点击显示 收起
1 From the Institute of Nutritional Physiology, Federal Research Centre for Nutrition and Food, Karlsruhe, Germany
2 Supported by a grant from the World Cancer Research Fund (2002-15). Schoenenberger Pflanzensäfte GmbH, Magstadt, Germany, provided the tomato and carrot juices. 3 Reprints not available. Address correspondence to B Watzl, The Institute of Nutritional Physiology, Federal Research Centre for Nutrition and Food, Haid-und-Neu-Str 9, 76131 Karlsruhe, Germany. E-mail: bernhard.watzl{at}bfe.uka.de.
ABSTRACT
Background: Whether different intakes of vegetables and fruit modulate immunologic markers is currently not known.
Objective: We investigated the effects of low, medium, and high intakes of vegetables and fruit on markers of immune functions, including nonspecific markers of inflammation.
Design: In a randomized controlled trial, nonsmoking men consumed a diet that included 2 servings/d of vegetables and fruit for 4 wk. The subjects were then randomly assigned to 1 of 3 groups to consume 2 servings/d, 5 servings/d, or 8 servings/d of carotenoid-rich vegetables and fruit for another 4-wk period. Plasma concentrations of vitamins C and E and carotenoids were measured. The assessment of immunologic and inflammatory markers included the number and activity of natural killer cells, secretion of cytokines, lymphocyte proliferation, and plasma C-reactive protein concentrations.
Results: The high intake (8 servings/d) of vegetables and fruit significantly increased total carotenoid concentrations in plasma compared with the low intake (2 servings/d; week 4 compared with week 8), whereas concentrations of vitamins C and E did not differ between week 4 and week 8. Immunologic markers were not significantly modulated. In contrast, C-reactive protein was significantly reduced at week 8 in the subjects who consumed 8 servings/d of vegetables and fruit compared with those who consumed 2 servings/d.
Conclusions: In healthy, well-nourished, nonsmoking men, 4 wk of low or high intakes of carotenoid-rich vegetables and fruit did not affect markers of immune function. However, a high intake of vegetables and fruit may reduce inflammatory processes, as indicated by the reduction of plasma C-reactive protein.
Key Words: Vegetables • fruit • natural killer cells • cytokines • C-reactive protein
INTRODUCTION
A high intake of vegetables and fruit (V+F) is associated with a reduced risk of several cancers (1). V+F are rich sources of nutrients, dietary fiber, and phytochemicals. Their biological mechanisms in the prevention of cancer, including modulation of immune functions, are only partially known. Although several nutrients and phytochemicals in V+F have been shown to modulate immune functions in humans, few studies have investigated the role of whole V+F consumption. No information is available as to whether a low intake compared with a high intake of V+F results in a modulation of immune functions in humans.
Among the different types of immune cells involved in the recognition of tumor cells are natural killer (NK) cells. Because of their production of immunoregulatory cytokines and their cytotoxic effects, NK cells are the primary effector cells in tumor cell elimination (2). Depletion of NK cells in vivo leads to enhanced tumor formation in several mouse tumor models (2). In addition, cancer patients who had recurrences within 2 y of surgery had significantly lower preoperative NK cell cytotoxicity than did recurrence-free patients (3). Data from a prospective cohort study indicate that in healthy humans, low NK cell cytotoxic activity is associated with an enhanced risk of epithelial cancer later in life (4). These data suggest that a high NK cell cytotoxicity is associated with lower cancer rates.
In earlier studies, we observed that the consumption of carotenoid-rich vegetable juices stimulated NK cell cytotoxicity, cytokine secretion, and lymphocyte proliferation (5, 6). However, this sort of stimulation was only seen in subjects who consumed a low-carotenoid diet (7). Studies with pure carotenoids also suggest that specific carotenoids, such as ß-carotene, affect lymphocyte and monocyte functions. Supplementation with pure ß-carotene stimulated lymphocyte proliferation in several human intervention studies (8–10). The lytic activity of NK cells was enhanced in elderly subjects who received long-term ß-carotene supplementation (11), and the percentage of NK cells was significantly increased after short-term supplementation (12). Besides ß-carotene, other carotenoids, including lycopene and lutein, have recently received more attention. Several studies showed that a high intake of these carotenoids is associated with a reduced incidence of prostate and lung cancer (13, 14). A high intake of tomato and tomato-based products was also consistently associated with a low risk of cancer in several anatomic sites (13). Numerous potentially immunomodulatory compounds are present in tomatoes, with lycopene being the major phytochemical (15). Only one study so far has looked at the immunomodulatory activity of pure lycopene. In contrast with ß-carotene, this carotenoid (15 mg/d) did not stimulate either monocyte surface molecule expression or tumor necrosis factor (TNF-) secretion in a human intervention study (16). Overall, these studies suggest an immunomodulatory potential of specific compounds that are present in V+F.
