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* Department of Cell Biology, Faculty of Biology, Complutense University, Madrid, Spain;
Department of Cell Biology, Faculty of Medicine, Complutense University, Madrid, Spain; and
Department of Biology, Imperial College of Science, Technology, and Medicine, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Eph family of receptors comprises several members of structurally related transmembrane receptor tyrosine kinases that interact with their ligands, called ephrins. These can be divided into two groups based on their sequence similarity and their preference for different ephrins. Eph A receptors bind GPI-anchored ligands, the ephrins A, whereas Eph B bind transmembrane ligands, the ephrins B (4, 5), that are phosphorylated after Eph-ephrin interaction (6). Eph-ephrin signaling mediates contact-dependent cell interactions demonstrated to be important for the migratory behavior of neurons and neural crest cells, the definition of rhombomeres and somites, tissue patterning, and blood vessel formation (7). Several studies have described a role for this family in the regulation of cell adhesion and the cytoskeleton (8). Presumably, Eph/ephrins could also be involved in the above-mentioned processes occurring in the thymus. However, there is very little information on their expression in the lymphoid organs, including the thymus. Eph A1 and B2 were detected by Northern analysis in the thymus (9, 10) and Eph B6 was cloned from mouse thymus (11), being expressed mainly by DP (CD4+CD8+) cells (12). Eph A2 is homogeneously expressed in the mouse thymus, as revealed by in situ hybridization (13), whereas in rat thymus it is detected at low levels by Northern blot (14), and in the human tonsils it has been reported in the dendritic-like cells of crypts (15). Eph A3, which seemed to be remarkably restricted to lymphoid tumor cell lines and not expressed in many other cell lines established from normal lymphoid and myeloid bone marrow cells, was detected by RT-PCR in thymocytes (16, 17, 18). Eph A4 and A7 seem to be significantly expressed in B lymphocytes but weakly expressed in both thymus and tonsil T cells (19). Eph B4 is expressed in both CD34+ and CD14+ cells of human cord blood but not in PMA-induced differentiated cells (20, 21). In addition, Eph B4 mRNA was detected in 68 of 70 continuous human leukemia or lymphoma cell lines (22). Ephrin A1 has not been detected by Northern blot in rat thymus, and in situ hybridization revealed expression associated with connective tissue septae in the later fetal stages of mouse thymus and scattered throughout the adult thymus (13), while ephrin A3 and ephrin B1 were detected by Northern blot (23). A membrane-bound form of ephrin A4 has been detected in peripheral T and B cells and a splice variant of this ephrin is expressed on stimulation of these cells. It is also detected by Northern blot in spleen, lymph nodes, peripheral lymphocytes, and fetal liver, and at lower levels in the thymus (15).
In the present study, we focus our attention on the expression of the Eph A family in developing and adult rat thymus. Our results demonstrate that these molecules are expressed in lymphoid and nonlymphoid cells in distinct but frequently overlapping patterns in both embryonic and adult rat thymus. More importantly, the supply of a soluble form of either Eph A or ephrin A to fetal thymus organ cultures (FTOCs) alters T cell differentiation, suggesting a physiological role for the Eph A members and their ligands in thymus function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wistar-Hannover rats (Harlan Iberica, Barcelona, Spain) were maintained in our animal facilities in conventional conditions. Rat fetuses were obtained from timed pregnancies. The day of finding a vaginal plug was designated day 0 of gestation.
