N-formyl Methionyl Leucyl Phenylalanine Research Articles

Effect of timosaponin E1 and E2 on superoxide generation induced by various stimuli in human neutrophils and on platelet aggregation in human blood

We have reported that six steroidal saponins isolated from Anemarrhenae rhizoma had various effects on stimulus-induced superoxide generation in human neutrophils. In this paper, two novel steroidal saponins, timosaponins E1 and E2 were isolated from Anemarrhenae rhizoma, and the effects of these steroidal saponins on superoxide generation in human neutrophils were investigated. Timosaponins E1 and E2 significantly inhibited N-formyl–methionyl–leucyl–phenylalanine (fMLP)-induced superoxide generation in a concentration-dependent manner, but not that induced by arachidonic acid (AA). On the other hand, both compounds enhanced superoxide generation induced by phorbol 12-myristate 13-acetate (PMA) in a concentration-dependent manner. The superoxide generation induced by PMA with timosaponins E1 and E2 was suppressed by staurosporine, an inhibitor of protein kinase C, but was not suppressed by genistein, an inhibitor of protein tyrosine kinase. Tyrosyl phosphorylation of a 58 kDa protein, which was increased by fMLP, was inhibited by timosaponins E1 and E2. Timosaponins E1 and E2 also inhibited the generation of a 47 kDa protein and platelet aggregation in human blood. The results suggest that protein tyrosine kinase participates in fMLP-mediated superoxide generation by timosaponin E1- and E2-treated human neutrophils.

INTERLEUKIN 10 SUPPRESSES PHAGOCYTIC AND ANTIHYPHAL ACTIVITIES OF HUMAN NEUTROPHILS

We investigated the effects of human interleukin 10 (IL-10) on the antibacterial and antifungal activities of human neutrophils (PMNs) against Staphylococcus aureus and Candida albicans. Incubation of PMNs from healthy volunteers with 20–100ng/ml of IL-10 at 37°C for 1h suppressed phagocytosis of serum-opsonized S. aureus (P=0.02) and blastoconidia of C. albicans (P<0.01). In contrast, 2–100ng/ml of IL-10 had no effect on superoxide anion production upon stimulation with phorbol myristate acetate, N-formylmethionyl leucyl phenylalanine, C. albicans blastoconidia or pseudohyphae; neither did it significantly affect conidiocidal or bactericidal activities of PMNs. However, 20–100ng/ml of IL-10 significantly decreased PMN-induced damage of C. albicans pseudohyphae (P=0.008). The suppression of phagocytic activity of PMNs against S. aureus and blastoconidia of C. albicans as well as the impairment of PMN-induced hyphal damage may have important implications for understanding the immunosuppressive profile of IL-10 in clinical usage.

Migration of neutrophils across human pulmonary endothelial cells is not blocked by matrix metalloproteinase or serine protease inhibitors.

It has long been speculated that neutrophils deploy proteases to digest subendothelial matrix as they migrate from the bloodstream. Direct evidence for the involvement of proteases in neutrophil transendothelial migration is, however, lacking. To address this issue we used transmission electron microscopy to verify the presence of continuous basal lamina beneath pulmonary endothelial cells grown on microporous filters, and then examined the effects of protease inhibitors on neutrophil migration through the endothelial cells and their associated subcellular matrix. Inhibitors of the two major matrix-degrading protease groups present in neutrophils, the matrix metalloproteinases (MMPs) and serine proteases, were assessed for their ability to modulate neutrophil transendothelial migration in response to the chemoattractant n-formylmethionyl leucylphenylalanine (FMLP). Neither the naturally occurring MMP inhibitor, tissue inhibitor of metalloproteinase-1, nor the hydroxamic acid-based inhibitors GM-6001, BB-3103, or Ro 31-9790 had any significant effect on FMLP-stimulated neutrophil migration across endothelial cells and associated basal lamina, with >/= 80% of neutrophils migrating through the system, even in the presence of inhibitors, at concentrations that totally inhibited all the gelatinase B (MMP-9) released upon stimulation with FMLP. Similarly, with serine protease inhibitors no significant inhibition of neutrophil migration was observed with a naturally occurring inhibitor, secretory leukocyte protease inhibitor, or a low molecular-weight synthetic inhibitor, Pefabloc SC. These results indicate that neither MMP nor serine protease digestion of sub-endothelial matrix is required for successful neutrophil transendothelial migration.

