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Infection and Immunity, May 2001, p. 3466-3471, Vol. 69, No. 5
Department of Medical Microbiology and
Immunology,1 Department of Infectious
Diseases,2 and Department of
Surgery,3 Sahlgrenska University Hospital,
Göteborg University, Göteborg, Sweden
Received 26 June 2000/Returned for modification 28 September
2000/Accepted 8 February 2001
The induction and dissemination of mucosal immune responses to
recombinant cholera toxin B subunit (rCTB) administered into the ileal
pouches of patients, who had been colectomized because of ulcerative
colitis, was analyzed. Biopsies from the duodenum and ileal pouch were
collected, along with peripheral blood and ileostomy fluids. Two
immunizations induced strong CTB-specific immunoglobulin A (IgA)
antibody-secreting cell (ASC) responses in the duodenum in five of five
patients, whereas weaker and less-frequent ASC responses were noted in
the ileal pouch. Intestine-derived CTB-specific IgA ASCs were found in
peripheral blood in three of the five patients. The vaccination also
induced significant IgA antitoxin titer rises in ileostomy fluid in all
of the patients. Increased production of gamma interferon in cell
cultures from the ileal pouch was found in four of five patients after
the vaccination. These results clearly indicate that rCTB administered
into the distal ileum is capable of inducing B-cell responses in the
"entire" small intestine and that homing of immunocompetent cells
occurs preferentially to the duodenum.
Induction of mucosal immune
responses has been studied mainly after oral administration of antigens
(11-16, 22). Mucosal immune responses are initiated by
uptake of antigens from mucosal surfaces into organized lymphoid
tissues located in the mucosa or in nearby lymph nodes, where
antigen-specific B cells are generated. B-cell immunoblasts recruited
at mucosal inductive sites subsequently enter the circulation and
migrate to local and distant mucosal tissues and glands, where terminal
differentiation occurs. This cellular migration is an important feature
of the mucosa-associated lymphoid tissue, since administration of
antigens in one mucosal region may generate secretory immunoglobulin A
(IgA) antibodies at distant mucosal sites (19, 20).
However, a number of studies have shown that local exposure to antigen
results in much higher levels of specific IgA antibodies in the region
of exposure than at distant sites (6-8).
In the present study, recombinant cholera toxin B subunit (rCTB) was
used as a model immunogen to assess the induction and dissemination of
mucosal immune responses after the administration of rCTB into the
ileal pouch of patients who had had colectomies due to ulcerative
colitis. Cholera toxin B subunit (CTB) is a well defined and potent
mucosal immunogen which can be safely administered to humans in the
form of the inactivated B-subunit-whole-cell (B-WC) cholera vaccine
(11, 12). Several studies have shown that rCTB gives rise
to strong IgA immune responses at various mucosal sites, especially
within the intestine (3, 12, 15, 17, 22). Recently, we
have also demonstrated that two oral doses of rCTB induced significant
CTB-specific IgA antibody responses in ileostomy fluid of patients
colectomized due to ulcerative colitis (14).
The aim of the present study was to examine whether CTB-specific immune
responses could be induced by antigen exposure in the distal ileum and
to determine to what extent such responses could disseminate to the
proximal small intestine. This was studied by collecting biopsies from
the ileal pouch and duodenum along with peripheral blood and ileostomy
fluid specimens from colectomized patients before and after the
administration of rCTB. The T-cell responses after vaccination were
also studied by assessing the cytokine production in ileostomy fluid
and cell supernatants from intestinal biopsies.
Study design.
Five adult patients (two women and three men),
aged 43 to 52 years, who had undergone colectomies due to ulcerative
colitis, were recruited from the regular follow-up program for patients with inflammatory bowel disease at the Department of Surgery of the
Sahlgrenska University Hospital in Göteborg. Continence surgery had been performed 5 to 12 years earlier by construction of a pelvic
pouch with an ileoanal anastomosis. The maximal extent of the small
bowel resection was limited to 10 cm of the distal ileum. All patients
were in general good health and had had no episodes of acute pouchitis
or signs of extraintestinal manifestations of ulcerative colitis for
the 3 years immediately preceding the study. None of the subjects had
previously been vaccinated against cholera. All subjects agreed to
participate in the study, which was undertaken with due approval from
the Human Research Ethical Committee of the Medical Faculty,
Göteborg University.
