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The Journal of Immunology, 2007, 178, 6058 -6065
Copyright © 2007 by The American Association of Immunologists, Inc.
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*Substance via MeSH

Nasal Cholera Toxin Elicits IL-5 and IL-5 Receptor {alpha}-Chain Expressing B-1a B Cells for Innate Mucosal IgA Antibody Responses1

Kosuke Kataoka*,{dagger}, Keiko Fujihashi*, Shinichi Sekine*, Tatsuya Fukuiwa*,{ddagger}, Ryoki Kobayashi*, Hideaki Suzuki*,§, Hideki Nagata{dagger}, Kiyoshi Takatsu, Satoshi Shizukuishi{dagger}, Jerry R. McGhee* and Kohtaro Fujihashi2,*

* Departments of Pediatric Dentistry and Microbiology, Immunobiology Vaccine Center, University of Alabama at Birmingham, Birmingham, AL 35294; {dagger} Department of Preventive Dentistry, Graduate School of Dentistry, Osaka University, Osaka, Japan; {ddagger} Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan; § Department of Dental Caries Control and Aesthetic Dentistry, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba, Japan; and Division of Immunology, Department of Microbiology and Immunology, Institute of Medical Sciences, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In this study, we examine whether native cholera toxin (nCT) as a mucosal adjuvant can support trinitrophenyl (TNP)-LPS-specific mucosal immune responses. C57BL/6 mice were given nasal TNP-LPS in the presence or absence of nCT. Five days later, significantly higher levels of TNP-specific mucosal IgA Ab responses were induced in the nasal washes, saliva, and plasma of mice given nCT plus TNP-LPS than in those given TNP-LPS alone. High numbers of TNP-specific IgA Ab-forming cells were also detected in mucosal tissues such as the nasal passages (NPs), the submandibular glands (SMGs), and nasopharyngeal-associated lymphoreticular tissue of mice given nCT. Flow cytometric analysis showed that higher numbers of surface IgA+, CD5+ B cells (B-1a B cells) in SMGs and NPs of mice given nasal TNP-LPS plus nCT than in those given TNP-LPS alone. Furthermore, increased levels of IL-5R {alpha}-chain were expressed by B-1a B cells in SMGs and NPs of mice given nasal TNP-LPS plus nCT. Thus, CD4+ T cells from these mucosal effector lymphoid tissues produce high levels of IL-5 at both protein and mRNA levels. When mice were treated with anti-IL-5 mAb, significant reductions in TNP-specific mucosal IgA Ab responses were noted in external secretions. These findings show that nasal nCT as an adjuvant enhances mucosal immune responses to a T cell-independent Ag due to the cross-talk between IL-5R{alpha}+ B-1a B cells and IL-5-producing CD4+ T cells in the mucosal effector lymphoid tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
For effective Ag- or pathogen-specific mucosal immune responses, live attenuated viral or bacterial delivery systems or mucosal adjuvants must generally be used. Adjuvants offer the advantage of eliciting mucosal as well as parenteral immune responses. A bacterial enterotoxin, native cholera toxin (nCT),3 as well as its nontoxic mutants (mutant CTs (mCTs)), are well-established mucosal adjuvants for the induction of both mucosal and systemic immunity to coadministered protein Ags (1, 2, 3, 4). Mucosal administration of vaccine together with nCT or nontoxic mCTs induces CD4+ Th2-type cells with characteristic plasma IgG1, IgG2b, IgE, and IgA, as well as mucosal secretory-IgA (S-IgA) Ab responses (1, 2, 3, 4).

Nasal delivery of Ag plus a mucosal adjuvant has emerged as perhaps the most effective way to induce both peripheral and mucosal immunity, including salivary S-IgA Ab responses. Again, most studies can be divided into those that use soluble vaccine components with mucosal adjuvants such as nCT and those that use attenuated bacterial and viral vectors, such as recombinant Salmonella or adenovirus vectors, respectively. In this regard, it has been shown that nasal immunization with the weak protein Ag OVA plus mCT elicited OVA-specific S-IgA Ab responses in the submandibular glands (SMGs) in addition to other mucosal effector lymphoid tissues (4, 5). Others have shown that mice given the nasal 40-kDa outer membrane protein of Porphyromonas gingivalis and nCT as mucosal adjuvant displayed significant levels of outer membrane protein-specific plasma IgG and IgA, as well as mucosal S-IgA Ab responses in saliva and nasal secretions (6).

