Gc-globulin (vitamin D-binding protein) enhances the neutrophil chemotactic activity of C5a and C5a des Arg.

Several serum proteins have been shown to be important in modulating leukocyte chemotaxis and inflammation. We investigated the possibility that the multifunctional serum protein Gc-globulin (vitamin D-binding protein) may also enhance the neutrophil chemotactic activity of complement-derived peptides. Purified Gc-globulin by itself did not induce chemotaxis of human neutrophils. However, as little as 0.01 nM Gc-globulin greatly enhanced the neutrophil chemotactic activity of C5a and its derivative, C5a des Arg over a wide concentration range. The effect was most pronounced at nonchemotactic doses of C5a (0.01 nM) and C5a des Arg (1 nM). Gc-globulin was unable to augment the neutrophil chemotactic activity of FMLP and leukotriene B4. This enhancing activity was not due to a nonspecific effect of anionic proteins since other purified serum proteins, of similar size and charge as Gc-globulin (alpha 1 acid glycoprotein, alpha 2 HS glycoprotein, alpha 2 histidine-rich glycoprotein), could not increase the chemotactic activity of C5a des Arg. Serum depleted of Gc-globulin by immunoaffinity chromatography totally lacked chemotactic enhancing activity for C5a des Arg. Gc-globulin-depleted serum activated with zymosan also had significantly less chemotactic activity than control- (sham-depleted) activated serum. Finally, radioiodinated C5a or C5a des Arg formed a 1:1 complex with purified Gc-globulin when analyzed by gel filtration chromatography. These results indicate that Gc-globulin is the major chemotactic enhancing factor in serum and may function as an up-regulator of the chemotactic activity of C5-derived peptides.

In vivo clearance and tissue distribution of C5a and C5a des arginine complement fragments in rabbits.

We have previously shown a marked difference in the inflammatory response to human C5a or C5a des arginine (Arg) instilled in rabbit lungs. These studies raised the question of where C5a and C5a des Arg are processes in vivo and what role neutrophils may play in the tissue distribution of these two mediators. After intravenous injection of purified, biologically active 125I-C5a or 125I-C5a des Arg, adult rabbits were serially bled and then killed at various time intervals. Although greater than 50% of the injected radioactivity was cleared from the circulation within 2 min for both mediators, C5a des Arg persisted in the circulation longer than C5a. C5a instillation caused an acute neutropenia, whereas C5a des Arg caused a less severe and more prolonged neutropenia, preceding a neutrophilic response observed with both mediators. Clearance of the mediators was primarily seen in the highly vascularized organs: the lung, spleen, liver, and kidney. A time-dependent accumulation was seen initially in the lung, followed by the spleen, liver, and kidney. Histologic examination showed a marked increase in the number of neutrophils within the lung and spleen. Depletion of circulating neutrophils by nitrogen mustard pretreatment of rabbits showed no change in the amount of labeled mediator bound in the lung, whereas splenic accumulation was dependent on the presence of neutrophils. These results indicate that C5a and C5a des Arg are rapidly removed from the circulation by specific accumulation in vascularized tissues. Clearance by the lung was not affected by neutrophil depletion, whereas clearance by the spleen was dependent on neutrophils. These experiments further suggest there are neutrophil-dependent and neutrophil-independent mechanisms involved in the removal of C5a and C5a des Arg from the circulation and that binding of C5 fragments in the pulmonary vasculature may precede and then induce neutrophil sequestration.

Additive effect of intravascular complement activation and brief episodes of hypoxia in producing increased permeability in the rabbit lung.

