ESR studies on oxygen consumption during the respiratory burst of human polymorphonuclear leukocytes

Oxygen consumption during the respiratory burst of human polymorphonuclear leukocytes (PMN) stimulated with phorbol myristate acetate (PMA) was studied with spin probe oxymetry and using the transition metal ion CrOX (potassium trioxalatochromate) as a widening agent. The experimental results demonstrated that during the respiratory burst of PMN stimulated with PMA, oxygen consumption was found mainly in the intercellular medium but no change of oxygen concentration was found in the intracellular medium.     

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.     

ESR studies on active oxygen radicals produced in the respiratory burst of human polymorphonuclear leukocytes

The mechanism and process of production of active oxygen radicals in the respiratory burst of polymorphonuclear leukocytes (PMN) stimulated with PMA (phorbol myristate acetate) was studied in this paper. The experimental results indicate that when the PMA was dilute enough or at the beginning of stimulation even when the PMA concentration was high, the spectrum of hydroxyl radical spin adducts, DMPO-OH, was dominant in the ESR spectra. However, at the maximum level of the respiratory burst, the spectrum of superoxide anion spin adducts, DMPO-OOH, was dominant.     

Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.     

Expression of cytolytic functions in HL-60 leukaemic cells after induction of polymorphonuclear leukocyte differentiation

Mature polymorphonuclear leukocytes (PMN) are capable of mediating phorbol myristate acetate (PMA)- and antibody (A)-dependent cellular cytotoxicity (DCC) against ox red blood cells (ORBC) by using oxidative means. The purpose of the present study was to investigate the acquirement of these cytotoxic functions during PMN ontogeny, using the promyelocytic HL-60 cell line as a model for PMN differentiation. HL-60 cells were induced to differentiate along the PMN pathway by exposure to dimethyl sulfoxide (DMSO). Uninduced HL-60 cells were found to be completely devoid of PMA-DCC and ADCC activity. DMSO-induced cells progressively acquired the capacity to kill ORBC and to undergo the activation of oxidative metabolic burst when triggered by PMA. Despite approximately 40% of them also were capable of binding IgG-sensitized ORBC, no ADCC activity and respiratory burst activation was observed: this finding indicates that maturing HL-60 cells require a more complete maturation than that induced by DMSO to actually exert ADCC. Together the results suggest that: a. the acquirement of both PMA-DCC and ADCC potential is a post-promyelocytic event; b. the cytotoxicity activating stimuli, PMA and IgG-coated targets, follow different post-receptor transductional pathways to trigger the effector cell lytic systems: only the PMA receptor-linked pathway develops during DMSO-driven differentiation of HL-60 cells.     

Beta-endorphin and related peptides suppress phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocytes

In the present study, the immunomodulatory effect of beta-endorphin (beta-E) and shorter pro-opiomelanocortin (POMC) fragments was evaluated by assessing their influence on respiratory burst in human polymorphonuclear leukocytes (PMN). The effect of the peptides (10(-17)M - 10(-10)M) on phorbol myristate acetate (PMA)-stimulated production of reactive oxygen metabolites was measured in a lucigenin-enhanced chemiluminescence (CL) assay. Both POMC peptides with opiate-like activity (i.e. alpha-endorphin (alpha-E), beta-E and gamma-endorphin (gamma-E] and their non-opioid derivatives (i.e. des-TYR1-beta-endorphin (dT beta E), des-TYR1-gamma-endorphin (dT gamma E), and des-ENK-gamma-endorphin (dE gamma E] were tested. With the exception of alpha-E, PMA-stimulated respiratory burst was suppressed by all POMC fragments tested. A U-shaped dose-response relation was observed. Doses lower than 10(-17)M and higher than 10(-8)M were without effect. beta-E and dT beta E both suppressed PMA-induced oxidative burst in human PMN at physiological concentrations (10(-16)M - 10(-10)M). gamma-E and dT gamma E proved to be less potent inhibitors, reaching maximal effect at higher concentrations (10(-12)M - 10(-10)M). DE gamma E exerted an even less pronounced but still significant suppressive effect at the concentration of 10(-10)M. None of the endorphins tested was shown to affect resting oxidative metabolism in the PMN. The modulatory effects of the opioid peptides could not be blocked by the opioid antagonist naloxone (10(-8)M). These data show that fragments derived from the POMC-precursor molecule modulate the activation of PMN by suppressing PMA-stimulated oxidative metabolism and that this activity does not involve a classical opiate-like receptor.     

Alterations in membrane fluidity of diabetic polymorphonuclear leukocytes.

