Circulating CD26 Is Negatively Associated with Inflammation in Human and Experimental Arthritis
2005; Elsevier BV; Volume: 166; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)62266-3
ISSN1525-2191
AutoresNathalie Busso, Nicolai Wagtmann, Christian Herling, Véronique Chobaz-Péclat, A. Bischof-Delaloye, Alexander So, Eric Grouzmann,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoDipeptidyl peptidase IV (DPPIV, CD26), a protease-cleaving N-terminal X-Pro dipeptide from selected proteins including some chemokines, is expressed both as a soluble form in plasma and on the cell surface of various immune and nonimmune cell types. To gain insights into the pathophysiological role of CD26 in arthritis, we explored DPPIV/CD26 expression during murine antigen-induced arthritis (AIA), an experimental model of arthritis. AIA induction led to reduced plasma DPPIV activity. In CD26-deficient mice, the severity of AIA was increased as assessed by enhanced technetium uptake and by increased histological parameters of inflammation (synovial thickness and exudate). We demonstrated that CD26 controls the in vivo half-life of the intact active form of the proinflammatory chemokine stromal cell-derived factor-1 (SDF-1). CD26-deficient mice exhibited increased levels of circulating active SDF-1, associated with increased numbers of SDF-1 receptor (CXCR4)-positive cells infiltrating arthritic joints. In a clinical study, plasma levels of DPPIV/CD26 from rheumatoid arthritis patients were significantly decreased when compared to those from osteoarthritis patients and inversely correlate with C-reactive protein levels. In conclusion, decreased circulating CD26 levels in arthritis may influence CD26-mediated regulation of the chemotactic SDF-1/CXCR4 axis. Dipeptidyl peptidase IV (DPPIV, CD26), a protease-cleaving N-terminal X-Pro dipeptide from selected proteins including some chemokines, is expressed both as a soluble form in plasma and on the cell surface of various immune and nonimmune cell types. To gain insights into the pathophysiological role of CD26 in arthritis, we explored DPPIV/CD26 expression during murine antigen-induced arthritis (AIA), an experimental model of arthritis. AIA induction led to reduced plasma DPPIV activity. In CD26-deficient mice, the severity of AIA was increased as assessed by enhanced technetium uptake and by increased histological parameters of inflammation (synovial thickness and exudate). We demonstrated that CD26 controls the in vivo half-life of the intact active form of the proinflammatory chemokine stromal cell-derived factor-1 (SDF-1). CD26-deficient mice exhibited increased levels of circulating active SDF-1, associated with increased numbers of SDF-1 receptor (CXCR4)-positive cells infiltrating arthritic joints. In a clinical study, plasma levels of DPPIV/CD26 from rheumatoid arthritis patients were significantly decreased when compared to those from osteoarthritis patients and inversely correlate with C-reactive protein levels. In conclusion, decreased circulating CD26 levels in arthritis may influence CD26-mediated regulation of the chemotactic SDF-1/CXCR4 axis. CD26, also known as dipeptidyl peptidase IV (DPPIV) (EC 3.4.14.5), is a multifunctional type II transmembrane glycoprotein.1De Meester I Korom S Van Damme J Scharpe S CD26, let it cut or cut it down.Immunol Today. 1999; 20: 367-375Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar This protein is expressed constitutively on epithelial cells, several types of endothelial cells and fibroblasts, as well as leukocyte subsets such as T, B, and natural killer lymphocytes and macrophages. A soluble form of CD26, lacking the cytoplasmic tail and transmembrane region is also found in plasma and other biological fluids.The enzyme activity of CD26 is capable of cleaving N-terminal dipeptides from polypeptides with either proline or alanine resides in the penultimate position. Distinct enzymes with similar DPP specificity have also been described.2Mentlein R Dipeptidyl-peptidase IV (CD26)—role in the inactivation of regulatory peptides.Regul Pept. 1999; 85: 9-24Crossref PubMed Scopus (1139) Google Scholar Several cytokines, hematopoietic growth factors, neuropeptides, and hormones share the X-Pro or X-Ala motif at their