Thymosin ß4 protects C57BL/6 mice from bleomycin-induced damage in the lung.

BACKGROUND: Thymosin ß4 (Tß4) was recently found at high concentration in the bronchoalveolar lavage fluid (BALF) of scleroderma patients with lung involvement. It has been hypothesized that Tß4 may exert a cyto-protective effect during lung injury because lower Tß4 levels were associated with interstitial lung disease progression. Moreover, Tß4 treatment prevented profibrotic gene expression in cardiac cells in vitro and in vivo. MATERIALS AND METHODS: In this study, we explored a putative Tß4 protective role in lung damage by utilizing a well-known in vivo model of lung fibrosis. C57BL/6 mice were treated with bleomycin (BLEO, 1 mg/kg) in the absence or presence of Tß4 (6 mg/kg delivered intraperitoneally on the day of BLEO treatment and for two additional doses). After sacrifice 1 week later, measurement of fluid and collagen content in the lung, BALF analysis, myeloperoxidase (MPO) activity assay, lung histology and IHC were performed. RESULTS: Compared with BLEO-treated mice, BLEO-treated mice who received Tß4 did not lose as much weight and had a higher survival rate. Moreover, BLEO-induced inflammation and lung damage were substantially reduced by Tß4 treatment, as demonstrated by the significant reduction in oedema, total collagen content, lung infiltration by leucocytes, MPO activity in lung homogenates, and histological evidence of the ongoing lung fibrosis. Results of IHC show a strong reactivity for Tß4 in the lung tissue of Tß4-treated mice. CONCLUSIONS: This is the first report that shows a Tß4 protective role in lung toxicity associated with BLEO in a mouse model. Future studies are needed to assess its putative antifibrotic properties.

Bradykinin differentiates human lung fibroblasts to a myofibroblast phenotype via the B2 receptor.

BACKGROUND: The identification of factors mediating the transition of lung fibroblasts into myofibroblasts is considered fundamental in the comprehension of abnormal reparative processes. Bradykinin, a mediator known for its proinflammatory action, is able to induce cytokine production and contractility in fibroblast cultures. OBJECTIVES: In this study the ability of bradykinin to drive fibroblast into a myofibroblast phenotype at the cellular and molecular level was evaluated. METHODS: alpha-Smooth muscle actin (alpha-SMA) expression and TGF-beta in bradykinin stimulated fibroblasts were tested by means of flow cytometry, Western blot, and RT-PCR. Cell proliferation and collagen production were evaluated by the colorimetric methylthiazol tetrazolium assay and sirius red assay, respectively. Which bradykinin receptor mediates the expression of alpha-SMA was evaluated using selective B1 and B2 blocking agents. Furthermore, the effect of bradykinin on extracellular signal-regulated kinase 1/2 phosphorylation was explored. RESULTS: Bradykinin caused in lung fibroblasts a significant increase in alpha-SMA at the cellular and molecular level. The B2 receptor was held responsible for this effect because a specific receptor antagonist had entirely blocked this effect. Bradykinin was able to induce fibroblast proliferation and collagen production. Bradykinin significantly activated mitogen-activated protein kinase pathway by phosphorylating extracellular signal-regulated kinase 1/2, whereas PD98059, a specific inhibitor, was able to block myofibroblast induction. Although bradykinin induced an increase of TGF-beta on fibroblasts, the blockage of this cytokine did not alter alpha-SMA expression. CONCLUSION: The data support the hypothesis that bradykinin may be involved in bronchial remodeling and lung fibrosis beyond its well recognized proinflammatory activity, also suggesting a new potential therapeutic strategy to control altered reparatory processes.

Interaction between human lung fibroblasts and T-lymphocytes prevents activation of CD4+ cells.