Although the role of nutrients for proper immune functions has been shown in many studies, the effects of whole V+F consumption on cancer-related immune function measurements have been studied little (1). Therefore, the objective of the present study was to conduct a human intervention study of the effects of V+F on intermediate markers of cancer risk, such as immune functions, with a focus primarily on NK cell functions and on nonspecific markers of inflammation.
SUBJECTS AND METHODS
Subjects
Sixty-four healthy men were recruited for the study through advertisements posted at local universities, at other institutions, and in regional newspapers. Exclusion criteria were smoking and use of dietary supplements or medication. All subjects were in good medical health, as was determined by a screening history and a medical examination. The study was approved by the Medical Ethical Committee of the Landesärztekammer Baden-Württemberg, and all participants gave their written consent.
Study design
The study was a randomized longitudinal trial of two 4-wk treatments (Figure 1) and was conducted during May and July 2003. The subjects were asked at recruitment to complete a previously validated food-frequency questionnaire, which was originally designed and applied in the German part of the European Prospective Investigation into Cancer and Nutrition Study (17). The subjects were randomly assigned to 1 of 3 dietary groups. During the first treatment period, all subjects were restricted to a maximum of 2 servings/d of V+F (1 serving = 100 g or 200 mL juice), and the V+F intake was recorded daily in a dietary record. Otherwise, the subjects were not restricted in their diets. During the second treatment period, all subjects received their lunchtime meals on weekdays under supervision at the Federal Research Centre for Nutrition and Food. V+F and juices for snacks, dinner, and breakfast the next morning were provided to the subjects. No other V+F, or products thereof, were allowed. On Fridays, V+F were provided as packages for weekend days with guidelines for storage, preparation, and consumption. Leftovers were brought back to the Institute on Mondays for registration. The V+F intake was also recorded daily during this study period. During the second 4-wk treatment period, the subjects were allowed to consume 2 servings/d (group 1), 5 servings/d (group 2), or 8 servings/d (group 3) of V+F in total. Vegetable intake included carrots, green beans, peas, broccoli, zucchini, tomatoes, kohlrabi, Brussels sprouts, red cabbage, cauliflower, spinach, corn, and salsifies. Salad intake included lettuce, tomatoes, carrots, corn, radishes, cucumbers, fennel, and cabbage. Fruit intake consisted of apples, pears, kiwis, bananas, peaches, nectarines, cherries, strawberries, and red currants. Macronutrient intake during weeks 4 and 8 was assessed with a validated 4-d food record (18). Blood samples from fasting subjects were collected at baseline, after 4 wk, and after 8 wk and were analyzed in a blinded fashion.
FIGURE 1.. Graphic presentation of the study design. Fasting blood samples were collected at baseline, after 4 wk, and after 8 wk.
Analysis of carotenoids and vitamins E and C in plasma
Plasma concentrations of carotenoids (- and ß-carotene, lycopene, lutein, zeaxanthin, and ß-cryptoxanthin) and vitamin E (-tocopherol) were analyzed by HPLC (19). Serum total vitamin C concentrations (sum of ascorbic acid and dehydroascorbic acid) were measured after protein precipitation, according to the method of Omaye et al (20). The supernatant fluid was treated with 2,4-dinitrophenylhydrazone-thiourea-copper sulfate reagent. The dehydroascorbic acid formed from the oxidation of ascorbic acid by copper was measured spectrophotometrically as the 2,4-dinitrophenylhydrazone derivative.
