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Originally published online as doi:10.1189/jlb.1106686 on March 5, 2007

Published online before print March 5, 2007
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(Journal of Leukocyte Biology. 2007;81:1434-1444.)
© 2007 by Society for Leukocyte Biology

Eosinophils develop in distinct stages and are recruited to peripheral sites by alternatively activated macrophages

David Voehringer*,1, N. van Rooijen{dagger} and Richard M. Locksley*,2

* Howard Hughes Medical Institute, Departments of Medicine and Microbiology/Immunology, University of California San Francisco, San Francisco, California, USA; and
{dagger} Department of Molecular Cell Biology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands

2 Correspondence: UCSF, Box 0654, S 1032B, 513 Parnassus Ave., San Francisco, CA 94143-0654, USA. E-mail: locksley{at}medicine.ucsf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eosinophils are associated with allergic diseases and helminth infections. Development of these cells and recruitment to peripheral tissues are only partially understood. Distinct stages of eosinophil development in fetal liver, bone marrow, and blood could be identified using IL-4 reporter mice and mAb against FIRE, Siglec-F, and CCR3. Immature eosinophils were present in the fetal liver and could reconstitute the eosinophil compartment in irradiated recipient mice. In adult mice, eosinophil maturation proceeded from CCR3– to CCR3+ cells in the bone marrow and was accompanied with changes in the transcriptional profile. Eosinophils appeared as activated cells in lung, thymus, lymph nodes, and Peyer’s patches but remained in a resting state in bone marrow, blood, and spleen. Mixed bone marrow chimeras revealed that recruitment to lung and peritoneum was dependent on Stat6 expression in noneosinophils. Alternatively activated macrophages contributed substantially to tissue recruitment of eosinophils, providing a novel basis for development of therapeutic approaches to lower tissue eosinophilia.

Key Words: maturation • recruitment


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eosinophils are associated with immune responses directed against parasites or allergens and contribute to immune pathology and parasite clearance [1 ]. Our current knowledge about eosinophil development is still limited and based mainly on in vitro cultures of human cord blood or mouse bone marrow cells using various cytokine cocktails. These studies revealed that IL-5 plays an important role during eosinophil maturation and prolongs their survival in peripheral tissues. IL-5 transgenic mice have greatly increased numbers of eosinophils [2 ]. However, IL-5 is not essential for eosinophil development, as IL-5-deficient and IL-5 receptor (IL-5R)-deficient mice have near-normal levels of eosinophils under homeostatic conditions as compared with wild-type mice [3 , 4 ]. Three transcription factors, Gata-1, PU.1, and c/EBP, regulate differentiation of multipotent precursor cells to mature eosinophils [5 ]. It is striking that deletion of a high-affinity binding site for Gata-1 in the proximal gata-1 promoter results in complete and selective loss of the eosinophil lineage [6 ].

Recruitment of eosinophils to peripheral sites can be mediated efficiently by chemokines. CCR3 is a chemokine receptor expressed mainly on mouse eosinophils and mediates their migration toward gradients of CCL11 (eotaxin-1) and CCL24 (eotaxin-2) [7 ]. The transcription factor Stat6 regulates expression of CCL11, CCL24, and CCL17 (thymus and activation-regulated chemokine) in the allergic lung, and Stat6-deficient mice are largely resistant to allergen- or parasite-induced lung eosinophilia [8 ]. We have shown previously that Stat6 expression was required in hematopoietic cells to mediate eosinophil recruitment to the lung [9 ]. However, eotaxins are not essential for eosinophil recruitment, as experiments using CCR3-deficient mice have shown that this receptor accounts for only half of the recruitment to the allergic lung [10 ]. Therefore, the synthesis or release of other eosinophil chemotactic factors, such as anaphylatoxins or arachidonate metabolites, might also be regulated by Stat6. Another possibility would be that Stat6 is required in eosinophils themselves, where it might regulate expression of chemokine receptors, as it has been shown for CD4 T cells [11 ].

Eosinophils can be identified by their characteristic staining with aniline dyes, first described by Paul Ehrlich in 1879, or by immunohistochemical staining for granular proteins such as major basic protein (MBP) or eosinophil peroxidase (EPX), which are highly expressed in eosinophils. However, these staining techniques cannot distinguish between different states of maturation or activation. Here, we describe surface markers that could be useful in phenotypic differentiation of eosinophil subsets by flow cytometry. Further, we analyze changes in gene expression profiles during maturation and activation of ex vivo-isolated eosinophils. Using mixed bone marrow chimeras, we show that Stat6 is not required in eosinophils themselves for their recruitment to lung and peritoneum. Finally, lack of macrophages resulted in greatly impaired eosinophil recruitment to lung and peritoneum, suggesting an important cross-talk between effector cells of the innate immune system during the establishment of inflammatory responses in peripheral tissues.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
IL-4 reporter mice (4 get mice) have been described and were used on a BALB/c background [12 ]. In brief, these mice carry an internal ribosome entry site-enhanced green fluorescent protein (eGFP) construct inserted after the stop codon of the IL-4 gene. IL-5 transgenic mice (Strain NJ.1638) on a BALB/c background were obtained from James Lee (Mayo Clinic, Scottsdale, AZ, USA) and crossed to 4 get mice [13 ]. Eosinophil-deficient {Delta}dblGATA mice were obtained from Craig Gerard (Harvard Medical School, Boston, MA, USA) and crossed to 4 get mice [14 ]. BALB/c mice, Stat6-deficient mice, IL-4R{alpha}-deficient mice, Ly5.1 mice (B6.SJL-Ptprca Pepcb/BoyJ), and osteopetrotic (op/op) mice (B6C3Fe a/a-Csf1op/J) [15 ] were obtained from The Jackson Laboratory (Bar Harbor, ME, USA).

