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Current Medicinal Chemistry, 2012, 19, 1741-1750. 1741. 1875-533X/12 ... radiation dose/quality and immune cell types investigated. In general, X-irradiation ...
Current Medicinal Chemistry, 2012, 19, 1741-1750

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Modulation of Inflammatory Immune Reactions by Low-Dose Ionizing Radiation: Molecular Mechanisms and Clinical Application F. Rödel*,1, B. Frey2, U. Gaipl2, L. Keilholz3, C. Fournier4, K. Manda5, H. Schöllnberger6, G. Hildebrandt5 and C. Rödel1 1

Department of Radiotherapy and Oncology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany

2

Department of Radiation Oncology, University Hospital Erlangen, Universitätsstraße 27, 91054 Erlangen, Germany

3

Department of Radiotherapy, Clinical Center Bayreuth, Preuschwitzer Straße 101, 95445 Bayreuth, Germany

4

Department of Biophysics, GSI Helmholtz für Schwerionenforschung, Planckstraße1, 64291 Darmstadt, Germany

5

Department of Radiotherapy and Radiation Oncology, University of Rostock, Südring 75, 18059 Rostock, Germany

6

Helmholtz Zentrum München, Institute of Radiation Protection, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany Abstract: During the last decade, a multitude of experimental evidence has accumulated showing that low-dose radiation therapy (single dose 0.5–1 Gy) functionally modulates a variety of inflammatory processes and cellular compounds including endothelial (EC), mononuclear (PBMC) and polymorphonuclear (PMN) cells, respectively. These modulations comprise a hampered leukocyte adhesion to EC, induction of apoptosis, a reduced activity of the inducible nitric oxide synthase, and a lowered oxidative burst in macrophages. Moreover, irradiation with a single dose between 0.5–0.7 Gy has been shown to induce the expression of X-chromosome linked inhibitor of apoptosis and transforming growth factor beta 1, to reduce the expression of E-selectin and L-selectin from EC and PBMC, and to hamper secretion of Interleukin-1, or chemokine CCL20 from macrophages and PMN. Notably, a common feature of most of these responses is that they display discontinuous or biphasic dose dependencies, shared with “non-targeted” effects of low-dose irradiation exposure like the bystander response and hyper-radiosensitivity. Thus, the purpose of the present review is to discuss recent developments in the understanding of low-dose irradiation immune modulating properties with special emphasis on discontinuous dose response relationships.

Keywords: Biphasic dose response, discontinuous dose dependency, immune modulation, inflammation, ionizing radiation, low-dose radiation therapy. 1. INTRODUCTION The interrelationship between ionizing radiation and the immune system is complex, multifactorial, and depends on the radiation dose/quality and immune cell types investigated. In general, X-irradiation with higher doses (e.g. single doses ≥ 2Gy) exerts pro-inflammatory effects and results in inflammatory processes as common toxicity of radiation therapy [1]. On the contrary, low-dose radiation therapy (LD-RT: single doses < 1 Gy) modulates a variety of inflammatory processes and clearly reveals anti-inflammatory properties [2]. Although LD-RT is clinically used since decades for the treatment of non-cancerous inflammatory and degenerative diseases [3, 4], underlying molecular mechanisms are far from being fully explored, at least in part because of their prominent discontinuous dose dependency and putative non (DNA)-targeted properties. The classical paradigm of radiation biology is based on the concept, that deposition of energy to the nucleus and as a consequence DNA damage is responsible for the biological consequences of radiation exposure. There is, based on recent findings, growing evidence for non-(DNA) targeted effects that challenged this classical concept. Among these findings bystander or out of field (abscopal) mechanisms, as well as responses that may be regarded as adaptive have been reported [5-7]. These effects arise either in cells that have received signals produced by irradiated cells or in cells that are non-clonal descendants of irradiated cells (radiation-induced genomic instability) [8]. These novel concepts take into consideration the complex intercellular communication and describe radiation responses on a tissue level or to the interaction of irradiated cells with the immune system [9]. Thus, the present review mainly elucidates the impact of a discontinuous or biphasic (which exhibits an area of low-dose *Address correspondence to this author at the Department of Radiation Therapy and Oncology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany; Tel: 0049 696301-6637; Fax: 0049 696301-5091; E-mail: [email protected] 1875-533X/12 $58.00+.00

stimulation and a range of high-dose inhibition) dose-response relationship in immune cell activity affected by low-dose irradiation (single dose 0.1-1 Gy) and its in vivo and clinical relevance. 2. MECHANISMS INVOLVED IN THE ANTI-INFLAMMATORY EFFICACY OF LOW-DOSE RADIATION During the last decades multiple efforts have been made for the molecular events following radiation exposure and subsequent irradiation-triggered pathways including induction of an inflammatory response. Initial events comprise direct damage to DNA or membrane lipids and the induction of oxidative stress [10], that in turn causes multiple modifications of essential cellular functions. In addition, activation of early signal transduction pathways by ionizing radiation to repair damaged DNA and to regulate cell cycle and cell death [11-13] is a common feature of the cellular radiation and inflammatory response. As there is substantial evidence for the involvement of nuclear transcription factors including TP53, specificity protein 1 (SP-1), activating protein 1 (AP-1), early growth response protein 1 (EGR-1), and nuclear factor kappa B (NF-κB) in both radiation response and inflammation [14, 15], they may also represent a crucial link between low-dose radiation and its anti-inflammatory properties. The NF-κB family comprises a heterogeneous group of homoor heterodimeric members of the Rel family including p50, p52, p65/RelA, c-Rel, and RelB [16, 17]. In a non-active status NF-κB is sequestered in the cytoplasm by inhibitor molecules of the IκB family (IκBα, IκBß, IκBγ/ΝΕΜΟ, IkB ε, p100 and p105) [16, 18], that in turn can be activated by a classical (also canonical) IκB polyubiquitination and 26S proteasomal degradation dependent pathway [17, 19, 20]. Additionally, two parallel cascades necessary for NF-κB activation by ionizing radiation have been reported. The first one depends on ataxia telangiectasia–mutated (ATM), which is activated by DNA double-strand breaks and the second depends on TP53-induced protein with a death domain (PIDD). Nevertheless, the phosphorylation of NF-κB essential modulator NEMO by ATM © 2012 Bentham Science Publishers

