By
From the Department of Molecular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
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Abstract |
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Histamine is considered one of the important mediators of immediate hypersensitivity and inflammation, and acts via G protein-coupled receptors. Here, we report that histamine may affect antigen receptor-mediated immune responses of T and B cells via a signal(s) from histamine H1 receptors (H1Rs). Histamine exhibited enhancing effects on the in vitro proliferative
responses of anti-CD3- or anti-IgM-stimulated spleen T and B cells, respectively, at the culture condition that the fetal calf serum was dialyzed before culture and c-kit-positive cells were
depleted from the spleen cells. In studies of histamine H1R knockout mice, H1R-deficient T
cells had low proliferative responses to anti-CD3
cross-linking or antigen stimulation in vitro.
B cells from H1R-deficient mice were also affected, demonstrating low proliferative responses
to B cell receptor cross-linking. Antibody production against trinitrophenyl-Ficoll was reduced in H1R-deficient mice. Other aspects of T and B cell function were normal in the H1R
knockout mice. H1R-deficient T and B cells showed normal responses upon stimulation with
interleukin (IL)-2, IL-4, CD40 ligand, CD40 ligand plus IL-4, and lipopolysaccharide. Collectively, these results imply that the signal generated by histamine through H1R augments antigen receptor-mediated immune responses, suggesting cross-talk between G protein-coupled
receptors and antigen receptor-mediated signaling.
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Introduction |
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Guanine-nucleotide binding (G)1 protein-coupled receptors (GPCRs) link to downstream signaling pathways through activation of heterotrimeric G proteins,
which are composed of three subunits, termed ,
, and
(1, 2) in an inactive state. Upon ligand/agonist binding,
GPCRs stimulate the
subunit of heterotrimeric G protein to release GDP and to bind GTP in its place. In the
GTP-bound form, a G
dissociates from a G
dimer,
each of which independently binds and activates target effectors. The
subunits that bind and hydrolyze GTP are
classified into four subfamilies based on sequence homology and shared effector molecules: G
s, G
i, G
q, and
G
12 (1). Intracellular signaling pathways regulated by GPCRs include the cAMP/protein kinase A pathway, the
phosphatidyl inositol/calcium/protein kinase pathway mediated by phospholipase C
(PLC
) and the mitogen-activated protein kinase (MAPK) pathway (4). Recent studies
have provided strong evidence that in some cell types activation of MAPK pathway by GPCR is tyrosine kinase dependent (5). The genetic and biochemical analysis clearly provides evidence that protein tyrosine kinase cascade bridges G protein and MAPK pathways in mammalian cells (9).
The G
subunits that regulate activity of PLC
belong to
the Gq class (G
q, G
11, G
14, G
15/16; references 10,
11). Tyrosine phosphorylation of the G
q/11 subunit by
protein tyrosine kinases (PTKs) contributes to GPCR-
mediated activation of Gq/11 (12, 13) followed by hydrolysis of phosphatidylinositol phosphates and production of inositol-1,4,5-triphosphate (IP3) and diacylglycerol. Tyrosine-phosphorylated G
q/11 has been shown to be more
active in stimulating PLC
in vitro (12).
Protein tyrosine phosphorylation is an important event
in the initiation of cellular responses triggered by antigen
receptors on both B and T cells (14). One of the initial
intracellular signaling events after cross-linking of the B cell
antigen-receptors (BCR) with antigens is the activation of
non-receptor type PTKs, such as Src family kinases (Lyn,
Blk, Fyn), Syk kinase, and Bruton's tyrosine kinase (Btk)
(15). Activation of the PTKs of the Src family such as Lck
and Fyn, followed by the activation of ZAP-70 kinase, has
been implicated in an initial step of TCR signal transduction (14). These protein tyrosine kinases rapidly phosphorylate a number of intracellular substrates and activate
signaling cascades that include activation of phospholipase
C-1 and 2 (PLC
1,
2), phosphatidylinositol 3 kinase
(PI3 kinase), and the Ras-MAPK pathway, which transmits further biochemical events that eventually regulate cell
cycle and gene expression.
