Granulocyte Suspensions Research Articles

Natural fluorescence of white blood cells: spectroscopic and imaging study

Autofluorescence has been proved to be an intrinsic parameter of biological substrates that may aid in both the characterization of the physiological state and the discrimination of pathological from normal conditions of cells, tissues and organs. In this work, the fluorescence properties of human white blood cells have been studied in suspension and on single cells at microscopy. The results indicate that suspensions of agranulocytes and granulocytes differ in the amplitude of the fluorescence signal on excitation at wavelengths in the range 250–370 nm. The differences are particularly enhanced when excitation is performed in the 250–265 nm range. Microspectrofluorometric analysis, performed on single cells, allows several leukocyte families to be characterized. Lymphocytes, monocytes, neutrophils and eosinophils can be distinguished according to the intensity and spectral shape of the autofluorescence emission in the visible range from 440 to 580 nm. Both the nature and extent of the differences change when the excitation wavelength is moved from 366 to 436 nm. Differences in the intrinsic metabolic engagement, rather than in the cell dimensions, seem to be responsible for the difference observed between the leukocyte populations. The results identify interesting perspectives for autofluorescence as a discriminating parameter in the differential counting of human white blood cells.

Metabolic response of granulocytes and platelets to synthetic vascular grafts: Preliminary results with an in vitro technique

The metabolism of granulocytes as well as platelets evoked by incubation with different synthetic vascular grafts was monitored during 6-h batch experiments using microcalorimetry. Standard knitted Dacron grafts, ePTFE-grafts, knitted Dacron grafts with collagen impregnation, and knitted Dacron grafts with external collagen-coating were used. The heat production per cell was calculated. A rapid increase of metabolic activity followed by a gradual decrease was demonstrated with both granulocyte suspension and platelet concentrate. Significant differences were obtained between the materials with a maximum response of Dacron grafts with collagen impregnation for both granulocyte and platelet response. The materials had different surface morphologies regarding cell adhesion after incubation as demonstrated with scanning electron microscopy with more pronounced adhesion on the collagen-impregnated grafts. The results suggest that microcalorimetry may be useful for the evaluation of cellular reactions on different biomaterials. However, further studies have to reveal the specificity of the reactions.

Eosinophils isolated with two different methods show different characteristics of activation

Eosinophils can be isolated from a mixed suspension of granulocytes by different procedures. We compared functional responses of human eosinophils purified according to two different principles: (1) an fMLP-induced difference in specific gravity between eosinophils and neutrophils and (2) selective removal of neutrophils by means of immunomagnetic beads coated with CD16 mAb. The results showed that eosinophils isolated with the CD16 beads method have a higher capacity to synthesize platelet activating factor (PAF) after stimulation with serumtreated zymosan (STZ) than eosinophils purified with the fMLP method. Binding of STZ and subsequent activation of the respiratory burst were also increased in CD16-isolated eosinophils. Furthermore, eosinophils isolated with the CD16 beads showed stronger chemotactic responses towards C5a and PAF. The difference in activity of these eosinophil preparations might be explained by a loss of the more active cells during the isolation with the fMLP method: only 30–60% of the eosinophils were recovered with this method, in contrast to a recovery of more than 95% with the CD16 beads method. Indeed, this ‘lost’ population of eosinophils, subsequently purified with CD16-coated beads, had a higher respiratory burst activity. The alternative explanation, i.e., an enhancement of eosinophil function by the beads method, appeared not to be valid, because repurification of fMLP-isolated eosinophils in the presence of fresh neutrophils and CD16-coated beads did not change the reactivity of the eosinophils. We conclude that the fMLP method leads to selective purification of eosinophils with a resting (or ‘unprimed’) phenotype.

