Effect of nitric oxide on staphylococcal killing and interactive effect with superoxide.

The role of reactive nitrogen intermediates (RNI) such as nitric oxide (.NO) in host defense against pyogenic microorganisms is unclear, and the actual interactive effect of RNI and reactive oxidative intermediates (ROI) for microbial killing has not been determined. Since, in nature, ROI and RNI might be generated together within any local infection, we evaluated the separate and interactive effects of .NO and O2- on staphylococcal survival by using a simplified system devoid of eukaryotic cells. These studies showed that prolonged exposure of staphylococci to .NO does not result in early loss of viability but instead is associated with a dose-related delayed loss of viability. This effect is abrogated by the presence of hemoglobin, providing further evidence that the effect is RNI associated. Superoxide-mediated killing also is dose related, but in contrast to RNI-mediated killing, it is rapid and occurs within 2 h of exposure. We further show that the interaction of .NO and O(2)- results in decreased O(2)--mediated staphylococcal killing at early time points. .NO, however, appears to enhance or stabilize microbial killing over prolonged periods of incubation. This study did not produce evidence of early synergism of ROI and RNI, but it does suggest that .NO may contribute to host defense, especially when ROI-mediated killing is compromised.

Biomaterial-neutrophil interactions: dysregulation of oxidative functions of fresh neutrophils induced by prior neutrophil-biomaterial interaction.

Biomaterial-associated infection results in increased morbidity and mortality, and may occur because of nonproductive premature activation of neutrophils resulting in impaired phagocyte function at the biomaterial surface in the event of bacterial challenge. To further explore the effects of this premature activation, we evaluated the supernatants of biomaterial associated neutrophils to determine whether soluble mediators were released, and the likely role of these mediators. We show that these supernatants contain a chemoattractant and thereby induce chemotaxis by fresh neutrophils. No evidence of enhanced oxidative free radical production by either unstimulated neutrophils or a primed response to other mediators occurs when neutrophils were incubated with these supernatants. We also examined the effect of adding fresh neutrophils to a biomaterial surface containing a previous inoculum of neutrophils, and observed that the fresh cells did not become stimulated to release reactive oxygen intermediates (ROI) and also exhibited impaired killing of staphylococci. These studies suggest that not only does the biomaterial surface activate the initial wave of neutrophils but that subsequent waves of neutrophils exhibit an impaired host-defense function. These results are consistent with the known impairment of host defense in the presence of biomaterials, and provide evidence for a long-term down-regulation of neutrophil function at biomaterial surfaces.

Mechanisms of biomaterial-induced superoxide release by neutrophils.

Biomaterial-centered infection is an important cause of the failure of prosthetic implants and organs. Because neutrophils mediate host defense against infection, the effect of biomaterials on neutrophil superoxide release and the mechanism of that effect were investigated using three materials commonly employed in surgical practice. The graft materials were expanded polytetrafluorethylene (PTFE), polyurethane and woven dacron. Polystyrene, a commonly used laboratory support vessel, was also studied. Both polystyrene and polyurethane were activating, but serum inhibitable, whereas PTFE was nonactivating, and woven dacron was not activating unless serum was present. The signaling mechanisms used by these materials demonstrated time and material dependency. Pertussis toxin inhibition of G protein-dependent activation had little or no effect on biomaterial induced activation, whereas FMLP-induced activation of the same biomaterial-associated cells was inhibited. Protein kinase C inhibition with staurosporine greatly inhibited polystyrene-induced activation, but had only a partial effect with polyurethane and even less effect with the activation associated with serum-treated woven dacron. These studies demonstrated that biomaterial contact-induced neutrophil activation differed from that described for cells in suspension, and showed that activation mechanisms on one material cannot be extrapolated to mechanisms on other materials.

Effect of low neutral endopeptidase expression on response to fMLP.

