Bronchoscopy and lung lavage induced bilateral pulmonary neutrophil influx and blood leukocytosis in dogs and monkeys

Investigators have previously determined that repetitive lung washes attract neutrophils to the lungs. They ascribed this attraction to the release of chemotactic factors. Our studies demonstrate that lavage of one lobe of a lung attracts neutrophhils to unwashed lobes as well; therefore, the attraction of neutrophils cannot be entirely due to the local release of chemotactic agents. Further experiments showed that influx of neutrophils also occurred after bronchoscopy without lung lavage but not after the anesthesia alone. These studies suggest that the lungs have mechanisms that regulate and control neutrophil influx both locally and contralaterally. It is not yet clear whether the controlling mechanisms represent an integrated system in the control of leukocyte traffic through the lungs. In addition, lung lavage and bronchoscopy without lung lavage cause an increase in the blood leukocyte count, with an increase in the number of young neutrophil forms. Lung wash concentrates cause a dramatic increase in the number of blood leukocytes when instilled into a bronchus. These data indicate that lungs contain a material that can stimulate the bone marrow to release leukocytes from its reserves.     

Inhibition of c-Jun N-terminal kinase limits lipopolysaccharide-induced pulmonary neutrophil influx

The influx of neutrophils into the lung is a sentinel event in LPS-induced acute lung inflammation. Previous studies have shown that systemic inhibition of p38 decreases LPS-induced neutrophil influx into the alveolar space but has no effect on pulmonary parenchymal neutrophil accumulation or on microvascular leak, indicating other pathways are important in LPS-induced acute lung inflammation. This study examined the role of c-Jun N-terminal kinase in LPS-induced acute lung inflammation. Systemic inhibition of c-Jun N-terminal kinase, with the specific c-Jun N-terminal kinase inhibitor SP600125, decreased the LPS-induced accumulation of neutrophils into the lung parenchyma and alveolar space. In addition, increases in microvascular leak after LPS exposure were diminished by c-Jun N-terminal kinase inhibition. To determine mechanisms by which systemic c-Jun N-terminal kinase inhibition decreased pulmonary neutrophil influx, LPS and tumor necrosis factor alpha (TNF-alpha-)-induced neutrophil actin assembly and retention were examined. Neutrophil actin assembly was decreased after LPS and TNF-alpha stimulation with SP600125 pretreatment, as well as LPS-induced neutrophil retention. Finally, c-Jun N-terminal kinase inhibition decreased Cdc42 activation after LPS or TNF-alpha stimulation, thereby providing one mechanism by which c-Jun N-terminal kinase inhibition decreased actin assembly, and thereby pulmonary neutrophil accumulation.     

Indomethacin inhibits the airway hyperresponsiveness but not the neutrophil influx induced by ozone in dogs

To determine whether oxygenation products of arachidonic acid may be involved in the airway hyperresponsiveness induced by ozone exposure, we studied whether ozone-induced hyperresponsiveness could be inhibited by the prostaglandin synthetase inhibitor, indomethacin, in dogs. Airway responsiveness was assessed with dose-response curves of acetylcholine aerosol versus pulmonary resistance in 2 sets of experiments: in one set, 5 dogs were given no indomethacin treatment and were studied both before and after ozone exposure (3.0 ppm, 2 h); in another set, the same dogs were studied before indomethacin treatment or ozone exposure and then during treatment (1 mg/kg every 12 h for 4 days) both before and after ozone exposure. On each occasion, we also determined the number of neutrophils in biopsies of the airway epithelium. When the dogs were not treated with indomethacin, ozone caused a marked increase in responsiveness to acetylcholine and a marked increase in the number of neutrophils in the airway epithelium. When the dogs were given indomethacin, responsiveness was no different during treatment than before treatment, but more importantly, responsiveness did not increase significantly after they were exposed to ozone. Interestingly, indomethacin treatment did not affect either the baseline number of epithelial neutrophils before ozone exposure or the increase in the number of neutrophils after exposure. The results suggest that oxygenation products of arachidonic acid that are sensitive to inhibition by indomethacin play a role in ozone-induced hyperresponsiveness without affecting the influx of neutrophils.     

