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Galactoside-specific plant lectin, Viscum album agglutinin-I induces enhanced proliferation and apoptosis of murine thymocytes in vivo
Immunology Letters 86 (2003) 23 /27 www.elsevier.com/locate/
Galactoside-specific plant lectin, Viscum album agglutinin-I induces enhanced proliferation and apoptosis of murine thymocytes in vivo Tibor Hajto´, Timea Berki *, Ferenc Boldizsa´r, Pe´ter Ne´meth Department of Immunology and Biotechnology, Faculty of Medicine, University of Pe´cs, Szigeti u´t 12, H-7643 Pe´cs, Hungary Received 30 July 2002; received in revised form 24 October 2002; accepted 8 November 2002
Abstract Galactoside-specific plant lectin, Viscum album agglutinin-I (VAA-I) has been shown to act as a biomodulator with proinflammatory and apoptosis-inducing effects, however its cellular targets and mechanism of immunobiological action in vivo are less well understood. Therefore, in the present work the short- and long-term in vivo effects of VAA-I on thymocyte subpopulations and peripheral T cells were tested using a murine (Balb/c) model. Cell surface CD4/CD8 staining and flow cytometry allowed us to follow the changes of thymocyte subpopulations: CD4 /CD8 / double negative (DN), CD4/CD8/ double positive (DP), CD4/ or CD8/ single positive (SP) and mature peripheral T cells after single or repeated injections with low doses of VAAI. The apoptosis of the cells was detected by flow cytometry using propidium iodide (PI) and Annexin V staining. To detect the short-term effects of the lectin, the animals were investigated 24 h after a single injection of 1 or 30 ng/kg body weight (BW) VAAI9/1 mg/kg Dexamethasone (DX). The total number of mature CD8/ SP thymocytes increased significantly with an enhancement of the ratio of apoptotic cells. In contrast, in the blood samples an elevated CD4/CD8 ratio was found. In the next trial, Balb/c mice were treated twice weekly with 1 or 30 ng/kg VAA-I9/1 mg/kg DX for 3 weeks. The total cell count of thymocytes showed significant increases after both doses of VAA-I, but an elevated percentage of apoptotic cells was found only after treatment with 30 ng/kg VAA-I. SP thymocytes revealed higher increases in lectin-induced apoptosis than DN or DP cells. In addition, both lectin doses significantly inhibited the DX-induced reduction of all thymocyte subpopulations investigated. In conclusion, our data suggest that VAA-I is able to modulate the maturation of thymocytes in vivo. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Viscum albumin agglutinin; Thymus; Apoptosis
1. Introduction Lectins are carbohydrate-binding proteins that are able to recognize and bind the glycan part of glycoconjugates (e.g. glycoproteins, glycolipids, oligo- and polysaccharides) in a sugar-specific manner [1]. In mistletoe plants, the galactoside-specific Viscum album agglutinin-I (VAA-I) is the most frequently investigated and important lectin [2,3]. It consists of two polypeptide subunits. Its A chain, with a molecular weight of 29 kDa because of its highly specific enzymatic efficacy, catalytically inhibits the protein synthesis in the 28S subunit of
* Corresponding author. Tel.: /36-72-536-291; fax: /36-72-536289. E-mail address: [email protected] (T. Berki).
rRNA [4 /7]. That is why VAA-I in common with other plant lectins, such as ricin, abrin and modeccin, belongs to the type II family of ribosome inactivating proteins (RIP) sharing numerous homologous structures [3,8]. The sugar-binding B chain of VAA-I with a molecular weight of 34 kDa is able to bind galactoside residues on the cell membrane preferring certain confirmations [9 / 11]. In low (not cytotoxic) doses, the B chain is responsible for the enhancing effect of VAA-I on the proinflammatory activity of natural immune system. After 24 h incubation with human PBMC, VAA-I (A and B chains in nanogram per milliliter concentrations) induced mRNA expression and enhanced secretion of proinflammatory cytokines [10,12]. Both in vitro and in vivo VAA-I stimulated the number and activity of natural killer (NK) cells [13,14] and induced elevated activation markers on the surface of monocytes and
0165-2478/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 2 ) 0 0 2 6 5 - 1
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macrophages [14,15]. Since proinflammatory effects have been reported to play an important role in the regulation of the thymus, in this study the in vivo effects of VAA-I on murine thymocyte development were first investigated. In vivo doses of VAA-I induced enhanced proliferation with a parallel increase in apoptosis of cells in the thymus. In addition, VAA-I was able to inhibit the Dexamethasone (DX)-induced reduction of thymocyte cell number.
