1 Department of Epidemiology and International Health, School of Public Health, University of Alabama at Birmingham, Birmingham, AL 35294, USA
2 Department of Biochemistry, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana
3 Department of Environmental Toxicology, Texas Tech, Lubbock, TX, USA
4 College of Veterinary Medicine, Faculty of Toxicology, Texas A&M, College Station, TX, USA
5 Peanut Collaborative Research Support Program, College of Agricultural and Environmental Sciences, University of Georgia, Griffin, GA, USA
Correspondence to: P. E. Jolly; E-mail: jollyp{at}uab.edu
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Abstract |
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Keywords: aflatoxin B1, cellular immunity, leukocyte phenotypes
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Introduction |
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Significant decreases in splenic CD4 (Th) cell numbers and IL-2 production have been observed in mice treated with AFB1 (20). Several studies show that AF might impair the function of macrophages in different animal species (2123). Phagocytoses of alveolar macrophages and tumor necrosis factor (TNF)- release were suppressed in rats and mice exposed to AF through the respiratory tract (24). AF exposure has also been shown to inhibit phygocytic cell function in normal human peripheral blood monocytes in vitro (25). AFB1 at concentrations
100 pg ml1 was cytotoxic to the monocytes, and concentrations of 0.51 pg ml1 inhibited monocyte phagocytic activity and intracellular killing of Candida albicans. Natural killer (NK) cell-mediated cytolysis of YAC-1 target cells in BALB/c mice has also been shown to be suppressed by AFB1 (26). More recently, a study conducted in weanling piglets exposed to low doses of AF showed that AF alters many aspects of humoral and cellular immunity in piglets and resulted in a reduction in primary and secondary immune responses and in antibody response in immunization with Mycobacterium agalactiae (27). The effect of AF on humoral immunity is not as evident as the effects on cell-mediated immunity, is less consistent in different species and seems to require higher doses of AF (20).
Although much data on the immune effects of AF are available from animal studies, there is a paucity of data on the effect of chronic consumption of AF-contaminated food on the human immune system. A recent study conducted in Gambian children reported that secretory IgA in saliva may be reduced by dietary levels of AF (28). The potential role of AFB1 in modifying the peripheral distribution of lymphocyte subsets and their function in humans has never been assessed. This information is urgently needed, especially as immune suppression is a significant global health problem and immune-suppressive/modulating diseases such as HIV/AIDS, malaria and other parasitic infections are rampant in developing tropical countries such as Ghana. In these countries, millions of people are chronically exposed to AF in their diet. We examined the distribution and function of lymphocyte subsets in Ghanaians in relation to AFB1albumin adduct levels in their plasma and present the first comprehensive report of the effect of AF on the cellular immune system in humans.
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Methods |
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Determination of AFB1albumin adduct levels in plasma by radioimmunoassay
AFB1albumin adduct levels in plasma of study participants were determined by radioimmunoassay (RIA) as published previously (3031). The assay measures AF that is covalently bound to albumin in peripheral blood and reflects AF exposure in the previous 23 months. Briefly, human plasma samples were concentrated by high-speed centrifugal filtration. The concentrated protein was re-suspended in PBS and the amount of human plasma albumin and total protein was determined. The total protein was then digested with Pronase (Calbiochem, La Jolla, CA, USA) after which the sample was mixed with ice-cold acetone and kept at 4°C for 1 h. The suspension was then centrifuged and the resulting supernatant containing the bound AF was decanted and dried in vacuo using a Savant Speed-Vac Concentrator. The RIA procedure (3031) was used to quantify AFalbumin adducts in duplicate human plasma protein digests, each containing 2 mg protein. Non-specific inhibition in the assay was determined by processing pooled normal human plasma standards obtained from Sigma (St Louis, MO, USA) and the average values of the background were subtracted from those of test samples in calculating AFB1albumin adduct levels. The standard curve for the RIA was determined using a non-linear regression method (32) and values were expressed as the amount of AFB1 per milligram of albumin (30).
