In addition to the adhesion machinery described in the preceding sections, leucocytes require orchestration from chemokines and other chemoattractant molecules not only to migrate through the interstitial spaces towards lymphatic vessels, but to guide them towards gaps in the basal lamina to trigger their arrest beneath the endothelium and to direct their polarity for diapedesis. Lymphatic endothelial cells themselves provide many of these cues through their synthesis of a wide range of different chemokines (reviewed in [52
]) including the immature/mature DC, monocyte and T cell attractants CCL2 (MCP-1), CCL5 (RANTES), CCL20 (MIP 1α) and CCL21 (secondary lymphoid chemokine), and the neutrophil attractants CXCL1 (Groα), CXCL2 (Groβ), CXCL5 (ENA-78) and CXCL8 (IL-8), all of which are upregulated upon stimulation with inflammatory cytokines [5
]. Hence in terms of chemotaxis, lymphatic vessels display a pleiotropic response to inflammation and can in principle attract the recruitment of almost any tissue migrating leucocyte subpopulation.
Of these many chemokines, the most widely studied is CCL21, which is expressed primarily in lymphatic endothelium, and its G protein coupled receptor CCR7 expressed by DC and certain other migratory leucocytes. Indeed CCR7 and its ligands has also been reported to direct the trafficking of effector memory T cells from skin and lung via
afferent lymphatics [10
] and just recently, the lymphatic trafficking of subpopulations of neutrophils in skin [7
]. Mice lacking CCR7 had delayed T cell immune responses and abnormal skin contact hypersensitivity responses in which Langerhans cells fail to exit the skin through afferent lymphatic vessels and migrate to draining lymph nodes [53
]. Similarly, exerting blockade of CCL21 by administering neutralizing mAbs impaired DC trafficking in skin lymphatics [55
]. Mice unlike humans express two different isoforms of the chemokine, CCL21ser and CCL21leu that differ by a single residue, are encoded by separate genes and localize exclusively to lymph node sinuses or peripheral lymphatics respectively [56
]. Notably, plt
mice, which carry a deletion of CCL21 leu, have a paucity of lymph node T cells and impaired immune responsiveness, consistent with a requirement for lymphatic endothelial derived CCL21 for entry and trafficking of antigen loaded DC.
Normally stored within intracellular vesicles and secreted at low levels by resting LEC, CCL21 is transcriptionally upregulated by inflammatory cytokines such as TNFα. After secretion, CCL21 induces both chemotaxis of DC towards lymphatic capillaries and ICAM-1 dependent adhesion to the endothelium via
conformational activation of β2 integrins on the DC [59
]. In common with other chemokines, CCL21 has a heparin-binding tract and is sequestered by heparan sulphate proteoglycans and collagen in the extracellular matrix (see below). Indeed this property appears to be critical in facilitating translymphatic migration as well as permitting microscopic imaging of secreted CCL21 in and around initial lymphatic capillaries in tissues.
Intravital imaging of resting and inflamed skin in the mouse footpad revealed the presence of discrete puncta of secreted CCL21 on the basolateral surface of initial lymphatics, close to the flap-like openings at button junctions, where it is tethered by collagen IV within the loose subendothelial matrix [60
]. Curiously, migrating DC were observed to extend filopodia towards these puncta after which they docked to the underlying lymphatic endothelium and transmigrated. In contrast, CCR7 deficient DC migrated past the CCL21 puncta and failed to dock [60
]. These findings suggest that CCL21 prompts the arrest and adhesion of DC at lymphatic vessels rather than exerting fluid phase chemotaxis, transiently docking the migrating cells next to perforations in the loose basal lamina from which they can initiate transendothelial migration.
A separate study using intravital microscopy to image DC trafficking in the more superficial layers of unstimulated mouse ear skin explants, revealed CCL21 to be distributed in the perilymphatic interstitium along a steep gradient that decayed with distance from the lymphatic capillary wall [61
]. Furthermore, migratory DC in the skin explants were observed to change from random to directional movement at a point some 90 µm from the vessel wall, the maximal effective distance that the gradient was estimated to extend from the vessel perimeter. Importantly, the CCL21 gradient was not disrupted by washing the explants or by short-term exposure to brefeldin A, an inhibitor of membrane re-cycling, but was flattened by adding exogenous CCL21 or by enzymatic digestion with heparitinase. The discrepancies in these two studies seem to indicate subtle differences in the distribution of CCL21 in the upper and lower dermal layers due to immobilization on different matrix components [60
]. However both studies imply a haptotactic rather than chemotactic function in which CCL21 signals adhesion close to the basolateral surface of lymphatic capillaries and subsequent transendothelial migration, presumably by activating β2 integrins in DC through CCR7 engagement and inside out signaling. How these features can be reconciled with a model of DC migration that relies purely on amoeboid movement is unclear [25
]. Indeed, conformational activation of β2 integrins by CCL21 has been shown to promote adhesion and transmigration across human LEC monolayers in vitro
]. Furthermore, depots of CCL21 have been observed next to the ICAM-1 microvilli that envelop transmigrating DCs in inflamed lymphatics, (as described above) and development of these protrusions is blocked by CCL21 neutralising mAbs [32
Besides CCL21, the chemokine and survival-inducing factor CXCL12 (SDF-1) has also been shown to direct trafficking of DC in lymphatic vessels. Expressed at low levels in resting LEC and normal skin and upregulated in inflamed skin lymphatics, CXCL12 is a potent chemoattractant for monocyte-derived DCs and stimulates basolateral-to-luminal transmigration across hDLEC monolayers in vitro
]. Furthermore, its receptor CXCR4 is highly expressed in cutaneous MHC class II+
DC and a synthetic CXCR4 antagonist (4-F-benzoyl-TN14003) was reported to impair migration of dermal DC and Langerhans cells to skin draining nodes during the sensitization phase of contact hypersensitivity in mice [63
]. In contrast, CXCL12 does not appear to control the exit of T cells to afferent lymphatic vessels in inflamed skin, despite expressing CXCR4 and retaining chemokine responsiveness [65
]. Hence CXCL12 may be rather specific for DC. Given some of the similarities with CCL21, and the fact that CXCL12 also binds heparan sulphate via
a tract of basic residues at the c-terminus, it is tempting to speculate that the two molecules regulate similar steps in DC transmigration through haptotactic guidance.