2. Materials and methods 2.1. Materials VAA-I was prepared from fresh aqueous extracts of the leaves and stems of mistletoe plants and purified on lactosylated agarose columns as described previously [13]. Endotoxin contamination in the samples used was B/0.5 pg/ml as determined by quantitative kinetic LAL assay. Dexamethasone (Oradexon, Organon) was purchased from N.V. Organon Oss Holland. Apoptosis was detected with propidium iodide (Sigma P 4170) and Annexin V-FITC (Pharmingen Cat No. 556420). For triple labeling experiments, the following mAbs were used: phycoerythrin (PE)-conjugated rat anti-mouse CD4 (L3T4, Pharmingen Cat No. 09005A) and CyChrome-conjugated rat anti-mouse CD8 (Ly-2 Pharmingen Cat. No. 553034) monoclonal antibodies. 2.2. Treatments of animals Eight male and eight female Balb/c mice used in the first study and the nine male and nine female in the second trial (6 weeks old, body weight :/20 g 9/10%) were obtained from Charles Rivers Laboratories. The animals were kept under standardized laboratory conditions. The mice were randomly assigned to six groups of three animals each in both studies. Three groups of three animals were treated subcutaneously with placebo (phosphate-buffered saline) or with VAA-I and/or with DX with the following doses: 1 or 30 ng/kg VAA-I alone, 1 or 30 ng/kg VAA-I combined with 1 mg/kg DX. As positive control, 1 mg/kg DX was given alone. In the first study, all animals were sacrificed 24 h after one subcutaneous injection. In the second trial, the subcutaneous injections were administered twice a week for 3 weeks. After the last injection (72 h), all animals were sacrificed by rapid decapitation. 2.3. Thymocyte preparation and staining Thymus glands were removed and placed in ice-cold PBS. Thymus tissue was homogenized in a glass/glass homogenizer and the suspension filtered through a nylon mesh filter. The thymocytes were washed in PBS
and the total cell number and viability were determined by counting in a hemocytometer using the Trypan blue dye exclusion test. Thymocytes (1 /106) in 100 ml binding buffer (PBS/0.1% NaN3/0.1% BSA) were labeled for the expression of CD4 and CD8 for 30 min on ice, after two washing steps in PBS, the cells were stored in PBS/0.1% PFA until flow cytometric analysis. For the detection of early apoptotic thymocyte subpopulations, after CD4/CD8 staining cells were labeled with Annexin V-FITC for 15 min in Annexin binding buffer. The samples were stored in 400 ml Annexin buffer until flow cytometric analysis. The late apoptotic cells were stained after fixation with 4% PFA in saponin buffer (0.1% Saponine/0.1% BSA/0.1% NaN3 in PBS) with 50 mg/ml of propidium iodide (PI Sigma P 4170) for 30 min at room temperature. 2.4. Flow cytometric acquisition and analysis The samples were analyzed in a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA), using the CellQuest software. Thymocytes were gated on forward and side scatter plots according to their size and granularity. The gate determined by the untreated thymocyte sample was used for all further measurements. Thymocytes were gated according to their CD4 and/or CD8 fluorescence. To determine the apoptotic cell number with the detection of the DNA content of the cells, PI incorporation into DNA was tested in FL-2 channel of the flow cytometer using a linear amplification. In addition, the early apoptotic cells after CD4/ CD8 staining were measured with Annexin V-FITC binding to membrane phosphatidylserine molecules of various thymocyte subpopulations. 2.5. Statistical analysis The results were analyzed with Student’s t-test and U test according to Wilcoxon, Mann /Whitney using the Statgraphics statistical package for IBM-compatible computers.
3. Results 3.1. Effect of VAA-I on thymocyte proliferation In the first trial, double negative (DN), double positive (DP), CD4/ and CD8/ single positive (SP) cells in the thymus as well as CD4/ and CD8/ cells in peripheral blood of Balb/c mice were investigated 24 h after a single lectin (VAA-I) injection. As shown in the first three columns of Fig. 1(A), a single injection of different doses of VAA-I did not cause significant alterations in the total thymocyte cell count or in the DN, DP and CD4/ cell number (Table 1A). Only the
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Fig. 1. Effect of VAA-I on thymocyte proliferation. Total numbers of thymocytes in Balb/c mice after treatment with 1 and 30 ng/kg lectin (VAA-I) and/or Dexamethasone (DX). The experiments were carried out 24 h after a single subcutaneous injection (A) or 72 h after the last injection of treatment twice weekly for 3 weeks (B). Each value represents the average of three animals (9/S.E.M.). The results of various subpopulations and statistical analysis are shown in Table 1.
Fig. 2. Effect of VAA-I on ratio between CD4/ and CD8/ cells. The mean ratios (9/S.E.M.) between CD4/ and CD8/ cells are shown in peripheral blood and in thymus 24 h after a single injection (A, C) or 72 h after the last injection of a 3 weeks treatment (B, D). Some 24 h after a single injection of both lectin doses, this ratio significantly decreased (C) in thymus (P B/0.01; P B/0.01) and increased (A) in blood (P B/0.01; P B/0.05). After 3 weeks (B, D), no significant differences between treated and control groups were found.
CD8/ thymocyte number revealed a significant (P B/ 0.0 25) elevation (70%) 24 h after 1 ng/kg VAA-I and after 30 ng/kg dose, this elevation was 44% (P /0.05). Therefore, the CD4//CD8/ ratio in thymus 24 h after the two different lectin doses fell 39 and 38% (P B/0.01; P B/0.01) (Fig. 2(C) and Table 1(A)). However, at the same time in peripheral blood, the CD4//CD8/ ratio elevated 74% after 1 ng/kg VAA-I and 55% following 30 ng/kg dose (P B/0.005; P B/0.01) as illustrated in Fig. 2(A).
