Luminol-independent chemiluminescence by phagocytes is markedly enhanced by dexamethasone,not by other glucocorticosteroids

The effect of several glucocorticosteroids on the generation of reactive oxygen species (ROS) was examined. The ROS assessed were O ₂ ^– , H₂O₂, OH·, and chemiluminescence (CL) (determined in the presence or absence of luminol), generated by both opsonized zymosan-stimulated neutrophils or monocytes and by the xanthine-xanthine oxidase system. Except for luminol-independent CL, only high concentrations (10^–4 M) of steroids could decrease each ROS. In contrast, luminol-independent CL generation in the phagocyte system was increased in a dose-dependent manner by the addition of dexamethasone, but not by any other steroid. Further, in lymphocyte cultures stimulated with Con A for four days, luminol-independent CL generation was demonstrated and enhanced by the addition of dexamethasone, although CL generation was not detected in the absence of dexamethasone. These findings provide evidence that CL does not always represent light specific to ROS, and they suggest the possibility that dexamethasone induces emission of light at sites of inflammation.     

Comparison of the effects of antioxidant non-steroidal anti-inflammatory drugs against myeloperoxidase and hypochlorous acid luminol-enhanced chemiluminescence

The interaction of myeloperoxidase (MPO) with H₂O₂ and Cl^– provides a potent antimicrobial/cytotoxic system for polymorphonuclear leukocytes (PMNs). MPO-related cytotoxicity may be associated with the formation of toxic oxidant MPO intermediates, HOCl, or both. MPO itself is able to oxidize drugs and cellular components. Non-steroidal anti-inflammatory drugs (NSAIDs) able to act as antioxidant free radical scavengers have recently been shown to inhibit luminol-enhanced chemiluminescence (CL) which results from the MPO–H₂O₂–Cl^– reaction. CL is a measure of the activity of this reaction. At that time it was not clear whether the source of CL which these NSAIDs affected was HOCl or components of the initial MPO–H₂O₂–Cl^– reaction. A NSAID antioxidant mechanism could affect MPO oxidant intermediates and HOCl.This study compares the effects of antioxidant NSAIDs, methylprednisone and free radical scavengers against MPO-based and NaOCl-based luminol-enhanced CL. Most NSAIDs which affected both MPO and NaOCl-CL appeared to share similar mechanisms, suggesting that MPO oxidant internediates and HOCl are susceptible to NSAID effects. However, most NSAIDs were more effective against MPO-CL. The effect of these NSAIDs against MPO-CL followed the profile of NSAIDs effective in previous studies against PMN-CL. One exception to this was methylprednisone, which has no effect on PMN or MPO-CL, yet inhibited NaOCl-CL. This and other data suggest that MPO and not HOCl-related reactions are a major source of PMN-CL. Less effective NSAIDs affected NaOCl-CL better than MPO-CL. While both HOCl and MPO oxidant intermediates may be affected by NSAIDs, it appears that MPO oxidant intermediates or MPO itself are the primary target for NSAID antioxidant free radical scavenging mechanisms. These antioxidant effects impair the major killing system of the PMN and may be NSAIDs' primary anti-inflammatory mechanism. Although our data suggests the production of superoxide anion and hydroxyl radical from the MPO–H₂O₂–Cl^– reaction, the actual presence or involvement of these free radical species is not confirmed herein.     

Antioxidation theory of non-steroidal anti-inflammatory drugs based upon the inhibition of luminol-enhanced chemiluminescence from the myeloperoxidase reaction

