The dynamics and associations of airway neutrophilia post lung transplantation.

UNLABELLED: Bronchoalveolar lavage (BAL) neutrophilia has been repeatedly observed in lung transplant recipients with established bronchiolitis obliterans syndrome (BOS). Little is known of the fluctuations in BAL and airway neutrophilic inflammation post-transplant. This prospective longitudinal study aimed to evaluate the dynamic changes of lung allograft neutrophils with time, immunosuppression, infection and BOS. A total of 28, initially healthy, BOS 0, lung transplant recipients underwent 134 bronchoscopic assessments, including BAL and endobronchial biopsies (EBB) (with immunohistochemistry) over 3-year follow up. Subsequently, 21 developed BOS 0p and 16 ultimately BOS. Compared to controls, there was early and persistent BAL neutrophilia (p < 0.05), contrasting with an initially normal EBB that shows a progressive increased airway wall neutrophil infiltrate. BAL neutrophilia (but not airway wall neutrophilia) was most striking when there was concomitant bronchopulmonary infection, particularly in the patients with BOS. Univariate and multivariate analyses suggested that BAL neutrophilia was linked to markers of infection while EBB neutrophilia was linked with coexistent inflammation with macrophages and lymphocytes. IN CONCLUSION: (i) BAL neutrophilia is predominantly associated with infection; (ii) Airway wall neutrophilia (as monitored by EBB) increases with time post-transplant and is not associated with infection; (iii) By itself, BOS is not the major contributor to BAL and EBB neutrophilia.

Inter-relationships between airway inflammation, reticular basement membrane thickening and bronchial hyper-reactivity to methacholine in asthma; a systematic bronchoalveolar lavage and airway biopsy analysis.

BACKGROUND: Asthma is accepted as a disease characterized by airway inflammation, with evidence that airway structural changes, or 'remodelling' occurs. There are few studies relating airway physiology, inflammation and remodelling, however. We have carried out a study of inter-relationships between airway inflammation, airway remodelling, reticular basement membrane (RBM) thickening, and bronchial hyper-reactivity (BHR), before and after high-dose inhaled corticosteroid (fluticasone propionate 750 microg b.d.), in a group of relatively mild but symptomatic, steroid naïve asthma patients. METHODS: Double-blind, randomized, placebo-controlled, parallel group study of inhaled corticosteroid (ICS) in 35 asthmatics, with bronchoalveolar lavage (BAL) and airway endobronchial biopsy (EBB) for inflammatory cell profiles and EBB for airway remodelling carried out at baseline, 3 and 12 months. RESULTS: At baseline RBM thickening was related to BAL mast cells and EBB eosinophil counts. In turn baseline log EBB EG2 eosinophil count, log%BAL epithelial cells and log RBM thickness explained 55% of the variability in BHR. CONCLUSION: We provide new information that airway inflammation, remodelling, and BHR in asthma are inter-related and improved by ICS therapy. Our data potentially support the need for early and long-term intervention with ICS even in relatively mild asthmatics, and the need to further assess the potential merit of longitudinal BHR testing in management of some patients, as this may reflect both airway inflammation and remodelling.

Effect of inhaled fluticasone propionate on BAL TGF-beta(1) and bFGF concentrations in clinically stable lung transplant recipients.