Determination of percentages of leukocyte immunophenotypes using flow cytometry
We determined the percentages of certain leukocyte immunophenotypes in PBMCs from the study participants. The percentages of T cells (CD3+), subsets of T cells (CD4+ and CD8+), B cells (CD19+), NK cells (CD3CD56+) and macrophages (CD14+) were measured by flow cytometry. CD8+ T cell subset classification has been proven to be useful in monitoring the immune system in several clinical situations (33). Therefore, we classified CD8+ T cell subsets into naive (CD8+CD45RA+CD27+), memory (CD8+CD45RACD27+) and effector (CD8+CD45RA+CD27) CD8 T cells by flow cytometry. Subtypes of NK cells CD3CD56brightCD16dim and CD3CD56dimCD16bright were determined. The percentages of activated marker CD69 on CD3+ T cells and CD19+ B cells were also measured.
PBMCs were incubated with combinations of FITC-, PE-, PerCP-labeled mAbs against CD3, CD4, CD8, CD14, CD16, CD19, CD56, CD45RA, CD27 and CD69 (BD PharMingen, San Diego, CA, USA) for 30 min at 4°C. Isotype-matched irrelevant FITC-, PE- and PerCP-labeled mAbs (BD PharMingen) were used as controls in the experiments. After washing the cells three times in PBS, cell fluorescence for each phenotype was analyzed using Becton Dickinson (San Diego, CA, USA) FACS and CELLQuest software.
Lymphoproliferative response of CD4+ T cells
We measured CD4+ T cell proliferative response using 5-bromo-2-deoxyuridine (BrdU, Sigma) incorporation assay by flow cytometry (34). PBMCs (1 x 106) were placed in 12 x 75-mm tissue culture tubes containing 2 ml of medium with or without PHA (Sigma). The cultures were incubated at 37°C in a humidified 5% CO2 atmosphere for 3 days. BrdU was added to the cells at a final concentration of 40 µM 16 h before the end of the 3-day incubation period. After incubation, cells were collected and stained with an anti-BrdU mAb conjugated to FITC (Becton Dickinson) together with a PE-coupled anti-CD4 mAb (Becton Dickinson) in the presence of deoxyribonuclease (Sigma). Thereafter, at least 10 000 cells were analyzed by flow cytometry.
Cytokine production by CD8+, CD4+ and CD3CD56+ cells
The cytokine profile of participants was analyzed using intra-cytoplasmic staining of cytokine in PBMC samples. This technique enables phenotypic characterization of cytokine-producing cells and, therefore, allows a more accurate and detailed comparison of the cytokine profile of blood lymphocytes at a single cell level.
CD4+ Th cytokine profile (IL-4, IFN-,) was examined after stimulation ex vivo with phorbol myristate acetate (PMA) and ionomycin. The proportions of CD4+ cells that stained positive for IFN-
and IL-4 were determined by a combination of cell-surface and intracellular mAb staining and analysis by flow cytometry. CD8+ T cell cytokine production (IFN-
, IL-4, perforin and granzyme A) was measured by intracellular cytokine staining and multiparameter flow cytometry. Also, we examined cytotoxic function of NK cells by detecting perforin and TNF-
expression in phenotypically defined NK cells (CD3CD56+).
For intracellular cytokine staining, PBMCs (1 x 106) were placed in 12 x 75-mm tissue culture tubes containing 2 ml of medium containing 0.5 µg each of CD28 and CD49d mAbs with or without PMA (Sigma). These cultures were incubated at 5° slants at 37°C in a humidified 5% CO2 atmosphere for 6 h. In the last 5 h, 10 µg ml1 of the secretion inhibitor, Brefeldin A (Sigma), was added. After incubation, the cells were collected in PBS and washed once with cold PBS containing 1% BSA. Cells were then re-suspended in 100 µl of staining buffer (PBS supplemented with 0.1% sodium azide and 1% fetal bovine serum (FBS) pH 7.4; BD PharMingen) and the phenotypic mAb (CD3, CD4, CD8 and CD56) and incubated at 4°C for 30 min. After staining, the cells were washed with PBS and re-suspended in 1 ml of fix/perm buffer (BD PharMingen). The cells were then fixed for 30 min at 4°C, washed, re-suspended in perm-staining buffer and incubated with cytokine antibodies (anti-IFN-, anti-IL-4, anti-perforin, anti-granzyme A and anti-TNF-
; BD PharMingen) for 30 min at 4°C. The cells were then washed with perm-staining buffer and re-suspended in fixative buffer (BD PharMingen) for flow cytometric analysis on a Becton Dickinson FACS using CELLQuest software.
