**3. 3,4-DHPEA-EA and chronic inflammation**

*Premise* **-** Reactive oxygen species (ROS) are produced in cells by several physiological and environmental stimulations, such as infections, ultraviolet radiation and pollutants, known collectively as oxidants. Interestingly, ROS have also been considered as risk and enhancer factors for autoimmune diseases (Filippin *et al.*, 2008) as there is a significant relation between the oxidative stress and such diseases (Filippin *et al.*, 2008; Avalos *et al.*, 2007). Rheumatoid arthritis (RA) is an autoimmune disease characterized by the sequestration of various leukocyte subpopulations within both the developing pannus and synovial space. The chronic nature of this disease results in multiple joint inflammation with subsequent destruction of joint cartilage and erosion of bone. While this disease has a worldwide distribution, its pathogenesis is not clearly understood (Harris, 1990). Type II collageninduced arthritis (CIA) in the mouse has proven to be a useful model of RA, as it possesses many of the cell and humoral immunity characteristics found in human RA (Holmdahl *et al.*, 1990). The pathogenesis of CIA is dependent upon the host's response to type II collagen challenge and the subsequent generation of antibodies that recognizes collagen rich joint tissue (Holmdahl *et al.*, 1990). The chronic activities initiated by immune complexes trigger a variety of cell-mediated and humoral events. Moreover, the recruitment and activation of neutrophils, macrophages, and lymphocytes into joint tissues and the formation of the pannus are hallmarks of the pathogenesis of both CIA and human RA. Recently, it has been demonstrated that interleukin (IL)-8, macrophage inflammatory protein (MIP)-lα, MIP-1β, and RANTES are differentially chemotactic for lymphocyte subsets (Taub *et al.*, 1993). Chemokines may play prominent roles in RA, as neutrophil and mononuclear cell stimulation and activation are prevalent in this disease. Concomitant with inflammation is the generation of ROS (Trichopoulou *et al.*, 2003)which increase oxidation of proteins and lipids, resulting in signals that trigger more inflammation. Thus, the study model was designed to evaluate the effects of 3,4-DHPEA-EA in a mice model of chronic inflammation (development of CIA in the mice). We have evaluated the following endpoints of the inflammatory process: (1) clinical score; (2) body weight; (3) inducible oxide nitric synthase (iNOS) and cyclooxygenase expression (COX-2); (4) nitrotyrosine formation and activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP); (5) cytokine and chemokines production; (6) neutrophil infiltration; (7) joint histopathology.

298 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

therapy of acute inflammation.

**3. 3,4-DHPEA-EA and chronic inflammation** 

MPO in the inflamed tissue (de la Puerta *et al.*, 2000). Various studies have demonstrated that PARP activation after single DNA strand breakage induced by ROS plays an important role in the process of acute lung injury (Szabo *et al.*, 1998). In this study we confirm the increase in PAR formation in the lung tissues from carrageenan-treated mice as well as that 3,4-DHPEA-EA treatment attenuates PARP activation. In this regard, several studies demonstrated that hydroxytyrosol, a hydrolysis product of 3,4-DHPEA-EA, also exerts an inhibitory effect on peroxynitrite-dependent DNA base modifications and tyrosine nitration (Deiana *et al.*, 1999). Similarly, Salvini *et al.* (2006) showed a 30% reduction of oxidative DNA damage in peripheral blood lymphocytes during intervention on postmenopausal women with virgin olive oil containing high amounts of phenols. In conclusion, concomitant with inflammation is the generation of free radicals, which increase oxidation of proteins and lipids, resulting in signals that trigger more inflammation. Taken together, the results of the present study enhance our understanding of the role of ROS generation in the pathophysiology of carrageenan-induced pleurisy implying that olive oil compounds such as 3,4-DHPEA-EA may be useful in the

