Phagocytosis of Borrelia recurrentis by blood polymorphonuclear leukocytes is enhanced by antibiotic treatment.

The removal of Borrelia spirochetes from the blood in relapsing fever was studied by examining patients' blood phagocytic cells with the Dieterle silver stain. Polymorphonuclear leukocytes ingested Borrelia at increased rates for several hours after antibiotic treatment, during which time the total numbers of circulating plasma spirochetes were decreasing. Incubation of infected blood at 37 degrees C for 2 h resulted in a progressive increase in phagocytosis. Addition of penicillin G and tetracycline to infected blood caused a further enhancement of phagocytosis. Electron microscopy of polymorphonuclear leukocytes revealed spirochetes in phagosomes. These results indicated that blood polymorphonuclear leukocytes have a prominent role in removing Borrelia from the plasma and suggested that antibiotics act by altering the surface of spirochetes to render them more susceptible to phagocytosis.     

Phagocytosis of Mycoplasma salivarium by human polymorphonuclear leukocytes and monocytes.

Mycoplasmacial activity was exhibited by human peripheal blood leukocytes in the absence of detectable specific antiserum. After incubation of varying concentrations of Mycoplasma salivarium with leukocytes, changes in colony-forming units (CFU) of this species per millilter occurred. The most noticeable decrease in CFU per milliliter was then the incubation mixtures contained five mycoplasmas per leukocyte. At this ratio, the mycoplasmacial min of incubation. Continued incubation demonstrated a tenfold decrease in CFU per milliliter by 4 h. Electron micrographs of incubated mixtures of human leukocytes and M. salivarium showed this mycoplasma to be phagocytized by monocytes and neutrophils whenever mutual contact or pseudopodial formation occurred. The process was continuous. Numerous phagocytic vacuoles developed which contained multiple ingested microorganisms. After the cytoplasmic granules of the leukocytes fused with the phagocytic vacuole, the phagocytized mycoplasmas became disrupted and unrecognizable.     

Phagocytosis and killing of staphylococci by human polymorphonuclear and mononuclear leucocytes.

The phagocytosis and killing of 3H-thymidine-labelled Staphylococcus aureus by polymorphonuclear leucocytes (PMNs) and monocytes (MNs) obtained from 50 health donors were evaluated. In addition, extracellular factors that might influence phagocytosis and killing were studied. The method described gave highly reproducible results. No significant difference was observed in the phagocytic and killing functions of a single donor's PMNs and MNs when studied several times in one day and longitudinally over a period of 1-12 weeks for six donors tested. Likewise, no signigicant difference in uptake and killing was observed when bacteria were opsonised with sera from 11 different normal donors. When Staph. aureus opsonised with normal serum was added to the leucocytes in a ratio of 10 bacteria: 1 leucocyte, the uptake by PMNs and MNs from 50 donors after 20 minutes' incubation was 85% +/- 7 standard deviation (SD) (range 75-98%) and 69% +/- 11 SD (range 54-90%), respectively. The rate of uptake by MNs in the first three minutes of the assay period was only 60% of that by PMNs.     

Phagocytosis of medically important yeasts by polymorphonuclear leukocytes.

Phagocytosis is a critical function of polymorphonuclear leukocytes in the control of mycotic infections. By using a modified fluorescence quenching assay to distinguish between attached and ingested organisms, we determined the percent phagocytosis of several medically important yeasts. The percentages of phagocytosis of serum-opsonized Candida albicans, Candida tropicalis, Candida parapsilosis, and Torulopsis glabrata were all comparable at 37 degrees C. By comparison, there was significantly less phagocytosis of Cryptococcus neoformans and Trichosporon beigelii isolates (P < 0.001). Thus, phagocytosis of C. albicans by polymorphonuclear leukocytes is comparable to that of species other than C. albicans but is significantly greater than that of the basidiomycetous yeasts T. beigelii and C. neoformans.     

