Biochemical and functional characterisation of macrophage stimulating factors secreted by mitogen-induced goldfish kidney leucocytes

Mitogen-stimulated goldfish kidney leucocytes secrete a number of different macrophage activation factors (MAF) that induce profound physiological changes in macrophages. MAF produced by goldfish kidney leucocytes was characterised using fast performance liquid chromatography (FPLC) and bioassays that measured MAF-induced respiratory burst (RB) and nitric oxide (NO) responses of activated macrophages. Mitogen-induced fish kidney leucocyte supernatants were fractionated using gel permeation FPLC (GP-FPLC) and the ability of different fractions to induce NO or RB measured. A MAF of M(r) 50 kD, that induced a potent nitric oxide response in both a long-term goldfish macrophage cell line (GMCL) and in in vitro-derived fish kidney macrophages (IVDKM) was identified. The GP-FPLC partially purified 50 kD MAF activity occasionally induced significantly higher nitric oxide production than that of the crude MAF preparations. This increase in the NO-inducing activity was due to segregation of the 50 kD MAF from a novel macrophage deactivating molecule of M(r) 10-12 kD present in crude MAF preparations. This 10-12 kD molecule was shown to inhibit nitric oxide production in cytokine-activated goldfish macrophages. Mitogen-induced fish kidney leucocyte supernatants contained two distinct MAFs that induced the respiratory burst in GMCL and IVDKM: the 50 kD and 30 kD proteins. The partially purified 30 kD MAF primed goldfish macrophage for increased RB activity after only 6 h of treatment, and continued to augment the RB activity after 24 h of stimulation. In contrast, the GP-FPLC partially purified 50 kD molecule also primed the RB after only 6 h of stimulation, but subsequently deprimed the RB after 24 h of stimulation, an effect similar to that observed for crude MAF preparations. The 50 kD MAF activity was further purified using chromatofocusing FPLC (C-FPLC) using basic pH gradients and was shown to consist of two distinct NO-inducing molecules (> pI 9.3). Mitogen-stimulated fish kidney leucocytes secrete several factors that profoundly affect the anti-microbial responses of teleost macrophages and which undoubtedly are responsible for regulating teleost macrophage function in vivo.     

Human phagocyte oxidative burst activation by BCG, M. leprae, and atypical mycobacteria: defective activation by M. leprae is not reversed by interferon gamma

The activation of the phagocyte oxidative respiratory burst by various mycobacteria was evaluated in an in vitro assay, by measuring the chemiluminescence, associated to the release of oxidizing species, generated by normal human whole blood phagocytes. All mycobacterium species, except Mycobacterium leprae, induced a significant chemiluminescence response. The strongest stimulus was provided by BCG, followed by M. triviale, M. chelonei, and M. fortuitum. M. kansasii, M. intracellulare, and M. lepraemurium elicited a weak response, although higher than that triggered by M. leprae. Both polymorphonuclear and mononuclear cells contributed to the whole blood cell chemiluminescence stimulated by mycobacteria, mononuclear cells being more efficient on a per cell basis. Phagocyte activation by recombinant interferon gamma did not improve M. leprae ability to trigger a significant chemiluminescence response. The failure of M. leprae and of some atypical mycobacteria to stimulate a strong phagocyte oxidative respiratory burst may have some relevance to their pathogenicity.     

Suppression of monocyte oxidative response by phenolic glycolipid I of Mycobacterium leprae

Mycobacterium leprae synthesizes a unique phenolic glycolipid (PGL-I) in abundant quantities. We studied the effect of PGL-I on the generation of superoxide anion (O2-) by stimulated human monocytes. Peripheral blood monocytes pretreated with PGL-I released less O2- when stimulated with M. leprae than did control monocytes. Monocytes pretreated with dimycocerosyl phthiocerol, mycoside A of Mycobacterium kansasii, or mycoside B of Mycobacterium microti, on the other hand, released O2- in quantities comparable to control monocytes in response to M. leprae stimulation. Monocyte O2- release in response to other stimuli of the oxidative metabolic burst, such as PMA, zymosan, Mycobacterium bovis Bacille Calmette-Guérin, or M. kansasii, was unaffected by lipid pretreatment. These findings demonstrate that PGL-I has a direct effect on monocyte O2- generation in response to M. leprae and suggest that PGL-I is a modulator of phagocytic cell function.     