INFLUENCE OF RECOMBINANT HUMAN ERYTHROPOIETIN ON NEUTROPHIL FUNCTION IN PREMATURE NEONATES

The in vivo influence of recombinant human erythropoietin (rhEpo) and iron on human neutrophil (PMN) antimicrobial function was assessed. A total of 21 preterm infants were randomized to receive either 200 U/kg/other day of rHuEPO+12 mg/kg/day of iron (EPO+high Fe, seven infants) or 200 U/kg/other day of rhEPO+4 mg/kg/day of iron (EPO+standard Fe, 9 infants) or 4 mg/kg/day of iron only (standard Fe, five infants). PMNs were isolated from blood of these infants 60±5 days after birth and from eight healthy adults. No differences between infants and adults were found in PMN random migration and chemotactic activity to N-formylmethionyl leucyl phenylalanine (FMLP), superoxide anion production in response to FMLP and phagocytosis ofStaphylococcus aureus. In contrast, percentage phagocytosis was significantly lower in EPO+standard Fe as compared to both EPO+high Fe and standard Fe groups (P<0.01). This modest impairment of phagocytic activity of neonatal PMNs found in association with administration of rhEPO and standard iron may be related to consumption of iron during rhEPO-enhanced erythropoiesis.

Tumor necrosis factor alpha enhances antifungal activities of polymorphonuclear and mononuclear phagocytes against Aspergillus fumigatus.

Invasive aspergillosis is a serious complication in immunocompromised patients. The effects of recombinant human tumor necrosis factor alpha (TNF-alpha) on antifungal activities of human neutrophils (polymorphonuclear leukocytes [PMNs]), human monocytes (MNCs), and rabbit pulmonary alveolar macrophages (PAMs) against Aspergillus fumigatus were studied. The percentage of PMN-induced hyphal damage was increased after 30 min of incubation of PMNs with 0.1 ng of TNF-alpha per ml at 37 degreesC (P = 0.043). At 0.1 to 10 ng/ml, TNF-alpha also increased superoxide anion (O2-) produced by PMNs in response to phorbol myristate acetate, N-formylmethionyl leucyl phenylalanine, and unopsonized hyphae (P < 0.01) but did not exert any effect on PMN phagocytosis of conidia in the presence of serum. By comparison, TNF-alpha induced only a slight increase in O2- production by MNCs in response to phorbol myristate acetate (P = 0.05) and no concomitant increase in the percentage of MNC-induced hyphal damage. Incubation of MNCs with TNF-alpha at 0.001 to 10 ng/ml for 2 days had no effect on phagocytosis or conidiocidal activity. By contrast, incubation of PAMs with TNF-alpha at 0.1 to 10 ng/ml for 2 days increased phagocytosis of conidia (P = 0.03). Thus, TNF-alpha augments the capacity of PMNs to damage Aspergillus hyphae, possibly through enhanced oxidative mechanisms, and increases PAM phagocytic activity against conidia. As such, TNF-alpha may have an important role in host defense against aspergillosis, and neutralization of its activity may be complicated by increased susceptibility to aspergillosis.