More than 200 species of bacteria populate the mucosal surfaces of the oral cavity, conjunctiva, and the gastrointestinal, genital, and upper respiratory tracts (7). The mucosal microbiota comprises some 1014 microorganisms, most of which reside in the large intestine. Effective mucosal protection for the host is achieved not only by Ag-specific but also by innate or natural S-IgA Abs. Indeed, oral, intestinal, and probably other mucosal bacteria are coated in vivo with Abs, particularly of the IgA isotype, that may prevent their adherence to the epithelial receptors but do not significantly interfere with their elimination and metabolism (8).

It is now known that B-1 B cells differ from conventional B cells in cell surface protein CD5 expression, anatomical localization, and functional characteristics (9, 10). With regard to the functional characteristics, B-1 B cells seem to differentiate primarily into Ab-producing plasma cells of all isotypes in response to polysaccharide. Although these responses can be enhanced/increased by T cells, they appear within 48 h of exposure to Ag and so are not dependent upon T cell help. B-1 B cells in the murine peritoneal cavity and lamina propria have been shown to develop from a common pool and may represent a lineage separate from that of conventional Peyer’s patch B cells (11). Other studies using transgenic mice have provided additional supportive evidence that intestinal IgA plasma cells are derived from B-1 B cells (12). Furthermore, it has been shown that intestinal mucosal IgA Abs with specificity for commensal bacteria are produced by B-1 B cells in a T cell-independent manner (13, 14). These studies clearly suggest that B-1 B cells are an important source of IgA-producing cells in the intestinal mucosal tissues.

Although it is in generally well known that in acquired immunity Ag-specific IgA Ab responses are regulated by CD4+ T cells and their derived cytokines, little information exists regarding the cellular and molecular mechanisms underlying the induction of innate S-IgA Ab responses in the oral and nasal mucosa. Like the intestinal lamina propria, the SMGs and nasal passages (NPs) act as mucosal effector lymphoid tissues, and both possess significant populations of B-1 B cells. We hypothesize that the oral and nasal innate immune systems can induce IgA Ab responses without direct T cell help. In this study, we examined whether nCT can be used as an adjuvant to enhance the induction of TNP-LPS-specific mucosal IgA Ab responses by B-1 B cells in the SMGs and NPs when a T cell-independent Ag (TI Ag), i.e., TNP-LPS is used.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female C57BL/6 mice (6–8 wk old) were purchased from the Frederick Cancer Research Facility (National Cancer Institute). These mice were transferred to microisolators, maintained in horizontal laminar flow cabinets, and provided sterile food and water as part of a specific pathogen-free facility. All of the mice used in these experiments were free of bacterial and viral pathogens.

Nasal immunization and sample collection

Mice were immunized nasally with 10 µg of TNP-LPS (Sigma-Aldrich) in the presence or absence of nCT (1 µg) (List Biological Laboratories) under anesthesia. Other groups of mice were given nasal PBS or nCT alone. On days 0 and 5, plasma and saliva were collected. Stimulated saliva was obtained following i.p. injection with 100 µg of sterile pilocarpine (15). When mice were sacrificed, nasal washes (NWs) were obtained by instillation of 1 ml of PBS on three occasions into the posterior opening of the nasopharynx with a hypodermic needle (5).

Analysis of TNP-specific Abs

TNP-specific Ab levels in plasma and mucosal external secretions were determined by ELISA as described previously (4, 5, 15). Briefly, 96-well Falcon microtest assay plates (BD Biosciences) were coated with 10 µg/ml TNP-BSA in PBS. After blocking with 1% BSA in PBS, 2-fold serial dilutions of samples were added and incubated overnight at 4°C. HRP-labeled goat anti-mouse µ, {gamma}, or {alpha} H chain-specific Abs (Southern Biotechnology Associates) were added to individual wells. For IgG Ab subclass analysis, biotinylated mAbs specific for IgG1, IgG2a, IgG2b, and IgG3 (BD Pharmingen) and peroxidase-conjugated goat anti-biotin Ab (Vector Laboratories) were used for detection. The color reaction was developed for 15 min at room temperature with 100 µl of 1.1 mM 2,2'-azino bis(3-ethylbenz-thiazoline-6-sulfonic acid). End point titers were expressed as the reciprocal log2 of the last dilution that gave an OD at 415 nm of 0.1 greater than background.

Enumeration of Ig-producing cells

Mononuclear cells from the spleen and nasopharyngeal-associated lymphoreticular tissues were isolated aseptically using a mechanical dissociation method that involved gentle teasing through stainless steel screens as described elsewhere (4, 5, 15). For isolation of NPs, a modified dissociation method based on a previously described protocol was used (5, 15). Mononuclear cells from SMGs were isolated by an enzymatic dissociation procedure with collagenase type IV (0.5 mg/ml; Sigma-Aldrich), followed by a discontinuous Percoll gradient centrifugation (Amersham Biosciences) (15, 16). Mononuclear cells obtained from mucosal lymphoid tissues and spleens were subjected to an ELISPOT assay to detect numbers of TNP-specific Ab-forming cells (AFCs). Briefly, 96-well nitrocellulose plates (Millititer HA; Millipore) were coated with 1 mg/ml TNP-BSA. The numbers of TNP-specific AFCs were quantified with the aid of a stereomicroscope, as well as an ImmunoSpot Analyzer reader (Cellular Technology) as described elsewhere (4, 5, 15, 16).

