Sirpa Jalkanen and Marko Salmi

University of Turku and National Public Health Institute, Turku, Finland

Physiological Lymphocyte Recirculation

A subset of vascular endothelial cells in peripheral lymph nodes and in organized lymphatic tissues in the gut (Peyer's patches and appendix) have differentiated to optimally support continuous lymphocyte trafficking into these tissues. They do not mediate extravasation of granulocytes in normal conditions. These vessels are postcapillary venules of the vascular tree and their endothelial cells are morphologically plump. To discriminate them from the flat-walled venules present in all organs, they are called high endothelial venules (HEV). The expression patterns of homing-associated molecules on HEV in peripheral lymph nodes and mucosa-associated lymphatic tissues differ, resulting in functional differences in the entry of lymphocyte populations into these two tissues. Lymphocytes also efficiently enter the spleen, but the mechanisms and molecules and even the contribution of vascular endothelium is not clear in lymphocyte homing into this organ. Vessels in nonlymphoid tissues support lymphocyte entrance only at a very low level and therefore, only occasional lymphocytes in search of microbes can be found in these tissues.

HEV are the most important site for lymphocyte entrance to organized lymphoid tissues. However, about 15 percent of lymphocytes enter the lymph nodes via afferent lymphatics. Lymphocytes leave the nodes via efferent lymphatic vessels. The vascular network guiding lymphocyte recirculation is schematically presented in Figure 1. Endothelium in lymphatics differs morphologically from endothelium in blood vessels, since lymphatic endothelium is flat and discontinuous. Although both of these two types of endothelia interact with lymphocytes, they have largely different molecular phenotypes.

Inflammation-Induced Changes

Inflammatory mediators induce molecular and morphological changes in endothelium, optimizing its capacity to efficiently mediate entry of different leukocyte populations to sites of inflammation. Several homing-associated molecules not normally present on flat endothelium appear on the surface of these vessels upon inflammation. In chronic inflammations the flat endothelial vessels morphologically transform to HEV-like vessels. Different leukocyte populations sequentially accumulate at the site of inflammation, lymphocytes being the last cell type to enter. Their main wave of entry is 2 to 3 days after the initiation of inflammation. In extreme chronic cases a nonlymphatic tissue can in fact start to resemble organized lymphatic tissues.

Molecular Events in Lymphocyte-Endothelial Cell Interaction

Lymphocyte interaction with endothelium is a dynamic process that takes place in a multistep fashion involving binding between several molecular pairs on lymphocyte and endothelial cell surface. The adhesive steps can be divided to the following phases: (1) tethering and rolling, (2) activation, (3) firm adhesion, and (4) transmigration. These steps are schematically depicted in Figure 2. All these steps have to be appropriately executed before a lymphocyte can enter the tissue. Molecular mechanisms mediating these steps have been traditionally divided into unique categories. However, depending on the hemodynamic conditions and on the vascular bed where the interaction takes place, one receptor-ligand pair can be involved in other phases as well.

Skin

Skin

Figure 1 Vascular network guiding lymphocyte recirculation. Transport of foreign material (pieces of microbes) into lymphoid organs takes place, for example, through the epithelium of the gut and via afferent lymphatics draining the skin. Bloodborne lymphocytes enter the organized lymphatic tissues (lymph nodes and Peyer's patches; PP) from the blood via the arterial tree, flow through the capillaries, and extravasate in the postcapillary high endothelial venules. Thereafter, the lymphocytes migrate through the tissue parenchyma in search of their cognate antigens. If the antigen is found, the lymphocytes get activated within the germinal centers (B cells) or outside the centers (T cells). Activated cells and cells that did not find their antigen leave the node by entering the lymphatic vessels and return via the efferent lymphatics back to the blood circulation. Activated cells then preferentially leave the blood in peripheral tissues, where the microbial insult took place, whereas the nonactivated lymphocytes continue their journey between blood and lymphoid organs. (see color insert)