Chemiluminescence response of phagocytes in E. coli induced experimental ascending pyelonephritis.

The mechanism of tissue injury at the cellular level by following the chemiluminescence response of various phagocytes in E. coli induced experimental pyelonephritis in mice was investigated. There was a marked increase in the capacity of various phagocytic cells viz; renal neutrophils and macrophages peritoneal macrophages, blood monocytes and neutrophils to produce reactive oxygens species through the respiratory burst activity as monitored by the chemiluminescence response. The chemiluminescence response was observed to be increased significantly (p less than 0.001) with increasing days post infection in all phagocytic cells. However, the quantity of total reactive oxygen species produced per million cells was much more in the renal and peritoneal macrophages as compared to blood monocytes and neutrophils. The peak chemiluminescence response time was observed to be decreased from 4 to 2 minutes with the progression of the diseases. The implications of these findings have been discussed.     

Xylanase production by Aspergillus nidulans

Abstract The effect of phagocyte activation by TNF-α on the ability to trigger a chemiluminescence (CL) response, associated with the release of oxidizing species was evaluated in healthy human mononuclear cells in the presence of Mycobacterium leprae . Recombinant TNF-α (r-TNF-α) increased the CL response of unstimulated M. bovis BCG- and PMA-stimulated cells but did not reverse the M. leprae defective activation of the human phagocyte oxidative burst. M. leprae was less well phagocytosed than M. bovis BCG but phagocytosis of mycobacteria was not altered by addition of r-TNF-α. The failure of activation of oxygen-free radical production might have some relevance to the pathogenesis of leprosy.     

An in vitro model for the lepromatous leprosy granuloma: fate of Mycobacterium leprae from target macrophages after interaction with normal and activated effector macrophages

The lepromatous leprosy granuloma is a dynamic entity requiring a steady influx of macrophages (Mphi) for its maintenance. We have developed an in vitro model to study the fate of Mycobacterium leprae in a LL lesion, with and without immunotherapeutic intervention. Target cells, consisting of granuloma Mphi harvested from the footpads of M. leprae-infected athymic nu/nu mice, were cocultured with normal or IFN-gamma-activated (ACT) effector Mphi. The bacilli were recovered and assessed for viability by radiorespirometry. M. leprae recovered from target Mphi possessed high metabolic activity, indicating a viable state in this uncultivable organism. M. leprae recovered from target Mphi incubated with normal effector Mphi exhibited significantly higher metabolism. In contrast, bacilli recovered from target Mphi cocultured with ACT effector Mphi displayed a markedly decreased metabolic activity. Inhibition by ACT Mphi required an E:T ratio of at least 5:1, a coculture incubation period of 3-5 days, and the production of reactive nitrogen intermediates, but not reactive oxygen intermediates. Neither IFN-gamma nor TNF-alpha were required during the cocultivation period. However, cell-to-cell contact between the target and effector Mphi was necessary for augmentation of M. leprae metabolism by normal effector Mphi as well as for inhibition of M. leprae by ACT effector Mphi. Conventional fluorescence microscopy and confocal fluorescence microscopy revealed that the bacilli from the target Mphi were acquired by the effector Mphi. Thus, the state of Mphi infiltrating the granuloma may markedly affect the viability of M. leprae residing in Mphi in the lepromatous lesion.     

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.     

Beta-endorphin and related peptides suppress phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocytes

In the present study, the immunomodulatory effect of beta-endorphin (beta-E) and shorter pro-opiomelanocortin (POMC) fragments was evaluated by assessing their influence on respiratory burst in human polymorphonuclear leukocytes (PMN). The effect of the peptides (10(-17)M - 10(-10)M) on phorbol myristate acetate (PMA)-stimulated production of reactive oxygen metabolites was measured in a lucigenin-enhanced chemiluminescence (CL) assay. Both POMC peptides with opiate-like activity (i.e. alpha-endorphin (alpha-E), beta-E and gamma-endorphin (gamma-E] and their non-opioid derivatives (i.e. des-TYR1-beta-endorphin (dT beta E), des-TYR1-gamma-endorphin (dT gamma E), and des-ENK-gamma-endorphin (dE gamma E] were tested. With the exception of alpha-E, PMA-stimulated respiratory burst was suppressed by all POMC fragments tested. A U-shaped dose-response relation was observed. Doses lower than 10(-17)M and higher than 10(-8)M were without effect. beta-E and dT beta E both suppressed PMA-induced oxidative burst in human PMN at physiological concentrations (10(-16)M - 10(-10)M). gamma-E and dT gamma E proved to be less potent inhibitors, reaching maximal effect at higher concentrations (10(-12)M - 10(-10)M). DE gamma E exerted an even less pronounced but still significant suppressive effect at the concentration of 10(-10)M. None of the endorphins tested was shown to affect resting oxidative metabolism in the PMN. The modulatory effects of the opioid peptides could not be blocked by the opioid antagonist naloxone (10(-8)M). These data show that fragments derived from the POMC-precursor molecule modulate the activation of PMN by suppressing PMA-stimulated oxidative metabolism and that this activity does not involve a classical opiate-like receptor.     