Flow cytometric analysis of B cell subsets

Aliquots of mononuclear cells (0.2–1.0 x 106 cells) from NPs and SMGs were stained with FITC-conjugated anti-mouse IgA mAbs (M18-254) and PE-labeled anti-mouse B220 (RA3-6B2). For the characterization of surface IgA (sIgA)+ B cells in the mucosal lymphoid tissues, B220+ B cells were purified using an AutoMACS system (Miltenyi Biotec) as described previously (15). Briefly, mononuclear cells were incubated with biotinylated anti-B220 mAb, followed by streptavidin-conjugated microbeads, and sorted to purity with the AutoMACS. This purified B cell fraction was comprised of >97% B220+, with >99% cell viability. Purified B cells were stained with FITC-conjugated anti-mouse IgA mAbs and PE-labeled anti-mouse CD23 (B3B4) (BD Pharmingen), allophycocyanin-tagged anti-CD5 mAbs (53-7.3) (BD Pharmingen), and biotinylated anti-IL-5R {alpha}-chain (anti-IL-5R{alpha}) mAbs (17, 18), followed by PerCP-Cy5.5-streptavidin. In some experiments, aliquots of mononuclear cells (0.2–1.0 x 106 cells) isolated from various tissues were stained with FITC-conjugated anti-CD5, PE-labeled anti-B220, and biotin-conjugated anti-IL-5R{alpha} mAbs, followed by PerCP-Cy5.5-streptavidin. The samples were then subjected to flow cytometric analysis (FACSCalibur; BD Biosciences) and used to set the lymphocyte gates. To exclude dead cells, 7-aminoactinomycin D solution (Via-Probe; BD Biosciences) was used. In brief, Via-Probe was added as a negative control sample (20 µl/106 cells) 10 min before flow cytometry analysis. The threshold of forward scatter was determined according to the point showing no cells at the FL3 channel. Using this method, >99.9% dead cells were excluded for the analyses of other samples.

Cytokine-specific ELISA

CD4+ T cells from NPs and SMGs were purified using an AutoMACS system (Miltenyi Biotec) as described previously (15). Briefly, mononuclear cells were incubated with biotinylated anti-CD4 mAb (GK 1.5) (BD Pharmingen), followed by streptavidin-conjugated microbeads, and sorted to purity with the AutoMACS. This purified T cell fraction was comprised of >97% CD4+ and the cells were >99% viable. This purified CD4+ T cell fraction was resuspended in RPMI 1640 (Mediatech) supplemented with HEPES buffer (10 mM), L-glutamine (2 mM), nonessential amino acids (10 ml/L), sodium pyruvate (10 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), gentamicin (80 µg/ml), and 10% FCS (complete RPMI 1640) (4 x 106 cells/ml) and cultured for 5 days The culture supernatants were collected, and the levels of cytokines were determined by IFN-{gamma}-, IL-2-, IL-4-, and IL-5-specific ELISA as described previously (15). The immunoplates (Nalge Nunc International) were coated with the respective anti-cytokine capturing mAb and blocked with 3% BSA in PBS. Serial 2-fold diluted samples and standards were added, and plates were incubated overnight at 4°C. The plates were washed, and the detection mAb was added. After an additional incubation overnight at 4°C, HRP-labeled goat anti-biotin Ab (Vector Laboratories) was added. The color reaction was developed at room temperature with 100 µl of 1.1 mM 2,2'-azino bis(3-ethylbenz-thiazoline-6-sulfonic acid). The detection limits for each cytokine were as follows: 106.3 pg/ml for IFN-{gamma}, 15.6 pg/ml for IL-2, 4.66 pg/ml for IL-4, and 39 pg/ml for IL-5.

