**6. Long-density granulocytes (LDG) and endothelial progenitor cells (EPC)**

Two studies (Denny et al., 2010, Hacbarth & Kajdacsy-Bella, 1986) have reported the presence of an abnormal subset of neutrophils in the peripheral circulation of SLE patients.

The Role of Neutrophils in Rheumatoid Arthritis – Experiments *In Vitro*: A Change of Conception? 67

It has been shown that neutrophils that have migrated to the site of inflammation can upregulate their production of chemokines, supporting the notion that, in this way, neutrophils participate in the regulation of leukocyte accumulation (Soehlein et al., 2009). In terms of production, the principal chemokine produced by neutrophils is IL-8, which activates neutrophils in an autocrine loop. IL-8 binds to CXCR2 expressed not just on neutrophils, but also monocytes. IL-8 also mediates adhesion both human neutrophils and

Adhesion of neutrophils to the endothelial cells results in rapid release of secretory vesicles: proteinase-3 and azurocidin (also known as cationic antimicrobial protein). Both azurocidin and proteinase-3 are strongly positively charged and may therefore act with negatively charged endothelial proteoglycans. Azuricidin was recently shown (Soehnlein et al., 2009) to induce monocyte extravasation, it has been demonstrated that depletion of neutrophils reduces the recruitment of inflammatory monocytes. Interestingly, this deficiency in recruitment can be almost completely rescued by the local application of the supernatant

Activated neutrophils are short-lived cells. Their apoptosis is a tightly regulated process involving ROI and pathogens. Once neutrophils migrate toward the site of inflammation, their life span increases because of the presence of survival signals in the inflammatory milieu. In response to pro-inflammatory signals, neutrophils not only extend their life span, but also release a web of DNA in which granule proteins are enweaved (Brinkmann et al., 2004). Exposure of granule proteins and entrapment within a net of DNA may contribute to creating a gradient of chemotactic stimuli relevant to monocyte recruitment. Apart from release of granule proteins, apoptotic neutrophils may release attraction signals leading to influx monocytes. In recent years, several apoptotic cell-derived "find-me" signals were identified. Among them is lysophosphatidylcholine (LPC), of which the latter has received much attention. LPC was identified (Kim et al., 2002) as an "eat-me" signal on the apoptotic cell surface. More recently, it has been shown that changes in membrane composition of apoptotic cells (negative surface charges) in initiate attractive signals for phagocytes including monocyte. It is feasible that apoptotic neutrophils generate such electric signals

In addition, neutrophil granule proteins enhance the production of ROI by monocytes (Soehnlein et a., 2008). Thus, various experimental setups provide evidence that the axis of neutrophils and inflammatory monocytes promotes and sustained inflammation. Taken together, the multifaceted action of neutrophils in recruiting and activating monocytes may offer a powerful target for interfering with the sustained inflammatory reponse in RA

The research conditions of most neutrophil experiments differ considerably from the physiological environment of the joint, i.e. the presence and concentration of cytokines such as TNF-α, IL-1β, IL-6, IL-8, IL-12, IL-17, IL-18, IL-23 and IFN-γ (McInnes et al., 2007), and physical factors such as viscosity, oxygen concentration, and pressure. Each of these differences can influence neutrophil metabolism and activation (Gajewski et al., 2006, 2010a; Cross et al., 2006). Reasoning that conditions imitating physiological environment are required for proper conclusions, we developed and used culture systems which more accurately simulated conditions in the joint (Gajewski, et al., 2010b). The proposal to

monocytes to the endothelium (Soehlein et al., 2009).

from activated human neutrophils (Soehlein et al., 2009).

resulting in electrotaxis of monocytes (Zhao et al., 2006).

(Soehnlein et al., 2009).

**8. Summary** 

Low density granulocytes (LDG) are present in PBMC preparations derived from lupus patients. LDGs display an activated phenotype, induce significant endothelial cytotoxicity and synthesize sufficient levels of type I IFNs to disrupt the capacity of endothelial progenitor cells (EPC) to differentiate into mature endothelial cells.

EPCs have been shown to play a role in the neovascularization that occurs in diseased tissues. Given the extensive neovascularization that occurs in RA, it was suggested by Denny (Denny et al., 2010) that EPCs are recruited to the arthritic synovium, where they might contribute to expansion of the synovial microcirculation. The VCAM-1/very late activation antigen 4 adhesive system critically mediated EPC adhesion to cultured RA fibroblasts. As was shown, in 3 diverse animal models used to investigate cell homing in arthritis, EPCs preferentially localized to inflamed synovium compared to normal synovium (Silverman et al., 2007). This correlates well with earlier observations that the number of EPCs per mm2 identified immunihistochemically in postsurgical human RA synovial tissue (ST) was elevated ~25-fold over the number of EPCs localized in normal ST (Ruger et al., 2004).

The findings provide evidence of a possible role of EPC in the synovial neovascularization that is critical to RA pathogenesis; and it may be suggested that neutrophils play a crucial role in this phenomenon. As was showed by Schruefer (Schruefer et al., 2004), neutrophils are critically involved in angiogenesis. Growing evidences indicates that angiogenesis can be initiated by inflammatory cytokines, including IL-8. Human neutrophils release a variety of proinflamatory cytokines, including IL-8, which was originally identified as a potent activator of human neutrophils (Baggiolini et al., 1989). Subsequently, IL-8 was shown to stimulate angiogenesis by promoting proliferation of endothelial cells, moreover IL-8 inhibits endothelial cell apoptosis and induces the upregulation of endothelial matrix metalloproteinase-2 and -9, which also play an important role in angiogenesis.

Neovascularization is a hallmark of diverse pathological conditions, including RA. Microcirculatory expansion occurs either through angiogenesis (the proliferation and branching off of pre-existing microvessels, by IL-8 from neutrophils), or by vasculogenesis (the de novo formation of blood vessels from circulating EPCs). This phenomenon may be restored by LDG depletion (Denny et al., 2010).

Inhibition of neovascularization and thus inhibition of the expansion of invasive tissue in RA is the desired effect. A clearer understanding of the role of neutrophils in biologic processes that guide EPCs to the angiogenic tissue and exert contradictory effects on mirocirculatory expansion may lead to the development of the novel tools to modulate these activities (Silverman et a., 2007).
