Outer membrane mutants of Salmonella typhimurium LT2 have lipopolysaccharide-dependent resistance to the bactericidal activity of anaerobic human neutrophils

The capacity of neutrophil polymorphonuclear granulocytes (PMNs) to phagocytize bacteria under anaerobic as well as aerobic conditions afforded the opportunity to compare the bactericidal activities of oxygen-independent and oxygen-dependent antimicrobial mechanisms in human PMNs challenged with Salmonella typhimurium LT2 and its lipopolysaccharide mutants (outer membrane mutants). Anaerobic human PMNs challenged with either opsonized LT2 or serum-treated zymosan failed to produce detectable superoxide anion (O2-) or to reduce nitroblue tetrazolium, although aerobic PMNs readily produced O2- in response to such challenge. Anaerobic PMNs killed these bacteria in an ordered fashion that appeared to be dependent on their lipopolysaccharide chemotype. As the carbohydrate content of the mutant lipopolysaccharide decreased, the bacteria became less resistant to the oxygen-independent bactericidal activity. The results resembled the ordered resistance to oxygen-independent killing observed with LT2 and its mutants in PMN-free systems with PMN granule proteins. Studies on the kinetics of killing showed these to be less rapid in anaerobic as compared with aerobic conditions. Opsonization increased the rate of phagocytosis, but such factors as opsonization and the rate of phagocytosis did not appear to affect intraleukocytic bactericidal capacity in that the resultant proportion of bacteria remaining viable after ingestion was similar regardless of which serum was used (normal serum, C6-deficient serum, C8-deficient serum, or no serum at all). The results are consistent with an active and substantial participation by oxygen-independent systems in the antimicrobial effects of neutrophils.     

Anaerobiosis increases resistance of Neisseria gonorrhoeae to O2-independent antimicrobial proteins from human polymorphonuclear granulocytes.

We investigated the in vitro resistance of Neisseria gonorrhoeae FA19 to the O2-independent antimicrobial systems of human polymorphonuclear leukocytes. Acid extracts of polymorphonuclear leukocyte granules (crude granule extracts) and a purified granule protein (57 kilodaltons) were, at low concentrations, bactericidal for gonococci under aerobic conditions that permitted growth. However, they were less effective under anaerobic conditions that imposed bacteriostasis. We found that adding sodium nitrite to reduced growth media permitted the growth of strain FA19 in an anaerobic environment. Under these conditions with nitrite, anaerobic cultures of strain FA19 were no more resistant to the crude granule extract and the 57-kilodalton protein than aerobic cultures. In contrast, Salmonella typhimurium SL-1004, a facultative anaerobe, was readily killed by both the crude granule extract and the 57-kilodalton antimicrobial protein regardless of the presence or absence of free molecular oxygen. This is the first demonstration that an isolated antimicrobial protein from polymorphonuclear leukocyte granules is active against bacteria under anaerobic conditions. Our results also indicated that the efficacy of human polymorphonuclear leukocyte O2-independent killing of N. gonorrhoeae may, in part, be inhibited by bacteriostatic conditions imposed by hypoxia.     

Oxygen-independent antimicrobial action in sphingosine-treated neutrophils

Sphingosine is reported to inhibit the oxidative burst and superoxide anion production of human polymorphonuclear neutrophils (PMN) phagocytosing in atmospheric oxygen (Wilson et al., 1986). We have confirmed its effect on superoxide production and examined the antimicrobial phagocytic capacity of PMN treated with sphingosine, comparing them with PMN, untreated but phagocytosing either under anaerobic conditions or in atmospheric oxygen. Sphingosine just like anaerobiosis partially inhibited, but did not eliminate, the bactericidal activity of PMN when compared to non-treated aerobic cells. In fact, sphingosine-treated PMN mimicked killing of Staphylococcus aureus (S. aureus) and Serratia marcescens (S. marcescens) due to anaerobic PMN. Moreover, our results with Salmonella typhimurium and sphingosine-treated cells duplicated results this laboratory published previously about comparative killing of Salmonella in aerobic versus anaerobic neutrophils. In these studies sphingosine-treated PMN took up bacteria as avidly as untreated PMN and retained their viability, as assessed by trypan blue exclusion. While sphingosine should not be completely substituted for anaerobic studies, it is a convenient screening reagent for the study of non-oxidative killing mechanisms of PMN. Results achieved with anaerobic and with sphingosine-treated cells suggest that O2-independent antimicrobial action is substantially more powerful than has been generally acknowledged.     

