Expression and Activity of CCR5 on THP‐1 Monocytes and Monocyte‐Derived Macrophages
Authors: Grindstaff Aliya S , Baer Robert W 
Published: April 2020

Macrophages represent more than 50% of tumor infiltrating cells and are known to express CC chemokine receptor CCR5. Though notable for its involvement in HIV infection, high expression of CCR5 has been found in tumor tissue of various cancers and has been associated with poor outcomes. This suggests CCR5 may play role in cancer progression. The purpose of this study is to further define the role of CCR5 stimulation in the behavior of macrophages and their predecessor cell, the monocyte. For this study, macrophages were derived from THP‐1 monocytes via treatment with phorbol 12‐myristate 13‐acetate (PMA) (160 nM for two days). THP‐1 monocyte and monocyte‐derived macrophage (MDM) CCR5 expression was confirmed using flow cytometry. Macrophage CCR5 expression was evaluated at 1, 2, 4, and 8 days post PMA treatment, showing an overall increase in CCR5 expression over the eight days. To establish appropriate stimulation of chemokine receptor CCR5, chemotaxis dose response curves were performed on THP‐1 monocytes using 8 μm pore, collagen‐1 coated trans‐well inserts. Chemokine ligand CCL4 was used as the chemoattractant for these studies due to its relative selectivity for CCR5. Experimental groups included: control cells in serum free medium (RPMI‐1640) and cells in CCL4 concentrations of 10 ng/mL, 30 ng/mL, 100 ng/mL, 300 ng/mL, and 1000 ng/mL in serum free medium. After 24 hours, cell concentration in the bottom chamber of each well was determined; cells were concentrated, resuspended in a known volume, and counted using a hemocytometer. Results showed a dose‐dependent chemotactic response to CCL4, reaching a plateau between 300 and 1000 ng/mL. To further evaluate the role of CCR5 in monocyte migration, chemotaxis studies similar to those above were carried out in the presence of CCR5 inhibitor maraviroc (MVC). Experimental groups included: control (serum‐free RPMI‐1640), CCL4 (100 or 300 ng/mL), MVC (5 μg/mL), and CCL4 & MVC (as previous). This assay was run for 24 hours and cells were counted as noted above. Results showed MVC attenuated chemotaxis both in the presence and absence of CCL4 treatment. The effect of CCL4 stimulation on monocyte and MDM CCR5 expression was examined using flow cytometry. Expression levels were measured after 3 and 18 hours of CCL4 treatment. Treatment concentrations included 100 ng/mL and 300 ng/mL for monocytes and 100 ng/mL for MDMs. Results showed no change in monocyte CCR5 expression after 3 or 18 hours when compared to untreated cells. For MDMs, there was no change in expression at 3 hours; however, CCR5 expression was modestly increased at 18 hours of CCL4 treatment. In summary, our findings show that THP‐1 CCR5 expression is affected by PMA monocyte‐to‐macrophage conversion. CCL4 effectively stimulates THP‐1 cell chemotaxis which is attenuated by CCR5 inhibition by MVC. Further development of this in‐vitro model should prove useful in determining the role of monocyte and macrophage CCR5 in cancer progression.Support or Funding InformationSupported by a Grant from KCOM Graduate Program Committee