| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
* Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden;
Tumor Immunology Laboratory, Department of Urology, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany; and
Institute for Molecular Immunology, GSF National Research Center for the Environment and Health, Munich, Germany
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CEACAM1, also known as CD66a, is an important multipotent signaling molecule (4) that regulates a variety of cellular activities such as cell proliferation (7), tumor growth (8, 9), apoptosis (10), angiogenesis (11), T cell cytotoxicity (12), dendritic cell function (13), granulocyte activation (14), and epithelial cell polarization (10). It has been demonstrated that CEACAM1 can act as a homophilic cell-cell adhesion molecule, binding to itself (4). The homophilic interaction represents the only species-independent physiological binding activity of the extracellular domain that has been unambiguously demonstrated thus far. However, CEACAM1 also functions as a microbial receptor. Thus, in human tissues CEACAM1 together with CEACAM3, CEA (CEACAM5), and CEACAM6 serves as the receptor for Opa protein-expressing gonococci and meningococci (15), and murine CEACAM1 is the receptor for mouse hepatitis virus (16). None of these ligands, or any monoclonal or polyclonal Abs analyzed thus far, cross-reacts with CEACAM1 from other species. In both rodents and humans, CEACAM1 is subject to differential splicing. The two major splice isoforms have four extracellular Ig domains but differ in their cytoplasmic domain and are denoted CEACAM1-L and CEACAM1-S, respectively, in which L denotes long and S denotes short (3, 4). The L cytoplasmic domain, which consists of 71- to 73-aa residues, have 2 phosphorylatable tyrosine residues that can recruit and activate SH2 domain-containing tyrosine kinases (17) and tyrosine phosphatases (18). The interactions with the tyrosine kinases and tyrosine phosphatases are believed to be important for the signaling activities of CEACAM1. The S cytoplasmic domain is 10–12 aa long and lacks phosphorylatable tyrosine residues. Soluble isoforms of CEACAM1 have also been found. Thus, PC12 cells express and secrete a fully glycosylated 4-Ig domain CEACAM1 lacking the transmembrane portion, due to an outsplicing of exons 6 and 7 (19).
In many CEACAM1-expressing cell types, which have been analyzed in depth, it has been found that CEACAM1-L and CEACAM1-S are coexpressed, although at varying ratios (7, 20). It has also been demonstrated that both the L and the S isoform can form dimers (21), which is believed to play an important role in the signal-regulating activities of CEACAM1 (4, 19). However, it is not known whether all CEACAM1-expressing cells express both the L and the S isoforms simultaneously, and under all functional states. With this in mind and because of the lack of expression of GPI-linked CEA family proteins in rats and mice, we initiated a comparative investigation of the expression of the L and S isoforms of CEACAM1 in leukocytes isolated from peripheral blood of rats, mice, and humans. Here we present data that show significant and striking differences in the CEACAM1 expression patterns in rodent and human leukocytes. Whereas rodent leukocytes express both the L and the S isoforms of CEACAM1, human leukocytes express only CEACAM1–4L and CEACAM1–3L, i.e., the long cytoplasmic domain isoforms having four or three extracellular Ig domains, respectively. The human leukocytes also express GPI-linked CEACAM8 and/or CEACAM6, which suggests that the GPI-linked CEA-related molecules may have functionally replaced the short cytoplasmic domains, CEACAM1-S, in these cells. Furthermore, we demonstrate that CEACAM1 and CEACAM8 are physically associated in human granulocytes and that CEACAM1 and CEACAM8 regulate the activation of extracellular signal-regulated kinases (Erk) in rodent and human granulocytes, respectively.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A hybridoma secreting the rat anti-mouse CEACAM1 mAb AgB10
