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The Journal of Nutrition Vol. 128 No. 7 July 1998,
pp. 1063-1069
in a Human Intestinal Epithelial Cell Line1,2
,, 3
* Department of Nutrition Sciences, Department of International Health and ** Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The secretory immunoglobulin A (IgA) antibody response to infections of mucosal surfaces requires transport of IgA from the basal to apical surface of mucosal epithelial cells by a specific transport protein, the polymeric immunoglobulin receptor (pIgR). We have tested the hypothesis that the vitamin A metabolite all-trans retinoic acid (RA) is required for the regulation of pIgR expression by the cytokines interleukin-4 (IL-4) and interferon-
(IFN-) in HT-29 cells, a well-differentiated human epithelial cell line derived from a colonic carcinoma. pIgR expression is upregulated by IFN- and IL-4 when HT-29 cells are grown in normal media, but this upregulation was significantly lower when cells were grown in vitamin A-depleted media. Treatment with RA at concentrations from 10
9 to 105 mol/L restored normal levels of pIgR expression. The percentages of cells expressing cell-surface pIgR after 24, 48 and 72 h of treatment with RA, IL-4 and IFN- were 66 ± 10, 90 ± 5 and 92 ± 1, respectively, significantly higher than the percentages seen without RA treatment, which were 32 ± 2.3, 72 ± 1.2 and 30 ± 7, respectively. In addition, the intensity of fluorescence of pIgR-positive cells was significantly higher in the RA-treated cultures than in the cultures without RA treatment. Similarly, pIgR mRNA levels (adjusted for 
-actin mRNA levels) in RA-supplemented cultures were 404, 105 and 949% higher at 24, 48 and 72 h, respectively, than were pIgR mRNA levels in identical cultures grown in the absence of RA. These data indicate that RA strongly interacts with IL-4 and IFN- to regulate pIgR expression in HT-29 cells, suggesting that vitamin A may be required for proper in vivo regulation of IgA transport in response to mucosal infections.
Vitamin A supplementation of young children living in areas where vitamin A deficiency is a significant public health problem decreases mortality from childhood infections (Fawzi et al.1993, Glaziou and Mackerras 1993). Although such supplementation programs often do not decrease the incidence of common illnesses such as diarrhea and respiratory tract infections, vitamin A deficiency has been associated with an increased risk of developing such infections (Bloem et al. 1990 Although several studies have examined the effects of vitamin A on development of the intestinal, antigen-specific IgA response Cell line.
The human colonic adenocarcinoma cell line HT-29 (Chintalacharuvu et al. 1991 Reagents.
Recombinant human IFN- Charcoal-treated serum.
FBS was absorbed with charcoal (Sigma) to deplete the serum-retinol content. Serum (500 mL) was absorbed with charcoal (10 g/L) by rotating for 1 h at room temperature. The serum was then centrifuged for 500 × g for 5 min at 4°C to pellet the charcoal and sterile-filtered with a 0.45-µm filter. This procedure was then repeated two more times. The retinol content of charcoal-absorbed serum was determined with the use of HPLC (Waters Model number 610, Novapak C-18 reverse-phase column, 100% methanol mobile phase and UV at 325 nm) as described (Stacewicz-Sapuntzakis et al. 1987 Cell culture protocol.
A typical experimental plan for cytokines and RA treatment was as follows: on d 1, cells were passed from DMEM containing 10% normal FBS to DMEM containing 20% charcoal-treated FBS at a 1:2 split. The medium was changed every 48 h. On d 6, cells (2 × 106 cells/well) were passed to six-well plates (Falcon, Franklin Lakes, NJ) and again cultured in DMEM containing 20% charcoal-treated FBS. On d 7, the medium was again changed to remove nonadherent cells and to initiate cytokine and RA treatments, which lasted from 24 to 72 h, when cells were processed for analysis by flow cytometry or Northern blot. Controls were carried with the same volume of diluting medium used for cytokines and RA. The stock concentration of IL-4 was 2.5 × 107 U/L in calcium and magnesium-free PBS (CMF-PBS) containing 10 g/L bovine serum albumin; that of IFN- Flow cytometric analysis of pIgR protein expression.