Attachment and ingestion of gonococci human neutrophils.

Previous studies have indirectly shown that type 1 gonococci are more resistant to phagocytosis by human neutrophils (PMN) than type 3 gonococci. Using phase contrast, fluorescent, and light microscopy, we directly quantitated PMN-gonococcal interaction, with emphasis on separating ingestion from attachment. PMN monolayers were incubated on slides with type 1 or type 3 gonococcal fluorescent antibody (FA). After methanol fixation, the FA-stained gonococci associated with PMN were cointed. Since the live PMN excludes FA, the FA-stained gonococci represent only extracellular gonococci. Methylene blue was then added to the smae slide to stain both ingested and surface attached gonococci. Using these methods, intracellular and extracellular cell-associated gonococci were quantitated under varying conditions. The numbers of methylene blue-stained cell-associated gonococci that were ingested were: with normal serum, 3.7 plus or minus 4.1 per cent for type 1 and 56.2 plus or minus 3.7 percent for type 3 (P smaller than 0.001); with heat-inactivated serum, 1.0 plus or minus 3.0 per cent for type 1 and 52.6 plus or minus 3.7 per cent for type 3 (P smaller than 0.001); with higher-titer anti-gonococcal antibody serum, 4.8 plus or minus 4.3 percent for type 1 and 64.0 plus or minus 1.6 per cent for type 3 (P smaller than 0.001). Thus, most type 3 organisms were ingested, but most type 1 gonococci were bound on the PMN surface.     

O antigen and lipid A phosphoryl groups in resistance of Salmonella typhimurium LT-2 to nonoxidative killing in human polymorphonuclear neutrophils.

We have compared the intraleukocytic survival of isogenic strains of Salmonella typhimurium, whose outer membrane lipopolysaccharide differed in O antigen and lipid A composition and whose susceptibility to nonoxidative antimicrobial granule proteins of human polymorphonuclear neutrophilis (PMN) could be established. We found that the order of resistance to the bactericidal activity of intact PMN of the three bacterial strains utilized closely resembled their ordered resistance to the purified human cationic antimicrobial 57,000-dalton protein (CAP57). LT-2, a smooth wild-type strain, was far more resistant than SH9178, its rough (Rb LPS) mutant. It was most significant that SH7426, a polymyxin B-resistant pmrA mutant of SH9178, not only was substantially more resistant to CAP57 and to intraphagocytic killing than SH9178 but also came close to being as resistant as LT-2. These experiments confirm earlier work that showed the importance of the glycosyl groups of O antigens of S. typhimurium for their resistance to O2-independent antimicrobial phagocytosis by PMN. The surprising result was that a rough strain, very susceptible to bactericide, became substantially more resistant when a mutation led to its lipid A phosphoryl groups being 100% substituted with amino pentoses. Yet unresolved is whether the protection is due to the loss of negative charges on the lipid A, the substitution of sugar molecules in vulnerable loci in the outer membrane, or both.     

