Although semaphorins were originally identified as axonal guidance cues that regulate cell motility and morphology, accumulating evidence indicates that they also function as immune-regulatory molecules. To date, most functional studies on semaphorins and their receptors have focused on their co-stimulatory effects on immune cells 17
. Although the nervous and immune systems have considerable crosstalk and overlap in their molecular repertories and machineries 36
, it is still unknown whether semaphorins function as guidance cues that physiologically regulate immune cell movement. Here we demonstrate that semaphorin signals are crucial for DC trafficking, particularly for the entry of DCs into the lymphatics. Furthermore, we highlighted a novel mechanism for DC transmigration across the lymphatics, in which Sema3A promotes actomyosin contraction at the trailing edge of migrating DCs so they can pass through narrow gaps.
To exit peripheral tissues, DCs must migrate towards and enter the initial lymphatics. Although it has been assumed that the migration of DCs into the lymphatics is an indolent process, we provide evidence that Sema3A expressed in lymphatics is crucially involved in DC transmigration. Furthermore, we found that plexin-A1 localizes at the trailing edge of migrating DCs, which is responsible for actomyosin contraction. Numerous factors, including chemokines 6,8
, inflammatory molecules 5,37,38
and adhesion molecules 7
, have been reported to participate in DC transmigration. However, in our study, neither chemokine responsiveness nor the expression of chemokine receptors was affected in the absence of plexin-A1. The adhesion of DCs to extracellular matrix (ECM) proteins or to lymphatic ECs was comparable between wild-type and Plxna1−/−
DCs and the expression levels of integrins were similar between wild-type and Plxna1−/−
DCs (data not shown). In addition, there were no differences in the secretion of pro-MMP9 and TNF-α between wild-type and Plxna1−/−
DCs (data not shown). Thus, these results not only indicate that DC transmigration is regulated by active mechanisms but also provide a novel mechanism for this process.
Plexin-A1 is a primary receptor component not only for soluble semaphorin Sema3A, in association with the Nrp1 receptor, but also for transmembrane-type semaphorins Sema6C and Sema6D. We here determine that Sema3A produced in the lymphatics functions as a ligand for the plexin-A1-Nrp1 receptor complex expressed in DCs, indicating that the Sema3A-Nrp1-plexin-A1 pathways play important roles in DC migration by mediating interactions between DCs and lymphatic ECs. However, it is still unclear why Sema3A, but not Sema6C or Sema6D, is involved in this process. One possible explanation is that signaling downstream plexin-A1 is modified by the presence of Nrp1 in the receptor. Alternatively, differential effect may be due to the fact that Sema3A is a soluble protein. In contrast, Sema6C and Sema6D are membrane-bound semaphorins, which may prevent them from functioning at critical interaction sites between transmigrating DCs and ECs.
Members of the Rho family of small GTPases, Rac1, Cdc42 and RhoA, regulate cell movement by altering actin assembly, adhesion and actomyosin contraction 39
. Among these molecules, Rac1 is required to generate actin-rich lamellipodial protrusions and integrin-mediated adhesion 40
. In contrast, RhoA activates ROCK and subsequently activates non-muscle myosin II, which promotes an actomyosin contractile force 41
. Mesenchymal cell movement depends on integrin-mediated traction forces. In contrast, amoeboid cell movement, particularly in three-dimensional environments does not require integrins,. Instead, ROCK and myosin II- dependent contraction, is crucially required for passage through narrow gaps 11
. Consistent with this, we found that Sema3A promoted actomyosin contraction by inducing MLC phosphorylation. Furthermore, this effect was attenuated by blocking ROCK activity, indicating that the Sema3A-induced effects on myosin II activity require RhoA-ROCK-mediated signals. In neurons, Sema3A induces the local translation of RhoA in neuronal dendrites 42
, and siRNA-mediated knockdown of RhoA blocked Sema3A-mediated growth cone collapse 43
. In addition, Sema3A induces MLC phosphorylation, and inhibition of myosin II activity blocks Sema3A-mediated axon retraction 34,35
. These findings indicate that Sema3A-mediated signals promote actomyosin contractile force through RhoA-dependent myosin II activation in both immune and neuronal cells.
DCs have to pass through different environments. In tissues such as fibroblastic reticular tissues and inner vessel walls, DCs use integrin-mediated attachment and contractile force for cell movement. By contrast, in constricted areas, DCs use myosin II-mediated actomyosin contractile force to move forward because such tissues confine and mechanically anchor cell bodies 44,45
. During DC transmigration, at least three sequential mechanisms may be required. First, DCs have to form a lamellipodial protrusion at the leading edge in response to chemokines, which are the driving signal for forward movement. Second, DCs must contract and squeeze their bodies by actomyosin contraction to pass through narrow gaps. Third, after DCs are exposed to the lumenal side, the trailing edge has to detach from ECs in order for these cells to enter the circulation. Here, we show that Sema3A acts on the rear side of DCs through plexin-A1 to promote DC migration. These findings indicate that Sema3A-mediated signals are involved not only in actomyosin contraction but also in disassembling adhesive components at the trailing edge during DC transmigration. Indeed, myosin II promotes a traction stress that facilitates detachment at the trailing edge 46,47
, which suggests that detachment can be induced by Sema3A-mediated actomyosin contraction. On the other hand, Sema3A can inhibit integrin-mediated adhesion by inducing the sequestration of phosphatidylinositol phosphate kinase type I isoform PIPKIγ661 from talin, a major component of focal adhesion 48
. In this context, it is plausible that Sema3A plays dual or integral roles in regulating actomyosin contraction and adhesion disassembly at the trailing edge during the course of DC transmigration.
In conclusion, our study not only shows the importance of Sema3A-mediated signal in DC trafficking, particularly in the passage through the lymphatics, but also provides a novel mechanism that promotes actomyosin contraction at the trailing edge of migrating cells. Since semaphorins are also expressed in vascular endothelial cells 14,16
, it is plausible that they play a role in leukocyte extravasation or cancer metastasis. Additional detailed studies are required to gain insight into these mechanisms which have the potential to regulate immune reactions in order to treat autoimmune, allergic and infectious diseases and inhibit cancer metastasis.