Pulmonary microvascular changes during hyperdynamic sepsis in an ovine model

Hyperdynamic sepsis (increased cardiac output and reduced peripheral vascular resistance) was created in sheep with chronic lung lymph fistulae (n = 8) by giving them a 30-min infusion of 1.5 micrograms/kg of endotoxin (LPS) iv. Four hours after LPS the cardiac output (CO) was reduced (6.56 +/- 0.43 to 4.96 +/- 0.33 liters/min) and lymph flow was increased (5.4 +/- 1.0 to 18.6 +/- 3.1 ml/h). Nine hours after LPS the CO output was increased (8.42 +/- 0.60 liters/min). Early cardiopulmonary changes were associated with a fall in neutrophils (PMNs) (2,667 +/- 748 to 450 +/- 90 cells/microliter) and an elevation of their chemiluminescence (CL), an indication of increased O2 free-radical formation in the blood (1,250 +/- 160 to 3,340 +/- 744 units/1,000 leukocytes). The granulocytic enzyme, aryl sulfatase, was increased in the lymph (0.19 +/- 0.03 to 0.37 +/- 0.05 microgram/h/mg protein) indicating degranulation (activation) of PMNs. When CO was increased (9 h after LPS), blood CL rose even higher (5,330 +/- 173 units/1,000 leukocytes) and CL in the lung lymph decreased (1,160 +/- 220 units/1,000 leukocytes). At this time, lymphatic aryl sulfatase had returned to baseline levels (0.25 +/- 0.02 microgram/h/mg protein). These data suggest that pulmonary microcirculatory injury produced by LPS may be the result of margination of PMNs in the lung and their release of permeability-inducing mediators. Later, as the CO increases, the PMNs or their lesion-producing mediators may be washed from the lung and the lung injury thus may be made less severe.     

Changes and significance of chemiluminescence of polymorphonuclear leukocytes in endotoxin-induced lung injury in conscious sheep]

The chemiluminescence (CL) of polymorphonuclear leukocytes and its relation with pulmonary microvascular permeability after endotoxin-induced lung injury in conscious sheep with lung lymph fistula were observed. Four hours after the injury the CL of PMNs increased from 0.27 cpm/PMN of baseline to 0.69 cpm/PMN (P < 0.05). The increment of the CL had positive correlation with the increment of lung lymph flow or permeability index (r = 0.632 0.638 P < 0.05), suggesting that the increase of pulmonary microvascular permeability after the endotoxin injury had relation with the increase of the respiratory tract of PMNs.     

Role of neutrophil adherence receptors (CD 18) in lung permeability following lower torso ischemia.

Ischemia and reperfusion of the lower torso lead to leukotriene- and neutrophil (PMN)-dependent lung injury characterized by lung PMN sequestration, increased permeability, and noncardiogenic edema. It is thought that PMNs require adhesion to endothelium to alter barrier function. This study tests the role of CD 18, the PMN adherence receptor, in mediating lung permeability after lower torso ischemia and reperfusion. Anesthetized rabbits (n = 9) underwent 3 hours of bilateral hind limb ischemia. Ten minutes after the release of the tourniquets, plasma leukotriene B4 levels increased to 395 +/- 85 pg/ml, higher than 129 +/- 35 pg/ml in controls (n = 9, p less than 0.01). At this time there was a reduction in circulating white blood cells (x 10(3)), 3.56 +/- 0.49/mm3 relative to 6.07 +/- 0.61/mm3 in controls (p less than 0.01). PMNs were sequestered in the hind limbs, indicated by increased myeloperoxidase activity of 1.06 +/- 0.19 units/g compared with 0.56 +/- 0.09 units/g in controls (p less than 0.05). Four hours after tourniquet release, PMNs were sequestered in the lungs, 52 +/- 4 PMNs per 10 high-power fields, a value higher than 31.5 +/- 3 PMNs per 10 high-power fields in controls; bronchoalveolar lavage fluid protein content increased to 554 +/- 90 micrograms/ml relative to 277 +/- 46 micrograms/ml in controls; and there was lung edema, measured by increased wet weight-to-dry weight ratios of 5.19 +/- 0.10, higher than 4.29 +/- 0.21 in controls (all p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiologic responses to small emboli and hemodynamic effects of changes in deformability of polymorphonuclear leukocytes in isolated rabbit lung.

