Heparin inhibition of human neutrophil and eosinophil-enriched leukocyte acid beta-glycerophosphatase

Heparin inhibited acid beta-glycerophosphatase (EC 3.1.3.2) from human blood leukocytes, eosinophil-enriched leukocytes, and neutrophils. The inhibition interfered in the hydrolysis of phosphorus from glycerophosphate, not in the formation or detection of colored complexes of phosphomolybdate in the second or color development step in two conventional assays. Heparin inhibited human hypereosinophilic syndrome leukocyte homogenate enzyme activity according to the equation: activity equals 0.946 - 0.087 ln heparin (units/assay) when heparin was varied from 1 to 100 units per assay. At 100 units of heparin per assay, 51% of the original activity remained. Enzyme activity was less in neutrophils than in eosinophils; moreover, the inhibition of neutrophil homogenate by heparin was considerably less than that seen in the eosinophil-enriched leukocyte preparations. In neutrophil homogenates containing 100 units of heparin per assay, 77.1% of activity without heparin was retained. When neutrophil lysates were utilized, less inhibition was observed: e.g., at 1 unit of heparin per assay, 91.7% enzyme activity was retained and at 1000 units, 76.2%; here, activity equals 0.289 - 0.007 ln heparin. The data allowed more precise consideration of the inhibition of acid beta-glycerophosphatase by heparin, and, while confirming quantitatively the greater content of acid beta-glycerophosphatase in eosinophil-enriched leukocyte preparations than in neutrophil preparations, provide experimental support for an acid beta-glycerophosphatase in human eosinophils, which is different from that in human neutrophils. It is more highly susceptible to heparin inhibition than acid beta-glycerophosphatase in human neutrophils from which it is apparently distinct.     

Selective release of extrahepatic lipoprotein lipase by polymetaphosphate

We have studied the lipase released into the circulation by polymetaphosphate injection into rats. Lipase release was in proportion to the dose injected. The post-polymetaphosphate lipase was almost completely inhibited by high salt concentrations or by addition of protamine sulfate to the assay system suggesting that this compound released lipoprotein lipase and not hepatic triglyceride lipase. The lipases released by polymetaphosphate and by heparin were compared using a heparin-sepharose affinity column technique which separates lipoprotein lipase from hepatic triglyceride lipase. While heparin released both lipoprotein lipase and hepatic triglyceride lipase, polymetaphosphate released almost exclusively lipoprotein lipase. Other experiments showed that neither polymetaphosphate nor heparin inhibited the hepatic lipase when added to the assay. These results suggest that lipoprotein lipase may be released by the negative charge on these high-charge polymers while hepatic triglyceride lipase release may require the specific sugar configuration of heparin.     

Acute Phase Immune Response to Exercise Coexists with Decreased Neutrophil Antioxidant Enzyme Defences

Long-duration or damaging exercise initiates reactions that resemble the acute phase response to infection and induces neutrophil priming for oxidative activity. Our objective was to establish the status of the antioxidant defences and of the oxidative equilibrium in the neutrophils of sportsmen prior to and after intense physical exercise. Nine voluntary male professional cyclists participated in this study. The exercise was a cycling mountain stage (171 km) and the cyclists took a mean &#45 SEM of 270 &#45 12 min to complete it. We determined the activities of catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), the levels and activity of superoxide dismutase (SOD), the concentrations of ascorbate, glutathione and glutathione disulphide (GSSG) and DNA levels in neutrophils. The cycling stage decreased enzyme activities expressed per DNA units: CAT (33%), SOD (38%), GPx (65%); increased ascorbate concentration in neutrophils and decreased the GSH/GSSG ratio and the enzyme activities expressed per DNA units. Neutrophils could contribute to plasma antioxidant defences against oxidative stress induced by exercise because they probably provide antioxidant enzymes and ascorbate.     

Acute phase immune response to exercise coexists with decreased neutrophil antioxidant enzyme defences

Long-duration or damaging exercise initiates reactions that resemble the acute phase response to infection and induces neutrophil priming for oxidative activity. Our objective was to establish the status of the antioxidant defences and of the oxidative equilibrium in the neutrophils of sportsmen prior to and after intense physical exercise. Nine voluntary male professional cyclists participated in this study. The exercise was a cycling mountain stage (171 km) and the cyclists took a mean ±SEM of 270 ±12 min to complete it. We determined the activities of catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), the levels and activity of superoxide dismutase (SOD), the concentrations of ascorbate, glutathione and glutathione disulphide (GSSG) and DNA levels in neutrophils. The cycling stage decreased enzyme activities expressed per DNA units: CAT (33%), SOD (38%), GPx (65%); increased ascorbate concentration in neutrophils and decreased the GSH/GSSG ratio and the enzyme activities expressed per DNA units. Neutrophils could contribute to plasma antioxidant defences against oxidative stress induced by exercise because they probably provide antioxidant enzymes and ascorbate.     

