dendritic cell trafficking to lymph nodes through lymphatic ... - Nature

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DENDRITICCELL TRAFFICKING TO LYMPH NODES THROUGH LYMPHATIC VESSELS Gwendalyn J. Randolph*, Veronique Angeli* and Melody A. Swartz‡ Abstract | Antigen-presenting dendritic cells often acquire foreign antigens in peripheral tissues such as the skin. Optimal encounter with naive T cells for the presentation of these antigens requires that the dendritic cells migrate to draining lymph nodes through lymphatic vessels. In this article, we review important aspects of what is known about dendritic-cell trafficking into and through lymphatic vessels to lymph nodes. We present these findings in the context of information about lymphatic-vessel biology. Gaining a better understanding of the crosstalk between dendritic cells and lymphatic vessels during the migration of dendritic cells to lymph nodes is essential for future advances in manipulating dendritic-cell migration as a means to fine-tune immune responses in clinical settings.

*Department of Gene and Cell Medicine, Icahn Research Institute, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1496, New York, New York 10029, USA. ‡ Swiss Federal Institute of Technology, CH-1015 Lausanne, Switzerland. Correspondence to G.J.R. e-mail: gwendalyn. [email protected] doi:10.1038/nri1670

Dendritic cells (DCs) are widely accepted to be the most potent and versatile antigen-presenting cell in the immune system, owing to their superior capacity for acquiring and processing antigens for presentation to T cells and their potential to express high levels of the co-stimulatory molecules that drive T-cell activation1,2. In addition to efficiently inducing the activation and proliferation of naive T cells, they fine-tune immune responses by instructing T-cell differentiation and polarization. DCs transmit a distinct set of instructions to T cells that is based on their state of differentiation or maturation, and these instructions programme outcomes that range from humoral to cytolytic to suppressive (regulatory) T-cell responses3. DCs are highly mobile and are present in the right place at the right time for the regulation of immunity. They are positioned as sentinels in the periphery, where they frequently encounter foreign antigens, and they readily relocate to secondary lymphoid organs, particularly lymph nodes, to position themselves optimally for encounter with naive or central memory T cells. The trafficking of DCs to lymph nodes through afferent lymphatic vessels is crucial for the execution of their functions, so we review what is known about

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this process and emphasize what should be focused on to fill the gaps in our understanding of DC migration. Lymphatic vessels: conduits for migrating DCs

To arrive in lymph nodes, DCs must migrate towards, and enter, lymphatic vessels. But much remains to be learned about how the lymphatic system might control and affect DC migration. Structure and function of lymphatic vessels. Lymph flow is unidirectional from the periphery to the heart: the lymph absorbs interstitial fluid from tissues and, eventually, returns it to the blood circulation at the thoracic duct, passing through the lymph nodes en route4,5. The initial lymphatic vessels are blind-ended structures with wide lumina and thin walls, and therefore more closely resemble the sinusoidal vessels of the liver and spleen than the capillaries of the circulatory system6,7. These structures have also been called lymphatic capillaries, terminal lymphatic vessels and absorbing lymphatic vessels. They are lined continuously with overlapping lymphatic endothelial cells that are surrounded by little or no basement membrane and are highly endocytic and permeable to proteins8. In the skin, they form a plexus

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Resting

Filling

Anchoring filament

Capillary plexus

Smoothmuscle cell

Pre-collecting ducts Flow To lymph node Contract Fill Collecting lymphatic vessel

Figure 1 | Lymphatic-vessel organization in the skin. Initial lymphatic vessels are organized as a polygonal capillary plexus below the epidermis. These vessels do not contain smooth-muscle cells but are attached to the surrounding matrix through anchoring filaments. Deeper in the skin, these vessels converge into pre-collecting ducts, which drain into collecting vessels. These are surrounded by smooth-muscle cells and participate actively in propelling the lymph towards the lymph node. The micrograph shows the regular pattern of initial lymphatic vessels in a mouse tail, as viewed through the epidermis after administration of fluorescent lymphatic tracers.

that runs parallel to, and just beneath, the epidermis, and this consists of a hexagonal or polygonal pattern of vessels that have highly variable diameters of 10–80 µm and are spaced ∼100 µm apart4,5,9 (FIG. 1). Formation of the lymph is thought to occur through bulk swelling of the interstitium (that is, through the accumulation of fluid that occurs as a normal part of the circulation as fluid leaves the blood capillary) or through events that cause strain on the extracellular matrix, such as respiration, arterial pulsations and skeletal motion. This, in turn, ‘opens’ the initial lymphatic vessel through small fibres known as anchoring filaments, which attach lymphatic endothelium to the extracellular matrix7. The sudden increase in intraluminal volume causes a small and temporary negative pressure that draws fluid in. As the lymphatic vessel fills, its overlapping cell–cell junctions close, and intraluminal pressure returns to baseline. These events create a ‘tissue pump’, by which small interstitial stresses draw fluid into the lymphatic vessel but prevent its leakage back into the interstitium7,10,11; the overlapping cell–cell junction has been referred to as the primary valve system of the lymphatic vessel4.

