Human CD62L(dim) Neutrophils Identified as a Separate Subset by Proteome Profiling and In Vivo Pulse-Chase Labelling

During acute inflammation three neutrophil subsets are found in the blood: neutrophils with a conventional segmented nucleus, neutrophils with a banded nucleus and T-cell-suppressing CD62L(dim) neutrophils with a high number of nuclear lobes. In this study (clinicaltrials.gov NCT01766414) we compared the in vivo kinetics and proteomes of banded, mature and hypersegmented neutrophils to determine whether these cell types represent truly different neutrophil subsets or reflect changes induced by LPS activation. Using in vivo pulse-chase labelling of neutrophil DNA with 6,6-(2)H2-glucose, we found that (2)H-labelled banded neutrophils appeared much earlier in blood than labelled CD62L(dim) and segmented neutrophils, which shared similar label kinetics. Comparison of the proteomes by cluster analysis revealed that CD62L(dim) neutrophils were clearly separate from conventional segmented neutrophils despite having similar kinetics in peripheral blood. Interestingly, the conventional segmented cells are more related at a proteome level to banded cells despite a two days difference in maturation time. The differences between CD62L(dim) and mature neutrophils are unlikely to be a direct result of LPS-induced activation, due to the extremely low transcriptional capacity of CD62L(dim) neutrophils and the fact that neutrophils do not directly respond to the low dose of LPS used in the study (2ng/kg bodyweight). Therefore, we propose CD62L(dim) neutrophils to be a truly separate neutrophil subset that is recruited to the bloodstream in response to acute inflammation.