Expression and functional effects of Eph receptor tyrosine kinase A family members on Langerhans like dendritic cells
© Munthe et al; licensee BioMed Central Ltd. 2004
Received: 16 November 2003
Accepted: 03 June 2004
Published: 03 June 2004
The Eph receptors are the largest receptor tyrosine kinase family. Several family members are expressed in hematopoietic cells. Previously, the expression of a member of this family, EphA2, was identified on dendritic like cells in tonsils. We therefore specifically examined the expression of EphA2 on in vitro generated dendritic cells.
In this study, expression of the EphA2 receptor was identified on in vitro generated Langerhans like dendritic cells compared to in vitro generated dendritic cells. We show that ligand induced engagement of the EphA2 receptor leads to receptor autophosphorylation indicating a functional receptor signaling pathway in these cells. We also observe phosphorylation and dephosphorylation of distinct proteins following ligand activation of EphA receptors. In co-stimulation assays, receptor-ligand interaction reduces the capacity of the Langerhans like dendritic cells to stimulate resting CD4+ T cells.
Engagement of EphA receptor tyrosine kinases on Langerhans like dendritic cells induces signaling as shown by tyrosine phosphorylation and dephosphorylation of distinct proteins. Furthermore this engagement renders the cells less capable of stimulating CD4+ T cells.
Immature dendritic cells are localized in tissues where they monitor the microenvironment and are characterized by their capacity to take up antigens. Dendritic cells must be activated by "danger signals" to become efficient antigen presenting cells [1–3]. This maturation process includes an efficient presentation of processed antigens by inducing cell surface expression of peptide loaded MHC molecules and an increased production of cytokines. Up-regulation of specific co-stimulatory and co-adhesive molecules, like CD80 and CD86, are also necessary to fully activate T cells. Finally, a potential to migrate to the lymph nodes is developed. The mature dendritic cell is thus equipped with a package of information that orchestrates the T cell response . Dendritic cells can be divided into several groups, with different cellular origins, localization and capacity to stimulate a primary T cell response . One group is the Langerhans cells, which are immature dendritic cells of myeloid origin resident in squamous epithelia, including skin and mucosa. These cells are characterized by high cell surface expression of CD1a and E-cadherin, in addition to the presence of Birbeck granules with langerin . Recently, it has been shown that Langerhans like cells can be generated in vitro from both adherent monocytes and CD34+ bone marrow cells in the presence of transforming growth factor β (TGF-β) [6, 7].
Previously, we have investigated the expression of Eph receptor tyrosine kinases and their ligands in lymphoid tissues [8, 9]. The Eph kinases are the largest known subfamily of receptor tyrosine kinases with 15 distinct members highly conserved from insects to man . The Eph receptor tyrosine kinases bind a family of ligands called ephrins, consisting of two subclasses, ephrin-A and ephrin-B . The six ephrin-A ligands are anchored to the membrane by a glycosylphosphatidylinositol (GPI)-tail, while the three ephrin-B ligands are transmembrane molecules. Members of both the Eph tyrosine kinases and the ephrin ligands mediate signaling after receptor-ligand interaction [11–17]. This bi-directional signaling are known to affect processes involving cellular interaction, like cell adhesion, cell migration and tissue border formation [16–18]. In particular, signaling through both the Eph kinases and the ephrin ligands have been shown to affect cellular adhesion through integrins [14, 19–22]. One receptor of the Eph family, EphA2, is expressed in rat intestine and skin , and in fetal mouse skin and the epithelial lining of the esophagus . Previously, we have shown the presence of EphA2 mRNA in several human hematopoietic tissues, and also identified protein expression in an adherent tonsil cell population with a dendritic appearance . The aim of this study was therefore to identify a dendritic cell population expressing EphA2 and further investigate its functional role.