Mixed bone marrow and fetal liver chimeras
Fetal liver cell suspensions were prepared from Embryonic Day 16.5 (E16.5) fetuses of 4 get/C57BL/6 mice and stained with Ter119 mAb. eGFP+Ter119– cells were sorted on a MoFlo cell sorter (Dako Cytomation, Fort Collins, CO, USA), mixed at different ratios with total bone marrow cells from wild-type Ly5.1 mice, and 3 x 105 total cells were transferred to lethally irradiated (1100 rad), wild-type Ly5.1 recipient mice. Bone marrow chimeras were generated by transfer of 106 bone marrow cells from Ly5.1 mice together with 106 bone marrow cells from Stat6-deficient mice (Ly5.2) into lethally irradiated recipient mice, which were given water containing antibiotics (2 g/l neomycin sulfate, 100 mg/l polymyxin B, Sigma-Aldrich, St. Louis, MO, USA) and SCID MD tablets (Bioserv, Frenchtown, NJ, USA), housed in the specific pathogen-free animal facility at University of California San Francisco (CA, USA) and used for experiments 7 weeks after reconstitution.

Nippostrongylus brasiliensis infection
Third-stage larvae (L3) of N. brasiliensis were recovered from the cultured feces of infected rats, washed extensively in saline (37°C), and injected (500 organisms) into mice s.c. at the base of the tail. Mice were provided antibiotic-containing water (2 g/l neomycin sulfate, 100 mg/l polymyxin B sulfate, Sigma-Aldrich) for the first 5 days after infection.

Flow cytometry
Single-cell suspensions of indicated organs were washed in FACS buffer (PBS/2% FCS/1 mg/ml sodium azide), incubated with anti-CD16/CD32-blocking antibody (2.4G2, BD PharMingen, San Diego, CA, USA) for 5 min at room temperature, and stained with the corresponding antibody mixtures. The following mAb and streptavidin were used: biotinylated anti-FIRE (a gift from I. Caminschi, Walter and Eliza Hall, Melbourne, Australia), PE-labeled anti-CCR3 (R&D Systems, Minneapolis, MN, USA), APC-labeled streptavidin (Molecular Probes, Eugene, OR, USA), APC-labeled anti-F4/80 (MF48005, Caltag Laboratories, Burlingame, CA, USA), biotinylated anti-CD34 (RAM34, eBioscience, San Diego, CA, USA), biotinylated anti-IgE (R35-72), biotinylated anti-CD8 (53-6.7), PerCP/Cy5.5-labeled anti-CD4 (L3T4), PE-labeled or purified anti-Siglec-F (E50-2440), biotinylated anti-CD62 ligand (CD62L; MEL-14), PE-labeled, anti-paired Ig-like receptor A/B (anti-Pir-A/B; 6C1), Alexa647-labeled anti-CCR3 (83103), PE-labeled Ter-119, biotinylated anti-Ly5.1 (A20), PerCP/Cy5.5-labeled anti-Ly5.2 (104), biotinylated anti-IL-4R{alpha} (M1), PE-labeled anti-CD11b (M1/70), biotinylated anti-Sca-1 (E13-161.7), APC-labeled anti-c-Kit (2B8), and APC-labeled goat antirat Ig. Antibodies were from BD PharMingen unless otherwise indicated. Cells were analyzed on FACSCalibur and LSRII instruments (BD Immunocytometry Systems, San Jose, CA, USA).

Microarray analysis
Total RNA was isolated from CD4–eGFP+FIRE+CCR3+ and CD4–eGFP+FIRE+CCR3– eosinophil populations sorted from bone marrow of naive 4 get mice, CCR3+ eosinophils sorted from spleens of naïve 4 get/IL-5 transgenic mice, and total eosinophils sorted from lungs of N. brasiliensis-infected 4 get mice, using the Total RNA isolation kit (Fluka, Buchs, Switzerland) and amplified by two rounds of in vitro transcription using the Amino Allyl MessageAmpTM aRNA kit (Ambion, Austin, TX, USA). Aminoallyl-UTP was incorporated during the second round of amplification, and 5 µg amplified RNA was coupled to Cy3 and Cy5 fluorescent dyes (CyScribeTM dye-labeling kit, Amersham Biosciences, Peapack, NJ, USA). Probes were hybridized to spotted glass oligonucleotide (70-mer) arrays, which cover just over 16,400 unique genes (Mouse Genome Set, Version 2.0, Qiagen, Germany), according to the following protocol: http://arrays.ucsf.edu/protocols/cdna_transcription_and_coupling.pdf. Slides were scanned on an Axon 4000B scanner using Genepix 3.0 software (Axon Instruments, Inc., Molecular Devices Corp., Union City, CA, USA) and normalized by "lowess" normalization on the pixel medians without background subtraction using Acuity 4.0 software (Axon Instruments, Inc.). "A" values indicate the total signal intensity of a given spot on the microarray and are calculated as A = 1/2*log2(R*G), where R and G give the intensity for the Cy5 and Cy3 channels, respectively. "M" values indicate the difference in gene expression on a log2 scale and are calculated as M = log2(R/G; e.g., M=1 indicates a twofold higher expression in the sample, which was labeled with the Cy5 dye, compared with the sample labeled with the Cy3 dye, and M=0 indicates equal expression in both samples). Genes with an M value of more than 1 or less than –1 and an A value of more than 8 in at least one of the arrays were further selected for analysis. The results have been deposited at the Gene Expression Omnibus (GEO) database with Accession Number GSE5411.