It has been shown that GTP exchange within Gq/11
and physical association of G
q/11 with CD3
are induced
upon cross-linking of the TCR by anti-CD3
antibody
(17). Moreover, it was demonstrated that upon TCR engagement G
q/11 is activated by a tyrosine kinase-dependent process that mediates both tyrosine phosphorylation of
immunoreceptor tyrosine activation motif (ITAM) on
CD3 molecules and IP3 generation through activation of
PLC
. Interestingly, tyrosine phosphorylation of TCR-
and CD3
chains as well as ZAP-70 were diminished upon anti-CD3 antibody triggering in cells transfected with a
function-loss mutant of G
11 (17). These data suggest the
involvement of the G
q/11 family in TCR signaling and a
reciprocal regulation between tyrosine kinases and G proteins during the initial stages of TCR-mediated signaling
(3). Activation of tyrosine kinases Pyk2 and Src may link
Gi- and Gq-coupled receptors to the MAPK pathway in
certain cell types (18, 19). The cross-linking of antigen receptors also induced Pyk2 activation in T cells (20). Thus,
Pyk2 may potentially serve as a convergence point for
TCR and GPCR signaling. Moreover, it was shown that
Gq-mediated signaling could trigger the translocation of
the transcription factor, nuclear factor (NF)-AT, to the nucleus upon TCR-mediated T cell activation (21). Among
chemokine receptors that are associated with GPCRs,
RANTES (regulated on activation, normal T cell expressed
and secreted) could activate ZAP-70, which plays a crucial
role in TCR-mediated signaling (22).
In B cells, Gq activates Btk (23), and G
subunits
have been also reported to bind and activate Btk (24, 25).
Thus, engagement of either the BCR or a Gq-linked
GPCR could activate Btk in B cells (3). Based on selective
gene targeting in an avian B lymphoma cell line, DT40,
MAPK activation through Gi-coupled receptors requires
Btk and Syk, whereas Gq-coupled receptors require Csk,
Lyn, and Syk (9).
Histamine is known to be a neurotransmitter (26), an inflammatory mediator, a factor in anaphylaxis, and a regulator of cardiac and gastrointestinal function. The pharmacological effects of histamine are mediated through three
types of membrane receptors, H1, H2, and H3 (27). The
H1R possesses all the structural features of GPCRs, including seven putative transmembrane domains, NH2-terminal glycosylation sites, and phosphorylation sites for protein kinase A and protein kinase C (28). It has been detected in
mammalian brain (29), smooth muscle from airway (31,
32), gastrointestinal tract (33), genitourinary system (34),
vascular smooth muscle (35), and lymphocytes (36, 37).
Upon H1R stimulation, various intracellular responses,
such as production of inositol phosphates, increase in Ca2+
influx, cyclic AMP and cyclic GMP accumulation, and
arachidonic acid release (27, 38), are induced through coupling of the H1Rs to the heterotrimeric G proteins (27, 39,
40). The primary mechanism by which the H1R produces
these functional responses in cells is the activation of PLC
through a pertussis toxin-insensitive G protein that is related to the Gq/11 family of G proteins (26, 27). It has
also been reported that the histamine-induced production of inositol phosphates was inhibited by an antibody against
G
q-like proteins (41). These data suggest the involvement
of a member of G
q family of the G proteins in H1R-
mediated signal transduction.
In this study, we have used H1R knockout mice (42) to demonstrate that histamine affects antigen receptor-mediated immune responses by T and B cells via signal(s) from histamine H1Rs, and we suggest the possibility of a reciprocal regulation between GPCRs and antigen receptor- mediated signaling.
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Materials and Methods |
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Generation of H1R/
Mice.
Genotype Determination by Southern Hybridization.
Genomic DNA (10 µg) was digested with EcoRI and subjected to agarose gel electrophoresis. DNA was transferred onto nucleic acid transfer membranes (Hybond TMCell Culture and Proliferation Assay.
