Altered function of synovial fluid granulocytes in patients with acute inflammatory arthritis

In rheumatoid arthritis (RA) a chronic inflammatory state exists in which the synovial fluid is periodically filled with large numbers of polymorphonuclear leukocytes (PMNs). Oxygen radicals produced by these cells have been implicated as mediators of tissue damage and may be directly involved in the pathogenesis of RA. We examined the production of oxygen radicals by synovial fluid PMNs (SFPMNs) and peripheral blood PMNs (PB-PMNs) by measuring chemiluminescence (CL) as well as Superoxide anion (O ₂ ^– ) release. Increased spontaneous CL in the presence of luminol and increased CL in response to phorbol myristate acetate (PMA) was observed in SF-PMNs when compared to PB-PMNs. When zymosan was used as the stimulus in the absence of luminol, a slightly lower CL response was observed in SF-PMNs as compared to PB-PMNs. No significant differences were observed in the generation of O ₂ ^– generation with any stimulus. Preincubation of normal PBPMNs in 10% synovial fluid enhanced the luminol-dependent spontaneous and PMA-stimulated CL as well as zymosan-stimulated CL. When O ₂ ^– release from normal PB-PMNs pretreated with 10% synovial fluid was compared to untreated controls, enhancement of spontaneous O ₂ ^– release was observed. PMA- and zymosan-stimulated responses did not differ significantly from controls. Increased spontaneous and PMA-stimulated release of myeloperoxidase (MPO) was also observed in normal PB-PMNs pretreated with synovial fluid. These findings may explain the increased luminol-dependent CL since this type of CL requires the presence of MPO. Our findings suggest that the enhanced chemiluminescence observed in normal PMNs treated with synovial fluids may be related to increases in spontaneous O ₂ ^– generation and myeloperoxidase release. Increased MPO release may account for enhanced CL observed in SF-PMNs.     

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.     

Endothelin-1 does not prime polymorphonuclear leukocytes for enhanced production of reactive oxygen metabolites

The in vitro effect of endothelin-1 (ET-1) on the capacity of polymorphonuclear leukocytes (PMNLs) to generate reactive oxygen species (ROS) was investigated. Human PMNLs were separated from healthy volunteers and preincubated for 10 min. at 37°C with varying concentrations (10^–7–10^–12 M) of ET-1. After subsequent stimulation with FMLP (10^–7 M) or opsonized zymosan (0.5 mg/ ml) the intra- and extracellular generation of ROS was assessed by luminol-amplified chemiluminescence, superoxide radical (·O ₂ ^– ) and hydrogen peroxide (H₂O₂) production.Results: ET-1 alone failed to stimulate ROS generation. Neither the capacity for extracellular generation of oxygen metabolites nor the production of ROS with an intracellular origin was changed after preincubation of PMNLs with ET-1. ET-1 did not cause a shift of the ·O ₂ ^– /H₂O₂ production ratio after stimulation of PMNLs with FMLP. These findings suggest that ET-1 in vitro does not prime human PMNLs for enhanced production of ROS.     

Properties of calcium ionophore-induced generation of superoxide anion by human neutrophils

Exposure of human neutrophils to the calcium ionophore, A23187, eventuates in a time- and concentration-dependent generation of Superoxide anion (O ₂ ^– ) in the presence but not absence of extracellular calcium. The selective requirement for calcium is demonstrated by the observation that magnesium caused a dose-related inhibition of A23187-stimulated O ₂ ^– generation. Preincubation of neutrophils with cytochalasin B prior to their interaction with A23187 results in a significant enhancement of O ₂ ^– production. The activity of the O ₂ ^– -generating system was maximum at 37°C and was significantly curtailed at lower and higher temperatures. A23187-induced O ₂ ^– generation was inhibited by the sulfhydryl reagents.N-ethyl maleimide (NEM) and iodoacetic acid (IA), and by the metabolic inhibitor 2-deoxy-D-glucose (2-DG) in the absence of glucose and cytochalasin B. Cyanide was inactive. Therefore, A23187 represents a useful pharmacologic probe for investigating the divalent cation and metabolic requirements of the neutrophil O ₂ ^– -generating system.     

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.     

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.     

