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.

C-reactive protein inhibits chemotactic peptide-induced p38 mitogen-activated protein kinase activity and human neutrophil movement.

Serum levels of the acute-phase reactant, C-reactive protein (CRP), increase dramatically during acute inflammatory episodes. CRP inhibits migration of neutrophils toward the chemoattractant, f-Met-Leu-Phe (fMLP) and therefore acts as an anti-inflammatory agent. Since tyrosine kinases are involved in neutrophil migration and CRP has been shown to decrease phosphorylation of some neutrophil proteins, we hypothesized that CRP inhibits neutrophil chemotaxis via inhibition of MAP kinase activity. The importance of p38 MAP kinase in neutrophil movement was determined by use of the specific p38 MAP kinase inhibitor, SB203580. CRP and SB203580 both blocked random and fMLP-directed neutrophil movement in a concentration-dependent manner. Additionally, extracellular signal-regulated MAP kinase (ERK) was not involved in fMLP-induced neutrophil movement as determined by use of the MEK-specific inhibitor, PD98059. Blockade of ERK with PD98059 did not inhibit chemotaxis nor did it alter the ability of CRP or SB203580 to inhibit fMLP-induced chemotaxis. More importantly, CRP inhibited fMLP-induced p38 MAP kinase activity in a concentration-dependent manner as measured by an in vitro kinase assay. Impressively, CRP-mediated inhibition of p38 MAP kinase activity correlated with CRP-mediated inhibition of fMLP-induced chemotaxis (r = -0.7144). These data show that signal transduction through p38 MAP kinase is necessary for neutrophil chemotaxis and that CRP intercedes through this pathway in inhibiting neutrophil movement.

Adenosine prevents permeability increase in oxidant-injured endothelial monolayers.

Adenosine is thought to prevent or reduce the increase in permeability, which is a hallmark of oxidant injury to endothelium. However, the effect of adenosine on endothelial cells directly exposed to oxidant species has not been demonstrated in vitro. By measuring the passage of Evan's blue dye-labeled albumin across confluent monolayers, we demonstrated the ability of adenosine (0.1-100 microM) to lower basal permeability of human umbilical vein endothelial cells in a concentration-dependent fashion and prevent the permeability increase induced by exposure of the cells to xanthine plus xanthine oxidase (X/XO). Whereas pretreatment of monolayers for 10 min with adenosine (10 and 100 microM) prevented the X/XO-induced permeability increase, these same concentrations of adenosine failed to increase intracellular adenosine 3',5'-cyclic monophosphate in X/XO-exposed cells. The protective effect of adenosine on endothelial monolayers was mimicked by adenosine amine congener and 5'-(N-ethylcarboxamido)adenosine but not by other agonists examined. Hence, the protective effect of adenosine against oxidant injury may include an adenosine 3',5'-cyclic monophosphate-independent mechanism by direct action of adenosine at A1 receptors on endothelial cells.

Increased gelatinolytic activity in bronchoalveolar lavage fluid in stable lung transplant recipients.

Proteolytic enzymes have been proposed to play a role in the pathogenesis of various inflammatory pulmonary diseases accompanied by parenchymal remodeling. To assess the role of inflammatory cells and proteolytic enzymes in the development of chronic allograft rejection after lung transplantation, bronchoalveolar lavage fluid (BALF) samples from clinically stable lung transplant (LT) recipients (i.e., without evidence of active infection or rejection), heart transplant (HT) recipients, and healthy volunteers (NL) were analyzed for total white blood cell (WBC) count and differential cell count, along with gelatinolytic/type IV collagenolytic activity. The LT group displayed a significantly increased total WBC count, neutrophil count, and percent neutrophils compared with the NL group, confirming the presence of inflammation. Furthermore, gelatin zymography revealed a significant increase in activity of the 72 and 92 kD gelatinases in the LT group compared with the NL group. A positive correlation existed between neutrophil counts and the increase in proteolytic activity. Immunosuppressive therapy did not account for the findings, since no significant difference in cell counts or proteolytic activity existed between the NL and HT control groups. These findings, together with those of others that relate chronic lung allograft dysfunction to an increase in BALF neutrophils and collagen matrix remodeling, collectively indicate that up-regulated proteolytic activity may have a role in chronic rejection after lung transplantation.