Splenic B cells were prepared by the treatment of spleen cells with anti-mouse Thy 1.2 mAb for 30 min on ice and then with complement for 30 min at 37°C for depletion of T cells. To remove the adherent cells, cells were incubated at 37°C for 1 h and nonadherent cells were collected by Ficoll-Hypaque density centrifugation. Red blood cells were lysed with NH4Cl lysis buffer. After washing, cell purity was measured by FACS® analysis. Splenic T cells were prepared by passing spleen cells through a nylon wool column, and erythrocytes were lysed with NH4Cl lysis buffer. Cells were suspended in RPMI 1640 medium containing 10% heat-inactivated FCS (), 20 µg/ml gentamycin, 0.05 mM 2-ME, and 2 mM L-glutamine, and cultured at a final concentration of 5 × 105 cells/ml in 200-µl/well aliquots in flat-bottomed 96-well tissue culture plates () at 37°C with 5% CO2 for 72 h. FCS was dialyzed twice against RPMI 1640 media at 4°C overnight. Various stimuli were added to the cells: LPS (Difco), goat F(ab)2 anti-mouse IgM (Southern Biotechnology Associates), rIL-4 (R & D Systems), soluble CD40 ligand (CD40L) (CD8 fusion protein contained in the culture supernatant [30% vol/vol] of a transfected myeloma cell line, provided by Dr. P. Lane, Basel Institute of Immunology, Basel, Switzerland), rIL-2 (R & D Systems), and goat anti-mouse CD3Depletion of c-kit-positive Cells.
Total spleen cells or purified B cells were suspended in serum-free RPMI 1640 media after red blood cell lysis. 107 cells/ml were incubated for 45 min on ice with 100 µl of 50 µg/ml rat IgG anti-c-kit mAb (clone ACK; a gift from Dr. S. Nishikawa, Kyoto University, Kyoto, Japan). After centrifugation at 4°C, cells were resuspended in 1 ml of serum-free media. Using a 21-gauge needle, 250 µl of 108 anti-rat IgG-coated beads (Dynabeads; AS) were added to the suspended cells and incubated for 5 min on ice. The incubated cells were centrifuged for 10 min at 4°C. After gentle pipetting, the Eppendorf tube was placed in front of a magnetic bar. The c-kit- positive cells were attached to the magnet. The nonattached cells were collected, washed, and finally resuspended in RPMI 1640 culture media containing 10% dialyzed FCS.Immunization with OVA and Antigen-specific T cell Proliferation.
The wild-type (+/+) and H1R knockout (Immunization with TNP-Ficoll and Measurement of Anti-TNP Antibody.
Wild-type and H1R knockout mice (8-10 wk of age) were immunized intraperitoneally once with 25 µg of TNP- Ficoll (a gift from Dr. S. Ono, Osaka University, Osaka, Japan) in 200 µl PBS. After 10 d, the serum was collected and anti-TNP IgM antibody titer was measured by ELISA. For ELISA, flat-bottomed 96-well probind assay plates () were coated with TNP-BSA at 4°C overnight. After blocking the nonspecific binding sites, serial dilutions of the sera were added (100 µl/well, in triplicate) and plates were incubated for 1 h at room temperature. After washing three times, horseradish peroxidase-conjugated anti-mouse IgM antibodies () were added and incubated at room temperature for 1 h. Finally, tetramethylbenzidine (TMB) peroxidase substrates (Kirkegaard and Perry Labs.) were added to each well and incubated for 5 min. The reaction was stopped by adding 1 M H3PO4. Absorbance at 450 nm was determined using a model 550 automatic microplate reader (Bio-Rad).Titration of Isotype-specific Serum Igs and Anti-OVA Antibodies.
The 96-well flat-bottomed probind assay plates () were coated with 10 µg/ml of isotype-specific goat anti-mouse antibodies overnight at 4°C. After washing uncoated antibodies, plates were blocked with 5% nonfat milk at room temperature for 1 h. The serially diluted sera from unimmunized mice were added to the wells and incubated at room temperature for 1 h. The HRP-labeled isotype-specific antibodies (Southern Biotechnology Associates) were applied to the plate and incubated for 2 h at room temperature. The TMB peroxidase substrate was added to each well for 5 min. Finally, the reaction was stopped by adding the stop solution (1 M H3PO4) and absorbance at 450 nm was measured using a model 550 automatic microplate reader (Bio-Rad Labs.). For determination of isotype-specific anti-OVA antibodies in sera that were collected 2 wk after second immunization with OVA (100 µg/mouse) attached to alum, the assay plates were coated first with OVA (10 µg/ml) in 200 µl/well of PBS and incubated overnight. After washing plates with PBS, they were blocked with 5% nonfat milk at room temperature for 1 h. Serially diluted sera from OVA-immunized mice were added to the wells of plates and incubated at room temperature for 1 h. HRP-labeled isotype-specific antibodies were used for determination of the antibody titer of each Ig subclass.Flow Cytometric Analysis.
