Dendritic cells and alveolar macrophages mediate IL-13–induced airway inflammation and chemokine production

Abstract

Background

IL-13 in the airway induces pathologies that are highly characteristic of asthma, including mucus metaplasia, airway hyperreactivity (AHR), and airway inflammation. As such, it is important to identify the IL-13–responding cell types that mediate each of the above pathologies. For example, IL-13’s effects on epithelium contribute to mucus metaplasia and AHR. IL-13’s effects on smooth muscle also contribute to AHR. However, it has been difficult to identify the cell types that mediate IL-13–induced airway inflammation.

Objective

We sought to determine which cell types mediate IL-13–induced airway inflammation.

Methods

We treated the airways of mice with IL-13 alone or in combination with IFN-γ. We associated the inhibitory effect of IFN-γ on IL-13–induced airway inflammation and chemokine production with cell types in the lung that coexpress IL-13 and IFN-γ receptors. We then evaluated IL-13–induced responses in CD11c promoter–directed diphtheria toxin receptor–expressing mice that were depleted of both dendritic cells and alveolar macrophages and in CD11b promoter–directed diphtheria toxin receptor– expressing mice that were depleted of dendritic cells.

Results

Dendritic cell and alveolar macrophage depletion protected mice from IL-13–induced airway inflammation and CCL11, CCL24, CCL22, and CCL17 chemokine production. Preferential depletion of dendritic cells protected mice from IL-13–induced airway inflammation and CCL22 and CCL17 chemokine production but not from IL-13–induced CCL11 and CCL24 chemokine production. In either case mice were not protected from IL-13–induced AHR and mucus metaplasia.

Conclusions

Pulmonary dendritic cells and alveolar macrophages mediate IL-13–induced airway inflammation and chemokine production. (J Allergy Clin Immunol 2012;129:1621-7.)

T_H2 cells contribute to the pathogenesis of asthma.¹ The T_H2 cytokine IL-13 is thought to be particularly important.² In asthmatic patients inhalation of an antagonist of IL-4 receptor (IL-4R) α, a shared receptor for IL-4 and IL-13, resulted in significant prevention of allergen-induced responses,³ and anti–IL-13 treatment significantly improved pulmonary function scores, particularly in a “high-T_H2” subset of asthmatic patients.⁴ In mice blockade of IL-13 in the airway prevented allergen-induced experimental asthma, including the development of airway hyper-reactivity (AHR), mucus metaplasia, and airway inflammation.⁵ Overexpression of IL-13 in the airway was sufficient to cause AHR and mucus metaplasia, as well as chemokine production and airway inflammation.^6–8 However, different cell types mediate different subphenotypes of IL-13–induced experimental asthma. In previous studies direct effects of IL-13 on airway epithelium were sufficient for the development of mucus metaplasia and AHR, but they were not sufficient for the development of airway inflammation.⁶ Recently, it was observed that IL-13’s effects exclusively on smooth muscle were sufficient for AHR, but they were not sufficient for the development of either mucus metaplasia or airway inflammation.⁹ The present study is the first that we know of to identify pulmonary dendritic cells and alveolar macrophages as important mediators of IL-13–induced airway inflammation.

We previously identified a large number of IL-13–induced chemokines associated with IL-13–induced airway inflammation. ⁷ However, we could not attribute their production to any particular cell type. In this study we associated the known inhibitory effect of IFN-γ on IL-13–induced airway inflammation, ¹⁰ with an inhibitory effect of IFN-γ on a restricted subset of IL-13–induced chemokines. This approach helped us to form the hypothesis that the cell types that mediate IL-13–induced airway inflammation coexpress IL-13 and IFN-γ receptors. Specifically, we observed that IFN-γ inhibited IL-13–induced airway inflammation in association with an inhibitory effect of IFN-γ on IL-13–induced CCL11 (eotaxin-1), CCL24 (eotaxin-2), CCL22 (macrophage-derived chemokine), and CCL17 (thymus and activation-regulated chemokine). In previous studies alveolar macrophages were identified as major producers of CCL11¹¹ and CCL24¹² in the allergic lung. In other studies pulmonary dendritic cells were identified as major producers of CCL22 and CCL17 in the allergic lung.^13–15 We confirmed that pulmonary dendritic cells and alveolar macrophages coexpressed IL-13 and IFN-γ receptors by means of flow cytometry. Therefore we hypothesized that pulmonary dendritic cells and alveolar macrophages mediate IL-13–induced airway inflammation.