Isolation of peripheral blood mononuclear cells, preparation of serum and plasma, and analysis of blood lipids and glucose
Venous blood samples were collected between 0700 and 0900 after the subjects had fasted overnight. Blood was collected from 21 subjects/d for 3 days into 9-mL tubes that contained lithium heparin. Plasma was collected after centrifugation at 1500 x g for 10 min at 4 °C. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation at 400 x g for 20 min at 20 °C, and the cells were counted with a Coulter Counter Z2 (Beckman Coulter, Krefeld, Germany). For serum preparation, blood was collected and the clotted blood was centrifuged at 1854 x g for 10 min at 4 °C. Autologous serum (AS) from each subject for the culture of PBMCs was heat-inactivated for 30 min at 56 °C. Enzymatic tests were applied for the measurement of serum total cholesterol, triacylglycerol, glucose (Roche, Mannheim, Germany), LDL cholesterol, and HDL cholesterol (WAKO Chemie, Neuss, Germany).
Lymphocyte proliferation
PBMCs were cultured at 1 x 109 cells/L in medium containing 10% AS and were stimulated by the T cell mitogen concanavalin A (5 mg/L; Sigma-Aldrich, Steinheim, Germany) for 72 h at 37 °C. AS was used instead of fetal bovine serum for the culture of PBMCs because it provides PBMCs with various plasma carotenoids throughout the length of the cell culture period. The proliferation of PBMCs was measured as previously described (5).
Quantification of cytokine secretion and of plasma C-reactive protein
PBMCs were cultured at 1 x 109 cells/L in medium containing 10% AS and were stimulated by 5 mg concanavalin A/L for 24 h at 37 °C [to measure the production of interferon (IFN) , interleukin (IL) 2, and IL-13] or by 1 µg lipopolysaccharide/L (Difco, Augsburg, Germany) for 24 h at 37 °C (to measure the production of TNF- and IL-12). Cell-free supernatant fluid was collected and stored at –80 °C until analyzed. IL-2 and IL-13 (R&D Systems GmbH, Wiesbaden, Germany) and IFN-, IL-12, and TNF- (Biosource GmbH, Solingen, Germany) were measured by enzyme-linked immunosorbent assays, as described by the manufacturers. Plasma C-reactive protein (CRP) concentrations were measured with a high-sensitivity CRP enzyme-linked immunosorbent assay kit according to the manufacturer's instructions (MP Biomedicals, Orangeburg, NY; distributed by ICN, Eschwege, Germany).
Lytic activity of NK cells
The lytic activity of NK cells against K562 target cells was measured by using 2 different ratios of PBMC to K562 (25:1 and 12.5:1) with a previously described flow cytometric method (21). The lytic activity was calculated as the percentage of dead target cells in the test samples minus the percentage of dead target cells in the control samples without effector cells.
Quantification of NK cells
The percentage of CD3–/CD56+ NK cells and of NK cells that expressed the natural cytotoxicity receptor NKp46 were measured by flow cytometry (FACSCalibur; BectonDickinson, Heidelberg, Germany). Whole blood samples (100 µL) were mixed with 10 µL fluorescein isothiocyanate-IgG1 antihuman CD3 antibody (BectonDickinson Pharmingen), 5 µL phycoerythrine (PE)-Cy5-IgG1 antihuman CD56 antibody (BectonDickinson Pharmingen), and 10 µL PE-IgG1 antihuman NKp46 antibody (IOTest; Beckman Coulter) and were incubated for 30 min in the dark at room temperature. Fluorescein isothiocyanate-IgG1 (BectonDickinson Pharmingen), PE-Cy5-IgG1 (BectonDickinson Pharmingen), and PE-IgG1 (Beckman Coulter) antimouse monoclonal antibodies were used as isotype controls. The tubes were then washed, and the red blood cells were lysed with fluorescence-activated cell sorting lysing solution (BectonDickinson). The stained cells were stored on ice in 1% paraformaldehyde (Sigma-Aldrich) and fluorescence was measured within 24 h.
