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*
Department of Pediatric Gastroenterology, St. Bartholomews and the Royal London School of Medicine, St. Bartholomews Hospital, London, United Kingdom;
Digestive Diseases Research Center, St. Bartholomews and the Royal London School of Medicine, The Royal London Hospital, London, United Kingdom; and
Department of Pediatric Gastroenterology, Royal Free Hospital, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, with negligible IL-4, IL-5, IL-10, or
TGF-ß. To help explain the PP T cell response to ßlg, we examined
IL-12 expression. Both IL-12p40 and -p35 transcripts were
abundantly expressed in PP, but not in adjacent normal ileal mucosa.
Immunoreactive IL-12p40-containing cells were present below the PP dome
epithelium. Furthermore, in culture, PP, but not paired PBMC,
spontaneously released IL-12p70. These results suggest that the human
response to oral Ags in the gut may be different from that in
rodents. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunological tolerance is commonly ascribed to deletion of Ag-reactive cells, anergy, or active suppression, and all three mechanisms have been shown to occur after feeding Ags (5). In rodents, feeding high doses of Ag seems to facilitate anergy/deletion of reactive T cells (6), whereas feeding low doses, probably akin to the physiological situation, induces regulatory cells (7). These regulatory T cells, which can be either CD4+ or CD8+ depending on the system, are primed in the Peyer’s patch (PP)4 of the small intestine and then migrate to the periphery (8, 9). When they re-encounter Ag they secrete immunoregulatory cytokines such as TGF-ß, IL-4, or IL-10, which then down-regulate newly initiated or ongoing Th1 cell responses (10). Human data on oral tolerance are more sparse. Volunteers fed keyhole limpet hemocyanin (KLH) and then immunized parenterally with KLH are comparatively unresponsive compared with volunteers who were not fed KLH (11). In addition, Weiner and colleagues have shown that short term T cell lines from the blood of multiple sclerosis patients fed myelin basic protein contain a higher frequency of lines that secrete TGF-ß than equivalent lines from MS patients who did not receive oral myelin basic protein (12). However, mechanistic human studies are lacking, in particular on the interaction of fed Ags with T cells in human PP.
PP are abundant in children, become rarer after puberty, and are difficult to see endoscopically in adults (13). In children, therefore, it is possible to identify and selectively biopsy PP during ileoscopy (14). It is unethical to feed children experimental Ags and then ask them to undergo endoscopy so that PP samples can be taken. Likewise, healthy adult volunteers cannot be fed Ags and the PP sampled at ileoscopy, because there is no guarantee that the PP would be visible. We have taken an alternative approach, based on the fact that certain foods, such as cow’s milk proteins are abundant in the diet, especially in children. We made the reasonable assumption that any general paradigm of immune responsiveness to orally administered Ags should apply to common dietary Ags as well as experimental Ags. We have therefore prospectively sampled ileal PP from patients undergoing ileoscopy and examined their PP mononuclear cell (PPMC) and PBMC in vitro recall responses to ß-lactoglobulin (ßlg) and casein of cow’s milk.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study received ethical approval from the East London and the City Health Authority. PP biopsies were sampled from the terminal ileum as described previously (14). In total, samples were obtained from 61 patients (34 males and 27 females; mean age, 11.3 years; range, 2.3–34.2 years). The majority of investigations were conducted to investigate suspected inflammatory bowel disease or rectal bleeding. In all cases, the ileum was macroscopically and histologically normal.