Pulmonary dendritic cells express CD11b at a high level,^16,17 and although macrophages in most organs also express CD11b at a high level,¹⁸ alveolar macrophages are an exception in that they express CD11b at a low level.^16,17 Both alveolar macrophages and pulmonary dendritic cells express CD11c at a high level.^16,17 Consistent with this, a previous study showed that both alveolar macrophages and pulmonary dendritic cells become depleted in the lungs of CD11c–diphtheria toxin receptor (DTR) mice.¹⁹ Furthermore, a previous study showed that dendritic cells, but not alveolar macrophages or granulocytes, become depleted in the lungs of CD11b-DTR mice.¹⁴ We confirmed these observations in both models, and on the basis of the responses of these mice to IL-13 in the airway, we concluded that pulmonary dendritic cells and alveolar macrophages mediate IL-13–induced airway inflammation potentially involving the production of CCL11, CCL24, CCL22, and CCL17.

METHODS

Mice

The experiments were approved by the Northwestern University Animal Care and Use Committee and complied with the “Guide for the care and use of laboratory animals” prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press (revised 1996). BALB/c wild-type and CD11c-DTR mice¹⁹ and FVB wild-type and CD11b-DTR mice¹⁸ were used.

Protocols

Mice received 3 cytokine treatments after achievement of anesthesia (45 mg/kg ketamine and 8 mg/kg xylazine administered intraperitoneally), each occurring 2 days apart with 10 µg of recombinant murine IL-13 or a mixture of 10 µg of recombinant murine IL-13 and 10 µg of recombinant murine IFN-γ (PeproTech, Rocky Hill, NJ) in 50 µL of PBS through the intratracheal route. Samples were harvested 2 days after the third cytokine treatment. CD11b-DTR mice were administered diphtheria toxin (DT) twice (25 ng/g administered intraperitoneally) 1 day apart, ending 1 day before the first cytokine treatment. CD11c-DTR mice were administered DT once (25 ng administered intratracheally) 1 day before the first cytokine treatment.

Samples

Bronchoalveolar lavage (BAL) fluid was collected through a tracheotomy by washing the lumen of the lung with 0.9 µL of PBS. One microliter of acridine orange (100 µg/µL) was added to 19 µL of BAL fluid, and a Cellometer AutoX4 (Nexcelom Bioscience, Lawrence, Mass) automated cell counter was used to count the numbers of nucleated cells. The lavage fluid was centrifuged (1200 rpm × 5 minutes), and the supernatant was removed and stored at −80°C. The cell pellet was resuspended in PBS, and cytospin preparations were stained for the identification of cells by means of light microscopy. In some mice the lung was fixed in 10% formalin, dehydrated in ethanol, embedded in paraffin blocks, cut into 5-µm sections, mounted on slides, and stained with hematoxylin and periodic acid–Schiff. In other mice the lung was homogenized in Trizol (Sigma, St Louis, Mo), and the isolated RNA was converted to cDNA by means of reverse transcription.

Transcripts

The Cyber Green method of real-time PCR was used to detect a preverified panel of chemokines and inflammatory markers (mouse chemokine array; SABiosciences, Frederick, Md). Preverified Taqman assays were used to detect dendritic cell–specific intercellular adhesion molecule–grabbing non-integrin (DC-SIGN), Ccl2, Ccl8, Ccl11, Ccl17, Ccl22, and Ccl24 (Applied Biosystems, Foster City, Calif). Copy numbers were normalized to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

ELISAs

BAL fluid was analyzed by using ELISAs (R&D Systems, Minneapolis, Minn) specific for CCL2, CCL8, CCL11, CCL17, CCL22, and CCL24.