N-terminus, including substance P, neuropeptide Y, endomorphin-2, GLP-1 (glucagon-like peptide), GIP (glucose-dependent insulinotropic polypeptide), RANTES (regulated on activation normal T cell expressed and secreted), eotaxin, MDC (monocyte-derived chemokine), and SDF-1 (stromal-derived factors).2Mentlein R Dipeptidyl-peptidase IV (CD26)—role in the inactivation of regulatory peptides.Regul Pept. 1999; 85: 9-24Crossref PubMed Scopus (1139) Google Scholar In many cases, CD26/DPPIV-mediated truncation of natural substrates has drastic effects on the biological activity or function. For instance, SDF-1 is a proinflammatory chemokine that stimulates chemotaxis in leukocytes by binding to its receptor CXCR4 (being itself only recognized by SDF-1).3Bluel CC Fuhlbrigge RC Casasnovas JM Aiuti A Springer TA A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1).J Exp Med. 1996; 184: 1101-1109Crossref PubMed Scopus (1267) Google Scholar In vitro, N-terminal processing of SDF-1 by CD26 reduced lymphocyte chemotaxis and CXCR4-signaling properties.4Proost P Struyf S Schols D Durinx C Wuyts A Lenaerts JP De Clercq E De Meester I Van Damme J Processing by CD26/dipeptidyl-peptidase IV reduces the chemotactic and anti-HIV-1 activity of stromal-cell-derived factor-1alpha.FEBS Lett. 1998; 432: 73-76Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar However, it is not known whether CD26 or other enzymes may truncate and inactivate SDF-1 in vivo, and if so what the functional consequences may be.CD26 may also potentially modulate immune responses by directly regulating lymphocytes.5Morimoto C Schlossman SF The structure and function of CD26 in the T-cell immune response.Immunol Rev. 1998; 161: 55-70Crossref PubMed Scopus (363) Google Scholar, 6Reinhold D Kahne T Steinbrecher A Wrenger S Neubert K Ansorge S Brocke S The role of dipeptidyl peptidase IV (DP IV) enzymatic activity in T cell activation and autoimmunity.Biol Chem. 2002; 383: 1133-1138Crossref PubMed Scopus (66) Google Scholar On human T cells, CD26 exhibits a co-stimulatory function;7Dang NH Torimoto Y Deusch K Schlossman SF Morimoto C Comitogenic effect of solid-phase immobilized anti-1F7 on human CD4 T cell activation via CD3 and CD2 pathways.J Immunol. 1990; 144: 4092-4100PubMed Google Scholar furthermore, CD26 plays an important role in the immune system via its ability to bind adenosine deaminase8Dong RP Tachibana K Hegen M Munakata Y Cho D Schlossman SF Morimoto C Determination of adenosine deaminase binding domain on CD26 and its immunoregulatory effect on T cell activation.J Immunol. 1997; 159: 6070-6076PubMed Google Scholar and mediates signaling by direct interaction with the cytoplasmic domain of CD45.9Ishii T Ohnuma K Murakami A Takasawa N Kobayashi S Dang NH Schlossman SF Morimoto C CD26-mediated signaling for T cell activation occurs in lipid rafts through its association with CD45RO.Proc Natl Acad Sci USA. 2001; 98: 12138-12143Crossref PubMed Scopus (143) Google Scholar Finally, CD26 was also involved in interactions with the extracellular matrix proteins, collagen and fibronectin.1De Meester I Korom S Van Damme J Scharpe S CD26, let it cut or cut it down.Immunol Today. 1999; 20: 367-375Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar The relative contribution of these effects of CD26 on lymphocytes, versus indirect effects because of possible cleavage of immunoregulating chemokines or cytokines, is not clear.In rheumatoid arthritis (RA), a chronic inflammatory autoimmune disease,10Firestein GS Evolving concepts of rheumatoid arthritis.Nature. 2003; 423: 356-361Crossref PubMed Scopus (2739) Google Scholar CD26 may have a dual role, on the one hand stimulating cellular immunity, and on the other hand inhibiting chemokine function. It has been previously reported that in RA patients the number of peripheral blood T lymphocytes expressing CD26 is increased.11Muscat C Bertotto A Agea E Bistoni O Ercolani R Tognellini R Spinozzi F Cesarotti M Gerli R Expression and functional role of 1F7 (CD26) antigen on peripheral blood and synovial fluid T cells in rheumatoid arthritis patients.Clin Exp Immunol. 