Abstract
Th1 cells exposed to Ag and the G1 blocker n-butyrate in primary cultures lose their ability to proliferate in Ag-stimulated secondary cultures. The ability of n-butyrate to induce anergy in Ag-stimulated, but not resting, Th1 cells was shown here to be blocked by cycloheximide. Subsequent experiments to delineate the nature of the protein apparently required for n-butyrate-induced Th1 cell anergy focused on the role of cyclin-dependent kinase (cdk) inhibitors p21Cip1 and p27Kip1. Normally, entry into S phase by Th1 cells occurs around 24 h after Ag stimulation and corresponds with relatively low levels of both p21Cip1 and p27Kip1. However, unlike control Th1 cells, anergic Th1 cells contained high levels of both p21Cip1 and p27Kip1 when examined 24 h after Ag stimulation. The increase in p21Cip1 observed in Ag-stimulated anergic Th1 cells appeared to be initiated in primary cultures. In contrast, the increase in p27Kip1 observed in these anergic Th1 cells appears to represent a re-expression of the protein much earlier than control cells following Ag stimulation in secondary cultures. The anergic Th1 cells contained functionally active cdk inhibitors capable of inhibiting the activity of both endogenous and exogenous cdks. Consequently, it appears that n-butyrate-induced anergy in Th1 cells correlated with the up-regulation of p21Cip1 and perhaps the downstream failure to maintain low levels of p27Kip1. Increased levels of both p21Cip1 and p27Kip1 at the end of G1 could prevent cdk-mediated entry into S phase, and thus help maintain the proliferative unresponsiveness found in the anergic Th1 cells.
Thelper 1 cells stimulated with Ag in the presence of the G1 blocker n-butyrate lose their ability to respond to Ag, even after n-butyrate has been removed from the cultures (1). Only those Th1 cells that are exposed to both Ag and n-butyrate are inactivated; Th1 cells exposed to n-butyrate alone are unaffected.
Experiments were initiated to investigate the molecular mechanism by which n-butyrate induced anergy in Th1 cells. These experiments were based on the following observations: 1) Th1 cells inactivated by exposure to n-butyrate and Ag are blocked in early G1 (1); 2) G1 cell cycle blockade can be induced by cyclin dependent-kinase inhibitors (CDKIs)3 (2, 3); and 3) n-butyrate promotes the expression of the p21Cip1/p27Kip1 family of CDKIs (4, 5, 6). It seemed possible that the ability of n-butyrate to induce proliferative unresponsiveness in Th1 cells was due to alterations in the levels of one or more members of the p21Cip1/p27Kip1 family of CDKIs.
Resting T cells contain a high level of p27Kip1 which is thought to maintain a quiescent state by suppressing the ability of cyclin-cyclin-dependent kinase (cdk) complexes to phosphorylate retinoblastoma protein (pRb), thereby suppressing T cell exit from G1 (2, 7). Following T cell activation and IL-2 synthesis, p27Kip1 is down-regulated, thus promoting pRb phosphorylation and S phase entry (2, 7, 8, 9). Although p27Kip1 is decreased in T cells following Ag stimulation, p21Cip1 is up-regulated. p21Cip1 is thought to play several roles in cell cycle progression; at low levels, such as those seen in late G1 phase, p21Cip1 has been shown to promote cyclin/cdk assembly, thus promoting phosphorylation of pRb. In contrast, higher levels of p21Cip1, such as those observed later in the cell cycle, are thought to inhibit cyclin/cdk activity, thus helping to end cell cycle progression. The normally inverse relationship between p21Cip1 and p27Kip1 results in a narrow window of time toward the end of G1 where neither CDKIs are present in high enough quantities to suppress cdk-mediated pRb phosphorylation and S phase entry. Cellular perturbations that alter the balance of p21Cip1 and p27Kip1 in Th1 cells could presumably alter normal cell cycle progression. This study was conducted to determine whether the loss of Ag-specific proliferation observed in n-butyrate-induced Th1 cell anergy was linked to alterations in p21Cip1 and/or p27Kip1.
