Thrombin-induced adherence of neutrophils to cultured endothelial monolayers: increased endothelial adhesiveness

We examined the effects of alpha-thrombin on the adherence of neutrophils to endothelial cell monolayers. Endothelial cells derived from the ovine pulmonary artery and ovine neutrophils were used. Thrombin (10(-8) M) resulted in a time-dependent increase in neutrophil adherence to the endothelium. The response was concentration-dependent with a maximal response at 10(-8) M. Thrombin did not induce neutrophil adherence either to plastic or to endothelial cell-derived matrix. The adherence response was inhibited in the presence of alpha-thrombin that had been inactivated with anti-thrombin III (1U:1U) or with hirudin (1 U/ml). However, the addition of either anti-thrombin III or hirudin simultaneously with alpha-thrombin to the cultured endothelial monolayers did not prevent neutrophil adherence. The monoclonal antibody MoAb 60.3, which precipitates a complex of four neutrophil surface glycoproteins (CDw18) was used to further characterize the reaction. MoAb 60.3 decreased the thrombin-induced adherence of neutrophils to the endothelial monolayer. Addition of 10(-8) M thrombin to the endothelial monolayer for 60 min, followed by washing the endothelium with fresh medium, caused resting neutrophils to adhere to the endothelial monolayers. MoAb 60.3 decreased neutrophil adherence to the washed endothelium. The factor(s) responsible for adherence was partially transferable. Medium obtained from incubating endothelial monolayers with thrombin (10(-8) M) for 60 min, adding hirudin to the medium to inactivate thrombin, and transferring it to untreated endothelial monolayers, elicited neutrophil adherence. The response was less than that obtained with thrombin alone (22.9 +/- 2.3% vs. 12.9 +/- 3.3%). The results indicate that the catalytic site of the thrombin molecule is responsible for the adherent activity. Thrombin elicits a rapid activation of endothelial cells with a response that involves the expression of endothelial adhesion sites and sites that interact with the neutrophil CDw18 adhesive glycoprotein complex. In addition, soluble transferable factor(s) which are generated by the endothelium also contribute to thrombin-induced neutrophil adherence.     

Thrombin-induced chemotaxis and aggregation of neutrophils

Thrombin-induced neutrophil chemotaxis and aggregation were studied using cells isolated from either human or sheep blood. Sheep neutrophils (10(8) cells/ml) exhibited maximum chemotactic migration towards 10(-8)M human alpha-thrombin, 10(-8)M gamma-thrombin (which lacks the fibrinogen site), and 10(-12)MD-Phe-Pro-Arg-CH2-alpha-thrombin (catalytically inactive thrombin). Chemotactic responses of the same magnitude were obtained with human neutrophils (10(8) cells/ml). The chemotactic responses to thrombin were comparable to those obtained with diluted (1:200 v/v) zymosan activated serum (ZAS) and 10(-11)M FMLP. Premixing of the thrombin forms with hirudin in 1:1 stoichiometric amounts abolished the chemotaxis but not chemokinesis Aggregatory responses of human and sheep neutrophils were comparable for ZAS, alpha-thrombin, and gamma-thrombin. The responses of both human and sheep neutrophils to D-Phe-Pro-Arg-CH2-alpha-thrombin were attenuated, indicating that the proteolytic site may be involved in the aggregatory response. The results suggest that thrombin-induced neutrophil chemotaxis and aggregation are mediated by different mechanisms, since chemotaxis is a catalytically independent response whereas aggregation is an active site independent response.     

Luminol dependent chemiluminescence of quartz and zymosan stimulated neutrophils

The effect of the opsonization by zymosan and quartz particles on the chemiluminescence was investigated on human neutrophil granulocytes. Opsonization of zymosan enhanced the chemiluminescence response, while opsonized quartz inhibited the chemiluminescence reaction. Calcium ionophore A 23187 treatment did not influence the chemiluminescence of quartz but the light signal in the presence of quartz decreased rapidly. In parallel experiments the protein pattern of zymosan treated neutrophils was investigated by high resolution two-dimensional polyacrylamide gel electrophoresis.     