Serum carboxypeptidase B: A spectrophotometric assay using protamine as substrate

A quantitative colorimetric assay for serum carboxypeptidase B (SCPB, anaphylatoxin inactivator, kininase I) is described. SCPB is known to possess an enzymatic specificity for cleaving COOH-terminallysyl and arginyl residues which is similar to the specificity of bovine pancreatic carboxypeptidase B. One function of SCPB involves the inactivation of C3a and C5a, the two complement derived anaphylatoxins. Since cobalt markedly enhances the activity of the enzyme, serum is treated with CoCl₂ before the SCPB assay is performed. Salmine, a protamine from salmon sperm, was selected as the substrate because it contains multiple COOH-terminal arginyl residues and is digested more rapidly by SCPB than other common substrates of carboxypeptidase B, including hippuryl-arginine and benzyl-glycylarginine. The kinetics for arginine release from salmine were first-order throughout the course of the assay and the colorimetric values obtained were related to micromols of arginine released. A unit of SCPB is defined as one nanomol of arginine released per minute per milliliter of serum. The range of SCPB activity in serum from healthy individuals was found to be 318 to 466 units. The medians of SCPB activity in sera obtained from patients with Dengue shock syndrome and with shock following intravenous dextran infusion were both lower than the mean SCPB activity of healthy individuals. SCPB levels in patients homozygous and heterozygous for cystic fibrosis were within the normal range.     

Characterization of human neutrophil ecto-protein kinase activity released by kinase substrates.

Although most studies of protein phosphorylation have focused on intracellular protein kinases, evidence for protein kinase activity on the surface of several types of cells has been described. Evidence was recently provided for the existence of ecto-protein kinase activity on the surface of human neutrophils. Evidence for three distinct ecto-protein kinase activities was detected, one that phosphorylates endogenous surface proteins, one that phosphorylates exogenous substrates in a cAMP-independent manner and is released in the presence of substrate, and a low level of activity of one that phosphorylates exogenous Kemptide in a cAMP-dependent manner. To begin to elucidate its role in neutrophil function, we have characterized several properties of the releasable ecto-protein kinase activity on human neutrophils. This enzyme activity was inhibited by impermeant stilbene disulfonic acids, which are known to alter neutrophil function, as well as by impermeant sulfhydryl reactive agents. Enzyme activity was detectable at physiologic concentrations of Mg2+, but was higher in the presence of Mn2+. Protein kinase activity was strongly inhibited by heparin, whereas trifluoperazine, cAMP, and cGMP had little effect on kinase activity. Protein kinase activity was selectively removed from the cell surface by incubation with the ecto-kinase substrates casein and phosvitin, but the enzyme was not released by phosphatidylinositol-specific phospholipase C. Repeated exposure of neutrophils to substrate depleted ecto-protein kinase activity from the cell surface, but activity was rapidly restored by incubation in buffer lacking substrate. The released protein kinase had a Km for ATP of approximately 0.5 microM and a pH maximum between 7.0 and 7.5. At least four ecto-protein kinase substrates were detected in serum; vitronectin was identified as one of these substrates by immunoprecipitation studies. Although the exact role of ecto-protein kinase activity in neutrophil function remains undefined, the identification of vitronectin as a serum substrate suggests that it interacts with a physiologically important substrate.     

Inhibition of phospholipase A2 by heparin

Phospholipase A2 (PLA2) is an important enzyme in the regulation of cell behavior. The hydrolysis of phosphatidylcholine in vitro catalyzed by porcine pancreatic PLA2 was inhibited by heparin. Other glycosaminoglycans inhibited PLA2 activity to a significantly lesser extent, with a pattern of inhibition: heparin much greater than chondroitin sulfate (CS)-C greater than CS-A greater than CS-B greater than keratan sulfate. Hyaluronic acid and heparan sulfate caused no inhibition. Heparin's ability to inhibit PLA2 activity did not depend on substrate concentration, but did depend on ionic strength, with inhibition decreasing with increasing ionic strength. Heparin inhibition also varied with pH, being more effective at pH 5-8 than at pH 10. As a consequence, heparin induced a shift of the pH optimum of PLA2 from 7 to 8. Histone IIA and protamine sulfate, heparin-binding proteins, reversed heparin-induced PLA2 inhibition. The concentration of heparin which inhibited PLA2 activity by 50% increased with increasing enzyme concentration. Furthermore, PLA2 bound to heparin-Affigel. The data indicate that the catalytic potential of PLA2 can be regulated by heparin or heparin-like molecules and that inhibition is contingent on the formation of a heparin-PLA2 complex.     

