Rapid killing of monocytes in vitro by Candida albicans yeast cells.

To study the interaction between Candida albicans blastoconidia and human phagocytes, we incubated peripheral leukocytes with fungi for 1 h at 37 degrees C and stained the cells with fluorescent vital stains ethidium bromide (EB) and fluorescein diacetate. Fungi that had been phagocytosed showed little staining; however, some leukocytes containing blastoconidia exhibited nuclear staining with EB, even though their cell membranes showed no signs of penetration by fungi. The number of EB-positive leukocytes was related to viability of the yeast cells and the temperature at which they were maintained before use. Because efforts to quantitate EB-positive leukocytes microscopically were frustrated by cell aggregation, we labeled the leukocytes with 51Cr and measured isotope release. We determined that leukocytes incubated with viable fungi released significantly more isotope than cells incubated alone or with killed blastoconidia. Furthermore, 51Cr release correlated directly with concentration of fungi in the assay, time of incubation, and temperature at which fungi were maintained before use. Using a number of isolates of C. albicans and several other species of Candida, we found that all exhibited cytotoxic activity against leukocytes, but the level of activity varied among organisms. Finally, we depleted or enriched peripheral leukocytes for specific cell populations and determined that only monocytes released more 51Cr after incubation with viable blastoconidia. Blastoconidia can lyse phagocytic cells through germination and penetration of cell membranes within 1 to 2 h, but the cytotoxic phenomenon we describe occurs within 15 to 30 min after yeast cells have been phagocytosed. Therefore, this capacity may represent a more immediate response by blastoconidia against phagocytosis and killing by monocytes.     

Alteration of polymorphonuclear leukocyte activity by viable Candida albicans.

The response of human polymorphonuclear leukocytes (PMN) to blastospores and pseudo-hyphae of the opportunistic fungus Candida albicans has been studied in vitro and in vivo. Of the fungicidal mechanisms elucidated thus far, the myeloperoxidase-hydrogen peroxide-halide system appears to be most effective against cells of this fungus. In our studies on the interaction between murine PMN and blastospores, we assayed the release of H2O2 by PMN incubated with viable or killed, unopsonized or opsonized blastospores by using two assay systems, lysis of murine erythrocytes and oxidation of scopoletin. Our results showed that PMN released increasing amounts of H2O2 when incubated with increasing numbers of opsonized or unopsonized killed blastospores, but released decreasing amounts of H2O2 when incubated with increasing numbers of opsonized or unopsonized viable blastospores. The oxidative metabolic burst by PMN in the presence of viable or killed blastospores was also measured by using reduction of nitroblue tetrazolium and chemiluminescence. Viable blastospores stimulated a stronger metabolic burst than killed blastospores, suggesting that PMN respond to live blastospores more vigorously than killed blastospores; however, live blastospores appear to alter or inhibit the release of H2O2 by PMN.     

Inhibition of Streptococcus mutans by the lactoperoxidase antimicrobial system.

Inhibition of bacterial metabolism by the lactoperoxidase (LP)-hydrogen peroxide (H2O2)-thiocyanate system was studied with representatives of serotypes a through g of Streptococcus mutans. The aims were to determine whether the amount of H2O2 released from these catalase-negative bacteria is sufficient to activate the LP system and whether these oral bacteria are resistant to inhibition by the LP system, which is active in human saliva. When the washed, stationary-phase cells were incubated aerobically with LP, thiocyanate, and glucose (Glc), greater than 90% inhibition of Glc utilization and lactate production was obtained with strains that released large amounts of H2O2 (BHT, FA-1, OMZ-176); 20 to 50% inhibition was obtained with strains that released about half as much H2O2 (B-13, Ingbritt); and no inhibition was obtained with strains that released only small amounts of H2O2 (AHT, HS-6, GS-5, LM-7, OMZ-175, 6715-15). Inhibition was most effective at pH 5, whereas release of H2O2 and accumulation of the inhibitor (hypothiocyanite ion) were highest at pH 8. With H2O2-releasing cells from early stationary phase, preincubation with Glc abolished inhibition, though it did not influence H2O2 release. Cells harvested 24 h later were depleted of sulfhydryl compounds. Inhibition of these cells was abolished by preincubation with Glc and certain sulfhydryl or disulfide compounds (reduced or oxidized glutathione, cysteine or cystine). This preincubation increased cell sulfhydryl content but had no effect on H2O2 release. All strains were inhibited when incubated with LP, thiocyanate, and added (exogenous) H2O2. Smaller amounts of H2O2 were required to inhibit at pH 5, and larger amounts were required to inhibit cells preincubated with Glc or with Glc and the sulfhydryl or disulfide compounds. The results indicate that pH, amount of H2O2, cell sulfhydryl content, and stored-carbohydrate content determine susceptibility to inhibition.     

