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Comparative Study
. 2011 Feb;12(2):167-77.
doi: 10.1038/ni.1984. Epub 2011 Jan 9.

Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5+ T(H)2 cells

Sabina A Islam  1 , Daniel S Chang, Richard A Colvin, Mike H Byrne, Michelle L McCully, Bernhard Moser, Sergio A Lira, Israel F Charo, Andrew D Luster
Affiliations
Comparative Study

Mouse CCL8, a CCR8 agonist, promotes atopic dermatitis by recruiting IL-5+ T(H)2 cells

Sabina A Islam et al. Nat Immunol. 2011 Feb.

Abstract

Mouse CCL8 is a CC chemokine of the monocyte chemoattractant protein (MCP) family whose biological activity and receptor usage have remained elusive. Here we show that CCL8 is highly expressed in the skin, where it serves as an agonist for the chemokine receptor CCR8 but not for CCR2. This distinguishes CCL8 from all other MCP chemokines. CCL8 responsiveness defined a population of highly differentiated, CCR8-expressing inflammatory T helper type 2 (T(H)2) cells enriched for interleukin (IL)-5. Ccr8- and Ccl8-deficient mice had markedly less eosinophilic inflammation than wild-type or Ccr4-deficient mice in a model of chronic atopic dermatitis. Adoptive transfer studies established CCR8 as a key regulator of T(H)2 cell recruitment into allergen-inflamed skin. In humans, CCR8 expression also defined an IL-5-enriched T(H)2 cell subset. The CCL8-CCR8 chemokine axis is therefore a crucial regulator of T(H)2 cell homing that drives IL-5-mediated chronic allergic inflammation.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Mouse CCL8 RNA and protein are detected in normal mouse skin. (a) Relative positioning of the six MCP-cluster chemokine genes on human chromosome 17 and mouse chromosome 11, modified from the Ensembl site. Black, known orthologs; blue, non-orthologs; red, presumed orthologs (focus of our study here). (b) Percent identity/percent similarity of amino acids using the GenBank sequence extending from the start codon to the stop codon, calculated using the EMBOSS Needleman-Wunsch algorithm. m, Mouse; h, human. (c) RNA hybridization blot comparing mRNA expression of MCP-family chemokines and CCL11 (eotaxin-1) in pooled organs of normal BALB/c mice, conducted once. (d) Representative immunofluorescence staining of normal wild-type (WT) C57BL/6 mouse skin and Ccl8−/−Ccl12−/− C57BL/6 mouse skin with a primary polyclonal antibody to mouse CCL8 and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Data are reflective of at least six independent experiments from three or more mice.
Figure 2
Figure 2
Mouse CCL8 induces migration and calcium flux in TH2-R2A cells. (a) Migration of mouse BMDMs to mouse CCL8 and CCL2. (b) Migration of NK cells to mouse CCL8, CCL2, CCL4 and CXCL11. (c) Migration of lymph node CD4+ and CD8+ T cells to mouse CCL8 and CXCL12. (d) Representative cytokine profiles and assays of TH1-R1 cell migration to mouse CCL8. Migration to mouse CXCL11 and CXCL12 was a positive control. (e) Representative cytokine profiles and assays of TH2-R1 cell migration to mouse CCL8. Migration to mouse CCL22 and CXCL12 was a positive control. Data in a–e are representative of three or more experiments. (f) Dose-response migration of TH2-R2A cells to mouse CCL8 (representative of more than 10 experiments) and PTX-mediated inhibition of mouse CCL8–induced migration (one of three independent experiments shown). Results in a–f are shown as mean ± s.e.m. (g) Analysis of TH2-R2A cells for calcium flux to 40 nM mouse CCL8 (downward arrow indicates time of addition); representative of eight separate experiments. (h) Analysis of dose-response of TH2-R2A cells to mouse CCL8 in calcium flux assays; representative of two independent experiments. Arrows pointing up indicate onset of control calcium flux response to ionomycin (1 μg/ml) at each concentration of chemokine.
Figure 3
Figure 3
