Pierre Larivée

Inflammatory Cytokine Production by Human Neutrophils Involves C/EBP Transcription Factors

A growing number of neutrophil-derived cytokines have proven to be crucial to various inflammatory and immune processes in vivo. Whereas C/EBP (CCAAT/enhancer-binding protein) transcription factors are important for neutrophil differentiation from myeloid precursors, we report herein that they also regulate cytokine production in mature neutrophils. All known C/EBP proteins but C/EBPγ are expressed in neutrophils; most isoforms localize to the nucleus, except for C/EBPα, which is cytoplasmic. Neutrophil stimulation does not alter the overall levels, cellular distribution, or turnover of C/EBP proteins; it also does not further induce the constitutive DNA-binding activity detected in nuclear extracts, consisting of C/EBPβ and C/EBPε. However, nuclear C/EBPβ is rapidly phosphorylated upon cell stimulation, suggesting that it can activate cytokine promoters. Indeed, the transactivation of an IL-8 promoter-luciferase construct in a human neutrophil-like cell line was impaired when its C/EBP or NF-κB sites were mutated. Overexpression of a C/EBP repressor also impeded IL-8 promoter transactivation, as well as the generation of IL-8, Mip-1α, and Mip-1β in this cellular model, whereas TNF-α generation was mostly unaffected. Finally, overexpression of a C/EBPβ mutant (T235A) as well as chromatin immunoprecipitation assays unveiled an important role for this residue in cytokine induction. This is the first demonstration that C/EBP factors are important regulators of cytokine expression in human neutrophils.

Inflammatory Cytokine Expression Is Independent of the c-Jun N-Terminal Kinase/AP-1 Signaling Cascade in Human Neutrophils

In the last decade, the ability of neutrophils to generate proinflammatory cytokines has become firmly established. Because neutrophils typically infiltrate inflammatory sites in large numbers, they could significantly contribute to the cytokine environment and even represent a substantial source of cytokines in chronic inflammatory disorders in which they predominate over other cell types. To date, however, most studies have focused on identifying which mediators are produced by neutrophils, as opposed to elucidating the molecular bases underlying this process. We previously showed that most stimuli of cytokine production in neutrophils also activate NF-κB in these cells. In this report, we turned our attention to another transcription factor that plays a central role in inflammation, AP-1. Among Jun/Fos proteins, only JunD and c-Fos are abundantly expressed in neutrophils, and they are mainly cytoplasmic. Both the cellular levels and distribution of the Jun/Fos proteins remain unaffected by various neutrophil stimuli, including those that are known to increase the corresponding mRNA transcripts. Similarly, c-Jun N-terminal kinase (JNK) 1 is overwhelmingly cytoplasmic in neutrophils and does not translocate to the nucleus upon cell activation. Although JNK is not activatable under most circumstances, specific conditions do allow its phosphorylation in response to TNF. However, no experimental condition (even those leading to JNK activation) resulted in the induction of genuine AP-1 complexes in neutrophils. Accordingly, the potent JNK inhibitor, SP 600125, failed to inhibit inflammatory cytokine gene expression in neutrophils. Collectively, our findings strongly suggest that the JNK/AP-1 signaling pathway has little or no impact on the generation of inflammatory mediators in neutrophils.

