**2.1.2 Biochemical markers**

Numerous studies have suggested an important role of oxidative stress (OS) in the pathogenesis of RA (Bauerova & Bezek, 1999; Bohanec et al., 2009). Several clinical studies as well as preclinical studies using animal models of RA have documented an imbalance in the body's redox homeostasis to a more pro-oxidative environment, suggesting that therapies

Modern Pharmacological Approaches to Therapies:

Substances Tested in Animal Models of Rheumatoid Arthritis 237

Strosova et al., 2008, 2009; Tastekin et al., 2007). In recent years, methods based on chemiluminescence and fluorescence measurements have been widely used for determination of ROS, RNS and lipid peroxidation metabolites. Fluorescent protein adducts are derivatives formed by reaction of secondary metabolites of lipid peroxidation (especially HNE and MDA) with free amino groups of proteins (Aldini et al., 2007; Requena et al., 1996). In murine and human serum, albumin protein fraction was identified as the most fluorescent fraction of proteins (Tsuchida et al., 1985). Several authors have suggested that protein adducts with secondary metabolites of lipid peroxidation also have immunogenic properties and may play a significant role in the pathogenesis of auto-immune diseases including RA (Kurien et al., 2006; Tuma, 2002). This highlights the importance of monitoring these adducts in auto-immune, chronic inflammatory diseases. The presence of oxidative damage in AA plasma was evaluated in our experiments also by measurement of fluorescence adducts in plasma. Two types of aldehydes were measured: HNE and MDA adducts of proteins (Biasi et al., 1995; Tsuchida et al., 1985). In rat AA, we were the first to monitor the HNE and MDA protein adducts in a time profile (Ponist et al., 2010). The level of HNE adducts was slightly increased already on day 7. The maximal level of HNE adducts was reached on day 14, and then it slowly decreased to the control level (day 28). Similar changes were recorded in the level of MDA adducts during the experiment, except that they were still significantly elevated on day 21 (Ponist et al., 2010). The time course of lipid peroxidation measured by the MDA protein adducts resembles the temporal pattern of ROS production by phorbol myristate acetate (PMA) - stimulated neutrophils measured by chemiluminescence of whole blood of arthritic animals, with maximum on day 14 and 21 (Nosal et al., 2007). Recent evidence from animal models of RA emphasizes the importance of neutrophils in the initiation and progression of AA (Cross et al., 2006). There are multiple experimental studies dedicated to neutrophil-generated chemiluminescence of whole blood (Arnhold et al., 1994; Cedergren et al., 2007; Miesel et al., 1996) and of synovial fluid (Arnhold et al., 1994; Cedergren et al., 2007), depending on the disease severity in patients with RA. An increase in whole blood chemiluminescence (2–8 fold) was shown in RA patients compared with healthy volunteers (Miesel et al., 1996). The results published by Nosal (Nosal et al., 2007) are in good agreement with this finding. Arthritic animals had significantly elevated spontaneous chemiluminescence from the seventh experimental day until the end of experiment (day 28). Neutrophils in whole blood of AA animals reacted excessively to stimulation with PMA and produced 6–9 times more ROS. The development of AA in rats was also accompanied with an increase in blood neutrophil count when compared with control animals (Nosal et al., 2007). Oxidative damage to the tissues in AA was demonstrated - ROS levels in the joint and the spleen were analysed by chemiluminiscence assessment (Drabikova et al., 2009). Measurements in the joint were completed by spectrophotometric analysis of myeloperoxidase activity in an experiment focused on evaluation of therapeutic effects of two stilbenoids in AA (Macickova et al., 2010). In this study, myeloperoxidase (MPO) activity in the hind paw joint homogenate of arthritic rats was approx. 3-times higher than in healthy controls. This finding is of importance as MPO is the most abundant enzyme in neutrophils. It is a marker of OS and ROS generated by MPO can deplete the NO level in vascular endothelium (Brennan & Hazen, 2003). MPO enhances the binding of leukocytes, including monocytes and neutrophils, to the endothelium (Johansson et al., 1997). Vascular endothelial cells are also capable of secreting various cytokines, which are potent chemoattractants for neutrophils. Both MPO and cytokines participate in the recruitment of cells into the area of

