Targeted Radiation Evokes Catecholamine Production Triggering Systemic Inflammatory Responses

Targeted irradiation (TR) is widely used for tumor treatment in the clinic. TR benefits tumor therapy through direct effects as well as poorly understood systemic (abscopal) effects. Recent studies suggest that the systemic innate and acquired immune responses to TR contribute to elimination of tumor cells, but also cause systemic inflammation with prolonged tissue injury that may result in secondary malignancies. To elucidate and eventually target the mechanisms underlying these systemic effects of TR, we utilized a murine model using the small animal radiation research platform (SARRP).

To define the dynamics of cytokine production and immune responses after TR, we administered local irradiation to a single tibia of 6-8 week old C57BL/6 male mice using a single dose of 15 Gy. We analyzed bone marrow (BM) and BM extracellular fluid (BMEF) from both the irradiated (TR) and non-irradiated, contralateral (CONT) tibiae at 2, 6, 48 hours, 1 and 3 weeks post-TR, performing phenotypic (flow cytometry) and cytokine analyses. As a tumor-bearing model, we utilized 3-4 weeks old C57BL/6 mice injected with Rhabdomyosarcoma (RMS) in one hind limb, and treated with (1) one dose i.p injection of 1mg/Kg Vincristine (Vin) as chemotherapy model, (2) 4.8GyX5times fractionated TR to the tumor area and (3) combination (TR+Vin) therapy. Analysis of peripheral blood (PB), BM, BMEF was performed 3 weeks after the final TR dose (n = 5-13 mice/time point).

We found that multiple inflammatory cytokines and chemokines, such as IL-1b, IL-18, CCL2, CCL3, CXCL2, CXCL9, CXCL10 were upregulated from very early phase (2hrs) up to 48hrs in BMEF of the radiated tibiae. Consistent with the dynamics of these cytokines, we observed influx of myeloid cells in both TR and CONT side and expansion of T cells peaking at 6hrs in BM. At the same time of these immune responses, Norepinephrine (NE) was elevated in BMEF even in CONT side. In the tumor-bearing model of RMS, fractionated TR eliminated the tumor while systemically expanding CD8+ cytotoxic T cells and reducing neutrophils. Vin alone did not eliminate the tumor and was associated with systemic decrease of lymphoid cells and expansion of neutrophils. In Vin+TR, tumor control and CD8+ cell expansion were restored, with normalization of neutrophils. These data suggest that TR in the setting of tumor differentially activates lymphoid and myeloid cells. Since recent studies showed catecholamine production from myeloid cells may augment cytokine production in the setting of infection, we hypothesized that BM myeloid cells respond to radiation-induced cell damage by producing catecholamines that trigger a systemic inflammatory response after TR. To test this hypothesis, we utilized standard long-term bone marrow cultures (LT-BM) that reproduce three-dimensional BM structures with myeloid-skewing in vitro, and irradiated them to look at inflammatory changes induced by radiation at 2, 6 and 24hrs. In this experimental model, 5Gy of radiation led to the elevation of NE along with the production of chemokines CCL2, CCL3, CXCL2, CXCL9 mostly peaking at 6hrs in the cell culture supernatants. In contrast, these responses could not be reproduced in spleen cultures, which also had a much lower baseline NE production compared to LT-BMs. These data indicate that radiation induced-chemokine elevations might come from myeloid cells stimulated by NE, independent of systemic innervation. To define the contribution of catecholamines to cytokine production in LT-BM, we directly stimulated culture-LT-BM with NE and Isoproterenol, a pan beta stimulant. While both agents showed similar effect and increased CXCL2, CXCL9, CCL2 and CCL3 at 6hrs, they decreased CXCL10 level, suggesting that catecholamine mostly stimulate myeloid cells but rather inhibit lymphoid activation through chemokine production.

Together, these data show that local irradiation initiates global immune responses, and identify local BM production of NE as its potential trigger. Blocking local catecholamine production in the bone marrow could therefore be a positive adjuvant to TR in tumor treatment by inhibiting unfavorable effects of radiation, such as chronic inflammation with systemic increases of neutrophils, while facilitating expansion and recruitment of the cytotoxic T cells which play an essential beneficial role in tumor immunity.