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Effect of whole-body x-irradiation on antigen-induced airway response in sensitized guinea pigs

First Dept of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Sendai, Japan

CORRESPONDENCE: G. Tamura, First Dept of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Sendai, Japan, 980-8574. Fax: 81 227177156

Keywords: adoptive transfer, CD4+ T-lymphocytes, eosinophil, guinea pig, late asthmatic response, x-irradiation

Received: January 20, 2000
Accepted July 27, 2000

	Abstract

TOP Abstract Methods Results Discussion Acknowledgements References

 
The objectives of this study were to test the hypothesis that x-irradiation inhibits the late asthmatic response (LAR) without influencing the early asthmatic response (EAR) and to examine the mechanism of the inhibitory effect.

Twenty sensitized guinea pigs were irradiated at a dose of 8 Gy. The next day, one-half of the animals were injected intravenously with spleen cells (2x10⁸) collected from unirradiated sensitized guinea pigs, whilst the other half were injected with vehicle only. Ten additional unirradiated sensitized guinea pigs also received vehicle only. Antigen inhalation challenge took place two days later. Pulmonary resistance was measured for 6 h after antigen exposure, and bronchoalveolar lavage and lung fixation were then undertaken. The area under the percentage pulmonary resistance curve 2–6 h after allergen inhalation was used for analysis of the LAR, while the maximal percentage change in pulmonary resistance was used for analysis of the EAR.

Irradiation abolished the LAR (364.4±49.4 versus 62.8±10.4) without inhibiting the EAR (229.3±27.2 versus 278.7±40.2) and significantly inhibited the accumulation of eosinophils and lymphocytes in the airways. Transfer of spleen cells restored the LAR (334.4±66.8) and the recruitment of cells to the levels seen in unirradiated sensitized guinea pigs. In addition, transfer of only CD4+ T-lymphocytes separated from the spleen cells restored the LAR (439.4±62.1) and the cell infiltration into the airways.

These inhibitory effects of x-irradiation were due to decreases in numbers of CD4+ T-lymphocytes.

Antigen inhalation challenge produces acute airway obstruction (the early asthmatic response, or EAR), which is often followed after 4–12 h by a second airway obstruction (the late asthmatic response; LAR) 1. While the EAR is generated by immunoglobulin (Ig)E-mediated activation of mast cells to release chemical mediators 2, the LAR depends on airway inflammation, which is generally characterized by the infiltration of eosinophils and lymphocytes into the airways 3, 4. Inflammatory cell progenitors originating from bone marrow are also thought to be involved in the airway inflammation of the LAR 5. Although bone marrow as a source of inflammatory cells is known to be sensitive to x-irradiation 6, as are leukocytes (especially lymphocytes) 7, the function and numbers of mast cells are not influenced even by lethal doses of x-irradiation 8, 9.Thus, x-irradiation could conceivably inhibit the LAR without influencing the EAR at all.

A guinea pig model of bronchial asthma has been developed in which both the EAR and the LAR follow allergen challenge 10. As previously reported, the LAR in this model is associated with histological changes, such as infiltration of eosinophils and mononuclear cells into the bronchial lumen wall, as well as the contraction of airway smooth muscle, the development of oedema of the airway wall, and the formation of mucus plugs in the bronchial lumen.

In the present study, using the model, it was examined whether whole-body x-irradiation blocks the LAR without affecting the EAR. In addition, the effects of adoptive transfer of sensitized guinea-pig spleen cells in general, and of CD4+ T-lymphocytes in particular, on the restoration of the LAR were evaluated. To the authors' knowledge, this is the first report that x-irradiation completely blocks the LAR without affecting the EAR at all.

	Methods

TOP Abstract Methods Results Discussion Acknowledgements References

 
Animals
Specific-pathogen-free male Hartley guinea pigs (Charles River, Kanagawa, Japan) weighing 160–180 g were housed in the animal research facility of Tohoku University School of Medicine. Animal care and handling were in accordance with the principles stated in the "Guide for the Care and Use of Laboratory Animals" 11. The experimental protocols described were approved by the Animal Care and Use Committee of Tohoku University.