BACKGROUND: Inhaled fluticasone propionate (FP) therapy decreases inflammation and sub-basement membrane thickness in asthmatic airways. Bronchiolitis obliterans syndrome (BOS) in lung transplant recipients (LTRs) involves progressive airway fibrosis and obliteration. Therefore, augmented immunosuppression may be of some benefit in treating BOS. In this study, we examined the effect of 3 months of treatment with high-dose inhaled FP on the concentrations of 2 fibrogenic factors, transforming growth factor (TGF)-beta(1) and beta fibrogenic growth factor (bFGF) in bronchoalveolar lavage (BAL) fluid from clinically stable LTRs. METHODS: We conducted a randomized, double-blind, placebo-controlled, parallel group study with inhaled FP (750 microg, twice/day for 3 months) in 28 LTRs (15 FP and 13 placebo). We recruited 23 healthy controls. We performed spirometry, bronchoscopy, and bronchoalveolar lavage procedures before treatment and after 3 months of treatment. We used commercially available enzyme-linked immunosorbent assay kits to measure BAL fluid TGF-beta(1) and bFGF concentrations. RESULTS: In LTRs before treatment, BAL TGF-beta(1) concentrations (but not bFGF concentrations), total cell counts, and neutrophil percentage increased compared with controls (p < 0.05). We found no significant differences between FP and placebo groups at baseline measurements. After treatment, BAL TGF-beta(1) concentrations significantly increased in the FP group (p = 0.03), but we found no difference between FP and placebo groups; BAL bFGF concentrations increased during treatment in both groups compared with controls (p < 0.05), but not significantly within either patient group (p > 0.05). We found a reverse correlation between forced expiratory volume in 1 second (FEV(1)) and BAL TGF-beta(1) concentration in the FP group (r = -0.53, p = 0.04), and between FEV(1) and BAL TGF-beta(1) concentration in the placebo group (r = -0.74, p = 0.004). Multivariable analysis indicated no significant independent effects of inhaled FP in either BAL TGF-beta(1) or bFGF concentrations. CONCLUSIONS: Bronchoalveolar fluid TGF-beta(1) concentrations increased in LTRs after transplantation and may correlate with the decrease in lung function. Inhaled FP added to conventional immunosuppression had no effect on TGF-beta(1) or bFGF production in BAL fluid.

Possible anti-inflammatory effect of salmeterol against interleukin-8 and neutrophil activation in asthma in vivo.

In-vitro data suggest that long-acting beta2-agonists may have a neutrophil-stabilising effect. A reduction in airway wall eosinophil number following introduction of salmeterol in persistent asthma has previously been described. There is currently interest in the "neutrophil system" in asthma, and thus the aim of the present study was to investigate the effect of salmeterol on interleukin (IL)-8, neutrophils and myeloperoxidase (MPO) in persistent asthma. In the same 12-week double-blind parallel-group placebo-controlled study as described previously, the effects on bronchoalveolar lavage fluid (BALF) IL-8, neutrophils and MPO of introducing salmeterol (50 microg b.i.d.) or giving additional inhaled corticosteroid (fluticasone 100 microg b.i.d.) in 45 subjects with persistent asthma already on low/moderate doses of inhaled corticosteroids were further investigated. At baseline, BALF IL-8 but not neutrophil or MPO levels were significantly raised in the asthmatic subjects compared to normal controls. MPO levels correlated strongly with IL-8 levels, and weakly with BALF neutrophil numbers in the asthmatics. Fluticasone treatment resulted in significantly elevated neutrophil numbers, but not MPO or IL-8 levels. In contrast, introducing salmeterol significantly reduced IL-8 and MPO levels, but did not affect BALF neutrophil numbers. Interestingly, salmeterol and fluticasone showed significantly contrasting effects on MPO and neutrophils, and there was a divergent effect on IL-8 levels that almost reached significance. Excessive interleukin-8 levels may be relevant to asthma pathogenesis, even in the setting of moderate-dose inhaled corticosteroid therapy. Reduction in interleukin-8 production and possibly stabilisation of airway neutrophil numbers may explain the greater clinical benefit of adding a long-acting beta2-agonist rather than merely increasing inhaled corticosteroid doses. Indeed, high-dose inhaled corticosteroid therapy alone may promote airway neutrophilia.

Effect of a long-acting beta2-agonist over three months on airway wall vascular remodeling in asthma.