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displays the convergence between the two radiation induced NF-κB activation pathways [15]. The liberated NF-κB dimers subsequently translocate into the nucleus and bind specific sequence elements in the enhancer/promoter regions of more than 150 effector genes implicated in the repair of damaged DNA and regulation of molecules involved in the execution or inhibition of cell death by apoptosis [17, 18]. In addition immunological relevant NF-κB target genes comprise molecules like cytokines (e.g. interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-α)), adhesion molecules (e.g. E-selectin) and enzymes (e.g. inducible nitric oxide synthase: iNOS) [21, 22]. Concerning low-dose irradiation, Prasad et al. (1994) were the first to report an activation of NF-κB with peak activity at eight hours and 36 hours following a 0.5 Gy exposure as demonstrated in 244B lymphoblastoid- and B16 melanoma cancer cells [23]. 3. DISCONTINUOUS DOSE DEPENDENCY IN ENDOTHELIAL CELLS As endothelial cells (EC) play a crucial role in the regulation of the local inflammatory process both by their ability to recruit blood derived leukocytes and their capacity to express a variety of cytokines/chemokines and growth factors [24], experiments were performed on the role of EC in the anti-inflammatory efficacy of LD-RT. In human Ea.Hy 926 EC, LD-RT prior to the stimulation by TNF-α resulted in a biphasic induction of NF-κB DNA binding and transcriptional activity with a first peak after 0.5 Gy and a second peak at doses higher than 2 Gy. In accordance with the data of Prasad et al. (1994), a comparable time dependency of induction with peaks at eight hours and at 24-30 hours after irradiation was observed [23, 25]. Based on these initial observations factors engaged in the pathway(s) of NF-κB activation in EC are worth to be determined. One such regulatory protein is X chromosomelinked inhibitor of apoptosis (XIAP) that, besides its anti-apoptotic properties, has been shown to enhance NF-κB activity, p65 nuclear translocation and to promote the degradation of NF-κB inhibitors [26, 27]. Thus, experiments were performed in stimulated Ea.Hy 926 EC on the impact of XIAP, apoptosis induction and NF-κB activity. Following irradiation, a discontinuous profile of XIAP expression was observed with a relative maximum at 0.5 Gy and 3 Gy which parallels a discontinuity in apoptosis induction and caspase3/7 activity in EC. RNA-interference (siRNA) mediated attenuation of XIAP resulted in an increased rate of apoptosis, and a hampered NF-κB transcriptional activity, indicating a direct interrelationship between these factors [28]. These data are further consistent with the observation of Winsauer and colleagues (2008), showing that XIAP interacts with and ubiquitinates MAP Kinase Kinase 2 (MEKK2). The latter has previously been reported to be associated with a second wave NF-κB activation and is essential for propagation of inflammatory processes [29]. Furthermore, functional consequences of XIAP-expression and altered NF-κB were evident after XIAP knockdown by small interfering RNA (siRNA). So a diminished PBMC/EC adhesion, normally observed after irradiation with 0.5 Gy, can be abrogated by XIAP attenuation. This effect may, at least in part, be driven by a reduction of the cytokine transforming growth factor beta1 (TGF-β1), a key player in the anti-inflammatory effects of low-dose irradiation [2]. Beside NF-κB, members of the c-fos and c-jun protein family that collectively form the homo- or heterodimeric AP-1 transcription factor complex [14], play a key role in the transcription of a variety of immune effector molecules, including TGF-β1. Using electrophoretic mobility shift (EMSA) and luciferase based transcriptional activity assays a biphasic induction of AP-1 was detected in Ea.Hy 926 ECs [30]. A pivotal molecular mechanism in the regulation of the inflammatory response is the secretion of cytokines. While

