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The Journal of Immunology, 2005, 175: 1276-1285.
Copyright © 2005 by The American Association of Immunologists

Mucosal B Cell Deficiency in IgA–/– Mice Abrogates the Development of Allergic Lung Inflammation1

Paul M. Arnaboldi*, Melissa J. Behr{dagger} and Dennis W. Metzger2,*

* Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY 12208; and {dagger} New York State Department of Health, Wadsworth Center, Albany, NY 12208


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have investigated the consequence of lack of IgA on host immunity using a murine model of allergic lung inflammation. Mice with a targeted disruption of the {alpha}-switch region and 5' H chain gene (IgA–/– mice), which lack total IgA, developed significantly reduced pulmonary inflammation with fewer inflammatory cells in lung tissue and bronchoalveolar lavage fluids, as well as reduced levels of total and IgG1 OVA-specific Abs and decreased IL-4 and IL-5 in bronchoalveolar lavage fluids compared with IgA+/+ controls, following allergen sensitization and challenge. This defect was attributable to fewer B cells in the lungs of IgA–/– mice. Polymeric IgR-deficient (pIgR–/–) mice, which lack the receptor that transports polymeric IgA across the mucosal epithelium where it is cleaved to form secretory IgA, were used to assess the contribution of secretory IgA vs total IgA in the induction of allergic lung inflammation. pIgR–/– and pIgR+/+ mice had comparable levels of inflammation, demonstrating that IgA bound to secretory component is not necessary for the development of allergic lung inflammation, although this does not necessarily rule out a role for transudated IgA in lung secretions because of "mucosal leakiness" in these mice. The results indicate that Ag-specific B cells are required at mucosal surfaces for induction of inflammation and likely function as major APCs in the lung for soluble protein Ags.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Humans synthesize ~66 mg/kg IgA/day, which is more than all other Ig isotypes combined (1). Of this, the majority (50–70%) is produced by submucosal B cells and transported into mucosal secretions by the polymeric IgR (pIgR)3 (1, 2). The pIgR is expressed on the basolateral surface of most mucosal epithelial cells, where it binds to polymeric IgA- and IgM-containing J chains (2). Following transcytosis of the Ig/receptor complex to the apical surface of the cell, the receptor is cleaved, releasing the Ig bound to the extracellular portion of the receptor, referred to as secretory component (SC) (2). SC-bound IgA, or secretory IgA (SIgA), is considered an important mediator of immunity at mucosal surfaces. As the majority of functions attributed to SIgA are noninflammatory, it is widely referred to as a neutralizing Ab (3, 4, 5). It has been shown to impede bacterial colonization of the mucosa and neutralize virus particles (3, 4, 5, 6), block the activity of bacterial enzymes and toxins (3, 4, 5, 7), and preserve tolerance to food Ags (3, 5, 8, 9). However, within tissues, IgA is capable of mediating the effector functions of several leukocyte populations through its interactions with CD89 (Fc{alpha}RI), including induction of cytokine production, phagocytosis, and Ab-dependent cellular cytotoxicity by neutrophils, monocytes, and macrophages (3, 5, 10) and degranulation of eosinophils (11). Thus, there is a certain degree of compartmentalization to the function of IgA in immunity (3, 5).

The development of IgA-deficient mice (IgA–/–) (12) has provided a model for the further characterization of the biological activities of IgA. Initial studies using these mice did not demonstrate an increased susceptibility of IgA–/– mice to challenge with infectious agents compared with IgA+/+ mice (7, 12, 13, 14, 15, 16, 17); however, more recent investigations have revealed subtle immune defects that could be linked to deficient APC function (18, 19). In the current study, we investigated the effect of IgA deficiency on the inflammatory response associated with allergic asthma using a murine model of allergic lung inflammation. The ability of SIgA to neutralize allergens and prevent sensitization is contentious; some reports (9, 20, 21) have demonstrated a negative correlation between IgA levels and allergic sensitization, whereas others have found no such association (22, 23, 24). Despite this, a possible link between IgA and the pathophysiology of established allergic asthma has been alluded to in a number of correlative human studies. Allergen-specific IgA can be isolated from the bronchoalveolar lavage (BAL) of asthmatics (25, 26) and is increased during periods of high-allergen exposure (27). IgA is capable of inducing degranulation of human eosinophils, the major cell type present in the inflammatory infiltrate of asthmatics (11). CD89 (Fc{alpha}R1) is up-regulated on eosinophils isolated from allergic patients, and IgA levels correlate with levels of eosinophil granule proteins in BAL fluids from asthmatics (28, 29, 30). Furthermore, Ag-specific IgA in BAL fluids has been correlated with stronger Ag-induced, late-phase asthmatic reactions (31). Although the ability of SIgA to neutralize Ags without the induction of inflammation is well established, the above results, although offering no specific mechanism of activity, suggest that IgA may enhance an ongoing mucosal inflammatory response.

We have now examined the ability of IgA–/– and pIgR–/– mice to respond to intranasal (IN) challenge in a murine model of allergic lung inflammation. Using this model, a defect in B cell expression was identified in the lungs of IgA–/– mice. Furthermore, it was found that although SIgA does not appear to contribute to the development of allergic lung inflammation following systemic (i.p.) sensitization and mucosal (IN) challenge, Ag-specific B cells appear to be an essential population for the development of respiratory inflammation induced by a soluble protein Ags (OVA), likely by functioning as APCs.


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

IgA–/– (C57BL/6 x 129) mice with a targeted disruption of the {alpha}-H chain and switch region were originally developed at the Baylor College of Medicine (12). IgA+/+ (C57BL/6 x 129) littermates, lacking the disrupting neomycin cassette, were used as controls. Mice deficient in expression of the pIgR (pIgR–/– mice, C57BL/6 x 129) were obtained from Dr. F.-E. Johansen (University of Oslo, Oslo, Norway) (32); wild-type (C57BL/6 x 129) littermates were used as controls. The mice were bred at Albany Medical College and the Institutional Animal Care and Use Committee approved all procedures concerning the use of these mice.

Induction of allergic lung inflammation

Allergic lung inflammation was induced as described previously (33). Mice were i.p. sensitized with 10 µg of OVA (Sigma-Aldrich) in alum (Reheis). Two weeks later, the mice were lightly anesthetized and IN challenged for 5 consecutive days with 100 µg of OVA in saline. Some groups also received 0.1 or 1.0 µg of IL-4 (R&D Systems) during i.p. sensitization on days 0, 1, and 2 to boost IgE production. Twenty-four hours after the final dose of OVA, sera were collected, and the mice were sacrificed. BAL fluids were collected by making an incision in the trachea and washing the lungs with 5 mM EDTA (Sigma-Aldrich) in saline. The BAL fluids were centrifuged at 300 x g for 5 min at 4°C, and the supernatants were stored at –20°C until analysis. Leakage of serum proteins into the lung compartment was monitored by measuring albumin levels in BAL fluid using Albustix (Bayer). Numbers of viable cells in BAL were determined by trypan blue exclusion, and the cells were mounted on slides for analysis by centrifugation at 110 x g for 5 min at room temperature. In addition, portions of spleen and lung tissues were snap frozen in liquid nitrogen and stored at –80°C for cytokine analysis. Other lung tissue samples were fixed in formalin (Fisher Scientific) or frozen embedded in OCT (Richard Allan Scientific) for histological analysis.

