Poly-MVA attenuates 7,12- dimethylbenz[a]anthracene initiated and croton oil promoted skin papilloma formation on mice skin.

OBJECTIVE: Chemopreventive agents which exhibit activities such as anti-inflammation, inhibition of carcinogen induced mutagenesis and scavenging of free radical might play a decisive role in the inhibition of chemical carcinogenesis either at the initiation or promotion stage. Many synthesized palladium (Pd) complexes tested experimentally for antitumor activity are found effective. Poly-MVA is a liquid blend preparation containing B complex vitamins, ruthenium with Pd complexed with alpha lipoic acid as the major ingredients. The antitumor effect of Poly-MVA was evaluated against 7,12-dimethylbenz[a] anthracene-initiated croton oil-promoted papilloma formation on mice skin. Skin tumor was initiated with a single application of 390 nmol of DMBA in 20 µl acetone. The effect of Poly-MVA against croton oil- induced inflammation and lipid peroxidation on the mice skin was also evaluated. Topical application of Poly-MVA (100 µl, twice weekly for 18 weeks) 30 minutes prior to each croton oil application, significantly decreased the tumor incidence (11%) and the average number of tumor per animals. Application of Poly-MVA (100 µl) before croton oil significantly (p < 0.05) protected the mouse skin from inflammation (36%) and lipid peroxidation (14%) when compared to the croton oil alone treated group. Experimental results indicate that Poly-MVA attenuate the tumor promoting effects of croton oil and the effect may probably be due to its anti-inflammatory and antioxidant activity.

Mitochondrial N-formyl methionine peptides contribute to exaggerated neutrophil activation in patients with COVID-19.

Neutrophil dysregulation is well established in COVID-19. However, factors contributing to neutrophil activation in COVID-19 are not clear. We assessed if N-formyl methionine (fMet) contributes to neutrophil activation in COVID-19. Elevated levels of calprotectin, neutrophil extracellular traps (NETs) and fMet were observed in COVID-19 patients (n = 68), particularly in critically ill patients, as compared to HC (n = 19, p < 0.0001). Of note, the levels of NETs were higher in ICU patients with COVID-19 than in ICU patients without COVID-19 (p < 0.05), suggesting a prominent contribution of NETs in COVID-19. Additionally, plasma from COVID-19 patients with mild and moderate/severe symptoms induced in vitro neutrophil activation through fMet/FPR1 (formyl peptide receptor-1) dependent mechanisms (p < 0.0001). fMet levels correlated with calprotectin levels validating fMet-mediated neutrophil activation in COVID-19 patients (r = 0.60, p = 0.0007). Our data indicate that fMet is an important factor contributing to neutrophil activation in COVID-19 disease and may represent a potential target for therapeutic intervention.

Novel structural determinants of the human neutrophil chemotactic activity of leukotriene B.

A specific 5(S),12(R)-dihydroxy-eicosa-6,8,10(trans/trans/cis), 14(cis)-tetraenoic acid, designated leukotriene B, is generated by the lipoxygenation and subsequent enzymatic hydration of arachidonic acid in a variety of leukocytes. Leukotriene B elicits a maximal human neutrophil chemotactic response in vitro which is similar in magnitude to those evoked by the chemotactic fragment of the fifth component of complement, C5a, synthetic formyl-methionyl peptides, and 5-hydroxy-eicosatetraenoic acid (5-HETE). The neutrophil chemotactic potency of purified leukotriene B, assessed by the 50% effective concentration of 6 x 10(-9) M, is equivalent to that of C5a, but is up to 100-fold greater than that of 5-HETE and of other natural di-HETE isomers. 5(S),12(R)-di-hydroxy-eicosa-6,8,10(all-trans),14(cis)-tetraenoic acid, which differs from leukotriene B only in having a trans-double bond in place of a cis-double bond in the triene portion of the molecule, and acetyl-leukotriene B are significantly less potent neutrophil chemotactic factors than leukotriene B, which indicates that both the conjugated double bonds and the free hydroxyl-group(s) are functionally critical determinants. The capacity of acetyl-leukotriene B to inhibit competitively and selectively the human neutrophil chemotactic response to equimolar concentrations of leukotriene B suggests the existence of a specific subset of receptors for this potent lipid mediator.

