Precursor B Cells Increase in the Lung during Airway Allergic Inflammation: A Role for B Cell-Activating Factor

<div><p>Background</p><p>B cells, key cells in allergic inflammation, differentiate in the bone marrow and their precursors include pro-B, pre-B and immature B cells. Eosinophil progenitor cells increase in the lung after allergen exposure. However, the existence and possible role of B cell precursors in the lung during allergic inflammation remains elusive.</p><p>Methods</p><p>A BALB/c mouse model of allergic airway inflammation was utilized to perform phenotypic and quantification analyses of pro-B and pre-B cells in the lung by flow cytometry. B cell maturation factors IL-7 and B cell-activating factor (BAFF) and their receptors (CD127 and BAFFR, BCMA, TACI, respectively) were also evaluated in the lung and serum. The effect of anti-BAFF treatment was investigated both <i>in vivo</i> (<i>i</i>.<i>p</i>. administration of BAFF-R-Ig fusion protein) and <i>in vitro</i> (colony forming cell assay). Finally, BAFF levels were examined in the bronchoalveolar lavage (BAL) of asthmatic patients and healthy controls.</p><p>Results</p><p>Precursor pro and pre-B cells increase in the lung after allergen exposure, proliferate in the lung tissue <i>in vivo</i>, express markers of chemotaxis (CCR10 and CXCR4) and co-stimulation (CD40, CD86) and are resistant to apoptosis (Bax). Precursor B cells express receptors for BAFF at baseline, while after allergen challenge both their ligand BAFF and the BCMA receptor expression increases in B cell precursors. Blocking BAFFR in the lung <i>in vivo</i> decreases eosinophils and proliferating precursor B cells. Blocking BAFFR in bone marrow cultures <i>in vitro</i> reduces pre-B colony formation units. BAFF is increased in the BAL of severe asthmatics.</p><p>Conclusion</p><p>Our data support the concept of a BAFF-mediated role for B cell precursors in allergic airway inflammation.</p></div

BAFF levels and BAFF receptors’ expression (BAFFR, BCMA, TACI) in B precursors BAFFR expression in the lung during allergic inflammation.

<p>(a) BAFF levels are increased during allergen exposure in the lung. (b) Total B cells (B220+SSC^low), pre/early pro-B (B220⁺CD43⁺BP-1^-) and pre-B cells (B220⁺CD43^-BP^-1⁺) express BAFFR and its expression increases in total B cells after allergen exposure. (c) Pre/early pro-B and pre-B cells express the BAFF receptor BCMA and its expression increases significantly in both B cell precursor populations. (d) Pre/early pro-B and pre-B cells also express the BAFF receptor TACI, however, no change in its expression is observed after allergen challenge. Data are presented as mean ± SEM, n = 5–8 mice/group, * <i>p</i> < 0.05, BAFF: B cell-activating factor, BAFFR: BAFF receptor, BCMA: B cell maturation antigen, TACI: transmembrane activator and calcium modulator and cyclophilin ligand interactor, % refers to the percentage of positive cells out of total cells of each population.</p

B cells precursors in the lung express markers of chemotaxis and functional activity.

<p>(a) CXCR4 expression is increased in pre-B cells (B220⁺CD43^-BP^-1⁺) compared to pre/early pro-B cells (B220⁺CD43⁺BP-1^-) and total B cells (B220+SSC^low). (b) CCR10 expression is increased in total B cells and pre-early pro-B cell precursors after allergen challenge. (c) CD86 and (d) CD40 activation markers are increased in pre-B cells in allergen challenged mice compared to controls and CD40 is increased in pre/early pro-B cells compared to total B cells. Data are presented as mean ± SEM, % refers to the percentage of positive cells out of total cells of each population, n = 5–8 mice/group, * <i>p</i> < 0.05.</p

Effects of <i>in vivo</i> and <i>in vitro</i> blocking of BAFF in the lung.

<p>Administration of BAFF-R-Ig fusion protein <i>in vivo</i> in OVA sensitized and challenged mice resulted in decreased eosinophilic inflammation in the a) BAL fluid and b) lung tissue, compared to its control protein, c) while the total number of lung B cells or d) B cell precursor populations remained unchanged. e) However, blocking BAFF resulted in decreased numbers of pre/early pro-B and pre-B cells undergoing proliferation in the lung. Data are presented as mean ± SEM, * p < 0.05, *** p < 0.001, n = 6–8 per group.</p

BAFF levels are increased in the BALF of patients with severe asthma.