Direct and alternative antimicrobial mechanisms of neutrophil-derived granule proteins

Polymorphonuclear leukocytes (PMN) contribute to bacterial clearance by uptake and intracellular killing of microbes. However, antimicrobial polypeptides are released extracellularly where they are enweaved in a chromatin web that traps and eliminates bacteria. In addition, PMN-derived antimicrobial polypeptides direct monocytes and macrophages to the site of infection and activate their antimicrobial armor. Increased expression of Fcγ receptors as well as opsonization of bacteria by PMN granule proteins support bacterial uptake by macrophages. PMN granule proteins also increase intracellular reactive oxygen species formation in macrophages. Finally, apoptotic PMN transfer parts of their antimicrobial peptides to macrophages, hence increasing killing of intracellular bacteria. Understanding mechanisms by which PMN granule proteins stimulate antimicrobial mechanisms in macrophages may open novel strategies in fighting bacterial infections.     

Interactions of Neisseria gonorrhoeae with adherent polymorphonuclear leukocytes

Neisseria gonorrhoeae causes severe exudative urethritis. The exudates from infected individuals contain large numbers of polymorphonuclear leukocytes (PMN) with ingested gonococci. The fate of N. gonorrhoeae within PMN has been a topic of debate for years. In this study, we examined the interactions of N. gonorrhoeae with PMN adherent to surfaces as a system that better models events during clinical disease. Using chemiluminescence to measure reactive oxygen species (ROS), we found that N. gonorrhoeae stimulated PMN to produce a respiratory burst. Different kinetics were seen when PMN were stimulated with opsonized zymosan particles. In addition, ROS were produced predominantly inside the PMN in response to gonococci. Laser scanning confocal microscopy and transmission electron microscopy showed that N. gonorrhoeae rapidly associated with PMN under these experimental conditions and was internalized. Some gonococci were cleared in the first 30 to 60 min after phagocytosis, but a majority of the population persisted for 6 h after phagocytosis. Quantification of viable organisms showed that a significant portion of the population resisted killing. The viability of this subpopulation remained unchanged for 2 h after phagocytosis. A significant increase of viable gonococci from 1 to 6 h was also observed, suggesting intracellular replication. Four different N. gonorrhoeae strains demonstrated the same capacity to resist PMN-mediated killing, whereas Escherichia coli was rapidly killed by PMN under the same conditions. Taken together, these findings suggest that a subpopulation of N. gonorrhoeae resists killing and replicates within PMN phagosomes in spite of NADPH oxidase activation.     

Bacterial iron enhances oxygen radical-mediated killing of Staphylococcus aureus by phagocytes.

It has been shown that increasing bacterial iron concentration enhances killing by hydrogen peroxide (H2O2) but not by polymorphonuclear granulocytes (PMN). It is possible that owing to the multiple bactericidal mechanisms of the PMN, differences in the killing rate of iron-loaded bacteria and control bacteria are obscured. We decided, therefore, to compare the killing of iron-loaded bacteria with that of control bacteria using human monocytes (MN), PMN, and PMN-derived cytoplasts. Incubation of Staphylococcus aureus with increasing concentrations of ferrous ammonium sulfate (0 to 1,000 microM) progressively increased the iron content in the bacteria (from 0.01 to 0.24 mumol of iron per 10(9) bacteria). Iron loading of the bacteria markedly increased their susceptibility to killing by H2O2. After 1 h of incubation with 1 mM H2O2, 95 +/- 2% of the iron-loaded bacteria were killed compared with 18 +/- 4% of the control bacteria (P less than 0.0001). Iron loading of bacteria did not alter their susceptibility to killing by human PMN. However, iron-loaded bacteria were more susceptible to killing by MN (after 12 min of incubation, 81 +/- 2 versus 74 +/- 2% killing; P less than 0.008) and to killing by PMN-derived cytoplasts (after 60 min of incubation, 52 +/- 8 versus 33 +/- 5%; P = 0.003) than the controls. Moreover, iron loading enhanced luminol-mediated chemiluminescence of MN, PMN, and PMN-derived cytoplasts. The hydroxyl radical scavenger thiourea inhibited H2O2-mediated killing of iron-loaded staphylococci as well as luminol-mediated chemiluminescence. These results suggest that alterations in intrinsic iron content increase killing of staphylococci by H2O2, MN, and PMN-derived cytoplasts by a free radical-mediated mechanism.     

