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The Journal of Immunology, 2001, 166: 6537-6544.
Copyright © 2001 by The American Association of Immunologists

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 on Murine Dendritic Cells Is a Potent Regulator of T Cell Stimulation1

Robert Kammerer*, Detlef Stober*, Bernhard B. Singer{dagger}, Björn Öbrink{dagger} and Jörg Reimann2,*

* Institute for Medical Microbiology and Immunology, University of Ulm, Ulm, Germany; and {dagger} Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are important APCs that play a key role in the induction of an immune response. The signaling molecules that govern early events in DC activation are not well understood. We therefore investigated whether DC express carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1, also known as BGP or CD66a), a well-characterized signal-regulating cell-cell adhesion molecule that is expressed on granulocytes, monocytes, and activated T cells and B cells. We found that murine DC express in vitro as well as in vivo both major isoforms of CEACAM1, CEACAM1-L (having a long cytoplasmic domain with immunoreceptor tyrosine-based inhibitory motifs) and CEACAM1-S (having a short cytoplasmic domain lacking phosphorylatable tyrosine residues). Ligation of surface-expressed CEACAM1 on DC with the specific mAb AgB10 triggered release of the chemokines macrophage inflammatory protein 1{alpha}, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 and induced migration of granulocytes, monocytes, T cells, and immature DC. Furthermore, the surface expression of the costimulatory molecules CD40, CD54, CD80, and CD86 was increased, indicating that CEACAM1-induced signaling regulates early maturation and activation of dendritic cells. In addition, signaling via CEACAM1 induced release of the cytokines IL-6, IL-12 p40, and IL-12 p70 and facilitated priming of naive MHC II-restricted CD4+ T cells with a Th1-like effector phenotype. Hence, our results show that CEACAM1 is a signal-transducing receptor that can regulate early maturation and activation of DC, thereby facilitating priming and polarization of T cell responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC)3 are the most potent APCs that can induce T cell responses (reviewed in Refs. 1, 2, 3). Immature, tissue-resident DC capture Ag and respond to other, not yet well-defined, signals derived from either the pathogen (e.g., LPS, bacterial DNA, viral double-stranded RNA) or from tissue lesions induced by the pathogen (e.g., stress proteins), before they develop into competent DC. To achieve this, they migrate to regional lymph nodes where they differentiate into presentation-competent DC. Hence, DC prime naive T cells remote from the site of initial Ag contact (4, 5). In the maturating process leading to presentation-competent cells, several signaling events triggered by soluble ligands as well as specific cell-cell interactions play a crucial role. An important theme in cell signaling is signal regulation by costimulation and coinhibition. In the immune system, a large number of both coinhibitory and costimulatory receptors are known, many of which belong to the Ig superfamily. Several of these receptors have immunoreceptor tyrosine-based activation motifs or immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic domains and operate by recruiting and activating SH2 domain-containing protein tyrosine kinases and protein tyrosine phosphatases. Among the ITIM-containing receptors signal-regulatory proteins and killer cell-inhibitory receptors are well known. Carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1), also known as CD66a, BGP, or C-CAM, is an ITIM-containing Ig superfamily receptor abundantly expressed on the cell surface. CEACAM1 is the receptor for a variety of microorganisms. Because it is involved in both cell-cell communication and pathogen-host interactions, we analyzed its putative expression and functional role in DC.

CEACAM1 is abundantly expressed in epithelia, vessel endothelia, granulocytes, macrophages, T cells, B cells, NK cells, and platelets (6, 7, 8, 9, 10, 11). It can mediate intercellular adhesion via homophilic binding (12, 13, 14). Work in many laboratories has demonstrated that CEACAM1 has important regulatory functions in cell proliferation, angiogenesis, apoptosis, immune responses, T cell cytotoxicity, differentiation, and polarization and lumen formation of epithelial cells (15, 16, 17, 18, 19, 20). It is down-regulated in many types of cancer (21, 22). CEACAM1-dependent signals can inhibit tumor growth in vivo (23). CEACAM1 can influence and regulate signal transduction. It is a molecular system composed of several splice isoforms that can influence cell signaling in both positive and negative ways. CEACAM1 probably operates by recruiting and activating either src-family kinases, or protein tyrosine phosphatases Src homology phosphatases 1 and 2 to the same tyrosine-phosphorylated ITIM motifs in the cytoplasmic domain of the isoform CEACAM1-L (24, 25, 26). The two major isoforms are CEACAM1-L and CEACAM1-S, which differ in their cytoplasmic domains. The cytoplasmic domain of CEACAM1-L consists of 73 amino acids and has 2 ITIM motifs with tyrosine residues that can be phosphorylated. CEACAM1-S has a cytoplasmic domain of only 10 amino acids and lacks ITIM motifs (27). CEACAM1-L and CEACAM1-S are coexpressed at different ratios in different cell types and in different functional states of cells of one lineage. Both isoforms can dimerize, and there is evidence that the S isoform can regulate the signaling activity of the L isoform (23, 28, 29).