Luminol-enhanced chemiluminescence after reaction of hydroperoxides with opsonized zymosan

Luminol-enhanced chemiluminescence (LEC) is very sensitive in detecting free radicals but relatively insensitive for hydroperoxides (hydrogen peroxide, tert-butyl hydroperoxide). However, in the presence of opsonized zymosan (often used for stimulation of phagocytic cells) hydroperoxides also induce LEC, suggesting that free radicals are produced under these conditions. Therefore careful interpretation with respect to the nature of the reactive species is necessary when LEC is used for characterization of zymosan-induced phagocytosis. We studied the properties of zymosan-induced LEC under different test conditions and with various inhibitors. Typical radical scavengers, e.g. nordihydroguaiaretic acid and superoxide dismutase, are strong inhibitors, indicating the importance of the superoxide anion. This system is useful for drug testing with respect to antioxidative or radical scavenging activity.     

Thrombin and histamine rapidly stimulate the phosphorylation of the myristoylated alanine-rich C-kinase substrate in human umbilical vein endothelial cells: evidence for distinct patterns of protein kinase activation.

Human alpha-thrombin and histamine each stimulates protein phosphorylation in human umbilical vein endothelial cells (HUVEC). We have identified the most prominent of these phosphoproteins by immunoprecipitation as the human homolog of the widely distributed myristoylated alanine-rich C-kinase substrate (MARCKS). Stimulation by 0.1-10 U/ml of alpha-thrombin produces a time-dependent, sustained (plateau 3-5 min) level of MARCKS phosphorylation. MARCKS phosphorylation requires thrombin catalytic activity but not receptor binding and is also seen in response to stimulation by a peptide, TR (42-55), that duplicates a portion of the thrombin receptor tethered ligand created by thrombin proteolytic activity. One micromolar histamine, like alpha-thrombin, produces sustained phosphorylation of MARCKS (plateau 3-5 min). In contrast, 100 microM histamine results in rapid but transient MARCKS phosphorylation (peak 1-3 min). HUVEC treated with 100 microM histamine for 5 min can be restimulated by alpha-thrombin but not fresh histamine, suggesting that the histamine receptor was desensitized. MARCKS phosphorylation can also be induced by several exogenous protein kinase C (PKC) activators and both alpha-thrombin- and histamine-induced MARCKS phosphorylation are inhibited by the PKC antagonist staurosporine. However, while prolonged PMA pretreatment ablates histamine-induced MARCKS phosphorylation, the ability of thrombin to induce MARCKS phosphorylation is retained. These findings provide evidence for agonist-specific pathways of protein kinase activation in response to thrombin and histamine in HUVEC.     

Effect of the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) on human neutrophil function: novel inhibition of neutrophil stimulus-response coupling?

Previously we had utilized the spin trap 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) to detect superoxide (.O2-) formation by human neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan. When N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A were substituted as the neutrophil stimulus spin trap evidence of neutrophil free radical production was not detected. Consequently, the hypothesis that DMPO interfered with neutrophil stimulus response coupling was examined. DMPO exhibited a concentration-related inhibition of neutrophil .O2- secretion (ferricytochrome C reduction) following exposure to six different stimuli. The extent of inhibition was stimulus dependent--large (FMLP, concanavalin A), moderate (PMA, opsonized zymosan, A23187), and mild (arachidonic acid). Inhibition was reversible. Onset was nearly instantaneous and was observed even if DMPO was added after stimulus-induced .O2- formation was ongoing. DMPO had only minimal effect on .O2- production by a cell-free NADPH-oxidase membrane preparation. DMPO also inhibited the neutrophil degranulation response for elastase and lactoferrin but not vitamin B12 binding protein. DMPO-mediated inhibition of neutrophil function was not related to alteration in stimulus binding (FMLP or concanavalin A). DMPO had minimal impact on the stimulus-induced rise in intracellular calcium. However, the presence of DMPO resulted in a concentration-dependent depolarization of the resting neutrophil membrane and blunting of the depolarization response to each stimulus examined. These data are of importance to investigators applying spin-trapping techniques to phagocytic cells and suggest DMPO could be used as a tool for investigating neutrophil stimulus-response mechanisms.     

Ethanol inhibition of the chemiluminescent response of stimulated macrophages.