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cytokines like IL-1, TNF-α or chemotactic factors like IL-8 and CCL20 activate the immune system in a pro-inflammatory manner [31, 32], anti-inflammatory peptides like the isoforms of TGF-ß1-3 or IL-10 down regulate and thus limit an inflammatory response [33, 34]. RNAse-protection assays performed at 24 hours after irradiation and stimulation of Ea.Hy 926 with TNF-α showed an elevated expression of TGF-β1 and Interleukin-6 (IL-6) whereas no induction of TNF-α/ß, Lymphotoxin, Interferon γ/ß, TGF-β2 nor TGF-β3 was detected under these conditions [35]. Furthermore, kinetic experiments revealed a biphasic dose and time dependency of TGF-β1 mRNA expression and protein secretion with maximum levels or peak expression after irradiation with 0.5 Gy at four hours and 24-30 hours after irradiation [36]. Additionally, several lines of evidence suggest that TGF-β1 plays a key role in mediating a hampered adhesion of leukocytes to EC [35, 37, 38]. In these studies adhesion of PBMC to EC was reduced to 40% – 50% of the control level at 4 hours and 24 hours after irradiation with a dose of 0.3-0.6 Gy and returned to control levels after irradiation with a dose > 1 Gy. This characteristic, however, was abolished by antiTGF-ß1 antibody pre-incubation. Additionally, at 12 hours after irradiation at 0.3 Gy an increased adhesion functionally coincided with a relative minimum of TGF-β1 secretion confirming a discontinuous behavior of leukocyte adhesion [36]. Finally, the expression of the cell adhesion molecule E-selectin on EC following low-dose irradiation was analyzed. LD-RT given within a few hours before the activation of ECs resulted in a reduced expression of E-selectin with a local minimum following a 0.3 Gy exposure, indicating an additional discontinuous dose response relationship in Ea.Hy 926 EC [35, 38]. 4. DISCONTINUOUS DOSE DEPENDENCY IN POLYMORPH NUCLEAR LEUKOCYTES The main cellular elements of the immune system comprise different subtypes of lymphocytes (B and T cells) as players of an antigen-specific effector response, as well as polymorphonuclear (PMN) and mononuclear leukocytes as main components of an innate host defense. Short-lived PMN (neutrophilic, eosinophilic and basophilic granulocytes) accumulate within hours at the site of inflammation and display a first line of immune defense [39]. Monocytes, unlike PMN, are long-lived and differentiate into tissue resident dendritic cells (DC) or macrophages [40]. The latter support a local inflammatory process by a plethora of functions like phagocytosis, antigen presentation, secretion of cytokines, release of reactive oxygen intermediates (ROIs) and the expression of enzymes like iNOS [41, 42]. Neutrophilic PMN infiltration has been implicated in the pathology of acute and chronic inflammatory diseases, such as rheumatoid arthritis [43, 44] in part by the secretion of chemotactic cytokines with the potential to amplify leukocyte infiltration [45]. Thus, the impact of LD-RT on chemokine production in PMNs was analyzed. As compared to CXCL8 and CCL18, CCL20 secretion was shown to be exclusively induced by a direct cell-cell contact between PMNs and EA.hy.926 ECs in a TNF-α-dependent manner. Furthermore, irradiation with doses between 0.5 and 1 Gy resulted in a discontinuous regulation and significant reduction of CCL20 production that parallels with a hampered PMN/EC adhesion with a highest level of reduction at a 0.7 Gy exposure [46]. Apoptosis displays a physiological endogenous cellular suicide program induced by a variety of endogenous and exogenous stimuli including ionizing irradiation or death receptor activation (e.g. TNF-α receptor) [47]. Moreover, apoptosis significantly impacts on cellular homeostasis, immune regulation and radiation response. Thus, Gaipl and colleagues irradiated isolated PMN with a single dose of 0.3–1.0 Gy two hours before stimulation with phorbol myristate acetate (PMA) and analyzed cell death by subG1 DNA content of these cells [48]. A discontinuous appearance of cell death

Modulation of Immune Reactions by Low-Dose Radiation

in irradiated PMN was observed, displaying a relative maximum at 0.3 Gy and minimum at 0.5 Gy, respectively. This discontinuity of cell death induction was coincident with the protein level of total cellular mitogen-activated protein (MAP) kinases and protein kinase B (or AKT), known to be involved in multiple cellular processes such as proliferation, metabolism, transcription and apoptosis [49]. 5. DISCONTINUOUS DOSE DEPENDENCY IN PERIPHERAL BLOOD MONONUCLEAR LEUKOCYTES Cells undergoing apoptosis contribute to the regulation of activated mononuclear cells in a paradox, thrombospondin receptor dependent manner by reducing the secretion of pro-inflammatory cytokines like TNF-α or IL-1. Instead, secretion of antiinflammatory cytokines like IL-10 is increased indicating immunesuppressive properties of apoptotic cells on activated monocytes [50]. Kern at al. (1999) were the first to report on a dose-dependent increase of apoptotic cells in peripheral blood mononuclear cells (PBMC) with a discontinuity (plateau or peak) between 0.3 Gy and 0.7 Gy in nine out of 10 blood donors [51], that contributes to a immunosuppressive microenvironment and anti-inflammatory characteristics of LD-RT. Additionally, the coincidence of a reduced PBMC/EC adhesion (as reported before) and induction of apoptotic cell death in PBMC prompted the group to investigate a putative link between apoptosis and the expression of adhesion molecules on the surface of the PBMC. They described a time dependent loss of L-selectin from leukocytes by proteolytic shedding that was associated with their early apoptotic phenotype [52]. Very recently, dose response and repair kinetics of serine 139 phosphorylated histone gamma-H2AX, a marker of radiation induced DNA double-strand breaks (DSBs) [53], was analyzed in whole blood and T-lymphocytes irradiated in vitro by X-rays (100 kV) and Co60 γ-rays. As compared to γ-irradiation a discontinuous behavior of the dose response with elevated values in the range of 0.2 - 0.3 Gy was reported for whole blood and less pronounced for isolated T-lymphocytes. Concerning repair kinetics, a delayed repair capacity was evident after X-ray irradiation (0.2 Gy) with 40% of γ-H2AX foci persisting 24 hours post-irradiation [54]. This may implicate a putative interrelationship between DNA-damage repair and a discontinuous dose response, that will be discussed in more detail in chapter 7. 6. DISCONTINUOUS DOSE DEPENDENCY IN MACROPHAGES AND DENDRITIC CELLS One hallmark of inflammatory macrophages is the expression of the enzyme iNOS which, through synthesis of nitric oxide (NO), is involved in numerous physiological and pathophysiological processes mostly in inflammatory conditions [55]. NO increases vascular permeability, contributes to oedema formation, and is involved in inflammatory pain [56, 57]. Low radiation doses (≤ 1 Gy), if applied 6-24 hours before or after stimulation with lipopolysaccharide (LPS) and IFN-γ, have been shown to inhibit the iNOS pathway and as a consequence NO production in murine RAW 264.7 macrophages without affecting iNOS mRNA expression [58]. This may, at least in part, contribute to the analgetic effects of low-dose irradiation and indicates that the inhibitory effect on iNOS protein activity is probably not regulated at the transcriptional level but by translational or post-translational modifications [59]. Tsukimoto et al. (2009) further examined signal transduction pathways in RAW 264.7 macrophage cells following irradiation with doses of 0.5 to 1 Gy from a 137Cs source. Dephosphorylation of both extracellular-signal-regulated kinases 1/2 (ERK1/2) and p38 MAPK was observed at 15 min after irradiation which was

