In vitro spontaneous osteoclastogenesis of human peripheral blood mononuclear cells is not crucially dependent on T lymphocytes.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 60, No. 4, April 2009, pp 1020–1025 DOI 10.1002/art.24413 © 2009, American College of Rheumatology In Vitro Spontaneous Osteoclastogenesis of Human Peripheral Blood Mononuclear Cells Is Not Crucially Dependent on T Lymphocytes Bernard Vandooren,1 Lode Melis,2 Eric M. Veys,2 Paul P. Tak,3 and Dominique Baeten1 physiologic freezing and thawing as a model for the activation of PBMCs, spontaneous osteoclastogenesis was significantly increased in cryopreserved versus fresh cells. Under these conditions, spontaneous osteoclastogenesis was not dependent on T lymphocytes, since it was not influenced by T cell depletion and persisted in purified CD14ⴙ cell cultures supplemented with M-CSF and RANKL. In contrast to studies with fresh PBMCs, spontaneous osteoclastogenesis under these conditions did not appear to be clearly different between healthy subjects and patients with arthritis. Conclusion. Spontaneous osteoclastogenesis in vitro is dependent on T lymphocytes or on the direct activation of monocytic cells, depending on the test conditions. This variability warrants better validation of the relevance of this functional test for in vivo osteoclastogenesis. Objective. In vitro spontaneous osteoclastogenesis from peripheral blood mononuclear cells (PBMCs) is increased in diseases with excessive bone loss. The purpose of this study was to reassess the role of T lymphocytes in this process. Methods. Fresh or cryopreserved PBMCs obtained from healthy subjects and from patients with rheumatoid arthritis, psoriatic arthritis, and nonpsoriatic spondylarthritis were cultured at high density and stained for tartrate-resistant acid phosphatase (TRAP). Resorption of mineralized matrix was assessed by a dentin disc assay. CD14ⴙ monocytes and CD3ⴙ T cells were selected using magnetically labeled antibodies. Results. Numerous multinucleated, TRAPⴙ, dentin-resorbing osteoclasts developed spontaneously from fresh PBMCs from healthy individuals. This process was abrogated by T cell depletion and was restored by exogenous macrophage colony-stimulating factor (MCSF) and RANKL, indicating the important role of T cells in spontaneous osteoclastogenesis in vitro. Using Destruction of juxtaarticular bone is a common complication of chronic inflammatory joint diseases such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA). The development of these focal bone erosions is critically dependent on osteoclasts, which are multinucleated cells specialized in the resorption of mineralized tissue. Osteoclasts originate from precursor cells of myeloid origin that circulate in the blood as CD14⫹CD11bhigh monocytes (1). Under physiologic conditions, the maturation and activation of osteoclasts are mediated by macrophage colony-stimulating factor (M-CSF) and RANKL expressed by osteoblasts and stromal cells. Under pathologic conditions such as arthritis, this process is markedly enhanced by proinflammatory cytokines such as tumor necrosis factor ␣ (TNF␣), interleukin-1␤ (IL-1␤), and IL-17 (2). Activated T cells as well as stromal cells, such as fibroblastlike synoviocytes, are important mediators of enhanced osteoclastogenesis since both can express RANKL and can produce proinflammatory cytokines (3). This publication reflects only the authors’ views. The European Community is not liable for any use that may be made of the information herein. Supported by The Netherlands Organization for Scientific Research, the Dutch Arthritis Association, and the European Community Sixth Framework Programme (project AutoCure). 1 Bernard Vandooren, MD, Dominique Baeten, MD, PhD: Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, and Ghent University Hospital, Ghent, Belgium; 2 Lode Melis, MSc, Eric M. Veys, MD, PhD: Ghent University Hospital, Ghent, Belgium; 3Paul P. Tak, MD, PhD: Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Dr. Vandooren and Mr. Melis contributed equally to this work. Address correspondence and reprint requests to Dominique Baeten, MD, PhD, Department of Clinical Immunology and Rheumatology, F4-218, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: D.L.Baeten@amc.uva.nl. Submitted for publication August 18, 2008; accepted in revised form January 7, 2009. 