Objectives (1) To compare the absolute T-cell numbers in bone marrow (BM) isolated from patients with rheumatoid arthritis (RA) and osteoarthritis (OA); (2) to measure the levels of soluble interleukin 15 (IL-15) and IL-7; (3) to analyse the expression of activation markers on T cells; (4) to analyse influence of IL-15 stimulation on T-cell proliferation.
Methods BM samples were obtained from patients undergoing joint replacement surgery. Concentrations of IL-15 and IL-7 were measured using specific ELISAs. The absolute number of T lymphocytes, their activation status and proliferation were evaluated by flow cytometry.
Results BM from patients with RA contained double the number of CD3 T cells in comparison with OA (6.1 vs 2.7 × 106 cells/ml, p<0.008). Ratio CD3CD4:CD3CD8 was increased in RA BM, clearly indicating accumulation of CD3CD4 cells. T cells obtained from patients with RA expressed higher level of early activation markers than from OA. Elevated levels of IL-15 were found in BM plasma from patients with RA in comparison with patients with OA (1304.5±956.3 pg/ml and 760±238.7 pg/ml respectively, p<0.01). These data were confirmed by immunohistochemistry of RA BM from regions proximal and distal to the joint. Although both CD3CD4 and CD3CD8 cells proliferated after IL-15 stimulation in vitro, CD3CD4 cells from patients with RA proliferated more vigorously than those from patients with OA, reflecting the composition of T-cell subsets in BM.
Conclusion These results suggest that locally overproduced IL-15 may be responsible for the activation and proliferation of T cells in situ, reflected by significantly increased number of activated T cells in RA BM, possibly contributing to the pathogenesis of RA.
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Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that leads to joint destruction.1 Although the pathogenesis of RA is still unclear, many activated cell types, including T cells, B cells, monocytes/macrophages, fibroblast-like synoviocytes, natural killer (NK) cells, mast cells and granulocytes found in inflamed joint synovial tissue and synovial fluid, probably acting in concert with released mediators of inflammation, proinflammatory cytokines and autoantibodies, contribute to the initiation and perpetuation of chronic joint destruction. Among these cell types, T cells are considered to be central for development of RA.2 In addition, there is a strong evidence that disrupted T-cell homoeostasis contributes greatly to joint pathology in RA.3 Although the source of T cells in affected joints is not entirely clear, the general assumption is that they represent lymph node activated memory cells that migrate through blood to inflamed tissue.4 5 In addition, some of these cells may represent naïve T cells that are activated and propagated in situ in lymphoid-like structures present in pannus of many patients with RA.
However, data indicate that bone marrow (BM), in addition to being a primary lymphoid organ responsible for haematopoiesis and a key homing/survival site for plasma cells, represents also a secondary lymphoid compartment contributing to T-cell homoeostasis and immunity.6 7 Memory T cells, both CD3CD8 and CD3CD4 cells, are known to reside in BM where, supported by locally produced survival factors, they maintain their antigenic memory with slow retention rate. These T cells represent a pool of longlasting, central memory cells ready for fast reaction for the next antigenic challenge in the periphery.8 9 In addition to serving as a storage site for these memory T cells, data indicate that under certain conditions BM acts also as a secondary lymphoid organ with an ability for antigen presentation exceeding that of lymph nodes.10 CD3CD4 T lymphocytes have been shown to have an important role in haematopoiesis where constant cognate antigen activation of BM CD4 cells is required for maintenance of normal haematopoiesis.11
Interestingly, although subchondral bone is directly affected by the destructive process in RA, relatively little is known about the role of BM in driving the activation and propagation of T cells in this disease.
In a previous study our group showed that TLR9 may participate in direct activation and proliferation of B cells isolated from BM, and therefore could have a role in the pathogenesis of RA.12 In this study we have analysed BM T-cell compartments in RA (a chronic autoimmune disease) and osteoarthritis (OA) (a relatively non-inflammatory disease). Determination of the absolute number of T-lymphocyte subpopulations in the BM of these patients, their activation status and response to interleukin 15 (IL-15), a key T-cell growth factor present in excess in RA, leads to the conclusion that RA BM represents important site of T-cell activation that probably contributes to the pathogenesis of RA.