Quantitative RT-PCR analysis for IL-5

To determine Th1 (IFN-{gamma} and IL-2)- and Th2 (IL-4 and IL-5)-type cytokine-specific mRNA levels, total RNA was isolated from purified CD4+ T and B220+ B cells using an acid guanidinium thiocyanate-phenol-chloroform extraction procedure. Aliquots of extracted RNA (25 µg/ml) were subjected to room temperature reaction and were treated with 1 µl of 10 µg/ml RNase H (Invitrogen Life Technologies). The levels of synthesized cDNA were measured and then the sample cDNA, and the external standards were amplified with Th1- or Th2-type cytokines primers along with SYBER Green I by using a LightCycler (Roche Applied Sciences). The specificity of PCR products was confirmed by a melting curve as well as by agarose gel electrophoresis. The concentration of sample cDNA was determined using linear-diluted external standards obtained by an identical PCR protocol with the LightCycler (15).

In vivo depletion of IL-5

To determine the role of IL-5 produced by CD4+ T cells in the induction of TNP-specific IgA Ab responses in the nasal and oral cavities, IL-5 production in mice given nasal TNP-LPS plus nCT was blocked by i.p. injections of anti-IL-5 mAb (TRFK4). A total of 400 µg of either mAb per mouse was injected at 2 days before and on the day of nasal immunization. The plasma of TRFK4 mAb-treated mice was subjected to IL-5-specific ELISA to confirm an absence of IL-5 production. As controls, mice were treated with rat IgG2a mAb. Plasma, NWs, and saliva from mAb-treated mice were collected and subjected to TNP-specific ELISA. Mononuclear cells from SMGs and NPs were subjected to flow cytometric analysis to measure levels of IL-5R{alpha} and sIgA expression by B-1a B cells.

Statistical analysis

The results are expressed as the mean ± 1 SEM. All mouse groups were compared with control mice using an unpaired Mann-Whitney U test with Statview software (Abacus Concepts) designed for Macintosh computers. Values of p < 0.05 or p < 0.01 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Induction of TNP-specific IgA Ab responses in external secretions

It is well known that nCT is the most effective mucosal adjuvant for the induction of both mucosal and systemic immune responses. However, the roles played by nCT in the induction and regulation of innate immunity to LPS, the major component of the outer membrane of Gram-negative bacteria known as T-independent Ag 1, remains unknown. In our initial experiment, we examined whether T cell-independent mucosal IgA, including S-IgA Ab responses, were up-regulated after nasal immunization with TNP-LPS plus nCT. Mice given nasal TNP-LPS plus nCT exhibited significantly higher levels of TNP-specific IgA Ab responses in NWs and saliva than did mice given the nasal TNP-LPS alone (Fig. 1A). The external secretion samples from naive mice did not contain detectable Ab levels (data not shown), whereas mice given nasal TNP-LPS plus nCT displayed high numbers of TNP-specific IgA AFCs in NPs and SMGs (Fig. 1B). These results clearly indicate that the nasal use of nCT as a mucosal adjuvant effectively enhances TNP-specific IgA Ab responses in mucosal lymphoid effector tissues.


Figure 1
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  		FIGURE 1. TNP-specific Ab responses in external secretions and mucosal lymphoid effector tissues. Groups of mice were given nasal TNP-LPS (10 µg) in the presence ({blacksquare}) or absence ({square}) of nCT (1 µg) as mucosal adjuvant. A, The levels of TNP-specific Ab titers in NWs and saliva were determined by TNP-specific ELISA 5 days after nasal immunization. B, Five days after nasal immunization, mononuclear cells isolated from SMGs and NPs were subjected to a TNP-specific ELISPOT assay to determine the number of AFCs present. The values are presented as the mean ± SEM of 25 mice for each group. **, p < 0.01 and *, p < 0.05 when compared with mice immunized with TNP-LPS alone.

 
TNP-specific plasma Abs in mice given the nasal TNP-LPS plus nCT

Because it has been shown that nasal nCT administration induces Ag-specific immune responses in systemic lymphoid tissues in addition to mucosal sites, TNP-specific plasma Ab responses were examined. Markedly higher levels of TNP-specific IgM and IgG Ab titers were seen in the plasma of mice given nasal TNP-LPS plus nCT than in that of mice given TNP-LPS alone (Fig. 2A). Although increased levels of plasma IgA Ab titers were seen, the responses were not significant as was noted for IgG and IgM isotypes. Among the IgG subclass Ab responses, TNP-specific IgG3 Ab levels were elevated significantly (Fig. 2A). To support these findings, the numbers of TNP-specific IgM and IgG AFCs were increased significantly in spleens of mice given nasal nCT as mucosal adjuvant than in those of control mice given TNP-LPS alone (Fig. 2B). The increased numbers of TNP-specific IgA AFCs was seen but did not reach statistical significance. Taken together, these results clearly show that nasal administration of nCT as mucosal adjuvant enhances innate immunity when TNP-LPS is used as an Ag.


