NEUTROPHIL-PLATELET INTERACTION
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1 NEUTROPHIL-PLATELET INTERACTION Johnny Nijm, MD; PhD Specialist in Internal medicine, Cardiology & Clinical Physiology Department of Medicine Diagnostic, Division of Clinical Physiology, County Hospital Ryhov, Jönköping & Department of Medical and Health Sciences, Division of Cardiovascular Medicine, Linköping University, Linköping, SWEDEN
2 NEUTROPHIL-PLATELET INTERACTION Sanofi-aventis AB & Bristol-Myers Squibb
3 Neutrophils develop from the same early precursors as Monocytes and Macrophages but in contrast to these cells, they have a very short life time (1-2 days) Neutrophils are the first inflammatory cells that appear in intimal lesions Neutrophils represent more than 50 % of the total circulating Leukocytes and play a pivotal role in innate immunity
4 Hematopoiesis
5 WHAT ARE WE DEALING WITH... Skumceller Aktiverade trombocyter Thrombocyte activation Monocyter T-celler Aktiverat endotel Intima Aktiverade T-celler Media Glatta muskelceller AUTOIMMUNITY AUTOANTIGEN
6 INFLAMMATION TNF-α INF-γ Th1 IL-6 IL-1 IL-2 IL-4 Th2 IL-10 ANTI INFLAMMATION
7 Neutrophils may contribute to plaque instability and rupture by mediating their effects through: The production of Proteases such as Cathapsins, MMPs and Elastase Cytokines like IL-1 beta, TNF-alpha, IL-8 Generation of Reactive Oxygen Species (ROS)
8 FURTHERMORE: Neutrophil activation in peripheral blood may be assessed by: CD11b up-regulation Neutrophil-platelet aggregates Elastase, and Myeloperoxidase (MPO) release Sanofi-aventis AB & Bristol-Myers Squibb
9 FACS plot showing the definition of neutrophil-platelet aggregates
10 Antal i medelvärde Antal i medelvärde antal i medelvärde Hundratal CELL POPULATIONS AND NEUTROPHIL-PLATELET AGGREGATES diff,2 ** * p < 0,05 ** p < 0,001 * Neutrofiler Lymfocyter Monocyter Eosinofiler Basofiler Kontroller SA UA CD41a+CD11b+CD45+ ** * Kontroller SA UA * p = 0,5 ** p < 0,01 S1 Expression of CD11b and CD41a (MFI) MFI CD11b Kontroll SA UA MFI CD41a ** Kontroll SA UA p = 0,001 Nijm J et al Am J Cardiol, 2005
11 NEUTROPHIL-PLATELET AGGREGATES CORRELATE TO INFLAMMATORY MARKERS. IL-6 pg/ml IL-1Ra pg/ml CD41a+CD11b+CD45+ cells/mm³ CRP mg/ml p<0,001 (r = 0,6) p<0,001 (r = 0,4) p<0,01 (r = 0,3) IL-6 pg/ml p<0,0001 (r = 0,5) p<0,01 (r = 0,3) IL-1Ra pg/ml p<0,05 (r = 0,2)
12 The number of neutrophils was significantly higher in the ACS patients compared to patients with stable disease However, the number of circulating neutrophilplatelet aggregates tended to be lower in ACS patients compared to stable CAD patients The expression of CD41a was significantly lower in the ACS group compared to the stable group, (probably due to the clopidogrel therapy).
13 Jochen Graff et al, Clinical Pharmacology & Therapeutics, 2005 showed that Clopidogrel inhibits the expression of platelet activation markers and leukocyte-platelets interaction Sanofi-aventis AB & Bristol-Myers Squibb
14 THE INTERACTION BETWEEN NEUTROPHILS AND PLATELETS IS MAINLY MEDIATED BY: P-selectin: on the surface of activated endothelial cells and platelets E-selectin: on the surface proteins of certain leukocytes (monocytes, granulocytes and T- lymphocytes) L-selectin: on leukocytes, acting as homing receptor for leukocytes to enter secondary lymphoid tissues
15 Beta I (CD11b/CD18) and beta II (CD41/CD61) Integrins regulate neutrophil transmigration across the vascular endothelium. Integrins exists in different conformations, from a low-affinity to a high-affinity state.