Specific binding of leukotriene B4 to a receptor on human polymorphonuclear leukocytes.

In this paper we have described the binding of nanomoler concentrations of [3H]leukotriene B4 (LTB4) to human polymorphonuclear leukocytes. Because up to 80% of the total [3H]LTB4 binding was blocked by excess (greater than 100 times) [14C]LTB4, the majority of binding is specific. Stereospecificity of the LTB4 binding is demonstrated by the diminished relative abilities of the 6-trans-and 12-epi-6-trans- isomers of LTB4 to block [3H]LTB4 binding. With these two isomers 3-10-fold higher than [14C]LTB4 concentrations were needed for equivalent inhibition of [3H]LTB4 binding. This difference is quantitatively less dramatic than the differences between these isomers in many in vitro functional assays such as chemokinesis, chemotaxis, and degranulation. Binding of [3H]FMLP is not blocked at greater than 100-fold excess of LTB4. The binding of [3H]LTB4 to cells appears to be essentially irreversible at 4 degrees C, but not at 37 degrees C where initially bound LTB4 is rapidly converted to metabolites which then enter the medium. These results suggest the presence of a saturable, stereospecific site for LTB4 on PMN. The association of LTB4 binding and the initiation of pharmacological responses to LTB4 will require further studies.

Mitochondrial N-formylmethionyl proteins as chemoattractants for neutrophils.

Mitochondria synthesize several hydrophobic proteins. Like bacteria, mitochondria initiate protein synthesis with an N-formylmethionine residue. Because N-formylmethionyl peptides have been found to be chemotactic for polymorphonuclear leukocytes (PMN), mitochondria isolated from cultured human cells and purified bovine mitochondrial proteins were tested for PMN chemotactic activity in vitro. Nondisrupted mitochondria were not chemotactic. However, intact mitochondria that had been incubated with a lysosomal lysate did stimulate PMN migration. Antibodies directed against two mitochondrial enzymes, cytochrome oxidase and ATPase, (both of which contain mitochondrially synthesized subunits) but not anti-C3 or anti-C5 decreased mitochondrially derived chemotactic activity. In addition, purified bovine mitochondrial N-formylmethionyl proteins stimulated PMN migration in vitro, whereas nonformylated mitochondrial proteins did not. Furthermore, the chemotactic activity of purified mitochondrial proteins and disrupted mitochondria was decreased by the formyl peptide antagonist butyloxycarbonyl-phenylalanine-leucine-phenylalanine-leucine-phenylalanine. Finally, disrupted mitochondria and purified mitochondrial proteins stimulated PMN-directed migration (chemotaxis), according to accepted criteria. In addition to other chemotactic factors, release of N-formylmethionyl proteins from mitochondria at sites of tissue damage, may play a role in the accumulation of inflammatory cells at these sites.

Polymorphonuclear leukocyte migration through human amnion membrane.

A new in vitro model has been developed for studying migration of human polymorphonuclear leukocytes (PMN) through living native cellular and matrix barriers. Human amnion membrane consists of a single layer of epithelium bound to a continuous basement membrane interfacing an avascular collagenous stroma. Living amnion was placed in plastic chambers with separate compartments on each side of the membrane. PMN were introduced on the epithelial side of the amnion, and a Millipore filter (Millipore Corp., Bedford, Mass.) was placed against the stromal side. In response to N-formylmethionyl-leucyl- phenylanlanine (FMLP) chemoattractant, PMN penetrated the full thickness of the amnion and were collected and counted on the filter. The rate of PMN traversal of the amnion was dependent on the concentration of FMLP (optimal at 10(-8)M) as well as the slope of the FMLP gradient across the amnion. The route of PMN migration was studied by transmission electron microscopy. PMN first attached to the epithelial surface, then infiltrated between intercellular junctions. PMN migrated around or through tight junction and hemidesmosome attachments. The PMN then penetrated the basement membrane and migrated through the dense collagenous stroma. The present amnion migration system has characteristics of the in vivo inflammatory state not described in any previous method for monitoring PMN migration in vitro. Prior methods have not used native epithelium, whole basement membrane, or collagenous stroma. PMN penetration of these barriers occurs in the normal inflammatory response and probably involves biochemical mechanisms not required for simple migration through the pores of an artificial filter. The amnion system can be useful for future biochemical and morphological studies of PMN penetration of these barriers and possible repair processes that may follow.

