Airway responses to antigen or methacholine are largely MC dependent.
In our chronic asthma model, OVA-sensitized wild-type (Kit+/+) mice exhibited increased enhanced respiratory pause (Penh) responses to aerosolized methacholine administered 24 hours after the eighth OVA challenge compared with responses in PBS-treated WBB6F1-Kit+/+ mice or OVA- or PBS-treated MC-deficient WBB6F1-KitW/W-v mice (Figure A). However, WBB6F1-KitW/W-v mice that had been selectively engrafted with WBB6F1-Kit+/+ bone marrow–derived cultured MCs (BMCMCs) (Kit+/+ BMCMCs→KitW/W-v mice) exhibited responses that were statistically indistinguishable from those in Kit+/+ mice (Figure A). Very similar results were obtained when such experiments were repeated using C57BL/6-Kit+/+, C57BL/6-KitW-sh/W-sh, and C57BL/6-Kit+/+ BMCMCs→KitW-sh/W-shmice (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI25702DS1).
Airway responses following i.n. OVA challenge in a mouse model of chronic asthma.
Thus, in this model, airway responses to methacholine appeared to be largely or entirely MC-dependent in either WB × C57BL/6 F1
) or C57BL/6 mice. Indeed, the role of MCs in the enhancement of Penh responses to methacholine in this chronic model appeared to be even greater than that which we reported in a more acute model of asthma, which also was elicited in mice that had been sensitized to OVA in the absence of artificial adjuvant (33
We also measured airway responses to antigen or PBS. Individual OVA-sensitized Kit+/+ or Kit+/+ BMCMCs→KitW/W-v mice exhibited a biphasic response to the ninth OVA challenge, with peaks of responsiveness at approximately 3 or 6 hours and at approximately 9 or 12 hours; the responses were substantially reduced by 24 hours after OVA challenge (Supplemental Figure 2). When data from individual mice were pooled, the biphasic response to OVA was still evident in OVA-sensitized Kit+/+ mice but not in OVA-sensitized Kit+/+ BMCMCs→KitW/W-v mice (Figure B). However, peak airway responses in these 2 groups were quite similar in magnitude. In contrast, there were no detectable responses to OVA in OVA-sensitized KitW/W-v mice, nor did any group of PBS-treated mice exhibit a response to PBS challenge (Figure B). Very similar results were obtained when the same experiments were performed in C57BL/6-Kit+/+, C57BL/6-KitW-sh/W-sh, and C57BL/6-Kit+/+ BMCMCs→KitW-sh/W-shmice (Supplemental Figure 1B).
Thus, airway responses induced by OVA challenge in OVA-sensitized KitW/W-vor KitW-sh/W-shmice, like Penh responses to methacholine after OVA challenge in such mice, were largely or entirely MC dependent.
To assess specifically airway responses distal to the trachea, we also performed invasive measurements of lung resistance (RL) and dynamic compliance (Cdyn) in mice treated with aerosolized methacholine 24 hours after the ninth challenge with OVA or PBS. OVA-sensitized Kit+/+ or Kit+/+ BMCMCs→KitW/W-v mice exhibited similar levels of AHR after OVA challenge whereas the OVA-sensitized MC-deficient KitW/W-v mice showed responses that were statistically indistinguishable from those of the PBS-treated control groups (Figure , C and D). Thus, our findings using invasive measurements of lung function to assess AHR in anesthetized, tracheostomized mice are basically in accord with those we obtained by using Penh to assess airway responses in conscious mice.
Antibody responses in this model are MC independent.
MCs can be activated by antigen to secrete mediators by both IgE/FcεRI- and IgG/FcγRIII-dependent mechanisms (14
). Such secreted products include cytokines that can regulate the production of IgE and other antibodies (23
). MCs can also express other functions that have the potential to influence the development of adaptive immune responses (23
). Accordingly, it has been proposed that MCs might function in part to enhance levels of antibody production during some acquired immune responses (23
We found that OVA sensitization resulted in the development of significant antigen-specific IgE and IgG1 responses, but the levels of these antibodies measured at the end of our experiments in groups of OVA-sensitized Kit+/+, KitW/W-v, or Kit+/+ BMCMCs→KitW/W-v mice were statistically indistinguishable (Supplemental Figure 3). Thus, the differences in airway responses to OVA or in the AHR to methacholine among OVA-sensitized Kit+/+, KitW/W-v, or Kit+/+ BMCMCs→KitW/W-v mice cannot be explained by differences in levels of OVA-specific IgE or IgG1 antibodies.
