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The Journal of Immunology, 2000, 165: 3868-3875.
Copyright © 2000 by The American Association of Immunologists

Functional Analysis of Ligand-Binding and Signal Transduction Domains of CD69 and CD23 C-Type Lectin Leukocyte Receptors1

David Sancho, Ana G. Santis, José L. Alonso-Lebrero, Fernando Viedma, Reyes Tejedor and Francisco Sánchez-Madrid2

Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Diego de León, Madrid, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD69 and CD23 are leukocyte receptors with distinctive pattern of cell expression and functional features that belong to different C-type lectin receptor subfamilies. To assess the functional equivalence of different domains of these structurally related proteins, a series of CD69/CD23 chimeras exchanging the carbohydrate recognition domain, the neck region, and the transmembrane and cytoplasmic domains were generated. Biochemical analysis revealed the importance of the neck region (Cys68) in the dimerization of CD69. Functional analysis of these chimeras in RBL-2H3 mast cells and Jurkat T cell lines showed the interchangeability of structural domains of both proteins regarding Ca2+ fluxes, serotonin release, and TNF-{alpha} synthesis. The type of the signal transduced mainly relied on the cytoplasmic domain and was independent of receptor oligomerization. The cytoplasmic domain of CD69 transduced a Ca2+-mediated signaling that was dependent on the extracellular uptake of Ca2+. Furthermore, a significant production of TNF-{alpha} was induced through the cytoplasmic domain of CD69 in RBL-2H3 cells, which was additive to that promoted via Fc{epsilon}RI, thus suggesting a role for CD69 in the late phase of reactions mediated by mast cells. Our results provide new important data on the functional equivalence of homologous domains of these two leukocyte receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
C-type lectins belong to a wide family of proteins characterized by a carbohydrate recognition domain (CRD).3 These molecules have been classified in seven subfamilies according to the overall architecture of the protein, the position of the CRD relative to other domains, and the degree of similarity of CRDs (1). Within the C-type lectin family, collectins and macrophage mannose receptors are important components of the innate immune response, whereas selectins mediate initial interactions between leukocytes and endothelium. Another subfamily of lectin receptors is involved in modulation of NK cell activity, and it could play an important role in the natural immune response (2).

CD69 and CD23 are type II transmembrane (TM) C-type lectins comprising a CRD-like domain linked by a neck (N) region and an N-terminal cytoplasmic (Cy) region (3, 4, 5). CD69, along with other receptors found mainly in NK cells such as CD94, are dimeric molecules that have been designated as group V or NK receptor domain (NKD) C-type lectins (2). Unlike other NKD lectins, CD69 is broadly expressed in bone marrow-derived cells (6), including activated mast cells (7). CD69 is a signal-transmitting receptor that seems to participate in cell activation, but its precise physiological role remains undetermined (6). Activating anti-CD69 mAbs can induce rise in intracellular free calcium ([Ca2+]i) activation of phospholipase A2 (PLA2), induction of cytokine secretion, and cell proliferation (8, 9, 10). CD23 belongs to the heterogeneous group II of type II lectins, and is characterized by an {alpha}-helical coiled-coil neck leading to trimer formation. This oligomeric form is important for its binding to IgE (11). The long neck is also necessary for proper orientation of lectin heads to interact with IgE (12). In addition to IgE, other ligands for CD23, such as CD21, CD11b, and CD11c, have been described (13, 14). There are two isoforms of CD23 differing by 6 aa in the Cy region, which determines different signaling properties. While CD23b only triggers increase in cAMP, CD23a is also able to signal through the phospolipase C (PLC) pathway (15, 16). CD23a, which is expressed exclusively in B cells, is involved in the control of IgE synthesis (17, 18, 19, 20). In this regard, a functional relationship between the IL-4R and CD23a at the level of adenylyl cyclase activation has been reported (21).

The homology between CD69 and CD23 and their genes suggests that they evolve from a common, although distant, ancestral domain (2, 22). However, the possible functional correspondence of these molecules has not been properly studied. To address this point, a series of chimeric molecules was constructed in which the CRD, N, or TM-Cy domains were exchanged. The RBL-2H3 rat mucosal mast cell line expressing these CD69/CD23 chimeric proteins was used as a model of mast cell activation (23) to study the structure-function relationship of these C-type lectins. Our results revealed the interchangeability between defined structural domains of these two proteins in terms of Ca2+ fluxes, serotonin release, and TNF-{alpha} synthesis. The type of signal transduced was dependent on the Cy domain. CD69 induced mast cell activation, acting through a pertussis toxin-sensitive G protein pathway. The activation signals transduced through Fc{epsilon}RI were enhanced by CD69. Our results suggest that CD69 has an important role in mast cell activation, and provide new important data on the functional equivalence of homologous domains of C-type lectins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of chimeric CD23/CD69 cDNAs