Degranulation, membrane addition, and shape change during chemotactic factor-induced aggregation of human neutrophils.

Neutrophils stimulated by the chemotactic factor formyl-methionyl-leucyl-phenyl-alanine (FMLP) undergo a transient change in surface properties that permits the cells to adhere more readily to surfaces and to each other. This transient change can be monitored by light scattering as stimulated neutrophils form aggregates while stirred in a platelet aggregometer. Maximum change in light scattering occurs within 1 min and correlates with an increase in the percentage of cells that are in aggregates of four or more cells and a decrease in the percentage of single cells. With time (3-5 min), small aggregates disappear and single cells reappear. The transient change in adhesiveness is accompanied by a persistent change in cell shape; the cells become polarized and protrude ruffles from one sector of the cell surface. During aggregation the cells adhere to one another with smooth sides together and ruffles pointed outward. During disaggregation the cells dissociate laterally with the simultaneous internalization of membrane in the region opposite the ruffles. Particle bound to the surface by charge (thorotrast, cationized ferritin) are concentrated and internalized in this region. The change in cell shape from round to ruffled occurs within seconds, suggesting that membrane is added to the cell surface from an intracellular store. We therefore quantified surface membrane by electron microscopy morphometry and measured a 25% increase within 10 s of adding FMLP. The source of new membrane appeared to be the specific granule membrane since the kinetics of granule discharge (between 30% and 50% of all release occurs in the first 10 s) correlate with the appearance of new membrane. Furthermore, the amount of membrane that appears at the cell surface at 10 s correlates with that lost from intracellular granules in that time. Chemotaxin-induced aggregation thus begins with granule discharge and membrane addition followed by protrusion of ruffles. Adherence is maximal at 60 s and the gradual loss of adhesiveness that follows is associated with uropod formation and enhanced endocytic activity.

Stimulation of tubulin tyrosinolation in rabbit leukocytes evoked by the chemoattractant formyl-methionyl-leucyl-phenylalanine.

Cellular tubulin is subject to a posttranslational modification involving the reversible addition to tyrosine through peptide linkage to the C-terminal glutamate of the alpha-chain. The synthetic peptide chemoattractant, N-formyl-methionyl-leucyl-phenylalanine, causes a specific, dose-dependent stimulation of tubulin tyrosinolation in rabbit leukocytes. This stimulation is prevented by carbobenzoxy-phenylalanyl-methionine, benzoyl-tyrosine ethylester, and nordihydroguaiaretic acid, which are all inhibitors of chemotaxis presumed to act via membrane-associated events. The combination of 3-deazaadenosine and homocysteine thiolactone, which inhibits phospholipid methylation, and quinacrine, an inhibitor of phospholipase A2, also abolishes the response to the peptide. Colchicine, however, which causes a marked disassembly of cellular microtubules in these cells and also inhibits chemotaxis, does not have any inhibitory effect on the basal or peptide-stimulated rate of tubulin tyrosinolation. In contrast, taxol, a microtubule-stabilizing agent, has an inhibitory effect on both the basal and peptide-stimulated tyrosine incorporation. Taxol also inhibits chemotaxis in rabbit leukocytes. The results strongly suggest the role of closely linked membrane-cytoskeleton interactions in leukocyte chemotaxis, in which tyrosinolation of tubulin may be functionally involved.

Alterations of new methylated phospholipid synthesis in the plasma membranes of macrophages exposed to chemoattractants.