Flow cytometry

Lungs were digested in DNase (0.25 mg/mL) and collagenase (2.5 mg/mL) for 1 hour at 37°C with gentle rotation and passed through a 200-µm filter into Dulbecco modified Eagle medium/10% FBS to identify IL-13 receptor (IL-13R)– and IFN-γ receptor (IFN-γR)–coexpressing cells. The cells were counted and resuspended in 90 µL of MACS running buffer into which 2.4G2 anti-FcεRI/III mAb (BD PharMingen, San Diego, Calif) was added to block nonspecific binding and either no beads (mock separation) or 10 µL of anti–MHC-II MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) per 10⁷ cells was added. After 30 minutes at 20°C with gentle rotation, aliquots were incubated with no antibodies, concentration-matched isotype control antibodies, or anti–MHC-II (allophycocyanin [APC]; Miltenyi Biotech), anti–IL-4Rα (phycoerythrin [PE]; BD PharMingen), anti–IFN-γRα (Biotin; BD PharMingen) conjugated to streptavadin–Alexa Fluor 488 (Invitrogen, Carlsbad, Calif), anti-CD11b (APC-Cy7; BD PharMingen), and anti-CD11c (PE-Cy7; BD PharMingen). After 30 minutes at 20°C with gentle rotation, the cells were washed, resuspended in 500 µL of MACS buffer, and passed through a 40-µm filter. The cells were run twice through an autoMACS separator using the possel-s mode, and the flow-through and bound fractions were counted and analyzed by using flow cytometry. For evaluation of the effects of DT treatment, anti-CD11b (APC-Cy7; BD PharMingen) and anti-CD11c (PE-Cy7; BD PharMingen) were used to detect dendritic cells, and anti-F4/80 (PE-Cy7; eBioscience, San Diego, Calif) and anti-CD204 (PE; eBioscience) were used were used to detect macrophages. 4′-6-Diamidino- 2-phenylindole dihydrochloride was used to gate on living cells. Fifty thousand events per sample were collected and analyzed with FACSDiva Software (BD Biosciences).

AHR

Airway reactivity to intravenous acetylcholine challenge was measured as previously described.²⁰ Mice were anesthetized with sodium pentobarbital (90 mg/kg), intubated, and mechanically ventilated. A laparotomy was performed to expose the abdominal vena cava, and decamethonium bromide (25 mg/kg administered intravenously) was administered for paralysis followed by acetylcholine (50 µg/kg administered intravenously) after which dynamic airway pressure was integrated for 5 minutes.

Statistics

ANOVA and the Tukey-Kramer post test were used. Means and SEs are shown. P values of .05 or less were considered statistically significant.

RESULTS

The numbers and types of cells in BAL fluid were compared between groups of mice that received PBS, IL-13, or an IL-13/IFN-γ mixture to the airway (Fig 1, A). Compared with PBS, there was no effect of IL-13 or IL-13/IFN-γ on the numbers of alveolar macrophages. However, IL-13 induced a significant increase in eosinophil and lymphocyte numbers and caused neutrophil numbers to trend higher. Mice treated with IL-13/IFN-γ had completely normalized eosinophil numbers. Both lymphocyte and neutrophil numbers trended lower but were not significantly lower in IL-13/IFN-γ–treated mice. No mucus metaplasia was observed in PBS-treated control animals (Fig 1, B). However, IL-13 caused severe mucus metaplasia (Fig 1, C), which was diminished in mice treated with IL-13/IFN-γ (Fig 1, D). These results confirm an inhibitory effect of IFN-γ on IL-13–induced airway inflammation¹⁰ and extend this observation to IL-13–induced mucus metaplasia.

FIG 1.

IFN-γ inhibits IL-13–induced airway inflammation and mucus metaplasia. A, Cell types and numbers retrieved in BAL fluid from PBS-treated (open bars), IL-13–treated (black bars), and IL-13/IFN-γ–treated mice (shaded bars). Eos, Eosinophils; Lymphs, lymphocytes; Macs, macrophages. B–D, Representative images of mucus metaplasia in PBS-treated (Fig 1, B), IL-13–treated (Fig 1, C), and IL-13/IFN-γ–treated (Fig 1, D) wild-type mice (n = 6 mice per group). *P ≤ .05 versus PBS. †P ≤ .05 versus IL-13.