Protamine, a substrate for thrombolytic agents and enzymes of similar specificity.

Protamine, a highly purified basic polypeptide of 4000 molecular weight containing 80–85% arginine, is a useful substrate for the assay of plasmin, activated plasminogen, and enzymes of similar specificity, e.g., urokinase, coagulation factor Xa, trypsin, and thrombin, and is also an excellent secondary substrate for activator assays of urokinase and streptokinase. The assays were performed manually, and also automated procedures for continuous multiple sample analyses were used. The relative sensitivities for various plasmin-like enzymes were: trypsin > plasmin > urokinase > factor Xa > thrombin. Using protamine with manual assay procedures, the amino-terminal groups of the enzyme-degraded protamine digestion products were detected and quantitated by the colorimetric ninhydrin or the fluorometric fluorescamine procedures, and using protamine with an automated system the ninhydrin method was used. Assigning the CTA casein assay for plasmin a nominal sensitivity of 0.1 (for 0.1 CTA unit of plasmin), the sensitivities of the various assay methods were casein, automated protamine, and manual protamine with ninhydrin, 0.1; manual protamine-trichloroacetic acid with fluorescamine, 0.005; and manual protamine direct fluorescamine, 0.0005. A unit of plasmin, based on the uptake of 1 μequiv base/min during hydrolysis of 0.4% protamine sulfate under standard conditions, is equal to approximately 1.7 × 10³ RFI units or 2.9 CTA units; or, 1 CTA unit of plasmin resulted in an average uptake of 0.346 μmol of base or equivalent bonds split per minute.     

Most of the coenzyme A in dormant spores of Bacillus megaterium is in disulfide linkage to protein.

Fibroblast-like stromal cells obtained from rat epididymal fat pads were grown in culture. It has been shown that these cells release lipoprotein lipase into the culture medium for approximately 10 hr upon exposure of the cells to heparin. Continued incubation of such heparin treated cells in the absence of heparin results in the replenishment of releasable lipase pools over a three day period. The released enzyme is inhibited by NaCl and protamine sulfate.     

Purification and characterization of a novel protamine kinase in HL60 cells

A protamine kinase from HL60 cells was purified to near homogeneity by DEAE-Sephacel, protamine-agarose, Hydroxylapatite, and S-200 chromatography. It was purified by 75.8-fold through four chromatographic steps, and 0.67% of total activity was recovered. The purified enzyme had an apparent molecular mass of 120 kDa and was activated by Mg(2+) or Mn(2+), but inhibited by Ca(2+). Neither phospholipid nor phorbol ester significantly affected the enzyme activity. Staurosporine was the most potent inhibitor of the enzyme among the protein kinase inhibitors tested, K(252a), H(7), heparin, and staurosporine. The purified protamine kinase exhibited a maximum velocity of 5,000 pmol/min/mg and K(m) of 1.3 mM for protamine sulfate as a substrate. Myelin basic protein and protamine sulfate served as the best substrates for the protamine kinase among those tested. The activity of the protamine kinase remained unchanged upon treatment with PMA, retinoic acid, dimethyl sulfoxide, or 1,25 dihydroxy vitamin D(3) for 15 min, while treatment with a differentiating agent, 1,25 dihydroxy vitamin D(3), for one week increased its activity. These results suggest that protamine kinase in HL60 cells is involved in the late stage of the macrophage-monocytic differentiation pathway and may play a role in maintenance of the differentiation after HL60 cells are committed.     

Lipolytic activity of whole isolated liver cells in aqueous suspension.

