Immunomodulatory Effects of Hypocrellin A on MHC-restricted Antigen Processing

Abstract

Hypocrellin A has gained much attention in recent years due to its light-induced antitumor, antifungal and antiviral activities. Here we report that hypocrellin A exerts immunomodulatory effects on MHC-restricted presentation of antigen. Hypocrellin A inhibited class II-MHC restricted presentation of exogenous antigen, but not class I MHC-restricted presentation of exogenous antigen, in dendritic cells. Hypocrellin A also inhibited the cytosolic pathway of endogenous antigen presentation. However, hypocrellin A did not inhibit the expression of class I and class II MHC molecules on dendritic cells (DCs), the phagocytic activity of DCs, or the H-2K(b)-restricted presentation of a synthetic peptide, SIINFEKL. These results show that hypocrellin A differentially modulates the MHC-restricted antigen presentation pathways.

Keywords: Hypocrellin A; Immunomodulator; MHC-restricted antigen presentation.

Figure 1

Effects of hypocrellin A on the MHC-restricted presentation of exogenous OVA. (A) DC2.4 cells were incubated with the indicated amounts of hypocrellin A for 2 h, and then biodegradable microspheres containing OVA (50µg/ml as OVA) were added to the cultures for 2 h. The cells were then fixed with paraformaldehyde, and the amounts of OVA peptides presented on MHC class I molecules were assessed by a LacZ T cell activation assay using OVA-specific CD8 T cell hybridoma B3Z cells. (B) BM-DCs generated from bone marrow cells of BALB/c mice were incubated with the indicated amounts of hypocrellin A for 2 h, and then biodegradable microspheres containing OVA (50µg/ml as OVA) were added to the cultures for 2 h. The cells were then washed, fixed with paraformaldehyde, and the amounts of OVA peptides presented on MHC class II molecules were assessed using OVA-specific CD4 T cell hybridoma DOBW cells.

Figure 2

Effects of hypocrellin A on the cytosolic pathway of endogenous antigen presentation. (A) DC2.4 cells were incubated with the indicated amounts of hypocrellin A for 2 h, washed, and then soluble OVA was loaded into cytosol by osmotic shock as described previously (13). After 2-h incubation, the amounts of H-2K^b-OVA peptide complexes were assessed using OVA-specific CD8 T cell hybridoma B3Z cells. (B) The indicated amounts of hypocrellin A were added to cultures of DC2.4 cells that had been infected with rVV-OVA (MOI 10). After 6 h, the DCs were washed, fixed with paraformaldehyde, and then the capacity to activate OVA-specific CD8 T cells was assessed using OVA-specific CD8 T cell hybridoma B3Z cells.

Figure 3

Hypocrellin A does not inhibit the expression levels of class I and class II MHC molecules or the presentation of a synthetic OVA peptide SIINFEKL. Hypocrellin A (2,000 nM) was added to cultures of DC2.4 cells for 2 h. The cells were harvested, washed, and then stained with anti-mouse H-2K^b monoclonal antibody (A) or anti-I-A^b monoclonal antibody (B). The shaded histograms represent the expression levels of class I and class II MHC molecules in the presence of hypocrellin A, and the thick lines represent the expression levels of class I and class II MHC molecules in the absence of hypocrellin A. The dotted lines represent isotype controls. (C) Indicated amounts of hypocrellin A were added to cultures of DC2.4 cells together with the antigenic peptide of OVA, SIINFEKL. The cells were harvested, washed, and then the amount of SIINFEKL-H-2K^b complex was measured by a LacZ T cell activation assay using OVA-specific CD8 T cell hybridoma B3Z cells.