Abstract
Human asthma is characterized by increased airway hyperreactivity to a variety of bronchoconstricting agents. Aberrant type 2 immune responses in the lung have been associated with airway hyperreactivity in both human asthma and in murine models of allergic airways disease. Despite their intrinsically elevated basal airway reactivity to smooth muscle constricting agents, A/J mice demonstrated no inherent inflammatory cell infiltration nor elevation of type 2 cytokines in the lung. Crossed bone marrow reconstitution experiments between A/J and MHC congenic B10.A mice revealed enhanced airway reactivity only in A/J recipients, irrespective of whether they had been reconstituted with A/J or B10.A hemopoietic cells. Further, A/J-derived bone marrow cells did not affect the reactivity of B10.A recipients. Although mice on RAG-deficient and IL-4-deficient backgrounds demonstrate substantial abrogation of allergen-induced airway hyperreactivity, these gene deletions had no impact on the elevated baseline reactivity when backcrossed onto A/J mice. Thus, in these mice, basal airway hyperreactivity is maintained independently of type 2 immunity induced by allergens.
Asthma, characterized by reversible episodes of airway narrowing in the presence of chronic airway inflammation, remains an increasingly prevalent disease of industrialized nations (1, 2). Accumulating data have associated the asthma phenotype in humans with the presence of an aberrantly active type 2 immune response in the lungs (3, 4, 5). Thus, activated Th2 cells—a polarized subset of CD4+ T cells that together release the cytokines IL-3, IL-4, IL-5, IL-9, IL-10, IL-13, and GM-CSF—and activated eosinophils are consistently present in bronchoalveolar lavage (BAL)3 and bronchial biopsies obtained from patients with asthma (6, 7, 8, 9, 10, 11). Together, cytokines and other mediators released by these activated cells are felt to orchestrate the recruitment of eosinophils and lymphocytes and the production of IgE that together contribute detrimentally to the chronic stimulation of mucus production and airway pathology that ultimately lead to clinical disease.
The hallmark of human asthma, airway hyperreactivity, refers to a reversible, intrinsically lower, threshold for airway narrowing in response to allergen or pharmacologic agents, such as acetylcholine and serotonin, that induce smooth muscle contractility. Genome-wide searches as well as individual candidate-gene studies among patients with allergic asthma have implicated a number of potential genes that are associated with type 2 immunity, including the cytokine gene cluster on human 5q (mouse chromosome 11) that contains IL-4, IL-13, and IL-5, as well as the high-affinity IgE receptor and the α-chain of the IL-4 receptor that is shared between IL-4 and IL-13 (12, 13, 14).
Mouse models of allergen-induced airways disease share with human asthma the association of type 2 immune responses in lung tissue and the development of pathologic and physiologic changes, including airway hyperreactivity (reviewed in Ref. 15). Recent experiments using mice transgenic for the human 5q cytokine cluster through incorporation of yeast artificial chromosomes demonstrated the remarkable conservation of control in this locus and increased hopes that these animal models might guide the search for human susceptibility genes (16). Classic studies initiated 10 years ago identified intrinsic differences in baseline airway hyperreactivity among inbred mouse strains (17, 18, 19, 20). Although a spectrum of reactivity was apparent, one strain, A/J, demonstrated remarkable airway hyperreactivity. Although early studies suggested a simple autosomal recessive inheritance underlying the phenotype (17, 18), subsequent studies have defined a complex polygenic trait (21, 22), one of which involves at least three major contributing loci (21).
Whereas the value of the A/J strain in identifying potential airway reactive susceptibility genes is apparent, few studies have attempted to demonstrate whether the adaptive immune response, so critically implicated in allergen-induced asthma, is involved in the baseline airway hyperreactivity that occurs spontaneously in this strain of mice. We have used baseline immunologic and T cell-depletion studies, crossed bone marrow chimeras, and genetic crosses of A/J mice to recombinase activating gene-1 (RAG)- and IL-4-deficient mice to demonstrate that cells of the adaptive immune system do not contribute to the intrinsically elevated airway hyperreactivity of these animals. Despite this finding, the aberrant baseline physiology in A/J mice contributes to incrementally greater responses to allergen sensitization, thus justifying efforts to identify contributory genes as asthma susceptibility loci.
