Microparticle and interleukin-1β production with human simulated compressed air diving

Abstract

Production of blood-borne microparticles (MPs), 0.1-1 µm diameter vesicles, and interleukin (IL)-1β in response to high pressure is reported in lab animals and associated with pathological changes. It is unknown whether the responses occur in humans, and whether they are due to exposure to high pressure or to the process of decompression. Blood from research subjects exposed in hyperbaric chambers to air pressure equal to 18 meters of sea water (msw) for 60 minutes or 30 msw for 35 minutes were obtained prior to and during compression and 2 hours post-decompression. MPs and intra-particle IL-1β elevations occurred while at pressure in both groups. At 18 msw (n = 15) MPs increased by 1.8-fold, and IL-1β by 7.0-fold (p < 0.05, repeated measures ANOVA on ranks). At 30 msw (n = 16) MPs increased by 2.5-fold, and IL-1β by 4.6-fold (p < 0.05), and elevations persisted after decompression with MPs elevated by 2.0-fold, and IL-1β by 6.0-fold (p < 0.05). Whereas neutrophils incubated in ambient air pressure for up to 3 hours ex vivo did not generate MPs, those exposed to air pressure at 180 kPa for 1 hour generated 1.4 ± 0.1 MPs/cell (n = 8, p < 0.05 versus ambient air), and 1.7 ± 0.1 MPs/cell (p < 0.05 versus ambient air) when exposed to 300 kPa for 35 minutes. At both pressures IL-1β concentration tripled (p < 0.05 versus ambient air) during pressure exposure and increased 6-fold (p < 0.05 versus ambient air) over 2 hours post-decompression. Platelets also generated MPs but at a rate about 1/100 that seen with neutrophils. We conclude that production of MPs containing elevated concentrations of IL-1β occur in humans during exposure to high gas pressures, more so than as a response to decompression. While these events may pose adverse health threats, their contribution to decompression sickness development requires further study.

Figure 1

Changes in microparticles. Blood borne MPs were quantified pre-, at- and post-exposures to 18 or 30 msw as described in Methods. Flow cytometric measurements were made to quantify the number of all 0.3 to 1 µm diameter Annexin V-positive particles (top frame), as well as those expressing proteins specific to certain cells: CD66b (mature neutrophils), CD41 (platelets), and CD31+/CD41−dim (endothelium). Data are mean ± SE, n is shown for each sample, * indicates significantly different from pre-exposure, p < 0.05, RM ANOVA.

Figure 2

Neutrophil activation. Surface proteins on neutrophils were quantified in blood samples as described in the caption for Fig. 1. Flow cytometry analyses of CD66b-positive cells (neutrophils) assessed surface expression of myeloperoxidase (MPO) and CD18, and platelet-specific CD41 as reflecting platelet-neutrophil interactions. Data are mean ± SE, n is shown for each sample, * indicates significantly different from pre-exposure, p < 0.05, RM ANOVA.

Figure 3

Representative Western blot of MPs and supernatant plasma fraction. Plasma samples from one research subject exposed to 30 msw were prepared as described in Methods to separate 10,000 MPs from suspending plasma. SDS buffer was added to the MPs pellet and supernatant fractions to maintain exact comparisons, and 20 µg protein was loaded to each lane. Data show entire, uncropped blots that had been cut so that individual antibodies could be used for probing pro- and mature forms of IL-1β, and actin obtained pre-, at- and post-pressure.

Figure 4

IL-1β in MPs. Data show the concentration of IL-1β as pg/1 million MPs from blood obtained as described in the caption for Fig. 1. Results in Fig. 3 and prior work have shown that virtually all blood-borne IL-1β is found within MPs. Data are mean ± SE, n is shown for each sample, * indicates significantly different from pre-exposure, p < 0.05, ANOVA.

Figure 5

MPs generation by neutrophils and platelets incubated ex vivo. Samples prepared from research subjects were incubated following the same pressure profiles as used in the simulated dive subjects, 180 kPa air for 1 hour or 300 kPa for 35 minutes, then decompressed and left at ambient pressure for 2 hours. At intervals the number of MPs was measured and plotted as number per 100 neutrophils (PMN) or platelets in the suspensions. Solid lines in the figure indicate samples that were kept at pressure for the indicated time and fixed immediately on decompression, dotted lines indicate samples incubated in air at ambient pressure. Data are mean ± SE, * indicates significantly different from pre-exposure, p < 0.05, ANOVA.

Figure 6

IL-1β in neutrophil MPs generated ex vivo. Data show the concentration of IL-1β as pg/1 million MPs in samples obtained as described in the caption for Fig. 5. Data are mean ± SE, * indicates significantly different from pre-exposure (labeled as Start), p < 0.05, ANOVA.