Alpha1-antitrypsin inhibits the ATP-induced release of monocytic interleukin-1β independent of its antiprotease activity

Introduction:

Alpha1-antitrypsin (AAT) is a well-described protease inhibitor. In addition to its anti-protease function, AAT exerts anti-inflammatory effects. Interleukin-1β (IL-1β) is a pro-inflammatory cytokine of innate immunity that plays an important role in host defense. Maturation and release of IL-1β must be tightly controlled, as increased systemic levels can cause life-threatening systemic inflammation. For example, patients that undergo cardiopulmonary bypass surgery (CPBS) show elevated IL-1β levels and frequently develop a systemic inflammatory response syndrome. During CPBS, the anti-protease activity of AAT is diminished by oxidation and by complex formation with proteases. Preliminary unpublished data from our laboratory indicated that AAT inhibits the ATP-induced IL-1β release from human monocytes by a novel signal transduction mechanism. To investigate, if the inhibitory activity of AAT depends on its anti-protease function, we compared different AAT preparations.

Materials and Methods:

Native AAT from the plasma of healthy persons and from CPBS patients was purified by affinity chromatography. Protein oxidation was performed with n-chlorosuccinimide, a method that was shown to inactivate the anti-protease function of AAT. Lipopolysaccharide-primed human monocytic U937 cells were stimulated with 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP), an agonist of ATP-receptor P2X7, for 30 minutes in absence and presence of different AAT preparations. Thereafter, the concentration of IL-1β was measured in cell culture supernatants by ELISA.

Results:

We demonstrated that physiological concentrations of native, purified AAT from healthy donors, CPBS patients and the AAT preparation Prolastin® dose-dependently inhibit BzATP-induced IL-1β release in U937 cells. Native AAT retains its anti-inflammatory activity upon oxidation, whereas its anti-protease function is lost. In contrast, Respreeza®, another commercial AAT preparation that is chemically reduced during purification, does not inhibit BzATP-induced IL-1β release. Oxidation reactivates the inhibitory function of Respreeza®. An inactivation of the inhibitory function of AAT on the IL-1β release could not be observed within the first 15 minutes of CPBS.

Conclusions:

Prolastin® and native AAT from healthy donors efficiently inhibit BzATP-induced IL-1β release from monocytic cells, which is independent of its anti-protease activity. Furthermore, the inhibitory effect of AAT is not inactivated within the first minutes of CPBS. However, later time-points of CPBS as well as the anti-protease activity of patient AAT need to be investigated.