Differential regulation of eosinophil chemokine signaling via CCR3 and non-CCR3 pathways

To investigate eosinophil stimulation by chemokines we developed a sensitiv e assay of leukocyte shape change, the gated autofluorescence/forward scatt er assay, Leukocyte shape change responses are mediated through rearrangeme nts of the cellular cytoskeleton in a dynamic process typically resulting i n a polarized cell and are essential to the processes of leukocyte migratio n from the microcirculation into sites of inflammation, We examined the act ions of the chemokines eotaxin, eotaxin-2, monocyte chemoattractant protein -1 (MCP-1), MCP-3, MCP-4 RANTES, macrophage inflammatory protein-1 alpha (M IP-1 alpha), and IL-8 on leukocytes in mixed cell suspensions and focused o n the responses of eosinophils to C-C chemokines, Those chemokines acting o n CCR3 induced a rapid shape change in eosinophils from all donors; of thes e, eotaxin and eotaxin-2 were the most potent. Responses to MCP-4 were qual itatively different, shoeing marked reversal of shape change responses with agonist concentration and duration of treatment, In contrast, MIP-1 alpha induced a potent response in eosinophils from a small and previously undesc ribed subgroup of donors via a non-CCR3 pathway likely to be CCR1 mediated, Incubation of leukocytes at 37 degrees C for 90 min in the absence of extr acellular calcium up-regulated responses to MCP-4 and MIP-1 alpha in the ma jority of donors, and there was a small increase in responses to eotaxin, M IP-1 alpha responsiveness in vivo may therefore be a function of both CCR1 expression levels and the regulated. efficiency of coupling to intracellula r signaling pathways. The observed up-regulation of MIP-1 alpha signaling v ia non-CCR3 pathways may play a role in eosinophil recruitment in inflammat ory states such as occurs in the asthmatic lung.