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Blood, 1 September 2006, Vol. 108, No. 5, pp. 1588-1594. Prepublished online as a Blood First Edition Paper on May 2, 2006; DOI 10.1182/blood-2005-12-012781.
IMMUNOBIOLOGY Alpha-4 integrins and VCAM-1, but not MAdCAM-1, are essential for recruitment of mast cell progenitors to the inflamed lungFrom the Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital and Department of Medicine, Harvard Medical School, Boston, MA; Molecular Immunology Program, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta; and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT.
Normal mouse lungs lack appreciable numbers of mast cells (MCs) or MC progenitors (MCp's), yet the appearance of mature MCs in the tracheobronchial epithelial surface is a characteristic of allergic, T-cell-dependent pulmonary inflammation. We hypothesized that pulmonary inflammation would recruit MCp's to inflamed lungs and that this recruitment would be regulated by distinct adhesion pathways. Ovalbumin-sensitized and challenged mice had a greater than 28-fold increase in the number of MCp's in the lungs. In mice lacking endothelial vascular cell adhesion molecule 1 (VCAM-1) and in wild-type mice administered blocking monoclonal antibody (mAb) to VCAM-1 but not to mucosal addressin CAM-1 (MadCAM-1), recruitment of MCp's to the inflamed lung was reduced by greater than 75%. Analysis of the integrin receptors for VCAM-1 showed that in  7 integrin-deficient mice, recruitment was reduced 73% relative to wild-type controls, and in either BALB/c or C57BL/6 mice, mAb blocking of  4, 1, or 7 integrins inhibited the recruitment of MCp's to the inflamed lung. Thus, VCAM-1 interactions with both 41 and 47 integrins are essential for the recruitment and expansion of the MCp populations in the lung during antigen-induced pulmonary inflammation. Furthermore, the MCp is currently unique among inflammatory cells in its partial dependence on 47 integrins for lung recruitment.
Mast cells (MCs) develop in tissues from bone marrow-derived MC progenitors (MCp's) drawn from the intravascular compartment. The intestine has an abundant supply of MCs, and MCp's constitutively home to this organ. In contrast, the naive mouse lung has few MCp's and lacks mature MCs beyond first- and second-generation bronchi.1-3 Nonetheless, MCs accumulate in the epithelial surfaces of both large and small bronchi of mice that are sensitized systemically with ovalbumin (OVA) and challenged repetitively by aerosol inhalation to generate chronic airway inflammation.3 Furthermore, MCs are required for the development of airway hyperresponsiveness to methacholine in protocols that lack adjuvant or otherwise limit the intensity of sensitization before challenge with OVA.4-6 In humans with bronchial asthma, MCs accumulate and degranulate in both the bronchial epithelium and airway smooth muscle,7-9 accompanied by increased numbers of MCp's in the blood.10 Treatment with humanized monoclonal antibody (mAb) that blocks access of serum IgE to the high-affinity Fc  RI receptor reduces the frequency of asthma exacerbations.11 These findings imply a pathophysiologic role for MCs and their expansion both in mouse models of airway inflammation and in the pathogenesis of human bronchial asthma. Although the small airways of the mouse have scant smooth muscle, little lamina propria, and negligible bronchial circulation compared with those of the human, an increment in intraepithelial MCs and leukocyte infiltrates around bronchovascular bundles is consistently observed in inflamed respiratory mucosal surfaces in both species, leading to the suggestion that the site of extravasation is via the associated microvasculature.12,13 It is thus likely that the pathways needed for incremental MC numbers are conserved. Moreover the low levels of MCp's and scarcity of mature MCs in the naive mouse lung suggest that MCp recruitment accounts for the MC accumulation in the bronchial mucosa with the induction of allergic inflammation. To date, no studies have addressed this issue.
