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Blood, Vol. 112, Issue 4, 1461-1471, August 15, 2008
Neutrophil secretion products pave the way for inflammatory monocytes
Blood Soehnlein et al.
112: 1461
Supplemental materials for: Soehnlein et al
Files in this Data Supplement:
- Table S1. Concentrations of IL-6R, MCP-1, and MCP-3 in the air pouch lavage fluid (PDF, 13 KB) -
After stimulation with PAF (10−6 M) for 12 hours, the air pouch was lavaged with 5 ml of 5 mM EDTA in ice-cold PBS. The cells were spun-down and IL-6R, MCP-1, and MCP-3 were measured in the cell-free supernatant by ELISA.
- Figure S1. PMN extravasation precedes monocyte recruitment (JPG, 40.5 KB)
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(A) Number of extravasated fluorescent monocytes and non-fluorescent PMN in M. cremaster of CX3CR1eGFP/+ mice quantified by intravital microscopy. One hour after baseline reading the muscle was superfused with PAF (10−7 M). The extravasation of PMN and fluorescent monocytes was recorded in the same fields for up to 4 hours. The 8 and 12 hour values were obtained after intrascrotal injection of PAF and subsequent exposure of the muscle at the indicated time points. (B) Number of PMN and monocytes in subcutaneous air pouch of C57BL/6 mice. The cells were harvested at indicated time points after stimulation with PAF (10−6 M) and differentiated by FACS analysis. Data are expressed as mean ± SD. n = 5 for each bar.
- Figure S2. Antibody depletion of PMN does not affect monocyte blood count (JPG, 29.5 KB)
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(A) CX3CR1eGFP/+ mice were injected with mAb RB6-8C5 (100 µg/mouse) and blood samples from the tail vein were stained with anti-Gr1 mAb at indicated time points. The figure displays representative plots for FSC vs. SSC and eGFP vs. Gr1 illustrating selective depletion of PMN. (B) Quantification of PMN (CD45+/Gr1+/eGFP−) and inflammatory (CD45+/Gr1+/eGFP+) and resident (CD45+/Gr1−/eGFP+) monocytes in peripheral blood following injection of RB6-8C5. Each data point represents the average of measurements in 4–6 mice.
- Figure S3. PMN depletion with the Ly6G-specific mAb 1A8 conveys similar results as PMN depletion with RB68C5 (JPG, 25.4 KB)
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(A) CX3CR1eGFP/+ mice were injected with mAb 1A8 (10 µg/mouse) and blood samples from the tail vein were stained with anti-Gr1 mAb at indicated time points. The figure displays representative plots for FSC vs. SSC and eGFP vs. Gr1 illustrating selective depletion of PMN. (B) Recruitment of monocytes into the air pouch was analysed in C57BL/6 mice 12 hours after injection of PAF (10−6 M). Comparisons are made between mice with intact WBC (ctrl), neutropenic mice (PMN depl), and neutropenic mice where PMN secretion was injected into the pouch (PMN depl + PMN sec). Neutropenia was induced by i.p. injection of 1A8. (C) Recruitment of Gr1+ inflammatory monocytes in the air pouch as described for B. ** indicates significant difference compared to ctrl and PMN depl + PMN sec.
- Figure S4. PMN secretion products specifically mobilize Gr1+ inflammatory monocytes (JPG, 32.9 KB)
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Cell recruitment into the air pouch was analyzed in CX3CR1eGFP/+ mice with intact WBC (left), mice that were made neutropenic (middle), as well as neutropenic mice locally injected with PMN-sec (right). Dot plots represent CD45+ cells. PMN depletion and injection of PMN-sec specifically affects recruitment of eGFPloGr1+ monocyte fraction.
- Figure S5. Recruitment of leukocyte subsets in DPPI−/− mice (JPG, 17.6 KB)
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Leukocyte recruitment to the air pouch was induced by injection of PAF (10−6 M) into mice with normal WBC, neutropenic mice, and neutropenic mice that were treated with PMN secretion. Leukocytes were harvested 12 hours after stimulation and differentiated using FACS. Charts represent the recruitment of PMN (A) and Gr1+ inflammatory monocytes (B) in wild type (white) and DPPI−/− (black) mice. ** indicates significant difference compared to ctrl and PMN depl + PMN sec. * indicates significant difference compared to PMN depl + PMN sec.
- Figure S6. Expression of formyl peptide receptors by murine inflammatory monocytes (JPG, 10.9 KB)
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Inflammatory monocytes were isolated from whole blood of C57BL/6 mice by FACS sorting. Subsequent PCR with specific primers for fpr1 and fpr2 and separation of PCR products by agarose gel electrophoresis gave distinct bands for fpr1 and fpr2. Displayed gel is representative of three independent experiments.
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