Involvement of reactive oxygen metabolites in the candidacidal activity of human neutrophils stimulated by muramyl dipeptide or tumor necrosis factor

In the presence of the adjuvant glycopeptide muramyl dipeptide (MDP), purified human PMN exhibited an enhanced capacity to kill Candida albicans cells at various cell ratios. A significant effect was obtained at 100 ng/ml MDP, and the maximum was reached at 1 micrograms/ml MDP. Recombinant human tumor necrosis factor (rHuTNF), a monokine that enhances host resistance to bacterial and fungal infections, also stimulated the candidacidal potency of PMN with a maximal effect at 10(-2) ng/ml rHuTNF. When MDP- or rHuTNF-stimulated PMN were cultured with yeast cells, the intracellular production of oxygen metabolites was enhanced. Pretreatment with inhibitors of oxidative burst demonstrated that the yeast cell killing by MDP-stimulated PMN was not affected by SOD but was inhibited by sodium azide, indicating the involvement of myeloperoxidase (MPO)-halide system in fungicidal mechanisms induced by MDP. When PMN were stimulated with rHuTNF, the killing of yeast cells was neutralized by iodoacetamide, showing that the candidacidal potency of stimulated-PMN was due to oxygen derivatives. Inhibition by sodium azide and sodium benzoate indicated that these oxygen metabolites could be derived from the MPO-halide system but also from hydroxyl radical production. Moreover, SOD partially inhibited the fungicidal potency of rHuTNF-stimulated PMN, thus indicating a possible reutilization of the released O2- anion for intracellular killing. Cytochalasin B abrogated the PMN fungicidal potency in all cases.     

Stimulation of human polymorphonuclear leukocyte oxidative metabolism by type 1 pili from Escherichia coli.

We compared the degree to which Escherichia coli phase variants which do (T1P+ E. coli) or do not (T1P- E. coli) express type 1 pili (T1P) stimulate human polymorphonuclear leukocyte (PMN) oxidative activity. Unopsonized T1P+ E. coli stimulated the release of 0.20 to 0.24 nmol of H2O2 per 10(6) PMN per min and the consumption of 1.4 to 4.0 nmol of O2 per 10(6) PMN per min; no measurable PMN oxidative activity was stimulated by unopsonized T1P- E. coli. In the presence of serum opsonins, T1P+ E. coli stimulated the release of 1.12 to 1.16 nmol of H2O2 per 10(6) PMN per min and the consumption of 5.0 to 6.0 nmol of O2 per 10(6) PMN per min, whereas T1P- E. coli stimulated the release of 0.42 to 0.43 nmol of H2O2 per 10(6) PMN per min and the consumption of 0.6 to 2.0 nmol of O2 per 10(6) PMN per min. Although unaggregated T1P did not stimulate PMN, latex beads coated with T1P (T1P-latex) stimulated alpha-methylmannoside-inhibitable, opsonin-independent PMN oxidative activity. The activity stimulated by either T1P+ E. coli or T1P-latex was susceptible to inhibition by cytochalasin B. Latex particles coated with bovine serum albumin or mannose-resistant pili did not stimulate PMN. These data indicate that T1P+ E. coli stimulate PMN oxidative metabolism more effectively than do T1P- E. coli and that a similar PMN oxidative response follows cellular stimulation by either unopsonized T1P+ or opsonized T1P- E. coli. Furthermore, T1P-latex faithfully mimics the ability of T1P+ E. coli to stimulate PMN oxidative metabolism. Such particles may be useful in further analyses of cellular responses to T1P+ E. coli.     

