Published online before print May 22, 2003
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* Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden; and
McGill Cancer Center, McGill University, Montreal, Canada
Correspondence: Eva Severinson, Karolinska Institutet, CMB, von Eulers väg 3 (Box 285), SE-171 77 Stockholm, Sweden. E-mail: eva.severinson{at}cmb.ki.se
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Key Words: rodent • B lymphocytes • coregulation • cellular proliferation • adhesion molecules
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The coreceptors that are mentioned above are largely B cell-specific. However, recent progress in the signaling field has indicated the existence of more general, signal-regulating systems that are expressed in a variety of different cell types. One such system is carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is also known as CD66a, biliary glycoproteins, or C-CAM [11 , 12 ]. CEACAM1 belongs to the Ig superfamily and is abundantly expressed in epithelia and vessel endothelia as well as in granulocytes, monocytes, natural killer (NK) cells, T cells, B cells, and dendritic cells (DCs) [11 , 13 , 14 ]. It can mediate cell–cell adhesion via homophilic binding [15 ], and it regulates cell proliferation [16 ], apoptosis [17 ], tumor growth [18 , 19 ], differentiation, and polarization of epithelial cells [17 ], angiogenesis [20 ], NK cell cytotoxicity [21 ], T cell cytotoxity [22 ], and T cell-mediated immune responses [22 23 24 25 26 ]. Two major splice variants of CEACAM1 differing in their cytoplasmic domains, CEACAM1-L and CEACAM1-S, occur [11 ]. In most CEACAM1-expressing cells, both isoforms are coexpressed but at various ratios [16 ]. The L-isoform but not the S-isoform has two phosphorylatable tyrosine residues in its cytoplasmic domain [11 ]. The more membrane-proximal tyrosine residue is part of an ITIM motif, whereas the membrane-distal one occurs in a slightly different sequence, which has been referred to as an immunoreceptor tyrosine-based switch motif [27 ]. After tyrosine phosphorylation, CEACAM1 can activate src-family tyrosine kinases [28 , 29 ] as well as the Src homology (SH)2 domain containing protein tyrosine phosphatases-1 and -2 [30 ]. It is interesting that stimulatory and inhibitory effects by CEACAM1 have been observed, e.g., on cell proliferation and on T cell activation [22 23 24 25 , 31 ]. This may be a result of differences in the balance between kinase and phosphatase activation by CEACAM1, which in turn might be regulated by the CEACAM1 isoform expression ratios [31 ].
In cells belonging to the immune system, various effects of CEACAM1 have been described in T cells, NK cells, and DCs [14 , 21 22 23 24 25 26 ]. However, its functional role in B cells has, until now, been completely unknown. We therefore undertook a study where we addressed the question of whether CEACAM1 might influence such basic functions in B cells as proliferation, Ig secretion, and homotypic cell adhesion. Using murine B lymphocytes and a monoclonal antibody (mAb) against CEACAM1 or CEACAM1-expressing fibroblasts, we found that perturbation of cell-surface CEACAM1 resulted in a strong and sustained costimulation of BCR-induced cell proliferation. Simultaneous antibody treatment of CEACAM1 and cross-linking of the BCR also induced lymphocyte function-associated antigen-1 (LFA-1)-mediated homotypic cell adhesion and enhanced Ig secretion. Thus, CEACAM1 is a potent regulator of BCR-mediated signaling in B cells.
