**2.2. Results**

*Effects of 3,4-DHPEA-EA on carrageenan-induced pleurisy –* When compared to lung sections taken from saline-treated animals (sham group Fig. 2A, see histological score 2D) histological examination of lung sections taken from mice treated with carrageenan revealed significant tissue damage and edema (Fig. 2B, see histological score 2D) as well as infiltration of neutrophils (PMNs) within the tissues (Fig. 2B). 3,4-DHPEA-EA (100 µM/kg) reduced the degree of lung injury (Fig. 2C, see histological score 2D). The pleural infiltration with PMN appeared to correlate with an influx of leukocytes into the lung tissue, thus we investigated the effect of 3,4-DHPEA-EA on neutrophil infiltration by measurement of MPO activity. MPO activity was significantly elevated at 4 h after carrageenan administration in vehicle-treated mice (Fig. 2E). Treatment with 3,4-DHPEA-EA significantly attenuated neutrophil infiltration into the lung tissue (Fig. 2E).

*Effects of 3,4-DHPEA-EA on the expression of adhesion molecules (ICAM-1, P-selectin) -* Staining of lung tissue sections obtained from saline-treated mice with anti-ICAM-1 antibody showed a specific staining along bronchial epithelium demonstrating that ICAM-1 is constitutively expressed (Fig. 3A). No positive staining for P-selectin was found in lung tissue sections from saline-treated mice (Fig. 3D). At 4 h after carrageenan injection, the ICAM-1 staining intensity increased in the vascular endothelium (Fig. 3B). Lung tissue sections obtained from carrageenan-treated mice showed positive staining for P-selectin localized in the vessels (Fig. 3E). No positive staining for ICAM-1 or P-selectin was observed in the lungs of carrageenan-treated mice treated with 3,4-DHPEA-EA (Fig. 3C and 3F respectively).

*Effects of 3,4-DHPEA-EA on the release of pro-inflammatory cytokine and nitrite–nitrate concentration -* When compared to sham animals, injection of carrageenan resulted in an increase in the levels of TNF-α and IL-1β in the pleural exudates (Fig. 4A,B). The release of TNF-α and IL-1β was significantly attenuated by treatment with 3,4-DHPEA-EA (Fig. 4A,B).

neutrophil infiltration into the lung tissue (Fig. 2E).

**2.2. Results** 

respectively).

4A,B).

hydrolysis of oleuropein extracted from olive leaves by means the patented method reported by Procopio *et al.* (2009). All other chemicals were of the highest commercial grade available. All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; Baxter, Italy, UK).

*Statistical evaluation* **-** 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. The results were analyzed by one-way ANOVA followed by a Bonferroni post-hoc test for multiple comparisons. A *p*-value less than 0.05 were considered significant and individual group means were then compared

with Student's unpaired t test. A *p*-value of less than 0.05 was considered significant.

*Effects of 3,4-DHPEA-EA on carrageenan-induced pleurisy –* When compared to lung sections taken from saline-treated animals (sham group Fig. 2A, see histological score 2D) histological examination of lung sections taken from mice treated with carrageenan revealed significant tissue damage and edema (Fig. 2B, see histological score 2D) as well as infiltration of neutrophils (PMNs) within the tissues (Fig. 2B). 3,4-DHPEA-EA (100 µM/kg) reduced the degree of lung injury (Fig. 2C, see histological score 2D). The pleural infiltration with PMN appeared to correlate with an influx of leukocytes into the lung tissue, thus we investigated the effect of 3,4-DHPEA-EA on neutrophil infiltration by measurement of MPO activity. MPO activity was significantly elevated at 4 h after carrageenan administration in vehicle-treated mice (Fig. 2E). Treatment with 3,4-DHPEA-EA significantly attenuated

*Effects of 3,4-DHPEA-EA on the expression of adhesion molecules (ICAM-1, P-selectin) -* Staining of lung tissue sections obtained from saline-treated mice with anti-ICAM-1 antibody showed a specific staining along bronchial epithelium demonstrating that ICAM-1 is constitutively expressed (Fig. 3A). No positive staining for P-selectin was found in lung tissue sections from saline-treated mice (Fig. 3D). At 4 h after carrageenan injection, the ICAM-1 staining intensity increased in the vascular endothelium (Fig. 3B). Lung tissue sections obtained from carrageenan-treated mice showed positive staining for P-selectin localized in the vessels (Fig. 3E). No positive staining for ICAM-1 or P-selectin was observed in the lungs of carrageenan-treated mice treated with 3,4-DHPEA-EA (Fig. 3C and 3F

*Effects of 3,4-DHPEA-EA on the release of pro-inflammatory cytokine and nitrite–nitrate concentration -* When compared to sham animals, injection of carrageenan resulted in an increase in the levels of TNF-α and IL-1β in the pleural exudates (Fig. 4A,B). The release of TNF-α and IL-1β was significantly attenuated by treatment with 3,4-DHPEA-EA (Fig.