Phagocytosis of Vibrio cholerae O139 Bengal by human polymorphonuclear leukocytes

Capsulated bacteria exhibit serum (complement) resistance and resistance to phagocytosis, which result in disseminated infections. Vibrio cholerae O139 strains possess a thin capsule and have been found to be partially serum resistant in a previous study. In the present study, compared to a standard capsulated Klebsiella pneumoniae strain, which showed total resistance to killing by phagocytosis, V. cholerae O139 strains were shown to be only partially resistant, with most strains showing <40% survival. These findings may explain the relative rarity of V. cholerae O139 bacteremia in cholera caused by this organism.     

Effect of staphylococcal alpha-toxin on phagocytosis of staphylococci by human polymorphonuclear leukocytes.

Forty species of anaerobes were screened for the ability to produce an ether-extractable mutagen which is present in the feces of 15 to 20% of individuals in populations at high risk for colon cancer. This mutagen can be produced in vitro by incubating the feces of these individuals anaerobically or by supplementing anaerobic broths with methanol extracts of the feces and incubating them with a dilute fecal inoculum. Of the anaerobes screened, strains of five species of Bacteroides (B. thetaiotaomicron, B. fragilis, B. ovatus, B. uniformis, and Bacteroides group 3452A) were capable of producing five- to eightfold increases in the concentration of mutagen. For in vitro production in broth, all producers required bile and the methanol extract for feces from a person who excretes the mutagen. Mutagen production appeared to be constitutive and occurred during the stationary phase of growth. Cell-free extracts were active and produced mutagen considerably faster than did whole cells. Our observations indicate that the excretion of this mutagen by certain people is dependent on the presence of some precursor of unknown origin. The mutagen-producing species of bacteria are among the most common of the intestinal microflora and were present in mutagen excreters and nonexcreters as well.     

Phagocytosis of virulent Porphyromonas gingivalis by human polymorphonuclear leukocytes requires specific immunoglobulin G.

No studies to date clearly define the interactions between Porphyromonas gingivalis and human peripheral blood polymorphonuclear leukocytes (PMN), nor has a protective role for antibody to P. gingivalis been defined. Using a fluorochrome phagocytosis microassay, we investigated PMN phagocytosis and killing of P. gingivalis as a function of P. gingivalis-specific antibody. Sera from a nonimmune rabbit and a healthy human subject were not opsonic for virulent P. gingivalis A7436, W83, and HG405; phagocytosis of these strains (but not 33277) required opsonization with hyperimmune antiserum (RaPg). Diluting RaPg with a constant complement source decreased proportionally the number of P. gingivalis A7436 cells phagocytosed per phagocytic PMN. Enriching for the immunoglobulin G fraction of RAPg A7436 enriched for opsonic activity toward A7436. An opsonic evaluation of 18 serum samples from adult periodontitis patients revealed that only 3 adult periodontitis sera of 17 with elevated immunoglobulin G to P. gingivalis A7436 were opsonic for A7436 and, moreover, that the serum sample with the highest enzyme-linked immunosorbent assay titer was most opsonic (patient 1). However, the opsonic activity of serum from patient 1 was qualitatively and not just quantitatively different from that of the nonopsonic human sera (but was less effective opsonin than RaPg). Strain variability was observed in resistance of P. gingivalis to phagocytosis, and opsonization was strain specific for some, but not all, strains tested. An evaluation of killing of A7436 revealed that serum killing and extracellular killing of P. gingivalis were less effective alone when compared with intracellular PMN killing alone.     

