Indirect macrophage responses to ionizing radiation: implications for genotype-dependent bystander signaling.

Coates PJ1, Rundle JK, Lorimore SA, Wright EG. Author information Abstract
In 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 GenotypeDependent Bystander Signaling
1. 2. 3. 4. Philip J. Coates, Jana K. Rundle, Sally A. Lorimore, and Eric G. Wright + Author Affiliations 1. Cancer Biology and Clinical Pathology, Division of Pathology and Neurosciences, Ninewells Hospital and Medical School, Dundee, United Kingdom 1. 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;

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In 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


The 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 genotypedependent 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

ANOVA and false-discovery rate assessments were also applied. have at least a change in absolute expression value of 80. 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. Microarray analysis. paraffin-embedded femurs that had been decalcified in EDTA (pH 7. Normalization and expression values were also calculated using RMA with the Affy packages available from Bioconductor 4 ( 27). For each experiment. cDNA synthesis used SuperScript III reverse transcriptase with oligo(dT) (Invitrogen). and are called present in at least one of the conditions tested. Statistical analysis used application of the Benjamini and Hochberg false discovery rate to Welsh t tests (corrected P < 0.000). Previous SectionNext Section Materials and Methods Macrophage culture and irradiation.001. Immunochemical assays. All reactions contained additional primers to amplify Gapdh as a housekeeping gene to allow normalization of samples for input cDNA. and results are presented only for dCHIP analysis. Histograms and boxplots of expression values both before and after normalization were inspected.) according to the methods recommended by the Medical Research Council (MRC) geneservice. CBA/Ca and C57BL/6 mice were bred in-house under conventional conditions. 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. All primer pairs amplify across at least one intron to ensure specificity. and RNA degradation plots were calculated using AffyRNAdeg.0). or on 4-μm sections of formalin-fixed. five flasks were exposed to 4 Gy γ -irradiation at a dose rate of 0. ref. and have an expression value >50 in at least one set of replicate samples. Polyclonal rabbit serum to murine Nos2 was obtained by immunizing with COOH-terminal peptide . GoSurfer2 and the NetAffx Gene Ontology Mining Tool 3 provided additional data and visual representations of Gene Ontology classifications. Immunohistochemical identification of antigens was performed on frozen sections of spleen fixed for 10 min in 50:50 methanol/acetone. Experiments were approved by local ethical review and followed the guidelines of the Medical Research Council and the Home Office (PPL 60/2841). 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.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. 1 After reverse transcription.101 gene probe sets representing 30. taken from C57BL/6 and CBA/Ca mice with or without previous whole-body exposure to 4 Gy γ-irradiation. Total RNA was extracted from bone marrow–derived macrophages using TRIzol (Invitrogen Ltd. United Kingdom). Changes in gene expression were identified as expression values that are at least 2-fold different at the lower 90% confidence interval. Antigens were identified with peroxidase avidin-biotin-complex (Vector Elite. Reverse transcription-PCR (RT-PCR) was performed in duplicate from bone marrow–derived macrophages prepared independently from those used for microarray analysis. Low or negative expression values were truncated at a value of 20 (10% of the median). are called “present” in at least one condition. Statistically significant ontology groupings were recorded as the default level of significance.45 Gy/min) or were sham irradiated.5 Gy/min at room temperature using a CIS Bio International 637 cesium irradiator and five were sham irradiated. Reverse transcription-PCR. cDNA was diluted 1:5 with sterile water and 1 μL was used for PCR with var ying numbers of cycles. have low variation between replicates [0 < median(SD/mean) < 0.3′ -diaminobenzidine as chromogen. P < 0. Genes were filtered to remove gene probes that are not changed across conditions (coefficient of variation 0.) and purified with RNeasy reagents (Qiagen Ltd. These arrays contain 45.759 individual transcripts.05). probes were hybridized to MOE-4302 GeneChips (Affymetrix) at the MRC geneservice (Cambridge. Cohorts of mice were whole body irradiated with the same source (dose rate 0.3 (PM/MM difference model. To produce sufficient mature macrophages for analyses and to reduce random variation in cultures derived from different individuals. Vector Labs) and 3.2 < SE/mean < 1. Microarray fluorescence signals were normalized and expression values were calculated using dCHIPv1. Similar results were obtained using expression values calculated by dCHIP or RMA. 26). synthesis of biotinylated cRNA and fragmentation of labeled cRNA.5].