The sensitivity of Porphyromonas gingivalis and Fusobacterium nucleatum to different (pseudo)halide-peroxidase combinations compared with mutans streptococci

Previous studies have shown that the peroxidase system with iodide is particularly effective against Actinobacillus actinomycetemcomitans. In the present study, the effects of iodide, chloride and thiocyanate in combinations with lactoperoxidase (LP) and myeloperoxidase (MP) on the viability of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and S. rattus were analysed. Bacteria were incubated in buffer solution containing peroxidase, substrate(s) and H2O2 (all in oral physiological concentrations), and plated after 0, 0.5 and 1 h. The oxidation product of iodide was the most bactericidal against all the bacteria tested. The effect was significantly weaker on mutans streptococci. Physiological concentrations of thiocyanate abolished the effects of LP-H2O2-iodide and MP-H2O2-iodide/chloride combinations. Thiocyanate-peroxidase systems have already been used in oral hygiene products. The incorporation of iodide into these products could make them much more potent against periodontal pathogens, and also help to prevent transmission of these pathogens from person to person via saliva.     

Susceptibility of Trichophyton quinckeanum and Trichophyton rubrum to products of oxidative metabolism.

Two dermatophyte strains, Trichophyton quinckeanum and Trichophyton rubrum, were highly susceptible to in vitro killing by components of the H2O2-peroxidase-halide system. Both strains were, however, resistant to relatively high concentrations of reagent H2O2 or H2O2 enzymatically generated by glucose and glucose oxidase, KI, or lactoperoxidase (LPO) alone. Resistance to hydrogen peroxidase killing was found to be in part due to the presence of endogenous catalase in the fungi; susceptibility was increased by pretreatment of the fungi with a catalase inhibitor. Kinetic studies using small quantities of reagent or enzymatically generated H2O2 and LPO-KI showed that the system was lethal for both fungal strains within 1 min. Furthermore, using the glucose-glucose oxidase-LPO-KI system, it was shown that catalase, superoxide dismutase and histidine scavengers of H2O2, superoxide anion and singlet oxygen, respectively, prevented the killing of fungus, whereas scavengers of hydroxyl radicals such as benzoate and mannitol had no effect. T. quinckeanum was found to contain large quantities of superoxide anion, as judged by the nitroblue-tetrazolium test. Consequently, the xanthine (or hypoxanthine) and xanthine oxidase system in which the main product is superoxide anion had no toxic effect on the fungus. The high sensitivity of dermatophytes to killing by the H2O2-peroxidase-halide system active in polymorphonuclear neutrophils and macrophages may account in part for fungal toxicity in vivo.     

Release and Functional Characterization of the Leukotriene D4-Metabolizing Enzyme (Dipeptidase) from Human Polymorphonuclear Leucocytes

Polymorphonuclear leucocytes released LTD4-dipeptidase activity in a time-, calcium-, and cell number-dependent fashion. The LTD4-dipeptidase released from polymorphonuclear leucocytes (PMN) by incubation with calcium (0.91 mM) was detectable up to a cell concentration of 1 X 10(6)/ml and increased with higher concentrations. Maximal LTD4-dipeptidase activity within the extracellular environment was detected after 15 min of incubation (2 X 10(7)/ml) in the presence of 2-4.5 mM calcium or after 30 min, when stimulation was carried out with 0.91 mM calcium. The activity of the released LTD4-dipeptidase was modulated by various metal ions and other compounds. The addition of Mn2+, Co2+, and Zn2+ (final concentration 1 mM) enhanced the LTD4-dipeptidase activity, while Cu2+ led to a complete inhibition. In the absence of exogenous calcium EDTA inhibited LTD4-dipeptidase. Calcium up to a concentration of 5 and 10 mM decreased the dipeptidase activity. The LTD4-dipeptidase is not affected by bestatin, leupeptin, or N-ethyl-maleinimide (NEM). The Km of LTD4-dipeptidase for LTD4 was 0.95 +/- 0.2 microM and Vmax was 737.5 +/- 112.5 pmol/min X mg protein (n = 3 +/- SEM). The highest LTD4-dipeptidase activity was obtained at physiological pH values. LTD4-dipeptidase activity can also be released from other cell types, but the enzyme activity from human PMN exceeded that of other cells (e.g. human lymphocytes/monocytes and basophils (LMB) and human lung cell suspension).     