We hypothesized that polymorphonuclear leukocytes (PMNs) exposed to lipopolysaccharide (LPS) or chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) would alter the pulmonary hemodynamics of buffer-perfused rabbit lung. Pulmonary arterial pressure (Ppa) was measured at baseline, at peak response, and at 30 min after PMN infusion in the perfusate (Ppa x time, PT product). Infusion of peritoneal-harvested PMNs resulted in a transient increase in both pulmonary vascular resistance (PVR) and lung weight. PVR also increased when glutaraldehyde-treated rabbit PMNs (GPMNs) or beads were infused. Upstream PVR (Pao-Pdo) remained high with the infusion of GPMNs and beads and returned to baseline only when PMNs were infused 30 min thereafter. FMLP-exposed PMNs increased the peak Ppa and PT product. Pretreatment with 3-isobutyl-1-methylxanthine (IBMX) blocked this increase in pressure, suggesting the release of vasoconstrictor(s) or a direct effect of FMLP. PMNs exposed to LPS increased peak Ppa and PT product with and without the addition of IBMX. Cytochalasin D treatment of PMNs prevented the increase in PT product, suggesting that actin polymerization of PMNs is involved. The effects of these agents on PMN rigidity were verified by means of 6.5-microm polycarbonate filters. PMN suspension treated with FMLP or LPS increased filter perfusion pressure and PT product. Cytochalasin D prevented these increases. These results suggest that, initially after injection, PMNs behave like small beads embolizing primarily the small arteries in the lung and that they then move distally through the vasculature. Exposure to FMLP or LPS alters PMN deformability and the ability of PMNs to pass through the pulmonary vasculature, resulting in increased pulmonary vascular resistance.     

全氟化碳腹腔注射对内毒素肺损伤大鼠中性粒细胞肺浸润的抑制作用

目的通过内毒素(脂多糖,LPS)导致的急性肺损伤(ALI)大鼠模型,探讨全氟化碳(perfluorocarbon,PFC,分子式为 C_8F_(18))腹腔预注射对内毒素导致 ALI 大鼠中性粒细胞(PMN)肺浸润的抑制作用。方法健康雄性 Wistar 大白鼠108只,随机分为正常对照组、PFC 对照组、LPS 组和PFC+LPS 组,每组分别在2、4、6 h 3个时间点各观察9只大鼠。行支气管肺泡灌洗,对支气管肺泡灌洗液(BALF)和血液进行白细胞计数和 PMN 分类,对病理组织进行 PMN 计数,免疫组化检测肺组织E-选择素和胞间黏附分子1(ICAM-1)。结果 (1)LPS 组 BALF 在2、4、6 h 白细胞计数分别为(1.98±0.21)、(2.98±0.43)(3.95±0.29)×10~6/L,PMN 分类分别为0.170±0.069、0.250±0.046、0.351±0.054,均较正常对照组升高[白细胞计数分别为(1.27±0.20)、(1.27±0.18)、(1.26±0.11)×10~6/L,PMN 分类分别为0.041±0.008、0.041±0.007、0.041±0.007,t 值为5.680～18.924,P 均<0.01],而 PFC+LPS 白细胞计数[(1.45±0.39)、(2.67±0.44)、(3.29±0.45)×10~6/L]、PNM分类(0.065±0.024、0.102±0.033、0.174±0.049)显著低于 LPS 组(t 值为-4.224～12.033,P均<0.01)。(2)与之相反,LPS 组血液白细胞计数在4、6 h 表达[(5.26±0.85)、(4.38±0.39)×10~9/L]显著低于正常对照组[(6.29±0.55)、(6.28±0.60)×10~9/L,t 值为-3.088、-7.946,P 均<0.01),而 PFC 预处理后升高。(3)PFC+LPS 组的肺组织 PMN[2、4、6 h 分别为(7.56±1.81)、(18.76±3.51)、(33.99±5.68)个/视野]、E-选择素(积分吸光度值表示,2、4、6 h 分别为867±128、1 161±227、1 922±424)和 ICAM-1(积分吸光度值表示,2、4、6 h 分别为208±78、283±67、625±85)均较 LPS 组[肺组织 PMN 2、4、6 h 分别为(10.78±0.92)、(31.55±3.00)、(54.14±5.49)个/视野,E-选择素2、4、6 h 分别为1 086±256、1 606±408、3 409±1 751,ICAM-12、4、6 h 分别为299±97、378±67、817±149]降低,差异均有统计学意义(t 值为-2.400～-11.480,P 均<0.01)。结论腹腔预注射 PFC 对内毒素导致肺损伤的 PMN 肺浸润有一定的抑制作用,可能与降低肺血管内皮细胞表达E-选择素和 ICAM-1有关。     

Lipoxygenase products induce neutrophil activation and increase endothelial permeability after thrombin-induced pulmonary microembolism