Materials and Methods
Animals and reagents
Male C57BL/10 mice at 6–8 wk of age were purchased from Harlan Sprague Dawley (Indianapolis, IN). Keyhole limpet hemocyanin (KLH) (Imject) was purchased from Pierce (Rockford, IL). The anti-p27Kip1 mAb (clone G173-524, mouse IgG1), anti-p21Cip1 mAb (clone SX118, mouse IgG1), the mouse IgG1 isotype standard (clone MOPC-21), the PE-anti-mouse CD4 (clone H129.19, rat IgG2a), and the FITC-anti-mouse IgG1 (clone G1-6.5, rat IgG2a) was purchased from PharMingen (San Diego, CA). The HRP-labeled rabbit anti-mouse IgG and HRP-labeled goat anti-rabbit IgG Ab was purchased from Transduction Laboratories (Lexington, KY). The anti-cdk2 Ab (rabbit IgG), anti-cdk4 Ab (rabbit IgG), anti-cdk6 Ab (rabbit IgG), and the anti-actin Ab (goat IgG) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-cyclin D2 mAb (clone34B1-3, rat IgG2a), the anti-cyclin D3 mAb (clone 18B6-10, rat IgG2a), the anti-cyclin E (rabbit IgG), and the HRP-labeled goat anti-rat IgG were also purchased from Santa Cruz Biotechnology. The Western blot recycling kit was purchased from Alpha Diagnostic (San Antonio, TX).
Th1 clones
The KLH-specific Th1 cells (clone D9) were developed as described previously (10), except that C57BL/10 mice were used and the Ag consisted of KLH. The Th1 clone is passed every 7–14 days using Ag (KLH, 25 μg/ml), irradiated syngeneic splenic APC, and IL-2-containing Con A-stimulated conditioned medium (Con A CM) as described elsewhere (10).
Inducing Th1 cell anergy
Th1 cells were incubated in primary cultures at 2.5 × 105 cells/ml along with 5 × 106/ml irradiated syngeneic spleen cells as APC, KLH (30 μg/ml), and 1 mM n-butyrate (Sigma, St. Louis, MO) as described previously (1). Control primary cultures received APC and either n-butyrate or Ag alone. In some experiments, the primary cultures also contained cycloheximide (0.25 μg/ml; Sigma). After incubation for 48 h at 37°C, the cells in the primary cultures were washed and reincubated in medium without n-butyrate. After a 0- to 3-day resting period, the Th1 cells were recultured at 2.5 × 105/ ml along with 5 × 106/ml irradiated syngeneic spleen cells as APC and KLH (30 μg/ml unless otherwise stated). After various time intervals in the secondary cultures, the Th1 cells were assessed for DNA synthesis (pulsed with [3H]TdR for 12 h), or passed over Ficoll-Hypaque to remove the irradiated APC and disrupted with lysing buffer containing 10 mM KCl, 10 mM HEPES, 1.5 mM MgCl2, 0.5% Nonidet P-40, 1 mM NaVO4, aprotinin (10 mg/ml), leupeptin (10 mg/ml), and 0.5 mM PMSF.
Western blotting and kinase assay
For Western blot analysis, Th1 cell lysates were analyzed, using equivalent amounts of protein (30–50 μg), on 12% SDS-polyacrylamide gels. The proteins were electrotransferred onto nitrocellulose (Amersham Life Sciences, Buckinghamshire, U.K.), and subsequently immunoblotted with different primary Abs (1–2 μg/ml) and appropriate secondary Abs, i.e., HRP-conjugated goat anti-mouse IgG (1:2000) or HRP-conjugated goat anti-rabbit IgG (1:2000) or HRP-conjugated goat anti-rat IgG (1:500). Immunodetection was performed by ECL using Hyperfilm ECL (Amersham Life Sciences). To test for appropriate protein loading, some blots were stripped with the Western blot recycling kit and reprobed with the anti-actin Ab. pRb phosphorylation status in Th1 cell lysates was analyzed using cell equivalents (2.5 × 105 cells/sample) disrupted in pRb lysing buffer containing 5% 2-ME, 10% glycerol, 0.625 mM Tris-HCl (pH 6.8), and 2% SDS. The proteins were resolved on 7.5% SDS-polyacrylamide gels and subsequently immunoblotted with mouse anti-pRb mAb (clone G3-245, mouse IgG1), purchased from PharMingen, and the appropriate secondary Ab.
CDKI activity was measured using a variation on a previously described approach (11). Briefly, cdk2 was immunoprecipitated from a lysate (100 μg/sample) of asynchronous EL4 cells using streptavidin-coated magnetic beads (Dynal, Great Neck, NY), biotinylated goat anti-rabbit IgG Fc Ab (Jackson ImmunoResearch, West Grove, PA), and rabbit anti-cdk2 Ab (Santa Cruz Biotechnology). The immune complexes were mixed with 100 μg of the lysate prepared from different Th1 cell preparations and incubated for 30 min at 30°C. The Th1 cell lysates were boiled before mixing. The immune complexes were next washed twice with lysing buffer and once with kinase buffer and tested for activity in a kinase assay kit (Upstate Biotechnology, Lake Placid, NY) using H1 as a substrate and 10 μCi of [γ -32P]ATP. The kinase reaction mixtures were incubated for 10 min at 30°C, transferred to phosphocellulose paper, and levels of γ-32P incorporation quantitated using a scintillation counter (Beckman LS6000TA; Beckman, Fullerton, CA). The results are presented as total cpm minus the background cpm measured in samples containing immunoprecipitated cdk2 but no substrate.
