Biomedical subjects

L Kobzik

Publications and source records attributed to L Kobzik.

At least 145 records · Page 8Linked to original sources

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

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

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

Selective down-regulation of alveolar macrophage oxidative response to opsonin-independent phagocytosis.

We have compared the oxidative response of alveolar macrophages (AM) during opsonin-dependent and independent phagocytosis by using multiparameter flow cytometry. The respiratory burst of AM during phagocytosis was quantitated by the intracellular oxidation of the nonfluorescent precursors dichlorofluorescin diacetate (DCFH) or hydroethidine (HE, a reduced precursor of ethidium) to their fluorescent (oxidized) counterparts. After loading freshly isolated normal hamster AM with DCFH or HE, red or green fluorescent beads, respectively, were added to the shaking cell suspensions. Ingestion of opsonized particles by AM caused a marked increase in oxidation of both DCFH and HE proportional to the number of beads ingested. In contrast, uptake of one to three unopsonized particles per cell led to inhibition of oxidative activity compared to control cells incubated without particles. AM ingesting four or more unopsonized particles showed some increase in oxidative metabolism, but far less than that with identical numbers of particles in opsonin-dependent ingestion. Similar results were obtained using fluorescent labeled staphylococcal bacteria. Using three-color flow cytometry to study cells ingesting both types of particles, cells first ingesting unopsonized beads were also found to have an inhibited oxidative response to subsequently ingested opsonized particles. The mitochondrial poison antimycin inhibited most of the intracellular oxidative response to either type of phagocytosis. The remaining antimycin-insensitive, membrane derived respiratory burst of AM was also substantially diminished after phagocytosis of unopsonized particles vs similar numbers of opsonized particles. The greatly increased mitochondrial respiration in AM during phagocytosis of opsonized particles may be related to bactericidal mechanisms. Killing of ingested Staphylococcus by AM was markedly impaired in the presence of antimycin. The results suggest that AM may ingest the numerous, unopsonized inert particles that are inhaled without generation of potentially toxic oxygen metabolites, while retaining the capacity to undergo a respiratory burst after ingesting opsonized particles and bacteria. The mechanism(s) for this distinct response may include generation of an inhibitor of intracellular oxidative metabolism.

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↗

PubMed1990

Benign pulmonary lesions that may be misdiagnosed as malignant.

Five benign pulmonary lesions that may be misdiagnosed as malignant tumors are reviewed. In three lesions, diagnostic problems arise when a spindle cell component is dominant and obscures other characteristic histologic features. In the inflammatory pseudotumor, correct diagnosis relies on recognition of the benign cytology of the spindle cells and identifying a typical admixture of plasma cell-rich inflammatory cells. For spindle cell carcinoids, useful diagnostic features are the organoid pattern, benign cytology, and neuroendocrine differentiation features of the spindle cells. Localized pleural mesothelioma (fibroma) is composed of benign spindle cells in a fibrocollagenous background; mesothelial differentiation is not present by ultrastructural or immunocytochemical analysis. In sclerosing hemangioma, a complex histology may suggest a number of malignancies. Observation of solid and papillary areas of benign tumor cells, as well as sclerosis of vessel walls and intervening areas, will allow correct diagnosis. Pseudolymphoma, a nodular benign lymphoid infiltrate, is distinguished by its polymorphous and polyclonal composition and numerous germinal centers.

Diagnosis, Differential↗

PubMed1990

Oxidative metabolism in the alveolar macrophage: analysis by flow cytometry.

