Kinetics of neutrophils in mice exposed to radiation and/or granulocyte colony-stimulating factor treatment.

Astronauts have the potential to develop the hematopoietic syndrome as a result of exposure to radiation from a solar particle event (SPE) during exploration class missions. This syndrome is characterized by a reduction in the number of circulating blood cells (cytopenias). In the present study the effects of SPE-like proton and γ radiation on the kinetics of circulating neutrophils were evaluated during a one-month time period using mice as a model system. The results revealed that exposure to a 2 Gy dose of either SPE-like proton or γ radiation significantly decreased the number of circulating neutrophils, with two nadirs observed on day 4 and day 16 postirradiation. Low circulating neutrophil count (neutropenia) is particularly important because it can increase the risk of astronauts developing infections, which can compromise the success of the mission. Thus, two granulocyte colony-stimulating factors (G-CSFs), filgrastim and pegfilgrastim were evaluated as countermeasures for this endpoint. Both forms of G-CSF significantly increased neutrophil counts in irradiated mice, however, the effect of pegfilgrastim was more potent and lasted longer than filgrastim. Using the expression of CD11b, CD18 and the production of reactive oxygen species (ROS) as markers of neutrophil activation, it was determined that the neutrophils in the irradiated mice treated with pegfilgrastim were physiologically active. Thus, these results suggest that pegfilgrastim could be a potential countermeasure for the reduced number of circulating neutrophils in irradiated animals.

Photohardening restores the impaired neutrophil responsiveness to chemoattractants leukotriene B4 and formyl-methionyl-leucyl-phenylalanin in patients with polymorphic light eruption.

A failure to induce immune suppression after UV exposure has been implicated in the pathogenesis of polymorphic light eruption (PLE). This immunological resistance has been linked to an impaired neutrophil infiltration into the skin following UV exposure. Therapeutic photohardening can restore this abnormal neutrophil infiltration in PLE skin and is thought to be responsible for the prophylactic efficacy. The aim of this study was to elucidate the pathogenic mechanism of the described neutrophil deficiency in PLE. Peripheral blood neutrophil responses to the chemoattractants leukotriene B4 (LTB(4)) and formyl-methionyl-leucyl-phenylalanin (fMLP) were investigated in vitro. Samples from 10 patients with PLE before and after 6 weeks of photohardening therapy were assessed. Flow cytometry was used to measure the changes associated with neutrophil activation. We found a significantly reduced neutrophil responsiveness to LTB(4) and fMLP in PLE patients, which was restored to normal levels after phototherapy. Indeed, PLE neutrophil responsiveness to these two chemoattractants after (but not before) phototherapy was similar to that of age- and sex-matched healthy control subjects. This indicates that an abnormal chemotactic potential to neutrophils is a crucial factor in the pathogenesis of PLE. Normalization following photohardening may therefore account for the therapeutic efficacy by restoring UV-induced neutrophil skin infiltration. Our results reveal a completely novel pathogenic mechanism involved in PLE and offer unique targets for therapy.

Transient infiltration of neutrophils into the thymus following whole-body X-ray irradiation in IL-10 knockout mice.

IL-10 is known to suppress the inflammatory responses in a variety of experimental models. Because we previously found that whole-body X-irradiation causes massive apoptosis in the thymus and transient infiltration of neutrophils, in this study, we examined whether or not IL-10 is involved in the regulation of neutrophil infiltration upon whole-body X-ray irradiation using IL-10 knockout mice. Although IL-10 was induced in the thymus on whole-body X-ray irradiation, apoptosis of thymocytes, neutrophil infiltration, and MIP-2 and KC production in the thymus were not affected by an IL-10 deficiency. Coculturing of bone marrow-derived macrophages with late apoptotic cells caused MIP-2 production, which was also not affected by an IL-10 deficiency. These results suggest the uniqueness of the inflammatory response induced by whole-body X-ray irradiation, which does not seem to be regulated by IL-10.

Influence of fractionated irradiation on neutrophilic granulocyte function.

BACKGROUND: A recent study has demonstrated that radiation therapy with single doses of up to 32 Gy has only a minor effect on neutrophilic granulocyte function. In clinical practice, by contrast, fractionated irradiation is applied. Therefore, the aim of the current study was to verify the influence of fractionated radiation therapy on granulocyte function. MATERIAL AND METHODS: Density gradient-purified granulocytes of voluntary healthy donors were used for all experiments. Granulocytes were kept in RPMI 1640 without fetal calf serum, incubated for 48 h and irradiated. Their function was assessed by measuring luminol-enhanced chemiluminescence after stimulation with phorbol myristate acid (PMA). All tests were performed at least five times. RESULTS: Relative changes (any reactive oxygen species [ROS] release before stimulation was defined as being equal to 100%) in ROS release increased after stimulation wit PMA (mean +/- SD): 0 Gy: 785 +/-, 462.2%; 2 Gy: 704.3 +/- 388.1%; 6 Gy: 1,360.3 +/- 710.5%; 12 Gy: 1,119.4 +/- 581.1%; 18 Gy: 1,087.3 +/- 622.4; 6 Gy (3 x 2 Gy): 279.4 +/- 201.1%; 12 Gy (6 x 2 Gy): 278.8 +/- 175.3%; 18 Gy (9 x 2 Gy): 84.2 +/- 41.5%. Comparing relative changes in ROS release after PMA stimulation, the differences between 0, 2, 6, 12, 18 Gy, and 6 Gy (3 x 2 Gy), 12 Gy (6 x 2 Gy), 18 Gy (9 x 2 Gy), and between 6 Gy (3 x 2 Gy), 12 Gy (6 x 2 Gy) and 18 Gy (9 x 2 Gy) proved to be significant (all p < 0.005). CONCLUSION: The study shows, that clinically used fractionated irradiation has an impact on granulocyte function, but contrary to common assumption, it is not to total dose itself but rather the fractionation which influences granulocyte function. This could have a major clinical impact on radiation treatment schemes especially for benign diseases or anti-inflammatory treatment.