which restore the redox balance may have beneficial effects on the disease process. The role of reactive oxygen species (ROS) in the etiology of RA is supported by numerous studies documenting that not only the damaging effects of ROS but also the role that ROS play in regulating various inflammatory processes contributes to the pathogenesis of the disease (Kunsch et al., 2005). The net effect of redox signaling is highly specific changes in gene expression and in the cellular phenotype. Therefore, by serving as second messengers, ROS/reactive nitrogen species are not only toxic agents but also mediators of physiological function (Giustarini et al., 2004; Poli et al., 2004). Considering these facts, we monitored parameters of OS together with the inflammatory marker plasma C-reactive protein (CRP). For the determination of rat plasma CRP concentration, the ELISA kit from Immunology Consultant Laboratories, Inc. (ICL) was used. CRP comparable to HPV was already significantly increased in arthritic rats on day 14 (Bauerova et al., 2010).

γ-Glutamyltransferase (GGT), as a non-specific marker of inflammation and OS, has been assessed in different cells and tissues of the lymphatic system — T-lymphocytes, macrophages, spleen and thymus tissue (Koner et al., 1997). The ectoenzyme form of GGT is not present on non-active peripheral T-lymphocytes, but its expression rises after activation of native T-lymphocytes. In other tissues, GGT is essential for "scavenging" glutathione metabolites (mostly γ-glutamyl) and their transport into cells, where GSH is synthesized *de novo* (Carlisle et al., 2003). GGT is an important component of inflammatory processes since its activity is closely connected with the overall antioxidant status of the organism. In our experiments, GGT in the joint from the hind paw (cartilage and soft tissue without bone) and in spleeen tissue was determined at the end of the experiment, on day 28. The activity of GGT was measured by the method of (Orlowski & Meister, 1970), modified by (Ondrejickova et al., 1993). We found that the activity of GGT was approximately 3–6 times higher in AA animals than in healthy controls in the spleen and 1.4–2.3 times higher in the joint (Bauerova et al., 2006, 2008b, 2009; Sotnikova et al., 2009). We assume that the increased activity of GGT in AA is a result of elevated systemic OS. The finding that the GGT activity is also elevated in peripheral joint tissue is in good agreement with clinical studies of patients with RA who had increased levels of GGT not only in the serum and urine but also in synovial fluid (Rambabu et al., 1990). In one of our studies we showed a good correlation between GGT activity in joint tissue and the HPV of arthritic animals (Bauerova et al., 2006). GGT expression has been detected in active lymphocytes that accumulate at the inflammation region, as observed in RA. Ishizuka et al. (2007) found that neutralizing antibodies against GGT had a therapeutic effect on joint destruction in collagen-induced arthritis in mice. Elevated expression and activity of GGT in joint tissue is a good marker for synovial inflammation and bone resorption. Substances able to reduce the activity and/or expression of GGT could be important for RA therapy. OS, a consequence of chronic inflammatory processes occurring in AA, increased after the experimental day 14, which was also the onset of clinical manifestations of the disease. OS increased the consumption of endogenous antioxidants in plasma and thus caused a lowering of the plasma antioxidant capacity, measured as the total antioxidant status with RANDOX® TAS kit (Bauerova et al., 2009, Mihalova et al., 2007). A frequently used marker of lipid peroxidation is malondialdehyde (MDA), assessed as an adduct with thiobarbituric acid (TBA). We used the substances reacting with thiobarbituric acid measured in plasma at 535nm (Brown & Kelly, 1996). Clinical studies have shown increased plasmatic levels of MDA in patients with RA (Baskol et al., 2005, 2006; Sarban et al., 2005). The level of MDA in the plasma of arthritic animals was also elevated (Bauerova et al., 2008b, 2009; He et al., 2006; Sotnikova et al., 2009;

