Biomedical subjects

D Shepro

Publications and source records attributed to D Shepro.

At least 37 records · Page 2Linked to original sources

PubMed1991

Neutrophil accumulations due to pulmonary thromboxane synthesis mediate acid aspiration injury.

Acid aspiration leads to lung injury associated with high levels of plasma thromboxane (Tx). This study tests the role of Tx synthesis by the aspirated lung segment in mediating local and remote neutrophil (PMN) sequestration, alveolar diapedesis, and permeability edema. Anesthetized rats underwent tracheostomy and insertion of a fine-bore cannula into the anterior segment of the left lung. This was followed by the installation of either 0.1 ml saline (n = 18) or the Tx synthase inhibitor OKY 046 (0.1 mg/kg in 0.1 ml, n = 18). Twenty minutes later either 0.1 ml 0.1 N HCL or 0.1 ml saline was similarly introduced (n = 18). Three hours later, in the saline-HCl group, the rise in Tx concentration in both plasma (1,340 pg/ml) and bronchoalveolar lavage (BAL) fluid (2,100 pg/ml) was higher than that in the saline-saline aspirated control group (350 and 115 pg/ml, respectively; both P less than 0.05). In the acid-aspirated lung PMN sequestration [87 PMN/10 high-power fields (HPF)] and diapedesis (96 x 10(4) PMN/ml in BAL) were higher than control values of 7 PMN/10 HPF and 3 x 10(4) PMN/ml (both P less than 0.05). Acid aspiration induced local permeability edema with a high protein concentration in BAL of 3,350 micrograms/ml and an increase in lung wet-to-dry weight ratio (W/D) of 6.6, both higher than control values of 482 micrograms/ml and 3.4, respectively (P less than 0.05). Leukosequestration in the aspirated side started at 30 min and was progressive over a 3-h monitoring period.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Role for tumor necrosis factor as mediator of lung injury following lower torso ischemia.

Ischemia and reperfusion of the ischemic lower torso lead to a neutrophil- (PMN) dependent lung injury characterized by PMN sequestration and permeability edema. This mimics the injury seen after infusion of tumor necrosis factor alpha (TNF), a potent activator of PMN and endothelium. This study tests whether TNF is a mediator of the lung injury after lower torso ischemia. Anesthetized rats underwent 4 h of bilateral hindlimb tourniquet ischemia, followed by reperfusion for 10 min, 30 min, 1, 2, 3, and 4 h (n = 6 for each time point). Quantitative lung histology indicated progressive sequestration of PMN in the lungs, 25 +/- 3 (SE) PMN/10 high-power fields (HPF) 10 min after reperfusion vs. 20 +/- 2 PMN/10 HPF in sham animals (NS), increasing to 53 +/- 5 PMN/10 HPF after 4 h vs. 23 +/- 3 PMN/10 HPF in sham animals (P less than 0.01). There was lung permeability, shown by increasing protein accumulation in bronchoalveolar lavage (BAL) fluid, which 4 h after reperfusion was 599 +/- 91 vs. 214 +/- 35 micrograms/ml in sham animals (P less than 0.01). Similarly, there was edema, shown by the lung wet-to-dry weight ratio, which increased by 4 h to 4.70 +/- 0.12 vs. 4.02 +/- 0.17 in sham animals (P less than 0.01). There was generation of leukotriene B4 in BAL fluid (720 +/- 140 vs. 240 +/- 40 pg/ml, P less than 0.01), and in three of six rats tested at this time TNF was detected in plasma, with a mean value of 167 pg/ml. TNF was not detectable in any sham animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Phalloidin attenuates postischemic neutrophil infiltration and increased microvascular permeability.

The aim of this study was to determine whether phalloidin (1 microM) or antamanide (1 microM), cyclic peptides that stabilize dense peripheral band and stress fiber F-actin in endothelium, would attenuate the increase in microvascular permeability induced by 4 h of ischemia and 30 min of reperfusion (I/R) in the isolated canine gracilis muscle. Changes in microvascular permeability (1 - sigma) were assessed by determining the solvent drag reflection coefficient for total plasma proteins (sigma) in muscles subjected to 4.5 h of continuous perfusion (nonischemic controls), I/R alone, I/R + phalloidin, or I/R + antamanide. Muscle neutrophil content was assessed by determination of myeloperoxidase (MPO) activity in tissue samples obtained at the end of the experiments. Fluorescent detection of nitrobenzoxadiazole-phallicidin in endothelial cell monolayers confirmed that phalloidin enters these cells. I/R was associated with marked increases in microvascular permeability and muscle neutrophil content (1 - sigma = 0.45 +/- 0.07; MPO = 8.9 +/- 0.5 units/g) relative to control (4.5 h continuous perfusion) preparations (1 - sigma = 0.12 +/- 0.03; MPO = 0.5 +/- 0.8 unit/g). These I/R-induced changes were largely prevented by administration of phalloidin (1 - sigma = 0.19 +/- 0.02; MPO = 0.8 +/- 0.4 U/g) or antamanide (1 - sigma = 0.07 +/- 0.11; MPO = 0.9 +/- 0.3 unit/g) at reperfusion. Similar results were obtained when phalloidin was administered before ischemia (1 - sigma = 0.24 +/- 0.04; MPO = 1.2 +/- 1.0 units/g). Although antamanide decreased superoxide production (by approximately 60%) and adherence to plastic (by approximately 75%) by activated neutrophils in vitro, phalloidin failed to alter these aspects of granulocyte function.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed1991

