MFG-E8 was first identified in the process of phagocytic clearance of apoptotic cells . More recently, it has been shown that MFG-E8 deficient mice show severe inflammatory imbalances, and an MFG-E8-mediated potential therapeutic benefit is evident in experimental inflammatory conditions [18, 20, 22–25, 29–32]. Previous studies have elucidated the role of MFG-E8 in diverse neoplastic and acute inflammatory diseases, yet this is the first study to investigate MFG-E8 expression in chronic pancreatitis.
In the present study, we demonstrated for the first time that MFG-E8 is significantly up-regulated in patients with chronic pancreatitis. Interestingly, and in contrast to acute inflammatory diseases where MFG-E8 is under-expressed in the acute phase and recombinant MFG-E8 seems beneficial, MFG-E8 expression in chronic pancreatitis is significantly higher than in normal pancreatic tissue. Our analysis further revealed that this overexpression is directly related to the presence of pain and associated with severe fibrosis in these patients. The connective link between the chemo-kine fractalkine and MFG-E8 that became evident in chronic pancreatitis further underlines the potential of MFG-E8 as a future therapeutic target in the treatment of painful chronic pancreatitis.
Long-lasting pancreatic pain is the most prominent symptom of chronic pancreatitis which has recently been suggested to be a mixed-type pain from both nociceptive and neuropathic mechanisms . Previous studies have demonstrated a key correlation between the extent of neural damage, neural plasticity, neural inflammatory cell infiltration (pancreatic neuritis), and the pain frequency and intensity of CP patients, thereby underlining the importance of neuropathic mechanisms in CP pain generation [27, 34]. In this study we were able to demonstrate that MFG-E8 correlates significantly with the presence of pancreatic pain in CP patients. This observed correlation may theoretically be explained by three different possible pathways: a) MFG-E8 expression may be induced by a yet unknown common factor that likewise induces pain in CP patients, b) MFG-E8 may have an analgesic potential and is therefore released by CP tissue as a reaction on a pain stimulus in the attempt to limit pain sensation. While these possibilities can currently not be completely ruled out, it seems more likely that c) MFG-E8 may influence the extent of neural alterations in chronic pancreatitis and thereby aggravate chronic pain sensation in these patients. While the exact mechanisms remain to be unravelled, a direct negative influence of MFG-E8 on neural inflammatory cell infiltration seems unlikely since MFG-E8 has been shown to rather attenuate neutrophil infiltration in acute lung injury . It is more likely that MFG-E8 modulates pain sensations by directly aggravating the extent of neural damage, possibly by MFG-E8 mediated phagocytosis of viable neurons and/or glial cells as it has very recently been shown for states of neuroinflammation . Accordingly, and consistent with findings in cancer therapy, MFG-E8 blockade - not treatment with recombinant MFG-E8 - may be a possible treatment for chronic pancreatic pain. This idea seems especially tempting since MFG-E8, as a secreted protein, may be rather easily targeted by appropriate blocking antibodies.
Our understanding of the cellular and molecular events leading to the development of pancreatic fibrosis has improved significantly in recent years, largely due to the isolation and characterization of pancreatic stellate cells (PSCs) which play a key role in fibrogenesis [5, 36]. Stellate cells are resident cells of the pancreas, located at the basolateral aspect of acinar cells. During inflammatory injury, PSC become activated by migrating mononuclear inflammatory cells to produce and secrete extracellular matrix proteins such as collagen, fibronectin, and laminin, but also pro-inflammatory cytokines and chemokines [37–40]. Our data show that MFG-E8 is highly expressed in human pancreatic stellate cells isolated from chronic pancreatitis tissue, suggesting that MFG-E8 is secreted in high levels by activated hPSC in the chronic state of pancreatitis. On the other hand, high MFG-E8 expression may be in indicator for enhanced hPSC turnover in chronic pancreatitis with a higher rate of phagocytosis of dying hPSCs. This is further underlined by our immunohistological findings that MFG-E8 immunoreactivity was noticeably increased in pancreatic areas neighboring tissue sections with increased fibrosis and ECM. While the ECM itself showed little to no immunoreactivity for MFG-E8 in our study, neighboring regions which are known to contain the activated pancreatic stellate cells showed the highest immunoreactivity . Furthermore, we were able to show that MFG-E8 expression significantly correlated with severe fibrosis in CP patients. According to our observed correlations with pain this correlation as such may be explained by different possible mechanisms. The most likely however is that MFG-E8 may aggravate fibrogenesis in CP patients, possibly by direct modulation of hPSC activation. This MFG-E8 overexpression in hPSC may be on the one hand an indicator of apoptosis, but on the other hand indicate enhanced growth signaling, as MFG-E8 was previously shown to induce PDGF signaling and regulate cyclin D1/D3 expression, especially in neoplastic cells. However, since smoking habits were unfortunately not recorded systematically in the medical history reports we cannot entirely exclude smoking as a systemic confounder influencing MFG-E8 expression in CP.
Another important finding of our study is the strong correlation of the expression of MFG-E8 and the chemokine fractalkine. Fractalkine is the sole member of the fourth class of chemokines and is known to mediate both chemotaxis and adhesion . Fractalkine can be released by proteolysis at a membrane-proximal region by TNF-α converting enzyme (TACE, ADAM17) and ADAM10 [42, 43]. It was furthermore reported to be involved in the pathogenesis and progression of numerous inflammatory diseases including chronic pancreatitis [21, 44–48]. Recent investigations by our group revealed that the expression of fractalkine strongly correlates with the degree of fibrosis, the severity of neural inflammatory cell infiltration (“pancreatic neuritis”), and the severity and frequency of pain in chronic pancreatitis . The correlation of MFG-E8 expression with fractalkine may therefore suggest an equally important role of MFG-E8 in chronic pancreatitis. This assumption is underlined by the fact that MFG-E8 was likewise significantly correlated to the presence of pain and severe fibrosis in chronic pancreatitis. At last, fractalkine expression seems to be closely linked to MFG-E8 expression since fractalkine stimulation of peritoneal macrophages and microglia has been reported to lead to MFG-E8 overexpression [19, 20]. Likewise, we were able to show for the first time that isolated human pancreatic stellate cells express MFG-E8 and show a significant increase in MFG-E8 expression after fractalkine stimulation. We therefore could illustrate that the connective link between MFG-E8 and fractalkine, which has been shown for microglia and macrophages in acute inflammatory conditions is similarly evident in hPSC in chronic pancreatitis. This connective link again underlines the significance of MFG-E8 as a potential therapeutic target.