Induction of Rheumatoid Arthritis and Response to Tyrosine Kinase Inhibitors

The goal of this work is to determine the role of the autoimmune cells in Rheumatoid arthritis (RA) induction and the immunomodulatory mechanism of therapy with Tyrosine Kinase Inhibitors (TKIs) in RA attenuation. B cells were isolated from naive DBA/1 mouse spleens and stimulated for 72 hours with LPS as a positive control forming a mouse model of RA in the presence or absence of 1–5 μM imatinib. B cell staging were assessed by adding 1 μCi of [ H ] thymidine for measuring proliferation in the final 18 hours of the stimulation, and a Beta plate scintillation counter was used to quantitate incorporated radioactivity. Samples of C1.MC/57.1 mast cells were stimulated with 100 ng/mL of Self Cell Factor (SCF) as a positive control of a mouse model of RA in the absence or presence of 1-5 μM of imatinib. Tumour Necrotic Factor (TNF) levels in culture supernatants from C1.MC/57.1 mast cells were measured by ELISA. The histologic grade ( G H ) and the level of TNF of the mouse model of RA were 1/10 and 10 times respectively those in the control one. This inverse proportion clarifies that RA disease is the result of big increase in TNF level perpetuating local inflammation and joint destruction leads to a major decrease in G H with the same ratio. The addition of 1 and 5 μ M doses of imatinib increased G H by 200% and 300% respectively while decreased TNF level to be 12.5% and 10% respectively of that in the mouse model of RA restoring rate of TNF level of normal tissue. This demonstrates that effective mitigation of symptoms of RA is the result of a significant increase in G H because of the cell cycle arrest resulting from the treatment of TKIs which leads to a significant reduction in the level of TNF but with a different ratio to increase G H unlike happened in incidence of RA.


Introduction
Rheumatoid arthritis (RA) is an autoimmune inflammatory disease affects many tissues and organs, but principally attacks flexible (synovial) joints. The exact mechanisms by which innate immune cells contribute to RA progression are not known yet. Mast cells, macrophages, and B cells contribute to RA pathogenesis and progression inducing synovial inflammation and joint destruction [1]. There is compelling evidence that number of mast cells in the human rheumatoid synovium is strongly correlated with the activity of RA [2]. In addition it is evident that macrophages infiltrate the synovium which is characteristically associated with the over-production and activity of TNF-α and other proinflammatory cytokines that potentiate inflammation in RA , transforming growth factor β (TGF β ) and platelet derived growth factor (PDGF) [3,4]. To date there is no good immunotherapy to treat or prevent the development of autoimmune diseases but only for attenuating symptoms and inhibiting disease progression. A recent study in an autoantibody induced mouse model of arthritis showed that Tyrosine Kinase Inhibitors (TKIs) inhibit a select set of tyrosine kinases that are directly implicated in the pathogenesis of RA [5]. Thus, TKIs is a one of those therapies that can treat autoimmune diseases inhibiting signaling pathways implicated in RA, including those mediated by the tyrosine kinases c-Fms and platelet-derived growth factor receptor (PDGFR) [6,7]. However, yet TKIs immunomodulatory mechanism of action is not fully understood. Therapy with TKIs modulates cytokine levels, inhibits T-cell activation and proliferation directly affecting the histologic grade (H G ) [8,9].
Recently, Moawad improved models of clinical and pathology based staging of the cellular kinematics' alterations enables to estimate the energy yield of drug doses and thus expecting the ability of those doses to inhibit the cell cycle progression in order to administer the appropriate dose and introduced an easy method allow more frequent monitoring to therapy response [8][9][10]. In an attempt to understand the immunological basis of RA, current approach tests the ability of TKIS to attenuate RA symptoms in a mouse model of RA.

Methods and Materials
Experiments were performed to determine the ability of imatinib to inhibit each of B cell proliferation and proinflammatory cytokines (TNF-α ) production in vitro as conducted and described by Paniagua et al [11]; Six-to eight-week-old male DBA/1 mice (The Jackson Laboratory) were housed at Stanford University and experiments performed under protocols approved by the Stanford University Committee of Animal Research and in accordance with NIH guidelines. .B cells were isolated from naive DBA/1 mouse spleens by negative selection with MACS beads (Miltenyi Biotec). Isolated B cells were stimulated for 72 hours with LPS (5 μg/ml; Sigma-Aldrich) in the presence or absence of 1-5 μM imatinib. For measurement of B cell proliferation, after 48 hours B cells were pulsed with 1 μCi [3H] thymidine (ICN Pharmaceuticals) for the final 18 hours of the stimulation, and a Beta plate scintillation counter (PerkinElmer) was used to quantitate incorporated radioactivity. For cytokine analysis; mouse mast cell line C1.MC/57.1 [12,13] were serum starved for 6-8 hours, pre incubated with imatinib for 2 hours, and stimulated for 10 minutes with SCF (100 ng/mL; Pepro Tech) in the presence of 0-5 μM imatinib, and after 48 hours culture supernatants were collected and analyzed for TNF-α using a bead-based cytokine assay. Pure imatinib was utilized for both of the in vitro B cell proliferation assays and mast cell cytokine release measurement. The induction of apoptosis by imatinib mesylate was investigated by annexin V (a marker for early apoptosis) or propidium iodide (a marker for cell death) using flow cytometry analysis as conducted by Juurikivi et al. [14].

