Polymorphonuclear Phagocytes Research Articles

The effect of Intralipid on mononuclear and polymorphonuclear phagocytes

Eight healthy subjects were given Intralipid, a soybean oil emulsion, 20% intravenously for 2 h. During the infusion a significant increase in the nitroblue tetrazolium-reduction of blood monocytes was noted. Preincubation of monocytes in vitro with Intralipid (20 to 100 mg/ml) for 30 min was found to increase the ability of the cells to migrate chemotactically and to phagocytize yeast particles. On the contrary, when neutrophilic granulocytes were preincubated with Intralipid in the same concentrations for 30 min. their nitroblue-tetrazolium-reduction, chemotactic and spontaneous locomotion, as well as their ingestion of yeast particles was depressed.

Ultrastructural features of phagocytosis and intracellular killing of Candida albicans by mouse polymorphonuclear phagocyte monolayers.

Phagocyte monolayers provided a simple method of following ultrastructural events associated with phagocytosis and intracellular killing of Candida albicans. Preformed monolayers of mouse polymorphonuclear (PMN) phagocytes attached to glass coverslips were incubated with blastospore phase C. albicans and then examined by scanning and transmission electron microscopy. Scanning electron microscopy revealed phagocytosis of C. albicans by mouse phagocytes. Ingestion of the organism was facilitated by the production of lamellipodia by the phagocytes. Transmission electron microscopy revealed complete phagocytosis of C. albicans and the fusion of lysosomal granules with loose and tight phagosomes. Ingested C. albicans remained structurally intact after 2 hr incubation in blastospore-free medium. However, cytoplasmic alterations were clearly evident, with a patchy loss of electron density. Alterations of the blastospore cell wall were also observed, with complete disruption of the plasma membrane but the wall remaining morphologically intact.

Outer membrane proteins of Neisseria gonorrhoeae associated with survival within human polymorphonuclear phagocytes.

The determinant(s) of gonococcal resistance to killing by human polymorphonuclear (PMN) phagocytes appear to be present in outer membrane vesicles (OMV) purified from lithium chloride extracts of Neisseria gonorrhoeae strain BS4 (agar) by wheat germ agglutinin (WGA) affinity chromatography. OMV neutralized the ability of antisera raised against whole gonococci to drastically reduce the capacity of strain BS4 (agar) to survive within PMN phagocytes. Furthermore, analysis by SDS-PAGE of OMV/WGA precipitates from lithium chloride extracts of strain BS4 (agar) and strain BSSH, which was more susceptible to phagocyte killing than strain BS4 (agar) and yielded OMV with poor antiserum-neutralizing activity, suggested that three proteins were associated with resistance to phagocyte killing.

Selective protection against conidia by mononuclear and against mycelia by polymorphonuclear phagocytes in resistance to Aspergillus. Observations on these two lines of defense in vivo and in vitro with human and mouse phagocytes.

By comparing natural immunity to Aspergillus fumigatus (AF) in vivo with the action of human or mouse phagocytes against AF in vitro, we delineated two sequential lines of defense against AF. The first line of defense was formed by macrophages and directed against spores. Macrophages prevented germination and killed spores in vitro and rapidly eradicated conidia in vivo, even in neutropenic and athymic mice. The second was the neutrophilic granulocyte (PMN), which protected against the hyphal form of AF. Human and mouse PMN killed mycelia in vitro. Normal, but not neutropenic mice, stopped hyphal growth, and eradicated mycelia. Either line of defense acting alone protected mice from high challenge doses. Natural immunity collapsed only when both the reticuloendothelial system and PMN were impaired. These findings are in keeping with the clinical observation that high doses of cortisone and neutropenia are the main risk factors for invasive aspergillosis. Cortisone inhibited the conidiacidal activity of mouse macrophages in vivo and of human or mouse mononuclear phagocytes in vitro. Cortisone damaged this first line of defense directly and not through the influence of T lymphocytes or other systems modifying macrophage function as shown in athymic mice and in vitro. In addition, daily high doses of cortisone in mice reduced the mobilization of PMN so that the second line of defense was also impaired. Thus, cortisone can break down natural resistance on its own. Myelosuppression rendered mice susceptible only when the first line of defense was overpowered by high challenge doses, by activated spores that cannot be killed by macrophages, or by cortisone suppression of the conidiacidal activity of macrophages. The host, thus, can call upon two independent phagocytic cell lines that form graded defense systems against aspergillus. These lines of defense function in the absence of a specific immune response, which seems superfluous in the control and elimination of this fungus.

