- Email: [email protected]
Effects of dietary wheat germ deprivation on the immune system in Wistar rats: a pilot study
International Immunopharmacology 2 (2002) 1495 – 1501 www.elsevier.com/locate/intimp
Effects of dietary wheat germ deprivation on the immune system in Wistar rats: a pilot study Roberto Chignola a,*, Corrado Rizzi a, Simone Vincenzi b, Tiziana Cestari c, Nadia Brutti d, Anna Pia Riviera d, Silvia Sartoris c, Angelo D.B. Peruffo a, Giancarlo Andrighetto c a
Dipartimento Scientifico e Tecnologico, Universita` di Verona, Strada Le Grazie 15, CV1, 37100 Verona, Italy b SOCADO s.r.l. Societa` Alimentare Dolciaria, Via Spagna 20, 37069 Villafranca, Verona, Italy c Dipartimento di Patologia, Sezione di Immunologia, Universita` di Verona, c/o Policlinico G.B. Rossi, 37100 Verona, Italy d Servizio di Immunologia Clinica, Azienda Ospedaliera di Verona, c/o Policlinico G.B. Rossi, 37100 Verona, Italy Received 22 April 2002; received in revised form 6 August 2002; accepted 6 August 2002
Abstract Bioactive molecules that can gain access to body tissues through the gastrointestinal tract may interact with immune regulatory circuits and effector functions. Among these are plant lectins, such as wheat germ (WG) agglutinin, which constitute common components of the human diet and target the immune system on a daily basis. Dietary bioactive molecules might be considered as immunomodulatory signals. To investigate the possible effects on the immune system of the long-term absence of such signals, two groups of rats were fed on a diet containing or deprived of WG. The WG-deprived diet induced a state of functional unresponsiveness in lymphocytes from primary and secondary lymphoid organs, as evaluated by in vitro stimulation with T cell mitogen phytohemoagglutinin (PHA) and B cell mitogen lypopolysaccarides (LPS). The unresponsive state of the immune cells could be reversed by injection of antigen emulsified in oil with inactivated mycobacteria (complete Freund’s adjuvant, CFA) Dietary signals can thus interact with the immune system possibly influencing its shaping during ontogenesis. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Wheat germ; Diet; Plant lectins; Immune system
1. Introduction Several dietary factors are known to affect various functions of the immune system and to interfere with immune regulatory circuits. Among these, the immunobiological activity of carbohydrate-binding proteins
*
Corresponding author. Tel.: +39-045-8027953. E-mail address: [email protected] (R. Chignola).
of vegetable origin (plant lectins) has long been recognized [1,2]. Plant lectins are able to modulate important immune mechanisms, including inflammatory reactions and effector functions [1 –7]. Different diet compositions, which may involve different lectins or different lectin concentrations, have been investigated in terms of their potential influences on human or animal health [1,2,8 –12]. These and other dietary components may target the immune system on a daily basis and hence should be
1567-5769/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 ( 0 2 ) 0 0 11 6 - 9
1496
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
considered as exogenous immunoregulatory signals. Due to the nutritional requirements of living organisms, dietary signals target the immune system during the whole life of an animal. The immune system might therefore be hypothesized to evolve and shape under the continuous stimulation of dietary signals that might add modulatory effects to those elicited by molecules produced by the immune system itself. If so, the absence of dietary immune-targeting signals might result in a different shaping of the immune system and/or in the alteration of some immune functions. To test this hypothesis, a pilot study was conducted with inbred Wistar rats. The rationale for a pilot study involving a relatively small group of rats stems from ethical issues that do not allow us to sacrifice a large number of animals to explore possible, but not probable, biological effects. Rats were fed for two generations with a diet containing wheat germ (WG) or with the same diet deprived of WG. WG was chosen as a model foodstuff interacting with the immune system due to its content in wheat germ agglutinin (WGA), a lectin known to influence several immune functions in vivo and in vitro [2– 6]. As a first general approach, the functional activity of the T- and B-lymphocyte compartments was analysed in the two groups of rats. This pilot study provides positive evidence for the potential role of dietary components in the shaping and/or in the tuning of immune system functions, and therefore provides a justification for more extensive studies on the immune system and its evolution in animals subjected to dietary restriction.