Figure 2
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  		FIGURE 2. Comparison of TNP-specific Ab responses in plasma and systemic lymphoid tissues. Groups of mice were immunized with TNP-LPS (10 µg) with ({blacksquare}) or without ({square}) nCT (1 µg) as mucosal adjuvant. A, The levels of anti-TNP IgM, IgG, IgA, and IgG subclass Abs in plasma were determined by TNP-specific ELISA. Five days later, plasma was collected and examined for TNP-specific Ab responses. The dotted line represents Ab levels in the plasma of naive mice. B, The mononuclear cells were isolated from the spleen and subjected to TNP-specific ELISPOT assay to determine the numbers of IgM, IgG, and IgA AFCs. The values are presented as the mean ± SEM of 25 mice for each group. **, p < 0.01 and *, p < 0.05 when compared with mice immunized with TNP-LPS alone.

 
Nasal administration of nCT induces B-1a B cells in mucosal effector lymphoid tissues

Because nasal administration of TNP-LPS with nCT as mucosal adjuvant elicits enhanced TNP-LPS-specific IgA Ab responses, especially in mucosal compartments, we next characterized the frequency of B cells and their sIgA expression in NPs and SMGs by flow cytometry. An analysis of the B220+ B cell populations of these various mucosal lymphoid tissues revealed no significant changes in the numbers of B220+ B cells even after nasal immunization with TNP-LPS plus nCT. However, increased numbers of sIgA+ B cells were seen in the SMGs and NPs of mice given nasal TNP-LPS plus nCT but not in control mice given TNP-LPS alone (Table I and Fig. 3).


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  		Table I. Frequencies of B cells subsets in SMGs and NPs of mice given nasal TNP-LPS plus nCTa

 

Figure 3
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  		FIGURE 3. Detection of sIgA+ B-1a B cells in mucosal effector tissues. Purified B cells from SMGs and NPs of naive mice and mice given nasal TNP-LPS plus nCT or TNP-LPS alone were stained with FITC-conjugated anti-IgA, PE-labeled anti-CD23, and allophycocyanin-tagged anti-CD5 mAbs. For sample analysis, the CD23-negative population was gated. The data represent a typical profile of 25 mice for each group. The percentage of sIgA+ B-1a B cells in total lymphocytes population was expressed in the upper right section of counter graph.

 
To further characterize sIgA+ B cells in SMGs and NPs, we assessed the expression of CD5 and CD23 molecules using flow cytometry. Interestingly, higher numbers of CD5 molecules expressing sIgA+ B-1a B cells were seen in mice nasally treated with TNP-LPS plus nCT than in those given TNP-LPS alone (Table I and Fig. 3). In addition, ~90% of this CD5+sIgA+ B cell subset did not express CD23 (Table I). These findings indicate that nCT as nasal adjuvant enhances the induction of TNP-specific mucosal IgA Ab responses by eliciting an increase in the numbers of B-1a B cells in SMGs and NPs.

Nasal nCT elicits IL-5R{alpha} expression by B-1a B cells

Because it has been reported that the majority of murine peritoneal B1 B cells express IL-5R{alpha} for IgA-producing cell differentiation, we next examined the frequency of IL-5R{alpha} expression by B-1a B cells in SMGs and NPs. Although B-1a B cells from SMGs and NPs of mice given nasal PBS or nCT exhibit some IL-5R{alpha} expression, this IL-5R{alpha}-expressing subset comprises only approximately one-third of the total B-1a B cell population (Fig. 4). Administered alone, nasal TNP-LPS increases the frequency of IL-5R{alpha}-expressing B-1a B cells; however, the magnitude of enhancement is limited. In contrast, coadministration of nasal TNP-LPS and nCT induces significant levels of IL-5R{alpha}-expressing B-1a B cells in SMGs and NPs. Indeed, ~90% of B-1a B cells in SMGs and two-thirds of those in NPs expressed IL-5R{alpha} when nCT was used as a nasal adjuvant (Fig. 4). Taken together, these results indicate that nasal immunization with TNP-LPS as Ag plus nCT as mucosal adjuvant preferentially induces IL-5R{alpha}+, sIgA+ B-1a B cells in SMGs and NPs.


Figure 4
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  		FIGURE 4. Comparison of IL-5R{alpha} expression by CD5+B220+ cells. Mononuclear cells were isolated from SMGs and NPs of mice given nasal TNP-LPS plus nCT ({blacksquare}), TNP-LPS ({square}), nCT (Figure 4), or PBS (Figure 4). A, The cells were then stained with FITC-conjugated anti-CD5, PE-labeled anti-B220, and biotin-conjugated anti-IL-5R{alpha} mAbs, followed by PerCP-Cy5.5-streptavidin. B, In some experiments, purified B cells were stained with FITC-conjugated anti-mouse IgA mAb, PE-labeled anti-mouse CD23 (BD Pharmingen), and biotinylated anti-IL-5R{alpha} mAbs, followed by PerCP-Cy5.5-streptavidin. The values are presented as the mean ± SEM of 25 mice for each group. **, p < 0.01 and *, p < 0.05 when compared with mice given TNP-LPS alone.

