Smoking Cessation in Mice Does Not Switch off Persistent Lung Inflammation and Does Not Restore the Expression of HDAC2 and SIRT1

Once COPD is established, pulmonary lesions can only progress and smoking cessation by itself is not sufficient to switch off persistent lung inflammation. Similarly, in former-smoker mice, neutrophil inflammation persists and lung lesions undergo progressive deterioration. The molecular mechanisms underlying disease progression and the inefficiency of smoking cessation in quenching neutrophilic inflammation were studied in male C57 Bl/6 mice after 6 months of rest from smoking cessation. As compared with the mice that continued to smoke, the former-smoker mice showed reduced expression of histone deacetylases HDAC2 and SIRT1 and marked expression of p-p38 MAPK and p-Ser10. All these factors are involved in corticosteroid insensitivity and in perpetuating inflammation. Former-smoker mice do show persistent lung neutrophilic influx and a high number of macrophages which account for the intense staining in the alveolar structures of neutrophil elastase and MMP-9 (capable of destroying lung scaffolding) and 8-OHdG (marker of oxidative stress). "Alarmins" released from necrotic cells together with these factors can sustain and perpetuate inflammation after smoking cessation. Several factors and mechanisms all together are involved in sustaining and perpetuating inflammation in former-smoker mice. This study suggests that a better control of COPD in humans may be achieved by precise targeting of the various molecular mechanisms associated with different phenotypes of disease by using a cocktail of drug active toward specific molecules

Severe Reduction in Number and Function of Peripheral T Cells Does Not Afford Protection toward Emphysema and Bronchial Remodeling Induced in Mice by Cigarette Smoke

8openThe protein Lck (p56(Lck)) is a Src family tyrosine kinase expressed at all stages of thymocyte development and is required for maturation of T cells. The targeted disruption of Lck gene in mice results in severe block in thymocyte maturation with substantial reduction in the development of CD4(+)CD8(+) thymocytes, severe reduction of peripheral T cells, and disruption of T-cell receptor signaling with defective function of T-cell responses. To investigate the role of T lymphocyte in the development of cigarette smoke-induced pulmonary changes, Lck(-/-) mice and corresponding congenic wild-type mice were chronically exposed to cigarette smoke, and their lungs were analyzed by biochemical, immunologic, and morphometric methods. Smoking mice from both genotypes showed disseminated foci of emphysema and large areas of goblet cell metaplasia in bronchial and bronchiolar epithelium. Morphometric evaluation of lung changes and lung elastin determination confirmed that mice from both genotypes showed the same degree of emphysematous lesions. Thus, cigarette smoke exposure in the presence of severe reduction in number and function of peripheral T cells does not influence the development of pulmonary changes induced by cigarette smoke. The data obtained suggest that innate immunity is a leading actor in the early development of pulmonary changes in smoking mice and that the adaptive immune response may play a role at later stages.openDe Cunto, Giovanna; Lunghi, Benedetta; Bartalesi, Barbara; Cavarra, Eleonora; Fineschi, Silvia; Ulivieri, Cristina; Lungarella, Giuseppe; Lucattelli, MonicaDE CUNTO, Giovanna; Lunghi, Benedetta; Bartalesi, Barbara; Cavarra, Eleonora; Fineschi, Silvia; Ulivieri, Cristina; Lungarella, Giuseppe; Lucattelli, Monic

Proteasi, antiproteasi e matrice interstiziale: studio di modelli murini di danno polmonare

Dottorato di ricerca in biologia e patologia delle matrici connettivali. 7. ciclo. A.a. 1991-94. Coordinatore I. Pasquali Ronchetti. Relatore G. LungarellaConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Purification and N-terminal amino-acid sequence analysis of rabbit neutrophil cathepsin G

