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*
Departments of Microbiology and Oral Biology, Immunobiology Vaccine Center, University of Alabama Medical Center, Birmingham, AL 35294;
Department of Internal Medicine, Division of Immunology, University of Cincinnati College of Medicine, Cincinnati, OH 45267;
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; and
§
Departimento di Clinica Medica, Immunologia e Malattie Infettive, Universita Degli Studi di Bari, Bari, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion both in vivo and
in vitro. Intranasal administration of IL-12 to mice nasally immunized
with the combined vaccine of TT and CT resulted in increased
TT-specific IgG2a and IgG3 Abs, while IgG1 and IgE Ab responses were
markedly reduced. This shift of the CT-induced immune response toward
Th1 type was associated with TT-specific CD4+ T cells
secreting IFN- and reduced levels of Th2-type cytokines (i.e., IL-4,
IL-5, IL-6, and IL-10). In contrast, intranasal IL-12 enhanced the
CT-induced serum IgG1 and IgE Ab responses in mice given the combined
vaccine orally. IFN- secretion by TT-specific CD4+ T
cells was also enhanced; however, Th2-type cytokine responses were
predominant. Mucosal secretory IgA responses to oral or nasal vaccines
were not affected by intranasal IL-12. Thus, intranasal IL-12 delivery
influences Th cell subset development in mucosal inductive sites that
are dependent on the route of vaccine delivery. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since most infectious diseases occur at mucosal sites of the
gastrointestinal (GI),4
respiratory, and genitourinary tracts, the development of strategies
for directed induction of Th1- or Th2-type responses by mucosal
vaccines is an important priority. Regulatory cytokines, i.e., IL-12
and IFN- vs IL-4, are effective for directing immunity through Th1
or Th2 pathways, respectively (3, 7) and could be combined with mucosal
adjuvants and delivery systems to elicit desired Th1- or Th2-type
responses in both mucosal and systemic immune compartments. In this
regard, Th2-type responses induced by the widely used mucosal adjuvant
cholera toxin (CT) (4, 8, 9) were down-regulated by both parenteral and
oral IL-12, while CD4+ T cell help for secretory IgA
(S-IgA) Ab responses to comucosally administered protein antigens was
not affected by orally administered IL-12 (5). IL-12 produced by APCs
(10, 11) is a potent stimulator of IFN- production by NK and T cells
(7, 12, 13) and preferentially supports Th1-type responses. Use of
IL-12 to direct Th1-type responses has important implications for
therapy (14, 15, 16, 17, 18). IL-12 has also been shown to induce T cell precursors
to develop into functional Th1-type cells (13, 19). However, it has
also been reported that IL-12 can exacerbate rather than suppress
Th2-type responses either in the absence of IFN- (20) or when
administered during ongoing Th2-type responses (21, 22).
Since IL-12 directs Th1-type and CT induces Th2-type responses to mucosal vaccines, it is important to define the mechanisms for these two major response pathways. Specifically, we studied both in vitro and in vivo effects of IL-12 on CT-mediated immune responses. Compelling evidence suggests that the nasal route is more effective for the induction of S-IgA responses in the GI, respiratory, and urogenital tracts (23, 24). Therefore, IL-12 was intranasally administered to mice given a nasal or oral vaccine with CT as mucosal adjuvant, and the results indicate that the initial stimulus in mucosal inductive sites determines whether Th1- or Th2-type responses predominate.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were obtained from Charles River Breeding Laboratories (Wilmington, MA) and were maintained in the animal facility of the University of Alabama at Birmingham Immunobiology Vaccine Center. Sterile food and water were provided ad libitum, and mice were used at 8–12 wk of age. Mice were pathogen free as determined by routine Ab screening against common mouse pathogens and histological analysis of organs and tissues. To ensure animal safety and compliance, we followed the guidelines proposed by the committee for the Care of Laboratory Animal Resources, Commission of Life Sciences, National Research Council.
