Bronchoalveolar Lavage Cell Populations Research Articles

Immunological Landscapes in Lung Transplantation: Insights from T Cell Profiling in BAL and PBMC.

Lung transplant recipients frequently encounter immune-related complications, including chronic lung allograft dysfunction (CLAD). Monitoring immune cells within the lung microenvironment is pivotal for optimizing post-transplant outcomes. This study examined the proportion of T cell subsets in paired bronchoalveolar lavage (BAL) and peripheral PBMC comparing healthy (n = 4) and lung transplantation patients (n = 6, no CLAD and n = 14 CLAD) using 14-color flow cytometry. CD4+ T cell proportions were reduced in CD3 cells in both PBMC and BAL, and positive correlations were discerned between T cell populations in peripheral PBMC and BAL, suggesting the prospect of employing less invasive PBMC sampling as a means of monitoring lung T cells. Furthermore, regulatory T cells (Tregs) were enriched in BAL when compared to peripheral PBMC for transplant recipients. A parallel positive correlation emerged between Treg proportions in BAL and peripheral PBMC, underscoring potential avenues for monitoring lung Tregs. Finally, the most promising biomarker was the Teff (CD8+Granzyme B+)-Treg ratio, which was higher in both the PBMC and BAL of transplant recipients compared to healthy individuals, and increased in the patients with CLAD compared to no CLAD and healthy patients. Conclusions: Distinct T cell profiles in BAL and peripheral PBMC underscore the significance of localized immune monitoring in lung transplantation. The Teff (CD8+granzyme B+)-Treg ratio, particularly within the context of CLAD, emerges as a promising blood and BAL biomarker reflective of inflammation and transplant-related complications. These findings emphasize the imperative need for personalized immune monitoring strategies that tailored to address the unique immunological milieu in post-transplant lungs.

Natural Killer Cells in Bronchoalveolar Lavage are Associated with Antibody-Mediated Rejection, CLAD, and Death in Lung Transplant Recipients

Natural killer (NK) cells mediate complement-independent cytotoxicity by binding donor specific antibodies (DSA) through the activating Fc receptor CD16A. We hypothesized that CD16+ NK cells in bronchoalveolar lavage (BAL) would be associated with antibody-mediated rejection (AMR) and subsequent chronic lung allograft dysfunction (CLAD). BAL was prospectively collected during 507 bronchoscopies from 195 lung transplant recipients. NK cell populations in BAL were assessed by flow cytometry. Samples with low total cell counts were excluded. AMR was graded according to ISHLT criteria. Associations between outcomes and NK cell groups were determined with generalized estimating equations. The impact of CD16+ NK cell exposure on CLAD-free survival as a time-dependent covariate was determined by Cox proportional hazards models adjusted for subject characteristics. Absolute NK cells were increased in BAL from subjects with higher numbers of HLA mismatches (p=0.006). Compared to subjects with 0-2 HLA mismatches (1x105cells/L), there were increased absolute CD16+ NK cells in subjects with 3-6 HLA mismatches (250%, p=0.01), 7-10 mismatches (560%, p<0.0001), and subjects with more than 10 mismatches (560%, p=0.001). However, BAL NK cell populations did not differ based on the presence of DSA at the time of bronchoscopy. Subjects with clinical AMR (definite, n = 6; probable, n = 12; possible, n= 18) had significantly increased CD16+ NK cells both in absolute numbers and as a percentage of NK cells in BAL compared to subjects with no AMR (Fig 1A, p<0.0001). Elevated absolute CD16+ NK cells were also associated with an increased risk for CLAD or death (Fig 1B, HR 1.3, CI 1.03-1.6, adjusted p=0.02). BAL CD16+ NK cells were associated with HLA donor and recipient mismatching, AMR, and subsequent risk of CLAD or death. NK cells may mediate allograft injury observed in AMR, and NK cell phenotyping may prove a useful adjunct in assessing AMR.

