Transiently increased circulating CD39+FoxP3+ Treg cells predict the clinical response to Methotrexate in Early Rheumatoid Arthritis.

OBJECTIVES: A subset of human circulating FoxP3+ regulatory T cells expresses CD39 (cTreg39+) and hydrolyses pro-inflammatory adenine nucleotides released at inflammatory foci, rendering the anti-inflammatory agent adenosine. Methotrexate (MTX), inhibiting ATIC, enhances the extrusion of adenine nucleotides and may help Treg39+ cells control inflammation. Therefore, we examined the relation of cTreg39+ cells with the effect of MTX in early Rheumatoid Arthritis (eRA). METHODS: Freshly isolated peripheral blood lymphocytes from 98 untreated eRA patients and 98 healthy controls (HC) were examined by cytometry. Twelve months (12m) after initiating MTX, 82 patients were clinically re-evaluated and cytometry was repeated in 40 of them. The effect of MTX on Treg cell potency was assessed in Treg/Tresp cocultures. RESULTS: The baseline (0m) cTreg39+ cell frequency was elevated in eRA above HC levels. Patients who reached low disease activity at 12 months (12m-LDA, DAS28-ESR≤ 3.2, n = 51) had presented with a significantly higher 0m cTreg39+ frequency vs those who did not (n = 31). The 0m cTreg39+ cutoff for attaining 12 m-LDA was 42.0% (Sensitivity=90.4%/Specificity=96.8%). At 12m, the cTreg39+ frequency was no longer elevated but its association with disease activity remained: it was still significantly higher in patients who had reached LDA vs those who had not. In vitro, MTX augmented the Treg39+ cell potency but had no effect on Treg39- cells. CONCLUSION: MTX cooperates with Treg39+ cells and the baseline cTreg39+ frequency predicts the response to MTX in eRA. In addition, the transiently elevated baseline cTreg39+ frequency in eRA may provide a slot for prompt MTX initiation.

Assessment of sequential combination of 5-fluorouracil-loaded-chitosan-nanoparticles and ALA-photodynamic therapy on HeLa cell line.

BACKGROUND: Natural polymers are used as components of nanoparticles (NPs) for drug delivery, as they provide targeted, sustained release and biodegradability. The purpose of this study was to increase the efficacy of the photodynamic therapy (PDT) by the combination of 5-aminolevulinic acid (ALA) with 5-fluorouracil-loaded-chitosan-nanoparticles (5-Fu-CNPs). METHODS: Nanoparticles based on chitosan (CNPs) were synthesized by the ionic crosslinking method via the TPP addition. 5-Fluorouracil (5-Fu), a first-line anticancer drug, was loaded into these 5Fu-CNPs, and they were assayed as controlled delivery formulation. HeLa cells were incubated in the presence of 5Fu-CNPs for 24h, next ALA was added to the culture medium and 4h later, to complete the PDT, light irradiation took place. Analysis of cell viability, reactive oxygen species (ROS) production, observation of the apoptosis by fluorescence microscopy followed by analysis of caspase-3 activity were carried out. RESULTS: Spherical 5Fu-CNPs with a mean diameter of 324±43nm, were successfully synthesized and characterized by TEM and DLS. 5-Fu incorporation was achieved successfully (12.3µg 5Fu/mg CNP) and the maximum 5-Fu release took place at 2h. The combined administration of 5Fu-CNPs and PDT mediated by ALA (ALA-PDT) led to an improved efficacy of the antineoplastic treatment by generation of great cytotoxicity inducted through an increased ROS production. HeLa cells were destroyed by apoptosis through activation of caspase pathway. CONCLUSIONS: This study proves that combination therapy (photodynamic "ALA"+chemical "5-Fu"+immunoadjuvant "chitosan") may be an effective approach for the treatment of cancer.

IL-15 expression on RA synovial fibroblasts promotes B cell survival.