The action of non-steroidal anti-inflammatory drugs (NSAIDS) has been ascribed to their ability to block the reaction of arachidonate with cyclooxygenase/peroxidase, thus inhibiting the cellular production of inflammation mediators such as prostaglandins and leukotrienes. However, this and other polymorphonuclear leukocyte (PMN) peroxidases such as myeloperoxidase (MPO) would still be capable of producing destructive oxidants which contribute to inflammation. Sulindac sulfide (Clinoril^® sulfide) has recently been shown to scavenge oxidant products of prostaglandin cyclooxygenase/peroxidase and MPO. The MPO–H₂O₂–Cl^– reaction is a potent antimicrobial/cytotoxic system which produces HOCl, a strong oxidant. MPO itself has the ability to oxidize drugs and cellular components, and may be the main oxidant in PMN defenses. An antioxidant/free radical scavenger action of NSAIDs against the MPO system could be a primary mechanism of their anti-inflammatory effects. Other antioxidant/free radical scavengers have anti-inflammatory effects.MPO activity has previously been quantified using chemiluminescence (CL). In this study, NSAIDs from various classes were tested for their ability to inhibit luminol-enhanced CL from MPO. The most potent NSAIDs against MPO-CL were BW755C, phenylbutazone, indomethacin and sulindac sulfide. Salicylates and arylacetic acid derivatives, such as naproxen, also decreased MPO-CL. These drugs are also effective against CL from PMNs, of which MPO may be a main source. This effect of NSAIDs on MPO suggests that NSAIDs may impair the killing mechanism of the PMN, preventing cell destruction and release of inflammation mediators. PMN MPO appears to be a target for the antioxidant/free radical scavenging effects of NSAIDs.     

Chemiluminescence in activated human neutrophils

Human neutrophils (PMNs) suspended in Hanks' balanced salt solution (HBSS), which are stimulated either by polycation-opsonized streptococci or by phorbol myristate acetate (PMA), generate nonamplified (CL), luminol-dependent (LDCL), and lucigenin-dependent chemiluminescence (LUCDCL). Treatment of activated PMNs with azide yielded a very intense CL response, but only a small LDCL or LUCDCL responses, when horse radish peroxidase (HRP) was added. Both CL and LDCL depend on the generation of Superoxide and on myeloperoxidase (MPO). Treatment of PMNs with azide followed either by dimethylthiourea (DMTU), deferoxamine, EDTA, or detapac generated very little CL upon addition of HRP, suggesting that CL is the: result of the interaction among H₂O₂, a peroxidase, and trace metals. In a cell-free system practically no CL was generated when H₂O₂ was mixed with HRP in distilled water (DW). On the other hand significant CL was generated when either HBSS or RPMI media was employed. In both cases CL was markedly depressed either by deferoxamine or by EDTA, suggesting that these media might be contaminated by trace metals, which catalyzed a Fenton-driven reaction. Both HEPES and Tris buffers, when added to DW, failed to support significant HRP-induced CL. Nitrilotriacetate (NTA) chelates of Mn²⁺, Fe²⁺, Cu²⁺, and Co²⁺ very markedly enhanced CL induced by mixtures of H₂O₂ and HRP when distilled water was the supporting medium. Both HEPES and Tris buffer when added to DW strongly quenced NTA-metal-catalyzed CL. None of the NTA-metal chelates could boost CL generation by activated PMNs, because the salts in HBSS and RPMI interfered with the activity of the added metals. CL and LDCL of activated PMNs was enhanced by aminotriazole, but strongly inhibited by diphenylene iodonium (an inhibitor of NADPH oxidase) by azide, sodium cyanide (CN), cimetidine, histidine, benzoate, DMTU and moderately by Superoxide dismutase (SOD) and by deferoxamine. LUCDCL was markedly inhibited only by SOD but was boosted by CN. Taken together, it is suggested that CL generated by stimulated PMNs might be the result of the interactions among, NADPH oxidase, (inhibitable by diphenylene iodonium), MPO (inhibitable by sodium azide), H₂O₂ probably of intracellular origin (inhibitable by DMTU but not by catalase), and trace metals that contaminate salt solutions. The nature of the salt solutions employed to measure CL in activated PMNs is critical.     

Lysosomal enzymes and metabolic activity of polymorphonuclear leukocytes from patients with systemic lupus erythematosus and from experimental animals after levamisole treatment

The activity of -d-glucuronidase (BDG) was lowered and the activity of myeloperoxidase (MPO) was elevated in polymorphonuclear leukocytes (PMNs) of SLE patients compared with normal subjects. After levamisole treatment, the activities of BDG, MPO, and lysozyme rose in PMNs of SLE patients. A higher activity of lysozyme was also observed in peritoneal PMNs of rabbits after levamisole administration. During incubation of human phagocytizing and non-phagocytizing PMNs, no effects of levamisole at concentrations of 10^–3 to 10^–5 M were observed on the release of lysosomal enzymes. These concentrations of levamisole also did not influence INT reductase activity and production of superoxide by normal human PMNs in the presence of zymosan particles. These findings suggest that levamisole might have an effect on the actual level of lysosomal enzymes in PMNs rather than on their release.     

