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The Journal of Immunology, 2004, 173: 3305-3309.
Copyright © 2004 by The American Association of Immunologists

TGF-{beta} Receptor Signaling Is Critical for Mucosal IgA Responses1

Stefan Borsutzky*, Balthazar B. Cazac{dagger}, Jürgen Roes2,{dagger} and Carlos A. Guzmán2,*

* Vaccine Research Group, Division of Microbiology, Gesellschaft für Biotechnologische Forschung-German Research Centre for Biotechnology, Braunschweig, Germany; and {dagger} Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Sciences, University College London, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TGF-{beta} receptor (T{beta}R) signaling is important for systemic IgA production; however, its contribution to IgA secretion at mucosal sites remained uncertain. This important question was addressed using mice lacking the T{beta}R in B cells (T{beta}RII-B). Although reduced, IgA-secreting cells and IgA were still present in the systemic and mucosal compartments. The adaptive immune response was investigated after oral or nasal immunization using adjuvants acting on different molecular targets, namely, the cholera toxin B subunit and the macrophage-activating lipopeptide-2. Efficient Ag-specific cellular and humoral responses were triggered both in controls and T{beta}RII-B mice. However, a significant reduction in Ag-specific IgG2b and increased levels of IgG3 were observed in sera from T{beta}RII-B mice. Furthermore, Ag-specific IgA-secreting cells, serum IgA, and secretory IgA were undetectable in T{beta}RII-B mice. These results demonstrate the critical role played by T{beta}R in Ag-driven stimulation of secretory IgA responses in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
At the mucosal barrier the body is confronted with potentially harmful Ags from the environment, food, or infectious agents. Therefore, the local immune system plays a critical role in the tight balance between tolerance and immune responsiveness. In this context, secretory IgA (sIgA)3 is thought to be an important effector mechanism for Ag neutralization, prevention of microbial attachment to the epithelium, elimination of excessive Ag load, and the overall maintenance of mucosal homeostasis (1, 2, 3, 4, 5).

Because of this important role of sIgA, many reports addressed the requirements for isotype switch to IgA and IgA secretion. Early in vitro studies demonstrated the importance of Th2 cytokines, since incubation with rIL-4 increased the frequency of membrane-anchored IgA-expressing (mIgA+) cells and coincubation with rIL-5 stimulated IgA secretion (6, 7). In vivo work revealed impaired IgA responses in IL-4 and IL-6 knockout (KO) mice (8, 9, 10). TGF-{beta} has also attracted significant attention, since in vitro tests showed that TGF-{beta} stimulates the isotype switch to IgA, as well as IgA secretion by LPS-stimulated mIgA– B cells from Peyer’s patches and spleen (11, 12, 13, 14, 15). Remarkably, a TGF-{beta}-independent mechanism of isotype switch to IgA has been proposed in µ+ murine B cells stimulated with LPS and all-trans-retinoic acid (16, 17), as well as in human mIgA1/2– B cells coincubated with vasoactive intestinal peptide and anti-CD40 mAb (18, 19).

To further elucidate the role of TGF-{beta} for IgA production in vivo, TGF-{beta}1 KO mice have been studied. These animals exhibited a partial IgA deficiency (20). However, the use of TGF-{beta}1 KO mice has a limited value for the assessment of the TGF-{beta}1 role on sIgA stimulation (20), since these mice show inflammatory disease, autoreactivity, and a life expectancy of only 5–6 wk (21, 22, 23). To circumvent these limitations, the ligand-binding chain of the TGF-{beta} receptor (T{beta}R) was selectively inactivated (knocked out) in B cells (T{beta}RII-B) by conditional mutagenesis (Cre/loxP) (24). This mutation leads to B cell hyperresponsiveness, expansion of B-1 cells, and a reduction in serum IgA (24). Intraperitoneal immunization of T{beta}RII-B mice using alum as adjuvant resulted in the stimulation of strong serum Ab responses, without detectable Ag-specific serum IgA (24). However, the relevance of the TGF-{beta} in mucosal production of IgA remained unclear.

