Volume 124, Issue 5, Pages 1249-1264 (May 2003) Platelets trigger a CD40-dependent inflammatory response in the microvasculature of inflammatory bowel disease patients  Silvio Danese, Carol de la Motte, Andreas Sturm, Jon D Vogel, Gail A West, Scott A Strong, Jeffry A Katz, Claudio Fiocchi  Gastroenterology  Volume 124, Issue 5, Pages 1249-1264 (May 2003) DOI: 10.1016/S0016-5085(03)00289-0

Figure 1 Flow cytometric analysis of CD40L and P-selectin expression by resting and activated platelets. Normal platelets were left untreated or activated with thrombin for different time periods (2, 5, 10, 15, 30, and 60 minutes), after which they were stained with the appropriate monoclonal antibody. The black curve represents the background signal from the isotype control. This figure is representative of 11 separate experiments. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 2 Enhanced expression of CD40L by circulating platelets in patients with IBD. Platelets from normal subjects (n = 11) and patients with clinically active CD (n = 12) and UC (n = 11) were left untreated or activated with thrombin for 2 minutes, after which the percentage of platelets expressing CD40L was assessed by flow cytometric analysis. The bars express the mean value of each group. ∗P < 0.05 for both activated CD and UC compared with activated normal platelets; ∗∗P < 0.01 for resting UC and resting CD compared with resting normal platelets. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 3 Enhanced expression of P-selectin by circulating platelets in patients with IBD. The percentage of platelets expressing P-selectin was assessed by flow cytometric analysis in unstimulated platelets from normal subjects (n = 11) and patients with clinically active CD (n = 12) and UC (n = 11). The bars express the mean value of each group. ∗P < 0.05 for both CD and UC compared with normal platelets. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 4 Detection of CD40L- and CD42b-expressing platelets in tissue sections of inflamed human colonic mucosa. Serial frozen sections, obtained from an inflamed colon resected from a patient with CD, were fluorescently labeled for detection of nuclei (DAPI, blue), platelets (CD42b with Alexa 488 secondary antibody, green), or CD40L (secondary FITC-conjugated antibody) and observed by confocal microscopy (63× objective). (A ) H&E staining (light microscopy). (B) Background control and DAPI staining of a serial section of C and D. (C ) CD42b staining in the blood vessel of the colonic mucosa. (D) CD40L staining of the same vessels. This figure is representative of 3 CD and 3 UC tissues. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 5 Capping of surface CD40L expressed by live thrombin-stimulated platelets. Platelets, treated with thrombin at room temperature and stained with a CD40L monoclonal antibody, were cocultured with HIMEC and observed by confocal differential interference contrast microscopy by using a 100× objective lens. (A ) CD40L-positive staining appears green on the live platelets. (B) CD40L-positive staining appears in platelet aggregates adherent to HIMEC. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 6 Modulation of HIMEC ICAM-1 and VCAM-1 expression by CD40L-positive platelets. HIMEC monolayers were left untreated (medium) or cocultured with resting or thrombin-activated platelets in the absence or presence of CD40L blocking antibody for 4 hours, after which HIMEC were stained with the appropriate antibodies and analyzed by flow cytometry. The black curve represents the background signal from the isotype control. Numbers represent the net percentage of positively stained cells. This figure is representative of 15 separate experiments. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 7 Enhanced up-regulation of HIMEC CAM expression by CD40L-positive IBD platelets (PLT). HIMEC monolayers were left untreated or cocultured with resting or thrombin-activated platelets from normal subjects (n = 7) and patients with active IBD (CD, n = 4; UC, n = 4) in the absence or presence of CD40L blocking antibody for 4 hours, after which HIMEC were stained with (A ) ICAM-1 or (B) VCAM-1 antibodies and analyzed by flow cytometry. The fraction of CD40L-induced up-regulation was 65% and 75% for ICAM-1 and 40% and 65% for VCAM-1 by normal and IBD platelets, respectively. Data represent the mean fluorescence intensity (MFI) and indicate the net percentage increase over spontaneous CAM expression. Data are expressed as mean ± SEM. ∗P < 0.05 for CD40L antibody-treated IBD platelets compared with untreated activated IBD platelets; ∗∗P < 0.01 for resting IBD compared with resting normal platelets. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 8 Enhanced HIMEC-derived IL-8 production by CD40L-positive IBD platelets (PLT). HIMEC monolayers were left untreated (medium) or cocultured with resting or thrombin-activated platelets from normal subjects (n = 10) and patients with active CD (n = 7) or UC (n = 7). Data are expressed as mean ± SEM. ∗P < 0.05 for resting CD and UC platelets compared with resting normal platelets and for activated CD and UC platelets compared with activated normal platelets. