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.     

Human polymorphonuclear leukocytes release leukotriene B4 during phagocytosis ofStaphylococcus aureus

We studied release of leukotriene B₄ (LTB₄) by human polymorphonuclear leukocytes (PMNs) during phagocytosis of staphylococci in the presence or absence of arachidonic acid. The 12×10⁷ PMNs incubated with 3×10⁹ opsonizedS. aureus and 50M arachidonic acid released 1.45±0.42 nmol LTB₄. No LTB₄ was detected after stimulation of PMNs withS. aureus or arachidonic acid by themselves. However, by increasing the concentration of arachidonic acid to 200 or 400M, 1.22±0.45 and 1.98±0.49 nmol LTB₄, respectively, was released by PMNs. The effect of different bacteria-PMN ratios on LTB₄ production was also studied. LTB₄ varied from 0.3 to 2.0 nmol when bacteria/PMN ratios increased from 5 to 50 (respectively) in the presence of 50 M arachidonic acid. Thus, phagocytizing PMNs produce LTB₄ in the presence of arachidonic acid, and its production is dependent on the number of bacteria phagocytized.     

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.     

In vitro generation of hydrogen peroxide and of superoxide anion by bovine polymorphonuclear neutrophilic granulocytes,blood monocytes,and alveolar macrophages

Studies were conducted to compare the capacity of bovine blood monocytes, polymorphonuclear granulocytes (PMNs), and alveolar macrophages (AMs) to generate hydrogen peroxide and Superoxide anion. Following stimulation with opsonized zymosan, bovine PMNs respond with an immediate and vigorous liberation of both oxygen species, generating 4.7 ± 0.3 nmol H₂O₂/10⁶ cell and 12.3 ± 1.8 nmol O₂ ^–/10⁶ cell during the initial 15 min. This is more than twice the amount generated by AMs (1.2 nmol H₂O₂/10⁶ cell; 2.5 nmol O₂/10⁶ cell) and blood monocytes (0.5 nmol H₂O₂/10⁶ cell; 2.1 nmolO₂ ^–/10⁶ cell) during the same period. However, AMs continue generating H₂O₂ and O₂ at a steady rate for a longer period and consequently produce amounts equal to those of PMNs when measured over a longer time span. Also, AMs can be stimulated with nonopsonized zymosan in contrast to PMNs. However, the AM population appears to comprise at least two subpopulations, which can be clearly distinguished by their capacity for generation of reactive oxygen species, and which correlate with their tendency for adherence to a plastic surface. In contrast to what has been found in other species, the bovine phagocytes were found to lack receptors for tuftsin and formylated oligopeptides, and thus remained unresponsive to these compounds. The in vitro activity of the three cell types was found to be very dependent on culture conditions, such as cell density and an adherent versus suspended state. In addition, a comparison with macrophages and PMNs elicited into the mammary gland suggest that in vivo factors can significantly influence the in vitro activities. The mammary gland cells have lower activity than blood and alveolar cells, even though they have been primed by chemotactic factors), and this is probably caused by milk components, i.e., the microenvironment. Our observations are discussed with respect to the results obtained from different laboratories, different species, and different cell types; emphasis is placed on the problem of drawing conclusions about in vivo functions of cells from parameters assayed in vitro.     

Neutrophil migration,oxidative metabolism,and adhesion in elderly and young subjects