Flow cytometric analysis was performed to determine the level of pIgR expression on the cell surface (Phillips et al. 1990 SDS-PAGE and immunoblot analysis.
Electrophoresis was performed in 10% polyacrylamide slab gels in Tris-HCl buffer as described (Laemmli 1970 RNA extraction and Northern blot analysis.
Total RNA was isolated from HT-29 cells (Chomczynski and Sacchi 1987
Statistical analysis.
Means were compared between two groups using Student's t test. Means were compared among groups of three or four treatments using ANOVA. After ANOVA, significant differences among means were determined using the least significant difference calculation (Snedecor and Cochran 1967 Selection of IL-4 and IFN- IL-4 and IFN- RA is required for stimulation of cell-surface expression of pIgR by IL-4 and IFN-
RA is required for upregulation of pIgR mRNA by IL-4 and IFN-
Effects of RA on IL-4 or IFN-
We have shown that IL-4- and IFN-
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Sommer et al. 1984), and at least two studies have shown that supplementation can decrease the risk of developing diarrhea (Biswas et al. 1994, Lie et al. 1993). The mechanism by which vitamin A deficiency might increase the risk of such infections is not certain. However, the secretory immunoglogulin A (IgA)4 system is the principal arm of the immune response that protects the upper respiratory and enteric tracts from reinfection by pathogenic organisms to which a specific immune response has already been elicited. This IgA response involves local production of IgA by plasma cells in mucosal sites followed by its transport onto mucosal surfaces or into glandular secretions such as saliva, bile and tears via the polymeric immunoglobulin receptor (pIgR) (Mestecky et al. 1991). Thus, an impaired IgA response could increase the risk of developing respiratory or enteric infections.
showing that it is specifically decreased by vitamin A deficiency the effect of vitamin A on pIgR expression by gut epithelial cells has not been specifically examined (Davis and Sell 1989, Puengtomwatanakul and Sirisinha 1986, Rombout et al.1992, Sirisinha et al. 1980, Wiedermann et al. 1993). Retinoic acid (RA) is a physiologically active metabolite of vitamin A, which acts by binding to the nuclear retinoic acid receptor (RAR) that regulates gene expression by binding to response elements upstream of affected genes (Mangelsdorf et al. 1994). Because vitamin A, or RA, is required for the normal differentiation of epithelial cells (DeLuca 1991), it is plausible that vitamin A deficiency could affect the secretory IgA response by decreasing pIgR expression. We tested this hypothesis in vitro by examining the effects of RA on pIgR expression by HT-29 cells, a human colonic adenocarcinoma cell line (Fogh and Trempe 1975). pIgR expression is stimulated in HT-29 cells by the T-cell cytokines interleukin-4 (IL-4) and interferon- (IFN-) (Denning 1996, Phillips et al. 1990), as well as by the monokine tumor necrosis factor-
(Kvale et al. 1988). These cytokines have been shown to be produced by lymphocytes isolated from the human intestinal lamina propria (Youngman et al. 1994). It was also shown that transforming growth factor- can upregulate pIgR expression in a rat intestinal epithelial cell line (McGee et al. 1991).
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
, Fogh and Trempe 1975, Huang et al. 1976, Kvale et al. 1988) was maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco Laboratories, Grand Island, NY) with low glucose concentrations (1 g/L), 1% penicillin, streptomycin, Fungizone mixture (Gibco), and 10-15% heat inactivated fetal bovine serum (FBS) (Gibco). Cells were propagated at 37°C in 5% CO2 and allowed to grow until confluent. For passage, cells were treated with 0.25% trypsin in 10 mmol/L EDTA (Gibco) for 3-4 min. Before passing the cells, cell viability was checked with 0.4% trypan blue in 8.5 g/L NaCl solution (trypan blue exclusion test). Cell viability always exceeded 95%.
and IL-4 expressed in Escherichia coli were purchased from Sigma Chemical (St. Louis, MO). Rabbit serum was purchased from Gibco and bovine serum albumin was purchased from ICN Immunochemical (Lisle, IL). Retinoic acid (all-trans) and other vitamin A analogs were purchased from Sigma and were diluted in 95% ethanol.