Materials and Methods
Mice
Six- to 8-wk-old A/J and MHC-matched B10.A/SgSnJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 mice deficient in T and B cells by disruption of the RAG-1 gene and C57BL/6 mice with deletion of the IL-4 gene were purchased from The Jackson Laboratory. RAG-deficient (23) and IL-4-deficient (24) mice were backcrossed six generations to A/J mice and intercrossed to generate N6 A/J RAG- and IL-4-deficient animals. Mice were typed for RAG deficiency using flow cytometric analysis to confirm the absence of peripheral blood T and B cells. Mice were typed for IL-4 deficiency by PCR analysis of tail DNA. Animals were housed in the University of California San Francisco pathogen-free animal facility.
Ag sensitization
Mice were immunized weekly for 3 wk with 25 μg chicken egg OVA (Sigma, St. Louis, MO) in 50 μl alum suspension s.c. at the base of the tail. Immunized mice received three aerosol exposures of 50 mg/ml OVA in PBS over 20 min, every other day, using a nose-only chamber adapted for mice (Intox Products, Albuquerque, NM) and coupled to a nebulizer (Aerotech II; CIS-US, Bedford, MA) as described (25). Airway reactivity was assessed 2 days after the final aerosol. Control mice were immunized with PBS in alum and exposed to a PBS aerosol.
Bone marrow reconstitution
Bone marrow cells were flushed from the tibia and femur of donor mice with RPMI 1640 supplemented with 10% FBS and antibiotics, passed through a 70-μm nylon mesh, washed, and resuspended at 2.5 × 107 cells/ml in PBS. Bone marrow cells (5 × 106) were injected i.v. into irradiated (900 rad) mice. Animals were maintained in pathogen-free conditions with antibiotic-supplemented water for 8 wk, at which time hemopoietic cell reconstitution was documented using flow cytometry.
T cell depletion
Where indicated, mice were injected i.p. with 1 mg anti-CD4 mAb (GK1.5) four times at 3-day intervals. Depletion of CD4+ T cells (<1% normal) was documented using flow cytometry. Airway reactivity was assessed 2 days after the final Ab treatment. Control mice received matched irrelevant mAb.
Airway reactivity
Airway reactivity to acetylcholine chloride (ACh) was measured as described (25). Briefly, mice were anesthetized with etomidate (28 μg/gm; Bedford Laboratories, CA). The trachea was cannulated and mice were ventilated with 100% oxygen at physiological rate and tidal volume using a rodent ventilator (Harvard Apparatus, South Natick, MA). Following paralysis to eliminate spontaneous respirations, mice were maintained inside a whole-body plethysmograph capable of measuring changes in air flow, as well as transthoracic pressures and resistance. After establishing a stable baseline for total lung resistance, ACh was administered i.v. over 1 s in escalating doses via the tail vein. Airway reactivity was expressed as the provocative concentration of ACh (in μg/g body weight) required to double baseline transthoracic resistance, designated PC200, as calculated by linear interpolation of appropriate dose-response curves. Significant differences were calculated using the logarithm of PC200 by ANOVA using reference to the specified control groups. Baseline pulmonary resistance in the absence of ACh did not differ among the various groups of mice studied.
Analysis of BAL
BAL cells were collected after instillation and withdrawal of three sequential 1-ml PBS aliquots through the tracheal cannula. Cells were washed, counted, and resuspended in RPMI 1640 with 10% FBS and antibiotics to a final concentration of 5 × 105 cells/ml. Aliquots (105 cells) were spun onto glass slides, air dried, fixed with methanol, and stained with Diff-Quik (Baxter Healthcare, Miami, FL). Eosinophils, macrophages/monocytes, and lymphocytes were enumerated based on morphology and staining characteristics and expressed as percentages of total BAL cells.
