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Department of Immunobiology, DNAX Research Institute, Palo Alto, CA 94304
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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), monocyte-chemotactic protein-1 (MCP-1), MCP-3, MCP-4,
and eotaxin. RANTES-induced PLD activation was augmented by GTP
S,
but not GDPßS, and inhibited by the protein kinase C inhibitor
bisindolylmaleimide, as well as the fungal metabolite brefeldin A, and
C3 exoenzyme (Clostridium botulinum),
implicating the activation of RhoA. RANTES also induced GTP-GDP
exchange of immunoprecipitated RhoA. RANTES-stimulated PLD activity was
dependent on an ADP-ribosylation factor(s), as assessed by inhibition
studies using a synthetic inhibitory peptide of the N-terminal 16 amino
acids of ADP-ribosylation factor 1. These studies indicate the
potential existence of a novel receptor-mediated mechanism for
activation of T cells by the chemokine RANTES. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and NAP-2; CXCR3, binding
IP-10 and monokine induced by IFN-; and CXCR4/fusin/LESTR, binding
SDF-1 (2, 8, 9, 10). Receptors binding members of the C-C family
include CCR-1 (affinity hierarchy:
MIP-1,2 RANTES), CCR-2
(MCP-1 through 5), CCR3 (eotaxin, RANTES, MCP-2, 3, 4), CCR-4 (TARC),
and CCR-5 (MIP-1ß, RANTES, MIP-1) (11, 12, 13, 14). While certain virally
encoded receptors may also bind a number of these chemokines (1, 2, 3, 11), a receptor for lymphotactin remains to be identified.
We have demonstrated recently that the chemokine RANTES is capable of
stimulating T lymphocyte activation via direct receptor-mediated
activation of dual signal-transduction pathways involving pertussis
toxin-sensitive heterotrimeric G proteins and tyrosine kinase enzymes
(15). Initially, RANTES stimulates an immediate transient of calcium
mobilization, followed by a more sustained flux through specific
membrane channels. Concurrently, there is rapid tyrosine
phosphorylation of intracellular proteins, including
pp125FAK, paxillin, and ZAP-70, but not TCR-
(16). One
manifestation of these signals is the secretion of IL-2 and the
up-regulation of numerous cell surface adhesion receptors (15, 17). The
Jurkat T cell line, however, does not respond to RANTES activation with
a rapid Ca2+ transient, nor does it migrate in response to
this chemokine (unpublished), making it a model system to use in
investigations of other RANTES-mediated signal-transduction events.
In our prior investigations of chemokine-induced signal transduction, we had demonstrated the potent induction of phospholipase D (PLD) activity by the C-X-C chemokine IL-8 in human T lymphocytes and T cell clones (18). PLD activation is now recognized as an important component of numerous cellular functions, including vesicular trafficking and cell transformation (19, 20, 21). While the existence of multiple isoforms of PLD has been postulated, the identification and cloning of the PLD enzyme in mammalian cells have only recently been described (22, 23). Membrane-associated PLD catalyzes the hydrolysis of phosphatidylcholine and is dependent on calcium, phosphoinositol bisphosphate, and the GTP-binding proteins ARF (ADP-ribosylation factors) and RhoA (21, 24, 25). Other nonmammalian isoforms of PLD have also been shown to preferentially hydrolyze phosphatidylinositol and phosphatidylethanolamine (19, 20, 21, 26). Most recently, Mayr et al. have demonstrated a novel phosphatidylethanolamine and phosphatidylserine-specific PLD in yeast that exhibits absolute dependence on calcium (27). While hydrolyzing phosphatidylcholine to phosphatidic acid (PA) and choline is known to be a direct consequence of numerous ligand-receptor interactions, the exact role of PA is as yet unclear. Of major importance is the finding that PLD is a vital component of the Golgi membrane and absolutely required in vesicular transport. Indeed the major function of the family of small GTP-binding proteins, the ARFs, is the activation of PLD (28, 29, 30). It is suggested that the generation of negatively charged lipid metabolites, including PA, is necessary for the docking of coatamer proteins to the lipid membrane in vesicle formation (31). These findings suggest a vital role for PLD activation in intracellular protein transport and vesicle formation.