Figure 1 Vascular network guiding lymphocyte recirculation. Transport of foreign material (pieces of microbes) into lymphoid organs takes place, for example, through the epithelium of the gut and via afferent lymphatics draining the skin. Bloodborne lymphocytes enter the organized lymphatic tissues (lymph nodes and Peyer's patches; PP) from the blood via the arterial tree, flow through the capillaries, and extravasate in the postcapillary high endothelial venules. Thereafter, the lymphocytes migrate through the tissue parenchyma in search of their cognate antigens. If the antigen is found, the lymphocytes get activated within the germinal centers (B cells) or outside the centers (T cells). Activated cells and cells that did not find their antigen leave the node by entering the lymphatic vessels and return via the efferent lymphatics back to the blood circulation. Activated cells then preferentially leave the blood in peripheral tissues, where the microbial insult took place, whereas the nonactivated lymphocytes continue their journey between blood and lymphoid organs. (see color insert)

ROLLING

ACTIVATION

FIRM ADHESION

TRANSMIGRATION

Select Ins, sialomuclns Others

Figure 2 The multistep cascade of lymphocyte extravasation. The bloodborne lymphocyte first makes transient and weak contacts with endothelial cells (rolling) using selectins and sialomucins. If the lymphocyte has the right chemokine receptor to interact with a chemokine presented by the endothelium, this molecular interaction leads to activation of integrins. Endothelial immunoglobulin superfamily counterparts bind to activated integrins, which results in firm adhesion of the lymphocyte to the vessel wall. Thereafter, the lymphocyte transmigrates into the tissues between or through the endothelial cells.

Ctiemnkines, Integrins,, 7 I'M receptors Ig su per family

The key molecular interplayers at each step are described in the following and summarized in Figure 3.

Tethering and Rolling

Freely flowing lymphocytes in the blood randomly make collisions with the vessel wall. They form transient and weak contacts with endothelium, which results in slowing of their velocity. This initial interaction is predominantly mediated by selectins and their sialomucin ligands. A typical feature in this interaction is that it involves binding of the lectin domain of a selectin to carbohydrates on sialomucin molecules. There are three members in the selectin family. They are L-selectin (CD62L) expressed on leukocytes, E-selectin (CD62E) present on endothelium, and P-selectin (CD62P) present on both platelets and endothelium.

The counter-receptors of L-selectin are sulfated, fucosy-lated, and sialylated carbohydrates (sialyl Lewis X-like structures) present on several protein backbones [for example, on glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1), CD34, podocalyxin, and even on a subset of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in Peyer's patches]. These molecules are collectively called peripheral lymph node addressins (PNAd). HEV in periph eral lymph nodes strongly express PNAd and thus guide L-selectin expressing lymphocytes efficiently into the nodes.

E-selectin is one of the inducible molecules found at sites of inflammation. Its expression becomes prominent within few hours after initiation of inflammation. E-selectin mediates lymphocyte entry, especially into inflamed skin. Cutaneous lymphocyte antigen CLA (a variant of P-selectin glycoprotein ligand, PSGL-1) serves as the lymphocyte ligand for E-selectin in the skin. In addition, E-selectin is also able to bind E-selectin ligand-1 (ESL-1) on leukocyte surface.

P-selectin is translocated within minutes from specific granules (Weibel-Palade bodies) to the surface of endothe-lium. Although it mainly binds granulocytes at an early phase of inflammation, it is also present in chronic inflammations due to new protein synthesis and binds to PSGL-1 on lymphocytes.

Endothelial cells at mucosa-associated lymphatic tissues have a unique phenotype, because they, unlike other normal endothelium in the body, express MAdCAM-1. MAdCAM-1 binds a4b7 integrin on lymphocytes and selectively mediates traffic of the a4b7 positive lymphocytes to mucosal lymphoid tissues. MAdCAM-1 also contributes to the firm adhesion step.

Figure 3 Molecules involved in lymphocyte extravasation. The most relevant endothelial glycoproteins mediating lymphocyte-endothelial cell interactions and their best characterized lymphocyte receptors are shown as receptor-ligand pairs. Their molecular families are also indicated, as well as the relative contribution of each receptor-ligand pair (the size of the black dot) in lymphocyte-endothelial cell interaction. (In addition to the molecules presented in this figure, there are individual reports describing involvement of many other molecules in the extravasation process). Ig, immunoglobulin; GAG, glucosaminoglycan.