RT-PCR analysis
Total RNA was isolated from CD4–eGFP+FIRE+CCR3+- and CD4–eGFP+FIRE+CCR3–-sorted eosinophils from the bone marrow of naïve 4 get mice using the Total RNA isolation kit (Fluka). cDNA was generated using the Superscript II® RT kit (Invitrogen, Carlsbad, CA, USA) and used as a template for quantitative RT-PCR analysis with the following primer pairs: hypoxanthine guanine phosphoribosyl transferase (HPRT): HPRT-1 (5'-gttggatacaggccagactttgttg-3') and HPRT-2 (5'-gagggtaggctggcctataggct-3'); Axl: Axl-1 (5'-ctgagattccaggacctaag-3') and Axl-2 (5'-tcgttaactggagcctcttg-3'); Ficolin A (FCNA): Fcna-1 (5'-ggagaaaggcgatacaggag-3') and Fcna-2 (5'-ttggactggtggcagttgtg-3'); transferrin (TRF): Trf-1 (5'-tctcgttgagaaaggagatg-3') and Trf-2 (5'-ccaggagtcgtgaggttgag-3'); MBP-2: MBP2-1 (5'-gagcgtctgctcttcatctg-3') and MBP2-2 (5'-gaacttccatcaacccatcg-3'); EPX: epx-1 (5'-catacatgaaggtggcatcg-3') and epx-2 (5'-ctctcgaaaccgtggtgatg-3'). PCR reactions were performed with 56°C annealing temperature and 60 s extension time at 72°C using the SYBR® Green Taq ReadyMixTM (Sigma-Aldrich) and Lightcycler PCR machine (Roche, Switzerland). Reactions were performed in triplicates and normalized to HPRT. Quantitative RT-PCR of sorted peritoneal macrophages (CD11b+F4/80+) was performed with the following primer pairs: Ym1: Ym1-1 (5'-tggaattggtgcccctacaa-3') and Ym1-2 (5'-aacttgcactgtgtatattg-3'); Fizz1: Fizz1-1 (5'-ccatagagattatcgtggag-3') and Fizz1-2 (5'-tggtccagtcaacgagtaag-3'); CCL24: CCL24-1 (5'-gctctgctacgatcgttg-3') and CCL24-2 (5'-agcaaacttggttctcactg-3'); arginase I: arg-1 (5'-gtatgacgtgagagaccacg-3') and arg-2 (5'-ctcgcaagccaatgtacacg-3'); HPRT-1 and HPRT-2 (see above). The annealing temperature was 61°C, and SYBR Green incorporation was read at 81°C for HPRT and 84°C for the other genes. The reactions were performed in triplicates, analyzed on a DNA Engine Opticon 2® system (MJ Research, Waltham, MA, USA), and normalized to HPRT.

Macrophage depletion with clodronate liposomes
Cl2MDP (clodronate) was a gift of Roche Diagnostics GmbH (Mannheim, Germany). Clodronate liposomes were generated as described [16 ]. Clodronate liposomes (200 µl) or PBS were injected i.p. 1 day before and 7 days after N. brasiliensis infection. Mice were analyzed on Day 11 after infection.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Constitutive IL-4 expression in fetal liver cells marks eosinophil precursors
Eosinophils are known to express low constitutive levels of IL-4 in mouse and man [17 , 18 ]. These cells appear as eGFP+ cells in sensitive IL-4 reporter mice (4 get mice) [12 , 19 ]. As it is currently unclear at what stage of development committed eosinophil precursors arise, fetal liver cells of 4 get mice were analyzed by flow cytometry. The first IL-4/eGFP+ cells could be identified at approximately E13.5, and a substantial population of ~1% of total fetal liver cells was observed at E16.5 (Fig. 1A and data not shown). These IL-4/eGFP+ cells were mainly c-Kit–Sca-1–CD34–CCR3–Siglec-F+ and not derived from the mother, as they were also present in fetuses from heterozygous matings, where only the father carried the 4 get reporter allele (Fig. 1A and data not shown). To assess whether this population of Ter119–IL-4/eGFP+ cells contained eosinophil-committed precursor cells, these cells were sorted to high purity, mixed at different ratios with total bone marrow cells from Ly5.1-congenic mice to facilitate reconstitution, and transferred into lethally irradiated Ly5.1 recipient mice. Eight weeks after reconstitution, these mixed chimeras were analyzed for the ratio of fetal liver cell-derived (Ly5.2+IL-4/eGFP+):bone marrow-derived (Ly5.1+) eosinophils in the peripheral blood by flow cytometry. Analysis of blood cells from mice that had received a 1:1 ratio of fetal liver:bone marrow cells showed that ~100 times more eosinophils were generated from the sorted fetal liver cells than from the cotransferred bone marrow cells (Fig. 1B) . A strong bias toward progeny of fetal liver cells was still observed when fetal liver and bone marrow cells were mixed at a ratio of 0.05:1 before reconstitution. Furthermore, all fetal liver-derived eosinophils remained IL-4/eGFP+ in reconstituted recipient mice (data not shown). The analysis of CD4+ T cells or total PBMC revealed a less-pronounced bias toward fetal liver cell-derived cells. This indicates that the IL-4/eGFP+-sorted fetal liver cells contained mainly eosinophil precursors. However, it remains unclear whether adult eosinophilopoiesis is dependent on a precursor population present in the fetal liver. Selective depletion of this population during development would be required to address this question.


Figure 1
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  		Figure 1. Eosinophil precursors in the fetal liver. (A) Single-cell suspensions of fetal liver cells from E16.5 fetuses of 4 get mice were stained for Ter119 (to exclude erythrocyte precursors), c-Kit, CD34, Sca-1, CCR3, Siglec-F, or CD8 (as isotype control) and analyzed by flow cytometry. Dot-plots are gated on Ter119-negative cells and are representative of three independent experiments. (B) Ter119–eGFP+ fetal liver cells (Ly5.2) were sorted to 98% purity, mixed with wild-type (Ly5.1) bone marrow cells at the indicated input ratios (Ly5.2:Ly5.1), and transferred to lethally irradiated recipient mice. The peripheral blood samples were analyzed 8 weeks after reconstitution for the ratio of eosinophils derived from fetal liver cells (Ly5.2):wild-type bone marrow (BM; Ly5.1) by staining for Ly5.1, Ly5.2, CD4, and CCR3 and gating on CD4–CCR3+SSChi cells within the Ly5.1+ or Ly5.2+ population. The Ly5.2:Ly5.1 ratio of CD4+ cells and total PBMC was analyzed in parallel. The experiment has been repeated with a 1:10 input ratio and similar results.