Histology

Formalin-fixed tissues were processed and embedded in paraffin blocks (Richard Allan Scientific). The blocks were sectioned (5-µm thickness) and stained with H&E (Fisher Scientific). The presence of inflammatory cells was assessed using standard light microscopy; inflammation manifested as infiltrates in the perivascular (PV) and peribronchiolar regions of the lung. Stained slides were coded and blindly examined by a pathologist without knowledge of the type of mice or treatment. The relative degree of inflammation was graded in a semiquantitative manner by assigning a score of 0–4 based upon the number, location, and size of the inflammatory infiltrates (0, no visible lesion; 1, multifocal inflammation; 2, locally extensive inflammation; 3, diffuse inflammation; and 4, severe diffuse inflammation).

Cryoembedded lung tissues were sectioned (8-µm thickness) at –20°C using a cryostat (Richard Allan Scientific). Cyanide-resistant eosinophil peroxidase (CN–EPO) staining was performed as described previously (34); eosinophils were enumerated by averaging numbers of stained cells in five randomly chosen fields per slide. For immunofluorescent staining of lung tissue, slides were fixed for 10 min in acetone (Sigma-Aldrich) and stored at –80°C until use. Before staining, sections were marked using a Pap Pen (Vector Laboratories), rehydrated in PBS for 5 min, and blocked using 2% BSA (Sigma-Aldrich) in PBS for 10 min at room temperature. Each acetone-fixed section was incubated with 50 µl of fluorescent Abs in a humidified chamber for 1 h at room temperature in the dark. Sections were washed and mounted using 90% glycerol in PBS and viewed with an Olympus DX-60 microscope. Fluorescent Abs were used at the following dilutions: 1/100 dilution of FITC-conjugated donkey anti-mouse IgM (µ-chain specific) (Jackson ImmunoResearch Laboratories), 1/100 dilution of FITC-conjugated goat anti-mouse IgA ({alpha}-chain specific) (Caltag Laboratories), 1/50 dilution of Texas red-conjugated goat anti-mouse IgG ({gamma}1, {gamma}2A, {gamma}2B, and {gamma}3 specific) (Jackson ImmunoResearch Laboratories).

Inflammatory cells isolated from BAL fluids were evaluated using a commercially available modified Wright-Giemsa stain (HEMA 3; Biochemical Sciences). Cytospins were stained according to the manufacturer’s instructions and examined under oil-immersion light microscopy.

Ab and cytokine levels

Total, IgG1, and IgA Ag-specific Ab levels were determined by ELISA. Ninety-six-well microtiter plates were coated with 10 µg/ml OVA overnight at 4°C, washed with 0.05% Tween 20 in PBS, and blocked with 10% FBS in PBS for 2 h at room temperature. Serum and BAL samples were serially diluted in 1% FBS in PBS and incubated on the OVA-coated plates overnight at 4°C. The plates were washed and incubated with alkaline phosphatase-conjugated detecting Ab (Southern Biotechnology Associates) for 5 h at 37°C or overnight at 4°C. After addition of substrate, the plates were analyzed in a microplate reader at 405 nm. Total IgE levels were determined in a similar manner using plates coated with 5 µg/ml goat anti-mouse IgE Ab (Southern Biotechnology Associates). Cytokine protein levels were determined in BAL samples using commercially available ELISA kits (BD Biosciences) according to the manufacturer’s instructions.

Bleomycin-induced inflammation

A total of 0.025 U of bleomycin sulfate (Gensia Sicor Pharmaceuticals) was diluted in 25 µl of normal saline and instilled IN. Nine days later, the mice were sacrificed, and BAL and lungs were isolated for histological analysis.

Cell transfers

Single-cell suspensions were prepared from the spleens of IgA+/+ and IgA–/– mice by passage through nylon mesh. RBCs, dead cells, and granulocytes were removed from the cell suspensions by gradient centrifugation on Lympholyte M (Cederlane Laboratories). A total of 5 x 106 splenocytes/ml was incubated in a 24-well culture plate with 10 µg/ml OVA for 72 h at 37°C (35). Following culture, cells were washed and resuspended in PBS. In some cases, CD4+ T cells were isolated or CD19+ B cells were depleted from whole splenocyte populations using anti-mouse CD4- or CD19-coated microbeads and an AutoMACs automated magnetic cell sorting system (Miltenyi Biotec), according to the manufacturer’s instructions. A total of 5 x 106 splenocytes, 1 x 106 CD4+ cells, or 3 x 106 CD19+ cell-depleted splenocytes were injected i.p. into IgA+/+ and IgA–/– mice in 200 µl of PBS. The number of CD4+ T cells and CD19+ cell-depleted splenocytes transferred correspond to the representation of these populations in the mouse spleen. Intranasal challenge with OVA was commenced 24 h after cell transfer as described above.

Flow cytometry

Single-cell suspensions were obtained from the lungs of IgA+/+ and IgA–/– mice by collagenase digestion (2.5 mg/ml collagenase D, 0.25 mg/ml DNaseI, and 1 mM MgCl2) (Roche) for 1 h at 37°C under constant agitation, followed by passage through a nylon mesh. The cells were then incubated with 2.4G2 anti-Fc{gamma}RI/III mAb at 4°C for 20 min to block nonspecific Ab binding, followed by incubation at 4°C for 30 min on an orbital shaker with tricolor-anti-mouse CD4, tricolor-anti-mouse CD8, FITC-anti-mouse CD11b, PE-anti-mouse CD19 (Caltag Laboratories), FITC-anti-mouse CD3, PE-anti-mouse CD11c, PE-anti-mouse DX5, PE-anti-mouse CD117 (BD Pharmingen), or FITC-anti-mouse Fc{epsilon}RI (eBioscience). Abs were titrated before use to determine the optimal staining concentrations. Binding of fluorescent Ab was detected using a FACSCanto or FACScan flow cytometer and analyzed using FACSDiva or CellQuest software, respectively (BD Biosciences). Ten thousand events per gate per sample were collected, and the data were reported as percentage of cells within the lymphocyte gate.

Statistics

Statistical comparisons between IgA+/+ and IgA–/– mice were performed using a two-tailed Students t test. Values of p < 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Recruitment of inflammatory cells into the lungs of sensitized mice following IN Ag challenge is decreased in IgA–/– mice

The most striking feature of murine allergic lung inflammation is the influx of inflammatory cells into the lung subsequent to allergen priming and challenge. After OVA sensitization and IN challenge, IgA+/+ mice developed profound inflammation (inflammatory score = 2.60 ± 0.53, n = 24) that manifested as PV and peribronchiolar cuffing (Fig. 1a, arrows), although areas of diffuse infiltrate were observed as well. Hypertrophy of the bronchial epithelium and plugging of some airways with mucus, inflammatory cells, and debris was also evident in the lungs of these mice (Fig. 1, a and b). By comparison, identically treated IgA–/– mice had a significantly diminished inflammatory response (inflammatory score = 1.50 ± 0.38, n = 24) (p < 0.01), in which fewer infiltrates were observed, primarily as PV cuffing with very little diffuse inflammation (Fig. 1, c and d). Furthermore, hypertrophy of the bronchial epithelium was mostly absent and plugging of airways was not seen (Fig. 1d). Eosinophils were detected in infiltrates from both groups of mice using a CN–EPO stain (Fig. 1, e and f); however, significantly (p < 0.01) greater numbers were detected within the infiltrates of IgA+/+ mice (307.05 ± 12.23 eosinophils/field) compared with IgA–/– mice (122.85 ± 32.64 eosinophils/field). Saline-sensitized and -challenged, saline-sensitized and OVA-challenged, and OVA-sensitized and saline-challenged mice were used as controls and demonstrated negligible inflammation (data not shown).



