Transcriptional down-regulation of suppressor of cytokine signaling (SOCS)-3 in chronic obstructive pulmonary disease.

BACKGROUND: Tobacco is a leading environmental factor in the initiation of respiratory diseases and causes chronic obstructive pulmonary disease (COPD). Suppressor of cytokine signaling (SOCS) family members are involved in the pathogenesis of many inflammatory diseases and SOCS-3 has been shown to play an important role in the regulation, onset and maintenance of airway allergic inflammation indicating that SOCS-3 displays a potential therapeutic target for anti-inflammatory respiratory drugs development. Since chronic obstructive pulmonary disease (COPD) is also characterized by inflammatory changes and airflow limitation, the present study assessed the transcriptional expression of SOCS-3 in COPD. METHODS: Real-time PCR was performed to assess quantitative changes in bronchial biopsies of COPD patients in comparison to unaffected controls. RESULTS: SOCS-3 was significantly down-regulated in COPD at the transcriptional level while SOCS-4 and SOCS-5 displayed no change. CONCLUSIONS: It can be concluded that the presently observed inhibition of SOCS-3 mRNA expression may be related to the dysbalance of cytokine signaling observed in COPD.

A novel C-type immulectin-3 from Manduca sexta is translocated from hemolymph into the cytoplasm of hemocytes.

Lectins interact with carbohydrates. They can function as pattern recognition receptors and play an important role in the innate immune system of animals. Previously, we have isolated two calcium-dependent (C-type) lectins, named immulectin-1 and -2, from the tobacco hornworm Manduca sexta. Both immulectin-1 and -2 stimulate prophenoloxidase activation in plasma. Here, we describe isolation and cDNA cloning of a novel member of immulectins, immulectin-3 (IML-3). IML-3, like immulectin-1 and -2, contains tandem carbohydrate-recognition domains (CRDs). The cDNA clone encoding IML-3 is 3802 bp long, with an open reading frame of 930 bp. This cDNA clone has an extremely long noncoding region at the 3' end that contains eight polyadenylation signal sequences. Northern analysis showed that a 5.0 kb IML-3 transcript was present in the fat body of control larvae (injected with saline) but not in the fat body of larvae injected with bacteria. However, a much more abundant 3.1 kb transcript was induced in the fat body of bacteria-injected larvae. IML-3 mRNA was not detected in hemocytes of control or bacteria-injected larvae. Recombinant IML-3 was expressed in bacteria and purified. It specifically bound to immobilized lipopolysaccharide (LPS) and lipoteichoic acid from bacteria, and to laminarin, a beta-1, 3-glucan. Binding of IML-3 to immobilized LPS was competed by excess free LPS. More importantly, IML-3 contains an anti-death-like motif in the carboxyl-terminal CRD. Endogenous IML-3 was detected in the cytoplasm of hemocytes, and FITC-labeled recombinant IML-3 was translocated from hemolymph into hemocytes. Coating of IML-3 onto agarose beads enhanced encapsulation of the beads.

Substance P mediates AP-1 induction in A549 cells via reactive oxygen species.

A common feature in asthma is the induction of reactive oxygen species (ROS) and the AP-1 transcription factor during the inflammatory process. AP-1 induction leads to an increased expression of pro-inflammatory cytokines. Also, higher levels of the pro-inflammatory neuropeptide substance P (SP) have been reported in bronchoalveolar-lavage fluid of asthmatics. Here, the role of SP on ROS induction and the downstream activation of AP-1 in A549 airway epithelial cells was investigated by dichloroflourescein-diacetate method and reporter gene assays. The SP-mediated AP-1 induction was dependent on extracellular calcium and ROS. The likely source of ROS are the mitochondria as rotenone inhibited AP-1 induction and the p47phox subunit of the NADPH oxidase complex, responsible for ROS generation in phagocytotic cells, was not expressed in A549 cells assayed by RT-PCR. This is consistent with results obtained from cells of murine bronchial epithelium, isolated by laser capture microdissection. In summary, this study provides evidence for an SP-mediated induction of AP-1, which may contribute to the expression of pro-inflammatory cytokines.

SMAD-signaling in chronic obstructive pulmonary disease: transcriptional down-regulation of inhibitory SMAD 6 and 7 by cigarette smoke.

Transforming growth factor-beta1 is a potent mediator of fibrosis stimulating the secretion of extracellular matrix proteins and is involved in airway remodeling in chronic obstructive pulmonary disease (COPD). Signals from the TGF superfamily are mediated by the SMAD group of transcription factors. Here, the expression of the regulatory SMAD2, 3, the co-SMAD4 and the inhibitory SMAD6 and 7 was assessed in bronchial biopsies of COPD patients and controls by quantitative RT-PCR. While SMAD2 was not expressed and SMAD3 and 4 displayed no change, the inhibitory SMAD6 and 7 were significantly down-regulated in COPD. To reveal the molecular basis of tobacco smoke-induced airway remodeling and to test whether it may interfere with intracellular SMAD signaling, the airway epithelial cell line A549 was incubated with cigarette smoke extract (1% and 10%) for 48 hours, which led to down-regulation of SMAD6 and 7 at both concentrations tested. It can be concluded that TGF-beta-mediated effects in COPD are influenced by a disturbed intracellular feedback mechanism of inhibitory SMADs. Also, the effects of non-volatile components in tobacco smoke may partly be regulated via a smoke-induced down-regulation of inhibitory SMADs.