General steps of the extravasation process

The extravasation is a multi-step process of the emigration of cells from the blood stream into the tissue. The most prominent types of extravasating cells are leukocytes, i.e. T lymphocytes, natural killer (NK) cells, neutrophil granulocytes and monocytes. These cells have to leave the blood vessels in order to reach tissue sites of inflammation, infection or injury. But not only the emigrating cells, the vascular endothelium takes actively part in the extravasation process, too. It has to deliver certain localization signals, so that the leukocytes know where and when to emigrate. Therefore, the endothelium changes dynamically its architecture including receptor expression and cell-cell contacts. In contrast to the important and physiological extravasation of leukocytes, tumor cells are disseminated by the circulation through the body during hematogenic metastasis development. Akin to leukocytes, tumor cells do not randomly leave the blood vessels, but are guided by certain signals that are also delivered by the vascular endothelium. Earliest observations on this have been documented more than hundred years ago by Paget, who regarded in his theory on breast cancer metastasis the cancer cells as a seed that can only grow at certain sites of the body, which he considered as the soil [1]. Balkwill and Mantovani worded the assumption that tumor cells might use chemokine gradients to spread around the body [2], therefore, tumor cells and leukocytes might use similar localization signals. The stromal cell-derived factor (SDF)-1α is such a chemokine, which is expressed in organs that are the first destination of breast cancer metastases [3]. We have elaborated this hypothesis on localization signals: ligands to G protein-coupled receptors (GPCRs), which are chemokines and neurotransmitters as well, might chemotactically guide tumor cells to their destination of metastasis formation [4].

The extravasation process is in principle divided into three sequential steps regardless the type of cells. In the first step, the cells slackly attach to the vascular endothelial cells. Due to this loose interaction, the extravasating cells are still pulled along with the blood stream, which results in a rolling motion of the cells on the vascular surface. In the second step, the cells tightly attach to the endothelial cells, whereas the receptors for this interaction are different from those involved in the rolling process. After attachment, the previously mostly spherical cells spread on the endothelium and actively transmigrate through the endothelial barrier. This third step of the extravasation process is called diapedesis.

In some descriptions of the extravasation process of leukocytes, two further differentiating steps are included: before the cells get in contact to the endothelium, they have to move to the fringe of the blood stream, which is termed 'margination'. There is an equilibrium between circulating and marginated leukocytes, whereas the ratio of these subpopulations changes under certain conditions [5]. At the margin of the blood vessel, the flow rate is much lower than in the centre, thus, the blood flow has a parabolic profile of variable steepness [6]. Therefore, the cells are slower at the fringe of the blood vessels, which relieves rolling and adhesion. A second additional step of the extravasation process is sometimes included between rolling and adhesion, where the leukocytes are activated by receptor-bound chemokines to attach tightly. This tight attachment is mediated by integrins, which are activated by chemokine signalling via the so-called 'inside-out' signalling. These mechanisms will be discussed in detail below together with the characterization of the rolling process, since these steps happen at the same time.

Leukocytes

Since the endothelia differ in their structural and functional features as well as in the hemodynamic pattern, there are differences in the way to recruit leukocytes [16]. In brain microvasculature, where high shear forces and low adhesion molecule expression exist, rolling seems to play a minor role. It was found that platelets function as a bridge connecting leukocytes and endothelium to mediate adhesion [16].

Generally, rolling of leukocytes is mainly mediated by selectins, also named as lectin cell adhesion molecules, which are expressed on the surface of both endothelial cells and leukocytes (Fig. ​(Fig.1;1; Tab. ​Tab.2).2). In contrast, tumor cells do not express selectins, but their respective ligands. The family of selectins comprises three members: the L-, P- and E-selectin (CD62L, P, E). All selectins are calcium-dependent single-chain transmembrane glycoproteins. Rolling of leukocytes on HEV is mainly mediated by L-selectin and its ligands. L-selectin ligands are different sulphated glycans, that are collectively known as peripheral lymph node adressin [17], and are constitutively expressed on HEV: GlyCAM-1 [18], CD34 [19], nepmucin [20], Sgp200 [21] and others. They can function as ligands for L-selectin when decorated with a specific carbohydrate structure known as 6-sulfo-sialyl Lewis X (sialic acidα2–3Galβ1–4 [Frucα1–3(sulfo-6)]GlcNAcβ1-R) [22]. This modification is mainly mediated by the HEV restricted N-acetylglucosamine-6-O-sulfotransferase (also known as GST-3 or LSST) [17]. LSST is constitutively expressed in HEV but recent studies have shown that it can be induced in other kinds of endothelium in case of inflammatory lesions, e.g. rheumatoid arthritis [23]. L-selectin was first defined as a homing receptor for lymphocytes, since it is a key regulator for the recruitment of naïve and central memory lymphocytes to the lymph node, a process that occurs constitutively. HEVs in mesenteric lymph nodes also express the mucosal adressin cell adhesion molecules (MadCAM) as a ligand for L-selectin [23], in order to mediate rolling and adhesion via the α4β7-integrin on leukocytes [7] (Fig. ​(Fig.11).