Time course of superoxide generation by leukocytes—The MCLA chemiluminescence system

This study was performed to examine the pattern of Superoxide (O ₂ ^– ·) generation from leukocytes using the O ₂ ^– · specific chemiluminescence (CL) method.Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (MCLA) was used as a CL probe. The appropriate conditions of the MCLA method was first determined for the evaluation of the time course of O ₂ ^– · generation by leukocytes. The time course of O ₂ ^– · generation obtained by the MCLA-CL system was compared with that by the luminol-dependent CL, electron spin resonance (ESR)/spin trapping, and cytochromec systems. Following stimulation by three different stimulants (PMA, OZ, FMLP), leukocytes continuously generated O ₂ ^– · for up to 5 h in the MCLA-CL system, irrespective of the kind of stimulation. The curves obtained by generation ceased more rapidly in the luminol-CL, ESR/spin trapping, and cytochromec systems. A 50% activity of the initial value was observed at 70 min in the MCLA-CL system, but 30, 10 and 35 min in the other systems, respectively. The CL or O ₂ ^– · generation value decreased to less than 1% (possible termination) at 300, 90, 120 and 180 min, respectively. With the exception of ESR studies with OZ, the cell viability was not significantly affected in any of the trials. These results indicate that leukocytes can generate O ₂ ^– · much longer than previously estimated and that the MCLA-CL-system is the most suitable system for the measurement of the O ₂ ^– · generation by leukocytes.     

Alterations in complement-induced shape change and stimulus-specific superoxide anion generation by neonatal calf neutrophils

Increased susceptibility of neonates to infection may be related to defects in newborn neutrophil (PMN) functional activities, including altered responses to complement fragments (Cf) and defective microbicidal activity. We therefore compared the kinetics of newborn and adult bovine PMN membrane shape change responses following stimulation with zymosan-activated plasma (ZAP) as a source of Cf. Measurement of PMN membrane shape change was a rapid, sensitive, and reproducible measure of Cf stimulation within a population of PMNs; a maximum of 67–85% of the PMNs exhibited easily detectable membrane ruffling, lamellipodia formation, and polarity within 2 min. Newborn PMNs exhibited significantly increased (P<0.01) membrane shape change at 20, 30, 60, 120, and 300 sec after Cf stimulation. A maximum of 85.8±3.2% of newborn PMNs exhibited such C-finduced shape changes by 120 sec, which was significantly greater (P<0.01) than the maximum stimulation (67.7±4.3%) attained with adult PMNs. These data indicate enhanced kinetics of induced newborn PMN membrane shape change in response to Cf stimulation. We also compared stimulus-specific superoxide anion (O ₂ ^– ) generation as a measure of respiratory burst activity after incubation of new-born and adult PMNs with soluble (phorbol myristate acetate, PMA) and particulate (opsonized zymosan, OZ) stimuli. When PMA was used as the stimulus, newborn PMNs generated significantly less O ₂ ^– (9.3±0.5 nmol O ₂ ^– /10⁶ PMN,P<0.05) than did adult PMNs (12.4±0.3 nmol O ₂ ^– )/10⁶ PMN). This finding was reversed when OZ was used as the stimulus; newborn PMNs generated significantly more O ₂ ^– (7.7±0.4 nmol O ₂ ^– /10⁶ PMN,P <0.05) than did adult PMN (5.5 ±0.5 nmol O ₂ ^– /10⁶ PMN). These findings collectively document biochemical and morphological differences between newborn and adult PMNs as determined by stimulusspecific O ₂ ^– generation and C_f-induced membrane shape change. Such differences may be important to neonatal disease susceptibility.     

