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Infection and Immunity, February 2000, p. 672-679, Vol. 68, No. 2
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Mucosally Induced Immunoglobulin E-Associated
Inflammation in the Respiratory Tract
Jerry W.
Simecka,1,*
Raymond J.
Jackson,2
Hiroshi
Kiyono,2,3 and
Jerry R.
McGhee2
Department of Molecular Biology and
Immunology, University of North Texas Health Science Center in Fort
Worth, Fort Worth, Texas 761071;
Department of Microbiology and Immunobiology Vaccine Center,
University of Alabama at Birmingham, Birmingham, Alabama
352942; and Department of Mucosal
Immunology, Research Institute for Microbial Diseases, Osaka
University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan3
Received 2 July 1999/Returned for modification 21 September
1999/Accepted 17 November 1999
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ABSTRACT |
The purpose of the present study was to determine the immunologic
responses, particularly immunopathologic reactions, associated with
nasal immunization with the mucosal adjuvant, cholera toxin (CT).
BALB/c mice were nasally immunized with tetanus toxoid (TT) combined
with CT, and the responses of these mice were determined. After nasal
immunization, mice produce a serum antibody response, primarily of the
immunoglobulin G (IgG) isotype of predominantly IgG1 subclass, against
both TT and CT. Along with the antibody responses, we also found that
inflammatory reactions, which could be potentially fatal, developed
within the lung. Furthermore, IgE responses were also induced after
nasal immunization, and these responses were associated with the
detection of interleukin 5 in the serum. Thus, nasal immunization with
TT plus CT likely results in the activation of Th2 cells, which may
contribute to serious immunopathologic reactions in the lung.
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INTRODUCTION |
Mucosal immunity constitutes the
first line of defense for the host and is a major component of
resistance against respiratory infections. The importance of mucosal
immunity, specifically secretory immunoglobulin A (S-IgA), in
controlling bacterial respiratory infections is exemplified in patients
with selective IgA deficiencies. These patients are more prone to
respiratory tract infections, including rhinosinusitis, otitis media,
tonsillitis, chronic pulmonary infections, and infectious asthma
(3-5, 25). Among the effector mechanisms of mucosal
immunity in bacterial disease, IgA can inhibit adherence or growth of
pathogenic bacteria (14, 15, 17, 34). The importance of
mucosal immunity, e.g., IgA, in resistance to respiratory disease is
probably best demonstrated for viral infections (7, 8, 26,
27). However, parenteral administration of vaccine does not
significantly promote immune responses within the upper respiratory
tract, despite development of significant serum antibody responses
(6). Circulating antibody, while effective against lower
respiratory tract infections, does not play a significant role in
protecting the upper respiratory tract (18, 30). However, systemic immunization is the route used for the current
Streptococcus pneumoniae and influenza vaccines, and results
from our laboratory clearly demonstrate that IgA responses in the upper
respiratory tract are not readily produced after systemic immunization
(L. Hodge, M. Marinaro, H. Jones, J. R. McGhee, H. Kiyono, and
J. W. Simecka, unpublished data). Therefore, generation of mucosal immunity is an obvious area in which notable improvement in vaccination against respiratory pathogens can be made.
Nasal immunization is anticipated to be an optimal route of
administration of vaccines against respiratory tract infections. Although oral immunization is an attractive approach to induce mucosal
immunity, it has had variable success in protection against upper
respiratory tract viral infections. For example, secondary nasal
immunization subsequent to primary oral immunization is required for
effective protection against viral respiratory disease (19).
