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Previous Article | Next Article 
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.
Induction and Distribution of Intestinal Immune Responses after
Administration of Recombinant Cholera Toxin B Subunit in the Ileal
Pouches of Colectomized Patients
Jan
Kilhamn,1,2
Hans
Brevinge,3
Marianne
Quiding-Järbrink,1
Ann-Mari
Svennerholm,1 and
Marianne
Jertborn1,2,*
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
 |
ABSTRACT |
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.
| |
TEXT |
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.
Each subject received two doses of an inactivated B-WC cholera vaccine
2 weeks apart; the first dose was given at least 3 days after preimmune
sampling of the specimens. The vaccine, containing 1.0 mg of rCTB and
1011 heat- and formalin-killed O1 vibrios per dose, was
produced by SBL Vaccin, Stockholm, Sweden (9). Each dose
of vaccine (3 ml) was suspended in 20 ml of phosphate-buffered saline
(PBS) and deposited into the ileal pouch, which had been emptied
immediately before the immunization. No coadministration of
bicarbonate buffer was needed, since the pH of the ileal pouch
secretion was found to be neutral. The participants remained resting
for 30 min, alternating between the supine and side positions, after
vaccine administration.
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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FIG. 1.
Frequencies of CTB-specific IgA ASCs in duodenal and
ileal pouch biopsies (A) and peripheral blood (B) before and 7 days
after two administrations of rCTB into the ileal pouch of colectomized
patients. The data are expressed as numbers of vaccine-specific IgA
ASCs per 104 IgA-secreting cells (A) or per 106
MNCs (B). The dotted lines indicate the level for a significant
response. The numbers correspond to the individual patients. For
patient 1, CTB-specific IgA ASCs in the ileal pouch after vaccination
could not be analyzed due to insufficient numbers of MNCs.
|
|
Studies of the compartmentalization of local immune responses indicate
that even though responses can be recorded at distant sites from that
of the antigen administration, the highest responses are generally seen
at the site of antigen administration (6-8). In the
present study, however, considerably stronger responses were seen in
the duodenum than at the site of CTB exposure. The weak IgA ASC
response found in the ileal pouch after vaccination might be explained
by differences in the distribution of IgA-secreting cells within the
small intestine. However, similar frequencies of IgA-secreting cells
were found in the intestinal biopsies; the geometric mean number of
IgA-secreting cells in duodenum was 19,000 per 105 MNCs
(range, 9,000 to 36,000), and in the ileal pouch 17,000 per
105 MNCs (range, 7,000 to 35,000). The frequencies of IgA
secreting cells in the duodenum and ileal pouches were similar in
biopsies obtained before and after vaccination. Another explanation for the decreased responsiveness to CTB at the inductive site could be that
there are differences in the expression of endothelial adhesion
molecules and/or chemokines within the small intestine. The mucosal
addressin cell adhesion molecule-1 (MadCAM-1), which is the only
characterized mucosa-specific addressin involved in lymphocyte
trafficking to mucosal lymphoid tissues, is localized to the intestinal
mucosa and gut-associated lymphoid tissue, and expression appears to be
increased at inflammatory foci associated with ulcerative colitis and
Crohn's disease (4). In spite of the weak response in the
ileal pouch, administration of rCTB in this location clearly resulted
in a strong efferent B-cell response. Thus, significant (>2-fold)
increases in CTB-specific IgA antibodies/total IgA were observed in the
ileostomy fluid in all of the five vaccinees. The geometric mean fold
increase of the antitoxin response was 9.5-fold on day 7 and 4.9-fold
on day 21 after the second vaccination (Table
1). However, the magnitude of the IgA
antitoxin responses was lower than that observed in colectomized
patients given CTB by the oral route (14).
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TABLE 1.
Antitoxin IgA antibody responses in ileostomy fluid
samples from colectomized patients before immunization and after two
doses of rCTB into the ileal pouch
|
|
Inflammation per se has been suggested to facilitate the induction of
immune responses (18). Recently, high frequencies of
vaccine-specific IgA ASCs were found locally in the inflamed gastric
mucosa of Helicobacter pylori-infected patients after oral
immunization with the B-WC cholera vaccine, whereas none of noninfected
controls responded to the vaccination in antrum (17). The
inflammation in the gastric mucosa of H. pylori-infected patients is characterized by a massive infiltration of lymphocytes and
neutrophils (31). Although a mild chronic lymphocytic
infiltration was observed in the lamina propria of the ileal pouches,
no evidence for an increased B-cell response following administration
of rCTB into the pouches was noted. One explanation for the variable
responsiveness to mucosal administration of antigen might be that the
recruitment of inflammatory cells in H. pylori-infected
gastric mucosa and ileal pouches differs.
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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FIG. 2.
IFN- production in cell supernatants of ileal pouch
MNCs from colectomized patients before and 7 days after administration
of rCTB into the ileal pouch. Results from unstimulated cultures are
shown before and after immunization, and in three patients, from whom
sufficient cell numbers were available, the data are also presented
after stimulation with 5 µg of CTB ml1 for ileal pouch
MNCs collected after vaccination. The numbers correspond to the
individual patients.
|
|
Administration of rCTB into the ileal pouch resulted in increased
production of IFN- 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.
| |
ACKNOWLEDGMENTS |
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.
| |
FOOTNOTES |
*
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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