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PARTIAL CHARACTERIZATION OF MOSQUITO LARVAE EXTRACT INDUCING DNA SYNTHESIS ALTERATIONS ON HUMAN MONONUCLEAR CELLS
Cell Biology International 2000, Vol. 24, No. 3, 175–181 doi:10.1006/cbir.1999.0489, available online at http://www.idealibrary.com on
PARTIAL CHARACTERIZATION OF MOSQUITO LARVAE EXTRACT INDUCING DNA SYNTHESIS ALTERATIONS ON HUMAN MONONUCLEAR CELLS J. R. RONDEROS1*, M. A. SALAS2, O. J. RIMOLDI3 and G. FINARELLI3 1
Ca´tedra de Histologı´a y Embriologı´a ‘B’, 2Ca´tedra de Fisiologı´a con Biofı´sica and 3INIBIOLP, Facultad de Ciencias Me´dicas, Universidad Nacional de La Plata, Calle 60 y 120, 1900 La Plata, Argentina Received 3 June 1999; accepted 20 November 1999
A crude mosquito larvae and dialysed extract alters the mitotic rate of several epithelial cell populations in normal young and adult hepatectomized mice. A crude extract also showed a biphasic effect on the proliferation of human mononuclear cells (MNCs), either stimulating or inhibiting them depending on the dose applied. In the present paper, we assayed the effect of the dialysed mosquito larvae extract and two different protein fractions on human MNCs. Analysis of cell viability after culture indicated that the extract did not have toxic effects. Our results show a dual response of the MNCs to the dialysed, as well as to the protein fraction, with the highest molecular weight inhibiting or stimulating proliferation, depending on the dose applied. The protein fraction with the lowest molecular weight (range between 12–80 kDa) showed only an 2000 Academic Press inhibitory effect on cell proliferation. K: mononuclear cells; lymphocytes; cell proliferation; mosquito; growth factors.
INTRODUCTION Cell growth is regulated by several factors, such as hormones, growth factors and cell surface receptor synthesis. Several of these factors, such as protooncogenes, have been maintained in nature throughout evolution and it is for this reason that distant organisms, such as insects and mammals, share some of these genes. In fact, proteins structurally related to transforming growth factor- (TGF-), platelet derived growth factor (PDGF) and epidermal growth factor (EGF) have been found in insects, nematodes, molluscs and echinoderms (Doctor et al., 1992; Franchini et al., 1996; Massague, 1990; Muskavitch and Hoffmann, 1990; Padgett et al., 1987; Ren et al., 1996). Evidence about the presence of a gene in Drosophila, which encodes a receptor of the TGF- superfamily, and TGF- receptors in molluscs was also provided (Kletsas et al., 1998; Tin Xie et al., 1994). Furthermore, the Drosophila decapentaplegic gene, which encodes a protein with high percentage of homology with TGF-, was reported as an imaginal *To whom correspondence should [email protected] 1065–6995/00/030175+07 $35.00/0
be
addressed.
E-mail:
disk cell synchronizer at G2/M interphase during Drosophila eye development (Penton et al., 1997). It has been demonstrated that in the mosquito Aedes aegipty larvae some cell populations exist which do not grow by hyperplasia (Tragger, 1937). This fact might be related to the existence of some molecule, or molecules inhibiting cell proliferation and perhaps inducing cell differentiation. In order to find growth factors homologous with vertebrates in insects, we have previously assayed a crude extract from other mosquito larvae species (Culex pipiens L.) on the mitotic rate of several epithelial cell populations of mice in vivo. From this, it has been demonstrated that crude extract of mosquito larvae has an inhibitory effect on the mitotic rate of partially hepatectomized adult mice hepatocytes (Ronderos and Echave Llanos, 1990). The same effect was seen on hepatocytes, enterocytes, sialocytes and tongue keratinocytes of young growing mice (Ronderos et al., 1994a; Ronderos et al., 1996). When the dialysed fraction of the extract (12 kDa cut-off pore membrane) was assayed on hepatocyte proliferation of hepatectomized adult mice, similar results were found, showing an association of the biological effect with 2000 Academic Press
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the macromolecular fraction of the extract (Ronderos et al., 1994a). The same was found when we analysed tongue keratinocyte proliferation (Ronderos et al., 1999). We have also studied the effect of different soluble fractions of the extract isolated by molecular exclusion gel permeation chromatography on regenerating liver hepatocytes (Ronderos et al., 1999). Three different fractions were analysed. The first one, including microsomal fraction, did not produce any effect. The other two fractions tested gave either a non-statistically significant increment or an inhibitory effect on the mitotic rate of this population. The last one had a molecular weight ranging between 80 and 12 kDa, peaking at a retention time of 25 kDa. In these experiments, treatments were applied before the DNA synthesis curve increased (Echave Llanos et al., 1971), and the sampling period took place 16–24 h after treatment, when the mitotic peak occurs (Surur et al., 1985). The time elapsed between treatments and animal death suggested an inhibitory effect of the mosquito extract on the cell cycle G1/S checkpoint. When the mitotic ratesampling period covered the dark phase of the circadian cycle (i.e. time of maximum DNA synthesis), a statistically significant increment was found in hepatocytes, nephrocytes and tongue keratinocytes of extract recipients. Taking the time elapsed from treatment administration until death into account (from 0 to 12 h), we suggested an effect of the extract at G2/M checkpoint of the cell cycle (Ronderos, 1996). In other experiments, treatments were applied after the DNA synthesis peak occurrence. The results were similar, showing a decrease in mitotic rates in hepatocytes, tongue keratinocytes and enterocytes (Ronderos et al., 1994b). In view of the results obtained on epithelial cell populations with Culex pipiens larvae extracts, and investigating the effect of the extract on other cell populations apart from the epithelial ones, we decided to assay the effect of the mosquito crude extract on the proliferation of human mononuclear cells (MNCs) activated with concanavalin-A (Con-A) (Ronderos et al., 1999). In these experiments the crude extract showed a dual effect, either inhibiting or stimulating lymphocyte proliferation depending of the dose applied. Furthermore, the biological activity of the crude extract was shown to be inactivated after heating at 90C for 30 min, suggesting the involvement of some macromolecule. In the present study we analysed the effect of the dialysed extract, as well as the two soluble fractions which previously proved to have a biological effect