Determination of monocyte phagocytic function
We examined phagocytosis of peripheral blood monocytes by flow cytometry using the method of Steinkamp et al. (35) with some modifications. The concentration of cell suspension was adjusted to 4 x 106 cells ml1 in medium without serum. Fluorescent latex particles (2.5% latex solids, 2.0 µm in diameter; Polyscience, Warrington, PA, USA) were diluted 10 times with medium and 4 µl of the suspension of these particles added to 1 ml of cell suspension. The mixture was allowed to react at 37°C for 90 min with gentle shaking in a water bath. The cells were then washed three times with cold PBS (pH 7.4) to terminate the reaction and stained with PE-labeled anti-CD14 mAb. The final pellet was re-suspended with PBS and applied to flow cytometry to analyze the phagocytosed fluorescent particles. The phagocytic rate, which represents the percentage of cells with ingested particles relative to the total number of CD14+ cells, was determined.
Statistical analysis
Data were entered and analyzed using Windows SPSS version 10.0 (SPSS Inc., Chicago, IL, USA). Statistical analysis was performed using the MannWhitney U-test. Correlation analyses were performed between the different immune variables and AFB1 levels. Multiple linear regression analysis was employed to assess the relationship of AFB1 levels in plasma with age, gender, percentage of T and B cells and other immune variables. Dummy variables were used to assess the effect of categorical variables or continuous variables where the relationship was not linear. A probability value of P < 0.05 was considered statistically significant.
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Results |
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Percentages of leukocyte phenotypes in relation to AFB1 levels
When the mean percentages of the various leukocyte subsets of the study group were examined according to high and low AFB1 levels, there were no differences in the percentages of CD3, CD4, CD8, CD14, CD19 and CD3CD56+ cells between the two groups (Fig. 2). Although the mean percentages of T (CD3+) and B (CD19+) cells were not significantly different between participants with high and low AFB1 levels, the mean percentages of CD3 and CD19 cells showing the CD69 activation marker (CD3+CD69+ and CD19+CD69+) were significantly lower (2.164 ± 1.24 and 6.92 ± 4.15, respectively) in participants with high AFB1 than in those with low AFB1 (3.905 ± 2.47 and 11.945 ± 7.07, respectively; P = 0.002 for both) (Fig. 3).
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For CD8+ T cells, there was no significant difference in the mean percentages of IFN-- and IL-4-expressing cells between the high-AFB1 and low-AFB1 groups (Table 1). However, the percentages of CD8+ T cells containing perforin and CD8+ cells containing both perforin and granzyme A were significantly lower in participants with high AFB1 compared with those with low AFB1 (Table 1, Fig. 4). There was no difference in percentages of cytotoxic effector (CD8+CD45RA+CD27), naive (CD8+CD45RA+CD27+) or memory (CD8+CD45RACD27+) CD8+ T cells in the two groups (Table 1).
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Correlation and multivariate analyses
By correlation analysis, factors that were significantly associated with higher AFB1 level included low CD3+CD69+ cells (r = 0.321, P = 0.016) and CD19+CD69+ cells (r = 0.334, P = 0.010). That is, study participants with high levels of AFB1 had significantly lower percentages of CD3+CD69+ and CD19+CD69+.