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.5.3466-3471.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Induction and Distribution of Intestinal Immune Responses after
Administration of Recombinant Cholera Toxin B Subunit in the Ileal
Pouches of Colectomized Patients
ABSTRACT
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Specimen collection. Mucosal biopsies (duodenum and ileal pouch), ileostomy fluids, and blood specimens were collected before the first immunization (day 0) and 7 days after the second vaccine dose. In addition, ileostomy fluids were collected 21 days after the second immunization. The duodenal biopsies were obtained under local anesthesia by using a gastrointestinal fibroscope and a pair of biopsy forceps (FB 24 K; Olympus, Stockholm, Sweden). On each occasion, 12 pinch biopsies, 1 to 2 mm in diameter, were collected approximately 4 cm distal to the pyloric sphincter. Five biopsies, 3 to 4 mm in diameter, were also collected from the ileal pouch by means of a pediatric rectoscope and rectal biopsy forceps (4 by 2.6 mm; Instrumenta AB, Partille, Sweden). The extent of inflammation in the duodenal and ileal pouch mucosa was assessed by light microscopy examination of formalin-fixed biopsy specimens.
Ileostomy fluids were collected within 3 h after the last emptying of the reservoir. A 50-ml portion of fluid was immediately chilled on ice, centrifuged, and treated with enzyme inhibitors as previously described (14). The ileostomy fluid was frozen at
70°C
until used. For determination of circulating vaccine-specific antibody-secreting cell (ASC) responses, 20 ml of heparinized venous
blood was collected from the patients before and 7 days after the last
vaccination. Serum specimens were obtained on the same occasions.
Detection of total and specific immunoglobulin-secreting
cells.
Intestinal mononuclear cells (MNCs) were isolated from
duodenal and ileal pouch biopsies using an enzymatic dispersion
technique as previously described (21). A pool of 12 duodenal biopsies yielded a mean of 1.4 × 106 viable
MNCs (range, 0.6 to 2.0 × 106), and a pool of 5 biopsies
from the ileal pouch 1.1 × 106 MNCs (range, 0.4 to
2.5 × 106). MNCs from heparinized venous blood were
isolated by standard gradient centrifugation on Ficoll-Paque (Pharmacia
Biotech AB, Uppsala, Sweden). Intestinal and peripheral blood cell
suspensions were assayed for numbers of IgA-secreting cells and
CTB-specific IgA ASCs by the enzyme-linked immunospot
(ELISPOT) technique described elsewhere (5, 27).
For the analyses of IgA-secreting cells, we added 50 to 500 intestinal
MNCs per well or 104 to 105 peripheral blood
MNCs per well. For the CTB-specific IgA ASC analyses, the corresponding
numbers were 104 to 105 intestinal MNCs per
well or 105 to 106 peripheral blood MNCs per
well. Spots were enumerated under low magnification (×40), and the
number of ASCs was calculated as the mean for four wells. IgA-secreting
cells in the intestine were expressed per 105 MNCs.
CTB-specific IgA ASCs were expressed per 104 IgA-secreting
cells in the intestine and per 106 MNCs in peripheral blood
to allow comparison with results in other studies (12, 17,
22). Vaccinees who as a mean had 
2 CTB-specific IgA ASCs per
104 IgA-secreting cells in their intestinal biopsies after
vaccination were considered as responders on the condition that they
had <1 CTB-specific IgA ASC per 104 IgA-secreting cells in
each of the four wells prior to immunization. The corresponding figure
for a response in peripheral blood was as a mean a postvaccination
value of 2 CTB-specific IgA ASCs per 106 MNCs. In no
instances were CTB-specific IgA ASCs detected when we examined the
highest available number of MNCs (106 cells) before immunization.
Antibody determinations. Levels of total IgA in ileostomy fluids were determined by a modified microplate enzyme-linked immunosorbent assay (ELISA) method as previously described (3, 30). The IgA antibody responses to cholera toxin in ileostomy fluid were studied by the GM1 ELISA method (14, 29). The specific IgA antitoxic activity in ileostomy fluid was determined by dividing the IgA ELISA antitoxin titer by the total IgA concentration (given in micrograms per milliliter) of the sample to adjust for variations in the IgA content in specimens collected from different persons and on different days. Based on previous calculations, a >2-fold increase in the mean IgA antitoxin titer/total IgA between pre- and postimmunization specimens was regarded as a significant response (1, 12). Serum antibody responses of IgA and IgG classes to cholera toxin were determined by the GM1 ELISA method (10, 11).
In vitro stimulation of MNCs.
MNCs from intestinal biopsies
were obtained according to methods described above. Circulating T cells
were separated from blood MNC suspensions by rosetting with
2-aminoethyliso-thiouroniumbromide (Sigma Chemical Co., St. Louis,
Mo.)-treated sheep red blood cells (26). The non-T-cell
fraction was then used as accessory antigen-presenting cells. According
to previous experiments, these cells consisted of approximately
one-third B cells (CD19+) and two-thirds monocytes
(CD14+) and contained <10% of T cells (25).
