Anthrax toxin blocks priming of neutrophils by lipopolysaccharide and by muramyl dipeptide.

We studied the pretreatment of human polymorphonuclear neutrophils (PMN) with purified preparations of the anthrax toxin components--protective antigen (PA), edema factor (EF), and lethal factor (LF)--and their effects on release of superoxide anion (O-2) after stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP). PMN isolated in the absence of lipopolysaccharide (LPS) (less than 0.1 ng/ml) released only small amounts of O-2 after FMLP stimulation; pretreatment with anthrax toxin had little effect. The release of O-2 was increased fivefold by prior treatment with 3 ng/ml LPS for 1 h at 37 degrees C, an effect referred to as priming. PMN were primed to an equivalent extent by treatment with 100 ng/ml N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]). Pretreatment of PMN with anthrax toxin components PA plus EF or PA plus LF inhibited priming by LPS or MDP, as shown by the reduction in the release of O-2 up to 90% relative to controls not treated with toxin; single toxin components were inactive. The inhibition was markedly reduced when priming with LPS or MDP was carried out before exposure to toxin. O-2 release after stimulation by phorbol myristate acetate was not increased by priming, and pretreatment with toxin did not inhibit O-2 release after this stimulus. Evidently, anthrax toxin inhibits the priming that is normally induced in PMN by bacterial products and is necessary for the full expression of antimicrobial effects.

Intracellular free calcium localization in neutrophils during phagocytosis.

Intracellular free calcium (Ca2+ i) is thought to be an important second messenger for phagocyte functions. The fluorescent indicator Quin2 was used to measure and visualize [Ca2+]i in human neutrophils during chemotaxis toward, and phagocytosis of, opsonized zymosan. In neutrophils migrating toward zymosan, [Ca2+]i was highest in the lamellipodium. Neutrophils ingesting opsonized zymosan had the highest [Ca2+]i in the pseudopods and periphagosomal cytoplasm. Most of the increase in [Ca2+]i was from extracellular sources. Regional increments in [Ca2+]i are strategically located to modulate such cellular functions as chemotaxis, oxidative activity, and degranulation.

Catalase, superoxide dismutase, and virulence of Staphylococcus aureus. In vitro and in vivo studies with emphasis on staphylococcal--leukocyte interaction.

Since oxygen-free polymorphonuclear neutrophils (PMN) cannot kill Staphylococcus aureus normally, the usual mechanisms for PMN bactericidal activity probably involve hydrogen peroxide or superoxide. Catalase can destroy hydrogen peroxide, and superoxide dismutase breaks down superoxide. Experiments were performed to study the influence of these enzymes (which are found in staphylococci) on virulence for mice or on leukocyte-bacterial interaction. 15 staphylococcal strains were injected i.p. into mice to quantitate virulence. There was good correlation between staphylococcal catalase activity and mouse lethality (r equals 0.88) but no correlation between staphylococcal superoxide dismutase activity and mouse lethality (r equals 0.14). Exogenous catalase (10,000 U/ml) increased the virulence of low-catalase staphylococci, but exogenous superoxide dismutase (200 mug/ml) did not alter the virulence of staphyloccal strains. C14=labeled high-catalase or low-catalase staphylococci were ingested equally well by PMN, with or without the addition of exogenous catalase. A high-catalase staphylococcal strain was killed relatively poorly by PMN, and addition of exogenous catalase (but not superoxide dismutase) decreased the ability of PMN to kill a low-catalase strain. Iodination of bacterial proteins by PMN is related to hydrogen peroxide, and a high-catalase staphylococcal strain was iodinated only 63% as much as a low-catalase strain. Addition of exogenous catalase decreased iodination of the low-catalase strain by 23%. These findings suggest that staphylococcal catalase protects intraphagocytic microbes by destroying hydrogen peroxide produced by the phagocyte. Thus, catalase may be a significant staphylococcal virulence factor.

Interaction of intraleukocytic bacteria and antibiotics.