Systemic complement activation with intravascularly administered cobra venom factor (CVF) or infusion of either zymosan-activated rabbit plasma or a fifth component of complement fragment with anaphylatoxin activity in the rabbit have not caused significant increases in bronchoalveolar lavage albumin in rabbits (Webster, R. O., G. L. Larsen, B. C. Mitchell, A. J. Goins, and P. M. Henson. 1982. Am. Rev. Respir. Dis. 125:335-340). To assess if another stimulus (hypoxia) acting in concert with complement activation can produce significant lung injury, rabbits were challenged with CVF alone, 10 min of 12% oxygen alone, or CVF followed by a 10-min exposure to 12% oxygen. Either stimulus alone caused no significant changes in arterial oxygen, pulmonary resistance, or dynamic compliance during the 240 min of observation after either stimulus, and neither stimulus alone caused increased albumin accumulation in bronchoalveolar lavage over a 30-min period at the end of the experiment. However, the combination of insults significantly altered arterial oxygen, pulmonary resistance, and dynamic compliance while also increasing albumin and neutrophils recovered by lavage. The increase in lavage albumin did not appear to be due to hemodynamic events in that the pulmonary artery pressure increased acutely after CVF infusion and again during the hypoxic exposure, but was normal when albumin accumulation in the lung was measured. Neutrophil depletion with nitrogen mustard abolished all of these changes induced by CVF plus hypoxia. In addition, meclofenamate pretreatment and infusion during the 4-h study abolished the increases in lavage albumin and neutrophils as well as the increase in pulmonary artery pressure after CVF. Meclofenamate pretreatment did not, however, block accumulation of albumin in the lung (interstitium). We conclude that complement activation, as an isolated event, will not cause a significant increase in lavage albumin in this model. However, combining complement activation with an episode of hypoxia will lead to an increase in lavage albumin that is dependent on the presence of neutrophils for its expression. Meclofenamate treatment will prevent increases in lavage albumin and neutrophils while not preventing albumin accumulation in the lung (interstitium), suggesting a product of the cyclooxygenase pathway of arachidonic acid metabolism is needed to produce movement of albumin and/or neutrophils across the alveolar epithelium in this model.

Adenosine inhibits fMLP-stimulated adherence and superoxide anion generation by human neutrophils at an early step in signal transduction.

The ability of physiological concentrations of adenosine to inhibit formylmethionylleucylphenylalanine (fMLP)-stimulated superoxide anion (O2-) generation, adherence and degranulation is well established in human neutrophils. However, the mechanism of inhibition remains to be determined. To better understand where adenosine blocks the fMLP signal transduction pathway, we examined the ability of adenosine to inhibit neutrophil adherence stimulated by phorbol myristate acetate (PMA), NaF, and A23187; these agents activate intermediate steps in fMLP signal transduction. Adenosine (0.1-100 microM) did not inhibit adherence mediated by these receptor-independent agonists or NaF- and A23187-mediated O2- production. Additionally, NaF and A23187 completely abrogated adenosine inhibition of fMLP-stimulated neutrophil adherence. We also found that pertussis toxin (5 and 10 microM) completely inhibited fMLP-induced neutrophil adherence and O2- generation, indicating that both processes are G protein mediated. Furthermore, fMLP-stimulated GTPase activity in neutrophil membrane preparations was significantly inhibited by adenosine (1 and 10 microM) or 5'-N-ethylcarboxamidoadenosine (1 microM) (NECA). These data indicate that adenosine inhibits a G-protein-dependent pathway of fMLP stimulation by uncoupling G proteins from the fMLP receptor. This may be a general mechanism of adenosine inhibition of cell-surface receptor-mediated signals as both fMLP- and C5a-stimulated neutrophil adherence were inhibited at similar concentrations.

Inhibition of neutrophil functions by platelets and platelet-derived products: description of multiple inhibitory properties.

The effect of platelets and their products on in vitro responses of human neutrophils to f-met-leu-phe (fMLP) and phorbol myristate acetate (PMA) was studied. Platelets produced a concentration-dependent inhibition of neutrophil superoxide anion (O2-) generation with maximum inhibition of the response to fMLP approaching 42.2 +/- 5.4% and that to PMA approaching 85.9 +/- 3.4%. Supernates of thrombin-activated platelets produced maximal inhibition of neutrophil O2- generation in response to fMLP (66.3 +/- 4.2%) at a ratio of 10 platelet equivalents (PEQ) to 1 neutrophil but failed to affect the cells' response to PMA. In contrast, lysate of sonicated platelets inhibited neutrophil O2- generation in response to both fMLP (59.6 +/- 2.0%) and PMA (90.1 +/- 1.3%). Biochemical characterization of platelet lysate identified at least two stimulus-specific inhibitory activities which differ in size, thermal stability and enzyme sensitivity. Platelet lysate also maximally inhibited neutrophil degranulation and chemotaxis at a ratio of 20 PEQ to 1 neutrophil. Our findings indicate that physiologically achievable levels of platelets or their products modulate the response of neutrophils in a stimulus-specific manner and are thereby capable of potentially limiting tissue damage which accompanies neutrophil activation during inflammation.