<p>BAFF levels were evaluated in the BALF of patients with asthma of varying severity and healthy control volunteers. Data are presented as mean ± SEM, * <i>p</i> < 0.05, BAFF: B cell-activating factor, BALF: bronchoalveolar lavage fluid, SA: severe asthmatics, MMA: mild-moderate asthmatics.</p

Lung and serum IL-7 and B precursors IL-7R expression during allergic inflammation.

<p>IL-7 levels in the a) lung and b) serum are not affected by allergen exposure. c) Expression of IL-7R was decreased upon allergic exposure. d) No differences were found in the intensity of the expression of the IL-7R in total B cells (B220+SSC^low) and pre-B cells (B220⁺CD43^-BP^-1⁺), while in pre/early pro-B cells (B220⁺CD43⁺BP-1^-) it was decreased during allergic inflammation. Data are presented as mean ± SEM, IL-7: interleukin-7, IL7R: interleukin-7 receptor, % refers to the percentage of positive cells out of total cells of each population, ns: not significant, n = 5–8 mice/group, * <i>p</i> < 0.05.</p

BALB/c mouse model of allergic airway inflammation.

<p>Schedule of OVA sensitization (8 μg each <i>i</i>.<i>p</i>.), OVA exposure (100 μg OVA in 25 μl PBS <i>i</i>.<i>n</i>. instillation each), BrdU administration (1 mg each <i>i</i>.<i>p</i>.) and tissue sampling in this model of allergic airway inflammation. All animals were sensitized to allergen, but control animals were exposed to PBS instead of OVA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161161#pone.0161161.ref013" target="_blank">13</a>]. In another set of experiments designed to evaluate the role of BAFF, OVA sensitized mice received <i>i</i>.<i>n</i>. BAFF-R-Ig fusion chimeric protein (7 μg), which binds to its receptor (BAFFR) and blocks its action, or its isotype control protein, one hour prior to OVA exposure on five consecutive days, as indicated by green arrows.</p

Establishment of allergic airway inflammation.

<p>(a) Total BAL fluid cell numbers were increased after OVA exposure. (b) BAL fluid eosinophils as a percentage of total BAL cells were increased after OVA/OVA exposure as compared to OVA/PBS exposure, while macrophages were decreased. (c) Total B cells (B220⁺SSC^low), newly produced B cells during allergen exposure (B220⁺SSC^lowBrdU⁺) and immature B cells (B220⁺SSC^lowCD93⁺) in various biological compartments harvested upon allergen exposure. B cell precursor population significantly decreases in the BM after OVA exposure, and increases at the same time in the spleen, the lung and the BAL fluid. Data are presented as mean ± SEM, n = 5–8 mice/group, * <i>p</i> < 0.05, <i>i</i>.<i>p</i>.: intraperitoneally, <i>i</i>.<i>n</i>.: intranasally, <i>i</i>.<i>c</i>.: isotype control, ND: not detectable.</p

Effects of <i>in vitro</i> blocking of BAFF on B cell precursor development.

<p>Blocking BAFF in an <i>in-vitro</i> model of bone marrow cell culture causes developmental arrest, as shown by the decreased number of pre-B cells colony forming units. Data are presented as mean ± SEM, * <i>p</i> < 0.05, results are representative of two independent experiments. BAFF: B cell-activating factor, I.C.: isotype control.</p

B cells precursors in the lung express markers of resistance to apoptosis and proliferation.

<p>(a) The apoptosis regulator Bax is downregulated in pre/early pro-B cells (B220⁺CD43⁺BP-1^-) upon allergen exposure. (b) Newly produced total B (B220+SSClow), pre/early pro-B and pre-B cells (B220⁺CD43^-BP^-1⁺) increase in the lung upon allergen exposure, as designated by increased expression of the thymidine analogue BrdU (5-bromo-29-deoxyuridine). (c) Pre/early pro-B cells and pre-B cells proliferate <i>in-situ</i> during allergen exposure. Data are presented as mean ± SEM, % refers to the percentage of positive cells out of total cells of each population, n = 5–8 mice/group, * <i>p</i> < 0.05.</p