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. Author manuscript; available in PMC: 2016 Oct 1.
Published in final edited form as: Arthritis Rheumatol. 2015 Oct;67(10):2634–2638. doi: 10.1002/art.39250

Role of the Neutrophil Chemorepellent Soluble Dipeptidyl Peptidase IV in Decreasing Inflammation in a Murine Model of Arthritis

Sarah E Herlihy 1, Monica L Brown 2, Darrell Pilling 1, Brad R Weeks 1, Linda K Myers 2, Richard H Gomer 1
PMCID: PMC4751999  NIHMSID: NIHMS756095  PMID: 26138693

Abstract

Objective

To determine whether an intraarticular injection of the neutrophil chemorepellent dipeptidyl peptidase IV (DPPIV; CD26) can attenuate inflammation and decrease the severity of arthritis in a murine model.

Methods

DBA/1 mice were immunized with type II collagen/Freund's complete adjuvant to produce collagen-induced arthritis (CIA). On day 25 postimmunization, recombinant human DPPIV (rhDPPIV) or phosphate buffered saline was injected intraarticularly, and arthritis severity scores were recorded 3 times per week. The hind legs of mice in both groups were fixed, decalcified, paraffin embedded, and sectioned. Pathologic scores for inflammation and neutrophil infiltration were recorded on a scale of 1–8, and the number of neutrophils was determined by morphometric cell counts. In addition, Mac-2–positive macrophages and articular damage were assessed using anti–Mac-2 antibodies and histologic staining, respectively.

Results

Injection of rhDPPIV reduced the mean score of arthritis severity in mice with CIA. DPPIV treatment reduced the overall extent of inflammation and articular damage around the arthritic joint and periarticular tissue, and also decreased neutrophil and macrophage infiltration.

Conclusion

A localized injection of the neutrophil chemorepellent DPPIV reduces inflammation and the severity of the disease in a murine model of arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and destruction of the joints (1). Neutrophils are an essential component of arthritis development. K/BxN mice lacking neutrophils are resistant to collagen-induced arthritis (CIA) and the spontaneous formation of arthritis (2). Conversely, neutrophils in RA patients are highly activated in the circulation, synovial fluid, and tissue (2,3). In the synovium, neutrophils allow deposition of antibodies, release reactive oxygen species, secrete chemokines that recruit neutrophils and other immune cells, and release neutrophil extracellular traps (NETs) (1–3). Changes in neutrophil chemotaxis and adhesion potentially preventing migration into the synovium are linked to remission of RA (1).

Dipeptidyl peptidase IV (DPPIV) is a serine protease present as both a membrane protein and a soluble protein in most body fluids (4). DPPIV activity is found in plasma, synovial fluid, and synovial tissue (5). In murine antigen-induced arthritis and CIA, the activity of plasma DPPIV is lower than that in naive mice, and DPPIV-deficient mice have an increased propensity for arthritis (6). Rats with CIA have decreased DPPIV activity in the plasma compared to CIA-resistant rats (5). Patients with inflammatory RA (characterized by high plasma levels of C-reactive protein) have lower plasma DPPIV activity than do patients with noninflammatory RA (characterized by lower plasma levels of C-reactive protein) (6). These results suggest that lower levels of DPPIV are correlated with an increased incidence and severity of arthritis.

We found that recombinant human DPPIV (rhDPPIV) functions as a chemorepellent of human and murine neutrophils, and that oropharyngeal administration of rhDPPIV decreases neutrophil numbers in the lungs in a murine model of pulmonary inflammation (7). In this study, we examined the effect of microinjecting rhDPPIV into the joint capsule in a murine model of arthritis.

MATERIALS AND METHODS

Immunization, arthritis induction, and measurement of severity of arthritis

Arthritis was induced in DBA/1 mice by immunization with type II collagen/Freund's complete adjuvant, as previously described (8). At 25 days after the initial immunization, the hind ankle tibiotarsal joint of each mouse was injected intraarticularly with either 1 μl of 2 μg/ml rhDPPIV (Enzo Lifesciences) in phosphate buffered saline (PBS) or 1 μl of PBS alone, as previously described (8). Titers of antibodies to type II collagen were analyzed 6 weeks after immunization (8). The severity of arthritis was scored 3 times per week, as previously described (8). Mice were killed on day 53, and the injected hind legs were fixed with 10% buffered formalin for 24 hours and decalcified with 15% EDTA for 3 weeks (9). Following decalcification, the legs were embedded in paraffin and sectioned lengthwise.