The effects of the low neutral endopeptidase (24.11/CD10) exhibited by cord blood neutrophils on response to the peptide mediator of cell function f-met-leu-phe (fMLP) were investigated. Oxidative radical release (superoxide and hydrogen peroxide) and chemotactic responses to fMLP were determined and compared to the responses of normal adult neutrophils. The effect of fMLP on CD10 expression as measured by flow cytometry also was evaluated. The data show that cord blood neutrophils produce increased amounts of O2- and H2O2 largely because of a prolonged reaction time to fMLP. In addition, adult polymorphonuclear neutrophil leukocytes increase the intensity of their expression of CD10 following fMLP stimulation, whereas cord blood CD10 expression does not change. Evaluation of chemotaxis demonstrated that cord blood neutrophils exhibited a shift in the fMLP dose-response relationship showing relatively better chemotaxis to lower concentrations. In support of this observation, the inhibition of endopeptidase on adult polymorphonuclear neutrophils leukocytes by phosphoramidon was associated with an augmentation of chemotaxis to 10(-9) and 10(-10) mol/L fMLP. These studies demonstrate that cord blood and adult neutrophils respond differently to fMLP and suggest that membrane endopeptidase plays a role in the observed response patterns. The low level of expression of CD10 on cord blood neutrophils and the failure to increase its expression after fMLP stimulation suggests that adult neutrophils have preformed intracellular CD10 that is not present in the newborn. We propose that the lack of endopeptidase on cord blood neutrophils together with other known features of immaturity may play a role in the overall compromised host defense exhibited by the newborn.

Biomaterial-induced alterations of neutrophil superoxide production.

Because periprosthetic infection remains a vexing problem for patients receiving implanted devices, we evaluated the effect of several materials on neutrophil free radical production. Human peripheral blood neutrophils were incubated with several sterile, lipopolysaccharide (LPS)-free biomaterials used in surgically implantable prosthetic devices: polyurethane, woven dacron, and velcro. Free radical formation as the superoxide (O2-) anion was evaluated by cytochrome c reduction in neutrophils that were exposed to the materials and then removed and in neutrophils allowed to remain in association with the materials. Neutrophils exposed to polyurethane or woven dacron for 30 or 60 min and then removed consistently exhibited an enhanced release of O2- after simulation via receptor engagement with formyl methionyl-leucyl-phenylalanine. Enhanced reactivity to stimulation via protein kinase C with phorbol myristate acetate, however, was not consistently observed. The cells evaluated for O2- release during continuous association with the biomaterials showed enhanced metabolic activity during short periods of association (especially with polyurethane and woven dacron). Although O2- release by neutrophils in association with these materials decreased with longer periods of incubation, it was not obliterated. These studies, therefore, show that several commonly used biomaterials activate neutrophils soon after exposure and that this activated state diminishes with prolonged exposure but nevertheless remains measurable. The diminishing level of activity with prolonged exposure, however, suggests that ultimately a depletion of reactivity may occur and may result in increased susceptibility to periprosthetic infection.

Neutrophil function in chronic neutrophilic leukemia: defective respiratory burst in response to phorbol esters.

Functional analyses were performed on neutrophils isolated from 6 patients from two institutions who displayed features of chronic neutrophilic leukemia (CNL). These neutrophils demonstrated a consistent deficiency (44 +/- 8% of control values) in superoxide anion (O2-) production in response to the phorbol ester, phorbol myristate acetate (PMA). O2- production in response to chemotactic peptides was near normal (82.3 +/- 10.7% of control values). Bacterial killing was normal in the two patients studied, and chemotaxis was diminished in response to zymosan-activated plasma and to high concentrations of chemotactic peptides in the patients studied. Cytosolic C kinase activity was decreased in one of the two patients studied. These results suggest that a deficient O2- release in response to PMA is a hallmark of neutrophils in CNL and may provide a diagnostic indicator of this condition.

Platelets and ATP prime neutrophils for enhanced O2- generation at low concentrations but inhibit O2- generation at high concentrations.

Platelets are currently thought to play a role in tissue injury and inflammatory states both directly and indirectly through their action on neutrophils (PMNs). Both stimulation and inhibition of PMN superoxide anion (O2-) production by platelets has been reported. To clarify these discrepant observations, we investigated the effects of wide ranges of platelet to PMN ratios as well as concentrations of ATP and ADP on human PMN O2- production. Platelets, at low platelet-to-PMN ratios (1:1 and 5:1), primed PMNs which were stimulated with either FMLP or PMA. However, at higher platelet-to-PMN ratios (25:1, 50:1, and 100:1), inhibition of O2- production was seen. ATP also had a biphasic effect on O2-production: low concentrations of ATP (1 x 10(-6) to 3.2 x 10(-4) M) increased O2-production and high concentrations of ATP (6.4 x 10(-4) M and above) inhibited O2-production. ADP, when added to stimulatory concentrations at ATP, also caused inhibition of O2- production. The incubation time for platelet-neutrophil interactions in vitro was also crucial. Short incubation periods lead to priming, whereas longer periods (greater than 5 min) lead to inhibition. We believe that these studies help to resolve the controversy over the effects of platelets upon the production of O2- by human PMNs and lend further support to the notion that platelets may modulate injury resulting from neutrophil activation.

