Stimulation Cells Is a Potent Regulator of T Cell Adhesion Molecule 1 On

Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 on Murine Dendritic Cells Is a Potent Regulator of T Cell Stimulation This information is current as of October 3, 2021. Robert Kammerer, Detlef Stober, Bernhard B. Singer, Björn Öbrink and Jörg Reimann J Immunol 2001; 166:6537-6544; ; doi: 10.4049/jimmunol.166.11.6537

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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,† Bjo¨rn O¨ brink,† and Jo¨rg Reimann2*

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 phosphorylat- able tyrosine residues). Ligation of surface-expressed CEACAM1 on DC with the specific mAb AgB10 triggered release of the Downloaded from chemokines macrophage inflammatory protein 1␣, 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, http://www.jimmunol.org/ thereby facilitating priming and polarization of T cell responses. The Journal of Immunology, 2001, 166: 6537–6544.

endritic cells (DC)3 are the most potent APCs that can known, many of which belong to the Ig superfamily. Several of induce T cell responses (reviewed in Refs. 1–3). Imma- these receptors have immunoreceptor tyrosine-based activation D ture, tissue-resident DC capture Ag and respond to other, motifs or immunoreceptor tyrosine-based inhibitory motifs (ITIM) not yet well-defined, signals derived from either the pathogen (e.g., in their cytoplasmic domains and operate by recruiting and acti- LPS, bacterial DNA, viral double-stranded RNA) or from tissue vating SH2 domain-containing protein tyrosine kinases and protein lesions induced by the pathogen (e.g., stress proteins), before they tyrosine phosphatases. Among the ITIM-containing receptors sig- by guest on October 3, 2021 develop into competent DC. To achieve this, they migrate to re- nal-regulatory proteins and killer cell-inhibitory receptors are well gional lymph nodes where they differentiate into presentation- known. Carcinoembryonic Ag-related cell adhesion molecule 1 competent DC. Hence, DC prime naive T cells remote from the (CEACAM1), also known as CD66a, BGP, or C-CAM, is an site of initial Ag contact (4, 5). In the maturating process leading ITIM-containing Ig superfamily receptor abundantly expressed on to presentation-competent cells, several signaling events triggered the cell surface. CEACAM1 is the receptor for a variety of micro- by soluble ligands as well as specific cell-cell interactions play a organisms. Because it is involved in both cell-cell communication crucial role. An important theme in cell signaling is signal regu- and pathogen-host interactions, we analyzed its putative expres- lation by costimulation and coinhibition. In the immune system, a sion and functional role in DC. large number of both coinhibitory and costimulatory receptors are CEACAM1 is abundantly expressed in epithelia, vessel endo- thelia, granulocytes, macrophages, T cells, B cells, NK cells, and *Institute for Medical Microbiology and Immunology, University of Ulm, Ulm, Ger- platelets (6–11). It can mediate intercellular adhesion via ho- many; and †Department of Cell and Molecular Biology, Karolinska Institutet, Stock- mophilic binding (12–14). Work in many laboratories has demon- holm, Sweden strated that CEACAM1 has important regulatory functions in cell Received for publication November 30, 2000. Accepted for publication March proliferation, angiogenesis, apoptosis, immune responses, T cell 21, 2001. cytotoxicity, differentiation, and polarization and lumen formation The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance of epithelial cells (15–20). It is down-regulated in many types of with 18 U.S.C. Section 1734 solely to indicate this fact. cancer (21, 22). CEACAM1-dependent signals can inhibit tumor 1 This work was supported by a grant from the Wilhelm Sander Stiftung (to J.R.), the growth in vivo (23). CEACAM1 