Immunization and treatment with recombinant murine IL-12 (rmIL-12)
For intranasal immunization, mice received 20 µg of tetanus toxoid (TT) and 1 µg of CT (in 20 µl; 10 µl per nostril) on days 0, 7, and 14. The oral immunization was performed as previously described (4, 5, 8, 25). Mice were orally given vaccine-grade TT alone (250 µg/mouse) (kindly provided by Dr. Patricia J. Freda Pietrobon, Connaught Laboratories, Swiftwater, PA) and with CT (10 µg/mouse; LIST Biologic Laboratories, Campbell, CA) on days 0, 7, and 14 (5, 6, 7). Fecal pellets and blood samples were collected at weekly intervals and processed as previously described (4, 5, 25). Specifically, fecal samples were weighed and dissolved in PBS containing 0.1% sodium azide by addition of 1 ml of PBS-sodium azide per 100 mg of fecal extract. Normalization of fecal sample was further confirmed by the analysis of protein content and total IgA levels. Vaginal washes were collected by gently flushing the vaginal canal with 150 µl of sterile PBS under anesthesia. Nasal washes were obtained by flushing the nasopharyngeal cavity with 100 µl of sterile PBS. Saliva was collected after i.p. injection of 0.1 mg of pilocarpine (Sigma, St. Louis, MO) in sterile PBS. After collection, nasal and vaginal washes and saliva samples were centrifuged for 5–10 min at 10,000 rpm, and supernatants were collected and stored at -70°C until assessed.
rmIL-12 was generously provided by Dr. Maurice K. Gately (Hoffmann-LaRoche, Nutley, NJ). The cytokine treatment was given to mice by the intranasal route on days 0, 3, 7, 10, 14, and 17, a frequency that resulted in optimal responses as determined in our preliminary experiments. The mice received the combined (TT plus CT) vaccine by either gavage or intranasal routes on days 0, 7, and 14, followed by intranasal IL-12-liposomes 10–15 min later. For in vivo treatment, 10, 100, or 1000 ng of rmIL-12 were complexed with preformed cationic liposomes (DOTAP; Boehringer Mannheim, Indianapolis, IN) as described previously (5).
Analysis of Ab isotypes and IgG subclasses
Specific Ab levels in nasal and vaginal washes, serum, and fecal
extracts were determined by endpoint ELISA (4, 25). Briefly, ELISA
plates (Microtest III; Becton Dickinson, Oxnard, CA) were coated with a
100-µl solution of TT (5 µg/ml; 1.25 limes flocculation
units/ml), and serial twofold dilutions of serum, fecal extracts,
saliva, or nasal or vaginal washes were added to individual wells.
Horseradish peroxidase (HRP)-conjugated goat anti-mouse µ-, -,
or 
-heavy chain-specific antisera (Southern Biotechnology
Associates, Birmingham, AL) were used to detect IgM, IgG, or IgA Abs.
For IgG subclass levels, biotin-conjugated, rat monoclonal
anti-mouse 1 (G1-7.3; 2 µg/ml), 2a (R19-15; 1 µg/ml),
2b (R12-3; 0.5 µg/ml), or 3 (R40-82; 1 µg/ml)
-chain-specific Abs (PharMingen, San Diego, CA), followed by
HRP-conjugated streptavidin (Life Technologies, Grand Island, NY), were
used as described previously (4, 5). The reactions were developed with
ABTS as substrate (Sigma), and the absorbance at 415 nm was
determined after 15 min. Endpoint titers were determined as the last
dilution exhibiting an optical density of 
0.1 when compared with
negative controls.
