have dramatically reduced nonopsonic interactions with neutrophils. Some gonococcal strains may resist killing by human neutrophils in vivo by such a mechanism.     

Gonococci possessing only certain P.II outer membrane proteins interact with human neutrophils.

We investigated the role of the protein II (P.II) family of gonococcal outer membrane proteins in the interaction of seven single P.II variants of Neisseria gonorrhoeae FA1090 with human neutrophils in vitro. The abilities of nonpiliated gonococci to adhere to and be killed by neutrophils and to stimulate luminol-dependent chemiluminescence (CL) depended on the possession of at least one P.II. Gonococci lacking P.II (i.e., P.II-) adhered poorly to and were not killed by neutrophils and induced only minimal CL. Although most P.II-containing (i.e., P.II+) variants adhered to, stimulated, and were readily killed by neutrophils, one variant, containing P.IIa, possessed none of these characteristics; it acted just like a P.II- variant. No correlation was found between the colony opacity phenotype and the interaction of gonococci with neutrophils. Data from CL experiments suggest that the stimulatory effect of P.II was dominant over that of pili; i.e., piliated P.II+ gonococci were much more stimulatory than piliated P.II- gonococci. The results indicate that most but not all P.II proteins mediate, in part or in full, the interaction of N. gonorrhoeae with human neutrophils, including adherence, stimulation of the neutrophil respiratory burst, and phagocytic killing.     

Role of anti-pilus antibodies in host defense against gonococcal infection studied with monoclonal anti-pilus antibodies.

Several monoclonal antibodies directed against gonococcal pili have been used to investigate the potential contribution of anti-pilus antibodies to host defense against gonococcal infection. Included were two antibodies (SM1 and SM2) which reacted with conserved determinants present on pili from all strains tested and others which exhibited antigenic specificity. Immunoblotting experiments revealed that antibodies SM1 and SM2 recognize epitopes on two different peptides derived by CNBr cleavage of alpha-pili from Neisseria gonorrhoeae P9-2. All antibodies used were capable of activating complement, as shown by their ability to bind Clq, and one type-specific antibody was effective in complement-mediated bactericidal killing. Antibodies directed against at least some pilus epitopes may therefore contribute to bactericidal activity during the course of natural infection. The opsonic effect of type-specific antibodies was demonstrated by their ability to stimulate luminol-dependent chemiluminescence of human polymorphonuclear leukocytes and promote phagocytic killing of variant P9-2. Phagocytic killing in the presence of each monoclonal antibody paralleled the increase in chemiluminescence, suggesting that for this variant killing was an inevitable consequence of the interaction of polymorphonuclear leukocytes with gonococci opsonized with anti-pilus antibodies. Antibody-mediated chemiluminescence of polymorphonuclear leukocytes was enhanced in the presence of human complement, and a weak opsonic effect was detected with one of the cross-reacting antibodies (SM1) when this system was used. Although cross-reacting antibody SM1 and type-specific antibody SM13 showed considerable differences in biological properties, they were of the same isotype and bound to native pili on intact gonococci in similar numbers and with similar avidity.     

Phagocytic and bactericidal activity of human neutrophils against two isolates of Group B streptococci Type Ic of differing pathogenicity.

The phagocytic and bactericidal activities of normal adult human neutrophils against 2 strains of Group B streptococci Type Ic of differing pathogenicity were examined. Both isolates were phagocytosed by the neutrophils in the presence of normal and homologous immune serum. However, the highly pathogenicity streptococci were killed less readily in the presence of immune serum than were the streptococci of low pathogenicity in the presence of immune or normal serum. This difference in killing ability was not due to a defect in phagocytosis by the neutrophils, but to a defect in bactericidal activity. The highly pathogenic streptococci were not killed in the presence of normal serum, but were readily phagocytosed by the neutrophils, in which they accumulated and eventually caused their destruction. The streptococci of low pathogenicity, however, were killed equally as well in the absence of specific antibody as in its presence. It is suggested that an in vitro assessment of neutrophil function against streptococci of differing pathogenicity for mice may provide a useful method by which the pathogenicity of streptococci for man can be compared.     

