Expression and translocation of Rac2 in eosinophils during superoxide generation

Abstract

Eosinophils induce tissue injury by releasing granule-associated cytotoxic proteins, lipid mediators and superoxide anions in response to appropriate stimuli. Superoxide generation associated with respiratory burst is largely dependent on the assembly of the NADPH oxidase complex in the membrane, consisting of membrane-bound cytochrome b558 and translocated p47phox and p67phox. The activation of this complex is critically dependent on the translocation of GTP-bound Rac1, or its homologue Rac2, from the cytosol to the membrane in neutrophils. Rac expression has not yet been fully characterized in eosinophils. We proposed that eosinophils translate and express Rac2 and its GDP-dissociation inhibitor, RhoGDI. Furthermore, we hypothesized that Rac2 translocates along with p47phox and p67phox proteins from the cytosol to the plasma membrane during respiratory burst. By reverse transcription-polymerase chain reaction analysis and sequencing of the amplified product, guinea-pig eosinophils were found to express Rac2 mRNA, exhibiting 93% homology with the human Rac2 sequence. Rac1 mRNA was also detected in eosinophils but not its translated product. In contrast, Rac2 protein expression was detected using a specific antibody. In subcellular fractions, Rac2 was found to translocate, along with p47phox and p67phox, from cytosol to plasma membrane-associated fractions following phorbol myristate acetate stimulation, while RhoGDI remained within cytosolic fractions. These findings suggest that Rac2 is preferentially expressed and activated in eosinophils, and is likely to be a crucial regulator of the release of reactive oxygen species from these cells during inflammatory reactions.

The Functional Role of Rho and Rho-Associated Coiled-Coil Forming Protein Kinase in Eotaxin Signaling of Eosinophils

“…However, in bone marrow-derived Rac2 Ϫ/Ϫ neutrophils, the superoxide production in response to IgG-coated SRBC or fMLP was significantly abrogated, suggesting the important role of Rac2 in the function downstream of chemoattractant and Fc␥R (42). In addition, Lacy et al (43) have shown that Rac2 translocates from cytosol to plasma membrane-associated fractions along with p47phox and p67phox in PMA-stimulated eosinophils.…”

Divergence of Mechanisms Regulating Respiratory Burst in Blood and Sputum Eosinophils and Neutrophils from Atopic Subjects

“…The molecular mechanisms associated with NADPH oxidase assembly and activation have been studied in more detail in neutrophils (5, 8, 10 -13) and cell-free assays (10,14) than in eosinophils (15)(16)(17)(18)(19). This complex is composed of five essential subunits, the membrane-bound cytochrome b 558 (a complex of two subunits, p22 phox and gp91 phox ) which associates with cytosolic subunits Rac, p47…”

“…Generation of extracellular O 2 -from cells in suspension was measured as previously described (19). Briefly, cells (1-2 ϫ 10 6 ) were suspended in 1-ml microcuvets containing supplemented PBS (PBS ϩ ), pH 7.4 (with 1.2 mM MgCl 2 , 5 mM KCl, 0.5 mM CaCl 2 , 5 mM glucose, and 0.1% BSA) and 50 M ferricytochrome c at 25°C.…”

“…Highly purified batches of eosinophils and neutrophils (Ն 99%) were subjected to total RNA extraction using a Qiagen RNeasy MiniKit (Qiagen, Mississauga, Canada) using 2 ϫ 10 6 cells per extraction (producing 0.2-1 g RNA) (19). Primer sequences used for detecting Rac1/2 mRNA were generated in our laboratory for Rac1 (product size, 575 bp), with forward and reverse sequences as follows: 2-24 bp, 5Ј-TGCAGGCCATCAAGT GTGTGGTG-3Ј and (554 -576 bp) 5Ј-CAACAGCAGGCATTTTCTCT TCC-3Ј.…”

“…Samples were subjected to acrylamide gel electrophoresis and transferred to Immobilon polyvinylidene difluoride membrane blots (19). Primary mouse mAbs to Rac (1/1000), p22…”