molecular-diffusion-plasma-membranes-primary-lymphocytes-measured

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Karolinska Institutet

The current international paradigm on the biological effects of radiation is based mainly on the effects of dose with some consideration for the dose rate. No allowance has been made for the potential influence of a changing dose rate (second derivative of dose), and the biological effects of exposing cells to changing dose rates have never been analyzed. This paper provides evidence that radiation effects in cells may depend on temporal changes in the dose rate. In these experiments, cells were moved toward or away from an X-ray source. The speed of movement, the time of irradiation, and the temperature during exposure were controlled. Here we report the results of the first experiments with TK6 cells that were exposed at a constant dose rate, at an increasing dose rate, or at a decreasing dose rate. The average dose rate and the total dose were same for all samples. Micronuclei were scored as the end point. The results show that the level of cytogenetic damage was higher in cells exposed to a decreasing dose rate compared to both an increasing and a constant dose rate. This finding may suggest that the second derivative of dose may influence radiation risk estimates, and the results should trigger further studies on this issue.

Cytogenetic damage in cells exposed to ionizing radiation under conditions of a changing dose rate.

There are conflicting data regarding the effect of culturing time of human peripheral blood lymphocytes on the yield of chromosomal aberrations induced by sparsely ionising radiation in the G0 phase of the cell cycle. While some authors find that the yield of aberrations does not change with time, others find increased frequencies of aberrations with harvesting time. The reasons for the conflicting results are not known, but the majority of studies were performed with lymphocytes of a single donor collected at one time point. We performed a study to verify if individual variability could be a confounding factor. As a positive control, lymphocytes were also exposed to high LET radiation (neutrons and alpha-rays), where an effect of harvesting time on the level of damage is expected to be seen. Blood was drawn from a total of 8 donors at two time points and exposed to X-rays, 6 MeV neutrons or alpha particles generated by an Am-241 source. Whole blood cultures were set up and micronuclei (Mn) were scored in binucleated cells harvested after 72, 96 and 120 h of culture time. The results show that in lymphocytes exposed to X-rays, the frequency of Mn was generally not influenced by the culture time while for both neutrons and alpha particles consistently increased micronucleus frequencies with culture time were detected. Some individual variability was detected and the conflicting results regarding the relationship between the yield of cytogenetic damage and lymphocyte culture time can, at least partly, be due to this variability.

The yield of radiation-induced micronuclei in early and late-arising binucleated cells depends on radiation quality.

In many exposure scenarios to ionising radiation, the dose rate is not constant. Despite this, most in vitro studies aimed at investigating the effects of ionising radiation are carried out exposing samples at constant dose rates. Consequently, very little data exist on the biological effects of exposures to changing dose rates. This may be due to technical limitations of standard irradiation facilities, but also to the fact that the importance of research in this area has not been appreciated. We have recently shown that cells exposed to a decreasing dose rate suffer higher levels of cytogenetic damage than do cells exposed to an increasing or a constant dose rate. To further study the effects of changing dose rates, a new device was constructed that permits the exposure of cell samples in tubes, flasks or Petri dishes to changing dose rates of X-rays. This report presents the technical data, performance and dosimetry of this novel device.

A new device to expose cells to changing dose rates of ionising radiation.

Exposure of humans to mixed fields of high- and low-linear energy transfer (LET) radiation occurs in many situations-for example, in urban areas with high levels of indoor radon as well as background gamma radiation, during airplane flights or certain forms of radiation therapy. From the perspective of health risk associated with exposure to mixed fields, it is important to understand the interactions between different radiation types. In most cellular investigations on mixed beams, two types of irradiations have been applied sequentially. Simultaneous irradiation is the desirable scenario but requires a dedicated irradiation facility. The authors have constructed a facility where cells can be simultaneously exposed to (241)Am alpha particles and 190-kV X-rays at 37°C. This study presents the technical details and the dosimetry of the setup, as well as validates the performance of the setup for clonogenic survival in AA8 Chinese hamster ovary cells. No significant synergistic effect was observed. The relative biological effectiveness of the alpha particles was 2.56 for 37 % and 1.90 for 10 % clonogenic survival.

Characterisation of a setup for mixed beam exposures of cells to 241Am alpha particles and X-rays.

Low temperature (hypothermia) during irradiation of cells has been reported to have a radioprotective effect. The mechanisms are not fully understood. This study further investigates the possible mechanisms behind hypothermia-mediated radioprotection.

Radioprotective effect of hypothermia on cells - a multiparametric approach to delineate the mechanisms.

