CD64-mediated specific elimination of M1-polarized macrophages : implications for therapeutic intervention in chronic inflammatory diseases

Macrophages are key players of the innate immunity and versatile cells that can adapt to microenvironmental signals resulting in distinct polarization states with altered phenotype and function. The dynamic ratio of M1 and M2 polarized macrophages is critical for the appropriate development and resolution of inflammation. In chronic diseases, this normal progression is arrested in the M1 dominant phase. In this thesis, the lack of available therapy strategies for treatment of chronic inflammatory diseases was addressed using CD64-targeted immunotoxins. Murine and human macrophages were polarized and characterized using a set of membrane and soluble markers. Among several other surface receptors including CD14, CD16 and MHCI/II, CD64 was unique due its increased expression in both murine and human M1 cells. M1 macrophages were also characterized by the increased production of pro-inflammatory cytokines such as IL-12, IL-6 and TNF-α. In contrast, CD206 and CD301 were more abundant on the surface of M2 macrophages and the pro-inflammatory cytokines listed above were suppressed. In vitro targeting experiments revealed the exclusive elimination of M1 type macrophages by three different hCD64-targeted proteins: the chimeric IT H22(sFv)-ETA' and the two fully human fusion proteins H22(scFv)-Angiogenin and Granzyme B-H22(scFv). Staining with AnnexinV-FITC confirmed that H22(scFv)-ETA' selectively induced apoptosis in M1 macrophages. M1-specific susceptibility was shown to be due to a reduced endosomal protease activity. These results were confirmed in vivo in a SLS-induced cutaneous chronic inflammation model in transgenic mice and ex vivo in a skin biopsy from a patient with atopic dermatitis. In addition, both populations showed phenotypic plasticity and after conversion into either type, only M1 polarization resulted in sensitivity towards the CD64-directed immunotoxin. In an attempt to replace the immunogenic bacterial toxin ETA' with a fully human effector protein, the ability of the human microtubule-associated protein tau (MAP) to act as a pro-apoptotic protein was investigated. Therefore, MAP was cloned in frame with H22(scFv), expressed in E. coli, and purified by standard chromatographic methods. Successful binding of H22(scFv)-MAP to the promyelocytic leukemia cell lines HL-60 and U937, and to polarized macrophages, was confirmed by flow cytometry. In vitro cell viability assays revealed the proliferation-dependent cytotoxicity of H22(scFv)-MAP. The in vivo performance was tested in the transgenic mouse model described above, revealing that H22(scFv)-MAP also selectively eliminated M1 macrophages, while leaving M2 macrophages unaffected. The successful killing of M1 macrophages by H22(scFv)-MAP, which confers cytotoxicity to proliferating cells only, suggested that fully differentiated and polarized tissue macrophages proliferate in situ. The local proliferation of M1 macrophages was confirmed by immunohistochemical analysis. In summary, these results show that CD64 is an appropriate target for therapeutic approaches focusing on M1 macrophages and attempting to alter their function or role in chronic inflammation. Specific targeting of M1 macrophages provides a therapeutic strategy to intervene in most chronic inflammatory diseases, e.g. rheumatoid arthritis, inflammatory bowel disease and chronic wounds