In vitro effects of platelet factor 4 on normal human neutrophil functions.

Platelet factor (PF4) prepared from human outdated platelets by heparinagarose affinity chromatography was confirmed to be chemotactic for human neutrophils and in a concentration-dependent fashion caused significant release of lysosomal enzymes (myeloperoxidase, lysozyme, beta-glucuronidase) from human neutrophils treated with cytochalasin B. Lysosomal enzyme release from PF4-stimulated neutrophils was rapid and reached a plateau by 1-3 min. PF4 did not cause release of the cytoplasmic enzyme lactate dehydrogenase which indicates that exocytosis of granule-containing lysosomal enzymes did not result from cytolysis. In contrast, superoxide anion generation from human neutrophils stimulated with PF4 was undetectable even at the highest PF4 concentration tested (2 X 10(-5) M). Pretreatment of neutrophils with PF4 caused significant increased adherence of neutrophils to plastic surfaces and cultured pulmonary artery endothelial cells. The concentration of PF4 that elicited neutrophil chemotaxis, lysosomal enzyme release and increased adherence is slightly higher than those concentrations found in normal human sera. However, the results suggest that PF4 may be an important mediator in neutrophil-platelet interactions and the induction of acute inflammation especially at sites of platelet microthrombi where the concentration of PF4 would be elevated.

Human monocyte chemotaxis to complement-derived chemotaxins is enhanced by Gc-globulin.

Gc-globulin has been found in bronchoalveolar lavage fluid in patients with chronic obstructive pulmonary disease (COPD) and adult respiratory distress syndrome (ARDS) and has been shown to enhance neutrophil chemotaxis to C5-derived peptides in vitro. We proposed that Gc-globulin may enhance the inflammatory response in lungs by influencing monocyte chemotaxis to C5-derived peptides as it does with neutrophils. Monocyte chemotaxis was measured in blind well chambers by a leading-front technique. Purified human Gc-globulin had no intrinsic chemotactic activity for monocytes at concentrations ranging from 1 fM to 1 microM. However, Gc-globulin, at concentrations as low as 10 pM, increased monocyte chemotaxis over 10-fold in a concentration-dependent fashion when added to non-chemotactic doses of C5a (0.1 nM) and C5a des Arg (0.5 nM). The chemotaxis-enhancing effect of Gc-globulin was specific for C5-derived peptides, as Gc-globulin did not enhance monocyte chemotaxis to other chemoattractants such as leukotriene B4 or formyl-Met-Leu-Phe. The enhancement of monocyte chemotaxis to C5-derived peptides by Gc-globulin was not a nonspecific effect of anionic proteins, as other serum proteins of similar size and charge did not enhance monocyte chemotaxis to C5a des Arg. These results indicate that Gc-globulin enhances the monocyte response to C5-derived peptides and, together with previous work, indicates that its presence in the airways of patients with COPD and ARDS may up-regulate the monocyte inflammatory response in the lungs.

Binding of Gc globulin (vitamin D binding protein) to C5a or C5a des Arg is not necessary for co-chemotactic activity.

Gc globulin (vitamin D binding protein) has been shown to augment significantly the leukocyte chemotactic activity of the activated complement peptides C5a and C5a des Arg. However, the mechanism of chemotaxis enhancement is not known. Natural C5-derived peptides contain a carbohydrate side chain that comprises approximately 25% of the mass of the 11-kDa peptides. Previous studies have demonstrated that Gc globulin binds to C5-derived peptides via sialic acid residues on this carbohydrate side chain. The necessity of this carbohydrate side chain for chemotaxis enhancement by Gc globulin was investigated by using both natural (glycosylated) and recombinant (carbohydrate-free) peptides. The dose-response curves of neutrophil chemotaxis to recombinant C5a or C5a des Arg plus Gc globulin were identical to those observed with the naturally derived peptides, despite the fact that natural C5a bound to Gc globulin while the recombinant C5a failed to bind this protein. Moreover, neutrophils pretreated with Gc globulin then washed before addition to the chemotaxis assay displayed significantly enhanced movement to C5a alone. These results indicate that the binding of C5a/C5a des Arg to Gc globulin is not necessary for their chemotactic activity to be augmented. Furthermore, these results demonstrate that the co-chemotactic activity of Gc globulin is generated on the cell surface, independent of C5a binding to its receptor.

