Using antibody-labeled fluorescence imaging techniques, we have shown in both ex vivo and in vivo models that ionizing radiation induces migration of two cutaneous dendritic cell populations, specifically epidermal Langerhans cells (LC) and dermal interstitial dendritic cells (iDC). Not only have we characterized and quantified the cellular migration of each population as a result of radiation exposure in a dose- and time-dependent manner, we have shown through intradermal injection of fluorescently conjugated antibodies that similar results could be achieved in live mice, suggesting a possible assay to monitor and diagnose degrees of radiation exposure. We also discovered an additional role for the immunostimulatory cytokine IL-12 and have shown that it can modify the migration induced by radiation when injected at the site of exposure. Taken together, these results suggest a novel in vivo approach to analyze cutaneous DC after radiation exposure, which may be useful in the field as a biomarker to aid in the triage of potential victims after a radiation event.
Although the depletion of LC as a result of radiation exposure has been documented previously (11
), improved visualization techniques, more specific antibodies, and further elucidation of the relationships between cutaneous immune cells and radiation provide motivation for further investigation. Using a specially constructed mouse jig, we were able to irradiate one ear while the remainder of the mouse including the contralateral ear remained shielded and acted as an internal self control. After radiation exposure, we saw a marked decrease in the density of iDC and LC via anti-MHC class II and anti-internal-Langerin staining, respectively, using fluorescence microscopy. Though the kinetics of the iDC and LC was qualitatively similar, there were subtle interesting differences that may reflect the degree of radioresistance between the cells. Regarding dose, it is clear for each cell type that the larger the dose, the more dramatic the loss in cells; however, the magnitude and initial rate of loss appears to differ between the populations (Figs. and ). Initially, the iDC have a more dramatic drop in overall density than the LC. These differences may be attributed to several possible factors: (a) LC must down-regulate E-cadherin mediated attachment to keratinocytes, whereas iDC do not, thus slowing the migration process (26
); (b) LC must traverse greater distances and navigate through the basement membrane zone that separates epidermis from dermis (27
); and finally (c) LC may be more radioresistant than iDC, as is evident from bone marrow chimeras and whole-body radiation experiments (9
). While each factor suggests interesting possible interpretations, additional studies to further elucidate the mechanism responsible for the differences in migratory rates between each DC population after irradiation need to be conducted before firm conclusions can be drawn. These studies are currently under way.
Upon examination of each population after radiation exposure, distinct morphological differences were evident. After irradiation, the remaining iDC appeared rounded with shorter projections than their unirradiated counterparts. These morphological changes are reasonable from a sterics standpoint, because they permit efficient trafficking through thick extracellular matrices toward draining lymphatics (27
). In contrast, LC were slightly larger with longer dendritic projections, which may seem counterintuitive but actually reflects similar morphological changes seen during contact hypersensitivity reactions triggered by the topical application of a reactive hapten such as dinitrofluorobenzene (28
). Similar to hapten-treated ears, irradiated ears also had gaps within the network of cells where LC were not present. The remaining LC appeared larger and with longer projections that extended over the gaps devoid of cells. Taken together, these morphological changes suggest an exodus from the cutaneous environment toward draining lymphatics as a result of radiation exposure similar to the migration seen from documented hypersensitivity experiments.
In addition to migration as a plausible explanation for the observed depletion of cutaneous DC after radiation exposure, it is reasonable to infer that some fraction of the cells may be dying as a result of the higher doses (15 Gy and 25 Gy). The iDC in particular () appear to be more radiosensitive, suffering greater initial decreases in overall density 2 and 4 days after exposure to 25 Gy and 15 Gy, respectively. However, the rates of depletion for the LC population () at days 2 and 4 do not appear to be as dramatic as for the iDC counterparts, suggesting again that LC are more radioresistant. Currently, we are experimenting with a modified TUNEL assay (29
), used to label and detect DSBs within DNA, to assess apoptosis in both dermal and epidermal tissues.
