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*
Departments of Microbiology and Oral Biology, Immunobiology Vaccine Center, University of Alabama, Birmingham, AL 35294; and
JCR Biopharmaceuticals, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The underlying mechanisms that result in the state of systemic
unresponsiveness after oral delivery of Ag are not fully understood;
however, the dose of Ag given has been shown to be an important factor
(14). For example, repeated low oral protein doses induce
cytokine-mediated active immune suppression characterized by the
presence of regulatory T cells, which include TGF-
-producing Th3
cells and IL-10-producing Tr1 cells (12, 15). In contrast,
a high oral Ag dose leads to T cell clonal deletion or anergy, which is
characterized by inhibition of both Ab and cell-mediated immune
responses. This cell-mediated immune or delayed-type hypersensitivity
(DTH) response is further characterized by reduced T cell
proliferation, contact hypersensitivity, and cytokine production
(10, 16, 17).
Oral tolerance has essentially been studied by initially feeding a protein Ag followed by systemic immunization with the same Ag given in CFA. In these experiments, a decline in serum IgG and IgE Ab responses together with diminished T cell responses are considered the hallmarks of oral tolerance (7, 8, 18). However, the systemic immunization with Ag and the potent adjuvant CFA does not allow one to determine whether tolerance extends to mucosal compartments. It is now well established that parenteral immunization elicits systemic immunity in the absence of mucosal immune responses and, thus, one cannot evaluate tolerance at the level of the GI tract mucosa (6). In general, oral delivery of soluble protein without adjuvant induces Ag-specific systemic immune unresponsiveness (1, 7, 18); however, oral tolerance can be abrogated and Ag-specific mucosal IgA Ab responses induced when oral proteins are given with cholera toxin (CT) as a mucosal adjuvant (19, 20). Based on these studies, mucosal immunization strategies using adjuvants such as CT and the related Escherichia coli labile toxin (LT) have been developed (6). One of the advantages of mucosal immunization is that this mode can elicit both systemic and mucosal immune responses (6). In addition, this strategy also provides a unique way to address the mechanisms of induction and regulation of mucosal immune responses represented by S-IgA Ab production.
Oral tolerance has continued to be defined as systemic unresponsiveness with the maintenance of mucosal Ab responses, and this goes back to studies performed over 20 years ago (2). This finding preceded our knowledge that mucosal adjuvants such as CT reverse oral tolerance and induce potent mucosal and systemic immune responses. Therefore, it was important to revisit this notion and to determine whether oral tolerance also influences mucosal immune responses, because oral tolerance has been mainly assessed by parenteral boosting with Ag in CFA. Furthermore, studies should address whether potent mucosal adjuvants such as CT can block existing oral tolerance and induce both systemic and mucosal Ab responses. In this study, we addressed these two important issues by gastric administration of OVA followed by an oral immunization protocol with OVA and CT as mucosal adjuvant.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were purchased from the Frederick Cancer Research Facility (National Cancer Institute, Frederick, MD). Upon receipt, mice were transferred to microisolators and maintained in horizontal laminar flow cabinets at the University of Alabama at Birmingham Immunobiology Vaccine Center. All mice were free of pathogenic bacteria and viruses as determined by Ab screening and routine histologic analysis of organs and tissues. All experiments were performed using mice between 8 and 14 wk of age.
Induction of oral tolerance
To induce Ag-specific immune unresponsiveness, mice were gastrically intubated with 50 mg of OVA (Fraction V; Sigma, St. Louis, MO) dissolved in 0.25 ml of PBS, while control mice were gastrically intubated with 0.25 ml of PBS. On days 7, 14, and 21 after intubation, mice were orally immunized with 1 mg of OVA and 10 µg of CT (List Biological Laboratories, Campbell, CA) as a mucosal adjuvant (21, 22). Both OVA- and B subunit of CT (CT-B)-specific B and T cell responses were determined 7 days after the final oral immunization (day 28).