Multiple linear regression analysis was employed to assess the relationship between AF levels and age, percentage of T and B cells and other variables. After adjusting for age and other immune parameters, the percentage of CD3+CD69+ and CD19+CD69+ cells showed strong negative associations with AFB1 levels (b = 0.574, P = 0.001 and b = 0.330, P = 0.032, respectively).
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Discussion |
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In this study, 100% of the participants had detectable AFB1albumin adducts. In Ghana, basic staples such as groundnuts, maize and other grains are contaminated with levels of AF that far exceed the 30 p.p.b. considered tolerable in foods for human consumption. Levels of AF up to 22 168 p.p.b. have been found in 71% of damaged groundnut kernels in market samples (36). While there are much available data on AF levels in staples such as peanuts and maize marketed and consumed in Ghana (3637), there are no data on dietary intake of AF by Ghanaians. Based on AFB1albumin adducts in our participants and previous work conducted by Wang et al. (38) on AF in high-risk populations in China, we estimated that our study participants consumed at least 10 µg of AFB1 in their foods daily.
When the percentages of the different leukocyte subsets were estimated according to high and low AFB1 levels, we found statistically significant differences between the groups in the percentages of CD3 and CD19 cells that express the CD69 activation marker and CD8+ T cells that express perforin and both perforin and granzyme A. The percentages of CD3+CD69+ and CD19+CD69+ in participants with high AFB1 were significantly lower than in participants with low AFB1. The activation marker CD69 (previously known as activation inducer molecule) is a 60-kDa glycosylated co-stimulatory molecule for T-lymphocyte proliferation which is expressed early after triggering of CD2 or CD3/TCR complex. T-lymphocyte activation via CD69+ will progress to proliferation, thus amplifying immune responses (39). In our study, the decreased number of activated T and B lymphocytes in participants with high AFB1 may indicate that the toxic effects of AF on lymphocytes may decrease their expression of the CD69 molecule, which is essential for a normal immune response against an infectious agent or a vaccine. The CD69 down-regulation on T lymphocytes at the cell level could partially contribute to the inability of these cells to mount immune responses.
The non-significant differences in the frequency of CD4+ IFN-- and IL-4-expressing cells observed in our study are similar to those found by Marin et al. (27) when they measured expression of mRNA regulatory cytokines in the blood of weanling piglets fed diets contaminated with AF for 30 days. The latter authors found no effect of AF on regulatory cytokines produced by either the Th1 or Th2 lymphocyte subsets (27). Similar results were also obtained in mice that received oral gavage with 30700 µg AF kg1 body weight (40) or in rats that received a weekly dose of 0.1 or 1 mg of AF by intra-gastric inoculation (41). Thus, the lack of effect of AF on regulatory cytokines produced by either the Th1 or the Th2 lymphocyte subsets in our results in humans is in agreement with results obtained in mice, rats and pigs that received AF (27, 4041).
We observed no significant difference in proliferation of CD4+ T cells between the two groups. Previous experiments in pigs showed that AF decreased lymphocyte proliferation (4243). Raisuddin et al. (44) found a dose-dependent suppression of the proliferative response of T and B cells in weanling rats fed with AFB1 for 4 weeks. However, a study using human cells and the MTT assay found that AFB1 at concentrations up to10 µg ml1 had no effect on lymphocyte proliferation (45). These results suggest that AFB1 could induce different T cell responses, depending on the type and amount of exposure to the toxin, the species involved and the method of exposure. Also, it may be possible that the changes observed in proliferation in vitro in relation to AFB1 levels could possibly be induced by acute aflatoxicosis. Our study participants are chronically exposed to subacute amounts of AF.
Effector-type CD8+ T cells can kill target cells by releasing a pore-forming protein (perforin) and serine proteases (granzymes) into the vicinity of target cell membranes. In response to antigen, this population of effector-type CD8+ cells with high levels of granzyme, perforin and Fas ligand expression that exert cytolytic activity contributes to the inhibition of pathogen spread through immediate lysis of infected cells. In our study, participants with high AFB1 levels had significantly lower levels of perforin-expressing and perforin- and granzyme A-expressing CD8+ T cells. This suggests that the CD8+ T cell function in individuals with high AFB1 level is impaired. This will definitely affect cellular immune function against infectious diseases.