We previously reported that constitutive transendothelial migration of MCp's to intestine and the development of mature MCs in that organ are absent in
Thus, we hypothesized that the minimal homing of MCp's to lung would be dramatically expanded by recruitment during antigen (Ag)-induced pulmonary inflammation and that the adhesion pathway(s) for this response would be different from that necessary for the abundant homing of MCp's to the intestine. Herein, we demonstrate a rapid increase in the number of MCp's in the lung with aerosolized antigen-induced inflammation. Furthermore, unlike intestinal homing, this recruitment is mediated by VCAM-1 interacting with the
Animals
All mice were 6- to 16-week-old males when used. BALB/c mice were obtained from Taconic Laboratories (Germantown, NY). The OVA sensitization and challenge protocol Groups of 2 to 3 mice received intraperitoneal injections of 10 µg OVA (A5503; Sigma-Aldrich, Grand Island, NY) adsorbed to 1 mg alum (77161; Pierce, Rockford, IL) in 200 µL of sterile Hanks balanced salt solution (HBSS) on days 0 and 7. Mice were challenged with 1% aerosolized OVA in HBSS for 30 minutes per day using a PARI nebulizer (PARI, Midlothian, VA). For most experiments, the mice were challenged on days 17 to 19 and were killed for the determination of MCp's on day 20. For time course experiments, mice were killed on day 20, approximately 20 hours after receiving the last daily challenge of the indicated number of challenges (Figure 1A). Antibodies and the blocking of homing protocol
The mAbs anti-VCAM-1 (429, rat-IgG2a), anti-MAdCAM-1 (MECA367, rat-IgG2a), anti- To evaluate the molecules important in pulmonary recruitment of MCp's, approximately 50 µg of isotype control or blocking mAb, in 100 µL of HBSS, was administered intraperitoneally to OVA/alum-sensitized mice. Injections were given daily starting on the first day of aerosolized OVA challenges. Mononuclear cell (MNC) preparation and MCp assessment Mice were killed by CO2 asphyxiation and both lungs and spleen were harvested. Lung and spleen were placed separately in 20 mL of RPMI 1640 complete (RPMI 1640 containing 100 U/mL penicillin, 100 µg/mL streptomycin, 10 µg/mL gentamicin, 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 10% heat-inactivated fetal calf serum; F2442, Sigma-Aldrich, Grand Island, NY) and were processed essentially as previously described.1,2 Briefly, the lungs, perfused with 10 mL of HBSS administered via the right ventricle, were removed, finely chopped with scalpels, and transferred to 50-mL plastic tubes with 30 mL of RPMI 1640 complete plus 1 mg/mL collagenase type 4 (Worthington, Lakewood, NJ). Three enzymatic digestions were carried out for approximately 20 minutes each at 37°C. The undigested tissue clumps were collected after each time period and subjected to another enzymatic digestion, while the liberated cells were pelleted, resuspended in 44% Percoll (P1644; Sigma-Aldrich, St Louis, MO), underlayed with a 67% Percoll layer, and spun at 400g for 20 minutes at 4°C.
The MNCs were harvested from the 44/67% Percoll interfaces, and cells from the lungs of the 3 digestions were pooled and washed in RPMI 1640 complete. The number of viable cells was determined by trypan blue dye exclusion on a hemocytometer. The cells were serially diluted 2-fold in RPMI 1640 complete, and 100 µL of each dilution was added to wells of standard 96-well flat-bottomed microtiter plates (3596; Corning, Corning, NY). Typically, 24 wells were plated for each cell concentration. Lung MNCs were plated beginning at 10 000 to 30 000 cells/well. Then, each well received 100 µL of  -irradiated (30 Gy) splenic feeder cells plus cytokines (recombinant mouse IL-3 at 20 ng/mL and recombinant mouse stem cell factor at 100 ng/mL). The cultures were placed in humidified 37°C incubators with 5% CO2 for 12 to 14 days, and wells containing MC colonies were counted with an inverted microscope. The MC colonies were easily distinguished as large colonies of nonadherent, small-to-medium-sized cells.17,18 The MCp concentration is expressed as the number of MCp's/106 MNCs isolated from the tissue. The number of MCp's/lung is derived by multiplying the concentration of MCp's by the MNC yield per lung for individual mice or divided by the number of mice when MNCs were pooled from more than one mouse. Histology The assessment of inflammation in mice was performed 1 day after the last of 3 daily challenges. Lung tissue was fixed in 4% paraformaldehyde overnight, washed in PBS, embedded, and sectioned as previously described.19 JB-4-embedded sections were stained for chloroacetate esterase to illustrate mast cells and neutrophils. Inflammatory infiltrates in the lung parenchyma were evaluated by counting the number of bronchovascular bundles with an inflammatory infiltrate out of a total of 20 such bundles randomly visualized in each section after Diff-Quick (Dade Behring, Newark, DE) staining. The extent of goblet cell hyperplasia in the bronchioles was assessed by a semiquantitative mucus scoring between 0 and 3. A score of 0 corresponds to absence of mucus overproduction, and a score of 3 represents blockage of the majority of bronchiolar lumena by mucus. For illustration of the histologic changes, digital images were acquired at room temperature using a Nikon DXM 1200 digital camera (Melville, NY) fitted onto a Leica DM LB-2 light microscope through a Leica C-mount adapter (0.63 x HC f2/3'') with ACT-1 software from the same manufacturer (Leica, Heidelberg, Germany). Images submitted were taken at 125 x magnification using a Leica HC plane 20x/0.5 numeric aperature objective lens. No further image processing was undertaken. Statistical analysis Data in the figures are expressed as the mean ± SEM, derived from 3 or more separate experiments with the values from pools of 2 to 3 mice or from 2 or more individual mice in each group. For the statistical calculations, each n is either the value from an individual mouse or the value from the cells pooled from 2 or 3 mice. Significance was determined using a 2-tailed Student t test. Values of P less than .05 were considered significant.
