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*
Department of Immunology and
Department of Cell Signaling, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94306; and
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305
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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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The first phase of this biphasic signal, which is transient in nature
and associated with chemotaxis, can be inhibited by the heterotrimeric
G
i protein inhibitor pertussis toxin (PTX).6
This is consistent with signaling through other C-C chemokine
receptors, such as the CCR-1 (7). By contrast, the second phase of this
signal, which comprises a sustained Ca2+ influx, is
insensitive to pertussis toxin PTX, but sensitive to the tyrosine
kinase inhibitor herbimycin A (HA). The second phase signal is
associated with cellular activation and has been shown by whole cell
patch clamp analyses to be similar to TCR-mediated early activation
events (6), a feature that suggests signaling apart from the
seven-transmembrane, G protein-linked chemokine receptors.
To identify and characterize the RANTES-responsive elements mediating the second phase signal, we have identified two cell lines that manifest only one of the two RANTES-induced signaling pathways. The monocytic cell line THP-1 responded to RANTES through a G protein-linked pathway, while the T cell line Jurkat responded through a tyrosine kinase-mediated pathway. Furthermore, we showed a direct correlation between CD3 expression and the RANTES response in the Jurkat cells, raising the possibility that RANTES may engage the TCR complex as a way of effecting cellular activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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THP-1 cells were obtained from the American Type Culture Collection (ATCC TIB-202) and the Jurkat cell lines were developed at DNAX. Both cells were grown in RPMI 1640 medium (JRH Biosciences) containing 10% heat-inactivated FBS, 25 mM HEPES (pH 7.5), 5 U/ml penicillin, and 5 µg/ml streptomycin.
Cytoplasmic Ca2+ mobilization assays
Cells were labeled with 3 µM indo-1 acetoxymethyl ester (Molecular Probes) in complete growth medium at a density of 107 cells/ml for 45 min at 20°C with gentle mixing. Cells were washed, resuspended at 107 cells/ml in HBSS (138 mM NaCl, 5 mM KCl, 5.6 mM D-glucose, 4 mM sodium bicarbonate) (Life Technologies) containing 1% FBS, and maintained at 20°C for up to 2 h. RANTES (R&D Systems) or mAb to human CD3 (UCHT1, Immunotech) was added to 106 cells in 2 ml of flux buffer (HBSS with 1.6 mM CaCl2, and 10 mM HEPES (pH 7.5)) and maintained at 37°C in an acrylic cuvette with constant stirring. Fluorescence measurements to determine the increases in cytoplasmic free Ca2+ concentration ([Ca2+]i) were performed with a Photon Technologies spectrofluorometer at an excitation wavelength of 350 nm (4 nm bandwidth) and simultaneous emission measurements at 400 and 490 nm (10 nm bandwidth). The ratio of 400 nm/490 nm was recorded at a rate of 2 Hz.
Pharmacologic inhibition of the chemokine response
Cells were incubated at a concentration of 106 cells/ml for 16 h in complete growth medium with either 10 µM HA (Calbiochem) or 100 ng/ml PTX (Calbiochem).
FACS analysis and Jurkat cell sorting
FACS analyses were performed using standard protocols. Briefly, cells were washed in PBS containing 1% BSA, resuspended at 2 x 105 cells per well in 96-well V-bottom plates (Costar) and incubated with FITC-conjugated human anti-CD3 mAb (UCHT1, Immunotech) for 30 min. They were washed three times and analyzed on a FACScan (Becton Dickinson). Acquisitions were based on the forward and side-scatter characteristics and 10,000 events were acquired. For FACS sorting, two independent acquisition gates were applied to the CD3-positive and CD3-negative populations respectively based on their FL1 fluorescence. 106 cells were collected for each population.
Overnight TCR stimulation of Jurkat T cells
Jurkat cells were incubated for 16 h at a density of 106 cells/ml in complete growth medium with the addition of 2.5 µg/ml human anti-CD3 mAb (UCHT1, Immunotech), or 2.5 µg/ml IgG1 isotype control Ab (Sigma).