In vitro uptake of polystyrene latex particles and parenteral fat emulsions by human granulocytes

Suspensions of human granulocytes were used to study the in vitro phagocytosis of polystyrene latex particles (1030 nm) as i.v. model drug carriers and of commercial fat emulsions for parenteral nutrition (Lipofundin, Intralipid). Chemiluminescence (CL) was employed to follow the time-dependent uptake of the particle. The total uptake was quantified by the area under the curve of the CL intensity/time profiles and comparitively by a modified fluorimetric assay. The data proved that a linear relationship existed between the AUC of the CL assay and the total mass of internalized polymer. The uptake of fat emulsions could not be followed directly by CL because the CL intensity signal was too weak. Their uptake could be quantified via the impairment of the phagocytic function of granulocytes after pre-incubation with emulsion. The polystyrene particles were used as test colloid. The phagocytic function was reduced up to 50% by 10% fat emulsions.

Alterations in Viscosity and Filterability of Whole Blood and Blood Cell Subpopulations in Diabetic Cats

Capillary closure and venous dilatation occur early in diabetic retinopathy, and altered blood rheology may play a role. For example previous studies have shown leukocytes are less deformable, are more activated, and occlude retinal capillaries more often in diabetic subjects. The purpose of this study was to determine if rheologic changes developed in diabetic eats, a model in which we have documented retinal capillary basement membrane thickening, microaneurysms, and intraretinal hemorrhage characteristic of early diabetic retinopathy. Viscosity of blood, plasma and purified erythrocyte suspensions was measured by rotational viscometry and plasma fibrinogen content was deterarmed by heat precipitation. Filterability of blood and purified erythrocyte, mononuclear leukocyte, and granulocyte suspensions was determined at constant flow, measuring the increase in pressure over 2 min relative to the pressure generated by buffer alone. Viscosity of whole blood and plasma, but not erythrocytes, was significantly elevated ( P < 0·005) in the diabetic cats over normals at all shear rates studied (450, 225 and 90 sec -1). Furthermore, fibrinogen levels were significantly elevated in diabetic cats compared to normals ( P < 0·004), and were positively correlated with the viscosity of whole blood and plasma. The filterability of mononuclear leukocytes and whole blood in diabetic cats was decreased 56% and 74% when compared to normals, P > 0·0006 and P < 0·025, respectively. In contrast, the filterability of granulocytes and erythrocytes was not significantly different between the two groups. These results suggest that the rheologic alterations in diabetes are numerous, and involve both cellular and plasma protein changes. The changes in the fibrinogen levels, plasma and whole blood viscosity, and mononuclear cell filterability may play important roles in the genesis of diabetic microangiopathy.

Electron paramagnetic resonance studies on cytochrome b-558 and peroxidases of pig blood granulocytes

Low-temperature electron paramagnetic resonance (EPR) spectrometry on granulocytes prepared from pig blood was carried out with concentrated cellular and subcellular fractions to characterize EPR signals of cytochrome b-588 (cyt b-558). A thick cell suspension (∼ 2 · 10 9 cells/ml), containing mostly neutrophils, showed typical high-spin EPR signals due to myeloperoxidase (MPO) and a low spin signal at a g value of around 3.2. A similar thick granulocyte suspension containing eosinophils showed not only these signals but also low spin heme signals at g values of 2.86, 2.13, and 1.66, which have been reported to be of cyt b-558 (Ueno et al. 1991, FEBS Lett. 281, 130–132). MPO and eosinophil peroxidase (EPO) were released from the membrane fractions with 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl, and then were highly concentrated, in which no cyt b-558 was detected by absorption spectra. The signal at a g value of 2.86 was found only in the EPO fraction, suggesting that this signal is derived from a low-spin form of an EPO-complex, but neither from MPO nor cyt b-558. The O 2 −-forming NADPH oxidase associated in the membranes was solubilized with heptylthioglucoside at 0°C and concentrated up to 45 μM cyt b-558 with no modification of the heme moiety confirmed by its O 2 −-generating activity and lack of carbon monoxide-binding capacity. Cyt b-558 showed an anisotropic signal at a g value of 3.2 ± 0.05, which was cyanide-insensitive and reducible with reductants. The signal intensity was concentration dependent, suggesting that the g = 3.2 signal is characteristic of the low-spin heme iron in cyt b-558.

