Cooperation between Th1 and Th2 cells in a murine model of eosinophilic airway inflammation (original) (raw)
Th1 and Th2 cells cooperate to promote eosinophilic inflammation. To evaluate potential roles of Th1 and Th2 cells in promoting eosinophilic airway inflammation, 107 OVA-specific DO11.10 Th1 cells, 107 Th2 cells, or 107 of each were transferred by intravenous injection to groups of unsensitized BALB/c mice. Control mice received an intravenous injection of sterile PBS alone. The following day, the mice received 30-minute challenges of nebulized 1% OVA in PBS, both in the morning and in the afternoon. Three days after the challenge, the mice were sacrificed and their airways were evaluated for inflammation by BAL and histology.
Without transfer of antigen-specific T cells, unsensitized mice showed few inflammatory cells and no eosinophils in their airways 3 days after challenge (Figure 1, a and b). Naive mice that received 107 Th1 cells before challenge with OVA aerosol mounted a dramatic inflammatory response, but the inflammation consisted primarily of mononuclear cells with only rare eosinophils observed. Interestingly, transfer of 107 Th2 cells did not support significant inflammation over background. In contrast, when Th1 and Th2 cells were transferred together, OVA challenge induced a strong eosinophilic inflammatory response. Similar results were obtained when 5 × 106 Th1 cells were mixed with 5 × 106 Th2 cells before intravenous transfer (data not shown). In fact, these patterns of cellular recruitment were observed across a broad range of numbers of transferred cells, from 106 to 2.5 × 107 cells of each Th phenotype (data not shown). These results indicate that Th1 and Th2 cells can cooperate to generate eosinophilic inflammation.
Synergy between Th1 and Th2 cells promotes eosinophilic airway inflammation. Here, 107 OVA-specific DO11.10 Th1 cells, 107 Th2 cells, or 107 of each were passively transferred to recipient mice by intravenous injection. The next day, in the morning and again in the afternoon, the mice were challenged with an aerosol of 1% OVA in sterile PBS. Three days after the challenge, the mice were sacrificed and BAL cells were collected. Shown is the average number of cells in each sample ± SD (n = 4 mice per group). (a) Total BAL nucleated cells. (b) BAL eosinophils. Similar results were obtained in 4 separate experiments. *Significantly different from all other groups (P < 0.05).
Presence of Th1 cells results in increased recruitment of Th2 cells. Th1 and Th2 cells have been reported to use distinct adhesion molecules for attachment to the vascular endothelium and for transmigration into tissues (42, 45). In addition, Th1 and Th2 cells have been shown to express different sets of chemokine receptors (46–49). To determine if Th1 and Th2 cells exhibit differential trafficking patterns within the lung after antigen challenge, we performed passive transfer experiments with DO11.10 Th1 and Th2 cells labeled with the fluorescent, membrane-intercalating dyes PKH67 (green) or PKH26 (red). After labeling, 107 cells were transferred to unsensitized BALB/c mice, and the mice were challenged with an OVA aerosol. Three days after challenge, the mice were sacrificed and frozen sections of the lungs were evaluated for the presence and location of fluorescent cells.
When infused into naive mice, Th1 and Th2 cells exhibited different trafficking patterns in the lung after the antigen challenge. Th1 cells were found clustered around vessels and airways throughout the lung tissue (Figure 2a). In contrast, Th2 cells were observed only as scattered individual cells. They did not cluster around the airways and vessels in the same manner as the Th1 cells (Figure 2b). This pattern was not simply due to decreased survival of the Th2 cells after transfer because, after aerosol challenge, numerous Th2 cells could be found in the paratracheal lymph nodes (not shown). In striking contrast to these observations, when Th2 cells were transferred together with Th1 cells, antigen challenge induced the recruitment of substantial numbers of both Th1 and Th2 cells. The Th2 cells were present in dense clusters around the airways and medium-and large-sized vessels of the lung in a pattern similar to that seen for the Th1 cells (Figure 2, c and d). In addition, numerous eosinophils were detected in close proximity to the areas of Th1 and Th2 cell clustering (Figure 2, e and f). Neither cell type accumulated in the lungs in the absence of antigen challenge.
Th1 cells promote recruitment of Th2 cells to the airways after the challenge. Cultured T cells (107) were labeled with the fluorescent dyes PKH26 (red) or PKH67 (green) and transferred to recipient mice. In the morning and again in the afternoon of the next day, the mice were challenged with an aerosol of 1% OVA in sterile PBS. Three days after the challenge, lung tissue was collected and frozen. Fluorescent cells in the tissue were detected directly by fluorescence microscopy of air-dried 10-μm sections. Where indicated, eosinophils in the tissue were detected by virtue of the cyanide-resistant peroxidase activity in their granules. For detection of eosinophils, lung sections were fixed in acetone and then incubated in a DAB solution containing 1.6 mg/mL KCN. Shown are examples of lung tissue from recipients of (a) 107 PKH26-labeled Th1 cells, (b) 107 PKH26-labeled Th2 cells (c–f), 107 PKH67-labeled Th1 cells, and 107 PKH26-labeled Th2 cells. (c) Single exposure demonstrating Th1 cells. (d) Single exposure revealing Th2 cells in the same section as c. (e) Double exposure demonstrating Th1 and Th2 cells in close proximity to one another. (f) Detection of eosinophils (brown DAB staining) in the same section as e. Similar results were seen in 2 separate experiments.