To identify chemokines associated with the inhibitory effect of IFN-γ on IL-13–induced airway inflammation, we used a chemokine-themed real-time PCR miniarray. Compared with PBS, IL-13 increased the expression of Ccl8, Ccr5, Inhba, Ccl2, FceR2a, and Itgb2, and IFN-γ did not inhibit the expression of these genes. However, IL-13 also increased the expression of Ccl17 and Ccl11, and the IL-13–induced expression of these genes was inhibited by IFN-γ (Table I). We extended this study by using ELISA of chemokines in BAL fluid. Because of its relatedness to CCL11 (eotaxin-1), we included CCL24 (eotaxin-2). As a consequence of detection of CCL17 (thymus and activation-regulated chemokine), we included CCL22 (macrophage-derived chemokine) because they are genetically linked,²¹ are both dendritic cell derived,^13–15 and both contribute to allergen-induced airway inflammation through their common receptor, CCR4.¹⁵ In agreement with their gene expression, IL-13 increased the production of CCL11, CCL24, CCL22, and CCL17 in the lung, and the IL-13–induced production of these chemokines was inhibited by IFN-γ (Fig 2, A–D). Also in agreement with their gene expression, IL-13 increased the levels of CCL8 and CCL2 in the lung, and the IL-13–induced production of these chemokines was not inhibited by IFN-γ (Fig 2, E and F). Thus IL-13– and IFNg– counterregulated airway inflammation was associated with IL-13 and IFN-γ counterregulated CCL11, CCL24, CCL22, and CCL17 chemokine production.

TABLE I.

Effect of IFN-γ on IL-13–induced chemokine gene expression

Gene	PBS vs IL-13	PBS vs IL-13/IFN-γ	IL-13 vs IL-13/IFN-γ	Fold induction, PBS vs IL-13
P values for genes induced by IL-13 and not inhibited by IFN-γ
Ccl8	.003	.015	.209	4.9
Ccr5	.007	.002	.292	3.2
Inhba	.015	.010	.532	2.6
Ccl2	.044	.050	.605	2.5
FceR2a	.022	.028	.557	2.4
Itgb2	.002	.009	.263	2.0
P values for genes induced by IL-13 and inhibited by IFN-γ
Ccl17	.002	.117	.021	9.6
Ccl11	.053	.694	.025	1.5

FIG 2.

IFN-γ inhibits a subset of IL-13–induced chemokines. Levels of CCL11 (A), CCL24 (B), CCL22 (C), CCL17 (D), CCL8 (E), and CCL2 (F) in BAL fluid from PBS-treated (open bars), IL-13–treated (black bars), and IL-13/IFNγ– treated wild-type mice (shaded bars) are shown (n = 6 mice per group). *P ≤ .05 versus PBS. †P ≤ .05 versus IL-13.

We hypothesized that cell types that coexpress IL-13 and IFN-γ receptors and that were known to produce CCL11, CCL24, CCL22, and CCL17 might mediate the counterregulatory effects of IL-13 and IFN-γ on airway inflammation. In previously published studies alveolar macrophages were identified as a major source of CCL11¹¹ and CCL24¹² in the allergic lung. In other studies dendritic cells were identified as a major source of CCL22 and CCL17 in the allergic lung.^13–15 Specifically, for MHC-II⁺ cells in the murine lung, dendritic cells have been defined as CD11b⁺/CD11c⁺ cells, and alveolar macrophages have been defined as CD11b⁻/CD11c⁺ cells.^16,17 Therefore to determine whether alveolar macrophages and pulmonary dendritic cells coexpressed IL-13 and IFN-γ receptors, we evaluated the expression of CD11b, CD11c, IL-4Rα and IFN-γRα on MHCII ⁺ cell–depleted and MHC-II⁺ cell–enriched lung cells from PBS- and IL-13–treated mice.

We used flow cytometry to determine the percentages of MHC-II⁺ cells in a single-cell suspension made from whole lungs after a mock and an anti–MHC-II magnetic bead–based separation procedure. After the mock separation procedure, the percentages of MHC-II⁺ cells in the flow-through and bound fractions were essentially the same, regardless of whether they were from the lungs of PBS- or IL-13–treated mice, and they comprised, on average, 2.9% of the total lung cell population. After MHC-II–targeted separation, the percentage of MHC-II⁺ cells in the flow-through fraction decreased to an average of 0.1%, and the percentage of MHC-II⁺ cells in the bound fraction increased to an average of 11.5% (see Table E1 in this article’s Online Repository at www.jacionline.org).

