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Inhibitors of dipeptidyl peptidase IV (DP IV, CD26) induces secretion of transforming growth factor-β1 (TGF-β1) in stimulated mouse splenocytes and thymocytes
Immunology Letters 58 (1997) 29 – 35
Inhibitors of dipeptidyl peptidase IV (DP IV, CD26) induces secretion of transforming growth factor-b1 (TGF-b1) in stimulated mouse splenocytes and thymocytes Dirk Reinhold a,*, Ute Bank a, Frank Bu¨hling a, Michael Ta¨ger a, Ilona Born b, Ju¨rgen Faust b, Klaus Neubert b, Siegfried Ansorge a a
Institute of Experimental Internal Medicine, Department of Internal Medicine, Otto-6on-Guericke-Uni6ersity Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany b Institute of Biochemistry, Department of Biochemistry and Biotechnology, Martin-Luther-Uni6ersity Halle-Wittenberg, 06099 Halle, Germany Received 10 February 1997; accepted 17 February 1997
Abstract Various studies have shown that the ectoenzyme dipeptidyl peptidase IV (DP IV, CD26), expressed on T, NK and B cells in the human immune system, is involved in the regulation of DNA synthesis and cytokine production. The DP IV/CD26 was found also on mouse splenocytes and thymocytes. Here, we show that the specific DP IV inhibitors Lys[Z(NO2)]-thiazolidide, Lys[Z(NO2)]-pyrrolidide inhibit DNA synthesis as well as production of IL-2, IL-6 and IL-10 of PHA-stimulated mouse splenocytes and Con A-stimulated mouse thymocytes. Most importantly, these inhibitors induce a three to fourfold increased secretion of latent transforming growth factor b1 (TGF-b1) by mitogen-stimulated mouse immune cells, as measured with a specific TGF-b1 enzyme-linked immunosorbent assay (ELISA). These data demonstrate that CD26 plays a role also in regulation of DNA synthesis and cytokine production by murine immune cells, that the enzymatic activity is required for mediating these effects, and that TGF-b1 might have key functions in these processes. © 1997 Elsevier Science B.V. Keywords: Dipeptidyl peptidase IV; CD26; Inhibitors; Transforming growth factor-b; Mouse immune cells
1. Introduction The dipeptidyl peptidase IV (DP IV, EC 3.4.14.3) is a transmembrane type II glycoprotein that is present on most mammalian cells [1,2]. In the immune system, this enzyme was found to be expressed by T lymphocytes, but also by B lymphocytes and NK cells [1,3,4]. DP IV is a serine peptidase that catalyzes the release of N-terminal dipeptides from oligo- and polypeptides preferentially with proline and with less efficiency alanine at the penultimate position
Abbre6iations: DP IV, dipeptidyl peptidase IV; Z(NO2), 4-nitrobenzyloxycarbonyl. * Corresponding author: Tel.: + 49 391 6713313; fax: +49 391 6713291. 0165-2478/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 7 8 ( 9 7 ) 0 2 7 1 6 - 8
[5]. In the plasma membrane, DP IV occurs as a dimer with a total molecular mass of 220–240 kD. An additional 175 kD monomer has been identified recently in serum [6]. At the 4th Workshop on Leukocytes Differentiation Antigens a number of monoclonal antibodies recognizing DP IV was subsumed under the term CD26 [7–9]. DP IV/CD26 is also known to be an adenosine desaminase (ADA) binding protein [10] that binds to collagen on the extracellular matrix [11–13] and is involved in CD3/T-cell receptor (TcR)-mediated signal transduction [14–19]. The enzyme has been shown to be an activation marker of various types of immune cells. Surface expression of CD26 is upregulated after mitogenic, antiCD3 or IL-2 stimulation of T cells, S. aureus protein
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D. Reinhold et al. / Immunology Letters 58 (1997) 29–35
Fig. 1. Influence of DP IV inhibitors on the enzymatic DP IV activity of mouse splenocytes (A) and thymocytes (B). Enzymatic DP IV activity was measured on freshly isolated intact murine splenocytes and thymocytes using the DP IV-specific substrate Gly-Pro-4p-nitroanilide in absence and presence (preincubation 30 min) of different concentrations of the DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide.
