Specific deactivation of monocyte and lymphocyte migration by antithrombin III

Background

Antithrombin III exerts direct effects on neutrophils by inhibiting chemokine-induced migration [1]. Whether ATIII directly affects the migratory behaviour of other types of leukocytes is unknown.

Methods

We investigated the effect of ATIII on spontaneous and chemokine-triggered migration using RANTES and interleukin-8 as attractants of lymphocytes, and RANTES and monocyte chemotactic peptide-3 as attractants of monocytes, in modified Boyden chamber micropore filter assays. Lymphocyte and monocyte populations from human peripheral blood were pure. Signaling of ATIII in migration of the leukocytes was studied by blocking signaling enzymes with staurosporine, GFX, wortmannin and rolipram. As ATIII, the concentrate Kybernin^®P and antibody purified ATIII thereof were used.

Results

Pretreatment of lymphocytes with ATIII slightly augmented random locomotion, chemotaxis toward optimal concentrations of RANTES or IL-8 was significantly inhibited by pretreatment of the cells with ATIII followed by washing. Significant inhibition of chemotaxis was seen at ATIII concentrations as low as 10 nU/ml. Exposure of lymphocytes to gradients of ATIII stimulated migration in the absence of additional chemokines. Pretreatment of monocytes with ATIII before triggering of directed migration revealed similar findings, with ATIII again being active at low concentrations. In the absence of chemokines, ATIII again activated monocytes' directed migration. This ATIII-induced augmentation of migration was used for investigating signaling events induced in the cells by preincubation with various enzyme blockers: in contrast to neutrophils, where ATIII effects are mediated by protein kinase C and cAMP, responses of monocytes were wortmannin- and rolipram-sensitive; lymphocytes were additionally affected by GFX.

Conclusion

ATIII directly affects monocyte and lymphocyte functions in vitro. ATIII inhibits chemokine-stimulated migration of the two peripheral blood mononuclear cell populations. Thus, cellular effects of ATIII may occur not only in neutrophils but also in other immune cell populations. Signal transduction may be cell type-dependent, as it differs between neutrophils, lymphocytes and monocytes. A specific pathway for direct cellular activation by ATIII is postulated.