In this study, we report on the activation of PLD in Jurkat cells by the C-C chemokine RANTES. We show that RANTES stimulates a potent and specific PLD activation that is dependent on prior activation of PKC. Additionally, the requirement for GTP, the reduction in PLD activation by the fungal metabolite Brefeldin A (BFA), and an inhibitory N-terminal peptide of mammalian ARF-1 indicate that the chemokine-induced activation of PLD in Jurkat is dependent on one or more ARFs. Evidence inferring a role for the activation of the monomeric GTP-binding protein RhoA is detailed from experiments using the C3 exoenzyme (Clostridium botulinum) as well as GDP/GTP exchange assays with immunoprecipitated RhoA. The activation of ARF- and Rho-regulated PLD suggests a potentially important role for RANTES not only as a proinflammatory stimulus, but also in the general physiology of T lymphocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640, phosphate-free RPMI (11877-032), DMEM, dialyzed FCS,
and BFA (Penecillum brefeldianum) were
obtained from Life Technologies (Grand Island, NY), and gentamicin,
GTP, and GDP were purchased from Sigma Chemical Co. (St. Louis, MO).
GTPS and GTPßS were from Boehringer Mannheim Corp. (Indianapolis,
IN); bisindolylmaleimide (GF109203X), Staphylococcus aureus
-toxin, and C3 exoenzyme (C. botulinum)
were from Calbiochem (San Diego, CA). [3H]oleic acid and
125I-RANTES were from DuPont NEN (Boston, MA);
[32P]orthophosphate (10 mCi/ml, 370 MBq/ml) was from
Amersham (Arlington Heights, IL); and PEI cellulose plates were from
Selecto Scientific (Norcross, GA). RhoA mAb (IgG1) was from Santa Cruz
Biotechnology (Santa Cruz, CA), and the PLD inhibitors SCH49209 and
SCH50672 (32) were kindly provided by Drs. R. Bryant and K. Pai
(Schering Plough Research Institute, Kenilworth, NJ). Inhibitory ARF
peptide (2–17 mARF1p; GNIFANLFKGLFGKKE) (33), a similar peptide,
but with scrambled peptide sequence (FLKEGKFALFGNINK), and an
N-terminal peptide of Gs (GCLGNSKTEDQRNEEK) were synthesized by
Research Genetics (Huntsville, AL). The chemokines RANTES, MIP-1ß,
MIP-1, MCP-1, and MCP-3 were purchased from R&D Systems
(Minneapolis, MN). Eotaxin and MCP-4 were purchased from PeproTech
(Rocky Hill, NJ). All other reagents were obtained through standard
suppliers.
PLD activity
The cell line Jurkat was maintained in RPMI 1640 containing 10% heat-inactivated FCS and gentamicin (50 µg/ml). Activation and analysis of PLD were performed as previously described (18). In brief, cells in DMEM containing dialyzed FCS were incubated overnight (37°C) with 50 mCi/ml of [3H]oleic acid. Labeled cells were harvested, washed twice, and resuspended in DMEM containing 130 mM ethanol and left to equilibrate for 15 min. Following equilibration, cells (5 x 106) were aliquoted into siliconized glass tubes containing agonists and/or antagonist and incubated for 40 min at 37°C with periodic shaking. After incubation, cells were rapidly pelleted and the medium was removed. Pellets were lysed with methanol:HCl (100:1, v/v):chloroform:0.25 M HCl (1:1:1, v/v) and vortexed for 10 to 15 s to disrupt the cell pellet. Addition of 100 µg 1,2-dioleoyl-sn-glycero-3-phosphoethanol as a tracer was followed by centrifugation to enable lipid-phase extraction. The dried phospholipids were resuspended in 20 µl of chloroform, and the reaction products were separated by TLC on silica gel 60 plates (Merck, Rahway, NJ) using a mobile phase of ethyl acetate:2,2,4-trimethylpentane:acetic acid:water (13:2:3:10, v/v). The radiolabeled phosphatidylethanol was located by staining the plate with iodine vapour (using tracer). The product was scraped from the plate, and radioactivity was assessed by liquid scintillation counting using Cytoscint (ICN Pharmaceuticals, Costa Mesa, CA). In experiments using BFA or PKC inhibitor (GF109203X), cells were preincubated with increasing concentrations of inhibitor for 20 min before assay. Cell viability, as assessed using trypan blue exclusion, was always greater than 98%.