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Initial lymphatic vessels converge into pre-collecting vessels, which are slightly larger ducts (100–220 µm in diameter) that drain the lymph to deeper regions of the dermis, into collecting vessels (FIG. 1). These collecting vessels are innervated and are surrounded by smoothmuscle cells, so they have intrinsic pumping activity 4,12,13. Flow of the lymph is regulated by these intrinsic pumps, as well as by passive extrinsic factors, such as movement of the surrounding tissue itself 4,13. The vessels consist of bulb-like segments, which are known as lymphangions, and these lymphangions are separated by valves and contract sequentially (FIG. 1). The valves in collecting lymphatic vessels allow unidirectional flow of molecules and cells, and they prevent transported cargo, molecular signals and leukocytes from flowing ‘upstream’ from the lymph node, to the periphery, instead of ‘downstream’4. So, molecules, such as chemokines, that are produced in the downstream lymph node would probably not be available to influence or communicate with DCs or lymphocytes that exit the peripheral tissue upstream. The interface of DCs with lymphatic vessels. At present, evidence indicates that DCs probably enter the lymph through initial lymphatic vessels14–18. It is uncertain whether DCs enter lymphatic vessels through gaps between neighbouring lymphatic endothelial cells, similar to the movement of fluid, or whether DCs squeeze themselves between closed junctions that separate lymphatic endothelial cells18. The molecular-weight cut-off for the entry of molecules or particulates to lymphatic vessels has not been precisely determined5,19, and it changes according to tissue conditions19. So, at present, it is difficult to determine whether DC passage across the lymphatic endothelium is passive, after DCs have approached the lymphatic vessel through chemotaxis, or whether molecular interactions occur between the endothelium and the DCs. Consistent with there being a requirement for molecular interactions between DCs and the lymphatic endothelium, there are data that point to a role for intercellular adhesion molecule 1 (ICAM1) — which is expressed not by DCs but by a peripheral cell type20 that might be lymphatic endothelial cells — in mediating the migration of DCs to lymph nodes20,21. Another adhesion molecule — junctional adhesion molecule 1 (JAM1), which is expressed by DCs and the lymphatic endothelium — also affects DC migration to lymph nodes22, because the absence of JAM1 expression by DCs facilitates their migration to lymph nodes22. However, it is not clear whether JAM1 expressed at the cell surface of DCs impedes their passage across the lymphatic endothelium, where it might support prolonged cell–cell interactions, or whether the increase in DC migration that is observed in the absence of JAM1 is associated with the positive regulation of β1-integrin expression by JAM1 REF. 23. The expression and activation of β1-integrins, which mediate the interaction of DCs with extracellular-matrix components24, might favour retention of DCs in the periphery.

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Table 1 | Assays to assess dendritic-cell migration Assay

Advantages

Disadvantages

References

Direct lymph-node cannulation: pseudo-afferent lymph for studies of multiple tissues in sheep, and mesenteric lymph in rats

Allows direct analysis of lymph DCs; allows steadystate analysis

Restricted to larger animals, for which there are few reagents available for DC characterization, as well as few genetically altered animals available

Ex vivo skin explants

Only assay readily available for analysis of naturally occurring human DC migration in a complex tissue; allows post-migratory analysis of DCs that have migrated; allows genetic studies using transgenic or gene-knockout mice

Poor viability of cultured tissue; evidence of discordance with in vivo studies in mice; lymphatic route of trafficking might not be the only means of emigration from cultures

16,17,20,45,121

FITC painting of skin

Allows genetic studies using transgenic or geneknockout mice; inducible with defined kinetics; readily quantifiable; elicits a robust migratory response; allows read-out of DC emigration from the epidermis in the same assay as DC appearance in the lymph node is assessed; coupled to induction of immune response for immunological read-out

Free FITC flows to lymph-node conduits; a few protocols might lead to DC labelling that does not indicate migration; a mixed population of DC types migrates, including Langerhans cells and poorly defined dermal DCs; does not allow direct analysis of DCs that enter or flow through the lymph

26,48,,49,117, 122,123

Interstitial injection of tumour-necrosis factor or lipopolysaccharide, or topical application of agents to skin

Allows genetic studies using transgenic or gene-knockout mice; coupled to induction of immune response for immunological read-out; inducible system that readily quantifies the rapid disappearance of DCs from the epidermis; limited studies in humans are possible

Does not allow direct analysis of DCs that enter or flow through the lymph; monitors DC disappearance from site of origin only

124–126

Particle-transport assay

Allows genetic studies using transgenic or geneknockout mice; coupled to induction of immune response for immunological read-out; inducible and quantifiable assay; allows distinct tracing of only monocyte-derived DCs; stable tracer within tracked cells

Does not allow direct analysis of DCs that enter or flow through the lymph; examines only monocyte-derived DCs; long experiments might result in passage of tracer between cells

118,127

Tagged protein uptake in lung, skin or other tissues

Allows genetic studies using transgenic or geneknockout mice; coupled to induction of immune response for immunological read-out

Does not allow direct analysis of DCs that enter or flow through the lymph; free flow of tracer to the lymph node might be a problem; mechanisms that induce migration are not well known (not known whether steady state or activation of DCs causes migration)

120,128

Adoptive transfer of DCs, such as those derived from bone marrow

Allows restriction of genetic modifiers to DC population; comparable to clinical protocols that transfer DCs to skin of patients

Does not allow direct analysis of DCs that enter or flow through the lymph; transferred cells migrate poorly

Gene-gun induction of DC migration

Coupled to induction of immune response for immunological read-out; allows restriction of genetic modifiers to DC population; traces DCs for full lifespan; potentially applicable to many species; allows genetic manipulation in various animals

Does not allow direct analysis of DCs that enter or flow through the lymph; mechanisms that induce migration in response to the gene gun are not known

35,38

46

129

DC, dendritic cell; FITC, fluorescein isothiocyanate.