Chemotactic factors have been shown to inhibit the methylation of phosphatidylethanolamine in macrophages without affecting total phospholipid synthesis. It would thus be anticipated that newly synthesized membranes of macrophages exposed to chemoattractants would have an increased ratio of phosphatidylethanolamine to its methylated derivatives. These ratios were measured directly in newly synthesized phospholipids of plasma membranes isolated from guinea pig peritoneal macrophages. The phosphatidylethanolamine: methylated phospholipid ratio in such plasma membranes was increased by 53 to 111% upon exposure of the cells to chemotactic factors. This increase was due to decreased synthesis of methylated phospholipids and not to altered formation of phosphatidylethanolamine or activation of phospholipases. Methylated phospholipid ratios were also studied in the leading front lamellipodia isolated from macrophages migrating under chemotactic and nonchemotactic conditions. The phosphatidylethanolamine:methylated phospholipid ratios were increased up to fourfold in lamellipodia of macrophages migrating towards chemotactic agents when compared to those from cells migrating randomly. Biophysical changes in the plasma membrane produced by an increase in the ratio of phosphatidylethanolamine:methylated phospholipids as a result of exposure of cells to chemoattractants may be required for sustained directed migration.

Analysis of the directed and nondirected movement of human granulocytes: influence of temperature and ECHO 9 virus on N-formylmethionylleucylphenylalanine-induced chemokinesis and chemotaxis.

The directed movement of human polymorphonuclear leukocytes (PMN) in a plane (Zigmond chamber assay) is described by a statistical model. We demonstrate that (a) the movement of a single cell is a superposition of a directed and a random movement, and (b) the degree of orientation, P1, of moving cells in a chemotactic gradient can be determined either by the time average of a single cell or by the average of movement of multiple cells at a fixed time (Ergoden hypothesis). However, an homogeneous cell population is a necessary condition. P1, which is identical with the McCutcheon index, is derived from the measured angular distribution function of moving cells. The statistical model allows one to distinguish between chemotaxis and chemokinesis. Applying this model to the temperature-dependent changes of cell movement, we found that P1 = 0.82 (37 degrees C) decreased to P1 = 0.4 (22 degrees C). The average speed of moving cells exhibits a very strong temperature-dependent variation from 30 microns/min (37 degrees C) to 5 microns/min (22 degrees C), indicating a different temperature dependence of chemotaxis and chemokinesis. At a fixed temperature (37 degrees C) the stability of the chemotactic gradient can also be checked by the angular distribution function. In addition, this model was applied to investigate the enteric cytopathogenic human orphan, strain 9 (ECHO 9) virus-induced disturbances of cell movement. We found: (a) The average speed of cell movement is not affected by the virus. (b) The degree of orientation is not affected for virus doses below a critical virus dose, ao (virus/PMN = 0.8:1). (c) The degree of orientation above this critical value exhibits a time- and virus-dose-dependence. (d) At a fixed viral dose, the time-dependent decrease of P1 is described by an exponential law (virus/PMN = 5:1, the characteristic time is 110 min). (e) This characteristic time investigated as a function of viral dose results in a logarithmic law analogous with the Weber-Fechner law. These findings indicate that only chemotactic and not chemokinetic response is disturbed by ECHO 9 virus.

Tubulin tyrosinolation in human polymorphonuclear leukocytes: studies in normal subjects and in patients with the Chediak-Higashi syndrome.

We have recently reported a specific dose-dependent stimulation of posttranslational incorporation of tyrosine into tubulin alpha-chains of rabbit peritoneal leukocytes as induced by the synthetic peptide chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). The present study reports a similar, specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN). When compared to normal PMN, both the resting and FMLP-stimulated levels of posttranslational tyrosine incorporation were two- to threefold higher in PMN of three patients with the Chediak-Higashi syndrome (CHS). The concentration of cellular tubulin and the specific activity of tubulin tyrosine ligase were similar in PMN of CHS patients and normal donors and resembled that of other non-neuronal cells. The high levels of tyrosine incorporation in PMN of CHS patients were normalized by the administration of ascorbate, both in vitro and in in vivo experiments. In vitro addition of ascorbate also inhibited the FMLP-induced stimulation of tyrosine incorporation in both normal and CHS cells. Normalization of higher levels of tyrosine incorporation in PMN of CHS patients and the inhibition of FMLP-induced stimulation of tubulin tyrosinolation in normal and CHS cells as observed with ascorbate could also be affected by other reducing agents such as reduced glutathione, cysteine, or dithiothreitol. These results suggest a possible relationship between cellular redox and tubulin tyrosinolation in PMN.

Activation of the rabbit polymorphonuclear leukocyte membrane "Na+, K+"-ATPase by chemotactic factor.