BACKGROUND: T lymphocytes are demonstrated to play an important role in several chronic pulmonary inflammatory diseases. In this study we provide evidence that human lung fibroblasts are capable of mutually interacting with T-lymphocytes leading to functionally significant responses by T-cells and fibroblasts. METHODS: Human lung fibroblast were co-cultured with PMA-ionomycin activated T-CD4 lymphocytes for 36 hours. Surface as well as intracellular proteins expression, relevant to fibroblasts and lymphocytes activation, were evaluated by means of flow cytometry and RT-PCR. Proliferative responses of T lymphocytes to concanavalin A were evaluated by the MTT assay. RESULTS: In lung fibroblasts, activated lymphocytes promote an increase of expression of cyclooxygenase-2 and ICAM-1, expressed as mean fluorescence intensity (MFI), from 5.4 +/- 0.9 and 0.7 +/- 0.15 to 9.1 +/- 1.5 and 38.6 +/- 7.8, respectively. Fibroblasts, in turn, induce a significant reduction of transcription and protein expression of CD69, LFA-1 and CD28 in activated lymphocytes and CD3 in resting lymphocytes. In activated T lymphocytes, LFA-1, CD28 and CD69 expression was 16.6 +/- 0.7, 18.9 +/- 1.9 and 6.6 +/- 1.3, respectively, and was significantly reduced by fibroblasts to 9.4 +/- 0.7, 9.4 +/- 1.4 and 3.5 +/- 1.0. CD3 expression in resting lymphocytes was 11.9 +/- 1.4 and was significantly reduced by fibroblasts to 6.4 +/- 1.1. Intracellular cytokines, TNF-alpha and IL-10, were evaluated in T lymphocytes. Co-incubation with fibroblasts reduced the number of TNF-alpha positive lymphocytes from 54.4% +/- 6.12 to 30.8 +/- 2.8, while IL-10 positive cells were unaffected. Finally, co-culture with fibroblasts significantly reduced Con A proliferative response of T lymphocytes, measured as MTT absorbance, from 0.24 +/- 0.02 nm to 0.16 +/- 0.02 nm. Interestingly, while the activation of fibroblasts is mediated by a soluble factor, a cognate interaction ICAM-1 mediated was demonstrated to be responsible for the modulation of LFA-1, CD28 and CD69. CONCLUSION: Findings from this study suggest that fibroblasts play a role in the local regulation of the immune response, being able to modulate effector functions of cells recruited into sites of inflammation.

Inhibitory effect of a leukotriene receptor antagonist (montelukast) on neurokinin A-induced bronchoconstriction.

BACKGROUND: Tachykinins are potent contractors of human airways producing a dose-related bronchoconstriction when administered by means of inhalation to asthmatic subjects. OBJECTIVE: The aim of this study was to examine the effective role played by leukotrienes (LTs) in neurokinin A (NKA)-induced bronchoconstriction in asthmatic patients. METHODS: To address this question, we investigated the protective effect of a selective cysteinyl LT receptor antagonist, montelukast, against inhaled NKA and determined LTE(4) excretion in the urine. RESULTS: Inhaled NKA in the absence of any drug treatment produced a concentration-related bronchospasm with a geometric mean provocative concentration required to produce a 15% decrease in FEV(1) from the postsaline baseline value (PC(15)) value of 290.9 microg/mL (+SE, 407.1 microg/mL; -SE, 207.84 microg/mL). Montelukast pretreatment significantly increased (P <.01) the PC(15) NKA value (708.8 microg/mL; +SE, 890.47 microg/mL; -SE, 564.15 microg/mL) in comparison with placebo (394.4 microg/mL; +SE, 491.88 microg/mL; -SE, 248.16 microg/mL) and produced a shift of the NKA concentration-response curve to the right in all the subjects studied. When compared with placebo, montelukast did not have a significant protective effect against methacholine challenge; the geometric mean PC(15) values obtained were 0.87 and 0.96 mg/mL with placebo and montelukast, respectively. Although we have not observed any increase in urinary LTE(4) excretion after NKA inhalation, we have shown that pretreatment of asthmatic subjects with montelukast elicits a significant protection against NKA-induced bronchoconstriction. CONCLUSION: In asthmatic subjects NKA-induced bronchoconstriction is indirectly caused by the release of LTs, and this mechanism could explain some of the antiasthmatic and anti-inflammatory effects of LT antagonists.