Inhibition of neutrophil superoxide anion generation by platelet products: role of adenine nucleotides.

Previously, we demonstrated the stimulus-specific ability of platelet lysate or release products to inhibit neutrophil functions in response to f-met-leu-phe (fMLP) or phorbol myristate acetate (PMA). The present study further characterizes these activities and identifies one of the platelet inhibitors of neutrophil superoxide anion (O2-) generation. Sephadex G-200 chromatography revealed two fMLP-specific inhibitory activities in platelet lysate. One component eluted with materials less than 13 kilodaltons (kD) molecular weight, whereas the second fractionated at the void volume (Mr greater than 200 kD). PMA-specific inhibitor(s) cofractionated at the void volume with the lysate's fMLP-inhibitory activity. Release products of activated platelets contained low-molecular-weight (less than 13 kD), fMLP-specific inhibitor activity but failed to demonstrate PMA-specific inhibition of neutrophil O2- generation. Biochemical characterization identified two distinct high-molecular-weight inhibitors in platelet lysate. Pretreatment with adenosine deaminase (ADA) completely abrogated the maximum inhibitory effect (43.8 +/- 7.5%) of the high-molecular-weight, fMLP-specific lysate component but failed to reduce maximum PMA-specific inhibition (93.0 +/- 2.0%) of neutrophil O2- generation. Likewise, while the lysate's fMLP-specific inhibitor was heat stable, heating reduced the PMA-specific inhibition 56.5 +/- 10.4%. Equal sensitivity to ADA, resistance to trypsin, and heat stability were demonstrated by the low-molecular-weight inhibitor(s) in platelet lysate and release products. Neither high- nor low-molecular-weight platelet inhibitors scavenged O2-. High-performance liquid chromatography (HPLC) of the low-molecular-weight inhibitory activity from both platelet lysate and release products revealed physiological levels of adenine nucleotides. Addition of pure adenine nucleotides at physiological levels (1-10 microM) mimicked the effectiveness of the platelet inhibitor. We conclude that stimulus-specific limitation of neutrophil O2- generation by platelet adenine nucleotides may represent a mechanism for reducing inadvertent tissue damage during inflammation.

Inhibition of cleavage of the third component of human complement (C3) by its small cleavage fragment, C3a: inhibition occurs with the classical-pathway, but not the alternative-pathway, C3 convertase.

Activation of the third component of complement (C), C3, is central to the functioning of the C system in inflammation. Cleavage of C3 by the C3 convertases of both the classical and alternative pathways results in the formation of two split products, C3b and C3a. C3a inhibited cleavage of C3 by the classical-pathway C3 convertase. The inhibition varied in a concn-dependent relationship, with a concn of approximately 40 micrograms/ml yielding 50% inhibition. Removal of the carboxy terminal arginine from the C3a did not alter the inhibition. C3a did not inhibit cleavage of C3 by the alternative C pathway C3 convertase, or cleavage of C5 by C5 convertase. The C3-cleaving capacity of EAC142oxy that had been previously incubated with C3a could be recovered completely by washing the cells, indicating that the C3a binding to the EAC42oxy cell must have been reversed without having had an effect on the amount of C2 bound. Ribonuclease, a molecule of similar size and charge to C3a, did not affect C3 cleavage and C3a inhibition was not reduced by providing a surface for non-specific adsorption of the C3a, suggesting that the effect of C3a on C3 cleavage was not mediated by non-specific interaction with cell surfaces. C3a inhibited the C3-cleaving capacity of the fluid-phase enzyme, C42oxy, to the same degree as it inhibited the cell-bound enzyme, EAC42oxy, indicating that the C3a must interact with the C42 complex directly. Inhibition of C3 cleavage by C3a is the first demonstration of product inhibition of a complement enzyme. It may provide another control of C3 activation.