Effect of hydrocortisone on oxygen free radical generation by mononuclear cells

Corticosteroids are known to exert antiinflammatory and immunosuppressive effects. Since reactive oxygen species (ROS) induce tissue damage and inflammation and since mononuclear cells (MNCs) generate ROS, we investigated whether corticosteroids inhibit ROS generation by MNCs when given systemically. A single dose of either 300 mg (n = 8) or 100 mg (n = 6) of hydrocortisone (HC) was injected intravenously into eight and six subjects, respectively. Blood samples were obtained before and sequentially after the injection. Following 300 mg HC, N-formylmethionyl leucyl phenylalanine (fMLP)-induced ROS generation, assayed by measuring chemiluminescence with luminol, decreased significantly at 0.5 hours and reached a nadir at 2 hours (8% of basal, P < .001); thereafter, it gradually recovered, but was still below baseline at 24 hours. Following the dose of 100 mg HC, ROS generation decreased significantly at 1 hour (nadir, 30% of basal; P < .01) and gradually recovered to near basal level at 8 hours. Serum cortisol concentrations were markedly elevated over basal and remained elevated throughout the first 8 hours of the experiment, returning to baseline at 24 hours. This inhibition of ROS generation by HC (and other glucocorticoids) may have a role to play in mediating the antiinflammatory action of corticosteroids.

Neutrophils exposed to bacterial lipopolysaccharide upregulate NADPH oxidase assembly.

Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more responsive to other stimuli, a phenomenon known as "priming." Since the mechanism of LPS-dependent priming is incompletely understood, we investigated its effects on assembly and activation of the NADPH oxidase. LPS pretreatment increased superoxide (O2-) generation nearly 10-fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). In a broken-cell O2--generating system, activity was increased in plasma membrane-rich fractions and concomitantly decreased in specific granule-rich fractions from LPS-treated cells. Oxidation-reduction spectroscopy and flow cytometry indicated LPS increased plasma membrane association of flavocytochrome b558. Immunoblots of plasma membrane vesicles from LPS-treated PMNs demonstrated translocation of p47-phox but not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three- to sixfold increase (compared with either agent alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O2- generation by intact PMNs. LPS treatment caused limited phosphorylation of p47-phox, and plasma membrane-enriched fractions from LPS- and/or fMLP-treated cells contained fewer acidic species of p47-phox than did those from cells treated with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respiratory burst.

Porphyromonas gingivalis infection of oral epithelium inhibits neutrophil transepithelial migration

Periodontal diseases are inflammatory disorders caused by microorganisms of dental plaque that colonize the gingival sulcus and, subsequently, the periodontal pocket. As in other mucosal infections, the host response to plaque bacteria is characterized by an influx of polymorphonuclear leukocytes (PMNs) to the gingival crevice. Neutrophil migration through the epithelial lining of the gingival pocket is thought to be the first line of defense against plaque bacteria. In order to model this phenomenon in vitro, we used the oral epithelial cell line KB and human PMNs in the Transwell system and examined the impact of Porphyromonas gingivalis-epithelial cell interactions on subsequent PMN transepithelial migration. We demonstrate here that P. gingivalis infection of oral epithelial cells failed to trigger transmigration of PMNs. Furthermore, it significantly inhibited neutrophil transmigration actively induced by stimuli such as N-formylmethionyl leucyl phenylalanine, interleukin-8 (IL-8), and the intestinal pathogen enterotoxigenic Escherichia coli. The ability of P. gingivalis to block PMN transmigration was strongly positively correlated with the ability to adhere to and invade epithelial cells. In addition, P. gingivalis attenuated the production of IL-8 and the expression of intercellular adhesion molecule 1 by epithelial cells. The ability of P. gingivalis to block neutrophil migration across an intact epithelial barrier may critically impair the potential of the host to confront the bacterial challenge and thus may play an important role in the pathogenesis of periodontal disease.