Impact of maternal use of asthma-controller therapy on perinatal outcomes

Background Asthma during pregnancy usually requires treatment with controller medications about which more safety information is needed. The objectives are to assess the impact of the use of long-acting β2-agonists (LABAs) and the dose of inhaled corticosteroids (ICSs) during pregnancy on the prevalence of low birth weight (LBW), preterm birth (PB) and small for gestational age (SGA). Methods A cohort of women with asthma giving birth from 1998 to 2008 was constructed from Québec (Canada) administrative databases. LBW was defined as weight <2500 g, PB as delivery before 37 weeks’ gestation and SGA as a birth weight below the 10th percentile. The impact of the use of LABAs and the dose of ICSs during pregnancy on the outcomes was determined with generalised-estimating-equation models. Results The cohort included 7376 pregnancies: 8.8% exposed to LABAs and 56.9% exposed to ICSs. All LABA users also received ICSs. The prevalence of LBW, PB and SGA was 7.7%, 9.5% and 13.5%, respectively. LABA use was not found to be associated with increased prevalence of LBW (OR 0.81; 95% CI 0.58 to 1.12), PB (OR 0.84; 95% CI 0.61 to 1.15) or SGA (OR 0.92; 95% CI 0.70 to 1.20). Mean ICSs doses >125 μg/day (fluticasone-equivalent) were associated with a non-significant trend of increased LBW, PB and SGA. Conclusions Despite the possibility of residual confounding due to uncontrolled or more severe asthma or smoking status, the use of LABA and low to moderate doses of ICSs were not associated with increased prevalence of perinatal outcomes. Additional research on higher ICSs doses is required to better evaluate their safety during pregnancy.

Signaling by the cysteinyl-leukotriene receptor 2. Involvement in chemokine gene transcription.

Cysteinyl-leukotrienes are involved in inflammation and act on at least two G-protein-coupled receptors, CysLT1 and CysLT2. However, the role of the CysLT2 receptor as well as its signaling remain poorly understood. Here we show that leukotriene (LT)C4 induced the production of the chemokine interleukin (IL)-8 in endothelial cells. To further study the signaling cascade involved, HEK293 cells were stably transfected with CysLT2 and used to study the transcriptional regulation of the IL-8 promoter. Stimulation of the cells with increasing concentrations of LTC4 resulted in a time- and concentration-dependent induction of IL-8 transcription and protein synthesis. Use of IL-8 promoter mutants with substitutions in their NF-κB, AP-1, or NF-IL-6 binding elements revealed an almost total requirement for NF-κB and AP-1 elements, and a lesser requirement for the NF-IL-6 element. Overexpression of dominant-negative IκBα prevented the IL-8 transactivation induced by LTC4. LTC4 stimulation induced NF-κB and AP-1 DNA binding, which involved the formation of a p50/p65 and a c-JUN·c-FOS complex, respectively. Transfection of the cells with a dominant negative (dn) form of PKCϵ prevented p65 phosphorylation, whereas dnPKCδ prevented AP-1 binding. Moreover, dnPKCδ, dnPKCϵ, and dnPKCζ prevented LTC4-induced IL-8 transcription in response to LTC4. Our data show for the first time that LTC4 can act via the CysLT2 receptor to transcriptionally activate chemokine production through induction of NF-κB and AP-1 transcription factors. These findings suggest the potential implication of CysLT2 in the inflammatory response through the modulation of chemokine gene transcription.

Enzyme activities of lung lavage in silicosis

The cytotoxic effect of quartz on lung cells has been well documented by in vitro and animal studies, but the pertinence of these findings to humans has not yet been documented. We measured lactate dehydrogenase (LDH) activities in the lung lavage of 24 long-term workers in the Québec granite industry and 25 control subjects. We found significant increases in LDH activities in the workers’ lung lavage, even in the absence of established silicosis (9 subjects). We looked at a similar observation in the sheep model of early silicosis, measured quartz content of lung lavage, and found significant correlation with LDH levels (R=0.64, p<0.001). All of the quartz particles in human and sheep lung lavage were in the alveolar macrophages. To test further the relationship of macrophage damage (cytotoxicity of quartz) we measured the release of LDH by sheep alveolar macrophage in 24 h cell culture under control conditions, exposure to inert dust, titanium, minusil-5 quartz, or aluminum-treated quartz. The LDH release was at control levels during titanium exposure and showed a significantly dose-related increase during quartz exposure. The latter cytotoxic effect was largely attenuated by aluminum treatment of quartz. These in vitro data agreed with previous reports. This study presents evidence of a cytotoxic effect of quartz inhalation in humans. The effect is related to the intensity of quartz retention in the lung macrophages; it is not a nonspecific dust exposure effect and can be attenuated by surface modification of the quartz.

Eosinophils in COPD Exacerbations Are Associated With Increased Readmissions.

