Pulmonary vascular response to leukotriene D4 in unanesthetized sheep: role of thromboxane.

We examined the pulmonary vascular response to an intravenous leukotriene D4 (LTD4) injection of (1 microgram X kg-1 X min-1 for 2 min) immediately followed by infusion of 0.133 microgram X kg-1 X min-1 for 15 min in awake sheep prepared with lung lymph fistulas. LTD4 resulted in rapid generation of thromboxane A2 as measured by an increase in plasma thromboxane B2 concentration. The thromboxane B2 generation was associated with increases in pulmonary arterial and pulmonary arterial wedge pressures while left atrial pressure did not change significantly. Pulmonary lymph flow (Qlym) increased (P less than 0.05) transiently from base line 6.87 +/- 1.88 (SE) ml/h to maximum value of 9.77 +/- 1.27 at 15 min following the LTD4 infusion. The maximum increase in Qlym was associated with an increase in the estimated pulmonary capillary pressure. The increase in Qlym was not associated with a change in the lymph-to-plasma protein concentration (L/P) ratio. Thromboxane synthetase inhibition with dazoxiben (an imidazole derivative) prevented thromboxane B2 generation after LTD4 and also prevented the increases in pulmonary vascular pressures and Qlym. We conclude that LTD4 in awake sheep increases resistance of large pulmonary veins. The small transient increase in Qlym can be explained by the increase in pulmonary capillary pressure. Thromboxane appears to mediate both the pulmonary hemodynamic and lymph responses to LTD4 in sheep.

Pulmonary microvascular response to LTB4: effects of perfusate composition.

We examined the effects of leukotriene B4 (LTB4) on pulmonary hemodynamics and vascular permeability using isolated perfused guinea pig lungs and cultured monolayers of pulmonary arterial endothelial cells. In lungs perfused with Ringer solution, containing 0.5 g/100 ml albumin (R-alb), LTB4 (4 micrograms) transiently increased pulmonary arterial pressure (Ppa) and capillary pressure (Pcap). Pulmonary edema developed within 70 min after LTB4 injection despite a normal Pcap. The LTB4 metabolite, 20-COOH-LTB4 (4 micrograms), did not induce hemodynamic and lung weight changes. In lungs perfused with autologous blood hematocrit = 12 +/- 1%; protein concentration = 1.5 +/- 0.2 g/100 ml), the increases in Ppa and Pcap were greater, and both pressures remained elevated. The lung weight did not increase in blood-perfused lungs. In lungs perfused with R-alb (1.5 g/100 ml albumin) to match the blood perfusate protein concentration, LTB4 induced similar hemodynamic changes as R-alb (0.5 g/100 ml) perfusate, but the additional albumin prevented the pulmonary edema. LTB4 (10(-11)-10(-6) M) with or without the addition of neutrophils to the monolayer did not increase endothelial 125I-albumin permeability. Therefore LTB4 induces pulmonary edema when the perfusate contains a low albumin concentration, but increasing the albumin concentration or adding blood cells prevents the edema. The edema is not due to increased endothelial permeability to protein and is independent of hemodynamic alterations. Protection at higher protein-concentration may be the result of LTB4 binding to albumin.

Mechanism of peptidoleukotriene-induced increases in pulmonary transvascular fluid filtration.