Trichinella spiralis infection affects p47(phox) protein expression in guinea-pig alveolar macrophages

To establish whether NADPH oxidase activation, responsible for previously demonstrated Trichinella spiralis-induced respiratory burst, results from assembling of membrane and cytosolic NADPH oxidase components and/or increased expression of the oxidase complex proteins, the superoxide anion production and expression of the regulatory p47(phox) subunit were measured in cultured alveolar macrophages obtained during T. spiralis infection of guinea pigs. The results demonstrate for the first time helminth parasite-infection-induced stimulation of NADPH oxidase p47(phox) subunit protein expression, with the effect being decreased by in vivo treatment with cyclosporin A, previously shown to inhibit T. spiralis infection-induced respiratory burst in guinea-pig alveolar macrophages. However, although the expression of the p47(phox) subunit protein remained induced during secondary infection, it was accompanied by superoxide anion production that was significantly suppressed in comparison with that observed during primary infection, suggesting suppressive action of T. spiralis on host's alveolar macrophage immune response, presumably connected with NADPH oxidase complex activity attenuation.     

Mechanisms of host defense against Candida species. II. Biochemical basis for the killing of Candida by mononuclear phagocytes.

We studied the biochemical basis of candidacidal activity by comparing the killing of Candida albicans, a serious pathogen, and Candida parapsilosis, a low-grade pathogen, by human monocytes (Mo) and monocyte-derived macrophages. Mo killed C. parapsilosis significantly better than C. albicans. The two species triggered the respiratory burst and release of myeloperoxidase (MPO) and beta-glucuronidase in Mo to an equivalent extent. In contrast to Mo, macrophages killed both species to an equivalent extent. Mo exhibited a greater candida-stimulated respiratory burst than did monocyte-derived macrophages, and the respiratory burst was required for the killing of both species. C. parapsilosis was killed much more easily than C. albicans by exposure to low concentrations of hypochlorite or monochloramine, MPO-dependent oxidants released by Mo but not macrophages, which lack MPO. With six different Candida strains there was a significant correlation between killing by Mo and susceptibility to hypochlorite (r = 0.926) or monochloramine (r = 0.981) (p less than 0.01 for each). Species differences in resistance to killing by Mo may be related to differences in sensitivity to MPO-derived oxidants, and the ability of C. albicans to resist the effects of these oxidants may be a virulence factor associated with this species.     

Stimulation of macrophage H2O2 release by resident thymocytes: effect of a soluble factor distinct from interferon-gamma

Activated T cells are known to stimulate macrophage oxidative metabolism and antimicrobial activity through release of interferon-gamma (IFN-gamma). In contrast, the role of nonactivated T cells in regulating macrophage effector functions is less well defined. We have previously reported that a low molecular weight soluble factor derived from resident (nonactivated) thymocytes enhances macrophage receptor-mediated phagocytosis. In the present study, we examined the capacity of resident murine thymocytes to stimulate the respiratory burst and microbicidal activity of peritoneal macrophages. Macrophages cultured for 1-2 days with cell-free thymocyte supernatant (TS) released two to three times more H2O2 in response to PMA or opsonized zymosan than did control macrophages. The H2O2-stimulating factor in TS was distinguished from IFN-gamma by its heat stability (100 degrees C, 20 min), approximate MW of 2400 Da (gel filtration high-pressure liquid chromatography), and absence of interferon activity in both antiviral and enzyme-linked immunosorbent assays. TS-treated macrophages, however, did not exhibit a greater capacity to kill or inhibit the intracellular growth of Toxoplasma gondii, indicating that the thymocyte factor did not fully activate macrophage microbicidal mechanisms. These data suggest that thymocytes can increase the respiratory burst capacity of macrophages in the absence of antigen-specific immune responses.     