RT-PCR
Total RNA from adult, fetal, or newborn rat thymus was purified using TRI reagent (Molecular Research Center, Cincinnati, OH). Total cDNA was synthesized with Superscript II RT polymerase (Life Technologies, Barcelona, Spain) according to the supplier’s instructions. cDNA was used as target in PCR amplifications performed with specific primers for the following: mouse Eph A1 (GenBank accession no. U18084), 5' (pos. 1035)-ctgcacagggagccttagac-3' and 5' (pos. 1412)-gctgaccaggagctagttgg; mouse Eph A2 (GenBank accession no. U07634), 5' (pos. 1061)-ccatgtcttgcacacgtcca-3' and 5' (pos. 1296)-acggacatcctcagaggact; rat Eph A3 (GenBank accession no. U69278), 5' (pos. 1088)-ggaggccggaaggatattac-3' and 5' (pos. 1482)-ctcgtctcttgctcctgctt-3'; mouse Eph A4 (GenBank accession no. X65138), 5' (pos. 1231)-ggactcaagacgaccagagt-3' and 5' (pos. 1780)-tactccgtcttcggctgatc-3'; rat ephrin A1 (GenBank accession no. D38056.1), 5' (pos. 30)-cgcgctatggagttccttt-3' and 5' (pos. 671)-cagggcaagcaaataccttc-3'; human ephrin A2 (GenBank accession no. NM_001405), 5' (pos. 462)-ctacatctctgccacgcctc-3'; mouse ephrin A2 (U14941), 5' (pos. 646)-cctgacactaggagcccaga-3'; mouse ephrin A3 (GenBank accession no. U92885.1), 5' (pos. 59)-tgtccgcactacaacagctc-3' and 5' (pos. 491)-tcccactgatgctcttctca-3'; mouse ephrin A4 (GenBank accession no. U90663.1), 5' (pos. 217)-gagctgggcttcaacgatta-3' and 5' (pos. 721)-tgacttggaaggtgtgcttg3'; and rat ephrin A5 (GenBank accession no. 69279), 5' (pos. 69)-tttctggtgctctggatgtg-3' and 5' (pos. 568)-acatcgaaaacacgatcacg-3', purchased from Amersham Pharmacia Biotech (Barcelona, Spain). The reaction was conducted for 35 incubation cycles for 45 s at 94°C, 45 s at 60°C, and 45 s at 72°C. The amplification products were analyzed by 1.5% agarose electrophoresis. As a control for cDNA quality we performed actin amplification.
Ephrin A3 PCR products were cloned into pCR II vector (Invitrogen, Groningen, The Netherlands) according to the supplier’s instructions and two different clones containing an insert of the correct size were sequenced in the DNA Sequencing Unit of Complutense University (Madrid, Spain).
Immunohistochemistry
Cryosections from adult and fetal rat thymuses were fixed in acetone for 10 min and air dried. Afterward, slides were incubated with either an anti-Eph A or an ephrin A rabbit antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min. Sections were later washed in PBS three times for 5 min and incubated with multiadsorbed biotin-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) Abs for 30 min. After washing in PBS, sections were incubated in an avidin-biotin peroxidase-conjugated solution (peroxidase Vectastain ABC kit; Vector Laboratories, Burlingame, CA) for another 30 min. The peroxidase reaction was developed with a 3,3'-diaminobenzidine kit from Vector Laboratories.
For immunofluorescence, slides were first incubated with anti-Eph A or ephrin A rabbit antiserum and mouse anti-pan-cytokeratin Ab (NCL pan-cytokeratin; Novocastra Laboratories, Newcastle-upon-Tyne, U.K.), then with biotin-conjugated donkey anti-rabbit IgG Ab, and, finally, with avidin-Texas red and multiadsorbed FITC-conjugated donkey anti-mouse IgG Ab. Each incubation step was conducted for 30 min at 4°C followed by three 5-min washes in PBS.
Sections were photographed with a Spot 2 digital camera on a Zeiss Axioplan microscope at the Servicio Comun de Investigacion (Faculty of Biology, Complutense University).
FTOCs
Sixteen-day-old fetal rat thymic lobes were cultured over polycarbonate membranes (Millipore Ibérica, Madrid, Spain) on serum-free cell culture medium (Serotec, Oxford, U.K.) containing 10-7 M Eph A-Fc or ephrin A1-Fc fusion proteins (R&D Systems, Oxon, U.K.) or purified human IgG-Fc fragments. After 6 days, lobes were processed for phenotypic analysis as detailed below.
