Introduction
Expression of the regulatory peptides, platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-β) are increased in synovial tissue and fluid of rheumatoid arthritis (RA) patients [
1‐
4]. PDGF has been implicated in RA pathogenesis, mainly through its function as a growth factor for fibroblast-like synoviocytes (FLS) [
3,
5]. In contrast, the actions of TGF-β are more complex. TGF-β plays a crucial role in maintaining immunological tolerance through the inhibition of lymphocytes and macrophages [
6]. On the other hand, it recruits and activates naive monocytes [
6], stimulates proliferation [
7] and induces aggrecanase synthesis [
8] by FLS. Systemic administration of TGF-β protects against development of collagen arthritis in mice [
9], whereas direct injection of TGF-β into rat joints leads to pronounced synovitis [
10].
In addition to these growth factors, chronically inflamed RA synovia contain a multitude of inflammatory mediators that may act in concert with each other. In this context, aggravating as well as mitigating effects of growth factors and cytokines on FLS have been demonstrated. For example, PDGF was reported to enhance IL1β-induced prostaglandin E2 production, while inhibiting collagenase synthesis [
11]. Also, PDGF was shown to induce synthesis of IL8 and MIP1α, along with IL1β, by FLS [
12], and also to synergize with TNFα to stimulate IL1β secretion, although these results are somewhat confusing since FLS are not typically considered a significant source of IL1β. On the other hand, TGF-β was earlier shown to inhibit TNFα-induced RANTES synthesis by FLS [
13]. A systematic study of the nature of the interaction among these mediators was not undertaken to date. Hence, the interplay between PDGF, TGF-β, and cytokines such as TNFα and IL1β on the activation of FLS remains unclear, albeit of potential significance considering the abundance of these proteins in the RA synovial environment.
Consequently, we set out to systematically determine the effect of PDGF and TGF-β, alone and in combination, on inflammatory biomarker expression and secretion by FLS. We describe significant potentiation by PDGF and TGF-β of the production of certain cytokines, chemokines, and matrix metalloproteinases (MMP) by FLS. This synergy was mediated by tyrosine-kinase receptor activation and dependent on PI3K, both of which are receiving attention as possible novel approaches to RA drug therapy.
Materials and methods
Reagents
Cytokines and TGF-β were obtained from R&D Laboratories (Minneapolis, MN, USA). Imatinib mesylate (LC Laboratories, Woburn, MA, USA) was dissolved in water. All other reagents, including PDGF-BB, were from Sigma (St. Louis, MO, USA) unless otherwise noted. Stock solutions in DMSO (1000×) of PD98059 and LY294002 were kept at -80°C.
Fibroblast-like synoviocytes (FLS)
FLS were cultured from the synovial tissues of RA patients undergoing arthroplastic surgery, as previously described [
14], after obtaining informed consent under approval from the University of California, San Diego Institutional Review Board, and maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with antibiotics, glutamine, and 10% fetal bovine serum. Passages 4 through 8 were used in experiments. Cells were subjected to a two- to three-day reduced serum condition (0.1% fetal bovine serum) prior to stimulation to minimize baseline activity.
Secreted protein assays
FLS supernatants at 24 hours following stimulation were assayed by ELISA for IL6 (eBioscience, San Diego, CA, USA), MMP1, and MMP3 (GE Healthcare Life Sciences, Piscataway, NJ, USA). Standard curves were constructed by regression line fitting on log(absorbance) vs log(concentration). Levels of cytokines and chemokines in supernatants were determined by Luminex multiplex analysis (BioRad Bio-Plex assays, Hercules, CA, USA) from four-parameter standard curve fits.
Gene expression assays
Messenger RNA for IL6, MIP1α, and MMP3 were quantified by real-time TaqMan quantitative Polymerase Chain Reaction (qPCR), using FLS cDNA, with GAPDH used as a housekeeper (all reagents from Applied Biosystems, Foster City, CA, USA). Resulting threshold cycle (Ct) data were normalized to standard curves constructed from cDNA from IL1β-stimulated FLS [
15], yielding cell equivalents. The ratio between the specific cytokine and GAPDH cell equivalents (relative expression units, REU) is reported.