Sample collection
Blood, fecal extracts, and saliva were collected on day 28. Blood was obtained by use of heparinized Natelson pipettes (Fisher Scientific, Pittsburgh, PA) placed into the supraorbital vein. The blood was centrifuged for 5 min at 5000 rpm, and the serum was collected and stored frozen at -20°C until assayed. Fecal pellets were weighed, added to microcentrifuge tubes, and PBS containing 0.01% sodium azide was used to adjust the volume (100 mg/ml). The pellets were homogenized by continuous shaking for 10 min with a Vortex (Scientific Industries, Bohemia, NY). Nondissolved particulate fecal debris was removed by centrifugation for 10 min at 14,000 rpm and supernatants were collected and stored frozen at -20°C. Saliva was obtained from mice following i.p. injection of 100 µl of 1 mg/ml pilocarpine (Sigma).
Lymphoid cell isolation
The spleen was removed aseptically and single-cell suspensions prepared by passage through sterile wire mesh screens as described previously (23, 24). Peyer’s patches were carefully excised from the intestinal wall and were dissociated using the neutral protease enzyme, collagenase type V (Sigma) in RPMI 1640 medium (Cellgro Mediatech, Washington, D.C.) to obtain single-cell preparations (23). Mononuclear cells in the lamina propria were isolated after removal of Peyer’s patches from the small intestine using a combination of enzymatic dissociation and discontinuous Percoll gradients (Pharmacia Fine Chemicals, Uppsala, Sweden) (23). Mononuclear cells in the interface between the 40 and 75% layers were collected, washed, and resuspended in RPMI 1640 medium containing 10% heat-inactivated FCS (Summit Biotechnology, Fort Collins, CO).
Detection of Ag-specific Ab responses in the serum, saliva, and fecal extracts
We have assessed Ab titers to both OVA and CT-B in serum,
saliva, and fecal extracts using an ELISA as described in detail
elsewhere (23, 24). CT-B was purified from Bacillus
brevis strains containing the plasmids for rCT-B
(25). Briefly, Falcon Microtest III assay plates (Becton
Dickinson, Oxnard, CA) were coated with a solution of OVA (1 mg/ml) or
with CT-B (5 µg/ml) in PBS and incubated overnight at 4°C. After
blocking with 1% BSA in PBS for 2 h at 25°C, serial dilutions
of serum or fecal extracts were added to each well. After incubation
for 2 h at 25°C, HRP-labeled goat anti-mouse µ, 
, or
heavy chain-specific Abs (Southern Biotechnology Associates,
Birmingham, AL) were added to wells and incubated for 2 h at
25°C. For analysis of IgG1 and IgG2a Ab subclasses, biotin-conjugated
mAbs specific for 1 (G1-6.5) or 2a (R19-15) (PharMingen, San
Diego, CA) were used. For detection, an HRP-labeled goat anti-biotin Ab
(Vector Laboratories, Burlingame, CA) was used. The color reaction was
developed with 1.1 mM 2,2'-azino-bis (3-ethylbenz-thiazoline-6-sulfonic
acid) (Sigma) in 0.1 M citrate-phosphate buffer (pH 4.2) containing
0.01% H2O2. Endpoint
titers were expressed as the last dilution yielding an OD at 415 nm
(OD415) of >0.1 U above negative control values
after a 15-min incubation.
