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Nutritional Immunology

Vitamin A Deficiency Increases the In Vivo Development of IL-10–Positive Th2 Cells and Decreases Development of Th1 Cells in Mice^1,2

U.S. Department of Agriculture, Western Human Nutrition Research Center at the University of California, Davis, and Nutrition Department, University of California, Davis, CA 95616

³To whom correspondence should be addressed. E-mail: cstephensen{at}ucdavis.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Vitamin A deficiency impairs both T helper type 1 (Th1)- and type 2 (Th2)-mediated immune responses, although Th2 responses seem to be principally affected. Multiple mechanisms are involved in this immune suppression, but the hypothesis that deficiency affects development of Th1/Th2 memory cell phenotype has not been tested directly in vivo. To do so, lymphocytes from DO11.10 T cell receptor (TCR)-transgenic mice were transferred to vitamin A–deficient or control BALB/c recipients. Recipients were then immunized with the cognate peptide antigen for the TCR-transgenic DO11.10 T cells (OVA_323–339). After 2–5 wk, the transferred OVA_323–339–specific T cells were identified from draining lymph nodes with the TCR-clonotypic antibody KJ1–26, and their Th1/Th2 phenotype was characterized by intracellular cytokine staining after in vitro stimulation with phorbol myristate acetate and ionomycin. The percentage of CD4⁺KJ1–26⁺ cells positive for IL-10 was 100% greater in vitamin A–deficient mice (3.49 ± 0.41%; mean ± SE) than in control mice (1.74 ± 0.37%). IL-4 did not differ between groups. In addition, the percentages of CD4⁺KJ1–26⁺ cells from vitamin A–deficient mice that were positive for interferon (IFN)- (8.8 ± 0.73%) and interleukin (IL)-2 (39.5 ± 3.1%) were both lower than the percentages in control mice (11.4 ± 0.67 and 47.0 ± 2.8%, respectively). Thus vitamin A deficiency, at the time of initial antigen exposure, enhances the development of IL-10–producing Th2 or T regulatory cells and diminishes the development of Th1 memory cells.

KEY WORDS: • rodent • Th1/Th2 • immunomodulators • transgenic/knockout • vitamin A deficiency

Population-based studies showed that vitamin A supplements decrease the risk of death from infectious diseases (1), presumably by restoring innate and adaptive immune functions compromised by vitamin A deficiency (2). However, some community and clinical studies found that supplements may increase the severity of respiratory infections (3,4) or the risk of vertical transmission of HIV (5). The reasons for these disparities are unclear but highlight the need to understand the mechanism by which vitamin A affects immune function.

Mechanistic studies are difficult to pursue in human subjects, and rodent models have provided valuable insights into the effect of vitamin A deficiency on immune function. For example, vitamin A deficiency decreases T-lymphocyte–mediated antibody responses (6,7), a finding that was replicated in humans (8). These and other studies indicate that immune responses mediated by T-helper type 2 (Th2)⁴ cells, such as IgG1, IgE, and IgA antibody responses, are impaired by vitamin A deficiency, whereas some aspects of Th1-mediated responses may be enhanced. In particular, production of interferon (IFN)- (a Th1 cytokine) on a per cell basis is increased during vitamin A deficiency and can be directly decreased by treatment with retinoic acid, the principal active metabolite of vitamin A (9,10). Because IFN- can impede the development of Th2 responses, this may be one mechanism by which vitamin A deficiency decreases T-lymphocyte–mediated antibody responses.

Because Th2 responses are diminished by vitamin A deficiency, it is logical to ask whether the number of Th2 memory cells may be lower in vitamin A–deficient mice. The answer to this question has implications for long-term immunity. For example, if the number of Th2 cells is decreased, this could mean that the memory response would remain impaired even after correction of vitamin A deficiency. Two principal approaches have been taken to address this question. First, lymphocyte populations from vitamin A–deficient and control mice were analyzed for the presence of cells (presumably T lymphocytes) capable of providing help to IgG1-producing B lymphocytes. Such functional studies indicate that vitamin A deficiency decreases the number of such cells, indirectly demonstrating a reduction in Th2 numbers (11) (12). A limitation of such studies is that increased production of IFN- could also have the same effect as decreased production of Th2 cytokines such as interleukin (IL)-4. Another study used an Elispot method to demonstrate that the number of IL-5 producing cells was decreased by vitamin A deficiency although the number of IFN- producing cells was not affected (10). Although the type of Th1/Th2 cytokine was identified in this study, the specific identity of the cells was not established.