We next evaluated the expression of IL-4Rα, IFN-γRα, CD11b, and CD11c on the MHC-II⁺ cell–depleted flow-through fraction and the MHC-II⁺ cell–enriched bound fraction of lung cells from PBS- and IL-13–treated mice. There were much greater numbers of IL-4Rα⁺/IFN-γRα⁺ cells detected among the MHC-II⁺ cell–enriched bound fraction compared with the MHC-II⁺ cell–depleted flow-through fraction (Fig 3, A). There were no differences detected between PBS- and IL-13–treated mice. However, we detected 2 major populations of IL-4Rα⁺/IFN-γRα⁺ cells in the MHC-II⁺ cell–enriched bound fraction that were only detected at low levels in the MHC-II⁺ cell–depleted flow-through fraction. One of these populations was comprised of CD11b⁺/CD11c⁺ cells, and the other population was comprised of CD11b⁻/CD11c⁺ cells (Fig 3, B). These cells fit the definition of dendritic cells and alveolar macrophages, respectively.^16,17 On the basis of their coexpression of IL-13 and IFN-γ receptor subunits, we concluded that pulmonary dendritic cells, alveolar macrophages, or both could be responsible for the counterregulatory effects of IL-13 and IFN-γ on airway inflammation and chemokine production.

FIG 3.

Dendritic cells and alveolar macrophages coexpress IL-13R and IFN-γR subunits. Numbers of IL-4Rα⁺/IFN-γRα⁺ cells (A) and their proportions that express CD11b and CD11c (B) from the lungs of PBS-treated (open bars) and IL-13–treated (shaded bars) wild-type mice in MHC-II⁺ cell–depleted flow-through and MHC-II⁺ cell–enriched bound fractions are shown (n = 6 mice per group). *P ≤ .05 versus flow-through.

In previously published studies pulmonary dendritic cells and alveolar macrophages became depleted in CD11c-DTR mice,¹⁹ and dendritic cells, but not alveolar macrophages, became depleted in the lungs of CD11b-DTR mice.¹⁴ In this study we used flow cytometry of whole lung cells to evaluate the percentages of CD11b⁺/CD11c⁺ cells as a marker of dendritic cells and the percentages of (F4/80)⁺/CD204⁺ cells as a marker of macrophages. The analysis occurred 1 day after the last DT treatment, corresponding to the time point of the first IL-13 treatment in subsequent studies. Although the depletions were not complete, in CD11c-DTR mice a single intratracheal treatment of DT caused an average 3-fold reduction in the percentages of dendritic cells, and an average 3-fold reduction in the percentages of macrophages was detected. In CD11b-DTR mice 2 intraperitoneal treatments of DT caused an average 2-fold reduction in the percentages of dendritic cells, and the percentages of macrophages were unaltered (see Fig E1 in this article’s Online Repository at www.jacionline.org).

In CD11c-DTR mice IL-13 increased the gene expression of the dendritic cell–specific marker DC-SIGN.^22,23 This result is indicative of IL-13–induced dendritic cell activation. However, DT treatment caused CD11c-DTR mice to have near ablation of baseline and IL-13–induced gene expression of DC-SIGN (Fig 4, A) and significantly lower numbers of alveolar macrophages in BAL fluid (Fig 4, B). These results are consistent with pulmonary dendritic cell and alveolar macrophage depletion in these mice.¹⁹ Furthermore, DT treatment protected these mice from IL-13–induced eosinophilic and lymphocytic airway inflammation but had no effect on neutrophil numbers (Fig 4, C). This result indicated that either dendritic cells, alveolar macrophages, or both mediated IL-13–induced eosinophilic and lymphocytic airway inflammation.

FIG 4.

Dendritic cell and alveolar macrophage targeting protects mice from IL-13-induced airway inflammation and chemokine production. A, Gene expression levels of DC-SIGN in whole lung. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. B–D, Numbers of alveolar macrophages (Macs; Fig 4, B); numbers of eosinophils (Eos), lymphocytes (Lymphs), and neutrophils (Neuts; Fig 4, C); and levels of chemokines (Fig 4, D) in BAL fluid from CD11c-DTR mice (n = 6 mice per group). *P ≤ .05 versus PBS. †P≤ .05 versus IL-13.

