The mechanisms neutrophils use to kill microorganisms have the potential to alter or injure normal tissues . Neutrophils’ Fcγ receptors illustrate this paradox. The best described role for neutrophils’ FcγRs is the optimization of the phagocytosis of IgG-opsonized pathogens [42–46]. However, these receptors are also involved in a variety of IgG-dependent processes including clearance of the naturally occurring immune complexes and the pathogenesis of auto-immune disorders such as RA . Although RFs are present in the serum of the majority of RA patients and are a predictor of disease severity, their potential role in FcγRs activation and in RA pathogenesis is poorly characterized [47, 48]. Yet, as IgG-IgM complexes (see Figure 6), RFs complexes represent potential ligands for FcγRs or for IgGs bound to FcγRs.
We observed that incubation of neutrophils with a preparation of RFs enhanced their ability to adhere to HUVECs. To our knowledge, this result represents the first observation of a biological effect of human RFs which could participate in the RA-associated vascular inflammation. In addition, we showed that activation of FcγRs of neutrophils with anti-human IgG antibodies which mimick the presence of RFs leads within 30 minutes to an extracellular release of lysosyme and superoxide anions concomitant with an enhancement of the adhesion of neutrophils to endothelial cells. These events may end up damaging the endothelium, a possibility supported by our observation that supernatants of neutrophils in which FcγRs have been cross-linked altered the phenotype of endothelial cells in an inflammatory direction (increased expression of ICAM-1 and production of IL-8). As IL-8 is a potent chemotactic mediator for neutrophils, the combined increase of IL-8 and ICAM-1 will lead to a positive activation loop of recruitment and adhesion of neutrophils with the potential to induce local vascular injury.
Few studies have characterized blood neutrophils of RA patients in spite of the demonstrated presence of circulating IC. Chronic peripheral activation of neutrophils in polyarticular juvenile RA has been reported and data suggest that blood neutrophils from RA patients have impaired FcγR-dependent bactericidal functions (superoxide production) [49, 50]. On the other hand, generation of mice with neutrophil-selective expression of FcγRIIa and FcγRIIIb indicate that, in a model of soluble immune complexes deposited in the vasculature, FcγRIIIb is responsible for slowing down neutrophils thereby facilitating adhesion to endothelial cells [13, 26]. Immobilized neutrophils may then activate FcγR-dependent cytotoxic functions and promote local endothelium damage. In innate immunity, by contrast, the vascular wall is rarely in contact with the toxic products of neutrophils as neutrophils leave the circulation by transendothelial migration. They arrive at the inflammation site where they release radical oxygen species and granular contents, most often in phagosome, thus preventing major damage to the cellular environment.
In humans, a similar mechanism of activation of FcγRs on circulating neutrophils has previously been described in anti-neutrophil cytoplasm autoantibody (ANCA)-associated diseases. These antibodies activate circulating neutrophils leading to vascular inflammation, a process in which FcγRs are directly involved. ANCAs are directed against enzymes stored in neutrophil granules but which have translocated to the plasma membrane in response to a minor inflammatory stimulus. The F(ab’)2 portion of ANCAs engages the antigen and the Fc portion engages FcγRs. This FcγR-mediated interaction is required for neutrophil activation [51, 52]. This mode of stimulation of neutrophils by ANCAs closely mimicks the mode of stimulation of RF proposed in the present study and argues for a role of RFs and RF-containing IC in RA-associated vascular inflammation. ANCAs-associated diseases, such as Wegener granulomatosis, clearly indicate that activation of circulated neutrophils can lead to vascular inflammation.
We showed that the circulating neutrophils possess membrane-bound IgGs which may be recognized by RFs leading to their activation. The mechanism of action of RF may require both the direct recognition of RF complexes by free FcγRs, such as HA-IgG and the binding of membrane-bound IgGs by RFs as pre-incubation of neutrophils with PPP enhances the RF-dependent activation of neutrophils.
Several data suggest that the affinity of FcγRIIa may be modulated by a priming signal such as fMLP, TNF or a mechanism including granular proteases [53, 54]. This is of particular relevance in the inflammatory context of arthritis where elevated concentrations of various cytokines and mediators are present and may activate circulating neutrophils. A cytokine-dependent increase of FcγRs affinity would influence the amount of membrane-bound IgGs and consequently the RF-dependent activation of neutrophils of RA patients. Of direct relevance to the present study, the efficiency of anti-TNF therapy is related to a loss of function of FcγRIIa . This effect may be mediated by the ability of TNF to modulate the functional responsiveness of FcγRIIa  and may, at least in part, explain the positive effects of anti-TNF therapy on endothelial dysfunction as previously proposed .
In the present study, we used a RF preparation purified from the sera of RA patients. This RF containing preparation is polyclonal, i.e., that it contained different populations of RFs (IgM and possibly IgG) with different specificities and affinities. The affinity of RFs for IgGs combined with the modulation of FcγR affinity for IgG by RA-associated cytokines will impact on the FcγR-dependent activation of neutrophils and consequently on the inflammation of the endothelium observed in RA patients. Further experiments using neutrophils from RA patients untreated or treated with anti-TNF therapy should help to understand not only the role of RA-associated cytokines on FcγR activity, but also the role of RF in the pathogenesis of RA.
In RA patients, high concentrations of RFs and the presence of extra-articular manifestations such as vasculitis are predictors of increased mortality, primarily from cardiovascular diseases . Patients with ANCA-associated diseases (Wegener granulomatosis) also present higher risks to develop cardiovascular diseases . On the other hand, FcγRs have been proposed to play a critical role in atherogenesis. Indeed, in apoE−/−mice (a model of human atherosclerosis), FcγRs appear to be important for the clearance of LDL-containing IC and apoE−/− mice deficient for the activating γ chain of FcγRs show less development and progression of atherosclerosis than apoE−/− mice which possess the γ chain of FcγRs . These data support a role for leukocytes’ FcγRs in cardiovascular diseases associated with auto-immune diseases.
Systemic lupus erythematosus (SLE) is a chronic inflammatory disease also characterized by systemic inflammation and the presence of auto-antibodies and RF. Patients with SLE have an increased mean intima media thickness and SLE represents a risk factor for cardiovascular disease [60, 61]. This indicates that the engagement of FcγRs of neutrophils and other immune cells may be involved in vascular dysfunction associated with various auto-immune inflammatory diseases.
Further examinations of neutrophil responses to RFs isolated from different patients are warranted. Indeed, the Ig composition, the affinity and the size of the RF complexes could influence neutrophil activation. Nevertheless, our results present a first report underlining the role of RFs in FcγR-dependent activation of circulating neutrophils and the potential consequences on systemic vascular inflammation. We previously characterized several of the pathways downstream of neutrophil’s FcγRs and we showed that both receptors cooperate for optimal FcγR-dependent responses [62, 63]. In particular, FcγRIIIb activation triggers a specific calcium signal . As FcγRIIIb is nearly exclusively expressed on human neutrophils, targeting this receptor or the FcγRIIIb-dependent calcium signal, may represent an interesting clinical avenue which could help control RF-associated systemic inflammation in RA patients.