RANTES equilibrium binding
A standard filtration binding protocol was employed (8). Briefly, 106 cells were incubated with approximately 0.1 nM 125I-RANTES (Dupont NEN) in the presence of increasing amounts of unlabeled RANTES competitor using the following buffer: 25 mM HEPES, 80 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA; adjusted to pH 7.4. The reactions were incubated for 2 h at 22°C before they were aspirated onto PEI-treated GF/C filters using a cell harvester (Packard Instrument Co.). The reactions were washed twice with the following buffer: 25 mM HEPES, 500 mM NaCl, 1 mM CaCl2, 5 mM MgCl2; adjusted to pH 7.4. Scintillant (MicroScint 10, Packard) was added to the filters, and the retained radioactivity measured using a TopCount scintillation counter (Packard). The data were then analyzed using IgorPro software (WaveMetrics).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A biphasic Ca2+ response was observed upon the
addition of RANTES to a final concentration of 1 µM (Fig. 1
A) in both the SPB21
CD4+ T cell clone and in PBL (6). This response comprised a
short transient spike of increased cytoplasmic Ca2+
followed by a more vigorous and sustained response accompanying the
opening of extracellular Ca2+ channels.
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B with 1A). This also highlighted a
difference in the kinetics of the two responses: the initiation of the
THP-1 response was more rapid that of the Jurkat response.
RANTES dose-response studies highlighted a second major difference
between the two cellular responses (Fig. 1, C and
D). Transient increases of cytoplasmic
Ca2+ were seen in THP-1 cells with as little as 0.1 nM
RANTES, which is consistent with known THP-1 in vitro chemotactic and
signal transduction characteristics and chemokine binding affinities
(KD values around 1 nM) (8, 9, 10). By contrast, the Jurkat
cells did not exhibit significant Ca2+ mobilization at less
than 100 nM RANTES; maximal stimulation was observed at 1 µM, while
only a minimal response was seen at 100 nM, with no response observed
at or below 10 nM RANTES. This closely mirrored the dose-response of
the RANTES-induced second phase signal in the SPB21 T cell clone.
Pharmacologic analysis of the RANTES-induced response
The pharmacologic sensitivities of the RANTES-induced cellular
responses in THP-1 and Jurkat cells were examined. PTX, which inhibits
heterotrimeric Gi protein signal transduction (11),
and HA, a selective inhibitor of the Src family of protein tyrosine
kinases (12), were used. HA treatment of THP-1 cells had no effect on
the RANTES-induced signal, while PTX inhibited the response (Fig. 2A). This was
consistent with G protein-linked signaling in these cells. By
contrast, the RANTES response in Jurkat cells was not affected by
the addition of PTX, while treatment with HA resulted in a marked
abrogation of signal (Fig. 2B). Thus the RANTES
response in Jurkat cells seemed transduced through a tyrosine kinase
pathway and not through a PTX-sensitive G protein pathway; suggesting
that RANTES is capable of signaling through two distinct and separable
pathways. The THP-1 cells exhibited a rapid transient response mediated
via G proteins; the Jurkat cells showed a delayed and sustained
response via tyrosine kinases. The Jurkat cells provided us with a
means to investigate this tyrosine kinase-linked RANTES response.
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To ascertain whether the RANTES-induced tyrosine kinase pathway
could be engaged by other chemokines, we tested chemokines of both
C-X-C and C-C classes on the Jurkat cells. Figure 3 shows the Jurkat cell
Ca2+ response to final concentrations of 1 µM
MIP-1, MIP-1ß, MCP-3, and PF-4. The C-C chemokines MIP-1 and
MIP-1ß, which are functionally and structurally closely related
to RANTES, had no effect on the Jurkat cells. MCP-3 and PF-4 resulted
in little, if any, resultant Ca2+ flux as compared with
RANTES. Furthermore, the chemokines MCP-1 and IL-8 also failed to
induce a Ca2+ flux in these Jurkat cells (data not shown).