The effect of ethanol on the luminol-dependent chemiluminescence and cAMP level in mice peritoneal macrophages was investigated. Ethanol was shown to inhibit the chemiluminescence of macrophages by acting both as a "trap" for active radicals and as a suppressor of the cellular functional activity. A short preincubation of macrophages with ethanol results in a dose-dependent decrease of the chemiluminescent response to the stimulatory agent (opsonized zymosan). Ethanol was also shown to induce a peakwise rise of the intracellular cAMP after a 2-min incubation. The observed effects are correlated both in time and concentration, which allows the presumption that inhibition of the functional activity of macrophages is mediated by the increase of the intracellular cAMP level.     

Inhibition of complement-mediated functions of human neutrophils by impermeant stilbene disulfonic acids

The impermeant labeling reagents 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic acid (SITS) inhibited in a concentration-related manner the enhanced generation of superoxide radicals (O2) by human neutrophils engaged in the phagocytosis of zymosan that had been opsonized in fresh serum, without altering the O2 generation by neutrophils exposed to zymosan opsonized in heat-decomplemented serum or to phorbol myristate acetate (PMA). That the stimulus specificity of the suppression of O2 generation by SITS and DIDS is predominantly attributable to an action on neutrophil plasma membrane receptors for complement was suggested by the similarity of the concentration dependence of the inhibition of the expression of neutrophil C3b receptors, as assessed by a rosetting assay. Washing neutrophils that had been pretreated with the covalent label DIDS failed to reverse either the suppression of C3b-dependent rosetting or the inhibition of O2 generation stimulated by opsonized zymosan. In contrast, pretreatment with DIDS and washing or erythrocytes bearing C3b and of opsonized zymosan did not inhibit their capacity to form rosettes and to stimulate O2 generation by neutrophils, respectively. In the same rosetting assay, the expression of IgG-Fc receptors was unaffected by SITS and DIDS. The rapid and apparently selective inhibition of the expression of neutrophil C3b receptors by noncytotoxic concentrations of the impermeant stilbene disulfonic acids may provide a means to analyze the complement dependence of other neutrophil effector functions.     

Whole blood luminol-enhanced chemiluminescence: Statistical analysis of the responses of different subjects

Whole blood luminol-enhanced chemiluminescence (CL) was studied using N-formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan particles as stimuli. The peak and the integral responses of CL were recorded. In the FMLP-induced CL the initial activation (1-minute values) was also studied, because it coincides with the extracellular production of oxy radicals. Correction factors based on neutrophil count and on haemoglobin concentration were found to decrease dispersion and shape the distributions of the CL responses close to normal in a study of 50 healthy adults. One-minute values were significantly lower in women than in men but there were no significant differences for peak or integral values between sexes. Depressed reaction is in accordance with the previous findings that phagocytic oxy radical production is depressed in female plasma. Thus, our results suggest that 1-minute value is a variable more sensitive than peak or integral value of the CL response.     

Influence of bioactive glasses on the respiratory burst metabolism of polymorphonuclear neutrophils]

BACKGROUND: Bio-glasses are bioactive materials used to coat implants. The immunological reaction to wear particles from such coatings has hardly been investigated. The generation of reactive oxygen species by polymorphonuclear neutrophils (PMN) on phagocytosis of particles might elicit further immune reactions. METHODS: The production of reactive oxygen species was investigated in whole blood in the form of chemiluminescence using the probes luminol and lucigenin. RESULTS: Bioglass particles stimulated PMN to generate free radicals as a function of the bioactivity of the composition. This activation was significantly reduced by prior soaking of the particles. Extracts of the bioactive glasses also inhibited the liberation of free radicals upon the stimulus opsonized zymosan. CONCLUSION: Our results show that despite the proven good biocompatibility of bioactive glasses, further in vivo checks in the early stages of the reaction are needed.     

Consumption of complement and activation of human neutrophils by an artificial immune complex: polyacrylic acid-IgG-polymer

