C57BL/6N (CD22 +/+) mice were initially obtained from Charles River (Wilmington, MA), and CD22 gene-deficient mice (backcrossed to C57BL/6J for greater than 20 generations before arrival) were obtained from Jackson Laboratory (Bar Harbor, ME). BALB/c mice, germ-free Swiss Webster (SW) mice and non-germ-free SW mice were obtained from Taconic Farms Inc (Hudson, NY). CD2-IL-5 transgenic (tg) mice were obtained from Dr. Colin Sanderson (Institute for Child Health Research, Perth, Australia) and then backcrossed onto the BALB/c background for at least 20 generations. In all experiments, we used 2-3-month-old, age- and gender-matched mice, which were housed in specific pathogen-free conditions at Cincinnati Children’s Hospital Medical Center (CCHMC) under IUACAC-approved protocols. All mice were housed in a room with an ambient temperature of 22°C and a 12-hour light cycle.
GI eosinophil isolation
Animals were euthanized by CO2 asphyxiation, and jejunums were excised 6 cm to 16 cm from the pylorus. Duodenum, ileum and colon were anatomically defined as 6cm distal to the pylorus, 10 cm proximal to the cecal sphincter and 5 cm distal to the cecum narrowing, respectively. The intestine segment was opened, washed with PBS and then subjected to EDTA buffer treatment for 15 minutes (1x HBSS with 10%FCS, 5mM EDTA, 40mM HEPES) at 37°C degree with agitation. Following a HBSS wash, tissues were minced into small pieces and treated with collagenase A (Roche) solution (2.4mg/mL in complete RPMI supplemented with 10% fetal calf serum) for 30 minutes at 37°C degrees with moderate agitation. The digest was passed through a 19-G needle three times and then two layers of gauze, and the filtered cell suspension was centrifuged at 300g for 5 minutes. After an additional wash with HBSS, the cells were suspended in appropriate volumes for flow cytometry staining or other experiments. The same procedure was applied to other solid tissues harboring eosinophils without modification, including the lung, stomach, and uterus.
Flow cytometry analysis of total GI lamina propria cells
Polychromatic flow cytometry compensation matrix setting was acquired in a pilot study with eosinophils from IL-5 tg mice. Single-cell suspensions of approximately 1 million total GI lamina propria cells were stained with markers for eosinophils and target proteins of interest using the 6 color FACS system. Antibodies/dyes (clone number) used in this study were purchased from Biolegend (CD22-AF647 OX-97 and isotype RTK-2071), Invitrogen (LIVE/DEAD Violet dye), BD (CD45-FITC 30-F11, Siglec F-PE E50-2440, CD11b-PECy7 M1/70, 7AAD) and R&D Systems (CCR3-PE 83103). Staining was performed on ice for 30 minutes in staining buffer (0.5%BSA, 0.01% NaN3 in 1x HBSS) with manufacturer-suggested titers, followed by a wash with the staining buffer. Stained cells were resuspended and subjected to analysis with the BD FACS Canto II flow cytometer. Raw flow cytometry data were analyzed by Flowjo software (Tree Star Inc., Ashland, OR). GI eosinophils were identified as 7AAD−CD45+Siglec-F+CD11b+ events. Quantification expression data were presented as ΔMFI (CD22 antibody MFI– isotype control MFI). For phosphoFACS, freshly isolated GI lamina propria cells were washed with PBS and rested in complete RPMI-1640 for 30 minutes and then kinetically subjected to the challenging media. Samples treated with vehicle media serve as control for baseline normalization. At the end of the incubation, cells were immediately fixed by adding equal volume of 4% formaldehyde and incubated at 37°C for 10 minutes, followed by ice-cold methanol permeabilization for 30 minute. Specific Thr202/Tyr204 p44/42 antibody (E10, Cell Signaling Technology, #4375) was used together with previously mentioned eosinophil markers to assess eosinophil MAPK activation. GI eosinophils were identified and gated as CD45-Siglec F-CD11b triple (+) events. Stimulated MFI values were normalized to vehicle treated samples for graphic presentation.
Flow-imaging analysis of GI eosinophils
Single-cell suspensions stained with different antibodies were prepared as described in the above-mentioned method and fixed with 2% formaldehyde in PBS. The antibody-stained samples were run through the ImageStreamX system (Amnis Corp. Seattle, WA) following the manufacturer’s instructions. Compensation matrix was established at the pixel level with single-stain controls. The image output was performed by analyzing the raw image data with the software of Amnis-IDEAS (Amnis Corp. Seattle, WA).
