Recent data indicate that bone marrow plays an important role not only as a primary lymphoid organ responsible for haemopoiesis, but also as a secondary lymphoid organ with capability of antigen presentation exceeding that of lymph nodes. Although in chronic inflammatory/immune disease, like rheumatoid arthritis (RA), bone marrow participates in the initiation and/or perpetuation of the disease, there is little information about the real number of lymphocyte subpopulations in the bone marrow of these patients and how they can be modulated by T-cell growth factors. IL-15 acting through IL-15 receptors (including high-affinity IL-15R alpha chain) is a key cytokine influencing the development of natural killer cells in bone marrow, and proliferation and maintenance of the memory CD3+ cell pool. However, there is no information about the levels of IL-15 in bone marrow.
To compare the real cell numbers within lymphocyte subsets in bone marrow isolated from RA and osteoarthritis (OA) patients. To measure the levels of soluble IL-15 and surface-expressed IL-15R alpha in bone marrow plasma and cells, respectively. To analyze the presence of memory and actively proliferating T cells in the bone marrow.
Bone marrow samples, obtained from RA and OA patients (mean age 53.1 ± 10.6 years and 54.3 ± 13.6 years, respectively) undergoing joint replacement surgery, were diluted four times in heparinized PBS. Bone marrow plasma samples were obtained by centrifugation and levels of IL-15 were measured using specific ELISA. The real number of lymphocytes stained for CD3+ and CD19+ were counted in the presence of TruCount beads using flow cytometry. Surface-expressed IL-15R was done on cells separated by gradient centrifugation, acid wash of surface-bound IL-15 and flow cytometric analysis. Surface expression of CD45RO was evaluated by flow cytometry. Cell proliferation was measured by intracellular expression of Ki-67.
There were twice as many T (CD3+) cells in RA in comparison with OA bone marrow (6.1 ± 2.8 versus 3.2 ± 1.6 cells × 106/ml bone marrow, P = 0.008). In contrast, only 42% of B (CD19+) cells present in OA were present in RA bone marrow (2.0 ± 0.9 versus 0.85 ± 0.3 cells × 106/ml bone marrow, P = 0.02). Interestingly, both CD3+CD4+ and CD3+CD8+ cells obtained from RA patients expressed a significantly higher level of CD45RO (P = 0.002 and P = 0.001, respectively), measured as the mean fluorescence intensity, than in OA. In addition, there was a tendency (although not statistically significant, P = 0.08) for a higher percentage of CD3+CD8+CD45RO+ cells in bone marrow from RA patients in comparison with OA patients (38.7% versus 28.1%). Interestingly, lymphocytes isolated from RA patients expressed a significantly higher level of surface IL-15R alpha chain (P = 0.01), indicating their activation status. Cells isolated from RA bone marrow bound significantly more Ki-67 than cells isolated from OA bone marrow, indicating that RA cells proliferated more vigorously There were elevated levels of IL-15 in bone marrow plasma from RA in comparison with OA patients (1304.5 ± 956.3 pg/ml and 760 ± 238.7 pg/ml, respectively, P = 0.01).
Lymphocytes obtained from RA patients expressed higher levels of IL-15R alpha and proliferated more rapidly than lymphocytes obtained from OA patients. It is likely that locally overproduced IL-15 is responsible for the elevated number of T cells in RA bone marrow. Higher density of CD45RO+ memory marker expressed on T cells from RA patients further supports the role of IL-15 as a known growth factor for memory T cells. A significantly lower B-cell number in RA than in OA suggests that these cells actively emigrate from RA bone marrow to peripheral blood and affected joints. Distinct subpopulations of mature lymphocytes present in bone marrow from RA and OA patients indicate that the bone marrow acts as a secondary lymphoid organ that actively contributes to the pathogenesis of RA.