was obtained from Drs. N. I. Kuprina and T. D. Rudinskaya
(Cancer Research Center, Moscow, Russia), and the mouse anti-mouse
CEACAM1 mAb CC1 was kindly provided by Dr. K. Holmes (University of
Colorado, Denver, CO). The hybridoma secreting the mouse anti-rat
CEACAM1 mAb 5.4 was generously provided by Dr. D. Hixson (Rhode Island
Hospital, Brown University, Providence, RI). The mouse mAb 5F4 against
human CEACAM1 was a gift from Dr. R. S. Blumberg (Brigham and
Women’s Hospital, Harvard Medical School, Boston, MA). The following
mouse mAbs (Sixth Leukocyte Typing Workshop, Osaka, Japan) were
obtained from the laboratory of Dr. F. Grunert (University of Freiburg,
Freiburg, Germany): 4/3/17 (specific for CEACAM1/CEA); 12/140/4
(specific for CEACAM1/CEA); 9A6 (specific for CEACAM6); 47 (specific
for CEACAM8); BEAR1 (specific for CD11b); and MEM48 (specific for
CD18). The CEACAM8-specific mAb 80H3 was from Coulter
International (Miami, FL). Human leukocytes do not express CEA
(CEACAM5); therefore the Abs 4/3/17 and 12/140/4 will specifically
detect CEACAM1 when leukocytes are analyzed. Abs were purified from
hybridoma supernatants by affinity chromatography on fast flow protein
G-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden). A fraction
of the purified Abs was coupled to biotin using a biotinylation kit
according to the manufacturer’s protocol (Sigma-Aldrich, St. Louis,
MO). FITC-labeled mouse anti-human CD3, rat anti-mouse CD3,
hamster anti-rat CD3, mouse anti-human CD19, rat anti-mouse
CD45R/B220, and hamster anti-rat CD45R/B220 Abs were obtained from
BD PharMingen (San Diego, CA). Streptavidin-PE was obtained from DAKO
(Copenhagen, Denmark). Abs against phosphorylated, activated Erk1/2
were obtained from New England Biolabs (NEB).
[
-32P]dCTP was from Amersham. As cDNA
templates for control PCR, we used pBlueScript plasmids (Stratagene, La
Jolla, CA) containing the complete coding sequences for rat CEACAM1-L
(22), rat CEACAM1-S (22), mouse CEACAM1-L
(23), mouse CEACAM1-S (23), and pcDNA/Neo
plasmids (Invitrogen, San Diego, CA) containing the complete coding
sequences for human CEACAM1–4L (24), human CEACAM1–3L
(24), human CEACAM1–4S (24), and human
CEACAM1–3S (24). The mouse and human CEACAM1 plasmids
were kindly provided by Dr. N. Beauchemin (McGill University, Montreal,
Canada) and by Dr. T. Barnett (Bayer Pharmaceutical Division, Berkeley,
CA), respectively.
Isolation of leukocytes from peripheral blood
Leukocytes were prepared from heparinized (5 U heparin/ml) peripheral blood from healthy donors, from BALB/c mice, and from Lewis rats (obtained from Charles River, Uppsala, Sweden) by sedimentation through Plasmasteril (Fresenius, Bad Homburg, Germany). PBMC and neutrophilic granulocytes (polymorphonuclear neutrophils (PMN)) were separated by centrifugation in Ficoll-Paque (Amersham Pharmacia Biotech). The erythrocytes in the pellet were lysed by repeated suspension in cold 0.2% NaCl for 20 s followed by washing with PBS. More than 95% of the remaining cells were PMN as judged from morphological criteria, with a viability of >97% as measured by trypan blue exclusion. The PBMC recovered from the top of the Ficoll-Paque cushion were separated into lymphocyte subpopulations by single-cell sorting in a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). The cells were labeled with anti-CD3-FITC for T cell sorting and either anti-CD19-FITC (human) or anti-CD45R/B220-FITC (rat and mouse) for B cell sorting. The purity of the isolated lymphocyte T and B subpopulations were 87–96% as determined with the FACSCalibur instrument using CellQuest software (BD Biosciences).