Results and Analysis
Data and results as shown by Paniagua et al [11]; Imatinib inhibited LPS-stimulated B cell proliferation in a dose-dependent fashion (P < 0.001) for concentrations of 1 μM and higher ( Table 1).
The incorporated radioactivity was quantified with Betaplate scintillation counter. The percentage of Labeled index(%Li) shown in table 1 represent the mean cpm ± SEM of quadruplicates and are representative of 3 independent experiments at statistical significance of P<0.05 by Student's t test, compared with cells stimulated in the absence of inhibitor. Further, TNF-α production by SCF-stimulated C1.MC/57.1 mast cells was significantly reduced by imatinib at a concentration of 1 μM, and restored the normal rate of control sample at 5 μM as shown in Table1.
As shown by Juurikivi et al [14]; the induction of apoptosis by imatinib mesylate was verified as determined by flow cytometry analysis at an imatinib concentration higher than 1 µ M (P < 0.001) [14].

Imatinib Inhibits in Vitro Proliferation of B Cells from Naive DBA/1 Mouse Spleens
Labeled index (Li) of 3

TKIs Inhibit Proinflammatory Cytokines Production
Measuring TNF levels by ELISA in culture supernatants from C1.MC/57.1 mast cells of control, +ve control and treated groups shows that imatinib at1-5 μM dramatically reduced mast cell production of TNF-α to levels similar to those in the unstimulated cell populations. Stimulating C1.MC/57.1 mast cells with SCF contribute to the pathogenesis of RA by producing proinflammatory cytokines where TNF level was increased 1000% from control sample (120 pg/mL) to the +ve control one (1200 pg/mL), while the addition of imatinib was able to decrease PDGFbb-induced TNF release by mast cells in treated groups as shown in table 1; Addition of 5 µ M dose of imatinib was able to restore normal level of TNF in control sample (120 pg/mL) diminishing RA symptoms to show that TKIs suppress PDGFbb-induced TNF production.

Discussion
The aim of this work is to differentiate normal tissue from that of RA induction and evaluate the ability of TKIs to inhibit autoimmune cells production of proinflammatory cytokines. Autoimmune B cell and mast cell C1.MC/57.1 cell lines were chosen for conducting staging assays and proinflammatory cytokine release measuring to determine the relation between G H and TNF-α level in RA tissue as human rheumatoid synovium is an autoimmune disease characterized by large number of mast cells correlates with the activity of the disease and contributes to the pathogenesis of experimental arthritis [2]. Tissue differentiation was conducted by pathologic staging system clarified that G H of RA tissue is in contrast to cancerous tissue less than that of normal counterpart as a result of the excess in cytokines production. New therapies that target specific pathways involved in RA pathogenesis are needed; here imatinib was shown robustly prevents and treats RA by selectively inhibiting a spectrum of signal transduction pathways central to the pathogenesis of RA. Imatinib abrogate PDGFR signaling in a mouse model of RA able to inhibit a narrow spectrum of tyrosine kinases including PDGFR α/β (IC50 = 0.1 μM), c-Fms (IC50 = 1.4 μM), and c-Kit (IC50 = 0.1 μM) as well as macrophage production of proinflammatory cytokines; PDGFR -induced TNF-α production [20][21][22]. Our in vitro data indicate that imatinib potently inhibits diverse cellular responses that synergize in inducing inflammation and thereby the formation of pannus tissue, which invades and destroys adjacent cartilage and bone in RA. Imatinib decreased C1.MC/57.1 mast cells proliferation as well as TNF production in response to PDGFR stimulation. As the mechanisms underlying the initiation and progression of RA remain undefined, our findings in staging the detected tissues suggest that RA induction in a tissue starts by local cell cycle disruption acts via increasing cell doubling time and consequently cell growth energy [15][16][17][18], increasing