16 THE AFFINITY STATE OF THE BETA2-INTEGRINS BEFORE AND AFTER STIMULATION EX VIVO Takagi J and Springer TA, Immunol. Rev. 2002
17 There was no activation of neutrophils in patients with stable CAD, expressed as CD18 or high affinity CD11b. Särndahl E, Nijm J et al, PLoS ONE 2007
18 The ROS production was reduced in neutrophils from patients with stable CAD. Särndahl E, Nijm J et al, PLoS ONE 2007
19 In conclusion: The number of neutrophil-platelet aggregates was higher in patients with stable CAD compared to healthy controls, suggesting a primed state of circulating neutrophils and increased thrombus susceptibility Neutrophils were not more activated in vivo than were cells in healthy controls, neither were the neutrophils in patients more prone to activation ex vivo In contrast, we obtained evidence for an impaired activation state in the patients, as assessed by decreased ROS production which may rather indicate an exhausted or frustrated neutrophils The clinical relevance of neutrophil dysfunction in CAD remains to be elucidated. Is it a marker of chronic inflammatory disease or a factor with impact on the progress of CAD? Ongoing studies on neutrophils from ACS patients might bring more light on their role in atherosclerosis
20 MMP-9 (ng/ml) However, a recent study, by Jönsson S et al, using IL-8 as a physiological activator, showed that MMP-9 release by neutrophils was significantly increased in stable CAD patients compared with controls, a result that might indicate neutrophil activation. At the same time the release of MMP-9, after a maximal stimulation with PMA, tended to be lower, a fact that might suggest an exhausted state of neutrophils 400 Neutrophil release of MMP Patients Control IL-8 Patients IL-8 Control Jönsson S et al, ESC 2010
21 Thank you for your attention ADP-Stimulated trombocyts. Sanofi-aventis AB & Bristol-Myers Squibb
22
23 The expression of annexin-1 was increased in neutrophils from patients with stable CAD. Särndahl E, Nijm J et al, Metabolism 2010
24 Matrixmetaloprotases, MMPs Group Name MMP Substrate Collagenases Gelatinases Fibroblast Neutrophil Collagenase-3 Collagenase-4 Gelatinase A Gelatinase B MMP-1 MMP-8 MMP-13 MMP-18 MMP-2 MMP-9 fibrillar collagen Gelatin, collagen IV, fibronectin, elastin, laminin Gelatin, elastin, fibronectin, vitronectin stromelysins Stromelysin 1 Stromelysin 2 Stromelysin 3 Matrilysins Matrilysin 1 Matrilysin 2 MMP-3 MMP-10 MMP-11 MMP-7 MMP-26 Gelatine, fibronectin, casein, laminin, elastin, MMP- 2/TIMP-2 Fibronectin, laminin, gelatine, aggrecan Fibronectin, vitronectin, laminin, gelatine, aggrecan Collagen IV, gelatine, fibronectin, fibrin, fibrinogen, type 1 gelatine Elastase Metalloelastase MMP-12 Elastin, gelatine, collagen IV, fibronectin, laminin, vitronectin, Proteoglycan Membrane Type Other MMPs MT1- MMP MT2- MMP MT3- MMP MT4 MMP MT5- MMP MT6- MMP Enamelysin MMP-14 MMP-15 MMP-16 MMP-17 MMP-24 MMP-25 MMP-19 MMP-20 MMP-22 MMP-23 MMP-27 MMP-28 Pro MMP-2, procollagenase 3 Pro MMP-2 Gelatine, TNF-α precursor, fibrin - Collagen IV, gelatine, laminin Collagen IV, gelatine, laminin, nidogen, tenacin, fibronectin, aggrecan Amelogenin Synthetic MMP substrate
25 Cluster of differentiation (CD) CD antigen Alternate name Function Cellular expression in blood CD3 Signalling component of T cells, part of a bigger complex which includes the T cell receptor. All T cells CD4 Co-receptor for MHC class II molecules. T helper cells, monocytes (weak) CD8 Co-receptor for MHC class I molecules. Cytotoxic T cells, subset of NK cells CD14 High-affinity receptor for lipopolysacharide. Monocytes CD19 B cell surface antigen B4 Involved in the regulation of B cells. Forms complex with complement receptor 2. B cells CD25 IL-2R a-chain In conjunction with IL-2Rb- and IL-2Rg, the CD25 antigen forms the high-affinity IL-2R complex. Activated T and B cells. CD45 Leukocyte common antigen Tyrosine phosphate, mediating signalling through B and T cell receptors All differentiated haematopoietic cells except erythrocytes and plasma cells CD62L L-selectin, LAM1 Mediates rolling interactions between leukocytes and endothelium B cells, T cells, monocytes, NK cells and granulocytes CD89 FcaR Neutrophil Fc receptor for IgA Neutrophils, monocytes CD11b Mac-1 a chain, complement receptor 3 Subunit of b2-integrin, associated with CD18. Granulocytes, monocytes, NK cells, subsets of T and B cells CD11b I-domain Conformation to the high-affinity state of b2-integrin As above (activated) CD18 Mac-1 b chain The second subunit of the b2-integrin, associated with CD11b CD41a Glycoprotein IIb Part of the glycoproteiniib/iiia complex which is the soluble receptor for fibrinogen Granulocytes, monocytes, NK cells, subsets of T and B cells Platelets
26 Platelets plays a major role in activating neutrophils and endothelial cells via cytokines, MMPs, TNF acting by CD40/CD40L Platelets secrete neutrophil and endothelial activators inducing production of the inflammatory cytokines found on APC and is required for their activation The binding of CD40L on Th cells to CD40 activates APC and induces a variety of downstream effects
27 Monocytes-Platelet interaction befor and after stimulation with TRA Lindmark et al, Arterioscler Thromb Vasc Biol 2000
28 INFECTION AS AN ACTIVATOR OF THE IMMUNE SYSTEM, BOTH IN A CHRONIC OR IN AN ACUTE MATTER CMV, EB, Chlamydia There is some data that links Adenovirus infection (subgroup) and the bacterial flora found in the colon with obesities (as a chronic inflammation in fat tissue) Other authors speculate of the possibility of a chronic Coxaccii virus B4 and the autoimmunity found in patients with type I diabetes There is documented data that showed a relationship between helicobacter pylori infection (after a non successful eradication) and a new onset of ACS during 6-12 months
29 WHAT ABOUT MEDICATION! There is data indicating that neutrophil-activating peptid-2 (NAP-2) activates both EC and leukocytes and could result in plaque rupture ASA-treatment results in decreased NAP-2 activity but do not influence CRP levels, whereas ACE-I does. Statin-treatment increase NAP-2 activity A study by Jochen Graff et al, Clinical Pharmacology & Therapeutics, 2005 showed that Clopedogrel inhibits the expression of platelet activation markers and the leukocyte-platelets interaction A possible explanation of these results is that the synthesis of vascular disease markers and inflammatory products such as scd40l and MMP-9 has been inhibited, and anti-inflammatory properties of Clopedogrel are likely to be a result of decreasing platelet activation
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