Detection of CEACAM1 by flow cytometry
To analyze the surface expression of CEACAM1 in the different leukocyte subpopulations, freshly isolated cells were stained with biotinylated anti-CEACAM1 Abs (50 µg/ml) in PBS containing 3% FCS for 1 h on ice, followed by washing with ice-cold PBS and incubation with streptavidin-PE at a dilution of 1/40. Background fluorescence was determined using isotype-matched immunoglobulins instead of specific primary Abs. Purified granulocytes were measured directly, whereas a two-color flow cytometric staining of the lymphocytes in the unfractionated PBMCs was performed. After the CEACAM1 staining, the PBMC were incubated with anti-CD3-FITC or anti-CD19-FITC or anti-CD45R/B220-FITC according to the recommendations of the manufacturer. The samples were measured in a FACSCalibur instrument, and the data were analyzed using CellQuest software. Dead cells were identified by propidium iodide staining (Sigma-Aldrich) and were excluded from the determinations.
RT-PCR for different CEACAM molecules
Total RNA was isolated from granulocytes, sorted
CD3+ T cells, and human
CD19+ and rodent
CD45R/B220+ B cells by guanidinium thiocyanate
extraction, using the Qiagen RNAeasy minikit (Qiagen, Valencia, CA).
Samples of RNA in a final volume of 20 µl were reverse transcribed by
Moloney murine leukemia virus reverse transcriptase (MBI Fermentas,
Hanover, MD) according to the manufacturer’s recommendation, using the
following oligonucleotide primers that specifically hybridize to the 3'
regions of the various CEACAMs: rat CEACAM1, 5'-GGCATTGAAGTTCAG-3';
mouse CEACAM1, 5'-ACAGTGTATGCGACG-3'; human CEACAM1,
5'-GTTGTTTCTGTCCC-3'; CEACAM6, 5'-CCAGTGGCTGAGTT-3'; CEACAM8,
CCAGTGGCTGAGTT-3'. The PCR were performed in a total volume of 30 µl
containing 5 µl of first-strand cDNA solution (or plasmid cDNA for
human, mouse, or rat CEACAM1-L or CEACAM1-S), 0.2 mM dNTPs, 3 U of
Taq DNA polymerase (Amersham Pharmacia Biotech), 3 µl of
10 x PCR buffer, and 0.6 µM concentrations of each of the PCR
primers. The reactions were 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, and 72°C for 60 s and an extension at 72°C for
10 min. The products were analyzed on 2.7% agarose gels in
Tris-borate-EDTA buffer and visualized by ethidium bromide staining.
The relative amounts of the PCR products were analyzed by scanning the
gels and determining the intensities in the ethidium bromide-stained
bands with Scan analyses software (Biosoft, Milltown, NJ). The PCRs
were performed with the primer combinations shown in Table I
. For CEACAM6 and CEACAM8
amplifications, a single primer set was used for each CEACAM,
respectively. For CEACAM1 amplifications, a common sense primer that
recognized both CEACAM1 splice variants equally well and two antisense
primers that were specific for the two spliced isoforms were used
(Table I and Fig. 4A). The antisense primers for the L
isoforms recognized the alternatively spliced exon 7 present only in
CEACAM1-L; the antisense primer for the S isoform was constructed to
anneal across the splice junction between exon 6 and exon 8. CEACAM1
PCRs were performed both in a conventional way with one sense and one
antisense primer and as a triple-primer PCR in which one sense primer
was used together with both antisense primers selectively recognizing
the L and the S isoforms, respectively. To test the ability of the
triple-primer PCR to return correct values of the input ratios, some
PCR were run with cDNA plasmids for rat CEACAM1-L and CEACAM1-S as
templates, and with the addition of
[-32P]dCTP in the deoxynucleotide mixture.