At present, there is no information available about how the transit of DCs to lymph nodes (after DCs have entered a lymphatic vessel) is affected as the DCs flow from initial lymphatic vessels to collecting lymphatic vessels. It is also unclear whether the intrinsic pump function of collecting vessels has an important role in DC transport through the lymph. It is expected that the rate of DC transit in the lymph would increase as the flow of the lymph increases, given that total leukocyte counts in the lymph increase in situations when the flow increases19, but it is not clear whether the number of DCs that arrive in the lymph node would also increase. Several molecules that are known to promote the migration of DCs to lymph nodes, such as histamine25 and cysteinyl leukotrienes26, also increase lymph flow and pumping13, whereas other molecules that negatively regulate the migration of DCs27, such


as platelet-activating factor, decrease lymph flow13. Indeed, whether all or only some of the DCs that enter peripheral lymphatic vessels after a given stimulus arrive in the lymph node is unknown. Current methods for the analysis of migration TABLE 1 generally involve the assumption, perhaps reasonably but without firm evidence, that the input of DCs into the lymph is directly and linearly related to the output recovered in the lymph node. Research has not yet addressed the alternative possibilities that DCs might sometimes migrate out of lymphatic vessels after they have entered or that they might accumulate for a period in these vessels and die before arrival in the lymph node. As discussed later, data from model systems, such as plt (paucity of lymphnode T cells) mice are consistent with, but do not prove, the idea that some DCs might enter lymphatic vessels but fail to arrive in lymph nodes.



REVIEWS vessels directly correlates with alterations in the magnitude of DC accumulation in lymph nodes (V.A. and G.J.R., unpublished observations). Initial lymphatic vessel Lymph-node subcapsule Penetrating terminal afferent Flow lymphatic vessel

Collecting lymphatic vessel

Lymphnode capsule

Dendritic cell

T-cell zone

Typical terminal lymphatic vessel in mice: branching and terminating in subcapsular sinus

B-cell follicle

Figure 2 | Terminal afferent lymphatic vessels in lymph nodes. Terminal afferent lymphatic vessels are seen as highly tree-like structures that emanate from the small number of afferent collecting vessels that connect with a lymph node. In mice, these vessels typically terminate in the subcapsular sinus (right), but they have been observed to penetrate deeply into the lymph node in other species (left). Because these terminal lymphatic vessels deliver dendritic cells to lymph nodes, their architecture might affect the efficiency and location of dendritic-cell entry.

TWOPHOTON MICROSCOPY

A fluorescence-imaging technique that takes advantage of the fact that fluorescent molecules can absorb two photons simultaneously during excitation, before they emit light. This technique greatly reduces photodamage of living specimens, improves depth of tissue penetration, allows distinct separation between excitation and emission wavelengths, and confines excitation to a discrete focal point. CONTACT ELICITATION

The inflammatory immune reactions that occur at the site of exposure after contact with a sensitizing antigen. These reactions occur after second and subsequent exposures to a particular sensitizing antigen, and they involve the recruitment and responses of effector T cells.

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DCs are delivered to the lymph node in pre-nodal lymphatic vessels of the collecting vessel type (FIG. 2). It is not clear how DCs make their way from there to the T-cell zone of the lymph-node cortex. As it is now possible to visualize the migration of DCs within lymph nodes, through the use of TWOPHOTON MICROSCOPY28,29, research on this topic is likely to be fruitful soon. The basic view of how lymphatic vessels converge with the structure of the lymph node, particularly in mice, is that only a few collecting vessels enter and terminate in the subcapsular sinus of the lymph node30 but that these few collecting vessels branch extensively at, and within, the sinus31 (FIG. 2). In other species, these branched terminal afferent lymphatic vessels penetrate deep into the structure of the lymph node31–34. Differences in how lymphatic vessels enter the lymph node and where they terminate in the lymph node could markedly influence not only where in the lymph node DCs enter but also how efficiently they enter the lymph node and interface with lymphocytes. Although mouse lymph nodes are thought to be simple with regard to lymphatic-vessel entry compared with the lymph nodes of other species, complexity of lymphatic-vessel structure can develop in mouse lymph nodes, and this expansion of lymphatic