). The retinol concentration of the charcoal-absorbed serum was consistently <20 g/L, compared with 
130 g/L for untreated serum. When 20% charcoal-treated FBS was used in the DMEM medium for growing cells, the final retinol concentration was ~1 × 108 mol/L. The charcoal-treated serum was stored at 
20°C.
was 5 × 106 U/L in CMF-PBS. The concentration of stock all-trans-RA was 104 mol/L in ethanol. Final concentrations of IL-4, IFN- and all-trans-RA were from 5 to 100 × 104U/L, 5 to 75 × 104 U/L, and 1012 mol/L to 105 mol/L, respectively. Sham-treated controls were treated with the appropriate volume of diluent.
). Single-cell suspensions were prepared as follows: monolayers of cells were washed first with cold CMF-PBS, trypsinized and pipetted vigorously to make a single-cell suspension. Cells were pelleted by centrifuging at 300 × g for 5 min at 4°C to remove trypsin, then resuspended in the appropriate medium (i.e., DMEM containing 20% charcoal-treated FBS, cytokines and all-trans RA). To allow re-expression of cell-surface pIgR protein cleaved by trypsin, cells were incubated for 2 h (5% CO2, 37°C) in suspension culture in 60-mm untreated Petri dishes (Falcon). Use of untreated tissue culture dishes prevented adherence of cells. Cells were then transferred to 15-mL Falcon tubes, washed with cold CMF-PBS, and incubated with fluorescein isothiocyanate (FITC)-labeled rabbit anti-human pIgR antibody (A495:A280 ratio = 0.63, final concentration 0.3 g/L; Dakopatts, Palo Alto, CA) in a final volume of 100 µL at 4°C for 30 min. Cells were then washed three times with cold CMF-PBS at 4°C and fixed with 10 g/L paraformaldehyde. Stained and fixed cells were examined directly on a Zeiss Model 18 microscope, using a 50-W mercury lamp, or were analyzed by flow cytometric analysis using a Becton Dickinson FACStar Model (Mountain View, CA) equipped with an Argon laser tuned to 488 nm. To confirm the specificity of the anti-pIgR antibody, we measured the nonspecific binding of an irrelevant antibody, an FITC-labeled rabbit anti-human immunoglobulin 
-chain antibody with an A495:A280 ratio of 0.58 (produced in our laboratory) at a final concentration of 0.30 g/L. No specific staining was seen by direct immunofluorescence analysis or by flow cytometry, nor did the intensity or pattern of staining change upon treatment with IL-4, IFN- or 106 mol/L RA.
). To determine the free or bound pIgR in HT-29 cells cultured as described, equal numbers of cells were treated with lysing buffer and electrophoresed under reducing conditions (1% -mercaptoethanol) at 0.5 mA/cm in a Bio-Rad minigel apparatus (St Louis, MO). Secretory IgA was used as a standard. Proteins were transferred to Immobilon-P (Millipore, Bedford, MA) (Burnette 1981). Membranes were reacted with biotinylated goat anti-secretory component followed by peroxidase-labeled neutravidin. The pIgR bands were visualized by the addition of the Supersignal (Pierce, Rockford, IL) chemiluminescent substrate and exposure to autoradiography film. Two experiments were performed with triplicate and quadruplicate samples for each of the four standard RA and cytokine treatments at 48 and 72 h.
