The ALPACA study: (In)Appropriate LAMA prescribing in asthma: A cohort analysis.

INTRODUCTION: Since long-acting muscarinic antagonists (LAMA) are only indicated as add-on therapy in subjects with moderate-to-severe asthma, there are concerns whether LAMA monotherapy is associated with worse asthma control. AIM: To study the prevalence of LAMA monotherapy and its potential association with severe asthma exacerbations (SAE) in patients with asthma. METHODS: A cohort study (2007-2017) in the IPCI primary care database, in asthma patients aged 6-50, using LAMA during follow-up. Respiratory prescriptions were retrieved from the electronic medical records based on ATC code. Asthma treatment periods were created and categorized as LAMA mono, dual (LAMA + ICS), or triple therapy (LAMA + ICS + LABA). Relative rates (RR) of SAE, adjusting for patient characteristics, were estimated to compare treatments. RESULTS: From a total of 66,508 asthma patients, 1236 (1.9%) LAMA users were identified. Median age was 41 years, 65.9% were females. LAMA users were responsible for 3596 LAMA treatment periods of which 1390 (38.7%) were LAMA monotherapy, 553 (15.4%) dual therapy and 1653 (46.0%) triple therapy. The RR of SAE during LAMA monotherapy compared to dual therapy was 1.5 (95% CI 0.6-3.8). In patients alternating between mono and dual therapy (but never triple therapy), the RR for LAMA monotherapy increased to 5.7 (95% CI 1.4-23.6). CONCLUSIONS: This observational study shows that when LAMA is prescribed, it is often prescribed without concurrent ICS (LAMA monotherapy). LAMA monotherapy was associated with an increased risk of exacerbations when not used concurrently with ICS. This emphasizes the importance that LAMA should never be prescribed without concomitant ICS use in patients with asthma.

The role of miR-155 in cigarette smoke-induced pulmonary inflammation and COPD.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent respiratory disease characterized by airflow limitation and chronic inflammation. MiR-155 is described as an ancient regulator of the immune system. Our objective was to establish a role for miR-155 in cigarette smoke (CS)-induced inflammation and COPD. We demonstrate increased miR-155 expression by RT-qPCR in lung tissue of smokers without airflow limitation and patients with COPD compared to never smokers and in lung tissue and alveolar macrophages of CS-exposed mice compared to air-exposed mice. In addition, we exposed wild type and miR-155 deficient mice to CS and show an attenuated inflammatory profile in the latter. Alveolar macrophages were sorted by FACS from the different experimental groups and their gene expression profile was analyzed by RNA sequencing. This analysis revealed increased expression of miR-155 targets and an attenuation of the CS-induced increase in inflammation-related genes in miR-155 deficient mice. Moreover, intranasal instillation of a specific miR-155 inhibitor attenuated the CS-induced pulmonary inflammation in mice. Finally, elastase-induced emphysema and lung functional changes were significantly attenuated in miR-155 deficient mice. In conclusion, we highlight a role for miR-155 in CS-induced inflammation and the pathogenesis of COPD, implicating miR-155 as a new therapeutic target in COPD.

Bone morphogenetic protein 6 (BMP-6) modulates lung function, pulmonary iron levels and cigarette smoke-induced inflammation.

Chronic obstructive pulmonary disease (COPD) is associated with abnormal inflammatory responses and airway wall remodeling, leading to reduced lung function. An association between the bone morphogenetic protein (BMP-6) locus and forced vital capacity has been found in a genome-wide association study. However, the role of BMP-6 in the pathogenesis of COPD remains unknown. The pulmonary expression of BMP-6 was analyzed in patients with COPD and in cigarette smoke (CS)-exposed mice. We evaluated lung function and histology in BMP-6 KO mice at baseline. We exposed BMP-6 KO mice to CS for 4 weeks and measured pulmonary inflammation and iron levels. Pulmonary mRNA levels of BMP-6 were decreased in smokers with and without COPD and in CS-exposed mice. Importantly, BMP-6 expression was lowest in severe COPD. Accordingly, protein levels of BMP-6 were decreased in patients with COPD. Lung function measurements demonstrated a decreased compliance and total lung capacity in BMP-6 KO mice, whereas lung histology was normal. Furthermore, BMP-6 KO mice displayed elevated iron levels and an aggravated CS-induced inflammatory response. These results suggest that BMP-6 is important for normal lung function and that downregulation of BMP-6-as observed in patients with COPD-contributes to pulmonary inflammation after CS exposure.