Challenge with aerosolized Ag increases the numbers of lung MCp's Under basal conditions, few MCp's2 or mature MCs3,20 are identified in the mouse lung relative to the numbers found in the bone marrow, spleen, and intestine. We evaluated the numbers of MCp's in the lung with and without the induction of pulmonary inflammation by exposure to aerosolized antigen in sensitized mice to determine whether an increase in MCp's occurred and the time frame of the response. BALB/c mice sensitized with OVA and alum on days 0 and 7, followed by daily challenges with aerosolized OVA beginning 1, 3, 5, or 7 days before the MCp assay on day 20 (Figure 1A), exhibited a dramatic increase in the number of MNCs isolated from the enzymatically dispersed lungs (Figure 1B). The MNC preparation was not contaminated with MCs, as fewer than one chloroacetate-positive MC could be detected per 5 million MNCs after 3 daily challenges. The average number of MNCs increased over the baseline value of 1.6 x 106 MNCs per mouse by 3.9-fold after 3 daily challenges (P < .05, n = 5) and progressed to 5.7-fold with 7 daily challenges (P < .05, n = 4). The concentration of MCp's isolated from the lungs also increased significantly, with a 4.8-fold increase in the mean number after 3 daily challenges (P < .05), with little further increase after 5 (6.6-fold, P < .05, n = 4) or 7 (6.8-fold) daily challenges (Figure 1C). The mean (± SE) increase in total MCp's per mouse lung relative to unchallenged controls was 2.4 ± 1.0-fold after 1 challenge, 28.1 ± 14.8-fold after 3 challenges, 42.2 ± 6.9-fold after 5 challenges, and 54.6 ± 16.1-fold after 7 challenges (Figure 1D). The increase in total lung MCp's relative to unchallenged controls was statistically significant after 3 and 5 days of challenges (P < .05). Histologic assessment confirmed that the 3 daily aerosol antigen challenges resulted in the presence of inflammation with increased numbers of eosinophils and MNCs around the bronchovascular bundles (Figure 2). BALB/c mice showed 45% ± 15% of the blood vessels with an inflammatory infiltrate (mean ± 0.5 range of 2 mice), 35% ± 15% of the bronchioles with an inflammatory infiltrate, and a relative mucus score of 2 for both mice. C57BL/6 mice, also assessed after 3 days, exhibited an inflammatory infiltrate around 35% ± 0% of the blood vessels (mean ± 0.5 range of 2 mice), and 17% ± 2.5% of the bronchioles, and a mucus score of 1 ± 0. The magnitude of the infiltrate is more striking in the BALB/c mice than in the C57BL/6 mice, as previously demonstrated by others, after sensitization and airway antigen challenge13,21,22 (reviewed in Boyce and Austen23). No increase in lung MCs was noted in either strain after 3 challenges (data not shown)
The increase in lung MCp's required active sensitization and challenge with the same antigen. Mice sensitized with alum alone or with an irrelevant protein (goat Ig) adsorbed to alum and challenged with OVA showed no increase in pulmonary MNCs or MCp's (data not shown). Given the dramatic increase in lung MCp's after only 3 daily challenges, we used this time point in the subsequent experiments in which we sought to evaluate the molecules on the surface of the MCp's and the endothelium that were involved in this recruitment. VCAM-1 deficiency and mAb blockade of VCAM-1 but not MAdCAM-1 reduces the recruitment of MCp's to the inflamed lung
In our previous studies,
We further evaluated the role of VCAM-1 and any role for MAdCAM-1 using blocking mAbs directed against these molecules individually. Sensitized C57BL/6 mice received the mAbs just before each challenge. Relative to nonchallenged controls, the number of MNCs/mouse lung increased 2- to 3-fold in mice receiving HBSS or MAdCAM-1 Ab (Figure 4A). Mice receiving anti-VCAM-1 showed a reduction in the number of MNCs recovered that was not statistically significant (41% ± 3.5%, mean ± SE, n = 4). The administration of anti-MAdCAM-1 (Figure 4B-C) did not reduce pulmonary recruitment of MCp's whereas mice receiving anti-VCAM-1 had significant reductions in MCp concentration (58% ± 12% reduction, mean ± SE, P < .05, n = 4; Figure 4B) and in total MCp's/mouse lung (75% ± 6.6% reduction, P < .01; Figure 4C).