INFLUENCE OF r-HuEPO ON NEUTROPHIL FUNCTION IN PREMATURE NEONATES. 76

Background: In neonates treated with recombinant human protein(rHuEPO) for anemia of prematurity low neutrophil (PMN) counts have been observed by certain investigators. In addition, the production of PMN by fetal haemopoietic progenitors appears to be down-modulated in vitro.Objective: To assess the possible influence of administration of r-HuEPO in association with iron on PMN antimicrobial function.Method: As part of a randomized controlled study, 17 premature neonates with BW<1300 gr, GA≤31 wk were randomized to receive either 200 U/kg/every other day rHuEPO + 12 mg/kg/d iron (EPO+high Fe, n=7) or 200 U/kg/every other day rHuEPO + 4 mg/kg/d iron (EPO + standard Fe, n=5) or 4 mg/kg/d iron only (standard Fe, n=5). All infants were given oral folate and vit E supplementation. PMN counts were measured during the treatment period and two weeks after discharge. PMN were isolated from venus blood of these neonates (mean ± SE 60±5 days after birth) and from 8 healthy young adults by dextran sedimentation/ficoll centrifugation. Random migration and chemotaxis to bacterial product analogue N-formyl methionyl leucyl phenylalanine (FMLP), phagocytic activity evaluated as% phagocytosis of Staphylococcus aureus bacteria (% of phagocytosing PMN) as well as weighted phagocytic index (WPI, index of average number of intracellular bacteria per PMN), and oxidative burst measured as O2-production in response to FMLP were evaluated. Results: The 3 groups of neonates had PMN counts 8.4±3.5, 9.8±3.1 and 7.6±1.8 (x 103/μl) respectively the day of discharge. No differences between neonates and adult controls were found in random migration and chemotactic activity as well as in O2-production. (O2- of neonates was 4.3±0.8 nmoles/0.5*106 PMN/15 min as compared to 2.4±0.7 of adults, p=0.11). Both% phagocytosis and WPI of neonates (90.2±2.2% and 3.0±0.1, respectively) were similar to those of adults(91.9±4.4% and 3.2±0.2). In addition, no significant differences were detected among EPO + high Fe EPO + standard Fe and standard Fe groups of neonates and adult controls in chemotaxis, O2-and WPI. In contrast, there were significant differences between EPO + high Fe and EPO + standard Fe as well as between EPO + standard Fe and standard Fe groups in% phagocytosis(93.6±3.3% vs. 81.8±4.0% vs. 93.8±1.8%, respectively, ANOVA with Bonferroni p<0.01). However, there was no increased incidence of infection in EPO + standard Fe group compared to the other neonates.Conclusion: A modest impairment of phagocytic activity of neonatal PMN was found to occur in association with administration of rHuEPO and standard iron as compared to iron alone and to administration of rHuEPO combined with high iron dose. This may be related to consumption of iron during rHuEPO-enhanced erythropoiesis.

C 2-ceramide primes specifically for the superoxide anion production induced by N-formylmethionylleucyl phenylalanine (fMLP) in human neutrophils

Activated sphingomyelinases release ceramide molecules believed to be involved in intracellular signalling. The present study investigated whether soluble C 2-ceramide modulates some of the effects of N-formylmethionylleucyl phenylalanine (fMLP) and other agonists on human neutrophils (or polymorphonuclear leukocytes-PMN); principally superoxide anion (O 2 −) production. The preincubation of PMN for 15 min with C 2-ceramide increased by up to almost 3-fold the amounts of O 2 − generated in response to 0.1 and 1 μM fMLP. Priming was detected at C 2-ceramide concentrations of 2 μM to 4 μM per million PMN. Though less potent than C 2-ceramide, C 6-ceramide ( N-hexanoylsphingosine) could prime for O 2 − generated in response to 0.1 μM fMLP, with maximal effects obtained at 10–20 μM. In contrast, micromolar concentrations of sphingosine, dihydroceramide, and ceramide-phosphate, failed to exert any potentiating effect on fMLP-induced O 2 − generation. As expected, TNF-α (1000 U/ml), also primed for fMLP-induced O 2 − production; however, the combination of TNF-α and C 2-ceramide showed no additive effect. Moreover, S. aureus sphingomyelinase (0.1 U/ml), was unable to reproduce the priming effects of C 2-ceramide and TNF-α. C 2-ceramide at 2 μM did not enhance the production of O 2 − induced by 100 nM recombinant human interleukin-8 (IL-8), leukotriene B 4 (LTB 4), platelet-activating factor (PAF) or 20 mM sodium fluoride (NaF). Furthermore, C 2-ceramide (2 μM) did not enhance the mobilization of calcium, the release of arachidonic acid or the accumulation of phosphatidylethanol, induced by 100 nM fMLP. This suggests that probably neither phospholipases C, A 2 or D (PLC, PLA 2, PLD) were involved in the priming effect by C 2-ceramide. However, C 2-ceramide inhibited in a dose-related manner the production of O 2 − induced by phorbol 12-myristate 13-acetate (PMA) and mezerein. Furthermore, PMA-stimulated PLD activity was also significantly reduced by a preincubation of PMN with C 2-ceramide. The priming O 2 − production by C 2-ceramide could involve yet unidentified mechanisms specific for fMLP, or it might imply that cytokines such as TNF-α have different mechanisms than C 2-ceramide.