*Premise* **-** Reactive oxygen species (ROS) are produced in cells by several physiological and environmental stimulations, such as infections, ultraviolet radiation and pollutants, known collectively as oxidants. Interestingly, ROS have also been considered as risk and enhancer factors for autoimmune diseases (Filippin *et al.*, 2008) as there is a significant relation between the oxidative stress and such diseases (Filippin *et al.*, 2008; Avalos *et al.*, 2007). Rheumatoid arthritis (RA) is an autoimmune disease characterized by the sequestration of various leukocyte subpopulations within both the developing pannus and synovial space. The chronic nature of this disease results in multiple joint inflammation with subsequent destruction of joint cartilage and erosion of bone. While this disease has a worldwide distribution, its pathogenesis is not clearly understood (Harris, 1990). Type II collageninduced arthritis (CIA) in the mouse has proven to be a useful model of RA, as it possesses many of the cell and humoral immunity characteristics found in human RA (Holmdahl *et al.*, 1990). The pathogenesis of CIA is dependent upon the host's response to type II collagen challenge and the subsequent generation of antibodies that recognizes collagen rich joint tissue (Holmdahl *et al.*, 1990). The chronic activities initiated by immune complexes trigger a variety of cell-mediated and humoral events. Moreover, the recruitment and activation of neutrophils, macrophages, and lymphocytes into joint tissues and the formation of the pannus are hallmarks of the pathogenesis of both CIA and human RA. Recently, it has been demonstrated that interleukin (IL)-8, macrophage inflammatory protein (MIP)-lα, MIP-1β, and RANTES are differentially chemotactic for lymphocyte subsets (Taub *et al.*, 1993). Chemokines may play prominent roles in RA, as neutrophil and mononuclear cell stimulation and activation are prevalent in this disease. Concomitant with inflammation is the generation of ROS (Trichopoulou *et al.*, 2003)which increase oxidation of proteins and lipids, resulting in signals that trigger more inflammation. Thus, the study model was designed to evaluate the effects of 3,4-DHPEA-EA in a mice model of chronic inflammation *Animals* **-** DBA/1J mice (9 weeks, Harlan Nossan, Italy) were used for these studies. 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 (Dlgs 116/92) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986).

*Experimental Design -* Mice were divided into the following four experimental groups: (i) CIA-Control; mice were subjected to collagen-induced arthritis (as described below) and administered 200 µl of 10% ethanol solution (i.p., vehicle for 3,4-DHPEA-EA) every 24 h, starting from day 25 (n = 20); (ii) CIA-3,4-DHPEA-EA; mice subjected to collagen-induced arthritis (as described below) were administered 3,4-DHPEA-EA (40 µM/kg, i.p.) every 24 h, starting from day 25 (n = 20); (iii) Sham-Control; mice subjected to an intradermal injection at the base of the tail of 100 µl of 0.01 M acetic acid instead of the emulsion containing 100 µg of CII, were treated with 200 µl of 10% ethanol solution (i.p., vehicle for 3,4-DHPEA-EA), every 24 h starting from day 25 (n = 20); (iv) Sham-3,4-DHPEA-EA; mice subjected to an intradermal injection at the base of the tail of 100 µl of 0.01 M acetic acid instead of the emulsion containing 100 µg of CII, were administered 3,4-DHPEA-EA (40 µM/kg, i.p.), every 24 h starting from day 25 (n = 20). The dose of 3,4-DHPEA-EA used here to reduce joint injury was chosen based on a previous study (Procopio A, *et al.*, 2009). Collageninduced arthritis (CIA) is induced in mice by two consecutive (interval 21 days) intradermal injection of 100 µl of the emulsion (containing 100 µg of bovine type II collagen) (CII) and complete Freund's adjuvant (CFA) at the base of the tail. Mice develop erosive hind paw arthritis with macroscopic clinical evidence of CIA as peri-articular erythema and edema in the hind paws. The incidence of CIA is 100% by day 27 in the CII challenged and the severity of CIA progressed over a 35-day period with a reabsorption of bone. The histopathology of CIA include erosion of the cartilage at the joint.

*Induction of CIA* - Bovine CII was dissolved in 0.01 M acetic acid at a concentration of 2 mg/ml by stirring overnight at 4°C. Dissolved CII was frozen at -70ºC until use. Complete Freund's adjuvant (CFA) was prepared by addition of Mycobacterium tuberculosis H37Ra at a concentration of 5 mg/ml. Before injection, CII was emulsified with an equal volume of CFA. CIA was induced as previously described (Szabo *et al.*, 1998). On day 1, mice were injected intradermally at the base of the tail with 100 µl of the emulsion containing 100 µg of CII. On day 21, a second injection of CII in CFA was administered.