Mucosal IgA production can be induced by the delivery of Ags via the mucosal route. However, rapid Ag clearance and poor penetration usually lead to poor immunogenicity. The coadministration of Ags with mucosal adjuvants such as cholera toxin or lipopeptides can overcome this problem (25, 26, 27, 28). We have recently reported the potent mucosal adjuvanticity of a synthetic derivative (S-[2,3-bispalmitoyloxypropyl] cysteinyl-GNNDESNISFKEK) of the Mycoplasma-derived macrophage-activating lipopeptide of 2 kDa (MALP-2). This molecule efficiently stimulates mucosal immune responses against codelivered Ags (29, 30). MALP-2, like the cholera toxin B subunit (CT-B), is a suitable amplification system to study the role of T{beta}R signaling for sIgA production during the stimulation of adaptive immune responses. Thus, we evaluated the immune response elicited in T{beta}RII-B mice immunized via oral or intranasal route in the presence or absence of the mucosal adjuvants CT-B and MALP-2. The obtained results establish the central role played by T{beta}R-mediated signaling in the stimulation of IgA responses at mucosal sites.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and cell cultures

The generation of T{beta}RII-B mice has been described elsewhere (24). Animals were maintained in vented cages under standard conditions according to institutional guidelines.

Histology

Intestinal tissues were fixed in Formalin before embedding in paraffin. Five-micrometer sections were stained with hematoxylin, followed by staining with a biotinylated polyclonal rat anti-mouse (Southern Biotechnology Associates, Birmingham, AL) and development using streptavidin-HRP (Sigma-Aldrich, St. Louis, MO), all following standard histochemical protocols.

Immunization protocols and sample collection

For intranasal immunization, groups of three to four mice (7–11 wk old) were immunized on days 1, 14, and 21 with 50 µg of {beta}-galactosidase ({beta}-gal; Roche, Mannheim, Germany) with or without 0.5 µg of MALP-2 (31) (20 µl/dose). Control animals received PBS. Serum samples were collected on days 0, 13, 20, and 31 and stored at –20°C. At day 31, mice were sacrificed and the final sampling was performed. Nasal (NL) and bronchial lavages (BL) were obtained by flushing the organs with a final volume of 0.2 and 1 ml of PBS supplemented with 5% FCS and 40 µM PMSF, respectively. Intestinal lavages were performed by using 1.5 ml of PBS supplemented with 0.1 mg/ml trypsin inhibitor (Sigma-Aldrich), 50 mM EDTA, 0.1% BSA, and 40 µM PMSF. The lavages were then centrifuged to remove debris (20 min at 13,000 x g and 4°C) and supernatants were stored at –70°C. Bone marrow and spleens were removed and pooled for the analysis of the cellular immune responses.

For CT-B-induced responses, mice were immunized orally with (4-hydroxy-3-nitrophenyl)-acetyl-chicken-{gamma}-globulin (NP) (1 mg/dose) in the presence of purified CT-B (10 µg; Sigma-Aldrich) on three occasions 10 days apart. Serum NP-specific IgA and IgG1 levels or total intestinal IgA levels were determined by ELISA as previously described (24).

T cell proliferation assays

Proliferation assays were performed in triplicate as previously described (29). Cells were grown in RPMI 1640 supplemented with 10% FCS, 100 U/ml penicillin, 1 mM L-glutamine, and 50 µg/ml streptomycin (Invitrogen Life Technologies, Karlsruhe, Germany) at 37°C in a humidified 5% CO2 atmosphere and were restimulated with {beta}-gal. The amount of incorporated [3H]thymidine was determined using a scintillation counter (Wallac 1450 MicroTrilux; PerkinElmer Wallac, Gaithersburg, MD). Results are expressed as the ratio of the mean [3H]thymidine uptake of the stimulated and the nonstimulated samples, a stimulation index of >3 was considered positive.