Ex vivo effects of macrophage colony-stimulating factor on human monocyte activity against fungal and bacterial pathogens

The ex vivo effects of macrophage colony-stimulating factor (M-CSF) on antifungal and antibacterial activities of human elutriated monocytes were studied. Cells were isolated prior to the initiation of therapy, on day 3 and at week 7, in six patients with an advanced malignancy receiving M-CSF in a phase I study. Superoxide anion production by monocytes in response to N-formyl methionyl leucyl phenylalanine was enhanced at day 3 of therapy ( P=0.011). In addition, at day 3, fungicidal activity against blastoconidia of Candida albicanswas enhanced by M-CSF treatment ( P=0.026), whereas antifungal activity against hyphae of Aspergillus fumigatuswas not significantly changed. Bactericidal activity against Staphylococcus aureuswas increased at day 3 ( P=0.004). By Northern blot analysis, M-CSF does not upregulate the expression of components of the NADPH-oxidase, the multi- component enzyme system responsible for generation of superoxide radicals by monocytes. Instead, the predominant effect of M-CSF on circulating monocytes is probably a post-transcriptional effect. In conclusion, these findings suggest that administration of M-CSF to patients may enhance microbicidal activities and thus may provide a useful adjunct to conventional antimicrobial therapy.

In vitro and ex vivo effects of cyclosporin A on phagocytic host defenses against Aspergillus fumigatus.

Because cyclosporin A (CsA) is extensively used as an immunosuppressive agent, its effects on phagocytic defenses against Aspergillus fumigatus were studied in vitro and ex vivo. After incubation with 10 to 250 ng of CsA per ml at 37 degrees C for 60 min, polymorphonuclear leukocytes (PMNs) exhibited unaltered superoxide anion (O2-) production in response to phorbol myristate acetate and N-formylmethionyl leucyl phenylalanine, whereas > or = 500 ng/ml significantly suppressed it (P < 0.01). Moreover, at < 250 ng of CsA per ml, PMNs exhibited no change in their capacity to damage unopsonized hyphae of A. fumigatus compared with controls, whereas at > or = 250 ng/ml, CsA suppressed the function (P < 0.01). Although neither CsA (250 ng/ml) nor hydrocortisone (10 micrograms/ml) suppressed PMN O2- production in response to phorbol myristate acetate and N-formylmethionyl leucyl phenylalanine, combination of the two agents reduced the function compared with that at the baseline (P < 0.05). Incubation of monocytes with 100 ng of CsA per ml for 1 or 2 days suppressed their antihyphal activity. No essential change in phagocytic activity of monocyte-derived macrophages (MDMs) against A. fumigatus conidia, tested as the percentage of phagocytosing MDMs and average number of MDM-associated conidia, was detected after 2 or 4 days of incubation with 10 to 1,000 ng of CsA per ml. Furthermore, in rabbits treated with CsA (up to 20 mg/kg of body weight per day intravenously for 7 days), neither O2- production and hyphal damage caused by PMNs or monocytes against hyphae nor phagocytosis of conidia by pulmonary alveolar macrophages was significantly suppressed. Thus, these results demonstrated that CsA within therapeutically relevant concentrations does not suppress antifungal activity of phagocytes except that of circulating monocytes. However, it may induce significant immunosuppression of phagocytes' antifungal function at relatively high concentrations in vitro, especially when combined with corticosteroids.