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L-subunit-specific mAb was purchased from the American Type Culture Collection (Manassas, VA). Anti-IgM mAb Ak13 [33
] was precipitated from hybridoma supernatants by ammonium sulfate (50% saturated) and coupled to cyanogen bromide-activated Sepharose beads (Pharmacia-Upjohn, Uppsala, Sweden) according to the manufacturer’s instructions. Coupling density was 2 mg mAb per 1 ml packed beads. In costimulation assays, beads were used at a concentration of 0.5% (bead vol/vol). A hybridoma-secreting anti-CEACAM1 (clone AgB10 [34
]) was a kind gift from Dr. T. D. Rudinskaya (Cancer Research Center, Moscow, Russia). Dr. Rolf Kemler (Max Plank Institute, Freiburg, Germany) kindly provided the hybridoma decompacting mAb (DECMA)-1, secreting isotype-matched, E-cadherin-specific IgG. Antibodies were purified from hybridoma supernatants by affinity chromatography on fast-flow protein G Sepharose (Amersham-Pharmacia Biotech, Uppsala, Sweden). AgB10 and DECMA-1 were used at 100 µg/ml in cell-stimulation experiments. Endotoxin levels of antibody preparations were determined using the Endosafe Gel-Clot assay (Charles River, Sulzfeld, Germany) and were found to be below detection limits, i.e., <0.03 endotoxin U/ml. Antibodies specific for CD45R/B220, CD3, CD11c, CD138 (syndecan-1), as well as isotype-matched control antibodies were obtained from PharMingen (San Diego, CA). Goat anti-mouse IgG3-fluorescein isothiocyanate (FITC), goat anti-mouse IgG2b-FITC, and goat anti-mouse IgG1-FITC were obtained from Southern Biotechnologies (Birmingham, AL).
Cells
CBA/JxC57BL/6 (F1) mice were obtained from Charles River (Uppsala, Sweden). In all experiments, male mice of 4–6 weeks of age were used. B cells were prepared from splenocyte suspensions after removal of T cells and Percoll gradient centrifugation as described previously [35
]. Routinely, cells prepared by this procedure consist of 80–93% B220-positive cells (a B cell marker), 0.1–5% CD3-positive cells (a T cell marker), and 2–8% Mac-1-positive cells (a granulocyte/macrophage/monocyte marker). The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 at a concentration of 0.5 x 106 cells/ml in RPMI-1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 IU/ml penicillin, 50 µg/ml streptomycin (all from Life Technologies, Paisley, Scotland), 5 µM 2-mercaptoethanol, and 10% of batch-selected fetal calf serum (Life Technologies). NIH-3T3 cells transfected with mouse CEACAM1-S have been described previously [36
]. Untransfected and CEACAM1-S-transfected NIH-3T3 cells were fixed in 95% ethanol, frozen at –20°C, transferred to RPMI-1640 medium, and used in B cell proliferation experiments.
Immunofluorescence
The surface expression of CEACAM1, CD3, CD11c, CD11b, CD19, B220, and CD138 (syndecan-1) was determined as follows: Cells were stained with primary antibody (20 µg/ml) in Earle’s balanced salt solution (EBSS; Life Technologies) for 1 h on ice, followed by washing in ice-cold EBSS and incubation with FITC-(Fab)2 mouse anti-rat IgG (Jackson Immunoresearch, West Grove, PA) at a dilution of 1/40. For intracellular staining, cells were fixed in 4% phosphate-buffered paraformaldehyde, washed in EBSS, permeabilized in 0.1% Saponin (Sigma-Aldrich), and incubated with FITC-coupled, primary antibodies in Saponin/EBSS for 1 h, followed by washing in Saponin/EBSS and EBSS. Labeled samples were analyzed by cytofluorimetry using a FACSCalibur (BD Biosciences, Belgium) instrument and CellQuest software.
Cell proliferation assays
Cell proliferation was determined as described previously [37
]. The activated B cells were pulsed with [3H]-thymidine (Amersham-Pharmacia Biotech; 2 µCi/ml final concentration) for time periods as specified in the figure legends. Plates from kinetic experiments were collected and kept at –20°C and were then processed as a set. Incorporated radioactivity was measured using a Wallac microplate scintillation counter (Wallac Oy, Turku, Finland).