We evaluated the reagents dichlorofluorescin (DCFH) and hydroethidine (HE) for use in flow cytometric analysis of the respiratory burst of alveolar macrophages and monocytes. DCFH and HE are non-fluorescent precursors which can be oxidized intracellularly to the fluorescent compounds dichlorofluorescein and ethidium. Alveolar macrophages (AMs) loaded with either DCFH or HE were analyzed after phorbol myristate acetate (PMA) stimulation. The results, expressed as fmol/cell oxidation product (DCF or ethidium) after fluorometric standardization of the flow cytometer, show that both DCFH (273 +/- 48, mean increase over control +/- SE, fmol/cell, N = 9) and HE (416 +/- 54, N = 11) detected the substantial respiratory burst of hamster AMs. Similar results were obtained with normal human AMs. By using multiparameter analyses, the oxidative response of AMs ingesting opsonized fluorescent latex beads was measured in subpopulations ingesting increasing numbers of particles. A graded increase in oxidation of both DCFH and HE was found in response to increasing phagocytosis. Ingestion of fluoresceinated staphylyococcal bacteria caused similar changes in HE-loaded AMs. Inhibition of respiration with antimycin showed that approximately 95% of the increased oxidative metabolism of hamster AMs ingesting opsonized beads or bacteria was mitochondrial. The remaining 5% (10-40 fmol/cell) is membrane-derived oxidative activity quantitatively similar to that measured in assays of extracellular release of H2O2. Monocytes loaded with either DCFH or HE showed substantial increases in fluorescence after PMA stimulation (mean % increase over control +/- SE at 30 min: 464 +/- 104, DCFH, 505 +/- 156, HE). While DCHF is known to measure H2O2, HE is less well characterized. Exposure of cells to an extracellular source of both superoxide anion (O2-) and H2O2, xanthine oxidase-xanthine, resulted in marked oxidation of intracellular HE. Addition of both superoxide dismutase and catalase blocked this oxidation, indicating that HE can detect both O2- and H2O2. These agents can be useful probes for precise analysis of oxidative metabolism during phagocytosis in AMs and other mononuclear phagocytes.

Animals↗

PubMed1989

The rapid induction by interleukin-2 of pulmonary microvascular permeability.

The clinical use of interleukin-2 (IL-2) is limited by severe cardiopulmonary dysfunction. This study examines the mechanism of respiratory failure related to IL-2, using sheep with chronic lung lymph fistulae. Awake animals were infused with an intravenous (I.V.) bolus of IL-2 10(5) U/kg (n = 5) or its excipient (EXC) control (n = 3), every 8 hours for 4 to 5 days. Cardiopulmonary function was monitored daily for at least one 8-hour period. Within 2 hours after each IL-2 administration, mean pulmonary arterial pressure (MPAP) rose. On Day 1, the mean rise was from 13 to 26 mmHg (p less than 0.05), and on Day 5, to 29 mmHg (p less than 0.05). MPAP returned to baseline levels after 2-3 hours. Pulmonary arterial wedge pressure was unchanged from 4 mmHg. There were transient falls in arterial oxygen tension, from 88 to 77 mmHg on Day 1 and to 73 mmHg (p less than 0.05) on Day 5. Lung lymph flow (QL) rose from 2.4 to 6.8 ml/30 minutes (p less than 0.05) on Day 1, and from 4.7 to 10.2 ml/30 minutes (p less than 0.05) on Day 5, whereas the lymph/plasma protein ratio increased on Day 1 from 0.69 to 0.83 (p less than 0.05) and from 0.63 to 0.71 (p less than 0.05) on Day 5. This documents an increase in pulmonary microvascular permeability. Thromboxane (Tx)B2 levels increased transiently after each IL-2 injection in plasma from 195 to 340 pg/ml (p less than 0.05) and in lung lymph from 222 to 772 pg/ml (p less than 0.05) on Day 1, and to similar levels on Day 5. There was a progressive rise in cardiac output from 5.7 to 8.6 1/minute (p less than 0.05) during the 5 days of infusion. Systemic blood pressure did not change. Temperature rose from 39.1 to 41.2 C (p less than 0.05), and shaking chills were common. There was a progressive fall in leukocyte count, from 8.4 to 3.2 X 10(3)/mm3 (p less than 0.05) by Day 5, reflecting a 77% fall in lymphocytes. Lung lymph lymphocyte counts rose, and lymphocyte clearance increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Vasodilating prostaglandins attenuate ischemic renal injury only if thromboxane is inhibited.