Plasma membrane fluidity of polymorphonuclear leukocytes was investigated in 28 patients with insulin dependent diabetes mellitus and 30 healthy controls. Membrane fluidity was measured by steady-state fluorescence anisotropy of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated into the plasma membrane. The fluorescence anisotropy values in resting (unstimulated) polymorphonuclear leukocytes from diabetic subjects were significantly higher than those of controls (0.318 +/- 0.003 vs 0.287 +/- 0.003, P less than 0.001). The addition of the respiratory burst stimulus phorbol myristate acetate induced a stable increase in fluorescence anisotropy values in both groups. Fluorescence anisotropy values of stimulated polymorphonuclear leukocytes from the diabetic and control groups were not significantly different (P greater than 0.05). These data demonstrate a decrease in plasma membrane fluidity of resting polymorphonuclear leukocytes obtained from diabetic subjects. This finding could be in part explained by an increase in their basal respiratory burst activity.     

Scavenging effect of schizandrins on active oxygen radicals

The reactive oxygen radicals produced from human polymorphonuclear leukocytes (PMN) stimulated with PMA (phorbol myristate acetate), hydroxyl radicals generated by a Fenton reaction, and superoxide anion radicals produced by irradiating solutions of riboflavin in the presence of EDTA have been taken as the models for production of oxygen radicals. With the use of the electron spin resonance spin trapping method, the scavenging effects of schizandrol A (solA) (5 x 10(-4) M) and schizandrin B (sinB) (5 x 10(-4) M) have been studied and compared with the effects of vitamin E (5 x 10(-4) M) and vitamin C (5 x 10(-4) M). It has been found that in cell system the scavenging effects of sinB and solA, as judged by ESR spin trappings, on hydrpxyl radicals (.OH) are greater than vitamin E and vitamin C and the scavenging effects on superoxide anion (O2) are greater than vitamin E but lower than vitamin C. With respect to the Fenton reaction, sinB has the strogest scavenging effect on .OH (77%) and solA has strong scavenging effect on .OH (63%), both of them larger than that of vitamin E (35%) and vitamin C (56%). In the riboflavin/EDTA system, the scavenging effect of sinB (46%) is smaller than that of vitamin C (96%) but larger than that of vitamin E (23%); the scavenging effect of solA is not obvious (14%). With the use of spin probe oximetry, the oxygen consumption during the respiratory burst of stimulated PMN has been measured when exposed to schizandrins. The experiment results demonstrated that they do not affect the activity of production of active oxygen radicals in the respiratory burst of PMN stimulated with PMA.     

Change in the chemiluminescence reactivity pattern during in vitro differentiation of human monocytes to macrophages

We determined the luminol-enhanced chemiluminescence (CL) of fresh human monocytes and monocytes cultured for 1-14 days in vitro, within hydrophobic membranes, using a variety of stimuli known to trigger the respiratory burst of phagocytes. It was assured that CL emerged from an adherent subpopulation of mononuclear cells; polymorphonuclear leukocytes (PMN) contaminating mononuclear leukocytes (MNL) contributed little, if anything, to the CL response of MNL. Typical response patterns were established for fresh monocytes triggered by phorbol 12-myristate 13-acetate (PMA), zymosan, the Ca2+ ionophore A 23187, antibody-coated erythrocytes and Sendai virus. Differentiation in vitro into macrophages was associated with a general decrease in magnitude of the CL peak, in an overproportional decrease of the A23187 triggered response and in a complete loss of the response to Sendai virus--a loss which could not be prevented by addition of myeloperoxidase (MPO). In contrast to monocyte CL, macrophage CL was resistant to sodium azide, indicating its MPO-independent origin. Macrophage-type reactivity was obtained at day 4 of culture. Activation of macrophages with recombinant interferon-gamma for the last 2 days of culture was associated with a quantitative (approx. threefold) increase of the CL signal, although qualitatively the same reactivity pattern was obtained as with control macrophages. In contrast to luminol-dependent CL, the lucigenin-dependent CL response of macrophages was greater than that of monocytes, an increase which was particularly prominent for PMA stimulation.     

