Integrins on neutrophils are dispensable for migration into three-dimensional fibrin gels

Summary

The innate immune system and the blood haemostasis system function cooperatively in many pathological conditions such as acute respiratory distress syndrome, deep venous thrombosis, ischaemia/reperfusion injury and cardiovascular disease. Infiltration of neutrophils into thrombotic substrates such as fibrin clots supports fibrinolysis, tissue damage and inflammation. Despite the importance of integrins in neutrophil attachment to fibrin-coated surfaces under flow conditions, little is known about their role in migration processes in shear free two-dimensional (2D) and three-dimensional (3D) fibrin(ogen) environments. Therefore, the present study was designed to study the role of functional integrins in mediating neutrophil migration on and in fibrin matrices. Time lapse video sequences of neutrophil chemokinesis and chemotaxis were made under conditions of active- or non-active integrins. Interestingly, migration of neutrophils on 2D fibrinogen coated surfaces and 3D fibrin matrices is independent of integrins as the response is not sensitive to αM-(CD11b) and β2-(CD18) blocking antibodies and/or chelation of Ca²⁺ and Mg²⁺ by EDTA in bivalent ion-free buffers. The blocking integrin antibodies were shown to be functionally active in regular adhesion assays. Our study shows that integrins are dispensable for migration on 2D and in 3D fibrin matrices, both when neutrophils enter into the fibrin matrix and when captured in the matrix.

