Novel Role of the CXC Chemokine Receptor 3 in Inflammatory Response to Arterial Injury by Johannes B.K. Schwarz, Nicolas Langwieser, Nicole N. Langwieser, Martin J. Bek, Stefan Seidl, Hans-Henning Eckstein, Bao Lu, Albert Schömig, Hermann Pavenstädt, and Dietlind Zohlnhöfer Circulation Research Volume 104(2):189-200 January 30, 2009 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. IP10-induced ROS generation and apoptosis of T cells is mediated by activation of mTORC1. a and c, Western blot analysis of phosphorylation of the mTORC1 downstream target p70S6K (p*p70S6K) in IP10-activated human (Jurkat) (a) and murine (HT-2) (c) T cells. Figure 1. IP10-induced ROS generation and apoptosis of T cells is mediated by activation of mTORC1. a and c, Western blot analysis of phosphorylation of the mTORC1 downstream target p70S6K (p*p70S6K) in IP10-activated human (Jurkat) (a) and murine (HT-2) (c) T cells. Equal protein loading was confirmed by the use of a monoclonal anti–β-actin antibody. Data shown are representative of 3 independent experiments. The histogram shows the fold increase in intensity relative to control (means±SEM; n=3). *P<0.05 vs percentage control. b and d, The effect of IP10 on ROS generation and apoptosis in human (b) and murine (d) T cells in the absence or presence of pharmacological mTORC1 inhibitors. IP10-induced ROS generation was measured by 2′,7′-dichlorofluorescein (DCF) fluorescence (means±SEM; n=3). *P<0.05. IP10-induced apoptosis was measured by TUNEL assay (means±SEM; n=3). *P<0.05 vs percentage control. Johannes B.K. Schwarz et al. Circ Res. 2009;104:189-200 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Enhanced expression of IP10, MIG, and CXCR3 on vessel injury Figure 2. Enhanced expression of IP10, MIG, and CXCR3 on vessel injury. a and b, Time course after femoral artery wire injury in WT mice. a, Representative images of noninjured and injured arteries stained with Elastica–van Gieson at days 0, 7, and 14 after vascular injury. Figure 2. Enhanced expression of IP10, MIG, and CXCR3 on vessel injury. a and b, Time course after femoral artery wire injury in WT mice. a, Representative images of noninjured and injured arteries stained with Elastica–van Gieson at days 0, 7, and 14 after vascular injury. Scale bar=100 μm. b, Quantification of intimal area after vascular injury (means±SEM; n>7 for each time point). *P<0.05. c, mRNA expression for IP10, MIG, and CXCR3 was analyzed at the time points indicated using quantitative real-time PCR. Expression levels were normalized to the housekeeping gene GAPDH for each sample (means±SEM; n=7). *P<0.05 vs baseline. IP10 (d) and MIG (e) protein concentration in serum samples after vascular injury were analyzed by ELISA (means±SEM; n=8). *P<0.05 vs baseline. Johannes B.K. Schwarz et al. Circ Res. 2009;104:189-200 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. CXCR3+ vascular cells represent T cells, ECs, and HPCs Figure 3. CXCR3+ vascular cells represent T cells, ECs, and HPCs. Paraffin-embedded sections of injured mouse femoral arteries were stained using double immunofluorescence with an antibody against CXCR3 (green) and an additional antibody (red) against CD3 for T cells, CD31 for ECs, c-kit for HPCs, or α-actin for SMCs. a, Representative sections at day 14 after vascular injury; arrows demonstrate colocalization of CXCR3 with cell type markers. Figure 3. CXCR3+ vascular cells represent T cells, ECs, and HPCs. Paraffin-embedded sections of injured mouse femoral arteries were stained using double immunofluorescence with an antibody against CXCR3 (green) and an additional antibody (red) against CD3 for T cells, CD31 for ECs, c-kit for HPCs, or α-actin for SMCs. a, Representative sections at day 14 after vascular injury; arrows demonstrate colocalization of CXCR3 with cell type markers. Scale bar=100 μm. b, Quantification of CXCR3+ cells, CD3+ T cells, CD31+ ECs, c-kit+ HPCs, and α-actin+ SMCs at different time points after vascular injury (means±SEM; n>7). *P<0.05. Johannes B.K. Schwarz et al. Circ Res. 2009;104:189-200 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. CXCR3 deficiency diminishes intimal hyperplasia after vascular injury. a and b, Morphometric analysis of intimal hyperplasia in CXCR3−/− mice compared to WT mice. a, Representative images of injured arteries stained with Elastica–van Gieson at day 7 and 14 after vascular injury. Figure 4. CXCR3 deficiency diminishes intimal hyperplasia after vascular injury. a and b, Morphometric analysis of intimal hyperplasia in CXCR3−/− mice compared to WT mice. a, Representative images of injured arteries stained with Elastica–van Gieson at day 7 and 14 after vascular injury. Scale bar=100 μm. b, Quantification of intimal area and lumen loss 14 days after vascular injury (means±SEM; n=8). *P<0.05 vs WT mice. c and d, Morphometric analysis of intimal hyperplasia in mice treated with control IgG or a neutralizing antibody cocktail against IP10 and MIG (anti-IP10/anti-MIG pAb). c, Representative images of injured arteries stained with Elastica–van Gieson at day 7 and 14 after vascular injury. Scale bar=100 μm. d, Quantification of intimal area and lumen loss 14 days after vascular injury (means±SEM; n=10). *P<0.05 vs control IgG–treated mice. Johannes B.K. Schwarz et al. Circ Res. 2009;104:189-200 Copyright © American Heart Association, Inc. All rights reserved.

