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The Journal of Immunology, 2000, 165: 1381-1386.
Copyright © 00 by The American Association of Immunologists

Functional Involvement of E-Cadherin in TGF-ß1-Induced Cell Cluster Formation of In Vitro Developing Human Langerhans-Type Dendritic Cells1

Elisabeth Riedl*,§, Johannes Stöckl{dagger}, Otto Majdic{dagger}, Clemens Scheinecker{ddagger}, Klemens Rappersberger§, Walter Knapp{dagger} and Herbert Strobl2,*

* Institute of Immunology, Vienna International Research Cooperation Center, Novartis Research Institute, Vienna, Austria; and {dagger} Institute of Immunology, {ddagger} Department of Internal Medicine III, Division of Rheumatology, and § Department of Dermatology I, University of Vienna, Vienna, Austria


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Epithelial Langerhans cells (LC) represent immature dendritic cells that require TGF-ß1 stimulation for their development. Little is known about the mechanisms regulating LC generation from their precursor cells. We demonstrate here that LC development from human CD34+ hemopoietic progenitor cells in response to TGF-ß1 costimulation (basic cytokine combination GM-CSF plus TNF-{alpha}, stem cell factor, and Flt3 ligand) is associated with pronounced cell cluster formation of developing LC precursor cells. This cell-clustering phenomenon requires hemopoietic progenitor cell differentiation, since it is first seen on day 4 after culture initiation of CD34+ cells. Cell cluster formation morphologically indicates progenitor cell development along the LC pathway, because parallel cultures set up in the absence of exogenous TGF-ß1 fail to form cell clusters and predominantly give rise to monocyte, but not LC, development (CD1a-, lysozyme+, CD14+). TGF-ß1 costimulation of CD34+ cells induces neoexpression of the homophilic adhesion molecule E-cadherin in the absence of the E-cadherin heteroligand CD103. Addition of anti-E-cadherin mAb or mAbs to any of the constitutively expressed adhesion molecule (CD99, CD31, LFA-1, or CD18) to TGF-ß1-supplemented progenitor cell cultures inhibits LC precursor cell cluster formation, and this effect is, with the exception of anti-E-cadherin mAb, associated with inhibition of LC generation. Addition of anti-E-cadherin mAb to the culture allows cell cluster-independent generation of LC from CD34+ cells. Thus, functional E-cadherin expression and homotypic cell cluster formation represent a regular response of LC precursor cells to TGF-ß1 stimulation, and cytoadhesive interactions may modulate LC differentiation from hemopoietic progenitor cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Epidermal Langerhans cells (LC)3 are bone marrow-derived immature dendritic cells (DC) that are highly specialized in Ag uptake and processing. LC form a three-dimensional network in suprabasal epidermal layers and stay in the epidermis for long periods of time, enabling them to fulfill a sentinel role in which they filter the surrounding tissue for foreign Ags and pathogens (1, 2, 3).

Differentiated epidermal LC share with epithelial cells of various organs high cell surface expression levels of the homophilic calcium-dependent cytoadhesion molecule E-cadherin (4, 5). LC to keratinocyte adhesion (4) as well as homotypic adhesion of in vitro generated murine LC (6) can be blocked by mAbs to E-cadherin, suggesting an important role for E-cadherin in LC adhesion (reviewed in Ref. 2). Among DC, only differentiated LC and a subpopulation of DC from skin-draining lymph nodes, which may be derived from LC, express E-cadherin (7). E-cadherin expression is markedly down-regulated upon migration and maturation of epidermal LC, and lower expression levels of E-cadherin on the surfaces of cultured LC correlate with decreased cell adhesiveness (4, 7, 8). Apart from LC-type DC, most other hemopoietic cells, with the exception of immature thymocytes (9, 10), murine dendritic epidermal T cells (11), and immature erythroid cells (12), lack E-cadherin expression.

The cytokine TGF-ß1 plays a key role during LC development and differentiation. Using an in vitro differentiation model of CD34+ hemopoietic progenitor cells, we recently demonstrated that development of LC from CD34+ progenitor cells in a serum-free culture system is absolutely dependent on TGF-ß1 stimulation (13). In vivo studies revealed that TGF-ß1-deficient mutant mice selectively lack epidermal LC (14), but possess functional LC precursors (15) and other myeloid cells and show normal bone marrow morphology (16). Bone marrow cells from TGF-ß1-deficient mice are capable, upon transplantation into normal mice, of differentiating into LC, suggesting that paracrine TGF-ß1 stimulation of LC precursors in peripheral organs is sufficient for LC differentiation in vivo (15). Furthermore, similar numbers of bone marrow-derived LC were identified in TGF-ß1-/- and control skin after engraftment onto nude mice. Additionally, epidermal-restricted TGF-ß type II receptor dominant negative transgenic mice contain normal numbers of LC (15). These observations suggest that the LC deficiency in TGF-ß1-/- mice reflects a requirement of LC or precursor cells for TGF-ß1 and is obviously not due to modulation of the cutaneous microenvironment. This model seems to be compatible with a critical role for endogenous TGF-ß1 in LC development. In line with this possibility, LC-type DC are known to synthesize TGF-ß1 abundantly (6, 17, 18), and a recent study showed that TGF-ß1-transduced DC show increased survival and persistence after transplantation into normal allogeneic mice (19). The mechanisms involved in TGF-ß1-dependent LC differentiation, however, remained unclear.

