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We have previously shown that the binding of integrins with extracellular matrix component fibronectin (Fn) can improve the ability of bronchial epithelial cells (BECs) in resisting oxidant injury by up-regulating the activity of catalase and increasing the content of GSH. However, the molecular mechanism or its signaling pathway of this protection is still unclear. In order to examine the intracellular signaling mechanism activated by Fn-integrin binding reaction, the present study investigated the mRNA expression of catalase in primary cultured rabbit BECs using RT-PCR based on a cell-injury model made with ozone exposure. The product bands of target gene CAT were checked with Southern blot and oligonucleotide probe hybridization. The results showed that Fn (10 microg/ml) promoted the catalase mRNA transcription (P<0.01). This effect was abolished either by protein-tyrosine kinase inhibitor genistein or calmodulin inhibitor W(7) (P<0.01). These results indicate that the promotion of catalase activity induced by Fn-integrin reaction is partly due to the elevation of catalase mRNA transcription, and that its signalling are possibly relevant to tyrosine phosphorylation or calmodulin pathway.

To explore the role of intrapulmonary neuropeptides in the development of airway hyperresponsiveness, we established an animal model of airway hyperresponsiveness (AHR) in rabbits by using ozone exposure. With the model, after test of the mechanics of respiration and bronchoalveolar lavage assay, the levels of vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) in the lungs were determined by radioimmunoassay, and the expression of mRNA coding receptors of these two neuropeptides was evaluated by reverse transcriptional-polymerase chain reaction (RT-PCR). At the same time, the distribution of VIP receptor-1 (VIPR1) and CGRP receptor-1 (CGRPR1) in lung tissues and its time-course were examined by in situ hybridization. The results showed: (1) in ozone-stressing groups, airway resistance increased significantly and typical inflammatory pathological changes were observed in pulmonary tissue slides, including neutrophil and eosinophil infiltration, mucus exudation and bronchial epithelial cells (BECs) shedding; (2) with elongation of ozone exposure, the levels of VIP and CGRP in the lungs increased at first, reaching a peak on d 2 to 4, then decreased slowly, and CGRP peaked somewhat earlier than VIP; (3) mRNA expression of the two neuropeptide receptors in the lungs changed in a similar manner like VIP and CGRP, but the high level of mRNA expression of VIPR1 lasted longer than that of CGRPR1; and (4) in situ hybridization for neuropeptide receptors demonstrated that, in unstressed control, VIPR1 and CGRPR1 positive cells appeared in the airway epithelium, pulmonary interstitial and focal areas of airway and vascular smooth muscles. With the elongation of ozone exposure, hybridization stained deeper and the majority of positive cells were located around the vessels and bronchus except a few in the alveoli. At 8 d, only a small number of positive cells were seen in the lungs. From the results, it is concluded that ozone-stressing can induce the development of AHR, in which VIP and CGRP may play important roles. That implies, through binding to CGRPR1, CGRP stimulates an early inflammation response which contributes in cleaning up of irritants, while VIP exerts a later dampening of pulmonary inflammation response. These two neuropeptides may play sequential and complementary roles in the development of AHR.

Intercellular adhesion molecule-1 (ICAM-1) is an important adhesion molecule leading to adhesion between cells; NF-kappaB, being universally distributed in the organism, is an important nuclear transcription factor leading to a rapid response to the stimuli. Line of evidence have shown that ICAM-1 transcription and NF-kappaB activation is an important step of inflammatory reaction. To testify that intrapulmonary regulatory peptides modulate inflammatory lesion of bronchial epithelial cells (BECs) through their effect on ICAM-1 expression and nuclear factor kappaB (NF-kappaB) activation, we used immunocytochemistry, RT-PCR, and electrophoretic mobility-shift assay (EMSA) to determine the ICAM-1 expression and NF-kappaB activity in BECs. The effects of NF-kappaB inhibitor MG-132 on ICAM-1 expression were also observed. The results showed that vasoactive intestinal peptide (VIP) and epidermal growth factor (EGF) decreased ICAM-1 expression in O(3)-stressed BECs, while endothelin-1 (ET-1) and calcitonin gene-related peptides (CGRP) increased ICAM-1 expression in resting BECs. MG-132 blocked ICAM-1 expression induced by O(3), ET-1 and CGRP. The results obtained by using EMSA confirmed that VIP and EGF restrained the activation of NF-kappaB in O(3)-stressed BECs; CGRP and ET-1 promoted activation of NF-kappaB. These observations indicate that VIP and EGF abated the injury by means of down-regulatory effects on ICAM-1 transcription and NF-kappaB activation, while ET-1 and CGRP enhanced the inflammation reaction by an up-regulatory effect. It is suggested that a developing and intensive airway inflammation correlates closely with a persistent expression of ICAM-1 and repeated activation of NF-kappaB.

