Signal-Regulatory Protein-α (SIRP- α) Expression Delineates Distinct Subsets in Monocytes/Macrophages in Normal Tissue and in B-Cell Non-Hodgkin Lymphoma

BACKGROUND

Monocytes and macrophages (mo/mΦ) are a key part of the composition of peripheral blood (PB) and tissues and increased numbers of mo/mΦ have been associated with patient outcome in non-Hodgkin lymphoma (NHL). Because CD14 is abundantly expressed on the surface of human mo/mΦ, it is often used to identify or isolate human mo/mΦ, and immunosuppressive CD14+HLA-DR^low monocytes have been shown to be increased in the peripheral blood of NHL patients. However, we have previously shown that CD14 expression on mo/mΦ is substantially lower than CD68 expression suggesting that many CD68⁺ mo/mΦ are CD14 negative, especially in spleen and lymph node tissues. To characterize both CD14⁺ and CD14^- mo/mΦ in PB and tissues, we isolated all mo/mΦ from B-cell NHL specimens and normal controls and assessed their phenotype and function.

METHODS

Human mo/mΦ were isolated by negative selection from PB and tissue biopsy specimens (B-cell NHL and normal tissues) using the immunomagnetic isolation (monocyte enrichment kit). Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by Giemsa stain and flow cytometry. Phagocytosis and migration assays were used to determine the function of isolated mo/mΦ. T cells were co-cultured with mo/mΦ and T cell proliferation was evaluated by CFSE staining and detected by flow cytometry.

RESULTS

Using a monocyte enrichment kit to isolate all mo/mΦ, the purity of isolated mo/mϕ was 85~99%, which was defined by the percentage of lineage-negative cells (i.e. cells without expression of CD3,CD19, CD20, and CD56). We found that these isolated mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. In contrast to PB, CD14 positive mo/mΦ constituted less than 40% of the tissue mo/mΦ from the isolated population. Furthermore, we found that the cell size from CD14⁺ mo/mΦ were larger than CD14^- mo/mΦ. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14⁺SIRP-α^high, CD14^-SIRP-α^dim and CD14^-SIRP-α^- cells. CD14⁺SIRP-α^high cells and CD14^-SIRP-α^dim cells typically constituted the population of larger cells, while CD14^-SIRP-α^- cells constituted the population of smaller cells. CD14^-SIRP-α^- cells lacked the typical phenotypic markers and had decreased phagocytic and migratory ability compared to CD14⁺SIRP-α^high and CD14^-SIRP-α^dim cells. Furthermore, we found that these 3 populations of mo/mΦ had a differential effect on activated T-cells and that the CD14^-SIRP-α^- cells appeared increased number in biopsy specimens from NHL when compared to normal tissues.

CONCLUSIONS

We have identified a unique population of small CD68+ mo/mΦ that lack expression of CD14, SIRP-α, and other FcγR markers. This subset of mo/mΦ is more prevalent in NHL tissues and has limited phagocytic and migratory functions. This CD14^-SIRP-α^- mo/mΦ subpopulation may play an inhibitory role in anti-cancer and inflammatory responses.