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  		FIGURE 1. Allergic lung inflammation is abrogated in IgA–/– mice. a–d, H&E-stained lung sections from OVA-sensitized and -challenged IgA+/+ and IgA–/– mice. A greater number of infiltrates (black arrows) were found in the lungs of IgA+/+ mice (a, x100) compared with IgA–/– mice (c, x100). Upon higher magnification, hypertrophy of the bronchial epithelium as well as plugging of some airways with mucous and debris was observed in IgA+/+ mice (b, x400) but not in IgA–/– mice (d, x400). Eosinophils were present in infiltrates from both groups of mice (b and d, black arrows). The results are representative of six experiments (n = 6–8 mice/group). e and f, CN–EPO staining of cryosections from OVA-sensitized and -challenged IgA+/+ (e, x200) and IgA–/– (f, x200) mice; eosinophils were stained brown and were found in greater numbers in the lungs of IgA+/+ mice compared with IgA–/– mice (black arrows). The results are representative of two experiments (n = 4–6 mice/group). g, Differential staining of inflammatory cells isolated from BAL fluids of OVA-sensitized and -challenged IgA+/+ and IgA–/– mice. The data are reported as mean ± SD and are representative of four experiments (n = 6–8 mice/group) (*, p < 0.05).

 
Similar to the responses observed in lung tissues, significantly more inflammatory cells were present in BAL fluids from IgA+/+ mice compared with IgA–/– mice (8.11 x 105± 3.10 x 105 total cells vs 2.91 x 105± 1.12 x 105 total cells; p < 0.005). Differential staining of BAL fluid inflammatory cell isolates revealed that eosinophils were the predominant cell population in both groups of mice, but again, there were significantly (p < 0.05) more eosinophils isolated from IgA+/+ mice than IgA–/– mice (Fig. 1g). Overall, eosinophils constituted 10- 20% less of the total inflammatory cell population isolated from BAL fluids of IgA–/– mice compared with IgA+/+ mice (data not shown). Lymphocyte, macrophage, and neutrophil numbers were comparable between both groups of mice (Fig. 1g).

To determine whether the diminished inflammatory response in IgA–/– mice was Ag specific or due to an inherent defect caused by disruption of the {alpha}-H chain, inflammation was chemically induced by IN challenge with bleomycin sulfate. Intranasal administration of bleomycin resulted in recruitment of neutrophils and macrophages to lung tissues of both groups of mice (Fig. 2). The development of inflammation was also reflected in the large numbers of inflammatory cells isolated from BAL fluids of IgA–/– and IgA+/+ mice (5.61 x 106 ± 0.01 x 106 vs 1.31 x 106 ± 0.53 x 106 total cells, respectively) (p < 0.05). These results indicate that IgA–/– mice are fully capable of manifesting a pulmonary inflammatory response and suggest that the defect observed in IgA–/– mice following OVA sensitization and challenge is a deficiency in responding to antigenic stimulation. Furthermore, the presence of significantly more inflammatory cells in BAL fluids from IgA–/– mice compared with IgA+/+ mice suggests that IgA plays different roles in inflammatory responses induced by different stimuli, e.g., Ag-specific vs Ag-nonspecific inflammatory responses.



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  		FIGURE 2. Bleomycin-induced lung inflammation occurs in IgA–/– mice. a–d, H&E-stained lung sections from IgA+/+ (a and b) and IgA–/– mice (c and d) 9 days after IN instillation of bleomycin sulfate. Black arrows represent neutrophil rich infiltrates (a and c, x100; b and d, x400). The results are representative of two experiments (n = 3–5 mice/group).

 
Local but not systemic Ab responses are diminished in IgA–/– mice

In addition to an influx of inflammatory cells into the lungs, murine allergic lung inflammation is typified by production of Th2-associated Abs (IgG1 and IgE). Similar to the decreased influx of inflammatory cells observed in the lungs of IgA–/– mice, local Ab production was abrogated in OVA-sensitized and -challenged IgA–/– mice compared with IgA+/+ mice (Fig. 3a). As expected, OVA-specific IgA production was only observed in IgA+/+ mice. However, levels of total and IgG1 OVA-specific Ab were also significantly (p < 0.05) reduced in BAL fluids of IgA–/– mice compared with IgA+/+ mice. In contrast, total and IgG1 OVA-specific serum Ab levels were comparable in sensitized and challenged IgA+/+ and IgA–/– mice (Fig. 3b). Again, OVA-specific serum IgA was only observed in IgA+/+ mice. These results suggest that immune defects in these mice are limited to the mucosal compartment, the primary site of IgA production.



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  		FIGURE 3. Local but not systemic Ab production is significantly greater in IgA+/+ mice compared with IgA–/– mice. OVA-specific Ab levels were measured by ELISA in BAL fluids (a) and sera (b) obtained from OVA sensitized and challenged IgA+/+ ({triangleup}) and IgA–/–mice ({square}). Total IgE was also quantitated (b). The data are reported as mean absorbance ± SD and are representative of four experiments (n = 6–8 mice/group) (*, p < 0.05).

 
Unlike other isotypes tested, levels of total serum IgE were significantly lower in IgA–/– mice compared with IgA+/+ mice (Fig. 3b). To determine whether decreased production of serum IgE was responsible for the diminished inflammatory response observed in these mice, both groups were sensitized in the presence of IL-4, the switch factor for IgE. Inclusion of IL-4 at the time of sensitization resulted in increased production of total IgE in the sera of IgA–/– mice to a level approximately half of that seen in IgA+/+ mice, without a concurrent increase in inflammation (data not shown). However, IgE levels were still significantly lower than those in IgA+/+ mice and could not be further increased with higher concentrations of IL-4. Thus, although these results suggest that IgE does not influence the development of inflammation in our model, we cannot completely exclude a role for this Ig isotype in enhancing inflammation in IgA+/+ mice. The data also indicate that disruption of the {alpha}-H chain has affected the ability of IgA–/– mice to produce Ig of this isotype. This confirms previous results of Harriman et al. (12), who found that naive IgA–/– mice have significantly lower levels of serum IgE compared with wild-type littermates.

The reduced inflammatory response in IgA–/– mice is due to lack of total IgA not SIgA

To establish whether the abrogated inflammatory response observed in IgA–/– mice is the result of a lack of total IgA or SIgA, we used pIgR–/– mice, which are unable to transcytose IgA across the mucosal epithelium. As a result, these mice lack SIgA but have 100-fold greater levels of serum IgA (32). OVA sensitization and challenge of pIgR+/+ and pIgR–/– mice resulted in a comparable influx of inflammatory cells into the lungs (Fig. 4, a and b, respectively) (pIgR+/+ inflammatory score = 2.33 ± 0.72, n = 12; pIgR–/– inflammatory score = 2.13 ± 0.49, n = 12) and BAL fluids (pIgR+/+ = 3.52 x 105 ± 1.97 x 105 total cells/lung and pIgR–/– = 3.93 x 105 ± 1.56x 105 total cells/lung). In addition, total and IgG1 OVA-specific Ab levels were similar in BAL fluids (Fig. 4c) and sera (Fig. 4d) from both groups of mice, as were serum levels of total IgE (Fig. 4d). The only differences observed between the mice were in levels of OVA-specific BAL and serum IgA, which were significantly higher in pIgR–/– mice compared with pIgR+/+ mice (Fig. 4, c and d). pIgR–/– mice do have some IgA in mucosal surfaces because disruption of the receptor leads to increased paracellular leakage of serum proteins into the mucosal lumen (32). Thus, although these results indicate that SC does not contribute to the development of allergic lung inflammation, we cannot completely exclude a role for IgA Abs in the respiratory tract.