which restore the redox balance may have beneficial effects on the disease process. The role of reactive oxygen species (ROS) in the etiology of RA is supported by numerous studies documenting that not only the damaging effects of ROS but also the role that ROS play in regulating various inflammatory processes contributes to the pathogenesis of the disease (Kunsch et al., 2005). The net effect of redox signaling is highly specific changes in gene expression and in the cellular phenotype. Therefore, by serving as second messengers, ROS/reactive nitrogen species are not only toxic agents but also mediators of physiological function (Giustarini et al., 2004; Poli et al., 2004). Considering these facts, we monitored parameters of OS together with the inflammatory marker plasma C-reactive protein (CRP). For the determination of rat plasma CRP concentration, the ELISA kit from Immunology Consultant Laboratories, Inc. (ICL) was used. CRP comparable to HPV was already

γ-Glutamyltransferase (GGT), as a non-specific marker of inflammation and OS, has been assessed in different cells and tissues of the lymphatic system — T-lymphocytes, macrophages, spleen and thymus tissue (Koner et al., 1997). The ectoenzyme form of GGT is not present on non-active peripheral T-lymphocytes, but its expression rises after activation of native T-lymphocytes. In other tissues, GGT is essential for "scavenging" glutathione metabolites (mostly γ-glutamyl) and their transport into cells, where GSH is synthesized *de novo* (Carlisle et al., 2003). GGT is an important component of inflammatory processes since its activity is closely connected with the overall antioxidant status of the organism. In our experiments, GGT in the joint from the hind paw (cartilage and soft tissue without bone) and in spleeen tissue was determined at the end of the experiment, on day 28. The activity of GGT was measured by the method of (Orlowski & Meister, 1970), modified by (Ondrejickova et al., 1993). We found that the activity of GGT was approximately 3–6 times higher in AA animals than in healthy controls in the spleen and 1.4–2.3 times higher in the joint (Bauerova et al., 2006, 2008b, 2009; Sotnikova et al., 2009). We assume that the increased activity of GGT in AA is a result of elevated systemic OS. The finding that the GGT activity is also elevated in peripheral joint tissue is in good agreement with clinical studies of patients with RA who had increased levels of GGT not only in the serum and urine but also in synovial fluid (Rambabu et al., 1990). In one of our studies we showed a good correlation between GGT activity in joint tissue and the HPV of arthritic animals (Bauerova et al., 2006). GGT expression has been detected in active lymphocytes that accumulate at the inflammation region, as observed in RA. Ishizuka et al. (2007) found that neutralizing antibodies against GGT had a therapeutic effect on joint destruction in collagen-induced arthritis in mice. Elevated expression and activity of GGT in joint tissue is a good marker for synovial inflammation and bone resorption. Substances able to reduce the activity and/or expression of GGT could be important for RA therapy. OS, a consequence of chronic inflammatory processes occurring in AA, increased after the experimental day 14, which was also the onset of clinical manifestations of the disease. OS increased the consumption of endogenous antioxidants in plasma and thus caused a lowering of the plasma antioxidant capacity, measured as the total antioxidant status with RANDOX® TAS kit (Bauerova et al., 2009, Mihalova et al., 2007). A frequently used marker of lipid peroxidation is malondialdehyde (MDA), assessed as an adduct with thiobarbituric acid (TBA). We used the substances reacting with thiobarbituric acid measured in plasma at 535nm (Brown & Kelly, 1996). Clinical studies have shown increased plasmatic levels of MDA in patients with RA (Baskol et al., 2005, 2006; Sarban et al., 2005). The level of MDA in the plasma of arthritic animals was also elevated (Bauerova et al., 2008b, 2009; He et al., 2006; Sotnikova et al., 2009;

significantly increased in arthritic rats on day 14 (Bauerova et al., 2010).