The PCR products were quantified by cutting out the ethidium
bromide-stained bands and counting in a 
-scintillation counter or by
analysis of the gels in a PhosphoImager (Molecular Dynamics,
Sunnyvale, CA).
|
|
Human granulocytes and IL-2-stimulated lymphocytes were solubilized, immunoprecipitated with mAb 4/3/17, and analyzed for CEACAM1 expression by Western blotting as described in Ref. 25 . For immunoblot analysis of CEACAM1 in rat and mouse cells, 2 x 106 freshly isolated granulocytes or lymphocytes were solubilized in 100 µl of lysis buffer containing 1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl (pH 7.5), 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml chymostatin, 10 µg/ml pepstatin A, and 1000 kIU/ml Trasylol (aprotinin). After centrifugation for 30 min at 15 000 x g, the supernatants were incubated overnight at 4°C with 10 µg/ml CEACAM1-specific mAbs (mAb 5.4, mAb CC1). BSA-saturated protein G-Sepharose was then used to collect the immune complexes. After thorough washing, the protein G-Sepharose beads were boiled for 5 min in 2x SDS sample buffer (250 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.01% bromphenol blue, 50 mM DL-DTT). The samples were electrophoresed on 8% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking of nonspecific binding with 1% skim milk powder in TBS, the membranes were incubated with primary anti-CEACAM1 Abs (10 µg/ml) and washed twice with TBS containing 0.1% Tween 20 (Merck, Rahway, NJ). HRP-coupled secondary Abs (DAKO) were added, and the filters were developed by ECL and documented using the Fuji gel documentation system.
For determination of activated Erk1/2 kinases, 107 rat or human granulocytes in 100 µl were incubated with mAbs (50 µg/ml in PBS) against CEACAM1 (mAb 5.4 for rat CEACAM1 and mAb 4/3/17 or mAb 5F4 for human CEACAM1), CEACAM6 (mAb 9A6), and CEACAM8 (mAb 80H3), respectively, for 5 min at room temperature. Isotype-matched Igs and PBS were used as controls. After centrifugation, the cell pellets were lysed in 50 µl of ice-cold 2x SDS sample buffer (see above), sonicated for 10 s, and boiled for 5 min. Twenty microliters of each sample were electrophoresed on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore). After blocking with 5% skim milk powder in TBS overnight at 4°C, the membranes were incubated with Abs specific for the phosphorylated form of activated Erk1/2 according to the manufacturer’s protocol (NEB). After two washings with TBS containing 0.05% Tween 20, the membranes were incubated with HRP-coupled goat anti-rabbit Abs (NEB), developed by ECL, and documented by the Fuji gel documentation system. Equal loading of samples in the electrophoresis step was checked by Amido Black staining of the membranes.
Sandwich ELISA
Microtiter plates (Nunc, Wiesbaden, Germany) were coated overnight at 4°C with 100 µl of solutions of various mAbs (10 µg/ml in PBS) of defined specificities. After being washed and blocked with 300 µl of 2% BSA in PBS, lysates of human granulocytes or HeLa-Neo cells (HeLa cells transfected with the neomycin resistance gene, kindly provided by Dr. F. Grunert, Department of Immunology, Freiburg University, Freiburg, Germany) were added, and the plates were incubated for 4 h at 4°C and washed thoroughly. The plates were then incubated with a second set of peroxidase- or biotin-conjugated Abs of defined specificities and were washed again. For detection of biotinylated Abs, the wells were then incubated with streptavidin-peroxidase (Pierce, Rockford, IL) and washed. Finally, peroxidase activity was analyzed using tetramethylbenzidine (Fluka, Buchs, Switzerland) as substrate. The reaction was stopped with 1 M H2SO4, and the OD was measured in an ELISA reader (SLT-Spectra, Salzburg, Austria) at 450 nm.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Pure populations of PMN and PBMC were isolated from healthy human
donors, rats, and mice. Analysis by flow cytometry showed that
granulocytes, CD3+ T cells, human
CD19+ B cells, and rodent
CD45R/B220+ B cells expressed significant amounts
of CEACAM1 on their surfaces (Fig. 1).