stimulation of B cells and IL-2 stimulation of NK cells [3,4,19–21]. Data from several groups have shown that DP IV seems to play an intergral role in the regulation of differentiation and growth of lymphocytes [14,19,20,22–28]. Using specific inhibitors of DP IV, it was demonstrated that DP IV is involved not only in the regulation of DNA synthesis, but also in production of various cytokines by human CD26 + -immune cells [25,29–32]. Recently, several groups have cloned cDNAs encoding human, mouse and rat CD26. Sequence analysis revealed a conservation up to 98% between different species [1]. The mouse thymocyte-activating molecule (THAM) was found to be the same as mouse DP IV/CD26 [33,34]. By flow cytofluorimetric analysis, CD26 expression was found on mouse thymocytes, splenocytes and bone marrow cells, but not on macrophages [33]. In the presence of the DP IV inhibitor Pro-boro-Pro the DNA synthesis and IL-2 production of transformed murine cell lines have been found to be suppressed [35]. However, information on the role of DP IV/CD26 on DNA synthesis and release of various ‘immunostimulatory’ and ‘immunoinhibitory’ cytokines on isolated non-transformed murine immune cells are missing as yet. In the present report, we demonstrate that the inhibitors of DP IV, Lys[Z(NO2)]-thiazolidide, and Lys[Z(NO2)]-pyrrolidide, inhibit the specific enzymatic
activity of freshly isolated murine splenocytes and thymocytes as well as the DNA synthesis, and production of IL-2, IL-6, and IL-10 by mitogen-stimulated splenocytes and thymocytes in a dose-dependent manner. Most importantly, these inhibitors are capable of inducing a three-fold increase in secretion of the immunoinhibitory cytokine TGF-b1. These data suggest a crucial role of TGF-b1 in regulating the DP IV/CD26 action in lymphocytes.
2. Materials and methods
2.1. Animals The 4 to 10 week old BALB/c mice were purchased from Harlan Winkelmann GmbH (Borchen, Germany) and housed in the animal facility at the University Magdeburg under pathogen-free conditions.
2.2. Cells Spleen and thymus were harvested aseptically and a single cell preparation was made by standard methods. Total splenocyte and thymocyte suspensions were isolated by density gradient centrifugation over FicollPaque gradients (Pharmacia LKB, Uppsala, Sweden), as described by Bøyum [36]. Splenocytes and thymocytes (106 cells/ml) were suspended in serum-free CG medium (Vitromex, Vilshofen, Germany).
D. Reinhold et al. / Immunology Letters 58 (1997) 29–35
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Fig. 2. Influence of DP IV inhibitors on DNA synthesis of PHA-stimulated mouse splenocytes (A) and Con A-stimulated mouse thymocytes (B). Murine cells (105 cells/100 ml) were incubated with PHA (1 mg/ml, splenocytes) or Con A (2 mg/ml, thymocytes) and different concentrations of the synthetic DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide. After 90 h, the cultures were pulsed with 3H-methylthymidine for a further 6 h. [3H]dThd incorporation in cpm is indicated as mean9S.D. from three different experiments. The values are expressed as %[3H]dThd incorporated in relation to control cultures without inhibitor ([3H]dThd incorporation in control cultures: 24 2009 3800 cpm on splenocytes and 73009 1200 cpm on thymocytes).
2.3. Enzymatic assay Enzymatic activity of DP IV was determined according to the method published by Scho¨n [37] using 1.6 mM Gly-Pro-4p-nitroanilide and 4 × 104 cells in the reaction mixture.
2.4. Proliferation assay Mice splenic cells and thymocytes (105 cells/100 ml) were stimulated in serum-free CG medium with phytohemagglutinin (PHA, 1 mg/ml; Murex, Burgwedel, Germany) or Concanavalin A (Con A, 2 mg/ml, Sigma Chemical, St. Louis, Mo) in the presence of different concentrations of the DP IV inhibitors Lys[Z(NO2)]thiazolidide, and Lys[Z(NO2)]-pyrrolidide [24,31]. After 90 h the cultures were pulsed for an additional 6 h with 3 H-methyl-thymidine ([3H-]dThd, 0.2 mCi per well; Amersham, Braunschweig, Germany). Cells were harvested onto glass fibre filters, and the incorporated radioactivity was measured by scintillation counting [31].
2.5. Cytokine induction Cells were induced to produce cytokines (IL-2, IL-6,
IL-10, TGF-b1) by stimulation with PHA or Con A. Briefly, 106 cells/ml were incubated in serum-free CG medium with 1 mg/ml PHA (splenocytes) or 2 mg/ml Con A (thymocytes) and different concentrations of the synthetic reversible inhibitors of DP IV, Lys[Z(NO2)]thiazolidide, and Lys[Z(NO2)]-pyrrolidide. After 2, 24 and 48 h, the cell culture supernatants were harvested for cytokine determination and stored at −70°C.
