**1. Introduction**

58 Rheumatoid Arthritis – Etiology, Consequences and Co-Morbidities

Xie, Y. D., Jin, L., and Yu, Q. W. (2007). *Xi bao yu fen zi mian yi xue za zhi = Chinese journal of* 

Xu, M., Mizoguchi, I., Morishima, N., Chiba, Y., Mizuguchi, J., and Yoshimoto, T. (2010).

Yago, T., Nanke, Y., Kawamoto, M., Furuya, T., Kobashigawa, T., Kamatani, N., and Kotake,

Yamada, N., Niwa, S., Tsujimura, T., Iwasaki, T., Sugihara, A., Futani, H., Hayashi, S.,

Yoshida, K., Yang, G. X., Zhang, W., Tsuda, M., Tsuneyama, K., Moritoki, Y., Ansari, A. A.,

Yoshimatsu, M., Kitaura, H., Fujimura, Y., Eguchi, T., Kohara, H., Morita, Y., and Yoshida,

Zelante, T., De Luca, A., Bonifazi, P., Montagnoli, C., Bozza, S., Moretti, S., Belladonna, M.

Okazaki, K., Lian, Z. X., Coppel, R. L., Mackay, I. R., and Gershwin, M. E. (2009).

L., Vacca, C., Conte, C., Mosci, P., Bistoni, F., Puccetti, P., Kastelein, R. A., Kopf, M.,

Okamura, H., Akedo, H., and Terada, N. (2002). *Bone* 30, 901.

*cellular and molecular immunology* 23, 536.

and Romani, L. (2007). *Eur J Immunol* 37, 2695.

S. (2007). *Arthritis Res Ther* 9, R96.

*Clin Dev Immunol* 2010.

*Hepatology* 50, 1494.

N. (2009). *Bone* 45, 1010.

Essential cells of innate immunity, neutrophils are often considered to be a homogenous population of terminally differentiated cells (Chakravarti et al., 2009). These cells represent the body's primary line of defence against invading pathogens such as bacteria, and constitute 40-60% of the white blood cell population. Neutrophils are short-lived polymorphonuclear phagocytes. They are known as first-responding inflammatory cells migrating towards the site of inflammation (Chakravarti et al., 2009**;** Edwards et al., 1997).

In the circulation of healthy adults, neutrophils exist in a resting state, which ensures that their toxic intracellular contents are not accidentally released to damage host tissues. Neutrophils become activated by agents that include bacterial products and cytokines or chemokines, such as TNF-α, IL-8 or IFN-γ. The primed neutrophils are then mobilized to the site of infection or inflammation and encounter activating signals to trigger bacterial killing. (Wright et al., 2010; Cascao et al., 2009)

It must be noted, that the functions of resting blood neutrophils and primed neutrophils may be very different. Thus, many of the regulatory functions of macrophages are shared by primed (but not resting) neutrophils (Wright et al., 2010). For this reason, *in vitro* experiments using freshly isolated blood neutrophils often fail to recognize the full functional repertoire and capacity of neutrophils.

#### **2. Oxidative metabolism of neutrophils**

In a chronic inflammatory process, such as rheumatoid arthritis (RA), large numbers of neutrophils are attracted across the synovial membrane, and become activated. The number of neutrophils in synovial fluid (SF) of patients with RA can reach 5x10-9 (Edwards et al., 1997). Neutrophils possess a range of potent proteinases and hydrolases, and have the ability to generate a series of reactive oxygen intermediates (ROI) via the combined activities of NADPH (reduced form) oxidase and myeloperoxidase (MPO) (Robinson et al., 1992; Duluray et al., 1990; Nurcombe et al., 1991a). If the neutrophils are not efficiently depleted, their production of inflammatory mediators, such as ROI, could prolong the inflammatory reaction. In fact, inappropriate release of ROI from activated neutrophils is responsible for joint damage observed in RA (Edwards et al., 1997). Beside ROI and inflammatory

The Role of Neutrophils in Rheumatoid Arthritis – Experiments *In Vitro*: A Change of Conception? 61

immune system as second messengers (Jones, 2006). Hitchon and El-Gabalawy propose that the physiological production of ROI by phagocytes in response to an antigen affects T-cell antigen interactions and possibly induces apoptosis in autoreactive arthritogenic T cells,

Although the presence of activated neutrophils in SF is well documented, it is still unknown how the neutrophils are activated, how they interact with other cells, and how long they