Differential response of human monocytes to Neisseria gonorrhoeae variants expressing pili and opacity proteins.

Experiments in vitro suggest that Neisseria gonorrhoeae surface variation plays a key role in gonococcal pathogenesis by providing the appropriate bacterial phenotypes to go through different stages of the infection. Here we report on the effects of phase and antigen variation of two major gonococcal adhesins, pili and opacity (Opa) outer membrane proteins, on the interaction of the gonococci with human monocytes. Using a set of recombinants of gonococcus strain MS11 that each express 1 of 11 genetically defined Opa proteins or a defined type of pilus, we found that both Opa proteins and pili promote bacterial phagocytosis by monocytes in the absence of serum and that this feature largely depends on the type of protein that is expressed. One of the Opa proteins (Opa[50]) strongly promoted uptake by monocytes but had little effect on the interaction with polymorphonuclear leukocytes under the conditions employed. Similarly, the phagocytosis-promoting effect of the pili was much more pronounced in monocytes than in neutrophils (4-fold versus 22-fold stimulation of uptake, respectively). Only a subpopulation of both types of phagocytes actively ingested bacteria, as has been observed during natural infections. Measurements of luminol-enhanced chemiluminescence demonstrated that phagocytosis of opaque but not piliated gonococci was accompanied by an increase in oxygen-reactive metabolites. These findings demonstrate that the monocyte response towards gonococci is highly dependent on the bacterial phenotype and differs from the neutrophil response. This diversity in bacterial behavior towards various types of human phagocytic cells underlines the biological impact of gonococcal surface variation and may explain previous contradictory results on this subject.     

Opsonins in normal mouse serum for the phagocytic killing of Proteus mirabilis by murine neutrophils.

An assay of phagocytic killing by murine neutrophils in homologous serum was used to determine the nature of the opsonins in normal mouse serum for phagocytic killing of Proteus mirabilis. Leucocytes from the peritoneal cavities of mice given an intraperitoneal inoculation of brain-heart infusion broth 3 h previously, phagocytosed and killed P. mirabilis in a 2-h assay in the presence of 10% serum from normal mice. The serum factors supporting phagocytic killing were heat-labile (50 degrees C or 56 degrees C for 30 min) and could be absorbed at 37 degrees C but not 4 degrees C by three different species of Gram-negative bacteria. The tested species of Gram-positive bacterium did not absorb the activity. At the end of the assays, greater than 90% of leucocyte-associated bacteria were associated with neutrophils. Leucocytes from unstimulated peritoneal cavities (less than I% neutrophils) did not kill bacteria in this assay, in contrast to leucocyte suspensions containing up to 98% neutrophils. These findings indicated that the phagocytic killing of P. mirabilis in this assay was mediated by neutrophils, and that complement fixation by the alternative pathway provided necessary opsonins in normal mouse serum.     

Opsonins in normal mouse serum for the phagocytic killing of Proteus mirabilis by murine neutrophils

An assay of phagocytic killing by murine neutrophils in homologous serum was used to determine the nature of the opsonins in normal mouse serum for phagocytic killing of Proteus mirabilis. Leucocytes from the peritoneal cavities of mice given an intraperitoneal inoculation of brain-heart infusion broth 3 h previously, phagocytosed and killed P. mirabilis in a 2-h assay in the presence of 10% serum from normal mice. The serum factors supporting phagocytic killing were heat-labile (50 degrees C or 56 degrees C for 30 min) and could be absorbed at 37 degrees C but not 4 degrees C by three different species of Gram-negative bacteria. The tested species of Gram-positive bacterium did not absorb the activity. At the end of the assays, greater than 90% of leucocyte-associated bacteria were associated with neutrophils. Leucocytes from unstimulated peritoneal cavities (less than I% neutrophils) did not kill bacteria in this assay, in contrast to leucocyte suspensions containing up to 98% neutrophils. These findings indicated that the phagocytic killing of P. mirabilis in this assay was mediated by neutrophils, and that complement fixation by the alternative pathway provided necessary opsonins in normal mouse serum.     

