Indirect macrophage responses to ionizing radiation: implications for genotype-dependent bystander signaling.Coates PJ1, Rundle JK, Lorimore SA, Wright EG.Author informationAbstractIn addition to the directly mutagenic effects of energy deposition in DNA, ionizing radiation is associated with a variety of untargeted and delayed effects that result in ongoing bone marrow damage. Delayed effects are genotype dependent with CBA/Ca mice, but not C57BL/6 mice, susceptible to the induction of damage and also radiation-induced acute myeloid leukemia. Because macrophages are a potential source of ongoing damaging signals, we have determined their gene expression profiles and we show that bone marrow-derived macrophages show widely different intrinsic expression patterns. The profiles classify macrophages derived from CBA/Ca mice as M1-like (pro-inflammatory) and those from C57BL/6 mice as M2-like (anti-inflammatory); measurements of NOS2 and arginase activity in normal bone marrow macrophages confirm these findings. After irradiation in vivo, but not in vitro, C57BL/6 macrophages show a reduction in NOS2 and an increase in arginase activities, indicating a further M2 response, whereas CBA/Ca macrophages retain an M1 phenotype. Activation of specific signal transducer and activator of transcription signaling pathways in irradiated hemopoietic tissues supports these observations. The data indicate that macrophage activation is not a direct effect of radiation but a tissue response, secondary to the initial radiation exposure, and have important implications for understanding genotype-dependent responses and the mechanisms of the hemotoxic and leukemogenic consequences of radiation exposure

Indirect Macrophage Responses to Ionizing Radiation: Implications for Genotype-Dependent Bystander Signaling

1. Philip J. Coates, 2. Jana K. Rundle, 3. Sally A. Lorimore, and 4. Eric G. Wright

+Author Affiliations1. Cancer Biology and Clinical Pathology, Division of Pathology and Neurosciences,

Ninewells Hospital and Medical School, Dundee, United Kingdom1. Requests for reprints:

Philip J. Coates, Cancer Biology and Clinical Pathology, Division of Pathology and Neurosciences, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom. Phone: 44-1382-633951; Fax: 44-1382-633952; E-mail:p.j.coates@dundee.ac.uk. 

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AbstractIn addition to the directly mutagenic effects of energy deposition in DNA, ionizing radiation is associated with a variety of untargeted and delayed effects that result in ongoing bone marrow damage. Delayed effects are genotype dependent with CBA/Ca mice, but not C57BL/6 mice, susceptible to the induction of damage and also radiation-induced acute myeloid leukemia. Because macrophages are a potential source of ongoing damaging signals, we have determined their gene expression profiles and we show that bone marrow–derived macrophages show widely different intrinsic expression patterns. The profiles classify macrophages derived from CBA/Ca mice as M1-like (pro-inflammatory) and those from C57BL/6 mice as M2-like (anti-inflammatory); measurements of NOS2 and arginase activity in normal bone marrow macrophages confirm these findings. After irradiation in vivo, but not in vitro, C57BL/6 macrophages show a reduction in NOS2 and an increase in arginase activities, indicating a further M2 response, whereas CBA/Ca macrophages retain an M1 phenotype. Activation of specific signal transducer and activator of transcription signaling pathways in irradiated hemopoietic tissues supports these observations. The data indicate that macrophage activation is not a direct effect of radiation but a tissue response, secondary to the initial radiation exposure, and have important implications for understanding genotype-dependent responses and the mechanisms of the hemotoxic and leukemogenic consequences of radiation exposure. [Cancer Res 2008;68(2):450–6]

Ionizing radiation

 

bystander effects

 

macrophages

 

IntroductionThe dogma that genetic alterations are restricted to directly irradiated cells has been challenged by observations in which effects of ionizing radiation, characteristically associated with the consequences of energy deposition in the cell nucleus, arise in nonirradiated cells. These so-called nontargeted effects ( 1, 2) are shown in cells that have received signals produced by irradiated cells (radiation-induced bystander effects) or that are the descendants of irradiated cells (radiation-induced genomic instability). Radiation-induced genomic instability is characterized by a number of delayed adverse responses, including chromosomal abnormalities, gene mutations, and cell death days and months postexposure. Similar effects, as well as responses that may be regarded as protective, have been attributed to bystander mechanisms ( 3, 4). The majority of studies of nontargeted effects use in vitro model systems and it is far from clear whether such mechanisms operate in vivo. However, plasma from X-irradiated patients can cause chromosome damage in cultured lymphocytes and similar activities have been obtained from atomic bomb survivors, Chernobyl liquidators, and from patients with chromosome instability syndromes and inflammatory disorders ( 2, 5). These effects are attributed to indirect acting mechanisms that generate clastogenic factors ( 6). These factors may be produced at sites of irradiation, often represent inflammatory-type tissue responses, and can mediate late tissue injury ( 7). These observations imply that the target for the biological effects of radiation is larger than the directly irradiated cell ( 8). Thus, after exposure in vivo, a case can be made for the tissue microenvironment contributing to delayed cell damage as a consequence of responses that are secondary to the initial radiation-induced injury. Because the responses of the hemopoietic system are major determinants of outcome following therapeutic, occupational, or accidental radiation exposures, we have been interested to determine the contribution of both direct and indirect effects to hemopoietic cell and tissue responses and have conducted studies to compare responses in CBA/Ca and C57BL/6 mice that differentially express nontargeted effects and are also, respectively, susceptible or resistant to radiation-induced acute myeloid leukemia ( 9).In previous studies of the in vivo response to a potentially leukemogenic dose of ionizing radiation, we showed the expected p53 signaling pathway responses in the first few hours after irradiation, albeit with some genotypic differences in the amount and timing of apoptosis; however, at 24 h, a marked genotype-dependent macrophage activation was consistent with indirect mechanisms resulting in a potentially damaging inflammatory-type microenvironment in the CBA/Ca hemopoietic system ( 10, 11). Inflammatory macrophages are a potent source of microenvironmental reactive nitrogen and oxygen species that can damage bystander cells and initiate tumor formation ( 12, 13). Macrophages are important components of the hemopoietic microenvironment that, in addition to their involvement in immunoregulatory and inflammatory processes, control tissue architecture dynamics and cell mobilization by the secretion of cytokines and chemokines and through cell-cell and cell-matrix interactions ( 14). More generally, they are versatile cells that respond to environmental cues and different subpopulations of tissue macrophages show differences in expression of a variety of receptors and regulatory cytokines ( 15, 16). Macrophages are increasingly recognized as contributing to tumorigenesis through the production of stimulatory or inhibitory molecules that affect tumor cell growth, formation of blood vessels, cellular adhesion, and tissue architecture (16). Moreover, tumorigenic agents often act not on the target cell for tumor initiation, but affect stromal cells that make up the microenvironment ( 17– 20). Tumor susceptibility is strongly influenced by genetic composition, and, in model systems, part of the genetic component lies in differences in inflammatory responses ( 21– 24), including differences in the genetics of macrophage function ( 25). Therefore, macrophages are likely to be major determinants of changes in tissue microenvironment that occur after the initial cellular damage responses and result in the potential for ongoing bystander-mediated damage in CBA/Ca but not in C57BL/6 hemopoietic tissues. To further investigate the phenotypes of macrophages in normal and irradiated hemopoietic tissues, we have determined gene expression profiles of C57BL/6 and CBA/Ca bone marrow–derived macrophages before and after irradiation in vitro, conducted confirmatory gene and protein expression studies, and used these in vitro results to guide in vivo experiments. We found that macrophages do not change their phenotypes as a result of direct irradiation but do so in the context of a tissue response: C57BL/6

macrophages have an intrinsic anti-inflammatory M2-like phenotype that is enhanced in irradiated tissue, whereas CBA/Ca macrophages are intrinsically M1-like and express a pro-inflammatory phenotype after irradiation in vivo. Overall, our data are consistent with macrophages having the potential for producing ongoing damage in CBA/Ca but not in C57BL/6 bone marrow and therefore contributing to the differential expression of nontargeted and delayed radiation effects.Previous Section Next Section