 
Analysis of eosinophil maturation by flow cytometry
Currently, three relatively useful surface markers expressed on murine eosinophils are the chemokine receptor CCR3, the F4/80-related receptor FIRE, and the inhibitory receptor Siglec-F [9 , 20 ]. Using flow cytometry and mAb against these receptors in combination with IL-4/eGFP expression allowed us to distinguish different stages of eosinophil development in vivo. Eosinophil precursors in the fetal liver were negative for CCR3; however, they expressed high levels of FIRE and low but detectable levels of Siglec-F (Fig. 2A ). In the bone marrow of adult mice, which were uninfected or analyzed after infection with the helminth parasite N. brasiliensis to induce eosinophilia, the level of FIRE expression started to decrease, whereas Siglec-F and CCR3 levels increased, indicated by the change of the mean fluorescence intensity of these markers. In the peripheral blood and spleen, eosinophils appeared as a homogenous population, which stained positively for all three markers. Eosinophils recovered from the lung tissue, through which the parasite larvae migrate, showed greatly reduced FIRE expression and also expressed less CCR3 as compared with eosinophils in the blood. In contrast, Siglec-F was increased further on lung eosinophils and might therefore serve as an activation marker for murine eosinophils in tissues (Fig. 2A) .


Figure 2
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  		Figure 2. Surface marker expression during maturation and activation. (A) Cells from the fetal liver of naïve mice, bone marrow, and blood of naïve mice and bone marrow, blood, and lung of N. brasiliensis(N.b.)-infected mice (Day 10) were stained for CD4 and FIRE, Siglec-F, or CCR3 and analyzed by flow cytometry. Gated CD4–SSChi cells, which represent eosinophils mainly, are displayed in the dot-plots. (B) Cells from indicated tissues of Day 10 N. brasiliensis-infected mice were stained for CD4, CD62L, and CCR3 or CD4, Pir-A/B, and CCR3. Gated CD4–GFP+SSChi cells (eosinophil gate) are displayed. (C) Cells from indicated tissues were stained for CD4, CD62L, and Siglec-F and gated as in B. All results are representative of several independent experiments. LN, Lymph nodes; PP, Peyer’s patches.

 
Eosinophils in the bone marrow could be separated phenotypically into CCR3– and CCR3+ cells. Double-staining revealed that during their maturation from CCR3– to CCR3+ cells, eosinophils up-regulated the expression of CD62L and Pir-A/B, a matched activating/inhibitory receptor pair with binding affinity for MHC Class I [21 ] (Fig. 2B) . It is interesting that double-staining for CD62L, which is down-regulated on the surface of activated eosinophils [9 ], and Siglec-F demonstrated that eosinophils in lymph node, Peyer’s patches, and thymus showed an activated phenotype (CD62Llo Siglec-Fhi), which was comparable with eosinophils in the lungs of parasite-infected animals. In contrast, eosinophils in bone marrow, blood, and spleen appeared to be CD62Lhi Siglec-Flo (Fig. 2C) . Eosinophils isolated from the peritoneum were partially activated, as they expressed intermediate levels of Siglec-F and CD62L (see also Go Go Go Fig. 6C ). To analyze whether modulation of CD62L and Siglec-F was a result of parasite infection, we compared expression of these markers in various tissues of noninfected animals and included normal BALB/c mice and eosinophil-deficient 4 get/{Delta}dblGATA mice as controls (Fig. 3 ). The frequency of eosinophil in different tissues was similar in 4 get as compared with control BALB/c mice, and this population was completely absent in 4 get/{Delta}dblGATA mice, confirming their identification as eosinophils. Like in parasite-infected mice, eosinophils isolated from the thymus showed an activated phenotype (Siglec-FhiCD62Llo), whereas eosinophils isolated from the peritoneum were activated only partially (Siglec-FintCD62Lint).


Figure 3
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  		Figure 3. Eosinophil populations in naïve 4 get and BALB/c mice. Single-cell suspensions of thymus, bone marrow, spleen, and peritoneum from naïve 4 get, BALB/c, and eosinophil-deficient 4 get/{Delta}dblGATA mice were stained for CD4, Siglec-F, and CD62L and analyzed by four-color flow cytometry. In each panel, the dot-plots on the left and in the middle are gated on total live cells, and the right dot-plots are gated on CD4/autofluorescence (CD4/autofl) –Siglec-F+ cells as indicated. The eosinophil population can be identified in the SSChiSiglec-F+ gate or in the CD4/autofluorescence–Siglec-F+ gate, as it is absent in eosinophil-deficient 4 get/{Delta}dblGATA mice. SSC, Side-scatter; PEC, peritoneal exudate cells.

 

Figure 4
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  		Figure 4. Summary of maturation and activation markers on eosinophils. Schematic representation of flow cytometric analysis of maturation and activation marker expression on ex vivo-isolated eosinophils. n.a., Not analyzed.

 

Figure 5
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  		Figure 5. Isolation and analysis of CCR3– and CCR3+ eosinophils from the bone marrow. (A) Bone marrow cells from naïve 4 get mice were stained for CD4, FIRE, and CCR3, sorted in CD4–eGFP+FIRE+CCR3+ and CD4–eGFP+FIRE+CCR3– cells, and stained with Diff-Quick. (B) Quantitative RT-PCR analysis of selected genes that appeared differentially expressed in immature (CCR3–, open bars) and mature (CCR3+, solid bars) eosinophils from the bone marrow based on the microarray experiments (Supplemental Fig. 1). Samples were run in triplicates and normalized to HPRT expression.