Thromboxane mediates diapedesis after ischemia by activation of neutrophil adhesion receptors interacting with basally expressed intercellular adhesion molecule-1.

Ischemic injury is characterized by neutrophil (PMN)--endothelial cell adhesion and diapedesis associated with thromboxane (TX) generation. Neutrophil-endothelial cell interaction is regulated in part by the leukocyte adhesion receptor CD 18 glycoprotein complex and the endothelial intercellular adhesion molecule-1 (ICAM-1). This study tests the role of TX in ischemia-induced diapedesis and evaluates whether the diapedesis is regulated by neutrophil or endothelial adhesion receptors. Plasma derived from rabbit hind limbs made ischemic for 3 hours (n = 6) and reperfused for 10 minutes had increased levels of TXB2 3,450 pg/ml, which was higher than sham rabbit (n = 6) values of 653 pg/ml (p less than 0.05). When introduced into abraded skin chambers placed on the dorsum of other normal rabbits (n = 6), this ischemic plasma induced 1,000 pg/ml of new TX synthesis and diapedesis of 1,235 PMN/mm3. The total TX concentration and PMN accumulations in blister fluid were correlated (r = 0.88, p less than 0.05). In contrast, sham rabbit plasma induced no TX synthesis and diapedesis of only 77 PMN/mm3 (p less than 0.05). Administration of 50 ng/ml of authentic TXB2 into blisters induced an accumulation of 453 PMN/mm3, which was higher than that in saline controls (18 PMN/mm3) (p less than 0.05). Pretreatment of normal rabbits used for the diapedesis assay (n = 4) with the TX synthetase inhibitor OKY 046 (2 mg/kg/hr) limited ischemic plasma and authentic TXB2 induced diapedesis to 142 and 76 PMN/mm3, respectively (both p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Modulation of pulmonary permeability in vivo with agents that affect the cytoskeleton.

In vitro studies show that the cytoskeleton of the microvascular barrier moderates polymorphonuclear leukocyte (PMN) diapedesis and the transport of macromolecules. This in vivo study tests indirectly whether the cytoskeleton of the pulmonary microvasculature responds similarly to agents known to reorganize the cytoskeleton to alter diapedesis and permeability in vitro. One of four agents, saline solution, cytochalasin B (which promotes actin microfilament disassembly), leukotriene (LT) B4 (PMN chemoattractant), or phalloidin (which promotes and stabilizes F-actin polymerization) was introduced into the bronchus of the anterior segment of the left lung of anesthetized rats (n = 152) through a tracheostomy and fine-bore cannula. Twenty minutes later, saline solution, cytochalasin B, LTB4, or phalloidin was similarly introduced. PMN accumulations (x 10(4) cells/ml), protein concentration in bronchoalveolar lavage fluid, and lung wet/dry weight ratios were measured 3 hours later. When the initial and secondary treatments were saline solution, PMN accumulations were 3 +/- 1 cells/ml and the protein level was 274 +/- 48 micrograms/ml. Secondary treatment of the saline-treated group with cytochalasin B or LTB4 led to increases in PMN to 18 +/- 2 and 9 +/- 2 cells/ml and protein to 960 +/- 50 and 840 +/- 40 micrograms/ml (p less than 0.05); secondary treatment with phalloidin was similar to that with saline solution. With saline solution used for both treatments, the wet/dry ratio was 3.4 +/- 0.2. Primary saline solution and secondary treatments with either cytochalasin B or LTB4 led to a similar increase in wet/dry ratios to 3.9 +/- 0.1 (p less than 0.05), whereas phalloidin was without effect (3.5 +/- 0.3). Initial cytochalasin B treatment followed by LTB4 led to increased PMN diapedesis (43 +/- 6 cells/ml and wet/dry ratio 4.3 +/- 0.2) (p less than 0.05). Secondary phalloidin treatment attenuated the cytochalasin B effect with counts of 12 +/- 2 PMN/ml, protein levels of 460 +/- 30 micrograms/ml, and a lower wet/dry ratio of 3.7 +/- 0.1 (p less than 0.05). Even more striking, phalloidin as initial treatment further reduced the cytochalasin B effect to 7 +/- 1 PMN/ml, whereas protein level was 490 +/- 60 micrograms/ml and wet/dry ratio was 3.5 +/- 0.1 (p less than 0.05). Further, phalloidin, given initially, attenuated the effect of secondary treatment with LTB4, resulting in fewer cells (4 +/- 2 PMN/ml), a lower wet/dry ratio (3.4 +/- 0.1), and protein level of 650 +/- 20 micrograms/ml (p less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Interleukin-2-induced lung injury is mediated by oxygen free radicals.