These data were consistent with results observed with the SPB21 T cell
clone (6), indicating that RANTES is unusual in its ability to activate
this tyrosine kinase signaling pathway.
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We next compared RANTES- and CD3-mediated signaling in Jurkat
cells. The addition of 1 µM RANTES or 2.5 µg/ml anti-CD3 mAb
(saturating concentration) resulted in Ca2+ fluxes of
similar magnitude in these Jurkat cells (Fig. 4). When a fivefold excess of the
extracellular Ca2+ chelator EGTA was added, the
Ca2+ release from intracellular stores was clearly
visible as a transient spike. This indicated that the sustained
Ca2+ fluxes seen in response to RANTES or anti-CD3 Ab
were attributable to Ca2+ entry from the extracellular
source. To test this hypothesis, we increased the extracellular
Ca2+ concentration from 1.6 mM to 10 mM approximately
70 s following stimulation. This increase in extracellular
Ca2+ concentration caused a temporary rise in the
Ca2+ flux followed by a rapid correction, presumably
through the closing of extracellular Ca2+ channels. This
phenomenon was seen with both RANTES and anti-CD3 mAb.
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We noted that not all Jurkat cell lines responded equally to
RANTES stimulation. We isolated a non-responding (NR) Jurkat cell line
that responded to RANTES in the Ca2+ mobilization
assay with less than 20% of the magnitude seen with the responding
Jurkats (Fig. 5A).
Strikingly, these NR Jurkat cells also had a marked attenuation of
their anti-CD3 response (Fig. 5B). FACS analyses
of CD3 expression on these two Jurkat populations revealed that 95% of
the responding Jurkats expressed CD3, while only 5% of the NR Jurkats
were CD3 positive (Fig. 5, C and D,
respectively). This suggested an association between CD3
expression and RANTES responsiveness.
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). While CD3
expression was dramatically different in these two populations, FACS
analyses of these two subpopulations using a variety of other cellular
markers (i.e., CD2, CD28, CD44) did not discern any other differences
(data not shown).
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). The responses to RANTES mirrored that of
anti-CD3 responses: CD3high Jurkats responded strongly
to RANTES, the CD3low Jurkats responded very weakly (Fig. 6). Thus RANTES appeared to require the presence of CD3 to induce a
Ca2+ signal in these Jurkat cells. Cross-modulation of RANTES and anti-CD3 induced signals
We examined whether anti-CD3 stimulation could down-regulate
the subsequent RANTES response. A mixed population of Jurkat cells,
incubated overnight with anti-CD3 mAb, were assayed for cytoplasmic
Ca2+ mobilization in response to RANTES or
anti-CD3 stimulation. Following the overnight anti-CD3 mAb
incubation, the surface expression of CD3 (as well as TCR) was
down-regulated as analyzed by FACS (using three different anti-CD3
mAbs and an anti-TCR /ß mAb for FACS analysis). Subsequently,
the cellular responses of these cells to both anti-CD3 mAb and
RANTES stimulation were significantly decreased (Fig. 7). Overnight incubation with an IgG1
isotype control did not down-regulate CD3 expression and had no effect
on subsequent cellular responses to RANTES and anti-CD3 mAb
stimulation (Fig. 7). These data strengthened the connection
between CD3 expression and RANTES responsiveness.
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To address the question of whether a RANTES-binding receptor was
expressed on these Jurkat cells, we employed equilibrium binding (Fig. 8). Displaceable binding with a
KD of 0.1 nM was observed on these cells; however, the
calculated receptor expression level was low, approximately 600 sites
per cell. These data then indicate that at least one chemokine receptor
is expressed upon these cells, but it is not at a level that could
easily explain the magnitude and kinetics of the resulting
RANTES-induced calcium flux.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At least three possible mechanisms may explain how RANTES might stimulate Jurkat T cells along a CD3-like signal transduction pathway. First, RANTES may signal by directly interacting with the TCR. Second, RANTES could transduce its signal through an unique receptor, such as a receptor tyrosine kinase or a seven transmembrane receptor coupled to PTX-insensitive G proteins with that receptor being coexpressed with CD3. A third possibility is that RANTES could bind to other cell surface moieties (such as extracellular matrix components), crosslinking the TCR through chemokine aggregation.