Interaction of granulocytes and endothelial cells upon stimulation with tumor necrosis factor-alpha: an ultrastructural study

By the production of microbicidal agents, such as reactive oxygen species, activated PMN are capable of inducing tissue damage in the host. TNF-alpha was recently shown to be a potent activator of PMN oxidative metabolism. To further evaluate the interaction between activated PMN with physiological target cells, the effect of human PMN on cultured bovine aortic and human umbilical vein endothelial cells (EC) upon stimulation with human TNF-alpha was investigated by ultrastructural techniques: Scanning and transmission electron microscopy (SEM and TEM resp.) and ultrastructural detection of H2O2 production. When isolated PMN were added to EC in the presence of recombinant human TNF-alpha (10(3) U/ml) the EC-monolayer was disrupted within 4 h and EC changed their shape by exhibiting a spindle-like structure. PMN were seen in the intercellular spaces. Release of H2O2 was observed at the surface of the PMN plasma membrane, the luminal part of the small intracytoplasmic vacuoles in the PMN as well as in the contact zone between PMN and EC, but not within the EC. Scavengers of reactive oxygen species, such as superoxide dismutase and catalase or D-mannitol failed to block the effect of TNF-alpha-stimulated PMN on EC. In contrast, addition of NaN3 (0.1 mM), an inhibitor of myeloperoxidase activity, almost completely inhibited the disruption of EC-monolayers. Subsequent addition of NaN3-insensitive horseradish peroxidase reconstituted the effect. The results obtained suggest that TNF-alpha-stimulated PMN effectively cause the disruption of EC monolayers by an adherence-dependent mechanism which is mediated by the release of myeloperoxidase. The results may be of major importance for the pathogenesis of inflammatory vascular reactions.     

Adsorption of peroxidase on titanium surfaces: A pilot study

The present study demonstrates the in vitro and in vivo adsorption of peroxidase onto titanium surfaces. Titanium foils (mean +/- SEM: 365 +/- 2 mm(2), n = 114) were incubated during 30 min with lactoperoxidase (4 mg in 5 mL 100 mM phosphate buffer pH 7). After 15 washings by H(2)O, titanium foils were incubated with o-phenylenediamine (6 mg/mL) and H(2)O(2) (7 mM) during 30 min. The reaction was then stopped by the addition of HCI 1M and the absorbance of the liquid phase was read on a spectrophotometer at 492 nm. In vitro adsorbed lactoperoxidase onto titanium surfaces was 0.70 +/- 0.05 ng/mm(2) (mean +/- SEM, n = 30). X-ray photoelectron spectroscopy confirmed the incorporation of protein nitrogen onto titanium surfaces: the nitrogen atomic percentage increased from 0.9 +/- 0.3 to 12.7 +/- 0.2% (n = 3) and from 3.7 +/- 0.1 to 14.4 +/- 0. 4% (n = 5) when titanium foils were incubated in the lactoperoxidase solution during 30 min and 24 h respectively. In vivo, oral peroxidases adsorbed on titanium healing abutments from 0.01 to 0.58 ng/mm(2) (n = 19) after 2 weeks in the oral environment.     