In the present study, we addressed the question whether vitamin A deficiency alters the development of Th1 and Th2 memory cells using an adoptive transfer model. Naïve T lymphocytes from DO11.10 /ß-T-cell receptor (TCR) transgenic mice (13) were transferred into vitamin A–deficient and control BALB/c mice. The DO11.10 TCR recognizes amino acids 323–339 of ovalbumin (OVA_323–339) and can be identified using the clonotypic monoclonal antibody KJ1–26 (14). After transfer, recipient mice were immunized with OVA_323–339 in incomplete Freund’s adjuvant (IFA) to stimulate development of memory T lymphocytes. The Th1/Th2 phenotype was determined 2–5 wk after immunization by intracellular cytokine staining. Vitamin A–deficient mice had a higher frequency of IL-10–positive Th2 memory [or T regulatory (Treg)] cells and a lower frequency of IFN-- and IL-2–positive Th1 memory cells than did control mice. A similar difference in supernatant cytokine concentrations was found in splenocyte cultures restimulated with OVA_323–339 peptide.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Mice. DO11.10 mice were a gift from Casey Weaver, University of Alabama at Birmingham, and were bred in our facility. BALB/c mice were purchased from Charles River. This research was approved by the U.C. Davis Institutional Animal Care and Use Committee.

    Diet protocol. Pregnant mice consumed a semipurified vitamin A–deficient diet (#88407; Teklad Diets) ad libitum beginning on gestation d 14. The number of pups per litter was balanced within 24 h of birth so that litter sizes were similar between diet groups and so that no litter had >5 pups. Half of the litters in each experiment were then switched to the control diet containing 4000 IU vitamin A/kg as retinyl palmitate (Teklad diet #88406). Pups were weaned at 21 d of age to the same diet fed to the dams. Serum retinol was measured at 7 wk of age in 4 mice per group to determine whether the mice fed the vitamin A–deficient diet had serum retinol that was <0.50 µmol/L and significantly lower than that of the control diet group. This protocol and the diet composition were described previously (15).

Two experiments with identical protocols were conducted. Intracellular cytokine staining data were combined from the 2 experiments after statistical comparison showed that the results from these experiments were essentially identical. Lymph node and spleen cultures were performed in the second experiment for measurement of cytokine concentrations by ELISA.

    Adoptive transfer and immunization. After vitamin A deficiency was confirmed using serum retinol concentration, mice in the 2 diet groups received 5 x 10⁶ CD4⁺KJ1–26⁺ cells by tail vein injection. Splenocytes and lymph node cells from DO11.10 mice were pooled and analyzed by flow cytometry, as described below, to determine the number of total cells to be injected. The CD4⁺KJ1–26⁺ cells were not separated before transfer. Cells were suspended in HBSS. The total injection volume was 0.15 mL.

Three days after transfer, OVA_323–339 peptide (Alpha Diagnostic International) was diluted in PBS and emulsified with incomplete Freund’s adjuvant (IFA; Sigma). The final injection volume was 0.3 mL/mouse and contained 300 µg peptide. Mice were injected s.c. in both hind footpads (50 µL per site) at the base of the tail (100 µL) and the back of the neck (100 µL).

    Cell culture and intracellular cytokine staining. Two to 5 weeks after immunization, the spleen and 6 draining lymph nodes (brachial, axillary, popliteal) were collected immediately after killing into cold HBSS containing 2% fetal bovine serum (FBS). Tissues were disrupted with a few strokes of a Duall glass tissue homogenizer (Kontes Glass) in HBSS:2% FBS and filtered using filter-top 5-mL tubes (Falcon #352063) to eliminate debris. Cells were centrifuged for 10 min at 200 x g at 4°C and resuspended in Russ-10 media, made as described (16). The retinol concentration in the lot of FBS used at 10% in the Russ-10 media was 0.77 ± 0.011 µmol/L; thus, the concentration of retinol in the cell culture media was 0.077 µmol/L. Splenocytes were treated with lysis buffer to eliminate erythrocytes (17). Cells were resuspend in Russ-10 at 2 x 10⁶ cells/0.5 mL, and 0.5 mL/well was placed in a 48-well plate followed by 0.5 mL of stimulation medium. Stimulation medium consisted of Russ-10 medium plus PMA (Sigma; 40 µg/L) and ionomycin (Sigma; 2 µmol/L). Cells were then cultured at 37°C in 5% humidified CO₂. Brefeldin A was added to block cytokine secretion after 2 h at a final concentration of 10 mg/L. Incubation was continued for an additional 3 h.

