Oxygen in the alveolar air space mediates lung inflammation in acute pancreatitis

During the early stages of acute pancreatitis, acute respiratory distress syndrome often occurs. This is associated with the release of proinflammatory mediators into the blood, but it remains unclear why these mediators induce inflammation especially in the lung. One of the first events occurring during the progression of acute pancreatitis is the induction of P-selectin expression in the endothelial cells of the lung. This expression has been associated with the generation of superoxide radicals by circulating xanthine oxidase. Because this enzyme needs molecular oxygen to perform the reaction, we have hypothesized that oxygen present in the alveolar space favors the generation of free radicals by xanthine oxidase and explains why P-selectin is expressed only in the lung. For this purpose, we evaluated the progression of the inflammatory process in rats with induced acute pancreatitis and one lung breathing nitrogen while the other lung continued breathing air. Acute pancreatitis was induced by intraductal administration of taurocholate and myeloperoxidase; P-selectin expression was measured 3 h after induction. Results indicated that, in the absence of oxygen in the alveolar space, the xanthine oxidase-dependent P-selectin expression did not occur and lung inflammation was significantly reduced.     

H(2)O(2) and PARS mediate lung P-selectin upregulation in acute pancreatitis

P-selectin and circulating xanthine oxidase are involved in the process of neutrophil infiltration into the lung associated with acute pancreatitis. This study investigated the mediators that trigger the upregulation of P-selectin in this process. Pancreatitis was induced in rats by intraductal administration of 5% sodium taurocholate. P-selectin expression was measured using radiolabeled antibodies. Neutrophil infiltration and PAF levels were also evaluated. The role of superoxide radical, H(2)O(2), or the enzyme poly (ADP-ribose) synthetase (PARS) on these processes was determined in groups of animals treated with the corresponding inhibitors. Pancreatitis was associated with an increase in P-selectin expression in the lung. Inhibition of PARS or H(2)O(2) abrogated P-selectin upregulation, PAF generation, and neutrophil recruitment. Superoxide dismutation prevented neutrophil recruitment and PAF generation, but had no effect on P-selectin expression. We conclude that during acute pancreatitis, upregulation of P-selectin in the pulmonary endothelium is triggered by H(2)O(2) and PARS activity.     

Role of extracellular superoxide in neutrophil activation: interactions between xanthine oxidase and TLR4 induce proinflammatory cytokine production

Reactive oxygen species (ROS) contribute to neutrophil activation and the development of acute inflammatory processes in which neutrophils play a central role. However, there is only limited information concerning the mechanisms through which extracellular ROS, and particularly cell membrane-impermeable species, such as superoxide, enhance the proinflammatory properties of neutrophils. To address this issue, neutrophils were exposed to superoxide generating combinations of xanthine oxidase and hypoxanthine or lumazine. Extracellular superoxide generation induced nuclear translocation of nuclear factor-kappaB (NF-kappaB) and increased neutrophil production of the NF-kappaB-dependent cytokines tumor necrosis factor-alpha (TNF-alpha) and macrophage inhibitory protein-2 (MIP-2). In contrast, there were no changes in TNF-alpha or MIP-2 expression when neutrophils lacking Toll-like receptor-4 (TLR4) were exposed to extracellular superoxide. Immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer (FRET) studies demonstrated association between TLR4 and xanthine oxidase. Exposure of neutrophils to heparin attenuated binding of xanthine oxidase to the cell surface as well as interactions with TLR4. Heparin also decreased xanthine oxidase-induced nuclear translocation of NF-kappaB as well as production of proinflammatory cytokines. These results demonstrate that extracellular superoxide has proinflammatory effects on neutrophils, predominantly acting through an TLR4-dependent mechanism that enhances nuclear translocation of NF-kappaB and increases expression of NF-kappaB-dependent cytokines.     