There are few data regarding the potential effects of antiasthma treatment on indices of airway remodeling, such as the increased subepithelial airway vascularity in patients with asthma. We studied 45 symptomatic subjects with asthma who were receiving treatment with low dose inhaled corticosteroids (ICS) (range 200-500 microg twice a day) and 28 normal subjects without asthma as a control population. Subjects underwent bronchoscopy with airway biopsy and subjects with asthma were then randomized to receive supplementary inhaled salmeterol 50 microg twice a day, fluticasone propionate 100 microg twice a day, or placebo for 3 mo in addition to their baseline ICS. Biopsy of the airway was then repeated. The biopsies were analyzed for vascular structures in the subepithelial lamina propria. Sufficient biopsy material was available for analysis of vascularity in 34 of the subjects with asthma and 28 of the normal subjects. We confirmed that airways of subjects with asthma had a significant increase in the number of vessels/mm2 of lamina propria compared with airways of normal subjects (524 +/- 137 vessels/mm2, n = 34 versus 425 +/- 130 vessels/mm2, n = 28; p = 0.004). There was a decrease in the density of vessels of lamina propria after treatment only in the salmeterol group compared with baseline (before, 535 +/- 153 vessels/mm2 versus after, 400 +/- 142 vessels/mm2; n = 12; p = 0.04). There was no significant change within the fluticasone (n = 11) or placebo (n = 11) treatment groups, but also no significant differences between the groups. Notably, no treatment was associated with increased airway wall vascularity. The demonstrated fall in vessel number within the salmeterol-treated group may suggest an advantageous effect of long-acting beta2-agonists on this manifestation of airway remodeling over the 3-mo time scale of this study, which is complementary to the action of ICS on airway vascularity.

Post-lung transplant bronchiolitis obliterans syndrome (BOS) is characterized by increased exhaled nitric oxide levels and epithelial inducible nitric oxide synthase.

In conditions characterized by airway inflammation, exhaled nitric oxide (eNO) levels are increased. Post-lung transplant bronchiolitis obliterans syndrome (BOS) is characterized by airway inflammation and development of progressive airway narrowing and fibrosis. We have previously shown that in stable lung transplant recipients (LTR), mean eNO levels were not elevated but were still related to the degree of airway neutrophilia within the group. The hypothesis now tested is that in BOS, eNO levels are increased in association with even greater airway neutrophilia and enhanced expression of inducible (iNOS) nitric oxide synthase in the bronchial epithelium. We determined eNO levels in 40 LTR in four groups: well and "stable": LTR (n = 20), BOS (n = 8), bacterial airway infection (BI, n = 6), and acute rejection (AR, n = 6). Following bronchoscopic sampling, we performed a quantitative assessment of iNOS and constitutive nitric oxide synthase (cNOS) expression in endobronchial biopsies by immunohistochemistry. Mean +/- SEM eNO levels in BOS and BI were significantly higher than in stable LTR (20 +/- 1.2 parts per billion [ppb] and 24.7 +/- 1.7 ppb versus 12.5 +/- 0.9 ppb; p < 0.01 for both). In AR, eNO levels (13.4 ppb +/- 0.5) were not different in stable LTR (p = 0.34). When compared with stable LTR, there was increased expression of iNOS in the bronchial epithelium and generally in the lamina propria (LP) in patients with BOS and BI. In AR, iNOS expression was increased but only in the LP in a perivascular distribution. Expression of cNOS was reduced in BOS but not in BI and AR compared with the stable group. Using regression analysis, only iNOS expression in the bronchial epithelium (r(2) = 0.77; p < 0.0001) and %BAL neutrophils (r(2) = 0. 79; p < 0.0001) were positively related to eNO in stable LTR and BOS. We conclude that epithelial iNOS appears to be the major source of eNO. Exhaled NO levels also appear to reflect the degree of airway neutrophilia in both stable LTR and BOS groups. This suggests that serial eNO measurements may be able to predict the early development of BOS.

Airway neutrophilia in stable and bronchiolitis obliterans syndrome patients following lung transplantation.