Cell aggregation assay
Activated B cells were gently washed and resuspended to single cells, as confirmed by counting in a hemocytometer. Sepharose beads were removed by gravity sedimentation for 5 min. The cell concentration was then adjusted to 1 x 106 cells/ml, and the suspensions were distributed into 5 ml Falcon tubes at 200 µl/tube. Samples were incubated at 37°C, 5% CO2 on a rotary shaker (100 rpm) for 30 or 60 min in the presence or absence of antibodies against LFA-1 (20 µg/ml). The percentage of cells in aggregates of two or more cells was determined in a hemocytometer by counting at least 200 cells in triplicate. Viable cells were discriminated by trypan blue exclusion.
Determination of secreted Ig
Ig secretion was determined in a sandwich enzyme-linked immunosorbent assay. Polystyrene plates (Immobilon, Nunc, Denmark) were coated overnight at 4°C with goat anti-mouse serum preabsorbed with rat, bovine, and human antigens (Jackson Immunoresearch). The plates were then blocked with 2% bovine serum albumin (Boehringer, Germany) in phosphate-buffered saline (PBS) for 2 h and were incubated with cell-culture supernatants in various dilutions for 1 h, followed by washing and incubation with horseradish peroxidase (HRP)-coupled goat anti-mouse Ig (Jackson Immunoresearch). Thereafter, 150 µl substrate buffer was added. The substrate buffer contained 150 µl 4% o-phenylendiamine (in methanol) and 6 µl 30% H2O2 in 15 ml phosphate/citrate buffer (2.19 g Na2HPO4, 1.47 g sodium citrate in 300 ml H2O, pH 5). The plates were incubated at room temperature for 10–15 min, and then the reaction was stopped by addition of 1 M H2SO4 (50 µl/well). Colored reaction products were quantified at 450 nm in a Termomax reader using the software SoftMax Pro (Molecular Devices, Sunnyvale, CA).
Reverse transcriptase (RT)-triple primer (tp)-polymerase chain reaction (PCR) for murine CEACAM1
Total RNA was isolated from stimulated and nonstimulated murine B cells by guanidinium thiocyanate extraction, using the Quiagen RNAeasy minikit (Quiagen, Valencia, CA). Samples with 3 µg RNA in a final volume of 20 µl were reverse-transcribed by Moloney murine leukemia virus RT (MBI Fermentas, Vilnius, Lithuania), according to the manufacturer’s recommendation, using a 15-mer oligonucleotide primer (RT-BP 46: 5'-ACA GTG TAT GCG ACG-3'), which specifically hybridizes to the 3' region of murine CEACAM1. The cDNA amplification was performed in a final volume of 50 µl containing 5 µl first-strand cDNA solution, 0.2 mM deoxynucleotide triphosphates, 5 units Taq DNA polymerase (Amersham-Pharmacia-Upjohn), 5 µl 10x PCR buffer, and 0.6 µM each PCR primer. A tp-PCR assay, described previously and evaluated [13
], was used. It was designed to quantify the ratios of mouse CEACAM1-L and CEACAM1-S in the following way: A forward primer that recognized both CEACAM1 splice variants equally well (FP46, 5'-GCC ATG CAG CCT CTA ACC CAC C-3') and two backward primers, which were specific for the two spliced isoforms, were used. The backward primer for the L isoform (BP43, 5'-CTG GAG GTT GAG GGT TTG TGC TC-3') recognized the alternatively spliced exon 7, present only in CEACAM1-L, and the backward primer for the S isoform (BP44, 5'-TCA GAA GGA GCC AGA CCC GCC-3') was constructed to anneal across the splice junction between exon 6 and exon 8. The PCR was initiated by heating the samples to 94°C for 60 s, followed by 30 cycles at 94°C for 45 s, 64°C for 45 s, 72°C for 60 s, and an extension at 72°C for 10 min. The PCR products were analyzed on 2.7% agarose gels in Tris-borate EDTA buffer and visualized by ethidium bromide staining. The ratios between CEACAM1-L and CEACAM1-S PCR products were quantified in a Fujifilm detector system.
