| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Mucosal Immunology, Research Institute for Microbial Diseases, and Departments of
Periodontology and Endodontology, and
Oral Microbiology, Faculty of Dentistry, Osaka University, Osaka, Japan; and
§
Department of Preventive Dentistry, Kagoshima University Dental School, Kagoshima, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cholera toxin (CT),3 an exotoxin produced by Vibrio cholerae, is the most widely known immunogen and adjuvant for studying mucosal immunity (1, 2, 3). It is a strong mucosal adjuvant for enhancement of Ag-specific mucosal IgA and systemic IgG responses to mucosally coadministrated protein Ag (4, 5). Previous studies have shown that CT elicits adjuvant responses by inducing Ag-specific Th2-type CD4+ T cells producing IL-4, IL-5, and IL-6, which are responsible for supporting Ag-specific IgA and IgG Ab production (4, 5). It has also been shown that CT can modulate a costimulatory molecule such as B-7 (6). For the development of effective mucosal vaccine, it is essential to consider the use of the adjuvant properties of CT.
Most mucosal diseases, especially chronic inflammation such as inflammatory bowel disease (7) and periodontal disease (8, 9, 10, 11), share immunological similarity in terms of their pathological features. Intensive investigations have been carried out to understand the molecular and cellular mechanisms of disease development. Although destruction of immunological homeostasis between the mucosal immune system and oral-intestinal microorganism is involved in the development of these inflammatory diseases, we still do not know the exact molecular and cellular pathological process that initiates their mucosal chronic inflammation. However, Porphyromonas gingivalis, black Gram-negative anaerobe, has been at least identified as one of the causative microorganisms for the development of chronic inflammation in periodontium (8, 11). In this study, our investigation was aimed to characterize a potential of nasal immunization for the induction of P. gingivalis-specific Th1 and Th2 cells and of B cell responses in both mucosal and systemic compartments. Further, our study was expanded to examine whether nasal vaccine-induced Ag-specific mucosal response can provide a first barrier against bacterial attachment to mucosal epithelial cells and subsequent production of inflammatory cytokines.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
BALB/c mice were obtained from Charles River Japan (Kanagawa, Japan). Mice were maintained in barrier-protected animal facilities under pathogen-free conditions using ventilated microisolater cages in the experimental animal facility of the Research Institute for Microbial Diseases, Osaka University.
Bacterial culture and Ag
P. gingivalis 381 (laboratory strain) was grown in GAM broth (Nissui, Tokyo, Japan) supplemented with hemin (5 mg/L; Wako Pure Chemical Industries, Osaka, Japan) and menadione (10 mg/L; Wako) in anaerobic atmosphere containing 5% CO2, 5% H2, and 90% N2 for 26 h at 37°C (12). Fimbriae of P. gingivalis 381 were prepared as described previously (12). In brief, cells were harvested by centrifugation at 8000 rpm for 15 min at 20°C. P. gingivalis were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing 0.15 M NaCl and 10 mM MgCl2, gently pipetted, and centrifuged at 8000 rpm for 15 min at 20°C. Solid ammonium sulfate was added to the supernatant to 40% saturation. The precipitate was collected and dialyzed, and then the supernatant was applied to a column (1.6 x 10) of DEAE-Sepharose Fast Flow (Pharmacia, Uppsala, Sweden). The column was eluted with a stepwise gradient of 0–0.15 M NaCl in 20 mM Tris-HCl buffer (pH 8.0), and solid ammonium sulfate was added to the fractions containing the fimbrial protein to 50% saturation. Precipitations were collected and dialyzed against 20 mM Tris-HCl buffer (pH 8.0). A single protein band of 41 kDa was observed following SDS-PAGE analysis as described previously (12).
Immunization
A group of mice were given nasal immunization with 10 µl of PBS containing P. gingivalis fimbriae (10 µg/mouse) and mucosal adjuvant CT (1 µg/mouse; Sigma Chemical, St. Louis, MO) (nasal vaccine). The other group of mice received oral vaccine containing a mixture of fimbriae (200 µg/mouse) and CT (20 µg/mouse) by an immunization protocol routinely used in our group (4). Each group of mice were nasally or orally immunized once per week for 3 consecutive weeks. Serum, saliva, nasal wash, and fecal samples were each collected at weekly intervals and monitored for IgM, IgG, and IgA anti-fimbriae Abs (13).
Detection of Ag-specific Ab production by ELISA
Ag-specific Ab titers in serum, saliva, and nasal wash were
determined by ELISA using modified method as described previously
(13, 14, 15). Polystyrene microtiter plates (Nunc, Roskilde,
Denmark) were coated with 5 µg/ml fimbriae in PBS. Wells were blocked
with Block Ace (SNOW BRAND, Sapporo, Japan). The samples were serially
diluted in 10% Block Ace-PBS and transferred to individual well.