After incubation, the contents of each well were then placed in 5 mL polystyrene tubes (Falcon) with 2 mL Dulbecco’s PBS containing 2% FBS and centrifuged at 200 x g for 10 min at 4°C. PBS:2% FBS was used for antibody dilutions and subsequent wash steps. The supernatant was then gently poured off and 1 µg F_c-block/million cells (Pharmingen #01241A) was added in a total volume of 10 µL/million cells. Cells were incubated for 15 min at 4°C. Antibodies for surface staining were then added in a total volume of 10 µL/million cells at a concentration of 0.2 µg/million cells and incubated for 20 min at 4°C in the dark. These antibodies included peridinin chlorophyll protein (PerCP)-labeled rat anti-CD4 (Pharmingen #553052), a PerCP-labeled rat IgG2a control antibody (Pharmingen #553933), allophycocyanin (APC)-labeled clonotypic TCR-specific mouse antibody KJ1–26 (Caltag #MM7505) and an APC-labeled mouse IgG2a control antibody (Caltag #MG2905). After surface staining, cells were washed once and then fixed with 0.5 mL 1% paraformaldehyde (PF) in calcium and magnesium-free PBS (1% PF). Cells were then stored at 4°C in the dark overnight.

On the following morning, cells were centrifuged at 200 x g at 4°C, the 1% PF buffer gently poured off, and 250 µL Cytofix/Cytoperm buffer (Pharmingen) was added. Cells were incubated at 4°C in the dark for 15 min. Cells were then washed once with 2 mL Perm/Wash buffer (Pharmingen) and stained with cytokine-specific and control antibodies (0.5 µg/million cells) diluted in Perm/Wash buffer (10 µL/million cells) for 60 min at 4°C in the dark. Cells were stained with phycoerythrin (PE)-labeled anti-IL-4 (Pharmingen #554435) or a PE-labeled control rat IgG1 (Pharmingen #553925) plus fluorescein (FITC)-labeled anti-IFN- (Pharmingen #554411) or FITC-labeled rat IgG1 control antibody (Pharmingen #553995). In addition, a separate tube of cells was stained with PE-labeled anti-IL-10 (Pharmingen #554467), or a PE-labeled control rat IgG2b (Pharmingen #553989) plus FITC-labeled anti-IL-2 (Pharmingen #554427), or a FITC-labeled rat IgG2b control antibody (Pharmingen #553988). Cells were then washed with 2 mL Perm/Wash buffer and resuspended in 0.5 mL 1% PF buffer.

Cells were analyzed using a FACSCalibur 4-color flow cytometer (Becton Dickenson) as outlined in Figure 1. Duplicate cultures were prepared from each mouse for each pair of cytokine antibodies, and a single culture was used for each isotype control pair; 200,000–300,000 events in the lymphocyte gate were collected for each culture. To correct for nonspecific staining, the percentage of cells positive with the appropriate isotype control antibody was subtracted from the percentage of cells positive with the cytokine-specific antibody. Means of the duplicates were determined and used for statistical analysis. Four mice (2 deficient and 2 control) were analyzed on a given day to help control for possible day-to-day variation. During wk 3 and 4, values for IL-4 from most mice were negative (after correction for the isotype control), and these values were adjusted to zero.

View larger version (61K): [in this window] [in a new window]   FIGURE 1 Flow cytometric analysis for identification of cytokine-producing CD4+KJ1–26+ T-cells in draining lymph nodes of BALB/c mice after adoptive transfer of lymphocytes from DO11.10 mice and immunization with OVA323–339 peptide. Cells were treated with PMA and ionomycin to stimulate cytokine production. Lymphocytes were identified by forward and side scatter (A, D). CD4+KJ1–26+ T cells from the lymphocyte gate were then identified as double-positive cells (B, E). The percentages of such double-positive cells positive for IL-10, IL-2 (C), IL-4 or IFN- (F) were then enumerated.

