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Cytotoxic effect of prolamin-derived peptides on in vitro cultures of cell line Caco-2: Implications for coeliac disease
Pergamon
0887-2333@4)002124
Toxic.in VifroVol. 9, No. 3, pp. 251-255,1995 Copyright 0 1995ElsevicrScienceLtd Printed in Great Britain. All rights reserved 0887-2333/9589.50+ 0.00
Cytotoxic Effect of Prolamin-derived Peptides on In V&o Cultures of Cell Line Caco-2: Implications for Coeliac Disease C. GIOVANNINI,
L. MAIURI* and M. DE VINCENZIt
Department of Metabolism and Pathological Biochemistry, Istituto Superiore di SanitP, Viale Regina Elena 299, 00161 Rome and *University Federico II of Naples, Via S. Panzini 5, 80131 Naples, Italy (Accepted
29 November
1994)
Abstract-The cytotoxic effects of various prolamin-derived peptides’on Caco-2 cells were investigated by measuring the alterations of several parameters at different stages of cell differentiation. The PT digest of bread wheat was active in inhibiting cell proliferation (by about 50%). whereas the other digests from durum wheat, maize and bovine serum albumin (BSA) did not affect the proliferating activity of cells. Compared with the control, colony-forming ability was inhibited by 20% by treatment with cereals that are toxic in coeliac disease (bread wheat, rye, oats and barley). BSA and maize peptides are devoid of this in vifroeffect. However, the decrease in alkaline phosphatase activity during Caco-2 cell differentiation was observed in the presence of bread wheat. This could be due to slowing down of the enterocytic differentiation of cells that are susceptible to interaction with toxic peptides. Therefore, long-term cultures of Caco-2 cells constitute a useful in vitro model to assess the ability of cereal proteins to damage the coeliac small intestine.
INTRODUCTION
The mechanism by which wheat gliadins produce damage to the coeliac small intestine is still unknown. Most investigators favour the hypothesis that deregulated immune responses to gliadin underlie the abnormality to coeliac disease (CD) patients (Strober, 1979). However, non-immunomediated cytotoxic activity of gliadin peptides on the small intestine and other cells might also be involved in the pathogenesis of mucosal damage, and evidence exists to support this hypothesis. Gliadin peptides reversibly inhibited development and morphogenesis of the very immature small intestine of 17-day-old rat foetuses, whereas they had no effect on in vitro cultured differentiated jejunum from 21-day-old rat foetuses. This therefore suggests that gliadin peptides may directly damage the small intestine mucosa only during an early phase of its morphogenesis (Auricchio et al., 1982; de Ritis et al., 1979). Another biological activity of gliadin peptides that is probably related to their toxicity for CD patients is their ability to agglutinate undifferentiated human K 562 cells, a line of chronic myeloid leukaemia (Auricchio et al., 1985).
tAuthor for correspondence. Abbreviations: AP = aDica1; BL = basolateral; BSA = bovine serum albumin; CD = coeliac disease; DMEM = Dulbecco’s modified Eagle’s medium; PBS = phosphate buffered saline.
The culture of organs from untreated CD patients has also been proposed as an in vitro model of CD (Falchuck et al., 1974; Jos et al., 1975). Gliadin peptides are able to inhibit the improvement of intestinal epithelium after in vitro culture. Both nonimmunomediated cytotoxic activity and immunological mechanisms could be responsible for this phenomenon. These three in vitro models have been used not only to study the mechanisms underlying the coeliac lesion, but also to identify toxic proteins and peptides. In in vivo and/or in vitro studies on the coeliac intestine, the toxic activity of a large series of proteins, mixtures of peptides and pure A-gliadin peptides appears to correlate very well with the cell agglutinating activity and the damaging effect on the in vitro developing foetal rat intestine. This suggests that unknown mechanism(s) related to the direct cytotoxic activity of gliadin peptides on undifferentiated cells may be involved in the pathogenesis of the intestinal lesion in CD. In this study we investigated the potentiality of another in vitro system as a possible model to study the mechanism of toxicity in CD-the long-term culture of Caco-2 cells, a line derived from human colon adenocarcinoma, which is capable of expressing some differentiated characteristics of small intestine enterocytes. The cytotoxic effects of various prolamin-derived peptides have been detected on Caco-2 cells by measuring the alterations of several parameters at different stages of cell differentiation. 251
252
C. Giovannini et al. MATERIALS AND METHODS