) and 20 µg per lane was run on an 10 g/L agarose gel using 0.66 mol/L formaldehyde as a denaturant in 1X MOPS (3-[N-morpholino]propanesulfonic acid) buffer (Sambrook et al. 1989). After running, the gel was soaked with several changes of diethylpyrocarbonate-treated water to remove the formaldehyde. RNA was then transferred to Nytran membrane (Schleicher and Scheull, Keene, NH) by using the Posiblot system (Stratagene, LaJolla, CA) and was cross-linked with the use of UV light (Stratalinker, Stratagene). The blot was prehybridized for 3 h at 42°C in 50% formamide, 6X SSPE buffer (Sambrook et al. 1989), 5X Deinhardt's reagent and 100 mg/L sheared herring sperm DNA. Probes were labeled with 32P-dCTP by the polymerase chain reaction (PCR) (Piskurich et al. 1995). A plasmid carrying a 3.9-kb XbaI-EcoRI insert containing a human pIgR cDNA clone was generously provided by Per Brandtzaeg and Peter Krajci (Krajci et al.1989) (GeneBank accession # M24559). The pIgR PCR primers spanned nucleotides 31 to 49 (5'-GCTAACCTCACCAACTTCC-3') and 513 to 531 (5'-GTCTTCATAAACCAGCTCG-3'). A plasmid (GeneBank accession # M10277) containing a human -actin genomic clone was obtained from the American Type Culture Collection (Cat. # 65129; Rockville, MD) (Adams et al. 1991). The -actin PCR primers spanned nucleotides 626 to 644 (5'-GCGTGACATTAAGGAGAAG-3') and 1069 to 1088 (5'-GTCATACTCCTGCTTGCTG-3'). Separate PCR labeling reactions were performed for each probe with 0.1 U Taq polymerase (Promega, Madison, WI) in 50 µL reaction by using the manufacturer's reaction buffer and 1.5 mmol/L MgCl2, 20 µmol/L each of dATP, dGTP and dTTP, 0.5 µmol/L 32P-dCTP (NEN, Boston, MA), 1 nmol/L plasmid template and 0.25 µmol/L of each primer. Reactions were carried out for 38 cycles using the following profile: 94°C, 1 min; 53°C, 1 min; and 72°C, 1 min. Unincorporated label was then removed by using a Chroma-Spin-100 column (Clontech Laboratories, Palo Alto, CA). The probe was added to the same prehybridization solution at ~1 GBq/L. Hybridization at 42°C continued for ~18 h. The blot was then washed twice in 2X SSPE containing 1 g/L SDS at 42°C for 10 min, twice in 0.1X SSPE containing 1 g/L SDS at 55 C for 15 min, and twice in 0.2X SSPE containing 1 g/L SDS at 24°C for 15 min. Autoradiographs were scanned and densities of bands determined with the Model GS-670 Imaging Densitometer (Bio-Rad, Hercules, CA).
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Fig 1.
Growth of HT-29 cells under standard experimental conditions. Cells were plated at a density of 2 ×105 cells/cm2 and were treated as follows: retinoic acid (RA) + interleukin-4 (IL-4) + interferon- (IFN-): 106 mol/L RA, 1 × 105 U/L IL-4 and 5 × 105 U/L IFN-; IL-4 + IFN-: 1 × 105 U/L IL-4 and 5 × 105 U/L IFN-; control: treated with the same volume of diluents used for the RA and cytokines. Values shown are means ± SD (n = 3 at each point). Symbols identified by different letters at the same time point represent means that are significantly different, P < 0.05.
).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
concentrations.
Previous work has shown that IL-4 and IFN- synergistically increase the expression of pIgR in HT-29 cells (Denning 1996, Phillips et al. 1990). To define conditions for subsequent experiments, HT-29 cells grown in 20% FBS were treated with IL-4 and IFN- each at concentrations of 0, 0.5, 1 and 5 × 105 U/L for 48 h. In these preliminary experiments, the peak of pIgR expression was achieved at IL-4 concentrations 1 × 105 U/L and IFN- concentrations 5 × 105 U/L. Thus 1 × 105 U/L IL-4 and 5 × 105 U/L IFN- were used in subsequent experiments. Under these conditions, ~50-80% of cells expressed cell-surface pIgR by flow cytometric analysis, whereas expression was typically <20% (although there was variation from experiment to experiment) without IL-4 and IFN- treatment (data not shown).
stimulate HT-29 cell growth.
Our next goal was to determine if treatment of HT-29 cultures with IL-4 and IFN-, both in the presence and absence of RA, affected HT-29 cell growth in vitamin A-depleted cultures. HT-29 cells grown in vitamin A-depleted medium for 6 d were passed into 6-well plates at a density of 2 × 106 cells per well (2 × 105 cells/cm2). On the following day IL-4, IFN- and RA were added and the growth monitored for 3 d. Growth was greater in the presence of the cytokines, whereas addition of RA with the cytokines did not further increase the number of cells in culture by 3 d (Fig. 1). Viability was consistently >95% with all treatments.
in HT-29 cells.