Biomaterial associated impairment of local neutrophil function.

The effect of biomaterials on neutrophil function was studied in vitro to determine if these materials activated neutrophils and to determine the subsequent response of these neutrophils to further stimulation. Two biomaterials--polyurethane, a commonly used substance, and Velcro pile (used in the Jarvik 7 heart)--were evaluated. Two control substances, polyethylene and serum-coated polystyrene, were used for comparison. Neutrophil superoxide release was measured following incubation with these materials for 10, 30, and 120 min in the absence of additional stimulation and after stimulation with formylmethionylleucylphenylalanine (fMLP) or phorbol myristate acetate (PMA). The authors observed that the incubation of neutrophils on both polyurethane and Velcro resulted in substantially increased superoxide release that was greater after the 10 min than after the 30 or 120 min association. These activated neutrophils exhibited a poor additional response to fMLP but responded well to PMA. The effect of implantation of the Novacor left ventricular assist device on peripheral blood neutrophil function was also evaluated. The peripheral blood neutrophils exhibited normal superoxide release and chemotaxis. These studies suggest that biomaterials may have a profound local effect on neutrophils, which may predispose the patient to periprosthetic infection, but that the reactivity of circulating neutrophils is unimpaired.

Endothelial cells inhibit receptor-mediated superoxide anion production by human polymorphonuclear leukocytes via a soluble inhibitor.

Confluent monolayers of bovine pulmonary artery endothelial cells (BPAE) or human umbilical vein endothelial cells (HUVE) inhibited by 80 to 90% the production of O2- by added human neutrophils (PMNs) stimulated by plasma membrane receptor-mediated activators (formylmethionylleucylphenylalanine [fMLP], opsonized zymosan, heat-killed Staphylococci), but not by non-plasma membrane receptor-mediated activators (phorbol myristate acetate and delta-hexachlorocyclohexane). Degranulation induced by fMLP was also inhibited by BPAE. Inhibition was not affected by eicosatetraynoic acid (ETYA) or indomethacin. To assess the role of cell-cell contact, 0.45-microns-pore culture plate inserts were employed to prevent PMN-endothelial cell contact during incubation. A similar amount of inhibition of stimulated PMNs superoxide production was seen as compared to PMN-endothelial incubations where contact occurred. A soluble component released by BPAE monolayers, when added to PMNs, duplicated the inhibition seen by BPAE-PMN co-incubation. Incubation of BPAE with adenosine deaminase did not reduce inhibition of O2- production compared to controls without adenosine deaminase. There was no evidence of endothelial scavenging of O2- generated by hypoxanthine-xanthine oxidase, and inhibition of endothelial superoxide dismutase did not diminish the inhibitory effort. We conclude that cell contact is not required for BPAE inhibition of fMLP-stimulated O2- production by PMN, and that scavenging of superoxide anion is not the mechanism. The inhibitor appears to be a polypeptide with an apparent molecular weight between 1,000 and 10,000 D and does not appear to be adenosine, an arachidonate metabolite, or superoxide dismutase. The mechanism may involve down-regulation of plasma membrane receptor-mediated activation of PMNs.

Inhibition of chemotaxis Ng-monomethyl-L-arginine: a role for cyclic GMP.

The metabolism of L-arginine to nitric oxide (NO) has been shown to be important for the effector functions of many cell types, including polymorphonuclear (PMN) leukocytes. Its effect appears to be mediated at least in part by NO stimulation of soluble guanylate cyclase. We evaluated the role of this pathway in two PMN effector functions: cell movement and microbial killing, using the competitive inhibitor of L-arginine conversion to NO, NG-monomethyl-L-arginine (NMA). We also evaluated the effect of additional L-arginine and dibutyryl cyclic guanosine monophosphate (cGMP) on any NMA-associated changes. Human peripheral blood neutrophils were used and the cells were incubated with and without NMA. Chemotaxis was evaluated using a 48-well micro-Boyden chamber. Microbial killing was evaluated using S aureus strains D2C and 502A. These studies demonstrated that chemotaxis to formyl-methionyl-leucyl-phenylalanine was markedly inhibited in NMA-treated cells. This inhibition could be overcome if L-arginine or dibutyryl cGMP were added with the NMA. In contrast, microbial killing of S aureus was unaffected by NMA. These observations support the hypothesis that the L-arginine metabolism to NO and its effect on the cGMP level may be important for the dynamic changes required for neutrophil chemotaxis.