Enhancement of polymorphonuclear leukocyte (PMN) function by OK-432

The influence of OK-432, a streptococcal preparation, on human polymorphonuclear leukocytes (PMN) was examined. OK-432 increased O₂⁻ generation by PMN in oral cancer patients, and sustained the production of O₂⁻ in patients on chemoradiotherapy. Enhanced O₂⁻ generation was also observed when PMN were cultured with 10⁻² KE/ml OK-432 for 1 h and then stimulated with phorbol myristate acetate or formyl-metionyl-leucil-phenylalanine (FMLP). In addition, PMN O₂⁻ generation was promoted by culture supernatants of peripheral blood mononuclear cells (PBMC) incubated with 10⁻³ or 10⁻² KE/ml OK-432. Furthermore, OK-432 (10⁻³−10⁻² KE/ml) enhanced the chemiluminescence of FMLP- and PMA-stimulated PMN. However, nitroblue tetrazolium reduction and myeloperoxidase activity were only minimally enhanced. Not only the candidacidal activity of PMN but also antibody-dependent cell-mediated cytotoxicity against Candida and Raji cells were enhanced in correspondence with the increased generation of reactive oxygen species.Culture of PMN or PBMC for 24 h with OK-432 resulted in a concentration-dependent increase in the substantial production of interleukin-1β, interleukin-6 and tumor necrosis factor-α. OK-432 also enhanced granulocyte-macrophage colony stimulating factor and gamma-interferon generation by leukocytes in a dose-dependent manner. Our research indicates that OK-432 enhances PMN function directly as well as via the promotion of cytokine production, and suggests that these effects of OK-432 could be beneficial in immunosuppressed patients.     

Relative roles of pneumolysin and hydrogen peroxide from Streptococcus pneumoniae in inhibition of ependymal ciliary beat frequency

Ciliated ependymal cells line the ventricular system of the brain and the cerebral aqueducts. This study characterizes the relative roles of pneumolysin and hydrogen peroxide (H(2)O(2)) in pneumococcal meningitis, using the in vitro ependymal ciliary beat frequency (CBF) as an indicator of toxicity. We have developed an ex vivo model to examine the ependymal surface of the brain slices cut from the fourth ventricle. The ependymal cells had cilia beating at a frequency of between 38 and 44Hz. D39 (wild-type) and PLN-A (pneumolysin-negative) pneumococci at 10(8) CFU/ml both caused ciliary slowing. Catalase protected against PLN-A-induced ciliary slowing but afforded little protection from D39. Lysed PLN-A did not reduce CBF, whereas lysed D39 caused rapid ciliary stasis. There was no effect of catalase, penicillin, or catalase plus penicillin on the CBF. H(2)O(2) at a concentration as low as 100 microM caused ciliary stasis, and this effect was abolished by coincubation with catalase. An additive inhibition of CBF was demonstrated using a combination of both toxins. A significant inhibition of CBF at between 30 and 120 min was demonstrated with both toxins compared with either H(2)O(2) (10 microM) or pneumolysin (1 HU/ml) alone. D39 released equivalent levels of H(2)O(2) to those released by PLN-A, and these concentrations were sufficient to cause ciliary stasis. The brain slices did not produce H(2)O(2), and in the presence of 10(8) CFU of D39 or PLN-A per ml there was no detectable bacterially induced increase of H(2)O(2) release from the brain slice. Coincubation with catalase converted the H(2)O(2) produced by the pneumococci to H(2)O. Penicillin-induced lysis of bacteria dramatically reduced H(2)O(2) production. The hemolytic activity released from D39 was sufficient to cause rapid ciliary stasis, and there was no detectable release of hemolytic activity from the pneumolysin-negative PLN-A. These data demonstrate that D39 bacteria released pneumolysin, which caused rapid ciliary stasis. D39 also released H(2)O(2), which contributed to the toxicity, but this was masked by the more severe effects of pneumolysin. H(2)O(2) released from intact PLN-A was sufficient to cause rapid ciliary stasis, and catalase protected against H(2)O(2)-induced cell toxicity, indicating a role for H(2)O(2) in the response. There is also a slight additive effect of pneumolysin and H(2)O(2) on ependymal toxicity; however, the precise mechanism of action and the role of these toxins in pathogenesis remain unclear.     