1994; 98: 252-256Crossref PubMed Scopus (59) Google Scholar, 12Gerli R Muscat C Bertotto A Bistoni O Agea E Tognellini R Fiorucci G Cesarotti M Bombardieri S CD26 surface molecule involvement in T cell activation and lymphokine synthesis in rheumatoid and other inflammatory synovitis.Clin Immunol Immunopathol. 1996; 80: 31-37Crossref PubMed Scopus (46) Google Scholar, 13Mizokami A Eguchi K Kawakami A Ida H Kawabe Y Tsukada T Aoyagi T Maeda K Morimoto C Nagataki S Increased population of high fluorescence 1F7 (CD26) antigen on T cells in synovial fluid of patients with rheumatoid arthritis.J Rheumatol. 1996; 23: 2022-2026PubMed Google Scholar By contrast, in serum as well as in synovial membrane from RA patients, DPPIV activity or CD26 antigen levels were reduced compared to controls.14Hagihara M Ohhashi M Nagatsu T Activities of dipeptidyl peptidase II and dipeptidyl peptidase IV in mice with lupus erythematosus-like syndrome and in patients with lupus erythematosus and rheumatoid arthritis.Clin Chem. 1987; 33: 1463-1465PubMed Google Scholar, 15Cordero OJ Salgado FJ Mera-Varela A Nogueira M Serum interleukin-12, interleukin-15, soluble CD26, and adenosine deaminase in patients with rheumatoid arthritis.Rheumatol Int. 2001; 21: 69-74Crossref PubMed Scopus (73) Google Scholar, 16Kamori M Hagihara M Nagatsu T Iwata H Miura T Activities of dipeptidyl peptidase II, dipeptidyl peptidase IV, prolyl endopeptidase, and collagenase-like peptidase in synovial membrane from patients with rheumatoid arthritis and osteoarthritis.Biochem Med Metab Biol. 1991; 45: 154-160Crossref PubMed Scopus (51) Google Scholar These studies in RA patients, and similar ones in experimental mouse models of RA17Fujita K Hagihara M Nagatsu T Iwata H Miura T The activity of dipeptidyl peptidase II and dipeptidyl peptidase IV in mice immunized with type II collagen.Biochem Med Metab Biol. 1992; 48: 227-234Crossref PubMed Scopus (5) Google Scholar suggested that decreased levels of CD26 are associated with severity of inflammation in RA. Paradoxically, it has been reported that pharmacological inhibition of DPPIV enzyme activity could reduce the progression of arthritis in an experimental rat model of RA,18Tanaka S Murakami T Nonaka N Ohnuki T Yamada M Sugita T Anti-arthritic effects of the novel dipeptidyl peptidase IV inhibitors TMC-2A and TSL-225.Immunopharmacology. 1998; 40: 21-26Crossref PubMed Scopus (42) Google Scholar, 19Tanaka S Murakami T Horikawa H Sugiura M Kawashima K Sugita T Suppression of arthritis by the inhibitors of dipeptidyl peptidase IV.Int J Immunopharmacol. 1997; 19: 15-24Crossref PubMed Scopus (91) Google Scholar suggesting that decreases in DPPIV-activity may alleviate inflammation under some circumstances, and based on these results it was proposed that DPPIV-inhibitors may be useful as treatment of RA.20Williams YN Baba H Hayashi S Ikai H Sugita T Tanaka S Miyasaka N Kubota T Dipeptidyl peptidase IV on activated T cells as a target molecule for therapy of rheumatoid arthritis.Clin Exp Immunol. 2003; 131: 68-74Crossref PubMed Scopus (22) Google Scholar However, these inhibitors may have inhibited not just CD26, but also other enzymes that exhibit similar enzyme activity. Thus, the relative roles of CD26 and other related enzymes in RA are not clear. It is also not clear whether the levels of circulating DPPIV-enzyme activity may itself influence the extent of inflammation in RA, or whether expression of this enzyme is down-modulated in inflammatory arthritis. Here we address these questions by analyzing the progression of antigen-induced arthritis (AIA) in CD26 gene knockout mice and wild-type control animals. AIA is a commonly used murine RA model, which is T- and B-cell-dependent, providing an ideal opportunity to dissect the potential role of CD26 in regulating the immune responses and/or in modulating through its peptidase activity crucial substrate for RA. We have finally extended the study of CD26 to patients with RA, in which chronic synovitis is prominent, and with osteoarthritis (OA), in which there is limited synovial inflammation.21Poole AR An introduction to the pathophysiology of osteoarthritis.Front Biosci. 