1020 SPONTANEOUS OSTEOCLASTOGENESIS BY HUMAN PBMCs IN VITRO Table 1. 1021 Characteristics of the RA, non-psoriatic SpA, and PsA patients* Age, mean ⫾ SD years Sex, no. male/female Disease duration, mean ⫾ SD years CRP, mean ⫾ SD mg/liter ESR, mean ⫾ SD mm/hour Swollen joint count, mean ⫾ SD Erosive disease Antibodies No. with RF No. with ACPA Medications No. taking DMARDs No. taking corticosteroids RA (n ⫽ 5) Non-psoriatic SpA (n ⫽ 5) PsA (n ⫽ 5) 62.0 ⫾ 12.3 0/5 8.9 ⫾ 12.8 19 ⫾ 20 31.8 ⫾ 16.2 6.4 ⫾ 4.1 4 26.8 ⫾ 10.8 2/3 1.3 ⫾ 1.6 16 ⫾ 19 13.2 ⫾ 16.0 1.6 ⫾ 1.3 0 59.4 ⫾ 15.5 4/1 5.2 ⫾ 4.5 15 ⫾ 12 17.6 ⫾ 11.8 3.4 ⫾ 2.3 1 4 2 0 0 0 0 3 2 2 0 1 0 * RA ⫽ rheumatoid arthritis; SpA ⫽ spondylarthritis; PsA ⫽ psoriatic arthritis; CRP ⫽ C-reactive protein; ESR ⫽ erythrocyte sedimentation rate; RF ⫽ rheumatoid factor; ACPA ⫽ anti–citrullinated protein antibody; DMARDs ⫽ disease-modifying antirheumatic drugs. Spontaneous osteoclastogenesis refers to the in vitro differentiation of peripheral blood mononuclear cells (PBMCs) into mature osteoclasts without the addition of exogenous mediators (4). Although there is no evidence that spontaneous osteoclastogenesis occurs in vivo, this functional assay has been proposed as a surrogate marker for the potential to form osteoclasts in vivo since it is increased in a variety of conditions associated with bone loss (4–7). Studies using the TNFtransgenic mouse model as well as clinical trials with TNF blockers indicate that enhanced spontaneous osteoclastogenesis in vitro is related to a TNF-mediated increase in myeloid precursor cells (2,4,8,9). Other studies, however, have emphasized the crucial role of activated T lymphocytes in this process (3,6,7). In the present study, we reassessed the mechanisms of spontaneous osteoclastogenesis in vitro with special focus on the role of T lymphocytes. PATIENTS AND METHODS Isolation of PBMCs. Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) density-gradient centrifugation was used to isolate PBMCs from the peripheral blood of healthy individuals and patients with RA who fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) classification criteria (10), patients with PsA who fulfilled the ClASsification of Psoriatic ARthritis criteria (11), and patients with non-psoriatic spondylarthritis (SpA) who fulfilled the European Spondylarthropathy Study Group classification criteria (12) (n ⫽ 5 patients per group). All patients had active disease, and none had been treated with TNF blockers. All patients gave written informed consent to participate in the study, and the study was approved by the Local Ethics Committee at Ghent University Hospital. Demographic and clinical features are shown in Table 1. Purification of CD14ⴙ monocytes and CD3ⴙ T lymphocytes. Fresh PBMCs were resuspended in ice-cold phosphate buffered saline plus 0.5% bovine serum albumin plus 2 mM EDTA and were incubated with magnetic microbeads that had been labeled for 15 minutes at 4°C with either anti-CD14 antibodies or anti-CD3 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany). After washing, the cell suspension was applied to a magnetic-activated cell sorter (MACS) column attached to a strong magnetic field (MACS separator; both from Miltenyi Biotec) and flushed 3 times with 500 l of buffer. The effluent was collected as the depleted cell fraction. After removal of the magnet, the adherent cells were eluted as the positively selected fraction. The purity of both fractions was ⬎90% in all experiments, as tested by flow cytometry. Physiologic freezing and thawing of PBMCs. Six million PBMCs were resuspended in 500 l of a mixture of 80% fetal calf serum (FCS) and 20% RPMI 1640 medium (both from Gibco Invitrogen, Carlsbad, CA). Subsequently, a mixture of 20% DMSO (VWR International, West Chester, PA) and 80% RPMI 