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Department of Dermatology, University Marselisborg Hospital, Aarhus University, Aarhus, Denmark;
Laboratory of Medical Allergology, National University Hospital, Copenhagen, Denmark; and
Department of Immunology, Tongji Medical University, Wuhan, Peoples Republic of China
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-bromocinnamylamino)ethyl)-5-isoquenilesulfonamide
(H-89), in T lymphocytes, indicating that eotaxin activates T
lymphocytes via a cAMP-signaling pathway. These results imply that the
CC chemokine eotaxin may be not only an important mediator for
eosinophils but also for T lymphocytes in terms of the cell migration,
accumulation, and recruitment that happen during the initiation and
development of inflammatory processes. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T lymphocytes from normal, nonallergic subjects were purified by nylon wool as previously described (14). For cell stimulation, lymphocytes were preincubated in RPMI 1640 containing IL-2 (10 ng/ml, PeproTech, NJ), and/or IL-4 (10 ng/ml, Schering-Plough, Kenilworth, NJ), and/or eotaxin (100 ng/ml, R&D Systems, Minneapolis, MN) with 10% FBS for 24 h before being subjected to the ensuing assays. For inhibition of signaling pathway of cells, IL-2- and IL-4-stimulated T lymphocytes were preincubated with H-89 (30 µM), pertussis toxin (PT, 1 µg/ml), or bisindolylmaleimide I (BIM I, 1 µM) for 30 min at 37°C before being subjected to the ensuing assays. These reagents were from Sigma (St. Louis, MO). For Ab blocking, the cells were preincubated with mouse anti-human Abs (anti-CD29, 4B4; anti-CD49a, HP2B6; and/or anti-CD49b, Gi9; 5 µg/ml, respectively (Coulter-ImmunoTech, Margency, France)) for 30 min at 4°C prior to assays. No cellular proliferation was seen in the cells stimulated with IL-2 (10 ng/ml) and IL-4 (10 ng/ml) within 24 h as detected by [3H]thymidine incorporation into DNA assay (our unpublished observations).
Flow cytometry
As previously described (6, 15) freshly isolated cells or T lymphocytes stimulated with either IL-2 and/or IL-4 were first incubated with a mouse anti-human CCR3 mAb (7B11) at 5 µg/ml or 5 µg/ml IgG2a isotype-matched mAb (Dako, Glostrup, Denmark) in PBS containing 2% human pooled AB serum and 0.1% sodium azide (staining buffer). After 20 min, the cells were washed twice with staining buffer and resuspended in 50 µl phycoerythrin (PE)-conjugated affinity-purified F(ab')2 rabbit anti-mouse mAb (Dako) for 20 min. The cells were then washed twice in staining buffer. For detection of ICAM-1, freshly isolated or cytokine-stimulated (24 h) T lymphocytes were incubated with mouse anti-human ICAM-1 mAb (Dako) for 20 min, followed by incubation with PE-conjugated affinity-purified F(ab')2 rabbit anti-mouse Ab (Dako) for 30 min. For detection of integrins, freshly isolated or cytokine-stimulated (24 h) T lymphocytes were incubated with different mouse anti-human Abs (anti-CD29, 4B4; anti-CD49a, HP2B6; anti-CD49b, Gi9; anti-CD29c, C3VLA3; anti-CD29d, HP2/1; or anti-CD49e, SAM1; 5 µg/ml, respectively (Coulter-ImmunoTech) for 20 min, followed by incubation with FITC-conjugated affinity-purified F(ab')2 rabbit anti-mouse Ab (Dako) for 30 min. Then the cells were labeled with PE-conjugated mouse anti-human CD3 mAb (Dako) for 15 min. All procedures were carried out at 4°C. The labeled cells were fixed with 1% paraformaldehyde. The analyses were performed with a flow cytometer (Coulter XL).
