Alpha-4 integrins and VCAM-1, but not MAdCAM-1, are essential for recruitment of mast cell progenitors to the inflamed lung

Abstract

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 beta7 integrin-deficient mice, recruitment was reduced 73% relative to wild-type controls, and in either BALB/c or C57BL/6 mice, mAb blocking of alpha4, beta1, or beta7 integrins inhibited the recruitment of MCp's to the inflamed lung. Thus, VCAM-1 interactions with both alpha4beta1 and alpha4beta7 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 alpha4beta7 integrins for lung recruitment.

Figure 1.

Time course for the increase in pulmonary MNCs and MCp's after OVA sensitization and challenge of BALB/c mice. (A) The protocol used for induction of pulmonary inflammation is depicted. Mice are sensitized intraperitoneally (IP Imm) with OVA and alum on days 0 and 7, and then challenged with aerosolized OVA daily, beginning on days 13, 15, 17, or 19, with assay on day 20. These mice received 7, 5, 3, or 1 daily challenges, respectively. (B) The mean number (± SEM) of lung MNCs recovered per BALB/c mouse is graphed for the various numbers of daily challenges as indicated. Values are means ± SE from 5 separate experiments, with following determinations (n) in each group: 0 (5); 1 day (3); 3 days (5); 5 days (4), 7 days (4). (C) The mean (± SE) MCp concentration (MCp's/10⁶ MNCs) in the lungs of the same mice as in panel B. (D) The mean (± SE) total number of MCp's per lung from the same mice as in panel B. ^*Indicates statistical significance (P < .05) as determined by the 2-tailed Student t test.

Figure 2.

Histology of BALB/c and C57BL/6 mouse lung after OVA sensitization and 3 days of aerosolized OVA challenge. Both strains show an inflammatory response around the bronchovascular bundles after 3 OVA challenges (arrows), the routine time for assessment of lung MCp's. The lung from the BALB/c mouse (left panel) shows much more leukocyte infiltration than does the lung from the C57BL/6 mouse (right panel).

Figure 3.

Effect of endothelial VCAM-1 deficiency on MCp recruitment to the inflamed lung. (A) Number of lung MNCs recovered per mouse from OVA-sensitized C57BL/6 (▪) 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.

Figure 4.

Effect of mAb to VCAM-1 and MAdCAM-1 on MCp recruitment to the inflamed lung. (A) Number of lung MNCs recovered per mouse from sensitized C57BL/6 mice not challenged with aerosolized OVA (No Challenge) or that had received injections of HBSS or the indicated mAb and then challenged with aerosolized OVA. Values are the mean ± SEM from 3 experiments with 4 determinations in each group. (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. ^*P < .05, ^**P < .01, as determined by the 2-tailed Student t test.

Figure 5.

Effect of β7 integrin deficiency on MCp recruitment to the inflamed lung. (A) Number of lung MNCs recovered per mouse from OVA-sensitized C57BL/6 (▪) and β7 integrin-deficient (□) mice with and without aerosolized OVA challenges. Values are the mean ± SEM from 6 separate experiments with 7 determinations in each group. (B) Concentration of MCp's (MCp's/10⁶ MNCs) in the lungs of the same mice. (C) Total number of MCp's per mouse in the lungs of the same mice. ^*P < .05, ^**P < .01, as determined by the 2-tailed Student t test.

Figure 6.

Histology of β7 integrin-deficient and C57BL/6 mouse lung after OVA sensitization and 3 days of aerosolized OVA challenge. The lung from the β7 integrin-deficient mouse (left panel) shows leukocyte infiltration around the bronchovascular bundles, similar to that observed in lung from the wild-type C57BL/6 mouse treated in parallel (right panel).

Figure 7.

Effect of mAb to α4, β1, β7, or α4B7 integrins on MCp recruitment to the inflamed lung in C57BL/6 mice. (A) Number of lung MNCs per mouse recovered from OVA-sensitized BALB/c mice not challenged (No Chall) or from mice that had received injections of HBSS or the indicated mAb and then challenged with aerosolized OVA. Values are the mean ± SEM from 6 separate experiments with following determinations in each group: HBSS (9); anti-α4 (3); anti-β1 (5); anti-β7 (3); anti-α4β7 (3). (B) Concentration of MCp's (MCp's/10⁶ MNCs) in the lungs of the same mice. (C) Total number of MCp's per mouse in the lungs of the same mice. ^*Indicates statistical significance (P < .05) as determined by the 2-tailed Student t test.

Figure 8.

Effect of mAb to α4, αE, β1, or β7 integrins on MCp recruitment to the inflamed lung of BALB/c mice. (A) Number of lung MNCs per mouse recovered from OVA-sensitized BALB/c mice not challenged (No Chall) or from mice that had been given injections of HBSS (HBSS) or the indicated mAb and then challenged with aerosolized OVA. Values are the mean ± SEM from 4 separate experiments with following determinations in each group: HBSS (6); anti-αE (3); anti-α4 (3); anti-β1 (5); anti-β7 (8). (B) Concentration of MCp's (MCp's/10⁶ MNCs) in the lungs of the same mice. (C) Total number of MCp's per mouse in the lungs of the same mice. ^*Indicates statistical significance (P < .05) as determined by the 2-tailed Student t test.