Ischemia-induced renal injury is prevented by inhibition of thromboxane (Tx) synthesis. This protection was believed to be secondary to a high prostaglandin (PG)/TxA2 ratio. This study tests whether increasing the PG/Tx ratio by administration of vasodilating PGs protects the reperfused ischemic kidney. Anesthetized rats underwent right nephrectomy and 45 minutes of left renal pedicle clamping. Beginning 10 minutes before clamp release, animals were treated intravenously with the following: saline placebo (n = 10); the cyclooxygenase inhibitor ibuprofen (Ibu), 12.5 mg/Kg in a bolus (n = 8); a stable analogue of prostacyclin (PGI2), 500 ng/kg/minute for 2 hours (n = 9); PGE1, 400 ng/kg/minute for 2 hours (n = 8); the combination Ibu and PGI2 (n = 8) or PGE1 (n = 8). In saline treated ischemic controls, 5 minutes after reperfusion plasma, thromboxane (TxB2) and 6-keto-PGF1 levels were 2537 and 317 pg/ml, respectively--higher than the TxB2 and 6-keto-PGF1 levels of 750 and 80 pg/ml, respectively, in nephrectomized but nonischemic sham controls (n = 7) (p less than 0.05). In ischemic control animals at 24 hours, creatinine levels were 4.6 mg/dl, relative to 0.9 ml/dl in sham animals (p less than 0.05); the weight of the left (L) ischemic kidney relative to the right (R) normal kidney was 118%, compared with 99% in sham animals (p less than 0.05); and renal histology of ischemic control animals at 24 hours showed acute tubular necrosis (ATN) relative to normal findings in sham animals. Pretreatment with Ibu led to: TxB2 and 6-keto-PGF1 levels of 116 and 40 pg/ml, lower than those of sham animals (p less than 0.05); creatinine levels of 4.6 mg/dl, L/R renal weight of 119%; and ATN similar to that of ischemic controls. Treatment with a PGI2 analogue or PGE1 was not protective and led to increases in TxB2, 6-keto-PGF1, creatinine, L/R renal weight, and ATN similar to that of ischemic controls. The combination of Ibu and either PGI2 or PGE1 led to: reduced levels of TxB2 and 6-keto-PGF1 (p less than 0.05); attenuated increases in creatinine to 2.2 and 2.3 mg/dl, respectively (p less than 0.05); and limited ATN (p less than 0.05). These data indicate that the vasodilating PG protect the ischemic reperfused kidney only when Tx is inhibited.

6-Ketoprostaglandin F1 alpha↗

PubMed1989

Leukotrienes but not complement mediate limb ischemia-induced lung injury.

Reperfusion after limb ischemia leads to sequestration of polymorphonuclear leukocytes (PMN) in the lungs and to leukocyte- (WBC) and thromboxane- (Tx) dependent respiratory dysfunction. This study examines the intermediary role of the chemoattractants leukotriene (LT)B4 and complement (C) fragments. Anesthetized sheep with chronic lung lymph fistulae underwent 2 hours of tourniquet ischemia of both hind limbs. In untreated controls (n = 7), 1 minute after tourniquet release, mean pulmonary artery pressure (MPAP) rose from 13 to 38 mmHg (p less than 0.05) and returned to baseline within 30 minutes. Pulmonary artery wedge pressure was unchanged from 3.6 mmHg. There were increases in plasma LTB4 levels from 2.46 to 9.34 ng/ml (p less than 0.01), plasma TxB2 levels from 211 to 735 pg/ml (p less than 0.05), and lung lymph TxB2 from 400 to 1005 pg/ml (p less than 0.05). C3 levels were 96% of baseline values. Thirty minutes after reperfusion, lung lymph flow (QL) increased from 4.3 to 8.3 ml/30 minutes (p less than 0.05), lymph/plasma protein ratio was unchanged from 0.6, and the lymph protein clearance increased from 2.6 to 4.6 ml/30 minutes (p less than 0.05), data consistent with increased microvascular permeability. WBC count fell within the first hour from 6853 to 3793/mm3 (p less than 0.01). Lung histology showed leukosequestration, 62 PMN/10 high-power fields (HPF) and proteinaceous exudates. In contrast to this untreated ischemic group, animals treated with the lypoxygenase inhibitor diethylcarbamazine (n = 5) demonstrated a blunted reperfusion-induced rise in MPAP to 17 mmHg (p less than 0.05). There were no increases in LTB4, TxB2, QL or lymph protein clearance (p less than 0.05). WBC count was unchanged and lung leukosequestration was reduced to 40 PMN/10 HPF (p less than 0.05). Decomplementation with cobra venom factor (n = 4) resulted in plasma C3 levels, 10% of baseline, but tourniquet release still led to pulmonary hypertension, elevated LTB4, TxB2 levels, and a decline in WBC count similar to that of untreated ischemic control animals. Histology showed 46 PMN/10 HPF sequestered in the lungs. Further, bilateral hind limb ischemia in either genetically sufficient (n = 10) or deficient (n = 10) C5 mice led to significant lung leukosequestration of 108 and 106 PMN/10 HPF, respectively, compared with 42 and 47 PMN/10 HPF in sham C5(+) and C5 (-) mice (n = 20) (p less than 0.01). These results suggest that the lung leukosequestration and increased microvascular permeability after lower torso ischemia are mediated by the chemotactic agent LTB4, but not by the complement system.