Enumeration of OVA-specific Ab-forming cells (AFCs)
Mononuclear cells from spleen and GI tract lamina propria were
subjected to OVA-specific enzyme-linked immunospot (ELISPOT) assay for
detection of IgM, IgG, and IgA AFCs (23, 24). Briefly,
96-well nitrocellulose plates (Millititer HA; Millipore, Bedford, MA)
were coated with 1 mg/ml of OVA in PBS overnight at 4°C. After
blocking with 1% BSA in PBS for 2 h at 37°C, cells were
suspended in RPMI 1640 medium containing 10% heat-inactivated FCS,
HEPES buffer (15 mM), L-glutamine (2 mM), penicillin (100
U/ml), streptomycin (100 µg/ml), and gentamicin (80 µg/ml)
(complete medium). Aliquots were then added to each well at appropriate
dilutions and incubated for 4 h at 37°C in an atmosphere of 5%
CO2 in air. The AFCs were detected with
HRP-labeled goat anti-mouse µ, , or heavy chain-specific
Abs (Southern Biotechnology Associates) and visualized by adding the
chromogenic substrate, 3-amino-9-ethylcarbazole (Moss, Pasadena, CA).
Spots representing individual AFCs were counted with the aid of a
dissecting microscope (SZH Zoom Stereo Microscope System; Olympus, Lake
Success, NY).
Measurement of DTH responses
We used a well-characterized technique to assess DTH responses in vivo (23, 24). Briefly, 10 µg of OVA in 20 µl of PBS was injected into the left ear pinna and PBS alone (20 µl) was administered to the right ear pinna as a control. Ear swelling was measured 24 h later with an upright dial thickness gauge (Peacock; Ozaki, Tokyo, Japan). The DTH response was expressed as the increase in ear swelling after OVA injection following subtraction of swelling in the control site injected with PBS.
CD4+ T cell proliferation assay
CD4+ T cells were purified by the magnetic activated cell sorter system (Miltenyi Biotec, Auburn, CA) as previously described (23, 24). Briefly, splenic or Peyer’s patch cells were incubated with biotin-conjugated anti-mouse CD4 mAb (GK 1.5) and subsequently with streptavidin-conjugated microbeads. The CD4+ T cell population was enriched after passage through a magnetized column. The isolated CD4+ T cells were >97% pure and >99% viable. Purified CD4+ T cells were cultured with 1 mg/ml of OVA in the presence of T cell-depleted irradiated (3000 rad) splenic feeder cells from naive syngeneic mice in 96-well tissue culture plates (Corning Glass Works, Corning, NY) for 5 days. An aliquot of 0.5 µCi of [3H]thymidine (Amersham, Arlington Heights, IL) was added during the last 18 h and amounts of [3H]thymidine incorporated into dividing cells was measured by scintillation counting.
Cytokine analysis
Cytokine levels in splenic or Peyer’s patch
CD4+ T cell culture supernatants were determined
by a cytokine-specific ELISA as described previously (23, 24). Culture supernatants were collected on day 2 for IL-2 and
on day 5 for IFN-, IL-4, IL-5, IL-6, and IL-10 for analysis of the
secreted cytokine, respectively. The immunoplates (Nunc, Naperville,
IL) were coated with monoclonal anti-IL-2 (JES6-1A12),
anti-IFN- (R4-6A2), anti-IL-4 (BVD4-1D11), anti-IL-5
(TRFK-5), anti-IL-6 (MP5-20F3), or anti-IL-10 (JES5-2A5)
(PharMingen). After blocking with 3% BSA in PBS, serial twofold
diluted samples and standards were added to wells and incubated
overnight at 4°C. The wells were washed and then incubated with
biotinylated monoclonal anti-IL-2 (JES6-5H4), anti-IFN-
(XMG1.2), anti-IL-4 (BVD6-24G2), anti-IL-5 (TRFK-4),
anti-IL-6 (MP5-32C11), or anti-IL-10 (JES5-16E5) mAbs for
detection, respectively. After incubation overnight at 4°C,
HRP-labeled goat anti-biotin Ab (Vector Laboratories) was added and
incubated for 1 h at 25°C. The color reaction was developed with
1.1 mM 2,2'-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) (Sigma)
in 0.1 M citrate-phosphate buffer (pH 4.2) containing 0.01%
H2O2. A mouse IL-10
immunoassay kit, Quantikine M (R&D Systems, Minneapolis, MN) was also
used to detect IL-10 in the culture supernatants. The minimal
detectable level for each cytokine was 1.95 pg/ml for IL-2, 156.25
pg/ml for IFN-, 4.69 pg/ml for IL-4, 1.95 pg/ml for IL-5, 39.06
pg/ml for IL-6, and 7.81 pg/ml for IL-10.