Determination of specific and unspecific Ig-secreting cells

The frequencies of either {beta}-gal-specific or total IgM-, IgG-, and IgA-secreting cells were determined by ELISPOT. Briefly, plates (Millipore, Bedford, MA) were coated (100 µl/well) with either {beta}-gal or isotype-specific capture Abs (Sigma-Aldrich) at 5 µg/ml in 0.05 carbonate buffer (pH 9.6). Serial dilutions of spleen or bone marrow cells in complete RPMI 1640 medium were incubated in quadruplicate for 6 h. After washing, the plates were incubated with 100 µl of biotinylated subclass-specific Abs (Sigma-Aldrich) overnight at 4°C. Plates were then washed and further incubated with 100 µl/well peroxidase-conjugated streptavidin (BD Pharmingen, Heidelberg, Germany) for 1 h. After additional washing, spot-forming units (SFU) were developed using 3-amino-9-ethyl-carbazole (Sigma-Aldrich) in 0.1 M acetate buffer (pH 5.0) and 0.05% H2O2 (30%). The reaction was stopped after 60 min and the spots were counted using a binocular microscope.

Detection of Ag-specific Ig

Ab titers in sera were determined by ELISA as previously described (32). End point titers were expressed as the reciprocal of the last dilution, which gave an OD at 405 nm of 0.1 U above the values of the negative controls after 15 min of incubation. Ag-specific Ig subclasses present in sera of immunized mice were measured using an isotype-specific ELISA (24, 29).

The amount of total and Ag-specific IgA or IgM present in lavages was determined as previously described (24, 29, 32). Serial dilutions of purified mouse IgA or IgM (Sigma-Aldrich) were used to generate standard curves. To compensate for variations in the efficiency of recovery of secretory Abs among animals, the results were normalized and expressed as percentage of {beta}-gal-specific IgA with respect to the total amount of IgA present in the sample.

Statistical analysis

Comparisons between two experimental groups were performed by using the double-sided Student’s t test. A value of p < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Few B cells from T{beta}RII-B mice produce and secrete IgA

The initial analysis of T{beta}RII-B mice revealed a virtually complete deficiency in IgA+ B cells in Peyer’s patches and a defective production of Ag-specific IgA in serum (24). Immunohistochemical analysis (Fig. 1, A and B) shows that in comparison to the controls (Fig. 1A), IgA-expressing plasma cells in the intestinal lamina propria are also substantially reduced, but few remain detectable in the mutant mice (Fig. 1B). In line with these findings, IgA-secreting cells are reduced 15-fold in both spleen (p < 0.001, Fig. 1C) and bone marrow (48 vs 642 SFU per 5 x 105 cells, p < 0.001) of the mutant mice. To determine whether the IgA levels at mucosal sites were also affected, total IgA in nasal, lung, and intestinal lavages were measured. The levels of sIgA in T{beta}RII-B mice were lower than in controls in all cases (Fig. 1D). However, the observed differences were statistically significant (p < 0.05) only for nasal and intestinal lavages.



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  		FIGURE 1. T{beta}R plays a critical role for the production of systemic and mucosal IgA. A and B, IgA expression in intestinal tissues of control (A, CD19cre/+) and T{beta}RII-B (B) mice. Plasma cells staining strongly for IgA are readily detected in the villi and lamina propria of controls, whereas only few (indicated by arrows) are apparent in the mutants. Also note the higher level of staining of epithelial cells in the controls, which produce large amounts of sIgA. Sections were counterstained with hematoxylin. C and D, Evaluation of total Ig production in T{beta}RII-B and control mice. Determination of IgM-, IgG-, and IgA-secreting cells by ELISPOT (C). Samples were tested in quadruplicate, results are expressed as average of SFU per total spleen cells. SEM are indicated by vertical lines. D, Analysis of the total amount of IgA present in mucosal lavages (NL, BL, and intestinal lavage (IL)). Results are expressed as micrograms per milliliter. SEM are indicated by vertical lines. ND, Not detectable. E, Analysis of the total amount of IgA present in the intestinal lavages of control, T{beta}RII-B, CD19cre/cre, and T{beta}RII-B/CD19cre/cre mice. Note the reduction in residual total IgA in lavages from mice lacking both T{beta}R and CD19 in B cells. *, Statistical significance at p < 0.05.

 
The absence of CD19 leads to a defect in B1 cells (33), which are known to contribute to IgA production (34, 35). To study the potential role played by B1 cells in the production of the residual sIgA, we determined the levels of total IgA in intestinal lavages from T{beta}RII-B/CD19 double KO animals. T{beta}RII-B mice homozygous for CD19(cre/cre) (being therefore CD19 deficient) show reduced levels of intestinal IgA (Fig. 1E). This suggests that B1 cells are indeed responsible for the production of the sIgA detected in T{beta}RII-B mice.