Addition of the synthetic chemotactic factor, formyl-methionyl-leucyl-phenylala-nine (F-Met-Leu-Phe) to medium containing magnesium, sodium, and potassium results in a doubling of the "Na+, K+"-ATPase activity of the plasma membrane fraction from polymophonuclear leukocytes (PMN). This activation is sensitive to ouabain inhibition and is dose dependent, maximal activity occuring at 10(-9)MF-Met-Leu-Phe. Equivalent activation was observed with the nonformylated derivative Met-Leu-Phe at 10(-9)M. The dipeptide, carbobenzoxy-methionylphenylalanine, which acts as an antagonist for F-Met-Leu-Phe, prevents the stimulation of the "Na+, K+"-ATPase by F-Met-Leu-Phe.

Phospholipid metabolism, calcium flux, and the receptor-mediated induction of chemotaxis in rabbit neutrophils.

Rabbit neutrophils were stimulated with the chemotactic peptide fMet-Leu-Phe in the presence of the methyltransferase inhibitors homocysteine (HCYS) and 3-deazaadenosine (3-DZA). HCYS and 3-DZA inhibited chemotaxis, phospholipid methylation, and protein carboxymethylation in a dose-dependent manner. The chemotactic peptide-stimulated release of [14C]arachidonic acid previously incorporated into phospholipid was also partially blocked by the methyltransferase inhibitors. Stimulation by fMet-Leu-Phe or the calcium ionophore A23187 caused release of arachidonic acid but not of previously incorporated [14C]-labeled linoleic, oleic, or stearic acids. Unlike the arachidonic acid release caused by fMet-Leu-Phe, release stimulated by the ionophore could not be inhibited by HCYS and 3-DZA, suggesting that the release was caused by a different mechanism or by stimulating a step after methylation in the pathway from receptor activation to arachidonic acid release. Extracellular calcium was required for arachidonic acid release, and methyltransferase inhibitors were found to partially inhibit chemotactic peptide-stimulated calcium influx. These results suggest that methylation pathways may be associated with the chemotactic peptide receptor stimulation of calcium influx and activation of a phospholipase A2 specific for cleaving arachidonic acid from phospholipids.

Chemotactic factor-induced release of membrane calcium in rabbit neutrophils.

The interaction of chemotactic factors (fMet-Leu-Phe and C5a) with rabbit neutrophils leads to rapid and specific release of membrane calcium, as evidenced by changes in the fluorescence of cell-associated chlorotetracycline. These two structurally different stimuli appear to interact with the same pool of membrane calcium.

Is cytosolic ionized calcium regulating neutrophil activation?

The concentration of cytosolic ionized calcium, [Ca2+]i, was measured in intact neutrophils by use of a fluorescent indicator trapped in the icytoplasm. A given rise of [Ca2+]i elicited by the chemotactic peptide formylmethionylleucylphenylalanine (FMLP) was associated with a much greater degree of superoxide generation and myeloperoxidase secretion than was the same or larger [Ca2+]i produced by a specific calcium ionophore, ionomycin, which bypasses cell surface receptors. Thus, FMLP appears to generate some important excitatory signal in addition to a rise in [Ca2+]i and exocytosis and superoxide generation in neutrophils may not be simply dependent on [Ca2+]i as is widely supposed.

Human neutrophil heterogeneity identified using flow microfluorometry to monitor membrane potential.