An artificial immune complex consisting of IgG covalently bound to polyacrylic acid (PAIGP) was prepared and investigated for its influence on a number of immunological reactions attributed to natural immune complexes. PAIGP consumed complement in a fast reaction. Complement consumption was complete after 10 min of incubation of guinea-pig serum with PAIGP. The concentration of PAIGP for 50% consumption was 2.3 micrograms/ml. PAIGP induced a chemiluminescence response in human peripheral polymorphonuclear leukocytes. This response was elicited in the absence and presence of serum and in whole blood. The response was maximal for leukocytes in the absence of serum and rather low in whole blood. The induction of chemiluminescence by PAIGP was inhibited by monoclonal antibodies to one of the Fc receptors of leukocytes (anti-Leu 11B), while unrelated antibodies had no influence on the chemiluminescence induced by PAIGP. PAIGP also stimulated the production of superoxide anion by polymorphonuclear leukocytes. The efficacy of PAIGP in stimulation of superoxide production was comparable to phorbol myristate acetate (PMA) and opsonized zymosan. PAIGP induced the discharge of elastase, a constituent of the azurophile granules of PMN leukocytes. Here, PAIGP was a rather weak stimulus compared to opsonized zymosan. PMA proved unable to induce elastase release. Thus, PAIGP induced a number of biological reactions usually brought about by naturally occurring antigen antibody complexes.     

Extracellular acidification induces human neutrophil activation

In the current work, we evaluated the effect of extracellular acidification on neutrophil physiology. Neutrophils suspended in bicarbonate-buffered RPMI 1640 medium adjusted to acidic pH values (pH 6.5-7.0) underwent: 1) a rapid transient increase in intracellular free calcium concentration levels; 2) an increase in the forward light scattering properties; and 3) the up-regulation of surface expression of CD18. By contrast, extracellular acidosis was unable to induce neither the production of H2O2 nor the release of myeloperoxidase. Acidic extracellular pH also modulated the functional profile of neutrophils in response to conventional agonists such as FMLP, precipiting immune complexes, and opsonized zymosan. It was found that not only calcium mobilization, shape change response, and up-regulation of CD18 expression but also production of H2O2 and release of myeloperoxidase were markedly enhanced in neutrophils stimulated in acidic pH medium. Moreover, extracellular acidosis significantly delayed neutrophil apoptosis and concomitantly extended neutrophil functional lifespan. Extracellular acidification induced an immediate and abrupt fall in the intracellular pH, which persisted over the 240-s analyzed. A similar abrupt drop in the intracellular pH was detected in cells suspended in bicarbonate-supplemented PBS but not in those suspended in bicarbonate-free PBS. A role for intracellular acidification in neutrophil activation is suggested by the fact that only neutrophils suspended in bicarbonate-buffered media (i.e., RPMI 1640 and bicarbonate-supplemented PBS) underwent significant shape changes in response to extracellular acidification. Together, our results support the notion that extracellular acidosis may intensify acute inflammatory responses by inducing neutrophil activation as well as by delaying spontaneous apoptosis and extending neutrophil functional lifespan.     

Luminol‐dependent chemiluminescence of human phagocyte cell lines: comparison between DMSO differentiated PLB 985 and HL 60 cells

The human promyelocytic leukemia HL 60 and PLB 985 cell lines can differentiate into terminally mature neutrophil‐like cells via dimethyl sulfoxide (DMSO) induction. In this study the luminol‐dependent chemiluminescence (LCL) of both neutrophil‐like cells was analayzed and compared in response to phorbol myristate acetate (PMA) and opsonized zymosan (OZ) stimulants. It was shown that, like human blood neutrophils, both neutrophil‐like cells expressed high levels of CD11b, but unlike human blood neutrophils these cells almost lack LCL‐detectable intracellular oxidase activity. By studying the pattern of activation to OZ and PMA and priming with GM‐CSF, we concluded that there is no difference between the percentage of differentiation and function of DMSO‐induced HL 60 and PLB 985. However, the LCL capacity (area under the curve) of DMSO induced PLB 985 cells was higher than that of HL 60 cells in response to both PMA and OZ, which implies a higher capacity to generate reactive oxygen species in PLB 985 cells. Copyright © 2009 John Wiley & Sons, Ltd.     

