**3. Neutrophils as therapeutic targets**

neutrophils. Experimental evidence indicates that citrullination is involved in the breakdown of immune tolerance and may generate neoantigens (neoAgs) that become additional targets during epitope spreading [80]. Citrullinated residues stimulate the production of anti-citrullinated protein antibodies (ACPAs) in predisposed individuals. It has been observed that ACPAs can be present for several years before any clinical signs of arthritis appear [81–83]. A substantial increase in the number and titer of many antibodies against posttranslationally modified proteins is also seen shortly before the onset of arthritis. Citrullinated Ags have increased immunogenicity and arthritogenicity, and their presence in arthritic joints corre-

Osteoclasts are dependent on citrullinating enzymes for their normal maturation and display citrullinated antigens on their cell surface in a non-inflamed state. In humans, the binding of ACPAs to osteoclasts in the bone compartment induces IL-8 secretion. In turn, IL-8 sensitizes and/or activates sensory neurons by binding to CXC chemokine receptor (CXCR) 1 and CXCR2 on peripheral nociceptors [87–90], producing IL 8 dependent joint pain that is associ-

IL-8 release contributes to the chemoattraction of neutrophils [49], which play critical roles in initiating and maintaining joint-inflammatory processes that have been described in experimental arthritis [36, 91]. However, the exact roles that neutrophils play in the posttranslational modification of proteins and disease initiation and progression in RA remain unclear. Recent evidence suggests that, among the various mechanisms by which neutrophils cause tissue damage and promote autoimmunity, aberrant formation of neutrophil extracellular

NETs are released during a process of cellular death named NETosis. NETosis occurs with neutrophils upon contact with bacteria, fungi [92], or under several inflammatory stimuli. This process is associated with changes in the morphology of the cells, which eventually lead to cell death with extrusion of NETs [93, 94]. This process requires calcium mobilization, reactive oxygen species (ROS) produced by NADPH oxidase, neutrophil chromatin decondensation mediated by neutrophil elastase (NE) and myeloperoxidase (MPO), and chromatin modification via the citrullination of histones by peptidyl arginine deiminase 4 (PAD4) [95–99]. NETs are a network of extracellular fibers, which contain nuclear compounds as DNA and histones and that are covered with antimicrobial enzymes and granular components, such as MPO, NE, cathepsin G, and other microbicidal peptides [93, 94]. In the extracellular environment, NET fibers entrap microorganisms, and their enzymes and granular substances reach locally high concentrations and are thus able to cleave virulence factors and

Although NETs play a key role in the defense against pathogens, they may cause undesirable effects to the host, which has increased the interest in the role of neutrophils and NETs in autoimmunity. Augmented NET formation was first described in preeclampsia and ANCA-associated vasculitis and followed by the description in a series of autoimmune conditions, including psoriasis, systemic lupus erythematosus (SLE), antiphospholipid antibody syndrome (APS), and RA [50, 100, 102–105]. Neutrophil extracellular traps are an obvious source of nuclear material. Among these are a range of cytoplasmic and extracellular citrullinated antigens, well-established

traps (NETs) could play important roles in the pathogenesis of RA [50].

lates with disease severity [80, 84–86].

94 Role of Neutrophils in Disease Pathogenesis

ated with ACPA-mediated bone loss.

kill microorganisms [95, 100, 101].

Animal models have been extensively used in studies of RA pathogenesis. Despite the inherent limitations of all animal models, several rodent models have greatly contributed to the overall knowledge of important processes/mediators in the generation of inflammation, cartilage destruction, and bone resorption. In addition, the pharmaceutical industry has used these models for testing potential anti-arthritic agents, leading to important advances in therapeutic interventions for this destructive disease [117]. Such models include collageninduced arthritis, collagen antibody-induced arthritis, zymosan-induced arthritis, the methylated BSA model, and genetically manipulated or spontaneous arthritis models such as the TNF-α-transgenic mouse, K/BxN mouse, and Skg mouse [118]. Many of these models show that neutrophils are the first immune cells to enter the arthritic joint, and that early measures of joint inflammation correlate with neutrophil infiltration [45, 119, 120]. In this section, we highlight pharmacological approaches targeting neutrophil recruitment and activity, which present a therapeutic benefit to patients with RA.