We examined the mechanism of the neutrophil (PMN)-dependent increase in pulmonary vascular permeability to protein after thrombin-induced pulmonary microembolism. Humoral factors that activate PMNs after thrombin-induced pulmonary microembolism were characterized in pulmonary lymph obtained from unanesthetized sheep challenged with intravenous infusion of alpha-thrombin. Time-dependent increases in PMN migration, aggregation, and superoxide anion (O2-) generation were induced by the pulmonary lymph obtained within 20 minutes after thrombin infusion. The pulmonary lymph neutrophil activating factors present in ether extracts of lymph had retention times of leukotriene B4 (LTB4) and monohydroxyeicosatetraenoic acids (HETEs) by high-performance liquid chromatography. The postthrombin lymph samples containing the LTB4 and HETEs increased PMN O2- generation and endothelial monolayer permeability to 125I-albumin in the presence of PMNs layered on the endothelial monolayers. Control lymph samples replete with LTB4, 5-HETE, and 15-HETE induced increases in PMN O2- generation and endothelial monolayer permeability to 125I-albumin in the presence of PMNs layered on the endothelial monolayers. Maximal increases in PMN O2- production and endothelial permeability occurred when LTB4, 5-HETE, and 15-HETE were coincubated with PMNs, indicating a synergistic action of these mediators in inducing PMN activation. Endothelial monolayer permeability to 125I-albumin did not increase with postthrombin lymph samples obtained after pretreatment with the 5-lipoxygenase inhibitor, L-651,392. The results indicate that lipoxygenase products generated in the lungs after thrombin-induced microembolism contribute to increased endothelial permeability secondary to PMN activation.     

Localized pulmonary neutrophil influx induced by lung lavage in sheep

The kinetics and distribution of neutrophil (PMN) influx into lung air spaces subsequent to bronchoalveolar lavage (BAL) were studied in adult sheep. The concentration of PMNs in BAL fluid peaked at 6 h and remained elevated for 48 h after the initial lavage. The response was observed primarily in the lung segment originally lavaged although minor changes were seen in BAL from immediately adjacent regions. No response was observed in the contralateral lung. Except for a transient decrease in pulmonary alveolar macrophages and monocytes at 6 h, changes in mononuclear cell populations (pulmonary alveolar macrophages, monocytes and lymphocytes) were minimal.     

Effects of recombinant human tumor necrosis factor alpha, lymphotoxin, and Escherichia coli lipopolysaccharide on hemodynamics, lung microvascular permeability, and eicosanoid synthesis in anesthetized sheep

We infused recombinant human tumor necrosis factor alpha (rhTNF alpha), lymphotoxin (rhLT), and Escherichia coli 0111:B4 lipopolysaccharide (LPS) into anesthetized sheep with a lung lymph fistula to compare their effects on systemic and pulmonary hemodynamics, lung lymph dynamics, and eicosanoid release. rhTNF alpha (25-150 micrograms/kg, n = 6 sheep), but not rhLT (25 micrograms/kg, n = 3), rapidly increased lung lymph and plasma levels of 6-keto-prostaglandin F1 alpha (6-k-PGF1 alpha) and caused profound systemic vasodilation and hypotension. Meclofenamate pretreatment (10 mg/kg) of three other sheep given 25 micrograms/kg rhTNF alpha prevented the increase of lymph and plasma 6-k-PGF1 alpha levels, systemic vasodilation, and the early (less than 2 hrs) but not the late (4-6 hours) hypotension caused by rhTNF alpha. LPS (1 micrograms/kg, n = 11) induced a briefer increase of lymph 6-k-PGF1 alpha levels than did rhTNF alpha while plasma 6-k-PGF1 alpha levels did not increase. LPS induced more gradual hypotension than did rhTNF alpha but did not cause systemic vasodilation. LPS and rhTNF alpha, but not rhLT, increased lymph thromboxane B2 (TXB2) levels during the first hour of study, whereas only LPS acutely increased plasma TXB2 levels. LPS caused acute pulmonary vasoconstriction and greater acute pulmonary artery hypertension than did either rhTNF alpha or rhLT. Whereas LPS-treated sheep required less fluid transfusion than rhTNF alpha-treated sheep to maintain mean systemic arterial pressure greater than 50 mm Hg, LPS infusion caused a greater increase of lung lymph protein clearance. rhTNF alpha caused minimal alterations of lung microvascular permeability. We conclude that eicosanoid mediators contribute importantly to differences of systemic and pulmonary hemodynamics caused by these agents in sheep. rhTNF alpha cannot account for all of the LPS-induced hemodynamic, lung lymph, and eicosanoid responses in sheep.     