Materials and MethodsMacrophage culture and irradiation. CBA/Ca and C57BL/6 mice were bred in-house under conventional conditions. Experiments were approved by local ethical review and followed the guidelines of the Medical Research Council and the Home Office (PPL 60/2841). To produce sufficient mature macrophages for analyses and to reduce random variation in cultures derived from different individuals, femoral bone marrow cells obtained from five individuals were pooled and grown in 10 T25 tissue culture flasks containing 10 mL modified α-Eagle's medium (Invitrogen) supplemented with 25% pretested horse serum and 25% pretested conditioned medium from the L929 cell line as a source of CSF-1. For each experiment, five flasks were exposed to 4 Gy γ-irradiation at a dose rate of 0.5 Gy/min at room temperature using a CIS Bio International 637 cesium irradiator and five were sham irradiated. Cohorts of mice were whole body irradiated with the same source (dose rate 0.45 Gy/min) or were sham irradiated.Microarray analysis. Total RNA was extracted from bone marrow–derived macrophages using TRIzol (Invitrogen Ltd.) and purified with RNeasy reagents (Qiagen Ltd.) according to the methods recommended by the Medical Research Council (MRC) geneservice. 1 After reverse transcription, synthesis of biotinylated cRNA and fragmentation of labeled cRNA, probes were hybridized to MOE-430-2 GeneChips (Affymetrix) at the MRC geneservice (Cambridge, United Kingdom). These arrays contain 45,101 gene probe sets representing 30,759 individual transcripts.Microarray fluorescence signals were normalized and expression values were calculated using dCHIPv1.3 (PM/MM difference model; ref. 26). Low or negative expression values were truncated at a value of 20 (10% of the median). Genes were filtered to remove gene probes that are not changed across conditions (coefficient of variation 0.2 < SE/mean < 1,000), are called “present” in at least one condition, have low variation between replicates [0 < median(SD/mean) < 0.5], and have an expression value >50 in at least one set of replicate samples.High-level analysis in dCHIP included hierarchical clustering by condition and gene to define arrays that are most similar to each other and genes that are similarly regulated. Changes in gene expression were identified as expression values that are at least 2-fold different at the lower 90% confidence interval, have at least a change in absolute expression value of 80, and are called present in at least one of the conditions tested. ANOVA and false-discovery rate assessments were also applied. Statistically significant ontology groupings were recorded as the default level of significance, P < 0.001. GoSurfer2 and the NetAffx Gene Ontology Mining Tool 3 provided additional data and visual representations of Gene Ontology classifications.Normalization and expression values were also calculated using RMA with the Affy packages available from Bioconductor 4 ( 27). Histograms and boxplots of expression values both before and after normalization were inspected; the percentage of probe sets in which the mean signal of the mismatch probes (MM) is greater than the corresponding perfect match (PM) were calculated; and RNA degradation plots were calculated using AffyRNAdeg. Statistical analysis used application of the Benjamini and Hochberg false discovery rate to Welsh t tests (corrected P < 0.05). Similar results were obtained using expression values calculated by dCHIP or RMA, and results are presented only for dCHIP analysis.Reverse transcription-PCR. Reverse transcription-PCR (RT-PCR) was performed in duplicate from bone marrow–derived macrophages prepared independently from those used for microarray analysis. cDNA synthesis used SuperScript III reverse transcriptase with oligo(dT) (Invitrogen). cDNA was diluted 1:5 with sterile water and 1 μL was used for PCR with varying numbers of cycles. All primer pairs amplify across at least one intron to ensure specificity. All reactions contained additional primers to amplify Gapdh as a housekeeping gene to allow normalization of samples for input cDNA.Immunochemical assays. Immunohistochemical identification of antigens was performed on frozen sections of spleen fixed for 10 min in 50:50 methanol/acetone, or on 4-μm sections of formalin-fixed, paraffin-embedded femurs that had been decalcified in EDTA (pH 7.0), taken from C57BL/6 and CBA/Ca mice with or without previous whole-body exposure to 4 Gy γ-irradiation. Antigens were identified with

peroxidase avidin-biotin-complex (Vector Elite, Vector Labs) and 3,3′-diaminobenzidine as chromogen. Polyclonal rabbit serum to murine Nos2 was obtained by immunizing with COOH-terminal peptide (VFSYGAKKGSALEEPKATRL) conjugated to keyhole-limpet hemocyanin with glutaraldehyde (Moravian Biotechnology). Specificity controls for this serum included preimmune serum applied at the same dilution and addition of peptide (1 μg/mL) to diluted serum before use for immunostaining. Both controls showed an absence of staining. Antibodies to CD14, Lcn2, Mfge8, and SR-A (Santa Cruz Biotechnology); goat anti-Aif1 (Abcam); rabbit anti–3-nitrotyrosine (Upstate Biotechnology); rat monoclonal antibodies to Dectin-1 and C1q (Serotec); Marco and CD68 (Hycult BioClone); rabbit monoclonal antibodies to phospho-Stat1 (Tyr701) and phospho-Stat3 (Tyr705; Cell Signalling); and to phospho-Stat5 (Tyr694), signal transducer and activator of transcription 6 (STAT6), and phospho-STAT6 (Tyr641; Abcam) were also used.Macrophage isolation. Macrophages were isolated from the femurs of cohorts of 4 to 10 CBA/Ca or C57BL/6 mice with or without 4 Gy whole-body γ-irradiation 24 h previously. Femurs were gently blown out with air pushed through 23G needles and cell clumps were digested with medium containing liberase (1.67 Wunsch units/mL) and 0.2 mg/mL DNase (Roche) for 30 min at 37°C. Cells were collected into lipopolysaccharide-free PBS containing 2 mmol/L EDTA and 0.5% bovine serum albumin (Invitrogen). Macrophages were positively selected using F4/80 followed by antirat microbeads (Miltenyi Biotec).Arginase activity. We used an adapted method from ref. 28 to increase sensitivity and allow measurements of urea production from primary macrophages. F4/80-positive macrophages (2 × 105) isolated from cohorts of mice were lysed in 50 μL 0.1% Triton X-100 for 10 min on ice and 50 μL 50 mmol/L Tris-HCl (pH 7.4) containing 10 mmol/L MnCl2were added. After enzyme activation at 55°C for 10 min, 25 μL of lysate were added to 25 μL of 0.5 mol/L L-arginine (pH 9.7) and incubated at 37°C for 2 h. The reaction was acidified with 200 μL of H2SO4/H3PO4/H2O (1/3/7 v/v), and 25 μL of 5% α-isonitrosopropiophenone (Sigma) were added and heated to 95°C for 1 h. Urea production was measured at 540 nm. Five thousand cultured macrophages were used and incubated for 1 h at 37°C.Previous Section Next Section

ResultsGenotype-dependent expression profiles and response to irradiation in vitro. Bone marrow–derived macrophages from both genotypes had the typical microscopic appearance of macrophages and expressed the macrophage marker F4/80. Contamination with other cell types was assessed by microarray analysis of lineage-specific transcripts that are highly expressed in B cells (CD19), T cells (CD3), granulocytes (myeloperoxidase), megakaryocytes (Itga2b/CD41), and erythrocytes (glycophorin A; ref. 29). All transcripts were either not expressed or expressed at very low levels, with <10% variation between strains or after irradiation. Thus, contaminating cell populations do not contribute to the expression profiles observed. Affymetrix analysis also showed no significant differences in cell cycle–related gene expression between genotypes, indicating that differences in cell cycle stage do not influence the results.Duplicate cultures from the two genotypes were either sham-irradiated or exposed to 4 Gy γ-irradiation and used for microarray analysis 24 h later, a time when in vivo alterations to macrophage activities are seen in hemopoietic tissues ( 10). Using ANOVA tests with Benjamini and Hochberg false-discovery correction for multiple testing (P < 0.05), no genes were identified as significantly different between irradiated and unirradiated macrophages in either strain, or when data from the two strains were combined. In contrast, 243 probe sets were identified as differentially expressed between genotypes regardless of irradiation status (∼1.5% of the expressed genes; Supplementary Table S1). Using the available Gene Ontology annotations, differentially expressed genes were found in all biological processes and cellular components and in each of the major categories of molecular function. Statistically significant associations were calculated (P< 0.001) for the 136 genes with annotation. Gene products involved in cell adhesion or phosphate transport, or that have lyase activity, function in the immune response or have scavenger receptor activities that were statistically significant (Supplementary Table S2). Expression differences were confirmed by RT-PCR of two independent sets of bone marrow–derived macrophages from the two strains with and without irradiation 6 or 24 h before collection, the 6-h time point representing the early response to radiation and the time at which in vivo macrophage responses are first observed ( 10). These data confirmed that expression levels are determined by the genotype of the macrophages and are not significantly altered by irradiation in vitro ( Fig. 1A ). We also did microarray analysis of macrophages collected 6 h after irradiation to examine earlier radiation-induced responses.

Using ANOVA with false discovery rate, no genes were statistically significantly changed and the Cdkn1a inhibitor of cyclin-dependent kinases that is induced in a p53-dependent manner after genotoxic stress was the only gene induced by at least 2-fold at 90% confidence in both genotypes. Expression values for genes involved in p53-mediated apoptosis were unaffected by radiation at either 6 or 24 h (Supplementary Table S3). These data indicating a lack of transcriptional activation of apoptotic pathways are in keeping with the radioresistant nature of bone marrow macrophages (cell counts showed >90% survival 24 h after irradiation).

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Figure 1.

RT-PCR and immunohistochemical verification of Affymetrix data. A, RT-PCRs were performed on cDNAs prepared from two independent sets of cultured macrophages from the two genotypes. Cells were either sham irradiated or exposed to 4 Gy γ-irradiation and collected 6 or 24 h later. B, immunohistochemistry of spleen frozen sections confirms genotype-dependent expression levels of the selected proteins.Genotype-dependent expression of genes involved in immune response, cellular adhesion, and oxidative stress. Gene Ontology analysis showed significant overrepresentations of genes that are involved in immune/defense responses or in regulating cell adhesion. Sixteen genes whose products are involved in cell motility or adhesion and 16 cytokines, genes regulated by cytokines, or cytokine receptors were identified as differentially expressed between the two strains ( Fig. 2 ); expression differences were confirmed for selected genes. The expression of these genes was not altered by irradiation  in vitro ( Fig. 1A). Ten genes involved in the production, removal, or response to reactive oxygen and/or nitrogen species were expressed in a genotype-dependent manner and were unchanged following irradiation in vitro ( Figs. 1A and 2).