1. Liver contains a lipase which catalyzes in vitro the hydrolysis of esters of short-chain normal primary alcohols and fatty acids. It is shown that this enzymatic activity can be measured by using intact liver cells as source of enzyme. During short-term incubations of suspensions of cells isolated from rat liver, the lipase acts as a membrane-bound enzyme and readily attacks [3H] oleoylethanol added as an emulsion into the bathing medium. The lipolytic reaction proceeds linearly for at least 20 min at 37 degrees C, at the pH optimum of 8.5. [3H] Oleic acid, a reaction product, is mostly retained in the medium and is used to monitor the lipolytic process. 2. In the presence of heparin, the bound lipase is released in the medium in amounts representing one-third to one half the total activity contained in the cells. This release is very rapid and associated in all cases with a concomitant release of lactate dehydrogenase activity. Such effects are consistent with the interpretation that heparin, at concentrations comprised between 10 and 100 mug per ml, causes alterations of the plasma membrane of the isolated cells, resulting in the dispersion of membrane-bound and cytoplasmtic material. This action of heparin is totally blocked by protamine sulfate (1 mg/ml). No specific effect of heparin directed towards the selective release of lipase could be demonstrated under these conditions. 3. During incubations in the presence of heparin, it was observed that the release of monoester lipase was quantitatively related to a simultaneous decrease in membrane-bound as well as in total monoester lipase activity measureable in the cells after homogenization. This, along with the reappearance of membrane-bound activity immediately after heparin withdrawal, suggest that under the experimental conditions, the membrane-bound enzyme is replaced from inside the cell in proportion of its release by heparin.     

Heparin potentiates in vivo neutrophil migration induced by IL-8

Chemokine IL-8 attracts neutrophils by a haptotactic gradient, made possible by its interaction with proteoglycans of the extracellular matrix. Heparan sulfate, but not heparin, potentiates the attraction exerted in vitro by IL-8. In the present study we first confirmed this in vitro phenomenon, observing that IL-8 activity was potentiated 100% by heparan sulfate, but not by heparin. Then, we evaluated the interference of heparan sulfate or heparin on in vivo neutrophil migration induced by IL-8. The activity of rat IL-8 (3.5 g/animal) preincubated with heparan sulfate (50 g/animal) or heparin (77 g/animal) was assayed on the rat dorsal air pouch. Contrary to in vitro experiments, heparin, but not heparan sulfate, potentiated the in vivo IL-8 activity two-fold. We investigated the relationship between this observation and that reported by others, that IL-8-induced migration depends on the presence of mast cells, which contain heparin-rich granules. We studied the neutrophil migration induced by IL-8 (3.5 g/animal) into the rat peritoneal cavity depleted of mast cells. Neutrophil migration was reduced by 32% when compared to that observed in normal animals. The response of depleted rats was reconstituted by preincubation of IL-8 with heparin (77 g/animal). These data suggest that heparin released from cytoplasmic granules may be the contribution of mast cells to IL-8-induced neutrophil migration.     

On the degradation of heparin by human blood platelets.

Human blood platelets are able to degrade heparin from different tissues and species. The main degradation product is an oligosaccharide. Low molecular weight components such as inorganic sulfate or monosaccharides, i.e. products released by exoenzymes are not detected. The in vitro degradation of heparin by the crude enzyme is observed at pHs below 6.5 with an optimum temperature around 37 degrees C. The presence of sulfate in the substrate structure is shown to be essential for the enzyme activity. Since the oligosaccharides formed have only 10 per cent of the anticoagulant activity of the heparins tested, it is conceivable that the platelet enzyme may play an important role in the inactivation of some of the biological properties of heparin.     

Regulation of serum glycosaminoglycan sulfotransferase activities: inhibition by sulfated glycosaminoglycans and activation by polyamines and basic peptides including a polylysine-containing segment of the c-Ki-ras 2 protein