can influence and regulate signal Swedish Medical Research Council (Project 05200; to B.O¨ ), and the Swedish Cancer Foundation (Project 3957; to B.O¨ ). transduction. It is a molecular system composed of several splice 2 Address correspondence and reprint requests to Dr. Jo¨rg Reimann, Department of isoforms that can influence cell signaling in both positive and neg- Medical Microbiology and Immunology, University of Ulm, Helmholtzstrasse 8/1, ative ways. CEACAM1 probably operates by recruiting and acti- D-89081 Ulm, Germany. E-mail address: [email protected] vating either src-family kinases, or protein tyrosine phosphatases 3 Abbreviations used in this paper: DC, dendritic cell; mDC, myeloid DC; BM, bone Src homology phosphatases 1 and 2 to the same tyrosine-phos- marrow; BMC, BM cell; FL, Flt3-ligand; MIP, macrophage inflammatory protein, MCP, monocyte chemoattractant protein; CEACAM1, carcinoembryonic Ag-related phorylated ITIM motifs in the cytoplasmic domain of the isoform cell adhesion molecule 1; FCM, flow cytometry; ITIM, immunoreceptor tyrosine- CEACAM1-L (24–26). The two major isoforms are CEACAM1-L based inhibitory motif; MHV, mouse hepatitis virus; CD40L, CD40 ligand; IRF, inhibitory receptor family; ILT, Ig-like transcript receptor; PIR, paired Ig-like recep- and CEACAM1-S, which differ in their cytoplasmic domains. The tor. cytoplasmic domain of CEACAM1-L consists of 73 amino acids

and has 2 ITIM motifs with tyrosine residues that can be phos- FCM analyses phorylated. CEACAM1-S has a cytoplasmic domain of only 10 Cells were suspended in PBS, 0.3% w/v BSA supplemented with 0.1% w/v amino acids and lacks ITIM motifs (27). CEACAM1-L and sodium azide. Unspecific binding of Abs to FcR was blocked by preincu- CEACAM1-S are coexpressed at different ratios in different cell bating cells with the anti-CD16/CD32 mAb 2.4G2 (1 ␮g mAb/106 cells; types and in different functional states of cells of one lineage. Both PharMingen, Hamburg, Germany). Cells were incubated with 0.5 ␮g/106 isoforms can dimerize, and there is evidence that the S isoform can cells of the relevant mAb for 30 min at 4°C, washed twice, and subse- quently incubated with a second-step reagent for 15 min at 4°C. Cells were regulate the signaling activity of the L isoform (23, 28, 29). washed twice and analyzed on a FACScan (Becton Dickinson, Mountain The only well-characterized physiological ligand interaction of View, CA). Dead cells were excluded by propidium iodide staining. The CEACAM1 is the homophilic binding to itself; this may represent following reagents and mAbs from PharMingen were used: PE-conjugated d d b b,d,k an important signal input via cell-cell interactions (29). Several anti-I-A /I-E ; PE-conjugated anti-I-A ; biotinylated anti-H-2D ; PE- conjugated anti-CD80 (B7-1); PE-conjugated anti-CD40; FITC-conjugated pathogenic microorganisms bind to CEACAM1 as their host cell anti-CD86 (B7-2); and PE-conjugated anti-CD11c. FITC-conjugated anti- receptor. Murine CEACAM1 is the major receptor for mouse hep- CD54 (ICAM-1) and PE-conjugated F4/80 were purchased from Cedarlane atitis viruses (MHV) (30, 31) and human CEACAM1 binds Esch- (Hornby, Ontario, Canada). We furthermore used the rat anti-mouse erichia coli, Salmonella typhimurium (32), Neisseria gonorrhoeae DEC205 (NLDC-145) mAb from Biozol (Eching, Germany) and the FITC- (33, 34), Neisseria meningitidis (35), and Haemophilus influenzae conjugated IgG1 mAb R3-34, PE-conjugated IgG1 mAb R3-34, and streptavidin Red 670 (Life Technologies, Eggenstein, Germany). (36). Both the physiological cell-cell interactions mediated by CEACAM1 and its binding to pathogens may have an influence on Cell culture and mAb purification the activation of the immune system. The mature B cell line L10 and the hybridomas producing the mAbs In this investigation, we show that both immature and mature Decma-1 (38) and AgB10 (39) were cultured