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Graphical representations of differentially expressed mRNAs identified by expression profiling.Black columns, expression levels in C57BL/6 macrophages; white columns, levels in CBA/Ca; bars,SE. A, five scavenger receptors are expressed at higher levels in CBA/Ca mice than in C57BL/6. B,four opsonins for apoptotic cells are more highly expressed in C57BL/6 than CBA/Ca. C,examples of differentially expressed cytokines. Four are more highly expressed in C57BL/6 and four are more highly expressed in CBA/Ca.Genotype-dependent expression of scavenger receptors and opsonins. Genes with scavenger receptor activity were overrepresented (P = 0.000036). Retrieval of expression values for other scavenger receptors revealed that CD14 was 2.72-fold altered between strains (90% confidence interval, 1.8-4.3) and myristolated alanine-rich C kinase substrate–like protein was 4.6-fold altered (range 3.1-6.6), although not included in this Gene Ontology classification. Five of the six differentially expressed scavenger receptors were present at a higher level in CBA/Ca mice ( Fig. 2). Elevated expression of CD14 and SR-A by CBA/Ca macrophages was confirmed by RT-PCR and immunohistochemical staining of frozen sections of spleen ( Fig. 1).Although there is no Gene Ontology group for this class of genes, four differentially expressed genes that are expressed at higher levels in C57BL/6 macrophages encode opsonins that enable macrophage recognition and engulfment of apoptotic cells ( Fig. 2). These results were confirmed for Mfge8 and C1qb by RT-PCR and immunohistochemistry of frozen sections of spleen ( Fig. 1). Of other apoptotic cell ligands, macrophages from both strains express high levels of Gas6 and moderate levels of serum amyloid-P ( Fig. 2).Genotype-dependent M1/M2 phenotype. Arginase 1 is expressed at 40-fold higher levels in C57BL/6 than CBA/Ca macrophages in vitro and is a reliable marker of M2 macrophage polarization in murine macrophages ( 30). Other markers of the M2 phenotype, Dectin1 (CLECSF12), IL-1Ra, and chitinase-like proteins (Ym1/2 and Chi3l3), are also elevated in C57BL/6 compared with CBA/Ca. Levels of Dectin1 protein were measured in vivo by immunostaining ( Fig. 1B) and on macrophages in vitro by fluorescence-activated cell sorting (FACS) analysis (not shown) and were higher in C57BL/6 than CBA/Ca in both assays. In contrast, CD14 and Marco show higher expression in CBA/Ca and are associated with innate activation phenotypes. Antibodies to Marco were insufficiently sensitive to identify this receptor in bone marrow macrophages by FACS, but CD14 is more highly expressed in CBA/Ca than C57BL/6 by immunohistochemistry ( Fig. 1B). These microarray and protein expression data indicate that macrophages from C57BL/6 have phenotypic characteristics of M2 alternatively activated macrophages, whereas the same cells derived from CBA/Ca have a phenotype in common with classically activated M1 macrophages. Irradiation of macrophages in vitrodid not alter the expression of these phenotypic markers (Supplementary Table S1; Fig. 1A).Macrophage responses to radiation in vivo. Although cultured macrophages do not change their gene expression profiles after irradiation in vitro, they possess intrinsic genotype-specific differences in their M1/M2 phenotypes. Previous investigations had shown macrophage activation 24 h postirradiation in vivo; therefore, to examine any alterations to macrophage phenotypes and inflammatory responses that are elicited as a consequence of irradiation in vivo, we measured Arginase 1 activities in purified macrophages freshly isolated from bone marrows of control and irradiated mice ( Fig. 3A). In keeping with the microarray and RT-PCR data, C57BL/6 cells from control mice show higher arginase activity than CBA/Ca (P = 0.01687). Macrophages isolated from bone marrow of C57BL/6 mice that were irradiated 24 h previously show 3.2-fold higher arginase activity compared with cells isolated at 24 h from sham-irradiated mice (P = 0.0014, control versus 4Gy) but there is no increase in arginase activity in macrophages isolated from irradiated compared with nonirradiated CBA/Ca bone marrow (P = 0.11628; Fig. 3A). This leads to an increase in the difference in arginase activity between irradiated CBA/Ca and C57BL/6 (2.6-fold higher in macrophages from unirradiated C57BL/6 mice compared with unirradiated CBA/Ca, increasing to an 8.4-fold higher activity in irradiated C57BL/6 compared with irradiated CBA/Ca).

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Figure 3.

Changes to M1 and M2 phenotypes of CBA/Ca and C57BL/6 mice after irradiation in vivo. A, arginase activity in macrophages purified from bone marrows of CBA/Ca or C57BL/6 24 h after whole-body 4 Gy γ-irradiation or sham irradiation. Data are shown relative to the level of sham-irradiated CBA/Ca mice. Macrophages from C57BL/6 have increased arginase activity after irradiation, whereas CBA/Ca do not. B to D, immunoperoxidase staining with the indicated antibodies in sections of bone marrow from sham-irradiated (0 h) mice or those irradiated 24 h previously (24 h). Positive staining is seen as a brown product and cell nuclei are counterstained blue.Nitric oxide (NO) is an effector of the innate immune system and its production is a characteristic feature of M1 macrophages. The normal method for measuring NO is the Griess reaction for nitrite but we found this method insufficiently sensitive to measure nitrite concentrations in primary bone marrow. Therefore, we assessed differences in NOS2 (the enzyme that synthesizes NO) by immunochemistry and found higher expression of this M1-associated enzyme in CBA/Ca than in C57BL/6. After whole-body irradiation, NOS2 is strongly repressed in C57BL/6 but not in CBA/Ca bone marrow ( Fig. 3B). Immunohistochemistry also showed preferential activation of STAT1 (a signal transducer and activator of transcription characteristic of pro-inflammatory M1 responses) in irradiated CBA/Ca bone marrow, whereas STAT6 (characteristic of M2 responses) showed increased activation in C57BL/6 but not in CBA/Ca ( Fig. 3C   and  D ).Previous Section Next Section

DiscussionBecause cultured macrophages retain the essential properties of primary cells ( 14), we have been able to use whole-genome microarrays to define the transcriptome of bone marrow–derived macrophages from two inbred strains of mice previously shown to be differentially susceptible to ongoing bone marrow damage and radiation-induced acute myelogenous leukemia and also to exhibit differences in macrophage activation 6 to 24 h after whole-body radiation ( 10). The microarray data of in vitro bone marrow–derived macrophages reveal widely different intrinsic expression profiles that reflect genetically determined differences and guided subsequent phenotypic studies in vivo. Notably, there are limited transcriptional responses of in vitro bone marrow–derived macrophages to direct irradiation. These data contrast with many previous studies of more radiosensitive primary cells, where even low doses of ionizing radiation exposure induce p53-mediated responses associated with induction of genes involved in growth arrest and/or apoptosis (31). However, radiation responses, including p53 stabilization, transcriptional activation, and apoptosis are dependent on cell and tissue type (reviewed in ref. 32). Importantly, even high-dose radiation does not induce p53-regulated proapoptotic genes in bone marrow–derived macrophages, consistent with their relatively radioresistant nature ( 33).The genotypic differences classify CBA/Ca mice as an M1-like strain and C57BL/6 as an M2-like strain. M1 and M2 functional subsets display pro-inflammatory versus anti-inflammatory and reparative patterns of function ( 15, 16) and after in vivo, but not after in vitro irradiation, C57BL/6 macrophages show

