Identification of proteases employed by dendritic cells in the processing of protein purified derivative (PPD)
© Mohamadzadeh et al; licensee BioMed Central Ltd. 2004
Received: 30 April 2004
Accepted: 02 August 2004
Published: 02 August 2004
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.
Dendritic cells (DC) are professional antigen presenting cells that induce primary antigen specific T cell responses  and exhibit all functional properties required to present exogenous antigen (Ag) to immunologically naïve T cells. These properties include: a) uptake of exogenous Ag via receptor-mediated endocytoses, b) processing of complex proteins into antigenic peptides, c) assembly of these peptides with MHC molecules, d) surface expression of MHC molecules as well as costimulatory molecules, including CD80, CD86, and CD40, e) secretion of T cell stimulatory cytokines, including IL-1β, IL-6, IL-8, TNF-α, and macrophage inflammatory protein (MIP)-1α and f) migration into draining lymph nodes .
In the present study, we sought to characterize the Ag processing capacity of DC, as well as the enzymes previously involved in this process. In this regard, several groups have previously reported that epidermal LC and splenic DC, both of which contain small numbers of non-DC contaminants, exhibit significant Ag processing capacities [3–12]. LC freshly obtained from skin are quite potent in their Ag processing capacity, but the majority of these LC lose this capacity as they "mature" during subsequent culture [3–6, 12]. On the other hand, other reports have shown that DC are less efficient than macrophages in Ag processing, with each employing different pathways for Ag processing [10, 13–16]. These differences suggest the possibility of unique pathways and requirements for Ag presentation by DC.
With respect to the mechanisms by which DC process complex protein Ags, chloroquine has been shown to inhibit this process; this suggests that Ag processing primarily occurs within acidic compartments [6–8], [10–12]. Macrophages and B cells have been reported to employ cathepsins B, D, and/or E for digesting protein Ag, including ovalbumin (OVA), hen egg white lysozyme (HEL), myoglobin, exogenous IgG, and Staphylococcus aureus nuclease [17–35]. These proteases may each exhibit differential pathways for activity; for example, macrophages appear to employ cathepsin D for the initial cleavage of myoglobin and cathepsin B for C-terminal trimming of resulting fragments . Little information, however, has been available with respect to proteases that are employed by DC for Ag processing. Thus, in the present study we sought to define the protease profiles produced by DC and then to identify which protease(s) would primarily mediate Ag processing in DC.
Materials and Methods
The XS52 DC cell line (a gift of Dr. Takashima, Dallas, Texas), a long-term DC line established from the epidermis of newborn BALB/c mice , were expanded in complete RPMI in the presence of 1 ng/ml murine rGM-CSF and 10% culture supernatants collected from the NS stromal cell line as described previously . Other phenotypic and functional features of this line are descibed elsewhere [23–25]. As responding T cells, we used the protein purified derivative (PPD)-reactive Th1 clone LNC.2F1 and the Th2 clone LNC.4K1 , both of which were kindly provided by Dr. E. Schmitt (Institute for Immunology, Mainz, Germany). As control cells, we also employed Pam 212 keratinocytes , 7–17 dendritic epidermal T cells (DETC) , J774 macrophages (ATCC, Rockville, MD), and BW5147 thymoma cells (ATCC).
Splenic DC were purified from BALB/c mice (Jackson Laboratories, Bar Harbor, ME) by a series of magnetic bead separations as before [24, 25]. Briefly, spleen cell suspensions were first depleted of B cells using Dynabeads conjugated with anti-mouse IgG. Subsequently, T cells were removed using beads coated with anti-CD4 (GK1.5) and anti-CD8 mAbs (3.155), and then macrophages were depleted using beads conjugated with F4/80 mAb. Finally, DC were positively sorted using beads coated with anti-DC mAb 4F7 . The resulting preparations routinely contained > 95% DC, as assessed by flow cytometry. DCs were propagated from bone marrow as described by Inaba et al. . Using magnetic beads, bone marrow cell suspensions were first depleted of B cells (with anti-mouse IgG), I-A+ cells (with 2G9 mAb, Pharmingen, San Diego, CA), and T cells (with GK1.5 and 3.155 mAbs). The remaining I-A- cells were then cultured in the presence of GM-CSF (10 ng/ml). The purity of bone marrow derived DC was more than 95% as determined by flow cytometry using anti-CD11c and anti-I-A antibody (not shown).