Several studies in animals and patients demonstrated that vaccination
by direct inoculation of the respiratory tract can be effective
(22, 28, 37). There also appears to be a significant protective advantage to the nasal route of immunization. Upper respiratory tract infection with the influenza virus was prevented in
mice nasally immunized with inactive influenza virus (23). In contrast, there was no noticeable protection after systemic immunization, as viral titers in samples recovered from nasal passages
were equivalent for naive (unimmunized) and subcutaneously immunized
mice. Another advantage of nasal immunization is the potential
generation of cross-protection between related serotypes of respiratory
pathogens. Mice previously infected with an aerosol of one strain of
influenza virus (e.g., H3N1) were resistant to infection with a
different, but cross-reactive, influenza virus (e.g., H3N2) (32,
33). In contrast, systemic immunization with live or inactive
virus did not provide protection from the cross-reactive influenza
virus. A similar cross-protection between different serotypes or
strains of pathogenic bacteria is also likely to be facilitated by the
generation of mucosal immune responses. Thus, the nasal route of
immunization has clear advantages over systemic routes in protecting
the upper respiratory tract from infection, including those caused by
cross-reactive pathogens. Importantly, the results obtained by nasal
immunization with the cold-adapted influenza virus vaccine (1,
13) establish the feasibility and effectiveness of this route of
vaccination in humans.
Immune responses, however, are not readily induced by antigen alone,
and to produce an effective immune response against respiratory pathogens at mucosal surfaces, intranasal immunization requires a safe
and potent adjuvant. Cholera toxin (CT), an exotoxin of Vibrio
cholerae, is the most common adjuvant for intranasal immunization. When intranasally coadministered with an antigen, there is a
significant enhancement in both mucosal and systemic immune responses
(19, 28, 37). Although CT seems to be an ideal adjuvant for
mucosal immunization, oral immunization with CT as a mucosal adjuvant results in IgE responses and hypersensitivity (20, 31).
These responses result from the ability of CT to preferentially induce Th2 responses which, through the action of interleukin 4 (IL-4), contributes to development of IgE responses (10). Thus,
there is the potential that intranasal immunization with CT can also induce IgE-associated reactions within the respiratory tract.
The purpose of the present study was to determine the extent of
immunologic responses, particularly potential immunopathologic reactions, associated with intranasal immunization with the mucosal adjuvant CT. For this study, BALB/c mice were intranasally immunized with tetanus toxoid (TT) combined with CT, and the responses of these
mice were determined. For immunization, TT was chosen since past
studies by members of our laboratory (16, 20) have described the immunologic reactions after oral immunization. After intranasal immunization, mice produced a serum antibody response against both TT
and CT, primarily antibodies of the IgG1 subclass. Although intranasal
immunization did induce a good immune response, we found that
inflammatory reactions which can be potentially fatal also developed
within the lung. Furthermore, IgE responses were also induced after
intranasal immunization, and these responses were associated with the
detection of IL-5 in the serum. Thus, intranasal immunization with TT
plus CT probably results in the activation of Th2 cells, which may
contribute to serious immunopathologic reactions.
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MATERIALS AND METHODS |
Animals.
Specific pathogen-free, female BALB/c mice were
obtained from the Frederick Cancer Research Facility (National Cancer
Institute, Frederick, Md.). F344 rats were from breeding colonies at
University of Alabama at Birmingham. Both groups of animals were from
colonies which were specific pathogen free, as determined by serologic and cultural tests for rodent viral and bacterial pathogens. Mice were
maintained in horizontal laminar flow cabinets and provided with
sterile food and water ad libitum during the experiment. All mice and
rats were used between 8 to 12 weeks of age. Prior to experimental
manipulation, mice were anesthetized with an intramuscular injection of
ketamine-xylazine. For intranasal immunization, mice were allowed to
inhale 20 µl of inoculum, which was placed upon the nares. If a
volume greater than 20 µl was needed, mice were given multiple
inocula (20 µl each), with a 5-min rest between each inoculation.
Collection of the serum samples was done by retroorbital bleeding.
Immunogens used.
CT was purchased from List Biological
Laboratories, Inc. (Campbell, Calif.). Patricia J. Freda Pietrobon
(Connaught Laboratories, Inc., Swiftwater, Pa.) kindly provided TT.
Histopathology.
For the collection of tissues for histologic
examination, anesthetized mice were sacrificed by exsanguination by
laceration of the femoral artery. The trachea and lungs were removed
intact. The lungs were gently inflated with buffered formalin by using a 3-ml syringe with a 20-gauge needle. The lungs were subsequently fixed in buffered formalin, and individual lung lobes were processed for paraffin embedding, sectioning, and hematoxylin and eosin staining.
