Effects of C-reactive protein on human neutrophil granulocytes challenged with N-formyl-methionyl-leucyl-phenylalanine and platelet-activating factor

The effects of C-reactive protein (CRP), the prototypical acute-phase reactant were studied on human polymorphonuclear leukocytes (PMNL) challenged with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF). CRP at 8-64 micrograms/ml concentrations inhibited degranulation and superoxide production by PMNL in time-, and dose-dependent manner and stabilized PMNL membranes against the lytic effect of lysophosphatidylcholine. CRP was also capable of binding PAF and in lesser extent fMLP. Furthermore, CRP, 32 micrograms/ml, diminished specific binding of [3H]-fMLP and [3H]-PAF to PMNL. These findings imply that CRP may play an important protective role during the early phase of acute inflammatory reactions.     

Selective release of lysosomal hydrolases from phagocytic cells by cytochalasin B

1. Cytochalasin B (10mug/ml) enhances the release of rabbit polymorphonuclear leucocyte lysosomal acid hydrolases induced by retinol (vitamin A alcohol). 2. This effect is seen at doses of the vitamin that cause selective release of acid hydrolases and those causing more general enzyme release indicated by the loss of lactate dehydrogenase. 3. Cytochalasin B (2-50mug/ml) has no effect on the release of sedimentable acid hydrolases of intact granules obtained from disrupted polymorphonuclear leucocytes. 4. Cytochalasin B (2-10mug/ml) causes a time- and dose-dependent release of mouse peritoneal macrophage acid hydrolases. 5. This effect is selective at all doses of cytochalasin B used, since no release of lactate dehydrogenase, malate dehydrogenase and leucine 2-naphthylamidase was detected. 6. Treatment with cytochalasin B at doses of up to 10mug/ml for as long as 72h did not significantly change the total activities of any of the enzymes measured. 7. The lack of toxicity of cytochalasin B was shown by dye-exclusion tests and its failure to release radioactive colloidal gold stored in secondary lysosomes.     

Platelet activating factor. Stimulation of the lipoxygenase pathway in polymorphonuclear leukocytes by 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine

1-O-Alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (AAGPC) triggered the release of [3H]arachidonate but not [14C]stearate from cellular phospholipids in cytochalasin B-treated rabbit polymorphonuclear leukocytes. Concentrations of AAGPC up to 20 nM caused a dose-dependent release and subsequent metabolism of the released [3H]arachidonic acid. Most of the release of the [3H]arachidonate had taken place within the first 2 min of stimulation. Phosphatidylinositol and phosphatidylcholine served as the sources of [3H]arachidonate with about 50% of the label coming from each pool. Challenge of cytochalasin B-treated polymorphonuclear leukocytes with AAPGC led to the production of [3H]hydroxyeicosatetraenoic acids and [3H]dihydroxyeicosatetraenoic acids. No significant production of [3H]prostaglandins or [3H]thromboxanes was detected. AAGPC also caused a dose-dependent degranulation of cytochalasin B-treated rabbit polymorphonuclear leukocytes as shown by the release of beta-glucuronidase and lysozyme. Both the AAGPC-stimulated production of arachidonate metabolites and the degranulation response were blocked by eicosatetraynoic acid and non-dihydroguaiaretic acid at similar inhibitor concentrations. These findings suggest the bioactions of AAGPC on polymorphonuclear leukocytes may be mediated by the release of arachidonic acid and the production of mono- and dihydroxyeicosatetraenoic acids.     

A novel dioxygenation product of arachidonic acid possesses potent chemotactic activity for human polymorphonuclear leukocytes

We have found that a novel dioxygenation product of arachidonic acid, 8(S),15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid (8,15-diHETE), possesses chemotactic activity for human polymorphonuclear leukocytes comparable to that of leukotriene B4. Authentic 8,15-diHETE, identified by gas chromatography-mass spectrometry, was prepared by treating arachidonic acid with soybean lipoxygenase and was purified by reverse-phase high performance liquid chromatography. Using a "leading front" assay, 8,15-diHETE exhibited significant chemotactic activity at a concentration of 5.0 ng/ml. Maximum chemotactic activity was observed at a concentration of 30 ng/ml. The 8,15-diHETE generated by mixed human leukocytes after stimulation with arachidonic acid and the calcium ionophore, A23187, exhibited quantitatively similar chemotactic activity. Two synthetic all-trans conjugated isomers of 8,15-diHETE, however, were not chemotactic at concentrations up to 500 ng/ml. In contrast to its potent chemotactic activity, 8,15-diHETE (at concentrations up to 10 micrograms/ml) was relatively inactive with respect to its ability to provoke either degranulation or generation of superoxide anion radicals by cytochalasin B-treated leukocytes. Both leukotriene B4 and 8,15-diHETE may be important mediators of inflammation.     