Figure 3 Molecules involved in lymphocyte extravasation. The most relevant endothelial glycoproteins mediating lymphocyte-endothelial cell interactions and their best characterized lymphocyte receptors are shown as receptor-ligand pairs. Their molecular families are also indicated, as well as the relative contribution of each receptor-ligand pair (the size of the black dot) in lymphocyte-endothelial cell interaction. (In addition to the molecules presented in this figure, there are individual reports describing involvement of many other molecules in the extravasation process). Ig, immunoglobulin; GAG, glucosaminoglycan.

Other molecules, such as vascular adhesion protein-1 (VAP-1) and hyaluronan presented on endothelium, are also involved in the lymphocyte rolling step, especially in inflammatory conditions. VAP-1 is an endothelial adhesin that also possess enzymatic activity catalyzing oxidation of amines to hydrogen peroxidase, aldehyde, and ammonium. These end products regulate the functional status of endothelial cells. Hyaluronan, on the other hand, serves as an endothelial cell counterstructure for lymphocyte CD44.

lial cells in different vascular beds have certain degree of tissue specificity regarding chemokine expression. One example is the skin, where endothelial CCL17 attracts CCR4 bearing lymphocytes.

Firm Adhesion

Members of the immunoglobulin superfamily, intercellular adhesion molecules (ICAM-1, CD54, and ICAM-2, CD 102) and vascular adhesion molecule (VCAM-1, CD 106) on vascular endothelium serve as ligands for activated integrins on lymphocytes. ICAM-1 and ICAM-2 bind to lymphocyte activation associated antigen (LFA-1, CD11a/CD18) and VCAM-1 adheres to a401 (CD49d/CD29) on lymphocytes. Expression of ICAM-1 and VCAM-1 is upregulated at sites of inflammation, whereas synthesis of ICAM-2 is not influenced by inflammatory mediators. ICAM-1, ICAM-2, and VCAM-1 do not confer tissue specificity in the homing process.

Transmigration

In successful cases firm adhesion leads to the transmigration phase. The active contribution of endothelial cells at this step can be seen as rapid changes in their cytoskeletal organization, which are controlled, for example, by small GTPases. It is still under debate whether lymphocytes transmigrate through the endothelial cell or whether they penetrate through intercellular junctions.

Molecules preferentially localized at the junction, such as, CD31 and junctional adhesion molecules A, B and C (JAM-A, B and C), are involved in lymphocyte transmigration. Also ICAM-1/LFA-1 and VCAM-1/a401 pairs as well as VAP-1 can contribute to this process. It is obvious that a transmigrating lymphocyte needs to use proteolytic mechanisms to get through the vessel wall. Those as well as rapid repair mechanisms required to close the transmigration path remain to be elucidated.

Importance of the Endothelial Homing-Associated Molecules

Activation

Chemokines present on endothelial cells and their receptors on lymphocytes are thought to be the key players in the activation step. Their interaction leads to activation of integrins. Typically chemokines are small soluble molecules that need to be presented by endothelial proteoglycans to be available for rolling lymphocytes. Chemokines can be classified structurally into four groups: CXC, CC, CX3C, and C (C is cysteine and X can be any amino acid). The most important chemokine presented by HEV-like vessels is CCL21. Also CCL19 is involved in activating lymphocytes binding to HEV Both bind CCR7 on lymphocytes. Endothe-

Modern technology has provided us with the ability to produce mice in which one or more endothelial adhesion molecule(s) have been deleted. Phenotypes of the knockout mice lacking ICAM-1, ICAM-2, P-selectin, E-selectin, CD31, VCAM-1, and carbohydrate decorations of PNAd have been reported. All these mice (excluding VCAM-1 deficient mice) show variable defects either in normal lymphocyte homing or leukocyte trafficking to sites of inflammation or in both of these functions. Mice lacking VCAM-1, in contrast, are embryonic lethal because of the essential role of VCAM-1 in formation of umbilical cord and placenta. In general, the mice lacking only one endothelial homing-associated molecule do not show very severe phe-

notype. However, when more molecules are simultaneously deleted, the mice are no longer able to respond efficiently to inflammatory stimuli. This illustrates the marked redundancy in the function of the endothelial homing-associated molecules.