Phagocytosis, chemoluminescence, and intracellular killing of fungi by phagocytes from subjects with deficiency of the second component of complement.

The capacity of phagocytes from animals or humans with complement component deficiency to ingest and kill Candida albicans has been much disputed. We show that peripheral blood polymorphonuclear leukocytes and mononuclear phagocytes from subjects with hereditary C2 deficiency (C2D) ingested C. albicans or Saccharomyces cerevisiae at an abnormally slow rate. After preincubating C. albicans in C2D plasma, the slow rate of phagocytosis was corrected and subsequent intracellular killing of C. Albicans was normal. A normal number of C2D phagocytes reduced nitroblue tetrazolium after stimulation with either phorbol myristate acetate or ingestion of C. albicans. The rate at which chemoluminescence was generated in response to C. albicans was abnormally slow, but peak chemoluminescence produced by C2D phagocytes in response to C. albicans was normal.

Characterization of the leucocytic infiltrate of rheumatoid synovium from tissue sections and synovial eluates.

Connective tissue diseases and rheumatoid arthritis (RA) are characterized by polyclonal B-cell hyperreactivity associated with an oligoclonal, disease-related pattern of autoantibody production. The underlying defects in immune regulations may be found either in the peripheral blood or locally at sites of lymphocyte/plasma cell infiltration. Our interest has focused on the local microenvironment of the inflammed joint as the main site for RA. The dynamic changes in histopathology-cellular infiltration and fibroblast proliferation- characteristic of RA-synovitis require separate analysis of the different states of the disease. In contrast to the morphological approach, a functional analysis of joint inflammation requires isolating the infiltrating cells. In this study, we report first on the distribution of mononuclear and polymorphonuclear (PMN) phagocytes in paraffin sections of different stages of RA-synovitis. Second, infiltrating lymphocytes were isolated from RA-synovium and the cellular eluates analysed for T-cell subpopulations.

Investigation of the determinants of the survival of Neisseria gonorrhoeae within human polymorphonuclear phagocytes.

In studies aimed at identifying the determinants responsible for the ability of gonococci to survive and grow within human phagocytes, the reduction of intracellular survival of phagocyte-resistant gonococci by prior treatment of the bacteria with homologous antiserum provided two indirect means of testing for possible determinants. First, surface washes of the resistant gonococci and fractions of these extracts were examined for ability to neutralize the above effect of antisera. Second, antisera raised to purified surface components of the resistant organisms were examined for ability to promote intracellular killing. A combination of these methods indicate that the aggressins were not pili but components of the outer membrane. A surface wash of a phagocyte-resistant, pilated strain, BS4 (agar), neutralized the activity of homologous antisera before and after centrifuging at 20 000 g, but pili separated from the supernatant did not. A corresponding supernatant from the surface wash of a phagocyte susceptible strain, BSSH, had little neutralizing ability. Antisera to pili failed to reduce intracellular survival of resistant gonococci, whereas antisera against outer membrane vesicles did so. Finally, the neutralizing activity of the surface wash supernatant was removed by centrifugation after treatment with wheat germ agglutinin, which precipitates outer membrane vesicles.

Antibody-mediated inhibition of phagocytosis in Leishmania donovani-human phagocyte interactions in vitro.

Amastigotes and promastigotes of Leishmania donovani were used as antigens for immunization of rabbits which produced anti-amastigote IgG demonstrable by indirect immunofluorescent tests. Effects of these and normal rabbit heat-inactivated sera on phagocytosis of amastigotes by a mixture of human mononuclear and polymorphonuclear phagocytes were studied in vitro. Parasites and phagocytes at different ratios were mixed and incubated at 37 degree C for various time periods in the presence of these sera at different dilutions. In all cases, phagocytosis of amastigotes was higher in neutrophils than in monocytes, and in most cases increased with time in both. The numbers of intracellular amastigotes were, however, consistently lower in cultures with anti-amastigote antiserum (anti-A) than in those with anti-promastigote antiserum (anti-P) or normal rabbit serum. The differential effect of anti-A and anti-P implies differences in antigenic determinants between these two leishmanial forms. A similar, but less pronounced, effect of anti-A was also observed by using opsonized chronic granulomatous disease (CGD) whose polymorphonuclear phagocytes were shown earlier to have only limited leishmanicidal activity. Results of kinetic study and those with CGD cells suggest that inhibition of phagocytosis rather enhanced leishmanicidal activity accounts for the anti-A-mediated decrease of intracellular amastigotes in human phagocytes. Blockade of phagocyte Fc receptors by soluble antigen-antibody immune complexes released during incubation is proposed as a possible mechanism of this inhibition for future investigation.