Total IgE levels and Ag-specific IgE Abs were determined by ELISA and a
passive cutaneous anaphylaxis (PCA) assay, respectively, as previously
described (4, 5). For total IgE measurements, Nunc-Immuno MaxiSorp
plates (Nalge Nunc International, Naperville, IL) were coated with a
rat monoclonal anti-mouse IgE Ab (R35-72; PharMingen), and the IgE
were detected in serum samples with a biotinylated rat
monoclonal anti-mouse IgE mAb (R35-92; PharMingen) followed by
HRP-conjugated streptavidin. For the PCA test, threefold dilutions of
mouse sera (starting at 1:10) were s.c. injected into the shaved backs
of Fisher rats (200–250 g) followed 16 h later by i.v. injection
of 200 µg of TT in 1 ml of 1% Evans blue dye in PBS. The rats were
killed 15–20 min later, and PCA endpoint titers were selected as the
last dilution resulting in a diameter of bluing of 5 mm in rat skin.
B cell enzyme-linked immunospot (ELISPOT) for IgA Ab-forming cells (AFC)
A previously described ELISPOT assay was used to determine
numbers of AFC in different organs (5, 25). Briefly, 96-well
nitrocellulose-based plates were coated with a 100-µl solution of TT
(5 µg/ml) diluted in PBS. After washing, serial fivefold dilutions of
cell suspensions (starting at 1 x 106 cells) were
added to the wells and incubated for 6 h. Individual AFC were
detected with peroxidase-labeled anti-mouse -chain-specific Ab
(1 µg/ml) (Southern Biotechnology Associates) and were visualized by
adding the chromogenic substrate 3-amino-9-ethylcarbazole (Moss,
Pasadena, MD). Individual AFC were counted with the aid of a stereo
microscope (SZH Zoom Stereo Microscope System; Olympus, Lake Success,
NY).
Assessment of Ag-specific CD4+ T cell responses
The isolation of cells from lungs, Peyer’s patches, and spleen, as well as purification of CD4+ T cells, was previously described (5, 8, 25). CD4+ T cells were restimulated in vitro according to previously described methods (4, 5, 8). Briefly, CD4+ T cells (2 x 106 cells/ml) were cultured with rIL-2 (10 U/ml, PharMingen) and T cell-depleted, irradiated spleen feeder cells from naive mice in flat-bottom 96-well (200 µl/well) or 24-well (1 ml/well) tissue culture plates (Corning Glass Works, Corning, NY) for proliferation and cytokine synthesis, respectively. To measure Ag-specific T cell proliferation, 0.5 µCi of tritiated [3H]thymidine (Amersham, Arlington Heights, IL) was added after 5 days of culture and 16 h before harvest. Approximately 10 TT-coated particles/cell were found to optimally stimulate CD4+ T cell cultures based on proliferative responses and cytokine levels produced in culture supernatants.
Cytokine measurement
Culture supernatants were harvested following 5 days of culture
and stored at -70°C before the assays. Cytokine levels in culture
supernatants and serum were determined by a sensitive ELISA using
appropriate combinations of mAbs described in our previous studies (5).
The IL-12 ELISA assay was performed with the 9A5 rat anti-mouse
IL-12 heterodimer Ab as coating Ab and the HRP-conjugate 5C3
anti-mouse IL-12p40 subunit Ab, both from Hoffmann-LaRoche.
Standard curves were generated using murine rIFN-, rIL-5, rIL-6, and
rIL-10 (Genzyme, Cambridge, MA); rIL-2 (PharMingen); rIL-4 (Endogen,
Boston, MA); and rIL-12 (Hoffmann-LaRoche). The ELISA used in this
study could detect 15 pg/ml of IFN-; 5 pg/ml of IL-2, IL-4, and
IL-5; 100 pg/ml of IL-6; 200 pg/ml of IL-10; and 25 pg/ml of IL-12.