Permeabilization of cells was conducted in glutamate buffer (120 mM
potassium glutamate, 20 mM potassium acetate, 3 mM magnesium chloride,
20 mM sodium HEPES, pH 7.4, 1 mM EGTA, and 1 mg/ml BSA) using -toxin
(34, 35). Cells were pelleted and resuspended in 20 ml glutamate buffer
containing 250 U/ml -toxin and incubated (37°C) for 20 min with
periodic shaking. Permeabilization was assessed using trypan blue.
Pelleted cells were then resuspended in glutamate buffer containing 0.1
µM free calcium and 130 mM ethanol and added to siliconized glass
tubes for assay. In assays incorporating GDPS or GTPßS,
nucleotides were added to permeabilized cells at the same time as
RANTES. In contrast, permeabilized cells were preincubated for 20 min
with ARF peptide, C3 exoenzyme, or PLD inhibitors before RANTES
stimulation. Again, cell viability, as assessed using trypan blue
exclusion, was consistently greater than 98%.
Chemokine-binding assay
Equilibrium binding of 125I-RANTES using a filtration protocol was employed. Briefly, 1 x 106 cells were incubated with 0.1 nM 125I-RANTES in the presence of various unlabeled chemokines in the following buffer for 3 h at 22°C: 25 mM HEPES, 80 mM NaCl, 1 mM CaCl2, 5 mM MgCl2, and 0.5% BSA, adjusted to pH 7.4. The reactions were then aspirated onto PEI-pretreated GF/C filters (Packard, Meriden, CT) using a 96-well cell harvester (Packard). The reactions were washed twice with the following buffer: 25 mM HEPES, 500 mM NaCl, 1 mM CaCl2, and 5 mM MgCl2, adjusted to pH 7.4. After the filters dried, 30 µl of scintillant (Microscint 10; Packard) was added and the resulting cpm were measured on a Packard Top count. The resulting data were analyzed using Igor Pro software (Wavemetrics, Lake Oswego, OR).
Assays of RhoA GDP/GTP exchange
To assess the activity of the small GTP-binding protein RhoA in the RANTES-induced PLD activation, assays of GDP/GTP exchange were performed (36). Jurkat T cells were resuspended at 5 x 107/ml in phosphate-free RPMI 1640/10% dialyzed FCS and incubated overnight with 0.5 mCi/ml of [32P]orthophosphate. Cells were pelleted and resuspended at 2 x 107/ml in assay buffer (PBS, 1 mM CaCl2, 1 mM MgCl2, and 1 mg/ml BSA). Samples (0.5-ml) were stimulated with RANTES for the indicated times, and cells were subsequently rapidly centrifuged at 4°C. Cells were lysed (50 mM HEPES buffer, pH 7.4, 1% Triton X-100, 150 mM NaCl, 5 mM MgCl2, 1 mg/ml BSA, containing protease and phosphatase inhibitors; 1 mM PMSF, 10 µg/ml each of aprotinin and leupeptin, 1 mM sodium orthovanadate, 1 mM EGTA, 100 µM ß-glycerophosphate, 10 mM sodium fluoride, 1 mM tetrasodium pyrophosphate). Lysates containing 100 µg of protein (as assessed using the bichinconinic acid reagent (Pierce, Rockford, IL) were incubated with 2 µg of anti-RhoA mAb (4°C, 1 h). Immune complexed Rho was captured using goat anti-mouse IgG-agarose (Sigma Chemical Co.; 4°C, 1 h). Immunoprecipitates were washed eight times in buffer containing 50 mM HEPES, pH 7.4, 500 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, and 0.005% SDS, and nucleotides were eluted with 5 mM EDTA, 2 mM DTT, 0.2% SDS, 0.5 mM GDP, and 0.5 mM GTP for 20 min at 68°C. Eluted nucleotides were separated on PEI cellulose plates run in 1.2 M ammonium formate/0.8 M HCl and visualized by autoradiography. Following removal of eluate, Laemmli sample buffer was added to the immunoprecipitates. After boiling for 5 min and electrophoresis on 16% Tris-glycine gels (NOVEX, San Diego, CA), the equivalence of RhoA immunoprecipitation was visualized by Western blot analysis using the same RhoA Ab. Blots were developed using enhanced chemiluminescence reagent (Amersham) and Biomax MR film (Eastman Kodak, Rochester, NY).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RANTES stimulation of Jurkat cells fails to induce a rapid initial
transient of Ca2+ (not shown), unlike classical
chemokine receptor-mediated signaling described for RANTES in other T
cell systems (15, 37). RANTES stimulation of Jurkat cells did, however,
result in a dose-dependent increase in [3H]PtE (Fig. 1
A), an indicator of
PLD activation (38). Initial experiments to determine the time course
of activation of PLD indicated that maximal cumulation of tritiated
product (PtE) occurred after 40 min (Ref. 18 and not shown); thus, all
additional experiments were performed for this length of time.