*Clinical assessment of CIA -* The development of arthritis in mice in all experimental groups was evaluated daily starting from day 20 after the first intradermal injection by using a macroscopic scoring system: 0 = no signs of arthritis; 1 = swelling and/or redness of the paw or one digit; 2 = two joints involved; 3 = more than two joints involved; and 4 = severe arthritis of

the entire paw and digits (Szabo *et al.*, 1998). Arthritic index for each mouse was calculated by adding the four scores of individual paws. Clinical severity was also determined by quantitating the change in the paw volume using plethysmometry (model 7140; Ugo Basile).

Oleuropein an Olive Oil Compound in Acute and Chronic Inflammation Models: Facts and Perspectives 301

analyzed. The extracts usually contained 0.2-1.5 mg protein/ml, as measured by protein assay kit (Pierce Chemical Co., Rockford, IL). The levels of MIP-1α and MIP-2 were quantified using a modification of a double ligand method, as previously described (Kasama *et al.*, 1994). Briefly, flat-bottomed 96- well microtiter plates were coated with 50 µl/well of rabbit anti-cytokine antibodies (1 µg/ml in 0.6 mol/liter NaCl, 0.26 mol/liter H3BO4 and 0.08 N NaOH, pH 9.6) for 16 h at 4°C, and then washed with PBS, pH 7.5, 0.05% Tween 20 (wash buffer). Nonspecific binding sites on microtiter plates were blocked with 2% BSA in PBS and incubated for 90 min at 37°C. Plates were rinsed four times with wash buffer, and diluted aqueous joint samples (50 µl) were added, followed by incubation for 1 h at 37°C. After washing of plates, chromogen substrate was added. The plates were incubated at room temperature to the desired extinction, after which the reaction was terminated with 50 µl/well of 3 M H2SO4 solution. The plates were then read at 490 nm in an ELISA reader.

*Myeloperoxidase (MPO) assay -* Neutrophil infiltration to the inflamed joints was indirectly quantitated using an MPO assay, as previously described for neutrophil elicitation (Mullane *et al.*, 1985). Tissue was prepared as described above and placed in a 50 mM phosphate buffer (pH = 6.0) with 5% hexadecyltrimethyl ammonium bromide (Sigma Chemical Co.). Joint tissues were homogenized, sonicated, and centrifuged at 12,000 *g* for 15 min at 4ºC. Supernatants were assayed for MPO activity using a spectrophotometric reaction with O-

*Materials -* 3,4-DHPEA-EA was obtained from Merck Biosciences (Calbiochem, Beecham, Nottingham, UK). Unless otherwise stated, other compounds were obtained from Sigma-Aldrich Company (Milan, Italy). All chemicals were of the highest commercial grade available. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter

*Data analysis -* 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 (histological or immunohistochemistry coloration) performed on different experimental days on the tissue sections collected from all the animals in each group. Data sets were examined by one- or two-way analysis of variance, and individual group means were then compared with *Student*'s unpaired *t* test. For the arthritis studies, Mann-Whitney U test (two-tailed, independent) was used to compare medians of the arthritic indices (Szabo *et al.*, 1998). A *p*-

*Effect of 3,4-DHPEA-EA on joint injury during experimental arthritis -* To imitate the clinical scenario of RA, mice were subjected to CIA. CIA developed rapidly in mice immunized with CII and clinical signs (periarticular erythema and edema) (Fig. 6B) of the disease first appeared in hind paws between 24 and 26 days post-challenge (Fig. 6D) leading to a 100% incidence of

This ELISA method consistently had a sensitivity limit of ~30 pg/ml.

dianisidine hydrochloride (Sigma Chemical Co.) at 460 nm.

value of less than 0.05 was considered significant.

**3.1. Results** 

Healthcare Ltd., Thetford, Norfolk, U.K.) or 10% ethanol (Sigma-Aldrich).

*Histological examination -* On day 35, animals were sacrificed while they were under anesthesia (sodium pentobarbital, 45 mg/kg, i.p), and paws and knees were removed and fixed in 10% formalin. The paws were then trimmed, placed in decalcifying solution for 24 h, embedded in paraffin, sectioned at 5 µm, stained with hematoxylin/eosin and Masson's trichrome stain and studied using light microscopy (Dialux 22 Leitz).