Bacteria that survive inside polymorphonuclear neutrophils (PMN) following phagocytosis are protected from the bactericidal action of most antibiotics. Two possible explanations are altered metabolism by intraleukocytic bacteria or failure of antibiotics to enter the phagosome. The oxygen consumption of intraleukocytic and extraleukocytic bacteria was measured as an index of bacterial metabolism. PMN respiration and bactericidal activity were suppressed with large doses of hydrocortisone and extraleukocytic bacterial oxygen consumption was abolished by the addition of lysostaphin. Intraleukocytic bacterial continued to consume oxygen suggesting that surviving ingested micro-organisms are metabolically active. Neither penicillin (which cannot kill intraleukocytic bacteria) nor rifampin (which can kill intraleukocytic bacteria) was bactericidal for staphylococci at 5 degrees C. Thus, rifampin is not uniquely able to kill "resting" bacteria.Intraleukocytic or extraleukocytic Staphylococcus aurens were incubated with [benzyl-(14)C]penicillin for 2 h at 37 degrees C. Live intraleukocytic bacteria bound only 13% as much penicillin as live bacteria incubated with killed PMN. To measure the penetration of antibiotics into PMN, [(14)C]rifampin and [(14)C]penicillin were measured in leukocyte pellets and in the supernatant fluid. The total water space in the pellets was quantitated using tritium water and the extracellular water space was measured using Na(235)SO(4). All penicillin associated with the cell pellet could be accounted for in extracellular water. Thus penicillin was completely excluded from the leukocytes. Rifampin was concentrated in the cell pellet 2.2 times when compared with the supernatant concentration. These studies suggest that a likely explanation for the survival of phagocytized bacteria in the presence of high concentrations of most antibiotics is the inability of the antibiotic to enter the phagocyte. Rifampin, which is highly lipid soluble, can enter leukocytes and kill intracellular bacteria.

Gonococcal interactions with polymorphonuclear neutrophils: importance of the phagosome for bactericidal activity.

Gonococci are capable of attaching to the surface of polymorphonuclear leukocytes (PMN). In this location they resist phagocytosis and are not killed by PMN. To delineate the factors involved in the survival of these gonococci, we investigated the interaction of virulent gonococci, which adhere to cells and resist phagocytosis, and avirulent gonococci, which are phagocytized and killed by PMN. In the presence of serum, both virulent and avirulent gonococci associate equally well with PMN and stimulate increases in oxidative metabolism. In the absence of serum virulent gonococci attached to PMN and stimulated PMN oxidative metabolism to a greater extent than avirulent gonococci which did not attach to PMN (P = 0.0009). Therefore, the survival of virulent gonococci attached to the PMN surface is not a result of failure to activate oxidative and bactericidal mechanisms. Both virulent and avirulent gonococci stimulated equivalent PMN specific granule release as measured by the appearance of lactoferrin in the media. Phagocytosis of avirulent gonococci stimulated significantly greater beta-glucuronidase release (P = 0.01) and myeloperoxidase-mediated iodination of protein (P = 0.001) by PMN than attachment of virulent gonococci. In the absence of serum neither type of gonococci stimulated beta-glocuronidase release or protein iodination by PMN. Thus, virulent gonococci fail to stimulate primary granule release by PMN. To further assess the role of attachment versus ingestion on the survival of gonococci, PMN were treated with cytochalasin B to block ingestion. Cytochalasin B-treated PMN were unable to kill either virulent or avirulent gonococci despite normal degranulation stimulated by the latter. The failure of PMN to kill surface-attached gonococci appears to be a consequence of the failure of PMN to enclose the virulent gonococci within a phagosome. The phagocytic vacuole thus plays a critical role in normal PMN bactericidal activity by providing a closed space in which the proper concentration of substances may be achieved to generate microbicidal activity.

The effect of an NADH oxidase inhibitor (hydrocortisone) on polymorphonuclear leukocyte bactericidal activity.

