The Multistep Paradigm

In order to ensure immunosurveillance of the body the recruitment of leukocytes from the blood into the different tissues needs to be a well controlled process directing the extravasation of the appropriate cell at the proper location at the right time. The multistep paradigm for leukocyte extravasation [2] has proven to be a valid framework for understanding the sequence of dynamic interactions between the circulating leukocyte and the endothelial cell during this process. The multistep paradigm postulates that an initial transient contact of the circulating leukocyte with the vascular endothelium, generally mediated by adhesion molecules of the selectin family and their respective carbohydrate ligands, slows down the leukocyte in the bloodstream. After an initial capture the leukocyte rolls along the vascular wall with greatly reduced velocity. The rolling leukocyte can now "sense" chemotactic factors from the family of chemokines presented on the endothelial surface. Chemokines bind to their respective serpentine receptors on the leukocyte surface. These receptors deliver a G protein-mediated pertussis toxin-sensitive signal into the cell resulting in the functional activation of adhesion molecules of the integrin family present on the leukocyte surface. Activation of integrins leads to an increase in their affinity and/or avidity. Only "activated" integrins are able to mediate the firm adhesion of leukocytes to the vascular endothe-lium by binding to their endothelial ligands from the immunoglobulin (Ig) superfamily of adhesion receptors. This ultimately leads to leukocyte diapedesis, through endothelial cell contacts involving molecules of the endothelial adherens and tight junctions [3]. Some reports even suggest that leukocytes extravasate by transcytosis through endothelial cells. Successful recruitment of circulating leukocytes into the tissue depends thus on the productive leukocyte/endothelial interaction during each of these sequential steps.

Capture and Rolling: Selectins and Selectin Ligands

Extravasation of leukocytes takes place in postcapillary venules. The process known as capture or tethering hereby represents the very initial contact of a white cell with the endothelium. The transiently captured leukocyte might begin to roll along the endothelial surface. Rolling velocities are between 5 and 40 mm/sec and thus below that of freely flowing cells within the same postcapillary venules where mean blood flow velocities range from 1,000 to 4,000 mm/sec (Figure 1).

Capture and rolling are usually mediated by the selectin family of adhesion molecules, which has only three members. Selectins recognize via their N-terminal lectin domain fucosylated and sialylated glycoprotein ligands. Leukocyte (L)-selectin is constitutively expressed on all granulocytes and monocytes and most lymphocytes. Platelet (P)-selectin is stored in a-granules of platelets and in Weibel-Palade bodies of endothelial cells and translocated to the cell surface within minutes upon their activation during inflammation. Endothelial (E)-selectin is not con-stitutively expressed but induced on endothelial cells by inflammatory stimuli.

As post-translational modifications rather than the protein itself are recognized by the selectins, there are many candidate ligands for selectins. P-selectin glycoprotein ligand 1 (PSGL-1) has been most extensively characterized at the cellular, molecular, and functional level. PSGL-1 is constitutively expressed on all lymphocytes, monocytes, eosinophils, and neutrophils. It is the major ligand for P-selectin but can also bind to E-selectin. L-selectin glycopro-tein ligands have been identified in high endothelial venules

High Endothelial Venules And Lymph NodesSelectin And Selectin Ligand

Figure 1 The multistep model of leukocyte endothelial interaction. Top: Lymphocyte/HEVs interactions in Peyer's patches (PP) or peripheral lymph nodes (PLN). Bottom: Neutrophil interaction with inflamed endothelium. *, Only T cells, unknown for B cells; #, only demonstrated in vitro. The velocities of free-flowing (noninteracting) cells and rolling leukocytes as well as other time estimates are derived from intravital microscopic observations published from the laboratories of Ulrich von Andrian, Eugene Butcher, and Klaus Ley. (see color insert)