Interleukin-2 therapy leads to respiratory dysfunction caused by increased vascular permeability. This study examines the role of oxygen-derived free radicals (OFR). Sheep (n = 6) with chronic lung lymph fistulae were given interleukin-2, 10(5) units/kg, as an intravenous bolus. The mean pulmonary artery pressure rose from 13 to 23 mm Hg (p less than 0.05) at 1 hour and remained elevated for 4 hours, although the pulmonary artery wedge pressure was unchanged at 4 mm Hg. Arterial oxygen tension fell from 88 to 77 mm Hg (p less than 0.05). Lung lymph flow rose from 2.2 to 6.4 ml/30 min (p less than 0.05) at 3 hours. This rise coincided with an increase in the lymph/plasma protein ratio from 0.67 to 0.77 (p less than 0.05) and lymph protein clearance from 1.5 to 4.4 ml/30 min (p less than 0.05), indicating increased lung microvascular permeability. Interleukin-2 led to transient increases in plasma thromboxane B2 from 168 to 388 pg/ml (p less than 0.05) and lung lymph thromboxane B2 from 235 to 694 pg/ml (p less than 0.05). The leukocyte count fell from 8156 to 4375/mm3 (p less than 0.05) primarily caused by a 78% drop in lymphocyte count. Platelet count declined from 292 to 184 X 10(3)/mm3 (p less than 0.05). Pretreatment with the hydroxyl radical scavenger dimethylthiourea, 1 gm/kg, intravenously, (n = 6) prevented the interleukin-2-induced increase in mean pulmonary artery pressure, lung lymph flow, lymph/plasma protein ration, lymph protein clearance, and thromboxane B2 levels in plasma and lung lymph. The arterial oxygen tension decreased from 85 to 80 mm Hg (p less than 0.05). The leukocyte count declined from 7854 to 6229/mm3 (p less than 0.05), but this was not as low nor as prolonged as the interleukin-2 group. Further, the decrease in platelet count was prevented (p less than 0.05). Interleukin-2 incubated with sheep or human leukocytes led to a dose-dependent increase in intracellular hydrogen peroxide production by neutrophils as measured by flow cytometry of dichlorofluorescein oxidation. These data indicate that interleukin-2 stimulates OFR generation and that OFR moderate the interleukin-2-induced increased lung permeability.

Animals↗

PubMed1990

Interleukin-2-induced lung permeability is mediated by leukotriene B4.

Interleukin (IL)-2 therapy leads to respiratory dysfunction due to increased vascular permeability. This study examines the role of the chemoattractant, immunomodulator, and permeability-promoting agent leukotriene (LT) B4 in this setting. Sheep with chronic lung lymph fistulae were given IL-2, 10(5) U/kg as an IV bolus (n = 6). Within 2 hours this led to a significant increase in LTB4 levels in both plasma and lung lymph. The mean pulmonary artery pressure (MPAP) rose while the pulmonary artery wedge pressure was unchanged. Arterial oxygen tension (PaO2) fell. Lung lymph flow (QL) was tripled (P less than 0.05) at 3 hours, coinciding with an increase in the lymph/plasma (L/P) protein ratio (P less than 0.05) resulting in an increase in the lymph protein clearance (P less than 0.05), data documenting increased microvascular permeability to protein. Mild leukopenia and thrombocytopenia (P less than 0.05) occurred. Body temperature rose and shaking chills were common. Pretreatment with the lipoxygenase inhibitor diethylcarbamazine (DEC; n = 6) reduced baseline plasma LTB4 levels and prevented the IL-2-induced increases in LTB4 in plasma and lung lymph (P less than 0.05). In contrast to IL-2 treatment alone, DEC blunted the increase in MPAP and prevented the rises in QL (P less than 0.05), L/P protein ratio (P less than 0.05), and lymph protein clearance (P less than 0.05). DEC also prevented the IL-2-induced leukopenia, the fall in platelet count, and the rise in body temperature (P less than 0.05, respectively). Infusion of IL-2 excipient control (n = 5) did not affect plasma or lymph LTB4 levels but there were mild increases in MPAP (P less than 0.05). The QL also rose but this occurred while the L/P protein ratio fell (P less than 0.05). Body temperature rose moderately. The PaO2, leukocyte, and platelet counts were unaffected. These data indicate that IL-2 administration leads to pulmonary dysfunction manifest by pulmonary hypertension and increased vascular permeability, events associated with LTB4 synthesis and prevented by DEC. Leukotriene B4 appears therefore to mediate the IL-2-induced lung injury.