 

Figure 6
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  		Figure 6. Stat6-dependent eosinophil recruitment to lung and peritoneum. (A) The frequency of eosinophils in blood, spleen, lung, and peritoneum of naïve or N. brasiliensis-infected wild-type 4 get mice or Stat6-deficient 4 get mice was analyzed by flow cytometry (CD4–eGFP+CCR3+SSChi). The graphs show combined results from two independent experiments. The Stat6-dependent increase of eosinophils in lung and peritoneum of infected mice was statistically significant (P<0.0001; Student’s t test). (B) Flow cytometric analysis of blood, PEC, and bronchoalveolar lavage (BAL) from N. brasiliensis-infected 4 get or Stat6-deficient 4 get mice. Single-cell suspensions were stained for CD4, IgE, and CCR3. Dot-plots displaying CD4 versus IL-4/eGFP are gated on total live cells, and dot-plots displaying CCR3 versus IgE are additionally gated on CD4–IL-4/eGFP+ cells (R1). Eosinophils are represented by CCR3+IgE– cells, and basophils are represented by CCR3–IgE+ cells. (C) PEC of N. brasiliensis-infected 4 get mice were double-stained for CD4 and FIRE, Siglec-F, or CCR3 or triple-stained for CD4, CCR3, and CD62L. Dot-plots are gated on CD4–SSChi cells. Results are representative of several independent experiments.

 
Taken together, we could distinguish different stages of eosinophil maturation and activation based on the expression levels of CCR3, Siglec-F, FIRE, CD62L, and Pir-A/B as summarized in Figure 4 .

Analysis of transcriptional changes during maturation and activation of eosinophils
The transition from CCR3– to CCR3+ cells in the bone marrow might mark a developmental stage at which novel, therapeutic drugs could interfere with eosinophil maturation. Morphologically, CCR3– eosinophils appeared to have less condensed chromatin as compared with CCR3+ eosinophils but already contained numerous cytoplasmic granules (Fig. 5A ). To analyze gene expression from both subsets of eosinophils on the transcriptional level, microarray analysis was performed with sorted CCR3+ and CCR3– eosinophil populations from bone marrow of naïve 4 get mice. To identify genes, which are induced or repressed during eosinophil activation, we compared gene expression profiles of mature eosinophils isolated from the spleen of IL-5 transgenic 4 get mice and eosinophils isolated from the lungs of N. brasiliensis-infected 4 get mice. The combination of both comparisons generated eight groups of genes: genes that are only induced during maturation, only induced during activation, induced during maturation and activation, repressed during maturation, repressed during activation, repressed during maturation and activation, induced during maturation and repressed during activation, and repressed during maturation and induced during activation (Supplemental Fig. 1 ). Genes, which were up- or down-regulated more than twofold, were included in the analysis. Supplemental Figure 1 shows a selection of genes, which were regulated on the transcriptional level during maturation and/or activation of eosinophils. It is interesting that the characteristic, eosinophil-specific effector genes, such as MBPs and EPX, were down-regulated during the transition from the CCR3–-to-CCR3+ stage in the bone marrow and were not reinduced during activation, and this was supported by quantitative RT-PCR using sorted populations from an independent experiment (Fig. 5B and Supplemental Fig. 1). Thus, early during development, eosinophils complete synthesis of their granular effector proteins and switch their transcriptional program to activate genes that might contribute to recognition of foreign material, regulate their activation, or mediate their recruitment to inflamed tissues.

A few of the most strongly up-regulated genes were notable. FCNA, a lectin that activates the complement cascade, was the most strongly induced gene during maturation (more than eightfold). Axl, an inhibitory receptor tyrosine kinase, was up-regulated during maturation and even further during activation. This receptor belongs to the Tyro 3 family and is expressed mainly in monocytes/macrophages. Mice, which lack all three Tyro 3 family members (Axl, Mer, and Tyro 3), develop severe lymphoproliferation and autoimmune phenotypes (reviewed in ref. [22 ]). TRF expression (but not TRF receptor expression; data not shown) was also up-regulated during maturation and activation, where it might facilitate iron scavenging in tissues. Efficient iron uptake is required to generate the prosthetic heme group of eosinophil-peroxidase and iron-sulfur clusters in various cytochromes. Up-regulation of FCNA, Axl, and TRF was confirmed by quantitative RT-PCR using independently generated cDNA samples (Fig. 5B) .

CCR3 mRNA expression was comparable between the CCR3– and CCR3+ eosinophil populations, suggesting that surface expression of this receptor might be regulated post-transcriptionally. However, down-regulation of CCR3 mRNA was observed in activated eosinophils consistent with reduced surface expression on eosinophils isolated from the lung (Supplemental Fig. 1 and Fig. 1 ). Pir-A, an activating receptor that can bind MHC Class I molecules [21 ], and paired Ig-like Type 2 receptor (PILR-ß), a DNAX activating protein of 12 kDa (DAP12)-associated, activating receptor specific for PILR-L, which is highly expressed in lung and spleen [23 ], were up-regulated during maturation and down-regulated during activation. The up-regulation of Pir-A could be confirmed by antibody staining (Fig. 1B) . The mAb used cannot distinguish between the activating receptor Pir-A and the inhibitory receptor Pir-B. However, as Pir-B expression did not change at the transcriptional level during maturation or activation, the antibody staining likely reflects Pir-A expression rather than Pir-B expression. The eosinophil-associated transcription factors Gata-1 and Gata-2 were highly expressed at equal levels in immature and mature eosinophils, whereas Spi-C (a member of the PU.1 family) and c/EBPß were up-regulated approximately twofold during maturation (Supplemental Fig. 1, a). This might indicate that Gata-1/Gata-2 are required at an earlier developmental step than Spi-C or c/EBPß. The IL-5R{alpha}-chain mRNA was only weakly expressed and slightly down-regulated during maturation (data not shown). Overall, expression profiling of eosinophil subsets has revealed several novel, target molecules with possible implications for therapeutic interventions.

