The ability of antigen, but not interleukin-2, to promote n-butyrate-induced T helper 1 cell anergy is associated with increased expression and altered association patterns of cyclin-dependent kinase inhibitors

Abstract

The ability of the cell cycle inhibitor n-butyrate to induce T helper 1 (Th1) cell anergy is dependent upon its ability to block the cell cycle progression of activated Th1 cells in G1. Results reported here show that although both interleukin (IL)-2 and antigen (Ag) push Th1 cells into G1 where they are blocked by n-butyrate, only the Ag-activated Th1 cells demonstrate functional anergy once the n-butyrate has been removed from the culture. Because n-butyrate-induced Th1 cell anergy has been linked to increased expression of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, mechanistic experiments focused on the role of these inhibitors. It was found that when Th1 cells were reincubated in Ag-stimulated secondary cultures, the Th1 cells previously exposed to Ag and n-butyrate (anergic Th1 cells) demonstrated a cumulative increase in p21Cip1 and p27Kip1 when compared with Th1 cells previously exposed to recombinant (r)IL-2 and n-butyrate (non-anergic Th1 cells). p27Kip1 in the anergic Th1 cells from the secondary cultures was associated with cyclin-dependent kinases (cdks). In contrast, p21Cip1 in the anergic Th1 cells, although present at high levels, did not associate significantly with cdks, suggesting that p21Cip1 may target some other protein in the anergic Th1 cells. Taken together, these findings suggest that Th1 cell exposure to Ag and n-butyrate, rather than IL-2 and n-butyrate, is needed to induce the cumulative increase in p21Cip1 and p27Kip1 that is associated with the proliferative unresponsiveness in anergic Th1 cells. In addition, p21Cip1 may inhibit proliferation in the anergic Th1 cells by some mechanism other than suppression of cdks that is unique to the induction of Th1 cell anergy.

Figure 1

n-Butyrate does not induce anergy in interleukin-2 (IL-2)-stimulated T helper 1 (Th1) cells. (a) Th1 cells (clone D9) were incubated for 48 hr in primary culture with antigen (Ag) alone, n-butyrate alone, Ag+n-butyrate, recombinant (r)IL-2 alone, rIL-2+n-butyrate, or rIL-2+n-butyrate+Ag. In addition, Th1 cells were stimulated with IL-2 or Ag + n-butyrate for 24, 48, or 72 hr. Following isolation from the primary cultures, the Th1 cells were fixed in 70% ethanol at 4° overnight. Th1 cells were then washed in phosphate-buffered saline (PBS), resuspended in RNAse (1 mg/ml) and propidium iodide (50 µg/ml), incubated for 20 min at room temperature in the dark, and analysed for their DNA content by flow cytometry. (b) Th1 cells were then isolated from the primary cultures and stimulated in secondary cultures with Ag for 48 hr. [³H]Thymidine ([³H]TdR) uptake by the Th1 cells was assessed and is presented as counts per minute (c.p.m.)±SD from a representative experiment. *The responses generated by the Th1 cells pretreated with Ag+n-butyrate or with rIL-2+Ag+n-butyrate and then restimulated with 50 or 150 µg/ml of keyhole limpet haemocyanin (KLH) in secondary cultures were determined by the Student's t-test to be statistically different at a P-value of 0·05 from their non-Ag control values. This experiment has been repeated twice with similar results.

Figure 2

Expression of G1 cell cycle regulatory proteins in interleukin-2 (IL-2)-stimulated Th1 cells. (a) Lysates were prepared from resting (R) T helper 1 (Th1) cells, or from Th1 cells stimulated for 24, 48, or 72 hr with IL-2. Expression of the various G1 cell-cycle regulatory proteins was assessed by immunoblotting. Equal loading of cell extracts was confirmed using an antibody (Ab) that recognized actin. (b). Lysates were prepared from resting Th1 cells, or from Th1 cells stimulated for 24, 48, or 72 hr with IL-2. Expression of p21^Cip1 and p27^Kip1 was assessed by immunoblotting. Densitometric analysis was performed, and the results are presented as percentage actin. This experiment was been repeated twice with similar results obtained on each occasion.

Figure 3

Effects of n-butyrate on cyclin-dependent kinase inhibitors (CDKIs) in interleukin-2 (IL-2)-stimulated T helper 1 (Th1) cells. Th1 cells were incubated in primary cultures containing IL-2+n-butyrate, or antigen (Ag)+n-butyrate, with or without IL-2. After 24 hr, the Th1 cells were isolated and lysates were prepared. p21^Cip1 and p27^Kip1 were immunoprecipitated, and the expression of p21^Cip1 and p27^Kip1, as well as their association with cyclin-dependent kinase 4 (cdk4), was assessed by immunoblotting. This experiment was been repeated, with similar results obtained on each occasion.