This asthma model is associated with increased numbers of MCs and MC-dependent lung inflammation.
OVA treatment resulted in significant increases in the numbers of MCs in the lungs of the WBB6F1
mice, including the appearance of intraepithelial MCs (Figures A and A); similar findings were observed in the C57BL/6-Kit+/+
mice (Supplemental Figure 4A and data not shown). These findings have also been reported in some patients with asthma (13
). OVA treatment also induced other features of allergic inflammation, such as prominent infiltrates of leukocytes, including lymphocytes and eosinophils (Figures , D and G, and , B and D); again, similar findings were observed in C57BL/6-Kit+/+
mice (Supplemental Figure 4, B and D).
Histology of lungs of OVA-sensitized/challenged mice 24 hours after the ninth OVA challenge.
Features of allergic inflammation in this chronic asthma model 24 hours after the ninth OVA or PBS challenge.
In certain settings, KitW/W-v
mice can develop MC populations by mechanisms that do not require normal signaling via the c-Kit receptor (11
). However, the lungs of OVA-treated KitW/W-v
mice (Figures B and A) or KitW-sh/W-sh
mice (Supplemental Figure 4A) remained completely devoid of MCs. In contrast, lung MC numbers in OVA- or PBS-treated Kit+/+
mice were statistically indistinguishable from those in Kit+/+
mice (Figures C and A); similar findings were observed in Kit+/+
mice (Supplemental Figure 4A).
In OVA-sensitized/challenged WBB6F1-Kit+/+ mice, the ninth OVA challenge resulted in a rapid rise in the levels of histamine detectable in the serum, with levels remaining significantly elevated over baseline (i.e., pre-OVA challenge) values even 24 hours after OVA challenge (Supplemental Figure 5 and Figure C). In OVA-sensitized/challenged mice, serum histamine measured 24 hours after the last OVA challenge was not only significantly elevated in Kit+/+ mice, but also, albeit to a lesser extent, in Kit+/+ BMCMCs→KitW/W-v mice; in contrast, only very low and statistically indistinguishable levels of serum histamine were detected in OVA- or PBS-treated KitW/W-v mice (Figure C); similar findings were observed in C57BL/6-Kit+/+, C57BL/6-KitW-sh/W-sh, and C57BL/6-Kit+/+ BMCMCs→KitW-sh/W-shmice (Supplemental Figure 4C). The corresponding values (serum histamine 24 hours after challenge) for OVA-sensitized, PBS-challenged WBB6F1-Kit+/+, WBB6F1-KitW/W-v, or WBB6F1-Kit+/+ BMCMCs→KitW/W-v mice were 31.4 ± 4.2, 6.0 ± 1.0, and 28.7 ± 4.6 nM, respectively. In naive WBB6F1-Kit+/+, WBB6F1-KitW/W-v, or WBB6F1-Kit+/+ BMCMCs→KitW/W-v mice, serum histamine levels were 25.9 ± 5.1, 5.1 ± 1.2, and 23.6 ± 3.4 nM, respectively. These results indicate that, in this setting, serum histamine is derived solely or largely from MCs and/or is derived from other cellular sources in an MC-dependent manner.
Kit+/+ and Kit+/+BMCMCs→KitW/W-v mice were also very similar in measurements of the inflammation associated with responses to OVA sensitization and challenge, with markedly increased numbers of inflammatory cells in the lungs versus only minimal responses in KitW/W-v mice (Figures , D–I, and , B and D). OVA-treated Kit+/+ or Kit+/+ BMCMCs→KitW/W-v mice also exhibited significant elevations in BAL fluid monocytes, macrophages, neutrophils, eosinophils, and lymphocytes (Figure D). While there were some differences in the number of individual types of leukocytes in the BAL fluid of antigen-challenged Kit+/+ versus Kit+/+ BMCMCs→KitW/W-v mice, taken together, the data in Figures , D–I, and , B and D, demonstrate that MCs are largely responsible for the antigen-induced leukocyte recruitment and chronic inflammation in this model. The same conclusion was supported when the same experiments were repeated using C57BL/6-Kit+/+, C57BL/6-KitW-sh/W-sh, and C57BL/6-Kit+/+ BMCMCs→KitW-sh/W-shmice (Supplemental Figure 4, B and D).