Chimeric CD23/CD69 gene constructs were obtained basically following the two-step rPCR protocol described previously (24). Briefly, the first step was performed on 100 ng of the first template (linear plasmid containing the appropriate CD69- or CD23-specific sequences) using 50 pmol of each primer: an outer one in the sense orientation and the chimeric oligonucleotide whose 3' half hybridized with the first template in the antisense orientation. In the second PCR, 100 ng of the second template (purified insert containing the specific sequences to make the chimerization) was mixed with 100 ng of the first PCR product. As template for CD69 sequences, the plasmid pORFII was used (3), whereas the oligonucleotides AIM6 and AIM7 were employed as outer primers (3). For CD23 sequences, a plasmid kindly provided by Dr. Hitoshi Kikutani (Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan), containing the cDNA encoding the "a" isoform was used. The oligonucleotides used were: CD23a(+), 5'-AGAATCCAAGCAGGACCGCC-3'; CD23a(-), 5'-GCTCTGGGCCTGGCTGTATC-3'. The sequences of the chimeric megaprimers were: IIA CD69 TM, 5'-AGCACACAGGAACAGGAACCTGAAGTCCCACGCCTGC-3' CD23 Cy; IIB CD69 CRD, 5'-AGAAAACATGGCTGTCTGAGGACACCTGCAACTCCATCC-3' CD23 N; IIIA CD23 CRD, 5'-GTTGCACACAAAGCCGCTTGGCATTGAGAATGTGTA-3' CD69 N; IIIB CD23 N, 5'-TTTTAGACTCTGTGTGGTGTCGCCCACTGATAAGGCAATG-3' CD69 T. To generate the construct CD69{Delta}Cy, the primer called AIM(-cyt), including in its 5' end consensus sequences for translation initiation, was used: AIM(-cyt), 5'-GCCAAAATGCATGAAGGGTCCTTCAAG-3'.

Each PCR product was cloned in the eukaryotic expression vector pCR3 (Invitrogen, Carlsbad, CA) and sequenced. The X construct corresponded to the religated pCR3 (mock) and was used as control in these studies.

Cell lines and transfections

COS-7 cells were cultured in DMEM supplemented with 10% of FCS. Cells (5 x 105) were transiently transfected with 10–15 µg of each CD69/CD23 chimeric construct as DEAE-dextran precipitates. Cells were used 48 h after transfection to perform immunofluorescence and iodination-immunoprecipitation assays. Rat basophilic leukemia (RBL-2H3) cells were grown in Eagle’s MEM with Earle’s salts supplemented with 10% FCS. This cell line was stably transfected with the chimeric cDNA constructs by using the DOTAP reagent (Boehringer Mannheim, Indianapolis, IN), as previously described (25). Transfectants were selected using 1 mg/ml G418 (Life Technologies, Gaithersburg, MD), and clones were obtained by culturing under limiting dilution conditions. The T lymphoblastoid cell line Jurkat was maintained in RPMI 1640 supplemented with 10% FCS. Stable transfections were performed by electroporation of 1–2 x 107 cells in RPMI using a BTX 600 electroporator with 2-mm cuvettes (126 V, 1700 µF, 72{Omega}). The different pCR3-derived plasmids (X, I, I{Delta}Cy, IIA, and IIB; 100 µg each), linearized by digestion with ScaI, were used for transfection. After 1 mg/ml G418 resistance selection, two limiting dilution cloning rounds were performed for isolation of positive clones.

Reagents and mAb

The anti-CD23 Bu38, anti-CD69 TP1/55, and anti-CD69 TP1/8 mAb have been described (10, 26). The P3X63 myeloma protein was used as negative control in immunofluorescence and immunoprecipitation studies. Sheep F(ab')2 anti-mouse IgG (SAM) and mouse monoclonal anti-dinitrophenyl IgE were purchased from Sigma (St. Louis, MO). The FITC-conjugated F(ab')2 rabbit anti-mouse IgG was from Dako (Glostrup, Denmark). PMA and Ca2+ ionophore A23187 were purchased from Sigma (St. Louis, MO). Pertussis toxin, wortmannin, and genistein were obtained from Calbiochem (La Jolla, CA).

Immunoprecipitation and flow cytometry assays

COS-7 cells harvested 48 h after transfection were radioiodinated (125I) in solution with chloroglycoluril (Iodogen; Pierce, Rockford, IL), as previously described (3). For immunoprecipitation, equal amounts of input radioactivity were mixed with 30 µl of purified anti-CD69 TP1/55 mAb, anti-CD23 Bu38 mAb, or P3X63 mAb directly conjugated to Sepharose (2 mg/ml). Immunoprecipitates were processed as previously described (10), and samples were subjected to SDS-10% PAGE under either reducing or nonreducing conditions.