Stat6-dependent eosinophil recruitment to the peritoneal cavity
Eosinophil recruitment to the lung of N. brasiliensis-infected mice is regulated by Stat6-dependent signals from bone marrow-derived, non-T cells [9 ]. The transcription factor Stat6 mediates signaling through the IL-4R and regulates expression of numerous genes involved in orchestration of Type 2 immunity in vivo. It was unexpected that Stat6 was also required for eosinophil recruitment to the peritoneal cavity of N. brasiliensis-infected mice, although the parasites do not migrate to the peritoneum (Fig. 6 ). Eosinophils isolated from the peritoneum expressed high levels of Siglec-F, consistent with an activated phenotype. However, in contrast to eosinophils isolated from the lung, peritoneal eosinophils maintained surface expression of FIRE, CCR3, and CD62L (Fig. 6C) . Thus, peritoneal eosinophils represent a phenotypically unique population of cells as compared with eosinophils found in other tissues, the function of which remains to be analyzed. In contrast, basophils (CD4–GFP+CCR3–IgE+), which accumulate in the lungs during N. brasiliensis infection or OVA-induced allergic inflammation [9 , 24 ], were almost absent in the BAL and the peritoneum, which indicates that these effector cells have a different migration potential as compared with eosinophils (Fig. 6B) .

Recruitment does not require direct recognition of IL-4/IL-13 by eosinophils
It remains unclear whether eosinophil recruitment requires Stat6 expression in eosinophils themselves. To address this, mixed bone marrow chimeras were generated with bone marrow from wild-type (Ly5.1) mice and Stat6-deficient (Ly5.2) mice. Chimeras were infected with N. brasiliensis 8 weeks after reconstitution and analyzed 9 days later for the ratio of wild-type:Stat6-deficient eosinophils in lung, blood, and peritoneum. As shown in Figure 7A and 7B , no significant bias toward accumulation of wild-type eosinophils in lung or peritoneum could be observed, indicating that Stat6 expression in eosinophils was not required. Further, expression of the IL-4R{alpha}-chain on splenic eosinophils was determined by flow cytometry. In contrast to CD4 T cells and non-CD4, noneosinophil cells, which expressed IL-4R{alpha} on the cell surface, no staining was observed on eosinophils, a finding consistent with previous observations using human cells [25 ] (Fig. 7C) . As eosinophils produce low amounts of IL-4, which could lead to autocrine down-regulation of the IL-4R, the experiment was repeated with cells from IL-4/IL-13-deficient mice with similar results (Fig. 7D) . Taken together, these data reveal that eosinophil recruitment does not require cell autonomous IL-4/IL-13 recognition. Rather, tissue recruitment of eosinophils, as shown here and elsewhere [9 ], requires another hematopoietic cell type, which is capable of IL-4/IL-13 recognition and Stat6-dependent signaling, presumably leading to production of additional products, which attract eosinophils.


Figure 7
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  		Figure 7. Analysis of Stat6 requirement and IL-4R{alpha} expression in eosinophils. (A) Mixed bone marrow chimeras were generated with total bone marrow cells from wild-type (WT; Ly5.1) and Stat6-deficient (Ly5.2) donor mice to analyze whether Stat6 expression in eosinophils was required for recruitment to effector sites. Chimeras were infected with N. brasiliensis 8 weeks after bone marrow reconstitution and analyzed on Day 9 after infection. Samples from blood, lung, and peritoneal exudates were stained for CCR3, Ly5.1, and Ly5.2. Dot-plots are gated on SSChiCCR3+ cells. (B) Combined results from five individual mice analyzed as described in A. (C) Splenocytes from N. brasiliensis-infected mice were stained for CD4, CCR3, and IL-4R{alpha}. The histograms were gated on the four populations indicated by the quadrants in the dot-plot, except eosinophils (Eos), which were gated on CD4–GFP+CCR3+ cells. Filled histograms show results from 4 get mice, and open histograms show results from IL-4R{alpha}-deficient 4 get mice. (D) Splenocytes from BALB/c mice (thin line), IL-4/IL-13-deficient mice (bold line), or IL-4R{alpha}-deficient (filled) mice were analyzed for IL-4R{alpha} expression on eosinophils (CD4–SSChiSiglec-F+), B cells, and CD4+ T cells.

 
Alternatively activated macrophages (AAM) regulate eosinophil recruitment in vivo
Stat6 has been shown to be required for the differentiation of monocytes to AAM, which might be involved in tissue repair and immunosuppression [26 ]. As macrophages are the main cell type in the peritoneum, and alveolar macrophages are positioned strategically to recognize pathogens in the lung, these cells remain a likely source for Stat6-dependent signals, which regulate eosinophil recruitment to peritoneum and lung, respectively. To analyze whether N. brasiliensis infection leads to Stat6-dependent differentiation of AAM in the peritoneum, peritoneal macrophages were isolated 9 days after N. brasiliensis infection and used for RT-PCR analysis. The AAM-associated genes Ym1, Fizz1, and arginase I were highly induced in a Stat6-dependent manner (Fig. 8A ). Of note, AAMwere induced, despite the fact that N. brasiliensis does not migrate through the peritoneum. Eosinophil chemotaxis can be mediated by CCL11 (eotaxin-1) and CCL24 (eotaxin-2), both ligands of CCR3, which are partially regulated by Stat6 in the lung [8 ]. Peritoneal macrophages did not express CCL11, and equal levels of CCL24 mRNA were observed in wild-type and Stat6-deficient macrophages, which makes it unlikely that eotaxins regulate eosinophil recruitment to the peritoneum. However, many other potent chemoattractants for eosinophils, including leukotriene B4 [27 ], PGD2 [28 ], platelet-activating factor (PAF) [29 ], or complement component C5a [30 ], have been described, which can be produced and released by activated macrophages.