Corticosteroids inhibit endotoxin-induced lung lymph neutrophil stimulating activity in sheep

The Adult Respiratory Distress Syndrome (ARDS) most frequently is the result of sepsis. Accumulation of neutrophils in lung interstitium is a well-documented phenomenon, but the nature of their presence remains obscure. We hypothesized that endotoxin causes the release of substances into lung lymph that activate neutrophils and that methylprednisolone may prevent sequestration and activation of neutrophils. We used the sheep lung lymph fistula-endotoxin model of ARDS to test this hypothesis. Unanesthetized animals were given either 0.5 microgram/kg of E. coli endotoxin intravenously alone or, on a different experimental day, an identical dose of endotoxin preceded by a 1 gm bolus of methylprednisolone plus a 1 gm/hr continuous infusion. Endotoxin infusion caused the release of substances into lung lymph that were capable of stimulating normal sheep neutrophils to aggregate, migrate, and release superoxide. This activity appeared within 1 hour of endotoxin and persisted for at least 4 hours. Pretreatment by methylprednisolone did not prevent the early activity but did significantly reduce such activity 3-4 hours after endotoxin, when the permeability defects caused by endotoxin are most pronounced. We speculate that endotoxin-stimulated production of humoral neutrophil-activating substances in the lung may play a role in the pathogenesis of acute lung injury.     

Cyclophosphamide induced early biochemical changes in lung lavage fluid and alterations in lavage cell function

The present investigation evaluated the changes in bronchoalveolar lavage fluid (BALF) biochemical constituents and indices of bronchoalveolar lavage cell functions to detect early lung injury in rats following intraperitoneal administration of cyclophosphamide (CP). Rats were exposed to a single intraperitoneal injection of CP (200 or 300 mg/kg body weight). Experimental and control rats were sacrificed at various time intervals (2, 3, 5, 7, 11, 21, and 42 days after cessation of exposure), and lung lavage was performed to examine several markers of lung injury. Biochemical analyses revealed dose-related increases in BALF angiotensin converting enzyme activity, total protein, lactate, lactate dehydrogenase, and N-acetyl--D-glucosaminidase (NAG) levels on days 2, 3, 5, 7, and dose-related increases in albumin, alkaline phosphatase, acid phosphatase, and lipid peroxidation on days 2, 3, 5, 7, and 11 after CP treatment. In contrast, reduced levels of ascorbic acid and glutathione (GSH) content were observed in lung lavage fluid. We also examined bronchoalveolar lavage cells for acid hydrolases (acid phosphatase, -glucuronidase, NAG) and GSH content. Activity of acid hydrolases was slightly elevated on day 2 and peaked on days 3, 5, and 7. However, lavage cell GSH content was decreased. Thus, measurements of pulmonary changes by analyzing lavage fluid and lavage cell functions seems to be a useful marker for assessing the early onset and development of CP-induced lung injury. Offprint requests to: G. Chandrakasan     

Hyaluronan (hyaluronic acid) in lung lavage of asbestos-exposed humans and sheep

The concentration of hyaluronan was measured in the bronchoalveolar lavage fluid (BALF) of 18 control subjects and 27 workers from the asbestos mills and mines of Québec, 9 without asbestosis and 18 with asbestosis. Hyaluronan was also measured in the BALF of 9 control sheep exposed to 100 ml phosphate-buffered saline (PBS) at 10 day intervals for 39 months, and 13 sheep exposed at the same intervals to 100 mg chrysotile in 100 ml PBS for 24 months. At month 24, the asbestos-exposed sheep were classified into 3 groups: (A) 4 sheep exposed to PBS alone, (B) 4 sheep exposed to 10 mg chrysotile asbestos every 10 days, and (C) 5 sheep exposed to 100 mg chrysotile asbestos every 10 days for 15 months. The BALF hyaluronan averaged 53.9 ± 7.4 ng/ml in human controls, 67.5 ± 10.3 ng/ml in asbestos-exposed workers without asbestosis, and 206 ± 83 ng/ml in workers with asbestosis (p < 0.05 vs. normal). In the control sheep, BALF hyaluronan was 34.7 ± 6.9 ng/ml, and it was 31.5 ± 17.8 ng/ml in the low-dosage asbestos-exposed group (A), 83.0 ± 27.7 ng/ml in the intermediate-dose group (B), and 248.0 ± 134.7 ng/ml in the high-dosage group (C) (p < 0.05 vs. controls). In contrast, the release of plasminogen activator, a protease that may play a role in limiting the fibrotic process, was increased in group A, but not in groups B and C. In conclusion, BALF hyaluronan constitutes an indicator of lung interstitial tissue changes that may reflect the activity of the fibrosing alveolitis associated with chronic asbestos exposure. Offprint requests to: A. M. Cantin     