Enzyme-linked immunocell spot (ELISPOT) assays
Lung cell suspensions were prepared by finely mincing the lungs (devoid of any lymph node or thymic tissue) and pressing the fragments through a 70-μm nylon mesh filter. RBC were lysed in hypotonic buffer, and the remaining cells were washed, counted, and resuspended at 107 cells/ml in RPMI 1640 with 10% FBS and antibiotics. The numbers of IL-4-producing cells were enumerated using ELISPOT assays as described (25). Briefly, lung cell suspensions were distributed in duplicate aliquots of 106 cells into 96-well microtiter plates that had been precoated with anti-murine IL-4 mAb (11B11). Serial 2-fold dilutions were prepared, and the plates were incubated undisturbed for 18 h at 37°C. After washing away the cells, biotinylated secondary anti-IL-4 mAb (BVD6-24G.2) was added. Captured IL-4 was revealed using streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch Laboratories, West Grove, PA) and developed using 5-bromo-4-chloro-indolyl-phosphate in 0.1 M 2-amino-2-methyl-1-propanol buffer (Sigma) suspended in 0.6% low-melt agarose. Individual blue spots were counted after solidification of the agar using inverted microscopy.
Serum IgE
Serum was prepared from whole blood collected after quantitation of airway reactivity. Total serum IgE was determined using a double mAb-based sandwich ELISA with Ab B.IE.3 as the capture Ab and biotinylated EM-95 as the detecting Ab. The plates were developed with streptavidin-conjugated alkaline phosphatase (Jackson ImmunoResearch), and the substrate was 5 mM p-nitrophenyl phosphate, disodium hexahydrate (Sigma) in 0.1 M 2-amino-2-methyl-1-propanol buffer (Sigma). Absorbance was quantitated at 405 nm (Bio-Tek Instruments, Burlington, VT) and normalized to concurrently developed standard controls.
Histopathology
Lungs were infused in situ with 1 ml of 3.7% formaldehyde in PBS through the tracheal cannula. The lungs were carefully removed and immersed in the same fixative with the trachea tied closed for 24 h. The tissues were embedded in paraffin, and 2- to 3-μm sections were cut and stained with hematoxylin and eosin.
Flow cytometric analysis
Conjugated mAbs for flow cytometric analysis included PE-anti-B220 and FITC-anti-CD8α (Caltag Laboratories, South San Francisco, CA), FITC-anti-Ly9.1 and PE-anti-CD4 (clone RM4-4) (PharMingen, San Diego, CA). The two anti-CD4 mAbs bind at distinct sites unaffected by the presence of the other. Stained cells were analyzed for surface expression using flow cytometry (FACScalibur; Becton Dickinson, Mountain View, CA).
Results
Airway hyperreactivity is increased in A/J as compared with MHC congenic B10.A mice
A/J mice display increased airway reactivity after challenge with a number of bronchoconstricting agents (17, 18, 19, 20). To compare airway reactivity in A/J mice with MHC congenic B10.A mice, cohorts of unimmunized animals were given escalating doses of ACh i.v. while maintained in a whole-body plethysmograph, enabling constant quantitation of transthoracic pressure and flow. A/J mice showed a marked increase in airway hyperreactivity as assessed by the significant decrease in the provocative ACh dose required to elicit a 200% increase in baseline airway resistance (PC200) (Fig. 1⇓). Thus, in agreement with prior studies, unimmunized A/J mice demonstrated ∼5-fold greater sensitivity to the airway constricting effects of ACh than did MHC-matched B10.A mice.
Airway resistance in response to escalating doses of i.v. ACh. After establishing a stable baseline for total lung resistance, ACh was infused i.v. in escalating doses into unimmunized, age-matched, B10.A and A/J mice. ACh injections are indicated by the numbered arrowheads, 1–5, and represent incremental doses of 0.03, 0.1, 0.32, 1.0, and 3.3 μg ACh per gram body weight as indicated by the horizontal black triangles. The concentration of ACh required to elicit a 200% increase from baseline airway resistance, or PC200, is indicated by the bold arrowhead. Results are representative of eight experiments involving over 30 animals in each group.