The current treatments available to RA patients include glucocorticoids, non-steroidal antiinflammatory drugs, and disease-modifying antirheumatic drugs. Only disease-modifying agents—and to some extent glucocorticoids—can impede or halt the inflammatory and destructive disease processes [121]. With a more complete understanding of the immuneinflammatory events that occur in the pathogenesis of RA, scientists have developed therapeutic strategies that include monoclonal antibodies and receptor constructs, which target specific soluble or cell-surface molecules of interest. Biological agents such as monoclonal antibodies and recombinant proteins that target TNF-α, CD20, CTLA-4 (cytotoxic T-lymphocyteassociated protein 4), and the IL-1 receptor as well as therapies based on the blockade of T-cell and B-cell functions have shown efficacy in controlling the physical signs and pain associated with RA [122, 123].

Many interventions used to treat RA exert inhibitory effects on neutrophil responses in inflammation. However, non-steroid anti-inflammatory drugs (NSAIDS), DMARDs, and biologics do not specifically target neutrophil function [124].

Most NSAIDs inhibit the action of the cyclo-oxygenase-1 and -2 (COX-1 and -2) enzymes, which metabolize arachidonic acid into inflammatory mediators of the prostaglandin family. NSAIDs have been shown to inhibit neutrophil adherence, decrease degranulation and oxidant production, inhibit neutrophil elastase activity, and induce neutrophil apoptosis [125–127]. Corticosteroids induce anti-inflammatory signals by several mechanisms; a major one may be to reduce the expression of cytokine-induced genes. They enter all cells and bind to the cytoplasmic steroid receptor, and then this complex translocates to the nucleus where it is recognized by specific DNA sequences. The major effect of binding to DNA is the suppression of transcription by opposing the activation of the transcription factors AP-1 and NF-κB [128]. Corticosteroids have been shown to inhibit neutrophil degranulation and ROS production, decrease production of inflammatory mediators, and prevent neutrophil adhesion and migration into RA joints [44, 129–131]. The most widely used DMARD in clinic settings is methotrexate, a compound that blocks folic acid metabolism. Its benefits in RA include the stimulation of neutrophil apoptosis [116], inhibition of the NF-κB pathway [132], and reduced adhesion molecule expression and LTB<sup>4</sup> production [133], consequently decreasing neutrophil recruitment and ROS production [134].

Anti-TNF-α therapies are also widely used for the treatment of RA patients. TNF primes the neutrophil respiratory burst, upregulates the expression of adhesion molecules, cytokines and chemokines, and at high local concentrations can stimulate ROS production in adherent neutrophils [135–138]. Three different TNF inhibitors are available for RA patients who fail to respond adequately to standard DMARD therapy. Infliximab and adalimumab are monoclonal antibodies against TNF, whereas etanercept is a TNFRII fusion protein. All three drugs sequester soluble TNF [139]. Reports regarding the direct effect of anti-TNF agents on neutrophils have been published, and these drugs have been shown to decrease the mobilization of neutrophils from the peripheral blood to inflamed joints [140], decrease *ex vivo* neutrophil ROS production [20], and reduce neutrophil chemotactic and adhesive properties [141].

Tocilizumab, a monoclonal antibody that blocks the soluble and tissue-expressed IL-6 receptor, is also proving to be a highly effective biologic agent in RA treatment [142]. Neutrophils are a major source of soluble IL-6 receptors, which they shed in large quantities when activated, and their accumulation in high numbers within the synovial joint could contribute significantly to IL-6 signaling within the synovium through trans-signaling [143]. *In vivo* therapeutic blockade of IL-6 with tocilizumab induces transient neutropenia caused by apoptosis or phagocytosis of apoptotic neutrophils but does not impair antibacterial neutrophil functions [144].