CL 316,243, a selective β3-adrenergic agonist, inhibits protein breakdown in rat skeletal muscle

The in vitro effect of CL 316,243 (CL), a selective ₃-adrenoceptor agonist in the rate of overall proteolysis, the activity of proteolytic systems (lysosomal, Ca²⁺-dependent, ATP-dependent, and ATP-independent) and in the process of protein synthesis was investigated in rat skeletal muscles. The rate of overall proteolysis in soleus muscle from rats incubated with CL (10^–4 and 10^–5 M) or epinephrine (10^–5 M) was significantly decreased. In vitro rates of maximal activity of Ca²⁺-dependent proteolysis in soleus muscles were decreased by about 41% in the presence of 10^–5 M CL. No change was observed in the activities of the lysosomal, ATP-dependent or ATP-independent proteolytic systems. The anti-proteolytic effect of CL or epinephrine was partially prevented by 10^–5 M SR 59230A, a selective ₃-adrenoceptor antagonist. The increase of proteolysis induced by food deprivation in soleus was abolished by in vitro addition of 10^–5 M CL. No change in proteolysis was observed in extensor digitorum longus (EDL) muscles incubated with any concentration of the ₃-adrenoceptor agonist tested. Rates of protein synthesis were not affected by 10^–4 M CL neither in soleus nor EDL. The data suggest that a ₃-adrenoceptor-mediated inhibition of Ca²⁺-dependent proteolysis participates of the antiproteolytic effect of catecholamines in oxidative muscles.     

Dissociation of phagocytosis,metabolic stimulation and lysosomal enzyme release in human leukocytes

In this paper studies are reported concerning the relationship between particle binding to the plasma membrane of human polymorphonuclear leukocytes (PMN's), phagocytosis, generation of oxidative metabolites, and the release of lysosomal enzymes by these cells. Superoxide (O ₂ ^– ) generation by, and lysosomal enzyme release from normal PMN's and cytochalasin B-treated cells were measured. We have found that neither phagocytosis nor lysosomal degranulation are prerequisites for enhanced O ₂ ^– generation. Cytochalasin B-treated PMN's, incapable of ingesting particles but still able to bind particles to membrane receptors, generated enhanced amounts of O ₂ ^– when treated with serum-treated zymosan (STZ), a C3b receptor stimulus, or with aggregated IgG (agg IgG), an Fc receptor stimulus. Moreover, the soluble stimulators complement component C5a, phorbol myristate acetate (PMA), and calcium ions in the presence of the ionophore A23187, also increased the O ₂ ^– production of these cells. In all cases a time and dose-dependent stimulation was found of both the O ₂ ^– generation and the lysosomal enzyme release, but there was no correlation between ability of any stimulus to provoke enzyme release and its ability to stimulate O ₂ ^– generation. When PMN's were preincubated with 5×10^–4 M hydrocortisone-Na-succinate, lysosomal enzyme exocytosis with the immune reactants was inhibited 16–35%. Hydrocortisone also inhibited O ₂ ^– generation, except when STZ was used as the stimulus. Thus, in the case of stimulation of functional processes of PMN's via the C3b receptor, hydrocortisone inhibits membrane fusion without interfering with one of the early biochemical events (O ₂ ^– production).Financially supported by a travel grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Inhibition by nonsteroidal antiinflammatory drugs of luminol-dependent human-granulocyte chemiluminescence and [3H]FMLP binding

A system is described to evaluate for nonsteroidal antiinflammatory drugs by means of luminol-dependent human-granulocyte chemiluminescence (CL) is described. The CL is produced using either opsonized zymosan (yeast cells) or the soluble chemotactic peptide f-Met-Leu-Phe as the perturbant of the granulocyte membrane. Using either system, the following drug effects 2×10^–5 M were noted: only sulindac sulfide, and not sulindac sulfone or sulindac, displayed marked inhibition of chemiluminescence, following the in vivo data regarding inflammatory effects. The 5-OH indomethacin metabolite was likewise inactive as an inhibitor of CL mirroring in vivo effects. MK(+)410, MK(–)830 and MK835 all showed approximately 50% inhibition of CL, displaying deviation from in vivo data. MK(+)830 markedly stimulated CL, 4–6 times the control (without drug), which is clearly different from its enantiomer, MK(–)830. The reasons for this behavior are unclear. However, receptor binding studies with [³H]FMLP were accomplished in the presence and absence of the various drugs at 2×10⁵ M that were effective inhibitors of chemiluminescence (CL). Indomethacin, MK(–)830 and MK(+)410 had equivalent percent control binding and percent control CL. Sulindac sulfide and MK(+)835 both had higher percent control binding than percent control CL, with MK(+)835 displaying apparent increased numbers of available receptors relative to control. MK(+)830, which produces large increases in CL, produced a minor effect on percent control binding. A direct relationship between binding and CL does not exist with each drug. Chemiluminescence is dependent on ion movement and oxidative metabolism and is a secondary event to agonist-receptor occupation.     