Our principal goal in these experiments was to define the effects of RA on cytokine-stimulated pIgR expression. We first confirmed that normal, cell-surface expression of pIgR could be stimulated by RA, IL-4 and IFN- using vitamin A-depleted culture media. HT-29 cells grown in vitamin A-depleted medium and treated for 48 h with 106 mol/L RA, 1 × 105 U/L IL-4 and 5 × 105 U/L IFN- were fixed in paraformaldehyde and examined by immunofluorescent antibody analysis using an FITC-labeled rabbit anti-pIgR antibody. Under these conditions, a smooth pattern of cell-surface fluorescence was seen on a majority of cells (Fig. 2A), as was expected for a cell-surface glycoprotein such as pIgR. Few positive cells were seen when IL-4, IFN- and RA were not added to the medium (sham treatment; Fig. 2C), nor was any surface staining seen when an irrelevant control antibody (FITC-labeled rabbit anti-human -chain antibody) was used to stain the cells treated with RA, IL-4 and IFN-.
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Fig 2.
Immunofluorescence (panels A and C) and phase contrast (panels B and D) photomicrographs of paraformaldehyde-fixed, HT-29 cells that were treated for 48 h with 10 6 mol/L retinoic acid (RA), 1 × 105 U/L interleukin-4 (IL-4) and 5 × 105 U/L interferon- (IFN-) (panels A and B) or were sham-treated (panels C and D).
on pIgR expression. Figure 3 shows flow cytometry profiles of pIgR expression from representative cultures treated as follows: A) sham-treated cultures, B) 106 mol/L RA, C) 1 × 105 U/L IL-4 and 5 × 105 U/L IFN-, and D) 106 mol/L RA, 1 × 105 U/L IL-4 and 5 × 105 U/L IFN-. Discrete populations of pIgR-positive and pIgR-negative cells can be readily distinguished. Neither RA alone nor IL-4 plus IFN- alone significantly increased the percentage of cells expressing pIgR or the fluorescence intensity of positive cells. Treatment with RA together with IL-4 and IFN- increased both the percentage of pIgR-positive cells and the intensity of fluorescence (Fig. 3D). These differences were shown to be significant and reproducible when compared in experiments using triplicate cultures for each treatment (Fig. 4). Sham-treated cultures typically showed a low percentage of cells expressing pIgR (~20% positive) and cultures treated with RA only showed no significant difference from this basal level, indicating that treatment of HT-29 cells with RA alone did not affect pIgR protein expression. Treatment of HT-29 cells with IL-4 and IFN- alone did increase the percentage of cells expressing pIgR to between 20 and 36% (Figs. 3 and 4). However, treatment with both RA and the combination of IL-4 and IFN- consistently caused surface pIgR expression in >50% of cells, significantly greater than when cultures were treated with IL-4 and IFN- alone. In addition, the positive cells in the cultures treated with RA plus IL-4 and IFN- showed a greater fluorescence intensity than did positive cells from the other three treatments (Fig. 4). This suggests that the number of cell-surface pIgR molecules expressed per cell is greater with RA treatment. These data clearly show that RA is required for IL-4 and IFN- to stimulate the maximum level of pIgR expression in HT-29 cells grown in vitamin A-depleted conditions when the cultures were examined 48 h after treatment.
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Fig 3.
Flow cytometry profiles (x-axis, fluorescence intenisty in arbitrary units; y-axis, number of cells) of polymeric immunoglogulin receptor (pIgR) expression from representative cultures of HT-29 cells treated for 48 h as follows: A) sham-treated cultures, B) 10 6 mol/L retinoic acid (RA), C) 1 ×105 U/L interleukin-4 (IL-4) and 5 × 105 U/L interferon- (IFN-), and D) 106 mol/L RA, 1 ×105 U/L IL-4 and 5 × 105 U/L IFN-. Positive and negative cells were differentiated by gating at a fluorescence intensity of 11 units, as indicated by the vertical lines in each panel. The percentage of cells positive in panels A through D are 21, 28, 31 and 73%, respectively. The dotted line in panel A represents unstained cells (0% positive).
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Fig 4.