1999; 4: D662-D670Crossref PubMed Google ScholarMaterials and MethodsAnimal StudiesMiceCD26-deficient mice on a C57BL/6 genetic background have been generated before.22Marguet D Baggio L Kobayashi T Bernard AM Pierres M Nielsen PF Ribel U Watanabe T Drucker DJ Wagtmann N Enhanced insulin secretion and improved glucose tolerance in mice lacking CD26.Proc Natl Acad Sci USA. 2000; 97: 6874-6879Crossref PubMed Scopus (478) Google Scholar All experiments were performed on females between 8 to 10 weeks of age at the start of the experiment. Age-matched wild-type female C57BL/6 mice were used as controls.Induction of Experimental ArthritisAIA was established as previously described.23Busso N Peclat V Van Ness K Kolodziesczyk E Degen J Bugge T So A Exacerbation of antigen-induced arthritis in urokinase-deficient mice.J Clin Invest. 1998; 102: 41-50Crossref PubMed Scopus (125) Google Scholar Briefly, mice were immunized at day 0 and 7 with 100 μg of mBSA (Sigma Chemical Co., Buchs, Switzerland) emulsified in 0.1 ml of complete Freund's adjuvant containing 200 μg of mycobacterial strain H37RA (Difco, Basel, Switzerland) by intradermal injection at the base of the tail. On day 0, 2 × 109 heat-killed Bordetella pertussis organisms (Berna, Bern, Switzerland) were also injected intraperitoneally as an additional adjuvant. Arthritis was induced at day 21 by intra-articular injection of 100 μg of mBSA in 10 μl of sterile phosphate-buffered saline (PBS) into the right knee, the left knee being injected with sterile PBS alone. Collagen-induced arthritis (CIA) was established as previously described.24Marty I Peclat V Kirdaite G Salvi R So A Busso N Amelioration of collagen-induced arthritis by thrombin inhibition.J Clin Invest. 2001; 107: 631-640Crossref PubMed Scopus (103) Google Scholar Institutional approval was obtained for these experiments.Isotopic Quantification of Joint InflammationJoint inflammation was measured by 99mTechnecium (Tc) uptake in the knee joint, as previously described.23Busso N Peclat V Van Ness K Kolodziesczyk E Degen J Bugge T So A Exacerbation of antigen-induced arthritis in urokinase-deficient mice.J Clin Invest. 1998; 102: 41-50Crossref PubMed Scopus (125) Google Scholar Briefly, mice were first anesthetized by methoxyflurane and then injected subcutaneously in the neck region with 10 μCi 99mTc. The accumulation of the isotope in the knee was determined by external γ counting after 15 minutes. The ratio of 99mTc uptake in the inflamed arthritic knee versus 99mTc uptake in the contralateral control knee was calculated. A ratio higher than 1.1 indicated joint inflammation.Histological Grading of ArthritisAt least 12 mice per group were sacrificed, the knees dissected and fixed in 10% buffered formalin for 7 days. Fixed tissues were decalcified for 3 weeks in 15% ethylenediaminetetraacetic acid, dehydrated, and embedded in paraffin. Sagittal sections (5 μm) of the whole knee joint were stained with safranin-O and counterstained with fast green/iron hematoxylin. Histological sections were graded independently by two observers unaware of animal genotype using the following parameters. Synovial membrane thickness, which reflects the degree of synovial inflammation and hyperplasia, was scored on a scale of 0 to 6 (0 = normal thickness to 6 = maximum thickness). Synovial cell exudate was scored from 0 to 6 according to the amount of inflammatory cells in synovial fluid (0 = no cells, 6 = maximal cell amount). Cartilage proteoglycan depletion, reflected by loss of safranin-O staining intensity, was scored on a scale of 0 (fully stained cartilage) to 6 (totally unstained cartilage) in proportion to severity. For each histopathological parameter, the score (mean ± SEM) of all slides was calculated.In Vitro T-Cell Proliferation AssayMice were sacrificed according to the experimental protocol. Inguinal lymph nodes and spleen were removed, and single cell suspensions were incubated in RPMI supplemented with β-mercaptoethanol, penicillin, streptomycin, and 1% autologous serum. Lymph node cells and spleen cells (2 × 105/200 μl/well) were plated in 96-well flat-bottom plates and stimulated with 0, 10, 50, and 250 μg/ml mBSA (Sigma). The cells were incubated at 37°C in 5% CO2 for 48 hours, then 1 μCi/well of [3H]-thymidine was added in cultures for 18 hours. The cells were harvested and [3H]-thymidine uptake was measured using a β-scintillation counter.Determination of Interferon (IFN)-γ Production in VitroLymph node and spleen cells were isolated and