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Platelets taken from allergen sensitised mice undergo enhanced chemotaxis towards MDC in vitro
Monocyte Derived Chemokine (MDC) is up-regulated in the lungs of asthmatic individuals (Bochner et al., 2003) and has been shown to induce platelet aggregation when co-incubated with sub-aggregatory levels of ADP (Abi-Younes et al., 2001). Platelets undergo chemotaxis to stimuli in vitro (Czapiga et al., 2005), and have been observed in bronchoalveolar lavage of allergen challenged mice (Pitchford et al. 2004), but no studies have so far been conducted to investigate platelet migration towards chemokines for which platelets display receptors for. The aim of this study was to determine whether platelets taken from ovalbumin (OVA) sensitised mice undergo chemotaxis towards MDC. Female BALB/C mice (n=6) w ere immunised on days 0 and 7 with OVA. (10 µg/mouse i.p. 0.4ml in 0.1M Al2(OH)3) or Alum alone. From days 14-18, mice were culled and cardiac bleeds performed using citrated syringes. Blood was centrifugated and the resulting platelet rich plasma was washed and resuspended in Tyrodes buffer at a density of 1x104cells µl-1. Some platelet suspensions were incubated for 10 minutes with 0.1 µM ADP) Transwell ChemoTx chambers (Neuroprobe Inc. MD USA) were prepared with concentrations of MDC ranging from 0-5000ng ml-1 in Tyrodes buffer, and a membrane placed over the top (2 µm pore diameter, 20,000 pores mm2). 24 µl of platelet suspension was then added to the surface of each well and incubated at 37°C for 90 minutes. After incubation, the filter was removed and platelet numbers in the bottom wells were counted by reading a haemocytometer using a microscope under phase contrast with x40 objective Data were analysed using one way ANOVA and Bonferroni multiple comparison tests between groups. After incubation, non stimulated platelets taken from sham immunised mice did not undergo chemotaxis to MDC, compared to platelets taken from sham sensitised mice stimulated with ADP, or platelets taken from OVA sensitised mice. (* p<0.05 figure 1). Whilst ADP did not enhance migration to MDC when platelets were taken from OVA sensitised mice, there was a significant difference in migration between ADP stimulated sham and OVA sensitised platelets (# p<0.05 figure 1). This study reveals the ability of ADP stimulated platelets to under chemotaxis to MDC, a process that is further enhanced by platelet sensitisation to allergen. Figure 1. Platelet Chemotaxis in response to MDC.
Abi-Younes, S. et al., (2001) Thromb. Res. 101, 279-289. |