Molecular mechanism of interaction of Mycobacterium tuberculosis with host macrophages under high glucose conditions

Mycobacterium tuberculosis has the potential to escape various cellular defense mechanisms for its survival which include various oxidative stress responses, inhibition of phagosome-lysosomes fusion and alterations in cell death mechanisms of host macrophages that are crucial for its infectivity and dissemination. Diabetic patients are more susceptible to developing tuberculosis because of impairement of innate immunity and prevailing higher glucose levels. Our earlier observations have demonstrated alterations in the protein profile of M. tuberculosis exposed to concurrent high glucose and tuberculosis conditions suggesting a crosstalk between host and pathogen under high glucose conditions. Since high glucose environment plays crucial role in the interaction of mycobacterium with host macrophages which provide a niche for the survival of M. tuberculosis, it is important to understand various interactive mechanisms under such conditions. Initial phagocytosis and containment of M. tuberculosis by macrophages, mode of macrophage cell death, respiratory burst responses, Mycobacterium and lysosomal co-localization were studied in M. tuberculosis H37Rv infected cells in the presence of varied concentrations of glucose in order to mimic diabetes like conditions. It was observed that initial attachment, phagocytosis and later containment were less effective under high glucose conditions in comparison to normal glucose. Mycobacterium infected cells showed more necrosis than apoptosis as cell death mechanism during the course of infection under high glucose concentrations. Co-localization and respiratory burst assay also indicated evasion strategies adopted by M. tuberculosis under such conditions. This study by using THP1 macrophage model of tuberculosis and high glucose conditions showed immune evasion strategies adapted during co-pathogenesis of tuberculosis and diabetes.     

In vitro interaction between persisting oocysts of Cryptosporidium parvum and murine resident peritoneal macrophages. I. Effect of parasite on macrophage capability for oxidative burst

Cryptosporidiosis in an opportunistic infection which poses a significant threat to the immunodeficient patients (including people with AIDS). The aim of the present work was to study whether oxidative burst, known as a nonspecific immune response of macrophages, may be modulated in vitro by persisting oocysts of a coccidian pathogen Cryptosporidium parvum. Live oocysts of C. parvum engulfed by murine perithoneal macrophages may persist within the phagosomes and retain their initial morphology for about 7 days following oocyst injection into macrophage monolayer. A short-term interaction of adherent macrophages with C. parvum oocyst suspension for 5-15 min caused oxidative burst in these macrophages. After a while, the intensity of this oxidative burst smoothly decreased to vanish within the following 2-6 h oocyst-macrophage cultivation. Dead oocysts caused no oxidative burst in macrophages. Co-cultivation of macrophages with oocysts for 1.0-1.5 days led to the appearance of macrophages, which had oocysts both contacting the cell surface and existing inside the phagosomes. In these macrophages the oxidative burst in response to the addition of a chemotactic peptide (fMLP) was considerably higher than in uninfected control cells. During a 1-4 day co-cultivation, the degree of oxidative burst caused by fMLP in macrophages containing only phagocytosed oocysts did not differ from that in the non-infected (oocyst-free) control. The data obtained enable us to propose that the products of oxidative burst in macrophages, formed in response to the interaction with C. parvum oocysts, do not kill the oocysts. These can survive for a long time in the phagosomes of macrophages. Such a long persistence of oocysts in phagosomes does not affect the capability of macrophages for oxidative burst in response to the action of fMLP.     

Oxygen metabolism and the microbicidal activity of macrophages.

Like neutrophils, phagocytizing macrophages undergo a "respiratory burst" in which significant quantities of oxygen are drawn into the cell. The consumed oxygen is not used in oxidative phosphorylation but, rather, in the formation of superoxide anion (O2) and H2O2. These oxygen metabolites and the products of their interaction, in particular hydroxyl radical (OH), have been implicated in the killing of ingested bacteria by neutrophils. Their role in macrophage microbicidal activity has not been fully defined. However, activated macrophages, which mediate increased resistance to infection in vivo, have a markedly increased capacity to generate O2 and H2O2 in vitro when stimulated by phagocytosis or surface perturbation. The enhanced capacity of activated macrophages to generate highly reactive oxygen metabolites during phagocytosis could contribute to the improved microbicidal and tumoricidal activity of these cells.     