Polymorphonuclear neutrophils (PMN) from patients with chronic granulomatous disease of childhood have impaired bactericidal activity and are deficient in diphosphopyridine nucleotide, reduced form of, (NADH) oxidase. Since hydrocortisone had been shown to inhibit NADH oxidation, experiments were undertaken to determine the effect of hydrocortisone on several parameters of human PMN function. The phagocytic and bactericidal capacity of PMN with or without hydrocortisone (2.1 mM) was determined by quantitation of cell-free, cell-associated, and total bacteria. Phagocytosis of Staphylococcus aureus and several gram-negative rods was unimpaired by the presence of hydrocortisone in the media. In contrast, killing of bacteria was markedly impaired by hydrocortisone. After 30 min of incubation, there were 20-400 times as many bacteria surviving in hydrocortisone-treated PMN as in simultaneously run controls without hydrocortisone. The defect of intracellular killing noted in the presence of hydrocortisone was not related to impaired degranulation. Quantitative kinetic studies of degranulation revealed no difference in the release of granule associated acid phosphatase in hydrocortisone-treated and control PMN after phagocytosis. Electron microscopy of PMN also indicated that the presence of hydrocortisone had no effect on the extent of degranulation after phagocytosis. These observations were confirmed by studies using histochemical techniques to detect lysosomal enzymes. After phagocytosis, hydrocortisone-treated PMN demonstrated less NADH oxidase activity, oxygen consumption, and hydrogen peroxide production than postphagocytic control PMN. In addition, Nitro blue tetrazolium dye reduction was diminished in hydrocortisone-treated PMN.Thus, impairment of NADH oxidase activity in normal human PMN by hydrocortisone results in reduced intracellular killing of bacteria, diminished postphagocytic oxygen consumption, decreased ability to reduce Nitro blue tetrazolium, and decreased hydrogen peroxide production. These abnormalities are similar to those seen in the PMN of patients with chronic granulomatous disease of childhood.

Interaction of polymorphonuclear neutrophils with Escherichia coli. Effect of enterotoxin on phagocytosis, killing, chemotaxis, and cyclic AMP.

Enterotoxigenic Escherichia coli are associated with noninflammatory diarrhea and stimulate adenylate cyclase activity of mammalian cells, thereby increasing intracellular cyclic adenosine 3',5'-monophosphate (cyclic AMP). Increased concentrations of cyclic AMP in polymorphonuclear neutrophils (PMN) inhibit phagocytosis, candidacidal activity, granule discharge, and chemotactic responsiveness. We examined the effect of enterotoxin on the interaction of human PMN with E. coli. Enterotoxigenic and nonenterotoxigenic strains, including serotypes of E. coli identical except for the presence or absence of the plasmid coding for enterotoxin production, were utilized. Enterotoxigenic and nonenterotoxigenic E. coli, tumbled with PMN, were phagocytized and killed (>97%) equally well, and these strains stimulated PMN hexose monophosphate shunt activity equivalently.However, a chemotaxis assay under agarose demonstrated that filtrates of 10 enterotoxigenic strains were less chemotactic for PMN by 15+/-2% total migration or 46+/-1% directed migration, when compared with 6 non-enterotoxigenic strains (P < 0.001). Inactivation of the enterotoxin by heat (65 degrees C for 30 min) or antibodies formed to E. coli enterotoxin eliminated the inhibitory effect of the enterotoxic filtrates for PMN chemotaxis. Addition of purified E. coli enterotoxin directly to the PMN decreased chemotaxis to E. coli filtrates by 32+/-2% (P < 0.001). These data suggest that the effect was due to the heat-labile enterotoxin. The phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (0.1 mM), which potentiates effects due to an increase in intracellular cyclic AMP, further decreased total PMN migration (random plus directed) toward enterotoxic filtrates to 46% of that to nonenterotoxic filtrates (P < 0.001). Addition of cholera toxin (1 mug/ml), which is similar to E. coli enterotoxin, to the PMN inhibited total migration toward nonenterotoxic filtrates by 16+/-2% (P < 0.001). Exogenous dibutyryl cyclic AMP (2 mM) inhibited total PMN migration toward E. coli filtrates by 32% (P < 0.001). PMN intracellular cyclic AMP levels increased by 220% after 2 h of incubation with purified E. coli enterotoxin. The decreased chemotactic attractiveness of enterotoxic E. coli filtrates appears to be related to the ability of enterotoxin to increase cyclic AMP in PMN. Enterotoxin production by E. coli may be advantageous to the microbe by decreasing its chemotactic appeal for PMN.