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concomitant with a significant increase in the expression of the MAPK phosphatase-1 (MKP-1) [60]. Since activated p38 MAPK mediates proinflammatory cytokine production, they further assayed the effect of radiation on TNF-α production, showing that production of the cytokine induced by LPS was significantly suppressed in 0.5 Gy irradiated cells. More recently, a discontinuous characteristic of IL-1ß secretion in macrophages differentiated from THP-1 monocytes and stimulated by LPS and monosodium urate crystals was published in a congress abstract. The authors report that low-dose X-irradiation resulted in a decreased secretion of the precursor and active (cleaved) form of the cytokine in a discontinuous dose-dependent manner, most pronounced after a 0.5 Gy or 0.7 Gy exposure, respectively. The decreased secretion of IL-1β correlated with a reduced nuclear translocation of the NF-κB subunit RelA (p65) in line with a decreased protein amount of up- (p38 MAPK) and downstream molecules (AKT), further confirming a role of NF-κB and the p38 MAP kinase pathway in the anti-inflammatory effects of low-dose irradiation [61]. Taken into consideration the intracellular regulation of IL-1β secretion by quality of irradiation, Conrad et al. (2009) reported that the expression of IL-1β only gets modulated when macrophages were activated by LPS [62]. Interestingly, the group indicated that LPS induced NO production and phagocytosis of beads as well as a decrease in IL-1β production was more pronounced after irradiation with 9.8 MeV/u carbon ions. These findings underscore an increased radiobiological effectiveness of heavy ion irradiation in activated macrophages that fosters further investigations. Activated inflammatory macrophages are an important source of ROIs during the inflammatory response when they mount an oxidative burst. Thus, Schaue et al. (2002) investigated the effect of LD-RT on the oxidative burst of RAW 264.7 macrophages after stimulation with PMA or zymosan. In their experiments irradiation with a dose of 0.3 Gy - 0.6 Gy significantly reduced oxidative burst activity and superoxide production again indicating a discontinuous dose dependency of macrophage immunological properties. The group therefore concluded that a diminished release of ROIs may contribute to the local therapeutic effect of LD-RT [63]. Jahns and coworkers were the first to report on an effect of lowdose irradiation on the maturation and cytokine release of human DCs and the functional consequences for co-cultured Tlymphocytes. They showed that in vitro irradiation of DCprecursors did neither influence surface marker (e.g. CD80, CD83, CD86) expression or cytokine secretion (e. g. TNF-α, IL-1, IL-6, IL-8; IL-10) of immature DCs and of mature DCs after LPS stimulation, nor change the capacity of the DCs to stimulate T-cell proliferation [64]. These results indicate that DCs may not display a main target of low-dose irradiation although an influence on other DC functions (e.g. homing) could not be excluded at present. 7. MECHANISMS UNDERLYING THE DISCONTINUOUS CHARACTERISTICS OF LOW-DOSE RESPONSES The molecular mechanisms responsible for the discontinuous dose response characteristic following low-dose irradiation as summarized in Table 1 remain elusive at present and may originate from an overlap of several processes. These are initiated at various thresholds, display different kinetics, and operate in a staggered manner. Furthermore, one can speculate, that a discontinuous dose response may be mechanistically related to the phenomenon of lowdose hyper-radiosensitivity (HRS) and induced radioresistance (IRR), which have been reported for cellular survival of mammalian cells at doses below 0.3 Gy and in the dose range of 0.3 Gy to 0.6 Gy [65, 66]. The current understanding in the regulation of these processes is that the HRS region (< 0.3 Gy) reflects an area of increased apoptotic cell death in cells that failed to undergo an ATM-dependent early G2-phase cell cycle arrest and thus proceed

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Table 1.

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Factors or Mechanisms that Display a Discontinuous or Biphasic Dose Dependency Following Low-Dose X-Irradiation of Inflammatory Cells

Inflammatory cell

Factor/Mechanism

Effect

References

Endothelial cells

Leukocyte adhesion

Reduced PBMC adhesion at 4 and 24 hours after irradiation with 0.5 Gy. Increased adhesion at 12 hours following 0.3 Gy.

[36, 37, 38]

TGF-β1

Time- and dose-dependent biphasic expression with local maxima at 4 hours and 2430 hours following irradiation with 0.5 Gy.