It was identified that addition of SF to neutrophils results in activation of neutrophils measured by a rapid chemiluminescence (CL) response (luminol- and lucigenin-dependent). Luminol-dependent CL is capable of monitoring both intracellular and extracellular ROI generation (NADPH oxidase and MPO), as luminol freely penetrates the neutrophil's cell membrane. Lucigenin-dependent CL, on the other hand, measures only the rate of extracellular ROI secretion (NADPH oxidase dependent) because lucigenin does not penetrate neutrophils, and light emission detected is independent of the activity of MPO. Pre-incubation of normal blood neutrophils in 10% SF enhanced the luminol- and lucigenin-CL, suggesting that both MPO and NADPH oxidase activity were activated in parallel during exposure to 10% SF from RA patients (Bender et al., 1986). Synovial fluid (20% concentration) isolated from RA patients activated blood neutrophils, leading to increase of luminol dependent CL over a 50 range (Nurcombe et al., 1991b). In contrast, the same fluid activated to a much lower range (two or three fold) of maximal rates of lucigenin dependent CL. All the mentioned reports were performed with SF in concentrations that did not exceed 20%. Other studies, confirmed that SF (used in concentration of 25%) produced rapid and

Our recent results (Gajewski et al., 2009), in contrast to earlier studies, indicate that higher concentrations of SF (up to 80%) have a quite different effect on lucigenin-dependent and luminol-dependent CL response. Increased concentrations of SF resulted in a reduction of luminol-dependent CL response and a very significant increase of lucigenin-dependent CL, reflecting extracellular ROI generation. This effect was observed irrespective of the stimulator used and whether neutrophils were isolated from SF or blood from either RA patients or healthy subjects. This indicates that increasing SF concentration results in higher extracellular ROI secretion and lower MPO-dependent ROI production. The promotion of extracellular release of ROI observed in this experiment is likely to be associated with the high concentrations of SF used, and raises the possibility that extracellular activity of neutrophils may be a general characteristic which prolongs the inflammatory process

Similar studies were performed by Bell et al. (1995). In these studies they examined the hypothesis that persistent inflammatory responses in RA may result from inhibition of neutrophils apoptosis by factors in SF. The effects of aging in culture and addition of SF on apoptosis was investigated using SF in a concentration range 0-75%. A significant effect of SF on promotion of apoptosis of synovial fluid neutrophils was observed at concentrations

It has been proposed that the process of hypoxic-reperfusion injury contributes to the persistence of synovitis in the inflamed joint. The generation of pathological, exercise

thereby preventing autoimmune responses (Hitchon & El-Gabalawy, 2004).

**3. Oxidative metabolism and apoptosis of neutrophils cultured in** 

parallel responses (luminol- and lucigenin-CL) in neutrophils.

persist at the site of inflammatory joint.

(Gajewski et al., 2009).

of 50% and above (Bell et al., 1995).

**physiological concentrations of SF, oxygen and cyclic loaded pressure** 

mediators, neutrophils are also an important source of proteolytic enzymes which play a role in degradation of articular structures. Wojtecka-Lukasik et al. were the first to isolate collagenase from peripheral blood neutrophils to a degree which allowed determination of its physicochemical properties and assessment of its effects on the biological activity of some drugs used in treatment of rheumatic diseases (Wojtecka-Lukasik et al., 1974). In addition to the active enzyme, its latent form and activator were discovered and described in the rheumatoid joint fluids (Dancewicz et al., 1978).

Neutrophils are able to form extracellular structures, named neutrophils extracellular traps (NETs). NETs are composed by nuclear components, such as chromatin DNA (i.e. histones anchored to this molecular backbone), and cytoplasmic components, such as granular peptides and enzymes. Upon activation, the induction of activation of NADPH oxidase was reported, suggesting that formation of NETs is ROI-dependent (Cascao et al., 2009). Neutrophils die upon release of these structures. However, this is a form of cell death different from apoptosis and necrosis, named "NETosis" (Steinberg et al., 2007). NETs represent an unconventional form of immune response, because these structures remain active even after the neutrophil's death. The presence of nucleic acid can contribute to the development of autoimmune diseases, such as systemic lupus erythematosus (SLE) in which there is an exacerbated reaction against the host DNA (Wartha et al., 2007).

As was shown by Wenthworth (Wenthworth et al., 2002), antibodies catalyze the generation of hydrogen peroxide from singlet molecular oxygen and water. This process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. Hydrogen peroxide production by antibodies alone was found to be not sufficient for bacterial killing. Wenthworth et al. suggested that the antibody-catalyzed water-oxidation pathway produced an additional molecular species with a chemical signature similar to that of ozone. This species is also generated during the oxidative burst of activated human neutrophils and during inflammation. These observations suggest that alternative pathways may exist for biological killing of bacteria that are mediated by potent oxidants previously unknown to biology (Wentworth et al., 2002).