Increased NKG2C+ Natural Killer Cells in Bronchoalveolar Lavage Precede CMV Viremia and Are Associated with CLAD or Death in Lung Transplant Recipients

Purpose Cytomegalovirus (CMV) mediates clinical outcomes in lung allograft recipients. The NKG2C receptor on natural killer (NK) cells is encoded by the KLRC2 gene and recognizes CMV with memory-like capabilities. We hypothesized that NKG2C+ NK cells in bronchoalveolar lavage (BAL) would be associated with CMV burden and decreased chronic lung allograft dysfunction (CLAD)-free survival. Methods BAL cells were prospectively collected from 130 lung transplant recipients at a single center, with a median 391 days follow up time. NKG2C+ and - NK cell populations in BAL were compared for markers of maturation (NKG2A, CD16, KIR), propagation (Ki67), and KLRC2 genotype (rs2734561) by Student's t-test or ANOVA. CMV viremia was defined as negative, low ( 1000 copies/mL). NKG2C+ NK cell association with CMV viremia was determined using generalized linear modeling, and association with CLAD-free survival was determined by Cox proportional hazards models adjusted for transplant characteristics, KLRC2genotype, CMV viremia, and CMV serostatus. Results BAL NKG2C+ NK cells were more mature (NKG2A-CD16+, 25.8% vs 18.4%, p Conclusion BAL NKG2C+ NK cells were more mature than NKG2C- NK cells and expression differed between KLRC2 genotypes. NKG2C+ NK cells increased prior to viremia and higher NKG2C+ NK cell frequencies were associated with worse CLAD-free survival. Measurement of NKG2C+ NK cells in BAL may be a useful tool for assessing CMV disease burden in this population.

Neutrophils mediate airway hyperresponsiveness after chlorine-induced airway injury in the mouse.

Chlorine gas (Cl2) inhalation causes oxidative stress, airway epithelial damage, airway hyperresponsiveness (AHR), and neutrophilia. We evaluated the effect of neutrophil depletion on Cl2-induced AHR and its effect on the endogenous antioxidant response, and if eosinophils or macrophages influence Cl2-induced AHR. We exposed male Balb/C mice to 100 ppm Cl2 for 5 minutes. We quantified inflammatory cell populations in bronchoalveolar lavage (BAL), the antioxidant response in lung tissue by quantitative PCR, and nuclear factor (erythroid-derived 2)-like 2 (NRF2) nuclear translocation by immunofluorescence. In vitro, NRF2 nuclear translocation in response to exogenous hypochlorite was assessed using a luciferase assay. Anti-granulocyte receptor-1 antibody or anti-Ly6G was used to deplete neutrophils. The effects of neutrophil depletion on IL-13 and IL-17 were measured by ELISA. Eosinophils and macrophages were depleted using TRFK5 or clodronate-loaded liposomes, respectively. AHR was evaluated with the constant-phase model in response to inhaled aerosolized methacholine. Our results show that Cl2 exposure induced neutrophilia and increased expression of NRF2 mRNA, superoxide dismutase-1, and heme-oxygenase 1. Neutrophil depletion abolished Cl2-induced AHR in large conducting airways and prevented increases in antioxidant gene expression and NRF2 nuclear translocation. Exogenous hypochlorite administration resulted in increased NRF2 nuclear translocation in vitro. After Cl2 exposure, neutrophils occupied 22 ± 7% of the luminal space in large airways. IL-17 in BAL was increased after Cl2, although this effect was not prevented by neutrophil depletion. Neither depletion of eosinophils nor macrophages prevented Cl2-induced AHR. Our data suggest the ability of neutrophils to promote Cl2-induced AHR is dependent on increases in oxidative stress and occupation of luminal space in large airways.

Allergen challenge in vivo alters rhinovirus-induced chemokine secretion from human airway macrophages

Allergen challenge in vivo alters rhinovirus-induced chemokine secretion from human airway macrophages