Phosphorylation of p22phox is mediated by phospholipase D-dependent and -independent mechanisms. Correlation of NADPH oxidase activity and p22phox phosphorylation

Human neutrophils participate in the host innate immune response, partly mediated by the multicomponent superoxide-generating enzyme NADPH oxidase. A correlation between phosphorylation of cytosolic NADPH oxidase components and enzyme activation has been identified but is not well understood. We previously showed that p22(phox), the small subunit of the membrane-bound oxidase component flavocytochrome b(558), is an in vitro substrate for both a phosphatidic acid-activated kinase and conventional protein kinase C isoforms (Regier, D. S., Waite, K. A., Wallin, R., and McPhail, L. C. (1999) J. Biol. Chem. 274, 36601-36608). Here we show that several neutrophil agonists (phorbol myristate acetate, opsonized zymosan, and N-formyl-methionyl-leucyl-phenylalanine) induce p22(phox) phosphorylation in intact neutrophils. To determine if phospholipase D (PLD) is needed for p22(phox) phosphorylation, cells were pretreated with ethanol, which reduces phosphatidic acid production by PLD in stimulated cells. Phorbol myristate acetate-induced phosphorylation of p22(phox) and NADPH oxidase activity were not reduced by ethanol. In contrast, ethanol reduced both activities when cells were stimulated by N-formyl-methionyl-leucyl-phenylalanine or opsonized zymosan. Varying the time of stimulation with opsonized zymosan showed that the phosphorylation of p22(phox) coincides with NADPH oxidase activation. GF109203X, an inhibitor of protein kinase C and the phosphatidic acid-activated protein kinase, decreased both p22(phox) phosphorylation and NADPH oxidase activity in parallel in opsonized zymosan-stimulated cells. Stimulus-induced phosphorylation of p22(phox) was on Thr residue(s), in agreement with in vitro results. Overall, these data show that NADPH oxidase activity and p22(phox) phosphorylation are correlated and suggest two mechanisms (PLD-dependent and -independent) by which p22(phox) phosphorylation occurs.     

Role of the epithelial layer in the generation of superoxide anion by the guinea-pig isolated trachea.

The lucigenin-dependent chemiluminescence generation by guinea-pig isolated tracheal two rings preparations was studied. Tracheal preparations stimulated with phorbol myristate acetate (PMA) or opsonized zymosan generated chemiluminescence. The total amount of chemiluminescence generated in 120 min was 754+/-63 mV x min for PMA and 4832+/-396 mV x min for zymosan. Generation of chemiluminescence was decreased by more than 50% when the tissues were co-incubated with superoxide dismutase (100 U/ml). Also, addition of direct donors of nitric oxide diminished chemiluminescence generation by zymosan-activated tracheal rings significantly by about 50%. However, the presence of the precursor or of inhibitors of nitric oxide synthase did not influence zymosan-induced chemiluminescence. Removal of the epithelial layer from tracheal rings caused an approximately 90% decrease in chemiluminescence response. However, isolated epithelial cell suspensions did not generate chemiluminescence. Histologic examination showed that the number of eosinophils in the tracheal tissue was reduced from 56+/-7 to 18+/-8 per mm basal membrane when the epithelial layer was removed. These results indicated that (1) superoxide anion formation can take place in the guinea-pig trachea, (2) eosinophils in the epithelial and submucosal layers of guinea-pig trachea are likely candidates for superoxide generation although other cell types can also be involved, and (3) besides relaxing airway smooth muscle, nitric oxide donors may also affect superoxide in the airways.     

Regulation of the growth and differentiation of a human monocytic cell line by lymphokines. I. Induction of superoxide anion production and chemiluminescence

The U937 human monocytic cell line was studied to determine its ability to generate a respiratory burst after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. U937 cells cultured in normal medium produced virtually no superoxide anion or chemiluminescence in response to either stimulus. In contrast, U937 cells cultured in medium containing soluble factors from activated lymphocytes produced significant O2- and chemiluminescence when stimulated with PMA or opsonized zymosan. The chemiluminescence in response to PMA was maximal in U937 cells precultured with these soluble factors for 3 days, whereas maximal responsiveness to opsonized zymosan was not observed until 5 to 6 days of lymphokine exposure. Although this ability to generate a respiratory burst persisted for a number of days in U937 cells that were subsequently recultured in normal medium, this responsiveness was gradually lost in the continued absence of these factors. The data indicate that the U937 monocytic cell line can be activated or induced to differentiate by soluble factors released by activated lymphocytes. In the process, these cells acquire the ability to generate a respiratory burst. The U937 cell line may serve as a useful model for the study of the ontogeny and regulation of the respiratory burst during human monocytic differentiation.     