[35, 36]

XIAP and apoptosis

Discontinuous expression of XIAP with highest value at 0.5 Gy in parallel to a reduced apoptotic fraction and caspase 3/7 activity.

[28]

NF-κB and AP-1

Biphasic characteristics of DNA-binding and transcriptional activity with peaks after 0.3 Gy (AP1), 0.5 and > 2 Gy and 8 hours and 24-30 hours post irradiation (NF-κB).

[25, 30, 36]

E-selectin

Reduced surface expression following a 0.3 Gy exposure.

[35, 38]

Apoptosis

Discontinuous induction and plateau of apoptosis in PBMC and PMN following irradiation with 0.3 Gy. Proteolytic shedding of L-selectin from apoptotic PBMC.

[48, 51, 52]

CCL20 chemokine

Irradiation with a dose between 0.5 and 1 Gy resulted in a discontinuous regulation and significant reduction of CCL20 release.

[46]

γ-H2AX

Biphasic behavior of the dose response. Elevated values at 0.3 Gy in whole blood and T-lymphocytes.

[54]

AKT kinase

Discontinuous course of apoptosis is coincident with the protein level of total cellular AKT kinase.

[48]

iNOS/NO activity

Inhibition of the iNOS pathway and NO production at doses < 1 Gy without affecting iNOS mRNA expression.

[58, 59]

ROI/oxidative burst

A single dose of 0.3 - 0.6 Gy significantly reduces oxidative burst activity and superoxide production.

[63]

IL-1, TNF-α,

Decreased secretion of IL-1β and TNF-alpha in a discontinuous dose-dependency, most pronounced after 0.5 or 0.7 Gy.

[60, 62]

AKT, p38 MAPK

Hampered expression of AKT and p38 MAPK phosphorylation at a dose of 0.3-0.5 Gy.

[60, 61]

Polymorphonuclear/Mononuclear cells

Macrophages

L-selectin

Abbreviations are: PMBC=peripheral blood mononuclear cells, TGF-β1 =transforming growth factor beta 1; PMN=polymorphonuclear cells; XIAP=X-chromosome linked inhibitor of apoptosis protein; AP-1=activator protein 1; iNOS=inducible nitric oxide synthase; ROI=reactive oxygen intermediates; MAPK=mitogen activated protein kinase; IL-1= interleukin1; TNF-α=tumor necrosis factor alpha.

through mitosis with damaged DNA. On the contrary, a transition to IRR originates from a change in the balance of G2-checkpoint induction, allowing time for repair of DNA-damage and increased cell survival. Corresponding to that notion, DNA double-strand breaks induced after exposure to very low-doses do not seem to be repaired, and cells containing residual damage will be removed by TP53-dependent apoptosis [67, 68]. Two seminal molecular studies [69, 70] have shown discontinuous dose-dependent radiation responses over the 0.1 to 1 Gy dose range, the most important being the activation of the critical DNA damage sensor ATM being (auto)phosphorylated after doses as low as 0.5 Gy. Once activated, ATM is responsible for initiating several signaling cascades, which are essential for cell cycle arrests and DNA damage repair. Notably, ionizing radiation also displays an important inducer of a ATM– IKK–NF-κB signaling pathway [20] that has been reported to affect cellular radiosensitivity [71]. An additional aspect to be involved in regulation of the characteristics of low-dose irradiation may be displayed by epigenetic alterations like DNA methylation, histone modification, and chromatin remodeling (reviewed in [72]). In that context, a whole body irradiation of mice with fractionated 0.5 Gy resulted in a decrease of histone H3-Lsy20 (tri)methylation in thymic tissue. This was accompanied by a decrease of global DNA methylation in parallel to a reduction of methyl CpG binding protein 2 (MECP2) and methyl binding domain protein 2 (MBD2) [73], that may alter gene expression as well as the accumulation of DNA damage as monitored by persistence of phosphohistone γ-H2AX foci in thymic tissue. Based on the data reported before, it is reasonable to assume that beside DNA (repair)-mediated and epigenetic mechanisms, a discontinuous dose effect relationship may also arise from a differential protein expression. Similar to this

hypothesis, Pluder and colleagues (2011) reported that exposure to Co60 γ rays of the human EC line EA.hy.926 resulted in rapid and time-dependent changes in the cytoplasmic proteome. Applying two dimensional gel electrophoresis (2D-DIGE technology), MALDI-TOF/TOF tandem mass spectrometry, and peptide mass fingerprint analyses 15 significantly differentially expressed proteins were identified of which 10 were up- and 5 downregulated. Pathways influenced by the low-dose exposures included the RhoA pathways, fatty acid metabolism and stress response [74]. Notably, a cell type-independent inhibitory effect of low-dose irradiation on the activity of the 26S proteasome at doses above 0.5 Gy has been reported [75]. As the 26S proteasome is involved in the degradation of all short-lived and 70 – 90% of the durable cellular proteins [76], this observation may also contribute to the regulation of the cellular proteome and to discontinuous effects as reported before. 8. MODELING OF DISCONTINUOUS DOSE DEPENDENCIES Although modeling of anti-inflammatory responses following low-dose irradiation has not yet been performed, modeling of discontinuous dose responses for other biological endpoints such as in vitro neoplastic transformation [77, 78], and lung cancer [79, 80] have already been successfully established. Three different teams of researchers have demonstrated that single low-doses of low linear energy transfer (LET) exposures delivered at low-dose rates can significantly reduce the risk of neoplastic transformation below the spontaneous level [81-85]. These groundbreaking observations argue for the induction of biological processes that protect the cell against naturally occurring

