**1.2 Adhesion molecules**

The recruitment and accumulation of monocytes and neutrophils at inflammatory sites is an essential step in the defense against invading microorganisms. The process of extravasation, when leukocytes slip through the endothelial cells and basement membrane into the underlying interstitium and further to the inflammatory site requires the expression of adhesion molecules on the endothelium. This serves to initiate leukocyte adherence by interaction between adhesion molecules on leukocytes and vascular endothelial cells (Johnson-Leger et al. 2000; van Buul & Hordijk 2004). The main families of adhesion molecules are the intercellular adhesion molecules (ICAMs); integrins, selectins and cadherins (calcium-dependent adherins) (Parkin & Cohen 2001).

The selectins, P-selectin (PADGEM, CD62P) and E-selectin (ELAM-1, CD62E), are membrane glycoproteins with a lectin-like domain that binds transiently to oligosaccharide molecules on passing leukocytes after cytokine-mediated activation of the endothelial cells. CD62L is present on circulating leukocytes (Janeway & Travers 2005). Selectin binding leads to tethering, which allows leukocytes to search the endothelium for the presence of activating factors. In a second step, leukocytes bind firmly to the endothelium, followed by the process of diapedesis (Albelda et al. 1994). The tighter adhesion is mediated by β2 integrins CD11a/CD18 (LFA-1) and CD11b (Mac-1 or CR3) expressed on leukocytes after a chemokine-mediated conformational change in the integrins. β2-integrins bind to intercellular adhesion molecules (ICAM-2 on resting endothelium and ICAM-1 on activated endothelium) (Adams & Shaw 1994; Gonzalez-Amaro & Sanchez-Madrid 1999; Janeway & Travers 2005). The β1- integrin very late antigen-4 (VLA-4) is present principally on mononuclear cells, mediating monocyte transmigration by binding to vascular adhesion molecules (VCAM-1) on activated endothelial cells (Chuluyan & Issekutz 1993).

### **1.3 Leukocyte adhesion and extravasation**

Leukocyte adhesion is made possible by the action of chemokines: small, structurally related molecules that interact with G-protein-coupled receptors. They perform activation of integrins in order to confer tight adhesion between leukocytes and endothelial cells, and promote the migration of adherent leukocytes across the endothelium and through the extracellular matrix (Adams & Shaw 1994). Chemokines are small molecules, divided into

by interacting with immune modulating cells (Cohen et al. 2001; Yamashiro et al. 2001; Cohen et al. 2003; Theilgaard-Monch et al. 2004). Neutrophil cytokine and chemokine production can be an important link between the innate and the adaptive immune responses. Cytokine-activated neutrophils produce and release multiple proinflammatory cytokines and chemokines, including IL-1, IL-8, monocyte chemotactic protein-1 (MCP-1/CCL2) and macrophage inflammatory protein-1α and 1β (MIP-1α/MIP-1β). MCP-1 and MIP-1α act as chemotactic and activating signals for mononuclear cells, especially monocytes, and for mobilization of other cell surface molecules involved in the adaptive

Chemokines attract neutrophils and monocytes from the circulation to the inflammatory/infectious site by first making the endothelium more adhesive to the circulating cells and then through a chemokine gradient through the tissue leading the way to the site of inflammation (Janeway & Travers 2005). Circulating monocytes that extravasate and get activated rapidly develop into mature macrophages with the principal

The recruitment and accumulation of monocytes and neutrophils at inflammatory sites is an essential step in the defense against invading microorganisms. The process of extravasation, when leukocytes slip through the endothelial cells and basement membrane into the underlying interstitium and further to the inflammatory site requires the expression of adhesion molecules on the endothelium. This serves to initiate leukocyte adherence by interaction between adhesion molecules on leukocytes and vascular endothelial cells (Johnson-Leger et al. 2000; van Buul & Hordijk 2004). The main families of adhesion molecules are the intercellular adhesion molecules (ICAMs); integrins, selectins and

The selectins, P-selectin (PADGEM, CD62P) and E-selectin (ELAM-1, CD62E), are membrane glycoproteins with a lectin-like domain that binds transiently to oligosaccharide molecules on passing leukocytes after cytokine-mediated activation of the endothelial cells. CD62L is present on circulating leukocytes (Janeway & Travers 2005). Selectin binding leads to tethering, which allows leukocytes to search the endothelium for the presence of activating factors. In a second step, leukocytes bind firmly to the endothelium, followed by the process of diapedesis (Albelda et al. 1994). The tighter adhesion is mediated by β2 integrins CD11a/CD18 (LFA-1) and CD11b (Mac-1 or CR3) expressed on leukocytes after a chemokine-mediated conformational change in the integrins. β2-integrins bind to intercellular adhesion molecules (ICAM-2 on resting endothelium and ICAM-1 on activated endothelium) (Adams & Shaw 1994; Gonzalez-Amaro & Sanchez-Madrid 1999; Janeway & Travers 2005). The β1- integrin very late antigen-4 (VLA-4) is present principally on mononuclear cells, mediating monocyte transmigration by binding to vascular adhesion

molecules (VCAM-1) on activated endothelial cells (Chuluyan & Issekutz 1993).