Here we demonstrate that LC development from CD34+ hemopoietic progenitor cells in response to TGF-ß1 stimulation is associated with pronounced homotypic cell cluster formation of cultured cells. This cell-clustering phenomenon is mediated by TGF-ß1-induced E-cadherin in cooperation with several constitutively expressed adhesion molecules. Cluster inhibition studies suggest that TGF-ß1-dependent cellular interactions may be functionally involved in LC differentiation from their precursor cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

The following murine mAbs were generated in our laboratory: VIAP (nonbinding isotype control Ab, calf intestine alkaline phosphatase specific), LZ-1 (human lysozyme), VIT6b (CD1a), 5E6 (CD11a), and 3B2/TA8 (CD99). CD14 mAb MEM-18 was provided by An der Grub (Kaumberg, Austria); CD34 mAb HPCA2 was obtained from Becton Dickinson (San Jose, CA); CD18 mAb TS1/18 was purchased from American Type Culture Collection (Manassas, VA); CD11b mAb 2LPM19C was obtained from Dako (Glostrup, Denmark). CD29 mAb 4B4, CD49d mAb HP2/1, CD49e mAb SAM1, and CD103 mAb 2G5 were obtained from Beckman Coulter (Fullerton, CA). E-cadherin mAb HECD-1 was purchased from Zymed (San Francisco, CA); CD31 mAb CLB-HEC/75 was purchased from CLB (Amsterdam, The Netherlands).

The following above-mentioned Abs were used in cell culture experiments. They were selected based on previously published functional studies: clone VIAP (control Ab, IgG1, purified from ascites using protein A chromatography) (20), clone 5E6 (CD11a, IgG1, purified from ascites fluid using protein A chromatography, cell adhesion-blocking mAb) (20), clone 3B2/TA8 (CD99, IgG1, purified from ascites fluid using protein A chromatography, mitogenic signaling mAb) (21), clone TS1/18 (CD18, IgG1, purified from hybridoma supernatant using protein A chromatography, cell adhesion-blocking mAb) (22), clone 4B4 (CD29, IgG1 purified from ascites fluid using ion exchange chromatography, cell adhesion-blocking mAb) (23), clone HP2/1 (CD49d, IgG1, purified from hybridoma supernatant using affinity purification, cell adhesion-blocking mAb) (24), clone SAM1 (CD49e, IgG1, purified from hybridoma supernatant using affinity purification, cell adhesion-blocking mAb) (25), clone HECD-1 (E-cadherin, IgG1, purified from ascites fluid using ammonium sulfate precipitation, cell adhesion-blocking mAb) (26) and clone CLB-HEC/75 (CD31, IgG1 purified from ascites fluid using affinity chromatography, cell adhesion-blocking mAb) (27). Before functional analysis, all Abs were dialyzed against serum-free cell culture medium (see below). Abs were added to cultures at a final concentration of 20 µg/ml. Ab preparations, which were used in cell culture experiments, were tested for endotoxin (LPS). All the mAbs tested contained <0.1 ng/ml LPS, with the exception of mAb HEC/75 (CD31), which contained low levels of endotoxin (3.5 ng/ml). Because all experiments were performed in serum-free medium, which does not contain significant amounts of LPS binding protein and soluble CD14, it is very unlikely that the observed functional effect of the mAb HEC/75 is due to LPS contamination.

Immunofluorescence staining procedures

For membrane staining, 50 µl of cells (107/ml) were incubated for 15 min at 4°C with 20 µl of conjugated mAb or unconjugated mAb. For staining using unconjugated mAb, FITC-conjugated F(ab')2 of sheep anti-mouse Ig Abs (An der Grub) were used as described previously (28). Cells were washed and analyzed by flow cytometry or were subjected to intracellular staining.

For suspension staining of the intracellular Ags, we used the commercially available reagent combination Fix&Perm (An der Grub) according to the manufacturer’s directions. Briefly, cells are fixed for 15 min at room temperature (50 µl of cells plus 100 µl of formaldehyde-based Fixation Medium). After one washing with PBS, pH 7.2, cells are resuspended in 50 µl of PBS and mixed with 100 µl of Permeabilization Medium plus 20 µl of fluorochrome-labeled Ab. After a further incubation for 15 min at room temperature, cells are washed again and analyzed by flow cytometry. Flow cytometric analyses were performed using a FACScan flow cytometer (Becton Dickinson).

Cord blood (CB) cells

CB samples were collected during normal full-term deliveries. Mononuclear cells were isolated within 10 h after collection using discontinuous Ficoll/Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. CD34+ cells were isolated from CB mononuclear cells using the MACS CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the instructions of the manufacturer. The purity of the CD34+ population exceeded 90%.

Primary cultures of purified CD34+ CB cells

Primary cultures of purified CD34+ CB cells were performed in 24-well plates (Costar, Cambridge, MA; 1 x 104 cells in 1 ml/well) at 37°C in a humidified atmosphere and in the presence of 5% CO2 as described previously (29). The serum-free medium X-VIVO 15 (BioWhittaker, Walkersville, MD) contained L-glutamine (2.5 mM), penicillin (125 U/ml), and streptomycin (125 µg/ml). Cultures were supplemented with optimized concentrations of the following human cytokines: Flt3 ligand (FL; 100 ng/ml; provided by Immunex, Seattle, WA), TGF-ß1 (0.5 ng/ml; purified from platelets; British Biotechnology, Abington, U.K.), rTNF-{alpha} (50 U/ml; Bender, Vienna, Austria), rGM-CSF (100 ng/ml; Novartis, Basel, Switzerland), and recombinant human stem cell factor (SCF; 20 ng/ml; Amgen, Thousand Oaks, CA).

