Effects of methylprednisolone on complement activation and leukocyte counts during cardiopulmonary bypass

Generation of the complement activation products C3dg and terminal complement complex (TCC) and numerical changes in peripheral granulocytes (PMN) and lymphocytes were assessed in patients undergoing aortocoronary bypass surgery with extracorporeal circulation (ECC). Fluid from bronchial lavage performed preoperatively and 4 hours postoperatively was analyzed for granulocyte elastase activity and PMN content. Ten of the 20 patients received methylprednisolone (30 mg/kg b.w.) immediately before ECC. No difference was found between them and the control group regarding C3dg and TCC, and both groups showed similar postoperative decrease of peripheral blood lymphocytes. The postoperative PMN count in peripheral blood was significantly higher in the methylprednisolone group than in the controls from 12 hours onwards. In bronchial lavage fluid the postoperative PMN count was unaltered in the methylprednisolone group, but significantly increased in the controls. No granulocyte elastase activity was found before or after surgery in either group. The results indicated that methylprednisolone does not affect complement activation during cardiopulmonary bypass, but increases the granulocytes in peripheral blood postoperatively.     

Complement activation following multiple injuries

Complement activation was evaluated by assay of plasma C3dg and the terminal complement complex (TCC) in 19 patients with multiple injuries. In the nine patients with thoracic involvement, statistically significant increase of plasma TCC was found at first sampling (average 90 min post-trauma), and of C3dg after 24 hours. Such increase was not found in the ten patients without thoracic involvement. Heightened granulocyte elastase activity was found in bronchial lavage fluid 90 min after the trauma in three patients with thoracic injury. Pulmonary insufficiency (pO2/FiO2 less than 16 kPa on intermittent positive-pressure ventilation) arose in four patients. All four had raised plasma levels of TCC or C3dg on arrival at the hospital. Six patients with complement activation did not show pulmonary insufficiency. Although the series was relatively small, the results indicate that thoracic injury is particularly associated with complement activation, and that complement activation alone does not suffice to produce post-traumatic pulmonary insufficiency.     

Biocompatibility of extracorporeal circulation. In vitro comparison of heparin-coated and uncoated oxygenator circuits

Oxygenator/tubing sets coated with endpoint-attached heparin were compared to uncoated sets in a dynamic model of extracorporeal circulation. Biocompatibility was assessed by evaluations of complement activation and platelet loss. The median concentration of C3 activation products increased gradually from 12 AU/ml at baseline to 65 AU/ml after 120 minutes in the uncoated sets and from 12 AU/ml to 19 AU/ml after 120 minutes in the coated sets (p less than 0.002). The median concentration of the terminal complement complex in the uncoated sets increased gradually from 3.1 AU/ml at baseline to 18.2 AU/ml after 120 minutes. In the coated sets the terminal complement complex reached a peak of 12.0 AU/ml at 15 minutes and returned to baseline values at 60 minutes. Median platelet loss at 120 minutes was 166 x 10(9) in the uncoated and 21 x 10(9) in the coated sets (p less than 0.01). Heparin coating thus improved biocompatibility by reducing both complement activation and platelet loss.     

A single dose of endotoxin activates neutrophils without activating complement

Both complement (C) and neutrophils (PMN) are activated in critically ill patients. To evaluate the role of endotoxin in this response, we studied C activation products and PMN cell surface receptors in seven normal subjects before and after endotoxin (USRef 20 U/kg) or saline solution administered on separate occasions. By 4 hours, with endotoxin only, all subjects had myalgia, headache, an increase in body temperature and heart rate, and leukocytosis that returned to normal by 24 hours. At the same time, PMN cell surface receptors for the complement opsonin C3b increased, as measured by indirect immunofluorescence, rising to 251 +/- 44% of baseline by 4 hours (p less than 0.01) and remaining elevated at 24 hours (237 +/- 16%, p less than 0.01). PMN receptors for iC3b increased to 308 +/- 49% of baseline by 4 hours (p less than 0.02) and returned to normal by 24 hours. There was no change in plasma of C3a desArg, C4a desArg, and C5a desArg (4 hours: mean C3a: 153.4 +/- 11.5 ng/ml versus 176.2 +/- 16.2 ng/ml for saline solution, p = ns; C4a: 159.6 +/- 32 ng/ml versus 151.4 +/- 21 ng/ml, p = ns; C5a: undetectable). To confirm the lack of C activation, we examined PMN chemotaxis (CTX) to C5a for any impairment caused by prior in vivo exposure to C5a. CTX to C5a was unaffected (4 hours: 109% +/- 22% of normal versus 114% +/- 10% for saline solution, p = ns). PMN CTX to formyl-methionyl-leucine-phenylalanine and PMN phagocytosis and killing of S. aureus were also unaffected by endotoxin. Thus, a single dose of endotoxin produced a subjective febrile illness and precipitated sustained PMN activation as indicated by increased PMN cell surface complement receptor number in the absence of C activation.     

