Astaxanthin, a Carotenoid, Stimulates Immune Responses by Enhancing IFN-γ and IL-2 Secretion in Primary Cultured Lymphocytes in Vitro and ex Vivo

Abstract

Astaxanthin, a potent antioxidant carotenoid, plays a major role in modulating the immune response. In this study, we examined the immunomodulatory effects of astaxanthin on cytokine production in primary cultured lymphocytes both in vitro and ex vivo. Direct administration of astaxanthin (70-300 nM) did not produce cytotoxicity in lipopolysaccharide (LPS, 100 µg/ mL)- or concanavalin A (Con A, 10 µg/ mL)-activated lymphocytes, whereas astaxanthin alone at 300 nM induced proliferation of splenic lymphocytes (p < 0.05) in vitro. Although astaxanthin, alone or with Con A, had no apparent effect on interferon (INF-γ) and interleukin (IL-2) production in primary cultured lymphocytes, it enhanced LPS-induced INF-γ production. In an ex vivo experiment, oral administration of astaxanthin (0.28, 1.4 and 7 mg/kg/day) for 14 days did not cause alterations in the body or spleen weights of mice and also was not toxic to lymphocyte cells derived from the mice. Moreover, treatment with astaxanthin significantly increased LPS-induced lymphocyte proliferation ex vivo but not Con A-stimulated lymphocyte proliferation ex vivo. Enzyme linked immunosorbent assay (ELISA) analysis revealed that administration of astaxanthin significantly enhanced INF-γ production in response to both LPS and Con A stimulation, whereas IL-2 production increased only in response to Con A stimulation. Also, astaxanthin treatment alone significantly increased IL-2 production in lymphocytes derived from mice, but did not significantly change production of INF-γ. These findings suggest that astaxanthin modulates lymphocytic immune responses in vitro, and that it partly exerts its ex vivo immunomodulatory effects by increasing INF-γ and IL-2 production without inducing cytotoxicity.

Keywords: Con A; IL-2; INF-γ; LPS; astaxanthin; immunomodulation; lymphocytes; mice.

Figure 1

Effects of astaxanthin on cell viability in lipopolysaccharide (LPS) and concanavalin A (Con A)-activated lymphocytes. Cells were treated with astaxanthin (70–300 nM) in the absence or presence of LPS (100 μg/mL) or Con A (10 μg/mL) for 48 h. (A–C) Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (n = 5). Data are presented as the mean ± SD and cell viability to solvent control (DMSO) group was 100%; * p < 0.05 compared with solvent control (DMSO).

Figure 2

Effects of astaxanthin on cell viability in LPS- and Con A-activated lymphocytes ex vivo. Mice were treated orally with astaxanthin (0.28–7 mg/kg/day), or vehicle (normal saline; NS) once daily for 14 consecutive days and then the total body weight (A) and spleen weight (B) were measured. Cells were collected and cultured with or without LPS or Con A for 48 h; (C–E) Cell viability was determined using a MTT assay (n = 5). All data are expressed as the mean ± SD (n = 4) and cell viability to solvent control (NS) group was 100%. * p < 0.05 compared with solvent control (NS); # p < 0.05 compared with LPS.

Figure 3

Isolation and primary cultivation of lymphocytes from spleen of BALB/c mice. (A) Mice were dissected and the spleen was removed using a forceps; (B) The spleen was directly placed in a sterile petri dish containing the Roswell Park Memorial Institute (RPMI) 1640 medium; (C,D) Single-cell suspensions were prepared by gently disrupting the spleen on a sterile wire mesh; (E) Red blood cells were lysed using the ACK (ammonium-chloride-potassium) lysis buffer, and centrifuged at 300× g for 5 min; (F) The lymphocyte pellets were collected and suspended with RPMI containing 5% heat-inactivated fetal bovine serum.