3nononeCathepsin G was isolated from granules of rabbit bloodstream leukocytes and purified to apparent homogeneity by a multi-step procedure consisting of ammonium sulphate precipitation, affinity chromatography on elastin-Sepharose, and finally by ion-exchange chromatography on a CM-52 column. The molecular weight of the enzyme, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), was 27,000. The first 24 N-terminal amino-acids were determined and showed 96%, 92% and 79% identity respectively to those of human, dog and rat cathepsin G. Despite the difference in the total amino-acid composition of cathepsin G between rabbit and other mammalian species, close similarities have been found in their substrate specificity and inhibition profile. The kcat/Km values of rabbit cathepsin G with Suc-Ala2-Pro-Phe-NA and Suc-Ala2-Pro-Leu-NA are quite similar to those reported for human cathepsin G under the same conditions. The inhibition profile of the isolated enzyme indicates that cathepsin G from rabbits, like that from other mammalians species belongs to the group of serine proteinases. Finally, like human cathepsin G, catalytically active rabbit enzyme is able to induce platelet aggregation.noneCAVARRA, E.; SANTUCCI, A.; LUNGARELLA, G.Cavarra, E.; Santucci, A.; Lungarella, G

sRAGE/mRAGE imbalance characterizes the cigarette smoke-induced lung changes in oxidant-sensitive DBA/2 mice.

INTRODUCTION: Unlike C57 Bl/6 (C57) mice, which develop emphysema without obvious fibrosis after chronic exposure to cigarette smoke (CS), DBA/2 mice develop emphysema associated with overt areas of fibrosis. This picture is similar to that described in man with the “Syndrome of Combined Pulmonary Fibrosis and Emphysema”. Thus, in mice like in men, pulmonary changes following to CS may be different and sustained by distinct pathogenic mechanisms. We analysed differences and similarities characterising the development of the CS−induced lung changes in alpha−1PI deficient C57 and DBA/2 mice, which are particularly sensitive to oxidants. METHODS: Mice were exposed to the smoke of 3 cigarettes / day, 5 days/week. At various times, lungs were analysed by morphology, morphometry, immunohistochemistry and biochemical techniques. RESULTS: At the different times after CS exposure, we found in lungs of C57 mice a decreased expression of the membrane bound receptor for advanced glycation end−products (mRAGE). On the contrary, we did not observed in DBA/2 mice significant changes in the expression of mRAGE. With regard to the soluble form of RAGE (sRAGE), we found after CS exposure a significant decrease of sRAGE in DBA/2 mice and no significant changes in C57 mice. These data indicate that a net balance in favour of mRAGE occurs in DBA/2 mice after CS exposure. CONCLUSIONS: RAGE has been recently implicated in programs responsible for acute and chronic inflammation and in lung fibrosis. RAGE signal may induce NF−kB activation and M−CSF hyper−expression promoting inflammation and macrophages activation via alternate pathway. sRAGE may counteract RAGE signal by acting as a “decoy receptor”. Our results suggest that an imbalance between mRAGE and sRAGE can play an important role in the onset of fibrotic lesions associated to lung emphysema after CS exposur

Different expression of cytokines and receptors in bleomycin-sensitive and resistant mice

The sensitivity to the fibrosis-inducing effect of bleomycin (BLM) varies considerably from species to species, and the variability of the response in different strains of mice is well documented. The reasons for such varied responses are not known. Recent evidence indicates that the up-regulated expression of cytokines and receptors may be involved. We evaluated the expression pattern of some cytokines and of their receptors in C57Bl/6J BLM-sensitive and Balb/C BLM-resistant mice. Animals from both strains received either saline (50µl), or BLM (0.1U/50µl) intratracheally. The mice were sacrificed at various times after the treatment. The lungs were analysed for cytokine and cytokine receptors mRNA by Ribonuclease Protected Assay. A significant increased expression (over saline-treated-animals) of TNF-alpha and IL-1 beta mRNA was observed in both strains at 8 hours. However, BLM resulted in an up-regulated lung expression for TNF-alpha and IL-1 receptors, only in C57Bl/6J sensitive animals. This profile is evident from 63 hours onward. In addition to TNF-alpha, BLM also resulted in the up-regulated expression of TGF-beta in the lungs of both strains at 8 hours, and in an enhanced expression of TGF-beta receptors I and II in only C57Bl/6J mice. The up-regulation of the TGF-beta receptor expression was preceded in this strain by an increased expression of IL-4, IL-13 and IL-13 receptor alpha1 at 8 hours after BLM. The difference we observed in the cytokine profile may offer an addidional explanation for the different fibrogenic response of the two mouse strains to BLM