1640 was slowly added to the suspension at 4°C, and the vials were transferred to a Nalgene cryovial container (Nalgene Nunc, Milwaukee, WI) at –80°C. For recovery of frozen cells, the cryovials were rapidly thawed in a 37°C water bath, and the cell suspension was gradually diluted to 1:10 in RPMI 1640 plus 10% FCS. Cell culture. A total of 700,000 PBMCs or 100,000 purified CD14⫹ cells were suspended in 300 l of complete RPMI 1640 and seeded into 16-well glass culture slides (Nunc/ Thermo Fisher Scientific, Rochester, NY). In coculture experiments, 300,000 purified CD3⫹ cells were added to 100,000 CD14⫹ cells. The medium was refreshed every 3 days by replacement of the upper 200 l of the culture medium for a total of 14 days. In specific experiments, the medium was supplemented with recombinant human RANKL and M-CSF (R&D Systems, Abingdon, UK). All experimental conditions were assayed in triplicate. Enumeration of osteoclasts. Osteoclasts were identified as tartrate-resistant acid phosphatase (TRAP)–positive multinucleated cells with at least 3 nuclei. TRAP staining was performed using a commercially available TRAP staining kit 1022 VANDOOREN ET AL Figure 1. T cell dependence of spontaneous osteoclastogenesis in cultures of fresh peripheral blood mononuclear cells (PBMCs) from healthy subjects. A, Numerous tartrate-resistant acid phosphatase–positive (maroon colored) multinucleated cells develop from the PBMCs of healthy subjects after 2 weeks of culture. B, Toluidine blue staining of a dentin disc shows multiple resorption lacunae (dark purple spots), indicating that these cells are genuine osteoclasts. C, Depletion of CD14⫹ monocytes abrogates spontaneous osteoclastogenesis by PBMCs from healthy subjects. D, Depletion of CD3⫹ T cells abrogates spontaneous osteoclastogenesis of PBMCs from healthy subjects, which is restored by exogenous macrophage colony-stimulating factor (M-CSF) and RANKL. E, Purified CD3⫹ lymphocytes are more potent than exogenous M-CSF and RANKL in inducing osteoclastogenesis in cultures of purified CD14⫹ monocytes from healthy subjects. Values in C–E are the mean and SD of 5 healthy subjects. ⴱ ⫽ P ⬍ 0.05. (Original magnification ⫻ 100 in A; ⫻ 40 in B.) according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO). The slides were subsequently counterstained with Gill’s hematoxylin (Sigma-Aldrich), and TRAP⫹ multinucleated cells were counted manually in 3 triplicate culture wells by an investigator (BV) who was blinded to the culture conditions. The TRAP⫹ multinucleated cells were confirmed to be osteoclasts by the dentin resorption assay. Briefly, 700,000 PBMCs were seeded on top of dentin discs (IDS, Boldon, UK) placed in a 96-well plate and cultured for 3 weeks. Then, the slides were washed 3 times and immersed in 70% sodium hypochlorite to remove adherent cells. The resorption lacunae were counterstained with toluidine blue (Sigma-Aldrich). Statistical analysis. Results are expressed as the mean ⫾ SD and were analyzed with Student’s t-test for between-group comparisons. P values less than or equal to 0.05 were considered statistically significant. RESULTS Spontaneous osteoclastogenesis in vitro in healthy control PBMCs. Since spontaneous osteoclastogenesis in vitro has been proposed as a surrogate marker in disorders associated with bone loss (5–7), we first tested whether significant spontaneous osteoclastogenesis could be observed with unfractionated PBMCs from healthy controls. As shown in Figure 1A, PBMCs from healthy subjects formed TRAP⫹ multinucleated cells after 2 weeks of culture, without the addition of exogenous factors. These cells were able to resorb bone on dentin slides (Figure 1B), indicating that they are genuine osteoclasts. They originated from peripheral blood monocytes, since CD14⫹ cell depletion nearly completely abrogated the appearance of TRAP⫹ multinucleated cells (Figure 1C). Promotion of spontaneous osteoclastogenesis in vitro by T lymphocytes. To test the role of T lymphocytes in spontaneous osteoclastogenesis, we first depleted CD3⫹ cells from the PBMCs. Consistent with