Background: A subset of patients with COPD demonstrates eosinophilic inflammation either in their sputum or blood. Previous studies regarding the association between increased blood eosinophil levels and poor readmission outcomes are conflicting. The goal of this study was to investigate outcomes following severe COPD exacerbations in patients with higher blood eosinophil levels. Methods: With an observational study design, data on hospitalizations for severe COPD exacerbation were retrospectively gathered. Patient health data previous to and up to 1 year following the index hospitalization were included. Patients were stratified into the eosinophilic group if the blood eosinophil level on admission was ≥ 200 cells/&mgr;L and/or ≥ 2% of the total WBC count. Clinical outcomes were 12‐month COPD‐related readmission, 12‐month all‐cause readmission, length of stay, and time to COPD‐related readmission. These outcomes were analyzed by using logistic, negative binomial, and Cox regression models. Results: A total of 167 patients were included; 55 had eosinophilia. Eosinophilia was associated with an increased risk of 12‐month COPD‐related readmission (OR, 3.59 [95% CI, 1.65–7.82]; P = .0013), an increased risk of 12‐month all‐cause readmission (2.32 [95% CI, 1.10–4.92]; P = .0277), and a shorter time to first COPD‐related readmission (hazard ratio, 2.74 [1.56–4.83]; P = .0005). The length of stay was not statistically different between eosinophilic and noneosinophilic patients. Sensitivity analyses using different eosinophilia definitions revealed a proportional increase in effect size with increasing eosinophil cell count definitions for predicting 12‐month readmissions. Conclusions: Blood eosinophil levels can be used as a biomarker in severe COPD exacerbations for predicting higher readmission rates.

Safety and Efficacy of HFA-134a Beclomethasone Dipropionate Extra-Fine Aerosol over Six Months

OBJECTIVE To compare the systemic safety and efficacy of hydrofluoroalkane beclomethasone dipropionate (HFA-BDP) extra-fine aerosol 800 microg/day with chlorofluorocarbon (CFC)-BDP 1500 microg/day. DESIGN Six-month, randomized, parallel-group, double-blind, double-dummy study. PATIENTS Patients (n=141) with moderate to severe asthma adequately controlled by CFC-BDP 1000 microg/day to 2000 microg/day. INTERVENTIONS Patients received CFC-BDP 1500 microg/day during a two-week run-in period and were then randomized to either HFA-BDP (n=70) or CFC-BDP (n=71). RESULTS Similar proportions of HFA-BDP and CFC-BDP patients had a 24 h urinary free cortisol values below the reference range at month 6 (15% versus 25%, P=0.35). Measures of adrenocorticotrophic hormone stimulation and morning plasma cortisol levels were also similar in each group. The frequency of skin bruising and oral candidiasis was low for both treatments. No change in intraocular pressure was reported for either treatment. Pulmonary function was similar in both groups; however, the onset of the first asthma exacerbation or increased asthma symptoms tended to be earlier for CFC-BDP than for HFA-BDP (P=0.076); 27% of CFC-BDP patients reported increased asthma symptoms, compared with 14% of HFA-BDP patients (P=0.095). CONCLUSIONS HFA-BDP 800 microg/day has a systemic adverse event profile comparable to that of CFC-BDP 1500 microg/day, and further control of asthma symptoms may be achieved after a switch from CFC-BDP 1500 microg/day to HFA-BDP 800 microg/day.

Relative perinatal safety of salmeterol vs formoterol and fluticasone vs budesonide use during pregnancy.