Role of C-reactive protein in acute lung injury.

Excessive inflammation caused by unregulated inflammatory processes can lead to disease. One example of this is seen in acute lung injury in which an individual is unable to regulate an inflammatory response in the lungs, with the net effect of self-induced tissue injury and loss of organ function. The acute-phase reactant, C-reactive protein, inhibits acute lung injury in animal models and, in this regard, acts as a major anti-inflammatory agent. Therefore, understanding the mechanism by which C-reactive protein elicits this inhibitory effect may provide important information about the design of therapeutic agents for the prevention or treatment of inflammation-mediated tissue injury and resultant loss of organ function.

C-reactive protein inhibits increased pulmonary vascular permeability induced by fMLP in isolated rabbit lungs.

Increased serum concentrations of C-reactive protein (CRP) have previously been shown to downregulate neutrophil (PMN) influx and vascular permeability changes in models of localized inflammation such as alveolitis [R. M. Heuertz, D. Xia, D. Samols, and R. O. Webster, Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L649-L654, 1994]. Experiments in isolated, buffer-perfused rabbit lungs were used to determine whether CRP attenuates vascular lung injury induced by PMNs stimulated with the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Peritoneal PMN were added to the perfusate of lungs from PMN-depleted rabbits. Stimulation with fMLP produced an immediate and transient rise in pulmonary artery pressure that peaked at 35-40 cmH2O. An increase in permeability occurred 60 min after fMLP, which was marked by a 70% increase (P < 0.05) in filtration coefficient and bronchoalveolar lavage (BAL) protein concentration. CRP pretreatment of PMNs prevented fMLP-induced increases in permeability and significantly reduced the BAL protein below levels in control lungs (P < 0.05). CRP pretreatment of PMNs did not alter the pulmonary arterial pressor response to fMLP and had no effect on the production of leukotrienes, thromboxane, prostacyclin, or superoxide anion induced by fMLP. The mechanism by which CRP protects lung tissue from vascular injury induced by activation of PMNs remains unclear.

Peptides derived from C-reactive protein inhibit neutrophil alveolitis.

C-reactive protein (CRP) is the classic acute phase reactant in humans, with serum levels elevated up to 1000-fold after the onset of inflammation. CRP inhibits chemotaxis of complement (C5a)-, LTB4-, IL-8-, and FMLP-stimulated neutrophils in vitro, and rabbits and transgenic mice with elevated serum CRP levels exhibit diminished neutrophil infiltration and vascular permeability in models of chemotactic factor-induced alveolitis. To evaluate the mechanism of CRP inhibition on chemoattractant-induced neutrophil inflammation in vivo, experiments were performed in mice infused with peptides of human CRP shown to inhibit C5a- and FMLP-stimulated neutrophil chemotaxis in vitro. After direct tracheal instillation of FMLP, mice previously injected via the retro-orbital plexus with CRP peptide 77-82 or 201-206 showed significant reductions (up to 90%) of neutrophils in the bronchoalveolar lavage fluid compared with vehicle-treated mice. Both CRP peptides also significantly (up to 55%) inhibited the increase in alveolar total protein levels. Control injections of native rabbit CRP (3 microM) inhibited neutrophil influx by 93% and protein leak by 55% in mice intratracheally instilled with FMLP. Despite similar levels of inhibition, approximately 10-fold more peptide by weight than native CRP was required. These data suggest that CRP degradation products at sites of tissue injury, in particular CRP peptides 77-82 and 201-206, are anti-inflammatory and can diminish lung injury by a reduction in neutrophil influx and protein leakage into alveoli following FMLP-induced inflammation.

Transgenic mice expressing rabbit C-reactive protein exhibit diminished chemotactic factor-induced alveolitis.