Granulocyte Adherence is Inhibited by Radiographic Contrast Media in Vitro

Different amounts of diatrizoate, ioxaglate, iohexol, iodixanol, NaCl 1,000 mOsm/kg, mannitol 1,098 mOsm/kg, and meglumine (meglumine concentrations corresponding to the content in the diatrizoate solutions) were added to either whole blood or a suspension of granulocytes in autologous plasma, and the adherence to nylon fibers was determined. At high concentrations all the investigated contrast media (CM) inhibited granulocyte adherence. The degree of inhibition was significantly greater when the ionic CM diatrizoate and ioxaglate were used, as compared with the nonionic media. Meglumine solutions at high concentrations also inhibited adherence but significantly less than diatrizoate solutions containing the same amount of meglumine. Diatrizoate showed the greatest inhibitory effect on granulocyte adherence, and significant inhibition could be detected even with a 1.25% solution.

A procedure for parallel isolation of white blood cells, granulocyte and purified neutrophil suspensions from the peripheral blood of cattle

A rapid method is described for parallel isolation of white blood cells, granulocytes and purified neutrophils from peripheral blood of normal cattle. The mean recovery (±S.D.) of white blood cells, granulocytes and purified neutrophils isolated from peripheral blood of 13 cows was 66.4±12.6%, 68.7±20.0% and 38.0±20.9%, respectively. The mean purity of the isolated granulocyte and neutrophil suspensions was 94.0±3.8% and 95.0±6.0%, respectively. Viability of isolated cells was more than 97%.

Separation of Leucocytes: Improved Cell Purity by Fine Adjustments of Gradient Medium Density and Osmolality

This paper briefly reviews commonly used procedures for separation of mononuclear cells (MNC) and granulocytes from human blood with X-ray contrast media as gradient material, and also presents new and modified procedures for leucocyte preparation. Standard techniques for human blood do not always yield satisfactory results with blood from other species. In general pure MNC are easily obtained (top fraction), but often the granulocyte fraction has a low purity, due to contamination with MNC that move to the bottom during centrifugation and contaminate the granulocyte suspension. Obviously the density distribution of MNC differs between species. However, the separation can be improved by fine adjustment of gradient medium osmolality. For this purpose we have used Nycodenz, a non-ionic X-ray contrast medium. A favourable property of Nycodenz solutions is that the osmolality and density can easily be varied over a broad range. The cells react promptly to a change of medium osmolality. In hypertonic medium the cells expel water, shrink, their density increases and they sediment faster, in spite of a smaller radius. Further, the cells may pass what was initially a density barrier. A hypotonic environment has the opposite effect. In the present work we were able to show that a slight change of medium osmolality clearly improved different techniques for separation of leucocyte subgroups. For instance, the Isopaque-Ficoll (IF) technique consistently yielded MNC and granulocytes of high purity with human blood. However, with blood from rabbits, rats and mice the granulocyte suspensions were contaminated by 40-60% MNC. By utilizing Nycodenz, and lowering the osmolality by 10-12 per cent (at constant density--1.077 g/ml) we obtained satisfactory separation of MNC as well as granulocytes with blood from these species. A problem in the routine separation of granulocytes (IF) is a high contamination of erythrocytes (2-5 per cell) in the granulocyte suspension. With a two-layer technique with Nycodenz solutions of different densities it was possible to separate granulocytes almost devoid of erythrocytes, after proper adjustment of osmolality. By appropriate combination of density and osmolality, Nycodenz was a suitable gradient material in other separation procedures as well, e.g. the separation of monocytes and mast cells. To facilitate the use of Nycodenz as a versatile gradient material, a computer program providing recipes for various Nycodenz solutions is included as an appendix.

