Migration of dendritic cells into lymphatics—The langerhans cell example: Routes, regulation, and relevance

Migration of Dendritic Cells into Lymphatics-The Langerhans Cell Example: Routes, Regulation, and Relevance Nikolaus Romani,’Gudrun Ratzinger,’Kristian Pfaller,* Willi Salvenmoser,3Hella StGssel,lFranzKoch,’and PatriziaStoitznerl Departments of Dermatology.’ Histology and Embryology,’ and Zoology.3 University of Innsbruck, A-6020 Innsbruck, Austria,

Dendriticcells are leukocytesof bone marrow origin. Theyare centralto the control of the immuneresponse.Dendriticcells are highly specializedin processingand presentingantigens(microbes,proteins)to helperT lymphocytes. Thereby,they critically regulatefurther downstreamprocessessuch as the developmentof cytotoxicT lymphocytes,the productionof antibodiesby B lymphocytes,or the activationof macrophages.A new field of dendriticcell biology is the study of their potentialrole in inducingperipheraltolerance.The immunogenic/tolerogenic potentialof dendriticcells is increasinglybeing utilized in immunotherapy,particularlyfor the elicitationof antitumor responses.Onevery importantspecializationof dendriticcells is their outstandingcapacityto migrate from sites of antigenuptaketo lymphoidorgans.Much has beenlearnedabout this processfrom studyingone particulartype of dendriticcell, namely,the Langerhanscell of the epidermis.Therefore,the migratorypropertiesof Langerhanscells are reviewed.Knowledgeaboutthis “prototypedendriticcell” may help researchersto understandmigrationof othertypes of dendriticceils. KEYWORDS: Langerhanscells, Dendriticcells, Migration,Cytokines,Electron microscopy. 12001Academcc Press

I. Introduction The field of dendritic cell research has experienced tremendous expansion in the past few years (Lotze and Thomson, 1999, 2001). This was mainly due to the

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development of new methods for the convenient generation of large numbers of dendritic cells (Caux et al., 1992; Sallusto and Lanzavecchia, 1994; Romani et al., 1994) and, thus, to the widespread recognition of the intriguing features of dendritic cells. Dendritic cells are leukocytes of bone marrow origin. They belong to the myeloid lineage of hematopoietic cells (Inaba et al., 1993). The picture may become more complex: it was reported that dendritic cells may also be of lymphoid origin (Shortman and Caux, 1997; Shortman, 2000). These possible differences in ontogeny also appear to be reflected in differences in function, namely, in the properties of dendritic cells to induce different qualities of helper T-lymphocyte responses (“Th 1 versus Th2” responses) (Rissoan et al., 1999; Pulendran et al., 1999). Clearly, these issues need to be studied more thoroughly before definitive answers can be given, especially in the light of recent data that emphasize the role of cytokines in the cell-fate conversion of lymphoid-committed progenitors (Kondo et al., 2000). The immunobiology of dendritic cells has been reviewed extensively in a number of excellent recent articles (Banchereau and Steinman, 1998; Schuler et al., 1997; Steinman et al., 1997, 1999; Banchereau et al., 2000; Bell et al., 1999a; Hart, 1997; Steinman. 1998). A. Immunogenic

Function of Dendritic

Cells

Dendritic cells are highly specialized antigen presenting cells. They have developed and optimized several functions that enable them to fulfill their prime task, i.e., to initiate primary immune responses. This functional spectrum comprises antigen uptake and processing capacities, which are exerted mostly in peripheral organs and tissues by immature or maturing dendritic cells, as well as T-cell sensitizing skills, which are performed in the lymphoid organs by terminally mature dendritic cells. Uptake of antigens is rendered efficient and preferentially directed toward microbial material by a number of recently defined pattern-recognition receptors, such as the lectin-like receptor DEC-205 (Jiang et al., 1995), DC-SIGN (Geijtenbeek et al., 2000a), Langerin (Valladeau et al., 2000) and others. Stimulation of T lymphocytes, particularly of naive T cells, is greatly helped by a set of highly expressed costimulatory and adhesive molecules on the surfaces of mature dendritic cells (Caux et al., 1994; Geijtenbeek et al., 2000b). The gap between the sites of antigen uptake and the sites of clonal T-cell activation is efficiently bridged by migratory dendritic cells that carry immunogenic complexes of MHC and antigenie peptides into the T-cell areas of lymphoid organs (Austyn, 1996; Steinman etal., 1997). Dendritic cells are superior to other antigen presenting cells in all three functional areas, i.e., antigen uptake/processing. migration, and T-cell stimulation. 6. Epidermal

Langerhans

Cells

Langerhans cells were-unknowingly-the very first dendritic cells to be described (Langerhans, 1868; Wolff, 1991). They are the dendritic cells of the

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epidermis. Many important features of Langerhans cell biology are compiled in two excellent books (not brand new but still informative) (Schuler, 1991; Mall 1995) and a recent specific review article (Maurer and Stingl, 1999). Besides Langerhans cells, the skin harbors a second population of dendritic cells, namely, dermal dendritic cells (Lenz et al., 1993; Nestle et al., 1993). Investigations on Langerhans cells have greatly contributed to the understanding of dendritic cell biology in general. The phenomenon of morphological, phenotypical, and functional dendritic cell maturation was first described with Langerhans cells by Schuler and Steinman (1985). This concept essentially maintains that the outstanding immunogenic functions of dendritic cells are regulated in a spatially and temporally well-coordinated fashion. Immature Langerhans cells are specialized for taking up and processing antigens; they are not yet equipped with the necessary molecules to activate T cells (Romani et al., 1989c, 199 1; Schuler and Steinman, 1985). They need some time to mature in the presence of what was originally thought to be keratinocyte products and is now known to be inflammatory cytokines (Heufler et al., 1988; WitmerPack et al., 1987; Koch et al., 1990). Mature Langerhans cells display an inverted functional profile: they are poor in taking up and processing antigens; they are very efficient, though, in stimulating resting, naive T cells (Schuler and Steinman, 1985; Romani et al., 1989c), even when they present only very few molecules of ligand for the T-cell receptor (Romani et al., 1989a). Immature dendritic cells are typically found in the tissues and organs of the body; mature dendritic cells occur mainly in the lymphatic organs. Langerhans cells share many features with other types of dendritic cells. They are characterized, however, by some specific markers. The morphological hallmark of Langerhans cells is an ultrastructural organelle, the Birbeck granule (Birbeck et al., 196 1). It is typically rod- or tennis racket-shaped, and it has a characteristic internal structure consisting of two perfectly parallel, juxtaposed membranes and a regular central striation, best described by Klaus Wolff some time ago (Wolff, 1967). It remained enigmatic for many years until Sem Saeland’s group in Dardilly, France, began to successfully solve the mystery. The molecules of the Birbeck granule are now being defined, the first one being Langerin. a lectin-like receptor that may serve antigen uptake. Old (“Lag”; Kashiharaetal., 1986) and new (“DCGM4”; Valladeau et al., 1999; Valladeau et al., 2000) monoclonal antibodies against Langerin are excellent tools to mark and trace Langerhans cells, and they are starting to be exploited for the investigation of Langerhans cell migration. For the sake of clarity in terminology, we should mention that we have come to the point where it is possible to generate Langerhans-like cells from progenitor cells in the blood or bone marrow in vitro. This is true for the human system, but it will not be long until mouse Langerhans cells can be generated as well. This can be done either by selection of precommitted precursor cells (Caux et al., 1996; Strunk et al., 1997) or by choosing a particular cytokine milieu (mainly TGFP 1) that allows the preferential outgrowth of Langerhans cell-like cells (Strobl et ul., 1996; Strobl and Knapp, 1999). These cells possess Birbeck granules and they share other phenotypical markers, such as E-cadherin, with Langerhans cells.

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Although it is very likely that they are identical to “real” Langerhans cells in all functional aspects, this remains to be demonstrated formally.

C. Importance

of Dendritic

Cell Migration

The deposition of antigen in the lymphoid organs is an absolute prerequisite for the generation of primary immune responses (Zinkernagel, 1996). It has been known for a long time that it is difficult to experimentally induce immune responses against soluble foreign antigens. This goal can be achieved much more efficiently if the antigen is mixed with inflammatory bacteria (“adjuvant”) or aggregated (Zinkernagel, 1996) or-still better-“loaded” ex viva onto dendritic cells that are then injected (Inaba et al., 1990). This property of dendritic cells has prompted Ralph Steinman to call them “nature’s adjuvant” (Steinman etal., 1990). Therefore, it appears that both naturally occurring and experimentally/therapeutically induced immune responses can only be successful if dendritic cells migrate.

II. Models

to Study

Langerhans

A. Contact

Hypersensitivity

Cell Migration

Dendritic cell migration was first observed in a contact hypersensitivity model. Silberberg-Sinakin noted Langerhans cells in the afferent lymphatics of the skin in response to the application of a contact allergen (Silberberg-Sinakin et al., 1976). Many key observations were made during the sensitization phase of contact hypersensitivity: emigration of Langerhans cells from the epidermis (Bergstresser et al., 1980) maturation of Langerhans cells as they begin to migrate (Aiba and Katz, 1990), tracing of contact sensitizer-charged dendritic cells to the lymph nodes (Macatonia et al., 1987) and determination of cytokine profiles in skin as Langerhans cells begin to migrate (Enk and Katz, 1992). A word of caution is needed here. The contact hypersensitivity model is often taken as a model for the migration of Lmzgerhuns cells. This is only half the truth. One must not forget that the dermis contains a resident population of interstitial, dermal dendritic cells (Lenz et al., 1993; Nestle et al., 1993). The fate of these cells in contact hypersensitivity has not been studied; certainly, however, they contribute to the phenomenon (Tse and Cooper, 1990). Thus, whenever cells are studied that arrive in lymph nodes in response to the epicutaneous application of contact sensitizers (Macatonia et al., 1987; Kripke et al., 1990) these may be Langerhans cells as well as dermal dendritic cells. Given the biological differences between CD34 progenitor-derived Langerhans-like cells and dermal dendritic-like cells (De Saint-Vis et al., 1998; Caux et al., 1997) one should not be surprised if the “real” Langerhans cells and

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dermal dendritic cells also differ in some properties, including properties related to migration. This issue has not been studied yet.

E3. Skin Explant Cultures This experimental model was originally established by Chris Larsen and colleagues (1990). The experimental setup is simple (Fig. 1). Whole skin or dispase-separated epidermis and/or dermis is placed onto culture medium, preferrably in 24-well plates (Ortner et al., 1996). Over a period of l-3 days dendritic cells migrate out of the explants into the culture medium. There, they can be collected and analyzed quantitatively and qualitatively. Moreover, epidermal and dermal sheets prepared from the remaining explant can be investigated by immunohistochemistry. In the dermis, one can observe characteristic string-like, nonrandom accumulations of strongly MHC class II-positive cells that Larsen et ul. called “cords” and that were eventually shown to represent migrating dendritic cells from both epidermis and dermis within the lumen of lymphatic vessels (see below; Lukas et al., 1996; Weinlich et al., 1998). The advantage of the model is that it looks specifically at migration of dendritic cells in the skin and that it allows the separate consideration of both Langerhans cells and dermal dendritic cells. This is in contrast to the contact hypersensitivity model, where the main readout is further downstream, namely, ear swelling responses. Two examples from our own work illustrate that this difference may be relevant. Some reports have described an impairment of contact hypersensitivity in mice rendered gene deficient for TNF-(lr or one or both receptors for TNF-(w (Wang et al., 1997; Wang et al., 1996). A migration defect of Langerhans cells has been concluded. When we set up skin explant cultures from the very same mice, we observed no reduction in the migratory capacity of Langerhans cells at all; Langerhans cells migrated from the epidermis equally well, if not better than in control mice (Stoitzner et al., 1999). Conversely, an unimpaired

FIG. 1 Schema of skin e.rplanf cultures. Explants of human or murine skin simply float on culture medium for a few days. Dendritic cells that emigrate can be enumerated and analyzed phenotypically or functionally. Epidermis (E) and/or dermis (D) may be investigated by immunohistochemistry. Epidermis and dermis may also be cultured separately. after dermo-epidermal separation by means of the enzyme dispase.