Insights in particulate matter-induced allergic airway inflammation: Focus on the epithelium.

Outdoor air pollution is a major environmental health problem throughout the world. In particular, exposure to particulate matter (PM) has been associated with the development and exacerbation of several respiratory diseases, including asthma. Although the adverse health effects of PM have been demonstrated for many years, the underlying mechanisms have not been fully identified. In this review, we focus on the role of the lung epithelium and specifically highlight multiple cytokines in PM-induced respiratory responses. We describe the available literature on the topic including in vitro studies, findings in humans (ie observations in human cohorts, human controlled exposure and ex vivo studies) and in vivo animal studies. In brief, it has been shown that exposure to PM modulates the airway epithelium and promotes the production of several cytokines, including IL-1, IL-6, IL-8, IL-25, IL-33, TNF-α, TSLP and GM-CSF. Further, we propose that PM-induced type 2-promoting cytokines are important mediators in the acute and aggravating effects of PM on airway inflammation. Targeting these cytokines could therefore be a new approach in the treatment of asthma.

Genes and pathways underlying susceptibility to impaired lung function in the context of environmental tobacco smoke exposure.

BACKGROUND: Studies aiming to assess genetic susceptibility for impaired lung function levels upon exposure to environmental tobacco smoke (ETS) have thus far focused on candidate-genes selected based on a-priori knowledge of potentially relevant biological pathways, such as glutathione S-transferases and ADAM33. By using a hypothesis-free approach, we aimed to identify novel susceptibility loci, and additionally explored biological pathways potentially underlying this susceptibility to impaired lung function in the context of ETS exposure. METHODS: Genome-wide interactions of single nucleotide polymorphism (SNP) by ETS exposure (0 versus ≥1 h/day) in relation to the level of forced expiratory volume in one second (FEV1) were investigated in 10,817 subjects from the Dutch LifeLines cohort study, and verified in subjects from the Swiss SAPALDIA study (n = 1276) and the Dutch Rotterdam Study (n = 1156). SNP-by-ETS exposure p-values obtained from the identification analysis were used to perform a pathway analysis. RESULTS: Fourty Five SNP-by-ETS exposure interactions with p-values <10-4 were identified in the LifeLines study, two being replicated with nominally significant p-values (<0.05) in at least one of the replication cohorts. Three pathways were enriched in the pathway-level analysis performed in the identification cohort LifeLines, i.E. the apoptosis, p38 MAPK and TNF pathways. CONCLUSION: This unique, first genome-wide gene-by-ETS interaction study on the level of FEV1 showed that pathways previously implicated in chronic obstructive pulmonary disease (COPD), a disease characterized by airflow obstruction, may also underlie susceptibility to impaired lung function in the context of ETS exposure.

Elevated GDF-15 contributes to pulmonary inflammation upon cigarette smoke exposure.

The molecular mechanisms underlying the pathogenesis of chronic obstructive pulmonary disease (COPD) are still unclear, however signaling pathways associated with lung development, such as the transforming growth factor (TGF)-ß superfamily, could be implicated in COPD. Growth differentiation factor (GDF)-15, a member of the TGF-ß superfamily, is involved in inflammation, mucus secretion, and cachexia. We analyzed the pulmonary expression of GDF-15 in smokers and patients with COPD, in cigarette smoke (CS)-exposed cultures of primary human bronchial epithelial cells (pHBECs), and in CS-exposed mice. Next, we exposed GDF-15 KO and control mice to air or CS and evaluated pulmonary inflammation. GDF-15 levels were higher in sputum supernatant and lung tissue of patients with COPD and smokers without COPD compared with never smokers. Immunohistochemistry revealed GDF-15 staining in the airway epithelium. Increased expression and secretion of GDF-15 was confirmed in vitro in CS-exposed pHBECs compared with air-exposed pHBECs. Similarly, GDF-15 levels were increased in lungs of CS-exposed mice. Importantly, GDF-15 deficiency attenuated the CS-induced pulmonary inflammation. These results suggest that increased GDF-15-as observed in lungs of smokers and patients with COPD-contributes to CS-induced pulmonary inflammation.

Increased levels of enzymes involved in local estradiol synthesis in chronic obstructive pulmonary disease.

