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First published online 22 July 2003
doi: 10.1242/jcs.00662

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Endothelial inflammation: the role of differential expression of N-deacetylase/N-sulphotransferase enzymes in alteration of the immunological properties of heparan sulphate

¹ Applied Immunobiology Research Group, Department of Surgery, University of Newcastle, The Medical School, Newcastle upon Tyne NE2 4HH, UK
² Institute of Pharmacy, Chemistry and Biomedical Science, University of Sunderland, Sunderland SR1 3SD, UK

View larger version (42K): [in a new window]   Fig. 1. Expression of NDST-1 and -2 transcripts by different cell types. Samples were run in a 1.5% agarose gel; for each sample 100 µg of total RNA (A), or 1 µg of poly(A)+ RNA (B) was run and blotted onto a nylon membrane. RNA was isolated from the cell types displayed at the top of the figure. Northern blot analysis was performed using a random prime synthesised probe using [32P]dCTP for NDST-1 (A) and an antisense probe labelled with [32P]UTP for NDST-2 (B). The bands showing 18S rRNA within the total RNA indicate the relative loading of each of the samples. The size of the RNA species was determined by co-electrophoresis of mRNA size standards (Promega).

View larger version (27K): [in a new window]   Fig. 2. Semi-quantitative analysis of NDST-1 and -2 transcript expression by the HMEC-1 cell line. Semi-quantitative RT-PCR analysis was carried out for HMEC-1 cells both 4 hours (A) and 16 hours (B) after stimulation with the pro-inflammatory cytokines TNF- and IFN-; the results were compared with data from resting cells. The RANTES transcript was also examined as a positive control and Gap-DH was examined as an internal standard. The gel images show the RT-PCR products generated from unstimulated (-) or stimulated (+) HMEC-1 cells with their respective Gap-DH products. The white bars in the graphs show the mean relative band intensity for the unstimulated control cells and are normalised to 100%. The black bars represent the mean relative band intensity for the cells stimulated with TNF- and IFN-. The P values above the bars show the significance of differences between the data sets.

View larger version (18K): [in a new window]   Fig. 3. Phenotypic analysis of the abundance of sulphated epitopes in HS. HMEC-1 cells were analysed by flow cytometry to measure the level of sulphation at 0, 16, 24, 48 and 72 hours after stimulation with TNF- and IFN-. (A) Representative flow cytometer fluorescence histograms showing the isotype control value (black) and data from time 0 (blue), 16 (green) and 72 hours (red); median values are also shown. (B) Summary results showing the increase in fluorescence due to binding of 10E4 antibodies to cytokine activated HMEC-1 cells; the dotted line indicates the upper level of isotype control fluorescence. Data points show the mean from triplicate determinations; the error bars indicate the s.e.m.

View larger version (11K): [in a new window]   Fig. 4. Analysis of the kinetics of GAG turnover. HMEC-1 cells were labelled with 3H-glucosamine for 24 hours and were then washed and cultured either with or without TNF- and IFN-. The cells were harvested after 4 hours and 16 hours, the GAGs were purified and incorporated 3H was measured. Bars represent the mean relative cpm for GAG derived from 105 cells. The sample at 0 hours (shaded) provides a measure of the initial level of incorporation. The white bars represent unstimulated cells and the black bars indicate cells stimulated with TNF- and IFN-. The P values above the bars indicate the significance of differences between the unstimulated and stimulated groups after 4 hours and 16 hours; the error bars show the s.e.m.

View larger version (34K): [in a new window]   Fig. 5. Analysis of proteoglycan core protein expression by western blotting. Lysates from HMEC-1 cells stimulated for 4 hours or 16 hours with TNF- and IFN- were compared with those from unstimulated cells. All lysates were digested in the presence of chondroitinase ABC and heparatinase. The cleavage products were separated by SDS-PAGE, electrotransferred to hybond-P membranes and probed by mAb 3G10. The figure is a composite generated from gels containing 8% (A) and 12% (B) acrylamide, as indicated. Blots were reprobed with an anti (a)-tubulin antibody to provide an internal protein loading control (C). Apparent molecular masses of the core proteins are indicated.

View larger version (57K): [in a new window]   Fig. 6. Immunofluorescence detection of RANTES binding to HMEC-1 cells. Scanning laser confocal microscopy was used to analyse the distribution of RANTES on the surface of HMEC-1 cells that had been cultured using a range of experimental conditions. (A) RANTES expression on the surface of resting HMEC-1 which had been treated with exogenous RANTES. (B) RANTES expression on the surface of HMEC-1 that had been stimulated with TNF- and IFN- and then treated with exogenous RANTES. (C) Expression of endogenous RANTES on the surface of HMEC-1 that had been stimulated with TNF- and IFN-. (D) RANTES expression on the surface of HMEC-1 cells that had been stimulated with TNF- and IFN- in the presence of chlorate and then treated with exogenous RANTES. (E) A constructed X-Z section through Fig. 6B confirming that binding of the exogenous RANTES occurs only on the apical surface of the cultured endothelial cells. (F) Summary data showing inverted mean fluorescence pixel intensity per cell for each of the four treatment groups; the resting and (cytokine) activated groups were treated with exogenous RANTES, the endogenous control group was cytokine-activated but received no exogenous RANTES and the chlorate control group was cytokine activated and received exogenous RANTES. The error bars show the s.e.m.

View larger version (12K): [in a new window]   Fig. 7. Radioligand quantification of RANTES binding to HMEC-1 cells. HMEC-1 cells were grown in the presence of medium alone (white bars), TNF- and IFN- (black bars) or TNF- and IFN- in the presence of 100 mM sodium chlorate (shaded bar). The cells were then treated with 125I-RANTES and the bound isotope was subsequently determined and expressed as counts per cell; data from the untreated control cells was normalised to 100% and the treated cells were measured relative to this value. The p value above the bars indicates the significance of differences between the unstimulated and stimulated groups after 4 hours and 16 hours; error bars show the s.e.m.

View larger version (16K): [in a new window]   Fig. 8. Comparison of leukocyte chemotaxis across monolayers of resting and activated HMEC-1. The number of migrant THP-1 cells on the lower surface of each transwell filter was assessed by counting the cells per high power microscope field. The bars show the mean number of cells in 10 fields per filter; the error bars represent the s.e.m.

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