Chemotaxis assay
The following human recombinant chemokines were studied: eotaxin
and MIP-1
(R&D Systems), and IL-8 (Dainippon Pharmaceuticals, Osaka,
Japan). The chemotaxis assay was a 48-well micro-chamber (Neuro Probe,
Bethesda, MD) technique (16). Briefly, chemokines were
diluted in RPMI 1640 with 0.5% pooled human serum and placed in the
lower wells (25 µl). A total of 50 µl of the cell suspension at
5 x 106 cells/ml were added to the upper well of the
chamber, which was separated from the lower well by a 5-µm pore size,
mouse collagen IV-coated, polycarbonate, polyvinylpyrrolidone-free
membrane (Nucleopore, Pleasanton, CA). The chamber was incubated for
120 min at 37°C in an atmosphere containing 5% CO2. The
membrane was then carefully removed, fixed in 70% methanol, and
stained for 5 min in Coomassie Brilliant Blue. The cells that migrated
and adhered to the lower surface of the membrane were counted by using
a light microscopy or by an ELISA reader (EL307C, Bio-Tek, Copenhagen,
Denmark), in which both methods of measurement showed identical
results. The results were expressed as chemotactic index, which are the
ratios between the numbers of migrating cells in the sample and in the
medium control (16). For blocking tests of IL-2- and
IL-4-stimulated T lymphocyte chemotaxis toward eotaxin, the cells were
preincubated with either anti-CCR3 mAb (10 µg/ml) or
with IgG2a isotype Ab (10 µg/ml) for 120 min at room temperature
before chemotaxis assay.
Adhesion assays
Adhesion assays were performed as described previously (17). Briefly, microtiter plates (96-well) were coated with laminin (20 µg/ml; Sigma) in PBS for 1 h at 37°C in a humidified atmosphere. Plates were washed with PBS and incubated with medium containing 0.2% BSA for 1 h in 5% CO2 to block nonspecific adhesion. Thereafter, single-cell suspensions were prepared in RPMI 1640 medium with 0.2% BSA at 4 x 105 cells/ml, and eotaxin was added at 100 ng/ml (or the chemokines indicated). The cell suspension was added at 100 µl per well in triplicate to 96-well plates, and incubated for 60 min (or indicated periods) at 37°C. Nonadherent cells were removed by washing with 0.2% BSA in PBS. Subsequently, the adherent cells were fixed with 1% formaldehyde and stained with 1% crystal violet. Crystal violet was then extracted by the addition of a 1:1 mixture of sodium citrate 0.1 M, pH 4.2/ethanol; absorbency was then read at 540 nm. Cells bound to collagen I (10 µg/ml) on a separate plate were used to represent 100% attachment. Background cell adhesion to 2% BSA-coated wells was subtracted from all readings. For inhibition assays, cells were preincubated with different pharmacological agents (at 37°C) or Abs (at 4°C) for 30 min prior to assays.
Aggregation assays
Aggregation assays were performed as described previously (18). Briefly, the cells were added at a concentration of 106/ml to 24-well culture plates. The cytokines were added in different combinations to RPMI 1640 culture medium with 10% FBS. After 24 h, the cells were observed, scored, and photographed using a Leitz microphotograph system. A semi-quantitative scoring method (18) was used: 0 for no aggregation; 1+ indicated that less than 10% of the cells were aggregated; 2+ indicated that more than 50% of the cells were aggregated; 3+ indicated that up to 90% of the cells were in small, loose clusters; and 4+ indicated more than 90% of the cells were aggregated in large clusters.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We examined the expression of CCR3 on human peripheral T
lymphocytes. The results from the flow cytometric analyses (Fig. 1
A) document that IL-2 and
IL-4 in combination induce the expression of CCR3 on T lymphocytes,
whereas there are no detectable CCR3+ cell fractions in
freshly isolated T lymphocytes (a). After 24-h incubation
with cytokine-free medium (b), there were still no flow
cytometric detectable CCR3+ cells (0%). IL-2
(c) or IL-4 (d) induced a small amount of CCR3
expression on T lymphocytes (5% or 17%, respectively). The
combination of IL-2 and IL-4 (e) induced a large amount of
CCR3+ cells (up to 85%). We also carried out a kinetic
study in terms of the expression of CCR3 on T lymphocytes stimulated
with IL-2 and IL-4. The results shown in Fig. 1B indicate
that a combination of IL-2 and IL-4 can significantly up-regulate the
expression of CCR3 on T lymphocytes from 0% (0 h) (a), 0%
(2 h) (b), over 11% (4 h) (c), 41.9% (8 h)
(d), 66.4% (16 h) (e) to 76.1% (24 h)
(f).
