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. 2008 Nov;57(11):3025-33.
doi: 10.2337/db08-0625. Epub 2008 Jul 15.

Increased expression of CCL2 in insulin-producing cells of transgenic mice promotes mobilization of myeloid cells from the bone marrow, marked insulitis, and diabetes

Andrea P Martin  1 , Sara Rankin, Simon Pitchford, Israel F Charo, Glaucia C Furtado, Sergio A Lira
Affiliations

Increased expression of CCL2 in insulin-producing cells of transgenic mice promotes mobilization of myeloid cells from the bone marrow, marked insulitis, and diabetes

Andrea P Martin et al. Diabetes. 2008 Nov.

Abstract

Objective: To define the mechanisms underlying the accumulation of monocytes/macrophages in the islets of Langerhans.

Research design and methods: We tested the hypothesis that macrophage accumulation into the islets is caused by overexpression of the chemokine CCL2. To test this hypothesis, we generated transgenic mice and evaluated the cellular composition of the islets by immunohistochemistry and flow cytometry. We determined serum levels of CCL2 by enzyme-linked immunosorbent assay, determined numbers of circulating monocytes, and tested whether CCL2 could mobilize monocytes from the bone marrow directly. We examined development of diabetes over time and tested whether CCL2 effects could be eliminated by deletion of its receptor, CCR2.

Results: Expression of CCL2 by beta-cells was associated with increased numbers of monocytes in circulation and accumulation of macrophages in the islets of transgenic mice. These changes were promoted by combined actions of CCL2 at the level of the bone marrow and the islets and were not seen in animals in which the CCL2 receptor (CCR2) was inactivated. Mice expressing higher levels of CCL2 in the islets developed diabetes spontaneously. The development of diabetes was correlated with the accumulation of large numbers of monocytes in the islets and did not depend on T- and B-cells. Diabetes could also be induced in normoglycemic mice expressing low levels of CCL2 by increasing the number of circulating myeloid cells.

Conclusions: These results indicate that CCL2 promotes monocyte recruitment by acting both locally and remotely and that expression of CCL2 by insulin-producing cells can lead to insulitis and islet destruction.

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Figures

FIG. 1.
FIG. 1.
Augmented levels of CCL2 in islets of Langerhans lead to an increased number of monocytes in blood, infiltrated islets, and spontaneous diabetes. A: Semiquantitative analysis of islet infiltrates in the pancreata from control and RIPCCL2 transgenic mice at 4 weeks of age (n = 8/group). B: Cumulative incidence of diabetes in RIPCL2 mice from lines 251 (n = 60), 10 (n = 40), 254 (n = 95), and 1 (n = 31) and from nontransgenic littermates (n = 20). C: Relative numbers of CD115+ cells in blood from wild-type and RIPCCL2 mice (n = 20 mice in each line; t test **P < 0.005, ***P < 0.0001). D: CCL2 levels in the serum of RIPCCL2 mice of lines 251, 10, 254, and 1 and wild-type littermates was analyzed by ELISA (n = 20/line; t test **P < 0.005, ***P < 0.0001).
FIG. 2.
FIG. 2.
CCL2 induces release of monocytes from the bone marrow. The perfusion of femoral bone marrow was performed as described in research design and methods. A: Representative dot plot of monocyte release from bone marrow induced by CCL2 perfusion. B and C: Relative (B) and absolute (C) number of monocytes (CD115+/Gr-1intermediate cells) after PBS (control) or CCL2 perfusion (n = 5 mice in each treatment; t test *P < 0.05, **P < 0.005).
FIG. 3.
FIG. 3.
Deletion of CCR2 abrogates peripheral monocytosis in RIPCCL2 mice. A and B: Relative number of CD115+Ly6C+ monocytes in the blood (A) and bone marrow (B) of wild-type, CCR2−/−, RIPCCL2/CCR2+/−, and RIPCCL2/CCR2−/− mice. Data are representative of one mouse in each group (n = 10/group). C: Immunostaining for CD45 (red) and insulin (green) in the pancreata of wild-type, RIPCCL2/CCR2+/−, and RIPCCL2/CCR2−/− mice at 8 weeks of age. Scale bars = 100 μm. (Please see http://dx.doi.org/10.2337/db08-0625 for a high-quality digital representation of this figure.)
FIG. 4.
FIG. 4.
Increased availability of circulating monocytes promotes spontaneous diabetes in RIPCCL2 mice with lower production of CCL2. A: Outline of the RIPCCL2 transgene and of the activator and responder transgenes in the Flt3L mice. In Flt3L mice, in the activator transgene (top), the human cytomegalovirus enhancer (hCMVe) was juxtaposed to the chicken β-actin promoter to control the expression of reverse tetracycline-controlled transactivator. The responder transgene encodes β-galactosidase and mFlt3L in opposite orientation. Transcription of the β-galactosidase and mFlt3L genes is strongly induced when DOX and reverse tetracycline-controlled transactivator are present. TRE, tetracycline-responsive element; CMV, minimal CMV promoter; and rglob, rabbit β-globin. RIPCCL2 mice from lines 251 and 10 were crossed with Flt3L mice to generate the RIPCCL2/Flt3L mice. B: Relative numbers of CD115+ cells in blood from untreated RIPCCL2/Flt3L mice (RIPCCL2/Flt3L −DOX, n = 13) and DOX-treated RIPCCL2/Flt3L (n = 21) mice from line 251 (t test). C: Relative numbers of CD115+ cells in blood from untreated RIPCCL2/Flt3L mice (RIPCCL2/Flt3L −DOX, n = 15) and DOX-treated RIPCCL2/Flt3L (n = 11) mice from line 10 (t test). D–G: Immunostaining for CD45 (red) and insulin (green) in the pancreata of untreated (−DOX; D and E) or treated (+DOX; F and G) RIPCCL2/Flt3L mice from line 251 (D and F) or line 10 (E and G). H: Cumulative incidence of diabetes in untreated RIPCCL2/Flt3L mice (RIPCCL2/Flt3L −DOX, n = 13), DOX-treated Flt3L mice (n = 10), and RIPCCL2/Flt3L mice (n = 21) from line 251. Note that DOX-treated RIPCCL2/Flt3L mice developed diabetes after weeks of treatment. I: Cumulative incidence of diabetes in untreated RIPCCL2/Flt3L mice (RIPCCL2/Flt3L −DOX, n = 15), DOX-treated Flt3L mice (n = 10), and RIPCCL2/Flt3L mice (n = 29) from line 10. Note that DOX-treated RIPCCL2/Flt3L mice developed diabetes after days of treatment. Scale bars = 100 μm. (Please see http://dx.doi.org/10.2337/db08-0625 for a high-quality digital representation of this figure.)
FIG. 5.
FIG. 5.
Diabetes in RIPCCL2 mice does not depend on T- and B-cells. Cumulative incidence of diabetes in RIPCL2/Rag+/− (n = 12) and RIPCL2/Rag−/− (n = 28) littermates from line 1 (P = 0.007).
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