Genome-wide microarray analysis on sorted eosinophils and qPCR validation
Lung and GI eosinophils were isolated as mention in earlier sections, with the pulmonary circulation being perfused prior to isolation procedures. Live eosinophils were sorted as DAPI−CCR3+Siglec-F+CD45+CD4−CD8a−CD19−B220−side scatter high (SSChigh) cells from 10 animals using FACS Aria (BD). Total RNA from sorted eosinophils was extracted by a standard Trizol RNA isolation (Invitrogen) and subsequently column-purified with a RNeasy Mini Kit (QIAGEN). mRNA integrity was validated by the Agilent 2100 bio-analyzer (Agilent technologies, Santa Clara, CA). Eosinophil mRNA was amplified and labeled with the WT-Ovation Pico RNA Amplification System (NuGen, San Carlos, CA) and subjected to the GeneChip Mouse Gene ST 1.0 Array chip(Affymetrix), which covers the whole mouse genome with 28853 probe sets. Microarray expression analysis was performed at CCHMC’s Chip Core facility, and expression data were analyzed by the software of Gene spring GX 11 (Agilent Technologies). Briefly, the Affymetrix raw expression values were first filtered with the threshold of 400. Differential expression between GI and lung eosinophils was identified by a >2-fold change and a p value < 0.01 with a false discovery rate (FDR) correction. The same mRNA sample used for microarray analysis was reverse transcribed with the iScript cDNA Synthesis Kit (Bio-Rad, 170-8891), and real-time PCR was performed with the iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) using a pair of primers spanning exons 4 and 5 of the CD22 transcript (forward 5’-GAAAATCCACCCGATACGTGC-3’; reverse 5’-TTGGAACGGTTTCTCCGAGAC-3’), resulting in a 115-bp amplicon.
OVA-induced GI inflammation model
As originally described by Brandt et al. (29
), animals of sensitive strains (littermates of BALB/c-C57BL/6 F4 backcross) were initially sensitized with 100 μg of OVA/Alum twice, 14 days a part under aseptic conditions. Beginning on day 28, OVA (50mg in saline) or saline was administered by oral gavage to the animal every other day for a total of 7 times. Mice were sacrificed 24 hours after the last OVA challenge. Eosinophils were then isolated from the inflamed GI tract for CD22 expression analysis.
Aspergillus-induced allergic lung model
Protein extract of 100 μg Aspergillus Fumigatus
(GREER, Lenoir, NC) was dissolved in sterile saline and administered into the airway of WT BALB/c mice by intranasal inhalation. A total of 9 challenges was given following a Monday-Wednesday-Friday regimen with 1 challenge/day for 3 weeks as previously reported (30
Major Basic Protein (MBP) immunostaining and GI eosinophil quantitative morphometric analysis
The jejunum tissue was fixed in 4% paraformaldehyde in PBS, embedded in paraffin, cut into 5 μm transverse sections, and immunostained with anti-MBP antibody, a kind gift of Dr. James Lee (Mayo Clinic, Scottsdale, AZ) following common immunohistochemistry procedures. For morphometric analysis, all of the MBP labeled eosinophils on the whole transverse jejunum section were enumerated as the number of total eosinophil for this transverse section. To acquire the specific area of laminar propria on the same section, the digital micrograph of the whole transverse section was used to calculate areas of manually outlined lamina propria by Image-Pro PLUS software. (Media Cybernetics, L.P.) The lamina propria eosinophil density was calculated by dividing the above two values into the unit of eosinophil number / mm2.
GI eosinophil turnover assay
This assay was adopted from a previous publication (8
). Briefly, animals were continuously fed with BrdU-containing water solution at a concentration of 80 mg/dL for 6 days, and GI eosinophil isolation was performed as described above. BrdU-positive eosinophils were detected using the BrdU APC Detection Kit from BD Pharmingen (BD Cat # 552598) in conjunction with the eosinophil markers CD45, Siglec-F and CD11b (as described above). The intranuclear staining was based on the manufacturer’s suggested protocols. After flow cytometry analysis, the gated eosinophil population was further gated by CD11c and BrdU with a no BrdU treatment control as an intensity reference. The CD11clow
sub-population represented newly migrated GI eosinophils(8
Statistical significance was analyzed using a two-tailed student t-test in all instances except for the OVA sensitization study, in which a 2-way ANOVA was used. Data are graphed as mean ± standard error of the mean (SEM).