The
VCAM-1 can interact with either the
To distinguish the contribution of 41, 47, and E7 integrins to the recruitment of MCp's after the induction of pulmonary inflammation, we used mAb directed to the individual components of the heterodimers 4, E, 1, and 7. In C57BL/6 mice, no inhibition of MNCs was noted (Figure 7A). The lung MCp concentration (per 106 MNCs; Figure 7B) and the total lung MCp's (Figure 7C) were significantly inhibited by mAb to 4, 1, 7, or 47 integrin. Thus, in the C57BL/6 strain, the recruitment of lung MCp's was inhibited by 83% ± 4.5% with mAb to 4 integrin (P < .05, n = 3), by 60% ± 2.8% with mAb to 1 integrin (P < .05, n = 5), by 67% ± 1.2% with mAb to 7 integrin (P < .05, n = 3), and by 43% ± 7% (P < .05, n = 3) with a mAb to a combinatorial epitope of the 47 integrin heterodimer. The inhibition of MCp recruitment to lung by anti-7 integrin mAb was very similar to that observed in the 7 integrin null strain, 67% versus 73%, respectively.
In BALB/c mice receiving intraperitoneal injections of mAb immediately before challenge with aerosolized OVA, no significant reduction of MNC yields was found (Figure 8A). In contrast, both the concentration of MCp's (Figure 8B) and the total number of pulmonary MCp's/mouse (Figure 8C) were consistently decreased in mice treated with anti- 4, anti-1, or anti-7 relative to mice given HBSS or the isotype-matched anti-E. Mice treated with anti-4 demonstrated significant reductions in both MCp concentration (73% ± 7.4%; mean ± SE, n = 3) and in total MCp's (65% ± 21%) relative to HBSS-treated mice (P < .05 for both measures). Mice treated with anti-1 had a 57% ± 15% reduction in MCp concentration (mean ± SE, n = 3) and a significant 62% ± 17% reduction in total MCp's (P < .05). Mice treated with anti-7 had a significant 42% ± 10% reduction in MCp concentration (mean ± SE, P < .05, n = 8) and a 26% ± 23% reduction in total lung MCp's. Mice given anti-E and mice deficient in E integrin (data not shown) showed no reduction in MCp recruitment.
The lung of the naive mouse has sparse MCp's1,2 and few submucosal or intraepithelial MCs in the bronchi, yet there is a distinct increase in intraepithelial MCs with sensitization and airway challenge with Ag.3,20 We therefore sought to determine whether this increment reflected de novo recruitment of blood-born MCp's via transendothelial migration to the lung. With systemic sensitization and various intervals for daily airway challenge before assay, we found that a small accumulation of pulmonary MCp's occurred after even a single challenge of a systemically sensitized mouse, with a large increase (> 28-fold) after 3 or more challenges (Figure 1). At day 3, there was a substantial cellular infiltration of the bronchovascular bundles and mucus in the airways, with the changes being greater in the BALB/c mice than in the C57BL/6 mice (Figure 2). The recovery of MNCs also was 2- to 4-fold greater in BALB/c than in C57BL/6 mice. The magnitude and rate of the increase in MCp's from baseline to day 3 suggests that the major contribution is from influx. The MCp's were identified by limiting dilution and clonal expansion from isolated pulmonary MNCs purified by density gradient centrifugation, and these preparations did not contain detectable mature MCs. Moreover, histologic analysis confirmed that the acute experimental conditions selected did not generate a reactive mastocytosis in the epithelium or submucosa, as was observed with chronic antigen exposure.3 Also, the efficacy of blocking antibody to the adhesion molecules involved in transendothelial migration of MCp's indicates that recruitment rather than proliferation accounts for the expanded lineage reservoir.
