The immunosuppressive effect of gossypol in mice is mediated by inhibition of lymphocyte proliferation and by induction of cell apoptosis

Abstract

Aim: To investigate the immunosuppressive effect of gossypol in mice both in vitro and in vivo.

Methods: The in vitro effect of gossypol on the proliferation of lymphocytes isolated from lymph nodes of BALB/c mice was determined by CFSE staining and by an MTS assay. Lymphocyte activation and lymphoblastic transformation were evaluated with immunostaining. Cell apoptosis was detected by Annexin-V and Hoechst 33342 staining. The in vivo immunosuppressive effect of gossypol on the DTH reaction was evaluated using a mouse DTH model induced by 2,4-dinitro-1-fluorobenzene (DNFB). The thickness of the ears was measured, and the histological changes of the mouse auricles were observed after hematoxylin-eosin staining. The proliferation capacity of lymphocytes from DTH mice was also assayed.

Results: In vitro, gossypol could significantly inhibit the proliferation of mouse lymphocytes stimulated with phorbol ester plus ionomycin in a dose-dependent manner. Although the expression of the early activation antigen CD69 was not affected, the lymphoblastic transformation of both T and B lymphocyte subsets was significantly suppressed by gossypol. Moreover, gossypol could induce apoptosis of lymphocytes, and the effect was time- and dose-dependent. In vivo, the DTH reaction in mice was markedly alleviated by gossypol injected intraperitoneally. Lymphocytes from drug-treated DTH mice had a reduced proliferation capacity as compared with lymphocytes from untreated DTH mice. Gossypol treatment also markedly reduced the number of infiltrated lymphocytes in the auricles of DTH mice.

Conclusion: Gossypol exhibited immunosuppressive effects in mice, probably by inhibition of lymphocyte proliferation and by induction of cell apoptosis.

Figure 1

Inhibitory effect of gossypol on the proliferation of mouse lymphocytes. (A) Flow cytometry analysis of CFSE-labeled cells. Lymphocytes were stained with CFSE and stimulated with PDB+Ion for 48 h in the presence of various concentrations of gossypol. (B) MTS assay. Cells were stimulated with PDB+Ion for 48 h in the presence of various concentrations of gossypol. At the end of the culturing period, 20 μL of the MTS solution was added to each well, and then the cells were incubated for another 4 h. The absorbance at 490 nm was measured using a microplate reader. Values are expressed as the mean±SD. ^bP<0.05, ^cP<0.01 vs PDB+Ion group. GOS: gossypol; GOS64: 64 μmol/L GOS; GOS32: 32 μmol/L GOS; GOS16: 16 μmol/L GOS; GOS8: 8 μmol/L GOS; GOS4: 4 μmol/L GOS.

Figure 2

Flow cytometry analysis of the effect of gossypol on CD69 expression and on lymphoblastic transformation of lymphocytes stimulated by PDB plus Ion for 24 h. One representative dot plot of each triplicate is presented. GOS: gossypol; GOS16: 16 μmol/L GOS; GOS8: 8 μmol/L GOS.

Figure 3

Flow cytometry analysis of apoptosis in mouse lymphocytes using Annexin-V staining. (A) Cells were stimulated with PDB+Ion for 8 h in the presence of various concentrations of gossypol. (B) Cells were stimulated with PDB+Ion for 24 and 48 h in the presence of 8 μmol/L gossypol. Values are the ratios of the percentage of cells in early apoptosis (Annexin V–PE positive/7-AAD negative) to the percentage of cells in late apoptosis (Annexin V–PE positive/7-AAD positive). One representative experiment of three independent experiments is presented.

Figure 4

The nuclear morphology of mouse lymphocytes stained with Hoechst 33342 (400×magnification). (A) Resting lymphocytes treated with gossypol for 24 h. (B) PDB+Ion-activated lymphocytes treated with gossypol for 24 h. Arrows show the apoptotic cells. GOS: gossypol.

Figure 5

Inhibitory effect of gossypol on the proliferation of lymphocytes from DTH model mice. Gossypol (25 mg·kg⁻¹·d⁻¹) was administered for 7 days by ip injection before the mice were sacrificed. Lymphocytes were isolated from lymph nodes and were stimulated with PDB plus Ion and then cultured for 48 h at 37°C in a humidified atmosphere of 5% CO₂. At the end of the culturing period, 20 μL of MTS solution was added to each well, and then the cells were incubated for another 4 h. The absorbance at 490 nm was measured using a microplate reader. Data are presented as the mean ±SD. n=6. ^bP<0.05 vs DTH group. GOS: gossypol.

Figure 6

Hematoxylin-eosin staining of mouse auricle tissue sections (×100). See Material and Methods for details. GOS: gossypol.

Figure S1

Effect of gossypol on the formation of cell aggregated colony.