Interaction of Campylobacter pyloridis with human immune defence mechanisms

The in-vitro susceptibility to host immune defence mechanisms of Campylobacter pyloridis was investigated. C. pyloridis was sensitive to antibody-dependent complement-mediated bactericidal activity of serum. The bacteria were phagocytosed and efficiently killed by polymorphonuclear neutrophils in the presence of serum opsonins. Serum opsonin depletion studies indicated that an intact classical complement pathway was required for optimal phagocytic killing.     

Stimulation of human neutrophil oxidative metabolism by nonopsonized Neisseria gonorrhoeae.

Nonopsonized gonococci possessing opacity-associated (Opa; previously PII) outer membrane proteins stimulate neutrophils to undergo a vigorous oxidative response when measured by luminol-dependent chemiluminescence (LDCL). In these studies, we characterized the mechanism of this stimulation. No gonococci that we tested induced measurable release of neutrophil superoxide anion (O2-) or hydrogen peroxide (H2O2) as measured by reduction of cytochrome c or the oxidation of scopoletin, respectively. Neutrophils pretreated with gonococci and then exposed to phorbol myristate acetate, the chemotactic peptide formylmethionylleucylphenylalanine, or opsonized zymosan released levels of neutrophil O2- and H2O2 comparable to controls, indicating that gonococci were not preventing or inhibiting neutrophil O2- or H2O2 release. To ascertain a possible explanation for these seemingly contradictory observations (i.e., induction of LDCL, but no release of O2- or H2O2), we further characterized the ability of Opa+ gonococci to stimulate LDCL. By using 1 mM azide and 4 U of horseradish peroxidase to monitor extracellular LDCL selectively and 2,000 U of catalase to monitor intracellular LDCL selectively, we determined that greater than 80% of total gonococcus-induced neutrophil LDCL occurred intracellularly. In addition, neutrophils stimulated with Opa+ gonococci showed a marked increase in O2 uptake and hexose monophosphate shunt activity. We conclude that Neisseria gonorrhoeae induces neutrophil oxidative metabolism without causing release of detectable amounts of reactive oxygen intermediates into the surrounding milieu. The gonococcus apparently directs oxidase assembly and activity to the phagolysosomal membrane. This could be a mechanism by which extracellular gonococci persist for extended periods in vivo in the presence of high concentrations of neutrophils.     

The effects of capsular polysaccharide on the capacity of serum to support the killing of type 3 Streptococcus pneumoniae by human neutrophils

To assess the effects of purified capsular polysaccharide from type 3 S. pneumoniae (PPS-3) on the capacity of serum to support pneumococcal killing by human neutrophils, varying concentrations of PPS-3 (0.01-100 micrograms/ml) were incubated (4 degrees C) with pooled serum for 30 min, and the resulting preparation, termed absorbed serum, was evaluated in bactericidal and phagocytic assays. The ability of serum to promote the killing of type 3 S. pneumoniae was significantly reduced at greater than or equal to 1.0 micrograms/ml PPS-3; similarly, serum absorbed with greater than or equal to 1.0 micrograms/ml PPS-2 failed to support the killing of type 2 S. penumoniae. However, the impact of these penumococcal polysaccharides was serotype specific, since the killing of type 3 S. pneumoniae was not impaired in serum absorbed with PPS-2, and the killing of type 2 S. pneumoniae was not attenuated in serum treated with PPS-3. The failure of serum absorbed with PPS-3 to promote the killing of type 3 S. pneumoniae was primarily a consequence of impaired bacterial ingestion; the reduction in phagocytosis was associated with parallel changes in superoxide anion release. The defects in phagocytosis and killing induced by PPS-3 were not associated with alterations in classical or alternative complement pathway activity; however, they were highly correlated with changes in serum antibody levels to the polysaccharide. The addition of polyclonal human IgG to serum treated with PPS-3 did not restore its capacity to support killing; however, preopsonization of the bacterium with the IgG preparation did partially correct the deficiency. Finally, neutrophils preincubated with serum containing greater than or equal to 10.0 micrograms/ml PPS-3 exhibited an impaired bactericidal activity against type 3 S. pneumoniae. Thus, these studies demonstrate that the presence of PPS-3 decreases the capacity of serum to support the killing of type 3 S. pneumoniae by absorbing immunoglobulins and by generating factors that interfere with neutrophil function.     