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Cells in the afferent lymph

Descriptions of DC emigration from peripheral tissues into lymphatic vessels preceded the identification of DCs as a distinct cell type. So, early characterization of the cellular content of the lymph described ‘veiled’ macrophages in the afferent lymph35. The first studies were carried out in cannulated lymphatic vessels of normal sheep and showed that, typically, ∼20% of the cells were veiled cells of the mononuclear phagocytic lineage, and most of the remaining cells were lymphocytes35. By the early 1980s, it was clear that the term veiled corresponded to the numerous cytoplasmic extensions for which DCs received their name and that cells trafficking in the afferent lymph were more frequently DCs than macrophages. This led to our current knowledge that DCs36–40, together with CD4+ memory T cells41, selectively and efficiently traffic through the lymph. Although it is now evident that historical descriptions of ‘macrophage’ transit through the lymph were often based on observations of mature DCs, the lymph also contains cells that closely resemble immature DCs, macrophages and/or monocytes. For example, in humans, the lymph draining the skin contains cells that express both CD14 and CD36, markers that are co-expressed by monocytes, macrophages and immature DCs but not by mature DCs; it also contains cells that express CD1a, a marker of epidermal DCs, which are known as Langerhans cells42. The characteristics of the cellular content of the afferent lymph that we have described so far pertain particularly to homeostatic conditions (that is, steadystate conditions). The relative restriction of cellular emigrants in the lymph to DCs and T cells does not apply in marked inflammatory states. Following 37 CONTACT ELICITATION , chronic inflammation induced by vigorous immunization14 or allograft transplantation43, a large flux of neutrophils (and even some erythrocytes) through the afferent lymph is observed. It is not known precisely how these cells gain entry to lymphatic vessels or whether neutrophils express specific lymphatic homing or adhesion molecules, but the presence of erythrocytes indicates non-specific and/or passive cell uptake into the lymph during inflammation. However, under both homeostatic and pro-inflammatory conditions, mouse DCs need to express their main lymph-nodehoming chemokine receptor, CC-chemokine receptor 7 (CCR7), to migrate to lymph nodes through peripheral lymphatic vessels44–46. So, the possibility that a common set of molecular events controls how different cell types enter the lymph seems unlikely, but this remains to be determined. Although neutrophils and macrophages can occasionally be found in the lymph, the biological consequence of these cells trafficking to draining lymph nodes is unknown, whereas it is clear that DCs traffic through lymphatic vessels to present antigen in lymph nodes. Therefore, here, we concentrate on the trafficking of DCs through the lymphatic system.



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b

FITC solution

Epidermis

a

FITC-labelled Langerhans cell

Langerhans cell

Blood vessel

Dermis

Monocytic precursor

FITC-labelled dermal DC

Monocyte Dermal DC

Particle

Lymphatic vessel

c

Figure 3 | Tracing dendritic-cell migration to lymph nodes. a | The diversity of dendritic-cell (DC) populations in the skin under homeostatic conditions is shown. Langerhans cells reside in the epidermis, and there is a distinct population of resident dermal DCs. In addition, precursors from the blood, including monocytes, constitutively traffic from the blood to the skin, where some become DCs. Under homeostatic conditions, the origins of the DCs present in lymphatic vessels are not fully defined, but these DCs include a small number of Langerhans cells. b | When fluorescein isothiocyanate (FITC) dissolved in a contact-sensitization solution is applied to the skin (known as FITC painting), labelling of Langerhans cells and resident dermal DCs occurs117. By contrast, 1 µm particles (red) that are injected into the skin are engulfed by newly recruited monocytes76,118. c | The left panel shows that, after FITC (green) painting, CD11c+ DCs (red) can be identified in the lymph node within 18 hours. The centre panel shows that, in mice with an impaired capacity for DC migration (a deficiency in multiple-drug-resistance-associated protein 1 (also known as ABCC1)26 in this case), free FITC that drains from the skin through the lymphatic vessels accumulates in lymph-node conduits119, but CD11c+ DCs are rarely heavily labelled, which is consistent with the concept that DCs migrating from the periphery bring in most of the peripheral antigens that are presented in the lymph node120. The right panel shows the accumulation of CD205+ DCs (red) containing microspheres (green) that were engulfed in the skin before transport to the T-cell zone of the draining lymph node.

Assays to study DC migration

Most of what is known about the mechanisms of DC migration to lymph nodes is derived from studies of the mobilization of DCs from the skin TABLE 1; FIG. 3). DCs in the skin are diverse: some are resident in the dermis; some are resident in the epidermis; and others are recruited from the blood and remain only transiently in the skin before entering lymphatic vessels. To a certain extent, different assays target different DC populations that emigrate from the skin to the lymph nodes TABLE 1; FIG. 3). The subpopulation that is confined to the epidermis, known as Langerhans cells, has been investigated in


the most detail with regard to migration to lymph nodes. It is often assumed that DCs found in the dermis and other tissue sites have similar basic properties to prototypical Langerhans cells, including the mechanism and regulation of migration. In terms of maturation and antigen presentation, however, the behaviour of Langerhans cells does not hold for all other types of lymphoid-organ DC in mice47. Therefore, it remains to be determined whether the molecular events involved in DC migration that we discuss here are widely applicable to a variety of organ systems or are largely restricted in their relevance to skin DCs.



REVIEWS Most of our knowledge of how DCs migrate from the skin is derived from the FLUORESCEIN ISOTHIOCYANATE FITCPAINTING ASSAY TABLE 1; FIG. 3), which involves epicutaneous application of FITC mixed with a CONTACT 48,49 SENSITIZER . The robust migration of DCs that is coupled to the induction of contact sensitivity in this assay probably accounts, in part, for it having a greater appeal than other assays. However, it is best used when compared side-by-side with other assays that test DC migration in the skin, as each type of assay has inherent advantages and disadvantages TABLE 1. Unfortunately, in contrast to direct analysis of afferent lymph DCs in larger animals, such as sheep and rats35,38, none of the assays that is used in mice to trace DC migration to lymph nodes can analyse DCs in afferent lymphatic vessels, owing to the small size of these vessels TABLE 1. Instead, conclusions about the events that regulate mouse DC migration through lymphatic vessels are based on the analysis of traced DCs that disappear from discrete peripheral sites, such as the epidermis, and then reappear in the draining lymph node. Consequently, there is a large gap in our understanding of how DCs directly interact with, enter and travel through lymphatic vessels to finally become positioned in a lymph node. That mouse models are more popular than rat models, despite considerable technical limitations, probably stems from the greater availability of genetically altered mice and reagents for characterizing mouse DCs. Molecules for migration expressed by DCs

