Mice (4–5 weeks old, 20–22 g) were purchased from Jackson Laboratories (Harlan Nossan, Italy). The animals were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M. 116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986).

Green tea extract (GTE) was a kind gift of Indena (Milano, Italy), and it was defined by the producer as having a polyphenolic content of 75 ± 5% with the major constituent being epigallocatechin-3-gallate at 62% and the minor ones being epicatechin-3-gallate, epigallocatechin and epicathechin.

Mice were anaesthetised with isoflurane and submitted to a skin incision at the level of the left sixth intercostals space. The underlying muscle was dissected and saline (0.1 ml) or saline containing 2%λ-carrageenan (0.1 ml) was injected into the pleural cavity. The skin incision was closed with a suture and the animals were allowed to recover. At 4 h after the injection of carrageenan, the animals were killed by inhalation of CO₂. The chest was carefully opened and the pleural cavity rinsed with 1 ml of saline solution containing heparin (5 U/ml) and indomethacin (10 μg/ml). The exudate and washing solution were removed by aspiration and the total volume measured. Any exudate, which was contaminated with blood, was discarded. The amount of exudate was calculated by subtracting the volume injected (1 ml) from the total volume recovered. The leukocytes in the exudate were suspended in phosphate-buffer saline (PBS) and counted with an optical microscope in a Burker's chamber after Blue Toluidine staining.

Mice were randomly allocated into the following groups: (i) CAR + saline group. Mice were subjected to carrageenan-induced pleurisy (N = 10), (ii) Green Tea group Same as the CAR + saline group but Green Tea (25 mg/kg i.p) were administered 1 h prior to carrageenan (N = 10), (iii) Sham+saline group. Sham-operated group in which identical surgical procedures to the CAR group was performed, except that the saline was administered instead of carrageenan (N = 10), (iv) Sham + Green Tea group. Same as the Sham+saline group but Green Tea (25 mg/kg i.p) were administered 1 h prior to carrageenan (N = 10). The doses of Green Tea used here to reduce acute lung injury have been reported by us to reduce the tissue injury caused by ischemia-reperfusion in the gut (dose-response curve study) (Muià et al submitted 2005).

Myeloperoxidase (MPO) activity, an indicator of polymorphonuclear leukocyte (PMN) accumulation, was determined as previously described 27. At 4 h after intrapleural injection of carrageenan lung tissues, were obtained and weighed. Each piece of tissue was homogenised in a solution containing 0.5% hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20,000 × g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM H₂O₂. The rate of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 μmol of peroxide min at 37°C and was expressed in mill units per gram weight of wet tissue.

TNF-α levels were evaluated in the exudates at 4 h after the induction of pleurisy by carrageenan injection. The assay was carried out by using a colorimetric, commercial ELISA kit (Calbiochem-Novabiochem Corporation, USA).

Nitrite/nitrate (NOx) production, an indicator of NO synthesis, was measured in pleural exudate. At the first nitrate in the supernatant was incubated with nitrate reductase (0.1 U/ml) and NADPH (1 mM) and FAD (50 μM) at 37°C for 15 min. Then followed another incubation with LDH (100 U/ml) and sodium pyruvate (10 mM) for 5 min. The nitrite concentration in the samples was measured by the Griess reaction, by adding 100 μl of Griess reagent (0.1% naphthylethylenediamide dihydrochloride in H₂O and 1% sulphanilamide in 5% concentrated H₂PO₄; vol. 1: 1) to 100 μl samples. The optical density at 550 nm (OD₅₅₀) was measured using ELISA microplate reader (SLT- Lab instruments Salzburg, Austria). Nitrate concentrations were calculated by comparison with OD₅₅₀ of standard solutions of sodium nitrate prepared in saline solution.

At 4 h after carrageenan administration, the lungs were fixed in 10% buffered formaldehyde and 8 μm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H₂O₂ in 60% methanol for 30 min. The sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Non-specific adsorption was minimised by incubating the section in 2% normal goat serum in phosphate buffered saline for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin. The sections were then incubated overnight with primary anti-ICAM-1 antibody (1:500), with 1:1000 dilution of primary antinitrotyrosine antibody (DBA), and anti-PAR antibody (1:500) or with control solutions. Controls included buffer alone or non-specific purified rabbit IgG.

To confirm that the immunoreaction for the nitrotyrosine was specific, some sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding specificity. To verify the binding specificity for PARS, sections were also incubated with only the primary antibody (no secondary) or with only the secondary antibody (no primary). In these situations, no positive staining was found in the sections, indicating that the immunoreaction was positive in all the experiments carried out.

Immunocytochemistry photographs (n = 5) were assessed by densitometry. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266).

Lung biopsies were taken at 4 h after injection of carrageenan. The biopsies were fixed for 1 wk in buffered formaldehyde solution (10% in PBS) at room temperature, dehydrated by graded ethanol and embedded in Paraplast (Sherwood Medical, Mahwah, N.J.). Tissue sections (thickness 7 μm) were deparaffinized with xylene, stained with trichromic Van Gieson, and studied using light microscopy (Dialux 22 Leitz). Blood was passed on the slide, fixed at 37°C, stained with May Grunward-Giensa, and studied using light microscopy.