**Figure 2.** Effect of 3,4-DHPEA-EA (Ole aglycone) on histological alterations of lung tissue 4 h after carrageenan-induced injury and on PMN infiltration in the lung. Lung sections taken from carrageenantreated mice treated with vehicle demonstrated edema, tissue injury (B) as well as infiltration of the tissue with neutrophils (B). Carrageenan-treated animals treated with 3,4-DHPEA-EA (C) demonstrated reduced lung injury and neutrophil infiltration. Section from sham animals demonstrating the normal architecture of the lung tissue (A). The histological score (D) was made by an independent observer. MPO activity, index of PMN infiltration, was significantly elevated at 4 h after carrageenan (CAR) administration in vehicle-treated mice (E), if compared with sham mice (E). 3,4- DHPEA-EA significantly reduced MPO activity in the lung (E). The figure is representative of at least 3 experiments performed on different experimental days. Data are expressed as mean ± S.E.M. from n = 10 mice for each group. \*\*, P < 0.01 versus sham group. ##, P < 0.01 versus carrageenan.

No significant increase of TNF-α and IL-1β exudates levels was found in the sham animal (Fig. 4A,B). NO levels were also significantly increased in the exudate obtained from mice administered carrageenan (Fig. 4C). Treatment of mice with 3,4-DHPEA-EA significantly reduced NO exudates levels (Fig. 4C). No significant increase of NO exudates levels was found in the sham animal (Fig. 4C).

Effects of 3,4-DHPEA-EA on carrageenan-induced nitrotyrosine formation, lipid peroxidation and poly-ADP-ribosyl polymerase (PARP) activation - Immunohistochemical analysis of lung sections obtained from mice treated with carrageenan revealed positive staining for nitrotyrosine (Fig. 5B). In contrast, no positive staining for nitrotyrosine was found in the lungs of carrageenan-treated mice, which had been treated with 3,4-DHPEA-EA (100 µM/kg) (Fig. 5C). In addition, at 4 hours after carrageenan-induced pleurisy, MDA levels were also measured in the lungs as an indicator of lipid peroxidation. As shown in Figure 5D, MDA levels were significantly increased in the lungs of carrageenan-treated mice. Lipid peroxidation was significantly attenuated by the intraperitoneal injection of 3,4-DHPEA-EA (Fig. 5D). At the same time point (4 h after carrageenan administration),

lung tissue sections were taken in order to determine the immunehistological staining for poly ADP-ribosylated proteins (an indicator of PARP activation). A positive staining for the PAR (Fig. 5F) was found primarily localized in the inflammatory cells present in the lung tissue from carrageenan-treated mice. 3,4-DHPEA-EA treatment reduced the degree of PARP activation (Fig. 5G). Please note that there was no staining for either nitrotyrosine (Fig. 5A) or PAR (Fig. 5E) in lung tissues obtained from the sham group of mice.

Oleuropein an Olive Oil Compound in Acute and Chronic Inflammation Models: Facts and Perspectives 295

**Figure 4.** Effect of 3,4-DHPEA-EA (Ole aglycone) on carrageenan-induced pro-inflammatory cytokine release and NO formation in the lung. TNF-α and Il-1β levels were significantly elevated at 4 h after carrageenan administration in vehicle-treated mice (A and B respectively), if compared with sham mice

respectively). Moreover nitrite and nitrate levels, stable NO metabolites, were significantly increased in the pleural exudates at 4 h after carrageenan administration (C) if compared with sham mice (C). 3,4- DHPEA-EA significantly reduced the carrageenan-induced elevation of nitrite and nitrate exudates levels (C). Data are expressed as mean ± S.E.M. from n 10 mice for each group. \*\*, P < 0.01 versus sham

(A and B respectively). 3,4-DHPEA-EA significantly reduced TNF-α and Il-1β levels (A and B

group. ##, P < 0.01 versus carrageenan.