2.6. ELISA for IL-2, IL-6, IL-10, and TGF-b1 The IL-2, IL-6, IL-10 levels of cell culture supernatants were determined with commercially available ‘sandwich’ enzyme-linked immunosorbent assays (ELISA) (Biosource, Camarillo, USA). Active TGF-b1 was measured with an ELISA as described by Danielpour using a mouse monoclonal anti-TGF-b1, b2, b3 antibody (Genzyme, Cambridge, MA) and a chicken anti-TGF-b1 antibody (R and D Systems, Minneapolis, MN) [38]. Samples were tested before and after transient acidification (reduction of the pH to 1.5 by addition of 5 N HCl for 30 min at 37°C and neutralization with 1.4 N NaOH in 0.7 M Hepes) [39].
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Fig. 3. Influence of DP IV inhibitors on IL-2, IL-6, and IL-10 production by PHA-stimulated mouse splenocytes. Murine splenocytes (106 cells/ml) were incubated with PHA (1 mg/ml) in presence and absence of the synthetic DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide (10-5 M). After 2, 24 and 48 h, supernatants were harvested, stored at − 70°C, and the cytokine levels were analyzed by enzyme immunoassays. Results are expressed as the mean of three different experiments. The S.D. was less than 20%.
3. Results and discussion
3.1. Enzymatic DP IV acti6ity The enzymatic activity of DP IV was studied on freshly isolated splenocytes and thymocytes incubated for a period of 30 min with different concentrations of the DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide. As shown in Fig. 1, the specific enzymatic activity of resting splenocytes was found to be 6.490.8 pkat/106 cells, of thymocytes 3.089 0.5 pkat/106 cells. The specific enzyme activity of these cells was found to be comparable with that described for human PBMC or purified T cells (3.490.1 pkat/106 cells and 4.59 0.4 pkat/106 cells, respectively [20]). The DP IV inhibitors were capable of suppressing significantly the enzymatic activity of these cell populations.
3.2. DP IV inhibitors suppressed DNA synthesis We have demonstrated elsewhere that the DNA synthesis of human PWM-stimulated PBMC was strongly inhibited by the synthetic competitive DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-piperidide [31]. A similar suppressive effect on proliferation of PWM-stimulated purified T cells was detectable after incubation with these two DP IV inhibitors as well as with Lys[Z(NO2)]-pyrrolidide. Neither thiazolidine, piperidine, nor pyrrolidine (all three are basic substances of the DP IV inhibitors) had an influence on DNA synthesis in these cell systems (unpublished data).
The effect of DP IV inhibitors on DNA synthesis of PHA-stimulated murine splenocytes and Con A-stimulated thymocytes was determined by incubation of viable cells for 96 h in the presence and absence of various concentrations of Lys[Z(NO2)]-thiazolidide, and Lys[Z(NO2)]-pyrrolidide. As shown in Fig. 2, the DNA synthesis of both murine cell systems was strongly suppressed. We found 65% inhibition on PHAstimulated splenocytes and 80% on Con A-stimulated thymocytes at an inhibitor concentration of 10 − 5 M.
3.3. DP IV inhibitors suppressed IL-2, IL-6, and IL-10 production To answer the question whether the suppressive effect of DP IV inhibitors on DNA synthesis of mitogenstimulated splenocytes and thymocytes correlates with a decrease in production and secretion of different cytokines, we measured the concentrations of IL-2, IL-6, and IL-10 in supernatants of PHA-stimulated splenocytes and Con A-stimulated thymocytes in the presence or absence of DP IV inhibitors (10 − 5 M). The concentrations of cytokines released after 2, 24 and 48 h were determined by enzyme immunoassays. As shown in Figs. 3 and 4, the production of all three cytokines in thymocytes was significantly lower in comparison to splenocytes. However, in both cell systems the DP IV inhibitors strongly suppressed the production of IL-2, IL-6 and IL-10 in a concentration-dependent manner. Recently, Flentke et al. [35] reported that the DP IV inhibitor Pro-boro-Pro inhibits antigen-induced prolif-
D. Reinhold et al. / Immunology Letters 58 (1997) 29–35
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Fig. 4. Influence of DP IV inhibitors on IL-2, IL-6, and IL-10 production by Con A-stimulated mouse thymocytes. Murine thymocytes (106 cells/ml) were incubated with Con A (2 mg/ml) in presence and absence of the synthetic DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide (10 − 5 M). After 2, 24 and 48 h supernatants were harvested, stored at − 70°C, and the cytokine levels were analyzed by enzyme immunoassays. Results are expressed as the mean of three different experiments. The S.D. was less than 20%.
