Endothelial AMs Involved in Leukocyte Trafficking

The AMs found on vascular ECs, with their corresponding ligands on the leukocyte, are usually distinguished by their contribution to rolling or firm adhesion (see Figure 2) [2]. The selectins (E-selectin, P-selectin) on the EC, and L-selectin on the leukocyte, are involved in initial tethering and rolling, while members of the immunoglobulin (Ig) superfamily of AMs, such as intercellular adhesion mole-cule-1 (ICAM-1) and cadherins, are mainly involved in adhesion and migration, respectively. However, it is possible that under certain circumstances, ICAMs and integrins can mediate rolling. The time course and magnitude of EC AM expression coordinates the recruitment of leukocytes to sites of inflammation. Thus, the rapid recruitment of rolling leukocytes results from L-selectin activation on leukocytes and rapid mobilization of preformed P-selectin to the EC surface. Leukocyte adhesion can also be fast because of the activation of b2-integrins on rolling leukocytes, which can interact with constitutively expressed ICAM-1, leading to firm adhesion and subsequent migration (see Figure 1). However, tissue-specific adhesion characteristics are apparent, such as in the liver sinusoidal microcirculation and the lung pulmonary circulation, where selectins are not required for leukocyte recruitment.

Rolling

Rolling is a transitory process that occurs in both nonin-flamed and inflamed vessels. As a result, many leukocytes "patrolling" the microvasculature will come into brief

Figure 2 Endothelial cell adhesion molecules and counterligands on leukocytes involved in cell trafficking. Activated EC express selectins that can bind to their carbohydrate receptors to allow leukocyte rolling under physiologic shear forces. Addressins expressed on high endothelial venules (HEVs) in lymph node vessels are expressed for lymphocyte homing; their peripheral vascular counterparts are less well defined. If activation occurs, leukocytes adhere to the endothelium via Ig-like superfamily proteins and integrins. Transendothelial migration is regulated by pECAM-1, cadherins, and other structures. (see color insert)

Figure 2 Endothelial cell adhesion molecules and counterligands on leukocytes involved in cell trafficking. Activated EC express selectins that can bind to their carbohydrate receptors to allow leukocyte rolling under physiologic shear forces. Addressins expressed on high endothelial venules (HEVs) in lymph node vessels are expressed for lymphocyte homing; their peripheral vascular counterparts are less well defined. If activation occurs, leukocytes adhere to the endothelium via Ig-like superfamily proteins and integrins. Transendothelial migration is regulated by pECAM-1, cadherins, and other structures. (see color insert)

contact with the endothelium. Sometimes, rolling leukocytes interact with other attached leukocytes in a process known as tethering. Approximately 80 percent of these cells will gently roll and detach from the endothelium and rejoin the circulation, while the remainder will roll more slowly and may adhere and flatten onto the endothelium. The first response to inflammatory stimuli that can be observed using intravital microscopy is an increase in the number of rolling cells, with a concurrent reduction in leukocyte velocity. Leukocytes will integrate chemokine and other signals on the EC surface while rolling along the endothelium until they reach a critical level of activation, resulting in firm adhesion.

Selectins and Their Ligands

The selectins are C-type lectins characterized by the presence of an N-terminal calcium-dependent carbohydrate recognition domain [3]. They are single-chain membrane glycoproteins that recognize counter-ligands usually containing sialylated and fucosylated carbohydrate residues, such as sialyl lewis x (Figure 2). There are three selectin AMs important in mediating initial tethering and rolling. Two selectins (E-selectin and P-selectin) are found on ECs, and one, L-selectin, is found on leukocytes.

L-selectin (Leukocyte-selectin) is involved mainly in the tethering of leukocytes to the endothelial surface, and proteolytic loss of expression of L-selectin, especially on granu-

locytes, occurs in conjunction with the activation of other AMs such as integrins. This soluble L-selectin can be detected in the serum and can competitively bind to endothelial ligands, blocking the interactions with leukocyte-bound L-selectin. The interaction of L-selectin with the EC is short-lived and usually with one of several ligands, such as CD34, mucosal addressins (e.g., MAdCAM-1), or vascular addressins.

