Figure 5. Large amounts of preformed CXCL5 in platelets and platelet origin for homeostatic CXCL5 in blood.
Comparison of plasma concentrations of CXCL5 (A) by retro-orbital bleeding (with coagulation) and vena cava bleeding (by injection of sodium citrate, without coagulation) in WT mice (n=4 mice/group) upon 107 CFU E.coli i.t. challenge, measured by ELISA. The plasma concentrations of CXCL1 (B), CXCL2 (C) and CXCL15 (D) by retro-orbital bleeding from naïve WT mice and Cxcl5−/− mice (n=4 mice/group) were measured by ELISA. CXCL5, but not CXCL1, is detected in inactivated platelets of naïve WT mice by western blot (E), and both preformed and secreted CXCL5 by thrombin stimulation from platelet is mostly CXCL5-93 form (F). R: recombinant CXCL5-93 (9.8 kD) and CXCL5-70 (7.6 kD); Fr/th: the supernatant after separated platelets were snap-frozen and thawed twice; thrombin: the supernatant after separated WT platelets were stimulated with 1 U/ml thrombin for 10 minutes at 37°C;WT or Mu: lysate of inactivated platelets in WT or mutant Cxcl5−/− mice. WT and Cxcl5−/− mice were reconstituted with BM from WT and Cxcl5−/− mice respectively, after 8 weeks, the citrated blood will be prepared from the mice in each group (n=4 mice/group). The plasma and red cells were separated, then the plasma CXCL5 concentrations (G) and erythrocyte-binding CXCL5 (H) were measured by ELISA. After incubation of the citrated blood with recombinant CXCL5 for 15 minutes, the plasma concentrations of CXCL5 (I) were measured. WW: BM from WT donor mice into WT recipient mice; WK: BM from WT donor mice into Cxcl5−/− recipient mice; KW: BM from Cxcl5−/− donor mice into WT recipient mice; KK: BM from Cxcl5−/− donor mice into Cxcl5−/− recipient mice. The amounts of plasma CXCL5 and erythrocyte-binding CXCL5 in citrated blood of Mpl−/− mice (J), Fog-1ki/ki mice (K) and their respective WT control mice were measured by ELISA. The plasma concentrations of CXCL4 and CXCL7 were measured in the citrated blood of Mpl−/− mice (M), Fog-1ki/ki mice (N) and their respective WT control mice as well. Only the CXCL7-74 (β-thromboglobulin) form is detected in platelets by Western blot (L). R: recombinant CXCL7-74 a.a. (8.2 kD). Data are representative of two independent experiments. N.D: not detected. *P<0.05; **P<0.01; ***P<0.001.