Animals↗

PubMed1990

Neutrophil adherence receptors (CD 18) in ischemia. Dissociation between quantitative cell surface expression and diapedesis mediated by leukotriene B4.

Neutrophils and eicosanoid chemoattractants are centrally involved with ischemia-reperfusion (I/R) injury. The CD 18 complex of adhesive glycoproteins, readily up-regulated by chemoattractants in vitro, is required for polymorphonuclear leukocyte (PMN) adherence to endothelium. This study tests whether CD 18 is up-regulated by ischemia in vivo and its role in mediating PMN diapedesis. Anesthetized rabbits underwent 3 h of bilateral hindlimb tourniquet ischemia (n = 16). Ten min after tourniquet release, levels of plasma leukotriene (LT)B4 increased to 390 +/- 62 pg/ml (mean +/- SE), higher than 134 +/- 26 pg/ml in control rabbits (n = 13, p less than 0.01). Aliquots of plasma were added to whole blood from normal rabbits (n = 6) for flow cytometric analysis of neutrophils with the CD 18 mAb R 15.7. Addition of I/R plasma failed to demonstrate an increase in surface expression of CD 18. Similarly, no CD 18 up-regulation was observed in vivo upon reperfusion in ischemic animals pretreated with mAb R 15.7 (n = 3). However, I/R plasma when introduced into plastic chambers taped atop dermabrasion sites in normal rabbits (n = 12) resulted in diapedesis, measured by the accumulation after 3 h of 1130 +/- 125 PMN/mm3 in the chambers relative to 120 +/- 31 PMN/mm3 with control plasma (p less than 0.01). Diapedesis in response to I/R plasma was abolished by pretreatment with mAb R 15.7 (less than 5 PMN/mm3, n = 6), was reduced by U 75,302, an LTB4 receptor antagonist (253 +/- 101 PMN/mm3, n = 6) (both p less than 0.01) and was not protein synthesis dependent. These results demonstrate that PMN diapedesis in response to I/R plasma is exclusively dependent upon the CD 18 glycoprotein complex by an LTB4-dependent mechanism, despite the fact that CD 18 is not up-regulated on circulating PMN in ischemia. These data indirectly indicate the functional importance of conformational changes of CD 18 in determining PMN adhesion.

Animals↗

PubMed1990

Expression of simple epithelial cytokeratins in bovine pulmonary microvascular endothelial cells.

Polypeptides of bovine aortic, pulmonary artery, and pulmonary microvascular endothelial cells, as well as vascular smooth muscle cells and retinal pericytes were evaluated by two-dimensional gel electrophoresis. The principal cytoskeletal proteins in all of these cell types were actin, vimentin, tropomyosin, and tubulin. Cultured pulmonary microvascular endothelial cells also expressed 12 unique polypeptides including a 41 kd acidic type I and two isoforms of a 52 kd basic type II simple epithelial cytokeratin microvascular endothelial cell expression of the simple epithelial cytokeratins was maintained in cultured in the presence or absence of retinal-derived growth factor, and regardless of whether cells were cultured on gelatin, fibronectin, collagen I, collagen IV, laminin, basement membrane proteins, or plastic. Cytokeratin expression was maintained through at least 50 population doublings in culture. The expression of cytokeratins was found to be regulated by cell density. Pulmonary microvascular endothelial cells seeded at 2.5 X 10(5) cell/cm2 (confluent seeding) expressed 3.5 times more cytokeratins than cells seeded at 1.25 X 10(4) cells/cm2 (sparse seeding). Vimentin expression was not altered by cell density. By indirect immunofluorescence microscopy it was determined that the cytokeratins were distributed cytoplasmically at subconfluent cell densities but that cytokeratin 19 sometimes localized at regions of cell-cell contact after cells reached confluence. Vimentin had a cytoplasmic distribution regardless of cell density. These results suggest that pulmonary microvascular endothelial cell have a distinctive cytoskeleton that may provide them with functionally unique properties when compared with endothelial cells derived from the macrovasculature. In conjunction with conventional endothelial cell markers, the presence of simple epithelial cytokeratins may be an important biochemical criterion for identifying pulmonary microvascular endothelial cells.

