Proteoglycans are complex molecules comprised of a core protein to which is attached one or more glycosaminoglycans. The proteoglycans are ubiquitous cell surface components of all mammalian cells, and are associated with cell membranes via three separate mechanisms: (1) as an integral membrane protein spanning the lipid bilayer, (2) partial insertion into the lipid bilayer of a phosphatidyl inositol component of the proteoglycan, or (3) by binding of a GAG side chain to specific plasma membrane receptors.
The development of periodontitis is associated with changes in the relative proportions of lymphocyte subpopulations within the gingival tissues. In gingivitis, there is an abundance of T-lymphocytes. However, in destructive periodontitis, there is a concomitant decrease in the number of T-lymphocytes and a relative increase in the number of B-lymphocytes and plasma cells. Although the expression of cell surface proteoglycans by lymphocytes located within periodontal tissues has not been examined, a number of important observations led to speculation that these cell surface molecules are likely to be important in the interactions of lymphocytes within the milieu of the inflamed periodontium. During the development of periodontitis, the migration and activation of lymphocyte subpopulations may involve the expression of lymphocyte cell surface proteoglycans.
The cell surface proteoglycans mediate cell-matrix interactions and have been implicated in lymphocyte migration and activation. Therefore, the aims of Chapter 2 were to examine lymphocyte expression of cell surface proteoglycans in periodontal tissues and to examine the percentage of peripheral blood B- and T-lymphocytes expressing syndecan-1 and to correlate these with various stages of periodontal inflammation. This study investigated the expression of syndecan-1, syndecan-4 and glypican by peripheral blood lymphocytes and by lymphocytes in various inflamed periodontal tissues. Gingival specimens from healthy, gingivitis or chronic periodontitis sites were stained using antibodies against B- and T-lymphocytes and also syndecan-1, syndecan-4 and glypican. Syndecan-1 expression by peripheral blood mononuclear cells (PBMC) from healthy, gingivitis and chronic periodontitis subjects was assessed using flow cytometry. Syndecan-1 was expressed by B-lymphocytes/plasma cells but not Tlymphocytes in both gingivitis and chronic periodontitis lesions, while syndecan-4 was expressed by both B-lymphocytes/plasma cells and T-lymphocytes in gingivitis and chronic periodontitis. Glypican was expressed only by macrophages. Stimulation of PBMC with mitogens and growth factors modulated syndecan-1 expression in both the T- and B-lymphocytes. Thus, cell surface proteoglycan expression by lymphocytes in periodontal inflammation is cell type specific and may be modulated by inflammation.
Cytokines are produced by activated lymphocytes, monocytes and fibroblasts, and cell surface PGs may capture cytokines and facilitate intracellular signaling, and modification of cell behavior. Secreted T-lymphocyte cytokines or direct contact between T- and B-lymphocytes might regulate T- and B-lymphocyte syndecan-1 expression. Accordingly, in Chapter 3, the aim was to examine the effect of cytokines and antigens on peripheral blood lymphocyte syndecan-1 expression in vitro. The exogenous addition of cytokines, (TGF-βl, IL-2, IL-4 and IL-lα and IL-lβ) and the antigens LPS and TT were used to examine the effect on T- and B- lymphocyte-syndecan-1 expression. The up-regulation of syndecan-1 expression by the peripheral blood B- and T-lymphocytes, exposed to transforming growth factor-pl (TGF-β1) at concentrations (4ng/mL and 2ng/mL) was statistically significant in relation to the respective controls. TGF-β1 is an important autocrine immuno-regulatory molecule, which limits clonal expansion and differentiation of normal B-lymphocytes, and may play a pivotal bi-functional role in determining T-lymphocyte mediated immune responses. TGF-βl induced syndecan-1 expression by B- and T-lymphocytes may have some beneficial effect in immune regulation, due to the many functions of syndecan-1 in the localization of cytokines and growth factors for cell function, and in control of lymphocyte migration. In the present study, IL-2 and IL-4, (known lymphocyte growth factors) failed to cause any significant increase in syndecan-1 expression. IL-2 can convey variable signals to T-lymphocyte subsets, altering cellular responses. The simultaneous synthesis of IL-2 and IL-4 by naïve and memory T-lymphocytes is synergistic for T-lymphocyte proliferation, yet restricts the nonspecific recruitment of the other cells within lymphoid tissue, resulting in subset specificity, which in-tum may regulate cell surface molecule expression. In the presence of the pro-inflammatory cytokines IL-1β or IL-lα from peripheral blood mononuclear cell cultures, the percentage of T-lymphocytes and B-lymphocytes expressing syndecan-1 was suppressed.
