Functions Of Human Polymorphonuclear Neutrophils Research Articles

Effect of Linezolid on the Phagocytic Functions of Human Polymorphonuclear Leukocytes

The effect of linezolid on the phagocytic and bactericidal functions of human polymorphonuclear neutrophils (PMN) against gram-positive cocci was evaluated. Preincubation (30 min; 37°C) of PMN with different concentrations of linezolid (2, 10 and 20 mg/l) did not significantly affect the phagocytosis of either Staphylococcus aureus (methicillin susceptible and resistant) or Enterococcus faecalis (vancomycin susceptible and resistant). Overnight exposure of vancomycin-resistant E. faecalis to 1/4 minimum inhibitory concentrations (MIC) of linezolid slightly increased phagocytosis by PMN. Preincubation of the other three strains with 1/4 MIC of linezolid did not affect phagocytosis by these cells. Preincubation of PMN (30 min; 37°C) using different extracellular concentrations of linezolid (2, 10 and 20 mg/l) did not affect their production of either superoxide or hydrogen peroxide radicals. In conclusion, linezolid did not affect the phagocytic and bactericidal functions of human PMN.

A novel calcium-dependent proapoptotic effect of annexin 1 on human neutrophils.

The glucocorticoid-inducible protein annexin (ANXA) 1 is an anti-inflammatory mediator that down-regulates the host response. Endogenously, ANXA1 is released in large amounts from adherent polymorphonuclear neutrophils (PMN) and binds to their cell surface to inhibit their extravasation into inflamed tissues. The present study determined the effects of exogenous ANXA1 on several functions of human PMN in vitro. Addition of 0.1-1 microM human recombinant ANXA1 to the PMN provoked rapid and transient changes in intracellular Ca2+ concentrations that were blocked by the Ca2+ channel inhibitor SKF-96365. Although ANXA1 did not affect oxidant production and only minimally affected PMN chemotactic properties, the ANXA1-promoted Ca2+ influx was associated with two important functional effects: shedding of L-selectin and acceleration of PMN apoptosis. The latter effect was confirmed using three distinct technical procedures, namely, cell cycle, Hoechst staining, and ANXA5 binding assay. ANXA1-induced PMN apoptosis was insensitive to inhibitors of L-selectin shedding, whereas it appeared to be associated with dephosphorylation of the proapoptotic intracellular mediator BAD. In conclusion, exogenous ANXA1 displayed selective actions on human PMN. We propose that the new proapoptotic effect reported here may be part of the spectrum of ANXA1-mediated events involved in the resolution of acute inflammation.

Ethanol-induced inhibition of cytokine release and protein degranulation in human neutrophils

Ethanol impairs immune responses in humans and animal models, in vivo and in vitro. In particular, ethanol inhibits some key functions of human polymorphonuclear neutrophils (PMN). We investigated the impact of ethanol on cytokine production by highly purified PMN. In a time- and concentration-dependent manner, ethanol inhibited the production of interleukin (IL)-8 protein and mRNA and also hindered tumor necrosis factor alpha (TNF-alpha) release by modulating the expression of the TNF-alpha-converting enzyme involved in TNF-alpha shedding. This disruption of PMN cytokine release by ethanol may contribute to the increased risk of infection in alcoholic patients. Degranulation of hepatocyte growth factor (HGF) was also impaired by a clinically relevant ethanol concentration (0.8%), an action that may delay the repair of alcoholic liver damage. These findings suggest that ethanol may modulate three major cytokines involved in alcoholic liver diseases, IL-8, TNF-alpha, and HGF, via three different mechanisms.

The stimulatory effects of nasal discharge from patients with perennial allergic rhinitis on normal human neutrophils are normalized after treatment with a new mixed formula of Chinese herbs

In our previous study, we found a new mixed formula of Chinese herbs containing Shin-yi-san+Xiao-qing-long-tang+Xiang-sha-liu-jun-zi-tang (9+3+3 g divided in three doses/day) was beneficial to the patients with perennial allergic rhinitis (AR) via complicated immunomodulatory effects on both mononuclear cells (MNC) and polymorphonuclear neutrophils (PMN). In the present study, we further determined the effects of nasal fluid from AR patients on the functions of human PMN before and after treatment with the mixed formula. We found the nasal discharge, but not serum, from AR group with high serum IgE (H-IgE, serum IgE >200 KIU/l) before treatment exerted many stimulating effects on normal PMN including delayed apoptosis, enhanced production of soluble intercellular adhesion molecule 1 (sICAM-1), interleukin 8 (IL-8) and prostaglandin E 2 (PGE 2), increased phagocytosis, and augmented cyclooxygenase 2 (COX-2) mRNA expression of PMN. However, these stimulating effects of nasal fluid on PMN were not found in low IgE group (L-IgE, serum IgE <200 KIU/l). These PMN-enhancing effects of H-IgE nasal fluid were abolished after 3-month treatment with the mixed Chinese herb formula. In conclusion, our results suggest that the new mixed herb formula treatment suppressed nasal mucosa inflammation by normalizing stimulatory effects of allergic nasal discharge of patients with H-IgE allergic rhinitis.

Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on N-formyl-methionyl-leucyl-phenylalanine-induced responses of polymorphonuclear neutrophils in vitro.

[Poly(ethyleneglycol)]-modified hemoglobin vesicles (PEG-HbV), a type of encapsulated hemoglobin, have been developed as artificial oxygen carriers and it is important to evaluate their blood compatibility. We studied the effects of PEG-HbV on human polymor phonuclear neutrophils (PMNs) in vitro, focusing on the functional responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP) as an agonist. The pretreatment of the PMNs with PEG-HbV up to a concentration of 60 mg/dl Hb did not affect the fMLP-triggered chemotactic activity. In parallel to these results, the fMLP-induced upregulation of CD11b (Mac-1) levels on the PEG-HbV-pretreated PMNs was comparable to that of untreated cells. Furthermore, the pretreatment of the PMNs with the PEG-HbV even at 600 mg/dl Hb did not affect the gelatinase B (Matrix methalloproteinase-9 (MMP-9)) release, suggesting that the fMLP-induced release of secondary and tertiary granules was normal. In addition, the fMLP-triggered superoxide production of the PMNs was unchanged by the pretreatment with the PEG-HbV at 600 mg/dl Hb. Thus, these results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on the fMLP-triggered functions of human PMNs.

Antioxidative properties of serotonin and the bactericidal function of polymorphonuclear phagocytes.

We investigated the antioxidative properties of platelet-released serotonin on the bactericidal function of polymorphonuclear neutrophils (PMN) since there is a surprising co-incidence of low blood serotonin and an increased rate of infections. The antioxidative properties of serotonin were demonstrated by its suppressive effects on phagocytosis-associated, luminol-enhanced chemiluminescence (CL). The bactericidal activity of PMN was determined by a microbiological assay using opsonized Staphylococcus aureus. Serotonin suppresses luminol-enhanced chemiluminescence in a dose-dependent manner indicating an interaction with reactive oxygen species, which are responsible for effective bacterial killing during the phagocytosis-associated "respiratory burst". The modulation of the bactericidal function of PMN by serotonin is complex and depends upon the amount of serotonin: at concentrations normally present at sites of tissue injury and consecutive thrombus formation (10(-6) to 10(-5) M) bacterial killing increases by about 50%. In contrast, at pharmacological concentrations (10(-3) to 10(-2) M) an adverse effect can be observed: the elimination of opsonized S. aureus is reduced by 30 to 90%. Exogenous serotonin is capable of modulating important biological functions of human PMN in vitro. At appropriate concentrations, the antibacterial defence improves significantly probably due to reduced autooxidation, whereas higher concentrations counteract an efficient bacterial killing.

Effect of RO 23-9424, cefotaxime and fleroxacin on functions of human polymorphonuclear cells and cytokine production by human monocytes.

The way in which an antibiotic interacts with host defences could influence the clinical outcome of many infectious diseases. The impact of RO 23-9424, a novel dual-action and extended-spectrum antibiotic, was studied on several functions of human polymorphonuclear neutrophils (PMNs). A significant (P < 0.05) increase of the superoxide (O2-) released by phorbol-myristate acetate (PMA) -stimulated PMN (10-100 mg/L) can be observed in the RO 23-9424 pre-treated cells. RO 23-9424, particularly at low dosages, showed an interesting but not statistically significant effect on PMN phagocytosis. Higher dosages of RO 23-9424 (50-200 mg/L) and fleroxacin (20-200 mg/L) significantly reduced PMN chemotaxis. Cytokine production by human monocytes were also evaluated after incubation with the antibiotic (100-200 mg/L) in both basal conditions and in response to endotoxin (lipopolysaccharide, LPS). In the LPS-treated cells, RO 23-9424 (100 mg/L) significantly (P < 0.05) enhanced the tumour necrosis factor-alpha (TNF-alpha) levels, compared with LPS controls after 4 h of incubation. RO 23-9424 (200 mg/L) was able to reduce in a dose-dependent way LPS-induced interleukin-1 beta (IL-1 beta) after 4 and 24 h of incubation. Interleukin-8 (IL-8) release was not significantly changed by RO 23-9424. Cefotaxime (200 mg/L) significantly (P < 0.05) increased the basal levels of IL-1 beta and reduced basal IL-8 concentration after 24 h of incubation. The lower concentration of cefotaxime reduced the LPS-stimulated IL-8 levels. Fleroxacin (100 mg/L) enhanced basal levels of IL-8. The potentiated PMN phagocytosis, the significantly enhanced O2- release by PMA-stimulated PMN and the dimetric changes of TNF-alpha and IL-1 beta appeared peculiar for RO 23-9424 and may have useful therapeutical implications.

