This investigation provides new insights into characteristics of the immune response in scleroderma and is the first to demonstrate a significant correlation between specific T cell subsets and distinct clinical manifestations. We used sensitive markers and specifically the CCR5/CRTH2 ratio of circulating T cells to reliably measure directly ex vivo the polarization of the underlying immune phenotype in a well-characterized group of patients with SSc.
We observed that a significant Th2/Tc2 bias is present among patients with scleroderma compared with healthy control subjects, and that both CD4+ and CD8+ T cells exhibit this “skewed” type 2 phenotype. Moreover, in patients with SSc, Th2/Tc2 polarization was significantly associated with the presence of ILD. This association was further confirmed by a strong linear correlation between a lower CCR5/CRTH2 T cell ratio (Th2/Tc2 bias) and worse FVC values. Although this was a cross-sectional analysis, we did have access to longitudinal lung function studies, allowing us to examine whether low CCR5/CRTH2 ratios were more likely to be observed in patients with active lung disease. Interestingly, the CCR5/CRTH2 T cell ratio was significantly lower in patients with SSc who had experienced a recent decline (>10%) in FVC values, suggestive of lung disease activity at the time of peripheral blood analysis. Although the CCR5/CRTH2 T cell ratio was not significantly associated with the DLCO value, an interesting finding became evident when the presence of pulmonary vascular disease (indicated by an abnormal eRVSP value as measured by echocardiography) was considered. In SSc patients with an eRVSP >35 mm Hg, a lower DLCO value was strongly associated with higher CCR5/CRTH2 ratios, suggestive of Th1/Tc1 T cell polarization, while those with an eRVSP <35 mm Hg exhibited an opposite correlation. Thus, the presence of ILD in our patients with SSc was associated with a Th2/Tc2 bias, with lower CCR5/CRTH2 ratios being even more prevalent in patients with progressive worsening of their lung function. In contrast, the presence of pulmonary vascular disease appears to be characterized by a dominant Th1/Tc1 circulating T cell phenotype.
Notably, although we observed that CCR5/CRTH2 ratios were generally lower in patients with dcSSc compared with patients with lcSSc, the modified Rodnan skin score (degree of skin disease) did not show any correlation with the polarization of T cells. We interpret this finding cautiously because of the cross-sectional design of our study and the high variability of skin involvement over time in scleroderma. In addition, although the lung function test is an excellent measure of ILD and its progression, we acknowledge that the echocardiographic determination of the eRVSP is not a definitive measure of pulmonary vascular disease or PAH. Most of our patients did not undergo right heart catheterization. Nevertheless, evaluating the eRVSP by echocardiography represents a widely accepted and validated screening method for determining the presence of pulmonary vascular disease (33
). Thus, the presence of a prevalent Th1/Tc1 T cell phenotype in patients with a high eRVSP may suggest a possible association with emerging or actual PAH, which will need to be further confirmed using direct measures (right heart catheterization) in longitudinal studies.
A dominant Th2 immune response in patients with SSc has been previously reported, but the prevalence of a Th1 or a mixed immune phenotype has also been described (19
). Importantly, the methods used to determine polarization of the immune response have been, in most cases, indirect and based on measuring highly variable parameters such as cytokine plasma levels or their mRNA expression within circulating cells or target tissues. In contrast to cytokine genes, CCR5 and CRTH2 are constitutively expressed in type 1 and type 2 T cells, thereby providing a more direct, stable measure of the relative prevalence of either subset. Furthermore, no study has specifically addressed and accurately analyzed the correlation between the polarized T cell phenotype and the underlying SSc-specific clinical manifestations. For these reasons, the clinical and pathogenetic relevance of a polarized immune response in SSc has not yet been fully elucidated.