Animals↗

PubMed1990

Inflammatory agonists that increase microvascular permeability in vivo stimulate cultured pulmonary microvessel endothelial cell contraction.

Bovine pulmonary microvessel endothelial cells grown on a flexible substrate contract upon the addition of angiotensin II, thrombin, bradykinin, and U44069, a stable analogue of thromboxane A2. All these agents promote inflammation and increase paracellular permeability in vivo or in vitro. The contractile response is mediated by intracellular and extracellular free calcium: the response is inhibited by TMB-8, an intracellular Ca2+ chelator, and EGTA. Contraction is inhibited by trifluoroperazine, a Ca2(+)-calmodulin antagonist, and by ML-7, an inhibitor of myosin light-chain kinase. Preincubation with PMA, a protein kinase C activator, prevents contraction by angiotensin II. The inactive analogue 4-alpha-phorbol 12,13-didecanoate does not inhibit contraction. In contrast cAMP, carbacyclin (a stable PGI2 analogue), and isoproterenol, agonists known to stabilize the microvascular barrier against inflammatory agents, relax pulmonary microvessel EC. This direct evidence of the contractile potential of microvessel endothelial cells lends support to the theory that endothelial contraction leads to increased junctional permeability.

Angiotensin II↗

PubMed1990

Modulation of cultured pulmonary microvessel and arterial endothelial cell barrier structure and function by serotonin.

Pulmonary microvessel endothelial cell and pulmonary artery endothelial cell monolayers in tissue culture were treated with serotonin (5-hydroxytryptamine; 5-HT) alone or in conjunction with histamine, bradykinin, the thromboxane analog U-46619, and the actin modulating agent cytochalasin B. After agent treatment, cross-sections through endothelial cell (EC) monolayers were examined by light microscopy and the percentage and widths of intercellular openings were quantitated. To correlate structural changes in the endothelial barrier with an alteration in permeability, EC monolayers cultured on micropore filters were assayed for transit of Evan's blue albumin (EBA) following treatment with vasoactive mediators. 5-HT was found to decrease the patency of endothelial junctions by up to 94%, compared to untreated monolayers, and to prevent or reverse the appearance of interendothelial gaps induced by histamine, bradykinin, U-46619, and cytochalasin B. The 5-HT effect was dose and time dependent, with a maximal increase in junctional apposition observed at a concentration of 10(-6) M for 30 min. This response was significantly blocked by the 5-HT antagonists LSD and ketanserin. The formation or reduction of interendothelial gaps by histamine, bradykinin, and U-46619 and by 5-HT, respectively, was positively correlated to changes in monolayer permeability to EBA. These results suggest that pulmonary edema caused by inflammatory mediators in part may be a consequence of transient increases in pulmonary EC junctional gaps, and that 5-HT may contribute to the homeostatic maintenance of endothelial barrier integrity.

Animals↗

PubMed1990

Ischemia activates neutrophils but inhibits their local and remote diapedesis.

Hindlimb ischemia and reperfusion results in local limb and distant lung injury. This study tests whether the mechanism of injury is by ischemia mediated polymorphonuclear leukocyte (PMN) activation and diapedesis. Anesthetized rabbits were subjected to three hours of hindlimb ischemia (n = 8) or sham ischemia (n = 4). PMN derived solely from the reperfused ischemic limb and assayed flow cytometrically displayed an oxidative burst of 135 /- 8 fentamoles dichlorofluorescein (fmDCF)/cell compared to preischemc levels of 74 +/- 14 fmDCF/cell (p less than 0.05). Additional aliquots of isolated neutrophils were treated with phorbol myristate acetate (PMA) 10(-7) M. In contrast to a 162% increase in oxidative burst before ischemia, neutrophils at ten minutes of reperfusion had an enhanced response to PMA of 336% (p less than 0.05). Plasma collected from the ischemic hindlimb at ten minutes of reperfusion when introduced into an abraded skin chamber or intratracheally induced diapedesis in nonischemic animals. PMN accumulations in the skin chamber were 1636 +/- 258 PMN/mm3 after three hours (n = 8) compared to 63 +/- 18 PMN/mm3 induced by sham plasma (n = 4, p less than 0.05). Introduction of ischemic plasma intratracheally into a lobar bronchus (n = 4) induced PMN accumulations after three hours, measured by bronchoalveolar lavage fluid of 19 +/- 2 X 10(4) PMN/mm3 compared to 5 +/- 1 X 10(4) PMN/mm3 with sham plasma (n = 4, p less than 0.05). Diapedesis was completely prevented (0-3 PMN/mm3, p less than 0.05) by introducing ischemic plasma into skin chambers in animals whose hindlimbs had been made ischemic (n = 6) or into chambers located on skin regions that had been previously made ischemic (n = 6). Similarly after hindlimb ischemia, lavage of the lung with ischemic plasma yielded few PMN 0-3/mm3 (p less than 0.05). These data indicate that ischemia and reperfusion lead to generation of a circulating component in plasma that causes an oxidative burst in PMN and inhibits their diapedesis but promotes diapedesis when applied extravascularly to a naive animal.