Single cell suspensions from freshly isolated red blood cell lysed thymocytes, splenocytes, and bone marrow were prepared, and 2 × 105 cells/ml were stained with mAbs for 30 min on ice in PBS containing 2% FCS and 0.5% NaN3. Cells were washed with PBS and analyzed by FACScan® with Cellquest software (). mAbs used for the staining were labeled with FITC or PE. mAbs to B220, CD5, CD4, and CD8 were obtained from , and anti-IgM and -IgD were from Southern Biotechnology Associates, Inc. Streptavidin red 670 () was used as a third color for FACS® analysis.Measurement of Cytokine Production.
Spleen cells (4 × 106/ well) were cultured in a 24-well culture plate (Costar Co.) with OVA at various concentrations (0, 5, 10, 20, 30, 40, or 50 µg/ ml) or in anti-CD3Immunoprecipitation and Western Blot Analysis.
After depletion of c-kit-positive cells from H1R+/+ spleen cell suspension, 107 cells of wild-type or H1R ![]() |
Results |
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Mouse spleen T and B cells were
initially cultured in medium containing 10% standard FCS
and stimulated with anti-CD3 (10 µg/ml) or anti-IgM
(10 µg/ml) antibodies in the presence of varying concentrations of histamine. Histamine had no effect on anti-CD3
or anti-IgM-stimulated T or B cell proliferation. To
eliminate any effect of preexisting histamine or its derivatives in FCS, the serum was dialyzed against RPMI 1640 culture medium before use, and c-kit-positive spleen cells,
which are presumably mast cells, were removed. Splenic T
(Fig. 1 A) or B cells (Fig. 1 B) were stimulated with anti-CD3
antibody or anti-IgM antibody, respectively, in the
presence of various concentration of histamine. Histamine
enhanced the proliferative responses of T and B cells at an
optimum concentration of 10 µM for both cell types by
about twofold. However, histamine did not show any effect on the proliferative responses unless the FCS was dialyzed before culture and c-kit-positive cells were depleted
from the spleen cells (data not shown).
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In contrast, the histamine-induced augmentation of antigen receptor-mediated proliferative responses of both T
and B cells was not observed in spleen cells obtained from
histamine H1R-deficient mice (Fig. 2) when cultured under the same conditions. These results indicate that histamine H1R-mediated signal(s) are responsible for the enhancing effect observed by the addition of histamine. It is
noteworthy that the level of thymidine incorporation of H1R/
splenic T and B cells upon antigen receptor cross-linking was very low when compared with that of spleen
cells from wild-type mice in the absence of histamine, as
shown in Fig. 1.
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Splenic T and B cells prepared from histamine H1R/
mice and normal littermates (+/+) were cultured in the
presence of varying concentrations of anti-CD3
(Fig. 3 A)
and anti-IgM (Fig. 3 B), respectively. After 72 h of culture,
proliferative responses were assayed by [3H]thymidine incorporation. The proliferative responses of T cells in
H1R
/
mice to anti-CD3
were five- to eightfold lower
than those of normal littermate controls. Similarly, responses to anti-IgM stimulation of B cells from H1R
/
mice were decreased three- to fourfold compared to wild-type mice.
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Both H1R/
and wild-type mice were immunized
twice with OVA to examine antigen-specific responses.
Splenic T cells prepared from immunized mice were cultured in the presence of varying concentrations of OVA
(Fig. 3 C). Proliferative responses to OVA antigen of
H1R
/
T cells were four- to sixfold reduced as compared
with the responses of the wild-type littermates. These data
indicated that TCR- and BCR-mediated proliferative responses of T or B cells from mice lacking the histamine
H1R were impaired, suggesting that a signal(s) from the
histamine H1R upon ligand binding may play an indispensable role in signaling pathways originating from TCR
and BCR complexes.
Impaired responses of splenic B
cells to anti-IgM stimulation in H1R/
mice suggested a
significant role for the signal derived from the histamine
H1R complex. H1R
/
and their wild-type littermates
were challenged with the T cell-independent antigen,
TNP-Ficoll (Fig. 4). After 10 d, sera were collected and
antibodies to the TNP hapten were measured. Anti-TNP
IgM antibody titer was low (about ninefold) in H1R
/
mice, indicating a crucial augmenting role for the interaction between ligands and H1Rs in responses triggered from
BCRs.