RANTES-induced PLD activity was observed following stimulation over the
concentration range of 0.03 to 1 nM RANTES, maximal activation being
between two- and fourfold above resting (unstimulated) controls (Fig. 1, A and B). Concentrations greater than 1
nM RANTES failed to give consistently higher values of
[3H]PtE. PMA, as expected, induced a large increase in
[3H]PtE (14,017 ± 2,317 cpm in unpermeabilized
cells; 12,592 ± 2,006 cpm increase in permeabilized cells);
however, there was little, if any, difference between cells from the
two experimental conditions.
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S augments RANTES-induced PLD activation
There was little, if any, difference between nonpermeabilized and
permeabilized cells (Fig. 1
, A and B),
which was to be expected due to the mild nature of the permeabilization
protocol and the rapid reversal of permeabilization in the presence of
free Ca2+. PLD has been shown to be dependent on the
activation of cofactors, including PIP2, ARFs, and the
GTP-binding protein Rho. In the presence of increasing concentrations
of the nonhydrolyzable analogue of GTP, GTPS, there was a
concomitant increase in the [3H]PtE product formed
following RANTES stimulation (Fig. 1C). Specificity
of this response was determined by the lack of increase in
[3H]PtE when cells were incubated in the presence of
GDPßS. Interestingly, the PLD activity was decreased in the presence
of this analogue.
RANTES dissociable binding to Jurkat cells
We used equilibrium binding to assess the presence and
affinity of a putative chemokine receptor for RANTES on these cells.
Preliminary experiments indicated that we could see dissociable RANTES
binding using a physiologic salt buffer, but not in the absence of salt
(a buffer generally used with CCR-3). Further characterization
indicated little sensitivity to different pH conditions of the buffer,
but rather peak total binding was observed with 80 mM NaCl in the
reaction buffer. As shown in Figure 2A, Jurkat cells express a
receptor that binds RANTES with an affinity of 0.1 nM and that is
expressed on these cells at approximately 600 sites per cell (over the
population). Consistent with CCR-5, MIP-1ß was capable of competing
RANTES binding with a Ki of 0.3 nM. Of note,
however, was the finding that MIP-1 was incapable of competing
125I-RANTES from the Jurkat receptor, a property not
consistent with binding characteristics ascribed to CCR-5. Heterologous
chemokine competitors, which were tested to confirm or deny the partial
expression of other characterized chemokine receptors on these cells,
were capable of completely displacing 125I-RANTES
from the Jurkat receptor (Fig. 2B).
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The activation of PLD by the chemokine IL-8 was shown to be PKC
dependent (18). To assess whether RANTES (a C-C chemokine) stimulates
PLD through a common mechanism, we analyzed the PKC dependence using
the specific, cell-permeant PKC inhibitor bisindolylmaleimide
(GF109203X; Ki = 10 nM). Increases in
[3H]PtE induced by 1 nM RANTES (maximal agonist
concentration) were reduced to basal levels in a dose-dependent manner
by the PKC inhibitor, with >50% inhibition occurring with 1 nM
concentration (Fig. 3). Although addition
of 10-8 M RANTES increased the maximal
[3H]PtE recovered in the presence of each concentration
of GF109203X, the maximal PLD activation was never greater than 75% of
control, and there was still almost complete inhibition in the presence
of 100 nM inhibitor (not shown).
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Using 1 µg/ml of the inhibitor BFA as an initial indication for
the role of small GTP-binding proteins in the RANTES-induced PLD
activation resulted in complete inhibition of the
[3H]PtE levels (Fig. 4A). There was a slight
increase in the basal levels of PLD activity in control (unstimulated)
samples in the presence of 1 µM BFA, possibly as a result of the
structural disintegration of the Golgi apparatus and other organelles
in the cytoplasm by BFA (39, 40). Inhibition of [3H]PtE
accumulation occurred with concentrations of BFA as low as 30 nM, with
maximal inhibition occurring in the presence of 1 µM BFA.
