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*
Gastroenterology Division, Departments of Medicine and
Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; and
Department of Microbiology, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The biliary glycoproteins (CD66a), or CEACAM1, of human, rat, and mouse are widely expressed and exhibit significantly different patterns of splicing. CEACAM1 is expressed by a wide variety of epithelial cell types (8), endothelial cells (9), and myeloid hemopoietic cells, including neutrophils and monocytes (10). The single human CEACAM1 gene generates 11 alternative splice products, eight of which encode transmembrane proteins. All these transmembrane forms contain an IgV-like N domain, followed by no IgC2-like domains (-1L and -1S forms), two IgC2-like domains called A1 and B (-3L and -3S forms), or three IgC2-like domains called A1, B, and A2 (-4L and -4S forms) (3). In addition, alternative splicing generates transmembrane forms with three extracellular domains in which the A2 domain is replaced by a serine-threonine rich, non-Ig-like, Alu sequence (A1, B, A1u; -3AL and -3AS forms). Each of these transmembrane isoforms generated by alternative splicing contains a long (73 aa) or a short (10 aa) cytoplasmic (cyt) tail. Differential splicing of the human CEACAM1 gene also generates three soluble forms that contain the N domain plus variable IgC2-like domains (the -3, -4C1, and -4C2 forms). The mouse Ceacam1 and -2 genes encode mouse CEACAM (mCEACAM) glycoproteins that exhibit high homology to the human homolog, including alternating splicing, which generates a limited number of transmembrane forms (3). The mouse Ceacam1 gene also exhibits alternative splicing, generating four different murine CEACAM1 glycoproteins that are composed of either four (-4L and -4S forms) or two (-2L and -2S forms) extracellular Ig domains containing either a long or a short cyt tail. Additionally, for the mouse, but not human, Ceacam1 gene, there are two allelic variants of the IgV-like N domain called CEACAM1a and CEACAM1b. Most inbred mouse strains, except SJL/J, express the CEACAM1a allele (3).
A major proven function of CEACAM1 is intercellular adhesion. Homophilic adhesion of the CEACAM1 has been linked to development of the intestine, placenta, muscle, tooth, and vascular systems and to the regulation of cell growth (11, 12, 13, 14). Heterophilic adhesion of CEACAM1 has also been observed with other CEACAM members (2), E-selectin through expression of the sialyl-Lewisx Ag by CEACAM1 (15), and cell surface structures of bacteria (including Escherichia coli, Salmonella, Hemophilus influenza, and Neisseria sp.) (7, 16, 17) and the murine coronavirus, murine hepatitis virus (5, 6). Such heterophilic interactions may be involved in regulating microbial colonization of the gut, leukocyte trafficking, bacterial phagocytosis, and the pathogenesis of coronavirus infections.
These regulatory functions associated with intercellular adhesion are highly associated with the ability of the long CEACAM1-associated cyt tail to deliver intracellular signals. The long cyt tail of CEACAM1 of mouse and rat, for example, has been shown to bind calmodulin and src-related tyrosine kinases (18, 19, 20). The latter leads to tyrosine phosphorylation of the two immunoreceptor tyrosine-based inhibition motifs (ITIM) contained within the long cyt tail of CEACAM1 and the binding to src homology 2 domain-containing protein tyrosine phosphatases (SHPs), such as SHP-1 and SHP-2 in epithelial cells (18, 19). Such attributes of CEACAM1 have been associated with inhibition of tumor cell growth (21, 22, 23). Whereas CEACAM5 (formerly called CEA) has been shown to be up-regulated in a wide variety of human epithelial tumors, CEACAM1 expression is commonly diminished (22, 24). As a corollary, transfection of CEACAM1 into tumors of the colon, breast, prostate, endometrium, and lung leads to inhibition of their growth, presumably through homophilic CEACAM1 interactions and recruitment of SHP-1 (23).
CEACAM1 has recently been reported on human B and T lymphocytes and NK
cells (25, 26, 27). Although CEACAM1 expression on B cells was
observed to be constitutive (25), the expression on T
cells was inducible and required prior activation (26, 27). Moller and colleagues (26) first reported that
CEACAM1, but not other CEACAM members, is expressed on a subset of
activated peripheral blood NK cells that are
CD16-CD56+ and on
conventional T cells using a panel of mAbs. Subsequently, the same
group reported confirmatory data (27) and showed that
CEACAM1 was expressed on activated CD4+ and
CD8+ T cells expressing either 
or 
TCR. Additionally, it was shown that immobilized and soluble
anti-CEACAM1 mAbs enhanced and inhibited the proliferation of human
T cells induced by anti-CD3 mAbs, respectively (27).