Determination of activated mitogen-activated protein (MAP) kinases
B cells (2x107) in a final volume of 100 µl were stimulated for 5 min as specified below. Following centrifugation for 30 s, the cell pellets were immediately lysed in 50 µl 2x sodium dodecyl sulfate (SDS) sample buffer (250 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DL-dithiothreitol, and 0.01% bromophenol blue), sonicated for 30 s, and boiled for 5 min. Equal volumes of each sample were electrophoresed on 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). After blocking with 5% skimmed milk powder in Tris-buffered saline (TBS) for 60 min at room temperature, the membranes were incubated with antibodies specific for the phosphorylated forms of stress-activated protein kinase (SAPK)/c-jun NH2-terminal kinase (JNK) or extracellular-regulated kinase (ERK)1/2 or p38, according to the manufacturer’s protocol (New England Biolabs, Beverly, MA). After washing twice with TBS containing 0.05% Tween 20, the membranes were incubated with HRP-coupled goat anti-rabbit antibodies (New England Biolabs), developed by enhanced chemiluminescence and documented using a Fujifilm detector system.
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Figure 1. Surface expression of CEACAM1 on freshly isolated and activated B cells. B lymphocytes were activated as indicated and stained with anti-CEACAM1 (20 µg/ml) and secondary antibodies and analyzed by cytofluorimetry as described in Materials and Methods. Dead cells were discriminated by staining with propidium iodide (PI) and excluded from the analysis. The results are representative of three independent experiments. The numbers correspond to the mean fluorescence intensity of the indicated gates. Solid curve, Fluorescence intensity with primary anti-CEACAM1 antibodies; stippled curve, fluorescence intensity without primary antibodies.
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1:1 was found in nonactivated cells or in cells stimulated by IL-4, antibodies to CEACAM1, or anti-IgM Sepharose, respectively. In contrast, the CEACAM1 L:S ratio was close to 3:1 in B cells activated by LPS or by anti-IgM Sepharose in combination with IL-4 or with antibodies against CEACAM1 (Fig. 2)
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Figure 2. Effect of different stimulations on the CEACAM1-L:S expression ratio. B cells were cultured with PBS (non activated), LPS, IL-4, IL-4 plus anti ()-IgM Sepharose, anti-CEACAM1, anti-CEACAM1 plus anti-IgM Sepharose, and the isotype-control antibody DECMA-1 plus anti-IgM Sepharose for 2 days. Total RNA was isolated and analyzed by RT-tp-PCR. The results are representative of three independent experiments.
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Figure 3. Stimulation of B cell proliferation by anti-CEACAM1 antibodies. (A) B lymphocytes were cultured for 48 h in plain medium or in medium containing LPS, IL-4, anti-IgM Sepharose, or anti-IgM Sepharose plus IL-4 in the absence (open bars) or presence (solid bars) of antibodies against CEACAM1 (100 µg/ml). After 48 h, the cells were pulsed with [3H]-thymidine for 16 h, and thymidine incorporation was measured by liquid scintillation as described in Materials and Methods. (B) B lymphocytes were cultured for various times and pulsed with [3H]-thymidine for the last 6 h. Thymidine incorporation was determined as in A. The cells were incubated in plain medium or in medium containing LPS, anti-CEACAM1, anti-IgM Sepharose plus IL-4, anti-IgM Sepharose plus DECMA-1, or anti-IgM Sepharose plus anti-CEACAM1. The results are representative of three independent experiments.
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Increasing concentrations of anti-CEACAM1 antibodies together with anti-IgM Sepharose caused a proportional increase in the stimulation of DNA synthesis. When similar experiments were performed in the presence of soluble anti-IgM instead of anti-IgM Sepharose, there was no stimulation of the DNA synthesis by anti-CEACAM1 antibodies. Furthermore, Sepharose beads carrying anti-CEACAM1 and -IgM antibodies did not stimulate the DNA synthesis differently than anti-IgM beads alone. Thus, costimulation of B cell–DNA synthesis seemed to require induced clustering of the B cell receptor complex but not of the surface-exposed CEACAM1.