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Splenic T cells of H1R/
and wild-type
mice were cultured in the presence of various concentration of IL-2. The thymidine incorporation was measured
72 h after culture and the level of the responses was not significantly changed (Fig. 5 A). Splenic B cells from H1R
/
and wild-type mice were cultured with IL-4 alone (Fig. 5
B) or in combination with CD40L (Fig. 5 C). Again, no
significant difference was observed in the proliferative potential of splenic B cells from wild-type or H1R
/
mice,
demonstrating that signals from histamine H1Rs do not
modulate signaling in CD40-mediated proliferative response. Response of B cells to the mitogen, LPS (Fig. 5 D),
and of T cells to the T cell mitogen, PHA, were also not
significantly affected by H1R deficiency (data not shown).
These data suggest that signals from H1Rs may not affect
the intracellular signals induced by mitogen, cytokines, or
costimulatory molecules, but exhibit augmenting effects on
antigen receptor-mediated responses.
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Mice were immunized
twice with OVA (100 µg per mouse) emulsified with 1 mg
Alum in 200 µl of PBS, and sera were collected 14 d after
the second immunization. Antibody titer of each Ig class
was measured by ELISA with isotype-specific antibodies. In unimmunized mice, serum level of each Ig subclass in
H1R/
mice was comparable to that of normal mice except for IgG3 class, which showed a slightly lower level in
mutant mice as shown in Fig. 6 A. After immunization, total IgG of antibodies to OVA in H1R
/
mice was comparable to that of normal littermates. But, as shown in Fig. 6
B, IgG3 and IgM subclasses of anti-OVA antibodies in H1R
/
mice were significantly lower than those of control mice (P < 0.01). IgG2a subclass of antibodies was also
somewhat low in mutant mice, but was not statistically significant. These data suggested that antibody production of
the IgM and IgG3 subclasses may be impaired in the mice
lacking H1Rs.
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Single cell suspension
of thymocytes, splenocytes, peritoneal exudated cells, and
bone marrow cells were stained with various FITC-conjugated mAbs. No difference in total cell numbers was observed in each organ. FACS® analysis showed no significant difference in numbers of total or T and B cell subpopulation distribution in the profiles of thymus, bone marrow, and spleen in the histamine H1R/
mice (data not
shown). Level of expression of IgM on B cells or CD3 on
T cells of H1R
/
mouse did not change and was comparable to that of wild-type littermates, although the numbers
of CD5+, B220+ cells in peritoneal cavity in histamine
H1R
/
mice were slightly decreased to about a half of
those of normal littermates (21.0 ± 8.6% versus 39.5 ± 9.1% of total peritoneal exudated cells). These data indicated that the absence of the histamine H1Rs does not affect the development and differentiation of mature T and B
lymphoid cells, but may affect the development of B1 cells
in the peritoneal cavity.
The low proliferative response of
T cell of H1R/
mice after triggering with anti-CD3
or
antigen (OVA) (Figs. 1 and 3) may suggest a low protein tyrosine phosphorylation in intracellular signaling events.
ZAP-70 kinase is known to play a crucial role in transduction of TCR-mediated signaling. Experiments were performed to investigate whether the ZAP-70 kinase activation is normally induced in H1R
/
mouse T cells (Fig. 7).
c-kit-positive cell-depleted spleen cells from wild-type or
H1R
/
mice were stimulated with anti-CD3
antibodies
in RPMI 1640 medium with dialyzed FCS. Whole cell lysates from anti-CD3
-stimulated spleen cells were immunoprecipitated with anti-ZAP-70 and blotted with antiphosphotyrosine antibodies. In wild-type mouse T cells, tyrosine phosphorylation of ZAP-70 kinase was induced
after stimulation with anti-CD3
(Fig. 7, left), and the tyrosine phosphorylation was strongly enhanced in the presence of histamine (10
5 M) (Fig. 7, middle). On the other
hand, phosphorylation of ZAP-70 in spleen T cells was
greatly reduced in the H1R
/
mouse (Fig. 7, right).
These results suggest a crucial role of signaling from the
histamine H1Rs in TCR-mediated protein tyrosine phosphorylation in the proximal signaling pathways.