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on the proliferation of human MNCs activated with Con-A in culture. MATERIALS AND METHODS Mosquito larvae extract Third and fourth instar larvae of mosquito Culex pipiens L. (Diptera: Culicidae) were obtained from an artificial colony. Larvae were separated from the colony, washed several times in fresh water to eliminate any breed plate residue, then filtered and weighed for extract preparation. Pooled mosquito larvae were homogenized in a Potter-Dual homogenizer in an ice-bath, and saline solution at 4C was added (2:10). The homogenate was centrifuged at 0C at 14,500g for 20 min in a Sorval RC5C centrifuge (crude extract) or ultracentrifuged at 100,000g with the aim of eliminating the fraction containing microsomes. After centrifugation, the supernatant was recovered and dialysed with a 12 kDa cut-off pore membrane in saline solution overnight at 4C. Mononuclear cells isolation Human heparinized blood was obtained from healthy adult donors. MNCs were isolated by centrifugation on a Ficoll gradient (Histopaque 1077; Sigma, St. Louis, MO, U.S.A.). After washing, cells were resuspended in RPMI medium supplemented with 2 m glutamine, 100 g/ml streptomycine, 100 U/ml penicillin, 2-ME 10 5 (all from Sigma) and 10% fetal bovine serum (Gen-Argentina). They were finally seeded into 96-well plates at a density of 105 cells/well. MNC viability was evaluated by Trypan blue exclusion test. MNC experimental design MNCs were cultured at 37C in a humidified atmosphere of 5% CO2 in air. The different treatments (5–6 wells/treatment) and the mitogen Con-A (5 g/ml) diluted in RPMI, were added simultaneously at the beginning of the culture. To evaluate lymphocyte proliferation, 3H-thymidine (1 Ci/well) were added 72 h after the start of the culture (time of maximum proliferation under Con-A activation) and 4 h before finishing the experiment. Cells were finally recovered on filters by the use of a cell harvester (Titerteck Cell Harvester, Flow Lab, Rockville, MD, U.S.A.). The radioactivity incorporated was measured in a liquid scintillation counter. All experiments were repeated
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at least twice, producing the same results. Consequently, the data presented here are from a representative experiment. Each step in purification included a positive control of the previous state of the extract (i.e. crude extract for dialysis assay; dialysis extract for F1 and F2 assays).
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The previously ultracentrifuged dialysed homogenate was applied to a Superose 6HR 10/30 column, equilibrated and eluted with buffer Tris-HCl 10 m, NaCl 0.15 , pH 8.0 at a flow rate of 0.5 ml/min. The effluent was monitored at 280 nm. The protein-containing fractions were pooled and concentrated by ultrafiltration (Centricom-10, Amicon, Beverly, MA, U.S.A.). The protein contents were determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.
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Statistical analysis Differences were analysed by one-way analysis of variance (ANOVA). For single comparisons, LSD multiple range test was applied. Only differences greater than P<0.05 were considered significant. RESULTS Analysis of MNC viability after mosquito larvae crude extract assay After 72 h of culture under basal conditions, three wells with and three without mosquito larvae crude extract (controls) were recovered. MNC viability was evaluated by Trypan blue exclusion test. There were no differences in cell viability between controls and cells receiving extract, showing no toxic effect of the extract. Effect of the ultracentrifuged supernatant of extract on MNC proliferation The supernatant of the ultracentrifuged crude extract was recovered and assayed on MNC proliferation. Treatments ranging from 1 to 0.001 mg/ml of the supernatant were applied to Con-A-activated MNCs at the beginning of the culture. 3H-thymidine incorporation was evaluated 72 h later (time of maximum proliferation of Con-A-activated MNCs). The results showed, as in previous experiments with crude extract, a dual response of MNCs. Doses from 1 to 0.1 mg/ml caused a decrease of proliferation, while doses
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Fig. 1. Dose-dependent proliferative response of Con-Aactivated MNC to supernatant of the ultracentrifuged mosquito larvae extract. Bars represent mean... 0= Controls without extract; *=statistically significant differences. Crude extract (1 mg/ml) showing an inhibitory effect was also included in the experiment (data not shown).
ranging between 0.01 and 0.001 mg/ml indicated that MNCs were entering S phase of the cycle (Fig. 1). Effect of the dialysed extract on MNC proliferation In order to discard steroids present in insects, mosquito larvae crude extract was dialysed (cut-off pore membrane 12 kDa). A range of doses similar to those used previously were applied. Again, a dual response was obtained, showing an inhibitory effect with the highest dose (0.5 mg/ml) and a stimulatory effect on Con-A-activated MNC proliferation, with a range of doses between 0.01 to 0.001 mg/ml (Fig. 2). Analysis of chromatographic fractions In view of the previous results achieved, we decided to test the soluble fractions obtained by molecular exclusion chromatography. The mosquito larvae crude extract, ultracentrifuged as described above and then dialysed (12 kDa cut-off pore membrane
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Fig. 2. Dose-dependent proliferative response of Con-Aactivated MNC to dialysed mosquito larvae extract. Bars represent mean... 0=Controls without extract; *=statistically significant differences. Crude extract (1 mg/ml) showing an inhibitory effect was also included in the experiment (data not shown).
against saline solution overnight), was applied to a gel permeation Superose 6B column and eluted with Tris-HCl saline buffer. Two main peaks were detected (F1 and F2 in Fig. 3). Effect of soluble fractions isolated by molecular exclusion chromatography on MNC proliferation A similar experimental design was applied to analyse the effect of both chromatographic fractions on MNC proliferation. The F1 assay showed an inhibitory effect only with the highest dose applied (0.5 mg/ml) and a stimulatory effect from doses ranging from 0.05 to 0.01 mg/ml. Doses lower than these had no effect on 3H-thymidine incorporation (Fig. 4). When we performed a dose-response assay using the lowest molecular weight fraction (F2), only an inhibitory effect was found. This effect was not achieved with the highest doses used (0.1 and 0.05 mg/ml), whereas a statistically significant inhibition appeared with doses ranging from 0.01 down to 0.0001 mg/ml (Fig. 5).
