When studying the pathogenesis of Dupuytren Disease, it is important to use appropriate controls. Many previous studies have compared cells from Dupuytren nodules or cords with cells from the fascia in the region of the carpal tunnel or the transverse carpal ligament from affected or normal individuals or uninvolved transverse palmar fibres from patients with Dupuytren Disease. However, this approach has limitations. The palmar fascia over the carpal tunnel is rarely affected by Dupuytren Disease in susceptible individuals, and the transverse carpal ligament is never involved; hence, it is possible that the constituent cells are inherently different (Satish et al. 2012). Furthermore, with the exception of nodules in Dupuytren Disease, fascia is sparsely populated by cells, and hence to obtain adequate numbers, most authors use cells to passage 5 (Bisson et al. 2003). However, we and others have shown that at passage 5 the phenotypes of myofibroblasts and normal human dermal fibroblasts tend to merge (Rehman et al. 2012; Verjee et al. 2010). In addition, whereas the cell of origin for Dupuytren myofibroblasts remains controversial, there is accumulating evidence that the adjacent tissues, including the peri-nodular fat and overlying dermis, make a significant contribution (Iqbal et al. 2012). Moreover, Dupuytren Disease is restricted to the palm of the hands in patients with a genetic predisposition. Therefore, it is important to avoid variations due to genetic and environmental factors. For these reasons, we compared dermal fibroblasts from palmar and non-palmar sites from the same group of patients, using palmar dermal fibroblasts from individuals without Dupuytren Disease as controls (Verjee et al. 2013). The restriction of Dupuytren Disease to the palm of genetically susceptible individuals suggests that there may be epigenetic regulation of precursor cells. Hence, it would be appropriate to compare cells from affected and unaffected regions of the same individual.

Several studies have sought to characterise the cells in Dupuytren Disease. We found that the majority of the myofibroblasts are concentrated in histological nodules in excised cords (Verjee et al. 2009), and within these nodules, approximately 90 % of the cells are α-SMA positive (Verjee et al. 2013). Several authors have demonstrated the presence of immune cells in nodules, including macrophages and T cells (Andrew et al. 1991; Baird et al. 1993; Meek et al. 1999; Sugden et al. 1993; Verjee et al. 2013). The group that did not find any CD68+ macrophages cells on immunostaining did not show their positive controls (Bianchi et al. 2015). In our view the data from several groups using immunostaining and FACS analysis of freshly disaggregated cells from Dupuytren Disease confirms the presence of immune cells in Dupuytren nodules, where the macrophages appear to localise predominantly in the vicinity of the blood vessels (Verjee et al. 2013).

A number of studies examining the expression of cytokines and growth factors by RT-PCR or immunohistochemistry identified IL1-α, IL-1β, TGF-β, FGF and VEGF in Dupuytren tissue (Baird et al. 1993; Berndt et al. 1995; Badalamente et al. 1996; Bianchi et al. 2015; Ratajczak-Wielgomas et al. 2012). We studied the cytokines in the supernatant secreted by freshly disaggregated cells from Dupuytren nodules, analogous to the experiments that led to the identification of TNF as a therapeutic target in rheumatoid arthritis (Brennan et al. 1989). Over a 24-h period, during which the inflammatory cells remained viable in culture without the addition of exogenous growth factors, we detected TGF-β1, TNF, IL-6 and GM-CSF; levels of IL-1β, IL-10 and IF-γ were very low (Verjee et al. 2013). We have subsequently found that the levels of secreted TNF remained unchanged over a range of cell concentrations (1.5 × 10⁵–1 × 10⁶ cells in 2 ml culture media). By passage 2 the inflammatory cells had been lost, and levels of TNF in cell culture supernatants fell to near zero, whilst levels of TGF-β1 increased over 2 fold, the latter through autocrine secretion by myofibroblasts in culture (Verjee et al. 2013). These findings highlight the importance of studying primary cells to identify potential therapeutic targets and may go some way towards explaining the lack of efficacy of all late phase clinical trials to date targeting TGF-β1 for fibrotic diseases (Hawinkels and Ten Dijke 2011; Varga and Pasche 2009). The published data suggest that Dupuytren Disease is a localised inflammatory disorder, and it has been suggested that all fibrosis occurs as result of preceding inflammation (Wick et al. 2013).

We compared the effects of the secreted cytokines on early passage palmar dermal fibroblasts from patients with Dupuytren Disease with control fibroblasts from the palmar skin of normal individuals and the non-palmar dermis of patients with Dupuytren Disease (Verjee et al. 2013). Predictably, TGF-β1 led to the differentiation of all three types of dermal fibroblasts into myofibroblasts but only at relatively high (1–10 ng/ml) concentrations. For comparison, freshly disaggregated cells from Dupuytren nodules in culture secreted 236 ± 248 pg/ml TGF-β1. TNF at concentrations of 50–100 pg/ml led to the differentiation of only palmar dermal fibroblasts from Dupuytren patients into myofibroblasts. At these levels, TNF had no effect on the control dermal fibroblasts, whilst higher concentrations (1–10 ng/ml) resulted in downregulation of their contractile activity, as previously reported (Goldberg et al. 2007). For comparison, freshly disaggregated nodular cells secreted 78 ± 26 pg/ml of TNF (Verjee et al. 2013).

The different responses to TNF of the various cell types may in part be related to enhanced expression of TNF receptors at both mRNA and protein level, in particular TNFR2 by palmar dermal fibroblasts from Dupuytren patients and by the Dupuytren myofibroblasts (Verjee et al. 2013).

A number of genetic studies have identified an association between the Wnt signalling pathway and Dupuytren Disease (Anderson et al. 2014; Dolmans et al. 2011). However, the expression of Wnts was found not to account for the dysregulated expression of β-catenin in Dupuytren Disease (O’Gorman et al. 2006). We found that only in palmar dermal fibroblast from Dupuytren patients, but not in control cells, there was crosstalk between TNF and canonical Wnt signalling pathways, whereby treatment of the cells with TNF led to inhibition of GSK-3β, which in turn leads to preservation of β-catenin from degradation (Fig. 8.2) (Verjee et al. 2013). The β-catenin translocates to the nucleus and upregulates the transcription of profibrotic genes, including for α-SMA and COL-1A1. The β-catenin is also a key component of intercellular adherens junctions. The crosstalk between TNF and Wnt signalling pathways has previously been shown in pre-adipocytes (Cawthorn et al. 2007; Hammarstedt et al. 2007).