A peroxidase-independent method for the quantitation of extracellular hydrogen peroxide generated by activated phagocytes in vitro.

A peroxidase-independent method for the determination of phagocyte-derived hydrogen peroxide in vitro is described. The method is based on the catalase-inhibitable oxidation of added cysteine. Human blood neutrophils (1 x 10(6)/ml) were coincubated with the myeloperoxidase (MPO) inhibitor, sodium azide (10 micrograms/ml), for 30 min at 37 degrees C followed by addition of phorbol 12-myristate 13-acetate (PMA, 10 ng/ml), a potent activator of membrane-associated oxidative metabolism. After incubation at 37 degrees C the cells were removed and the supernatants aliquoted and incubated with and without catalase (500 U/ml) for 10 min at room temperature followed by addition of cysteine (50 microM). Thereafter the catalase-inhibitable, H2O2-mediated oxidation of added cysteine was assayed spectrophotometrically following the addition of the thiol-reactive agent 5,5'-dithiobis-(2-nitrobenzoic acid), and the H2O2 concentration determined using a standard curve constructed from difference data based on the oxidation of cysteine by H2O2 (0-200 nmol). The average amount of H2O2 produced by 10(6) PMA-activated neutrophils was 47 +/- 7 nmol/30 min. This sensitive assay procedure is likely to be particularly useful for investigating the effects of pharmacological agents and anti-oxidant nutrients on H2O2 production by activated phagocytes, since these agents are generally unsuited for use in peroxidase-based assays.     

Stimulus-dependent inhibition of superoxide generation by prostaglandins

Infiltrating phagocytes generate superoxide anion (O2-) and prostaglandin (PG) at sites of inflammation. Thus PG-O2- interactions may be important to the initiation and control of inflammation. PGE1, PGE2, and PGD2 inhibit O2- generation (as measured by superoxide dismutase-inhibitable reduction of ferricytochrome c) in a dose-dependent manner (10(-6)-10(-9) M) when human peripheral blood polymorphonuclear leucocytes (PMN) are stimulated with 10(-7) M of the chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). These PG did not alter O2- generation when PMN were stimulated with 0.1 microgram/ml phorbol myristate acetate (PMA) or 1 mg/ml serum-treated zymosan (STZ). Increments of cyclic AMP (cAMP) (peak: fourfold) in PGE1, PGE2, and PGD2 treated PMN stimulated with PMA or STZ (in which O2- was not reduced) were similar to those in PG-treated PMN stimulated with FMLP (in which O2- was reduced markedly). High concentrations of theophylline and dibutyryl cAMP reduced FMLP and STZ stimulated O2- generation but had no effect on PMA stimulation, suggesting that the stimuli induce different sensitivities to the effects of cellular cAMP. PGF2 alpha had little effect on O2- generation or cAMP levels regardless of the stimulus. PGE1 did not inhibit binding of FML(3H)P to PMN and did not scavenge O2- anions. Therefore the effect of PG on O2- production is dependent on the specific stimulator and an increased concentration of cAMP in activated PMN is by itself not sufficient to limit O2- generation induced by all stimuli.     

Suppression of human lymphocyte proliferation by activated neutrophils or H2O2: surviving cells have an altered T helper/T suppressor ratio and an increased resistance to secondary oxidant exposure