Increased Apoptosis in the Alveolar Microenvironment of the Healthy Human Lung

Apoptosis represents a physiological clearance mechanism in human tissues. The role of apoptosis has not been examined in normal lung cell populations, such as alveolar macrophages and polymorphonuclear cells. What is the percentage, as well as the role, of apoptosis in the alveolar microenvironment of the healthy human lung? Bronchoalveolar lavage was obtained from 21 volunteers without lung disease. The specimens were analyzed using: Annexin V binding, DNA laddering, light microscopy and immunohistochemistry for bcl-2 expression. Apoptosis of the total bronchoalveolar lavage cell population was 51.2%. Both alveolar macrophages and polymorphonuclear cells had a high apoptotic rate (62.1% and 48.3%, respectively) as determined by Annexin V binding. These findings were further confirmed using morphological criteria for apoptosis and gel electrophoresis for DNA fragmentation. In the majority of the individuals examined, (8 out of 21), the bcl-2 gene was expressed in the lymphocyte population mainly. The percentage of apoptosis in lung cells of healthy humans is high. Apoptosis plays a key role in normal lung cell death. It appears to be the mechanism that opposes cell proliferation by eliminating, aged or damaged cells thus facilitating the process of lung remodeling.

Effect of corticosteroid and bronchodilator therapy on bronchoalveolar lavage cytology following intrapulmonary blood inoculation

Intrapulmonary blood causes an inflammatory response. Modifying this inflammatory response may be useful in the treatment of exercise induced pulmonary haemorrhage (EIPH). To evaluate the efficacy of 3 treatments, an oral bronchodilator, inhaled steroid and oral steroid in modifying the cellular response to intrapulmonary blood inoculation. Experimental design was a randomised block divided into 4 experimental segments of 2 weeks. Eight normal horses were randomly assigned to pairs and treatments. Autologous blood was inoculated into the lungs on 4 occasions over an 8 week period. For each treatment, a bronchoalveolar lavage (BAL) sample was collected on Day 0 and then 2 randomly selected segmental bronchi (one in each lung) were inoculated with blood (50 ml). BAL fluid was collected from one inoculated bronchus on Day 3 and from the other on Day 10. From BAL samples, total RBC and total and differential WBC were determined. Results for percentages and absolute cell counts were expressed as mean +/- s.e. and analysed using one, 2 and 3 way analysis of variance. All treatments significantly reduced the number of erythrophages at Day 3 (P = 0.02) Treatment with oral prednisolone resulted in a significantly higher percentage of macrophages (P = 0.03) and also reduced the relative and absolute number of haemosiderophages in BAL compared to the other 2 treatments at Day 10 (P = 0.07). We concluded that treatments altered the dynamics of cell populations in BAL following blood inoculation. Whether this alters the evolution of lung lesions following episodes of EIPH and indeed whether there are benefits for equine lungs following episodes of EIPH, remain to be determined. Future studies into treatments for EIPH may benefit from incorporating a variety of dose rates for oral prednisolone and a longer period of evaluation. Such studies may also benefit from the use of cell activation markers. The concomitant use of pulmonary function testing and cytology in the field may assist in ascertaining whether treatments influence the performance and welfare of athletic horses subsequent to episodes of EIPH.

Comparison of Induced Sputum and Bronchoalveolar Lavage in Lung Transplant Recipients

Chronic lung rejection is characterized by obliterative bronchiolitis (OB) diagnosed based on spirometric criteria reflecting an already advanced process. Biologic markers such as bronchoalveolar lavage (BAL) neutrophilia or increased levels of chemokines (interleukin-8, RANTES [regulated on activation: normal T cell expressed and secreted]) have been proposed as early diagnosis tools. However, BAL is too invasive to be used as a routine strategy. Induced sputum (IS), however, is a non-invasive method of recovering bronchial cells. The aim of this study was to compare BAL and IS differential cellular counts as well as IL-8 and RANTES levels between patients with bronchiolitis obliterans syndrome (BOS), recipients with good outcome and well-preserved lung function (non-BOS) and non-transplanted controls. We compared 34 BAL and IS findings obtained consecutively from 34 lung transplant recipients (LTRs), including 22 non-BOS and 12 BOS patients. IS results were compared with 19 samples from non-transplanted controls. IS was well tolerated. There was no correlation between BAL and sputum cell populations. BAL neutrophils and IL-8 levels were increased in BOS, and these parameters were positively correlated. Moreover, BAL neutrophils and IL-8 levels were both negatively correlated with respiratory function. Sputum evaluation allows discrimination of BOS from non-BOS by the presence of higher neutrophil and eosinophil counts. Moreover, IS neutrophils and eosinophils were both correlated with lung function parameters. In contrast to BAL, IL-8 level in sputum was not a useful predictive marker of BOS development. IS RANTES levels were higher in BOS than in healthy recipients and correlated significantly with IS eosinophils. IS and BAL provide different but complementary data. In this study, IS appeared to be a useful, non-invasive tool for LTR monitoring. Furthermore, IS provides new insights into BOS pathogenesis, especially with regard to implication of eosinophils and its chemokine, RANTES, at the bronchial level.