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contact hypersensitivity response was observed in a mouse that lacks an important matrix metalloproteinase (MMP-9) (Wang, M., et al., 1999). Using the very same mice we observed a strongly inhibited migration of cutaneous dendritic cells from the skin of these animals (Ratzinger et al., manuscript in preparation, 200 I ). There is another important difference between the skin explant model and contact hypersensitivity model (and also the injection model; see below). The application of a contact sensitizer or the injection of some cytokine into the skin sets in motion a cascade of events from a quiescent steady state. In contrast, the mere preparation of the skin explant and placing it into culture medium already sets off this cascade of inflammatory events. Addition or neutralization/blockade of mediators or molecular interactions will only modify an ongoing process. These pros and cons for one or the other approach have to be considered when designing migration experiments. Skin explant cultures may also serve as a simple method to obtain populations of substantially enriched mature dendritic cells (Ortner et al., 1996; Pope et al., 1995). If epidermis and dermis are separated before the onset of culture, one may obtain mature Langerhans cells and mature dermal dendritic cells side-by-side (Fig. 2). Notably, these cells have not undergone any enzymatic treatment such as standard trypsinization used for the procurement of epidermal cell suspensions (Romani et al., 1997; Koch et al., 2001).

C. Other Models 1. Transplantation Skin grafting is rarely used for studies of Langerhans cell migration. Larsen et al. (1990) have investigated the density of Langerhans cells in mouse skin transplant. They noted an emigration of Langerhans cells from the epidermis of the graft. Xenogeneic skin grafts (human skin onto nude mice) have yielded some interesting insights. The classical work by Krueger and colleagues (1983) has shown that human Langerhans cells reside in the graft for long periods of time but that some of the cells also leave the epidermis. Hoefakker et al. (1995) have extended these findings in that they applied a fluorescent contact allergen onto the human skin graft. They demonstrated an enhanced emigration of Langerhans cells in response to the sensitizer and, moreover, they observed antigen-bearing human MHC class II-positive cells (presumably in part Langerhans cells) in the draining murine lymph node, thereby emphasizing the migratory capacity of these cells. 2. In viva Administration

of Cytokines

This approach is frequently taken. Jon Austyn’s group in Oxford has for the first time shown the crucial importance of inflammatory mediators (LPS and TNF-o) for the migration of-among others-Langerhans cells (Roake et&., 1995). These

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FIG. 2 Lunger-ham cells ohrained,from skin explant cultures. Epidermal sheets were prepared by means of dispase from human skin explants. They were cultured for 48 hr. Cells that had emigrated into the culture medium are viewed by phase contrast in the upper picture. Note that Langerhans cells are enriched and they extend distinct, thin cytoplasmic “veils.” lmmunotluorescence staining in the lower picture shows that virtually all MHC class II-positive cells (i.e., Langerhans cells; green fluorescence) coexpress the Birbeck granule-associated molecule Langerin as detected by monoclonal antibody “Lag” (red fluorescence). (See color insert.)

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authors had administered the mediators systemically. The injection of TNF-(IU directly into the skin leads to the emigration of a substantial part of Langerhans cells from the epidermis both in mice (Cumberbatch et al., 1994) and in humans (Cumberbatch et al., 1999). 3. Artificial

Substrates

An interesting model for the investigation of dendritic cells or Langerhans cells, in particular, was recently developed and refined by Fried1 and coworkers (1998). They monitored the migration of dendritic cells including Langerhans cells (pathways, speed, chemotactic responses) in three-dimensional collagen lattices by image analysis tools (Gunzer et al., 2000). This model may be useful for gaining insights into the regulation of Langerhans cell migration specifically through the dermal meshwork. 4. Genetically Modified Animals Gene knockout mice and transgenic mice are widely used for studies on the migration of dendritic cells, including Langerhans cells. The models outlined above are applied to various types of knockout mice. The “problem” with knockout mice is that they often develop compensatory mechanisms and therefore show no defects. There is only one knockout mouse that has a very striking phenotype: TGFP 1 genedeficient mice have absolutely no Langerhans cells in the epidermis (Borkowski et al., 1996, 1997).

III. Other

Forms

of Langerhans

Cell Migration

For the sake of completeness we should briefly mention that Langerhans cell migration may be viewed from two additional standpoints. Neither of them will be considered in detail in this review. First, Langerhans cell precursors from the bone marrow need to make their way from the blood stream through endothelial cells and connective tissue into the epidermis, their final destination (at least until challenged by inflammation). Little is known about the regulation of this process except two key points: Langerhans cell precursors appear to be recruited from the blood and guided into the epidermis by the chemokine MIP-3o/CCL20 in a specific manner. MIP-3a is produced by keratinocytes of the epidermis and Langerhans cell precursors express the appropriate chemokine receptor, CCR6 (Dieu et al., 1998; Charbonnier et al., 1999; Dieu-Nosjean et al., 2000). In addition, TGFP 1 plays a critical role. Knockout mice that lack this cytokine have absolutely no Langerhans cells in their epidermis (Borkowski et al., 1996, 1997). The immigration of dendritic cell precursors into the skin has been visualized in real

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time by a fascinating new video technique (Robert et al., 1999): fluorescencetagged, intravenously injected CD1 l+/CDla+ precursors, that had recently been described to be likely candidates for Langerhans cell precursors in the blood (Ito et al., 1999) extravasated into the dermis, dependent on the selectin expression of the dermal blood vessels; the epidermis was not specifically investigated in that study. The second aspect is rarely thought of. Langerhans cells must not rest in the epidermis without moving; rather, they need to move like a runner on a running machine. Keratinocytes are constantly dividing and moving upward within the epidermis. Langerhans cells, in contrast, normally stay in their suprabasal location and are not sloughed off like the dead stratum comeum cells. This implies that the cellular contacts between Langerhans cells and surrounding keratinocytes must be of dynamic nature so that Langerhans cells can “run against the current.” Virtually nothing is known about this type of “relative migration.” Clearly, E-cadherin, the molecule mediating the binding of Langerhans cells to keratinocytes (Tang et al., 1993; Blauvelt et al., 1995; Udey, 1997; Jakob et al., 1999) must be involved. Another important type of migration in the skin is also not dealt with here. It is the migration of monocytes across endothelial barriers and further into the lymph nodes. This model was established by Gwen Randolph who could show that monocytes can turn into dendritic cells upon transmigration through endothelium in vitro (Randolph et al., 1998b), but also in an in viva setting (Randolph et al., 1999).

IV. Routes

of Langerhans

Cell Migration

The path that Langerhans cells take to migrate from the epidermis to the lymph nodes has been well defined for the most part. After loosening the intercellular contacts to keratinocytes (E-cadherin), Langerhans cells begin to move actively. This becomes evident by scanning electron microscopy of skin explant cultures (Fig. 3) where one can see that Langerhans cells literally “squeeze” out between surrounding keratinocytes, rather than just fall out from a disintegrating epidermis. The first major obstacle for a migrating Langerhans cell is the basement membrane. This process can also be visualized in skin explant cultures, both by light (Fig. 4) and transmission electron microscopy (Fig. 5). Also the next part of the way appears to be a difficult one. Scanning electron microscopy illustrates impressively, that the Langerhans cell must make quite an effort to make its way through the dense dermal network of collagen fibrils (Fig. 6). Finally, the migrating Langerhans cell enters the dermal lymphatic vessel (Fig. 7). By electron microscopy one can sometimes observe large gaps in the endothelial layer that surrounds the lymph vessel (Fig. 8). Whether dendritic cells enter preferentially (or only) through these gaps or whether they enter (also) between tightly connected endothelial cells has not been studied. The only model in which to investigate this question would be the skin

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FIG. 3 A migratoq murinr Lungerhans cell “craw1~ out” from the epidrrmi.~. Scanning electron micrograph of a Langerhans cell emigrating from an epidermal sheet cultured for 2 days. A view of the basal side is presented; basement membrane is absent. Note that the layer of basal keratinocyte5 appears intact. Langerhans cells seem to “squeeze out” between the keratinocyteh. The Langerhans cells possess pronounced cytoplasmic processes, so-called “veils.” These structures indicate that the migratory Langerhans is mature. Final magnification, 2.800x; bar = IO km.

explant model in the mouse. Only there would one find enough dendritic cell-filled lymph vessels (“cords”) to make an ultrastructural evaluation feasible. The further pathway has not been directly observed. Presumably, migrating dendritic cells are carried away by the lymph current (Fig. 9). They are deposited in the subcapsular sinus of the lymph nodes and from there they migrate to the T-cell areas. Although generally assumed and most likely, it has never been formally proven that epidermal Langerhans cells really arrive in the draining lymph nodes. This is due to the fact that cell tracking experiments cannot be performed in humans where specific markers for Langerhans cells, which are discriminatory for other types of dendritic cells, specifically dermal dendritic cells, are obtainable. Such markers are not yet available for mice and rats. (With the development of antimouse Langerin antibodies by Sem Saeland’s group this will change very soon: J. Valladeau, J.-J. Pin, F. Koch, N. Romani, S. Lebecque, and S. Saeland, manuscript

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FIG. 4 A migrrrtoyy humnn Lan~erhans cell ctw~c~ the basement membrane. In this cryostat section of a skin explant that has been cultured for 2 days the collagenous part of the basement membrane is immunohistochemically identified with antibodies against collagen type IV (in blue: alkaline phosphatase), whereas Langerhans cells are stained with antibodies to MHC class II molecules (in red: peroxidase). The epidermis is above the basement membrane, the dermis beneath it. The Langerhans cell appears to penetrate the basement membrane at one point. (See color insert.)

in preparation, 2001). The common assay is painting a fluorescent contact allergen (e.g., FITC) onto the skin and then isolating FITC-bearing dendritic cells from the draining lymph nodes. Using this approach, Langerhans cells clearly disappear from the epidermis and FITC-bearing dendritic cells appear in the nodes. These cells were often assumed to be Langerhans cells: they may, however, also be dermal dendritic cells, at least in part. A long time ago, thorough investigators (Duijvestijn and Kamperdijk, 1982; Kamperdijk et al., 1978) noted cells with Birbeck granules in lymph nodes (Schuler et al., 1991). If this organelle is assumed to be an invariable tag for Langerhans cells, it is legitimate to conclude that Birbeck granule-containing cells have physically come from the epidermis. On the other hand, one cannot rule out that Birbeck granules may be induced outside the epidermal microenvironment (Schuler et al., 1991); clearly, Birbeck granules can form in dendritic cells derived from CD34+ progenitor cells in the absence of an epidermal milieu (Caux et al., 1996; Strobl et al., 1996). Macatonia et al. (1987), in their classical experiments, have used electron microscopy to look at dendritic cells in draining lymph nodes following painting of a fluorescent contact allergen onto the skin. Indeed, they found that a portion of allergen-bearing dendritic cells

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FIG.5 A migratory murine Langrrhans cell CYOSS~Sthe basement membrane. This transmission electron micrograph is from a skin explant that has been cultured for 2 days. It shows the very rare occasion in which a Langerhans cell (asterisk) is still within the epidermis; however, it has made a hole into the basement membrane (arrows) and already “sticks one leg” through it into the dermis. Final magnification, 6.600~: bar = 3 p,m.

also displayed Birbeck granules, suggesting that they may have come from the epidermis. Such important observations would need to be quantified. This is very cumbersome by electron microscopy and, therefore, nobody has done it. Again, such experiments will be possible soon with the advent of Langerhans cell-specific antibodies for the mouse (see above). E-cadherin was shown to be another marker specific for Langerhans cells. By means of this marker Borkowski and coworkers provided good evidence that a

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FIG. 6 A migrcrtop murinr dmdritic cell “struggles ” through the collugenous mrshwork ofthr dernris. Scanning electron microscopy of a dermal sheet from a whole skin explant cultured for 2 days. This micrograph shows how dense the dermal collagen network is, and suggests that moving through it is probably an active and energy-consuming proces. Scanning electron microscopy does not allow us to distinguish whether these cells are Langerhans cells or dermal dendritic cells. Final magnification, 13.000x: bar = 2 km.

substantial portion of dendritic cells in skin-associated lymph nodes originated in the epidermis. The percentage of E-cadherin-expressing dendritic cells increased on application of a contact sensitizer, strongly suggesting that this may indeed reflect newly arrived Langerhans cells (Borkowski et al., 1994). E-cadherin was

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FIG. 7 Cutaneous dendritic cells including marq Lmgerhans cells do not enter blood vessels. This human skin explant was cultured for 2 days and subsequently double stained for MHC class II (green fluorescence), identifying dendritic cells, and mAb PAL-E (red fluorescence), identifying specifically blood endothelial cells. Note that dendritic cells accumulate in vessel-like structures that do not stain with the marker for blood endothelia. Electron microscopy confirmed the conclusion that these vessels were lymph vessels (Fig. 9). (See color insert.)

not expressed on dendritic cells from other sources such as spleen or mesenterial lymph nodes.