cultured with or without the presence of 0 to 250 μg/ml of mBSA. The culture supernatants were harvested after 72 hours for determination of IFN-γ levels. Quantification of cytokine levels production was performed by an enzyme-linked immunosorbent assay (ELISA) for murine IFN-γ (Amersham Pharmacia, Dubendorf, Switzerland).In Vivo Lymph Node Cell ProliferationMice were injected intraperitoneally with 0.2 ml of 5 mg/ml of bromodeoxyuridine (BrdU, Sigma Chemical Co.) 1 hour before sacrifice. At the end of the experiment, inguinal lymph nodes were dissected. Lymph node paraffin sections were subjected to BrdU immunohistochemistry. Briefly, paraffin sections, treated with 3% H2O2 to block endogenous peroxidase and incubated with 1 N HCl for 30 minutes at 42°C, were incubated for 60 minutes with a mouse anti-BrdU biotinylated primary antibody (anti-BrdU, Zymed, Basel, Switzerland) at room temperature. The slides were washed in PBS and incubated for 30 minutes with avidin-biotin-peroxidase complex (Vector Laboratories, Seruion, Switzerland) at room temperature. After washing, diaminobenzidine was added as a chromogen, and the slides were then counterstained with hematoxylin. Proliferating cells, having incorporated BrdU, had brown-stained nuclei. Quantification of stained lymph node cells was performed by morphometry. Briefly, noncounterstained tissue sections, magnified 61 times through a microscope (Reichert Jung, Germany), were scanned using a color Kappa CF15/3 camera (Kappa, Germany) and a Semper 6P image analysis software (Synoptics, UK). The results were expressed as the ratio between the number of pixels associated to immunoreactive regions and to the total area examined.Measurement of Serum Levels of Anti-mBSA AntibodiesFor determination of anti-mBSA IgG, 96-well plates (Maxisorp-Nunc; Life Technologies AG, Basel, Switzerland) were coated overnight at 4°C with 1% bovine serum albumin (BSA) in PBS. After four washings with TTBS (50 mmol/L Tris, pH 7.4, 140 mmol/L NaCl, containing 0.05% Tween 20), 100 μl of serum, serially diluted in 1% gelatin/PBS (final dilutions 1/100, 1/200, 1/400) were incubated for 2 hours at room temperature. Wells were washed four times. Then, 100 μl/well of alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma Chemical Co.) diluted 1/500 in TTBS was added for 30 minutes. After four washings with TTBS, color was developed with 100 μl/well of p-nitrophenylphosphate (Sigma Chemical Co.) and the reaction stopped by adding 25 μl/well of 3 mol/L NaOH. Plate reading was performed at 405 nm and results calculated according to a standard curve with a reference serum.Mass Spectrometric Analysis of SDF-1 in PlasmaPlasma pooled from wild-type and CD26−/− mice were analyzed by antibody-capture surface enhanced laser desorption ionization time-of-flight–mass spectrometry (SELDI-MS). The anti-SDF-1 monoclonal antibody MAB310 (R&D Systems, Minneapolis, MN) was coupled to a PS20 chip (Ciphergen, Dumbarton Circle, CA) at a concentration of 200 ng/ml in 0.01 mol/L PBS, pH 7.4. The total volume on each spot was 4 μl. After the coupling the chip was treated with ethanolamine to block remaining activated coupling sites. The chip was subsequently incubated overnight with mouse plasma diluted 1:1 in a PBS buffer, pH 7.4 with fish gelatin and bovine IgG as protein stabilizing additives. The total volume of plasma/buffer mixture was 60 μl. After this incubation, chips were washed and covered with matrix (a-cyano-cinnamic acid saturated in 0.1% trifluoroacetic acid/50% acetonitrile). The chips were subjected to MS analysis on the Ciphergen PS-II protein chip system. Mass spectra were obtained with a laser intensity of 230, a sensitivity of 10, and an average of 90 shots. The PBS II system was calibrated with LMW calibrator kit obtained from Ciphergen. Positive controls consisted of mouse plasma spiked with recombinant SDF-1α (460-SD, R&D Systems), and analyzed immediately or after a 1-hour incubation at room temperature.Assessment of CXCR4 by ImmunohistochemistryParaffin knee joint sections were deparaffinized, saturated with 5% BSA and 20% normal rabbit serum for 1 hour at room temperature, and treated with 3% H2O2 for 10 minutes to block endogenous peroxidases. The slides were then incubated overnight