Strosova et al., 2008, 2009; Tastekin et al., 2007). In recent years, methods based on chemiluminescence and fluorescence measurements have been widely used for determination of ROS, RNS and lipid peroxidation metabolites. Fluorescent protein adducts are derivatives formed by reaction of secondary metabolites of lipid peroxidation (especially HNE and MDA) with free amino groups of proteins (Aldini et al., 2007; Requena et al., 1996). In murine and human serum, albumin protein fraction was identified as the most fluorescent fraction of proteins (Tsuchida et al., 1985). Several authors have suggested that protein adducts with secondary metabolites of lipid peroxidation also have immunogenic properties and may play a significant role in the pathogenesis of auto-immune diseases including RA (Kurien et al., 2006; Tuma, 2002). This highlights the importance of monitoring these adducts in auto-immune, chronic inflammatory diseases. The presence of oxidative damage in AA plasma was evaluated in our experiments also by measurement of fluorescence adducts in plasma. Two types of aldehydes were measured: HNE and MDA adducts of proteins (Biasi et al., 1995; Tsuchida et al., 1985). In rat AA, we were the first to monitor the HNE and MDA protein adducts in a time profile (Ponist et al., 2010). The level of HNE adducts was slightly increased already on day 7. The maximal level of HNE adducts was reached on day 14, and then it slowly decreased to the control level (day 28). Similar changes were recorded in the level of MDA adducts during the experiment, except that they were still significantly elevated on day 21 (Ponist et al., 2010). The time course of lipid peroxidation measured by the MDA protein adducts resembles the temporal pattern of ROS production by phorbol myristate acetate (PMA) - stimulated neutrophils measured by chemiluminescence of whole blood of arthritic animals, with maximum on day 14 and 21 (Nosal et al., 2007). Recent evidence from animal models of RA emphasizes the importance of neutrophils in the initiation and progression of AA (Cross et al., 2006). There are multiple experimental studies dedicated to neutrophil-generated chemiluminescence of whole blood (Arnhold et al., 1994; Cedergren et al., 2007; Miesel et al., 1996) and of synovial fluid (Arnhold et al., 1994; Cedergren et al., 2007), depending on the disease severity in patients with RA. An increase in whole blood chemiluminescence (2–8 fold) was shown in RA patients compared with healthy volunteers (Miesel et al., 1996). The results published by Nosal (Nosal et al., 2007) are in good agreement with this finding. Arthritic animals had significantly elevated spontaneous chemiluminescence from the seventh experimental day until the end of experiment (day 28). Neutrophils in whole blood of AA animals reacted excessively to stimulation with PMA and produced 6–9 times more ROS. The development of AA in rats was also accompanied with an increase in blood neutrophil count when compared with control animals (Nosal et al., 2007). Oxidative damage to the tissues in AA was demonstrated - ROS levels in the joint and the spleen were analysed by chemiluminiscence assessment (Drabikova et al., 2009). Measurements in the joint were completed by spectrophotometric analysis of myeloperoxidase activity in an experiment focused on evaluation of therapeutic effects of two stilbenoids in AA (Macickova et al., 2010). In this study, myeloperoxidase (MPO) activity in the hind paw joint homogenate of arthritic rats was approx. 3-times higher than in healthy controls. This finding is of importance as MPO is the most abundant enzyme in neutrophils. It is a marker of OS and ROS generated by MPO can deplete the NO level in vascular endothelium (Brennan & Hazen, 2003). MPO enhances the binding of leukocytes, including monocytes and neutrophils, to the endothelium (Johansson et al., 1997). Vascular endothelial cells are also capable of secreting various cytokines, which are potent chemoattractants for neutrophils. Both MPO and cytokines participate in the recruitment of cells into the area of