Flow cytometric analysis of protein expression was performed using a FACScan cytofluorometer (Becton Dickinson Immunocytometry Systems, San Jose, CA), as previously described (27). Measurement of [Ca2+]i was performed by flow cytometry using the calcium-sensitive fluorocrome Fluo-3/AM (Molecular Probes, Eugene, OR), as previously described (28). In brief, cells (2 x 106/ml) were loaded for 30 min at 37°C with 2 µg/ml of Fluo-3/AM. Then they were washed twice in HBSS, acquired during the assay using the FACScan cytofluorometer, and analyzed using CellQuest software (Becton Dickinson). Density plot profiles are represented. In these assays, 2 x 106 Fluo-3-loaded cells were stimulated with the anti-CD69 TP1/8 mAb (10 µg/ml) in a final volume of 1 ml, followed by cross-linking with 20 µg/ml SAM. Cross-linking was not required in cells stimulated with the anti-CD23 Bu38 mAb (10 µg/ml) to induce Ca2+ signaling.

Serotonin release

RBL cells (106 cells/ml) were labeled with 2 µCi/ml [3H]serotonin (American Radiolabelled Chemicals, St. Louis, MO) at 37°C for 1 h, washed, and incubated for an additional hour. Then, cells were washed again, seeded at a density of 2 x 105 cells/well in 96-well flat-bottom plates in the presence of PMA (2 ng/ml), and incubated with 20 µg/ml purified mAbs for 1 h at 37°C in a final volume of 50 µl. Serotonin release was stopped by adding 150 µl ice-cold medium. One hundred microliters of cell-free supernatants were mixed with 5 ml Ecoscint H scintillation fluid (National Diagnostics, Atlanta, GA) and counted in a beta counter. For determination of the total amount of [3H]serotonin incorporated, 100 µl of 1% SDS, 1% Nonidet P-40 were added to the remaining cell suspension in each well, and then 100 µl were counted as mentioned above. All experiments were run by triplicate. For positive control, wells precoated with 20 µg/ml of IgE were used. Serotonin release was calculated by the following formula: % serotonin release = ((cpm sample) - (cpm spontaneous)/(cpm total) - (cpm spontaneous)).

TNF-{alpha} production assay

RBL transfectants (2 x 105 cells/well) were cultured in the presence of PMA (2 ng/ml) and 10 µg/ml of the activating mAbs in a final volume of 100 µl. Where indicated, SAM was used as cross-linker (20 µg/ml). After 12 h, plates were centrifuged (200 x g, 3 min), and cell-free supernatants were collected and assayed for TNF-{alpha}. For positive control, wells precoated with 20 µg/ml of IgE were used. TNF-{alpha} was measured by a cytotoxicity assay on TNF-{alpha}-sensitive L929 cells, as described (29). Briefly, 3 x 104 L929 cells were plated in each well of 96-well tissue culture plates. After 20 h of incubation at 37°C, medium was replaced by serial 2-fold dilutions of cell-free supernatants from RBL cells. Supernatants were diluted in complete medium containing 1.5 µg/ml actinomycin D (Sigma). After 20 h of incubation at 37°C, medium was removed, adherent cells were stained with 0.2% crystal-violet (Sigma) in 1% ethanol for 15 min, wells were washed and dried, and the absorbance (550 nm) was measured.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Construction, expression, and structural analysis of CD69 and CD23 chimeras

Chimeric CD69/CD23 proteins were generated using different and reciprocal combinations of Cy tail, N region, and CRD (Fig. 1Goa). These regions were defined by us through a comparative analysis of amino acid sequences of different C-type lectins. The limits assigned for the Cy, TM, N, and CRD regions were for CD69 the aa 38, 64, and 77, respectively, and for CD23 the residues 23, 47, and 155. These domains were combined by a two-step rPCR method (24), and the four chimeric constructs depicted in Fig. 1Goa were generated.



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  		FIGURE 1. Structural analysis of CD69/CD23 chimeric receptors. a, Schematic representation of CD69/CD23 chimeric proteins studied. The Cy, TM, N, and CRD of CD69 and CD23 are indicated and were exchanged to generate chimeras IIA, IIB, IIIA, and IIIB. b, Immunoprecipitation analysis of CD69/CD23 chimeric proteins. COS-7 cells were transiently transfected with the different constructs, radioiodinated (125I), and immunoprecipitated, as described in Materials and Methods. Samples separated by SDS-PAGE under reducing (A) and nonreducing (B) conditions are shown. Each pair of lanes correspond to the construction indicated in the presence (+) of the mAb specific against the corresponding CRD (anti-CD69 TP1/55 or anti-CD23 Bu38 mAb) or the P3X63 mAb used as negative control (-). X represents the religated pCR3 (mock).