Monoclonal Ab stimulation experiments of CD34+ cells

Primary cultures of CD34+ cells were set up exactly as described above. After 24 h cells were harvested and replated in duplicate in 96-well plates containing identical growth media at a cell density of 1 x 104 cells/well. Cultures were supplemented with 20 µg/ml mAb when indicated, and plates were incubated at 4°C to allow Ab binding. Afterward, cells were transferred to 37°C and cultured for 7 days. On days 3 and 7, cultures were analyzed for cell cluster formation by phase contrast microscopy using a scoring system previously established in our laboratory (20) with slight modifications. Scores ranged from 0 to 4; 0 indicated that <10% of the cells were in aggregates, 1 represented 10–50% aggregated cells, 2 indicated that about 50–75% of the cells were aggregated, 3 indicated 75- 90% of the cells were aggregated, and 4 indicated that >90% of the cells were found in large aggregates. On day 7 cells were harvested and analyzed by flow cytometry (see above).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TGF-ß1-induced LC development from CD34+ progenitor cells is associated with cell cluster formation

We recently established a serum-free culture system for the efficient generation of Birbeck granule Ag Lagbright, CD1abright LC from human CD34+ progenitors and observed that LC generation is strictly dependent on addition of TGF-ß1 to the culture medium (29) containing the classical DC cytokines GM-CSF plus TNF-{alpha}, SCF, and FL (30, 31, 32).

As shown in Fig. 1Go (upper panel), TGF-ß1-dependent LC growth observed in this defined culture system is associated with the formation of large cell clusters. In the absence of TGF-ß1 (Fig. 1Go, lower panel) cells do not form or form only few, very small, cell clusters and do not differentiate into CD1a+ LC (Fig. 1Go, lower panel).



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  		FIGURE 1. DC generated in the presence of TGF-ß1 in defined serum-free medium show specific features of epidermal LC and form large cell clusters. Purified CD34+ cells were cultured for 7 days in serum-free medium supplemented with the cytokines GM-CSF plus TNF-{alpha}, SCF, and FL in the presence or the absence of TGF-ß1. Cells were harvested and analyzed for cell morphology (see Materials and Methods) and the expression of informative molecules. Typical microscopic appearance of generated cells in the presence or the absence of TGF-ß1 (left) (original magnification, x20). Diagrams show correlated expression of E-cadherin (center) or the monocyte marker molecule lysozyme (LZ) vs CD1a by generated cells in the presence (upper panel) or the absence (lower panel) of TGF-ß1 (right).

 
Cell cluster formation in TGF-ß1-supplemented cultures starts on day 4 of culture. By day 7 the vast majority of cultured cells in the presence of TGF-ß1 reside within large aggregates. These cell clusters can be easily disrupted by pipetting, but secondary cluster formation occurs within 24 h upon replating of generated cells in identical growth medium at 37°C. Incubation of cells at 4°C or addition of EDTA (10 mM) to the cultures completely prevents secondary cell cluster formation of LC (data not shown).

In line with the selective generation of LC in the presence of TGF-ß1, only a minor portion of cultured cells show monocytic features (LZ+, CD1a-; Fig. 1Go, upper right). Conversely, a large portion of cells generated in TGF-ß1-nonsupplemented cultures develops into LZ+CD1a- monocytic cells (Fig. 1Go, lower right). These data suggest an association of TGF-ß1-induced cluster formation and LC differentiation.

TGF-ß1 induces expression of the homophilic adhesion molecule E-cadherin

As described above, TGF-ß1-induced LC differentiation is strictly associated with cell cluster formation, suggesting regulation of adhesion molecule expression by TGF-ß1. The homophilic epithelial adhesion molecule E-cadherin is known to be expressed in vivo not only by epithelial cells but also by LC (5), and mediates high affinity binding of LC to keratinocytes (4). As shown in Fig. 1Go, the vast majority of CD1a+ cells in vitro generated in the presence of TGF-ß1 strongly express E-cadherin. Cells generated in the absence of TGF-ß1 remain E-cadherin-, except for a small E-cadherin+CD1a- subset.

Fig. 2Go shows representative expression profiles of a panel of cytoadhesion molecules by cells generated in the presence or the absence of TGF-ß1. As shown, CD103 (HML-1, integrin {alpha}E chain), which associates with the ß subunit of the heterodimer integrin {alpha}Eß7, a heteroligand of E-cadherin (33), could not be detected on the surface of generated cells. Most cells generated in the presence or the absence of TGF-ß1 express the ß1 integrins CD49d/CD29 (VLA-4) and CD49e/CD29 (VLA-5) as well as the ß2 integrin CD11a/CD18 (LFA-1); <10% of cells express the ß2 integrin {alpha}-chain CD11b (data not shown). Additionally a subset of generated cells expresses CD31 (PECAM-1), and most cells express CD99 (MIC-2/E2). All these latter molecules are already expressed at the CD34+ progenitor cell stage (34), and their expression pattern is not significantly affected by TGF-ß1 stimulation. In contrast, E-cadherin is clearly induced by TGF-ß1 stimulation and cannot be detected on the cell surface of CD34+ hemopoietic progenitors (12).



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  		FIGURE 2. Analysis of cytoadhesion molecule expression by generated cells. Cells were generated in defined serum-free medium in the presence or the absence of TGF-ß1 (see Materials and Methods). On day 7 cells were harvested and stained for expression of a panel of adhesion molecules. Overlay histograms show total generated cells analyzed for the expression of the adhesion molecules as indicated (open histograms) vs isotope-matched negative (filled histograms) controls. Data are representative of three experiments.

