Published online before print November 30, 2007
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(SDF)-induced human T cell chemotaxis becomes phosphoinositide 3-kinase (PI3K)-independent: role of PKC-
Department of Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee, USA
1Correspondence: Department of Pharmacology, University of Tennessee Health Science Center, 874 Union Avenue, Memphis, TN 38163, USA. E-mail: bsharp{at}utmem.edu
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(SDF-1) is the exclusive ligand for the chemokine receptor CXCR4. This receptor plays a pivotal role in immune responses, the pathogenesis of infection such as HIV, and cellular trafficking. However, the signaling mechanisms regulating SDF-driven T cell migration are not well defined. In this study, we determined the role of PI3K and protein kinase C- 
(PKC-) in SDF-induced human T cell migration in fresh versus cultured T cells. Purified human T cells (fresh vs. 48 h in media, unstimulated or activated by anti-CD3+anti-CD28) were used. Western blots showed that SDF induced phospho-(p)-Akt [threonine (Thr)308 and serine 473], a proxy for PI3K activity, in fresh cells and p-PKC- in 48 h unstimulated cells. LY294002 (PI3K inhibitor) reduced SDF-induced chemotaxis in fresh cells by 51%, whereas it minimally affected chemotaxis in 48 h unstimulated or activated cells. However, a specific PKC- inhibitor, pseudosubstrate for PKC-, reduced chemotaxis in 48 h unstimulated and stimulated T cells by 72% and 87%, respectively. Thus, chemotaxis becomes independent of PI3K signaling in human T cells cultured for 48 h. Under these conditions, PKC- is phosphorylated (Thr538) by SDF, and chemotaxis becomes largely PKC--dependent.
Key Words: Akt • LY294002 • pseudosubstrate • CXCR4
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(SDF-1), also known as CXCL12, is a member of the CXC or -chemokine subfamily. It is the natural ligand for the chemokine receptor CXCR4, the coreceptor for the X4 HIV envelope [4
5
6
]. SDF-1 and CXCR4 are expressed in a large number of tissues [6
7
8
9
] including T and B lymphocytes and monocytes. SDF-1 has been shown to act as a potent chemoattractant for lymphocytes [10
].
PI3K has been shown to mediate the chemotaxis of T lymphocytes induced by SDF-1 [11
]. Chemokines such as MCP-1 and SDF-1 stimulate PI3K, leading to the formation of phosphatidyl 3,4,5-triphosphate (PIP3) and in T cells, to the activation of Akt {protein kinase B (PKB) [12
, 13
]}. PIP3 interacts with the pleckstrin homology domain of Akt, causing its translocation to the inner leaflet of the plasma membrane where two critical residues [threonine (Thr)308 and serine (Ser)473], required for activation of Akt, are phosphorylated by 3-phosphoinositide-dependent kinase 1 (PDK-1) [14
, 15
].
The signal transduction pathways required for chemotaxis may vary according to the specific cellular phenotype. In the case of platelet-derived growth factor (PDGF), Higaki et al. [16
] have shown that PDGF-induced chemotaxis is independent of PI3K activity in vascular smooth muscle cells and Swiss 3T3 cell types, whereas PDGF-induced amino acid uptake and glucose incorporation are dependent on PI3K. More recently, Carnevale and Cathcart [17
] demonstrated the involvement of a classical PKC, PKC-β, in the signaling pathway for human monocyte chemotaxis in response to MCP-1. Other investigators have also emphasized the pivotal role of PKC isoforms in chemotaxis. Using PKC inhibitors and the specific pseudosubstrate for PKC-
or a PKC- overexpression cell line, Petit et al. [18
] have shown that the SDF-1-induced migration of CD34 cells from umbilical cord blood and pre-B acute lymphoblastic leukemia cells is PKC--dependent.
The PKC family consists of more than 12 serine threonine kinases that vary in tissue distribution, subcellular translocation, and function [19
]. A member of the novel class of PKC isotypes whose activation is diacylglycerol-dependent and calcium-independent, PKC- is predominantly expressed in T lymphocytes and to a lesser extent, in B cells [20
, 21
]. PKC- is critical to the activation of T lymphocytes [22
]. During antigen presentation, PKC- is the only PKC isoform that is rapidly recruited to the central domain of the plasma membrane interface formed by a T cell and an APC [23
, 24
]. Its recruitment to this site is essential for the induction of IL-2 transcription [25
, 26
]. PKC- activates NF-
B and AP-1, transcription factors that are directly involved in IL-2 transcription [27
28
29
]. Indeed, the phosphorylation of Thr-538 (p-Thr538) in the activation loop of PKC- is critical for the activation of NF-B [30
]. Recently, it has been reported that SDF-1-induced chemotaxis was dependent on NF-B [10
]. Therefore, we queried the potential involvement of PKC- in SDF-1-induced chemotaxis of T cells.