Activation of MCs via IgE or, in the mouse, IgG1 antibodies requires signaling through the FcRγ chain (FcRγ) that is shared by FcεRI and FcγRIII (14
). We found that levels of FcR
γ mRNA in lungs obtained 24 hours after the last challenge with OVA or PBS were highly upregulated in OVA-treated Kit+/+
mice but not in MC-deficient KitW/W-v
mice (Figure A); similar results were obtained in C57BL/6-Kit+/+
, and C57BL/6-Kit+/+
mice (Supplemental Figure 6). Although FcRγ is not restricted to MCs (41
), these results are in accord with our findings that levels of tissue MCs and other hematopoietic cells are much more markedly increased in the lungs of OVA-challenged as opposed to PBS-challenged wild-type, Kit+/+
, or Kit+/+
mice than in OVA-challenged MC-deficient KitW/W-v
mice (Figures , A–I, and , A, B, and D; Supplemental Figure 4, A, B, and D). We also quantified levels of a transcript which is thought to be restricted to T cells, i.e., T cell–associated GTPase
), and found that levels of this mRNA were also highly upregulated in the lungs of OVA-treated Kit+/+
mice but not in MC-deficient and KitW/W-v
mice (Figure B).
Lung mRNA levels of genes encoding (A
)T cell–specific GTPase
24 hours after the ninth OVA or PBS challenge.
Taken together, these quantitative RT-PCR results are consistent with our histological findings, which showed that OVA treatment was associated with increased numbers of MCs (Figures , A and C, and A; Supplemental Figure 4A) and striking lung infiltrates of lymphocytes, as well as of other leukocytes (Figure , D, F, G, and I, and data not shown).
MC-dependent enhancement of airway goblet cell hyperplasia, mucin gene expression, and collagen deposition.
Quantitative RT-PCR analysis demonstrated a highly MC-dependent upregulation of expression of genes encoding mucins 5AC and 5B, which are the major components of mucus secreted by goblet cells and submucosal glands, respectively (7
) (Figure , A and B; Supplemental Figure 7, A and B). These findings are consistent with our histological observations, which revealed that the striking increases in numbers of mucus-secreting goblet cells in the airway epithelium of OVA-treated mice were largely or fully MC-dependent (Figures , J–L, and C; Supplemental Figure 7C).
Airway goblet cell numbers and mucin gene expression in this chronic asthma model 24 hours after the ninth OVA or PBS challenge.
Masson trichrome staining revealed that OVA treatment resulted in enhanced deposition of collagen in the airways of Kit+/+ or Kit+/+ BMCMCs→KitW/W-v mice but had little or no such effect in the airways of KitW/W-v mice (Figure , J–L); similar results were observed in C57BL/6-Kit+/+, C57BL/6-KitW-sh/W-sh, and C57BL/6-Kit+/+ BMCMCs→KitW-sh/W-shmice (data not shown). Much of this enhanced collagen deposition was localized in the airways immediately below the airway epithelium, a location that also exhibited what appeared to be an increased amount of airway smooth muscle (Figure , J and L). Quantification of lung hydroxyproline in WBB6F1 mice confirmed that the OVA-induced increase in lung collagen in this model was predominantly MC-dependent (Figure D).
MC expression of FcRγ is required for optimal expression of enhanced airway responses, numbers of airway MCs, airway inflammation, and mucin gene expression.
MCs can be activated to release mediators in response to ligand-dependent engagement of multiple distinct cell surface receptors and signaling pathways (10
). However, antigen-dependent activation of MCs via either IgE/FcεRI or IgG1/FcγRIII requires signaling mediated by the γ chain (FcRγ) common to both of these receptors (14
). To examine the extent to which FcεRI/FcγRIII-mediated MC activation is required for expression of our chronic asthma model, we compared the expression of various features of our model in KitW/W-v
mice that had been engrafted with C57BL/6-FcR
MC expression of FcRγ in BMCMCs→KitW/W-v mice had no detectable effects on the levels of total or antigen-specific IgE or IgG1 antibodies (Supplemental Figure 8). However, OVA treatment induced little or no enhancement of Penh responses to methacholine in FcRγ–/– BMCMCs→KitW/W-v mice, but substantial enhancement of the responses was observed in FcRγ+/+ BMCMC→KitW/W-v mice (Figure A). Similarly, OVA challenge of OVA-sensitized mice induced a much stronger airway response in FcRγ+/+ BMCMC→KitW/W-v mice than in FcRγ–/– BMCMCs→KitW/W-v mice (Figure B). Very similar results were obtained in C57BL/6-FcRγ+/+ versus C57BL/6-FcRγ–/– BMCMCs→KitW-sh/W-shmice (Supplemental Figure 9), except that the FcRγ–/– BMCMCs→KitW-sh/W-shmice, in contrast to the FcRγ–/– BMCMCs→KitW/W-v mice, exhibited no detectable enhancement of Penh in response to OVA challenge (compare with Supplemental Figure 9B and Figure B).
