The oxidative burst reaction in mammalian cells depends on gravity

Abstract

Gravity has been a constant force throughout the Earth's evolutionary history. Thus, one of the fundamental biological questions is if and how complex cellular and molecular functions of life on Earth require gravity. In this study, we investigated the influence of gravity on the oxidative burst reaction in macrophages, one of the key elements in innate immune response and cellular signaling. An important step is the production of superoxide by the NADPH oxidase, which is rapidly converted to H2O2 by spontaneous and enzymatic dismutation. The phagozytosis-mediated oxidative burst under altered gravity conditions was studied in NR8383 rat alveolar macrophages by means of a luminol assay. Ground-based experiments in "functional weightlessness" were performed using a 2 D clinostat combined with a photomultiplier (PMT clinostat). The same technical set-up was used during the 13th DLR and 51st ESA parabolic flight campaign. Furthermore, hypergravity conditions were provided by using the Multi-Sample Incubation Centrifuge (MuSIC) and the Short Arm Human Centrifuge (SAHC). The results demonstrate that release of reactive oxygen species (ROS) during the oxidative burst reaction depends greatly on gravity conditions. ROS release is 1.) reduced in microgravity, 2.) enhanced in hypergravity and 3.) responds rapidly and reversible to altered gravity within seconds. We substantiated the effect of altered gravity on oxidative burst reaction in two independent experimental systems, parabolic flights and 2D clinostat / centrifuge experiments. Furthermore, the results obtained in simulated microgravity (2D clinorotation experiments) were proven by experiments in real microgravity as in both cases a pronounced reduction in ROS was observed. Our experiments indicate that gravity-sensitive steps are located both in the initial activation pathways and in the final oxidative burst reaction itself, which could be explained by the role of cytoskeletal dynamics in the assembly and function of the NADPH oxidase complex.

Figure 1

Special equipment for experiments in simulated and in real microgravity. Inside view of a pipette clinostat for parallel operation of up to 10 pipettes (Design J. Hauslage, DLR, Cologne) (A), of the PMT clinostat with the photomultiplier, sample cuvette and motor (B), parabolic flight aircraft Airbus A300B2-103 (C) and flight profile (D).

Figure 2

Influence of parabolic flight on the ROS production visualized by luminol kinetics in the PMT clinostat (non-rotating) during the parabolic flights. Cells were stimulated by opsonized zymosan as indicated. Luminescence was measured in counts per second (CPS). Two independent measurements during one flight day are shown in blue (stimulation before parabola 0) and green (before parabola 16). 1 g control experiment was performed on ground after each flight using the same experiment hardware, in flight-analogueconditions. Unstimulated control cells (PBS control) were not activated with zymosan.

Figure 3

Influence of changing g-forces during parabolic flight on the ROS production visualized by luminol kinetics in the PMT clinostat (non-rotating). Cells were activated by opsonized zymosan stimulation before the first parabola. Luminescence was measured in counts per second (CPS). A. Detailed ROS production during parabolas 6–10 (blue) is shown in correspondence to g-profile (grey, kindly provided by Novespace). B. Detailed ROS production in response to altered gravity during parabola 6.

Figure 4

Influence of changing g-forces during parabolic flight and simultaneous clinorotation on the ROS production visualized by luminol kinetics in the PMT clinostat. Cells were activated by opsonized zymosan stimulation before the first parabola and clinorotated for 11 parabolas at 60 rpm. Luminescence was measured in counts per second (CPS). Detailed ROS production (blue) is shown in correspondence to g-profile (grey, kindly provided by Novespace). The clinorotated interval is grey shaded.

Figure 5

Influence of changing g-forces during parabolic flight and clinorotation on the ROS production visualized by luminol kinetics in the PMT clinostat. Cells were activated by opsonized zymosan stimulation before parabola 16 and clinorotated in μg at 60 rpm for 8 parabolas. In parabolas 24 and 25 clinorotation was performed during μg and hyper-g and rotation was then stopped. Luminescence was measured in counts per second (CPS). Detailed ROS production (blue) is shown in correspondence to g-profile (grey, kindly provided by Novespace). The clinorotated intervals are grey shaded.

Figure 6

Influence of two different clinorotation profiles on the ROS production visualized by luminol kinetics in the PMT clinostat. Cells were activated by opsonized zymosan stimulation beforehand. Luminescence was measured in counts per second (CPS). A Profile 60Stop (60 rpm for 20 min, 1 g for 30 min, blue) in comparison to 1 g (no rotation) (red). B Profile Stop60 (1 g for 20 min, 60 rpm for 30 min, green) in comparison to 1 g (no rotation) (red).

Figure 7

Influence of clinorotation on the phagocytosis index determined by the incorporation of FITC-zymosan. Cells were incubated with FITC-zymosan and directly subjected to rotation at 60 rpm for the indicated time periods. After rotation, phagocytosis was measured in relative fluorescent units (RFU) using a microplate reader. * p<0.05.

Figure 8

ROS production during clinorotation, phagocytosis and sedimentation. A. Influence of clinorotation on the phagocytosis-mediated and phagocytosis-independent ROS production determined by the NBT assay. Cells were incubated with NBT-zymosan (white bars) or NBT-PBS (black bars) and directly subjected to rotation at 60 rpm for the indicated time periods. After rotation, ROS production was measured in relative optical density (OD) at 630 nm using a microplate reader. B. Influence of clinorotation and sedimentation on the phagocytosis-mediated ROS production determined by the NBT assay. Cells were incubated with NBT-zymosan (white bars) with or without methyl cellulose (MC) and directly subjected to rotation at 60 rpm for 60 min. Sedimentation effects in control cells were minimized using a supplementation of 0.3% MC. After rotation, ROS production was measured in relative optical density (OD) at 630 nm using a microplate reader. **p<0.01; ***p<0.001.

Figure 9

Influence of previous clinorotation in comparison to permanent 1 g exposure on the ROS production visualized by luminol kinetics in a microplate reader. Cells were activated by opsonized zymosan directly after clinorotation (w/zymosan) or left untreated (w/o zymosan). Luminescence was measured in relative luminescence units (RLU) per minute. A. Cells were clinorotated for 30 min previous to measurement. B. Cells were clinorotated for 24 h previous to measurement.

Figure 10

Influence of centrifugation on the Short Arm Human Centrifuge (SAHC) on the ROS production visualized by luminol kinetics in the PMT clinostat. Cells were activated by opsonized zymosan stimulation beforehand and subjected to 1.8 g, 3 g or to 1 g generated by the PMT clinostat within the SAHC. Luminescence was measured in relative light units (RLU). Data reflects one representative experiment of at least three replicates.

Figure 11

Influence of centrifugation on the Multi Sample Incubator Centrifuge on the phagocytosis-mediated superoxide production determined by the NBT assay. Cells were incubated with NBT-zymosan and directly subjected to rotation at 1.8 g and 3 g for 45 min. After centrifugation, ROS production was measured in relative optical density (OD) at 630 nm using a microplate reader (Promega). *p<0.05.

Figure 12

Simulation of μg-phases of a parabolic flight by a clinorotation profile and influence on the ROS production visualized by luminol kinetics in the PMT clinostat. Cells were activated by opsonized zymosan stimulation beforehand. Luminescence was measured in counts per second (CPS). Blue, ROS production by cells subjected to the parabolic flight simulation (PF profile); red, ROS production by cells subjected to 1 g (no rotation profile); grey, rotation profile in rounds per minute (rpm).