 
Chimeric and wild-type (wt) constructs were transiently transfected in COS cells, and their cell surface expression was corroborated by indirect immunofluorescence staining (not shown). Cell surface proteins of transiently transfected COS cells were immunoprecipitated and showed the expected molecular mass bands for the monomeric forms of each construct under reducing conditions: I (28 kDa), IIA (26.5 kDa), IIB (40 kDa), IIIA (32 kDa), IIIB (50 kDa), and IV (45 kDa) (Fig. 1Gob, A). Interestingly, only the chimeric proteins that contained the N region of CD69 (I, IIA, and IIIA) were able to form disulfide-linked dimers, as revealed by the increase in molecular mass observed for these chimeras under nonreducing conditions (Fig. 1Gob, B). Since there is a single Cys residue (Cys68) in the N region of CD69, these data point out Cys68 as the amino acid responsible for dimerization. In addition, the chimeras IIB, IIIB, and IV containing the CD23 neck appear to contain trimers, while these are not found in the chimeras containing the CD69 neck (Fig. 1Gob). These results suggest that the N region of CD23 is responsible for the trimer formation.

Functional interchangeability of the Cy and lectin domains of CD69 and CD23

To perform the functional analysis of the chimeras, stable transfectants were obtained in the RBL-2H3 mast cell line. This cell line provides a suitable model of signaling associated to functional responses triggered through a variety of receptors (23, 25, 30, 31). Fig. 2Goa shows the similar expression levels in RBL-2H3 transfectants of the chimeras bearing the CRD of CD23 (IIIA, IIIB, and IV). The chimera IIB with the extracellular region of CD69 (CD23 N/CD69 CRD) showed a higher expression compared with CD69 wt. In contrast, it was not possible to obtain membrane expression of the chimera IIA in the RBL system (not shown).



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  		FIGURE 2. Expression and intracellular calcium mobilization through the chimeric CD69/CD23 receptors in RBL-2H3 stable transfectants. a, Expression of chimeric constructs in RBL-2H3 stable transfectants. The bold line corresponds to anti-CD69 TP1/55 (X, I, IIB) or anti-CD23 Bu38 (IIIA, IIIB, IV), and thin line represents the immunofluorescence intensity of the negative control P3X63. b and c, Stable transfectants of RBL-2H3 were loaded with Fluo-3/AM, as indicated in Materials and Methods, and [Ca2+]i levels were estimated by flow cytometry. b, R-X, R-I, and R-IIB transfectants were preincubated 5 min in the presence of anti-CD69 TP1/8 mAb, which did not have any effect on [Ca2+]i by itself (not shown), and then started the measurement of [Ca2+]i as linear fluorescence intensity (LFI). After 100–120 s, to determine the basal level of [Ca2+]i, the SAM (20 µg/ml) was added (arrows) as a cross-linker. c, The [Ca2+]i of R-X, R-IIIA, R-IIIB, and R-IV transfectants was measured, and after 100–120 s, the anti-CD23 Bu38 mAb was added (arrows). A representative experiment of five is shown. Data are represented as density plot profiles.

 
Signaling through CD69 and CD23 has been shown to involve an increase in [Ca2+]i (15, 32). We therefore assessed the functionality of CD69/CD23 chimeras in Ca2+ flux assays. Cross-linking of CD69 CRD-containing receptors in R-I and R-IIB transfectants resulted in a rise in [Ca2+]i, which was higher in the R-IIB chimera bearing the Cy tail of CD23 (Fig. 2Gob). Cells expressing chimeras with the extracellular portion of CD23 increased [Ca2+]i levels in response to the anti-CD23 mAb without the addition of SAM. The Ca2+response was lower in magnitude and less persistent in the chimeras IIIA and IIIB containing CD69 Cy than in CD23 wt (IV) (Fig. 2Goc). Assays using anti-CD69 mAb with the transfectants expressing CD23 CRD or, conversely, anti-CD23 mAb with the cells expressing CD69 CRD, did not trigger any [Ca2+]i signal (not shown). These results imply that the Cy, TM, N, and CRD domains from CD69 and CD23 are functionally interchangeable, and suggest a higher activity of CD23 Cy in triggering [Ca2+]i fluxes.

Ca2+ flux signaling is dependent exclusively on CD23 or CD69 Cy domain

To elucidate whether the Ca2+ fluxes induced through CD69 and CD23 Cy domains were dependent on extracellular Ca2+, mobilization assays were performed in the presence of EGTA. The [Ca2+]i rise triggered through CD69 wt was blocked in the presence of EGTA (Fig. 3Goa). Accordingly, Ca2+ fluxes promoted through chimeras containing CD69 Cy tail (IIIA and IIIB) were mostly inhibited by EGTA (Fig. 3Gob). However, the Ca2+ signaling induced through the CD23 Cy-bearing chimera (IIB) was not modified by EGTA (Fig. 3Goa). A similar result was obtained with CD23 wt, which displayed a more transient, but similar level of [Ca2+]i rise. Therefore, Ca2+ influx induced through CD69 Cy is dependent on extracellular Ca2+, whereas CD23 Cy induces Ca2+ release mainly from intracellular stores. In addition, Ca2+ signaling relies on the Cy domain, but neither on the agonist mAb employed nor the extracellular domain of the chimeric lectin receptor.