The only well-characterized physiological ligand interaction of CEACAM1 is the homophilic binding to itself; this may represent an important signal input via cell-cell interactions (29). Several pathogenic microorganisms bind to CEACAM1 as their host cell receptor. Murine CEACAM1 is the major receptor for mouse hepatitis viruses (MHV) (30, 31) and human CEACAM1 binds Escherichia coli, Salmonella typhimurium (32), Neisseria gonorrhoeae (33, 34), Neisseria meningitidis (35), and Haemophilus influenzae (36). Both the physiological cell-cell interactions mediated by CEACAM1 and its binding to pathogens may have an influence on the activation of the immune system.

In this investigation, we show that both immature and mature murine (myeloid and lymphoid) DC express CEACAM1 on the surface in vivo and in vitro. Specific binding of a mAb to surface CEACAM1 expressed by immature DC stimulates their maturation including the release of cytokines and chemokines. We demonstrate that CEACAM1 facilitates priming of naive CD4+ T cells by Ag-bearing DC in vitro and favors a Th1-type differentiation. These data show that CEACAM1 takes part in the signaling scenario that leads to the induction of Th1 immune responses.


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

BALB/cJ (H-2d) and C57BL/6J (H-2b) mice were obtained from Bomholtgard (Ry, Denmark) and kept under standard pathogen-free conditions in the animal colony of Ulm University (Ulm, Germany). BALB/c-derived DO11.10 TCR-transgenic mice were kindly provided by D. Loh (Roche, Nutley, NJ). Female mice were used at 10–16 wk of age.

Generation of myeloid DC (mDC) from murine bone marrow (BM)

The in vitro generation of mDC from murine bone marrow has been described (37). Briefly, bone marrow cells (BMC) obtained from femurs and tibiae were depleted of CD4+ and CD8+ T cells, B220+ B cells, and MHC class II+ maturing myeloid cells by MACS sorting following the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). These BMC were cultured at a density of 106 cells/ml in UltraCulture medium (BioWhittaker, Verviers, Belgium) supplemented with 5 ng/ml GM-CSF and 10 ng/ml Flt3 ligand (FL) (PeproTech, Rocky Hill, NJ), 2 mM glutamine, and antibiotics. BMC from C57BL/6 mice were cultured in serum-free medium; BMC from BALB/c mice required low FCS supplements (0.5% v/v) to the medium to maintain viability and support expansion of mDC. Cultures were incubated at 37°C in humidified air supplemented with 5% CO2. On days 3 and 5, cells were fed by medium exchange. On day 7 of culture, nonadherent cells were harvested; CD11c+ cells were purified by magnetic bead separation (Miltenyi Biotec) and replated in UltraCulture medium supplemented with GM-CSF, FL, glutamine, and antibiotics.

CD11c+ cells were analyzed by flow cytometry at day 8–10 of culture. DC harvested from day 8 cultures were seeded into 96-well round-bottom plates at 2 x 105 cells/well in UltraCulture medium with GM-CSF, FCS, and antibiotics. These cultures were stimulated with the indicated cytokines and Abs. In some experiments, 6.6 x 104 cells/well CD40 ligand (CD40L)-transfected J558L cells (or negative control nontransfected J558L cells) were added. After 48–72 h incubation, supernatants were harvested, and cytokine release was detected by ELISA.

Isolation and purification of CD4+ T cells

Single-spleen cell suspensions were passed through nylon wool columns. CD4+ T cells were selected from the nonadherent cell population by MACS. The purity of the isolated CD4+ T cell subset was >96% as determined by flow cytometry (FCM).