V. Regulation

of Langerhans

Cell Migration

The pathway of Langerhans cells described above is highly regulated. It may be helpful to accompany a Langerhans cell on its way from the epidermis and to consider the regulatory mechanisms at each leg of the journey. Our contemplations extend two recent review articles on Langerhans cell migration (Kimber et al., 2000; Wang et al., 1999b).

A. Exit from the Epidermis Langerhans cells reside within the suprabasal layer of the epidermis. They are physically connected to their keratinocyte neighbors by homotypic E-cadherin bonds.

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FIG. 8 Dermul lyph vessel.c,~equenr~~ have lqye ,qqw. Through such gaps Langerhans cells might enter. This example is from a human skin explant. Final magnification, 19.000x: bar = I pm.

These bonds appear as “adherens junctions” by electron microscopy (Jakob et al., 1999). They need to be broken up before the Langerhans cell can move. E-cadherin expression on Langerhans cells was shown to be down-regulated on maturation in vitro (Tang et al., 1993; Blauvelt et al., 1995). E-cadherin on Langerhans cells was also reduced in response to the application of contact sensitizers (Schwarzenberger and Udey, 1996), that is, in response to migration-inducing substances. Further dissection of this process revealed inflammatory cytokines IL- 1B and TNF-CI as responsible cytokines (Schwarzenberger and Udey, 1996). Thus, it would appear that the action of inflammatory cytokines makes the Langerhans cells “ready to go.” Observations in human Langerhans cell histiocytosis, a proliferative disease of Langerhans cells (Lieberman et al., 1996; Egeler and Nesbit, 1995), seem to confirm the experimental data: the dissemination of Langerhans cell histiocytosis

FIG. 9 Migrcrtory murinr dendritic w//s crcc~umulorein md truval fhrough a /.wnphatic vr.s.vel.This electron micrograph is from a whole skin explant that has been cultured for 2 days. Note the wide lymphatic vessel beneath the epidermis (lefthand side). It is lined by a one-layered thin endothelium and is filled with cells that display pronounced “veils.” i.e., thin and long cytoplasmic extensions. This is characteristic for mature dendritic cells. Several of these cella cells contain small Birbeck granules indicating that they are Langerhans cells. Final magnification, I .230x; bar = I5 pm.

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cells was negatively correlated with E-cadherin expression on these cells as determined by immunohistochemistry on patients’ biopsies (Geissmann et al., 1997). In other words, the less E-cadherin the cells expressed, the more they broke away from the original (proliferating) cell aggregate and spread out. Recent experiments indicate that CD40-CD40 ligand (CD 154) interactions may play a role (Moodycliffe et ul., 2000). Emigration of Langerhans cells from the epidermis in the contact hypersensitivity model is markedly inhibited in CD40 ligand knockout mice. The authors discuss that this may be due the missing ligation of CD40 on keratinocytes that in turn would trigger TNF-a production by keratinocytes. The cellular source for the CD40 ligand remains unclear though. The fact that Langerhans cells disappear from the epidermis on antigenic challenge has been observed repeatedly. In the contact hypersensitivity model Paul Bergstresser and colleagues (1980) first revealed that the numbers of Langerhans cells in the epidermis drop l-2 days after application of contact sensitizers. This was confirmed several times by others (Botham etal., 1987; Weinlich et al., 1998) also in the skin explant model, where this drop in Langerhans cells is even more pronounced (Larsen et al., 1990), and in grafted pieces of skin (Larsen et al., 1990).

B. Crossing

of the Basement

Membrane

Basement membranes are intricately organized structures that connect epithelia with underlying connective tissue (Timpl, 1996). The electron-dense “lamina densa” consists mainly of type IV collagen and it represents a physical barrier for migrating cells. This implies that enzyme systems must be operative that help to “make holes” into this membrane where the cells can to go through (Fig. 5). There is ample evidence that the matrix metalloproteinase (MMP) enzyme system is critically involved in the penetration of basement membranes by migrating cells (Murphy and Gavrilovic, 1999; Shapiro, 1998); some MMPs, e.g., MMP-9 and MMP-2, possess a substrate specificity for collagen type IV. There is now also good evidence that the MMPs play an important role in the migration of Langerhans cells through the basement membrane and further through dermal connective tissue. Kobayashi ef al. ( 1994) have first shown that human Langerhans cells can invade artificially reconstituted basement membranes. Eventually, it was demonstrated that Langerhans cells express MMPs (Uchi et al., 1998; Kobayashi et al., 1999; Ratzinger et al., 2000). These MMPs, in particular MMP-9, are functionally involved in the migration of Langerhans cells as may be concluded from some recent sets of data: injection of blocking anti-MMP-9 antibodies into the skin before the application of a contact sensitizer prevented the emigration of Langerhans cells from the epidermis (Kobayashi et al., 1999). Inhibitors of MMPs dramatically reduce the numbers of dendritic cells (including Langerhans cells) that emigrate from skin explants (Lebre et al., 1999; Ratzinger et al., 2000). Finally, migration of Langerhans cells in skin explants from mice deficient for gelatinase B/MMP-9

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is greatly diminished (Ratzinger et al., 2000). The MMP-9 system is activated by the same inflammatory cytokines (TNF-o, IL-IB) (Zhang et al., 1998) that also mediate the down-regulation of E-cadherin, one step earlier (see above). It has not been directly shown that this is also the case in skin, but it is very likely, based on the following observations: Kobayashi et al. (1994) had originally noted that Langerhans cells invade basement membranes in a greatly enhanced manner when treated with contact sensitizing agents that are well known to induce TNF-cx and IL- 1B and other inflammatory mediators (Enk and Katz, 1992; Enk et al., 1993; Rambukkana et al., 1996). Some preliminary experiments using mice that are deficient for various components of the plasminogen activator/urokinase enzyme system (Chapman, 1997) suggest that this enzymatic cascade may also be involved in penetration of the basement membrane (P. Stoitzner, B. Binder, et al., unpublished observations, 1998). Dendritic cells (albeit from the blood) do express the urokinase receptor, CD87 (Ebner et al., 1998). On human cord blood-derived dendritic cells, this molecule was recently shown to be of functional importance in the transmigration through endothelia and the invasion into extracellular matrix (Ferrer0 et ul., 2000). Whether or not components of the plasminogen system are expressed specifically in or on Langerhans cells is not known. Preliminary data indicate that CD87 may indeed be expressed in a focal pattern on Langerhans cells in human skin explant cultures (N. Sepp, unpublished data, 1996).

C. Moving through

the Dermal Connective

Tissue

As can be seen from Fig. 6, the way from the dermal side of the basement membrane to the closest lymphatic vessel is not a straightforward one. The picture illustrates that there is still a bit of work to do for the migrating Langerhans cell until it may enter the lymph vessel (Fig. 8), relax and get carried away by the lymphatic stream (Fig. 9). Movement through the collagenous and elastic fiber meshwork is again regulated by various molecular interactions. Clearly, the above-mentioned MMP and plasminogen activator multienzyme systems play a role. Integrin molecules are expressed on Langerhans cells (Aiba et al., 1993; Romani et al., 1989b; Teunissen et al., 1990) and are therefore expected to be important. Indeed, this has been shown directly. Price et al. (1997) were able to strongly inhibit the migration of Langerhans cells in skin explants and in viva in response to intradermal injection of TNF-IX with antibodies to a (~6integrin. Antibodies to (~4intergrins had no effect on the migration. Many other integrin molecules have not yet been studied in that regard. The work of Price et al. furthermore indicates that integrins are also involved in the transmigration of Langerhans cells through the basement membrane. When they blocked the (Y6integrin with antibodies, they observed that Langerhans cells in the epidermis were hardly reduced in numbers

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but they were rounded and had retracted their dendrites. This suggested that they were detached from keratinocytes and about to leave the epidermis, but could not get across the basement membrane. CD44 is a receptor molecule for hyaluronic acid, an important component of the dermal matrix. This molecule is expressed on migrating Langerhans cells and it is functional: the addition of blocking antibodies to skin explant cultures could strongly inhibit the emigration of Langerhans cells from the epidermis, again suggesting that this molecule also helps in traversing the basement membrane (Weiss et al., 1997). The CD44 molecules involved in migration are up-regulated on maturation of dendritic cells.

D. Entry into Lymphatic

Vessels

Almost nothing is know about the molecules that are involved in the process of “intravasation,” that is, the entry of Langerhans cells into the lymph vessels. The vessel wall of superficial dermal lymphatics is very thin; only one layer of thin endothelial cells, which is frequently interrupted by wide gasp (Fig. S), delimits the lumen. It has not been studied whether the selectins, CD3 1, integrins, or other molecules play a role. Clearly, selectins are necessary for the interaction of dendritic cells with skin blood endothelial cells (Robert et al., 1999). The reason for the paucity of data on this particular point is the fact that there are no well-established lymph endothelial cell lines that could be used for appropriate studies in vitro. One set of recent data, however, may bear on this point. Gwen Randolph’s group found that antagonists (antibodies, drugs) to the P-glycoprotein (MDR-I), a well-known transporter that mediates effIux of chemotherapeutic agents from the intracellular milieu and thereby contributes to drug resistance, strongly inhibited transmigration of dendritic cells through (blood) endothelial cells from the ablumenal to the lumenal side (Randolph et al., 1998a). This would mimic the entry of a dendritic cell into the vessel. The MDR-1 anatagonists also inhibited efflux of Langerhans cells from the epidermis and the up-regulation of MHC class II on these Langerhans cells. Recently, leukotrienes were shown to be carried by these transporters and to promote optimal chemotaxis to MIP-3@CCL19 (Robbiani et rzl., 2000).

E. Arrival

in Lymphatic

Organs

Very little is known about this last leg of the journey of a Langerhans cell. It is presumably “spilled into” the lymph node in a subcapsular sinus. Most likely, adhesive interactions (selectins, integrins, etc.) are again important in order to get from there to the T-cell area where it finally ends up as an interdigitating cell that finds and binds antigen-specific T cells.

256 F. Driving and Directing of Langerhans Cells

NIKOLAUS ROMANIETAL.

