Monocytes Infiltrate the Pancreas via the MCP-1/CCR2 Pathway and Differentiate into Stellate Cells

<div><p>Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP)⁺CD45^– cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl₄). Because the vast majority of EGFP⁺CD45^– cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs). EGFP⁺ PaSCs were also observed in CCl₄-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) increased in the pancreas of CCl₄-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2) and Ang II type 1 receptor (AT1R), were expressed on Ly6C^high monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II <i>in vitro</i>. Irbesartan inhibited not only their <i>in vitro</i> chemotaxis but also <i>in vivo</i> migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP⁺F4/80⁺CCR2⁺ monocytic cells and EGFP⁺ PaSCs in the pancreas of CCl₄-treated chimeric mice receiving EGFP⁺ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP⁺ PaSCs in injured mice. We propose that CCR2⁺ monocytes migrate into the pancreas possibly via the MCP-1/CCR2 pathway and give rise to PaSCs.</p></div

Experimental design.

<p>(A) Clones from a CD34^–c-kit⁺Sca-1⁺lineage^– (CD34^–KSL) cell or a total of 2×10⁶ bone marrow-total nucleated cells (BM-TNCs) isolated from enhanced green fluorescent protein (EGFP)-transgenic mice were transplanted into lethally irradiated C57BL/6J-Ly5.1 mice. Two months after BM transplantation, mice were intraperitoneally injected with CCl₄ or olive oil twice a week for 12 weeks. (B) C57BL/6J-Ly5.1 mice were intraperitoneally injected with CCl₄ twice weekly for 5 weeks. Ly6C⁺ monocytes, peripheral blood (PB)-TNCs, or a PB-Ly6C⁺ cell-depleted population isolated from EGFP-transgenic mice were adoptively transferred into CCl₄-treated mice at 24 hours after each injection of CCl₄ for 2 weeks. (C) C57BL/6J-Ly5.1 mice were intraperitoneally injected with CCl₄ twice weekly for 5 weeks. They were fed chow containing irbesartan or normal chow for 2 weeks. Ly6C⁺ monocytes isolated from EGFP-transgenic mice were adoptively transferred into CCl₄-treated mice at 24 hours after each injection of CCl₄ for 2 weeks. (D) C57BL/6J-Ly5.1 mice that received 2×10⁶ EGFP⁺ BM-TNCs were fed chow containing irbesartan or normal chow for 6 weeks. In another group of mice, RS504393 or vehicle was subcutaneously administered once a day for 6 weeks. One week after the initiation of irbesartan or RS504393 treatment, CCl₄ treatment was started and continued for 5 weeks.</p

Hematopoietic engraftment in mice transplanted with clonal cells derived from a single CD34^–KSL cell.

<p>EGFP, enhanced green fluorescent protein; CD34^–KSL cell,</p><p>CD34^–c-kit⁺Sca-1⁺lienage^– cell.</p

MCP-1 and angiotensinogen production in pancreas of CCl₄-treated mice and their receptor expression on monocytes.

<p>(A) The number of engrafted EGFP⁺ cells and the percentages of CD45^–GFAP⁺ cells among EGFP⁺ cells in the pancreases of mice that received Ly6C^highc-kit^– cells, PB-TNCs, or a PB-Ly6C⁺ cell-depleted population isolated from EGFP-transgenic mice are shown. Data are the means ± SD of three mice. (B) Total RNA isolated from the pancreases from four untreated mice, four olive oil-treated mice, and three CCl₄-treated mice was analyzed by RT-PCR for mRNA expression of MCP-1 and angiotensinogen. The level of mRNA was normalized to that of GAPDH mRNA. Data are the means ± SD of three or four mice per group. *<i>P</i><0.05 versus untreated mice or olive oil-treated mice. (C) Total RNA from Ly6C⁺ monocytes, which were isolated from the BM of naive EGFP mice, was analyzed by RT-PCR for mRNA expression of CCR2, AT1Ra, and AT1Rb. Representative examples of CCR2, AT1Ra, and AT1Rb mRNA expression are shown in the left panel. The expression of CCR2 and AT1R on Ly6C⁺ monocytes was evaluated by single-color flow cytometry. Representative examples of histograms for CCR2 and AT1R expression are shown in the center and right panel, respectively. Black lines indicate isotype control staining.</p

Effect of irbesartan on hematopoietic lineage cell infiltration into the CCl₄-injured pancreas.