To measure endogenous cdk2 activity, cdk2 was immunoprecipitated from 100 μg of Th1 cell lysate/sample and tested in the kinase assay kit as described above. The results are presented as total cpm minus the background cpm measured in samples containing immunoprecipitated cdk2 but no substrate. In addition, some of the immunoprecipitated cdk2 was resolved on 12% SDS-polyacrylamide gels and immunoblotted with anti-p21Cip1 or anti-p27Kip1 Ab.
DNA analysis
Th1 cells were incubated in primary cultures at 2.5 × 105 cells/ml along with 5 × 106/ml irradiated syngeneic spleen cells as APC, and KLH (100 mg/ml). At various time intervals after Ag stimulation, the Th1 cells were isolated from the primary cultures and fixed in prechilled 70% ethanol at 4°C overnight. The fixed Th1 cells were next washed in PBS, resuspended in 1 ml of PBS containing RNase (1 mg/ml; Sigma) and propidium iodide (50 μg/ml; Sigma), incubated for 20 min in the dark at room temperature, and analyzed by flow cytometry using a FACScalibur (Becton Dickinson, Mountain View, CA). The data were analyzed using the ModFit DNA analysis program.
Flow cytofluorometric analysis of cell cycle regulatory proteins
The Th1 cells were stained with PE-anti-CD4 mAb (2.5 μg/ml for 30 min at 4°C) and then fixed in 2% formaldehyde overnight at 4°C. The fixed Th1 cells were next washed three times with PBS and reincubated for 5 min at 4°C in 0.5 ml of a 0.25% solution of Triton X-100 to permeabilize the cells. Following one wash in 1× PBS, the cells were stained overnight at 4°C with either mouse anti-p27Kip1 mAb (40 μg/ml) or control mouse IgG1 (40 μg/ml), then incubated for 1 h at 4°C with FITC-goat anti-mouse IgG (10 μg/ml), and analyzed on a FACScan (Becton Dickinson). The samples were gated on the PE-positive Th1 cells, and the results are presented as FITC histograms or as FITC vs forward scatter (FCS) dot plots.
Results
Characterization of n-butyrate-induced anergy in KLH-specific Th1 cells
The ability of n-butyrate to induce anergy in murine Th1 cells was first demonstrated in human γ-globulin-specific Th1 cells derived from A/J mice (H-2a) (1). Experiments were conducted here to confirm that n-butyrate-induced proliferative unresponsiveness could also be induced in KLH-specific Th1 cells derived from C57BL/10 mice. Similar to earlier results, Th1 cells exposed in primary cultures to both Ag and n-butyrate, unlike Th1 cells pretreated with Ag or n-butyrate alone, lost their ability to proliferate in response to Ag in secondary cultures that did not contain n-butyrate (Fig. 1⇓). The Ag-specific Th1 cell unresponsiveness could be demonstrated regardless of whether the Th1 cells were rested after the primary cultures. Although the anergic Th1 cells lost their ability to respond to Ag stimulation, they retained their ability to respond to IL-2, demonstrating that their loss of proliferative capacity was specific for Ag and not due to generalized suppression or a decrease in viability.
KLH-specific Th1 cells are susceptible to n-butyrate-induced anergy. Th1 cells (KLH-specific clone D9) were incubated for 48 h in primary culture with Ag alone (▨), n-butyrate alone ([□), or with n-butyrate and Ag (▪). After resting for 0 (A) or 72 h (B), the Th1 cells were restimulated with Ag or IL-2-containing Con A CM in secondary cultures for 2 days. [3H]TdR uptake by the Th1 cells was assessed and is presented as cpm ± SD from a representative experiment. ∗, This value was determined by Student’s t test to be statistically different, with p = 0.05 from both control values.
To test whether protein synthesis was required for n-butyrate-induced Th1 cell anergy, subsequent experiments were conducted using the protein synthesis inhibitor cycloheximide. As shown in Fig. 2⇓, Th1 cells exposed in primary cultures to both n-butyrate and Ag lost their ability to proliferate in response to Ag, but not IL-2, in secondary cultures. In contrast, the addition of cycloheximide to primary cultures of Th1 cells exposed to both Ag and n-butyrate suppressed the ability of n-butyrate to induce Ag-specific proliferative inactivation in Th1 cells. Cycloheximide by itself, or in conjunction with Ag, did not suppress the ability of the Th1 cells to respond to a subsequent stimulation with Ag (data not shown), even though cycloheximide treatment suppressed Ag-induced proliferation in the primary cultures. This result suggests that n-butyrate-induced Th1 cell anergy requires protein synthesis.