Objective. To evaluate neutrophil functions in the elderly.Methods. We investigated the PMN migration in vivo and PMN superoxide production and adhesion in response to a variety of compounds; PMN have been isolated both from blood and from a skin experimental exudate (obtained by Senn's skin window technique) of 25 normal elderly and of 25 normal young control subjects.Results. No difference was found in PMN migration in vivo (62.9±21.3×10⁶ and 65.5±9.1×10⁶ PMN/cm²/24 hours in elderly and young subjects respectively), neither were different the adhesion under basal condition and after some stimuli and the superoxide production in basal condition and in response to STZ and PMA in two groups. In elderly subjects superoxide production, in response to fMLP, markedly resulted lower than in young controls both by circulating PMNs (3.6±2.7 and 9.3±3.3 nMOLES O ₂ ^– /10⁶ PMN respectively, p<0.0001) and by exudate PMNs (13.6±4.3 and 19.4±6 nMOLES O _2– ^{10 6} PMNs respectively, p<0.005).Conclusion. Many PMN functions in the elderly do not differ from young people, suggesting that the overall defense function of these cells is not affected by aging. The only parameter that we have found to be different between the two groups is the poor superoxide production after fMLP stimulus of PMNs. The stimulus- and function-specificity of this defect in PMNs from elderly subjects indicates the existence of a dysregulation of the signal transduction pathway distal to fMLP receptor and proximal to NADPH oxidase activation.     

Neutrophils from asthmatics exhibit diminished responsiveness to 2-chloroadenosine which is reversed by theophylline

We have shown previously that neutrophils (PMNs) from patients with asthma have a more potent stimulated respiratory burst than normals and that their respiratory burst is significantly less suppressed with exposure to 2-chloroadenosine (2-CADO). The present studies investigated the basis of this defect in responsiveness to 2-CADO. PMNs obtained from asthmatics either not on theophylline (minus theophylline) or taking theophylline (plus theophylline) generated significantly more superoxide in response to 2×10^–8 M FMLP (2.08±0.36 nmol/5×105 PMNs (minus theophylline) (P<0.01 compared to controls) vs. 2.16±0.44 (plus theophylline) (P<0.01) as compared to controls (1.05±0.17 nmol). In the presence of FMLP (2×10^–8 M), PMNs from the minus theophylline cohort had less 2-CADO (10^–6 M) -mediated suppression of superoxide generation as compared to controls (38.3±3.8% vs. 67±3.8%; (P<0.001). The plus theophylline group exhibited suppression values similar to controls (64.5±7.2%). Theophylline, in the presence of a physiological concentration of 2-CADO (0.1M) accentuated the suppression of the respiratory burnt in normals (74.1±5.9%, 80.1±4.9% (P<0.02) and 84.7±3.8% (P<0.02) at 0, 10, and 100M, respectively). PMNs from asthmatics not taking theophylline demonstrated suppression values of 46.2±6%, 53.8±6.6% (P=NS), and 63.2±7.1% (P<0.01), respectively. Resting PMNs from normal controls generated 0.97±0.20 pmol cAMP/10⁷ cells compared to 2.83±0.75 pmol in the pressnce of 0.1M 2-CADO. The combination of 2-CADO and theophylline (10–100M) produced cAMP concentrations not significantly different from that observed with 2-CADO alone. These findings support the existence of a novel cAMP-independent adenosine receptor in PMNs. The specific binding of 10^–8 M³H-labeled 2-CADO (in cpm) was 10,358 ± 1502 (P < 0.001 compared to controls), 5468 ± 843 (NS compared to controls), and 3751 ± 477 in the plus theophylline group, minus theophylline group, and controls, respectively. Such up-regulation of specific binding may represent the effects of theophylline as shown by the specific binding of [³H]2-CADO in PMNs from normal controls exposed to 10 M theophylline for 30 min (6013 ± 969) compared to unexposed PMNs (3768 ± 656; P < 0.05). These data support an antiinflammatory mechanism of action for theophylline in the therapy of asthma and suggest that this may occur through potentiation of the antiinflammatory mediator adenosine.This research was supported by NIH grants AI24843 and AI24848.     

Effect of ATP,carbachol and other agonists on intracellular calcium activity and membrane voltage of pancreatic ducts