A wave of elevated intracellular free calcium spreads through human neutrophils during phagocytosis of zymosan.

The cytosolic concentration of free calcium ([Ca2+]i) plays an important role in the control of many neutrophil functions. In this study, we characterize the early rapid subcellular changes in [Ca2+]i that occur in adherent neutrophils during phagocytosis of zymosan particles, using both dual- and single-excitation wavelength Fura-2 ratio imaging. We observed a wave of elevated cytosolic calcium that began shortly after zymosan contact and propagated from the region of neutrophil contact with the zymosan throughout the cell at a rate of approximately 17 microns/s at 31 degrees C. The wave was initiated by both opsonized and unopsonized zymosan and occurred independently of extracellular calcium. Multiple characteristics of the [Ca2+]i signal (including the absolute and regional [Ca2+]i and wave properties such as amplitude, frequency, duration, and topography) may be responsible for the differential regulation of cellular functions in the neutrophil.

Tumor necrosis factor-alpha decreases neutrophil chemotaxis to N-formyl-1-methionyl-1-leucyl-1-phenylalanine: analysis of single cell movement.

Tumor necrosis factor-alpha (TNF-alpha), a cytokine produced by mononuclear cells in response to endotoxin, inhibits neutrophil chemotaxis. We analyzed the effects of TNF-alpha on the orientation and movement of individual neutrophils in a chemoattractant gradient. Neutrophils, treated or untreated with TNF-alpha, were observed migrating in a gradient of the chemotactic peptide N-formyl-1-methionyl-1-leucyl-1-phenylalanine (fMLP) on a specially constructed chamber (Zigmond bridge). The movement of these cells was videotaped, digitized, and then tracked using a newly designed computer algorithm. The data obtained from this algorithm were then utilized to calculate distance traveled, speed and ability to polarize and migrate in a directed manner for each individual cell. TNF-alpha-treated cells behaved like cells not exposed to fMLP in that they failed to orient in a chemotactic gradient and moved in a manner similar to randomly migrating cells. This study provides unique observations of the effect of TNF-alpha on multiple parameters of PMN migration.

Both recombinant interleukin-1 (beta) and purified human monocyte interleukin-1 prime human neutrophils for increased oxidative activity and promote neutrophil spreading.

Both purified human monocyte interleukin-1 and recombinant interleukin-1 (beta) primed neutrophils for increased superoxide production and chemiluminescence in response to f-met-leu-phe. In addition, purified human monocyte interleukin-1 and recombinant interleukin-1 (beta) altered neutrophil shape. Recombinant interleukin-1 (alpha) used at the same concentration of interleukin-1 (beta) did not prime neutrophils for increased superoxide production after stimulation with f-met-leu-phe. Interleukin-1 expressed by monocytes in response to endotoxin stimulation could act as a modulator of neutrophil function.

Reversible Pelger-Huët anomaly associated with ibuprofen therapy.

A patient had a case of acquired-reversible pseudo-Pelger-Huët anomaly associated with a hypersensitivity reaction. Seven weeks after the illness, the hematologic anomaly was no longer present.

Polymorphonuclear leukocytes: dedicated professional phagocytes.

Polymorphonuclear leukocytes are termed professional phagocytes because they are specially equipped to seek and destroy invading microorganisms. Polymorphonuclear leukocytes are formed in the bone marrow and released into the circulation, where they are transported to the tissues. At sites of tissue invasion by microorganisms, humoral factors are released that induce these cells to leave the bloodstream and enter the tissues. Chemotactic substances guide polymorphonuclear leukocytes to the infecting organisms. Antibody and complement can function as opsonins and enhance the ability of polymorphonuclear leukocytes to engulf microbes. Ingested organisms are killed by oxidative or nonoxidative systems. Defects in the various aspects of polymorphonuclear leukocyte function may be found in patients with recurrent, severe, or unusual infections. Evaluation of selected patients with recurrent infections should include tests of polymorphonuclear leukocyte function.