5 mol/L L-arginine (pH 9.4) containing 10 mmol/L MnCl2were added. no genes were identified as significantly different between irradiated and unirradiated macrophages in either strain. goat anti-Aif1 (Abcam). Using ANOVA with false discovery rate.2 mg/mL DNase (Roche) for 30 min at 37°C.001) for the 136 genes with annotation. 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. Antibodies to CD14. the 6-h time point representing the early response to radiation and the time at which in vivo macrophage responses are first observed ( 10). Cells were collected into lipopolysaccharide-free PBS containing 2 mmol/L EDTA and 0. Femurs were gently blown out with air pushed through 23G needles and cell clumps were digested with medium containing liberase (1. Previous SectionNext Section Results Genotype-dependent expression profiles and response to irradiation in vitro. signal transducer and activator of transcription 6 (STAT6). rat monoclonal antibodies to Dectin-1 and C1q (Serotec). Mfge8. We also did microarray analysis of macrophages collected 6 h after irradiation to examine earlier radiation-induced responses. Gene products involved in cell adhesion or phosphate transport. Marco and CD68 (Hycult BioClone).1% Triton X -100 for 10 min on ice and 50 μL 50 mmol/L Tris-HCl (pH 7. The reaction was acidified with 200 μL of H2SO4/H3PO4/H2O (1/3/7 v/v). megakaryocytes (Itga2b/CD41). Thus. Statistically significant associations were calculated (P< 0. contaminating cell populations do not contribute to the expression profiles observed. with <10% variation between strains or after irradiation. Abcam) were also used. granulocytes (myeloperoxidase). 25 μL of lysate were added to 25 μL of 0. and phospho-STAT6 (Tyr641. rabbit anti–3-nitrotyrosine (Upstate Biotechnology). 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. and to phospho-Stat5 (Tyr694). rabbit monoclonal antibodies to phospho-Stat1 (Tyr701) and phospho-Stat3 (Tyr705. Using the available Gene Ontology annotations. a time when in vivo alterations to macrophage activities are seen in hemopoietic tissues ( 10). In contrast. 28 to increase sensitivity and allow measurements of urea production from primary macrophages.7) and incubated at 37°C for 2 h. function in the immune response or have scavenger receptor activities that were statistically significant (Supplementary Table S2). We used an adapted method from ref. 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 . T cells (CD3). Macrophage isolation. ref. or when data from the two strains were combined. 243 probe sets were identified as differentially expressed between genotypes regardless of irradiation status (∼1. Lcn2. and SR-A (Santa Cruz Biotechnology). Cell Signalling). 1A ). After enzyme activation at 55°C for 10 min. These data confirmed that expression levels are determined by the genotype of the macrophages and are not significantly altered by irradiation in vitro ( Fig.(VFSYGAKKGSALEEPKATRL) conjugated to keyhole-limpet hemocyanin with glutaraldehyde (Moravian Biotechnology). Arginase activity.5% of the expressed genes.5% bovine serum albumin (Invitrogen). Supplementary Table S1). Urea production was measured at 540 nm. Contamination with other cell types was assessed by microarray analysis of lineage-specific transcripts that are highly expressed in B cells (CD19). 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. Using ANOVA tests with Benjamini and Hochberg false-discovery correction for multiple testing (P < 0. Both controls showed an absence of staining. Bone marrow–derived macrophages from both genotypes had the typical microscopic appearance of macrophages and expressed the macrophage marker F4/80. All transcripts were either not expressed or expressed at very low levels. 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. and erythrocytes (glycophorin A. and 25 μL of 5% αisonitrosopropiophenone (Sigma) were added and heated to 95°C for 1 h.67 Wunsch units/mL) and 0. Five thousand cultured macrophages were used and incubated for 1 h at 37°C. differentially expressed genes were found in all biological processes and cellular components and in each of the major categories of molecular function. Affymetrix analysis also showed no significant differences in cell cycle – related gene expression between genotypes.05). 29). indicating that differences in cell cycle stage do not influence the results. Macrophages were positively selected using F4/80 followed by antirat microbeads (Miltenyi Biotec). or that have lyase activity. F4/80-positive macrophages (2 × 105) isolated from cohorts of mice were lysed in 50 μL 0.

1A). RT-PCRs were performed on cDNAs prepared from two independent sets of cultured macrophages from the two genotypes. expression differences were confirmed for selected genes. 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). Cells were either sham irradiated or exposed to 4 Gy γ-irradiation and collected 6 or 24 h later. Gene Ontology analysis showed significant overrepresentations of genes that are involved in immune/defense responses or in regulating cell adhesion. Genotype-dependent expression of genes involved in immune response. removal. Sixteen genes whose products are involved in cell motility or adhesion and 16 cytokines. Ten genes involved in the production.was the only gene induced by at least 2-fold at 90% confidence in both genotypes. 1A and 2). cellular adhesion. or response to reactive oxygen and/or nitrogen species were expressed in a genotype-dependent manner and were unchanged following irradiation in vitro ( Figs. or cytokine receptors were identified as differentially expressed between the two strains ( Fig. and oxidative stress. View larger version:  In this page  Figure 1. The expression of these genes was not altered by irradiation in vitro ( Fig. 2 ). B.  In a new window Download as PowerPoint Slide RT-PCR and immunohistochemical verification of Affymetrix data.  In a new window Download as PowerPoint Slide . Expression values for genes involved in p53-mediated apoptosis were unaffected by radiation at either 6 or 24 h (Supplementary Table S3). A. View larger version:  In this page  Figure 2. immunohistochemistry of spleen frozen sections confirms genotype-dependent expression levels of the selected proteins. genes regulated by cytokines.

we measured Arginase 1 activities in purified macrophages freshly isolated from bone marrows of control and irradiated mice ( Fig. Dectin1 (CLECSF12). 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. 3A).Graphical representations of differentially expressed mRNAs identified by expression profiling. 1A). Four are more highly expressed in C57BL/6 and four are more highly expressed in CBA/Ca. 1B). Genes with scavenger receptor activity were overrepresented (P = 0. macrophages from both strains express high levels of Gas6 and moderate levels of serum amyloid-P ( Fig. although not included in this Gene Ontology classification. .0014. are also elevated in C57BL/6 compared with CBA/Ca. In contrast.Black columns. but CD14 is more highly expressed in CBA/Ca than C57BL/6 by immunohistochemistry ( Fig. IL-1Ra. 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. B. Levels of Dectin1 protein were measured in vivo by immunostaining ( Fig. Macrophage responses to radiation in vivo. Fig.4-fold higher activity in irradiated C57BL/6 compared with irradiated CBA/Ca). C. Five of the six differentially expressed scavenger receptors were present at a higher level in CBA/Ca mice ( Fig. 1). Other markers of the M2 phenotype. 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.3) and myristolated alanine-rich C kinase substrate–like protein was 4. Of other apoptotic cell ligands. Genotype-dependent M1/M2 phenotype. 1.four opsonins for apoptotic cells are more highly expressed in C57BL/6 than CBA/Ca. expression levels in C57BL/6 macrophages. 2).01687). Although cultured macrophages do not change their gene expression profiles after irradiation in vitro. therefore. bars. 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). 2). they possess intrinsic genotype-specific differences in their M1/M2 phenotypes. These microarray and protein expression data indicate that macrophages from C57BL/6 have phenotypic characteristics of M2 alternatively activated macrophages.examples of differentially expressed cytokines. Genotype-dependent expression of scavenger receptors and opsonins. Previous investigations had shown macrophage activation 24 h postirradiation in vivo. In keeping with the microarray and RT-PCR data.1-6. Macrophages isolated from bone marrow of C57BL/6 mice that were irradiated 24 h previously show 3.000036).2-fold higher arginase activity compared with cells isolated at 24 h from shamirradiated mice (P = 0. Irradiation of macrophages in vitrodid not alter the expression of these phenotypic markers (Supplementary Table S1. to examine any alterations to macrophage phenotypes and inflammatory responses that are elicited as a consequence of irradiation in vivo. five scavenger receptors are expressed at higher levels in CBA/Ca mice than in C57BL/6. 2). control versus 4Gy) but there is no increase in arginase activity in macrophages isolated from irradiated compared with nonirradiated CBA/Ca bone marrow ( P = 0.6).72-fold altered between strains (90% confidence interval.SE. 1). CD14 and Marco show higher expression in CBA/Ca and are associated with innate activation phenotypes.8-4. This leads to an increase in the difference in arginase activity between irradiated CBA/Ca and C57BL/6 (2. Antibodies to Marco were insufficiently sensitive to identify this receptor in bone marrow macrophages by FACS. C57BL/6 cells from control mice show higher arginase activity than CBA/Ca (P = 0. whereas the same cells derived from CBA/Ca have a phenotype in common with classically activated M1 macrophages. and chitinase-like proteins (Ym1/2 and Chi3l3). These results were confirmed for Mfge8 and C1qb by RT-PCR and immunohistochemistry of frozen sections of spleen ( Fig. increasing to an 8. 3A). Retrieval of expression values for other scavenger receptors revealed that CD14 was 2. Although there is no Gene Ontology group for this class of genes. A. levels in CBA/Ca.6-fold altered (range 3. Fig.6-fold higher in macrophages from unirradiated C57BL/6 mice compared with unirradiated CBA/Ca.11628. white columns.