Xanthine oxidase and activated neutrophils cause oxidative damage to skeletal muscle after contractile claudication

We previously showed oxidative damage and edema within skeletal muscle after contractile claudication. To investigate the sources of this oxidative damage in the gastrocnemius muscle, we administered allopurinol (Allo, to inhibit xanthine oxidase) and cyclophosphamide (Cyclo, to deplete neutrophils) before inducing contractile claudication in male Sprague Dawley rats. Contractile claudication (ligated stimulated, LS) caused a significant increase in xanthine oxidase activity [sham ligated stimulated (SS) = 2.57 +/- 0.07; LS = 3.22 +/- 0.07] and neutrophil infiltration (SS = 0.47 +/- 0.03; LS = 0.91 +/- 0.10) compared with controls (SS), and this was associated with increased lipid peroxidation, protein oxidation, muscle damage, and edema. Pretreatment with Allo attenuated the increase in xanthine oxidase activity and attenuated lipid hydroperoxides (control LS = 12.85 +/- 0.50; Allo LS = 9.96 +/- 0.71), muscle damage, and neutrophil infiltration (control LS = 0.91 +/- 0.10; Allo LS = 0.61 +/- 0.07). This latter finding suggests that xanthine oxidase-derived oxidants are chemotactic to neutrophils. Pretreatment with Cyclo reduced neutrophil infiltration (control LS = 0.91 +/- 0.10; Cyclo LS = 0.55 +/- 0.02) and attenuated lipid peroxidation (control LS = 12.85 +/- 0.50; Cyclo LS = 6.462 +/- 0.62), protein oxidation (control LS = 2.59 +/- 0.47; Cyclo LS = 1.77 +/- 0.60), muscle damage, and edema. Together, these data indicate that contractile claudication causes an increase in xanthine oxidase activity and neutrophils in muscle and that inhibition of these oxidant sources protects against oxidative stress, muscle damage, and edema.     

Rapid upregulation of endothelial P-selectin expression via reactive oxygen species generation

Endothelial cell ICAM-1 upregulation in response to TNF-alpha is mediated in part by reactive oxygen species (ROS) generated by the endothelial membrane-associated NADPH oxidase and occurs maximally after 4 h as the synthesis of new protein is required. However, thrombin-stimulated P-selectin upregulation is bimodal, the first peak occurring within minutes. We hypothesize that this early peak, which results from the release of preformed P-selectin from within Weibel-Palade bodies, is mediated in part by ROS generated from the endothelial membrane-associated xanthine oxidase. We found that this rapid expression of P-selectin on the surface of endothelial cells was accompanied by qualitatively parallel increases in ROS generation. Both P-selectin expression and ROS generation were inhibited, dose dependently, by the exogenous administration of disparate cell-permeable antioxidants and also by the inhibition of either of the known membrane-associated ROS-generating enzymes NADPH oxidase or xanthine oxidase. This rapid, posttranslational cell signaling response, mediated by ROS generated not only by the classical NADPH oxidase but also by xanthine oxidase, may well represent an important physiological trigger of the microvascular inflammatory response.     

Translational inhibition of E-selectin expression stimulates P-selectin-dependent neutrophil recruitment

Although known for its role in hemostasis, there is a growing body of evidence that thrombin can induce leukocyte recruitment and contribute to the inflammatory response. An in vitro parallel-plate flow chamber was used to systematically examine thrombin-induced neutrophil interactions with human endothelium. Stimulation of endothelial cells with thrombin (1 U/ml) resulted in an immediate, P-selectin-dependent increase in neutrophil rolling and adhesion that was comparable in magnitude to optimal levels of histamine (the classical inducer of P-selectin). However, thrombin, but not histamine, induced a delayed (4 h) E-selectin-dependent rolling similar to that of tumor necrosis factor-alpha, suggesting that thrombin has the unique ability to recruit neutrophils by an early P-selectin and a delayed E-selectin pathway. Surprisingly, inhibition of E-selectin expression with the general protein synthesis inhibitor cycloheximide induced P-selectin expression 4 h after thrombin stimulation. Cycloheximide and thrombin (4 h) induced sufficient P-selectin-dependent rolling to recruit as many neutrophils as were recruited with 4 h of stimulation with thrombin alone. Histamine in the presence of cycloheximide or cycloheximide alone did not evoke the P-selectin response at 4 h, suggesting that this was not due to direct cycloheximide induction of P-selectin. Treatment of endothelium with tumor necrosis factor-alpha (an E-selectin inducer) and cycloheximide also eliminated E-selectin expression but, much like thrombin, induced P-selectin expression and neutrophil recruitment. In conclusion, inhibition of E-selectin via protein synthesis inhibition activates the protein synthesis-independent pathway of P-selectin expression to support adequate leukocyte recruitment.     