    Cell culture and cytokine ELISA. Lymph node and spleen cells were cultured at 2 x 10⁶ cells/mL in Russ-10 media containing either 6 mg/L OVA_323–339 or an equal volume of diluent. Cultures were then placed in wells of a 48-well (0.5 mL) plate (Nunc) and cultured at 37°C in 5% humidified CO₂ for 7 d. Individual wells were harvested at 2, 4 or 5, and 7 d. Data from d 4 and 5 were analyzed together as a single day. Cells were pelleted by centrifugation at 200 x g at 4°C for 10 min. The supernatant was frozen for later analysis. IL-2, IL-4, IL-10, and IFN- were measured in cell culture supernatants by capture ELISA using antibodies, purified standards, and protocols suggested by the manufacturer (Pharmingen). The limit of detection of the 4 ELISAs was 0.03 µg/L.

    Statistical analysis. Statistical analysis was performed with the SigmaStat program (Jandel Scientific). A two-tailed P-value of 0.05 was used to determine statistical significance unless otherwise indicated. Serum retinol concentrations were compared between diet groups by 2-way ANOVA to control for gender, and then by Student’s t test because gender was not a significant factor. Body weights were measured weekly, and weights for males (M) and females (F) were compared separately between diet groups by 2-way repeated-measures ANOVA to control for changes in weight over time.

The percentage of CD4⁺KJ1–26⁺ cells in lymph nodes, the percentage of cells producing cytokines, and the concentration of cytokines from lymph node cultures were compared by two-way ANOVA with mice categorized by time after immunization and gender. Gender was a significant factor for predicting the percentage of CD4⁺KJ1–26⁺ cells in lymph nodes; thus, diet groups were compared separately for males and females by 2-way ANOVA controlling for time after immunization. Gender was not a significant predictor for cytokine-positive cells and cytokine concentrations in lymph node cultures. Thus, these variables were compared between diet groups using 2-way ANOVA to control for time after immunization. Cytokine concentrations from lymph node cultures were compared between diet groups only on days when an antigen-specific response occurred. When the OVA_323–339–treated culture had a significantly higher cytokine concentration (P < 0.05 by one-tailed t test) than did the unstimulated culture from the same mouse, the response was considered antigen specific.

Data from antigen-stimulated and unstimulated splenocyte cultures were available from 24 mice (deficient, n = 6 F, 6 M; control diet n = 6 F, 6 M) from the second experiment. Equal numbers of observations were available in each diet group on all culture days (d 2, 4/5 and 7) for OVA_323–339–stimulated as well as unstimulated cultures, for both males and females. Because of this balance, 3-way ANOVA could be used to compare cytokine concentrations among these groupings. The initial analysis simultaneously compared males vs. females, days in culture, and stimulated vs. unstimulated cultures. Cytokine concentrations did not differ between males and females; thus, the data from males and females were analyzed together to determine the effects of diet, days in culture, and antigen-specific stimulation.

Backwards, stepwise regression analysis was used to identify significant predictors of the percentage of cytokine-producing CD4⁺KJ1–26⁺ cells in lymph nodes using the categorical variables "gender" (female = 1, male = 0), "vitamin A deficiency" (control diet = 0, deficient diet = 1) and "experiment" (first experiment = 1, second experiment = 2), as well as the continuous variable "days postimmunization."

Results of 2- and 3-way ANOVAs are presented as means ± SE. Summary statistics and the results of t tests and 1-way ANOVA are presented in the text and figures as means ± SD.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Confirmation of vitamin A deficiency using serum retinol. Serum retinol was measured at 7 wk of age (n = 2 M and 2 F/diet group in each experiment). In both experiments, serum retinol was higher in control mice (1.15 ± 0.49 and 0.91 ± 0.37 µmol/L) than in mice fed the vitamin A–deficient diet (0.36 ± 0.11 µmol/L, P = 0.020; 0.30 ± 0.071 µmol/L P = 0.018, respectively).

