It has long been thought that the major function of proteases in the inflammatory milieu is to cause tissue damage by degrading extracellular matrix proteins. However, it has become clear that extracellular proteases play an important regulatory role in controlling local inflammatory processes that extends beyond their established destructive capability (1, 2). Dipeptidyl peptidase I (DPPI; also known as cathepsin C) is a widely expressed lysosomal cysteine protease belonging to the papain gene family. DPPI is critical for the activation of multiple serine proteases, including cytotoxic lymphocyte–associated granzymes (3), neutrophil-derived elastase, cathepsin G, proteinase 3 (4), and mast cell chymase (5).
We previously demonstrated that DPPI−/− mice were protected against acute arthritis induced by passive transfer of monoclonal antibodies against type II collagen (CII) (4). In this model of acute arthritis, mice were injected intravenously with 4 mg of a cocktail of 4 separate monoclonal antibodies specific for the main arthrogenic determinant of CII, followed by the injection of lipopolysaccharide, a strong inducer of proinflammatory cytokines (6). Although this model provided insights into the role of DPPI in acute inflammation, the disease is transient and is distinct histologically from the chronic disease seen in human rheumatoid arthritis (RA). In addition, using the passive antibody transfer model, we were unable to evaluate the role of DPPI in cellular and humoral immunity to collagen.
In the current study, we extend our observations to a model of more chronic inflammation. Collagen-induced arthritis (CIA) is a model of inflammatory arthropathy that shares many features with human RA. Here, we established that despite having normal humoral and cellular immunity to bovine CII, the majority of DPPI−/− mice were highly resistant to the development of CIA. In addition, adoptive transfer of CII-sensitized splenocytes from immunized WT animals induced arthritis in wild-type (WT) mice but not in DPPI−/− mice. Taken together, these results suggest that DPPI regulates a critical step in the development of arthritis that is independent of T cell and B cell functions.
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- MATERIALS AND METHODS
CIA in DPPI−/−mice. WT and DPPI−/− mice backcrossed to a DBA/1J strain for 10 generations were immunized with 200 μg of bovine CII in CFA on day 0, followed by 200 μg of bovine CII in IFA on day 21. In WT mice, clinical arthritis was usually detected 5–7 days after the second immunization. The cumulative incidence of disease was 93% in WT mice (n = 14) compared with 26% in DPPI−/− mice (n = 14) (Figure 1A). In a few DPPI−/− mice, mild redness and swelling developed following the booster injection, but the inflammation resolved without further progression. Some DPPI−/− mice had delayed onset of clinically evident arthritis manifested by redness and swelling in 1 or 2 digits (Figure 1D, bottom left panel). On day 44, the mean ± SD arthritis index in the DPPI−/− group was 0.43 ± 0.85, compared with 7.37 ± 3.5 in WT mice (P < 0.001) (Figure 1B). In addition, DPPI−/− mice had significantly reduced paw thickness (mean ± SD 3.08 ± 0.35 mm versus 3.71 ± 0.56 mm in WT mice; P < 0.01), as reflected by the measurements obtained on day 42 (Figure 1C).
Figure 1. Collagen-induced arthritis in wild-type (WT) and dipeptidyl peptidase I–deficient (DPPI−/−) mice. A, Disease incidence in type II collagen (CII)–immunized mice, expressed as the percentage of mice in which clinical arthritis developed (n = 14 per genotype). B, Disease activity, as scored on a scale of 0–3 for each paw (maximum possible score per mouse = 12), and expressed as the mean ± SD cumulative arthritis index score over time. C, Paw thickness, as measured across the ankle, on day 42 (the peak of arthritis). Values are the mean and SD. ∗ = P < 0.01. D, Hind paws of WT and DPPI−/− mice were processed for histologic analysis on day 42 after immunization. Note the marked swelling of the entire paw, including all digits, in the WT mouse (top left). In contrast, DPPI−/− mice showed no clinical arthritis (middle left) or disease as manifested by redness and swelling in the digits only (bottom left). Hematoxylin and eosin–stained sections of joints from WT mice showed intense inflammatory infiltrates and severe bony destruction (top right, arrowheads). In contrast, most DPPI−/− mice had normal joint histology, with a thin synovial layer (middle right, arrow) and no inflammatory infiltrate. In DPPI−/− mice in which arthritis developed, significant leukocyte infiltration and focal bone erosions can be seen (bottom right, arrowhead). E, IgG1 and IgG2a anti-CII antibody titers were measured by enzyme-linked immunosorbent assay, as described in Materials and Methods. Data are expressed as the mean and SD relative changes in antibody titers (fold increase) in immunized mice (n = 5 per genotype) compared with preimmunization levels.
