Fig 1: In vitro and in vivo PMN migration.(A) Migration of neutrophil cell lines through transwells seeded with mouse endothelial cells. PMNs migrated into the bottom chamber in response to MIP-2 were counted at different time points as indicated. Pooled results from at least 4 independent experiments are presented as mean ± SEM. Itgam−/− and wild type C57BL/6 neutrophil cell lines were used as controls. CD11b-deficient PMNs, known to have weaker endothelial interactions, migrated faster than the C57BL/6 and the hCD11b expressing cell lines (p<0.001 at 60 mns and p<0.05 at 90 mns). Statistical analysis by Bonferroni's multiple comparison test. (B) Time course of the migration of freshly isolated human 77R/R or 77R/H neutrophils through a HUVEC layer in response to MIP-2. Pooled results from at least 4 independent experiments are presented as mean ± SEM. (C, D) In vivo peritoneal migration of hCD11b-77R and hCD11b-77H PMN cells lines following i.p. injection of MIP-2 (C) and thioglycollate (D). The two hCD11b expressing PMN lines were labelled with DDAO or CFSE and adoptive transferred at a 1∶1 ratio into C57BL/6 mice. Absolute numbers of labelled PMNs recovered from the peritoneum are shown. Data of one out of at least 3 independent experiments are presented. Bars indicate means.
Fig 2: Cytokine response.Monocytes (A), DCs (B) were stimulated with 2 µg/ml and 10 µg/ml of TLR7/8 ligand (R848) respectively for 24 h. Cytokines quantified using a bead multiplex assay. Closed symbols: 77R/R cells, open symbols: 77R/H-77H/H cells. Each dot represents a single individual, bars denote means. No significant differences between the two CD11b genotypes. Statistical analysis by paired t test. (C, D) Modulation of TLR7/8-induced cytokine release by hiC3b-coated beads. Monocytes (C), DCs (D) were fed with hiC3b-coated beads one hour prior to R848 stimulation. The cytokine changes between the samples with and without CR3 pre-engagement with iC3b are shown with the p values indicated. Data are expressed as mean+/−SEM. The cytokine responses of 77R/R cells (black column) and 77R/H-77H/H cells (white columns) were not statistically different in paired assays. IL, interleukin; TNF-α, tumour necrosis factor alpha; IP-10, Interferon gamma-induced protein 10.
Fig 3: Cell surface expression of CD11b on different cell populations.The expression was quantified by flow cytometry using ICRF44 (A) and CBRM1/5 (B) antibodies. The latter only recognises the headpiece of CD11b in its active state. Data are presented in mean fluorescence intensity (MFI), closed symbols 77R/R donors, open symbols 77R/H-77H/H donors. The two groups were not statistically different. Bars indicate means.
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