Macrophages Engulf Tumor B Cells Coated With Rituximab More Effectively Under The Hypoxic Condition

Growing evidence has been accumulated indicating that the tumor microenvironment plays a pivotal role in the pathogenesis of various tumors. Macrophages are one of the most important players in such a microenvironment, and those residing in tumor tissues are called “Tumor-Associated Macrophages (TAM)”. The TAM content predicts survival in follicular lymphoma patients following treatment, and a high TAM count was associated with a poor clinical course after conventional chemotherapy before the rituximab era. However, immunochemotherapy with rituximab is able to circumvent the unfavorable outcome associated with a high TAM count. This finding thus favors the hypothesis that TAM may act synergistically with anti-CD20–targeted therapy.

Hypoxia is a key feature of the microenvironment of cancer cells, and actively participates in tumor progression. In this study, we investigated the impact of hypoxia on the interaction between rituximab and TAM.

Macrophages were prepared by culturing isolated monocytes from adult volunteers at the Hokkaido Blood Bank in the presence of M-CSF for seven days in a humidified incubator containing 20% O₂, 5% CO₂ and 75% N₂ (normoxic macrophages) or 1% O₂, ⁵% CO₂ and 94% N₂(hypoxic macrophages). We used CD20-positive B cells from patients with chronic lymphocytic leukemia (CLL B cells) as target cells. When CLL B cells were opsonized with rituximab, the cell-binding assay showed that the hypoxic macrophages bound more effectively than normoxic macrophages (29.2±0.6% and 18.5±0.9%, respectively, p<0.01). We then performed phagocytosis assays using standard cytospin techniques and microscopic evaluation of stained phagocytic cells. Again, the percentage of phagocytosis of CLL B cells in the presence of rituximab was significantly higher under hypoxic conditions compared to those cultured under normoxic conditions (22.5±1.3% and 13.9±0.9%, respectively, p<0.01).

We next investigated the expression of Fc receptors on macrophages cultured in the different oxygen conditions. A flow cytometric analysis revealed that the expression levels of CD16, but not CD32 or CD64, were enhanced on hypoxic macrophages compared to normoxic macrophages, and this upregulation of CD16 was considered to contribute to the increased binding and phagocytosis of macrophages under hypoxic conditions.

The phenotype and functional activities of macrophages can be modulated by a number of cytokines and microbial products. Among these cytokines, IL-10 was reported to be the best inducer of macrophages with high phagocytic activity. When IL-10 was added in the culture 24 hours before the phagocytosis assay, the phagocyte rate of hypoxic, but not normoxic, macrophages was significantly enhanced. Intriguingly, the expression levels of CD16 on hypoxic macrophages were upregulated by IL-10 treatment, and this upregulation was not seen in the normoxic macrophages.

Taken together, these findings indicate that the hypoxic microenvironment facilitated the rituximab-mediated binding and phagocytic activity of TAM through the upregulation of CD16 expression. IL-10 could therefore be a possible effector modulator that enhances the activity of rituximab in the hypoxic microenvironment.