Caco -2 cell cultures The Caco-2 cells were a gift from Dr J. Sambuy of the Istituto Nazionale della Nutrizione, Italy. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Bio Whittaker, Walkersville, MD, USA) with 4.5 g glucose/litre, supplemented with I % (v/v) non-essential amino acids (Flow), 0.58 mg L-glutamine/ml (Flow), 5 U penicillin/ml and 5 pg streptomycin/ml (Flow) and 10% (v/v) foetal bovine serum (Flow), and were cultured at 37°C in a humidified atmosphere of 5% CO? in air. The cells were seeded at a density of 3 x lo5 in 25 cm* tissue culture flasks (Falcon) and routine renewal of cell stocks was carried out twice a week by removing cells with a solution containing 0.25% (w/v) trypsin and 0.02% (w/v) EDTA, in calcium-free and magnesiumfree phosphate buffered saline solution (PBS-). For each experiment, cells were grown in inserts (25 mm diameter) composed of a cyclopore polyester membrane (Falcon) (pore size 0.45 pm). These chambers were inserted into wells of six-well plates (Falcon). In this type of arrangement, the growing environment was divided into two compartmentsthe apical (AP) and the basolateral (BL) compartment-separated by a polyester membrane. This culture system simulates an in air10 situation, with the upper surface corresponding to the luminal enterocyte surface and the lower surface corresponding to the compartment that is in contact with extracellular matrix and vasa. The polyester membrane is permeable to the medium components and metabolites. However, when cells reach confluency, they start to differentiate, producing a tight impermeable monolayer that prevents passive diffusion of compounds from the apical to the basolateral compartment (Fig. 1). Cells were seeded at 6 x IO4 per insert, 2 ml culture medium were placed on the membrane and 3 ml of the same medium were placed under the membrane in the well of the culture plate. The cells were kept on filters for up to 23 days. Cereals and reagents Pure hexaploid wheat (Triticum aestivum, var. S. Pastore), tetraploid wheat (Triticum durum, var. Adamello), maize (Zea mays, var. B. 73) barley (Hordeum vulgare, var. Arma), rye (Secale cereale. var. 500-2), and oats (Avena sativa, var. Astra) were kindly supplied by the Istituto Sperimentale per la Cerealicoltura in Rome. Prolamins and their digest Prolamine fractions were extracted as described previously by Auricchio et al. (1982). The protein fractions were submitted to peptic-tryptic sequential digestion according to de Ritis (de Ritis et al., 1979) to obtain the corresponding PT digest. The peptides were heated for 30min at 100°C.
6
B _ e
FL E 7.0
-
8 a 6.0 In .f 5.0 -
-
.t ,; 4.0 -
I.0 ‘M
E
s 5 3.0 2
2 2.0 z 5
10
15
20
25
Time in culture (days) Fig. I. Caco-2 differentiation. To determine [3H]mannitol permeability (O), cells were seeded in membrane inserts, and were exposed to 0.5 mCi/ml in the AP compartment of the chambers for 2 hr. The radioactivity present in the AP compartment and in the BL compartment were then counted and the results expressed as a percentage of the radioactivity found in the BL compartment compared with that found in the AP compartment on different days of culture. Each point represents the mean of two experiments performed in duplicate. For assessment of alkaline phosphatase activity (0) the enzyme activity on different days of culture was determined using a calorimetric method and expressed as IU/mg protein per dish. Each curve point represents the mean of three experiments performed in duplicate f SEM. Proliferation of undifferentiated cells 4 hr after seeding, the cells were exposed ent cereal PT digests, final concentration medium, and incubated for 24, 48, 72 and the end of the incubations the cells were with trypsin and EDTA and counted using counter.
to differ1 mg/ml 96 hr. At detached a Coulter
Viability test At confluence (day 7 of culture), the medium was removed and fresh medium containing 1 mg/ml bread wheat PT digest was added. After 48 hr the cells were washed twice with PBS, solubilized in 1 ml of 0.1 M NaOH and their total protein content determined by the method of Lowry et al. (1951). Colony-forming ability For the colony-forming ability studies, the cell cultures were detached and subcultivated in 60 x 15 mm tissue culture dishes with 500 cells per dish for 14 days; the medium was renewed once, after 1 wk. The colonies produced were subsequently fixed with methyl alcohol, stained with methylene blue and the number of visible colonies scored. Enzyme activity For the alkaline phosphatase assay, on day 5 of culture the medium was removed and fresh medium containing 1 mg bread wheat PT digest/ml was added and renewed every 2 days. At different days of
Cytotoxic activity of cereals on Caco-2 cells culture, the cells were dissolved in a solution containing 0.25% sucrose and 1% deoxycholate. The enzyme activity was assayed according to a calorimetric method (Bessey er al., 1946) using an assay kit from Boehringer Mannheim GmbH diagnostica.
