Despite the fact that naive lymphocytes are intrinsically non-motile under standard in vitro
, these cells constitutively enter lymphoid organs. Co-culture of lymphocytes with HECs promotes the efficient passage of lymphocytes across the endothelial layer, suggesting HECs express a ‘lymphocyte migration stimulus’33,34
. Although a soluble factor, which may correspond to this activity has been described34
, the biochemical identity and mechanism of action of this ‘stimulus’ have not been defined. We report here that ATX is a strong candidate for the postulated lymphocyte motility stimulus.
Following up our initial gene profiling analysis of HECs10
, we found that that murine lymphoid organs expressed abundant transcripts for ATX and confirmed that HECs were a particularly rich source. The expression of ATX protein in lymph nodes HEVs was verified by immunocytochemistry and biochemical analysis of isolated HECs. We also found ATX expression in HEV-like vessels in two different models of lymphoid neogenesis. A number of proteins expressed by HEVs (including selectin ligand scaffolds, fucosyltransferases, sulfotransferases, and chemokines), which are critically involved in the lymphocyte homing cascade, are elements of an HEV differentiation program4
. Lymphotoxin signaling is required for this program during organogenesis and for the induction of HEV-like vessels at sites of lymphoid neogenesis4,35
. Interestingly, Enpp2
(which encodes ATX) is one of a limited subset of genes that is suppressed in lymph nodes by blockade of lymphotoxin signaling36
, suggesting that it may be an element of the HEV differentiation program.
Our finding that ATX was secreted by HECs in an apical orientation indicated a possible parallel with ‘arrest’ chemokines. These are secreted by HEVs, become associated with apical receptors, and exert their activities on adherent lymphocytes7
. Noting a potential α4
-binding motif (LDV) in the sequence of ATX, we asked whether ATX could bind to lymphocytes via α4
. Indeed, we found that Jurkat cells and primary human T cells, activated in three different ways, exhibited α4
-dependent adhesion to immobilized ATX. This finding suggests that ATX could be targeted to HEV-adherent lymphocytes whose integrins have been activated during the arrest step of the recruitment cascade. A number of α4
ligands are known, including fibronectin, VCAM-1 and osteopontin37
. ATX also possesses an RGD sequence, suggesting that ATX may be able to partner with other integrins26
. Further experiments are needed to define the nature of the divalent-cation dependent ATX receptors on murine lymphocytes. It is conceivable that the versatility of ATX may include the ability to bind simultaneously to both HEV and lymphocyte, which could help bridge the lymphocyte to the endothelium.
To arrive at a rationale for how ATX might influence lymphocyte behavior, we examined the effects of LPA on primary T cells. LPA was our focus because many of the biological activities of ATX are attributable to its production of this phospholipid13
. Moreover, ATX accounts for the basal concentrations of LPA in the blood14,15
. Several previous reports had investigated the responses of lymphocytes (usually lymphoma populations) to LPA19,28,31,38–40
. Most pertinent to the present study are the rapid actions of LPA in inducing chemokinesis of Jurkat cells31
and in promoting shape change and invasiveness of mouse lymphoma lines28
. Our experiments with primary lymphocytes are consistent with these previous cell line studies. Thus, we showed that LPA induced actin polymerization in suspended cells and was chemokinetic in a transwell assay. The dose-response curves were similar to those reported for LPA in other bioassays17,20,31
. Importantly, the optimal concentration for LPA in our experiments (≈1 µM) exceeds basal values for blood plasma both in mouse and human13–15
. Interestingly, we found that the chemokinetic effect of LPA was additive with the chemoattractant effects of CXCL12 and CCL21, suggesting the possibility of cooperative interactions between LPA and chemokine signaling in migrating lymphocytes.
Our strongest functional results came from short-term homing experiments. We showed that intravenous injection of an enzymatically-inactive form of ATX blocked homing of blood-borne lymphocytes to lymph nodes, spleen and Peyer’s patches. Our model posits that the inactive ATX would interfere with the function of endogenous ATX by displacing it from a limited number of binding sites on lymphocytes (and possibly HECs), thus exerting a dominant negative effect. The endogenous ATX is proposed to emanate from HEVs. For spleen, the proposed sources are marginal zones, which surround white pulp regions and are known sites of lymphocyte entry. The net result of this competition from the inactive ATX would be reduced amounts of locally produced LPA and consequently a reduction in lymphocyte entry. Whether LPA is functioning to affect cell motility, cell adhesion (for example, post-arrest affects on LFA-1), cell shape, or transendothelial migration during the complex process of entry remains to be investigated. It should be noted that locally produced LPA at the lymphocyte–HEV nexus could also have effects on HEV function during lymphocyte recruitment, as LPA is capable of eliciting responses in endothelial cells41
The activity of ATX in regulating lymphocyte migration may extend beyond the entry phase, since we detected ATX in stromal regions of lymphoid organs. Lymphocytes exhibit considerable motility within lymphoid organs, much of it apparently random in direction3,21,42,43
. CCR7 and its chemokine ligand CCL21 account for a portion of T cell motility, probably through a chemokinetic effect44–47
. LPA is a plausible candidate for an additional motility-stimulating factor within lymphoid organs. We found that treatment of mice with inactive ATX caused a reduction in the distance between labeled lymphocytes and the nearest HEVs. Whether this effect is attributable to delayed entry into the lymph node or reduced motility within the node is not known. With regard to a possible ATX contribution to lymphocyte egress from lymph nodes, we did not detect ATX in LYVE-1 positive vessels (not shown). Finally, it should be noted that ATX may be stably tethered to lymphoid stromal elements and thus could provide an adhesive substrate for migrating lymphocytes.
LPA signals cells through 5 known GPCRs (LPA1–5
), the first 3 of which are members of the EDG family to which the S1P receptors also belong19,48,49
. Lymphocytes predominantly express LPA1 40,48
, LPA2 38,48
with subset preferences. Unlike chemokine GPCRs, which primarily signal through Gi
, LPA receptors can utilize a variety of G-protein families including the Gi
. We found that the LPA-induced chemokinesis of lymphocytes was blocked by pertussis toxin, indicating the involvement of Gi
proteins in this response. However, pertussis toxin has no effect on lymphoma invasiveness, whereas G-proteins of the Gq
families are implicated28
. The LPA signaling pathways involved in lymphocyte migration into and within lymphoid organs remain to be studied.
Lymphocyte recruitment into lymphoid organs is a multistep process. Its molecular elucidation over the past 20 years represents a triumph of immunology. Here, we propose a novel step in this process involving the regulation of lymphocyte migration by the ectoenzyme ATX. Our model of ATX action lends itself to testing with genetically engineered mice and pharmacological agents. As inhibitors of lymphocyte exit from lymphoid organs are of clinical value for achieving immunosuppression19
, inhibitors of the proposed ATX pathway may also be of therapeutic value in some settings by preventing entry of lymphocytes into secondary lymphoid organs or into induced tertiary lymphoid organs at sites of chronic inflammation50