eration and IL-2 production in murine T-cell lines. Here, we could show that our DP IV inhibitors suppress the DNA synthesis and the production of IL-2, IL-6 and IL-10 in mitogen-stimulated murine splenocytes and thymocytes, indicating that this effect is a more common phenomenon. Taken together, these data convincingly show that DP IV/CD26 plays a cruical role in regulation of DNA synthesis and cytokine production also in murine immune cells and its role is a more general one in the immune systems of different species.
3.4. DP IV inhibitors stimulated secretion of latent TGF-b1 Various reports have suggested that the multifunctional cytokine transforming growth factor-b1 (TGFb1) is involved in the regulation of the cytokine network and the cytokine-mediated communication between immune cells [40 – 44]. TGF-b1 is known to have similar effects on the proliferation and cytokine production of immune cells, as has been described for the DP IV inhibitors we applied [43–45]. To determine whether the ‘immunoinhibitory cytokine’ TGF-b1 is involved in the DP IV inhibitor-induced suppression of DNA synthesis and cytokine production, TGF-b1 production of PHAstimulated splenocytes and Con A-stimulated thymo-
cytes were studied in presence or absence of the DP IV inhibitors (Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]pyrrolidide, 10 − 5 M). TGF-b1 concentrations were determined using an enzyme immunoassay. Samples were tested before (active TGF-b1) and after transient acidification (latent TGF-b1) of the supernatants. 1209 30 pg/ml (splenocytes) and 200940 pg/ml (thymocytes) were found in acid treated cell culture supernatants of PHA-stimulated splenocytes and Con A-stimulated thymocytes obtained after 24 h of incubation (Fig. 5). Measurement of untreated culture supernatants revealed that only a small amount (10–20%) of the TGF-b1 was present in the active 25 kDa form (data not shown). Interestingly, the DP IV inhibitors were capable of inducing a strong increase in secretion of latent TGF-b1 on both PHA-stimulated splenocytes and Con A-stimulated thymocytes after a period of 24 h (Fig. 5). In respect of these findings it is conceivable that the suppressive effect of DP IV inhibitors on DNA synthesis as well as cytokine production (IL-2, IL-6 and IL-10) by mitogen-stimulated mouse splenocytes and thymocytes may result from the induction of the inhibitory cytokine TGF-b1. It remains for additional experiments to determine whether this is the only mechanism responsible for the effects mediated by DP IV inhibitors.
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D. Reinhold et al. / Immunology Letters 58 (1997) 29–35
Fig. 5. Influence of DP IV inhibitors on production of latent TGF-b1 in PHA-stimulated splenocytes and Con A-stimulated thymocytes. 106 cells/ml were incubated with PHA (splenocytes, 1 mg/ml) or Con A (thymocytes, 2 mg/ml) and the synthetic DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide (10 − 5 M). After 24 h supernatants were harvested, stored at − 70°C and the TGF-b1 concentrations were measured with an enzyme immunoassay. Samples were tested after transient acidification (latent TGF-b1). Results are expressed as mean9S.D. of three independent experiments.
In summary, our findings show that CD26 plays a role in regulation of DNA synthesis and cytokine production also in mouse immune cells and that the enzymatic activity is required for mediating these effects. We conclude that at least some of the immunosuppressive effects of DP IV inhibitors are mediated by TGFb1 which is secreted by immune cells after inhibition of the enzymatic activity of DP IV.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft, SFB 387, grant No. A4 and A5. The authors thank Ms Silke Mo¨hring for the skillful technical assistance.