Animals↗

PubMed1989

Postischemic renal injury is mediated by neutrophils and leukotrienes.

Neutrophils have been implicated as central mediators in myocardial and skeletal muscle ischemia-reperfusion injury. This study tests whether these cellular elements and the chemoattractant leukotriene (LTB4) play a role in postischemic renal failure. Anesthetized rats underwent 45 min of left renal pedicle clamping. Five minutes after reperfusion, LTB4 levels were elevated to 1.42 ng/ml (P less than 0.05); thromboxane (Tx)B2 was 2,840 pg/ml, higher than 503 pg/ml in sham controls (P less than 0.05); renal artery blood flow was 67% of preclamping values at 1 min of reperfusion compared with 111% in sham (P less than 0.05). At 24 h, creatinine levels were 4.6 mg/dl (P less than 0.05). At 24 h, creatinine levels were 4.6 mg/dl (P less than 0.05); histology showed acute tubular necrosis (ATN). Neutrophil depletion by rabbit antiserum (n = 8) led during reperfusion to reduced LTB4 and TxB2 levels, 1.04 ng/ml and 1.043 pg/ml (P less than 0.05); increased renal blood flow of 174% (P less than 0.05); reduced creatinine levels of 1.8 mg/dl (P less than 0.05); and limited ATN. Pretreatment with diethycarbamazine prevented the increases in LTB4 and TxB2 (P less than 0.05), increased renal blood flow (P less than 0.05), minimized creatinine increase to 1.7 mg/dl (P less than 0.05), and reduced ATN. These data indicate that neutrophils and LTB4 play a role in ischemia-induced Tx synthesis and mediate postischemic renal injury.

6-Ketoprostaglandin F1 alpha↗

PubMed1989

Thromboxane A2 mediates increased pulmonary microvascular permeability following limb ischemia.

Lower torso ischemia and reperfusion lead to respiratory dysfunction characterized by pulmonary hypertension and increased lung microvascular permeability. This is associated with lung leukosequestration and thromboxane (TX) generation. This study tests the role of elevated TX levels following muscle ischemia in mediating remote lung injury. Anesthetized sheep prepared with chronic lung lymph fistulae underwent 2 hours of bilateral hind limb tourniquet ischemia. In untreated controls (n = 7), 1 minute after reperfusion there was a transient increase in plasma immunoreactive (i)-TXB2 levels from 211 to 735 pg/ml (p less than 0.05), and at 30 minutes, lung lymph i-TXB2 levels rose from 400 to 1,005 pg/ml (p less than 0.05). At 1 minute, the mean pulmonary arterial pressure (MPAP) increased from 13 to 38 mm Hg (p less than 0.05) and pulmonary microvascular pressure (Pmv) from 7 to 18 mm Hg (p less than 0.05). Lung lymph flow (QL) rose from 4.3 to 8.3 ml/30 min (p less than 0.05), the lymph/plasma (L/P) protein ratio was unchanged from 0.6, and the lymph protein clearance increased from 2.6 to 4.6 ml/30 min (p less than 0.05). Two hours after reperfusion, neutrophils were observed sequestered in lung capillaries and proteinaceous exudates were found in alveoli in contrast to sham-operated animals (n = 3). To maximize lung vascular surface area and achieve a pressure independent L/P protein ratio a left atrial balloon was inflated during one group of ischemia-reperfusion experiments (n = 5). This resulted in a baseline rise in MPAP to 20 mm Hg (p less than 0.05); a 4.3-fold increase in QL (p less than 0.05), a decrease in the L/P ratio from 0.70 to 0.28 (p less than 0.05) and a protein reflection coefficient (sigma d) of 0.72. During reperfusion the L/P ratio rose to 0.49 (p less than 0.05) and the sigma d decreased to 0.51 (p less than 0.05), documenting an increase in lung microvascular permeability. In contrast to untreated ischemic controls, inhibition of TX synthetase with OKY 046 (n = 6) reduced plasma i-TXB2 levels to 85 pg/ml (p less than 0.05) but also increased i-6-keto-PGF1 alpha levels to 78 pg/ml relative to 15 pg/ml in untreated controls (p less than 0.05). OKY 046 prevented the increase in MPAP, Pmv, QL, and lymph protein clearance (p less than 0.05). Lung histology was normal in distinction to the leukosequestration in untreated ischemic controls.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1989