Staurosporine, a protein kinase inhibitor, up-regulates the stimulation of human neutrophil respiratory burst by N-formyl peptides and platelet activating factor

Staurosporine (STAR), a potent protein kinase C (PKC) antagonist, was found to modulate the chemoattractant-induced respiratory burst of human polymorphonuclear leukocytes (PMNs) according to drug concentration. Low STAR concentrations from 10 to 200 nM potentiated the N-formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet activating factor (Paf)-induced respiratory burst, affecting both the initial rate and the total amount of superoxide anion generated. The maximal increase occurred in the presence of 100 nM STAR and optimal fMLP concentration and reached 60-100% of control values. Above 250 nM, STAR inhibited the respiratory burst with an IC50 of 360 and 320 nM for fMLP and Paf, respectively. The respiratory burst induced by PKC activators such as phorbol myristate acetate or phorbol 12, 13 dibutyrate was inhibited effectively by STAR, with a low IC50 (25 nM) for both stimuli. Thus, the use of low STAR concentrations points to two possible roles of PKC in the regulation of NADPH oxidase activity, i.e. a positive regulation in phorbol ester-treated cells and a negative regulation in chemoattractant-stimulated PMNs.     

Polymorphonuclear leukocyte-mediated, antibody-dependent, cellular cytotoxicity against tumor cells: dependence on oxygen and the respiratory burst.

Experiments were done to determine 1) whether the respiratory burst of superoxide anion (O2-) production in polymorphonuclear leukocytes (PMN) is triggered during antibody-dependent killing of tumor cells and 2) whether O2- production is essential for cytotoxicity. Three parameters of the respiratory burst (1-14C-glucose oxidation, oxygen consumption, and O2- release) were increased 2.5- to 7.3-fold during killing of antibody-primed tumor cells by human PMN. Added catalase and superoxide dismutase did not inhibit lysis, possibly because these enzymes were unable to diffuse into the inter-plasma-membrane space between killer and target cells. Evidence for an O2- requirement for cytotoxicity was the fact that concentrations of amobarbital or phenylbutazone sufficient to inhibit the cyanide-insensitive respiration of PMN also inhibited cytotoxicity. Also, hypoxic conditions inhibited cytotoxicity from 29 to 73%. The requirement for oxygen was most likely related to O2- generation and not mitochondrial respiration since cyanide and azide, which inhibit mitochondrial respiration, increased cytotoxicity.     

Sesquiterpene lactone from Wunderlichia crulsiana inhibits the respiratory burst of leukocytes triggered by distinct biochemical pathways

The sesquiterpene lactone tubiferin was chemically purified from the brazilian native plant Wunderlichia crulsiana and identified by NMR and GC/MS data. Its ability to inhibit the respiratory burst of peritoneal inflammatory polymorphonuclear leukocytes (PMN) stimulated upon addition of phorbol miristate acetate (PMA), opsonized zymosan (OZ), and N-formyl-methionyl-leucyl-phenylalanine (fMLP) was evaluated. The tubiferin inhibition was more pronounced when PMN were stimulated through the protein kinase C pathway (PMA) compared to the alternative complement pathway (OZ). The inhibition when PMN were triggered by a chemoattractant stimulus (fMLP) was similar to that achieved with OZ-stimulated phagocytes. Tubiferin showed dose-dependent effects on the PMN respiratory burst triggered by the three different substances, and also decreased substantially the carrageenan-induced mice paw edema.     

Human neutrophil laminin receptors: activation-dependent receptor expression

Activated human polymorphonuclear leukocytes (PMN) isolated from peripheral blood specifically bind 125I-laminin after stimulation with phorbol 12-myristate 13-acetate (PMA) or f-Met-Leu-Phe (FMLP) at 37 degrees C. Changes in laminin receptor expression are stimulus dose dependent at both chemotactic (10(-10) M to 10(-6) M) concentrations of FMLP, and secretory (greater than 5 ng/ml) levels of PMA. In the presence of cytochalasin B (5 micrograms/ml), 10(-7) M FMLP activation stimulates specific laminin binding, with an apparent Kd = 3.9 X 10(-9) M and 6.47 X 10(5) binding sites/cell, reaching equilibrium within 10 min at 4 degrees C. This observed activation-dependent change in laminin receptor expression is not due to interference by endogenous laminin, because no fluorescein-visualized anti-laminin antibody bound to cells without added glycoprotein, regardless of the level of activation. Levels of neutrophil lysozyme release, which show a PMA dose dependence similar to that of receptor binding activity, suggest that granule-plasma membrane fusion may be significant during increases in receptor expression. A lack of receptor stimulation by PMA from a granule-deficient patient or in granule-depleted cytoplasts from normal donors additionally supports this hypothesis. Electroblot transfer and autoradiography of subcellular fractions from unstimulated PMN reveals the presence of a 68,000 dalton laminin-binding component in the secondary/tertiary granule (beta) fraction, which may represent an intracellular laminin receptor pool.     