2. Materials and methods 2.1. Animals, diet composition and feeding procedures In order to test the long-term effects of dietary signal deprivation on the immune system, 1-month-old Wistar rats (from our own breeding colony) were fed with WG-deprived diet (see below) and allowed to breed. Newborns were continuously fed with the same diet. WG-deprived and WG-containing diets both contained the following common ingredients selected to provide animals with a balanced nutritional base: pork liver, powdered carrots (New Foods Industry, Verona, Italy),
coconut flour and sugar. These ingredients were mixed in proportion to yield the following formula (percent of wet weight of single components over total wet weight of the diet): proteins 7.8 –8.2%, fat 5.1 –5.7% carbohydrates 22.7 –24.8%. To this basic feedstuff, 15% (w/w) WG or 20% pork liver plus 6% sugar were added to obtain WG-containing or WG-deprived diets, respectively. WG, containing 0.3 F 0.2% (w/w) WGA [13], was used in this pilot study instead of purified WGA to obtain a nutritional condition resembling a normal animal diet. All experiments involving rats were carried out according to current Italian laws (D.L. 116, 1992) in agreement with the directions mandated by 86/609/CEE on animal experimentation. 2.2. Cell preparations and in vitro stimulation assays Spleen, thymus, mesenteric lymphnodes, inguinal lymphnodes and bone marrow were collected from sacrificed 3-month-old rats of the second generation. Cells were washed twice in cold RPMI medium supplemented with 10% foetal bovine serum (FBS) and then counted. Cell availability was evaluated by Trypan blue exclusion. Cells were subjected to stimulation assays with PHA (from Phaseolus vulgaris, Sigma, St. Louis, MO, USA) and LPS (lipopolysaccharides, Sigma). Three independent variables were considered in the stimulation assays: (1) number of plated cells (range 2.5 104 – 2 105/well, (2) PHA and LPS concentration (range 40 –0 or 4– 0 Ag/ml, respectively) and (3) time of stimulation with PHA or LPS (range 12 –120 h). In all the above assays, cell proliferation was measured by 3H-thymidine incorporation by pulsing cells with 0.5 ACi of methyl-3H-thymidine at 2 Ci/ mmol for 12 h at 37 jC. The radioactivity of four replicates was averaged and the standard deviation was calculated. In stimulation experiments with mitogens, each one of the three experimental conditions was assayed by collecting cells from two rats selected at random in the group of animals fed with WGcontaining or with WG-deprived diet. Data from different rats were numbered as R1 –R12. 2.3. Analysis of in vitro stimulation data The in vitro stimulation assays described above provided us with three sets of curves, each set con-
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
sisting of stimulation curves for T and B cells from five lymphoid organs, where cell proliferation was considered as a function of cell number, of PHA or LPS concentration and of time. Exactly 1940 data points of mean lymphocyte stimulation were obtained. To quantitatively analyse the data, the Area Under the stimulation Curves (AUC) was considered. AUC represents a measure of the total amount of stimulation and is given in units of incorporated radioactivity (cpm) multiplied by cell number, PHA or LPS concentration (Ag/ml) or time units (days), respectively. It should be noted that the AUC of control unstimulated cells (referred to as ‘‘Control’’ in Table 1, Fig. 1, top and bottom panels) could not be calculated in doseresponse assays with PHA or LPS. In fact, cell proliferation of unstimulated control cells corresponded to the single-point cell proliferation measure evaluated in the absence of mitogens, and the area beneath a single-point measure is meaningless. The mean AUC F S.D. was calculated for each set of Table 1 Average fold inhibition of total cell proliferation of PHA- or LPSstimulated cells and of unstimulated control cells in rats fed with WG-deprived diet Sa Group A
Group B Group C
b
c
Control + PHA + LPS + PHA + LPS Control + PHA + LPS
d
7.28 16.16 19.2 1.93 4.63 1.72 5.46 7.19
T
ML
IL
BM
2.09 3.31 2.95 1.34 1.36 3.67 4.58 3.37
1.92 7.0 2.34 11.0 2.52 3.45 39.73 6.78
2.42 21.02 6.78 1.85 0.96 1.75 8.22 2.04
0.55 1.22 0.53 6.07 4.16 0.89 0.79 0.64
a Symbols are as follows: S = spleen, T = Thymus, ML = Mesenteric Lymphnodes, IL = Inguinal Lymphnodes, BM = Bone Marrow. b Groups are as follows: A = total cell proliferation as a function of cell number, B = total cell proliferation as a function of PHA or LPS concentration, C = total cell proliferation as a function of time. Total cell proliferation is the Area Under the Curve estimated as described in Materials and methods. c The term Control refers to total cell proliferation measured for unstimulated cells as a function of cell number or time of mock stimulation. d Values represent the ratio between average total proliferation measured for cells drawn from rats fed with WG-containing/WGdeprived diets, and therefore are a non-dimensional measure of the inhibition of cell proliferation in WG-deprived rats (or equivalently of the increase in cell proliferation in the other group of rats). Total stimulation levels were measured as described in Materials and methods.
1497
curves by using the trapezoidal rule implemented in the math tools of SigmaPlot software (Jandel Scientific). 2.4. Cytofluorimetric assays Cells from 1-year-old rats of the second generation fed with WG-deprived or normal feedstuff (i.e., commercially available feedstuff for rat maintenance, Altromin-MT, Rieper, Bolzano, Italy) were obtained from primary and secondary lymphoid organs as described above and subjected to cytofluorimetric analysis using the following antibodies: OX34 (antiCD2), OX35 (anti-CD4), OX8 (anti-CD8) and OX6 (anti-Ia), fluorescein isothiocyanate (FITC)-conjugated antibody to the k/E chains of rat immunoglobulins (Sigma). OX34, OX8 and OX6 hybridomas were from the European Collection of Animal Cell Cultures (Centre for Applied Microbiology and Research, Salisbury, UK) whereas OX35 hybridoma was a kind gift of Dr. Margot Zo¨ller (Department of Tumour Progression and Immune Defence, German Cancer Center, Heidelberg, Germany). In indirect assays, bound antibodies were revealed using an FITC-conjugated goat F(abV)2 anti-mouse antibody (Immunotech, Marseille, France). Controls were obtained by incubating cells with secondary antibody only. Cell-associated fluorescence was analyzed by flow cytometry using an Epics XL cytofluorimeter (Hialeah, FL, USA). 2.5. Immunization assays and antibody measurements Rats were immunized using as antigen a haptenized protein carrier (KLH – FITC) known to stimulate an antibody response dependent on T – B cooperation. FITC (Sigma) was conjugated to KLH (Keyhole Limpet Hemocyanin, Sigma) following standard procedures described elsewhere [14]. The FITC/protein ratio of the conjugate was determined spectrophotometrically and was 6.5 Ag/mg. Rats were injected three times at 1-week intervals with 100 Al PBS solution of KLH – FITC at 0.66 mg/ml emulsified in complete (CFA, first injection) or incomplete (IFA, second and third injection) Freund’s adjuvant (1/1 v/v). Antibodies against protein carrier and hapten were determined in standard ELISA assays by measuring in diluted serum samples (1:10 serial dilution) the reactivity against
1498
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
Fig. 1. Stimulation assays. Three representative experiments are shown. In all panels, R1 – R12 refer to cells drawn from rats 1 – 12. Black bars indicate response levels of mock-treated cells, grey bars cells stimulated with PHA and dark-grey bars cells stimulated with LPS. The response levels are expressed as AUC in indicated units as described in Materials and methods. Top panel: spleen cells stimulated at fixed PHA or LPS concentrations and fixed time; middle panel: bone marrow cells stimulated with different PHA or LPS concentrations at fixed cell number and time of stimulation; bottom panel: cells from inguinal lymphnodes stimulated for different times at fixed cell number and PHA or LPS concentration.