To investigate the influence of vasoactive intestinal peptide (VIP) on chemotaxis of bronchial epithelial cells (BECs). Rabbit chemotactic migration of primary BEC was assessed in a blind-well Boyden chamber. Radioimmunoassay and radio-ligand affinity analysis were used for determining VIP secretion and vasoactive intestinal peptide receptor (VIPR) expression. The results showed: (1) the method for determining chemotaxis of BECs by using insulin as chemotactic factor was stable and reproducible (r=0.9703, P<0.01). (2) VIP (0.001-1 micromol/L) elicited chemotaxis of BECs which was substantial and concentration-dependent. The effects of VIP were inhibited by W-7 and H-7 (P<0.01). (3) Heat stress enhanced the secretion of VIP (P<0.01) and upregulated the expression of VIPR on BECs (P<0.05). These results indicate that VIP in the lungs may play an important role in the repair of damaged epithelium, accelerating restoration of the airway to its normal state. Calmodulin and protein kinase C may be involved in the signal transduction of VIP effects.

To explore the role of regulatory peptides in the secretion of bronchial epithelial cells (BECs), we observed the effects of four peptides, i.e.vasoactive intestinal peptide (VIP), epidermal growth factor (EGF), endothelin-1 (ET-1), and calcitonin gene-related peptide (CGRP), on the secretion of ILs from unstimulated or O3-stressed BECs. The results of the experiments showed that VIP exerted an inhibitory effect on the secretion of IL-1 and IL-8 from unstimulated and O3-stressed BECs, VIP also decreased the secretion of IL-5 from O3-stressed BECs; EGF promoted secretion of IL-1 and IL-8 from unstimulated BECs, but decreased the secretion of ILs from O3-stressed BECs; ET-1 and CGRP enhanced the secretion of IL-1, IL-5, and IL-8 from unstimlated BECs, CGRP also increased the secretion of ILs from O3-stressed BECs. The results obtained demonstrate that intrapulmonary regulatory peptides modulate the secretion of ILs from BECs, and may play an important part in transduction of inflammatory signals.

To explore the roles of regulatory peptides in the process of various anaphylactic inflammation of the airway, we observed the influence of four peptides, i.e., vasoactive intestinal peptide (VIP), epidermal growth factor (EGF), endothelin-1 (ET-1), and calcitonin gene-related peptide (CGRP), on the adhesion of eosinophil (EOS) to unstimulated and O(3)-stressed bronchial epithelial cells (BEC). From the experiments we observed that VIP and EGF decreased EOS adherence to O(3)-stressed BEC and downregulated airway inflammation; ET-1 and CGRP increased the adhesion of EOS to BEC in the inflammatory process; and CGRP aggravated O(3)-stressed reactions. The effects of ET-1 and CGRP were inhibited by W(7)and H(7). Anti-ICAM-1 antibody inhibited the adhesion of EOS to BEC, which brings to light that EOS adherence to BEC may be related to the expression of ICAM-1 of BEC.

Antioxidant activity of bronchial epithelial cells (BECs) plays an essential role in preventing the airway epithelium integrity from damage in structure and function. Integrin expressed by BECs is the receptor of extracellular matrix such as fibronectin (Fn), and it is involved in modulation of proliferation, differentiation and metabolism of the cells. In order to test the hypothesis that integrin-ligand binding reaction supports the ability of cells to withstand oxidant attack, the present study evaluated the antioxidant activity of primary cultured rabbit BECs treated with fibronectin or its sequence Arg-Gly-Asp (RGD peptide), by determining changes in the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) and in the level of glutathione (GSH). The results are as follows: (1) Fn (10 micrograms/ml) increased significantly the activity unit of GSH-Px (P < 0.05, n = 5), which was inhibited by calmodulin-inhibitor W7 (10(-5) mol/L) (P < 0.05). Both Fn (5-20 micrograms/ml) and RGD (15-60 micrograms/ml) showed a dose-dependent upregulatory effect (respectively r = 0.93 and r = 0.73). (2) Treatment with Fn increased SOD activity (P < 0.01, n = 7), which was abolished by W7 (P < 0.01). (3) Catalase activity was also stimulated by Fn (P < 0.05, n = 6) and reversed by W7 (P < 0.01). (4) A dose-dependent increase of GSH level was observed in both Fn (r = 0.82) and RGD treatment (r = 0.84). The data suggest that the binding of integrin with extracellular matrix can upregulate activity of antioxidant enzymes, and increase the content of GSH and improve the ability of BECs to resist oxidant injury.

In this study, it was observed that ozone (O3) exposure has cytotoxic effects on cultured airway epithelial cells, which was positively related with exposure duration. Both the production of malondialdehyde (MDA) and the 3H release in the exposure group were much higher than the control (P < 0.01), suggesting that lipoperoxidation occurring in the cell membrane was responsible for the cellular injury observed under O3 attack. EGF at low concentration (5 ng/ml) showed cytoprotective effect on airway epithelial cells exposed to O3 as shown by attenuated MDA production and decrease of 3H release and cytotoxic index (P < 0.01). Pretreatment with EGF could reverse the depletion of intracellular GSH content as a result of O3 exposure and increase the total glutathionein the cells. The above results suggest that the cytoprotection of EGF on airway epithelial cells may be related to its promotive effect on GSH synthesis.