Antioxidative activity of flavonoids in stimulated human neutrophils

The release and production of oxidative products generated by the respiratory burst under the influence of natural flavonoids: quercetin, kaempferol and isorhamnetin derivatives have been studied in the polymorphonuclear neutrophils (PMNs) from healthy human donors. Flavonoids were tested in vitro at concentration range 1-100 microM. The antioxidative potential of flavonoids was compared to the activity of a food preservative, butylated hydroxyanisole. Two methods were applied to the measurement of the PMNs respiratory burst: flow cytometry using dichlorofluorescein diacetate and luminol-dependent chemiluminescence. It was found that the studied products decreased the neutrophil hydrogen peroxide production in concentration-dependent manner. The highest degree of inhibition was registered for concentration of 100 microM, although in the chemiluminescence method the metabolic activity inhibition was more prominent. Antioxidative activity of flavonoids depended on the number of hydroxyl groups. These results provide useful data for establishing methods used to assess the respiratory burst in phagocytic cells.     

Subclinical Infection in Leprosy

Lymphocyte transformation has been used to study the immune response to Mycobacterium leprae among contacts and non-contacts of leprosy patients. Of 26 subjects living in a leprosy endemic area for less than two months none responded to M. leprae; 24% of subjects who had lived in an endemic area for more than a year gave a positive response to M. leprae; more than 50% of individuals with occupational contact of leprosy for more than a year responded; and about 50% of contacts of tuberculoid and treated lepromatous patients responded to M. leprae, while only 22% (4/18) of contacts of lepromatous patients treated for less than six months responded.It seems that leprosy is more highly infectious than is indicated by the prevalence of the disease and that a subclinical infection commonly follows exposure to M. leprae. The relatively low response found in contacts of active lepromatous patients suggests that in these contacts “superexposure” to M. leprae can bring about a decrease in host resistance.     

Enzymes for purine synthesis and scavenging in pathogenic mycobacteria and their distribution in Mycobacterium leprae

Three enzymes catalysing the synthesis of four intermediates (phosphoribosylglycinamide, phosphoribosylaminoimidazole-succinocarboxamide, phosphoribosylaminoimidazole-carboxamide and AMP) in the purine biosynthetic pathway were detected in extracts of Mycobacterium microti and M. avium, even when the organisms had been grown in mice. However only one of the three enzymes, adenylosuccinate AMP-lyase (catalysing the synthesis of the last two of the four intermediates listed above) was detected in M. leprae. Phosphoribosyltransferases, which convert adenine, guanine and hypoxanthine to the corresponding nucleoside monophosphates, and adenosine kinase were the major enzymes for purine scavenging in all mycobacteria studied. In contrast to enzymes in the synthetic pathway, evidence for metabolic regulation of the purine-scavenging enzymes was obtained. In particular, 20-80-fold differences in the activities of guanine phosphoribosyltransferase and adenosine kinase were observed when M. microti was grown in media with or without purines, or in mice. In M. leprae, activities of all phosphoribosyltransferases were low in comparison with activities in M. microti and M. avium (specific activity less than 2% when comparisons were made between extracts of host-grown mycobacteria). However, activity of adenosine kinase was higher in host-grown M. leprae than in host-grown M. microti or M. avium.     

Bovine dendritic cells are more permissive for Mycobacterium bovis replication than macrophages, but release more IL-12 and induce better immune T-cell proliferation