Thromboxane receptor blockade prevents pulmonary hypertension induced by heparin-protamine reactions in awake sheep

We used competitive thromboxane A2-prostaglandin endoperoxide receptor blockade (SQ 30,741) as a probe to evaluate the role of thromboxane in ovine pulmonary vasoconstriction associated with protamine reversal of heparin anticoagulation. Control heparin-protamine reactions induced rapid release of thromboxane into arterial plasma (more than 1 ng/ml plasma), a 2.5-fold increase of pulmonary artery pressure, a 20% decrease of PaO2, and a 30% reduction in arterial white blood cell concentration. After giving SQ 30,741 despite similar thromboxane release into arterial plasma after heparin-protamine challenge, acute pulmonary hypertension was significantly reduced when 94% of pulmonary vascular smooth muscle thromboxane receptors were occupied with SQ 30,741 (p less than 0.01 at 1 minute after protamine versus control heparin-protamine reaction) and was completely abolished by a 10 mg/kg i.v. bolus (p less than 0.0001 at 1 minute after protamine versus control). Peripheral leukopenia was not affected by SQ 30,741 prophylaxis, but hypoxemia was prevented. We conclude that thromboxane causes pulmonary vasoconstriction in ovine heparin-protamine-induced pulmonary hypertension. Pulmonary vasoconstriction and hypoxemia can be completely prevented by thromboxane receptor blockade.     

Luminal neutrophil migration in ileoanal pouches studied by whole gut lavage

OBJECTIVE: White cell scintigraphy has shown that neutrophils migrate into the gut wall and lumen in ileoanal pouches. We aimed to establish whether whole gut lavage fluid can be used to investigate intestinal neutrophil migration in ileoanal pouches. DESIGN: A prospective single centre study recruiting consecutive patients. METHODS: Whole gut lavage with polyethylene glycol electrolyte solution was performed in 56 (32 men, 24 women) ileoanal pouch patients who had undergone colectomy for ulcerative colitis; the first clear effluent was collected, processed and stored at -70 degrees C. The fluid was assayed for neutrophil granulocyte elastase using a specific colorimetric assay, IgG, albumin, alpha1-antitrypsin, haemoglobin and cytokines IL-1beta and IL-8 using previously described techniques. Patients' disease activity was characterized following pouchoscopy and biopsy. RESULTS: Patients with pouchitis had significantly higher levels of granulocyte elastase in whole gut lavage fluid compared with those without pouchitis. Patients with detectable granulocyte elastase had higher pouchoscopy score, more severe mucosal neutrophil infiltration and protein loss and bleeding. These patients had significantly higher levels of cytokines IL-1beta and IL-8 in the whole gut lavage fluid, compared with patients with undetectable granulocyte elastase. CONCLUSION: Whole gut lavage fluid samples may provide a useful investigative tool to study mucosal inflammation and luminal neutrophil migration in ileoanal pouches.     

Dibutyryl cyclic AMP attenuates lung responses induced by endotoxin in conscious sheep.

Dibutyryl cyclic AMP (DBcAMP) could inhibit the production of prostanoids and modulate the pulmonary vascular responses induced by endotoxin. Diffuse lung injury after endotoxemia in sheep is accompanied by the production of prostanoids and an increase in endothelial permeability. To determine whether exogenous DBcAMP could prevent the endotoxin responses, we measured pulmonary hemodynamics, gas exchange, and lung lymph responses to an intravenous infusion of Escherichia coli endotoxin (1.0 micrograms/kg over 30 min) in unanesthetized sheep in the presence and absence of DBcAMP (30 micrograms/kg/min) infused intravenously for 6 h beginning 1 h before endotoxin infusion or for 4.5 h after 30 min of treatment with endotoxin infusion. We also measured circulating leukocytes and lung lymph and plasma concentrations of thromboxane B2 (TXB2) and prostacyclin (6-keto-PGF1 alpha) metabolites by radioimmunoassay. DBcAMP infusion before endotoxin infusion decreased endotoxin-induced pulmonary hypertension and hypoxemia and markedly attenuated the increased lung lymph flow and lymph protein clearance. DBcAMP after endotoxin only attenuated the increased lung lymph flow and lymph protein clearance. DBcAMP treatment both before and after endotoxin infusion blocked endotoxin-induced increases in lung lymph and plasma TXB2 and 6-keto-PGF1 alpha. DBcAMP did not affect the number of circulating leukocytes. Although DBcAMP alone did not affect the pulmonary and systemic hemodynamics and lung lymph balance, the potential that DBcAMP directly modulates the pulmonary vascular responses to endotoxin as a vasodilator could be expected. We conclude that DBcAMP infusion attenuates lung dysfunction caused by endotoxemia, possibly by preventing prostanoid release and modulating the pulmonary vascular responses.     