in a humidified atmosphere Downloaded from murine (myeloid and lymphoid) DC express CEACAM1 on the with 5% CO2 at 37° in RPMI supplemented with 10% heat-inactivated ␮ surface in vivo and in vitro. Specific binding of a mAb to surface FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 g/ml streptomy- cin. The supernatants were collected, and the Abs were affinity-purified on CEACAM1 expressed by immature DC stimulates their matura- a HiTrap protein G column according to the manufacturer’s instructions tion including the release of cytokines and chemokines. We dem- (Amersham Pharmacia Biotech, Uppsala, Sweden). Endotoxin content in onstrate that CEACAM1 facilitates priming of naive CD4ϩ T cells the Ab preparations was Ͻ0.03 endotoxin U/ml as determined using the by Ag-bearing DC in vitro and favors a Th1-type differentiation. Endosafe Gel-Clot Assay (Charles River, Sulzfeld, Germany). These data show that CEACAM1 takes part in the signaling sce- Cytokines and cytokine detection by ELISA http://www.jimmunol.org/ nario that leads to the induction of Th1 immune responses. The following recombinant mouse cytokines were obtained from Pepro- Tech: IL-4, IL-6, IL-10, IL-18, monocyte chemoattractant protein 1 (MCP- Materials and Methods 1), macrophage inflammatory protein 2 (MIP-2), TNF-␣, GM-CSF, and ␣ Mice FL. IL-12 p40 and MIP-1 were purchased from R&D Systems (Wiesba- den, Germany). IFN-␥ and IL-12 p70 were obtained from PharMingen. BALB/cJ (H-2d) and C57BL/6J (H-2b) mice were obtained from Bomholt- Cytokines released into culture supernatants were detected by a con- gard (Ry, Denmark) and kept under standard pathogen-free conditions in ventional double-sandwich ELISA. For detection and capture, the follow- the animal colony of Ulm University (Ulm, Germany). BALB/c-derived ing mAbs (from PharMingen) were used: mAb R4-6A2 and biotinylated DO11.10 TCR-transgenic mice were kindly provided by D. Loh (Roche, mAb XMG1.2 were used for IFN-␥; mAb 2H5 and biotinylated mAb 4E2/ by guest on October 3, 2021 Nutley, NJ). Female mice were used at 10–16 wk of age. 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; Generation of myeloid DC (mDC) from murine bone marrow mAb C17.8 was used for detection in both cases. Extinction was analyzed (BM) at 405/490 nm on a TECAN microplate ELISA reader (TECAN, Crailsheim, Germany) using EasyWin software (TECAN). The detection The in vitro generation of mDC from murine bone marrow has been de- limits of the cytokine ELISAs were: 0.2 pg/ml for IL-4; 1 pg/ml for MIP-2; scribed (37). Briefly, bone marrow cells (BMC) obtained from femurs and 10 pg/ml for IL-12p40, MIP-1␣, and MCP-1; 20 pg/ml for IL-12 p70 and tibiae were depleted of CD4ϩ and CD8ϩ T cells, B220ϩ B cells, and MHC IFN-␥; and 60 pg/ml for IL-18. class IIϩ maturing myeloid cells by MACS sorting following the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Chemotaxis assay 6 These BMC were cultured at a density of 10 cells/ml in UltraCulture Chemotaxis was assayed by an in vitro two-chamber migration assay fol- medium (BioWhittaker, Verviers, Belgium) supplemented with 5 ng/ml lowed by FCM. Cells in 100 ␮l complete medium were added to the upper GM-CSF and 10 ng/ml Flt3 ligand (FL) (PeproTech, Rocky Hill, NJ), 2 chamber of Falcon Transwells (6.5-mm diameter, 3-␮m pore size, poly- mM glutamine, and antibiotics. BMC from C57BL/6 mice were cultured in ethylen-terephtalat membrane; Becton Dickinson, Heidelberg, Germany), serum-free medium; BMC from BALB/c mice required low FCS supple- and chemotactic substances were added to the lower chamber to form a ments (0.5% v/v) to the medium to maintain viability and support expan- chemotactic gradient. A total of 1 ϫ 106 cells were incubated for4hinthe sion of mDC. Cultures were incubated at 37°C in humidified air supple- upper chamber of the Transwell. After cells were collected in suspension, mented with 5% CO2. On days 3 