Patients and methods
Tissue specimens were obtained from patients with RA and from patients with OA as a control. Peripheral blood (PB) and BM samples were obtained from the same patients during total hip replacement surgery. Included were 65 patients with RA (51 women and 14 men, median age 53.5, range 24–72) and 63 patients with OA (43 women and 20 men, median age 55.4, range 24–74). Thirty-three patients with RA were treated both with methotrexate and steroids, five received only methotrexate and 17 received only steroids. The remaining patients were receiving non-steroidal anti-inflammatory drugs.
Additionally, six BM samples obtained during orthopaedic surgery from patients previously studied by flow cytometry were randomly selected for histopathological examination.
BM trephine biopsy specimens obtained from iliac crest of 98 patients with RA were retrieved from the files of Department of Pathology, Institute of Hematology. Fifteen patients with RA with Larsen score 1 or 2 (median age 51 years, range 44–74) and associated with mild unexplained haematological abnormalities, such as anaemia, leucopenia, thrombocytopenia, eosinophila, monoclonal gammapathy or spenomegaly, were selected for histopathological examination. Their final haematological diagnosis did not account for the observed changes in the BM. Therefore, we suggested that these changes were related to RA.
Patients fulfilled the American College of Rheumatology revised criteria for RA or for OA. All individuals gave informed consent and the study was approved by the Institute of Rheumatology Ethics Committee.
Additional data describing patients, reagents and detailed methods, including staining procedures for immunohistochemistry, flow cytometry and ELISA, are published in the online supplementary material.
Data were analysed using Statistica vol. 6.0 software. Comparisons between the groups of patients with RA and OA were analysed by the two-tailed Mann–Whitney U test. Correlation between the number of CD3 cells and erythrocyte sedimentation rate (ESR) was assessed using a Spearman test. p Values <0.05 were considered significant. Data are shown in the text as the median, min-max. Data are shown in the figures as the median. The top of the box represents the 75th centile and the bottom of the box the 25th centile.
Elevated numbers of CD3 cells are present in RA BM
The absolute number of BM CD3 cells was counted using Trucount tubes and flow cytometry. There was a significant increase in the absolute number of CD3 cells per microlitre of femur BM from RA (median value 6154, range 1248–1253) compared with OA (median value 2694, range 983–9090) patients (p<0.008, figure 1A). These results suggested: enhanced infiltration of T cells from the periphery to RA BM, increased proliferation in situ and/or decreased outflow of T cells from RA BM in comparison with OA. The experiments described below were designed to clarify these possibilities. The number of CD3 cells showed a strong inverse correlation with ESR (R spearman=–0.5412) only in the group of patients with RA (figure 1C).
Higher proportions of helper CD3CD4 than cytotoxic CD3CD8 are present in RA BM
To further understand which main subpopulations account for observed T-cell accumulation in RA BM, the ratio of CD3CD4 helper to CD3CD8 cytotoxic T-lymphocyte subpopulations was analysed (figure 1B). A higher content of CD3CD4 than CD3CD8 (ratio: CD3CD4:CD3CD8=1.10±0.25) was found in RA, while fewer CD3CD4 than CD3CD8 (ratio: CD3CD4:CD3 CD8=0.76±0.17 were present in OA BM (p<0.004). Thus, our data indicate that helper CD3CD4 cells accumulate in RA BM.
Activation status of CD3 T cells in BM and PB of patients with RA and OA
To estimate the activation status of BM CD3CD4 and CD3CD8 lymphocytes, the expression of CD69 and HLA-DR activation markers were compared between samples isolated from patients with RA and OA (figures 2 and 3). The percentage of early activation marker CD69 was significantly increased on both CD3CD4 and CD3CD8 T-lymphocyte subsets in patients with RA. The median values for CD3CD4CD69 were 2.8% (RA) and 0% (OA, p<0.05). Since CD69 expression is characteristic for cells accumulation in the sites of inflammation, these results suggest a continuing inflammatory process activating both CD3CD4 and CD3CD8 subpopulations in RA BM.13
HLA-DR represents a late activation marker on T cells. Surprisingly, there was a tendency (but not statistically significant) to lower the expression of HLA-DR on CD3CD4 and CD3CD8 cells obtained from RA BM. Interestingly however, in both RA and OA groups the expression of HLA-DR was higher on BM CD3 cells than on PB CD3 cells (figures 2 and 3, p<0.05).
