Glioma-associated hyaluronan induces apoptosis in dendritic cells via inducible nitric oxide synthase: implications for the use of dendritic cells for therapy of gliomas. 
As a means of enhancing immunity to gliomas, we investigated local delivery of rat, bone marrow-derived dendritic cells (DCs) into rat 9L gliosarcoma tumors and into 9L tumors induced to undergo apoptosis by gamma knife radiosurgery.  Contrary to other tumors, local delivery of DCs had no therapeutic effect on 9L gliomas, even when tumor apoptosis was induced via radiosurgery, which leads to efficient "loading" of the DCs with tumor antigen.  To determine whether antigen-presenting cells, such as DCs, were viable in tumors, we carried out multiparametric staining of 9L tumors, using phycoerythrin-conjugated OX6 (MHC class II) or OX62 (DC specific) and FITC-labeled Val-Ala-Asp-fluoromethyl ketone (FITC-VAD-FMK; activated caspases).  It was determined that DCs were undergoing apoptosis in these tumors.  We therefore sought to determine which glioma cell surface receptors or components of the extracellular matrix in gliomas influenced DC viability.  Hyaluronan (HA) is a major component of glioma extracellular matrix and has been found to support tumor cell migration and metastasis.  However, its influence on the immune system, and particularly on DCs, via its receptor CD44 is not well documented.  Using reverse transcription-PCR, Northern blot, and Western blot analyses, we determined that HA stimulated production of inducible nitric oxide synthase (iNOS) in DCs.  NO production by HA-stimulated DCs was then verified biochemically.  NO production was dependent on the size of HA; intermediate HA fragments had the greatest capacity to induce NO production in DC, whereas completely digested HA oligosaccharides failed to induce NO.  Furthermore, N-monomethyl-L-arginine, an inhibitor of iNOS, completely blocked HA-induced NO production by DCs.  Because induction of NO results in the induction of apoptosis in macrophages as well as other cells, DCs treated with HA were examined for apoptosis in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP biotin nick-end labeling assays.  It was demonstrated that HA induced apoptosis in DCs and that induction of apoptosis was dependent on the production of NO because it was entirely inhibited by N-monomethyl-L-arginine.  Using flow cytometric analyses with FITC-VAD-FMK, which is specific for activated caspases, we also determined that induction of apoptosis in DCs with HA could be titrated.  Coincubation of 9L tumor cells with DCs was found to induce apoptosis in DCs as indicated by fluorescent staining with FITC-VAD-FMK.  Specificity of this reaction for CD44-HA interactions was determined by pretreatment of DCs with anti-CD44 or pretreatment of 9L tumor cells with hyaluronidase, which blocked the induction of apoptosis in DCs.  These data indicate that HA expressed by gliomas may contribute to their immunosuppressive effects by promoting apoptosis among professional antigen-presenting cells such as DCs via iNOS induction after CD44-HA interactions.