Figure 4

Expression of cyclin-dependent kinase inhibitors in anergic and non-anergic T helper 1 (Th1) cells following restimulation with antigen (Ag). (a) Th1 cells were incubated for 48 hr in primary culture with interleukin-2 (IL-2)+n-butyrate, or with Ag+n-butyrate (with or without IL-2). These Th1 cells were then restimulated with Ag for 24 hr, at which time they were lysed. Some Th1 cells (resting) were left untreated before being lysed. Expression of p21^Cip1 and p27^Kip1 was assessed by immunoblotting. (b) Densitometric analysis was performed, and the cumulative expression of p21^Cip1 and p27^Kip1 is presented as percentage actin. This experiment was repeated three times, with similar results obtained on each occasion.

Figure 5

Expression of G1 cell-cycle regulatory proteins in anergic and non-anergic T helper 1 (Th1) cells following restimulation with antigen (Ag). Th1 cells were incubated for 48 hr in primary culture with interleukin-2 (IL-2)+n-butyrate, or with Ag+IL-2+n-butyrate. The Th1 cells were then restimulated with Ag for 24 hr, at which time they were lysed. Expression of cyclin-dependent kinase (cdk)2, cdk4, cdk6, cyclin D2, cyclin D3, and cyclin E was assessed by immunoblotting. Equal loading of protein was confirmed by staining with an anti-actin antibody.

Figure 6

(a) Endogenous retinoblastoma protein (pRb) levels in anergic and non-anergic T helper 1 (Th1) cells following restimulation with antigen (Ag). Lysates were prepared from resting Th1 cells, or from Th1 cells exposed in primary cultures to Ag alone, n-butyrate alone, Ag+n-butyrate, interleukin-2 (IL-2) alone, IL-2+n-butyrate, Ag+n-butyrate, or IL-2+Ag+n-butyrate, and which had been restimulated with Ag for 24 hr. These lysates were immunoblotted with anti-pRb Ab. Densitometric analysis was performed, and the results are presented as integrated density value (IDV). (b) Activity of cyclin-dependent kinase inhibitors (CDKIs) in anergic and non-anergic Th1 cells following restimulation with Ag. Lysates were prepared from resting Th1 cells, or from Th1 cells exposed in primary cultures to n-butyrate alone, Ag+n-butyrate, IL-2+n-butyrate, or IL-2+Ag+n-butyrate, and then recultured with Ag for 24 hr. The Th1 cell lysates, either intact or depleted of both p21^Cip1 and p27^Kip1 by immunoprecipitation, were mixed in equal protein amounts with cdk2 immunoprecipitated from lysates of asynchronous EL4 cells for 30 min, and then tested for activity in an H1 kinase assay. The results are presented as total counts per minute (c.p.m.) minus the background c.p.m. measured in samples containing immunoprecipitated cdk2 but no substrate. This experiment was repeated with similar results obtained. (c) Representative experiment demonstrating the immunodepletion of p27^Kip1 and p21^Cip1 from the Th1 cell lysates (treated with IL-2) confirmed by Western blotting.

Figure 7

(a) Association of cyclin-dependent kinase (cdk)4 with p21^Cip1 and p27^Kip1 in anergic and non-anergic T helper 1 (Th1) cells following restimulation with antigen (Ag). Lysates were prepared from Th1 cells exposed in primary cultures to interleukin-2 (IL-2)+n-butyrate, or from Th1 cells exposed in primary cultures to Ag+n-butyrate (with or without IL-2) that had been restimulated with Ag for 24 hr. p27^Kip1 or p21^cip1 (200 µg/sample) was immunoprecipitated from these lysate preparations and analysed via Western blotting for expression of cdk4. (b) Association of p21^Cip1 cyclin D2, and cyclin D3 with cdk2, cdk4, and cdk6 in anergic and non-anergic Th1 cells following restimulation with Ag. Lysates were prepared from Th1 cells exposed in primary cultures to IL-2+n-butyrate, or from anergic Th1 cells exposed in primary cultures to Ag+n-butyrate (with or without IL-2) that had been restimulated with Ag for 24 hr. cdk2, cdk4, or cdk6 (200 µg/sample) were immunoprecipitated from these lysates and analysed via Western blotting for expression of p21^Cip1 cyclin D2 and cyclin D3.