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The results in Fig. 2A
show that IL-8 and MIP-1 can significantly induce chemotactic
migration of freshly isolated T lymphocytes, whereas eotaxin shows no
chemotactic effect. However, Fig. 2B shows that 10 ng/ml of
eotaxin induces a potent migration of T lymphocytes, which have been
stimulated with IL-2 and IL-4 for 24 h; IL-8 and MIP-1 still
show chemotactic activities. We found that a concentration range from
10 ng/ml to 100 ng/ml of eotaxin induced a significant chemotactic
activity, whereas lower (
1 ng/ml) or higher (
1 µg/ml)
concentrations appeared to have little or no reproducible migratory
activity, thus yielding a typical bell-shaped dose-dependent chemotaxis
response curve. To confirm these findings, we used anti-CCR3 mAb to
block the activity of eotaxin for IL-2- and IL-4-stimulated T
lymphocytes. The anti-CCR3 mAb can completely block the chemotaxis
of IL-2- and IL-4-stimulated T lymphocyte toward eotaxin, whereas
isotype Ab has no blocking effect (Table I). The results of checkerboard analysis
(19) demonstrate that migration of IL-2- and
IL-4-stimulated T lymphocytes toward eotaxin is chemotactic, but not
chemokinetic (data not shown).
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We next examined the effects of eotaxin, IL-2, and/or IL-4 on the
expression of ICAM-1 on T lymphocytes (Fig. 3A). Eotaxin can significantly
increase the expression of ICAM-1 on T lymphocytes in association with
IL-2 and IL-4. There is about 18% ICAM-1+ cells in freshly
isolated T lymphocytes (a); 15% in cytokine-free 24
h-cultured T lymphocytes (b); 20% in IL-2-stimulated T
lymphocytes (c); 22% in IL-4-stimulated T lymphocytes
(d); 30% in eotaxin-stimulated T lymphocytes
(e); 19% in IL-2- and IL-4-stimulated T lymphocytes
(f); 30% in IL-2- and eotaxin-stimulated T
lymphocytes (g); 14% in IL-4- and eotaxin-stimulated T
lymphocytes (h); and about 90% in IL-2-, IL-4-, and
eotaxin-stimulated T lymphocytes (i). We also carried out
the kinetic study in terms of the expression of ICAM-1. The results
shown in Fig. 3B indicate that a combination of eotaxin,
IL-2, and IL-4 can significantly up-regulate the expression of ICAM-1
on T lymphocytes within 8 h (d) by 58% despite the
fact that there are 19% ICAM-1-positive cells in freshly isolated T
lymphocytes (a) and about 20% ICAM-1-positive cells after
2-h stimulation with a combination of eotaxin, IL-2, and IL-4
(b). After 4 h (c), the ICAM-1 expression is
31%. The ICAM-1 expression has been up-regulated to more than 80% by
16 h (e) and to 97% by 24 h
(f).
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To examine whether eotaxin plays a role in T lymphocyte adhesion,
we performed aggregation tests of T lymphocytes stimulated with IL-2,
IL-4, and/or eotaxin. The results shown in Fig. 4 indicate that a strong aggregation is
observed among T lymphocytes that have been stimulated with a
combination of eotaxin with IL-2 and IL-4 (h) similar to
LPS-stimulated T lymphocytes (i). Neither IL-2, IL-4, nor
eotaxin alone induces such a phenomenon (a–e).
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We next examined the expression of some integrins on T lymphocytes
stimulated with the combination of eotaxin, IL-2, and IL-4. Since we
had already demonstrated that eotaxin in association with IL-2 and IL-4
could significantly induce T lymphocyte chemotaxis and aggregation, we
therefore conducted further experiments to investigate whether this
combination would enhance the expression of integrins on T lymphocytes.
The results from the flow cytometric analyses in Figure 5 indicate that eotaxin can significantly
increase the expression of certain integrins on T lymphocytes in
association with IL-2 and IL-4. There is about 53% CD29+
cells in freshly isolated T lymphocytes (a), 43%
CD49a+ (b), 14% CD49b+
(c), 39% CD49c+ (d), 16%
CD49d+ (e), and 35% CD49e+
(f), respectively. After 24-h culture in a medium
containing eotaxin, IL-2, and IL-4, the expression of CD29, CD49a, and
CD49b has been selectively and substantially up-regulated. There is
about 95% CD29+ cells in eotaxin-stimulated T lymphocytes
(g), 89% CD49a+ (h), and 72%
CD49b+ (i), respectively. No significant changes
have been observed regarding the expression of CD49c, CD49d, and CD49e:
36% CD49c+ cells in eotaxin-stimulated T lymphocytes
(j), 24% CD49d+ (k), and 36%
CD49b+ (l), respectively.