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The activity of BFA and GTPS, in the inhibition and activation
of PLD, respectively, was consistent with a role for the ARF family of
small GTP-binding proteins (28, 29, 30). To assess the contribution of ARFs
to the PLD activation stimulated by RANTES, an inhibitory peptide of
human ARF1 (IC50 = 75 µM in ARF-dependent assay of
ADP ribosylation of Gs) was used in subsequent assays (33).
Nonmyristylated ARF N-terminal peptide, but not the same peptide with
scrambled sequence, was able to inhibit the RANTES-induced PLD
activity dose dependently at concentrations from 1 to 100 µM (Fig. 4B). In contrast, a control peptide corresponding to
the N terminus of Gs was ineffective in inhibiting RANTES-induced
PLD (not shown).
RhoA activation mediates the RANTES-induced PLD response
Since Rho has been shown to be a necessary cofactor for PLD
activity, we analyzed the effect of C3 exoenzyme (C.
botulinum), a specific inhibitor of Rho (41, 42), on
PLD activation induced by 1 nM RANTES. Figure 4C
demonstrates that preincubation of cells with C3 exoenzyme dose
dependently inhibited the RANTES-induced PLD activation. To
further characterize the role of Rho, GDP/GTP exchange on
immunoprecipitated Rho was investigated. Figure 4D
demonstrates that there is a basal level of [32P]GTP
present in the immunoprecipitates from unstimulated cells. This is most
likely a reflection of the intrinsic activation of this immortalized
cell line. RANTES stimulates rapid exchange of GDP for GTP on RhoA,
with significant elevation in [32P]GTP as early as
30 s. The increase in GTP continued to increase up to a maximum at
5 min in this assay system. In addition, this increase was seen with
concentrations of 10-9 M, the concentration most effective
in stimulating PLD activation. The loading of equal amounts of RhoA in
the samples was indicated by Western blot analysis of eluted immune
complexed Rho from the samples, as described in Materials and
Methods.
SCH49209 and SCH56072 inhibit PLD activity in Jurkat
In a final analysis, the specificity of the PLD activity was
determined by the use of PLD inhibitors SCH49209 and SCH56072. Reported
as inhibitors of receptor-stimulated PLD and PLA2
(IC50 = 2 µM for FMLP-stimulated PLD activity in HL-60
cells) (32), these compounds were analyzed over the concentration range
(0.1 nM-3 µM). Figure 5 shows that
there was a potent dose-dependent inhibition of PLD activity stimulated
by 1 nM RANTES. Half-maximal inhibition occurred using 1 µM of
compound, and there was no significant difference between the
analogues.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
MIP-1ß, MCP-1, MCP-3, lymphotactin) that induces as robust a response
as seen in this study, although the others listed were capable of low
levels of activity (not shown). Binding analyses have revealed a receptor for RANTES, expressed at a low level on these Jurkat cells. Heterologous competition analyses demonstrated inhibitory activity in the order of potency of RANTES > MIP-1ß = MCP-3 > MCP-4 > eotaxin > MCP-1, indicating a unique specificity in ligand binding. Additionally, the buffer conditions utilized in these experiments are inconsistent with binding to known receptors. In addition, only CCR-4 has been shown to be present on Jurkat, while CCR-1, 2, 3, and 5 have not been detected (43). It is feasible that there is more than one receptor, although the complete displacement obtained with each ligand in isolation points away from this hypothesis. Alternatively, Jurkat cells may express cell surface moieties that modify the binding characteristics (hence, the experimental procedures required to observe them) of the known receptors. Finally, it is not inconceivable that there is a novel, previously unrecognized CCR, which may explain this unique binding pattern and the relative specificity of RANTES over other chemokines in eliciting this signal-transduction pathway in Jurkat cells.
We have demonstrated that activation of PLD in Jurkat conforms to the standard requirements for the activity of the cofactors ARF and RhoA. Use of the fungal metabolite BFA as an inhibitor clearly indicated a role for intact Golgi and ARF proteins. The mode of activation of BFA is thought to be through the destruction of vesicular and Golgi membranes (39, 40), in addition to inhibition of ARF-guanine nucleotide exchange factor activity on ARFs (44). Our studies have demonstrated that BFA can completely inhibit the RANTES-induced PLD activation. Exactly which ARF family members are activated and/or the most important cofactors awaits further characterization in this system. Use of the N-terminal peptide of ARF1 does, however, implicate this family member in the activation of Jurkat PLD. In addition, the potency of the ARF peptide in these experiments is similar to that reported (33), indicating the utility of this inhibitory peptide in determining ARF dependency. Further information concerning the identity of ARF family members in Jurkat may help to determine the specificity of the mechanisms involved in RANTES-mediated PLD activation.