Percentage of cells expressing polymeric immunoglobulin receptor (pIgR) determined by flow cytometry analysis of triplicate cultures of HT-29 cells treated for 48 h as follows: control: sham-treatment; retinoic acid (RA): 10 6 mol/L RA; Cyt: 1 × 105 U/L interleukin-4 (IL-4) and 5 × 105 U/L interferon- (IFN-); RA + Cyt: 106 mol/L RA, 1 ×105 U/L IL-4 and 5 × 105 U/L IFN-. Bars and error bars represent the mean ± 1 SD. Bars identified by different letters represent means that are significantly different, P < 0.05. The mean ± SD intensities of positive cells from the control, RA, Cyt and RA + Cyt cultures were 60 ± 2, 57 ± 5, 70 ± 6 and 105 ± 7 arbitrary intensity units, respectively (least significant difference by ANOVA, P < 0.05 = 11, n = 3 replicates per treatment).
at concentrations 109 mol/L. In one representative experiment (n = 2 observations per experiment) we found that the mean percentages of positive cells in cultures treated with 0, 1012, 109 and 106 mol/L RA were 50, 50, 64 and 82%, respectively, whereas the mean intensities of positive cells were 77, 71, 94 and 162 intensity units, respectively. Significant increases (P < 0.05) in both the fluorescence intensity of positive cells and the percentage of cells expressing pIgR were consistently seen at RA concentrations 108 mol/L in this and other experiments.
. As shown in Figure 5, the percentage of cells expressing pIgR in cultures treated with RA plus IL-4 and IFN- was 206% of the level seen in cultures treated with IL-4 and IFN- alone 24 h after treatment, 125% of that level 48 h after treatment and 307% of the cytokine-only level 72 h after treatment. Similarly, the intensity of positive cells in the cultures treated with RA, IL-4 and IFN- was greater at all time points than was the intensity of positive cells in the IL-4 and IFN- treatments. At 24, 48 and 72 h, the intensities in the RA plus IL-4 and IFN- treatments were 129, 189 and 335% of the levels seen in the cytokine-only cultures, respectively. The percentage of cells expressing pIgR in the sham-treated cultures stayed low and was always lower than that in both the cytokine-only and RA plus IL-4 and IFN- treatments at all time points. However, the intensity of fluorescence of positive cells in the sham-treatment cultures was not significantly different from that seen in the cytokine-only treatment at 24 and 72 h, whereas the cytokine-only treatment did show significantly greater fluorescence intensity at the 48-h time point. These data confirm that RA allows IL-4 plus IFN- to stimulate pIgR expression in a greater percentage of HT-29 cells at all time points and also suggests that the level of pIgR expression per cell is increased by RA treatment. We also examined pIgR expression at 48 and 72 h by immunoblot analysis in total cellular extracts and found that total cellular pIgR levels (cell surface plus cytoplasmic) was modulated by RA treatment essentially as shown in the flow cytometric analysis presented in Figure 5 (data not shown).
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Fig 5.
Percentage of cells expressing polymeric immunoglobulin receptor (pIgR) (lower panel) and intensity of fluorescence of positive cells (upper panel) determined by flow cytometry analysis of triplicate cultures of HT-29 cells treated for 24 to 72 h as follows: control: sham-treatment; interleukin-4 (IL-4) + interferon- (IFN-): 1 × 105 U/L IL-4 and 5 × 105 U/L IFN-; retinoic acid (RA) + IL-4 + IFN-: 106 mol/L RA, 1 × 105 U/L IL-4 and 5 × 105 U/L IFN-. The data shown are means ± 1 SD. Symbols identified by different letters at the same time point represent means that are significantly different, P < 0.05.
.