The acute phase protein, C-reactive protein (CRP), can increase more than a thousandfold during acute inflammatory states, and it is known to modulate neutrophil-mediated inflammatory responses. We have previously shown that CRP inhibits chemotaxis of C5a-stimulated neutrophils in vitro and that rabbits with elevated CRP blood levels exhibit diminished pulmonary vascular permeability and neutrophil infiltration in a model of alveolitis. To study the effect of CRP on alveolitis induced by different chemoattractants, transgenic mice capable of expressing rabbit CRP in a dietary-inducible fashion were treated with inflammatory doses of the chemoattractants. Intratracheal installation of FMLP (8 x 10(-10) mol), LTB4 (2 x 10(-11) mol), or IL-8 (5 x 10(-12) mol) in normal CF1 mice resulted in significant (p<0.05) influx of neutrophils and protein into the alveolar space. Transgenic mice with elevated plasma levels of CRP showed significantly (p<0.05) diminished infiltration of neutrophils into bronchoalveolar lavage fluid (BALF) and significant reduction in BALF protein compared with that in normal mice. Rabbit CRP (10 to 500 micrograms/ml) inhibited in vitro neutrophil chemotaxis in a concentration-dependent fashion when stimulated by the various chemoattractants examined. These data show that rabbit CRP can modify both in vivo and in vitro neutrophil responses to several classes of chemoattractants and that CRP has a significant protective effect in alveolitis by reducing neutrophil influx and protein leakage into the lung.

Bidirectional effects of hepatic ischemia/reperfusion on E. coli-induced TNF-alpha gene expression.

We tested the hypothesis that gram-negative bacteremia (GNB) and brief (30 min) reductions in the hepatic O2 supply by low-flow ischemia differentially modulate tumor necrosis factor-alpha (TNF-alpha) gene expression owing to sequence-specific activation of cyclooxygenase vs. complement (C) pathways. Buffer-perfused Sprague-Dawley rat livers (n = 82) were studied over 180 min after intraportal 10(9) live E. coli serotype 055:B5 (EC) or 0.9% NaCl (NS) at t = 0. Compared with EC and NS controls receiving constant-flow perfusion, sequential GNB and ischemia/reperfusion (I/R) were studied in EC + 30 I/R and NS + 30 I/R livers, in which 30 min of ischemia (I) beginning 0.5 h after EC or NS was followed by 120 min of reperfusion (R). This sequence was reversed in 30 I/R + EC and 30 I/R + NS groups. Bacterial clearance, bioactive and antigenic TNF-alpha, prostaglandin E2 (PGE2), and hepatic O2 uptake and performance were serially assessed. Venous TNF-alpha increased in EC controls to peak at 155 +/- 29 U/ml after 180 min (P < 0.001 vs. NS controls) as did hepatic TNF-alpha mRNA. Both TNF-alpha transcripts and protein levels were markedly attenuated in EC + 30 I/R (P < 0.001 vs. EC) despite equivalent EC clearance by Kupffer cells. Indomethacin (10(-5) M) decreased I/R-induced PGE2 secretion and restored TNF-alpha to control levels. In contrast, TNF-alpha levels in 30 I/R + EC perfusates exceeded those of EC + 30 I/R livers (P < 0.05) and were indistinguishable from EC controls. Allopurinol pretreatment but not heat inactivation of C or infusion of soluble human complement receptor type 1 inhibited TNF-alpha production in 30 I/R + EC organs. These results identify a novel sequence-dependent interaction whereby hepatic O2 deprivation after GNB downregulates TNF-alpha via generation of cyclooxygenase metabolites, whereas ischemia preceding GNB increases cytokine expression via reactive O2 species but not C activation.

Co-chemotactic effect of Gc-globulin (vitamin D binding protein) for C5a. Transient conversion into an active co-chemotaxin by neutrophils.