The pancreatic duct has been regarded as a typical cAMP-regulated epithelium, and our knowledge about its Ca²⁺ homeostasis is limited. Hence, we studied the regulation of intracellular calcium, [Ca²⁺]_i, in perfused rat pancreatic ducts using the Ca²⁺-sensitive probe fura-2. In some experiments we also measured the basolateral membrane voltage, V _bl, of individual cells. The resting basal [Ca²⁺]_i was relatively high, corresponding to 263±28 nmol/l, and it decreased rapidly to 106±28 nmol/l after removal of Ca²⁺ from the bathing medium (n=31). Carbachol increased [Ca²⁺]_i in a concentration-dependent manner. At 10 mol/l the fura-2 fluorescence ratio increased by 0.49±0.06 (n=24), corresponding to an increase in [Ca²⁺]_i by 111±15 nmol/l (n=17). ATP, added to the basolateral side at 0.1 mmol/l and 1 mmol/l, increased the fluorescence ratio by 0.67±0.06 and 1.01±14 (n=46; 12), corresponding to a [Ca²⁺]_i increase of 136±22 nmol/l and 294±73 nmol/l respectively (n= 15; 10). Microelectrode measurements showed that ATP (0.1 mmol/l) hyperpolarized V _bl from –62±3 mV to-70±3 mV, an effect which was in some cases only transient (n=7). This effect of ATP was different from that of carbachol, which depolarized V_bl. Applied together with secretin, ATP delayed the secretin-induced depolarization and prolonged the initial hyperpolarization of V _bl (n=4). Several other putative agonists of pancreatic HCO ₃ ^– secretion were also tested for their effects on [Ca²⁺]_i. Bombesin (10 nmol/l) increased the fura-2 fluorescence ratio by 0.24±0.04 (n=8), neurotensin (10 nmol/l) by 0.25±0.04 (n=6), substance P (0.1 mol/l) by 0.22±0.06 (n=6), and cholecystokinin (10 nmol/l) by 0.14±0.03 (n=7). Taken together, our studies show that Ca²⁺ homeostasis plays a role in pancreatic ducts. The most important finding is that carbachol and ATP markedly increase [Ca²⁺]_i, but their different electrophysiological responses indicate that intracellular signalling pathways may differ.Preliminary reports of the present study have been presented at the 72nd Meeting of the German Physiological Society, March 1993     

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.     

Specific binding of 1-[3H]-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoryl choline (platelet-activating factor,PAF) by human polymorphonuclear neutrophils

1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) is a potent activator of polymorphonuclear neutrophil (PMN) aggregation, exocytosis and chemotaxis. Specific desensitization of PMN to PAF suggests a receptor-mediated interaction. The binding of 1-[³H]-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (³H-PAF) to human PMN and platelets was analysed and compared. Binding was saturable at 0.6 nM and 0.1 nM for 2×10⁶ PMN and 5×10⁷ platelets, respectively. The time course of binding at 22°C and 37°C for both cell types reached the plateau at 2 min. The averageK _d was 45.0±1.7 nM (mean ±1 SD of 4 experiments) for PMN (27.391±1381 sites for PMN) and 20.1±6.3 nM (4 experiments) for platelets (1577±461 sites for platelets). The Scatchard plot analysis revealed two distinct binding sites both on PMN and platelets: a high affinity binding site and a non-saturable binding site.This work was supported by C.N.R. Rome grant no. 81.00089.04.     

Function and stimulus-specific effects of phorbol 12-myristate 13-acetate on human polymorphonuclear neutrophils: Autoregulatory role for protein kinase C in signal transduction

Exposure of polymorphonuclear neutrophils (PMNs) to phorbol 12-myristate 13-acetate (PMA) resulted in a concentration-dependent (1–10 ng/ml) inhibition of granule exocytosis induced with the receptor-specific ligands,N-formylmethionyl-leucyl-phenylalanine (FMLP), pepstatin A, 5(S),12(R)-dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (LTB₄), and acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), PMA exerted a marginal inhibitory effect on calcium ionophore A23187-induced PMN degranulation, and the PMA analog, 4-phorbol 12, 13-didecanoate (4-PDD), was inactive. However, PMA potentiated AGEPC, pepstatin A, FMLP, LTB₄, and A23187-stimulated Superoxide anion (O ₂ ^– ) production. The mobilization of intracellular sequestered calcium (Ca²⁺) by the receptor-specific ligands, as reflected by a rise in the cytosolic-free Ca²⁺ concentration ([Ca²⁺]_i) in PMNs loaded with the CA²⁺-sensitive dye, Fura-2, was suppressed by PMA. A protein kinase C (PKC) inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) reversed the PMA-mediated inhibition of PMN degranulation and intracellular CA²⁺ mobilization. However, another, but less potent PKC inhibitor,N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), had no effect on the inhibition of PMN activation by PMA.     