FCM analyses

Cells were suspended in PBS, 0.3% w/v BSA supplemented with 0.1% w/v sodium azide. Unspecific binding of Abs to FcR was blocked by preincubating cells with the anti-CD16/CD32 mAb 2.4G2 (1 µg mAb/106 cells; PharMingen, Hamburg, Germany). Cells were incubated with 0.5 µg/106 cells of the relevant mAb for 30 min at 4°C, washed twice, and subsequently incubated with a second-step reagent for 15 min at 4°C. Cells were washed twice and analyzed on a FACScan (Becton Dickinson, Mountain View, CA). Dead cells were excluded by propidium iodide staining. The following reagents and mAbs from PharMingen were used: PE-conjugated anti-I-Ad/I-Ed; PE-conjugated anti-I-Ab; biotinylated anti-H-2Db,d,k; PE-conjugated anti-CD80 (B7-1); PE-conjugated anti-CD40; FITC-conjugated anti-CD86 (B7-2); and PE-conjugated anti-CD11c. FITC-conjugated anti-CD54 (ICAM-1) and PE-conjugated F4/80 were purchased from Cedarlane (Hornby, Ontario, Canada). We furthermore used the rat anti-mouse DEC205 (NLDC-145) mAb from Biozol (Eching, Germany) and the FITC-conjugated IgG1 mAb R3-34, PE-conjugated IgG1 mAb R3-34, and streptavidin Red 670 (Life Technologies, Eggenstein, Germany).

Cell culture and mAb purification

The mature B cell line L10 and the hybridomas producing the mAbs Decma-1 (38) and AgB10 (39) were cultured in a humidified atmosphere with 5% CO2 at 37° in RPMI supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. The supernatants were collected, and the Abs were affinity-purified on a HiTrap protein G column according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Uppsala, Sweden). Endotoxin content in the Ab preparations was <0.03 endotoxin U/ml as determined using the Endosafe Gel-Clot Assay (Charles River, Sulzfeld, Germany).

Cytokines and cytokine detection by ELISA

The following recombinant mouse cytokines were obtained from PeproTech: IL-4, IL-6, IL-10, IL-18, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 2 (MIP-2), TNF-{alpha}, GM-CSF, and FL. IL-12 p40 and MIP-1{alpha} were purchased from R&D Systems (Wiesbaden, Germany). IFN-{gamma} and IL-12 p70 were obtained from PharMingen.

Cytokines released into culture supernatants were detected by a conventional double-sandwich ELISA. For detection and capture, the following mAbs (from PharMingen) were used: mAb R4-6A2 and biotinylated mAb XMG1.2 were used for IFN-{gamma}; mAb 2H5 and biotinylated mAb 4E2/MCP1 were used for MCP-1; mAb C15.6 or mAb RedT/G297-289 were used as coating Abs for IL-12 p40 and IL-12 p70 ELISA, respectively; mAb C17.8 was used for detection in both cases. Extinction was analyzed at 405/490 nm on a TECAN microplate ELISA reader (TECAN, Crailsheim, Germany) using EasyWin software (TECAN). The detection limits of the cytokine ELISAs were: 0.2 pg/ml for IL-4; 1 pg/ml for MIP-2; 10 pg/ml for IL-12p40, MIP-1{alpha}, and MCP-1; 20 pg/ml for IL-12 p70 and IFN-{gamma}; and 60 pg/ml for IL-18.

Chemotaxis assay

Chemotaxis was assayed by an in vitro two-chamber migration assay followed by FCM. Cells in 100 µl complete medium were added to the upper chamber of Falcon Transwells (6.5-mm diameter, 3-µm pore size, poly-ethylen-terephtalat membrane; Becton Dickinson, Heidelberg, Germany), and chemotactic substances were added to the lower chamber to form a chemotactic gradient. A total of 1 x 106 cells were incubated for 4 h in the upper chamber of the Transwell. After cells were collected in suspension, 0.5 ml 5 mM EDTA was added to the lower chamber for 15 min at 37°C to detach adherent cells such as monocytes and granulocytes from the bottom of the wells. Detached cells were combined with the previously collected suspension cells for cell counting. Migrated granulocytes, monocytes, and lymphocytes were counted by FCM by gating on appropriate populations of cells using forward scatter and side scatter channels. CD4+ and CD8+ T cells were detected with FITC- or PE-conjugated mAbs to CD4 and CD8. Each chemotaxis experiment was performed in duplicate. The results from three independent experiments were pooled. For statistical analyses, nested ANOVA was performed to compare the mean counts of cells migrating toward supernatants of stimulated vs unstimulated DC. When ANOVA detected a statistically significant difference in mean counts, the Tukey method of multiple comparisons was applied. A p value of <0.05 was considered to be statistically significant for all analyses.