Activation of phospholipid methyltransferase by glucagon in rat hepatocytes

Glucagon produces a time- and dose-dependent activation of phospholipid methyltransferase activity in isolated rat hepatocytes. Half-maximal effect is caused by a dose of glucagon of 1 x 10(-10) M. This activation is due to an increase of the Vmax value of the enzyme, without affecting the Km value for S-adenosylmethionine. Exogenous cyclic AMP added to isolated rat hepatocytes mimics the effect of glucagon, and the activation of phospholipid methyltransferase by a nonsaturating concentration of glucagon is spontaneously reversible within 40 min of incubation.     

Priming of human polymorphonuclear leukocytes with granulocyte-macrophage colony-stimulating factor involves protein kinase C rather than enhanced calcium mobilisation.

Pretreatment of human polymorphonuclear leukocytes with the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) enhances leukotriene biosynthesis in response to a receptor agonist (e.g. N-formyl-methionyl-leucyl-phenylalanine, fMLP) or a Ca(2+)-ionophore (e.g. ionomycin). This priming effect could be traced back to an elevated release of arachidonic acid from the phospholipid pools and hence an increased leukotriene biosynthesis by 5-lipoxygenase. Preincubation of polymorphonuclear leukocytes with GM-CSF did not influence the basal intracellular Ca2+ level and does not enhance cytosolic free calcium after stimulation with fMLP or ionomycin. Only a small increase in the second Ca2+ phase after receptor agonist stimulation was found. However, the Ca(2+)-threshold level necessary for the liberation of arachidonic acid by phospholipase A2 was decreased from 350-400 nM calcium in untreated cells to about 250 nM calcium in primed cells. This allows phospholipase A2 to be activated by a release of calcium from intracellular stores and by ionomycin concentrations which are ineffective in untreated cells. Protein biosynthesis inhibitors like actinomycin D (10 micrograms/ml) and cycloheximide (50 micrograms/ml) had no effect on the enhanced leukotriene biosynthesis in primed cells after stimulation with ionomycin. However, staurosporine (200 nM), an inhibitor of protein kinase C totally abolished the priming effect of GM-CSF after stimulation with ionomycin. The priming effect of GM-CSF could be mimicked by phorbol myristate acetate (PMA; 1 nM) and no additive or synergistic effect was found on leukotriene biosynthesis by simultaneous pretreatment with PMA and GM-CSF and stimulation with either fMLP or ionomycin. These results provide evidence that the enhanced arachidonic acid release in GM-CSF-primed polymorphonuclear leukocytes after stimulation with either fMLP or ionomycin involves activation of protein kinase C which, by a still unknown mechanism, reduces the Ca2+ requirement of phospholipase A2.     

Characterization and biologic properties of 5,12-dihydroxy derivatives of eicosapentaenoic acid, including leukotriene B5 and the double lipoxygenase product

Leukotriene B5 (LTB5) and three stereoisomers were prepared biosynthetically from eicosapentaenoic acid and compared with the analogous derivatives of arachidonic acid for their chemotactic and aggregating effects on human neutrophilic polymorphonuclear leukocytes. Leukotriene B4 (LTB4), LTB5, and the 6-trans-diastereoisomers of each were generated by activating polymorphonuclear leukocytes with the calcium ionophore A23187 in the presence of 14C-labeled and unlabeled arachidonic acid or 14C-labeled and unlabeled eicosapentaenoic acid, respectively. The double lipoxygenase products, (5S,12S)-6-trans-8-cis-LTB4 and (5S,12S)-6-trans-8-cis-LTB5, were generated from 5S-hydroxyeicosatetraenoic acid and racemic 5-hydroxyeicosapentaenoic acid intermediates by incubation with platelet sonicates. The products of each reaction were isolated by reverse-phase-high performance liquid chromatography and identified by their retention times relative to the appropriate totally synthetic standards, ultraviolet absorption spectra, immunoreactivity in a radioimmunoassay for LTB4, and, for all but the double lipoxygenase products, by incorporation of radiolabel from the specific polyunsaturated fatty acid source. When the concentration of LTB5 eliciting maximum chemotactic response of human polymorphonuclear leukocytes, 50 ng/ml (1.5 X 10(-7) M), and that eliciting a maximum aggregation response, 20 ng/ml (5.9 X 10(-8) M), were compared with the interpolated values of LTB4 eliciting comparable effects, the potency of LTB5 relative to LTB4 was approximately 1:8 as a chemotactic agent and about 1:20 as an aggregating agent. The double lipoxygenase products and the resolved 6-trans-diastereoisomers of the pentaene and tetraene series were about 2 logs less active as chemotactic factors than LTB4 and only (5S,12S)-6-trans-8-cis-LTB4 had even minimal aggregating activity.     