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Histologic analysis confirmed that DPPI−/− mice were more protected against severe inflammation compared with WT mice. Evaluation of diseased joints from WT mice revealed extensive inflammatory infiltrates accompanied by severe bone and joint erosions (Figure 1D, top right panel). In contrast, most of the DPPI−/− mice exhibited normal joint morphology, with no inflammatory infiltrates (Figure 1D, middle right panel). DPPI−/− mice in which clinical disease developed exhibited synovitis with focal bone erosions; however, there was some preservation of the joint architecture (Figure 1D, bottom right panel). Overall, 87% of the joints from WT mice showed erosive changes (n = 31 joints; mean ± SD severity score 2.1 ± 1.2) compared with 11% of the joints from DPPI−/− mice (n = 27 joints; mean ± SD severity score 0.2 ± 0.7 [P < 0.001 versus WT]). In addition, 90% of the joints from WT mice showed inflammation (mean ± SD severity score 2.4 ± 1.0) compared with 20% of the joints from DPPI−/− mice (mean ± SD severity score 0.4 ± 0.9; P < 0.001). Taken together, these results suggest that the majority of DPPI−/− mice are resistant to the development of CIA; however, once disease is initiated in a joint, DPPI−/− mice are not entirely protected against synovial inflammation and bony erosions.
Humoral immunity to collagen in DPPI−/−mice. Humoral immunity to collagen, specifically IgG2a production, has been shown to play an essential role in the initiation of disease in CIA. In the model of passive transfer of anti-CII antibody, the response of DPPI−/− mice to CII immunization could not be evaluated. Due to the lysosomal localization of DPPI, it has been proposed that DPPI might play a role in antigen processing that is very similar to the action of cathepsin S and cathepsin L (8, 9). To evaluate whether DPPI−/− mice responded normally to collagen immunization, serum levels of IgG1 and IgG2a anticollagen antibodies were measured 7 days after the booster injection was administered. The levels of IgG1 and IgG2a anticollagen antibodies were equivalent in WT and DPPI−/− mice (Figure 1E). These results suggest that DPPI does not participate in the presentation of CII, and that the mechanism that confers resistance to the development of CIA in DPPI−/− mice is not attributable to diminished humoral immunity.
Cellular immunity to collagen in DPPI−/−mice. To assess cellular immunity to collagen in DPPI−/− mice, in vitro T cell proliferation was measured. WT and DPPI−/− mice were killed on day 44, and their draining inguinal lymph nodes were harvested and pooled. T cell proliferation was measured in lymph node cells cultured in the presence of 50 μg/ml of collagen or 5 μg/ml of Con A. The values for CII- or Con A–induced proliferation were similar for both WT and DPPI−/− cells (Figure 2A). These results suggest that cellular immunity to collagen was not altered in the absence of DPPI.