    Body weights of vitamin A–deficient and control mice. Body weights were compared by diet for the 45 mice in the 2 experiments (including 5 unimmunized controls). In the first experiment (deficient diet; n = 4 M, 6 F; control diet; n = 3 M, 5 F), body weights were compared at 8, 9, 10 and 11 wk of age and mice were killed for analysis beginning at 11 wk of age. Males did not differ (P = 0.41) on the basis of diet, but there was an interaction of diet with age (P = 0.011) reflecting the fact that male mice fed the deficient diet began the study slightly heavier than control mice (mean of 24.3 vs. 23.4 g) but ended the study slightly lighter. Body weights did not differ in females due to diet (P = 0.68). In the second adoptive transfer study (deficient diet; n = 6 M, 8 F; control diet; n = 7 M, 6 F) body weights were compared at 5, 7, 8, 9, and 10 wk of age. Mice were killed beginning at 10 wk of age for analysis. Males did not differ (P = 0.81) on the basis of diet, but there was an interaction of diet with age (P = 0.013), again reflecting the fact that male mice fed the deficient diet began the study slightly heavier than control mice (mean of 22.2 vs. 20.7 g) but ended the study slightly lighter (24.6 vs. 25.5 g). Body weights did not differ in female mice due to diet (P = 0.062), although the deficient females were slightly heavier than the control mice at all time points.

    Percentages of CD4⁺-KJ1–26⁺ donor cells in draining lymph nodes. After adoptive transfer, CD4⁺-KJ1–26⁺ cells accounted for 3.2 ± 0.8% of total CD4⁺ cells in the lymph nodes of unimmunized mice fed the vitamin A–deficient diet (n = 2 M, 3 F). After immunization, the percentage of CD4⁺-KJ1–26⁺ cells decreased from 3.2 ± 1.2% (n = 9 F, 9 M) during wk 3 to 1.9 ± 1.0% (n = 6 F, 6 M) during wk 4 and remained stable at 1.6 ± 0.8% during wk 5 (n = 7 F, 3 M) (P = 0.002, wk 3 vs. 5; P = 0.015, wk 3 vs. 4). The percentages did not differ on the basis of diet, but male recipients had higher percentages of CD4⁺-KJ1–26⁺ cells than females at all time points: 3.5 vs. 2.5% in wk 3, 2.3 vs. 1.4% in wk 4 and 1.9 vs. 1.3% in wk 5 (P = 0.019 for gender comparison by 2-way ANOVA, comparing weeks and gender). Staining with anti-CD62L demonstrated that the mean percentage of CD4⁺-KJ1–26⁺ cells with a memory phenotype (CD62L_lo) was 73.5 ± 5.4% after immunization (n = 11 mice).

    Intracellular cytokine staining of CD4⁺-KJ1–26⁺ donor cells from draining lymph nodes. The mean percentages of cytokine-positive cells in unimmunized mice (n = 2 M, 3 F; vitamin A–deficient diet) were as follows: IL-4, 0.27 ± 0.40%; IFN-, 0.81 ± 0.32%; IL-10, 0.20 ± 0.30%; and IL-2, 22.9 ± 7.4%.

Vitamin A deficiency increased the percentage of memory T lymphocytes positive for the Th2 cytokine IL-10 (P = 0.003) (Fig. 2A). The overall percentage of IL-10 positive cells was 100% higher in vitamin A–deficient mice (3.49 ± 0.41%) than in control mice (1.74 ± 0.37%). This difference occurred during wk 3 (90% higher), wk 4 (170%), and wk 5 (70%) after immunization. This positive association of the percentage IL-10–positive cells with vitamin A deficiency was also present in the multiple regression analysis (Table 1). The overall percentage of IL-4–positive cells was quite low throughout the experiment, and the diet groups did not differ (P = 0.27; Fig. 2B, Table 1).

View larger version (26K): [in this window] [in a new window]   FIGURE 2 The effect of vitamin A deficiency on the percentage of antigen-specific CD4+KJ1–26+ T lymphocytes producing the Th2 cytokines IL-10 (A) and IL-4 (B), and the Th1 cytokines IL-2 (C) and IFN- (D). Percentages were determined by intracellular cytokine staining of draining lymph node cells treated with PMA and ionomycin nodes after adoptive transfer of lymphocytes from DO11.10 mice and immunization with OVA323–339 peptide. The numbers of vitamin A–deficient and control mice in wk 3, 4 and 5 were 9, 6, and 6, and 9, 6 and 4, respectively. Diet treatments were significantly different for IL-10 and IFN-, as indicated by the asterisks (*). Values are means ± SE.

    Cytokine concentrations after antigenic stimulation of draining lymph node cells. Draining lymph node cells not needed for flow cytometric analysis were used to assess antigen-specific cytokine production by ELISA (n = 10 control and 8 deficient mice). Unstimulated control cultures were included when cells were available (n = 5). An antigen-specific response did not occur for IL-4 but was present for IL-2 on d 2 and for IFN- and IL-10 on both d 4/5 and 7. Cytokine concentrations did not differ between the vitamin A–deficient and control groups (data not shown).

