The experiments reported in this study provide new information with respect to complex Ag processing by DC. First, the long-term DC line, XS52 DC, was capable of processing PPD into immunogenic peptides, in the complete absence of other cell types. Although previous studies using several different DC preparations have documented similar results (3–12), this is the first report validating the Ag processing capacity of DC, in the absence of contaminating cells. Second, we have characterized the protease profiles expressed by DC. XS52 DC, 4F7+ splenic DC, and bone marrow-derived DC, all exhibited significant protease activities for cathepsins B, C, D/E, H, J, and L, BLT esterase, and chymotrypsin. Thus, DC possess the capacity to produce a family of protease activities. Finally, pepstatin A, but not other protease inhibitors, abrogated almost completely the ability of XS52 DC to digest native PPD into an antigenic product, suggesting an important role for pepstatin A-sensitive proteases (most likely cathepsin D and/or E) during Ag processing by DC. Taken together, these results reinforce the concept that DC are fully capable of processing complex protein Ag into antigenic peptides.
As described before, macrophages and B cells have been reported to employ cathepsins B, D, and E primarily to digest complex protein Ag, such as ovalbumin (OVA), hen egg white lysozyme (HEL), myoglobin, exogenous IgG, and Staphylococcus aureus nuclease (17–22). Here we report that DC also employ cathepsin D and/or E to digest PPD into an immunogenic Ag-product. This conclusion is supported by several lines of evidence: a) pepstatin A, but not other protease inhibitors, completely blocked the presentation of intact PPD by XS52 DC to PPD-reactive Th1 and Th2 clones, whereas it did not affect the presentation of PPD fragments; b) pepstatin A pretreatment inhibited cathepsin D/E activity selectively among the DC-associated protease activities; and c) all tested DC preparations expressed cathepsin D mRNA constitutively. In this regard, DC isolated from the mouse thoracic duct have been reported to produce neglible, if any, cathespin D immunoreactivity (assessed by immunofluorescence staining), whereas peritoneal macrophages produced relatively large amounts . Also comparable levels of cathepsin D/E activity were detected in extracts from bone marrow-derived DC and from bone marrow-derived macrophages (data not shown). This discordance may reflect differences in the DC preparations tested and/or in the assays employed to detect cathepsin D. Nevertheless, our observations indicate that DC employ cathepsin D/E to degrade some protein Ag, with the implication that pepstatin A and other cathepsin D/E inhibitors  may be useful to prevent and even to treat unwanted hypersensitivity reactions against such protein Ag.
It is important to emphasize that different protein Ag may be degraded by different proteases in DC. Moreover, DC isolated from different tissues or in different maturational states may employ different proteases. For example, murine DC isolated from the thoracic are unable to digest human serum albumin effectively , and murine splenic DC purified following overnight culture have failed to degrade KLH significantly into a TCA-soluble form . Moreover, several reports document that LC lose their Ag processing capacity as they mature in culture [3–6, 12]. Thus, it will be interesting to compare DC from different tissues and in different states of maturation for their protease profiles and susceptibilities to pepstatin A treatment. We believe that the experimental system described in this report will provide unique opportunities to study the function of proteases and the regulation of their production in DC.