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Fibroblasts from different sites may promote or inhibit recruitment of flowing lymphocytes by endothelial cells.

McGettrick HM, Smith E, Filer A, Kissane S, Salmon M, Buckley CD, Rainger GE, Nash GB - Eur. J. Immunol. (2009)

Bottom Line: Interestingly, co-culture with dermal fibroblasts caused a marked reduction in cytokine-induced adhesion, while synovial fibroblasts had variable effects depending on their source.Exogenous IL-6 was also found to inhibit response to TNF-alpha+IFN-gamma.Normal stromal fibroblasts appear to regulate the cytokine-sensitivity of vascular endothelium, while fibroblasts associated with chronic inflammation bypass this and develop a directly inflammatory phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cardiovascular Sciences, The Medical School, The University of Birmingham, Birmingham, UK.

ABSTRACT
We examined the hypothesis that stromal fibroblasts modulate the ability of endothelial cells (EC) to recruit lymphocytes in a site-specific manner. PBL were perfused over HUVEC that had been cultured with fibroblasts isolated from the inflamed synovium or the skin of patients with rheumatoid arthritis or osteoarthritis, or from normal synovium, with or without exposure to the inflammatory cytokines TNF-alpha+IFN-gamma. Fibroblasts from inflamed synovium, but no others, caused unstimulated HUVEC to bind flowing lymphocytes. This adhesion was supported by alpha(4)beta(1)-VCAM-1 interaction and stabilised by activation of PBL through CXCR4-CXCL12. Antibody neutralisation of IL-6 during co-culture effectively abolished the ability of EC to bind lymphocytes. Cytokine-stimulated EC supported high levels of lymphocyte adhesion, through the presentation of VCAM-1, E-selectin and chemokine(s) acting through CXCR3. Interestingly, co-culture with dermal fibroblasts caused a marked reduction in cytokine-induced adhesion, while synovial fibroblasts had variable effects depending on their source. In the dermal co-cultures, neutralisation of IL-6 or TGF-beta caused partial recovery of cytokine-induced lymphocyte adhesion; this was complete when both were neutralised. Exogenous IL-6 was also found to inhibit response to TNF-alpha+IFN-gamma. Normal stromal fibroblasts appear to regulate the cytokine-sensitivity of vascular endothelium, while fibroblasts associated with chronic inflammation bypass this and develop a directly inflammatory phenotype. Actions of IL-6 might be pro-inflammatory or anti-inflammatory, depending on the local milieu.

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Related in: MedlinePlus

Effect of co-culture on the gene transcriptional profiles of EC. EC were cultured alone or with human RA dermal or synovial fibroblasts (HDF or HSF respectively) for 24 h in the absence of exogenous cytokines. Endothelial mRNA was isolated and analysed using microarray technology on an 850 human gene array. Data was normalised (sample/reference) and then analysed by TIGR Multiple Experiment Viewer allowing statistical analysis of microarrays (SAM) and hierarchical cluster analysis (HCA). The SAM false-positive level was set at 0%. (A) List of 18 genes identified by SAM to vary significantly between the three culture-conditions. (B) HCA based on the 18 genes identified. The colour scale from green to red for each gene represents the natural logarithm of the gene expression, relative to the value for the reference sample tested with it. The different culture conditions showed distinct segmentations, as indicated by the coloured tree (blue-brown-yellow) across the top, associated with them. Comparisons between the different culture conditions were done on three occasions, each using different primary EC isolates and different arrays. In each array, the individual genes were tested in triplicate.
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fig06: Effect of co-culture on the gene transcriptional profiles of EC. EC were cultured alone or with human RA dermal or synovial fibroblasts (HDF or HSF respectively) for 24 h in the absence of exogenous cytokines. Endothelial mRNA was isolated and analysed using microarray technology on an 850 human gene array. Data was normalised (sample/reference) and then analysed by TIGR Multiple Experiment Viewer allowing statistical analysis of microarrays (SAM) and hierarchical cluster analysis (HCA). The SAM false-positive level was set at 0%. (A) List of 18 genes identified by SAM to vary significantly between the three culture-conditions. (B) HCA based on the 18 genes identified. The colour scale from green to red for each gene represents the natural logarithm of the gene expression, relative to the value for the reference sample tested with it. The different culture conditions showed distinct segmentations, as indicated by the coloured tree (blue-brown-yellow) across the top, associated with them. Comparisons between the different culture conditions were done on three occasions, each using different primary EC isolates and different arrays. In each array, the individual genes were tested in triplicate.