Statistics
The data are expressed as the mean ± 1 SD and mouse groups
were compared with control mice using Student’s t test with
StatView software (Abacus Concepts, Berkeley, CA) designed for
Macintosh computers. For statistical analysis of cytokine levels below
the detection limit, one-half of the minimal detectable levels (e.g.,
IFN- = 78.13 pg/ml) were recorded and analyzed. A p
value of <0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is now well established that oral immunization with protein Ags
such as OVA with CT as mucosal adjuvant induces immune responses in
both the systemic and mucosal compartments (23). However,
parenteral immunization with OVA and the adjuvant CFA induces systemic
but not mucosal immune responses. We have used a well-established oral
immunization protocol of OVA plus CT to assess whether prior OVA
feeding affected both systemic and mucosal B cell responses. Groups of
C57BL/6 mice were given oral PBS or a high dose of OVA and subsequently
orally immunized with OVA and CT on three occasions at weekly
intervals. We first determined whether the high oral dose of OVA
affected serum OVA-specific Ab responses after oral immunization.
OVA-fed mice showed significantly reduced serum IgG Ab responses
compared with those of PBS-fed mice (Fig. 1
A; p <
0.05). In addition, serum anti-OVA IgA Ab responses were
dramatically reduced in OVA-fed mice (Fig. 1A;
p < 0.05). In contrast, IgM Ab levels were comparable
between the two groups (Fig. 1A). These results clearly show
that both serum IgG and IgA Ab responses are down-regulated by feeding
of a high dose of OVA. Furthermore, the data suggest that use of CT as
oral adjuvant cannot reverse this state of oral tolerance in the
systemic immune compartment.
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B). This result shows
that reduced serum Ab responses in mice fed OVA were OVA specific.
It has been shown that CT induces Th2-type responses to coadministered
Ags given orally and the Th2 cells provide help for B cells producing
IgG1, IgA, and IgE Abs (21, 22, 23). We assessed serum IgG1
and IgG2a anti-OVA Abs to determine the potential influence of oral
immunization with OVA and CT on induction of particular serum IgG Ab
subclasses. Consistent with previous reports, oral challenge with OVA
and CT induced brisk IgG1 and low IgG2a Ab responses in control,
PBS-fed mice. In OVA-fed mice, OVA-specific IgG1 but not IgG2a Ab
responses were significantly reduced, indirectly suggesting that
OVA-specific Th2-type responses induced by CT as adjuvant were
effectively suppressed by prior OVA feeding (Fig. 2A; p <
0.05). The reduction of IgG1 Ab responses observed in OVA-fed mice
compared with those in PBS-fed mice were OVA-specific, because IgG1
anti-CT-B Ab responses were elevated and essentially identical in
the two mouse groups (Fig. 2B).
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Because it has been shown that oral immunization with OVA plus CT
as mucosal adjuvant elicited significant Ag-specific mucosal IgA Ab
responses, we further determined whether oral tolerance affects mucosal
Ab responses in saliva and the GI tract. Interestingly, fecal IgA Ab
responses specific for OVA were dramatically reduced in OVA-fed mice
compared with PBS-fed mice (Fig. 3A; p <
0.05). In contrast, significant levels of CT-B-specific IgA Ab
responses were induced in comparable amounts in fecal extracts of mice
regardless of OVA feeding (Fig. 3B). The levels of salivary
OVA-specific IgA Abs were undetectable in both groups of mice, although
CT-B-specific IgA Ab responses were comparably induced at low levels in
both mouse groups (data not shown). These results clearly indicated
that mucosal IgA Ab responses were also susceptible to oral tolerance
induction. Furthermore, the data suggest that use of CT as oral
adjuvant cannot reverse this state of oral tolerance in mucosal
compartments.