    Cytokine concentrations after antigenic stimulation of spleen cells. Concentrations of the Th2 cytokines IL-4 and IL-10 were both higher in antigen-stimulated splenocyte cultures from vitamin A–deficient mice than in cultures from control mice, whereas concentrations of the Th1 cytokines IL-2 and IFN- were both lower (Fig. 3). Although IL-10 concentrations did not differ by diet group when all days were compared (P = 0.41), within-day comparisons showed that the IL-10 concentration on d 4/5 was 19% higher in cultures from deficient mice than in cultures from control mice (P = 0.043) (Fig. 3A). The IL-4 concentration on d 2 was higher in cultures from the deficient mice than from the control mice (P < 0.001) (Fig. 3B) but subsequent days did not differ. On both d 2 (P = 0.01) and d 4/5 (P < 0.001), IL-2 concentrations were lower in cultures from vitamin A–deficient mice than in those from control mice (Fig. 3C), whereas IFN- concentrations were lower in cultures from deficient mice than from control mice (P < 0.001) on all days (Fig. 3D).

View larger version (24K): [in this window] [in a new window]   FIGURE 3 The effect of vitamin A deficiency on the concentration of the Th2 cytokines IL-10 (A) and IL-4 (B) and the Th1 cytokines IL-2 (C) and IFN- (D) in supernatants from splenocyte cultures from vitamin A–deficient and control mice following adoptive transfer of lymphocytes from DO11.10 mice and immunization with OVA323–339 peptide. Cells were restimulated with OVA323–339 peptide (OVA) or were treated with a vehicle control (no OVA). Diet treatments were significantly different for all cytokines on the days indicated by asterisks (*). Values are means ± SE, n = 12 deficient and 12 control.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

To minimize the effect of antigen-presenting cells and bystander cells on the assessment of Th1/Th2 phenotype after immunization, we chose to stimulate T-cell cytokine production directly using PMA and ionomycin. This approach was taken because vitamin A has multiple effects on antigen-presenting cell development and function. For example, vitamin A deficiency can increase IL-12 production by antigen-presenting cells and IFN- production by T cells (10). On the other hand, retinoic acid treatment during in vitro development of dendritic cells from peripheral blood monocytes can also result in the development of IL-12–producing antigen-presenting cells that promote Th1 development (25). Using PMA and ionomycin to stimulate cytokine production directly bypassed these potentially confounding effects on assessment of Th1/Th2 phenotype. Many stimuli could have been used to stimulate T-cell cytokine production directly. We evaluated anti-CD3 plus anti-CD28 antibodies in addition to PMA plus ionomycin and chose the latter because anti-CD3/anti-CD28 stimulation interfered with identification of CD4⁺KJ1–26⁺ cells by flow cytometry, presumably due to internalization of the TCR complex after antibody binding.

Immunization with the OVA_323–339 peptide transformed naïve DO11.10 T lymphocytes into memory cells, as shown by CD62L staining and Th1/Th2 cytokine profiles. We chose to immunize with the peptide not only to provide a potent antigenic stimulus for the adoptively transferred transgenic T lymphocytes, but also to minimize the response by host lymphocytes to other epitopes that might occur had we used intact ovalbumin protein as the antigen. The percentage of CD4⁺KJ1–26⁺ cells expressing Th1/Th2 cytokines from unimmunized mice after adoptive transfer was very low and increased after immunization, as expected with the development of a memory cell phenotype. The percentage of cells positive for the Th1 cytokine IFN- was 10%, although the percentage of IL-2–positive cells was much higher. Higher percentages of IFN-–positive cells (20–40% of CD4⁺KJ1–26⁺ cells) occurred in our laboratory after 2 immunizations using CFA followed by IFA, or using alum, or after in vitro stimulation with IL-12 treatment (60%), but the lower percentage response seen here was reproducible and appears to represent a robust memory response to a single immunization. Previous work comparing ovalbumin protein to OVA_323–339 immunization in BALB/c mice showed similar T-cell cytokine recall responses (24), supporting our conclusion that a peptide immunization produced a recall response similar to that induced by intact protein. The percentage of cells positive for the Th2 cytokine IL-4 was quite low throughout the study. Similar percentages were found using 2 immunizations, or after oral immunization with cholera toxin as an adjuvant (data not shown), although never as high as that after in vitro stimulation with IL-4 (30%). The percentage of IL-10–positive cells increased from wk 3 to 5 after immunization, as might be expected for a regulatory cytokine that can dampen T-cell–mediated immune responses (26).