Forces behind Migration

Up to now we have discussed mainly which molecular interactions may be involved in releasing the Langerhans cell from its epidermal neighbors, enabling it to “melt” holes into the basement membrane in order to get through it, and allowing it to move through the connective tissue. But how does the Langerhans cell know where to go? What, in the first place, tells it to go “down” in the epidermis? The Langerhans cell does not wander around in the dermal thicket without any sense of direction. What guides it toward the lymphatic vessels? The answer to these questions is simple: chemokines (Rossi and Zlotnik, 2000). However, it is very difficult-if not impossible-to answer in detail the question of how the actions of the various (known and not yet known) chemokines are coordinated. This has been attempted in a few excellent recent reviews (Kimber et al., 2000; Cyster, 1999; Sallusto and Lanzavecchia, 1999; Sozzani et al., 1999). Yet, some key chemokines that are relevant for the migration of Langerhans cells have emerged. Mature dendritic cells (Dieu et al., 1998) including Langerhans cells (Yanagihara et al., 1998; Saeki et al., 1999) express chemokine receptor-7 (CCR-7) which exclusively binds the chemokine macrophage inflammatory protein-3B (MIP-3B), also termed “Epstein-Barr virus-induced molecule I ligand chemokine” (ELC) and the “secondary lymphoid tissue chemokine” (SLC). According to the recently introduced nomenclature for chemokines (Zlotnik and Yoshie, 2000) these chemokines are designated CCL 19 and CCL2 1, respectively. Immature dendritic cells do not express CCR-7. These two chemokines may act at two levels. First, endothelial cells contain, and probably also secrete, SLC/CCL21 (Saeki et al., 1999). This may direct Langerhans cells toward the vessels. In a skin explant model, these authors and others (Stoitzner et al., 2001) could demonstrate a strong chemoattracting capacity of this chemokine for cutaneous dendritic cells including Langerhans cells. Second, MIP-3B/CCL19 also attracts Langerhans cells in the skin explant model (Kellermann et al., 1999). This chemokine was localized to the T-cell-rich areas of the lymph nodes (Dieu et al., 1998). Therefore, it appears that in viva MIP-3B/CCL 19 draws those Langerhans cells that are discharged from the afferent lymph into the subcapsular sinus toward the T-cell-rich areas. Recently, another group of chemokines was identified as a player in this game: mice rendered deficient for the chemokine receptor CCR2, that binds MCP (macrophage chemoattractant protein) chemokines showed normal emigration of Langerhans cells from the epidermis into the dermis; they did not move on to the lymph nodes though, but presumably accumulated in dermal lymphatics. This suggests that CCR2-binding chemokines attract Langerhans cells to the dermal vessels and/or to the T-cell areas in the nodes (Sato et al., 2000). CCRS and its ligand MIP-la had less pronounced effects. Not only chemokines but also cytokines with chemoattracting properties seem to be involved. We have evidence that IL- I6 enhances the emigration of Langerhans cells from the epidermis (Stoitzner et ul.,

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2001). In viva, this cytokine may be provided by T cells that happen to be in the dermis.

VI. Relevance

of Langerhans

Cell Migration

A. Generally in Vim Many experiments have shown the importance of migrating dendritic cells for the generation of immunity. The necessity for migrating Langerhans cells was demonstrated in the classical contact hypersensitivity experiments of Paul Bergstresser and colleagues. Essentially, they found that the depletion of Langerhans cells from the epidermis by literally “pulling them out” with adhesive tape (“tape stripping”) abrogated the development of contact hypersensitivity (Toews et al., 1980; Streilein et al., 1982). Epithelia that are naturally devoid of Langerhans cells such as the central portion of the cornea of the eye (Streilein et al., 1979) are not immunogenic when transplanted (Peeler and Niederkorn, 1987). 1. Langerhans Cell Migration

and Tumor Immunity

Although hard to prove, one may assume that migrating dendritic cells including Langerhans cells are crucial for a well-functioning “immune surveillance.” This may be particularly important in the case of tumors. Many studies deal with the question, whether and to what extent tumors are populated with dendritic cells, in the case of carcinomas with Langerhans cells (Bell et al., 1999b; Sprinzl et al., 2000; Wischatta et al., 2000). Correlations with the clinical course are attempted. It is generally assumed that the more dendritic cells a tumor contains, the better it will be presented to T cells and, as a consequence, the better the prognosis will be (Goldman etal., 1998; Saito et&., 1998). Due to the relatively low numbers of cases in some studies and to the difficulties in accurately quantifying small numbers of cells in immunohistochemistry, such correlations need to be viewed with caution. Nevertheless, experimental data support the clinical observations: when tumor cells were transduced with MIP-3oYCCL20 (Fushimi et al., 2000), the chemokine that specifically attracts Langerhans cells precursors (Carramolino et al.. 1999; Dieu et al., 1998), an influx of dendritic cells into the tumors was noted, and, moreover, growth of these transduced tumors was significantly inhibited. Another cytokine that appears to make Langerhans cells migrate into the epidermis is IL- 12. This is in contrast to flt-3 ligand, which leads to the accumulation of dendritic cells in the dermis when given systemically (Esche et al., 1999). Tumors were not looked at in these studies. Thus, the tumor’s cytokine milieu probably determines whether it harbors many or few dendritic cells, in particular, Langerhans cells. The tumors’ cytokine milieu not only determines whether Langerhans cell precursors come into the tumor but also whether tumor-resident Langerhans cells are

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able to do their job; that is, to take up tumor antigens, process them, and migrate to the draining lymph nodes in order to elicit a successful antitumor response. It has repeatedly been described that tumors try to escape from being presented to the immune system by elaborating immunosuppressive cytokines such as IL- 10 (Chen et al., 1994; Dummer et al., 1996), VEGF (Saito et al., 1998; Gabrilovich et al., 1996), TGF-B (Bodmer et al., 1989), or others. For IL-10 it has been shown that it acts (negatively) on migrating Langerhans cells (Wang et al., 1999a). Nothing is known about the other cytokines in that particular regard. 2. Langerhans Cell Migration

and Microbial Infections

The immune system has evolved as a defense against infectious organisms, mainly microbes. Langerhans cells are residents of the most peripheral tissue of the body and are therefore predestined to serve in the uptake, processing, and transport of the microorganisms. A few examples underscore that this occurs efficiently in viva. Heidrun Moll’s group was among the very first to trace Langerhans cells along their path from the epidermis to the lymph nodes. They could show that Langerhans cells take up Leishmania organisms and carry them through the dermis to the draining nodes (Blank et al., 1993; Moll et al., 1993). Very recently, Sally SchlessingerFrankel and coworkers showed that Langerhans cells can be efficiently infected by Dengue virus and that they carry the virus from the epidermis into the culture medium in skin explant cultures (Wu et al., 2000).

E3. In Vaccination

Protocols

for Immunization

As mentioned earlier, dendritic cells are being used in clinical trials with the aim of generating effective antitumor responses (Schuler and Steinman, 1997; Fong and Engleman, 2000; Morse and Lyerly, 1999; Timmerman and Levy, 1999). First results are published, and they are encouraging (Thurner et al., 1999; SchulerThurner et al., 2000). In most studies ex viva-generated, antigen-laden dendritic cells are administered intracutaneously, be it subcutaneously or intradermally. The intravenous route has also been taken. The assumption is that dendritic cells placed into the skin migrate to the draining lymph nodes. This assumption has been proven many times starting from the classical experiments of Fossum (1988) and Austyn et al. (1988) to more recent models of immunization against model antigens (Inaba et al., 1990) or tumors (Zitvogel et al., 1996; Celluzzi et al., 1996; Lappin et al., 1999). Even though injected dendritic cells can be recovered from the draining lymph nodes, this process is far from being efficient in quantitative terms. Very few studies have investigated the fate of radioactively (typically indium) labeled, injected dendritic cells in human patients undergoing immunotherapy. Interestingly, most of the injected radioactively labeled cells remained at the injection site; only a minor percentage made its way to the node (Morse et al., 1999). This is basically the same as previously found in the mouse (Inaba et al., 1992;

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Lappin et al., 1999). These data emphasize the need for a better understanding of the regulation of dendritic cell migration in order to be able to improve it. Some recent data appear promising in that regard: Josien and colleagues studied mouse dendritic cells that had been pretreated with a TRANCE, tumor necrosis factor [TNF]-related activation-induced cytokine, a new TNF family member predominantly expressed in T cells (Wong et ~zl., 1997). They noted that intradermally injected, fluorescently labeled TRANCE-treated bone marrow dendritic cells migrated to the draining lymph nodes strikingly better than untreated control dendritic cells (Josien et al., 2000). Thus, it appears that there are ways to therapeutically improve dendritic cell migration and, as a consequence, immunogenicity. With regard to Langerhans cells it remains to be determined whether these epidermal dendritic cells have particular migration properties as compared to dendritic cells from other, experimentally more convenient sources.

C. For the Maintenance

of Induction

of Tolerance

Peripheral tolerance mechanisms are important to keep in check unwanted immune responses against antigens that have escaped central tolerance in the thymus. These are mainly molecules that are not present in the thymus and for which T cells can therefore not be negatively selected. One mechanism by which peripheral tolerance operates is T-cell anergy (Steinman et al., 2000). If T cells are presented antigen, i.e., MHC/peptide complexes (“signal 1”) in the absence of costimulatory signals (“signal 23, an antigen-specific anergic state of the T-cells ensues. How could Langerhans cells accomplish a tolerogenic function? Most of our current evidence holds that Langerhans cells mature as they migrate (Weinlich et al., 1998). Therefore, they would present antigen together with costimulation (e.g., CD80, CD86) and, as a consequence, they would immunize. Two tolerance-inducing scenarios that may involve migrating Langerhans cells are presently being discussed. Some evidence for each of them has been corroborated. First, MacPherson’s group has recently shown in the rat that dendritic cells may also migrate without concomitant maturation (Huang et al., 2000). These immature migratory dendritic cells were collected from the lymph, and they were found to carry antigenic material, in particular apoptotic cells, from the tissue they originally resided in (here, the gut). The mere uptake of apoptotic cells by dendritic cells in the absence of inflammatory mediators does not lead to their maturation (Sauter et al., 2000). Upon arrival in the lymph nodes such dendritic cells might tolerize rather than sensitize antigen-specific T cells. It has not been investigated whether small subsets of migrating Langerhans cells remain immature or whether some low-level Langerhans cell migration occurs also under noninflammatory conditions. The second scenario is based on much of Kayo Inaba’s and Ralph Steinman’s work (Steinman et al., 2000; lnaba et al., 1998, 2000). Immature dendritic cells can efficiently take up and present apoptotic cells. As discussed above, they may migrate without maturation to the nodes and induce tolerance directly. The main

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problem with this idea is that in the absence of inflammatory stimuli the ligands for the T-cell receptor (i.e., MHC/peptide complexes) are not formed efficiently (Inaba et al., 2000). A hypothesis was put forward (Steinman et al., 2000): dendritic cells carry antigenic MHC/peptide complexes from the periphery to the nodes. These dendritic cells are short lived and they die by apoptosis. Their apoptotic “corpses” are taken up by a subset of dendritic cells specialized for inducing tolerance rather than immunization. Stiss and Shortman (1996) have provided some evidence for such a subpopulation. In the human, dendritic cells derived from plasmacytoid precursors (“DC2”; Rissoan et al., 1999) may represent this subset. Nothing is known about this with regard to Langerhans cells.

VII. Concluding

Remarks

Much is now known about the emigration of Langerhans cells from the epidermis and their entry into lymphatic vessels and organs. The route of migration seems well defined. The regulation is complex and less well understood. Yet, one common denominator for all steps are injhmmatory cytokines, in particular TNF-(-w and IL- 1l3. These mediators, that would be induced by a microbial attack in vivo: Detach the Langerhans cell from the surrounding keratinocytes (via downregulation of E-cadherin). l Up-regulate enzyme molecules (MMPs) that allow it to penetrate the basement membrane. l Up-regulate adhesion molecules (integrins, CD44) that facilitate its movement through basement membrane and interstitial tissue. . Up-regulate chemokines that attract it to the lymph vessels and further into the T-cell area of the lymph node (MIP-3P/CCL19, SLCKCL21). l Up-regulate the respective chemokine receptors (CCR7, CCR2) on Langerhans cells. l

Understanding the “fine-tuning” of Langerhans cell migration by chemokines will still take some time and many experiments. In closing, we would like to emphasize the importance of learning more about the regulatory mechanisms in migration. This will enable us to improve dendritic cell migration in vivo and, as a consequence, the immunogenicity of therapeutically administered dendritic cells.