In these studies, we first determined the role of PI3K in SDF-1-induced chemotaxis by human T cells in different activation states: fresh versus cultured versus stimulated (anti-CD3+anti-CD28). In so doing, we compared the efficacy of the PI3K inhibitor LY294002 (LY) in blocking SDF-1-induced chemotaxis with the ability of SDF-1 to stimulate Akt phosphorylation (a proxy for PI3K activity) by T cells obtained in these three different activation states. We found that LY was only effective in fresh T cells, yet SDF-1 stimulated Akt phosphorylation in all groups of T cells. Therefore, we hypothesized that PKC- mediates SDF-1-induced chemotaxis of cultured T cells and that SDF-1 would induce the phosphorylation of PKC-. We found that two different PKC antagonists significantly inhibited SDF-1-induced chemotaxis of cultured T cells: rottlerin, a class-specific PKC inhibitor (IC50, 3–6 µM, for novel class PKC isoforms including PKC- vs. IC50, 30–42 µM, for classical PKC- and -β), and myristoylated PKC- pseudosubstrate, a specific inhibitor of PKC-. Additionally, SDF-1 induced the phosphorylation of PKC-. Therefore, the role of PI3K in mediating SDF-1-induced chemotaxis of T cells depends on the activation state of the cells; PKC- is involved in cultured cells, where chemotactic signaling no longer depends on PI3K.
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(Thr538 in the activation loop), were purchased from Cell Signaling Technology (Beverly, MA, USA). The following kinase inhibitors were from Calbiochem (San Diego, CA, USA): Go6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole], PKC- inhibitor (myristoylated pseudosubstrate peptide), rottlerin, LY [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], wortmannin (W), and PD98059. Fluorescent anti-OKT3 was from BD Biosciences PharMingen (San Diego, CA, USA).
Cell isolation and purification
Buffy coats were obtained from anonymous, healthy male donors (Lifeblood Biological Service, Memphis, TN, USA), and T cells were isolated using the T cell enrichment cocktail from StemCell Technologies Inc. (Vancouver, BC, Canada). Briefly, 50 µl human T cell enrichment cocktail was added to 1 ml buffy coat and incubated for 20 min. Samples were diluted with PBS plus 2% FBS, mixed gently, layered over RosetteSep DM-L (StemCell Technologies), and centrifuged at 1200 g for 20 min at 22°C. Enriched cells at the interface were collected, washed with PBS plus 2% FBS, centrifuged at 500 g, and mixed with alkaline lysing buffer (0.15 M NH4Cl, 0.01 M KHCO3, 0.01 M sodium EDTA, pH 7.4). Cells were resuspended in RPMI 1640 containing 10% FBS. T cell purity was determined by flow cytometry using anti-OKT3. As determined by anti-OKT3, T cell purity was consistently >97%.
Cell culture
T cells were maintained under three conditions: Freshly obtained T cells (3x106/ml) were suspended in RPMI 1640 containing 0.2% BSA for 20 min; T cells were incubated for 48 h in complete medium containing RPMI 1640 with 10% FBS, 100 IU/ml penicillin, and 0.1% streptomycin; and activated T cells were cultured for 48 h in complete medium containing 1 µg/ml anti-CD28 (BD Biosciences PharMingen) in flasks with immobilized anti-OKT3 (35 ng/cm2, eBioscience, San Diego, CA, USA). After 48 h, activated T cells and quiescent cells in complete medium were subsequently incubated in serum-free medium for 16 h; cell viability was consistently >96%.
Chemotaxis assay
Chemotaxis was performed in 96-well Multiscreen-minimal inhibitory concentration plates with 5 µm porosity polycarbonate membranes (Millipore, Billerica, MA, USA). Prior to chemotaxis assays, T cells were treated with different inhibitors or controls for 20–60 min. Each sample (50 µl; 150,000 cells) was then loaded in the upper compartment of a well in which the lower chamber contained 150 µl serum-free medium ± SDF-1, 5 nM (R&D Systems, Minneapolis, MN, USA). Plates were incubated for 2 h at 37°C in 5% CO2, and then 100 ml cellular suspension from each of the lower compartments was transferred to a 96-well plate to determine the number of migrated cells, which was measured using the CellTiter-Glo luminescent assay (Promega, Madison, WI, USA), using a BioTek FLx800 microplate reader (BioTek Instruments, Winooski, VT, USA); the luminescence of an unknown samples was compared with the emission from a standard serial dilution of a known number of cells.