Beta-glucuronidase release from leukocytes in children

Summary The release of -glucuronidase from polymorphonuclear leukocytes (PMNs) is important in the killing of bacteria and in producing tissue damage in acute inflammation. To investigate the effects of various diseases or drugs on degranulation, we studied the kinetics of -glucuronidase release from PMNs exposed to opsonized zymosan. PMNs of children with bacterial infections demonstrated increased degranulation. Within 5, 15, and 30 min the PMNs released 19±3%, 23±3%, and 26±3% of total -glucuronidase compared to 12±2%, 15±2%, and 16±2% of total -glucuronidase of control PMNs. Viral infections induced a significant delay of -glucuronidase release from PMNs. Maintenance therapy of acute lymphoblastic leukemia with 6-mercaptopurine and methotrexate, as well as administration of vincristine, diminished the degranulation. After 5, 15, and 30 min the PMNs released 8±1%, 10±1%, and 11±1%, as well as 6±3%, 8±2%, and 9±2% of total -glucuronidase. This study demonstrated that bacterial infections stimulate -glucuronidase release by PMNs. In contrast, cytostatic drugs inhibit lysosomal enzyme release, increasing the susceptibility to bacterial infections. The total enzyme activities were unchanged.Abbreviations c-AMP cyclic Adenosinemonophosphate - c-GMP cyclic Guanosinemonophosphate - PMNs polymorphonuclear Leukocytes Supported by DFG Ri 275/6-1Dedicated to Prof. Dr. E. Gladtke on the occasion of his 60^th birthday     

Fuel utilisation during prolonged low-to-moderate intensity exercise when ingesting water or carbohydrate

Previously, we examined the effects of carbohydrate (CHO) ingestion on glucose kinetics during exercise at 70% of maximum O₂ uptake ( O_2,max). Here we repeat those studies in heavier cyclists (n=6 per group) cycling for 3 h at a similar absolute O₂ uptake but at a lower (55% of O_2,max) relative exercise intensity. During exercise, the cyclists were infused with a 2-³H-glucose tracer and ingested U-¹⁴C glucoselabelled solutions of either flavoured water (H₂O) or 10 g/100 ml glucose polymer, at a rate of 600 ml/h. Two subjects in the H₂O trial fatigued after 2.5 h of exercise. Their rates of glucose appearance (R _a) declined from 2.9±0.6 to 2.0±0.1 mmol/min (mean ± SEM) and, as their plasma glucose concentration [Glu] declined from 4.7±0.2 to below 3.5±0.2 mM, their rates of glucose oxidation (R _ox) and fat oxidation plateaued at 2.7±0.4 and 1.7±0.1 mmol/min respectively. In contrast, all subjects completed the CHO trial. Although CHO ingestion during exercise reduced the final endogenousR _a from 3.4±0.6 to 0.9±0.3 mmol/min at the end of exercise, it increased totalR _a to 5.5±0.5 mmol/min (P<0.05). A higher totalR _a with CHO ingestion raised [Glu] from 4.3±0.3 to 5.3±0.1 mM and acceleratedR _ox from 3.5±0.2 to 5.9±0.2 mmol/min after 180 min of exercise (P<0.05). The increased contribution to total energy production from glucose oxidation (34±1 vs. 20±1 %) decreased energy production from fat oxidation from 51±2 to 40±5% (P=0.08) and produced patterns of glucose, muscle glycogen (plus lactate) and fat utilisation similar to those during exercise at 70% of ( O_2,max). Thus, CHO ingestion is necessary to sustain even prolonged, low to moderate intensity exercise and when ingested, it suppresses the higher relative rates of fat oxidation usually observed at exercise intensities less than 60% of O_2,max.     

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.     