To eliminate the possibility that the labeling dyes were affecting the migration of the transferred cells, we confirmed these results using the anti-clonotype antibody KJ1-26 and intracellular cytokine staining followed by flow cytometry to identify the DO11.10 transgenic Th1 and Th2 cells in the BAL. We transferred 107 Th1 cells, 107 Th2 cells, or 5 × 106 of each to groups of 4 wild-type BALB/c mice. The mice were challenged and BAL cells were collected 3 days later. Cells from groups of identically treated mice were pooled and cultured for 6 hours in T-cell medium containing monensin, PMA, and ionomycin. The cells were stained for CD4 and the transgenic DO11.10 TCR (43), as well as for intracellular IFN-γ as a marker for Th1 cells and IL-4 as a marker for Th2 cells. When 107 Th1 cells were transferred, more than 9.2 × 103 CD4+ KJ1-26+ IFN-γ+ were recovered (Figure 3). When the same number of Th2 cells was transferred, only 9.2 × 102 CD4+ KJ1-26+ IL-4+ cells were recovered in the BAL. In contrast, when only 5 × 106 Th2 cells were transferred together with 5 × 106 Th1 cells, substantial numbers of both IFN-γ+/IL-4– (4.9 × 103) and IFN-γ–/IL-4+ (3.2 × 103) cells were recovered in the BAL. These data, along with the results of transfer of fluorescently labeled cells, support the hypothesis that, over this time course, Th2 cells alone are poorly recruited to the airways. Th1 cells, in contrast, are competent for recruitment. Furthermore, Th1 cells can induce alterations in the lung microenvironment that then potentiate Th2 cell recruitment.
Flow cytometric analysis of Th1 and Th2 cell recruitment to the airways after challenge. Groups of 4 mice received (a) no transferred cells, (b) 107 DO11.10 Th1 cells, (c) 107 DO11.10 Th2 cells, or (d) 5 × 106 Th1 cells plus 5 × 106 Th2 cells. The mice were challenged, and BAL cells were collected as described in Figure 1. Cells from each group were pooled and then stimulated for 6 hours with PMA and ionomycin in the presence of monensin. Aliquots were stained with anti-CD4 and the anti-clonotypic antibody KJ1-26 to mark the transferred transgenic T cells. The cells were then fixed, permeabilized, and stained for intracellular IFN-γ and IL-4 as markers for Th1 and Th2 differentiation before analysis by flow cytometry. IFN-γ and IL-4 staining are shown for cells within the CD4+ KJ1-26+ gate. The numbers of cytokine-producing Th1 (IFN-γ+ IL-4–) and Th2 (IFN-γ– IL-4+) cells are indicated. Similar results were seen in 3 separate experiments.
Adoptively transferred Th1 and Th2 cells modulate expression of adhesion molecules in the lung. To determine whether transfer of Th cells resulted in changes in adhesion-molecule expression, we analyzed expression of the adhesion molecules ICAM-1 and VCAM-1 in the lungs after cell transfer and challenge. Th1 cells, Th2 cells, or both were transferred to mice, and the next day the mice were challenged with an aerosol of 1% OVA as described previously. Lung tissue was collected 48 hours later and analyzed by immunohistochemistry for ICAM-1 and VCAM-1 expression. ICAM-1 was detected at high levels in unchallenged animals and was induced to even higher levels in animals that had received Th1 cells (data not shown). ICAM-1 expression was strongest in the alveoli and was not seen in the larger vessels that were surrounded by inflammatory infiltrates. Thus, it seems unlikely that ICAM-1 mediates the increased recruitment of Th2 cells or eosinophils seen after antigen challenge. In contrast, VCAM-1 was expressed only at low levels in mice receiving no transferred cells and in recipients of Th2 cells alone (Figure 4, a and c); however, in recipients of Th1 cells, VCAM-1 was induced strongly in the medium- and large-sized vessels of the lungs (Figure 4b). When Th1 and Th2 cells were transferred together, VCAM-1 induction was even stronger, and eosinophils could be seen in the tissue adjacent to the VCAM-1–positive vessels (Figure 4d). This suggests that Th1 cells promote Th2 cell recruitment and subsequent eosinophil recruitment through induction of endothelial VCAM-1.