Fcα R expression on polymorphonuclear leukocyte and superoxide generation in IgA nephropathy

Superoxide (O2-) production and Fc alpha R antigen expression of circulating polymorphonuclear leukocytes (PMNL) isolated from patients with IgA nephropathy (IgAN) and non-IgA mesangial proliferative glomerulonephritis (PGN) and healthy volunteers were investigated to establish their biological importance in the immunopathogenesis of mesangial proliferative glomerulonephritis. PMNL from both patient groups showed increased O2- production when stimulated with N-formyl methionyl leucyl phenylalanine (FMLP) and phorbol myristate acetate (PMA). The increased O2- generation demonstrated a positive correlation with the degree of proteinuria. Aggregated IgA caused enhanced O2- production only in patients with IgAN who also showed a significant correlation with proteinuria. Increased expression of Fc alpha R on circulating PMNL was observed in IgAN patients as determined by flow cytometric analysis. The amount of Fc alpha R on PMNL was positively correlated with O2- generation triggered with IgA aggregates. These results suggest that: 1. Circulating PMNL may potentially be participating in the pathogenesis of glomerular injury in mesangial proliferative glomerulonephritis, and 2. IgA aggregates/immune complexes may contribute to the immunopathogenesis of IgAN through augmenting the Fc alpha receptor-mediated generation of superoxide anion.

Respiratory burst reaction changes with age in rat peritoneal macrophages

The respiratory burst reaction, estimated as O ·− 2 production, has been studied in rat peritoneal macrophages of different age (3, 12 and 24 months). To stimulate NADPH oxidase, the enzyme responsible for the respiratory burst, various stimuli that act in different ways have been used: PMA (phorbol myristate acetate), Con-A (concanavalin A) and N-FMLP ( N-formyl-methionyl-leucyl-phenylalanine). All produced a decrease in response with age, with that from PMA being the greatest. The PMA-induced decrease in the O ·− 2 production may be related to the inactivation of NADPH-producing enzymes such as glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase that we have found with age. Glutathione reductase, an enzyme that participates in the maintenance of the redox status in the cell, also showed an age-related decrease. Enzymes that participate in oxygen species scavenging, such as glutathione peroxidase and Cu/Zn superoxide dismutase, did not change with age, although an age-related decrease in catalase activity was found.

Prevention of corticosteroid-induced suppression of human polymorphonuclear leukocyte-induced damage of Aspergillus fumigatus hyphae by granulocyte colony-stimulating factor and gamma interferon