 
CD4+ T cells are the major source of IL-5 production

Because cytokine signals are essential for B cell activation and differentiation, we first separated whole SMG cells into CD3+ T cell and CD3– cell populations and cultured them for 5 days. When the culture supernatants were examined for IL-5 production, CD3+ T cells from mice given nCT as nasal adjuvant contained significantly higher levels of IL-5 (328 ± 57.2 pg/ml) than those from mice nasally immunized TNP-LPS alone (160 ± 40.2 pg/ml). In contrast, CD3– cell subsets contained essentially the same levels of IL-5 (TNP-LPS alone; 72.5 ± 29.1 pg/ml, TNP-LPS plus nCT; 88.4 ± 21.5 pg/ml). We next assessed the levels of Th1- and Th2-type cytokine production by CD4+ T cells isolated from the SMGs and NPs, finding that those from mice given nasal TNP-LPS plus nCT exhibited significantly higher levels of IL-5 production than did those from mice given TNP-LPS alone (Table II). In addition, higher levels of IFN-{gamma}, IL-2, and IL-4 production were seen in the SMGs and NPs of mice given nCT as nasal adjuvant than in those given TNP-LPS alone, although there are no statistically significant differences (Table II). No differences in other Th2-type (IL-6 and IL-10) cytokines were seen (data not shown). These results were further confirmed by quantitative real-time PCR. CD4+ T cells from the SMGs and NPs of mice given nasal nCT contained significantly higher levels of IL-5-specific mRNA than did those from mice immunized nasally with TNP-LPS alone (Table II). In addition, higher levels of IFN-{gamma}-, IL-2-, and IL-4-specific mRNA were detected in CD4+ T cells from the SMGs and NPs of mice given nCT as nasal adjuvant than in those from mice given TNP-LPS alone (Table II).


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  		Table II. Th1- and Th2-type cytokine profiles for CD4+ T cells from the SMGs and NPs of mice given nasal TNP-LPSa

 
IL-5 synthesis by CD4+ T cells is essential for TNP-specific mucosal IgA Ab responses

Thus far, our findings indicate that CD4+ T cells from SMGs and NPs provide an essential cytokine, i.e., IL-5, for B-1a B cell differentiation and for the subsequent induction of TNP-specific mucosal IgA Ab responses. To test whether the help of IL-5-producing CD4+ T cells is required for the induction of TNP-specific mucosal immunity, mice given nasal TNP-LPS plus nCT were treated with anti-IL-5 mAb (TRFK4). Significant reductions in TNP-specific mucosal IgA Ab responses were seen in saliva and NWs; however, plasma anti-TNP-LPS IgG Ab levels were essentially identical with those of untreated mice given nasal TNP-LPS plus nCT (Fig. 5A). These results were further confirmed at the cellular level by using TNP-specific ELISPOT assays (Fig. 5A). Importantly, the same frequency of sIgA+ B-1a B cells was noted in the SMGs and NPs of both anti-IL-5 mAb-treated mice given nasal TNP-LPS plus nCT and untreated mice. Furthermore, treatment with anti-IL-5 mAb did not affect IL-5R{alpha} expression by sIgA+ B-1a B cells in the SMGs and NPs (Fig. 5B). These results clearly show that IL-5-producing CD4+ T cells are required for the induction of TNP-LPS-specific mucosal IgA Ab responses.