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  		FIGURE 3. Effect of EGTA on intracellular calcium mobilization through the chimeric CD69/CD23 receptors in the RBL-2H3 stable transfectants. a and b, RBL-2H3 transfectants were loaded with Fluo-3/AM, as indicated in Materials and Methods, and [Ca2+]i was estimated by flow cytometry in the presence of EGTA (1 mM). a, Stable transfectants R-X, R-I, and R-IIB were preincubated with the anti-CD69 TP1/8 mAb, and then the measurement of [Ca2+]i as linear fluorescence intensity (LFI) started. After 100–120 s to determine the basal level of [Ca2+]i, the SAM (20 µg/ml) was added (arrows) as a cross-linker. b, The [Ca2+]i of the stable transfectants R-X, R-IIIA, R-IIIB, and R-IV was measured, and after 100–120 s, the anti-CD23 Bu38 mAb (10 µg/ml) was added (arrows). A representative experiment of three is shown, which was conducted in parallel and with the same batches of cells as in Fig. 2Go. Data are represented as density plot profiles.

 
To further demonstrate that the Cy domain of CD69 mediates Ca2+ signaling, a truncated CD69 construct lacking the Cy domain (I {Delta}Cy) was transfected in RBL-2H3 cells. Since membrane expression of this truncated construct was not observed, we looked for another cellular system to analyze it. Thus, Jurkat T cell clones expressing the truncated, wt, and chimeric forms of CD69 were obtained and studied (Fig. 4Goa). A consistent rise in [Ca2+]i was triggered by anti-CD69 mAb in CD69 wt-transfected Jurkat cells, but this response was completely absent in the Jurkat cells transfected with the truncated form of CD69 (Fig. 4Gob). In contrast, the chimeras IIA and IIB, containing the CD23 Cy tail, mediated a strong [Ca2+]i rise. These results show that the Cy domain of CD69 is necessary to trigger Ca2+ signaling, and further demonstrate the different Ca2+ signal efficiency of CD69 and CD23 Cy domains.



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  		FIGURE 4. Expression and intracellular calcium mobilization through CD69/CD23 chimeric proteins in Jurkat T cells. a, Expression of the construct I (CD69 wt), I {Delta}Cy (truncated form of CD69 Cy region), IIA (CD23a Cy/CD69 TM N CRD), and IIB (CD23a Cy TM N/CD69 CRD) in Jurkat stable transfectants. X represents the religated pCR3 plasmid (mock). The bold line corresponds to anti-CD69 TP1/55 mAb, and the thin line to P3X63 negative control. b, Transfected Jurkat cells were loaded with Fluo-3/AM, as indicated in Materials and Methods, and [Ca2+]i was estimated by flow cytometry. Cells were preincubated with the anti-CD69 TP1/8, and then the measurement of [Ca2+]i as linear fluorescence intensity (LFI) started. After 100–120 s to determine the basal level of [Ca2+]i, the SAM (20 µg/ml) was added (arrows) as a cross-linker. A representative experiment of three performed is shown. Data are represented as density plot profiles.

 
Different activation pathways are promoted by the Cy domains of CD69 and CD23 in RBL-2H3 cells

The RBL-2H3 cell line provides a suitable experimental model to study both the functional responses triggered through CD69 and its relation with other activation pathways in mast cells. The rise of [Ca2+]i in mast cells is associated to their degranulation, with serotonin release, and synthesis of cytokines, including TNF-{alpha} (23, 25, 31). We found that activation of protein kinase C and cross-linking of the stimulating mAb were required for optimal serotonin release (not shown). Under these conditions, the chimeric receptors containing the Cy tail of CD23 induced about 2-fold higher serotonin release than those bearing the Cy region of CD69 (Fig. 5Go). On the other hand, anti-CD69 mAb induced a high production of TNF-{alpha} through CD69 wt (I), whereas cells expressing the chimera IIB (CD23 Cy) synthesized lower levels of this cytokine than mock-transfected cells (Fig. 6Goa). These differences were less pronounced when activation was triggered through chimeric receptors containing the CRD of CD23. In contrast, with the very low levels of TNF-{alpha} synthesis induced through the CD23 Cy tail of the chimera IIB, CD23 wt was able to induce TNF-{alpha} synthesis, although at lower degree than that induced via CD69 (Fig. 6Gob). These results suggest that CD69 has a major role in the induction of inflammatory cytokine production by mast cells.