BACKGROUND Recent asthma guidelines endorse the safety of long-acting β2-agonists (LABAs) and of mild and moderate doses of inhaled corticosteroids (ICSs) when required to control asthma during pregnancy, yet do not state a preferred medication within each class. OBJECTIVE To estimate the relative perinatal safety with the use of salmeterol and formoterol (LABAs) and that of fluticasone and budesonide (ICSs) during pregnancy. METHODS A subcohort of pregnancies from asthmatic women was selected from health care administrative databases of Quebec, Canada. Low birth weight (LBW) was defined as weight less than 2,500 g, preterm birth (PB) as delivery before 37 weeks of gestation, and small for gestational age (SGA) as a birth weight below the 10th percentile. The effect of treatment with salmeterol vs formoterol and fluticasone vs budesonide on the outcomes was determined with generalized estimating equation models. RESULTS The LABA and ICS subcohorts were composed of 547 (385 salmeterol and 162 formoterol users) and 3,798 (3,190 fluticasone and 608 budesonide users) pregnancies, respectively. No statistically significant differences were observed for LBW (odds ratio [OR], 0.91; 95% confidence interval [CI], 0.44-1.88), PB (OR, 1.11; 95% CI, 0.56-2.23), and SGA (OR, 1.16; 95% CI, 0.67-2.02) newborns between women exposed to salmeterol vs formoterol or between women exposed to fluticasone vs budesonide (LBW: OR, 1.08; 95% CI, 0.76-1.52; PB: OR, 1.07; 95% CI, 0.78-1.49; and SGA, OR: 1.10; 95% CI, 0.85-1.44). CONCLUSION This study does not provide evidence of greater perinatal safety for one LABA or one ICS over the other.

Benefits of low-dose inhaled fluticasone on airway response and inflammation in mild asthma

RATIONALE Current guidelines suggest that asthma should be controlled with the lowest dose of maintenance medication required. OBJECTIVES To evaluate the effects of a low dose of inhaled corticosteroid compared to a placebo, on airway inflammation and responsiveness in patients with mild symptomatic asthma. METHODS In this randomized double-blind, placebo-controlled, parallel group study, we looked at the influence of inhaled fluticasone propionate 250 microg/day for 3 months followed by 100 microg/day for 9 months on airway inflammation and methacholine responsiveness in non-smoking subjects with mild allergic asthma. Subjects were evaluated at baseline and 3, 6, 9 and 12 months after treatments; a 2-week evaluation of respiratory symptoms and peak expiratory flow measurements was done before each visit. RESULTS Fifty-seven subjects completed the 3-month study period. Airway responsiveness, expressed as the PC20 methacholine, increased by 0.27 and 1.14 doubling concentrations, respectively, in placebo-treated (n=33) and in fluticasone-treated (n=24) asthmatic subjects (p=0.03). An additional improvement in PC20 up to 2.16 doubling concentrations was observed in the fluticasone-treated group during the 9-month lower-dose treatment (p=0.0004, end of low-dose period compared with placebo). Sputum eosinophil counts decreased after 3 months of fluticasone 250 microg/day compared with placebo (p<0.0001) and remained in the normal range during the 9-month lower-dose treatment. Respiratory symptoms and peak expiratory flows did not change significantly throughout the study in both groups. CONCLUSION In mild asthma, keeping a regular minimal dose of ICS after asthma control has been achieved, may lead to a further reduction in airway responsiveness and keep sputum eosinophil count within the normal range.

Are Improvements Maintained after In-Home Pulmonary Telerehabilitation for Patients with Chronic Obstructive Pulmonary Disease?

This study investigated if improvements can be maintained over 24 weeks when in-home pulmonary telerehabilitation is combined with asynchronous self-management education for Chronic Obstructive Pulmonary Disease (COPD). Twenty-three community-living elders with moderate to very severe COPD participated in a pre/post-intervention study. Over 8 weeks, they had access to self-learning capsules on self-management, received 15 in-home teletreatment sessions and were encouraged to gradually engage in unsupervised sessions. Participants were assessed before the intervention (T1), immediately after the intervention (T2), and 6 months later (T3). Outcome measures were (1) exercise tolerance (6-minute walk test [6MWT]), Cycle Endurance Test [CET]), and (2) quality of life (Chronic Respiratory Questionnaire [CRQ]). Although there were significant improvements after 8 weeks of pulmonary telerehabilitation on the 6MWT, CET and three of four CRQ domains, none of these improvements were maintained after 6 months and scores returned to their baseline values (all p values > 0.05 when comparing T3 with T1). While pulmonary telerehabilitation is possible and has a positive impact on patients with moderate to very severe COPD, improvements were not maintained in the long-term even when physical therapy was accompanied by self-management education.