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    • Finally there are some other caveats

    2018-10-25

    Finally, there are some other caveats to be considered. Because of the time required to purify and radiolabel blood eosinophils and neutrophils ex vivo it was logistically impossible to reinfuse the labelled cells earlier than 5–6h after blood sampling. Full processing and labelling of reinfused cells prior to commencing challenge would have required a 24h protocol. Consequently we timed our scans to coincide with the period during the peak of LAR, commencing 6h after allergen challenge, but we were unable to gather any data earlier than this. It is conceivable that precise timing of the scans may be critical here: there is some evidence, for example, that neutrophil influx into allergen-challenged airways is early and transient (Koh et al., 1993; Kelly et al., 2000; Nocker et al., 1999). For the same reason, the whole blood used for neutrophil/eosinophil isolation from the subjects was obtained just prior to, rather than after allergen challenge. It is possible that the challenge procedure itself might have altered the functions of populations of circulating eosinophils and neutrophils available for labelling. In designing future studies of real time tracking of net granulocyte influx and efflux from the lungs of asthmatics, and especially those involving allergen challenge or other forms of induced bronchoconstriction, these considerations must be taken into account. As a readout of neutrophil activation, both ex vivo and following the isolation procedure, we monitored the percentages of these cells expressing CD11b (CD11bHigh) above a predetermined fluorescence intensity. We chose CD11b because it is an integrin component implicated in cellular adhesion, leukoaggregation and pulmonary sequestration of granulocytes although neutrophil migration, as distinct from adhesion more clearly depends on co-expression of CD18 in the integrin heterodimer (Vedder and Harlan, 1988). What we found was that the mean percentages of blood neutrophils expressing CD11b above this threshold directly ex vivo were already elevated in the three groups of patients with sglt inhibitors (to 36% in Group 1, 45% in Group 2 and 57% in Group 3) as compared (retrospectively (Lukawska et al., 2014)) with healthy volunteers (~0.5%) even following isolation. This is likely to reflect priming by underlying chronic inflammation (Mann and Chung, 2006; Kämpe et al., 2011). Following the isolation and radiolabelling processes, these increased further to >98% in all three groups of asthmatics. Nevertheless, this had no significant effect, within the resolution of our measurements, on the net kinetics of influx and efflux of these cells into and out of the lungs, not only in the patients with asthma but also (retrospectively (Lukawska et al., 2014)) in the healthy volunteers: when we compared the kinetics of net lung neutrophil influx/efflux in healthy volunteers (CD11bHigh 1.1±0.8%) and all three asthma groups (CD11bHigh >98%) we uncovered no significant differences. Similarly, as a readout of eosinophil activation we monitored the percentages of these cells expressing CD69. CD69 is an activation marker on subgroups of T cells as well as eosinophils, and has also been implicated in delaying the egress of T cells from lymph nodes (Zhi et al., 2011). In this case we found that the mean percentages of eosinophils expressing CD69 were not significantly different in the 3 groups of asthmatic patients (1.9% in Group 1, 3% in Group 2 and 2.5% in Group 3) and retrospectively (Lukawska et al., 2014) in healthy volunteers (~1.8%) directly ex vivo, while the process of isolation and radiolabelling significantly increased the mean percentages of eosinophils expressing CD69 in all 3 groups of asthmatic patients (to 40% in Group 1, 40% in Group 2 and 34% in Group 3) but not healthy volunteers (~4.8%). One could speculate that this might have been higher still if the procedures had been performed in the asthmatics following allergen challenge. As with neutrophils, this had no significant effect, within the resolution of our measurements, on the net kinetics of influx and efflux of these cells into and out of the lungs in the asthmatic patients. The only significant difference we were able to observe (retrospectively) was a greater net retention time of eosinophils in the Group 1 asthmatic patients as compared with the healthy volunteers. Consequently, although it is appropriate to be concerned about the possible effects of cellular isolation and labelling on cellular functions and properties in vivo, and although we chose what we perceived as likely relevant functional markers, expression of neither CD11b on neutrophils nor CD69 on eosinophils appeared to alter their migration kinetics in this study. The factors which govern net cellular migration into and out of organs in vivo are likely highly complex and variable, and as yet relatively poorly understood, and in time it may be possible to identify more appropriate markers to “screen” for possible effects of cellular isolation on these processes. We do not believe, however, that this reservation detracts from the intrinsic value of our observations and of the process, and indeed further experiments may result in more appropriate markers of “activation” which influence cellular migration to be defined.