The Role of Polymorphonuclear Leucocytes in the Pulmonary Dysfunction Induced by Complement Activation

To determine the role of polymorphonuclear leucocytes (PMNs) in the pulmonary reaction induced by complement activation, pigs were infused with complement-activated plasma (CAP), cell-free supernatant from PMNs activated in vitro, or washed PMN aggregates produced in vitro. Infusion of CAP resulted in transient peripheral leucopenia, a reversible rise in pulmonary vascular resistance (PVR) and decreased arterial oxygen tension (PaO2). Indomethacin did not influence the CAP-induced drop in PMN count or the accumulation of PMNs in the lung, but significantly counteracted the rise in PVR and fall in PaO2. Antihistamines did not prevent the cellular or pulmonary reactions to CAP infusion. Methylprednisolone did not inhibit the decrease in PMN count, but modified the pulmonary reaction to CAP, although it did not prevent the rise in PVR to the same extent as indomethacin; it counteracted the fall in PaO2. Infusion of supernatant from activated PMNs did not influence the PMN count, but caused a reversible increase in PVR and a drop in PaO2. Indomethacin counteracted the pulmonary reaction to this infusion. Infusion of washed PMN aggregates did not result in any cellular or physiological changes. These findings suggest that the pulmonary reaction induced by complement activation is mediated by humoral components generated and/or released during activation of PMNs. Arachidonic acid metabolites play an important role and it is likely that substance(s) released from activated PMNs trigger prostanoid synthesis in other cells. It is conceivable, however, that PMNs exposed to activated complement factors also directly synthesize and release arachidonic acid metabolites.     

Negative-pressure acute tracheobronchial hemorrhage and pulmonary edema

Negative-pressure pulmonary edema is a well-known complication of an acute upper airway obstruction, which may rarely present as acute alveolar hemorrhage in cases of severe capillary stress failure. Hemorrhage from the central airways has also been reported as a rare manifestation of acute tracheobronchial injury, associated with severe disruption of the bronchial vasculature due to highly negative inspiratory pressure. In this clinical report, we describe a case of both acute tracheobronchial and alveolar hemorrhage in a young man, occurring immediately after extubation due to laryngospasm, diagnosed by bronchoscopy with bronchoalveolar lavage (BAL), measurement of the pulmonary edema fluid/plasma protein ratio, and by thoracic computed tomography (CT) scan. We propose that the patient experienced severe postobstructive negative-pressure pulmonary edema, related to increased permeability of the alveolar capillary membrane and a parallel loss of integrity of the bronchial vascular network. Our findings suggest that both changes in the bronchial circulation and mechanical stress failure of the more distal alveolar-capillary system may be induced by severe and acute upper-airway obstruction.     

Complement activation and increased systemic and pulmonary vascular resistance indices during infusion of postoperatively drained untreated blood