Either activated neutrophils (PMNs) or nanomole amounts of reagent hydrogen peroxide (H2O2) were found to cause catalase-reversible suppression of concanavalin A (Con A)-stimulated human lymphocyte proliferation. Suppression required PMN activation and occurred with PMN/lymphocyte ratios found in peripheral blood. Inhibition by reagent H2O2 occurred with 10-40 nmol H2O2/1 X 10(6) lymphocytes, a noncytolethal amount which is produced readily by PMA-activated PMNs. Lymphoblasts recovered from suppressed cultures were two- to fourfold less sensitive than control lymphoblasts to a second exposure to H2O2. These relatively H2O2-resistant lymphoblasts also scavenge H2O2 at higher rates than do control lymphoblasts. Progeny lymphocytes from suppressed cultures contain an unusually high percentage of T helper cells with a concomitant decrease in T suppressors. These studies demonstrate a potential immunoregulatory role for phagocyte-released oxidants, especially H2O2, and suggest a possible additional mechanism to explain the excess of T helpers observed in situations of chronic inflammation.     

肾上腺素对中性粒细胞释放PGE_2和TXB_2的影响

目的 :观察肾上腺素对体外中性粒细胞在A2 31 87刺激下对释放PGE2 和TXB2 的影响。方法 :用无菌的 1%糖元注入大鼠腹腔 ,收集腹水 ,提取PMN。将PMN分成PMN组、A2 31 87组和A2 31 87加ADR三组 ,并用RIA法分别测定上清中PGE2 和TXB2 的含量 ,上述试验过程重复 3次。结果 :PMN释放PGE2 和TXB2 的含量 ( x±s,pg/5× 10 6 细胞 ) ,各组分别为 :( 1)PMN正常对照组 :60 .3± 3 4 .5 ,5 7.4± 12 .3 ;( 2 )PMN +A2 31 87组 ;93 .3 4± 3 2 .6,10 1.9± 3 1.2 ;( 3 )PMN +A2 31 87+ADR组 :15 6.6± 2 8.5 ,169.8± 48.2。且 ( 1)组和 ( 2 )组相比 ,( 2 )组二者的含量显著高于 ( 1)组 (P <0 .0 5 ) ;( 1)组、( 2 )组和 ( 3 )组比 ,且 ( 3 )组二者的含量显著高于 ( 1)组、( 2 )组 (P <0 .0 5 )。结论 :ADR促进由A2 31 87诱导的PMN释放PGE2 和TXB2 含量的增加 ,可能是ADR直接影响了还氧酶活性 ,推测与开启Ca+ + 通道有关。     

Role of histamine in the chemotactic deactivation of polymorphonuclear leukocytes following incubation with formylmethionyl peptides.

To clarify the mechanism of chemotactic deactivation of polymorphonuclear neutrophils (PMN) following incubation with the synthetic dipeptide N-formylmethionyl phenylalanine (FMP), we tested the hypothesis that histamine, which is released from leukocytes during incubation with FMP, could explain this inhibition. Human PMN were incubated in the presence or absence of FMP (10(-7) to 10(-5) M) and histamine measured fluorometrically in the supernatant. Washed PMN were then tested in Boyden chambers against FMP (10(-5) M) and other chemoattractants. Incubation of leukocytes with FMP caused a nonpreferential PMN deactivation which was proportionally and kinetically related to FMP-induced histamine release. No histamine release or chemotactic deactivation was observed in the absence of Ca2+ and Mg2+. The inhibitory effect of the peptide was significantly prevented by an H2 blocker or when using basophil-depleted PMN suspensions. Histaminase abolished the capacity of FMP-incubated leukocyte supernatants to decrease PMN chemotaxis. Preincubation of leukocytes with anti-IgE or a sensitizing allergen caused a significant PMN chemotactic deactivation. These results show that histamine which is released during leukocyte incubation with FMP, contributes, at least in part, to the chemotactic deactivation of PMN.     

Capacity of recombinant gamma interferon to activate macrophages for Salmonella-killing activity.