The purpose of this study was to analyse the cytogenetic effect of exposing human peripheral blood lymphocytes (PBL) to a mixed beam of alpha particles and X-rays. Whole blood collected from one donor was exposed to different doses of alpha particles ((241)Am), X-rays and a combination of both. All exposures were carried out at 37 °C. Three independent experiments were performed. Micronuclei (MN) in binucleated PBL were scored as the endpoint. Moreover, the size of MN was measured. The results show that exposure of PBL to a mixed beam of high and low linear energy transfer radiation led to significantly higher than expected frequencies of MN. The measurement of MN size did not reveal any differences between the effect of alpha particles and mixed beam. In conclusion, a combined exposure of PBL to alpha particles and X-rays leads to a synergistic effect as measured by the frequency of MN. From the analysis of MN distributions, we conclude that the increase was due to an impaired repair of X-ray-induced DNA damage.

Micronuclei in human peripheral blood lymphocytes exposed to mixed beams of X-rays and alpha particles.

Gamma-H2AX foci in cells exposed to a mixed beam of X-rays and alpha particles.

Modern radiotherapy treatment modalities are associated with undesired out-of-field exposure to complex mixed beams of high and low energy transfer (LET) radiation that can give rise to secondary cancers. The biological effectiveness of mixed beams is not known. The aim of the investigation was the analysis of chromosomal damage in human peripheral blood lymphocytes (PBL) exposed to a mixed beam of X-rays and alpha particles. Using a dedicated exposure facility PBL were exposed to increasing doses of alpha particles (from (241)Am), X-rays and a mixture of both. Chromosomal aberrations were analysed in chromosomes 2, 8 and 14 using fluorescence in situ hybridisation. The found and expected frequencies of simple and complex aberrations were compared. Simple aberrations showed linear dose-response relationships with doses. A higher than expected frequency of simple aberrations was only observed after the highest mixed beam dose. A linear-quadratic dose response curve for complex aberrations was observed after mixed-beam exposure. Higher than expected frequencies of complex aberrations were observed for the two highest doses. Both the linear-quadratic dose-response relationship and the calculation of expected frequencies show that exposure of PBL to mixed beams of high and low LET radiation leads to a higher than expected frequency of complex-type aberrations. Because chromosomal changes are associated with cancer induction this result may imply that the cancer risk of exposure to mixed beams in radiation oncology may be higher than expected based on the additive action of the individual dose components.

Complex aberrations in lymphocytes exposed to mixed beams of (241)Am alpha particles and X-rays.

Cytokines have the potential to drastically augment immune cell activity. Apart from altering the expression of a multitude of proteins, cytokines also affect immune cell dynamics. However, how cytokines affect the molecular dynamics within the cell membrane of immune cells has not been addressed previously. Molecular movement is a vital component of all biological processes, and the rate of motion is, thus, an inherent determining factor for the pace of such processes. Natural killer (NK) cells are cytotoxic lymphocytes, which belong to the innate immune system. By fluorescence correlation spectroscopy, we investigated the influence of cytokine stimulation on the membrane density and molecular dynamics of the inhibitory receptor Ly49A and its ligand, the major histocompatibility complex class I allele H-2D(d), in freshly isolated murine NK cells. H-2D(d) was densely expressed and diffused slowly in resting NK cells. Ly49A was expressed at a lower density and diffused faster. The diffusion rate in resting cells was not altered by disrupting the actin cytoskeleton. A short-term stimulation with interleukin-2 or interferon-α + β did not change the surface density of moving H-2D(d) or Ly49A, despite a slight upregulation at the cellular level of H-2D(d) by interferon-α + β, and of Ly49A by IL-2. However, the molecular diffusion rates of both H-2D(d) and Ly49A increased significantly. A multivariate analysis revealed that the increased diffusion was especially marked in a subpopulation of NK cells, where the diffusion rate was increased around fourfold compared to resting NK cells. After IL-2 stimulation, this subpopulation of NK cells also displayed lower density of Ly49A and higher brightness per entity, indicating that Ly49A may homo-cluster to a larger extent in these cells. A faster diffusion of inhibitory receptors could enable a faster accumulation of these molecules at the immune synapse with a target cell, eventually leading to a more efficient NK cell response. It has previously been assumed that cytokines regulate immune cells primarily via alterations of protein expression levels or posttranslational modifications. These findings suggest that cytokines may also modulate immune cell efficiency by increasing the molecular dynamics early on in the response.

Elina Staaf

Department of Microbiology,
Tumor and Cell Biology,
Department of Microbiology, Tumor and Cell Biology

Karolinska Institutet

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Elina Staaf

Department of Microbiology,Tumor and Cell Biology,Department of Microbiology, Tumor and Cell Biology

Karolinska Institutet

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Department of Microbiology,
Tumor and Cell Biology,
Department of Microbiology, Tumor and Cell Biology