Modern Pharmacological Approaches to Therapies:

**2.1.3 Immunological markers** 

Substances Tested in Animal Models of Rheumatoid Arthritis 239

comparable to protein carbonyls in plasma. This novel finding emphasizes the systemic role of OS in chronic inflammatory diseases such as AA, with oxidatively modified proteins, not

RA is associated with elevated levels of IL-1 in the synovium. IL-1 is closely related to inflammation and articular damage in several arthritis models and it is therefore generally accepted that IL-1 has a pivotal role in the pathophysiology of RA. In particular, IL-1 is a potent stimulator of synoviocytes, chondrocytes and osteoblasts. Moreover, IL-1 is a key mediator of synovial inflammation and pannus formation (Dinarello & Moldawer, 2002). It has a severe impact on different cell populations and exerts biological effects, e.g. increased synthesis of acute phase reactants. IL-1α is secreted by monocytes/macrophages activated via TNF-α and/or bacterial endotoxin. Furthermore, IL-1α markedly potentiates the toxic effect of TNF-α in animal experiments (Waage, et al., 1991). In the AA model used in our experiments, IL-1α was significantly increased in plasma on day 14 and also on day 28 (Bauerova et al., 2007, 2009; Bauerova et al., 2010a). The course of plasma levels of both proinflammatory cytokines IL-1α and TNF-α in AA was very similar, with the maximum on day 14 and with decreasing levels on days 21 and 28 in comparison to day 14 (Bauerova et al., 2009). These results are of importance as TNF-α controls the gene expression of various cytokines and chemokines in different cell types engaged in the host immune response to infection and triggers the cascade of cytokines acting in the inflammatory response. The efficient biological activities of TNF-α include direct activation of T- and B-lymphocytes, macrophages, and natural killer cells, release of acute-phase proteins, and endothelial cell activation. The activated monocyte or macrophage represents the primary source for TNF-α, especially after IFN-γ priming. TNF-α is a key regulator of other pro-inflammatory cytokines such as IL-1α, IL-6, and IL-8. Further, we followed the course of monocyte chemoattractant protein 1 (MCP-l) (Bauerova et al., 2009). This chemokine is mainly expressed by macrophages in response to a wide range of cytokines, e.g. TNF-α and IL-1. In this experiment, the significant maximum of MCP-1 plasma level measured on day 21 and the following decrease is in close association with kinetics of both TNF-α and IL-1α. According to the target cell specificity, MCP-1 was postulated to play a pathognomonic role in various diseases with monocyte cell infiltration. MCP-1 is a member of the CC chemokine subfamily that modulates monocyte chemotaxis both *in vitro* (Oppenheim et al., 1991) and *in vivo* (Rollins, 1996; Volejnikova et al., 1997). MCP-1 displays chemotactic activity for monocytes and basophils but not for neutrophils or eosinophils. Expression of MCP-1 has been detected in a number of pathologic conditions associated with monocyte aggregation, including atherosclerosis, arthritis, and glomerulonephritis (Rollins, 1996). The synovial fluid (SF) and serum MCP-1 concentrations are significantly higher in RA patients. This suggests that MCP-1 is mainly produced locally by activated cells, where it may exacerbate and sustain inflammation by attracting proinflammatory leukocytes, predominantly monocytes (Stankovic et al., 2009). Substances that can suppress the production of MCP-1 have shown beneficial effects in animal models of arthritis (Guglielmotti et al., 2002; Inoue et al., 2001). A completely different picture was revealed for IL-4. The level of this antiinflammatory cytokine was increasing with time with the maximum observed on day 28 in AA animals (Bauerova et al., 2009). IL-4 is a pleiotropic cytokine produced by mature Th2 cells and mastocyte- and/or basophil-derived cells. IL-4 has marked inhibitory effects on the expression and release of monocyte-derived pro-inflammatory cytokines, e.g. IL-1, TNF-α,

only in directly affected tissues (cartilage, bone and skeletal muscle).

inflammation. Lefkowitz et al. (1999) reported that MPO may be an important mediator in the inflammatory response.