Ceruloplasmin inhibits carbonyl formation in endogenous cell proteins.

Exposure of cells to oxygen radicals results in cellular injury and protein oxidation. Ceruloplasmin is a plasma antioxidant that increases in concentration during inflammation. Therefore, the ability of ceruloplasmin to protect endothelial cells from neutrophil-mediated injury was investigated. The inhibition of protein oxidation by ceruloplasmin was also examined in neutrophil and endothelial cell proteins by analysis of carbonyl formation. In addition, the iron oxidation state was measured to determine the effect of ceruloplasmin ferroxidase activity in oxygen-radical generating systems. Ceruloplasmin significantly (p < .01) inhibited neutrophil-mediated cytotoxicity of endothelial cells by 48%. Carbonyl formation in phorbol myristate acetate (PMA)-stimulated neutrophil proteins was also significantly (p < .01) reduced by ceruloplasmin from 0.172 +/- 0.028 to 0.086 +/- 0.004 mole carbonyl/mole protein. Even though ceruloplasmin itself had a threefold increase in carbonyl formation (0.452 +/- 0.010 vs. 0.146 +/- 0.018 mole carbonyl/mole protein) in the presence of PMA-stimulated compared with unstimulated neutrophils, no loss of functional activity was detected. In xanthine oxidase-treated endothelial cells, ceruloplasmin significantly (p < .05) reduced carbonyl formation from 0.132 +/- 0.010 to 0.097 +/- 0.009 mole carbonyl/mole protein. Ceruloplasmin also significantly (p < .01) oxidized iron when added to PMA-activated neutrophils, thereby decreasing Fe(II) from 98 +/- 8 to 7 +/- 2 microM. Similarly, ceruloplasmin added to xanthine oxidase/hypoxanthine reactions resulted in significant (p < .01) iron oxidation, decreasing Fe(II) from 99 +/- 1 to 15 +/- 3 microM. The ability of ceruloplasmin to protect both endothelial cells and endogenous neutrophil and endothelial cell proteins from oxidative injury suggests that it may be important in regulating cellular and protein damage by oxygen radicals during inflammation.

Pulmonary microvascular alterations and injury induced by complement fragments: synergistic effect of complement activation, neutrophil sequestration, and prostaglandins.

Fragments of C5 that are generated at, or administered to, extravascular sites in the pulmonary parenchyma induced neutrophil infiltration, edema, tissue damage, and a complete inflammatory response. Generation of C5 fragments within the vascular system induced leukocyte sequestration in the pulmonary vasculature, but without detectable increased vascular permeability or neutrophil migration. By contrast, the combination of short episode of hypoxemia with the intravascular C5 activation led significant increases in pulmonary vascular permeability, mild endothelial alterations, and emigration of neutrophils. Infusion of 10 micrograms PGE2 into animals in which intravascular complement had been activated produced changes in the lungs that were similar to, though less severe than, the combination of hypoxia and complement activation.

Role of neutrophils in xanthine/xanthine oxidase-induced oxidant injury in isolated rabbit lungs.

Reactive oxygen species have been shown to play an important role in the pathogenesis of lung injury. This study was designed to clarify the role of intrapulmonary neutrophils in the development of xanthine/xanthine oxidase (X/XO)-induced lung injury in isolated buffer-perfused rabbit lungs. We measured microvascular fluid filtration coefficient (K(f)) and wet-to-dry weight ratio to assess lung injury. X/XO induced a significant increase in K(f) and wet-to-dry weight ratio in neutrophil-replete lungs, whereas the lung injury was attenuated in neutrophil-depleted lungs. A neutrophil elastase inhibitor, ONO-5046, also attenuated the lung injury. In addition, X/XO induced a transient pulmonary arterial pressure (P(pa)) increase. The thromboxane inhibitor OKY-046 attenuated the P(pa) increase but did not alter the increase in permeability. Neutrophil depletion reduced the K(f) increase but had no effect on the P(pa) increase. These results suggest that intrapulmonary neutrophils activated by X/XO play a major role in development of the lung injury, that neutrophil elastase is involved in the injury, and that the X/XO-induced vasoconstriction is independent of intrapulmonary neutrophils.