whereas CBA/Ca do not. 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. Macrophages from C57BL/6 have increased arginase activity after irradiation. NOS2 is strongly repressed in C57BL/6 but not in CBA/Ca bone marrow ( Fig. However. there are limited transcriptional responses of in vitro bone marrow–derived macrophages to direct irradiation. 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. A. Importantly. 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. and apoptosis are dependent on cell and tissue type (reviewed in ref. Therefore. B to D. 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). 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. 3C and D). radiation responses. whereas STAT6 (characteristic of M2 responses) showed increased activation in C57BL/6 but not in CBA/Ca ( Fig. including p53 stabilization.  In a new window Download as PowerPoint Slide Changes to M1 and M2 phenotypes of CBA/Ca and C57BL/6 mice after irradiation in vivo. 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. arginase activity in macrophages purified from bone marrows of CBA/Ca or C57BL/6 24 h after whole-body 4 Gy γirradiation or sham irradiation. transcriptional activation. even high-dose radiation does not induce p53-regulated proapoptotic genes in bone marrow– derived macrophages. Data are shown relative to the level of sham-irradiated CBA/Ca mice.View larger version:  In this page  Figure 3. 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. Notably. M1 and M2 functional subsets display pro-inflammatory versus anti-inflammatory and reparative patterns . 3B). Nitric oxide (NO) is an effector of the innate immune system and its production is a characteristic feature of M1 macrophages. Previous SectionNext Section Discussion Because cultured macrophages retain the essential properties of primary cells ( 14). These data contrast with many previous studies of more radiosensitive primary cells. 32). After whole-body irradiation. 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).

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. in M2 macrophages. 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. because C57BL/6 is considered a prototypic Th1/M1 strain ( 34. whereas the M1 phenotype associates with pro-inflammatory conditions ( 15. whereas STAT6 activation is characteristic of M2 responses ( 16. those classifications are based on the responses of C57BL/6 macrophages to classic pro-inflammatory stimuli such as lipopolysaccharide and IFN. respectively. Although these characteristics are not altered by direct irradiation of bone marrow–derived macrophages in vitro. although T-cell activities are regulated through interactions with macrophages and other antigen-presenting cells. whereas arginase activity is higher in C57BL/6. These radiationinduced changes therefore enhance the differences between genotypes.30). 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. 16) and after in vivo. Previous studies had shown that macrophage activation in irradiated hemopoietic tissues was associated with phagocytic clearance of apoptotic cells. 35). 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. Although little is known about the signaling pathways activated or repressed by distinct receptors with or without particular opsonins.of function ( 15. although proinflammatory/anti-inflammatory M1/M2 and the corresponding Th1/Th2 phenotypes are regulated through interdependent cytokine signaling pathways ( 34). The straindependent expression of these molecules implies that the engulfing cells in the two genotypes use different pathways for recognition and clearance. Conversely. but not after in vitro irradiation. a powerful inflammatory mediator. Most notably for our studies relating to delayed and indirect effects of irradiation. our data obtained by studying bone marrow macrophages in vivo show that genetically determined default M1/M2 phenotypes are independent of Th1/Th2 characteristics. or . STAT1 activation is characteristic of pro-inflammatory M1 responses. metabolism of arginine through NOS2 produces citrulline and NO. primary Th1/Th2 phenotypes are determined by the genotype of the T cells and not the antigen-presenting cell ( 36). Thus. metabolism of arginine through Arginase 1 reduces NOS2 activity and simultaneously produces polyamines and proline. indicating that apoptotic cell engulfment is one of these signals ( 10). 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. 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. macrophage phenotypes are generated by complex interactions between intrinsic characteristics and responses to external stimuli that differ according to different genetic components. 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. differential responses have been described ( 30). M2 phenotypes associate with reduced inflammation and improved tissue repair after injury. chemical. C57BL/6 macrophages show enhanced M2 activities and those from CBA/Ca mice enhanced M1 activities. each phenotype is also independently determined by genotypic influences. Hemopoietic tissues of CBA/Ca have more NOS2 protein than C57BL/6. M1-like or M2-like. Therefore. 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. 30). because C57BL/6 are intrinsically Th1 yet their bone marrow macrophages are intrinsically M2 in vivo. In M1 macrophages. Thus. consistent with CBA/Ca macrophages having intrinsic M1 properties and the same cells from C57BL/6 exhibiting an M2 phenotype. However. whereas C57BL/6 lose NOS2 and increase arginase activity after irradiation. where CBA/Ca bone marrow macrophages maintain NOS2 levels and arginase activity. which act as antioxidants and stimulate tissue regeneration ( 16. 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. and in fact those same studies show that C57BL/6 macrophages in vitro in the absence of stimuli exhibit M2 characteristics ( 34). 16. 30). Similarly. Similarly. there are genotype-dependent alterations in vivo. These observations may be regarded as surprising. Importantly.