Mentions: The functional responses induced or modified by co-culture were not clearly linked to changes in the expression of single genes (either for adhesion molecules, chemokines or IL-6 receptors). Nevertheless, IL-6 was implicated in distinct and diverse effects for the different co-cultures, suggesting that the underlying state of the EC may have been altered under the different culture conditions. To investigate this possibility, we used DNA microarray technology to examine changes in the transcriptional profiles of EC cultured alone or with the different fibroblasts from RA donors. The microarray analysed 850 immune-response genes (associated with adhesion and chemokine receptors, cytokines, apoptosis, cell cycle and intracellular signalling pathways). Our aim was to test whether the cells had taken on separate genomic ‘inflammatory’ phenotypes, rather than to identify specific genes that might be linked to altered function. Indeed, at a 0% false-positive level, ‘statistical analysis of microarrays’ (SAM) indicated that there were 18 genes that varied significantly between the three culture-conditions (Fig. 6A). Subsequent hierarchical clustering analysis (HCA) based on these genes showed distinct segmentation between the transcriptional profiles of the endothelial mono- and co-cultures (Fig. 6B). Interestingly, EC co-cultured with synovial fibroblasts displayed a transcriptional phenotype further removed from EC cultured alone than EC co-cultured with dermal fibroblasts (Fig. 6). This trend was borne out by individual comparisons of culture conditions, where 79 genes showed significant differences between EC cultured alone or with synovial fibroblasts, and only two genes were significantly different between EC cultured alone or with dermal fibroblast (data not shown). Thus, microarray analysis indicated that EC cultured with RA synovial fibroblasts had developed a separate phenotype from EC cultured with dermal fibroblast and from those in mono-culture.


Fibroblasts from different sites may promote or inhibit recruitment of flowing lymphocytes by endothelial cells.

McGettrick HM, Smith E, Filer A, Kissane S, Salmon M, Buckley CD, Rainger GE, Nash GB - Eur. J. Immunol. (2009)