Although the expression of CEACAM1 by activated human T cells was
recently confirmed by two additional groups in mucosal and peripheral
tissues (28, 29), both a negative (28) and a
positive (29) costimulatory role was ascribed to the cell
surface expression of this molecule in human T cells as defined in
vitro, respectively. Such inconsistencies may relate to the complex
splicing patterns attributable to CEACAM1 (3) and the
methodologic approaches used, among other possibilities.
The in vivo function of CEACAM1 in T cell-dependent models has not, however, yet been examined in rodents that express highly related homologs (3). In fact, previous studies have concluded that mouse T cells are CEACAM1a negative (30, 31). Given the significant homologies that exist between human and mCEACAM1, we have re-examined the expression and function of mCEACAM1a on T lymphocytes to determine the immunoregulatory function of this molecule in this species, if any. In this report we show that cell surface expression of mCEACAM1a can be detected after T cell activation, including ligation of the TCR/CD3 complex. Moreover, we provide evidence that mCEACAM1a is present in resting T cells and is rapidly mobilized to the cell surface after T cell activation. In association with this, we observed that the nonphosphorylated and phosphorylated long cyt tails of mCEACAM1a differentially associate with adaptor protein 1 and SHP-1, respectively. Finally, mCEACAM1a ligation with an anti-CEACAM1a-specific mAb was associated with inhibition of T cell activation in vitro and in vivo as defined in an oxazolone-mediated, delayed-type hypersensitivity (DTH) model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse mAb specific for mCEACAM1a (CC1; mouse IgG1) and polyclonal rabbit anti-mCEACAM1a Ab have been previously described (5, 6, 32, 33). Normal rabbit and mouse control Abs, anti-mouse IgG1-FITC, anti-CD3-PE, anti-CD3-PerCP, anti-CD3, anti-CD28, anti-CD4-PE, anti-CD8-PE, anti-CD19-FITC, anti-CD16/32, and anti-CD69-biotin were purchased from BD PharMingen (San Diego, CA). Anti-SHP-1 Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell preparations
Spleen mononuclear cells were prepared from 6-wk-old male BALB/c or C57BL/6 mice (Charles River Breeding Laboratories, Wilmington, MA) and activated with either Con A (2.5 µg/ml) or plate-bound anti-CD3 mAb by standard methods. To purify T cells from spleen, negative selection was performed using Dynabeads (Dynal Biotech, Lake Success, NY) containing CD19 and CD11b mAbs, resulting in preparations that contained <2% B cell contamination based upon FACS analysis of anti-CD19-stained samples. A mouse T cell lymphoma line, 4A2 (ATCC T1B-154), and a mouse B cell line, SP20 (ATCC CRL-1581), were purchased from American Type Culture Collection (Manassas, VA).
FACS analysis
FACS was performed using a FACSort (BD Biosciences, San Diego,
CA) after staining by standard methods (34). Briefly, the
cell suspensions were incubated with an anti-FcR Ab
(anti-CD16/32, 2.4G2) to prevent nonspecific binding by the
secondary Ab for 1 h and were washed twice in suspension medium
(SM; PBS without calcium, but with 1% FBS). The cell suspension was
incubated with the primary Ab for 1 h at a concentration of 1–10
µg/ml, washed twice with SM, and labeled with secondary Ab, followed
by two washes with SM before suspension in PBS containing 0.5%
paraformaldehyde. FACS was then performed. Control staining with an
irrelevant isotype-matched Ab was performed for every FACS analysis. To
identify mCEACAM1 on splenic T cells by two- and three-color staining,
the cell suspension was stained with the
mCEACAM1a mAb, CC1, as the primary Ab, followed
by an anti-mouse IgG1-FITC conjugate as the secondary Ab and
staining with an anti-CD3-PE conjugate and/or a biotin-labeled
anti-CD69, -CD4, or -CD8 mAb, with development of the latter with
tricolor-conjugated avidin (BD PharMingen).