enhanced M2 activities and those from CBA/Ca mice enhanced M1 activities. Changes in macrophage activity after irradiation in a tissue context in vivo but not as isolated cells in vitro is consistent with a lack of a direct radiation effect and in vivo responses being due to some kind of signal(s) derived secondarily from other cells in response to radiation exposure. Previous studies had shown that macrophage activation in irradiated hemopoietic tissues was associated with phagocytic clearance of apoptotic cells, indicating that apoptotic cell engulfment is one of these signals ( 10). A plausible explanation for genotype-dependent differences in macrophage activation following engulfment of radiation-induced apoptotic cells is the expression levels of certain scavenger receptors and opsonins. The strain-dependent expression of these molecules implies that the engulfing cells in the two genotypes use different pathways for recognition and clearance. Although little is known about the signaling pathways activated or repressed by distinct receptors with or without particular opsonins, differential responses have been described ( 30).A major finding of our study is the genotypic differences that classify macrophages obtained from bone marrow of CBA/Ca or C57BL/6 mice as, respectively, M1-like or M2-like. In M1 macrophages, metabolism of arginine through NOS2 produces citrulline and NO, a powerful inflammatory mediator. Conversely, in M2 macrophages, metabolism of arginine through Arginase 1 reduces NOS2 activity and simultaneously produces polyamines and proline, which act as antioxidants and stimulate tissue regeneration ( 16,30). Thus, the balance between NOS2 versus arginase activity is a key determinant of the pro-inflammatory M1 phenotype or the anti-inflammatory and protective environment associated with M2 macrophages. Hemopoietic tissues of CBA/Ca have more NOS2 protein than C57BL/6, whereas arginase activity is higher in C57BL/6, consistent with CBA/Ca macrophages having intrinsic M1 properties and the same cells from C57BL/6 exhibiting an M2 phenotype. These observations may be regarded as surprising, because C57BL/6 is considered a prototypic Th1/M1 strain ( 34, 35). However, those classifications are based on the responses of C57BL/6 macrophages to classic pro-inflammatory stimuli such as lipopolysaccharide and IFN, and in fact those same studies show that C57BL/6 macrophages in vitro in the absence of stimuli exhibit M2 characteristics ( 34). Similarly, analysis of microarray data provided in a study of the transcriptional responses of macrophages to lipopolysaccharide ( 35) reveals M2 features in unstimulated C57BL/6 macrophages. Importantly, our data obtained by studying bone marrow macrophages in vivo show that genetically determined default M1/M2 phenotypes are independent of Th1/Th2 characteristics, because C57BL/6 are intrinsically Th1 yet their bone marrow macrophages are intrinsically M2 in vivo. Similarly, although T-cell activities are regulated through interactions with macrophages and other antigen-presenting cells, primary Th1/Th2 phenotypes are determined by the genotype of the T cells and not the antigen-presenting cell ( 36). Therefore, although pro-inflammatory/anti-inflammatory M1/M2 and the corresponding Th1/Th2 phenotypes are regulated through interdependent cytokine signaling pathways ( 34), each phenotype is also independently determined by genotypic influences. Thus, macrophage phenotypes are generated by complex interactions between intrinsic characteristics and responses to external stimuli that differ according to different genetic components.Most notably for our studies relating to delayed and indirect effects of irradiation, M2 phenotypes associate with reduced inflammation and improved tissue repair after injury, whereas the M1 phenotype associates with pro-inflammatory conditions ( 15, 16, 30). Although these characteristics are not altered by direct irradiation of bone marrow–derived macrophages in vitro, there are genotype-dependent alterations in vivo, where CBA/Ca bone marrow macrophages maintain NOS2 levels and arginase activity, whereas C57BL/6 lose NOS2 and increase arginase activity after irradiation. These radiation-induced changes therefore enhance the differences between genotypes, with C57BL/6 becoming more M2-like than previously whereas CBA/Ca retain an M1 phenotype within the bone marrow and do not induce M2 characteristics. We also show phosphorylation of STAT1 but not STAT6 in hemopoietic tissues of CBA/Ca mice in response to radiation and STAT6 but not STAT1 in C57BL/6 mice. STAT1 activation is characteristic of pro-inflammatory M1 responses, whereas STAT6 activation is characteristic of M2 responses ( 16, 30). These observations provide further evidence for tissue-dependent responses following irradiation and indicate genotype-dependent differences in the overall production of potentially damaging (CBA/Ca) and protective (C57BL/6) hemopoietic microenvironmental responses. These responses can be considered in terms of “danger” signals that mobilize the innate and acquired immune system to maintain the integrity of the body following exposure to a variety of pathologic, chemical, or physical agents and have been suggested to act to mediate local tissue recovery or mediate damaging bystander effects (reviewed in ref. 37).

Taken together, our data show altered macrophage activity after irradiation in vivo but notin vitro. The genotype-specific expression profiles indicate intrinsic differences in bone marrow macrophage activities, including M1/M2 phenotypes that govern their regulatory functions in immunity and hemopoiesis. Although macrophages do not respond to direct irradiation, their activities are altered in vivo after irradiation, with CBA/Ca hemopoietic tissues showing an M1-like phenotype that associates with potentially damaging inflammatory-type responses, compared with the induction of an anti-inflammatory phenotype and tissue reparative response seen in C57BL/6 tissues. These complex differences in macrophage function are likely to contribute to the medium and long-term outcomes of radiation exposure in the hemopoietic system by their involvement in the delayed and nontargeted effects exhibited in vivo ( 38– 40). Importantly, a genotype-dependent, radiation-induced genomic instability phenotype in vivo need not necessarily be a reflection of intrinsically unstable cells but the responses to ongoing production of damage as a consequence of a persisting inflammatory-type response secondary to the initial radiation-induced injury.Previous Section Next Section

AcknowledgmentsGrant support: Leukaemia Research Fund specialist programme grant 0214.The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Tom Freeman and the LRF microarray service for advice on gene expression profiling.

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Footnotes Note: Supplementary data for this article are available at Cancer Research Online

(http://cancerres.aacrjournals.org/). MIAME-compliant microarray data are available from Array Express (http://www.ebi.ac.uk/arrayexpress),

accession number E-MEXP-1291. ↵1 http://geneservice.co.uk ↵2 http://biosun1.harvard.edu.complab/gosurfer/ ↵3 http://www.affymetrix.com/analysis/netaffx/ ↵4 http://www.bioconductor.org/ Received August 8, 2007. Revision received October 25, 2007. Accepted October 25, 2007.

©2008 American Association for Cancer Research.Previous Section

 

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Inflammatory-type responses after exposure to ionizing radiation in vivo: a mechanism for radiation-induced bystander effects?

Sally A Lorimore, Philip J Coates, Gillian E Scobie, Gordon Milne and Eric G Wright

Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, UK

Correspondence to: P J Coates, Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, UK. E-mail: p.j.coates@dundee.ac.uk

Abstract

Haemopoietic tissues exposed to ionizing radiation are shown to exhibit increased macrophage activation, defined by ultrastructural characteristics and increased lysosomal and nitric oxide synthase enzyme activities. Macrophage activation post-irradiation was also associated with enhanced respiratory burst activities and an unexpected neutrophil infiltration. Examination of p53-null mice demonstrated that macrophage activation and neutrophil infiltration were not direct effects of irradiation, but were a consequence of the recognition and clearance of radiation-induced apoptotic cells. Increased phagocytic cell activity was maintained after apoptotic bodies had been removed. These findings demonstrate that, contrary to expectation, recognition and clearance of apoptotic cells after exposure to radiation produces both a persistent macrophage activation and an inflammatory-type response. We also demonstrate a complexity of macrophage activation following radiation that is genotype dependent, indicating that the in vivo macrophage responses to radiation damage are genetically modified processes. These short-term responses of macrophages to radiation-induced apoptosis and their genetic modification are likely to be important determinants of the longer-term consequences of radiation exposure. Furthermore, in addition to any effects attributable to immediate radiation-induced damage, our findings provide a mechanism for the production of damage via a 'bystander' effect which may contribute to radiation-induced genomic instability and leukaemogenesis. Oncogene(2001) 20, 70857095.

Keywords

ionizing radiation; macrophage; inflammation; genetics; bystander

Introduction

Until recently, it has been generally accepted that the genotoxic and carcinogenic consequences of radiation exposure are due to the damage inflicted directly by the radiation, producing irreversible changes during DNA replication, or during the processing of the DNA damage by enzymatic repair processes. However, there is now considerable evidence that cells that were not themselves irradiated but were the progeny of cells exposed to ionizing radiation many cell divisions previously may express delayed gene mutations and a variety of chromosomal aberrations. These effects are generally referred to as radiation-induced genomic instability. Although the mechanism for these delayed effects of ionizing radiation is unclear, excessive production of reactive oxygen species has been implicated (reviewed by Wright, 1998;Iyer and Lehnert, 2000; Little, 2000). The paradigm of genetic alterations being restricted to direct DNA damage following radiation exposure has also been challenged by studies showing that nuclear damage can be observed after targeted cytoplasmic irradiation using the newly developed microbeam irradiators (Prise et al., 1998; Wu et al., 1999). Chromosomal damage has also been observed in cells that were not themselves irradiated but were in the neighbourhood of irradiated cells (Prise et al., 1998; Zhou et al., 2000) and cytotoxic effects can be observed in the medium of irradiated cells when the cell-free medium is subsequently transferred to non-irradiated cells (Mothersill and Seymour, 1997; Lehnert and Goodwin, 1997). Finally, irradiated cells secrete growth-inhibitory molecules both in vitro and in vivoin a p53-dependent manner (Komarova et al., 1998). These observations have given rise to the notion that ionizing radiation can induce so-called 'bystander effects' where the irradiated cells transfer a signal to non-irradiated cells. Again, the mechanism for such bystander-mediated effects are unclear, although reactive oxygen species have been implicated (Narayanan et al., 1997; Wu et al., 1999; Lyng et al., 2000). We have recently established a link between these two indirect radiation effects by demonstrating that genomic instability in haemopoietic cells can be induced by unexpected interactions between irradiated and non-irradiated cells i.e. by a bystander mechanism (Lorimore et al., 1998; Watson et al., 2000). These untargeted effects of radiation clearly pose a major challenge to current views of the mechanisms of radiation-induced DNA damage and radiation-induced malignancy.

Malignancies, and in particular myeloid leukaemias, are a major health consequence of exposure to ionizing radiation and interactions between the haemopoietic microenvironment and the target stem cells as well as

the damage to the haemopoietic stem cells themselves have been implicated by both clinical and experimental observations. Thus, whilst uncommon, patients treated for haemopoietic disorders by bone marrow transplantation following preparative whole body irradiation may relapse with disease in the donor-derived cells (reviewed in McCann et al., 1993; Giralt and Champlin, 1994). Irradiation has also been shown to induce leukaemic transformation of non-irradiated stem cells transplanted into syngeneic mice (Duhrsen and Metcalf, 1990). These findings may reflect the altered characteristics of the stem cell microenvironment after irradiation, since irradiated haemopoietic stromal cells release mutagenic reactive oxygen species, produce different sets of adhesion molecules and growth factors, and alter the overall growth and phenotypic characteristics of co-cultured non-irradiated stem cells (Greenberger et al., 1996).

