Double immunostaining of leukemia cells using anti-IL-2Rα and anti-CD13 antibodies in a representative patient with IL-2Rα⁺CD4⁺AML.

<p>The horizontal axis indicates the logarithmic scale of FITC-conjugated anti- IL-2Rα binding, and the vertical axis PE-conjugated anti-CD13 binding. IL-2Rα and CD13 dual-positive cells are shown in the right upper quadrant (B).</p

Relationship between IL-2Rα expression levels and OS.

<p>Kaplan—Meier estimates of OS for non-M3 patients ≤60 years old are shown according to the expression levels of IL-2Rα (sites/cell).</p

Correlation of cytokine receptor level with clinicalclinical and cellular features in patients with AML.

<p>Data are given as the mean levels of cytokine receptor expression (sites/cell) ± standard error (number of patients analyzed).</p><p>* <i>p</i> < 0.05.</p><p>**<i>p</i> < 0.01.</p><p>Correlation of cytokine receptor level with clinicalclinical and cellular features in patients with AML.</p

Effect of IL-2Rα expression on cytogenetic-risk classification.

<p>Kaplan—Meier estimates of OS for AML patients ≤60 years old are shown based on the 3 distinct cytogenetic-risk groups. (A) The intermediate-risk group was clearly divided into IL-2Rα⁺ patients and IL-2Rα⁻ patients. (B) Revised survival curves show that the original favorable-risk group, the intermediate-risk group in which IL-2Rα⁺ patients were excluded, and the adverse-risk group in which IL-2Rα⁺ patients in the intermediate-risk group were included.</p

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

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

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

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

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