Determination of protease activities
Cells were lysed in 0.1% Triton X-100 in 0.9% NaCl; extracts were then examined for protease activities using the following substrates: a) Z-Arg-Arg-βNA (for cathepsin B, at pH 6.0), b) denatured hemoglobin (cathepsin D/E, pH 3.0), c) Arg-βNA (cathepsin H, pH 6.8), d) Z-Phe-Arg-MCA (cathepsin J, pH 7.5), e) Z-Phe-Arg-MCA (cathepsin L, pH 5.5), f) Gly-Phe-βNA (DPPI or cathepsin C, pH 5.5), g) BLT ester (BLT esterase, pH 7.5), and h) Suc-Ala-Ala-Pro-Phe-SBz and Suc-Phe-Leu-Phe-SBz (chymotrypsin-like proteases, pH 7.5). Samples were incubated at the indicated pH and enzymatic activities were assessed by colorimetric or fluorogenic changes . Enzymatic activities were expressed as nmol/min/mg soluble protein, in which protein concentrations were measured by the bicinchoninic acid method using bovine serum albumin as a standard .
Ag presentation and T cell stimulation assays
XS52 DC were γ-irradiated (2000 rad) and then pulsed for 8 hr with 100 μg/ml of PPD (kindly provided by Dr. E. Schmitt, Mainz, Germany) in the presence of each of the following inhibitors (or vehicle controls): a) pepstatin A (100 μg/ml, Sigma, St. Louis, MO), b) DCI 100 μM, Sigma), c) E-64 (100 μM, Sigma), d) DMSO (1%), and e) NH4CL (15 mM). Subsequently, the XS52 cells were washed 3 times with PBS to remove unbound PPD and then cultured in 96 round-bottom well-plates (104 cells/well) with either the PPD-reactive Th1 or Th2 clone (105 cells/well) in the presence of the same inhibitor at the above concentration. In some experiments, XS52 DC were pulsed overnight with PPD in the presence of an inhibitor and then fixed with 0.05% glutaraldehyde in PBS for 30 seconds at 4°C; the fixation reaction was stopped by adding 0.1 M L-lysine. These XS52 cells were then washed with PBS and examined for their ability to activate Th1 or Th2 clones in the absence of protease inhibitors. In order to determine the mechanism of action for pepstatin A, XS52 cells were pulsed in its presence with PPD either in a native form or following digestion with trypsin-conjugated sepharose beads (Pierce, Rockford, IL) for 15 minutes at 37°C. We also examined the effect of added pepstatin A on the capacity of XS52 cells to activated allogeneic T cells isolated from CBA mice (Jackson Laboratories). Samples were pulsed for 18 hr with 1 μCi of 3H-thymidine and then harvested using an automated cell harvestor.
mRNA expression for cathepsin D was examined by RT-PCR. RNA isolation, reverse-transcription, and cDNA amplification were carried out as previously described . The following primers were designed based on the published sequence of murine cathepsin D : 5'-GGTCAGAGCAGGTTTCTGGG-3' and 5'-GCTTTAAGCTTTGCTCTCTTCGGG-3'. After 25 cycles of amplification, PCR products were analyzed in 1% agarose gel electrophoresis containing 2 μg/ml ethidium bromide. Other experimental conditions, including primer sequences for the β-actin control, are described elsewhere .
DC exhibit several different protease activities and they process the complex protein Ag PPD into antigenic peptides
Protease Profiles Expressed by Several DC Populations
Bone Marrow DC
BW5147 Thymoma Cells
133 ± 392
123 ± 3.3
121 ± 3.3
1.5 ± 0.08
34 ± 7
16 ± 4
0.4 ± 0.1
34 ± 6
30 ± 14
22 ± 2
1.6 ± 0.1
2.8 ± 0.8
3.5 ± 0.7
1 ± 0.2
0.9 ± 0.2
26 ± 0.3
0.7 ± 0.3
3.0 ± 0.1
0.2 ± 0.09
14 ± 0.6
26 ± 11
19 ± 0.6
0.6 ± 0.08
25,000 ± 400
58,000 ± 4,000
21,300 ± 100
1,900,000 ± 61,000
310,000 ± 10,800
1,150,000 ± 10,200
12,000 ± 100
433,000 ± 2,900
134,000 ± 7,300
360,000 ± 6,100
3,400 ± 600
Pepstatin A inhibits the capacity of XS52 DC to present native PPD to T cells
Functional role of cathepsin D/E in the processing of PPD by XS52 DC
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.
Dr. Mohamadzadeh is the major contributor (15%) of the experimental data and a rough draft of the paper. The next three intermediate authors' contributed remaining data and advice. Dr. Luftig was the overall individual who directed the several drafts and contributed to providing a new set of references to the manuscript.
This work was supported by NIH grant DA016029 (MM) and Tulane base grant RR00164 (MM). The authors would like to thank Dr. M. J. McGuire (UTSMC, Dallas, Texas) for his support and the fruitful discussions.
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