In the second long-term trial, Balb/c mice were treated with the same doses of VAA-I lectin/DX twice a week for 3 weeks. After the last injections (72 h), the same investigations as after 24 h were carried out. As the first three columns of Fig. 1(B) show, the total thymocyte cell count in the thymus increased significantly after both lectin doses (P B/0.05 and P B/0.025, respectively). As demonstrated in Table 1(B), with the exception of CD4/ cells, all investigated thymocyte subpopulations (DN, DP and CD8/ cells) revealed significant elevation after long-term treatment with 30 ng/kg VAA-I (P B/
Table 1 Thymus
Ctrl
VAA 1 ng
VAA 30 ng
VAA 1 ng /DX
VAA 30 ng /DX
DX
A Effect of single dose VAA-I and DX treatment on thymocyte subpopulations /10 (S.E.M.) DN 2.4 (0.4) 3.0 (0.5) 4.7 (0.1) 1.6 (0.4) DP 117.8 (7.5) 97.1 (1.3) 104.5 (13) 64.4 (14) CD4/ 12.5 (1.4) 12.7 (1.7) 10.5 (1.2) 9.0 (1.7) 4.9 (1.3) 8.4 (1.2)* 7.1 (1.7) 7.0 (2.8) CD8/
1.8 55.7 8.2 6.9
1.2 48.1 5.4 2.3
(0.1)* (5.4)* (0.2)* (0.03)*
B Effect of long-term VAA-I and DX treatments on thymocyte subpopulations /106 (S.E.M.) DN 3.2 (0.7) 3.5 (0.2) 4.7 (0.24)* 4.8 (1.6)# DP 48.8 (14.7) 69.7 (15.9) 101 (18.0)* 54.4 (16)# CD4/ 7.2 (1.4) 9.4 (0.9) 10.6 (2.1)* 7.6 (2.0)# 2.3 (0.3) 3.6 (0.08)* 4.4 (0.1)* 3.8 (0.9)# CD8/
30 (0.8)# 53.7 (16.7)# 6.3 (1.8)# 5.0 (3.0)
0.9 10.2 2.2 0.9
(0.3)* (1.5)* (0.3)* (0.3)*
6
(0.6) (13.0) (2.4) (2.9)
The average absolute numbers (9/S.E.M.) of double negative (DN), double positive (DP), CD4/ and CD8/ cells are compared after various treatments. * Indicate that in comparison with negative control a P value B/0.05. # Relates to statistical significance (P B/0.05) when comparing lectin groups with positive control animals that were treated with Dexamethasone (DX) alone.
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0.01; P B/0.05 and P B/0.0025, respectively). 1 ng/kg lectin also caused an increase in all cell populations, but significant growth could be measured only in CD8/ thymocyte population, indicating that CD8/ thymocytes in both short- and long-term studies were found to be more susceptible to lectin-induced proliferation in thymus. 3.2. VAA-I inhibited the Dexamethasone-induced thymocyte reduction Since it is well known that DX causes considerable reduction of thymocyte count parallel to the lectin induced alterations, we also investigated the effects of VAA-I treatment on short (24 h) and long-term (twice a week, for 3 weeks) DX (1 mg/kg BW) therapy. As expected, DX treatment alone in both cases induced significant reduction in total number of thymocytes (P B/0.05) (fourth columns of Fig. 1A, B). This DXinduced reduction of thymocyte cell count was significantly less if DX was injected in combination with VAAI (fifth and sixth columns of Fig. 1A, B). As shown in Table 1(B), all investigated thymocyte subpopulations (DN, DP, SP) showed significant elevation if DX was combined with VAA-I for 3 weeks. 3.3. Effect of VAA-I on apoptosis of murine thymocytes in vivo In the first trial, 24 h after a single injection of 1 and 30 ng/kg VAA-I, the percentage of apoptotic cells rose 1.9- and 2.2-fold (P B/0.07 and P B/0.05, respectively; see Fig. 3A). This elevation in apoptosis was higher than that caused by 1 mg/kg DX treatment, suggesting a possible additive effect of VAA-I on DX. To investigate
Fig. 3. Effect of VAA-I on thymocyte late apoptosis. Mean percentage (9/S.E.M.) of apoptotic cells in thymus 24 h after a single injection of lectin (A) and after 3 weeks treatment (B). Some 30 ng/kg lectin induced significant enhancement in both trials (P B/0.05; P B/0.01).
the initial phase of apoptosis in a more sensitive manner, the phosphatidylserine expression on the surface of thymocyte subpopulations by Annexin V binding was also determined. As shown in Fig. 4, 24 h after a single injection of 30 ng/kg VAA-I CD4/ and CD8/ SP thymocyte subpopulations showed 2-and 1.7-fold enhancement in frequency of apoptosis compared to negative control values (P B/0.05 and P B/0.01, respectively). In the second trial, only 30 ng/kg VAA-I (72 h after the last injection of a treatment for 3 weeks) caused (see Fig. 3B) a significant increase (54%) in percentage of apoptotic thymocytes (P B/0.01). The VAA-I induced elevated number of DN and DP thymocytes with increased apoptosis may reflect on faster proliferation and enhanced maturation (positive and negative selection) of these immature cells resulting in an increased number of mature SP cells.
4. Discussion Our studies showed that in agreement with previous in vitro findings [12], VAA-I can act in vivo on the regulation of thymus function inducing both proliferation and apoptosis of thymocytes. The short- and longterm effects of VAA-I on thymus proliferation revealed responses of both mature and immature cells. These effects, at least in part, may be related to proinflammatory activity of VAA-I [12], since IL-1b, IL-6, TNFa and
Fig. 4. Effect of VAA-I on early apoptosis of thymocyte subpopulations. Mean augmentation [(experimental value/negative control value)/S.E.M.] of apoptotic thymocyte subpopulations 24 h after a single injection of VAA-I or DX. All investigations were carried out with Annexin V staining of mouse thymocyte subpopulations. 30 ng/kg lectin induced significant increases in apoptosis of CD4/ and CD8/ SP subpopulation (P B/0.05 and P B/0.01).