Cycloheximide blocks n-butyrate-induced anergy. Th1 cells (KLH-specific clone D9) were incubated for 48 h in primary culture containing various combinations of Ag, n-butyrate, and cycloheximide. The Th1 cells were then restimulated with Ag or IL-2-containing Con A CM in secondary cultures for 24 or 48 h. [3H]TdR uptake by the Th1 cells was assessed and is presented as cpm ± SD from a representative experiment. ∗, This value was determined by Student’s t test to be statistically different, with p = 0.05 from control (Th1 cells exposed to n-butyrate alone in primary cultures) values. This experiment has been repeated with similar results.
Expression of G1 cell cycle regulatory proteins in Ag-activated Th1 cells
To begin to examine CDKIs as candidates for the proteins required for n-butyrate-induced Th1 cell anergy, experiments were conducted to measure the baseline levels of CDKI and other cell cycle regulatory proteins in resting and Ag-activated normal Th1 cells. cdk6 and cdk4 were detected in resting Th1 cells, and the level of these proteins did not vary appreciably during the cell cycle (Fig. 3⇓). In contrast, cdk2 was not detected in resting Th1 cells, but appeared in the Th1 cells by 12 h after Ag stimulation. None of the G1 cyclins were detected in appreciable amounts in resting Th1 cells, but were found in Ag-stimulated Th1 cells. Cyclin D2 appeared first, followed by cyclin E and cyclin D3.
Resting Th1 cells contain cdk inhibitory activity. A, Lysates were prepared from resting Th1 cells or Th1 cells stimulated for 12, 24, 36,48, 60, or 72 h with Ag. Expression of the various G1 cell cycle regulatory proteins was assessed by immunoblotting. Equal loading of cell extracts was confirmed using an Ab that recognized actin. B, Resting Th1 cells, or Th1 cells stimulated for 2 days with Ag, were stained with PE-anti-CD4 and control mouse Ig or mouse anti-p27Kip1 mAb followed by FITC-labeled anti-mouse Ig. The results are presented as FSC vs FITC dot plots after gating on the PE+ Th1 cells. [3H]TdR uptake by the Th1 cells was also assessed and is presented as cpm ± SD: resting Th1 cells = 77 ± 25; Ag-stimulated Th1 cells = 27,208 ± 5,274.
In terms of CDKIs, p21Cip1 was not found in the resting Th1 cells, but was detected at low levels by 12 h after Ag stimulation, reaching its highest level at 72 h after Ag activation (Fig. 3⇑A). In contrast to p21Cip1, p27Kip1 was found at a high level in resting Th1 cells, but was dramatically down-regulated by 12 h after Ag stimulation. p27Kip1 levels in the Th1 cells remained low until 60 h after Ag stimulation.
Levels of p27Kip1 in the Th1 cells was also examined by cytofluorometric analysis to confirm that down-regulation of this protein was associated with cell activation. Multiparameter analysis showed that resting Th1 cells contained high levels of p27Kip1, but were uniformly low in terms of FSC (Fig. 3⇑B). In contrast, Th1 cells activated in response to Ag stimulation or exogenous IL-2 increased in size as measured by FSC, but down-regulated expression of p27Kip1.
DNA analysis of Ag-activated Th1 cells was also conducted to correlate the expression of regulatory proteins with cell cycle progression. Almost 90% of Ag-activated Th1 cells remained in G0-G1 12 h after stimulation. However, by 24 h after Ag stimulation, 40% of the Th1 cells had entered S phase, and, by 36 h poststimulation, some of the Th1 cells could be found in G2-M and some had already reentered G0-G1. Close to 100% of the Ag-activated Th1 cells had regained the G0-G1 phase of the cell cycle by 72 h after stimulation (data not shown). Taken together these data suggest a single-cell division by the Ag-activated Th1 cells could be accomplished somewhere between 24 and 36 h. It also suggests that the cessation of DNA synthesis, which occurred by 72 h, did not necessarily correspond with complete senescence, since the levels of cell cycle regulatory proteins found in the Th1 cells 72 h after Ag stimulation (Fig. 3⇑) did not correlate with those found in true resting cells.