Ovarian cancer: a solid tumor with evidence of normal cellular immune function but abnormal B cell function.

Immunologic assays of B and T lymphocyte function were performed on 21 patients with epithelial ovarian cancer prior to either chemotherapy or radiotherapy. The results were compared to similar studies on 12 age-matched normal women. The total peripheral blood lymphocyte counts, proportion of E rosette positive cells, stimulation of T cells by phytohemagglutinin and concanavalin A, recall skin tests, and the ability to have a primary delayed hypersensitivity response to keyhold limpet hemocyanin did not differ between patients and controls. However, patients with ovarian cancer had statistically significant reduction in surface immunoglobulin positive cells, proliferative response to pokeweed mitogen and primary antibody response to keyhole limpet hemocyanin. In contrast to results in patients with other solid tumors, these data indicate that untreated patients with ovarian cancer have evidence of normal cellular immune function but abnormal B cell function.

Invasive aspergillosis of the lung and pericardium in a nonimmunocompromised 33 year old man.

In a 33 year old man with no discernible immunologic defect, invasive aspergillosis developed in both the pericardium and lung with marked granulomatous reaction. The patient received 2 g of intravenous amphotericin B over eight weeks, with partial regression of the pulmonary infiltrate and disappearance of symptoms. However, five months later, he returned with marked progression of his disease. Evaluation of host defense, including granulocyte and lymphocyte function, was normal. The patient was given an additional 3g of amphotericin B over nine weeks with marked improvement in symptoms and chest roentgenogram. At six-month follow-up, he was asymptomatic with a stable radiographic appearance. A recurrence in symptoms and the pulmonary infiltrate was noted two months later. He was treated with an additional course of amphotericin and currently is receiving ketoconazole in hopes of suppressing the infection. We could find no immune impairment to explain the severe pulmonary and pericardial disease due to Aspergillus flavus in this young man.

Pneumonia treated with imipenem/cilastatin.

In an open, prospective, multicenter trial the efficacy and tolerance of imipenem/cilastatin for the treatment of bacterial pneumonia was investigated. Forty-three adults were studied: 29 with nosocomial and 14 with community-acquired infections. Significant underlying disease was present in 91 percent of patients. Nosocomial infection was frequently associated with endotracheal intubation (48 percent), prior antibiotic therapy (48 percent), and recent surgery (31 percent). Most frequent sputum isolates included Pseudomonas aeruginosa (10, all nosocomial), Hemophilus influenzae (10), Escherichia coli (eight), Staphylococcus aureus (seven), and Streptococcus pneumoniae (six). Treatment with imipenem/cilastatin was associated with clinical cure in 93 percent of patients. Two of three failures and one superinfection occurred in association with isolates of Pseudomonas aeruginosa resistant to imipenem. Overall, six of 10 strains of Pseudomonas aeruginosa isolated prior to therapy developed resistance to imipenem after an average of 10 days of therapy. Adverse effects occurred in nine patients (21 percent) and included one case of pseudomembranous colitis. Monotherapy with imipenem/cilastatin of serious lower respiratory tract infections was relatively safe and highly effective with the exception of disease associated with P. aeruginosa.

Adenosine modulation of tumor necrosis factor-alpha-induced neutrophil activation.