The regulatory mechanisms for the glycosaminoglycan sulfotransferases in fetal calf serum were investigated. The enzymes examined were those which transfer sulfate from 3'-phosphoadenosine 5'-phosphosulfate to 1) position 6 of the internal N-acetylgalactosamine units of chondroitin, 2) position 6 of galactose units of keratan sulfate, and 3) position 2 (an amino group) of glucosamine units of heparan sulfate. The former two enzymes were activated by spermidine, spermine, protamine, and poly L-lysine. All the enzymes were strongly inhibited by heparin and dextran sulfate, whereas only the chondroitin 6-O-sulfotransferase was inhibited by sulfated galactosaminoglycans. The inhibition of this enzyme by the sulfated glycosaminoglycans was abolished by polylysine, indicating that the activation by polylysine is partly due to the neutralization of endogenous acidic inhibitors, including sulfated glycosaminoglycans. Affinity chromatographic studies demonstrated that heparin specifically binds to the three enzymes, which have anionic isoelectric points, and that chondroitin 6-sulfate, spermine, and polylysine bind to the former two enzymes under physiological conditions. Thus, the activation by spermine and polylysine as well as the inhibition by sulfated glycosaminoglycans also appears to occur through their binding to the enzymes. Studies with synthetic lysine oligomers and an affinity-purified (approximately 700-fold) fraction containing the former two enzymes indicated that the pentamer is the minimum unit required for the activation. A synthetic peptide, containing six consecutive lysines at the carboxy terminus of the human c-Ki-ras 2 protein, also regulated the two enzyme activities at micromolar concentrations. The possible physiological implications of the observed effects of these regulatory substances on the glycosaminoglycan sulfotransferases are discussed in relation to glycosaminoglycan synthesis during the proliferation, differentiation, and transformation of cells. The possibility of sulfated glycosaminoglycans being enzyme regulators is also discussed.     

Properties of cyclic nucleotide phosphodiesterase from lingual taste papillae]

Phosphodiesterase activity is estimated in extracts and partially purified preparations from functionally different parts of bovine tongue. The enzyme activity varied from 4.0 to 10.4 nmole/mg of protein/min. Properties of phosphodiesterase from circumvallate papillae are studied, the pH optimum being 8.0--8.5, Km for cAMP--1.5.10(-4) M and for cGMP--6.5.10(-5) M. The enzyme activity did not change after the treatment with trypsin, protamine sulphate (0.01--1.0%), heparin (0.01--1.0) and taste agents: L-leucine (from 1.10(-2) M to 1.10(-5) M), quinine (from 4.10(-3) M to 4.10(-8) M) and D-glucose (from 1.10(-1) M to 1.10(-4) M). The protein inhibitor of the enzyme, isolated from retina external rod-cell segments considerably suppressed phosphodiesterase activity, and the protein activator from brain tissue stimulated it insignificantly. Thermostable protein modulators, which inhibit or activate (depending on experimental conditions) phosphodiesterase activity, are isolated from circumvallate papillae.     

Effect of leukopenia on pulmonary hypertension after heparin-protamine in pigs.

Heparin neutralization by protamine after cardiac surgery and cardiopulmonary bypass may be associated with complement activation, transient leukopenia, thromboxane A2 release, and severe pulmonary hypertension. The role of leukocytes in the heparin-protamine reaction was studied in leukopenic pigs (n = 9) and a control group (n = 8). Leukopenia was induced by pretreatment with cyclophosphamide (30 mg.kg-1.day-1) for 6-7 days. During general anesthesia and after catheterization, baseline recordings of hemodynamics were performed and blood samples were withdrawn. Heparin (250 IU/kg) was injected and measurements were repeated after 10 min. Protamine sulfate (100 mg) was then infused over 2 min and measurements were performed after 2, 5, and 15 min. Prostanoid concentrations were measured by radioimmunoassays. In additional in vitro experiments, the release of thromboxane B2 from washed platelets and leukocytes after heparin-protamine stimulation was measured. Pretreatment with cyclophosphamide reduced leukocyte counts by 95.5% and the number of neutrophils by greater than 99.9%. Protamine infusion increased mean pulmonary arterial pressure by 74 and 46% and pulmonary vascular resistance by 185 and 384% in control and leukopenic animals, respectively. Thromboxane B2 concentrations increased in both groups. Stimulation by heparin, protamine, or heparin and protamine in sequence did not induce any thromboxane A2 release from washed blood cells. It is concluded that leukocytes do not contribute to pulmonary hypertension after heparin-protamine.     

Reduction in rat mesentery mast cell staining after degranulation by protamine. A competitive antagonism.

Degranulation of rat mesentery mast cells by increasing concentrations of protamine causes a parallel decrease in the numbers of mast cells stained with toluidine blue or with berberine sulfate. No decrease in mast cell numbers occurs when degranulation is inhibited. Since protamine does not enter into non stimulated mast cells, these results suggest that this reduction in mast cell numbers is caused by the binding of protamine to the anionic sites of heparin of exocytosed granules thereby preventing their staining. There seems to be a competitive antagonism between protamine and toluidine blue at the anionic sites of heparin for increasing concentrations of toluidine blue progressively reverse the reduction in mast cell numbers.     

