Micheler Ricardo Richardson

Leukocytes migrate to sites of inflammation where they participate in host defensive and/or tissue destructive activities. Many of these activities are mediated via activation of cell surface chemoattractant receptors which couple to G-proteins. Prolonged stimulation of chemoattractant receptors by proinflammatory factors such as interleukin-8 (IL-8), the fifth fragment of the complement system (C5a), formyl peptides (fMLP) and platelet activating factor (PAF) results in diminished ability of the ligands to induce cellular responses, a process termed desensitization. Interestingly, groups of chemoattractants are capable of cross-desensitizing each others' responses suggesting cross-regulation of classes of receptor. Our laboratory has sought to understand how chemoattractant receptor-mediated cellular responses are regulated in inflammatory cells.

We have developed a model system using a rat basophilic leukemia cell line (RBL-2H3). RBL-2H3 is a very responsive and biologically relevant cell line which possesses the machinery to induce most of the cellular responses stimulated by chemoattractants in neutrophils. We have successfully expressed epitope tagged receptors for fMLP, C5a and PAF in this cell line. As for the native receptors in neutrophils, the expressed receptors, upon against stimulation, mediated biochemical and functional responses such as generation of inositol phosphates, Ca2+ mobilization, secretion of granules and actin polymerization. One advantage of this system is the expressed receptors can be easily immunopurified using the commercially available epitope-tag specific antibody (12CA5). Another advantage of this system is the RBL-2H3 cell can be easily permeabilized to introduce into the cell not only small peptide molecules but also proteins such as PKC isozymes and antibodies while retaining its functional properties. These characteristics make the RBL-2H3 cell as versatile and useful system suitable to carry out experiments aimed at defining the molecular mechanism(s) of chemoattractant receptor regulation.

Key Words: Leukocytes, Chemoattractant Receptors, Signal Transduction, G-protein, Phosphorylation, Desensitization