We examined the effects of leukotrienes C4 (LTC4) and D4 (LTD4) (1 microgram) on the pulmonary vascular filtration coefficient, a measure of vessel wall conductivity to water, and the alterations in pulmonary vascular resistance (PVR) in isolated-perfused guinea pig lungs. We also assessed whether LTC4 and LTD4 increased the permeability to albumin in cultured monolayers of pulmonary artery endothelial cells. In Ringer-perfused and blood-perfused lungs, LTC4 resulted in increases in pulmonary arterial pressure (Ppa) and the pulmonary capillary pressure (Pcap) measured as the equilibration pressure after simultaneous pulmonary arterial and venous occlusions. Pulmonary venous resistance (Rv) increased to a greater extent than arterial resistance (Ra) in both Ringer-perfused and blood-perused lungs challenged with LTC4. The greater increase in PVR in blood-perfused lungs corresponded with a greater elevation of lung effluent thromboxane B2 (TxB2) concentration. The LTC4-stimulated increase in PVR was prevented by pretreatment with meclofenamate (10(-4) M). LTD4 also induced rapid increases in Ppa and Pcap in both Ringer-perfused and blood-perfused lungs; however, Ppa decreased before stabilizing at a pressure higher than base line. The increases in Rv with LTD4 were greater than Ra. The LTD4-stimulated increases in Ra and Rv also paralleled the elevation in TxB2 concentration. As with LTC4, the increases in Ppa, Pcap, PVR, and TxB2 concentration were greater in blood-perfused than in Ringer-perfused lungs. Pretreatment with meclofenamate reduced the magnitude of the initial increase in Ppa, but did not prevent the response.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary microvascular responses to arachidonic acid in isolated perfused guinea pig lung.

We examined the effects of arachidonic acid (AA) on pulmonary hemodynamics and fluid balance in Ringer- and blood-perfused guinea pig lungs during constant-flow conditions. Mean pulmonary arterial (Ppa), venous (Pv), and capillary pressures (Pcap, estimated by the double-occlusion method) were measured, and arterial (Ra) and venous resistances (Rv) were calculated. Bolus AA injection (500 micrograms) caused transient increases (peak response 1 min post-AA) in Ppa, Pcap, and Rv without affecting Ra in both Ringer- and blood-perfused lungs. The response was sustained in blood-perfused lungs. AA had no effect on the capillary filtration coefficient in either Ringer- or blood-perfused lungs. AA stimulated the release of thromboxane B2 and 6-ketoprostaglandin F1 alpha in both Ringer- and blood-perfused lungs, but the responses were sustained only in the blood-perfused lungs. Meclofenamate (1.5 X 10(-4) M), a cyclooxygenase inhibitor, abolished the AA-induced pulmonary hemodynamic responses in both Ringer- and blood-perfused lungs, whereas U-60257 (10 microM), a lipoxygenase inhibitor, attenuated the response only in the blood-perfused lungs. In conclusion, AA does not alter pulmonary vascular permeability to water in either Ringer- or blood-perfused lungs. AA mediates pulmonary venoconstriction and thus contributes to the rise in Pcap. The venoconstriction results from the generation of cyclooxygenase-derived metabolites from lung parenchymal cells and blood-formed elements. Lipoxygenase metabolites may also contribute to the vasoconstriction in the blood-perfused lungs.

Effect of BI-L-239, A-64077 and MK-886 on leukotriene B4 synthesis by chopped guinea pig lung and on antigen-induced tracheal contraction in vitro.

The 5-lipoxygenase (5-LO) inhibitors BI-L-239 and A-64077 were compared with the 5-LO translocation inhibitor MK-886 for the ability to inhibit leukotriene B4 (LTB4) biosynthesis by chopped (1 mm3) guinea pig lung. LTB4 synthesis by ovalbumin-sensitized chopped lung tissue was determined after stimulation with either calcium ionophore (A23187) or antigen. With A23187 stimulation, MK-886 was more potent (IC50 = 0.39 +/- 0.23 microM, mean +/- SEM, p < 0.01) than BI-L-239 (IC50 = 2.48 +/- 0.46 microM) or A-64077 (IC50 = 4.68 +/- 0.70 microM) and BI-L-239 was more potent than A64077 (p < 0.02). Thus, the order of potency was MK-886 > BI-L-239 > A-64077 for inhibition of calcium ionophore-induced LTB4 generation. There was no significant differences in potency of the compounds in chopped lung stimulated with antigen: IC50 for LTB4 synthesis by A-64077 = 3.31 +/- 1.70 microM, for BI-L-239 = 9.06 +/- 4.94 microM, and for MK-886 = 13.33 +/- 7.91 microM. The ability of these compounds to inhibit contraction of tracheal tissue from actively sensitized guinea pigs in response to antigen was also determined in the presence of indomethacin (15 micrograms/ml), mepyramine, and atropine (5 micrograms each/ml). Both 5-LO inhibitors inhibited antigen-induced contraction, with IC50 values for BI-L-239 and A-64077 of 1.58 and 4.35 microM respectively. MK-886 was ineffective at inhibiting antigen-induced tracheal contraction in vitro at concentrations up to 30 microM. In summary, these compounds inhibit antigen-induced and A23187-induced leukotriene biosynthesis in guinea pig tissue. These 5-LO inhibitors were similarly effective at inhibiting antigen-induced tracheal contraction where MK-886 was ineffective.

