Identification of proteases employed by dendritic cells in the processing of protein purified derivative (PPD)

Abstract

Dendritic cells (DC) are known to present exogenous protein Ag effectively to T cells. In this study we sought to identify the proteases that DC employ during antigen processing. The murine epidermal-derived DC line Xs52, when pulsed with PPD, optimally activated the PPD-reactive Th1 clone LNC.2F1 as well as the Th2 clone LNC.4k1, and this activation was completely blocked by chloroquine pretreatment. These results validate the capacity of XS52 DC to digest PPD into immunogenic peptides inducing antigen specific T cell immune responses. XS52 DC, as well as splenic DC and DCs derived from bone marrow degraded standard substrates for cathepsins B, C, D/E, H, J, and L, tryptase, and chymases, indicating that DC express a variety of protease activities. Treatment of XS52 DC with pepstatin A, an inhibitor of aspartic acid proteases, completely abrogated their capacity to present native PPD, but not trypsin-digested PPD fragments to Th1 and Th2 cell clones. Pepstatin A also inhibited cathepsin D/E activity selectively among the XS52 DC-associated protease activities. On the other hand, inhibitors of serine proteases (dichloroisocoumarin, DCI) or of cystein proteases (E-64) did not impair XS52 DC presentation of PPD, nor did they inhibit cathepsin D/E activity. Finally, all tested DC populations (XS52 DC, splenic DC, and bone marrow-derived DC) constitutively expressed cathepsin D mRNA. These results suggest that DC primarily employ cathepsin D (and perhaps E) to digest PPD into antigenic peptides.

Figure 1

XS52 DC are capable of presenting native PPD effectively to T cells: (A) XS52 DC were γ-irradiated and then pulsed for 8 hr with the indicated concentrations of PPD. The PPD-reactive Th1 clone (diamonds) or Th2 clone (squares) (10⁵ cells/well) was cultured for 2 days with PPD-pulsed XS52 cells (10⁴ cells/well). (B) Following a 3 hr incubation with or without chloroquine (100 μM), XS52 DC were pulsed with PPD (100 μg/ml) in the presence or absence of chloroquine (100 μM) and then examined for their capacity to activate the PPD-specific Th1 and Th2 clones. Data shown are the mean ± SD (n = 3) of ³H-thymidine uptake. Baseline proliferation of γ-irradiated XS52 DC alone was <300 cpm.

Figure 2

Pepstatin A inhibits the capacity of XS52 DC to present native PPD: (A) γ-irradiated XS52 DC were pulsed with PPD (100 μg/ml) in the presence or absence of each protease inhibitor (100 μg/ml pepstatin A, 100 μM DCI, or 100 μM E-64) or vehicle alone (1% DMSO or 15 mM NH₄Cl). XS52 DC were then cultured for 2 days with the PPD-reactive Th1 or Th2 clone in the continuous presence of the same inhibitor or vehicle alone. Data shown are the mean ± SD (n = 3) of ³H-thymidine uptake in three representative experiments. (B) XS52 DC were incubated with each of protease inhibitor (100 μg/ml pepstatin A, 100 μM DCI, or 100 μM E-64) or vehicle alone (1% DMSO or 15 mM NH₄Cl) for 16 hrs. Subsequently, cells were harvested and their viability was measured by trypan blue.

Figure 3

Failure of pepstatin A to inhibit the Ag presenting capacity of PPD-pulsed and fixed XS52 DC: (A) γ-irradiated XS52 DC were pulsed with PPD and then fixed with paraformaldehyde (left panels). Alternatively, XS52 DC were first fixed and then pulsed with PPD. Subsequently, the XS52 DC were cultured with the PPD-specific Th1 or Th2 clone in the presence or absence of pepstatin A. Data shown are the mean ± SD (n = 3) of ³H-thymidine uptake. (B): Allogeneic splenic T cells isolated from CBA mice (5 × 10⁵ cells/well) were cultured for 4 days with the indicated numbers of γ-irradiated XS52 DC in the presence or absence of pepstatin A. Data shown are the mean ± SD (n = 3) of ³H-thymidine uptake. (C): γ-irradiated XS52 DC were pulsed for 8 hr with either native PPD or trypsin-digested PPD in the presence or absence of pepstatin A. XS52 DC were then cocultured for 4 days with PPD-reactive Th1 or Th2 clones in the presence or absence of pepstatin A. Cocultures were then pulsed for 18 hr with ³H-thymidine and then harvested using a β-counter.

Figure 4

Pepstatin A Inhibits selectively the cathepsins D/E. XS52 DC were pretreated for 60 min with each of protease inhibitors or vehicles. After extensive washing, the cells were extracted and subsequently examined for protease activities. Data shown are % inhibition compared with untreated control cells.

Figure 5

DC constitutively express cathepsin D mRNA. Total RNA isolated from the indicated cell types were subjected to RT-PCR analysis for cathepsin D and β-actin. Data are shown, including bone marrow DC and macrophages, as well as 4F7⁺ splenic DC (splDC), products after 25 cycles of amplification.