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Fig. 3. Chromatographic profile of mosquito larvae extract eluted through a Superose 6 HR 10/30 molecular exclusion column.
Effect of the crude extract on basal MNC proliferation Previous experiments showed that mosquito larvae crude extract has a dual effect on Con-A-activated MNCs: inhibiting or stimulating DNA synthesis depending on the dose applied. As Con-A modifies the biological activity of monocytes, modulating TGF- release (Assoian et al., 1987), we decided to assay the effect of the crude extract on MNC proliferation under basal conditions (i.e. without Con-A). Dose-response treatments ranging from 1 to 0.01 mg/ml of mosquito larvae crude extract were applied to MNCs under basal conditions at the beginning of the culture. After 72 h, MNC proliferation was evaluated by 3H-thymidine incorporation. Contrary to Con-A-activated conditions,
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Fig. 4. Dose-dependent proliferative response of Con-Aactivated MNC to F1 soluble fraction obtained by molecular exclusion chromatography of mosquito larvae extract after ultracentrifugation and dialysis. Bars represent mean... 0=Controls without extract; *=statistically significant differences. Dialysed extract (0.5 mg/ml) showing an inhibitory effect was also included in the experiment (data not shown).
results showed only an inhibitory effect of mosquito larvae extract at the range of doses applied, which were the same as those used for the Con-A-activated MNC assay (Fig. 6).
DISCUSSION As shown above, a statistically significant alteration in the proliferation process of MNCs appears after treatment with both mosquito larvae dialysed extract and soluble fractions obtained by the use of molecular exclusion chromatography. Both dialysed extract and the soluble fraction with the highest molecular weight (F1) cause a biphasic response of MNCs, inhibiting and stimulating cell proliferation depending on the dose applied. The F2 fraction, with the lowest molecular weight within the range 10–80 kDa, and a maximum relative absorbance (at 280 nm) which peaks at a molecular weight level of 25 kDa (Ronderos et al., 1999), only has an inhibitory effect. Surprisingly,
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Fig. 5. Dose-dependent proliferative response of Con-Aactivated MNC to F2 soluble fraction obtained by molecular exclusion chromatography of mosquito larvae extract after ultracentrifugation and dialysis. Bars represent mean... 0=Controls without extract; *=statistically significant differences. Dialysed extract (0.5 mg/ml) showing an inhibitory effect was also included in the experiment (data not shown).
this effect is not evident with the highest doses applied (0.1 and 0.05 mg/ml), but it is significant with doses as low as 0.0001 mg/ml. Previous experiments demonstrated that the biological effect of the dialysed extract disappeared when it was heated at 90C for 30 min, suggesting that some protein is involved in the observed results (Ronderos et al., 1999). As proliferation on MNC in the present study was measured by 3H-thymidine incorporation, the results indicate, as in previous studies, an effect of the extract on G1/S interphase of the cell cycle (Ronderos and Echave Llanos, 1990; Ronderos et al., 1994a; Ronderos et al., 1996). Looking for messengers which can regulate cell proliferation, both TGF-1, which modulates cell proliferation acting on late G1 phase cyclindependent kinases (Reddy et al., 1994), and other members of the superfamily appear as possible candidates to be present in our extract. They have been suggested as mediators for the regulation of DNA synthesis and differentiation in epithelial, as well as lymphocyte populations (Ahuja et al., 1993; Altiok et al., 1994; De Jong et al., 1994; Fox et al.,
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Members of this growth factor superfamily have also been linked to the regulation of endocrine function, particularly with respect to pituitary hormones (Jin et al., 1997; De et al., 1996; Murata and Ying, 1991; Pastorcic et al., 1995). We have found that the dialysed extract from mosquito larvae also has a specific modulatory effect on rat pituitary hormone release. It inhibits both prolactin and GH, and stimulates LH and TSH release (unpublished results). Finally, our results suggest the existence of some known or unknown peptidic molecule in Culex pipiens L. mosquito larvae, modulating lymphocyte proliferation behaviour in humans.
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ACKNOWLEDGEMENTS 100
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Thanks are due to Mrs Gabriela Simonetto for her skilful technical assistance and to the members of the Insect Pathology Laboratory (CEPAVE-UNLP) for mosquito larvae supply. 0
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Fig. 6. Dose-dependent proliferative response MNC in basal conditions (without Con-A) to crude mosquito larvae extract. Bars represent mean... 0=Controls without extract; *=statistically significant differences.
1992, 1993; Lee and Rich, 1993; Reinhold et al., 1994; Ying and Becker 1995; Kay and Fausto, 1997). Experiments tending to confirm the presence of TGF-1 in the mosquito larvae F2 fraction are currently in progress. The behaviour of the MNCs was different according to whether treatments included lectin activation or not. The dose-response assay of the crude extract under basal conditions showed only an inhibitory response. As we have shown, the same treatment induced a biphase effect on Con-Aactivated MNC proliferation. Furthermore, in our experiments, the rate of lymphocyte proliferation inhibition changes in basal and lectin activated assays, being greater in the latter. Assoian et al. (1987) reported that lectins do not affect TGF- mRNA expression but increase its secretion by peripheral blood monocytes and alveolar macrophages. Thus, the TGF- released by monocytes could synergise the inhibitory effect of the extract. With respect to the biphase effect, a similar response has been described for several other messengers, which can elicit opposite effects on the same cell population depending on the concentration used (Jin et al., 1997; Moses et al., 1990; Qian et al., 1996).