FLUORESCEIN ISOTHIOCYANATE PAINTING ASSAY

(FITC painting). An experimental assay of contact sensitization in mice. In this assay, the contact-sensitizing substances are dibutyl phthalate and the fluorochrome FITC, which also functions as a migration tracer. The application of this mixture to the skin, in an equal volume of acetone, is often called painting. CONTACT SENSITIZATION

The initial reaction that occurs after the first exposure to a ‘sensitizer’ hapten or antigen. This step requires dendritic-cell migration to lymph nodes to prime contact-antigen-specific T cells.

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The mobilization of DCs from the periphery to the lymph nodes (FIG. 2) is regulated by the ‘gate-keeper’ chemokine receptor CCR7. After it was recognized that CCR7 expression is induced together with the maturation of human DCs50–53, while the expression of other chemokine receptors is downregulated50,53, study of CCR7-deficient mice revealed a marked defect in DC migration to lymph nodes44,45. Evidence that DCs themselves need to express CCR7 to migrate to lymph nodes, rather than the possibility that the absence of CCR7 on another cell type indirectly affects DC trafficking, came from adoptive transfer of CCR7deficient DCs to normal CCR7+ hosts46. Transfer of CCR7-deficient DCs results in the recovery of less than one-tenth the number of DCs from the lymph node compared with transfer of CCR7+ DCs46. Expression of CCR7 alone, however, is not sufficient for DC migration, as it can be expressed in a biologically ‘insensitive’ state such that CCR7+ DCs either fail to undergo chemotaxis towards CCR7 ligands54 or require a high concentration of CCR7 ligands before they respond26. Signals that are expected to be found at sites of inflammation — including the lipid mediators cysteinyl leukotrienes and prostaglandin E2 REFS 26,54,55, and the ADP-ribosyl cyclase CD38 REF. 56 — are required to sensitize CCR7 to its ligands CC-chemokine ligand 19 (CCL19) and CCL21 REFS 57,58. The mechanisms by which these mediators alter CCR7 functionality is not known, but they probably trigger signalling events that, in turn, alter the signalling cascades that are engaged when CCR7 binds its ligands59.

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The CCR7 promoter has not been characterized, and little is known about how CCR7 gene expression is regulated. Although its expression typically correlates with the upregulation of MHC and co-stimulatorymolecule expression that occurs when DCs mature50–53, there are stimuli that can induce CCR7 expression by DCs independently of maturation. For example, the uptake of apoptotic cells by DCs induces CCR7 expression and DC chemotaxis in response to CCR7 ligands, but it does not induce DC maturation 60 . At the molecular level, activation of the signalling adaptor protein DAP12 (DNAX activation protein 12) induces CCR7 expression without full DC maturation61. This might be relevant to the control of DC migration under steady-state conditions, because homeostatic accumulation of DCs in peripheral sites (including the skin and gut mucosa) is observed in mice that lack DAP12 REFS 62,63. DC migration from the periphery to the T-cell zone of lymphoid organs is a process that is distinct not only from DC maturation but also from other events that require reorganization of the plasma membrane. For example, RAC1 was recently shown to be required in vivo for phagocytosis by DCs, including the uptake of apoptotic cells, but absence of this RHO (RAS homologue) GTPase in DCs did not affect either migration from the skin to the lymph nodes or movement from the marginal zone to the white pulp of the spleen64. This failure to uncover a role for RAC1 is likely to be explained by the possibility that RAC2 could instead function to promote migration or by the possibility that RAC1 and RAC2 have redundant functions in the support of DC migration, because the absence of both RAC1 and RAC2 strongly impairs the extension of dendrites by DCs and the mobilization of DCs to lymph nodes65. Integrins are involved in the migration of many cell types. Data indicate that the integrin lymphocyte function-associated antigen 1 (LFA1; αLβ2) is a mediator of DC migration to lymph nodes21, which is consistent with studies indicating that DCs that lack β2-integrin are partially impaired in their migration to lymph nodes20. Pairing of the α6-integrin chain with an undefined β-integrin subunit mediates DC escape from the epidermis, probably through binding laminin in the basement membrane of the epidermis66. Potential roles for other integrins in DC migration from peripheral sites have not yet been determined. Consequently, intrinsic regulators of integrin activation also affect DC migration from the periphery to the lymph nodes. For example, activation through chemokine receptors stimulates the activity of RAP1 REF. 67 and its effector RAPL68, which leads RAP1 to form a complex with LFA1 and α4β1-integrin, and this facilitates the adhesive activity of these, and possibly other, integrins. Chemokines and DC entry to lymphatic vessels

The prevailing model to explain how CCR7+ DCs arrive at peripheral lymphatic vessels is that they respond to a chemotactic gradient of CCR7 ligands, CCL19 and/or CCL21, that originates from the