Halothane reduces the early lipopolysaccharide-induced lung inflammation in mechanically ventilated rats

Several studies suggest that anesthetics modulate the immune response. The aim of this study was to investigate the effect of halothane and thiopental on the lung inflammatory response. Rats submitted or not to intratracheal (IT) instillation of lipopolysaccharides (LPS) were anesthetized with either halothane (0. 5, 1, or 1.5%) or thiopental (60 mg. kg(-1)) and mechanically ventilated for 4 h. Control rats were treated or not by LPS without anesthesia. Lung inflammation was assessed by total and differential cell counts in bronchoalveolar lavage fluids (BALF) and by cytokine measurements (tumor necrosis factor-alpha [TNF-alpha], interleukin-6 [IL-6], macrophage inflammatory protein-2 [MIP-2], and monocyte chemoattractant protein-1 [MCP-1]) in BALF and lung homogenates. In the absence of LPS treatment, neither halothane nor thiopental modified the moderate inflammatory response induced by tracheotomy or mechanical ventilation. Cell recruitment and cytokine concentrations were increased in all groups receiving IT LPS. However, in halothane-anesthetized rats (halothane > or = 1%), but not in thiopental-anesthetized rats, the LPS-induced lung inflammation was altered in a dose-dependent manner. Indeed, when using 1% halothane, polymorphonuclear leukocyte (PMN) recruitment was decreased by 55% (p < 0.001) and TNF-alpha, IL-6, and MIP-2 concentrations in BALF and lung homogenates were decreased by more than 60% (p < 0.001) whereas total protein and MCP-1 concentrations remained unchanged. The decrease of MIP-2 (observed at the protein and messenger RNA [mRNA] level) was strongly correlated to the decrease of PMN recruitment (r = 0.73, p < 0.05). This halothane-reduced lung inflammatory response was transient and was reversed 20 h after the end of the anesthesia. Our study shows that halothane > or = 1%, delivered during 4 h by mechanical ventilation, but not mechanical ventilation per se, alters the early LPS-induced lung inflammation in the rat, suggesting a specific effect of halothane on this response.     

Combined monitoring of thoracic duct and lung lymph during E. coli sepsis in awake sheep

A thoracic duct lymph fistula in combination with a lung lymph fistula in the awake sheep was used to evaluate effects of thoracic lymph diversion during a septic insult and to monitor systemic and local changes in the lung and gastrointestinal tract. Live Escherichia coli 10(9) kg-1 b.w. were infused in 9 sheep. After sepsis, arterial pressure, cardiac output, partial pressure of oxygen, leukocytes and platelets decreased significantly compared to baseline values. Pulmonary arterial pressure increased significantly throughout the experiment with peak values at 44 +/- 4 mmHg after 15 minutes. Lung lymph flow (QL) (n = 6) increased from 23 +/- 0.5 to 11.2 +/- 2.4 ml/30 minutes after 60 minutes. QL then decreased but remained elevated. Lymph to plasma protein concentration ratio (L/P) in lung lymph decreased from 0.62 +/- 0.02 during baseline to 0.47 +/- 0.04 after 60 minutes. L/P then increased and was, after 150 minutes, no longer different from baseline. These lung lymph data favor increased pulmonary microvascular permeability during sepsis. Lymph flow in the thoracic duct (QT) (n = 9) increased from 34.2 +/- 6 to 58.3 +/- 9 ml/30 minutes during the first 30 minutes after bacterial infusion. QT was, after 90 minutes, back to baseline but then progressively increased. L/P in thoracic lymph steadily increased from 0.56 +/- 0.03 to 0.78 +/- 0.04. Thromboxane B2 and 6-keto PGF1 alpha in thoracic duct and lung lymph increased significantly after bacterial infusion and remained elevated thereafter. Combined monitoring of thoracic duct and lung lymph enabled comparison of systemic and pulmonary reactions in septic sheep.     