Pulmonary microcirculatory responses to leukotrienes B4, C4 and D4 in sheep.

The pulmonary microvascular responses to leukotrienes B4, C4, and D4 (total dosage of 4 micrograms/kg i.v.) were examined in acutely-prepared halothane anesthetized and awake sheep prepared with lung lymph fistulas. In anesthetized as well as unanesthetized sheep, LTB4 caused a marked and transient decrease in the circulating leukocyte count. Pulmonary transvascular protein clearance (pulmonary lymph flow X lymph-to-plasma protein concentration ratio) increased transiently in awake sheep, suggesting a small increase in pulmonary vascular permeability. The mean pulmonary artery pressure (Ppa) also increased. In the acutely-prepared sheep, the LTB4-induced pulmonary hemodynamic and lymph flow responses were damped. Leukotriene C4 increased Ppa to a greater extent in awake sheep than in anesthetized sheep, but did not significantly affect the pulmonary lymph flow rate (Qlym) and lymph-to-plasma protein concentration (L/P) ratio in either group. LTD4 increased Ppa and Qlym in both acute and awake sheep; Qlym increased without a significant change in the L/P ratio. The LTD4-induced rise in Ppa occurred in association with an increase in plasma thromboxane B2 (TxB2) concentration. The relatively small increase in Qlym with LTD4 suggests that the increase in the transvascular fluid filtration rate is the result of a rise in the pulmonary capillary hydrostatic pressure. In conclusion, LTB4 induces a marked neutropenia, pulmonary hypertension, and may transiently increase lung vascular permeability. Both LTC4 and LTD4 cause a similar degree of pulmonary hypertension in awake sheep, but had different lymph flow responses which may be due to pulmonary vasoconstriction at different sites, i.e. greater precapillary constriction with LTC4 because Qlym did not change and greater postcapillary constriction with LTD4 because Qlym increased with the same rise in Ppa.

Effects of exogeneous leukotrienes on the pulmonary circulation.

This review illustrates the diverse effects of LTB4 and the peptidoleukotrienes (LTC4 and LTD4) on pulmonary hemodynamics and lung fluid balance. LTB4 had a lesser effect on pulmonary hemodynamics compared to the peptidoleukotrienes. The small increases in pulmonary artery pressure and pulmonary vascular resistance were the result of precapillary constriction associated with increases in effluent thromboxane concentration. On the other hand, peptidoleukotrienes resulted in marked increases in thromboxane concentrations, pulmonary artery pressure, pulmonary capillary pressure, and pulmonary vascular resistance. Increased thromboxane generation probably contributes to the noted hemodynamic alterations following peptidoleukotriene administration. Moreover, in the isolated perfused guinea pig lung, there was also a direct effect of LTD4 on pulmonary hemodynamics which was not blocked by cyclooxygenase inhibition. This latter, resistant vasoconstrictor response is analogous to the LTD4-mediated pulmonary vasoconstriction in some species which is not dependent on cyclooxygenase (Kadowitz et al., 1984). The primary site of vasoconstriction with LTD4 was in the postcapillary vessels in contrast to the LTB4 response. LTB4 increased lung vascular permeability directly (perhaps by a direct endothelial contracture) and may also contribute to increase in permeability via neutrophil activation. However, the neutrophil dependent permeability-increasing response could not be demonstrated in the isolated perfused guinea pig lung; thus, this response requires further study. In contrast, LTC4 and LTD4 had no effect on lung vascular permeability in the awake sheep, isolated perfused guinea pig lung, and the cultured bovine pulmonary endothelial monolayer system.