with a primary goat anti-mouse CXCR4 antibody at 6.6 μg/ml (anti-CXCR4; Abcam, Cambridge, UK) at 4°C. Bound antibodies were visualized using a secondary anti-goat biotinylated antibody followed by avidin-biotin-peroxidase complex (Vector Laboratories) at room temperature. After washings, diaminobenzidine was added as a chromogen, and CXCR4 positivity graded independently by two observers unaware of animal genotype on a scale of 0 to 6 (0 = no staining at all to 6 = maximum of staining).Human StudiesPatientsPlasma were collected from patients with diagnosis of OA (n = 26, 15 females and 11 males; mean age, 63.2 ± 3.7 years) and RA (n = 41, 26 females and 15 males; mean age, 58.9 ± 2.2 years). Synovial fluids were collected from patients with OA (n = 22, 14 females and 8 males; mean age, 71.7 ± 2.48 years) and RA (n = 17, 9 females and 8 males; mean age, 56 ± 3.28). OA patients were diagnosed by clinical and radiological criteria and RA patients fulfilled at least four of the seven American Rheumatism Association revised criteria for RA.Plasma and Synovial Fluid SamplingPlasma and synovial fluid samples were collected into citrated Sarstedt Monovette tubes (Numbrecht, Germany). Anti-coagulated plasma and synovial fluid were centrifuged at 2000 × g for 15 minutes and the supernatants aliquoted and stored at −80°C until assayed.Laboratory ParametersC-Reactive protein (CRP) was measured by nephelometry.CD26 ELISAA sandwich ELISA, recognizing human CD26 (Bender Medsystems, Vienna, Austria) was used.Determination of DPPIV ActivityDPPIV activity was determined according to Scharpé and colleagues25Scharpe S De Meester I Vanhoof G Hendriks D van Sande M Van Camp K Yaron A Assay of dipeptidyl peptidase IV in serum by fluorometry of 4-methoxy-2-naphthylamine.Clin Chem. 1988; 34: 2299-2301PubMed Google Scholar with the following modifications: DPPIV activity was determined on 1-, 2.5-, and 5-μl samples (plasma, synovial fluid, or tissue extracts) fluorometrically using Gly-Pro-AMC (Novabiochem, Lucerne, Switzerland) at 5 mmol/L final concentration for 60 minutes at 37°C under agitation in an Eppendorf thermomixer in 25 μl of 100 mmol/L Tris-HCl, pH 8. The reaction was stopped by the addition of 2.5 μl of pure acetic acid. The incubation mixture was recovered in 3 ml of water. A blank value was obtained by incubating the substrate in the absence of the enzyme. A standard curve was determined by using AMC fluorescence measurement on a Perkin-Elmer LS-5 fluorometer (λ excitation, 370 nm; λ emission, 460 nm). DPPIV activities were expressed as nmol of substrate converted per minute per ml.Statistical AnalysisData are reported as mean values ± standard error of the mean (SEM). The Wilcoxon/Kruskal-Wallis (rank sum test) for unpaired variables was used to compare differences between groups with non-Gaussian distribution. The unpaired Student's t-test was used to compare the groups with normally distributed values. A level of P < 0.05 was considered as statistically significant. Correlation between parameters were analyzed by linear regression. All statistical calculations were performed using the JMP package (JMP version 4.02; SAS Institute, Cary, NC).ResultsDecreased Plasma DPPIV Activity in Murine Models of ArthritisAIA and CIA are recognized to be murine models that recapitulate some of the features of RA. We asked if in these animal models there was a decrease in DPPIV activity associated with the establishment of arthritis. Plasma from naive mice (before immunization) and of arthritic mice were collected and DPPIV activity measured. In both AIA and CIA there was a small but significant reduction in DPPIV activity in the diseased state (Figure 1, a and b, respectively). Most of this peptidase activity is accounted for by CD26 because we observed a reduction of more than 80% in plasma from CD26-deficient mice (results not shown).Effect of CD26 Deficiency on the Severity of AIATo explore whether CD26 deficiency had an effect on the course of AIA, we measured the levels of knee joint inflammation according to the ratio of 99mTc uptake in the inflamed arthritic joint over that of the nonarthritic contralateral knee joint at different time points up to day 14 after the onset of arthritis (Figure 2). The results showed that the