Monocytes Infiltrate The Pancreas Via MCP-1/CCR2 Signaling and Differentiate Into Pancreatic Stellate Cells During Pancreatic Injury

We previously reported that monocytes could differentiate into hepatic stellate cells during carbon tetrachloride (CCl₄) injury. Pancreatic stellate cells (PaSCs), which play a central role in the development of pancreatic fibrosis, share many morphological and functional characteristics with those of hepatic stellate cells. Although PaSCs have been thought to be of mesenchymal origin, some authors have recently reported that they are derived from bone marrow (BM) cells during inflammation. Meanwhile, both monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) are thought to be involved in the development of pancreatic fibrosis. We hypothesized that PaSCs are also derived from monocytes and investigated the mechanism of how monocytes enter the injured pancreas, focusing on MCP-1 and Ang II.

First, we generated chimeric mice by transplantation of clonal populations of cells derived from single lineage negative Sca-1⁺c-kit⁺CD34^- cells or total-nucleated cells, which were sorted from the BM of enhanced green fluorescent protein (EGFP)-transgenic mice, into lethally irradiated C57BL/6 mice. Since 2 months after BM transplantation, the chimeric mice revealing high-level multilineage engraftment were intraperitoneally injected with CCl₄ twice a week. Next, we intravenously transferred Ly6C^highc-kit^– monocytes, which were isolated from the BM of EGFP-transgenic mice, into non-irradiated CCl₄-treated mice. We analyzed the presence of EGFP⁺ PaSCs in the injured pancreas using immunofluorescence and laser confocal scanning microscopy. Finally, we investigated the effect of Ang II type 1 receptor (AT1R) antagonist, irbesartan, which is also an antagonist of MCP-1 receptor, CC chemokine receptor 2 (CCR2) on the occurrence of monocyte-derived PaSCs in the pancreases of CCl₄-treated mice.

After 12 weeks of injury by CCl₄, EGFP⁺CD45^- cells were detected in the pancreases of chimeric mice. They expressed PaSC-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin. Next, we found EGFP⁺ PaSCs in CCl₄-treated mice with adoptively transferred Ly6C^high monocytes but not other lineages. MCP-1 and Ang II were highly produced in the pancreas of CCl₄-treted mice and their receptors CCR2 and AT1R were respectively expressed on Ly6C^high monocytes isolated from the BM of EGFP-transgenic mice. Monocytes migrated toward MCP-1, but not Ang II, and irbesartan inhibited their chemotaxis in vitro. Irbesartan treatment significantly reduced the numbers of EGFP⁺CCR2⁺F4/80⁺ macrophages and EGFP⁺ PaSCs in the pancreas of CCl₄-treated mice. We observed that a specific CCR2 antagonist RS504393 inhibited the monocyte migration toward MCP-1 in vitro and the occurrence of EGFP⁺ PaSCs in the injured mice like irbesartan.

We propose that CCR2⁺ monocytes migrate into the injured pancreas via the MCP-1/CCR2 pathway and differentiate into PaSCs during inflammation.

Masuya:Dainippon-Sumitomo Pharmaceutical: Research Funding.