Role of O2- in neutrophil recruitment into sites of dermal and pulmonary vasculitis

Using analogous models of acute dermal vasculitis and alveolitis in rats, we have examined the role of oxygen-derived metabolities in the tissue damage associated with neutrophil influx into sites of immune complex deposition. In the lung, as previously reported, catalase and deferoxamine are highly protective, while superoxide dismutase (SOD) has a transient protective effect. The xanthine oxidase inhibitors, allopurinol, and lodoxamide, are also protective. In the skin, neither catalase (which has been covalently linked to the antibody) nor deferoxamine is protective, suggesting that H2O2 and iron are not absolutely required for the development of dermal vasculitis. In the skin, SOD, as well as the inhibitors of xanthine oxidase, have protective effects. These data suggest that the neutrophil-mediated pathways of immune complex injury in the dermal and pulmonary microvascular compartments are fundamentally different. As a measurement of neutrophil accumulation, measurements of myeloperoxidase in tissue extracts have been employed. In both the lung and skin, the protective effects of SOD and the xanthine oxidase inhibitors are paralleled by reductions in neutrophil influx into sites of injury. In contrast, catalase and deferoxamine have no effect on neutrophil accumulation. These data suggest that vascular beds in rat skin and lung are fundamentally different with respect to mechanisms of acute immune complex mediated injury. The data also provide evidence that O2- contributes significantly to the accumulation of neutrophils.     

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.     

Role of superoxide in hemorrhagic shock-induced P-selectin expression

Superoxide has been implicated in the regulation of endothelial cell adhesion molecule expression and the subsequent initiation of leukocyte-endothelial cell adhesion in different experimental models of inflammation. The objective of this study was to assess the contribution of oxygen radicals to P-selectin expression in a murine model of whole body ischemia-reperfusion, i.e., hemorrhage-resuscitation (H/R), with the use of different strategies that interfere with either the production (allopurinol, CD11/CD18-deficient or p47(phox)-/- mice) or accumulation [intravenous superoxide dismutase (SOD), mutant mice that overexpress SOD] of oxygen radicals. P-selectin expression was quantified in different regional vascular beds by use of the dual-radiolabeled monoclonal antibody technique. H/R elicited a significant increase in P-selectin expression in all vascular beds. This response was blunted in SOD transgenic mice and in wild-type mice receiving either intravenous SOD or the xanthine oxidase inhibitor allopurinol. Mice genetically deficient in either a subunit of NADPH oxidase or the leukocyte adhesion molecule CD11/CD18 also exhibited a reduced P-selectin expression. These results implicate superoxide, derived from both xanthine oxidase and NADPH oxidase, as mediators of the increased P-selectin expression observed in different regional vascular beds exposed to hemorrhage and retransfusion.     

Hsp72 induces inflammation and regulates cytokine production in airway epithelium through a TLR4- and NF-kappaB-dependent mechanism

Heat shock proteins are generally regarded as intracellular proteins acting as molecular chaperones; however, Hsp72 is also detected in the extracellular compartment. Hsp72 has been identified in the bronchoalveolar lavage fluid (BALF) of patients with acute lung injury. To address whether Hsp72 directly activated airway epithelium, human bronchial epithelial cells (16HBE14o-) were treated with recombinant Hsp72. Hsp72 induced a dose-dependent increase in IL-8 expression, which was inhibited by the NF-kappaB inhibitor parthenolide. Hsp72 induced activation of NF-kappaB, as evidenced by NF-kappaB trans-activation and by p65 RelA and p50 NF-kappaB1 binding to DNA. Endotoxin contamination of the Hsp72 preparation was not responsible for these effects. Next, BALB/c mice were challenged with a single intratracheal inhalation of Hsp72 and killed 4 h later. Hsp72 induced significant up-regulation of KC, TNF-alpha, neutrophil recruitment, and myeloperoxidase in the BALF. A similar challenge with Hsp72 in TLR4 mutant mice did not stimulate the inflammatory response, stressing the importance of TLR4 in Hsp72-mediated lung inflammation. Last, cultured mouse tracheal epithelial cells (MTEC) from BALB/c and TLR4 mutant and wild-type mice were treated ex vivo with Hsp72. Hsp72 induced a significant increase in KC expression from BALB/c and wild-type MTEC in an NF-kappaB-dependent manner; however, TLR4 mutant MTEC had minimal cytokine release. Taken together, these data suggest that Hsp72 is released and biologically active in the BALF and can regulate airway epithelial cell cytokine expression in a TLR4 and NF-kappaB-dependent mechanism.     