Effects of n-butyrate on G1 cell cycle regulatory proteins in Th1 cells
If n-butyrate-induced Th1 cell anergy is due to an up-regulation of CDKIs, it might be expected that Th1 cells isolated from primary cultures containing n-butyrate would demonstrate increased levels of p21Cip1 and p27Kip1. When testing this possibility, it was found that similar to Ag-activated Th1 cells, Th1 cells treated with n-butyrate alone down-regulated p27Kip1 (Fig. 4⇓). Th1 cells treated with both Ag and n-butyrate also down-regulated rather than up-regulated their level of p27Kip1 albeit at a slower rate than control Th1 cells, retaining a significant amount of p27Kip1 even after 24 h following stimulation. In terms of p21Cip1 it was found that, unlike Ag-activated Th1 cells, Th1 cells treated with n-butyrate alone failed to up-regulate p21Cip1. In contrast to Th1 cells treated with n-butyrate alone, Th1 cells treated with n-butyrate and Ag exhibited a rapid up-regulation of p21Cip1. Taken together, the results showed that Th1 cells treated with both n-butyrate and Ag down-regulated p27Kip1 more slowly than control Th1 cells treated with either n-butyrate or Ag alone and increased their levels of p21Cip1.
Expression of CDKIs by n-butyrate-treated Th1 cells. Th1 cells were incubated in primary cultures containing n-butyrate alone or n-butyrate and Ag. After 12, 24, 36, or 48 h, the Th1 cells were isolated and lysates prepared. Expression of p21Cip1 and p27Kip1 was assessed by immunoblotting. Equal loading of cell extracts was confirmed using an Ab that recognized actin. This experiment has been repeated with similar results.
Expression of CDKIs in Ag-stimulated anergic Th1 cells
If CDKIs help maintain the anergy observed in Th1 cells tolerized by exposure to n-butyrate and Ag, one or more of these proteins should be present at high levels in the anergic Th1 cells even after Ag stimulation. Consequently, experiments were conducted to correlate the loss of Ag-specific proliferative capacity in the Th1 cells with the levels of CDKIs and other cell cycle regulatory proteins. Control Th1 cells (treated with n-butyrate alone in primary cultures) and anergic Th1 cells (treated with both n-butyrate and Ag in primary cultures) were compared at 0, 12, 24, and 48 h after Ag stimulation in secondary cultures. Since p27Kip1 had been shown to be down-regulated in Th1 cells treated with either n-butyrate or with n-butyrate and Ag, it was not surprising that both groups of these Th1cells contained low levels of p27Kip1 at the initiation of secondary cultures (Fig. 5⇓). The level of p27Kip1 in control Th1 cells was subsequently increased, but only after 48 h of Ag stimulation. In contrast to control Th1 cells, the anergic Th1 cells contained high levels of p27Kip1 by as early as 24 h after Ag stimulation. Expression of p21Cip1 also differed between control and anergic Th1 cells. Th1 cells exposed to n-butyrate alone in primary cultures contained their highest level of p21Cip1 at 48 h after Ag stimulation. In contrast, anergic Th1 cells maintained high levels of p21Cip1 from the outset of Ag restimulation. Thus, Ag-stimulated anergic Th1 cells, unlike Ag-stimulated control Th1 cells, contained significant amounts of p27Kip1 and p21Cip1 at a time (i.e., 24 h poststimulation) which would otherwise represent the main stage of S phase entry.
Expression of CDKIs in anergic Th1 cells following restimulation with Ag. Th1 cells were incubated for 48 h in primary culture with n-butyrate alone or with n-butyrate and Ag. The Th1 cells were then restimulated with Ag for 0, 24, or 48 h, at which time they were lysed. A, Expression of p21Cip1 and p27Kip1 in the restimulated control (C) Th1 cells (exposed to n-butyrate alone in primary cultures) and restimulated anergic (A) Th1 cells (exposed to both n-butyrate and Ag in primary cultures) was assessed by immunoblotting. Equal loading of cell extracts was confirmed using an Ab that recognized actin. B, [3H]TdR uptake by the Th1 cells in the secondary cultures was assessed after 48 h, and the results are presented as cpm ± SD. ∗, This value was determined by Student’s t test to be statistically different, with p = 0.05 from that of the controls. This experiment has been repeated twice with similar results.
G1 blockade due to lack of cdk activity could reflect a decrease of cdk proteins as well as an increase in CDKIs. Consequently, levels of G1 cdks were also examined in the Ag-restimulated anergic Th1 cells during the apparently crucial first 24 h. In agreement with the earlier finding that cdk4 and cdk6 levels remained fairly constant during the cell cycle, and levels of these proteins did not appear to differ between control and anergic Th1 cells isolated from Ag-stimulated secondary cultures (Fig. 6⇓A). Even cdk2, which is not highly expressed in resting Th1 cells, was found in similar levels in both control and anergic Th1 cells at 24 h after Ag stimulation.