Figure 5
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  		FIGURE 5. IL-5 production is essential for TNP-specific Ab responses. TNP-specific Ab responses in mice treated with anti-IL-5 mAb were analyzed. Mice were nasally immunized as described in the Fig. 1 legend. Two days before and at the day of immunization, groups of mice were injected with 200 µg of anti-IL-5 mAb (TRFK4) ({blacksquare}) or control isotype-matched rat IgG2a mAb ({square}) by the i.p. route. A, The levels of anti-TNP IgA Abs in saliva and NWs and the levels of anti-TNP IgG Abs in plasma were determined by TNP-specific ELISA. Mononuclear cells from spleen SMGs and NPs were subjected to TNP-specific ELISPOT assays. B, Mononuclear cells from SMGs and NPs in TRFK4-treated ({blacksquare}) or control rat IgG2a-treated ({square}) mice given TNP-LPS plus CT were stained with FITC-conjugated anti-IgA, PE-labeled anti-CD23, and allophycocyanin-tagged anti-CD5 and biotin-conjugated anti-IL-5R{alpha} mAbs, followed by PerCP-Cy5.5-streptavidin. The values are presented as the mean ± SEM of 15 mice. *, p < 0.05 when compared with mice treated with control rat IgG2a mAb.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although nCT is widely accepted as a nasal adjuvant to coadministered protein Ag, its efficacy at enhancing innate-type immune responses has not yet been established. To shed light on this issue, we began by investigating whether the nasal administration of nCT could enhance TI Ag-specific mucosal immune responses. In this study, we showed that nasal administration of TI Ag, i.e., TNP-LPS, results in the induction of TI Ag-specific mucosal IgA Abs in addition to plasma IgM and IgG3 Ab responses. Importantly, coadministration of nCT as nasal adjuvant elicited significantly elevated titers of TNP-specific mucosal IgA Abs in external secretions, including saliva and NWs. At the same time, increased numbers of TI Ag-specific IgA, IgG, and IgM AFCs were induced in both mucosal and systemic compartments.

We next investigated the cellular and molecular mechanisms involved in the induction of elevated LPS-specific immune responses. Our study is the first to show that nasal immunization with TNP-LPS plus nCT results in increased numbers of IL-5R{alpha}+sIgA+, B-1a B (CD5+) cells for the induction of TI Ag-specific mucosal IgA Ab responses in SMGs and NPs. Furthermore, we showed enhancement of IL-5 synthesis by CD4+ T cells in SMGs and NPs and a reduction in TNP-specific mucosal IgA Ab responses by blocking IL-5 production. Taken together, our results show that nCT used as a nasal adjuvant induced significantly high titers of TI Ag-specific mucosal IgA Ab responses and up-regulated interactions between IL-5R{alpha}+sIgA+ B-1a B cells and IL-5-producing CD4+ T cells in the SMGs and NPs.

At least two B cell subsets, B-1 and B-2 B cells are present in the mouse and human periphery. It is now well accepted that B-1 B cells, a minor subset comprising ~5% of the total B cell population, arise during fetal development and have a restricted receptor repertoire. These B-1 B cells were first identified by surface expression of the protein CD5 and were also characterized by high levels of IgM, IgA, and IgG3 of low affinity and broad specificity for polysaccharides from Gram-positive bacteria and LPS from Gram-negative bacteria (19, 20, 21). It was previously reported that two distinct lineages of sIgA+ B cells developed from B-1 and B-2 B cells are involved in the induction of S-IgA Abs for mucosal immunity (22, 23). Although peritoneal B-1 B cells and splenic marginal zone B cells play a central role in the induction of IgA immune responses to TI Ags (13, 14), the roles played by B-1 B cells in the induction of TI Ag-specific IgA Ab responses in the oral-nasopharyngeal mucosa remains unclear. To bridge this gap in our knowledge base, we examined the population of sIgA+CD5+ (B-1a) B cells from the SMGs and NPs of mice given nasal TNP-LPS or TNP-LPS plus nCT. Our results clearly show higher populations of B-1a B cells in the SMGs and NPs of mice given TNP-LPS and nCT than in control mice (Table I). We also noted elevated TNP-LPS-specific mucosal IgA Ab levels in saliva and NWs and an increase in the numbers of IgA-producing AFCs in SMGs and NPs (Fig. 1). Taken together, these findings suggest that the B-1a B cells in SMGs and NPs play key roles in the induction of TI Ag-specific mucosal IgA Ab responses in the oral-nasopharyngeal mucosa in addition to the gastrointestinal tract.

The B-1 B cell subset has been shown to constitutively express IL-5R{alpha} (17, 24) and to exhibit modest levels of proliferation and differentiation when treated with IL-5 (25, 26, 27). These studies clearly indicate that IL-5 plays a central role in B-1 B cell activation. Furthermore, studies with IL-5 transgenic and knockout mice have demonstrated a key role for IL-5 in the development and maintenance of the B-1 B cell population (22, 28, 29). Drawing on these studies, we examined IL-5R{alpha} expression by B-1a B cells and IL-5 production by mononuclear cells from SMGs and NPs. Our results clearly showed that B-1a B cells in the SMGs and NPs of mice given nasal TNP-LPS plus nCT expressed higher levels of IL-5R{alpha} than those seen in control mice. In addition, the use of nCT as nasal adjuvant increases IL-5 production by these mucosal effector tissues. Interestingly, our findings further show that CD4+ T cells are the main IL-5-producing cells in the SMGs and NPs, suggesting that nCT adjuvant activity has two arms. The first arm consists of nCT, which directly acts on B-1a B cells to increase their numbers and up-regulate IL-5R{alpha} expression. The second involves nCT enhancing IL-5 production by CD4+ T cells. In general, it has been shown that protein Ags (such as OVA or tetanus toxoid)-specific Ab responses induced by nCT as mucosal adjuvant are mediated by IL-4-producing, Ag-specific CD4+ T cells (3, 30, 31). Although our previous studies indeed showed that IL-4 production was up-regulated when nCT is used as a nasal adjuvant, IL-5 produced by CD4+ T cells was shown to be the key player in the induction of TNP-specific mucosal IgA Ab responses because anti-IL-5 mAb treatment eliminated TNP-specific mucosal IgA Ab responses. Thus, our study is the first to show that the adjuvanticity of nCT for TI Ag-specific mucosal IgA Ab responses is dependent on IL-5.