Interestingly, in a manner similar to UV radiation, LC as well as iDC migrate from the site of exposure to draining lymph nodes (in this case the auricular lymph node) after exposure to ionizing radiation (24
). The reasons for this migration and a plausible mechanism remain unknown; however, we are currently exploring a variety of assays to further elucidate this phenomenon. One explanation could be the induction of apoptosis of keratinocytes resulting from a variety of DNA lesions such as double-strand breaks (DSBs) caused by radiation exposure (5
). In the case of UVB radiation, keratinocytes develop cyclobutane pyrimidine dimers (CPD) within their DNA, which triggers death receptors that further activate the pathways to apoptosis in an effort to protect the body from potentially malignant mutations (25
). This process is known as sunburn cell (SBC) formation (30
), and the same damage has been reported within LC (25
). In an effort to save some of these SBC, the nucleotide excision repair (NER) pathway is activated to remove CPD and repair the DNA. Schwarz et al
. have shown that NER can be induced by administration of the immunostimulatory cytokine IL-12 (25
) and that it can further limit the number of SBC (24
) as well as LC with CPD in draining lymph nodes (25
). Perhaps the same can be inferred for ionizing radiation, which does not create CPD but rather makes DSBs within DNA.
Our studies with rIL-12 administration would suggest that not only does the cytokine trigger NER, it may play a role in initiating DSB repair pathways, which could explain the recovery seen when compared to PBS-treated/irradiated mice. In addition to the possible retention induced by rIL-12, cells deemed unsalvageable
by repair mechanisms as a result of radiation exposure might trigger LC to engulf the apoptotic bodies and migrate to draining lymph nodes, as is the case for UVB-radiation-induced damage (32
). Although it is well established that apoptotic bodies are engulfed and cleared by phagocytic cells such as LC and iDC (33
), it remains unclear what “self” signals are modified by UV radiation and, in this case, ionizing radiation to cause such a phenomenon (6
Another explanation for the migration of LC may be attributed to cytokines and cadherins that control and regulate mobilization of LC. Upon antigen recognition by LC, IL-1β is up-regulated internally and acts in an autocrine fashion through the IL-1 receptor on both LC as well as neighboring keratinocytes (35
). The cytokine TNF-α is then secreted by keratinocytes and acts back on the LC as a secondary messenger to cause down-regulation of E-cadherin and other adhesion molecules (35
). Thus mobilization is initiated, and the LC are permitted to traffic to the draining lymph node. In the event of exposure to radiation, perhaps trauma induced upon neighboring keratinocytes causes a faulty release of TNF-α or simply down-regulates the strong adhesive cadherins, permitting LC to traffic. Additional analysis of secreted IL-1β and TNF-α as well as their expression patterns from both LC and keratinocytes after radiation exposure need to be conducted to elucidate the role of cytokines in inducing this migration. Studies aimed toward exploring E-cadherin and other adhesion molecules would also be relevant for this proposed mechanism.
Although the ability to monitor changes in cutaneous dendritic cell populations for use as a potential biomarker appears promising, additional research needs to be conducted, namely in the area of local compared to whole-body ionizing radiation exposure. Whereas the extremities can withstand over 20 Gy, there have been no reported survivors of victims exposed to 10 Gy whole body (4
). Therefore, we are expanding our research to further elucidate the mechanisms and signaling pathways involved in this marked migration as a result of low-dose exposure. Currently, we are attempting to delineate among five low-dose groupings of radiation exposure including 1–2, 2–4, 4–6, 6–8 and 8–10 Gy, where well-defined clinical symptoms and manifestations have been reported (4
In summary, we have demonstrated a novel approach using fluorescently conjugated antibodies to monitor a unique cellular response to ionizing radiation. We have also shown that cutaneous tissue samples can be fluorescently stained in vivo to monitor changes in the density of two morphologically distinct antigen-presenting cells including Langerhans cells and interstitial dendritic cells. These findings were confirmed by ex vivo analysis using whole-mount histology, further showing the usefulness and range of the application.