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D. Reinhold et al. / Immunology Letters 58 (1997) 29–35 [28] Torimoto, Y., Dang, N.H., Tanaka, T., Prado, C., Schlossman, S.F., Morimoto, C. (1992) Mol. Immunol. 29, 183. [29] Scho¨n, E., Eichmann, E., Jahn, S., Kopp, J., Volk, H.D., Ansorge, S. (1988) Biol. Ztbl. 107, 141. [30] Plana, M., Vinas, O., DeLa Calle-Martin, O., Lozano, F., InglesEsteve, J., Romero, M., Alberola-Ila, J., Vilella, R., Vives, J. (1991) Eur. J. Immunol. 21, 1085. [31] Reinhold, D., Bank, U., Bu¨hling, F., Neubert, K., Mattern, T., Ulmer, A.J., Flad, H.-D., Ansorge, S. (1993) Immunobiol. 188, 403. [32] Reinhold, D., Bank, U., Bu¨hling, F., Ka¨hne, T., Kunz, D., Faust, J., Neubert, K., Ansorge, S. (1994) Immunobiology 192, 121. [33] Vivier, I., Marguet, D., Naquet, P., Bonicel, J., Black, D., Xiao-Yan Li, C., Bernard, A.-M., Gorvel, J.-P., Pierres, M. (1991) J. Immunol. 147, 447. [34] Marguet, D., Bernard, A.-M., Vivier, I., Darmoul, D., Naquet, P., Pierres, M. (1992) J. Biol. Chem. 267, 2200. [35] Flentke, G.R., Munoz, E., Huber, B.T., Plaut, A.G., Kettner, C.A., Bachovchin, W.W. (1991) Proc. Natl. Acad. Sci. USA 88, 1556.
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Inhibitors of dipeptidyl peptidase IV (DP IV, CD26) induces secretion of transforming growth factor-b1 (TGF-b1) in stimulated mouse splenocytes and thymocytes Dirk Reinhold a,*, Ute Bank a, Frank Bu¨hling a, Michael Ta¨ger a, Ilona Born b, Ju¨rgen Faust b, Klaus Neubert b, Siegfried Ansorge a a
Institute of Experimental Internal Medicine, Department of Internal Medicine, Otto-6on-Guericke-Uni6ersity Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany b Institute of Biochemistry, Department of Biochemistry and Biotechnology, Martin-Luther-Uni6ersity Halle-Wittenberg, 06099 Halle, Germany Received 10 February 1997; accepted 17 February 1997
Abstract Various studies have shown that the ectoenzyme dipeptidyl peptidase IV (DP IV, CD26), expressed on T, NK and B cells in the human immune system, is involved in the regulation of DNA synthesis and cytokine production. The DP IV/CD26 was found also on mouse splenocytes and thymocytes. Here, we show that the specific DP IV inhibitors Lys[Z(NO2)]-thiazolidide, Lys[Z(NO2)]-pyrrolidide inhibit DNA synthesis as well as production of IL-2, IL-6 and IL-10 of PHA-stimulated mouse splenocytes and Con A-stimulated mouse thymocytes. Most importantly, these inhibitors induce a three to fourfold increased secretion of latent transforming growth factor b1 (TGF-b1) by mitogen-stimulated mouse immune cells, as measured with a specific TGF-b1 enzyme-linked immunosorbent assay (ELISA). These data demonstrate that CD26 plays a role also in regulation of DNA synthesis and cytokine production by murine immune cells, that the enzymatic activity is required for mediating these effects, and that TGF-b1 might have key functions in these processes. © 1997 Elsevier Science B.V. Keywords: Dipeptidyl peptidase IV; CD26; Inhibitors; Transforming growth factor-b; Mouse immune cells
1. Introduction The dipeptidyl peptidase IV (DP IV, EC 3.4.14.3) is a transmembrane type II glycoprotein that is present on most mammalian cells [1,2]. In the immune system, this enzyme was found to be expressed by T lymphocytes, but also by B lymphocytes and NK cells [1,3,4]. DP IV is a serine peptidase that catalyzes the release of N-terminal dipeptides from oligo- and polypeptides preferentially with proline and with less efficiency alanine at the penultimate position
Abbre6iations: DP IV, dipeptidyl peptidase IV; Z(NO2), 4-nitrobenzyloxycarbonyl. * Corresponding author: Tel.: + 49 391 6713313; fax: +49 391 6713291. 0165-2478/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 7 8 ( 9 7 ) 0 2 7 1 6 - 8
[5]. In the plasma membrane, DP IV occurs as a dimer with a total molecular mass of 220–240 kD. An additional 175 kD monomer has been identified recently in serum [6]. At the 4th Workshop on Leukocytes Differentiation Antigens a number of monoclonal antibodies recognizing DP IV was subsumed under the term CD26 [7–9]. DP IV/CD26 is also known to be an adenosine desaminase (ADA) binding protein [10] that binds to collagen on the extracellular matrix [11–13] and is involved in CD3/T-cell receptor (TcR)-mediated signal transduction [14–19]. The enzyme has been shown to be an activation marker of various types of immune cells. Surface expression of CD26 is upregulated after mitogenic, antiCD3 or IL-2 stimulation of T cells, S. aureus protein
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D. Reinhold et al. / Immunology Letters 58 (1997) 29–35
Fig. 1. Influence of DP IV inhibitors on the enzymatic DP IV activity of mouse splenocytes (A) and thymocytes (B). Enzymatic DP IV activity was measured on freshly isolated intact murine splenocytes and thymocytes using the DP IV-specific substrate Gly-Pro-4p-nitroanilide in absence and presence (preincubation 30 min) of different concentrations of the DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide.
