FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disease characterized by persistent synovitis and hyperplasia of the joint synovium, development of pannus, and invasion of leukocytes into the joint followed by destruction of local articular components such as cartilage and bone [1, 2]. In the RA synovium a variety of cell types can be found, specifically T cells, B cells, macrophages and dendritic cells (DCs) [3, 4]. DCs derive from two sources: stem cells in the bone marrow, and precursor cells found in the circulation. In humans there are four major groups of DCs so far characterized: myeloid DCs (mDCs), plasmacytoid DCs (pDCs), migratory DCs such as Langerhans cells and dermal DCs, and monocyte-derived DCs (mo-DC) [5]. Although DCs represent a relatively small subset of immune cells, they are widely distributed throughout lymphoid and nonlymphoid tissues [6].

DCs have a crucial role in the initiation of primary immune responses. Individuals with autoimmune disease show a high number of aberrantly activated DCs either in circulation or in the autoimmune lesions, secreting large amounts of proinflammatory cytokines that mediate inflammation and differentiation of pathogenic T-helper type 1 and T-helper type 17 cells [7]. Rheumatoid synovial DCs have been described as having a more mature, differentiated phenotype, expressing high levels of HLA-DR, CD86 and nuclear RelB, and have been observed to associate with T cells in perivascular mononuclear cell aggregates surrounding the postcapillary venules, and in germinal center-like structures [8]. In addition, the RA synovium contains abundant immature mDCs and pDCs that express cytokines (interleukin (IL)-12, IL-15, IL-18, and IL-23), HLA class II molecules, and costimulatory molecules that are necessary for T-cell activation and antigen presentation [9]. In the synovial fluid (SF), DCs exhibit a semi-mature phenotype showing low levels of CD80 and CD83 expression [9]. An important sequel of continued antigenic stimulation via DCs is the formation of lymphoid structures at the site of inflammation. By coordinating the recruitment and/or activation of other immune cells, DCs can drive the generation of ectopic lymphoid tissues, as in the case of inflamed synovia in RA and systemic lupus erythematosus [10].

FMS-related tyrosine kinase 3 ligand (Flt3L) is crucial for steady-state pDC and mDC development. Mice lacking Flt3L have reduced numbers of DCs [11], as do mice that are deficient in signal transducer and activator of transcription 3 [12], which is an important molecule in the Flt3L signaling cascade. Conversely, administration of Flt3L to mice or humans leads to a dramatic increase in DC numbers both in lymphoid and nonlymphoid organs [13]. Flt3L is abundantly expressed in most human tissues, as a membrane-bound form and/or as a secreted form. Flt3L is initially synthesized as a membrane-bound protein, which must be cleaved to become a soluble growth factor. The extracellular domain alone has been shown to be sufficient for bioactivity [14]. Ectodomain shedding of Flt3L is metalloproteinase dependent and is mediated by tumor necrosis factor-converting enzyme (TACE) [15], a type 1 membrane protein belonging to a large family of transmembrane metalloproteases (a disintegrin and metalloprotease domain gene family) that was originally identified as the enzyme responsible for the cleavage of pro-tumor necrosis factor (TNF) alpha [16], but also has numerous additional substrates and functions, including a critical role in activating the ligands of the epidermal growth factor receptor, and in the modulation of immune reactions [17]. The receptor for Flt3L, CD135 is a transmembrane receptor tyrosine kinase expressed in bone marrow cells during the early stages of hematopoiesis [18], where it is involved in the control of maintenance, expansion, mobilization and differentiation of progenitor cells [19]. CD135 is required for DC homeostasis, and inhibition of CD135-mediated signals results in fewer DCs [20]. The effects of CD135 deficiency are most evident in the periphery, where this receptor is essential for the homeostatic expansion of DC progenitor populations in lymphoid organs [21].

Flt3L has been shown to accumulate in RA SF and induces arthritis when injected into healthy mouse knee joints. In addition, administration of Flt3L worsens experimental arthritis, while tyrosine kinase inhibitors that target CD135 alleviate experimental arthritis in mice models [22, 23]. Given the relevance of Flt3L/CD135 in early hematopoiesis and DC generation, and possible involvement in RA, we characterized in detail the expression of both receptor and ligand in RA patients in comparison with healthy individuals (HI) and non-RA disease controls.

In the present study we show in RA patients the expression of Flt3L and its receptor in three different compartments: blood, SF and ST. Flt3L was significantly elevated in RA SF compared with paired serum, confirming previous observations [23]. In addition, we reported for the first time that RA serum contains significantly elevated levels of Flt3L compared with HI serum. Moreover, we observed a higher expression of Flt3L (mRNA) in STs of RA patients compared with gout patients. RA and gout samples were matched in terms of macrophage numbers and IL-6 and IL-8 expression. This might indicate that Flt3L levels might reflect disease specificity more than just inflammation.

