Permeabilidad de la membrana a los diferentes iones

In the human body DC continuously migrate from the periphery into lymphatic tissues to orchestrate the immune system both in the presence and the absence of ‘danger’ signals. In an effort to maximize the migration of DC for cellular therapy it is indispensable to know how DC migration is initiated and what factors are essential for the cell to move towards the draining lymph node and enter the T cell-rich areas.

3.1 Emigration of DC from peripheral tissues towards lymph nodes

In a steady state, immature myeloid DC mainly reside in peripheral tissues where they continuously sample the environment for ‘danger’ signals. When they sense such signals the DC rapidly become activated, mature and emigrate from the tissue. Langerhans cells (LC) migrate from the epidermis to the draining lymph node upon application of proinflammatory mediators on the skin, such as contact allergens (23; 24) or toll-like receptor (TLR) ligands like CpG (25) and lipopolysaccharide (LPS) (26; 27). Critical factors are the local production of tumor necrosis factor  (TNF) and interleukin (IL)-1

and of prostaglandin E2 (PGE2). Indeed, direct injection of TNF (28-30) or IL-1 (31; 32) into the

dermis of mice or human skin explants induces the emigration of LC (reviewed in (33)). Similarly, neutralizing antibodies to TNF and IL-1 inhibit the spontaneous migration of DC from skin explants in mice and human (34). Interestingly, TNF effects were neutralized by anti-IL-1 and vice versa (32; 34; 35), demonstrating that both cytokines are required for the initiation of DC migration. That the emigration upon proinflammatory stimuli is very rapid is illustrated by the fact that lung DC and LC migrate within 2 hours to draining lymph nodes after viral inoculation (36) or upon the application of the proinflammatory cytokines, respectively (30; 32). Importantly, TNF effects were dose dependent: low concentrations (50 U/ml) of TNF augmented LC migration, whereas high doses (5000 U/ml) of the same cytokine inhibited LC migration in human skin explant (34).

Proinflammatory mediators exert their effects on different levels: while they act directly on the DC by inducing DC maturation and initiation of DC migration to the draining lymph nodes, they also stimulate the local environment to produce factors, such as chemokines that promote emigration of DC and recruitment of circulating immune cells. Furthermore, LPS, IL-1, and TNF also induce the

production of PGE2 (37), which has proven to be required for DC migration and increases the

responsiveness of monocyte-derived DC of the lymph node homing receptor, CCR7 (38-41). When applying DC therapy, we may benefit from these effects by stimulating the migration of injected DC in situ. By applying proinflammatory stimuli to the site where ex vivo generated DC will be injected, the local environment will be conditioned such that the emigration of the injected cells is favored. Indeed, Martín-Fontecha et al. (42) described that in mice conditioning of the injection site increased the migration of subcutaneously injected BM-DC up to 10-fold. The footpath of mice was pretreated by injection of TNF or IL-1 or with DC, respectively 8 or 24 hours before vaccination. As a result 5- to 10 fold more fluorescently-labeled DCs were recovered from the draining lymph nodes. Accordingly, the T cell responses were up to 10 fold enhanced. The authors hypothesized that local inflammation induced by the injection of DC or pro-inflammatory cytokines conditioned the environment to promote rapid migration of DC towards the lymph nodes. To support this, they showed that the