Persistent increases of BAL neutrophils as a predictor of mortality following lung transplant

STUDY OBJECTIVES: To evaluate whether findings from surveillance bronchoscopy predict survival following lung transplantation. DESIGN: Retrospective review and analysis of 498 bronchoscopies with transbronchial biopsy (TBB) and BAL performed in 34 patients after lung transplantation. SETTING: University-based, tertiary referral medical center. PATIENTS: Thirty-four patients after lung transplantation. The mean age at transplantation was 49+/-9 years; 20 (59%) were female. Twenty-four (71%) underwent single and 10 (29%) underwent bilateral lung transplantation. The most common pretransplantation diagnostic groups were emphysema/COPD without concomitant alpha1-antiprotease deficiency (n = 13) and other obstructive disease processes (n = 10). INTERVENTIONS: Over follow-up, subjects underwent multiple bronchoscopies with TBB and BAL. The median number per subject was 15 (25 to 75% range 13 to 17). MEASUREMENTS AND RESULTS: We calculated the overall median BAL WBCs and median percent neutrophils (polymorphonuclear leukocytes [PMNs]) among all of the BALs performed for each subject. We then calculated the mean +/- SD of those median values. We used Cox proportionate hazards to assess mortality risk. The median overall follow-up observation period for the cohort was 560 days. There were 11 deaths during this period. Twenty-four subjects (71%) had acute rejection (AR) grades 2 to 4 (mild to severe), and nine (27%) had obliterative bronchiolitis (OB) diagnosed by TBB at any point. The mean value for BAL WBCs was 366+/-145 x 10(3) per milliliter; for percentage PMNs, the mean was 7+/-10%. Adjusting for age, gender, single vs bilateral lung transplantation, pretransplantation diagnostic group, presence of AR, presence of OB, BAL WBC concentration, and lymphocyte CD4/CD8 ratio, PMN percent was a significant predictor of mortality (p = 0.02). CONCLUSIONS: Ongoing inflammation manifested by an increased percentage PMNs over repeated bronchoscopies predicts mortality following lung transplantation. Biopsy data alone may be insufficient to identify posttransplantation patients at risk of poor outcome.     

急性肺损伤大鼠多形核白细胞L选择素变化及其在肺内扣押中的作用

目的 探讨急性肺损伤(ALI)大鼠循环血多形核白细胞(PMN)L选择素表达的变化及其于肺内扣押中的作用.方法 通过静脉注射内毒素(5 mg/kg)复制大鼠ALI模型,56只大鼠随机分为7组,每组8只.分别为(1)内毒素5 min组;(2)内毒素15 min组;(3)内毒素30 min组;(4)内毒素60 min组;(5)fucodin干预5 min组;(6)Fucodin干预15 min组静脉注射fucodin 5 mg/kg后,立即静脉注射内毒素5 mg/kg;(7)正常对照组静脉注射等量生理盐水替代内毒素.用免疫荧光间接法和流式细胞仪检测大鼠ALI过程中PMN L选择素蛋白表达的变化;用髓过氧化酶(MPO)分析法及组织学检查定量ALI过程中PMN于肺内的扣押量.结果 (1)PMN L 选择素的表达于静脉注射内毒素后5 min为7.8±1.6,与对照组(10.5±2.1)比较差异有显著性(P<0.05),静脉注射内毒素后15 min(2.9±0.5)、30 min(3.5±0.7)和60 min(4.9±0.7)与对照组比较差异更具显著性(P<0.01).(2)大鼠肺组织MPO活力于静脉注射内毒素后5 min [(0.359±0.074) U/mg肺组织]、15 min [(0.490±0.069) U/mg肺组织]、30 min [(0.565±0.111 ) U/mg肺组织]、60 min [(0.710±0.112) U/mg肺组织]与对照组[(0.069±0.011) U/mg肺组织]比较差异有显著性(P<0.01);fucodin干预5 min组[(0.391±0.071)U/mg肺组织]和对应时相点的内毒素组[(0.359±0.074) U/mg肺组织]比较差异无显著性,fucodin干预15 min组[(0.396±0.061) U/mg肺组织]和对应时相点的内毒素组[(0.490±0.069) U/mg肺组织]比较差异有显著性(P<0.05).结论 (1)正常状态下L选择素在PMN表面呈结构性表达,内毒素致伤后PMN L选择素表达迅速减少,伤后15 min时最低,其后呈回升趋势.(2)内毒素性ALI大鼠PMN肺内的早期扣押可能是L选择素非依赖性的,但L选择素对维持肺PMN的持续扣押仍然是重要的.     