Keywords

Migration - 3D matrix - fibrin - neutrophils - integrins - chemotaxis

• References

• 1 Ware LB. Pathophysiology of acute lung injury and the acute respiratory distress syndrome. Semin Respir Crit Care Med 2006; 27: 337-349.
• 2 Frangogiannis NG. Targeting the inflammatory response in healing myocardial infarcts. Curr Med Chem 2006; 13: 1877-1893.
• 3 Distelmaier K, Adlbrecht C, Jakowitsch J. et al. Local complement activation triggers neutrophil recruitment to the site of thrombus formation in acute myocardial infarction. Thromb Haemost 2009; 102: 564-572.
• 4 Mestas J, Ley K. Monocyte-endothelial cell interactions in the development of atherosclerosis. Trends Cardiovasc Med 2008; 18: 228-232.
• 5 Plow EF. The contribution of leukocyte proteases to fibrinolysis. Blut 1986; 53: 1-9.
• 6 Mosesson MW. Fibrinogen and fibrin structure and functions. J Thromb Haemost 2005; 3: 1894-1904.
• 7 Altieri DC, Bader R, Mannucci PM. et al. Oligospecificity of the cellular adhesion receptor Mac-1 encompasses an inducible recognition specificity for fibrinogen. J Cell Biol 1988; 107: 1893-1900.
• 8 Wright SD, Weitz JI, Huang AJ. et al. Complement receptor type three (CD11b/CD18) of human polymorphonuclear leukocytes recognizes fibrinogen. Proc Natl Acad Sci USA 1988; 85: 7734-7738.
• 9 Loike JD, Sodeik B, Cao L. et al. CD11c/CD18 on neutrophils recognizes a domain at the N terminus of the A alpha chain of fibrinogen. Proc Natl Acad Sci USA 1991; 88: 1044-1048.
• 10 Kuijper PH, Gallardo TH, van der Linden J. et al. Neutrophil adhesion to fibrinogen and fibrin under flow conditions is diminished by activation and L-selectin shedding. Blood 1997; 89: 2131-2138.
• 11 Kuijper PH, Gallardo TH, Lammers JW. et al. Platelet and fibrin deposition at the damaged vessel wall: cooperative substrates for neutrophil adhesion under flow conditions. Blood 1997; 89: 166-175.
• 12 Lishko VK, Burke T, Ugarova T. Antiadhesive effect of fibrinogen: a safeguard for thrombus stability. Blood 2007; 109: 1541-1549.
• 13 Lindbom L, Werr J. Integrin-dependent neutrophil migration in extravascular tissue. Semin Immunol 2002; 14: 115-121.
• 14 Hogg N, Laschinger M, Giles K. et al. T-cell integrins: more than just sticking points. J Cell Sci 2003; 116: 4695-4705.
• 15 Loike JD, Cao L, Budhu S. et al. Differential regulation of beta1 integrins by chemoattractants regulates neutrophil migration through fibrin. J Cell Biol 1999; 144: 1047-1056.
• 16 Gonzalez AL, El-Bjeirami W, West JL. et al. Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis. J Leukoc Biol 2007; 81: 686-695.
• 17 Schmalstieg FC, Rudloff HE, Hillman GR. et al. Two-dimensional and three-dimensional movement of human polymorphonuclear leukocytes: two fundamentally different mechanisms of locomotion [corrected]. J Leukoc Biol 1986; 40: 677-691.
• 18 Sixt M, Hallmann R, Wendler O. et al. Cell adhesion and migration properties of beta 2-integrin negative polymorphonuclear granulocytes on defined extracellular matrix molecules. Relevance for leukocyte extravasation. J Biol Chem 2001; 276: 18878-18887.
• 19 Zigmond SH, Slonczewski JL, Wilde MW. et al. Polymorphonuclear leukocyte locomotion is insensitive to lowered cytoplasmic calcium levels. Cell Motil Cytoskeleton 1988; 9: 184-189.
• 20 Friedl P, Entschladen F, Conrad C. et al. CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize beta1 integrin-independent strategies for polarization, interaction with collagen fibers and locomotion. Eur J Immunol 1998; 28: 2331-2343.
• 21 Doerschuk CM, Tasaka S, Wang Q. CD11/CD18-dependent and -independent neutrophil emigration in the lungs: how do neutrophils know which route to take?. Am J Respir Cell Mol Biol 2000; 23: 133-136.
• 22 Mizgerd JP, Kubo H, Kutkoski GJ. et al. Neutrophil emigration in the skin, lungs, and peritoneum: different requirements for CD11/CD18 revealed by CD18-deficient mice. J Exp Med 1997; 186: 1357-1364.
• 23 Lammermann T, Bader BL, Monkley SJ. et al. Rapid leukocyte migration by inte-grin-independent flowing and squeezing. Nature 2008; 453: 51-55.
• 24 Koenderman L, Kok PT, Hamelink ML. et al. An improved method for the isolation of eosinophilic granulocytes from peripheral blood of normal individuals. J Leukoc Biol 1988; 44: 79-86.
• 25 Langereis JD, Prinsen BH, de Sain-van der Velden MG. et al. A 2D-DIGE approach to identify proteins involved in inside-out control of integrins. J Proteome Res 2009; 8: 3824-3833.
