Role of heat shock protein 70 and sulphatases 1 and 2 in apoptosis induced by cytotoxic cells of the immune system

Intracellular heat shock protein 70 (HSP70) belongs to the stress response system and can protect cells from different apoptotic stimuli. Extracellular HSP70 on the other hand can activate cells of the innate and adaptive immune system. Acute overexpression of intracellular HSP70 could even increase the susceptibility of melanoma cells to cytotoxic T-lymphocytes (CTLs) that use the granule-exocytosis pathway for killing (Dressel et al. 1999). To decipher the molecular pathway of this increased susceptibility to CTLs, the effect of the acute overexpression of HSP70 on gene expression was analysed in cells, in which Hsp70 is under the control of a tetracycline-inducible promoter. The results indicated that the acute overexpression of HSP70 does not have a major impact on the regulation of other genes. Thus, the effects of HSP70 on CTL-mediated killing are most likely regulated on the protein level.To reduce the complexity of CTL-induced apoptosis, further experiments were performed with granzyme (Gr)B, a component of the cytotoxic granules of CTLs and NK cells, that has been shown to interact with HSP70. Acute overexpression of HSP70 significantly increased the percentage of melanoma cells with fragmented DNA and of melanoma cells with reduced mitochondrial cytochrome c content after GrB-induced apoptosis. These results suggest that HSP70 is not inhibiting GrB-induced apoptosis but can slightly enhance the progression of apoptosis in some cells. In staurosporine-induced apoptosis, in contrast, the acute overexpression of HSP70 partly protected the same cells from early apoptosis.Heparan sulphates (HSs) are involved in GrB-binding to target cells of CTLs and Raja et al. (2005) could show that a suppression of sulphation of HSs on the cell surface led to a reduced uptake of GrB and diminished GrB and CTL-induced apoptosis. A deficiency of the Sulf1 and Sulf2 genes, encoding for sulphatases 1 and 2, which modify the sulphation pattern of cells, could therefore have an effect on the efficiency of CTL-mediated apoptosis due to the fact that Sulf-deficient cells show increased sulphation patterns on HS proteoglycans (PGs). Mouse embryonic fibroblasts (MEFs) from Sulf1 and Sulf2 double knock-out mice were used as targets for CTLs. Their susceptibility to granule-exocytosis-dependent lysis was slightly but significantly increased. The slight change could be attributed to the deficiency of the Sulf1 gene. GrB-induced apoptosis was not significantly altered suggesting no major contribution of Sulf1 and Sulf2 genes to the control of susceptibility of target cells in GrB and CTL-induced apoptosis.However, during these experiments it turned out that the deficiency of Sulf1 and Sulf2 or both drastically increased the infection of these cells with adenovirus (AdV) type 5 that was used to deliver the GrB. Target cell infection was determined by green fluorescence protein (GFP) expression since the AdV contained a GFP expression construct. This increase was not explained by a different expression of receptors known to be involved in the uptake of AdV (coxsackie and adenovirus receptor (CAR) and integrin αv) on the Sulf-deficient MEFs. A digestion of HSPGs by heparinases II and III further increased the efficacy of infection. Similarly, a genetic defect of HSPGs in CHO cells was associated with an increased infection rate.These results suggest that HSPGs could function as decoy receptors for AdV channeling them into a non-infectious pathway rather than functioning as co-receptors for infection.