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A; p <
0.05). In particular, IgG AFCs in spleen from OVA-fed mice were
dramatically decreased (
5/106 cells) compared
with those from PBS-fed mice (70/106 cells)
(Fig. 4A; p < 0.05). We also assessed the
frequency of OVA-specific AFCs in the lamina propria of mice given
either oral PBS or OVA because this is the major mucosal effector site
of the GI tract. Of importance, OVA-specific IgA AFCs were markedly
diminished in OVA-fed mice compared with control mice
(400/106 cells and
3500/106 cells, respectively). Furthermore,
significant reductions in OVA-specific IgG AFCs (Fig. 4B;
p < 0.05) were also seen. This provides direct
evidence that mucosal IgA Ab responses are susceptible to oral
tolerance. These findings clearly indicate that OVA-specific B cell
unresponsiveness is induced for both IgG and IgA Abs in systemic
tissues and for IgA Ab responses in mucosal effector sites after a
single high oral dose of Ag.
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We next determined whether a single high oral dose of OVA affected
both systemic and mucosal T cell responses induced by subsequent oral
challenge with OVA plus CT as adjuvant. We first examined OVA-specific
DTH responses in mice given either oral PBS or OVA. OVA-specific DTH
responses were induced in mice fed PBS before oral immunization with
OVA and CT as adjuvant (Fig. 5). In
contrast, significantly lower OVA-specific DTH responses were observed
in OVA-fed mice (Fig. 5; p < 0.05). To further
characterize T cell responses in these mice, we assessed OVA-specific T
cell proliferative responses in vitro. Splenic
CD4+ T cells from mice fed PBS before oral
immunization with OVA plus CT showed marked OVA-specific proliferative
responses, although the responses were variable among individual mice
(stimulation indices ranging from 3.5 to 9.6) (Fig. 6A). In contrast, splenic
CD4+ T cells from mice fed OVA before oral
immunization were unresponsive to OVA (stimulation indices ranging from
0.9 to 2.1) (Fig. 6A). We also examined
CD4+ T cell responses in Peyer’s patches of
these mice because this tissue is the major mucosal inductive site for
immune responses in the GI tract. Of interest,
CD4+ T cells from Peyer’s patches of mice given
oral OVA were essentially unresponsive to the fed Ag (stimulation
indices ranging from 1.1 to 1.9) (Fig. 6B). In contrast,
Peyer’s patch CD4+ T cells from PBS-fed mice
exhibited significant OVA-specific proliferative responses (stimulation
indices ranging from 1.7 to 4.9) (Fig. 6B). These results
indicate that a high oral dose of OVA induces OVA-specific T cell
unresponsiveness in mucosal inductive sites in addition to the systemic
compartments after subsequent oral challenge with OVA and CT.
Furthermore, the data suggest that use of CT as oral adjuvant cannot
reverse this state of T cell unresponsiveness.
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It is well established that a single high oral dose of protein
induces Ag-specific clonal anergy and/or deletion to subsequent
systemic challenge with the same Ag. This was characterized by
reductions in T and B cell responses and cytokine production by
CD4+ T cells (6, 8). It was
important to determine whether a single high dose of oral OVA would
influence cytokine responses induced by oral immunization. Splenic
CD4+ T cells from mice fed PBS before oral
challenge with OVA and CT produced high levels of the Th2-type
cytokines IL-4, IL-5, IL-6, and IL-10, but essentially no IL-2 or
IFN- (Th1-type) in response to OVA (Fig. 7, A and B). In
contrast, these dominant Th2-type cytokine responses by splenic
CD4+ T cells were markedly decreased when mice
were fed a high dose of OVA (Fig. 7B; p <
0.05). In addition, up-regulation of Th1-type cytokine responses were
not observed in OVA-fed mice and these cytokine levels remained below
detection in these mice. To assess the effects of tolerance on the
mucosal T cell cytokine responses, we obtained Peyer’s patch
CD4+ T cells from mice fed PBS or OVA. Of
interest, both Th1- and Th2-type cytokine responses were below
detectable levels in Peyer’s patch CD4+ T cells
from mice given oral OVA (Fig. 8, A and B). In contrast, Peyer’s patch
CD4+ T cells from PBS-fed mice exhibited
significant OVA-specific Th2-type but not Th1-type cytokine responses
(Fig. 8, A and B).