NK cells are one component of the innate immune system and have the ability to both lyse target cells and provide an early source of immunoregulatory cytokines (46). NK cells can be divided into subsets based on their cell-surface density of CD56. The majority of human NK cells has low-density expression of CD56 (CD56dim) and express high levels of FcR III (CD16), whereas a smaller percentage of NK cells are CD56brightCD16. A study conducted by Cooper et al. (47) suggests that the major function of the CD56bright NK cells during the innate immune response in vivo might be to provide macrophages and other antigen-presenting cells with early IFN-
and other cytokines, promoting a positive cytokine feedback loop and efficient control of infection. Functional CD56+ NK cells can be generated in vitro from CD34+ bone marrow progenitors and they need IL-2 for full expression (48). The high-affinity form of IL-2R is expressed on the CD56bright NK subset and the latter is highly sensitive to IL-2 (44). In our study, we found a lower percentage of CD56bright NK cells in those with high AFB1. This suggests that AF may cause a decline in the availability of IL-2 and CD56bright NK cells that express the high-affinity IL-2R.
Exposure of human peripheral blood monocytes to different concentrations of AFB1 in vitro showed that AFB1 significantly impaired the phagocytic and microbicidal activity of the monocytes (24). Previous studies conducted in mice on the effect of AF on alveolar macrophages after aerosol inhalation or intra-tracheal instillation of AF by rats and mice (25) or on peritoneal macrophages isolated from mice fed AF in their diet (23) also showed that AF suppressed macrophage phagocytic activity. However, a study that investigated the effect of AF fed in the diet to weanling pigs found that AF did not alter the relative number of monocytes in peripheral blood similar to our study in humans (27). We also found no significant difference in phagocytosis by monocytes from our study participants in relation to AFB1 levels. Further studies need to be conducted to determine the effects of dietary AF exposure on human monocytes/macrophages.
In summary, we describe the differential subset distributions and functional alterations of specific lymphocyte subsets between study participants with high and low levels of AFB1. The major changes in the constitution of lymphocyte subsets are a decrease in activated T cells and B cells and significantly lower levels of perforin- and granzyme A-expressing CD8+ cytotoxic T cells in those with high AFB1 compared with those with low AFB1. These alterations in immunological parameters in participants with high AFB1 levels could result in impairments in cellular immunity in these individuals that could decrease their resistance to infections. Immune suppression by a toxicant can result from various mechanisms such as decreased protein and/or DNA synthesis, changes or loss in enzymatic activity and changes in metabolism or cell cycles, which may result in apoptosis or necrosis (49). Our findings should be considered exploratory, given the cross-sectional design of this study and because there may be other variables not considered such as other mycotoxins that may affect immune cell distribution and function. Other mycotoxins, such as fumonsins, ochratoxins, gliotoxin, patulin, zearaleone and trichothecenes including deoxynivalenol, have been reported to modulate the immune system (45, 5052). The co-occurrence of fumonsins and AFs has been shown in maize from Ghana (53) and co-occurrence of fumonsins, trichothecenes and AF demonstrated in maize from China (54). Further clarification of the immune effects of mycotoxins and of other possible immune correlates of AF exposure might be afforded by additional investigation.
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Acknowledgements |
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Abbreviations |
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AF | aflatoxins |
AFB1 | aflatoxin B1 |
BrdU | 5-bromo-2-deoxyuridine |
FBS | fetal bovine serum |
FITC | fluorescein isothiocyanate |
MTT | (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide; Thiazolyl blue) |
PBS | Phosphate buffered saline |
PE | polyethylene |
PerCP | peridinin chlorophyll a protein |
PMA | phorbol myristate acetate |
RIA | radioimmunoassay |
TNF | tumor necrosis factor |
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Notes |
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Received 26 October 2004, accepted 22 March 2005.
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References |
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