 
Inhibition of cell cluster formation by mAbs to CD99 or CD18 abrogates LC differentiation and cells develop into a monocytic pathway

To analyze whether TGF-ß1-induced LC differentiation and cell cluster formation are functionally linked, we performed cell cluster inhibition experiments using blocking mAbs to adhesion molecules and studied whether cell cluster inhibition may affect LC differentiation in TGF-ß1-supplemented LC generation cultures.

Addition of mAb 3B2/TA8 or TS1/18, specific for CD99 or the common ß-chain of ß2 integrins (CD18), respectively, strongly inhibited LC cluster formation when added on day 1 to TGF-ß1-supplemented LC generation cultures. As shown in Fig. 3Go, cultures supplemented with mAbs TS1/18 or 3B2/TA8 contained no or only very small cell clusters.



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  		FIGURE 3. Typical morphology of cells generated in the presence of TGF-ß1 and various mAbs to adhesion molecules. Cells were generated in cultures supplemented with TGF-ß1 plus GM-CSF, TNF-{alpha}, SCF, and FL for 7 days as described in Materials and Methods. On day 1 cultures were supplemented with mAbs of the following specificity: A, control mAb; B, CD99; C, CD18; and D, E-cadherin. The representative microscopic appearance of generated cells is shown (original magnification, x20; n = 4).

 
Phenotypic analysis of day 7 cells generated in the presence of the CD99 mAb 3B2/TA8 revealed that only very low percentages of cells express the LC marker molecule CD1a (5.0 ± 3.5%; n = 3), and that, in turn, high percentages of cells express the monocyte marker molecule LZ (35) and lack CD1a (46.8 ± 8.8%; n = 3; p = 0.002 compared with control cultures: 13.0 ± 4.1% LZ+ cells; Fig. 4Go). Thus, mAb 3B2/TA8 binding to CD99 not only inhibits cluster formation, but the overall differentiation pattern of CD34+ progenitor cells switches from LC to monocytes. Very similar effects on differentiation were observed in cultures containing the CD18 mAb TS1/18 (14.7 ± 2.9% of CD1a+ cells and 55.6 ± 8.8% of LZ+ CD1a- cells; Fig. 4Go). A shift in the lineage differentiation pattern from LC to monocytic cells was confirmed by the observation that substantial percentages of cultured cells in the presence of mAbs 3B2/TA8 or TS1/18 express the monocyte marker molecule CD14 in the absence of CD1a (33.7 ± 9.1 and 24.5 ± 8.8%, respectively; n = 3; see Fig. 4Go). The numbers of cells recovered on day 7 from Ab-supplemented cultures initiated with 1 x 104 CD34+ cells on day 0 did not differ significantly (mAb 3B2/TA8, 20.8 ± 4.7 x 104; mAb TS1/18, 18.1 ± 4.8 x 104; control mAb VIAP, 25.1 ± 4.9 x 104; n = 4). The differentiation pattern and morphology of cells generated in these cultures resemble those of cells cultured in the absence of TGF-ß1 (Fig. 1Go) (29). Thus, addition of mAbs to these cytoadhesion molecules inhibits TGF-ß1-induced cell cluster formation and LC differentiation.



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  		FIGURE 4. Phenotype of cells generated in TGF-ß1-supplemented cultures in the presence of mAbs to adhesion molecules. CD34+ cells were cultured in defined serum-free medium containing the cytokines TGF-ß1 plus GM-CSF, TNF-{alpha}, SCF, and FL. Parallel cultures were supplemented with mAbs to E-cadherin, CD18, or CD99 or with negative control Abs; (see Materials and Methods). Dot plots show LZ vs CD1a expression or CD14 vs CD1a expression, respectively, of total day 7 generated cells in the presence of mAbs as indicated. Markers were set according to negative control staining. Data are representative of four experiments.

 
Inhibition of cell cluster formation by mAbs to CD31 or LFA-1 abrogates LC differentiation and inhibits cell proliferation

Addition of mAb CLB-HEC/75 or 5E6, specific for CD31 or LFA-1, respectively, also inhibited TGF-ß1-induced cell cluster formation when added to TGF-ß1-supplemented LC generation cultures of CD34+ cells. This effect was associated with strong inhibition of cell proliferation. As shown in Fig. 5Go cell numbers of cultured cells continuously decreased from days 0 to 3 to 7 in cultures supplemented with either of these mAbs. We previously demonstrated that TGF-ß1 exerts an anti-apoptotic effect on LC progenitor cells when added to GM-CSF- plus TNF-{alpha} and SCF-supplemented cultures of CD34+ cells (36). Decreased viability of LC precursors in the presence of these LC cluster-blocking mAbs may, therefore, reflect a role of cell clustering in mediating an anti-apoptotic activity of TGF-ß1 on cultured CD34+ cells.



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  		FIGURE 5. Time kinetics of total cell numbers in LC generation cultures supplemented with mAbs to CD11a, CD31, or E-cadherin. CD34+ cells (1 x 104) were cultured in defined serum-free medium containing the cytokines TGF-ß1 plus GM-CSF, TNF-{alpha}, SCF, and FL. Parallel cultures were supplemented with mAbs to CD11a, CD31, or E-cadherin or negative control Abs (see Materials and Methods). Cell numbers were determined on days 3 and 7. Data are representative of three experiments.