Th1 and Th2 cells cooperate to induce high levels of endothelial VCAM-1 expression and tissue eosinophilia. Mice received transferred T cells and were challenged as described in Figure 1. Two days after the challenge, the mice were sacrificed and lung tissue was collected and frozen. Sections of tissue were then cut and stained for the presence of VCAM-1 (red) and eosinophils (brown) as described in Methods. Shown are representative sections of lung from mice that received (a) no transferred cells, (b) Th1 cells, (c) Th2 cells, or (d) Th1 and Th2 cells. Similar results were seen in 4 separate experiments.
TNF-α and VCAM-1 contribute to Th-induced eosinophilic inflammation. The preceding experiments suggest that Th1 cells supply a factor that supports the recruitment of Th2 cells into the airway and that VCAM-1 may contribute to this process. To identify Th1 products that contribute to this synergy, we assessed the effects of neutralizing anti–IFN-γ, anti–TNF-α, and anti–VCAM-1 mAb’s on eosinophil and Th2 cell recruitment. Naive BALB/c mice were treated with 107 Thy1.1 DO11.10 Th1 and 107 Thy1.2 DO11.10 Th2 cells by intravenous infusion. The next day, groups of 4 unsensitized mice were injected intravenously with 500 μg of control antibody (anti–glutathione-_S_-methyl transferase PIP), anti–IFN-γ (H22), anti–TNF-α (TN3), or anti–VCAM-1 (MK2.7). The mice were then challenged with an aerosol of 1% OVA in the morning and afternoon. The day after the challenge, the mice receiving the anti–VCAM-1 antibody were injected with a second dose of antibody. All of the mice were sacrificed on the second day after the challenge, and BAL cells were collected. Anti-TNF-α– and anti-VCAM-1–treated mice had significantly decreased airway eosinophilia, whereas mice that received anti–IFN-γ antibody showed increased airway eosinophilia compared with the control antibody–treated animals (Figure 5a). Similar results were seen in 2 separate experiments.
Neutralization of IFN-γ, TNF-α, or VCAM-1 alters eosinophil and Th2 cell recruitment. Mice received 107 Thy1.1 Th1 cells and 107 Thy1.2 Th2 cells by intravenous injection. On the following day, groups of 4 mice received injections of anti–glutathione-_S_-methyl transferase (control antibody), anti–TNF-α, anti–VCAM-1, or anti–IFN-γ antibodies and then were challenged with an aerosol of OVA as described in Figure 1. The day after the challenge, the anti-VCAM-1–treated group received an additional injection of antibody. Two days after the challenge, the mice were sacrificed and BAL cells were collected and counted. (a) The number of eosinophils in the BAL. Shown are the average values ± SEM for 2 pooled experiments. (b) The ratio of transferred Th2 to Th1 cells in the BAL. An aliquot of the BAL cells from each animal was stained for CD4, KJ1-26, and Thy1.2 and analyzed by flow cytometry. The average ratio of CD4+KJ1-26+Thy1.2+ to CD4+KJ1-26+Thy1.2– cells is shown. *Significantly different from control antibody–treated group (P < 0.05).
Recruitment of OVA-specific Th1 and Th2 cells to the lungs was also assessed by flow cytometry using allotypic markers present on the transferred cells (Thy1.1 for Th1 and Thy1.2 for Th2). BAL cells were stained with anti-CD4, KJ1-26, and anti-Thy1.2, and then analyzed by flow cytometry. Transferred Th2 cells were CD4+ KJ1-26+ Thy1.2+ and transferred Th1 cells were CD4+ KJ1-26+ Thy1.2–. There was a clear correlation between the degree of eosinophil inflammation in the lungs and the degree of Th2 recruitment (Figure 5, a and b). Mice treated with anti–IFN-γ had higher proportions of Th2 cells relative to Th1 cells in their airways, whereas mice treated with anti–TNF-α or anti–VCAM-1 had lower proportions of Th2 cells recruited to their airways.
Finally, we assessed the effect of neutralizing TNF-α antibodies on vascular VCAM-1 expression. Mice treated with anti–TNF-α antibody showed greatly reduced VCAM-1 staining and decreased tissue eosinophilia when compared with mice treated with the control antibody (Figure 6). Together, these results suggest that Th1 cells promote VCAM-1 expression and subsequent Th2 and eosinophil recruitment through a pathway using TNF-α.
Neutralizing antibody against TNF-α inhibits VCAM-1 expression and tissue eosinophilia. Mice were treated as described in Figure 5. After BAL cells were harvested, the lung tissue was collected and frozen. Sections of lung were then stained for the presence of VCAM-1 (red staining) and eosinophils (brown staining). (a) Section from a mouse treated with anti–TNF-α, and (b) section from a mouse treated with anti–glutathione-_S_-methyl transferase (control antibody). Similar results were seen in 2 separate experiments.