Flow cytometry
FTOCs cell suspensions were stained for 20 min in PBS 1% FCS
with specific mAbs against rat CD4 (OX38), CD8
(OX8),
TCR
(R73), or TCR
(V65), labeled with PE, FITC, or CyChrome
(BD PharMingen, San Diego, CA), or annexin V (annexin V-FLUOS; Roche
Molecular Biochemicals, Barcelona, Spain). For cell cycle analysis,
after surface labeling, cells were permeabilized with 30% EtOH for 10
min on ice, washed in PBS, and stained with 7-amino actinomycin D
(Sigma-Aldrich, Madrid, Spain). Stained cells were analyzed in a
FACScan (BD Biosciences, San Jose, CA) at the Servicio Comun de
Investigacion (Faculty of Biology, Complutense University).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because previous data had indicated that some Eph receptors were
expressed in thymus, we analyzed the expression of Eph A1, A2, A3, and
A4 mRNA in adult and embryonic rat thymus by RT-PCR using specific
primers corresponding to the extracellular domain of these molecules. A
unique PCR product of the expected size was obtained for the four
studied Ephs. All of them were expressed from the earliest stage of
development at which the rat thymus can be dissected (day 15) to the
later embryonic stages (day 18) as well as in the adult organ (Fig. 1
).
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A). In some cases these
more intensely stained areas surrounded blood vessels. Apart from its
expression on thymocytes (Fig. 2A, inset), double
immunofluorescence staining for Eph A1 in combination with cytokeratin
was conducted to identify possible Eph A1-positive thymic epithelial
cells. Both cytokeratin-positive and -negative cells were stained with
the anti-Eph A1 antiserum (Fig. 2C). Eph A2 staining
showed a more restricted pattern mainly circumscribed to the medulla
(Fig. 2D). Nevertheless, groups of strongly stained cells
were found inside and around both blood vessels and trabeculae
(Fig. 2D) that corresponded to cytokeratin-negative cells
(Fig. 2, E and F). Eph A3 was weakly detected
mainly in the medulla and subcapsulary areas. Within the medulla the
staining was not homogeneous, consisting of stained regions, largely
associated to blood vessels, and nonstained regions. Large ramified Eph
A3+ cells occurred scattered throughout the
thymic cortex in a pattern that resembled the morphology and
distribution of ED2 macrophages previously reported by us and others
(24, 25) in the cortical region of rat thymus (Fig. 2G, inset). Eph A4 expression was found mainly in
the cortex, staining pattern corresponded to a cortical
epithelial meshwork (Fig. 2J), and double fluorescence
immunostaining confirmed that Eph A4-positive cells principally
corresponded to cytokeratin-positive cells (Fig. 2, K and
L). Some Eph A4-positive epithelial cells occurred in the
medulla as well. Both connective tissue trabeculae and blood vessels
were also stained with anti-Eph A4 antiserum (Fig. 2K).
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Several ephrins A are expressed in adult thymus
To determine the possible occurrence in rat thymus of ligands for
these Eph receptors, we performed RT-PCR amplification of total adult
thymus RNA with specific primers for ephrins A1, A2, A3, A4, and A5,
obtaining a PCR product of the expected size for ephrins A1, A2, and
A5. Ephrin A4 was amplified at low levels, and for ephrin A3 we
obtained a smaller-sized band than expected from the described
sequences of its human and mouse orthologs (Fig. 3). When sequenced, the amplified
fragment lacked a fragment corresponding to the fourth exon of the
human ephrin A3 gene (GB 11432199) and was identical to the
splice variant previously described (26). The lack of exon
4 does not imply a frameshift, and the predicted protein from the
obtained nucleotide sequence is, therefore, homologous to mouse and
human sequences except for the gap corresponding to the missing part of
the sequence. This missing part of the sequence does not affect either
the GPI linkage domain or the domain homologous to the Eph A2
metalloprotease cleavage site (27).