Elevated levels of IL-15 and IL-15-triggered proliferation of T cells in RA BM
Increased number of T cells in RA BM may reflect their enhanced proliferation in situ. IL-15 is a key T-cell growth factor, contributing to NK and T-cell homoeostasis in BM.8 14 IL-7 is also an important cytokine that can contribute to T-cell proliferation/homoeostasis. Therefore experiments were performed to compare RA and OA BM samples by evaluation of (i) the concentration of IL-15 and IL-7, and (ii) the proliferation of these cells triggered by exogenous IL-15. Importantly, we found higher a concentration of IL-15 in BM plasma of patients with RA (median value 868, range 600–1581 pg/ml) than OA (median value 744, range 428–962 pg/ml) patients. The difference was highly significant with p<0.007 (figure 4A). Surprisingly, there were no difference in IL-7 concentration between the groups of patients with OA and RA (figure 4B).
IL-15 triggered proliferation of both CD3CD4 and CD3CD8 T cells in RA, but only CD3CD8 in OA BM cells cultured for 6 days in vitro as judged by 5,6 carboxyfluorescein diacetate, succinimidyl ester (CFSE) dilution (figure 5).
Taken together these data suggest that in RA BM, IL-15 is present in concentrations high enough to provide locally survival/proliferation signals for T cells, especially of the helper (CD3CD4) subset. The composition of T-cell subsets in RA BM seems to reflect increased proliferation of these subpopulations in response to IL-15 in vitro.
Immunohistochemistry of RA BM
In nine of 15 BM trephine biopsy specimens of patients with early RA (Larsen score 1–2) lymphocytic inflammatory infiltrates had a follicular pattern (figure 6A). In five cases we observed structures resembling primary and secondary lymphoid follicles with the presence of B cells (CD20) (figure 6B) surrounded by T cells (mainly CD4) (figure 6C). Only a few CD8 T cells were present (figure 6D). In all samples there were also a few dispersed CD3 cells, which accounted for an average of 10% of the total nucleated cells count (range 5–15%) cells. In 13/15 studied patients immunostaining with CD69 demonstrated positive cytoplasmic and membrane expression of this antigen on T and B cells, especially in lymphoid follicles (figure 6E), but also in dispersed lymphocytes. Expression of IL-15 was evident in lymphoid nodule cells (figure 6F).
In a group of six patients with advanced RA (Larsen score 4–5) the BM morphological and immunohistochemical characteristics were similar to those in the group with early RA (Larsen score 1–2). In three of six patients a follicular pattern of lymphocytic inflammatory infiltrates was observed (figure 6G). B cells were present in the centre of these nodules (figure 6H) surrounded by T cell expressing predominantly CD4 (figure 6I), and only a few T cells expressing CD8 (figure 6J). Expression of IL-15, and CD69 had a similar pattern to that in the group with early RA (figure 6 K and L).
These immunohistological data generally confirm the presence of activated lymphocytes, and characteristic shift toward CD3CD4 T cells in RA BM.
Although cellular composition of synovial fluid and synovial membranes in patients with RA and OA has been studied in great detail, much less is known about BM in these diseases. Recent data indicate that this compartment may have an important role in the initiation and perpetuation of the chronic inflammation characteristic of patients with RA. In this report we analysed the composition of lymphocyte subsets and their activation status in the epiphyseal femoral BM which, although not considered to be a major haematopoietic organ in adults, is adjacent to inflamed/destroyed joint, and therefore may participate in the inflammatory process in RA. The BM from patients with OA served as a control of non-inflammatory diseases.