The variation in syndecan-1 expression was more prominent for B-lymphocytes following antigen stimulation, than for T-cells. Stimulation with LPS from P.gingivalis or TT resulted in more peripheral blood B- and T-cells showing a decrease in syndecan-1 expression, compared to the control. When cytokines were combined with TGF-pl (4ng/mL), the percentage of peripheral blood B-lymphocytes expressing syndecan-1 increased significantly (TGF-β1+ LPS, TGF-β1 + IL-4, TGF-βl + TT), whereas Tlymphocytes expressing syndecan showed a significant up- regulation only with TGF-βl + IL-4.
In Chapter 4, syndecan-1 mRNA expression and the effect of cytokine stimulation on syndecan-1 messenger expression in peripheral blood mononuclear cells, and various cell lines were examined. A syndecan-1 specific transcript of 416 bp was detected in PBMC, and transcription was not affected by stimulation with TGF-β1. The syndecan-1 specific transcript was not observed in pure T-lymphocytes sorted from the fresh PBMC. While the Jurkat cell line, a human malignant T-lymphocyte cell line, showed syndecan-1 gene expression, this was not affected by stimulation for five days with cytokines. SPLCL, a lymphoblastoid B-cell line, also expressed syndecan-1 mRNA, and no change in gene expression was observed following stimulation with cytokines. From the present observations and the literature, it appears that syndecan-1 expression may be related to the activation status of the cell. While syndecan-1 expression by B-lymphocytes has already been established in the literature, this is the first demonstration of syndecan-1 gene expression by T-lymphocytes.
In Chapter 5, the effect of TGF-pl induced syndecan-1 expression on the adhesion response to type-I collagen by peripheral blood mononuclear cells and the lymphoblastoid B-cell lines SPLCL and J. Staples were investigated. While PBMC following stimulation with TGF-β1 (4ng/mL) showed a significant increase in adhesion response after wash one, the subsequent washes showed no significant alteration in adhesion to collagen type-I compared to the control. This may either point to a disruption of the other adhesion molecules by TGF-β1, or it may indicate syndecan-1 up-regulation with an increase in the glycosaminoglycan chains, which does not contribute to increased adhesion property of the cells. The adhesion assay conducted with the lymphoblastoid B-cell line SPLCL showed a dramatic increase in adhesion to type-I collagen following stimulation with TGF-βl at concentrations of 4ng/mL and 2ng/mL, compared to the control, when cultured for 24 hours at 2% FCS and 10% FCS. The J. Staples cell line only showed significant adhesion to type-I collagen when cultured in 2% FCS for 24 hours following stimulation with TGF-βl at 4ng/mL. This may indicate different molecular forms of syndecan-1 have different affinities for collagen. The glycosaminoglycan content of syndecan may also determine the affinity of syndecan for matrix ligands. Furthermore, different syndecan-1 isoforms can display functional differences, such as binding to extra-cellular matrix proteins, binding to specific cell adhesion receptors, and binding of growth factors. Syndecan-1 expression during B-cell differentiation may control B-lymphocyte localization within specific microenvironments, and modulate signaling pathways. These pathways may be activated in cell-matrix interactions, and lead to cytoskeletal reorganization and cell motility.
In conclusion, the results from this series of studies suggest a cytokine influence on T- and B-lymphocyte expression of syndecan-1, which may be useful for identifying deviations in syndecan-1 expression in immuno-pathogenic disorders. Future studies should be directed at investigating the role of secreted T/B-lymphocyte cytokines in the control of T/B-lymphocyte syndecan-1 expression in periodontal inflammation, and the subset variation associated with syndecan-1 expression. Whether syndecan-1 has a role in immuno-regulatory events in arresting disease progression, by lymphocyte isotype or subset switching, differentiation, apoptosis or mediation via cytokine locali2ation, should be further investigated. An understanding of the mechanism by which the syndecan-TGF-β1 axis regulates the immune response will contribute to the understanding of immune mediators that maintain the fine control over normal immune responses.