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, A–C). Ephrin A3 immunostaining was found
throughout the thymus, with a slightly higher intensity in the medulla
(Fig. 4, G–I). Both cortical and medullary
thymocytes expressed ephrin A3, but also some epithelial cells were
ephrin A3 positive (Fig. 4, H and I). Ephrin A5
staining principally occurred in the thymic medulla and areas
surrounding connective tissue trabeculae (Fig. 4, J–L). A weaker staining was present in the
cortex with no or low stained regions around some blood vessels. At the
cell level, stained cells corresponded mainly to thymocytes (Fig. 4, K and L). Ephrin A2 staining was less intense and
partially overlapped the pattern of ephrin A5 (Fig. 4, D–F). The strongest staining
occurred in the medulla, whereas the thymic cortex showed a weak
expression. Ephrin A4 was weakly expressed in the rat thymic medulla
(data not shown). We immunohistologically studied the expression of
ephrins A2 and A5 during thymic ontogeny. Both ephrins were detected,
as for Eph receptors, in the embryonic thymus of 16-day-old fetal rats
(data not shown).
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To analyze a possible role of Eph A in thymocyte development we cultured 16-day postcoitum fetal thymic lobes for 6 days in the presence of Eph A1-Fc, Eph A2-Fc, or Eph A3-Fc, fusion proteins consisting of the Eph A extracellular domain and a human IgG Fc region. Exogenous Eph A-Fc proteins compete with the endogenous Eph A receptors for the binding of their ligand(s); therefore, the endogenous Eph-ephrin interactions and, consequently, receptor activation are prevented.
In treated lobes we found that the absolute number of cells were
significantly reduced in all analyzed experimental conditions (Fig. 5A). Flow cytometry analysis
of the different thymic cell subpopulations defined by the expression
of CD4/CD8 cell markers revealed a significant reduction in the number
of cells in the four determined cell subpopulations. This reduction was
especially important in the DP
(CD4+CD8+) cell
subpopulation. Furthermore, treated cultures showed a reduced
proportion of the smaller thymocytes (Fig. 5B). However,
TCR expression was not reduced, and although the absolute numbers
of TCRhigh-expressing cells were reduced,
the percentage of these in the treated lobes increased compared with
control ones (Fig. 5B). Thus, within the DP
(CD4+CD8+) and
CD4-CD8+ cell populations,
the percentages of TCR-/low thymocytes
were reduced as compared with controls, while those of
TCRhigh cells were increased. The
proportion of TCR-expressing cells was not reduced either (data
not shown). These changes in the cell content of treated lobes were
less evident when Eph A2 was added to the medium. In contrast, the
addition of ephrin A1, which can interact with most Eph A, to the
culture medium resulted in a more drastic effect on cell survival and a
more reduced proportion of DP
(CD4+CD8+) cells than in
the cases where Eph A-Fc was added. These results indicate that
disrupting Eph A interactions resulted in lower cell numbers and that,
in the overall reductions of cell numbers, the most affected
populations were the immature
CD8+CD4- and DP
(CD4+CD8+) cells.
However, this did not affect the ability of these cells to further
develop to TCRhigh-expressing cells (both
CD4 SP and CD8 SP).
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A). On the contrary, analysis
of the proportion of cycling thymocytes showed a small reduction in the
percentage of cycling cells from treated lobes that was only
statistically significant in those supplied with Eph A3-Fc. This
reduction mainly occurred in the TCR- cell
compartment (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As mentioned above, our results demonstrate the expression in rat thymus of Eph A1, A2, A3, and A4, although their respective patterns of expression are different. Eph A1 is broadly expressed in both thymic epithelial cells and thymocytes. Previously, Eph A1 had been demonstrated in thymus by Northern blot (9). Eph A2 had been detected at low levels in rat thymus by this same technique (14), but by in situ hybridization on sections of mouse thymus it appeared homogeneously expressed through the organ (13). We have found Eph A2 expression to be quite restricted to thymic medulla and some perivascular and peritrabecular areas. Eph A3 expression has been reported in leukemia cells, and by PCR it can be detected in thymocytes (16, 17, 18). In agreement with this, our study demonstrates that Eph A3, like Eph A1, is expressed on thymocytes from the subcapsulary region, cortex, and medulla. It is also expressed on epithelial cells of the thymic medulla. Eph A4 expression, which had been reported in B cells (19), is restricted in the rat thymus to the epithelial cells of both cortex and medulla. In agreement with these results, expression of Eph A1 and A3, but not A2 and A4, has been found by RT-PCR on isolated highly purified thymocyte subpopulations defined by the expression of CD4/CD8 cell markers (data not shown).