and 5, cells were fed by medium ϩ 0.5 ml 5 mM EDTA was added to the lower chamber for 15 min at 37°C exchange. On day 7 of culture, nonadherent cells were harvested; CD11c to detach adherent cells such as monocytes and granulocytes from the cells were purified by magnetic bead separation (Miltenyi Biotec) and re- bottom of the wells. Detached cells were combined with the previously plated in UltraCulture medium supplemented with GM-CSF, FL, glu- collected suspension cells for cell counting. Migrated granulocytes, mono- tamine, and antibiotics. ϩ cytes, and lymphocytes were counted by FCM by gating on appropriate CD11c cells were analyzed by flow cytometry at day 8–10 of culture. populations of cells using forward scatter and side scatter channels. CD4ϩ DC harvested from day 8 cultures were seeded into 96-well round-bottom and CD8ϩ T cells were detected with FITC- or PE-conjugated mAbs to ϫ 5 plates at 2 10 cells/well in UltraCulture medium with GM-CSF, FCS, CD4 and CD8. Each chemotaxis experiment was performed in duplicate. and antibiotics. These cultures were stimulated with the indicated cytokines The results from three independent experiments were pooled. For statistical ϫ 4 and Abs. In some experiments, 6.6 10 cells/well CD40 ligand (CD40L)- analyses, nested ANOVA was performed to compare the mean counts of transfected J558L cells (or negative control nontransfected J558L cells) cells migrating toward supernatants of stimulated vs unstimulated DC. were added. After 48–72 h incubation, supernatants were harvested, and When ANOVA detected a statistically significant difference in mean cytokine release was detected by ELISA. counts, the Tukey method of multiple comparisons was applied. A p value of Ͻ0.05 was considered to be statistically significant for all analyses. Isolation and purification of CD4ϩ T cells Detection of CEACAM1 splice variants by RT-PCR Single-spleen cell suspensions were passed through nylon wool columns. CD4ϩ T cells were selected from the nonadherent cell population by Total mRNA was isolated from 5 ϫ 106 cells using the RNeasy kit (Qia- MACS. The purity of the isolated CD4ϩ T cell subset was Ͼ96% as de- gen, Hilden, Germany). The RNA yield was quantified photometrically and termined by flow cytometry (FCM). 1 ␮g mRNA was used for cDNA syntheses by reverse transcription using The Journal of Immunology 6539 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 (␤-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 5Ј-AGCGTCAGGAGGAGCAACTCAA and 3Ј-AGAAGAAGGGGCT GAAGTTGGC, 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 ␤-actin mRNA was amplified using the 5Ј- primer ATGGATGACGATATCGCT and the 3Ј-primer ATGAGG TAGTCTGTCAGGT. Ten microliters of each PCR product were analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. Results Expression of CEACAM1 by murine DC We generated CD11cϩ 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 Downloaded from from day 7 cultures, 50–70% had the CD11cϩ/CD11bϩ/CD4Ϫ/ CD8Ϫ/E-cadherinϩ surface phenotype of mDC. Surface expression of MHC-II molecules and CD40, CD80, and CD86 costimulator molecules was low or undetectable in most CD11cϩ cells from day 7 cultures. FIGURE 1. Expression of CEACAM1 by murine DC. A, The histo- We used the mAb AgB10 specific for the extracellular domain grams show the log fluorescence intensity of CEACAM1 on the surface of http://www.jimmunol.org/ of murine CEACAM1 to detect CEACAM1 expression on the cell DC labeled with either fluorochrome-conjugated mAb AgB10 (bold pro- surface of DC (39). Nonadherent CD11cϩ mDC harvested from files) or an isotype control mAb (gray profiles). Only CD11cϩ cells are 7-day BMC cultures expressed low but detectable levels of shown. The percents of positive cells are indicated. In this setting, 99% of CEACAM1 (Fig. 1A). The expression of this marker was up-reg- the isotype control Ab stained cells were negative. B, mRNA was isolated ϩ ϫ 6 ϩ ulated 3- to 5-fold when purified, nonadherent CD11c mDC were from 5 10 purified CD11c DC (harvested from day 7 or day 10 cul- subcultured without a maturation-inducing agent (IL-4, TNF-␣, tures), the B cell line L10 (positive control), and NIH 3T3 cells (negative control). Using primers specific for the A2 domain and the 3Ј-end of LPS, or CD40L) for another 3 days in GM-CSF and FL (Fig. 1A). CEACAM1, two mRNA products were amplified: the 321-bp product The expression level on mDC harvested from day 8–10 cultures