Each lung lobe was examined for histopathologic changes by light microscopy.
TT- and CT-specific antibody ELISA.
TT- and CT-specific
antibody titers in sera were determined by enzyme-linked immunosorbent
assay (ELISA), as previously described (16). Briefly, Falcon
Microtest III assay plates (Becton Dickinson, Oxnard, Calif.) were
coated with optimal concentrations of TT (100 µl at 5 µg/ml) or the
B subunit of CT (CT-B) (100 µl at 5 µg/ml) in phosphate-buffered
saline (PBS). After overnight incubation at 4°C, the plates were
washed three times with PBS-0.05% Tween 20 and blocked with
PBS-0.05% Tween 20 supplemented with 10% goat serum (Life
Technologies, Gibco BRL, Gaithersburg, Md.) for 2 h at room
temperature. Serum samples were serially diluted with PBS-0.05% Tween
20-10% goat serum, and 100 µl was placed in duplicate into wells of
the antigen-coated plates. After overnight incubation at 4°C, the
plates were washed four times with PBS-0.05% Tween 20. The secondary
antibodies (biotinylated anti-mouse IgM, IgG, or IgA stock reagents at
0.5 mg/ml; Southern Biotechnology Associates, Birmingham, Ala.) were
diluted to 1:4,000 in PBS-0.05% Tween 20-10% goat serum, and 100 µl was added to the appropriate wells. After a 5-h incubation at room
temperature, the plates were again washed four times with PBS-0.05%
Tween 20, and a 1:2,000 dilution in PBS-0.05% Tween 20-10% goat
serum of peroxidase-conjugated streptavidin (Neutravidin; Southern
Biotechnology Associates) was added to the wells (100 µl). The plates
were incubated at room temperature for 2 h, and the plates were
washed twice with PBS-0.05% Tween 20 and twice with PBS. The
reactions were developed at room temperature by the addition of 100 µl of 1.1 mM ABTS [2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid)
in 0.1 M citrate-phosphate buffer, pH 4.2, containing 0.01% H2O2] in each of the wells. Endpoint titers
were expressed as the reciprocal dilution of the last dilution which
yielded an optical density at 415 nm of more than 0.1 U above the
optical density of the negative controls after a 20-min incubation.
For IgG subclasses, plates were washed three times with PBS and blocked
with PBS-10% goat serum-1% bovine serum albumin. The plates were
washed three times with PBS-0.05% Tween 20, and serial dilutions of
serum starting at 1:64 in blocking buffer were added in duplicate.
After 2 h of incubation at 37°C, plates were washed and
incubated for 1 h at 37°C with biotinylated monoclonal
subclass-specific antibodies (anti-IgG1 [02232D clone A85-1, 1 µg/ml], anti-IgG2a [02012D clone R19-15, 1 µg/ml], anti-IgG2b
[02032D clone R-12-3, 0.5 µg/ml], and anti-IgG3 [02062D clone
4082, 1 µg/ml]; PharMingen, San Diego, Calif.). The reaction
products were visualized as described above.
Determination of total serum IgE.
Total serum IgE was
determined by an ELISA. Plates were coated with capture anti-IgE
monoclonal antibody (PharMingen) at 2 µg/ml. After blocking
with 10% rat serum in PBS, serial dilutions of murine IgE standard
(PharMingen) or serum samples in 3% bovine serum albumin in PBS were
added to the wells in duplicate. After overnight incubation at 4°C,
biotinylated anti-IgE monoclonal antibody (PharMingen) was added to the
wells at a concentration of 4 µg/ml and incubated for 4 h at
room temperature. The reaction product was visualized with neutralite
avidin-horseradish peroxidase (Southern Biotechnology Associates),
followed by addition of substrate 3,3',5,5'-tetramethylbenzidine (Moss,
Inc., Pasadena, Md.). The absorbance readings (450 nm) obtained from
the individual serum samples were converted to micrograms of IgE per
milliliter by reference to a standard curve produced with dilutions of
a standard preparation of murine IgE for each assay. The detection
limits for these assays were 125 ng/ml.