BACKGROUND: The bronchiolitis obliterans syndrome (BOS) remains the major constraint on the long term success of lung transplantation. Neutrophils have been associated with fibrosing lung conditions and have been noted to be increased in the bronchoalveolar lavage (BAL) fluid of patients with BOS. METHODS: This study was undertaken to examine neutrophil accumulation in the BAL fluid, airway wall and lung parenchyma, as well as levels of interleukin (IL)-8 in the BAL fluid, in normal controls and lung transplant recipients with and without BOS. Bronchoscopic examination included endobronchial biopsy (EBB), BAL fluid, and transbronchial biopsy (TBB) sampling. Tissue neutrophils were identified by neutrophil elastase staining on 3 microm paraffin biopsy sections and quantified by computerised image analyser. IL-8 levels were measured in unconcentrated BAL fluid by ELISA. RESULTS: Compared with controls, airway wall neutrophilia was increased in both stable lung transplant recipients and those with BOS (p<0.05). BAL neutrophils and IL-8 levels were also increased in both groups of transplant recipients compared with controls (p<0.01), the levels being significantly higher in the BOS group (p<0.01). Neutrophil numbers in the lung parenchyma were not significantly different between the two groups of lung transplant recipients. CONCLUSION: Increased levels of neutrophils are present in the airway wall and BAL fluid of lung transplant recipients with and without BOS. BAL fluid levels of IL-8 are also increased, raising the possibility that neutrophils and/or IL-8 may play a part in the pathogenesis of BOS following lung transplantation.

Bronchoalveolar lavage cellular profiles in lung transplantation: the effect of inhaled corticosteroids.

UNLABELLED: Bronchiolitis Obliterans Syndrome (BOS) remains a major cause of long term morbidity and mortality in lung transplantation, and occurs despite significant immunosuppression. Airway inflammation is thought to precede the development of BOS. OBJECTIVES: To examine the effect of inhaled corticosteroids on airway inflammation and the development of BOS in lung transplant recipients. METHODS: 30 patients were recruited and randomised in a double blind fashion to receive either 750 micrograms Fluticasone propionate (FP) twice daily or an identical appearing placebo for 3 months. BAL cell counts and differentials were performed at time 0 and after 3 months treatment. Lung function was assessed at each time point using spirometry. RESULTS: 24 patients were felt to be stable and free from infection at both time points and thus included in the analysis. There was a significant reduction in total cell count in BAL fluid after treatment with 3 months FP compared to 3 months placebo, however no change in cell differentials nor lung function was found. DISCUSSION: Despite a reduction in total cell numbers in BAL fluid, lung function was not altered over the 3 months of treatment. It may be that longer treatment is required to see an effect.

In stable lung transplant recipients, exhaled nitric oxide levels positively correlate with airway neutrophilia and bronchial epithelial iNOS.

In conditions characterized by airway inflammation, exhaled nitric oxide (eNO) levels are increased. Variable degrees of airway inflammation are present in stable lung transplant recipients (LTR), and may lead to airway remodeling and chronic graft dysfunction. The hypothesis tested is that in stable LTR, eNO concentrations would reflect the expression of inducible (iNOS) (but not constitutive [cNOS] nitric oxide synthase) in the bronchial epithelium as well as the degree of airway inflammation. We determined eNO concentrations in 20 stable LTR, free of infection, rejection, or obliterative bronchiolitis (OB). At routine bronchoscopy, we measured the differential cell count on bronchoalveolar lavage (BAL) and a quantitative assessment of iNOS and cNOS expression in endobronchial biopsies by immunohistochemistry. Mean +/- SEM eNO concentrations in stable LTR were not significantly different from control subjects (13 +/- 0.7 ppb versus 14.2 +/- 0.49; p = 0.42). Percent BAL neutrophils was 11.5 +/- 3.2 which was significantly higher than in a group of local control subjects (1.7 +/- 0.6; p < 0.001). The bronchial epithelium and lamina propria contained abundant iNOS but cNOS was present only in the lamina propria. Using regression analysis, percent BAL neutrophils (r(2) = 0.82; p < 0.0001) and iNOS expression in the bronchial epithelium (r(2) = 0.75; p < 0.0001), but not in the lamina propria (r(2) = 0.16; p = 0.08), were positively predictive of eNO. There was an inverse relationship between cNOS and eNO. We conclude that eNO concentrations although normal for the group, still reflect the degree of airway inflammation in stable LTR. Epithelial iNOS appears to be the major source of eNO and expression of cNOS may be downregulated with increasing iNOS expression.

Airway vascular changes in lung allograft recipients.