Statistics
The results are expressed as the mean ± 1 SD. Statistical
significance (p 
0.05) was determined by
Student’s t test and by ANOVA followed by the Fisher least
significant difference test. For statistical analysis, cytokine levels
below the detection limit were recorded as one-half the detection limit
(e.g., for IFN-, 7.5 pg/ml).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis in vitro
Addition of IL-12 to splenocyte cultures resulted in high levels
of IFN-, while addition of CT did not induce IFN- secretion (Fig. 1
A). The combination of CT or
the nontoxic B subunit of CT (CT-B) and IL-12 inhibited the
IL-12-mediated IFN- production (Fig. 1). Preincubation of splenocyte
cultures with CT or CT-B for 24 h before addition of IL-12 almost
abolished the IFN- secretion, and CT exhibited a greater inhibitory
effect than CT-B. Conversely, preincubation with IL-12 followed by CT
or CT-B either slightly diminished or failed to impair the ability of
IL-12 to induce IFN- secretion (Fig. 1). These results, and similar
data obtained with splenic CD4+ T cells and Peyer’s
patches and lung-associated lymphoid tissue T cells (data not shown),
show that the binding of CT to lymphocytes alters the ability of IL-12
to induce IFN- when CT is presented either before or together with
IL-12.
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synthesis in vivo
It remained possible that the reciprocal effects of CT and IL-12
on IFN- synthesis could be due to culture conditions and did not
reflect the effects in vivo. Therefore, IL-12 was administered by
parenteral or nasal routes, and subsequent serum IL-12 and IFN-
levels were evaluated. Parenteral or nasal administration of 10 ng of
IL-12 did not result in detectable serum IL-12 levels (data not shown).
Enhanced serum IL-12 and subsequent IFN- levels were achieved when
100 ng of IL-12 was administered by either route, and these effects
were greater with 1000 ng of IL-12 per dose (Fig. 2, A and B).
Further, serum IL-12 levels reached after intranasal administration of
1000 ng of IL-12 were 
20% of those achieved by i.p. administration
of this cytokine. Parenteral vs nasal IL-12 treatment also differed in
kinetics of serum IL-12 achieved. In this regard, serum IL-12 levels
peaked later after the nasal treatment than after parenteral IL-12
administration (Fig. 2, A and B). Both i.p. and
intranasal delivery of 1000 ng of IL-12 resulted in large increases in
serum IFN- levels; however, i.p. IL-12 delivery resulted in serum
IFN- levels that were fourfold higher than those reached after
intranasal administration of IL-12 (Fig. 2, C and
D).
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(data not shown). Further, the increased serum IFN- levels
induced by nasal or parenteral administration of 1000 ng of IL-12 were
not affected by CT administered by parenteral or nasal routes,
respectively. In contrast, IL-12-induced serum IFN- production was
significantly reduced when IL-12 and CT were administered by the same
route. In fact, the i.p. injection of CT and IL-12 or administration of
CT 24 h before IL-12 by the same parenteral route reduced by
90–100% the levels of IL-12-induced serum IFN- (Fig. 2
E). Intranasal coadministration of CT and IL-12 resulted in
lower inhibition (44–56% inhibition) of IL-12-induced serum IFN-
levels (Fig. 2F). These observations suggest that both
parenteral and mucosal CT inhibit serum IFN- responses to
coadministered IL-12 and that the regulatory effects of mucosally
delivered CT on IL-12 are less dramatic than those of parenteral CT. Ab responses in mice intranasally immunized with a combined vaccine and nasally treated with IL-12-liposomes
The regulatory effects of the mucosal adjuvant CT (a Th2 inducer)
and IL-12 (a Th1 inducer) were further investigated by analyzing Ab
responses of mice intranasally immunized with TT and CT as adjuvant.
This combined vaccine resulted in serum anti-TT Ab responses of all
isotypes, including prominent IgG1 and IgG2b subclass responses (Fig. 3). Nasal coadministration of 10 ng of
IL-12 did not affect CT-induced serum anti-TT Ab responses (data
not shown). On the other hand, a 100-ng IL-12 dose induced IgG2a and
inhibited IgE Ab responses (Fig. 3). TT-specific IgM, IgG, and IgA Ab
responses were unchanged when 1000 ng of IL-12 was intranasally
coadministered in mice. However, this nasal IL-12 dose significantly
reduced both total and TT-specific IgE Ab responses and shifted the
profile of IgG subclasses toward increased IgG2a and IgG3 and decreased
IgG1 Ab responses when compared with mice given the intranasal combined
vaccine only (Fig. 3). Titers of TT-specific S-IgA Abs in nasal and
vaginal washes or saliva were not significantly affected by the
IL-12 treatment (Fig. 3).