Following 2 h of incubation, the plates were washed and reacted
with detection Ab consisting of a 1/4000 dilution of biotinylated goat
anti-mouse-µ, -
, or -
(Southern Biotechnology Associates,
Birmingham, AL). A 1/2000 dilution of detection enzyme, horseradish
peroxidase-streptavidin (Life Technologies, Gaithersburg, MD), was then
added to the plate. The reaction was developed by 50 µl/well of
3,3',5,5'-tetramethylbenzidine (Moss, Pasadena, MD) for 15 min at room
temperature, and the development reaction was terminated by the
addition of 50 µl/well of 0.5 N HCl. Optical density was recorded by
a plate reader (Model 450, Bio-Rad Laboratories, Hercules, CA) at 450
nm. Endpoint titers were expressed as the reciprocal log 2 of the last
dilution of sample that gave an O.D. value of >0.1.
Isolation of mononuclear cells
Mononuclear cells from submandibular glands (SMG), nasal passage (NP), nasopharyngeal-associated lymphoreticular tissue (NALT), Peyer’s patch (PP), and lamina propria (LP) of intestine and spleen (SP) were isolated as previously described (15, 16, 17). In brief, mononuclear cells from NALT and SP were isolated by the mechanical dissociation method using gentle teasing through stainless steel screens. NP, SMG, PP, and LP mononuclear cells were isolated by the enzymatic dissociation procedure with collagenase type IV (Sigma).
ELISPOT assay
Cells were analyzed for fimbriae-specific Ab production at the
single-cell level using Ag and an isotype-specific enzyme-linked
immunospot (ELISPOT) assay as previously described (12, 18). Briefly, 96-well filtration plates with a nitrocellulose
base (Millititer HA, Millipore, Bedford, MA) were coated with 25
µg/ml fimbriae (12). Single-cell suspensions of
mononuclear cells from different tissues were added at varying
concentrations and then incubated at 37°C for 4 h in air with
5% CO2 and 95% humidity. After incubation and washing,
detection Abs consisting of 1 µg/ml horseradish peroxidase-labeled
goat anti-mouse-µ, -, or - (Southern Biotechnology) were
then added to the plate. The spots were developed by
3-amino-9-ethylcarbazole (Moss) and counted under a dissecting
microscope.
Purification of CD4+ T cells
CD4+ T cells was purified by a previously described method with minor modification (4). Briefly, single-cell suspensions were prepared and incubated in complete medium at 37°C for 4 h in air with 5% CO2 to remove adherent cells. The CD3+ T cell-enriched fraction was obtained by negative panning on a petri plate coated with F(ab')2 goat anti-mouse Ig to avoid T cell stimulation (4). Single-cell suspensions were incubated with anti-mouse CD4 (L3T4)-coupled microbeads (Miltenyi Biotec, Sunnyvale, CA), and CD4+ T cells were obtained with a magnetic activated cell sorter column (Miltenyi Biotec). This procedure yielded fractions of >95% purified CD4+ T cells.
Analysis of Ag-specific T cell responses
For analysis of Ag-specific T cell responses, NALT, NP, and SMG
CD4+ T cells were suspended in complete medium, cultured at
a density of 1 x 106 cells/ml in the presence of
different concentrations of fimbriae (1–100 µg/ml) and T
cell-depleted and irradiated (3000 rad) splenic feeder cells (1 x
106 cells/ml) in flat-bottom 96-well microculture plates
(Costar, Cambridge, MA). Among different concentrations of fimbriae
tested, a dose of 100 µg/ml gave the best Ag-specific T cell
proliferation response. Culture supernatants of Ag-stimulated T cells
were examined for the production of IFN-, IL-2, IL-4, IL-5, IL-6,
and IL-10 by cytokine-specific ELISA. Ag-specific CD4+ T
cell-derived cytokines were measured with murine cytokine ELISA kits
(Amersham, Arlington Heights, IL).
RT-PCR for cytokine-specific mRNA
For detection of cytokine-specific mRNA (IFN-, IL-2, IL-4,
IL-5, IL-6, and IL-10) in Ag-specific CD4+ T cells, a
standard RT-PCR amplification protocol was used (4, 19).
CD4+ T cells were cultured at a density of 1 x
106 cells/ml in the presence of 100 µg/ml fimbriae and T
cell-depleted and irradiated (3000 rad) splenic feeder cells (1 x
106 cells/ml). After 2 days of incubation, CD4+
T cells were purified by magnetic activated cell sorting to obtain
total RNA using Trizol reagent (Life Technologies).