A detailed analysis of cellular components in paired blood and SF revealed that monocytes are the major cell population that expresses extracellular Flt3L. Monocytes are bone marrow-derived cells that mediate essential regulatory and effector functions in innate and adaptive immunity [32]. Circulating PB monocytes migrate into tissues, where they differentiate into different effector cells such as macrophages, DC and osteoclasts [33] that are of importance in RA pathology. The percentage of circulating CD14⁺ Flt3L⁺ monocytes is increased in RA patients, compared with HI. Moreover, in RA SF the percentage of Flt3L-expressing monocytes is significantly higher compared with paired PB. Importantly, RA SF monocytes have a superior capacity to express Flt3L on a per cell basis compared with PB (circulating) monocytes. Mobilization of preformed Flt3L from intracellular stores rather than de novo synthesis might be responsible for increased Flt3L levels at the site of inflammation. Flt3L is a crucial growth factor for DC differentiation that leads to increased numbers of these cells both in mice and humans [13]. Interestingly, Flt3L can substitute for macrophage colony-stimulating factor in support of osteoclast differentiation and function [34], raising the possibility of a direct role for Flt3L in bone damage in RA.

The expression of the receptor for Flt3L, CD135, is elevated in RA PB monocytes compared with HI PB monocytes. In addition, we show that RA SF monocytes express higher levels of CD135 compared with paired PB monocytes. Coexpression of CD135 and its ligand in the same cell (in this case by RA monocytes) suggests a possible autocrine stimulatory mechanism, as already reported for primary acute myeloid leukemia (AML) [35].

TACE is the major sheddase for Flt3L [15]. Flt3L is primarily produced as a membrane-bound protein [36] and soluble Flt3L is generated through ectodomain shedding [15]. TACE has been implicated in RA due to its role in processing membrane-bound TNF to its soluble form [37]. Previous studies have demonstrated a central role for TNF in RA, and early preclinical studies indicated that inhibition of TACE was beneficial for patients with arthritis [38]. We observed that in RA ST the main sources of TACE were macrophages (both CD68⁺ and CD163⁺ populations), CD55⁺ fibroblast-like synoviocytes, activated endothelial cells or infiltrating monocytes (CD31⁺) and B cells (CD19⁺). As soluble Flt3L levels are highly dependent on TACE activity, the above-mentioned cells might contribute to local levels of soluble Flt3L. Since the TACE expression level (mRNA) in RA and gout STs was the same and Flt3L levels were increased in RA ST, it is tempting to speculate that TACE biological activity in RA might be elevated compared with gout patients. This observation cannot be attributed to a differential degree of ST inflammation between RA and gout STs since the expression of the inflammatory cytokines IL-6 and IL-8 and macrophages numbers (CD68⁺ and CD163⁺ cells) was similar. Macrophages are critically involved in the pathogenesis of RA [39]. Not only do they produce a variety of proinflammatory cytokines and chemokines, but macrophages also contribute to cartilage and bone destruction [40]. In addition, it has been reported that the number of macrophages in RA ST correlates with bone damage and that increased numbers of macrophages are an early hallmark of active disease [3]. One of the main features of macrophages is their high plasticity during development. The nomenclature of general macrophage polarization has been proposed in the last decade, in which M1 (classical, inflammatory) and M2 (alternative, anti-inflammatory) refer to the two extremes of a spectrum of possible macrophage activation status [41]. M1 macrophages are mainly present in RA and are characterized by a proinflammatory phenotype, producing high levels of TNF, IL-1, IL-6, IL-12, reactive oxygen species, and low levels of IL-10 [42]. Here we show for the first time that, in addition to the above-mentioned inflammatory mediators, Flt3L might be considered a specific marker for IFNγ-differentiated macrophages. Polarizing cytokines such as IFNγ might contribute to the high levels of Flt3L found in RA synovium by shifting the macrophage polarization into a M1-like phenotype. Overall, these data suggest that in RA, in addition to circulating monocytes, IFNγ-differentiated macrophages might be an important source of Flt3L.

Sublining macrophages are a reliable biomarker for response to therapy in RA [43]. We have previously shown that oral prednisolone, an effective therapy in RA, was associated with a reduction in macrophage infiltration in ST [26]. In this study we observed a marked reduction of Flt3L serum levels in RA patients after prednisolone treatment, and a significant correlation between the Disease Activity Score in 28 joints and Flt3L serum levels. In addition, in the responder group of RA patients treated with adalimumab we observed a trend toward reduced serum levels of Flt3L. The reduction of Flt3L serum levels observed after effective treatment might reflect the reduction in the numbers of the main source(s) of Flt3L: circulating monocytes and/or ST macrophages. Flt3L has recently been outlined within a panel of preclinical biomarkers of predictive value for the development of RA [44]. Flt3L levels might therefore be valuable as a potential biomarker of inflammation or response to treatment.

The Flt3L/CD135 axis is increased in RA patients compared with HI and disease controls, and is responsive to both prednisolone and adalimumab treatment. Collectively these data suggest that the Flt3L/CD135 axis might be important in RA pathophysiology.