253
*
1
I
Permeability
Mannitol permeability was performed using [3H]mannitol (NEN; sp. act.: 30 Ci/mmol), 0.5 pCi/ ml, added to the apical compartment of the cell culture system. After 2 hr of incubation, radioactivities in the apical and in the basal compartments were counted in a liquid scintillation spectrometer. RESULTS
In order to standardize and optimize experimental conditions, the cell line was characterized as described below. The growth curve was obtained both by counting the cell number and determining the protein content per dish at different days in culture. The resulting curves (data not shown) are similar: the experimental growth phase continues until day 7 in culture; subsequently a plateau, Go phase, is reached and this is maintained thereafter. In order to study cellular differentiation, we considered two parameters-tight junction formation and alkaline phosphatase activity (Fig. 1). The tight junctions formed were measured by [3H]mannitol permeability. The results were expressed as the percentage of radioactivity found in the basolateral
24
48
72
96
Exposure period (hr) Fig. 3. Cytotoxic effects of prolamine-derived peptides from bread wheat determined as colony-forming ability. Caco-2 cells after day 19 of culture were exposed to 1mg PT digest/ml for 24, 48, 72 and 96 hr. Cells were subsequently detached and re-seeded at low density (500 cells per 60 mm dish). After 14 days, the culture medium was removed and colonies were stained. The columns represent the percentage of visible colonies found in treated Caco-2 cells compared with controls. Values are of five experiments performed in duplicate (+ SEM). Asterisks indicate significant differences from controls (*P < 0.05; **P< 0.01; Student’s f-test).
**
1050
24
48
12
96
Exposure period (hr) Fig. 2. Effect of cereal peptide derivatives on undifferentiated Caco-2 cells. 4 hr after seeding, cells were exposed to 1 mg different cereal PT-digests (0, durum wheat, var. Adamello; 0, maize; n , bread wheat, var. S. Pastore) for 24-96 hr (0, control; A, BSA). The curves were obtained from the means of three experiments performed in duplicate.
1Barley
Rye
Oats
Durum wheat
Fig. 4. Colony-forming ability of Caco-2 cells exposed to 1 mg cereal prolamine-derived peptides/ml. Each column represents the mean percentage of colonies found after 96 hr treatments compared with control data. Values are means of three experiments performed in duplicate (k SEM). Asterisks indicate significant differences from controls (*P< 0.05; **Pc 0.01; Student’s r-test).
C. Giovannini el al.
254
compartment compared with that found in the apical compartment on different days of culture. We observed that tight junction forming is a process that depends on the day of culture (the lowest value of radioactivity was reached after day 18 of culture). Cell differentiation was also studied by measuring alkaline phosphatase activity (Fig. 1). This parameter was expressed as IU/mg protein and increased with time until reaching a maximum on day 18 of culture, in agreement with results obtained also for tight junction formation. The effect of prolamin-derived peptides from cereals such as bread wheat, durum wheat, maize, rye, oats and barley were investigated on several cellular endpoints of Caco-2 cells at different times of culture. In actively proliferating cells, the PT protein digest of durum wheat, maize and BSA were inactive in inhibiting the Caco-2 cell proliferation. Otherwise. a growth inhibition close to 50% was observed in the presence of bread wheat gliadin peptides (Fig. 2). In addition, durum wheat peptides, only when tested at high concentration (2 mg/ml) are able to exert a growth inhibition close to 15%. At confluency (day 7 of culture), exposure for 48 hr to bread wheat gliadin peptides reduced viability, determined as mg protein per dish compared with controls. by about 30% (data not shown). Unlike other cell lines, which lose their proliferating ability after differentiation, if they are reseeded at low density differentiated Caco-2 cells have the peculiarity of being able to start to proliferate. In this way, cytotoxicity can also be studied on differentiated cells using normal cloning techniques. Differentiated cells were exposed to bread wheat PT peptides for 24,
14
Time in culture
I8
(days)
Fig. 5. Effect of prolamine-derived peptides from bread wheat (0) and durum wheat (8) on alkaline phosphatase activity on days 9, 14 and 18 of culture, compared with controls (D) on the same days of culture. Enzyme activity is expressed as IU/mg protein per dish. Each point is obtained from averages of two different experiments in triplicate. Bars represent SEM. Asterisks indicate significant differences from the controls (*P < 0.05: **P < 0.01; Student’s r-test).