Moreover, in the scale of systemic OS parameters, phagocytosis, oxidative burst and metabolic activity of rat granulocytes isolated from peripheral blood were monitored. Flow cytometric analysis was used for these measurements, according to the method published by Kronek et al. (2010) and modified for the model of AA (Bauerova et al., 2010a). Interestingly, increased production of ROS by neutrophils recorded by whole blood chemiluminiscence measurements emerged already in an early phase of disease, on the day 7. We therefore decided to investigate this finding more precisely using flow cytometry. Another reason was that the changes in neutrophils occur before the clinical parameter HPV starts to be increased. Due to arthritis, both phagocytosis and oxidative burst were already significantly increased on experimental day 7. Metabolic activity of neutrophils as the percentage of double positive cells (simultaneously phagocytotic and positive for oxidative burst) was decreased. This finding could be explained by an increased number of "arthritic" neutrophils, which are positive only for oxidative burst and therefore are not counted as double positive cells (Bauerova et al., 2010a). Further we analyzed in plasma the level of one of the most important endogenous antioxidants in rats – coenzyme Q9 (CoQ9). Significant changes in the levels of CoQ9 and/or CoQ10 have been noted in a wide variety of diseases in both animal and human studies. These changes may be caused by impairment in CoQ biosynthesis or excessive utilization of CoQ by the body, or any combination of these processes (Bauerova et al., 2008a; Littarru et al., 1991). In this experiment, we focused on evaluating the CoQ9 plasmatic levels as the dominant form of CoQ in rats. Its concentration is about 10 times higher than the concentration of CoQ10 (Dallner & Sindelar, 2000). In AA the arthritis process increases significantly the level of CoQ9 in comparison with healthy controls. Evidently, the arthritic processes stimulate the synthesis of CoQ9 and its transport to plasma. In addition to monitoring lipid peroxidation, also protein oxidation was followed up in AA. Arthritis, similarly to many other diseases, is accompanied by oxidative damage of plasma proteins induced by the action of free radicals. Protein carbonyls (aldehydes and ketones) are produced directly by oxidation or *via* reactions with other molecules generated by the oxidation process. The assay of protein carbonyls as biomarkers of OS in various diseases is with advantage used in diagnostics because of the relatively early formation and relative stability of carbonylated proteins (Dalle-Donne et al., 2003). The ability of certain compounds to reduce the amount of carbonyls is considered as one of the indirect proofs of their antioxidant activity. In our AA experiments, enzyme linked immunosorbent assay (ELISA) was used most frequently for quantitative determination of protein carbonyls in plasma (Buss et al., 1997). The first measurement of protein carbonyls in our experiments with AA was performed in a study with carboxymethyl (1/3)-b-D-glucan isolated from *Saccharomyces cerevisiae* administered to arthritic rats (Kogan et al., 2005). In this study, the content of carbonyls in the arthritic animals increased significantly in comparison with healthy controls and protein carbonyl determination in plasma was performed according to the method described by (Levine et al., 1990) and modified in agreement with the previously applied experimental conditions (Bauerova et al., 2002). Also in the following experiments with AA we found significant damage of proteins caused by oxidative stress accompanying arthritis (Bauerova et al., 2005b; Strosova et al., 2009). In addition to determination of protein carbonyls in plasma, we performed an assay of carbonyls in brain tissue and applied it as a marker of antioxidative properties of carnosine evaluated for monotherapy of AA (Ponist et al., 2011). Protein carbonyls in brain tissue homogenates were significantly elevated, comparable to protein carbonyls in plasma. This novel finding emphasizes the systemic role of OS in chronic inflammatory diseases such as AA, with oxidatively modified proteins, not only in directly affected tissues (cartilage, bone and skeletal muscle).