Ontogeny of inflammatory cell responsiveness

Newborn calves, like human infants, are uniquely susceptible to bacterial infections. Part of this increased susceptibility may be related to defects in newborn polymorphonuclear leukocyte (PMN) defensive functions. It remains unclear whether reported deficits in newborn PMN function represent maturational disorders or are manifestations of some form of perinatal suppression phenomenon. We therefore compared the ability of bovine newborn PMNs (less than 24 h old), newborn PMNs (7–10 days of age), fetal PMNs (210–220 days gestational age), and adult PMNs to generate superoxide anion (O ₂ ^– ) as an indicator of respiratory burst activity. Citrated biood was collected, and PMNs were isolated to greater than 95% purity and 98% viability. O ₂ ^– generation was measured as the superoxide dismutase-inhibitable (10 g/ml) reduction of ferricytochrome c (2 mg/ml) after activation of PMNs with phorbol myristate acetate (PMA, 2 g/ml) to directly stimulate protein kinase C. The reaction kinetics were measured (37°C, 550 nm) using a spectrophotometer and chart recorder for continuous monitoring. O ₂ ^– generation was measured for 5 min after the initial lag period and the total nanomoles of O ₂ ^– generated calculated using the extinction coefficient for ferricytochromec. Newborn PMNs (N=10) generated significantly less O ₂ ^– (5.7 ±0.8 nmol O ₂ ^– /10⁶ cells/5 min,P < 0.01) than did adult PMNs (N=14) (9.6 ±2.1 nmol O ₂ ^– /10¹⁰ cells/5 min) or fetal PMNs (N=4) (10.7 ±0.7 nmol O ₂ ^– /10⁶ cells/5 min). PMNs from 7-to 10-day-old calves (N=9) generated almost identical amounts of O ₂ ^– as newborn PMNs (5.7 ±1.6 nmol O ₂ ^– /10⁶ ceils/5 min). There was no difference in measured lag time period between new-born and adult PMNs, but fetal PMNs had significantly reduced (P < 0.01) mean lag time. The data indicated that bovine newborn PMNs have a decreased ability to generate O ₂ ^– in response to PMA stimulation, which persists for at least 7–10 days, and that this functional decrement may be a manifestation of some form of perinatal PMN suppression phenomenon rather than a developmental abnormality since fetal PMNs produced O ₂ ^– as well as adult PMNs.     

Eicosanoid production in rheumatoid synovitis

Leukotriene B₄ (LTB₄) synthesis in rheumatoid synovitis was studied using peripheral and synovial fluid polymorphonuclear leukocytes (PMNs) and rheumatic synovial lining cells. No differences were found in LTB₄ synthesis between peripheral PMNs from healthy volunteers and rheumatoid arthritis patients. When peripheral and synovial PMNs from the same RA patient were compared, arachidonic acidinduced LTB₄ synthesis in synovial fluid PMNs was increased 1.7–7.2 fold, whereas the response to Ca ionophore A23187 stimulation was similar. This suggests 5-lipoxygenase stimulating factor(s) in inflamed joints. Rheumatic synovial lining cells in a primary cell culture produced small amounts of LTB₄, the concentrations being less than 0.1 per cent of those of prostaglandin E₂ (PGE₂). PGE₂ synthesis in synovial cells was increased when arachidonic acid or interleukin-1 was added to the culture, whereas LTB₄ production remained unaltered. The present results suggest that in inflamed joints LTB₄ originates mainly from PMNs whereas synovial lining cells are the source for PGE₂.     

Role of myeloperoxidase in respiratory burst of human polymorphonuclear leukocytes

A comparative study of the respiratory burst [monitored as superoxide (O₂ ^–) production] of normal and myeloperoxidase (MPO) -deficient poiymorphonuclear leukocytes (PMNs) was carried out on 11 MPO-deficient subjects that represent the largest sample of this kind ever studied. The rate of O₂ ^– production by isolated PMNs and whole blood from normal and MPO-deficient subjects was comparable during the initial 30–40 min of incubation with serum-treated zymosan (STZ). Afterwards, the amount of O₂ ^– produced became progressively higher in MPO-deficient cells at least until 120 min incubation with STZ. On the contrary the rate of O₂ ^– production by both cell types in response to 4--phorbol-12-myristate-13-acetate (PMA) was the same. The PMNs of four MPO-deficient subjects were tested for their ingestion ability by counting the number of ingested particles on toluidine blue-stained sections of epoxy-embedded PMN suspensions. Both cell types ingested STZ particles at a comparable rate at early postphagocytic times, whereas on prolonged incubation MPO-deflcient PMNs ingested more STZ particles than normal PMNs. These results suggest that the ingestion capacity of normal cells may undergo a more rapid deterioration than that of MPO-deficient cells during incubation with STZ. Evidence for a higher deterioration of normal PMNs with respect to MPO-deficient PMNs was obtained also from studies on the effect of storage on O₂ ^– generation. After standing at melting ice temperature for 3 h, normal PMNs produced less O₂ ^– than MPO-deficient PMNs in response to PMA, and the difference in O₂ ^– production by the two cell types in response to STZ was evident at earlier postphagocytic periods than with freshly isolated cells. Taken all together these results suggest that normal PMNs and MPO-deficient PMNs do not intrinsically differ in O₂ ^– generating potential and that the difference in the respiratory burst observed during phagocytosis may be accounted for by a more marked deterioration, in normal PMNs, of one or more functions related to the respiratory burst.     

