Figure 1.
60Co-radiation induced expression of IL-1ß, IL-18, and IL-33 in mouse tissues.
Mouse thymi, spleens, and BM were collected after 1, 3, 6, and 9 days after 0, 8 and 10 Gy of 60Co-TBI. Their homogenates were generated in PBS, and for each assay an equally determined amount of total protein was applied for quantitative detection of IL-1β, IL-18, and IL-33 by ELISA assay. After normalization, the results were displayed as the cytokine levels measured in 1 mg of tissue homogenates. (A) and (B) show the levels of IL-1β, IL-18, and IL-33 in thymus and spleen homogenates, respectively. Results were from a total of three experiments, N = 6/group in each experiment; *p<0.05, **p<0.01; mean ± SD. (C) shows the levels of IL-18 from six BM cell lysates combined and tests were performed in duplicate.
Figure 2.
Immunoblotting detection of IL-18 and IL-33 expression in mouse spleen cells after 60Co-TBI.
Analysis of protein expression of active IL-18 and IL-33 was performed by western blot. SDS-PAGE was conducted with 120 µg of spleen cell homogenates from individual mice, three mice per group. The mature sizes of IL-18 and IL-33 are approximately 18 and 33 KDa, respectively.
Figure 3.
Elevation of IL-18 and IL-6 in mouse serum after 60Co-TBI exposure.
Mouse sera from (A) 0, 5, 7, 8, 9, 10, or 12 Gy TBI mice were collected 1, 3, 6, 9 and 13 days after irradiation. Serum IL-18 concentrations were determined by ELISA assay, which specifically detects the bioactive form of mouse IL-18. ELISA was conducted using 50–100 µl of serum per individual sample. The values represent the data from four independent experiments (6 mice/group in each experiment, N = 24). *p<0.05, **p<0.01; mean ± SD. (B) ROC curve of serum IL-18 concentration 1 day after gamma-radiation. The area under the curve (AUC) was 0.96 (95% CI 0.91–1.00; P = 4.84×10−8). Statistical data of the AUC at 95% CI and AVOVA p-values in comparison of IL-18 concentrations in sham- and 1, 3, 6, 9 and 13 day post-irradiated samples are presented in table 1. (C) Mouse serum from 0 Gy control, and 8, or 10 Gy TBI mice were collected at 4, 8, and 24 h after irradiation. Serum IL-6 concentrations were determined by ELISA using 50–100 µl serum per individual sample. Values represent data from three independent experiments (6 mice/group in each experiment, N = 18). *p<0.05, **p<0.01; mean ± SD.
Table 1.
Performance evaluation of circulating IL-18 as a radiation biomarker in mouse serum.
Figure 4.
Cytokine antibody array in mouse serum after TBI.
A cytokine array using a total of 62 cytokines and chemokines (listed in table 2) was used to examine the pooled mouse serum collected 1, 3, and 6 days post-8 Gy irradiation. The unirradiated pooled serum (0 Gy) served as the baseline level. (A) Blots show duplicate measurements for each cytokine from control and three indicated time points of 8 Gy irradiated samples. Positions of G-CSF (2D), IL-10 (3E) and IL-12p40/70 (3F) are circled. Net light intensity images were detected using Image Gauge software. (B) The levels of cytokines and chemokines were graphed as the percentage of the positive control (representing approximately 100%) for each blot.
Table 2.
Cytokine antibody array map.
Figure 5.
IL-18 levels in serum of Rhesus macaques in response to 60Co-TBI exposure.
Sera were collected from five animals (3 females and 1 male) before and after 7 Gy of TBI. ELISA was performed using 100 µl of serum per individual sample. Using samples from pre-radiation as control, levels of IL-18 from same animals were evaluated at 2 and 4 days after TBI exposure. Radiation significantly increased IL-18 levels in the sera of NHPs on day 2 post-TBI, P = 0.021.
Figure 6.
IL-18 levels in plasma of Gottingen minipigs in response to 60Co-TBI exposure.
(A) Levels of IL-18 in plasma samples from individual animals collected before and 3 h and 1, 2, 3 and 7 days after 1.6 Gy irradiation and measured by ELISA. (B) Significant differences are shown at 1 and 3 days after 1.6 Gy. N = 4 *p<0.05; mean ± SD. (C) Levels of IL-18 were evaluated in pooled samples at different time points including pre-radiation, 3 hours, 2 days and 3 days after 1.78 Gy TBI. ELISA was performed using 100 µl of combined serum sample from each indicated time point.
Figure 7.
Discriminant analysis of IL-18, ALC, and ANC/ALC ratio in peripheral blood of irradiated mice, NHPs and minipigs.
(A) After TBI of 0, 5, 8 and 10 Gy, the three markers from peripheral blood, IL-18, ALC, and ANC/ALC ratio were evaluated in mice at 1, 3, and 7 day post-irradiation (+1 d, +3 d, and+7 d). (B) The levels of IL-18, ALC, and ANC/ALC ratio from the same blood samples of individual NHP obtained before and after radiation (+2 days and +4 days) were compared. (C) The levels of IL-18, ALC, and ANC/ALC ratio from the same blood samples of individual minipigs obtained before and after radiation (+3 h, +1 day and +3 days) were compared. Statistical differences of three combined markers between groups were determined by multivariate analysis of variance (MANOVA). Changes in biomarker levels were significant (Wilks Lambda p<0.001) as shown in each individual panel for all combinations and time points.