**Figure 3.** Effect of 3,4-DHPEA-EA (Ole aglycone) on the immunohistochemical localization of ICAM-1 and P-selectin in the lung after carrageenan injection. No positive staining for ICAM-1 was observed in lung sections taken from sham mice (A). Lung sections taken from carrageenan-treated mice showed intense positive staining for ICAM-1 along the vessels (B). The degree of positive staining for ICAM-1 was markedly reduced in lung sections obtained from mice treated with 3,4-DHPEA-EA (C). No positive staining for P-selectin was observed in lung sections taken from sham mice (D). Lung sections taken from carrageenan-treated mice treated with vehicle showed intense positive staining for Pselectin along the vessels (E). The degree of positive staining for P-selectin was markedly reduced in tissue sections obtained from mice treated with 3,4-DHPEA-EA (F). The figure is representative of at least three experiments performed on different experimental days.

mice.

lung tissue sections were taken in order to determine the immunehistological staining for poly ADP-ribosylated proteins (an indicator of PARP activation). A positive staining for the PAR (Fig. 5F) was found primarily localized in the inflammatory cells present in the lung tissue from carrageenan-treated mice. 3,4-DHPEA-EA treatment reduced the degree of PARP activation (Fig. 5G). Please note that there was no staining for either nitrotyrosine (Fig. 5A) or PAR (Fig. 5E) in lung tissues obtained from the sham group of

**Figure 3.** Effect of 3,4-DHPEA-EA (Ole aglycone) on the immunohistochemical localization of ICAM-1 and P-selectin in the lung after carrageenan injection. No positive staining for ICAM-1 was observed in lung sections taken from sham mice (A). Lung sections taken from carrageenan-treated mice showed intense positive staining for ICAM-1 along the vessels (B). The degree of positive staining for ICAM-1 was markedly reduced in lung sections obtained from mice treated with 3,4-DHPEA-EA (C). No positive staining for P-selectin was observed in lung sections taken from sham mice (D). Lung sections taken from carrageenan-treated mice treated with vehicle showed intense positive staining for Pselectin along the vessels (E). The degree of positive staining for P-selectin was markedly reduced in tissue sections obtained from mice treated with 3,4-DHPEA-EA (F). The figure is representative of at

least three experiments performed on different experimental days.

**Figure 4.** Effect of 3,4-DHPEA-EA (Ole aglycone) on carrageenan-induced pro-inflammatory cytokine release and NO formation in the lung. TNF-α and Il-1β levels were significantly elevated at 4 h after carrageenan administration in vehicle-treated mice (A and B respectively), if compared with sham mice (A and B respectively). 3,4-DHPEA-EA significantly reduced TNF-α and Il-1β levels (A and B respectively). Moreover nitrite and nitrate levels, stable NO metabolites, were significantly increased in the pleural exudates at 4 h after carrageenan administration (C) if compared with sham mice (C). 3,4- DHPEA-EA significantly reduced the carrageenan-induced elevation of nitrite and nitrate exudates levels (C). Data are expressed as mean ± S.E.M. from n 10 mice for each group. \*\*, P < 0.01 versus sham group. ##, P < 0.01 versus carrageenan.

Oleuropein an Olive Oil Compound in Acute and Chronic Inflammation Models: Facts and Perspectives 297