Like other mucosal effector lymphoid tissues, SMGs contain a high frequency of B-1a B cells (23). All of those B-1a B cells express CD5, and the majority of sIgA+ B cells are B-1a B cells (23). However, it remains unclear how and where SMGs sIgA+ B-1a B cell switching occurs, although IgA isotype class switching occurs only in organized mucosa-associated tissues such as nasopharyngeal-associated lymphoreticular tissue, Peyer’s patches, and isolated lymphoid follicles (32). Murine B-1 B cells are capable of isotype switching as well as proliferation and differentiation in the presence of IL-5, even in the absence of CD40-CD40L signaling (33). Furthermore, our studies show that the SMGs contain significant numbers of sIgM+IgA– B-1a B cells. It is possible that nCT-induced IL-5 synthesis by CD4+ T cells induces B-1a B cell IgA isotype class switching in SMGs. In addition, we postulate that dendritic cells (DCs) in the SMGs play a key role in the induction of B-1a B cell IgA class switching. Studies showing that DCs induced CD40-independent Ig class switching support our contention (34, 35, 36). DCs directly interact with B cells through the B cell activation factor of the TNF family (BAFF), also called lymphocyte stimulator protein (BLyS), and a proliferation-inducing ligand (APRIL) to induce sIgA+ B cells (36). In this regard, we are currently testing whether SMG DCs induce sIgA+ B-1a B cell switching. Because it has been shown that B-1 B cells express receptors for these ligands, BAFF-R and transmembrane activator and CAML interactor, respectively (37), we are also testing the potential roles of SMG DCs for B-1a B cell activation.

In summary, the current study shows that nCT induces enhanced oral-nasopharyngeal immune responses to LPS via the IL-5-IL-5R signaling pathway. The adjuvanticity of nCT in TNP-LPS-specific mucosal IgA Ab production is clearly due to the induction of IL-5R{alpha}+IgA+ B-1a B cells and IL-5-producing CD4+ T cells in the SMGs and NPs. The current findings that nCT enhances innate mucosal immunity in addition to acquired immunity suggests that the potent adjuvant activity of nontoxic CT mutants may be important for the induction of both innate and acquired immune responses in the oral and nasal cavities.


   Acknowledgments
 
We thank Dr. Hiroshi Kiyono of the Department of Mucosal Immunology, University of Tokyo, for his scientific discussion and critiques. Furthermore, we thank Dr. Shigetada Kawabata and Dr. Yutaka Terao of the Department of Oral Molecular Microbiology and Hiroshi Yamazaki at the Center for Medical Research and Education, Osaka University Graduate School of Medicine, for their technical assistance. We also acknowledge Dr. Kimberly K. McGhee for her editorial assistance in the preparation of the manuscript and Sheila D. Turner for the final preparation of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work is supported by National Institutes of Health Grants DE 12242, AI 43197, AI 18958, and AG 025873, as well as by Grants-in-Aid C17592179 and A17209066 from the Ministry of Education, Science, Sports, and Culture of Japan. Back

2 Address correspondence and reprint requests to Dr. Kohtaro Fujihashi, Departments of Pediatric Dentistry and Microbiology, Immunobiology Vaccine Center, University of Alabama at Birmingham, 845 19th Street South BBRB 761, Birmingham, AL 35294. E-mail address: kohtarof{at}uab.edu Back

3 Abbreviations used in this paper: nCT, native cholera toxin; AFC, Ab-forming cell; DC, dendritic cell; IL-5R{alpha}, IL-5R {alpha}-chain; mCT, mutant CT; NP, nasal passage; NW, nasal wash; S-IgA, secretory IgA; sIgA, surface IgA; SMG, submandibular gland; TI Ag, T cell-independent Ag; TNP-LPS, trinitrophenyl-LPS. Back

Received for publication November 9, 2006. Accepted for publication March 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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