RA also increased steady-state pIgR mRNA levels (Fig. 6), in addition to increasing pIgR protein expression. The maximum effect of RA on expression of pIgR mRNA was also seen 72 h after treatment with RA, IL-4 and IFN-. Total cellular RNA extracts were analyzed by Northern blot analysis using probes for pIgR and for -actin. As shown in Figure 6, pIgR mRNA was clearly identified 24, 48 and 72 h after treatment with RA plus IL-4 and IFN-. pIgR mRNA bands were typically not identified in the RA-only and sham treatments at any time point. Thus the intensities of the pIgR bands relative to -actin (pIgR:-actin ratios) were 
0.38 (range, 0-0.38) at all time points in the RA-only and sham-treated cultures, representing little or no detectable pIgR mRNA (as seen in Fig. 6). In the cultures treated with IL-4 and IFN-, the pIgR:-actin ratios at 24, 48 and 72 h were 0.27, 2.03 and 0.49, respectively, showing significant increases (above the sham or RA treatment) in pIgR mRNA expression at 48 and 72 h. In the cultures treated with RA plus IL-4 and IFN-, the pIgR:-actin ratios were 1.09, 2.14 and 4.65, respectively, representing significant increases at all time points. Using these ratios, we see that the level of steady-state pIgR mRNA in the RA plus IL-4 and IFN- cultures at 24, 48 and 72 h was 404, 105 and 949% greater, respectively, than that seen in the cultures treated with IL-4 and IFN- only. Expression of pIgR mRNA was examined at each time point in three to five independent experiments. The relative expression of pIgR in the RA plus IL-4 and IFN- treatment was always greater than that seen in the IL-4 plus IFN- treatment at 24 and 72 h, with no consistent difference being evident at 48 h (data not shown).
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Fig 6.
Northern blot analysis of 10 µg of total RNA from HT-29 cells treated for 24 to 72 h with the indicated treatment or combination of treatments: retinoic acid (RA): 10 6 mol/L; interleukin-4 (IL-4) + interferon- (IFN-): 1 × 105 U/L IL-4 and 5 × 105 U/L IFN-. Values on the left indicate molecular mass markers for single-stranded RNA. The locations of the polymeric immunoglobulin receptor (pIgR) and -actin mRNA bands are identified by arrows.
-induced pIgR upregulation.
Although IL-4 and IFN- act synergistically to increase pIgR expression, the effect of each cytokine when administered independently is not equally dependent on RA. Treatment of HT-29 cells with 5 and 7.5 × 105 U/L of IFN- in the absence of added RA significantly increased the percentage of cells expressing pIgR, to levels 216 and 190%, respectively, of that seen in sham-treated control (Fig. 7). In parallel cultures treated with 106 mol/L RA, expression was not significantly higher in the 5 x 105 U/L culture, and was only slightly higher in the 7.5 × 105 U/L culture. In contrast, treatment of HT-29 cells with 1 × 105 or 1 × 106 U/L of IL-4 in the absence of added RA did not alter the percentage of HT-29 cells expressing pIgR, compared with the sham-treated control. However, treatment with both RA and IL-4 increased fluorescence intensity to levels 188 and 288% of that seen in the parallel cultures without RA, respectively (Fig. 7). These data suggest that the ability of HT-29 cells to respond to IL-4 stimulation requires the presence of RA, whereas responsiveness to IFN- can be augmented by, but is not dependent on, RA.
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Fig 7.
Percentage of cells expressing polymeric immunoglobulin receptor (pIgR) determined by flow cytometry analysis of triplicate cultures of HT-29 cells treated for 48 h with the indicated concentrations of interleukin-4 (IL-4) and interferon- (IFN-) in the presence (cross-hatched bar) and absence (open bar) of 106 mol/L retinoic acid (RA). Bars and error bars represent the mean ± 1 SD. Bars identified by different letters represent means that are significantly different, P < 0.05.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
-mediated pIgR mRNA and protein expression by HT-29 cells is impaired under vitamin A-depleted culture conditions, but is restored by the addition of physiologic concentrations of RA. Flow cytometric and Northern blot analyses revealed a pattern of increasing pIgR protein and mRNA expression from 0 through 48 h, followed by a plateau lasting at least through 72 h. This pattern has also been reported in HT-29 cells when a quantitative immunoblot method was used to measure total cellular pIgR (Denning 1996). Although addition of IL-4 and IFN- alone did stimulate variable levels of pIgR mRNA and protein expression at the 48 h time point in some experiments (Fig. 5), this level of expression may be due to the slow conversion of the low concentration of retinol that was still present in the charcoal-depleted culture medium (108 mol/L) into RA, rather than to an RA-independent mechanism, although our current experimental methods do not rule out the latter possibility. To completely rule out a vitamin A-independent mechanism would require that these cells be cultured in serum-free medium. Our attempts to culture HT-29 cells under such conditions were not successful.