Leishmanicidal mechanisms of human polymorphonuclear phagocytes.

Human phagocytes isolated from peripheral blood were infected in vitro with Leishmania donovani amastogotes derived from infected hamster spleens. Phagocytosis of the parasites occurred without specific opsonization and the phagocytic efficiency of various cell types was in the order: neutrophil greater than monocyte greater than eosinophil. Light and electron microscopy showed that amastigotes were often degraded by polymorphonuclear phagocytes, but not by monocytes. Ultrastructural cytochemistry for lysosomal enzymes indicated lysosomephagosome fusion after the intracellular entry of the parasites. Reaction products for H2O2 were also detected in parasitophorous vacuoles of all cell types. Exposure of amastigotes to polymorphonuclear phagocytes at 37 degree C resulted in fewer promastigotes emerging subsequently at 27 degree C than in controls without phagocytes. By the same assay method, polymorphonuclear phagocytes from patients with chronic granulomatous disease showed limited leishmanicidal activity. A mixture of phagocyte enzyme extract, H2O2 and Cl-, Br- or I- at pH 5 showed leishmanicidal activity. The leishmanicidal mechanisms of these cells are, thus, attributable to their myeloperoxidase-H2O2-halide microbicidal system and oxygen metabolites generated by the phagocytosis-induced respiratory burst. A lower level of these microbicidal activities associated with monocytes may account for the ability of amastigotes to survive in these cells.

Degradation in vivo of articular cartilage in rheumatoid arthritis by leucocyte elastase from polymorphonuclear leucocytes.

Using a specific substrate, no leucocyte elastase activity could be detected in 55 synovial fluids, including 29 from patients with rheumatoid arthritis (RA). However, a high percentage of samples contained phagocytic inclusions of elastase, alpha 1-proteinase inhibitor (alpha 1-PI) and alpha 2-macroglobulin (alpha 2-MG) in both the polymorphonuclear (PMN) and mononuclear phagocytes. Immunofluorescence and indirect peroxidase-antiperoxidase staining of articular cartilage (ACA) from 52% of 21 patients with RA and one with juvenile RA (JRA) showed presence of elastase in the superficial layer of microscopically intact but proteoglycan depleted pannus-free ACA. In histologically altered pannus-free RA-ACA superficial elastase deposits were found in 24% of the cases. Adjacent ACA sections contained IgG, C3, alpha 1-PI and rarely alpha 2-MG. RA-ACA below or surrounded by pannus showed close contact with intact and decaying PMN in 62% and 48% of the cases, respectively. ACA specimens from patients with degenerative disease and systemic lupus were negative. These findings strongly suggest that PMN leucocyte elastase is operative in the degradation of RA-ACA and JRA-ACA, and that this activity is largely dependent upon the presence of entrapped immune complexes in such cartilage.

Dissociation of opsonized particle phagocytosis and respiratory burst activity in an Epstein-Barr virus-infected myeloid cell line.

A continuous tissue culture cell line (Karpas line 120), derived from a patient with acute myeloblastic leukemia, not only demonstrates myeloblastic morphology and in vitro expression of several myeloid-specific biochemical markers but also contains Epstein-Barr virus (EBV) nuclear antigen. The present studies demonstrate EBV-genome-specific DNA within the total cellular DNA by molecular hybridization, thus establishing the presence of stable viral genome integration. The cells demonstrate complex coordinated myeloid functions including ingestion, degranulation, and respiratory burst activity. Line 120 cells show a respiratory burst (superoxide and hydrogen peroxide generation and hexosemonophosphate shunt activity) in response to soluble (phorbol myristate acetate) and particulate (latex beads) stimuli, as do normal granulocytes. They ingest complement-opsonized particles (lipopolysaccharide-oil droplets, zymosan, and bacteria), and degranulate in response to them. However, unlike normal granulocytes, the line 120 cells do not demonstrate respiratory burst activity in response to these complementopsonized particles. The dissociation between ingestion of complement-opsonized particles and activation of oxygen-dependent bactericidal activity severely impairs bacterial killing as compared with normal polymorphonuclear phagocytes.