The genotype-specific expression profiles indicate intrinsic differences in bone marrow macrophage activities. 159: 567–80. Taken 2007. with CBA/Ca hemopoietic tissues showing an M1-like phenotype that associates with potentially damaging inflammatory-type responses. ↵ Morgan WF. 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).com/analysis/netaffx/ ↵4 http://www. 2007. Revision received October 25. our data show altered macrophage activity after irradiation in vivo but notin Radiat Res2003. 2007. a genotype-dependent. Previous SectionNext Section Footnotes          Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres. We thank Tom Freeman and the LRF microarray service for advice on gene expression profiling. This article must therefore be hereby marked advertisement in accordance with 18 U.bioconductor. Section 1734 solely to indicate this fact. their activities are altered in vivo after irradiation. accession number ↵1 http://geneservice. Although macrophages do not respond to direct irradiation.complab/gosurfer/ ↵3 http://www. Non-targeted and delayed effects of exposure to ionizing radiation: I.harvard. Medline .physical agents and have been suggested to act to mediate local tissue recovery or mediate damaging bystander effects (reviewed in Received August 37).  ©2008 American Association for Cancer Research.ebi. compared with the induction of an anti-inflammatory phenotype and tissue reparative response seen in C57BL/6 tissues. 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. MIAME-compliant microarray data are available from Array Express (http://www. The costs of publication of this article were defrayed in part by the payment of page charges.affymetrix. including M1/M2 phenotypes that govern their regulatory functions in immunity and hemopoiesis. Previous SectionNext Section Acknowledgments Grant support: Leukaemia Research Fund specialist programme grant 0214. Previous Section References 1.S.aacrjournals. Radiationinduced genomic instability and bystander effects in vitro. Accepted October ↵2 http://biosun1.C. Importantly.

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Inflammatory-type responses after exposure to ionizing radiation in vivo: a mechanism for radiation-induced bystander effects? Sally A Lorimore. recognition and clearance of apoptotic cells after exposure to radiation produces both a persistent macrophage activation and an inflammatory-type response. contrary to expectation. Furthermore. genetics. Macrophage activation post-irradiation was also associated with enhanced respiratory burst activities and an unexpected neutrophil infiltration. Dundee DD1 9SY. bystander . Ninewells Hospital and Medical School. Increased phagocytic cell activity was maintained after apoptotic bodies had been removed. Keywords ionizing radiation. Philip J Coates. Gordon Milne and Eric G Wright Department of Molecular and Cellular Pathology. Dundee DD1 9SY. Department of Molecular and Cellular Pathology. indicating that the in vivo macrophage responses to radiation damage are genetically modified processes.coates@dundee. E-mail: p. but were a consequence of the recognition and clearance of radiation-induced apoptotic cells. Ninewells Hospital and Medical School. University of Dundee. in addition to any effects attributable to immediate radiation-induced damage. 70857095. UK Correspondence to: P J Coates. These findings demonstrate our findings provide a mechanism for the production of damage via a 'bystander' effect which may contribute to radiationinduced genomic instability and leukaemogenesis. Scotland. macrophage. Scotland. Examination of p53-null mice demonstrated that macrophage activation and neutrophil infiltration were not direct effects of irradiation. Oncogene(2001) 20. We also demonstrate a complexity of macrophage activation following radiation that is genotype dependent. 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 Abstract Haemopoietic tissues exposed to ionizing radiation are shown to exhibit increased macrophage activation.j. defined by ultrastructural characteristics and increased lysosomal and nitric oxide synthase enzyme activities. Gillian E Scobie. inflammation. University of Dundee. UK.

. 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. Malignancies. These effects are generally referred to as radiation-induced genomic instability. 1998. 1999). Although the mechanism for these delayed effects of ionizing radiation is unclear.. 1998. excessive production of reactive oxygen species has been implicated (reviewed by Wright. and in particular myeloid leukaemias. Chromosomal damage has also been observed in cells that were not themselves irradiated but were in the neighbourhood of irradiated cells (Prise et al. Finally. are a major health consequence of exposure to ionizing radiation and interactions between the haemopoietic microenvironment and the target stem cells as well as .. 1998. 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. the mechanism for such bystander-mediated effects are unclear.. Wu et al.Introduction Until recently. producing irreversible changes during DNA replication. Little. irradiated cells secrete growth-inhibitory molecules both in vitro and in vivoin a p53-dependent manner (Komarova et al. or during the processing of the DNA damage by enzymatic repair processes. However.. 2000).e.. 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. by a bystander mechanism (Lorimore et al. 1998.. Lyng et al. 2000).Iyer and Lehnert. although reactive oxygen species have been implicated (Narayanan et al. Lehnert and Goodwin. 2000). 1997. Again. 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. it has been generally accepted that the genotoxic and carcinogenic consequences of radiation exposure are due to the damage inflicted directly by the radiation. Wu 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.. Zhou et al. 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. 1999. 1997. 2000.. Watson et al. 1997)..