Blast cells were quantified using an EPICS XL flow cytometer equipped with an argon laser (excitation: 488 nm). In each sample from the upper and lower migration chamber, blast cells were identified by side-scatter, and cell flow was stopped after 1 min (at least 105 events). Microscopic determination confirmed the accuracy of the flow cytometric quantitation.
Cell membrane preparation
Cells were washed briefly with PBS, lysed using 500 ml radioimmunoprecipitation assay (RIPA) buffer [1x PBS, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, and a mixture of protease and phosphatase inhibitors], and then sonicated three times (2–3 s each) on ice. Lysates were centrifuged at 120,000 g for 1 h at 4°C. Pellets were resuspended in RIPA buffer containing 0.1% Triton X-100 and sonicated briefly, and then protein concentrations were determined using the bicinchoninic acid protein assay kit. Membranes were boiled for 5 min, and 5–10 µg protein was loaded on 11% SDS polyacrylamide gels (1.5 mm depth).
Western immunoblotting
SDS polyacrylamide gels were transferred to a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA) over 16 h at 24°C. Membranes were briefly washed with TBS (20 mM Tris-HCl, 137 mM NaCl, 0.05% Tween-20, 0.05% NP-40, pH 7.7) and then blocked in TBS containing 5% nonfat dry milk for 2 h. They were washed three times with TBS and incubated overnight at 4°C with primary antibody. Membranes were washed extensively and incubated with a peroxidase-tagged secondary antibody (Pierce, Rockford, IL, USA) for 1 h at room temperature, and chemiluminescence was detected (Super Signal ULTRA, Pierce).
Statistical analyses
One-way ANOVA was performed, and post-hoc testing was done using the Bonferroni test. Differences were considered significant at P < 0.05. Values are expressed as mean ± SEM.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-induced chemotactic responses by human T cells in different activation states-induced chemotaxis by human T cells in different activation states: fresh versus cultured in complete medium alone versus stimulated (anti-CD3+anti-CD28). In so doing, we compared the efficacy of the PI3K inhibitors, LY and W, in blocking SDF-1-induced chemotaxis by T cells under these three conditions. In the presence of LY for 60 min, the viability of fresh cells was 98 ± 0.65. As expected, SDF-1 (5 nM) stimulated the chemotaxis of freshly isolated human T cells by more than 400% of control (Fig. 1
). Chemotaxis was significantly inhibited by W (100 nM) and LY (20 and 50 µM; F=12.5, P<0.0001; P=0.02 and P<0.001 for W and LY, respectively), although LY was more effective (LY20 and LY50 vs. W: P=0.04 and P=0.007, respectively). However, 45% of the chemotactic response persisted in presence of the higher concentration of LY. We also determined whether signaling through the MAPKs, ERK1/2, is required for SDF-1-induced chemotaxis in fresh T cells. Pretreatment with PD98059 (30 µM), which inhibits activation of ERK1/2, did not affect chemotaxis.
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Figure 1. Effects of PI3K inhibitors on SDF-1-induced chemotaxis of freshly isolated human T cells. As described in Materials and Methods, 3 x 106/ml T cells were pretreated with vehicle (C), 100 nM W (W100), 20 or 50 µM LY (LY20, LY50), or 30 µM PD98059 (PD30) for 60 min prior to measuring the chemotactic response to 5 nM SDF-1 for 2 h. Control (C/C) samples were incubated with medium alone. Results are expressed as percent of control (mean±SEM). The data are from four to five different subjects per treatment, each performed in quadruplicate. Preliminary studies showed that SDF, 2.5–5.0 nM, induced near-maximal (90% of peak) chemotaxis by purified T cells. In response to SDF, the migratory fraction was 39.5 ± 4.0%. C/SDF significantly increased chemotaxis compared with C/C (*), and W/SDF or LY/SDF was significantly less than C/SDF (**; F=19.65, P<0.0001; P<0.0001 for C/C vs. C/SDF; P=0.020 for C/SDF vs. W/SDF; P<0.001 for C/SDF vs. LY20/SDF or LY50/SDF).