As with many responses to radiation, the development of radiogenic leukaemia is strongly influenced by genetic factors (Wright, 1998). In mouse model systems, CBA/Ca mice characteristically develop myeloid leukaemia after exposure to ionizing radiation and their haemopoietic cells are also susceptible to radiation-induced chromosomal instability. In contrast, C57BL/6 mice are resistant to both the development of radiation-induced myeloid leukaemia and to radiation-induced chromosomal instability in haemopoietic cells. Because interactions between tissue stroma and stem cells appear to be important in determining the overall consequences of radiation, we are investigating both the short- and long-term effects of radiation on haemopoietic tissues. Here we report unexpected macrophage activation combined with neutrophil infiltration following whole body irradiation, effects that persist long after the initial radiation insult. We also show that the degree of these responses is genotype dependent and their inflammatory nature suggests the potential for ongoing damage after the initial radiation insult.

Results

Macrophage ultrastructure following in vivo irradiation

Electron microscopical studies of macrophages before and 24 h after irradiation identified both increased cell membrane ruffling and increased lysosome number and size in the irradiated tissues, the classic ultrastructural features of macrophage activation (Adams and Hamilton, 1992) (Figure 1). Many of the lysosomal components of the cells were seen to be secondary and tertiary lysosomes, indicative of degradation

of phagocytosed material.

Lysosomal and respiratory burst activity in macrophages following in vivo irradiation

In view of the morphological data suggesting macrophage activation post-irradiation, we investigated whether there was any evidence for increased enzyme activity in these cells. Quantitative spectrophotometric studies of lysosomal acid  -galactosidase activity in the spleens and bone marrow of three individual mice showed mean increases of fourfold (0.11 to 0.45) in the spleen and of threefold (0.13 to 0.4) in the bone marrow 24 h after irradiation with 4 Gy  -rays. At this dose, there was a mean reduction in tissue cellularity of 82 and 74% in the spleen and bone marrow, respectively. Histochemical staining showed that the increase in acid  -galactosidase activity localized to cells with the distribution and morphological characteristics of acid phosphatase-positive macrophages (Figure 2ad).

To investigate the time at which increased lysosomal enzyme activity is first seen, we measured enzyme activities spectrophotometrically in spleen cell suspensions from three individual mice at various times after 4Gy irradiation. Mean levels of enzyme activity were similar to control unirradiated levels at times up to and including 4 h post-irradiation, but were significantly raised by 6 h. Enzyme levels continued to increase, reaching more than four times the control level at 24 h post-irradiation (Figure 3a). Interestingly, in control mice positive cells were seen at the margins of the red and white pulp and these represented only a small percentage of the total macrophage content as determined by acid-phosphatase staining. At 6 and 9 h post-irradiation, the proportion of macrophages staining positively for acid  -galactosidase had increased, and the macrophages were seen to be increased in size by acid phosphatase staining (Figure 2a,b). Unlike control spleen, acid  -galactosidase staining was observed in both red and white pulp 6 to 9 h post-irradiation (Figure 2c,d) and acid  -galactosidase-positive macrophages in the white pulp contained multiple pyknotic nuclei, indicating that these macrophages had recently ingested apoptotic cells (Figure 2g). By 24 h the staining was again predominant in red pulp areas. Similarly, in unirradiated bone marrow, only a small proportion of the acid phosphatase-positive macrophages showed acid  -galactosidase activity, but after irradiation most macrophages became strongly positive for acid  -galactosidase (data not shown).

We also exposed triplicate mice to differing doses of  -irradiation and measured levels of acid  -galactosidase in spleens 24 h later. There was no significant increase above the control enzyme activity after exposure

to 0.25 or 0.5 Gy (36 and 48% mean reduction in tissue cellularity respectively). Significantly increased activity was evident at doses equal to or greater than 1 Gy and for the relatively small decrease in tissue cellularity from 65% at 1 Gy to 73% at 2 Gy, a 2.5-fold increase in enzyme activity was measured (Figure 3b).

Additional evidence for functional activation of macrophages in both the spleen and bone marrow after irradiation was provided by a standard luminescence assay for the production of superoxide (characteristic of the respiratory burst that occurs in phagocytic cells) in the absence of exogenous stimulation. Luminescence was measured from three mice in duplicate over a period of 1 h in bone marrow or spleen suspensions. Values represent the amount of light emitted at each time point measured and are given in arbitrary units (a.u.). Without any radiation exposure, phagocytes from C57BL/6 mice show a low level of constitutive respiratory burst activity that is increased from 125 to 250 a.u. 24 h after 4 Gy  -irradiation. After stimulation with PMA cells from unirradiated control mice showed a greater respiratory burst when compared to cells removed from mice exposed to 4 Gy  -irradiation 24 h previously (910 vs 740 a.u. respectively).

Increased nitrotyrosine following in vivo irradiation

Nitric oxide (NO) production is a characteristic feature of activated macrophages, which induce nitric oxide synthase 2 (NOS2, or iNOS) as part of the inflammatory process. Nitrotyrosine is a product of the reaction between reactive nitrogen species and peptides or proteins, and formation of nitrotyrosine in vivo is associated with the expression of NOS2 (Wink et al., 1998). By immunohistochemistry we found nitrotyrosine to be present in a small percentage of cells in unirradiated spleen with the distribution and cellular morphology of macrophages. After irradiation, both the number of positive cells and the intensity of the reaction product were greatly increased (Figure 2h,i). These data indicate that exposure to ionizing radiation induces the expression of NOS2 in tissue macrophages in vivo, providing further evidence for radiation-induced macrophage activation.

Radiation-induced macrophage activation is associated with neutrophil infiltration

Further investigations of the tissue response to apoptosis revealed an unexpected accumulation of neutrophils in the red pulp of irradiated spleens. Neutrophils were identified by the characteristic morphology of these cells and neutrophil infiltration was confirmed by immunostaining for myeloperoxidase (Figure 4af). Neutrophil accumulation was first

seen 6 h after radiation exposure, and the number of neutrophils increased up to 24 h post 4 Gy  -irradiation. Neutrophils were seen both within the red pulp and also at the margins of the blood vessels in the red pulp. This latter feature of neutrophil margination is a classic sign of an acute inflammatory response, during which neutrophils are recruited from the bloodstream to the affected site due to the local production of chemoattractant molecules. Consequently, these observations clearly indicate an ongoing neutrophil infiltration into the spleen following irradiation, the timing of which coincides with the increased macrophage activity we have also demonstrated.

Increased macrophage activation following irradiation is associated with p53-dependent apoptosis

The data above indicate that increased macrophage activity is a consequence of radiation exposure. The observations that macrophages which contained multiple intracellular apoptotic bodies were both  -galactosidase-positive and contained detectable amounts of nitrotyrosine, together with the finding that enzyme activities begin to rise concurrently with the peak number of apoptotic cells suggested that there might be a link between macrophage activation and apoptosis, rather than activation being a direct effect of radiation exposure. To test whether macrophage activation is a direct effect of irradiation exposure, we measured enzyme activities in the spleens of three individual p53-/- mice, where radiation-induced apoptosis is absent due to the lack of p53 function. In these mice, acid  -galactosidase was not increased above control 24 h after 4 Gy irradiation, although a mean 4.5-fold increase was seen in three identically treated p53+/+ mice of the same genetic background (Figure 4g). Similarly, immunohistochemical examination of nitrotyrosine in spleens from p53-null mice showed no detectable increase in nitrotyrosine immunoreactivity after radiation.

These data are compatible with our hypothesis that macrophage activation following radiation exposure is due to radiation-induced apoptosis, but might also indicate a direct p53-mediated pathway of macrophage activation. To test whether the increased macrophage activity is dependent on p53 function, we examined acid  -galactosidase activity in a classical situation of physiological apoptosis during embryonic tissueremodelling. Previous studies have shown that apoptosis in the developing footplate of the mouse can be easily demonstrated in vivo by acridine orange staining, and the apoptotic cells are contained within F4/80-positive macrophages (Wood et al., 2000). The forelimbs of unirradiated wild type and p53-null E13.5 mouse embryos each demonstrated a clear temporal and spatial association between high apoptosis (acridine orange-positive cells) and intense

acid  -galactosidase staining in individual cells within the interdigital zones of the footplate (Figure 4h,i). In contrast, before (E12.5) or after (E14.5) the extensive apoptotic remodelling phase of footplate development, both acridine orange and acid  -galactosidase showed minimal staining in both p53-null and wild type animals. These footplate data clearly demonstrate that increased macrophage acid  -galactosidase activity occurs in the presence of high numbers of apoptotic cells and is independent of p53 function.

Taken together with the nitrotyrosine data, these observations indicate that increased macrophage activity is induced by apoptosis, rather than being a direct effect of radiation. Additionally, because neutrophil infiltration was not apparent in p53-/- irradiated spleens, inflammation after irradiation is also not a direct effect of radiation exposure (Figure 4e).