Extracellular Hsp72, an endogenous DAMP,is released by virally infected airway epithelial cells and activates neutrophils via Toll-like receptor (TLR)-4

Background

Neutrophils play an important role in the pathophysiology of RSV, though RSV does not appear to directly activate neutrophils in the lower airways. Therefore locally produced cytokines or other molecules released by virally-infected airway epithelial cells are likely responsible for recruiting and activating neutrophils. Heat shock proteins (HSPs) are generally regarded as intracellular proteins acting as molecular chaperones; however, HSP72 can also be released from cells, and the implications of this release are not fully understood.

Methods

Human bronchial epithelial cells (16HBE14o-) were infected with RSV and Hsp72 levels were measured by Western blot and ELISA. Tracheal aspirates were obtained from critically ill children infected with RSV and analyzed for Hsp72 levels by ELISA. Primary human neutrophils and differentiated HL-60 cells were cultured with Hsp72 and supernatants analyzed for cytokine production. In some cases, cells were pretreated with polymyxin B prior to treatment with Hsp72. IκBα was assessed by Western blot and EMSA''s were performed to determine NF-κB activation. HL-60 cells were pretreated with neutralizing antibody against TLR4 prior to Hsp72 treatment. Neutrophils were harvested from the bone marrow of wild type or TLR4-deficient mice prior to treatment with Hsp72.

Results

Infection of 16HBE14o- with RSV showed an induction of intracellular Hsp72 levels as well as extracellular release of Hsp72. Primary human neutrophils from normal donors and differentiated HL-60 cells treated with increasing concentrations of Hsp72 resulted in increased cytokine (IL-8 and TNFα) production. This effect was independent of the low levels of endotoxin in the Hsp72 preparation. Hsp72 mediated cytokine production via activation of NF-κB translocation and DNA binding. Using bone marrow-derived neutrophils from wild type and TLR4-mutant mice, we showed that Hsp72 directly activates neutrophil-derived cytokine production via the activation of TLR4.

Conclusion

Collectively these data suggest that extracellular Hsp72 is released from virally infected airway epithelial cells resulting in the recruitment and activation of neutrophils.     

Effects of L-carnitine on neutrophil-mediated ischemia-reperfusion injury in rat stomach

Reactive oxygen metabolites play an important role in ischemia-reperfusion related gastric injury. Primary sources of reactive oxygen metabolites seem to be the xanthine/xanthine oxidase system and neutrophils accumulating within the reperfused tissue. Tissue myeloperoxidase activity is an important index of neutrophil accumulation. The purpose of the present study was to clarify the effect of L-carnitine on the accumulation of neutrophils and neutrophil-induced gastric mucosal damage in rats exposed to ischemia-reperfusion. Rats were randomly divided into three groups: sham-operated, ischemia-reperfusion and ischemia-reperfusion plus L-carnitine groups. Ischemia was induced by clamping the celiac artery for 30 min and then reperfusion was established for 60 min. Gastric injury was assessed by measuring myeloperoxidase activity in gastric tissue. The neutrophil accumulation and hemorrhagic lesions due to ischemia-reperfusion in gastric mucosa were ascertained in a histological study. L-Carnitine (100 mg kg(-1)) administrated intravenously 5 min before ischemia significantly reduced both the gastric injury and myeloperoxidase activity compared with the ischemia-reperfusion group. The results suggest that L-carnitine provides marked protection against ischemia-reperfusion-related gastric injury which could be due to its ability to reduce neutrophil accumulation in ischemic tissue.     