We hypothesized that adenosine, known to be release from inflammatory sites, could lessen the potentially damaging activity of neutrophils (PMN) primed by tumor necrosis factor-alpha (TNF alpha) at sites of infection. We investigated the effect of adenosine on PMN primed with cell-free medium from mononuclear leukocytes (MNL) that had been treated with lipopolysaccharide (LPS) yielding a conditioned medium rich in TNF alpha and on PMN primed with recombinant human TNF alpha (rhTNF alpha). LPS (10 ng/mL) minimally primed PMN, but LPS-MNL-conditioned medium increased PMN chemiluminescence in response to f-Met-Leu-Phe (fMLP) 1242% compared with unprimed PMN. LPS-MNL-conditioned medium contained adenosine (approximately 30 nM). Converting the adenosine in the LPS-MNL-conditioned medium to inosine with adenosine deaminase (ADA) or blocking adenosine binding to PMN with the adenosine receptor antagonist 1,3-dipropyl-8-(phenyl-p-acrylate)-xanthine (BW A1433U) resulted in a near doubling of chemiluminescence. The LPS-MNL-conditioned medium contained TNF alpha (836 pg/mL; approximately 1 U/mL). Recombinant human TNF alpha (1 U/mL) primed PMN for a 1033% increase in chemiluminescence. Added adenosine decreased rhTNF alpha-primed PMN chemiluminescence (IC50 approximately 100 nM), and adenosine (100 nM) decreased both superoxide and myeloperoxidase release from rhTNF alpha-primed fMLP-stimulated PMN. The activity of adenosine was counteracted by ADA and BW A1433U, and the modulating effect of adenosine was on the primed response rather than on priming per se. Thus, physiological concentrations of adenosine reduce the effects of recombinant human TNF alpha and native human TNF alpha (released from LPS-treated MNL) on PMN activity. Endogenous adenosine may preclude or minimize damage to infected tissue by damping the TNF alpha-primed PMN oxidative response.

Early events in target-cell lysis by cytotoxic T cells.

Using ratio-imaging fluorescence microscopy, we have investigated the changes in intracellular Ca2+ [( Ca2+]i) that occurred in cytotoxic T lymphocytes (CTL) upon target-cell binding. This process resulted in a rapid increase in [Ca2+]i, which was localized in the region of the CTL in contact with the target cell. This increase was mediated both by influx from the external medium as well as by release from intracellular stores. Although the magnitude of the initial increase in [Ca2+]i was not dependent upon the presence of extracellular Ca2+, influx was necessary for sustained elevation of [Ca2+]i. Inasmuch as target-cell lysis by the CTL clone used is dependent on extracellular Ca2+, this suggests that a prolonged elevation of [Ca2+]i is necessary for lytic function. It was also shown that the increase in [Ca2+]i and its subsequent decay show several pulsations. The mechanism by which these variations are generated and their possible function is not known. Finally, a role for K+ efflux in the control of the antigen-induced increase in [Ca2+]i was demonstrated. Thus it is becoming clear that signal transduction in CTL is remarkably complex, involving several ionic species and second messengers.

Adjuncts to antibacterial therapy.

Strategies of augmenting or attenuating the immune system's response to infection are promising potential methods to enhance antibacterial therapy. Many of these new therapies are natural cytokines, such as the hematopoietic growth factors and interferons, which upregulate the immune response. Other examples include replacement therapies, such as immune globulin, and downregulators of the immune response, such as corticosteroids. In the near future it may be possible to adjust the host response to microbial infection to maximally inhibit the microbe while minimizing inflammatory damage.

The importance of penetration of antimicrobial agents into cells.

Phagocytes may shelter intracellular pathogens from the otherwise lethal effects of many antibiotics. Research in conditions such as chronic granulomatous disease, tuberculosis, legionellosis, and experimental staphylococcal infection underscores the principle that an antibiotic must enter the cell in order to be active against an intracellular microorganism. Demonstrating entry does not guarantee activity, however. The microenvironment and intracellular distribution of the pathogen and antimicrobial agent, and interactions between agent, pathogen, and host cell all contribute to determining the treatment result.

The Jeremiah Metzger Lecture. Microbial defenses against killing by phagocytes.

Phagocytes are a key feature of defense against microorganisms. Phagocyte function is a complex system with many intricately involved components. Each of these components provides microorganisms with a target for countermeasures against phagocytes. We have discussed examples and purported mechanisms for microbial defenses against the steps involved in killing by phagocytes. Understanding the interplay of these host and pathogen factors leads to a better understanding of both normal host defenses and pathogenesis of disease.