To apply the same amount of synthesized cDNAs from CD4+ T cells, the amounts of synthesized cDNAs labeled with digoxigenin (DIG) were measured with a chemiluminescent image analyzer as follows. Purified RNA was transcribed using DIG DNA Labeled Mix (Boehringer Mannheim, Indianapolis, IN) which incorporates DIG-labeled dUTP every 20–25 nucleotides during reverse transcription. DIG-labeled, synthesized cDNA and a series of diluted DIG-labeled control cDNA (Boehringer Mannheim) were dot blotted onto the nucleic acid transfer membrane (Amersham) and cross-linked by UV cross-linker (Spectronics, Westbury, NY). The membrane was subjected to blocking with 1% Blocking reagent (Boehringer Mannheim) in 0.15 M NaCl and 0.1 M maleic acid for 30 min, followed by an additional 30 min of incubation with 7.5 U/L of alkaline phosphatase-conjugated anti-DIG Abs (Boehringer Mannheim) in 1% Blocking reagent. The membrane was then incubated with 1% chemiluminescent substrate for alkaline phosphatase, CSPD (TROPIX, Bedfold, MA) in 100 mM Tris-HCl, 100 mM NaCl, and 50 mM MgC2. The developed chemiluminescent signals on the membrane were exposed to the imaging screen for 18 h and then characterized by the image analyzer (Molecular Imager system; Bio-Rad). The images on the screen were extracted by a laser scanner, and the amounts of synthesized cDNA samples were quantitated using the image analyzer.
For amplification of cDNA, a PCR program of 1 min at 95°C and 1 min
at 60°C was used. The oligonucleotide primers specific for IL-5
(sense, 5'-GAA AGA GAC CTT GAC ACA GCT G-3'; antisense, 5'-GAA CTC TTG
CAG GTA ATC CAG G-3'), IL-6 (sense, 5'-GAA CAA CGA TGA TGC ACT TGC
AG-3'; antisense, 5'-AAA TCG GTG AGG AAG AAC TG-3') and IL-10 (5'-ATG
CAG GAC TTT AAG GGT TAC TTG GGT T-3'; antisense 5'-TAA AGC CTC TCT CCA
TGT TTG CTG CTC CAA A-3') were prepared according to published results
(20). The sequences of the other primers including
ß-actin, IFN-, IL-2, and IL-4 used for this study were already
described in our previous paper (21). Following 35 cycles
of amplification, the PCR products were then electrophoresed on a 1.8%
agarose gel and visualized by ethidium bromide staining (0.2
µg/ml).
Generation of fimbriae-specific mouse IgA mAb
Female BALB/c mice were nasally immunized with fimbriae and CT as described above. From 3 to 4 days after final immunization, mononuclear cells isolated from SMG were fused with X63Ag8.653 myeloma cells by an established method. Hybridoma cells producing mouse anti-P. gingivalis fimbriae Abs were screened by using Ag-specific ELISA method as described above. Positively reacting hybridomas were selected and subsequently cloned three times by limiting dilution. The isotypes of the selected mAbs were determined using a mouse mAb isotyping kit (Amersham). Hybridoma cells were cultured with 500 ml of serum-free medium (CELGROSSER-H, Sumitomo Pharm., Osaka, Japan). Culture supernatants were harvested and mixed with solid ammonium sulfate to 50% saturation. The precipitate was collected and dialyzed in PBS.
Cell culture
The KB cell line (derived from a human oral epidermoid carcinoma) obtained from the American Type Culture Collection (ATCC CCL17, Manassas, VA) was maintained in DMEM (Nikken Seibutsu, Kyoto, Japan) supplemented with 10% FCS and 50 µg/ml gentamicin in 5% CO2 atmosphere at 37°C. KB cells (4.5 x 104 or 1 x 105) were seeded (200 µl or 1 ml) into tissue culture on 96-well or 48-well microtiter plates (IW AKI, Chiba, Japan) 24 h before the experiment.