Animals↗

PubMed1990

Tumor necrosis factor-alpha mediates acid aspiration-induced systemic organ injury.

Acid aspiration-induced systemic organ injury is mediated by the sequestration of activated neutrophils (PMN). In other settings cytokines have been shown to increase neutrophil-endothelial adhesion, a requisite for injury. This study tests whether the systemic leukosequestration and permeability following localized aspiration is mediated by tumor necrosis factor (TNF)-alpha-induced synthesis of an adhesion protein. Anesthetized rats underwent tracheostomy and insertion of a fine-bore cannula into the anterior segment of the left lung. This was followed by the instillation of either 0.1 mL 0.1 N HCI (n = 18) or 0.1 mL saline in control rats (n = 18). Localized aspiration induced generalized pulmonary leukosequestration with 95 PMN/10 high-power fields (HPF) in the aspirated lung and 46 PMN/10 HPF in the nonaspirated lung, higher than control values of 7 PMN/10 HPF and 5 PMN/10 HPF in saline- and nonsaline-aspirated sides, respectively (p less than 0.05). The leukosequestration was associated with permeability edema shown by increased protein concentrations in bronchoalveolar lavage (BAL) of 3900 micrograms/mL in the aspirated and 2680 micrograms/mL in the nonaspirated side, higher than saline with 482 micrograms/mL and 411 micrograms/mL, respectively (p less than 0.05). There was generalized pulmonary edema following aspiration measured by increase in wet-to-dry weight ratios (w/d) of 6.6 in the aspirated and 5.1 in the nonaspirated lung, higher than control values of 3.5 and 3.4, respectively (p less than 0.05). Localized aspiration led to systemic leukosequestration documented by increases in myeloperoxidase activity (units/g tissue) of 2.2 and 1.7 in heart and kidney, higher than control values of 0.3 and 0.4, respectively (p less than 0.05). This event was associated with edema of these organs with w/d ratios of 4.6 and 4.3, relative to control values of 3.0 and 3.4 (p less than 0.05). Treatment of animals (n = 18) 20 minutes after aspiration with anti-TNF-alpha antiserum (rabbit anti-murine) but not normal rabbit serum (n = 18) reduced lung leukosequestration in the aspirated and nonaspirated segments (61 and 32 PMN/10HPF), BAL protein concentration (1490 and 840 micrograms/mL), and w/d ratio (4.3 and 3.7) (all p less than 0.05). In the heart and kidney there were reductions in myeloperoxidase activity (0.7 and 0.6) and w/d ratio (3.5 and 3.6) (both p less than 0.05). Treatment of rabbits (n = 18) with the protein synthesis inhibitor cycloheximide, 0.2 mg/kg/hr was as effective as TNF-alpha antiserum in modifying aspiration injury.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Involvement of thromboxane and neutrophils in multiple-system organ edema with interleukin-2.