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-induced chemotaxis is regulated by PI3K in human T cells cultured with complete medium or activated with anti-OKT3 and anti-CD28 for 48 h. In both T cell activation states, SDF-1-induced chemotaxis was not significantly reduced by PI3K inhibition (i.e., W, 100 nM, or LY, 20 µM) or by MEK inhibition (Figs. 2
and 3
). Figure 3
shows that chemotaxis by activated T cells was marginally reduced (27%; P<0.05) by the higher concentration (50 µM) of LY; however, no significant reduction was observed in cells cultured in complete medium (Fig. 2)
.
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Figure 2. Effects of PI3K inhibitors on SDF-1-induced chemotaxis of T cells cultured in medium for 48 h. T cells (3x106/ml) were pretreated with vehicle, 100 nM W, 20 or 50 µM LY, or 30 µM PD98059 for 60 min prior to measuring the chemotactic response to 5 nM SDF-1 for 2 h. Control (C/C) samples were incubated with medium alone. Results from eight subjects, measured in quadruplicate, are expressed as percent of control (mean±SEM). The migratory fraction responding to SDF-1 was 46.5 ± 2.3%. SDF increased chemotaxis, and W or LY failed to inhibit this response (F=13.01, P<0.0001; *, P<0.0001, for C/C vs. C/SDF).
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Figure 3. Effects of PI3K inhibitors on SDF-1-induced chemotaxis by T cells cultured with anti-OKT3 + anti-CD28 for 48 h. T cells (3x106/ml) were pretreated with vehicle, 100 nM W, 20 or 50 µM LY, or 30 µM PD98059 for 60 min prior to measuring the chemotactic response to 5 nM SDF-1 for 2 h. Control (C/C) samples were incubated with medium alone. Results from six subjects, measured in quadruplicate, are expressed as percent of control (mean±SEM). The migratory fraction responding to SDF-1 was 37.9 ± 3.5%. SDF increased chemotaxis (F=13.96, P<0.0001; *, P<0.0001, for C/C vs. C/SDF; **, P<0.05, for LY50/SDF vs. C/SDF).
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SDF-1 stimulates Akt phosphorylation by human T cells in different activation states
As SDF-1-induced chemotaxis depends on PI3K in fresh T cells, and Akt, a major downstream effector of PI3K, is a proxy for PI3K activity, we measured the phosphorylation of Akt stimulated by SDF-1 [11
, 31
]. We also compared the phosphorylation of Akt in fresh T cells to T lymphocytes cultured in medium ± anti-CD3 + anti-CD28. p-Thr308 and p-Ser473 Akt were measured; the former is required for the catalytic activity of Akt, and the latter modulates this activity [32
]. To minimize the basal phosphorylation of Akt, T cells cultured in medium ± anti-CD3 + anti-CD28 were starved overnight prior to treatment with SDF-1. Stimulation for 5 min with 5 nM SDF-1 (C/SDF) markedly increased the phosphorylation of membrane-associated Akt at Thr308 and Ser 473 (Figs. 4A
5A,
and 6A
). Pretreatment with the specific PI3K inhibitor, LY at 20 or 50 µM (LY20 or -50/SDF), significantly reduced the phosphorylation of Akt at both sites under all three T cell conditions (Figs. 4B
5B
and 6B)
. In general, LY, 50 µM, was not significantly more effective than LY, 20 µM. However, W appeared to be less effective than LY at p-Ser473, and it did not significantly diminish p-Ser473 Akt in activated cells (Fig. 6B)
. Inhibition of MEK had no effect on SDF-1-induced phosphorylation of Akt. Comparison of the effects of LY on chemotaxis versus Akt phosphorylation demonstrated that blockade of PI3K significantly reduced Akt phosphorylation by SDF-1-stimulated T cells under all three conditions, whereas LY only inhibited the chemotaxis of fresh T cells.