Neisseria gonorrhoeae catalase is not required for experimental genital tract infection despite the induction of a localized neutrophil response

Neisseria gonorrhoeae produces several antioxidant defenses, including high levels of catalase, which may facilitate the persistence during an inflammatory response via neutralization of H2O2 produced by phagocytes. In vivo testing of the role of catalase in gonococcal survival is critical since several physiological factors impact interactions between N. gonorrhoeae and polymorphonuclear leukocytes (PMNs). Here we assessed the importance of gonococcal catalase in a surrogate model of female genital tract infection. Female BALB/c mice were treated with 17-beta estradiol to promote susceptibility to N. gonorrhoeae and inoculated intravaginally with wild-type gonococci or a catalase (kat) deletion mutant. A localized PMN influx occurred in an average of 43 and 81% of mice infected with wild-type or kat mutant gonococci, respectively, and PMNs associated with numerous wild-type or catalase-deficient bacteria were observed in vaginal smears. The combined results of six experiments showed a significant difference in the number of days wild-type bacteria were recovered compared to the catalase-deficient gonococci. However, there was much variability between experiments, and we found no correlation between PMN influx, colonization load, and clearance of wild-type or kat mutant bacteria. Estradiol treatment did not impair bacterial uptake, the luminol-dependent chemiluminescence response, or the killing capacity of isolated murine PMNs against N. gonorrhoeae or Staphylococcus aureus. Our data suggest N. gonorrhoeae is not significantly challenged by H2O2 produced by PMNs in the murine lower genital tract; alternatively, redundant defense mechanisms may protect the gonococcus from reactive oxygen species during infection.     

Innate immunity against Granulibacter bethesdensis, an emerging gram-negative bacterial pathogen

Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G. bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components (e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived in human serum concentrations of ≥90%, indicating a high degree of serum resistance. Consistent with the clinical host tropism of G. bethesdensis, CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, reduced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethesdensis was sensitive to H(2)O(2) but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance to O(2)-independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the relative inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms likely play an important role in G. bethesdensis pathogenesis.     

Effects of anaerobiosis and aerobiosis on interactions of human polymorphonuclear leukocytes with the dental plaque bacteria Streptococcus mutans, Capnocytophaga ochracea, and Bacteroides gingivalis.

Human polymorphonuclear leukocytes (PMN) were able to generate and release superoxide anions upon stimulation of Streptococcus mutans, Bacteroides gingivalis, and Capnocytophaga ochracea when incubated aerobically but not when incubated anaerobically. Lysozyme release and phagocytosis by PMN were independent of oxygen, and no difference between PMN incubated aerobically or anaerobically was observed (PMN stimulated by B. gingivalis released 7.6% total lysozyme when aerobic and 6.9% when anaerobic). There were variations in lysozyme release and phagocytosis for the three organisms, particularly for phagocytosis. B. gingivalis and C. ochracea yielded lower phagocytosis values than those for S. mutans, e.g., at 1 h 67% of the initial inoculum of S. mutans was phagocytosed (versus only 40% for B. gingivalis). Transmission electron microscopy showed that both S. mutans and B. gingivalis were internalized into classical phagolysosomes. In contrast, C. ochracea showed two forms of internalization; C. ochracea either formed a classical phagolysosome or was tightly bound in the cytoplasm with no surrounding cell membrane. Intracellular killing of S. mutans and C. ochracea was unaffected by anaerobiosis, but killing of C. ochracea was much lower than that of S. mutans (1 x 10(7) to 2 x 10(7) bacteria killed compared with 5.1 x 10(7) bacteria killed at 6 h). In contrast, a greater number of B. gingivalis was killed in the presence of oxygen (5.3 x 10(7) bacteria were killed when aerobically incubated and 1.9 x 10(7) bacteria were killed when anaerobically incubated). These results suggest that the ability to survive anaerobically may enable some bacteria to evade PMN killing; however, abnormal phagocytosis may represent a more efficient way to evade both oxygen-dependent and -independent killing mechanisms, leading to enhanced virulence of the organism.     