*Immunohistochemical localization of nitrotyrosine, Poly ADP Ribose (PAR), iNOS, and COX-2 -* On day 35, the joints were trimmed, placed in decalcifying solution for 24 h 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 minimized 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. Sections were incubated overnight with 1) anti-rabbit polyclonal antibody directed at iNOS (1:1000 in PBS, v/v) (DBA, Milan, Italy) or 2) anti-COX-2 goat polyclonal antibody (1:500 in PBS, v/v) or 3) anti- nitrotyrosine rabbit polyclonal antibody (1:1000 in PBS, v/v) or 4) with anti-PAR goat polyclonal antibody rat (1:500 in PBS, v/v) or 5). Controls included buffer alone or non-specific purified rabbit IgG. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG (for nitrotyrosine and iNOS) or with a biotin-conjugated goat anti-rabbit IgG (for PAR and COX-2) and avidin-biotin peroxidase complex. In order 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 PAR, COX-2 and iNOS, some 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 by using Optilab Graftek software on a Macintosh personal computer.

*Measurement of cytokines* **-** Tumor Necrosis Factor-α (TNF-α) levels were evaluated in the plasma from CIA and sham mice as previously described (Cuzzocrea *et al.*, 2006). The assay was carried out using a colorimetric commercial ELISA kit (Calbiochem-Novabiochem Co., Milan, Italy) with a lower detection limit of 10 pg/ml.

*Measurement of chemokines -* Levels of chemokines MIP-1α and MIP-2 were measured in the aqueous joint extracts. Briefly, joint tissues were prepared by first removing the skin and separating the limb below the ankle joint. Joint tissues were homogenized on ice in 3 ml lysis buffer (PBS containing: 2 mM PMSF, and 0,1 mg/ml [final concentration], each of aprotinin, antipain, leupeptin, and pepstatin A) using Polytron (Brinkinarm Instr., Westbury, NY). The homogenized tissues were then centrifuged at 2,000 g for 10 min. Supernatant were sterilized with a millipore filter (0.2 µm) and stored at –80oC until analyzed. The extracts usually contained 0.2-1.5 mg protein/ml, as measured by protein assay kit (Pierce Chemical Co., Rockford, IL). The levels of MIP-1α and MIP-2 were quantified using a modification of a double ligand method, as previously described (Kasama *et al.*, 1994). Briefly, flat-bottomed 96- well microtiter plates were coated with 50 µl/well of rabbit anti-cytokine antibodies (1 µg/ml in 0.6 mol/liter NaCl, 0.26 mol/liter H3BO4 and 0.08 N NaOH, pH 9.6) for 16 h at 4°C, and then washed with PBS, pH 7.5, 0.05% Tween 20 (wash buffer). Nonspecific binding sites on microtiter plates were blocked with 2% BSA in PBS and incubated for 90 min at 37°C. Plates were rinsed four times with wash buffer, and diluted aqueous joint samples (50 µl) were added, followed by incubation for 1 h at 37°C. After washing of plates, chromogen substrate was added. The plates were incubated at room temperature to the desired extinction, after which the reaction was terminated with 50 µl/well of 3 M H2SO4 solution. The plates were then read at 490 nm in an ELISA reader. This ELISA method consistently had a sensitivity limit of ~30 pg/ml.

*Myeloperoxidase (MPO) assay -* Neutrophil infiltration to the inflamed joints was indirectly quantitated using an MPO assay, as previously described for neutrophil elicitation (Mullane *et al.*, 1985). Tissue was prepared as described above and placed in a 50 mM phosphate buffer (pH = 6.0) with 5% hexadecyltrimethyl ammonium bromide (Sigma Chemical Co.). Joint tissues were homogenized, sonicated, and centrifuged at 12,000 *g* for 15 min at 4ºC. Supernatants were assayed for MPO activity using a spectrophotometric reaction with Odianisidine hydrochloride (Sigma Chemical Co.) at 460 nm.

*Materials -* 3,4-DHPEA-EA was obtained from Merck Biosciences (Calbiochem, Beecham, Nottingham, UK). Unless otherwise stated, other compounds were obtained from Sigma-Aldrich Company (Milan, Italy). All chemicals were of the highest commercial grade available. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, U.K.) or 10% ethanol (Sigma-Aldrich).

*Data analysis -* 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 (histological or immunohistochemistry coloration) performed on different experimental days on the tissue sections collected from all the animals in each group. Data sets were examined by one- or two-way analysis of variance, and individual group means were then compared with *Student*'s unpaired *t* test. For the arthritis studies, Mann-Whitney U test (two-tailed, independent) was used to compare medians of the arthritic indices (Szabo *et al.*, 1998). A *p*value of less than 0.05 was considered significant.