REVIEWS lymphatic vessel. In an early study of these chemokines, CCL21 was observed to be expressed on small lymphatic vessels in the liver69. The genes encoding CCL19 and CCL21 have been duplicated and modified during evolution such that there is now more than one gene encoding each chemokine. In mice, a chemokine gradient towards the lymphatic vessel probably partly comprises CCL21-Leu (which contains a leucine residue at position 65), because the expression of this form of CCL21 has been shown on vessels in the skin that seem to be lymphatic vessels70. Furthermore, CCL21-Leu is generally known to be distributed in peripheral tissues but not in lymph nodes70,71. It is distinguished from CCL21-Ser, which is encoded by a separate gene and contains a serine residue in place of the leucine residue71. In contrast to CCL21-Leu, CCL21-Ser is expressed in lymph nodes but not in afferent lymphatic vessels or by other cell types in the periphery70,71 (FIG. 4a).

a

In a naturally occurring mouse mutant, the plt mouse, some of the genes that encode CCL19 and the CCL21 variants are absent, as part of a chromosomal abnormality71–74, and DC migration to lymph nodes is greatly impaired in these mice after FITC painting72, lipopolysaccharide administration75 or microsphere injection76. It is now evident that plt mice lack expression of CCL19 protein73,74 but retain expression of peripherally expressed CCL21-Leu, apparently on lymphatic vessels70, while lacking lymph-node CCL21Ser71 (FIG. 4b). As well as the defect in DC migration that has been reported for plt mice, there is other evidence that CCL19 and CCL21 are each required for the mobilization of peripheral DCs to the lymph node26,77,78. For example, CCL19 is required to prime allogeneic T cells in vivo79, whereas other studies indicate that CCL21 is also required for T-cell priming78. CCL19, but not CCL21, participates in the extension of dendrites by DCs80, which in turn might affect migratory responses.

b

Initial lymphatic vessel

CCL21-Leu

CCL19-non-expressing DC

CCL21-Leu

CCL19-expressing DC

Collecting lymphatic vessel

Lymph-node subcapsule Lymph-node capsule

CCL21-Ser

T-cell zone

B-cell follicle

Figure 4 | Expression of CC-chemokine-receptor-7 ligands and fate of dendritic cells in wild-type and plt mice. a | The CC-chemokine receptor 7 (CCR7) ligand CC-chemokine ligand 19 (CCL19) is expressed by dendritic cells (DCs) after their maturation. In mice, there are two known functional genes that encode CCL21. One form of CCL21 — CCL21-Leu (purple) — is expressed in the periphery, at a minimum by initial lymphatic vessels. The other form of CCL21 — CCL21-Ser (red) — is expressed in lymph nodes, including in the terminal lymphatic vessels that are present in the subcapsular sinus. Which of these CCL21 gene products is expressed by collecting lymphatic vessels (whether either or both) is not clear. b | Functional CCL19 expression by DCs is abrogated in plt (paucity of lymph-node T cells) mice. In addition, CCL21-Ser, but not CCL21-Leu, is absent. Peripheral DCs migrate poorly to the T-cell zone of lymph nodes in plt mice, but some DCs aberrantly accumulate at the subcapsular sinus.




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Static

Flow

Figure 5 | Potential mechanism of flow-enhanced autologous chemotaxis or secreted matrixmetalloproteinase redistribution. Shown is an idealized steady-state concentration profile of a secreted protease or chemokine surrounding a cell (centred in the red ring) of 15 µm in diameter and under interstitial flow of 4 µm per second, with a diffusion coefficient of 7 × 10–6 cm2 per second. (Interactions with the extracellular matrix and transient changes are not considered.) The colour indicates the concentration range of the secreted molecule, from 100% of the secreted concentration (red) to 0% (dark blue).

Although these chemokines clearly support DC and T-cell migration and facilitate T-cell priming, at least some immune responses can eventually by-pass a requirement for the CCR7-ligand–CCR7 pathway 81. From the current scientific literature, it is not possible to be sure that either CCL19 or CCL21 functions according to the model in which they are expressed by lymphatic endothelium to promote entry of DCs to the lymphatic vessel. Regarding CCL19, it has not been determined whether this chemokine is expressed by lymphatic vessels. Regarding CCL21, plt mice usually express CCL21 (CCL21-Leu) in the lymphatic vessels but still have a deficit in the appearance of skin DCs72 and monocyte-derived DCs76 in the lymph node. It is not known whether DCs enter the lymphatic vessels in normal numbers in plt mice. But if they do enter the lymphatic vessels in response to CCL21-Leu, they cannot be recovered from the lymph nodes in plt mice, although the appearance of a few DCs in or near the lymph-node subcapsular sinus has been observed. This is consistent with the possibility that, in plt mice, some DCs arrive at the lymph node but do not proceed to the T-cell zone82 (FIG. 4b). However, if most of the DCs in plt mice arrive at the subcapsular sinus in a normal manner and simply fail to mobilize to the T-cell zone, as has been argued83, one would expect the total number of traced DCs in lymph nodes to be relatively normal and only the distribution of DCs to be altered. Because this is not the case, and instead there are far fewer traced DCs observed in whole lymph-node suspensions72, it seems most probable that the main defect in DC migration in plt mice is upstream of the lymph-node subcapsular sinus, but it is uncertain at what point upstream of the sinus the defect occurs. Because initial lymphatic vessels seem to express CCL21-Leu70, and the subcapsular-sinus lymphatic vessels that are, ultimately, continuous with those in the periphery express CCL21-Ser74, somewhere on the