We report elevated number of CD3 T cells in the BM of RA in comparison with patients with OA. This finding clearly indicates that BM is a site of T-cell accumulation that might account for BM oedema observed during development of RA.15 A similar study by Tomita et al reported increased mononuclear contents in iliac BM but not in tibial epiphyseal BM.16 However, these authors did not study femoral BM. Therefore, although both studies indicate differences of lymphocyte subpopulations in BM from patients with RA and OA, we could not directly compare their results with ours. Another possible reason for the different results is that Tomita et al used density gradient centrifugation with the analysis done on buffy coat cells, while we studied the whole BM using Trucount beads. We believe that the method used by our group may better reflect the real values.
Our more detailed analysis showed an accumulation of CD3CD4 T cells in RA BM. This finding, confirming a previous report of an increased ratio CD3CD4:CD3CD8 cells,17 extends our knowledge of BM as a site for memory CD3CD8 cells and also for CD3CD4 T cells.9 Increased numbers of CD8 T cells in RA BM in patients with MRI BM oedema have been also recently reported.18 However, these authors did not analyse CD4 T cells. Corresponding to our findings is a report of lymphoid aggregates on the subchondral side of the joint in established RA, which was found by Bugatti et al.19 These authors also suggested that inflammation may develop independently of the expansion of synovial tissue.19
Surprisingly, we found that T-cell numbers correlated inversely with ESR. A possible explanation might be that during systemic inflammation reflected by highly elevated ESR, T cells migrate from BM to the joint cavity through canals connecting BM and synovium. It has been shown that during the development of collagen-induced arthritis, enlargements of these canals and migration of cells from BM takes place in the early stages of the disease.20
Another characteristic finding in the femoral BM of patients with RA was a significantly higher expression of early T-cell activation markers: CD69. As CD69 is expressed only in areas with persistent inflammation, this observation strongly support the hypothesis that the inflammatory process occurs in the BM.13
Interestingly, there was a tendency (although not statistically significant) for a lower percentage of HLA-DR cells in BM from patients with RA in comparison with patients with OA. Again, a possible explanation for this tendency is that recently activated T cells with higher expression of HLA-DR migrated to the synovium.
To further understand which T-cell growth factor contributes to increased T-cell numbers and their activation in RA BM we measured the concentrations of IL-15 and IL-7 in BM plasma. IL-15, in addition to its role as a key cytokine for development and maintenance of NK cells in BM, is a potent T-cell growth factor and stimulator of CD69 expression.9 14 21 Importantly, we found an elevated level of IL-15, but not IL-7, in RA in comparison with OA BM plasma. Moreover, IL-15 significantly stimulated in vitro proliferation of CD3CD4 and CD3CD8 cells from both RA and OA groups of patients. However, the stimulating effect of IL-15 was more profound for CD3CD4 cells from patients with RA than for these cells from the OA group. These results suggest that owing to high local concentration, IL-15, but not IL-7, is probably the cytokine responsible for accumulation of CD3CD4 in RA BM.
Interestingly, there are data which suggest that at high concentrations IL-15 may stimulate monocytes/macrophages to activate autoreactive CD4 T cells that are characteristic of RA.22
Immunohistochemistry of RA BM confirmed the presence of recently activated T cells. Interestingly, characteristic structures of germinal centres were present in advanced RA and also in early RA BM (Larsen score 1–2), indicating that the immune response takes place in BM in the early stages of the disease. In addition, the presence of similar lymphoid structures in RA BM from both proximal and distal to joint regions further suggests that pathological immune processes are characteristic for RA BM.
In conclusion, our results suggest that locally overproduced IL-15 supports T-cell proliferation and survival and may thus be one factor contributing to the activation and proliferation of T cells in situ. There is some evidence that BM may also be important in the pathogenesis of other autoimmune diseases such as chronic colitis and lupus.23 24 Certainly, BM is also a site for important pathology driving joint damage, and distinct subpopulations of mature lymphocytes present in BM from patients with RA and OA indicate that BM acts as an organ that actively contributes to the pathogenesis of RA.
Funding European Community FP6 Project LSHB-CT-018661 AutoCure; grant No 2P05A 176 29 from the Polish Ministry of Science.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the Institute of Rheumatology Ethics Committee.
Provenance and peer review Not commissioned; externally peer reviewed.