 
E-cadherin ligation allows LC generation in the absence of cell cluster formation

Addition of mAb HECD-1 specific for E-cadherin on day 1 to TGF-ß1-supplemented cultures of CD34+ cells strongly inhibits cell cluster formation (Fig. 3Go). Phenotypic analyses revealed that a large percentage of cells generated after 7 days, however, express high levels of CD1a. As shown in the representative phenotypic analyses in Fig. 4Go, E-cadherin mAb-supplemented cultures contained reproducibly even higher percentages of CD1a+ cells than control cultures (47.3 ± 2.9 vs 41.8 ± 2.1%; n = 4).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we describe a tight association of homotypic cell cluster formation and LC differentiation of cultured hemopoietic progenitor cells in response to TGF-ß1 costimulation. CD34+ CB hemopoietic progenitor cells stimulated in serum-free cultures in the presence of the basic cytokine combination GM-CSF plus TNF-{alpha}, SCF, and FL for 7 days fail to form cell clusters and predominantly develop into monocytic cells. Addition of TGF-ß1 to these short term cultures results in pronounced cell cluster formation and a concomitant shift in the differentiation pattern of stimulated CD34+ cells from monocytic progeny to LC-type DC. This LC differentiation-linked cell-clustering phenomenon starts on day 4 after culture initiation of purified CD34+ cells and thus obviously requires the pregeneration of a committed precursor cell population. These data suggest that cell clustering may mark an early branching point at which monocyte/LC precursor cells start to differentiate into LC. In support of this assumption, we previously observed that CD1a+ LC in TGF-ß1-supplemented cultures seem to develop from a day 4 generated early monopoietic precursor cell population (13) identifiable by the expression of lysozyme in the absence of CD14 (35). Furthermore, recent studies demonstrated that even later stage CD14+ precursor cells can be induced to differentiate into LC in response to TGF-ß1 costimulation (37, 38, 39), thus supporting this possibility.

Homophilic E-cadherin interactions between developing LC precursor cells seem to be critically involved in the TGF-ß1-induced, early appearing, cell-clustering phenomenon of developing LC precursor cells described herein. First, large proportions of cells from TGF-ß1-supplemented cultures express E-cadherin at high levels, whereas most cells from TGF-ß1-nonsupplemented parallel cultures lack E-cadherin expression (with the exception of a small E-cadherindim subset). Second, physical separation of clustered cells from TGF-ß1-supplemented LC generation cultures (by 1 x g sedimentation) results in strong enrichment of E-cadherin+ CD1a+ cells (data not shown). Third, addition of a blocking anti-E-cadherin mAb on day 1 to the cultures inhibits TGF-ß1-induced cell cluster formation. Furthermore, cells generated in TGF-ß1-supplemented LC generation cultures lack expression of CD103, a previously characterized heterophilic E-cadherin ligand (33). Thus, the cell cluster phenomenon described herein obviously involves homotypic LC precursor cell interactions, which are mediated by E-cadherin adhesion. Given the fact that E-cadherin has previously been described as a reliable surface marker molecule for developing LC-type DC in cultures of CD34+ cells (30), these observations seem to support above-mentioned tight linkage of cell cluster formation and LC differentiation in our culture model.

Ab blockage experiments revealed that in addition to E-cadherin, several constitutively expressed adhesion molecules are functionally involved in TGF-ß1-induced homotypic LC precursor cell clustering. We demonstrate that addition of mAbs to CD11a (LFA-1), CD18, CD31, or CD99 to day 1 cultures of CD34+ cells inhibits TGF-ß1-induced LC precursor cell clustering. Interestingly, in contrast to E-cadherin, all these latter adhesion molecules were previously shown to be constitutively expressed by freshly isolated CD34+ cells (reviewed in Ref. 34), and we observed that their expression intensities by cultured cells were only slightly modulated by TGF-ß1 costimulation.

The identification of several cell cluster-blocking mAbs allowed us to study a possible functional linkage of cell cluster formation and LC differentiation. Ab addition experiments indeed seem to argue in favor of this possibility. We observed that inhibition of LC precursor cell clustering by addition of certain mAbs to TGF-ß1-supplemented progenitor cell cultures selectively inhibits LC generation. For example, addition of mAb TS1/18 (CD18) or the mAb 3B2/TA8 (CD99) inhibited LC development, and cells developed into LZ+CD14+CD1a- monocytic cells. From this differentiation pattern these cells strikingly resembled cells generated in parallel cultures in the absence of TGF-ß1 (Fig. 1Go). Thus, inhibition of cell cluster formation by mAbs to these adhesion molecule systems seemed to be associated with abrogation of the effects of TGF-ß1 on the differentiation pattern of cultured cells. These data suggest, but do not formally prove, a functional linkage of cell cluster formation and LC differentiation in our culture model system. We cannot exclude the possibility that functional activation of certain adhesion receptors in response to mAb ligation may inhibit LC differentiation. Consistent with this latter possibility, mAb 3B2/TA8 directed against CD99 was previously demonstrated to induce a mitogenic cosignal for T lymphocytes (21), and overexpression of CD99 promotes cell cycling of B cell lines, whereas antisense blockage enforces differentiation of these cells (40). With regard to a possible role of CD99 signaling in modulating TGF-ß1-dependent progenitor cell differentiation, CD34+ abundantly express CD99 molecules (34). Thus, future studies should analyze further the functional consequences of CD99 signaling on cytokine-induced lineage differentiation of CD34+ cells.