Gc-globulin (vitamin D binding protein) has been shown to augment significantly the leukocyte chemotactic activity of the activated C peptides C5a and C5adesArg (i.e., the co-chemotactic effect). However, the mechanism of chemotaxis enhancement is not known. To investigate the role that the neutrophil plays in this process, cells were co-incubated with Gc-globulin for up to 45 min and washed, and their subsequent chemotactic response to a suboptimal concentration of C5a alone was measured during a 30-min assay. The generation of co-chemotactic activity during the preincubation period was time dependent, showed minimal activity for the first 10 min and a steep rise from 10 to 20 min, and was maximal and stable at 30 min. The binding of radiolabeled Gc-globulin by neutrophils at 37 degrees C mirrored this time-dependent generation of C5a co-chemotactic activity, with stable cellular levels achieved between 30 and 45 min at 36 +/- 4 fmol (2 +/- 0.1 ng)/10(6) cells. The binding of radiolabeled Gc-globulin and the generation of co-chemotactic activity were dependent upon physiologic temperatures (37 degrees C) and levels of Ca2+ (1.3 mM) and Mg2+ (0.8 mM), and were inhibited by an Ab to Gc-globulin. Finally, the C5a co-chemotactic activity of Gc-globulin would decay rapidly if neutrophils were washed and then incubated a second time at 37 degrees C before chemotaxis to C5a. These results demonstrate that neutrophils bind exogenous Gc-globulin and generate C5a co-chemotactic activity in a time-, temperature-, and divalent cation-dependent manner. Moreover, this activity is transient if neutrophils lack a continuous supply of Gc-globulin.

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 C5a des Arg-induced neutrophil alveolitis in transgenic mice expressing C-reactive protein.

C-reactive protein (CRP) is the classic acute phase reactant in man with serum levels elevated up to 1,000-fold after the onset of inflammation. CRP inhibits chemotaxis of complement (C5a)-stimulated neutrophils in vitro and rabbits with elevated serum CRP levels exhibit diminished neutrophil infiltration and vascular permeability in a model of C5a-induced alveolitis. To specifically evaluate the effect of CRP on C5a-induced neutrophil inflammation in vivo, experiments were performed in transgenic mice capable of expressing rabbit CRP in an inducible fashion. After direct instillation of a known inflammatory agent (C5a des Arg) into the airways, transgenic mice with high plasma levels of CRP showed significantly diminished infiltration of neutrophils into bronchoalveolar lavage fluid (BALF) and a significant reduction of BALF total protein levels compared with normal mice. These data indicate that CRP can diminish lung injury by a reduction in neutrophil influx and protein leakage into alveoli following complement-induced inflammation.

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.

Effect of phenytoin on acute lung injuries in unanesthetized sheep.

OBJECTIVE: To determine if the intravenous administration of phenytoin attenuates or prevents acute experimental lung injury. DESIGN: Placebo-controlled, longitudinal animal investigative study. SETTING: University research laboratory. SUBJECTS: Sixteen yearling female lambs weighing 30 +/- 3 kg. INTERVENTION: After administration of anesthesia, the animals were endotracheally intubated and mechanically ventilated. Using sterile techniques, four thoracotomies were performed. Through the left fourth intercostal space, cannulas for pressure measurements were inserted directly into the main pulmonary artery and left atrium. An ultrasound flow cuff for determination of cardiac output was placed around the main pulmonary artery. Through the left tenth intercostal space, the diaphragmatic and mediastinal parietal pleura were widely cauterized. Through the right tenth intercostal space, the caudal mediastinal lymph node was identified and divided at the caudal margin of the right pulmonary ligament, and a 1- to 2-cm portion of the node distal to the ligament was resected. The diaphragmatic and mediastinal parietal pleura were widely cauterized. Through the right sixth intercostal space, the efferent duct (or ducts) was identified, ligated at the site of entry into the thoracic duct, and cannulated. The lymph cannula was brought to the outside of the thorax through a separate stab wound. MEASUREMENTS AND MAIN RESULTS: Unanesthetized sheep were studied 7 to 10 days after surgery. Hemodynamic, lung fluid balance, and arterial blood variables were measured in uninjured sheep and in sheep injured by intravenous infusions of Escherichia coli endotoxin (1 microgram/kg iv over 30 mins), air bubbles (0.056 to 0.074 mL/kg/min over 4 hrs), or oleic acid (0.06 mL/kg over 1 hr). The sheep were studied when untreated and after pretreatment with phenytoin. We found that the expected increase in protein-rich lung lymph flow with injuries, resulting from increased microvascular permeability in the lungs, was attenuated by phenytoin when the lungs were injured by endotoxin or air bubbles. In contrast, phenytoin had no effect on oleic acid-induced lung injury or on uninjured lungs. CONCLUSIONS: Phenytoin attenuates acute lung injuries in sheep that are thought to be caused by stimulation of host inflammatory responses (e.g., endotoxin and air bubbles), but has no effect on direct injuries to the lungs (e.g., oleic acid). A plausible mechanism for this finding is phenytoin inhibition of polymorphonuclear leukocyte function.

