CXCL17 is a major chemotactic factor for lung macrophages

Abstract

Chemokines are a superfamily of chemotactic cytokines that direct the movement of cells throughout the body under homeostatic and inflammatory conditions. The mucosal chemokine CXCL17 was the last ligand of this superfamily to be characterized. Several recent studies have provided greater insight into the basic biology of this chemokine and have implicated CXCL17 in several human diseases. We sought to better characterize CXCL17's activity in vivo. To this end, we analyzed its chemoattractant properties in vivo and characterized a Cxcl17 (-/-) mouse. This mouse has a significantly reduced number of macrophages in its lungs compared with wild-type mice. In addition, we observed a concurrent increase in a new population of macrophage-like cells that are F4/80(+)CDllc(mid). These results indicate that CXCL17 is a novel macrophage chemoattractant that operates in mucosal tissues. Given the importance of macrophages in inflammation, these observations strongly suggest that CXCL17 is a major regulator of mucosal inflammatory responses.

Figure 1. CXCL17's Expression Pattern In Germ Free Mouse Mucosal Tissues Demonstrates That CXCL17 is a hybrid (Both an Inflammatory and Homeostatic Chemokine)

Q-PCR was used to determine CXCL17 expression levels in tissue samples from germ free (GF) and Specific Pathogen Free (SPF) mice, which were compared to tissues from mice housed in a standard vivarium. These data suggest that CXCL17 is expressed under inflammatory conditions in many mucosal tissues, but has homeostatic expression in the colon and trachea. X-axis, tissues; y-axis, CXCL17 expression units; Sm Intest, small intestine.

Figure 2. Intraperitoneal Injection of rmCxcl17 Increases Recruitment of Macrophages 48 Hours Post Injection

After injecting rmCxcl17 into the peritoneal cavities of three mice, a significant increase in macrophages (Mφ), but not dendritic cells (DCs) was observed (F.-H.) compared to mice injected with PBS vehicle (C.-E.). The specific cell populations were determined by staining the cells with cell specific antibodies (F4/80, macrophages; CDllc, DCs; Gr-1, granulocytes). This experiment was performed twice with n=3 mice per group. See also Supplementary Figure 2.

Figure 3. CXCL17 is An Important Chemoattracting Factor In The Lung

A. Single cell suspensions of lung cells from WT and Cxcl17^-/- mice showed differences in their chemotactic activity in response to recombinant Cxcl17. The Cxcl17^-/- mouse lung cells demonstrate a significant reduction in the number of chemotaxing cells compared to WT mouse lung cells. Treatment with Bordatella pertussis toxin (PTX) reduced chemotaxis of WT lung cells. This experiment was performed with n=3 mice per group. **p=0.03. B. The endothelial cells surrounding blood vessels of the lung stain positive for CXCL17. The arrows show positive CXCL17 staining at the luminal facing side of the respiratory mucosa and the endothelial cells.

Figure 4. Altered Lung Macrophage Populations Observed In the Cxcl17^-/- Mice Compared to WT Mice

Cells collected from lungs of WT (A.) and Cxcl17^-/- mice (B.) were stained with fluorophore conjugated antibodies for analysis by flow cytometry. When we analyzed the macrophage populations (A-C.) we observed a significant decrease in the percentage of alveolar macrophages in Cxcl17^-/- lungs compared to WT mouse lungs (C.) *p=0.05. We also observed a significant increase in a previously unidentified population of macrophage-like cells that are F4/80⁺CDllc^mid (A-B., D.) *p=0.04. A-B are representative FACS plots from two separate experiments with n=at least 4 mice per group. IMs, interstitial macrophages; AMs, alveolar macrophages; DCs, dendritic cells.

Figure 5. The Expression of Two Lung Specific Macrophage Markers Is Significantly Reduced in Cxcl17^-/- Mice

The expression of lung macrophage specific markers (36), (37), (35) were measured using qPCR in single cell suspensions from WT and Cxcl17^-/- lungs. The expression of two markers, ABCC3 (A.) and NR1D1 (B.) was significantly reduced in the cells isolated from Cxcl17^-/- mouse lungs. ABCC3 *p=0.02; NR1D1 *p=0.03. Each experiment was performed twice with n=2 mice per group. Y axis, relative expression units.