Cefdinir-induced modification of the susceptibility of bacteria to the antibacterial activity of human serum and polymorphonuclear neutrophils

The effect of serum on the bactericidal activity of cefdinir, and the ability of the antibiotic to modify the interaction of bacteria with human polymorphonuclear neutrophils were assessed. In the presence of antibiotic, serum-resistantEscherichia coli were sensitised to the bactericidal activity of normal human serum. Cefdinir enhanced opsonophagocytic killing ofEscherichia coli andStaphylococcus aureus at suprainhibitory concentrations. Significant potentiation of killing occurred with the combination of inhibitory concentrations of cefdinir, neutrophils and sub-optimal levels of serum opsonins. Pre-exposure ofEscherichia coli, but notStaphylococcus aureus, to cefdinir enhanced phagocytic uptake and killing of the antibiotic-damaged bacteria. These results indicate that cefdinir-mediated phenotypic modification ofEscherichia coli renders the bacteria susceptible to serum antibacterial activity and phagocytic uptake and intracellular killing.     

An assessment of the factors contributing to the killing of type 3 Streptococcus pneumoniae by human polymorphonuclear leukocytes in vitro

To characterize the factors that contribute to the killing of type 3 S. pneumoniae, human neutrophils were obtained from healthy donors and incubated with viable organisms. In contrast to prior observations with other pneumococcal serotypes, killing was not detected when 10(6) colony forming units (cfu) were incubated at 37 degrees C for 2-4 hours with 10(6) neutrophils in the presence of 20-80% fresh autologous serum; further, pneumococcidal activity was not found when preopsonized bacteria and primed neutrophils were employed in the standard assay. However, when the bacterium to cell ratio was reduced to 1:100 and 1:1000, microbicidal action was detected; a 10-fold reduction in the number of viable bacteria was observed when 2 x 10(3) cfu were incubated with 2 x 10(6) neutrophils and 80% autologous serum at 37 degrees C for 4 hours. To assess the effects of serum factors on killing, bactericidal assays were performed in the presence of normal human serum (NHS), heat-inactivated human serum (HIHS) and absorbed human serum (AHS); heating reduced and absorption eliminated the capacity of serum to support killing. Studies performed with mutanolysin, an enzyme that lyses type 3 pneumococci, demonstrated that the effects of HIHS and AHS on bactericidal activity were highly correlated with alterations in the ability of the sera to support phagocytosis. Studies of neutrophil activation revealed changes in the production of superoxide anion that correlated well with phagocytosis and killing; however, the results of assays of leukotriene B4 generation and degranulation (beta-glucuronidase and lactoferrin release) were more variable. In mixing experiments, the capacity of HIHS to support killing was normalized with NHS; however, the ability of AHS to promote killing was not restored with HIHS or NHS. Thus, these studies demonstrate the relatively limited capacity of human serum to support the killing of type 3 pneumococci, and they emphasize the importance of killing assays in assessing interactions between the bacterium and neutrophils.     