REFERENCES A SS, P F, Y H, B JE, B DT, 1993. Effect of transforming growth factor- on early and late activation events in human T cells. J Immunol 150: 3109–3118. A A, J M, M T, K E, 1994. Biphasic effect of transforming growth factor- on EpsteinBarr virus-induced activation of human tonsillar B cells. Immunol Letts 40: 111–115. A RK, F BE, S HC, M PJ, M DK, R EW, R R, S MB, 1987. Expression and secretion of type transforming growth factor by activated human macrophages. Proc Natl Acad Sci USA 84: 6020–6024. D A, M TE, S RC, B N, S DK, 1996. Pituitary lactotrope express transforming growth factor (TGF) type II receptor mRNA and protein and contains 125I-TGF1 binding sites. J Endocrinol 149: 19–27. D J R, V L RAW, R FW, S C, D P, M MD, 1994. Differential effect of transforming growth factor-1 on the activation of human naive and memory CD4 + T lymphocytes. Intl Immunol 6: 631–638. D JS, J PD, R KE, V M, H FM, 1992. Sequence, biochemical characterization, and developmental expression of a new member of the TGF- superfamily in Drosophila melanogaster. Dev Biol 151: 491–505. E L JM, D’A M, S M, B R, S JM, 1971. Circadian variations in DNA synthesis, mitotic activity and cell size of hepatocyte population of young immature male mice growing liver. Virchows Archiv Abt B Zellpath 8: 309–317. F FE, C RJ, F HC, L RJ, M JS, N PC, 1992. Transforming growth factor- inhibits
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human T-cell proliferation through multiple targets. Lymph Cyt Res 11: 299–305. F FE, F HC, D R, C S, N PC, 1993. Evidence that TGF- can inhibit human T-lymphocyte proliferation through paracrine and autocrine mechanisms. Cell Immunol 150: 45–58. F A, K D, O E, 1996. Immunocytochemical evidence of PDGF- and TGF--like molecules in invertebrate and vertebrate immunocytes an evolutionary approach. Histochem J 28: 599–603. J L, Q X, K E, S N, S BW, K K, Y WF J, L RV, 1997. Transforming growth factor- receptor II and p27Kip1 expression in nontumorous and neoplastic human pituitaries. Amer J Pathol 151: 509–519. K MA, F N, 1997. Liver regeneration: prospects for therapy based on new technologies. Mol Med Today 3: 108–115. K D, S D, F A, O E, 1998. PDGF and TGF- induce cell shape changes in invertebrate immunocytes via specific cell surface receptors. EJCB 75: 362–366. L HM, R S, 1993. Differential activation of CD8-positive T cells by transforming growth factor-1. J Immunol 151: 668–677. L OH, R NJ, F AL, R RJ 1951. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275. M J, 1990. The transforming growth factor- family. Annu Rev Cell Biol 6: 597–641. M HL, Y EY, P JA, 1990. TGF- Stimulation and inhibition of cell proliferation: new mechanistic insights. Cell 63: 245–247. M T, Y S, 1991. Transforming growth factor- and activin inhibit basal secretion of prolactin in a pituitary monolayer culture system. Proc Soc Exp Biol Med 198: 599–605. M AT, H FM, 1990. Homologous of vertebrate growth factors in Drosophila melanogaster and other invertebrates. Curr Top Dev Biol 24: 289–328. P RW, S J RD, G WM, 1987. A transcript from Drosophila pattern gene predicts a protein homologous to the transforming growth factor- family. Nature 325: 81–84. P M, D A, B N, V W, S DK, 1995. Reduction in the expression and action of transforming growth factor 1 on lactotropes during estrogeninduced tumorigenesis in the anterior pituitary. Cancer Res 55: 4892–4898. P A, S SB, H FM, 1997. Regulation of cell cycle synchronisation by decapentaplegic gene during Drosophila eye development. Science 275: 203–206.
181
Q X, J L, G JP, L RV, 1996. Transforming growth factor- and p27 expression in pituitary cells. Endocrinology 137: 3051–3060. R KB, H BA, H PH, 1994. Inhibition of G1 phase cyclin dependent kinases by transforming growth factor- 1. J Cell Biochem 56: 418–425. R D, B U, B F, L U, U AJ, F HD, A S, 1994. Transforming growth factor-1 (TGF-1) inhibits DNA synthesis of PWM-stimulated PBMC via suppression of IL-2 and IL-6 production. Cytokine 6: 382–388. R P, L C, J R, A PS, P D, R DL, 1996. Control of C. elegans larval development by neuronal expression of a TGF- homologue. Science 274: 1389–1391. R JR, E L JM, 1990. Inhibitory effect of mosquito larvae extract on the mitotic rate of hepatocytes of young growing and adult partially hepatectomised mice. Com Biol 9: 133–142. R JR, S AO, A CA, E L JM, 1994a. Inhibition of mouse parenchymal cells proliferation by mosquito larvae extract. Cell Mol Biol 40: 1183–1188. R JR, O A, S AO, E L JM, 1994b. Further action of mosquito larvae extract on the mitotic rate of mouse cell populations. BioCell 18: 51–57. R JR, 1996. Early effect of mosquito larvae extract on mouse cells proliferation in vivo. Cell Biol Intl 20: 249–254. R JR, R FL, O A, 1996. Mosquito larvae extract induces mitotic rate alterations of mice tongue keratinocytes along a circadian rhythm period. Cell Biol Intl 20: 567–572. R JR, R O, S MA, B RR, 1999. Partial characterization of mosquito larvae extract modulating mouse and human cell proliferation. Cell Biol Intl 23: 219–226. S JM, M FR, B AF, E L JM, 1985. Variations in DNA synthesis and mitotic indices in hepatocytes and sinusoid litoral cells of adult intact male mouse along a circadian time span. Chronobiol Intl 2: 161–168. T X, F AL, P RW, 1994. The Drosophila saxophone gene: a serine-threonine kinase receptor of the TGF- superfamily. Science 263: 1756–1759. T W, 1937. Cell size in relation to the growth and metamorphosis of the mosquito, Aedes aegypti. J Exp Zool 76: 467–489. Y SY, B A, 1995. Inhibin and activin modulate transforming growth factor--induced immunosuppression. J Biomed Sci 2: 237–241.