48, 72 and 96 hr and then detached, counted and seeded at low density to measure the colony-forming ability. The results are reported in Fig. 3. We observed that bread wheat-derived peptides caused a 20% inhibition of colony-forming ability as compared with the control. In Fig. 4 the effects of all the cereal-derived peptides tested on the colony-forming ability are reported and compared at the end of the exposure period (96 hr). It is possible to see that, whereas durum wheat has no effect, all the other cereals (rye, oats and barley) have a very similar cytotoxic effect, which is comparable with that exerted by bread wheat. Moreover, we observed that total alkaline phosphatase activity was reduced in Caco-2 cells treated with bread wheat gliadin peptides but not in the cells exposed to gliadin peptides from durum wheat. The results (Fig. 5) showed that there was a significant difference between cells treated with bread wheat and control cells. This seems to indicate that there is a delay of about 4 days in the differentiating process of the cells treated with bread wheat. DlSClJSSlON
An in vitro biological activity of gliadin peptides has been previously demonstrated in different experimental models: gliadin peptides, as well as prolamin peptides from cereal toxic to the coeliac small intestine, are able to inhibit the in vitro differentiation of foetal rat intestine (Auricchio et al., 1984a). to damage in vitro the intestinal mucosa from untreated coeliac patients (Auricchio et al., 1982), and to agglutinate undifferentiated K 562 (S) cells (Auricchio et al., 1984b). Conversely, prolamin peptides from rice and maize, which are non-toxic for the coeliac intestine. are devoid of such an in vitro effect (Auricchio et al., 1984a). The relationship between this in vitro effect and the mechanism underlying the coeliac lesion is far from being completely understood. It probably reflects only some properties of gliadin peptides related to their capacity to induce damage in CD intestinal mucosa. Whatever the mechanism, these in vitro models have been demonstrated to be useful for the identification of cereals and cereal proteins toxic to the coeliac intestine. In the study reported here we have shown that the long-term culture of the Caco-2 cell line is a further in vitro model to identify cereals toxic to the intestinal mucosa of CD patients. Caco-2 cells undergo full differentiation with enterocyte-like features, both structurally (microvilli, tight junctions) and functionally (brush border-associated enzymes, capacity of transport across the surface membranes) (Matsumoto et al., 1990; Pinto et al., 1989; Rousset, 1986). Moreover, these cells are able to maintain, even at late stages of differentiation, some features that are characteristic of foetal colonic cells (Grasset et al., 1984). We have demonstrated that a PT digest of
Cytotoxic activity of cereals on Caco-2 cells gliadin from bread wheat is able to inhibit Caco-2 cell proliferation and this effect is evident in undifferentiated and actively proliferating cells. However, cell viability experiments clearly show that PT digest of bread wheat is cytotoxic, both for cells at confluency and differentiated cells. A delayed differentiation time has been demonstrated, as well as a significant decrease of alkaline phosphatase activity after longterm exposure to gliadin peptides. Moreover, gliadin peptides are able to induce inhibition of the colonyforming ability of Caco-2 cells reseeded at low density after differentiation. This effect seems to be specific for gliadin peptides and for prolamin peptides derived from cereals that are toxic to CD patients. Rye, barley and oats have a very similar cytotoxic activity, which is comparable with that obtained with bread wheat, whereas BSA and maize-derived prolamin are devoid of such an in vitro effect. Because of the very good correlation between the toxicity to the coeliac intestine and the ability to exert such in vitro adverse effects of different cereal prolamin peptides, this model may be put forward as a new test to identify cereals and cereal proteins toxic to the intestine in CD. This model may be useful not only to identify putative toxic amino acid sequences of gliadin but also to facilitate understanding of the mechanism by which gliadin peptides are able to exert a direct cytotoxic effect on the small intestine enterocytes of CD patients. Acknowledgements-We thank Mrs Francesca Maialetti for excellent technical assistance in biochemical preparations.
REFERENCES
Auricchio S., Cardelli M.. de Ritis G. and De Vincenzi M. (1984a) An in vitro animal model for the study of cereal components toxic in celiac disease. Pediatric Research 18, 1372-1378. Auricchio S., de Ritis G., De Vincenzi M., Mancini E., Minetti M., Sapora 0. and Silano V. (1984b) Agglutinating activity of gliadin derived peptides from bread wheat: implication for celiac disease pathogenesis. Biochemical and Biophysical Research Communications 121, 428-433.
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Auricchio S., de Ritis G., De Vincenri M., Ckcorsio P. and Silano V. (I 982) Effectsof gliadin peptides prepared from hexaploid and tetraploid wheat on cultures of intestine from rat foetuses and coeliac children. Pediatric Research 16, 1004-1010. Auricchio S., de Ritis G., De Vincenzi M. and Silano V. (1985) Toxicity mechanisms of wheat and other cereals in celiac disease and related enteropathies. JournaI of Pediatric Gastroeneterology and Nutrition 4, 923-930.
Bessey 0. A., Lowry 0. H. and Brock M. J. (1946) A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum, Journal of Biological Chemistry 164, 321-329. de Ritis G., Occorsio P., Auricchio S., Gramenzi F., Morisi G. and Silano V. (1979) Toxicity of wheat flour proteins and protein-derived peptides for in vitro developing intestine from rat fetus. Pediatric Research 13, 1255-1261.