RhoA has been shown to be necessary for PLD activation; however, there have been reports that Rac family members may also be important as cofactors (23, 45). In our investigations of RANTES-induced activation of cytoskeletal components (19), we consistently immunoprecipitated RhoA and Rac with activated pp125FAK, suggesting activation of these small GTP-binding proteins. Our preliminary evidence from the assays reported in this study suggests that Rac is not activated (in terms of GDP/GTP exchange; not shown). In addition, Rac is a poor substrate for C3 (41, 42). Our studies on the activation of FAK were, however, performed on T cells and T cell clones that exhibit classical heterotrimeric G protein-mediated calcium flux profiles and are known to express the receptors CCR1 and 5 (K.B.B., unpublished). Jurkat T cells do not respond to RANTES with rapid Ca2+ transients (possibly due to the low level of receptor expression), and it is likely that the signal-transduction mechanisms activated in normal T cells attain greater complexity with the additional component of immediate, store-released calcium flux.
That RANTES-induced PLD activation occurs downstream of PKC activation is not surprising. Previous analyses in T cells, using anti-CD3 and IL-8, have clearly demonstrated a PKC dependency of the PLD activation (18, 46). Additional systems, using agonists binding to seven-transmembrane-type receptors, have shown agonist-stimulated PLD activation that was PKC dependent (47, 48, 49, 50, 51). Thus, it is not unwarranted that RANTES should be working through a similar mechanism. Once again, exactly where the PKC activation is derived is unknown. It is feasible that the cumulative PLD response measured in this study is dependent on cyclical PKC activation; thus, inhibition of PKC would ultimately result in decreased product of PLD activity. Dephosphorylation by the enzyme PA phosphohydrolase can generate diacylglycerol, which leads to PKC activation. Our present studies, however, await more precise kinetic measurements of PKC activity.
The role of PLD activation in T cells is still unclear. It has been postulated that PA, the major metabolite, can induce activation of the AP-1 transcription factor (52). Indeed, we have demonstrated recently the potent activity of RANTES in stimulating T cell proliferation (15), as well as the up-regulation of numerous cell surface receptors (17). The nuclear activation events leading to cell proliferation, being in turn governed by the regulation of AP-1, may in fact be a direct consequence of the PA generation from RANTES-induced PLD activation. The inhibitors used in this study to inhibit PLD have been shown to inhibit FMLP-induced PLA2 activity in HL-60 cells, but not A23187-induced [3H]arachidonic acid release, suggesting some selectivity on receptor-induced PLA2 in certain cell systems. RANTES-mediated PLD activation appears to occur via binding to a specific receptor; however, we have not investigated RANTES-stimulated PLA2 activation in Jurkat. Since PLA2 has been shown to stimulate Rho activation in bradykinin-stimulated cell lines (53), but not PLD activity due to tyrosine kinase activation (32), it is interesting to speculate that these inhibitors are functioning upstream of the phospholipases themselves, possibly at the GTP-binding proteins or their guanine nucleotide exchange factors.
Perhaps of greater relevance, when one considers the activation of ARFs
and Rho, is the control of vesicular trafficking. Little is known to
date of the receptor fate following agonist (chemokine) ligation. It is
not improbable that ligand-mediated receptor endocytosis of chemokine
receptors is linked somehow to the efficient assembly of coatamer
proteins. Efficient coatamer binding to membranes has been linked to
the presence of high concentrations of negatively charged lipids in the
membrane bilayer. The highly negatively charged PA has been shown to
provide such an environment (31). Indeed, constitutive PLD activity in
certain membrane preparations has been shown to be sufficient to allow
vesicle formation in the absence of ARF activation (54). Furthermore,
the ARF family of proteins has been implicated in the retrograde
transport of proteins between Golgi and endoplasmic reticulum (44),
making this RANTES-induced activation of PLD a likely candidate
mechanism for receptor down-regulation. Interestingly, numerous
chemokines of the C-C family (MIP-1, MIP-1ß, MCP-1, MCP-3) and
lymphotactin of the C family are capable of inducing PLD activation in
normal peripheral blood T and B lymphocytes (K.B.B., unpublished). It
is not unlikely, therefore, that chemokine-induced PLD activation may
be responsible for receptor down-regulation as a common mechanism.