Here, we present the selective expression of EphA2 on in vitro generated Langerhans like dendritic cells. Functional signaling through EphA receptors is revealed by induced tyrosine phosphorylation and dephosphorylation of distinct proteins. Ligation of EphA receptors with a ligand reduces the capacity of Langerhans like dendritic cells to stimulate resting CD4+ T cells.
Results and Discussion
In vitro generated Langerhans like dendritic cells (LLDC) express the EphA2 receptor tyrosine kinase
By PCR, the expression of other EphA members in LLDC was also tested (EphA1, A2, A3, A4, A5, A7, A8). In addition to EphA2, expression of EphA1 could also be detected (data not shown). In this study, we have not investigated the expression of EphA1 at the protein level. Thus, we cannot exclude that EphA1 is involved in the functional effects observed after ligand binding presented below.
During the preparation of this manuscript, the expression of EphA2 was reported on epidermis residing Langerhans cells .
Ephrin-A induces signaling through EphA receptor kinases in LLDC
To further investigate the signaling capacity of EphA receptors, cells were incubated with bead coupled ephrin-A1-Fc for 10, 20 and 30 min, and then fractionated into cytosol and membrane fractions followed by SDS-PAGE separation, blotting and hybridization with an anti-phosphotyrosine antibody. Distinct differences in the protein tyrosine phosphorylation pattern were observed after ephrin-A1-Fc receptor ligation in the membrane fraction (figure 3B). In particular, a protein with approximately molecular mass of 70 kDa was phosphorylated upon 20 and 30 min incubation with ephrin-A1, while dephosphorylation of a protein with approximately molecular mass of 35 kDa was observed already after 10 min incubation (figure 3B). Several proteins have been reported to be phosphorylated after ephrin ligation of EphA receptors like Fak, paxillin and p130Cas , or dephosphorylated like Fak and paxillin [30, 31]., depending on the cell system applied. None of these proteins correlate with the estimated molecular mass of the phosphorylated and dephosphorylated proteins identified in LLDC (figure 3B). The transient phosphorylation and dephosphorylation patterns observed after stimulation of Eph receptors involve distinct phosphatases. Shp-2 can associate with EphA2 and might be involved in dephosphorylation of FAK and paxillin leading to dissociation of the EphA2-FAK complex . Low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) can be recruited to EphB2 receptor complexes after ephrin ligation . Whether these phosphatases are involved in EphA signaling in LLDC awaits further studies.
Engagement of Eph receptors does not influence the cell surface expression of accessory- and adhesion molecules of LLDC
Effect of engagement of EphA receptors on LLDC on the proliferative response of T cells in co-culture assays
Based on the observation that EphA activation led to changes in the tyrosine phosphorylation pattern; we investigated if EphA receptor ligation had any consequence for the capacity of the LLDC to activate T cells. An allogeneic assay was performed with increasing numbers of LLDC or dendritic cells in co-culture with 50 000 CD4+ T cells in the presence of either immobilized ephrin-A4-Fc or CD19-Fc (control protein). T cell proliferation was measured as thymidine uptake on day 6–7. For this experiment ephrin-A4 was chosen since we observed binding of ephrin-A1-Fc to T cells, as shown by others, but only weak or no binding of ephrin-A4-Fc (data not shown).
Also a syngeneic antigen presentation assay, using the recall antigen PPD from mycobacterium, was performed. Increased numbers of LLDC, on either immobilized ephrin-A4-Fc or CD19-Fc, were co-cultured with 50000 CD4+ T cells in the presence of PPD. Also here, ligation of EphA receptors with immobilized ephrin-A4 resulted in reduced T cell proliferation indicating that the reduction in the stimulatory capacity of the LLDC is independent of the presented antigen. As shown for the allogeneic assay (figure 5A), the effect was most pronounced at a high T cell: LLDC ratio (figure 5C). Thus, the reduction in T cell proliferation is most pronounced when sub-optimally stimulating the T cell.