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way to the lymph node, lymphatic vessels must begin to express CCL21-Ser (which is encoded by the gene that plt mice lack) instead of CCL21-Leu. Where does this transition take place, and do lymphatic vessels need to express DC attractants (such as CCL21) for the entire length of the vessel to promote DC arrival in the lymph node? In humans, there is evidence that, similar to initial lymphatic vessels, collecting lymphatic vessels express CCL21 REF. 84, but the role of collecting-vessel- or subcapsular-sinus-expressed CCL21 in the delivery of DCs to lymph nodes has not been explored. It is clear, therefore, that DC migration from the periphery to the lymph nodes depends on CCR7 and its ligands; however, although the hypothesis that lymphatic-vessel expression of CCR7 ligands guides DCs towards these vessels for subsequent entry is appealing, this has not been shown. When DCs become activated for maturation and migration, they begin to secrete chemokines, including CCL19 REFS 8587, so it is possible that DCs might use autocrine mechanisms of CCR7-dependent migration to lymph nodes. However, the popular, but as yet unproven, concept that leukocytes migrate along gradients of chemokines in vivo seems to counter this idea, and the question arises of how autologous chemokines could lead to efficient migration into lymphatic vessels. Here, it is of interest to consider the possibility that the biophysical environment can influence the complex signalling events that direct DC trafficking towards lymphatic vessels. Because initial lymphatic vessels drain interstitial fluid, there would be a small flow field around any migrating DC (similar to other migrating cells), and this field would necessarily be orientated in the direction of the nearest initial lymphatic vessel88,89. These flow fields are sometimes referred to as pre-lymphatic channels4. Depending on the velocity of flow and the size of the molecule, this flow field is likely to alter the distribution of any cell-secreted molecule and bias it downstream of the cell89. The larger the molecule, the slower its diffusion coefficient and the stronger the effect of interstitial flow on its transport away from the cell in the direction of flow. The local extracellular gradients of small chemokines (such as CCL19), which have diffusion coefficients in the order of 10–6 cm2 per second, will be weakly influenced by typical interstitial flow velocities, which are in the order of 1 µm per second90 near the cell. But convection in vivo will skew the transport of these molecules for distances that are greater than the diameter of a cell89 (FIG. 5). In turn, any asymmetry in the local gradient might help to drive chemotaxis of the cell to autocrine factors in the direction of flow or towards the lymphatic vessel, as this mechanism would be expected to lead to a higher occupancy of chemokine receptors at the leading edge of the migrating cell relative to the trailing rear, even when secretion of the chemokine itself is not polarized. However, it has not yet been explored whether asymmetry in autologous chemokine gradients can induce directed cell migration.



REVIEWS Along the same lines of reasoning, interstitial flow can skew secreted (soluble) matrix proteinases in the direction of the lymphatic vessel (FIG. 5). Because DC migration to lymph nodes is known to require matrix metalloproteinase 2 (MMP2) and MMP9 REF. 91, which have molecular weights in the range 72–92 kDa, this effect would be markedly greater than that seen for small chemokines. This would lead to preferential matrix degradation downstream (towards the lymphatic vessel) and therefore to preferential ease of migration towards the lymphatic vessel91. DC-subpopulation-specific mediators. In general, the assays that are in use at present for the study of DC migration focus on skin-resident DCs (such as Langerhans cells) or DCs derived in vitro from bonemarrow precursors TABLE 1, and they rarely take into account the possibility that different migratory events occur in the mobilization of different DC subsets to the lymph node47. Several DC subsets have been described in mice, but it remains difficult to identify their respective blood precursors and to trace the migratory routes and pathways of each subset as it is recruited into peripheral tissues and then homes to lymph nodes through lymphatic vessels. For example, the subset of DCs known as plasmacytoid DCs92 can be recruited to the skin by chemokines93 or chemokine-like molecules94,95, but the homing of plasmacytoid DCs to the lymph nodes through lymphatic vessels has not been traced. Other studies have characterized molecules that are used by the monocyte-derived subset of DCs as they migrate to lymph nodes. Similar to skin DCs, monocyte-derived DCs heavily rely on the CCR7ligand–CCR7 pathway for mobilization to lymph nodes76, but other mediators that regulate migration to lymph nodes seem to more specifically influence this subset. For example, CCR8 mediates migration of monocyte-derived DCs to lymph nodes, but it does not affect migration of skin-derived DCs that respond to FITC painting76.

node and results in them ‘spilling over’ into the efferent lymph104. In the same study, the contact sensitizer 2,4,6-trinitrochlorobenzene (TNCB), which also induces migration of skin DCs, did not result in the appearance of DCs in the efferent lymph104. In another study, subcutaneous beryllium, functioning as a potent adjuvant, led to substantial numbers of macrophages and DCs entering the efferent lymph, even while the lymph node itself underwent considerable remodelling, including a fibrotic response105. Although these conditions are not physiological, the possibility that other strong adjuvants induce a similar response, even to a lesser degree, cannot be eliminated. Calculations of DC migration to lymph nodes do not typically take into account the loss of DCs through the efferent lymph, because the output of DCs in the efferent lymph, even in cases in which stimulants such as DMBA are used, would have only minimal impact on these calculations. In the presence of DMBA, only ∼10% of the DCs that entered the lymph node exited by the efferent route104, and this proportion is substantially lower under other conditions101–103. However, the biological consequences of releasing DCs into the bloodstream, which would occur because of the convergence of the efferent thoracic lymph duct with the bloodstream, might markedly affect the induction of immune responses in distal tissues and the spread of pathogens that DCs might harbour. For example, in some cases, injection of DCs into the skin can lead to priming of T cells in the spleen, a result that strongly implies that DCs can access the bloodstream from the skin or skin-draining lymph nodes106. Furthermore, Mycobacterium tuberculosis, Salmonella spp. and antigens derived from these bacteria spread from the initial sites of bacterial entry to distal organs, such as the spleen, through a haematogenous route107,108. This dissemination probably involves being carried by antigen-presenting cells that first migrate to the lymph nodes and then enter the efferent lymph to gain access, ultimately, to the blood107.

