Leukocyte adhesion in CXCL1-stimulated cremaster muscle venules.
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The number of adherent cells (mean ± SEM) was assessed in exteriorized cremaster muscle venules of RAGE-/- mice (10 venules in 6 mice), Icam1mice (11 venules in 5 mice), untreated (12 venules in 7 mice), anti-LFA-1 antibody (10 venules in 4 mice) or isotype antibody (9 venules in 3 mice) treated wild type mice before and five minutes after systemic injection of CXCL1 (keratinocyte-derived chemokine, KC; 600 ng/mouse).
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Leukocyte adhesion (number of adherent cells/mm2) in mouse cremaster muscle venules and leukocyte transmigration (perivascular neutrophils/mm2 surface area) in cremaster muscle whole mounts in response to glucose during trauma-induced inflammation.
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To dissect the role of the chemokine pathway in this model, WT mice were either pretreated with 4 μg PTx 3 h prior to cremaster exteriorization and glucose stimulation or coinjected with 600 ng CXCL1 and glucose and compared to injection of normal saline.
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Flow Cytometric Analysis
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For flow cytometric analysis of surface markers and intracellular cytokines, enriched C4+ T cells, and single-cell suspensions of lungs were stained with optimal concentrations of the following specific antibodies against surface molecules and intracellular cytokines: C8 α-V450, C4-V500, C11c-FITC, C62L-APC, Gr-1-APC, IFNγ-APC from , , , , and MHCII(I-A/I-E)-eFluor780 from eBioscience, C44-FITC, and IFNγ-PE fromand IL-22-PE from& .
- Fluorescence intensity was measured by using a FacsCantoII® flow cytometer equipped with a 405 nm, 488 nm and 633 nm laser.
- Analysis was performed utilizing the FCSExpress4 program (DeNovo™ Software) gated on leukocytes identified by the forward-scatter/side-scatter profile and further characterization as specified in the figures.
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In vivo experiments
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For adoptive transfer studies, classical monocytes were isolated from BM by FACS sorting using antibodies to CD45, CD115, and Gr1.
- After labelling the monocytes with CFSE, 106 cells were adoptively transferred by tail vein injection to .
- Twenty-four hours after transfer, aortas and hearts of recipient mice were collected for further analysis.
- For monocyte mobilization studies, rmCXCL1/KC was injected i.v.
- at 40 µg/kg.
- After 1 h, blood was drawn and mice were sacrificed to harvest bones and spleens.
- Serum CXCL1 in mice was neutralized by daily administration of 5 µg of anti-CXCL1 antibody (clone 124014) or IgG isotype control (clone 54447, both& ) for 1 week and for 3 consecutive weeks every other day.
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Elimination of 12/15-LO activity alters chemokine homeostasis.
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CXCL1 and CXCL2 concentrations in the plasma was measured in WT mice (black bars) and Alox15mice (white bars) under baseline conditions and 3 h after LPS inhalation (n = 3 to 4).
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Sdc-3 deletion reduces neutrophil recruitment in CXCL1-injected joints.
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Sdc-3 null and wild-type mice were injected intra-articularly with 3 μg per knee joint of recombinant murine CXCL1 or PBS and after 4 hours processed for histology.
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