Interleukin-2 (IL-2) produces toxicity characterized by generalized edema within 24 hours. This study tests whether the rate of IL-2 administration modulates the onset of edema and examines thromboxane (Tx) and neutrophils as possible mediators of this event. Recombinant human IL-2, 10(5) U (n = 7), 10(6) U (n = 9), or vehicle (n = 8) were given to anesthetized rats intravenously during a period of 1 hour. At 6 hours edema, as measured by increase in wet to dry weight (w/d) ratio, was present in the heart, liver, and kidney, with 10(5) U IL-2 and in the lung, heart, liver and kidney, with 10(6) U IL-2, relative to values with vehicle-infused controls (all p less than 0.05). With a 1-hour infusion of 10(6) U IL-2, there was an increase in plasma thromboxane (Tx)B2 level to 1290 +/- 245 pg/mL, higher than 481 +/- 93 pg/mL in control rats (p less than 0.05); lung polymorphonuclear leukocyte (PMN) sequestration of 53 +/- 7 PMN/10 higher-power fields (HPF) relative to 23 +/- 2 PMN/10 HPF in controls (p less than 0.05); and increased bronchoalveolar lavage (BAL) fluid protein concentration of 1970 +/- 210 micrograms/mL relative to 460 micrograms/mL in controls (p less than 0.05). When 10(6) U IL-2 was given as a 1-minute intravenous bolus (n = 9), edema was not demonstrated, plasma TxB2 levels were similar to controls, there was no leukosequestration, and BAL protein levels were normal. These data indicate that a constant infusion but not the rapid bolus administration of IL-2 produces in rats multiple-system organ edema, increased plasma TxB2, sequestration of PMNs, and microvascular permeability. These findings may explain the early toxicity seen in patients given high-dose IL-2 in cancer treatment.

6-Ketoprostaglandin F1 alpha↗

PubMed1990

Attenuation of acid aspiration edema with phalloidin.

Acid aspiration leads to pulmonary endothelial and epithelial cell (EC/EpC) injury characterized by increased permeability and polymorphonuclear (PMN) leukocyte diapedesis. Actin in the EC/EpC cytoskeleton has been shown to play a significant role in maintenance of the microvascular junction barrier. This study tests indirectly whether the development of permeability and diapedesis following acid aspiration is via disruption of the pulmonary cytoskeleton. Manipulation was achieved by the actin microfilament assembler phalloidin. Anesthetized rats (n = 88) underwent segmental lung installation of 0.1 ml saline or phalloidin (2 x 10(-6) M). Twenty minutes later 0.1 N HCl, saline, or phalloidin was introduced. After 3 h there was an increase in wet-to-dry weight (W/D) ratio of 6.6 and 5.1 in the HCl-injected and noninjected sides, protein concentration, 3,970 and 2,530 micrograms/ml, and accumulation of 93 PMN/ml (x 10(4] in bronchoalveolar lavage (BAL) of the HCl-injected lung. These values were higher than control animals. Local pretreatment with phalloidin attenuated acid-induced localized but not generalized permeability with reduction in W/D ratio, BAL protein concentration, and diapedesis (P less than 0.05). Acid injection into airways also led to elevated thromboxane B2 and leukotriene B4 levels in plasma and BAL (P less than 0.05) and generalized lung leukosequestration, events not affected by phalloidin. Taken together, these data suggest that acid aspiration lung injury is determined largely by loss of integrity of the pulmonary EC/EpC cytoskeleton with resultant loss of barrier function.

Administration, Inhalation↗

PubMed1990

Ischemia-induced neutrophil activation and diapedesis is lipoxygenase dependent.

Ischemia and reperfusion lead to eicosanoid- and neutrophil (PMN)-dependent injury. This study tests the role of ischemia-induced lipoxygenase activity in mediating PMN activation and diapedesis. Anesthetized rabbits (n = 8) underwent 3 hours of bilateral hindlimb ischemia. At 10 minutes of reperfusion, leukotriene B4 (LTB4) levels in femoral venous effluent were 0.49 +/- 0.05 ng/ml compared with 0.04 +/- 0.07 ng/ml in sham-treated animals (n = 10) (p less than 0.05). Intracellular H2O2 production of circulating PMNs assayed flow cytometrically by dichlorofluorescein (DCF) oxidation, increased from a preischemic value of 74 +/- 14 femtomoles DCF/cell to 135 +/- 8 fmol DCF/cell (p less than 0.05). PMNs were treated with phorbol myristate acetate (PMA), 10(-7) mol/L. In contrast to a 162% increase in H2O2 production before ischemia, PMNs at 10 minutes of reperfusion had an enhanced response to PMA of 336% (p less than 0.05). Addition of authentic LTB4 (0.5 ng/ml) to PMN from sham-treated animals led to their activation, manifest by an oxidative burst, 127 +/- 12 fmol DCF/cell, and an enhanced response of 337% to PMA stimulation. To study diapedesis, plasma collected at 10 minutes of reperfusion was introduced into plastic chambers taped atop skin abrasions in rabbits (n = 8). After 3 hours, 1610 +/- 246 PMN/mm3 accumulated and LTB4 levels in blister fluid were 0.83 +/- 0.03 ng/ml, higher than values of 44 +/- 23 PMN/mm3 (p less than 0.05) and 0.04 +/- 0.03 ng LTB4/ml (p less than 0.05) with saline solution and 68 +/- 16 PMN/mm3 (p less than 0.05) and 0.19 +/- 0.02 ng/ml (p less than 0.05) with nonischemic plasma. The introduction of LTB4, 3.3 ng/ml, into the chambers resulted in an accumulation of 536 +/- 352 PMN/mm3 (p less than 0.05). Pretreatment of animals before hindlimb ischemia (n = 5) with the lipoxygenase inhibitor diethylcarbamazine abolished PMN activation (51 +/- 12 fmol DCF/cell) and ischemic plasma-induced diapedesis into the plastic chamber (38 +/- 18 PMN/mm3). Pretreatment of nonischemic animals (n = 13) used for the dermabrasion bioassay with diethylcarbamazine abolished diapedesis into the plastic chambers induced by ischemic plasma (n = 5) (32 +/- 24 PMN/mm3) or LTB4 (n = 3) (36 +/- 28 PMN/mm3). These data indicate that PMN activation after reperfusion of ischemic tissue is mediated by a lipoxygenase product, perhaps LTB4, and that both reperfusion plasma and authentic LTB4 induce diapedesis by stimulating de novo lipoxygenase activity.