Unique characteristics of superoxide production by human eosinophils in eosinophilic states

Eosinophils from patients with peripheral blood eosinophilia and human neutrophils from normal subjects and patients with neutrophilia produced superoxide anion (O₂ ^–) in vitro at similar rates in the absence of stimulation and exhibited comparably increased rates of O₂ ^– production during the initial 1 h of incubation with opsonized zymosan. In the presence of opsonized zymosan, the rate of O₂ ^– production by eosinophils was constantly high for 3 h, whereas the rate of production by neutrophils fell by more than 65% after 1 h. Consequently, the amount of superoxide produced by phagocytizing leukocytes was twofold higher for eosinophils than for neutrophils at 3 h. O₂ ^– production by cell-free sonicates of zymosan-stimulated eosinophils and neutrophils exhibited the same preference for NADPH over NADH. One mM sodium azide significantly decreased the generation of O₂ ^– by phagocytizing eosinophils, but lacked an effect on neutrophils. The prolonged release of O₂ ^– by eosinophils engaged in phagocytosis may contribute both to their unique microbicidal profile and to the capacity of eosinophils to injure host tissues in some eosinophilic syndromes.     

Kojic acid scavenges free radicals while potentiating leukocyte functions including free radical generation

The effects of kojic acid, a compound that suppresses melanogenesis and is widely consumed in the Japanese diet with the belief that it is beneficial to health, were investigated on several aspects of leukocyte function. Kojic acid significantly decreased the levels of reactive oxygen species (ROS) (O ₂ ^– ), H₂O₂, OH) generated by neutrophil and by a cell-free ROS-generating system. In contrast, it significantly enhanced neutrophil phagocytosis and ROS generation, and lymphocyte proliferation stimulated by phytohemagglutinin. In addition, calcium concentration, [Ca²⁺]i in human neutrophils was increased in the presence of kojic acid. These results suggest that kojic acid is a favorable agent in terms of host defense in that it enhances a number of activities of leukocytes, but scavenges ROS excessively released from cells or generated in the tissues or blood vessels that are potentially injurious to host tissues.     

Dexamethasone increases ROS production and T cell suppressive capacity by anti-inflammatory macrophages

Macrophages have been demonstrated to suppress T cell responses by producing reactive oxygen species (ROS) leading to the subsequent induction of T regulatory cells in a ROS-dependent manner. Macrophages may therefore be instrumental in downregulating T cell responses in situations of exacerbated immune responses. Here we investigated the effect of immunosuppressive drugs on ROS production by macrophage subsets and the subsequent effects on T cell activation. Macrophage types 1 and 2 were differentiated with GM-CSF or M-CSF, in presence or absence of dexamethasone, cyclosporine A, FK506, rapamycin, or mycophenolic acid. The ROS producing capacity of fully differentiated Mph was highest in anti-inflammatory Mph2 and not affected by exposure to immunosuppressive drugs. However, presence of rapamycin during Mph2 differentiation decreased the ROS production of these cells. In contrast, other immunosuppressive drugs, with dexamethasone being the most potent, increased the ROS producing capacity of Mph2. Intriguingly although the ROS producing ability of Mph1 was unaffected, dexamethasone strongly increased the ROS producing capabilities of dendritic cells. Both at the mRNA and protein level we found that dexamethasone enhanced the expression of NOX2 protein p47^phox. Functionally, dexamethasone further enhanced the capacity of Mph2 to suppress T cell mediated IFN-γ and IL-4 production. In vivo, only in rats with normal ROS production (congenic DA.Ncf1^E3/E3) it was observed that dexamethasone injection resulted in long-lasting upregulation of ROS production by macrophages and induced higher levels of Treg in a ROS-dependent manner. In conclusion, we show that the anti-inflammatory drug dexamethasone increases the ROS producing capacity of macrophages.     