Figure 1 The multistep model of leukocyte endothelial interaction. Top: Lymphocyte/HEVs interactions in Peyer's patches (PP) or peripheral lymph nodes (PLN). Bottom: Neutrophil interaction with inflamed endothelium. *, Only T cells, unknown for B cells; #, only demonstrated in vitro. The velocities of free-flowing (noninteracting) cells and rolling leukocytes as well as other time estimates are derived from intravital microscopic observations published from the laboratories of Ulrich von Andrian, Eugene Butcher, and Klaus Ley. (see color insert)

(HEVs) of secondary lymphoid organs and are collectively known as the peripheral node addressins (PNAd) and recognized by the monoclonal antibody MECA-79. L-selectin mediates the rolling of naive lymphocytes on PNAd in HEVs of peripheral lymph nodes. Mice lacking L-selectin have very small peripheral lymph nodes due to the failure of their lymphocytes to roll on HEVs at this site. Evidence for the importance of selectins for effective neutrophil recruitment into inflamed tissues is derived from mice lacking either selectins or selectin ligands and from patients suffering from leukocyte adhesion deficiency II (LADII). These patients fail to incorporate fucose into selectin ligands and thus lack functional selectin ligands. Interestingly, the defect does not affect a biosynthetic enzyme. The defective gene in LAD II is a GDP fucose transporter in the Golgi membrane that provides the fucosyltransferases with their substrate [4]. As a consequence of this defect, leukocytes lacking selectin ligands cannot be captured to endothelium and patients suffer from bacterial infections.

Besides the adhesion receptor family of selectins and their ligands, on lymphocytes, a4-integrins have been shown to support capture and rolling. a4-integrin mediated rolling is rather slow and was measured to be around 10 mm/sec. a4b1 (VLA-4) mediated capture of T lym-phoblast on its vascular ligand VCAM-1 is required for successful recruitment of activated T cells into the central nervous system [5], whereas a4b7 (LPAM-1) mediated capture and rolling via MAdCAM-1 on HEVs in mucosal associated lymphatic tissue is required for the recruitment of a4p7-integrinP°sitive gut homing lymphocytes to these sites. One key to the participation of a4-integrins in capture and rolling of lymphocytes is their localization on the micro-villous processes on the surface of circulating cells, which they share with L-selectin.

Activation: Chemokines and Chemokine Receptors

In order to stop a rolling leukocyte, further signals are required. This is illustrated by in vivo observations that both neutrophils and lymphocytes can use L-selectin to roll in HEVs of peripheral lymph nodes; however, only lymphocytes have the ability to stop [6]. This points to the necessity of selective signals that specifically trigger adhesion of lymphocytes but not of neutrophils in HEVs.

Chemokines have been recognized to trigger adhesion of leukocytes to the endothelium under shear. Chemokines are a family of about 50 low-molecular-weight chemotactic cytokines (8 to 14kDa) that can bind to and signal through seven transmembrane spanning G-protein coupled receptors expressed on leukocytes but also on other cell types. Chemokines share sequence homologies and are divided into four groups (CC, CXC, CX3C, and C) based on the orientation of conserved cysteines in their amino termini [7]. The chemokine receptors are grouped accordingly into four receptor families (CCR, CXCR, CX3CR, and CR). Chemokine receptors preferentially signal via Gai-proteins, which can be blocked by pertussis toxin. The expression of chemokine receptors varies greatly among different leukocyte subsets and can change upon lymphocyte activation. Furthermore, chemokines are differentially expressed in different tissues. Many chemokines have been shown to bind to the surface of endothelial cells. This is due to the fact that most chemokines have highly charged amino acid residues that can mediate binding to heparan sulfate and other glycosaminoglycans (GAGs).