PARTIAL CHARACTERIZATION OF MOSQUITO LARVAE EXTRACT INDUCING DNA SYNTHESIS ALTERATIONS ON HUMAN MONONUCLEAR CELLS J. R. RONDEROS1*, M. A. SALAS2, O. J. RIMOLDI3 and G. FINARELLI3 1
Ca´tedra de Histologı´a y Embriologı´a ‘B’, 2Ca´tedra de Fisiologı´a con Biofı´sica and 3INIBIOLP, Facultad de Ciencias Me´dicas, Universidad Nacional de La Plata, Calle 60 y 120, 1900 La Plata, Argentina Received 3 June 1999; accepted 20 November 1999
A crude mosquito larvae and dialysed extract alters the mitotic rate of several epithelial cell populations in normal young and adult hepatectomized mice. A crude extract also showed a biphasic effect on the proliferation of human mononuclear cells (MNCs), either stimulating or inhibiting them depending on the dose applied. In the present paper, we assayed the effect of the dialysed mosquito larvae extract and two different protein fractions on human MNCs. Analysis of cell viability after culture indicated that the extract did not have toxic effects. Our results show a dual response of the MNCs to the dialysed, as well as to the protein fraction, with the highest molecular weight inhibiting or stimulating proliferation, depending on the dose applied. The protein fraction with the lowest molecular weight (range between 12–80 kDa) showed only an 2000 Academic Press inhibitory effect on cell proliferation. K: mononuclear cells; lymphocytes; cell proliferation; mosquito; growth factors.
INTRODUCTION Cell growth is regulated by several factors, such as hormones, growth factors and cell surface receptor synthesis. Several of these factors, such as protooncogenes, have been maintained in nature throughout evolution and it is for this reason that distant organisms, such as insects and mammals, share some of these genes. In fact, proteins structurally related to transforming growth factor- (TGF-), platelet derived growth factor (PDGF) and epidermal growth factor (EGF) have been found in insects, nematodes, molluscs and echinoderms (Doctor et al., 1992; Franchini et al., 1996; Massague, 1990; Muskavitch and Hoffmann, 1990; Padgett et al., 1987; Ren et al., 1996). Evidence about the presence of a gene in Drosophila, which encodes a receptor of the TGF- superfamily, and TGF- receptors in molluscs was also provided (Kletsas et al., 1998; Tin Xie et al., 1994). Furthermore, the Drosophila decapentaplegic gene, which encodes a protein with high percentage of homology with TGF-, was reported as an imaginal *To whom correspondence should [email protected] 1065–6995/00/030175+07 $35.00/0
be
addressed.
E-mail:
disk cell synchronizer at G2/M interphase during Drosophila eye development (Penton et al., 1997). It has been demonstrated that in the mosquito Aedes aegipty larvae some cell populations exist which do not grow by hyperplasia (Tragger, 1937). This fact might be related to the existence of some molecule, or molecules inhibiting cell proliferation and perhaps inducing cell differentiation. In order to find growth factors homologous with vertebrates in insects, we have previously assayed a crude extract from other mosquito larvae species (Culex pipiens L.) on the mitotic rate of several epithelial cell populations of mice in vivo. From this, it has been demonstrated that crude extract of mosquito larvae has an inhibitory effect on the mitotic rate of partially hepatectomized adult mice hepatocytes (Ronderos and Echave Llanos, 1990). The same effect was seen on hepatocytes, enterocytes, sialocytes and tongue keratinocytes of young growing mice (Ronderos et al., 1994a; Ronderos et al., 1996). When the dialysed fraction of the extract (12 kDa cut-off pore membrane) was assayed on hepatocyte proliferation of hepatectomized adult mice, similar results were found, showing an association of the biological effect with 2000 Academic Press
176
the macromolecular fraction of the extract (Ronderos et al., 1994a). The same was found when we analysed tongue keratinocyte proliferation (Ronderos et al., 1999). We have also studied the effect of different soluble fractions of the extract isolated by molecular exclusion gel permeation chromatography on regenerating liver hepatocytes (Ronderos et al., 1999). Three different fractions were analysed. The first one, including microsomal fraction, did not produce any effect. The other two fractions tested gave either a non-statistically significant increment or an inhibitory effect on the mitotic rate of this population. The last one had a molecular weight ranging between 80 and 12 kDa, peaking at a retention time of 25 kDa. In these experiments, treatments were applied before the DNA synthesis curve increased (Echave Llanos et al., 1971), and the sampling period took place 16–24 h after treatment, when the mitotic peak occurs (Surur et al., 1985). The time elapsed between treatments and animal death suggested an inhibitory effect of the mosquito extract on the cell cycle G1/S checkpoint. When the mitotic ratesampling period covered the dark phase of the circadian cycle (i.e. time of maximum DNA synthesis), a statistically significant increment was found in hepatocytes, nephrocytes and tongue keratinocytes of extract recipients. Taking the time elapsed from treatment administration until death into account (from 0 to 12 h), we suggested an effect of the extract at G2/M checkpoint of the cell cycle (Ronderos, 1996). In other experiments, treatments were applied after the DNA synthesis peak occurrence. The results were similar, showing a decrease in mitotic rates in hepatocytes, tongue keratinocytes and enterocytes (Ronderos et al., 1994b). In view of the results obtained on epithelial cell populations with Culex pipiens larvae extracts, and investigating the effect of the extract on other cell populations apart from the epithelial ones, we decided to assay the effect of the mosquito crude extract on the proliferation of human mononuclear cells (MNCs) activated with concanavalin-A (Con-A) (Ronderos et al., 1999). In these experiments the crude extract showed a dual effect, either inhibiting or stimulating lymphocyte proliferation depending of the dose applied. Furthermore, the biological activity of the crude extract was shown to be inactivated after heating at 90C for 30 min, suggesting the involvement of some macromolecule. In the present study we analysed the effect of the dialysed extract, as well as the two soluble fractions which previously proved to have a biological effect