(characteristic of the CEACAM1-L splice variant); and the 268-bp product by guest on October 3, 2021 was lower than that of B cells, which are known to display high (characteristic for CEACAM1-S splice variant). Amplification of a mRNA CEACAM1 surface expression (Fig. 1A) (9). fragment of ␤-actin indicated that comparable amounts of RNA were am- The expression of CEACAM1 by murine mDC was confirmed plified. C, CEACAM1 expression by CD11cϩ DC isolated from inguinal by RT-PCR (Fig. 1B). The primer pair used amplifies both the L lymph nodes or spleen of C57BL/6J mice. Both CD8␣ϩ and CD8␣Ϫ DC and the S cytoplasmic isoforms. CD11cϩ DC from day 7 and 9 express CEACAM1 (bold profiles). BMC cultures expressed both CEACAM1 isoforms (Fig. 1B). The presence of the L isoform indicated that CEACAM1 expressed on the surface of mDC can mediate signaling via its ITIM motifs. pression was, however, not preferentially up-regulated in DC with We isolated CD11cϩ cells from spleen and lymph nodes of a more mature phenotype, i.e., high surface expression of MHC-II, BALB/c and C57BL/6 mice to test whether DC express CD40, CD80, and CD86 molecules (Fig. 2). CEACAM1 is thus CEACAM1 in vivo. A significant proportion of freshly isolated expressed on the surface of early DC developing in vitro from BM CD8␣ϩ and CD8␣Ϫ DC from both spleen and lymph nodes ex- progenitors, is up-regulated to a limited extent on DC during their pressed CEACAM1 on the surface (Fig. 1C). The level of in vitro differentiation, but is not preferentially expressed by ma- CEACAM1 surface expression by freshly isolated, murine DC was ture, presentation-competent mDC. comparable with the level of CEACAM1 surface expression by We tested whether surface expression of CEACAM1 by DC can DC generated in vitro (compare Fig. 1A and Fig. 1C). DC gener- be modulated by proinflammatory cytokines that induce DC mat- ated in vitro or in vivo thus express CEACAM1 on the cell surface. uration, i.e., TNF-␣, IL-4, IFNs, or LPS. When DC were treated with these agents from day 7 to day 10 of culture, surface expres- CEACAM1 surface expression by maturing mDC sion of MHC II and costimulator (CD40, CD54, CD80, and CD86) When nonadherent CD11cϩ DC were purified from day 5 BMC molecules was strikingly induced or up-regulated (data not cultures and cultured for a further 5 days with GM-CSF and FL, a shown). In contrast, the surface expression of CEACAM1 was not subset of 20–30% of the cells showed surface phenotype changes modulated (data not shown). Hence, CEACAM1 expression is not indicating “spontaneous” maturation. This was evident by up-reg- up-regulated by exogenous cytokines during maturation of DC. ulated surface expression of CD40, CD54, CD80, and CD86 costimulator and of MHC II molecules (Fig. 2). CEACAM1 surface CEACAM1-dependent signaling of mDC triggers release of expression was also up-regulated between day 5 and day 10 of chemokines that attract granulocytes, monocytes, T cells, and culture (Fig. 1A). This “spontaneous” up-regulation of CEACAM1 DC surface expression was seen in C57BL/6-derived DC growing in Because early signaling in the immune system involves production serum-free cultures, and in BALB/c-derived DC cultured in FCS- and release of chemokines, we investigated the secretion of MIP- supplemented medium (data not shown). CEACAM1 surface ex- 1␣, MIP-2, and MCP-1 by DC stimulated by CEACAM1 ligation 6540 CEACAM1 REGULATES DC FUNCTION Downloaded from http://www.jimmunol.org/ FIGURE 2. CEACAM1 and costimulator/MHC molecule surface coex- pression by murine DC. Cells were labeled with mAb AgB10 and a FITC- Ј 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 Ͼ98% of isotype control mAb-stained cells were in the lower left quadrant. Data are representative of three experiments using independent DC cultures. by guest on October 3, 2021