The CEACAM1 expression in rat, mouse, and human leukocytes was
confirmed by Western blotting, which showed that granulocytes and
lymphocytes of both mouse and rat origin expressed CEACAM1 molecules
with identical size, corresponding to an apparent molecular mass of 140
kDa (Fig. 2). In contrast, human
granulocytes and lymphocytes expressed CEACAM1 with different apparent
molecular masses, 160 and 140 kDa, respectively, due to differences in
glycosylation (Fig. 2).
|
|
The expression ratios of the two isoforms, CEACAM1-L and
CEACAM1-S, were determined by RT-PCR using primers that could
discriminate between the splice variants giving rise to the long and
short cytoplasmic domains, respectively. The specificity of the primers
was tested using full length cDNA for the L and S isoforms
of CEACAM1 from the respective species (Fig. 3). Complete specificity was shown under
the conditions used for the PCR, yielding products of the expected
nucleotide lengths. Because human cells express splice variants of
CEACAM1 that have either three or four Ig domains, the PCR primers
amplified L and S splice isoforms of both the three Ig domain isoforms
(CEACAM1–3L and CEACAM1–3S), and the four Ig domain isoforms
(CEACAM1–4L and CEACAM1–4S) (Fig. 3A). Mice and rats do
not express the three Ig domain splice variants that lack Ig-domain 4,
and therefore only one band for the L isoform and S isoform,
respectively, was amplified by the PCR primers that were used (Fig. 3, B and C).
|
A). The cDNAs of the two
isoforms accordingly compete for the sense primer, which results in a
ratio of the products that corresponds to the input ratios of the
templates. By using specified ratios of CEACAM1-L and CEACAM1-S cDNA as
templates and radioactive nucleotide precursors, we could demonstrate
that the procedure worked and indeed gave the same ratio of the
products as the ratio of the templates in the input mixture (Fig. 4B). Furthermore, determining the ratio of the products by
quantifying the fluorescence signals of the ethidium bromide-stained
electrophoresis bands gave values almost identical with the ones
determined from the radioactivity of the separated bands. By applying
the triple-primer PCR assay to reverse transcribed RNA preparations, we
found that granulocytes, B cells, and T cells of rats and mice
expressed both the L and the S isoforms of CEACAM1 (Fig. 4C). In all of these cell types the L isoform was expressed
to a higher extent than the S isoform, with a L:S ratio of 2.2–2.9. In
striking contrast, however, granulocytes, B lymphocytes, and T
lymphocytes from human peripheral blood did not express any trace of
the S isoforms as analyzed by RT-triple primer-PCR (Fig. 5A). This was confirmed when
the PCR was repeated with specific primers for either the L or the S
isoforms (Fig. 5B). Thus, human leukocytes only express the
L isoforms (CEACAM1–3L and CEACAM1–4L) of CEACAM1.
|
It is well documented that human granulocytes express two
GPI-linked CEACAM molecules, CEACAM6 and CEACAM8 (14), but
the expression of GPI-linked CEACAM molecules has not been reported in
normal lymphocytes. We therefore analyzed purified populations of human
granulocytes, B lymphocytes, and T lymphocytes for expression of
CEACAM6 and CEACAM8 by RT-PCR. We could confirm the expression of
CEACAM6 and CEACAM8 in granulocytes (Fig. 6). In addition, we found that both B
lymphocytes and T lymphocytes expressed CEACAM6, whereas no signals
were found for CEACAM8 expression in these cells (Fig. 6).
|
CEACAM1 is a signal-regulating cell surface molecule. One of the
pathways that are regulated by CEACAM1 in epithelial cells is the
Erk/MAP kinase pathway (I. Scheffrahn, B. B. Singer, and B.