Polymorphonuclear leukocyte heterogeneity in neonates and adults

We have used a mouse monoclonal antibody (31D8) to determine whether differences in neutrophil (PMN) subpopulations might help explain decreased PMN chemotaxis in neonates compared with that of adults. 31D8 has been shown to bind heterogeneously to adult PMNs. Approximately 80% of the PMNs that strongly bind 31D8 (31D8 "bright") are the same cells that depolarize and migrate chemotactically when stimulated with the chemoattractant N-formyl-methionylleucylphenylalanine, while the 20% that weakly bind 31D8 fail to similarly respond. All neonatal PMNs bound 31D8 heterogeneously. There was a smaller population of 31D8 "bright" cells in neonates at birth (76% +/- 6%, n = 45) compared with that of neonates at three to 15 days of age (82% +/- 5%, n = 10, P less than 0.002) and both were smaller than that of adults (88% +/- 4%, n = 45, P less than 0.001 and P less than 0.001). Neonatal cord PMNs, which traversed a micropore filter in a modified Boyden chemotaxis chamber in the presence of a chemoattractant, had an increased percentage of 31D8 "bright" cells (89% +/- 7%) than did PMNs which remained above the filter (82% +/- 7%, n = 10, P = 0.034). PMN chemotaxis was less in neonates at birth (32.7 +/- 4.5 micron) than at three to six days of age (36.8 +/- 11.3 micron) and both were decreased compared with that of adults (69.1 +/- 12.4 micron, P less than 0.001 and P less than 0.001). These findings indicate that decreased PMN chemotaxis in neonates may be due in part to a smaller PMN subpopulation of highly motile cells.     

The effect of anesthesia and surgery on diaphragmatic lymph vessel flow after endotoxin in sheep

Increases in diaphragmatic lymph vessel flow (Qdi) may be important in preventing ascites because diaphragmatic lymph vessels drain the peritoneal space. However, lymphatic vessel function may be depressed in anesthetized, open chested animals. To test this hypothesis, we cannulated diaphragmatic lymph vessels in five sheep which were anesthetized with 1-2% halothane. We performed a thoracotomy and cannulated a diaphragmatic lymph vessel in each sheep. Then we infused 0.75-1.0 micrograms/kg of E. coli endotoxin intravenously and we measured Qdi and the lymph protein concentration for 2-4 hrs. The data were compared to previously reported data for five unanesthetized sheep (J. Appl. Physiol. 62:706-710, 1987). At baseline Qdi = 0.8 +/- 0.7 (SD) in the anesthetized sheep and 1.0 +/- 0.8 ml/hr in the unanesthetized sheep. After endotoxin, Qdi increased to 4.5 +/- 3.1 ml/hr in the unanesthetized sheep (p less than 0.05) but Qdi did not change in the anesthetized sheep. However, the lymph protein concentration increased similarly in each group, indicating that endotoxin caused the same degree of injury in each group. Our results indicate that diaphragmatic lymph vessel function is depressed in anesthetized, open chested sheep.     

Effect of calcium antagonist gallopamil on antigen-induced early and late bronchoconstrictor responses in allergic sheep

Calcium channel blockers have been shown to modify acute antigen-induced bronchoconstriction in allergic animals and in some human subjects with bronchial asthma; however, the effects of calcium antagonists on late-phase bronchial responses are not known. In this investigation, we studied the effects of calcium antagonist gallopamil on antigen-induced early- and late-phase bronchial responses in sheep allergic to Ascaris suum antigen. In 11 unsedated sheep, specific lung resistance (SRL) was measured before, immediately after, and serially up to 8 h after inhalation challenge with A. suum antigen. On different experiment days, at least 3 wk apart, the sheep were pretreated with gallopamil, administered either as an aerosol (5 mg, n = 10; 10 mg, n = 9) or as an intravenous injection (20 micrograms/kg, n = 10), and the antigen challenge was repeated. For the control experiments (n = 11), mean +/- SE SRL increased immediately after antigen challenge (early response) to 533 +/- 72% of baseline and again 6 to 8 h after challenge (late response) to 304 +/- 31% of baseline (p less than 0.05). Neither intravenously administered nor aerosolized gallopamil had a significant effect on baseline SRL. Both aerosolized and intravenously administered gallopamil blunted the early response and completely blocked the late-phase bronchoconstrictor response (p less than 0.05). Mean SRL during early phase increased to 208 +/- 39% and 245 +/- 39% of baseline with 5 mg and 10 mg of aerosolized gallopamil and to 191 +/- 35% of baseline with intravenously administered gallopamil, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Augmentation of glutathione in the fluid lining the epithelium of the lower respiratory tract by directly administering glutathione aerosol.