Effects of Zymosan-activated Human Granulocytes on Isolated Human Airways

In asthma a temporal association exists between the late allergic reaction (LAR), the influx of granulocytes into the airway wall, and an increase in bronchial responsiveness. We therefore tested the hypothesis that activated human granulocytes constrict isolated human airways and increase their sensitivity to cholinergic stimuli. Bronchial rings were dissected from 23 lung tissue specimens collected at thoracotomy and studied isotonically in organ baths. Airways were incubated with 1, 2, 5, 10, or 20 x 10(6) granulocytes from normal or atopic donors. Activation of the cells with serum-treated zymosan (STZ, 0.2 mg/ml), which itself did not alter baseline airway caliber, resulted in a bronchoconstriction proportional to the number of zymosan-activated granulocytes (ZAG) present (rs = 0.79, p less than 0.001). This contraction was reduced by about 70% with the leukotriene C4/D4 receptor antagonist FPL 55712 (11.5 microM; p less than 0.001) or with the lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM; p less than 0.001). The scavengers of activated oxygen molecules superoxide dismutase (300 U/ml) and bovine catalase (5,000 U/ml), the cyclooxygenase inhibitor indomethacin (10 microM), or the histamine (H1) receptor antagonist mepyramine (2.8 microM) had no effect. Granulocyte suspensions from atopic donors contained more eosinophils (p less than 0.001), and the magnitude of the contraction to 10 x 10(6) ZAG was related to the proportion of eosinophils (rs = 0.66, p less than 0.01). The sensitivity of the airways to methacholine was unchanged in the presence of 1, 2, or 5 x 10(6) ZAG and decreased with 10 or 20 x 10(6) ZAG (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Stable isotope dilution analysis of cystine in granulocyte suspensions as cysteine: A powerful method for the diagnosis, the follow-up, and treatment of patients with cystinosis

A stable isotope dilution method was developed for the determination of cystine in granulocytes. Granulocytes were isolated from blood samples of treated cystinosis patients. Cystine in the granulocyte suspension was decoupled from proteins and converted to cysteine by treatment with a tri-butyl phosphine solution. Tertiary butyldimethyl silyl derivatives were prepared and analyzed by gas chromatography/mass spectrometry. Selective ion monitoring was carried out at m/ z 304.3 (M-159 and m/ z 406.4 (M-57) for the natural, and at m/ z 306.3 and 408.4 for the labelled compound. [3,3,3',3'- 2H]-DL-cystine was used as internal standard for the isotope dilution analysis. Concentrations of cystine in granulocytes could be accurately measured. There was a distinct difference in cystine concentrations in healthy individuals and treated patients.

Two‐layer gradient separation of granulocytes for functional tests

Two gradient separation techniques were compared with the dextran sedimentation method for the separation of granulocytes from blood. Both techniques gave adequate yield and excellent purity, and flow cytometric analysis of surface membrane markers gave no indication of subset selection during the procedure. Cells separated by the three methods behaved comparably in functional tests including random migration, chemotaxis and chemiluminescence. The gradient separation techniques are rapid and efficient methods for the preparation of near 100% pure granulocyte suspensions for functional studies.

The appearance of hypodense eosinophils during interleukin-2 treatment

Based on membrane receptors, metabolic activity, and cell density, human eosinophils (EOSs) are a heterogeneous population of leukocytes. EOS heterogeneity translates into biologic significance, since low density cells can be metabolically more active and thus more capable of causing tissue injury. Efforts to identify mechanisms that lead to the development of hypodense EOSs have found that an in vitro exposure to cytokines reduces cell density and is associated with increased cell activity. Consequently, we evaluated the effect of an in vivo administration of interleukin-2 (IL-2) on the cell counts and density of circulating EOSs in six patients who received IL-2 as cancer biologic-modifier therapy. To determine the pattern of EOS density in relationship to IL-2 treatment, granulocyte suspensions were isolated from peripheral blood and then centrifuged over multiple discontinuous density Percoll gradients. During IL-2 treatment, the percentage of circulating hypodense EOSs increased significantly ( p < 0.01) until nearly all (97.6 ± 1.6%) EOSs were hypodense (density <1.095 gm/ml). Similarly, the absolute blood EOS counts significantly increased throughout treatment. On completion of IL-2 therapy, the EOS counts and density distribution returned to pretreatment values. In contrast, no increase in blood EOS counts was observed in similar patients receiving interferon (γ or β) therapy. Our observations support a hypothesis that IL-2, either directly or, more likely, through the generation of other factors, participates in a change in EOS density that may, in turn, establish a subpopulation of cells with altered metabolic activity.