PCA assay.
The passive cutaneous anaphylaxis (PCA) assay was
used to compare the levels of TT-specific reagenic antibody in serum
samples (20). Sera were serially diluted with PBS. A volume
of 0.1 ml of each diluted sample was injected intradermally in the back of ether-anesthetized rats. Amounts of 1 ml of 1% Evan's blue and 200 µg of TT were injected intravenously the next day. After 10 min, the
highest dilution of sera with a positive (blue) reaction was considered
the PCA titer.
Cytokine detection by ELISA.
Cytokine levels in sera were
determined by cytokine-specific ELISAs. Anti-murine IL-4, IL-5, and
gamma interferon (IFN-
) capture antibodies, biotinylated
anti-cytokine antibodies, and recombinant cytokines, for use as
standards, were purchased from PharMingen. The ELISA was performed on
serum samples according to manufacturer's recommendations. Diluted
(1:3) serum samples were placed in wells coated with anti-IL-4 (11B11),
-IL-5 (TRFK5), or -IFN- (XMG1.2) capture antibody and incubated
overnight at 4°C. The reaction products were visualized with
biotinylated anti-IL-4 (BVD6-24G2), -IL-5 (TRFK4), or -IFN- (R4-6A2)
antibody and neutralite avidin-horseradish peroxidase (Southern
Biotechnology Associates), followed by the addition of substrate
3,3',5,5'-tetramethylbenzidine (Moss, Inc.). The absorbance readings
(450 nm) obtained from the individual serum samples were converted to
amounts of cytokine by reference to a standard curve produced with
dilutions of recombinant murine cytokine for each assay. The detection
limits for IL-4, IL-5, and IFN- were 15 pg/ml, 1.2 U/ml, and 9.3 U/ml, respectively.
Statistical analysis.
Statistical analysis was performed
with the SYSTAT program (Systat, Inc., Evanston, Ill.). Antibody titers
were transformed to common logarithms prior to statistical analysis.
The data were analyzed by analysis of variance, followed by post-hoc
tests for multigroup comparisons, as needed. The data were also
analyzed by Student's t test or an unpaired Mann-Whitney U
test. A probability (P) of 0.05 or less was accepted as significant.
| |
RESULTS |
Intranasal immunization results in pathologic changes.
To
examine the development of host responses after intranasal immunization
with TT and CT as the adjuvant, anesthetized mice were intranasally
given 250 µg of TT combined with 10 µg of CT. These doses were
previously shown to be effective for oral immunization (16).
Seven days later, mice were intranasally immunized a second time with
the same doses of TT and CT.
Intranasal immunization of mice with these high doses of TT and CT
resulted in potentially fatal, pathologic changes in the
lungs. In the
initial experiment, three out of five animals died
within 10 min of the
second intranasal immunization with a full
dose of antigen (250 µg of
TT plus 10 µg of CT), even though the
mice seemed to tolerate the
first immunization well. By gross
examination, lungs of the dead
animals did not deflate even after
removal from the chest cavity.
Histologic examination of the lungs
of the animals that died revealed
the presence of edema and an
increase in the number of macrophages in
the alveoli (Fig.
1).
Furthermore, there
was evidence of an infiltration of mononuclear
cells into the submucosa
of the pulmonary airways.
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FIG. 1.
Histopathologic changes in lungs of mice intranasally
immunized with TT plus CT. BALB/c mice were intranasally immunized with
TT in combination with CT on days 0 and 7. (a) The alveoli of mice, who
died after receiving a high dosage of TT (250 µg) and CT (10 µg)
for the second immunization, contained large numbers of macrophages and
edema (magnification, 360×). Also, thickening of the alveolar walls
was evident. (b) In mice receiving one-third of the primary dose for
their second inoculation, there was a massive infiltration of cells
around the airways and blood vessels (magnification, 36×). The
infiltration also involved alveoli surrounding the vessels and
airways.