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Figure 1

Receptors involved in the rolling of leukocytes and tumor cells. The endothelium is shown in green, leukocytes in blue and tumor cells in red.

In endothelium different to HEV, additionally P-and E-selectin regulate the rolling process of lymphocytes: P-selectin and L-selectin lead to rolling and initial localization of leukocytes and platelets, whereas E-selectin enhances the subsequent recruitment of leukocytes. P- and E-selectin both bind to PSGL-1 (P-selectin glycoprotein ligand-1), which is expressed on all blood lymphocytes, monocytes and neutrophils. P-selectin is preformed and stored in Weibel-Palade bodies of the endothelium and α-granules of platelets; exposure of endothelial cells to inflammatory cytokines, histamine, thrombin and other proinflammatory mediators leads to a rapid upregulation of P-selectin [24].

E-selectin is expressed within the first hours after exposure to inflammatory mediators, with the exception of the skin and bone microvessels, where it is constitutively expressed [25,26]. E-selectin ligands, e.g. CD43 and PSGL-1, are proteins that are decorated with sialyl Lewis X (sLeX) or sialyl Lewis A (sLeA). Decorated PSGL-1 is called cutanous lymphocyte antigen (CLA), that regulates skin homing of activated T cell subtypes [25]. HCELL (hematopoietic cell E-/L-selectin ligand) is a glycoform of CD44 that is expressed natively only on stem cells. It has the highest affinity of all natively expressed E-selectin ligands [27].

Furthermore, we have identified N-cadherin as a molecule involved in the rolling process of neutrophil granulocytes on pulmonary microvasculature [28]. Cadherins are calcium-dependent homophilic cell-cell adhesion molecules in any solid tissue of the body [29]. The cadherin family comprises several members with a distinct tissue expression patterns. Classical cadherins are the epithelial (E-), neural (N-), placental (P-), retinal (R-) and vascular endothelial (VE-) cadherin [29]. As the name says, N-cadherin is expressed in neural but also in other mesenchymal and connective tissues [30]. Besides N-cadherin, only the vascular endothelial (VE)-cadherin has yet been identified to play a role in the extravasation process of leukocytes, since it is expressed in endothelial adherens junctions [31]. However, it is so far not clear, how leukocytes manage crossing of that barrier [32].

Leukocytes

Integrins are the main adhesion molecules of leukocytes. During the rolling step, integrins are activated, and then bind to their counterparts on the endothelial cells to mediate a tight attachment (or arrest) of the leukocytes. Integrins are single transmembrane cell adhesion molecules consisting of two non-covalently associated α and β chains. In vertebrates 18 α and 8 β subunits form 24 known αβ dimmers, whereas β2 and β7 integrins are exclusively expressed on leukocytes [65]. Chemokines are the most powerful physiological activators of integrin-mediated adhesion. Within less than a second they regulate integrin affinity and avidity in a cell-type specific manner by inside-out signalling [66,67]. This signalling induces integrins to undergo conformational changes from a bent low-affinity state to an extended intermediate- or high-affinity state resulting in the opening of the ligand-binding pocket [68]. However, chemokine-stimulated inside-out signalling induces conformational changes in only a fraction of integrins [51]. The intracellular signalling cascades leading from GPCR activation to integrin activation are still incompletely understood, since different cell types, species and integrins use different signalling steps. A linear signal-transduction pathway would be a simplification of the complex network between chemoattractant-binding and integrin-adhesion [69]. Key regulatory events are the phosphorylation of the cytoplasmic domains [49,50], as well as binding of talin [70].