Detection of CEACAM1 splice variants by RT-PCR

Total mRNA was isolated from 5 x 106 cells using the RNeasy kit (Qiagen, Hilden, Germany). The RNA yield was quantified photometrically and 1 µg mRNA was used for cDNA syntheses by reverse transcription using the Reverse Transcription System (Promega, Mannheim, Germany). The reverse transcription product was amplified by PCR for 25 cycles with taq polymerase (Qiagen). PCR conditions were 95°C for 1.5 min, annealing at 60°C (CEACAM1) and at 58°C ({beta}-actin) for 1.1 min, extension at 72°C for 1.5 min, and final extension at 72°C for 15 min. The primers used were 5'-AGCGTCAGGAGGAGCAACTCAA and 3'-AGAAGAAGGGGCTGAAGTTGGC, complementary to both sides of exon 7. Thus, an amplified fragment of 268 bp is expected from the short cytoplasmic isoform, and a fragment of 321 bp is expected from the long cytoplasmic isoform. A 569-bp fragment from the {beta}-actin mRNA was amplified using the 5'-primer ATGGATGACGATATCGCT and the 3'-primer ATGAGGTAGTCTGTCAGGT. Ten microliters of each PCR product were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CEACAM1 by murine DC

We generated CD11c+ mDC from BM progenitors in cultures supplemented with GM-CSF and FL. mDC developed from C57BL/6 -derived marrow progenitors in serum-free cultures; BALB/c-derived BMC cultures required 0.5% v/v FCS supplementation for the in vitro generation of mDC. In nonadherent cells harvested from day 7 cultures, 50–70% had the CD11c+/CD11b+/CD4-/CD8-/E-cadherin+ surface phenotype of mDC. Surface expression of MHC-II molecules and CD40, CD80, and CD86 costimulator molecules was low or undetectable in most CD11c+ cells from day 7 cultures.

We used the mAb AgB10 specific for the extracellular domain of murine CEACAM1 to detect CEACAM1 expression on the cell surface of DC (39). Nonadherent CD11c+ mDC harvested from 7-day BMC cultures expressed low but detectable levels of CEACAM1 (Fig. 1GoA). The expression of this marker was up-regulated 3- to 5-fold when purified, nonadherent CD11c+ mDC were subcultured without a maturation-inducing agent (IL-4, TNF-{alpha}, LPS, or CD40L) for another 3 days in GM-CSF and FL (Fig. 1GoA). The expression level on mDC harvested from day 8–10 cultures was lower than that of B cells, which are known to display high CEACAM1 surface expression (Fig. 1GoA) (9).



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  		FIGURE 1. Expression of CEACAM1 by murine DC. A, The histograms show the log fluorescence intensity of CEACAM1 on the surface of DC labeled with either fluorochrome-conjugated mAb AgB10 (bold profiles) or an isotype control mAb (gray profiles). Only CD11c+ cells are shown. The percents of positive cells are indicated. In this setting, 99% of the isotype control Ab stained cells were negative. B, mRNA was isolated from 5 x 106 purified CD11c+ DC (harvested from day 7 or day 10 cultures), the B cell line L10 (positive control), and NIH 3T3 cells (negative control). Using primers specific for the A2 domain and the 3'-end of CEACAM1, two mRNA products were amplified: the 321-bp product (characteristic of the CEACAM1-L splice variant); and the 268-bp product (characteristic for CEACAM1-S splice variant). Amplification of a mRNA fragment of {beta}-actin indicated that comparable amounts of RNA were amplified. C, CEACAM1 expression by CD11c+ DC isolated from inguinal lymph nodes or spleen of C57BL/6J mice. Both CD8{alpha}+ and CD8{alpha}- DC express CEACAM1 (bold profiles).

 
The expression of CEACAM1 by murine mDC was confirmed by RT-PCR (Fig. 1GoB). The primer pair used amplifies both the L and the S cytoplasmic isoforms. CD11c+ DC from day 7 and 9 BMC cultures expressed both CEACAM1 isoforms (Fig. 1GoB). The presence of the L isoform indicated that CEACAM1 expressed on the surface of mDC can mediate signaling via its ITIM motifs.

We isolated CD11c+ cells from spleen and lymph nodes of BALB/c and C57BL/6 mice to test whether DC express CEACAM1 in vivo. A significant proportion of freshly isolated CD8{alpha}+ and CD8{alpha}- DC from both spleen and lymph nodes expressed CEACAM1 on the surface (Fig. 1GoC). The level of CEACAM1 surface expression by freshly isolated, murine DC was comparable with the level of CEACAM1 surface expression by DC generated in vitro (compare Fig. 1GoA and Fig. 1GoC). DC generated in vitro or in vivo thus express CEACAM1 on the cell surface.