Flow cytometric characterization of cell populations in bronchoalveolar lavage and bronchial brushings from patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a serious, chronic inflammatory disease of the airway associated with cigarette smoking. Leucocytes are involved in the inflammatory process in the airways in COPD. There is a need for accurate characterization of cellular populations in bronchoalveolar lavage (BAL) due to variation in the predominant cell types reported, which were investigated mostly with manual counting techniques. Bronchial brushings and BAL were obtained from human subjects undergoing fiber optic bronchoscopy. Flow cytometry was applied to identify various cell types. Quenching of autofluorescence of BAL-derived alveolar macrophages was achieved with eta-octyl beta-D-galactopyranoside and crystal violet. Comparisons of cell counts obtained with flow cytometric and manual counting methods were performed. Correlation analysis showed that manual cell counting methods overestimated the percentage of macrophages when compared with flow cytometric methods (R2 = 0.54). There was also a small tendency by manual counting to underestimate the percentage of lymphocytes and neutrophils. Using flow cytometry, the percentage and absolute numbers of alveolar macrophages and lymphocytes in BAL were not significantly different between patients with COPD and control subjects. The percentage and absolute numbers of neutrophils were higher in BAL from patients with moderate to severe COPD. This novel flow cytometric assay for identification of various cell types from heterogenous samples of BAL and bronchial brushing will allow further investigation of cell characteristics, such as cytokine production and receptor expression, and an accurate evaluation of apoptosis for different cell types and provide a rationale for urgently required effective treatments for COPD.

Up-regulation of IL-8 secretion by alveolar macrophages from patients with fibrosing alveolitis: a subpopulation analysis.

Neutrophil accumulation in the lower respiratory tract of patients with fibrosing alveolitis is thought to be facilitated by IL-8, a neutrophil chemoattractant primarily secreted by mononuclear phagocytes. The aims of this study were: (i) to explore IL-8 secretion by lung and blood mononuclear phagocytes in subjects with cryptogenic fibrosing alveolitis, systemic sclerosis with and without fibrosing alveolitis, sarcoidosis and normal individuals; (ii) to examine IL-8 secretory heterogeneity in alveolar macrophages and peripheral blood monocytes; and (iii) to correlate alveolar macrophage phenotypic profile to IL-8 secretion. We observed that more monocytes secreted IL-8 than autologous macrophages and that there was heterogeneity in the in vitro IL-8 secretion by alveolar macrophages and peripheral blood monocytes. IL-8 secretion by alveolar macrophages was significantly higher in subjects with fibrosing alveolitis compared with subjects without fibrosing alveolitis, due to a higher percentage of IL-8-secreting alveolar macrophages in the fibrotic group both in the absence (P < 0.002) and presence of lipopolysaccharide (LPS) (P < 0.04) and correlated with bronchoalveolar lavage neutrophil percentage. Using the MoAbs RFD1, RFD7 and RFD9, that distinguish subsets of alveolar macrophages, we have been able to identify associations between secretion of IL-8 and smaller cells and the cells identified by the MoAb RFD7. In situ hybridization of the bronchoalveolar lavage cell population revealed that alveolar macrophages are the predominant source of IL-8 in the lung. We conclude that there is an increased number of IL-8-secreting alveolar macrophages in the lungs of patients with fibrosing alveolitis, and IL-8 secretion by these cells is associated with specific phenotypic profile expression.