Glutathione (GSH), a cysteine-containing tripeptide, functions as an antioxidant, provides cells with cysteine, and is required for optimal function of the immune system. Because the epithelial-lining fluid (ELF) of the lower respiratory tract normally contains high GSH levels and lung ELF GSH deficiency states can exist, we evaluated the feasibility of augmenting lung ELF GSH levels by (i) administering GSH to sheep i.v. and by direct aerosolization and then (ii) measuring the GSH levels in lung ELF, lung lymph, venous plasma, and urine. When GSH (600 mg) was administered i.v. (n = 11), GSH levels in venous plasma, lung lymph, and ELF rose, but only transiently, suggesting the i.v. route would not deliver adequate GSH to the alveolar epithelial surface. For directly administering GSH to the lung by the aerosol route, in vitro studies were first conducted to show that greater than 50% of a GSH solution could be converted to droplets less than 3 microns in aerodynamic diameter without oxidizing the GSH. To target functional GSH to the lower respiratory tract, an aerosolized solution of GSH (600 mg) was administered to sheep (n = 12). Significantly, the GSH level in ELF increased 7-fold at 30 min (preaerosol, 45.7 +/- 10 microM; 30-min postaerosol, 337 +/- 64 microM; P less than 0.001). The ELF GSH levels remained above baseline at 1 hr (P less than 0.01), returning toward baseline over a 2-hr period. In contrast, GSH levels in lung lymph, venous plasma, and urine were not significantly increased during the period--i.e., aerosol therapy selectively augmented the GSH levels only at the lung epithelial surface. Thus, functional GSH can be delivered by aerosol to directly augment the ELF GSH levels of the lower respiratory tract. Such an approach may prove useful in treating a variety of lung disorders.     

Lipopolysaccharide-induced lung injury in mice. I. Concomitant evaluation of inflammatory cells and haemorrhagic lung damage

Intratracheal instillation of lipopolysaccharide (LPS) induces an inflammatory response characterized by infiltration of polymorphonuclear neutrophils (PMNs) into the extracellular matrix and by the release of mediators that play a fundamental role in lung damage. In the present study, we developed a mouse model which allows correlation of the inflammatory response and haemorrhagic tissue injury in the same animal. In particular, the different steps of the inflammatory response and tissue damage were evaluated by the analysis of three parameters: myeloperoxidase (MPO) activity in the parenchyma, reflecting PMNs accumulation into the lung, inflammatory cells count in the bronchoalveolar lavage fluid (BALF), reflecting their extravasation, and total haemoglobin estimation in BALF, a marker of haemorrhagic tissue damage consequent to PMNs degranulation. In our experimental conditions, intra-tracheal administration of 10 microg/mouse of LPS evoked an increase of MPO activity in the lung at 4 h (131%) and 6 h (147%) from endotoxin challenge. A significant increase of PMNs in the BALF was noticed at these times with a plateau between the 12nd and 24th h. PMN accumulation produced a time-dependent haemorrhagic lung damage until 24 h after LPS injection (4 h: +38%; 6 h: +23%; 12 h: +44%; 24 h: +129% increase of haemoglobin concentration in the BALF vs. control). Lung injury was also assessed histopathologically. Twenty-four hours after the challenge, diffuse alveolar haemorrhage, as well as PMN recruitment in the interstitium and alveolus were observed in the LPS group. This model was pharmacologically characterized by pretreatment of LPS-treated mice with antiinflammatory drugs acting on different steps of the . We demonstrated that: a) betamethasone (1, 3, 10, 30 mg/kg p.o.) reduced in a dose-dependent manner the MPO activity, the number of inflammatory cells and, at the same time, lung injury; b) pentoxifylline, a TNFalpha production inhibitor (200 mg/kg i.p.), inhibited PMN extravasation and lung haemorrhage but it was not able to reduce MPO activity in the lung; c) L-680,833, an anti-elastase compound (30 mg/kg po), decreased significantly only the haemorrhagic lung damage; d) indomethacin, a non steroidal antiinflammatory drug (5 mg/kg p.o.), did not show any effect on any of the parameters considered. In conclusion, our in vivo mouse model is a practical alternative to animal models of ARDS (Adult Respiratory Distress Syndrome) recently described and it permits to dissect and to characterize the different steps of PMNs infiltration and, at the same time, the damage caused by their activation.     

Impact of carbon monoxide on cardiopulmonary dysfunction after smoke inhalation injury