It is important to emphasize that because of the relatively low cell yield from individual PP biopsies (2–3 million cells), it was not possible to perform all the assays on the same samples. Thus, the proliferation assays used samples from nine patients (five males and four females; mean age, 13.3 years; range, 7.2–34.3 years). Of these, four had polyps, three had irritable bowel syndrome (IBS), 1 had distal colitis, and 1 had Crohn’s colitis in remission. Biopsies from 19 patients were used for the enzyme-linked immunospot (ELISPOT) assay (15 males and 4 females; mean age, 9.3 years; range, 3.3–17.8 years). In this group of patients, 8 had colonic polyps, 3 had IBS, 1 had Crohn’s disease (CD), 1 had Peutz-Jeghers syndrome, and 6 had no abnormality. Subsequent to this initial study we also used ELISPOT to examine the response of PP cells from six children with colonic Crohn’s disease. These comprised four boys and two girls (mean age, 10.8 years; range, 7.8–14.7 years). FACS investigations used samples from 11 patients (three males and eight females; mean age, 17.3 years; range, 14.2–24 years), six diagnosed with IBS, three with hemorrhoids, and two with polyps. For the RT-PCR studies, samples from seven subjects were used (three males and four females; mean age, 9.2 years; range, 2.2–13.2 years); four had polyps, two had IBS, and one had ulcerative colitis. For the study of IL-12 RT-PCR, five patients were studied (three males and two females; mean age, 10.8 years; range, 4.3–15.1 years). Two of these patients were normal, one had colonic CD, one had polyps, and one had cystic fibrosis. Samples from four normal patients (three males and one female; mean age, 9.4 years; range, 2.3–13.8 years) were used to study TGF-ß, whereas samples from six patients (two males and four females; mean age, 9.6 years; range, 5.4–16.0 years) were used to study IL-12p70. Four of these patients were normal, one had indeterminate colitis, and one had ulcerative colitis.
Isolation of PPMC and PBMC
Selective biopsy of one or two PP was made during ileal endoscopy from each patient (14). Freshly isolated PP biopsies were incubated for 30 min at 37°C in calcium- and magnesium-free HBSS containing 1 mM EDTA (Sigma Chemical Aldrich, Dorset, U.K.) to remove the epithelium. Following dissection with hypodermic needles, single-cell suspensions were prepared by collagenase digestion as previously described (14). Each PP biopsy yielded between 2–3 x 106 PPMC. In addition to PP biopsies, 1–3 ml of peripheral blood was taken from each patient. Mononuclear cells were isolated in a Ficoll-Hypaque (Pharmacia, Piscataway, NJ) density gradient using standard procedures. The buffy coat was removed carefully following centrifugation and washed twice in RPMI 1640 medium containing 10% FCS (all from Sigma). Cells were counted and assessed for viability.
Proliferation assays
Freshly isolated PPMC and PBMC were plated in 96-well
flat-bottom plates at 105 lymphocytes/well in
RPMI 1640 medium containing 10% FCS. PBMC and PPMC were incubated in
the presence or the absence of ßlg (Sigma; 500 µg/ml), PHA (10
µg/ml; Sigma), or anti-CD3 Ab cross-linked by Fc-transfected L
cells (50 µl UCHT-1 supernatant with Fc-transfected L cells at
2 x 104/well) for 48 h at 37°C in a
5% CO2-95% O2
water-saturated atmosphere. Preliminary experiments revealed that PP
responses were optimal at 100–500 µg/ml ßlg. Cells were pulsed
with [3H]thymidine (1 µCi) for 16 h
before being harvested onto filters. The filters were immersed in
scintillation fluid, and incorporated radioactivity was measured on a
beta counter. We were concerned that FCS might contain Ags to which PP
T cells would be sensitized; however, preliminary experiments revealed
that the PP responses to ßlg were identical whether cells were
cultured in FCS or pooled AB serum, and background counts in medium
with FCS alone were always low.
Enumeration of cytokine-secreting cells by ELISPOT assay
PPMC or PBMC cultures were stimulated with ßlg, casein
(Sigma), PHA, or anti-CD3 and harvested after 48 h. ELISPOTs
for IFN--, IL-4-, IL-5-, and IL-10-secreting cells were performed as
previously described (15). The frequency of spot-forming
cells was calculated per 105 total viable cells.
The majority of the counts were assessed blindly by one of the authors
(S.N.), and selected samples were confirmed by other co-authors.