Phagocytosis of virulent and avirulent leptospires by guinea-pig and human polymorphonuclear leukocytes in vitro

Chemiluminescence (CL) and electron microscopy were used to study the phagocytosis of both virulent and avirulent strains of Leptospira interrogans serovar copenhageni by guinea-pig and human polymorphonuclear leukocytes (PMN). A significant CL response was observed when guinea-pig PMN were incubated with virulent leptospires in the presence but not in the absence of specific immune serum. This response was markedly enhanced by the addition of guinea-pig complement. Phagocytosis was confirmed by the observation of intracellular leptospires in guinea-pig PMN by electron microscopy. The phagocytosis of avirulent leptospires by guinea-pig PMN and of both virulent and avirulent leptospires by human PMN required the presence of both specific immune serum and complement. Thus the ability of leptospires to resist phagocytosis by PMN in the absence of immune serum does not appear to be a major determinant of virulence.     

Phagocytosis and binding via complement receptors by salivary polymorphonuclear leukocytes

The ability of oral polymorphonuclear leukocytes (PMNs) to phagocytoseCandida albicans cells and bindSalmonella typhi via complement receptors was investigated. A significantly higher percent of oral PMNs could phagocytose and bind via complement receptors as compared to peripheral blood PMNs. While treatment of peripheral blood PMNs with the donor's saliva caused an increase in the number of complement-receptor bearing cells, as well as a partial increase in phagocytosis, PMNs treated with gingival crevicular fluid (CF) showed a decrease both in phagocytosis and binding. The complexity of environmental conditions and factors, and its role in PMN functions in inflammatory sites is discussed.This study was supported by a research grant obtained from the joint research fund of the Hebrew University-Hadassah School of Dental Medicine, Founded by the Alpha Omega Fratenity, Jerusalem, Israel, and by U.S. Public Health Service Grant No. DE 03995.     

Influence of antibiotics on fibronectin binding and phagocytosis of staphylococci by human polymorphonuclear leukocytes

The effect of subbactericidal concentrations of cell wall and protein synthesis-inhibiting antibiotics on fibronectin-staphylococcal interactions was examined. Exposure of staphylococci to protein synthesis inhibitors resulted in a dose-dependent decrease in fibronectin's ability to bind to and promote the phagocytosis of treated organisms by human polymorphonuclear leukocytes. Cell wall inhibitors, on the other hand, had little or no effect on fibronectin binding or phagocytosis. Serum remained an effective opsonin despite antibiotic modulation of fibronectin binding site expression.     

Activity of polymorphonuclear leukocytes in the presence of sulfide.

Polymorphonuclear leukocytes (PMN) isolated from human blood were exposed to various levels of hydrogen sulfide. The effect on respiratory burst, myeloperoxidase activity, and capacity to phagocytose and kill bacteria were studied. A 1-h exposure of the PMN to 1 mM sulfide did not decrease their myeloperoxidase activity or their capacity to initiate a respiratory burst. Actually the products of the respiratory burst rapidly oxidized sulfide. The phagocytosis and killing of bacteria in the presence of 1 mM sulfide was only decreased to a minor extent. Myeloperoxidase in cell extract was, however, almost completely inhibited by 1 microM sulfide. These results indicate that hydrogen sulfide does not easily permeate PMN. PMN may be able to function in infected sites with high sulfide levels such as in the gingival pockets of periodontal disease. In the oxygenated areas of these sites the PMN may actually help in the detoxification of sulfide.     

Opsonization of Staphylococcus aureus protects endothelial cells from damage by phagocytosing polymorphonuclear leukocytes.

When phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes (PMN) takes place on the surface of cultured human endothelial cells, the endothelial monolayers are damaged by lysosomal enzymes that are released by the PMN. Because PMN can phagocytose opsonized as well as unopsonized staphylococci on an endothelial surface, we studied the role of bacterial opsonization in the damage caused to the endothelium. Phagocytosis of unopsonized S. aureus was accompanied by greater damage (expressed as the percentage of the endothelial cells detached from the culture plates) of the monolayers than was phagocytosis of opsonized S. aureus: 52 +/- 10% and 24 +/- 7%, respectively, after 30 min of phagocytosis and 73 +/- 5% and 50 +/- 6%, respectively, after 60 min of phagocytosis. When correlated to the amount of phagocytosis, this difference was even greater (uptake was 35 +/- 4% for unopsonized S. aureus and 56 +/- 5% for opsonized S. aureus after 30 min and 42 +/- 3% and 60 +/- 5%, respectively, after 60 min). Total release of lysozyme and myeloperoxidase and generation of superoxide anion were the same during phagocytosis of opsonized or unopsonized staphylococci. Adherence of PMN to the endothelial cells was greater during phagocytosis of unopsonized S. aureus: 42 +/- 4% verus 27 +/- 3% during phagocytosis of opsonized staphylococci. Possibly, increased adherence of the PMN resulted in a locally higher concentration of enzymes which induced more damage. We conclude that opsonization of bacteria not only improves bacterial uptake, but also protects bystander cells from damage by the phagocytosing PMN.     