The lung samples have been collected in liquid nitrogen and stored at -80°C until use. Nuclear extracts have been prepared according to 28 in the presence of 10 μg/ml leupeptin, 5 μg/ml antipain and pepstain, and 1 mM PMSF (Sigma-Aldrich Company, Milan, Italy). Protein concentration in the nuclear extracts was determined using the method of 29. Ten μg of nuclear extract have been incubated at room temperature for 20 min with (2–5 × 10⁴cpm) of ³²P-labeled double stranded oligonucleotide, containing the STAT-1 binding site (sis-inducible factor-binding recognition element, SIE/M67) from the c-Fos promoter (5'-GTCGACATTTCCCGTAAATCG-3'), the PARP-1 binding site (5'-TTCCTTGCCCCTCCCATTTTTC-3') from the Reg promoter 30 or the SP-1 consensus sequence (5'GGGGCGGGGC-3', Santa Cruz Biotechnology, CA) in a 15 μl of binding reaction buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 10% glycerol, 0.5 mM DTT, 0.1 mM EDTA, 2 μg poly(dI-dC), 1 μg salmon sperm DNA). Products have been fractioned on a non denaturing 5% polyacrilamide gel in TBE (Tris-Borate-EDTA buffer, 0.5X). The intensity of the retarded bands has been measured with a Phosphorimager (Molecular Dynamic, Milan, Italy).

Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company (Milan, Italy). Primary monoclonal ICAM-1 (CD54) for immunoistochemistry was purchases by Pharmingen. Reagents and secondary and nonspecific IgG antibody for immunohistochemical analysis were from Vector Laboratories InC. Primary monoclonal anti-poly (ADP-ribose) antibody was purchased by Alexis. All other chemicals were of the highest commercial grade available. All stock solutions were prepared in non pyrogenic saline (0.9% NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, U.K.).

All values in the figures and text are expressed as mean ± standard error (s.e.m.) of the mean of n observations. For the in vivo studies n represents the number of animals studied. In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments performed on different experimental days. The results were analysed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. A p-value less than 0.05 was considered significant.

Histological examination of lung sections revealed significant tissue damage (Fig. 1B). Thus, when compared with lung sections taken from saline-treated animals (Fig. 1A), histological examination of lung sections of mice treated with carrageenan showed oedema, tissue injury (Fig 1B), and infiltration of the tissue with neutrophils (PMNs) (Fig. 1B1). GTE significantly reduced the degree of injury as well as the infiltration of PMNs (Fig. 1C). Furthermore, injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterized by the accumulation of fluid (oedema) that contained large amounts of PMNs (Fig. 2A,B). Oedema and PMNs infiltration in pleural cavity were attenuated by the i.p. injection of GTE (Figs. 2A,B).

The levels of TNF-α were significantly elevated in the exudates from vehicle-treated mice at 4 h after carrageenan administration (Fig. 3). In contrast, the levels of this pro-inflammatory cytokine was significantly lower in carrageenan-treated mice treated with GTE (Fig. 3). No significant increased of TNF-α levels was observed in the exudates of sham-operated mice.

The above histological pattern of lung injury appeared to be correlated with the influx of leukocytes into the lung tissue. Therefore, we investigate the role of GTE on the neutrophils infiltration by measurement of the activity of myeloperoxidase. Myeloperoxidase activity was significantly elevated (p < 0.001) at 4 h after carrageenan administration in vehicle-treated mice (Fig. 4). In Mice treated with green tea extract lung myeloperoxidase activity was significantly reduced (p < 0.01) in comparison to those of vehicle-treated mice (Fig. 4).

Staining of lung tissue sections obtained from saline-treated mice with anti-ICAM-1 antibody showed specific staining along bronchial epithelium, demonstrating that ICAM-1 is constitutively expressed (data not shown). At 4 h after carrageenan injection, the staining intensity substantially increased along the bronchial epithelium (see arrows, Fig. 5A, 6). Sections from GTE-treated mice did not reveal any up-regulation of constitutively expressed ICAM-1, which was normally expressed in the epithelium (see arrows, Fig. 5B, 6). To verify the binding specificity for ICAM-1, some sections were also incubated with only the primary antibody (no secondary). In these situations, no positive staining was found in the sections, indicating that the immunoreaction was positive in all the experiments carried out.

The levels of NO_x were significantly (P < 0.01) increased in the exudate from carrageenan-treated mice (Fig. 7). In contrast, levels of NO_x were significantly lower in the exudate of mice treated with GTE (Fig. 7).

At 4 h after carrageenan injection, lung sections were taken in order to determine the immunohistological staining for nitrotyrosine or PARS. Sections of lung from saline-treated mice did not reveal any immunoreactivity for nitrotyrosine or PARS within the normal architecture (data not shown). A positive staining for nitrotyrosine (Fig. 6, 8A) and PARS (Fig. 6, 8C) was localized primarily in the vessels and in the bronchial epithelium. GTE reduced the staining for both nitrotyrosine (Fig. 6, 8B) and PARS (Fig. 6, 8D). Therefore, no differences between groups were shown for SP-1 DNA binding activity (data not shown). It was also shown the DNA binding capacity of PARP-1 to the promoter sequence of the Reg gene 30. The retarded bands of the carraggeenan-treated mice were reduced in comparison to those of vehicle-treated or GTE pre-treated mice (Fig. 9A, B)

To examine the molecular mechanisms responsible for mediating the anti-inflammatory effects of GTE we measured, by EMSA, the changes in activation of the transcription factors STAT-1 and SP-1. DNA-binding activity of STAT-1 was significantly elevated at 4 h after carrageenan administration in vehicle-treated mice (Fig. 10A, B). In Mice treated with green tea extract lung STAT-1 activity was similar to those of sham-operated group and significantly reduced in comparison to those of vehicle-treated mice (Fig. 10A, B).