As anti-CEACAM1 antibodies may not be a physiological stimulus for cell-surface-expressed CEACAM1, we investigated whether homophilic trans-binding to CEACAM1 presented by a contacting cell would have similar effects on B cell proliferation. To that end, we substituted the anti-CEACAM1 antibodies for fixed and killed CEACAM1-expressing NIH-3T3 cells in B cell [3H]-thymidine incorporation experiments. As demonstrated in Figure 4 , CEACAM1-expressing NIH-3T3 cells, but not untransfected NIH-3T3 cells, strongly stimulated DNA synthesis in the presence of anti-IgM Sepharose beads. Without anti-IgM beads, the CEACAM1-expressing fibroblasts had no effect. Thus, CEACAM1–CEACAM1 homophilic binding is a strong, coregulatory stimulus for B cells.
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Figure 4. Stimulation of B cell proliferation by CEACAM1-expressing cells. B lymphocytes were cultured for 48 h in plain medium or medium containing anti-IgM Sepharose, together with various numbers of fixed and killed CEACAM1-S-expressing NIH-3T3 fibroblasts (•) or untransfected NIH-3T3 fibroblasts ( ), respectively. After 48 h, the cells were pulsed with [3H]-thymidine for 16 h, and thymidine incorporation was measured by liquid scintillation as described in Materials and Methods. ( ) B cells incubated with CEACAM1-expressing NIH-3T3 cells in the absence of anti-IgM Sepharose; ( ) B cells incubated with untransfected NIH-3T3 cells in the absence of anti-IgM Sepharose; ( ) B cells incubated in the absence of NIH-3T3 cells or anti-IgM Sepharose. The presented data are the mean ± SD of triplicate incubations.
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Figure 5. Surface expression of lineage-specific markers on isolated splenic B cells activated by anti-IgM Sepharose plus anti-CEACAM1 or by LPS for 4 days. Cells analyzed for syndecan-1 were cultured for 5 days. Staining and analysis by cytofluorimetry for the indicated surface antigens were performed as described in Materials and Methods. Dead cells were discriminated by staining with PI and excluded from the analysis. The results are representative of three independent experiments. Solid curve, fluorescence intensity with the indicated, primary antibodies; stippled curve, fluorescence intensity with a nonspecific, control antibody.
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Activation by anti-IgM Sepharose plus anti-CEACAM1 leads to increased cell adhesion
As CEACAM1 is an adhesion molecule, it was of interest to analyze the adhesive properties of the primary B lymphocytes. Formation of homotypic aggregates in cell culture is a common feature of activated polyclonal B cells, and stimulation with LPS, anti-CD40, or anti-IgM in the presence of IL-4 leads to increased adhesiveness. As previously reported [40
], the adhesive mechanisms include up-regulation of the ß2 integrin, LFA-1-mediated interactions. Anti-IgM alone did not stimulate proliferation and concomitant aggregation. However, increased aggregation was observed in cultures activated by anti-IgM Sepharose plus anti-CEACAM1 (data not shown). Thus, it was important to investigate whether this aggregation involved LFA-1-dependent adhesion mechanisms or was mediated by other adhesive systems. To address LFA-1-dependent adhesion, cell aggregates were disrupted, and cells were reaggregated in the presence or absence of antibodies against LFA-1 as described in Materials and Methods. Reaggregated cells were analyzed after 30 and 60 min of incubation and were compared with LPS-activated B cells treated in a similar manner (Fig. 6
). We found no significant difference in the levels of reaggregation when cells were activated by LPS or by anti-IgM Sepharose plus anti-CEACAM1. Reaggregation was strongly inhibited by antibodies against LFA-1 (Fig. 6)
, whereas antibodies against CEACAM1 had no effect (data not shown). This does not rule out a role for CEACAM1 as an adhesion molecule in B cells. However, the low levels of remaining LFA-1-independent adhesion indicate that it would account for only a minor part of the observed cell aggregation. We conclude that B cell aggregation induced by anti-IgM Sepharose plus anti-CEACAM1 primarily is a result of activation of LFA-1.