KLH and bovine serum albumin (BSA) –FITC, respectively. As a positive control, a monoclonal anti-FITC antibody (clone FL-D6; Sigma) was also used in ELISA assays. Negative controls were obtained by
measuring the reactivity against the same antigens in sera of non-immunized animals belonging to the two dietary groups ( < 5% of the maximal reactivity measured in the sera of immunized animals).
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
1499
Data like those shown in Fig. 1 are difficult to compare owing to the different scale of the vertical axis between different assays and, in the same assay, between different analysed cell compartments. A nondimensional measure of the inhibition of PHA or LPS stimulation of cells from rats fed with WG-deprived diet is therefore given in Table 1. The inhibition of the stimulatory effects of PHA and of LPS was abrogated in dose-response assays (Group B in Table 1) carried out with cells from thymuses, inguinal lymphnodes and for spleen cells stimulated with PHA. That is to say, cells in these organs were less influenced by the different dietary conditions since varying the concentration of the stimuli restored their proliferative capacity. The stimulatory effects of PHA and of LPS were instead not restored in cells from mesenteric lymphnodes or in B cells of the spleen, at least under the present experimental conditions. The functional state of unresponsiveness of these cells persisted even at the higher PHA and LPS concentrations used in the experiments. It is noteworthy that even unstimulated control cells from WG-deprived rats appear to
3. Results and discussion 3.1. Dietary effects on lymphocytes stimulation Rats were fed for two generations with a controlled diet deprived in WG. No alterations of common physiologic parameters (e.g., loss of body weight, growth, fur status) were observed during the course of our experiments in animals fed with both WGdeprived and WG-containing diets as compared to animals fed with common feedstuff. Cells from primary and secondary lymphoid organs collected from 12 rats of the second generation were stimulated in vitro with PHA or LPS to evaluate whether dietary conditions could have altered the responsiveness of the T- and/or the B-cell compartments, respectively. Representative results, expressed as total amount of stimulation as described in Materials and methods, are given in Fig. 1. Overall, PHA or LPS treatments stimulated cells from WG-deprived rats to a lower extent than cells from rats fed with WG-containing diet.
Table 2 Cell composition of primary and secondary lymphoid organs collected from rats fed with WG-deprived diet or with normal feedstuff as evaluated by cytofluorimetry WG-deprived diet S
Normal feedstuff
T
ML
IL
CD2
3.4 9.5 8.4
28.2 12.7 10.38
46.3 38 37.1
55.4 49.9 69.5
1.8 2.28 1.63
CD4
1.9 6.2 4.4
24.9 ND 4.53
34.2 33 22.7
39.3 41.8 51.8
3.84 7.08 3.13
CD8
1.8 6.8 4.9
23.4 7.7 3.92
18.8 13.7 18.9
14.3 10.9 35.3
2.11 1.98 1.83
Ia
2.3 4.6 2.9
9.1 ND 5.83
10.4 11 4.6
9.2 10.4 13.9
1.39 1.78 2.33
20.63 31.42 15.62
9.91 7.57 11.4
58.93 62.85 45.17
39.01 40.46 9.26
36.5 29.25 12.85
k–E
BM
S
T
ML
IL
BM
12.9 12 11.5 10 7.8 9.1 6.8 5 4.7 6.4 2.1 4.2 2.5 0 0 5.3 22.12 18.27 20.83 25.85
52.52 62.61 8.29 11.44 49.92 54.81 7.59 9.34 40.52 45.51 6.59 4.68 21.72 22.31 3.7 6.84 2.72 2.12 1.49 5.28
60.4 60.6 25.5 41.6 45.2 44.4 17.4 33.1 14.4 17.9 3.2 21.5 7.6 6.8 3 17.5 36.31 31.7 16.07 48.3
62.6 62.9 43 32.9 45.5 47.7 30.3 27.6 15.8 20.5 25.2 15.7 9.8 3.3 6.7 22.1 21.73 33.2 32.72 7.3
2.42 2 0.28 ND 4.8 6.9 4.32 ND 2.11 2.1 0 ND 1.12 4 0 ND 10.08 9.19 15.07 ND
Values represent percentages of cells expressing the indicated cell marker. Within the same set, values in different rows correspond to measurements carried out in different animals. Symbols are as in Table 1.
1500
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
display less spontaneous proliferation than control cells from rat fed with WG-containing diet (see, e.g. Fig. 1). This observation further support the hypothesis concerning the interplay between immune cells and dietary signals. Not surprisingly, the higher suppressive effects were observed for cells from mesenteric lymphnodes draining the gastrointestinal tract. The strategy followed here did not allow us to test the statistical significance of the experimental data. This experimental approach was thought to increase the chances of establishing possible effects of dietary restriction on immune cells. The suppressive effects of WG-deprived diet were nonetheless observed in all independent assays (see, e.g. Table 1). 3.2. WG-deprived diet did not alter the cell composition of primary and lymphoid organs To rule out the possibility that the unresponsiveness of lymphocytes induced by a WG-deprived
diet could be simply explained in terms of a decreased number of T and B cells in the organs of these animals and to eliminate possible ‘‘immunotoxic’’ long-term effects of the diet, the cellular content of primary and secondary lymphoid organs was evaluated by flow cytometry. The results are shown in Table 2. The percentages of CD2 +, CD4 +, CD8 + cells (T cells), of antibody-k – Echains + cells (B cells) and of class-II + cells (antigen presenting cells) were comparable in animals fed with WG-deprived or with normal feedstuff, within the limits of experimental variability among different animals. This is most evident for the mesenteric and inguinal lymphnodes where the higher inhibitory effects of the diet on T- and Bcell stimulation were observed. Therefore, the inhibitory effects reported in Fig. 1 and in Table 1 cannot be explained in terms of possible differences in cell composition of primary and secondary lymphoid organs or in terms of some immunotoxic effects induced by a WG-deprived diet.