In nine healthy young patients, operated on for thoracic scoliosis, a pulmonary artery catheter was inserted for the study of haemodynamic variables and blood sampling during autologous transfusion of postoperatively drained blood. At 1-3 h after wound closure, 10 ml kg/body weight of drained untreated blood from the wound was collected and recirculated over a l-h period. The concentration of the complement activation product, C3bc, increased from a mean of 5.4 (SD 1.5) AU ml-1 before infusion to 11.1 (3.9) AU ml-1 during infusion and then returned to 7.8 (2.8) AU ml-1 after infusion. The concentration of the terminal complement complex (TCC) increased from 0.5 (0.2) to 1.3 (0.5) AU ml-1 and was reduced to 0.7 (0.3) AU ml-1 after infusion. Only TCC exceeded the reference values which are 14 AU ml-1 for C3bc and 1.0 AU ml-1 for TCC. Pulmonary vascular resistance index concomitantly increased from a mean of 130 (SD 52) to 195 (88) dyn s cm-5 m-2 and was reduced to 170 (86) dyn s cm-5 m-2 after infusion. Systemic vascular resistance index increased from a mean of 1238 (SD 403) to 1349 (473) dyn s cm-5 m-2 and returned to 1196 (401) dyn s cm-5 m-2 after infusion. White blood cell count (WCC) increased from 14.4 (4.3) x 10(9) litre-1 before infusion to 17.8 (7.2) x 10(9) litre-1 during and after infusion. No change in platelet count during infusion was observed. There were no differences in WCC or platelet count between mixed venous or peripheral arterial blood. Pulmonary and systemic vascular resistance indices may be influenced by activated complement in drained untreated blood when it is recirculated.      

Pre-treatment of donor with 1-deamino-8-d-arginine vasopressin could alleviate early failure of porcine xenograft in a cobra venom factor treated canine recipient.

OBJECTIVE: Unlike cardiac or renal xenotransplants, the depletion of complement using cobra venom factor (CVF) does not improve pulmonary xenograft survival. Several cases suggest that the swine von Willebrand factor (vWF) may play a major role in presenting a different pathogenesis of pulmonary xenograft dysfunction from other organs. To evaluate the role of vWF and the complement system in mediating hyperacute vascular injury of pulmonary xenografts and elucidate pathogenesis of the injury, we performed swine-to-canine orthotropic single lung xenotransplantation after pre-treatment of 1-deamino-8-d-arginine vasopressin (DDAVP) and CVF. METHODS: We set up three groups for lung xenotransplantation: group I served as the control group; group II, recipients pre-treated with CVF; group III, donors pre-treated with DDAVP (9 mg/kg, 3 days)/recipients pre-treated with CVF (60 u/kg). Hemodynamic data, coagulation and complement system parameters, and grafted lung pathologies were examined serially for 3h after transplantation. RESULTS: DDAVP infusion reduced the vWF content in swine lung tissue in vivo (7.7+/-2.4 AU/mg vs 16.0+/-5.6 AU/mg, P < 0.0001). Infusion of CVF 24 h prior to transplantation effectively depleted the recipient's serum C3 and complement hemolytic activity below the detectable range. Regardless of the use of CVF, both groups I and II transplanted with unmodified grafts showed an immediate drop in leukocytes and platelet counts after transplantation. However, in group III, in recipients transplanted with DDAVP pre-treated swine lung, the platelet count did not decrease after transplantation (P = 0.0295). The decrease of plasma antithrombin and fibrinogen tended to be attenuated in group III. Light microscopic examination revealed extensive vascular thromboses in both capillary and larger vessels, as well as early pulmonary parenchymal damage in groups I and II, but were rarely observed in group III. CONCLUSIONS: Complement inhibition alone was not enough to alleviate intravascular thrombosis, the main pathology in pulmonary xenotransplantation. Pre-infusion of DDAVP to the donor animal was effective in preventing platelet sequestration and attenuated intravascular thrombosis. It is suggested that the strategies targeting vWF would be promising for successful pulmonary xenotransplantation.     

Reduced complement activation with heparin-coated oxygenator and tubings in coronary bypass operations.

Complement activation after cardiopulmonary bypass is correlated with postoperative organ dysfunction. Heparin coating of the entire blood-contact surface of the cardiopulmonary bypass circuit has proved to reduce complement activation in vitro. A membrane oxygenator and tubing setup coated with functionally active heparin was compared with an uncoated, otherwise identical setup in 20 patients undergoing routine coronary bypass operations. The concentrations of C3 activation products and the terminal complement complex were measured in sensitive and specific enzyme immunoassays. Peak concentrations of C3 activation products were 90.1 (74.7 to 107.4) AU/ml (medians and 95% confidence intervals) and 52.4 (35.7 to 76.4) AU/ml with the uncoated and coated setups, respectively (p = 0.02). The corresponding concentrations of the terminal complement complex were 26.2 (20.1 to 37.5) AU/ml and 13.7 (11.1 to 25.1) AU/ml (p = 0.03). Blood loss from the mediastinal drains during the first 12 postoperative hours was 533 (416 to 975) ml in patients treated with the uncoated setup and 388 (313 to 579) ml in the coated treatment group (p = 0.06) and was significantly correlated with peak concentrations of the terminal complement complex (p = 0.01). There were no differences in neutrophil counts nor platelet numbers between the treatment groups. The approximate 45% reduction in complement activation with the heparin-coated cardiopulmonary bypass device indicates a substantial improvement of biocompatibility.     

