Chondroitin sulphate structure affects its immunological activities on murine splenocytes sensitized with ovalbumin

Abstract

Chondroitin sulphate (CS) is a glycosaminoglycan widely distributed in animal tissues, which has anti-inflammatory and chondroprotective properties. We reported previously that chondroitin 4-sulphate (CS-A) up-regulates the antigen-specific Th1 immune response of murine splenocytes sensitized with ovalbumin in vitro, and that CS suppresses the antigen-specific IgE responses. We now demonstrate that a specific sulphation pattern of the CS polysaccharide is required for the Th1-promoted activity, as other polysaccharides such as dextran and dextran sulphate do not significantly induce this activity. While the presence of some O-sulpho groups appear to be essential for activity, CS-A, and synthetically prepared, partially O-sulphonated CS, induce higher Th1-promoted activity than synthetically prepared, fully O-sulphonated CS. CS-A induces an activity greater than chondroitin sulphate B (CS-B) or chondroitin 6-sulphate (CS-C). In addition, chondroitin sulphate E (CS-E) induces greater activity than CS-A or CS-D. These results suggest that the GlcA(beta1-3)GalNAc(4,6-O-disulpho) sequence in CS-E is important for Th1-promoted activity. Furthermore, rat anti-mouse CD62L antibody, an antibody to L-selectin, inhibits the Th1-promoting activity of CS. These results suggest that the Th1-promoted activity could be associated with L-selectin on lymphocytes. These findings describe a new mechanism for the anti-inflammatory and chondroprotective properties of CS that may be useful in designing new therapeutic applications for CS used in the treatment of immediate-type hypersensitivity.

Figure 1. Dose response of CS on the cytokine production of murine splenocyte in vitro

BALB/c mice (n=5) were intraperitoneally injected on day 0 and day 13 with 20 μg of OVA and 2 mg of Al(OH)₃ at a total volume of 400 μl. Splenocytes (5.0×10⁶ cells/ml) were collected on day 14 and were co-cultured with OVA (final concentration 100 μg/ml). The amounts of cytokines in the supernatant were measured by ELISA. *P<0.05, **P<0.01 (significantly different from control value). Error bars represent means±S.D. for 6 wells.

Figure 2. Chemical structure of polysaccharides

Figure 3. Effects of polysaccharide on the cytokine production of murine splenocyte in vitro

BALB/c mice (n=5) were intraperitoneally injected on day 0 and day 13 with 20 μg of OVA and 2 mg of Al(OH)₃ in a total volume of 400 μl. Splenocytes (5.0×10⁶ cells/ml) were collected on day 14 and were co-cultured with OVA (final concentration 100 μg/ml). The samples (each at a final concentration of 1 μg/ml), or saline in vitro were added to the 24-well culture cluster in triplicate at 37 °C in a CO₂ incubator for 3 days. The amounts of cytokines in the supernatant were measured by ELISA. *P<0.05, **P<0.01 (significantly different from control value). Error bars represent means±S.D. for 6 wells.

Figure 4. ¹H-NMR spectra of CS-A and CS derivatives

(a) Intact CS-A, (b) partially O-sulphated CS-A, (c) fully O-sulphated CS-A, (d) de-O-sulphated CS-A recorded in ²H₂O at 333 K (or 60 °C). The letters in the spectra refer to the corresponding residues in the structures. Assignments were determined using 2D NMR [27].

Figure 5. Effects of CS-A and CS derivatives on the cytokine production of murine splenocyte in vitro

BALB/c mice (n=5) were intraperitoneally injected on day 0 and day 13 with 20 μg of OVA and 2 mg of Al(OH)₃ in a total volume of 400 μl. Splenocytes (5.0×10⁶ cells/ml) were collected on day 14 and were co-cultured with OVA (final concentration 100 μg/ml). The samples (each at a final concentration of 1 μg/ml), or saline in vitro were added to the 24-well culture cluster in triplicate at 37 °C in a CO₂ incubator for 3 days. The amounts of cytokines in the supernatant were measured by ELISA. *P<0.05, **P<0.01 (significantly different from control value). Error bars represent means±S.D. for 6 wells.

Figure 6. Effects of various CS on the cytokine production of murine splenocyte in vitro

BALB/c mice (n=5) were intraperitoneally injected on day 0 and day 13 with 20 μg of OVA and 2 mg of Al(OH)₃ in a total volume of 400 μl. Splenocytes (5.0×10⁶ cells/ml) were collected on day 14 and were co-cultured with OVA (final concentration 100 μg/ml). The samples (each at a final concentration of 1 μg/ml), or saline in vitro were added to the 24-well culture cluster in triplicate at 37 °C in a CO₂ incubator for 3 days. The amounts of cytokines in the supernatant were measured by ELISA. *P<0.05, **P<0.01 (significantly different from control value). Error bars represent means±S.D. for 6 wells.

Figure 7. CS receptor associated with Th1-promoted activity on splenocytes in vitro

BALB/c mice (n=5) were intraperitoneally injected on day 0 and day 13 with 20 μg of OVA and 2 mg of Al(OH)₃ in a total volume of 400 μl. Splenocytes (5.0×10⁶ cells/ml) were collected on day 14 and were co-cultured with OVA (final concentration 100 μg/ml). The samples (each at a final concentration of 1 μg/ml), or saline in vitro were added to the 24-well culture cluster in triplicate at 37 °C in a CO₂ incubator for 3 days. The amounts of cytokines in the supernatant were measured by ELISA. *P<0.05, **P<0.01 (significantly different from control value). Error bars represent means±S.D. for 6 wells.

Figure 8. Effect of spiked CS-A on binding to L-selectin

Splenocytes (5.0×10⁶ cells/ml) were cocultured with OVA (final concentration 100 μg/ml) and/or the rat anti-mouse CD62L antibody (final concentration 2 μg/ml) in triplicate at 37 °C in a CO₂ incubator for 3 days. The colleted cells were then staining with FITC-labelled anti anti-mouse CD62L antibody (MEL-14) and analysed on FACSCalibur™ flow cytometer (Becton Dickinson). (A) control, (B) after culture with CS, and (C) after culture with CS and the rat anti-mouse CD62L antibody.