125I-labeled peptide mapping of some heat-modifiable proteins of the gonococcal outer membrane

Gonococci from opaque colonies have cell wall outer membrane proteins that are lacking from organisms which form transparent colonies. These "colony opacity-associated" proteins are among a group of "minor" proteins that exhibit heat modification of their apparent subunit molecular sizes, are easily extracted by deoxycholate, have apparent subunit molecular weights varying from 24,000 to 29,000 and are exposed on the surfaces of gonococci. Other minor proteins found on gonococci are the "leukocyte association proteins," whose presence correlates with reactivities of gonococci with human neutrophils. Several of the colony opacity-associated proteins and leukocyte association proteins were subjected to 125I-peptide mapping of protein bands separated by polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. The structural similarities and differences among these heat-modifiable surface proteins were studied, as well as their similarities with the major protein of the gonococcal outer membrane. A relatively high apparent degree of structural homology is found among the heat-modifiable proteins from different strains of opaque colony gonococcal forms. There is also some apparent structural homology for 125I-peptides of heat-modifiable versus major proteins of the gonococcal outer membrane.     

Luminol-amplified chemiluminescence: a sensitive method for detecting the carrier state in chronic granulomatous disease.

Patients with chronic granulomatous disease have a marked defect in neutrophil oxidative metabolism and microbicidal activity. Asymptomatic mothers of males with the disease can usually be identified as heterozygous carriers by intermediate leukocyte function. Most mothers of females with the disease, however, have normal leukocyte function, and the pattern of genetic transmission in these families has been difficult to establish. Of 14 mothers of males and females with chronic granulomatous disease, 10 had been found previously to have intermediate values for neutrophil bactericidal activity, oxygen consumption, hexose monophosphate shunt activity, and Nitro Blue Tetrazolium reduction, and 4 had normal in viro leukocyte function. In the present study, 4 of these 14 mothers had normal neutrophil bactericidal activity, 3 had normal zymosan-stimulated chemiluminescence, but none had normal luminol-amplified zymosan-stimulated chemiluminescence. The presence of luminol (5-amino-2,3-dehydro-1,4-phthalazinedione) in the phagocytic mixtures markedly increased the sensitivity of the assay, permitting detection of subtle defects in leukocyte oxidative metabolism in three previously unidentifiable carriers of the disease. Thus, luminol-amplified chemiluminescence appears to be one of the most sensitive methods available for detection of chronic granulomatous disease heterozygotes; the simplicity and reproducibility of the microtechnique permit evaluation of leukocyte function in infants and newborns.     

Effect of polymorphonuclear neutrophils on serum bactericidal activity againstStreptococcus pneumoniae after amoxicillin administration

The effect of phagocytic killing on serum bactericidal activity againstStreptococcus pneumoniae was investigated 0, 1.5, 8 and 12 h after a single 875 mg oral dose of amoxicillin in healthy adults. Killing curves were determined with polymorphonuclear neutrophils (PMN), serum or PMN plus serum. Global killing (i. e. intracellular and extracellular killing) over 3 h of incubation was expressed as the area under the killing curve (AUKC; log cfu × h/ml). Amoxicillin did not affect the activity of PMN alone. For serum alone, the AUKC of post-administration samples (with supra-inhibitory amoxicillin concentrations) was significantly lower than in baseline samples. For serum plus PMN, significant bactericidal activity of serum was still found in samples after antibiotic concentrations had reached sub-inhibitory levels.     

Evaluation of a fluorochrome assay for assessing the bactericidal activity of neutrophils in human phagocyte dysfunctions

We evaluated a method for the assessment of the phagocytic and bactericidal activity of human peripheral neutrophils against Staphylococcus aureus Cowan I, which is a modified version of the acridine orange staining technique originally described by Smith and Rommel (1977). The modification consisted of the use of free leukocyte suspensions rather than coverglass adhered leukocytes in order to avoid two main problems: the inefficient neutrophil adherence to glass that can be observed in specimens from patients with certain functional phagocyte defects, and the risk of selecting among neutrophils. An additional advantage of the modified procedure is that it permits a uniform bacteria: phagocyte ratio in different cell samples. The method was tested on 25 healthy adults and on four children with functional phagocytic defects (chronic granulomatous disease of infancy, Chediak-Higashi syndrome, and Rothmund-Thomson syndrome associated to persistent neutropenia and low chemotactic response). The neutrophils of all four patients showed a low bactericidal activity, with percent values of intracellular killed bacteria below the mean +/- 2 SD range observed in the healthy population at all incubation times tested (5, 15 and 30 min). A significant reduction in phagocytosis index and in % killed unopsonized S. aureus was observed in relation to bacteria treated with a pool of normal human serum. These results demonstrate the high sensitivity of the method, which could be used to determine intrinsic and extrinsic functional alterations in human neutrophils.     

