C57BL/6 and Myd88-/- mice were used at 8-10 weeks of age. C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Myd88-/- mice were maintained in our laboratory animal resource facility (LARC) at the Oklahoma Medical Research Foundation.
Recombinant mouse IL-7, stem cell factor (SCF), Flt-3 ligand (FL), M-CSF, GM-CSF, recombinant mouse CD14/Fc chimera protein, and recombinant human CD14 were purchased from R&D Systems (Minneapolis, MN). LPS from Escherichia coli 055:B5 was purchased from Sigma-Aldrich (St. Louis, MO). A synthetic lipopeptide Pam3CSK4 was purchased from InvivoGen (San Diego, CA).
Antibodies and flow cytometry
The following Abs for flow cytometry were purchased from eBioscience (San Diego, CA): biotinylated anti-TLR4-MD-2 (clone MTS510), biotinylated anti-RP105 (clone RP/14), biotinylated anti-MD-1 (clone MD113), biotinylated anti-CD14 (clone Sa2-8), PE-conjugated anti-CD62L (clone MEL-14), allophycocyanin (APC)-conjugated anti-F4/80 (clone BM8), anti-c-Kit (clone ACK2), phycoerythrin (PE)-Cy5-conjugated anti-Sca-1 (clone D7), biotinylated anti-Flk-2 (clone A2F10), purified anti-mouse CD115 (CSF-1R, clone AFS98), purified anti-mouse GM-CSF (clone MP1-22E9), and purified anti-mouse TNFα (clone MP6-XT22).
The following Abs for flow cytometry were purchased from BD Pharmingen (San Diego, CA): FITC-conjugated anti-CD2 (clone RM2-5), anti-CD3ε (clone 145-2C11), anti-CD8α (clone 53-6.7), anti-CD45R (B220; clone RA3-6B2), anti-Ly6G (clone RB6-8C5), anti-CD11b/Mac-1 (clone M1/70), anti-TER119, anti-CD34 (clone RAM34), anti-CD11c (clone HL3), PE-conjugated IL-7Rα (clone SB/119), anti-CD19 (clone 1D3), anti-Ly6G (clone RB6-8C5), anti-CD11c (clone HL3), anti-FcγR2/3 (clone 2.4G2), anti-AA4.1, anti-CD86 (clone GL1), APC-conjugated anti-CD45R (clone RA3-6B2), anti-CD11b/Mac-1 (clone M1/70), biotin-conjugated anti-VCAM-1 (clone 429, MVCAM.A) PE-conjugated streptavidin and PE Texas-Red-conjugated streptavidin. For analyzing cultured cells by flow cytometry, 7-AAD (BD Pharmingen) was always used to exclude dead cells.
Flow cytometry analyses was conducted on a FACSCan™, FACSCalibur™ or FACSAria™ (Becton Dickinson & Co., Mountain View, CA), and the data were analyzed with FlowJo software (Treestar, San Carlos, CA).
Establishment of the anti-mouse TLR4 monoclonal antibody (clone UT49)
A full description of the preparation and validation of this reagent will be published elsewhere. Briefly, TLR4-deficient mice were intraperitoneally injected four times at a week intervals with 1x107 Ba/F3 cells expressing mouse TLR4 and mouse MD-2. Three days after the last injection, mice were euthanized and spleens were removed. Spleen cells were dispersed and fused with SP2/O cells using a standard fusion protocol with polyethylene glycol 1500 (Roche, Basel, Switzerland). Hybridoma cells were selected in hypoxanthine/aminopterine/thymidine medium and screened by flow cytometry with Ba/F3 cells expressing mouse TLR4-MD-2 complex and parent Ba/F3 cells. Biotin-conjugated this antibody was used for flow cytometry.
Isolation of hematopoietic stem and progenitor cells
Bone marrow cells were harvested and enriched for lineage-negative cells by incubation with antibody to CD11b/Mac-1 (clone M1/70), anti-Ly6G (clone RB6-8C5), anti-CD45R (B220; clone RA3-6B2), anti-CD19 (clone 1D3) and anti-TER119, followed by negative selection using the MACS cell separation system (Miltenyi Biotec, Auburn, CA). For sorting of Flk-2- or Flk-2+ LKS+ (Lin- IL-7Rα- c-Kithi Sca-1+), LKS-(Lin- IL-7Rα- c-Kithi Sca-1-) and CLP (Lin- IL-7Rα+ c-Kitlo Sca-1lo), these partially lineage-depleted cells were further stained with FITC-conjugated lineage markers; anti-CD2, anti-CD3ε, anti-CD8α, anti-CD45R, anti-Ly6G, anti-CD11b/Mac-1, anti-TER119, PE-conjugated IL-7Rα, APC-conjugated anti-c-Kit, PE-Cy5-conjugated anti-Sca-1, and biotinylated anti-Flk-2 combined with PE Texas-Red conjugated streptavidin. For sorting of CMP (Lin- IL-7Rα- c-Kithi Sca-1- CD34+ FcγR2/3lo), GMP (Lin- IL-7Rα- c-Kithi Sca-1- CD34+ FcγR2/3hi) and MEP (Lin- IL-7Rα- c-Kithi Sca-1- CD34- FcγR2/3lo), the sorted LKS- cells were further stained with FITC-conjugated anti-CD34 and PE-conjugated anti-FcγR2/3. Cells were sorted on a FACSAria™ (Becton Dickinson & Co.) The sort gates and the post sort analyses are illustrated in .
