Figure 3.

Effects of anti–IL-8:IL-8 immune complexes and LPS on expression and activation of Bruton’s tyrosine kinase (Btk) in purified human blood neutrophils. (A) Western blot analysis of whole-cell lysates of neutrophils incubated with medium (Med) or anti–IL-8:IL-8 complexes (IC) or LPS for 5 minutes, or LPS for 2 hours, or “primed” with LPS for 2 hours and stimulated with IC for 5 minutes, using specific antibodies against a kinase-domain Btk (upper blot) or total Btk (middle blot) and actin (bottom blot). The graph depicts the fold increase in the amount of Btk compared with medium. (B) Western blot analysis of whole-cell lysates of neutrophils incubated with medium (Med) or anti–IL-8:IL-8 complexes or LPS for 5 minutes, or LPS for 2 hours, or “primed” with LPS for 2 hours and stimulated with IC for 5 minutes, using specific antibodies against phosphorylated Btk (pBtk) and actin. The graph depicts the fold increase in the amount of pBtk compared with medium. (C) Btk or pBtk (green) is visualized in purified human blood neutrophils, using laser confocal microscopy. P < 0.001, for LPS (5 minutes) and LPS/IC versus medium, and for IC versus LPS/IC for pBtk. P = 0.003 for Btk. Representative cells (in white squares above) are enlarged to show membrane localization of pBtk (arrows). The vertical bar chart depicts levels of Btk or pBtk after treatment with various compounds (as already described). More than 5,000 cells were evaluated. Representative data from three experiments are shown.