The effect of azelastine on neutrophil and eosinophil generation of superoxide

Azelastine is a new investigational drug used to treat rhinitis and asthma. In addition to described actions that include inhibition of immunologic release of histamine from mast cells and basophils, blockade of smooth muscle contraction to various spasmogenic mediators, and relaxation of airway smooth muscle, azelastine has been ascribed anti-inflammatory properties. To understand further the mechanisms by which azelastine may regulate inflammation, its effect on neutrophil and eosinophil superoxide (O 2 -) generation was evaluated. Purified suspension of neutrophils (>95% pure) and eosinophils (>85% pure) were isolated from human peripheral blood samples by continuous density gradients of Percoll. The isolated granulocyte suspensions (1 × 10 5 cells) were added to 96-well microtiter plates in the presence of cytochrome c, activated by either N-formyl-methionyl-leucyl-phenylalanine, phorphol myristate actetate, calcium ionophore A23187, or opsonized zymosan particles; O 2 - generation was measured by the reduction of cytochrome c. We found that azelastine significantly inhibited both neutrophil and eosinophil generation of O 2 - in a dose-dependent fashion (10 −7 to 10 −5 mol/L) with each activator except zymosan. Furthermore, the degree to which azelastine suppressed O 2 - was similar in neutrophils and eosinophils. Thus, azelastine, in concentrations achieved therapeutically, inhibited granulocyte generation of O 2 -. This anti-inflammatory effect may also be beneficial in the treatment of asthma.

Glucagon Degrading Activity

To evaluate the effect of glucagon degrading activity (GDA) on radioimmunoassay (RIA) of glucagon, I measured GDAs in plasma, serum, lysed red blood cells (RBC), and suspension of mononuclear cells and granulocytes. Serum levels of GDA in patients with various diseases were also examined. 1. Serum GDA values in normal subjects (control) measured by TCA precipitation method were 4.6 +/- 2.2% (mean +/- S.D.). GDA values in plasma treated with citrate and those in serum treated with aprotinin were not different from those in nontreated serum. GDAs in plasma treated either with EDTA and aprotinin or with EDTA alone were significantly lower than those in control serum, but the values in plasma treated with heparin were markedly higher than those in control serum. 2. GDAs in RBC lysate and suspension of mononuclear cells and granulocytes remained low up to the concentration of 23 X 10(4) RBC/microliters and 5000 mononuclear cells or granulocytes/microliters, respectively. GDAs in RBC lysate and mononuclear cells were markedly suppressed by the treatment with EDTA, whereas GDAs in granulocytes were inhibited by the treatment with aprotinin. 3. Markedly high values of GDA were obtained in serum of patients with pancreas diseases, liver diseases, renal diseases and hyperthyroidism. However, in four patients these elevated levels were restored to normal value after recovery from these disorders. The elevated GDA values in serum were suppressed to normal value by the addition of EDTA and aprotinin. 4. On Bio-Gel P-6 column chromatography, 125I-glucagon incubated with patient serum containing high GDA values revealed several peaks eluted after 125I-glucagon. 5. In healthy subjects, immunoreactive glucagon (IRG) levels in nontreated serum were not different from those in plasma treated either with EDTA and aprotinin or with heparin. 6. In patients showing high serum GDA levels, serum GDA levels were not significantly related to IRG levels in plasma treated with EDTA and aprotinin. These results indicate that a series of treatment of blood samples before assay: addition of EDTA and aprotinin to the blood samples, immediate separation of plasma from blood cells, and storage at 4 degrees C is recommended to avoid breakdown of glucagon by GDAs.