Localization and quantitation of the vitamin D binding protein (Gc-globulin) in human neutrophils.

Plasma-derived vitamin D binding protein (DBP) is an important physiologic regulator of the neutrophil chemotactic response to activated complement. A cell-associated form of DBP has been observed in numerous cell types. We now report that mature, circulating human neutrophils also contain cell-associated DBP. Immunofluorescence studies of normal untreated neutrophils showed the presence of DBP on the cell surface. Western blotting of detergent-soluble neutrophil lysates with a polyclonal anti-DBP showed two major immunoreactive bands, one with an apparent molecular weight of 56 Kd (identical to purified plasma-derived DBP) and a second less prominent band at 12 to 14 Kd. Quantitation of the immunoreactive bands by video densitometry indicated that normal human neutrophils contain 1.5 +/- 0.8 ng DBP/10(6) cells (n = 9). Immunoprecipitation of detergent-soluble lysates with the polyclonal anti-DBP showed only the 56-Kd form by Western blotting. In contrast, a monoclonal anti-DBP immunoprecipitated the 12 to 14 Kd form of DBP from lysates of surface-radioiodinated cells. Western blots of subcellular fractions showed that immunoreactive bands were found in the specific (secondary) granule and plasma-membrane fractions. In addition, pretreatment of neutrophils with 10 nmol/L phorbol myristate acetate (PMA) resulted in approximately a 50% reduction in the amount of DBP in both the specific granule and plasma-membrane fractions. Finally, analysis of the cell-free supernates showed that DBP was spontaneously released into the extracellular milieu: moreover, this release was enhanced if the cells were first stimulated with C5a, formyl-norleucyl-leucyl-phenylalanine (fNLP) or PMA.

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.

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.

Rabbits with elevated serum C-reactive protein exhibit diminished neutrophil infiltration and vascular permeability in C5a-induced alveolitis.

In previous studies, we have shown that C-reactive protein (CRP) inhibits chemotaxis of neutrophils to complement fragments in vitro. To evaluate the effect of CRP on C5a-induced inflammation in vivo, a rabbit model of alveolitis was used. Rabbits pretreated with subcutaneous injections of croton oil had serum CRP increase from undetectable levels to 270 +/- 70 micrograms/ml 48 hours later. Rabbits were intubated and C5a des arg (10 micrograms/ml) instilled directly into the lungs via an endotracheal tube. Four to six hours later, the animals were killed and bronchoalveolar lavage performed. Rabbits pretreated with croton oil had significantly (P < 0.01) reduced C5a des arg-stimulated neutrophil infiltration (30 +/- 5%) into alveolar air spaces compared to untreated rabbits (64 +/- 9%). Increased numbers of total leukocytes in the alveolar washes coincided with increased neutrophil numbers whereas alveolar macrophages remained unchanged in all groups. Rabbits pretreated with croton oil also had a significant decrease (P < 0.05) in total protein (320 +/- 50 micrograms/ml) in lavage fluid after C5a instillation compared with untreated animals (850 +/- 140 micrograms/ml). In vitro, rabbit CRP (50 micrograms/ml) added to normal rabbit serum significantly (P < 0.05) inhibited chemotaxis of human neutrophils by 41%. Finally, direct intravenous pretreatment of rabbits with purified CRP also significantly reduced C5a-induced alveolitis. The CRP-C5a group had 33 +/- 10% neutrophil infiltration, a significant (P < 0.01) reduction from the C5a group (71 +/- 6%). The total protein content of the CRP-C5a rabbits was 986 +/- 165 micrograms/ml in the lavage fluid, which was significantly (P < 0.05) lower than the C5a group (1645 +/- 363 micrograms/ml). Therefore, CRP inhibits the development of neutrophil alveolitis and protein leakage in vivo and inhibits neutrophil chemotaxis in vitro. These data indicate that CRP offers a protective effect in neutrophil-mediated lung injury by reducing neutrophil influx and protein leak.

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.