Measurement of pulmonary resistance
The method of measuring pulmonary resistance (Rl) has been described previously 12, 13. In brief, a pneumotachograph coupled to a differential pressure transducer was connected to the endotracheal tube cannulated into a trachea to measure airflow; volume data were obtained by electrical integration of the flow signal with an integrator. Oesophageal pressure was measured with a water-filled polyethylene tube and a pressure transducer. Airway opening pressure was measured with a pressure transducer connected between the endotracheal tube and the pneumotachograph. The Rl during spontaneous breathing for each animal was calculated by the method of Mead and Whittenberger 14.

Irradiation
Whole-body irradiation was generated with a therapy machine (model SHT250M-3; Shimazu, Tokyo, Japan), which was operated at 200 kV and 10 mA with a 0.5-mm copper and 1.0-mm Aluminium filter. The absorbed dose rate was measured at 0.722 Gy·min^–1 at the abdomen of the guinea pigs.

Experimental protocol 1
Ascaris suum extract was prepared as previously described 12. As shown in figure 1, 40 guinea pigs were injected intraperitoneally on days 1 and 14 with a mixture of 20 µg of A. suum protein and 20 mg of silica suspended in 1 mL of physiological saline. On day 21, the guinea pigs inhaled an aerosol of A. suum extract (1.25 mg·mL^–1 in physiological saline) for 30 s through a face mask. The aerosol was generated with an ultrasonic nebulizer (NE-U06; Omron, Kyoto, Japan) and was administered at a constant flow of 10 mL·s^–1. On day 25, 20 animals were irradiated at a dose of 8 Gy. On day 26, spleen cells (2x10⁸) collected from 10 of the unirradiated guinea pigs were injected intravenously into 10 of the irradiated guinea pigs (designated the R-T group), and vehicle only was injected into the other 10 irradiated animals (designated the R group). The remaining 10 unirradiated guinea pigs were also injected with vehicle only and were designated the C group. On day 28, the guinea pigs in the R-T, R, and C groups were challenged first with an aerosol of physiological saline and then with an aerosol of A. suum extract (2.5 mg·mL^–1 in physiological saline). The aerosols were directed at a constant airflow (10 mL·s^–1) into a cylindrical adaptor (volume=10 mL) connected to the endotracheal tube. Each guinea pig inhaled the aerosol for 15 s. Baseline Rl was defined as the pulmonary resistance measured after saline aerosol inhalation. Subsequently, Rl was measured at 5, 10 and 15 min, and then on the hour, until 6 h after antigen inhalation. Airway secretions, if any, were removed with a paper string before each Rl measurement. Immediately after the last Rl measurement, the guinea pigs were deeply anaesthetized with an additional dose of urethane and killed by exsanguination from the abdominal aorta. Bronchoalveolar lavage (BAL) was then performed and was followed by lung fixation.

On day 25 (before irradiation), day 26 (before spleen-cell or vehicle injection), day 27, and day 28 (before challenge), blood was collected from each animal's ear, and leukocytes in the sample were counted with a haemocytometer. Differential cell counts in blood smears stained with Wright-Giemsa were determined by counting >500 leukocytes at a magnification of x1,000 (oil immersion).

To examine the effect of the irradiation on total and differential cell counts in BAL and bone marrow, 15 additional guinea pigs were immunized in the manner described above. Five of the animals (the unchallenged-R group) were irradiated on day 25 and underwent BAL without antigen inhalation challenge on day 28. The other 10 animals (the unchallenged-C group) underwent BAL without irradiation or antigen inhalation challenge on day 28. After BAL, bone marrow was collected by the method of Nanba et al. 15 from all five animals in the unchallenged-R group and from half of those in the unchallenged-C group. Total and differential cell counts in bone marrow were determined as described above for peripheral blood samples.