Penh responses following i.n. OVA antigen challenge inKitW/W-v
mice that had been engrafted withFcR
In accord with these results, we found that expression of FcRγ by adoptively transferred MCs significantly enhanced the ability of such MCs to orchestrate most of the other features of this chronic asthma model in the recipient KitW/W-v
mice (Figures and ; Supplemental Figures 10 and 11). Indeed, OVA-induced increases in numbers of lung MCs appeared to be entirely dependent on the expression of FcRγ by the adoptively transferred MCs (Figure A and Supplemental Figure 10A). These results strongly suggest that an effect mediated through MC-FcRγ, perhaps including the ability of IgE antibodies to promote MC survival or proliferation even in the absence of known antigen (44
), contributed to the increased numbers of MCs observed in this setting.
Features of allergic inflammation in this chronic asthma model 24 hours after the ninth OVA or PBS challenge inFcRγ–/–
Airway goblet cell numbers and mucin gene expression in this chronic asthma model 24 hours after the ninth OVA or PBS challenge inFcRγ–/–
mice versus (more ...)
However, in some other features of the response that were strongly expressed in FcRγ+/+ BMCMCs→KitW/W-vor FcRγ+/+ BMCMCs→KitW-sh/W-shmice, OVA treatment also induced significant, albeit relatively modest, responses in the FcRγ–/– BMCMCs→KitW/W-vor FcRγ–/– BMCMCs→KitW-sh/W-shmice. For numbers of lung eosinophils (Figure B and Supplemental Figure 10B), numbers of most types of leukocytes present in the BAL fluid 24 hours after OVA challenge (Figure D and Supplemental Figure 10D), and levels of mRNA of genes encoding mucin 5AC or 5B (Figure , B and C; Supplemental Figure 11, B and C), FcRγ, and T cell GTPase (Supplemental Figure 12, A and B), the responses in the FcRγ–/– BMCMCs→KitW/W-vor FcRγ–/– BMCMCs→KitW-sh/W-shmice, although significant compared with responses in the PBS-challenged mice, were significantly lower, and in many cases at least 50% lower, than those in the OVA-challenged FcRγ+/+ BMCMCs→KitW/W-vor FcRγ+/+ BMCMCs→KitW-sh/W-shmice. These results indicate that FcRγ-independent mechanisms of MC activation can contribute to the development of these features of this chronic asthma model but that optimal responses require MC expression of FcRγ.
FcRγ-independent mechanisms of MC activation can significantly contribute to elevations of serum histamine and increased numbers of airway goblet cells. Serum histamine levels were significantly lower in OVA-treated FcRγ–/– versus FcRγ+/+ BMCMCs→KitW/W-v mice, but the reduction was only approximately 19% (Figure C). Given that virtually all of the elevation of serum histamine in this model is MC dependent (Figure B), the most straightforward interpretation of this finding is that in WBB6F1 mice, OVA-treatment either induced MC histamine release by FcRγ-independent mechanisms or (less likely, we think) that such mechanisms permit MCs to promote histamine release from other sources. Notably, in mice on the C57BL/6 background, FcRγ-independent mechanisms appear to be less important than FcRγ-dependent mechanisms in regulating OVA-induced histamine release (Supplemental Figure 10C).
The increased numbers of goblet cells induced in OVA-treated versus PBS-treated FcRγ–/– BMCMCs→KitW/W-v mice reached levels that were nearly as high as, and were statistically indistinguishable from, the levels induced in the corresponding FcRγ+/+ BMCMCs→KitW/W-v mice (Figure A). In contrast, the enhanced levels of Muc5ac and Muc5b mRNA in OVA-challenged mice were significantly reduced in FcRγ–/– versus FcRγ+/+ BMCMCs→KitW/W-v mice (Figure , B and C). FcRγ-independent mechanisms appeared to contribute significantly both to the increases in goblet cell numbers and to the enhanced levels of Muc5ac and Muc5b mRNA in OVA-challenged C57BL/6 mice as well (Supplemental Figure 11).
Our findings indicate that FcRγ-independent activation of MCs can contribute significantly to the enhanced goblet cell numbers (especially in mice on the WBB6F1 background) and to the markedly increased mucin gene expression that develops in a largely MC-dependent manner in this chronic asthma model.