Human eosinophil chemotaxis and selective in vivo recruitment by sphingosine 1-phosphate

Abstract

Sphingosine 1-phosphate (S1P) is a sphingolipid mediator that is involved in diverse biological functions. Local administration of S1P causes inflammation coupled to a large eosinophil (EO) recruitment in the rat-paw tissue. The inflammatory response is accompanied by an increase in S1P receptors, namely S1P(1), S1P(2), S1P(3), and by an enhanced expression of CCR3, which is the main chemokine receptor known to be involved in EO function. Human EOs constitutively express S1P(1) and, at a lower extent, S1P(2), S1P(3) receptors. S1P in vitro causes cultured human EO migration and an increase in S1P receptor mRNA copies and strongly up-regulates CCR3 and RANTES (regulated on activation, normal T cell-expressed and secreted) message levels; in particular CCR3 is up-regulated 18,000-fold by S1P. A blocking anti-CCR3 Ab inhibits S1P-induced chemotaxis, implying that S1P acts as specific recruiting signal for EOs not only through its own receptors but also through CCR3. These results show that S1P is involved in EO chemotaxis and contribute to shed light on the complex mechanisms underlying EO recruitment in several diseases such as asthma and some malignancies.

Fig. 1.

S1P local administration causes edema formation and EO infiltration. (A) Subplantar administration of S1P (1, 3, and 10 μg per paw) causes a dose-dependent edema. Control animals received the vehicle used to dissolve S1P. *, P < 0.05; ***, P < 0.001 vs. control. (B) The histology shows that a large EO infiltrate (×10) is present in rat paw 1 h after the injection of S1P (10 μg). (C) Higher magnification (×40) of the area delimited by the square in B, where eosinophilia is clearly visible. Data are expressed as mean ± SEM.

Fig. 2.

S1P increases S1PRs and CCR3 message in paw tissue. (A) S1P injection (10 μg) increases mRNA levels of S1P₁, S1P₂, and S1P₃ receptors in the rat-paw tissue. Receptor expression reaches its maximum at the 1-h time point (***, P < 0.001 vs. its respective basal level). At the 5-h time point, when the edema heals, whereas S1P₂ and S1P₃ expression returns to basal level, S1P₁ expression is significantly lower than its basal level (###, P < 0.01). (B) CCR3 and RANTES mRNA levels are undetectable in control tissue. After S1P injection (10 μg) CCR3 mRNA is strongly up-regulated (***, P < 0.001), as opposed to RANTES at the 1-h time point. CCR3 mRNA levels decrease significantly at the 5-h time point, when the edema heals (***, P < 0.001 vs. 1 h).

Fig. 3.

S1P-induced EO migration. Dose-dependent migration of EOs after incubation with S1P (A); S1P-induced migration (10^–8 M) is reduced significantly by anti-CCR3 neutralizing Ab (8 μg/ml) (B). Eotaxin has been used as a positive control. ***, P < 0.001 vs. MEM; ##, P < 0.01 vs. S1P. (C) Representative photos of EO chemotaxis in vitro. Eotaxin (a;8 μg/ml) or S1P (b;40ng/ml) causes EO chemotaxis, and incubation with anti-CCR3 antibodies reduces S1P-induced chemotaxis significantly (c).

Fig. 4.

S1P increases S1PRs expression on human EOs. Dose-dependent increase in S1P₁, S1P₂, and S1P₃ expression on human purified EOs after incubation with MEM or S1P after 12 h. Incubation for 18 h causes a loss of expression. *, P < 0.05; **, P < 0.01 vs. MEM; #, P < 0.05 vs. S1P₁ basal level.

Fig. 5.

S1P increases the message for CCR3 and RANTES on human EOs. Dose-dependent increase in mRNA copies of CCR3 and RANTES after incubation for 12 h with S1P. Longer time of incubation (18 h) causes a loss in expression. *, P < 0.01; ***, P < 0.001 vs. their relative basal values.