Granulocyte macrophage-colony stimulating factor levels in bronchoalveolar lavage fluid from patients with chronic bronchitis

Background and objectives: Although inflammatory changes are found throughout the airways of patients with chronic bronchitis (CB), the mechanisms of the pathogenesis of CB are still unclear. The aim of this study was to investigate airways inflammation in patients with and without an exacerbation of CB. Granulocyte macrophage-colony stimulating factor (GM-CSF) levels in serum and bronchoalveolar lavage (BAL) fluid were assayed. Materials and methods: 40 CB patients and 10 never smoking, age and sex matched controls were studied. 20 of the CB patients were studied under baseline conditions (B), and 20 during an exacerbation (E) of CB. Bronchoscopy and bronchoalveolar lavage (BAL) with cytological analysis were performed for 7 males from CB(E) group, and 6 males from CB(B) group. The levels of GM-CSF were determined in sera and in BAL supernatants by a solid phase enzyme immunoassay. Results: There was high significant elevation of serum GM-CSF in both CB (E) and CB(B) groups in comparison with the control group (P<0.0001 & P=0.002 respectively). Both serum and BAL GM-CSF levels were elevated in CB(E) group in comparison with CB(B) group (P=0.0001 & P=0.009 respectively). Also there was significant elevation of BAL neutrophils, eosinophils and lymphocytes in CB(E) in comparison with CB(B) (P<0.05, P<0.01 and P<0.01 respectively). There was only significant positive correlation between BAL GM-CSF and BAL neutrophils count in CB(E) group, while no significant results were detected in CB(B) group. Conclusion: During exacerbations of CB there were changes in the cell populations in BAL of patients consistent with a recruitment of polymorphonuclear leucocytes, eosinophils and lymphocytes in the airway lumen. These recruited different inflammatory cells could work together toward the production of airway abnormalities and lung destruction. The locally elevated levels of BAL GM-CSF might be a cause and /or a result suggesting a role for this cytokine in the inflammatory processes of chronic bronchitis. The elevated levels of serum GM-CSF in chronic bronchitis may be due to the interference of other cytokines, microenvironmental factors in bone marrow and/or other factors.

Granulocyte macrophage-colony stimulating factor levels in bronchoalveolar lavage fluid from patients with chronic bronchitis

Background and objectives: Although inflammatory changes are found throughout the airways of patients with chronic bronchitis (CB), the mechanisms of the pathogenesis of CB are still unclear. The aim of this study was to investigate airways inflammation in patients with and without an exacerbation of CB. Granulocyte macrophage-colony stimulating factor (GM-CSF) levels in serum and bronchoalveolar lavage (BAL) fluid were assayed. Materials and methods: 40 CB patients and 10 never smoking, age and sex matched controls were studied. 20 of the CB patients were studied under baseline conditions (B), and 20 during an exacerbation (E) of CB. Bronchoscopy and bronchoalveolar lavage (BAL) with cytological analysis were performed for 7 males from CB(E) group, and 6 males from CB(B) group. The levels of GM-CSF were determined in sera and in BAL supernatants by a solid phase enzyme immunoassay. Results: There was high significant elevation of serum GM-CSF in both CB (E) and CB(B) groups in comparison with the control group (P<0.0001 & P=0.002 respectively). Both serum and BAL GM-CSF levels were elevated in CB(E) group in comparison with CB(B) group (P=0.0001 & P=0.009 respectively). Also there was significant elevation of BAL neutrophils, eosinophils and lymphocytes in CB(E) in comparison with CB(B) (P<0.05, P<0.01 and P<0.01 respectively). There was only significant positive correlation between BAL GM-CSF and BAL neutrophils count in CB(E) group, while no significant results were detected in CB(B) group. Conclusion: During exacerbations of CB there were changes in the cell populations in BAL of patients consistent with a recruitment of polymorphonuclear leucocytes, eosinophils and lymphocytes in the airway lumen. These recruited different inflammatory cells could work together toward the production of airway abnormalities and lung destruction. The locally elevated levels of BAL GM-CSF might be a cause and /or a result suggesting a role for this cytokine in the inflammatory processes of chronic bronchitis. The elevated levels of serum GM-CSF in chronic bronchitis may be due to the interference of other cytokines, microenvironmental factors in bone marrow and/or other factors.

Nitric oxide production by rat bronchoalveolar macrophages or polymorphonuclear leukocytes following intratracheal instillation of lipopolysaccharide or silica.