Experimental protocol 2
To determine whether or not restoration of the LAR depends on the adoptive transfer of T cells, CD4+ T-lymphocytes were isolated from spleen cells of sensitized guinea pigs. Mononuclear cells (MNCs) were isolated from a crude spleen-cell preparation by density-gradient centrifugation on Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden). The isolated MNCs were resuspended in 2 mL of Hanks' balanced salt solution (HBSS; Gibco BRL, Rockville, MD, USA) with 2% foetal bovine serum (FBS; Gibco BRL). Thereafter, CD4+ T-lymphocytes were separated from CD4– MNCs by magnetic cell sorting (MACS), as described by Miltenyi et al. 16. In brief, 10⁶ MNCs were incubated first at 4°C for 15 min with 10 µL of mouse monoclonal antibody (mAb) to guinea pig CD4+ T-cells (Serotec Ltd., Oxford, UK) and then at 4°C for 15 min with microbeads directly conjugated with rat mAb to mouse IgG1 (Miltenyi Biotec, Auburn, CA, USA) (20 µL of magnetic microbeads per 10x10⁶ cells). Labelled cells were subsequently removed by MACS and attached to a column in a magnetic field. After collection of negatively selected cells with an additional 20 mL of HBSS plus 2% FBS, the MACS column was removed from the magnet, and positively selected cells were eluted by flushing with 30 mL of HBSS plus 2% FBS. Consequently, 3–7x10⁶ CD4+ T-lymphocytes and 20–30x10⁶ CD4– MNCs were obtained from each sensitized guinea pig.

On day 26 (i.e. 24 h after whole-body irradiation of the immunized guinea pigs from protocol 1 at a dose of 8 Gy) CD4+ T-lymphocytes and CD4– MNCs were adoptively transferred to six animals each. On day 28, antigen inhalation challenge and BAL were performed as described above.

Bronchoalveolar lavage and lung fixation
The methods used for BAL and lung fixation have been described previously 13. In brief, the lung was lavaged twice with 5 mL of physiological saline and then inflation-fixed with periodate-lysine paraformaldehyde via the trachea at 25 cmH₂O pressure. Total cell numbers in BAL fluid were counted with a haemocytometer. A 100-µL aliquot was cytocentrifuged with Cytospin 2 (Shandon, Inc., Pittsburgh, PA, USA). Differential cell counts were obtained from centrifuged preparations stained with Wright-Giemsa by counting >1,000 cells at a magnification of x1,000 (oil immersion). The fixed lungs were cut into sections 3 µm thick, stained with Diff-Quik solution I (Baxter, McGraw Park, IL, USA), and counterstained with haematoxylin. Three bronchioles were selected randomly from each section, and eosinophils (stained red) located in the bronchiolar wall (including smooth muscle and a cuff of 50 µm outside the muscle) were counted with a video micrometer (VM-30; Olympus, Tokyo, Japan) in a blind fashion.

Statistical analysis
The area under the percentage Rl curve (1 unit = 1%·h^–1) 2–6 h after allergen inhalation was used for analysis of the LAR, while the maximal percentage change in Rl was used for analysis of the EAR. All results were expressed as mean±sem values. Data obtained from more than three groups were analysed with the Dunn test after ANOVA. Data obtained from two groups were analysed with Welch's t-test. In all analyses, a p-value <0.05 was considered significant.