T. Hajto´ et al. / Immunology Letters 86 (2003) 23 /27
IFNg are known to play an important role in proliferation and apoptosis of thymocytes [16 /21]. The correct judgment as to whether there is a direct effect of the lectin on the thymocytes or it is a consequence of the lectin-induced proinflammatory cytokines has yet to be determined. VAA-I was also found to modulate protein synthesis and to induce apoptosis in various eukaryotic cells in culture. The apoptotic effect of VAA-I is dose-dependent and in this action the caspase-3 is involved, at least in part [22]. In vitro VAA-I can induce a characteristic programmed cell death between 10 and 1000 ng/ml after 24 h in various cells of immune origin [22,23]. In the present in vivo study, a dose as low as 1 ng/kg appears during the DX therapy to be more effective in reconstitution of lymphoid tissues than 30 ng/kg VAA-I. However, the higher lectin dose (30 ng/kg BW) was more effective on the enhanced proliferation rate and percentage of apoptotic thymocytes. This observation indicate that the VAA-I induced apoptosis may be a consequence of the enhanced selection steps in contrast to the DX induced direct apoptosis of immature thymocytes. Our finding that long-term VAA-I can inhibit the DX induced reduction of thymocyte number, may indicate the importance of studying the effect of lectin on the treatment of various diseases with glucocorticoid hormone therapy. Further investigations are in progress to clarify the effect of VAA-I on steroid resistance.
Acknowledgements We would like to thank Margit von Lessing for supporting this research.
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References [1] N. Sharon, Biol. Cell 51 (1984) 239 /246. [2] P. Ziska, H. Franz, in: T.C. Bog Hansen, J. Breborowicz (Eds.), Lectins, vol. IV, Walter de Gruyter, USA, 1985, pp. 473 /480. [3] J.B. Dietrich, G. Ribereau-Gayon, M.L. Jung, H. Franz, J.P. Beck, R. Anton, Anticancer Drug 3 (1992) 507 /511. [4] H. Franz, Oncology 43 (1986) 23 /34. [5] Y. Endo, K. Tsurugi, H. Franz, FEBS Lett. 231 (1988) 378 /380. [6] K. Sandvig, S. Olsnes, J. Biol. Chem. 257 (1982) 7504 /7513. [7] A. Wiedlocha, K. Sandvig, H. Walzel, C. Radzikowsky, S. Olsnes, Cancer Res. 51 (1991) 916 /920. [8] S. Olsnes, F. Stirpe, K. Sandvig, A. Pihl, J. Biol. Chem. 257 (1982) 13263 /13270. [9] R.T. Lee, H.-J. Gabius, Y.C. Lee, Carbohydr. Res. 254 (1994) 269 /276. [10] K. Hostanska, T. Hajto, G. Spagnoli, J. Fischer, H. Lentzen, R. Herrmann, Nat. Immunol. 14 (1995) 295 /304. [11] H. Walzel, H. Bremer, H.-J. Gabius, in: H.-J. Gabius, S. Gabius (Eds.), Lectins and Glycobiology, Springer, New York, 1993, pp. 357 /361. [12] T. Hajto, K. Hostanska, K. Frei, C. Rordorf, H.-J. Gabius, Cancer Res. 50 (1990) 3322 /3326. [13] T. Hajto, K. Hostanska, H.-J. Gabius, Cancer Res. 49 (1989) 4803 /4808. [14] T. Hajto, K. Hostanska, K. Weber, H. Zinke, J. Fischer, U. Mengs, H. Lentzen, R. Saller, Nat. Immunol. 16 (1998) 34 /46. [15] K. Weber, U. Mengs, T. Schwarz, T. Hajto, K. Hostanska, T.R. Allen, R. Weyhenmeyer, H. Lentzen, Arznem. Forsch. 48 (1998) 497 /502. [16] J. Deman, M. Van Meurs, E. Claassen, C. Humblet, J. Bonvier, M. Defresne, Immunology 89 (1996) 152 /157. [17] B. Von Patay, H. Loppnow, J. Feindt, B. Kurz, R. Mentlein, J. Neuroimmunol. 86 (1998) 182 /189. [18] M. Pezzano, D. Philip, S. Stephenson, Y. Li, V. Reid, R. Maitta, J.C. Guyden, Cell Immunol. 169 (1996) 174 /184. [19] H.G. Durkin, B.H. Waksman, Immunol. Rev. 182 (2001) 33 /57. [20] M. Artico, C. Cavallotti, M. Camerini, D. Cavallotti, Cytokine 15 (2001) 261 /265. [21] J.G. Baseta, O. Stutman, J. Immunol. 165 (2000) 2621 /2630. [22] A. Savoie, V. Lavastre, M. Pelletier, T. Hajto, K. Hostanska, D. Girard, J. Leukoc. Biol. 68 (2000) 845 /853. [23] K. Hostanska, T. Hajto, J. Fischer, U. Mengs, K. Weber, H. Lentzen, R. Saller, Cancer Detect. Prevent. 23 (1999) 511 /523.