Falchuck Z. M., Gebhard R. L., Sessoms C. and Strober W. (1974) An in virro model of gluten sensitive enteropathy. Journal of Clinical Invesligation 53, 487-500.
Grasset E., Pinto M., Dussaulx E., Zweibaum A. and Desjeux J.-F. (1984) Epithelial properties of human colonic carcinoma cell Caco-2: electrical parameters. American Journal of Physiology 247, C2604267.
Jos J., Lenoir G. and Rey J. (1975) In vitro pathogenetic studies of coeliac disease. Effect of protein on coeliac intestinal biopsy specimens maintained in culture for 48 hours. Scandinavian Journal of Gastroenterology 10, 121-128. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, 265-275. Matsumoto H., Erickson R. H., Fum J. R., Yoshioca M., Gum E. and Kim Y. S. (1990) Biosynthesis of alkaline phosphatase during differentiation of the human colon cancer cell line Caco-2. Gastroenterology 98, 1199-1207. Pinto M., Robine-Leon S., Appay M. D., Kedinger M., Triadou N., Dussaulx E., Lacroix B., Simon-Asmann P., Haffen K., Fogh J. and Zweibaum A. (1983) Enterocytelike differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biology of the Cell 41, 323-330.
Rousset M. (1986) The human colon carcinoma cell lines HT-29 and Caco-2: two in vitro models for the study of intestinal differentiation. Biochemie 68, 1035-1040.
Strober W. (1979) An immunological theory of gluten-sensitive enteropathy. In Perspectives in Coeliac Disease. Edited by B. McNichol, C. F. McCarthy and P. F. Fottrel. pp. 156-169. MTP Press, Lancaster.
0887-2333@4)002124
Toxic.in VifroVol. 9, No. 3, pp. 251-255,1995 Copyright 0 1995ElsevicrScienceLtd Printed in Great Britain. All rights reserved 0887-2333/9589.50+ 0.00
Cytotoxic Effect of Prolamin-derived Peptides on In V&o Cultures of Cell Line Caco-2: Implications for Coeliac Disease C. GIOVANNINI,
L. MAIURI* and M. DE VINCENZIt
Department of Metabolism and Pathological Biochemistry, Istituto Superiore di SanitP, Viale Regina Elena 299, 00161 Rome and *University Federico II of Naples, Via S. Panzini 5, 80131 Naples, Italy (Accepted
29 November
1994)
Abstract-The cytotoxic effects of various prolamin-derived peptides’on Caco-2 cells were investigated by measuring the alterations of several parameters at different stages of cell differentiation. The PT digest of bread wheat was active in inhibiting cell proliferation (by about 50%). whereas the other digests from durum wheat, maize and bovine serum albumin (BSA) did not affect the proliferating activity of cells. Compared with the control, colony-forming ability was inhibited by 20% by treatment with cereals that are toxic in coeliac disease (bread wheat, rye, oats and barley). BSA and maize peptides are devoid of this in vifroeffect. However, the decrease in alkaline phosphatase activity during Caco-2 cell differentiation was observed in the presence of bread wheat. This could be due to slowing down of the enterocytic differentiation of cells that are susceptible to interaction with toxic peptides. Therefore, long-term cultures of Caco-2 cells constitute a useful in vitro model to assess the ability of cereal proteins to damage the coeliac small intestine.
INTRODUCTION
The mechanism by which wheat gliadins produce damage to the coeliac small intestine is still unknown. Most investigators favour the hypothesis that deregulated immune responses to gliadin underlie the abnormality to coeliac disease (CD) patients (Strober, 1979). However, non-immunomediated cytotoxic activity of gliadin peptides on the small intestine and other cells might also be involved in the pathogenesis of mucosal damage, and evidence exists to support this hypothesis. Gliadin peptides reversibly inhibited development and morphogenesis of the very immature small intestine of 17-day-old rat foetuses, whereas they had no effect on in vitro cultured differentiated jejunum from 21-day-old rat foetuses. This therefore suggests that gliadin peptides may directly damage the small intestine mucosa only during an early phase of its morphogenesis (Auricchio et al., 1982; de Ritis et al., 1979). Another biological activity of gliadin peptides that is probably related to their toxicity for CD patients is their ability to agglutinate undifferentiated human K 562 cells, a line of chronic myeloid leukaemia (Auricchio et al., 1985).
tAuthor for correspondence. Abbreviations: AP = aDica1; BL = basolateral; BSA = bovine serum albumin; CD = coeliac disease; DMEM = Dulbecco’s modified Eagle’s medium; PBS = phosphate buffered saline.
