Activated platelets promote increased monocyte expression of CXCR5 through prostaglandin E2-related mechanisms and enhance the anti-inflammatory effects of CXCL13  Bente Halvorsen, Linda M. Smedbakken, Annika E. Michelsen, Mona Skjelland, Vigdis Bjerkeli, Ellen Lund Sagen, Kjetil Taskén, Bjørn Bendz, Lars Gullestad, Sverre Holm, Erik A. Biessen, Pål Aukrust  Atherosclerosis  Volume 234, Issue 2, Pages 352-359 (June 2014) DOI: 10.1016/j.atherosclerosis.2014.03.021 Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Regulation of CXCR5 in THP-1 monocytes. The figure shows the effect of LPS (5 ng/ml), Pam3Cys (1 μg/ml), releasate from un-stimulated (uPRL) and thrombin-activated (sPRL) platelets, and the non-selective β adrenergic agonist isoproterenol (isoprot, 20 μM) on mRNA levels of CXCR5 in THP-1 monocytes after culturing for 6 h. Prior to the experimental start, the cells were cultured with (left) or without (right) TNFα (5 ng/ml) for 96 h mRNA levels were assessed by real-time RT-PCR in relation to the control gene β-actin and represent delta CT values. Data are mean ± SEM (n = 4–8). *p < 0.05 and ***p < 0.001 versus controls (vehicle) without TNFα pre-activation. ###p < 0.001 versus controls (vehicle) with TNFα pre-activation. Atherosclerosis 2014 234, 352-359DOI: (10.1016/j.atherosclerosis.2014.03.021) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 The platelet-mediated increase in CXCR5 expression in THP-1 monocytes involves PGE2, cAMP, RANTES and NFκB. The upper panels show the effect of different concentrations of PGE2 (A) and the selective EP2 receptor agonist butaprost without PGE2 (B) on CXCR5 mRNA levels in THP-1 monocytes after culturing for 6 h. Panel C shows the effect of butaprost (250 μM) in comparison with the effect of the selective agonist for the PGD2 receptor DP (i.e., BW 245C, 200 nM) and the PGF2α receptor FP (i.e., Fluprostenol, 200 nM). Panel E and F show the effect of releasate from thrombin-activated platelets (sPRL) on the mRNA levels of CXCR5 in THP-1 monocytes with or without pre-incubation of the THP-1 cells for 30 min with Rp-8-Br-cAMP (Rp, 1000 μM), a selective antagonist of the cAMP-dependent activation of PKA (E), or with or without co-culturing with neutralizing antibodies (Ab) against RANTES or isotype-matched control antibodies (F), after culturing for 6 h. Rp-8-Br-cAMP had no effect when given alone. For comparisons, the effect of releasate from un-stimulated platelets (uPRL) is also shown. Panel G shows the effect of uPRL and sPRL on the mRNA levels of CXCR5 in THP-1 monocytes, with or without pre-incubation of the THP-1 cells for 30 min with the NFκB inhibitor Bay-11-7082 (12 μM), after culturing for 6 h. In panel A–C and E-G, THP-1 cells were pre-activated by TNFα (5 ng/ml) for 96 h before experimental start. Panel D shows the effect of PGE2 (100 ng/ml) and butaprost (250 μM) without pre-incubation with TNFα. mRNA levels were measured by real-time RT-PCR in relation to the control gene β-actin and represent delta CT values. Data are mean ± SEM (n = 6–8). *p < 0.05, **p < 0.01 and ***p < 0.001 versus controls (vehicle). #p < 0.05 and ###p < 0.001 versus sPRL without Rp-8-Br-cAMP (E), anti-RANTES Ab (F) or the NFκB inhibitor (Bay) (G). Atherosclerosis 2014 234, 352-359DOI: (10.1016/j.atherosclerosis.2014.03.021) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Releasate from thrombin-activated platelets potentates the effect of CXCL13 on CXCR5. The panels show the effect of CXCL13 (200 ng/ml) on CXCR5 (A–B), IL-10 (C–D), TGFβ (E–F) and arginase-1 (G–H) in THP-1 monocytes (left panels) and primary monocytes from five healthy individuals (right panels) with and without platelet releasate from un-stimulated (uPLR) and thrombin activated (sPLR) platelets after culturing for 6 h. In all experiments, THP-1 cells, but not the primary monocytes, were pre-activated by TNFα (5 ng/ml) for 96 h before experimental start. mRNA levels were measured by real-time RT-PCR in relation to the control gene β-actin and represent delta CT values. Data are mean ± SEM (n = 6). #p < 0.05 and ##p < 0.01 versus CXCL13 and sPRL given alone. Atherosclerosis 2014 234, 352-359DOI: (10.1016/j.atherosclerosis.2014.03.021) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 The regulation of CXCR5 in primary monocytes. The figure shows the effect of LPS (5 ng/ml), Pam3Cys (1 μg/ml), PGE2 (200 ng/ml) and butaprost (250 μM) (A) and releasate from un-stimulated (uPRL) and thrombin-activated (sPRL) (B) platelets on mRNA levels of CXCL13 in primary monocytes from 5 healthy donors after culturing for 6 h. Panel C shows the effect of thrombin-activated platelets (sPRL) with or without co-culturing with neutralizing antibodies (Ab) against RANTES or isotype-matched control antibodies on the expression of CXCR5 after culturing for 6 h. Prior to the experimental start, the cells were pre-activated with TNFα (5 ng/ml) for 48 h mRNA levels were assessed by real-time RT-PCR in relation to the control gene β-actin and represent delta CT values. Data are mean ± SEM (n = 5). *p < 0.05, p < 0.01 and ***p < 0.001 versus controls (vehicle). #p < 0.05 versus sPRL without anti-RANTES Ab. Atherosclerosis 2014 234, 352-359DOI: (10.1016/j.atherosclerosis.2014.03.021) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Strong immunostaining of CXCR5 in thrombus material. The panels show double immunofluorescent staining of CXCR5 (green), CD41 (thrombocytes, red) and nucleus (DAPI, blue) in thrombus material removed from the site of plaque rupture in STEMI patients undergoing PCI. The last panel is a merge of the three above. Pictures are obtained at 400x magnification. The panels show double immunofluorescent staining of CXCR5 (green), CD41 (thrombocytes, red) and nucleus (DAPI, blue) in thrombus material removed from the site of plaque rupture in STEMI patients undergoing PCI. The last panel is a merge of the three above. Pictures are obtained at 400× magnification. The Figure presents one representative patient out of seven and all panels are from the same thrombus from one patient. Atherosclerosis 2014 234, 352-359DOI: (10.1016/j.atherosclerosis.2014.03.021) Copyright © 2014 Elsevier Ireland Ltd Terms and Conditions