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We have found that eotaxin, IL-2, and IL-4 can selectively and
highly up-regulate the expressions of CD29, CD49a, and CD49b on T
lymphocytes. Our study was therefore expanded to explore what role
these integrins are playing in adhesion of T lymphocytes. As shown in
Figure 6, eotaxin, in association with
IL-2 and IL-4, can induce significant adhesion in T lymphocytes in the
absence of anti-integrin Abs (anti-CD29, anti-CD49a, and
anti-CD49b). None of these anti-integrin Abs alone can prohibit
the adhesion of T lymphocytes induced by eotaxin. The combination of
anti-CD29 mAb and anti-CD49a or anti-CD49b mAb partly
inhibits adhesion of T lymphocytes induced by eotaxin, IL-2, and IL-4
by approximately 50%. However, the combination of anti-CD49a and
anti-CD49b mAbs completely blocks the adhesion of T lymphocytes
induced by eotaxin, IL-2, and IL-4. A similar pattern of blocking
effect on eotaxin-induced adhesion of T lymphocytes has also been
observed for the combination of anti-CD29, anti-CD49a, and
anti-CD49b mAbs. In some of the experiments in which fibronectin
replaced laminin as substratum, similar results were obtained in terms
of the blocking effects of anti-CD49a and anti-CD49b mAbs (our
unpublished observations). Interestingly, eotaxin cannot induce freshly
isolated T lymphocytes to adhere to laminin substratum within 60 min,
whereas other chemokines, RANTES, MIP-1, and IL-8, stimulate
adhesion in T lymphocytes (Fig. 7A). The results in Figure 6
imply that eotaxin induces an adhesion of T lymphocyte via the
up-regulation of some adhesion molecules, not only ICAM-1, but also
certain integrins (CD29, CD49a, and CD49b). In the pathway of this
reaction, CD49a and CD49b may be very important ligands.
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A, RANTES stimulates
rapid adhesion of T lymphocytes as well as does MIP-1. IL-8, a
representative of CXC chemokines, causes a later adhesion in T
lymphocytes. In contrast, eotaxin cannot induce T lymphocytes to adhere
to laminin substratum within 60 min in the absence of IL-2 and IL-4. As
shown in Figure 7B, eotaxin induces a significant later
adhesion of IL-2- and IL-4-stimulated T lymphocytes, whereas IL-8
appears to lose the ability of induction of T lymphocyte adhesion after
60 min. This phenomenon is coordinating our previous observation
(16, 20). The patterns of RANTES and MIP-1 to induce
adhesion of IL-2- and IL-4-stimulated T lymphocytes are not
significantly distinct from those seen in the absence of IL-2 and
IL-4. Involvement of cAMP-signaling pathway in eotaxin-induced adhesion of T lymphocytes
In order to explore which signaling pathways are involved in
eotaxin-induced adhesion in T lymphocytes, we examined whether the
interference with different signaling pathways could affect the
adhesion of T lymphocytes induced by eotaxin (Fig. 8). We pretreated the T lymphocytes for
30 min at 37°C with PT (1 µg/ml), a specific inhibitor of certain G
proteins (21), although a small PT-resistant component can
usually be detected (22, 23, 24); H-89 (30
µM), a selective inhibitor of cAMP-dependent protein kinase
(25); or BIM I (1 µM), a selective inhibitor of protein
kinase C (26), respectively. As previously shown, eotaxin,
IL-2, and IL-4 can induce significant adhesion of T lymphocytes in the
absence of inhibitors of signal transduction pathways. The dose of PT
employed (1 µg/ml) induces an incomplete inhibition of the
eotaxin-induced adhesion in T lymphocytes. Interestingly, H-89
completely prevents eotaxin-induced adhesion of T lymphocytes. In
contrast, BIM I cannot prohibit the eotaxin-induced adhesion of IL-2-
and IL-4-stimulated T lymphocytes. Thus, these results strongly
indicate that the cAMP-signaling pathway is involved in the events of
adhesion of T lymphocytes caused by eotaxin, IL-2, and IL-4
stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eotaxin is a recently described CC chemokine and has been implicated in
animal and human eosinophilic inflammatory states. Cellular sources of
eotaxin are bronchial epithelial cells, T lymphocytes, macrophages, and
eosinophils themselves (27). The importance of this
chemokine was widely recognized due to its activation and attraction of
leukocytes, including eosinophils and basophils. For most chemokines,
including CXC, CC, and C chemokines (28, 29), their
chemotactic activities for T lymphocytes have been well described.