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  		FIGURE 4. SIgA is not required for the development of allergic lung inflammation. a and b, H&E-stained lung sections from OVA-sensitized and -challenged pIgR+/+ (a, x100) and pIgR–/– (b, x100) mice. The black arrows indicate areas of cellular infiltrate. c and d, OVA-specific Ab levels in BAL fluids (c) and sera (d) from pIgR+/+ ({triangleup}) and pIgR–/– ({square}) mice. The data are reported as mean absorbance ± SD and are representative of two independent experiments (n = 6–8 mice/group) (*, p < 0.05).

 
Th2 lung cytokine levels are reduced in IgA–/– mice compared with IgA+/+ mice

Th2 cytokine production is another hallmark of allergic asthma and murine allergic lung inflammation. Accordingly, expression of various cytokines and chemokines (including IL-4, IL-5, IL-10, IL-13, TGF-{beta}, and IFN-{gamma}) were determined over several time points during i.p. sensitization and IN challenge of IgA+/+ and IgA–/– mice. mRNA was isolated from spleens of mice 24 and 48 h after i.p. sensitization and 24 h after days 1, 3, and 5 of IN challenge. Cytokine message levels were then determined by RT-PCR, real-time RT-PCR, and nylon membrane gene arrays. Paradoxically, all cytokine and chemokine mRNA expression levels were similar between IgA+/+ and IgA–/– mice for all time points studied (data not shown), despite significant differences in the inflammatory and Ab responses observed between these groups of mice. However, analysis of protein in BAL fluids revealed significantly higher concentrations of IL-4 and IL-5 in IgA+/+ mice compared with IgA–/– mice at early time points during IN challenge (Fig. 5). These levels correlated with increased inflammation in the lungs of IgA+/+ mice compared with IgA–/– mice at the different time points assayed (data not shown).



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  		FIGURE 5. Th2 cytokine production is greater in the lungs of IgA+/+ mice compared with IgA–/– mice after IN OVA challenge. BAL fluids were isolated from naive mice and 24 h after IN challenge with OVA on days 1, 3, and 5. IL-4 and IL-5 levels were determined by ELISA. The data are reported as mean pg/ml ± SE and are representative of two experiments (n = 3 replicates/mouse, 3 mice/group) (*, p < 0.05).

 
Diminished numbers of B cells are found in the lungs of IgA–/– mice

Whole lung tissue was analyzed for the expression of various leukocyte populations to determine whether the defect in inflammation observed in IgA–/– mice could be associated with a specific cell type. Leukocyte populations were isolated from the lungs of IgA+/+ and IgA–/– mice 24 h after the final IN challenge with OVA and were enumerated by flow cytometry using various cell surface markers (Table I). Although fewer numbers of cells were isolated from the lungs of IgA–/– mice (3.84 x 106 ± 2.39 x 106) compared with IgA+/+ mice (9.44 x 106 ± 1.06; values shown are representative of four experiments (n = 3 mice/group)), the percent expression of each cell population was equivalent, with the exception of B cells. Significantly more B cells were present in the lungs of IgA+/+ mice compared with IgA–/– mice following IN challenge (Table I) (p < 0.05). Furthermore, analysis of naive mice revealed significantly fewer B cells to be present in the lungs of naive IgA–/– mice (12.1 ± 7.6%) compared with naive IgA+/+ mice (22.0 ± 4.9%) (p < 0.05). We then analyzed expression of IgA+, IgM+, and IgG+ B cells in lung cryosections of IgA+/+ and IgA–/– mice 24 h after the final IN challenge with OVA via immunofluorescent tissue staining. Although lung sections from IgA–/– mice were devoid of IgA+ B cells, many IgA+ B cells were detected in IgA+/+ mice (Fig. 6a). IgM staining was limited primarily to dense areas of tissue representing infiltrates, and the level of staining per infiltrate appeared to be similar between both groups of mice (Fig. 6b), although quantitative analysis was not performed. However, because more infiltrates were present in the lungs of IgA+/+ mice, these animals also likely contained more IgM+ cells. However, the most striking observation was the complete lack of IgG+ cells in the lungs of IgA–/– mice. IgG+ cells were prevalent in lung sections from IgA+/+ mice following development of respiratory inflammation (Fig. 6c). Lung sections from IgA–/– mice were almost completely devoid of these cells (Fig. 6c), a finding that correlates with very low levels of IgG in BAL fluids obtained from these mice (Fig. 3a). Thus, disruption of the {alpha}-H chain led to subsequent immunological defects, including reduced production of other isotypes and reduced expression of pulmonary B cells.


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  		Table I. Leukocyte populations in the lungs of IgA+/+ and IgA–/– mice 24 h after the final IN challenge with OVAa

 


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  		FIGURE 6. Expression of IgG+ cells is decreased in the lungs of IgA–/– mice compared with IgA+/+ mice. Lung cryosections were stained with FITC-labeled Ab against mouse IgA (a, x200) or IgM (b, x200) or with Texas red-labeled Ab against mouse IgG (c, x200). The sections were counterstained with 4',6'-diamidino-2-phenylindole (a–c, lower panels). Overlays of 4',6'-diamidino-2-phenylindole-stained images denote placement of cells within tissue sections (a–c, lower panels). The results are representative of images from two experiments (n = 3 mice/group).

 
Ag-specific B cells are required for enhanced inflammation in IgA+/+ mice

To determine whether B cells are required for the enhanced inflammatory response observed in IgA+/+ mice, cell transfer experiments were performed using sensitized splenocytes or CD4+ T cells as described previously (35). Splenocytes were isolated from 14-day sensitized IgA+/+ and IgA–/– mice and cultured with OVA for 72 h. Whole splenocyte populations or CD4+ T cells isolated from cultured splenocytes were then injected i.p. into naive mice. The recipients were challenged IN with OVA, and the lungs were examined by histology for inflammation. Transfer of IgA+/+-sensitized splenocytes into naive IgA+/+ mice resulted in profound inflammation (inflammatory score = 3.25 ± 1.07, n = 8), similar to that seen in directly sensitized and challenged IgA+/+ mice (Fig. 7a). As expected, this was significantly greater (p < 0.05) than the moderate inflammatory response observed in the IgA–/–->IgA–/– cell transfer group (inflammatory score = 1.83 ± 1.00, n = 9) (Fig. 7a). In contrast, transfer of isolated CD4 T cells from sensitized IgA+/+ cells into IgA+/+ recipients resulted in a decreased inflammatory response (inflammatory score = 1.58 ± 0.38, n = 6) comparable to that observed in the IgA–/–->IgA–/– group (inflammatory score = 1.58 ± 0.38, n = 6) (Fig. 7b). This indicates that isolation of CD4+ T cells from whole splenocytes resulted in loss of some cell type responsible for the enhanced inflammatory response observed in IgA+/+ mice. To determine the importance of Ag-specific B cells in this response, whole splenocyte populations depleted of CD19+ cells were transferred into naive mice. Depletion of CD19+ cells from the splenocyte population before transfer resulted in a significantly attenuated inflammatory response in the IgA+/+->IgA+/+ group (inflammatory score = 1.83 ± 0.76, n = 3), similar to that observed in the IgA–/–->IgA–/– group (inflammatory score = 1.67 ± 0.58, n = 3) (Fig. 7c). Differential staining of BAL fluid cells revealed that eosinophil numbers mirrored the inflammatory responses observed by tissue staining (data not shown). Transfer of naive cells followed by IN challenge did not result in the recruitment of inflammatory cells to the lungs of any group of mice. Thus, Ag-specific B cells are required for the enhanced inflammatory response observed in allergen-sensitized and -challenged IgA+/+ mice.