Interestingly, mAb HEC/75 or 5E6 (directed against CD31 or CD11a, respectively) blocked TGF-ß1-induced LC precursor cell clustering of CD34+ cells, and this effect was associated with inhibition of cell proliferation early during culture. It is possible that CD31 and/or CD11a interactions are involved in initial TGF-ß1-induced cell cluster formation of CD34+ cells and may be functionally involved in mediating an anti-apoptotic (36) or (pro-) proliferative effect (39) of TGF-ß1 on LC precursor cells. Consistent with this possibility, both CD11a and CD31 molecules have previously been implicated in cell adhesion of hemopoietic progenitor cells. We previously demonstrated that mAb 5E6 (specific for CD11a; used in the present study) blocks CD34 ligation-induced homotypic cell clustering of the hemopoietic progenitor cell line KG1a (20). Lastres et al. (27) observed that CD31 mAb HEC/75 blocks TGF-ß1-induced cell clustering of the promonocytic cell line U-937. In line with our observations, CD31 is expressed at high levels by virtually all CD34+ cells, and transmigration of CD34+ cells (41) or a subset of CD34+ DC precursors (42) through vascular endothelial layers is dependent on CD31 adhesion.

As mentioned above, mAb HECD-1 directed against E-cadherin inhibited TGF-ß1-induced LC precursor cell cluster formation, but in contrast to the above cell cluster-blocking mAbs, outgrowth of CD1a+ LC was not impaired. These observations thus seem to argue against a functional involvement of cell clustering in LC differentiation. We cannot, however, rule out the possibility that E-cadherin ligation by mAb may functionally alter the differentiation pattern of cultured cells, eventually leading to enhancement of LC generation. Consistent with this possibility, E-cadherin has previously been implicated in the differentiation processes of epithelial (43) and hemopoietic progenitor cells (10, 12). Additionally, mAb ligation of E-cadherin on the surface of epithelial cell lines was previously shown to mimic homophilic E-cadherin ligation in inducing transmembrane signaling in epithelial cell lines (44, 45, 46).

In conclusion, cell cluster formation represents a regular response of LC precursor cells to TGF-ß1 stimulation, and TGF-ß1-dependent cellular interactions may modulate LC development and differentiation. This differentiation model is optimally suited for studying the possible functional involvement of E-cadherin in LC differentiation using gene transfer experiments.


   Acknowledgments
 
We thank A. Renner for his invaluable contribution in cell separation. We are indebted to all the collaborating nurses and doctors of the gynecology departments at Sozialmedizinisches Zentrum Ost and Kaiser Franz Josef Spital (Vienna, Austria) for providing cord blood samples. Additionally we thank Dr. A. Barth for critically reading the manuscript.


   Footnotes
 
1 This work was supported by the Interdisziplinares Cooperations-Projekt program of the Austrian Ministry for Research and Transport. Back

2 Address correspondence and reprint requests to Dr. Herbert Strobl, Institute of Immunology, University of Vienna, Borschkegasse 8A, A-1090 Vienna, Austria. Back

3 Abbreviations used in this paper: LC, Langerhans cells; SCF, stem cell factor; FL, Flt3 ligand; DC, dendritic cells; CB, cord blood. Back