Acute respiratory distress syndrome induced by rifampicin with high levels of neutrophil and eosinophil products in bronchoalveolar lavage fluid

We reported a case with acute respiratory distress syndrome (ARDS) caused by rifampicin during therapy for pulmonary tuberculosis. A high level of eosinophil cationic protein in bronchoalveolar lavage fluid (BALF) was detected as well as interleukin-8 and neutrophil elastase. Based on these results together with the positive result of the drug lymphocyte-stimulating test, we concluded that rifampicin was the causative drug leading to ARDS. Corticosteroid therapy resulted in clinical improvement and resolution of the pulmonary infiltrates on the chest radiograph without the recurrence of pulmonary tuberculosis.     

Localized pustulosis induced by betalactams.

Localized forms of pustular drug eruptions related to antibiotics are uncommon and their mechanism is still unknown. We describe herein a patient who developed numerous pin-head pustules without erythema in the peribuccal area after ingestion of ceftibuten and amoxicillin. The relationship with these drugs was confirmed by single-blind oral challenges. The following tests were performed: prick and intradermal tests with benzylpenicilloyl polylysine, minor determinant mixture, benzylpenicillin and amoxicillin; patch tests were also carried out with benzylpenicillin, amoxicillin, cloxacillin, cefuroxime, ceftriaxone, cefazolin, ceftibuten and cefaclor. All cutaneous tests were negative. Controlled single-blind challenge tests were performed with amoxicillin, cefadroxil, ceftibuten, cefuroxime, cefaclor, erythromycin and ciprofloxacin. All betalactam antibiotics tested gave a positive reaction, with good tolerance of other antibiotics; this would appear to indicate a specific mechanism of hypersensitivity and not an unspecific reaction to wide spectrum antibiotics.     

大容量全肺灌洗治疗重度肺泡蛋白沉积症五例分析

目的　评价大容量全肺灌洗治疗重度肺泡蛋白沉积症的临床疗效及安全性。方法采用全身麻醉下双腔气管插管 ,大容量分次肺泡灌洗 ,负压回收灌洗液并记录出入量 ,计算回收率。以患者症状的改善及胸片或胸部CT及肺功能、动脉血气的改变评价疗效。结果　 5例患者的症状均得到明显改善 ,胸片显示大部分阴影消失 ,肺功能及动脉血气有明显好转。 5例患者无一例出现严重并发症。结论　大容量全肺灌洗治疗重度肺泡蛋白沉积症疗效高、安全性好     

Long-term durable benefit after whole lung lavage in pulmonary alveolar proteinosis.

Whole lung lavage (WLL) is still the gold-standard therapy for pulmonary alveolar proteinosis (PAP). The few studies on the duration of the effect of WLL, belonging to a rather remote period, show significant but transient benefits. In 21 patients with idiopathic PAP, the duration of any benefit and, in 16 of them, the time course of lung function improvement (at baseline, 1 week, 6 months, 1 yr and then every 2 yrs after WLL) were evaluated. The present WLL technique takes longer, is invasively monitored and partially modified with respect to past techniques. More than 70% of patients remained free from recurrent PAP at 7 yrs. The bulk of the improvement in spirometric results was almost completely gained in the immediate post-WLL period due to the efficient clearance of the alveoli. At a median of 5 yrs, recovery of diffusing capacity of the lung for carbon monoxide was incomplete (75 +/- 19% of the predicted value) and there were residual gas exchange abnormalities (alveolar to arterial oxygen tension difference 3.6 +/- 1.5 kPa (27 +/- 11 mmHg)) and exercise limitation, probably explained by engorgement of lymphatic vessels. In conclusion, whole lung lavage for idiopathic pulmonary alveolar proteinosis is currently a safe procedure in an experienced setting, and provides long-lasting benefits in the majority of patients.     