Activated neutrophils increase microvascular permeability in skeletal muscle: role of xanthine oxidase

To determine the role of xanthine oxidase in the microvascular dysfunction produced by activated granulocytes, we examined the effect of xanthine oxidase depletion or inhibition on the increase in microvascular permeability produced by infusion of the neutrophil activator phorbol myristate acetate (PMA). Changes in vascular permeability were assessed by measurement of the solvent drag reflection coefficient for total plasma proteins (sigma) in rat hindquarters subjected to PMA infusion in xanthine oxidase-replete and -depleted animals, in animals pretreated with the xanthine oxidase inhibitor oxypurinol, and in animals depleted of circulating neutrophils by pretreatment with antineutrophil serum (ANS). Xanthine oxidase depletion was accomplished by administration of a tungsten-supplemented (0.7 g/kg diet) molybdenum-deficient diet. In animals fed the tungsten diet, muscle total xanthine dehydrogenase plus xanthine oxidase activity was decreased to less than 10% of control values. Estimates of sigma averaged 0.84 +/- 0.04 in control hindquarters, whereas PMA infusion was associated with a marked increase in microvascular permeability (decrease in sigma to 0.68 +/- 0.03). PMA infusion also caused an increase in the amount of the radical-producing oxidase form of xanthine oxidase (from 3.9 +/- 0.05 to 5.6 +/- 0.4 mU/g wet wt). ANS pretreatment attenuated this permeability increase (sigma = 0.77 +/- 0.04) and diminished the rise in xanthine oxidase activity (4.9 +/- 0.5 mU/g wet wt). Xanthine oxidase depletion with the tungsten diet or pretreatment with oxypurinol had no effect on this neutrophil-mediated microvascular injury (sigma = 0.69 +/- 0.06 and 0.67 +/- 0.03, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Xanthine oxidase activity in mouse pancreas: effects of caerulein-induced acute pancreatitis

The development of acute pancreatitis involves a number of pathophysiological changes which result in pancreatic tissue damage. Data from several models of acute pancreatitis suggest that the in vivo conversion of the enzyme xanthine dehydrogenase to xanthine oxidase may cause tissue damage by the subsequent generation of oxygen-derived free radical products. In the present studies, acute pancreatitis was induced in mice by the administration of supramaximal secretory doses of caerulein, a cholecystokinin analogue. Pancreatic xanthine oxidase activity was observed to occur in the dehydrogenase form in both control and treated mice. Artifactual conversion to the oxidase form could be induced by exclusion of 2-mercaptoethanol and phenylmethylsulfonyl fluoride from the buffer during tissue preparation. These data indicate that no significant conversion of xanthine dehydrogenase to oxidase is associated with this model of acute pancreatitis in mice.     

Hsp72 overexpression accelerates the recovery from caerulein-induced pancreatitis

Background and Aims

Heat shock protein (Hsp) 72 is a molecular chaperone which is upregulated in response to a variety of stress situations and has a general cytoprotective function. Increased Hsp72 levels were implicated in protection from acute pancreatitis; a hypothesis which was not tested in a transgenic mouse model yet.

Methods

To analyze the role of Hsp72 during acute pancreatitis, well-characterized transgenic animals overexpressing rat Hsp72 (Hsp72 mice) under the control of the ß-actin promoter were subjected to caerulein- and L-arginine-induced acute pancreatitis. The severity of experimental pancreatitis was determined via serum lipase levels, morphometric evaluation and quantification of pancreatic edema/inflammation.

Results

Hsp72 mice displayed ∼100-times Hsp72 overexpression, but no changes in the remaining chaperones. Robust Hsp72 signal was observed in pancreatic acini, but not in islets or ductal cells. In both models, elevated Hsp72 did not protect from development of acute pancreatitis and the pancreatitis-associated lung injury, but accelerated recovery from caerulein-induced tissue injury (lower lipase levels, edema, inflammation and necrosis 36 h after caerulein administration). The observed protective function of Hsp72 in caerulein-induced pancreatitis is likely due to an attenuated NF-κB signalling.

Conclusions

Hsp72 overexpression accelerates the recovery from acute pancreatitis and may represent a potential treatment strategy.     