Immunized A/J mice develop enhanced airway hyperreactivity to aerosolized Ag
To compare Ag-dependent airway hyperreactivity between A/J and B10.A mice, groups of mice were immunized three times with OVA and then challenged three times with OVA-containing aerosol. Two days after the third aerosol, mice were anesthetized and ventilated in a whole-body plethysmograph. As assessed by airway resistance in response to ACh, immunized A/J mice developed a further 4-fold increased reactivity, consistent with allergen-induced models of airway hyperreactivity. Similarly, immunized B10.A mice developed substantial airway hyperreactivity after sensitization and airway challenge with OVA (Fig. 2⇓A).
Airway hyperreactivity and the immune response in control and immunized A/J and B10.A mice. Groups of four to eight B10.A and A/J mice were challenged with PBS (Saline) or OVA. A, PC200 in response to escalating doses of ACh. B, Numbers of IL-4-producing cells in the lungs of control and immunized mice as determined by ELISPOT analysis. C, Serum IgE levels in control and immunized A/J and B10.A mice as assessed by ELISA. D, Percentage eosinophils in total BAL cells. In each case, results are representative of at least two experiments. Bars depict means and SEs of the means. Significant differences (p < 0.05) between control and OVA groups are indicated by an asterisk.
Ag-induced airway reactivity in B10.A mice was comparable to baseline airway reactivity in A/J mice (Fig. 2⇑A). This raised the possibility, considered by others (26, 27, 28, 29), that A/J mice develop enhanced responsiveness to environmental airway allergens that results in immune-mediated elevations in baseline airway reactivity. Because airway hyperreactivity in Ag-driven systems is accompanied by type 2 immune responses, we assessed resting A/J mice for evidence of active immune responses in the lungs. First, using ELISPOT assays to determine numbers of IL-4-producing cells from whole lung preparations, no increase was observed in unimmunized A/J mice. Further, the numbers of IL-4-producing cells in the lung that appeared after OVA challenge was comparable in A/J and B10.A mice (Fig. 2⇑B). Second, serum IgE, a sensitive index of chronic IL-4- and IL-13-mediated stimulation, was low in unimmunized A/J mice and not significantly different from B10.A mice. With OVA challenge, IgE levels actually increased to greater levels in B10.A mice (Fig. 2⇑C). Finally, there was no histologic evidence of active inflammatory injury in the lungs of resting A/J mice as assessed either by enhanced numbers of eosinophils in BAL (Fig. 2⇑D) or by microscopic examination of lung tissues (Fig. 3⇓A). OVA-immunized A/J and B10.A mice demonstrated cardinal features of allergic lung disease (Fig. 3⇓, B and C), including peribronchiolar infiltration with lymphocytes and eosinophils and the appearance of numerous eosinophils in BAL (Fig. 2⇑D). If anything, the extent of cellular inflammation was more pronounced in comparably immunized B10.A mice. This difference is unlikely to reflect a greater OVA-specific precursor T cell frequency in A/J, as compared with B10.A, mice, because mice immunized with OVA developed similar numbers of CD4+ cytokine-producing T cells in the regional lymph nodes (5 × 105 and 7 × 105 total cells in A/J and B10.A mice, respectively).
Lung histology in naive A/J and immunized A/J and B10.A mice. A, A/J lung after saline aerosol. No cellular infiltrate is apparent in the bronchiolar airways or alveolar spaces. B, A/J lung after OVA aerosol. Periarterial space is filled with a lymphocyte and eosinophil rich infiltrate. C, B10.A lung after OVA aerosol. The periarterial space is filled with an inflammatory infiltrate that spreads between two adjacent bronchiolar airways. All sections stained with hematoxylin and eosin and viewed at ×100 magnification.
Thus, despite the marked elevation in airway reactivity in A/J mice, we could find little evidence for chronic activation of type 2 immune responses under resting conditions. In contrast, immunization and aerosol challenge in both A/J and B10.A mice resulted in highly stereotyped type 2 immune responses, including elevations in the numbers of IL-4-producing cells, in eosinophils, in tissue infiltration by lymphocytes and eosinophils, and in serum IgE.