Effect of co-culture on the gene transcriptional profiles of EC. EC were cultured alone or with human RA dermal or synovial fibroblasts (HDF or HSF respectively) for 24 h in the absence of exogenous cytokines. Endothelial mRNA was isolated and analysed using microarray technology on an 850 human gene array. Data was normalised (sample/reference) and then analysed by TIGR Multiple Experiment Viewer allowing statistical analysis of microarrays (SAM) and hierarchical cluster analysis (HCA). The SAM false-positive level was set at 0%. (A) List of 18 genes identified by SAM to vary significantly between the three culture-conditions. (B) HCA based on the 18 genes identified. The colour scale from green to red for each gene represents the natural logarithm of the gene expression, relative to the value for the reference sample tested with it. The different culture conditions showed distinct segmentations, as indicated by the coloured tree (blue-brown-yellow) across the top, associated with them. Comparisons between the different culture conditions were done on three occasions, each using different primary EC isolates and different arrays. In each array, the individual genes were tested in triplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig06: Effect of co-culture on the gene transcriptional profiles of EC. EC were cultured alone or with human RA dermal or synovial fibroblasts (HDF or HSF respectively) for 24 h in the absence of exogenous cytokines. Endothelial mRNA was isolated and analysed using microarray technology on an 850 human gene array. Data was normalised (sample/reference) and then analysed by TIGR Multiple Experiment Viewer allowing statistical analysis of microarrays (SAM) and hierarchical cluster analysis (HCA). The SAM false-positive level was set at 0%. (A) List of 18 genes identified by SAM to vary significantly between the three culture-conditions. (B) HCA based on the 18 genes identified. The colour scale from green to red for each gene represents the natural logarithm of the gene expression, relative to the value for the reference sample tested with it. The different culture conditions showed distinct segmentations, as indicated by the coloured tree (blue-brown-yellow) across the top, associated with them. Comparisons between the different culture conditions were done on three occasions, each using different primary EC isolates and different arrays. In each array, the individual genes were tested in triplicate.
Mentions: The functional responses induced or modified by co-culture were not clearly linked to changes in the expression of single genes (either for adhesion molecules, chemokines or IL-6 receptors). Nevertheless, IL-6 was implicated in distinct and diverse effects for the different co-cultures, suggesting that the underlying state of the EC may have been altered under the different culture conditions. To investigate this possibility, we used DNA microarray technology to examine changes in the transcriptional profiles of EC cultured alone or with the different fibroblasts from RA donors. The microarray analysed 850 immune-response genes (associated with adhesion and chemokine receptors, cytokines, apoptosis, cell cycle and intracellular signalling pathways). Our aim was to test whether the cells had taken on separate genomic ‘inflammatory’ phenotypes, rather than to identify specific genes that might be linked to altered function. Indeed, at a 0% false-positive level, ‘statistical analysis of microarrays’ (SAM) indicated that there were 18 genes that varied significantly between the three culture-conditions (Fig. 6A). Subsequent hierarchical clustering analysis (HCA) based on these genes showed distinct segmentation between the transcriptional profiles of the endothelial mono- and co-cultures (Fig. 6B). Interestingly, EC co-cultured with synovial fibroblasts displayed a transcriptional phenotype further removed from EC cultured alone than EC co-cultured with dermal fibroblasts (Fig. 6). This trend was borne out by individual comparisons of culture conditions, where 79 genes showed significant differences between EC cultured alone or with synovial fibroblasts, and only two genes were significantly different between EC cultured alone or with dermal fibroblast (data not shown). Thus, microarray analysis indicated that EC cultured with RA synovial fibroblasts had developed a separate phenotype from EC cultured with dermal fibroblast and from those in mono-culture.

Bottom Line: Interestingly, co-culture with dermal fibroblasts caused a marked reduction in cytokine-induced adhesion, while synovial fibroblasts had variable effects depending on their source.Exogenous IL-6 was also found to inhibit response to TNF-alpha+IFN-gamma.Normal stromal fibroblasts appear to regulate the cytokine-sensitivity of vascular endothelium, while fibroblasts associated with chronic inflammation bypass this and develop a directly inflammatory phenotype.

View Article: PubMed Central - PubMed

Affiliation: Centre for Cardiovascular Sciences, The Medical School, The University of Birmingham, Birmingham, UK.

ABSTRACT
We examined the hypothesis that stromal fibroblasts modulate the ability of endothelial cells (EC) to recruit lymphocytes in a site-specific manner. PBL were perfused over HUVEC that had been cultured with fibroblasts isolated from the inflamed synovium or the skin of patients with rheumatoid arthritis or osteoarthritis, or from normal synovium, with or without exposure to the inflammatory cytokines TNF-alpha+IFN-gamma. Fibroblasts from inflamed synovium, but no others, caused unstimulated HUVEC to bind flowing lymphocytes. This adhesion was supported by alpha(4)beta(1)-VCAM-1 interaction and stabilised by activation of PBL through CXCR4-CXCL12. Antibody neutralisation of IL-6 during co-culture effectively abolished the ability of EC to bind lymphocytes. Cytokine-stimulated EC supported high levels of lymphocyte adhesion, through the presentation of VCAM-1, E-selectin and chemokine(s) acting through CXCR3. Interestingly, co-culture with dermal fibroblasts caused a marked reduction in cytokine-induced adhesion, while synovial fibroblasts had variable effects depending on their source. In the dermal co-cultures, neutralisation of IL-6 or TGF-beta caused partial recovery of cytokine-induced lymphocyte adhesion; this was complete when both were neutralised. Exogenous IL-6 was also found to inhibit response to TNF-alpha+IFN-gamma. Normal stromal fibroblasts appear to regulate the cytokine-sensitivity of vascular endothelium, while fibroblasts associated with chronic inflammation bypass this and develop a directly inflammatory phenotype. Actions of IL-6 might be pro-inflammatory or anti-inflammatory, depending on the local milieu.

Show MeSH
Related in: MedlinePlus