All of the above findings are in support of the view that 3,4-DHPEA-EA attenuates the degree of acute inflammation in the mouse. What, then, is the mechanism by which ole reduces acute inflammation? One consequence of increased oxidative stress is the activation and inactivation of redox-sensitive proteins (Bowie & O'Neill, 2000). Recent studies have observed that the acute consumption of olive oil decreased the activation of NF- κB system on mononuclear cells from healthy men (Perez-Martinez *et al.*, 2007) and that 3,4-DHPEA-EA, trans-resveratrol, and hydroxytyrosol incubated with human umbilical vein endothelial cells inhibit LPS-triggered NF- κB and AP-1 activation (Carluccio *et al.*, 2003). Moreover, various experimental evidence have clearly suggested that NF- κB plays a central role in the regulation of many genes responsible for the generation of mediators or proteins in acute lung inflammation associated with carrageenan administration (Cuzzocrea *et al.*, 2006) such us TNF-α, IL-1β, nitric oxide synthase inducible (iNOS) and COX-2. By inhibiting the activation of NF- κB, the production of joint destructive inflammatory mediators may be reduced as well. In this regard, Miles *et al.*, demonstrated that ole glycoside significantly decreased the concentration of IL-1β in LPSstimulated human whole blood cultures. Therefore, this study also demonstrates that 3,4- DHPEA-EA attenuates the TNF-α and IL-1β production in the lung of carrageenan-treated mice. In addition, recent studies also showed that the potential cardioprotective activity of oleuropein in acute cardiotoxicity induced by doxorubicin treatment was determined in vivo in rats (Andreadou *et al.*, 2007) by inhibiting lipid peroxidation products, decreasing oxidative stress and reducing iNOS in cardiomyocytes. We show here that NO levels, evaluated as NO2/NO3 and MDA levels which is the products of lipid peroxidation, were increased at 4h after carrageenan injection while 3,4-DHPEA-EA decreased the levels of NO and MDA. For many years, much attention has been paid to the effects of NO in respiratory diseases 26 but recently, the focus has been shifted toward RNS in general, and to peroxynitrite (ONOO−) in particular (Sadeghi-Hashjin *et al.*, 1998). To probe the pathological contributions of ONOO− to acute lung injury we have used the appearance of nitrotyrosine staining in the inflamed tissue. We have observed here that the immunoassaying of nitrotyrosine is reduced in the lung of carrageenan-treated mice and treated with 3,4-DHPEA-EA. Therefore, the inhibition of nitrotyrosine formation by oleuropein described in the present study is most likely attributed to the strong antioxidant activity of ole. During inflammation initiation, circulating leukocytes must first be able to adhere selectively and efficiently to vascular endothelium. This process is facilitated by induction of vascular cell adhesion molecules on the inflamed endothelium, such as vascular cell adhesion molecule VCAM-1, ICAM-1, E-selectin demonstrated that 3,4- DHPEA-EA was a more potent inhibitor of adhesion molecule expression on cultured human endothelial cells than was the glycoside(Carluccio *et al.*, 2003). Furthermore, the absence of an increased expression of the adhesion molecule in the lung from CAR mice treated with 3,4- DHPEA-EA was correlated with the reduction of leukocyte infiltration as assessed by the specific granulocyte enzyme MPO and with the attenuation of the lung tissue damage as evaluated by histological examination. Several studies also showed that in the auricular edema induced by either arachidonic acid (AA) or 12-O-tetradecanoylphorbol acetate (TPA), the topical application of the olive oil compounds such as ole also produced an inhibition of the

**2.3. Discussion** 

**Figure 5.** Effect of 3,4-DHPEA-EA (Ole aglycone) on carrageenan-induced nitrotyrosine formation, lipid peroxidation and PARP activation in the lung. No staining for nitrotyrosine is present in lung section from sham mice (A). Lung sections taken from carrageenan-treated mice treated with vehicle showed positive staining for nitrotyrosine, localized mainly in inflammatory cells (B). There was a marked reduction in the immunostaining for nitrotyrosine in the lungs of carrageenan-treated mice treated with 3,4-DHPEA-EA (C). Malondialdehyde (MDA) levels, an index of lipid peroxidation, were significantly increased in lung tissues 4 h after carrageenan administration (D), if compared with lung from sham mice (D). 3,4-DHPEA-EA significantly reduced the carrageenan-induced elevation of MDA tissues levels (D). Lung sections taken from carrageenan-treated mice showed positive staining for PAR (F). There was a marked reduction in the immunostaining for PAR in the lungs of carrageenan-treated mice treated with 3,4-DHPEA-EA (G). Lung section from sham mice showed no staining for PAR (E). The figure is representative of at least 3 experiments performed on different experimental days. Data are expressed as mean ± S.E.M. from n 10 mice for each group. \*\*, P < 0.01 versus sham group. ##, P < 0.01 versus carrageenan.

#### **2.3. Discussion**

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

**Figure 5.** Effect of 3,4-DHPEA-EA (Ole aglycone) on carrageenan-induced nitrotyrosine formation, lipid peroxidation and PARP activation in the lung. No staining for nitrotyrosine is present in lung section from sham mice (A). Lung sections taken from carrageenan-treated mice treated with vehicle showed positive staining for nitrotyrosine, localized mainly in inflammatory cells (B). There was a marked reduction in the immunostaining for nitrotyrosine in the lungs of carrageenan-treated mice treated with 3,4-DHPEA-EA (C). Malondialdehyde (MDA) levels, an index of lipid peroxidation, were significantly increased in lung tissues 4 h after carrageenan administration (D), if compared with lung from sham mice (D). 3,4-DHPEA-EA significantly reduced the carrageenan-induced elevation of MDA tissues levels (D). Lung sections taken from carrageenan-treated mice showed positive staining for PAR (F). There was a marked reduction in the immunostaining for PAR in the lungs of carrageenan-treated mice treated with 3,4-DHPEA-EA (G). Lung section from sham mice showed no staining for PAR (E). The figure is representative of at least 3 experiments performed on different experimental days. Data are expressed as mean ± S.E.M. from n 10

mice for each group. \*\*, P < 0.01 versus sham group. ##, P < 0.01 versus carrageenan.