Stimulation of neutrophil oxidative metabolism by indomethacin

Human neutrophils exposed to indomethacin demonstrate an enhanced capacity for superoxide ion (O ₂ ^– ) generation when stimulated with opsonized zymosan. Enhancement is not seen with indomethacin-treated cells exposed to solube oxidative stimuli. To further investigate this phenomenon, O ₂ ^– generation, chemiluminescence, and phagocytosis were assessed in human neutrophils preincubated with indomethacin. Zymosan-stimulated O ₂ ^– release was increased from 150 to 300% of controls in neutrophils exposed to 400 g/ml. indomethacin. Enhancement was not reversed by removal of indomethacin from the medium prior to addition of the stimulus and was dose-dependent at drug concentrations of 5 to 400 /ml. Neutrophils exposed to methacin alone also generated more O ₂ ^– than control cells, although this increment was not sufficient to account for the degree of enhancement seen when indomethacintreated cells were exposed to zymosan. Neutrophil cehmiluminescence induced by zymosan was also increased by exposure to indomethacin, and at a drug concentration of 400 g/ml (1.1 mM), enhancement randed from 253 to 333% of controls. As was observed with O ₂ ^– generation, chemiluminescence of neutrophils was increased in the presence of indomethacin alone, although, to a degree far less than was seen when drug-treated cells were stimulated with zymosan. Phagocytosis of radiolabeledS. aureus by neutrophils incubated with indomethacin was increased 13±5% over controls (P<0.01,n=5), but was unaltered by incubation of cells with the buffer used to solubilize the drug. The modest degree of enhancement of phagocytosis suggests that increased particle uptake is not the sole mechanism of oxidative enhancement. The data are in keeping with the hypothesis that indomethacin has a direct effect on the neutrophil plasma membrane and/or the O ₂ ^– -forming oxidase.     

Intracellular pH during secretion in the perfused rabbit mandibular salivary gland measured by31P NMR spectroscopy

Intracellular pH (pH_i) was measured in the isolated, perfused rabbit mandibular salivary gland by³¹P NMR spectroscopy. In the unstimulated gland perfused with HCO ₃ ^– /CO₂-buffered Ringer's solution, pH_i was 7.27±0.01. Continuous stimulation with acetylcholine elicited dose- and time-dependent changes in pH_i. 10^–6 mol/l acetylcholine caused a brief intracellular acidosis (–0.19±0.06 pH units) followed by an increase in pH_i to a more alkaline steady-state value (7.33±0.02). In the absence of perfusate HCO ₃ ^– or in the presence of 10^–4 mol/l DIDS (4,4-diisothiocyanatostilbene-2,2-disulphonic acid), the transient acidosis was abolished and pH_i increased rapidly to give a sustained alkalosis (7.49±0.03 and 7.44±0.03 respectively). In the presence of 10^–3 mol/l amiloride, the response to acetylcholine was a rapid decrease in pH_i to 7.02±0.02. The data suggest that, during perfusion with HCO ₃ ^– /CO₂-buffered solutions, stimulation with acetylcholine results in a transient loss of HCO ₃ ^– from the acinar cells (causing a transient acidosis), and, independently, the activation of Na⁺–H⁺ exchange (causing a sustained alkalosis). In the unstimulated gland, DIDS and the HCO ₃ ^– -free perfusate caused decreases in pH_i to 7.12±0.02 and 7.04±0.01 respectively. In contrast, amiloride had little effect. The relatively high value of pH_i maintained by the unstimulated gland is therefore probably not due to Na⁺–H⁺ exchange.     

AlF 4 − and vanadate stimulate chloride secretion in rabbit colon by a Ca2+-dependent mechanism

Stripped rabbit descending colon mucosae were studied in vitro in modified Ussing chambers to determine the effects of AlF ₄ ^– and vanadate on Cl^– transport. Serosal additions of AlF ₄ ^– and vanadate increase short-circuit current (I _SC) and tissue conductance, while luminal addition of the agents is ineffective. Addition of aluminium potentiates the effect of NaF on I _SC. AlF ₄ ^– and vanadate increase serosal-to-mucosal flux of ³⁶Cl without affecting mucosal-to-serosal flux. The effects of these agents on I _SC are markedly inhibited by serosal addition of bumetanide and depend on the presence of Na⁺ in the serosal bathing solution. The effects of AlF ₄ ^– and vanadate on I _SC are dependent on the presence of Ca²⁺ in the bathing solution, and are completely inhibited by indomethacin, but the effect of forskolin is not affected by the removal of Ca²⁺ from the bathing solution and the addition of indomethacin. AlF ₄ ^– and vanadate significantly increase the level of inositol phosphate metabolites. The results indicate that AlF ₄ ^– and vanadate increase Cl^– secretion in the rabbit colon via an increase in prostaglandin synthesis which is mediated by the increase of intracellular Ca²⁺ concentrations.     