The effect of recombinant human granulocyte macrophage colony-stimulating factor on neutrophil activation in patients with refractory carcinoma.

Patients with refractory carcinoma were treated with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) by intravenous (IV) infusion. During the period of treatment, studies of polymorphonuclear leukocyte superoxide (O2-) release in response to formylmethionylleucylphenylalanine (fMLP) and phorbol myristate acetate (PMA) and studies of chemotaxis in response to fMLP and C5a were performed. We observed that patients receiving rhGM-CSF in vivo exhibited primed O2- release after stimulation both with fMLP and PMA. Chemotaxis, however, was not enhanced by the treatment. These data suggest that host defenses may be enhanced by this treatment and that rhGM-CSF may be a useful therapeutic adjunct in compromised patients.

ATP induces transient elevations of [Ca2+]i in human neutrophils and primes these cells for enhanced O2- generation.

ATP, when added to human polymorphonuclear neutrophils (PMNs) at concentrations similar to those attained extracellularly at sites of platelet thrombus formation (0.1 to 20 microM), causes an enhancement of N-formyl(methionyl)leucylphenylalanine (FMLP)-stimulated superoxide anion (O2-) generation. However, ATP by itself is an ineffective agonist for O2- generation by PMNs. The ATP-induced enhancement of O2- generation is associated with a shortened lag time in the response of PMNs to FMLP without a change in the median effective dose for FMLP, suggesting that signal transduction, rather than altered receptor affinity, is responsible for the enhanced oxidative response. Maximum enhancement of O2- generation is detected as early as 15 seconds and is maintained for at least 10 minutes. Of various nucleotides and nonhydrolyzable-ATP analogs test d, only ATP, UTP, and ITP were found to cause enhanced O2- generation by PMNs. Addition of ATP to quin2-loaded PMNs, in the absence of other stimuli, elicits a dramatic rise in [Ca2+]i which reaches a maximum of 500 to 800 nM at 30 seconds and slowly returns to baseline over 5 minutes. This ATP-induced rise in intracellular free Ca2+ concentration is correlated with the enhancement of FMLP-stimulated O2- generation both with respect to dose and nucleotide specificity. Stimulated Ca2+ uptake, rather than mobilization of intracellular Ca2+ stores, appears to be primarily responsible for the rise in intracellular free Ca2+ concentration. These studies indicate that an ATP-induced rise in intracellular free Ca2+ concentration, although insufficient by itself to elicit O2- generation by PMNs, is associated with a priming of PMNs for enhanced O2- generation when stimulated by other agonists.

Inhibition of cell movement and associated changes by hexachlorocyclohexanes due to unregulated intracellular calcium increases.

Hexachlorocyclohexanes (HCCHs) are potent stimulators of polymorphonuclear leukocyte (PMN) oxidative metabolism and of mobilization of calcium from intracellular stores. It was of interest, therefore, to evaluate the effect of HCCHs on PMN orientation and chemotaxis and to determine their effectiveness as chemotaxins. Chemotaxis was evaluated using micro-Boyden chambers, f-actin was quantitated by nitrobenzoxadiazole (NBD)-phallacidin fluorescence, and microtubules were quantitated by observing the concanavalin A (Con A) capping phenomenon. We also evaluated changes in intracellular calcium [Ca2+]i using quin 2 fluorescence. We found that the HCCH isomers were not chemotaxins and that the HCCH isomers that stimulated O2- formation (delta and gamma HCCH) inhibited chemotaxis. This effect was associated with inhibition of orientation. In addition, we found extensive inhibition of both f-actin and Con A cap formation. These effects of HCCH on cell function were associated with marked increases of [Ca2+]i. This work suggests that non-receptor-mediated increases of [Ca2+]i associated with HCCH have divergent effects on cell function and suggests that physiologic responses of PMNs requiring cytoskeletal alterations, such as chemotaxis, depend on the controlled responses of receptor-mediated stimulation.