Macrophage activation is a genetically modified process

We wanted to determine whether there was any evidence for genetic modification of the responses we had found, because we have previously reported genetic differences in radiation-induced chromosomal instability in two inbred strains of mice, C57BL/6 and CBA/Ca (Watson et al., 1997). The results described above had all been obtained using the C57BL/6 strain. When measured using the quantitative spectrophotometric assay, mean levels of acid  -galactosidase activity were higher in spleen and bone marrow from C57BL/6 animals than in CBA/Ca mice, the levels in spleen cell suspensions being induced fourfold for C57BL/6 compared to 2.5-fold for CBA/Ca 24 h after whole body irradiation with 4Gy  -irradiation. One possible explanation for these results was that there are higher percentages of macrophages in the C57BL/6 strain, thereby giving higher average levels of lysosomal enzyme activity. To investigate this, we performed acid phosphatase staining together with immunohistochemistry and FACS analysis using a monoclonal antibody against the mouse macrophage marker, F4/80. By acid phosphatase staining and immunohistochemistry of paraffin sections, the distribution and numbers of macrophages were indistinguishable between the two genotypes. By quantitative FACS analysis of cell suspensions, the percentages of F4/80-positive macrophages were also seen to be similar in the two genotypes, with or without prior exposure to radiation (6.2% for both CBA/Ca and C57BL/6 without irradiation; 1.6% for C57BL/6 and 1.2% for CBA/Ca 24 h after 4 Gy  -irradiation). Because F4/80 measures monocytic precursors as well as resident macrophages, we quantitated macrophage numbers in bone marrow morphologically by electron microscopy, which showed that after irradiation the two strains

contained similar numbers of macrophages (260.4 per grid unit area for C57BL/6 versus 22.50.87 per grid unit area for CBA/Ca). Taken together, these data show that the relatively higher levels of acid  -galactosidase in lysates of spleen and bone marrow cell suspensions of C57BL/6 mice are not simply due to the presence of increased numbers of macrophages in these samples, but must instead be due to an increased level of enzyme activity per cell. Histochemical staining for acid  -galactosidase on frozen sections of spleen or bone marrow taken from C57BL/6 and CBA/Ca mice showed only a small proportion of macrophages positive in unirradiated tissues with more stained cells present in C57BL/6 than in CBA/Ca mice. At 9 h post-irradiation with 4 Gy, the number of positively stained cells and the intensity of staining was increased in both strains and was greater in C57BL/6 mice than in CBA/Ca (Figure 2cf). This genotypic difference in staining was maintained up to 24 h post-irradiation.

We have previously demonstrated genetic differences in PMA-induced superoxide generation between unirradiated C57BL/6 and CBA/Ca bone marrow cells (Watson et al., 1997). We have now extended that study to assess superoxide generation in spleen and bone marrow cells with and without radiation and in the presence or absence of exogenous stimulation with PMA. Unlike the results in C57BL/6 mice, in the absence of exogenous stimulation with PMA phagocytic cells from unirradiated CBA/Ca bone marrow and spleen had no detectable superoxide activity. Furthermore, even after radiation (which induces a further increase in the respiratory burst of C57BL/6 mice) there was no detectable superoxide generation in CBA/Ca animals. In contrast to the undetectable endogenous respiratory burst activity of CBA/Ca mice, exogenous stimulation of the cells with PMA led to a greater respiratory burst than that seen in C57BL/6 mice, in both normal and irradiated CBA/Ca tissues (Figure 5).

We also studied the in vivo production of NO and the accumulation of neutrophils in the two genotypes by immunohistochemical staining for nitrotyrosine or myeloperoxidase in sections of spleen. We noted that whilst both strains showed an induction of nitrotyrosine-modified proteins after radiation exposure, the staining was less intense in irradiated CBA/Ca animals compared to C57BL/6 mice, although the distribution of staining was similar (Figure 2hk). Neutrophil accumulation in the two strains was quantitated by counting the number of myeloperoxidase-positive polymorphs in the red pulp with or without exposure to 4 Gy  -irradiation 24 h previously. Again, neutrophil accumulation was seen to occur in both strains, and there was no significant difference between the strains (29.21.2 and 32.01.9, P=0.1098, increasing to 82.13.6 and 88.44.1, P=0.0786;

for C57BL/6 and CBA/Ca respectively).

These data indicate complex genotypic differences in radiation-induced macrophage activation and neutrophil infiltration in these two inbred strains of mice. Since we have shown that induction of macrophage activities is correlated with radiation-induced apoptosis, and it is known that the level of apoptosis after radiation exposure varies between different mouse strains (Nomura et al., 1992), one possible explanation for these results would be that there are higher levels of apoptosis in C57BL/6 mice than in CBA/Ca mice, leading to higher enzyme activities in the former. To investigate this possibility, we counted apoptotic cells at various times after irradiation in the spleens of duplicate animals. In both strains, radiation-induced apoptotic cells are first seen 2 h after radiation, their numbers peak at 6 h and have begun to decrease by 9 h post-irradiation, reaching near to control values by 24 h. Quantitative analysis showed there to be an approximately 1.5-fold higher level of apoptosis in C57BL/6 mice after radiation (C57BL/6; 91142 and CBA/Ca; 61940 apoptotic cells per high power field, 6 h after 4 Gy). This resulted in a greater reduction in tissue cellularity at 24 h for C57BL/6 than for CBA/Ca; 82 and 72% respectively.

These data might suggest that the genotypic differences in radiation-induced macrophage activation we had observed were due simply to differing levels of radiation-induced apoptosis. To investigate the relationship between cell death and macrophage activation we turned to the DBA/2 strain, which is known to be particularly resistant to radiation-induced apoptosis (Nomura et al., 1992). Spectrophotometric measurements in spleen lysates from these mice 24 h after 4 Gy irradiation showed that the levels of acid  -galactosidase were approximately twofold higher than the corresponding CBA/Ca level. In contrast, measurements of apoptosis showed twofold lower levels of cell death in DBA/2 mice. Therefore, genotypic differences in the induction of macrophage lysosomal enzyme activity following irradiation are not directly related to genotypic differences in apoptosis.

Discussion

We have shown that macrophage activation and inflammatory-type responses in the haemopoietic system are early consequences of exposure to ionizing radiation in vivo. This conclusion is based on the finding of tissue neutrophil infiltration, the ultrastructural characteristics of tissue macrophages, their increased lysosomal and NOS enzyme activities and the enhanced respiratory burst activities indicating increased phagocytic cell activity. Dose response and time course

studies, together with the absence of these responses in irradiated p53-null mice, indicated that the mechanism for this response is the recognition and phagocytosis of radiation-induced apoptotic cells.

Our electron microscopic investigations of unirradiated haemopoietic tissues demonstrated a bimodal distribution of macrophages in which cells had few or many secondary lysosomes, but all cells showed minimal membrane ruffling. Those cells with many secondary lysosomes may reflect the ongoing phagocytic clearance of apoptotic cells as a part of normal cell turnover in the rapidly proliferating haemopoietic system. Indeed, phagocytosis of apoptotic cells is a feature of normal steady-state haemopoiesis (Necas et al., 1998), with physiological levels of apoptosis quantitatively similar to those associated with apoptosis after exposure to the doses of radiation used in our studies. However, steady-state apoptosis in vivo is inconspicuous and difficult to measure because of the extremely rapid phagocytosis of the dead cells and their degradation beyond histological identification. Twenty-four hours after irradiation all macrophages had larger and increased numbers of secondary lysosomes together with very extensive membrane ruffling, reflecting the increased activity of tissue phagocytes and demonstrating that essentially all macrophages in haemopoietic tissues are involved in the recognition and phagocytosis of radiation-induced apoptotic cells. The distributions of nitrotyrosine and acid  -galactosidase positive cells confirms the presence of macrophage heterogeneity in control tissues and shows that following radiation exposure the majority of these cells induce these enzyme activities. The morphological identification of macrophage activation and increased macrophage enzyme activities were also associated with increased phagocytic activity (measured by respiratory burst assays), and with neutrophil infiltration.

The time and dose response experiments and irradiation of p53-null mice indicate that increased macrophage activities and neutrophil infiltration are a function of the apoptotic process, rather than being a direct consequence of radiation exposure. However, although there are significant reductions in tissue cellularity at doses up to 0.5 Gy, lysosomal enzyme activities were within the control range and significant increases in acid  -galactosidase activity were seen only at doses of 1 Gy and above. Furthermore, the greatest enzyme activity was not coincident with the initial increase in apoptosis (first seen at 2 h post-irradiation) and increased up to 24 h, during which time apoptosis had declined almost to basal levels. Thus, acid  -galactosidase activity is not simply related to either radiation dose or resultant cell loss. Neutrophil infiltration of the splenic red pulp was also observed at 6 h (coincident with the first increases in macrophage enzyme activity) and

increased up to 24 h post-irradiation. As the resolution of inflammation is known to occur by apoptosis and rapid phagocytosis of neutrophils (Giles et al., 2000), the continued increase in enzyme activity in the red pulp might be due to ongoing phagocytosis of infiltrating apoptotic neutrophils. However, electron micrographs of tissues at 24 h post-irradiation showed only a few macrophages contained identifiable apoptotic bodies whereas many had secondary lysosomes containing degraded cellular material; this is not consistent with the phagocytosis of recently produced apoptotic cells, but the data are compatible with phagocytosis of apoptotic cells produced by the initial radiation damage resulting in persistent macrophage activation and neutrophil accumulation in the red pulp, a conclusion supported by a recent report of neutrophil infiltration in the thymus after irradiation (Uchimura et al., 2000). Whilst it would be expected that the haemopoietic cell death resulting from irradiation requires rapid phagocytic clearance, the increase in enzyme activity after phagocytosis, the length of time that activated macrophages persist in bone marrow and spleen and the significant neutrophil infiltration would not be expected (reviewed by Giles et al., 2000; Gregory, 2000).