P-selectin (platelet-selectin), originally named granule-external membrane protein, is a 140-kDa glycoprotein con-stitutively expressed in a-granules of platelets and the Weibel-Palade bodies of ECs. P-selectin is thought to be the predominant AM responsible for the rolling phase of the leukocyte adhesion cascade. Stimulation of ECs with inflammatory mediators such as histamine and thrombin results in translocation and expression of P-selectin on the cell surface within minutes. Reactive oxygen species (ROS), sulfidopeptide leukotrienes, and complement products can also induce fast mobilization of P-selectin onto the EC surface. P-selectin surface expression is transient and is recycled by reinternalization. P-selectin expression can also be transcriptionally regulated by cytokines such as interleukin-4 (IL-4) and IL-13. Thus, it is possible that P-selectin is important in Th2-mediated inflammatory diseases. In contrast, tumor necrosis factor (TNF), IL-1, and lipopolysac-charide (LPS), which induce other AMs on ECs, do not induce P-selectin gene transcription or expression. Like all the selectins, P-selectin binds to sialyl lewis x molecules but more specifically binds to P-selectin glycoprotein ligand-1 (PSGL-1), a homodimeric structure and member of the sialomucin family of AMs found on all circulating leukocytes. P-selectin can also bind to CD24 and other ligands. The interactions between P-selectin and PSGL-1 cause leukocyte-EC contacts and rolling along the venular endothelium. The P-selectin-EC associations are longer than the L-selectin-EC interactions and result in slower leukocyte rolling velocities. The signal transduction pathways that follow P-selectin binding to its ligand involve the activation of mitogen-activated kinases, Src kinases, and increases in intracellular calcium. All these pathways can lead to other downstream events such as integrin activation, shape change, and further ROS production.

E-selectin (endothelial-selectin) expression is protein synthesis dependent, occurring on the cell surface approximately 4 hours after activation with TNF, IL-1, or LPS. Eosinophils and neutrophils bind equally well to P-selectin, whereas eosinophils form fewer primary tethers to E-selectin. E-selectin has similar, possibly redundant functions to P-selectin, but more importantly, may be important in converting rollers to adherent cells, as E-selectin mediates even slower rolling than P-selectin. The off-rate of E-selectin is similar to that of P-selectin; thus, it is likely that E-selectin or its ligand is more highly expressed than P-selectin and PSGL-1. In addition, because its peak expression is around 4 hours, E-selectin could functionally support rolling when P-selectin expression is reduced or when P-selectin is internalized. E-selectin is capable of binding to

PSGL-1 and other sialic acid-containing glycoproteins, but the primary E-selectin ligands in humans are glycolipid structures termed myeloglycans. In mice, the identified ligand of E-selectin is a glycoprotein known as E-selectin ligand-1 (ESL-1).

Studies in knockout animals have revealed the importance of L-selectin and P-selectin in the initial induction of rolling, while all selectins can support stable rolling. However, in E-selectin and P-selectin double-deficient mice, L-selectin can sufficiently produce rolling that results in activation and adhesion, suggesting that overlap and redundancy occur in selectin function. Interestingly, E-selectin and b2 integrin AMs (together with ICAMs) cooperate to control the time that a leukocyte takes to roll and firmly adhere. Further, these double-deficient mice exhibit signs of early lethality. This suggests that E-selectin operates "downstream" from P-selectin, more toward the firm adhesion step of the cascade.

In summary, selectins are either constitutively expressed, or inducible with inflammatory mediator or cytokine exposure. These AMs all play roles in early stages of extravasation involving tethering and rolling, with varying degrees of attachment. This allows temporary interaction of patrolling leukocytes in the circulation with vascular ECs, allowing them to react to stimuli (e.g., chemokines) presented on the EC surface. This, in turn, causes leukocyte activation and subsequent extravasation.

Addressins

In secondary lymphoid tissues (such as lymph nodes and Peyer's patches), the entry site for naive T and B lymphocytes is known as the High Endothelial Venule (HEV), because of its cuboidal shape. The ECs of HEV express specific AMs that allow lymphocyte homing into the lymphoid tissue, while other cells, such as granulocytes, may enter the HEV but do not stop. Murine HEVs selectively bind to an antibody, MECA-79, that recognizes a carbohydrate epitope not found on ECs of postcapillary venules or large vessels in the spleen or thymus. These carbohydrate epitopes bind to L-selectin, are partly responsible for the tissue-specific trafficking of lymphocytes, and are called vascular addressins [4, 5]. The peripheral node addressins (PNAds) include Gly-CAM-1 (glycosylation-dependent cell AM), CD34, podoca-lyxin, and Sgp200, whereas mucosal or mesenteric addressin is thought to be MAdCAM-1 (Mucosal Addressin cell AM-1). The adhesion molecules are important in the homing of lymphocytes and dendritic cells to lymphoid tissues for antigen presentation.

Firm Adhesion

Firm adhesion of leukocytes on the vascular endothelium occurs after the activation of leukocyte AMs by chemokines and other stimuli presented on the EC surface. All firmly adherent leukocytes are exclusively recruited from the rolling pool. The AMs involved in firm adhesion are mainly the members of the Ig superfamily of AMs and their integrin ligands.

Ig Superfamily of AMs

This is a wide family of AMs, consisting of Ig-like domains characterized by intrachain disulfide bonds. Together with their ligand counterparts, they play major roles in leukocyte adhesion and the regulation of firm adhesion. Members include ICAM (intercellular AM)-1, -2, and -3 and VCAM-1 (vascular cell AM-1).