A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: its use in the simultaneous fluorimetric assay of cellular activation processes

A fluorimetric assay for the indirect determination of superoxide production during the respiratory burst of stimulated polymorphonuclear leukocytes was described. The method allowed the determination of submicromolar concentrations of superoxide, and was sufficiently sensitive that first-derivative kinetic analysis of the respiratory burst could be quickly analyzed. Conditions for the simultaneous fluorimetric analysis of superoxide production and intracellular calcium fluxes were described.     

Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals

With the use of the spin trapping methods, the scavenging effects of the extracts of green tea and other natural foods are studied. In stimulated polymorphonuclear leukocytes (PMN) system, water extract fraction 6 (F6) from green tea and green tea polyphenols (GTP) have the strongest scavenging effect on the active oxygen radicals, much stronger than vitamin C (Vc) and vitamin E (VE). Rosemary antioxidants (RA) and Curcumin (Cur) have weaker scavenging effects than Vc, but stronger than VE. In Fenton Reaction, Cur has the strongest scavenging effect (69%) on hydroxyl radicals. In irradiation, riboflavin system F6(74%) and GTP(72%) have very strong scavenging effects that are weaker than Vc, but much stronger than VE (23%). With the use of spin probe oxymetry, the oxygen consumption in respiratory burst of stimulated PMN were measured when the antioxidants existed in these systems. The results demonstrated that these antioxidants did not affect the respiratory burst of human polymorphonuclear leukocytes stimulated with PMA.     

Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions

Cross-linking of the neutrophil-beta 2- or beta 3-related leukocyte response integrins by extracellular matrix (ECM) proteins or monoclonal antibodies (mAb) stimulates cytoskeletal rearrangement leading to cell spreading and respiratory burst. Tyrosin phosphorylation of a variety of proteins and activation of the Src family kinases within polymorphonuclear leukocytes (PMN) have recently been implicated in the intracellular signaling pathways generated by leukocyte integrins (Yan, S.R., L. Fumagalli, and G Berton. 1995. J. Inflammation. 45:217-311.) To directly test whether these functional responses are dependent on the Src family kinases p59/61hck and p58c-fgr, we examined adhesion- dependent respiratory burst in PMNs isolated from hck -/-, fgr -/-, and hck -/- fgr -/- knockout mice. Purified bone marrow PMNS from wild-type mice released significant amounts of O2- when adherent to fibrinogen-, fibronectin-, or collagen-coated surfaces, in the presence of activating agents such as tumor necrosis factor (TNF) or formyl- methionyl-leucyl-phenylalanine, as described for human PMNs. PMNs from hck-/-fgr-/- double-mutant mic, however, failed to respond. This defect was specific for integrin signaling, since respiratory burst was normal in hck-/-fgr-/-PMNs stimulated by immune complexes or PMA. Stimulation of respiratory burst was observed in TNF-primed wild-type PMN plated on surfaces coated with murine intracellular adhesion molecule-1 (ICAM-1), while hck-/-fgr-/- PMNs, failed to respond. Direct cross-linking of the subunits of beta 2 and beta 2 integrins by surface-bound mAbs was elicited O2- production by wild-type PMNs, while the double-mutant hck- /-fgr-/- cells failed to respond. Photomicroscopy and cell adhesion assays revealed that the impaired functional responses of hck-/-fgr-/- PMNs were caused by defective spreading and tight adhesion on either ECM protein- or mAb-coated surfaces. In contrast, hck-/-or fgr-/-single mutant cells produced O2- at levels equivalent to wild-type cells on ECM protein, murine ICAM-1, and antiintegrin mAb-coated surfaces. Hence, either p59/61 hck and p 58c-fgr is required for signaling through leukocyte beta 2 and beta 3 integrins leading to PMN spreading and respiratory burst. This is the first direct genetic evidence of the importance of Src family kinases in integrin signaling within leukocytes, and it is also the best example of overlapping function between members of this gene family within a defined signal transduction pathway.     

Diacylglycerol generation and phosphoinositide turnover in human neutrophils: effects of particulate versus soluble stimuli