Ligand binding studies shed some light on these different possibilities. First, the data suggest that a high affinity receptor for RANTES is expressed on Jurkat cells, although the expression level is low. While Jurkat cells do have signaling responses to RANTES in a manner consistent with a pathway through this high affinity receptor (15), neither the binding affinity nor the receptor expression level are consistent with the magnitude and kinetics of the responses reported here. In addition, in our experience low expression levels (less than a thousand sites per cell) of chemokine receptors on cells of this type normally do not give rise to significant cytoplasmic calcium flux in response to chemokine stimulation.
RANTES aggregation appears to be a dynamic process with protein
aggregating and dissociating rapidly at equilibrium, and is dependent
upon protein concentration and buffer conditions (data not shown). It
is possible that glycosaminoglycans binding sites for RANTES exist on
the surface of Jurkat cells, allowing the chemokine to attach to the
cell surface via relatively low affinity interactions, but promoting
subsequent aggregation of RANTES into higher order aggregates. This
could explain the effect observed in the experiment depicted in Figure 8 upon addition of unlabeled RANTES at concentrations greater than 10
nM. Also, we have noted that monoclonal antibodies specific for RANTES
will react with the cell surface of Jurkat cells which have been
pre-incubated with the chemokine, and that the intensity of antibody
staining increases with increasing amounts of RANTES added to the cells
(data not shown). A cell surface aggregation process involving tethered
RANTES could result in the cross-linking of CD3/TCR complexes in a type
of molecular latticework, not dissimilar to what has been hypothesized
for CD4 (16), resulting in the activation of tyrosine kinase signaling
downstream of the TCR.
A wide variety of cell types express chemokines, either
constitutively or under appropriate stimulation (e.g., TNF-,
IFN-
, or IL-1ß). RANTES, for example, is expressed by leukocyte
subpopulations, fibroblasts, and endothelial cells (4). During an
inflammatory reaction, a significant chemokine release is thought to
occur within the immunologic microenvironment. It is possible that
chemokines from a transient release could be sequestered within local
environment and presented to responding target cells. In this way, the
local chemokine concentration of RANTES might well exceed 100 nM in
vivo. Chemokine concentration gradients, where the concentration is
directly proportional to the distance from the site of inflammatory
reaction, may exist to serve two distinct purposes. At a distance away
from the inflammatory site, the lower concentrations of chemokine could
serve a chemoattractant role for leukocytes populations. Closer to the
inflammatory site, however, higher chemokine concentrations (at least
of RANTES) may serve to activate specific populations of responding
cell. Consistent with a T cell priming role, RANTES activation would
leave the CD3/TCR surface expression intact, perhaps facilitating
specific Ag recognition subsequently.
In summary, we have shown that chemokine activation might be mediated through the action of molecules other than seven-transmembrane G protein-coupled receptors. Such knowledge may be useful in evaluating the roles of chemokines in inflammatory pathologies and in the design of therapeutic agents.
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Acknowledgments |
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Footnotes |
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2 Current address: Departments of Immunology and Surgery, Imperal College School of Medicine, Hammersmith Hospital, DuCane Rd., London W12 ONN, U.K.
3 Current address: Department of Hematopoietic Growth Factors, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan.
4 Current address: Neurocrine Biosciences, 3050 Science Park Rd., San Diego, CA 92121.
5 Address correspondence and reprint requests to Dr. Thomas J. Schall, ChemoCentryx c/o MMRI, 325 East Middlefield Road, Mountain View, CA 94043.
6 Abbreviations used in this paper: PTX, pertussis toxin; MIP-1,ß, macrophage inflammatory protein -1,ß; MCP-1,2,3, macrophage chemotactic protein -1, 2, 3; PF-4, platelet factor 4; HA, herbimycin A.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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