ICAM-1 is found constitutively at low levels on the EC surface, but is upregulated upon exposure to inflammatory cytokines (e.g., TNF, IFN-g, and IL-1) with peak expression after about 24 hours. Of note, treatment of ECs with IFN-g selectively increases the expression of ICAM-1, with no effect on other AMs. ICAM-1 contains five Ig-like extracellular domains, where the first Ig-like domain (on the amino-terminal end) is the recognition site for fibrinogen, an extracellular matrix protein, and the integrin lymphocyte associated function antigen (LFA-1 or CD11a/CD18). The third Ig-like domain recognizes another integrin, macrophage antigen-1 (Mac-1 or CD11b/CD18). Mac-1, found on granulocytes and monocytes, binds ICAM-1 with high affinity. As described, LFA-1 can also bind to ICAM-1, but with lower affinity than Mac-1. ICAM-1 has another interesting function, in that it is the exploited receptor of most strains of rhinovirus and the malarial parasite. The ligation of ICAM-1 can cause structural changes in junctional EC cytoskeleton proteins without causing EC injury or retraction. These structural proteins have been shown to dissociate within minutes of leukocyte-EC adhesion. This could be linked to subsequent events in transmigration.

ICAM-2 is a truncated form of ICAM-1, with only two Ig-like domains. LFA-1 binds to ICAM-2 with higher affinity than to ICAM-1. ICAM-2 is highly and constitutively expressed on ECs, without further expression after stimulation. It is found preferentially at endothelial borders, and thus could be important in transmigration. ICAM-3 is found on resting leukocytes, but can be induced in ECs. It contains five Ig-like extracellular domains, similar to ICAM-1, and binds to LFA-1 and adP2, another P2 integrin found on most leukocytes.

VCAM-1 is found at very low levels, if at all, on nonacti-vated ECs, but is upregulated substantially after long-term exposure to cytokines. It is expressed on the surface of IL-1 and TNF activated EC, and similar to the ICAMs, binds to integrins. However, it binds to a different class of integrins with Pj and P7 subunits. The most common ligand for the binding of VCAM-1 is very late antigen-4 (VLA-4, CD49d/CD29), found on eosinophils, basophils, mast cells, lymphocytes, and monocytes, but not neutrophils. VLA-4-VCAM-1 binding has been shown to mediate leukocyte rolling and adhesion. In particular, VCAM-1 ligation is essential for eosinophil and monocyte migration, and exposure of ECs to the Th2 dependent cytokines IL-4 and IL-13 (and TNF and IL-1 synergistically) selectively increases VCAM-1 expression, indicating the potential importance of VCAM-1 in Th2-mediated diseases such as allergy and asthma.

In summary, the Ig superfamily of adhesion molecules and integrins plays an extremely important role in the adhesion step of the recruitment cascade. ICAM-1 is important in adhesion of essentially all leukocytes, while VCAM-1 is the Ig-like adhesion molecule involved in adhesion of all leukocytes except neutrophils.

Transmigration

The final step of cell recruitment involves transmigration between adjacent ECs at tricellular junctions, a pause in the wall, and subsequent penetration through the basement membrane. During the adhesion phase, a disruption of EC-cadherin complexes occurs, inducing a loss of lateral junction localization, leading to migration between ECs [6]. PECAM-1 (platelet endothelial cell adhesion molecule-1, CD31) is another member of the Ig super family and is vitally important in the transmigration step of neutrophil recruitment. It has six Ig-like domains as well as immunore-ceptor tyrosine-based inhibitory motifs. PECAM-1 is in especially high density at the gap junction area of ECs, but is evenly distributed on platelets and leukocytes. Because of its concentration at the intercellular junctions of the vascular endothelium, it is thought to be a crucial AM to guide adherent cells to the migration site. PECAM-1 mediates endothelial transmigration through binding to itself (homophilic) or to other ligands (glycosaminoglycans such as CD44). PECAM-1 is also involved in the migration through the basement membrane and subendothelial matrix. PECAM-1 is critical for neutrophil (but not eosinophil) transendothelial migration, and the first Ig-like domain is important for this step. Cells treated with anti-PECAM-1 antibodies, or PECAM-IgG constructs comprising domain 1, arrest on ECs but do not transmigrate. Furthermore, the 6th Ig-like domain is thought to be important in the migration across the basement membrane. In this case, as there is no PECAM-1 expressed in the basement membrane, PECAM-1 is thought to bind to an as yet unidentified structure in the basal lamina. It may be possible that PECAM-1 is more involved in migration across the basement membrane, as leukocytes from PECAM-1-deficient mice do migrate through ECs but are delayed in their migration across the basement membrane. Altogether the process of leukocyte recruitment from the circulation into the tissue involves many AMs on the leukocyte as well as the EC. Thus, the process requires a step-by-step activation and deactivation (or deadhesion) of each and every constituent for successful leukocyte extravasation.

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Coping with Asthma

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