Fig. 2. Antibody response to KLH – FITC in rats fed with WG-containing diet (open symbols) or WG-deprived diet (filled symbols). Specific antibodies in the sera of immunised rats were determined in ELISA assays. Left panels: total IgM antibodies against KLH carrier (upper panel) or FITC hapten (lower panel); Right panels: total IgG antibody against KLH carrier (upper panel) or FITC hapten (lower panel). In all panels, different symbols refer to different immunised rats. Antibody titres are expressed as Optical Density units corrected for serum dilution.
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
3.3. Specific antibody production as a function of the diet The immune response to repeated injections of a haptenized-protein antigen was evaluated in the sera of rats fed with WG-containing or WG-deprived diet by quantitating, in time, the specific antibody production to both hapten and protein carrier (Fig. 2). Both IgM and IgG production was measured. No significant differences were observed between rats as a function of the different dietary conditions. CFA/IFA treatment results in the mobilization of cytokines promoting the shift between type-I and type-II responses, as well as in other not yet well defined potentiating effects on the immune response [15]. Thus, CFA/IFA treatment might be able to activate the cells involved in the specific antibody response by providing the extra stimulus necessary to overcome the state of unresponsiveness induced by a WG-deprived diet. These considerations open several questions on whether the humoral immune response to antigen in the absence of CFA might be dependent on the diet. Intriguingly, lectins might act as natural adjuvant. Studies of this possibility deserve attention. Alternative explanations for the results shown in the present pilot study include possible imbalance of regulatory cells and/or of cytokines as a consequence of dietary restrictions. It is not clear from these data if the functional unresponsiveness of lymphocytes from WG-deprived animals is biologically relevant and if it might be reversed with an introduction of a normal diet. Nonetheless, the data strongly suggest that dietary signals interact with the immune system. Whether this interaction results in an imbalance of immune regulatory circuits remains to be elucidated.
Acknowledgements This work was supported in part by grant from the Italian Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica, Cofin99 ‘‘Un approccio multidisciplinare alla regolazione della risposta immunitaria’’. New Foods Industry (Bussolengo, Verona) is gratefully acknowledged for kindly providing us with some dietary components used in this study.
1501
References [1] Kilpatrick DC. Lectins in immunology. In: Pusztai A, Bardocz S, editors. Lectins—biomedical perspectives. Basingstroke: Taylor and Francis; 1995. p. 155 – 82. [2] Kilpatrick DC. Immunological aspects of the potential role of dietary carbohydrates and lectins in human health. Eur J Nutr 1999;38:107 – 17. [3] Muraille E, Pajak B, Urbain J, Leo O. Carbohydrate-bearing cell surface receptors involved in innate immunity: interleukin12 induction by mitogenic and nonmitogenic lectins. Cell Immunol 1999;191:1 – 9. [4] Haas H, Falcone FH, Schramm G, Haisch K, Gibbs BF, Klauche J, et al. Dietary lectins can induce in vitro release of IL-4 and IL-13 from human basophils. Eur J Immunol 1999;29:918 – 27. [5] Toribio ML, De la Hera A, Pereira P, De Landazuri MC. Modulation of cytolitic T cell function by lectins. J Immunol 1985;134:2179 – 84. [6] Greene WC, Goldman CK, Marshall ST, Fleisher TA, Waldmann TA. Stimulation of immunoglobulin biosynthesis in human B cells by wheat germ agglutinin. J Immunol 1985;127: 799 – 804. [7] Tchernychev B, Wilchek M. Natural human antibodies to dietary lectins. FEBS Lett 1996;397:139 – 42. [8] Bounous G, Shenouda N, Kongshavn PA, Osmond DG. Mechanism of altered B-cell response induced by changes in dietary protein type in mice. J Nutr 1985;115:1409 – 17. [9] Bounous G, Kongshavn PA. Differential effect of dietary protein type on the B-cell and T-cell immune responses in mice. J Nutr 1985;115:1403 – 8. [10] Pusztai A, Ewen SWB, Grant G, Brown DS, Stewart JC, Peumans WJ, et al. Antinutritive effects of wheat-germ agglutinin and other N-acetylglucosamine-specific lectins. Br J Nutr 1993;70:313 – 21. [11] Lavelle EC, Grant G, Pusztai A, Pfu¨ller U, O’Hagan DT. Mucosal immunogenicity of plant lectins in mice. Immunology 2000;99:30 – 7. [12] Watzl B, Neudecker C, Hansch GM, Rechemmer G, PoolZobel BL. Dietary wheat germ agglutinin modulates ovalbumin-induced immune responses in Brown Norway rats. Br J Nutr 2001;85:483 – 90. [13] Lis H, Sharon N. Lectins in higher plants. In: Stumpf PK, Conn EE, editors. The biochemistry of plants. New York: Academic Press; 1981. p. 371 – 94. [14] Chignola R, Anselmi C, Franceschi A, Pasti M, Candiani C, Tridente G, et al. Sensitivity of human leukemia cells in exponential or stationary growth phase to immunotoxins. Role of intracellular processing events. J Immunol 1994;152:2333 – 43. [15] Yip HC, Karulin AY, Tary-Lehmann M, Hesse MD, Radeke H, Heeger PS, et al. Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response. J Immunol 1999;162:3942 – 9.