stimulation of B cells and IL-2 stimulation of NK cells [3,4,19–21]. Data from several groups have shown that DP IV seems to play an intergral role in the regulation of differentiation and growth of lymphocytes [14,19,20,22–28]. Using specific inhibitors of DP IV, it was demonstrated that DP IV is involved not only in the regulation of DNA synthesis, but also in production of various cytokines by human CD26 + -immune cells [25,29–32]. Recently, several groups have cloned cDNAs encoding human, mouse and rat CD26. Sequence analysis revealed a conservation up to 98% between different species [1]. The mouse thymocyte-activating molecule (THAM) was found to be the same as mouse DP IV/CD26 [33,34]. By flow cytofluorimetric analysis, CD26 expression was found on mouse thymocytes, splenocytes and bone marrow cells, but not on macrophages [33]. In the presence of the DP IV inhibitor Pro-boro-Pro the DNA synthesis and IL-2 production of transformed murine cell lines have been found to be suppressed [35]. However, information on the role of DP IV/CD26 on DNA synthesis and release of various ‘immunostimulatory’ and ‘immunoinhibitory’ cytokines on isolated non-transformed murine immune cells are missing as yet. In the present report, we demonstrate that the inhibitors of DP IV, Lys[Z(NO2)]-thiazolidide, and Lys[Z(NO2)]-pyrrolidide, inhibit the specific enzymatic
activity of freshly isolated murine splenocytes and thymocytes as well as the DNA synthesis, and production of IL-2, IL-6, and IL-10 by mitogen-stimulated splenocytes and thymocytes in a dose-dependent manner. Most importantly, these inhibitors are capable of inducing a three-fold increase in secretion of the immunoinhibitory cytokine TGF-b1. These data suggest a crucial role of TGF-b1 in regulating the DP IV/CD26 action in lymphocytes.
2. Materials and methods
2.1. Animals The 4 to 10 week old BALB/c mice were purchased from Harlan Winkelmann GmbH (Borchen, Germany) and housed in the animal facility at the University Magdeburg under pathogen-free conditions.
2.2. Cells Spleen and thymus were harvested aseptically and a single cell preparation was made by standard methods. Total splenocyte and thymocyte suspensions were isolated by density gradient centrifugation over FicollPaque gradients (Pharmacia LKB, Uppsala, Sweden), as described by Bøyum [36]. Splenocytes and thymocytes (106 cells/ml) were suspended in serum-free CG medium (Vitromex, Vilshofen, Germany).
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Fig. 2. Influence of DP IV inhibitors on DNA synthesis of PHA-stimulated mouse splenocytes (A) and Con A-stimulated mouse thymocytes (B). Murine cells (105 cells/100 ml) were incubated with PHA (1 mg/ml, splenocytes) or Con A (2 mg/ml, thymocytes) and different concentrations of the synthetic DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide. After 90 h, the cultures were pulsed with 3H-methylthymidine for a further 6 h. [3H]dThd incorporation in cpm is indicated as mean9S.D. from three different experiments. The values are expressed as %[3H]dThd incorporated in relation to control cultures without inhibitor ([3H]dThd incorporation in control cultures: 24 2009 3800 cpm on splenocytes and 73009 1200 cpm on thymocytes).
2.3. Enzymatic assay Enzymatic activity of DP IV was determined according to the method published by Scho¨n [37] using 1.6 mM Gly-Pro-4p-nitroanilide and 4 × 104 cells in the reaction mixture.
2.4. Proliferation assay Mice splenic cells and thymocytes (105 cells/100 ml) were stimulated in serum-free CG medium with phytohemagglutinin (PHA, 1 mg/ml; Murex, Burgwedel, Germany) or Concanavalin A (Con A, 2 mg/ml, Sigma Chemical, St. Louis, Mo) in the presence of different concentrations of the DP IV inhibitors Lys[Z(NO2)]thiazolidide, and Lys[Z(NO2)]-pyrrolidide [24,31]. After 90 h the cultures were pulsed for an additional 6 h with 3 H-methyl-thymidine ([3H-]dThd, 0.2 mCi per well; Amersham, Braunschweig, Germany). Cells were harvested onto glass fibre filters, and the incorporated radioactivity was measured by scintillation counting [31].