With the inhalation of smoke, there are both cardiopulmonary changes and elevated levels of carbon monoxide (CO). We hypothesize that these changes in cardiopulmonary function are the result of a histotoxic hypoxia associated with CO poisoning. This hypothesis was tested in chronically instrumented sheep (n = 19). Piezoelectric crystals were attached to the left ventricle for the measurement of its external minor and major diameters in addition to wall thickness. A pressure transducer was placed in the left ventricle via the apex. The caudal-mediastinal lymph node was also cannulated. After a five-day recovery period, six sheep (smoke group) were insufflated with four series of 16 breaths (700 ml/breath) of cotton smoke, and five sheep (control group) were insufflated with air using a modified bee smoker (smoke group: COHb, 90 +/- 6%; control group: COHb, 6 +/- 1%). Eight sheep (CO group) were ventilated with 2% CO in air to reach a COHb of 90% (COHb, 92 +/- 1%). In the smoke group, lung lymph flow reached 42 +/- 10 ml/hr at 24 hours after smoke insufflation (baseline, 6 +/- 1 ml/hr). The maximum elastance of the left ventricle (end-systolic pressure-volume ratio), a sensitive index of myocardial contractility, was significantly decreased from a baseline of 6.5 +/- 0.9 to 3.3 +/- 0.7 mm Hg/ml. In the control and CO group, neither lung lymph flow nor maximum elastance varied from the baseline value. We conclude that the cardiopulmonary dysfunction after smoke inhalation does not occur after a similar exposure to CO. Initial CO poisoning alone is not a causative factor of cardiopulmonary dysfunction after smoke inhalation.     

Circulating human peripheral blood granulocytes synthesize and secrete tumor necrosis factor alpha.

Circulating peripheral blood polymorphonuclear neutrophils (PMNs) have long been considered terminally differentiated cells that do not synthesize or secrete protein. However, work of others and ourselves has shown that PMNs can secrete the cytokine interleukin 1. In the present study we investigated whether circulating PMNs are capable of synthesizing and secreting another cytokine, tumor necrosis factor alpha (TNF-alpha). Highly purified (greater than 99% granulocytes) PMNs were isolated from normal human volunteer blood and cultured with or without bacterial lipopolysaccharide (LPS) for up to 24 hr. Cell culture supernatants were collected and tested for TNF-alpha, and total RNA was isolated from cells at various times after stimulation and assessed for TNF-alpha mRNA by Northern blot techniques. The results showed that message for TNF-alpha was produced after 60 min of in vitro stimulation with LPS and was maximal at about 4 hr. TNF-alpha was secreted into the supernatant of unstimulated PMNs from two different donors during 24 hr of culture (35-50 pg/ml), but significantly more (160-190 pg/ml) was secreted by PMNs when stimulated with LPS. PMNs from six other normal volunteers showed significant LPS-stimulated secretion of TNF at 60-180 min of culture. The secreted product also had biological activity against the TNF-sensitive L-M cell line, confirming that PMNs can make and secrete immunologically and biologically active TNF. Since it is also possible for monocytes to synthesize and secrete TNF, the amount of TNF secreted by a monocyte population equal to 20% of the PMNs cultured was measured. The results showed that monocytes at a concentration 20 times that potentially contaminating the PMN populations cultured could not produce as much TNF (unstimulated, 26-65 pg/ml; stimulated, 32-87 pg/ml). The PMN must now be considered a cell capable of altering the acute inflammatory response and modulating the immune response through the synthesis and release of cytokines.     

The role of cellular maturation in neutrophil heterogeneity

Previous studies have shown that many neutrophil (PMN) characteristics are heterogeneous but the origin of PMN heterogeneity is unknown. It is unclear if PMN functional heterogeneity is secondary to maturational differences or due to distinct subpopulations of cells that possess different functional capacities. The PMN 31D8 antigen is a useful probe for evaluation of PMN subpopulations. The majority of PMNs (approximately 85%) exhibit a high intensity fluorescence after 31D8 monoclonal antibody (MoAb) labeling (31D8 enriched or "bright" PMNs) as determined by flow cytometric analysis. These cells are more functional than cells with low intensity fluorescence (31D8 diminished or "dull" PMNs). Various immunologic, clonogenic and functional techniques were used to study the expression of the 31D8 antigen in HL-60 cells and myeloid cells in order to evaluate antigenic and functional heterogeneity during morphologic maturation. The results of this study indicate that the percentage of 31D8 antigen positive (31D8 antigen enriched and diminished) bone marrow cells increases from 20 +/- 11% in myeloblast cells to 68 +/- 10% in promyelocytes, 93 +/- 2% in myelocytes and 99 +/- 1% in bands and PMNs. 31D8 antigen enriched cells first appear at the myelocyte stage (32 +/- 10%) and increase in bands (52 +/- 13%), marrow PMNs (62 +/- 13%) and peripheral blood PMNs (88 +/- 4%). These data indicate that the heterogeneous expression of 31D8 antigen in PMNs is due, at least in part, to maturational differences within the PMN population and raise the possibility that other heterogeneously expressed PMN characteristics are also maturationally derived. They also suggest that 31D8 antigenic expression may be a more precise indicator of myeloid functional maturation than maturation as identified by cellular morphology.