Measurement of the TNF- –308 promotor polymorphism
Genomic DNA was extracted from whole blood that was preserved in EDTA with the GenomicPrep Blood DNA Isolation Kit (Amersham, Freiburg, Germany), according to the manufacturer's directions. The fragment containing the TNF*2 polymorphism was amplified with sense 5AAT AGG TTT TGA GGG CCA TG 3 and antisense 5ATC TGG AGG AAG CGG TAG TG 3 primers (22). DNA samples were amplified in a 50-µL solution that contained 10 nmol of each deoxynucleotide triphosphate, 5 µL 10x reaction buffer [100 mmol KCl/L, 100 mmol (NH4)2SO4/L, 200 mmol Tris/HCl/L pH 8.8, 20 mmol MgSO4/L, and 1% Triton X-100], 200 ng of each primer, 500 ng DNA sample, and 2.5 U Taq polymerase (New England Biolabs, Ipswich, MA) for one cycle at 94 °C for 4 min, 60 °C for 30 s, and 72 °C for 60 s; followed by 39 cycles at 94 °C for 60 s, 60 °C for 30 s, and 72 °C for 60 s; followed by one cycle at 72 °C for 5 min. The polymerase chain reaction products were digested at 37 °C with NcoI to detect the TNF*2 allele and then were subjected to a 5% agarose gel electrophoresis (MoSieve; Peqlab, Erlangen, Germany).
Statistical analyses
Results are reported as means ± SDs. To assess the efficiency of randomization, the differences between the groups at baseline were analyzed with an analysis of variance. We used a 2-factor analysis of variance containing 3 time points and 3 groups to analyze the data. The number of servings (2, 5, or 8) was included as the main effect, and the changes in immune variables over time (week 4 compared with both baseline and week 8) were the dependent variables. Analyses were also run with only the 4 wk and 8 wk data, and the results were not significantly different from the results with all 3 time points. When significant effects of serving numbers were observed with repeated-measures analysis of variance, the differences were analyzed with a post hoc Dunnet's test. To assess correlations between immunologic markers, Pearson's correlation coefficients were computed. Values of P < 0.05 were considered significant. All statistical calculations were performed with the STATVIEW program (1998; SAS Institute, Cary, NC).
RESULTS
Subjects, baseline measurements, and dietary intake
Sixty-three subjects completed the study, and one subject dropped out because of personal reasons. Age distribution and body mass index at baseline are shown in Table 1. The habitual dietary intakes of the subjects in the 3 groups at baseline are also presented in Table 1. No significant differences were seen between the 3 groups at baseline. It was observed that fruit juice consumption previous to the trial was high in all groups.
View this table:
TABLE 1. Baseline characteristics and habitual daily dietary intake in the 3 groups at the start of the study1
The numbers of servings of V+F consumed during the 2 study periods are presented in Table 2. During weeks 1 and 4, all subjects consumed 2 servings/d of V+F. During weeks 5–8, the subjects who were asked to consume 5 and 8 servings/d significantly increased their intakes compared with the low-intake group (Table 2). Body weight, body mass index, and total energy and macronutrient intakes did not change significantly during the intervention (Table 3). Blood glucose and lipid profiles were also not significantly different (Table 4).
View this table:
TABLE 2. Number of servings of vegetables and fruit, including juices, consumed daily1
View this table:
TABLE 3. Body weight, BMI, and daily macronutrient intake in subjects who consumed 2, 5, or 8 servings/d of vegetables and fruit for 4 wk (weeks 5–8)1
View this table:
TABLE 4. Lipid profiles and blood glucose in subjects who consumed 2, 5, or 8 servings/d of vegetables and fruit for 4 wk (weeks 5–8)1
Compliance of the subjects with the study protocol was controlled by measuring plasma concentrations of vitamin C and carotenoids. No significant differences in the concentrations of vitamins C and E and total carotenoids were seen between the groups at baseline and after 4 wk of the low V+F diet (Table 5). Plasma total carotenoid concentrations at the end of the study were significantly higher in the subjects who consumed 5 and 8 servings/d than in the subjects who consumed 2 servings/d (P < 0.01), which confirmed compliance of the study subjects. Plasma concentrations of vitamins C and E did not differ significantly between the groups throughout the study period.
View this table:
TABLE 5. Plasma concentrations of vitamin C, vitamin E, and total carotenoids in subjects who consumed 2, 5, or 8 servings/d of vegetables and fruit for 4 wk (weeks 5–8)1
Ex vivo cytokine production
The ex vivo production of cytokines by lipopolysaccharide-activated PBMCs (TNF- and IL-12) and concanavalin A–activated PBMCs (IFN-, IL-2, and IL-13) was not significantly modulated by the V+F intervention (Table 6). The percentages of subjects with the TNF*1/TNF*1, TNF*1/TNF*2, and TNF*2/TNF*2 genotypes were 65%, 32%, and 3%, respectively. TNF- production was unrelated to either TNF*1 or TNF*2 genotype (data not shown).