The critical relationship between free radicals and degrees of ischemia: evidence for tissue intolerance of marginal perfusion.

UNLABELLED: Skin-flap ischemia has been associated with the presence of free radicals. In this study, two enzyme systems involved in free-radical metabolism were used to compare a distal skin flap to a skin graft. Forty-two rats were divided into several test groups. A 10 X 3 cm dorsal rat flap was used, and tissue biopsies for xanthine oxidase and malonyldialdehyde (MDA) were obtained 2.5, 5.5, and 8.5 cm from the base of the flap at the hours given. In group I (control), the flap was outlined but not elevated, and biopsies were obtained. In group II, the flap was elevated, and biopsies were obtained at 6 hours. In group III, the flap was elevated, the distal 4 X 3 cm was amputated and replaced as a full-thickness skin graft, and biopsies were obtained at 6 hours. In group IV, the flap was elevated, and biopsies were obtained at 12 hours. In group V, the flap was treated as in group III, and biopsies were obtained at 12 hours. In group VI, the flap was elevated, and biopsies were obtained at 24 hours. In group VII, the flap was treated as in group III, and biopsies were obtained at 24 hours. RESULTS: Xanthine oxidase was significantly higher in all distal biopsies compared to proximal biopsies. Xanthine oxidase also increased with time. Malonyldialdehyde increased over time as well as with distance from the flap base. Distal flap biopsies at 24 hours had greatly increased levels of malonyldialdehyde compared to skin grafts (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Transient chemotactic defect in a child with elevated IgE.

Leukocyte function was studied in a child with elevated IgE and many infections. At age 7 months, chemotaxis was decreased, but improved first toward formylated peptides and finally toward complement. Polarization and nitroblue tetrazolium reduction also were transiently impaired. Functional and clinical improvements were concomitant although IgE remained elevated.

Augmentation of skin flap survival with allopurinol.

Although allopurinol is primarily known as an effective medication for gout, it has been shown to enhance tissue survival in a wide range of ischemic conditions. The study reported here investigated the effects of allopurinol on flap survival in a dorsal rat model. In a preliminary study, animals were given varying doses of allopurinol (0 to 400 mg per kilogram). A clinically efficacious dose was established upon conclusion of the test period by laboratory determinations and necropsy data. Other animals were divided into 3 groups: 1 (saline control), N = 11; 2 (50 mg per kilogram of allopurinol daily), N = 10; 3 (100 mg per kilogram of allopurinol qd), N = 11. Flaps were raised and necrosis assessed at 8 days. Flaps treated with allopurinol 100 mg per kilogram had significantly better survival than the controls (p less than 0.001) and 50 mg per kilogram (p less than 0.01). Allopurinol 50 mg per kilogram had no effect on flap survival.

Simultaneous evaluation of terminal deoxynucleotidyl transferase and myeloperoxidase in acute leukemias using an immunocytochemical method.

The classification of acute leukemia is important for the selection of optimal therapy. Classification often rests on morphologic, cytochemical, and immunologic criteria, and the marker enzyme terminal deoxynucleotidyl transferase (TdT) has been considered to be a reliable indicator of lymphoblastic leukemias. Because TdT-positive cells sometimes are seen in leukemias otherwise identified as myeloblastic, the authors evaluated blasts identified as myeloid by the presence of myeloperoxidase (MPO) for the simultaneous expression of TdT. The blasts in the bone marrow aspirate or peripheral blood of unselected patients with hematologic malignancies were evaluated and 60 cases are shown. The French-American-British system and, in some patients, cytochemical and immunologic studies were used to classify the leukemias. The authors demonstrated that blasts simultaneously contained MPO and TdT in 29% of patients with acute myeloblastic leukemia and 3% of patients with acute lymphocytic leukemia (ALL). This finding supports the hypothesis that TdT is an expression of cell primitivity rather than a marker for lymphoblastic cells.

Free radicals: basic concepts concerning their chemistry, pathophysiology, and relevance to plastic surgery.

This paper is an attempt to present important concerns in the rapidly expanding field of free radicals to a plastic surgical audience. Mechanisms and a systematic approach to free-radical pathology are presented, with several illustrative areas discussed more deeply.