Inhibitor treatment
Before activating the spleen cells and assessment by FACS, the cell suspension was treated with actinomycin D, cycloheximide, or brefeldin A for 30 min at maximum concentrations of 200 µM, 10 ng/ml, and 20 µM, respectively.
GST long cyt tail fusion protein and GST pull-down
mRNA were prepared by poly(A) selection, and cDNA were prepared from murine spleen cells by standard methods (35); the long cyt tail of mCEACAM1a (aa 420–492) was cloned by RT-PCR using forward (5'-GCGGATCCTATTTCCTCTATTCCAGGAAG-3') and reverse (5'-CCGGAATTCTCACTTCTTTTTTACTTCTGA-3') primers incorporating BamHI and EcoRI restriction sites, respectively, and subcloned into the pGEX-4T vector (Amersham Pharmacia Biotech, Piscataway, NJ). TheGST-mCEACAM1cyt fusion protein was synthesized and purified using the GST fusion protein system (Amersham Pharmacia Biotech) and modified to contain phosphorylated tyrosine residues using the TK-Phos System (Stratagene, La Jolla, CA). Lysates were prepared by solubilizing cells in immunoprecipitation buffer containing 100 mM NaCl, 10 mM Tris (pH 7.8), 1 mM EDTA, protease inhibitors, and 1% Nonidet P-40 as previously described (36, 37), and GST pull-downs were performed with the fusion proteins followed by Western blotting with an Ab specific for SHP-1 by standard methods (36, 37). mRNA and cDNA were also used to clone the cDNA encoding AP47 (µ1 chain of adaptor protein 1) using forward(5'-CCAAGCTTATCTCCGCCAGCGCCGTCTAC-3') and reverse (5'-TACTCGAGTCACTGGGTCCGGAGCTGATAATC-3') primers incorporating HindIII and XhoI sites, respectively, and subcloned into the pCDNA3.1 expression vector (Invitrogen, Carlsbad, CA). In vitro transcription and translation were performed using the TNT kit (Promega, Madison, WI) in the presence of [35S]cysteine/methionine (Amersham Pharmacia Biotech) (36). In vitro synthesized AP47 was pulled down with either the tyrosine-phosphorylated or the nonphosphorylated GST-CEACAM1cyt fusion protein, the immunoprecipitates were resolved by SDS-PAGE under reducing conditions, and autoradiography was performed.
MLR and cell proliferation assay
Spleen cells from C57BL/6 were used as the responder cells, and spleen cells from BALB/c, which were treated with mitomycin C (100 µg/ml; Sigma-Aldrich, St. Louis, MO) for 90 min, followed by extensive washing with complete medium, were used as the stimulator cells. The responder cells and stimulator cells were mixed in equal ratios in a total volume of 200 µl at a cell concentration of 1 x 106 cells/ml and cultivated in 96-well, round-bottom microtiter plates for 3 days at 37°C in 5% CO2. Subsequently, [3H]thymidine (Amersham Pharmacia Biotech) was added 5 h before harvesting (38). Incorporation of [3H]thymidine was assessed by scintillation using a 96-well plate reader (Packard Instrument, Meriden, CT). For cell proliferation assays, plate-bound anti-CD3- and anti-CD28-specific Abs were prepared according to a standard protocol, described previously (38), in either the presence or the absence of varying concentrations of the anti-mCEACAM1a mAb CC1. Freshly prepared mouse T cells from spleen were incubated in 96-well, flat-bottom microtiter plates for 3 days at 37°C in 5% CO2. Incorporation of [3H]thymidine was assessed by scintillation as described above.
Immunohistochemical staining
Cytospins of purified mouse T cells were prepared with a cytocentrifuge (Cytofuge2; StatSpin, Norwood, MA) onto silane-coated glass slides and air-dried, followed by fixation and permeabilization in acetone for 30 s. After dehydration with alcohol, staining was performed with a polyclonal rabbit anti-mCEACAM1a Ab or control rabbit serum using the avidin biotin complex kit (Vector Laboratories, Burlingame, CA) for visualization of the primary Ab.