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Figure 6. Reaggregation of B lymphocytes. Aggregation of B lymphocytes activated by anti-IgM Sepharose plus anti-CEACAM1 was determined as described in Materials and Methods in the presence (hatched bars) or absence (solid bars) of antibodies against LFA-1. The levels of aggregation were compared with those observed in cultures of B lymphocytes activated by LPS in the presence (open bars) or absence (vertical-striped bars) of antibodies to LFA-1. There were no cells aggregated at time 0. The results are representative of two experiments.
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Next, we investigated whether there was any production of IgG1, IgG2b, or IgG3, which would indicate that Ig class recombination had occurred. Whereas stimulation with LPS or LPS plus IL-4 resulted in significant, intracellular staining for these Ig (data not shown), we did not detect intracellular staining for any of these Ig classes after stimulation by anti-IgM Sepharose plus anti-CEACAM1. Thus, we conclude that costimulation by anti-CEACAM1 antibodies did not result in Ig class-switching.
Anti-CEACAM1 activates the SAPK/JNK pathway
Activation of MAP kinase pathways is crucial in cell proliferation. To test whether the costimulatory effect of CEACAM1 may work through MAP kinase activation, we analyzed antibody-stimulated B cells with antibodies specific for the activated, phosphorylated forms of ERK1/2, SAPK/JNK, and p38, respectively. As demonstrated in Figure 7
, ERK1/2 was active to a similar extent in cells that were incubated with anti-CEACAM1, anti-IgM Sepharose, anti-CEACAM1 plus anti-IgM Sepharose, isotype-control IgG, or isotype-control IgG plus anti-IgM Sepharose. No activation of p38 was observed under any of these conditions. However, anti-CEACAM1 and anti-CEACAM1 plus anti-IgM Sepharose but not anti-IgM Sepharose alone specifically activated SAPK/JNK. The specific JNK activation was confirmed by a kinase assay (data not shown).
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Figure 7. Activation of signal-transduction pathways by CEACAM1. B cells were incubated for 5 min at room temperature with the indicated agents. Aliquots of whole-cell lysates were subjected to SDS-PAGE, and activation of endogenous SAPK/JNK, ERK1/2, and p38 was monitored by immunoblotting with activation-specific antiphospho-SAPK/JNK, antiphospho-ERK1/2, or antiphospho-p38 antibodies, respectively. Equal protein loading was confirmed by subsequent amido black staining of the membranes. Similar results were obtained in three experiments. IgM, IgM-Sepharose; Isotype control, DECMA-1.
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Adhesion and adhesion-mediated signaling play central roles in the biology and activation of lymphocytes. Increased homotypic adhesion after polyclonal activation of B lymphocytes in vitro has been observed in many situations and may reflect what is going on in germinal centers that are compact structures of lymphoid tissue highly enriched in B cells. Different adhesion molecules may participate in homotypic B cell interactions, but the adhesion pair LFA-1/intercellular adhesion molecule-1 plays a major function [38 , 41 ]. The present data show that CEACAM1 now can be added to the components that activate LFA-1-dependent adhesion. That CEACAM1 itself did not contribute significantly to the homotypic adhesion under these conditions can be explained by the fact that the adhesive properties of CEACAM1 are under cellular control, via regulation of the CEACAM1 monomer–dimer equilibrium [42 ]. A similar situation has previously been reported in neutrophilic granulocytes, where CEACAM1-mediated activation also induced LFA-1-dependent adhesion [43 ]. An interesting, functional consequence of up-regulated LFA-1-dependent adhesion in B lymphocytes is that it can regulate cell survival [44 ] and Ig secretion [40 ].