Öbrink, unpublished observations). We therefore wanted to know
whether the Erk pathway could be influenced by CEACAM molecules also in
leukocytes. To that end, we investigated whether Erk1 and Erk2 could be
activated by CEACAM Abs added to human and rat granulocytes,
respectively. The mAb 5.4 against rat CEACAM1 had a strong activation
effect on Erk1/2 in rat granulocytes (Fig. 7), whereas neither mAb 4/3/17 (Fig. 7)
nor mAb 5F4 (data not shown) against human CEACAM1 activated Erk1/2 in
human granulocytes. Erk1/2 in human granulocytes could, however, be
significantly activated by the mAb 80H3 against CEACAM8 (Fig. 7). The
mAb 9A6 against CEACAM6, in contrast, did not affect Erk1/2 activation
differently than the Abs against CEACAM1 (Fig. 7).
|
The finding that Ab perturbation of the GPI-linked CEACAM8 caused
activation of Erk1/2 prompted us to investigate whether CEACAM8 might
be associated with CEACAM1, because this would offer an explanation for
the signaling activity of CEACAM8. To that end, we designed a sandwich
ELISA that could detect bimolecular complexes of membrane-bound
proteins. In this method, a capturing Ab was used to bind one molecular
species to a microtiter plate. A second Ab with a different specificity
was then used to detect molecules that were physically associated with
the captured protein. Using Abs against different epitopes in the same
molecule, we showed that this sandwich ELISA could capture and detect
both CEACAM1 and CEACAM8 (Table II). Its
ability to detect bimolecular complexes was demonstrated for the
2 integrin CD11b/CD18, where anti-CD11b
was used as capturing Ab, and anti-CD18 was used as detecting Ab
(Table II). The specificity in detecting bimolecular complexes was
demonstrated using anti-CD11b as capturing Ab and anti-CEACAM8
as detecting Ab, which did not give any signal either in granulocytes
or in HeLa-Neo cells. We could then unambiguously demonstrate that
CEACAM8 solubilized from human granulocytes was physically associated
with CEACAM1, using anti-CEACAM1 as capturing Ab and
anti-CEACAM8 as detecting Ab (Table II).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CEACAM1-L has signaling or signal-regulating abilities due to the presence of phosphorylatable tyrosine residues in immunoreceptor tyrosine-based inhibitory motif sequences (4) and immunoreceptor tyrosine-based inhibitory motif-related switch sequences (28), which can recruit and activate src family kinases or SH2 domain-containing protein tyrosine phosphatases. CEACAM1-S lacks these tyrosine-containing sequences, but it can regulate the signaling activities of CEACAM1-L, probably via dimer formation with the L isoform (4, 19). This raises the question whether CEACAM1-mediated signaling or signal regulation is different in human vs rodent leukocytes. In this context, it is interesting that human cells, in contrast to rodent cells, express several CEACAM proteins that are associated with the cell surfaces via GPI anchors. No satisfactory explanation to the functions of these GPI-linked CEACAM molecular species have been given so far. However, it has been reported that both CEACAM6 and CEACAM8 can form complexes with CEACAM1 in human granulocytes (29), and we demonstrated unambiguously that CEACAM8 is physically associated with CEACAM1 in these cells. Thus, it seems plausible that these GPI-linked CEACAM proteins have functionally replaced the short cytoplasmic domain isoform CEACAM1-S in human leukocytes.