CEACAM1 surface expression by maturing mDC

When nonadherent CD11c+ DC were purified from day 5 BMC cultures and cultured for a further 5 days with GM-CSF and FL, a subset of 20–30% of the cells showed surface phenotype changes indicating "spontaneous" maturation. This was evident by up-regulated surface expression of CD40, CD54, CD80, and CD86 costimulator and of MHC II molecules (Fig. 2Go). CEACAM1 surface expression was also up-regulated between day 5 and day 10 of culture (Fig. 1GoA). This "spontaneous" up-regulation of CEACAM1 surface expression was seen in C57BL/6-derived DC growing in serum-free cultures, and in BALB/c-derived DC cultured in FCS-supplemented medium (data not shown). CEACAM1 surface expression was, however, not preferentially up-regulated in DC with a more mature phenotype, i.e., high surface expression of MHC-II, CD40, CD80, and CD86 molecules (Fig. 2Go). CEACAM1 is thus expressed on the surface of early DC developing in vitro from BM progenitors, is up-regulated to a limited extent on DC during their in vitro differentiation, but is not preferentially expressed by mature, presentation-competent mDC.



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  		FIGURE 2. CEACAM1 and costimulator/MHC molecule surface coexpression by murine DC. Cells were labeled with mAb AgB10 and a FITC-conjugated donkey anti-rat IgG F(ab')2 reagent. PE-conjugated mAb were used to detect CD11c, CD40, CD80, or I-Ab. Biotinylated mAb and streptavidin-PE were used to detect CD86 or H-2Kb. Nonspecific FcR binding of Abs was blocked by preincubating cells with anti-CD16/CD32 mAb. Quadrants were set so that >98% of isotype control mAb-stained cells were in the lower left quadrant. Data are representative of three experiments using independent DC cultures.

 
We tested whether surface expression of CEACAM1 by DC can be modulated by proinflammatory cytokines that induce DC maturation, i.e., TNF-{alpha}, IL-4, IFNs, or LPS. When DC were treated with these agents from day 7 to day 10 of culture, surface expression of MHC II and costimulator (CD40, CD54, CD80, and CD86) molecules was strikingly induced or up-regulated (data not shown). In contrast, the surface expression of CEACAM1 was not modulated (data not shown). Hence, CEACAM1 expression is not up-regulated by exogenous cytokines during maturation of DC.

CEACAM1-dependent signaling of mDC triggers release of chemokines that attract granulocytes, monocytes, T cells, and DC

Because early signaling in the immune system involves production and release of chemokines, we investigated the secretion of MIP-1{alpha}, MIP-2, and MCP-1 by DC stimulated by CEACAM1 ligation (Fig. 3GoA). Signaling through CEACAM1 strikingly enhanced the low "spontaneous" MIP-1{alpha} and MIP2 release by DC. Ligation of surface CEACAM1 was more potent in triggering release of these chemokines by DC than the other stimuli tested. It also enhanced the release of MCP-1 although to a lesser extend. Isotype-matched control mAb, mAb to E-cadherin (DC generated in the BMC cultures expressed the adhesion molecule E-cadherin on the cell surface), or heat-inactivated mAb AgB10 (to exclude a contaminating LPS effect) had no measurable effect on chemokine release by DC (Fig. 3GoA and data not shown). The chemokine response of DC to CEACAM1 ligation was rapidly inducible and showed a different kinetic for MIP-1{alpha} and MIP-2 (Fig. 3GoB).



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  		FIGURE 3. CEACAM1-dependent signaling of DC induces chemokine release. A, DC (5 x 104 cells/well) were stimulated with mAb AgB10 (10 µg/ml), an isotype-matched control mAb (10 µg/ml), the anti-E-cadherin mAb Decma-1 (10 µg/ml), LPS (1 µg/ml), or CD40 ligation (CD40L) from day 7 to day 9 of culture. Chemokine release into the supernatants was determined by ELISA. B, DC (5 x 104 cells/well) were stimulated with anti ({alpha})-CEACAM1 or anti-E-cadherin ({alpha}-E-Cad.) mAb, and the release of the chemokines MIP-1{alpha} and MIP-2 was determined after 2.5, 5, 7.5, and 24 h of culture. C, Migration of leukocytes induced by supernatants conditioned by DC stimulated with 10 µg/ml AgB10 or 10 µg/ml isotype control mAb. Migration of the cells was analyzed using a Transwell system. d, Migration of splenic T cells toward different concentrations of MIP-1{alpha} was used as a positive control. Migrating cells were counted and identified by FCM using forward scatter and side scatter channels (granulocytes, monocytes, lymphocytes) and specific mAb (to identify CD4+ and CD8+ T cells). Results are mean values ± SEM of three experiments (*, p < 0.05; **, p < 0.01).