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We have previously reported that oral administration of IL-12
resulted in negligible serum IL-12 that could shift Th2-type responses
to vaccine co-orally administered with CT (5). Since intranasal IL-12
delivery results in significant levels of serum IL-12, we addressed the
regulatory effects that intranasal IL-12 would have on the oral
combined vaccine of TT and CT. Interestingly, both TT-specific serum
IgG and IgA Ab responses were significantly enhanced when mice orally
immunized with the combined vaccine were nasally treated with 1000 ng
of IL-12 (Fig. 4A). The
enhancing effect of intranasal IL-12 resulted in increased TT-specific
serum IgG1 and IgG2b Ab levels and higher IgG2a Abs (Fig. 4B). Total and TT-specific IgE Ab responses were also
increased by the intranasal IL-12 treatment (Fig. 4C),
demonstrating that intranasal IL-12 can enhance systemic Th2-type
responses induced by the combined oral vaccine. TT-specific IgA AFC in
the intestinal lamina propria and S-IgA Abs in external secretions were
not significantly affected in mice given intranasal IL-12 and oral
combined vaccine (Fig. 4, D and E).
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). CT-induced IgG1 and IgG2b subclasses
were also up-regulated by delayed parenteral IL-12, and a lower but
significant increase in IgG2a Abs was also noted (Table I). In contrast
to the delayed parenteral IL-12 treatment, the effects of early
parenteral IL-12 treatment were restricted to stimulation of IgG2a and
IgG3 and inhibition of IgE Ab responses, thus suggesting an important
role for the timing of IL-12 treatment on the outcome of immune
responses (Table I).
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Th cell subsets responsible for the divergent Ab responses
following intranasal IL-12 administration to mice that received either
intranasal or oral combined vaccines were further determined by
analysis of cytokines secreted by TT-specific CD4+ T cells.
While TT-specific CD4+ T cells induced by the intranasal
vaccine alone produced only negligible levels of Th1-type cytokines
(IFN- and IL-2), these two cytokines were induced by intranasal
administration of IL-12-liposomes (Fig. 5). This IL-12 treatment also
significantly reduced IL-4 and IL-5 and slightly affected IL-6 and
IL-10 secretion by systemic (i.e., splenic) and mucosal (i.e., lung)
TT-specific CD4+ T cells (Fig. 5).
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). However, in contrast to mice given
the intranasal vaccine, those receiving the combined oral vaccine
showed no alteration in IL-4, IL-5, and IL-6 secretion by TT-specific
CD4+ T cells from systemic (i.e., spleen) or from mucosal
(i.e., Peyer’s patches) inductive sites following intranasal
IL-12 administration (Fig. 6). Further, IL-10 levels were significantly
increased in culture supernatants of TT-specific CD4+ T
cells in the latter group (Fig. 6). Thus, while Th1-type responses were
induced in all groups of mice receiving intranasal IL-12, Th2-type
responses were unaltered and IL-10 was significantly increased in mice
given the oral vaccine.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most IL-12 effects are mediated through IFN- synthesis (7), and CT
has been shown to exert its mucosal adjuvant effect via IL-4 and
Th2-type responses (4, 9). Therefore, in an attempt to elucidate the
reciprocal regulation of IL-12 (a Th1 inducer) and CT (a Th2 inducer)
and to understand its role in subsequent immunity to mucosal vaccines,
we began by measuring IL-12-induced IFN- secretion in the presence
or absence of CT. Addition of CT to total splenocyte or
CD4+ T cell cultures inhibited the ability of IL-12 to
induce IFN-. Further, taking advantage of the efficiency of the
intranasal delivery of IL-12 to achieve significant serum levels of
this cytokine, we were able to show an inhibitory effect of CT on
IFN- secretion that is normally induced by IL-12. These observations
clearly demonstrate the ability of CT to inhibit IL-12-induced IFN-
production both in vitro and in vivo and support the greater
effectiveness of the intranasal route when compared with the oral route
for the induction of mucosal immunity (23, 24).