<p>EGFP⁺ BM-TNC-transplanted mice were fed a normal chow diet or an irbesartan-containing diet and treated with CCl₄ for 6 weeks. (A) Numbers of EGFP⁺F4/80⁺ monocytic cells, EGFP⁺Ly6G⁺ neutrophils, EGFP⁺B220⁺ B cells, and EGFP⁺CD3ε⁺ T cells in the pancreas of both groups. Data are the means ± SD of three mice per group. *<i>P</i><0.05 versus mice fed a normal chow. (B) Panels show EGFP as green, CCR2 as red, F4/80 as blue, and the merged images of EGFP, CCR2, F4/80, and DIC images. White triangles, the yellow triangle, and the yellow asterisk indicate EGFP⁺F4/80⁺CCR2⁺ cells, an EGFP⁺F4/80⁺CCR2^– cell, and an EGFP⁺F4/80⁻CCR2⁺ cell, respectively. Scale bars, 30 µm. (C). Numbers of EGFP⁺F4/80⁺CCR2⁺ cells and EGFP⁺F4/80⁺CCR2^– cells in the pancreas of both groups. Data are the means ± SD of three mice per group. *<i>P</i><0.05 versus mice fed a normal chow. HPF, high power field.</p

PaSC expression markers in donor-derived cells from injured pancreas of mice transplanted with clonal cells derived from a single CD34^–KSL cell or BM-TNC.

<p>PaSC, pancreatic stellate cell; CD34^–KSL cell, CD34^–c-kit⁺Sca-1⁺lienage^– cell; BM-TNCs, bone marrow-total nucleated cells; EGFP, enhanced green fluorescent protein; GFAP, glial fibrillary acidic protein; α-SMA, α-smooth muscle actin; SD, standard deviation. We analyzed the pancreases of two mice transplanted with clonal cells derived from a single CD34^–KSL cell and three mice transplanted with BM-TNCs.</p

Inhibitory effect of irbesartan on Ly6C⁺ monocyte migration toward MCP-1.

<p>Chemotaxis of Ly6C⁺ monocytes toward MCP-1, Ang II, or MIP-1α was investigated using transfilter assays. (A) Photos show representative micropore membrane images with or without MCP-1 (1 nmol/l) in the lower chamber of a 24-well transwell plate. (B) Ly6C⁺ monocytes (2×10⁵) were seeded in the upper chamber of a 24-well transwell plate, and various concentrations of MCP-1 or Ang II (0.1 and 1 nmol/L or 1 and 10 µmol/L, respectively) were placed in the lower chamber. Data are the means ± SD of three wells and representative of two independent experiments. *<i>P</i><0.05; **<i>P</i><0.01 versus control. (C) Effect of various concentrations of irbesartan on Ly6C⁺ monocyte migration towards MCP-1 (1 nmol/l). Data are the means ± SD of three wells and representative of two independent experiments. *<i>P</i><0.05; **<i>P</i><0.01 versus control. (D) Comparison of irbesartan and RS504393 for Ly6C⁺ monocyte migration. Ly6C⁺ monocytes were treated with the indicated concentrations of irbesartan or RS504393 for 1 hour and subjected to MCP-1- or MIP-1α-induced chemotaxis. Data are the means ± SD of three wells and representative of two independent experiments. *<i>P</i><0.05; **<i>P</i><0.01 versus control. (E) Effects of irbesartan on migration of adoptive transferred EGFP⁺ monocytes into the pancreas of CCl₄-treated mice. Number of engrafted EGFP⁺ cells in 10 sections of pancreas from mice treated with or without irbesartan is shown. Data are the means ± SD of three different mice per group. *<i>P</i><0.05 versus control mice fed a normal chow.</p

Expression of PaSC-associated antigens in EGFP⁺ cells in pancreas of mice receiving clonal cell populations derived from a single EGFP⁺ hematopoietic stem cell.

<p>(A) Pancreases from CCl₄-treated mice that received clones from a CD34^–KSL cell isolated from EGFP-transgenic mice were examined immunohistochemically. Panels show EGFP as green, TO-PRO-3 nuclei stain as blue, differential interference contrast (DIC) image, and the combined merged image. Scale bars, 30 µm. (B) Panels show EGFP as green, vimentin, GFAP, desmin, procollagen-I or α-SMA as red, CD45 as blue, and the merged images of these markers together with DIC images. White triangles indicate EGFP⁺CD45^–vimentin⁺, EGFP⁺CD45^–GFAP⁺, EGFP⁺CD45^–desmin⁺, EGFP⁺CD45^–procollagen-I⁺ or EGFP⁺CD45^–α-SMA⁺ cells. Scale bars, 30 µm.</p

Effect of irbesartan on the occurrence of hematopoietic cell-derived PaSCs in pancreas of CCl₄-treated mice.

<p>EGFP⁺ BM-TNC-transplanted mice were fed a normal chow diet or an irbesartan-containing diet for 6 weeks and treated with CCl₄ for 5 weeks. (A) Numbers of EGFP⁺CD45^–vimentin⁺, EGFP⁺CD45^–desmin⁺, EGFP⁺CD45^–GFAP⁺, EGFP⁺CD45^–procollagen-I⁺, and EGFP⁺CD45^–α-SMA⁺cells in the pancreas of both groups. Data are the means ± SD of three mice per group. *<i>P</i><0.05 versus mice fed normal chow. (B) Comparison of irbesartan and RS504393 in the occurrence of EGFP⁺CD45^–GFAP⁺ cells in the pancreas of CCl₄-treated mice. Data are the means ± SD of three mice per group. *<i>P</i><0.05 versus mice fed normal chow. HPF, high power field.</p