Effects of dietary wheat germ deprivation on the immune system in Wistar rats: a pilot study Roberto Chignola a,*, Corrado Rizzi a, Simone Vincenzi b, Tiziana Cestari c, Nadia Brutti d, Anna Pia Riviera d, Silvia Sartoris c, Angelo D.B. Peruffo a, Giancarlo Andrighetto c a
Dipartimento Scientifico e Tecnologico, Universita` di Verona, Strada Le Grazie 15, CV1, 37100 Verona, Italy b SOCADO s.r.l. Societa` Alimentare Dolciaria, Via Spagna 20, 37069 Villafranca, Verona, Italy c Dipartimento di Patologia, Sezione di Immunologia, Universita` di Verona, c/o Policlinico G.B. Rossi, 37100 Verona, Italy d Servizio di Immunologia Clinica, Azienda Ospedaliera di Verona, c/o Policlinico G.B. Rossi, 37100 Verona, Italy Received 22 April 2002; received in revised form 6 August 2002; accepted 6 August 2002
Abstract Bioactive molecules that can gain access to body tissues through the gastrointestinal tract may interact with immune regulatory circuits and effector functions. Among these are plant lectins, such as wheat germ (WG) agglutinin, which constitute common components of the human diet and target the immune system on a daily basis. Dietary bioactive molecules might be considered as immunomodulatory signals. To investigate the possible effects on the immune system of the long-term absence of such signals, two groups of rats were fed on a diet containing or deprived of WG. The WG-deprived diet induced a state of functional unresponsiveness in lymphocytes from primary and secondary lymphoid organs, as evaluated by in vitro stimulation with T cell mitogen phytohemoagglutinin (PHA) and B cell mitogen lypopolysaccarides (LPS). The unresponsive state of the immune cells could be reversed by injection of antigen emulsified in oil with inactivated mycobacteria (complete Freund’s adjuvant, CFA) Dietary signals can thus interact with the immune system possibly influencing its shaping during ontogenesis. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Wheat germ; Diet; Plant lectins; Immune system
1. Introduction Several dietary factors are known to affect various functions of the immune system and to interfere with immune regulatory circuits. Among these, the immunobiological activity of carbohydrate-binding proteins
*
Corresponding author. Tel.: +39-045-8027953. E-mail address: [email protected] (R. Chignola).
of vegetable origin (plant lectins) has long been recognized [1,2]. Plant lectins are able to modulate important immune mechanisms, including inflammatory reactions and effector functions [1 –7]. Different diet compositions, which may involve different lectins or different lectin concentrations, have been investigated in terms of their potential influences on human or animal health [1,2,8 –12]. These and other dietary components may target the immune system on a daily basis and hence should be
1567-5769/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 7 - 5 7 6 9 ( 0 2 ) 0 0 11 6 - 9
1496
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
considered as exogenous immunoregulatory signals. Due to the nutritional requirements of living organisms, dietary signals target the immune system during the whole life of an animal. The immune system might therefore be hypothesized to evolve and shape under the continuous stimulation of dietary signals that might add modulatory effects to those elicited by molecules produced by the immune system itself. If so, the absence of dietary immune-targeting signals might result in a different shaping of the immune system and/or in the alteration of some immune functions. To test this hypothesis, a pilot study was conducted with inbred Wistar rats. The rationale for a pilot study involving a relatively small group of rats stems from ethical issues that do not allow us to sacrifice a large number of animals to explore possible, but not probable, biological effects. Rats were fed for two generations with a diet containing wheat germ (WG) or with the same diet deprived of WG. WG was chosen as a model foodstuff interacting with the immune system due to its content in wheat germ agglutinin (WGA), a lectin known to influence several immune functions in vivo and in vitro [2– 6]. As a first general approach, the functional activity of the T- and B-lymphocyte compartments was analysed in the two groups of rats. This pilot study provides positive evidence for the potential role of dietary components in the shaping and/or in the tuning of immune system functions, and therefore provides a justification for more extensive studies on the immune system and its evolution in animals subjected to dietary restriction.