	Results

TOP Abstract Methods Results Discussion Acknowledgements References

 
Time course of pulmonary resistance
Since there were no significant differences in baseline Rl values among the C, R, and R-T groups (198.8± 22.9, 189.0±14.5, and 151.0±9.6, respectively), changes in Rl in these three groups after antigen challenge are shown as percentages relative to each baseline value (fig. 2). In all three groups, an EAR was evident after antigen inhalation, and Rl returned approximately to the baseline value in the next hour. Thereafter, however, %Rl for the R group did not increase at all, while %Rl for the C and R-T groups gradually increased again 4–5 h after antigen inhalation, a change reflecting the LAR. Maximal increases in %Rl during the EAR among the C, R, and R-T groups were 229.3±27.2, 278.7±40.2, and 270.6±46.5, respectively, and the area under the curve 2–6 h after allergen inhalation among the C, R, and R-T groups were 364.4±49.4, 62.8±10.4, and 334.4±66.8, respectively. Thus, whole-body irradiation at a dose of 8 Gy almost completely blocked the LAR without changing the EAR, and adoptive transfer of spleen cells from sensitized animals restored the LAR.

View larger version (22K): [in this window] [in a new window]   Fig. 2.— Time course for changes in pulmonary resistance (Rl) after Ascaris challenge in the C group (), the R group (•), and the R-T group () (10 animals each; see text and fig. 1 legend for definition of groups)). Although there was no significant difference in %Rl at EAR among these three groups, %Rl at late asthmatic response was significantly smaller for the R group than for C and R-T groups. Values are expressed as mean±sem.

View larger version (22K): [in this window] [in a new window]   Fig. 3.— Time course for changes in pulmonary resistance (Rl) after Ascaris challenge in animals that received either CD4+ T-lymphocytes (•) or CD4– mononuclear cells (MNCs) () (six animals each). Although there was no significant difference in %Rl at early asthmatic response between the two groups, %Rl at late asthmatic response was significantly greater for animals given CD4+ T-lymphocytes than for those given CD4– MNCs. Values are expressed as mean±sem.

View larger version (65K): [in this window] [in a new window]   Fig. 4.— Photomicrographs of lung samples obtained from guinea pigs in a) the C group; b) the R group; and c) the R-T group (see text or fig. 3 legend for definition of groups), 6 h after antigen exposure. Remarkable eosinophil infiltration (stained red) is apparent in the animals from the C and R-T groups. The lung was stained with Diff Quik solution I and haematoxylin (original magnification;x200).

View larger version (14K): [in this window] [in a new window]   Fig. 5.— Changes in guinea-pig peripheral-blood leukocyte (a) total cells; c) neutrophils; b) mononuclear cells; and d) eosinophils) counts from day 25 to day 28, i.e. in the first 72 h after irradiation. There were 10 animals per group. Values are expressed as mean±sem. Total cells, mononuclear cells, and eosinophils significantly decreased in number after x-irradiation (8 Gy); neutrophil counts decreased significantly only after 72 h. (): c group; (•): R group; (): R-T group; *: p<0.05, **: p<0.01, and ***: p<0.005 versus the C group at each time point. the (R-T group) at each time point. For definitions of groups see text or fig. 1 legend.

	Discussion

TOP Abstract Methods Results Discussion Acknowledgements References

In a preliminary study using doses of 2, 4, 8, and 12 Gy, the effect of x-irradiation on decreases in guinea-pig peripheral-blood leukocyte counts were evaluated. It was found that the number of leukocytes, especially MNCs, drastically decreased 3 days after all doses of irradiation, and that a dose of 8 Gy gave a submaximal effect. In 1955, Rosenthal 17 reported that myeloid and erythroid cells in bone marrow disappeared 3 days after irradiation with 6 Gy and that these cell populations did not regenerate thereafter in normal guinea pigs. In light of preliminary findings and the results reported by Rosenthal 17, 8 Gy was selected for the irradiation dose in the present study. Counts of MNCs, eosinophils, and neutrophils in peripheral blood significantly decreased during the 72 h after irradiation, and adoptive transfer of spleen cells did not alter these decreases at all. In addition, as expected, numbers of erythroid cells, immature leukocytes, lymphocytes, and eosinophils in bone marrow significantly decreased over this interval. No guinea pig died as a result of irradiation during this study.

