IntroductionNeutrophils are indispensable for host defense. 1 In addition, these cells play a detrimental role in the pathogenesis of many acute and chronic inflammatory diseases. They can cause tissue damage through aspecific activation of their repertoire of antimicrobial mechanisms. Neutrophils also inform and shape subsequent immunity 2 and can prolong inflammation by release of cytokines 3 and chemokines. 4 There is an emerging concept that neutrophils directly influence adaptive immune responses through pathogen shuttling to draining lymph nodes, 5,6 antigen presentation, 7 and modulation of T helper 1/T helper 2 responses. 8 Along this line, neutrophils have been reported to be an important component of myeloid-derived suppressor cells mediating lymphocyte suppression in various experimental models of acute 9 and chronic inflammation. 10 Targeting neutrophils in disease has mainly been focused on limiting their damaging capacity or directing their cytotoxic machinery to tumors. 11 Their immune modulatory functions have received little attention as potential targets in inflammatory diseases. This may at least in part be due to the current paradigm that these functions are of limited importance because of the generally accepted short circulatory half-life of neutrophils. Neutrophil lifespans have mainly been assessed by determination of ex vivo lifespans in culture (Ͻ 24 hours) and by transfer studies of ex vivo-manipulated neutrophils. The latter studies showed an estimated circulating half-life of approximately 8 hours in humans. 12 Ex vivo manipulation has been shown to have dramatic effects on neutrophil redistribution in vivo. 13 In mice, half-lives of 8 to 10 hours were reported when neutrophils were labeled in vivo. 14 In contrast, ex vivo labeling in mice showed that after transfer 90% of labeled neutrophils were cleared from the circulation within 4 hours, resulting in a half-life of less than 1.5 hours. 15 These differences between in vivo and ex vivo labeling strengthen our hypothesis that neutrophil transfer experiments may lead to an underestimation of neutrophil lifespan. The activation during ex vivo manipulation has probably led to retention in the lungs, 16 liver, spleen, and bone marrow (BM), 15 which may drastically reduce their circulatory half-life. To circumvent the complications introduced by ex vivo manipulation, we labeled the neutrophil pool in vivo in healthy mice and humans by administration of 2 H 2 O in drinking water. Acquisition of label and appearance of labeled neutrophils in the circulation is characterized by (1) the rate of division in the mitotic pool (MP) in the BM, (2) the transit time of newly formed neutrophils through the postmitotic pool (PMP) in the BM, and (3) the delay in mobilization of neutrophils from the PMP to the blood. With the use of a combination of gas chromatography and mass spectrometry the fraction of 2 H-labeled adenosine in the DNA of the proliferating neutrophil pool was measured, and the kinetics of the neutrophil pool was determined. Study des...

Key Points• Life span estimates can be sensitive to the duration of stable isotope label administration, explaining discrepancies in the literature.• Multiexponential models are needed to obtain reliable leukocyte life span estimates.Quantitative knowledge of the turnover of different leukocyte populations is a key to our understanding of immune function in health and disease. Much progress has been made thanks to the introduction of stable isotope labeling, the state-of-the-art technique for in vivo quantification of cellular life spans. Yet, even leukocyte life span estimates on the basis of stable isotope labeling can vary up to 10-fold among laboratories. We investigated whether these differences could be the result of variances in the length of the labeling period among studies. To this end, we performed deuterated water-labeling experiments in mice, in which only the length of label administration was varied. The resulting life span estimates were indeed dependent on the length of the labeling period when the data were analyzed using a commonly used single-exponential model. We show that multiexponential models provide the necessary tool to obtain life span estimates that are independent of the length of the labeling period. Use of a multiexponential model enabled us to reduce the gap between human T-cell life span estimates from 2 previously published labeling studies. This provides an important step toward unambiguous understanding of leukocyte turnover in health and disease. (Blood. 2013;122(13):2205-2212

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cells because of manipulation during exogenous labeling or abnormal homing conditions in the previous studies.We propose that there is an alternate interpretation of the data of Pillay et al. The bone marrow is a delay compartment that was not measured experimentally by Pillay et al. Using a multicompartment model, with the pool of marrow neutrophils serving as the precursor input for a blood plasma pool, we performed simulations using data from Dancey et al. 2 In this model, progenitor cells incorporate label as they proliferate, and then mature in the bone marrow before being released into the blood pool, where the labeled granulocytes are sampled experimentally ( Figure 1A).We found that the model can be solved with many sets of rate constants that produce similar model fits to the specific activity of granulocytes measured in the blood. There are 2 solutions that are particularly notable. In solution 1, the blood pool is the rate-limiting kinetic step, and the result is a blood half-life of 3.5 days, consistent with the results presented by Pillay et al ( Figure 1B). Note that the specific activity in the marrow rises earlier and decays faster than that in the blood because the latter step is rate-limiting. For solution 2, if one imposes a blood neutrophil half-life time of 7.5 hours, consistent with literature values (Figure 1C), proliferation and differentiation of neutrophils through the marrow become the rate-limiting kinetic step. In this situation, kinetics in the delay compartment are responsible for both the lag in the first appearance of labeled neutrophils in blood and the shape of the blood specific activity curve. Moreover, the specific activity in the bone marrow is nearly equilibrated with that in the blood under this solution, because the former is rate-limiting.The authors' published data in mice show that the 2 H-enrichment of mature neutrophils in the marrow matches well with that in the blood, suggesting that the measured appearance of labeled cells in the blood reflects their kinetics in the marrow. These results are consistent with flux of labeled populations through the marrow being the dominant kinetic step in the system. Thus, we believe that deuterium measurement of neutrophil kinetics yields results that are, in fact, quite harmonious with previous reports and likely reflect primarily kinetics of cellular flux through the bone marrow compartment. Response The in vivo half-life of human neutrophilsWe have read the comments raised by Tofts and colleagues and Li and colleagues with great interest. We share their surprise about our finding that-under normal homeostatic conditions-peripheral blood neutrophils have an average life span of 5.4 days, and we welcome their constructive replies to this important topic. Tofts et al correctly show that an alternative model in which neutrophils have an infinitely short life span in the peripheral blood provides an equally good fit to our deuterium data. However, their model is compatible with our data only if neutrophils spend approximately 11 ...