Novel transcellular interaction: conversion of granulocyte-derived leukotriene A4 to cysteinyl-containing leukotrienes by human platelets.

Human platelets dose-dependently converted exogenous leukotriene A4 to leukotriene C4 and efficiently metabolized this compound to leukotrienes D4 and E4. Neither of these compounds were produced after stimulation of human platelet suspensions with ionophore A23187. After LTA4 incubation of subcellular fractions, formation of leukotriene C4 was exclusively observed in the particulate fraction and was separable from the classical glutathione S-transferase activity. This suggested the presence of a specific leukotriene C4 synthase in human platelets. Addition of physiological amounts of autologous platelets to human granulocyte suspensions significantly increased ionophore A23187-induced formation of leukotriene C4. In contrast, the production of leukotriene B4 was decreased. After preincubation of platelets with [35S]cysteine, 35S-labeled leukotriene C4 was produced by A23187-stimulated platelet-granulocyte suspensions, strongly indicating a transcellular biosynthesis of this compound.

Endogenous and Exogenous Adenosine Inhibit Granulocyte Aggregation Without Altering the Associated Rise in Intracellular Calcium Concentration

Abstract The effects of adenosine, adenosine deaminase (ADA), and an irreversible ADA inhibitor 2′-deoxycoformycin (DCF) on granulocyte aggregation in response to four different stimuli: the synthetic chemotaxin N-formyl-met-leu-phe (FMLP), zymosan-activated plasma (ZAP), the calcium ionophore A23187, and phorbol myristate acetate (PMA) were studied. Adenosine inhibited granulocyte aggregation in response to 10(- 7) mol/L FMLP in a dose-dependent fashion; inhibition in the presence of 1 mumol/L adenosine was 25% +/- 3% (SD) and was 50% (the maximal inhibition observed) with 1 mmol/L adenosine. Quantitatively similar results were obtained when ZAP or A23187 was used as the aggregant but the response to PMA was not affected. ADA not only reversed the inhibition due to adenosine but actually augmented the aggregation to FMLP by 118% +/- 9%. Similar results were obtained with ZAP and A23187 but not with PMA. These effects of ADA depended on its enzymatic activity as they could be blocked by preincubation with DCF. Fluorescent measurement of intracellular calcium in fura-2 loaded granulocyte suspensions established that neither adenosine nor ADA affected subsequent FMLP-stimulated calcium responses. Adenosine, therefore, may inhibit granulocyte responsiveness by blocking signal transduction at a point after calcium entry/mobilization but before activation of protein kinase C. Furthermore, the augmentation of responses seen with ADA suggests that endogenous adenosine may be a physiologic autocrine regulator of granulocyte function.

Endogenous and exogenous adenosine inhibit granulocyte aggregation without altering the associated rise in intracellular calcium concentration

The effects of adenosine, adenosine deaminase (ADA), and an irreversible ADA inhibitor 2'-deoxycoformycin (DCF) on granulocyte aggregation in response to four different stimuli: the synthetic chemotaxin N-formyl-met-leu-phe (FMLP), zymosan-activated plasma (ZAP), the calcium ionophore A23187, and phorbol myristate acetate (PMA) were studied. Adenosine inhibited granulocyte aggregation in response to 10(-7) mol/L FMLP in a dose-dependent fashion; inhibition in the presence of 1 mumol/L adenosine was 25% +/- 3% (SD) and was 50% (the maximal inhibition observed) with 1 mmol/L adenosine. Quantitatively similar results were obtained when ZAP or A23187 was used as the aggregant but the response to PMA was not affected. ADA not only reversed the inhibition due to adenosine but actually augmented the aggregation to FMLP by 118% +/- 9%. Similar results were obtained with ZAP and A23187 but not with PMA. These effects of ADA depended on its enzymatic activity as they could be blocked by preincubation with DCF. Fluorescent measurement of intracellular calcium in fura-2 loaded granulocyte suspensions established that neither adenosine nor ADA affected subsequent FMLP-stimulated calcium responses. Adenosine, therefore, may inhibit granulocyte responsiveness by blocking signal transduction at a point after calcium entry/mobilization but before activation of protein kinase C. Furthermore, the augmentation of responses seen with ADA suggests that endogenous adenosine may be a physiologic autocrine regulator of granulocyte function.

