**2.1.3 Immunological markers**

238 Rheumatoid Arthritis – Treatment

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

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,

the inflammatory response.

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,

Modern Pharmacological Approaches to Therapies:

information about administration of antioxidants in AA.

**3.1 Natural substances isolated from plants** 

Substances Tested in Animal Models of Rheumatoid Arthritis 241

As resulted also from our previous experiments (Bauerova et al., 2005a, 2005b, 2008a, 2008b, 2009, Drabikova et al., 2009; Drafi et al., 2010; Jancinova et al., 2009; Kogan et al., 2005; Macickova et al., 2010; Ponist et al., 2010; Sotnikova et al., 2009; Strosova et al., 2008, 2009), all performed in the AA model, substances with antioxidant properties have a high potency to be used in therapy of RA. They decreased the progression of AA when administered to rats with AA over the period of 28 days. For our experiments, we chose substances with antioxidative properties and low toxicity. These antioxidative substances were synthetic antioxidants, as pyridoindol derivatives, and natural substances possessing antioxidative properties. We chose compounds and extracts isolated from plants, polysaccharides isolated from yeast and mushrooms and finally also endogenous antioxidants. An overview of some selected results is given below along with new unpublished data to provide complex

The world of plants is an unlimited source of compounds with healing effects, including antiinflammtory, antioxidative and immunomodulating properties. We chose some of them for our experiments with AA. Figure 1 compares all these plant ingredients concerning their effect on the basic clinical parameter – change of hind paw volume (HPV), together with selected parameters of OS as plasmatic TBARS and GGT assessed in spleen and joint tissue homogenates. We compared the effect of *Boswellia serrata* extract (Bo), *Arctostaphylos uva-ursi* extract (UV), *Zingiber officinale* extract (Zg) in combination with two previous extracts (Bo-UV-Zg), sesame oil in combination with *Arctostaphylos uva-ursi* extract (Bo-So), arbutin (Ar), curcumin (CU), Pycnogenol® (PYC) and two stilbenoids – pinosylvin (PIN) and pterostilbene (PTE). The compounds and extracts were all given per os in a single dose immediately after induction of AA and were administered daily until the end of the experiment – experimental day 28. The experimental protocol was approved by the Ethics Committee of the Institute of Experimental Pharmacology and Toxicology and by the Slovak State Veterinary and Food Administration. AA was induced by a single intradermal injection of heat-inactivated *Mycobacterium butyricum* in incomplete Freund's adjuvant (Difco Laboratories, Detroit, MI, USA) to male Lewis rats. The injection was performed near the tail base. All experiments included healthy animals (HC), arthritic animals not treated (AA), arthritic animals treated with the compounds/extracts studied. The oral daily doses used were 30 mg/kg b.w for AA-PIN and AA-PTE, 10 mg /kg b.w for A-PYC, 50 mg/kg b.w for AA-UV, AA-Bo, AA-Bo-So, AA-Ar and AA-CU, 50+25+25 mg/kg b.w for the mixture AA-Bo-UV-Zg and 0.1 ml/kg b.w. for sesame oil. For statistical analysis of the obtained data the same procedure was applied in all experimental cases. The data for all parameters are expressed as arithmetic mean ± S.E.M. For significance calculations unpaired Student`s *t*-test was used with \**p*<0.05 (significant), \*\**p*<0.01 (very significant), \*\*\**p*<0.001 (extremely significant). The arthritis group was compared with healthy control animals (\* symbol). The treated arthritis groups were compared with untreated arthritis (+-symbol). In each experimental group 8–10 animals were used. In Figure 1 the reduction of HPV and OS parameters is illustrated in relation to untreated arthritic rats (100% represented by dot-anddash line). The situation for AA is complicated due to the dominant involvement of Th 1 driven autoimmune etiopathology. OS in this animal model occurs as a reaction to autoimmune processes. Under these conditions, control of OS is of secondary importance, although it could enhance immunomodulatory therapy of RA (Bauerova et al., 2011). Figure 1 clearly shows that plant-related treatment is not enough for successful improvement of

IL-6, IL-8, and MIP- 1α. It was shown to suppress macrophage cytotoxic activity, parasite killing, and macrophage-derived nitric oxide production (Vannier, et al., 1992). In our experiments, detection of plasma IL-1α, IL-4, TNF-α, and MCP-1 was done by the flowcytometric (Cytomics FC 500, Beckman Coulter Inc. Fullerton, USA) fluorescent beadbased multiplex assay Rat Cytokine Flow Cytomix Multiplex (Bender Med System, GmbH., Austria). Additionally for determination of IL-1α in plasma an ELISA kit from Bender MedSystems or from R&D Systems Quantikine® was used and to as assessed MCP in plasma by Instant ELISA kit from eBioscience®.