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Cytokine production in response to OVA or anti-CD3 cross-linking was assessed by using spleen cells from
the wild-type and H1R
/
mice that had been immunized
with OVA. Cells were cultured in presence of various concentration of OVA (5-50 µg/ml) or in anti-CD3
-coated plates for 72 h. The culture supernatants were collected to
determine the amount of IL-2, IL-4, and IFN-
. The results are shown in Table I. Compared with the level of cytokines produced by wild-type mouse spleen cells upon
stimulation with OVA antigen or anti-CD3
cross-linking,
the amounts of IL-2 and IFN-
produced by spleen cells
from H1R
/
mice was significantly reduced, whereas the
IL-4 level was not decreased, but rather was enhanced.
These results suggest that the production of Th1-derived
cytokines, which is triggered by TCR signals, might be
more dependent on the signal(s) from histamine H1Rs than
Th2-type cytokine production, and that Th2 activation might be negatively regulated by the signal(s) from histamine H1Rs.
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Discussion |
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In this study, we describe the effect of histamine H1R
signals on antigen receptor-mediated T and B cell signaling. Histamine caused a dose-dependent increase in the
proliferation of splenic T and B cells induced by cross-linking of antigen receptors with anti-CD3 and anti-IgM, respectively. The enhanced response was only observed if the
population of c-kit-positive cells, which includes mast
cells, was depleted before culture, and if the FCS was dialyzed before use as a media supplement. In vitro T cell proliferative responses triggered by anti-CD3 antibody or antigen stimulation with OVA were markedly decreased in
mice lacking H1R. Anti-IgM-stimulated proliferation of B
cells was also impaired in H1R
/
mice. In contrast, the
responses to IL-2, LPS, IL-4, CD40L, and CD40L plus IL-4
were unaffected by the absence of H1R. These results strongly suggest a significant contribution of signal(s) from the histamine H1R to the antigen receptor-mediated signaling pathways that induce proliferative responses in T
and B cells.
TCR signaling has been reported to act on Gq, and results in its physical association with CD3
(17). Activation
of protein tyrosine kinases could result in G
q and G
q/11
phosphorylation, thus augmenting GPCR-mediated G
q/11
signaling (12, 13). Therefore, G
q/11 could mediate both
tyrosine phosphorylation of CD3 and IP3 generation upon
TCR engagement. The cross-linking also induced Pyk2
activation in T cells, which may potentially serve as a convergence point for TCR and GPCR signaling (18). It is
noteworthy that the G
q protein appears to associate with
the histamine H1R as a signal transducer (26, 27, 41). Gq-mediated signaling also triggers the translocation of the
transcription factor nuclear factor (NF)-AT to the nucleus,
which is involved in IL-2 gene activation, providing another mechanism by which TCR and GPCR-linked signaling pathways may communicate with each other (21). It has been reported that the cells derived from the human
Jurkat T cell line, which carried a function-deficient mutant of G
q/11, displayed diminished tyrosine phosphorylation of TCR/CD3
and
chains, as well as ZAP-70,
upon anti-CD3 antibody triggering (17). Reduced tyrosine
phosphorylation of ZAP-70 was also observed in H1R
/
spleen T cells after cross-linking of TCR by anti-CD3 antibody (Fig. 7). Moreover, addition of histamine enhanced
tyrosine phosphorylation of ZAP-70 in anti-CD3-stimulated T cells of wild-type mice. Present data prove the
speculation that signals from GPCR may regulate the
TCR-mediated ZAP-70 activated tyrosine phosphorylation. Thus, the low T cell proliferative responses observed
in the H1R
/
T cells may suggest a possible interaction
between TCR and GPCR signaling triggered by H1R. Interestingly, not only proliferative responses, but also production of cytokines, such as IL-2 and IFN-
, were impaired in H1R
/
spleen T cells as compared with T cells
of the normal littermates upon cross-linking of TCR with
anti-CD3 or antigen (OVA) in vitro (Table I). On the
other hand, in the case of IL-4 production was not reduced, but rather was enhanced. These results suggested that H1R-mediated signal(s) may play a crucial role in antigen-receptor signaling mainly in Th1-type T cells, and it
might be possible that signal(s) from H1R may function as
a feedback regulator for activation of Th2-type T cells.