Immunohistochemistry and pathologic scoring

Sections were stained with hematoxylin and eosin (H&E), trichrome, or Safranin O (10), and the tibiotarsal joints and periarticular tissue were each subjectively evaluated for overall inflammation, deposition of collagen, appearance of cartilage, and the presence of neutrophils by a veterinary pathologist (BRW) who was blinded with regard to the sample group. The pathologic scores for inflammation and neutrophil infiltration were set on a scale of 1–8, in which a score of 1 indicates none to rare, 2 indicates minimum, 3 indicates minimum to mild, 4 indicates mild, 5 indicates mild to moderate, 6 indicates moderate, 7 indicates moderate to severe, and 8 indicates severe (11).

For cell counts, neutrophils were identified in H&E-stained sections on the basis of a characteristic staining pattern and nuclear morphology, as determined using a 60 × 1.4–numerical aperture oil immersion objective. Two 260-μm fields of view in the synovial cell exudate were examined for each section.

To detect DPPIV, sections were deparaffinized and antigens were retrieved using antigen-unmasking solution (pH 6; Vector) at 98°C for 20 minutes. The sections were then stained with 5 μg/ml mouse anti-human DPPIV (BioLegend), as previously described (7), with detection using a Mouse-on-Mouse immunodetection kit (Vector), following the manufacturer's instructions. Tissue sections stained with DPPIV antibodies were analyzed with NIH ImageJ software. The percentage area of tissue stained with DPPIV antibodies was quantified as a percentage of the total area of the tissue, as described previously (12,13).

To detect macrophages, deparaffinized sections were stained with 5 μg/ml anti–Mac-2 monoclonal antibodies (clone M3/38, rat IgG2a; BioLegend), and detected with 1 μg/ml F(ab′)2 biotin-conjugated donkey anti-rat antibody (Jackson ImmunoResearch), followed by streptavidin alkaline phosphatase (Vector). Isotype-matched mouse and rat irrelevant antibodies (BioLegend) were used as controls.

Statistical analysis

Groups were compared using Student's unpaired 2-tailed t-test or the Mann-Whitney U test, calculated with GraphPad Prism software. P values less than 0.05 were considered statistically significant.

RESULTS

Reduction in the severity of arthritis by rhDPPIV

We previously observed that creating a concentration gradient of rhDPPIV in the lungs of mice, with the high side of the gradient in the alveoli and the low side in the circulation, repelled neutrophils out of the lungs and/or prevented neutrophils from entering the lungs (7). To test the hypothesis that a localized injection of rhDPPIV into a joint capsule, creating a localized gradient of rhDPPIV that was high in the joint space and lower outside the joint capsule, could alleviate arthritis, rhDPPIV was microinjected intraarticularly into the hind legs of mice 25 days following an immunization that induced arthritis. In mice, the serum DPPIV concentration is ~400 ng/ml (4,6). The joint was injected with 1 μl of 2 μg/ml rhDPPIV. Assuming that the volume of the tibiotarsal joint capsule is ~1 ml, injecting 1 μl of 2,000 ng/ml rhDPPIV into the joint would cause the joint DPPIV concentration to be higher than the serum DPPIV concentration.

The severity of arthritis decreased with rhDPPIV treatment (Figure 1A). The mean scores of arthritis severity in the rhDPPIV-treated mice were significantly lower than those in PBS-treated mice beginning at 39 days post-immunization (Figure 1A). Moreover, rhDPPIV-treated mice showed significantly less synovial inflammation compared to PBS-treated mice on day 53, as determined by pathologic scores and histologic assessment (Figures 1B–D). Remarkably, traces of rhDPPIV could still be detected in areas of the tibiotarsal joint (3 of the 5 tibiotarsal joints) on day 53 (Figure 2). These results suggest that intraarticular microinjection of rhDPPIV reduces the severity of arthritis and the overall extent of synovial inflammation.

Figure 1.

Figure 1

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Alleviation of collagen-induced arthritis (CIA) following treatment with recombinant human dipeptidyl peptidase IV (rhDPPIV). A, On day 25 postimmunization, mice were injected intraarticularly with rhDPPIV in phosphate buffered saline (PBS) (n = 12; Arthritic + DPPIV) or with an equal volume of PBS alone (n = 11; Arthritic). The mice were then evaluated for signs of arthritis 3 times per week. Values are the mean±SEM severity scores. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 versus PBS-treated mice with CIA, by t-test. B, On day 53, PBS-injected (n = 3) and rhDPPIV-injected (n = 5) mice with CIA were killed and the synovial cell exudate was scored for the extent of inflammation on a scale of 0–8. Values are the mean±SEM. ** = P < 0.01 by t-test. C and D, Histologic assessment was performed on day 53 using hematoxylin and eosin–stained leg sections from PBS-injected (C) and rhDPPIV-injected (D) mice with CIA. Representative sections are shown. Bar = 100 μm.

