Synthesis and release of platelet-activating factor by stimulated human mononuclear phagocytes

Platelet-activating factor (PAF) is a potent phospholipid mediator that may participate in inflammatory responses by virtue of its ability to activate platelets, leukocytes, and vascular cells. We examined the synthesis and release of PAF by human peripheral blood monocytes (PBM) isolated by countercurrent elutriation. PAF was produced after stimulation by calcium ionophore A23187 (IoA), opsonized zymosan (OpsZ), and PMA with a relative order of potency IoA much greater than OpsZ greater than PMA. The portion of PAF subsequently released from the cell was dependent on the specific agonist, the time of incubation, and the presence of albumin. Under optimal conditions, PBM released 67, 49 and 32% of the total PAF produced in response to IoA, OpsZ, and PMA, respectively. Changes in PAF metabolism were observed in PBM that were examined after short term adherence or differentiation into macrophages. Adherent PBM accumulated and released less PAF than suspended monocytes, and monocyte-derived macrophages produced less PAF than the parent PBM. The ability of monocytes to release significant amounts of newly synthesized PAF from the cell is unusual among human cell types, which in general retain the vast majority of the lipid, and may be of particular pathophysiologic importance.     

Overproduction, purification, and characterization of beta-1,3-glucanase type II in Escherichia coli.

An Escherichia coli recombinant system produced soluble and full-length beta-1,3-glucanase type II (BglII) cloned from the yeast-lytic actinomycete Oerskovia xanthineolytica. The expression system was designed to produce recombinant BglII with a six-histidine peptide fused to the carboxy end of the protein. The expression level was optimized to produce 30% of total protein of E. coli as the recombinant protein, releasing 75% to the extracellular space. The 43-kDa recombinant protein was purified by IMAC to homogeneity and its molecular and biochemical characteristics were studied, showing that there are no important functional differences with those properties described for the BglII purified from O. xanthineolytica.     

Leukotriene B4 generation by human monocytes and neutrophils stimulated by uropathogenic strains of Escherichia coli

The generation of the 5-lipoxygenase product, leukotriene B4 (LTB4) by human mononuclear phagocytes (monocytes) following incubation with 25 different uropathogenic strains of Escherichia coli correlated with the haemolytic activity of the strains (r = 0.572, P less than 0.01). LTB4 generation by human neutrophils (PMN), however, was unrelated to this haemolytic potential (r = 0.164). In contrast, both prelabelled monocytes and PMN were stimulated by haemolytic strains of E. coli and by haemolytic culture supernatants to release significant amounts of [3H]arachidonic acid. There was a significant correlation between haemolytic activity and [3H]arachidonic acid release generated by individual strains from monocytes (r = 0.804, P less than 0.001) and PMN (r = 0.888, P less than 0.001). In addition, nonhaemolytic strains but not their culture supernatants were capable of causing slow release of both [3H]arachidonic acid and LTB4 from PMN and mononuclear cells. These results suggest that both the possession of haemolytic activity, and the direct interaction of bacteria with the leukocyte surface are mechanisms by which uropathogenic strains of E. coli may cause the release and metabolism of arachidonic acid. In addition, there was synergistic augmentation by nonhaemolytic bacteria of the PMN LTB4 response to haemolytic culture supernatants or to low doses of the calcium ionophore A23187. These results support an ionophore-like mechanism for the activation of the cell by haemolysin. LTB4 generation by PMN incubated with haemolytic supernatants was also augmented by particulate zymosan in a manner dependent on the dose of zymosan, suggesting that the direct interaction of E. coli with PMN may involve an activation mechanism similar to that for zymosan. These results demonstrate differing responses of peripheral mononuclear cells and PMN from the same donors to identical strains of E. coli and suggest that the generation of the potent chemotactic agent LTB4 in response to E. coli infection in vivo need not depend solely on the elaboration of cytotoxic haemolysins by individual strains.     

Characterization of two beta-1,3-glucosyltransferases from Escherichia coli serotypes O56 and O152

The O antigens of outer membrane-bound lipopolysaccharides (LPS) in gram-negative bacteria are oligosaccharides consisting of repeating units with various structures and antigenicities. The O56 and O152 antigens of Escherichia coli both contain a Glc-beta1-3-GlcNAc linkage within the repeating unit. We have cloned and identified the genes (wfaP in O56 and wfgD in O152) within the two O-antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid [GlcNAc-alpha-PO3-PO3-(CH2)11-O-phenyl] was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode glucosyltransferases. Enzyme products from both glucosyltransferases were isolated by high-pressure liquid chromatography and analyzed by nuclear magnetic resonance. The spectra showed the expected Glc-beta1-3-GlcNAc linkage in the products, confirming that both WfaP and WfgD are forms of UDP-Glc: GlcNAc-pyrophosphate-lipid beta-1,3-glucosyltransferases. Both WfaP and WfgD have a DxD sequence, which is proposed to interact with phosphate groups of the nucleotide donor through the coordination of a metal cation, and a short hydrophobic sequence at the C terminus that may help to associate the enzymes with the inner membrane. We showed that the enzymes have similar properties and substrate recognition. They both require a divalent cation (Mn2+ or Mg2+) for activity, are deactivated by detergents, have a broad pH optimum, and require the pyrophosphate-sugar linkage in the acceptor substrate for full activity. Substrates lacking phosphate or pyrophosphate linked to GlcNAc were inactive. The length of the aliphatic chain of acceptor substrates also contributes to the activity.     

