Hemin induces human neutrophil chemotaxis and actin polymerization.

(A) The effect of hemin on neutrophil chemotaxis was evaluated in a Boyden chamber. To stimulate PMN chemotaxis, we added either 10⁻⁷ M N-formyl-methionyl-leucyl-phenylalanine, 3 μM hemin (H3), 3 μM hemin + 10 nM BIM (H3 + BIM), or 3 μM hemin + 50 nM calphostin C (H3 + CC) to the bottom wells. Some groups were stimulated to migrate in the presence of 1% human serum albumin (+HSA). Neutrophils were added to the top wells and incubated for 1 hour at 37°C under 5% CO₂. After that, the filter was removed from the chamber and processed for neutrophil quantification as described in “Materials and methods.” Results are expressed as mean ± SD and are representative of 3 different experiments performed in triplicate for each sample. *P < .01. (B) Neutrophils were treated for 1 hour with 10 μM hemin in the presence (hemin + BIM) or absence (hemin) of 10 nM BIM or 100 nM PMA (PMA). The control group received only vehicle as treatment (control). After that, cells were lysed and the presence of actin in the Triton-soluble cytosolic fraction (G) and in the Triton-insoluble cytoskeleton fraction (F) was analyzed in each group by Western blot.