Titrimetric method for determination of O-desulfated heparin in physiological samples using protamine-sensitive membrane electrode as endpoint detector

O-Desulfated heparin (ODSH) is a promising new anti-inflammatory agent for the prevention of reperfusion injury following myocardial infarction or stroke. This partially desulfated heparin derivative has less anticoagulant activity than unfractionated heparin but retains the inherent anti-inflammatory properties of heparin. Thus, ODSH could be administered at the high doses needed to achieve desired anti-inflammatory function without risk of hemorrhage. However, given the very low anticoagulant activity of this species, traditional methods for heparin determination in clinical samples might not be well suited for ODSH measurements. In this article, a novel titrimetric method for detection of ODSH in buffer and plasma is described using a protamine-sensitive polymer membrane electrode as the detector. Titrations of ODSH with the heparin antagonist protamine yield sharp endpoints with sensitivity to ODSH in the micrograms per milliliter range for plasma samples. The stoichiometry for protamine interaction with ODSH is determined to average 1.39 microg protamine/microg ODSH in plasma. This technology is further applied to a toxicokinetic study of ODSH in an animal model, demonstrating the ability to detect the changes in ODSH concentrations in biological samples.     

Biochemical analysis of the activation of adherent neutrophils in vitro

The role played by neutrophil oxidative responses in host defense and injury is an area of active investigation. In order to study neutrophil responses in vitro, methods are required for cell purification, enumeration, and quantification of activation responses, which mimic the in vivo situation as closely as possible. In this communication (and its companion paper, Albertine et al., 1988) improved methods for all of these tasks are described and applied to investigate neutrophil structure-function relationships in vitro and in vivo. Human neutrophils were purified by using a series of platelet-poor plasma-Percoll gradients (51, 62, 76 and 80% in Percoll). This modification of previously published procedures results in consistently successful neutrophil purification and has allowed us to purify neutrophils from bronchoalveolar lavage fluid as well as blood. Activation of human and sheep neutrophils (superoxide anion production) was quantitated by the reduction of ferricytochrome c using a microtiter plate reader to measure the increase in absorbance at 550 nm from adherent neutrophils. Adherence of neutrophils was quantitated by measurement of LDH in cells lysed with Triton X-100 using a new method which uses readily available commercial reagents and can quantitate the LDH content of as few as 5000 neutrophils (or the LDH released from 5% of 100,000 neutrophils). Assay conditions for superoxide anion were optimized, limitations both in assay design and instruments used to measure OD were explored and enumerated, and these methods were used to quantitate sheep and human neutrophil activation responses. Using methods described in Albertine et al. (1988) for fixing neutrophils in microtiter wells after assay of their functional capacity, we have studied the same cells functionally and morphologically. We have used these techniques to study blood and alveolar neutrophils from a patient with acute respiratory failure. His alveolar neutrophils displayed 67% of the activation response as peripheral neutrophils (4.31 +/- 0.12 nmol superoxide released per 250,000 neutrophils at 60 min vs. 6.38 +/- 0.18 in blood, P less than 0.01) and structural changes which suggested previous activation in vivo. These studies demonstrate that similar morphological changes are observed in neutrophils activated with phorbol myristate acetate in vitro, as are observed in cells which have been activated by pathophysiologic processes in vivo.     

Leishmania donovani: surface membrane acid phosphatase blocks neutrophil oxidative metabolite production

We show that a purified preparation of the prominent tartrate-resistant acid phosphatase (E.C.3.1.3.2), isolated from the external surface of the intracellular parasite Leishmania donovani (promastigote form), inhibits toxic oxidative metabolite production of neutrophils. Preincubation of a neutrophil suspension (2.5 X 10(6) cells/ml) for 15 min at 37 C with 250 units (1 unit equals 1 nmole of 4-methylumbelliferyl phosphate cleaved per hr at pH 5.5) of the acid phosphatase in Krebs-Ringer phosphate buffer (pH 7.4) decreased O2 consumption, O2- production, and H2O2 production of N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)-stimulated neutrophils to 15-25% of control values. The acid phosphatase also affected concanavalin A-stimulated O2-production by neutrophils, but had no effect on the rate of phorbol myristic acetate-stimulated O2- production, chemotactic peptide binding, degranulation, or membrane depolarization. Addition of an acid phosphatase inhibitor (Complex E; (NH4)6[P2Mo18O62] X 9H2O) to suspensions of opsonized promastigotes and neutrophils resulted in a threefold or greater enhancement of O2- production. These results suggest a possible pathophysiologic role for the acid phosphatase of L. donovani promastigotes.