Previous studies of neutrophil nitroblue tetrazolium dye reduction in response to endotoxin and rosetting of IgG-coated erythryocytes have suggested functional heterogeneity of peripheral blood neutrophils. In the following study we utilized flow microfluorometry and the membrane potential-sensitive fluorescent dye 3-3'-dipentyloxacarbocyanine to assess the heterogeneity of neutrophils upon activation by a variety of stimuli. Unstimulated neutrophils from normal subjects exhibited a unimodal distribution of fluorescence, suggesting that all the cells possessed the same resting membrane potential. As neutrophils aged (>5 h), some cells lost fluorescence producing a bimodal distribution. In studies with fresh cells, the secretagogue phorbol myristate acetate (20 ng/ml) stimulated a uniform loss of fluorescence (apparent depolarization). The chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) (0.1 muM) caused the neutrophils to assume and maintain (for > 30 min) a bimodal fluorescence distribution in which 65+/-5% of the neutrophils first decreased and then increased fluorescence (apparent depolarization/partial repolarization), and 35+/-5% of the cells exhibited either an increase in fluorescence (apparent hyperpolarization) or no change. Treatment of neutrophils with cytochalasin B before stimulation caused the cells to respond homogeneously to f-Met-Leu-Phe. Additional studies using neutrophils from patients with chronic granulomatous disease, which exhibit abnormal membrane potential responses, indicated that this defect affected all such neutrophils uniformly. These observations demonstrate the need to investigate the physiological significance of the heterogeneity of neutrophil function and indicate that the f-Met-Leu-Phe-induced changes in membrane potential observed in bulk population cell studies are the summation of two different responses.

Motility and adhesiveness in human neutrophils. Redistribution of chemotactic factor-induced adhesion sites.

Human peripheral blood neutrophils obtained from healthy adults were examined in vitro. We assessed the effects of sequential stepwise increases in the concentration of the chemotactic dipeptide N-formyl-l-methionyl-l-phenylalanine (f-Met-Phe) on neutrophil attachment to serum-coated glass, detachment from serum-coated glass and the distribution on the cell surface of binding sites for albumin-coated latex beads. The initial exposure to f-Met-Phe resulted in increased adhesiveness and binding of latex beads in a random pattern over the cell surface. The second exposure to f-Met-Phe resulted in decreased adherence, detachment of neutrophils from serum-coated glass, and movement of binding sites for latex beads to the uropod. Enhanced adhesiveness and redistribution of binding sites were blocked by 0.1 mM N-alpha-p-tosyl-l-lysine chloromethyl ketone, a concentration that did not reduce the change in cellular shape caused by f-Met-Phe. Cytochalasin B (5 mug/ml) blocked the redistribution of binding sites as well as the change in shape. The third exposure to f-Met-Phe was given along with the latex beads. The stimulus was stopped after 2 min by fixing cells in suspension with glutaraldehyde. If the third exposure was at a concentration higher than the second, the beads were bound in the region of the lamellipodia in 70% of the cells. If lower, binding to the lamellipodia was found in a significantly smaller proportion of cells (13%). The results support the concept that neutrophils develop a polarized distribution of f-Met-Phe-induced adhesion sites in response to increasing concentrations of f-Met-Phe, and these sites flow from the region of the lamellipodia to the uropod.

Activation of the respiratory burst enzyme in human polymorphonuclear leukocytes by chemoattractants and other soluble stimuli. Evidence that the same oxidase is activated by different transductional mechanisms.

Chemoattractant-receptor coupling triggers several biologic responses in phagocytic cells including activation of the respiratory burst. Prior evidence in intact cells implied that stimulation of the respiratory burst by chemoattractants was by a mechanism different from other soluble agents suggesting the possibility that different oxidative enzymes were responsible. We now show that the chemoattractants N-formyl-methionyl-leucyl-phenylalanine and a split fragment of the fifth component of complement (C5a) stimulate an NADPH oxidase activity, measured in the 50,000-g particulate fraction from human polymorphonuclear leukocytes (PMN). Levels of oxidase activity stimulated by the chemoattractants were both time and dose dependent and required the presence of cytochalasin B during stimulation. In contrast, activation by two nonchemotactic stimuli, the ionophore A23187 and phorbol myristate acetate (PMA), did not require cytochalasin B. Temporal patterns of oxidase activation suggested that different stimuli follow different transductional pathways. Chemoattractant-mediated activation was immediate (no lag); peaked by 45 s and declined rapidly to approximately 50% of maximal by 2 min. In contrast, activation by A23187 or PMA had a 15-30-s lag and increased more slowly. Stimulation by A23187 peaked at 5 min, then declined. Stimulation by PMA plateaued at 20 min and did not decline by 90 min. Comparison of Km values for NADPH and NADH obtained by Lineweaver-Burk analysis of the oxidase activity stimulated by N-formyl-methionyl-leucyl-phenylalanine, A23187, and PMA suggested that the same enzyme was activated by all stimuli. Thus, chemoattractants and other soluble stimuli appear to activate the same respiratory burst enzyme in PMN but they utilize different transductional mechanisms and are regulated differently.