Opsonization of Listeria monocytogenes type 4b by human adult and newborn sera

We studied the requirements for opsonization of Listeria monocytogenes type 4b with chemiluminescence and bactericidal assays and electron microscopy. Preopsonization with 3% adult serum had good opsonic activity (27,300 +/- 11,000 [standard deviation] counts, chemiluminescence assay), while 3% newborn cord serum was not opsonically active (820 +/- 530 counts, P less than 0.001 versus adult serum). In addition, organisms opsonized with cord serum were not killed (0% bacterial killing) and were less frequently visualized intracellularly on electron micrographs (0 to 4 bacteria per cell) than organisms opsonized with adult serum (70% killing and 10 to 20 bacteria per cell). Opsonic requirements for L. monocytogenes type 4b at low concentrations of serum were studied in detail with Sepharose-protein A-treated adult serum to obtain immunoglobulin G (IgG) and IgM fractions and zymosan-absorbed and C4 inactivator-treated serum to obtain alternative and classical complement pathway-deficient sera, respectively. In the presence of complement, IgM was opsonically active (59% of control) while IgG was not (6% of control). In addition, classical complement activity was required for efficient opsonization (greater than 100% of control) while the alternative complement pathway was unnecessary (3% of control). Since IgM is absent and classical complement activity is low in neonatal serum and at the common sites of neonatal Listeria infection, the requirement for IgM and classical complement activity for efficient opsonization of L. monocytogenes type 4b at low serum concentrations may be a factor in the pathogenesis of neonatal disease.     

Role of Peroxide in Phagocytic Killing of Pneumococci

Two mutants of a pneumococcus type I with diminished peroxide production were selected from a population of nitrosoguanidine-treated cells. White cells of normal patients killed the mutant pneumococci as well as the otherwise isogenic wild-type strain. In patients studied with chronic granulomatous disease, however, the peroxide-poor strain was killed far less well than the wild type. These studies indicate that the removal of a peroxide-generating system in the phagocytic vacuole specifically brings forth the killing defect in chronic granulomatous disease.     

A rapid, inexpensive and easily quantified assay for phagocytosis and microbicidal activity of macrophages and neutrophils.

The objective of the present study was to develop a technique to quantitate the phagocytic and intracellular microbicidal activity in different populations of phagocytes, i.e. neutrophils and macrophages. Elicited peritoneal neutrophils and macrophages as well as alveolar macrophages and adherent splenic macrophages were used as representative cell types. The method to assess intracellular killing was based upon dye uptake and concentration by a dead micro-organism; methylene blue was used as the indicator dye and the test organism was Saccharomyces cerevisiae. Phagocytosis was measured by counting, microscopically, the number of ingested yeast (Saccharomyces cerevisiae) within the neutrophils or macrophages. A number of killed yeast, i.e., those which took up the dye, were readily visualized. The temporal pattern of phagocytosis and killing was determined concurrently. Splenic macrophages demonstrated the slowest phagocytic activity whereas neutrophils and alveolar macrophages manifested a similar phagocytic activity. Peritoneal macrophages exhibited a continuous increase in activity throughout the test period. Microbicidal activity was similar for all 4 cells types. The new technique for measuring phagocytosis and killing provides a rapid, inexpensive and easily quantified assay for assessing discrete phagocytic cell functions.