Animals↗

PubMed1989

Role of thromboxane in interleukin 2-induced lung injury in sheep.

Interleukin (IL)-2 administration leads to respiratory dysfunction due to increased vascular permeability. This study examines the role of thromboxane (Tx)A2 in IL-2 induced lung injury in sheep with chronic lung lymph fistulae. This preparation enables evaluation of permeability prior to the development of gross edema. IL-2, 10(5) units/kg (n = 6), or its excipient control (n = 5) was given as an i.v. bolus over 2 min. After 2 h of IL-2 administration, plasma TxB2 increased from 168 to 388 pg/ml (P less than 0.05) and lung lymph TxB2 from 235 to 694 pg/ml (P less than 0.05). Mean pulmonary artery pressure (MPAP) rose from 13 to 29 mm of Hg (P less than 0.05) at 30 min and remained elevated for 4 h while the pulmonary artery wedge pressure was unchanged at 4 mm of Hg. Arterial oxygen tension (PaO2) fell from 88 to 77 mm of Hg (P less than 0.05). Lung lymph flow (QL) rose from 2.2 to 3.8 ml/30 min (P less than 0.05) at 1 h and to 6.4 ml/30 min at 3 h. This rise coincided with an increase in the lymph/plasma (L/P) protein ratio from 0.67 to 0.77 (P less than 0.05). In contrast, the non-IL-2-infused sheep (n = 3) recruitment of the lung vasculature by left atrial balloon inflation led to a rise in QL from 2.4 to 8.2 ml/30 min, whereas the L/P ratio declined from 0.62 to 0.25, suggesting that the protein-rich lymph flow after IL-2 administration reflected increased microvascular permeability. In further proof of an increase in permeability, IL-2 administration into sheep (n = 2) with an inflated left atrial balloon led, after a pressure-independent L/P protein ratio had been achieved, to an increase in L/P protein ratio and decrease in protein reflection coefficient. At 2 h after IL-2, the blood leukocyte count fell from 8156 to 4375/mm3 (P less than 0.05) primarily due to a 73% drop in lymphocytes. The platelet count declined from 292 to 184 x 10(3)/mm3 (P less than 0.05). Body temperature rose from 38.9-40.3 degrees C (P less than 0.05), and shaking chills were common. Pretreatment with the Tx synthetase inhibitor OKY 046 (n = 7) lowered baseline plasma and lymph TxB2 levels to 22 and 52 pg/ml (P less than 0.05) and prevented the IL-2-induced increase in plasma and lung lymph TxB2 (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Reperfusion pulmonary edema.

Reperfusion following lower-torso ischemia in humans leads to respiratory failure manifest by pulmonary hypertension, hypoxemia, and noncardiogenic pulmonary edema. The mechanism of injury has been studied in the sheep lung lymph preparation, where it has been demonstrated that the reperfusion resulting in pulmonary edema is due to an increase in microvascular permeability of the lung to protein. This respiratory failure caused by reperfusion appears to be an inflammatory reaction associated with intravascular release of the chemoattractants leukotriene B4 and thromboxane. Histological studies of the lung in experimental animals revealed significant accumulation of neutrophils but not platelets in alveolar capillaries. We conclude that thromboxane generated and released from the ischemic tissue is responsible for the transient pulmonary hypertension. Second, it is likely that the chemoattractants are responsible for leukosequestration, and, third, neutrophils, oxygen-derived free radicals, and thromboxane moderate the altered lung permeability.

Animals↗