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Figure 4. Effects of PI3K inhibitors on SDF-1-induced phosphorylation of Akt in freshly isolated T cells, which were pretreated with 100 nM W, 20 or 50 mM LY, or 30 mM PD98059 for 60 min and then stimulated with 5 nM SDF-1 for 5 min. Membrane lysates were prepared as described in Materials and Methods. (A) p-Akt was determined by Western immunoblotting with anti-p-Ser473 Akt (top blot) and anti-p-Thr308 Akt (third blot). These blots were reprobed with an antibody against total Akt (second and bottom blots). The data are from a single representative subject. (B) The densitometric analysis of immunoblots from four subjects, expressed as percent of control (mean±SEM). C/SDF increased p-Ser473 and p-Thr308 Akt compared with C/C (*; F=31.12, P<0.0001, and F=4.97, P<0.015, respectively; P<0.0001 and P<0.01 for p-Ser473 and p-Thr308 Akt, respectively), and W100/SDF or LY20 and -50/SDF significantly reduced C/SDF-induced phosphorylation of Akt (**; P<0.0001 and P<0.01, for p-Ser473 and p-Thr308 Akt, respectively).
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Figure 5. Effects of PI3K inhibitors on SDF-1-induced phosphorylation of Akt in T cells cultured in medium for 48 h. T cells were pretreated with 100 nM W, 20 or 50 mM LY, or 30 mM PD98059 for 60 min and then stimulated with 5 nM SDF-1 for 5 min. Membrane lysates were prepared as described in Materials and Methods. (A) p-Akt was determined by Western immunoblotting with anti-p-Ser473 Akt (upper blot) and anti-p-Thr308 Akt (lower blot). The data are from a single representative subject. (B) The densitometric analysis of immunoblots from 10 subjects, expressed as percent of control (mean±SEM). C/SDF increased p-Ser473 and p-Thr308 Akt compared with C/C (*; F=11.16, P<0.0001, and F=4.96, P<0.0001, respectively; P<0.0001 and P=0.0002 for p-Ser473 and p-Thr308 Akt, respectively), and W100/SDF or LY20 and -50/SDF significantly reduced C/SDF-induced phosphorylation of Akt (**; P<0.0002, for all comparisons except P=0.0035 for p-Ser473 of W100/SDF).
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Figure 6. Effects of PI3K inhibitors on SDF-1-induced phosphorylation of Akt in T cells cultured with anti-OKT3 + anti-CD28 for 48 h. T cells were pretreated with 100 nM W, 20 or 50 mM LY, or 30 mM PD98059 for 60 min and then stimulated with 5 nM SDF-1 for 5 min. (A) p-Akt was determined by Western immunoblotting with anti-p-Ser473 Akt (top blot) and anti-p-Thr308 Akt (third blot). The data are from a single representative subject. (B) The densitometric analysis of immunoblots from three subjects, expressed as percent of control (mean±SEM). C/SDF increased p-Ser473 and p-Thr308 Akt compared with C/C (*; F=3.78, P<0.003, and F=6.59, P<0.0001, respectively; P<0.002 and P=0.0001 for p-Ser473 and p-Thr308 Akt, respectively), and W100/SDF or LY20 and -50/SDF significantly reduced C/SDF-induced phosphorylation of Akt (**, P<0.01 and P<0.0005, for p-Ser473 and p-Thr308, respectively).
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is involved in SDF-1-induced chemotaxis by human T cells-induced T cell chemotaxis may depend on the activation of NF-B and on PKCs, we evaluated the potential role of PKC-, the predominant isoform in T lymphocytes and the one that activates NF-B [10
20
, 26
, 27
]. First, we tested Go6976, a cell-permeable inhibitor of PKC-, -β, and -
(classical isoforms) at nM concentrations, which is ineffective at blocking the Ca2+-independent PKC isoforms 
, 
, , and , even at µM concentrations [33
]. Pretreatment of T cells cultured in medium with 0.6 µM Go6976 failed to inhibit SDF-1-induced chemotaxis [Go6976 (G)/SDF vs. C/SDF; Fig. 7
]. Similarly, Go6976 did not reduce SDF-1-induced chemotaxis by fresh T cells [chemotactic responses (percentage of control): SDF-1=343.0±77.8; Go6976+SDF-1=381.3±68.5]. These results indicate that classical PKC isoforms are not involved in SDF-1-induced chemotaxis.
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Figure 7. Effects of PKC inhibitors on SDF-1-induced chemotaxis of T cells cultured in medium for 48 h. T cells were pretreated with Go6983 (600 nM), a selective inhibitor for Ca2+-dependent PKC- and -β; rottlerin, 10 µM (R10; for 1 h), a specific inhibitor of novel PKC isoforms including PKC- and -; or a PKC- pseudosubstrate inhibitor, 10–15 µM (PS10, PS15; for 20 min), prior to measuring the chemotactic response to 5 nM SDF-1 for 2 h. Control (C/C) samples were incubated with medium alone. Results from four to seven subjects, measured in quadruplicate, are expressed as percent of control (mean±SEM). SDF significantly increased chemotaxis, and R10 or PS reduced this response (F=12.72, P<0.0001; *, P<0.0001, for C/C vs. C/SDF; **, P<0.0001, for C/SDF vs. R10 or PS10 and -15/SDF).