Expression of cdks and cyclins in anergic Th1 cells following restimulation with Ag. Th1 cells were incubated for 48 h in primary culture with n-butyrate alone or with n-butyrate and Ag. The Th1 cells were then restimulated with Ag for 0, 12, or 24 h, at which time they were lysed. A, Expression of cdk2, cdk4, and cdk6 in the restimulated control (C) Th1 cells (exposed to n-butyrate alone in primary cultures) and restimulated anergic (A) Th1 cells (exposed to both n-butyrate and Ag in primary cultures) was assessed by immunoblotting. Equal loading of cell extracts was confirmed using an Ab that recognized actin. B, Expression of cyclin D2 and D3 in the control Th1 cells (exposed to n-butyrate alone in primary cultures) and anergic Th1 cells (exposed to both n-butyrate and Ag in primary cultures) was assessed by immunoblotting following Ag restimulation for 24 h.
Alternatively, loss of cdk activity and subsequent G1 blockade could result from abnormal fluctuations in the levels of the D-type cyclins. Western blot analysis revealed that the levels of cyclin D2 did not appear to differ between the control and anergized Th1 cells restimulated with Ag (Fig. 6⇑B). In contrast to these results, levels of cyclin D3 did appear to be reduced in the anergized Th1 cells when compared with controls following Ag restimulation.
Anergic Th1 cells contain CDKI activity
In addition to measuring protein levels of p21Cip1 and p27Kip1 in anergic Th1 cells, the lysates prepared from anergic Th1 cells that had been incubated in Ag-stimulated secondary cultures for 24 h were also tested for functional CDKI activity. The method used to demonstrate CDKI function is based on the ability of p21Cip1 and p27Kip1 to suppress the kinase activity of exogenous cdks (11). In accordance with their activated status, Ag-stimulated nontolerized Th1 cells did not contain substantial cdk2-suppressive activity: lysates from Ag-stimulated Th1 cells, either previously untreated, or exposed to butyrate in primary cultures, were unable to suppress the activity of preformed exogenous cdk2 complexes immunoprecipitated from lysates of EL4 cells (Fig. 7⇓A). In contrast, lysates from Ag-restimulated anergic Th1 cells contained high levels of CDKI activity capable of inhibiting exogenous cdk2 kinase activity.
CDKI activity in anergic Th1 cells restimulated with Ag. A, Lysates were prepared from Ag-stimulated Th1 cells or from control Th1 cells (exposed to n-butyrate alone in primary cultures) or anergic Th1 cells (exposed to both n-butyrate and Ag in the primary cultures) that had been restimulated with Ag for 24 h. cdk2 immunoprecipitated from lysates of asynchronous EL4 cells was mixed with equal protein amounts of the Th1 cell lysates or with lysing buffer alone for 30 min and then tested for activity in an H1 kinase assay. B, Lysates prepared from control Th1 cells or anergic Th1 cells that had been restimulated with Ag were resolved by SDS-PAGE and immunoblotted with anti-pRb Ab. Densitometric analysis was performed, and the results are presented as integrated density value (IDV). C, Lysates were prepared from control Th1 cells or anergic Th1 cells that had been restimulated with Ag. cdk2 was immunoprecipitated from the lysates and tested for activity in an H1 kinase assay. cdk2 immunoprecipitated from Ag-stimulated Th1 cells routinely demonstrated kinase activity 3- to 4-fold higher than cdk2 immunoprecipitated from resting Th1 cells in this assay (data not shown). In addition, cdk2 immunoprecipitated from restimulated control or anergic Th1 cells was resolved by SDS-PAGE and immunoblotted with anti-p21Cip1 or anti-p27Kip1 Ab.
To confirm that the CDKI activity demonstrated in anergic Th1 cells was specifically relevant to G1 cell cycle progression, endogenous pRb phosphorylation was evaluated. Lysates from Ag-stimulated control Th1 cells (treated with n-butyrate alone in primary cultures) exhibited increased levels of pRb phosphorylation, indicative of normal cdk activity (Fig. 7⇑B). In contrast, lysates derived from Ag-stimulated anergic Th1 cells (treated with both n-butyrate and Ag in primary cultures) demonstrated lower levels of pRb phosphorylation, suggestive of decreased cdk activity, as compared with controls. Correlated with the decreased pRb phosphorylation in the anergic Th1 cells was a decrease in endogenous cdk2 activity as compared with controls (Fig. 7⇑C). In addition, the cdk2 immunoprecipitated from the anergic restimulated Th1 cells associated to a greater degree with both p21Cip1 and p27Kip1 than did cdk2 immunoprecipitated from control restimulated Th1 cells. Taken together, these results demonstrate that anergic Th1 cells exhibit functional CDKI activity, capable of inhibiting both endogenous and exogenous cdk activity, in association with increased levels of CDKI proteins.
