Figure 1.
HPSE mediates release of pro-inflammatory cytokines from human peripheral blood mononuclear cells.
Quantitation of IL-8, IL-6, IL-1β, TNF, IL-10 and IL-12p70 released by human PBMCs after treatment with 10 µg/mL exogenous endotrap-purified HPSE for 16 h, using a human pro-inflammatory CBA assay. Data represent the mean ±SEM of triplicate samples, representative of three independent experiments. *p = <0.05, **p = <0.001 unpaired, two-tailed Student's t-test.
Figure 2.
HPSE induces gene expression of pro-inflammatory cytokines from human peripheral blood mononuclear cells.
Expression of IL-8, TNF, IL-1β, IL-6, IL-10 and IL-12p35 mRNA by RT-PCR in PBMCs incubated in the presence or absence of 10 µg/ml HPSE for 4, 12 or 24 h. Data are expressed as the mean level of expression normalised to ubc e2d2, data represent the mean ±SEM of triplicate samples, results are representative of three independent experiments. *p = <0.05, **p = <0.001 unpaired, two-tailed Student's t-test.
Figure 3.
Proteinase-K treated HPSE results in a reduced cytokine response in PBMCs.
HPSE (1 µg) was treated with proteinase-K agarose beads (80 µg/ml) for 30 min at 37°C before removal of proteinase-K agarose beads and SDS-PAGE analysis. (A) Western blot analysis for HPSE confirms that proteinase-K treatment dramatically reduced intact HPSE levels in the sample. (B) HPSE activity assay on identically treated samples of either HPSE alone, proteinase K alone, or HPSE treated with proteinase K. Data represent the mean ±SEM (n = 3). (C) Expression of cytokines after stimulation with proteinase-K treated HPSE in IL-1β, IL-6, IL-10, IL-8 and TNF in PBMCs isolated from human whole blood. Data represent the mean ±SEM of triplicate samples, results are representative of three independent experiments. *p = <0.05, **p = <0.001; unpaired, two-tailed Student's t-test.
Figure 4.
HPSE cleaves HS from the cell surface, and soluble HS leads to cytokine release.
(A) The ability of HPSE to cleave HS in in vitro conditions was examined by treating cells with either the positive control of PK (100 µg/mL) or HPSE (10 µg/mL) for 1 h before staining for the presence of cell-surface HS, and analysing by flow cytometry. MFI of representative overlaid histograms of untreated (grey-filled curve), HPSE treated (empty black line), or PK treated cells (dotted line). (B) Endotrap-purified HS fragments (50 µg/mL) were added to PBMCs for the indicated time, before the supernatant was used in a human CBA pro-inflammatory assay to quantify cytokine release. Data represent the mean ±SEM of triplicate samples, results are representative of three independent experiments. *p = <0.05, **p = <0.001; unpaired, two-tailed Student's t-test.
Figure 5.
HPSE-induced cytokine release is dependent on the enzymatic activity of heparanase.
(A) HPSE (1 ng) was treated with the enzymatic inhibitors heparin (10 ng) or OGT 2115 (20 ng) for 10 min at 37°C. HPSE activity assays were performed on treated samples of either HPSE alone, buffer or inhibitors alone, or HPSE treated with inhibitors. Data represent the mean ±SEM (n = 3). (B) Expression of cytokines (IL-8, IL-6, IL-1β, TNF and IL-10) by human PBMCs isolated from whole blood after stimulation with heparanase (10 µg/ml) treated with enzymatic inhibitors heparin (100 µg/ml) or OGT 2115 (200 µg/ml) for 24 h. Data represent the mean ±SEM of triplicate samples, results are representative of three independent experiments. *p = <0.05, **p = <0.001; unpaired, two-tailed Student's t-test.
Figure 6.
HPSE dependent cytokine release is via a MyD88-dependent pathway.
Mouse splenocytes from WT and MyD88−/− mice were isolated and treated with HPSE (10 µg/mL) or LPS (100 nM) for the indicated time, before the supernatant was analysed using the CBA assay for the mouse pro-inflammatory cytokines MCP-1, IL-6 and TNF. Data represent the mean ±SEM of triplicate samples, results are representative of three independent experiments. *p = <0.05, **p = <0.001; unpaired, two-tailed Student's t-test.
Figure 7.
HPSE signals via TLR4 to upregulate cytokine expression.
Mouse splenocytes isolated from WT and TLR4−/− mice were treated with HPSE (10 µg/mL) or LPS (100 nM) for the indicated time, before the supernatant was analysed via a mouse CBA pro-inflammatory cytokine assay for MCP-1, IL-6, and TNF. Statistical analysis was performed on WT and TLR4−/− HPSE treated cells, as well as TLR4−/− LPS and HPSE treated cells. Data represent the mean ±SEM (n = 5). *p = <0.05, **p = <0.001; unpaired, two-tailed Student's t-test.
Figure 8.
HPSE-treated cells exhibit NF-κB pathway activation.
SV40T immortalised MEFS infected with pTRH1-NF-κB-GFP were treated with HPSE (10 µg/mL) or LPS (100 nM) for the indicated time and analysed by immunofluorescence flow cytometry. (A) Representative overlaid histograms of HPSE treated cells for 0 h (grey-filled curve), 1 h (empty black line), 2 h (dotted line) and 3 h (dashed lines). (B) MFI calculated from the histograms. Data represent the mean ±SEM (n = 6). * = p<0.05, ** = p<0.001; unpaired, two-tailed Student's t-test.
Figure 9.
Model of cytokine upregulation in response to HPSE.
Schematic model of the proposed function of HPSE in releasing pro-inflammatory cytokines. (1) HPSE cleaves and solubilises HS fragments from the cell surface of immune cells. (2) HS fragments then signal through MyD88-dependent receptors, of which TLR4 is one, and this leads to NF-κB cleavage and activation. (3) NF-κB-dependent upregulation of cytokine production and release, including IL-8, IL-10, IL-6, TNF and IL-1β. (4) Cytokines are involved in inflammatory events including chemoattraction and increased antibody production. (5) These events contribute to the induction of an inflammatory response.
Table 1.
qPCR oligonucleotide sequences.