Quantification of cytokine mRNA by quantitative competitive RT-PCR
A competitive quantitative RT-PCR was used as previously described (16). The DNA construct pHCQ1, which encodes the primer sites for various cytokines, was donated by Dr. M. Kagnoff (University of California, San Diego, CA). PBMC and PPMC were incubated in the presence or the absence of ßlg (500 µg/ml) or PHA (10 µg/ml) for 48 h. The cells were harvested, and total RNA was extracted using TRIzol (Life Technologies, Poole, U.K.) and chloroform, followed by isopropanol precipitation. Quantitative competitive RT-PCR was then conducted exactly as described previously (16).
RT-PCR for IL-12/p40, p35, and GAPDH
Total RNA was extracted from freshly isolated PP and ileal mucosal samples as described above. RT-PCR were performed in a total volume of 50 µl in the presence of 1 U of Taq DNA polymerase (Promega, Madison, WI), 200 µmol of dNTPs (Amersham), and 25 pmol/l 5' and 3' primers. PCR primers (Genosys, Cambridge, U.K.) were previously described (17). The reaction was terminated after 35 cycles (17).
Immunostaining
Frozen sections (6 µm) of PP and adjacent ileal mucosa from the same patient were stained with a goat polyclonal anti-IL-12p40 Ab (25 µg/ml; Autogene Bioclear, Calne, U.K.), goat IgG at equivalent concentration (Ig control), or without primary Ab (background control). Ab binding was localized by alkaline phosphatase immunohistochemistry using alkaline phosphatase-conjugated rabbit anti-goat IgG (Dako, Ely, U.K.) as described previously (18).
Flow cytometry
PPMC and PBMC were incubated in the presence or the absence of ßlg (500 µg/ml) or anti-CD3 mAb for 72 h. The cells were then harvested, counted, and aliquoted at 5 x 105 cells/FACS tube. mAbs to the following surface markers were added to each FACS tube at appropriate concentrations according to the manufacturer’s instructions: CD4-peridinin chlorophyll protein, CD8-allophycocyanin, and CD25-FITC (Becton Dickinson, Franklin Lakes, NJ); and CCR4-FITC and CCR5-PE (Wako Chemicals, Tokyo, Japan). Samples were then analyzed on a FACScan (Becton Dickinson, Mountain View, CA), and data were analyzed using WinMDI software.
ELISA
Freshly isolated PBMC and PPMC from four normal patients at 106 cells/ml were incubated in the presence or the absence of ßlg (500 µg/ml) for 48 h in RPMI 1640 containing 10% FCS and then washed and cultured for an additional 72 h in RPMI 1640 supplemented with a serum replacement reagent (HL-1, BioWhittaker, Ashby de la Zouch, U.K.). The presence of TGF-ß1 in the cell culture supernatants was measured using a TGF-ß1 Quantikine ELISA kit (R&D Systems, Abington, U.K.), with a lower limit of sensitivity of 5 pg/ml. Acid treatment of the samples was used to activate latent TGF-ß1 in the supernatant. To investigate IL-12 production, freshly isolated PPMC and PBMC from the same patient were cultured for 24 h in the presence or the absence of staphylococcus enterotoxin B (SEB) at 1 µg/ml. Supernatants were collected, and the concentrations of IL-12p70 were determined using a Biotrak ELISA kit (Amersham, Little Chalfont, U.K.) according to the manufacturer’s instructions.