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 9 Effect of CD40L blockade on activated platelet (PLT)-induced IL-8 production by HIMEC. HIMEC monolayers were cocultured with thrombin-activated platelets from normal subjects (n = 10) and patients with active CD (n = 7) or UC (n = 7) in the presence of CD40L blocking antibody for 4 hours. Data are expressed as mean ± SEM. ∗P < 0.05 for CD40L antibody-treated activated CD and UC platelets compared with untreated autologous activated platelets; ∗∗P < 0.01 for CD40L antibody-treated activated normal platelets compared with untreated autologous activated platelets. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 10 Immunoblotting of CD40-dependent p38 phosphorylation in platelet (PLT)-stimulated HIMEC. HIMEC monolayers were left alone (medium) or cocultured with resting, activated platelets or sCD40L. After the indicated time points, platelets were completely removed by extensive washings, HIMEC were lysed, and total proteins were extracted for hybridization with a phospho-p38-specific polyclonal antibody. The extract is a commercially supplied positive control. This figure is representative of 4 separate experiments. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 11 Inhibition of activated platelet (PLT)-induced HIMEC IL-8 production by the p38 MAP kinase inhibitor SB203580. HIMEC monolayers were cultured alone (medium) or cocultured with activated platelets or sCD40L before or after pretreatment with p38 MAP kinase inhibitor SB203580. The presence of dimethyl sulfoxide, used for SB203580 dilution, had no effect on IL-8 production. Data are expressed as mean ± SEM of 4 separate experiments. ∗P < 0.05 for inhibitor pretreated compared with untreated platelet-stimulated HIMEC; ∗∗P < 0.001 for inhibitor pretreated compared with untreated sCD40L-stimulated HIMEC. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 12 Enhanced RANTES levels in IBD platelet (PLT)-HIMEC cocultures. (A ) HIMEC monolayers were left untreated (medium) or cocultured with resting or thrombin-activated platelets from normal subjects (n = 8) and patients with active CD (n = 7) or UC (n = 7) for 4 hours. (B) Autologous platelets were activated as described in A but in the absence of HIMEC monolayers. Data are expressed as mean ± SEM. (A and B) ∗P < 0.05 for resting CD and UC platelets compared with resting normal platelets and for activated CD and UC platelets compared with activated normal platelets. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 13 Source of RANTES in platelet (PLT)-HIMEC cocultures. HIMEC monolayers were left untreated (medium) or stimulated with sCD40L, IL-1β, activated platelets, or degranulated platelets for 4 hours. In the same experiments, autologous platelets were also activated in the absence of HIMEC and their cell-free supernatants collected. Data are expressed as mean ± SEM of 3 separate experiments. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 14 Flow cytometric analysis of RANTES bound to a single HIMEC suspension. Confluent HIMEC monolayers grown on coverslips were treated with thrombin-activated platelet supernatants or recombinant RANTES for 60 minutes, rinsed, and gently trypsin-treated to generate a single cell suspension. The black curve represents the background signal from the isotype control. This figure is representative of 4 separate experiments. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 15 Detection of RANTES bound to the surface of HIMEC incubated with platelet-derived supernatants and recombinant RANTES. Confluent HIMEC monolayers grown on coverslips were treated with thrombin-activated platelet supernatants or recombinant RANTES for 5–60 minutes. After rinsing and fixation, HIMEC were fluorescently labeled for detection of RANTES (Alexa 488 secondary antibody, green) and nuclei (DAPI, blue) and observed by confocal microscopy (20× objective). (A) Isotype control. (B) Unstimulated HIMEC. (C) Supernatants from stimulated platelets. (D) Recombinant RANTES. This figure is representative of 4 separate experiments after 60 minutes of incubation. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)

Figure 16 Inhibition of RANTES-mediated T-cell adhesion to HIMEC by the RANTES receptor antagonist Met-RANTES. HIMEC monolayers were left untreated (medium) or stimulated with activated platelets (PLT), recombinant RANTES, or activated platelet-derived cell-free supernatants for 1 hour. MOLT4 cells, preincubated or not with Met-RANTES, were added to the HIMEC monolayers for 1 hour, and nonadherent cells were removed by washing. The number of adherent cells in each experimental condition was expressed as mean ± SEM of 4 separate experiments. ∗P < 0.01 for Met-RANTES pretreated compared with untreated MOLT4 cells. Gastroenterology 2003 124, 1249-1264DOI: (10.1016/S0016-5085(03)00289-0)