(Fig. 3A). Signaling through CEACAM1 strikingly enhanced the low “spontaneous” MIP-1␣ and MIP2 release by DC. Ligation of FIGURE 3. CEACAM1-dependent signaling of DC induces chemokine ϫ 4 surface CEACAM1 was more potent in triggering release of these release. A, DC (5 10 cells/well) were stimulated with mAb AgB10 (10 ␮g/ml), an isotype-matched control mAb (10 ␮g/ml), the anti-E-cadherin chemokines by DC than the other stimuli tested. It also enhanced mAb Decma-1 (10 ␮g/ml), LPS (1 ␮g/ml), or CD40 ligation (CD40L) the release of MCP-1 although to a lesser extend. Isotype-matched from day 7 to day 9 of culture. Chemokine release into the supernatants control mAb, mAb to E-cadherin (DC generated in the BMC cul- was determined by ELISA. B, DC (5 ϫ 104 cells/well) were stimulated tures expressed the adhesion molecule E-cadherin on the cell sur- with anti (␣)-CEACAM1 or anti-E-cadherin (␣-E-Cad.) mAb, and the re- face), or heat-inactivated mAb AgB10 (to exclude a contaminating lease of the chemokines MIP-1␣ and MIP-2 was determined after 2.5, 5, LPS effect) had no measurable effect on chemokine release by DC 7.5, and 24 h of culture. C, Migration of leukocytes induced by superna- (Fig. 3A and data not shown). The chemokine response of DC to tants conditioned by DC stimulated with 10 ␮g/ml AgB10 or 10 ␮g/ml CEACAM1 ligation was rapidly inducible and showed a different isotype control mAb. Migration of the cells was analyzed using a Transwell kinetic for MIP-1␣ and MIP-2 (Fig. 3B). system. d, Migration of splenic T cells toward different concentrations of ␣ Chemokines stimulate the migration of leukocytes. We analyzed MIP-1 was used as a positive control. Migrating cells were counted and identified by FCM using forward scatter and side scatter channels (gran- cellular migration in a Boyden chamber assay. From nonfraction- ϩ ulocytes, monocytes, lymphocytes) and specific mAb (to identify CD4 ated spleen cell populations, supernatants conditioned by DC stim- and CD8ϩ T cells). Results are mean values Ϯ SEM of three experiments .(p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء) ulated through CEACAM1 ligation attracted granulocytes and monocytes (Fig. 3C, a). From splenic T cell populations, the migration of both CD4ϩ and CD8ϩ T cells was enhanced 2-fold by supernatants from CEACAM1-stimulated DC (Fig. 3C, b). dium with the mAb AgB10. Either isotype-matched control mAb CEACAM1-stimulated DC also enhanced the migration of DC or the mAb Decma-1 specific for murine E-cadherin was added to (Fig. 3C, c). Hence, CEACAM1-dependent signals of DC recruit the medium in control cultures. The anti-CEACAM1 mAb AgB10 myeloid and lymphoid cells into the immune response, most likely but neither the anti-E-cadherin mAb Decma-1 nor an isotype- through increased chemokine release. matched control mAb up-regulated surface expression of CD40, CD54, CD80, and CD86 costimulator molecules by DC that was Ligation of surface CEACAM1 on DC by mAb AgB10 induces comparable with that induced by TNF-␣ treatment (Fig. 4). Induc- maturation tion of DC maturation may result either directly through Purified CD11cϩ DC harvested from day 7 BMC cultures were CEACAM1 signaling or indirectly through cytokines/chemokines cultured for 2 days in serum-free GM-CSF/FL-supplemented me- released by CEACAM1-triggered signals. Binding of the anti- The Journal of Immunology 6541

CEACAM1 Ab did not trigger release of TNF-␣ by DC (data not shown).