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  		FIGURE 5. Serotonin release by the RBL-2H3 CD69/CD23 stable transfectants. RBL-2H3 transfectants were loaded with [3H]serotonin, as indicated under Materials and Methods. a, R-X, R-I, and R-IIB transfectants were incubated in the presence (anti-CD69) or absence (control) of anti-CD69 TP1/8 mAb (10 µg/ml) for 10 min in the presence of PMA (2 ng/ml), and then SAM was added. Samples were maintained 40 min at 37°C in 96-well microculture plates before collecting the supernatants and measurement of [3H]serotonin release. b, R-X, R-IIIA, R-IIIB, and R-IV stable transfectants were assayed in the presence (anti-CD23) or the absence (control) of anti-CD23 Bu38 mAb (10 µg/ml) for 10 min, with PMA (2 ng/ml), and then SAM (20 µg/ml) was added and the samples treated as in a. As a positive control, the 96-well microculture plates were precoated with IgE (20 µg/ml). Samples were analyzed by triplicate, and the arithmetic mean ± SE of five independent experiments is expressed as percentage of specific serotonin release relative to the specific release promoted by IgE as the positive control. *, p < 0.05 compared with control without mAb. **, p < 0.05 compared with R-I + anti-CD69 (a) or R-IIIA and R-IIIB + anti-CD23 (b).

 


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  		FIGURE 6. TNF-{alpha} production by RBL-2H3 CD69/CD23 transfectants. a, R-X, R-I, and R-IIB stable transfectants were incubated (anti-CD69) or not (untreated/IgE) with the anti-CD69 TP1/8 mAb (10 µg/ml) for 20 min in the presence of PMA (2 ng/ml), and then SAM (20 µg/ml) was added and the assay continued for 12 h, before collecting the cell-free supernatants to determine TNF-{alpha} production, as described in Materials and Methods. b, R-X, R-IIIA, R-IIIB, and R-IV stable transfectants were treated (anti-CD23/anti-CD23 + IgE) or not (untreated/IgE) with the anti-CD23 Bu38 mAb (10 µg/ml) for 20 min in the presence of PMA (2 ng/ml), and then SAM (20 µg/ml) was added and the assay continued for 12 h, before measuring TNF-{alpha} levels in the cell-free supernatant, as indicated in Materials and Methods. Where indicated (IgE), the 96-well microculture plates were precoated with IgE (20 µg/ml). The arithmetic mean ± SE of five independent experiments is shown. *, p < 0.05, and **, p < 0.01, compared with control without mAb.

 
We next analyzed the modulation of the signals transduced via Fc{epsilon}RI by CD69/CD23. An additive effect of Fc{epsilon}RI and either CD69 wt or chimeric receptors bearing the Cy tail of CD69 on TNF-{alpha} synthesis was observed (Fig. 6Go). In marked contrast, stimulation through chimera IIB, containing the Cy tail of CD23, significantly inhibited the TNF-{alpha} production induced via Fc{epsilon}RI, further suggesting that chimera IIB activates an inhibitory signal on TNF-{alpha} synthesis (Fig. 6Go, a and b). This behavior depended on the type of extracellular domain, since CD23 wt (IV) did not show any inhibitory effect on Fc{epsilon}RI-induced TNF-{alpha} production.

To elucidate possible signaling pathways involved in the induction of serotonin release and TNF-{alpha} production, RBL-2H3 clones expressing CD69 CRD (I and IIB) or CD23 CRD (IIIA, IIIB, and IV) were triggered with their specific mAb in the presence of different inhibitors. Pertussis toxin, which interferes with heterotrimeric Gi protein-mediated signaling, inhibited the signal transduction through CD69 Cy, but not through CD23 Cy (Fig. 7Go, a and b). In contrast, the phosphatidylinositol-3 kinase inhibitor wortmannin did not exert any significant effect, and the Tyr-kinase inhibitor genistein only promoted a slight inhibition of serotonin release (Fig. 7Goa). These results suggest that, under our experimental conditions, CD69-mediated signal transduction events involve a pertussis toxin-sensitive G protein pathway.