More recently (Yamashita et al., 2008), it was discovered that four amino acids themselves (tryptophan, methionine, cysteine and histidine) are able to catalyze the production of an oxidant with the chemical signature of ozone from singlet oxygen in the water-oxidation pathway. The resultant oxidant with the chemical signature of ozone exhibited significant bactericidal activity in human neutrophils. These results also suggest that an oxidant with the chemical signature of ozone produced by neutrophils might potentiate a host defence system, when the host is challenged by high doses of infectious agents. These findings provide biological insights into the killing of bacteria by neutrophils (Yamashita et al., 2008).

From a different point of view, it is believed that ROI function as second messengers (Hitchon & El-Gabalawy, 2004). Typically, second messengers are short-lived molecules that at the time of activation of a receptor, act specifically on effectors to alter their activity transiently. Indeed, ROI can be generated at the time of receptor activation and they are short-lived, as the other second-messengers (Fillipin et al., 2008). ROI produced by phagocytes are critical for protection against invading microorganisms but also seem to have important physiological roles in priming the immune system. It has been demonstrated that exposure to ROI down-regulate the activity of T lymphocytes: ROI produced by phagocytes also seem to have essential physiological roles in priming the

mediators, neutrophils are also an important source of proteolytic enzymes which play a role in degradation of articular structures. Wojtecka-Lukasik et al. were the first to isolate collagenase from peripheral blood neutrophils to a degree which allowed determination of its physicochemical properties and assessment of its effects on the biological activity of some drugs used in treatment of rheumatic diseases (Wojtecka-Lukasik et al., 1974). In addition to the active enzyme, its latent form and activator were discovered and described in the

Neutrophils are able to form extracellular structures, named neutrophils extracellular traps (NETs). NETs are composed by nuclear components, such as chromatin DNA (i.e. histones anchored to this molecular backbone), and cytoplasmic components, such as granular peptides and enzymes. Upon activation, the induction of activation of NADPH oxidase was reported, suggesting that formation of NETs is ROI-dependent (Cascao et al., 2009). Neutrophils die upon release of these structures. However, this is a form of cell death different from apoptosis and necrosis, named "NETosis" (Steinberg et al., 2007). NETs represent an unconventional form of immune response, because these structures remain active even after the neutrophil's death. The presence of nucleic acid can contribute to the development of autoimmune diseases, such as systemic lupus erythematosus (SLE) in which

As was shown by Wenthworth (Wenthworth et al., 2002), antibodies catalyze the generation of hydrogen peroxide from singlet molecular oxygen and water. This process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. Hydrogen peroxide production by antibodies alone was found to be not sufficient for bacterial killing. Wenthworth et al. suggested that the antibody-catalyzed water-oxidation pathway produced an additional molecular species with a chemical signature similar to that of ozone. This species is also generated during the oxidative burst of activated human neutrophils and during inflammation. These observations suggest that alternative pathways may exist for biological killing of bacteria that are mediated by potent oxidants previously unknown to

More recently (Yamashita et al., 2008), it was discovered that four amino acids themselves (tryptophan, methionine, cysteine and histidine) are able to catalyze the production of an oxidant with the chemical signature of ozone from singlet oxygen in the water-oxidation pathway. The resultant oxidant with the chemical signature of ozone exhibited significant bactericidal activity in human neutrophils. These results also suggest that an oxidant with the chemical signature of ozone produced by neutrophils might potentiate a host defence system, when the host is challenged by high doses of infectious agents. These findings provide biological insights into the killing of bacteria by neutrophils (Yamashita et al.,

From a different point of view, it is believed that ROI function as second messengers (Hitchon & El-Gabalawy, 2004). Typically, second messengers are short-lived molecules that at the time of activation of a receptor, act specifically on effectors to alter their activity transiently. Indeed, ROI can be generated at the time of receptor activation and they are short-lived, as the other second-messengers (Fillipin et al., 2008). ROI produced by phagocytes are critical for protection against invading microorganisms but also seem to have important physiological roles in priming the immune system. It has been demonstrated that exposure to ROI down-regulate the activity of T lymphocytes: ROI produced by phagocytes also seem to have essential physiological roles in priming the

there is an exacerbated reaction against the host DNA (Wartha et al., 2007).

rheumatoid joint fluids (Dancewicz et al., 1978).

biology (Wentworth et al., 2002).

2008).

immune system as second messengers (Jones, 2006). Hitchon and El-Gabalawy propose that the physiological production of ROI by phagocytes in response to an antigen affects T-cell antigen interactions and possibly induces apoptosis in autoreactive arthritogenic T cells, thereby preventing autoimmune responses (Hitchon & El-Gabalawy, 2004).