2. Materials and methods 2.1. Animals, diet composition and feeding procedures In order to test the long-term effects of dietary signal deprivation on the immune system, 1-month-old Wistar rats (from our own breeding colony) were fed with WG-deprived diet (see below) and allowed to breed. Newborns were continuously fed with the same diet. WG-deprived and WG-containing diets both contained the following common ingredients selected to provide animals with a balanced nutritional base: pork liver, powdered carrots (New Foods Industry, Verona, Italy),
coconut flour and sugar. These ingredients were mixed in proportion to yield the following formula (percent of wet weight of single components over total wet weight of the diet): proteins 7.8 –8.2%, fat 5.1 –5.7% carbohydrates 22.7 –24.8%. To this basic feedstuff, 15% (w/w) WG or 20% pork liver plus 6% sugar were added to obtain WG-containing or WG-deprived diets, respectively. WG, containing 0.3 F 0.2% (w/w) WGA [13], was used in this pilot study instead of purified WGA to obtain a nutritional condition resembling a normal animal diet. All experiments involving rats were carried out according to current Italian laws (D.L. 116, 1992) in agreement with the directions mandated by 86/609/CEE on animal experimentation. 2.2. Cell preparations and in vitro stimulation assays Spleen, thymus, mesenteric lymphnodes, inguinal lymphnodes and bone marrow were collected from sacrificed 3-month-old rats of the second generation. Cells were washed twice in cold RPMI medium supplemented with 10% foetal bovine serum (FBS) and then counted. Cell availability was evaluated by Trypan blue exclusion. Cells were subjected to stimulation assays with PHA (from Phaseolus vulgaris, Sigma, St. Louis, MO, USA) and LPS (lipopolysaccharides, Sigma). Three independent variables were considered in the stimulation assays: (1) number of plated cells (range 2.5 104 – 2 105/well, (2) PHA and LPS concentration (range 40 –0 or 4– 0 Ag/ml, respectively) and (3) time of stimulation with PHA or LPS (range 12 –120 h). In all the above assays, cell proliferation was measured by 3H-thymidine incorporation by pulsing cells with 0.5 ACi of methyl-3H-thymidine at 2 Ci/ mmol for 12 h at 37 jC. The radioactivity of four replicates was averaged and the standard deviation was calculated. In stimulation experiments with mitogens, each one of the three experimental conditions was assayed by collecting cells from two rats selected at random in the group of animals fed with WGcontaining or with WG-deprived diet. Data from different rats were numbered as R1 –R12. 2.3. Analysis of in vitro stimulation data The in vitro stimulation assays described above provided us with three sets of curves, each set con-
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
sisting of stimulation curves for T and B cells from five lymphoid organs, where cell proliferation was considered as a function of cell number, of PHA or LPS concentration and of time. Exactly 1940 data points of mean lymphocyte stimulation were obtained. To quantitatively analyse the data, the Area Under the stimulation Curves (AUC) was considered. AUC represents a measure of the total amount of stimulation and is given in units of incorporated radioactivity (cpm) multiplied by cell number, PHA or LPS concentration (Ag/ml) or time units (days), respectively. It should be noted that the AUC of control unstimulated cells (referred to as ‘‘Control’’ in Table 1, Fig. 1, top and bottom panels) could not be calculated in doseresponse assays with PHA or LPS. In fact, cell proliferation of unstimulated control cells corresponded to the single-point cell proliferation measure evaluated in the absence of mitogens, and the area beneath a single-point measure is meaningless. The mean AUC F S.D. was calculated for each set of Table 1 Average fold inhibition of total cell proliferation of PHA- or LPSstimulated cells and of unstimulated control cells in rats fed with WG-deprived diet Sa Group A
Group B Group C
b
c
Control + PHA + LPS + PHA + LPS Control + PHA + LPS
d
7.28 16.16 19.2 1.93 4.63 1.72 5.46 7.19
T
ML
IL
BM
2.09 3.31 2.95 1.34 1.36 3.67 4.58 3.37
1.92 7.0 2.34 11.0 2.52 3.45 39.73 6.78
2.42 21.02 6.78 1.85 0.96 1.75 8.22 2.04
0.55 1.22 0.53 6.07 4.16 0.89 0.79 0.64
a Symbols are as follows: S = spleen, T = Thymus, ML = Mesenteric Lymphnodes, IL = Inguinal Lymphnodes, BM = Bone Marrow. b Groups are as follows: A = total cell proliferation as a function of cell number, B = total cell proliferation as a function of PHA or LPS concentration, C = total cell proliferation as a function of time. Total cell proliferation is the Area Under the Curve estimated as described in Materials and methods. c The term Control refers to total cell proliferation measured for unstimulated cells as a function of cell number or time of mock stimulation. d Values represent the ratio between average total proliferation measured for cells drawn from rats fed with WG-containing/WGdeprived diets, and therefore are a non-dimensional measure of the inhibition of cell proliferation in WG-deprived rats (or equivalently of the increase in cell proliferation in the other group of rats). Total stimulation levels were measured as described in Materials and methods.
1497
curves by using the trapezoidal rule implemented in the math tools of SigmaPlot software (Jandel Scientific). 2.4. Cytofluorimetric assays Cells from 1-year-old rats of the second generation fed with WG-deprived or normal feedstuff (i.e., commercially available feedstuff for rat maintenance, Altromin-MT, Rieper, Bolzano, Italy) were obtained from primary and secondary lymphoid organs as described above and subjected to cytofluorimetric analysis using the following antibodies: OX34 (antiCD2), OX35 (anti-CD4), OX8 (anti-CD8) and OX6 (anti-Ia), fluorescein isothiocyanate (FITC)-conjugated antibody to the k/E chains of rat immunoglobulins (Sigma). OX34, OX8 and OX6 hybridomas were from the European Collection of Animal Cell Cultures (Centre for Applied Microbiology and Research, Salisbury, UK) whereas OX35 hybridoma was a kind gift of Dr. Margot Zo¨ller (Department of Tumour Progression and Immune Defence, German Cancer Center, Heidelberg, Germany). In indirect assays, bound antibodies were revealed using an FITC-conjugated goat F(abV)2 anti-mouse antibody (Immunotech, Marseille, France). Controls were obtained by incubating cells with secondary antibody only. Cell-associated fluorescence was analyzed by flow cytometry using an Epics XL cytofluorimeter (Hialeah, FL, USA). 2.5. Immunization assays and antibody measurements Rats were immunized using as antigen a haptenized protein carrier (KLH – FITC) known to stimulate an antibody response dependent on T – B cooperation. FITC (Sigma) was conjugated to KLH (Keyhole Limpet Hemocyanin, Sigma) following standard procedures described elsewhere [14]. The FITC/protein ratio of the conjugate was determined spectrophotometrically and was 6.5 Ag/mg. Rats were injected three times at 1-week intervals with 100 Al PBS solution of KLH – FITC at 0.66 mg/ml emulsified in complete (CFA, first injection) or incomplete (IFA, second and third injection) Freund’s adjuvant (1/1 v/v). Antibodies against protein carrier and hapten were determined in standard ELISA assays by measuring in diluted serum samples (1:10 serial dilution) the reactivity against
1498
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
Fig. 1. Stimulation assays. Three representative experiments are shown. In all panels, R1 – R12 refer to cells drawn from rats 1 – 12. Black bars indicate response levels of mock-treated cells, grey bars cells stimulated with PHA and dark-grey bars cells stimulated with LPS. The response levels are expressed as AUC in indicated units as described in Materials and methods. Top panel: spleen cells stimulated at fixed PHA or LPS concentrations and fixed time; middle panel: bone marrow cells stimulated with different PHA or LPS concentrations at fixed cell number and time of stimulation; bottom panel: cells from inguinal lymphnodes stimulated for different times at fixed cell number and PHA or LPS concentration.
