Platelets are essential for leukocyte recruitment in allergic inflammation Simon C. Pitchford, BSca, Hiroshi Yano, PhDa, Rebecca Lever, PhDa, Yanira Riffo-Vasquez,

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Presentation transcript:

Platelets are essential for leukocyte recruitment in allergic inflammation Simon C. Pitchford, BSca, Hiroshi Yano, PhDa, Rebecca Lever, PhDa, Yanira Riffo-Vasquez, PhDa, Silvia Ciferri, PhDb, Mark J. Rose, BSca, Silvia Giannini, BScb, Stefania Momi, PhDb, Domenico Spina, PhDa, Brian O'Connor, MDc, Paolo Gresele, MDb, Clive P. Page, PhDa Journal of Allergy and Clinical Immunology Volume 112, Issue 1, Pages 109-118 (July 2003) DOI: 10.1067/mai.2003.1514 Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 1 Platelet-leukocyte aggregates in the blood of asthmatic patients. A, The percentage of circulating leukocytes positive for platelet markers was measured in whole blood drawn from allergic asthmatic patients before (open bar) and after (filled bar) allergen challenge or after challenge with saline solution (hatched bar) . B, The relative index of the number of platelets per leukocyte was assessed again before and after challenge with allergen or saline solution. C, CD11b MFI was measured on leukocytes involved in aggregates with platelets in circulating blood and is expressed as a percentage of the level observed before challenge. All data are expressed as mean ± SE values (n = 8). *P < .05. Journal of Allergy and Clinical Immunology 2003 112, 109-118DOI: (10.1067/mai.2003.1514) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 2 Representative dot plots of GP1b fluorescence on circulating leukocytes in asthmatic blood before allergen challenge (A) , 8 hours after allergen challenge (B) , 24 hours after allergen challenge (C) , and 8 hours after saline challenge (D) . Journal of Allergy and Clinical Immunology 2003 112, 109-118DOI: (10.1067/mai.2003.1514) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 3 Representative dot plots of CD11b fluorescence on circulating leukocytes in asthmatic blood: gating strategy to identify leukocytes expressing CD11b (A) and CD11b expression on leukocytes before allergen challenge (B) , 8 hours after allergen challenge (C) , 24 hours after allergen challenge (D) , and 8 hours after saline challenge (E) . Journal of Allergy and Clinical Immunology 2003 112, 109-118DOI: (10.1067/mai.2003.1514) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 4 Effect of platelet depletion and restoration on pulmonary leukocyte recruitment in allergic mice. C57BL/6 mice, sham sensitized or sensitized to OVA, were subsequently exposed to aerosolized antigen. BAL was carried out 24 hours after the start of the last day of challenge, and BAL fluid was analyzed for eosinophil (A) and lymphocyte (B) numbers. In some experiments animals were rendered thrombocytopenic by means of busulfan (BUS) administration before sensitization. Groups of thrombocytopenic animals were injected intravenously with either PPP or PRP obtained from sensitized donor animals before inhaled antigen challenge. A further group of thrombocytopenic animals was administered NPRP. Numbers of animals per group are indicated in parentheses. Data are expressed as means ± SE. *P < .05, **P < .01. Journal of Allergy and Clinical Immunology 2003 112, 109-118DOI: (10.1067/mai.2003.1514) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 5 Effect of immune-mediated platelet depletion on pulmonary leukocyte recruitment in allergic mice. C57BL/6 mice, sham sensitized or sensitized to OVA, were subsequently exposed to aerosolized antigen. BAL was carried out 24 hours after the start of the last day of challenge, and BAL fluid was analyzed for eosinophil (A) and lymphocyte (B) numbers. Some animals received intravenous APAS or CS 30 minutes before allergen challenge on the first 2 days of exposure. Numbers of animals per group are indicated in parentheses. Data are expressed as means ± SE. *P < .05. Journal of Allergy and Clinical Immunology 2003 112, 109-118DOI: (10.1067/mai.2003.1514) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 6 Platelet-leukocyte aggregates in the blood of allergic mice. C57BL/6 mice, sham sensitized or sensitized to OVA, were subsequently exposed to aerosolized antigen. Blood sampling was carried out 24 hours after the start of the last day of challenge and analyzed for the presence of platelets attached to leukocytes (A) , the intensity of CD11b expression on leukocytes attached to platelets (B) , and the intensity of CD11b expression on circulating leukocytes irrespective of platelet attachment (C) . Some animals received intravenous APAS or CS 30 minutes before allergen challenge on the first 2 days of exposure. Numbers of animals per group are indicated in parentheses. Data are expressed as means ± SE (***P < .001). Also shown are representative dot plots of CD41 and CD11b fluorescence on circulating leukocytes in blood from allergic mice: gating strategy to identify leukocytes (1) from other blood cells (Ai) ; circulating platelet-leukocyte aggregates on sham-sensitized (Aii) and OVA-sensitized (Aiii) mice; and CD11b expression on circulating leukocytes of sham-sensitized (Bi) , OVA-sensitized (Bii) , and OVA plus APAS-sensitized (Biii) mice. Journal of Allergy and Clinical Immunology 2003 112, 109-118DOI: (10.1067/mai.2003.1514) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 7 Effect of platelets on PMN adhesion to HUVECs. A, Adhesion of chromium 51-labeled PMNs to HUVEC monolayers was assessed in the absence and presence of autologous platelets. **P < .01. B, fMLP-stimulated PMN adhesion to HUVECs was examined with and without platelets. **P < .01 represents the significance of the effect of fMLP in the absence of platelets (X) . §§P < .01 represents the significance of the effect of platelets (2 × 105 [open circles] , 5 × 106 [filled circles] , or 1.1 × 108 [triangles] per well) in the presence of indicated concentrations of fMLP. C, The effect of platelets on adhesion of PMNs to tissue-culture plastic (ie, in the absence of HUVECs) was tested. D, Platelets were pretreated with wortmannin and then washed before being introduced (2.3 × 107 [hatched bars] or 5.6 × 108 [filled bars] per well) to the adhesion assay. The effects of platelets, both untreated and wortmannin treated, on PMN adhesion to HUVECs are shown (**P < .01). In all cases data represent the percentage of PMNs added (2 × 105 cells per well) adherent to HUVECs at the end of the assay and are expressed as mean ± SE values from 4 to 6 separate experiments, each carried out in triplicate. Journal of Allergy and Clinical Immunology 2003 112, 109-118DOI: (10.1067/mai.2003.1514) Copyright © 2003 Mosby, Inc. Terms and Conditions

