Mononuclear phagocytes and marker modulation: when CD16 disappears, CD38 takes the stage

Abstract

We have read with great interest the article from Döbel et al, 1 which shows the ability of freshly separated 6-sulfo LacNac (slan)-positive dendritic cells (slanDCs) to internalize immunoglobulin G (IgG)-opsonized particles and aggregated IgG via FcgRIIIA/CD16-dependent phagocytosis. SlanDCs are a subset of "nonclassical monocytes" expressing CD16.2,3 Schäkel et al 4 have previously elegantly demonstrated that these cells in vitro undergo rapid spontaneous maturation, becoming potent producers of interleukin-12, and that these events in vivo are restrained by CD47 expressed on the membranes of erythrocytes; now, the same group reveals that metalloproteasedependent shedding of the low-affinity IgG receptor CD16 accompanies these changes. 1 We noted the downregulation of CD16 in all CD16-expressing mononuclear myeloid cells in experiments analyzing the cytokine response to Toll-like receptor (TLR) 4 and TLR7/8 agonists in lithium-heparinized whole blood (and thus in the presence of erythrocytes) (Figure 1A-C). As shown in Figure 1C, this modulation is concomitant with the production of tumor necrosis factor a (TNFa). Thus, microbial stimuli can bypass the restraining effect mediated by erythrocytes on the maturation of nonclassical monocytes.Extending the observations of Belge et al 5 who proposed HLA-DR as a stable marker for nonclassical monocytes, to track the loss of CD16 in monocyte subsets we developed a flow cytometric strategy which identifies nonclassical monocytes, together with other monocyte and myeloid DC subsets, taking advantage of the differential expression pattern of CD38, thus bypassing the need for CD16 as a critical selection marker. Nonclassical monocytes express CD38 at very low levels, unlike other monocytes and DC subsets ( Figure 1D-E), consistent with their proposed developmental derivation from classical monocytes 3 and their well-differentiated phenotype. Because CD38 expression is stable, at least following short-term stimulation ( Figure 1F), we have exploited this strategy to monitor CD16 downregulation in nonclassical monocytes and, as a control, CD62L shedding in classical monocytes 6 after stimulation with TLRs. As shown in Figure 1G, the metalloprotease inhibitor Ilomastat (GM6001) dose-dependently prevents CD16 and CD62L shedding induced by the brief incubation with resiquimod (R848, a TLR7/8 agonist).In conjunction with the results presented by Döbel et al, 1 our data thus demonstrate that the process of ectodomain shedding of 2 distinct surface receptors occurs in the same circumstances and by overlapping mechanisms in both nonclassical monocytes (and slanDCs) and classical monocytes, underscoring the similarities between the activation-dependent enzymatic machinery of 2 closely related monocyte family members. Furthermore, we show that TLR engagement can drive a response that overcomes the inhibition exerted by red blood cells in nonclassical monocytes. Our data extend the findings from Döbel et al not only to nonclassical monocytes other than slanDCs, but also to the whole...

Monocyte Subsets Are Differentially Lost from the Circulation during Acute Inflammation Induced by Human Experimental Endotoxemia

“…However, the larger number of measurements in our study allowed for a better overview of the loss of cells during the first hours after LPS injection and of the increased CM numbers 8 h after LPS injection. Furthermore, the multidimensional FACS analysis allowed us to demonstrate that our findings are unlikely to be due to the loss of CD14 or CD16 expression, as was described in vitro [18] .…”

“…NCM have been shown to lose CD16 expression after stimulation with LPS in vitro [18] . Therefore, we identified the 3 monocyte subsets without the use of the classical markers CD14 and CD16.…”

IL-12–producing monocytes and HLA-E control HCMV-driven NKG2C+ NK cell expansion

“…However, we did not detect CD16 expressionconsidered to represent an "inflammatory" monocyte markeron IL-12 + CD14 + cells. A possibility is that CD16 was lost due to cellular activation via TLRs, as previously shown (29). We therefore defined the IL-12-producing subpopulation as HLA-DR + , CD38 lo , and CD120b + , which is consistent with the phenotype of inflammatory monocytes (29,30).…”

Multi‐site reproducibility of a human immunophenotyping assay in whole blood and peripheral blood mononuclear cells preparations using CyTOF technology coupled with Maxpar Pathsetter, an automated data analysis system

“…Subclassification of monocytes into Classical, Transitional, and Non‐classical used CD14 and CD38 (see Table ) instead of the more traditional CD14 and CD16 (Picozza, Battistini, & Borsellino, ). The patterns produced by CD14 and CD38 were found to classify analogous subpopulations while improving the overall reproducibility of the results (data not shown).…”

“…Non-classical used CD14 and CD38 (see Table 2) instead of the more traditional CD14 and CD16 (Picozza, Battistini, & Borsellino, 2013).…”