BACKGROUND: In asthma there has been increasing interest in the contribution of airway microvasculature to airway wall thickness and lumenal narrowing. Post-lung transplant, the survival of the donor airway is generally dependent on mixed-venous blood flow from pulmonary artery collaterals associated with the discontinuation of the bronchial circulation. This may lead to an altered vasculature of the airways post transplant, which may contribute to airflow limitation. METHODS: Endobronchial biopsies were taken from the lower lobe sub-carinae in 22 lung transplant recipients (LTR), 8 with Bronchiolitis Obliterans Syndrome (BOS), 14 without, and 14 controls. Seven microm frozen sections were stained for type IV collagen with a monoclonal antibody, using an indirect immunoperoxidase method. Bronchial vessels were identified by typical staining of type IV collagen in the true basement membrane supporting the endothelium. The number of vessels per mm2 of submucosa to a depth of 150 microm below the basement membrane, the percent vascularity and average vessel size were quantified using a computerised image analyser. RESULTS: Compared to the controls, a higher percent vascularity was found in LTR both with and without BOS (p < 0.05). In the BOS group, the percent best FEV1.0 decreased exponentially, in association with increased airway vessel size (r2 = 0.67, p = 0.01). CONCLUSIONS: These findings suggest that increased airway vascularity is a feature of the allograft airways post transplant. This may be a result of the relative hypoxia and hypercarbia in the blood supplying the airways from the pulmonary artery collaterals or of the chronic inflammatory process in the airways. These changes in vascularity could contribute to airflow limitation in BOS.

Vascularity in asthmatic airways: relation to inhaled steroid dose.

BACKGROUND: There is an increase in vascularity in the asthmatic airway. Although inhaled corticosteroids (ICS) are an effective anti-inflammatory treatment in asthma, there are few data on any effects on structural changes. METHODS: Endobronchial biopsy specimens from seven asthmatic subjects not receiving ICS and 15 receiving 200-1500 microg/day beclomethasone dipropionate (BDP) were immunohistochemically stained with an anti-collagen type IV antibody to outline the endothelial basement membrane of the vessels. These were compared with biopsy tissue from 11 non-asthmatic controls (four atopic and seven non-atopic). RESULTS: There was a significant increase in the density of vessels (number of vessels/mm2 of lamina propria) in the asthmatic subjects not on ICS compared with non-asthmatic controls (mean 485 (interquartile range (IQR) 390-597) versus 329 (IQR 248-376) vessels/mm2, p<0.05; 95% CI for the difference 48 to 286). There was no significant difference between asthmatic subjects on ICS and those not on ICS or control subjects in the number of vessels/mm2 (mean 421 (IQR 281-534)). However, patients who received >/=800 microg/day BDP tended to have a reduced number of vessels/mm2 compared with patients not on ICS and those receiving

Endobronchial biopsy and bronchoalveolar lavage in stable lung transplant recipients and chronic rejection.

We have obtained endobronchial biopsies (EBB), bronchoalveolar lavage (BAL), and transbronchial biopsies (TBB) in 17 stable lung transplant recipients (sLTR), 8 subjects with physiologic evidence of chronic rejection (BOS), and 9 normal subjects. A striking finding was the marked neutrophilia in BAL samples from patients with BOS, in the carefully screened absence of infection. A statistically higher neutrophil count was also present in the sLTR group relative to the normal group. Median BAL neutrophil count in BOS was 100 x 10(3)/ml, range 13-1,661 10(3)/ml (p < 0.001 relative to normal subjects and sLTR). Median BAL neutrophil count in sLTR was 7 x 10(3)/ml, range 1-81 10(3)/ml (p < 0.01 relative to normal subjects). Normal subjects had a median BAL neutrophil count of 3 x 10(3)/ml, range 1-7 10(3)/ml. There was evidence of a predominance of CD8 lymphocytes in BAL from sLTR and BOS with a lower CD4/CD8 ratio in both compared to normal subjects (p < 0.05). EBB mononuclear cell counts, class II major histocompatibility complex expression, and T-cell activation markers were normal in BOS, in contrast to the sLTR group. Our data may be consistent with BOS, representing a relative resolution of an active mononuclear cell chronic inflammation, perhaps at the expense of airway fibrosis. The relevance of the BAL neutrophilia and its role in BOS pathogenesis need further longitudinal investigation.