 
Chemokines stimulate the migration of leukocytes. We analyzed cellular migration in a Boyden chamber assay. From nonfractionated spleen cell populations, supernatants conditioned by DC stimulated through CEACAM1 ligation attracted granulocytes and monocytes (Fig. 3GoC, a). From splenic T cell populations, the migration of both CD4+ and CD8+ T cells was enhanced 2-fold by supernatants from CEACAM1-stimulated DC (Fig. 3GoC, b). CEACAM1-stimulated DC also enhanced the migration of DC (Fig. 3GoC, c). Hence, CEACAM1-dependent signals of DC recruit myeloid and lymphoid cells into the immune response, most likely through increased chemokine release.

Ligation of surface CEACAM1 on DC by mAb AgB10 induces maturation

Purified CD11c+ DC harvested from day 7 BMC cultures were cultured for 2 days in serum-free GM-CSF/FL-supplemented medium with the mAb AgB10. Either isotype-matched control mAb or the mAb Decma-1 specific for murine E-cadherin was added to the medium in control cultures. The anti-CEACAM1 mAb AgB10 but neither the anti-E-cadherin mAb Decma-1 nor an isotype-matched control mAb up-regulated surface expression of CD40, CD54, CD80, and CD86 costimulator molecules by DC that was comparable with that induced by TNF-{alpha} treatment (Fig. 4Go). Induction of DC maturation may result either directly through CEACAM1 signaling or indirectly through cytokines/chemokines released by CEACAM1-triggered signals. Binding of the anti-CEACAM1 Ab did not trigger release of TNF-{alpha} by DC (data not shown).



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  		FIGURE 4. CEACAM1-dependent signals up-regulate surface expression of costimulator molecules by DC. DC (106 cells/ml) were stimulated from day 7 to day 9 of culture with either 10 µg/ml mAb AgB10 (bold profiles; left), or 10 ng TNF-{alpha} (bold profiles; right), or 10 µg/ml isotype-matched control mAb (gray solid profiles in both panels). In FCM analyses, FITC-conjugated mAb to CD54 or CD86 or PE-conjugated mAb to CD11c, CD40, or CD80 were used. Results are from a representative experiment of four independent experiments.

 
Signaling through CEACAM1 triggers release of IL-6 and IL-12

We tested whether mDC stimulated by mAb AgB10 release cytokines. Purified mDC from both mouse strains spontaneously released low amounts of IL-12 p40 (0.3–1.0 ng/106 cells/ml), IL-12 p70 (20–60 pg/106 cells/ml), and IL-18 but no IFN-{gamma} or IL-6 into the supernatant during a 2- to 3-day incubation (Fig. 5Go and data not shown). CEACAM1-mediated stimulation of DC strongly enhanced the release of IL-6, IL-12 p40, and IL-12 p70. Ligation of CEACAM1 was as effective as LPS in stimulating release of IL-6 by DC (Fig. 5GoA). CEACAM1-dependent signals were more efficient than TNF-{alpha} or LPS in triggering release of IL-12 p70 but less potent than CD40 ligation (Fig. 5GoA). Release of IL-18 or IFN-{gamma} by DC was not induced by CEACAM1 ligation (data not shown). Treatment of DC with isotype-matched control mAb, the anti-E-cadherin mAb Decma-1, or heat-inactivated mAb AgB10 did not stimulate IL-6 or IL-12 release by DC (Fig. 5GoB and data not shown). We detected no synergy among CEACAM1-, CD40-, or TNF-{alpha}-dependent signals in stimulating the release of IL-6, IL-12, or IL-18 release by DC (Fig. 5GoC and data not shown).



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  		FIGURE 5. CEACAM1-dependent signals stimulate IL-6, IL-12 p40, and IL-12 p70 release by DC. A, CD11c+ mDC were stimulated for 2 days with 1 µg/ml LPS, 10 ng/ml TNF-{alpha}, CD40 ligation (CD40L), 10 µg/ml mAb AgB10, 10 µg/ml mAb Decma-1, or 10 µg/ml isotype-matched control mAb. The concentration of each reagent chosen led to optimal stimulation of the cells. Supernatants were harvested, and the cytokine concentration was determined by ELISA. {alpha}-E-Cad., Anti-E-cadherin. B, Heat inactivation (h.i.) of the anti ({alpha})-CEACAM1 mAb destroyed its capacity to induce IL-12 p70 release, indicating that LPS contamination is not responsible for the induction of IL-12 p70 release. C, Costimulation of DC with TNF-{alpha} or CD40 ligation plus CEACAM1-dependent signals does not induce a synergistic cytokine release response, demonstrated here with IL-12 p40 release. Similar data were observed when IL-6 or IL-12 p70 release was measured (data not shown).