The mice consuming the vitamin A–deficient diet in this study presented biochemical evidence of deficiency by the time of immunization, as indicated by low serum retinol levels, but they were not so deficient that body weight was affected. Although the rate of weight gain in vitamin A–deficient males was lower than in males fed the control diet, the potential confounding effect of inanition on immune function was avoided in this study because significant weight differences were not present at the time of immunization.

A principal finding of this study was that vitamin A deficiency doubles the frequency of antigen-specific, IL-10–producing CD4⁺ T cells. The magnitude of the difference between the diet groups increased over time and may represent a relative increase in abundance of T cells, Th2 or Treg, capable of downregulating immune responses (26). This is a novel observation and may represent a mechanism by which vitamin A deficiency could diminish the immune response to infectious or inflammatory diseases. This could include Th2-mediated responses, which are diminished by vitamin A deficiency (2), as well as Th1-mediated immune responses, which have received less attention, such as cytotoxic T-lymphocyte or delayed-type hypersensitivity responses (27–29). Research on regulatory T cells is in its infancy in many regards, and even the identification of which T cells are "regulatory T cells," i.e., induced vs. naturally occurring Treg cells, Tr1 cells, or Th3 cells, is still a point of contention (26,30,31). Thus, it is not clear whether the IL-10—positive cells identified in this study fall into this category of regulatory T cells. Clearly, some CD4⁺ T-cell types with regulatory activity act by producing IL-10 that downregulates immune responses. Thus, it will be of interest to determine whether the IL-10–positive cells identified here do indeed play a regulatory role.

How vitamin A deficiency affects the development of IL-10–producing T cells is not clear. The development of Treg cells, which produce IL-10 and little IL-2 (as seen in the present study), is facilitated by an early exposure to IL-10. However, previous work demonstrated lower antigen-specific IL-10 production in immunized, vitamin A–deficient mice (10). In addition, work from this laboratory showed no effect of retinoic acid treatment on IL-10 production during development of DO11.10 memory T cells cultured in vitro (16). Further characterization of these cells is required to clarify their role in immune suppression caused by vitamin A deficiency.

Vitamin A–deficient mice had 23% fewer IFN-—positive and 16% fewer IL-2–positive cells in draining lymph nodes than did mice consuming the control diet with adequate vitamin A. These differences indicate that vitamin A deficiency decreased development of Th1 memory cells, although the magnitude of the decrease was not large. Previous work using an Elispot method to enumerate IFN-—positive cells in vitamin A–deficient and control mice after immunization showed no difference between the diet groups, but the variability of the Elispot method (based on reported SE from that study) was such that a 20% difference would not have been detected (10). In vitro studies from our laboratory (16) with naïve DO11.10 T cells indicated increased Th2 development with 9-cis but not all-trans retinoic acid, although other groups reported increased Th2 development with all-trans retinoic acid using similar methods (32,33). The present findings suggest that in vitro methods may not adequately recapitulate the in vivo effects of vitamin A deficiency on Th1/Th2 development. For example, direct addition of retinoic acid to cell culture may by-pass the normal regulation of retinoic acid production that exists in vivo (34), thereby producing a temporary excess in the in vitro studies. Thus, in vitro treatment with retinoids may be more representative of a condition of vitamin A excess vs. deficiency, rather than adequacy vs. deficiency. This question could be addressed by assessing the effect of high-level dietary vitamin A on Th1/Th2 development using the DO11.10 adoptive-transfer model.

When cultures from draining lymph nodes were restimulated with peptide antigen, IFN- and IL-2 concentrations did not differ, even though the frequencies of IFN-–producing CD4⁺KJ1–26⁺ cells differed between the diet groups. However, the IL-2 concentration in the deficient cultures at 2 d was 21% lower than in the control cultures. This value is similar to the difference seen with intracellular cytokine staining; however, given the SD in these ELISA data, a much larger sample size (n = 64) would be required to identify a 20% difference as significant (assuming power = 0.80 and P-value = 0.05 for Student’s t test). A similarly large sample size would be required to detect a 23% difference in IFN- concentrations. Cytokines produced by non-CD4⁺KJ1–26⁺ cells would also be detected by ELISA, but not by flow cytometry, and may also be a factor in explaining the different findings with the 2 methods.