Statistical analysis
In each group data points were first analyzed to determine whether they conformed to a sample from a normally distributed population. Comparison between all groups was made by two-tailed Mann Whitney U test; p < 0.05 was considered significant. Expression of IL-12p70 in PP and PBMC were compared by Fisher’s test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first analyzed the in vitro proliferative responses of PPMC and
PBMC to ßlg. Only weak responses were obtained with PBMC (mean
stimulation index (SI), 2.6; range, 0–20.4); however, in every case
PPMC responded better than PBMC from the same patient (Fig. 1
a), with a mean SI of 11.3
(range, 3.5–59.6). In response to PHA, however, identical vigorous
responses were obtained, with mean SIs of 89.5 (range, 31.6–291.0) for
PBMC and 66.5 (range, 15.4–204.1) for PPMC. We also analyzed the
numbers of CD25+ cells in ßlg-stimulated
cultures of PBMC and PPMC. Very few
CD4+/CD25+ or
CD8+/CD25+ cells were seen
in samples of PBMC stimulated with ßlg; however, in PPMC cultures,
CD4+/CD25+ cells and
CD8+/CD25+ cells were
abundant (Fig. 1, b and c). We also analyzed CCR4
and CCR5 expression in the ßlg-stimulated cultures (Fig. 2). Very little change was seen in PBMC;
however, in PPMC, ßlg induced a large increase in
CD4+ and CD8+ cells
expressing the Th1-associated chemokine receptor CCR5 (19, 20).
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The cytokine profile of the ßlg-responsive cells was evaluated
by ELISPOT and quantitative competitive RT-PCR. Only very weak ELISPOT
responses were obtained with PBMC stimulated with ßlg (Fig. 3, a and b),
although PHA (not shown) and cross-linked anti-CD3 elicited
vigorous responses, with an excess of IFN--secreting cells (Fig. 3c). In PPMC, however, ßlg consistently elicited a greater
ELISPOT response than PBMC from the same patient, and the response was
dominated by IFN- (Fig. 3a). ßlg failed to elicit an
IL-4 response (Fig. 3b). The numbers of IL-5- and
IL-10-secreting cells were also extremely low in ßlg-stimulated PPMC
(<40/105 cells in all patients). When sufficient
cells were available, PPMC were incubated with casein for 48 h,
and the numbers of IFN-- and IL-4-secreting cells were determined.
The number of IFN--secreting cells was significantly greater in the
casein-treated PPMC compared with the control cultures (casein-treated
PPMC: mean, 110.8 ± 37.5; range, 6–430; control: 12.2 ±
2.3; range, 2–20; p < 0.05; n = 11).
Casein failed to elicit an IL-4 response (casein-stimulated cultures:
mean, 6.7 ± 1.4; range, 1–20; control: mean, 3.3 ± 0.9;
range, 0–15). To establish whether colonic inflammation could alter
the PPMC response to ßlg, we analyzed the responses of cells from six
patients with CD. In the six Crohn’s patients the IFN- ELISPOT
response was 202 ± 22 spot-forming cells
(SFC)/105 cells. As is shown in Fig. 3a, the mean IFN- ELISPOT response of PPMC in this
heterogeneous group of patients was 140 ± 25
SFC/105 cells. No IL-4 ELISPOT response to ßlg
was seen in the Crohn’s patients. Thus we consider it unlikely that
colonic inflammation is itself responsible for the dominant IFN-
response to ßlg in PP.
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transcripts were
detectable, but low (mean 4,050 ± 1,298 transcripts/µg RNA) in
PBMC stimulated with ßlg. However, they were abundant in PPMC (mean
47,320 ± 3,984 transcripts/µg RNA) stimulated with ßlg (Fig. 4). In the same samples, IL-4, IL-5, and
IL-10 transcripts were around or below the level of detection (1000
transcripts/µg RNA). PHA-stimulated PPMC produced abundant
transcripts for IL-4, IL-5, and IL-10 (IL-4, 21,260 ± 6,579
transcripts; IL-5, 29,979 ± 2,231 transcripts; IL-10, 4,249
± 1,487 transcripts). To assess TGF-ß1 production, PPMC and PBMC
were stimulated with ßlg for 2 days and then washed and cultured for
3 days with ßlg in serum-free medium. Analysis of the supernatants by
ELISA, however, failed to detect any TGF-ß1.
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IL-12 is one of the main factors that drive naive T cells along
the Th1 pathway (19). To explore whether IL-12 is produced
in PP, we first analyzed transcripts for both IL-12p40 and -p35
subunits in fresh PP and adjacent normal ileum samples. Transcripts for
both IL-12 subunits were abundantly expressed in PP, but not in
adjacent mucosa (Fig. 5). This is
consistent with previous studies showing that IL-12 is undetectable in
normal human small intestine (17, 20, 21).
