Since September 11, 2001, both private and government-sponsored publications have suggested that, after either a large-scale radiological or nuclear event, there could be a rapid exhaustion of local resources and that successful management of any medical response will therefore require the ability to acquire biodosimetry for individuals and appropriately direct treatment (28
). Treatment of those who have received doses that will induce acute lethal or severe responses will be of paramount importance. However, subsequent late effects may be seen not only among the survivors of acute injury but also in the vast majority of the population that received lower, sublethal doses. These may also require attention and possibly the use of mitigating therapies. Of the tissues/organs that could exhibit such late effects and therefore require preventative strategies, the lung has been identified as a prime candidate since both lethal and nonlethal radiation-induced pulmonary injuries have been seen after accidental exposures (7
) and in the Japanese A-bomb survivors (29
Similar to work from other investigators looking at normal tissue radiobiology, previous studies from our group using higher doses relevant to radiotherapy have shown that the lung responds rapidly, within hours, to radiation injury with increased expression of proinflammatory cytokines (18
). This observation has led us to hypothesize that the immediate alteration in cytokine expression initiates and orchestrates a cascade of events involving an interaction between the injured tissues and infiltrating inflammatory cells (19
), which culminates in the pulmonary late pathology. If this is true, such a sequence of events could be mitigated through interference in the cytokine-inflammation chain of communication, a supposition that was indirectly supported when we previously demonstrated the successful reduction of lung late effects through administration of lovastatin, a potent chemokine inhibitor (9
However, application of such a hypothesis to the search for a mitigating regimen in the aftermath of a terrorist event holds a great deal of uncertainty since it is unclear whether the pulmonary response to low-dose radiation would result in a similar and immediate up-regulation of cytokine expression, with its subsequent inflammatory consequences, and a further complicating unknown, how total-body compared to whole-lung irradiation would affect such a response. Therefore, one of the aims of our project within the University of Rochester-based Center for Medical Countermeasures against Radiation (CMCR) was to assess pulmonary injury after either a localized or a systemic injury in terms of the tissue cytokine and inflammatory cell response. In addition, since other groups within the CMCR network are developing biodosimetric techniques for use as triage (31
), we also looked at circulating proinflammatory cytokine levels with respect to their potential use as indicators and/or measures of radiation exposure.
Our earlier research led us to a working hypothesis that the early expression of IL1B is a key event in the initiation of the pulmonary response to radiation injury. Our current results continued to indicate that there is a significant increase in the early expression of IL1B, but not IL1A, in the lung at all dose levels within 1 h of irradiation. IL1B message levels remained elevated at the higher doses (>5 Gy) until 6 h postirradiation, then gradually declined, returning to base levels by 24 h. We also demonstrated a significant increase in the expression of the receptor IL1R2, but not IL1R1, that was again seen at all doses within 1 h of irradiation, with message levels remaining elevated at the higher doses at 6 h postirradiation and returning to base levels by 24 h. Since IL1R2 has been shown to be functionally inactive (32
) and a number of investigators have suggested that IL1
regulation may occur partially through a receptor-ligand balance (34
), we postulate that the IL1B-IL1R2 expression seen in this dose range may be part of a normal wound healing response and/or a tissue compensation mechanism.
The data also indicated a rapid increase in the level of expression of the chemokine receptors CCR1 and CXCR2. We considered this significant since the two receptors, in particular CXCR2, are associated with the regulation of neutrophils to sites of local inflammation (36
). Indeed, a dose-dependent and temporally associated infiltration of neutrophils was seen in the lungs. This finding was significant because the murine pulmonary response to radiation has not been considered to be neutrophil driven. Although some investigators have suggested a critical role for the CXC chemokine receptor after other forms of lung injury (37
), we believe that the transient nature of the neutrophil response and the temporal relationship with the observed apoptosis (compare and ) suggest that these events are part of a normal wound healing response.