Interaction and intracellular killing of Candida albicans blastospores by human polymorphonuclear leucocytes, monocytes and monocyte-derived macrophages in aerobic and anaerobic conditions.

Polymorphonuclear leucocytes (PMN), monocytes and monocyte-derived macrophages were capable of interacting with opsonized C. albicans in both aerobic and anaerobic conditions. Superoxide anion release by these cells was inhibited in anaerobic conditions while lysozyme release and phagocytosis were equally efficient in both aerobic and anaerobic conditions. All cell types tested were capable of intracellular killing of C. albicans and this appeared to be maximum at 6 h for monocytes and macrophages and 24 h for PMN. Monocytes killed the lowest number of organisms, 1 x 10(6), and the killing was similar for aerobic and anaerobic conditions. In contrast, PMN and macrophages demonstrated greater killing of C. albicans in aerobic conditions compared with anaerobic conditions; PMN killed 1.9 x 10(6) organisms and macrophages 3 x 10(6) when incubated anaerobically. Inhibitors of oxygen metabolism decreased intracellular killing of C. albicans by macrophages and PMN in aerobic but not anaerobic conditions. The oxygen reaction products involved in the killing of C. albicans appeared to be different however: macrophage killing was decreased by superoxide anion and hydrogen peroxide inhibitors. PMN killing was decreased by superoxide anion, hydrogen peroxide, hypochlorous acid and hydroxyl radical inhibitors. The present study shows that although monocytes, macrophages and PMN function similarly in their interaction with C. albicans, they appear to use different oxygen reactive products for the intracellular killing of C. albicans.     

Relationship between extracellular stimulation of intracellular killing and oxygen-dependent microbicidal systems of monocytes.

Human monocytes require serum components immunoglobulin G, C3/C3b, and B/Bb to exert optimal microbicidal action against ingested microorganisms. The present study was performed to find out whether these factors act by enhancing oxygen-dependent antimicrobial mechanisms. Serum enhanced oxygen consumption and superoxide production by monocytes before phagocytosis, but did not further increase these processes in monocytes that had recently ingested bacteria. Furthermore, serum did not boost iodination during intracellular killing by monocytes. Phorbol myristate acetate, N-formyl-methyonyl-leucyl-phenylalanine, concanavalin A, and concanavalin A-Sephadex all stimulated the conversion of O2 to H2O2 by monocytes, but only concanavalin A augmented intracellular killing. Reactive oxygen intermediates generated by cell-free enzymes (xanthine oxidase or glucose oxidase) in concentrations comparable to those accumulating extracellularly during incubation of monocytes containing bacteria with phorbol myristate acetate did not promote intracellular killing. The presence of catalase during phagocytosis inhibited killing, but had no effect on killing in the postphagocytic state. Monocytes deprived of glucose for 24 h showed markedly impaired O2 consumption, O2- generation, and bacterial killing; all of these effects were rapidly reversed by restoration of glucose. It is concluded that both an intact respiratory burst and extracellular serum factors are necessary for optimal killing of intracellular Staphylococcus aureus by human monocytes. Serum does not appear to act by enhancing the respiratory burst, but rather to have a separate, synergistic role, the biochemical basis of which is unknown.     