In order to examine the ability of chemokines to induce adhesion in the context of the multistep recruitment cascade, many investigators have employed in vitro flow chamber assays that mimic blood flow. Using these assay systems, chemokines have been demonstrated to trigger arrest of rolling lymphocytes on purified ligands or on endothelial cells under shear and have therefore been called "arrest chemokines" [8]. The chemokine CCL21 (SLC) was shown to trigger the arrest of T lymphocytes rolling on purified L-selectin ligands on ICAM-1. CCL21 is constitutively expressed on HEVs in peripheral lymph nodes. As only T lymphocytes but not neutrophils possess CCR7, the receptor for CCL21, it becomes plausible that only lymphocytes but not neutrophils can be arrested in HEVs in vivo. The importance of the CCL21/CCR7 chemokine/chemokine receptor interaction for successful T-cell homing to peripheral lymph nodes is exemplified by the lack of T cells within peripheral lymph nodes of CCR7-deficient mice or in mice that, because of a spontaneous mutation, lack expression of CCL21. Other chemokines have been characterized triggering the arrest of monocytes on inflamed endothelium [8]. Thus the chemokine/ chemokine receptor system has a great impact in the specificity of leukocyte extravasation at the level of cell arrest.

Firm Adhesion: Integrins and Ig Superfamily Members

Leukocyte integrins form a family of noncovalently linked ab-heterodimeric transmembrane proteins and are normally expressed on circulating leukocytes in a relatively inactive state. Upon chemokine receptor engagement, inte-grins become functionally activated within seconds, triggering leukocyte arrest on the endothelium. With the exception of the fact that a pertussis toxin-sensitive G-protein signal is delivered into the cell by the chemokine receptors, the downstream signals leading to integrin activation, commonly referred to as "inside-out-signaling," are as yet unknown. "Functional activation" of integrins on the leukocyte surface is achieved by an affinity increase through conformational changes, an avidity increase by integrin clustering, or a combination of both effects [9].

Each class of leukocytes displays a particular pattern of integrins that can change in a signal- and time-dependent fashion. For example resting human T lymphocytes express P1-, P2-, and b7-integrins on their surface, whereas neutrophils express mostly b2-integrins, and to a lesser degree also b1- and b3-integrins. All leukocytes, however, express at least one member of the b2-integrin family, which is restricted to the leukocyte lineage and therefore referred to as the leukocyte integrins. The |2-integrins are a family of four receptors, namely LFA-1 (aL|2; CD 11 a/CD 18), Mac-1 (aM|2; CD11b/CD18), p150,95 (ax|2; CD 11c/ CD 18), and aD|2 (CD11d/CD18). Especially LFA-1 and Mac-1 are involved in leukocyte extravasation by mediating adhesion to intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 on endothelial cells, whereas aD|2-integrin was shown to bind to vascular cell adhesion molecule 1 (VCAM-1).

Involvement of LFA-1 in lymphocyte homing to peripheral lymph nodes was first demonstrated by Alf Hamann (in 1988) by antibody inhibition studies and was more recently confirmed by intravital microscopy, where it could be demonstrated that LFA-1 mediates the activation-dependent adhesion of lymphocytes on HEVs at this site [6].

The importance of | 2-integrins in leukocyte recruitment is exemplified by an inherited autosomal recessive disease in man, called leukocyte adhesion deficiency-I (LAD-I). LAD-I is the result of mutations in the | 2 integrin subunit inhibiting cell surface expression and/or function of all | 2-integrins. Patients suffer from recurring soft-tissue infections, impaired wound healing, and gingivitis. The CD18 "knockout" mouse has similar problems to human LAD-I patients. The disorder is primarily considered a failure of neutrophil function, extravasation of which relies most heavily on | 2-integrins, because they lack significant levels of other integrins.

Monocytes and lymphocytes can use other integrins, such as the a4-integrins, when the |2-integrins are missing. There are two a4-integrins, VLA-4 (a4|1; CD49d/CD29) and a4|7-integrin. a4|7 is the lymphocyte receptor for mucosal addressin cell adhesion molecule -1 (MAdCAM-1). MAdCAM-1 is expressed on postcapillary venules in the lamina propria of the gut, and on HEVs of the intestine associated lymphoid tissues, especially the Peyer's patches and the mesenteric lymph nodes, thus directing trafficking of gut homing lymphocytes expressing its lymphocyte receptor a4|7 to these otteo. MAdCAM-1 is also found to be upregulated above constitutive levels in the intestinal lamina propria of patients suffering from ulcerative colitis or Crohn's disease and contributes to the maintenance of these chronic inflammatory diseases. Constitutive expression of VCAM-1 on endothelial cells is low or absent with the exception of the central nervous system, where constitutive VCAM-1 is involved in mediating the firm adhesion of a4-integrin positive T lymphoblasts in postcapillary venules [5].

