Paul R. Eynott

Distribution and function of the peptide transporter PEPT2 in normal and cystic fibrosis human lung

Background: Aerosol administration of peptide based drugs has an important role in the treatment of various pulmonary and systemic diseases. The characterisation of pulmonary peptide transport pathways can lead to new strategies in aerosol drug treatment. Methods: Immunohistochemistry and ex vivo uptake studies were established to assess the distribution and activity of the β-lactam transporting high affinity proton coupled peptide transporter PEPT2 in normal and cystic fibrosis human airway tissue. Results: PEPT2 immunoreactivity in normal human airways was localised to cells of the tracheal and bronchial epithelium and the endothelium of small vessels. In peripheral lung immunoreactivity was restricted to type II pneumocytes. In sections of cystic fibrosis lung a similar pattern of distribution was obtained with signals localised to endothelial cells, airway epithelium, and type II pneumocytes. Functional ex vivo uptake studies with fresh lung specimens led to an uptake of the fluorophore conjugated dipeptide derivative d-Ala-l-Lys-AMCA into bronchial epithelial cells and type II pneumocytes. This uptake was competitively inhibited by dipeptides and cephalosporins but not ACE inhibitors, indicating a substrate specificity as described for PEPT2. Conclusions: These findings provide evidence for the expression and function of the peptide transporter PEPT2 in the normal and cystic fibrosis human respiratory tract and suggest that PEPT2 is likely to play a role in the transport of pulmonary peptides and peptidomimetics.

Allergen-induced inflammation and airway epithelial and smooth muscle cell proliferation: role of Jun N-terminal kinase.

Chronic cellular inflammation and airway wall remodelling with subepithelial fibrosis and airway smooth muscle (ASM) cell hyperplasia are features of chronic asthma. Jun N‐terminal kinase (JNK) may be implicated in these processes by regulating the transcriptional activity of activator protein (AP)‐1. We examined the effects of an inhibitor of JNK, SP600125 (anthra [1,9‐cd] pyrazole‐6 (2 H)‐one), in a model of chronic allergic inflammation in the rat. Rats sensitised to ovalbumin (OA) were exposed to OA‐aerosol every third day on six occasions and were treated with SP600125 (30 mg kg−1 b.i.d; 360 mg in total) for 12 days, starting after the second through to the sixth OA exposure. We measured eosinophilic and T‐cell inflammation in the airways, proliferation of ASM cells and epithelial cells by incorporation of bromodeoxyuridine (BrdU), and bronchial responsiveness to acetylcholine. SP600125 significantly reduced the number of eosinophils (P<0.05) and lymphocytes (P<0.05) in bronchoalveolar lavage fluid, suppressed eosinophilic (P<0.05) and CD2+ T‐cell (P<0.05) infiltration within the bronchial submucosa, and the increased DNA incorporation in ASM (P<0.05) and epithelial cell incorporation (P<0.05). SP600125 did not alter bronchial hyper‐responsiveness observed after chronic allergen exposure. Pathways regulated by JNK positively regulate ASM cell proliferation and allergic cellular inflammation following chronic allergen exposure.

Role of nitric oxide in allergic inflammation and bronchial hyperresponsiveness.

The role of nitric oxide (NO) in allergic inflammation and bronchial hyperresponsiveness is unclear. We studied a selective prodrug nitric oxide synthase (NOS)-2 inhibitor, L-N(6)-(1-iminoethyl)lysine 5-tetrazole amide (SC-51). In ovalbumin-sensitized and challenged rats, exhaled NO levels increased by 3 h following challenge (3.73 +/- 0.74 ppb; P < 0.05), peaking at 9 h (11.0 +/- 2.75; P < 0.01) compared to saline controls (1.87 +/- 0.26; P < 0.05 and 2.81 +/- 0.18; P < 0.01). Immunoreactive lung NOS2 expression was increased in ovalbumin-challenged rats compared with ovalbumin-sensitized, saline-challenged rats at 8 h post-challenge. SC-51 (10 mg/kg; p.o.) inhibited allergen-induced increase in exhaled NO levels to 1.3 +/- 0.17 ppb. SC-51 inhibited bronchial hyperresponsiveness in ovalbumin-sensitized and challenged rats (P < 0.05). In sensitized non-exposed rats, SC-51 increased bronchial responsiveness (P < 0.05). SC-51 reduced the allergen-induced increase in bronchoalveolar lavage neutrophils, but caused a nonsignificant reduction in bronchial mucosal eosinophil numbers. NO generated through NOS2 contributes to allergen-induced bronchial hyperresponsiveness but not to bronchial eosinophilia, indicating that these are independently expressed.