Interleukin 1 production by human lung tissue. I. Identification and characterization

Crucial to the development of inflammatory infiltrates is the localized production of mediators which promote adherence of leukocytes to the vascular endothelium. Previous in vitro studies, using monolayers of cultured human vascular endothelial cells (VEC), have identified various agents which promote the acquisition of adhesiveness in VEC for polymorphonuclear leukocytes. In the present studies, we report that human lung fragments cultured for 4 to 24 hr release a factor which acts on VEC to promote adherence of polymorphonuclear leukocytes. Adhesiveness in VEC stimulated by lung fragment culture supernatants was time- and dose-dependent. This adherence-promoting factor appears to be a mixture of the alpha and beta forms of interleukin 1 (IL-1) and has the following properties: 1) it is heat-labile; 2) it is not inactivated by polymyxin B; 3) it has mobility on Sephadex G-75 column chromatography corresponding to apparent m.w. of approximately 15,000, 30,000, and 70,000 (a pattern observed previously for IL-1); 4) it has activity in the thymocyte costimulation IL-1 assay, but no interleukin 2 activity, and 5) it is neutralized by anti-human IL-1 antisera but not by anti-human tumor necrosis factor antiserum. Production and release of IL-1 in vivo may play a role in the development of inflammatory infiltrates in human lung and other tissues by acting on endothelium to promote the localized adherence of leukocytes.     

Cleavage of CD14 on human gingival fibroblasts cocultured with activated neutrophils is mediated by human leukocyte elastase resulting in down-regulation of lipopolysaccharide-induced IL-8 production

Activated polymorphonuclear leukocytes (PMNs) release various types of proteases and express them on the cell surface. The proteases play important roles in PMN-mediated events. In the present study, flow cytometric analysis revealed that CD14 expression on human gingival fibroblasts (HGF) was markedly reduced by PMA-activated PMNs in a coculture system. We found that this reduction was caused by both secreted and cell surface proteases produced by activated PMNs. A protease responsible for the reduction was found to be human leukocyte elastase (HLE) secreted from the activated PMNs by use of various protease inhibitors, although HLE was only partially involved in CD14 reduction caused by cell-bound molecule(s) on fixed PMNs. Analysis with purified HLE revealed a time- and dose-dependent reduction of CD14 on HGF, and complete reduction was observed by 20 microg/ml HLE treatment for 30-60 min, but the other molecules such as CD26, CD59, CD157, and MHC class I on HGF were only slightly reduced. This reduction of CD14 resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD14 on fixed HGF and also on purified cell membranes. As a result of CD14 proteolysis, IL-8 production by HGF was suppressed when triggered by 10 ng/ml LPS, but not by IL-1alpha, indicating that HLE inhibited a CD14-dependent cell activation. These findings suggested that activated PMNs have a potential negative feedback mechanism for HGF function at the inflammatory site, particularly in periodontal tissues.     

