RA is characterized by chronic infiltration of T lymphocytes and B lymphocytes, plasma cells, and macrophages into the synovial tissue of joints [13
]. High levels of proinflammatory cytokines are invariably detectable in RA synovia with severe lymphocyte infiltration [14
], suggesting that a signal(s) directing prolonged cytokine synthesis is present within the synovial tissues of RA patients. We previously established several RA-SNC cells that produce a large amount of proinflammatory cytokines on direct contact with lymphocytes [7
]. In the present study, we focused on the mechanisms of the cell contact-mediated cytokine production by the RA-SNC cells, and examined, in particular, the adhesion pathways involved in the cell contact and the subsequent transmigration of the lymphocytes. We also attempted to verify whether cell adhesion or transmigration, or both, is important for the production of the proinflammatory cytokines by the RA-SNC cells.
To assess the relative contributions of cell binding and the subsequent transmigration of lymphocytes in this phenomenon, we took advantage of transmigration-defective MC/car cells pretreated with C3 transferase. The present results suggest that lymphocyte adhesion itself is sufficient, and that transmigration is not required, for induction of a high level of cytokine production by RA-SNC77 cells. In addition, since an anti-VLA-4 mAb did not affect cytokine production by RA-SNC77 cells at all, but significantly inhibited MC/car cell binding, our results suggest that one or more VLA-4-independent adhesion pathway is involved in the enhanced cytokine production by RA-SNC77 cells. Adhesion molecules previously identified in synovial tissues in RA patients, such as VAP-1 [19
] and activated leucocyte cell adhesion molecule [21
], do not appear to be involved in the binding of lymphocytes to RA-SNC77 cells, since flow cytometric analysis indicated a lack of expression of these adhesion molecules in RA-SNC77 cells (Takeuchi et al
., unpublished observation). Other undefined adhesion molecules may therefore play a key role in inducing the enhanced proinflammatory cytokine production by RA-SNC77 cells.
We found that the VLA-4-dependent adhesion pathway was involved in both binding and transmigration of MC/car cells to a cloned stromal cell line, RA-SNC77. VCAM-1, a functional ligand for VLA-4, however, did not appear to contribute to these cellular interactions, suggesting that RA-SNC77 cells express an alternative ligand(s) for VLA-4. Other investigators have also reported the involvement of a VLA-4-dependent/VCAM-1-independent adhesion pathway in the interaction between bone marrow stromal cells and leukocytes [23
]. Previously identified ligands for VLA-4 include VCAM-1 [25
] and the CS-1 isoform of fibronectin [26
]. The CS-1 isoform of fibronectin, which has been reported to be expressed in synovial tissues in RA patients [27
], was detected at mRNA levels in RA-SNC77 cells, and it may function as a ligand for VLA-4, although further study is required to verify this issue.
In the inflamed RA synovial tissue, various inflammatory cytokines other than IL-6 or IL-8 are readily detected [18
]. Certain proinflammatory cytokines, such as tumor necrosis factor alpha and IL-1, may participate in the dysregulated production of multiple cytokines in the RA synovial tissues. Although production of tumor necrosis factor alpha and IL-1 by RA-SNC cells is limited to low levels even after stimulation with lymphocytes [7
], such regulatory cytokines may contribute the cell contact-dependent production of IL-6 and IL-8 observed in this study. Enhanced expression of proteolytic enzymes, such as cathepsins, matrix metalloproteases [30
], and aggrecanases [32
], is also seen in the inflamed RA synovial tissue, and these proteolytic enzymes are thought to be involved in the cartilage and joint destruction. Whether there is any functional link between the lymphocyte adhesion-driven cytokine production and the enhanced expression of the proteolytic enzymes in the RA synovium merits future investigation.
Burger et al
] recently demonstrated that, other than specialized nurse-like stromal cells, conventional fibroblast-like synoviocytes and IL-4-stimulated dermal fibroblast-like cells also can support pseudoemperipolesis of Blymphoid cells. They also found that, irrespective of their origin, the ability of fibroblastic cells to support B-cell pseudoemperipolesis is dependent on their expression of SDF-1 and VCAM-1, both of which are also detected in RA-SNC77 cells. These findings suggest that the specialized nurse-like cells and conventional fibroblast-like cells share some functional similarities to support B-cell pseudoemperipolesis while the nurse-like cells established from synovial tissues of patients with RA are distinct from other stromal cells derived from non-RA patients in both morphology and cellular functions, particularly proinflammatory cytokine production [7
]. Further comparative studies are needed to characterize fibroblastic-stromal cells and nurse-like cells at molecular levels.
In summary, the present results indicate that lymphocyte binding per se is critical for enhanced proinflammatory cytokine production by RA-SNC77 cells. While transmigration of lymphocytes underneath the RA-SNC cells did not appear to play a significant role in the production of IL-6 and IL-8, this biological process may be involved in the production of other cytokines and/or proteinases. This nurse-like cell activity, which is seen in stromal cells isolated from the synovia of RA patients but not in those from disease-free controls, may alternatively influence the effector functions of infiltrated lymphocytes in RA synovia. Although VLA-4 is involved in both lymphocyte adhesion to and transmigration beneath RA-SNC cells through the interaction with non-VCAM-1 ligand(s), the VLA-4-independent adhesion pathway appears to be important for the cell contact-induced cytokine production by RA-SNC77 cells. Further investigation to identify the adhesion receptors necessary for cell contact-dependent activation of the nurse-like stromal cells may lead to novel therapeutic strategies through regulating the functions of the nurse-like stromal cells in RA patients.