Adenosine prevents phorbol ester injury in rabbit lungs: role of leukotrienes and TNF.

The objective of this study was to determine whether adenosine (ADO) prevents phorbol myristate acetate- (PMA) induced lung injury by modulating peptidoleukotrienes (LT) and/or tumor necrosis factor (TNF) production. PMA significantly increased pulmonary vascular resistance (PVR, 275 +/- 4 to 447 +/- 30 cmH2O.1-1.min) and microvascular filtration coefficient.(Kf, 0.024 +/- 0.002 to 0.040 +/- 0.006 g.min-1.cmH2O-1) in isolated blood-perfused rabbit lungs. ADO (5 mumol/min) blocked the increases in PVR (257 +/- 9 to 283 +/- 26) and Kf (0.028 +/- 0.005 to 0.018 +/- 0.002). After PMA (30 min), perfusate levels of LTC4 + LTD4 increased by 15.3 +/- 2.1 pg/ml; LTE4 increased by 15.1 +/- 4.1 pg/ml. ADO reduced the increase in LTC4 + LTD4 to 2.7 +/- 6.1 pg/ml, but total LT increased by 31.9 +/- 16.6 pg/ml, implying that ADO enhanced the conversion of LTC4 and LTD4 to LTE4. MK-886 (L663,536), an LT synthesis inhibitor, blocked the increase in total LT (6.1 +/- 13.9 pg/ml) but did not reduce the PMA-induced increase in Kf (0.022 +/- 0.003 to 0.035 +/- 0.005) or PVR (238 +/- 11 to 495 +/- 21). After PMA administration, perfusate TNF levels were not different from the 10-fold increase observed in control experiments and were not reduced by ADO or MK-886. TNF production was independent of perfusate blood components and presumably due to low levels of endotoxin in the perfusate (70-90 ng/ml). These results indicate that ADO does not protect against PMA-induced acute lung injury by altering circulating levels of LT or TNF.

Cyclooxygenase inhibition prevents PMA-induced increases in lung vascular permeability.

The effect of cyclooxygenase inhibition in phorbol myristate acetate (PMA)-induced acute lung injury was studied in isolated constant-flow blood-perfused rabbit lungs. PMA caused a 51% increase in pulmonary arterial pressure (localized in the arterial and middle segments as measured by vascular occlusion pressures), a 71% increase in microvascular permeability (measured by the microvascular fluid filtration coefficient, Kf), and a nearly threefold increase in perfusate thromboxane (Tx) B2 levels. Cyclooxygenase inhibition with three chemically dissimilar inhibitors, indomethacin (10(-7) and 10(-6) M), meclofenamate (10(-6) M), and ibuprofen (10(-5) M), prevented the Kf increase without affecting the pulmonary arterial pressure increase or resistance distribution changes after PMA administration. The specific role of TxA2 was investigated by pretreatment with OKY-046, a specific Tx synthase inhibitor, or infusion of SQ 29548, a TxA2 receptor antagonist; both compounds failed to protect against either the PMA-induced permeability or the vascular resistance increase. These results indicate that cyclooxygenase-mediated products of arachidonic acid other than TxA2 mediate the PMA-induced permeability increase but not the hypertension.

Cyclooxygenase inhibition prevents PMA-induced increase in pulmonary vascular permeability to albumin.

In a previous study, we demonstrated that phorbol myristate acetate-(PMA) induced injury in isolated blood-perfused rabbit lungs was characterized by increased pulmonary vascular resistance (PVR) and permeability to water as measured by fluid filtration coefficient (Kf). The Kf increase was prevented by pretreatment with three cyclooxygenase inhibitors, indomethacin, ibuprofen, and meclofenamate. Other studies have shown that PMA causes a decrease in pulmonary vascular surface area, probably due to the increase in arterial resistance. Measurement of Kf requires increased microvascular pressure, and therefore Kf estimates the permeability of the entire vascular bed. Thus the permeability of the flowing vessels may be overestimated by Kf. In this study, we chose to investigate the effect of PMA on vascular permeability to protein by measuring albumin leak. Because this measurement does not require a hydraulic stress, it is more likely to reflect the permeability of flowing vessels. PMA administration (5 x 10(-8) M) caused significant increases in both PVR and 125I-labeled bovine serum albumin leak. Cyclooxygenase inhibition with indomethacin, ibuprofen, or meclofenamate prevented the PMA-induced increase in albumin leak without affecting the PVR increase. These results suggest that cyclooxygenase-mediated products of arachidonic acid mediate the PMA-induced increase in vascular permeability to both water and protein.