Neutrophil activation after burn injury: contributions of the classic complement pathway and of endotoxin

We attempt to elucidate the mechanisms of neutrophil (PMN) activation after burn injury. We previously reported prolonged elevations of PMN cell surface complement (C) opsonin receptor levels after burn trauma with a corresponding period of depressed PMN chemotaxis to C5a, which suggests that the C product, C5a, was responsible for PMN activation. However, a lack of direct correlation of C activation with C receptor levels soon after injury raised the possibility of a second PMN-activating substance. We therefore investigated the effect of endotoxin (LPS) on the expression of the C receptors (CR1 and CR3) by normal human PMNs. Concentrations from 0 to 50 ng/ml of LPS 026:B6 caused a dose response increase in the PMN surface expression of CR1 and CR3 as assessed by monoclonal antibody binding and indirect immunofluorescence. The relative CR1-dependent fluorescence rose from a mean of 50 to 385 and CR3 from 50 to 300. Chelation by ethylenediaminetetra acetic acid (EDTA) did not influence this dose response, thus ruling out the possibility of C activation by LPS--an inference supported by the lack of complement activation observed with these concentrations of LPS in normal serum. A similar dose response was obtained in the absence of other cell types or serum, which implies a direct effect that mimicked that of C5a. To determine the mechanism of the later, prolonged C activation after burn injury, we next examined C activation products in 22 patients with burn injuries. Elevations of plasma C3a desArg were present and persisted for 50 days. Elevations were at maximum levels on days 9 through 13 postburn (mean +/- standard error of mean [SEM], 496 +/- 47 ng/ml versus normal 113 +/- 32; p less than 0.01). These were accompanied by elevations of C4a desArg (917 +/- 154 ng/ml versus normal 424 +/- 50; p less than 0.01), which are indicative of classic pathway activation. Finally, we examined PMN function, phagocytosis and percentage killing of Staphylococcus aureus, and found PMN function to be unaltered in the 22 patients. Thus PMN activation after burn injury appears to be caused by LPS soon after injury and by C5a later after injury and affects only selected PMN functions.     

Effects of zymosan-activated plasma and phorbol myristate acetate on isolated, perfused rabbit lungs

The effects of complement activation on pulmonary vascular permeability are disputed. In rabbit lungs perfused with autologous blood, zymosan activated plasma (ZAP) induced a moderate increase in pulmonary vascular resistance (PVR), but did not detectably change the vascular permeability within 2 h. The stronger neutrophil granulocyte (PMN) activator, phorbol myristate acetate (PMA), usually gave larger PVR increases and also increased pulmonary vascular permeability. Lungs from neutropenic animals, similarly perfused and given PMA, showed unchanged PVR reactions but had no apparent increase in vascular permeability. Lungs perfused with cell-free medium and given PMA displayed modest PVR increases, and no measurable permeability change. The lung preparatory procedure itself markedly influenced leukocyte circulation. Exsanguination of lung donors decreased the concentration of circulating PMN significantly, and they virtually disappeared from the perfusate within minutes after start of lung perfusion. PMN-mediated effects must therefore have been caused by cells already sequestered in the lungs. We conclude that ZAP does not induce an increased pulmonary vascular permeability in isolated, perfused rabbit lungs, in contrast to PMA. The permeability effects of PMA appear to be PMN dependent.     

Hyaluronic acid in bronchoalveolar lavage fluid in patients with sarcoidosis: relationship to lavage mast cells.