Transendothelial Migration

Firm adhesion of the leukocyte to the endothelial surface is a prerequisite for diapedesis. The |2-integrins LFA-1 and Mac-1 and their endothelial ligand ICAM-1 have been implied to be involved in transendothelial migration (TEM) of T lymphocytes. In fact, endothelial cells lacking both ICAM-1 and ICAM-2 no longer support TEM of T cells in vitro. The apparent dual role of ICAM-1 in mediating firm adhesion and TEM can be assigned to different parts of the molecule. Whereas the extracellular domain of endothelial ICAM-1 suffices to mediate T-cell adhesion, the cytoplasmic domain is required to mediate TEM of T cells, probably by inducing Rho-signaling within the endothelial cells. Activation of Rho leads to cytoskeletal rearrangements within the endothelium, which are necessary to allow the passage of leukocytes across the endothelial cell wall. The actin cytoskeleton within the endothelium is anchored at cell-matrix interaction sites but also in cell-to-cell contacts forming adherens and tight junctions. Migrating leukocytes have been found to induce a delocalization of the vascular endothelial (VE)-cadherin from the endothelial adherens junctions by a yet unidentified mechanism, which potentially disrupts the endothelial adherens junction in a defined region and allows the leukocyte to exit via the opened gap. On the other hand, direct manipulation of adherens junctions causing molecular disorganization and an increase in vascular permeability shows no influence on TEM of leukocytes in vitro, demonstrating that TEM of leukocytes is not directly correlated to changes in endothelial permeability. Besides VE-cadherin other molecules were found to be concentrated at the lateral borders of endothelial cells and have been implicated in the process of transendothelial migration of leukocytes. These include the Ig-superfamily members platelet endothelial cell adhesion molecule 1 (PECAM-1) and the junctional adhesion molecules (JAMs: JAM-A, JAM-B, and JAM-C) and probably a unique molecule CD99. All of these molecules are potentially capable of homophilic interactions, which are thought to be involved in the establishment of the endothelial cell-to-cell contacts. PECAM-1, CD99, JAM-A, and JAM-C have also been demonstrated to be expressed on leukocytes. Therefore leukocyte extravasation through the endothelial cell junctions is often pictured as a zipper-like model, where the leukocyte on its passage transiently replaces the homophilic molecular interactions usually occurring in between the endothelial cells. This concept is supported by the findings that antibodies blocking homophilic interactions of PECAM-1 arrest leukocytes at the apical surface of endothelial cells. On the other hand, PECAM-1 deficient mice do not reveal any major defect in leukocyte extravasation, indicating that a requirement of PECAM-1 is not obligatory for TEM. Additionally, antibody inhibition studies have implied the JAMs to be involved in leukocyte extravasation. Interestingly, all members of the JAM family are able to bind to either | 2- or | 1-integrins on activated leukocytes, further supporting their possible involvement in leukocyte endothelial interactions.

Observations arguing for an alternative transcellular route for extravasating leukocytes through endothelial cells may suggest that their might be more than one molecular mechanism or route for leukocytes to move through the endothelial cell barrier. A number of very well performed studies documented leukocyte extravasation by sparing endothelial cell-to-cell contacts, suggesting that leukocytes traverse the endothelial cell proper [10]. In fact, Go wans and colleagues, who discovered the postcapillary venules within the lymphoid organs as the exit sites of circulating lymphocytes in the early 1960s [11] were even convinced that the specialized endothelial cells in HEVs engulf lymphocytes by mechanisms resembling phagocytosis, thus allowing their passage from the bloodstream to the lymphoid tissue. Thus, although certain stimuli may trigger a transcytotic pathway of leukocyte TEM, based on the current experimental evidence the general belief is that leukocytes traverse the endothelium through small gaps at intercellular junctions. The orchestration of molecular signals exchanged between the leukocyte and the endothelium during this process remains to be determined.