Splenocyte cultures from vitamin A–deficient mice in the present study had lower antigen-specific IFN- and IL-2 concentrations and higher IL-4 and IL-10 concentrations than did cultures from control mice. When data from all 3 poststimulation days were considered, the IFN- and IL-2 concentrations in cultures from the deficient mice were 41 and 60% of the levels in the control cultures, whereas the concentration of IL-4 was 181% of control levels (using data from d 2). These deficits for IFN- and IL-2 are greater than the 20% lower frequency of IFN-– and IL-2–positive CD4⁺KJ1–26⁺ cells in lymph nodes from deficient mice, but the direction of the difference is the same. It is possible that the differences in splenocyte cultures reflect even greater differences in frequencies of cytokine-producing CD4⁺KJ1–26⁺ cells than in draining lymph nodes. Higher IL-10 concentrations occurred in splenocyte cultures from deficient mice on 1 of 3 d in culture, which is consistent with the higher frequency of IL-10–producing CD4⁺KJ1–26⁺ cells in lymph nodes from deficient mice, although the magnitude of the difference is smaller.

Earlier studies showed that vitamin A deficiency increases antigen-specific production of IFN- in antigen-restimulated lymphocyte cultures on an absolute and per cell basis (10,35). The higher rate of IFN- was diminished by the addition of exogenous vitamin A. In contrast, in the present study, vitamin A deficiency decreased the production of IFN- in restimulated splenocyte cultures. One possible explanation for this phenomenon is that the use of 10% FBS in the culture media in the present study produced a less Th1-biased environment in vitro than in previous studies using serum-free medium, due to the presence of vitamin A or other nutrients or hormones in FBS that support lymphocyte and antigen-presenting cell function. Differences between the DO11.10 adoptive transfer system and previous methods may also be involved. For example, previous studies immunized with intact proteins or infectious agents, whereas the present study used a peptide. Because intact proteins require processing by antigen-presenting cells before presentation by major histocompatibility complex molecules, and these processes are affected by vitamin A (36), initial antigen presentation to naïve T lymphocytes could have been less affected by vitamin A deficiency in the present study than in previous studies. In addition, the present study used IFA as the adjuvant, whereas previous studies have used adjuvants such as CFA, which has greater Th1-inducing activity (23). Use of such an adjuvant may interact with vitamin A deficiency to affect Th1/Th2 development.

In summary, our data demonstrate that vitamin A deficiency at the time of initial antigen exposure significantly enhanced the development of IL-10–producing Th2 or Treg cells while diminishing the development of Th1 memory cells. This observation adds to our knowledge of how vitamin A deficiency affects Th1/Th2-mediated immune responses and suggests a novel mechanism by which vitamin A deficiency may diminish such responses. Such decreases in immune function may contribute to the increased risk of death from infectious diseases that is a hallmark of human vitamin A deficiency.

	ACKNOWLEDGMENTS

 
The authors thank Daniel Hwang, Darshan Kelley, and Susan Zunino for reading the manuscript and making helpful suggestions, Alina Wettstein for performing retinol analysis, and Pat Bucy for help with setting up the adoptive transfer model.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 04, April 2004, Washington, DC [Stephensen, C. B., Jiang, X. & Freytag, T. L. (2004) Vitamin A deficiency increases development of IL-10-positive Th2 or Treg cells. Experimental Biology meeting abstracts (accessed at http://www.biosis-select.org/faseb/index.html). FASEB J. 18: 37.2 (abs.)].

² Supported by U.S. Department of Agriculture National Research Initiative grant #97–35200–4229, USDA CRIS Project #5306–51530–006-00D and National Institutes of Health grant #1 R01 AI 50863.

⁴ Abbreviations used: APC, allophycocyanin; CFA, complete Freund’s adjuvant; F, female; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; IFA, incomplete Freund’s adjuvant; IFN, interferon; IL, interleukin; M, male; OVA_323–339, peptide comprised of amino acids 323 through 339 of ovalbumin; PE, phycoerythrin; PerCP, peridinin chlorophyll protein; PF, paraformaldehyde; PMA, phorbol myristate acetate; TCR, T cell receptor; Th1, T helper type 1; Th2, T helper type 2; Treg, T regulatory cell.

Manuscript received 7 May 2004. Initial review completed 2 June 2004. Revision accepted 7 July 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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