KLH and bovine serum albumin (BSA) –FITC, respectively. As a positive control, a monoclonal anti-FITC antibody (clone FL-D6; Sigma) was also used in ELISA assays. Negative controls were obtained by
measuring the reactivity against the same antigens in sera of non-immunized animals belonging to the two dietary groups ( < 5% of the maximal reactivity measured in the sera of immunized animals).
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
1499
Data like those shown in Fig. 1 are difficult to compare owing to the different scale of the vertical axis between different assays and, in the same assay, between different analysed cell compartments. A nondimensional measure of the inhibition of PHA or LPS stimulation of cells from rats fed with WG-deprived diet is therefore given in Table 1. The inhibition of the stimulatory effects of PHA and of LPS was abrogated in dose-response assays (Group B in Table 1) carried out with cells from thymuses, inguinal lymphnodes and for spleen cells stimulated with PHA. That is to say, cells in these organs were less influenced by the different dietary conditions since varying the concentration of the stimuli restored their proliferative capacity. The stimulatory effects of PHA and of LPS were instead not restored in cells from mesenteric lymphnodes or in B cells of the spleen, at least under the present experimental conditions. The functional state of unresponsiveness of these cells persisted even at the higher PHA and LPS concentrations used in the experiments. It is noteworthy that even unstimulated control cells from WG-deprived rats appear to
3. Results and discussion 3.1. Dietary effects on lymphocytes stimulation Rats were fed for two generations with a controlled diet deprived in WG. No alterations of common physiologic parameters (e.g., loss of body weight, growth, fur status) were observed during the course of our experiments in animals fed with both WGdeprived and WG-containing diets as compared to animals fed with common feedstuff. Cells from primary and secondary lymphoid organs collected from 12 rats of the second generation were stimulated in vitro with PHA or LPS to evaluate whether dietary conditions could have altered the responsiveness of the T- and/or the B-cell compartments, respectively. Representative results, expressed as total amount of stimulation as described in Materials and methods, are given in Fig. 1. Overall, PHA or LPS treatments stimulated cells from WG-deprived rats to a lower extent than cells from rats fed with WG-containing diet.
Table 2 Cell composition of primary and secondary lymphoid organs collected from rats fed with WG-deprived diet or with normal feedstuff as evaluated by cytofluorimetry WG-deprived diet S
Normal feedstuff
T
ML
IL
CD2
3.4 9.5 8.4
28.2 12.7 10.38
46.3 38 37.1
55.4 49.9 69.5
1.8 2.28 1.63
CD4
1.9 6.2 4.4
24.9 ND 4.53
34.2 33 22.7
39.3 41.8 51.8
3.84 7.08 3.13
CD8
1.8 6.8 4.9
23.4 7.7 3.92
18.8 13.7 18.9
14.3 10.9 35.3
2.11 1.98 1.83
Ia
2.3 4.6 2.9
9.1 ND 5.83
10.4 11 4.6
9.2 10.4 13.9
1.39 1.78 2.33
20.63 31.42 15.62
9.91 7.57 11.4
58.93 62.85 45.17
39.01 40.46 9.26
36.5 29.25 12.85
k–E
BM
S
T
ML
IL
BM
12.9 12 11.5 10 7.8 9.1 6.8 5 4.7 6.4 2.1 4.2 2.5 0 0 5.3 22.12 18.27 20.83 25.85
52.52 62.61 8.29 11.44 49.92 54.81 7.59 9.34 40.52 45.51 6.59 4.68 21.72 22.31 3.7 6.84 2.72 2.12 1.49 5.28
60.4 60.6 25.5 41.6 45.2 44.4 17.4 33.1 14.4 17.9 3.2 21.5 7.6 6.8 3 17.5 36.31 31.7 16.07 48.3
62.6 62.9 43 32.9 45.5 47.7 30.3 27.6 15.8 20.5 25.2 15.7 9.8 3.3 6.7 22.1 21.73 33.2 32.72 7.3
2.42 2 0.28 ND 4.8 6.9 4.32 ND 2.11 2.1 0 ND 1.12 4 0 ND 10.08 9.19 15.07 ND
Values represent percentages of cells expressing the indicated cell marker. Within the same set, values in different rows correspond to measurements carried out in different animals. Symbols are as in Table 1.
1500
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
display less spontaneous proliferation than control cells from rat fed with WG-containing diet (see, e.g. Fig. 1). This observation further support the hypothesis concerning the interplay between immune cells and dietary signals. Not surprisingly, the higher suppressive effects were observed for cells from mesenteric lymphnodes draining the gastrointestinal tract. The strategy followed here did not allow us to test the statistical significance of the experimental data. This experimental approach was thought to increase the chances of establishing possible effects of dietary restriction on immune cells. The suppressive effects of WG-deprived diet were nonetheless observed in all independent assays (see, e.g. Table 1). 3.2. WG-deprived diet did not alter the cell composition of primary and lymphoid organs To rule out the possibility that the unresponsiveness of lymphocytes induced by a WG-deprived
diet could be simply explained in terms of a decreased number of T and B cells in the organs of these animals and to eliminate possible ‘‘immunotoxic’’ long-term effects of the diet, the cellular content of primary and secondary lymphoid organs was evaluated by flow cytometry. The results are shown in Table 2. The percentages of CD2 +, CD4 +, CD8 + cells (T cells), of antibody-k – Echains + cells (B cells) and of class-II + cells (antigen presenting cells) were comparable in animals fed with WG-deprived or with normal feedstuff, within the limits of experimental variability among different animals. This is most evident for the mesenteric and inguinal lymphnodes where the higher inhibitory effects of the diet on T- and Bcell stimulation were observed. Therefore, the inhibitory effects reported in Fig. 1 and in Table 1 cannot be explained in terms of possible differences in cell composition of primary and secondary lymphoid organs or in terms of some immunotoxic effects induced by a WG-deprived diet.