Neutrophils (PMNs) are a critical line of defense against Aspergillus fumigatus infection. Increased frequency of invasive aspergillosis has been observed in patients receiving corticosteroids, suggesting a deleterious effect of these compounds on PMN antifungal function. To investigate this hypothesis and to determine the potential preventive utility of granulocyte colony-stimulating factor (G-CSF) and gamma interferon (IFN-gamma), the effects of hydrocortisone (HCS) and dexamethasone (DXS) on PMN-induced damage of Aspergillus fumigatus hyphae were studied with or without pretreatment of PMNs with G-CSF and IFN-gamma. PMNs treated with HCS (> or = 3,000 microM) or DXS (> or = 10 microM) during a 2-h colorimetric tetrazolium metabolic assay (using methylthiotetrazolium) showed suppressed percentage of hyphal damage (P < 0.02). In addition, both HCS (> or = 30 microM) and DXS (> or = 1 microM) significantly suppressed oxidative burst measured as superoxide anion release by PMNs in response to opsonized and nonopsonized hyphae as well as to N-formylmethionyl leucyl phenylalanine. Pretreatment of PMNs with G-CSF (4,000 U/ml) and/or IFN-gamma (100 and 1,000 U/ml) for 90 min prevented the suppression of hyphal damage that occurred in the presence of HCS (3,000 microM; P < 0.01) or DXS (10 microM; P < or = 0.001). G-CSF (4,000 U/ml) and IFN-gamma (100 U/ml) combined had an additive effect on increasing the antifungal activity of HCS-treated but not of DXS-treated PMNs compared with IFN-gamma alone (P = 0.015). Thus, these findings reveal that corticosteroids impair PMN function in response to A. fumigatus and that G-CSF and IFN-gamma prevent this impairment.

Synthesis and Biological Evaluation of a New N4-(N-Formyl Peptide)- 2′,3′-Dideoxy-3′-Thiacytidine as Anti-Hiv Prodrug

Abstract The synthesis of a new analogue of 2′,3′-dideoxy-3′-thiacytidine 9 covalently linked to an N-formyl methionyl leucyl phenylalanine peptide is described. This new prodrug analogue has been tested on the one hand as activator of human polymorphonuclear leukocytes (an EC50 value of 1.8 10−5 M was determined from dose-response curve for superoxide production) and on the other hand as inhibitor of the syncitium formation caused by HIV-1 in MT4-cells (IC50 = 8.0± 0.8 μM). In so far as this new prodrug possesses these two biological properties, it represents a useful “chemical-head” capable of targeting specific receptors located on leukocytes membranes.

Enhancement of oxidative response and damage caused by human neutrophils to Aspergillus fumigatus hyphae by granulocyte colony-stimulating factor and gamma interferon

Invasive aspergillosis is a serious fungal infection caused by the proliferation and invasion of Aspergillus hyphae in tissue. Neutrophils (PMNs) are the most important line of defense against Aspergillus hyphae. To investigate the role of granulocyte colony-stimulating factor (G-CSF) and gamma interferon (IFN-gamma) against Aspergillus fumigatus, we studied the effects of the two cytokines on the oxidative burst and the capacity of normal human PMNs to damage hyphae of the organism. G-CSF enhanced PMN oxidative burst measured as superoxide anion (O2-) production in response to N-formylmethionyl leucyl phenylalanine, serum opsonized hyphae, and nonopsonized hyphae by 75, 37, and 24%, respectively, compared with control PMNs (P < 0.015). IFN-gamma also induced increases of 52, 71, and 96%, respectively, in response to the same stimuli (P < 0.006). In addition, the capacity of PMNs to damage hyphae as measured by the 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MMT) colorimetric metabolic assay was significantly enhanced by G-CSF and IFN-gamma (P < 0.01 and < 0.05, respectively). The enhancement was achieved irrespective of serum opsonization of the hyphae, suggesting upregulatory actions of the two cytokines on signal pathways specific for opsonized and nonopsonized hyphae. The combination of the two cytokines exhibited an additive effect at the higher concentrations compared with the effects of the cytokines alone (P < 0.05). Pretreatment of PMNs with protein synthesis inhibitors showed that IFN-gamma activates PMN function through transcriptional regulation, whereas the effect of G-CSF does not require new proteins. These in vitro effects suggest modulatory roles for G-CSF and IFN-gamma in the host defense against Aspergillus hyphae irrespective of serum opsonization and a potential utility of the cytokines as adjuncts for the prevention and possible treatment of invasive aspergillosis.

Suppression of polymorphonuclear leukocyte bactericidal activity by suramin.