Fig. 8 Effect of platelets on PMN activation. A, The effect of platelets on MPO release from PMNs was assessed in the presence of HUVEC monolayers. *P < .01, **P < .01. B, MPO release from fMLP-stimulated PMNs, again in the presence of HUVECs, was examined with and without platelets. **P < .01 represents the significance of the effect of fMLP in the absence of platelets (X) . §P < .05 represents the significance of the effect of platelets (2 × 105 [open circles] , 5 × 106 [filled circles] , or 1.1 × 108 [triangles] per well) in the presence of indicated concentrations of fMLP. C, The effect of platelets on MPO release from PMNs incubated on tissue-culture plastic (ie, in the absence of HUVECs) was examined. *P < .05. D, Platelets were pretreated with wortmannin and then washed before being introduced (2.3 × 107 [hatched bars] or 5.6 × 108 [filled bars] per well) to the mixed cell assay. The effects of platelets, both untreated and wortmannin treated, on MPO release are shown (*P < .05). In all cases MPO release is expressed as a percentage of the total MPO remaining at the end of the assay in identical wells incubated in the same manner before lysis of the total content and measurement of MPO. Data shown are mean ± SE values from 4 to 6 separate experiments, each carried out in triplicate. Journal of Allergy and Clinical Immunology 2003 112, 109-118DOI: (10.1067/mai.2003.1514) Copyright © 2003 Mosby, Inc. Terms and Conditions