INTRODUCTION: Steroid hormones are involved in lung development, pulmonary inflammation, and lung cancer. Estrogen signaling and exposure may play a role in pulmonary disorders, including COPD. In both genders, estrogens can be generated locally in the lungs and this contributes importantly to the tissue exposure to these steroids. OBJECTIVE: To characterize and assess differences in localization of estrogen receptors and enzymes involved in the local generation of estrogens in COPD. METHODS: Estrogen Receptor alpha (ERα/ESR1), Estrogen Receptor beta (ERß/ESR2) and G-protein-coupled estrogen receptor 1 (GPER) were explored by real-time (RT)-PCR analysis (mRNA expression), immunohistochemistry and western blotting in controls and COPD patients. mRNA expression of the enzymes involved in the local estrogen generation - i.e. aromatase (CYP19A1), 17beta-hydroxysteroid dehydrogenases (17ß-HSDs) 1, 2, 4, 5, 7 and 12, steroid sulfatase (STS) and sulfotransferase (SULT1E1) - were analyzed by RT-PCR. RESULTS: ERα, ERß and GPER were expressed in lung tissue, but no differences were observed between patients and controls. The main enzymes involved in local estrogen generation were also present in both normal and COPD lung tissue. In lungs of COPD patients compared with controls, we observed increased expression of the enzymes 17ß-HSD type 1 and aromatase (positive association), both involved in the local synthesis of active estrogens. CONCLUSION: All ER subtypes are present in the lung. The shift in local mRNA level of estrogen metabolic enzymes suggests that exposure to estrogens is involved in the pathogenesis of COPD.

Targeted Therapy for Older Patients with Uncontrolled Severe Asthma: Current and Future Prospects.

Severe asthma in the elderly places a high burden on affected individuals and society. Emerging therapies target specific phenotypes of the asthma disease spectrum, and can be beneficial for older asthmatics, albeit their response might be altered due to age-related characteristics. Paradoxically, these characteristics are often ground for exclusion from clinical trials. The question thus arises how the senior asthmatic population can successfully enter the era of targeted therapy. Therefore, we highlight characteristics of this population relevant to effective treatment, and review the evidence for targeted therapy in elderly patients. For targeted therapy it is important to account for aging, as this affects the distribution of phenotypes (e.g. late-onset asthma, non-eosinophilic asthma) and may alter biomarkers and drug metabolism. Elderly asthmatics suffer from age-related comorbidities and subsequent polypharmacy. A systematic search into targeted asthma therapy yielded no randomized clinical trials dedicated to older asthmatics. Post hoc analyses of the anti-immunoglobulin E agent omalizumab indicate similar efficacy in both younger and older adults. Conference abstracts on anti-interleukin-5 and anti-interleukin-13 therapy suggest even more pronounced effects of targeted treatments in late-onset disease and in asthmatic patients 65 years or older, but full reports are lacking. For non-eosinophilic asthma in the elderly, there is not yet high-level evidence for targeted therapy, but macrolides may offer a viable option. In conclusion, there is a gap in knowledge regarding the effect of older age on the safety and efficacy of targeted asthma therapy. Further investigations in the elderly are needed, with special emphasis on both late-onset asthma and therapeutics for non-eosinophilic asthma.

COPD is associated with reduced pulmonary interstitial expression of pentraxin-3.

Pentraxin (PTX)3 is involved in antimicrobial defence, apoptotic cell clearance and extracellular matrix stability. As these processes are altered in chronic obstructive pulmonary disease (COPD), we aimed to investigate PTX3 expression in patients with this disease. PTX3 expression was quantified by immunohistochemical staining of lung tissue from never-smokers, smokers without COPD, and in patients with COPD of Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I, II and III-IV. mRNA expression was examined in total lung tissue by quantitative RT-PCR. PTX3 concentration was measured in induced sputum and plasma by ELISA. PTX3 is mainly localised in the interstitium of the small airways and alveolar walls. There were no significant differences in pulmonary, sputum and plasma PTX3 expression between study groups. However, PTX3 expression in small airways correlated significantly with forced expiratory volume in 1 s (r = 0.35, p = 0.004). In the alveolar walls, PTX3 expression correlated significantly with carbon monoxide transfer coefficient (r = 0.28, p = 0.04). In sputum, PTX3 levels were highly correlated with the number of neutrophils. Finally, systemic levels of PTX3 tended to be lower in severe COPD compared with mild COPD. In COPD, airflow limitation and reduced transfer coefficient for carbon monoxide are associated with lower pulmonary interstitial expression of PTX3.

Tiotropium Handihaler and the risk of cardio- or cerebrovascular events and mortality in patients with COPD.