The replacement hypothesis is supported by our demonstration that the activation of the Erk/MAP kinase pathway could be regulated by CEACAM proteins in granulocytes. In rat granulocytes, Erk1/2 was strongly activated by an Ab directed against the N-terminal Ig-domain of CEACAM1, which confirmed the signal-regulating activity of CEACAM1 in this cell type. However, neither of two different mAbs against the N-terminal Ig-domain of human CEACAM1 activated Erk1/2 in human granulocytes. This discrepancy might be due to recognition of different epitopes in human and rat CEACAM1 by these Abs but could also be due to participation of CEACAM1-L in different types of complexes in rat and human granulocytes. The latter possibility is supported by our finding that Abs against CEACAM8 strongly activated Erk1/2 in human granulocytes. GPI-linked cell surface proteins do not have any cytoplasmic domains and can therefore trigger transmembrane signals only by complexing with other signaling proteins. Our finding that CEACAM8 occurs in a complex with CEACAM1 suggests that the signal triggered by perturbation of CEACAM8 is mediated by CEACAM1-L. Complexes between CEACAM1-L and CEACAM8 may therefore play a similar role in transmembrane signaling in human granulocytes as complexes between CEACAM1-L and CEACAM1-S in rat or mouse granulocytes. Interestingly, Abs against CEACAM6 did not cause any significant activation of Erk1/2, although both CEACAM6 and CEACAM8 can complex with CEACAM1 in human granulocytes (28). CEACAM1/CEACAM6 and CEACAM1/CEACAM8 complexes in human granulocytes therefore seem to have different functions. This suggests that human granulocytes have a more varied repertoire in terms of signal regulation than rodent granulocytes, which lack the GPI-linked CEACAM-molecules.
The activation of Erk1/2 by CEACAM Abs clearly shows that these cell surface molecules can regulate signaling activities in both rodent and human granulocytes. However, it seems unlikely that anti-CEACAM Abs are the physiological triggering substances of such signaling. Several different CEACAM molecules can act as homophilic cell adhesion molecules (4), and therefore it is possible that CEACAM-mediated signaling activities under physiological and pathological conditions are triggered by leukocyte adhesive events. This might either be adhesion between different cells or CEACAM-mediated binding of microbes to the cell surface. However, unactivated leukocytes are nonadhesive cells that require some kind of activation to become adhesive. This has clearly been demonstrated for cell surface-exposed integrins that are inactive in unactivated leukocytes and platelets but become adhesive when the cells are exposed to various activating agents (30). A similar situation seems to prevail for the CEACAM proteins. It is a challenge for future work to unravel the mechanisms of CEACAM activation and the molecular events that trigger CEACAM-mediated cell signaling.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Björn Öbrink, Department of Cell and Molecular Biology, Box 285, Karolinska Institutet, SE-17177 Stockholm, Sweden. E-mail address: bjorn.obrink{at}cmb.ki.se
3 Abbreviations used in this paper: IgSF, Ig superfamily; CEA, carcinoembryonic Ag; CEACAM, CEA-related cell adhesion molecule; PMN, polymorphonuclear neutrophil; Erk, extracellular signal-regulated kinase; MAP, mitogen-activated protein kinase; NEB, New England Biolabs; FP, forward primer; BP, backward primer.
Received for publication December 14, 2001. Accepted for publication March 5, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B. J. Biol. Chem. 276:24331.This article has been cited by other articles:
|
|
 |
|