The luminol-amplified chemiluminescence of neutrophils and monocytes in patients with gastric cancer after intraoperative radiotherapy using radiosensitizer sanazole.

The effect of electron-beam intraoperative radiotherapy (IORT) with radiosensitizer sanazole (drug AK-2123) on the functional activity of neutrophils and monocytes in patients with gastric cancer was studied with the use of luminol-amplified chemiluminescence. Sanazole was administered intravenously in a dose of 1.2 g/m2, 30 min before IORT. After resection of the tumor a single dose of 10 Gy was given to the field of regional lymph collectors and the bed of the removed tumor applying a small-size betatron MIB-6E. Cells were separated and chemiluminescence was estimated on the 1-st day before and on the 3-d day and 2-d week after IORT with sanazole. Insignificant effect on the zymosan- or phorbol-12 -myristate-13-acetate (PMA)-stimulated chemiluminescence was observed in this group, although there was found a considerable increase of chemiluminescence of neutrophils in response to zymosan and PMA in 5 from 8 patients, and chemiluminescence of monocytes--in 3 from 8 patients. Correlation between individual indices of zymosan- and PMA-stimulated chemiluminescence of neutrophils before IORT with sanazole was not high, r = 0.71, but it increased significantly to r = 0.91 on the 3-d day and to r = 0.96 on 2-d week after treatment. Correlation between individual indices of stimulated chemiluminescence of monocytes before IORT with sanazole was r = 0.90, and increased to r = 0.99 on the 3-d day and to r = 0.96 on the 2-d week after treatment. So, it is indicative of a correcting effect of the combined treatment including IORT with sanazole administration on the functional activity of neutrophils and monocytes, and the absence of functional disturbances of these cells.

Suppression by radiosensitizer AK-2123 of early phorbol myristate acetate-stimulated chemiluminescence response of human neutrophils induced by gamma-irradiation (short communication).

The influence of gamma-irradiation and radiosensitizer AK-2123 on secretion of reactive oxygen species (ROS) of human neutrophils was studied. gamma-Irradiation (from 5 to 25 Gy) of the neutrophil suspension inhibited spontaneous chemiluminescence and activated phorbol-12-myristate-13-acetate (PMA)-stimulated chemiluminescence. The time of maximum PMA-stimulated chemiluminescence amplitude was decreased with the dose range from 2 to 25 Gy. The addition of radiosensitizer AK-2123 depressed the early PMA-stimulated chemiluminescence response to gamma-irradiation. The obtained results suggest that this effect is connected with influence of AK-2123 on ion canals of neutrophils and may prevent the radiation-induced damage of blood cells.

Urocanic acid suppresses the activation of human neutrophils in vitro.

Exposure to ultraviolet (UV) light impairs the function of inflammatory cells. Urocanic acid (UCA) in an stratum corneum has been suggested as a mediator in the immunosuppression of lymphoid cells detected after irradiation with UVB (UV wavelengths 280-320 nm). In this study, we examined the effects of the two UCA isomers, trans and cis UCA on human polymorphonuclear leukocytes, neutrophils. It was found that treatment of cells with either trans of cis UCA isomers inhibited the opsonized zymosan-induced respiratory burst activity, measured with luminol-enhanced chemiluminescence assay. Both isomers were also able to partially block the up-regulation of complement receptors 1 (CR1; CD35) and 3 (CR3; CD11b/ CD18) in N-formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated neutrophils. These results indicate that the isomerization of trans UCA to cis UCA is not essential for the action of UCA on neutrophils. Neither of the UCA isomers were found to induce cyclic AMP (cAMP) formation in 3-isobutyl-1-methylxanthine treated cells, suggesting that the activation of adenylate cyclase cAMP system is not involved in UCA provoked suppression of neutrophils. It is concluded that the function of UCA may be protective, to suppress the activation of human neutrophils in inflamed, sunburned epidermis.

[Chemiluminescence, blood lipid peroxidation and neutrophil activity during the hypoxic training of persons subjected to ionizing radiation exposure]. / Khemiliuminestsentsiia, perekisnoe okislenie lipidov krovi i aktivnost' neitrofilov pri gipoksicheskoi trenirovke u lits, podvergshikhsia vozdeistviiu ioniziruiushchego izlucheniia.

Free radical processes and some indices of antibacterial defense system have been examined in 29 male residents of Chernobyl area during adaptation to periods of intermittent hypoxia. 18 men (the experimental group) were exposed to normobaric isocapnic progressive hypoxia during 10 days of three daily 5-7 min sessions with 15 min breaks, and 11 men (control group) were exposed to air breathing. All subjects were divided into two subgroups with initial high (1) and low (2) level of blood chemiluminescence (ChL). Patients of the 1 subgroup were characterized with high oxygen-generated activity of neutrophils (OGA) and high malondialdehyde (MDA) concentration. After hypoxic training (HT) there was a decrease of spontaneous and initiated ChL and MDA. Patients of the 2 subgroup were characterised with low level both spontaneous and initiated ChL, low MDA concentration and low phagocytosing activity of neutrophils. After HT there was significant rise of initiated ChL and MDA concentration up to normal level with the increasing of neutrophil phagocytosing activity. We suggest that HT causes the normalizing effect on free radical processes in subjects who were exposed to radiation influences.