BACKGROUND: Tiotropium has been associated with an increased risk of mortality and/or cardiovascular events. Recent data from RCTs suggests tiotropium Handihaler to be safe, but its safety has not yet been fully investigated under real-life circumstances. METHODS: We conducted 2 nested case-control studies in a COPD cohort from the Dutch IPCI database. In the first case-control study, cases had a cardiovascular or cerebrovascular endpoint (CCVE): stroke and transient ischemic attack (TIA), myocardial infarction, heart failure and/or ventricular arrhythmia. In the second, cases were all patients who died. Cases were matched to controls on age, sex and index date. Conditional logistic regression analysis was used to calculate adjusted odds ratios (OR(adj)) with 95% confidence intervals (CI) for tiotropium vs. long-acting beta-agonists (LABA). RESULTS: Within a cohort of 6788 COPD patients, 784 CCVE's and 1032 deaths were reported. Compared to current LABA use, use of tiotropium Handihaler was neither associated with an increased risk of a CCVE (OR(adj) 0.89, 95% 0.55-1.44) nor with an increased risk of death (OR(adj) 0.79, 95% CI 0.49-1.28). CONCLUSIONS: In real life, use of tiotropium Handihaler in COPD patients is not associated with an increased risk of a CCVE or mortality compared to LABA.

Role of IL-1α and the Nlrp3/caspase-1/IL-1ß axis in cigarette smoke-induced pulmonary inflammation and COPD.

Cigarette smoke (CS), the primary risk factor of chronic obstructive pulmonary disease (COPD), leads to pulmonary inflammation through interleukin-1 receptor (IL-1R)I signalling, as determined using COPD mouse models. It is unclear whether interleukin (IL)-1α or IL-1ß, activated by the Nlrp3/caspase-1 axis, is the predominant ligand for IL-1RI in CS-induced responses. We exposed wild-type mice (treated with anti-IL-1α or anti-IL-1ß antibodies), and IL-1RI knockout (KO), Nlrp3 KO and caspase-1 KO mice to CS for 3 days or 4 weeks and evaluated pulmonary inflammation. Additionally, we measured the levels of IL-1α and IL-1ß mRNA (in total lung tissue by RT-PCR) and protein (in induced sputum by ELISA) of never-smokers, smokers without COPD and patients with COPD. In CS-exposed mice, pulmonary inflammation was dependent on IL-1RI and could be significantly attenuated by neutralising IL-1α or IL-1ß. Interestingly, CS-induced inflammation occurred independently of IL-1ß activation by the Nlrp3/caspase-1 axis. In human subjects, IL-1α and IL-1ß were significantly increased in total lung tissue and induced sputum of patients with COPD, respectively, compared with never-smokers. These results suggest that not only IL-1ß but also IL-1α should be considered as an important mediator in CS-induced inflammation and COPD.

Effect of different asthma treatments on risk of cold-related exacerbations.

Common colds often trigger asthma exacerbations. The present study compared cold-related severe exacerbations during budesonide/formoterol maintenance and reliever therapy, and different regimens of maintenance inhaled corticosteroids (ICS), with or without long-acting ß(2)-agonists (LABA), and with as-needed short-acting ß(2)-agonists (SABA) or LABA. Reported colds and severe exacerbations (defined by oral corticosteroid use and/or hospitalisation/emergency room visit) were assessed for 12,507 patients during 6-12 months of double-blind treatment. Exacerbations occurring ≤14 days after onset of reported colds were analysed by a Poisson model. The incidence of colds was similar across treatments. Asthma symptoms and reliever use increased during colds. Budesonide/formoterol maintenance and reliever therapy reduced severe cold-related exacerbations by 36% versus pooled comparators plus SABA (rate ratio (RR) 0.64; p=0.002), and for individual treatment comparisons, by 52% versus the same maintenance dose of ICS/LABA (RR 0.48; p<0.001); there were nonsignificant reductions versus higher maintenance doses of ICS or ICS/LABA (RR 0.83 and 0.72, respectively). As-needed LABA did not reduce cold-related exacerbations versus as-needed SABA (RR 0.96). Severe cold-related exacerbations were reduced by budesonide/formoterol maintenance and reliever therapy compared with ICS with or without LABA and with as-needed SABA. Subanalyses suggested the importance of the ICS component in reducing cold-related exacerbations. Future studies should document the cause of exacerbations, in order to allow identification of different treatment effects.