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  		FIGURE 7. Enhanced allergic lung inflammation in IgA+/+ mice requires Ag-specific B cells. a, H&E-stained lung sections (x100) after transfer of sensitized IgA+/+ splenocytes into naive IgA+/+ mice (IgA+/+->IgA+/+) and sensitized IgA–/– splenocytes into naive IgA–/– mice (IgA–/–->IgA–/–). The results are representative of three experiments (n = 3 mice/group). b, H&E-stained lung sections (x100) after transfer of sensitized IgA+/+ CD4+ cells into naive IgA+/+ mice (IgA+/+->IgA+/+) and sensitized IgA–/– CD4+ cells into naive IgA–/– mice (IgA–/–->IgA–/–). Results are representative of two experiments (n = 3 mice/group). c, H&E-stained lung sections (x100) after transfer of sensitized IgA+/+ splenocytes depleted of CD19+ cells into naive IgA+/+ mice (IgA+/+->IgA+/+) and CD19+ cell-depleted sensitized IgA–/– splenocytes into naive IgA–/– mice (IgA–/–->IgA–/–) (n = 3 recipient mice/group). Black arrows indicate inflammatory infiltrates.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Several studies have suggested that IgA plays a role in the pathology of allergic inflammation in asthmatic patients (25, 26, 27, 28, 29, 30, 31). In the present study, using a murine model of allergic lung inflammation, we demonstrate that IgA–/– mice have reduced pulmonary inflammation following sensitization and IN challenge with soluble OVA. However, this defect appears to be linked to the absence of Ag-specific B cells within the pulmonary compartment of IgA–/– mice, rather than a lack of SIgA, as pIgR–/– mice developed inflammation similar to pIgR+/+ mice, although using this model we could not completely exclude a role for IgA Abs that may have transudated from serum into the respiratory tract. An earlier study by Arulanandam et al. (18) showed that the absence of IgA in IgA–/– mice results in defective APC function. Furthermore, Mbawuike et al. (19) demonstrated altered Th1 cell function in IgA–/– mice that might also be attributable to a defect in APC function. The present findings suggest that this defect exists because of a lack of B cells in the lung and that B cells at this site play an important role as APCs.

Sensitization and challenge of IgA–/– mice with OVA resulted in an abrogated inflammatory response compared with IgA+/+ mice, with markedly reduced recruitment of cells to both the tissue and airways, and decreased levels of allergen-specific Ab and Th2 cytokines in BAL fluids. Numbers of eosinophils, the dominant cell type present in allergic lung infiltrates, were significantly diminished in the lungs of IgA–/– mice, likely the result of decreased IL-5 production because IL-5 is a hemopoietic and chemotactic factor for these cells (37). In addition, OVA-specific Ab levels were found to be significantly lower in BAL fluids from IgA–/– mice while serum Ab levels were similar. Ongoing work in our laboratory has demonstrated that Abs found in BAL fluids after IN vaccination are produced by resident pulmonary B cells (D. Albu et al., manuscript in preparation). Thus, decreased levels of allergen-specific Ab in BAL fluids of IgA–/– mice likely reflect a lack of Ag-specific B cells at the site of inflammation, which is consistent with our data demonstrating defective expression of IgG+ B cells in the lungs of these mice.

Total and IgG1 allergen-specific serum Ab levels were similar between both groups of mice, indicating normal systemic B cell responses. However, decreased levels of IgE were observed in IgA–/– mice compared with IgA+/+ mice. Total Ig, rather than IgE Ab, is typically used as a marker for increased isotype production in allergy models because the low level of IgE production coupled with uptake by the high-affinity Fc{epsilon}RI usually renders free IgE Ab undetectable via conventional ELISA. Administration of IL-4, the switch factor for IgE, during sensitization resulted in an increase in total serum IgE production to a level approximately half of that observed in IgA+/+ mice, without a concurrent increase in inflammation, suggesting that IgE is not critical for the development of allergic inflammation in our model. These results corroborate other studies using IgE and mast cell deficient mice, which imply that this Ab isotype and its interaction with mast cells does not contribute to the development of allergic lung inflammation in mice (38, 39). However, as we were unable to induce similar levels of IgE production in IgA+/+ and IgA–/– mice, we cannot fully exclude a role for this isotype in the development of allergic lung inflammation. These data also indicate that disruption of the {alpha}-H chain affects class switching to other isotypes as well. This is in accordance with Harriman et al. (12), who demonstrated significantly lower levels of serum IgE in naive IgA–/– mice compared with IgA+/+ mice. Thus, disruption of IgA leads to a generalized defect in class switching; curiously, this is a phenomenon that is also observed in many IgA-deficient patients and a defect that one would not expect in a system with such a dissimilar etiology from human IgA deficiency (8, 9, 40).

Th2 cytokine levels were significantly lower in IgA–/– mice compared with IgA+/+ mice at early time points during IN challenge with OVA but were similar at later times. This correlates with an earlier influx of inflammatory cells into the lungs of IgA+/+ mice compared with IgA–/– mice, whereas the diminished level of cytokine production during the IN challenge of IgA–/– mice is indicative of the weaker inflammatory response observed in these mice. It is unclear why a discrepancy exists between mRNA transcript and protein levels. There may be a difference in posttranscriptional regulation of cytokine production and/or secretion between IgA+/+ and IgA–/– mice. Alternatively, the isolation of total lung mRNA for transcript analysis, rather than specific cell types, may have muted subtle differences in expression making the ELISA a more sensitive method for detecting differences in cytokine levels between the two groups of mice. In either case, there is a distinct difference in levels of cytokine produced during IN challenge of IgA+/+ and IgA–/– mice.

Bleomycin challenge was used to confirm that the reduced inflammatory response observed in allergen-exposed IgA–/– mice was a deficiency in mounting an Ag-specific immune response. Bleomycin induces inflammation in the lungs and skin due to the lack of the enzyme bleomycin hydrolase in these tissues (41). Although the inflammatory response observed after bleomycin challenge is primarily mediated by neutrophils and macrophages, unlike allergic lung inflammation which involves significant eosinophil infiltration, the resultant response demonstrated that IgA–/– mice are capable of mounting pulmonary inflammation. However, the recruitment of significantly more inflammatory cells to the lungs of IgA–/– mice relative to IgA+/+ mice indicates that IgA and/or mucosal B cells play very different roles in mediating inflammation induced by different stimuli. IgA is widely considered to be an anti-inflammatory Ab at mucosal sites, where it is involved in the neutralization and clearance of bacterial, viral, and other Ags (5). However, as administration of bleomycin sulfate does not induce Ag-specific Abs, any anti-inflammatory effect of IgA in this system would not occur through neutralization but through an, as of yet, unknown mechanism.

Analysis of pulmonary leukocyte populations in IgA+/+ and IgA–/– mice revealed significantly fewer B lymphocytes in the lungs of IgA–/– mice in the presence or absence of allergic lung inflammation. In addition, immunofluorescent tissue staining demonstrated a complete absence of IgG staining cells in lung sections from IgA–/– mice following the induction of allergic lung inflammation, whereas many IgG+ cells were present in the lungs of IgA+/+ mice. This finding correlates with the negligible amounts of IgG1 found in the airways, although it is unclear why disruption of the {alpha}-H chain in IgA–/– mice results in a lack of IgG+ B cell expression in the lungs. In a recent study, Uren et al. (42) also described disrupted mucosal B cell homeostasis in pIgR–/– mice, which contain 100-fold greater levels of serum IgA than pIgR+/+ mice. However, the altered B cell homeostasis manifested in this case as an increased number of IgA+ plasmablasts in the lamina propria and spleen (42); the mechanism for this defect was not determined. As IgA–/– mice have decreased numbers of B cells at mucosal sites, it is likely that IgA production influences B cell homeostasis; the mechanism(s) responsible for this defect are currently being investigated.