Serum-free, stromal cell-free cell cultures
Round-bottomed 96-well plates or flat-bottomed 24-well plates (Corning Inc.) were used for these cultures. Sorted cells were cultured with X-VIVO15 medium (Biowhittaker, Walkersville, MD) or StemPro-34 SFM medium (Invitrogen, Carlsbad, CA). X-VIVO15 medium, that seemed to be optimal for lymphoid cultures, contained 1% detoxified bovine serum albumin (Stem Cell Technologies, Vancouver, Canada), 5×10-5 M 2-mercaptoethanol (2-ME), 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. StemPro-34 SFM medium containing 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin was usually used for myeloid cultures. The concentrations of cytokines were IL-7, 1 ng/ml; FL, 100 ng/ml; SCF, 20 ng/ml, M-CSF, 20 ng/ml; GM-CSF, 20 ng/ml. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2 in air.
Single cell culture on OP9 stromal cells
Sorted CLPs were cultured with X-VIVO15 in the presence of SCF (20 ng/ml), FL (100 ng/ml) and IL-7 (1 ng/ml) with or without LPS (10 μg/ml), or Pam3CSK4 (1 μg/ml). After 24 hr, cultured cells were harvested and washed with medium three times. A single cell sorting for the cultured cells was then conducted on a FACSAria™ and a single cell was plated on OP9 stromal cells in 96-well plate and cultured for 10 days in the presence of SCF (20 ng/ml), FL (100 ng/ml) and IL-7 (1 ng/ml). The conditioning medium for OP9 was MEMα medium (Invitrogen) containing 20% FCS, 5×10-5 M 2-ME, 2 mM L-glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin. Positive wells (more than 30 cells) were determined by microscopic observation. Cells were harvested and then stained with mAbs to CD19, CD11c, and Mac-1 and analyzed by flow cytometry. Cells were also stained with mAb to VCAM-1 to exclude stromal cells. The OP9 stromal cell line was kindly provided by Dr. Shin-Ichi Hayashi (Tottori University, Japan).
In vitro BrdU incorporation
Sorted Flk-2- LKS+ cells (10,000 cells/well) were cultured with or without LPS (10 μg/ml) or Pam3CSK4 (1 μg/ml) in the presence of SCF (20 ng/ml) for 50 hr, pulsing with 10 μM 5-bromo-2’-deoxyuridine (BrdU) for the final 18 hr. The cells were then stained with anti-BrdU. BrdU flow kit was purchased from BD Pharmingen. Analyses by flow cytometry were conducted on a FACSCan™.
Interaction of LPS with the TLR4-MD-2 complex
Whole bone marrow cells from C57BL/6 mice were cultured in RPMI1640 medium (Mediatech, Inc. Herndon, VA) containing 10% FCS, 5×10-5
M 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin and stimulated with or without 1 μg/ml LPS for 1 hr. Then cells were harvested and stained with mAbs to TLR4-MD-2, Mac-1, lineage markers (CD2, CD3e, CD8a, CD45R/B220, CD11b/Mac-1, Ly6G, TER119), Sca-1, and c-Kit. Alternatively, C57BL/6 mice were intravenously or intraperitoneally injected with PBS or 100 μg LPS. After 1 hr, whole bone marrow cells were harvested from femurs and tibiae, and stained with mAbs to TLR4-MD-2, Mac-1, lineage markers, Sca-1, and c-Kit. The MTS 10 reagent is unique in detecting a conformation dependent epitope on TLR4-MD-2 (Akashi et al., 2003
Cytospins of cultured progenitor cells were stained with Giemsa-May-Grünwald (Sigma Diagnostics, St. Louis, MO), mounted in Immersol (Zeiss, Thornwood, NY), and analyzed in OMRFs Biological Imaging Facility on a Zeiss Axioplan 2i microscope with a 100x/1.4 NA Plan Achromat objective. Photomicrographs were made using an AxioCam MRc color camera (Zeiss), and AxioVision 3.1 software (Zeiss).
Semi-quantitative RT-PCR analysis of gene expression
The mRNAs were isolated from sorted cells using MicroPoly(A) Pure (Ambion, Austin, TX). The cDNAs were then prepared from DNase I-treated mRNA using oligo-dT and Moloney murine leukemia virus reverse transcriptase (Invitrogen). PCR reactions were conducted in buffer containing 200 μM dATP, dGTP, dTTP, 100 μM dCTP and 0.5 μCi [α 32P] dCTP. Aliquots were removed at cycle 25, 28 and 31 for β-actin and cycles 32, 35 and 38 for all others to insure that PCR remained within the exponential range of amplifications. Five micro liter aliquots were denatured in a formamide-loading buffer and applied to a 6 % polyacrylamide gel containing 7 M urea. Incorporation of [α 32P] dCTP into PCR product bands was quantified by PhosphoImager (Molecular Dynamics, Sunnyvale, CA). The primer sequences for each gene are given in .
The statistical significance of differences between group means was determined with the Student’s t-test. P values less than 0.05 were considered significant.