All of the above findings are in support of the view that 3,4-DHPEA-EA attenuates the degree of acute inflammation in the mouse. What, then, is the mechanism by which ole reduces acute inflammation? One consequence of increased oxidative stress is the activation and inactivation of redox-sensitive proteins (Bowie & O'Neill, 2000). Recent studies have observed that the acute consumption of olive oil decreased the activation of NF- κB system on mononuclear cells from healthy men (Perez-Martinez *et al.*, 2007) and that 3,4-DHPEA-EA, trans-resveratrol, and hydroxytyrosol incubated with human umbilical vein endothelial cells inhibit LPS-triggered NF- κB and AP-1 activation (Carluccio *et al.*, 2003). Moreover, various experimental evidence have clearly suggested that NF- κB plays a central role in the regulation of many genes responsible for the generation of mediators or proteins in acute lung inflammation associated with carrageenan administration (Cuzzocrea *et al.*, 2006) such us TNF-α, IL-1β, nitric oxide synthase inducible (iNOS) and COX-2. By inhibiting the activation of NF- κB, the production of joint destructive inflammatory mediators may be reduced as well. In this regard, Miles *et al.*, demonstrated that ole glycoside significantly decreased the concentration of IL-1β in LPSstimulated human whole blood cultures. Therefore, this study also demonstrates that 3,4- DHPEA-EA attenuates the TNF-α and IL-1β production in the lung of carrageenan-treated mice. In addition, recent studies also showed that the potential cardioprotective activity of oleuropein in acute cardiotoxicity induced by doxorubicin treatment was determined in vivo in rats (Andreadou *et al.*, 2007) by inhibiting lipid peroxidation products, decreasing oxidative stress and reducing iNOS in cardiomyocytes. We show here that NO levels, evaluated as NO2/NO3 and MDA levels which is the products of lipid peroxidation, were increased at 4h after carrageenan injection while 3,4-DHPEA-EA decreased the levels of NO and MDA. For many years, much attention has been paid to the effects of NO in respiratory diseases 26 but recently, the focus has been shifted toward RNS in general, and to peroxynitrite (ONOO−) in particular (Sadeghi-Hashjin *et al.*, 1998). To probe the pathological contributions of ONOO− to acute lung injury we have used the appearance of nitrotyrosine staining in the inflamed tissue. We have observed here that the immunoassaying of nitrotyrosine is reduced in the lung of carrageenan-treated mice and treated with 3,4-DHPEA-EA. Therefore, the inhibition of nitrotyrosine formation by oleuropein described in the present study is most likely attributed to the strong antioxidant activity of ole. During inflammation initiation, circulating leukocytes must first be able to adhere selectively and efficiently to vascular endothelium. This process is facilitated by induction of vascular cell adhesion molecules on the inflamed endothelium, such as vascular cell adhesion molecule VCAM-1, ICAM-1, E-selectin demonstrated that 3,4- DHPEA-EA was a more potent inhibitor of adhesion molecule expression on cultured human endothelial cells than was the glycoside(Carluccio *et al.*, 2003). Furthermore, the absence of an increased expression of the adhesion molecule in the lung from CAR mice treated with 3,4- DHPEA-EA was correlated with the reduction of leukocyte infiltration as assessed by the specific granulocyte enzyme MPO and with the attenuation of the lung tissue damage as evaluated by histological examination. Several studies also showed that in the auricular edema induced by either arachidonic acid (AA) or 12-O-tetradecanoylphorbol acetate (TPA), the topical application of the olive oil compounds such as ole also produced an inhibition of the

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 therapy of acute inflammation.

Oleuropein an Olive Oil Compound in Acute and Chronic Inflammation Models: Facts and Perspectives 299

(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

*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

*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

*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

*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

production; (6) neutrophil infiltration; (7) joint histopathology.

histopathology of CIA include erosion of the cartilage at the joint.

CII. On day 21, a second injection of CII in CFA was administered.

regulations (O.J. of E.C. L 358/1 12/18/1986).