In vivo assessment of DTH response
C57BL/6 mice were injected i.p. with either the CC1 mAb (1 mg in PBS) or the control IgG1 mAb. Twenty-four hours after mAb treatment mice were presensitized by painting 150 µl of the haptenating agent, 3% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; Sigma-Aldrich), a classic low m.w. chemical hapten, dissolved in 100% ethanol onto a shaved abdomen (39, 40). Five days after presensitization, 1% oxazolone in 20 µl of 100% ethanol or ethanol alone as a control was painted on the right and left ears, respectively, in either the presence or the absence of a second i.p. injection of the CC1 or control IgG1 mAbs administered 24 h before ear challenge. Ear swelling was measured before and 24 h after the ear challenge with a dial thickness gauge (Mitutoyo, Kanagawa, Japan). DTH responses were expressed as the increase in ear swelling after oxazolone painting on the ear following subtraction of the thickness before the challenge for the control (IgG1 mAb) and anti-CEACAM1a (CC1 mAb)-treated groups. A fragment (5 mm2) of the center portion of the ear from six mice in each group was assessed after paraffin embedding by standard H&E staining, and three sections from each tissue block were examined.
Statistics
Data were expressed as the mean ± SEM and were analyzed using a two-tailed Student’s t test for independent samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Confirming previous observations, mCEACAM1a
could not be detected on resting splenic T cells using the CC1 mAb
(30). However, when mouse splenocytes from C57BL/6 animals
were activated with Con A, mCEACAM1a was rapidly
up-regulated on the cell surface of CD3+ cells,
as defined by two-color FACS. mCEACAM1a
expression was detected by 0.5 h after T cell activation, with
expression peaking at 12–24 h, by which time all T cells exhibited
mCEACAM1a expression on the cell surface (Fig. 1
A) with persistence for up to
72 h (data not shown). This was in contrast to CTLA-4, which was
up-regulated after 72 h as previously described by others
(41). mCEACAM1a could be detected on
large (blast-like) CD3+ cells as assessed by
forward scattering analysis and overlapped nearly completely with the
expression of CD69, which was observed early after T cell activation
(data not shown). Identical observations were made after anti-CD3
stimulation of purified T cells from spleen and were confirmed with a
second polyclonal rabbit anti-mCEACAM1a Ab
(data not shown). mCEACAM1a could be detected on
the cell surface of both CD4+ and
CD8+ T cells as defined by three-color FACS with
anti-mCEACAM1a, anti-CD3, and either
anti-CD4 or anti-CD8 mAbs (Fig. 1, B and
C). By 2 h of activation with Con A, 48–53% of T
cells, as defined by anti-CD3 staining, expressed
CEACAM1a (Fig. 1B). Of these, 36% of
the CD4+ T cells (Fig. 1B) and 37% of
the CD8+ T cells (Fig. 1B) expressed
mCEACAM1a, suggesting that
mCEACAM1a was regulated similarly in both T cell
subsets. These results were confirmed by examining
mCEACAM1a expression on the
CD4+ and CD8+ subsets after
gating on the CD3+ cells which showed
mCEACAMa expression on blastic CD4+ and
CD8+ cells (Fig 1C and data not shown). These
studies indicate that murine CEACAM1a, like human
CEACAM1 (26, 27, 28, 29), is expressed on the cell surface of
activated, but not resting, CD4+ and
CD8+ T cells.
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We subsequently observed that actinomycin D, an inhibitor of
DNA-dependent RNA synthesis, was unable to inhibit the cell surface
expression of mCEACAM1a on T cells activated by
Con A (Fig. 2A,
,
although it could inhibit the expression of CD69 (Fig. 2A, 
) in a
dose-dependent manner. Identical findings were made with cycloheximide,
an inhibitor of protein synthesis, and after direct anti-CD3
stimulation (data not shown). These results indicate that the
expression of mCEACAM1a on T cells requires
neither de novo transcription nor translation, suggesting that
mCEACAM1a is presynthesized in resting T cells.