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1 plays an
important role in the induction and regulation of oral tolerance, we
also measured TGF-1 levels in culture
supernatants of splenic and Peyer’s patch CD4+ T
cells from mice fed PBS or OVA. Interestingly,
TGF-1 in the culture supernatants of
OVA-stimulated splenic and Peyer’s patch CD4+ T
cells in vitro was below detectable levels, although we performed
TGF-1-specific ELISA using a highly sensitive
luminometric assay (data not shown). This suggests that T cell
production of TGF-1 was not a major factor in
mucosal and systemic unresponsiveness after oral delivery of a large
dose of OVA. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oral tolerance has been characterized as the state of Ag-specific systemic unresponsiveness with the maintenance of mucosal S-IgA Ab responses. This concept came from the initial finding that oral administration of a streptococcal Ag or OVA to mice resulted in suppression of Ag-specific systemic immune responses in the presence of reduced but significant salivary S-IgA Ab responses (2). Notably, the levels of salivary S-IgA Abs in Ag-fed mice were low, and mucosal immune responses were not induced in systemically challenged control mice (2). Thus, we must reconsider the concept of oral tolerance and address whether mucosal immune responses are susceptible to oral tolerance. To date, nearly all studies of oral tolerance have been assessed with a systemic immunization protocol after oral administration of Ag; however, by using this method, the susceptibility of mucosal immune responses to oral tolerance remains undefined. In this study, we directly addressed this issue by use of a well-established oral immunization protocol with OVA plus CT as mucosal adjuvant. The main advantage to mucosal immunization is that this mode can elicit marked Ag-specific mucosal S-IgA Abs as well as serum Ab responses and one can evaluate the influence of oral Ag on both mucosal and systemic immunity concurrently. To this end, our results provide the first direct evidence that oral tolerance down-regulates mucosal as well as systemic immunity.
In this study, we were unable to detect OVA-specific salivary IgA Abs even in PBS-fed mice after oral immunization, although CT-B-specific IgA Ab responses were comparably induced in both PBS- and OVA-fed mouse groups. This is most likely due to the weak immunogenicity of OVA because oral immunization with tetanus toxoid and CT induced tetanus toxoid-specific IgA Ab responses in saliva (26). In addition, oral immunization is suboptimal for induction of salivary IgA Ab responses. In this regard, it has been shown that nasal immunization is superior to oral immunization for the induction of Ag-specific immune responses in the upper respiratory tract and in the salivary gland (27). Furthermore, nasal immunization was shown to be the most effective way to elicit Ag-specific Ab responses in mucosal compartments including the nasal passages and the oral cavity, while oral immunization is much more effective for induction of mucosal IgA responses in the GI tract (28).