Alternatively activated macrophages and impaired phagocytosis of S. aureus in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by biased Th2 inflammation and CRS without nasal polyps (CRSsNP) by a Th1 immune response. Colonization by Staphylococcus aureus is increased in CRSwNP. We aimed to determine macrophage phenotypes in nasal mucosa of CRSwNP and CRSsNP and to examine phagocytosis of S. aureus in these pathologies. METHODS: Macrophage phenotyping was performed by immunohistochemical staining on nasal mucosa sections from 28 patients; in addition flow cytometry analysis was performed. Tissue homogenate protein levels of IFN-γ, IL-5, IL-6, IL-1ß, TGF-ß, eosinophil cationic protein (ECP) and total IgE were analyzed and correlated with macrophage subtypes. Phagocytosis of S. aureus was analyzed by flow cytometry. Survival of S. aureus in Thp1 cells in the presence of polarizing cytokines was studied in vitro. RESULTS: By immunohistochemical analysis more M2 macrophages were present in CRSwNP than in CRSsNP. This also was positively correlated with increased levels of IL-5, ECP and locally produced IgE and decreased levels of IL-6, IL-1ß and IFN-γ. FACS analysis of dissociated nasal tissue confirmed the presence of increased numbers of M2 macrophages (CD206(+) HLADR(+) CD14(+) CD11c(+) CD20(-) ) in CRSwNP as compared to controls, while the number of M1 macrophages (CD206(-) HLADR(+) CD14(+) CD11c(int) CD16(-) CD20(-) ) was not different. Phagocytosis of S. aureus by human tissue derived macrophages was reduced in CRSwNP as compared to macrophages from the control inferior turbinates. CONCLUSIONS: Decreased phagocytosis of S. aureus and an M2 activation phenotype in CRSwNP could potentially contribute to persistence of chronic inflammation in CRSwNP.

Plasmacytoid dendritic cells in pulmonary lymphoid follicles of patients with COPD.

Plasmacytoid dendritic cells (pDCs) are professional antigen-presenting cells with antiviral and tolerogenic capabilities. Viral infections and autoimmunity are proposed to be important mechanisms in the pathogenesis of chronic obstructive pulmonary disease (COPD). The study aimed to quantify blood dendritic cell antigen 2-positive pDCs in lungs of subjects with or without COPD by immunohistochemistry and flow cytometry, combined with the investigation of the influence of cigarette smoke extract (CSE) on the function of pDCs in vitro. pDCs were mainly located in lymphoid follicles, a finding compatible with their expression of lymphoid homing chemokine receptors CXCR3 and CXCR4. pDC accumulated in the lymphoid follicles and in lung digests of patients with mild to moderate COPD, compared with smokers without airflow limitation and patients with COPD Global Initiative for Chronic Obstructive Lung disease (GOLD) stage III-IV. Exposing maturing pDC of healthy subjects to CSE in vitro revealed an attenuation of the expression of co-stimulatory molecules and impaired interferon-α production. Maturing pDC from patients with COPD produced higher levels of tumour necrosis factor (TNF)-α and interleukin (IL)-8 compared to pDC from healthy subjects. CSE significantly impairs the antiviral function of pDCs. In COPD, a GOLD stage dependent accumulation of pDC in lymphoid follicles is present, combined with an enhanced production of TNF-α and IL-8 by maturing pDCs.

Hedgehog-interacting protein is a COPD susceptibility gene: the Rotterdam Study.

The Hedgehog signalling pathway plays an important role in lung morphogenesis and cellular responses to lung injury. A genome-wide association study has demonstrated that two single nucleotide polymorphisms (SNPs) near the Hedgehog-interacting protein (Hip) gene, SNP identifiers rs1828591 and rs13118928, are associated with risk of chronic obstructive pulmonary disease (COPD). The aim of the present study was to validate the observed association between genetic variation near the Hip gene and COPD, and to investigate whether risk estimates were modified by smoking behaviour. The association between the Hip gene SNPs and COPD was investigated in the Rotterdam Study by logistic regression analyses, adjusted for several covariates. In addition, an association meta-analysis was performed that included data from the genome-wide association study on COPD. Both SNPs were significantly associated with risk of COPD (OR 0.80; 95% CI 0.72-0.91). Homozygosity for the minor G allele resulted in a decreased risk of COPD of approximately 40% (95% CI 0.47-0.78). There was a significant interaction with the number of pack-years of smoking (p = 0.004). The meta-analysis yielded an odds ratio for COPD of 0.80 per additional G allele (p = 3.4 x 10(-9)). Genetic variation near the Hip gene was significantly associated with risk of COPD, depending on the number of pack-years of smoking.

Lymphoid follicles in (very) severe COPD: beneficial or harmful?