Dendritic cells strictly regulate the quantity of a T cell response. Adhesion molecules are involved in the synapse formation and adhesion between antigen presenting cells and T cells influence the activation. In addition, rearrangements of the actin cytoskeletal in dendritic cells are necessary for optimal stimulation of resting T cells . Bi-directional signaling through the Eph receptors and ephrin ligands influence both the shape and the adhesive properties of cells, by affecting integrin affinity and the cytoskeleton [16, 18]. In addition, MAPK activation has been shown to result in inhibition of adhesion to extracellular matrix . MAPK activation has also been shown to influence activation and maturation of dendritic cells [39, 40]. Thus, one may speculate that a reduced contact between the LLDC and the T cell, possibly through integrin deactivation, inhibits stimulation of T cells, although we did not observe any difference in expression of β2 integrins (CD18, CD11a and CD11c). In contrary, a recent report presents results that indicates increased adhesion of EphA2 expressing dendritic cells derived from CD34+ progenitors to fibronectin coated surfaces in the presence of ephrin-A3 through β1 integrin activation .
Alternatively, the activation of Eph kinases induces translocation to caveolae-like domains, thus; engagement of EphA receptors on LLDC with immobilized ligand may induce ectopic raft aggregates, and thereby destabilize the immunological synapse [41, 42]. We have not tested if secretions of soluble factors important for T cell activation are affected after EphA receptor activation on LLDC. Thus we cannot exclude that altered secretion might lead to the observed reduced T cell proliferation.
Cross-linking of EphA receptors on LLDC does not change the cytokine profile indicative of a Th1 or Th2 response
Expression of differentiation and activation markers on re-stimulated CD4+ cells
IFN-g, % pos
IL4, % pos
CD25, % pos
Functional activation of CD4+ T cells induces the expression of cytokines indicative for Th1 or Th2 responses. Different factors such as the number and type of antigen presenting cells, co-stimulatory molecules and the duration of T cell receptor stimulation influence the polarization of the T cell response, although cytokines are the dominant regulators [43, 44]. To investigate if EphA ligation had a selective effect on polarization of effector cell, autologous CD4+ T cells were cultured one week with LLDC in the presence of either immobilized ephrin-A4-Fc or CD19-Fc, before re-stimulation with TPA and ionomycin. The T cells were then analyzed for the expression of cytokines indicative of either a Th1 (IFN-γ) or a Th2 response (IL-4) by flow cytometry. Although EphA receptor ligation reduced the number of T cells expressing the indicated cytokines on a general basis compared to CD19-Fc, no selective reduction in either of the Th1 or Th2 populations were seen (table 1). This demonstrates that EphA receptor signaling in LLDC does not influence these functional properties of CD4+ T cells.
Since the reduction in proliferation of CD4+ T cells is more pronounced at a high CD4+: LLDC ratio, the differentiation status of these cells in a mixed lymphocyte reaction was also studied with higher numbers of LLDC. In accordance with what is reported in the literature, we observed in our system an increased number of T cells producing cytokines, especially the Th1 cytokine IFN-γ, when decreasing the CD4+: LLDC ratio (table 1). In conclusion, engagement of EphA receptors on LLDC does not seem to affect the polarization of CD4+ T cells, which may indicate that the effect is through inhibition of adhesion.
