Leukocyte Adhesion Cascade

For a leukocyte to leave the blood stream and to enter an organ, a number of sequential steps must occur, often referred to as the “leukocyte adhesion cascade”.  This is broadly segmented into 5 different stages:  margination, tethering and rolling, exposure of the leukocytes to chemoattractants in soluble form and/or presented by endothelial cells, firm adhesion, spreading and diapedesis or extravasation.

 

Selectins

Selectins and selectin ligands are presented by leukocytes and endothelial cells.  PSGL-1 binds to P-selectin but can also bind to E- an dL-selectin.  ESL-1 is a specific ligand for E-selectin.  L-selectin has many specific ligands:  CD34, MadCAM, GlyCAM and PNAD.  Further ligands for E-, P- and L-selectin may be found in the glossary section of Cellix’s website.

 

Chemoattractants and their receptors

Binding of chemoattractants to receptors on the rolling leukocyte induces firm adhesion of the cell to the endothelial lining and subsequent migration.  It is thought that an important part of the chemoattractants are presented by glycoproteins on the endothelium and this has been shown for IL-8 [43].  Chemoattractants are largely segmented into two classes:  classical chemoattractants and chemokines.  Both groups bind to GTP-binding proteins (G-protein)-coupled seven spanning transmembrane receptors (or serpentine receptors).  These receptors are characterized by the presence of a G-protein that consists of an α and a βg subunit.  Upon receptor activation, the G-protein exchanges GDP for GTP resulting in dissociation of the α from the βg subunit.  This process leads to activation of phospholipase C (PLC) and this enzyme hydrolyses its substrate phosphatidylinositol (4,5)- biphosphate (PIP2) into inositol (1,4,5) trisphophate (IP3) and diaclyglycerol (DAG).  IP3 binds to its receptor on intracellular Ca2+ in the cytosol, whereas DAG activates protein kinase C (PKC) [44].  Ca2+, PKC and other intracellular messengers that are activated control a broad array of cellular processes such as chemotaxis, differentiation, proliferation and survival [45].

 

Classical Chemoattractants

Classical chemoattractants activate most leukocyte subsets.  These chemoattractants can be divided into at least 3 different groups:  bacterial peptides such as fMLP, serum factors such as C5a and C3a, and bioactive lipids such as LTB4 and PAF.

 

Chemokines

The chemokine family can be divided into four groups:  the CCL, CXCL, CX3CL and CL chemokines that bind to their receptors CCR, CXCR, CX3CR and CR, respectively [46].  All chemokines share structural elements including conserved cysteine residues that form disulfide bonds in the tertiary structure.  The CCL, CXCL and CX3CL have four cysteines that form disulfide bonds with no, one or three amino-acids (X) in between the cysteines, respectively.  The C group only has one disulfide bond.  Of each of the subgroups CL and CXC3L, only one member is known today, lymphotactin [47] and fractalkine/neurotactin [48], respectively.  The number of chemokines in the CCL (28 members) and CXCL (16 members) groups has grown extensively and they have overlapping functions, although some specificity is present [49].  Besides the structural classification of chemokines, a classification according to function can also be made.  Part of the chemokines are continuously expressed in the body for direct homing o leukocytes, thereby securing immuno-surveillance.  An example is the stromal cell derived factor (SDF-1, CXC chemokine), which plays a major role in homing of stem cells to the bone marrow [50].  Furthermore, secondary lymphoid-tissue chemokine (SLC) is expressed by lymph nodes and spleen and attracts lympocytes to these sites [51].  Other chemokines are expressed upon an inflammatory reaction, and might therefore be called inflammatory chemokines.  Generally, it is thought that CCL chemoattractants are more chemotactic for monocytes, eosinophils, lymphocytes and basophils whereas the CXCL chemoattractants are probably more specific for neutrophils and to a lesser extent for basophils and T-cells [52].  One reason why chemokines have overlapping functions is that different chemoattractants can bind to the same receptor and that one chemokine can bind to distinct chemokine receptors.  In this respect RANTES, eotaxin and MCP2 and MCP3 can all bind to CCR3 [53;54] and RANTES binds to CCR1, CCR3 and CCR5 [55].

 

Firm Adhesion and Spreading

Activation of the rolling cell by chemoattractants induces firm adhesion and subsequent spreading of the cell.  Firm adhesion is mediated by integrins on the leukocytes that bind to their counter ligands on the endothelium belonging to the immunoglobulin superfamily.  For example, the integrins VLA-4, MAC-1 and LFA-1 are important for firm adhesion to VCAM-1, ICAM-1 and ICAM-2, respectively. 

 

Adhesion counter-receptors belonging to the Ig superfamily members

Immunoglobulins are plasma proteins including all antibody molecules and are characterized by an immunoglobulin domain.  Members of the immunoglobulin superfamily share structural and genetic features with immunoglobulin molecules and contain at least one immunoglobulin domain.  The vascular endothelium expresses molecules of the immunoglobulin superfamily, which act as counter-receptors for leukocyte integrins.  Three immunoglobulin type ligands are present on the endothelium, of which ICAM-2 is constitutively present and ICAM-1 and VCAM-1 are induced by cytokine stimulation.