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-␥ or IL-6 into the supernatant during a 2- to 3-day incubation (Fig. 5 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. 5A). CEACAM1-dependent signals were more efﬁ- cient than TNF-␣ or LPS in triggering release of IL-12 p70 but less potent than CD40 ligation (Fig. 5A). Release of IL-18 or IFN-␥ 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. 5B and data not Downloaded from shown). We detected no synergy among CEACAM1-, CD40-, or TNF-␣-dependent signals in stimulating the release of IL-6, IL-12, or IL-18 release by DC (Fig. 5C and data not shown).

Stimulation of DC through CEACAM1 enhances their T cell-

stimulatory function http://www.jimmunol.org/ 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 CD4ϩ T cells from TCR-transgenic DO11.10 donor mice (40) for 5 days with OVA- FIGURE 4. CEACAM1-dependent signals up-regulate surface expres- pulsed DC. The OVA-pulsed DC were pretreated with either mAb sion of costimulator molecules by DC. DC (106 cells/ml) were stimulated AgB10 to signal through CEACAM1 or an isotype-matched con- from day 7 to day 9 of culture with either 10 ␮g/ml mAb AgB10 (bold trol Ab. IL-4 and IFN-␥ release by T cells was measured after 5 ␣ ␮ profiles; left), or 10 ng TNF- (bold profiles; right), or 10 g/ml isotype- days of culture. As shown in Fig. 6, CEACAM1-stimulated, OVA- by guest on October 3, 2021 matched control mAb (gray solid profiles in both panels). In FCM analyses, presenting DC stimulated IFN-␥ release by specifically primed FITC-conjugated mAb to CD54 or CD86 or PE-conjugated mAb to CD4ϩ T cells. Release of IL-4 by in vitro-primed CD4ϩ T cell CD11c, CD40, or CD80 were used. Results are from a representative experiment of four independent experiments. populations was strikingly reduced when these cells were cocultured with CEACAM1-stimulated, OVA-presenting DC. Hence,

FIGURE 5. CEACAM1-dependent signals stimulate IL-6, IL-12 p40, and IL-12 p70 release by DC. A, CD11cϩ mDC were stimulated for 2 days with 1 ␮g/ml LPS, 10 ng/ml TNF-␣, CD40 ligation (CD40L), 10 ␮g/ml mAb AgB10, 10 ␮g/ml mAb Decma-1, or 10 ␮g/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. ␣-E-Cad., Anti-E-cadherin. B, Heat inactivation (h.i.) of the anti (␣)-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, Costimu- lation of DC with TNF-␣ or CD40 ligation plus CEACAM1-dependent signals does not induce a synergistic cytokine release response, demon- strated here with IL-12 p40 release. Similar data were observed when IL-6 or IL-12 p70 release was measured (data not shown). 6542 CEACAM1 REGULATES DC FUNCTION

DC activated through CEACAM1-dependent signals facilitate CEACAM1-S can both form homodimers and heterodimers (28). priming of Th1-like CD4ϩ T cell responses. Dimerization is regulated by the cells via mass action (i.e., the expression levels of the CEACAM1 isoforms), intracellular pro- Discussion cesses, and homophilic CEACAM1 interactions between adjacent We show that murine DC express two major splice isoforms of the cells (28). Thus, the monomer/dimer status can be regulated in a signal-regulating cell adhesion molecule CEACAM1 and that dynamic way and would accordingly reflect the functional state of CEACAM1-triggered signaling facilitates T cell priming. A com- the cell. It is believed that the monomer/dimer status of bination of enhanced recruitment of responding and presenting CEACAM1 regulates the recruitment and activation of src-family cells enhanced Ag presentation as a result of increased MHC and kinases and protein tyrosine phosphatases, and consequently the costimulator molecule expression, and increased release of cyto- CEACAM1-regulated signaling. Thus, any process including ho- kines may be involved in facilitating the efficient priming and Th1- mophilic cell-cell binding that alters the monomer/dimer equilib- biased polarization of CD4ϩ T cell responses by DC stimulated by rium of CEACAM1 would trigger a change in CEACAM1-medi- CEACAM1-mediated signals. ated cell signaling. Also, CEACAM1 might occur in a CEACAM1 is related to the inhibitory receptor family (IRF) nonadhesive form at the cell surface at certain states of the mono- because it contains ITIM motifs in the cytoplasmic domain similar mer/dimer equilibrium. Hence, homophilic