Suramin is a polyanionic compound with potent antineoplastic properties. Because polymorphonuclear leukocytes (PMNs) are a crucial component of host defenses against bacteria and fungi, the effects of suramin on PMN function were studied in vitro. PMNs from healthy donors were incubated with concentrations of suramin of 1 to 1,000 micrograms/ml (within and exceeding the therapeutic range) for 30 min, and PMN functional parameters were subsequently assessed. Suramin had no effect on viability, chemotaxis to N-formylmethionyl leucyl phenylalanine, phagocytosis of Candida albicans, or superoxide anion production in response to phorbol myristate acetate and formylmethionyl leucyl phenylalanine. Fungicidal activity against C. albicans blastoconidia was unaffected at a suramin concentration of < 500 micrograms/ml, whereas at higher concentrations a slight suppression was observed (P = 0.04). Bactericidal activity against Staphylococcus aureus was significantly suppressed by concentrations of > or = 100 micrograms/ml (P < 0.01). Phagocytosis of S. aureus was also significantly impaired at > or = 10 micrograms/ml (P < 0.05). The presence of 10% human serum during pretreatment did not abrogate the suramin-induced suppression of bactericidal activity. Treatment of PMNs with granulocyte colony-stimulating factor (4,000 U/ml) for 30 min prior to the addition of suramin (250 micrograms/ml) improved the bactericidal defect (P = 0.02). The PMN functional impairment may be related to increased susceptibility to bacterial infections, and granulocyte colony-stimulating factor may improve the defect.

Diminished actin polymerization by neutrophils from newborn infants.

During the newborn period, abnormalities of neutrophil (PMN) function predispose infants to serious bacterial disease. Actin is a major contributor to PMN shape change and motile behavior. To determine the mechanism underlying defects in newborn granulocyte polarity and chemotaxis, we investigated actin polymerization by cord blood PMN from healthy term infants and adult controls. F-actin (filamentous) content was quantified in the resting state and after stimulation by fluorescence-activated cell-sorter analysis of nitrobenzoxadiazole-phallacidin-stained cells. PMN from newborn infants demonstrated similar basal F-actin levels when compared with adults. N-formyl methionyl leucyl phenylalanine induced a marked increase in actin polymerization that was maximal at 30 s in both neonates and adults and that then declined slowly (depolymerization) over the following 10 min. However, the F-actin content of PMN from newborn infants was significantly diminished when compared with adults at 30 and 60 s after N-formyl methionyl leucyl phenylalanine stimulation (p < 0.05). Both the rate and dose response of N-formyl methionyl leucyl phenylalanine-induced actin polymerization were similar for adult and neonatal PMN. PMN from newborn infants also demonstrated significantly diminished actin polymerization when compared with adults 60 s after stimulation with platelet-activating factor (p < 0.05). Decreased concentrations of F-actin may help explain the observed abnormalities of PMN polarity and chemotaxis in healthy newborn infants.

Fibronectin binding and neutrophil aggregation in burn injury.

Fibronectin (Fn) plays an important role in the adhesive function of many cells including neutrophils (PMN). We examined the hypothesis that activated PMN develop binding sites for fibronectin which allows for the aggregation of contiguous PMN. Because PMN adhesive function is altered in acute burn injury, we also investigated the role of Fn in the aggregation of PMN from subjects with acute thermal injury. The chemotactic peptide, n-formylmethionyl leucyl phenylalanine, induced rapid binding of radioiodinated plasma Fn to PMN. Significant binding of Fn was detected as early as 30 sec poststimulus and maximal binding occurred at 5 min. Fn binding was only partially reversible and nonsaturable. The chemotactic peptide induced aggregation and binding of Fn to PMN with similar kinetics, concentration dependence, temperature, and cation requirements. In burn patients, PMN demonstrated a significant decrease in chemotactic peptide-induced aggregation which was associated with decreased binding of Fn. Alterations in the binding of Fn to PMN may be responsible, in part, for diminished aggregation responses of PMN in the early stages of thermal injury.