Histaminergic receptors modulate the coronary vascular response in isolated guinea pig hearts. Role of nitric oxide

Objective: The effects of histamine and of histamine receptor agonists and antagonists on the coronary outflow and on the generation of nitric oxide (NO) were evaluated on isolated guinea pig hearts.Methods: Isolated guinea pig hearts were perfused for 50 min in a Langendorff apparatus with histamine (10^–7– 10^–8 M), in the absence or in the presence of N^G-monome-thyl-L-arginine (L-NMMA, 10^–4 M), a NO synthase inhibitor and of triprolidine (310^–8 M) and cimetidine (10^–7 M), H₁ receptor and H₂ receptor antagonists, and with trifluoromethyl-phenylhistamine (TFMPH, 10^–7 M) and dimaprit (10^–7 M), H₁ and H₂ receptor agonists. The effects of (R)--methylhistamine (10^–7 M), a H₃ receptor agonist and of FUB 181 (10^–7 M), a H₃ receptor antagonist, were studied in the presence of bradykinin (10^–7 M).Results: Histamine increases the coronary outflow and the generation of NO in a concentration-dependent fashion. The effects were completely abolished by blocking NO-synthase (NOS) with L-NMMA (10 ^–4 M). The effects were also abolished by cimetidine (10 ^–7 M), H ₂ receptor antagonist, and only scarcely affected by triprolidine (310 ^–8 M), H ₁ receptor antagonist. The effects were reproduced by dimaprit (10 ^–7 M), H ₂ receptor agonist, and only scarcely by TFMPH (10 ^–7 M), a selective H ₁ receptor agonist. Bradykinin (10 ^–7 M) produces a sustained coronary dilation paralleled by a marked increase in the generation of NO; the effects were significantly reduced by L-NMMA. The stimulation of H ₃ receptors by (R)--methylhistamine (10 ^–7 M) significantly reduced both effects, which reverted to normal with FUB 181 (10 ^–7 M), an H ₃ receptor antagonist.Conclusion: These results suggest that, in isolated guinea pig hearts, histamine produces coronary dilation through an H ₂ /H ₃ -dependent mechanism involving the generation of nitric oxide.Received 3 December 2002; returned for revision 8 April 2003; accepted by M. Parnham 26 May 2003     

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.     

Occupancy of adenosine receptors on human neutrophils inhibits respiratory burst stimulated by ingestion of complement-coated particles and occupancy of chemoattractant but not Fc receptors

Chemoattractants are generated at inflammatory loci that not only induce neutrophils (PMNs) to leave the vasculature but also stimulate PMNs to release potentially toxic agents (e.g., H₂O₂, O ₂ ^– or OH). We have recently demonstrated that endothelium releases adenosine which, when bound to a specific receptor on the PMN surface, inhibits release of toxic oxygen metabolites from stimulated PMN. To determine whether occupancy of adenosine receptors modulates generation and release of oxygen metabolites, we have studied the effect of 2-chloroadenosine on O ₂ ^– generation and O₂ consumption in response to opsonized zymosan particles (STZ) and immune complexes (IC). 2-Chloroadenosine inhibits, in a dose-dependent fashion, Of generation by neutrophils that have been exposed to C3b-coated particles (STZ). Inhibition of Of generation is similar in the presence or absence of cytochalasin B (IC₅₀=53 ±19 and 16 ±5nM, respectively,P=NS). Since occupancy of adenosine receptors might inhibit only externalization but not generation of oxygen metabolites, we studied the effect of 2-chloroadenosine on oxygen consumption by activated neutrophils. 2-Chloroadenosine inhibited O₂ consumption stimulated by STZ and the surrogate bacterial chemoattractant FMLP; however, inhibition of O₂ consumption varied with the presence or absence of cytochalasin B. In contrast, when neutrophils were stimulated by immune complexes, 2-chloroadenosine only minimally inhibited O ₂ ^– release and O₂ consumption (10 ± 5 and 5 ± 4% inhibition, respectively). Thus, occupancy of adenosine receptors inhibits O₂ consumption in parallel with inhibition of O ₂ ^– release. These results support the hypothesis that ingestion of complement-opsonized particles stimulates the respiratory burst by a mechanism different from that by which the respiratory burst is stimulated after occupancy of Fc receptors. Moreover, these observations suggest that endothelium, by releasing adenosine, prevent activated neutrophils from damaging the microvasculature at inflammatory loci. In contrast, deposition of immune complexes in vessel walls leads to vascular damage because endothelial cells are incapable of preventing attack by immune complex-stimulated neutrophils.This research was supported by grants from the U.S. Public Health Service (AI-10343 and HL29034) and the Veterans Administration.Dr. Cronstein is the recipient of a Clinical Investigator Award (K11-AR-01490) and was a fellow of the Arthritis Foundation. Dr. Broekman was an Established Investigator of the American Heart Association.     