Consistent with this, mCEACAM1a could be detected
in lysates of splenic T cells by Western blot analysis. Fig. 2B shows that when protein lysates of total splenocytes and
purified T cells were subjected to Western blot analysis with a rabbit
anti-mCEACAM1a polyclonal Ab, two bands with
molecular masses of 
120 and 65 kDa, consistent with the
four-extracellular domain (Fig. 2B, 
) and
two-extracellular domain (Fig. 2B, 
) isoforms of
mCEACAM1a, respectively, could be detected. As a
confirmation of this result, cytospins prepared from purified spleen T
cells were permeabilized with acetone and stained with a rabbit
anti-mCEACAM1a-specific Ab. As shown in Fig. 2C, a punctate, reddish-brown staining pattern was
detectable with the specific polyclonal Ab (Fig. 2B,
left panel), but not the isotype-matched control Ab (Fig. 2B, right panel), in virtually all cells
examined. Although the cellular location of
mCEACAM1a remains to be defined, interestingly,
we observed that the activation-induced expression of
mCEACAM1a on the cell surface was not affected by
brefeldin A treatment, which blocksprotein trafficking in the
biosynthetic pathway (data not shown). These results strongly support
the hypothesis that mCEACAM1a is presynthesized
in resting T cells, which has not previously been appreciated in human
T cells (26, 27, 28, 29), and either rapidly exported to and/or
stabilized upon the cell surface after activation.
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The factors that regulate the function and cellular sorting
behavior of mCEACAM1a in T cells are unknown.
Previous studies in non-T cells have shown that the long cyt tail of
mCEACAM1a can associate with SHP-1 and SHP-2
through binding to the two ITIMs present in the cyt tail when
phosphorylated, an event that has been linked to the inhibitory
functions of this molecule (2, 18, 19, 23, 42). Moreover,
our studies described above, which show that
mCEACAM1a is present in resting T cells but is
only detected on the cell surface after activation, suggest that the
sorting of mCEACAM1a is an actively regulated
process. These observations caused us to examine whether the long cyt
tail of mCEACAM1a could associate with SHP-1 in T
cells and bind the adaptor protein 1 complex that regulates the
cellular sorting of transmembrane proteins between the
trans-Golgi network (TGN) and the endolysosomal system
(43). Therefore, we used a
GST-CEACAM1a-cyt tail fusion protein to show that
the phosphorylated, but not the nonphosphorylated, cyt tail could pull
down SHP-1 from protein lysates derived from splenic T cells, a mouse T
cell lymphoma line (4A2), and a mouse B cell line (SP20; Fig. 3A, arrowhead); these results
were consistent with previous studies in mouse epithelial cells
(18, 19).
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, 1, µ1 (AP47), and 
1
chains that link transmembrane proteins to clathrin. This is effected
through association between tyrosine-based motifs within the cyt tail
of transmembrane proteins and the µ1 (AP47) chain of adaptor protein
1 and between clathrin and the 1 subunit of adaptor protein 1
(43). Binding of the µ1 chain of adaptor protein 1 to
this tyrosine-based motif, which consists of YXX
(tyrosine-amino
acid-amino acid-amino acid containing a bulky hydrophobic side chain),
regulates protein transport between the TGN and the endolysosomal
system. The YXX motif only binds adaptor protein 1 in the
nonphosphorylated state (43) and is contained in the long
cyt tail of mCEACAM1a. We thus examined the
association between the GST-mCEACAM1a-cyt tail
fusion protein and the µ1 (AP47) chain generated by in vitro
translation in the presence of
[35S]cysteine-methionine from the respective
gene that we cloned using specific primers and RT-PCR amplification. As
shown in Fig. 3
B, we observed that the nonphosphorylated,
but not the phosphorylated, GST-mCEACAM1a-cyt
tail fusion protein was capable of associating with AP47. This
association between the nonphosphorylated cyt tail of
mCEACAM1a and adaptor protein 1 has not been
previously reported. Taken together, these results suggest that
phosphorylation and dephosphorylation of the
mCEACAM1a cyt tail in T cells may regulate its
reciprocal association with SHP-1 and adaptor protein 1 and its
consequent cellular localization and function. Ligation of mCEACAM1a regulates T cell function in vitro and in vivo
The physiologic role of mCEACAM1a in the
immune response has yet to be elucidated. The association between
mCEACAM1a and SHP-1 in T cells established above
and the presence of two ITIMs within the cyt tail suggested a potential
immunoregulatory role. Therefore, we examined the effects of the murine
CEACAM1a-specific mAb, CC1, in the context of T
cell activation. mAb CC1, but not control IgG1, significantly
suppressed the allo-MLR between C57BL/6 and BALB/C spleen cells (Fig. 4A). These results were not
due to a toxic effect of the CC1 mAb based upon both trypan blue
exclusion and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide assays (data not shown). We further examined the response of
mouse spleen T cells to plate-bound anti-CD3 and anti-CD28 mAbs
in the presence or the absence of soluble
anti-mCEACAM1a mAb, CC1. As shown in Fig. 4B, left panel, cross-linking of
mCEACAM1a with the CC1 mAb significantly
inhibited the proliferation of splenic T cells induced by the
anti-CD3 and anti-CD28 mAbs in a concentration-dependent manner
compared to the isotype-matched Ab control (Fig. 4B,
right panel). Either increased mAb CC1 or decreased
anti-CD3 and CD28 mAbs resulted in increased inhibition.