Hyaluronate (hyaluronic acid), a potential marker for activated pulmonary fibroblasts, appears in increased concentrations in bronchoalveolar lavage fluid from patients with sarcoidosis. The mechanisms underlying fibroblast proliferation are largely unknown but activated alveolar T lymphocytes and macrophages probably play a part; the mast cell is also important for fibroblast proliferation. This study was designed to determine whether there is any association between pulmonary mast cells in lavage fluid, which are known to be increased in patients with sarcoidosis, and signs of pulmonary fibroblast activation. A strong correlation was found between lavage fluid hyaluronate and recovered mast cells (r = 0.72, p less than 0.001). Moreover, mast cell and hyaluronate estimations correlated inversely with lung volume and transfer factor for carbon monoxide, and both indices increased with advancing radiological sarcoid stage. Macrophage and granulocyte counts were normal in lavage fluid from patients with sarcoidosis and were not related to lavage fluid hyaluronate or laboratory signs of the disease in the lungs. Lymphocytes were recovered in increased numbers (p less than 0.001) and were related to the lavage fluid mast cells and hyaluronate. It is concluded that in sarcoidosis release of hyaluronate into the airways is related to the degree of lung disease and to the local inflammatory reaction in the lung as defined by increased numbers of mast cells and lymphocytes in lavage fluid. The findings may reflect a link between the immune system, activation of mast cells, and a pulmonary fibroblast proliferation.     

虫草多糖预先给药对大鼠内毒素性急性肺损伤的影响

目的 评价虫草多糖预先给药对大鼠内毒素性急性肺损伤的影响.方法 雄性成年SD大鼠40只,体重190～220 g,随机分为5组(n=8):虫草多糖预先给药组(CP1-3,组)采用灌胃法分别给予虫草多糖1、2、3 g/ks,1次/12 h,连续6次,于最后1次给药后2 h时经股静脉注射内毒素5 mg/kg;急性肺损伤组(ALI组)以生理盐水1ml/100 g代替虫草多糖,其余方法同CP组;对照组(C组)以生理盐水1 ml/100 g代替虫草多糖和内毒素,其余方法同CP组.静脉注射内毒索后6 h时处死大鼠,计算肺湿干重比(W/D)、肺渗透指数(PPI);行支气管肺泡灌洗,对支气管肺泡灌洗液(BALF)行白细胞(WBC)和多形核白细胞(PMN)计数;测定肺组织及BALF肿瘤坏死因子α(TNF-α)水平,并行肺组织病理学评分.结果 与C组比较,ALI组和CP1～3组肺W/D、PPI、WBC和PMN计数、肺组织病理学评分、肺组织和BALF TNNF-α水平均升高(P<0.05或0.01);与ALI组比较,CP1组上述指标差异无统计学意义(P>0.05),CP2,3组上述指标降低(P<0.05);与CP2组比较,CP3组PMN计数、肺组织病理学评分、肺组织和BALFTNF-α水平降低(P<0.05).结论 虫草多糖预先给药可减轻大鼠内毒素性急性肺损伤,且呈剂量依赖性,其机制可能与虫草多糖降低肺组织炎性反应有关.     

Complement activation with bubble and membrane oxygenators in aortocoronary bypass grafting

Thirty-three patients admitted for coronary bypass grafting were randomized to cardiopulmonary bypass with a bubble oxygenator (Cobe or Polystan) or a membrane oxygenator (SciMed). Plasma concentrations of C3 activation products and the terminal complement complex were measured using enzyme immunoassays. Both variables increased almost linearly after onset of cardiopulmonary bypass, with maximal concentrations at closure of the sternum. From a baseline of 7.5 to 12.0 arbitrary units (AU)/mL (medians), the concentrations of C3 activation products increased by 117.5 AU/mL (Cobe), 120.5 AU/mL (Polystan), and 213.3 AU/mL (SciMed). The increase in the membrane group was significantly higher than in the two bubble oxygenator groups (p less than 0.01). From a baseline of 0.9 to 1.3 AU/mL, the concentrations of terminal complement complex increased by 5.4 AU/mL (Cobe), 6.6 AU/mL (Polystan), and 7.7 AU/mL (SciMed) (differences not significant). The higher C3 activation caused by the membrane oxygenator may be explained by differences in flow profile and surface area in contact with blood. The study cannot confirm the general assumption that membrane oxygenators lead to lower complement activation than do bubble oxygenators.     