Our in vitro studies show that the inhibitory effect of CT is dependent
to a large extent on the binding of the CT-B subunit. Thus, the
inhibitory effect may not primarily involve the adenylate cyclase
system mediated by the A subunit of CT (28, 29). One possible mediator
for the inhibitory effect of CT and CT-B on IL-12-induced IFN-
secretion is CT-induced prostaglandin E2 (PGE2)
(30, 31) which can inhibit IFN- production by T cells (32). In fact,
it has been reported that PGE2 inhibited cytokine
production in Th1 cells but not in Th2 cells (33) and that IFN-
production by CD4+ T cells was largely regulated by the
ratio of IL-12 to PGE2 during T cell activation (33). An
important finding from our in vitro studies was the inability of either
CT or IL-12 to reciprocally reverse the ongoing effects of the other
mediator. These observations suggest that both the timing and the route
of CT and IL-12 administration, and the subsequent levels of these
molecules that reach the inductive sites, are critical for the cytokine
environment and the resulting differentiation of T helper subsets in
vivo.
The consequences of the reciprocal regulation of intranasally
administered CT and IL-12 on mucosal and systemic immune responses were
further investigated in mice that received intranasal IL-12 and
intranasal or oral combined vaccine (CT plus TT). We have previously
shown that despite negligible serum levels of IL-12 achieved after oral
administration of this cytokine, this treatment shifted systemic
Th2-type responses induced by oral CT as mucosal adjuvant toward a Th1
type (5). Specifically, oral and parenteral IL-12 down-regulated IL-4
and Ag-specific IgG1 and IgE Ab responses, while IgG2a and IgG3 Abs, as
well as IFN-, were enhanced (5). However, only oral IL-12 preserved
mucosal S-IgA Ab responses (5). Interestingly, intranasally delivered
IL-12 affected Ab responses to an intranasal vaccine differently from
the way it affected Ab responses to an oral vaccine containing CT as
adjuvant. Intranasal coadministration of IL-12 and the combined vaccine
shifted CT-induced systemic Th2-type responses toward a Th1 type (i.e.,
down-regulation of IgG1 and IgE Ab responses and enhanced IgG2a and
IgG3), but preserved S-IgA Ab responses. Conversely, intranasal IL-12
enhanced Th2-type responses induced by the oral combined vaccine with
CT as adjuvant and promoted IgG2a Ab responses. Thus, divergent
profiles of Ab responses to either intranasal or oral combined vaccine
with CT were induced by intranasal IL-12 despite induction of IFN-
(i.e., a Th1-type cytokine), which provides support for IgG2a Abs.
Another striking finding of the present study was the ability of
intranasal IL-12 to enhance oral CT-induced Th2-type immune responses
and to up-regulate all Ig isotype and IgG subclass Ab responses. This
observation is in contrast to the known ability of IL-12 to promote the
development of Th1 type and to down-regulate Th2-type responses (7, 13, 19). However, IL-12 has also been shown to exacerbate rather
than to suppress Th2-type responses either in the absence of IFN-
(20) or when administered during ongoing Th2-type responses (21, 22).
In our system, temporal factors related to the compartmentalized
delivery of oral combined vaccine and intranasal IL-12 during the
initiation of the immune response could explain both why the Th2
phenotype was not reversed and why intranasal IL-12 enhanced the oral
CT-induced Th2-type responses. In this regard, the GI and respiratory
tracts display distinct anatomy and physiology (34, 35, 36). Further, the
central role of timing in the reciprocal regulation of CT and IL-12 was
supported by the observation that delayed parenteral administration of
IL-12 induced Th1-associated IgG2a and IgG3 but did not inhibit IL-4
and Th2-associated IgE and IgG1 Abs to an oral vaccine with CT as
adjuvant (Table I).