Natural killer T cells and CD8+ T cells are dispensable for T cell-dependent allergic airway inflammation.

Natural killer T cells and CD8 + T cells are dispensable for T cell–dependent allergic airway inflammation

Mitogen-activated protein kinase signalling pathways in IL-1β-dependent rat airway smooth muscle proliferation

Asthma is associated with abnormal airway smooth muscle (ASM) growth that may contribute to airway narrowing and hyperresponsiveness. We investigated the role of mitogen‐activated protein kinase (MAPK) pathway in IL‐1β induced ASM proliferation in the rat. Rat tracheal ASM cells were dissociated and maintained in culture. We examined the effect of selective MAPK inhibitors, SB239063 (a p38 MAPK inhibitor), U0126 (a mitogen‐activated and extracellular regulated kinase kinase, MEK‐1, inhibitor which inhibits downstream extracellular regulated kinase, ERK, activity), and SP600125 (a c‐jun N‐terminal kinase, JNK, inhibitor) on IL‐1β‐induced proliferation. Proliferation of ASM cells was significantly increased following exposure to IL‐1β in a dose‐dependent manner. p38, JNK and ERK MAPKs were activated by IL‐1β in a time‐dependent manner, with peak activation time at 30, 60 min and at 6 h, respectively. This activation was inhibited by their respective inhibitors. SP600125 (20 μM) had no effect on IL‐1β‐induced ERK and p38 phosphorylation. SB239063, U0126 and SP600125 dose‐dependently inhibited IL‐1β‐dependent proliferation at doses that inhibit the activities of p38, ERK and JNK MAPKs, respectively. No additive or synergistic effects were observed on proliferative responses with any combination of these compounds. In conclusion, the three major MAPK pathways, ERK as well as the p38 MAPK and JNK pathways, are independent regulators of IL‐1β‐dependent proliferation of rat ASM.

Effect of an inhibitor of Jun N-terminal protein kinase, SP600125, in single allergen challenge in sensitized rats

Jun N‐terminal kinase (JNK) has been implicated in the pathogenesis of inflammatory diseases including asthma. We examined the effect of SP600125 (anthra [1,9‐cd] pyrazol‐6 (2H)‐one), a novel inhibitor of JNK in a model of asthma. Brown‐Norway rats were sensitized to ovalbumin and treated with SP600125 intraperitoneally (90 mg/kg in total). SP600125 inhibited allergen‐induced, increased activity of phosphorylated c‐jun but not of phosphorylated‐MAPKAPK2, indicative of activation of p38 MAPK, in the lung. SP600125 inhibited macrophage (P < 0·04), lymphocyte (P < 0·05), eosinophil (P < 0·04) and neutrophil (P < 0·005) numbers in bronchoalveolar lavage. Eosinophil and T‐cell accumulation in the airways, mRNA expression for interleukin‐1β, tumour necrosis factor‐β, interleukin‐3, interleukin‐4 and interleukin‐5, serum levels of allergen‐specific immunoglobulin E and bronchial hyperresponsiveness were not affected by SP600125. Selective inhibition of JNK reduced inflammatory cell egress into the airway lumen after single allergen exposure. The role of JNK mitogen‐activated protein kinase activation may be limited in the pathogenesis of bronchial hyperresponsiveness after single allergen exposure.