 
Stimulation of DC through CEACAM1 enhances their T cell-stimulatory function

The secretion of IL-12 by DC in response to CEACAM1 suggests that these DC can efficiently prime Th1 T cell responses. To test this hypothesis, we cocultured naive splenic CD4+ T cells from TCR-transgenic DO11.10 donor mice (40) for 5 days with OVA-pulsed DC. The OVA-pulsed DC were pretreated with either mAb AgB10 to signal through CEACAM1 or an isotype-matched control Ab. IL-4 and IFN-{gamma} release by T cells was measured after 5 days of culture. As shown in Fig. 6Go, CEACAM1-stimulated, OVA-presenting DC stimulated IFN-{gamma} release by specifically primed CD4+ T cells. Release of IL-4 by in vitro-primed CD4+ T cell populations was strikingly reduced when these cells were cocultured with CEACAM1-stimulated, OVA-presenting DC. Hence, DC activated through CEACAM1-dependent signals facilitate priming of Th1-like CD4+ T cell responses.



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  		FIGURE 6. CEACAM1-mediated signals facilitate Th1 CD4+ T cell priming. Naive CD4+ T cells from OVA-specific TCR-transgenic D011.10 BALB/c mice were stimulated with syngeneic OVA-pulsed DC pretreated or not pretreated with mAb AgB10. Release of IFN-{gamma} and IL-4 was determined by ELISA. Results are mean values ± SEM of triplicates (of a representative experiment of three independent experiments).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We show that murine DC express two major splice isoforms of the signal-regulating cell adhesion molecule CEACAM1 and that CEACAM1-triggered signaling facilitates T cell priming. A combination of enhanced recruitment of responding and presenting cells enhanced Ag presentation as a result of increased MHC and costimulator molecule expression, and increased release of cytokines may be involved in facilitating the efficient priming and Th1-biased polarization of CD4+ T cell responses by DC stimulated by CEACAM1-mediated signals.

CEACAM1 is related to the inhibitory receptor family (IRF) because it contains ITIM motifs in the cytoplasmic domain similar to NK cell-inhibitory receptors or Ig-like transcript receptors (ILT). Identification of CEACAM1 expression on DC is important, because the only members of the IRF that have been described on murine DC thus far are the paired Ig-like receptors (PIR) (41). In contrast, human DC express several members of the IRF, namely ILT, leukocyte-associated Ig-like receptor, DC immunoreceptor, and signal-regulatory protein (42, 43, 44, 45). Recent findings indicate that some of these molecules are involved in Ag uptake and presentation by human DC (46). To our knowledge, our work is the first to demonstrate that receptors of the IRF transmit maturation-inducing signals to DC that facilitate T cell priming.

Perturbation of DC-expressed CEACAM1 by Abs recognizing its extracellular domain induced chemokine secretion, particularly MIP-1{alpha} and MIP-2. Chemokine release associated with the development and activation of DC (47, 48, 49, 50, 51, 52, 53, 54, 55) facilitates recruitment of presenting, regulating, and responding myeloid and lymphoid cells into an immune response; e.g., MIP-1{alpha} attracts DC and MIP-2 recruits granulocytes to the site of inflammation (53). In agreement with these observations, we found that Ab-induced CEACAM1 signaling in DC triggered migration of granulocytes, monocytes, T cells, and DC toward the CEACAM1-stimulated DC. Thus, it appears that the early expression of CEACAM1 on immature DC facilitates the rapid recruitment of other cell subsets into an immune response around the nucleus of the emerging response, the DC. Interestingly, the pattern of chemokine release was unique for CEACAM1 signaling (MIP-1{alpha} >> MCP-1) compared with chemokine responses triggered by LPS or TNF-{alpha}. Thus, the activating receptor used to signal DC may determine which types of immune cells are preferentially recruited into the emerging immune response. CEACAM1-mediated signaling stimulated an up-regulation of the surface expression of MHC and costimulator molecules on DC and enhanced their IL-12 and IL-6 release. IL-12 p70 (the bioactive form of IL-12) release by DC in response to CEACAM1 perturbation was greater than the IL-12 p70 release triggered by LPS or TNF-{alpha} but lower than the IL-12 p70 release triggered by CD40 ligation, which is the most potent IL-12-inducing stimulus yet identified (56, 57). These data identify CEACAM1 as an efficient inducer of IL-12 release by DC.