To confirm the observed protective effect of dendritic cell and alveolar macrophage depletion on IL-13–induced airway inflammation, we measured the levels of chemokines in BAL fluid. In CD11c-DTR mice IL-13 increased the production of CCL11, CCL24, CCL22, and CCL17 chemokines. DT treatment protected these mice from the IL-13–induced chemokine response (Fig 4, D).We extended this study to the effects of IL-13 on mucus metaplasia and AHR. In previous studies these pathologies were attributed to IL-13’s effects on airway epithelial and smooth muscle cells.^6,9 As expected, we observed that the development of IL-13–induced AHR and mucus metaplasia was not prevented by DT treatment of CD11c-DTR mice (see Fig E2 in this article’s Online Repository at www.jacionline.org).

In CD11b-DTR mice IL-13 increased the gene expression of DC-SIGN, and DT treatment almost abolished the baseline and IL-13–induced gene expression of DC-SIGN in the lung (Fig 5, A). However, the numbers of alveolar macrophages in BAL fluid were unchanged (Fig 5, B). In combination, these results are consistent with dendritic cell but not alveolar macrophage depletion in CD11b-DTR mice.¹⁴ IL-13 induced significantly increased numbers of eosinophils, lymphocytes, and neutrophils. DT treatment protected CD11b-DTR mice from the development of eosinophilic and lymphocytic airway inflammation but had no effect on neutrophil numbers (Fig 5, C).

FIG 5.

Dendritic cell targeting protects mice from IL-13–induced airway inflammation and a subset of IL-13–induced chemokines. A, Gene expression levels of DC-SIGN in whole lung. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. B–D, Numbers of alveolar macrophages (Macs; Fig 5, B); numbers of eosinophils (Eos), lymphocytes (Lymphs), and neutrophils (Neuts; Fig 5, C); and levels of chemokines (Fig 5, D) in BAL fluid from CD11b-DTR mice (n = 6 mice per group). *P ≤ .05 versus PBS/PBS. †P ≤ .05 versus PBS/IL-13.

To confirm the protective effect of dendritic cell depletion on IL-13–induced airway inflammation, we evaluated the levels of IL-13–induced chemokines, including CCL22, CCL17, CCL11, and CCL24, in BAL fluid. IL-13 increased the levels of all of the chemokines assayed. DT treatment protected CD11b-DTR mice from IL-13–induced CCL22 and CCL17 but not from IL-13–induced CCL11, and there was a not quite significant inhibition of IL-13–induced CCL24 (Fig 5, D), which is potentially indicative of a partial role for dendritic cells in CCL24 production and/or some depletion of alveolar macrophages in the CD11b-DTR stain. IL-13–induced AHR and mucus metaplasia were not prevented by DT treatment of CD11b-DTR mice (data not shown). We conclude that pulmonary dendritic cells and alveolar macrophages cooperatively mediate the development of IL-13–induced airway inflammation and chemokine production.

DISCUSSION

In summary, IFN-γ inhibited IL-13–induced airway inflammation in association with an equivalent effect on CCL11, CCL24, CCL22, and CCL17 chemokine production. On the basis of their coexpression of IL-13 and IFN-γ receptors, pulmonary dendritic cells and alveolar macrophages were considered potential mediators. CD11c-DTR mice were depleted of both dendritic cells and alveolar macrophages, and these mice were protected from IL-13–induced airway inflammation in association with protection from CCL11, CCL24, CCL22, and CCL17 production. In comparison, CD11b-DTR mice were depleted of pulmonary dendritic cells, and these mice were also protected from IL-13–induced airway inflammation. In the case of CD11b-DTR mice, the protection from IL-13–induced airway inflammation occurred in association with protection from IL-13–induced CCL22 and CCL17 chemokine production, but these mice were not protected from IL-13–induced CCL11 production, and the majority of IL-13–induced CCL24 chemokine production was preserved. These results are consistent with previous observations that pulmonary dendritic cells are a major source of allergen-induced CCL22 and CCL17^13–15 and that alveolar macrophages are a major source of allergen-induced CCL11 and CCL24.^11,12