Endothelial selectin blockade attenuates lung permeability of experimental acid aspiration

BACKGROUND: A central role for the polymorphonuclear leukocyte (PMN) in experimental acid aspiration has been demonstrated by the observation that PMN depletion reduced pulmonary vascular permeability. This study investigates the role of recombinant soluble P-selectin glycoprotein ligand-immunoglobulin fusion protein (rPSGL-Ig), a P- and E-selectin antagonist in moderating acid aspiration lung injury. METHODS: Tracheostomy tubes were placed in male C57BL/6 mice and 0.1 N HCl was instilled into the trachea at 2 mL/kg after intravenous injection of (125)I-albumin. After 4 hours the lung vascular permeability index (PI) and PMN accumulation in the bronchoalveolar lavage fluid were assessed. RESULTS: PI in neutropenic mice was 63% reduced compared with the untreated group and similar to the PI of mice treated with 1 mg/kg rPSGL-Ig before acid aspiration. PMN count of 19 +/- 5 in the bronchoalveolar lavage fluid in rPSGL-Ig treated mice was significantly less than the untreated group PMN count of 586 +/- 72. The respective PI in mice treated with rPSGL-Ig (1/2) hour and 1 hour after acid aspiration was 45% and 39% reduced compared with the untreated group. CONCLUSIONS: Endothelial selectin blockade is as effective as PMN depletion in moderating acid aspiration induced lung permeability. Delayed antiselectin therapy can decrease lung injury.     

Conventional Mechanical Ventilation Is Associated with Bronchoalveolar Lavage-induced Activation of Polymorphonuclear Leukocytes: A Possible Mechanism to Explain the Systemic Consequences of Ventilator-induced Lung Injury in Patients with ARDS

Background: Protective ventilatory strategies have resulted in a decreased mortality rate in acute respiratory distress syndrome, but the underlying mechanisms remain unclear. The authors hypothesized that (1) mechanical ventilation modulates activation of polymorphonuclear leukocytes (PMNs), (2) the consequent release of proteinases is correlated with a systemic inflammatory response and with multiple organ dysfunction, and (3) these deleterious effects can be minimized by a protective ventilatory strategy.

Methods: Human PMNs were incubated with bronchoalveolar lavage fluid obtained from patients at entry or 36 h after randomization to ventilation with either a conventional (control) or a lung-protective strategy. PMN oxidant production and surface expression of adhesion molecules and granule markers, including CD18, CD63, and L-selectin, were measured by flow cytometry. Extracellular elastase activity was quantified using a fluorescent substrate.

Results: Bronchoalveolar lavage obtained from both groups of patients at entry showed similar effects on PMN oxidant production and expression of surface markers. At 36 h, exposure of PMNs to bronchoalveolar lavage fluid from the control group resulted in increased PMN activation as manifested by a significant increase in oxidant production, CD18, and CD63 surface expression, and shedding of L-selectin. By contrast, these variables were unchanged at 36 h in the lung-protective group. There was a significant correlation between the changes of the variables and changes in interleukin-6 level and the number of failing organs.     

Ipratropium Decreases Airway Size in Dogs by Preferential M sub 2 Muscarinic Receptor Blockade In Vivo

Background: Two major groups of drugs are available to prevent bronchoconstriction: beta-agonists and muscarinic blocking agents. Ipratropium is the most commonly used anti-cholinergic agent to treat chronic obstructive pulmonary disease. The authors studied anti-muscarinic agents to determine if they are as effective bronchodilators as beta-adrenergic agents and if not to identify the mechanism of their reduced effectiveness.

Methods: Six anesthetized dogs were studied using high-resolution computed tomography to measure changes in the cross-sectional area of conducting airways induced by cumulative doses of ipratropium with and without gallamine, a selective M2 muscarinic receptor blocker, and after metaproterenol.

Results: Metaproterenol dilated the airways and ipratropium constricted the airways. Ipratropium in concentrations of 0.01 and 0.1 mg/ml constricted the airways to 22 +/- 2% and 20 +/- 3% of control, respectively (P < 0.01), whereas larger concentrations caused bronchodilation. After complete blockade of the M2 receptors by pretreatment with intravenous gallamine, the bronchoconstrictor effect of ipratropium was abolished, and ipratropium dilated the airways by 16 +/- 8% and 27 +/- 10% of pre-gallamine baseline after doses of 0.01 and 0.1 mg/ml, respectively (P < 0.01).     