• 26 Ulfman LH, Kuijper PH, van der Linden JA. et al. Characterization of eosinophil adhesion to TNF-alpha-activated endothelium under flow conditions: alpha 4 integrins mediate initial attachment, and E-selectin mediates rolling. J Immunol 1999; 163: 343-350.
• 27 Batschelet E. Recent statistical methods for orientation data. In: Animal Orientation and Navigation. Washington, DC: Government printing office; 1972. pp. 61-91.
• 28 Mardia KV. Statistics of Directional Data. London: Academic Press; 1972
• 29 Arendse MCBA. Magnetic orientation in the semi-terrestrial amphipod, Orchestia cavimana, and its interrelationship with photo-orientation and water loss. Physiological Entomology 1981; 6: 333-342.
• 30 Dransfield I, Cabanas C, Craig A. et al. Divalent cation regulation of the function of the leukocyte integrin LFA-1. J Cell Biol 1992; 116: 219-226.
• 31 Friedl P, Borgmann S, Brocker EB. Amoeboid leukocyte crawling through extra-cellular matrix: lessons from the Dictyostelium paradigm of cell movement. J Leukoc Biol 2001; 70: 491-509.
• 32 Alblas J, Ulfman LH, Hordijk P. et al. Activation of RhoA and Rock are essential for detachment of migrating leukocytes. Mol Biol Cell 2001; 12: 2137-2145.
• 33 Worthylake RA, Lemoine S, Watson JM. et al. RhoA is required for monocyte tail retraction during transendothelial migration. J Cell Biol 2001; 154: 147-160.
• 34 Smith A, Bracke M, Leitinger B. et al. LFA-1-induced T cell migration on ICAM-1 involves regulation of MLCK-mediated attachment and ROCK-dependent detachment. J Cell Sci 2003; 116: 3123-3133.
• 35 Woolf E, Grigorova I, Sagiv A. et al. Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces. Nat Immunol 2007; 8: 1076-1085.
• 36 Heit B, Tavener S, Raharjo E. et al. An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients. J Cell Biol 2002; 159: 91-102.
• 37 Berger M, Budhu S, Lu E. et al. Different G(i)-coupled chemoattractant receptors signal qualitatively different functions in human neutrophils. J Leukoc Biol 2002; 71: 798-806.
• 38 Ulfman LH, Alblas J, van Aalst CW. et al. Differences in potency of CXC chemokine ligand 8-, CC chemokine ligand 11-, and C5a-induced modulation of inte-grin function on human eosinophils. J Immunol 2005; 175: 6092-6099.
• 39 Malawista SE, de Boisfleury CA. Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes (PMN) in the presence of EDTA: PMN in close quarters require neither leukocyte integrins nor external divalent cations. Proc Natl Acad Sci USA 1997; 94: 11577-11582.
• 40 Malawista SE, de Boisfleury CA, Boxer LA. Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes from a patient with leukocyte adhesion deficiency-1: normal displacement in close quarters via chimneying. Cell Motil Cytoskeleton 2000; 46: 183-189.
• 41 Malawista SE, de Boisfleury CA. Functions of neutrophil motility of human blood: locomotion and chemotaxis in simplified systems. Bull Acad Natl Med 1998; 182: 1011-1021.
• 42 Mandeville JT, Maxfield FR. Effects of buffering intracellular free calcium on neutrophil migration through three-dimensional matrices. J Cell Physiol 1997; 171: 168-178.
• 43 Mandeville JT, Lawson MA, Maxfield FR. Dynamic imaging of neutrophil migration in three dimensions: mechanical interactions between cells and matrix. J Leukoc Biol 1997; 61: 188-200.
• 44 De Bruyn P. The amoeboid movement of the mammalian leucocyte in tissue culture. Anat Rec 1946; 95: 117-192.
• 45 Brown AF. Neutrophil granulocytes: adhesion and locomotion on collagen substrata and in collagen matrices. J Cell Sci 1982; 58: 455-467.
• 46 Bouma BN, Van Mourik JA. Unraveling the mystery of von Willebrand factor. J Thromb Haemost 2006; 4: 489-495.
• 47 Coffer PJ, Koenderman L. Granulocyte signal transduction and priming: cause without effect?. Immunol Lett 1997; 57: 27-31.
• 48 Kakkar AK, Lefer DJ. Leukocyte and endothelial adhesion molecule studies in knockout mice. Curr Opin Pharmacol 2004; 4: 154-158.
• 49 Baran KW, Nguyen M, McKendall GR. et al. Double-blind, randomized trial of an anti-CD18 antibody in conjunction with recombinant tissue plasminogen activator for acute myocardial infarction: limitation of myocardial infarction following thrombolysis in acute myocardial infarction (LIMIT AMI) study. Circulation 2001; 104: 2778-2783.
• 50 Yonekawa K, Harlan JM. Targeting leukocyte integrins in human diseases. J Leukoc Biol 2005; 77: 129-140.
• 51 Doerschuk CM. Leukocyte trafficking in alveoli and airway passages. Respir Res 2000; 1: 136-140.