Isolated intestinal MNCs (2 × 105 cells per well)
were cultured in duplicate together with accessory cells (2 × 104 cells per well) in round-bottom 96-well plates (Nunc,
Roskilde, Denmark) in Iscove medium (Biochrom KG, Berlin, Germany)
supplemented with 5% of human AB+ serum, 3 µg of
L-glutamine (Biochrom KG) ml
1, and 100 µg
of gentamicin (Schering-Plough AB, Stockholm, Sweden) ml1. Thereafter, the T-cell cultures were incubated
without further additives or stimulated by CTB at final concentrations
of 5 and/or 10 µg ml1. Ten micrograms of
phytohemagglutinin (Murex Diagnostics, Ltd., Temple Hill, United
Kingdom) ml1 was used as a positive control to assess
polyclonal T-cell activation. The culture medium was collected after
48 h, pooled, and stored at 70°C until further analyzed for
cytokine content.
Cytokine detection.
The concentrations of interleukin-4
(IL-4), IL-10 and gamma interferon (IFN-
) in ileostomy fluids and
cell supernatants from intestinal biopsies and peripheral blood were
determined by different ELISAs. Ninety-six-well plates (Nunc) were
coated with mouse anti-human monoclonal antibodies specific for the
respective cytokine in 0.05 M carbonate buffer (pH 9.6) at 4°C
overnight. After blocking of the remaining binding sites of the plates
with 1% bovine serum albumin (BSA) in PBS at room temperature for
1 h, cell culture supernatants or ileostomy fluid specimens
diluted in PBS-Tween (0.05%) containing 0.1% BSA were added to the
individual wells and incubated at 4°C overnight. The cytokine
concentrations were determined by stepwise addition of biotinylated
antibodies reacting with the respective cytokine, peroxidase-labeled
extravidin (Sigma Chemical Co.), and o-phenylenediamine
substrate (Sigma Chemical Co.). The coating and detecting antibodies to
IL-4 and IL-10 were purchased from Pharmingen (San Diego, Calif.), and
the IFN--specific reagents were from Chromogenix (Mölndal,
Sweden). Standard curves were constructed using recombinant human
cytokines obtained from Genzyme (Cambridge, Mass.).
B-cell responses in the intestine. CTB is a potent immunogen which has been used in numerous studies to assess the induction of immune responses at various mucosal sites (3, 12, 15, 17, 22, 23). Large numbers of CTB-specific IgA ASCs in the duodenum of healthy volunteers have been demonstrated after oral immunization with the B-WC cholera vaccine (17, 22). Rectal and intratonsillar immunization of healthy humans with CTB have also induced substantial IgA ASC responses to CTB at the site of antigen exposure (9a, 23).
In the present study, suspensions of MNCs obtained from enzymatically dispersed duodenal and ileal pouch biopsies were assayed for CTB-specific IgA ASCs after administration of rCTB in the ileal pouch of colectomized patients. Prior to immunization, <1 CTB-specific IgA ASC per 104 IgA-secreting cells were found in the duodenum and the ileal pouch of all patients. Two administrations of rCTB into the ileal pouch induced weak CTB-specific IgA ASC responses in the distal ileum in two of four patients (one postvaccination specimen had to be excluded from the analysis due to insufficient numbers of ileal pouch MNCs) (Fig. 1A). Interestingly, all of the five vaccinees responded with substantial increases in CTB-specific IgA ASCs per 104 IgA-secreting cells in the duodenum, and the magnitude of the response had a mean value of 95-fold (Fig. 1A). Thus, considerably stronger CTB-specific IgA ASC responses were observed in the duodenum than at the site of CTB exposure, i.e., in the ileal pouch of colectomized patients. The response in the duodenum was most likely not due to a gastrointestinal reflux of CTB, since radiologic evaluations in colectomized patients with continent ileostomy reservoirs have shown a maximum reflux of 20 cm after volume infusions of 100 to 300 ml into the reservoirs (2). As the total volume administered into the empty ileal pouch was no more than 23 ml in the present study, retrograde migration of antigen seems highly unlikely. Furthermore, we have recently shown that installation of CTB into the small intestine approximately 30 cm beyond the pyloric sphincter does not result in any detectable reflux as determined by coadministration of C13-labeled polyethylene glycol, as well as antigen analysis of gastric fluid for CTB (M. Quiding-Järbrink, H. Lönroth, I. Ahlstedt, J. Holmgren, and A.-M. Svennerholm, unpublished results). Our findings indicate the possibility of inducing B-cell responses within the gastrointestinal tract of humans without the presence of antigen at the effector site. The high frequencies of CTB-specific IgA ASCs found in the duodenum of all the colectomized patients clearly show that immunocompetent cells initially activated in the distal ileum have the capacity to disseminate to a more proximal part of the small intestine. The observed homing of ASCs to the duodenum might be due to an upregulated expression of mucosa-specific adhesion molecules in the duodenal mucosa, resulting in an increased migration of plasma cell precursors into the duodenal mucosa.