Proinflammatory roles of T-cell receptor (TCR)gammadelta and TCRalphabeta lymphocytes in a murine model of asthma.

The role of lymphocytes bearing alphabeta or gammadelta T-cell receptors (TCRs) was assessed during the acute allergic response in a mouse model of asthma. The inflammatory immune response to ovalbumin (OVA) was characterized in wild-type C57BL/6J mice and congenic TCRbeta(-/-) and TCRdelta(-/-) mice by evaluation of airway eosinophilia, histopathology, serum immunoglobulin (Ig)E levels, and in vivo airway responsiveness to methacholine. OVA-challenged wild-type mice demonstrated marked pulmonary inflammation, evidenced by airway eosinophilia (68 +/- 7 x 10(4) cells), peribronchial lympho-plasmocytic infiltration, and elevated serum IgE (4.9 +/- 0.6 microg/ml). These responses were markedly attenuated in TCRdelta(-/-) animals (5.0 +/- 1.0 x 10(4) eosinophils and 1.6 +/- 0. 3 microg/ml IgE) and were completely absent in TCRbeta(-/-) mice (< 1 x 10(3) eosinophils and 0.38 +/- 0.21 microg/ml IgE). Similar results were observed in mice treated with anti-TCRgammadelta or anti-TCRalphabeta monoclonal antibodies. Airway responsiveness to aerosolized methacholine was also reduced in challenged TCRdelta(-/-) animals relative to challenged wild-type mice. These results demonstrate that acute allergic airway responses are dependent upon intact TCRalphabeta and TCRgammadelta lymphocyte function and that TCRgammadelta cells promote acute airway sensitization.

The effects of an anti-CD18 antibody (R15.7) in antigen-induced airway hyperresponsiveness (AH) and cell influx in guinea pigs.

Aerosol ovalbumin challenge (OA) of sensitized guinea pigs induced airway hyperreactivity (AH) to i.v. acetylcholine (Ach) and serotonin (5-HT) 24 hr post OA. Bronchoalveolar lavage fluid 24 hrs after OA showed increased leukocytes compared to unsensitized unchallenged animals. Treatment with monoclonal antibody R15.7 (3 mg/kg i.v.,) 1 hr prior and 4 hours after OA prevented the induction of AH to Ach but not to 5-HT and reduced influx of leukocytes. We conclude: 1) antigen inhalation induces an increase in AH with an increase in proinflammatory cell influx and 2) treatment with anti-CD18 antibody inhibits cell influx and airway hyperreactivity.

Repeated measurement of intestinal permeability as an assessment of colitis severity in HLA-B27 transgenic rats.