Beyond the lymph node

Immunomodulation and DC trafficking

It is widely thought that, after DCs migrate to, and integrate into, the lymph node, they die locally96–98 and do not leave through efferent lymphatic vessels, which is in contrast to the movement of T cells36,99,100. This idea is partly based on the early failure to identify DCs (or macrophages) in the efferent lymph. However, although DCs might be rare in the efferent lymph, they are not absent. If one accepts that cells in the lymph that were originally called macrophages are, in fact, DCs, then reports from the late 1970s and early 1980s show that normal efferent lymph contains macrophages and DCs but that these cells typically comprise less than 0.1% of total efferent cellular output101–103. Under conditions in which DC migration from the periphery is strongly induced, such as inflammation, the efferent output of DCs can, however, increase substantially. Epicutaneous application of the carcinogenic chemical 7,12-dimethylbenz(a)anthracene (DMBA) greatly augments the migration of DCs to the lymph

So far, we have discussed the various molecules that regulate the migration of DCs to lymph nodes, and we have discussed how DCs interface with the lymphatic system during their migration. In several clinical settings, it would be appealing to take advantage of the knowledge that we have gained about the migratory properties of DCs to design strategies for their delivery to lymph nodes to effect immunomodulation: for example, for induction of an antitumour immune response109. Indeed, several clinical trials have been carried out or are in progress to determine the efficacy of therapeutic vaccines that use ex vivo-matured DCs as the main component109. Administration of these DCs to patients is typically carried out by intradermal adoptive transfer110, which resembles the assay for studying the migration of bone-marrow-derived DCs to lymph nodes TABLE 1. As is the case for bone-marrow-derived DCs, only a small proportion of the injected DCs subsequently migrate to the lymph node110, and this poor




REVIEWS migratory response might directly limit the efficacy of the vaccine109,110. In mouse models, conditioning the site of injection with adjuvants or cytokines, or previous injection of DCs themselves, is an efficacious means of enhancing the migration of transferred DCs to lymph nodes46. The mechanism of this enhancement might involve increased expression of CCL21 by lymphatic vessels46, but there is no direct evidence to confirm this possibility. If more of the events that regulate DC migration into lymphatic vessels were defined, a wider range of rational approaches to improve the migration of DCs could be evaluated. In contrast to trials that aim to treat cancer, which attempt to boost immunity by transferring mature, migratory DCs, other diseases and clinical protocols — including autoimmunity and transplantation — might benefit from tipping the balance in favour of inducing immune tolerance109. Considering the evidence that relatively immature DCs can induce and/or maintain peripheral tolerance111, clinical methods to promote the mobilization of immature DCs that display antigens of interest to lymph nodes seem attractive. Although it is clear that relatively immature DCs can migrate through the lymph42,112, our knowledge of how their emigration is regulated is more limited than our understanding of the migration of mature DCs. At present, there are no known methods for selectively inducing the emigration of immature DCs. Potential alternative strategies to dampen immune responses in the setting of autoimmunity or transplantation by interfering with the migration of mature DCs should be regarded with caution. When DCs can still

1.

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mature but fail to migrate to lymph nodes normally, inflammation might occur in the periphery and in the draining lymph nodes27. Indeed, the failure of DCs to migrate to lymph nodes normally might exacerbate local immune and inflammatory responses27, because the activated DCs remain in peripheral tissue and can produce pro-inflammatory mediators and attractants for other leukocytes, thereby sustaining the local reaction. It is possible that inflammatory pathologies in the skin might be inherently associated either with DCs that are not competent to migrate or with lymphatic vessels that cannot direct DC migration113. Finally, controlling DC migration in the context of transplantation could be particularly challenging, because CCR7 is probably not required for the passage of DCs from a transplanted tissue to a secondary lymphoid organ. Following transplantation, DCs can enter the bloodstream directly from the transplant, apparently by passing into blood vessels directly114,115. Given that CCR7 ligands are only rarely expressed by the endothelium of blood vessels116, it is unlikely that this trafficking occurs through CCR7-dependent entry to lymphatic vessels. The molecules that mediate ‘reverse transmigration’ into the blood have not been defined, so the feasibility of interfering with DC re-entry to the bloodstream is low at present. On the whole, therefore, modulating immunity for clinical benefit by controlling the mobilization of DCs from tissues to lymphoid organs is an approach that holds promise, but fulfilment of this promise will require considerably more research to achieve a broader understanding of the applicable mechanisms that regulate DC migration.

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Acknowledgements We thank M. Fleury for producing figure 5.

Competing interests statement The authors declare no competing financial interests.

Online links DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene CCL19 | CCL21 | CCR7 | ICAM1 | JAM1 | LFA1 | MMP2 | MMP9 Access to this interactive links box is free online.