[3H]Thymidine-labeled bacterial adhesion assay
The assay of adhesion of P. gingivalis to KB cells was performed by the modified method of Mintz and Five-Taylor (22) as described previously. For radiolabeling of bacteria, the cells were cultured with 5 µCi of [3H]thymidine per ml (Amersham) for 24 h at 37°C in an anaerobic atmosphere containing 5% CO2, 5% H2, and 90% N2. Unincorporated [3H]thymidine was removed by centrifugation followed by the washings with PBS at 6000 rpm. The labeled 108 bacteria were suspended with 200 µl of DMEM containing fimbriae-specific IgA mAbs (50 µg/ml), purified mouse IgA (50 µg/ml, Pharmingen, San Diego, CA), or 10% saliva from mucosally immunized mice with fimbriae and CT and then added to the KB cell monolayer for 1.5 h of incubation at 37°C. The monolayers were rinsed four times with PBS and solubilized by incubation with 0.5 M NaOH-0.1% SDS overnight at 37°C. Solubilized cells were harvested, and the amount of radioactivity present was determined using the TopCount Microplate Scintillation Counter (Packard Instrument, Meriden, CT).
Measurement of cytokine produced by KB cells
For detection of inflammatory cytokines produced by P. gingivalis-infected KB cells, IL-6-, IL-8-, and MCP-1-specific ELISAs were used. P. gingivalis (5 x 108 bacteria) were incubated with 1 ml of DMEM in the presence or absence of fimbriae-specific IgA mAbs (50 µg/ml). P. gingivalis were added to the monolayer of KB cells and incubated with or without Ag-specific mAb at 37°C for 1.5 h. The monolayers were rinsed four times with PBS, replaced with fresh DMEM, and cultured for an additional 24 h. Culture supernatants were harvested and then subjected to analyses of IL-6, IL-8, and MCP-1 production by human cytokine ELISA kits (BioSource International, Camarillo, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In our initial experiment, mice were nasally or orally immunized
with fimbriae (10 or 200 µg) in the presence of mucosal adjuvant CT
(1 or 20 µg) for the analysis of Ag-specific Abs in serum and
secretions. In serum, a comparable level of Ag-specific Abs was induced
in both groups. In the case of saliva and nasal wash, high levels of
fimbriae-specific IgA responses were noted in mice received nasal
vaccine. In contrast, lower levels of fimbriae-specific IgA responses
were seen in mice immunized with oral vaccine. In fecal extracts, the
levels of fimbriae-specific IgA in nasally immunized mice were much
weaker than those detected in the oral vaccine group. These findings
suggest that nasal vaccine containing 10 µg of fimbriae and
1 µg of CT is an effective immunization regimen for the induction of
Ag-specific IgA Abs in saliva and nasal wash, but not intestinal
secretion (Fig. 1
).
|
). Further, it was shown that
mice receiving nasal vaccine had elevated numbers of fimbriae-specific
IgA AFC in NP and NALT. However, nasal vaccination induced low numbers
of Ag-specific IgA in intestinal LP. In comparison to nasal vaccine,
oral vaccine effectively induced a high frequency of fimbriae-specific
IgA AFC in intestinal LP. These findings demonstrate that nasal
immunization with 10 µg of fimbriae and 1 µg of CT is effective for
the induction of localized Ag-specific IgA responses in nasal and oral
compartments, while oral immunization with 200 µg of fimbriae and 20
µg of CT results in compartmentalized Ag-specific IgA responses in
intestine-associated tissues.
|
Inasmuch as high levels of fimbriae-specific IgA responses were
induced in mucosal immune compartments by nasal vaccination, it was
important to examine the nature of fimbriae-specific T cell responses
(e.g., Th1 and Th2 type) induced in these nasally immunized mice. To
characterize Ag-specific Th1 and Th2 responses, CD4+ T
cells were isolated from NALT, NP, and SMG of mice given nasal vaccine
and restimulated with fimbriae in vitro. Culture supernatants from
fimbriae-stimulated CD4+ T cells were then examined for the
presence of Th1 and Th2 cytokines by ELISA (Fig. 3). High levels of both Th1 (e.g.,
IFN-, IL-2) and Th2 (e.g., IL-5, IL-6, and IL-10) cytokines were
detected in the culture supernatant harvest from in vitro
fimbriae-stimulated CD4+ T cells isolated from mucosal
effector tissues such as NP and SMG. In contrast, CD4+ T
cells isolated from NALT, an IgA-inductive site for the upper
respiratory tract, predominantly produced Th2 cytokines such as IL-4
but not other cytokines. To confirm these observations at molecular
levels, Th1 and Th2 cytokine-specific RT-PCR was performed by using RNA
samples extracted from the other aliquot of fimbriae-stimulated
CD4+ T cells (Fig. 4). In NP
and SMG, both Th1 and Th2 cytokine-specific mRNAs were expressed by in
vitro restimulated Ag-specific CD4+ T cells. Among an array
of Th1 and Th2 cytokines, PCR products representing IFN-, IL-6, and
IL-10 resulted in high intensity bands. IL-4-specific mRNA was detected
more prominently in NALT than in NP or SMG. These cytokine-specific
mRNA results further confirmed the findings obtained by ELISA (Fig. 3).