Ephrin expression is largely restricted to rat thymocytes rather than to thymic epithelial cells. Subcapsulary thymocytes, presumably double negative (CD4-CD8-) cells, express ephrins A1, A2, A3, and A5, whereas medullary thymocytes express all the ephrins studied. Remarkably, DP (CD4+CD8+) cells that occupy the thymic cortex express only ephrin A3 and, weakly, ephrin A5. In contrast, only ephrin A1 and A2 are significantly expressed on thymic epithelial cells. Ephrin A1 occurs in both subcapsulary and inner cortex, but not in the thymic medulla, and ephrin A2 occurs in the medullary epithelial cells. Ephrins A3 (23, 28) and A4 (15) but not A1 (23) had been previously found in the thymus by Northern blot. In addition, ephrin A1 was detected by in situ hybridization in the connective tissue trabeculae of later fetal thymus and scattered throughout the thymic parenchyma in the adult organ (10).
Remarkably, the sequence of the amplified form of ephrin A3 lacks the region corresponding to ephrin A3 gene exon 4 and represents an alternatively splicing form (26). However, we could not find in the rat thymus an mRNA corresponding to the whole form.
Taken together, our results indicate that ephrins A are largely expressed on rat thymocytes, whereas Eph expression is principally restricted to thymic epithelial cells. However, Eph A1 and ephrin A1 are expressed in both thymocytes and thymic epithelial cells, and ephrin A3 expression is quite homogeneously distributed through the thymus structure. On the contrary, other members of the family occur only in restricted thymic regions and/or cell types. Furthermore, during the movement of thymocytes throughout the different thymic regions (subcapsulary region, cortex, medulla) that occurs along with T cell maturation, the pattern of Eph/ephrin expression on thymocytes is changing. Double negative (CD4-CD8-) thymocytes, which occupy the subcapsulary area and the mature SP cells of thymic medulla, express Eph A1 and A3 and most of the studied ephrins. On the contrary, in the DP (CD4+CD8+) cell compartment of the cortex, both Ephs and ephrins are down-regulated, and expression of ephrins A1 and A2 disappears and that of Eph A3 and ephrin A5 decreases.
The overall patterns of Eph/ephrins A in the rat thymus cannot be matched to complementary patterns of Eph/ephrins in the major defined thymic regions (i.e., subcapsule, cortex, and medulla) and are overlapping. Overlapping patterns of Eph-ephrin expression have been found in other systems. Thus, retinal axons express Eph A4 and Eph A5 uniformly (29, 30) and express ephrin A2 and ephrin A5 in a nasal to temporal gradient (31, 32). The overlap in these expression patterns leads to a persistent phosphorylation of Eph A4 in nasal retinal axons (29). In the rat thymus, the expression of the studied Eph and ephrin A is particularly important in the thymic medulla, a dynamic compartment from which mature thymocytes migrate through blood vessels to the periphery. In this respect, the overlapping expression of Eph/ephrins could contribute to thymocyte motility.
Remarkably, the Eph A4 and ephrin A1 expression patterns in the cortex are both coincident with the cortical epithelial meshwork. Given the known role of Eph family in controlling cell adhesion (for review, see Refs. 8 and 33), it could be implicated in preventing the collapse of thymic epithelium maintaining the three-dimensional structure of thymic stroma necessary to house thymocyte development, or in controlling the adhesion-repulsion balance, which allows the thymocyte-epithelium interactions.