Since divergent profiles of serum Ab responses were induced by
intranasal IL-12 in mice that received combined intranasal as opposed
to oral vaccine, we analyzed cytokines secreted by TT-specific
CD4+ T cells to determine Th cell responses that regulate
these effects. Increased Th1-type secretion (e.g., IL-2 and IFN-) by
TT-specific CD4+ T cells was induced by intranasal IL-12 in
mouse groups receiving either the intranasal or the oral combined
vaccine. This finding is consistent with the potential of IL-12 to
stimulate IFN- and to lead to Th1-type responses (7, 13).
Interestingly, intranasal IL-12 exhibited distinct effects on Th2-type
cytokine secretion by TT-specific CD4+ T cells in mice
receiving the intranasal and the oral combined vaccine. In fact,
Th2-type cytokines were down-regulated by intranasal IL-12 in mice
given the nasal combined vaccine, while Th2-type cytokine secretion
(i.e., IL-4, IL-5, and IL-6) by TT-specific CD4+ T cells
either was not reduced (i.e., for IL-4, IL-5, and IL-6) or was enhanced
(i.e., for IL-10) in mice given the combined vaccine by the oral route.
Thus, IL-12 delivered at a distant mucosal site failed to inhibit
Th2-type cells triggered by orally administered CT. In this regard,
IL-12 was reported to be unable to reverse the phenotype of
differentiated Th2-type cells (13, 19, 37) or to prevent Th2-type
differentiation in vivo in the presence of an overwhelming IL-4/IL-10
response (38). Although IL-12 was previously reported to stimulate
IFN- and IL-10 secretion (21), our results are the first to
demonstrate that mucosally administered IL-12 can both enhance mucosal
CT-induced Th2-type responses and simultaneously induce Th1-type
responses. Activated B cells were reported to respond to IL-12 with
increased cell growth through IFN- production (39). Further, it has
been recently shown that IL-12 can act as an adjuvant for humoral
immunity through IFN--dependent and -independent mechanisms (40).
This might explain the previously reported enhanced IgE production
following IL-12 treatment (22) and our results when mice were orally
immunized.
We have shown that mucosal administration is an efficient way to
deliver a regulatory cytokine, e.g., IL-12 to specific mucosal
inductive sites. In this regard, the differential effect of oral vs
intranasal administration of IL-12 on serum IL-12 and IFN- levels
provides additional ways to target desired effects on the mucosal vs
systemic immune compartments. Our study provides important new evidence
that mucosally administered IL-12 can shift or enhance Th2-type
responses to a mucosal vaccine with CT as adjuvant. Our results clearly
show that timing and mucosal route of vaccine and regulatory cytokine
administration are critical factors for the dual effect of IL-12 and
the subsequent induction of targeted immune responses. These
observations have important implications for the optimization of
mucosal vaccine regimens employing cytokines.
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Acknowledgments |
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Footnotes |
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2 M.M. and P.N.B. contributed equally to this work.
3 Address correspondence and reprint requests to: Dr. Jerry R. McGhee, The Immunobiology Vaccine Center, The University of Alabama, 761 BBRB, 845 19th Street South, Birmingham, AL 35294-2170. E-mail address:
4 Abbreviations used in this paper: GI, gastrointestinal; CT, cholera toxin; S-IgA, secretory IgA; rmIL-12, recombinant murine IL-12; TT, tetanus toxoid; HRP, horseradish peroxidase; PCA, passive cutaneous anaphylaxis; ELISPOT, enzyme-linked immunospot; AFC, Ab-forming cells; CT-B, nontoxic B subunit of CT.