Neutrophil responses to intravascular pneumococcal sonicate

Leukopenia and pulmonary leukostasis are prominent features in patients succumbing to pneumococcal (PNC) infections. We examined mechanisms involved in recruitment of polymorphonuclear neutrophils (PMNs) into pulmonary capillaries and alveolae after PNC sonicate injection. We showed that by 15 min postinjection, PMN chemotactic activity was found in bronchoalveolar lavage (BAL) fluids and increased with time until the end point of the study at 90 min. Accompanying the increased chemotactic activity in BAL fluids was a decrease in circulating PMNs more pronounced in the femoral artery (FA) then the pulmonary artery (PA). Super-oxide anion (O ₂ ^– ) production by peripheral PMNs was depressed following PNC sonicate injection, and comparison of FA and PA showed that FA PMNs produced less O ₂ ^– than PA PMNs. PA PMNs also showed enhanced random migration when compared to the depressed random migration of FA PMNs. This study demonstrated that an intravascular challenge of PNC sonicate was associated with increased chemotactic activity for PMNs in BAL fluid. Fewer PMNs and altered PMN function resulted from passage through the pulmonary microvasculature after PNC sonicate injection.Supported in part by USDHHS grant CA 20819 from the National Cancer Institute and from the Veterans Administration Research Service.     

Lipoteichoic acid-antilipoteichoic acid complexes induce superoxide generation by human neutrophils

Human neutrophils (PMNs) which have been incubated with lipoteichoic acid (LTA) from group A streptococci generated large amounts of Superoxide (O ₂ ^– chemiluminescence and hydrogen peroxide when challenged with anti-LTA antibodies. Cytochalasin B further enhanced O ₂ ^* generation. The onset of Of generation by the LTA-anti-LTA complexes was much faster than that induced by BSA-anti-BSA complexes. LTA-treated PMNs generated much less O ₂ ^* when challenged with BSA complexes, suggesting that LTA might have blocked, nonspecifically, some of the Fc receptors on PMNs. PMNs treated with LTA-anti-LTA complexes further interacted with bystander nonsensitized PMNs resulting in enhanced Of generation, suggesting that small numbers of LTA-sensitized PMNs might recruit additional PMNs to participate in the generation of toxic oxygen species. Protelolytic enzyme treatment of PMNs further enhanced the generation of O ₂ ^– by PMNs treated with LTA-anti-LTA. Superoxide generation could also be induced when PMNs and anti-LTA antibodies interacted with target cells (fibroblasts, epithelial cells) pretreated with LTA. This effect was also further enhanced by pretreatment of the target cells with proteases. PMNs incubated with LTA released lysosomal enzymes following treatment with anti-LTA antibodies. The amounts of phosphatase,-glucoronidase,N-acetylglucosaminidase, mannosidase, and lysozyme release by LTA-anti-LTA complexes were much smaller than those released by antibody or histone-opsonized streptococci, suggesting that opsonized particles are more efficient lysosomal enzyme releasers. However, since the amounts of O ₂ ^– generated by the LTA complexes equaled those generated by the opsonized particles, it is assumed that the signals for triggering a respiratory burst and lysosomal enzyme secretion might be different.Supported by a research grant from Dr. S. M. Robbins of Cleveland Ohio, and by grants HL-288442, HL-31963 and GM-29507, from the National Institutes of Health, Bethesda, Maryland, and by grant IM-432 from the American Cancer Society.     