The omega-hydroxylation of arachidonic acid by human polymorphonuclear leukocytes

Incubations of [1-14C]arachidonic acid with unstimulated human polymorphonuclear leukocytes resulted in the formation of four new metabolites in a previously described reverse-phase HPLC system. Three of these metabolites were largely suppressed in a CO/O2 (80/20, by vol.) atmosphere indicating a cytochrome-P450-dependent monooxygenase reaction. In agreement with this assumption is their NADPH/O2-dependent formation in the microsomal fraction. One metabolite was identified by gas chromatography/mass spectrometry analysis as omega-hydroxy-arachidonic acid and the two others were secondary products identified as omega-carboxy-arachidonic acid and 5,20-dihydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid. Since the affinity for arachidonate of the omega-monooxygenase was quite low and the presence of LTB4 suppressed the omega-hydroxylation of arachidonate, we conclude that the known LTB4 omega-monooxygenase is responsible for the formation of omega-hydroxy-arachidonate. It is unlikely, however, that significant concentrations of these metabolites are formed by activated polymorphonuclear leukocytes in vivo. The fourth metabolite remains tightly associated with the leukocytes but has not been further characterized.     

Regulation of phosphatidic acid phosphohydrolase activity during stimulation of human polymorphonuclear leukocytes.

Phosphatidic acid phosphohydrolase (PPH) activity was determined in human polymorphonuclear leukocytes (PMNs) by measuring the hydrolysis of [32P]phosphatidic acid (PA) added to cell sonicates. Enzyme activity was localized primarily to a soluble fraction. Soluble and particulate activities required magnesium and were inhibited by calcium, N-ethylmaleimide, sphingosine, and propranolol. The activity in unstimulated PMNs was 0.64 +/- 0.11 nmol of PA hydrolyzed.mg protein-1.min-1 in particulate and 4.20 +/- 0.42 in soluble fractions. Stimulation of PMNs with 1 microM f-Met-Leu-Phe (FMLP) for 10 min caused a slight decrease in soluble activity and a small increase in the activity of particulate fractions. Preincubation with 10 microM cytochalasin B for 5 min before FMLP stimulation markedly enhanced both of these changes. The effect of FMLP plus cytochalasin B was rapid (less than 10 s), whereas the calcium ionophore A23187 (1 microM) and phorbol myristate acetate (100 ng/ml) caused slower and smaller changes in enzyme activity. These results indicate that after chemoattractant stimulation; PPH activity decreases in the soluble fraction and increases in the particulate fraction suggesting that PPH may participate in signal transduction in the PMN.     

Inhibitory effect of linoleyl-hydroxamic acid on the oxidation of linoleic acid by 12-lipoxygenase from porcine leukocytes]

Linoleic acid oxidation by 12-lipoxygenase from porcine leukocytes has been studied as affected by linoleyl-hydroxamic acid. Linoleyl-hydroxamic acid has been found to be an effective inhibitor of porcine leucocyte 12-lipoxygenase. Aerobic preincubation of 12-lipoxygenase with 0.1-6 microM of linoleyl-hydroxamic acid led to a time- and dose-dependent inhibition of the enzyme. The inhibitor's concentration able to induce a 50% loss of the enzyme activity with and without 15-min preincubation were 3.5 and 0.65 microM, respectively. Experimental results obeyed a kinetic scheme, which supposed 2 extra substrate molecules bounding with the enzyme-substrate complex in the presence of linoleyl-hydroxamic acid.     

Stimulation of neutrophil leukocyte chemotaxis by a cloned cytolytic enterotoxin of Aeromonas hydrophila

A cytolytic enterotoxin produced by Aeromonas hydrophila, isolate SSU, has been cloned in our laboratory. This enterotoxin lysed rabbit red blood cells, destroyed Chinese hamster ovary cells, caused fluid secretion in rat ligated ileal loops, inhibited the phagocytic function of mouse phagocytes, and was lethal to mice when injected intravenously. In this study, the effect of this cytolytic enterotoxin on the chemotaxis of human leukocytes (cell line RPMI 1788) was examined. This toxin, at concentrations from 92.5 to 370 ng/ml, significantly stimulated the chemotactic activity of human leukocytes in a dose-dependent fashion. The stimulation of leukocyte chemotaxis elicited by cytolytic enterotoxin was abolished when the toxin was neutralized, heated, or exposed to low pH values. This stimulatory effect also was inhibited by various concentrations of pertussis toxin. These results demonstrated that cytolytic enterotoxin may stimulate increased chemotaxis of human leukocytes, and suggest that human leukocytes may possess cytolytic enterotoxin receptors which may be coupled to pertussis toxin-sensitive G-protein.     