Galactoside-specific plant lectin, Viscum album agglutinin-I induces enhanced proliferation and apoptosis of murine thymocytes in vivo Tibor Hajto´, Timea Berki *, Ferenc Boldizsa´r, Pe´ter Ne´meth Department of Immunology and Biotechnology, Faculty of Medicine, University of Pe´cs, Szigeti u´t 12, H-7643 Pe´cs, Hungary Received 30 July 2002; received in revised form 24 October 2002; accepted 8 November 2002
Abstract Galactoside-specific plant lectin, Viscum album agglutinin-I (VAA-I) has been shown to act as a biomodulator with proinflammatory and apoptosis-inducing effects, however its cellular targets and mechanism of immunobiological action in vivo are less well understood. Therefore, in the present work the short- and long-term in vivo effects of VAA-I on thymocyte subpopulations and peripheral T cells were tested using a murine (Balb/c) model. Cell surface CD4/CD8 staining and flow cytometry allowed us to follow the changes of thymocyte subpopulations: CD4 /CD8 / double negative (DN), CD4/CD8/ double positive (DP), CD4/ or CD8/ single positive (SP) and mature peripheral T cells after single or repeated injections with low doses of VAAI. The apoptosis of the cells was detected by flow cytometry using propidium iodide (PI) and Annexin V staining. To detect the short-term effects of the lectin, the animals were investigated 24 h after a single injection of 1 or 30 ng/kg body weight (BW) VAAI9/1 mg/kg Dexamethasone (DX). The total number of mature CD8/ SP thymocytes increased significantly with an enhancement of the ratio of apoptotic cells. In contrast, in the blood samples an elevated CD4/CD8 ratio was found. In the next trial, Balb/c mice were treated twice weekly with 1 or 30 ng/kg VAA-I9/1 mg/kg DX for 3 weeks. The total cell count of thymocytes showed significant increases after both doses of VAA-I, but an elevated percentage of apoptotic cells was found only after treatment with 30 ng/kg VAA-I. SP thymocytes revealed higher increases in lectin-induced apoptosis than DN or DP cells. In addition, both lectin doses significantly inhibited the DX-induced reduction of all thymocyte subpopulations investigated. In conclusion, our data suggest that VAA-I is able to modulate the maturation of thymocytes in vivo. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Viscum albumin agglutinin; Thymus; Apoptosis
1. Introduction Lectins are carbohydrate-binding proteins that are able to recognize and bind the glycan part of glycoconjugates (e.g. glycoproteins, glycolipids, oligo- and polysaccharides) in a sugar-specific manner [1]. In mistletoe plants, the galactoside-specific Viscum album agglutinin-I (VAA-I) is the most frequently investigated and important lectin [2,3]. It consists of two polypeptide subunits. Its A chain, with a molecular weight of 29 kDa because of its highly specific enzymatic efficacy, catalytically inhibits the protein synthesis in the 28S subunit of
* Corresponding author. Tel.: /36-72-536-291; fax: /36-72-536289. E-mail address: [email protected] (T. Berki).
rRNA [4 /7]. That is why VAA-I in common with other plant lectins, such as ricin, abrin and modeccin, belongs to the type II family of ribosome inactivating proteins (RIP) sharing numerous homologous structures [3,8]. The sugar-binding B chain of VAA-I with a molecular weight of 34 kDa is able to bind galactoside residues on the cell membrane preferring certain confirmations [9 / 11]. In low (not cytotoxic) doses, the B chain is responsible for the enhancing effect of VAA-I on the proinflammatory activity of natural immune system. After 24 h incubation with human PBMC, VAA-I (A and B chains in nanogram per milliliter concentrations) induced mRNA expression and enhanced secretion of proinflammatory cytokines [10,12]. Both in vitro and in vivo VAA-I stimulated the number and activity of natural killer (NK) cells [13,14] and induced elevated activation markers on the surface of monocytes and
0165-2478/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 4 7 8 ( 0 2 ) 0 0 2 6 5 - 1
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macrophages [14,15]. Since proinflammatory effects have been reported to play an important role in the regulation of the thymus, in this study the in vivo effects of VAA-I on murine thymocyte development were first investigated. In vivo doses of VAA-I induced enhanced proliferation with a parallel increase in apoptosis of cells in the thymus. In addition, VAA-I was able to inhibit the Dexamethasone (DX)-induced reduction of thymocyte cell number.