binding may not occur to NK cell-inhibitory receptors or Ig-like transcript receptors between CEACAM1-expressing adjacent cells, unless CEACAM1 (ILT). Identification of CEACAM1 expression on DC is important, is activated by alteration of its supramolecular organization. The because the only members of the IRF that have been described on mechanisms for Ab-induced CEACAM1 signaling may accord- murine DC thus far are the paired Ig-like receptors (PIR) (41). In ingly be different in different cellular states depending on the state contrast, human DC express several members of the IRF, namely of the monomer/dimer equilibrium of CEACAM1. Abs against the Downloaded from ILT, leukocyte-associated Ig-like receptor, DC immunoreceptor, extracellular domain may alter the monomer/dimer equilibrium ei- and signal-regulatory protein (42–45). Recent findings indicate ther by direct action or indirectly, by interfering with CEACAM1 that some of these molecules are involved in Ag uptake and pre- homophilic binding under conditions when this occurs. sentation by human DC (46). To our knowledge, our work is the Although we do not know whether there are other physiological first to demonstrate that receptors of the IRF transmit maturation- ligands for CEACAM1, it is well known that CEACAM1 acts as inducing signals to DC that facilitate T cell priming. receptor for several microbial pathogens. Thus, MHV uses http://www.jimmunol.org/ Perturbation of DC-expressed CEACAM1 by Abs recognizing CEACAM1 as its receptor (9, 30, 60), and in humans CEACAM1 its extracellular domain induced chemokine secretion, particularly serves as the receptor for N. gonorrhoeae, N. meningitidis, H. in- MIP-1␣ and MIP-2. Chemokine release associated with the devel- fluenzae, and several strains of the genus Salmonella and E. coli opment and activation of DC (47–55) facilitates recruitment of (32–36). Pathogens may therefore trigger CEACAM1-mediated presenting, regulating, and responding myeloid and lymphoid cells signals in DC in vivo. Priming IL-12 release by DC and of their into an immune response; e.g., MIP-1␣ attracts DC and MIP-2 competence to respond to (CD40-dependent) T cell-derived sig- recruits granulocytes to the site of inflammation (53). In agreement nals in vivo is under the control of exogenous microbial factors with these observations, we found that Ab-induced CEACAM1 (61). It could thus be speculated that pathogen-mediated signal by guest on October 3, 2021 signaling in DC triggered migration of granulocytes, monocytes, T input through CEACAM1 is an important priming event for T cell cells, and DC toward the CEACAM1-stimulated DC. Thus, it ap- responses that work in concert with homophilic cell-cell interac- pears that the early expression of CEACAM1 on immature DC tions to regulate the CEACAM1 supramolecular organization. facilitates the rapid recruitment of other cell subsets into an im- On the basis of the present results and the arguments given mune response around the nucleus of the emerging response, the above, we propose the following scenario for the role of DC. Interestingly, the pattern of chemokine release was unique for CEACAM1 during an immune response. The first event would be CEACAM1 signaling (MIP-1␣ ϾϾ MCP-1) compared with che- the recognition of a pathogen that binds to DC via CEACAM1. mokine responses triggered by LPS or TNF-␣. 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-␣ 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 ho- ϩ FIGURE 6. CEACAM1-mediated signals facilitate Th1 CD4 T cell mophilic binding is a major mode of signal input through cells priming. Naive CD4ϩ T cells from OVA-specific TCR-transgenic D011.10 surface-expressed CEACAM1 (29, 59). However, available data BALB/c mice were stimulated with syngeneic OVA-pulsed DC pretreated indicate that it may not be the homophilic interaction per se but or not pretreated with mAb AgB10. Release of IFN-␥ and IL-4 was de- rather the supramolecular organization on the cell surface that is termined by ELISA. Results are mean values Ϯ SEM of triplicates (of a important for CEACAM1-regulated signaling. CEACAM1-L and representative experiment of three independent experiments). The Journal of Immunology 6543

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