Deletion of CD4+ T cells does not alleviate airway hyperreactivity in A/J mice
CD4+ T cells have been demonstrated to be both necessary and sufficient to mediate airway hyperreactivity in murine models of allergic airways disease (15, 30, 31, 32). Despite our inability to document activated type 2 immune responses in unimmunized A/J mice, it remained possible that circulating CD4+ T cells were mediating airways disease by some unclear pathway. To assess this possibility, A/J mice were depleted to <1% of normal CD4+ T cells using mAb given over a 2-wk period (Fig. 4⇓A). CD4-depleted and A/J mice given matched irrelevant control Ab were compared with B10.A mice using airway reactivity to escalating doses of ACh (Fig. 4⇓B). Despite CD4+ T cell depletion, A/J mice displayed airway hyperreactivity that was no different from A/J mice given control mAb. Thus, CD4+ T cells do not mediate baseline airway hyperreactivity in A/J mice.
Airway hyperreactivity in CD4+ T cell-depleted mice. A, Flow cytometric analysis of spleen cells after treatment of nonimmunized A/J mice with control and anti-CD4 mAb. B, Groups of four to six nonimmunized A/J mice treated with control (ctrl Ab) or anti-CD4 mAb, or nonimmunized B10.A mice, were anesthetized and ventilated for determination of PC200 in response to ACh. Bars represent means and SEs of the means. Significant differences (p < 0.05) between the untreated B10.A and the anti-CD4-treated A/J mice are indicated by an asterisk. Results are representative of two experiments.
Airway hyperreactivity of A/J mice is not dependent on bone marrow-derived cells
Bone marrow radiation chimeras were constructed between A/J and B10.A mice to evaluate the role of hemopoietic cells in mediating baseline airway hyperreactivity. The Ly9.1 surface Ag, present on A/J but not on B10.A hemopoietic cells, was used to mark donor-derived bone marrow cells. As a control, bone marrow cells were used to reconstitute each mouse strain with its own donor cells. Analysis 8 wk after reconstitution confirmed that the majority of hemopoietic cells were donor derived, although reconstitution was more complete from A/J to B10.A than vice versa (Fig. 5⇓).
Flow cytometric analysis of spleen cells from bone marrow-reconstituted radiation chimeras. Eight weeks after reconstitution, spleen cells from untreated A/J mice (A), irradiated B10.A recipients reconstituted with A/J bone marrow cells (B), untreated B10.A mice (C), and irradiated A/J recipients reconstituted with B10.A bone marrow cells (D) were stained with mAb to Ly9.1, B220, and CD4 to mark hemopoietic cells of the A/J lineage, B cells, and Th cells, respectively. A/J hemopoietic cells are Ly9.1+ whereas B10.A cells are Ly9.1−.
Mice reconstituted with homologous or heterologous bone marrow cells were analyzed for baseline airway hyperreactivity after 8 wk, at which time all mice appeared well and without apparent disease. Although there was some variability in the absolute PC200, B10.A remained relatively unresponsive to ACh as compared with A/J mice. After irradiation and bone marrow reconstitution, all mice demonstrated enhanced PC200 as compared with unmanipulated mice (Fig. 6⇓). There was no evidence for graft-vs-host disease in the lungs or elsewhere, and no pulmonary infiltrations by inflammatory cells was apparent in any of the mice. Despite these consistent findings, A/J mice, whether recipients of their own bone marrow or B10.A bone marrow, demonstrated significantly increased airway hyperreactivity (decreased PC200) as compared with B10.A recipients. In a comparable way, B10.A recipient mice displayed decreased responsiveness to ACh regardless of whether they had been reconstituted with A/J or B10.A cells. Thus, in this model, we could find no evidence suggesting that bone marrow-derived cells contribute to the intrinsic elevation in airway reactivity of resting A/J mice.