Exposure of the lung to lipopolysaccharide (LPS) or silica results in an activation of alveolar macrophages (AMs), recruitment of polymorphonuclear leukocytes (PMNs) into bronchoalveolar spaces, and the production of free radicals. Nitric oxide (NO) is one of the free radicals generated by bronchoalveolar lavage (BAL) cell populations following either LPS or silica exposure. The purpose of the present study was to assess the relative contributions of AMs and PMNs to the amounts of NO produced by BAL cells following intratracheal (IT) instillation of either LPS or silica. Male Sprague Dawley rats (265-340 g body wt.) were given LPS (10 mg/100 g body wt.) or silica (5 mg/100 g body wt.). BAL cells were harvested 18-24 h post-IT and enriched for AMs or PMNs using density gradient centrifugation. Media levels of nitrate and nitrite (NOx; the stable decomposition products of NO) were then measured 18 h after ex vivo culture of these cells. Following IT exposure to either LPS or silica, BAL cell populations were approximately 20% AMs and approximately 80% PMNs. After density gradient centrifugation of BAL cells from LPS- or silica-treated rats, cell fractions were obtained which were relatively enriched for AMs (approximately 60%) or PMNs (approximately 90%). The amounts of NOx produced by the AM-enriched fractions from LPS- or silica-treated rats were approximately 2-4-fold greater than that produced by the PMN-enriched fractions. Estimations of the relative contribution of AMs or PMNs to the NOx produced indicated that: (i) following LPS treatment, 75%-89% of the NOx was derived from AMs and 11%-25% from PMNs; and (ii) following silica treatment, 76%-100% of the NOx was derived from AMs and 0-24% from PMNs. Immunohistochemistry for inducible NO synthase on lung tissue sections supported these findings. We conclude that AMs are the major source of the NO produced by BAL cells during acute pulmonary inflammatory responses to LPS or silica.

Beyond our pages

Beyond our pages

Expression of bcl-2 Protein in Bronchoalveolar Lavage Cell Populations from Patients with Idiopathic Pulmonary Fibrosis

To investigate changes in the expression of the antiapoptotic protein bcl-2 in bronchoalveolar lavage fluid (BALF) cell populations in patients with idiopathic pulmonary fibrosis (IPF). Ten patients with IPF underwent fiberoptic bronchoscopy and bronchoalveolar lavage (BAL) in the area of maximal radiographic shadowing (based on high-resolution computed tomography findings). Results were compared with those of 10 normal people in the control group. Cellular bcl-2 expression was identified using an immunoperoxidase staining method. A statistically significant (P < .001) increase in the expression of bcl-2 in BALF neutrophils and eosinophils was observed in patients with IPF as compared with controls. BAL macrophages exhibited only a slight (statistically insignificant) increase in bcl-2 expression in IPF patients. No bcl-2 expression was observed in BAL lymphocytes from IPF patients in contrast to the control group. The overexpression of bcl-2 on BALF neutrophils and eosinophils, cells that characterize the special cellular profile of alveolitis in IPF, could be one of the pathophysiologic mechanisms of this disease.

Evidence for oxidative stress in bronchiolitis obliterans syndrome after lung and heart-lung transplantation. The Munich Lung Transplant Group.

Bronchiolitis obliterans syndrome (BOS) is the most serious long-term sequel of lung or heart-lung transplantation (H/LTX). Neutrophilia in the lower respiratory tract is a prominent feature of BOS. Because polymorphonuclear leukocytes (PMN) are capable of releasing large quantities of reactive oxygen species, we measured indicators of oxidative stress and glutathione levels representing antioxidant defense in H/LTX patients (HLTX, n=6; double-LTX, n=7; single-LTX, n=9). We analyzed 19 bronchoalveolar lavage (BAL) samples from 13 non-BOS patients (nine female, four male: age 39+/-4 years) and 17 BAL samples from nine BOS patients (five female, four male: age 33+/-2 years). PMN were the predominant BAL cell population in BOS (61.7+/-7.8% vs. 12.3+/-3.4%, P<0.001). Myeloperoxidase activity in the epithelial lining fluid and oxidized methionine residues in BAL-derived proteins were elevated in BOS (8.6+/-1.6 U/ml vs. 2.2+/-0.6 U/ml, P<0.01; and 12.6+/-1.1% vs. 7.7+/-0.8%, P<0.001, respectively). In addition, the concentration of reduced glutathione in epithelial lining fluid was decreased in BOS (162.6+/-20.1 microM vs. 345.8+/-57.1 microM, P<0.01), whereas the proportion of oxidized glutathione was increased (13.9+/-2.0O% vs. 6.7+/-1.2%, P<0.001). PMN, myeloperoxidase, and oxidized methionine residues were inversely correlated, whereas reduced glutathione was positively correlated with forced expiratory volume in 1 sec (P<0.05 to P<0.001). We conclude that excessive oxidative stress and a lack of glutathione are associated with BOS after H/LTX and may play relevant roles in the development of this disorder.