The current treatments available to RA patients include glucocorticoids, non-steroidal antiinflammatory drugs, and disease-modifying antirheumatic drugs. Only disease-modifying agents—and to some extent glucocorticoids—can impede or halt the inflammatory and destructive disease processes [121]. With a more complete understanding of the immuneinflammatory events that occur in the pathogenesis of RA, scientists have developed therapeutic strategies that include monoclonal antibodies and receptor constructs, which target specific soluble or cell-surface molecules of interest. Biological agents such as monoclonal antibodies and recombinant proteins that target TNF-α, CD20, CTLA-4 (cytotoxic T-lymphocyteassociated protein 4), and the IL-1 receptor as well as therapies based on the blockade of T-cell and B-cell functions have shown efficacy in controlling the physical signs and pain associated

Many interventions used to treat RA exert inhibitory effects on neutrophil responses in inflammation. However, non-steroid anti-inflammatory drugs (NSAIDS), DMARDs, and biologics

Most NSAIDs inhibit the action of the cyclo-oxygenase-1 and -2 (COX-1 and -2) enzymes, which metabolize arachidonic acid into inflammatory mediators of the prostaglandin family. NSAIDs have been shown to inhibit neutrophil adherence, decrease degranulation and oxidant production, inhibit neutrophil elastase activity, and induce neutrophil apoptosis [125–127]. Corticosteroids induce anti-inflammatory signals by several mechanisms; a major one may be to reduce the expression of cytokine-induced genes. They enter all cells and bind to the cytoplasmic steroid receptor, and then this complex translocates to the nucleus where it is recognized by specific DNA sequences. The major effect of binding to DNA is the suppression of transcription by opposing the activation of the transcription factors AP-1 and NF-κB [128]. Corticosteroids have been shown to inhibit neutrophil degranulation and ROS production, decrease production of inflammatory mediators, and prevent neutrophil adhesion and migration into RA joints [44, 129–131]. The most widely used DMARD in clinic settings is methotrexate, a compound that blocks folic acid metabolism. Its benefits in RA include the stimulation of neutrophil apoptosis [116], inhibition of the NF-κB pathway [132], and reduced

Anti-TNF-α therapies are also widely used for the treatment of RA patients. TNF primes the neutrophil respiratory burst, upregulates the expression of adhesion molecules, cytokines and chemokines, and at high local concentrations can stimulate ROS production in adherent neutrophils [135–138]. Three different TNF inhibitors are available for RA patients who fail to respond adequately to standard DMARD therapy. Infliximab and adalimumab are monoclonal antibodies against TNF, whereas etanercept is a TNFRII fusion protein. All three drugs sequester soluble TNF [139]. Reports regarding the direct effect of anti-TNF agents on neutrophils have been published, and these drugs have been shown to decrease the mobilization of neutrophils from the peripheral blood to inflamed joints [140], decrease *ex vivo* neutrophil ROS production [20], and reduce neutrophil chemotactic and adhesive

production [133], consequently decreasing neutro-

with RA [122, 123].

96 Role of Neutrophils in Disease Pathogenesis

do not specifically target neutrophil function [124].

adhesion molecule expression and LTB<sup>4</sup>

properties [141].

phil recruitment and ROS production [134].

Despite the clinical efficacy of these therapies, many patients do not exhibit significant responses or discontinue treatment because of adverse effects. In addition, the limited availability of biological agents in developing countries, the need for parenteral administration of these products, and the high cost restrict access to such therapies for many RA patients worldwide, and this promotes a continuous search for new therapeutic targets and the development of new drugs [145]. Due to these limitations, interest has grown in the use of alternative treatments and herbal therapies for arthritis patients [146, 147] (**Table 1**).


**Table 1.** Current therapeutic targets for arthritis and their effect on neutrophils.