2.5. Cytokine induction Cells were induced to produce cytokines (IL-2, IL-6,
IL-10, TGF-b1) by stimulation with PHA or Con A. Briefly, 106 cells/ml were incubated in serum-free CG medium with 1 mg/ml PHA (splenocytes) or 2 mg/ml Con A (thymocytes) and different concentrations of the synthetic reversible inhibitors of DP IV, Lys[Z(NO2)]thiazolidide, and Lys[Z(NO2)]-pyrrolidide. After 2, 24 and 48 h, the cell culture supernatants were harvested for cytokine determination and stored at −70°C.
2.6. ELISA for IL-2, IL-6, IL-10, and TGF-b1 The IL-2, IL-6, IL-10 levels of cell culture supernatants were determined with commercially available ‘sandwich’ enzyme-linked immunosorbent assays (ELISA) (Biosource, Camarillo, USA). Active TGF-b1 was measured with an ELISA as described by Danielpour using a mouse monoclonal anti-TGF-b1, b2, b3 antibody (Genzyme, Cambridge, MA) and a chicken anti-TGF-b1 antibody (R and D Systems, Minneapolis, MN) [38]. Samples were tested before and after transient acidification (reduction of the pH to 1.5 by addition of 5 N HCl for 30 min at 37°C and neutralization with 1.4 N NaOH in 0.7 M Hepes) [39].
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Fig. 3. Influence of DP IV inhibitors on IL-2, IL-6, and IL-10 production by PHA-stimulated mouse splenocytes. Murine splenocytes (106 cells/ml) were incubated with PHA (1 mg/ml) in presence and absence of the synthetic DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide (10-5 M). After 2, 24 and 48 h, supernatants were harvested, stored at − 70°C, and the cytokine levels were analyzed by enzyme immunoassays. Results are expressed as the mean of three different experiments. The S.D. was less than 20%.
3. Results and discussion
3.1. Enzymatic DP IV acti6ity The enzymatic activity of DP IV was studied on freshly isolated splenocytes and thymocytes incubated for a period of 30 min with different concentrations of the DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide. As shown in Fig. 1, the specific enzymatic activity of resting splenocytes was found to be 6.490.8 pkat/106 cells, of thymocytes 3.089 0.5 pkat/106 cells. The specific enzyme activity of these cells was found to be comparable with that described for human PBMC or purified T cells (3.490.1 pkat/106 cells and 4.59 0.4 pkat/106 cells, respectively [20]). The DP IV inhibitors were capable of suppressing significantly the enzymatic activity of these cell populations.
3.2. DP IV inhibitors suppressed DNA synthesis We have demonstrated elsewhere that the DNA synthesis of human PWM-stimulated PBMC was strongly inhibited by the synthetic competitive DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-piperidide [31]. A similar suppressive effect on proliferation of PWM-stimulated purified T cells was detectable after incubation with these two DP IV inhibitors as well as with Lys[Z(NO2)]-pyrrolidide. Neither thiazolidine, piperidine, nor pyrrolidine (all three are basic substances of the DP IV inhibitors) had an influence on DNA synthesis in these cell systems (unpublished data).
The effect of DP IV inhibitors on DNA synthesis of PHA-stimulated murine splenocytes and Con A-stimulated thymocytes was determined by incubation of viable cells for 96 h in the presence and absence of various concentrations of Lys[Z(NO2)]-thiazolidide, and Lys[Z(NO2)]-pyrrolidide. As shown in Fig. 2, the DNA synthesis of both murine cell systems was strongly suppressed. We found 65% inhibition on PHAstimulated splenocytes and 80% on Con A-stimulated thymocytes at an inhibitor concentration of 10 − 5 M.