CCR8 is required for mouse CCL8-induced TH2-R2A cell migration. (a) CC-chemokine receptor mRNA enrichment measured by QPCR in TH2-R2A cells that migrated to mouse CCL8 in Transwell assays relative to medium alone; representative of three experiments. (b) Kinetics of mouse Ccr8 and Ccl1 mRNA induction in TH2-R2A cells generated by repeat rounds of polarization and subsequent activation with antibodies to CD3 and CD28. Data are normalized to β2-microglobulin and presented on a scale of 0 to 100; representative of three experiments. (c) Comparison of Ccr8 mRNA expression in leukocyte subsets assayed for mouse CCL8 migration; representative of two to five experiments. (d) Comparison of chemotaxis of wild-type (WT), Ccr8-, Ccr2- and Ccr5-deficient TH2-R2A cells to mouse CCL8 or mouse CCL1; control migration to the CCR4 agonist CCL22 is also shown. One of three experiments is shown. (e) Dose-dependent inhibition of wild-type TH2-R2A cell migration to mouse CCL8 using a neutralizing polyclonal mouse CCR8 antibody (nAb), and migration to control CCL22 chemokine at various concentrations of antibody. One of two experiments is shown. Results in d and e are shown as mean ± s.e.m.
Figure 4
Figure 4
Mouse CCL8 is a specific agonist of mouse and human CCR8. (a) Dose-response chemotaxis assay of mouse Ccr8–transfected Baf/3 cells to mouse CCL8 and CCL1. Untransfected Baf/3 cells migrated only to CXCL12 (data not shown). (b) Calcium flux of mouse Ccr8–transfected cells to mouse CCL8 but not to the CCR4 agonist mouse CCL22. Mouse CCL8 did not induce calcium flux of untransfected Baf/3 cells, but positive control CXCL12 did. (c) Dose-response calcium flux of mouse Ccr8–transfected cells to mouse CCL8. (d) Cross-desensitization of mouse Ccr8–transfected cells to mouse CCL8 and CCL1. (e) Dose-response chemotaxis assay of human CCR8 receptor–transfected 4DE4 cells to mouse CCL8 and CCL1. Nontransfected 4DE4 cells did not migrate to either ligand (data not shown). (f) Specificity of mouse CCL8–induced calcium flux signaling in human CCR8–transfected cells, as shown by desensitization to repeat signaling by mouse CCL8; lack of flux of untransfected 4DE4 cells to mouse CCL8. (g) Human CCL8 did not induce calcium flux in CCR8-transfected cells. (h) Cross-desensitization of human CCR8–transfected cells by mouse CCL8 and human CCL1. Data are representative of at least three independent experiments (error bars (a–e), s.e.m).
Figure 5
Figure 5
Ccr8−/− and Ccl8−/−Ccl12−/− mice have decreased skin inflammation in a model of chronic atopic dermatitis. Histological analysis of skin was done on day 50, 24 h after the last of three 1-week rounds of topical sensitization with PBS or OVA. (a) Hematoxylin and eosin (H&E) staining of wild-type (WT) and Ccr8−/− mice sensitized with PBS or OVA. Far right, immunohistochemical analysis of CD3+ T cells in sensitized skin of wild-type and Ccr8−/− mice. Data are representative of three to seven experiments. (b) H&E staining of wild-type and Ccl8−/−Ccl12−/− mice sensitized with PBS or OVA. Far-right panels are at higher magnification, showing eosinophils and characteristic spongiosis of keratinocytes in wild-type sensitized mice; representative of three experiments. (c) H&E staining of OVA-sensitized Ccr4−/− and Ccl12−/− mice; representative of two experiments. (d) Skin thickness and leukocyte counts in wild-type and Ccr8−/− mice shown as mean ± s.e.m.; n = 6–8 mice per group. *P < 0.00001, **P = 0.004, ***P = 0.002, ****P = 0.02 and *****P = 0.005 for OVA-sensitized Ccr8−/− versus wild-type mice. HPF, high-powered field. (e) Skin thickness and leukocyte counts in OVA-sensitized wild-type, Ccl8−/−Ccl12−/−, Ccl12−/− and Ccr4−/− mice. *P = 0.0004, **P = 0.02, ***P < 0.00001 and ****P < 0.00001 for gene-deficient versus wild-type mice. Data are shown as mean ± s.e.m.; n = 6–9 mice per group. (f) H&E-stain of OVA-sensitized wild-type mice treated with CCL1-neutralizing antibody (left) or isotype control (right) during last week of epicutaneous sensitization. (g) Skin thickness and leukocyte counts in mice from f; n = 4–5 mice per group in two independent experiments. Scale bar = 100 μm in all photomicrographs except for far-right panels in b, where scale bar = 20 μm.
Figure 6
Figure 6
Ccr8−/− mice have decreased production of IL-5, IL-25 and eosinophil-active chemokines in allergen-sensitized skin. (a) QPCR measurements of transcripts for eosinophil-attracting chemokines. (b) Summary of QPCR measurements of chemokine mRNA expression in skin biopsies of wild-type (WT; *P = 0.01, **P = 0.0004 and ***P = 0.003 for PBS versus OVA) and Ccr8−/− (*P = 0.04 and **P = 0.03 for PBS versus OVA) mice after topical sensitization. (c) QPCR measurements of transcripts for TH2 cytokines. NS, not significant. (d) Serum OVA-specific IgE and IgG1 concentrations measured by enzyme-linked immunosorbent assay (ELISA) in sensitized mice. (e) QPCR of transcripts for CCR8 