Phagocytosis of opsonized yeast induces tumor necrosis factor-alpha mRNA accumulation and protein release by human polymorphonuclear leukocytes.

In this report we show that phagocytosis of yeast particles opsonized with IgG (Y-IgG) by human polymorphonuclear cells (PMN) results in the selective induction of tumor necrosis factor (TNF-alpha) messenger RNA (mRNA) and release of its mature protein. Lipopolysaccharide (LPS) was also found able to induce TNF-alpha secretion by PMN, but was a less potent stimulus compared with Y-IgG. There was no evidence of interleukin-6 (IL-6) gene expression in PMN after phagocytosis of Y-IgG or in response to LPS, whereas IL-6 mRNA expression and secretion were induced by either stimulus in monocytes. These findings demonstrate that a physiological function such as phagocytosis modulates the gene expression for a cytokine in PMN and shed new light on the understanding of the pathogenesis of the inflammatory process.     

Electron microscopic and autoradiographic study of the interaction of human polymorphonuclear leukocytes and staphylococci

Leukocyte suspensions of normal subjects and patients with burns and wounds were incubated with Staphylococcus epidermidis and incorporation of 3H-uridine into leukocytes and bacteria was examined by electron microscopic autoradiography. As the maturation of the precursor cells of polymorphonuclear leukocytes (PMNL) progressed the level of RNA synthesis in them was found to decrease gradually and in PMNL RNA synthesis ceased completely. If PMNL take part in phagocytosis, however, RNA synthesis is found in a considerable number of them. The results indicate that RNA synthesis in PMNL is associated with the formation and functional condition of primary and secondary granules. Certain conditions affecting the viability of staphylococci ingested by phagocytes are described.     

Injury to endothelial cells by phagocytosing polymorphonuclear leukocytes and modulatory role of lipoxygenase products.

Phagocytosis of microorganisms by polymorphonuclear leukocytes (PMN) is accompanied by inadvertent extracellular release of microbicidal products; this could result in tissue damage. We investigated whether PMN damages endothelial cells when phagocytosis of Staphylococcus aureus occurs on the endothelial surface and how this damage might be modulated. Damage was assayed by the measurement of cell detachment or cell lysis of cultured endothelial cells that were radiolabeled with 51Cr. Uptake of bacteria was accompanied by nonlytic detachment of endothelial cells from the monolayer. This effect was inhibited by alpha-1-antitrypsin but remained unaffected by scavengers of toxic oxygen species. During phagocytosis, PMN adhered to the endothelial cells. Adherence could be prevented by inhibition of the lipoxygenase pathway of arachidonic acid metabolism of the PMN with nordihydroguaiaretic acid. This inhibition also resulted in a marked decrease of the detaching activity of the PMN. The addition of exogenous leukotriene B4 during phagocytosis greatly enhanced the damage to the endothelial monolayer. These results indicate that phagocytosis of staphylococci by PMN is accompanied by injury to endothelial cell monolayers due to released lysosomal proteases and that products of the lipoxygenase pathway of PMN play a modulatory role in this injury.     