Leukocyte trafficking and myocardial reperfusion injury in ICAM-1/P-selectin-knockout mice

P-selectin and intercellular adhesion molecule-1 (ICAM-1) mediate early interaction and adhesion of neutrophils to coronary endothelial cells and myocytes after myocardial ischemia and reperfusion. In the present study, we examined the physiological consequences of genetic deletions of ICAM-1 and P-selectin in mice. In wild-type mice, after 1 h of ischemia followed by reperfusion, neutrophil influx into the area of ischemia was increased by 3 h with a peak at 24 h and a decline by 72 h. ICAM-1/P-selectin-deficient mice showed a significant reduction in neutrophils by immunohistochemistry or by myeloperoxidase activity at 24 h but no significant difference at 3 h. Infarct size (area of necrosis/area at risk) assessed 24 h after reperfusion was not different between wild-type and deficient mice after 30 min and 1 h of occlusion. Mice with a deficiency in both ICAM-1 and P-selectin have impaired neutrophil trafficking without a difference in infarct size due to myocardial ischemia-reperfusion.     

Neutrophil-induced changes in the biomechanical properties of endothelial cells: roles of ICAM-1 and reactive oxygen species

This study evaluated the changes in the biomechanical properties of endothelial cells (ECs) induced by neutrophil adhesion and the roles of ICAM-1 and reactive oxygen species (ROS) in modulating these changes. Neutrophil adherence to 24-h TNF-alpha-activated pulmonary microvascular ECs induced an increase in the apparent stiffness of ECs within 2 min, measured with magnetic twisting cytometry. An anti-ICAM-1 Ab blocked the EC stiffening response without inhibiting neutrophil adherence. Moreover, cross-linking ICAM-1 mimicked the stiffening response induced by neutrophils. The neutrophil-induced increase in the apparent stiffness of ECs was inhibited with 1% DMSO (a hydroxyl radical scavenger), allopurinol (a xanthine oxidase inhibitor), or deferoxamine (an iron chelator), suggesting that ROS may be involved in mediating the EC stiffening response. The cellular sources of ROS were determined by measuring the oxidation of dichlorofluorescein. Neutrophil adherence to TNF-alpha-activated ECs induced ROS production only in ECs, and not in neutrophils. This ROS production in ECs was completely prevented by the anti-ICAM-1 Ab and partially inhibited by allopurinol. These results suggest that ICAM-1-mediated signaling events during neutrophil adherence may activate xanthine oxidase, which in turn mediates the ROS production in ECs that leads to stiffening. ROS generated in ECs on neutrophil adherence appear to mediate cytoskeletal remodeling, which may modulate subsequent inflammatory responses.     

Proinflammatory effects of copper deficiency on neutrophils and lung endothelial cells

Dietary copper deficiency increases the accumulation of circulating neutrophils in the rat lung microcirculation. This process includes neutrophil adhesion to, migration along, and emigration though the vascular endothelium. The current study was designed to examine the role of copper in each of these steps. Neutrophils were isolated from rats fed either a copper-adequate (CuA, 6.1 microg Cu/g diet) or copper-deficient diet (CuD, 0.3 microg Cu/g diet) for 4 weeks. First, transient and firm adhesion of neutrophils to P-selectin in a flow chamber showed there were more adhered CuD neutrophils than CuA ones. This effect is probably caused by the increased expression of CD11b that was observed in the current study. Second, the evaluation of neutrophil migration under agarose showed that the CuD neutrophils moved farther than the CuA group in response to IL-8 but not fMLP; this suggests an increased sensitivity to a CD11/CD18-independent signalling pathway. Third, the contractile mechanism of endothelial cells was studied. Elevated F-actin formation in Cu-chelated lung microvascular endothelial cells suggests that neutrophil emigration may be promoted by enhanced cytoskeletal reorganization of the endothelium during copper deficiency. Combined, these results support the theory that dietary copper deficiency has proinflammatory effects on both neutrophils and the microvascular endothelium that promote neutrophil-endothelial interactions.     

Isolation and characterization of a cDNA clone for a novel serine-rich neutrophil protein.

A cDNA expression library from pig blood neutrophils was immunoscreened with a rabbit antiserum raised against a 32 kDa neutrophil membrane phosphoprotein. Previous work indicated this protein as a component of the superoxide-forming NADPH oxidase enzyme complex (1,2). Only one cDNA clone (B+) was highly positive. The B+ clone contained a 1109 bp insert, with an open reading frame encoding for 284 amino acids. The deduced B+ amino acid sequence contained a 72 amino acid domain with proline and glutamine repeats and two domains extremely enriched with serine residues. The isolated cDNA hybridizes with a 3.1 kb mRNA expressed in pig and human leukocytes.