Up until now, few studies have addressed the induction of oral tolerance in mucosal immune compartments. In one study, oral priming with keyhole limpet hemocyanin (KLH) followed by an oral boost with KLH and CT as adjuvant was found to result in markedly diminished mucosal Ab responses to KLH (29). Similar reductions in Ag-specific Ab responses were reported in OVA-fed mice before oral immunization with OVA and LT as adjuvant (30). These results are in excellent agreement with our findings; however, these studies were mainly focused on the adjuvant properties of CT and LT and detailed analyses of systemic and mucosal immune responses were not performed. Furthermore, and more importantly, T cell responses in systemic and mucosal compartments were not addressed in these studies. In contrast, our previous study indicated that oral administration of trinitrobenzene sulfonic acid (TNBS) could modify systemic and mucosal anti-trinitrophenyl Ab responses in experimental hapten-induced colitis, which could be induced by intracolonic sensitization with TNBS in 50% ethanol (31). Thus, oral TNBS protected mice from development of TNBS-induced colitis. Furthermore, striking decreases in the numbers of trinitrophenyl-specific IgM, IgG, and IgA AFCs occurred in caudal lymph node, and IgM and IgG but not IgA AFCs decreases were seen in the colonic lamina propria. These results suggested that although oral administration with TNBS inhibited hapten-specific IgG responses in mice with colitis, Ag-specific IgA Ab responses were less affected. It should be noted that the TNBS hapten model is useful for studies of colonic inflammation; however, it is not ideal to determine whether oral tolerance blocks induction of mucosal IgA Ab responses to protein Ags because the hallmark of this response is colonic inflammation. More recently, it was reported that OVA-specific fecal IgA Ab responses induced by intraperitoneal challenge with OVA in CFA was marginally suppressed by prior OVA feeding in normal BALB/c mice (32). As is well documented, mucosal tissues form an integrated and connected network at the tissue, cell, and molecular levels and mucosal immune responses are regulated by unique pathways termed the common mucosal immune system (CMIS) in mucosal inductive tissues (e.g., Peyer’s patches) and effector sites (e.g., lamina propria) (33). However, the induction of IgA Ab responses in these studies were considered to be a CMIS-independent event and were not initiated in the major mucosal inductive tissues, the Peyer’s patches. In addition, the peritoneal cavity is a major source of B-1 cells, which are distinguishable from conventional B cells (B-2 cells) by cell surface markers; of note, B-1 cells express Mac-1 and are B220low, whereas B-2 cells are B220high and Mac1- (34, 35). In addition, B-1 cells, which are derived from the peritoneal cavity, tend to migrate into lamina propria effector sites of the GI tract and differentiate into IgA-producing plasma cells through a T cell-independent mechanism (36, 37). In contrast, Ag-specific IgA Ab production induced by CT in mucosal effector tissues originates from Th cell-dependent B-2 cells (38, 39). In this study, we showed that prior OVA feeding clearly suppressed both systemic and mucosal immune responses elicited by oral challenge with OVA and CT, indicating that oral administration of a soluble protein Ag blocks the induction of CMIS-dependent mucosal immune responses, including IgA Ab production in the GI tract lamina propria.
As is well documented, CT is an excellent mucosal adjuvant for induction of both systemic and mucosal immune responses, including S-IgA Ab responses in mucosal compartments, and nontoxic derivatives are being evaluated for clinical use (6, 20, 21). Because CT exhibits severe toxicity for humans in its native form, mutants of CT that lack ADP-ribosyltransferase activity and diarrheagenicity but retain adjuvanticity have been developed by using molecular biological techniques to resolve this problem (23). Another possible serious side effect of CT is that mucosal administration of CT may abrogate already established tolerance and lead to mucosal hypersensitivity to food Ags and commensal bacteria. In this study, we showed that a single high-dose feeding of protein Ag induces a state of Ag-specific immune unresponsiveness in mucosal compartments, and CT does not break already established tolerance in either mucosal or systemic compartments. In this regard, our data suggest that CT cannot reverse tolerance to Th2-mediated hypersensitivity responses. Assessment as to whether CT abrogates the maintenance of oral tolerance established by repeated low-dose feeding of Ag is under investigation. In addition, further analyses of oral tolerance in mucosal immune compartments followed by Th1-type immune responses seen when LT is used as mucosal adjuvant or when attenuated Salmonella expressing OVA would provide interesting information on mucosal immune responses in the induction and regulation of oral tolerance.