It has been reported that a Gq activates Btk (23), and
the G
subunit binds and activates Btk (24, 25), which
implicates G
q in B and mast cell function. Thus, engagement of either the BCR or a Gq-linked GPCR could activate Btk in B cells. The xid (CBA/N) mice carry a point
mutation in the pleckstrin homology domain of Btk. These
mice exhibit reduced numbers of mature B cells, reduced
serum levels of IgM and IgG3, and low proliferative responses to anti-IgM and LPS stimulation in vitro (43), and also lack CD5+ B-1 cells in the peritoneal cavity. The
H1R
/
mice showed normal differentiation pattern of B
and T cells, and the numbers of mature B and T cells were
within normal range. However, the response to anti-IgM
cross-linking was reduced (Fig. 2) and serum levels of IgG3
and IgM class antibodies were low (Fig. 6). These data suggest that signal(s) from the G protein coupled to the histamine H1Rs may contribute through Btk to the activation
of B cells induced by antigen receptor signaling, but not
during B cell maturation or in the case of a mitogenic response. Interestingly, the proportion of CD5+ B-1 cells
was found to be decreased in the peritoneal cavity of the
H1R
/
mice to half of that of normal littermates, suggesting the impaired activation of Btk in B1 cells in the H1R
gene knockout mice. All these findings related to GPCR-mediated signaling lead us to postulate that ligand-GPCR
binding triggers the tyrosine phosphorylation of members
of the antigen receptor complex, thereby modifying antigen receptor signaling. Furthermore, activation of the histamine H1R may produce functional signals in cells via the
Gq/11 family of G proteins and communicate with antigen
receptor-mediated signaling, which results in the augmentation of proliferative responses induced by cross-linking of
the TCR and BCR.
Proliferative responses of H1R/
T and B cells in response to stimulation with anti-CD3 or anti-IgM antibodies were lower than those of control littermates in the absence of histamine (Fig. 1 versus Fig. 2). This may suggest
the existence of an endogenous or exogenous H1R ligand(s) other than histamine, which may also affect antigen
receptor-mediated activation of the lymphocytes, and thus
the lack of H1R results in more pronounced effects. One of the possibilities is that a signal(s) from histamine H2Rs
may play a role in H1R
/
mice since there was a report
that the signal(s) from H2R appeared to suppress the production of certain types of cytokines, such as IL-10 and
IL-12, from Th cells (44). Therefore, it is possible to speculate that the H1R-mediated signal(s) may be augmented in
H1R
/
T cells. To discover the role of histamine H2R in
immune response in H1R
/
mice, the spleen T and B cells
of H1R
/
mice were stimulated with anti-CD3
and anti
IgM, respectively, in the presence of varying concentrations
of an H2 antagonist, famotidine, (10
4 M to 10
7 M). The
addition of Famotidine revealed a 20-40% reduction of proliferative responses of both T and B cells (data not
shown). These data implicate the part of dependency of the
remaining proliferative responses of H1R
/
T and B cells
on the H2R-mediated signaling, but low proliferative responses of H1R
/
T and B cells was not due to the suppression through the H2R-mediated signaling. Alternatively, H1R knockout mice may carry an unknown genetic polymorphism that causes a low response to antigen receptor-mediated signaling, since the background of
H1R
/
mice and their control littermates is not homogenous, although they have been backcrossed to C57BL/6
for more than five generations.
Mast cells were once considered mere effector cells for IgE-mediated hypersensitivity that released pharmacologically active mediators, including histamine. However, in recent years, with the realization that mast cells also secrete cytokines, the involvement of this cell type in regulating the immune response has become appreciated (45, 46). Our studies indicate that the mast cell product histamine may play an even more direct role in acquired immunity than was previously suspected.
Although the precise molecular mechanism remains obscure, further detailed investigation of the interaction of the H1R with the G proteins should reveal the importance of the cross-talk between GPCR-mediated and antigen receptor-mediated signaling pathways.
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Footnotes |
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Address correspondence to Takeshi Watanabe, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Phone: 81-92-642-6835; Fax: 81-92-632-1499; E-mail: watanabe{at}bioreg.kyushu-u.ac.jp
Received for publication 28 September 1998 and in revised form 1 December 1998.
We express our sincere thanks to Dr. Peter Burrows for reading the manuscript and for his helpful comments. We also thank Dr. M. Nakashima for his invaluable assistance with the experiments. ![]() |
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