Cell transfer studies were performed in an effort to determine whether the enhanced inflammatory response observed in IgA+/+ mice compared with IgA–/– mice is dependent upon the presence of Ag-specific B cells. Transfer of whole splenocyte populations from IgA+/+->IgA+/+ mice resulted in a profound inflammatory response that was visibly more severe than the response seen in IgA–/–->IgA–/– mice. However, removal of Ag-specific B cells from the transferred population, either by isolating CD4+ T cells or by depleting CD19+ B cells, resulted in a loss of the response observed in IgA+/+ mice. Transfer of purified CD19+ B cells was not performed as splenocyte populations depleted of CD4+ cells have been shown previously to be incapable of transferring disease to naive animals (35). Unfortunately, we could not perform IgA+/+->IgA–/– and IgA–/–->IgA+/+ cell transfers because the IgA–/– mice are not on a pure inbred background. It is not clear what role B cells play in the response, i.e., if they enhance the response through local Ab production and/or whether they function as APCs. B cells are potent Ag-specific APCs, especially for soluble protein Ags and/or in cases of limited Ag concentrations (43, 44, 45, 46, 47). They can rapidly take up, catabolize, and present Ags and have been shown to play an important role in optimizing Ag presentation to T cells (44, 47). Furthermore, lack of B cells during presentation has been shown to result in reduced T cell responses (44, 45, 47, 48, 49). Dendritic cells (DCs) are likely responsible for the limited inflammatory response observed in IgA–/– mice; however, the lack of adjuvant during IN challenge in this model would cause suboptimal DC activation and maturation. As it has been reported that alveolar macrophages are poor APCs (50), B cells may be a critical APC population within the respiratory tract. This would explain previous data demonstrating that IN immunization of IgA–/– mice with vaccine + adjuvant resulted in protective immunity, whereas vaccination of these mice in the absence of a DC-stimulating adjuvant resulted in a loss of protection (18). We have found that equivalent numbers of IgA–/– and IgA+/+ CD45+ APC populations can stimulate similar levels of cell proliferation and cytokine production cells by CD4+ T cells purified from OT-II mice, which are specific for OVA (our unpublished observations). Thus, there is no inherent defect in the ability IgA–/– B cells to present Ags; rather, our data indicate that reduced B cell numbers in the lungs of IgA–/– mice leads to defective APC activity. Under normal conditions, such as in IgA+/+ mice, Ag-specific B cells likely traffic through mucosal effecter sites, including the lungs, where they encounter their cognate Ags and, with DCs, initiate activation of memory T cells.

It has been reported previously, using B cell-deficient (µMT) mice, that B cells do not contribute to the induction of murine allergic lung inflammation. However, it should be recognized that µMT mice are not fully deficient in B cells. Several studies have demonstrated the presence of IgA-, IgG-, and IgE-producing B cells in the absence of membrane IgM and IgD expression (51, 52, 53). Furthermore, Macpherson et al. (51) demonstrated that a significant number of IgA+ cells are present in an immune effector site, i.e., the lamina propria of µMT mice. In addition, it has been suggested that B cells are critical for normal secondary organ lymphorganogenesis (45, 54, 55, 56). Lack of B cells during development may result in the formation of compensatory mechanisms that might not occur when only a subpopulation of B cells is missing. Evidence for this comes from experiments by Rivera et al. (45), demonstrating that the ability of µMT APCs to induce functional immunity is profoundly reduced when these cells were placed in a normally structured immune system by reconstituting irradiated C57BL/6 mice with µMT bone marrow. This suggests that the lack of B cells during development in µMT mice resulted in formation of a compensatory pathway that allows adequate APC function and that such a mechanism was not functional in a wild-type environment (45). In addition, while some studies using B cell-deficient mice found that B cells do not contribute to the development of normal immune responses, others have demonstrated defects in CD4+ T cell function generated in the absence of B cells (43, 44, 45, 46, 48, 49, 57). It is possible that differences in immunization protocols, the types of Ags and adjuvants used (43), as well as the background strain of mice (45), are partially responsible for the discrepancies in experimental findings between different groups.

We believe that the lack of mucosal B cells and deficient allergic lung inflammation observed in IgA–/– mice correlates with the previously reported defect in APC function in these mice (18). In that study, Arulanandam et al. (18) noted that a defect in APC function was observed when attempting to elicit protection from lethal influenza challenge via IN immunization with a protein subunit vaccine. However, the defect was not observed when mice were immunized with the vaccine plus a strong adjuvant (IL-12). Similarly, IgA–/– mice do not have an increased susceptibility to infection with influenza virus (13, 19, 58), HSV-2 (16), Shigella flexneri (17), or Helicobacter pylori (15), following immunization with a strong adjuvant or attenuated bacteria/viruses. However, under such conditions, DCs and macrophages would be adequately stimulated to present Ags to T cells, diminishing the need for B cells to function as APCs. In the absence of DC-activating adjuvants, such as in the allergic lung inflammation model used here, B cells may play an essential role in enhancing immune responses. Our findings imply that in the absence of Ag-specific B cells at mucosal sites, there is a need for DC-activating adjuvants for efficacious immunization. It has been reported that some IgA-deficient humans also have defects in mucosal B cell numbers (59, 60); it will be of interest to determine whether such a defect is responsible for the increased incidence of infectious sinopulmonary disease observed in these individuals (8, 9).


   Acknowledgments
 
We thank Dr. Maria Lopez for her guidance in operating the flow cytometer and evaluating flow cytometry data and the Immunology Core of the Center for Immunology and Microbial Disease for histology services.


   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 was supported by National Institutes of Health Grant AI41715 and by Philip Morris USA, Inc., and by Philip Morris International. P.M.A. was supported by National Institutes of Health Training Grant AI49822. Back

2 Address correspondence and reprint requests to Dr. Dennis Metzger, Center for Immunology and Microbial Disease, Albany Medical College, Mail Code 151, 47 New Scotland Avenue, Albany, NY 12208. E-mail address: metzged{at}mail.amc.edu Back

3 Abbreviations used in this paper: pIgR, polymeric IgR; SC, secretory component; SIgA, secretory IgA; BAL, bronchoalveolar lavage; IN, intranasal; PV, perivascular; CN–EPO, cyanide-resistant eosinophil peroxidase; DC, dendritic cell. Back