INTRODUCTION: The purpose of this study was to examine the role of RA Synovial Fibroblast (RASFib) IL-15 expression on B cell survival. METHODS: Magnetically sorted peripheral blood memory B cells from 15 healthy subjects were cocultured with RASFib. RESULTS: RASFib constitutively expressed membrane IL-15. Survival of isolated B cells cultured for 6 days, below 5%, was extended in coculture with RASFib to 52+/-8% (p<0.001). IL-15 neutralizing agents but not isotype controls, reduced this rate to 31+/-6% (p<0.05). Interestingly, rhIL-15 had no effect on isolated B cells but significantly increased their survival in coculture with RASFib. In parallel, B cell IL-15R chains were upregulated in cocultures. BAFF and VCAM-1, that are expressed on RASFib, were tested as potential candidates involved in upregulating B cell IL-15R. Culture of B cells in the presence of rhBAFF or rhVCAM-1 resulted in significantly increased survival, together with upregulation of all three IL-15R chains; in parallel, rhIL-15 potentiated the anti-apoptotic effect of BAFF and VCAM-1. Both BAFF and VCAM-1 neutralizing agents downmodulated the effect of RASFib on B cell survival and IL-15R expression. In parallel, rhIL-15 had a lower effect on the survival of B cells cocultured with RASFib in the presence of BAFF or VCAM-1 neutralizing agents. Peripheral blood B cells from 15 early RA patients demonstrated an upregulated IL-15R and increased survival in cocultures. CONCLUSION: IL-15 expression on RASFib significantly contributes to the anti-apoptotic effect of RASFib on B cells. IL-15 action is facilitated by BAFF and VCAM-1 expressed on RASFib, through an upregulation of IL-15R chains.

A dual action of rheumatoid arthritis synovial fibroblast IL-15 expression on the equilibrium between CD4+CD25+ regulatory T cells and CD4+CD25- responder T cells.

We previously described that fibroblast-like cells from the synovium of rheumatoid arthritis patients (RASFib) constitutively express intracellular and surface IL-15, which induces activation of cocultured T cells. Our objective was to study the effect of RASFib IL-15 expression on the function of human CD4(+)CD25(+) regulatory T cells (Treg). RASFib, through their constitutive IL-15 expression, were able to induce the proliferation of human Tregs stimulated through their TCR, and at the same time potentiated their suppressive action on the cytokine secretion of CD4(+)CD25(-) responder T cells (Tresp). In parallel, constitutive RASFib IL-15 expression mediated an up-regulated response of Tresp. Subsequently, total CD4(+) T cells, containing natural proportions of Treg and Tresp, secreted an increased amount of pathogenic cytokines when cocultured with RASFib despite the presence of proliferating Treg with superior regulatory potency. In summary, RASFib IL-15 exerts a dual action on the equilibrium between Treg and Tresp by potentiating the suppressive effect of Treg while augmenting the proinflammatory action of Tresp; the result is a shift of the Treg/Tresp balance toward a proinflammatory state. This alteration of the Treg/Tresp equilibrium is not observed in the presence of osteoarthritis synovial fibroblasts or dermal fibroblasts, which do not constitutively express surface IL-15. Additionally, Treg with superior suppressive potency were present in the peripheral blood and the synovial fluid of RA patients, but this enhanced immunoregulatory activity was not able to overcome the increased secretion of pathogenic cytokines by RA-Tresp, indicating that rheumatoid arthritis patients demonstrate an altered Treg/Tresp equilibrium in vivo.

Peripheral blood T lymphocytes from patients with early rheumatoid arthritis express RANKL and interleukin-15 on the cell surface and promote osteoclastogenesis in autologous monocytes.

OBJECTIVE: To investigate the osteoclastogenic potential of T cells from the peripheral blood (PB) and synovial fluid (SF) of patients with rheumatoid arthritis (RA) on autologous monocytes, and to study the cytokines implicated in this process. METHODS: T cells and monocytes were isolated from the PB of 20 healthy subjects and 20 patients with early RA, and from the SF of 20 patients with established RA. Autologous T cell/monocyte cocultures were established in the absence of exogenous cytokines or growth factors in order to examine spontaneous ex vivo osteoclast differentiation by tartrate-resistant acid phosphatase staining and calcified matrix resorption activity. RESULTS: Surface RANKL was expressed on freshly isolated T cells from the PB of patients with early RA and the SF of patients with established RA. In addition, surface interleukin-15 (IL-15) was detected on freshly isolated T cells and monocytes from the PB of patients with early RA and the SF of patients with established RA. Autologous T cell/monocyte cocultures derived from the SF of patients with established RA and from the PB of patients with early RA, but not from the PB of healthy controls, resulted in osteoclast differentiation that was significantly inhibited by osteoprotegerin (OPG) and by neutralizing monoclonal antibodies to IL-15, IL-17, tumor necrosis factor alpha (TNFalpha), and IL-1beta. OPG, anti-TNFalpha, and anti-IL-1beta demonstrated a cooperative inhibitory effect. At 1-year followup, surface RANKL and IL-15 and ex vivo osteoclastogenesis were no longer observed on PB T cells or monocytes from patients with early RA in whom clinical remission had been achieved with treatment. CONCLUSION: T cells are important contributors to the pathogenesis of bone erosions in RA through interaction with osteoclast precursors of the monocyte/macrophage lineage.