Our study used a sensitive, reliable, and easily reproducible experimental assay and measured ex vivo the polarization of circulating T cells, using very specific surface markers. The SSc patient population analyzed was relatively large, clinically well characterized, and selected independently of any laboratory study or disease status. These factors confer strength and relevance to our findings and draw attention to the measurement of evolving T cell polarization (the CCR5/CRTH2 T cell ratio) as a potential novel and sensitive approach to monitor the course of disease manifestations in scleroderma and particularly in lung disease. Our findings suggest the possibility that the CCR5/CRTH2 ratio will not change over time in patients with SSc in whom lung disease is stable, while it may decrease, marking a shift toward a higher Th2/Tc2 bias, just before or at the time of onset of active alveolitis. Furthermore, a greater type 2 skewing of circulating T cells at the time of diagnosis or early in the disease course may identify patients who are at higher risk of ILD developing or in whom ILD will progress to more severe disease. Indeed, a group of patients in our study had normal results of pulmonary function tests and exhibited a relatively low CCR5/CRTH2 ratio (). It will be of great interest to follow up these patients prospectively and to confirm the predictive value of measuring a polarized T cell response before lung disease becomes manifest.
This is the first report to describe the detection of an opposite pattern of T cell polarization in association with SSc-related ILD or pulmonary vascular disease. The relevance of a Th2/Tc2-polarized immune response in the pathogenesis and progression of SSc lung fibrosis is plausible, although it remains to be confirmed whether such bias is present within early inflammatory lung infiltrates during ILD initiation. With regard to SSc vasculopathy, previous studies have implicated both cellular and humoral immune effector pathways and have demonstrated that T cells can cause microvascular injury directly through cell-mediated cytotoxicity or by inducing proinflammatory molecules on the endothelial cell surface (36
). Recently, new data have shown that the endothelium and inflammatory cells infiltrating vessel walls in lesional skin and lung biopsy specimens from patients with SSc exhibit up-regulation of IFNγ
-inducible molecules that are implicated in vascular smooth muscle migration/proliferation as well as proinflammatory cytokine secretion (38
Thus, it is possible that a Th1/Tc1 microenvironment is functionally relevant for generation of the vascular disease seen in scleroderma. Further studies are needed to determine whether the polarized immune responses observed in the context of specific SSc lung disease manifestations are causally implicated or whether they result from abnormal lymphocyte trafficking secondary to the specific tissue damage affecting different lung compartments in SSc (i.e., interstitium or vascular bed). Other disease models exist in which extreme immune polarization toward a Th1 or Th2 response can be associated with different disease manifestations and mediate distinct outcomes. For example, in human schistosomiasis an initial acute proinflammatory Th1 response continues until a transition toward a chronic Th2 polarization occurs, with promotion of perioval granuloma formation (39
). When this Th1-to-Th2 shift is balanced and controlled, the disease tends to be mild. Conversely, a persistent Th1-type response correlates with a more severe systemic infection, while an excessive Th2-dominated tissue environment is associated with development of severe liver fibrosis (40
Our study provides new insight into potential pathogenetic pathways in scleroderma and may have important clinical implications, particularly in terms of the management and treatment of lung involvement. Clinicians caring for patients with SSc are challenged to identify those with active lung disease, who predictably will experience progressive loss of lung function. In addition, potentially toxic immunosuppressive treatments are administered and monitored based almost exclusively on crude clinical responses, with significant attendant morbidity and mortality. Measuring the CCR5/CRTH2 T cell ratio in the peripheral blood of patients with SSc in order to quantify polarization of the immune response may prove to be a useful measurable biologic marker to monitor disease activity and allow early identification of patients who will evolve toward a more aggressive and life-threatening disease phenotype. In addition, this assay has the potential of a broader application to other autoimmune conditions associated with ILD, including, for example, the antisynthetase syndrome in myositis, systemic lupus erythematosus lung disease, and mixed connective tissue disorder. Finally, specific interventions to rebalance skewed type 1 or type 2 T cell responses in patients with developing PAH or ILD may represent a novel therapeutic strategy.
In summary, we have demonstrated that specific manifestations of lung disease in patients with SSc are strongly correlated with opposite patterns of circulating T cell polarization, as measured by the CCR5/CRTH2 T cell ratio. A Th2/Tc2-polarized immune response is associated with ILD and particularly with active lung disease. We speculate that polarized T cell effectors may have a crucial role in the development and/or progression of lung disease in SSc. Further characterization and prospective measurement of polarized type 1/type 2 immune effectors in SSc are needed to confirm these data and to allow the identification of more dependable biomarkers of lung disease activity and accurate predictors of outcome, and potentially to open the door to novel disease-specific therapeutic targets.