On the basis of the experiments reported here, it is not possible to determine whether alveolar macrophages also mediate IL-13–induced airway inflammation through CCL11, CCL24, or both because no protocol was available for selectively depleting alveolar macrophages. However, compared with CD11c-DTR mice, we observed that in CD11b-DTR mice CCL11 and CCL24 production remained increased in the context of protection from IL-13–induced airway inflammation. This could indicate that CCL11 and CCL24 chemokine production by alveolar macrophages is not sufficient for the development of IL-13–induced airway inflammation. However, this conclusion should be made with caution because the baseline levels of dendritic cells and alveolar macrophages and the IL-13–induced inflammatory responses were quantitatively different in the 2 strains. This might be due to genetic background differences or the effects of genetic engineering. Furthermore, the idea that CCL11 and CCL24 are dispensable for IL-13–induced airway inflammation is in contrast to a previous finding that CCR3, the receptor for CCL11 and CCL24, is necessary for IL-13–induced airway inflammation.²⁴ Another consideration is that there appears to be a complex mechanistic interaction between CCL11 and CCL24 chemokines and CCL22 and CCL17 chemokines. This is evidenced by a murine study in which CCR3, the receptor for CCL11 and CCL24, was observed to be important for early allergen-induced T_H2 cell recruitment into the airway, and then subsequently CCR4, the receptor for CCL22 and CCL17, became important.²⁵

The present study confirmed a previous report that IL-13–induced airway inflammation was inhibited by IFN-γ.¹⁰ IL-13 in the airway is sufficient to induce eosinophilic and lymphocytic airway inflammation^10,26 and to augment allergen-induced airway inflammation.²⁷ Conversely, IL-13 gene deletion protects mice from allergic airway inflammation.²⁸ The inhibitory effects of IFN-γ on the IL-13–induced inflammatory responses that we observed are supported by previous findings that IFN-γ in the airway inhibits allergen-induced airway inflammation^10,27,29 and that IFN-γR deletion augments allergic airway inflammation.³⁰ There are diverse mechanisms by which IL-13 can augment (as previously reviewed by Kuperman and Schleimer²), and IFN-γ can suppress allergic airway inflammation (as reviewed by Teixeira et al³¹). However, the present study, which identified pulmonary dendritic cells and alveolar macrophages as IL-13R– and IFN-γR–coexpressing cells, highlights a novel possibility that the local balance of IL-13 and IFN-γ in the airway counterregulates allergic airway inflammation by counterregulating the levels of chemokines produced by pulmonary dendritic cells and alveolar macrophages.

Previous studies using CD11b-DTR and CD11c-DTR mice with adoptive transfer of in vitro polarized T_H2 cells supported the conclusion that antigen presentation by pulmonary dendritic cells to effector/memory T_H2 cells led to production of T_H2 cytokines and the development of allergic airway inflammation.^14,19 Furthermore, it was observed that dendritic cell–derived CCL17 and CCL22 was critical for allergen-induced airway inflammation. ¹⁵ However, in allergen-driven models of airway inflammation such as these, it is difficult to dissect the contribution by dendritic cells that occurs as a consequence of their ability to present antigen and thus trigger cytokine production from the responsiveness of dendritic cells to the cytokines that are consequently produced. In this study allergen was not used to drive the inflammatory response, making the data easier to interpret. In combination with the above studies, our findings support a model in which dendritic cell–mediated antigen presentation to memory/effector T_H2 cells induces their production of IL-13 and then the IL-13 helps to establish allergic inflammation by causing pulmonary dendritic cells and alveolar macrophages to produce chemokines.

Clinical implications: This is study might lead to the development of novel cell-targeted asthma therapies.

Abbreviations used

AHR

Airway hyperreactivity

APC

Allophycocyanin

BAL

Bronchoalveolar lavage

DC-SIGN

Dendritic cell–specific intercellular adhesion molecule–grabbing nonintegrin

DT

Diphtheria toxin

DTR

Diphtheria toxin receptor

IFN-γR

IFN-γ receptor

IL-4R

IL-4 receptor

IL-13R

IL-13 receptor

PE

Phycoerythrin