The simplest interpretation of our findings is that the recognition and engulfment of large numbers of apoptotic cells post-irradiation by macrophages (and possibly other resident non-professional phagocytes that are recruited to deal with the increase in apoptosis) induces a cascade of signalling mechanisms within the phagocytic cells that in turn lead to the features of macrophage activation and neutrophil infiltration we have identified. This hypothesis is supported by the time course of acid  -galactosidase and nitrotyrosine accumulation. At 6 h post-irradiation only a few macrophages showed detectable staining, and these contained multiple apoptotic bodies. The proportion of positively stained macrophages increased over time and by 24 h, when very few apoptotic bodies were visible by light microscopy, there were further increases in positively staining macrophages, indicating considerable temporal heterogeneity in the increase in enzyme activities after phagocytosis of apoptotic cells post-irradiation.

A more conventional explanation for our findings would be that macrophage activation and neutrophil infiltration result either from direct effects of ionizing radiation on macrophages and/or from a failure to efficiently clear apoptotic cells leading to the presence of secondarily necrotic cells. These necrotic cells would accumulate to appreciable levels only when the phagocytic capacity of the tissue had been overwhelmed, such as when a large number of apoptotic cells were produced within a short time frame, as is the case following radiation exposure. Necrosis, unlike apoptosis, is well known to produce an

inflammatory response, and this difference is one of the most commonly quoted characteristics that distinguish necrotic cell death from spontaneous apoptotic cell death in vivo (Wyllie et al., 1980). Studies in vitro have shown that phagocytosis of necrotic cells causes a pro-inflammatory response, whereas phagocytosis of apoptotic cells results in the production of both pro- and anti-inflammatory cytokines, providing a molecular mechanism to account for these differences (Giles et al., 2000;Gregory, 2000). In our experiments performed in vivo, analysis of apoptotic cells shows them to be clustered and within macrophages, but our histological studies cannot rule out that a few necrotic cells may be present. If increased macrophage activities were caused solely by phagocytosis of necrotic cells, macrophage activation and neutrophil infiltration should be directly related to the number of cells killed by irradiation for the equivalent number of macrophages. Analysis of different genetic backgrounds indicates that this is not the case, since the levels of induced acid  -galactosidase in DBA/2 mice are intermediate between those of CBA/Ca and C57BL/6 mice, even though DBA/2 mice have the lowest apoptotic index of the three strains. Furthermore, there is a similar degree of neutrophil infiltration but different levels of apoptosis between C57BL/6 and CBA/Ca mice. These genotypic differences in apoptosis, induced macrophage activity and neutrophil infiltration demonstrate that the in vivo inflammatory-type response to radiation is not simply related to the number of apoptotic cells and therefore cannot be explained solely on the basis of secondary necrotic cells accumulating as a result of a macrophage clearance deficit.

The influence of genetic factors on macrophage responses following in vivoexposure to ionizing radiation is an additional important finding of our study. Phagocytes from C57BL/6 mice showed a low constitutive superoxide production that was not observed in cells obtained from CBA/Ca mice and this genotype difference was more marked after irradiation. However, phagocytes from CBA/Ca mice produced a greater rate and total amount of inducible superoxide both with and without irradiation than those from C57BL/6 mice. In both strains, prior irradiation reduced the maximal ability of cells to respond to PMA. In addition, acid  -galactosidase activity was higher in C57BL/6 mice, and was more induced in this strain after radiation. Furthermore, although both C57BL/6 and CBA/Ca mice showed increased nitrotyrosine staining after radiation, the levels were higher in C57BL/6 than in CBA/Ca, compatible with higher induction of NOS2 activity in the former strain. Previous studies in vitro have shown that irradiation potentiates the production of NO by macrophages when they are subsequently stimulated by lipopolysaccharide or interferon-gamma, by a process involving induction of tumour necrosis factor alpha

(McKinney et al., 1998). NO is pleiotropic and its effects depend on concentration and target cell. Thus, NO can be either pro- or anti-apoptotic, and can either downregulate or upregulate p53 activity (Brune et al., 1996; Brockhaus and Brune, 1999). NO can also directly induce the DNA-dependent protein kinase, a key enzyme for repair of DNA damage resulting in enhanced protection to the direct toxic effects of NO and also protecting from subsequent DNA damaging agents (Xu et al., 2000). Finally, NO is well known as an immunoregulatory molecule and can be either pro-or anti-inflammatory (Nathan and Shiloh, 2000). Whilst it has been suggested that high dose irradiation may directly induce NO production by macrophages (Gorbunov et al., 2000), our data indicate that macrophage activation as a consequence of cell death contributes significantly to the production of NO in vivo.

The precise pathway(s) responsible for the observations of an inflammatory-type response following irradiation are unclear at the present time, but presumably result from the activation of macrophages following recognition and phagocytosis of apoptotic cells and the consequent production and release of cytokines and other stimulatory molecules. The recognition of apoptotic cells by macrophages is highly complex, and involves at least seven distinct molecular families. The outcome for the macrophage of apoptotic cell recognition is further complicated by cells being at different stages of apoptosis and by variation of phagocytic responses in different sub-populations of macrophages (Giles et al., 2000). Cellular interactions of macrophages involve adhesion molecules, cytokines, prostaglandins and glucocorticoids (Giles et al., 2000; Gregory, 2000) and there are multiple pathways by which macrophages may be activated, many markers used to define activation and an activated macrophage may not necessarily exhibit all such markers (Adams and Hamilton, 1992; Handel-Fernandez and Lopez, 2000). As haemopoietic tissues are complex due to the variety of cell types and heterogeneity of radiation responses, dissecting the cellular interactions and responses underlying our findings by the analysis of cytokine production and target cell response would present a major challenge. Furthermore, the conventional hypothesis that secondary necrosis or direct effects of ionizing radiation may induce macrophage activation cannot be ruled out. However, since genetic factors markedly influence macrophage responses, the solution to this problem is best investigated in vivo by genetic linkage analysis. Indeed, preliminary observations of (C57BL/6xCBA/Ca) F1 animals have shown that the F1 mice have an acid  -galactosidase response that is intermediate between the two parental strains, but is more similar to C57BL/6 than to CBA/Ca. Our ongoing analysis of N2 animals (CBA/CaxF1) has revealed a complex pattern of responses, with less than 10% of the animals showing an F1-

like response. These data indicate that at least three genetic loci probably contribute to the overall phenotypic response (Silver, 1995). In contrast, preliminary studies of the increased respiratory burst following radiation show that this phenotype is dominant for the CBA/Ca strain, indicating that the two responses to irradiation are controlled by different sets of genes. Studies using other inbred mouse strains also indicate that increased superoxide production and increased lysosomal enzyme activity 24 h post-irradiation in haemopoietic tissue are not linked. Whilst the respiratory burst is generally seen in conjunction with phagocytosis, dissociation of phagocytosis from the respiratory burst can be observed (Yamamoto and Johnston, 1984) and the data indicate that the strain-dependent differences in phagocytic cell function, as a consequence of apoptosis, reflect the complexity of factors that regulate macrophage biology.

In addition to demonstrating that an inflammatory-type response is an unexpected part of the response to ionizing radiation, our data are important for a fuller understanding of the long-term effects of radiation on haemopoietic and other tissues. Inflammatory responses can contribute to the development of leukaemia and may be particularly important in the development of radiation-induced leukaemias (Walburg et al., 1968;Yoshida et al., 1993). There is also evidence that radiation-induced genomic instability can be induced by an indirect mechanism (Lorimore et al., 1998;Watson et al., 2000) and that in both haemopoietic tissue (Watson et al., 1997) and mammary epithelium (Ponnaiya et al., 1997), there is genotype-dependent expression of the instability phenotype. The finding of a more efficient apoptotic response to DNA damage in C57BL/6 than in CBA/Ca haemopoietic tissues is consistent with our previous findings of genotype-dependent apoptotic responses in irradiated urinary epithelium (Mothersill et al., 1999). Taken together, the data support the hypothesis that there is an inverse relationship between the more effective recognition of damage and expression of an instability phenotype. Our findings also provide a plausible bystander mechanism for the unexpected interactions between irradiated and unirradiated haemopoietic cells producing genomic instability both in vitroand in vivo (Lorimore et al., 1998; Watson et al., 2000) as activated macrophages are known to produce clastogenic factors via the intermediacy of superoxide and NO, and are able to produce gene mutations, DNA base modifications, DNA strand breaks, and cytogenetic damage in neighbouring cells, all features associated with radiation-induced chromosomal instability (Wright, 1998).

Materials and methods

Animals and irradiation

CBA/Ca, C57BL/6, DBA/2 and p53+/+ or -/- mice (Jacks et al., 1994) were used in this study in accordance with the guidance issued by the Medical Research Council and Home Office Project Licence Number PPL 30/1272. All mice were bred and housed on site. Mice were  -irradiated at 0.45 Gy min-1 using a CIS bio international IBL 637 Caesium irradiator. Most experiments employed a whole body dose of 4 Gy  -rays, a non-lethal but effective leukaemogenic dose (Mole et al., 1983).

Preparation of cell suspensions

Whole spleen cell suspensions were produced by mechanical dissociation through a steel gauze with 2.5 ml ice cold 10 mM phosphate buffered saline, pH 7.4 (PBS), followed by syringing through a 21G needle three times. Bone marrow suspensions were prepared by removal of both femurs, which were cut at either end to allow collection of cells and flushed three times using a 21G needle into a total volume of 2 ml cold HBSS containing 0.1% BSA. Tissue cellularity was assessed by diluting bone marrow and spleen suspensions in 3% acetic acid and counting nucleated cells with a haemocytometer. Reduction in tissue cellularity represents the decrease in total cell number of irradiated tissues compared to unirradiated controls.