Figure 2. Humoral and T cell response to CII immunization in WT and DPPI−/− mice. A, Lymph nodes were harvested on day 44 and cultured in the presence of CII or concanavalin A (Con A) for 72 hours. Proliferation was measured by 3H-thymidine incorporation (n = 5 per genotype). B–D, Spleens were harvested on day 44 and cultured in the presence of CII or Con A for 48 hours. The levels of tumor necrosis factor α (TNFα) (B), interleukin-1β (IL-1β) (C), and IL-6 (D) in the supernatant were measured using a specific enzyme-linked immunosorbent assay (ELISA). E–H, Cells from paws were prepared as outlined in Materials and Methods. The average number of cells recovered from each DPPI−/− mouse paw was significantly lower compared with the number of cells obtained from WT mouse paws (n = 16 paws per genotype) (E). These cells were cultured without additional stimulus for 24 hours, and the spontaneous release of TNFα (F), IL-1β (G), and IL-6 (H) was measured using a specific ELISA. Values are the mean and SD. ∗ = P < 0.01. See Figure 1 for other definitions.
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Cytokine production in CII-stimulated spleen cells. It is well recognized that the production of proinflammatory cytokines is critical for the development and perpetuation of arthritis. To evaluate whether DPPI−/− splenocytes have a defect in their ability to produce cytokines, spleen cells from WT and DPPI−/− mice were harvested on day 44 after immunization and cultured in the presence of 50 μg/ml of CII or 5 μg/ml of Con A. The levels of TNFα, IL-1β, and IL-6 released from splenocytes were measured after 48 hours in culture. WT and DPPI−/− mouse splenocytes cultured in the presence of CII or Con A produced equivalent levels of TNFα (Figure 2B). IL-1β production by sensitized splenocytes in response to CII stimulation was minimal in both genotypes. Although we detected a 50% reduction in IL-1β production by DPPI−/− mouse splenocytes stimulated with Con A (Figure 2C), this decrease was not reproducible in subsequent experiments (data not shown). In contrast, IL-6 production was increased by 50% and 35% in DPPI−/− mouse splenocytes cultured in the presence of CII and Con A, respectively (Figure 2D). Taken together, these results indicate that DPPI−/− mouse splenocytes have no significant intrinsic defect in their ability to produce and release proinflammatory cytokines.
Cytokine production by joint mononuclear cells. We next isolated mononuclear cells from the paws of WT and DPPI−/− mice in order to determine the spontaneous release of TNFα, IL-1β, and IL-6 after 24 hours of in vitro culture. Similar to the results of the histologic analysis, we recovered 3-fold more mononuclear cells from the paws of WT mice than from the paws of DPPI−/− mice (n = 16 paws per genotype; P < 0.01) (Figure 2E). However, the spontaneous release of TNFα and IL-6 from WT and DPPI−/− mouse mononuclear cells was not different on a single-cell basis (Figures 2F and H). In these in vitro cell cultures, IL-1β was barely detectable (Figure 2G). The normal production of proinflammatory cytokines by these mononuclear cells is consistent with the inflammation observed in DPPI−/− mice with clinically manifest arthropathy.
Adoptive transfer of arthritis using splenocytes from CII-sensitized WT mice. Previous studies have shown that the transfer of splenocytes from arthritic mice into naive recipients induces arthritis. Depletion of CD4+ T cells inhibits the transfer of arthritis, while depletion of CD8+ T cells enhances the onset of arthritis (10). To directly evaluate whether the resistance of DPPI−/− mice to CIA depends on subtle defects in T lymphocyte functions, we performed adoptive transfer of splenocytes from CII-immunized, arthritic WT mice into naive WT and DPPI−/− mice, followed by immunization with CII. In naive WT mice that received 107 splenocytes from CII-immunized WT DBA/1J mice, moderate arthritis developed after 6–8 days (mean ± SD arthritis severity score 5 ± 2) (Table 1). In contrast, DPPI−/− mice were completely resistant to arthritis development after splenocyte transfer, although the increases from baseline in anti-CII antibody titers were equivalent in both genotypes (Table 1). These results indicate that resistance to CIA in DPPI−/− mice was independent of T cell and B cell functions.
Table 1. Adoptive transfer of arthritis by splenocytes from CII-immunized WT DBA/1J mice*
|Mean ± SD arthritis severity score†||5 ± 2||NA|
|Day at onset of arthritis||6–8||NA|
|Mean ± SD anti-CII antibody titer‡||34 ± 13||59 ± 25|