The presence of several Eph receptor tyrosine kinases and ephrin ligands in cells involved in immune responses suggest a functional role for these genes in immunity [8, 41, 45, 46]. However, the molecular mechanism still remains elusive. Here we specifically present expression of the EphA2 receptor tyrosine kinase on monocyte derived LLDC. The activation of EphA receptor tyrosine kinases on these cells reduces the potential of monocyte derived LLDC to activate CD4+ T cells. The outcome of signaling through Eph receptors has been demonstrated to be either increased adhesion or decreased adhesion dependent on the cell system studied. Although the mechanism for the reduced T cell proliferation remains unknown, one might speculate that it is due to a reduced interaction between the LLDC and T cells, or that EphA ligation inhibits activation and maturation of the LLDC. CD4+ T cells acquire an Eph ligand, ephrin-A4, after stimulation with CD3/CD28, and thus these cells might interact with Langerhans cells in vivo e.g. in lymph nodes through this ligand. Interestingly, several Eph receptors and ephrin ligands are ectopically expressed in cancers, e.g. both EphA2 and ephrin-A1 are over-expressed in aggressive melanomas [47, 48]. It is tempting to speculate that the influence of Eph and ephrin interactions in immunity play a role in the etiology of cancer, in addition to the increased angiogeneic potential and the effect on migration of metastatic cells .
Antibodies, cytokines and fusion proteins
Monoclonal anti-EphA2 was a kind gift from Dr. R.A Lindberg  and polyclonal α-EphA2 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine antibodies: PY99 (Santa Cruz) and 4G10 (Upstate Biotechnology, Lake Placid, NY). Secondary antibody for Western blot hybridization: α-mouse-HRP (DAKO, Copenhagen, Denmark). Antibodies used for flow cytometry analysis: α-CD25 (DAKO), α-CD80 (Becton Dickinson, SanJose, CA), α-CD83 (Immunotech, Marseilles, France), α-CD86 (PharMingen, San Diego, CA), α-HLA-DR (Becton Dickinson), α-E-Cadherin (Zymed Laboratories Inc, San Francisco, CA), phycoerytrin-labeled (PE) – or Fluorescein (FITC)-labeled (F) α-mouse Ig polyclonal antibody (Ig-RPE and Ig-RF, Southern Biotechnology Associates, Birmingham, AL, USA), α-IL-4-PE and α-INFγ-FITC (PharMingen). IL-4 was a kind gift from Schering-Plough Research Institute (Kenilworth, J), TGF-β1 was obtained from Habersham Pharmacia Biotech (Uppsala, Sweden) and GM-CSF was obtained from Roche (Mannheim, Germany). Fusion proteins of the extracellular part of either CD19 (negative control protein) or ephrin-A4 fused to the mouse constant and hinge region of IgG2b heavy chain is previously described in . Ephrin-A1-Fc fusion protein was generated as described in , with gene specific primers.
Cell isolation and treatments
Mononuclear cells were obtained from Buffy coat from normal, healthy donors using the lymph prep kit (Nycomed Pharma, Oslo, Norway). Platelet numbers were reduced by 8 min. centrifugation at 180 g. 20 × 106 mononuclear cells were seeded per well in a 6-well plate (Costar Corp., Cambridge, MA), and non-adherent cells were removed by extensive washing after incubation at 37°C for two hours. Cells were grown for one week in RPMI 1640, supplemented with 10% FCS, 200 ng/ml GM-CSF and 100 ng/ml IL-4. For the generation of LLDC, 10 ng/ml rhTGF-β1 were added in addition. Fresh medium supplemented with cytokines was added at day 2–3. Any remaining B and T cells were depleted with anti-CD19 and anti-CD3 coated Dynabeads (Dynal Biotech, Oslo, Norway) before experiments with LLDC. CD4+ T cells were isolated using anti-CD4 coated Dynabeads (Dynal) and resting CD4+ T cells were obtained using negative depletion with anti-MHC class II Dynabeads (Dynal). CD3/CD28 stimulation of CD4+ T cells was performed with 2 Dynabeads (Dynal) per T cell. All cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5 % CO2.