Taken together, these findings suggested that nasal immunization
induces Ag-specific IFN--producing Th1 type and IL-5-, IL-6-, and
IL-10-secreting Th2 type CD4+ T cells in mucosal effector
sites (e.g., NP and SMG) while IL-4-producing Th2 type cells are
predominantly induced in IgA-inductive tissue (e.g., NALT).
|
|
To investigate whether nasal vaccine-induced Ag-specific IgA Abs
can inhibit bacterial attachment to oral epithelial cells, we developed
a fimbriae-specific mAb-producing cell line from SMG isolated from mice
nasally immunized with fimbriae and CT. In our initial experiment, it
was shown that saliva from nasally immunized mice possessed some
ability to reduce the attachment rate of P. gingivalis on
epithelial cells (Fig. 5). To directly
demonstrate the inhibitory function of fimbriae-specific IgA Abs,
Ag-specific IgA mAbs were generated using B cells isolated from SMG of
nasally vaccinated mice as a fusion partner source. Following extensive
subcloning and screening from 87 clones, 4 hybridomas (5G4, 2D3, 2B2,
and 5H3) with heavy and 
light chains were selected and used for
this experiment. When P. gingivalis organisms were treated
with fimbriae-specific IgA mAbs, inhibition of attachment to KB cells
was noted (Fig. 5). Although inhibition ability of bacteria to adhere
to KB cells differed among these four fimbriae-specific IgA mAbs
(30–70%), mAb 5G4 gave the highest inhibition titer at a
concentration of 50 µg/ml IgA Abs. These results suggest that nasal
immunization can induce immunologically active fimbriae-specific IgA
Abs possessing ability to inhibit bacterial attachment to epithelial
cells.
|
To further examine the immunological effect of nasal
vaccine-induced fimbriae-specific IgA, we next investigated whether
blockage of bacterial attachment to epithelial cells by
fimbriae-specific IgA led to the reduction of inflammatory cytokine
production. When levels of IL-6, IL-8, and MCP-1 production were
examined as inflammatory cytokines, increased secretions of those
cytokines were observed when KB cells were incubated with P.
gingivalis. However, pretreatment of bacteria with
fimbriae-specific IgA resulted in the partial blockage of inflammatory
cytokine production by epithelial cells (Fig. 6). Thus, secretions of IL-6, IL-8, and
MCP-1 were reduced to 50–70, 43–65, and 28–56%, respectively. These
results suggest that fimbriae-specific IgA induced by nasal vaccination
can contribute as a protective Ab for the reduction of inflammatory
cytokine production by epithelial cells (Fig. 6).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and 2). In
contrast, oral immunization with higher doses of fimbriae (200 µg)
and CT (20 µg) resulted in the induction of Ag-specific IgA Abs
predominantly in intestinal secretions. These findings suggest that a
compartmentalized mucosal immune system is involved for such a
distinctively separated dominant IgA response induced by nasal and oral
immunization. Thus, nasal immunization is most effective for the
induction of Ag-specific Ab response in the mucosal compartment of the
upper part of body including nasal passage and oral cavity, while oral
immunization is more effective in inducing intestinal immune responses.
To this end, it was recently shown that nasal immunization is superior
to oral administration for the induction of Ag-specific immune
responses in the upper respiratory tract of human
(23). For the development of a mucosal vaccine, our present findings provide an important implication where one could select from two immunization regimens (e.g., oral or nasal) for the induction of the most optimal Ag-specific immune response in the intestinal tract or nasal-oral cavity, depending on the initial invasion site of different pathogenic microorganisms. Oral vaccine might be more suitable for various enteric infectious diseases caused by V. cholera, Salmonealla, pathogenic Escherichia coli, Shigella, etc. In contrast, nasal vaccine would be beneficial for respiratory infectious diseases caused by influenza virus, adenovirus, respiratory syncytial virus, Streptococcus pneumoniae, etc. To this end, it has been shown that nasal immunization with influenza Ag and mucosal adjuvant CT (or heat-labile enterotoxin E. coli.) induced protective immunity (24). Most recently, nasal vaccine containing PspA from S. pneumoniae and mucosal adjuvant mutant CT resulted in the generation of Ag-specific S-IgA and serum IgG responses that provide protection against the bacterial challenge (25). In addition to these respiratory infections, our present findings provided supportive evidence that nasal vaccination is also an effective immunization regimen for the induction of Ag-specific immune responses in the oral region. The oral cavity is immunologically considered to be a unique organ in which the mucosal and systemic origin of S-IgA and IgG Abs simultaneously provide two layers of protection via saliva and gingival crevicular fluids, respectively. Since high levels of fimbriae-specific S-IgA and systemic IgG are induced in the oral cavity by nasal immunization, a concept of nasal vaccine can be applied toward the development of a new immunoprophylaxis therapy in order to control infectious diseases in oral cavity.