indicative of degradation of phagocytosed material. effects that persist long after the initial radiation insult. As with many responses to radiation. the classic ultrastructural features of macrophage activation (Adams and Hamilton. 1998). Many of the lysosomal components of the cells were seen to be secondary and tertiary lysosomes. we are investigating both the short. 1990).. CBA/Ca mice characteristically develop myeloid leukaemia after exposure to ionizing radiation and their haemopoietic cells are also susceptible to radiation-induced chromosomal instability. 1992) (Figure 1). produce different sets of adhesion molecules and growth factors. since irradiated haemopoietic stromal cells release mutagenic reactive oxygen species.the damage to the haemopoietic stem cells themselves have been implicated by both clinical and experimental observations. In contrast. Giralt and Champlin. In mouse model systems. 1993. C57BL/6 mice are resistant to both the development of radiation-induced myeloid leukaemia and to radiation-induced chromosomal instability in haemopoietic cells. whilst uncommon. 1996). . Thus. 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. 1994). 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. and alter the overall growth and phenotypic characteristics of co-cultured nonirradiated stem cells (Greenberger et al.and long-term effects of radiation on haemopoietic tissues. 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. Irradiation has also been shown to induce leukaemic transformation of non-irradiated stem cells transplanted into syngeneic mice (Duhrsen and Metcalf.. These findings may reflect the altered characteristics of the stem cell microenvironment after irradiation. the development of radiogenic leukaemia is strongly influenced by genetic factors (Wright. Because interactions between tissue stroma and stem cells appear to be important in determining the overall consequences of radiation. Here we report unexpected macrophage activation combined with neutrophil infiltration following whole body irradiation.

Enzyme levels continued to increase. acid -galactosidase staining was observed in both red and white pulp 6 to 9 h post-irradiation (Figure 2c. the proportion of macrophages staining positively for acid galactosidase had increased. There was no significant increase above the control enzyme activity after exposure to 0. Similarly. reaching more than four times the control level at 24 h post-irradiation (Figure 3a). respectively.13 to 0. At this dose. At 6 and 9 h post-irradiation. we investigated whether there was any evidence for increased enzyme activity in these cells. Significantly increased activity was evident at . and the macrophages were seen to be increased in size by acid phosphatase staining (Figure 2a.4) in the bone marrow 24 h after irradiation with 4 Gy -rays. We also exposed triplicate mice to differing doses of -irradiation and measured levels of acid -galactosidase in spleens 24 h later. Unlike control spleen. in unirradiated bone marrow.25 or 0. By 24 h the staining was again predominant in red pulp areas. 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.b).11 to 0. but after irradiation most macrophages became strongly positive for acid -galactosidase (data not shown).45) in the spleen and of threefold (0.Lysosomal and respiratory burst activity in macrophages following in vivo irradiation In view of the morphological data suggesting macrophage activation post-irradiation. but were significantly raised by 6 h.d) and acid galactosidase-positive macrophages in the white pulp contained multiple pyknotic nuclei. Interestingly. Quantitative spectrophotometric studies of lysosomal acid -galactosidase activity in the spleens and bone marrow of three individual mice showed mean increases of fourfold (0. indicating that these macrophages had recently ingested apoptotic cells (Figure 2g). we measured enzyme activities spectrophotometrically in spleen cell suspensions from three individual mice at various times after 4Gy irradiation.5 Gy (36 and 48% mean reduction in tissue cellularity respectively). there was a mean reduction in tissue cellularity of 82 and 74% in the spleen and bone marrow. 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 2ad). Mean levels of enzyme activity were similar to control unirradiated levels at times up to and including 4 h postirradiation. only a small proportion of the acid phosphatase-positive macrophages showed acid -galactosidase activity. To investigate the time at which increased lysosomal enzyme activity is first seen.

Nitrotyrosine is a product of the reaction between reactive nitrogen species and peptides or proteins. respectively). and formation of nitrotyrosine in vivo is associated with the expression of NOS2 (Wink et al. phagocytes from C57BL/6 mice show a low level of constitutive respiratory burst activity that is increased from 125 to 250 a. 1998). and the number of neutrophils increased up to 24 h post 4 Gy -irradiation. 24 h after 4 Gy -irradiation. or iNOS) as part of the inflammatory process. Neutrophils were seen both within the red pulp and also at the margins . Neutrophil accumulation was first seen 6 h after radiation exposure.u. 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. 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.5-fold increase in enzyme activity was measured (Figure 3b). 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. 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. a 2.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. Without any radiation exposure. After irradiation.u.i). Values represent the amount of light emitted at each time point measured and are given in arbitrary units (a. which induce nitric oxide synthase 2 (NOS2. These data indicate that exposure to ionizing radiation induces the expression of NOS2 in tissue macrophages in vivo.. Increased nitrotyrosine following in vivo irradiation Nitric oxide (NO) production is a characteristic feature of activated macrophages. both the number of positive cells and the intensity of the reaction product were greatly increased (Figure 2h. Luminescence was measured from three mice in duplicate over a period of 1 h in bone marrow or spleen suspensions. Neutrophils were identified by the characteristic morphology of these cells and neutrophil infiltration was confirmed by immunostaining for myeloperoxidase (Figure 4af).u. providing further evidence for radiation-induced macrophage activation.).

These data are compatible with our hypothesis that macrophage activation following radiation exposure is due to radiation-induced apoptosis. The observations that macrophages which contained multiple intracellular apoptotic bodies were both galactosidase-positive and contained detectable amounts of nitrotyrosine. we examined acid galactosidase activity in a classical situation of physiological apoptosis during embryonic tissueremodelling. during which neutrophils are recruited from the bloodstream to the affected site due to the local production of chemoattractant molecules.of the blood vessels in the red pulp. 2000). and the apoptotic cells are contained within F4/80-positive macrophages (Wood et al. but might also indicate a direct p53-mediated pathway of macrophage activation. Similarly. 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.i). To test whether the increased macrophage activity is dependent on p53 function. although a mean 4. In these mice.5) the extensive apoptotic remodelling phase of .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.5-fold increase was seen in three identically treated p53+/+ mice of the same genetic background (Figure 4g).5) or after (E14. where radiation-induced apoptosis is absent due to the lack of p53 function.. In contrast. This latter feature of neutrophil margination is a classic sign of an acute inflammatory response. before (E12. 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. these observations clearly indicate an ongoing neutrophil infiltration into the spleen following irradiation. rather than activation being a direct effect of radiation exposure. The forelimbs of unirradiated wild type and p53-null E13. Previous studies have shown that apoptosis in the developing footplate of the mouse can be easily demonstrated in vivo by acridine orange staining. To test whether macrophage activation is a direct effect of irradiation exposure. acid -galactosidase was not increased above control 24 h after 4 Gy irradiation. Consequently. we measured enzyme activities in the spleens of three individual p53-/mice. immunohistochemical examination of nitrotyrosine in spleens from p53-null mice showed no detectable increase in nitrotyrosine immunoreactivity after radiation. the timing of which coincides with the increased macrophage activity we have also demonstrated.