Building upon earlier findings from our group that identified circulating markers that appeared predictive of late pulmonary injury (40
), we analyzed plasma from animals at these early times, looking at a panel of proteins. Since the realization that circulating systemic markers could be used as a relatively simple assessment tool, clinicians and scientists alike have attempted to identify specific markers for a wide array of uses, including markers that may be predictive for radiation-induced late effects. This research has given rise to a number of circulating markers that are potentially predictive for radiation-induced lung effects, including transforming growth factor β (42
), IL6 and IL1A (41
). One of the proteins that we had identified in our earlier clinical study, IL6, demonstrated a highly significant and discriminatory increase after doses above 2 Gy at 6 h postirradiation ( and ), although there was no corresponding change in IL6 mRNA expression in the lungs (additional analysis also demonstrated no change in liver tissue; data not shown). This contrasted with the changes in expression of keratinocyte chemoattractant, KC/CXCL1, which also showed a significant, dose-related increase in the plasma at 6 h postirradiation. KC/CXCL1, a neutrophil chemotactic factor, is a ligand for CXCR2 and has been shown to increase in expression after other pulmonary injuries (44
); therefore, the CXCL1/CXCR2 relationship may be a key factor that is responsible for the increased recruitment of neutrophils into the lung (). However, since there is no current correlation between these early and transient increases in expression and the development of pulmonary late effects, such increases may only serve as surrogates of injury and not necessarily targets for mitigation. Overall, these findings were thought to support the potential use of IL6 and KC recognition in the circulation as part of a temporally related biodosimetry panel indicating radiation exposure; however, it is unclear whether their expression is specific to radiation injury rather than being part of a stress response. In addition, the differential response between pup and adult mouse lungs after radiation injury tempers enthusiasm for their use since the marker expression may relate to different end points in different segments of the population.
An important observation made as part of this study was the overall differential response between the adult and neonate animals. It has been demonstrated that, under many circumstances, children respond differently than adults to radiation injury, for example after radiation treatment (22
). Since this differential is often exhibited as an apparent change in radiation sensitivity, it suggests that this population may be at altered or even increased risk of developing radiation-induced late effects. However, apart from case studies of overexposure in children, the majority of the available data in this population are limited to the monitoring that took place after the nuclear power accident at Chernobyl, which has focused on the subsequent carcinogenesis and genomic changes (45
). A small Ukrainian epidemiological study also suggested that children living around Chernobyl demonstrated higher rates of respiratory tract illness than those reported in the area prior to the incident (46
This latter finding provides support for the contention that the pediatric lung may be an organ that is especially at risk from radiation injury since it has been shown by many investigators that children's lungs are highly susceptible to damage from exposure to many environmental toxicants (47
). This is principally a result of the protracted maturation of the respiratory system, which in humans extends from the embryonic phase through to adolescence (50
). Besides an immature respiratory system at birth, children possess unique differences in their physiology and behavioral characteristics compared to adults. These are believed to augment the vulnerability of their developing lungs to perturbations by environmental toxins. Indeed, our results demonstrated that the early changes in IL1B and IL1R2 expression that were observed in the adult animals after irradiation could not be detected in mouse pups until they had reached 28 days of age (), which coincides with the time of weaning, although the detection of apoptosis in the lungs of all age groups () indicated that pulmonary injury occurred to a similar extent. This finding is of concern since work from our group has previously shown that damage to the lung during the developmental period may act as a priming agent for a subsequent injury, triggering sensitization to a secondary stimulus (51
), suggesting that factors that disrupt the developmental events may have significant long-term consequences.
Although rapid and similar increases in the level of expression of CXCL1 were seen in the plasma of both mouse pups and adults (), the increase was not coincident with neutrophil infiltration in the mouse pups; infiltration was seen only in the adult animals (). Therefore, our findings emphasize the need to recognize that radiation-induced late effects in young animals (and by extension, human children) may differ from adult responses and support the contention that current models do not sufficiently predict for late effects in a pediatric population (52
). There is also an increased expression of proinflammatory cytokines within the pulmonary tissue in the TBI animals compared to the whole-lung-irradiated animals, despite the fact that the tissue itself would have received the same dose. This suggests a systemic influence on the local tissue response that could support an altered immune response. If true, it may be worth speculating that such an alteration may affect the lung's ability to respond to later challenges and that this may be exacerbated further in the pediatric population. Therefore, further study is required to assess whether any of these early proinflammatory markers play a role in the induction of late effects in this low-dose model and how age may affect not only the initial proinflammatory cytokine response but also the subsequent communications between injured parenchyma and infiltrating inflammatory cells; these studies are ongoing.