Endothelial Homing-Associated Molecules as Drug Targets

As endothelial cells in different vascular beds are at a key position to control lymphocyte accumulation in the inflamed tissues, homing-associated molecules on endothelial cells are potential targets when new anti-inflammatory drugs are developed. All homing-associated endothelial molecules mentioned in this chapter have been targeted with promising results in in vivo animal studies utilizing a variety of inflammatory models. Unfortunately, in clinical trials many of them have not fulfilled the expectations set based on the animal studies. However, many of them are still being tested in clinical trials. The excellent results obtained by targeting a4-integrin (present mainly on lymphocytes) in multiple sclerosis suggest that by targeting endothelial molecules equally good efficacy may be obtained in harmful inflammations.

Summary

Endothelial cells at different vascular beds are important in regulating lymphocyte entrance into the organs. Therefore, they are at a key position determining what types of immune reactions take place in different tissues of the body. Adhesion molecules present on the endothelium serve as counter-receptors for homing receptors expressed on circulating lymphocytes. The molecular signature of vascular endothelial cells in different tissues vary, and only those lymphocytes having the right repertoire of homing receptors for the given endothelial counter-molecules are able to enter a particular tissue. Inflammation induces and upregulates several homing-associated adhesion molecules on endothe-lium, allowing extensive traffic of massive number of lymphocytes into the tissues. Accumulation of lymphocytes can cause devastating symptoms in harmful inflammations as for example in diabetes, rheumatoid arthritis, and inflammatory bowel diseases. Therefore, homing-associated molecules at vascular endothelium are potential targets for drug development aiming at blocking abnormal lymphocyte trafficking.

Glossary

Chemokines: Small attractant molecules.

Extravasation: The process in which a cell migrates from the vasculature into the tissue site.

High endothelial venules: Specialized postcapillary vessels mediating lymphocyte entrance into the lymphoid organs.

Homing: Migration of recirculating lymphocytes from the bloodstream to particular lymphoid sites.

Lymphocyte recirculation: Continuous trafficking of a subset of white blood cells between the blood and lymphoid organs.

Further Reading

Butcher, E. C. (1991). Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity. Cell 67, 1033-1036. This classical review introduces the concept of the multistep adhesion cascade in leukocyte-endothelial cell interaction. Gowans, J. L., and Knight, E. J. (1964). The route of re-circulation of lymphocytes in rat. Proceedings of the Royal Society of London. 159, 257-282. This is the pioneering work that demonstrated the recirculation routes of lymphocytes and is considered as the beginning of the modern lymphocyte homing field. Hogg, N., Henderson, R., Leitinger, B., McDowall, A., Porter, J., and Stanley, P. (2002). Mechanisms contributing to the activity of integrins on leukocytes. Immunological Reviews 186, 164-171. Johnston, B., and Butcher, E. C. (2002). Chemokines in rapid leukocyte adhesion triggering and migration. Seminars in. Immunology 14, 83-92.

Lowe, J. B. (2003). Glycosylation in the control of selectin counter-receptor structure and function. Immunological Reviews 186, 1936.

Miller, D. H., Khan, O. A., Sheremata, W. A., Blumhardt, L. D., Rice, G. P., Libonati, M. A., Willmer-Hulme, A. J., Dalton, C. M., Miszkiel, K. A., and O'Connor, P. W. (2003). International Natalizumab Multiple Sclerosis Trial Group. A controlled trial of natalizumab for relapsing multiple sclerosis. New England Journal of Medicine 348, 15-23. The first clinical breakthrough using antibody therapy against an adhesion molecule.

Muller, W. A. (2003). Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trends in Immunology. 24, 327-334, 2003. Salmi, M., and Jalkanen, S. (1997). How do lymphocytes know where to go: Current concepts and enigmas of lymphocyte homing. Advances in. Immunology 64, 139-218. A comprehensive review of the history and progress in the field of lymphocyte—endothelial cell interactions. http://www.ncbi.nlm.nih.gov/PR0W/guide/45277084.htm A data bank and systematic nomenclature for molecules (CD classification). http://cytokine.medic.kumamoto-u.ac.jp/ A data bank for chemokines.

Capsule Biography

Sirpa Jalkanen, M.D., Ph.D., is Professor of the Finnish Academy and Professor of Immunology at University of Turku.

Marko Salmi, M.D., Ph.D., is Laboratory Director (Cell Trafficking) at National Public Health Institute in Turku. They have long experience in the field of leukocyte trafficking.

They lead the National Center of Excellence called Cell Trafficking that studies different aspects of cell migration in physiological and pathological conditions such as in harmful inflammations and cancer spread.

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