Antigen presentation assays
Round-bottom 96-well tissue culture plates were coated with 10 ug/ml α-mouse-IgG in 50 mM Tris-HCl pH 9.5, blocked with 0.1% BSA, and finally incubated with 20 ug/ml of the indicated fusion proteins in PBS. Indicated numbers of dendritic cells were seeded along with 50000 CD4+ cells in RPMI-1640 medium supplemented with 5% FCS and antibiotics. In addition, for autologue presentation assay, 2 ng/ml PPD ("purified protein derivative") from Mycobacterium tuberculosis (Veterinary Institute, Oslo, Norway) were added. At day 6–7, the cells were pulsed with 3.7 × 104 Bq/well 3H-thymidine ON. The cells were harvested in a 96-well harvester (Packard, Meriden, CT), and incorporated thymidine was measured with a Top Count liquid scintillation counter (Packard). All experiments were performed in triplicate.
LLDC were stimulated with ephrin-A1-Fc for the indicated times, then washed in ice-cold phosphate-buffered saline (PBS) and resuspended in buffer A (5 mM Tris-HCl, pH 8,0.5 mM EDTA, 75 mM sucrose and proteinase inhibitors) and sonicated four times 15 s. Nuclei were pelleted by centrifugation at 400 g for 5 min 4°C in a microsentrifuge. The supernatants were centrifuged at 32 000 g for 30 min at 4°C in a Beckmann centrifuge. The supernatants was collected and used as the cytosol fraction. The membrane pellets were washed three times with PBS, solubilised in buffer A containing 1 % Triton X-100 for 15 min at 4°C and then centrifuged at 10 000 g for 10 min at 4°C in a microsentrifuge. Supernatants were used as the membrane fraction.
Phosphorylation, immunoprecipitation and Western blot
LLDC were serum starved (1% FBS) over night and washed in PBS. Prewarmed cells were then incubated with Ephrin-A1-Fc coated on anti-mouse Ig magnetic beads (five beads/cell; Dynal, Oslo, Norway) for the indicated times at 37°C before resuspension in buffer A, sonication and fractionation into cytosol and membrane fractions as described above (subcellular fractionation) or lysis in lysis buffer (PBS, 1% NP40, aprotinin (Sigma, St.Louis, MO) and 0.5% phosphatase inhibitor cocktail II (Sigma) on ice for 30 minutes. Protein concentrations were estimated by Ponceau red (Sigma) staining of dot blots on nitrocellulose membranes (Schleicher and Schuell GmbH, Dassel, Germany) by comparison with proteins of known concentration. 1 ug polyclonal antibody was used to precipitate the EphA2 receptor over night at 4°C, and the antibody-complex was captured with protein-G sepharose (Pharmacia). After three times washing with TBS + 0.1% Tween20, captured proteins were eluted by boiling in 3 × SDS sample buffer. PAGE was performed with indicated lysates or immunoprecipitates. The Western blot was immunoblotted with indicated antibodies and visualized (ECL+, Amersham). The filter was stripped and reprobed with polyclonal α-EphA2.
Cell staining and flow cytometry analysis
Cell surface staining
Cells were pre-incubated with 0.1 mg/ml human aggregated gamma globulin to block unspecific staining, then incubated with 20 ug/ml fusion protein or antibody for 30 min. at 4°C. The cells were washed twice, and stained with the indicated anti-mouse conjugated secondary antibody. 10000 cells were analyzed.
CD4+ T cells co-incubated with LLDC in the presence of immobilized fusion protein were re-stimulated with 2,5 ug/ml TPA and 250 ng/ml ionomycin for 5 hour, and 10 ug/ml brefeldinA (Sigma) was added the last 2–3 hours. Intracellular staining was performed using the Fix and Perm cell permeabilization kit (Caltag laboratories), as described by the manufacturer. 104 cells were analyzed by a FACSCalibur flow cytometer, and the analyses were performed with CELLQuest software (Becton Dickinson). 10000 cells were analyzed.
We thank Schering-Plough Research for kindly providing the IL4, Dr. R.A. Lindberg for providing monoclonal EphA2-antibody, Helena Hauge for expert technical assistance and Professor Steinar Funderud for critically reading the manuscript. This work was supported by the Norwegian Research Council and the Norwegian Cancer Society.
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