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as well as radiation-induced alterations leading to cell transformation. Based on such experimental evidence Schöllnberger et al. (2004, 2005) assumed that low-doses of low-LET radiation delivered at low-dose rates can induce protective mechanisms in the target cells of human lung cancer and that induced DNA repair and radical scavenger activities can also repair and prevent DNA lesions produced through endogenous processes. These authors applied mechanistic multistage cancer models to perform sensitivity studies related to the changes in cellular defense mechanisms necessary to produce a dose dependent lifetime probability for lung cancer that deviates from a linear no-threshold (LNT) type response [79, 80]. The most well-known mechanistic cancer model is the MVK model of Moolgavkar, Venzon and Knudson, also referred to as two-stage model [86, 87]. This model describes the age-dependence of cancer incidence and mortality and has been tested extensively [88-91]. In this model, normal stem cells (N-cells) are converted to initiated cells (I-cells) at rate µ1 (Fig. 1). I-cells may divide symmetrically at rate α and die or differentiate at rate β. I-cells may also divide asymmetrically into one malignant cell (M-cell) and another I-cell at rate µ2. An M-cell develops into a full grown tumor after a lag-time, tlag. The MVK model is stochastic: it describes the above mentioned transitions between N-cells, I-cells, and M-cells with stochastic coupled differential equations. The lag-time is usually treated deterministically: for lung cancer, for example, a constant value of 5 years is used [79, 80, 92]. I-cells are mutated cells. Mutations can be caused by ionizing radiation and by endogenous processes. Therefore, µ1 was parameterized into radiation-dependent factors and into terms that do not depend on dose or dose rate but on the expected numbers of simple (e.g. SSBs) and complex lesions (e.g. DSBs) created by endogenous processes. Values for most of these quantities including misrepair probabilities are available in the scientific literature and have been summarized in Schöllnberger et al. (2004). For low-dose exposures it can be assumed that µ2 does not depend on dose or dose rate [90]. Based on experimental evidence (e.g. [93-95]), Schöllnberger et al. (2004, 2005) assumed that low-doses of low-LET radiation induce DNA repair mechanisms and radical scavengers. Two dosedependent Gaussian-shaped scaling functions were used to simulate various increased levels of DNA repair and scavenging activities. Rate µ 1 was divided by these functions to account for changes in the probability of lesion misrepair and for changes in the radical scavenging capacity of a cell (and hence the initial yield of DNA damage) as a function of dose rate [79, 80]. For values of these scaling functions greater than one, radiation is less effective at inducing genomic instability and cell transformation (i.e., reduces the rate µ1). The exact tumor incidence formula in closed form developed for the stochastic two-stage model by Kopp-Schneider et al. (1994) [96] has been applied by Schöllnberger et al. (2005). The lifetime probability for lung cancer versus total absorbed dose delivered in 75 years has been calculated for various levels of repair

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and scavenger induction. For doses comparable to background radiation levels, the results suggest that endogenous DNA damage accounts for as much as 54 to 78% of the predicted lung cancers. For a lifetime dose of 1 Gy, endogenous processes may still account for as much as 21% of the predicted cancers [79]. The results suggest that radiation must induce changes in DNA repair of at least 50% of the baseline value in order to produce cumulative probability levels for lung cancer outside the range expected for endogenous processes and background radiation. For scavengers the changes must be at least 200% to lead to significant deviations in the dose-response curves for lifetime probability for lung cancer [79]. In the current model, distinct U-shaped curves are only produced when both the accuracy of DNA repair and the capacity for radical scavenging are enhanced about three-fold. These studies challenged the classical paradigm of radiation protection that DNA damage determines the risk. This concept is more and more challenged by a new paradigm as reported before: the response to the damage (including intra- and intercellular signals) determines the risk. The mechanisms implemented into the stochastic MVK model and into a deterministic multistage cancer model [79, 80] reflect this novel view: Low-LET radiation causes DNA-damage (represented by the radiation-dependent components in µ1); low doses of low-LET radiation stimulate various cellular defense mechanisms such as DNA repair and radical scavenging activities (that is the dose-dependent response to the damage represented by the scaling functions) which can subsequently repair radiation-induced DNA-lesions and also reduce the impact of damage formed through endogenous cellular processes. Thus, in principle it should be possible to apply and/or adapt the mathematical techniques described before to model discontinuous dose dependencies related to immune responses such as those described in the present review. 9. ANIMAL MODELS TO STUDY THE EFFECT OF LOWDOSE IRRADIATION A first series of animal studies on the effects of low-dose ionizing irradiation on osteoarthritis was performed by von Pannewitz in the early thirties of the last century by electrocoagulation of the knee joint cartilage or by mechanical bone destruction. He reported an improvement of the clinical symptoms, joint swelling and pain in the irradiated animals. Importantly, he could not detect any effect on degenerative changes or structural integrity [97]. Using an intra-articular injection of granugenol, inactivated mycobacterium tuberculosis or papain, Budras et al. (1986), Trott et al. (1995) and Fischer et al. (1998) induced an acute arthritis in rabbit knees. In these models five weekly fractions of 1.5 Gy or 1.0 Gy reduced the inflammatory proliferation of the synovial cover cells, the synthesis of synovial fluid, and thus swelling of the joint [98-100].

