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*
Leukocyte Biology Section, Biomedical Sciences Division, Imperial College School of Medicine, South Kensington, London, United Kingdom; and
LeukoSite, Inc., Cambridge, MA 02142
To investigate eosinophil stimulation by chemokines we developed a
sensitive assay of leukocyte shape change, the gated
autofluorescence/forward scatter assay. Leukocyte shape change
responses are mediated through rearrangements of the cellular
cytoskeleton in a dynamic process typically resulting in a polarized
cell and are essential to the processes of leukocyte migration from the
microcirculation into sites of inflammation. We examined the actions of
the chemokines eotaxin, eotaxin-2, monocyte chemoattractant protein-1
(MCP-1), MCP-3, MCP-4, RANTES, macrophage inflammatory protein-1
(MIP-1), and IL-8 on leukocytes in mixed cell suspensions and
focused on the responses of eosinophils to C-C chemokines. Those
chemokines acting on CCR3 induced a rapid shape change in eosinophils
from all donors; of these, eotaxin and eotaxin-2 were the most potent.
Responses to MCP-4 were qualitatively different, showing marked
reversal of shape change responses with agonist concentration and
duration of treatment. In contrast, MIP-1 induced a potent response
in eosinophils from a small and previously undescribed subgroup of
donors via a non-CCR3 pathway likely to be CCR1 mediated. Incubation of
leukocytes at 37°C for 90 min in the absence of extracellular calcium
up-regulated responses to MCP-4 and MIP-1 in the majority of donors,
and there was a small increase in responses to eotaxin. MIP-1
responsiveness in vivo may therefore be a function of both CCR1
expression levels and the regulated efficiency of coupling to
intracellular signaling pathways. The observed up-regulation of
MIP-1 signaling via non-CCR3 pathways may play a role in eosinophil
recruitment in inflammatory states such as occurs in the asthmatic
lung.
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