Received for publication March 22, 2005. Accepted for publication May 12, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Mestecky, J., I. Moro, M. Kerr, J. M. Woof. 2005. Mucosal immunoglobulins. J. Mestecky, and M. E. Lamm, and W. Strober, and J. Bienenstock, and J. R. McGhee, and L. Mayer, eds. In Mucosal Immunology Vol. 1: 153-181 Academic Press, San Diego. .
  2. Johansen, F. E., R. Braathen, P. Brandtzaeg. 2000. Role of J chain in secretory immunoglobulin formation. Scand. J. Immunol. 52: 240-248.[Medline]
  3. van Egmond, M., C. A. Damen, A. B. van Spriel, G. Vidarsson, E. van Garderen, J. G. van de Winkel. 2001. IgA and the IgA Fc receptor. Trends Immunol. 22: 205-211.[Medline]
  4. Phalipon, A., B. Corthesy. 2003. Novel functions of the polymeric Ig receptor: well beyond transport of immunoglobulins. Trends Immunol. 24: 55-58.[Medline]
  5. Russell, M. W., M. Kilian. 2005. Biological activities of IgA. J. Mestecky, and M. E. Lamm, and W. Strober, and J. Bienenstock, and J. R. McGhee, and L. Mayer, eds. Mucosal Immunology 267-289 Academic Press, San Diego. .
  6. Hodge, L. M., M. Marinaro, H. P. Jones, J. R. McGhee, H. Kiyono, J. W. Simecka. 2001. Immunoglobulin A (IgA) responses and IgE-associated inflammation along the respiratory tract after mucosal but not systemic immunization. Infect. Immun. 69: 2328-2338.[Abstract/Free Full Text]
  7. Lycke, N., L. Erlandsson, L. Ekman, K. Schon, T. Leanderson. 1999. Lack of J chain inhibits the transport of gut IgA and abrogates the development of intestinal antitoxic protection. J. Immunol. 163: 913-919.[Abstract/Free Full Text]
  8. Burrows, P. D., M. D. Cooper. 1997. IgA deficiency. Adv. Immunol. 65: 245-276.[Medline]
  9. Cunningham-Rundles, C.. 2001. Physiology of IgA and IgA deficiency. J. Clin. Immunol. 21: 303-309.[Medline]
  10. Monteiro, R. C., J. G. J. van de Winkel. 2003. IgA Fc receptors. Annu. Rev. Immunol. 21: 177-204.[Medline]
  11. Abu-Ghazaleh, R. I., T. Fujisawa, J. Mestecky, R. A. Kyle, G. J. Gleich. 1989. IgA-induced eosinophil degranulation. J. Immunol. 142: 2393-2400.[Abstract]
  12. Harriman, G. R., M. Bogue, P. Rogers, M. Finegold, S. Pacheco, A. Bradley, Y. Zhang, I. N. Mbawuike. 1999. Targeted deletion of the IgA constant region in mice leads to IgA deficiency with alterations in expression of other Ig isotypes. J. Immunol. 162: 2521-2529.[Abstract/Free Full Text]
  13. Mbawuike, I. N., S. Pacheco, C. L. Acuna, K. C. Switzer, Y. Zhang, G. R. Harriman. 1999. Mucosal immunity to influenza without IgA: an IgA knockout mouse model. J. Immunol. 162: 2530-2537.[Abstract/Free Full Text]
  14. O’Neal, C. M., G. R. Harriman, M. E. Conner. 2000. Protection of the villus epithelial cells of the small intestine from rotavirus infection does not require immunoglobulin A. J. Virol. 74: 4102-4109.[Abstract/Free Full Text]
  15. Blanchard, T. G., S. J. Czinn, R. W. Redline, N. Sigmund, G. Harriman, J. G. Nedrud. 1999. Antibody-independent protective mucosal immunity to gastric Helicobacter infection in mice. Cell. Immunol. 191: 74-80.[Medline]
  16. Parr, M., G. R. Harriman, E. Parr. 1998. Immunity to vaginal HSV-2 infection in immunoglobulin A knockout mice. Immunology 95: 208-213.[Medline]
  17. Way, S. S., A. C. Borczuk, M. B. Goldberg. 1999. Adaptive immune response to Shigella flexneri 2a cydC in immunocompetent mice and mice lacking immunoglobulin A. Infect. Immun. 67: 2001-2004.[Abstract/Free Full Text]
  18. Arulanandam, B. P., R. H. Raeder, J. G. Nedrud, D. J. Bucher, J. Le, D. W. Metzger. 2001. IgA immunodeficiency leads to inadequate Th cell priming and increased susceptibility to influenza virus infection. J. Immunol. 166: 226-231.[Abstract/Free Full Text]
  19. Zhang, Y., S. Pacheco, C. L. Acuna, K. C. Switzer, Y. Wang, X. Gilmore, G. R. Harriman, I. N. Mbawuike. 2002. Immunoglobulin A-deficient mice exhibit altered T helper 1-type immune responses but retain mucosal immunity to influenza virus. Immunology 105: 286-294.[Medline]
  20. Burks, A. W., Jr, R. W. Steele. 1986. Selective IgA deficiency. Ann. Allergy 57: 3-13.[Medline]
  21. Ostergaard, P. A.. 1980. Clinical and immunological features of transient IgA deficiency in children. Clin. Exp. Immunol. 40: 561-565.[Medline]
  22. Van Asperen, P. P., M. Gleeson, A. S. Kemp, A. W. Cripps, S. B. Geraghty, C. M. Mellis, R. L. Clancy. 1985. The relationship between atopy and salivary IgA deficiency in infancy. Clin. Exp. Immunol. 62: 753-757.[Medline]
  23. Plebani, A., V. Monafo, A. G. Ugazio, C. Monti, M. A. Avanzini, P. Massimi, G. R. Burgio. 1987. Comparison of the frequency of atopic diseases in children with severe and partial IgA deficiency. Int. Arch. Allergy Appl. Immunol. 82: 485-486.[Medline]
  24. Brasher, G. W., P. D. Bourland. 1975. The role of IgA in the pathogenesis of atopy. Ann. Allergy 34: 137-140.[Medline]
  25. Kitani, S., K. Ito, T. Miyamoto. 1985. IgG, IgA, and IgM antibodies to mite in sera and sputa from asthmatic patients. Ann. Allergy 55: 612-620.[Medline]
  26. Nahm, D. H., H. Y. Kim, H. S. Park. 1998. Elevation of specific immunoglobulin A antibodies to both allergen and bacterial antigen in induced sputum from asthmatics. Eur. Respir. J. 12: 540-545.[Abstract]
  27. Reed, C. E., M. Bubak, S. Dunnette, J. Blomgren, M. Pfenning, P. Wentz-Murtha, N. Wallen, M. Keating, G. J. Gleich. 1991. Ragweed-specific IgA in nasal lavage fluid of ragweed-sensitive allergic rhinitis patients: increase during the pollen season. Int. Arch. Allergy Appl. Immunol. 94: 275-277.[Medline]
  28. Monteiro, R. C., R. W. Hostoffer, M. D. Cooper, J. R. Bonner, G. L. Gartland, H. Kubagawa. 1993. Definition of immunoglobulin A receptors on eosinophils and their enhanced expression in allergic individuals. J. Clin. Invest. 92: 1681-1685.
  29. Peebles, R. S., Jr, M. C. Liu, N. F. Adkinson, Jr, L. M. Lichtenstein, R. G. Hamilton. 1998. Ragweed-specific antibodies in bronchoalveolar lavage fluids and serum before and after segmental lung challenge: IgE and IgA associated with eosinophil degranulation. J. Allergy Clin. Immunol. 101: 265-273.[Medline]
  30. Nahm, D. H., H. S. Park. 1997. Correlation between IgA antibody and eosinophil cationic protein levels in induced sputum from asthmatic patients. Clin. Exp. Allergy 27: 676-681.[Medline]
  31. Peebles, R. S., Jr, R. G. Hamilton, L. M. Lichtenstein, M. Schlosberg, M. C. Liu, D. Proud, A. Togias. 2001. Antigen-specific IgE and IgA antibodies in bronchoalveolar lavage fluid are associated with stronger antigen-induced late phase reactions. Clin. Exp. Allergy 31: 239-248.[Medline]