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B-cell responses in peripheral blood. Monitoring of different homing receptors on circulating ASCs induced by various routes of immunization has clearly shown that ASCs of the IgA isotype assayed 7 days after antigen administration by the oral or rectal routes largely represent cells of gut origin (13, 24). After administration of rCTB into the distal part of the small intestine, circulating CTB-specific IgA ASCs were detected in peripheral blood in three of the five colectomized patients, but the magnitude of the response was modest (Fig. 1B). The frequency and magnitude of the ASC responses were lower than those observed in healthy volunteers given CTB by the oral (12) or rectal (9a) route. According to the concept of a common mucosal immune system, the destination of these circulating B cells initially activated in the gut is the lamina propria and other mucosal tissues distant from the initial site of antigen exposure (19, 20).
Also in serum, significant increases in IgA antitoxin titer were found in three of five vaccinees, and four of them developed IgG antitoxin responses. The magnitudes of the increases in serum antitoxin titer among the responders were 14-fold for IgA and 3.3-fold for IgG. The seroconversion rate to CTB was similar to what has been noted after oral vaccination (14).Cytokine responses in the intestine.
The human intestinal
mucosa seems to be a rich source of cells producing IFN-. High
frequencies of cells spontaneously secreting IFN- have been found in
duodenal biopsies from healthy volunteers (22). In
patients with ulcerative colitis, mucosal biopsies from uninflamed
ileoanal pouches have been shown to contain slightly increased numbers
of IFN--producing cells (28). In the present study, all
of the patients had detectable amounts of IFN- in the supernatants
of unstimulated cells isolated from the ileal pouch (Fig.
2) and duodenal (not shown) biopsies
before immunization. The IFN- production from ileal pouch and
duodenal cells was similar, i.e., the mean IFN- concentration ± 1 standard deviation was 156 ± 89 ng ml1 for
ileal pouch cells (Fig. 2) and 137 ± 59 ng ml1 for
duodenal cells. Secretion of IL-4 and IL-10, on the other hand, was
found more often and in higher levels in unstimulated cell cultures
from the duodenum (246 ± 196 and 277 ± 199 ng
ml1, respectively) than from the ileal pouch (20 ± 14 and 34 ± 27 ng ml1, respectively).
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in unstimulated cultures of ileal pouch cells for
three of the five patients (Fig. 2). These findings are consistent with
a previous report showing increased numbers of IFN--producing cells
in the duodenal mucosa of healthy volunteers after oral immunization
with the B-WC cholera vaccine (22). Stimulation with CTB
of ileal pouch cells collected after immunization induced a further
increase in IFN- secretion in the three patients from whom
sufficient numbers of cells were available for analyses (Fig. 2). We
believe that this further increase in IFN- production is a result of
an antigen-specific stimulation. Histopathological examinations of the
biopsies revealed no difference in the inflammatory score before and
after administration of rCTB. In contrast to our findings in the ileal
pouch, duodenal cells from one vaccinee only (subject 4) showed an
increase in IFN- production after immunization, and no further
increase was observed after stimulation with CTB. The production of
IL-4 and IL-10 was only increased in the supernatant of unstimulated
duodenal cells from one patient (subject 5) after vaccination; in
cultures of ileal pouch cells increased amounts of IL-10 were also
found in the same vaccinee. Prevaccination ileostomy fluids rarely
contained detectable amounts of the cytokines studied, and
administration of rCTB into the ileal pouch only induced increased
levels of IFN-, IL-4, and IL-10 in one of the patients (subject 3).
In conclusion, the present study clearly shows that it is possible to
induce B-cell responses within the small intestine without the actual
presence of antigen at the effector site. Administration of rCTB into
the ileal pouches of colectomized patients resulted in considerably
stronger CTB-specific IgA ASC responses in the duodenum than at the
site of antigen exposure, suggesting that the homing of immunocompetent
B cells occurs preferentially to the duodenum. The observation that the
cytokine response was limited to the induction site suggests a lower
extent of distribution for the T-cell response than the B-cell response.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Swedish Medical Research Council (16X-09084).
We are grateful to Elisabeth Lindholm and Harriet Törnqvist for collecting the specimens and to Marie Bengtsson and Ingela Ahlstedt for skillful technical assistance. The support from the staff at the Gastroenterology Unit at Sahlgrenska University Hospital is also gratefully acknowledged.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Göteborg University, Guldhedsgatan 10, SE-413 46 Göteborg, Sweden. Phone: 46-31-342 46 14. Fax: 46-31-82 69 76. E-mail: marianne.jertborn{at}microbio.gu.se.
Editor: J. D. Clements
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Infection and Immunity, May 2001, p. 3466-3471, Vol. 69, No. 5
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.5.3466-3471.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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