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In subsequent experiments, mice were given one-third of the primary
dose of TT plus CT for their secondary immunization (83.3
µg of TT
plus 3.33 µg of CT). Although mice given the lower secondary
dose of
TT plus CT survived, there were obvious clinical signs
(e.g., lack of
activity, ruffled fur, and wasting) in mice after
immunization. Three
days after the secondary immunization, the
lungs of the mice were
removed and inspected. As described above,
the lungs did not deflate as
did those from naive mice or mice
given TT alone. By histologic
examination, there was a dramatic
infiltration of mononuclear cells
around every airway and blood
vessel in lungs of mice immunized with TT
plus CT (Fig.
1). Similar
histopathologic reactions were found in lungs
of mice given CT
alone.
Serum antibody responses after intranasal immunization with TT plus
CT.
Antigen-specific serum antibody responses were evaluated in
mice intranasally immunized with TT plus CT. Mice were intranasally immunized with either TT (250 µg) alone or TT plus CT (250 µg of TT
plus 10 µg of CT). A group of naive mice were included as controls.
Seven days later, the mice were reimmunized with a one-third dose (83.3 µg of TT plus 3.33 µg of CT [TT plus CT] or 83.3 µg of TT [TT
alone]) of the initial inoculum. Three days after the secondary
immunization, serum was collected from each of the mice, and titers of
antibody against TT and CT in the serum samples were measured by an ELISA.
Antibody responses to TT and CT were detected in the serum of mice 3 days after the secondary immunization (10 days after
primary
immunization). The anti-TT-specific IgG and IgM antibody
responses were
present in serum samples from immunized mice (Table
1). However, only low levels of IgG
against CT were present,
and anti-CT-specific IgM responses were
undetectable. The immune
responses of the IgA isotype to either antigen
were undetectable
at this time point. Antibody to either antigen was
not detected
in serum from unimmunized (naive) mice.
Kinetics and subclass of serum antibody responses after intranasal
immunization with TT and CT.
To further examine antibody responses
in mice after intranasal immunization, we compared the development of
serum antibody responses in mice immunized with TT alone and TT in
combination with CT. Mice were immunized on days 0 (a full dose of 250 µg of TT with or without 10 µg of CT), 7 (one-third dose), and 14 (one-third dose), and serum samples were collected on days 7, 14, and
21. Serum antibody titers were determined by endpoint ELISA assays for
each of the antibody isotypes.
Mice immunized with TT plus CT developed greater anti-TT IgG responses
than mice immunized with TT only (Fig.
2). At 10 days
after immunization, serum
IgG responses against TT were close
to 10-fold higher than when CT was
included as an adjuvant. TT-specific
IgM responses were greater in mice
given TT plus CT (titer of
4,096 at 10-day time point) than in mice
immunized with TT alone
(titer of 512 at 10-day time point). Anti-TT
IgA responses were
low (titer of 256) or undetectable in the sera from
either group
of immunized mice.
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FIG. 2.
Anti-TT IgG responses after intranasal immunization.
Mice were intranasally immunized with TT alone or TT in combination
with CT (TT + CT) on days 0, 7, and 14. Serum samples were taken
at 7, 10, 14, and 21 days after the primary immunization. Serum
antibody titers were determined by a TT-specific IgG ELISA. Sera from
animals (n = 9 for each time point except for day 10 [n = 3]) were pooled.
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Mice immunized with TT alone had no antibody response to CT. However,
mice immunized with TT plus CT developed significant
anti-CT IgM
antibody responses by 7 days after immunization, which
plateaued by day
14 (Fig.
3). Anti-CT IgG responses were
present
at 7 days after infection, but continued to increase throughout
the course of these experiments. Serum IgA responses against CT
began
to appear 14 days after primary immunization.
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FIG. 3.
Anti-CT antibody responses after intranasal immunization
with TT plus CT. Mice were intranasally immunized with TT in
combination with CT (TT + CT) on days 0, 7, and 14. Serum samples
were at 7, 10, 14, and 21 days after the primary immunization. Serum
antibody titers were determined by a CT-B-specific ELISA. Sera were
pooled from animals (n = 9 for each time point except
for day 10 [n = 3]).