Human T cells constitutively express IL-15 that promotes ex vivo T cell homeostatic proliferation through autocrine/juxtacrine loops.

Homeostatic proliferation of T cells in vivo is responsible for the maintainance of the T cell pool, and IL-15 is a pivotal cytokine implicated in this process. Known cell sources providing physiologically active IL-15 are monocytes/macrophages, dendritic cells, and stromal cells. T lymphocyte expression of functionally active IL-15 and its possible role in T cell biology have not been investigated. In this study, we demonstrate that human T cells constitutively express IL-15 that acts through autocrine/juxtacrine loops to promote ex vivo homeostatic T cell proliferation.

Rheumatoid arthritis synovial fluid fibroblasts express TRAIL-R2 (DR5) that is functionally active.

OBJECTIVE: To examine fibroblasts grown from the synovial fluid of rheumatoid arthritis (RA) patients for TRAIL-R2 expression, and for susceptibility to apoptosis induced by an agonistic anti-TRAIL-R2 monoclonal antibody (mAb). METHODS: The expression of TRAIL-R2 (DR5) was determined by flow cytometry on fibroblasts grown from the synovial fluid of patients with RA, osteoarthritis (OA), seronegative arthritis, and unclassified monarthritis or oligoarthritis, and on fibroblasts from the synovial membrane of RA and OA patients. Susceptibility to apoptosis mediated by an agonistic anti-TRAIL-R2 mAb was determined by alamar blue bioassay, fluorescence microscopy (annexin V/propidium iodide staining), and caspase activation. RESULTS: Fibroblasts grew from 35 of 50 RA synovial fluid samples, of which 26 were DR5(+) (mean [+/-SD] fluorescence intensity [MFI] 18.74 +/- 2.5). Fibroblasts also grew from 15 of 30 seronegative arthritis synovial fluid samples, 28 of 40 OA synovial fluid samples, and 8 of 20 unclassified monarthritis or oligoarthritis synovial fluid samples; all of these were DR5- (MFI 0.32 +/- 0.02). All 10 of the fibroblast lines from joint replacement surgery or synovectomy specimens of RA patients were DR5(+) (MFI 20.3 +/- 3.2). All fibroblast lines from the synovium of 10 OA patients were DR5-, as were fibroblasts from the skin of 5 healthy subjects. DR5(+) fibroblast cultures underwent apoptosis when treated in vitro with an agonistic anti-DR5 antibody. CONCLUSION: Fibroblasts grown from the synovial fluid and synovial membrane of RA patients express TRAIL-R2 that is functionally active. An agonistic anti-TRAIL-R2 antibody that does not induce hepatocyte toxicity may be an alternative strategy for treatment of RA.

IL-15 and the initiation of cell contact-dependent synovial fibroblast-T lymphocyte cross-talk in rheumatoid arthritis: effect of methotrexate.

To characterize the molecules responsible for synovial fibroblast-T lymphocyte (TL) cross-talk in rheumatoid arthritis (RA), synovial fibroblasts from patients with established RA (RASFibs) were cocultured with TLs from peripheral blood of early RA patients (RAPBTL). TLs from peripheral blood of healthy controls and from synovial fluid of RA served as controls. Adhesion molecules and cytokines were determined by flow cytometry, ELISA, and real-time PCR. RAPBTL (n = 20) induced an up-regulation of ICAM-1, intracellular IL-8, IL-6, IL-15, and surface IL-15 in cocultured RASFibs. In turn, RAPBTL showed an up-regulation of TNF-alpha, IFN-gamma, IL-17, CD25, and CD69 expression. Responses seen with TLs from peripheral blood of healthy controls (n = 20) were significantly lower, whereas responses with TLs from synovial fluid of RA (n = 20) were maximal. Blocking Abs to IL-15 and CD54, but not an isotype-control Ab, down-regulated the increased TL cytokine and activation marker expression. Abs to CD69, CD11a, IL-17, TNF-alpha, and IFN-gamma significantly decreased the up-regulation of RASFib cytokine and CD54 expression. Cocultures using 0.4- micro m inserts did not result in up-regulation of surface molecules or cytokines. Methotrexate significantly inhibited RASFib/TL cross-talk signals and decreased adhesion of TL to RASFibs. In summary, RASFib production of IL-15 induces the proinflammatory cytokines TNF-alpha, IFN-gamma, and IL-17 in cocultured TLs through a cell contact-dependent mechanism. In turn, these cytokines stimulate the expression of IL-15, IL-8, and IL-6 in RASFibs, thereby creating a feedback loop that favors persistent synovial inflammation. Methotrexate seems to disrupt this loop by decreasing cell adhesion.