The culture of organs from untreated CD patients has also been proposed as an in vitro model of CD (Falchuck et al., 1974; Jos et al., 1975). Gliadin peptides are able to inhibit the improvement of intestinal epithelium after in vitro culture. Both nonimmunomediated cytotoxic activity and immunological mechanisms could be responsible for this phenomenon. These three in vitro models have been used not only to study the mechanisms underlying the coeliac lesion, but also to identify toxic proteins and peptides. In in vivo and/or in vitro studies on the coeliac intestine, the toxic activity of a large series of proteins, mixtures of peptides and pure A-gliadin peptides appears to correlate very well with the cell agglutinating activity and the damaging effect on the in vitro developing foetal rat intestine. This suggests that unknown mechanism(s) related to the direct cytotoxic activity of gliadin peptides on undifferentiated cells may be involved in the pathogenesis of the intestinal lesion in CD. In this study we investigated the potentiality of another in vitro system as a possible model to study the mechanism of toxicity in CD-the long-term culture of Caco-2 cells, a line derived from human colon adenocarcinoma, which is capable of expressing some differentiated characteristics of small intestine enterocytes. The cytotoxic effects of various prolamin-derived peptides have been detected on Caco-2 cells by measuring the alterations of several parameters at different stages of cell differentiation. 251
252
C. Giovannini et al. MATERIALS AND METHODS
Caco -2 cell cultures The Caco-2 cells were a gift from Dr J. Sambuy of the Istituto Nazionale della Nutrizione, Italy. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Bio Whittaker, Walkersville, MD, USA) with 4.5 g glucose/litre, supplemented with I % (v/v) non-essential amino acids (Flow), 0.58 mg L-glutamine/ml (Flow), 5 U penicillin/ml and 5 pg streptomycin/ml (Flow) and 10% (v/v) foetal bovine serum (Flow), and were cultured at 37°C in a humidified atmosphere of 5% CO? in air. The cells were seeded at a density of 3 x lo5 in 25 cm* tissue culture flasks (Falcon) and routine renewal of cell stocks was carried out twice a week by removing cells with a solution containing 0.25% (w/v) trypsin and 0.02% (w/v) EDTA, in calcium-free and magnesiumfree phosphate buffered saline solution (PBS-). For each experiment, cells were grown in inserts (25 mm diameter) composed of a cyclopore polyester membrane (Falcon) (pore size 0.45 pm). These chambers were inserted into wells of six-well plates (Falcon). In this type of arrangement, the growing environment was divided into two compartmentsthe apical (AP) and the basolateral (BL) compartment-separated by a polyester membrane. This culture system simulates an in air10 situation, with the upper surface corresponding to the luminal enterocyte surface and the lower surface corresponding to the compartment that is in contact with extracellular matrix and vasa. The polyester membrane is permeable to the medium components and metabolites. However, when cells reach confluency, they start to differentiate, producing a tight impermeable monolayer that prevents passive diffusion of compounds from the apical to the basolateral compartment (Fig. 1). Cells were seeded at 6 x IO4 per insert, 2 ml culture medium were placed on the membrane and 3 ml of the same medium were placed under the membrane in the well of the culture plate. The cells were kept on filters for up to 23 days. Cereals and reagents Pure hexaploid wheat (Triticum aestivum, var. S. Pastore), tetraploid wheat (Triticum durum, var. Adamello), maize (Zea mays, var. B. 73) barley (Hordeum vulgare, var. Arma), rye (Secale cereale. var. 500-2), and oats (Avena sativa, var. Astra) were kindly supplied by the Istituto Sperimentale per la Cerealicoltura in Rome. Prolamins and their digest Prolamine fractions were extracted as described previously by Auricchio et al. (1982). The protein fractions were submitted to peptic-tryptic sequential digestion according to de Ritis (de Ritis et al., 1979) to obtain the corresponding PT digest. The peptides were heated for 30min at 100°C.
6
B _ e
FL E 7.0
-
8 a 6.0 In .f 5.0 -
-
.t ,; 4.0 -
I.0 ‘M
E
s 5 3.0 2
2 2.0 z 5
10
15
20
25
Time in culture (days) Fig. I. Caco-2 differentiation. To determine [3H]mannitol permeability (O), cells were seeded in membrane inserts, and were exposed to 0.5 mCi/ml in the AP compartment of the chambers for 2 hr. The radioactivity present in the AP compartment and in the BL compartment were then counted and the results expressed as a percentage of the radioactivity found in the BL compartment compared with that found in the AP compartment on different days of culture. Each point represents the mean of two experiments performed in duplicate. For assessment of alkaline phosphatase activity (0) the enzyme activity on different days of culture was determined using a calorimetric method and expressed as IU/mg protein per dish. Each curve point represents the mean of three experiments performed in duplicate f SEM. Proliferation of undifferentiated cells 4 hr after seeding, the cells were exposed ent cereal PT digests, final concentration medium, and incubated for 24, 48, 72 and the end of the incubations the cells were with trypsin and EDTA and counted using counter.