rather than being a direct effect of radiation. 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. because we have previously reported genetic differences in radiation-induced chromosomal instability in two inbred strains of mice. 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. The results described above had all been obtained using the C57BL/6 strain. which showed that after irradiation the two strains contained similar numbers of macrophages (260. we performed acid phosphatase staining together with immunohistochemistry and FACS analysis using a monoclonal antibody against the mouse macrophage marker.87 per grid unit area for CBA/Ca). Additionally. these data show that the relatively higher levels of acid -galactosidase in lysates of spleen . To investigate this.footplate development.. One possible explanation for these results was that there are higher percentages of macrophages in the C57BL/6 strain. 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.2% for both CBA/Ca and C57BL/6 without irradiation. the levels in spleen cell suspensions being induced fourfold for C57BL/6 compared to 2.2% for CBA/Ca 24 h after 4 Gy irradiation). because neutrophil infiltration was not apparent in p53-/.irradiated spleens. 1.50. C57BL/6 and CBA/Ca (Watson et al. Taken together.4 per grid unit area for C57BL/6 versus 22.6% for C57BL/6 and 1. both acridine orange and acid -galactosidase showed minimal staining in both p53-null and wild type animals. we quantitated macrophage numbers in bone marrow morphologically by electron microscopy. Because F4/80 measures monocytic precursors as well as resident macrophages. the distribution and numbers of macrophages were indistinguishable between the two genotypes. these observations indicate that increased macrophage activity is induced by apoptosis.5-fold for CBA/Ca 24 h after whole body irradiation with 4Gy -irradiation. Taken together with the nitrotyrosine data. inflammation after irradiation is also not a direct effect of radiation exposure (Figure 4e). By quantitative FACS analysis of cell suspensions. 1997). mean levels of acid galactosidase activity were higher in spleen and bone marrow from C57BL/6 animals than in CBA/Ca mice. thereby giving higher average levels of lysosomal enzyme activity. When measured using the quantitative spectrophotometric assay. By acid phosphatase staining and immunohistochemistry of paraffin sections. F4/80.

Furthermore.. in both normal and irradiated CBA/Ca tissues (Figure 5). P=0. P=0. 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 2hk).13.9.01. increasing to 82. 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.2 and 32. 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. but must instead be due to an increased level of enzyme activity per cell.1098. Again. 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 2cf). for C57BL/6 and CBA/Ca respectively). 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. In contrast to the undetectable endogenous respiratory burst activity of CBA/Ca mice. neutrophil accumulation was seen to occur in both strains.6 and 88.1. At 9 h post-irradiation with 4 Gy.0786. This genotypic difference in staining was maintained up to 24 h postirradiation. in the absence of exogenous stimulation with PMA phagocytic cells from unirradiated CBA/Ca bone marrow and spleen had no detectable superoxide activity.21. Unlike the results in C57BL/6 mice.and bone marrow cell suspensions of C57BL/6 mice are not simply due to the presence of increased numbers of macrophages in these samples.44. 1997). and there was no significant difference between the strains (29. These data indicate complex genotypic differences in radiation-induced macrophage activation and neutrophil infiltration in these two inbred . We have previously demonstrated genetic differences in PMA-induced superoxide generation between unirradiated C57BL/6 and CBA/Ca bone marrow cells (Watson et al. exogenous stimulation of the cells with PMA led to a greater respiratory burst than that seen in C57BL/6 mice. 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. 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.

This resulted in a greater reduction in tissue cellularity at 24 h for C57BL/6 than for CBA/Ca. This conclusion is based on the finding of tissue neutrophil infiltration. 61940 apoptotic cells per high power field. 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. 1992). leading to higher enzyme activities in the former. indicated that the mechanism for this response is the recognition and phagocytosis of radiation-induced apoptotic cells. which is known to be particularly resistant to radiation-induced apoptosis (Nomura et al. their numbers peak at 6 h and have begun to decrease by 9 h post-irradiation. Therefore. To investigate the relationship between cell death and macrophage activation we turned to the DBA/2 strain. To investigate this possibility. one possible explanation for these results would be that there are higher levels of apoptosis in C57BL/6 mice than in CBA/Ca mice. the ultrastructural characteristics of tissue macrophages. In both strains.strains of mice. we counted apoptotic cells at various times after irradiation in the spleens of duplicate animals. 91142 and CBA/Ca. genotypic differences in the induction of macrophage lysosomal enzyme activity following irradiation are not directly related to genotypic differences in apoptosis. their increased lysosomal and NOS enzyme activities and the enhanced respiratory burst activities indicating increased phagocytic cell activity. radiation-induced apoptotic cells are first seen 2 h after radiation. 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. Quantitative analysis showed there to be an approximately 1. 6 h after 4 Gy). together with the absence of these responses in irradiated p53-null mice.. reaching near to control values by 24 h. 82 and 72% respectively. and it is known that the level of apoptosis after radiation exposure varies between different mouse strains (Nomura et al. Dose response and time course studies.5fold higher level of apoptosis in C57BL/6 mice after radiation (C57BL/6. measurements of apoptosis showed twofold lower levels of cell death in DBA/2 mice. These data might suggest that the genotypic differences in radiationinduced macrophage activation we had observed were due simply to differing levels of radiation-induced apoptosis. Since we have shown that induction of macrophage activities is correlated with radiation-induced apoptosis..