Our earlier study on the mechanism of basal MCp homing to the intestine suggested that ongoing transendothelial influx was the major mechanism maintaining the baseline reservoir in this organ.1,2 MCp's were absent in the small intestine of
As both 41 and 47 integrins mediate binding to VCAM-1, we evaluated which of these integrins was involved in recruitment of MCp's to inflamed lung. Although the 7 integrin-deficient mice did not differ from wild-type C57BL/6 mice in their low baseline levels of lung MCp's, the incremental recruitment of MCp's was strikingly attenuated in the null strain (Figure 5). These 7 integrin-deficient mice had no decrement in any of the other parameters of sensitization and inflammation monitored (Figure 6). The blocking of MCp recruitment by mAbs in wild-type C57BL/6 mice showed that specificity for 4, 1, or 7 integrins markedly suppressed total lung MCp recruitment, with inhibition by the anti-7 integrin being comparable to that observed in the 7 integrin-deficient mice (Figure 7). In wild-type BALB/c mice, blocking of MCp recruitment was significant for total lung MCp's when the mAbs were directed to 4 and 1 integrins, with some effect for 7 integrin and none for E (Figure 8). When expressed as inhibition of recruited MCp's per 106 MNCs, the effects of mAbs to 4 and 7 integrins were significant, with a substantial effect with mAb to 1 integrins and none for mAb to E integrin. The 41 integrin has been clearly implicated in recruitment of other leukocytes to sites of allergic inflammation,27,32 but we know of no other leukocyte in which 47 plays such a prominent role in trafficking to the lung. The fact that mAb to heterodimeric 47 integrin failed to similarly suppress the elicitation of MCp's in lung (Figure 7) may be due to differences in epitopes blocked by binding of the various mAbs. Importantly, blockade of the E integrin subunit, the only other partner for 7, had no effect at all, further supporting the involvement of the 47 dimer.
Just as integrin-dependent transendothelial migration regulates basal MCp homing to the intestine, we now find that integrin-dependent transendothelial migration is the basis for inflammation-induced expansion of MCp's in the lung, a tissue that normally contains very few cells of this lineage in laboratory strains of mice. This recruitment of MCp's likely reflects the critical dependence on the induction of VCAM-1, as the constitutive MAdCAM-1 is not expressed by the pulmonary endothelium. The induction of VCAM-1 was sufficiently rapid to permit recruitment of MCp's within 72 hours of antigen challenge, during the initiation of Th2 cell-type pulmonary inflammation. Subsequent amplification of the inflammatory response by mucosal Th2 cells may permit proliferation and maturation of interepithelial MCs. Notably, whereas
Dr Abonia's current address is Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave, ML7028, Cincinnati, OH 45229-3039.
Submitted December 9, 2005; accepted April 19, 2006.
Prepublished online as Blood First Edition Paper, May 2, 2006; DOI 10.1182/blood-2005-12-012781.
Supported by grants AI 057991, HL 036110, AI 031599, AI 052353, AI 048802, and AI 047379 from the National Institutes of Health and the P. E. Lindahl Fund from The Royal Swedish Academy of Sciences.
J.P.A., K.F.A., and M.F.G. designed the experiments; J.P.A., J.H., T.J., T.S., Y.X., and M.F.G. performed the experiments and analyzed the data; P. K. and R.A.F. provided vital reagents; and J.P.A., J.A.B., K.F.A., and M.F.G. wrote the manuscript.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Michael F. Gurish, Brigham and Women's Hospital, Smith Building, Room 624, 1 Jimmy Fund Way, Boston, MA 02115; e-mail: mgurish{at}rics.bwh.harvard.edu.
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Abstract
Introduction
) and VCAM-1-deficient (
) mice with and without aerosolized OVA challenges. Values are the mean ± SEM from 3 separate experiments with MNCs pooled from 2 to 3 mice in each experiment. (B) Concentration of MCp's (MCp's/106 MNCs) in the lungs of the same mice. (C) Total number of MCp's per lung in the same mice. *Indicates statistical significance (P < .05) as determined by the 1-tailed Student t test.