to differ1 mg/ml 96 hr. At detached a Coulter
Viability test At confluence (day 7 of culture), the medium was removed and fresh medium containing 1 mg/ml bread wheat PT digest was added. After 48 hr the cells were washed twice with PBS, solubilized in 1 ml of 0.1 M NaOH and their total protein content determined by the method of Lowry et al. (1951). Colony-forming ability For the colony-forming ability studies, the cell cultures were detached and subcultivated in 60 x 15 mm tissue culture dishes with 500 cells per dish for 14 days; the medium was renewed once, after 1 wk. The colonies produced were subsequently fixed with methyl alcohol, stained with methylene blue and the number of visible colonies scored. Enzyme activity For the alkaline phosphatase assay, on day 5 of culture the medium was removed and fresh medium containing 1 mg bread wheat PT digest/ml was added and renewed every 2 days. At different days of
Cytotoxic activity of cereals on Caco-2 cells culture, the cells were dissolved in a solution containing 0.25% sucrose and 1% deoxycholate. The enzyme activity was assayed according to a calorimetric method (Bessey er al., 1946) using an assay kit from Boehringer Mannheim GmbH diagnostica.
253
*
1
I
Permeability
Mannitol permeability was performed using [3H]mannitol (NEN; sp. act.: 30 Ci/mmol), 0.5 pCi/ ml, added to the apical compartment of the cell culture system. After 2 hr of incubation, radioactivities in the apical and in the basal compartments were counted in a liquid scintillation spectrometer. RESULTS
In order to standardize and optimize experimental conditions, the cell line was characterized as described below. The growth curve was obtained both by counting the cell number and determining the protein content per dish at different days in culture. The resulting curves (data not shown) are similar: the experimental growth phase continues until day 7 in culture; subsequently a plateau, Go phase, is reached and this is maintained thereafter. In order to study cellular differentiation, we considered two parameters-tight junction formation and alkaline phosphatase activity (Fig. 1). The tight junctions formed were measured by [3H]mannitol permeability. The results were expressed as the percentage of radioactivity found in the basolateral
24
48
72
96
Exposure period (hr) Fig. 3. Cytotoxic effects of prolamine-derived peptides from bread wheat determined as colony-forming ability. Caco-2 cells after day 19 of culture were exposed to 1mg PT digest/ml for 24, 48, 72 and 96 hr. Cells were subsequently detached and re-seeded at low density (500 cells per 60 mm dish). After 14 days, the culture medium was removed and colonies were stained. The columns represent the percentage of visible colonies found in treated Caco-2 cells compared with controls. Values are of five experiments performed in duplicate (+ SEM). Asterisks indicate significant differences from controls (*P < 0.05; **P< 0.01; Student’s f-test).
**
1050
24
48
12
96
Exposure period (hr) Fig. 2. Effect of cereal peptide derivatives on undifferentiated Caco-2 cells. 4 hr after seeding, cells were exposed to 1 mg different cereal PT-digests (0, durum wheat, var. Adamello; 0, maize; n , bread wheat, var. S. Pastore) for 24-96 hr (0, control; A, BSA). The curves were obtained from the means of three experiments performed in duplicate.
1Barley
Rye
Oats
Durum wheat
Fig. 4. Colony-forming ability of Caco-2 cells exposed to 1 mg cereal prolamine-derived peptides/ml. Each column represents the mean percentage of colonies found after 96 hr treatments compared with control data. Values are means of three experiments performed in duplicate (k SEM). Asterisks indicate significant differences from controls (*P< 0.05; **Pc 0.01; Student’s r-test).
C. Giovannini el al.
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compartment compared with that found in the apical compartment on different days of culture. We observed that tight junction forming is a process that depends on the day of culture (the lowest value of radioactivity was reached after day 18 of culture). Cell differentiation was also studied by measuring alkaline phosphatase activity (Fig. 1). This parameter was expressed as IU/mg protein and increased with time until reaching a maximum on day 18 of culture, in agreement with results obtained also for tight junction formation. The effect of prolamin-derived peptides from cereals such as bread wheat, durum wheat, maize, rye, oats and barley were investigated on several cellular endpoints of Caco-2 cells at different times of culture. In actively proliferating cells, the PT protein digest of durum wheat, maize and BSA were inactive in inhibiting the Caco-2 cell proliferation. Otherwise. a growth inhibition close to 50% was observed in the presence of bread wheat gliadin peptides (Fig. 2). In addition, durum wheat peptides, only when tested at high concentration (2 mg/ml) are able to exert a growth inhibition close to 15%. At confluency (day 7 of culture), exposure for 48 hr to bread wheat gliadin peptides reduced viability, determined as mg protein per dish compared with controls. by about 30% (data not shown). Unlike other cell lines, which lose their proliferating ability after differentiation, if they are reseeded at low density differentiated Caco-2 cells have the peculiarity of being able to start to proliferate. In this way, cytotoxicity can also be studied on differentiated cells using normal cloning techniques. Differentiated cells were exposed to bread wheat PT peptides for 24,
14
Time in culture
I8
(days)
Fig. 5. Effect of prolamine-derived peptides from bread wheat (0) and durum wheat (8) on alkaline phosphatase activity on days 9, 14 and 18 of culture, compared with controls (D) on the same days of culture. Enzyme activity is expressed as IU/mg protein per dish. Each point is obtained from averages of two different experiments in triplicate. Bars represent SEM. Asterisks indicate significant differences from the controls (*P < 0.05: **P < 0.01; Student’s r-test).