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Immunostaining was then used to localize IL-12 in the PP. In PP, cells
positive for IL-12p40 protein were abundant in the subepithelial dome
area of the tissue (Fig. 6A).
However, no immunoreactivity was detectable in the ileal mucosa (Fig. 6C). No staining was seen with control goat IgG.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Notwithstanding these caveats, the results presented here are unexpected given the large amount of rodent data suggesting that mucosal T cell responses are of the non-Th1 type. It is, however, fair to say that the molecular basis for the non-Th1 responses described in rodents after feeding Ags is still not fully understood. Many studies have used cholera toxin or related molecules as oral adjuvants to elicit responses (23, 24), and it is still not clear why CT or its derivatives have this biological activity. There is a suggestion that agents such as CT elevate B7.1 and B7.2 expression on APCs (25), and although there is some evidence that manipulation of B7.1 or B7.2 can alter Th1/Th2 responses to fed Ags in vivo (26), precise details of the changes in PP elicited by CT are not known.
We can, however, be reasonably certain that one of the major determinants of Th1-Th2-Th3 differentiation is the cytokine environment at initial sensitization. There are no studies on IL-12 expression in murine PP, but it is highly likely that the microenvironment in the PP of a 5- to 8-wk-old specific pathogen-free mouse will be different from that in the PP from a human child or adolescent. Here we report that freshly obtained PP, but not adjacent ileal mucosa, contains transcripts for both IL-12 subunits. In addition, we show that cultured PPMC, but not autologous PBMC, spontaneously release IL-12p70, the functionally active dimer, suggesting compartmentalization of IL-12 expression and release in PP. The amount of IL-12 produced by PPMC was relatively small, but was comparable to that measured in cultures of lamina propria mononuclear cells isolated from Crohn’s disease, a Th1-associated gastrointestinal disease (17). Since IL-12 is very potent in inducing its biological activities when present at low concentrations (i.e., 1 pg/ml), it is likely that the IL-12 levels found in PP are sufficient to drive the local Th1 response. The abundant IL-12 in ileal PP in humans that we have demonstrated here is probably due to the resident bacterial flora in the ileum being transported across the follicle-associated M cells into the dome area. It is interesting in this context that IL-12 immunoreactivity was most abundant in the region of the PP immediately below the dome epithelium. This is also consistent with the demonstration PPMC are functionally responsive to SEB, which enhances IL-12 secretion.
There are, however, other data to reinforce the idea that there are
major differences in the types of T cell responses elicited at mucosal
surfaces between rodents and man. In vitro activated lamina propria T
cells in rodents have been reported to make IFN-, IL-4, and IL-5 in
approximately equal amounts (27). However, in normal LPL
in humans, the in vitro response is dominated by IFN-
(28). Indeed, in previous studies from both us and others,
the spontaneous production of cytokines by freshly isolated lamina
propria T cells is dominated by IFN- (29, 30), and in
our previous study of freshly isolated PP T cells, we also found an
excess of IFN--secreting cells (14).
It is important to emphasize that these present data are not in conflict with the idea that oral tolerance is a biologically important phenomenon in humans, only with the idea that one of the putative pathways of oral tolerance, generation of regulatory cells in PP, may be less important in humans than in rodents. It is very well established that soluble dietary proteins enter the circulation, and these may be tolerogenic (31). Even in rodents, the similarity between oral tolerance and tolerance induced by systemic administration of soluble Ags has been highlighted (32), and there is the well-established observation that sera taken from mice given Ag orally a few hours previously is highly effective at inhibiting subsequent Th1 responses (33). Indeed, there is a large body of literature showing that systemic administration of soluble Ag can induce clonal anergy, clonal deletion, or immune deviation (34). Feeding Ags, therefore, may be an acceptable means of delivering soluble Ags into the systemic circulation, where they can function as immune modulators.