Acknowledgments The work on dendritic cell migration in the senior author’s lab over the years has been supported by grants from the Austrian Science Fund (FWF grants P-9967.Med and P- I2 I63-Med). We thank Konrad Eller, Department of Zoology, for expert low-power photography with the electron microscope. We appreciate the continuous support, discussions, and encouragement by Drs. Peter Fritsch, chairman

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of the Department of Dermatology, University of Innsbruck, and Ralph M. Steinman, Laboratory for Cellular Physiology and Immunology, The Rockefeller University, New York.

References Aiba, S., and Katz, S. 1. (1990). Phenotypic and functional characteristics of in vivo activated Langerham cells. J. lmmmunol.145, 2791-2796. Aiba. S., Nakagawa, S., Orawa, H., Miyake, K., Yagita. H., and Tagami. H. (1993). Upregulation of 4 integrin on activated Langerhans cells: Analysis ofadhesion molecules on Langerhans cells relating to their migration from skin to draining lymph nodes. J. I~n~esr.Denncrtol. 100, 143-147. Austyn, J. M. (1996). New insights into the mobilization and phagocytic activity of dendritic cells. J. Exp. Med. 183, 1287-1292. Austyn, J. M.. Kupiec-Weglinski, J. W., Hankins, D. F., and Morris, P. J. (1988). Migration patterns of dendritic cells in the mouse. Homing to T cell-dependent areas of spleen and binding within marginal zone. J. Exp. Med. 167,646-65 I. Banchereau. J.. and Steinman. R. M. (I 998). Dendritic cells and the control of immunity. N~~t~~rr ILmd.) 392,245-252. Banchereau, J.. Briere. E. Caux, C., Davoust, J., Lebecque, S., Liu, Y. T.. Pulendran, B., and Palucka. K. (2000). Immunobiology of dendritic cells. Annu. Rev. lmmuno/. 18,767-8 11. Bell, D., Young, J. W., and Banchereau, J. (I 999a). Dendritic cells. Arh Immunol. 72, 255-324. Bell. D.. Chomarat. P.. Broyles, D., Netto, G., Harb. G. M., Lebecque. S., Valladeau, J.. Davoust. J., Palucka, K. A., and Banchereau, J. (1999b). In breast carcinoma tissue, immature dendritic cells reside within the tumor. whereas mature dendritic cells are located in peritumoral areas. J. Eup. Med. 190, 1417-1425. Bergstresser, P. R., Toews, G. B., and Streilein, J. W. (1980). Natural and perturbed distributions of Langerhans cells: Responsesto ultraviolet light, heterotopic skin grafting. and dinitrofluorobenzene sensitization. J. Invest. Drrmatol. ‘75, 73-77. Birbeck, M. S., Breathnach, A. S., and Everall, J. D. (1961). An electron microscopic study of basal melanocytes and high level clear cells (Langerhans cell) in vitiligo. J. Itwest.Drrmatol.37,5 l-64. Blank, C., Fuchs. H., Rappersberger, K., Rollinghoff, M.. and Mall, H. (I 993). Parasitism of epidermal Langerhans ceils in experimental cutaneous Leishmaniasis with Leishmania major. .I. I@ct. Dis. 167,418-425. Blauvelt, A.. Katz, S. I., and Udey, M. C. (I 9Y5). Human Langerhans cells express E-cadherin. J. Invest. Dermutol. 104, 293-296. Bodmer, S., Strommer, K., Frei, K., Siepl, C.. De Tribolet, N., Heid, I., and Fontana, A. (1989). Immtmosuppression and transforming growth factor-B in glioblastoma. Preferential production of transforming growth factor-p 2. J. hnzwo/. 143, 3222-3229. Borkowski, T. A., Van Dyke, B. J., Schwarzenberger, K., McFarland, V. W.. Farr, A. G., and Udey. M. C. (1994). Expression of E-cadherin by murine dendritic cells: E-cadherin as a dendritic cell differentiation antigen characteristic ofepidermal Langerhans cells and related cells. Eur J. Immune/. 24,2761-2714. Borkowski, T. A., Letterio, J. J., Farr. A. G.. and Udey. M. C. (1996). A role forendogenous transforming growth factor-p 1 in Langerhans cell biology: The skin of transforming growth factor-p 1 null mice is devoid of epidermal Langerhans cells. J. Exp. Med. 184, 2417-2422. Borkowski, T. A., Letterio, J. J., Mackall, C. L.. Saitoh, A., Wang, X. J., Roop, D. R., Cress, R. E., and Udey, M. C. (I 997). A role for TGF 1 in Langerhans cell biology-Further characterization of the epidermal Langerhans cell defect in TGF I null mice. .I. C/in. Invest.100,575-58 I Botham, P. A., Rattray. N. J., Walsh, S. T., and Riley, E. J. (I 987). Control of the immune response to contact sensitizing chemicals by cutaneous antigen-presenting cells. Br: J. Drtwmtol. 117, I-9.

262

NIKOLAUSROMANI ETAL.

Carramolino, L., Kremer, L., Goya, I., Varona, R., Buesa, J. M., Gutierrez, J., Zaballos, A., Martinez, A., and Mkquez, G. (1999). Down-regulation of the chemokine receptor CCR6 in dendritic cells mediated by TNF-o and IL-4. J. Leukocyte Biol. 66,837-844. Caux, C., Derutter-Dambuyant. C., Schmitt, D.. and Banchereau, J. (1992). GM-CSF and TNF-a cooperate in the generation of dendritic Langerhans cells. Natare (Land.) 360,258-26 I, Caux, C., Vanbervliet, B.. Massacrier, C., Azuma, M.. Okumura, K.. Lanier. L. L., and Banchereau, J. (1994). B7O/B7-2 is identical to CD86 and is the major functional ligand for CD28 expressed on human dendritic cells. J. Exp. Med. 180, 184 l-1 847. Caux, C., Vanbervliet, B.. Massacrier, C., Dezutter-Dambuyant. C., De Saint-%. B., Jacquet, C., Yoneda, K., Imamura, S.. Schmitt, D., and Banchereau, J. (1996). CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF-o. J. Esp. Med. 184,695-706. Caux, C., Massacrier, C., Vanbervliet, B., Dubois, B., Durand, I., Cella, M., Lanzavecchia, A., and Banchereau, J. (1997). CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor-u. 2. Functional analysis. Blood 90, 14.58-1470. Celluzzi. C. M., Mayordomo, J. I., Storkus, W. J., Lotre, M. T., and Falo, L. D. (1996). Peptide-pulsed dendritic cells induce antigen-specific, CTL-mediated protective tumor immunity. J. Exp. Med. 183, 283-287. Chapman, H. A. (1997). Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration. Curr: @in. Cell ho/. 9,7 14-724. Charbonnier, A. S., Kohrgruber. N., Kriehuber, E., Sting], G., Rot, A., and Maurer, D. (1999). Macrophage inflammatory protein 3 is involved in the constitutive trafficking of epidermal Langerhans cells. J. Exp. Med. 190, 1755-1767. Chen, Q., Daniel, V., Maher, D. W., and Hersey, P. (1994). Production of IL-IO by melanoma cells: Examination of its role in immunosuppression mediated by melanoma. Int. J. Cancer56, 755-760. Cumberbatch, M., Fielding, I., and Kimber, 1. (1994). Modulation of epidermal Langerhans’ cell frequency by tumour necrosis factor-o. Immuno1og.v 81,395401. Cumberbatch, M., Griffiths, C. E., Tucker. S. C., Dearman, R. J., and Kimber, 1. (1999). Tumour necrosis factor-alpha induces Langerhans cell migration in humans. BI: .I. Dermntol. 141, 192-200. Cyster, J. G. (1999). Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs. J. Exp. Med. 189,447-450. De Saint-Us, B., Fugier-Vivier, I., Massacrier, C.. Gaillard, C., Vanbervliet, B., Aiit-Yahia, S., Banchereau, J., Liu. Y. J., Lebecque, S., and Caux, C. (1998). The cytokine prohle expressed by human dendritic cells is dependent on cell subtype and mode of activation. J. Immunol. 160, 1666-1676. Dieu, M. C.. Vanbervliet, B., Vicari, A., Bridon, J. M., Oldham, E., Ait-Yahia, S., Briere, F., Zlotnik, A., Lebecque, S., and Caux, C. (1998). Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J. E?rp.Med. 188, 373-386. Dieu-Nosjean, M. C., Massacrier, C., Homey, B., Vanbervliet, B., Pin, J. J.. Vicari, A., Lebecque, S., Dezutter-Dambuyant, C.. Schmitt, D., Zlotnik, A., and Caux, C. (2000). Macrophage inflammatory protein-3 alpha is expressed at inflamed epithelial surfaces, and is the most potent chemokine known in attracting Langerhans cell precursors. J. Exp. Med. 192,705-718. Duijvestijn, A. M., and Kamperdijk, E. W. A. (1982). Birbeck granules in interdigitating cells of thymus and lymph node. (abstract). Cell Biol. Int. Rep. 6,655. Dummer, W.. Bastian, B. C., Ernst, N., Schanzle, C.. Schwaaf, A., and Brocker, E. B. (1996). Interleukin- 10 production in malignant melanoma: Preferential detection of IL- I O-secreting tumor cells in metastatic lesions. hr. J. Cancer 66, 607-610. Ebner, S., Lenz, A., Reider, D., Fritsch, P., Schuler, G., and Romani, N. (1998). Expression of maturation-/migration-related molecules on human dendritic cells from blood and skin. lmmunobiology 198,568-587.

MIGRATIONOFDENDRITIC CELLSINTOLYMPHATICS Egeler, R. M., and Nesbit, M. E. (1995). Langerhans cell histiocytosis and other disorders ofmonocytehistiocyte lineage. Crit. Rev Oncol. Hematol. l&9-35. Enk, A. H., and Katz, S. 1. (1992). Early molecular events in the induction phase of contact sensitivity. Proc. Nurl. Acad. Sci. USA 89, 1398-1402. Enk, A. H., Angeloni, V. L.. Udey, M. C.. and Katz, S. 1. (1993). An essential role for Langerhans cell-derived IL-ID in the initiation of primary immune responses in skin. J. f?nntunol. 150, 3698-3704.

Esche. C., Subbotin, V. M.. Hunter, O., Peron, J. M., Maliszewski, C.. Lotre, M. T., and Shurin, M. R. (1999). Differential regulation of epidermal and dermal dendritic cells by IL- 12 and Flt3 Ii&and. J. Invest. Drrmnlol. 113, 1028-l 032. Ferrero, E., Vettoretto, K., Bondanra, A.. Villa, A., Resnati, M., Poggi, A., and Zocchi, M. R. (2000). uPA/uPAR system is active in immature dendritic cells derived from CDl4+CD34’ precursors and is down-regulated upon maturation. J. Immunol. 164, 7 12-7 18. Fong, L., and Engleman, E. G. (2000). Dendritic cells in cancer immunotherapy. Anrzu. Rev Immunol. 18,245-273. Fossum, S. (1988). Lymph-borne dendritic leucocytes do not recirculate, but enter the lymph node paracortex to become interdigitating cells. Stand. J. fmmunol. 27,97-106. Fried], P.. Zanker, K. S., and BrGcker, E. B. (1998). Cell migration strategies in 3-D extracellular matrix: Differences in morphology, cell matrix interactions, and integrin function. Microsc. Res. Tech. 43, 369-378.