Figure 2.

Figure 2

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Immunohistochemical staining for rhDPPIV in mice with CIA. A–C, Leg joint sections from a PBS-injected mouse (A) and an rhDPPIV-injected mouse (B) with CIA were stained with anti-human DPPIV antibodies. As a control, a leg joint section from an rhDPPIV-treated mouse with CIA was stained with isotype-matched mouse irrelevant antibodies (Ab) (C). Bar = 200 μm. D, The percentage area stained with anti-human DPPIV antibodies was quantified as a percentage of the total area of the joint. Symbols represent individual joints; the horizontal bar indicates the sample mean. See Figure 1 for other definitions.

Reduction in neutrophil infiltration by DPPIV

We previously found that rhDPPIV is a neutrophil chemorepellent (7). To determine whether rhDPPIV reduced the accumulation of neutrophils in the arthritic joints of mice, the presence of neutrophils was assessed both qualitatively with a pathologic score and quantitatively by cell counts. Mice treated with rhDPPIV accumulated significantly fewer neutrophils than did PBS-treated mice, as indicated by the mean pathologic scores of neutrophil infiltration (Figure 3A). When the number of neutrophils per mm2 was calculated using morphometric neutrophil counts, again rhDPPIV-treated mice were found to accumulate significantly fewer neutrophils than PBS-treated mice (Figure 3B). These data suggest that intraarticular microinjection of rhDPPIV reduces the number of infiltrated neutrophils in the joints of mice with CIA.

Figure 3.

Figure 3

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Reduction in neutrophil density by rhDPPIV. A, The synovial cell exudate in hematoxylin and eosin–stained leg sections from PBS-injected mice with CIA (n = 3; Arthritic) and rhDPPIV-injected mice with CIA (n = 5; Arthritic + DPPIV) was scored for neutrophil infiltration on a scale of 0–8. B, Neutrophils in the synovial cell population were independently identified by morphology and counted. Values are the mean±SEM. ** = P < 0.01; *** = P < 0.001, by t-test. See Figure 1 for definitions.

Reduction in synovial macrophage counts and articular damage by DPPIV

To determine whether rhDPPIV reduced the accumulation of other inflammatory cells in the arthritic joints of mice, the presence of macrophages was assessed by anti–Mac-2 antibody staining. Compared to PBS-treated mice, rhDPPIV-treated mice had fewer Mac-2–positive macrophages in the synovial tissue (Figures 4A–D). These data suggest that intraarticular microinjection of rhDPPIV reduces the number of macrophages in the joints of mice with CIA.

Figure 4.

Figure 4

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Reduction in Mac-2–positive macrophage density by rhDPPIV in mice with CIA. A, and B, Representative leg joint sections from a PBS-injected mouse (A) and an rhDPPIV-injected mouse (B) with CIA were stained with anti–Mac-2 antibodies. Boxed areas indicate representative areas of the synovium used to count Mac-2–positive cells. Bar = 200 μm. C, A leg joint section from a PBS-treated mouse with CIA was stained with control antibodies. D, Mac-2–positive cells were counted in the synovium of PBS-injected mice (n = 3) and rhDPPIV-injected mice (n = 5) with CIA. Values are the mean±SEM. * = P < 0.05 by Mann-Whitney U test. See Figure 1 for definitions.

To determine whether rhDPPIV reduced the extent of articular damage, joints were assessed by staining with either trichrome or Safranin O. The joints of rhDPPIV-treated mice showed attenuated inflammation and lower levels of synovial hyperplasia as compared to PBS-treated mice (Figures 5A and B). However, Safranin O staining indicated that there was little difference in the articular proteoglycan content between rhDPPIV-treated mice and PBS-treated mice (Figures 5C and D). These data suggest that intraarticular microinjection of rhDPPIV reduces inflammation in the synovium without affecting the extent of articular damage in mice with CIA.

Figure 5.