3.3. DP IV inhibitors suppressed IL-2, IL-6, and IL-10 production To answer the question whether the suppressive effect of DP IV inhibitors on DNA synthesis of mitogenstimulated splenocytes and thymocytes correlates with a decrease in production and secretion of different cytokines, we measured the concentrations of IL-2, IL-6, and IL-10 in supernatants of PHA-stimulated splenocytes and Con A-stimulated thymocytes in the presence or absence of DP IV inhibitors (10 − 5 M). The concentrations of cytokines released after 2, 24 and 48 h were determined by enzyme immunoassays. As shown in Figs. 3 and 4, the production of all three cytokines in thymocytes was significantly lower in comparison to splenocytes. However, in both cell systems the DP IV inhibitors strongly suppressed the production of IL-2, IL-6 and IL-10 in a concentration-dependent manner. Recently, Flentke et al. [35] reported that the DP IV inhibitor Pro-boro-Pro inhibits antigen-induced prolif-
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Fig. 4. Influence of DP IV inhibitors on IL-2, IL-6, and IL-10 production by Con A-stimulated mouse thymocytes. Murine thymocytes (106 cells/ml) were incubated with Con A (2 mg/ml) in presence and absence of the synthetic DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide (10 − 5 M). After 2, 24 and 48 h supernatants were harvested, stored at − 70°C, and the cytokine levels were analyzed by enzyme immunoassays. Results are expressed as the mean of three different experiments. The S.D. was less than 20%.
eration and IL-2 production in murine T-cell lines. Here, we could show that our DP IV inhibitors suppress the DNA synthesis and the production of IL-2, IL-6 and IL-10 in mitogen-stimulated murine splenocytes and thymocytes, indicating that this effect is a more common phenomenon. Taken together, these data convincingly show that DP IV/CD26 plays a cruical role in regulation of DNA synthesis and cytokine production also in murine immune cells and its role is a more general one in the immune systems of different species.
3.4. DP IV inhibitors stimulated secretion of latent TGF-b1 Various reports have suggested that the multifunctional cytokine transforming growth factor-b1 (TGFb1) is involved in the regulation of the cytokine network and the cytokine-mediated communication between immune cells [40 – 44]. TGF-b1 is known to have similar effects on the proliferation and cytokine production of immune cells, as has been described for the DP IV inhibitors we applied [43–45]. To determine whether the ‘immunoinhibitory cytokine’ TGF-b1 is involved in the DP IV inhibitor-induced suppression of DNA synthesis and cytokine production, TGF-b1 production of PHAstimulated splenocytes and Con A-stimulated thymo-
cytes were studied in presence or absence of the DP IV inhibitors (Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]pyrrolidide, 10 − 5 M). TGF-b1 concentrations were determined using an enzyme immunoassay. Samples were tested before (active TGF-b1) and after transient acidification (latent TGF-b1) of the supernatants. 1209 30 pg/ml (splenocytes) and 200940 pg/ml (thymocytes) were found in acid treated cell culture supernatants of PHA-stimulated splenocytes and Con A-stimulated thymocytes obtained after 24 h of incubation (Fig. 5). Measurement of untreated culture supernatants revealed that only a small amount (10–20%) of the TGF-b1 was present in the active 25 kDa form (data not shown). Interestingly, the DP IV inhibitors were capable of inducing a strong increase in secretion of latent TGF-b1 on both PHA-stimulated splenocytes and Con A-stimulated thymocytes after a period of 24 h (Fig. 5). In respect of these findings it is conceivable that the suppressive effect of DP IV inhibitors on DNA synthesis as well as cytokine production (IL-2, IL-6 and IL-10) by mitogen-stimulated mouse splenocytes and thymocytes may result from the induction of the inhibitory cytokine TGF-b1. It remains for additional experiments to determine whether this is the only mechanism responsible for the effects mediated by DP IV inhibitors.
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Fig. 5. Influence of DP IV inhibitors on production of latent TGF-b1 in PHA-stimulated splenocytes and Con A-stimulated thymocytes. 106 cells/ml were incubated with PHA (splenocytes, 1 mg/ml) or Con A (thymocytes, 2 mg/ml) and the synthetic DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide (10 − 5 M). After 24 h supernatants were harvested, stored at − 70°C and the TGF-b1 concentrations were measured with an enzyme immunoassay. Samples were tested after transient acidification (latent TGF-b1). Results are expressed as mean9S.D. of three independent experiments.
In summary, our findings show that CD26 plays a role in regulation of DNA synthesis and cytokine production also in mouse immune cells and that the enzymatic activity is required for mediating these effects. We conclude that at least some of the immunosuppressive effects of DP IV inhibitors are mediated by TGFb1 which is secreted by immune cells after inhibition of the enzymatic activity of DP IV.
Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft, SFB 387, grant No. A4 and A5. The authors thank Ms Silke Mo¨hring for the skillful technical assistance.
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