The ability of recombinant gamma interferon (rIFN-gamma) to activate macrophages for Salmonella-killing activity was kinetically examined in relation to phagosome-lysosome fusion and H2O2 generation. Resident peritoneal macrophages of BALB/c mice incubated with 10(2) to 10(3) U of rIFN-gamma per ml for 12 h exhibited enhanced bactericidal activity against Salmonella typhimurium, although H2O2 generation was unaltered. In contrast, macrophages incubated with equal doses of rIFN-gamma for 48 h showed both an enhanced Salmonella-killing activity and an increased generation of H2O2. To evaluate Salmonella-killing activities of macrophages, intracellular bacteria were assayed at 0, 2, and 8 h after infection. During the initial 2 h of infection, 12-h-activated macrophages, as well as the unstimulated control macrophages, showed a decline in bacterial population at the same rate. Over the next 6 h of infection, however, the number of viable bacteria in activated macrophages remained unchanged, whereas the number of bacteria in control macrophages significantly (P less than 0.05) increased. Similar results were obtained in 48-h-activated macrophages. On the other hand, macrophages incubated with 10 to 10(3) U of rIFN-gamma exhibited enhanced fusion of lysosomes to Salmonella-containing phagosomes in both the 12-h- and 48-h-stimulated stages. Moreover, when 48-h-activated macrophages were incubated concomitantly with superoxide dismutase and catalase, Salmonella-killing activity was not affected. These results indicate that rIFN-gamma per se is able to activate peritoneal macrophages to induce Salmonella-killing activity and suggest that increased phagosome-lysosome fusion followed by an oxygen-independent killing mechanism is primarily responsible for the enhanced Salmonella-killing activity in rIFN-gamma-activated macrophages.     

Neutrophils Injure Bronchial Epithelium after Ozone Exposure

Neutrophil (PMN) influx is an early, prominent finding in the airways of humans after experimental inhalation of ozone (O3), however the potential for PMN to contribute to epithelial injury in this setting is unknown. Bronchial epithelial cells of the human BEAS 2B R1.4 cell line or primary human bronchial epithelial cells underwent DNA labeling by incubation with BrdU. Monolayers were exposed to O3 (0.05 to 1 ppm) or filtered air for 60 min., and subsequently incubated with PMN for 2 h. Epithelial cell cytolysis was significant only in BEAS exposed to O3 and co-cultured with PMN. Apoptosis was maximal in BEAS exposed to O3 + PMN. Primary bronchial epithelial cells were resistant to injury; no cytolysis was detected, and apoptosis was detected only after treatment with 10 mM H₂O₂. Neutrophils may increase damage to the respiratory epithelium after O3 exposure, but primary bronchial epithelial cells are resistant to PMN and ozone induced injury.     

Respiratory burst facilitates the digestion of Escherichia coli killed by polymorphonuclear leukocytes.

We examined factors that may limit degradation of bacterial protein of Escherichia coli S15 killed by polymorphonuclear leukocytes (PMN). Both human and rabbit PMN degraded up to 40% of [14C]amino acid-labeled protein of ingested and killed E. coli in 2 h as determined by loss of acid-precipitable radioactivity. In contrast, equally bactericidal broken-PMN preparations or isolated granules degraded only about 10% of bacterial protein regardless of pH. To determine whether activation of the respiratory burst contributes to digestion, we compared degradation by intact PMN in room air and under N2. Depletion of O2 by N2 flushing had no effect on the bactericidal activity of either human or rabbit PMN but reduced degradation by approximately 50%. Protein degradation during phagocytosis was also reduced in the presence of cyanide or azide, inhibitors of myeloperoxidase (MPO). PMN of two patients with chronic granulomatous disease ingested and killed E. coli S15 as well as did normal PMN but degraded bacterial protein as did normal PMN incubated under N2. The low degradative activity of PMN disrupted by sonication could be raised to nearly the level of intact PMN incubated in room air by preincubation of the PMN with 10(-7) M formyl-methionyl-leucyl-phenylalanine (fMLP) before sonication and by pretreatment of E. coli with MPO. Depletion of O2 or chloride during these preincubations with formyl-methionyl-leucyl-phenylalanine respectively, virtually abolished and markedly diminished stimulation of bacterial protein degradation. We conclude that enhanced MPO-mediated O2 metabolism of intact PMN plays a role in the digestion of killed E. coli.