The production of total and Ag-specific IgA after oral stimulation with CT-B is affected in T{beta}RII-B mice

It is known that oral immunization with CT-B as adjuvant leads to increased levels of IgA, probably due to an increased IgA class switching on B cells through TGF-{beta} signaling (36). Thus, we performed immunization studies in T{beta}RII-B mice using CT-B as amplification signal. The defect in the production of total sIgA was strikingly apparent in T{beta}RII-B mice, in contrast to the WT controls (Fig. 2A). In line with earlier data, Ag-specific serum IgA was undetectable (Fig. 2B). We also observed a modest increment in the level of serum IgG1 in T{beta}RII-B mice. However, the differences were not statistically significant (p > 0.05, Fig. 2C).



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  		FIGURE 2. The production of total and Ag-specific IgA after oral stimulation with CT-B is affected in T{beta}RII-B mice. A, Levels of total IgA in intestinal lavages after oral immunization with the hapten-protein conjugate (4-hydroxy-3-nitrophenyl)-acetyl-chicken-{gamma}-globulin and CT-B as adjuvant. Note the failure of the T{beta}RII-B mice to boost total IgA upon CT-B stimulation. B, Although NP-specific IgA levels remained below the detection limit in intestinal lavages from all immunized animals (data not shown), NP-specific IgA is readily detectable in serum of controls, but not of the mutant mice. C, Production of serum NP-specific IgG1 is not significantly affected in the mutants (p > 0.05).

 
Humoral immune responses are altered after intranasal immunization with {beta}-galactosidase in T{beta}RII-B mice

In agreement with the previously reported hyper-{gamma}-globulinemia and B cell hyper responsiveness (24), higher numbers of IgM- and IgG-secreting cells were detected in the spleens of T{beta}RII-B mice (Fig. 1C). To establish whether this apparent defect in production of sIgA represented a more general deficiency, we evaluated the immune responses elicited after immunization via the intranasal route. To this end, we assessed whether the T{beta}R deficiency affected B cell responses after intranasal administration of {beta}-gal (50 µg/dose) alone or coadministered with MALP-2 (0.5 µg/dose) as adjuvant. The induction of strong Ag-specific proliferative responses in spleen cells against {beta}-gal in vitro required coadministration of MALP-2 (Fig. 3A). In the absence of this adjuvant, responses were barely detectable. No significant differences (p > 0.3) were observed in control and T{beta}RII-B mice, confirming the adequate activation of T cells. Intranasal immunization with {beta}-gal alone induced readily detectable serum IgG titers in both T{beta}RII-B mice and controls (Fig. 3B). However, coadministration of MALP-2 boosted the responses ~10-fold (p < 0.05). Although the IgG titers in T{beta}RII-B mice were always slightly higher than those of control animals, the differences were not statistically significant (p > 0.4).



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  		FIGURE 3. {beta}-gal-specific immune responses stimulated in T{beta}RII-B and control mice. Animals were immunized by the intranasal route with {beta}-gal (50 µg) alone or coadministered with MALP-2 (0.5 µg) on days 1, 14, and 21. A, Anti-{beta}-gal T cell proliferative responses from spleen cells restimulated in vitro for 4 days with different concentrations of {beta}-gal. Results are expressed as the ratio between values (average of triplicates) from stimulated and nonstimulated samples (stimulation index). SEM are indicated by vertical lines. B, Kinetics of anti-{beta}-gal IgG responses in sera from vaccinated mice. Results are expressed as the reciprocal geometric mean end point titer. Immunizations are indicated by the arrows.