CT has been shown to be an effective mucosal adjuvant for
supporting induction of Ag-specific mucosal and systemic immune
responses (1, 2, 3). Although the exact mode of the CT
immunomodulator function is still not well understood, recent
accumulated evidence suggests that mucosally coadministrated CT is
involved in the induction and regulation of Ag-specific Th1 and Th2
responses. It has been shown that CT inhibited IL-2 production and
proliferation of Th1 cells but failed to down-regulate IL-4 production
and associated Th2 cell proliferation (26). Further,
orally administered CT has been shown to induce Ag-specific Th2
responses predominantly (4, 27). Our results also showed
that nasal immunization with fimbrial protein and CT induces dominant
Th2-type responses in NALT (Fig. 3). Among an array of Th2-type
cytokines, a selective production of IL-4 was noted. This finding
suggests that these IL-4-producing Th2-type CD4+ T cells
may provide a molecular environment for preferential immunogloblin
class switching for IgA in NALT, an example of IgA-inductive tissue,
since IL-4 has been shown to support TGF-ß-induced IgA-specific class
switching (28).
In NP and SMG, mucosal effector sites for the production of Ag-specific
S-IgA Abs, Ag stimulation resulted in the induction of both Th1- and
Th2-type cytokines. Interestingly, among different Th2 type cytokines,
a group of IgA-enhancing cytokines that has been shown to induce
differentiation of IgA-committed B cells to plasma cells such as IL-5,
IL-6, and IL-10 (29, 30, 31) was preferentially produced by
fimbriae-stimulated CD4+ T cells isolated from NP and SMG.
In addition, the induction of Th1-type cytokines including IFN- and
IL-2 by these Ag-specific CD4+ T cells may lead to creation
of an optimal molecular environment for the maximum production of
Ag-specific S-IgA Ab synthesis in NP and SMG. IFN- has been shown to
enhance the induction of secretory component, an important poly-Ig
receptor for the formation and transport of S-IgA by epithelial cells
(32), while IL-2 can synergistically support IgA enhancing
cytokine (e.g., IL-5) induced IgA B cell differentiation
(33). Thus, it is possible that the coexistence of
selected Th1 and Th2 cytokines produced by Ag-specific CD4+
T cells may provide a highly effective immunological environment for
the production of Ag-specific S-IgA responses in mucosal effector
tissues such as NP and SMG.
It was shown that P. gingivalis was capable of
adhering to and invading epithelial cells (e.g., KB cells and human
gingival epithelial cells) (34, 35). Further, fimbriae
have been shown to play a central role for the initial step of
adherence to and invasion of oral epithelial cells (36).
If one can induce Ag-specific immune responses against P.
gingivalis fimbriae, it might be possible to disturb cell to cell
interaction between prokaryotic and eukaryotic cells leading to
prevention of initial invasion by the pathological microorganism. A
previous study has shown that mAb against fimbriae of P.
gingivalis could almost block its adhesion to epithelial cells in
vitro (37). Further, IgM isotype of mAb specific to
hemagglutinating adhesion also inhibited cell to cell interaction
between P. gingivalis and KB cells (38). These
findings further supported a potential idea that induction of
fimbriae-specific Ab leads to the inhibition of P.
gingivalis attachment to mucosal epithelial cells. Our present
study provides strong evidence that nasal vaccine containing fimbriae
and mucosal adjuvant CT can induce high levels of Ag-specific S-IgA, in
addition to serum-derived IgG Abs in external secretions (e.g.,
saliva), which can then interfere with bacterial attachment to
epithelial cells (Fig. 5).
To determine the role of fimbriae-specific IgA Abs as an
immunoprophylaxis molecule, a panel of Ag-specific mAbs was generated
from SMG of mice nasally immunized with or without fimbriae and CT. It
was shown that fimbriae-specific IgA mAbs were established from SMG of
nasally immunized mice but not from the control group. These
fimbriae-specific IgA mAbs possessed capability to inhibit binding of
P. gingivalis to KB cells (Fig. 5). These findings further
support the notion that immunological actions of salivary IgA are
thought to inhibit adherence or growth of microorganisms on soft
tissues (e.g., epithelial cells) and hard tissues (e.g., teeth).