Production and secretion of Igs are the prime functions of mature B lymphocytes. Although entering antigen-induced proliferation, B lymphocytes also undergo Ig class-switching if they encounter proper stimuli. However, costimulation via CEACAM1 only led to increased Ig secretion and did not trigger class-switching. This was in agreement with the finding that costimulation by anti-CEACAM1 did not lead to differentiation of the B cells into typical plasma cells, as judged by the lack of induction of syndecan-1. Thus, CEACAM1 seems to affect early but not late events in B cell differentiation.
Activation of B cells leading to proliferation and differentiation is a multistep process, which includes clustering of the BCR and other receptors at the cell surface, signal transduction, and cytoskeletal rearrangements. Although cross-linking of the BCR complex promotes signaling events, it is a weak mitogenic stimulus when triggered alone. However, simultaneous signals from other receptors, e.g., CD19/CD21, the IL-4 receptor, or CD40, generate strong, proliferative responses [2 , 45 ]. It is interesting that the costimulatory effect on proliferation mediated by CEACAM1 was much more pronounced, peaked later, and lasted longer than the responses induced by costimulation with IL-4 or by LPS, which is a commonly used mitogen for B cells.
The details of the molecular mechanisms, whereby coactivation of CEACAM1 induces cell proliferation, are unknown. However, from other cellular systems, it is known that a crucial part of CEACAM1-mediated signal transduction is tyrosine phosphorylation of the cytoplasmic domain of CEACAM1-L, which then can recruit and activate src-family kinases and SH2 domain-containing, cytoplasmic protein tyrosine phosphatases [28 29 30 ]. However, the links from the CEACAM1-mediated activation of tyrosine kinases/phosphatases to downstream signaling pathways are not known. It is also not known how the balance between tyrosine kinase and tyrosine phosphatase activation is regulated by CEACAM1. An interesting observation that we have made previously is, however, that there is a correlation between the proliferative status of epithelial cells and the expression ratio of the CEACAM1 cytoplasmic domain isoforms, and we have suggested that the balance of activation of the tyrosine kinases and phosphatases may be regulated by the CEACAM1 isoform-expression ratio [16 ]. An increased ratio of CEACAM1-L-to-CEACAM1-S correlated with cell proliferation, whereas a decreased ratio was seen in quiescent cells [16 ]. It was therefore of great interest to see that mitogenic stimulation of the splenic B cells, including that triggered by anti-IgM Sepharose plus anti-CEACAM1, altered the CEACAM1-L:CEACAM1-S expression ratio from 1:1 to close to 3:1. To our knowledge, these results represent the first demonstration that a similar transition in the expression ratio of CEACAM1-L-to-CEACAM1-S, as observed in proliferating epithelial cell lines, also occurs in nontransformed, primary cells that enter proliferation.
In previous work, we have found that CEACAM1-induced signaling regulates the MAP kinases 1,2 pathway in granulocytes [13 ] and in epithelial cells (Inka Scheffrahn, B. Singer, B. Öbrink, unpublished results). However, in the splenic B cells, ERK1/2 was already constitutively active under the experimental conditions used. Instead, stimulation by anti-CEACAM1 alone and by anti-IgM Sepharose plus anti-CEACAM1 activated the MAP kinase JNK, whereas the activity of the MAP kinase p38 was unaffected. Thus, the JNK pathway might be engaged in the enhanced coactivation of the BCR signaling by CEACAM1, but its activation is not sufficient, as a similar activation of JNK was observed by anti-CEACAM1 alone. Therefore, CEACAM1 costimulation of the BCR response must also induce other, presently unknown, cellular events.