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lymph node chemokine CCL21 is up-regulated in the local lymphatics. These data show that conditioning of the skin has potential in increasing DC migration to lymph nodes after i.d. or s.c. injection. Skin conditioning prior to vaccination with mature DC has not yet been described in humans, but is a promising opportunity to improve DC migration. When proinflammatory mediators act directly on the DC, DC migration to the draining lymph nodes is likely initiated by the induction of DC maturation. With the use of radio-labels it was shown that immature, monocyte-derived DC are not able to migrate to subsequent lymph nodes upon injection in patients. Stimulating the maturation of immature DC after i.d. injections may improve migration and circumvents the need for in vitro maturation. Prins et al. showed that the combination of Imiquimod (containing TLR7 ligand) and s.c. injection of immature DC dramatically increased both the persistence and the trafficking of DC into the draining lymph nodes of mice (43). In mice, migration of immature DC was enhanced from 1.8 to 6.5% when cells were injected into skin that was pretreated with Imiquimod. Importantly, although migration of mature DC injected in untreated skin was higher (10%), the CTL responses induced after injection of immature DC in pretreated skin were superior to those induced by mature DC alone (20). In follow-up studies, Nair et al. injected immature DC intradermally in human skin pretreated with Imiquimod. Application of Imiquimod onto the injection site every other day for three times before injection of immature DC increased migration of DC compared to immature DC alone. Again the percentage of migrating cells did not exceed that of injections with mature DC (20). Application of Imiquimod before injection of immature DC for cellular therapy seems therefore a valid alternative to circumvent ex vivo maturation of DC, but not for amplifying the percentage of migrating DC. Its effect may increase after optimization of the timing and dose of Imiquimod, for example to apply the agent not before but after the injection of the immature cells, so that Imiquimod can directly stimulate the injected DC. Interestingly, it has been described that DC that migrate into the lymph nodes are not uniquely responsible for the activation of specific T cells. They can transfer antigen to resident DC and thereby spread antigens to a larger pool of DC to increase priming efficiency (44; 45). This would mean that, when antigen loading is sufficient, only a small number of cells need to reach the draining lymph node. How antigens are being transferred to lymphoid DC and what the requirements are for the maturation state of the immigrating DC to induce effective T cell responses is yet unknown. Addressing these questions may renew the insights on the type of DC and method of antigen-loading that are most appropriate for DC immunotherapy. 3.2 DC phenotype

Homing to specific tissues is controlled by the combined expression of different chemokine receptor and specific adhesion molecule receptors. After stimulation with proinflammatory stimuli, DC maturation is initiated and many molecules that are important for their change in function are either up- or down-regulated. Not only must DC change from an endocytic sentinel into a professional antigen presenting cell, but it also needs to detach from its local environment, migrate through surrounding tissues, enter the lymphatics and subsequent the draining lymph node and in particular penetrate the T cell area. To emigrate from the periphery towards the draining lymph nodes, DC rapidly change their repertoire of chemokine receptor and adhesion molecules and the expression of many other migration related proteins, such as matrix metalloproteases and cytoskeletal proteins. In this section an overview will be presented of chemokine receptors, adhesion molecules and MMP that were found essential in lymph node homing of endogenous DC.

3.2.1 Chemokine receptors

The dominant mediator in the mobilization of DC to lymph nodes via lymphatics is CCR7 (46-48). Constitutive expression of ligands CCL19 and CCL21 is found at the luminal site of high endothelial venules and in the T cell rich areas of secondary lymphoid tissues, such as tonsil, spleen and lymph

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is increased during inflammation (42). The expression of CCR7 is essential for both the entry of DC into lymphatic vessels at peripheral sites and the entry into T cell rich areas of lymphoid tissues in both steady state and under inflammatory conditions (48). Besides CCR7 several other chemokine receptors play a role in DC homing from the periphery to draining lymph nodes but appear less important. Lung myeloid DCs are dependent on CCR5 and its ligand CCL5 for migration and

maturation, since these events are significantly impaired in CCL5-/- and CCR5-/- mice. However, this

effect seems to be mediated via CCR7 as in these mice the failure of DC to migrate to draining lymph nodes is associated with impaired up-regulation of CCR7 (36). Similarly, emigration of skin-DC (50) and lung-DC (51) to the draining LN was only partially blocked in CCR8-deficient mice. Migration of plasmacytoid DC matured with CpG for 24 hour from peripheral blood into the lymph nodes was described to depend on the CXCR3 ligand CXCL9 (10). But also pDC express high levels of CCR7 and gain responsiveness to CCR7 upon maturation (10; 52), suggesting that CCR7 is also important in lymph node homing of pDC. Overall, it appears that CCR7 is the main receptor for lymph node homing and that targeting from the periphery to the lymph node is promoted by additional chemokine receptors. For cellular therapy, chemokine-mediated migration of DC may be enhanced in two ways, by manipulating the local chemokine expression or by manipulating the expression of chemokine receptors by the DC itself. As described in the previous section, conditioning of the skin injection site, increased the expression of CCL21 on the local lymphatics (42). Inducing the expression of chemokines, such as CCL21, on (local) lymphatics before or together with the DC injection may improve the recruitment of CCR7-expressing cells to afferent lymphatics and eventually the draining lymph nodes. The chemokine receptor expression profile of DC is dependent on DC culture and

maturation methods. The addition of PGE2 as maturation stimulus enhances CCR7 expression and its

activation, and drives effective migration of monocyte-derived DC towards CCR7 ligands CCL19 and 21 (38-40; 53). Another approach could be to introduce the expression of chemokine receptors in the ex vivo generated DC, which will be discussed below.