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  		FIGURE 7. Effect of different inhibitors on serotonin release and TNF-{alpha} production by CD69/CD23 transfectants. a, R-X, R-I, R-IIB, R-IIIA, R-IIIB, and R-IV stable transfectants were loaded with [3H]serotonin, as indicated in Materials and Methods. Then cells were preincubated for 30 min in the presence of pertussis toxin (PTx, 0.5 µg/ml), wortmannin (WMN, 0.2 µM), genistein (GEN, 20 µg/ml), or medium (control/{alpha}-CD69 or {alpha}-CD23). After that, they were incubated in the presence ({alpha}-CD69 or {alpha}-CD23) or the absence (control) of the specific mAb (10 µg/ml) for 10 min, before adding the SAM (20 µg/ml). The samples were further maintained 40 min at 37°C in 96-well microculture plates before collecting the supernatant and measuring [3H]serotonin release. For positive control, the 96-well microculture plates were precoated with IgE (20 µg/ml). Samples were analyzed by triplicate and the arithmetic mean ± SE of two independent experiments is expressed as percentage of specific serotonin release relative to the specific release promoted by IgE-positive control. *, p < 0.05 compared with control treated with mAb. b, R-X, R-I, R-IIB, R-IIIA, R-IIIB, and R-IV stable transfectants were preincubated for 30 min in the presence of pertussis toxin (PTx, 0.5 µg/ml), wortmannin (WMN, 0.2 µM), genistein (GEN, 20 µg/ml), or medium (control/{alpha}-CD69/{alpha}-CD23), and then treated ({alpha}-CD69 or {alpha}-CD23) or not (control) with the specific mAb (10 µg/ml) for 20 min in the presence of PMA (2 ng/ml). After that, SAM (20 µg/ml) was added and the assay continued for 12 h, before measuring TNF-{alpha} levels in the cell-free supernatant, as indicated in Materials and Methods. The arithmetic mean ± SE of three independent experiments is shown. *, p < 0.05, compared with the treatment with the specific mAb ({alpha}-CD69 or {alpha}-CD23).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CD69 and CD23 are type II TM proteins with CRD-like domains linked by N regions of various lengths to N-terminal Cy tails. Gene duplication and additional exon shuffling could give rise to different families of receptors by combination of extracellular domains with distinct Cy tails (22). CD69 and other receptors mainly expressed by NK cells belong to the group V subfamily of C-type lectins, which has been renamed as NKD C-type lectin-like NK receptor domains (2). Recently, the crystal structure of the extracellular domain of CD94 has provided a structural model for other NKD lectins, such as CD69 (33). In CD69, Cys68 and Cys85 are located in the N region, and these residues are good candidates to mediate the dimerization of the receptor. It has been previously reported using purified recombinant soluble CD69 CRD generated in bacteria that dimerization is more efficient in proteins containing the N region with Cys68 than in constructs lacking this residue, although the contribution of Cys85 was not ruled out (34). Our data using an eukaryotic expression system reveal that Cys68 is responsible for CD69 dimerization, since the replacement of the short N region of this protein (aa 64–76) by that of CD23 abolished its capacity to dimerize. Notably, both the construct lacking the Cy domain of CD69 and the one that bears the short CD23 Cy domain and the rest of CD69 were not expressed in the plasma membrane of RBL-2H3 cells, although they were detected in intracellular compartments. These data point out the importance of the CD69 Cy domain, which would not be substituted by the Cy domain of CD23 in the RBL-2H3 heterologous system, on the adequate transport of CD69 to the plasma membrane, as described for other proteins (35).

The structure of CD23 is characterized by an {alpha}-helical coiled-coil stalk which, as suggested by our data and previous reports, seems to mediate trimer formation, allowing an efficient interaction with IgE (11, 12). However, our results show that CD69/CD23 neck exchange in the IIIA and IIIB chimeras does neither affect the recognition by mAbs nor signaling triggering through these chimeric receptors. Therefore, the stalk region does not seem to influence the signaling through the Cy domain of CD69 or the domain recognized by mAb, which would be different from that of IgE binding.

The interchangeability of defined structural domains from CD69 and CD23 supports at the functional level the molecular evolution hypothesis established for this superfamily based on protein and gene similarities (2, 22). The described chimeras demonstrate the independence of the ligand-binding and signal transduction domains in these proteins, and confirm the functional adequacy of the modules defined by structural approaches in C-type lectins (1, 2). In addition, the CD23/CD69 chimeric constructs allowed us to analyze the role of the distinct domains in signal transduction. In this regard, the chimeric receptors with the Cy tail of CD69 mediated a Ca2+ response that was dependent on extracellular Ca2+. In contrast, the Ca2+ mobilization induced through the Cy region of CD23 was dependent mainly on intracellular stores, which leads to a higher secretory response (36). This behavior was independent of the type of CRD, or N domain in the chimera, thus confirming that the Cy-TM domain is fully responsible for signal transduction. In addition, our data indicate that although the short Cy domain of CD69 lacks of clearly established signaling motifs, it is necessary for signal transduction.

An early report showed that the few molecules of CD69 present in resting T cells were associated with a 39-kDa G protein that was ADP ribosylated by pertussis toxin, an interaction that could not be demonstrated in activated T cells expressing high levels of CD69 (37). Our studies with specific inhibitors show that both serotonin release and TNF-{alpha} synthesis are clearly affected by pertussis toxin in chimeras containing the Cy domain of CD69, thus confirming the involvement of a pertussis toxin-sensitive G protein in the signal transduction pathways triggered through CD69. In this regard, the activation of mast cells through a pertussis toxin-sensitive pathway independent of Fc{epsilon}RI has been reported (38). Nevertheless, CD69-mediated serotonin release and TNF-{alpha} synthesis were not completely inhibited by pertussis toxin, thus suggesting that other pathways could be involved. In this regard, besides mediating an extracellular Ca2+ influx, CD69 cross-linking activates cytosolic PLA2 in platelets and monocytes (8, 9). On the other hand, our data show a partial inhibition of serotonin release by the tyrosine kinase inhibitor genistein. Receptor-associated protein tyrosine kinases, such as Syk, appear to be good candidates to interact with the Cy domain of CD69, participating in the signals triggered in mast cells. Syk is able to induce the PLC/protein kinase C and mitogen-activated protein kinase cascades, which are involved in the regulation of the secretory pathway and PLA2 activation (23). Accordingly, CD69-mediated extracellular signal-related kinase activation has been described recently (39). These data support a common CD69 early signaling machinery operative in different cell lineages (6).