In vitro and in vivo activation of human mononuclear phagocytes by interferon-gamma. Studies with normal and AIDS monocytes

To determine the potential immunotherapeutic role of interferon-gamma (IFN-gamma) as a mononuclear phagocyte-activating agent, we examined the effector cell function of peripheral blood monocytes from healthy donors and acquired immunodeficiency syndrome (AIDS) patients after either in vitro and/or in vivo treatment with recombinant (r) IFN-gamma. When assayed immediately after a 24-hr in vitro pulse with 300 U/ml, normal and AIDS monocytes behaved similarly with little augmentation of their intrinsically high levels of H2O2 release and activity against Toxoplasma gondii; in contrast, activity toward the more resistant intracellular pathogen, Leishmania donovani, was appreciably enhanced by rIFN-gamma. In addition, upon testing 4 to 6 days after in vitro pulsing, both normal and AIDS monocytes showed clear evidence of persistent activation in all three assays. The capacity of IFN-gamma to similarly activate monocytes in vivo was confirmed in all ten treated AIDS patients by examining cells before and after 24-hr infusions of 0.03 and 0.5 mg of rIFN-gamma/square meter (M2) of body surface area. For postinfusion monocytes tested after 1 day in culture, H2O2 release and antitoxoplasma activity were essentially unchanged, but antileishmanial effects were augmented. After 5 to 7 days in culture, monocytes from treated patients showed 3.2- to 5.9-fold increases in H2O2-releasing capacity and increases of 49 to 68% and 35 to 61% in intracellular activity against T. gondii and L. donovani, respectively. These results indicate that the human monocyte can be induced by rIFN-gamma to express signs of both immediate and persistent activation and suggest that, as a direct activator of mononuclear phagocytes, rIFN-gamma may also have potential as an immunotherapeutic agent for patients with intracellular infections.     

Superoxide production by phagocytes. Another look at the effect of cytochrome c on oxygen uptake by stimulated neutrophils.

The widely held view that stimulated phagocytes liberate $\text{O}{}_2{}^{\mathrm{?}}$ into the extracellular medium is supported by the alterations in oxygen uptake which occur when ferricytochrome $\text{c}$ is added to a suspension of zymosan-treated neutrophils. An explanation consistent with this view is provided for some previously reported results $\text{(}\text{FEBS Lett}\text{. 100}$, 27) which initially appeared to conflict with the notion that $\text{O}{}_2{}^{\mathrm{?}}$ is released by phagocytes.     

Structure of beta-1,3-xylooligosaccharides generated from Caulerpa racemosa var. laete-virens beta-1,3-xylan by the action of beta-1,3-xylanase

Recently we reported the molecular cloning and characterization of a novel beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 [Kiyohara et al. (2005) Biochem. J. 388, 949-957]. We report here the structural analysis of oligosaccharides generated from beta-1,3-xylan of a siphonous green alga, Caulerpa racemosa var. laete-virens, by the action of beta-1,3-xylanase. The enzyme degraded the polysaccharide producing oligosaccharides with different R(f)s on TLC (EX2-EX5). Sugar component, linkage, and MALDI-TOF-MS analyses revealed that EX2 and EX3 were Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,3-Xyl, respectively. On the other hand, EX4 was a mixture of Glc-1,3-Xyl-1,3-Xyl, Xyl-1,4-Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,4-Xyl, while EX5 was a mixture of tetra-saccharides containing 3-substitued Glc in addition to the same components of EX4. Branching was not likely present in EXOs prepared from the polysaccharide by the enzyme. These results strongly suggest that the C. racemosa beta-1,3-xylan is a linear heteropolysaccharide containing 1,3-Glc and 1,4-Xyl both of which are thought to be located within a beta-1,3-Xyl chain and linked via covalent bonds. This report indicates the usefulness of the enzyme for the structural analysis of beta-1,3-xylan.     