2. Materials and methods 2.1. Materials VAA-I was prepared from fresh aqueous extracts of the leaves and stems of mistletoe plants and purified on lactosylated agarose columns as described previously [13]. Endotoxin contamination in the samples used was B/0.5 pg/ml as determined by quantitative kinetic LAL assay. Dexamethasone (Oradexon, Organon) was purchased from N.V. Organon Oss Holland. Apoptosis was detected with propidium iodide (Sigma P 4170) and Annexin V-FITC (Pharmingen Cat No. 556420). For triple labeling experiments, the following mAbs were used: phycoerythrin (PE)-conjugated rat anti-mouse CD4 (L3T4, Pharmingen Cat No. 09005A) and CyChrome-conjugated rat anti-mouse CD8 (Ly-2 Pharmingen Cat. No. 553034) monoclonal antibodies. 2.2. Treatments of animals Eight male and eight female Balb/c mice used in the first study and the nine male and nine female in the second trial (6 weeks old, body weight :/20 g 9/10%) were obtained from Charles Rivers Laboratories. The animals were kept under standardized laboratory conditions. The mice were randomly assigned to six groups of three animals each in both studies. Three groups of three animals were treated subcutaneously with placebo (phosphate-buffered saline) or with VAA-I and/or with DX with the following doses: 1 or 30 ng/kg VAA-I alone, 1 or 30 ng/kg VAA-I combined with 1 mg/kg DX. As positive control, 1 mg/kg DX was given alone. In the first study, all animals were sacrificed 24 h after one subcutaneous injection. In the second trial, the subcutaneous injections were administered twice a week for 3 weeks. After the last injection (72 h), all animals were sacrificed by rapid decapitation. 2.3. Thymocyte preparation and staining Thymus glands were removed and placed in ice-cold PBS. Thymus tissue was homogenized in a glass/glass homogenizer and the suspension filtered through a nylon mesh filter. The thymocytes were washed in PBS
and the total cell number and viability were determined by counting in a hemocytometer using the Trypan blue dye exclusion test. Thymocytes (1 /106) in 100 ml binding buffer (PBS/0.1% NaN3/0.1% BSA) were labeled for the expression of CD4 and CD8 for 30 min on ice, after two washing steps in PBS, the cells were stored in PBS/0.1% PFA until flow cytometric analysis. For the detection of early apoptotic thymocyte subpopulations, after CD4/CD8 staining cells were labeled with Annexin V-FITC for 15 min in Annexin binding buffer. The samples were stored in 400 ml Annexin buffer until flow cytometric analysis. The late apoptotic cells were stained after fixation with 4% PFA in saponin buffer (0.1% Saponine/0.1% BSA/0.1% NaN3 in PBS) with 50 mg/ml of propidium iodide (PI Sigma P 4170) for 30 min at room temperature. 2.4. Flow cytometric acquisition and analysis The samples were analyzed in a FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA), using the CellQuest software. Thymocytes were gated on forward and side scatter plots according to their size and granularity. The gate determined by the untreated thymocyte sample was used for all further measurements. Thymocytes were gated according to their CD4 and/or CD8 fluorescence. To determine the apoptotic cell number with the detection of the DNA content of the cells, PI incorporation into DNA was tested in FL-2 channel of the flow cytometer using a linear amplification. In addition, the early apoptotic cells after CD4/ CD8 staining were measured with Annexin V-FITC binding to membrane phosphatidylserine molecules of various thymocyte subpopulations. 2.5. Statistical analysis The results were analyzed with Student’s t-test and U test according to Wilcoxon, Mann /Whitney using the Statgraphics statistical package for IBM-compatible computers.
3. Results 3.1. Effect of VAA-I on thymocyte proliferation In the first trial, double negative (DN), double positive (DP), CD4/ and CD8/ single positive (SP) cells in the thymus as well as CD4/ and CD8/ cells in peripheral blood of Balb/c mice were investigated 24 h after a single lectin (VAA-I) injection. As shown in the first three columns of Fig. 1(A), a single injection of different doses of VAA-I did not cause significant alterations in the total thymocyte cell count or in the DN, DP and CD4/ cell number (Table 1A). Only the
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Fig. 1. Effect of VAA-I on thymocyte proliferation. Total numbers of thymocytes in Balb/c mice after treatment with 1 and 30 ng/kg lectin (VAA-I) and/or Dexamethasone (DX). The experiments were carried out 24 h after a single subcutaneous injection (A) or 72 h after the last injection of treatment twice weekly for 3 weeks (B). Each value represents the average of three animals (9/S.E.M.). The results of various subpopulations and statistical analysis are shown in Table 1.
Fig. 2. Effect of VAA-I on ratio between CD4/ and CD8/ cells. The mean ratios (9/S.E.M.) between CD4/ and CD8/ cells are shown in peripheral blood and in thymus 24 h after a single injection (A, C) or 72 h after the last injection of a 3 weeks treatment (B, D). Some 24 h after a single injection of both lectin doses, this ratio significantly decreased (C) in thymus (P B/0.01; P B/0.01) and increased (A) in blood (P B/0.01; P B/0.05). After 3 weeks (B, D), no significant differences between treated and control groups were found.
CD8/ thymocyte number revealed a significant (P B/ 0.0 25) elevation (70%) 24 h after 1 ng/kg VAA-I and after 30 ng/kg dose, this elevation was 44% (P /0.05). Therefore, the CD4//CD8/ ratio in thymus 24 h after the two different lectin doses fell 39 and 38% (P B/0.01; P B/0.01) (Fig. 2(C) and Table 1(A)). However, at the same time in peripheral blood, the CD4//CD8/ ratio elevated 74% after 1 ng/kg VAA-I and 55% following 30 ng/kg dose (P B/0.005; P B/0.01) as illustrated in Fig. 2(A).