Previously, eotaxin was considered only as an eosinophil-specific CC
chemokine, and assumed to be involved in eosinophilic inflammatory
diseases such as atopic dermatitis, allergic rhinitis, asthma, and
parasitic infections (30). Its expression is stimulus and
cell specific (30). Ponath et al. (5)
reported that eotaxin acts exclusively on eosinophils and has no effect
on neutrophils, monocytes, or T lymphocytes. Recently, researchers
observed that mature CD4+ T lymphocytes were absolutely
required for OVA-induced eosinophil accumulation since lung
eosinophilia was prevented in CD4+-deficient mice
(31). Eotaxin, but not MIP-1 and RANTES, is one of the
molecular links between Ag-specific T lymphocyte activation and the
recruitment of eosinophils into the airways (32). It was
also reported that selective eosinophilic recruitment during allergic
lung inflammation results from a sequential accumulation of certain
leukocyte types, particularly T lymphocytes, and relies on the presence
of both eosinophilic chemoattractants and adhesion receptors
(31). More interestingly, in contrast to Ponath’s
observation, Sallusto et al. showed that CCR3 is expressed on
Th2-derived cells, in which eotaxin induced intracellular calcium
increase and chemotaxis (13). Our results show that
freshly isolated T lymphocytes have no chemotactic response to eotaxin,
but IL-2- and IL-4-stimulated T lymphocytes express a large amount of
CCR3, so that eotaxin can induce potent chemotactic migration of T
lymphocytes. It should also be noticed that IL-2 or IL-4 alone induced
a small amount of T lymphocytes to express CCR3 (5% and 17%,
respectively). Our results not only support Sallusto’s findings, but
also specify the necessity of IL-2 and IL-4 in the responsiveness of T
lymphocytes to eotaxin. Thus, IL-2 and IL-4, which are typical
representatives for Th1-derived and Th2-derived cytokines,
respectively, may play very important roles in the eotaxin-mediated
cascades of biological or pathophysiological events in T lymphocytes in
terms of induction of expression of CCR3 and up-regulation of ICAM-1
and several integrins on these cells.
In knockout mice of IFN-
or IL-4 genes, researchers showed that
there is a balance and a network among Th1-derived cytokines (IL-2,
IFN-, and TNF-); Th2-derived cytokines (IL-4, IL-5, IL-10, and
IL-13), and chemokines (eotaxin, RANTES, and monocyte chemotactic
proteins) regarding different types of inflammation and its
relationship to local and regional cytokine expression
(33). Our results, which show that IL-2 and IL-4 together,
but only marginally alone, induce the expression of CCR3 on T
lymphocytes, and that eotaxin in combination with IL-2 and IL-4
enhances the expression of ICAM-1 and several integrins on T
lymphocytes, resulting in adhesion and aggregation, strongly suggest
that there are a balance and collaboration between Th1-derived and
Th2-derived cytokines and chemokines during activation, migration,
adhesion, accumulation, and recruitment of T lymphocytes, which are
crucial steps in inflammation.