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There was a qualitative difference in IgG subclass responses between
mice immunized with TT alone and mice given TT plus CT
(Table
2). Serum IgG1 antibody responses
developed in mice immunized
with TT alone, but no other IgG subclasses
were detected. In contrast,
mice immunized with TT plus CT produced
TT-specific responses
by all IgG subclasses, although IgG1
predominated. Similarly,
IgG1, IgG2a, IgG2b, and IgG3 responses against
CT were present
in mice intranasally immunized with TT plus CT.
However, it should
be noted that the highest titers of TT- and
CT-specific IgG1 subclass
responses were noted when mice were
intranasally immunized with
TT in the presence of CT. Anti-CT antibody
was not detected in
mice immunized with TT alone.
Intranasal immunization with TT combined with CT results in an IgE
response.
We examined the sera from control and immunized mice for
the levels of total IgE and the presence of TT-specific reagenic antibody. In mice intranasally immunized with TT plus CT, the levels of
total IgE in sera were higher than those found in sera from naive mice
(Table 3). The average level of total IgE
in two experiments was 2,200 ng of IgE/ml of serum from immunized mice.
In contrast, IgE was not detected in control (unimmunized) mice. As the
detection limits of these assays, the control mice had levels of IgE in
serum that were less than 125 ng of IgE/ml of serum. Thus, there was a
minimum of a 17-fold increase in total IgE of the immunized mice above
that of the control mice after intranasal immunization. Furthermore, we
were able to detect the presence of specific reagenic antibody,
presumably of the IgE class, by the PCA assay (Table 3).
We also measured the levels of total IgE and PCA antibody titers in
mouse serum at various times after primary immunization.
By ELISA, the
amount of total serum IgE increased 7 days after
primary immunization
with TT plus CT, whereas serum IgE levels
in mice given TT alone were
not above those in serum from naive
mice (Fig.
4). Levels of total IgE increased at
subsequent time
points in the sera of both immunized groups of mice (TT
only and
TT plus CT). However, the TT-plus-CT immunized mice had
dramatically
higher amounts of IgE in their sera than the TT-only
immunized
mice. There was a transient increase in the TT-specific
reagenic
antibody response, presumably of the IgE class, in the serum
of
these mice, which peaked at day 10 after immunization with TT
plus
CT (Table
4). In contrast, there was no
TT-specific reagenic
antibody detected by PCA in the sera of mice
immunized with TT
alone.
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FIG. 4.
Total IgE in serum after intranasal immunization. Mice
were intranasally immunized with TT alone or TT in combination with CT
(TT + CT) on days 0, 7, and 14. Serum samples were obtained 7, 10, 14, and 21 days after the primary immunization. Total IgE in pooled
serum samples was determined by ELISA. Sera from unimmunized mice
(control sera) contained 143 ± 25.2 (mean ± standard
deviation) ng of IgE/ml. Sera were pooled from animals (n = 9 for each time point except for day 10 [n = 3]).
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IL-5 is detected in sera from mice immunized with TT plus CT.
Serum IL-4, IL-5, or IFN- levels were measured in mice intranasally
immunized with TT plus CT. Mice were intranasally immunized with TT
plus CT (250 µg of TT plus 10 µg of CT). A group of naive mice were
included as controls. Seven days later, the mice were reimmunized with
a one-third dose (83.3 µg of TT plus 3.33 µg of CT) of the initial
inoculum, as done in earlier experiments. Three days after the
secondary immunization, serum was collected from each of the mice, and
cytokine levels in the serum samples were measured by ELISA.
We were unable to detect IL-4 or IFN-
in the sera of immunized mice
at 10 days after the primary immunization. We found an
average of 4.16 U of IL-5/ml of these serum samples (Table
5).
However, IL-5 levels in serum from
naive mice were below the detection
limit of the assay (<1.2 U/ml).
| |
DISCUSSION |
Although nasal immunizations with live vaccines are currently done
in clinical trials (1, 9), there are significant advantages
with using inactive vaccine antigens, in contrast to live vaccines, for
nasal immunization. Inactive vaccines should not be a safety issue for
individuals immunocompromised due to disease, chemotherapy, or age.