The inactivation of the polymorphonuclear leukocyte by non-steroidal anti-inflammatory drugs

When human neutrophils (PMNs) are activated by appropriate stimuli, they aggregate, generate Superoxide anion (O ₂ ^? ) and secrete lysosomal enzymes. Pre-incubation of PMNs in vitro with the cyclo-oxygenase (COx) inhibitor piroxicam (50μM) before stimulation with the chemotactic peptide f-met-leu-phe (FMLP, 10^?7 M) inhibited all of these responses. The COx inhibitor ibuprofen inhibited FMLP-induced aggregation and lysozyme secretion, leaving O ₂ ^? generation unaffected. Binding of³H-FMLP was inhibited by piroxicam. When the plant iectin concanavalin A (Con-A, 30μg/ml) or the tumor promoter phorbol myristate acetate (PMA, 50μml) was used as a stimulus, ibuprofen had no effect on PMN response, while piroxicam inhibited only O ₂ ^? generation. To determine whether such inhibition might also occur in vivo, we tested neutrophil aggregation and O ₂ ^? generation in response to FMLP in 26 normal subjects. These subjects were then administered therapeutic doses of piroxicam (20 mg/ day), ibuprofen (2400 mg/day) or indomethacin (100 mg/day), and neutrophil functions were retested after 3 days. Piroxicam inhibited FMLP-induced aggregation by 31% (5.2 cm²/min versus 3.6cm²/min,P<0.004) and O ₂ ^? generation by 35% (15.8 nmol cytochrorae c reduced versus 10.2 nmol,P<0.002). Ibuprofen inhibited FMLP-induced aggregation by 44% (5.2 versus 3.0,P<0.03) but had no effect on O ₂ ^? production. Indomethacin inhibited FMLP-induced aggregation (6,4 versus 2.9,P<0.01) but had no effect on O ₂ ^? generation. These studies suggest that: (a) multiple pathways exist for the activation of the neutrophil, since inhibition by non-steroidal anti-inflammatory drugs (NSAIDs) is stimulus-dependent; (b) NSAIDs have cellular effects that are not dependent on COx inhibition; (c) unique effects on cellular function may account for the clinical variability in responsiveness to different NSAIDs.     

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.     

Effects of HMGB1 on PMN apoptosis during LPS-induced acute lung injury

Objective

To observe the effects of intravenous injection of HMGB1 inhibitor sodium butyrate on changes in apoptosis of PMN during LPS-induced acute lung injury in rats and HMGB1 in vitro on human circulating PMN apoptosis, in order to clarify the role of HMGB1 in the pathogenesis of acute lung injury.

Methods

(1) LPS-induced acute lung injury rat model was developed by LPS infusion. At different time-points after LPS challenge in the presence or absence of sodium butyrate (SB), the rat tissue sample, peripheral blood PMNs and BALF were collected. RT-PCR was applied to examining rat lung tissue HMGB1 mRNA expression level, and Western blotting analysis was adopted to determine expression of rat lung tissue HMGB1 protein. PMN apoptotic changes were determined by flow cytometric (FCM) analysis, Giemsa staining and TdT-mediated dUTP nick end labeling (TUNEL) method. (2) Separated and purified human circulating PMN were coincubated for 24 h with different doses of HMGB1 (0, 10, 100, 1000 ng/ml, respectively) at 37 °C in 5% CO₂. PMN apoptosis rate was determined by flow cytometric (FCM) analysis and by TdT-mediated dUTP nick end labeling (TUNEL) method.