Figure 8
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  		Figure 8. Macrophage-dependent eosinophil recruitment. (A) Peritoneal macrophages from wild-type and Stat6-deficient mice were sorted on Day 9 after N. brasiliensis infection and subjected to quantitative RT-PCR analysis with primer pairs specific for CCL24 (eotaxin-2), arginase I, Fizz1, Ym1, and HPRT. (B) Analysis of eosinophil recruitment to lung and peritoneum in wild-type and op/op mice on Day 9 after N. brasiliensis infection. The number of eosinophils is based on total cell counts and flow cytometric analysis of eosinophils. Four mice per group were analyzed. Only the data from the lung were statistically significant (P<0.05; Student’s t test). (C) Mice were given 300 µl clodronate liposomes (Clodro) or PBS i.p. 1 day before and 7 days after N. brasiliensis infection. Blood eosinophilia and total number of eosinophils in the peritoneum were analyzed by flow cytometry on Day 11 after N. brasiliensis infection. The graphs show combined results from two independent experiments.

 
To determine whether macrophages were required for eosinophil recruitment to the peritoneum or lung, wild-type and macrophage-deficient op/op mice were infected with N. brasiliensis, and recruitment of eosinophils was analyzed on Day 11 after infection. Greatly reduced numbers of eosinophils were recovered from the peritoneum and lungs of op/op mice [15 ] as compared with wild-type mice (Fig. 8B) . Similarly, when macrophages were depleted with clodronate liposomes before N. brasiliensis infection, highly reduced eosinophil recruitment was observed (Fig. 8C) . Thus, AAM promote eosinophil recruitment to the lung and peritoneal cavity during helminth infection.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We describe different stages of eosinophil development using flow cytometry and microarray analysis. In IL-4 reporter mice (4 get mice), IL-4-expressing eosinophil precursors were present in the fetal liver, and these cells were able to reconstitute the eosinophil lineage in lethally irradiated recipient mice (Fig. 1) . Thus, eosinophils are one of the first IL-4-expressing cells during ontogeny, as T cells are not present at that stage of development. Mast cell precursors in the fetal liver did not express IL-4 and acquired IL-4 expression only after in vitro culture with stem cell factor [17 ]. In the human immune system, eosinophilopoiesis was observed in the fetal liver as early as 5 weeks after gestation [31 ]. The finding that classical stem cell markers (c-Kit, CD34, and Sca-1) were absent on fetal liver eosinophils (Fig. 1A) indicates that these cells have already committed to the eosinophil lineage, although they retained their reconstitution potential after transfer into lethally irradiated recipient mice (Fig. 1B) . Although fetal and adult hematopoiesis are probably not linked directly, these results show that eosinophilopoiesis starts at an early stage of development.

In adult mice, a recent study identified an early eosinophil precursor cell (IL-5R{alpha}+CD34+c-Kitlo), which gave rise to mature eosinophils during in vitro culture [32 ]. Our attempts to stain for IL-5R{alpha} using commercially available antibodies were unsuccessful. However, the microarray experiment showed low but equal expression of IL-5R{alpha} in CCR3– and CCR3+ eosinophil subsets in the bone marrow, suggesting that this receptor is expressed early during commitment to the eosinophil lineage (data not shown). The transition from immature (CCR3–CD62LloPir-A/Blo) to mature (CCR3+CD62LhiPir-A/Bhi) eosinophils might be a useful drug target, as only the CCR3+ eosinophils can respond to eotaxin, which has been shown to induce mobilization of eosinophils from the bone marrow [33 , 34 ]. Immature and mature eosinophils are morphologically, almost indistinguishable; however, they differentially express a large number of genes (Fig. 5 and Supplemental Fig. 1). The mRNAs for classical, eosinophil-associated granular proteins such as MBPs and EPX were down-regulated during maturation, confirming a previous report [35 ] (Fig. 5B and Supplemental Fig. 1). This result could provide an explanation for the recent finding that despite an increase in eosinophil numbers in patients with eosinophilic esophagitis, mRNAs for MBP and EPX were not increased in the affected tissue [36 ]. Therefore, eosinophils finish synthesis of their effector molecules, which are stored in cytoplasmic granules, before they up-regulate CCR3 and are licensed to leave the bone marrow. At this stage of development, they also acquire expression of activating receptors including Pir-A and PILR-ß and increase the expression of the signaling adaptor molecule DAP12 to be ready to respond to activating signals in peripheral tissues (Supplemental Fig. 1). The increased expression of the inhibitory receptor Siglec-F might counterbalance other activating receptors to prevent uncontrolled degranulation, although this needs to be analyzed in future experiments.

FCNA expression was strongly induced in eosinophils during maturation. This secreted protein is not well characterized but has been described in monocytes/macrophages and can mediate activation of the complement cascade [37 ]. It is interesting that in vitro studies have shown that larvicidal activity of eosinophils depends on complement [38 ]. However, only the L3 stage is susceptible to C3 deposition [39 ]. IL-5 transgenic mice with high numbers of tissue resident eosinophils have been shown to kill helminth larvae at the site of inoculation (although eosinophils are not required for elimination of adult helminths from the intestine) [40 ]. Further, eosinophil-deficient mice are more susceptible to reinfection with N. brasiliensis as compared with wild-type mice [24 ]. It is possible that FCNA released from eosinophils in the vicinity of migrating L3 helminth larvae contributes to host protection against these parasites.

Eosinophils in lymph node, thymus, and Peyer’s patches appeared activated upon isolation. This could reflect recruitment of activated eosinophils from peripheral tissues or their local activation within lymphoid tissues. It is currently unclear how eosinophils enter lymph nodes or Peyer’s patches, although their presence in these lymphoid organs has been described [41 , 42 ]. Intratracheal instillation of eosinophils leads to their trafficking to draining lymph nodes [43 ]. The role of eosinophils with an activated phenotype in the thymus, even in naïve mice, requires further investigation (Fig. 3) . Eosinophils in lung-draining lymph nodes were shown to express MHC Class II together with costimulatory molecules and could induce proliferation of antigen-specific T cells in vitro [44 ]. Therefore, eosinophils in lymph nodes could perhaps provide a positive-feedback signal from inflamed tissues and promote expansion of antigen-experienced Th2 cells in local draining lymph nodes.