Modulation by phorbol myristate acetate of arachidonic acid release and leukotriene synthesis by human polymorphonuclear leukocytes stimulated with A23187

Phorbol myristate acetate augmented the release of 3H-AA and the synthesis of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by human polymorphonuclear leukocytes stimulated by A23187. PMA alone had no effect. Enhancement of the response to A23187 was not seen when the inactive phorbol ester 4-alpha phorbol didecanoate was added with A23187. These data are consistent with the hypothesis that activation of protein kinase C enhances AA release and metabolism in stimulated polymorphonuclear leukocytes.     

Role of functional groups of human plasma and luminol in scavenging of NaOCl and neutrophil-derived hypochlorous acid

Hypochlorous acid HOCl/OCl^? and other oxidants derived from stimulated polymorphonuclear leukocytes are involved in tissue damage during a number of pathological processes. In order to obtain more detailed information on possible reactions of HOCl/OCl^? the effects of both NaOCl and PMN-derived hypochlorous acid on functional groups of amino acid solutions and human plasma are studied. In valine and lysine solutions NaOCl diminishes the number of amino groups in a molar ratio of 1:1 between NaOCl and amino groups. In cysteine and methionine samples the decrease of amino groups starts only after all sulfhydryl or thioether groups are oxidized by NaOCl. If freshly prepared human plasma is treated with increasing amounts of NaOCl all plasma SH groups are oxidized first, then probably the thioether groups and only after this the amino groups are affected. Furthermore, it was found, that the reactivity of luminol against NaOCl is similar to that of amino groups. Increasing amounts of SH groups of components of human plasma are oxidized by incubation with PMA-stimulated polymorphonuclear leukocytes dependent on the incubation time. Plasma amino groups are not affected under the same experimental conditions. The addition of plasma to FMLP-stimulated PMN in the presence of luminol decreases that part of chemiluminescence caused by extracellularly generated hypochlorous acid. Plasma samples pretreated with NaOCl cause a lower inhibition of light generation in FMLP-stimulated PMN only when more than 4 · 10^?8 mol NaOCl per mg protein are used to pretreat plasma. It is assumed that in the development of tissue injuries caused by infiltrated PMN the following sequence of damage occurs in accessible tissue regions. First, the sulfhydryl groups are oxidized, then the thioether groups, and only after this amino and other target groups are affected.     

Studies on the mechanism of PMN activation III. by lymphokines

The influence of a guinea pig lymphokine preparation on the oxidative metabolism of human and guinea pig granulocytes of various sources was investigated. A dose-dependent increase of the oxidative burst following lymphokine challenge was observed. It occurred in unstimulated guinea pig peripheral polymorphonuclear leukocytes (PMN) and in prestimulated PMN obtained from the peritoneal cavity after glycogen injection as well. The lymphokine effect on the oxidative metabolism is not species-restricted because the guinea pig lymphokine preparation elicits an oxidative burst in human PMN, too. The increase caused by lymphokines is nearly of the same order of magnitude as that obtained with zymosan.     

Differences in the effect of arachidonic acid on polymorphonuclear and mononuclear leukocyte function

Incubation of human polymorphonuclear leukocytes with arachidonic acid resulted in a stimulation of the oxidative metabolism of the cells. Upon stimulation with 80 microM arachidonic acid, neutrophils (5 X 10(6) cells/ml) produced superoxide (53 +/- 8 nmol/5 X 10(6) cells per 15 min), generated chemiluminescence (1211 100 +/- 157 000 cpm) and consumed oxygen (20 +/- 1 nmol/10(6) cells per 5 min). The stimulation of the cell metabolism could be reduced 40-60% by prior incubation of the cells with 10 microM indomethacin. Incubating polymorphonuclear leukocytes with arachidonic acid also resulted in a diminished chemotaxis towards an attractant, a decreased uptake of opsonized staphylococci and aggregation of the cells. This may be due to inhibitory products of arachidonic acid metabolism and toxic oxygen species produced during stimulated oxidative metabolism. The effects of arachidonic acid are specific for neutrophils, as mononuclear phagocytes only produced 17 +/- 8 nmol superoxide/5 X 10(6) cells per 15 min and generated 27 000 +/- 15 000 cpm chemiluminescence when stimulated with 80 microM arachidonic acid. When monocytes and neutrophils were stimulated with particles such as opsonized staphylococci, the amount of superoxide produced, oxygen consumed and chemiluminescence generated were similar. The phagocytic activity of the monocytes was also not affected by prior incubation with arachidonic acid. We conclude that in contrast to monocytes, neutrophil metabolism can be stimulated with arachidonic acid and this stimulation resulted in a decreased phagocytic activity of these cells.     