Results

(1) The percentage of apoptosis of PMN in rat model of LPS-induced ALI was gradually decreased as compared with that of normal control. The PMN apoptosis-initiation time and non-survival time in rat BALF prolonged significantly as compared with that of normal control. The injured rat lung tissue HMGB1 mRNA and protein expression was upregulated 6–24 h after LPS exposure; SB intervention significantly ameliorated the upregulation. In addition, the morphologic examination indicated that the edema severity and pathological changes of lung tissues were excessively aggravated in rats after LPS administration. By comparison, SB treatment diminished the severity of lung damage. Combined with lung HMGB1 expression level, the above changes indicate that the pathological changes of lung tissue were related to the injured lung HMGB1 expression, as well as apoptotic changes in PMN. (2) After coincubation of HMGB1 with human circulating PMNs, TUNEL and flow cytometry were performed. The study revealed that PMN apoptosis ratios was (40.53 ± 4.12) % in control group (PMNs + RPMI 1640 medium), (19.05 ± 2.44) % in LPS group (PMNs + RPMI 1640 medium + 10 μg/ml LPS), (40.52 ± 2.73) % in HMGB1-1 group (PMNs + RPMI 1640 medium + 10 ng/ml HMGB1), (34.89 ± 1.15) % in HMGB1-2 group (PMNs + RPMI 1640 medium + 100 ng/ml HMGB1), and (18.77 ± 3.02) % in HMGB1-3 group (PMNs + RPMI 1640 medium + 1 000 ng/ml HMGB1). There was statistical significance. Meanwhile, PMN TUNEL positive rate was (31.42 ± 4.40) %, (31.39 ± 3.80) %, (25.62 ± 2.46) %, and (17.98 ± 3.20) % in control group, HMGB1-1 group, HMGB1-2 group and HMGB1-3 group, respectively. The inhibitory effect was HMGB1 dose-depended as compared with that of control group.

Conclusion

After LPS challenge, high expression of rats' lung HMGB1 mRNA occurs at a later phase, but keeps for a long time. Sodium butyrate (SB) treatment attenuated LPS-induced PMN apoptosis delay and inhibition, and down-regulated HMGB1 mRNA expression of injured lung. HMGB1 in vitro inhibited human circulating PMN apoptosis markedly, and the inhibitory effect was HMGB1 dose-depended. The results demonstrated that HMGB1 may play an important role as a modulator in apoptotic changes in PMN during LPS-induced ALI. It concludes that HMGB1 may contribute to the development of PMN apoptotic changes during LPS-induced acute lung injury.     

The effect of adherence on the generation of reactive oxygen species by human neutrophilic granulocytes

Human neutrophilic granulocytes (PMN) suspended in protein containing salt solution or adherent on protein coated nylon fibers were tested for the production of H₂O₂ and O ₂ ^– in response to various PMN stimulants. Upon stimulation with the chemotactic factors formyl-methionyl-leucyl-phenylalanine, C5a and platelet activating factor, the non-chemotactic ionophore A23187, and the chemotaxis inhibitors tumor necrosis factor (TNF) and lymphotoxin (TNF ) adherent PMN produced considerably more reactive oxygen metabolites than suspended cells. The relative amounts of the two metabolites varied with the stimulus and its concentration, TNF and TNF favoring H₂O₂ production, C5a eliciting more O ₂ ^– than H₂O₂ and the other active stimulants being in between. Leukotriene B₄ and a novel monocytederived chemotaxin were inactive in releasing either oxygen derivative from adherent or suspended PMN. The data indicate that attachment of PMN to endothelial cells or to connective tissue substances can strongly enhance its ability to respond to a given stimulus with the production of reactive oxygen metabolites. The findings may in part explain the priming phenomenon since many PMN-priming mediators increase the cells' adherence.     

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.     

The effect of secretagogues on ion conductances of in vitro perfused,isolated rabbit colonic crypts