previous reports (5–7), T cell depletion significantly suppressed the formation of TRAP⫹ multinucleated cells (Figure 1D). Addition of 40 ng/ml of exogenous recombinant RANKL to T cell–depleted cultures largely, but not completely, restored the appearance of TRAP⫹ multinucleated cells (P ⬍ 0.05) (Figure 1D), indicating an important, although not exclusive, role of RANKL expression by T cells. The contribution of other factors was confirmed in purified CD14⫹ monocytes cultures, where the addition of T cells was more potent than SPONTANEOUS OSTEOCLASTOGENESIS BY HUMAN PBMCs IN VITRO 1023 Figure 2. T cell dependence of spontaneous osteoclastogenesis in cultures of cryopreserved peripheral blood mononuclear cells (PBMCs). A, Cryopreservation significantly enhances spontaneous osteoclastogenesis by PBMCs. B, Spontaneous osteoclastogenesis by cryopreserved PBMCs is abrogated by CD14⫹ monocyte depletion. C, Cryopreservation significantly enhances osteoclastogenesis by purified CD14⫹ cells cultured in the presence of exogenous macrophage colony-stimulating factor (M-CSF) and RANKL. D, Depletion of CD3⫹ T cells and/or addition of exogenous M-CSF and RANKL does not influence spontaneous osteoclastogenesis by cryopreserved PBMCs. E, Cryopreserved PBMCs from healthy controls (HC) and from patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), and non-psoriatic spondylarthritis (SpA) display the same levels of spontaneous osteoclastogenesis. Values in A–D are the mean and SD of 5 healthy subjects; values in E are individual data points for 5 subjects per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.001. exogenous M-CSF (10 ng/ml) and RANKL (40 ng/ml) for the induction of TRAP⫹ multinucleated cells (Figure 1E). Whereas these experiments were performed exclusively with PBMCs from healthy control subjects, the findings are consistent with studies of PBMCs from patients with arthritis (4,7). Spontaneous osteoclastogenesis in vitro not crucially dependent on T lymphocytes. Whereas these data confirm that T lymphocytes promote spontaneous osteoclastogenesis by RANKL expression and production of inflammatory cytokines, we next assessed whether T lymphocytes are absolutely required for this process by assessing spontaneous osteoclastogenesis under different conditions. Physiologic freezing and thawing of PBMCs from healthy subjects, a process which affects the T cell cytokine production (13), significantly enhanced the induction of TRAP⫹ multinucleated cells by 102 ⫾ 61% (mean ⫾ SD) (P ⬍ 0.001) (Figure 2A). This process was dependent on monocytes, since it was abrogated by depletion of CD14⫹ cells (Figure 2B) and could be reproduced in purified CD14⫹ cell cultures supplemented with M-CSF and RANKL (Figure 2C). Of particular interest, T cell depletion with or without the addition of exogenous RANKL did not affect spontaneous osteoclastogenesis in thawed PBMCs from healthy subjects (Figure 2D), indicating that the role of T lymphocytes is redundant under these particular culture conditions. Moreover, the previously observed increase in spontaneous osteoclastogenesis in vitro in cells from patients with erosive arthritis (4,7) was lost under these conditions, since cultures of cryopreserved PBMCs from healthy subjects showed significant numbers of TRAP⫹ multinucleated cells, which were not fundamentally different between healthy subjects and patients with arthritis (Figure 2E). However, the latter results should be interpreted with caution, since larger cohorts are needed to exclude smaller differences between RA and PsA as prototypical forms of destructive arthritis and with non-psoriatic SpA as a nonerosive form of chronic inflammatory joint disease. DISCUSSION Although there is no evidence of spontaneous osteoclastogenesis in vivo, this phenomenon has been reported to occur in cultures of PBMCs from patients 1024 affected by diseases in which there is pronounced bone loss (4–7) and has been proposed to reflect the propensity of circulating osteoclast precursor cells to differentiate into mature osteoclasts in vivo. This intriguing phenomenon