Airway hyperreactivity in bone marrow reconstituted radiation chimeras. Groups of four to eight irradiated A/J and B10.A recipients were reconstituted with either A/J or B10.A bone marrow cells and compared with untreated A/J and B10.A mice 8 wk after reconstitution as indicated. Bars depict mean and SEs of the mean PC200 as determined by responsiveness to escalating doses of ACh. Significant differences (p < 0.05) between the A/J and B10.A recipient groups given either A/J or B10.A donor bone marrow, respectively, are indicated by an asterisk. Results are representative of two independent experiments.
Airway hyperreactivity of A/J mice is independent of T and B cells or IL-4
Despite our inability in implicating immune dysfunction in the airway hyperreactivity of resting A/J mice, it remained possible that prolonged developmental effects mediated by the immune system might produce chronic airway changes that persisted even after immune cell depletion in adult life. We took a genetic approach to this possibility by crossing A/J mice to RAG-deficient and IL-4-deficient mice. Both of these mice have been demonstrated to have abrogated airway responses following Ag challenge, with RAG-deficient mice having a more complete attenuation of the response than IL-4-deficient mice (31, 33, 34, 35). For the RAG backcross, C57BL/6 RAG-1-deficient mice were crossed six generations to A/J, and the progeny were intercrossed to create N6 A/J RAG-deficient mice. Similarly, C57BL/6 IL-4-deficient mice were crossed six generations to A/J, and the progeny were intercrossed to create N6 A/J IL-4-deficient mice. The success of the backcross was confirmed by PCR analysis of tail DNA to confirm the presence of the targeted but not the wild-type IL-4 allele and by flow cytometry to confirm the absence of T and B cells in the RAG-deficient A/J mice.
A/J RAG-deficient and IL-4-deficient mice were analyzed for reactivity to ACh and compared with control A/J and B10.A mice (Fig. 7⇓). Despite the complete absence of T and B cells from birth, or the complete inability to produce the cytokine IL-4, the backcrossed A/J mice displayed airway hyperreactivity comparable to wild-type A/J mice and to control backcrossed N6 A/J mice. Although IL-13 can mediate airway hyperreactivity in the OVA model in the absence of IL-4 (33, 36), we could confirm no increase in IL-13 mRNA in A/J, A/J RAG-deficient, A/J IL-4-deficient, or B10.A mice in the absence of airway sensitization with allergen (data not shown). Thus, in this model, we could define no contributions from the adaptive immune system that contribute to the underlying airway hyperreactive phenotype of A/J mice.
Airway hyperreactivity in IL-4 and B and T cell-deficient mice. Unimmunized groups of four to eight IL-4-deficient and T and B cell-deficient (rag−/−) A/J mice, wild-type A/J, B10.A, and pooled N6 A/J littermate control mice were anesthetized and ventilated for determinations of PC200 in response to escalating doses of ACh. Bars depict means and SEs of the means. Significant differences (p < 0.05) between the mutant (IL4- and rag-deficient) mice and B10.A mice are represented by an asterisk. Results are representative of three experiments.
Discussion
The role of the adaptive immune system in mediating allergen-induced airway hyperreactivity, despite some modest controversy, seems well established (reviewed in Ref. 15). In this model, CD4+ T cells differentiate preferentially to a type 2 phenotype, leading to the activation of various effector cells and the elaboration of reactive agents that together result in the classic cell and tissue responses that characterize asthma. Many of these downstream effector functions seem to depend upon release of IL-4 and IL-13 from Th2 cells, which can mediate eosinophil recruitment, mucus hyperplasia, and airway hyperreactivity by a mechanism dependent on signaling through IL-4Rα (33, 37, 38, 39) and activation of Stat6 (40, 41). Despite this knowledge, the contributions of adaptive immunity to the maintenance of baseline airway reactivity to bronchoconstricting agents remain less well studied. Various transgenic mice that express cytokines in the lung have demonstrated effects on baseline airway hyperreactivity (42, 43, 44, 45), but whether this reflects the physiologic state is unknown.