Furthermore. 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. 1998).. 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. 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. However. 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. although there are significant reductions in tissue cellularity at doses up to 0. 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. 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. As the resolution of inflammation is known to occur by apoptosis and rapid phagocytosis of neutrophils (Giles et al. and with neutrophil infiltration. 2000). The morphological identification of macrophage activation and increased macrophage enzyme activities were also associated with increased phagocytic activity (measured by respiratory burst assays). but all cells showed minimal membrane ruffling.5 Gy. during which time apoptosis had declined almost to basal levels. rather than being a direct consequence of radiation exposure.Our electron microscopic investigations of unirradiated haemopoietic tissues demonstrated a bimodal distribution of macrophages in which cells had few or many secondary lysosomes. 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. phagocytosis of apoptotic cells is a feature of normal steady-state haemopoiesis (Necas et al. acid -galactosidase activity is not simply related to either radiation dose or resultant cell loss. Indeed. the continued increase in enzyme activity in the red pulp might be due to ongoing phagocytosis of infiltrating apoptotic neutrophils. with physiological levels of apoptosis quantitatively similar to those associated with apoptosis after exposure to the doses of radiation used in our studies.. Thus. However. Twenty-four hours after irradiation all macrophages had larger and increased numbers of secondary lysosomes together with very extensive membrane ruffling. electron micrographs of tissues at 24 h post- . However.

Studies in vitro have shown that phagocytosis of necrotic cells causes a pro-inflammatory response. 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. the increase in enzyme activity after phagocytosis. and these contained multiple apoptotic bodies. as is the case following radiation exposure. 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.and anti-inflammatory . The proportion of positively stained macrophages increased over time and by 24 h. this is not consistent with the phagocytosis of recently produced apoptotic cells. 2000). 2000). Necrosis. 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. These necrotic cells would accumulate to appreciable levels only when the phagocytic capacity of the tissue had been overwhelmed. 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. whereas phagocytosis of apoptotic cells results in the production of both pro..irradiation showed only a few macrophages contained identifiable apoptotic bodies whereas many had secondary lysosomes containing degraded cellular material. unlike apoptosis. there were further increases in positively staining macrophages. a conclusion supported by a recent report of neutrophil infiltration in the thymus after irradiation (Uchimura et al. At 6 h post-irradiation only a few macrophages showed detectable staining.. when very few apoptotic bodies were visible by light microscopy. 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. is well known to produce an inflammatory response. indicating considerable temporal heterogeneity in the increase in enzyme activities after phagocytosis of apoptotic cells post-irradiation. such as when a large number of apoptotic cells were produced within a short time frame. 1980). Gregory. Whilst it would be expected that the haemopoietic cell death resulting from irradiation requires rapid phagocytic clearance.

and can either downregulate or upregulate p53 activity (Brune et al. 1996. 2000. there is a similar degree of neutrophil infiltration but different levels of apoptosis between C57BL/6 and CBA/Ca mice. compatible with higher induction of NOS2 activity in the former strain. NO can also directly induce the DNA-dependent protein kinase. Analysis of different genetic backgrounds indicates that this is not the case.. NO can be either pro. 1999).. Thus.Gregory.. NO is pleiotropic and its effects depend on concentration and target cell. 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. providing a molecular mechanism to account for these differences (Giles et al. but our histological studies cannot rule out that a few necrotic cells may be present. since the levels of induced acid -galactosidase in DBA/2 mice are intermediate between those of CBA/Ca and C57BL/6 mice. 2000). The influence of genetic factors on macrophage responses following in vivoexposure to ionizing radiation is an additional important finding of our study. These genotypic differences in apoptosis. by a process involving induction of tumour necrosis factor alpha (McKinney et al. even though DBA/2 mice have the lowest apoptotic index of the three strains. the levels were higher in C57BL/6 than in CBA/Ca. In addition. In our experiments performed in vivo. although both C57BL/6 and CBA/Ca mice showed increased nitrotyrosine staining after radiation. macrophage activation and neutrophil infiltration should be directly related to the number of cells killed by irradiation for the equivalent number of macrophages. prior irradiation reduced the maximal ability of cells to respond to PMA. acid -galactosidase activity was higher in C57BL/6 mice. 1998). In both strains. Furthermore. Brockhaus and Brune. 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 .or antiapoptotic. Furthermore. analysis of apoptotic cells shows them to be clustered and within macrophages.cytokines. 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. However. If increased macrophage activities were caused solely by phagocytosis of necrotic cells. and was more induced in this strain after radiation. 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. 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.

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. As haemopoietic tissues are complex due to the variety of cell types and heterogeneity of radiation responses. our data indicate that macrophage activation as a consequence of cell death contributes significantly to the production of NO in vivo. many markers used to define activation and an activated macrophage may not necessarily exhibit all such markers (Adams and Hamilton.. Finally. 2000. Cellular interactions of macrophages involve adhesion molecules. However. In contrast. NO is well known as an immunoregulatory molecule and can be either pro-or anti-inflammatory (Nathan and Shiloh. and involves at least seven distinct molecular families. 2000). 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.. but is more similar to C57BL/6 than to CBA/Ca. preliminary studies of the increased respiratory burst following radiation show that this phenotype is dominant for the CBA/Ca strain. since genetic factors markedly influence macrophage responses. 2000). the solution to this problem is best investigated in vivo by genetic linkage analysis. The precise pathway(s) responsible for the observations of an inflammatory-type response following irradiation are unclear at the present time. The recognition of apoptotic cells by macrophages is highly complex. dissecting the cellular interactions and responses underlying our findings by the analysis of cytokine production and target cell response would present a major challenge.(Xu et al.. the conventional hypothesis that secondary necrosis or direct effects of ionizing radiation may induce macrophage activation cannot be ruled out. Our ongoing analysis of N2 animals (CBA/CaxF1) has revealed a complex pattern of responses. indicating that the two responses to irradiation are controlled by different sets of genes. 2000) and there are multiple pathways by which macrophages may be activated. 2000). 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. Handel-Fernandez and Lopez. cytokines. Gregory. Studies using other inbred mouse strains also indicate that increased superoxide production and increased lysosomal enzyme activity 24 h post-irradiation in haemopoietic tissue . 1995). prostaglandins and glucocorticoids (Giles et al. Furthermore. 2000). with less than 10% of the animals showing an F1like response. 1992.. 2000). These data indicate that at least three genetic loci probably contribute to the overall phenotypic response (Silver. Whilst it has been suggested that high dose irradiation may directly induce NO production by macrophages (Gorbunov et al. Indeed.