Recently a third chemokine CX3CL1 (fractalkine) and its sole receptor CX3CR1 have also been reported to direct leucocyte trafficking in lymphatics. Unusually, CX3CL1 shares the dual properties of an adhesion molecule and a conventional chemoattractant, in both cases through interaction with its receptor expressed in cells of the CD14+ monocyte/macrophage/DC lineage and subsets of tissue resident DCs and epidermal Langerhans cells [66
]. Synthesized as a type I integral membrane protein with an extracellular domain containing a novel C-X-X-X-C chemokine motif and a mucin-like stalk, the full-length molecule induces tight integrin-independent adhesion of leucocytes to blood vascular endothelium [66
]. The soluble chemokine form is generated by cleavage of membrane-anchored CX3CL1 with the disintegrin and metalloproteinases ADAM10 and ADAM17 and promotes conventional integrin-mediated chemotaxis [73
]. Significantly, lymphatic endothelial cells were shown to express CX3CL1 only after activation with inflammatory cytokines and it is the soluble chemokine rather than the membrane-bound form that predominates [75
]. Notably, CX3CL1 was also found to mediate transmigration of DC across LEC monolayers, and disruption of CX3CR1 in these cells results in impaired entry to lymphatics and delayed trafficking to draining lymph nodes. However CX3CL1 appears to have a more subtle effect on migration and unlike CCR7-/-
DC do not accumulate at the basolateral surface of lymphatic capillaries during skin contact hypersensitivity responses in mice. The polarity of CX3CL1 secretion by HDLEC is similar to that of CCL21 and the majority is released from the basolateral surface of the endothelium in vitro
]. However, CX3CL1 does not bind heparin sulphate and so cannot establish similar haptotactic gradients near lymphatic capillaries. It may therefore assist in directing DC migration close to the basolateral surface of the vessel, perhaps guiding cells towards ICAM-1 rich microvillar projections in inflamed tissues.
Interestingly, the activity of CCL21, CXCL12 and several other C-C chemokines in directing leucocyte entry is also regulated by a group of recently discovered atypical chemokine receptors that includes ACKR1 (Duffy antigen), ACKR2, formerly known as D6, ACKR3 (CXCR7) and ACKR4 (CCRL1). These non-signalling receptors endocytose their chemokine ligands and thus regulate their levels in the extracellular milieu. In the case of ACKR2, which binds as many as eighteen different CC chemokines, the receptor prevents their accumulation in lymphatic endothelium during inflammation that would otherwise cause inappropriate logjamming of recruited leucocytes at the vessel surface [76
]. In contrast, CXCR7, which binds CXCL12 and CCRL1, which binds CCL21 are thought to control cell migration by shaping chemotactic gradients of these chemokines near lymphatic vessels and between the floor and ceiling of lymph node subcapsular sinuses [79
]. Undoubtedly, further investigation into these novel receptors will reveal additional subtleties in the way that leucocyte chemotaxis is regulated by lymphatic endothelia.
Besides conventional chemokines, a number of other chemotactic agents can also direct leucocyte trafficking via
lymph. For example the semaphorins, a group of membrane bound and secreted chemorepellants that direct axonal guidance via
their receptors, the plexins and neuropilins were reported to play a critical role during transmigration by regulating the contraction and squeezing of DC that allows their migration through the lymphatic endothelium to the vessel lumen. In particular, the soluble semaphorin Sema 3A was shown to be secreted by LEC and to bind neuropilin 1 at the posterior surface of transmigrating DC, delivering a signal via
RhoA and its downstream effector kinase ROCK for actomyosin contraction and amoeboid movement as opposed to adhesion [81
]. Hence semaphorins appear to mediate diapedesis and are likely to participate in this important event under both resting (integrin-independent) and inflammatory (integrin-dependent) conditions.
Finally, the lipid mediator sphingosine 1 phosphate and its receptor S1P1 that regulate lymphocyte egress in the lymph node medulla have been shown also to control leucocyte transit across lymphatic vessels in the tissues. Dermal lymphatic vessel endothelial cells were reported to release sphingosine 1 phosphate in response to inflammatory stimuli, and its interaction with T cells led to their arrest via
β2 integrin mediated adhesion in the underlying parenchyma [82