In the second long-term trial, Balb/c mice were treated with the same doses of VAA-I lectin/DX twice a week for 3 weeks. After the last injections (72 h), the same investigations as after 24 h were carried out. As the first three columns of Fig. 1(B) show, the total thymocyte cell count in the thymus increased significantly after both lectin doses (P B/0.05 and P B/0.025, respectively). As demonstrated in Table 1(B), with the exception of CD4/ cells, all investigated thymocyte subpopulations (DN, DP and CD8/ cells) revealed significant elevation after long-term treatment with 30 ng/kg VAA-I (P B/
Table 1 Thymus
Ctrl
VAA 1 ng
VAA 30 ng
VAA 1 ng /DX
VAA 30 ng /DX
DX
A Effect of single dose VAA-I and DX treatment on thymocyte subpopulations /10 (S.E.M.) DN 2.4 (0.4) 3.0 (0.5) 4.7 (0.1) 1.6 (0.4) DP 117.8 (7.5) 97.1 (1.3) 104.5 (13) 64.4 (14) CD4/ 12.5 (1.4) 12.7 (1.7) 10.5 (1.2) 9.0 (1.7) 4.9 (1.3) 8.4 (1.2)* 7.1 (1.7) 7.0 (2.8) CD8/
1.8 55.7 8.2 6.9
1.2 48.1 5.4 2.3
(0.1)* (5.4)* (0.2)* (0.03)*
B Effect of long-term VAA-I and DX treatments on thymocyte subpopulations /106 (S.E.M.) DN 3.2 (0.7) 3.5 (0.2) 4.7 (0.24)* 4.8 (1.6)# DP 48.8 (14.7) 69.7 (15.9) 101 (18.0)* 54.4 (16)# CD4/ 7.2 (1.4) 9.4 (0.9) 10.6 (2.1)* 7.6 (2.0)# 2.3 (0.3) 3.6 (0.08)* 4.4 (0.1)* 3.8 (0.9)# CD8/
30 (0.8)# 53.7 (16.7)# 6.3 (1.8)# 5.0 (3.0)
0.9 10.2 2.2 0.9
(0.3)* (1.5)* (0.3)* (0.3)*
6
(0.6) (13.0) (2.4) (2.9)
The average absolute numbers (9/S.E.M.) of double negative (DN), double positive (DP), CD4/ and CD8/ cells are compared after various treatments. * Indicate that in comparison with negative control a P value B/0.05. # Relates to statistical significance (P B/0.05) when comparing lectin groups with positive control animals that were treated with Dexamethasone (DX) alone.
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T. Hajto´ et al. / Immunology Letters 86 (2003) 23 /27
0.01; P B/0.05 and P B/0.0025, respectively). 1 ng/kg lectin also caused an increase in all cell populations, but significant growth could be measured only in CD8/ thymocyte population, indicating that CD8/ thymocytes in both short- and long-term studies were found to be more susceptible to lectin-induced proliferation in thymus. 3.2. VAA-I inhibited the Dexamethasone-induced thymocyte reduction Since it is well known that DX causes considerable reduction of thymocyte count parallel to the lectin induced alterations, we also investigated the effects of VAA-I treatment on short (24 h) and long-term (twice a week, for 3 weeks) DX (1 mg/kg BW) therapy. As expected, DX treatment alone in both cases induced significant reduction in total number of thymocytes (P B/0.05) (fourth columns of Fig. 1A, B). This DXinduced reduction of thymocyte cell count was significantly less if DX was injected in combination with VAAI (fifth and sixth columns of Fig. 1A, B). As shown in Table 1(B), all investigated thymocyte subpopulations (DN, DP, SP) showed significant elevation if DX was combined with VAA-I for 3 weeks. 3.3. Effect of VAA-I on apoptosis of murine thymocytes in vivo In the first trial, 24 h after a single injection of 1 and 30 ng/kg VAA-I, the percentage of apoptotic cells rose 1.9- and 2.2-fold (P B/0.07 and P B/0.05, respectively; see Fig. 3A). This elevation in apoptosis was higher than that caused by 1 mg/kg DX treatment, suggesting a possible additive effect of VAA-I on DX. To investigate
Fig. 3. Effect of VAA-I on thymocyte late apoptosis. Mean percentage (9/S.E.M.) of apoptotic cells in thymus 24 h after a single injection of lectin (A) and after 3 weeks treatment (B). Some 30 ng/kg lectin induced significant enhancement in both trials (P B/0.05; P B/0.01).
the initial phase of apoptosis in a more sensitive manner, the phosphatidylserine expression on the surface of thymocyte subpopulations by Annexin V binding was also determined. As shown in Fig. 4, 24 h after a single injection of 30 ng/kg VAA-I CD4/ and CD8/ SP thymocyte subpopulations showed 2-and 1.7-fold enhancement in frequency of apoptosis compared to negative control values (P B/0.05 and P B/0.01, respectively). In the second trial, only 30 ng/kg VAA-I (72 h after the last injection of a treatment for 3 weeks) caused (see Fig. 3B) a significant increase (54%) in percentage of apoptotic thymocytes (P B/0.01). The VAA-I induced elevated number of DN and DP thymocytes with increased apoptosis may reflect on faster proliferation and enhanced maturation (positive and negative selection) of these immature cells resulting in an increased number of mature SP cells.
4. Discussion Our studies showed that in agreement with previous in vitro findings [12], VAA-I can act in vivo on the regulation of thymus function inducing both proliferation and apoptosis of thymocytes. The short- and longterm effects of VAA-I on thymus proliferation revealed responses of both mature and immature cells. These effects, at least in part, may be related to proinflammatory activity of VAA-I [12], since IL-1b, IL-6, TNFa and
Fig. 4. Effect of VAA-I on early apoptosis of thymocyte subpopulations. Mean augmentation [(experimental value/negative control value)/S.E.M.] of apoptotic thymocyte subpopulations 24 h after a single injection of VAA-I or DX. All investigations were carried out with Annexin V staining of mouse thymocyte subpopulations. 30 ng/kg lectin induced significant increases in apoptosis of CD4/ and CD8/ SP subpopulation (P B/0.05 and P B/0.01).