raises two crucial questions: Which mechanisms are driving this process in vitro, and how relevant are these mechanisms to bone destruction in vivo? From a mechanistic point of view, it is still unclear to what extent this phenomenon depends on RANKL expression by T cells or on soluble factors such as TNF (5). The increased spontaneous osteoclastogenesis in vitro in PBMCs from patients with metastatic cancers (5), periodontitis (6), and PsA (7) has been demonstrated to be T cell–dependent, since it was completely abrogated by T cell depletion. Our findings are consistent with this concept, since T cell depletion abrogated spontaneous osteoclastogenesis in vitro and could be partially compensated for by exogenous RANKL in cultures of PBMCs from healthy donors. Previous studies in diseases such as PsA have indicated that T cells contribute to this in vitro phenomenon both by RANKL expression and by the production of proinflammatory cytokines, since it could be blocked by osteoprotegerin (OPG) as well as TNF inhibition (4). In contrast, T cells contribute mainly by cytokine production and not by RANKL expression in metastatic diseases, since the spontaneous osteoclastogenesis was abrogated by TNF blockade but not by OPG (5). Since T cells become activated by physiologic freeze–thawing, we used this system to reassess the contribution of activated T cells (13). Whereas the appearance of TRAP⫹ multinucleated cells was clearly increased under these culture conditions as compared with fresh PBMCs, the major finding of the present study is that T cells were completely redundant in the spontaneous osteoclastogenesis of frozen PBMCs. The increased, T cell–independent osteoclastogenesis under these conditions is caused by a direct effect on monocytes, since we obtained similar results with purified CD14⫹ mononuclear cells supplemented with exogenous M-CSF and RANKL. These data indicate that T cells are not an absolute requirement for spontaneous osteoclastogenesis and that this process can be strongly induced by direct activation of monocytes. This activation could be due to monocyte-derived cytokines in an autocrine loop (14) or to RANKL expression by non–T cells (15). In a first attempt to address these possibilities, we repeated the experiments using cryopreserved PBMCs from healthy donors in the presence of blocking antibodies against either TNF␣, IL-1␤, or RANKL. However, VANDOOREN ET AL blocking these factors had no effect on spontaneous osteoclastogenesis in our model (data not shown). Therefore, the exact molecular mechanisms of spontaneous osteoclastogenesis in vitro under these conditions remain to be unraveled. A second important issue raised by our findings is the relevance of spontaneous osteoclastogenesis in vitro for osteoclastogenesis in vivo in inflammatory arthritis. Our experiments with frozen PBMCs and CD14⫹ monocytes clearly indicate the intrinsic ability of healthy donor peripheral monocytes to differentiate into osteoclasts and seem to indicate that the increased spontaneous osteoclastogenesis in arthritis (4,7) using fresh PBMCs can not be reproduced under freeze–thaw conditions. Although larger patient cohorts are needed to confirm or exclude subtle differences between RA and PsA as prototypical erosive diseases and SpA as nonerosive arthritis, these data raise two important issues. First, this functional assay does not appear to be a reliable surrogate marker for large clinical trials in which cryopreservation is a critical step in avoiding experimental variation. Second, differences in spontaneous osteoclastogenesis appear to be related to the culture conditions, suggesting a critical role of extrinsic factors, rather than an intrinsic increase in osteoclast precursors, in the peripheral blood monocyte population (5,8). The extent to which the in vitro expression of these factors reflects the in vivo situation in the inflamed joint remains in question (16,17). AUTHOR CONTRIBUTIONS Dr. Baeten had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study design. 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