Disparities in TLR5 expression and responsiveness to flagellin in equine neutrophils and mononuclear phagocytes

As sentinel cells of the innate immune system, neutrophils and mononuclear phagocytes use specific TLRs to recognize the conserved molecular patterns that characterize microbes. This study was performed to compare the responses of equine neutrophils and mononuclear phagocytes to LPS and flagellin, components of bacteria that are recognized by TLR4 and TLR5, respectively. Neutrophils and mononuclear phagocytes isolated from healthy horses were incubated in vitro with LPS, flagellin, or pronase-inactivated flagellin in the presence or absence of polymyxin B. Production of reactive oxygen species and expression of mRNA for proinflammatory cytokines were used as readouts for activation of neutrophils; production of TNF-α was used for the mononuclear cells. Western blot analysis and flow cytometry were used to detect TLR5 protein in both cell types. Although the neutrophils responded to both LPS and flagellin by producing reactive oxygen species and expressing mRNA for proinflammatory cytokines, flagellin had no stimulatory effect on monocytes or macrophages. Although both neutrophils and monocytes expressed mRNA for TLR5, it appeared to be translated into protein only by the neutrophils. Incubation with neither LPS nor IFN-γ altered TLR5 expression by the monocytes. These findings indicate that flagellin has disparate effects on neutrophils and mononuclear phagocytes isolated from horses, a species that is exquisitely sensitive to the TLR4 ligand, LPS, and that equine mononuclear phagocytes, unlike corresponding cells of other mammalian species, lack surface expression of TLR5 and do not respond to flagellin.     

In vitro determination of phagocytosis and intracellular killing of Aspergillus species by mononuclear phagocytes

We investigated phagocytosis and intracellular killing of clinical and environmental isolates of Aspergillus spp. by human monocyte-derived macrophages (MDMs). Serial pathogens such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus were examined with a microbiological assay. Phagocytosis for resting conidia of Aspergillus spp. was similar for all isolates tested. During 30 min of incubation phagocytosis ranged from 49.9% to 85.5% for clinical isolates and from 40.3% to 87.1% for environmental isolates. MDMs killed A. fumigatus, A. flavus and A. terreus conidia after ingestion for 120 min, as shown by a decrease in colony forming units (cfu) count of intracellular fungi. The killing index for all isolates of Aspergillus spp., ranged from 12.1 ± 1.1% to 90.3 ± 10.4%; isolate-dependent (P < 0.01) differences against the fungicidal action of MDMs were observed. In conclusion, significant differences were noted for killing indices between several strains of Aspergillus spp. whereas phagocytosis was similar for all isolates tested in vitro. No differences were observed within environmental and clinical isolates.     

Variability of Photodynamic Killing in Escherichia coli and Avoidance of Variability with Agar

Photodynamic killing of Escherichia coli in acridine orange is influenced by the composition of the containing vessel, and after high kill the variance between replicate suspensions is greater than attributable solely to sampling and plating. Addition of agar minimizes both phenomena, but a higher illumination dose is required to produce the same degree of killing.     

Increased nitroblue tetrazolium dye reduction by human neutrophils stimulated by interferon in vitro

Human leukocyte interferon enhanced nitroblue tetrazolium dye (NBT) reduction by human neutrophils (PMNs). Increase in NBT reduction paralleled increase in interferon dose. When human leukocyte interferon was heated to 60 C or 80 C for 30 min, both the antiviral activity and the effect on NBT reduction decreased. Human leukocyte interferon neutralized with anti-human leukocyte interferon serum showed no effect on NBT reduction. A human fibroblast interferon preparation also enhanced NBT reduction. The species dependency of interferon was shown in NBT reduction as well as in antiviral activity.     

Experimental salmonellosis. VII. In vitro transfer of cellular immunity by ribosomal fraction of mouse mononuclear phagocytes

Sato, Ichiei (Gunma University, Maebashi, Japan), and Susumu Mitsuhashi. Experimental salmonellosis. VII. In vitro transfer of cellular immunity by ribosomal fraction of mouse mononuclear phagocytes. J. Bacteriol. 90:1194-1199. 1965.-The mononuclear phagocytes (termed monocytes) of mice hyperimmunized with live vaccine of Salmonella enteritidis inhibited the intracellular growth of virulent strain 116-54 of S. enteritidis. Also, the monocytes withstood the degeneration of cells caused by the phagocytosis of bacteria in the absence of immune serum in the tissue culture medium, termed cellular immunity. When the nonimmune monocytes were incubated with the ribosomal fraction of immune monocytes, obtained from the abdominal cavity of mice hyperimmunized with live vaccine of S. enteritidis, they acquired cellular immunity, but the monocytes did not acquire immunity when ribosomal fractions from normal mouse monocytes or from the monocytes of mice immunized with killed vaccine of S. enteritidis were used. The transfer agent present in the ribosomal fraction of immune monocytes was inactivated by treatment with ribonuclease but not with deoxyribonuclease or with trypsin.     

Kinetics of superoxide production by stimulated neutrophils.