Bovine dendritic cells (DCs) were obtained by incubating blood monocytes with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The ability of DCs to phagocytose and allow the replication of virulent Mycobacterium bovis in vitro was studied, and compared with bovine blood monocyte-derived macrophages. In addition, the release of cytokines by M. bovis-infected DCs was assessed. DCs were shown to phagocytose M. bovis efficiently, and allowed a more substantial replication of M. bovis when compared to macrophages, as assessed by the metabolic activity of intracellular bacteria. During the course of M. bovis infection, it was found that macrophages released substantial amounts of pro-inflammatory factors such as tumour necrosis factor-alpha (TNF-alpha), nitric oxide (NO) and interleukin-1 beta (IL-1 beta). M. bovis-infected DCs released much smaller quantities of NO, IL-1 beta and TNF-alpha (5- to 10-fold lower amounts), when compared to macrophages. Treating cells with interferon-gamma (IFN-gamma) before and during the in vitro infection process was shown to increase the release of NO, TNF-alpha and IL-1 beta by M. bovis-infected macrophages, but not by M. bovis-infected DCs. M. bovis-infected macrophages released more interleukin-10 (IL-10) than infected DCs. Treating cells with IFN-gamma/LPS was shown to reduce M. bovis metabolic activity in infected macrophages, but had no such impact on M. bovis metabolic activity in infected DCs. A variety of T-cell-derived cytokines (IFN-gamma, GM-CSF, IL-4) had no impact on the replication of M. bovis in infected DCs. On the other hand, DCs infected with M. bovis sustained a more efficient replication of autologous sensitized T lymphocytes compared to M. bovis-infected macrophages. M. bovis-infected DCs released more substantial amounts of interleukin-12 (IL-12) than similarly infected macrophages. These data suggest a complementary role for DCs and macrophages with regard to bacteriostatic activity and induction of an efficient immune response against M. bovis.     

Rat macrophage treatment with lipopolysaccharide leads to a reduction in respiratory burst product secretion and a decrease in NADPH oxidase affinity

The effect of LPS on the respiratory burst in resident rat peritoneal macrophages has been examined. Rat macrophages secreted high levels of both O2- and H2O2 in response to triggering with phorbol esters, opsonized zymosan, and immune complexes. After culture in vitro with LPS these macrophages exhibited a marked diminution in their capacity to secrete high levels of respiratory burst products. The LPS-mediated loss of secretory activity was apparent after 2 hr of exposure to LPS and was inhibitable by polymyxin B in a dose-dependent fashion. The effect was not selective for any triggering agent type as inhibition of secretory activity occurred after triggering with PMA, zymosan and immune complexes. PGE2 added at levels secreted by the macrophages in response to LPS also inhibited respiratory burst product secretion. In addition, indomethacin prevented the LPS-mediated inhibition of secretion. Because the inhibition of secretion was common to all triggering agents tested, this suggested that the basis for the impaired secretion was at a level other than the receptor for the triggering agent. Both LPS and PGE2 treatment of the macrophages increased the Km of the oxidase for NADPH (1.7- to 2.3-fold) without affecting significantly the Vmax of the enzyme. These data suggest that stimulation of rat peritoneal macrophages by LPS results in an impaired ability to secrete respiratory burst products as a result of a PGE2-mediated decrease in NADPH oxidase affinity and that this alteration is independent of alterations in tumoricidal activity.     

Adenosine regulates the respiratory burst of cytokine-triggered human neutrophils adherent to biologic surfaces

The effect of adenosine on the respiratory burst was investigated using human neutrophils adherent to serum-coated surfaces. Adenosine caused complete suppression of the respiratory burst elicited by TNF-alpha, FMLP, or CSF for granulocytes; partial suppression of the response to CSF for granulocytes/macrophages, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, or uncoated polystyrene surfaces; and no suppression of the response to PMA. In most experiments, 4.7 x 10(-7) M and 2.5 x 10(-8) M adenosine caused 50% suppression of H2O2 release in response to TNF-alpha and FMLP, respectively, and 10 microM caused 100% suppression. Preexposure of neutrophils to ADP blocked the inhibitory effect of adenosine. With adherent neutrophils, there is a prolonged lag period in the onset of the respiratory burst in response to cytokines. Adenosine was fully suppressive if its addition was delayed past the first third of this lag period, or if it was removed during the last third of the lag period. A 10-min pulse with adenosine was most inhibitory when delivered in the middle third of the lag period. Dihydrocytochalasin B abolished the suppressive effect of adenosine on H2O2 release in response to FMLP. Thus adenosine, at concentrations found in human plasma, is a potent but selective inhibitor of the respiratory burst of adherent human neutrophils in response to physiologic, soluble stimuli, and ADP is a potentially physiologic counter-suppressant. Adenosine appears to exert most of its effect during a discrete interval within the lag period before onset of the respiratory burst, and may affect the coupling of agonist receptors to the cytoskeleton.