Leukocyte adhesion is made possible by the action of chemokines: small, structurally related molecules that interact with G-protein-coupled receptors. They perform activation of integrins in order to confer tight adhesion between leukocytes and endothelial cells, and promote the migration of adherent leukocytes across the endothelium and through the extracellular matrix (Adams & Shaw 1994). Chemokines are small molecules, divided into

**1.3 Leukocyte adhesion and extravasation** 

immune response (Yamashiro et al. 2001; Kobayashi 2008).

**1.2 Adhesion molecules** 

function of phagocyting microorganisms (Janeway & Travers 2005).

cadherins (calcium-dependent adherins) (Parkin & Cohen 2001).

Fig. 1. Leukocyte adhesion to the endothelium, subsequent extravasation and transmigration through a chemotactic gradient in the interstitium towards a site of inflammation.

Fig. 2. Neutrophil adhesion, extravasation and transmigration.

**Chemokines Receptor Functions References** 

cells.

**CCL2** CCR2 Chemotactic factor and

Induces neutrophil CD11b/CD18 up-

regulation, transmigration and activation. Stimulates the release of MMP-9/NGAL. Binding to the receptor causes a

(Zeilhofer & Schorr 2000; Drost & MacNee 2002; Adams & Lloyd

(Adams & Lloyd 1997; Jiang et al. 1992; Jiang et al. 1994; Ikeda et al. 2002; de Lemos et al. 2003; Pawlak et

(Rezzonico et al. 2001; Adams & Lloyd 1997; Ramos et al. 2005; Weber et al. 2000)

(Yan et al. 2001; Alberts et al. 2002; Brogden & Guthmiller 2002; Van Den Steen et

al. 2003)

al. 2004)

1997)

reconformation of integrins, which allows neutrophils to bind to the endothelial

activator of monocytes and macrophages. Produced by

inflammatory cells. Induces

Released from monocytes

CD11b/CD18. Promotes the recruitment of inflammatory cells. Chemotactic factor for both monocytes and neutrophils. Activates macrophages by

many different

up-regulation of CD11b/CD18 and facilitates monocyte adhesion to endothelial cells. Associated with chronic and acute inflammation, as well as with the acute coronary

syndrome.

after trigging of

up-regulation of CD11b/CD18.

cytokines.

Table 1. Chemokines analyzed in our study and their respective functions.

enzymes degrade the extracellular matrix and promote leukocyte transmigration. Marker of neutrophil activation and release of reactive oxygen species. Regulates chemokine activity by cleaving of chemokines and

MMP-9 and proteolytic

receptor α and β

**CCL3** CCR1 and CCR5

**IL-8 (interleukin-8) CXCL8** IL-8

**MCP-1 (monocyte chemotactic protein-1)** 

**MIP-1α (macrophage inflammatory protein-1α)** 

**MMP-9/NGAL (matrix** 

**metalloproteinase-9 in complex with neutrophil gelatinaseassociated lipocalin)** 

CXC (α-chemokines) and CC (β-chemokines) depending on the positions of two cysteine residues (C) relative to other amino acids (X) (Charo & Ransohoff 2006). Chemokines are produced by inflammatory cells after stimulation with proinflammatory cytokines or bacterial products, and there are both soluble and membrane-bound chemokines, with various functions (Parkin & Cohen 2001). Some of the chemokines and cytokines analyzed in our study, and their respective functions, are listed in Table 1 and Table 2.

Leukocyte binding to endothelial cells induces production of signaling molecules in the endothelial cells and activation of NADPH oxidase in leukocytes. NADPH oxidase promotes production of reactive oxygen species that break down the barrier to leukocyte passage between the endothelial cells and through the basement membrane (van Buul & Hordijk 2004). PECAM-1 plays an important role in transendothelial migration of leukocytes, by inducing phosphorylation of tyrosine in junctional proteins which leads to loss of cell-cell adhesion (van Buul & Hordijk 2004).

When neutrophils extravasate, they produce enzymes (i.e. elastase and other proteases such as matrix metalloproteinase-9, MMP-9) that break down extracellular matrix proteins and in this way promote leukocyte migration through the interstitium (Hermant et al. 2003).

The final step of the transmigration is the chemokine concentration gradient, which guides leukocytes through the interstitium and towards the inflammatory site. CXCL8 (IL-8) and CCL2 (MCP-1) act as chemotactic factors for neutrophils and monocytes, respectively. They bind to proteoglycans in the extracellular matrix and to similar molecules on the leukocytes (Janeway & Travers 2005).

Neutrophils and monocytes in blood normally express a low amount of CD11b on their surface. Following chemokine-mediated activation of the cells, CD11b is mobilized on the cell surface and the molecules are activated in order to display their functions (Adams & Shaw 1994; Albelda et al. 1994; Adams & Lloyd 1997; Gonzalez-Amaro & Sanchez-Madrid 1999). Mobilization of CD11b is important in the process of leukocyte transmigration, phagocytosis and complement activation as a response to inflammation/infection (Bainton et al. 1987; Borregaard et al. 1987; Miller et al. 1987).