Received for publication February 22, 2000. Accepted for publication May 23, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Steinman, R.. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9:271.[Medline]
  2. Udey, M. C.. 1997. Cadherins and Langerhans cell immunobiology. Clin. Exp. Immunol. 107:(Suppl. 1):6.
  3. Strobl, H., E. Riedl, C. Bello-Fernandez, W. Knapp. 1998. Epidermal Langerhans cell development and differentiation. Immunobiology 198:588.[Medline]
  4. Tang, A., M. Amagai, L. G. Granger, J. R. Stanley, M. C. Udey. 1993. Adhesion of epidermal Langerhans cells to keratinocytes mediated by E-cadherin. Nature 361:82.[Medline]
  5. Blauvelt, A., S. I. Katz, M. C. Udey. 1995. Human Langerhans cells express E-cadherin. J. Invest. Dermatol. 104:293.[Medline]
  6. Jakob, T., A. Saitoh, M. C. Udey. 1997. E-cadherin-mediated adhesion involving Langerhans cell-like dendritic cells expanded from murine fetal skin. J. Immunol. 159:2693.[Abstract]
  7. Borkowski, T. A., B. J. Van Dyke, K. Schwarzenberger, V. W. McFarland, A. G. Farr, M. C. Udey. 1994. Expression of E-cadherin by murine dendritic cells: E-cadherin as a dendritic cell differentiation antigen characteristic of epidermal Langerhans cells and related cells. Eur. J. Immunol. 24:2767.[Medline]
  8. Schwarzenberger, K., M. C. Udey. 1996. Contact allergens and epidermal proinflammatory cytokines modulate Langerhans cell E-cadherin expression in situ. J. Invest. Dermatol. 106:553.[Medline]
  9. Lee, M.-G., S. O. Sharrow, A. G. Farr, A. Singer, M. C. Udey. 1994. Expression of the homophilic adhesion molecule E-cadherin by immature thymocytes and thymic epithelial cells. J. Immunol. 152:5653.[Abstract]
  10. Muller, K. M., C. J. Luedecker, M. C. Udey, A. G. Farr. 1997. Involvement of E-cadherin in thymus organogenesis and thymocyte maturation. Immunity 6:257.[Medline]
  11. Lee, M. G., A. Tang, S. O. Sharrow, M. C. Udey. 1994. Murine dendritic epidermal T cells (DETC) express the homophilic adhesion molecule E-cadherin. Epithelial Cell Biol. 3:149.[Medline]
  12. Armeanu, S., H.-J. Bühring, M. Reuss-Borst, C. A. Müller, G. Klein. 1995. E-cadherin is functionally involved in the maturation of the erythroid lineage. J. Cell Biol. 131:243.[Abstract/Free Full Text]
  13. Strobl, H., E. Riedl, C. Scheinecker, C. Bello-Fernandez, W. F. Pickl, K. Rappersberger, O. Majdic, W. Knapp. 1996. TGF-ß1 promotes in vitro development of dendritic cells from CD34+ hemopoietic progenitors. J. Immunol. 157:1499.[Abstract]
  14. Borkowski, T. A., J. J. Letterio, A. G. Farr, M. C. Udey. 1996. A role for endogenous transforming growth factor ß 1 in Langerhans cell biology: the skin of transforming growth factor ß1 null mice is devoid of epidermal Langerhans cells. J. Exp. Med. 184:2417.[Abstract/Free Full Text]
  15. Borkowski, T. A., J. J. Letterio, C. L. Mackall, A. Saitoh, X.-J. Wang, D. R. Roop, R. E. Gress, M. C. Udey. 1997. A role for TGFß1 in Langerhans cell biology. J. Clin. Invest. 100:575.[Medline]
  16. Boivin, G. P., B. A. O’Toole, I. E. Orsmby, R. J. Diebold, M. J. Eis, T. Doetschman, A. B. Kier. 1995. Onset and progression of pathological lesions in transforming growth factor-ß1-deficient mice. Am. J. Pathol. 146:276.[Abstract]
  17. Gruschwitz, M. S., O. P. Hornstein. 1992. Expression of transforming growth factor type ß on human epidermal dendritic cells. J. Invest. Dermatol. 99:114.[Medline]
  18. de Saint-Vis, B., I. Fugier-Vivier, C. Massacrier, C. Gaillard, B. Vanbervliet, S. Ait-Yahia, J. Banchereau, Y. J. Liu, S. Lebecque, C. Caux. 1998. The cytokine profile expressed by human dendritic cells is dependent on cell subtype and mode of activation. J. Immunol. 160:1666.[Abstract/Free Full Text]
  19. Lee, W. C., C. Zhong, S. Qian, Y. Wan, J. Gauldie, Z. Mi, P. D. Robbins, A. W. Thomson, L. Lu. 1998. Phenotype, function, and in vivo migration and survival of allogeneic dendritic cell progenitors genetically engineered to express TGF-ß. Transplantation 66:1810.[Medline]
  20. Majdic, O., J. Stöckl, W. F. Pickl, J. Bohuslav, H. Strobl, C. Scheinecker, H. Stockinger, W. Knapp. 1994. Signaling and induction of enhanced cytoadhesiveness via the hematopoietic progenitor cell surface molecule CD34. Blood 83:1226.[Abstract/Free Full Text]
  21. Waclavicek, M., O. Majdic, T. Stulnig, M. Berger, R. Sunder-Plassmann, G. J. Zlabinger, T. Baumruker, J. Stoeckl, C. Ebner, W. Knapp, et al 1998. CD99 engagement on human peripheral blood T cells results in TCR/CD3 dependent cellular activation and allows for Th1-restricted cytokine production. J. Immunol. 161:4671.[Abstract/Free Full Text]
  22. Smith, C. W., R. Rothlein, B. J. Hughes, M. M. Mariscalco, H. E. Rudloff, F. C. Schmalstieg, D. C. Anderson. 1988. Recognition of an endothelial determinant for CD18-dependent human neutrophil adherence and transendothelial migration. J. Clin. Invest. 82:1746.
  23. Davis, L. S., N. Oppenheimer-Marks, J. L. Bednarczyk, B. W. McIntyre, P. E. Lipsky. 1990. Fibronectin promotes proliferation of naive and memory T cells by signaling through both the VLA-4 and VLA-5 integrin molecules. J. Immunol. 145:785.[Abstract]
  24. Weller, P. F., T. H. Rand, S. E. Goelz, G. Chi-Rosso, R. R. Lobb. 1991. Human eosinophil adherence to vascular endothelium mediated by binding to vascular cell adhesion molecule 1 and endothelial leukocyte adhesion molecule 1. Proc. Natl. Acad. Sci. USA 88:7430.[Abstract/Free Full Text]