We used A/J mice, a strain with inherently increased baseline airway hyperreactivity to pharmacologic bronchoconstricting agents, to assess the role of the adaptive immune system in mediating this physiologic response. Despite markedly increased baseline airway reactivity as compared with MHC congenic B10.A mice, we could find no evidence that cells of the adaptive immune system contribute to resting airway responsiveness. This was assessed by multiple independent strategies, including the acute depletion of CD4+ T cells, the crossed bone marrow reconstitutions between A/J and B10.A mice, and the genetic approach involving crossing A/J mice to RAG- and IL-4-deficient backgrounds. The baseline hyperreactivity of A/J mice was essentially unaltered by each of these approaches. Earlier studies similarly confirmed no affect of deletion of the T cell compartment on the resting airway reactivity of C57BL/6 mice, which, aside from MHC genes, are genetically closely related to the B10.A mice studied here (31). Further, there existed no histologic evidence to support an immune-mediated mechanism for maintaining baseline reactivity in A/J mice. In contrast to immunized B10.A mice with comparable airway hyperreactivity (Fig. 2⇑A), naive A/J mice displayed no cellular infiltrates into pulmonary tissue and no evidence for intrinsic activation of type 2 immune responses. The data suggest an intrinsic mechanism for airway hyperreactivity in A/J mice that makes this a valuable model for investigating contributions of nonimmune genes to airway physiology.
Our inability to implicate hemopoietic cells in baseline airway hyperreactivity in A/J mice was in contrast to a prior study that used a similar approach using crossed bone marrow chimeric mice. Importantly, however, the prior study used F1 A/J × C57BL/6 mice as the hyperreactive parental strain as compared with C57BL/6 control animals (26). F1 mice were used to minimize contributions by graft-vs-host disease in chimeric animals, but the discriminatory differences in airway physiology were smaller in F1 as compared with parental A/J mice. We used MHC congenic B10.A mice to both minimize the potential for graft-vs-host disease among recipients while maximizing the discrimination in airway physiologic parameters as compared with A/J mice. We could discern no evidence for graft-vs-host disease in reconstituted mice using histologic criteria, but observed a consistent change in the baseline airway physiology in all irradiated mice, irrespective of the source of the donor marrow cells. Despite these baseline changes, which were consistent within groups of mice, it was clear that A/J recipients demonstrated resting airway hyperreactivity that was elevated comparably whether reconstituted with A/J or B10.A-derived bone marrow cells. Conversely, B10.A recipients displayed relatively normal airway hyperreactivity even after reconstitution with A/J-derived cells.
Acute reconstitution experiments leave open the possibility that developmental processes mediated by hemopoietic cells, and specifically by immune T cells, could induce chronic airway structural alterations leading to airway hyperreactivity that might not be readily reversed in adult life. We, in agreement with a prior report (28), could demonstrate no effect on baseline airway physiology mediated by acute depletion of CD4+ T cells, but potential chronic effects of lymphocytes have not been investigated. We used a genetic approach by crossing A/J mice to RAG-1-deficient and IL-4-deficient mice because, in each case, animals on these backgrounds have demonstrated profound deficiencies in the ability to mount airway responses to aerosolized Ag, including OVA (31, 33, 34, 35). In each case, the crossing in of A/J-derived genes resulted in increased baseline airway hyperreactivity in the N6 generation, at which point over 98% of the genome was A/J derived. Airway hyperreactivity of control littermates was indistinguishable from parental A/J mice. However, in contrast to parental A/J mice the backcrossed mutant mice developed similar airway hyperreactivity in the complete absence of B and T cells (RAG-1-deficient) or IL-4 (IL-4-deficient). Thus, any contributions of lymphocytes or IL-4 to baseline airway hyperreactivity in A/J mice would seem to be nonessential.