Pr and Gd antigens on human B and T lymphocytes and phagocytes

Pr 1h, Pr 2, and Pr 3 and Gd antigens were detected on the membrane of human peripheral blood and tonsillary B and T lymphocytes, thymocytes, polymorphonuclear phagocytes, and monocytes using bithermic cytotoxicity and immunofluorescence assays. Generally anti-Pr and anti-Gd had weaker cytotoxic activity, as compared to anti-i and anti-I cold agglutinins. Anti-Pr 1h IgA (R.K.) examined by us had very weak cytotoxicity against peripheral blood lymphocytes but bound to more than 70% of the cells. It reduced E and EAC rosetting capacity of the cells and killed strongly tonsillary B cells, thymocytes, and cord blood polymorphonuclears. Anti-Pr 1h IgM was much more cytotoxic to peripheral blood lymphocytes and to other cells. Anti-Pr 2 had very weak cytotoxic activity and bound to only about half of peripheral blood lymphocytes. Anti-Pr 3 was more cytotoxic to peripheral blood lymphocytes and expressed preferential reactivity against peripheral B and tonsillary B cells. Anti-Gd CA had quite a strong cytotoxic activity against all cells tested.

The intracellular survival and growth of gonococci in human phagocytes.

In reassessment of previous tests for intracellular survival, results have been confirmed and additional evidence obtained indicating that some gonococci can survive and multiply in human phagocytes. Use was made of the ability of penicillin to penetrate phagocytes and to kill only actively growing organisms. In microscopic counts on 33 urethral exudate smears, an average of 49% of gonococci were associated with polymorphonuclear phagocytes. The organisms were unevenly distributed amongst the phagocytes, with most cells uninfected and some containing large numbers. Many phagocytes also remained uninfected in tests in vitro with low gonococcal inocula although experiments with large inocula showed that most phagocytes could ingest gonococci. It is proposed that ingestion of one gonococcus may stimulate the phagocytes to take up more. Phagocytes were killed and disintegrated after ingesting large numbers of gonococci and similar effect in vivo may be responsible for the large clumps of organisms seen in urethral exudate. These results underline the probable importance in the pathogenesis of gonorrhoea of intracellular survival in phagocytes.

Demonstration of Intracellular Growth of Gonococci in Human Phagocytes using Spectinomycin to Kill Extracellular Organisms

The use of spectinomycin to kill extracellular bacterial in phagocytosis tests with gonococci and human polymorphonuclear phagocytes allowed the demonstration of a greater degree of intracellular survival and growth than in previous tests using penicillin.

Decreased Mononuclear and Polymorphonuclear Chemotaxis in Human Newborns,Infants, and Young Children

A new method, chemotaxis under agarose gel, was used to assess the directed motility of polymorphonuclear (PMN) and mononuclear (MN) phagocytes of 21 newborns, 71 infants and children, and 50 adults. This assay requires only small quantities of cells and is rapid, easy, reproducible, and provides a permanent record. The chemotactic substance was zymosan-activated human serum. Monocyte chemotaxis in the newborn was approximately 50% of adult control values, using the Boyden chamber (8.1 ± 2 cells per high-power field[HPF] [SE] in newborns compared to 17 ± 3 cells per HPF in adults), and 25% of adult control values, using the agarose method (50 ± 10 cells in newborns compared to 216 ± 15 cells in adults). Both are significant differences (P < .005). MN chemotaxis remains extremely low through age 5, and remains moderately reduced until age 10. PMN chemotaxis in the newborn was 82 ± 21 cells compared to adult controls of 300± 42 cells, also a significant difference (P < .05). PMN chemotaxis remains markedly depressed through age 2. Thereafter, PMN chemotaxis increases but remains significantly less than in adults until age 16. These chemotactic defects may play an important role in depressed delayed hypersensitivity skin tests, diminished inflammatory reactions, and increased susceptibility to infection present in the newborn and young infant.

Resistance of Neisseria gonorrhoeae grown in vivo to ingestion and digestion by phagocytes of human blood.

Attempts to study quantitatively the phagocytosis of gonococci from urethral pus failed because of the small numbers of organisms and technical difficulties. However, gonococci from chambers implanted subcutaneously in guinea pigs, which were similar to gonococci from urethral pus in their resistance to killing by human serum, were obtained in sufficient quantities for comparison in phagocytosis tests with the in vitro grown strains from which they were derived. Microscopic and viable counts of gonococci in phagocytes showed that in vivo grown organisms (strain BSV) were readily phagocytosed by human polymorphonuclear phagocytes. There was little difference betweee to ingestion. There was, however, a marked difference in the intracellular survival of strains BSV and BS during the first hour of phagocytosis. Whereas BSV organisms survived well, many BS organisms were killed. Subsequently, strain BSV and the survivors of the strain BS inoculum responded similarly to the intracellular bactericidins. These results were supported by electron microscopy of infected phagocytes. Resistance of gonococci in vivo to ingestion and digestion by human phagocytes seem to be important facets of the pathogenesis of gonorrhoea.