The integrin receptor family is subdivided into 8 groups according to the β chains. The following groups are most widely studied: β1 (CD29) = very late activation antigen (VLA) integrins, which mediate mainly cell-matrix interactions; β2 (CD18) = leukocyte integrins, which mediate mainly cell-cell interactions; β3 (CD61) with various adhesive functions, and β7 integrins mediating cell-cell interactions (Tab. ​(Tab.2).2). Those integrins that are most important in leukocyte arrest belong to the β1 and β2 subfamilies [33,51]: the αLβ2 (lymphocyte function-associated antigen (LFA)-1) and αMβ2 (Mac-1) are both ligands for ICAM-1/-2 (Fig. ​(Fig.2),2), whereas αLβ2 can also bind to the junctional adhesion molecule-A (JAM-A), and αMβ2 to JAM-C on the endothelium, which will be explained in detail in the diapedesis section. The α4β1 integrin VLA-4 is the ligand for the vascular cell adhesion molecule (VCAM)-1. Neutrophil granulocytes express VLA-4 only in case of sepsis [71]. ICAM-1/-2 (CD54/CD102) are expressed on surface of resting endothelium at low levels, but ICAM-1 becomes upregulated at sites of inflammation, e.g. in response to the tumor necrosis factor (TNF)α or interleukin (IL)-1β [65]. VCAM-1 (CD106) is also upregulated in response to inflammatory mediators. VCAM-1 is described as a key molecule in monocyte recruitment to early lesions in experimental models of atherogenesis [72,73]. Thus, on the side of the endothelium, several members of the immunoglobuline (Ig) superfamily are involved in the adhesion of leukocytes [74]. Furthermore, integrins that are expressed on endothelial cells are involved in leukocyte adhesion, too. The integrins α5β1 (VLA-5) and αvβ3 on the endothelium bind to L1-CAM, which is a molecule of the Ig superfamily expressed on lymphocytes [33,75,76].

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Figure 2

Receptors involved in the adhesion of leukocytes and tumor cells to endothelial cells. The endothelium is shown in green, leukocytes in blue and tumor cells in red.

There is a growing body of evidence, that galectins are involved this part of the extravasation. Galectins are defined by their ability to recognize β-galactose because of their conserved carbohydrate-recognition domains (CRDs) of about 130 amino acids. Fifteen mammalian galectins have been identified, which can be subdivided into three groups: those with one CRD, those with two CRDs, and chimera galectins with a tail of short tandem repeats fused to one single CRD [77,78]. Galectins can be secreted or, depending on the cell-type and activation state, be localized in the nucleus, the cytoplasm or the cell surface, where they interact with appropriately glycosylated proteins [79]. The most extensively studied function of galectins is their involvement in regulation of apoptosis (galectin-3 has anti-apoptotic function, galectin-1 is a pro-apoptotic receptor) [80,81], but during the past ten years it became evident that galectins are modulators of the immune response (pro- and anti-inflammatory functions) [82], as well as modulators of cell migration, adhesion and angiogenesis [78].

Galectin-1 is a non-covalent homodimer of two single CDR molecules, expressed in cultured endothelia of different origin as human aorta, umbilical vein, pulmonary artery, and mouse liver, brain and lung [78]. The activation of endothelial cells, e.g. with lipopolysaccharides, or tumor-conditioned media results in a rapid increase of galectin-1 expression, and it is constitutively expressed in tumor-associated endothelial cells [83,84]. Galectin-1 has anti-inflammatory properties. Peretti et al. observed that endogenous galectin-1 inhibits the adhesion of leukocytes to the endothelium, in the case of neutrophil granulocytes this was due to a suppression of Mac-1 upregulation [85-87]. Thus, galectin-1 seems more to function as a signalling molecule in the regulation of integrins than to act as a classical adhesion molecule. Accordingly, the upregulation of galectin-1 in tumor-associated endothelial cells is postulated as a tumor escape mechanism from immune response [88].

The expression of galectin-3 on endothelial cells is reported in vitro in human umbilical vein and dermal microvasculature as well as mouse lung, brain and liver [78]. Galectin-3 is upregulated in endothelial cells of cancer tissue as found in vivo and in a murine tumor model of hepatocellular carcinoma [89]. Nieminen et al. recently found a role for galectin-3 in the β2 integrin-independent adhesion of neutrophil granulocytes to lung endothelium in case of Streptococcus infection [90].

Galectin-9 was originally identified as a chemoattractant for eosinophil granulocytes [91]. Recently, it was identified as a receptor for the T_helper1 lymphocyte-specific cell-surface molecule Tim-3, thus negatively regulating T_helper1 lymphocyte responses [92]. Galectin-9 is expressed in various endothelial cells but so far mainly studied in the context of viral infections.