An important issue for future analyses is how CEACAM1 perturbation triggers signaling and how this is induced in vivo. The only well-characterized physiological ligand for CEACAM1 is CEACAM1 itself. The N-terminal Ig domain mediates reciprocal homophilic binding (58), which is responsible for the cell-cell adhesion activity of CEACAM1. It has been proposed that homophilic binding is a major mode of signal input through cells surface-expressed CEACAM1 (29, 59). However, available data indicate that it may not be the homophilic interaction per se but rather the supramolecular organization on the cell surface that is important for CEACAM1-regulated signaling. CEACAM1-L and CEACAM1-S can both form homodimers and heterodimers (28). Dimerization is regulated by the cells via mass action (i.e., the expression levels of the CEACAM1 isoforms), intracellular processes, and homophilic CEACAM1 interactions between adjacent cells (28). Thus, the monomer/dimer status can be regulated in a dynamic way and would accordingly reflect the functional state of the cell. It is believed that the monomer/dimer status of CEACAM1 regulates the recruitment and activation of src-family kinases and protein tyrosine phosphatases, and consequently the CEACAM1-regulated signaling. Thus, any process including homophilic cell-cell binding that alters the monomer/dimer equilibrium of CEACAM1 would trigger a change in CEACAM1-mediated cell signaling. Also, CEACAM1 might occur in a nonadhesive form at the cell surface at certain states of the monomer/dimer equilibrium. Hence, homophilic binding may not occur between CEACAM1-expressing adjacent cells, unless CEACAM1 is activated by alteration of its supramolecular organization. The mechanisms for Ab-induced CEACAM1 signaling may accordingly be different in different cellular states depending on the state of the monomer/dimer equilibrium of CEACAM1. Abs against the extracellular domain may alter the monomer/dimer equilibrium either by direct action or indirectly, by interfering with CEACAM1 homophilic binding under conditions when this occurs.

Although we do not know whether there are other physiological ligands for CEACAM1, it is well known that CEACAM1 acts as receptor for several microbial pathogens. Thus, MHV uses CEACAM1 as its receptor (9, 30, 60), and in humans CEACAM1 serves as the receptor for N. gonorrhoeae, N. meningitidis, H. influenzae, and several strains of the genus Salmonella and E. coli (32, 33, 34, 35, 36). Pathogens may therefore trigger CEACAM1-mediated signals in DC in vivo. Priming IL-12 release by DC and of their competence to respond to (CD40-dependent) T cell-derived signals in vivo is under the control of exogenous microbial factors (61). It could thus be speculated that pathogen-mediated signal input through CEACAM1 is an important priming event for T cell responses that work in concert with homophilic cell-cell interactions to regulate the CEACAM1 supramolecular organization.

On the basis of the present results and the arguments given above, we propose the following scenario for the role of CEACAM1 during an immune response. The first event would be the recognition of a pathogen that binds to DC via CEACAM1. This would activate the DC, resulting in the secretion of chemokines, which would recruit cellular components of the innate immune system to the site of infection. In addition, this primary CEACAM1-mediated contact leads to maturation of the DC and their migration to lymphoid organs, where they interact with the adaptive immune system. At this stage, cells expressing CEACAM1, e.g., T cells and B cells, may provide further signal inputs through CEACAM1 into DC. This signaling, in combination with other signals provided by cell-cell contacts (e.g., CD40-CD40L) then stimulate cytokine release by DC, which regulate the Th1/Th2 balance of the response.


   Acknowledgments
 
The expert technical assistance of Tanja Güntert, Anja Müller, Tom Krieg, and Claude Oehninger is gratefully acknowledged. We thank Drs. N. I. Kuprina and T. D. Rudinskaya for making the AgB10 hybridoma available to us.


   Footnotes
 
1 This work was supported by a grant from the Wilhelm Sander Stiftung (to J.R.), the Swedish Medical Research Council (Project 05200; to B.Ö), and the Swedish Cancer Foundation (Project 3957; to B.Ö). Back

2 Address correspondence and reprint requests to Dr. Jörg Reimann, Department of Medical Microbiology and Immunology, University of Ulm, Helmholtzstrasse 8/1, D-89081 Ulm, Germany. E-mail address: joerg.reimann{at}medizin.uni-ulm.de Back

3 Abbreviations used in this paper: DC, dendritic cell; mDC, myeloid DC; BM, bone marrow; BMC, BM cell; FL, Flt3-ligand; MIP, macrophage inflammatory protein, MCP, monocyte chemoattractant protein; CEACAM1, carcinoembryonic Ag-related cell adhesion molecule 1; FCM, flow cytometry; ITIM, immunoreceptor tyrosine-based inhibitory motif; MHV, mouse hepatitis virus; CD40L, CD40 ligand; IRF, inhibitory receptor family; ILT, Ig-like transcript receptor; PIR, paired Ig-like receptor. Back

Received for publication November 30, 2000. Accepted for publication March 21, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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