Vascular resistance and Kf in normal and PMA-injured rabbit lungs: effects of adenosine.

The effects of adenosine (ADO) on pulmonary vascular resistance (PVR) distribution, vascular compliance (C), and permeability were determined in normal and PMA-injured isolated rabbit lungs perfused with a 1:1 mixture of 6% albumin in Krebs-Henseleit buffer and autologous blood. ADO or vehicle was continuously infused into the reservoir at 1,4, or 5 mumol/min after a 1-mumol bolus of ADO or vehicle. The capillary filtration coefficient (Kf) and arterial, venous, and double occlusion pressures were measured at baseline and 30 min after phorbol myristate acetate (PMA; 4 x 10(-8) M) or vehicle. Perfusate differential and total leukocyte counts as well as adenine nucleotides, 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 (TxB2) concentrations were determined at each measurement period. ADO was recovered as hypoxanthine and inosine in the perfusate. ADO alone did not alter PVR, C, Kf, or TxB2 but reduced 6-keto-PGF1 alpha levels. PMA induced an increase in Kf (0.024 +/- 0.002 to 0.040 +/- 0.006 g.cmH2O-1.min-1, P less than 0.05) that was completely blocked by 4 or 5 mumol/min ADO. PVR increased by 63 +/- 11% after PMA, primarily in the arteries and arterial and venous microvessels. The postcapillary resistance increase was blunted by 4 mumol/min ADO; 5 mumol/min ADO prevented the PVR increase in all segments. ADO did not affect the initial adherence of neutrophils in the lung or the PMA-induced 87 +/- 2% decrease in circulating leukocytes (greater than 98% lymphocytes) or threefold increase in TxB2 levels. These results suggest that protection by ADO is not mediated by the altering of cyclooxygenase products or by leukocyte adherence.

Cyclooxygenase inhibitors prevent ethchlorvynol-induced injury in rat and rabbit lungs.

The effect of three chemically dissimilar cyclooxygenase inhibitors on ethchlorvynol-(ECV) induced acute lung injury was studied in isolated buffer-perfused rat and blood-perfused rabbit lungs. ECV caused the microvascular fluid filtration coefficient (Kf) to increase by greater than threefold in the rat lungs and twofold in the rabbit lungs. ECV caused increased pulmonary vascular resistance (PVR) and microvascular pressure measured by the double occlusion technique (Pdo) compared with the vehicle control group in the rat experiments. However, ECV had no effect on PVR or Pdo in the rabbit experiments. Pretreatment with the cyclooxygenase inhibitors indomethacin, ibuprofen, and meclofenamate prevented the increase in microvascular permeability in both the rat and rabbit lung preparations. The cyclooxygenase inhibitors also prevented the ECV-induced PVR and Pdo increases in the rat lungs but had no effect on PVR or Pdo in the rabbit lungs. These results indicate that cyclooxygenase products of arachidonate metabolism mediate the ECV-induced Kf increase in both isolated rat and rabbit lungs.

Rapid micromeasurement of neutrophil exocytosis.

A simple method is described for the visual detection of neutrophil granule exocytosis. The microassay measures release of myeloperoxidase from azurophilic granules following neutrophil stimulation and can be accomplished in less than 30 min. The assay is sensitive, reproducible from day to day, and does not require separation of leukocytes from the reaction before enzyme assay. The procedure is also applicable to the rapid measurement of the suppression of degranulation by antiinflammatory agents.

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-alpha (rTNF) stimulated increased generation of superoxide anion (O2-) 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 O2- in response to C5a and f-Met-Leu-Phe (FMLP). Recombinant TNF did not enhance O2- 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.