Oxygen-derived active species and superoxide radical in particular are generated and excreted upon granulocyte activation and are instrumental in host defense against bacterial and fungal infections. Associated with the activation of neutrophils is an apparent transitory oxy-radical production. Evidence from independent methods has previously suggested that radical production peaks shortly following neutrophil stimulation and decays within minutes. However, since neutrophil function in the body might reasonably be expected to last beyond the few minutes following stimulation, cessation of the production of oxy-radicals is unexpected. In an attempt to reconcile this discrepancy, the formation kinetics of superoxide by stimulated human neutrophils was reinvestigated by three independent methods: electron spin resonance, chemiluminescence, and ferricytochrome c reduction. The present results demonstrate that under appropriate experimental conditions stimulated neutrophils have the capacity to produce superoxide for several hours. The reasons for the previously reported "apparent" ephemeral nature of oxy-radical formation are discussed.     

Respiratory burst activated by Escherichia coli in human neutrophils primed with different lipopolysaccharides

Endotoxic shock is a dangerous complication of infection caused by gram-negative bacteria. Searching for agents capable to block the effects of endotoxins is a fundamental scientific and medical problem. Here we studied the effects of neutrophil priming with non-toxic lipopolysaccharide from Rhodobacter capsulatus (LPS _{Rb. caps.} ) and toxic LPS from Escherichia coli (LPS _{E. coli} ) on the respiratory burst evoked in neutrophils by opsonized E. coli. We found that preincubation of neutrophils with each of the LPS increased ROS production by neutrophils as compared to non-primed neutrophils. Subsequent incubation of neutrophils with LPS _{Rb. caps.} and then with LPS _{E. coli} practically abolished the effects of both endotoxins.     

Piroxicam enhances phagocytosis of Escherichia coli by human polymorphonuclear neutrophils

Human polymorphonuclear neutrophils were exposed to piroxicam, 1.5 uM, 15 uM and 150 uM during phagocytosis of radiolabelled Escherichia coli in vitro. Preincubation of the cells for 30 minutes before phagocytosis stimulated the uptake of E. coli at all concentrations. The elimination of substances of bacterial origin from the neutrophis in the postingestion phase was, however, not influenced by piroxicam.     

Abnormal in vitro proliferation of splenic mononuclear phagocytes from autoimmune motheaten mice

Motheaten mice develop combined immunodeficiency and fatal autoimmune disease that follow autosomal recessive inheritance. In splenocyte cultures of motheaten mice, supplemented with 5% normal serum proliferating cells (MP) were present exhibiting morphologic characteristics of mononuclear phagocytes at light and electron microscopic levels. The macrophage nature of these cells was confirmed by the lack of Thy-1 antigen and immunoglobulins; the expression of Mac-1 antigen, FcR for IgG, and Ia antigens on their cell surfaces; their ability to phagocytize EA and adhere to plastic; the presence of nonspecific esterase and lysomal enzymes in their cytoplasm; and the pattern of peroxidase localization similar to monocyte-derived macrophages. MP from motheaten mice exponentially grew in culture in the absence of exogenous growth factors with a doubling time of approximately 76 hr. Although these cells were present in splenocyte cultures of normal controls, their number did not increase during the culture period under the same conditions. The addition of dextran sulfate further enhanced the proliferation of MP from motheaten mice, and induced exponential growth of these cells from normal controls, reaching only the level of unstimulated cells from motheaten mice. Radioautographic analysis demonstrated that MP substantially contributed to the elevated spontaneous and dextran sulfate-induced DNA synthesis in splenocyte cultures. Therefore, the in vitro abnormality of MP may be indicative of in vivo aberrancies of macrophages from motheaten mice and lends credence for investigating the role of macrophages in immunodeficiency and autoimmunity that develop very early in motheaten mice.     

Deoxyribonucleic acid repair in vitro by extracts of Escherichia coli.

Deoxyribonucleic acid (DNA) from bacteriophage T7 has been used to monitor the capacity of gently lysed extracts of Escherichia coli to perform repair resynthesis after ultraviolet (UV) irradiation. Purified DNA damaged by up to 100 J of UV radiation per m2 was treated with an endonuclease from Micrococcus luteus that introduces single-strand breaks in irradiated DNA. This DNA was then used as a substrate to study repair resynthesis by extracts of E. coli. It was found that incubation with the extract and exogenous nucleoside triphosphates under suitable assay conditions resulted in removal of all pyrimidine dimers and restoration of the substrate DNA to its original molecular weight. Repair resynthesis, detected as nonconservative, UV-stimulated DNA synthesis, was directly proportional tothe number of pyrimidine dimers introduced by radiation. The repair mode described here appears to require DNA polymerase I since it does no occur at the restrictive temperature in polA12 mutants, which contain a thermolabile polymerase. The addition of purified DNA polymerase I to extracts made from a polA mutant restores the ability to complete repair at the restrictive temperature.