Alveolar clearance in horses with chronic obstructive pulmonary disease

Abstract Objective To assess sensitivity of scintigraphic alveolar clearance rate as an indicator of alveolar epithelium damage in horses. Animals 5 healthy horses (group A) and 5 with chronic obstructive pulmonary disease (COPD; group B). Procedure Horses underwent clearance rate (k [%/min]) determination. Clearance rate of group-B horses was determined after remission of the disease following 2 months at pasture (remission 1), stabling in a controlled environment (remission 2), and during crisis induced by exposure to moldy hay and straw. Methacholine challenge test was performed at each investigation period to determine nonspecific pulmonary airway hyperresponsiveness. Pulmonary function tests (PFT) also were performed, and cell populations in bronchoalveolar lavage (BAL) fluid were determined on another occasion. Results Group-B horses had significantly faster mean clearance rate during crisis (k = 4.30 ± 0.95%/min), compared with that for remission 1 (k = 1.98 ± 0.55%/min), which did not differ from the rate in group-A horses (k = 1.95 ± 0.33%/min). Despite lack of clinical signs of COPD during remission when stabled in a controlled environment, an intermediate value was found (k = 3.20 ± 0.72%/min). Conclusions This technique allowed grading of lung damage induced by COPD, whereas use of PFT and determination of BAL fluid cell populations failed to differentiate between remission 1 and remission 2. Clinical Relevance Determination of alveolar clearance rate by use of scintigraphy is a sensitive indicator of lung damage. A modified clearance rate was found despite the lack of clinical and functional changes. (Am J Vet Res 1999;60:495-500)

Tumour necrosis factor-alpha expression and cell recruitment in Sephadex particle-induced lung inflammation: effects of dexamethasone and cyclosporin A.

1. Tumour necrosis factor-alpha (TNF-alpha) is a cytokine with diverse properties consistent with a possible role in inflammatory disease. We investigated whether TNF-alpha is induced during the progression of lung inflammation elicited by a particulate non-antigenic stimulus, and whether pharmacological control of TNF-alpha expression influences recruitment of specific inflammatory cell types. 2. A single intravenous injection of Sephadex particles into rats led to extensive granulomatous inflammation in lung alveolar and bronchial tissue that peaked in intensity after 24-72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively. 3. Messenger RNA encoding TNF-alpha was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF-alpha protein was localized to mononuclear cells at all times points pre- and post-Sephadex administration. 4. Treatment of rats with dexamethasone significantly reduced the Sephadex-induced recruitment of mononuclear cells, neutrophils and eosinophils into the bronchoalveolar cavity, and significantly reduced TNF-alpha mRNA expression by BAL cells. 5. Treatment of rats with cyclosporin A was without effect on Sephadex-induced elevations of mononuclear cell numbers and expression of TNF-alpha, but did reduce significantly recruitment of neutrophils and eosinophils to BAL cell populations. 6. These results show that a sequential asthma-like recruitment of neutrophils, eosinophils and mononuclear cells into lung tissue can be induced by single exposure to a non-antigenic stimulus. Pharmacological and histological studies reveal that mononuclear cell mobilization relates closely to induced TNF-alpha expression, whereas mobilization of neutrophils and eosinophils appears secondary to expression of the cytokine.