Eosinophil recruitment has been shown to be regulated by Stat6-dependent genes in hematopoietic non-T cells [9 ]. A recent study suggested that eosinophil precursors in the bone marrow respond directly to IL-4 [45 ]. However, we found no surface expression of the IL-4R{alpha}-chain on eosinophils, and mixed bone marrow chimeras showed that Stat6 expression was not required in eosinophils to mediate their recruitment to lung or peritoneum (Fig. 7) . Therefore, it seems unlikely that IL-4 or IL-13 acts directly on eosinophils or their precursors to induce maturation or recruitment.

The peritoneal cavity appeared to be a major site for eosinophil accumulation during N. brasiliensis infection, although this parasite does not migrate through the peritoneum. A transient increase of eosinophils in the peritoneum has also been described during the acute phase of infection with a strictly enteric nematode parasite, Heligmosomoides polygyrus [46 ]. Injection (i.v. or s.c.) of Sephadex 200 or PAF, respectively, resulted in accumulation of eosinophils in the peritoneal cavity [47 , 48 ], which indicates that eosinophils migrate to the peritoneum, even in the absence of a direct, inflammatory stimulus at that site. However, this migratory response after N. brasiliensis infection was reduced in Stat6-deficient mice (Fig. 6) , which suggests that migration to the peritoneum is mediated by an active chemotactic process, which is regulated by Stat6-dependent factors. Mixed bone marrow chimeras showed that Stat6 was not required in eosinophils, which excludes the possibility that a Stat6-dependent chemotactic receptor in eosinophils was involved (Fig. 7) . In naïve mice, eosinophil numbers in the peritoneum were similar in wild-type and Stat6-deficient mice, suggesting that homeostatic migration to the peritoneum is controlled by Stat6-independent mechanisms (Fig. 6A) . Another study showed that i.p. administration of macrophage migration-inhibitory factor from Brugia malayi leads to IL-4-dependent accumulation of AAM and eosinophils in the peritoneum, which further supports the concept of macrophage-mediated eosinophil recruitment [49 ]. It is interesting that basophils were not recuited to the peritoneum or BAL. The identity of these cells is based on nuclear morphology, expression of the high-affinity IgE receptor, and microarray analysis of highly purified basophils, isolated from the lung of N. brasiliensis-infected mice [9 ]. Sorted mouse basophils lack the typical granules present in human basophils detected in blood smears, which might be explained by induction of degranulation during the sorting and staining procedures.

The differentiation of monocytes to AAM is a Stat6-dependent process, and the absence of these cells largely prevented eosinophil recruitment to lung and peritoneum (Fig. 8) . It is currently unclear whether AAM differentiate in tissues, such as the lung and peritoneum, or whether they are recruited to these sites from blood as differentiated cells. Furthermore, the cellular source of IL-4 or IL-13, which is critically required for the differentiation of AAM, is unknown. Mast cells, basophils, eosinophils, NKT cells, and Th2 cells have the potential to produce these cytokines; however, adoptive transfer experiments or generation of conditional, IL-4/IL-13-deficient mice will be required to identify the critical cell type(s) that mediate the differentiation of AAM in vivo.

Taken together, we present several novel findings regarding eosinophil development and recruitment in vivo. Although the findings presented here were obtained with a parasite infection model, they can probably be generalized for other Type 2 immune responses, as they reflect a stereotypic immune response, which includes Stat6-dependent lung eosinophilia and the induction of AAM. A better understanding of these processes is critically required to develop more efficient drugs to cure diseases associated with tissue eosinophilia, such as allergic asthma, atopic dermatitis, and gastrointestinal disorders.


   ACKNOWLEDGEMENTS
 
This work was supported in part by National Institutes of Health grant AI30663 and the Howard Hughes Medical Institute, the Sandler Asthma Basic Research Center (to R. M. L.), and the Emmy Noether Program of the Deutsche Forschungsgemeinschaft (grant Vo944/2 to D. V.). The authors have no conflicting financial interests. We thank N. Flores, C. McArthur, and L. Stowring for excellent technical assistance.


   FOOTNOTES
 
1 Current address: Institute for Immunology, University of Munich, Goethestrasse 31, D-80336 Munich, Germany. Back

Received November 21, 2006; revised January 27, 2007; accepted January 29, 2007.


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S. B. S. King, A. M. Knorn, C. Ohnmacht, and D. Voehringer
Accumulation of Effector CD4 T Cells during Type 2 Immune Responses Is Negatively Regulated by Stat6
J. Immunol., January 15, 2008; 180(2): 754 - 763.
[Abstract] [Full Text] [PDF]

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H.-B. Wang, I. Ghiran, K. Matthaei, and P. F. Weller
Airway Eosinophils: Allergic Inflammation Recruited Professional Antigen-Presenting Cells
J. Immunol., December 1, 2007; 179(11): 7585 - 7592.
[Abstract] [Full Text] [PDF]

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C. Ohnmacht, A. Pullner, N. van Rooijen, and D. Voehringer
Analysis of Eosinophil Turnover In Vivo Reveals Their Active Recruitment to and Prolonged Survival in the Peritoneal Cavity
J. Immunol., October 1, 2007; 179(7): 4766 - 4774.
[Abstract] [Full Text] [PDF]

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P. Loke, I. Gallagher, M. G. Nair, X. Zang, F. Brombacher, M. Mohrs, J. P. Allison, and J. E. Allen
Alternative Activation Is an Innate Response to Injury That Requires CD4+ T Cells to be Sustained during Chronic Infection
J. Immunol., September 15, 2007; 179(6): 3926 - 3936.
[Abstract] [Full Text] [PDF]