Scar collagen deposition in the airways of allografts of lung transplant recipients.

Collagen subtype deposition has not been studied in the airways of transplanted lungs. As part of rejection, a series of immunologic insults results in a remodeling of the allograft. In chronic rejection, changes in the airway leading to obliterative bronchiolitis syndrome (OBS) are particularly important. To better understand the mechanism of OBS occurring in chronic lung rejection, we investigated deposition of three fibrillar collagens (type I, III, V) in airway biopsies of lung allografts taken from 10 clinically well lung transplant recipients (wLTR) and eight lung transplant recipients (LTR) with OBS (OBLTR) using an immunoperoxidase method. Collagen III deposition and the ratio of collagen type III to type I were found to be significantly increased in OBLTR compared with wLTR (p < 0.05), and the latter correlated inversely with both FEF(25-75) (r = -0.69; p < 0.05) and FEV1 (r = 0.62; p = 0.05) in OBLTR. This suggests that an increased proportion of collagen III in the airway walls of transplanted lungs might be an early signal of the progression to terminal chronic lung allograft dysfunction. The changes in the ratio of type III to type I collagen in the airways of lung allografts may provide important insights into the process of airway remodeling in chronic lung rejection.

Immunopathological changes in the airways of stable lung transplant recipients.

BACKGROUND: Pathological obliterative bronchiolitis, characterised by inflammation and occlusion of airways, is a serious complication of lung transplantation. Endobronchial biopsy (EBB) provides a means of examining transplanted airways. This study aimed to investigate the role of EBB samples in revealing early signals of airway injury. METHODS: In 18 stable lung transplant recipients with close to maximal lung function (median FEV1, best after transplantation 100%, interquartile range 98-100%) EBB samples were taken simultaneously with transbronchial biopsy samples and bronchoalveolar lavage (BAL) fluid (median 195 days after transplantation). OCT embedded specimens were snap frozen on an isopentane slurry made with liquid nitrogen and 7 microns sections were stained with monoclonal antibodies using a three stage immunoperoxidase method. RESULTS: Compared with nine non-transplanted control subjects, EBB specimens from the stable transplant group had significantly increased CD8 positivity (median 53 versus 27 cells/mm basement membrane, p = 0.04; 95% CI for the difference 1 to 46)) and increased HLA-DR positivity (median 84 versus 26 cells/mm basement membrane, 95% CI for the difference 6 to 115). There was an increase in CD68 positive cells in the EBB specimens from transplant recipients of borderline significance (median 92 versus 68, p = 0.08, 95% CI for the difference 1 to 84). CD3, CD4, and CD25 counts were similar in the two groups. EBB findings were not influenced by age, sex, indication for transplant, immunosuppression doses or levels, nor the presence of airway commensals in the BAL fluid. CONCLUSIONS: EBB is practicable in a transplant setting and provides information about bronchial inflammatory changes. It is likely that there is ongoing inflammation, possibly rejection mediated, even in healthy lung transplant recipients despite triple immunosuppression.

Airway versus transbronchial biopsy and BAL in lung transplant recipients: different but complementary.

Lung transplantation is now an established therapeutic intervention for end-stage cardiopulmonary disease in humans. Chronic rejection, in the form of bronchiolitis obliterans syndrome (BOS), remains the commonest cause of morbidity and mortality in those surviving more than 3 months. The pathology of BOS involves airway changes. We have evaluated the potential for endobronchial biopsies (EBB) to complement existing sampling methods used in allograft monitoring and have compared the results of EBB findings with those of bronchoalveolar lavage (BAL) and transbronchial biopsy (TBB) in 18 clinically stable patients. We found that all the EBB had inflammatory cells present but that only five TBB specimens had evidence of inflammation, with airway material being present in 78% of the TBB. Paired BAL and EBB yielded different results, with no correlations between total macrophages, lymphocytes, CD4+ cells or CD8+ cells. We conclude that endobronchial biopsies are potentially useful as an additional sample for the monitoring of inflammation in lung allografts, since they yield different, and potentially complimentary, information to bronchoalveolar lavage and transbronchial biopsy.