There are a number of studies concerning chemokine-induced adhesion in
T lymphocytes. Secondary lymphoid-tissue chemokine (SLC), a recently
identified CC chemokine and specific binding to CCR7 (34, 35), induces firm adhesion of naive T lymphocytes via ß2
integrin binding to the counter receptor, ICAM-1, as a necessary step
for lymphocyte recruitment (36). Four chemokines, SDF-1,
SLC (6-C-kine), MIP-3, and MIP-3ß, induce adhesion in T cell
subsets to ICAM-1 linked to the Gi subunit of a heterotrimeric
GTP-binding protein, then trigger rapid integrin-dependent adhesion to
arrest rolling cells (37). Eotaxin up-regulates the
quantitative level of CD11b and CD18, and increases the adhesion to
fibronectin in eosinophils preincubated in vitro with IL-5
(38). Eotaxin potentially induces eosinophil accumulation
in vivo, being dependent on 4 integrin/vascular cell adhesion
molecule-1 and ß2 integrin/ICAM-1 adhesion pathways
(39). Weber et al. demonstrated that CC chemokines MCP-1,
RANTES, and MIP-1ß can differentially and selectively regulate
avidity of integrins sharing common ß subunits. Transient activation
and deactivation of very late Ag (VLA)-4 serve to facilitate
transendothelial diapedesis, whereas late and prolonged activation of
VLA-5 mediates subsequent interactions with the basement membrane and
extracellular matrix (40). Springer and coworkers
(41) studied the ability of MCP-1, RANTES, and MIP-1ß to
induce binding of T cells; they documented that these chemokines are
the most important, not in initiating integrin-dependent firm adhesion
in T lymphocytes to the vascular wall, but rather, in subsequent
adhesive interactions during migration into tissue. Recently, Springer
and coworkers (42) found the first evidence that
endothelial cell-derived chemokines can activate firm adhesion through
4 and ß2 integrins, even in the presence of shear flow. In the
present study, we have demonstrated that neither eotaxin alone nor
IL-2 + IL-4 without eotaxin induces adhesion (Fig. 7A),
whereas the combination of the three cytokines in fact do so (Fig. 7B). Moreover, the adhesion can be completely blocked by Abs
to the CD49a + CD49b (Fig. 6). It can therefore be inferred that
all three cytokine are, in fact, necessary for integrin-mediated
adhesion. We showed that these inductions of chemotaxis and aggregation
of T lymphocytes by eotaxin are dependent on the IL-2- and IL-4-induced
expression of CCR3 on T lymphocytes. We also showed that eotaxin in
association with IL-2 and IL-4 can significantly and selectively
up-regulate the expression of ICAM-1 and certain integrins (CD29,
CD49a, and CD49b) to induce adhesion and aggregation of T lymphocytes.
Thus, these results strongly suggest that when IL-2 and IL-4 induce the
expression of CCR3 on T lymphocytes, eotaxin may subsequently activate
T lymphocytes via CCR3 in terms of inductions of chemotaxis,
aggregation, and expression of ICAM-1 and certain integrins. It would
be noticed that an exact role of ICAM-1 in the cascade of T lymphocyte
adhesion remains speculative. However, further investigation into this
issue, for example, the blocking test by ICAM-1 mAb, will be
interesting. Generally, our results imply that a selective recruitment
of T lymphocytes to sites of inflammation is controlled by regulation
of cytokines, chemokines, and adhesion molecules. Eotaxin stimulates
circulating T lymphocytes to recruit from the blood to the tissue by
triggering later integrin-dependent adhesion. On the other hand, it
will be very interesting to investigate whether eotaxin regulates
endothelial cells to arrest T lymphocytes rolling under flow
conditions, and whether eotaxin modulates the avidity of integrins on T
lymphocytes (41).
The cAMP-dependent pathway appears to be involved in the mobility of
human T lymphocyte surface molecules (43). MIP-1 was
reported to induce an increase in intracellular cAMP level in the
megakaryocytic leukemia cell line (44). Our results that a
selective inhibitor of cAMP-dependent protein kinase H-89 can
completely block eotaxin-induced adhesion in T lymphocytes indicate
that cAMP is directly involved in eotaxin-induced adhesion in T
lymphocytes. On the other hand, chemokine receptors are coupled to G
proteins and their activation results in prominent changes in cell
migration and adhesion. Recently, it was demonstrated (45)
that chemotaxis of T lymphocytes induced by CC chemokines is dependent
on activation of Gi and the release of Gß dimers and that
Gi-coupled receptors not traditionally associated with chemotaxis
can mediate directed migration when they are expressed in hemopoietic
cells. RANTES was reported to induce biphasic mobilization of
Ca2+ in T cells mediated by, initially, a G protein-coupled
pathway, and subsequently, a protein tyrosine kinase (46).