Toxic effects of fatty acid anilides on the oxygen defense systems of guinea pig lungs and erythrocytes

Toxic oil syndrome (TOS) is caused by ingestion of denatured edible oils. Even though the etiology and pathogenesis of this disease are not fully known, it is quite clear that generation of free radicals caused by ingestion of fatty acid anilides is responsible for the pathogenetic mechanism in many TOS patients. Fatty acid anilides may also alter the free radical status of lungs and erythrocytes; this possibility may shed some light on understanding toxic oil syndrome. The present study describes the effects of oral administration of fatty acid anilides on the activities of major enzymes involved in the oxygen defense systems of lungs and erythrocytes. Feeding fatty acid anilides caused an increase in the superoxide dismutase (SOD) activity in erythrocytes, whereas it caused a decrease in the SOD activity in lungs. GSH-Px activity was not significantly changed in erythrocytes but was decreased in lungs. Although the activity of catalase was increased only by a higher dose in the erythrocytes, it was not affected in the lung at any dosage. Even though the ingestion of fatty acid anilides caused an increase in the SOD activity in the erythrocytes and a decrease in the SOD activity in the lungs, there was an increase in the lipid peroxidation in both cases. The increase in lipid peroxidation in erythrocytes is probably caused by the accumulation of H₂O₂, and that in the lungs is due to the accumulation of superoxide anion.     

Inactivation of phosphofructokinase by glucagon in rat hepatocytes.

Kinetic evidence of a time- and dose-dependent inactivation of phosphofructokinase by glucagon in isolated rat hepatocytes is reported. This inactivation, which persists after gel filtration of a cell-free extract on Sephadex G-25 and after 400-fold purification of the enzyme on agarose-ATP, is observed when the enzyme activity is measured at subsaturating concentrations of fructose 6-phosphate, while there is no change in Vmax. Phosphofructokinase inactivation by glucagon parallels the known inactivation of pyruvate kinase L and activation of glycogen phosphorylase alpha. Exogenous cyclic AMP mimics the effect of this hormone. Half-maximal effect for both phosphofructokinase and pyruvate kinase L is caused by a similar dose of glucagon (1 x 10(-10) M). The inactivation of phosphofructokinase by nonsaturating concentration of glucagon is reversed spontaneously within 40 min of incubation and this reversion is accelerated by insulin.     

Role of functional groups of human plasma and luminol in scavenging of NaOCl and neutrophil-derived hypochlorous acid.

Hypochlorous acid HOCl/OCl- and other oxidants derived from stimulated polymorphonuclear leukocytes are involved in tissue damage during a number of pathological processes. In order to obtain more detailed information on possible reactions of HOCl/OCl- the effects of both NaOCl and PMN-derived hypochlorous acid on functional groups of amino acid solutions and human plasma are studied. In valine and lysine solutions NaOCl diminishes the number of amino groups in a molar ratio of 1:1 between NaOCl and amino groups. In cysteine and methionine samples the decrease of amino groups starts only after all sulfhydryl or thioether groups are oxidized by NaOCl. If freshly prepared human plasma is treated with increasing amounts of NaOCl all plasma SH groups are oxidized first, then probably the thioether groups and only after this the amino groups are affected. Furthermore, it was found, that the reactivity of luminol against NaOCl is similar to that of amino groups. Increasing amounts of SH groups of components of human plasma are oxidized by incubation with PMA-stimulated polymorphonuclear leukocytes dependent on the incubation time. Plasma amino groups are not affected under the same experimental conditions. The addition of plasma to FMLP-stimulated PMN in the presence of luminol decreases that part of chemiluminescence caused by extracellularly generated hypochlorous acid. Plasma samples pretreated with NaOCl cause a lower inhibition of light generation in FMLP-stimulated PMN only when more than 4.10(-8) mol NaOCl per mg protein are used to pretreat plasma. It is assumed that in the development of tissue injuries caused by infiltrated PMN the following sequence of damage occurs in accessible tissue regions. First, the sulfhydryl groups are oxidized, then the thioether groups, and only after this amino and other target groups are affected.