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and -, whereas the IC50 values are 30–42 µM for classical PKC and 80–100 µM for atypical PKC. Therefore, in T cells, rottlerin is an antagonist of PKC- [34
] and PKC- [35
]. The two experiments shown in Figures 7
and 8
demonstrate that rottlerin, 10 µM, significantly reduced SDF-1-induced chemotaxis in T cells cultured in medium (Figs. 7
and 8
: F=12.7, P<0.0001; F=22.7, P<0.0001, respectively; C/SDF vs. R10/SDF, P<0.0001, in both figures). Rottlerin also reduced chemotaxis by fresh T cells [chemotactic responses (percentage of control): C/SDF-1=343.0±77.8 vs. R10/SDF-1=57.3±23.2, P=0.0006; F=11.8, P=0.0003]. Thus, SDF-1-induced chemotaxis by fresh T cells depends on PI3K and PKC. Cell viability after treatment with rottlerin was: 98.0% ± 1.2 in fresh T cells; 97.2% ± 0.8 in cultured cells.
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Figure 8. Effects of PI3K and PKC inhibitors and their combinations on SDF-1-induced chemotaxis by T cells cultured in medium for 48 h. T cells were pretreated with LY, 20 µM; LY, 50 µM; rottlerin, 10 µM; and LY20 + R10 or LY50 + R10 for 1 h prior to measuring the chemotactic response to 5 nM SDF-1 for 2 h. Control (C/C) samples were incubated with medium alone. Results from three to four subjects, measured in quadruplicate, are expressed as percent of control (mean±SEM). SDF significantly increased chemotaxis, and R10 reduced this response (F=22.61, P<0.0001; *, P<0.0001, for C/C vs. C/SDF; **, P<0.0001, for C/SDF vs. R10/SDF or R10+LY/SDF).
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is specifically involved in chemotaxis by using the N-myristoylated pseudosubstrate of PKC- [36
]. Figure 7
shows that SDF-1-induced T cell chemotaxis was significantly reduced (72%) by 15 µM myristoylated pseudosubstrate of PKC- (F=12.7, P<0.0001; P<0.0001 for comparison of C/SDF vs. PS15/SDF). The viability of cells pretreated with 15 µM pseudosubstrate was 90%. As a result of the toxicity of PS in fresh T cells, the role of PKC- could not be evaluated directly. In summary, the efficacy of rottlerin and of PS in the inhibition of SDF-1-induced T cell chemotaxis indicates that PKC- mediates the chemotactic effects of SDF-1 in cultured human T cells.
Additional studies were initiated to determine whether signaling through PI3K could be detected if PKC- were inhibited during SDF-1-induced chemotaxis by T cells cultured in medium (Fig. 8)
. T cells were pretreated with LY ± rottlerin (R10). As expected, R10 alone significantly reduced chemotaxis (R10/SDF), whereas LY, 20 or 50 µM, was ineffective (LY20 or -50/SDF). Neither concentration of LY enhanced the efficacy of R10. Thus, the involvement of PI3K in SDF-1-induced chemotaxis by cultured T cells cannot be detected despite concomitant blockade of PKC-.
Studies were also conducted to evaluate the role of PKC- in chemotaxis by activated T cells and T cell blasts. Figure 9
shows that rottlerin and 10–15 µM of the pseudosubstrate of PKC- inhibited SDF-1-induced chemotaxis by T cells (e.g., total T cells) cultured for 48 h with anti-OKT3 + anti-CD28 (F=27.53, P<0.0001; P<0.0024 for C/SDF vs. R10/SDF or PS10 and -15/SDF). The chemotaxis of blast T cells (all groups expressed relative to the total T cell control group) showed a fold increase induced by SDF-1 (C/C vs. C/SDF) that was similar to the fold change observed in total T cells. Blast cell chemotaxis was inhibited to a similar extent by the pseudosubstrate of PKC- (F=40.57, P<0.0001; P<0.0001 for C/SDF vs. PS10 and -15/SDF).