The role of nonhemopoietic cell factors in allergen-mediated airway hyperresponsiveness was illustrated here by the enhanced reactivity of A/J mice to sensitization with OVA as compared with B10.A mice with the same amount of Ag challenge (Fig. 2⇑A). Similar findings were noted in comparing A/J and C3H mice after sensitization with OVA or SRBC (29). Thus, subsequent type 2 inflammation resulted in incrementally greater airway hyperreactivity at more modest levels of immune cell recruitment, as evident by microscopic examination (Fig. 3⇑). Understanding the mechanisms for such interaction with the immune response may have great implications for the role of innate factors in contributing to susceptibility to allergic airways disease.
These findings raise questions regarding the mechanism(s) that underlie intrinsic airway hyperreactivity in A/J mice. Differences in airway caliber or number were not apparent in our studies (H. Hadeiba, R. M. Locksley, data not shown). The finding that high-dose cyclosporin A could attenuate airway hyperreactivity in A/J mice is intriguing (28). The ubiquitous distribution of calcineurin, the target of cyclosporin A, in all tissues, will require definition of the amounts and affinities of this phosphatase in discrete lung cell types. Indeed, the recent incrimination of calcineurin-dependent pathways in myocardial hypertrophy and signaling through the myocyte angiotensin receptor point out previously unappreciated roles for phosphatases of this class outside of the immune system (46). Other possibilities include the role of the autonomic nervous system control of airway smooth muscle as determined by a combination of β2-adrenergic and postganglionic muscarinic receptors (reviewed in Ref. 47). Of the various airway muscarinic receptor subtypes, the two most implicated in human asthma are designated subtypes II (M2) and III (M3). M2 receptors—present on postganglionic nerves—inhibit acetylcholine release via a negative feedback loop. Some evidence has been provided for dysfunctional M2 receptors in both human asthma (48) and in animal models of allergic airway disease (49). M3 receptors—present on airway smooth muscle, submucosal glands, and epithelial and endothelial cells—mediate airway smooth muscle contraction and mucus hypersecretion (47). Some evidence also suggests that coupling of muscarinic receptors to signal-transducing G proteins might be more efficient in A/J mice, although the subtypes of muscarinic receptors involved and their distribution will require further study (50). We did note modestly enhanced expression of M3 receptor mRNA in lung tissues extracted from A/J, as compared with B10.A, mice (H. Hadeiba, R. M. Locksley, data not shown), but further study will be required to quantitate the overall stoichiometry of the various muscarinic receptor subtypes to discern any relationship with baseline airway hyperresponsiveness. Although recent studies have defined a role for the integrin αvβ6 in mediating lung tissue homeostasis (51), baseline histologic studies failed to provide evidence for aberrant cell infiltration in mediating the physiologic changes in A/J lung.
These studies reaffirm the importance of the A/J strain in elucidating intrinsic factors that modulate airway hyperreactivity that arise independent of the acquired immune system. The aggravated phenotype demonstrated by A/J mice, with a proportionately greater airway response following immunization, points out the capacity of such genetic tendencies to aggravate subsequent immune-mediated airways disease. A/J mice will provide not only a valuable strain for classical genetic analysis to elucidate nonimmune genes that might contribute to asthma susceptibility, but will remain a stringent test for examining the ability of immune interventions to alleviate successfully the induced allergic response.
Acknowledgments
We gratefully acknowledge the assistance and advice of D. J. Fowell and J. Cyster at the University of California, San Francisco, and N. Flores-Wilson for expert animal care.
Footnotes
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↵1 This work was supported by National Institutes of Health Grants HL56385 and AI26918 and the Howard Hughes Medical Institute.
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↵2 Address correspondence and reprint requests to Dr. Richard M. Locksley, University of California San Francisco, Box 0654, C-443, 521 Parnassus Avenue, San Francisco, CA 94143. E-mail address: locksley{at}medicine.ucsf.edu
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↵3 Abbreviations used in the paper: BAL, bronchoalveolar lavage; ACh, acetylcholine chloride; ELISPOT, enzyme-linked immunocell spot; PC200, provocative dose of acetylcholine required to generate a 200% increase in baseline airway resistance; RAG, recombinase-activating gene.
- Received November 23, 1999.
- Accepted February 14, 2000.
- Copyright © 2000 by The American Association of Immunologists
References
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