Changes in lung structure and cellular division induced by tracheal obstruction in fetal sheep

Increased expansion of the fetal lung, caused by obstruction of the fetal trachea, is a potent stimulus for growth and structural development of the fetal lung. Our aim was to analyze the changes in lung structure induced by fetal tracheal obstruction and to identify cell types that contribute to the growth response. Fetal sheep were exposed to 2, 4, or 10 days of tracheal obstruction (TO) and on day 128 of gestation (term "147 d"), were injected with 3H-thymidine 8 hours before tissues were collected. The right lung was fixed at 20 cm H2O and prepared for stereological and autoradiographic analysis. Alveolar wall thickness (7.8 +/- 0.3 microns vs 5.5 +/- 0.4 microns) and percent tissue space (27.9 +/- 0.9% vs 21.4 +/- 2.8%) were increased at 2 days of TO, but were not different from control at 4 and 10 days. The luminal surface area of the right lung increased gradually from 2.4 +/- 0.2 m2/kg in control fetuses to 3.6 +/- 0.4 m2/kg following 10 days of TO and this increase was accompanied by an increase in alveolar number (control: 808 x 10(6) +/- 81.9 x 10(6) vs 10d obstruct: 1254 x 10(6) +/- 63 x 10(6). Alveolar diameter increased at 2 days of TO (51.8 +/- 1.4 microns vs 43.8 +/- 1.9 microns), but was not increased further at 4 or 10 days. The percentage of dividing cells was increased at 2 days of TO (12.64 +/- 3.39% vs 1.73 +/- 0.31%), remained elevated at 4 days (5.01 +/- 0.27%), but had returned to control by day 10. The increase at 2 days was due to division of type II epithelial cells, fibroblasts, and endothelial cells. We conclude that increased expansion of the fetal lung induces time-dependent changes in lung structure and cell division rates; these include a transient increase in alveolar wall thickness, a rapid increase in alveolar number, and a gradual increase in luminal surface area. The latter is probably caused by an increase in alveolar number rather than an increase in the alveolar size.     

Whole lung lavage: the salvage therapy for pulmonary alveolar proteinosis

A 53-year-old school teacher presented with progressive exertional breathlessness and dry cough of three months duration. His diagnosis was confirmed as pulmonary alveolar proteinosis on open lung biopsy. In about three months, the disease progressed to life threatening respiratory failure. He was subjected to whole lung lavage (WLL) as a salvage therapy. The technical details of WLL performed on this patient are described. At six months follow up, he was clinically and functionally stable and leading a near normal life.     

Improvement in alveolar macrophage function after therapeutic lung lavage in pulmonary alveolar proteinosis

Alveolar macrophage (AM) function was investigated in 4 cases of pulmonary alveolar proteinosis (PAP). Therapeutic bronchoalveolar lavage (BAL) with a flexible bronchofiberscope was done 7-13 times during periods of 4-24 weeks. In the initial BAL, the total cell count was only 0.18-0.92 x 10(5)/ml. Also low AM, lymphocytosis and eosinophilia values were noted. The phagocytic activity of AM was only 3-14% and nitroblue tetrazonium (NBT) reduction of AM was under 5% before therapeutic BAL. However, the phagocytic activity was from 25-63% and NBT reduction from 15-35% after therapeutic BAL, AM function with periods of remission of PAP was normal but AM function decreased during relapse and exacerbation of PAP. After therapeutic BAL, two patients have been in remission for three years, one patient with incomplete restoration of AM function relapsed after one year, and one patient without restoration of AM function showed further deterioration after developing pulmonary tuberculosis. These findings indicate that the defect in AM function found in PAP is reversible with therapeutic BAL, and that the severity of PAP is closely associated with AM function.     

同期双侧序贯全肺灌洗术治疗肺泡蛋白沉积症3例并文献复习

目的探讨肺泡蛋白沉积症的诊断及同期序贯全肺灌洗的疗效。方法总结本院3例肺泡蛋白沉积症的临床资料及诊治经过,并复习相关文献。结果肺泡蛋白沉积症临床表现以胸闷、呼吸困难为主,CT表现为双肺弥漫性渗出或磨玻璃影,伴小叶间隔增厚,肺功能表现为弥散功能下降,肺泡灌洗液和肺组织活检PAS染色阳性可确诊,全肺灌洗为主要治疗方法。结论肺泡蛋白沉积症临床表现不典型,确诊需行支气管肺泡灌洗和肺活检,同期双肺序贯灌洗值得推广。