levels of 99mTc uptake on days 1, 3, 7, and 14 were higher in CD26−/− mice than in CD26+/+ mice, but only results on day 7 reached a significant difference (day 7, 1.42 ± 0.06 versus 1.63 ± 0.07; P < 0.05). This increased joint inflammation at day 7 was associated with an increase in serum amyloid A, an acute-phase response protein, which was more elevated in serum of CD26-deficient mice (499 ± 151 μg/ml versus 350 ± 59 μg/ml in control mice), although this increase was not statistically significant.Figure 2Time course of knee joint inflammation in CD26-deficient mice with AIA. Time course of knee joint inflammation in CD26-deficient mice with AIA was measured by external γ counting of 99mTc uptake on days 1, 3, 7, and 14 after antigen-challenge into the right knee. Results are expressed as the ratio of 99mTc uptake in the right (R) arthritic knee joint over the left (L) noninflamed knee joint. For each time point, mean of ratios is represented by a horizontal bar. Joint inflammation was significantly increased at day 7 in CD26−/− mice versus the control group (P < 0.05). Control mice at days 1, 3, 7 (n = 22), and at day 14 (n = 6), and CD26-deficient mice at days 1, 3, 7 (n = 24), and at day 14 (n = 11).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then examined the histological features of arthritic knee joints from CD26−/− and CD26+/+ mice at day 10 and 20 after arthritis onset (Figure 3, only results at day 20 are shown). In both mouse strains, overt signs of synovitis were seen in the joints injected with mBSA. In contrast, no signs of inflammation were present in contralateral knees injected with PBS (data not shown). The synovial thickness was graded independently by two observers unaware of animal genotype. At day 10 of AIA there was a trend toward a higher synovial inflammation score in CD26−/− mice as compared with controls, although results did not reach statistical significance (4.37 ± 0.34 versus 3.62 ± 0.31, P = 0.07). At day 20, there was a clear difference in the thickness of synovial membrane, which was more inflamed in CD26−/− than in CD26+/+ mice [Figure 3, 3.4 ± 0.08 in CD26−/− mice (n = 21) versus 2.2 ± 0.1 in control mice (n = 18), P = 0.03]. In addition, the abundance of inflammatory synovial fluid (exudates) in the joints of CD26-deficient mice was significantly increased (2.38 ± 0.08 versus 0.94 ± 0.09, P = 0.01). The effect of AIA on articular cartilage degradation was evaluated according to the proteoglycan content, as demonstrated by safranin-O staining. In contrast to the results on synovial inflammation, cartilage damage scorings were not significantly different in CD26−/− compared to control mice. These results suggested that CD26 deficiency exacerbates inflammation in AIA.Figure 3Histological features of knee joints from control and CD26-deficient mice with AIA. a: Histologies of whole knee joint sections of control wild-type (WT) and of CD26−/− (KO) mice with AIA. Safranin-O-stained sections of arthritic knee joints were performed at day 20 after arthritis induction. Note the difference of thickness of synovial membrane, which is thicker in CD26−/− mice than in the control arthritic mice. b: Synovial membrane thickness, cell exudate, and cartilage damage were scored histologically using an arbitrary scale from 0 to 6, at day 20 after arthritis induction. Results are expressed as mean + SEM (WT mice, n = 18; CD26−/− mice, n = 21). Statistical significance was tested by the Wilcoxon rank sum test. *, P < 0.05 was considered significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of CD26 Deficiency on Humoral and Cellular Responses of AIATo investigate potential mechanisms whereby CD26 deficiency may contribute to inflammation in AIA, we first examined the humoral immune response by measuring the serum levels of total immunoglobulins and of specific anti-mBSA antibodies by ELISA in naive mice and in immunized mice at day 10 and 20 after the onset of arthritis. Anti-mBSA antibodies were undetectable in nonimmunized mice (data not shown). In arthritic mice, the circulating levels of anti-mBSA antibodies were similar in CD26−/− and CD26+/+ mice [46.8 ± 9 in CD26−/− mice (n = 21) versus 50.5 ± 10.1 in control mice (n
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