Evaluation of neutrophil activity and circulating immune complexes levels in diabetic patients

We have studied chemiluminescence produced by neutrophils stimulated by opsonized zymosan in insulin dependent (IDD) and non insulin dependent (NIDD) diabetic patients. Chemiluminescence was evaluated as the integral and maximum peak, total time and time to maximum peak of the response curve to opsonized zymosan. These values were then compared with circulating immune complexes (CIC) and glucose levels. Both IDD and NIDD patients had significantly higher values of chemiluminescence and CIC than normal controls. We also observed that patients who had the highest values of CIC and chemiluminescence levels were the ones with clinical microvascular complications.     

Effect of prostacyclin on pulmonary vascular response to thrombin in awake sheep

We determined the effects of infusion of prostacyclin (PGI2) and 6-alpha-carba-PGI2 (6-cPGI2), a stable PGI2 analogue, on pulmonary transvascular fluid and protein fluxes after intravascular coagulation induced by thrombin. Studies were made in control awake sheep prepared with lung lymph fistulas (n = 6) and in similarly prepared awake sheep pretreated with either 6-cPGI2 (n = 5) or PGI2 (n = 5). Both prostacyclin compounds (500 ng X kg-1 X min-1) were infused intravenously. All groups were challenged with 80 U/kg thrombin. Pulmonary arterial pressure (Ppa), pulmonary vascular resistance (PVR), pulmonary lymph flow (Qlym), lymph protein clearance (Qlym X lymph/plasma protein concentration ratio), and neutrophil and platelet counts were determined. In vitro tests assessed sheep neutrophil chemotaxis and chemiluminescence and platelet aggregation. In both 6-cPGI2 and PGI2 groups, the increases in Qlym after thrombin were less than those in the control group. The increase in lymph protein clearance in the 6-cPGI2 group was the same as that in control, whereas the increase in clearance in the PGI2 group was reduced. PVR and Ppa increased to a greater extent in the 6-cPGI2 group than in the control group, whereas the increases in PVR and Ppa were inhibited in the PGI2 group. Neutrophil and platelet counts decreased after thrombin in PGI2 and 6-cPGI2 groups, as they did in the control group. Neither 6-cPGI2 altered neutrophil chemotaxis induced by thrombin and chemiluminescence induced by opsonized zymosan. Both prostacyclin compounds inhibited platelet aggregation induced by ADP or thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-adrenergic receptor function and oxygen radical production in bovine pulmonary alveolar macrophages

Reactive oxygen species production by bovine pulmonary alveolar macrophages was evaluated by a chemiluminescence assay utilizing luminol and opsonized zymosan. Incubation with dobutamine (5 x 10(-8) and 5 x 10(-7) M) or isoproterenol (5 x 10(-8) and 5 x 10(-7) M) prior to zymosan challenge significantly (p less than 0.05) increased the time for chemiluminescence to begin, and significantly decreased the level of maximum chemiluminescence. The agonists' inhibitory effects on maximum chemiluminescence were significantly reduced by pre-incubation with the appropriate antagonist (atenolol at 1 x 10(-6) M for dobutamine; and propranolol at 1 x 10(-6) M for isoproterenol). Salbutamol at 1 x 10(-6) M significantly reduced the level of maximum chemiluminescence only, but did not increase the time for chemiluminescence to begin. This effect was significantly reduced by the presence of the beta 2-antagonist ICI 118,551 at 1 x 10(-6) M. The results reveal the presence of beta 1- and beta 2-adrenoceptors on bovine pulmonary alveolar macrophages, and suggest that these receptors are important in the regulation of reactive oxygen species production by these cells.     

The acute phase protein serum amyloid A primes neutrophils

We studied here the effect of the acute phase protein serum amyloid A (SAA) on the oxidative burst of neutrophils. Incubation of neutrophils with SAA increased the rate of oxygen uptake and the production of reactive oxygen species of neutrophils activated with opsonized zymosan (OZ). The increment in the neutrophil oxidative burst was dependent on SAA concentration in the range of 3-33 microg protein ml(-1) and was observed only in the presence of a relatively low amount of OZ (1 x 10(6) particles ml(-1)). SAA did not affect oxygen consumption and reactive oxygen production triggered by other stimuli, such as f-Met-Leu-Phe, phorbol myristate acetate or non-opsonized zymosan. Our finding points to a priming effect of SAA probably associated with mobilization of receptors for opsonized particles and strengthens the role of SAA as an effector of neutrophil functions in inflammation.