Studies in normal and chronic granulomatous disease neutrophils indicate a correlation of tubulin tyrosinolation with the cellular redox state.

A specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN) is induced by the synthetic peptide chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), and this stimulation of tyrosinolation in PMN is completely inhibited in the presence of various reducing agents. Further studies to characterize the mechanism of stimulation of tyrosinolation in PMN have revealed that conditions that inhibited the respiratory burst in stimulated PMN, e.g., an anaerobic atmosphere, or addition of antioxidants such as cysteamine, azide, or 2,3-dihydroxybenzoic acid, also inhibited the peptide-induced stimulation of tyrosinolation in these cells. Moreover, the sulfhydryl reagent, N-ethylmaleimide, depressed tyrosinolation in resting PMN and completely inhibited the fMet-Leu-Phe-induced stimulation. In contrast, addition of diamide, which preferentially oxidizes cellular glutathione, significantly stimulated tyrosinolation both in resting and fMet-Leu-Phe-stimulated PMN. Furthermore, resting levels of tyrosinolation in seven patients with chronic granulomatous disease (CGD), whose oxidative metabolism is severely depressed, were 35-45% lower (P less than 0.01). Most strikingly, PMN from CGD patients failed to respond to fMet-Leu-Phe or the Ca2+-ionophore A23187, which also induced stimulation of tyrosinolation in normal resting PMN. Methylene blue normalized the depressed tyrosinolation in resting CGD PMN, although it did not increase tyrosinolation in stimulated PMN. These results are consistent with the idea that the characteristic activation of the oxidative metabolism and the associated changes in the redox state in stimulated PMN are coupled to the induction of stimulation of tubulin tyrosinolation in these cells.

Role of cell surface contact in the kinetics of superoxide production by granulocytes.

The complement-derived anaphylatoxin C5a and a putative analogue of bacterial chemotactic factor (N-formyl-methionyl-leucyl-phenylalanyl [fMLP]), as well as bacterial lipid A, all stimulate human granulocyte (PMN) adhesiveness and superoxide (O-2) production in a concentration-dependent manner. Since attachment of particulate matter to the PMN membrane is an early event in the triggering of respiratory burst of these cells, we further examined how adherence might modulate the release of O-2 induced by soluble mediators of inflammation. We found that both the quantity and kinetics of O-2 production depend on prior attachment of the cells to a surface. In stirred suspensions of PMN, fMLP induces only a short burst (2.5 min) of O-2 release associated with reversible PMN aggregation. The magnitude, but not the time course, of both these responses depend on the fMLP concentration. Unlike the short respiratory response of cells in suspension, PMN allowed to settle onto stationary petri dishes, then overlaid with fMLP, rapidly spread and attach to the surface where they remain and release O-2 throughout the 60-min test period. Prolonged O-2 release also follows fMLP stimulation in suspensions of PMN pretreated with cytochalasin B, in which case aggregation becomes irreversible during the 20-min burst. If fMLP is slowly infused into a suspension of cells at 37 degrees C or if PMN are challenged at 0 degrees C, and then warmed to 37 degrees C, O-2 release greatly decreases or becomes undetectable. Suspended PMN do not respond to a second challenge by the same stimulus regardless of the rate or temperature at which the first stimulus was added, a phenomenon formerly described as desensitization. However, if PMN challenged with fMLP in suspension undergo the short respiratory response and then are later placed in petri dishes, they adhere and resume production of O-2 without further stimulation. Chemotactic factor-induced adherence and O-2 release of PMN on a surface is entirely independent of either the mode of activation or prior O-2 release during preincubation in suspension. Human C5a also promotes PMN adherence and prolonged O-2 release in petri dishes. Furthermore, lipid A increases O-2 release and adherence of settled PMN, but fails to elicit either response from suspended PMN. These results indicate that cell surface contact plays an essential role in triggering the respiratory burst of PMN activated by soluble stimuli. This long-lasting O-2 release by chemotactic factor-stimulated PMN may play a significant role in inflammatory reactions when PMN become adherent in vivo.