Survivin but not Fms-like tyrosine kinase 3 ligand is up-regulated before the onset of rheumatoid arthritis: a pilot study

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation of joint synovial tissue subsequently leading to the destruction of cartilage and bone. RA is considered a multifactorial disease with both genetic and environmental factors contributing to disease development[1–3]. However, the aetiopathogenic factors leading to disease development are not fully understood.

We, and others, have previously shown that anti-citrullinated protein antibodies of several fine specificities, as well as cytokines, can be detected several years before onset of disease, suggestive of an upregulation of immune system years before the onset of symptoms indicating a joint disease[4–8].

The ability to predict the development of RA in nonsymptomatic individuals is as yet limited, and therefore there is a need for additional biomarkers to be identified. Survivin is an intracellular protein with anti-apoptotic and cell-cycle regulatory functions, and Fms-like tyrosine kinase 3 ligand (Flt3L) is involved in the function of cells of the immune system[9, 10]. High levels of survivin and Flt3L in blood and synovial fluid of patients with RA are implicated in the pathogenesis of joint inflammation[11–13]. Flt3L has recently been highlighted within a panel of preclinical biomarkers highly predictive for the development of RA[8].

In the present study, using blood samples from the Medical Biobank of Northern Sweden, we analyzed the levels of the two proteins, survivin and Flt3L, in presymptomatic individuals and matched controls together with samples taken at the time of diagnosis. The results were related to previously published concentrations of cytokines and chemokines[5].

In this pilot study we investigated the presence of survivin and Flt3L in plasma samples from presymptomatic individuals, population controls and RA patients collected at the time of diagnosis. Survivin and Flt3L have recently emerged as biomarkers of joint damage and poor response to anti-rheumatic treatment[17, 18]. The present study addressed the question of whether the levels of these proteins were altered before the onset of RA symptoms, and whether they were associated with other inflammatory markers; for example, cytokines, chemokines and anti-CCP2 antibodies analyzed at the same time points. The levels of survivin in the samples collected years before onset of RA symptoms were significantly increased compared with matched controls. The levels of survivin were prominently high in the anti-CCP-positive presymptomatic individuals. In the these individuals, survivin was most strongly associated with IL-9 and IL-12 (that is, the cytokines predisposing and assuring formation of Th1 and Th17 proinflammatory T-cell subsets) and also IL-2, the cytokine regulating the formation of effector T cells. The concentration of Flt3L was most evidently increased in RA patients and to a lower extent in the presymptomatic individuals. In RA patients, the levels of Flt3L correlated to the cytokines of the Th1, Th2 and regulatory T-cell lineages and monocyte chemoattractant protein-1, known among the major chemoattractants produced by the inflamed synovia.

Survivin has been shown to be crucial at the early stages of T-cell development, where it is required for the formation of a functional T-cell receptor[19, 20]. Survivin is also suggested important for intracellular transfer of signals from the co-stimulatory molecules during T-cell activation[21], and for the formation of memory immune responses[22]. In the context of arthritis, extracellular survivin has been found in the blood and synovial fluid of RA patients[11, 23]. The proportion of survivin-positive patients varied between 60% in patients with early RA and 28% in a cohort of established and treated RA patients[11, 13]. A prospective study of the Swedish patient cohort with early RA showed that high levels of survivin measured at the first visit to a rheumatologist were predictive for a severe cause of the disease[13]. Indeed, extracellular survivin was consistently associated with the development and progression of joint damage in RA and also distinguished patients with a therapy-resistant disease, nonresponders to biological treatment and those with a low rate of disease remission[11, 13, 18, 23]. High levels of survivin were also detected in the rheumatoid synovia where the expression of survivin correlated with the synovial infiltration with macrophages and memory T cells and with the low rate of apoptosis. These findings suggested a key function for survivin in the regulation of invasive properties of fibroblasts in the inflamed rheumatic joint[24–26]. The result of our study is not consistent with these findings, since the concentration of survivin was increased before the patients presented any symptoms of joint disease. Of course, we cannot rule out early synovitis in symptomless individuals, although results from other studies show a rather late involvement of the synovia in the antibody-positive patients with joint complains[27].

The functional effects of Flt3L are mediated and occur through interaction with Fms-like tyrosine kinase 3, its receptor tyrosine kinase. Fms-like tyrosine kinase 3/Flt3L signaling has recently been shown critical for the development of early B-cell progenitors and dendritic cells, and for the expansion of induced regulatory T cells[10, 28]. Blockade of Flt3L signaling using a small-molecule Fms-like tyrosine kinase 3 inhibitor ameliorates antigen-induced arthritis[29]. The experimental analysis of common biological processes linked high expression of survivin and Flt3L in RA patients and showed that Fms-like tyrosine kinase 3/Flt3L signaling was required for the expression of survivin in vivo[15]. In this case, the expression of survivin in leukocytes would appear as a downstream event of Flt3L signaling, and is in contradiction to the present observation of survivin as a predictor of disease development. Interestingly, survivin is increased in the preclinical phase of RA and showed association with another established predictor of RA, anti-CCP antibodies. Additionally, survivin was associated with the pattern of regulatory cytokines (IL-9, IL-12) already at the presymptomatic stage of RA and potentially supported formation of T-regulatory cells and proinflammatory Th1 and Th17 cell subsets with known pathogenic importance for RA[30, 31].

Our recent study showed that the pattern of cytokines could be helpful in distinguishing presymptomatic individuals from controls or RA patients[5]. In the presymptomatic individuals, the RA-specific autoantibody production, including anti-CCP and IgM-RF, was strongly related to the T-cell cytokines. These cytokines are essential for the control of antibody production in the steps of engagement of antigen-presenting macrophages and B cells (that is, IL-13 and IL-4[32]), for the maturation of induced T-regulatory cells and Th17 cells (that is, IL-9[30]), and for the differentiation of follicular B cells and formation of germinal centers (that is, IL-4[33]).

There are two major limitations within this study: a relatively small size of the tested cohort, and a broad variation of the time interval between the blood samples collected before the onset of the disease and the RA diagnosis. These samples were not collected on a regular basis but were retrieved from the existing bank of blood donors. Stratification of the studied subjects reduced the numbers in each group and made additional complications for the detection of statistical significance.

In summary, increased levels of survivin in patients were detected years before the onset of RA symptoms, particularly in those individuals positive for anti-CCP antibodies. The high levels of survivin appear to be more related to the cytokines produced by the Th1 and Th17 subsets. These results suggest a role for survivin at the early presymptomatic stages of RA.