Fig. 2. Antibody response to KLH – FITC in rats fed with WG-containing diet (open symbols) or WG-deprived diet (filled symbols). Specific antibodies in the sera of immunised rats were determined in ELISA assays. Left panels: total IgM antibodies against KLH carrier (upper panel) or FITC hapten (lower panel); Right panels: total IgG antibody against KLH carrier (upper panel) or FITC hapten (lower panel). In all panels, different symbols refer to different immunised rats. Antibody titres are expressed as Optical Density units corrected for serum dilution.
R. Chignola et al. / International Immunopharmacology 2 (2002) 1495–1501
3.3. Specific antibody production as a function of the diet The immune response to repeated injections of a haptenized-protein antigen was evaluated in the sera of rats fed with WG-containing or WG-deprived diet by quantitating, in time, the specific antibody production to both hapten and protein carrier (Fig. 2). Both IgM and IgG production was measured. No significant differences were observed between rats as a function of the different dietary conditions. CFA/IFA treatment results in the mobilization of cytokines promoting the shift between type-I and type-II responses, as well as in other not yet well defined potentiating effects on the immune response [15]. Thus, CFA/IFA treatment might be able to activate the cells involved in the specific antibody response by providing the extra stimulus necessary to overcome the state of unresponsiveness induced by a WG-deprived diet. These considerations open several questions on whether the humoral immune response to antigen in the absence of CFA might be dependent on the diet. Intriguingly, lectins might act as natural adjuvant. Studies of this possibility deserve attention. Alternative explanations for the results shown in the present pilot study include possible imbalance of regulatory cells and/or of cytokines as a consequence of dietary restrictions. It is not clear from these data if the functional unresponsiveness of lymphocytes from WG-deprived animals is biologically relevant and if it might be reversed with an introduction of a normal diet. Nonetheless, the data strongly suggest that dietary signals interact with the immune system. Whether this interaction results in an imbalance of immune regulatory circuits remains to be elucidated.
Acknowledgements This work was supported in part by grant from the Italian Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica, Cofin99 ‘‘Un approccio multidisciplinare alla regolazione della risposta immunitaria’’. New Foods Industry (Bussolengo, Verona) is gratefully acknowledged for kindly providing us with some dietary components used in this study.
1501
References [1] Kilpatrick DC. Lectins in immunology. In: Pusztai A, Bardocz S, editors. Lectins—biomedical perspectives. Basingstroke: Taylor and Francis; 1995. p. 155 – 82. [2] Kilpatrick DC. Immunological aspects of the potential role of dietary carbohydrates and lectins in human health. Eur J Nutr 1999;38:107 – 17. [3] Muraille E, Pajak B, Urbain J, Leo O. Carbohydrate-bearing cell surface receptors involved in innate immunity: interleukin12 induction by mitogenic and nonmitogenic lectins. Cell Immunol 1999;191:1 – 9. [4] Haas H, Falcone FH, Schramm G, Haisch K, Gibbs BF, Klauche J, et al. Dietary lectins can induce in vitro release of IL-4 and IL-13 from human basophils. Eur J Immunol 1999;29:918 – 27. [5] Toribio ML, De la Hera A, Pereira P, De Landazuri MC. Modulation of cytolitic T cell function by lectins. J Immunol 1985;134:2179 – 84. [6] Greene WC, Goldman CK, Marshall ST, Fleisher TA, Waldmann TA. Stimulation of immunoglobulin biosynthesis in human B cells by wheat germ agglutinin. J Immunol 1985;127: 799 – 804. [7] Tchernychev B, Wilchek M. Natural human antibodies to dietary lectins. FEBS Lett 1996;397:139 – 42. [8] Bounous G, Shenouda N, Kongshavn PA, Osmond DG. Mechanism of altered B-cell response induced by changes in dietary protein type in mice. J Nutr 1985;115:1409 – 17. [9] Bounous G, Kongshavn PA. Differential effect of dietary protein type on the B-cell and T-cell immune responses in mice. J Nutr 1985;115:1403 – 8. [10] Pusztai A, Ewen SWB, Grant G, Brown DS, Stewart JC, Peumans WJ, et al. Antinutritive effects of wheat-germ agglutinin and other N-acetylglucosamine-specific lectins. Br J Nutr 1993;70:313 – 21. [11] Lavelle EC, Grant G, Pusztai A, Pfu¨ller U, O’Hagan DT. Mucosal immunogenicity of plant lectins in mice. Immunology 2000;99:30 – 7. [12] Watzl B, Neudecker C, Hansch GM, Rechemmer G, PoolZobel BL. Dietary wheat germ agglutinin modulates ovalbumin-induced immune responses in Brown Norway rats. Br J Nutr 2001;85:483 – 90. [13] Lis H, Sharon N. Lectins in higher plants. In: Stumpf PK, Conn EE, editors. The biochemistry of plants. New York: Academic Press; 1981. p. 371 – 94. [14] Chignola R, Anselmi C, Franceschi A, Pasti M, Candiani C, Tridente G, et al. Sensitivity of human leukemia cells in exponential or stationary growth phase to immunotoxins. Role of intracellular processing events. J Immunol 1994;152:2333 – 43. [15] Yip HC, Karulin AY, Tary-Lehmann M, Hesse MD, Radeke H, Heeger PS, et al. Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response. J Immunol 1999;162:3942 – 9.