Fushimi, T., Kojima, A., Moore, M. A. S., and Crystal, R. G. (2000). Macrophage inflammatory protein 301 transgene attracts dendritic cells to established murine tumors and suppresses tumor growth. J. Clirl. Invesr. 105, 1383-1393. Gabrilovich, D. I., Chen. H. L., Cunningham, H. T., Meny, G. M., Nadaf, S.. Kavanaugh, D., and Carbone, D. P. (1996). Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells. Nurture Med. 2, 1096-I 103. Geijtenbeek, T. B. H., Kwon, D. S., Torensma, R., Van Vliet. S. J.. Van Duijnhoven, G. C. E, Middel, J., Cornelisaen, 1. L. M. H., Nottet, H. S. L. M., KewalRamani, V. N., Littman, D. R., Figdor, C. G., and Van Kooyk, Y. (2000a). DC-SIGN, a dendritic cell-specific HIV-l-binding protein that enhances tmns-infection of T cells. Cell 100, 587-597. Geijtenbeek, T. B. H., Torensma. R., Van Vliet, S. J., Van Duijnhoven, G. C. F., Adema. G. J., Van Kooyk, Y., and Figdor, C. G. (2000b). Identification of DC-SIGN, a novel dendritic cell-specific ICAM- receptor that supports primary immune responses. Cell 100,.575-585. Geissmann, E, Emile. J. F., Andry, P.. Thomas, C., Fraitag, S., De Prost, Y., and Brousse, N. (1997). Lack of expression of E-cadherin is associated with dissemination of Langerhans’ cell histiocytosis and poor outcome. J. Pathol. 181, 301-304. Goldman, S. A., Baker, E., Weyant. R. J., Clarke, M. R.. Myers. J. N.. and Lotze, M. T. (1998). Peritumoral CDla-positive dendritic cells are associated with improved survival in patients with tongue carcinoma. Arch. Otolar?;ngol. Head Neck Surg. 124, 641-646. Gunzer, M., Fried]. P., Niggemann, B., BrGcker. E. B., KBmpgen, E., andZLnker, K. S. (2000). Migration of dendritic cells within 3-D collagen lattices is dependent on tissue origin, state of maturation, and matrix structure and is maintained by proinflammatory cytokines. J. Leukocyre Biol. 67, 622629.

Hart, D. N. J. (1997). Dendritic cells: Unique leukocyte populations which control the primary immune response. Blood 90,3245-3287. Heufler, C., Koch, F.. and Schuler, G. (1988). Granulocyte-macrophage colony-stimulating factor and interleukin-1 mediate the maturation of murine epidermal Langerhans cells into potent immunostimulatory dendritic cells. J. Exp. Med. 167,70@705. Hoefakker, S., Balk, H. P., Boersma, W. J. A., Van Joost, T., Notten, W. R. F., and Claassen, E. (1995). Migration of human antigen-presenting cells in a human skin graft onto nude mice model after contact sensitization. immunology 86, 296-303.

264

NIKOLAUS ROMANI ETAL.

Huang, F. P., Platt. N., Wykes, M., Major, J. R., Powell, T. J.. Jenkins, C. D., and MacPherson, G. G. (2000). A discrete subpopulation of dendritic cells transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes. J. Exp. Med. 191,435%443. Inaba, K., Metlay. J. P., Crowley, M. T., and Steinman, R. M. (1990). Dendritic cells pulsed with protein antigens in vitro can prime antigen-specific. MHC-restricted T cells in situ. J. Ex/J. Mrd. 172,63 I-640. Inaba, K., Steinman, R. M., Pack, M. W., Aya, H.. Inaba. M., Sudo, T., Wolpe, S., and Schuler, G. (1992). Identification of proliferating dendritic cell precursors in mouse blood. J. EXLJ.Med. 175, 1157-l 167. Inaba, K., Inaba, M., Deguchi, M., Hagi, K., Yasumizu, R.. Ikehara, S., Muramatsu, S., and Steinman, R. M. (I 993). Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class B-negative progenitor in mouse bone marrow. Puoc. Ntrrl. Arcid. Sci. USA 90,3038-3042. Inaba, K.,Turley, S.. Yamaide. F., Iyoda, T., Mahnke, K., Inaba. M., Pack, M., Subklewe, M., Sauter, B., Sheff, D., Albert, M., Bhardwaj, N., Mellman, I., and Steinman, R. M. (1998). Efficient presentation of phagocytosed cellular fragments on the major histocompatibility complex class II products of dendritic cells. .I. E.xp. Med. 188, 2 163-2 173. Inaba, K., Turley, S., Iyoda, T., Yamaide. F.. Shimoyama, S., Sousa, C. R. E., Germain, R. N., Mellman, I., and Steinman, R. M. (2000). The formation of immunogenic major histocompatibility complex class II-peptide ligands in lysosomal compartments of dendritic cells is regulated by inflammatory stimuli. .I. &J. Med. 191,927-936. Ito, T., Inaba, M., Inaba, K., Toki, J., Sogo. S., Iguchi, T., Adachi, Y., Yamaguchi, K., Amakawa, R., Valladeau, J., Saeland, S., Fukuhara. S., and Ikehara, S. (1999). A CDla’/CDI Ic’ subset of human blood dendritic cells is a direct precursor of Langerhans cells. J. /mnunol. 163, 1409-1419. Jakob, T., Brown, M. J., and Udey. M. C. (1999). Characterization of E-cadherin-containing junctions involving skin-derived dendritic cells. J. Invesr. Dennurol. 112, 102-108. Jiang, W., Swiggard, W. J., Heufler, C., Peng. M., Mirza, A.. Steinman, R. M., and Nussenaweig, M. C. (1995). The receptor DEC.205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing. Nature (Land.) 375, 151-155. Josien. R., Li, H. L.. Ingulli, E., Sarma. S., Wong, B. R., Vologodskaia, M., Steinman, R. M., and Choi, Y. (2000). TRANCE, a tumor necrosis factor family member. enhances the longevity and adjuvant properties of dendritic cells in viva. J. Exp. Med. 191,495-SO I. Kamperdijk, E. W. A., Rdaymakers, E. M., de Leeuw, J. H. S., and Hoefsmit, E. C. M. (1978). Lymph node macrophagea and reticulum cells in the immune response. I. The primary response to parathyphoid vaccine. Cell Tissue Rec. 192, l-23. Kashihara, M., Ueda, M., Horiguchi, Y.. Furukawa. F.. Hanaoka. M., and Imamura. S. (1986). A monoclonal antibody specifically reactive to human Langerhans cells. J. Invesf. Dermarol. 87,602612. Kellermann, S. A., Hudak. S., Oldham, E. R., Liu, Y. J., and McEvoy. L. M. (1999). The CC chemokine receptor-7 ligands 6Ckine and macrophage inllammatory protein-3P are potent chemoattractants for in vitro- and in vivo-derived dendritic cells. J. Immune/. 162, 3859-3864. Kimber, I., Cumberbatch, M., Dearman, R. J., Bhushnan, M., and Grifhths, C. E. M. (2000). Cytokines and chemokines in the initiation and regulation of epidermal Langerhana cell mobilization. BI: .I. Dermatol. 142,401-412. Kobayashi, Y., Staquet, M.-J., Dezutter-Dambuyant. C., and Schmitt, D. (1994). Development of motility of Langerhans cell through extracellular matrix by in vitro hapten contact. Eur: J. Immunol. 24, 2254-2257. Kobayashi. Y., Matsumoto, M.. Kotani, M., and Makino, T. (1999). Possible involvement of matrix metalloproteinaae-9 in Langerhana cell migration and maturation. J. Immunol. 163,5989-5993. Koch, F., Heufler, C.. Kampgen, E., Schneeweiss, D., Bock, G., and Schuler. G. (1990). Tumor necrosis factor alpha maintains the viability of murine epidermal Langerhans cells in culture but in contrast

MIGRATION OFDENDRITIC CELLS INTO LYMPHATICS

265

to granulocyte/macrophage colony-stimulating factor without inducing their functional maturation. J. Exp. Med. 171, 159-171.

Koch, E, Klmpgen, E., Schuler, G., and Romani, N. (2001). Isolation, enrichment and culture of murine epidermal Langerhans cells. In “Dendritic Cell Protocols” (A. .I. Stagg and S. Robinson Eds.) in press. Humana Press. Totowa, NJ. Kondo, M., Scherer, D. C., Miyamoto. T.. King, A. G., Akashi, K., Sugamura, K., and Weissman, 1. L. (2000). Cell-fate conversion of lymphoid-committed progenitors by instructive actions of cytokines. Nurure (Lorzd.) 407,3X3-386.

Kripke, M. L., Munn, C. G., Jeevan, A., Tang, J.-M.. and Bucana. C. (I 990). Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization. J. lmmunol. 1452833-283X. Krueger, G. G., Daynes, R. A., and Mansoor, E. (1983). Biology of Langerhans ceils: Selective migration of Langerhans cells into allogeneic and xenogeneic grafts on nude mice. Pmt. Narl. Acad. Sci. USA 80, 1650-I 654. Langerhans, P. (186X). iiber die Nerven der menschlichen Haut. Virchoms Arch. [A] 44,325-337. Lappin, M. B., Weiss, J. M., Delattre, V., Mai, B., Dittmar, H., Maier, C., Manke, K., Grabbe, S., Martin, S., and Simon, J. C. (1999). Analysis of mouse dendritic cell migration in viva upon subcutaneous and intravenous injection. Immunology 98, 181-188. Larsen, C. P., Steinman, R. M.. Witmer-Pack, M., Hankins, D. E, Morris, P. J.. and Austyn, J. M. (I 990). Migration and maturation of Langerhans cells in skin transplants and explants. .I. E.xp. Med. 172, 1483-1493. Lebre, M. C., Kalinski, P., Das, P. K., and Everts. V. (1999). Inhibition of contact sensitizerinduced migration of human Langerhans cells by matrix metalloproteinase inhibitors. Arch. Drrmatol. Res. 291,447-452. Lenz, A., Heine, M., Schuler, G.. and Romani, N. (1993). Human and murine dermis contain dendritic cells. Isolation by means of a novel method and phenotypical and functional characterization. J. Clin. lnvrst.

92, 2587-2596.

Lieberman, P. H., Jones, C. R., Steinman, R. M., Erlandson, R. A., Smith, J., Gee, T., Huvos, A., Garin-Chesa, P., Filippa, D. A., Urmacher, C., Gangi, M. D., and Sperber. M. (1996). Langerhans cell (eosinophilic) granulomatosis-A clinicopathologic study encopassing 50 years. Am. J. Surg. Pathol. 20.5 19-552. Lotze, M. T., and Thomson, A. W. (Eds.) (1999). “Dendritic Cells: Biology and Clinical Applications.” Academic Press, San Diego. Lo&e, M. T., and Thomson. A. W. (Eds.) (2001). “Dendritic Cells: Biology and Clinical Applications,” 2 Ed. Academic Press, San Diego. Lukas, M., Stiissel, H., Hefel, L., Imamura, S., Fritsch, P., Sepp, N. T., Schuler, G., and Romani, N. (I 996). Human cutaneous dendritic cells migrate through dermal lymphatic vessels in a skin organ culture model. J. Invest. Dermarol. 106, 1293-1299. Macatonia. S. E., Knight, S. C., Edwards, A. J., Griffitha, S., and Fryer, P. (I 987). Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. J. Exp. Med. 166, 1654-1667. Matsuno, K., and Ezaki, T. (2000). Dendritic cell dynamics in the liver and hepatic lymph. ht. Reu Cytol. 197, X3-136. Maurer, D., and Stingl, G. (1999). Dendritic cells in the context of skin immunology. In “Dendritic Cells: Biology and Clinical Applications.” (M. T. Lotze and A. W. Thomson Eds.). pp. 1 I l-122. Academic Press, San Diego. Mall, H., Fuchs, H., Blank, C., and Railinghoff, M. (1993). Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen-specific T cells. EUK J. Immunol. 23,1595-l 601. Mall, H. (Ed.) (1995). “The Immune Functions of Epidermal Langerhans Cells,“pp. l-193. SpringerVerlag, Berlin.