Effects of plasticizers and plastic bags on granulocyte function during storage.

The influence of the plasticizers, di-(2-ethylhexyl)phthalate (DEHP) and tri-(2-ethylhexyl)trimellitate (TOTM), on granulocyte function was examined. Polyvinyl chloride (PVC) bags with DEHP (DEHP-PVC) leaked DEHP into plasma, but TOTM did not dissolve in plasma under the same conditions. Glow discharge treatment inhibited the leakage of DEHP from DEHP-PVC bags. Depending on the amount of DEHP added into granulocyte suspension, chemotaxis and bactericidal activity decreased, but cell counts and phagocytosis were not affected. During storage for 24 h at 22 degrees C, granulocyte function decreased greatly in DEHP-PVC, but was well maintained in the bags which did not leak plasticizers, TOTM-PVC and glow-discharged DEHP-PVC.

Oxidation of salicylates by stimulated granulocytes: evidence that these drugs act as free radical scavengers in biological systems.

Although salicylates have been used for centuries as treatment of inflammatory diseases, the mechanism of action of these drugs is still not clear. Aspirin (acetylsalicylic acid) and other nonsteroidal anti-inflammatory drugs (NSAID) inhibit prostaglandin biosynthesis, a property that appears to explain part of their anti-inflammatory activity. However, this mechanism does not appear to explain the anti-inflammatory properties of salicylic acid, which is a major metabolite of ASA in vivo. Results of prior studies in our laboratory have established that benzoic acid, the parent compound of the salicylate group of drugs, is decarboxylated and hydroxylated by the hydroxyl free radical (OH.) produced by stimulated granulocytes. These observations suggested that salicylates might be similarly metabolized by granulocytes. If so, the capacity of salicylates to rapidly react with OH. might relate directly to their known anti-inflammatory properties. Preliminary experiments established that salicylic acid and aspirin were decarboxylated by the hydroxyl free radical generated by the enzyme system xanthine-xanthine oxidase. We then studied the metabolism of salicylates by human granulocytes. Unstimulated granulocyte suspensions did not oxidize ASA or salicylic acid. However, suspensions stimulated by opsonized zymosan particles rapidly oxidized both substrates in pharmacological concentrations. The rate of oxidation of salicylic acid was 16-fold higher than benzoic acid, whereas the rate of oxidation of ASA was four-fold higher. The reaction was oxygen dependent and could be inhibited by known hydroxyl scavengers, particularly dimethylthiourea. The reaction could also be inhibited by superoxide dismutase and azide, indicating that O-2 and heme or an iron-dependent enzyme were required for the reaction. The reaction was not impaired by compounds known to react with the HOCL and the chloramines generated by stimulated PMN. Furthermore, salicylic acid in high concentrations did not impair the HMPS pathway, the production of O-2 or the production of H2O2 by granulocytes. These data provide evidence that salicylates are rapidly oxidized by the hydroxyl free radical produced by granulocytes and not O-2, H2O2, or HOCL. This capacity of salicylates to react rapidly and selectively react with OH. may directly relate to their anti-inflammatory properties. In addition, results of our experiments indicate that stimulated granulocytes acquire the capacity to metabolize these drugs. Therefore, several metabolites of salicylates may be produced at a site of inflammation, all of which may have altered biological activity compared with the parent compound.