Destruction of cultured vascular endothelial cells and red blood cells by immune-activated polymorphonuclear leukocytes

The cytotoxicity of human polymorphonuclear leukocytes (PMNs) against autologous red blood cells (RBC) and cultured vascular endothelial cells (EC) was investigated. PMNs were activated by phorbol myristate acetate (PMA) together with immune stimuli such as immune complexes and aggregated IgG. In the standard⁵¹Cr-release assay, in which PMA concentration was 5 ng/ml and effector versus target ratio was 5, 76.7% and 34.2% specific⁵¹Cr release was observed from RBC and EC, respectively. Significant levels of⁵¹Cr were released, albeit to a lesser degree, when PMNs were stimulated by immune stimuli. Further experiments which employed various scavengers of oxygen radicals suggested that hydrogen peroxide was the most potent mediator of this cytotoxicity; the implications of these in vitro observations with the pathogenesis of immune vasculitis are of clinical interest.Supported by Manabe Medical Foundation.     

Bioactive glasses induce chemiluminescence by human polymorphonuclear leukocytes.

The effect of bioactive glasses on human polymorphonuclear leukocytes (PMNLs) were studied in vitro by a chemiluminescence (CL) assay. Eight different glasses were chosen. All glasses induced a rapid CL response by human PMNLs, which proved to be dose dependent. The CL response also seemed to depend on the durability of the glasses. The least durable glass caused the highest CL response, and highly durable glasses caused only low CL responses by the cells.     

Oxidation of glucosamine by human polymorphonuclear leukocytes

When exposed to a phagocytic stimulus (opsonized zymosan), human polymorphonuclear leukocytes (PMNs) produced¹⁴CO₂ from [1-¹⁴C]glucosamine at a rate 10–25% of that produced from glucose under the same conditions. The production of CO₂ from glucosamine by intact PMNs was inhibited by glucose and dependent upon activation of the hexosemonophosphate shunt (HMPS). However, the metabolic pathways for the oxidation of glucose and glucosamine by PMNs are not identical. This is suggested by the fact that glucose-6-phosphate dehydrogenase, the initiating enzyme for the HMPS, did not utilize glucosamine-6-phosphate as a substrate. In addition, glucosamine was not oxidized by sonically disrupted PMNs whereas oxidation of glucose by the same preparation was increased sevenfold over intact cells. Taken together, the data suggest that PMNs oxidize glucosamine by converting it to a compound compatible with the enzymes of the HMPS. This conversion requires intact PMNs and/or an as yet unidentified cofactor.     

Phagocytosis and killing of Actinobacillus pleuropneumoniae by alveolar macrophages and polymorphonuclear leukocytes isolated from pigs.

To study the cellular response of phagocytic cells to Actinobacillus pleuropneumoniae, we investigated whether porcine alveolar macrophages (AM) and polymorphonuclear leukocytes (PMN) are able to phagocytize and intracellularly kill A. pleuropneumoniae in vitro. Bacterial cultivation methods of A. pleuropneumoniae were used to assess in vitro phagocytosis and the ability to kill. A specific-pathogen-free pig was killed, blood was collected, and PMN were isolated and counted. The AM were isolated by lung lavage of the same animal and counted. In addition, convalescent-stage serum was collected from a specific-pathogen-free pig that was infected with A. pleuropneumoniae. Both porcine AM and porcine PMN effectively phagocytized A. pleuropneumoniae in the presence of convalescent-stage pig serum. PMN killed 90 to 99% of the bacteria intracellularly, whereas AM did not. Because A. pleuropneumoniae produces exotoxins that kill porcine AM and porcine PMN, we incubated equal amounts of bacteria and phagocytic cells and tested the viability of the cells 120 min later. In the presence of convalescent-stage pig serum, A. pleuropneumoniae was toxic to AM but not to PMN. Probably in porcine AM, intracellular released toxins of A. pleuropneumoniae lessen the ability of the cell to kill the bacterium. Consecutive lysis of AM and release of viable A. pleuropneumoniae may initiate the characteristic porcine pleuropneumonia.