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on the proliferation of human MNCs activated with Con-A in culture. MATERIALS AND METHODS Mosquito larvae extract Third and fourth instar larvae of mosquito Culex pipiens L. (Diptera: Culicidae) were obtained from an artificial colony. Larvae were separated from the colony, washed several times in fresh water to eliminate any breed plate residue, then filtered and weighed for extract preparation. Pooled mosquito larvae were homogenized in a Potter-Dual homogenizer in an ice-bath, and saline solution at 4C was added (2:10). The homogenate was centrifuged at 0C at 14,500g for 20 min in a Sorval RC5C centrifuge (crude extract) or ultracentrifuged at 100,000g with the aim of eliminating the fraction containing microsomes. After centrifugation, the supernatant was recovered and dialysed with a 12 kDa cut-off pore membrane in saline solution overnight at 4C. Mononuclear cells isolation Human heparinized blood was obtained from healthy adult donors. MNCs were isolated by centrifugation on a Ficoll gradient (Histopaque 1077; Sigma, St. Louis, MO, U.S.A.). After washing, cells were resuspended in RPMI medium supplemented with 2 m glutamine, 100 g/ml streptomycine, 100 U/ml penicillin, 2-ME 10 5 (all from Sigma) and 10% fetal bovine serum (Gen-Argentina). They were finally seeded into 96-well plates at a density of 105 cells/well. MNC viability was evaluated by Trypan blue exclusion test. MNC experimental design MNCs were cultured at 37C in a humidified atmosphere of 5% CO2 in air. The different treatments (5–6 wells/treatment) and the mitogen Con-A (5 g/ml) diluted in RPMI, were added simultaneously at the beginning of the culture. To evaluate lymphocyte proliferation, 3H-thymidine (1 Ci/well) were added 72 h after the start of the culture (time of maximum proliferation under Con-A activation) and 4 h before finishing the experiment. Cells were finally recovered on filters by the use of a cell harvester (Titerteck Cell Harvester, Flow Lab, Rockville, MD, U.S.A.). The radioactivity incorporated was measured in a liquid scintillation counter. All experiments were repeated
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at least twice, producing the same results. Consequently, the data presented here are from a representative experiment. Each step in purification included a positive control of the previous state of the extract (i.e. crude extract for dialysis assay; dialysis extract for F1 and F2 assays).
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The previously ultracentrifuged dialysed homogenate was applied to a Superose 6HR 10/30 column, equilibrated and eluted with buffer Tris-HCl 10 m, NaCl 0.15 , pH 8.0 at a flow rate of 0.5 ml/min. The effluent was monitored at 280 nm. The protein-containing fractions were pooled and concentrated by ultrafiltration (Centricom-10, Amicon, Beverly, MA, U.S.A.). The protein contents were determined by the method of Lowry et al. (1951) using bovine serum albumin as standard.
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Statistical analysis Differences were analysed by one-way analysis of variance (ANOVA). For single comparisons, LSD multiple range test was applied. Only differences greater than P<0.05 were considered significant. RESULTS Analysis of MNC viability after mosquito larvae crude extract assay After 72 h of culture under basal conditions, three wells with and three without mosquito larvae crude extract (controls) were recovered. MNC viability was evaluated by Trypan blue exclusion test. There were no differences in cell viability between controls and cells receiving extract, showing no toxic effect of the extract. Effect of the ultracentrifuged supernatant of extract on MNC proliferation The supernatant of the ultracentrifuged crude extract was recovered and assayed on MNC proliferation. Treatments ranging from 1 to 0.001 mg/ml of the supernatant were applied to Con-A-activated MNCs at the beginning of the culture. 3H-thymidine incorporation was evaluated 72 h later (time of maximum proliferation of Con-A-activated MNCs). The results showed, as in previous experiments with crude extract, a dual response of MNCs. Doses from 1 to 0.1 mg/ml caused a decrease of proliferation, while doses
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Fig. 1. Dose-dependent proliferative response of Con-Aactivated MNC to supernatant of the ultracentrifuged mosquito larvae extract. Bars represent mean... 0= Controls without extract; *=statistically significant differences. Crude extract (1 mg/ml) showing an inhibitory effect was also included in the experiment (data not shown).
ranging between 0.01 and 0.001 mg/ml indicated that MNCs were entering S phase of the cycle (Fig. 1). Effect of the dialysed extract on MNC proliferation In order to discard steroids present in insects, mosquito larvae crude extract was dialysed (cut-off pore membrane 12 kDa). A range of doses similar to those used previously were applied. Again, a dual response was obtained, showing an inhibitory effect with the highest dose (0.5 mg/ml) and a stimulatory effect on Con-A-activated MNC proliferation, with a range of doses between 0.01 to 0.001 mg/ml (Fig. 2). Analysis of chromatographic fractions In view of the previous results achieved, we decided to test the soluble fractions obtained by molecular exclusion chromatography. The mosquito larvae crude extract, ultracentrifuged as described above and then dialysed (12 kDa cut-off pore membrane
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Fig. 2. Dose-dependent proliferative response of Con-Aactivated MNC to dialysed mosquito larvae extract. Bars represent mean... 0=Controls without extract; *=statistically significant differences. Crude extract (1 mg/ml) showing an inhibitory effect was also included in the experiment (data not shown).
against saline solution overnight), was applied to a gel permeation Superose 6B column and eluted with Tris-HCl saline buffer. Two main peaks were detected (F1 and F2 in Fig. 3). Effect of soluble fractions isolated by molecular exclusion chromatography on MNC proliferation A similar experimental design was applied to analyse the effect of both chromatographic fractions on MNC proliferation. The F1 assay showed an inhibitory effect only with the highest dose applied (0.5 mg/ml) and a stimulatory effect from doses ranging from 0.05 to 0.01 mg/ml. Doses lower than these had no effect on 3H-thymidine incorporation (Fig. 4). When we performed a dose-response assay using the lowest molecular weight fraction (F2), only an inhibitory effect was found. This effect was not achieved with the highest doses used (0.1 and 0.05 mg/ml), whereas a statistically significant inhibition appeared with doses ranging from 0.01 down to 0.0001 mg/ml (Fig. 5).