Serum-treated, or "opsonized" zymosan (OZ), a particulate material which can be phagocytized by polymorphonuclear leukocytes, activates the superoxide-generating respiratory burst in these cells. The use of dual wavelength spectroscopy in the present studies has allowed accurate continuous monitoring of superoxide generation (cytochrome c reduction) upon cellular activation by this turbid material; activation occurs after a short lag period (about 20 s) which is similar to the lag seen after activation with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Unlike the fMLP response which terminates after about 90 s, superoxide generation in response to OZ continues beyond 10 min, and is similar in this regard to the response seen with the protein kinase C activator phorbol myristate acetate (PMA). OZ and fMLP, but not PMA, also activate receptor-linked phospholipase C mechanisms as judged by the appearance of inositol trisphosphate (IP3) (as well as other inositol phosphates) and diacylglycerol (DAG), with the latter measured by a mass assay. The appearance of these potential mediators corresponded to the loss of phosphoinositides, in particular phosphatidylinositol 4,5-bisphosphate (PIP2). The magnitude of DAG and inositol sugar generation as well as the breakdown of PIP2 was considerably greater using OZ than with fMLP. In addition, while fMLP resulted in a transient increase in IP3 and DAG, OZ resulted in a sustained elevation of these molecules. With both agonists, the onset and duration of generation of putative mediators corresponded to the period of generation of O2-, consistent with a role for DAG and/or IP3 in the activation of the respiratory burst.     

Priming of the respiratory burst of neutrophils by diacylglycerol. Independence from activation or translocation of protein kinase C

Preincubation of neutrophils with certain agonists may "prime" the cells to cause increased responses to a second stimulus ("primed stimulation"). We used two approaches to examine the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in priming and stimulation by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and N-formyl-Met-Leu-Phe (fMLP): inhibition of protein kinase C by 1-(5-isoquinolinesulfonyl)-piperazine (C-I) and measurement of protein kinase C translocation induced by priming and stimulatory concentrations of OAG. C-I had little effect on stimulation or primed stimulation by fMLP, suggesting that fMLP invokes events independent of protein kinase C. C-I equally inhibited stimulation and primed stimulation by PMA. Direct stimulation by OAG was inhibited, but priming and primed stimulation by OAG was unaltered by C-I. OAG concentrations greater than or equal to 100 microM caused translocation of protein kinase C, in correlation with direct stimulation of the respiratory burst. Lower OAG concentrations (10-30 microM) primed to stimulation by fMLP and, conversely, stimulated neutrophils primed with fMLP, yet did not cause translocation of protein kinase C. The data are compatible with previous assumptions that PMA and OAG directly stimulate polymorphonuclear neutrophil leukocytes by translocation and activation of protein kinase C. However, priming and primed stimulation by OAG apparently invoke distinct transduction mechanisms other than protein kinase C translocation.     

Studies on opsonized zymozan, FMLP, carbachol, PMA and A23187 stimulated respiratory burst of human PMNLs

The O2- production as a marker of the respiratory burst was investigated under various stimulations in polymorphonuclear leukocytes of healthy young and aged subjects. Stimulation of the respiratory burst in the cells of elderly by specific agents (opsonized zymozan, N-formyl-methionyl-leucyl-phenylalanine, carbachol) resulted in a diminished response while it remained unchanged on the effect of non-specific stimulation (A23187, phorbol myristate acetate) comparing to young subjects. To elucidate the postreceptor signal transduction mechanism involved in respiratory burst stimulation various inhibitors were used as follows: neomycin (for phospholipase C enzyme), mepacrine (for phospholipase A2 enzyme) and pertussis toxin (for GTP binding regulatory protein). The results suggest that phospholipase C as well as phospholipase A2 could be involved in the postreceptor signal transduction depending on the stimulus, but the impairment of the pertussis toxin sensitive GTP binding protein with aging might explain the decrease response of the respiratory burst after stimulating the different receptors.     

DNA damage in human leukocytes after ischemia/reperfusion injury

Leukocytes have been shown to play an important role in the development of tissue injury after ischemia and reperfusion (I/R). In the present study, the effects of tourniquet-ischemia on induction of DNA damage in peripheral leukocytes and on respiratory burst of neutrophils in humans were examined. The DNA damage was measured as increased migration of DNA using the single-cell gel-electrophoresis technique (comet assay). Intracellular production of reactive oxygen species by neutrophils was measured flow-cytometrically using dihydrorhodamine 123 as indicator. Postischemic, significantly increased migration of DNA was found in leukocytes of 20 patients (tourniquet-ischemia of the lower limb for 65-130 min, anterior-cruciate-ligament-reconstruction) and in 10 experiments (1 volunteer, repeated tourniquet-ischemia of the upper limb for 60 min, no operation). DNA effects were most pronounced 5-30 min after tourniquet release, and then declined over a 2 h period, but did not return to preischemic baseline values. A similar time course showed the oxidative status of unstimulated granulocytes during reperfusion. Simultaneously, opposing changes were measured in formyl peptide (f-MLP)- or phorbol ester (PMA)-stimulated granulocytes, which showed a significantly declined respiratory burst reaction after tourniquet-release indicating preactivation of neutrophils by IR. Our data suggest that IR induces genotoxic effects in human leukocytes presumably in response to oxidative stress during reperfusion.