266

NIKOLAUSROMANIETAL.

Moodycliffe, A. M., Shreedhar, V., Ullrich, S. E., Walteracheid. J., Bucana, C., Kripke, M. L., and Flares-Romo. L. (2000). CD40-CD40 ligand interactions in viva regulate migration of antigenbearing dendritic cells from the skin to draining lymph nodes. J. Exp. Med. 191,201 I-2020. Morse, M. A., and Lyerly. H. K. (1999). Dendritic cell-based immunization for cancer therapy. Ads Exp. Med. Biol. 465,335-346. Morse, M. A., Coleman, R. E., Akabani, G., Niehaua, N., Coleman, D.. and Lyerly, H. K. (1999). Migration of human dendritic cells after injection in patients with metastatic malignancies. Cancer Res. 59, 56-58. Murphy, G., and Gavrilovic, J. (1999). Proteolysis and cell migration: Creating a path‘? Curr: @in. Cell Biol. 11, 614-621. Nestle, F. O., Zheng, X.-G.. Thompson, C. B., Turka. L. A., and Nickoloff, B. J. ( 1993). Characterization of dermal dendritic cells obtained from normal human akin reveals phenotypic and functionally distinctive subsets. J. Inmuno/. 151,6535-6545. Ortner, U.. Inaba, K., Koch, F.. Heine, M., Miwa, M., Schuler, G., and Romani, N. (1996). An improved isolation method for murine migratory cutaneous dendritic cells. J. lmmunol. Methods 193, 71-79. Peeler. J. S., and Niederkorn, J. Y. (1987). Effect of Langerhans cells on cytotoxic lymphocyte-T responses to major and minor alloantigens expressed on heterotopic cornea1 allografts. Tr~lnsplnntatim Proc. 19, 3 16-320. Pope. M., Betjes. M. G. H.. Hirmand, H., Hoffman, L., and Steinman, R. M. (1995). Both dendritic cells and memory T lymphocytes emigrate from organ cultures of human skin and form distinctive dendritic-T-cell conjugates. J. Inwst. Derwmto/. 104, I l-17. Price. A. A., Cumberbatch, M., Kimber. I., and Ager, A. (1997). 016integrins are required for Ldngerhans cell migration from the epidermis. J. Exp. Med. 186, 172551735. Pulendran, B.. Smith. J. L., Caspary. G.. Brasel, K., Pettit, D., Maraskovsky, E., and Maliszewski, C. R. (1999). Distinct dendritic cell subsets differentially regulate the class of immune response in \i~w. Proc. Nut/. Acad. Sci. USA 96, IO36- IO4 I Rambukkana. A., Pistoor. F. H. M.. Bos. J. D.. Kapsenberg, M. L., and Das, P. K. (1996). Effects of contact allergens on human Langerhans cells in skin organ culture: Migration, modulation of cell surface molecules. and early expression of interleukin-I B protein. Lrih. Immt. 74,422-436. Randolph, G. J.. Beaulieu. S.. Pope. M., Sugawara, I., Hoffman, L., Steinman, R. M., and Muller, W. A. (1998a). A physiologic function for p-glycoprotein (MDR-I) during the migration of dendritic cells from skin via afferent lymphatic vessels. Proc. Nut/. Acctd. Sci. USA 95,6924-6929. Randolph. G. J.. Beaulieu, S., Lebecque, S., Steinman, R. M., and Mullet-, W. A. (199813).Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking. Science 282,480483. Randolph, G. J., Inaba, K.. Robbiani, D. E, Steinman, R. M., and Muller. W. A. ( 1999). Differentiation of phagocytic monocytes into lymph node dendritic cells in vivo. Immunin 11,753-761, Ratzinger, G., Stoitzner, P., Lutz. M. B., Schuler, G., Senior. R. M., Shipley, J. M.. Fritach. P., and Romani, N. (2000). Effect of matrix metalloproteinaaes on the migration of murine cutaneous dendritic cells (abstract). Arch. Dermatol. Rex 292, 74. Riasoan. M. C., Soumelis, V.. Kadowaki, N., Grouard, G., Briere. F., Malefyt, R. D., and Liu, Y. J. (1999). Reciprocal control of T helper cell and dendriticcell differentiation. Science 283, I 183-l 186. Roake. J. A., Rao. A. S., Morris, P. J., Larsen, C. P., Hankins. D. F.. and Austyn, J. M. (1995). Dendritic cell loss from nonlymphoid tissues after systemic administration of lipopolysaccharide. tumor necrosis factor, and interleukin I. J. Exp. Med. 181,2237-2248. Robbiani, D. F., Finch, R. A., Jager, D., Muller, W. A., Sartorelli, A. C., and Randolph, G. J. (2000). The leukotriene Cq transporter MRPI regulates CCL19 (MIP-3B, ELC).dependent mobilization of dendritic cells to lymph nodes. Cell 103,757-76X. Robert, C., Fuhlbrigge, R. C., Kieffer, J. D., Ayehunie, S., Hynes, R. O., Cheng, G. Y., Grabbe, S., Von Andrian, U. H., and Kupper, T. S. (1999). Interaction of dendritic cells with skin endothelium: A new perspective on immunosurveillance. J. Exp. Med. 189,627-635.

MIGRATION OFDENDRITIC CELLS INTO LYMPHATICS

267

Romani, N., Inaba, K., Pure, E., Crowley. M., Witmer-Pack, M., and Steinman, R. M. (1989a). A small number of anti-CD3 molecules on dendritic cells stimulates DNA synthesis in mouse T lymphocytes. J. Exp. Med. 169, 1153-l 168. Romani, N., Lenz, A., Glassel, H.. St&se], H., Stanzl. U., Majdic, 0.. Fritsch, P., and Schuler, G. (1989b). Cultured human Langerhans cells resemble lymphoid dendritic cells in phenotype and function. .I. Invest. Dermcrrol. 93,600-609. Romani. N., Koide, S., Crowley. M., Witmer-Pack, M., Livingstone. A. M., Fathman, C. G.. Inaba, K., and Steinman, R. M. (1989~). Presentation ofexogenous protein antigens by dendritic cells to T cell clones: Intact protein is presented best by immature epidermal Langerhans cells. .I. Exp. Med. 169, 1169-I 178. Romani, N.. Witmer-Pack, M., Crowley, M., Koide. S.. Schuler, G., Inaba, K.. and Steinman. R. M. (199 I). Langerhans cells as immature dendritic cells. /?I “Epidermal Langerhans Cells” (G. Schuler. Ed.), pp. 19 l-216. CRC Press, Inc., Boca Raton, FL. Romani. N., Gruner, S.. Brang, D., Kampgen, E.. Lenz, A.. Trockenbacher, B.. Konwalinka, G., Fritsch. P. O., Steinman, R. M.. and Schuler, G. (1994). Proliferating dendritic cell progenitors in human blood. J. E.xp. Med. 180, 83-93. Romani, N., Bhardwaj, N., Pope. M., Koch, F., Swiggard, W. J., O’Doherty, U.. Witmer-Pack, M. D., Hoffman, L., Schuler, G., Inaba. K., and Steinman, R. M. (1997). Dendritic Cells. In “Weir’s Handbook of Experimental Immunology,” 5th Ed. (L. A. Herzenberg, D. M. Weir. L. Herzenberg, and C. Blackwell, Eds.). pp. 156. I-156.14. Blackwell Science, Oxford. Rosloniec, E. E, Vitez, L. J.. Buus. S., and Freed, J. H. (1990). MHC class II-derived peptides can bind to class II molecules, including self molecules. and prevent antigen presentation. J. Exp. Med. 171, 1419-1430. Rosai, D., and Zlotnik, A. (2000). The biology of chemokines and their receptors. Awm. Rev. Immunol. l&217-243. Saeki, H., Moore. A. M., Brown. M. J., and Hwang, S. T. (1999). Cutting edge: Secondary lymphoidtissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes. J. lmmunol. 162, 2472-2475 Saito, H., Tsujitani, S., Ikeguchi, M.. Maeta, M., and Kaibara, N. (1998). Relationship between the expression of vascular endothelial growth factor and the density of dendritic cells in gastric adenocarcinoma tissue. BI: J. Cancer 78, 1.573-1577. Sallusto, E, and Lanzavecchia. A. (1994). Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor-o. J. Exp. Med. 179, 1109-l I 18. Sallusto, E, and Lanzavecchia, A. (1999). Mobilizing dendritic cells for tolerance. priming, and chronic inflammation. J. Ex~. Med. 189,61 1-614. Sato, N., Ahuja, S. K., Quinones. M., Kostecki, V., Reddick, R. L., Melby. P. C., Kuziel. W. A.. and Ahuja. S. S. (2000). CC chemokine receptor (CCR) 2 is required for Langerhans cell migration and localization of T helper cell type I (Thl)-inducing dendritic cells: Absence of CCR2 shifts the Lrishmar~iu major-resistant phenotype to a susceptible state dominated by Th2 cytokines. B cell outgrowth, and sustained neutrophilic inflammation. J. Exp. Med. 192, 205-218. Sauter, B., Albert, M. L., Francisco, L., Larsson, M., Somersan. S.. and Bhardwaj, N. (2000). Consequences of cell death: Exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells. induces the maturation of immunostimulatory dendritic cells. J. E.tp, Med. 191, 423433. Schuler-Thurner, B., Dieckmann, D., Keikavoussi, P., Bender. A., Maczek, C., Jonuleit, H., Roder, C., Haendle, I.. Leisgang, W.. Dunbar, R., Cerundolo. V., Von den Driesch, P.. Knop, J., Briicker, E. B., Enk, A., Kampgen, E., and Schuler, G. (2000). Mage-3 and influenza-matrix peptide-specific cytotoxic T cells are inducible in terminal stage HLA-A2. I + melanoma patients by mature monocytederived dendritic cells. J. Immunol. 165, 3492-3496. Schuler, G. (Ed.) (1991). “Epidermal Langerhans Cells.” I Ed. CRC Press, Inc., Boca Raton, FL.

268

NIKOLAUSROMANIETA.