  25. Blase, L., A. Merling, S. Engelmann, P. Moller, R. Schwartz-Albiez. 1996. Characterization of cell surface-expressed proteochondroitin sulfate of pre-B Nalm-6 cells and its possible role in laminin adhesion. Leukemia 10:1000.[Medline]
  26. Shimoyama, Y., S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, O. Abe. 1989. Cadherin cell-adhesion molecules in human epithelial tissues and carcinomas. Cancer Res. 49:2128.[Abstract/Free Full Text]
  27. Lastres, P., N. Almendro, T. Bellon, J. A. Lopez-Guerrero, R. Eritja, C. Bernabeu. 1994. Functional regulation of platelet/endothelial cell adhesion molecule-1 by TGF-ß1 in promonocytic U-937 cells. J. Immunol. 153:4206.[Abstract]
  28. Stöckl, J., O. Majdic, W. F. Pickl, A. Rosenkranz, E. Prager, E. Gschwantler, W. Knapp. 1995. Granulocyte activation via a binding site near the C-terminal region of complement receptor type 3 {alpha}-chain (CD11b) potentially involved in intramembrane complex formation with glycosylphophatidylinositol-anchored Fc{gamma}RIIIB (CD16) molecules. J. Immunol. 154:5452.[Abstract]
  29. Strobl, H., C. Bello-Fernandez, E. Riedl, W. F. Pickl, O. Majdic, S. T. Lyman, W. Knapp. 1997. Flt3 ligand in cooperation with transforming growth factor-ß1 potentiates in vitro development of Langerhans-type dendritic cells and allows single-cell dendritic cell cluster formation under serum-free conditions. Blood 90:1425.[Abstract/Free Full Text]
  30. Caux, C., B. Vanbervliet, C. Massacrier, C. Dezutter-Dambuyant, B. de Saint-Vis, C. Jacquet, K. Yoneda, S. Imamura, D. Schmitt, J. Banchereau. 1996. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF{alpha}. J. Exp. Med. 184:695.[Abstract/Free Full Text]
  31. Szabolcs, P., M. A. S. Moore, J. W. Young. 1995. Expansion of immunostimulatory dendritic cells among the myeloid progeny of human CD34+ bone marrow precursors cultured with c-Kit ligand, granulocyte-macrophage colony-stimulating factor, and TNF {alpha}. J. Immunol. 154:5851.[Abstract]
  32. Maraskovsky, E., K. Brasel, M. Teepe, E. R. Roux, S. D. Lyman, K. Shortman, H. J. McKenna. 1996. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. J. Exp. Med. 184:1953.[Abstract/Free Full Text]
  33. Cepek, K. L., S. K. Shaw, C. M. Parker, G. J. Russell, J. S. Morrow, D. L. Rimm, M. B. Brenner. 1994. Adhesion between epithelial cells and T lymphocytes mediated by E-cadherin and the {alpha}Eß7 integrin. Nature 372:190.[Medline]
  34. Knapp, W., H. Strobl, C. Scheinecker, C. Bello-Fernandez, O. Majdic. 1995. Molecular characterization of CD34+ human hematopoietic progenitor cells. Ann. Hematol. 70:281.[Medline]
  35. Scheinecker, C., H. Strobl, G. Fritsch, B. Csmarits, O. Krieger, O. Majdic, W. Knapp. 1995. Granulomonocyte-associated lysosomal protein expression during in vitro expansion and differentiation of CD34+ hemopoietic progenitor cells. Blood 86:4115.[Abstract/Free Full Text]
  36. Riedl, E., H. Strobl, O. Majdic, W. Knapp. 1997. TGF-ß1 promotes in vitro generation of dendritic cells by protecting progenitor cells from apoptosis. J. Immunol. 158:1591.[Abstract]
  37. Geissmann, F., C. Prost, J. P. Monnet, M. Dy, N. Brousse, O. Hermine. 1998. Transforming growth factor ß1, in the presence of granulocyte/macrophage colony-stimulating factor and interleukin 4, induces differentiation of human peripheral blood monocytes into dendritic Langerhans cells. J. Exp. Med. 187:961.[Abstract/Free Full Text]
  38. Jaksits, S., E. Kriehuber, A. S. Charbonnier, K. Rappersberger, G. Stingl, D. Maurer. 1999. CD34+ cell-derived CD14+ precursor cells develop into Langerhans cells in a TGF-ß1-dependent manner. J. Immunol. 163:4869.[Abstract/Free Full Text]
  39. Caux, C., C. Massacrier, B. Dubois, J. Valladeau, C. Dezutter-Dambuyant, I. Durand, D. Schmitt, S. Saeland. 1999. Respective involvement of TGF-ß and IL-4 in the development of Langerhans cells and non-Langerhans dendritic cells from CD34+ progenitors. J. Leukocyte Biol. 66:781.[Abstract]
  40. Kim, S. H., E. Y. Choi, Y. K. Shin, T. J. Kim, D. H. Chung, S. I. Chang, N. K. Kim, S. H. Park. 1998. Generation of cells with Hodgkin’s and Reed-Sternberg phenotype through downregulation of CD99 (Mic2). Blood 92:4287.[Abstract/Free Full Text]
  41. Yong, K. L., M. Watts, N. Shaun Thomas, A. Sullivan, S. Ings, D. C. Linch. 1998. Transmigration of CD34+ cells across specialized and nonspecialized endothelium requires prior activation by growth factors and is mediated by PECAM-1 (CD31). Blood 91:1196.[Abstract/Free Full Text]
  42. Ferrero, E., A. Bodanza, B. E. Leone, S. Mancini, A. Poggi, M. R. Zocchi. 1998. CD14+ CD34+ peripheral blood mononuclear cells migrate across endothelium and give rise to immunostimulatory dendritic cells. J. Immunol. 160:2675.[Abstract/Free Full Text]
  43. Larue, L., C. Antos, S. Butz, O. Huber, V. Delmas, M. Dominis, R. Kemler. 1996. A role for cadherins in tissue formation. Development 122:3185.[Abstract]
  44. Kinch, M. S., L. Petch, C. Zhong, K. Burridge. 1997. E-cadherin engagement stimulates tyrosine phosphorylation. Cell. Adhes. Commun. 4:425.[Medline]
  45. Levenberg, S., B. Z. Katz, K. M. Yamada, and B. Geiger. 1998. Long-range and selective autoregulation of cell-cell or cell-matrix adhesions by cadherin or integrin ligands. J. Cell. Sci. 111.
  46. Levenberg, S., A. Yarden, Z. Kam, B. Geiger. 1999. p27 is involved in N-cadherin-mediated contact inhibition of cell growth and S-phase entry. Oncogene 18:869.[Medline]



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