In conclusion, we have demonstrated the potent and specific activation of PLD by a C-C chemokine, RANTES, in a model T cell system, the Jurkat T cell line. While the receptor(s) mediating this effect awaits characterization, it is clear that activation of this signal-transduction pathway, either in physiologic or inflammatory contexts, by RANTES, will have profound effects on the regulation of cell activity. Additionally, activation of this pathway in vivo may have important implications in chronic inflammation.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; ARF, adenosine diphosphate-ribosylation factor; BFA, Brefeldin A; 125I-RANTES, 125I-labeled RANTES; MCP, monocyte-chemotactic protein; PA, phosphatidic acid; PEI, polyethyleneimine; PKC, protein kinase C; PLA, phospholipase A; PLD, phospholipase D; PtE, phosphatidylethanol.
Received for publication July 23, 1997. Accepted for publication October 27, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 T. L.-Y. Chang, C. J. Gordon, B. Roscic-Mrkic, C. Power, A. E. I. Proudfoot, J. P. Moore, and A. Trkola Interaction of the CC-Chemokine RANTES with Glycosaminoglycans Activates a p44/p42 Mitogen-Activated Protein Kinase-Dependent Signaling Pathway and Enhances Human Immunodeficiency Virus Type 1 Infectivity J. Virol., March 1, 2002; 76(5): 2245 - 2254. [Abstract] [Full Text] [PDF]
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 M. A. Wetzel, A. D. Steele, T. K. Eisenstein, M. W. Adler, E. E. Henderson, and T. J. Rogers {micro}-Opioid Induction of Monocyte Chemoattractant Protein-1, RANTES, and IFN-{gamma}-Inducible Protein-10 Expression in Human Peripheral Blood Mononuclear Cells J. Immunol., December 1, 2000; 165(11): 6519 - 6524. [Abstract] [Full Text] [PDF]
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R. M. Richardson, B. C. Pridgen, B. Haribabu, and R. Snyderman Regulation of the Human Chemokine Receptor CCR1. CROSS-REGULATION BY CXCR1 AND CXCR2 J. Biol. Chem., March 24, 2000; 275(13): 9201 - 9208. [Abstract] [Full Text] [PDF]
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A. Trkola, C. Gordon, J. Matthews, E. Maxwell, T. Ketas, L. Czaplewski, A. E. I. Proudfoot, and J. P. Moore The CC-Chemokine RANTES Increases the Attachment of Human Immunodeficiency Virus Type 1 to Target Cells via Glycosaminoglycans and Also Activates a Signal Transduction Pathway That Enhances Viral Infectivity J. Virol., August 1, 1999; 73(8): 6370 - 6379. [Abstract] [Full Text]
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D. J. Dairaghi, R. A. Fan, B. E. McMaster, M. R. Hanley, and T. J. Schall HHV8-encoded vMIP-I Selectively Engages Chemokine Receptor CCR8. AGONIST AND ANTAGONIST PROFILES OF VIRAL CHEMOKINES J. Biol. Chem., July 30, 1999; 274(31): 21569 - 21574. [Abstract] [Full Text] [PDF]
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C. J. Gordon, M. A. Muesing, A. E. I. Proudfoot, C. A. Power, J. P. Moore, and A. Trkola Enhancement of Human Immunodeficiency Virus Type 1 Infection by the CC-Chemokine RANTES Is Independent of the Mechanism of Virus-Cell Fusion J. Virol., January 1, 1999; 73(1): 684 - 694. [Abstract] [Full Text] [PDF]
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 M. Stamnes, G. Schiavo, G. Stenbeck, T. H. Sollner, and J. E. Rothman ADP-ribosylation factor and phosphatidic acid levels in Golgi membranes during budding of coatomer-coated vesicles PNAS, November 10, 1998; 95(23): 13676 - 13680. [Abstract] [Full Text] [PDF]
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M. Wong, S. Uddin, B. Majchrzak, T. Huynh, A. E. I. Proudfoot, L. C. Platanias, and E. N. Fish RANTES Activates Jak2 and Jak3 to Regulate Engagement of Multiple Signaling Pathways in T Cells J. Biol. Chem., March 30, 2001; 276(14): 11427 - 11431. [Abstract] [Full Text] [PDF]
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