  E. Klaile, O. Vorontsova, K. Sigmundsson, M. M. Muller, B. B. Singer, L.-G. Ofverstedt, S. Svensson, U. Skoglund, and B. Obrink The CEACAM1 N-terminal Ig domain mediates cis- and trans-binding and is essential for allosteric rearrangements of CEACAM1 microclusters J. Cell Biol., November 16, 2009; 187(4): 553 - 567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Muller, E. Klaile, O. Vorontsova, B. B. Singer, and B. Obrink Homophilic adhesion and CEACAM1-S regulate dimerization of CEACAM1-L and recruitment of SHP-2 and c-Src J. Cell Biol., November 16, 2009; 187(4): 569 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. J. Riley, K. P. Engelhardt, J. W. Saldanha, W. Qi, L. S. Cooke, Y. Zhu, S. T. Narayan, K. Shakalya, K. D. Croce, I. G. Georgiev, et al. Design and Activity of a Murine and Humanized Anti-CEACAM6 Single-Chain Variable Fragment in the Treatment of Pancreatic Cancer Cancer Res., March 1, 2009; 69(5): 1933 - 1940. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Semiramoth, A. Gleizes, I. Turbica, C. Sandre, R. Gorges, I. Kansau, A. Servin, and S. Chollet-Martin Escherichia coli type 1 pili trigger late IL-8 production by neutrophil-like differentiated PLB-985 cells through a Src family kinase- and MAPK-dependent mechanism J. Leukoc. Biol., February 1, 2009; 85(2): 310 - 321. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Chen, L. Chen, S.-W. Qiao, T. Nagaishi, and R. S. Blumberg Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 Inhibits Proximal TCR Signaling by Targeting ZAP-70 J. Immunol., May 1, 2008; 180(9): 6085 - 6093. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Yu, E. M. C. Chow, H. Wong, J. Gu, O. Mandelboim, S. D. Gray-Owen, and M. A. Ostrowski CEACAM1 (CD66a) Promotes Human Monocyte Survival via a Phosphatidylinositol 3-Kinase- and AKT-dependent Pathway J. Biol. Chem., December 22, 2006; 281(51): 39179 - 39193. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Zhang, O. Schwartz, M. Pantelic, G. Li, Q. Knazze, C. Nobile, M. Radovich, J. He, S.-C. Hong, J. Klena, et al. DC-SIGN (CD209) recognition of Neisseria gonorrhoeae is circumvented by lipooligosaccharide variation J. Leukoc. Biol., April 1, 2006; 79(4): 731 - 738. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Klaile, M. M. Muller, C. Kannicht, B. B. Singer, and L. Lucka CEACAM1 functionally interacts with filamin A and exerts a dual role in the regulation of cell migration J. Cell Sci., December 1, 2005; 118(23): 5513 - 5524. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 A. L. Servin Pathogenesis of Afa/Dr Diffusely Adhering Escherichia coli Clin. Microbiol. Rev., April 1, 2005; 18(2): 264 - 292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Lucka, M. Fernando, D. Grunow, C. Kannicht, A. K. Horst, P. Nollau, and C. Wagener Identification of Lewis x structures of the cell adhesion molecule CEACAM1 from human granulocytes Glycobiology, January 1, 2005; 15(1): 87 - 100. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. B. Thorp and T. M. Gallagher Requirements for CEACAMs and Cholesterol during Murine Coronavirus Cell Entry J. Virol., March 15, 2004; 78(6): 2682 - 2692. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Chen, H. Iijima, T. Nagaishi, A. Nakajima, S. Russell, R. Raychowdhury, V. Morales, C. E. Rudd, N. Utku, and R. S. Blumberg Carcinoembryonic Antigen-Related Cellular Adhesion Molecule 1 Isoforms Alternatively Inhibit and Costimulate Human T Cell Function J. Immunol., March 15, 2004; 172(6): 3535 - 3543. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 H. Iijima, M. F. Neurath, T. Nagaishi, J. N. Glickman, E. E. Nieuwenhuis, A. Nakajima, D. Chen, I. J. Fuss, N. Utku, D. N. Lewicki, et al. Specific Regulation of T Helper Cell 1-mediated Murine Colitis by CEACAM1 J. Exp. Med., February 17, 2004; 199(4): 471 - 482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Greicius, E. Severinson, N. Beauchemin, B. Obrink, and B. B. Singer CEACAM1 is a potent regulator of B cell receptor complex-induced activation J. Leukoc. Biol., July 1, 2003; 74(1): 126 - 134. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
400 bp is supercoiled CEACAM1–4L
that on reamplification with the same primer set gave a band of 506 bp.
B, Regular RT-PCR of RNA isolated from human
granulocytes. The cDNA was amplified by PCR with either FP49 and BP60
(L) or FP49 and BP59 (S) as primers. The latter primer combination was
also used with CEACAM1–4S as template, which demonstrated that this
primer combination indeed could amplify a product of 466 bp. Only
products corresponding to the L isoforms, but no products corresponding
to the S isoforms, were amplified from the granulocyte RNA.