-mediated upregulation of pIgR expression. We have not specifically examined the mechanisms behind this observation in this publication, but the options seem clear. The presence of a retinoic acid response element (RARE) upstream of the pIgR gene, acting in conjunction with cytokine response elements, could explain this interaction. The pIgR region of human genomic DNA has been cloned, and the 5' sequences are currently being analyzed for functional regulatory elements (Krajci et al. 1992). The outcome of this analysis may indicate whether RA directly regulates pIgR mRNA expression. Other mechanisms of regulation are also possible. Stimulation of mRNA expression via cytokine receptors requires at least two steps, binding of the cytokine to its cell-surface receptor and receptor-mediated signal transduction to regulate gene expression. Although pIgR expression in response to each cytokine administered independently is quite weak, we found that cell-surface pIgR expression stimulated by IL-4 was impaired to a greater extent in vitamin A-depleted culture conditions than was the upregulation induced by IFN-. This suggests that an IL-4-dependent step may be the principal point in pIgR expression that is sensitive to RA. One possibility is that RA is required for IL-4 receptor expression. There is ample precedent for such a mechanism. For example, RA regulates the expression of both the and chains of the human IL-2 receptor (Bhatti and Sidell 1994, Sidell et al. 1993). We have also found (Stephensen, C., unpublished observations) consensus sequences for RARE (Mangelsdorf et al. 1994) upstream of the murine IL-4 receptor gene (Wrighton et al. 1992), suggesting that RA may directly regulate IL-4 receptor expression, although the function of these RARE has not been directly demonstrated. It is also possible that RA is required for IL-4-or IFN--mediated signal transduction, as has been shown in HL-60 cells (Linnekin et al. 1992). In lymphocytes, both IL-4 and IFN- regulate gene expression via Janus kinases and the activity of separate protein transcription factors (signal transducers and activators of transcription) (Ihle et al. 1994). Although these mechanisms have not been extensively investigated in HT-29 cells, the synergistic action of IL-4 and IFN- on pIgR expression is diminished by inhibitors of tyrosine kinase activity, suggesting that similar mechanisms are involved (Denning 1996).
, Puengtomwatanakul and Sirisinha 1986, Rombout et al. 1992, Sirisinha et al. 1980, Wiedermann et al. 1993), suggesting that total secretory IgA levels may be decreased due to decreased pIgR expression, resulting in decreased IgA transport into the bile and intestinal tract. However, vitamin A deficiency seems to have the opposite effect on IgA secretion and pIgR expression at other sites. Total salivary IgA concentrations are significantly increased in vitamin A-deficient mice, although the antigen-specific response is diminished (Stephensen et al. 1996). Furthermore, vitamin A-deficient mice have higher levels of salivary gland pIgR mRNA expression than do control mice (Gangopadhyay et al. 1996), suggesting that IgA transport was increased rather than decreased in the salivary glands. This suggests that there may be a fundamental difference in the regulation of pIgR expression between the gut and salivary glands, either in the amount and types of cytokines produced or in the way in which pIgR-expressing epithelial cells respond to cytokine stimulation.
).
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FOOTNOTES |
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, interferon-; IgA, immunoglobulin A; interleukin-4 (IL-4); MOPS, 3-[N-morpholino]propanesulfonic acid; PCR, polymerase chain reaction; pIgR, polymeric immunoglobulin receptor; RA, retinoic acid; RAR, retinoic acid receptor; RARE, retinoic acid response element.
Manuscript received 27 October 1997. Initial reviews completed 7 January 1998. Revision accepted 23 February 1998.
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ACKNOWLEDGMENTS |
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We thank Sharon Blount for technical assistance in the conduct of these studies and Rose Kulhavy for anti-pIgR antibody preparation as well as technical assistance.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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enhances secretory component and major histocompatibility complex class I antigen expression on rat IEC- 6 intestinal epithelial cells.
Cytokine
1991;
3:543-550[Medline] activity in supernatants from stimulated human intestinal mononuclear cells prevents upregulation of the polymeric Ig receptor in an intestinal epithelial cell line.
J. Immunol.
1994;
153:675-681[Abstract]
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