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Fig. 3. Chromatographic profile of mosquito larvae extract eluted through a Superose 6 HR 10/30 molecular exclusion column.
Effect of the crude extract on basal MNC proliferation Previous experiments showed that mosquito larvae crude extract has a dual effect on Con-A-activated MNCs: inhibiting or stimulating DNA synthesis depending on the dose applied. As Con-A modifies the biological activity of monocytes, modulating TGF- release (Assoian et al., 1987), we decided to assay the effect of the crude extract on MNC proliferation under basal conditions (i.e. without Con-A). Dose-response treatments ranging from 1 to 0.01 mg/ml of mosquito larvae crude extract were applied to MNCs under basal conditions at the beginning of the culture. After 72 h, MNC proliferation was evaluated by 3H-thymidine incorporation. Contrary to Con-A-activated conditions,
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Fig. 4. Dose-dependent proliferative response of Con-Aactivated MNC to F1 soluble fraction obtained by molecular exclusion chromatography of mosquito larvae extract after ultracentrifugation and dialysis. Bars represent mean... 0=Controls without extract; *=statistically significant differences. Dialysed extract (0.5 mg/ml) showing an inhibitory effect was also included in the experiment (data not shown).
results showed only an inhibitory effect of mosquito larvae extract at the range of doses applied, which were the same as those used for the Con-A-activated MNC assay (Fig. 6).
DISCUSSION As shown above, a statistically significant alteration in the proliferation process of MNCs appears after treatment with both mosquito larvae dialysed extract and soluble fractions obtained by the use of molecular exclusion chromatography. Both dialysed extract and the soluble fraction with the highest molecular weight (F1) cause a biphasic response of MNCs, inhibiting and stimulating cell proliferation depending on the dose applied. The F2 fraction, with the lowest molecular weight within the range 10–80 kDa, and a maximum relative absorbance (at 280 nm) which peaks at a molecular weight level of 25 kDa (Ronderos et al., 1999), only has an inhibitory effect. Surprisingly,
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Fig. 5. Dose-dependent proliferative response of Con-Aactivated MNC to F2 soluble fraction obtained by molecular exclusion chromatography of mosquito larvae extract after ultracentrifugation and dialysis. Bars represent mean... 0=Controls without extract; *=statistically significant differences. Dialysed extract (0.5 mg/ml) showing an inhibitory effect was also included in the experiment (data not shown).
this effect is not evident with the highest doses applied (0.1 and 0.05 mg/ml), but it is significant with doses as low as 0.0001 mg/ml. Previous experiments demonstrated that the biological effect of the dialysed extract disappeared when it was heated at 90C for 30 min, suggesting that some protein is involved in the observed results (Ronderos et al., 1999). As proliferation on MNC in the present study was measured by 3H-thymidine incorporation, the results indicate, as in previous studies, an effect of the extract on G1/S interphase of the cell cycle (Ronderos and Echave Llanos, 1990; Ronderos et al., 1994a; Ronderos et al., 1996). Looking for messengers which can regulate cell proliferation, both TGF-1, which modulates cell proliferation acting on late G1 phase cyclindependent kinases (Reddy et al., 1994), and other members of the superfamily appear as possible candidates to be present in our extract. They have been suggested as mediators for the regulation of DNA synthesis and differentiation in epithelial, as well as lymphocyte populations (Ahuja et al., 1993; Altiok et al., 1994; De Jong et al., 1994; Fox et al.,
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Members of this growth factor superfamily have also been linked to the regulation of endocrine function, particularly with respect to pituitary hormones (Jin et al., 1997; De et al., 1996; Murata and Ying, 1991; Pastorcic et al., 1995). We have found that the dialysed extract from mosquito larvae also has a specific modulatory effect on rat pituitary hormone release. It inhibits both prolactin and GH, and stimulates LH and TSH release (unpublished results). Finally, our results suggest the existence of some known or unknown peptidic molecule in Culex pipiens L. mosquito larvae, modulating lymphocyte proliferation behaviour in humans.
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ACKNOWLEDGEMENTS 100
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Thanks are due to Mrs Gabriela Simonetto for her skilful technical assistance and to the members of the Insect Pathology Laboratory (CEPAVE-UNLP) for mosquito larvae supply. 0
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Fig. 6. Dose-dependent proliferative response MNC in basal conditions (without Con-A) to crude mosquito larvae extract. Bars represent mean... 0=Controls without extract; *=statistically significant differences.
1992, 1993; Lee and Rich, 1993; Reinhold et al., 1994; Ying and Becker 1995; Kay and Fausto, 1997). Experiments tending to confirm the presence of TGF-1 in the mosquito larvae F2 fraction are currently in progress. The behaviour of the MNCs was different according to whether treatments included lectin activation or not. The dose-response assay of the crude extract under basal conditions showed only an inhibitory response. As we have shown, the same treatment induced a biphase effect on Con-Aactivated MNC proliferation. Furthermore, in our experiments, the rate of lymphocyte proliferation inhibition changes in basal and lectin activated assays, being greater in the latter. Assoian et al. (1987) reported that lectins do not affect TGF- mRNA expression but increase its secretion by peripheral blood monocytes and alveolar macrophages. Thus, the TGF- released by monocytes could synergise the inhibitory effect of the extract. With respect to the biphase effect, a similar response has been described for several other messengers, which can elicit opposite effects on the same cell population depending on the concentration used (Jin et al., 1997; Moses et al., 1990; Qian et al., 1996).