Western blot
FLS extracts were prepared in RIPA buffer with Complete Protease Inhibitors (Roche Applied Science, Indianapolis, IN, USA), denatured in sample buffer and 0.1 M dithiotreitol, and fractioned on Invitrogen (Carlsbad, CA, USA) NuPage 4 to 12% precast gels. Following blotting to polyvinylidene fluoride (PVDF) membranes and blocking with 5% dry milk, blots were probed with antibodies against phospho- or total p38, JNK, Erk, or Akt, as well as with secondary anti-rabbit-IgG-HRP (all Cell Signaling Technologies, Danvers, MA, USA). GAPDH was used as a gel loading control (antibody from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were developed with Immun-Star WesternC ECL substrate (BioRad, Hercules, CA, USA) and imaged on a VersaDoc imaging system (BioRad), using QuantityOne software (Hercules, CA, USA) for image capture and densitometry.
Statistical analysis
Data are reported as mean and standard error of the mean (SEM). Protein secretion and gene expression data in single time-point experiments were analyzed by one-way ANOVA followed by Tukey-Kramer's post-hoc test comparing all groups, or by Dunnett's post-hoc test comparing control to all others, as appropriate. Time course data were analyzed by two-way ANOVA followed by contrast testing. Student's t-test was used to examine synergistic effects of growth factors and cytokines. Real-time qPCR data were log-transformed prior to analysis.
Discussion
The chronically inflamed rheumatoid synovium is a complex environment with various cellular subtypes, cytokines, growth factors, chemokines, proteases and mechanical phenomena interacting with each other over time. Animal models may provide valuable insights into disease processes, but are limited in their ability to demonstrate specific target mediated effects that correspond to observations in RA. In addition, the typical rat and mouse models utilized, albeit useful in many ways, do not fully recapitulate human disease [
16]. Studies of synovial tissue
ex vivo can provide a snapshot of cellular activity in RA, and the accumulation of these observations provide insight into disease pathogenesis.
In vitro studies of isolated human synovial cells can illuminate dynamic disease-specific cellular mechanisms. However, complete recapitulation of the RA synovial complexity
in vitro is impractical if not impossible. Typical
in vitro studies involve stimulating or activating cells, blocking signaling pathways and observing disease-relevant gene expression or proliferative outcomes. Interestingly, such studies have demonstrated what appear to be unresolved opposing effects of various mediators known to be present in the rheumatoid synovium. In this study we attempt to incrementally close the gap between cells and tissue by evaluating the role of peptide mediators historically identified as growth factors (PDGF and TGF-β) in providing a context for the response of FLS to inflammatory cytokines.
The surprising and novel central finding of these studies is the significant and striking synergistic effect of a combination of PDGF and TGF-β on cytokine-induced FLS secretion of selected inflammatory mediators (IL6, IL8, MIP1α and MMP3), while leaving some other mediators unaltered. Both PDGF and TGF-β induce proliferation of FLS [
3,
5,
7], and cytokine-induced growth of FLS is potentiated by PDGF [
17] and TGF-β [
7]. Therefore, a potential reason for the synergistic effect of growth factors and cytokines on secretion of inflammatory mediators by FLS could simply be that a higher number of FLS are present after growth factor activation. This is unlikely to provide an explanation for our findings, however, for two reasons. First, FLS are slow growing cells [
14] and the relatively short incubation times employed in the current studies (3 h for mRNA, 24 h for protein secretion) make it unlikely that a significantly higher number of FLS could have been generated. Second, in the mRNA expression studies, all data were normalized to GAPDH for the purpose of controlling for cell numbers. Since the mRNA and protein results essentially mirrored each other (compare for example Figures
2 and
4, and Figures
5a and
5b), the underlying reason for the synergy of the two growth factors along with cytokines on FLS is unlikely to be simply an effect on cell number.