The mechanisms of oral tolerance induction have been assessed in several models; however, most of these studies only determined whether systemic unresponsiveness was induced. In this study, we have now investigated whether both systemic and mucosal immune responses induced by oral immunization are influenced by prior Ag feeding and we have clearly shown that both systemic and mucosal B and T cell responses were dramatically reduced by a single high oral dose of OVA before oral immunization with OVA and CT. Our results should now change the established notion of oral tolerance that has been defined as suppression of Ag-specific systemic immune responses in the presence of mucosal S-IgA Ab responses. Our results provide a new concept for oral tolerance that oral delivery of protein Ag results in a state of both systemic and mucosal immune unresponsiveness to a subsequent encounter with the same Ag. Our studies also indicate that CT, a potent mucosal adjuvant, does not abrogate already established oral tolerance. Finally, this study has established a new model system to assess orally induced unresponsiveness in both the systemic and mucosal immune compartments.
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Acknowledgments |
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1-specific luminometric assay and Sheila D. Turner
for final preparation of this manuscript. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Jerry R. McGhee, Department of Microbiology, Immunobiology Vaccine Center, University of Alabama, 761 Bevill Biomedical Research Building, 845 19th Street South, Birmingham, AL 35294-2170.
3 Abbreviations used in this paper: S-IgA, secretory IgA; GI, gastrointestinal; DTH, delayed-type hypersensitivity; CT, cholera toxin; LT, labile toxin; CT-B, B subunit of CT; AFC, Ab-forming cell; ELISPOT, enzyme-linked immunospot; KLH, keyhole limpet hemocyanin; TNBS, trinitrobenzene sulfonic acid; CMIS, common mucosal immune system.
Received for publication October 11, 2000. Accepted for publication December 22, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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release after oral tolerization are distinct from encephalitogenic epitopes and mediate epitope-driven bystander suppression. J. Immunol. 151:7307.[Abstract]
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T cell-deficient mice have impaired mucosal immunoglobulin A responses. J. Exp. Med. 183:1929. T cells regulate mucosally induced tolerance in a dose-dependent fashion. Int. Immunol. 11:1907.This article has been cited by other articles:
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 D. Alignani, B. Maletto, M. Liscovsky, A. Ropolo, G. Moron, and M. C. Pistoresi-Palencia Orally administered OVA/CpG-ODN induces specific mucosal and systemic immune response in young and aged mice J. Leukoc. Biol., June 1, 2005; 77(6): 898 - 905. [Abstract] [Full Text] [PDF]
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D. W. Smith and C. Nagler-Anderson Preventing Intolerance: The Induction of Nonresponsiveness to Dietary and Microbial Antigens in the Intestinal Mucosa J. Immunol., April 1, 2005; 174(7): 3851 - 3857. [Abstract] [Full Text] [PDF]
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K. Kataoka, J. R. McGhee, R. Kobayashi, K. Fujihashi, S. Shizukuishi, and K. Fujihashi Nasal Flt3 Ligand cDNA Elicits CD11c+CD8+ Dendritic Cells for Enhanced Mucosal Immunity J. Immunol., March 15, 2004; 172(6): 3612 - 3619. [Abstract] [Full Text] [PDF]
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 H. Kato, K. Fujihashi, R. Kato, T. Dohi, K. Fujihashi, Y. Hagiwara, K. Kataoka, R. Kobayashi, and J. R. McGhee Lack of oral tolerance in aging is due to sequential loss of Peyer's patch cell interactions Int. Immunol., February 1, 2003; 15(2): 145 - 158. [Abstract] [Full Text] [PDF]
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) or PBS (
) before oral
challenge. Mice were fed 0.25 ml PBS or 50 mg of OVA on day 0. On days
7, 14, and 21 after oral feeding, these mice were orally immunized with
1 mg of OVA and 10 µg of CT as a mucosal adjuvant. Ag-specific Ab
responses were determined 7 days after the final challenge. The results
are expressed as the mean titer ± SD for 20 mice in each
experimental group. *, p < 0.05 compared with
control mice.