Although these Ag-specific IgA mAbs generated from mice receiving nasal
vaccine have been shown to inhibit adhesion of P. gingivalis
to KB cells, these effects did not result in complete blockage of
bacterial adhesion (Fig. 5). These findings suggest that other
bacterial surface molecules in addition to fimbriae might be involved
in the bacterial adhesion. To this end, bacterial cell surface
components such as hemagglutinin and capsule have been shown to be
important molecules for adherence to epithelial cells
(11). For the purpose of maximum inhibition of P.
gingivalis attachment to epithelial cells, one might consider
additional P. gingivalis cell adhesion molecules for the
induction of Ag-specific S-IgA Ab responses by nasal immunization.
In this study, we also aimed to examine the possible influences of
fimbriae-specific IgA Abs on the production of inflammatory cytokines
(e.g., IL-6, IL-8, and MCP-1) by P. gingivalis-stimulated KB
cells. Since these cytokines are known as proinflammatory molecules, it
was important to study the potential role of fimbriae-specific IgA
antibody for the inhibition of these proinflammatory cytokine
syntheses. To this end, IL-6 has been shown to be a multifunctional
cytokine playing a central role in host defense mechanisms
(39). IL-8 is known as the principal chemoattractor for
neutrophils, and MCP-1 has an essential role in recruiting and
activating monocytes (40, 41). Expression of these
cytokines is enhanced by mucosal epithelial cells after bacterial
infection (42). Our results also demonstrated that
incubation of KB cells together with P. gingivalis results
in the up-regulation of IL-6, IL-8, and MCP-1 synthesis (Fig. 6). As
shown in the results demonstrated in Fig. 6, mAbs specific to fimbriae
of P. gingivalis could inhibit the production of these
inflammatory cytokines. These findings further emphasize the importance
of the generation of Ag-specific IgA Ab, which possesses the ability to
block the bacterial attachment to epithelial cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Hiroshi Kiyono, Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.
3 Abbreviations used in this paper: CT, cholera toxin; AFC, Ab-forming cells; ELISPOT, enzyme-linked immunospot assay; LP, small intestinal lamina propria; NALT, nasopharyngeal-associated lymphoreticular tissue; NP, nasal passage; SP, spleen; PP, Peyer’s patch; DIG, digoxigenin; SMG, submandibular glands; S-IgA, secretory IgA; MCP-1, monocyte chemoattractant protein-1.
Received for publication August 12, 1998. Accepted for publication December 8, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-producing T cells, T cells receptor expression, and IgA plasma cells from mouse salivary gland-associated tissues. J. Immunol. 148:2030.[Abstract]
and ß T cells. Eur. J. Immunol. 25:2743.[Medline]
This article has been cited by other articles:
|
|
 |
|
  K. Takamura, S. Fukuyama, T. Nagatake, D.-Y. Kim, A. Kawamura, H. Kawauchi, and H. Kiyono Regulatory Role of Lymphoid Chemokine CCL19 and CCL21 in the Control of Allergic Rhinitis J. Immunol., November 1, 2007; 179(9): 5897 - 5906. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Taubman, X. Han, K. B. LaRosa, S. S. Socransky, and D. J. Smith Periodontal Bacterial DNA Suppresses the Immune Response to Mutans Streptococcal Glucosyltransferase Infect. Immun., August 1, 2007; 75(8): 4088 - 4096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Hagiwara, Y. I. Kawamura, K. Kataoka, B. Rahima, R. J. Jackson, K. Komase, T. Dohi, P. N. Boyaka, Y. Takeda, H. Kiyono, et al. A Second Generation of Double Mutant Cholera Toxin Adjuvants: Enhanced Immunity without Intracellular Trafficking. J. Immunol., September 1, 2006; 177(5): 3045 - 3054. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Medaglini, A. Ciabattini, A. M. Cuppone, C. Costa, S. Ricci, M. Costalonga, and G. Pozzi In Vivo Activation of Naive CD4+ T Cells in Nasal Mucosa-Associated Lymphoid Tissue following Intranasal Immunization with Recombinant Streptococcus gordonii. Infect. Immun., May 1, 2006; 74(5): 2760 - 2766. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Hajishengallis, S. Arce, C.M. Gockel, T.D. Connell, and M.W. Russell Immunomodulation with Enterotoxins for the Generation of Secretory Immunity or Tolerance: Applications for Oral Infections Journal of Dental Research, December 1, 2005; 84(12): 1104 - 1116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Zhou, T. Desta, M. Fenton, D. T. Graves, and S. Amar Cytokine Profiling of Macrophages Exposed to Porphyromonas gingivalis, Its Lipopolysaccharide, or Its FimA Protein Infect. Immun., February 1, 2005; 73(2): 935 - 943. