Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain inovalbumin-immunized mice

Abstract

We previously reported that CA074, a specific inhibitor of cathepsin B, modulates specific immune responses from the T helper 2 (Th2) type to Th1 type in BALB/c mice infected with Leishmania major. In the present study, we found that a similar type of immune deviation was also induced in mice immunized with ovalbumin (OVA). However, treatment of mice with pepstatin A, a specific cathepsin D inhibitor, suppressed the OVA-specific proliferation of lymphocytes and blocked the development of both Th1 and Th2 cellular responses. These inhibitors did not appear to have any direct influence in vitro on functions of naive lymphocytes. OVA antigen (47 000 MW) was digested mainly into 40 000 MW protein in vitro by lysosomal proteases from naive BALB/c mice, and its digestion was markedly inhibited by the addition of CA074, but not by addition of pepstatin A, during incubation. However, pepstatin A strongly suppressed the degradation of the major histocompatibility complex class II-associated invariant chain (Ii) molecule in vivo and in vitro. Thus, cathepsin B appears to process antigens directed to preferential activation of Th2 cells, while cathepsin D may be responsible for the degradation of Ii, the processing of which is essential in initiating the antigen-specific activation of Th1 and Th2 CD4+ T cells. These lysosomal proteases may have different functions in regulating immune responses.

Figure 1

T helper (Th) phenotype in mice treated with cathepsin inhibitors. Mice were immunized intraperitoneally (i.p.) with ovalbumin (OVA) adsorbed with alum. CA074 or pepstatin A (0·25 mg of each/mouse) was injected i.p. 2 hr before and after immunization and every 12 hr thereafter for 10 days. Frequencies of splenic interleukin (IL)-4, IL-5 and interferon-γ (IFN-γ)-secreting cells from naive, untreated, CA074- or pepstatin A-treated mice were determined by using an enzyme-linked immunospot (ELISPOT) assay. Spots were counted, and the results are expressed per 10⁶ cells. Standard deviations (SD) were < 10% of total spots counted for each cytokine. Results are representative of three separate experiments.

Figure 2

Levels of ovalbumin (OVA)-specific antibodies in mice treated with cathepsin inhibitors. Mice were treated as indicated in the Materials and methods, and serum titres of antigen-specific immunoglobulin G2a (IgG2a) and immunoglobulin E (IgE) from untreated, CA074- or pepstatin A-treated animals (n = 6) were assessed by using enzyme-linked immunosorbent assay (ELISA) 10 days after immunization. Results are representative of five separate experiments.

Figure 3

The delayed-type hypersensitivity (DTH) response in mice treated with cathepsin inhibitors. Mice (n = 3–5) were challenged subcutaneously (s.c.) with 10 μg of ovalbumin (OVA) in alum in the left hind footpad 10 days after immunization with OVA. The size of the footpad swelling in naive, untreated, CA074- or pepstatin A-treated mice was measured by comparing the swollen footpad with the non-swollen footpad 24 hr after challenge. The results of the DTH reaction are representative of three individual experiments.

Figure 4

Flow cytometry analysis of activated splenocytes in mice treated with cathepsin inhibitors. Cells from untreated (upper panel), CA074- (middle panel), or pepstatin A- (lower panel) treated mice were evaluated by fluorescence-activated cell sorter (FACScan) using the double-staining method. These panels were shown as the percentage of Thy-1·2⁺ blastoid cells. FSC, forward scatter.

Figure 5

Ovalbumin (OVA)-specific or non-specific proliferative response of splenocytes in mice treated with cathepsin inhibitors. Mice were immunized and treated as indicated in the Materials and methods, and cells were isolated and cultured for 72 hr with OVA (a), concanavalin A (Con A) (b) or anti-CD3 (b). Proliferation was measured by [³H]thymidine incorporation. The data are means of triplicate wells ± SD and are representative of five separate experiments. c.p.m., counts per minute; incorp., incorporation.

Figure 6

In vitro digestion of ovalbumin (OVA) by lysosomal proteases. Five micrograms of OVA was digested with 10 μg of mitochondria/lysosome (ML) fraction in the absence (lane 3) or in the presence of CA074 (lanes 4 and 5) or pepstatin A (lanes 6 and 7) (each at 1 μg/ml) for 3 hr at 37°. The OVA digests were then stained with Coomassie Brilliant Blue following separation by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The untreated OVA control is shown in lane 1, and control lysosomes in lane 2. Filled arrowheads indicate the lysosomal band, and the open arrowhead indicates the band of digested OVA.

Figure 7

Degradation of invariant chain (Ii) molecules in cathepsin inhibitor-treated A20 cells (a) and BALB/c mice (b). (a) Cells (10⁸) were incubated for 4 hr in the absence (lane 1) or in the presence of CA074 (lane 2), pepstatin A (lane 3), or leupeptin (lane 4) (each at 100 μg/ml). The cells were then lysed and immunoprecipitated with In-1 (anti-Ii) monoclonal antibody (mAb), as described in the Materials and methods. Immunoprecipitates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained using silver-staining reagents. In-1 mAb only is shown in lane 5. IgH is the H chain of immunoglobulin, and IgL is the L chain of immunoglobulin. (b) BALB/c mice were treated with CA074 or pepstatin A for 10 days. Splenocytes were collected and blocked with normal mouse serum for 30 min at 37° and then stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse Ii mAb (solid lines) Dotted lines indicate the unstained control.