3.2.2 Adhesion molecules

Adhesion molecules play a pivotal role in successive steps in the process of migration. Therefore, their expression profile is tightly controlled. A switch in adhesion molecule expression profile is required to allow detachment of resident DC from existing interactions and to enable migration through the lymphatic system and into the lymph node. Thus far, no extensive analysis of the molecules implicated in transendothelial migration of DC into the lymphatic system has been carried out (54), but a variety of adhesion molecules is involved in the emigration of DC through the

extracellular matrix (ECM) and in emigration of LC from skin. In mice LC, 6 and 4 integrins are

oppositely regulated during migration to lymph nodes. 6 integrin is up-regulated and contributes to migration of LC (55). When the interaction between 6 and laminin was blocked, the emigration of LC from the epidermis was completely abrogated, although the cells had lost their cellular processes

and seemed to have detached from neighboring keratinocytes. By contrast, blocking of 4 integrin

had no effect on LC migration (55). In addition, ICAM-1 and LFA-1 (L2) were found to play a

significant role in contact hypersensitivity-induced migration of LC to regional lymph nodes (56). Also non-integrins are regulated during DC migration and maturation. In mice CD44 is described as an adhesion molecule that binds to ECM-compound hyaluronic acid, which is expressed on the leading edge of migrating cells. Migrating and lymph node LC and DC express different CD44 splicing variants (57). CD44 can also bind the matrix metalloprotease MMP9 to the cells surface and mediate degredation of extracellular matrix (58). Blocking of CD44 inhibited the migration of LC from the epidermis. Furthermore, it prevented binding of both activated LC and DC to the T cell rich areas of lymph nodes (57). Another example of adhesion molecules regulated during DC migration are the syndecans (SDC), a family of transmembrane proteoglycans. In human skin explants, SDC1 and 4 (59)

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have been implicated in the migration of LC. SDC1 was down-regulated and SDC4 was strongly up- regulated during LC emigration and within the first hours of LPS-induced DC maturation (59). Blocking SDC4 decreased DC motility. Thus, SDC1 may be involved in stable adhesive interactions with the ECM, whereas SDC4 seems involved in trafficking through the ECM. Lymph node homing from the peripheral blood is dependent on L-selectin, and is required for efficient rolling and attachment on HEV. L-selectin is expressed on a subset of circulating leukocytes, such as naive T cells (60) and pDC (61), but not on DC of the myeloid lineage. Next to L-selectin, pDC depend on the inducible endothelial adhesion molecule E-selectin to enter lymph nodes (10). CD44, which is a ligand for E-selectin, may therefore also play a role in pDC migration. In addition to chemokine responsiveness, manipulation of the expression of adhesion molecules on ex vivo generated DC may improve the migratory capacities of the cellular vaccine.

3.2.3 Matrix metalloproteases

To move through the physical obstacles, such as basement membranes and ECM, they express and secrete matrix metalloproteases (MMP). Upon triggering with IL-1 and TNF, murine LC increase the expression of MMP9, (57; 62). Human immature monocyte derived-DC secrete MMP1, 2, 3 and 9 (63; 64), and their inhibitors (TIMP1 and 2) (63; 64), which are down-regulated upon stimulation with TNF (64). The importance of MMP in DC migration through tissues is demonstrated in MMP9- deficient mouse, where DC migration through tracheal epithelial tight junctions was impaired (65). Furthermore, in human skin explants antibodies against MMP2 and 9 and inhibitors of MMP markedly reduced the migration of human LC and dermal DC (66; 67). Also, up-regulation of genes encoding matrix metalloproteinases (MMP), such as MMP1, 10 and 12 correlated with enhanced migratory capacity of DC measured in Matrigel (68). Thus, MMP are essential for effective migration through extracellular matrix. In brief, DC need to express appropriate chemokine receptors, adhesion molecules, and other migration-related proteins in addition to high T cell stimulatory capacities. To achieve maximal DC migration after injection, we need to exploit this information for the development of better DC or DC vaccination protocols.