Penetration of penicillin into human phagocytes containing Neisseria gonorrhoeae: intracellular survival and growth at optimum concentrations of antibiotic.

Phagocytes obtained from fresh human buffy coat (predominantly polymorphonuclear phagocytes) or from human buffy coat which had been incubated on a glass surface for 1 to 3 days (predominantly mononuclear phagocytes) were allowed to ingest gonococci, and then incubated with penicillin. More intracellular gonococci were killed at high than at low penicillin concentrations, indicating that penicillin penetrated the phagocytes. This was supported by autoradiography experiments with radiolabelled penicillin. A pilated, small-colony-forming gonococcal strain survived and multiplied for at least 15 h in polymorphonuclear phagocytes which were incubated with penicillin at the optimum concentration for killing the extracellular bacteria but not the intracellular ones; whereas a non-pilated, large-colony-forming strain survived for only 10 h. The former strain survived for at least 6 h in similar experiments with mononuclear phagocytes. Intracellular survival and in growth may be an important facet of the pathogenicity of gonococci.

Non-glial phagocytes within the degenerating optic nerve of the newt (Triturus viridescens).

Two non-glial phagocytes were found to participate along with ependymoglial cells in Wallerian degeneration of the severed optic nerve of the newt (Triturus viridescens). The first type of non-glial cell (polymorphonuclear phagocyte) was positively identified as a neutrophil and participates in the early stages of degeneration. Cells of this type make a brief appearance, reaching a peak by the second postoperative day (2 p.o.d.), and quickly diminish until few can be found by 4 p.o.d. Neutrophils invade the degenerating optic nerve from surrounding connective tissue spaces, most likely, through channels which penetrate the nerve parenchyma. The second type of non-glial cell is an invading mononuclear phagocyte which exhibits characteristics of microglial cells reported in other vertebrate species. Such cells appear in the nerve much later than the neutrophils and towards the end of Wallerian degeneration (6-10 p.o.d.). Their mode of entry and exit appears to be the same as that reported for neutrophils. The neutrophils and microglial-like, mononuclear phagocytes may serve to supplement the histolytic action of the ependymoglial cells, picking up scattered fragments of degenerating myelin and axons.

The influence of homogeneous cold agglutinins on polymorphonuclear and mononuclear phagocytes

Homogeneous IgM cold agglutinins of anti-I and anti-i types were cytotoxic to the cord and adult polymorphonuclear leukocytes and mononuclear phagocytes. An assay in which the cells were incubated with cold agglutinins and fresh rabbit serum as a source of complement for the first hour at 4°C, and for the second hour at 25°C was employed. The killing of polymorphonuclear leukocytes varied from 7% to 76% and was consistently higher using anti-i cold agglutinins. A certain proportion of polymorphonuclear leukocytes that survived the exposure to cold agglutinins lost their phagocytic activity, while others that were still able to phagocytize engulfed a smaller number of particles than control cells. We conclude that cold agglutinins are cytotoxic in vitro to polymorphonjclear and mononuclear phagocytes and that they decrease the phagocytic activity of the surviving cells. The susceptibility of phagocytes to the cytotoxic action of cold agglutinins of both anti-i and anti-I types implies the existence of Ii antigens on their surface.

Electron microscopy of the in vivo internalization of virulent Chlamydia psittaci 6BC strain.

The internalization of virulent Chlamydia psittaci 6BC particles by wandering mononuclear phagocytes in the peritoneal cavity of intraperitoneally inoculated mice occurred asynchronously, i.e., fragile reticulate bodies (RB) appeared to be more readily phagocytized than the rigid elementary bodies (EB). Early damage of mononuclear phagocytes occurred after internalization of chlamydiae. This was followed by a decreased uptake of particles, and may explain the relatively long persistence (up to 6 h after inoculation) of free, extracellular, "swollen", and RB-like particles. Internalized particles within phagolysosomes showed varying degrees of disintegration. The subsequent influx of polymorphonuclear phagocytes and monocytes into the inflammed peritoneal cavity may explain the rapid disappearance of chlamydiae and their antigens from the peritoneal fluid. The alteration in ultrastructure of peritoneal cells and chlamydial parasites during the inflammatory process are discussed.