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Stromal cells differentially regulate neutrophil and lymphocyte recruitment through the endothelium.

McGettrick HM, Buckley CD, Filer A, Rainger GE, Nash GB - Immunology (2010)

Bottom Line: Stromal fibroblasts modify the initial recruitment of leucocytes by endothelial cells (EC), but their effects on subsequent transendothelial migration remain unclear.Fibroblasts had little effect on tumour necrosis factor-α-induced transendothelial migration of neutrophils, but tended to increase the efficiency of migration away from the endothelium.To avoid contact, we cultured EC and fibroblasts on separate 3-μm pore filters one above the other.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, Medical School, The University of Birmingham, Birmingham, UK. h.m.mcgettrick@bham.ac.uk

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Effect of fibroblasts on the expression of E-selectin or vascular cell adhesion molecule 1 (VCAM-1) by endothelial cells (EC). Co-cultures were formed for 24 hr before treatment with tumour necrosis factor-α plus interferon-γ (TNF + IFN) for a further 24 hr. In (a) and (b), endothelial messenger RNA was isolated from co-cultures treated with TNF + IFN and the gene expression of E-selectin (a) or VCAM-1 (b) was assessed by quantitative polymerase chain reaction. Values were expressed as relative expression units (REU) compared with β-actin. In (c) and (d), EC from cytokine-treated co-cultures were labelled using antibodies against (c) E-selectin or (d) VCAM-1 and surface expression was assessed by flow cytometry (expressed as mean fluorescence intensity; MFI). Data are mean ± SEM from three independent experiments.
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fig06: Effect of fibroblasts on the expression of E-selectin or vascular cell adhesion molecule 1 (VCAM-1) by endothelial cells (EC). Co-cultures were formed for 24 hr before treatment with tumour necrosis factor-α plus interferon-γ (TNF + IFN) for a further 24 hr. In (a) and (b), endothelial messenger RNA was isolated from co-cultures treated with TNF + IFN and the gene expression of E-selectin (a) or VCAM-1 (b) was assessed by quantitative polymerase chain reaction. Values were expressed as relative expression units (REU) compared with β-actin. In (c) and (d), EC from cytokine-treated co-cultures were labelled using antibodies against (c) E-selectin or (d) VCAM-1 and surface expression was assessed by flow cytometry (expressed as mean fluorescence intensity; MFI). Data are mean ± SEM from three independent experiments.

Mentions: We previously reported that adhesion of flowing lymphocytes to EC that had been treated with TNF-α + IFN-γ was mainly supported through α4β1-integrins binding to VCAM-1 on the EC, with a contribution from E-selectin; stabilization of this attachment required activation of lymphocytes through CXCR3 binding its ligands (CXCL9, CXCL10 and/or CXCL11).8 Using qPCR, we found that co-culture had no significant effect on mRNA levels for E-selectin, and tended to increase mRNA for VCAM-1 for EC treated with TNF-α + IFN-γ (Fig. 6a,b). As judged by flow cytometry, surface expression of E-selectin and VCAM-1 on the EC was not significantly different for cytokine-treated EC cultured alone or with fibroblasts (Fig. 6c,d). The RT-PCR for the CXCR3 ligands CXCL9, CXCL10 and CXCL11 showed their up-regulation in EC by the cytokines, but no difference in levels between EC cultured alone or with fibroblasts (data not shown). Hence, the functional loss observed in co-cultures could not be explained by changes in expression of endothelial adhesion receptors and chemokines identified as active in this model.


Stromal cells differentially regulate neutrophil and lymphocyte recruitment through the endothelium.

McGettrick HM, Buckley CD, Filer A, Rainger GE, Nash GB - Immunology (2010)

Effect of fibroblasts on the expression of E-selectin or vascular cell adhesion molecule 1 (VCAM-1) by endothelial cells (EC). Co-cultures were formed for 24 hr before treatment with tumour necrosis factor-α plus interferon-γ (TNF + IFN) for a further 24 hr. In (a) and (b), endothelial messenger RNA was isolated from co-cultures treated with TNF + IFN and the gene expression of E-selectin (a) or VCAM-1 (b) was assessed by quantitative polymerase chain reaction. Values were expressed as relative expression units (REU) compared with β-actin. In (c) and (d), EC from cytokine-treated co-cultures were labelled using antibodies against (c) E-selectin or (d) VCAM-1 and surface expression was assessed by flow cytometry (expressed as mean fluorescence intensity; MFI). Data are mean ± SEM from three independent experiments.
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Related In: Results  -  Collection

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fig06: Effect of fibroblasts on the expression of E-selectin or vascular cell adhesion molecule 1 (VCAM-1) by endothelial cells (EC). Co-cultures were formed for 24 hr before treatment with tumour necrosis factor-α plus interferon-γ (TNF + IFN) for a further 24 hr. In (a) and (b), endothelial messenger RNA was isolated from co-cultures treated with TNF + IFN and the gene expression of E-selectin (a) or VCAM-1 (b) was assessed by quantitative polymerase chain reaction. Values were expressed as relative expression units (REU) compared with β-actin. In (c) and (d), EC from cytokine-treated co-cultures were labelled using antibodies against (c) E-selectin or (d) VCAM-1 and surface expression was assessed by flow cytometry (expressed as mean fluorescence intensity; MFI). Data are mean ± SEM from three independent experiments.
Mentions: We previously reported that adhesion of flowing lymphocytes to EC that had been treated with TNF-α + IFN-γ was mainly supported through α4β1-integrins binding to VCAM-1 on the EC, with a contribution from E-selectin; stabilization of this attachment required activation of lymphocytes through CXCR3 binding its ligands (CXCL9, CXCL10 and/or CXCL11).8 Using qPCR, we found that co-culture had no significant effect on mRNA levels for E-selectin, and tended to increase mRNA for VCAM-1 for EC treated with TNF-α + IFN-γ (Fig. 6a,b). As judged by flow cytometry, surface expression of E-selectin and VCAM-1 on the EC was not significantly different for cytokine-treated EC cultured alone or with fibroblasts (Fig. 6c,d). The RT-PCR for the CXCR3 ligands CXCL9, CXCL10 and CXCL11 showed their up-regulation in EC by the cytokines, but no difference in levels between EC cultured alone or with fibroblasts (data not shown). Hence, the functional loss observed in co-cultures could not be explained by changes in expression of endothelial adhesion receptors and chemokines identified as active in this model.

Bottom Line: Stromal fibroblasts modify the initial recruitment of leucocytes by endothelial cells (EC), but their effects on subsequent transendothelial migration remain unclear.Fibroblasts had little effect on tumour necrosis factor-α-induced transendothelial migration of neutrophils, but tended to increase the efficiency of migration away from the endothelium.To avoid contact, we cultured EC and fibroblasts on separate 3-μm pore filters one above the other.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, Medical School, The University of Birmingham, Birmingham, UK. h.m.mcgettrick@bham.ac.uk

Show MeSH
Related in: MedlinePlus