Decreased production of nitric oxide by human neutrophils during septic multiple organ dysfunction syndrome

The objective of this study was to determine nitric oxide (NO) and superoxide anion release (O ₂ ^– ) by neutrophils (PMNs) in the septic multiple organ dysfunction syndrome (MODS) and to compare them with the response of normal cells to lipopolysaccharide (LPS) and cytokines. NO production was measured by the release of nitrites in the medium, its maximal production rate by a modified oxyhemoglobin assay and O ₂ ^– by standard methods. Normal cells were incubated with LPS, gamma interferon (IFN-), or tumor necrosis factor (TNF-) alone or in combination. Results showed that PMN release of both NO and O ₂ ^– was reduced in septic samples; in contrast, an association of LPS, IFN-, and TNF- promoted maximal NO release by normal cells (40–50%). We conclude that while interaction of normal PMNs with cytokines increases NO and O ₂ ^– release, progression of sepsis to a multiple organ dysfunction impairs these responses in both functions.     

Influence of neutrophil cationic proteins on generation of superoxide by human polymorphonuclear cells during phagocytosis

The cationic proteins from neutrophyl lysosomes have been shown to modulate phagocytic activity of granulocytes. The present study reports the effects of the cationic protein fractions on the generation of O ₂ ^– by human PMNs during phagocytosis. Human PMNs were reacted win different phagocytic stimuli in the presence and absence of lysosomal cationic proteins and the amount of O ₂ ^– generated was determined by superoxide dismutase inhibitable reduction of cytochromec. Total cationic protein extract from neutrophil lysosomes enhanced O ₂ ^– generated by PMNs during the phagocytosis of IgG-coated latex beads and opsonized zymosan particles. The analysis of the fractions of cationic proteins obtained from a Sephadex G-75 column showed that the O ₂ ^– generation-enhancing activity was associated with the proteins eluted in fractions III and IV. A protein fraction mainly eluted in void volume inhibited the cytochromec reduction by O ₂ ^– formed during phagocytosis. This was due to the presence of superoxide dismutase-like activity since O ₂ ^– generated by the xanthine-xanthine oxidase system was also inhibited by this fraction. The cationic protein fractions III and IV from the Sephadex G-75 column were further subfractionated. Although the O ₂ ^– -enhancing activity was eluted in the same fractions as chymotrypsin activity, there was no quantitative correlation between the amount of O ₂ ^– generation and chymotrypsin activity. Moreover, commercial chymotrypsin did not enhance O ₂ ^– generation. Electrophoretic analysis of the isolated protein fractions suggests that O ₂ ^– generation enhancing protein (SGEP) is different from lysozyme or chymotrypsin and probably represents previously undescribed protein.     

Polyl-histidine

Poly-l-histidine (PHSTD) of molecular weight 26,000 induced the generation of large amounts of Superoxide (O ₂ ^– ) and hydrogen peroxide (H₂O₂) in human neutrophils (PMNs). Despite its low solubility at neutral pH, PHSTD was bound very rapidly to the PMN surfaces. Maximal generation of O ₂ ^– took place with 4–5 ×10^–6 M of PHSTD, starting after a lag of about 25 sec and proceeding for 15–17 min at a rate of 150 nmol/10⁷ PMNs/min, suggesting that this polycation is one of the most potent stimulators of O ₂ ^– generation known, PHSTD was found to be non-toxic for PMNs even at millimolar concentrations. Generation of O ₂ ^– by PHSTD depended on extracellular calcium; it was inhibited by calcium channel blockers and by trifluoperazine, and it triggered a sharp rise in intracellular calcium as determined by the Quin 2 fluorescence technique. The generation of both O ₂ ^– and H₂O₂ by PHSTD was partially inhibited by cytochalasin B or (CYB, CYE). On the other hand, CYB markedly enhanced the generation of both O ₂ ^– and H₂O₂ following stimulation of PMNs either by PHSTD, polyarginine, histone, or by antibody-opsonized group A streptococci. Electron microscopic analysis and NBT reduction tests revealed that both PHSTD and PHSTD-opsonized streptococci were avidly phagocytosed by PMNs. Since CYB totally inhibited internalization of both PHSTD and the PHSTD-opsonized streptococci, it was suggested that these agents stimulated oxygen radical generation mainly on the leukocyte surfaces. Complexes (CX) formed between PHSTD and polyanethole sulfonate (a strong polyanion) or between histone and the polyanion mimicked immune CX in their ability to trigger the generation of large amounts of O ₂ ^– which were inhibited by CYB. Generation of O ₂ ^– and chemiluminescence either by PHSTD or by PHSTD-opsonized streptococci were markedly inhibited by poly-l-glutamate, suggesting that PHSTD acted as a cationic agent which interacted via electrostatic forces with some negatively charged sites in the leukocyte membrane. Generation of H₂O₂ by PHSTD was also markedly inhibited by deoxyglucose, KCN, DASA, as well as by the lipoxygenase inhibitors nordihydroguaiaretic acid, phenidone, and propylgallate. On the other hand, cyclooxygenase inhibitors such as aspirin, indomethacin, and piroxicam were inactive, suggesting that arachidonic acid metabolism via lipoxygenase pathway might have been involved in the activation by PHSTD of the NADPH oxidase in PMNs. PHSTD may mimic the effects of antibodies both as an opsonin and as a potent stimulator of the respiratory burst in PMNs and may thus serve as a model for further study of leukocyte-bacteria interactions in infectious and inflammatory sites and of the pathogenicity of immune complexes.Supported by a research grant from Dr. S. M. Robbins of Cleveland, Ohio.     