View this table:
TABLE 6. Cytokine concentrations in supernatant fluid of peripheral blood mononuclear cells activated ex vivo with mitogens1
Activity of NK cells and lymphocyte proliferation
The percentage of CD56+ NK cells in PBMCs and the percentage of NK cell cytotoxicity against K562-target cells were not significantly affected by the dietary intervention (Table 7). The expression of the natural cytotoxicity receptor NKp46, which is restricted to resting and activated NK cells, did not differ significantly between the groups at the end of the intervention trial. No significant correlation between NK cell activity and the expression of NKp46 independent of V+F intakes was measured at any time, whereas the percentage of CD56+ NK cells significantly correlated with NK cell cytotoxicity in all groups (data not shown). Lymphocyte proliferation was also not significantly changed by the dietary intervention (Table 7).
View this table:
TABLE 7. Natural killer (NK) cell cytotoxicity (25:1), percentage of peripheral blood mononuclear cells expressing the NK cell marker CD56 and the natural cytotoxicity receptor NKp46+, and lymphocyte proliferation1
Plasma CRP concentrations
Plasma CRP concentrations were measured after 4 wk of low V+F intake and after an additional 4 wk of 2, 5, or 8 servings/d of V+F. After 4 wk of a low V+F intake, no significant differences in plasma CRP concentrations were observed between groups. However, shifting from a low intake to 8 servings/d of V+F significantly reduced plasma CRP concentrations compared with the subjects who continued to consume a low amount of V+F (P < 0.05) (Figure 2). After 4 wk of 8 servings/d of V+F, plasma CRP concentrations were inversely correlated with plasma ß-carotene (r = –0.514, P = 0.016) and -carotene (r = –0.413, P = 0.062) concentrations. No significant correlation was observed with the other carotenoids.
FIGURE 2.. Mean (±SEM) change in plasma C-reactive protein (CRP) concentrations between weeks 4 and 8 in subjects who consumed 2 servings/d of vegetables and fruit for 4 wk (low intake) followed by 2, 5, or 8 servings/d of vegetables and fruit for 4 wk. n = 21 per group. *Significantly different from the 2 servings/d group at week 8, P < 0.05 (Dunnet's post hoc test). CRP concentrations (in mg/L) at week 4 were 1.51 ± 0.39 (2 servings/d group), 0.85 ± 0.21 (5 servings/d group), and 1.41 ± 0.40 (8 servings/d group), P = 0.424 (ANOVA).
DISCUSSION
The main objective of the present study was to investigate whether low or high V+F intakes affect cancer-related immune functions in healthy, nonsmoking men. Our study indicated that short-term restriction in the intake of V+F as well as short-term intervention with high amounts of V+F in well-nourished, nonsmoking men has no major effects on immune functions.
The intake of V+F (which included fruit juices) of our study subjects at baseline was above the German average, when compared with consumption data provided by the European Prospective Investigation into Cancer and Nutrition Study (23). The high plasma vitamin C and total carotenoid concentrations at baseline reflect this high intake. After 4 wk of low intake, vitamin C and total carotenoid concentrations were still high. Only after 8 wk of a low V+F intake did vitamin C concentrations tend to decline. In contrast, medium and high intakes significantly increased plasma total carotenoid concentrations at the end of the study, which confirmed observations made by others (24).
