Analysis of Heparanase expression in human natural killer cells

Abstract

Natural Killer (NK) cells are cells of the innate immune system important for tumour and viral clearance. Lack of functional human NK cells results in severe and recurrent viral infections in humans. Migration of NK cells requires movement through the extracellular matrix (ECM) and basement membrane (BM). The ECM and BM are structural scaffolds which provide shape and support to cells and tissues. Migration to target sites requires the degradation of the ECM and BM. Heparanase (HPSE) is a {u03B2}3-D-endoglucoronidase which cleaves the heparan sulphate (HS) side chains of heparan sulphate proteoglycans (HSPGs), a major component of the ECM and BM. Expression of HPSE by NK cells may facilitate migration to target sites, and ultimately affect NK function. The expression of HPSE has been documented in several leukocyte cells, but not NK cells. This study investigated whether heparanase is expressed by human NK cells. Utilizing immunofluorescence flow cytometry and quantitative RT-PCR (qRT-PCR), it is shown that heparanase protein as well as mRNA are constitutively expressed in resting human NK cells. Activation and proliferation of NK cells after culturing with certain target cells and interleukin 2 (IL-2) results in upregulation of HPSE mRNA and protein expression. Expression of HPSE mRNA and protein did not show the functionality of the HPSE protein. Utilizing a heparanase functional assay, it is shown that heparanase activity is upregulated, on a per cell basis, in activated NK (a-NK) cells compared to fresh NK (f-NK) cells. The intracellular distribution of HPSE in NK cells was also of interest. Investigation into the intracellular distribution of HPSE of f-NK and a-NK was done by confocal microscopy. Analysis by confocal microscopy indicates that heparanase has a broad intracellular distribution within NK cells, and is not restricted to lysosomes based on CD107a staining. In addition, HPSE is not associated with Granzyme B based on CD107a staining. This is the first report of heparanase expression in human NK cells. The cell surface calcium independent mannose 6-phosphate receptor (CIMPR) has recently been shown to bind HPSE and facilitate cellular migration after binding. Data in Chapter 4 show that human NK cells do not express any cell surface CIMPR and may not bind HPSE on the cell surface. The HPSE protein expressed by NK cells is functional, but the assessment was done with lysed NK cells so it was not known if live NK cells could utilize HPSE. Chapter 5 describes the ability of live NK cells to use heparanase to degrade ECM in a {u00B3}{u2075}S-ECM degradation assay. Data is also presented in Chapter 5 which describes the relevance of HPSE for human NK migration to human tumours in a mouse model. Inhibition of HPSE had no effect on the migration of human NK cells to human tumours in vivo. This thesis presents strong evidence that HPSE is expressed human NK cells and HPSE is both functional and utilised. Show more