RAFTK, a novel tyrosine kinase, appears to provide a functional bridge
for the transmission of CCR5 receptor signaling to the cytoskeleton and
nucleus (47). Our results that a specific inhibitor of
certain G proteins can partially prevent eotaxin-induced adhesion in T
lymphocytes indicate that eotaxin-induced adhesion in T lymphocytes
requires partially the activation of the G protein-signaling pathway.
Leukocytes express multiple chemoattractant receptors that can trigger
adhesion and direct their migration. Cross-talk between chemoattractant
receptors and their signaling pathways may help target leukocyte
migration in the context of complex chemoattractant arrays in vivo
(48). Our results are not astonishing findings since it
has been described that other adhesion receptors such as CD2 are also
linked to cAMP-dependent signaling (49). This interesting
point deserves further investigation. It also deserves a further
understanding in terms of a clearer signal transduction mechanism
associated with chemokine-mediated adhesion in T lymphocytes.
In summary, the present study provides useful insights into novel mechanisms of the actions of eotaxin concerning eotaxin-induced T lymphocyte chemotaxis and adhesion, IL-2- and IL-4-induced CCR3 expression, and eotaxin-enhanced expression of ICAM-1 and certain integrins on T lymphocytes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint request to Dr. Tan Jinquan, Laboratory of Medical Allergology, National University Hospital, DK-2200 Copenhagen N., Denmark. E-mail address:
3 Abbreviations used in this paper: CCR, CC chemokine receptor; BIM I, bisindolylmaleimide I; H-89, N-(2-(-bromocinnamylamino)ethyl)-5-isoquenilesulfonamide; MIP, macrophage inflammatory protein; PT, pertussis toxin; SLC, secondary lymphoid-tissue chemokine; VLA, very late Ag; PE, phycoerythrin.
Received for publication September 24, 1998. Accepted for publication December 28, 1998.
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References |
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  E. Forbes, M. Hulett, R. Ahrens, N. Wagner, V. Smart, K. I. Matthaei, E. B. Brandt, L. A. Dent, M. E. Rothenberg, M. Tang, et al. ICAM-1-dependent pathways regulate colonic eosinophilic inflammation J. Leukoc. Biol., August 1, 2006; 80(2): 330 - 341. [Abstract] [Full Text] [PDF]
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 H. B. Kwak, S. W. Lee, H. M. Jin, H. Ha, S. H. Lee, S. Takeshita, S. Tanaka, H.-M. Kim, H.-H. Kim, and Z. H. Lee Monokine induced by interferon-{gamma} is induced by receptor activator of nuclear factor {kappa}B ligand and is involved in osteoclast adhesion and migration Blood, April 1, 2005; 105(7): 2963 - 2969. [Abstract] [Full Text] [PDF]
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 D. Tao, L. Shangwu, W. Qun, L. Yan, J. Wei, L. Junyan, G. Feili, J. Boquan, and T. Jinquan CD226 Expression Deficiency Causes High Sensitivity to Apoptosis in NK T Cells from Patients with Systemic Lupus Erythematosus J. Immunol., February 1, 2005; 174(3): 1281 - 1290. [Abstract] [Full Text] [PDF]
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T. Jinquan, H. H. Jacobi, C. Jing, A. Millner, E. Sten, L. Hviid, L. Anting, L. P. Ryder, C. Glue, P. S. Skov, et al. CCR3 Expression Induced by IL-2 and IL-4 Functioning as a Death Receptor for B Cells J. Immunol., August 15, 2003; 171(4): 1722 - 1731. [Abstract] [Full Text] [PDF]
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T. Jinquan, L. Anting, H. H. Jacobi, C. Glue, C. Jing, L. P. Ryder, H. O. Madsen, A. Svejgaard, P. S. Skov, H.-J. Malling, et al. CXCR3 Expression on CD34+ Hemopoietic Progenitors Induced by Granulocyte-Macrophage Colony-Stimulating Factor: II. Signaling Pathways Involved J. Immunol., October 15, 2001; 167(8): 4405 - 4413. [Abstract] [Full Text] [PDF]
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), MIP-1
), and IL-8 (
). The illustrated data are from a single
representative experiment of six performed. All results were determined
as described in Materials and Methods and expressed as
chemotactic index (C.I.) and based on triplicate determination of
chemotaxis on each concentration of chemoattractant. C.C. indicates
chemokine concentrations (ng/ml).
), and IL-8 (
), or control (