Angiotensin-converting enzyme affects the presentation of MHC class II antigens

Abstract

Antigen processing and presentation through the MHC class II pathway is critical for activating T helper cells. Angiotensin-converting enzyme (ACE) is a carboxyl peptidase expressed by antigen-presenting cells. By analysis of ACE null (knockout), wild-type, and ACE-overexpressing (ACE10) mice and the antigen-presenting cells derived from these mice, we found that ACE has a physiological role in the processing of peptides for MHC class II presentation. The efficiency of presenting MHC class II epitopes from ovalbumin (OVA) and hen egg lysosome is markedly affected by cellular ACE levels. Mice overexpressing ACE in myeloid cells have a much more vigorous CD4⁺ T-cell and antibody response when immunized with OVA. ACE is present in the endosomal pathway where MHC class II peptide processing and loading occur. The efficiency of MHC class II antigen presentation can be altered by ACE overexpression or ACE pharmacological inhibition. Thus, ACE is a dynamic participant in processing MHC class II peptides. Manipulation of ACE expression by antigen-presenting cells may prove to be a novel strategy to alter the immune response.

FIGURE 1. The effects of ACE on ovalbumin MHC class II antigen presentation in vitro

The efficiency of ACE KO, WT and ACE10 macrophages in presenting OVA_329–339, when fed with no antigen, OVA protein and OVA_329–339 peptide was evaluated by upregulation of CD69 (A) and secretion of IL-2 (B) by OT-II T cells. n=6. *P<0.05, **P<0.01, ***P<0.001. This figure, and all figures, show the standard error of the mean (SEM).

FIGURE 2. The effects of ACE on macrophage surface molecules and pinocytosis

(A) Expression of MHC class II molecular I-A^b and co-stimulatory factors CD80 and CD86 on the surface of macrophages from ACE KO, WT and ACE10 mice. N=5. (B) Pinocytosis of fluorescein conjugated OVA by ACE KO, WT and ACE10 macrophages was measured by flow cytometric analysis of mean fluorescence intensity (MFI). n=5. *P<0.05, **P<0.01, ***P<0.001.

FIGURE 3. The effects of ACE on HEL MHC class II antigen presentation in vitro

The efficiency of ACE KO, WT and ACE10 macrophages in presenting HEL-associated epitopes by I-A^b was measured by the secretion of IL-2 from their corresponding hybridomas. n=6. *P<0.05, **P<0.01, ***P<0.001.

FIGURE 4. ACE over-expression enhances OVA presentation to CD4⁺ T cells in vivo

WT and ACE10 mice were immunized with OVA-CFA. The spleens and draining lymph nodes (LN) were harvested 9 days later. OVA_329–339-specific CD4⁺ T cell activation was evaluated by production of the cytokines IFN-γ (A) and IL-17A (B). n=6–7. WT and ACE 10 mice were immunized with OVA-CFA (C) or OVA-Alum (D) followed by a boost of OVA alone 14 days later. Seven days after the boost, the titers of anti-OVA IgG subtypes were evaluated by ELISA. n=5. *P<0.05. **P<0.01, ***P<0.001.

FIGURE 5. ACE is present in MHC class II pathway and its catalytic activity is required for antigen processing

One hour after macrophage phagocytosis was induced with latex beads, ACE co-localized in the compartments of MHC class II (A), early endosome (B) and late endosome/lysosome (C). Stars indicate latex beads which were pinpointed under bright field. (A-C) Representative pictures of 3 independent experiments are shown. (D) WT macrophages were transfected with constructs encoding either wild-type ACE (ACE) or catalytic domain mutated ACE (mACE). Their presentation efficiency of OVA_323–339 following OVA administration was assessed by IL-2 secretion of OT-II T cells. n=6. *P<0.05.

FIGURE 6. MHC II antigen presentation is altered by ACE inhibition

(A) Macrophages purified from WT and ACE10 mice were pre-treated with or without lisinopril for 2 hours and then co-incubated with OVA. OVA_323–339 presentation was assessed through surface CD69 upregulation (top) or IL-2 secretion (bottom) by OT-II T cells. (B) WT and ACE10 mice were treated with lisinopril for 7 days followed by s.c. immunization of OVA-CFA. After another 9 days of lisinopril treatment, the spleens and the draining lymph nodes were harvested and the OVA_323–339-specific T cell responses were evaluated for IFN-γ (top) and IL-17 (bottom) production. n=6–7. *P<0.05. **P<0.01, ***P<0.001, NS: Not significant.