A number of studies (reviewed in Ref. 1) have highlighted the role of TGF-ß1-secreting T cells in the down-regulation of immune responses. We therefore attempted to determine whether PPMC stimulated with ßlg secreted TGF-ß1 using protocols involving serum-free medium previously shown by others to be optimal (35). We were unable to detect TGF-ß1 protein by ELISA. We are, however, reluctant to exclude a role for TGF-ß1 in the PP T cell response to oral Ags because TGF-ß1 is rather difficult to detect, and we may not have optimized the culture conditions. In addition, in animals specific feeding regimens can be used to elicit immunoregulatory cells in PP, whereas in our studies Ag exposure was not quantified.
One of the difficulties in analyzing responses to dietary food Ags in humans is that it is difficult to be precise about the dose. ßlg makes up about 10% of the protein in cow’s milk, and it has been estimated that the average adult ingests 18.5 g of cow’s milk protein/day, with increasing amounts in children (36). Therefore, it is likely that the average daily consumption of ßlg is at least 1.85 g/day. For a 70-kg individual this is a daily dose of approximately 26 mg/kg, which in mice would be considered a low dose. In addition, since cow’s milk proteins are found in a variety of foods, the consumption of ßlg in humans is probably continuous rather than as a bolus. ßlg is not present in human milk, so there is little possibility that there is tolerance because it is related to a human self Ag.
The final piece of new data from this work is that the PP Th1 response to ßlg was associated with a large increase in the number of cells expressing CCR5. Previous immunohistologic studies showed that CCR5+ cells dominate in the lamina propria (37), and it is well established that lamina propria T cells are derived from PP T cells. In addition, it has been shown that infection of macaques with CCR5 tropic strains of chimeric SIV, but not CCR4 tropic strains, leads to a dramatic loss of lamina propria T cells (38). Given that HIV/SIV replication is most abundant in lymphoid tissues (39), of which there is an abundance in the gut, it is probable that the depletion of CCR5+ cells in the lamina propria is a direct consequence of depletion of CCR5+ T cells responding to luminal Ags in the PP.
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Acknowledgments |
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Footnotes |
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2 S.N. and C.M. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Thomas T. MacDonald, Center for Infection, Allergy, Inflammation, and Repair, Level E, South Academic Block, Mail Point 813, Southampton General Hospital, Tremona Road, Southampton, United Kingdom SO16 6YD.
4 Abbreviations used in this paper: PP, Peyer’s patch; KLH, keyhole limpet hemocyanin; ßlg, ß-lactoglobulin; PPL, PP lymphoyte; PPMC, PP mononuclear cells; IBS, irritable bowel syndrome; CD, Crohn’s disease; ELISPOT, enzyme-linked immunospot; SFC, spot-forming cells; SEB, staphylococcal enterotoxin B; SI, stimulation index.
Received for publication April 4, 2000. Accepted for publication August 4, 2000.
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References |
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, IL-4, IL-5 and IL-10 productions by ELISPOT and quantitative reverse transcriptase-PCR in human Peyer’s patches. Cytokine 10:627.[Medline]
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, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J. Immunol. 157:1261.[Abstract]
and interleukin-4, -5 and -10 in the blood and duodenal mucosa of children with cow’s milk hypersensitivity. Pediatr. Res. 42:629.[Medline]
and interleukin 4 by human intraepithelial and lamina propria gut lymphocytes. Gut 42:643.This article has been cited by other articles:
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  I. Peluso, D. Fina, R. Caruso, C. Stolfi, F. Caprioli, M. C. Fantini, G. Caspani, E. Grossi, L. Di Iorio, F. M. Paone, et al. Lactobacillus paracasei subsp. paracasei B21060 Suppresses Human T-Cell Proliferation Infect. Immun., April 1, 2007; 75(4): 1730 - 1737. [Abstract] [Full Text] [PDF]
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CD3) cross-linked
by Fc