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A, the CC1 mAb-treated mice
exhibited a 64.3% decrease in ear swelling compared to that in mice
treated with the control mAb (p = 0.017) when
the anti-CEACAM1a-specific mAb was
administered both before initial oxazolone sensitization and at the
time of the secondary ear challenge exposure. However, blockade of the
DTH response required that the anti-CEACAM1a
mAb be present at the time of oxazolone-specific T cell priming,
because administration of the anti-CEACAM1a
mAb at this time resulted in a 65.3% decrease in ear swelling
(p = 0.0079; Fig. 5B). However,
administration of the CC1 mAb at the time of the secondary ear
challenge exposure alone was unable to significantly inhibit the DTH
response (Fig. 5B). Although administration of the CC1 mAb
at this latter time point resulted in a 30.6% inhibition of ear
swelling, this did not reach statistical significance
(p = 0.1829), indicating that the effect was
marginal. The inhibition of skin swelling observed in association with
the DTH response was shown to be due to a dramatic diminution in the
lymphoid infiltrates observed within the dermis and thickness of the
epithelial layer imposed by the CC1 (Fig. 5C, right
panel), but not control (Fig. 5C, left
panel), mAb. Anti-CD3 immunostaining of the tissue sections
revealed that the lymphoid subset affected by the CC1 mAb was indeed
derived from the T cell compartment (data not shown). Therefore, our
studies support an important role for mCEACAM1a
as an activation-induced, cell surface molecule involved in regulating
Ag-driven T cell responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the current report we now extend these observations to mouse T cells and provide information on the novel behavior of mCEACAM1a in this cellular population. In contrast to epithelial cells and other cell types, which constitutively express mCEACAM1a on the cell surface (1, 2, 7, 8, 9, 10), mCEACAM1a expression on the cell surface of mouse T cells is revealed only upon activation, as has been shown with human T cells (26, 27, 28, 29). Furthermore, our studies suggest that although mCEACAM1a cannot be detected on the cell surface of resting T cells, it is already present in this cell type before activation. Upon activation, we have observed that mCEACAM1a is rapidly detected on the cell surface, and its expression on the cell surface requires neither de novo transcription nor translation. These studies suggest three possible scenarios. First, it cannot be excluded that the epitope recognized on mCEACAM1a by the CC1 mAb is exposed only after T cell activation, although the mCEACAM1a molecule is present on the cell surface of resting T cells. Against this possibility is the fact that we observed activation-induced expression of mCEACAM1a with two different Abs. Second, mCEACAM1a is preformed and retained in an as yet to be defined intracellular compartment and is released from this compartment for transport to the cell surface upon activation. Alternatively, mCEACAM1a is normally directed to the cell surface but is rapidly internalized due to recycling from this cellular location and is only stabilized upon the cell surface as a consequence of activation. Our observation that mCEACAM1a can associate with adaptor protein 1, as shown in this study, is consistent with either scenario. CTLA-4, for example, which is retained intracellularly before T cell activation, associates with adaptor protein 1 and adaptor protein 2 and is inhibited from this association by tyrosine phosphorylation (45, 46). Therefore, it is possible that interactions between the nonphosphorylated cyt tail of mCEACAM1a and the µ1 (AP47) chain of adaptor protein 1 directs mCEACAM1a to an as yet to be defined intracellular location where it is retained. Based upon our observations that brefeldin A does not inhibit T cell activation-induced cell surface expression of mCEACAM1a, this putative compartment would be predicted to be beyond the TGN, consistent with the known function of adaptor protein 1, which is to direct transmembrane proteins from the TGN to the endolysosomal system (43). T cell activation may recruit src-related tyrosine kinases that are known to associate with and phosphorylate the long cyt tail of CEACAM1a (47). In this case CEACAM1a would be forced to release from adaptor protein 1 binding and potentially be transported to the cell surface. Phosphorylation of the mCEACAM1a cyt tail would, on the other hand, allow for an association with SHP-1 and/or SHP-2, as we have shown in this study and has been previously shown in epithelial cells (18, 19). Such an association is likely to occur on the cell surface (48) wherein the inhibitory functions of mCEACAM1a isoforms containing the long cyt tail could be asserted. As noted above, an alternative and related scenario is the possibility that mCEACAM1a is rapidly recycling between the cell surface and endosomes independent of TCR/CD3 complex signaling through reciprocal interactions between adaptor protein 2 and cellular phosphatases such as SHP-1 and the nonphosphorylated and phosphorylated cyt tails, respectively. In this case T cell activation would be predicted to serve in the stabilization of mCEACAM1a on the cell surface. These hypotheses are not mutually exclusive; they need to be subjects of future investigation and obviously do not apply to mCEACAM1a isoforms that express the short cyt tail.