Purine catabolism in polymorphonuclear neutrophils. Phorbol myristate acetate-induced accumulation of adenosine owing to inactivation of extracellularly released adenosine deaminase.

Since physiological concentrations (0.1-1 microM) of adenosine influence the functions of human polymorphonuclear neutrophils (PMNs), we investigated the metabolism of adenosine in suspensions of stimulated and unstimulated PMNs. Stimulation with phorbol myristate acetate (PMA, 1 microM), but not by zymosan (0.5 mg/ml) or N-formyl-methionyl-leucyl-phenylalanine (fMLP, 1 microM), provoked an accumulation of endogenous adenosine at a rate of 2.3 +/- 1.0 amol/cell per minute. A similar accumulation was observed with both unstimulated and stimulated PMNs after the addition of deoxycoformycin (dCF, 1-100 microM), an inhibitor of adenosine deaminase. Exogenous adenosine (10 microM) was deaminated at a rate of 9.8 +/- 3.7 amol/cell per minute in control or zymosan or fMLP-stimulated PMN suspensions. This deamination was nearly completely suppressed when the PMNs had been stimulated with PMA. In contrast, the activity of adenosine deaminase in PMN lysates (231 +/- 72 amol/cell per minute) was not modified by PMA stimulation. alpha, beta-Methyleneadenosine 5'-diphosphate (AMPCP, 2.5 mM), an inhibitor of membranous ecto-5'-nucleotidase, profoundly inhibited endogenous adenosine accumulation under all conditions. PMA stimulation also provoked an inactivation of extracellular adenosine deaminase, purine nucleoside phosphorylase, and lactate dehydrogenase in PMN suspensions. We concluded that PMNs, even when not stimulated, continuously produce adenosine by dephosphorylation of extracellularly released adenylates; and that stimulation of PMNs by PMA causes adenosine accumulation owing to the inactivation of adenosine deaminase released by broken cells.

Modulation of phagocytosis-associated respiratory burst by human cystatin C: Role of the N-terminal tetrapeptide LysProProArg

Cystatin C, a cysteine protease inhibitor, has recently been suggested to be a potent regulator in inflammatory processes. Human cystatin C was isolated from the urine of one patient suffering from tubular disorders and was tested for its effects on two functions of human polymorphonuclear neutrophils (PMN): O 2 − release and phagocytosis. Slow-form or (des 1–4) cystatin C and fast-form or (des 1–8) cystatin C differed by the presence in (des 1–4) cystatin C only of the N-terminal tetrapeptide LysProProArg. Whereas (des 1–8) cystatin C did not seem to interfere with PMN functions at physiological concentrations, (des 1–4) cystatin C induced an inhibition of PMN phagocytosis-associated respiratory burst in response to opsonized zymosan particles. The inhibition may be attributed to the tetrapeptide LysProProArg which has been synthesized and shown to have the same inhibitor effects, at concentrations similar to those required for (des 1–4) cystatin C. These results support a potential role for cystatin C as a modulator during inflammation.

Influence of Diesel Soot Particles and Sulfite on Functions of Polymorphonuclear Leukocytes

Aqueous suspensions of diesel soot particles in combination with sulfite influence certain functions of human polymorphonuclear neutrophils in vitro. Chemiluminescence, generated after activation by opsonized zymosan as well as oxygen uptake were decreased, whereas phagocytosis was increased. An enhancement of degranulation could not be observed. The single substances show little or no effects on the above properties. The results indicate that combinations of air pollutants such as diesel soot and sulfite may modulate vital functions of activated leukocytes in vivo.

Activation of human polymorphonuclear neutrophil functions by interferon-gamma and tumor necrosis factors.

Recombinant human interferon-gamma (rHuIFN-gamma) and natural human tumor necrosis factor beta (nHuTNF-beta) (previously called lymphotoxin), purified to homogeneity, were used to assess their effects on certain functions of human polymorphonuclear neutrophils (PMN) in vitro. The treatment of PMN with 100 U of either rHuIFN-gamma or nHuTNF-beta for 20 min significantly increased their ability to phagocytize 1.5-microns latex beads as detected by flow cytometry. Preparations of recombinant human TNF-beta (rHuTNF-beta) showed activities similar to those of its natural counterpart in activating phagocytosis. In addition, a significant enhancement in PMN-mediated antibody-dependent cellular cytotoxicity was observed after treatment for 2 hr with IFN gamma and both TNF-alpha and TNF-beta. The enhancement by treatment with a combination of rHuIFN-gamma and nHuTNF-beta exceeded the enhancement caused by either agent alone. We also show that although lipopolysaccharide (LPS) is a potent stimulator of PMN function, polymyxin B can block LPS-induced but not lymphokine-induced activation. These data demonstrate new activities for both TNF-alpha and TNF-beta in augmenting the phagocytic and cytotoxic activities of PMN.