The mechanisms by which leukocytes exit the tissues via lymph and migrate to draining lymph nodes have remained unclear despite their central importance for efficient generation of the immune response. The afferent lymphatics must cater for both low-level trafficking of APCs during normal immune surveillance and the increase in DC, effector memory T cell, and neutrophil trafficking that is triggered by tissue inflammation (for review see reference 3
). In this manuscript, we set out to define the mechanisms underlying these processes using a combination of in vitro studies with primary dermal LECs and in vivo studies with a mouse model of oxazolone-induced skin inflammation. Specifically, we showed that cultured primary HDLECs and MDLECs respond to the cytokines TNF-α and TNF-β (lymphotoxin-α), and to a lesser extent IL-1, by rapidly and reversibly up-regulating expression of the leukocyte adhesion molecules ICAM-1, VCAM-1, and E-selectin, together with synthesis and release of chemotactic agents, including the key inflammatory CC chemokines CCL5 (RANTES), CCL2 (MCP-1/JE), and CCL20 (MIP-3α). In addition, we demonstrated the induction of ICAM-1 and VCAM-1 expression in afferent lymphatic vessels draining the skin of oxazolone-treated mice in vivo and presented evidence that administration of ICAM-1– and VCAM-1–neutralizing antibodies blocked exit of CD11c-positive skin DCs via afferent lymphatics. Finally, we presented evidence from in vitro lymphatic transmigration assays with MDDCs that ICAM-1 and VCAM-1 mediate inflammation-induced transmigration by promoting leukocyte-endothelial adhesion.
The induction of CAM expression in activated lymphatic vessel endothelium reveals an unexpected similarity with the blood vasculature, where up-regulation of these same molecules in inflamed postcapillary venules promotes leukocyte transmigration by mediating firm adhesion (for review see reference 2
). In hemovascular transmigration, ICAM-1 and VCAM-1 promote stable adhesion of leukocytes to the apical endothelial membrane surface after their initial capture from blood flow and tethering on E- and P-selectin (50
). Binding via ICAM-1 and VCAM-1 then allows the adherent leukocytes to crawl toward intercellular junctions (51
), where additional interactions with the homophilic adhesion molecules CD31 (platelet/endothelial cell adhesion molecule 1) and CD99, together with junctional molecules such as junctional adhesion molecule 1 (JAM-1) and VE-cadherin, promote diapedesis (52
). The prediction from our own experiments that ICAM-1 and VCAM-1 would mediate lymphatic vascular transmigration through a similar mechanism of leukocyte adhesion was confirmed by our finding that LPS-treated MDDCs bind to TNF-activated LECs in static assays and that the interaction was blocked by CAM-neutralizing mAbs using similar concentrations to those that blocked transmigration. There is also the likelihood that events downstream of this CAM-mediated adhesion might be similar to those in the hemovasculature, given that primary HDLECs express similar junctional molecules, including VE-cadherin and JAMs, and that LEC monolayers can form both tight and adherens junctions in vitro (43
). Furthermore, mice with targeted deletion of the gene for JAM-1
) display abnormalities in DC trafficking to lymph nodes, consistent with a role for the molecule in lymphatic vessel diapedesis (55
). Detailed functional studies of these molecules in lymphatics are therefore clearly warranted.
A key question regarding entry of leukocytes to the afferent lymphatics is whether transmigration occurs at the distinctive overlapping junctions found within some initial lymphatics, at conventional interendothelial junctions, or through the endothelial cell body. It is interesting to note that docking structures containing ICAM-1 and VCAM-1 that were shown to mediate leukocyte transcellular/paracellular migration in hemovascular endothelial cells have also been observed in cells resembling a lymphatic phenotype (56
). Hence, it may be that leukocytes can transmigrate lymphatic endothelium using more than one mechanism, and it will be of major interest to determine the preferred route for individual leukocyte populations and the factors influencing such choice in future experiments.
It is generally assumed that most leukocyte traffic through afferent lymphatics involves transmigration in the basolateral to luminal direction. Nevertheless, it is also possible that reverse migration in a luminal to basolateral direction might occur. Such a notion is supported by our finding that ICAM-1 and VCAM-1 are expressed on both the upper and lower faces of the endothelial membrane in primary HDLECs in vitro and that activated HDLECs promote CAM-dependent migration of MDDCs equally in both directions. If this phenomenon occurs in vivo, it raises the intriguing possibility that some leukocytes might exit the afferent lymphatics and reenter at different points within inflamed tissue, a process that might increase the efficiency of immune surveillance. Bidirectional migration across activated lymphatic sinus endothelium could also be envisaged to play roles in trafficking within inflamed lymph nodes.
Finally, besides identifying mechanisms by which adhesion molecules facilitate transmigration of activated lymphatics, we also identified several chemokines that could potentially direct the process. Although existing evidence indicates that the major chemokine for directing lymphatic entry of mature DC and memory T cell entry is CCL21 (also known as secondary lymphoid chemokine, or slc
), which binds the G protein–coupled receptor CCR7 (5
), it seemed likely to us that other chemoattractants might also be involved. As indicated by the results of gene chip microarray analyses and chemokine ELISAs presented in this manuscript, it is now clear that activated HDLECs synthesize a large number of different chemoattractants, including the T cell/monocyte chemokines CCL20 (MIP-3α), CCL5 (RANTES), CCL2 (MCP-1), and CX3
CL1 (fractalkine). Release of these chemokines in vivo might be envisaged to promote lymph node trafficking of those newly extravasated monocytes and immature DCs bearing cognate CCR2, CCR5, CCR6, and CX3CR that enter skin and other tissues in response to inflammation and that subsequently mature into professional APCs (57
). Moreover, secretion into lymph could have long-range effects on cell trafficking in the blood vasculature (59
). Thus, “activated” LECs may well be the source of CCL2 (MCP-1) in inflamed skin that was shown recently to be rapidly transported via afferent lymph to the luminal surface of draining lymph node high endothelial venues, where it triggered integrin-mediated arrest and recruitment of monocytes from the blood circulation (60
). Overall, the broad range of chemoattractants that we observed in cytokine-stimulated LECs, including both monocyte/T cell chemokines and neutrophil chemokines such as CXCL2 (GROβ) and CXCL5 (ENA-78), suggests a far greater role for lymphatics in coordinating inflammatory leukocyte recruitment than has previously been appreciated.
In conclusion, we have shown for the first time that inflamed lymphatic endothelium promotes the exit of leukocytes from tissue to afferent lymph through newly induced expression of the adhesion molecules ICAM-1 and VCAM-1, which were previously thought to be specific for blood vessel transmigration. These findings reveal an overlap between the traffic signals within the blood and lymphatic circulations and identify the process of lymphatic transmigration as a potential target for antiinflammatory drug therapy.