Electron microscopy

Spleens were removed, divided into pieces of 0.2 cm maximum dimension and fixed in 3% glutaraldehyde for at least 1 h. Bone marrow plugs were prepared by expelling the bone contents by flushing. Tissues were post-fixed in osmium tetroxide before embedding in epoxy resin according to standard procedures. Ultrathin sections were stained with lead citrate and uranyl acetate and were examined using a Phillips EM208S transmission electron microscope. Macrophages were identified by their characteristic ultrastructural morphology and quantitated by counting the number of macrophages per unit area.

Histochemical detection of lysosomal enzyme activities

Acid phosphatase positive cells were identified in frozen sections of spleen and bone marrow plugs. Sections were dried at room temperature for 1 h or longer and fixed in 65% acetone; 8% formalin in citrate buffer, pH 3.6 for 30 s at room temperature, followed by washing in distilled water. Slides were incubated in a solution of napthol AS-B1 phosphoric acid and freshly diazotized fast garnet GBC in 0.1 M acetate,

pH 5.2 (Sigma, UK), washed in distilled water and counterstained with haematoxylin. Acid  -galactosidase activity was identified by fixing frozen sections of adult tissues or whole embryonic fore-limbs in ice cold 0.5% gluteraldehyde in PBS for 15 min. Samples were washed in PBS containing 2 mM MgC12 and in detergent solution (40 mM citric acid/sodium phosphate, pH 4.0; 0.01% v/v NP-40; 0.01% w/v C24H39O4Na; 100 mM NaCl; 2 mM MgCl2), then incubated at 37°C in detergent solution containing 1 mg/ml X-Gal, 5 mM K3Fe(CN)6 and 5 mM K4Fe(CN)6, washed and counterstained with neutral fast red.

Quantitative analysis of acid  -galactosidase activity

An equal volume of red cell lysis buffer pH 7.4 (155 mM NH4C1; 11.9 mMNaHCO3; 0.1 mM EDTA) was added to 5106 nucleated splenic or bone marrow cells suspended in PBS and left for 10 min on ice. The cells were washed twice in PBS and nucleated cells lysed on ice for 10 min in 1 ml 0.1% Triton X-100 in water. After spinning at 14 000 g for 10 min at 4°C, 800  l of lysate was added to 200  l of 5 assay buffer (40 mM citric acid/sodium phosphate, 100 mM NaC1; 2 mM Mg C12; pH 4.0). Two hundred  l of either 4 mg/ml ortho-nitrophenyl- -D-galactopyranoside (ONPG) in 1 assay buffer or 1 assay buffer alone was then added and the samples were incubated for 1 h at 37°C before 300  l 1 M sodium carbonate was added to stop the reaction. Samples were read in a spectrophotometer at 420 nm and the control value (minus ONPG) subtracted from the readings of the test samples to give the measured absorbance due to acid  -galactosidase activity.

Measurement of superoxide generation (respiratory burst)

Wells of a chemiluminescence microtitre plate (Dynatech Laboratories, Billinghurst, Sussex, UK) were coated with 200  l of Hanks' buffered saline solution containing 20 mM HEPES, pH 7.4 and 0.1 g/100 ml BSA (HBSS/BSA; Life Technologies, Paisley, UK) overnight at 4°C then washed three times with HBSS. Bone marrow or spleen cells were suspended at 2106/ml in HBSS/BSA and 100  l of each sample was added to each of four wells. Fifty  l of luminol (Sigma, Dorset, UK) (134  g/ml in HBSS/BSA) was added to each well. Finally, 50  l HBSS/BSA10 nMphorbol 12-myristate 13-acetate (PMA) (Sigma) was added and chemiluminescence measured in each of the duplicate wells with or without PMA at regular intervals for up to 1 h in a Microlumat LB96P luminometer.

FACS and immunohistochemistry

Assessment of macrophage numbers was performed by FACS analysis

of spleen and bone marrow cell suspensions. The F4/80 macrophage-specific rat monoclonal antibody (AMS Biotechnology, Oxon, UK) was diluted 1/10 in PBS containing 5% normal rabbit serum and 5% normal mouse serum (Harlan Sera-Lab Ltd, Leicestershire, UK) and 10  l were added to 1106cells in PBS plus rabbit and mouse sera. Following three washes, FITC-conjugated affinity purified anti-rat immunoglobulins, pre-absorbed with mouse immunoglobulins (Vector Laboratories, Peterborough, UK) diluted 1/100 in PBS plus sera were added and incubated for 30 min. Cells were analysed by flow cytometry using a FACSVantage SE (Becton Dickinson, UK). Cells incubated in the absence of the primary F4/80 monoclonal antibody were used as a negative control to gauge levels of endogenous fluorescence and non-specific binding of the fluorochrome.

For immunohistochemistry, bone marrow plugs or bisected spleens were fixed in neutral buffered 10% formalin or in Methacarn solution (60% methanol; 30% chloroform; 10% glacial acetic acid), processed to paraffin wax and 4  m sections cut for immunocytochemistry. Sections were incubated overnight at 4°C in the appropriate primary antibody. Macrophages were identified in Methacarn tissues using F4/80 diluted 1/100 in 5% normal rabbit serum. Nitrotyrosine was identified using an immunoaffinity purified rabbit polyclonal antibody (Upstate Biotechnology, Lake Placid, NY, USA) diluted to 1  g/ml in PBS containing 3% BSA. Neutrophils were identified using a polyclonal rabbit serum to myeloperoxidase (Dako, UK) diluted 1/2000 in 5% normal swine serum. Following washes in PBS, antigens were detected using biotinylated rabbit anti-rat or swine anti-rabbit immunoglobulins (Dako Ltd, UK) diluted 1/500 in PBS containing 3% BSA, followed by a pre-formed complex of avidin-biotin-peroxidase (Dako) each for 30 min at room temperature. Immunoreactive sites were revealed with hydrogen peroxide in the presence of diaminobenzidine and cell nuclei were counterstained with haematoxylin.

Quantitation of apoptosis and neutrophil infiltration

Apoptotic cells were quantitated in 4  m sections of formalin fixed, paraffin processed tissues stained with haematoxylin and eosin. Apoptosis was identified by the characteristic morphological features of nuclear condensation, condensed chromatin margination at the nuclear periphery, cytoplasmic blebbing and nuclear fragmentation (Wyllie et al., 1980). Neutrophils were identified in haematoxylin/eosin stained paraffin sections by their characteristic nuclear morphology and by myeloperoxidase immunocytochemistry. Quantification of apoptotic cells or neutrophils was performed by counting the number of cells present in a minimum of 15 high power fields (at least 10 000 apoptotic

or myeloperoxidase-positive polymorphonuclear cells) with the aid of an eyepiece graticule. Results are presented as the mean number per high power fieldthe standard error of the mean. For acridine orange staining embryonic forelimbs were incubated in 1  g/ml acridine orange in PBS for 30 min at 37°C washed in PBS and viewed by confocal microscopy (Wood et al., 2000).

Acknowledgements

The authors thank R Mitchell, S Macfarlane, J Gibbs, N Kernohan, M Boylan and R Pleass. The research was supported by grants from the Medical Research Council, the European Commission and the Leukaemia Research Fund.

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Interactions of Apoptotic Cells with Macrophages in Radiation-Induced Bystander Signaling

Shubhra Rastogi 1, Michael Boylan , Eric G. Wright and Philip J. CoatesMedical Research Institute, Ninewells Hospital and Medical School, University of

Dundee, DD1 9SY, United Kingdom1Address for correspondence: Jacqui Wood Centre, Medical Research Institute, Ninewells

Hospital and Medical School, University of Dundee, DD1 9SY, UK; e-mail: s.rastogi@dundee.ac.uk.

Nontargeted effects that result in ongoing cellular and tissue damage show genotype-dependency in murine models with CBA/Ca, but not C57BL/6, exhibiting sensitivity to induced genomic instability. In vivo, radiation exposure is associated with genotype-dependent macrophage activation, and these cells are a source of bystander signaling involving cytokines and reactive oxygen and nitrogen species. The mechanisms responsible for macrophage activation and production of damaging bystander signals after irradiation are unclear. Macrophages from CBA/Ca exhibit an M1 (proinflammatory) phenotype compared to the M2 (anti-inflammatory) phenotype of C57BL/6 macrophages. Using the murine RAW264.7 macrophage-like cell line, we show that the ability of macrophages to interact with apoptotic cells and their responses to interaction varies significantly according to macrophage phenotype. Nonstimulated and M2 macrophages induce anti-inflammatory markers arginase and TGFβ after engulfment of apoptotic cells. In contrast, M1 macrophages do not induce anti-inflammatory responses, but express the proinflammatory markers NOS2, IL-6, TNFα, superoxide and NO, able to contribute to a damaging microenvironment. Macrophages stimulated with both inflammatory and anti-inflammatory agents prior to exposure to apoptotic cells induce a mixed response. The results indicate a complex cross-talk between macrophages and apoptotic cells and demonstrate that phagocytic clearance of apoptotic cells induced by genotoxic stress can produce microenvironmental responses consistent with the induction of a chromosomal instability phenotype in sensitive CBA/Ca mice with M1 macrophage activation, but not in resistant C57BL/6 mice with M2 macrophage activation. Modulation of macrophage phenotypes may represent a novel approach for reducing the nontargeted effects of radiation.