Increased Expression of an Inhibitor of NFkappaB, IkappaBalpha, in APCS Endows Them With the ACAID–Inducing Property

Abstract

Abstract: : Purpose: ACAID–inducing APCs were found to express increased levels of IkappaBalpha which is an inhibitor of proinflammatory transcription factor NFkappaB. Up–regulated message of IkappaBalpha correlated with increased nuclear IkappaBalpha protein, in APCs exposed to TGFß2. We examined if this increased expression of this inhibitor is involved in the ACAID–promoting effects of TGFß2. Methods: Macrophage hybridoma #59 cells stably expressing anti–IkappaBalpha siRNA were pulsed with ovalbumin (OVA) and cultured in the presence of TGFß2 (5 ng/ml). The cells were examined (a) in vitro for their ability to express IkappaBalpha protein (using western blot) and their ability to secrete IL–12 in culture supernatants (using ELISA); and (b) in vivo for their capacity to suppress OVA–specific delayed hypersensitivity (DTH) when injected into naive mice immunized subsequently with OVA plus complete Freunds’ adjuvant. Similarly #59 ectopically overexpressing IkappaBalpha were tested for their ability to suppress DTH. NFkappaB activity of #59, #59 expressing siRNA and #59 overexpressing IkappaBalpha was tested by measuring nuclear levels of subunits p50 and p65 using TransAm chemiluminescence assay (Active Motif, CA). Results: APCs expressing anti– IkappaBalpha siRNA failed to express increased nuclear levels of IkappaBalpha in response to TGFß 2 treatment as against control APCs infected with an empty vector. These siRNA expressing APCs also failed to inhibit IL–12 secretion as seen in control APCs treated with TGFß 2. Also, unlike the control APCs treated with TGFß 2, anti– IkappaBalpha siRNA expressing APCs failed to suppress ova–specific DTH response in vivo . On the other hand, APCs overexpressing IkappaBalpha suppressed ova–specific DTH responses similar to TGFß2–treated APCs. Moreover, nuclear levels of NFkappaB p50 subunit were found significantly decreased after 18 hrs of TGFß2 exposure of APCs (#59). However, anti– IkappaBalpha siRNA expression in APCs prevented this decrease whereas nuclear p50 levels of IkappaBalpha overexpressing APCs were decreased as compared to their control APCs. Conclusions: TGFß2 exposure of APCs induces increased expression of IkappaBalpha which by inhibiting proinflammatory transcription factor NFkappaB prevents secretion of IL–12. This expression of IkappaBalpha in APCs is critical to their ACAID–inducing phenotype.

Keywords: ACAID • antigen presentation/processing • gene/expression