Priming of human neutrophil functions by tumor necrosis factor: Enhancement of superoxide anion generation,degranulation, and chemotaxis to chemoattractants C5a and f-Met-Leu-Phe

Recombinant human tumor necrosis factor- (rTNF) stimulated increased generation of Superoxide anion (O ₂ ^– ) by human neutrophils in a concentration-dependent fashion. Preincubation of human neutrophils with rTNF (2.2–2200 units/ml) for 10 min enhanced the subsequent generation of O ₂ ^– in response to C5a and f-MetLeu-Phe(FMLP). Recombinant TNF did not enhance O ₂ ^– generation by neutrophils stimulated with phorbol myristate acetate (PMA). Recombinant TNF alone failed to induce release of myeloperoxidase (MPO) and lysozyme by neutrophils. However, it did enhance the release of MPO and lysozyme by neutrophils stimulated with C5a and FMLP, but not with PMA. Although rTNF alone (0.001–50,000 units/ml) was not chemotactic for neutrophils, preincubation of neutrophils with rTNF (0.001–0.1 units/ml) enhanced the chemotactic activity of suboptimal concentrations of C5a (0.1 nM) and FMLP (5 nM). Neutrophils treated with high concentrations of rTNF (100–10,000 units/ml) showed inhibition of random movement and of chemotaxis induced by C5a or FMLP. We conclude from these studies that rTNF primes neutrophils for enhanced responses to subsequent stimuli and thus may augment the inflammatory response by increased oxidant production and lysosomal enzyme release and promote down-regulation of chemotactic movement.     

Patients with adult respiratory distress syndrome (ARDS) demonstrate in vivo neutrophil activation associated with diminished binding of neutrophil-specific monoclonal antibody 31D8

Neutrophils (PMNs) from patients with adult respiratory distress syndrome (ARDS) were assessed for light scattering, membrane potential, and phagocytic responses using fluorescent probes and flow cytometry to evaluate individual cells. Qualitative and quantitative oxidant responses were measured by nitroblue tetrazolium (NBT) and cytochromec reduction assays, respectively. The results were correlated with the proportion of cells binding the PMN subset-specific monoclonal antibody 31D8. Despite an increased forward scatter signal (4.3±1.6 vs. 1.3±1.1 ARDS vs. control,P=0.041) and spontaneous NBT test (12.6±4.7% vs. 2.5 ±0.8% positive, ARDS vs. control,P=0.033) indicating in vivo priming of ARDS PMNs, there were no significant differences between ARDS and control PMNs in assays of stimulated membrane potential, NBT, and O·₂ ^– production or phagocytosis. However, positive correlations between the degree of prestimulus forward light scatter and subsequent O·₂ ^– production to FMLP (r=0.673,P=0.006) and between the percentage of bands and the O·₂ ^– response to PMA (r=0.660,P =0.003), suggest that the great variability of the ARDS PMN functional responses may relate to varying degrees of in vivo cell priming and/or deactivation. ARDS PMNs demonstrated a significantly lower percentage of 31D8 positive cells (73.4 ±7.5% vs. 94.5±1.6%,P=0.012) and a lower level of 31D8 staining when compared to normals (60.1±10.4% of control level,P=0.001). The lower 31D8 expression did not directly correlate with any functional parameter tested or with the proportion of immature cells. However, patients receiving an intravenous PGE₂1-infusion demonstrated a significant increase in 31D8 staining relative to controls and inhibition of PMA-stimulated O·₂ ^– production. The data suggest that the function of PMNs from ARDS patients varies widely and reflects great in vivo variation in cell priming. While the mechanism responsible for the lowered expression of the 31D8 antigen and its apparent modulation by PGE₁ is unknown, 31D8 may be an indirect marker for in vivo stress factors that regulate the preferential release of a structurally distinct PMN subset from the bone marrow.This work was supported in part by NIH grant P30-DK35747, a University of California, Davis, Dean's Research Grant, and The Upjohn Company.     