The function of CEACAM1 on T cells remains controversial from studies in human T lymphocytes that have ascribed both stimulatory and inhibitory properties to this molecule (27, 28, 29). In this report we have shown that an anti-CEACAM1a-specific mAb, CC1, inhibits the activation of mouse T cells in vitro. Whether these results are due to blockade of a costimulatory signal provided by mCEACAM1a or stimulation of an inhibitory signal delivered by mCEACAM1a cannot be concluded. In addition, it must be noted that signaling through Ab-mediated ligation is not physiological such that firm conclusions about the function of mCEACAM1a on T cells must await studies with native ligands. In the allo-MLR, it is possible that the CC1 mAb is blocking an activation signal provided by interactions between mCEACAM1a on the activated T cell and the counterligand, presumably mCEACAM1a (49, 50), on the APC. Alternatively, cross-linking of mCEACAM1a by the CC1 mAb may directly stimulate an inhibitory pathway, as suggested by our results showing that CC1 mAb inhibits the response of purified spleen T cells to anti-CD3- and anti-CD28-stimulated proliferation. Regardless, these observations are likely to be biologically relevant, because we observed that the mCEACAM1a-specific mAb, CC1, inhibited a DTH response to oxazolone in vivo. Interestingly, and potentially consistent with the fact that mCEACAM1a was observed to be an early activation Ag, the inhibition of the DTH response was observed only during the phase of T cell priming and not the effector phase in vivo as shown in this work. Arguably, these results are more consistent with blockade of a crucial positive signal delivered by an APC such as a dendritic cell (49) to an mCEACAM1a-bearing T cell rather than to either direct stimulation of an inhibitory signal by the mAb on an effector T cell or blockade of T cell recruitment into the affected tissues through interactions between T cells and endothelial cells (9). Future studies must be aimed at defining the specific mechanism by which mCEACAM1a is capable of regulating T cell responses in vivo.
In summary, in contrast to previous studies that have concluded that mouse T cells do not express mCEACAM1a, we have observed that mouse T cells are capable of expressing mCEACAM1a on the cell surface under conditions of T cell activation. Cell surface expression of mCEACAM1a is likely to be physiologically important in regulating T cell responses during the early phases of T cell activation in vivo. These studies thus mark mCEACAM1a as an important immunoregulatory molecule on mouse T cells.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard S. Blumberg, Gastroenterology Division, Department of Medicine, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. E-mail address: rblumberg{at}partners.org
3 Abbreviations used in this paper: CEA, carcinoembryonic Ag; CEACAM, CEA-related cell adhesion molecule; cyt, cytoplasmic; DTH, delayed-type hypersensitivity; ITIM, immunoreceptor tyrosine-based inhibition motif; mCEACAM, mouse CEACAM; PSG, pregnancy-specific glycoprotein; SHP, src homology 2 domain-containing protein tyrosine phosphatase; SM, suspension medium; TGN, trans-Golgi network.
Received for publication September 11, 2001. Accepted for publication November 20, 2001.
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References |
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-glycosides of mannose. Infect. Immun. 59:2051.2-microglobulin. J. Biol. Chem. 14:9289.
/CD3 complex, but not CD28, interact with clathrin adaptor complexes AP-1 and AP-2. J. Immunol. 163:1868.This article has been cited by other articles:
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