T-Cell-Derived Cytokines Drive Fibroblast Matrix Deposition
A characteristic feature of SSc is the presence of a persistent inflammatory infiltrate, including T cells, associated with regions of fibrosis and matrix remodelling. Fibrosis occurs when the homeostatic balance is altered between the deposition of extracellular matrix and the proteolytic breakdown of the matrix. Mediators that alter either the deposition or breakdown of the matrix will lead to a perturbation of matrix leading to fibrosis. The cellular and molecular factors that govern this alteration in matrix deposition remain largely unknown. There is currently a strong impetus to identify factors that induce matrix deposition, with a major focus on TGF-β, however, we argue that the role of T-cell-derived cytokines are just, if not more, important. A widely held assumption is that monocytes are the predominant source of pro-fibrotic mediators responsible for fibrosis, however, Th2 cells are a potent source of pro-fibrotic mediators and elucidation of their co-operative roles will yield new drug targets.
Classic Th2 cytokines include IL-4, IL-5 and IL-13. IL-4 is a kDa-multifunctional cytokine produced by activated Th2 T cells. The gene for IL-4 has been found to be clustered on chromosome 5q particularly close to IL-13 gene. It normally promotes humoral immunity by inducing immunoglobulin production and isotype switching and is involved in the differentiation of naïve CD4 T cells towards a Th2 phenotype. Antibody-mediated depletion of CD3 T cells in bleomycin mice decreased fibrosis along with reduced IL-4 secretion. Elevated levels of IL-4 have been demonstrated in SSc tissue both at the mRNA level and protein level. Luzina et al. have demonstrated that CD8 cytotoxic T cells are activated and secrete high levels of IL-4 in SSc patients. Moreover, IL-4 has been shown to induce tenascin, a large extracellular matrix protein, in both normal and SSc fibroblasts. Also direct stimulation of human fibroblasts with IL-4 leads to a significant increased expression of collagen and fibronectin, and SSc-derived fibroblasts are more sensitive to such induction. In the Tsk model, treatment with antibodies against IL-4 prevented the induction of dermal fibrosis. The Tsk mouse harbours a chromosome 2 mutation that produces an increase in skin and internal organ deposition of collagen. Targeted mutation of the IL-4R-α gene in Tsk mice leads to attenuated skin fibrosis, collagen content and, interestingly, anti-topo I antibodies. Indeed, elevated levels of IL-4 and IL-6 have been demonstrated in the sera of SSc patients, suggesting systemic release of IL-4. Some of the strongest evidence for a pro-fibrotic role of IL-4 is the fact that genetic deletion of the IL-4 gene in mice subsequently challenged with bleomycin leads to reduced fibrosis and fibroblast collagen production compared with wild-type animals. Indeed, it has been demonstrated that both IL-4-secreting cells (T cells) and CD40 ligation on the fibroblasts drives a synergistic increase in fibroblasts activation and proliferation. This suggests that as well as the pro-fibrotic effects of IL-4, cells expressing CD40 ligand (CD154) help synergize the activation response. CD154 is primarily expressed on activated T cells and is found in greater abundance in CD4 Th cells in the peripheral blood of SSc patients. Indeed, anti-CD154 antibodies suppress skin fibrosis in the bleomycin animal model of fibrosis, and this was, at least partly, dependent on blockade of monocyte chemoattraction protein-1 (MCP-1) and RANTES.
IL-13 is an immunoregulatory cytokine also produced predominantly by activated Th2 cells. IL-13 is an important mediator in the pathogenesis of asthma and airway remodelling, IgE antibody production and mastocytosis, and is directly pro-fibrotic. IL-13 shares many functional activities with IL-4 because both share a common IL-4Rα chain receptor and signal through STAT-6. Although both IL-4 and IL-13 have similar and overlapping functions they both have non-redundant functions. IL-4 is important for polarization of naïve CD4 T cells. IL-13, however, is not necessary, as IL-13Rα1 has not been demonstrated on T cells. Indeed, IL-13 was found to be directly fibrotic in a hepatic fibrosis model, and deletion of IL-13 was associated with much reduced fibrosis and collagen deposition; it was clear from the double IL-4–IL-13 knockout mice that IL-13 was the predominant pro-fibrotic mediator in a natural model of hepatic fibrosis induced by infection with Schistosoma mansoni. Interestingly, in the same infection hepatic fibrosis model IL-13-dependent fibrosis was evident, yet was TGF-β and Smad-3 independent, thus indicating that IL-13 is directly driving fibrosis that is independent of the classic TGF-β pathway of fibrosis. Targeted pulmonary overexpression of IL-13 using genetic approaches in mice results in lung fibrosis. Using the bleomycin model of SSc it was demonstrated that IL-13 is increased in the pathogenesis of the disease and neutralization with IL-13 antibodies attenuates this effect. In both normal and keloid fibroblasts, IL-13 resulted in direct up-regulation of collagen production that was similar in magnitude to TGF-β-stimulated production. Addition of IL-13 also reduced MMP levels and increased tissue inhibitor of metalloproteinase 1 (TIMP-1) expression, thereby altering the extracellular matrix remodelling in favour of increased deposition. IL-13 has also been shown to be an absolute requirement for the development of cutaneous fibrosis mediated by IL-33, as genetic deletion of IL-13, but not of IL-4, totally abrogates IL-33-mediated fibrosis and collagen content, and this was independent of TGF-β1 expression.