Thus, despite the known promiscuity of Eph-ephrin interactions, some observed expression patterns could contribute to keeping certain thymic domains separate, while others could promote cell motility.
The expression of Eph and ephrins in the rat thymus would suggest a
role for this family of molecules in thymic function. To test this
possibility, we examined in vitro thymocyte development in FTOCs
supplied with Eph A-Fc fusion proteins. In these experimental
conditions, the Eph A-Fc proteins induce a significant reduction in the
total number of cells isolated from the thymic lobes. This reduction
affects all the thymocyte subsets, especially the DP
(CD4+CD8+) cells.
Furthermore, annexin V staining demonstrates that the diminished cell
number is accompanied by increased proportions of dead cells
principally within the DP
(CD4+CD8+) cell compartment
and the immature CD8+ thymocytes. However, there
are not significant changes in the proportion of cycling cells that
could contribute to explaining the changes found in the treated lobes.
Besides, the addition of ephrin A-Fc to the cultures results in a more
drastic effect on cell survival, and the percentage of DP
(CD4+CD8+) cells is more
reduced than in the cultures receiving Eph A-Fc proteins. These
findings indicate that disrupting Eph A family interactions results in
the death of thymocytes, mainly at the DP
(CD4+CD8+) stage, but it
does not abolish the capacity of surviving cells to further develop to
TCRhigh SP (both
CD4+CD8- and
CD4-CD8+) cells.
However, it is difficult to understand how the Eph/ephrin system may be working in situ. On the one hand, if as has been reported for ephrin A5 (34), Eph-ephrin interactions could result in intracellular signaling within the ligand-expressing cells, the addition of Eph A-Fc fusion proteins to FTOCs not only blocks Eph A activation but also could activate the ephrin A signaling pathway. On the other hand, the addition of a soluble ephrin A-Fc to the cultures would activate the Eph A receptors. Thus, in our experimental model, the addition of either Eph A-Fc or ephrin A-Fc to FTOCs results in a similar effect, which could be interpreted as the effect induced by restricted unilateral and delocalized activation of one of the two interacting members of the Eph-ephrin pair.
In addition, the fact that a single receptor may functionally interact with more than one ligand and vice versa adds another level of complexity to the system. As several Eph A are expressed in thymus, and the addition of Eph A1-Fc, Eph A2-Fc, or Eph A3-Fc fusion proteins to FTOCs results in a similar effect, our results would actually reflect the additive effect of the Eph A fusion protein on all Eph A members expressed in the thymus. Thus, the phenotypical changes observed in T cell maturation after the addition of the Eph A/ephrin A soluble forms to FTOCs could be not directly dependent on thymocytes themselves but mediated through signals provided by other thymic nonlymphoid cell populations, which also express Eph/ephrins A. Therefore, we must consider that the observed effects may not be due to a direct role of Eph-ephrin interactions on cell survival but may rather be a consequence of disrupting the correct interactions of thymocytes with the nonlymphoid cell components of the microenvironment, largely thymic epithelial cells. As a consequence of this altered thymus functionality, fewer numbers of thymocytes are able to develop and many of them die because of this failure to differentiate.
Therefore, more detailed definition of the expression patterns of both Eph receptors and ligands in the thymus is necessary to allow a better identification of the cell interactions occurring in each thymic domain and also in vitro models in which individual pairs may be examined, to establish the specific role of the different Eph/ephrin members in thymus physiology.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Agustín G. Zapata, Department of Cell Biology, Faculty of Biology, Complutense University, 28040 Madrid, Spain. E-mail address: Zapata{at}bio.ucm.es
3 Abbreviations used in this paper: DP, double positive; SP, single positive; FTOC, fetal thymus organ culture.
Received for publication October 2, 2001. Accepted for publication April 29, 2002.
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