Discussion
At one level Th1 cell proliferation is under the control of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol phospholipase Cγ1 signaling pathways that lead from TCR cross-linking to the production of IL-2. However, cell division in Th1 cells, as in all eukaryotic cells, is also controlled by cdks which must be activated at specific time points during the cell cycle to phosphorylate different sets of proteins, such as pRb and histones, that are required for orderly cell cycle progression. Cdks are negatively regulated by their association with different CDKIs, most notably p21Cip1 and p27Kip1.
We have demonstrated here that unlike control Th1 cells, Th1 cells tolerized by exposure to n-butyrate and Ag contained high levels of the CDKI p27Kip1 when restimulated with Ag for 24 h in the absence of n-butyrate. Normally, p27Kip1, although present in resting Th1 cells is down-regulated following activation, and is not seen in Th1 cells again until 48–60 h after Ag stimulation. p21Cip1 was also found at much higher concentrations in anergic Th1 cells as compared with control Th1 cells when examined 24 h after Ag stimulation in secondary cultures. In nontolerized Th1 cells, S phase entry peaks at 24 h after Ag stimulation, a time period at which p21Cip1 or p27Kip1 are normally present at undetectable or low levels. The unusually high concentrations of p21Cip1 and p27Kip1 in the anergic Th1 cells at this crucial time in the cell cycle would severely inhibit the ability of the anergic Th1 cells to enter S phase.
The likelihood that the p21Cip1 and p27Kip1 found in the anergic Th1 cells was capable of suppressing proliferation in the Th1 cells was confirmed functionally: lysates from Ag-stimulated anergic Th1 cells, unlike lysates from Ag-stimulated control Th1 cells, exhibited decreased cdk2 activity and pRb phosphorylation. The decrease in endogenous cdk2 activity observed in the anergic Th1 cells correlated with an increased association of cdk2 with both p21Cip1 and p27Kip1. Additionally, lysates from restimulated anergic Th1 cells suppressed the kinase activity of exogenous cdk2 complexes. Taken together, these results suggest that even following Ag activation, anergic Th1 cells, but not control Th1 cells, contain CDKI activity capable of suppressing the activity of both endogenous and exogenous cdks.
In addition to examining the levels of CDKIs in Ag-stimulated control and anergic Th1 cells, we also determined the direct effects of n-butyrate on CDKIs in primary cultures. In the present study, unlike Th1 cells treated with n-butyrate alone, Th1 cells treated with both n-butyrate and Ag, strongly up-regulated p21Cip1. Up-regulation of p21Cip1 in T cells has been shown by others to follow the addition of IL-2 (2). However, more recently it has been shown that p21Cip1 was up-regulated in T cells costimulated with anti-CD3 and anti-CD28, and this up-regulation was only partially IL-2 dependent (9). Since the Th1 cells exposed in primary cultures to both Ag and n-butyrate are presented with ligands for both the TCR and CD28, it is perhaps not surprising that these Th1 cells are capable of activating p21Cip1. Although this has not yet been documented in T cells, p21Cip1 in other cell types has been shown to be mediated by different, but overlapping MAPK-dependent and MAPK-independent signaling pathways following cell activation (12, 13). These pathways can increase levels of p21Cip1 by stimulating transcription and/or by stabilizing p21Cip1 mRNA (14, 15). n-Butyrate-induced up-regulation of p21Cip1, which has been documented in fibroblasts, appears to occur at the transcriptional level (16, 17). Perhaps n-butyrate and TCR/CD28 stimulation stimulate p21Cip1 by different pathways in T cells, resulting in a synergistic, or at least additive, effect on p21Cip1 expression. Th1 cells treated with n-butyrate alone did not up-regulate p21Cip1. However, since p21Cip1 expression appears to depend on activation-induced signaling pathways and has only been induced by n-butyrate when it is added to cultures of actively dividing cells (17, 18), this finding is not surprising.
Similar to Th1 cells treated with Ag alone, Th1 cells treated with Ag and n-butyrate in primary cultures down-regulated p27Kip1. This finding suggests that the G1 blockade observed in Th1 cells isolated from primary cultures containing both Ag and n-butyrate is probably due to the rapid and powerful up-regulation of p21Cip1, rather than an increase, or lack of decrease, in the level of p27Kip1. Differences between these findings and those reported by Boussiotis et al. (19), in which they show that Th1 cell stimulation with Ag in the absence of costimulation appears to increase p27Kip1 expression but has no affect on p21Cip1, may reflect differences in the systems used to induce Th1 cell anergy . Alternatively, the apparent discrepancy between the two studies may reflect differences in the methods used to illustrate that CDKIs prevent proliferation in restimulated Th1 cells. Boussiotis et al. (19), rather than demonstrating p27Kip1 expression in restimulated anergic Th1 cells, showed that forced intracellular expression of p27Kip1 prevented Th1 cells from proliferating in response to Ag . In our system, the role of p27Kip1 in maintaining Th1 cell anergy appears to be primarily restricted to its early re-expression in Ag-stimulated secondary cultures. This finding suggests the normal degradation of p27Kip1 may operate in the primary cultures of n-butyrate-treated Th1 cells, but may still be altered, in that it allows p27Kip1 to accumulate again sooner in the secondary cultures than it would during normal cell cycle progression. The possible effects of n-butyrate on the posttranslational regulation of p27Kip1 are presently being clarified to determine the likelihood of this possibility.