Our immunologic measurements focused on NK cell functions, such as cytokine production and cytotoxic activity. NK cell activity contributes to the elimination of tumor cells (2). V+F-associated constituents that may modulate this activity could in part explain the cancer-preventive effect that is associated with a high intake of V+F. NK cells produce many immunoregulatory cytokines, including IFN- and TNF- (25); conversely, cytokines that are produced by other immune cells, such as IL-2 and IL-12, modulate NK cell functions (26). Therefore, we included IFN-, TNF-, IL-2, IL-12, and IL-13 in our immunologic measurements. None of the cytokines was significantly affected by the dietary intervention. A study showed that the effect of fish oil on TNF- production was associated with the TNF- polymorphism (27). This prompted us to study whether the potential effects of different V+F intakes on TNF- production depended on the TNF- polymorphism. In our study, however, TNF- production was independent of the TNF*1 and TNF*2 genotype and was not significantly affected by V+F intakes. Similar frequencies of the TNF*1 and TNF*2 alleles to the ones found in the present study were observed by others (27). We previously showed that an intervention with vegetable juices can stimulate the production of IL-2 and TNF- in carotenoid-depleted subjects, whereas a low intake of carotenoid-rich food by itself reduced TNF- production ex vivo (5, 6). In contrast, such vegetable juices did not modulate cytokine production in well-nourished elderly subjects with no dietary restrictions and with high plasma vitamin C and carotenoid concentrations (7). These studies suggest that the additional intake of vegetable juices stimulates cytokine production only in subjects with low-carotenoid diets, which could explain the findings of the present study.
No significant changes in the number of CD3–/CD56+ NK cells, the expression of NKp46, and NK cell cytotoxicity were observed between the subjects with a high or low V+F intake. NKp46 belongs to a group of natural cytotoxicity receptors whose surface density correlates with their ability to kill various tumor cells (28). Again, our earlier studies suggest that the good nutritional status of our study subjects, even after 4 wk of low V+F intake, may have inhibited any immunomodulatory effects (6, 7). However, in both a cross-sectional and a prospective epidemiologic study, an increased intake of green vegetables was associated with higher NK cell cytotoxicity (4, 29).
CRP is a nonspecific indicator of inflammatory processes, and its plasma concentration highly correlates with cardiovascular disease risk (30, 31). Recently, a prospective study showed that plasma CRP concentrations are elevated in persons who subsequently develop colon cancer (32). The major effect observed in our study was the reduction in plasma CRP concentrations within 4 wk in subjects who consumed 8 servings/d of V+F compared with the subjects who consumed 2 servings/d. CRP was inversely associated with ß-carotene and -carotene, which suggests that increased V+F intake is related to the observed changes in CRP. Other factors that are known to affect inflammation and plasma CRP concentrations, such as blood glucose and lipids, did not differ significantly between the groups. Epidemiologic studies suggest that a high intake of V+F, dietary fiber, vitamin C, or carotenoids, as well as a prudent diet, is inversely associated with plasma CRP concentrations (33–37), which supports the outcome of the present study. Our intervention study is the first to show that plasma CRP concentrations can be modulated by the consumption of V+F.
One limitation of the present study was that an intervention with 5 or 8 servings/d of V+F not only results in an increased supply of plant-derived nutrients, but also may induce changes in macronutrient intake, such as fat and carbohydrates (38, 39). The analysis of the food records, however, indicated that the macronutrient intakes were similar in all groups. This suggests that the observed changes in CRP concentrations were related to the V+F intake.
In conclusion, our study suggests that a short-term low intake of V+F does not significantly modulate immune functions in well-nourished, nonsmoking men. Having 5 or 8 servings/d of carotenoid-rich V+F in such a diet for a period of 4 wk also does not significantly change immune functions. In well-nourished persons, longer periods of low V+F intakes are probably required to induce changes in immunologic functions, as was suggested by the minor changes in plasma vitamin C after 8 wk of 2 servings/d. However, reduced plasma CRP concentrations in the subjects who consumed 8 servings/d of V+F indicate that inflammatory processes are reduced in subjects who consume high amounts of V+F despite similar micronutrient intakes in all groups.
ACKNOWLEDGMENTS
We thank F Kratzer, S Demirel, T Gadau, E Hoch, M Giorgi-Kotterba, S Merkel, S Müller, G Schultheiss, U Stadler-Prayle, P Koch, and K Appenzeller for their excellent technical assistance; the subjects for taking part in this study; and L Korn for his statistical consultation.
BW developed the initial idea, measured immunologic markers, and drafted the manuscript. JM and AB recruited and checked the volunteers, planned and scheduled meals and V+F allowances, oversaw the kitchen personnel, and handled, collected, and stored all specimens. JM performed the analysis of the food frequency questionnaire and dietary records. SEK measured the carotenoids and vitamins C and E in plasma. SWB measured the genetic polymorphism. AB measured CRP concentrations. BW and AB performed the statistical analyses. All coauthors participated in critically revising the manuscript. None of the authors had any conflicts of interest.
REFERENCES