Increased bronchoalveolar granulocytes and granulocyte/macrophage colony-stimulating factor during exacerbations of chronic bronchitis

Although inflammatory changes are found throughout the airways of patients with chronic bronchitis, the mechanisms of the pathogenesis of chronic bronchitis are still unclear. The aim of this study was to investigate airways inflammation in patients with and without an exacerbation of bronchitis. Thirteen chronic bronchitic patients and nine normal subjects were studied. Eight of the patients were studied under baseline conditions (B), and five during an exacerbation of bronchitis (E). Bronchoscopy and bronchoalveolar lavage (BAL) with cytological analysis were performed, and the levels of granulocyte/macrophage colony-stimulating factor (GM-CSF) were determined in sera and in BAL supernatants by a solid phase enzyme immunoassay. Compared with patients under baseline conditions, chronic bronchitic patients with an exacerbation had increased numbers of BAL neutrophils (10+/-3 and 83+/-18x10(3) cells x mL(-1), respectively; p<0.0001) and of BAL eosinophils (1.9+/-0.5 and 6.7/-1.9x10(3) cells x mL(-1), respectively; p=0.014). Patients with chronic bronchitis, as a whole, had significantly increased levels of BAL GM-CSF compared to control subjects (36+/-5 and 19+/-4 pg x mL(-1), respectively; p=0.035), and similar levels of serum GM-CSF. Serum levels of GM-CSF were markedly increased in chronic bronchitic patients with an exacerbation, as compared with patients under baseline conditions (1.4+/-0.4 and 13+/-1 pg x mL(-1), respectively; p <0.0001). BAL levels of GM-CSF were also increased in chronic bronchitic patients with an exacerbation (25+/-5 and 54+/-8 pg x mL(-1), respectively; p=0.009). During exacerbations of chronic bronchitis there are changes in the cell populations in bronchoalveolar lavage of patients consistent with a recruitment of polymorphonuclear leucocytes in the airway lumen. The increased levels of granulocyte/macrophage colony-stimulating factor might suggest a role for this cytokine in the inflammatory processes of chronic bronchitis.

Effects of particle exposure and particle-elicited inflammatory cells on mutation in rat alveolar epithelial cells

To investigate mechanisms underlying development of lung adenomas and carcinomas in rats exposed to poorly soluble particles the relationships between particle exposure, inflammation and mutagenesis in rat alveolar type II cells were characterized. Rats were exposed to saline or saline suspensions of 10 and 100 mg/kg of alpha-quartz, carbon black or titanium dioxide by intratracheal instillation. Fifteen months after exposure, bronchoalveolar lavage (BAL) cells were characterized as to number and type and lung histopathology performed. The alveolar type II cells were isolated and cultured in 6 thioguanine (6TG) containing media to select for mutation in the hprt gene. The potential contribution of lung inflammatory cells to in vivo mutagenic responses, were evaluated by co-culturing BAL cells with the rat alveolar epithelial cell line, RLE-6TN for 24 h and the RLE-6TN cells selected for 6TG resistance. Neutrophilic inflammation was detected in all rats exposed to 10 and 100 mg/kg of alpha-quartz and carbon black and 100 mg/kg titanium dioxide; epithelial hyperplasia was observed in rats exposed to 10 and 100 mg/kg of alpha-quartz and 100 mg/kg carbon black. Hprt mutation frequency was increased in alveolar type II cells from rats exposed to 10 and 100 mg/kg of alpha-quartz, 100 mg/kg carbon black and 100 mg/kg titanium dioxide. In vitro exposure of RLE-6TN cells to BAL cells from rats treated with 10 and 100 mg/kg of alpha-quartz or 100 mg/kg carbon black increased hprt mutant frequency. Both macrophage and neutrophil enriched BAL cell populations were mutagenic to RLE-6TN cells, however, the mutagenic activity appeared greatest for neutrophils. Addition of catalase to BAL cell:RLE-6TN co-cultures inhibited the increase in hprt mutation frequency. These studies demonstrate exposure of rats to doses of particles producing significant neutrophilic inflammation is associated with increased mutation in rat alveolar type II cells. The ability of particle-elicited macrophages and neutrophils to exert a mutagenic effect on epithelial cells in vitro supports a role for these inflammatory cells in the in vivo mutagenic effects of particle exposure. The inhibition of BAL cell-induced mutations by catalase implies a role for cell-derived oxidants in this response.