Because the G1 blockade associated with anergy induction in Th1 cells could potentially be caused by alterations in other G1 cell cycle regulatory components, these possibilities were also explored. Levels of cdk2, cdk4, and cdk6 did not appear to differ between control and anergic Th1 cells during the critical first 24 h following Ag restimulation. Expression of the D-type cyclins was also examined in a similar manner, since reduced cyclin D3 expression and/or increased cyclin D2 expression can reportedly induce G1 blockade (20, 21). Although no evidence of excess cyclin D2 expression was observed in anergic vs control Th1 cell lysates in this study, levels of cyclin D3 did appear to be reduced in comparison to those of controls following Ag restimulation. Miyatake et al. (22) have shown T cell growth arrest associated with decreased levels of cyclin D3 was only partially reversed by subsequently overexpressing the protein, suggesting that D3-associated kinase activity is inhibited by additional mechanisms, such as suppression by CDKIs . Thus, although reduced cyclin D3 expression may play a role in G1 blockade during anergy induction, it seems more likely that the abnormal expression of CDKIs p27Kip1, and especially p21Cip1, are primarily responsible for the observed G1 blockade and anergy induction in Th1 cells.
Although the anergic Th1 cells fail to proliferate to Ag, in association with the accumulation of CDKIs, the anergic Th1 cells retain their ability to proliferate to IL-2. Presumably, this is because exogenous IL-2 can alter the balance of CDKIs found in the anergic Th1 cells. For example, IL-2 has been shown to stimulate the production of cyclin E and cdk2, which together phosphorylate p27Kip1, thus making the CDKI susceptible to ubiquitination and proteasome-dependent degradation (23). Since IL-2 is not produced by the anergic Th1 cells stimulated by Ag, this effect would only occur when the anergic Th1 cells were stimulated with exogenous IL-2. If the proliferative unresponsiveness of the anergic Th1 cells requires the increased expression of both p21Cip1 and p27Kip1, IL-2-induced down-regulation of p27Kip1 could destroy this balance and allow the CDKI levels to decrease sufficiently for cdk-induced pRb phosphorylation, and S phase entry, to occur. It is important to note that the n-butyrate-induced up-regulation of CDKIs and inhibition of IL-2 synthesis in anergic Th1 cells do not necessarily represent separate mechanisms. For example, the block in IL-2 production may be related to the growth arrest caused by the abnormal increases in CDKIs observed here, and thus, both pathways regulating T cell proliferation may contribute to the anergic phenotype. This possibility is presently being investigated.
Taken together, the results suggest that the proliferative unresponsiveness observed in Th1 cells tolerized by exposure to n-butyrate and Ag is maintained by high levels of both p21Cip1 and p27Kip1 which are normally inversely expressed. Together, these two CDKIs are present at high levels at a time point that is crucial for S phase entry, and which normally is characterized by low levels of both p27Kip1 and p21Cip1. The induction of p21Cip1 and the prevention of p27Kip1 down-regulation has similarly been shown to induce T cell unresponsiveness in association with sustained activation of the Raf–mitogen-activated protein/extracellular signal-related kinase–extracellular signal-related kinase pathway induced by treatment with anti-CD3 mAb (20). The findings described here do not conflict with the numerous reports documenting the negative effects of anergy induction on the signaling pathways and transcription factors required for IL-2 production (24, 25, 26, 27). Rather, it just suggests that proliferative unresponsiveness in Th1 cells may be mediated at more than one level, and that these different pathways regulating T cell proliferation may be connected by as yet undisclosed mechanisms.
Footnotes
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1 This work was supported by a grant from the Arkansas Chapter of the Arthritis Foundation and Grant MCB-9817191 from the National Sciences Foundation.
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↵2 Address correspondence and reprint requests to Dr. Kathleen Gilbert, Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205. E-mail address: gilbertkathleenm{at}exchange.uams.edu
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↵3 Abbreviations used in this paper: CDKI, cyclin-dependent kinase inhibitor; cdk, cyclin-dependent kinase; pRb, retinoblastoma protein; KLH, keyhole limpet hemocyanin; MAPK, mitogen-activated protein kinase; FSC, forward scatter.
- Received May 31, 2000.
- Accepted October 25, 2000.
- Copyright © 2001 by The American Association of Immunologists
References
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