Characterization of folate receptor positive monocytes/macrophages and folate receptor-mediated specific targeting in inflammatory diseases

Wei Xia, Purdue University


Macrophages have been shown to play an integral role in inflammatory diseases such as rheumatoid arthritis. Previous work has shown that a subset of macrophages under inflammatory conditions expresses a folate receptor (FR) that can mediate internalization of folate-linked molecules. This thesis work characterizes the subset of macrophages that express FR and further investigate whether monocytes, the precursors to activated macrophages, also express FR. FR expression was observed on a subset of highly activated peritoneal and synovial macrophages, but not on other infiltrating immune cells or non-activated resident macrophages. Cell surface markers that co-express with FR were identified and antigens characteristic of an activated state (CD80, CD86, Ly-6C/G) were found to correlate with the expression of FR on macrophages. Further, functional analysis indicated that FR+ macrophages produced tumor necrosis factor-alpha (TNF- α) and reactive oxygen species (ROS), and production of ROS correlated linearly with expression of FR. These results suggest that FR constitutes a marker for macrophage activation. FR was then demonstrated to not only mediate binding but also internalization of folate-linked molecules. Uptake of folate-conjugates by by FR+ macrophages was observed in an in vivo murine peritonitis model. Further, using this model, a folate-drug was shown to be able selectively kill FR+ macrophages. Translating from murine model to human arthritis patient, synovial macrophages collected from arthritic patients were found to bind folate-linked dyes. Moreover, a folate-linked radioimaging agent was shown to image inflamed joints of rheumatoid arthritic patients. Taken together, these data suggest that FR expressed on macrophages can be targeted with folate-linked drugs without promoting drug uptake by non-activated macrophages. A subpopulation of human peripheral blood monocytes was also found to expresses FR, albeit at a lower level than activated macrophages. Surface antigen characterization studies illustrated that FR was preferably expressed on classic CD14HighCD16¯ monocytes. Furthermore, it was found that these FR+ monocytes were recruited into the synovial joints of arthritic patients and display an activated phenotype (expressing CD86, HLA-DR, and CD163). Preliminary clinical data also suggest that the numbers of peripheral FR+ monocytes could be reduced by a folate-hapten immunotherapy directed against FR. Finally, a subset of FR+ monocytes/macrophages that also expressed stem/progenitor markers CD34, CD133, and CD117 was detailed. The studies of FR+ monocytes and FR+ progenitor-like cells point to a dual targeting mechanism of folate-drugs to not only FR+ activated macrophages but also FR+ activated monocytes and possibly FR+ "inflammation stem cells" in the treatment of inflammatory diseases.




Low, Purdue University.

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