Principal cells of cortical collecting ducts of the rat are not a route of transepithelial Cl− transport

The rat cortical collecting duct (CCD) exhibits high rates of NaCl reabsorption when stimulated by mineralocorticoid and antidiuretic hormone (ADH). The present study was undertaken to determine if there is significant transcellular Cl^– movement across the principal cells of the rat CCD. CCDs were dissected from kidneys of rats that had been injected with deoxycorticosterone (5 mg, i.m.) 2–9 days prior to the experiment. The ducts were perfused in vitro with identical perfusing and bathing solutions, except that 200 pmol.l^–1 ADH was added to the bathing solutions. The basolateral membrane voltage (PD_bl) of principal cells was –77±1 mV and the luminal membrane voltage (PD₁) was –68±1 mV (mean ± SEM, n=124). Separate impalements with single-barrelled Cl^–-selective microelectrodes gave an apparent intracellular Cl^– activity of principal cells of 17±2 mmol.l^–1. Transepithelial PD and PD_bl were unaffected by luminal furosemide, hydrochlorothiazide (HCT), 4-acetamido-4-isothiocyanostilbene2,2-disulphonic acid, (SITS), or the Cl^– channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB); bath addition of SITS or the Cl^– channel blocker diphenylamino-2-carboxylic acid; or replacement of bath HCO ₃ ^– by Cl^–. The intracellular Cl^– activity (a _cell ^Cl ) also remained unchanged with the addition of HCT, SITS or the Cl^– channel blockers to either the perfusing or bathing solutions, or with replacement of the bathing solution HCO ₃ ^– . With Cl^– replacement in both solutions, a _cell ^Cl decreased to 9 mmol.l^–1, but not until after 4–6 min, indicating a very low rate of Cl^– transport in these cells, even under conditions of maximal stimulation of NaCl reabsorption by mineralocorticoid plus ADH. The remaining a _cell ^Cl could be attributed to interference with the Cl^– selective electrodes by other cytosolic anions. We conclude that a _cell ^Cl of principal cells in the rat CCD is not far above passive equilibrium, and that these cells do not contribute significantly to transepithelial Cl^– reabsorption, which must occur by alternative routes such as the paracellular pathway, and/or through intercalated cells.Parts of this study were presented at the 65th meeting of the Deutsche Physiologische Gesellschaft at Würzburg, Federal Republic of Germany, 1988     

Role of Thromboxane A2 in the Induction of Apoptosis of Immature Thymocytes by Lipopolysaccharide

Lipopolysaccharide (LPS) causes apoptotic deletion of CD4⁺ CD8⁺ thymocytes, a phenomenon that has been linked to immune dysfunction and poor survival during sepsis. Given the abundance of thromboxane-prostanoid (TP) receptors in CD4⁺ CD8⁺ thymocytes and in vitro evidence that thromboxane A₂ (TXA₂) causes apoptosis of these cells, we tested whether enhanced generation of TXA₂ plays a role in LPS-induced thymocyte apoptosis. Mice injected with 50 μg of LPS intraperitoneally displayed a marked increase in generation of TXA₂ and prostaglandin E₂ in the thymus as well as apoptotic deletion of CD4⁺ CD8⁺ thymocytes. Administration of indomethacin or rofecoxib inhibited prostanoid synthesis but did not affect thymocyte death. In contrast, thymocyte apoptosis in response to LPS was significantly attenuated in TP-deficient mice. These studies indicate that TXA₂ mediates a portion of apoptotic thymocyte death caused by LPS. The absence of an effect of global inhibition of prostanoid synthesis suggests a complex role for prostanoids in this model.