**5. Long-lived dedifferentiated neutrophils**

In the clinical setting it was observed, that neutrophils are present in high number in the synovial tissues during the initial stages of RA, and are described to persist in the SF during the course of this disease (Cascao et al., 2009). Patients in an active disease state may have in the SF cellular infiltrate up to 90% of neutrophils (Edwards et al., 1997).

Neutrophils that enter a joint are exposed to multiple factors such as cytokines and extreme physical conditions (low oxygen concentration, high pressure conditions). Recent achievements in neutrophil research indicate that certain inflammatory conditions induce a phenotyphic switch in circulating neutrophils toward a resident neutrophil with different functions (Chakravarti et al., 2009).

Neutrophils isolated from RA patients contain high levels of class II MHC RNA (Cross et al., 2003). It was identified that neutrophils isolated from SF express different types and numbers of surface receptors like CD49, CD80, CD83, CD86, HLA-DR, have increased cell surface ICAM-1 (Iking-Konert et al., 2005).

There are many differences in protein expression between SF and blood-derived neutrophils in RA patients. Neutrophils from RA SF have mobilized pre-formed molecules from intracellular stores to the cell surface and activated genes expression resulting from

The observed opposite effects may suggest that different subtypes of muscarinic receptors are expressed on neutrophils presented in blood and synovial fluid, respectively. Because there are five distinct genes encoding muscarinic cholinergic receptors subtypes, we investigated the expression of mRNA encoding these receptors subtypes (i.e. m1-m5) in neutrophils isolated from healthy blood donors and patients with RA. Our results demonstrated for the first time the presence of m3, m4 and m5 muscarinic receptor subtypes in human blood neutrophils (Bany et al., 1999). The lack of mRNA for m4 muscarinic receptor subtype in neutrophils isolated from SF, may contribute to the opposite responses to cholinergic stimuli observed in neutrophils from blood and SF (Gajewski et al., 1997, Bany

These findings were partially confirmed by Tracey (Tracey, 2002). The molecular dovetail between the cholinergic nervous system and the innate immune system is a nicotinic αbungarotoxin-sensitive macrophage acetylcholine receptor. Exposure of human macrophages, but not peripheral blood monocytes, to nicotine or acetylcholine inhibits the release of TNF-α, IL-1 and IL-8 in response to endotoxin. Tissue macrophages, but not circulating monocytes, produce most of the TNF-α which appears systemically during an excessive inflammatory response. Interaction between the macrophage cholinergic receptor and its ligand inhibits the synthesis of pro-inflammatory cytokines (TNF-α, IL-1 and IL-18) but not anti-inflammatory cytokines (such as IL-10). Acetylcholine inhibits the expression of TNF-α protein in macrophages, but not the induction of TNF-α mRNA levels, indicating that activation of the cholinergic receptor transduces intracellular signals that inhibit cytokine synthesis at a post-transcriptional stage. As compared with macrophages, monocytes are refractory to the cytokine-inhibiting effects of acetylcholine: only supraphysiological concentrations of cholinergic agonist inhibit cytokine synthesis in monocytes

Further studies on the "reprogramming" of blood neutrophils (contradictory effects of adrenergic and cholinergic systems) into other cell types, SF neutrophils, (both systems inhibiting CL response) are expected to yield new insight into event related to RA therapy.

In the clinical setting it was observed, that neutrophils are present in high number in the synovial tissues during the initial stages of RA, and are described to persist in the SF during the course of this disease (Cascao et al., 2009). Patients in an active disease state may have in

Neutrophils that enter a joint are exposed to multiple factors such as cytokines and extreme physical conditions (low oxygen concentration, high pressure conditions). Recent achievements in neutrophil research indicate that certain inflammatory conditions induce a phenotyphic switch in circulating neutrophils toward a resident neutrophil with different

Neutrophils isolated from RA patients contain high levels of class II MHC RNA (Cross et al., 2003). It was identified that neutrophils isolated from SF express different types and numbers of surface receptors like CD49, CD80, CD83, CD86, HLA-DR, have increased cell

There are many differences in protein expression between SF and blood-derived neutrophils in RA patients. Neutrophils from RA SF have mobilized pre-formed molecules from intracellular stores to the cell surface and activated genes expression resulting from

the SF cellular infiltrate up to 90% of neutrophils (Edwards et al., 1997).

et al., 1999).