α

N

μ1

μ2

I

M

tlag

T

β Fig. (1). Conceptual view of the model of Moolgavkar, Venzon and Knudson (MVK model) modified according to [89]. Normal stem cells (N-cells) are converted to initiated cells (I-cells) at rate µ1. I-cells may divide symmetrically at rate α and die or differentiate at rate β. I-cells may also divide asymmetrically into one malignant cell (M-cell) and another I-cell at rate µ2 . An M-cell develops into a full grown tumor after a lag-time, tlag .

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Rödel et al.

The effects of LD-RT on histomorphological progression of adjuvant induced arthritis in rats were investigated in the years 2000-2004 [101-103]. In these experiments, local irradiation of the arthritic joints with daily fractionated doses of 5 x 0.5 Gy or 5 x 1.0 Gy displayed an anti-inflammatory effect and reduces clinical symptoms, if given at the days 15-19 after induction of experimental arthritis. A histopathological analysis revealed a significant reduction of cartilage and bone destruction with minimal effect on the number of inflammatory cells in the periarticular tissue. Additionally, the histologically observed prevention of the experimental disease appears to be related to the modulation of iNOS activity with a reduction of the iNOS score by 45-50% [103]. In proceeding analyses a carrageenan air pouch model was developed in NMRI mice to analyse the expression of inflammatory cytokines TNF-α, IL-1, iNOS, heme oxygenase-1 (HO-1), cyclooxygenase-2 (Cox2) and heat shock protein-70 (HSP-70) in an acute phase (6 hours after induction) of inflammation. In this model, IL-1 and iNOS expression was attenuated by irradiation concomitant with an increased expression of HO-1 and HSP-70 further confirming a functional modulation of cytokines and the iNOS-system by in vivo irradiation [104]. Interestingly, a discontinuous modulation of leukocyte adhesion was reported by Arenas et al. (2006) in C57/Bl6 mice following irradiation with single doses of 0.1, 0.3, and 0.6 Gy. In this model, a significant reduction of LPS induced leukocyte adhesion in murine intestinal venules (as monitored by intravital microscopy) was demonstrated with the highest extent following a 0.3 Gy exposure [105]. This behavior mechanistically correlated with increased levels of circulating TGF-β1, as neutralization of the cytokine partially restored leukocyte adhesion and thus confirms the in vitro results as described before. More recently, Frey et al. (2009) used a transgenic mouse model [106] to examine the effects of LD-RT on Rheumatoid Arthritis. In this model, transgenic mice express the human cytokine TNF-α and develop a chronic polyarthritis at an age of 4– 6 weeks which is characterized by synovial inflammation, cartilage damage, and bone erosion. In line with proceeding analyses as described above, the group observed a significant temporal improvement of the clinical progression of disease in terms of grip strength and joint swelling when mice were irradiated at the beginning of the disease with 0.5 Gy in five fractions within one week [107]. Table 2.

In conclusion, the in vivo data convincingly confirm antiinflammatory effects of LD-RT and may display suitable platforms for an intensified research of the underlying mechanisms. 10. CLINICAL APPLICATION OF LOW-DOSE RADIATION THERAPY Beside the very recent successes in exploring molecular and cellular mechanisms, low-dose radiotherapy of acute and chronic inflammatory diseases and painful degenerative joint disorders is a well accepted conservative treatment in Germany and other European countries [3, 4]. It has been traditionally used in the clinical settings as early as 1898, when Sokoloff first reported on pain relieve in patients with arthritis treated with low-dose Xirradiation [108]. Treatment schedules and doses in clinical applications have been established empirically in the 30ths of the last century [97] recommending single doses of 0.3-1.0 Gy in 4-5 fractions for acute and 1-3 fractions for chronic diseases per week to total doses of 3-5 Gy and 12 Gy, respectively [109]. Due to publications on late harmful side effects and on epidemiological studies that reported increased mortality from leukaemia and aplastic anaemia in patients with ankylosing spondylitis, LD-RT is considered unfashionable in some countries [110, 111]. The turn away from LD-RT was further encouraged by the availability of effective non-steroidal or steroidal drugs. Nevertheless, these therapies also display numerous side effects and a considerable number of patients does not respond properly, if at all. Even though the carcinogenic risk of low-dose irradiation is still a matter of controversial debates, improved patient radiation protection and recent progress in the development of predictive objectives for the response to anti-inflammatory radiotherapy may help to reconsider it as an alternative option even in these countries. In 2004 a patterns-of-care study performed in Germany was published with 37410 patients treated for degenerative or hyperproliferative disorders like impingement of the shoulder joint (rotator cuff syndrome), tennis/golfer´s elbow, plantar fasciitis (painful heel spur), osteoarthritis or Dupytren’s disease [3]. Concerning the most important clinical endpoints pain relief, complete response and long time analgetic effects, LD-RT is reported to result in a 33-100%, a 47-100% and a 12-89% efficacy, respectively [112-116]. An actual patterns-of-care study [117] further described that in 2010 more than 4500 patients with

Summary of Actual Patterns of Care Studies and Clinical Investigations of Low-Dose Anti-Inflammatory and Analgesic Radiation Therapy in Germany

First author

Disease

Number of patients

Main findings

Reference

Micke et al.

Painful heel spur syndrome (plantar fasciitis)

7947

Pain reduction for at least 3 months in 70%, persistent pain reduction in 65% of the patients. No radiogenic acute or chronic side effects.

[112]

Mücke et al.

Painful/refractory Gonarthrosis

502

Significant prognostic factors for pain relief are a single treatment series, age >58 years and high voltage photons.

[120]

Heyd et al.