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Figure 9. Effects of PKC inhibitors on SDF-1-induced chemotaxis of T cells and T cell blasts cultured with anti-OKT3 + anti-CD28 for 48. T cells were pretreated with rottlerin, 10 µM (for 1 h), or a PKC- pseudosubstrate inhibitor, 10–15 µM (for 20 min), prior to measuring the chemotactic response to 5 nM SDF-1 for 2 h. Control (C/C) samples were incubated with medium alone. Results in total T cell and blast cell groups, obtained from three to five subjects and measured in quadruplicate, are all expressed as percent of the total T cell control group (mean±SEM). The migratory fraction of blast cells responding to SDF-1 was 47.1 ± 5.3% of the total blast population. SDF significantly increased chemotaxis, and PS abolished this response in total T cell and blast cell populations (total T cells: F=27.53, P<0.0001; *, P<0.0001, for C/C vs. C/SDF; **, P<0.0024, for C/SDF vs. R10 or PS10 and -15/SDF; blast cells: F=40.57, P<0.0001; *, P<0.0001, for C/C vs. C/SDF; **, P<0.0001, for C/SDF vs. PS10 and -15/SDF).
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at Thr538 by SDF-1 was evaluated by immunoblotting membrane versus cytosolic fractions from cultured T cells (Fig. 10
). SDF-1 induced p-PKC- in a time-dependent manner. The effect of SDF-1 on the level of p-PKC- was detected in the cytosolic fraction at 1 min, reaching maximum levels at 3 min. In the membrane fraction, increased phosphorylation also was evident by 1 min; phosphorylation was maximal by 5–10 min and similar to control by 15 min.
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Figure 10. Time course of the induction of p-PKC- by SDF-1 in T cells cultured in medium for 48 h. T cells were treated with 5 nM SDF-1 for the indicated time intervals. Thereafter, cell membrane lysates and cytosol were obtained, and p-PKC- was detected with anti-p-Thr-538 PKC-. SDF-1 phosphorylated PKC- in a time-dependent manner. This result from a single subject is representative of three separate experiments.
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appear to depend on the PDK-1. As our studies have shown that SDF-1-induced chemotaxis by T cells cultured in medium alone is unaffected by PI3K antagonists, we directly determined whether one of these agents can inhibit the SDF-1-induced phosphorylation of PKC-. Figure 11
shows that neither LY 20 nor 50 µM reduced the level of p-PKC- stimulated by SDF-1 in cytosol or membrane. [Note: For the membrane fraction, the control (C/C) is shown in Lane 2.]
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Figure 11. Effect of a PI3K inhibitor on the p-PKC- induced by SDF-1. T cells were pretreated with LY, 20 µM or 50 µM, for 1 h prior to stimulation with 5 nM SDF-1 for 5 min. Thereafter, cell membrane lysates and cytosol were obtained, and p-PKC- was detected with anti-p-Thr-538 PKC-. Inhibition of PI3K had no effect on the levels of p-PKC-. This experiment with a single subject is representative of two separate studies.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the current studies of multiple human donors demonstrated that signaling through PI3K is only involved in chemotaxis by fresh T cells [11
, 13
]. Under these conditions, inhibition of PI3K significantly reduced the chemotactic response but only by 
50%. In contrast, we found that rottlerin, a selective inhibitor of novel PKC isoforms including PKC- and -, was effective at reducing chemotaxis by 75% in fresh T cells. Rottlerin also inhibited chemotaxis by 60% in T cells cultured in medium alone (Fig. 7)
, which were insensitive to PI3K antagonists. In these cells, the myristoylated pseudosubstrate of PKC- reduced chemotaxis by 72%. Additionally, the phosphorylation of PKC- was induced by SDF-1. In summary, these studies demonstrate that the involvement of PI3K in SDF-1-induced chemotaxis depends on the state of the T cells. The PI3K/Akt pathway is activated by SDF-1 in all three T cell states (i.e., fresh, cultured in medium, and activated) studied herein, yet only in freshly obtained T cells is SDF-1-induced chemotaxis partially dependent on PI3K. Chemotaxis by cultured T cells becomes PI3K-independent and largely PKC--dependent.