T. Hajto´ et al. / Immunology Letters 86 (2003) 23 /27
IFNg are known to play an important role in proliferation and apoptosis of thymocytes [16 /21]. The correct judgment as to whether there is a direct effect of the lectin on the thymocytes or it is a consequence of the lectin-induced proinflammatory cytokines has yet to be determined. VAA-I was also found to modulate protein synthesis and to induce apoptosis in various eukaryotic cells in culture. The apoptotic effect of VAA-I is dose-dependent and in this action the caspase-3 is involved, at least in part [22]. In vitro VAA-I can induce a characteristic programmed cell death between 10 and 1000 ng/ml after 24 h in various cells of immune origin [22,23]. In the present in vivo study, a dose as low as 1 ng/kg appears during the DX therapy to be more effective in reconstitution of lymphoid tissues than 30 ng/kg VAA-I. However, the higher lectin dose (30 ng/kg BW) was more effective on the enhanced proliferation rate and percentage of apoptotic thymocytes. This observation indicate that the VAA-I induced apoptosis may be a consequence of the enhanced selection steps in contrast to the DX induced direct apoptosis of immature thymocytes. Our finding that long-term VAA-I can inhibit the DX induced reduction of thymocyte number, may indicate the importance of studying the effect of lectin on the treatment of various diseases with glucocorticoid hormone therapy. Further investigations are in progress to clarify the effect of VAA-I on steroid resistance.
Acknowledgements We would like to thank Margit von Lessing for supporting this research.
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References [1] N. Sharon, Biol. Cell 51 (1984) 239 /246. [2] P. Ziska, H. Franz, in: T.C. Bog Hansen, J. Breborowicz (Eds.), Lectins, vol. IV, Walter de Gruyter, USA, 1985, pp. 473 /480. [3] J.B. Dietrich, G. Ribereau-Gayon, M.L. Jung, H. Franz, J.P. Beck, R. Anton, Anticancer Drug 3 (1992) 507 /511. [4] H. Franz, Oncology 43 (1986) 23 /34. [5] Y. Endo, K. Tsurugi, H. Franz, FEBS Lett. 231 (1988) 378 /380. [6] K. Sandvig, S. Olsnes, J. Biol. Chem. 257 (1982) 7504 /7513. [7] A. Wiedlocha, K. Sandvig, H. Walzel, C. Radzikowsky, S. Olsnes, Cancer Res. 51 (1991) 916 /920. [8] S. Olsnes, F. Stirpe, K. Sandvig, A. Pihl, J. Biol. Chem. 257 (1982) 13263 /13270. [9] R.T. Lee, H.-J. Gabius, Y.C. Lee, Carbohydr. Res. 254 (1994) 269 /276. [10] K. Hostanska, T. Hajto, G. Spagnoli, J. Fischer, H. Lentzen, R. Herrmann, Nat. Immunol. 14 (1995) 295 /304. [11] H. Walzel, H. Bremer, H.-J. Gabius, in: H.-J. Gabius, S. Gabius (Eds.), Lectins and Glycobiology, Springer, New York, 1993, pp. 357 /361. [12] T. Hajto, K. Hostanska, K. Frei, C. Rordorf, H.-J. Gabius, Cancer Res. 50 (1990) 3322 /3326. [13] T. Hajto, K. Hostanska, H.-J. Gabius, Cancer Res. 49 (1989) 4803 /4808. [14] T. Hajto, K. Hostanska, K. Weber, H. Zinke, J. Fischer, U. Mengs, H. Lentzen, R. Saller, Nat. Immunol. 16 (1998) 34 /46. [15] K. Weber, U. Mengs, T. Schwarz, T. Hajto, K. Hostanska, T.R. Allen, R. Weyhenmeyer, H. Lentzen, Arznem. Forsch. 48 (1998) 497 /502. [16] J. Deman, M. Van Meurs, E. Claassen, C. Humblet, J. Bonvier, M. Defresne, Immunology 89 (1996) 152 /157. [17] B. Von Patay, H. Loppnow, J. Feindt, B. Kurz, R. Mentlein, J. Neuroimmunol. 86 (1998) 182 /189. [18] M. Pezzano, D. Philip, S. Stephenson, Y. Li, V. Reid, R. Maitta, J.C. Guyden, Cell Immunol. 169 (1996) 174 /184. [19] H.G. Durkin, B.H. Waksman, Immunol. Rev. 182 (2001) 33 /57. [20] M. Artico, C. Cavallotti, M. Camerini, D. Cavallotti, Cytokine 15 (2001) 261 /265. [21] J.G. Baseta, O. Stutman, J. Immunol. 165 (2000) 2621 /2630. [22] A. Savoie, V. Lavastre, M. Pelletier, T. Hajto, K. Hostanska, D. Girard, J. Leukoc. Biol. 68 (2000) 845 /853. [23] K. Hostanska, T. Hajto, J. Fischer, U. Mengs, K. Weber, H. Lentzen, R. Saller, Cancer Detect. Prevent. 23 (1999) 511 /523.