Inflammation is a main pathogenetic factor in the development and progression of chronic obstructive pulmonary disease (COPD). Recently, it has become clear that not only the innate, but also the specific immune response plays a role. A striking finding, in particular in lungs of patients with severe COPD, often with a predominant emphysema phenotype, is the presence of B-cell follicles. As seen in other tissues, these follicles are the result of lymphoid neogenesis. The finding of oligoclonality in B-cell follicles in COPD suggests that they play a role in local antigen specific immune responses. To date, it is not known which antigens may be involved; microbial antigens, cigarette smoke-derived antigens and antigens from extracellular matrix breakdown products have been suggested. Consequently, the pathogenetic role of this follicular B-cell response is not yet clear. It might be protective against microbial colonisation and infection of the lower respiratory tract and, therefore, beneficial, or it could be of a more harmful (autoimmune) nature, directed against lung tissue components. It is necessary to determine the specific antigen(s) and to explore the exact role of the COPD related B-cell response in order to include modulation of this response and develop therapeutic options.

Decreased FOXP3 protein expression in patients with asthma.

BACKGROUND: T-regulatory cells (T(reg)) are important in balancing immune responses and maintaining peripheral tolerance. Current evidence suggests that asthma is characterized by a relative deficiency in T(reg), allowing T helper 2 cells to expand. In this study, we aimed to evaluate circulating T(reg), defined by the protein FOXP3, in both control subjects and patients with stable asthma. METHODS: Peripheral blood mononuclear cells (PBMC) of control (n = 14) and asthmatic patients (n = 29) were labeled for CD4, CD25, and intracellular FOXP3 and analyzed using flow cytometry. In CD3/CD28 stimulated PBMC, the effects of dexamethasone on the transcription factors T-bet, GATA-3, FOXP3, and RORc2 and representative cytokines were studied. RESULTS: In control subjects and asthmatic patients, numbers of peripheral blood CD4(+)CD25(high) and CD4(+)CD25(high)FOXP3(+) T-cells were similar. However, FOXP3 protein expression within CD4(+)CD25(high) T-cells was significantly decreased in asthmatic patients. There was a tendency for increased FOXP3 expression within CD4(+)CD25(high) T-cells in glucocorticosteroid-treated patients when compared to steroid-naive asthmatic patients. In stimulated PBMC, dexamethasone treatment increased the anti-/proinflammatory transcription ratios of FOXP3/GATA-3, FOXP3/T-bet, and FOXP3/RORc2. CONCLUSION: Asthmatic patients have decreased FOXP3 protein expression within their CD4(+)CD25(high) T(reg). Our findings also suggest that treatment with inhaled glucocorticosteroids in asthmatics might increase this FOXP3 protein expression within the CD4(+)CD25(high) T-cell population.

Role of lymphotoxin-alpha in cigarette smoke-induced inflammation and lymphoid neogenesis.

In chronic obstructive pulmonary disease (COPD), chronic inflammation is accompanied by peribronchial lymphoid aggregates. Lymphotoxin (LT)-alpha, crucial in secondary lymphoid organogenesis, may be involved in lymphoid neogenesis. We examined cigarette smoke (CS)-induced pulmonary lymphoid neogenesis and inflammation in vivo in LTalpha knockout (LTalpha(-/-)) and wild-type (WT) mice and studied the expression of lymphoid chemokines by lung fibroblasts in vitro. T-cell numbers (in bronchoalveolar lavage fluid (BALF) and lungs) and lymphoid aggregate numbers were significantly higher in air-exposed LTalpha(-/-) mice than in WT animals, and increased upon chronic CS exposure in both genotypes. In contrast, local immunoglobulin A responses upon chronic CS exposure were attenuated in LTalpha(-/-) mice. CXC chemokine ligand (CXCL) 13 and CC chemokine ligand (CCL) 19 mRNA in total lung and CXCL13 protein level in BALF increased upon CS exposure in WT, but not in LTalpha(-/-) mice. In vitro lymphotoxin-beta receptor (LTbetaR) stimulation induced CXCL13 and CCL19 mRNA in WT lung fibroblasts. Furthermore, in vitro exposure to CS extract upregulated CXCL13 mRNA expression in WT, but not in LTbetaR(-/-), lung fibroblasts. In this murine model of COPD, CS induces pulmonary expression of lymphoid chemokines CXCL13 and CCL19 in a LTalphabeta-LTbetaR-dependent fashion. However, LTalpha is not required for CS-induced pulmonary lymphocyte accumulation and neogenesis of lymphoid aggregates.