Figure 5

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Reduction in joint inflammation by rhDPPIV. Immunohistochemical staining was performed using trichrome (A and B) or Safranin O (C and D) in representative leg joint sections from PBS-injected (A and C) and rhDPPIV-injected (B and D) mice with CIA. Asterisks indicate areas of synovial hyperplasia; arrows indicate areas of articular cartilage. Bars = 200 μm. See Figure 1 for definitions.

DISCUSSION

Neutrophil accumulation is detrimental to the joint in RA (1,3). In addition to recruiting other inflammatory cells to the joint, neutrophils release proteases, reactive oxygen species, and NETs that damage the synovial tissue (1,3). We found that rhDPPIV acts as a chemorepellent of human neutrophils (7), and other researchers found that RA patients have low serum DPPIV activity (6). In this study, we found that in a murine CIA model, microinjection of rhDPPIV into the joint capsule decreased the severity of arthritis, attenuated inflammation, and reduced the synovial neutrophil and macrophage density.

Decreased levels of DPPIV in both the synovial fluid and synovial membranes of patients with RA suggest that lower levels of DPPIV are correlated with an increased incidence and severity of arthritis (6,14,15). In addition, the results of a large cohort study suggested that DPPIV inhibitors may reduce the risk of autoimmune diseases, including arthritis (16). However, there are several reports indicating that patients with type 2 diabetes receiving systemic DPPIV inhibitors can develop a polyarthritis (17,18). Whether systemic DPPIV inhibitors can lower serum DPPIV activity to a point where the synovial fluid DPPIV activity is greater than the circulating levels, thus generating a reverse gradient of DPPIV, is open to speculation.

DPPIV acts on a variety of molecules that regulate leukocyte migration and activation (19,20). Whether the reduced accumulation of neutrophils and macrophages in the DPPIV-treated mice indicates the direct effect of DPPIV in its role as a chemorepellent (7), and/or its role in cleaving chemoattractants, the outcome is a reduced inflammatory cell number and less tissue damage. However, we have previously found that DPPIV does not appear to act as a chemorepellent for monocytes and macrophages, but is a direct chemorepellent for neutrophils in vitro and in vivo (7). To determine whether DPPIV acts directly on neutrophils to inhibit migration or acts via its protease activity will require the generation of recombinant DPPIV that lacks enzymatic activity.

Synovial fluid has approximately one-third to one-half less DPPIV activity than that in the serum (4). Dipeptidyl protease activity is mostly accounted for by DPPIV, as DPPIV-deficient mice display a >80% reduction in DPPIV activity (6). The dipeptidyl protease activity in mice corresponds to ~475 ng/ml DPPIV in the serum and ~250 ng/ml in the synovial fluid (4,6). We previously found that many gradients of DPPIV cause chemorepulsion of neutrophils (7). These gradients included 0–1,000 ng/ml (0–11.7 nM), 400–1,000 ng/ml (4.7–11.7 nM), and 800–2,000 ng/ml (9.4–23 nM) (7). The normal and the post–rhDPPIV injection serum to synovial fluid gradients of DPPIV fall within this range. Remarkably, the effects of a single rhDPPIV injection on day 25 persisted to at least day 53. This could be attributable either to a sufficient amount of the injected rhDPPIV remaining in the synovium, and continuously acting as a neutrophil chemorepellent, or to a shorter time effect of rhDPPIV breaking a vicious cycle of inflammation and neutrophil recruitment, allowing some resolution to occur. We did detect DPPIV on day 53 in some of the rhDPPIV-injected joints, but it is not possible to determine the actual concentration.

Although DPPIV inhibitors have been tested as therapeutic agents for RA, there is controversy about whether inhibiting all DPPIV activity is desirable (4). We previously found that inhibiting DPPIV activity also reduces its ability to act as a human neutrophil chemorepel-lent (7), and therefore the results in this study suggest that reducing DPPIV neutrophil chemorepellent activity in the serum, while preserving the activity in the synovial fluid (if this could be done), might be useful to reduce arthritis severity. Alternatively, direct injection of rhDPPIV into an inflamed joint, as we did in this murine model, might be useful as a therapeutic strategy.

Footnotes

Texas A&M University has filed a patent application for the use of soluble dipeptidyl peptidase IV (DPPIV) as a neutrophil repellent. If the DPPIV intellectual property were to be licensed, Drs. Herlihy, Pilling, and Gomer could receive a share of the royalties through Texas A&M University.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Gomer had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Herlihy, Myers, Gomer.

Acquisition of data. Herlihy, Brown, Pilling.

Analysis and interpretation of data. Herlihy, Brown, Pilling, Weeks, Myers, Gomer.

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