One challenge in understanding CEACAM1 signaling is that positive and negative effects have been observed. Thus, stimulation and inhibition of T cell activation [22 23 24 25 ] and of epithelial cell proliferation [31 ] have been observed when cell surface-expressed CEACAM1 has been perturbed by antibodies. It is not known which criteria determine if CEACAM1 will have a positive or a negative effect on cell signaling, but we have suggested that crucial factors are the CEACAM1-L-to-CEACAM1-S ratios, CEACAM1 dimer interactions, calmodulin binding, and serine/threonine phosphorylation of the cytoplasmic domains of CEACAM1 [11 , 16 , 31 , 42 ]. As already mentioned, anti-CEACAM1 antibodies are not the most likely agents that regulate CEACAM1 signaling activity in vivo. As no other physiological ligand for the extracellular domain of CEACAM1 than CEACAM1 itself has been identified so far, homophilic CEACAM1 trans-binding might be the dominant, extracellular, regulatory event. CEACAM1 is expressed on epithelial and endothelial cells as well as on platelets, granulocytes, macrophages, DCs, T cells, and B cells, and accordingly, there are plenty of opportunities for trans-homophilic interactions to occur in a B cell regulatory scenario.
Another question related to CEACAM1-positive and -negative effects on cell signaling is how CEACAM1 influences the activity of the BCR complex. The effects of anti-CEACAM1 antibodies or CEACAM1-expressing cells do not tell if CEACAM stimulates or inhibits the BCR. If CEACAM1 is constitutively associated with the BCR, anti-CEACAM1 antibodies or homophilic CEACAM1–CEACAM1 binding might dissociate it from the BCR and release a putative, inhibitory effect, thereby activating the BCR. If, conversely, antibodies or homophilic binding stimulate the activity of CEACAM1, this might lead to a signal that works in synergy with the BCR signal. Further work is needed to answer these questions.
Received December 6, 2002; accepted March 24, 2003.
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 M. Pantelic, Y.-J. Kim, S. Bolland, I. Chen, J. Shively, and T. Chen Neisseria gonorrhoeae Kills Carcinoembryonic Antigen-Related Cellular Adhesion Molecule 1 (CD66a)-Expressing Human B Cells and Inhibits Antibody Production Infect. Immun., July 1, 2005; 73(7): 4171 - 4179. [Abstract] [Full Text] [PDF]
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 M. M. Muller, B. B. Singer, E. Klaile, B. Obrink, and L. Lucka Transmembrane CEACAM1 affects integrin-dependent signaling and regulates extracellular matrix protein-specific morphology and migration of endothelial cells Blood, May 15, 2005; 105(10): 3925 - 3934. [Abstract] [Full Text] [PDF]
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 E. Hemmila, C. Turbide, M. Olson, S. Jothy, K. V. Holmes, and N. Beauchemin Ceacam1a-/- Mice Are Completely Resistant to Infection by Murine Coronavirus Mouse Hepatitis Virus A59 J. Virol., September 15, 2004; 78(18): 10156 - 10165. [Abstract] [Full Text] [PDF]
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C. Ramakrishna, C. C. Bergmann, K. V. Holmes, and S. A. Stohlman Expression of the Mouse Hepatitis Virus Receptor by Central Nervous System Microglia J. Virol., July 15, 2004; 78(14): 7828 - 7832. [Abstract] [Full Text] [PDF]
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R. Mansson, P. Tsapogas, M. Akerlund, A. Lagergren, R. Gisler, and M. Sigvardsson Pearson Correlation Analysis of Microarray Data Allows for the Identification of Genetic Targets for Early B-cell Factor J. Biol. Chem., April 23, 2004; 279(17): 17905 - 17913. [Abstract] [Full Text] [PDF]
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 U. Sundberg, N. Beauchemin, and B. Obrink The cytoplasmic domain of CEACAM1-L controls its lateral localization and the organization of desmosomes in polarized epithelial cells J. Cell Sci., March 1, 2004; 117(7): 1091 - 1104. [Abstract] [Full Text] [PDF]
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