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.     

Mast cells and leukotrienes mediate neutrophil sequestration and lung edema after remote ischemia in rodents.

Reperfusion of ischemic hindlimbs leads to leukotriene B4 (LTB4) and polymorphonuclear neutrophil (PMN)-dependent lung injury. Pulmonary mast cells are capable of synthesizing LTB4 and are potential mediators of this inflammatory response. This study tests their role in PMN sequestration and pulmonary edema after hindlimb ischemia. Anesthetized, mast cell-sufficient mice (n = 8) or their congeneic mast cell-deficient strain (n = 8) were subjected to 3 hours of hindlimb ischemia. After another 3 hours of reperfusion, plasma LTB4 levels rose to 651 pg/ml, higher than sham ischemic control (n = 8) values of 202 pg/ml (p less than 0.05). At this time there was sequestration of neutrophils in the pulmonary microcirculation (54 PMN/10 high-power fields [HPF]) and an increase in lung wet/dry weight ratio (W/D) of 4.4. Both these values were higher (p less than 0.05) than those in sham ischemic animals that showed sequestration of 18 PMN/10 HPF and a lung W/D of 3.1. In contrast, mast cell-deficient mice showed an attenuation of ischemia- and reperfusion-induced rise in plasma LTB4 (507 pg/ml), fewer sequestered neutrophils (34 PMNs/10 HPF), and a reduction in lung W/D to 3.9 (all p less than 0.05). To test the role of lung LTB4 in determining PMN sequestration, rats (n = 78) were subjected to 3 hours of hindlimb ischemia. After 3 hours of reperfusion, plasma and bronchoalveolar lavage (BAL) LTB4 concentrations rose to 956 and 211 pg/ml, respectively--higher than sham values of 460 and 121 pg/ml (both p less than 0.05). After 4 hours, plasma LTB4 levels had returned to baseline, whereas BAL LTB4 had increased further to 658 pg/ml, indicating lung origin. Treatment of other rats by localized lung lavage of the lipoxygenase inhibitor diethylcarbamazine (80 mg/kg in 0.1 ml twice) prevented the ischemia- and reperfusion-induced rise in BAL LTB4 (267 pg/ml) and limited local neutrophil sequestration (from 51 PMN/10 HPF after saline aspiration to 36 PMN/10 HPF) and lung W/D (from 4.5 to 4.1) (all p less than 0.05). The data indicate that after hindlimb ischemia pulmonary mast cells and localized LTB4 synthesis mediate, in part, the lung inflammatory response.     

Phospholipid content of bronchoalveolar lavage fluid in granite workers with silicosis in Quebec.

BACKGROUND--Some of the prominent features of silicosis are hyperplasia and hypertrophy of epithelial type II cells, which in experimental animals are often accompanied by accumulation of phospholipids in the lung. METHODS--The total phospholipid content of lung lavage fluid and its composition in 28 granite stone cutters with long term exposure to silica dust (23 with radiological silicosis) was compared with that of lavage fluid in 15 normal volunteers, 15 patients with untreated idiopathic pulmonary fibrosis, and 19 patients with untreated stage 2 or 3 sarcoidosis. All lavage fluid was obtained at the time of first pulmonary investigation, which also included lung function tests. RESULTS--In the normal subjects total phospholipid content was 1.13 (0.16) micrograms phosphorus/ml of lung lavage, in the patients with idiopathic pulmonary fibrosis 0.52 (0.07) microgram/ml (p < 0.05), and in the patients with sarcoidosis 1.02 (0.20) microgram/ml composition being in the range reported in humans. In the patients with silicosis total phospholipid content was significantly decreased to an average of 0.46 (0.08) microgram/ml compared with the findings in normal subjects and patients with sarcoidosis. Within the group exposed to silica changes in total phospholipid content did not correlate with the severity of the radiographic disease, changes in lung function, the cellularity of lung lavage fluid, or hyaluronate concentrations. The secretory capacity of rat epithelial type II cells was not significantly different when cultured with bronchoalveolar lavage fluid from all four groups of subjects. CONCLUSIONS--Total phospholipid content in lung lavage fluid was significantly reduced in granite workers with radiological evidence of lung disease, but showed no correlation with radiological or functional markers of disease severity.