Alteration of bacteria induced by subinhibitory concentrations of cefixime: consequences on bactericidal activity of human polynuclear neutrophils]

Subinhibitory concentrations of most parenteral cephalosporins have been reported to alter bacterial infectivity and, in particular, to increase the susceptibility of altered bacteria to the killing effects of polymorphonuclear neutrophils (PMN). Few data on this issue are available for oral cephalosporins. This study investigated the effects of sub-MIC concentrations of the new oral cephalosporin cefixime on two bacterial targets, i.e., S. aureus 209P (MIC 20 mg/l) and E. coli K12 (MIC 0.15 mg/l). After overnight incubation (18 hours) with 10 or 5 mg/l cefixime, susceptibility of S. aureus to the killing effects of PMNs was increased two-fold as compared with control organisms and susceptibility to the O2-independent PMN bactericidal system (PMN extract) was also increased. In contrast, the susceptibility of E. coli to PMN and to cell-free bactericidal systems was identical for cefixime-exposed strains (0.1 and 0.05 mg/l) and for unexposed controls. However, cefixime-exposed E. coli were filamentous, suggesting that bactericidal efficacy in terms of the bacterial mass eliminated was enhanced in exposed strains. These data show that low levels of cefixime are capable of producing major alterations in susceptible and resistant bacteria and of increasing their susceptibility to PMN. These effects may be relevant in vivo, in particular when low concentrations of antibiotics persist over long periods in infected sites.     

The Fate of Gonococci in Polymorphonuclear Leucocytes: An Electronmicroscopic Study of the Natural Disease

The fate of gonococci in polymorphonuclear leucocytes (PMN) from men with acute gonorrhoea was studied using the electronmicroscope to look for ultrastructural damage in the bacteria. Our observations demonstrate that PMN degranulate normally into vacuoles containing gonococci and that obviously degenerate forms were present in older vacuoles. Morphologically intact gonococci were common in vacuoles containing evidence of recent degranulation suggesting that these organisms had only recently been phagocytosed. When urethral pus was suspended in tissue culture medium containing 1 μg/ml penicillin there were no “persisters” presumably because the intracellular organisms were killed by the PMN. Timed in vitro experiments using a proven virulent gonococcus demonstrated that gonococci degenerate within 30 to 60 min after phagocytosis.     

Neutrophils migrate across intestinal epithelium using beta2 integrin (CD11b/CD18)-independent mechanisms

Recruitment of polymorphonuclear leucocytes (PMN) across the intestinal epithelium is dependent on specific adhesion molecules and chemoattractants diffusing from the intestinal lumen. The present understanding is that in response to fMLP, PMN migration across a T84 colon carcinoma monolayer is dependent on the beta(2) integrin, Mac-1 (CD11b/CD18). To further understand PMN transepithelial migration, we sought to determine whether migration to C5a, IL-8 and LTB(4) was similarly Mac-1-, or even CD18-dependent. T84 epithelial cell monolayers growing on Transwell filters were used in combination with radiolabelled peripheral blood PMN. The number of migrated PMN was established by the amount of radioactivity recovered from the well after the migration period. Monoclonal antibodies were used to block integrin function. Whereas essentially all migration to fMLP across T84 monolayers was prevented by anti-CD18 antibody, significant migration to C5a, IL-8 or LTB(4) persisted despite anti-CD18 antibody, indicating PMN are capable of beta(2) integrin-independent transepithelial migration. An antibody to CD11b but not CD11a blocked migration to an extent similar as with anti-CD18. CD18-independent PMN migration to C5a occurred only in the basolateral-to-apical direction across epithelial cells. Co-stimulation of PMN with C5a and fMLP or IL-8 plus LTB(4) and fMLP still resulted in CD18-independent migration. Thus CD18 use during PMN migration across this model epithelium is a function of the chemoattractant inducing migration. The finding of CD18-independent migration mechanisms needs to be considered when developing antiadhesion molecule strategies to reduce or reverse intestinal inflammation.     