Painful heel spur syndrome

130

RT is an effective treatment option, irrespective of treatment with a total dose of 3 Gy (single 0.5) or 6 Gy (single 1 Gy).

[121]

Niewald et al.

Periarthritis of the shoulder

141

Pain relief and improvement of motility was 69% and 89%, respectively, after 4.5 months (median) and 73% after 3.9 years with virtually no side effects.

[113]

Betz et al.

Early-stage Dupuytren's contracture

135

RT is effective in prevention of disease progression (59-87%) in a follow up of 13 years. RT improves patient’s symptoms in early-stages with minor late toxicity.

[115]

Mücke et al.

Painful/refractory Gonarthrosis

4544/year

Median pain reduction for at least 3 months in 60%, at least 12 months in 40% of the patients.

[117]

Heyd et al.

Plantar fibromatosis (Morbus Ledderhose)

24

Complete remission of cords or nodules in 33%, reduced number in 54% and unchanged in 12.1% of the patients. Pain relief was achieved in 68.4% of the patients.

[122]

Adamietz et al.

Calcifying tendonitis of the shoulder joint

102

Pain relief was achieved in 82% at a follow-up of 18 months. Sonographic classification (Farin Type III) is predictive for response.

[114]

Abbreviations are: RT= radio- therapy.

Modulation of Immune Reactions by Low-Dose Radiation

Endothelial cells

Current Medicinal Chemistry, 2012 Vol. 19, No. 12

Leukocytes

1747

Macrophages

Fig. (2). Advanced model showing the modulation of immune cell activity and factors involved in the anti-inflammatory efficacy of LD-RT. Irradiation with doses of 0.3-0.5 Gy resulted in a decreased expression of E-selectin on the surface of activated EC, a modulation of XIAP expression and as a consequence in a decreased apoptosis, increased NF-κB activity, increased TGF-β1 expression and a reduced PMBC/EC adhesion. In leukocytes (granulocytes and monocytes) an induction of apoptosis, a proteolytic shedding of L-selectin as well as a reduced secretion of the chemokine CCL20 were evident after lowdose irradiation. In stimulated macrophages a reduced activity of the iNOS-pathway concomitant with reduced levels of NO, a lowered ROI mediated oxidative burst, decreased secretion of the pro-inflammatory cytokines IL-1β and TNF-α as well as a hampered expression of AKT kinase and p38 MAPK may contribute to the anti-inflammatory effects.

osteoarthritis of the knee received LD-RT demonstrating an increased acceptance of this treatment (95% referral for radiotherapy). A detailed summary of additional recent clinical investigations in Germany is given in Table 2. Importantly, the development of reliable predictive objectives for the response to low-dose radiotherapy is seriously needed. In that context, a predictive value of pretreatment sonographic classification of calcifying tendonitis for the outcome after LD-RT has recently been reported by Adamietz and colleagues [114]. According to that, a European Society for Radiotherapy and Oncology (ESTRO) conference in Nice 2007 [118] aimed to categorize the indication of radiotherapy for non-malignant disease in accepted, only acceptable in clinical trials and non-acceptable indications. Additionally, a randomized trial was recommended to prove the effectiveness of LD-RT as compared to NSAID treatment or steroid injections especially in younger patients. This study was initiated in 2008 in Germany [119].

Stiftung Bamberg, Germany, and the Thomas-Wildey-Institut e.V., Munich, Germany. ABBREVIATIONS AP-1

=

activator protein 1

ATM

=

ataxia telangiectasia–mutated

DC

=

dendritic cell

DSBs

=

DNA double-strand breaks

EC

=

endothelial cell

EMSA

=

electrophoretic mobility shift assay

HRS

=

hyper-radiosensitivity

I-cells

=

initiated cells

IL-1

=

interleukin-1

IRR

=

induced radioresistance

CONCLUSIONS

iNOS

=

inducible nitric oxide synthase

Although considerable progress has been achieved during the last two decades in the understanding of the molecular mechanisms being prominent after a low-dose exposure (pictured in Fig. 2), a multitude of unresolved questions exists that foster further investigations. For instance, is there a real interrelationship between HRS/IRR and the immune modulation by low-dose irradiation or do these responses differ with respect to mechanisms and factors involved? Moreover, as inflammatory diseases are characterized by complex (patho)physiological networks, ongoing research will also focus on additional immunological mechanisms and cellular components and a putative relation to tumor immunobiology. In the long run, these efforts may give extended insight in the radiobiology of low-dose radiation exposure and may not only help to improve radiation therapy of non-malignant diseases but also radiation protection and treatment of cancer.

LD-RT

=

low-dose radiation therapy

LET

=

linear energy transfer

LNT

=

linear no-threshold

LPS M-cell MAPK N-cells NF-κB NO PBMC PMN ROI RT TNF-α TGF-β1

= = = = = = = = = = = =

lipopolysaccharide malignant cell mitogen activated protein kinase normal stem cells nuclear factor kappa B nitric oxide peripheral blood mononuclear cells polymorphonuclear cells reactive oxygen intermediates radiotherapy tumor necrosis factor alpha transforming growth factor beta 1

XIAP

=

X-chromosome linked inhibitor of apoptosis protein

ACKNOWLEDGEMENT This work was supported by the European Commission under contracts FP6-036465 (NOTE), and FP7-249689 (European Network of Excellence, DoReMi), by the Doktor Robert Pfleger-

1748 Current Medicinal Chemistry, 2012 Vol. 19, No. 12

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Received: November 01, 2011

Revised: November 29, 2011

Accepted: November 30, 2011

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