Role of cathepsin S in ozone-induced airway hyperresponsiveness and inflammation

Ambient ozone has been linked to the worsening of symptoms of patients with obstructive diseases such as chronic obstructive pulmonary disease (COPD) and asthma. We investigated the role of cathepsin S on ozone-induced airway hyperresponsiveness (AHR) and inflammation, using the selective cathepsin S inhibitor, Compound A. Balb/c mice were exposed to ozone at a concentration of 3 ppm or air for 3 h, following administration by gavage of Compound A or vehicle. Bronchoalveolar lavage (BAL) was performed 3 h and 20-24 h following exposure, AHR was measured at 20-24 h only. Ozone exposure, compared to air exposure increased BAL cathepsin S levels, AHR and BAL inflammatory cells. Compound A (30 mg kg(-1) p.o.) dosing compared to vehicle dosing inhibited ozone-induced AHR (-logPC100 vehicle: -0.70+/-0.12, n=8 vs. cathepsin S inhibitor: -1.30+/-0.06, P<0.001, n=8) at 20-24 h and BAL neutrophilia at 3 h and 20-24 h (P<0.05, n=6). Ozone exposure increased levels of BAL cytokines IL-6, TNF-alpha and IFN-gamma. Compound A reduced IL-6 at 3 h and 20-24 h (P<0.05, n=5) and TNF-alpha, at 20-24 h (P<0.05, n=6). These data indicate an important role for cathepsin S in the regulation of ozone-induced AHR and neutrophil cell recruitment and suggest that cathepsin S may be a target in the treatment of oxidative stress-induced AHR and inflammation.

Effects of cyclosporin A and a rapamycin derivative (SAR943) on chronic allergic inflammation in sensitized rats

Immunomodulators such as cyclosporin A (CsA) and SAR943 (32‐deoxorapamycin) inhibit single allergen‐induced allergic inflammation such as eosinophilic and lymphocytic infiltration and mRNA expression for interleukin (IL)‐4 and IL‐5. We examined the effects of CsA and SAR943, administered orally, on asthmatic responses in a rat model of chronic allergic inflammation. Sensitized Brown‐Norway (BN) rats were exposed to ovalbumin (OVA) aerosol every third day on six occasions. CsA (5 mg/kg/day), SAR943 (2·5 mg/kg/day) or vehicle (Neoral™) was administered orally, once a day, from days 10 to 21 (a total of 12 doses). We measured eosinophilic and T‐cell inflammation in the airways, proliferation of airway cells by incorporation of bromodeoxyuridine (BrdU) and bronchial responsiveness to acetylcholine. CsA had no effects, while SAR943 inhibited airway smooth muscle (ASM, P < 0·05) and epithelial cell (P < 0·01) BrdU incorporation, and the number of CD4+ T cells (P < 0·05), without effects on BHR. ASM thickness was not significantly increased following chronic allergen exposure. Therefore, CsA and SAR943 have no effect on chronic eosinophilic inflammation, while SAR943, but not CsA, had a small effect on the proliferation of ASM and epithelium.

Resolution of allergic airways inflammation but persistence of airway smooth muscle proliferation after repeated allergen exposures

Background Chronic inflammation in asthmatic airways can lead to characteristic airway smooth muscle (ASM) thickening and pathological changes within the airway wall.

Transcriptional down-regulation of neurotrophin-3 in chronic obstructive pulmonary disease.

Abstract Chronic obstructive pulmonary disease (COPD) leads to progressive development of airflow limitation and is characterised by cough, mucus hypersecretion and inflammatory changes. These characteristic features of the disease may be modulated by neural mediators such as neurotrophins (NT). Here we examined the expression and transcriptional regulation of neurotrophins in bronchial biopsies of COPD patients and compared the data to control biopsies. Histology revealed characteristic changes in the COPD tissues, including remodelling of the epithelial lining. RT-PCR demonstrated the mRNA expression of neurotrophins in all biopsies. Immunohistochemistry confirmed the expression of different proteins. To assess changes in the transcriptional expression level, quantitative real-time PCR was carried out and revealed differential mRNA expression of neurotrophins, with marked down-regulation of NT-3 mRNA expression and constant levels of nerve growth factor (NGF), brain-derived nerve factor (BDNF), and NT-4/5 mRNA expression. The present data on neurotrophin-specific transcriptional down-regulation of NT-3 in human COPD indicate a pathophysiological role for neurotrophins in COPD.