 
To determine whether isotype switching is affected in T{beta}RII-B mice, we determined the end point titers of the different {beta}-gal-specific Ig isotypes in sera from immunized animals. Mainly IgG1 followed by IgG2a > IgG2b > IgG3 were induced in both groups by immunization with {beta}-gal/MALP-2 (Fig. 4A). In comparison to the controls, sera from T{beta}RII-B mice contained significantly less IgG2b (p < 0.05) and virtually no IgA (p < 0.01; Fig. 4, A and B). ELISPOT analysis of splenocytes confirmed the absence of IgA-producing plasma cells (<1 SFU per 5 x 105 cells, p < 0.005), whereas production of IgM and IgG Abs in the spleen was unaffected by T{beta}R deficiency (Fig. 4C). Significantly, the virtually complete defect in the generation of Ag-specific IgA was also apparent in the nasal and bronchoalveolar lavages (p < 0.01, Fig. 4D), whereas {beta}-gal-specific IgM titers were not affected (data not shown).



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  		FIGURE 4. Analysis of {beta}-gal-specific humoral immune responses stimulated in T{beta}RII-B and control mice. Animals were immunized by the intranasal route with {beta}-gal (50 µg) coadministered with MALP-2 (0.5 µg) on days 1, 14, and 21. A, Anti {beta}-gal IgG isotypes present in sera of immunized mice. Results are expressed as the reciprocal of the geometric mean end point titer. B, Evaluation of the levels of {beta}-gal-specific IgA in sera. Results are expressed as the reciprocal of the geometric mean end point titer. C, Determination of {beta}-gal-specific IgM-, IgG-, and IgA-secreting cells in spleen. Results are expressed as SFU per total cells (average of quadruplicates). D, Evaluation of the {beta}-gal-specific sIgA responses in T{beta}RII-B and control mice. Ag-specific IgA in NL and BL were quantified by ELISA. Results are expressed as the percentage of {beta}-gal-specific IgA with respect to the total IgA. The total mean IgA values for the T{beta}RII-B and controls were 0.02 and 0.737 µg/ml in NL and 0.696 and 2.38 µg/ml in BL, respectively. SEM are indicated by vertical lines. ND, Not detectable. *, Statistical significance at p < 0.05.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mucosal membranes represent a place in which the balance between tolerance and immune responses is tightly regulated. IgA secreted by plasma cells in mucosal tissues can bind to polymeric Ig receptors present on the membrane of epithelial cells, initiating a process of transcytosis and release of polymeric IgA into the lumen of the mucosal tissues (37). Representing the most abundant Ig isotype in humans, an important role of IgA in host protection has long been postulated (2, 38). The isotype switch to IgA and its secretion can be induced by TGF-{beta}-dependent and TGF-{beta}-independent mechanisms (11, 12, 13, 14, 15, 16, 17, 18, 19). Making use of a mouse model lacking T{beta}R selectively in B cells (24), the present study shows that the efficient production of Ag-specific mucosal IgA is driven by T{beta}R signaling (Figs. 2 and 4).

The number of IgA-producing cells was reduced by an order of magnitude in the lamina propria (Fig. 1, A and B) and spleen (Fig. 1C) of T{beta}RII-B mice with respect to that of controls. Interestingly, the levels of IgA in mucosal lavages were less affected than what was expected, as demonstrated by the immunohistochemistry (Fig. 1, B and D). In contrast, it has been found that the concentrations of total IgA depend on the age of T{beta}RII-B mice (24). Thus, differences may reflect, at least in part, the fact that the mice were older when sacrificed for the histological analysis. However, it is clear that there is a residual production of IgA in T{beta}RII-B mice. Whether the presence of residual IgA is attributable to incomplete receptor deletion and escape of small numbers of T{beta}R-expressing B cells or whether it may reflect a T{beta}R-independent mechanism remains uncertain.

B1 cells represent a self-renewing population of B cells that depend on positive selection through BCR signaling and the presence of antigenic determinants, including autoantigens (39). Notably, these cells contribute significantly to the total pool of IgA (34, 35). Small numbers of T{beta}R-positive B1 cells, which may have escaped T{beta}R deletion, could undergo TGF-{beta}-induced differentiation into IgA-producing plasma cells. The reduced levels of total intestinal IgA in T{beta}RII-B(fl/fl)/CD19(cre/cre) (T{beta}R/CD19-deficient) mice could thus result from the absence of B1 cells (Fig. 1E). Alternatively, enhanced receptor deletion in the presence of two copies of the CD19-cre locus could explain the effect. However, the virtually complete absence of Ag-specific IgA responses upon challenges with mucosal adjuvants highlights the essential role of T{beta}R signals in the generation of Ag-specific mucosal immunity.