Furthermore, in the case of viral vectors, immune responses against the
vector can limit the effectiveness of immunization (35),
preventing the widespread utilization of these vectors for use in
multiple vaccines. Notably, the ability to immunize with an inactive
vaccine also allows the use of a variety of vaccine antigens, including
polysaccharide-protein conjugates, which may prove impossible to
produce with a viral vector. This should allow for the adaptation of
this vaccination approach against numerous bacterial and viral
respiratory pathogens. Thus, intranasal immunization with inactive
vaccines has many attractive features that should result in an
extremely powerful approach to prevent respiratory infection and disease.
The purpose of the present study was to determine the extent of
immunologic responses, particularly immunopathologic reactions, associated with intranasal immunization with the mucosal adjuvant CT.
For this study, BALB/c mice were intranasally immunized with TT
combined with CT, and the responses of these mice were determined. For
immunization, TT was chosen since studies by members of our laboratory
(16, 20) described the immunologic reactions against this
immunogen after oral immunization. However, the doses of TT and CT,
although ideal for oral immunization, are probably too high for
intranasal immunization. In ongoing studies (data to be published),
similar results were obtained with a different antigen (influenza
vaccine) and lower doses of CT.
It is clear that CT is an effective adjuvant for the intranasal
immunization of mice. In our studies, CT promoted the development of
specific serum antibody responses after intranasal immunization with
TT. The levels of anti-TT IgG were about four to eight times higher in
sera from mice immunized with TT combined with CT than in mice
immunized with TT alone. Others have demonstrated similar results with
other immunogens, such as Sendai virus and respiratory syncytial virus
(19, 28, 37). We found that IgG1 responses were particularly
enhanced after intranasal immunization, which is consistent with
results obtained after oral immunization of healthy mice with TT and CT
(24).
Although intranasal immunization is an effective route of immunization,
we found that potentially fatal, pathologic changes occurred in the
lungs after antigen and CT were deposited in the lower respiratory
tract. At higher doses of CT and TT, some mice died a short time after
the secondary immunization, and this was associated with edema and an
increase in the number of macrophages in the alveoli. Although they
rarely died, mice given lower secondary doses of TT plus CT also had
clinical and pathologic signs. The lungs of these mice remained
inflated even after their removal. This suggests that the airways were
constricted and/or blocked with exudate, which thereby inhibited
airflow. Histologically, we confirmed that there was a dramatic
infiltration of mononuclear cells almost exclusively around pulmonary
airways and vessels. To illustrate the magnitude of the cellular
response, we were able to recover more than six times as many cells
from the lungs of these mice as from naive mice (data not shown). These
effects were seen only in mice inoculated with TT plus CT and not in
those given TT alone. In addition to the pathologic effects, there was a large, transient increase in the TT-specific IgE level present in
sera from mice immunized with TT plus CT, but not in mice immunized with TT alone. These effects are not unique to TT, as a similar phenomenon of IgE responses associated with peribronchial and perivascular cell infiltration occurs with a different antigen (e.g.,
influenza vaccine) in combination with lower doses (0.1 µg) of CT
(Hodge et al., unpublished data). Overall, the changes in the lung,
increases in cell numbers from respiratory tissues, and the production
of antigen-specific reagenic antibody (IgE) suggest that an
IgE-associated hypersensitivity response developed in the lung after
the intranasal immunization of mice with a mixture of TT and CT, but
not after the intranasal immunization with TT alone.
The immunopathologic effects of intranasal immunization with TT plus CT
are probably associated with the activation of Th2 cells. Th2 cells are
characterized by their support of humoral immunity through the
secretion of selected cytokines, such as IL-4, IL-5, and IL-6
(21). Murine Th2 cells preferentially promote the production
of IgG1 over other IgG subclasses, and IL-4 produced by Th2 cells aids
in the development of IgE responses (10, 21). In the present
studies, the early and predominant production of antigen-specific IgG
of the IgG1 subclass and IgE responses are consistent with the
activation of Th2 cells after intranasal immunization with TT plus CT.