Comparison of group I and II soluble phospholipases A2 activities on phagocytic functions of human polymorphonuclear and mononuclear phagocytes

Soluble phospholipase A₂ (PLA₂) purified from rheumatoid synovial fluid (group II) and repurifiedNaja naja venom PLA₂ (group I) were compared for their influence on phagocytic activity of human polymorphonuclear (PMN) and mono-nuclear (MO) phagocytes. Group II PLA₂ reduced chemotaxis, adhesiveness, and intracellular bactericidal activity (ICBA) and induced release of muramidase from PMNs. Group I PLA₂ suppressed chemotaxis, and enhanced ICBA but had no influence on other phagocytic functions. Group II PLA₂ purified from synovial fluid or from placenta caused marked spontaneous superoxide generation followed by inhibition of phagocytosis-induced burst of energy. Group INaja naja and porcine pancreatic PLA₂ had no effect on superoxide generation. Group II but not group I PLA₂ reduced markedly ICBA of monocytes. It may be concluded that human group II soluble PLA₂, in concentrations comparable to those present in inflamed joints or in sera of patients with active arthritis or septic shock, causes spontaneous formation of the oxygen radical superoxide and release of lysosomal enzymes, and suppresses conventional phagocytic activities of PMNs and monocytes. Marked differences between group I and group II PLA₂s may mean that these enzymes exert different influences on cell membrane.     

Effects of histamine agonists and antagonists on luminol-dependent chemiluminescence of granulocytes

Histamine inhibited luminol-dependent chemiluminescence (CL) of granulocytes in a dose-dependent manner, with an ID₅₀ of about 3×10^–5 M. Dimaprit, a selective H₂-agonist, produced a histamine-like effect. Furthermore, cimetidine, ranitidine, and TZU 0460, which are selective H₂-antagonists, but not mepyramine, a selective H₁-antagonist, blocked the inhibitory effect of histamine on CL. Thus it may be concluded that the inhibitory effect of histamine is mediated via histamine H₂-receptors. H₁- and H₂-antagonistsper se, except at extremely high concentrations, had no effect on CL of granulocytes.     

Effect of piroxicam therapy on granulocyte function and granulocyte elastase concentration in peripheral blood and synovial fluid of rheumatoid arthritis patients

The effect of piroxicam therapy (20 mg/day for 15 days) on various polymorphonuclear granulocyte (PMN) responses and on PMN elastase concentration was investigated in nine patients with active rheumatoid arthritis. Peripheral biood and synovial fluid samples were collected before starting therapy and 12 h after the last dose of the drug. All patients were evaluable for peripheral blood analysis and six for synovial fluid analysis. Piroxicam therapy had no effect on PMN random migration and phagocytosis, while it significantly reduced both FMLP-induced aggregation and FMLP-induced chemotaxis. This seems mainly due to an effect on FMLP binding, as no differences were observed after therapy in PMA- and PHA-induced aggregation as well as in serum-induced chemotaxis. In contrast, a marked impairment of NBT test and PMA- and FMLP-induced superoxide anion (O₂ ^–) production was found after piroxicam therapy. This effect was as evident in peripheral blood as in synovial fluid PMN. Also, a significant reduction in synovial fluid PMN number and synovial fluid PMN elastase concentration (elastase- ₁-proteinase complex) was found after treatment. It is concluded that piroxicam may act at different sites on various PMN responses-Its effect on O₂ ^– generation and PMN elastase concentration in synovial fluid may have an important role in reducing destruction of arthritic joint tissue.