Signaling through CD23a involves the triggering of two main pathways: the first one involves the association with a pertussis toxin-insensitive G protein and the src-related kinase Fyn that results in PLC activation (15). This appears to be the main pathway involved in serotonin release and TNF-{alpha} production, and our data indicate that it is strongly stimulated by activating anti-CD23 mAbs. The second pathway triggered by CD23 ligation is the generation of cAMP (21). Notably, our results show a different behavior in TNF-{alpha} production between the chimera IIB (CD23 Cy TM N/CD69 CRD) and CD23 wt (IV). Whereas chimera IIB failed to trigger TNF-{alpha} synthesis in response to anti-CD69, CD23 wt induced a significant production, although at lower degree than that induced via chimeric receptors containing the Cy domain of CD69. These data suggest that the signaling pathways preferentially induced through CD23 are dependent on the nature of the stimulus or the ligand. This is of particular interest since several ligands have been described for CD23 (5, 13, 14). We have analyzed the modulation by CD69/CD23 of the signals transduced via Fc{epsilon}RI, which has been extensively used for the study of both the Ig superfamily killer cell-inhibitory and CD94/NKG2 receptors (25, 30, 31). Interestingly, anti-CD69 stimulation through chimera IIB, bearing the Cy tail of CD23, inhibited the TNF-{alpha} production induced via Fc{epsilon}RI, whereas this effect was not observed in the anti-CD23-stimulated construct IV (CD23 wt). These results further support that different stimuli may trigger distinct signaling pathways through the Cy tail of CD23. Thus, it is feasible that the signal triggered by anti-CD69 mAb through the chimera IIB preferentially induces the production of cAMP, which may account for the suppressive effect on TNF-{alpha} production. These data concur with previous reports on the inhibition via CD23 of IL-4-mediated B cell activation (21). The inhibitory role of CD23 is further supported by studies on the suppressive effect of CD23 on IgE synthesis observed in CD23-deficient and transgenic mice (17, 18, 19, 20).

The signaling through Fc{epsilon}RI and other receptors in RBL-2H3 rat mucosal mast cells has been extensively studied and their linkages with functional responses established (23, 25, 30, 31). Our data using this experimental cell model demonstrate that CD69 is able to induce a moderate serotonin release and a high TNF-{alpha} production, compared with Fc{epsilon}RI. Furthermore, our results show that the effects induced by IgE and CD69 on TNF-{alpha} production are partially additive. These data suggest that CD69 may act as a stimulatory molecule in mast cells. Thus, CD69 could be involved in the induction of the late phase reactions mediated by mast cells through TNF-{alpha} release. These reactions have a key role in type I hypersensitivity responses and are involved in the modulation of neutrophil influx, and bacterial clearance in experimental models of acute septic peritonitis (40, 41). In this regard, it is feasible that CD69 could interact, through its CRD, with carbohydrates from bacteria, participating in the innate mechanisms of immune response. The definition of the elusive ligands of CD69 will uncover molecular interactions of potential general relevance for the activation and function of hemopoietic cells, including mast cells.


   Acknowledgments
 
We thank R. González-Amaro, M. López-Cabrera, M. Carretero, and M. Vicente-Manzanares for critical reading of the manuscript, and M. Vitón for expert technical assistance.


   Footnotes
 
1 This work was supported by Grants SAF 99/0034-C01 and 2FD97-0680-C02-02 from the Spanish Ministerio de Educación y Cultura, and 08.1/0011/97 from the Comunidad Autónoma de Madrid. Back

2 Address correspondence and reprint requests to Dr. Francisco Sánchez-Madrid, Servicio de Inmunología, Hospital de La Princesa, C/Diego de León, 62, E-28006, Madrid, Spain. Back

3 Abbreviations used in this paper: CRD, carbohydrate recognition domain; [Ca2+]i, intracellular free calcium; Cy, cytoplasmic; N, neck; NKD, NK receptor domain; PLA2, phospholipase A2; PLC, phospholipase C; SAM, sheep anti-mouse; TM, transmembrane; wt, wild-type. Back

Received for publication November 8, 1999. Accepted for publication July 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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