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Shikina, T. Hiroi, K. Iwatani, M. H. Jang, S. Fukuyama, M. Tamura, T. Kubo, H. Ishikawa, and H. Kiyono IgA Class Switch Occurs in the Organized Nasopharynx- and Gut-Associated Lymphoid Tissue, but Not in the Diffuse Lamina Propria of Airways and Gut J. Immunol., May 15, 2004; 172(10): 6259 - 6264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Guthrie, S. Y. C. Wong, B. Liang, L. Hyland, S. Hou, E. A. Hoiby, and S. R. Andersen Local and Systemic Antibody Responses in Mice Immunized Intranasally with Native and Detergent-Extracted Outer Membrane Vesicles from Neisseria meningitidis Infect. Immun., May 1, 2004; 72(5): 2528 - 2537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. C. Lavelle, A. Jarnicki, E. McNeela, M. E. Armstrong, S. C. Higgins, O. Leavy, and K. H. G. Mills Effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent J. Leukoc. Biol., May 1, 2004; 75(5): 756 - 763. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Hagiwara, J. R. McGhee, K. Fujihashi, R. Kobayashi, N. Yoshino, K. Kataoka, Y. Etani, M.-N. Kweon, S. Tamura, T. Kurata, et al. Protective Mucosal Immunity in Aging Is Associated with Functional CD4+ T Cells in Nasopharyngeal-Associated Lymphoreticular Tissue J. Immunol., February 15, 2003; 170(4): 1754 - 1762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Sakaue, T. Hiroi, Y. Nakagawa, K. Someya, K. Iwatani, Y. Sawa, H. Takahashi, M. Honda, J. Kunisawa, and H. Kiyono HIV Mucosal Vaccine: Nasal Immunization with gp160-Encapsulated Hemagglutinating Virus of Japan-Liposome Induces Antigen-Specific CTLs and Neutralizing Antibody Responses J. Immunol., January 1, 2003; 170(1): 495 - 502. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Okuda, I. Takahashi, J.-K. Kim, N. Ohta, K. Iwatani, H. Iijima, Y. Kai, H. Tamagawa, T. Hiroi, M.-N. Kweon, et al. Development of Colitis in Signal Transducers and Activators of Transcription 6-Deficient T-Cell Receptor {alpha}-Deficient Mice: A Potential Role of Signal Transducers and Activators of Transcription 6-Independent Interleukin-4 Signaling for the Generation of Th2-Biased Pathological CD4+{beta}{beta}T Cells Am. J. Pathol., January 1, 2003; 162(1): 263 - 271. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hiroi, H. Goto, K. Someya, M. Yanagita, M. Honda, N. Yamanaka, and H. Kiyono HIV Mucosal Vaccine: Nasal Immunization with rBCG-V3J1 Induces a Long Term V3J1 Peptide-Specific Neutralizing Immunity in Th1- and Th2-Deficient Conditions J. Immunol., November 15, 2001; 167(10): 5862 - 5867. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kunisawa, T. Nakanishi, I. Takahashi, A. Okudaira, Y. Tsutsumi, K. Katayama, S. Nakagawa, H. Kiyono, and T. Mayumi Sendai Virus Fusion Protein-Mediates Simultaneous Induction of MHC Class I/II-Dependent Mucosal and Systemic Immune Responses Via the Nasopharyngeal-Associated Lymphoreticular Tissue Immune System J. Immunol., August 1, 2001; 167(3): 1406 - 1412. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sharma, K. Honma, R. T. Evans, D. E. Hruby, and R. J. Genco Oral Immunization with Recombinant Streptococcus gordonii Expressing Porphyromonas gingivalis FimA Domains Infect. Immun., May 1, 2001; 69(5): 2928 - 2934. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Sabirov, S. Kodama, T. Hirano, M. Suzuki, and G. Mogi Intranasal Immunization Enhances Clearance of Nontypeable Haemophilus influenzae and Reduces Stimulation of Tumor Necrosis Factor Alpha Production in the Murine Model of Otitis Media Infect. Immun., May 1, 2001; 69(5): 2964 - 2971. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Thakur, J. Kyd, M. Xue, M. D. P. Willcox, and A. Cripps Effector Mechanisms of Protection against Pseudomonas aeruginosa Keratitis in Immunized Rats Infect. Immun., May 1, 2001; 69(5): 3295 - 3304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
T. Hiroi, M. Yanagita, N. Ohta, G. Sakaue, and H. Kiyono IL-15 and IL-15 Receptor Selectively Regulate Differentiation of Common Mucosal Immune System-Independent B-1 Cells for IgA Responses J. Immunol., October 15, 2000; 165(8): 4329 - 4337. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