(Tracey, 2002).

**5. Long-lived dedifferentiated neutrophils** 

functions (Chakravarti et al., 2009).

surface ICAM-1 (Iking-Konert et al., 2005).

enhanced transcription and translation (Quayle et al., 1997). Consequently, several gene products, such as IL-8 and MMP-9, are up-regulated, allowing not only the up-regulation of cell function but also development of new cellular responses, such as antigen-presentation to T cell via activated MHCII expression (Cascao et al., 2009). Neutrophils isolated from the SF of RA patients expressing MHCII, CD80 and CD86 are able to stimulate T-cell proliferation (Wright et al., 2010). Indeed, the levels of expression of MHCII and costimulatory molecules on neutrophils from SF have been reported to be equivalent to or greater than the levels of expression on monocytes and B cells (Sandilands et al., 2005). Apart from their ability to stimulate T-cells in this way, it is also possible that neutrophils can expose cryptic epitopes, as they possess different proteases than other antigenpresenting cells. Thus, their function within SF could be different from that of other antigenpresenting cells (Wright et al., 2010).

Inflammatory reprogramming may increase neutrophil viability. As was shown, 8-17% neutrophils of the global neutrophil population have the potential to persist for more than 72 h under inflammatory conditions (Chakravarti et al., 2008). This is in contrast to the circulating neutrophils whose survival is measured in hours. The mechanism of this persistence remains unknown, but it seems that the protein kinases are largely implicated in survival of these long-lived neutrophils (Cronstein et al., 1992). The phospholipids metabolic pathway leading to leukotriene B4 (LTB4) synthesis also illustrates differences between these long-lived neutrophils and circulating neutrophils. As was shown, a significant amount of the 5-lipoxygenase (5-LO) is localized to the nuclear membrane in long-lived neutrophils, in basal conditions, a phenomenon absent in circulating neutrophils (Chakravarti et al., 2008).

As was mentioned, neutrophils exposure to SF (50%) induces transdifferentiation of neutrophils into dendritic like cells (Iking-Konert et al., 2005). When the neutrophils were cultured with TNF-α, IFN-γ and IL-4, the resultant cells had morphologic, cytochemical, and phenotypic features of macrophages. In contrast to the starting population, they were negative for myeloperoxidase, specific esterase and lactoferrin. It appears that, in response to the cytokines present in SF, postmitotic neutrophils can become macrophages (Araki et al., 2004).

Neutrophils are known to phagocytose invading pathogens and harmful particles. However, in the study of Rydel-Tormanen et al. (Rydel-Tormanen et al., 2006) it was demonstrated that neutrophils are also able to engulf apoptotic neutrophils or cell debris resulting from secondary necrosis of neutrophils. Previously, neutrophils phagocytosing apoptotic cells and nuclei have been described in blood smears from patients with systemic lupus erythematosus (SLE), a feature called LE cells (Bohm, 2004). Moreover, in inflammatory foci, apparently viable neutrophils with phagosomes enclosing were found with what appeared to be whole apoptotic neutrophils and apoptotic nuclei. Neutrophils may thereby contribute to clearance and resolution of inflammation, thus acting as a back up system in situations when the macrophages clearance system is insufficient and/or overwhelmed. It is apparent that neutrophils have the abilities needed to mimic macrophage behavior and express most, if not all, surface receptors used by macrophages in the process of phagocytosis, suggesting the mechanisms to be similar in the two types of cells (Rydell-Tormanen et al., 2006).