REFERENCES A SS, P F, Y H, B JE, B DT, 1993. Effect of transforming growth factor- on early and late activation events in human T cells. J Immunol 150: 3109–3118. A A, J M, M T, K E, 1994. Biphasic effect of transforming growth factor- on EpsteinBarr virus-induced activation of human tonsillar B cells. Immunol Letts 40: 111–115. A RK, F BE, S HC, M PJ, M DK, R EW, R R, S MB, 1987. Expression and secretion of type transforming growth factor by activated human macrophages. Proc Natl Acad Sci USA 84: 6020–6024. D A, M TE, S RC, B N, S DK, 1996. Pituitary lactotrope express transforming growth factor (TGF) type II receptor mRNA and protein and contains 125I-TGF1 binding sites. J Endocrinol 149: 19–27. D J R, V L RAW, R FW, S C, D P, M MD, 1994. Differential effect of transforming growth factor-1 on the activation of human naive and memory CD4 + T lymphocytes. Intl Immunol 6: 631–638. D JS, J PD, R KE, V M, H FM, 1992. Sequence, biochemical characterization, and developmental expression of a new member of the TGF- superfamily in Drosophila melanogaster. Dev Biol 151: 491–505. E L JM, D’A M, S M, B R, S JM, 1971. Circadian variations in DNA synthesis, mitotic activity and cell size of hepatocyte population of young immature male mice growing liver. Virchows Archiv Abt B Zellpath 8: 309–317. F FE, C RJ, F HC, L RJ, M JS, N PC, 1992. Transforming growth factor- inhibits
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human T-cell proliferation through multiple targets. Lymph Cyt Res 11: 299–305. F FE, F HC, D R, C S, N PC, 1993. Evidence that TGF- can inhibit human T-lymphocyte proliferation through paracrine and autocrine mechanisms. Cell Immunol 150: 45–58. F A, K D, O E, 1996. Immunocytochemical evidence of PDGF- and TGF--like molecules in invertebrate and vertebrate immunocytes an evolutionary approach. Histochem J 28: 599–603. J L, Q X, K E, S N, S BW, K K, Y WF J, L RV, 1997. Transforming growth factor- receptor II and p27Kip1 expression in nontumorous and neoplastic human pituitaries. Amer J Pathol 151: 509–519. K MA, F N, 1997. Liver regeneration: prospects for therapy based on new technologies. Mol Med Today 3: 108–115. K D, S D, F A, O E, 1998. PDGF and TGF- induce cell shape changes in invertebrate immunocytes via specific cell surface receptors. EJCB 75: 362–366. L HM, R S, 1993. Differential activation of CD8-positive T cells by transforming growth factor-1. J Immunol 151: 668–677. L OH, R NJ, F AL, R RJ 1951. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275. M J, 1990. The transforming growth factor- family. Annu Rev Cell Biol 6: 597–641. M HL, Y EY, P JA, 1990. TGF- Stimulation and inhibition of cell proliferation: new mechanistic insights. Cell 63: 245–247. M T, Y S, 1991. Transforming growth factor- and activin inhibit basal secretion of prolactin in a pituitary monolayer culture system. Proc Soc Exp Biol Med 198: 599–605. M AT, H FM, 1990. Homologous of vertebrate growth factors in Drosophila melanogaster and other invertebrates. Curr Top Dev Biol 24: 289–328. P RW, S J RD, G WM, 1987. A transcript from Drosophila pattern gene predicts a protein homologous to the transforming growth factor- family. Nature 325: 81–84. P M, D A, B N, V W, S DK, 1995. Reduction in the expression and action of transforming growth factor 1 on lactotropes during estrogeninduced tumorigenesis in the anterior pituitary. Cancer Res 55: 4892–4898. P A, S SB, H FM, 1997. Regulation of cell cycle synchronisation by decapentaplegic gene during Drosophila eye development. Science 275: 203–206.
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Q X, J L, G JP, L RV, 1996. Transforming growth factor- and p27 expression in pituitary cells. Endocrinology 137: 3051–3060. R KB, H BA, H PH, 1994. Inhibition of G1 phase cyclin dependent kinases by transforming growth factor- 1. J Cell Biochem 56: 418–425. R D, B U, B F, L U, U AJ, F HD, A S, 1994. Transforming growth factor-1 (TGF-1) inhibits DNA synthesis of PWM-stimulated PBMC via suppression of IL-2 and IL-6 production. Cytokine 6: 382–388. R P, L C, J R, A PS, P D, R DL, 1996. Control of C. elegans larval development by neuronal expression of a TGF- homologue. Science 274: 1389–1391. R JR, E L JM, 1990. Inhibitory effect of mosquito larvae extract on the mitotic rate of hepatocytes of young growing and adult partially hepatectomised mice. Com Biol 9: 133–142. R JR, S AO, A CA, E L JM, 1994a. Inhibition of mouse parenchymal cells proliferation by mosquito larvae extract. Cell Mol Biol 40: 1183–1188. R JR, O A, S AO, E L JM, 1994b. Further action of mosquito larvae extract on the mitotic rate of mouse cell populations. BioCell 18: 51–57. R JR, 1996. Early effect of mosquito larvae extract on mouse cells proliferation in vivo. Cell Biol Intl 20: 249–254. R JR, R FL, O A, 1996. Mosquito larvae extract induces mitotic rate alterations of mice tongue keratinocytes along a circadian rhythm period. Cell Biol Intl 20: 567–572. R JR, R O, S MA, B RR, 1999. Partial characterization of mosquito larvae extract modulating mouse and human cell proliferation. Cell Biol Intl 23: 219–226. S JM, M FR, B AF, E L JM, 1985. Variations in DNA synthesis and mitotic indices in hepatocytes and sinusoid litoral cells of adult intact male mouse along a circadian time span. Chronobiol Intl 2: 161–168. T X, F AL, P RW, 1994. The Drosophila saxophone gene: a serine-threonine kinase receptor of the TGF- superfamily. Science 263: 1756–1759. T W, 1937. Cell size in relation to the growth and metamorphosis of the mosquito, Aedes aegypti. J Exp Zool 76: 467–489. Y SY, B A, 1995. Inhibin and activin modulate transforming growth factor--induced immunosuppression. J Biomed Sci 2: 237–241.