We report on the development of a method for repeated monitoring of mucosal permeability that allows assessment of the severity of colitis and evaluation of treatment efficacy in HLA-B27 transgenic rats. We determined the extent to which intestinal permeability related to stool condition, colon weight, and histological pathology in precolitic and diseased rats up to 29 weeks old. Intestinal permeability was measured by the urinary excretion of iodixanol at 24 h after oral administration. Mean permeability values increased significantly with age in HLA-B27 rats but remained decreased in the background strain Fischer-344 (F-344) control animals. Macroscopic evaluation of HLA-B27 rat colons between 20 and 24 weeks old showed colonic thickening with colonic wet weights increased from 3.4+/-0.13 mg/kg b.wt. in F-344 rats to 6.79+/-0.73 mg/kg b.wt. (p<.05) in HLA-B27 rats. Histological examination of HLA-B27 rat colons confirmed the colonic inflammation as a chronic active mononuclear cell infiltrate. The increase in colon weight was associated with an increase in permeability: 1.16+/-0.17 mg iodixanol versus 5.37+/-1.3 mg of iodixanol in F-344 and HLA-B27 rats, respectively. Three weeks treatment of HLA-B27 rats with cyclosporin A, but not sulfasalazine, showed a dose-dependent decrease in mucosal permeability and colon weight. Neither treatment improved stool condition. We conclude that the measurement of intestinal permeability by iodixanol excretion is a useful biochemical marker that is associated with increases in colonic weight and histological evaluation of inflammation. These data indicate that this technique may be valuable for diagnostic and evaluation purposes in preclinical models of inflammatory bowel disease.

Reduction of antigen-induced airway hyperreactivity and eosinophilia in ICAM-1-deficient mice.

A murine model of asthma is described in which we examined the role of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of airway reactivity, pulmonary eosinophilia, and inflammation. We sensitized wild-type control [C57BL/6J, (+/+)] and ICAM-1 knockout [C57BL/6J-ICAM-1, (-/-)] mice to ovalbumin (OVA), and challenged them with OVA delivered by aerosol (OVA-OVA) to induce a phenotype consistent with an asthmatic response. Bronchial responsiveness to methacholine and counts of cell numbers and measurements of eosinophil content and cytokine levels in bronchoalveolar lavage fluid (BALF) were significantly attenuated in ICAM-1(-/-) mice as compared with (+/+) mice. We also showed that the absence of ICAM-1 had no significant affects on the production of serum IgE antibody, but did have an effect on ex vivo lymphocyte proliferation. Additionally, immunohistochemistry: (1) revealed increased staining for vascular cell adhesion molecule-1 (VCAM-1) after antigen challenge in the ICAM-1(-/-) mice but not in the ICAM-1(+/+) controls; and (2) confirmed the presence of alternatively spliced forms of ICAM-1 in the lungs of ICAM-1(-/-) mice. Thus, despite the availability of alternate adhesion pathways in ICAM-1(-/-) mice, the absence of ICAM-1 prevented eosinophils from entering the airways. In summary, we found that the ICAM-1 knockout mice exhibited a significantly inhibited response to aerosol antigen challenge for most of the parameters examined, and conclude that ICAM-1 is an important ligand mediating T-cell proliferation in response to antigen, eosinophil migration into the airways, and the development of airway hyperreactivity (AHR) in allergen-sensitized and -challenged mice.

Human eosinophil major basic protein augments bronchoconstriction induced by intravenous agonists in guinea pigs.

The direct effect of intratracheal (IT) administration of human major basic protein (MBP) on pulmonary inspiratory pressure (PIP), and the effect on agonist-induced change in PIP, were determined in anesthetized, ventilated guinea pigs. 500 micrograms MBP increased PIP from 24.1 +/- 4.3 to 49.8 +/- 7.4 cmH2O (p < 0.002, n = 10). Maximum PIP was achieved within 30 min after 500 micrograms MBP. The direct PIP response to 250 micrograms MBP was not different from vehicle. The PIP responses to intravenous (IV) acetylcholine (Ach) and 5-hydroxytryptamine (5-HT) were measured before and after administration of 250 micrograms MBP (n = 12). MBP caused a modest, but significant potentiation of the increase in PIP induced by 1, 3 and 10 micrograms/kg Ach (24, 32 and 28%, respectively, p < 0.02) and to 1 microgram/kg 5-HT (43% p < 0.02). We conclude that MBP at a dose that does not directly affect inspiratory pressure is capable of augmenting the PIP response to IV Ach and 5-HT in vivo.