ligands. (f) Representative immunofluorescence analysis of mouse CCL8 protein expression in PBS-sensitized epidermis (epi) and dermis (derm) and OVA-sensitized epidermis and dermis of wild-type and Ccr8−/− mice from two experiments; n = 4 mice per group. (g, h) QPCR measurements of CCR4 and CCR10 ligands in skin biopsies of wild-type and Ccr8−/− mice after topical sensitization. (i) TH2 cytokine production by DLN cells after ex vivo stimulation with OVA protein and CD3, measured by ELISA (n = 8–10 mice per group). Pooled data from three experiments are shown. Results in b and i are shown as mean ± s.e.m. All data were obtained 50 d after the initiation of sensitization and are reflective of at least three experiments, except for f.
Figure 7
Figure 7
Competitive in vivo homing of adoptively transferred OVA-specific wild-type and Ccr8−/− TH2 and TH1 cells in OVA-sensitized mice. OVA(323–339) peptide–specific TCR-transgenic (OTII) Thy1.1 and Ccr8−/− OTII Thy1.2 TH2 cells were transferred into OVA-sensitized Thy1.1 × Thy1.2 mice on day 41. Separate mice received OTII Thy1.1 TH1 cells and Ccr8−/− OTII Thy1.2 TH1 cells. Twenty-four hours later, recipient mice underwent topical sensitization with OVA for 96 h, and sensitized skin, DLN and spleen cells were analyzed by flow cytometry. (a) CD4+ T cells recovered from organs at time of collection. Top, mice that received TH2 cells; bottom, mice that received TH1 cells. (b) Homing index was calculated as the ratio of wild-type Thy1.1 to Ccr8−/− Thy1.2 cells, corrected for input ratio at time of transfer. Summary of competitive in vivo homing of wild-type and Ccr8−/− TH2 (left; n = 13–16 mice) and TH1 (right; n = 6 mice) cells from three or more experiments. (c) In vivo proliferation of transferred OTII Thy1.1 wild-type and OTII Thy1.2 Ccr8−/− TH2 cells in response to antigen was assessed by injecting sensitized recipient mice with BrdU intraperitoneally 24 h before organ collection in experiments set up as above. Left and middle, representative BrdU staining of wild-type and Ccr8−/− T cells isolated from lymph nodes. Right, summary of three experiments comparing in vivo proliferation of wild-type and Ccr8−/− T cells measured by BrdU uptake; n = 12 mice. (d) Amounts of TH1 cell–active (Cxcl9 and Cxcl10) and TH2–cell active (Ccl1, Ccl8, Ccl17 and Ccl22) chemokine mRNA in unsensitized and OVA-sensitized wild-type day-50 skin DLNs and spleen; n = 6 mice. ND, not detected. (e) Comparison of steady-state mRNA expression of Ccl1, Ccl8 and Ccl21 in lymph nodes draining various organs in normal 6- to 8-week-old mice; pooled data from n = 3–7 mice are shown as mean ± s.e.m. (f) Ccl1 and Ccl8 mRNA expression in organ library of representative C57BL/6 mouse.
Figure 8
Figure 8
Mouse CCL8–responsive TH2-R2A cells are enriched for IL-5, IL-25R, TNF and OX40. (a) Flow cytometry of IL-4 and IL-5 by ICS in TH2-R1, TH2-R2, and TH2-R2A cells; representative of six experiments. Below each plot is the chemotactic index of the various TH2 cells to mouse CCL8 compared to medium alone in migration assays. (b) Gata3 mRNA expression in paired TH2-R1 and TH2-R2 cells from four independent experiments; *P = 0.02. (c) Correlation of Ccr8 and Tnfrsf4 (OX40) mRNA expression in TH2-R2A cells. (d) Flow cytometry of TNF and IL-9 by ICS in TH2-R2A cells; representative of three to six experiments. (e) Enrichment of TH2-associated cytokines and receptors in TH2-R2A cells that migrated to mouse CCL8 and CXCL12 relative to medium in Transwell assay; schematic of assay shown on right. Representative of at least three experiments. (f) QPCR analysis of Tnf and Tnfrsf4 mRNA expression in skin biopsies of wildtype and Ccr8−/− mice after topical sensitization. Pooled data from two to three independent experiments with n = 5–9 mice per group are shown; *P = 0.03 and **P = 0.03 in OVA-sensitized wild-type versus Ccr8−/− mice. (g) Flow cytometry of IL-5 and IL-4 by ICS after phorbol myristate acetate–ionomycin activation of peripheral blood CD4+ T cells from healthy human donors. Data from same sample were gated on CCR8+CD4+ T cells (left) and bulk total CD4+ T cells (right); reflective of data from seven donors. (h) Summary of IL-5 and IL-4 cytokine production by circulating fresh CD4+ T cell subsets from healthy human donors. Results are shown as mean ± s.e.m., reflecting data from n = 3–7 donors.
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