Received for publication April 7, 1998. Accepted for publication September 4, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production by NK cell stimulatory factor (NKSF): characterization of the responder cells and synergy with other inducers. J. Exp. Med. 173:869. production and diminished interleukin 4 inhibition of such priming. Proc. Natl. Acad. Sci. USA 90:10188.. J. Immunol. 154:3999.[Abstract]
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 C. P. Bradney, G. D. Sempowski, H.-X. Liao, B. F. Haynes, and H. F. Staats Cytokines as Adjuvants for the Induction of Anti-Human Immunodeficiency Virus Peptide Immunoglobulin G (IgG) and IgA Antibodies in Serum and Mucosal Secretions after Nasal Immunization J. Virol., January 15, 2002; 76(2): 517 - 524. [Abstract] [Full Text] [PDF]
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C. Fromantin, B. Jamot, J. Cohen, L. Piroth, P. Pothier, and E. Kohli Rotavirus 2/6 Virus-Like Particles Administered Intranasally in Mice, with or without the Mucosal Adjuvants Cholera Toxin and Escherichia coli Heat-Labile Toxin, Induce a Th1/Th2-Like Immune Response J. Virol., November 15, 2001; 75(22): 11010 - 11016. [Abstract] [Full Text] [PDF]
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H. F. Staats, C. P. Bradney, W. M. Gwinn, S. S. Jackson, G. D. Sempowski, H.-X. Liao, N. L. Letvin, and B. F. Haynes Cytokine Requirements for Induction of Systemic and Mucosal CTL After Nasal Immunization J. Immunol., November 1, 2001; 167(9): 5386 - 5394. [Abstract] [Full Text] [PDF]
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H. Kato, K. Fujihashi, R. Kato, Y. Yuki, and J. R. McGhee Oral Tolerance Revisited: Prior Oral Tolerization Abrogates Cholera Toxin-Induced Mucosal IgA Responses J. Immunol., March 1, 2001; 166(5): 3114 - 3121. [Abstract] [Full Text] [PDF]
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C. Tang, M. D. Inman, N. van Rooijen, P. Yang, H. Shen, K. Matsumoto, and P. M. O'Byrne Th Type 1-Stimulating Activity of Lung Macrophages Inhibits Th2-Mediated Allergic Airway Inflammation by an IFN-{{gamma}}-Dependent Mechanism J. Immunol., February 1, 2001; 166(3): 1471 - 1481. [Abstract] [Full Text] [PDF]
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T. Koga, J. R. McGhee, H. Kato, R. Kato, H. Kiyono, and K. Fujihashi Evidence For Early Aging in the Mucosal Immune System J. Immunol., November 1, 2000; 165(9): 5352 - 5359. [Abstract] [Full Text] [PDF]
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J. Skok, J. Poudrier, and D. Gray Dendritic Cell-Derived IL-12 Promotes B Cell Induction of Th2 Differentiation: A Feedback Regulation of Th1 Development J. Immunol., October 15, 1999; 163(8): 4284 - 4291. [Abstract] [Full Text] [PDF]
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P. N. Boyaka, M. Marinaro, R. J. Jackson, S. Menon, H. Kiyono, E. Jirillo, and J. R. McGhee IL-12 Is an Effective Adjuvant for Induction of Mucosal Immunity J. Immunol., January 1, 1999; 162(1): 122 - 128. [Abstract] [Full Text] [PDF]
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, 
) or 1000 ng (•, 
) of rmIL-12-liposomes on day 0
by either the i.p. (A and C) or
intranasal (B and D) route. Mice were
bled both before rmIL-12 administration and 30 min and 2, 24, 48, and
96 h thereafter, and serum IL-12 and IFN-
) or 1000
ng (
) of rmIL-12-liposomes, and Ab titers were determined by ELISA
on day 21 following initial immunization. A, Isotype of
serum Abs. B, Serum IgG subclasses. C,
TT-specific IgE Ab titers that were measured at day 14 by PCA.
D, IgA Ab titers. The results are expressed as
mean ± SD and are representative of three separate experiments
(five mice per group).