Several secretagogues were used in this study, including those which enhance intracellular cyclic adenosine monophosphate (cAMP) production, as well as others which elevate intracellular Ca²⁺ activity and are known to increase Cl^– secretion in the intact colon and in colonic carcinoma cell lines. They were examined with respect to their effects on electrophysiological properties in isolated rabbit distal colonic crypts. Crypts were dissected manually and perfused in vitro. Transepithelial voltage (V _te), transepithelial resistance (R _te), membrane voltage across the basolateral membrane (V _bl), and fractional basolateral membrane resistance (FR _bl), were estimated. Basolateral prostaglandin E₂ (PGE₂, 0.1 mol/l), vasoactive intestinal peptide (VIP, 1 nmol/l) and adenosine (0.1 mmol/l) induced an initial depolarisation and a secondary partial repolarisation of (V _bl). In the case of adenosine, the initial depolarization of (V _bl) was by 31±2 mV (n=47).R _te fell significantly from 16.4±3.6 to 14.2±3.7 ·cm² (n= 6), andFR _blincreased significantly from 0.11±0.02 to 0.51±0.10 (n=6). In the second phase the repolarisation of (V _bl) amounted 11±2 mV (n=47) and a steadystate (V _bl) of –51±2 mV (n=47) was reached.R _te fell further and significantly to a steady-state value of 12.4±3.8 ·cm² (n=6) andFR _bl fell significantly to 0.42±0.13 (n=6). In 30% of the experiments, a transient hyperpolarisation of (V _bl) by 8±2 mV (n=14) was seen during wash out of adenosine. In the presence of adenosine, but not under control conditions, lowering of luminal Cl^– concentration from 120 to 32 mmol/l depolarised (V _bl) significantly by 8±1 mV (n=9). Basolateral ATP and ADP (0.1 mmol/l) led to a short initial depolarisation followed by a sustained and significant hyperpolarisation by 6±2 mV (n=27) and 5±4 mV (n=8), respectively. Carbachol (CCH) hyperpolarised (V _bl) in a concentration-dependent manner. At 100 mol/l (bath) the hyperpolarisation was by 14±2 mV (n=11) andFR _bl fell slightly. Neurotensin (10 nmol/l), isoproterenol (10 mol/l) and uridine 5-triphosphate (UTP, 0.1 mmol/l) had no effect. It is concluded that PGE₂, VIP and adenosine upregulate sequentially a luminal Cl^– conductance and a basolateral K⁺ conductance by increasing intracellular cAMP concentration. Ca²⁺ mobilising hormones such as ATP, ADP, and CCH increase the basolateral K⁺ conductance, while the effect on luminal Cl^– conductance appears to be very limited.     

Recombinant interleukin-1β interacts with high-affinity receptors to activate neutrophil leukotriene B4 synthesis

The capacity of interleukin-1 (IL-1) to function as a neutrophil (PMN) activator has been the subject of controversy. While IL-1 purified from mononuclear cell supematants induced PMN activation, these observations have not been confirmed with recombinant IL-1. To document a cellular basis for a putative PMN-IL-1 interaction, we investigated the presence of IL-1 receptors on the PMN. Using an [³⁵S]methionine-labeled preparation, specific binding of IL-1 to PMNs was demonstrated. Through Scatchard analysis PMNs were calculated to have a mean of 469 ±337 receptors per PMN with an affinity(K _d) of 0.32±0.09 nM. As IL-1 frequently activates arachidonic acid metabolism in other cell types, we investigated eicosanoid production as a putative consequence of the IL-1-PMN interaction. HPLC analysis of extracted supernatants of IL-1-treated PMNs demonstrated the release of leukotriene B₄ (LTB₄), its oxidative products, and 5-hydroxyeicosatetraenoic acid (5-HETE). Production of LTB₄ was quantified using a commercial RIA. LTB₄ secretion increased from 17.2±1.1 to 96.7+-16.4 ng, also with 10.0 ng of IL-1. In time-course studies, it was shown that maximal eicosanoid secretion required a 30-min incubation with IL-1. These observations confirm the proinflammatory activity of IL-1 on neutrophils and resolve the controversy concerning a direct effect of IL-1 on neutrophils. In conclusion, recombinant IL-1 interacts with neutrophils through the presence on the PMN of a high-affinity receptor and results in the secretion of arachidonate metabolites.This work was presented in part at the American Federation for Clinical Research National Meeting, Washington, D.C., May 1988 (Clin. Res. 36:436A). This work was supported by USPHS NIH grants A125173 (L. J. R.), HL33961 (L. J. R.), and A124843 (L. B.). L. J. R. is the recipient of an Allergic Diseases Academic Award (A100595). L. B. is the recipient of a Burroughs Wellcome Developing Investigator Award.