  32. Johansen, F. E., M. Pekna, I. N. Norderhaug, B. Haneberg, M. A. Hietala, P. Krajci, C. Betsholtz, P. Brandtzaeg. 1999. Absence of epithelial immunoglobulin A transport, with increased mucosal leakiness, in polymeric immunoglobulin receptor/secretory component-deficient mice. J. Exp. Med. 190: 915-922.[Abstract/Free Full Text]
  33. Alouani, S., P. Juillard, Y. Chvatchko. 2000. Murine model of allergic lung inflammation. Methods Mol. Biol. 138: 285-293.[Medline]
  34. Horton, M. A., K. A. Larson, J. J. Lee, N. A. Lee. 1996. Cloning of the murine eosinophil peroxidase gene (mEPO): characterization of a conserved subgroup of mammalian hematopoietic peroxidases. J. Leukocyte Biol. 60: 285-294.[Abstract]
  35. Wise, J. T., T. J. Baginski, J. L. Mobley. 1999. An adoptive transfer model of allergic lung inflammation in mice is mediated by CD4+CD62LlowCD25+ T cells. J. Immunol. 162: 5592-5600.[Abstract/Free Full Text]
  36. Kamath, A. T., C. E. Sheasby, D. F. Tough. 2005. Dendritic cells and NK cells stimulate bystander T cell activation in response to TLR agonists through secretion of IFN-{alpha}{beta} and IFN-{gamma}. J. Immunol. 174: 767-776.[Abstract/Free Full Text]
  37. Foster, P. S., S. P. Hogan, M. Yang, J. Mattes, I. G. Young, K. I. Matthaei, R. K. Kumar, S. Mahalingam, D. C. Webb. 2002. Interleukin-5 and eosinophils as therapeutic targets for asthma. Trends Mol. Med. 8: 162-167.[Medline]
  38. Mehlhop, P. D., M. van de Rijn, A. B. Goldberg, J. P. Brewer, V. P. Kurup, T. R. Martin, H. C. Oettgen. 1997. Allergen-induced bronchial hyperreactivity and eosinophilic inflammation occur in the absence of IgE in a mouse model of asthma. Proc. Natl. Acad. Sci. USA 94: 1344-1349.[Abstract/Free Full Text]
  39. Takeda, K., E. Hamelmann, A. Joetham, L. D. Shultz, G. L. Larsen, C. G. Irvin, E. W. Gelfand. 1997. Development of eosinophilic airway inflammation and airway hyperresponsiveness in mast cell-deficient mice. J. Exp. Med. 186: 449-454.[Abstract/Free Full Text]
  40. Asano, T., H. Kaneko, T. Terada, Y. Kasahara, T. Fukao, K. Kasahara, N. Kondo. 2004. Molecular analysis of B cell differentiation in selective or partial IgA deficiency. Clin. Exp. Immunol. 136: 284-290.[Medline]
  41. Sleijfer, S.. 2001. Bleomycin-induced pneumonitis. Chest 120: 617-624.[Abstract/Free Full Text]
  42. Uren, T. K., F. E. Johansen, O. L. Wijburg, F. Koentgen, P. Brandtzaeg, R. A. Strugnell. 2003. Role of the polymeric Ig receptor in mucosal B cell homeostasis. J. Immunol. 170: 2531-2539.[Abstract/Free Full Text]
  43. Constant, S., N. Schweitzer, J. West, P. Ranney, K. Bottomly. 1995. B lymphocytes can be competent antigen-presenting cells for priming CD4+ T cells to protein antigens in vivo. J. Immunol. 155: 3734-3741.[Abstract]
  44. Linton, P. J., J. Harbertson, L. M. Bradley. 2000. A critical role for B cells in the development of memory CD4 cells. J. Immunol. 165: 5558-5565.[Abstract/Free Full Text]
  45. Rivera, A., C. C. Chen, N. Ron, J. P. Dougherty, Y. Ron. 2001. Role of B cells as antigen-presenting cells in vivo revisited: antigen-specific B cells are essential for T cell expansion in lymph nodes and for systemic T cell responses to low antigen concentrations. Int. Immunol. 13: 1583-1593.[Abstract/Free Full Text]
  46. Kurt-Jones, E. A., D. Liano, K. A. HayGlass, B. Benacerraf, M. S. Sy, A. K. Abbas. 1988. The role of antigen-presenting B cells in T cell priming in vivo: studies of B cell-deficient mice. J. Immunol. 140: 3773-3778.[Abstract]
  47. Byersdorfer, C. A., R. J. Dipaolo, S. J. Petzold, E. R. Unanue. 2004. Following immunization antigen becomes concentrated in a limited number of APCs including B cells. J. Immunol. 173: 6627-6634.[Abstract/Free Full Text]
  48. Langhorne, J., C. Cross, E. Seixas, C. Li, T. von der Weid. 1998. A role for B cells in the development of T cell helper function in a malaria infection in mice. Proc. Natl. Acad. Sci. USA 95: 1730-1734.[Abstract/Free Full Text]
  49. Homann, D., A. Tishon, D. P. Berger, W. O. Weigle, M. G. von Herrath, M. B. Oldstone. 1998. Evidence for an underlying CD4 helper and CD8 T cell defect in B cell-deficient mice: failure to clear persistent virus infection after adoptive immunotherapy with virus-specific memory cells from muMT/muMT mice. J. Virol. 72: 9208-9216.[Abstract/Free Full Text]
  50. Leemans, J. C., T. Thepen, S. Weijer, S. Florquin, N. van Rooijen, J. G. van de Winkel, T. van der Poll. 2005. Macrophages play a dual role during pulmonary tuberculosis in mice. J. Infect. Dis. 191: 65-74.[Medline]
  51. Macpherson, A. J., A. Lamarre, K. McCoy, G. R. Harriman, B. Odermatt, G. Dougan, H. Hengartner, R. M. Zinkernagel. 2001. IgA production without µ or {delta} chain expression in developing B cells. Nat. Immunol. 2: 625-631.[Medline]
  52. Orinska, Z., A. Osiak, J. Lohler, E. Bulanova, V. Budagian, I. Horak, S. Bulfone-Paus. 2002. Novel B cell population producing functional IgG in the absence of membrane IgM expression. Eur. J. Immunol. 32: 3472-3480.[Medline]
  53. Seagal, J., E. Edry, H. Naftali, D. Melamed. 2004. Generation and selection of an IgG-driven autoimmune repertoire during B-lymphopoiesis in Ig micro-deficient/lpr mice. Int. Immunol. 16: 905-913.[Abstract/Free Full Text]
  54. Golovkina, T. V., M. Shlomchik, L. Hannum, A. Chervonsky. 1999. Organogenic role of B lymphocytes in mucosal immunity. Science 286: 1965-1968.[Abstract/Free Full Text]
  55. Fu, Y. X., D. D. Chaplin. 1999. Development and maturation of secondary lymphoid tissues. Annu. Rev. Immunol. 17: 399-433.[Medline]
  56. Tumanov, A. V., D. V. Kuprash, M. A. Lagarkova, S. I. Grivennikov, K. Abe, A. N. Shakhov, L. N. Drutskaya, C. L. Stewart, A. V. Chervonsky, S. A. Nedospasov. 2002. Distinct role of surface lymphotoxin expressed by B cells in the organization of secondary lymphoid tissues. Immunity 17: 239-250.[Medline]
  57. Liu, Y., Y. Wu, L. Ramarathinam, Y. Guo, D. Huszar, M. Trounstine, M. Zhao. 1995. Gene-targeted B-deficient mice reveal a critical role for B cells in the CD4 T cell response. Int. Immunol. 7: 1353-1362.[Abstract/Free Full Text]
  58. Benton, K. A., J. A. Misplon, C. Y. Lo, R. R. Brutkiewicz, S. A. Prasad, S. L. Epstein. 2001. Heterosubtypic immunity to influenza A virus in mice lacking IgA, all Ig, NKT cells, or {gamma}{delta} T cells. J. Immunol. 166: 7437-7445.[Abstract/Free Full Text]
  59. Brandtzaeg, P., G. Karlsson, G. Hansson, B. Petruson, J. Bjorkander, L. A. Hanson. 1987. The clinical condition of IgA-deficient patients is related to the proportion of IgD- and IgM-producing cells in their nasal mucosa.