Figure 5. CXCR3 deficiency reduces recruitment of T cells, leukocytes, and HPCs. a and b, Immunohistochemical analysis of the cellular composition of intimal tissue from WT and CXCR3−/− mice 14 days after femoral artery injury. a, Representative sections of immunohistological stainings. Figure 5. CXCR3 deficiency reduces recruitment of T cells, leukocytes, and HPCs. a and b, Immunohistochemical analysis of the cellular composition of intimal tissue from WT and CXCR3−/− mice 14 days after femoral artery injury. a, Representative sections of immunohistological stainings. Scale bar=100 μm. b, Quantitative analysis of immunohistochemistry for T cells (CD3), leukocytes (CD45), HPCs (c-kit), and SMCs (α-actin) (means±SEM; n=8). *P<0.05 vs WT mice. c and d, Immunohistochemical analysis of intimal tissue from mice treated with control IgG or anti-IP10/anti-MIG pAb after arterial injury. c, Representative sections of immunohistological stainings. Scale bar=100 μm. d, Quantitative analysis of immunohistochemistry (means±SEM; n=8). *P<0.05 vs control IgG–treated group. Johannes B.K. Schwarz et al. Circ Res. 2009;104:189-200 Copyright © American Heart Association, Inc. All rights reserved.

Figure 6. CXCR3 deficiency decreases recruitment of CD4+CD103+ and CD8+CD103+ regulatory T cells. a, Double immunofluorescence staining of frozen arteries sections from WT and CXCR3−/− mice 7 days after arterial injury for CD4 (red) and CD103 (green). Figure 6. CXCR3 deficiency decreases recruitment of CD4+CD103+ and CD8+CD103+ regulatory T cells. a, Double immunofluorescence staining of frozen arteries sections from WT and CXCR3−/− mice 7 days after arterial injury for CD4 (red) and CD103 (green). Shown are representative images. Arrows demonstrate colocalization of CD4 with CD103. Scale bar=100 μm. b, Quantification of the amount of CD4+ cells, CD4+CD103+ cells, and CD4+CD103− cells (means±SEM; n>8). *P<0.05 vs WT mice. c, Double immunofluorescence staining for CD8 (red) and CD103 (green). Shown are representative images. Arrows demonstrate colocalization of CD8 with CD103. Scale bar=100 μm. d, Quantification of the amount of CD8+ cells, CD8+CD103+ cells, and CD8+CD103− cells (means±SEM; n>8). *P<0.05 vs WT mice. Johannes B.K. Schwarz et al. Circ Res. 2009;104:189-200 Copyright © American Heart Association, Inc. All rights reserved.

Figure 7. CXCR3-dependent activation of mTORC1 after arterial injury Figure 7. CXCR3-dependent activation of mTORC1 after arterial injury. mTORC1 activation in injured arteries was measured by immunofluorescence staining against p*p70 S6 kinase 14 days after vascular injury in WT and CXCR3-deficient mice (a) and in mice treated with control IgG or neutralizing anti-IP10/anti-MIG pAb (b). Figure 7. CXCR3-dependent activation of mTORC1 after arterial injury. mTORC1 activation in injured arteries was measured by immunofluorescence staining against p*p70 S6 kinase 14 days after vascular injury in WT and CXCR3-deficient mice (a) and in mice treated with control IgG or neutralizing anti-IP10/anti-MIG pAb (b). Shown are representative images. Scale bar=100 μm. Statistical quantification of p*p70 S6 kinase+ intimal cells after vascular injury: means±SEM; n=8; *P<0.05 vs control. Johannes B.K. Schwarz et al. Circ Res. 2009;104:189-200 Copyright © American Heart Association, Inc. All rights reserved.

Figure 8. Injury-mediated generation of ROS and induction of apoptosis is diminished in CXCR3−/− mice. a, Generation of ROS was detected in situ by staining femoral arteries with the superoxide-sensitive dye dihydroethidine. Figure 8. Injury-mediated generation of ROS and induction of apoptosis is diminished in CXCR3−/− mice. a, Generation of ROS was detected in situ by staining femoral arteries with the superoxide-sensitive dye dihydroethidine. Shown are representative sections. Scale bar=100 μm. Statistical quantification of ROS production in injured femoral arteries after 7 days: means±SEM; n>7; *P<0.05 vs WT. TUNEL analysis (b) and caspase-3 staining (c) were performed in WT and CXCR3−/− mice 14 days after vascular injury to quantify apoptotic cell death. Shown are representative sections. Scale bar=100 μm. Quantification of the immunostaining for TUNEL+ and caspase-3+ cells (means±SEM; n>8). *P<0.05 vs WT. d and e, mTORC1-dependent induction of ROS and apoptosis in SMCs. d, Effect of IP10 on ROS generation and apoptosis in the absence or presence of pharmacological mTORC1 inhibitors was analyzed in SMCs. ROS generation in SMCs was measured by DCF fluorescence. Induction of apoptosis in SMCs was measured by TUNEL assay (means±SEM; n=3). e, Effect of a cell-free, conditioned media from IP10-stimulated T cells on ROS generation and apoptosis of SMCs was analyzed in the presence or absence of free radical scavengers (NAC) or pharmacological mTORC1 inhibition (means±SEM; n=3). *P<0.05 vs percentage control. Johannes B.K. Schwarz et al. Circ Res. 2009;104:189-200 Copyright © American Heart Association, Inc. All rights reserved.