Furthermore, significant levels of IL-5, a cytokine produced by Th2
cells (21), were detected in sera from immunized mice. These
observations are consistent with our previous studies describing the
preferential activation of Th2 cells after oral immunization with TT
plus CT (20). Although Th2 cells are mediators of humoral
immunity, Th2-cell responses also appear to contribute to the lung
pathology associated with viral diseases (12), asthma (29), cryptogenic fibrosing alveolitis (36), and
adverse reactions associated with vaccination against respiratory
viruses (11). Some of these effects may be due to
Th2-cell-induced IgE production, which is supported by earlier studies
demonstrating that pulmonary challenge of rats sensitized with
antigen-specific IgE produces a peribronchial and alveolar infiltration
of mononuclear cells (2). Thus, our results are consistent
with the idea that the activation of Th2 cells is responsible for the
intense immunopathologic reactions observed in mice intranasally
immunized with TT combined with CT.
The inclusion of CT most likely has a qualitative effect on immune
responses after intranasal immunization, in addition to its adjuvant
effects. In contrast to results for mice given TT alone, IgG2a
responses, particularly against CT, were also found in serum from mice
intranasally immunized with TT plus CT. As the production of IgG2a is
mediated by IFN- production, a product of Th1 cells (19),
this suggests that intranasal immunization with CT results in the
activation of Th1 cells. In support, ongoing studies suggest that both
Th1 and Th2 cells are activated in lungs after intranasal
immunization with the adjuvant, CT, whereas intranasal immunization
with antigen alone results in a predominantly Th2 response. Thus, the
selection of adjuvants used for intranasal immunization could have a
significant effect on the nature of immunity generated and influence
the potential for inflammatory reactions associated with immunization.
However, additional studies are needed to determine if and how T-helper
subset activation contributes to these adverse immunologic reactions.
In summary, it is clear that CT is an effective adjuvant for intranasal
immunization with TT in mice. However, our results also demonstrate
that intranasal immunization with CT as an adjuvant can result in the
development of IgE responses which may contribute to potentially fatal,
pathologic changes in the lungs of mice. Most likely, this precludes
the use of CT as a mucosal adjuvant for man, especially in atopic
individuals. The present studies used relatively high doses of antigen
and CT, and lower doses do reduce, but do not eliminate, these adverse
reactions without compromising immunogenicity (data to be published).
In addition, other adjuvants need to be examined for their adjuvant
activity. However, it is possible, given the preferential production of IL-4 by lung cells (data to be published), that IgE production will be
a major component of any response to a soluble antigen given by this
route. As locally produced cytokines are likely to be quickly absorbed
or inactivated, the lack of IL-4 and IFN- in serum does not
necessarily reflect their lack of production in the respiratory tract
after intranasal immunization. By employing this immunization model, we
will be able to more fully characterize the activation of
CD4+ T cells and cytokine production, which promote the
development of IgE responses and recruitment of cells to the lungs.
Future studies can then determine if these responses can be
beneficially altered by treatment with recombinant cytokines or other
modulators that downregulate IgE production or cellular recruitment,
leading to vaccine-adjuvant combinations which induce appropriate
protective immune responses.
| |
ACKNOWLEDGMENTS |
We would like to thank the other members of Mucosal Immunization
Research Group, including Gail Cassell, Mariarosario Marinara, and
Michell Coste for their support and useful discussion. We also
appreciate the review of the manuscript and useful comments by Lisa
Hodge and Harlan Jones. We also thank Patricia J. Freda Pietrobon and
Connaught Laboratories, Inc. for the generous supply of tetanus toxoid.
We also appreciate the excellent technical support by Padma Patel and
Haifa Al-Khatib.
This work was supported by the American Lung Association of Texas
(J.W.S.) and Public Health Service grant AI15128 from the National
Institutes of Health (J.R.M.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Biology and Immunology, University of North Texas Health
Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107. Phone:
(817) 735-2116. Fax: (817) 735-2118. E-mail:
jsimecka{at}hsc.unt.edu.
Editor:
T. R. Kozel
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Abstract
Introduction