Schuler, G., and Steinman, R. M. (1985). Murine epidermal Langerhans cells mature into potent immunostimulatory dendritic cells in vitro. J. Ex~. Med. 161, 526-546. Schuler. G., and Steinman, R. M. (1997). Dendritic cells as adjuvants for immune-mediated resistance to tumors. J. Exp. Med. 186, 1183-l 187. Schuler, G., Romani, N.. Stossel, H., and Wolff, K. (1991). Structural organization and biological properties of Langerhans cells. In “Epidermal Langerhans Cells” (G. Schuler. Ed..) pp. 87-137. CRC Press, Inc., Boca Raton, FL. Schuler, G., Thumer, B., and Romani, N. (1997). Dendritic cells: From ignored cells to major players in T-cell-mediated immunity. Int. Arch. Allergy Immunol. 112, 317-322. Schwarzenberger. K., and Udey, M. C. (1996). Contact allergens and epidermal proinflammatory cytokines modulate Langerhans cell E-cadherin expression in situ. J. Invest. Dermutol 106, S53558. Shapiro, S. D. (1998). Matrix metalloproteinase degradation of extracellular matrix: Biological consequences. Curr: @in. Cell Biol. 10,602-608. Shortman, K. (2000). Dendritic cells: Multiple subtypes, multiple origins, multiple functions. lmmunol. Cell Biol. 78, 161-165. Shortman, K., and Caux, C. (1997). Dendritic cell development: Multiple pathways to nature’s adjuvants. Stem Cells 15,409Al9. Silberberg-Sinakin, I., Thorbecke, G. J., Baer. R. L., Rosenthal, S. A., and Berezowsky, V. (1976). Antigen-bearing Langerhans cells in skin, dermal lymphatics and in lymph nodes. Cell. Immunol, 25,137-151. Sozzani, S., Allavena, P., Vecchi, A., and Mantovani, A. (1999). The role of chemokines in the regulation of dendritic cell trafficking. J. Leukocyte Binl. 66, l-9. Sprinzl, G. M., Hussl, B., Obrist, P., Yoneda, K., Thumfart, W. F., Romani, N., and Schrott-Fischer, A. (2000). Dendritic cells in precancerous lesions of the larynx. Laygoscope 110, 13-18. Steinman, R. M. (1998). Dendritic cells. In “Fundamental Immunology,” 4 Ed. (W. E. Paul, Ed.), pp. 547-573. Lippincott-Raven, Philadelphia. Steinman, R. M., Metlay, J., Bhardwaj, N., Freudenthal, P., Langhoff, E., Crowley, M., Lau, L.. WitmerPack, M., Young. J. W., Pure, E., Romani, N., and Inaba, K. (1990). Dendritic cells: Nature’s adjuvant. In “Immunogenicity” (C. A. Janeway, Jr.. J. Sprent, and E. Sercarz, Eds.), Vol. 113, pp. 155-165. Alan R. Liss Inc., New York. Steinman, R. M., Pack, M., and lnaba, K. (1997). Dendritic cells in the T-cell areas of lymphoid organs. Immunol. Rev 156,25-37. Steinman, R. M., Inaba, K., Turley, S., Pierre, P., and Mellman, I. (1999). Antigen capture, processing, and presentation by dendritic cells: Recent cell biological studies. Human Immunol. 60, 562567. Steinman, R. M., Turley, S., Mellman, I., and Inaba, K. (2000). The induction of tolerance by dendritic cells that have captured apoptotic cells. J. Exp. Med. 191,41 1416. Stoitzner, P., Zanella, M., Ortner, U.. Lukas, M., Tagwerker, A., Janke, K., Lutz, M. B., Schuler, G., Echtenacher, B., Ryffel, B., Koch, F., and Romani, N. (1999). Migration of Langerhans cells and dermal dendritic cells in skin organ cultures: Augmentation by TNF-a and IL- 1B. J. Leukocyte Biol. 66,4622470. Stoitzner, I?, Ratzinger, G., Koch, F., Janke, K., Scholler, T., Kaser, A., Tilg, H., Cruikshank, W. W., Fritsch, P., and Romani, N. (2001). Interleukin-16 supports the migration of Langerhans cells, partly in a CD4-independent way. J. Invest. Dermatol. in press. Streilein, J. W., Toews, G. B., and Bergstresser, P. R. (1979). Cornea1 allografts fail to express Ia antigens. Nature 282,326-327. Streilein, J. W., Lonsberry, L. W., and Bergstresser, P. R. (1982). Depletion of epidermal Langerhans cells and Ia immunogenicity from tape-stripped mouse skin. J. Exp. Med. 155, 863-87 I. Strobl, H., and Knapp, W. (1999). TGF-Bl regulation of dendritic cells. Microbes Infect. 1, 12831290.

MIGRATION OFDENDRITIC CELLS INTO LYMPHATICS

269

Strobl, H., Riedl, E., Scheinecker, C., Bello-Fernandez. C., Pick], W. F., Rappersberger, K., Majdic, O., and Knapp, W. (1996). TGF-PI promotes in vitro development of dendritic cells from CD34+ hemopoietic progenitors. J. Immunol. 157, 1499%1507. Strunk, D., Egger, C., Leitner, G., Hanau, D., and Stingl, G. (1997). A skin homing molecule defines the Langerhans cell progenitor in human peripheral blood. J. Exp. Med. 185, 1 13 l-l 136. Siiss, G., and Shortman, K. (1996). A subclass of dendritic cells kills CD4 T cells via Fas Fasligandinduced apoptosis. J. Exp. Med. 183, 1789-1796. Tang. A., Amagai, M., Granger, L. G., Stanley, J. R., and Udey, M. C. (1993). Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin. Narure (Land.) 361, 82-85. Teunissen. M. B. M., Wormmeester, J., Krieg, S. R., Peters. P. J., Vogels, I. M. C.. Kapsenberg, M. L., and Bos, J. D. (1990). Human epidermal Langerhans cells undergo profound morphological and phenotypical changes during in vitro culture. J. Inve.yr. Dermrrrol. 94, 166-173. Thurner. B., Haendle, I., Rader. C.. Dieckmann, D.. Keikavoussi, P., Jonuleit, H.. Bender, A., Maczek, C., Schreiner, D., den Driesch. P., BrGcker, E. B., Steinman, R. M., Enk, A., Klmpgen, E., and Schuler. G. (1999). Vaccination with Mage-3A 1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastasesin advanced stage IV melanoma. J. Eup. Med. 190, 1669-1678. Timmerman, J. M., and Levy. R. (1999). Dendritic cell vaccines for cancer immunotherapy. Amzu. Rea Med. 50,507-529.

Timpl, R. (1996). Macromolecular organization of basement membranes. Curr: @in. Cell Biol. 8, 6 18-624. Toews. G. B., Bergstresser, P. R., and Streilein, J. W. (1980). Epidermal Langerhans cell density determines whether contact hypersensitivity or unresponsiveness follows akin painting with DNFB. J. lmmunol. 124,445-453. Tue, Y., and Cooper. K. D. (1990). Cutaneous dermal Ia+ cells are capable of initiating delayed type hypersensitivity responses. J. Invrsf. Dermatol. 94,267-272. Uchi, H., Imayama, S., Kobayashi, Y., and Furue. M. (1998). Langerhans cells express matrix metalloproteinase-9 in the human epidermis. J. Invest. Dermcuol. 111, 1232-1233. Udey, M. C. ( 1997). Cadherins and Langerhans cell immunobiology. Clin. Exp. fmmunol. 107 (Suppl. I), 6-8. Valladeau, J., Duvert-Frances, V., Pin, J. J., Dezutter-Dambuyant, C., Vincent, C., Massacrier, C., Vincent. J.. Yoneda, K., Banchereau, J., Caux, C.. Davoust, J., and Saeland, S. (1999). The monoclonal antibody DCGM4 recognizes Langerin, a protein specific of Langerhans cells, and is rapidly internalized from the cell surface. ELK J. Immunol. 29,2695-2704. Valladeau, J., Ravel, O., Dezutter-Dambuyant. C., Moore, K.. Kleijmeer, M., Liu, Y., Duvert-Frances, V., Vincent, C., Schmitt, D., Davoust, J., Caux, C.. Lebecque. S., and Saeland, S. (2000). Langerin, a novel C-type lectin specific to Langerhans cell.\, is an endocytic receptor that induces the formation of Birbeck granules. Immunily 12, 7 1-8 1. Wang, B. H.. Kondo, S., Shivji, G. M., Fujisawa. H., Mak, T. W., and Sauder. D. N. (1996). Tumour necrosis factor receptor II (~75) signalling is required for the migration of Langerhans’ cells. Immunology

88, 284-288.

Wang, B. H., Fujisawa, H., Zhuang, L. H., Kondo, S.. Shivji, G. M., Kim. C. S.. Mak, T. W., and Sauder, D. N. (1997). Depressed Langerhans cell migration and reduced contact hypersensitivity response in mice lacking TNF receptor ~7.5. J. Immunol. 159,6148-6155. Wang, B. H., Zhuang. L. H., Fujisawa, H., Shinder, G. A., Feliciani, C., Shivji. G. M., Suzuki, H., Amerio, E., Toto, P., and Sauder, D. N. (1999a). Enhanced epidermal Langerhans cell migration in IL-IO knockout mice. J. Immunol. 162,277-283. Wang, B. H., Amerio. P., and Sauder, D. N. (1999b). Role of cytokines in epidermal Langerhans cell migration. J. Leukocyre Bin/. 66,33-39. Wang, M., Qin, X. J., Mudgett, J. S., Ferguson, T. A., Senior, R. M., and Welgus, H. G. (1999). Matrix metalloproteinase deficiencies affect contact hypersensitivity: Stromelysin- 1 deficiency prevents the

270

NIKOLAUSROMANIETAL.

response and gelatinase B deficiency prolongs the response. Proc. Nntl. Acad. Sci. USA 96,68856889. Weinlich, G., Heine, M., St&se], H., Zanella, M., Stoitzner, P., Ortner. U.. Smolle, J., Koch. F., Sepp, N. T., Schuler. G., and Romani, N. (1998). Entry into afferent lymphatics and maturation in situ of migrating cutaneous dendritic cells. J. Invest. Dermatol. 110,441-448. Weiss, J. M., Sleeman, J.. Renkl, A. C., Dittmar. H., Termeer, C. C.. Taxis. S., Howells, N.. Hofmann, M., Kiihler. G., Schopf, E., Ponta, H., Herrlich, P., and Simon, J. C. (1997). An essential role for CD44 variant isoforms in epidermal Langerhans cell and blood dendritic cell function. J. Cc/l Bid. 137, 1137-l 147. Wischatta, M., Sprinal, G. M.. Gunkel, A. R., Hussl, B., Romani, N.. and Schrott-Fischer. A. (2000). Dendritic cells in selected head and neck tumors, Ann. Otol. Rhinol. Laryzgol. 109,56-62. Witmer-Pack, M. D., Olivier, W.. Valinsky, J., Schuler, G., and Steinman, R. M. (1987). Granulocyte/ macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells. J. Enp. Med. 166, 1484-1498. Wolff, K. (1967). The fine structure of the Langerhans cell granule. .I. Cell Biul. 35,466&473. Wolff. K. (1991). The fascinating story that began in 1868. In “Epidermal Langerhans Cells” (G. Schuler, Ed.), pp. 1-21. CRC Press, Inc., Boca Raton. FL. Wong, B. R., Josien, R., Lee, S. Y., Sauter, B., Li, H. L., Steinman, R. M., and Choi, Y. W. (1997). TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine). a new TNF family member predominantly expressed in T cells. is a dendritic cell-specific survival factor. J. Exp. Med. 186,2075-2080. Wu, S. J. L.. Grouard-Vogel, G., Sun, W., Mascola, J. R.. Brachtel, E., Putvatana, R., Louder, M. K., Filgueira, L., Marovich, M. A.. Wong, H. K., Blauvelt, A., Murphy. G. S., Robb, M. L., Innes, B. L., Birx, D. L., Hayes, C. G., and Frankel, S. S. (2000). Human skin Langerhans cells are targets of dengue virus infection. Nature Med. 6, 8 16-820. Yanagihara. S., Komura, E., Nagafune, J., Watarai, H., and Yamaguchi, Y. (1998). EBIl/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation. J. Immunol. 161,3096-3102. Zhang, Y. A., McCluskey, K., Fujii, K., and Wahl. L. M. (I 998). Differential regulation of monocyte matrix metalloproteinase and TIMP- I production by TNF-granulocyte-macrophage CSF, and IL- 1 through prostaglandin-dependent and -independent mechanisms. J. Immunol. 161,3071-3076. Zinkernagel, R. M. (1996). Immunology taught by viruses. Science 271, 173-178. Zitvogel, L., Mayordomo, J. I., Tjandrawan, T.. Deleo, A. B., Clarke, M. R., Lotze, M. T.. and Storkus, W. J. (1996). Therapy of murine tumors with tumor peptide-pulsed dendritic cells: Dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J. hp. Med. 183, 87-97. Zlotnik. A., and Yoshie, 0. (2000). Chemokines: A new classification and their role in immunity. Immuni~ 12, 121-127.