Since the multistep cascade of leukocyte recruitment was put forward [2], we have come a long way in our understanding of the traffic signals involved in leukocyte recruitment from the blood into tissue. However, although many of the players involved have been characterized, we still lack detailed mechanistic insights into their orchestrated function. How can chemokines trigger integrin activation and leukocyte arrest in seconds? Where and how do leukocytes penetrate the vascular wall without increasing vascular permeability? How are shear forces involved in leukocyte adhesion and TEM? Filling in the gaps in our understanding of the multistep cascade of leukocyte/endothelial interaction in the near future promises exciting new insights into this well-regulated process, which directs the trafficking of the correct cells at the right time to the right place in order to fight infections and disease.


1. Kubes, P. (2002). Introduction: The complexities of leukocyte recruitment. Semin. Immunol. 14, 65-72.

2. Butcher, E. C. (1991). Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity. Cell 67, 1033-1036. In this minireview Eugene Butcher puts forward the idea of the multistep cascade of leukocyte-endothelial interaction, based on his observations and ideas.

3. Vestweber, D. (2002). Regulation of endothelial cell contacts during leukocyte extravasation. Curr. Opin. Cell Biol. 14, 587-593.

4. Luhn, K., Wild, M. K., Eckhardt, M., Gerardy-Schahn, R., and Vestweber, D. (2001). The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter. Nat. Genet. 28, 69-72.

5. Vajkoczy, P., Laschinger, M., and Engelhardt, B. (2001). Alpha4-integrin-VCAM-1 binding mediates G protein-independent capture of encephalitogenic T cell blasts to CNS white matter microvessels. J. Clin. Invest. 108, 557-565.

6. Warnock, R. A., Askari, S., Butcher, E. C., and von Andrian, U. H. (1998). Molecular mechanisms of lymphocyte homing to peripheral lymph nodes. J. Exp. Med. 187, 205-216.

7. Zlotnik, A., and Yoshie, O. (2000). Chemokines: Anew classification system and their role in immunity. Immunity 12, 121-127. This review focuses on several areas of chemokine biology of particular interest to immunologists and gives a comprehensive list of known human chemokines. Most importantly, it introduces the new nomenclature for chemokines, which is based on the chemokine receptor nomenclature in use.

8. Ley, K. (2003). Arrest chemokines. Microcirculation 10, 289-295.

9. Kim, M., Carman, C. V., and Springer, T.A. (2003). Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins. Science 301, 1720-1725.

10. Feng, D., Nagy, J. A., Pyne, K., Dvorak, H. F., and Dvorak, A. M. (1998). Neutrophils emigrate from venules by a transendothelial cell pathway in response to FMLP. J. Exp. Med. 187, 903-915.

11. Gowans, J. L., and Knight, E. J. (1964). The route of recirculation of lymphocytes in the rat. Proc. R. Soc. Lond. B Biol. 159, 257. This paper describes the discovery of the specialized high endothelial venules (HEVs) in lymph nodes as the sites where circulating lymphocytes leave the bloodstream and enter the lymphatic tissue.

Capsule Biography

After heading a research group at the Max-Planck Institute in Münster, Germany, Dr. Engelhardt became director of the Theodor Kocher Institute at the University of Bern, Switzerland, in November 2003. Her research focuses on leukocyte trafficking into the central nervous system.

Dr. Vestweber became head of the Institute of Cell Biology at the University of Münster in 1994 and in 2001 became the founding director of the new Max-Planck-Institute for Molecular Biomedicine in Münster. His work focuses on the extravasation of leukocytes into inflamed and lymphoid tissue.

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