Development of surface membrane characteristics of "premedullary" macrophages in organ cultures of embryonic rat and hamster lungs.

A replicating population of non-monocyte-derived free cells appears in organ-cultured embryonic rat lungs, indistinguishable from alveolar macrophages by classical criteria such as ultrastructure, lysosomal enzyme cytochemistry, and phagocytic behavior. We demonstrate similar events in cultured embryonic hamster lungs and development of macrophage-associated properties on the plasmalemma of these cells in both species. Immunoperoxidase localizations were obtained using monoclonal antibodies against alveolar macrophage antigen (HAM1) in hamsters, and rat macrophage antigen (ED1) and leukocyte-common antigen (OX1) in rats. Fc and C3b receptors were identified in both species by immune rosetting. HAM1 staining, perinuclear in rare cells at explantation, gains definitive surface localization 3-4 days later as cells prepare to emerge through the pleura. ED1 and OX1 cytoplasmic staining first occurs after 24 hr, increases as macrophages multiply and congregate beneath the pleura, and translocates to the plasmalemma of emerged cells. Some glass-adherent cells from lung explants have Fc receptors. The proportion rises sharply for 24 hr and equals fully emerged cells (90-95%) by days 3-4. At first phagocytosis is slow to follow Fc receptor binding, but ingestion time decreases to 3-10 min as macrophages mature. A minority of emerged macrophages bind complement-opsonized erythrocytes, which are rarely taken up. These properties are shared by alveolar macrophages of adults.

Animals↗

PubMed1989

Thromboxane mediates the ischemia-induced neutrophil oxidative burst.

Indirect evidence exists that the reperfusion of ischemic tissue activates white blood cells. Thus local and systemic reperfusion injuries are prevented by making animals leukopenic or by inhibiting white blood cell lung entrapment by blocking thromboxane A2 generation. This study tests directly whether ischemia and reperfusion activates neutrophils, as measured by their oxidative burst, and whether thromboxane mediates this event. Anesthetized rats underwent 4 hours of bilateral hind limb tourniquet ischemia followed by 60 minutes of reperfusion. Plasma thromboxane B2 levels increased to 2750 pg/ml at 5 minutes of reperfusion, higher than the sham control (n = 36) value of 370 pg/ml (p less than 0.01). In untreated ischemic animals (n = 30) the intracellular H2O2 production of circulating neutrophils, as assayed flow cytometrically by dichlorofluorescein oxidation, increased from a preischemic value of 133 to a peak of 251 femtomoles dichlorofluorescein/neutrophil at 5 minutes of reperfusion (p less than 0.01). Treatment of neutrophils with phorbol myristate acetate (PMA) 10(-7) mol/L led to a 91% increase in neutrophil H2O2 production before ischemia, and 5 minutes after reperfusion there was an enhanced response to PMA of 222% (p less than 0.01). Pretreatment of animals with the thromboxane-synthetase inhibitor OKY 046 (n = 36) prevented ischemia-induced thromboxane generation, neutrophil H2O2 production (p less than 0.05), as well as the enhanced response to PMA stimulation (p less than 0.05). Treatment with the thromboxane-receptor antagonist SQ 29,548 (n = 36) did not affect the increase in plasma thromboxane levels after ischemia but was as effective as OKY 046 in preventing the ischemia-induced increase in neutrophil H2O2 production and the enhanced response to PMA stimulation. These data indicate that lower-torso ischemia leads to neutrophil activation, manifest by H2O2 production, an event mediated by thromboxane.

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