CT-B and MALP-2 exert their immune-enhancing effects via different molecular pathways. CT-B binds to monosialogangliosides on epithelial cells (40, 41) and has been shown to induce TGF-{beta}-dependent class switching and IgA secretion by murine B cells in vitro (36). MALP-2 is a ligand of TLR-2/6 heterodimers and activates NF-{kappa}B (42, 43). After stimulation with MALP-2, immune cells, such as macrophages and dendritic cells, release proinflammatory cytokines like TNF-{alpha}, IL-1, IL-6, IL-12, and chemokines like MIP-1/2 and MCP-1 (44, 45). Since both CT-B and MALP-2 act on different molecular targets and since the induced IgA responses are suppressed in T{beta}RII-B mice, TGF-{beta} signaling seems to play a central role for the induction of IgA (Figs. 2 and 4). However, we cannot rule out the possibility that TGF-{beta}-independent mechanisms of IgA switching may exist during natural infections.

A profound shift in Ig isotype distribution in T{beta}RII-B mice has been previously reported (24). However, in this report T{beta}R-deficient animals did not show any significant changes in the level of IgG2b. This is in contrast to in vitro studies demonstrating the isotype switch of LPS-stimulated B cells to IgG2b and IgA by TGF-{beta} (46). In the present study, the administration of Ag via the mucosa resulted in a pronounced reduction of IgG2b with an accompanying loss of IgA in the mutants (Fig. 3), in agreement with previous in vitro work. Thus, the effects of TGF-{beta} on B cells are most apparent at mucosal sites. This is supported by the fact that mucosally administered Ag induces TGF-{beta} expression by macrophages and Ag-specific CD4+ T cells in the lamina propria (47).

TGF-{beta} is a negative regulator of immune responses involved in oral tolerance (48, 49) and our data demonstrate that TGF-{beta} stimulates IgA production in vivo. Interestingly, IgA has been postulated to be the main effector mechanism of the mucosal immune system against infections (2). However, there is a growing body of evidence challenging the absoluteness of the theory that protection against infections is the only function of IgA (50, 51, 52, 53, 54). Recent studies have revealed the importance of IgA for the maintenance of mucosal homeostasis in mice (4, 5). In line with these findings, the observed correlation of IgA deficiency in humans with mucosal inflammatory diseases further suggests a regulatory function of sIgA (55, 56). The function of TGF-{beta} is consistent with the role of IgA as noninflammatory isotype, which not only neutralizes Ag but also supports the homeostatic effects of TGF-{beta}. This would minimize the risk of inappropriate inflammatory responses, which may lead to clinical manifestations such as inflammatory bowel disease (5, 49)


   Acknowledgments
 
We are particularly grateful to Werner Müller for critical discussions, to Peter F. Mühlradt for providing MALP-2, and to Urte Jäger and Elena Reinhard for their commitment and excellent technical help. Finally, we thank Thomas Ebensen for outstanding support and for critical reading of the manuscript.


   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 was in part supported by the Wellcome Trust. Back

2 Address correspondence and reprint requests to Dr. Carlos A. Guzmán, Vaccine Research Group, Division of Microbiology, Gesellschaft für Biotechnologische Forschung-German Research Centre for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. E-mail address: cag{at}gbf.de or to Dr. Jürgen Roes, Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Sciences, University College London, 46 Cleveland Street, London W1T 4JF, U.K. E-mail address: j.roes{at}ucl.ac.uk Back

3 Abbreviations used in this paper: sIgA, secretory IgA; mIgA+, membrane anchored IgA expressing; KO, knockout; T{beta}R, TGF-{beta} receptor; T{beta}RII-B, TGF-{beta} receptor deficiency in CD19+ cells; MALP-2, macrophage-activating lipopeptide-2; CT-B, cholera toxin subunit B; {beta}-gal, {beta}-galactosidase; SFU, spot-forming unit; NL, nasal lavage; BL, bronchial lavage; NP, (4-hydroxy-3-nitrophenyl)-acetyl-chicken-{gamma}-globulin. Back

Received for publication December 5, 2003. Accepted for publication June 29, 2004.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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