A previous study of activated human T cells, derived from PBMCs cultured for 9–12 days in the presence of IL-2, demonstrated that 10–100 nM SDF-1 stimulated a marked increase in phosphorylated Akt within 1 min; levels had declined toward the baseline by 20 min after the 10-nM concentration of SDF-1 [13
]. Similarly, we observed a fivefold increase of p-Ser473 Akt in activated human cells after 5 min of SDF-1 (5 nM). Nevertheless, the activation of PI3K was largely unrelated to SDF-1-induced chemotaxis, which was only reduced by 27% in response to LY, 50 µM, in activated cells and was unaffected by LY, 20 µM. A recent report provides novel insight into the function of the PI3K signaling cascade that is stimulated by SDF-1 in quiescent and activated T cells. Kumar et al. [37
] reported that SDF-1 induces the physical interaction of CXCR4 with the TCR, and this depends in part on PI3K activity and is independent of TCR activation. This close association of CXCR4 and the TCR enables SDF-1 to stimulate the tyrosine kinase ZAP-70, resulting in prolonged activation of ERKs [13
, 38
]. Additionally, ZAP-70 has been shown to enhance T cell chemotaxis to SDF-1 [39
, 40
].
A subset of proteins essential to the T cell activation cascade that is located within the multiprotein signaling complex [supramolecular activation complex (SMAC)] in the immunological synapse, a contact site formed by the T cell and APC, is most probably involved in signaling by the associational complex formed by CXCR4 and the TCR [41
]. Formation of this associational complex is induced by SDF-1 and does not appear to require concomitant activation of the TCR. The scaffold protein, Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), present within the SMAC at the immunological synapse, contains protein interaction domains that recruit and mediate binding to phospholipase C (PLC)1 and other proteins. As SLP-76 is required for the prolonged activation of ERKs induced by CXCR4 signaling in T cells [38
], SLP-76 is probably located close to the associational complex of CXCR4 and the TCR. ZAP-70, which interacts with ITAM, such as those contained in TCR subunits within the SMAC, also requires ITAM domains for signaling by SDF-1 through the CXCR4-TCR complex [37
]. Moreover, within the SMAC, ZAP-70 is known to transduce downstream signals by phosphorylating substrates such as SLP-76 [42
]. The rapid recruitment of PKC- to the immunological synapse requires PLC1 and SLP-76 [27
], and the activation of PKC- requires diacylglycerol cleaved from the membrane by PLC1 [43
]. Therefore, PLC1 and SLP-76 appear to be required for the recruitment and activation of PKC- by the CXCR4-TCR complex, and this process might involve ZAP-70 through its effect on SLP-76.
In T cells, the catalytic activity of PKC- depends on PDK-1-induced phosphorylation at Thr538 [44
]. Additionally, the membrane localization of PKC- to lipid rafts is dependent on PDK-1, whose activity is regulated to a large extent by PI3K [45
]. Although we observed that SDF-1-induced chemotaxis was independent of PI3K in cultured T cells, LY was efficacious at inhibiting the phosphorylation of Akt stimulated by SDF-1 under these conditions. Thus, the dependence of SDF-1-induced chemotaxis on PKC-, despite the failure of a PI3K antagonist to inhibit chemotaxis, suggests that the activation of PKC- by PDK-1 may be largely independent of PI3K in quiescent T cells or those activated by anti-CD3 + anti-CD28. Indeed, results from the current studies are consistent with this view. We demonstrated that inhibition of PI3K had no effect on the phosphorylation of PKC- induced by SDF-1 in T cells cultured in complete medium alone. Similarly, the PDK-1-dependent phosphorylation of conventional PKC isoforms is known to be independent of PI3K, although activation loop phosphorylation of the atypical isoform PKC- is moderately sensitive to PI3K inhibitors [46
]. In accord with these findings, activated PI3K does not appear to be required for the PKC--dependent chemotaxis of quiescent or activated T cells induced by SDF-1, although SDF-1 does stimulate PI3K activity in these cells.
In summary, SDF-1 signals through PI3K in fresh, cultured, and activated human T cells, yet only in fresh T cells does SDF-1-induced chemotaxis depend in part on PI3K. In fresh T cells, chemotaxis is also dependent on PKC, as evident by the efficacy of rottlerin. In T cells cultured in medium with or without activation, SDF-1-induced chemotaxis becomes independent of PI3K signaling. Experiments with T cells cultured in medium alone or with anti-CD3 + anti-CD28 demonstrated that SDF-1-induced chemotaxis is largely dependent on PKC-, which is phosphorylated in a PI3K-independent manner by SDF-1.
Received June 20, 2007; revised October 16, 2007; accepted November 5, 2007.
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