 

 

 

Integrins

Integrins are heterodimeric proteins that consist of an α subunit that is non-covalentlyh bound to a β subunit.  At least 8 different β subunits (β1-8) and 18 different α subunits (αL,M,X,D, α1-11, αE, αIIb, αV) have been described on human cells that combine to form 24 known adhesion receptors [56] (Hemler Keystone 2001) (Table 1).  The integrins that are important for leukocyte adhesion to endothelium are all four members of the β2 integrin family; αLβ2 (CD11a/CD18, LFA-1),  αMβ2 (CD11b/CD18, Mac-1, CR3),  αXβ2 (CD11c/CD18, p150,95),  αDβ2 (CD11dCD18) and two members of the β1 and β7 integrin family, α4β1 (VLA-4) and α4β7, respectively.  αLβ2 is expressed on the surface of all leukocytes although the highest expression is found on lymphocytes.  αMβ2 and αXβ2 are predominantly expressed on granulocytes, monocytes and macrophages.  αDβ2 was characterized as an integrin present on foam cells [57] but also appears to be present on a subset of leukocyte populations [58].  αLβ2 binds to ICAM-1, -2 and -3, whereas αMβ2 binds to ICAM-1 [59], C3bi [60], fibrinogen [61], heparin [62], LPS [63], factor X [64] and an unknown molecule [65].  αDβ2 and α4β1 integrins both bind to VCAM-1 [66;67].  The importance of functional β2 integrins in host defense mechanisms is illustrated by the rare genetic disease LAD I (Leukocyte adhesion deficiency I) [68;69].  These patients have no expression of β2 integrins on their leukocytes and suffer from recurrent bacterial infections.  The only permanent therapy for this disorder is allogeneic bone marrow transplantation of which graft versus host disease is the major drawback [70].

On non-activated, resting cells, integrins maintain a conformationally inactive state.  An exception of this characteristic accounts for the α4β1 integrin that is present on resting cells and is able to bind to its ligand VCAM-1 [71-73].  Upon stimulation by chemoattractants or other stimuli integrins change their conformation leading to increased affinity and/or avidity.  This process is called “inside-out” signalling [74-76].  The binding, however, of integrins to their ligands in itself may lead to activation of intracellular signalling pathways; this process is called “outside-in” signalling.

 

Margination

Margination involves the movement of free flowing leukocytes from the centre of the blood vessel to the wall of the blood vessel.

 

Tethering and Rolling

Leukoctye migration to inflammatory sites occurs mainly in the postcapillary venules.  The blood flow at these sites roughly varies between 3-6 cm/s (shear stress of 0.5-2 dyne/cm2).  Cells flowing with these velocities can tether (i.e. a short transient interaction) on the endothelium that lines the vessel walls.  Subsequently, they roll with a speed that is around a thousand times lower than the speed of free flowing leukocytes in the venule.  The moment a cell is rolling, it endures a force of the blood stream called “shear stress”.  A certain minimal shear stress is required for rolling interactions to occur (4;5).  The first interaction between leukocytes and endothelial cells preceding the rolling phase is called “primary tethering”.  Additional recruitment of cells to the surface involves the tethering of fast flowing leukocytes to an already bound leukocyte.  These homotypic interactions or “secondary tethering” facilitate the subsequent formation of clusters or strings of cells on the vessel wall.  The molecules involved in tether and rolling interactions are the selectins and their ligands which are mucin-like carbohydrate structures that are sialylated, fucosylated and/or sulphated.

 

Migration

Chemoattractant-induced activation of leukocytes leads to firm adhesion and spreading on the endothelium.  Subsequently, cells crawl to the site where endothelial cells connect to each other and migrate in between them to the inflammatory site.  It was shown that outside-in signalling by adhesion of neutrophils to endothelial receptors increased the endothelial permeability by disorganizing endothelial cell-cell adherent junctions and thus facilitating migration [82].  Another mechanism for passing the endothelial layer is the observation that neutrophils do not crawl in between endothelial cells but migrate through an endothelial cell [83].  When cells have passed the endothelium, they interact with the extracellular matrix (ECM) [84].  For neutrophils β1- and β3- but to a lesser extent β2-integrins are essential for adhesion to ECM proteins such as fibronectin, collagen I and IV, laminin, vitronectin and tenascin [85].  Finally, cells enter the site of inflammation.

 

 The Process of Migration

The spread cell has a typical tear-drop resembling shape.  The broad and flat edge is the leading front of the cell, also called the lamellipodium, while the narrow end at the rear of the cell is called uropod [86].  Signals both from chemoattractants and from integrin crosslinking lead to extension of the lamellipodium membrane, a process called protrusion.  Filopodia are other protrusions and resemble thin cylindrical needle-like projections.  Protrusion is associated with an increased polarization of filamentous actin (F-actin), and in fibroblasts it has been shown that in the protruding lamellipodium focal adhesion formation takes place.  Focal adhesions are structures of clustered integrins associated intracellularly with structural and signalling molecules [87].  In fibroblasts, focal adhesions were shown to increase in size as the cell moves over them, and when they reach the rear of the cell, they are left behind [88;89] and/or are recycled [90].  In leukocytes smaller structures than focal adhesions are present that are called focal complexes or adhesisomes, and these appear to have the same characteristics [91].