Intracellular Survival of Leishmania major in Neutrophil Granulocytes after Uptake in the Absence of Heat-Labile Serum Factors

The role of polymorphonuclear neutrophil granulocytes (PMN) in defense against the intracellular parasite Leishmania is poorly understood. In the present study, the interaction of human PMN with Leishmania major promastigotes was investigated in vitro. In the presence of fresh human serum, about 50% of PMN phagocytosed the parasites within 10 min and the parasite uptake led to PMN activation, resulting in the killing of most ingested parasites. Heat inactivation of the serum markedly reduced the rate of early parasite phagocytosis, suggesting a role of complement components in the early uptake of LEISHMANIA: However, over 50% of PMN were able to ingest parasites in the presence of heat-inactivated serum if the coincubation was extended to 3 h. After 3 h, 10% of the PMN were found to internalize Leishmania even under serum-free conditions. These findings indicate that PMN possess mechanisms for both opsonin/complement-dependent and -independent uptake of LEISHMANIA: Both pathways of uptake could be partially blocked by anti-CR3 antibody. Mannan-binding lectin was found not to be involved in this process. When phagocytosed in the absence of opsonin, the majority of Leishmania parasites survived intracellularly in PMN for at least 1 day. These data suggest a dual role of PMN in the early response to L. major infection. On the one hand, PMN can rapidly eliminate the intracellular parasites, and on the other hand, Leishmania can survive intracellularly in PMN. These data, together with the finding that intact parasites were seen in PMN isolated from the skin of infected mice, suggest that PMN can serve as host cells for the intracellular survival of Leishmania within the first hours or days after infection.     

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.     

Elucidation of the phagocytosis mechanism with the aid of luminous bacteria

Phagocytosis of the luminous bacterium Vibrio cholerae var. albensis caused a similar decrease both in viable count and in the in-vivo luminescence. These effects of polymorphonuclear neutrophil leukocytes (PMN) were oxygen-dependent processes. Exposure of PMN to oxygen caused a prompt decrease in the luminescence of bacteria that had been ingested in anaerobic conditions. Cell-free supernates from active PMN suspensions caused a decrease in luminescence and as much as 10% of the killing could be attributed to extracellular killing. Similarly, bacteria entrapped on a membrane filter showed a decrease in luminescence upon addition of active PMN, even though they could not be ingested.     

Roles of antibody and complement in the bactericidal activity of mouse peritoneal exudate neutrophils

The contributions of complement and antibody to phagocytosis and, as a separate process, intracellular killing of Proteus mirabilis, were investigated using mouse peritoneal exudate neutrophils. Phagocytosis of P. mirabilis was promoted by both immune mouse (IMS) and normal mouse (NMS) sera. Opsonization by IMS promoted significantly greater phagocytosis than did NMS, as did NMS compared with heated IMS (HIMS). The ability of NMS to opsonize P. mirabilis for both phagocytosis and phagocytic killing was diminished by chelation with EGTA and abolished by chelation with EDTA. This suggested that fixation of complement by both alternative and classical pathways provided optimal opsonization of this organism in NMS. In order to study intracellular killing as a process separate from phagocytosis, peritoneal exudate cell suspensions were exposed to P. mirabilis, previously incubated with 1% NMS, 1% IMS, 10% HNMS (heated normal mouse serum) or 10% HIMS, followed by centrifugation of the phagocyte-bacteria mixtures on Percoll density gradients. Populations of neutrophils containing viable intracellular bacteria, and relatively free of extracellular bacteria (less than 7% of total) were recovered in washed suspensions of cells fractionated at densities greater than 1.069 g/ml. For P. mirabilis that had been opsonized with 1% NMS before phagocytosis, the continued presence of extracellular serum was necessary for intracellular killing. NMS stimulated significantly greater intracellular killing than did HNMS, which stimulated some intracellular killing compared with the absence of serum, in which no killing occurred. IMS was similar to NMS in its ability to stimulate intracellular killing. EGTA partially blocked the stimulation of intracellular killing by NMS, and EDTA abolished it. These findings suggested that (as for optimal opsonization) complement activated via both alternative and classical pathways was responsible for optimal stimulation of intracellular killing.