To clarify whether sensitized lymphocytes can restore the LAR in this model, spleen cells from immunized guinea pigs were transfused into irradiated animals. Although cellular traffic was not defined after the transfer in this study, Berman et al. 18 have indicated that it takes 18–72 h for the distribution of lymphocytes to stabilize, and Schuyler et al. 19 have challenged animals 2 days after adoptive transfer in another model of cell-mediated pulmonary disease. Therefore, the presented protocol, in which animals were challenged 2 days after adoptive transfer, seems adequate to evaluate the function of transferred cells. This cellular supplementation almost completely restored the Rl increase during the LAR as well as the accumulation of eosinophils and lymphocytes in the airways. Because some investigators have reported that transfer of specific V beta T-cell subsets from sensitized mice increases airway responsiveness in naive recipients 20, 21 and that depletion of CD4+ T-lymphocytes completely prevents both antigen-induced airway hyperreactivity and recruitment of eosinophils in mice 22, the role of CD4+ T-lymphocytes separated from spleen cells of sensitized, unirradiated animals in the LAR and in airway infiltration by eosinophils was further examined. The transfer of CD4+ T-lymphocytes into irradiated animals restored the LAR and cell accumulation. Therefore, the authors suspected that CD4+ T-lymphocytes played a critical role in the development of the LAR and in cell recruitment into airways in the guinea-pig asthma model.

Allergen exposure of irradiated animals 48 h after transfer of CD4+ T-lymphocytes resulted in a level of eosinophil recruitment into the airways as high as that in animals that were merely sensitized; however, irradiation with 8 Gy significantly decreased eosinophil counts in both peripheral blood and bone marrow immediately before challenge, and the transfer of spleen cells did not alter these decreases. In 1979, Muller and Muntener 23 reported that irradiation caused extensive destruction of lymphocytes in the circulation and that this destruction was followed by the disappearance of eosinophils from the circulation and was paralleled by an increase in the number of eosinophils in the lymphatic organs. Still earlier, in 1953, Cronkite 24 reported that mature eosinophils might be resistant to irradiation. Therefore, it is possible that the decrease in circulating eosinophils in the guinea pigs after irradiation may have been due to the redistribution of these cells into organs rather than to their destruction. In addition, it was found that small numbers of eosinophils remained in the bone marrow 3 days after irradiation. Since some investigators have reported a possible role for Th2 cytokines in late-phase lung inflammation in allergic mice, 25–27 it is believed that transferred CD4+ T-lymphocytes may have recruited eosinophils into the airways from the lymphatic organs and the bone marrow by releasing Th2 cytokines such as interleukins 4 and 5.

To conclude, in this model, x-irradiation abolishes the late asthmatic response, with significant inhibition of eosinophil infiltration into the airways after antigen inhalation challenge and this effect is due to the destruction of CD4+ T-lymphocytes. It is suggested that the late asthmatic response and cell recruitment into the airways are mediated by CD4+ T-lymphocytosis in the guinea-pig asthma model.

View larger version (22K): [in this window] [in a new window]   Fig. 1.— Flow diagram of protocol 1 (—) and 2 (- -). Numbers represent day number. C group (n=10) unirradiated guinea pigs injected with vehicle only; R group (n=10) irradiated guinea pigs injected with vehicle only; R-T group (n=10) irradiated guinea pigs injected with spleen cells from unirradiated guinea pigs. n=6 for CD4– MNCs and CD4+ T-lymphocytes; i.p.: intraperitoneally; MNCs: Mononuclear cells; Rl: pulmonary resistance; BAL: bronchoalveolar lavage.

	Acknowledgements

TOP Abstract Methods Results Discussion Acknowledgements References

 
The authors are grateful to Y. Hosoi for his technical guidance and discussion concerning x-irradiation. Thanks are also due to N. Sakai for his skillful assistance in bone marrow analysis.

	References

TOP Abstract Methods Results Discussion Acknowledgements References

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Permissions Request Permissions Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Liu, Y. Articles by Shirato, K. Search for Related Content PubMed PubMed Citation Articles by Liu, Y. Articles by Shirato, K.