To our knowledge, this report is the first to establish a synergy of the combined effects of PDGF and TGF-β on cytokine-induced gene expression in FLS. The underlying signaling mechanisms are not entirely clear. However, the effect is receptor-mediated as demonstrated by the reversing action of imatinib mesylate, also known as Gleevec. This compound is a moderately selective tyrosine kinase inhibitor that targets several classes of receptor kinases including abl [
18], c-kit [
19], c-fms (the M-CSF receptor) [
20], and PDGF receptor kinases [
18,
21]. In FLS, imatinib blocks PDGF-induced proliferation and phosphorylation of downstream targets of PDGF receptor stimulation [
22‐
25]. Due to its inhibition of abl, imatinib also has a role in TGF-β induced signaling and fibrogenesis in cultured fibroblasts [
26,
27]. Hence, the reversal of the growth factor-induced synergy by imatinib indicates involvement of specific growth factor signaling pathways.
With respect to common signaling pathways in fibroblasts, both PDGF and TGF-β are known to activate the PI3K [
28,
29] and the Ras-Raf-MEK-ERK pathways [
30,
31]. Indeed, both Akt and ERK were phosphorylated for at least four hours by 2GF treatment of FLS, making them attractive signaling candidates. The testing of this hypothesis was complicated by the fact that the PI3K inhibitor used (LY294002) had significant effects on IL6 expression induced by TNFα alone, as earlier reported [
32] and similar to earlier published results where IL17 was used to induce IL6 [
33]. To circumvent this problem, we took advantage of the fact that a short pulse of 2GF, separated in time from the TNFα stimulation, was capable of potentiating TNFα-induced IL6 expression to the same extent as continuous incubation with 2GF without affecting signaling in FLS stimulated with TNFα alone. In this system, LY294002 added before 2GF and removed prior to the addition of TNFα significantly blocked the synergy, demonstrating a PI3K role. The ERK pathway, however, did not appear to play a role, at least at levels distal to MEK1. Thus, PI3K constitutes a pharmacological target of interest for synovitis mediated by this mechanism. Indeed, studies antagonizing PI3K signaling have shown promise in animal models of arthritis. Gene transfer of a negative regulator of PI3K signalling, PTEN, ameliorates collagen arthritis [
34] and in murine models of arthritis, inhibitors of the gamma isoform PI3K have been shown to reduce joint destruction [
35]. Notably, this particular isoform was recently demonstrated to be specifically upregulated in human RA FLS [
36].
These findings, in addition to demonstrating novel synergistic effects of growth factors and cytokines on FLS, may also have clinical implications. In particular, the effect of imatinib is of interest, since this compound is already in clinical use for Philadelphia chromosome-positive hematological malignancies [
37] as well as for gastrointestinal stromal tumor [
38]. A few case reports exist [
39,
40] of imatinib mesylate as a successful treatment for refractory RA, with reductions in swollen joint counts and CRP observed. In addition, a phase II study of imatinib in RA has been completed (Clinicaltrials.gov identifier # NCT00154336), however the results have not yet been made publicly available. In animal models, imatinib limits joint inflammation in mouse collagen arthritis [
23,
41] and rat adjuvant arthritis [
25], and reduces joint destruction in collagen arthritis in rats [
42]. Additionally, in preliminary studies in our laboratory, imatinib limited the arthritis induced by K/BxN serum transfer (data not shown), a murine model in which the adaptive immune system has been bypassed. The precise mechanism of imatinib in RA is not known and could involve downregulation of the function of a number of cell types, as shown
in vitro: T and B lymphocytes [
23,
43], macrophages [
20,
44], osteoclasts [
42], and mast cells [
23,
45]. The studies described herein provide yet another potential explanation for the effect of imatinib in arthritis: inhibition of a two-legged response by FLS, which require both a cytokine and growth factors to become activated to its fullest potential.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SR designed and performed the experiments and statistical analysis, and prepared the manuscript. MPC provided the animal model and edited the manuscript; and DLB conceived of the study, designed experiments, and edited the manuscript.