48, 72 and 96 hr and then detached, counted and seeded at low density to measure the colony-forming ability. The results are reported in Fig. 3. We observed that bread wheat-derived peptides caused a 20% inhibition of colony-forming ability as compared with the control. In Fig. 4 the effects of all the cereal-derived peptides tested on the colony-forming ability are reported and compared at the end of the exposure period (96 hr). It is possible to see that, whereas durum wheat has no effect, all the other cereals (rye, oats and barley) have a very similar cytotoxic effect, which is comparable with that exerted by bread wheat. Moreover, we observed that total alkaline phosphatase activity was reduced in Caco-2 cells treated with bread wheat gliadin peptides but not in the cells exposed to gliadin peptides from durum wheat. The results (Fig. 5) showed that there was a significant difference between cells treated with bread wheat and control cells. This seems to indicate that there is a delay of about 4 days in the differentiating process of the cells treated with bread wheat. DlSClJSSlON
An in vitro biological activity of gliadin peptides has been previously demonstrated in different experimental models: gliadin peptides, as well as prolamin peptides from cereal toxic to the coeliac small intestine, are able to inhibit the in vitro differentiation of foetal rat intestine (Auricchio et al., 1984a). to damage in vitro the intestinal mucosa from untreated coeliac patients (Auricchio et al., 1982), and to agglutinate undifferentiated K 562 (S) cells (Auricchio et al., 1984b). Conversely, prolamin peptides from rice and maize, which are non-toxic for the coeliac intestine. are devoid of such an in vitro effect (Auricchio et al., 1984a). The relationship between this in vitro effect and the mechanism underlying the coeliac lesion is far from being completely understood. It probably reflects only some properties of gliadin peptides related to their capacity to induce damage in CD intestinal mucosa. Whatever the mechanism, these in vitro models have been demonstrated to be useful for the identification of cereals and cereal proteins toxic to the coeliac intestine. In the study reported here we have shown that the long-term culture of the Caco-2 cell line is a further in vitro model to identify cereals toxic to the intestinal mucosa of CD patients. Caco-2 cells undergo full differentiation with enterocyte-like features, both structurally (microvilli, tight junctions) and functionally (brush border-associated enzymes, capacity of transport across the surface membranes) (Matsumoto et al., 1990; Pinto et al., 1989; Rousset, 1986). Moreover, these cells are able to maintain, even at late stages of differentiation, some features that are characteristic of foetal colonic cells (Grasset et al., 1984). We have demonstrated that a PT digest of
Cytotoxic activity of cereals on Caco-2 cells gliadin from bread wheat is able to inhibit Caco-2 cell proliferation and this effect is evident in undifferentiated and actively proliferating cells. However, cell viability experiments clearly show that PT digest of bread wheat is cytotoxic, both for cells at confluency and differentiated cells. A delayed differentiation time has been demonstrated, as well as a significant decrease of alkaline phosphatase activity after longterm exposure to gliadin peptides. Moreover, gliadin peptides are able to induce inhibition of the colonyforming ability of Caco-2 cells reseeded at low density after differentiation. This effect seems to be specific for gliadin peptides and for prolamin peptides derived from cereals that are toxic to CD patients. Rye, barley and oats have a very similar cytotoxic activity, which is comparable with that obtained with bread wheat, whereas BSA and maize-derived prolamin are devoid of such an in vitro effect. Because of the very good correlation between the toxicity to the coeliac intestine and the ability to exert such in vitro adverse effects of different cereal prolamin peptides, this model may be put forward as a new test to identify cereals and cereal proteins toxic to the intestine in CD. This model may be useful not only to identify putative toxic amino acid sequences of gliadin but also to facilitate understanding of the mechanism by which gliadin peptides are able to exert a direct cytotoxic effect on the small intestine enterocytes of CD patients. Acknowledgements-We thank Mrs Francesca Maialetti for excellent technical assistance in biochemical preparations.
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