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. 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. the data support the hypothesis that there is an inverse relationship between the more effective recognition of damage and expression of an instability phenotype... 1997). DNA base modifications.Watson et al. 1968. a non-lethal but effective leukaemogenic dose (Mole et . reflect the complexity of factors that regulate macrophage biology. Inflammatory responses can contribute to the development of leukaemia and may be particularly important in the development of radiation-induced leukaemias (Walburg et al. There is also evidence that radiation-induced genomic instability can be induced by an indirect mechanism (Lorimore et al. DNA strand breaks.. 1999). Materials and methods Animals and irradiation CBA/Ca.45 Gy min-1 using a CIS bio international IBL 637 Caesium irradiator. 1998).. All mice were bred and housed on site. 1993).mice (Jacks et al. 1984) and the data indicate that the strain-dependent differences in phagocytic cell function. Watson et al.. our data are important for a fuller understanding of the long-term effects of radiation on haemopoietic and other tissues. DBA/2 and p53+/+ or -/. 1998. 1997) and mammary epithelium (Ponnaiya et al. dissociation of phagocytosis from the respiratory burst can be observed (Yamamoto and Johnston. there is genotype-dependent expression of the instability phenotype. 1998. all features associated with radiation-induced chromosomal instability (Wright. 2000) and that in both haemopoietic tissue (Watson et al.. and cytogenetic damage in neighbouring cells. Most experiments employed a whole body dose of 4 Gy -rays. Whilst the respiratory burst is generally seen in conjunction with phagocytosis.Yoshida et al..are not linked. 2000) as activated macrophages are known to produce clastogenic factors via the intermediacy of superoxide and NO.. 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. Mice were irradiated at 0. as a consequence of apoptosis... Taken together. C57BL/6. and are able to produce gene mutations. In addition to demonstrating that an inflammatory-type response is an unexpected part of the response to ionizing radiation.

Bone marrow suspensions were prepared by removal of both femurs. then incubated at 37°C in detergent solution containing 1 mg/ml X-Gal. washed in distilled water and counterstained with haematoxylin. UK). Electron microscopy Spleens were removed. Tissues were post-fixed in osmium tetroxide before embedding in epoxy resin according to standard procedures. 1983). Histochemical detection of lysosomal enzyme activities Acid phosphatase positive cells were identified in frozen sections of spleen and bone marrow plugs. pH 3.01% v/v NP-40. 100 mM NaCl. Tissue cellularity was assessed by diluting bone marrow and spleen suspensions in 3% acetic acid and counting nucleated cells with a haemocytometer. pH 5. 0.1 M acetate. 0.5 ml ice cold 10 mM phosphate buffered saline.01% w/v C24H39O4Na. Ultrathin sections were stained with lead citrate and uranyl acetate and were examined using a Phillips EM208S transmission electron microscope. Bone marrow plugs were prepared by expelling the bone contents by flushing. followed by syringing through a 21G needle three times.2 cm maximum dimension and fixed in 3% glutaraldehyde for at least 1 h. 2 mM MgCl2). 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. Preparation of cell suspensions Whole spleen cell suspensions were produced by mechanical dissociation through a steel gauze with 2. pH 4. 8% formalin in citrate buffer.6 for 30 s at room temperature. pH 7. 5 mM K3Fe(CN)6 and 5 . Macrophages were identified by their characteristic ultrastructural morphology and quantitated by counting the number of macrophages per unit area. Sections were dried at room temperature for 1 h or longer and fixed in 65% acetone.2 (Sigma.1% Slides were incubated in a solution of napthol AS-B1 phosphoric acid and freshly diazotized fast garnet GBC in 0. divided into pieces of 0. 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. Samples were washed in PBS containing 2 mM MgC12 and in detergent solution (40 mM citric acid/sodium phosphate. followed by washing in distilled water.4 (PBS).0.. Reduction in tissue cellularity represents the decrease in total cell number of irradiated tissues compared to unirradiated controls.

Finally. Sussex.4 (155 mM NH4C1. pH 7. 2 mM Mg C12. 50 l HBSS/BSA10 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. 0. After spinning at 14 000 g for 10 min at 4°C. UK) diluted 1/100 in PBS plus sera were added and incubated for 30 min. washed and counterstained with neutral fast red. Dorset. Bone marrow or spleen cells were suspended at 2106/ml in HBSS/BSA and 100 l of each sample was added to each of four wells. UK) overnight at 4°C then washed three times with HBSS. Oxon. FITC-conjugated affinity purified anti-rat immunoglobulins.4 and 0. Fifty l of luminol (Sigma.0).mM K4Fe(CN)6. Billinghurst. Following three washes. FACS and immunohistochemistry Assessment of macrophage numbers was performed by FACS analysis of spleen and bone marrow cell suspensions. UK) were coated with 200 l of Hanks' buffered saline solution containing 20 mM HEPES.1 g/100 ml BSA (HBSS/BSA. pH 4. Cells were analysed by flow cytometry using a . UK) (134 g/ml in HBSS/BSA) was added to each well.1 mM EDTA) was added to 5106 nucleated splenic or bone marrow cells suspended in PBS and left for 10 min on ice. Leicestershire. 11. Quantitative analysis of acid -galactosidase activity An equal volume of red cell lysis buffer pH 7. Measurement of superoxide generation (respiratory burst) Wells of a chemiluminescence microtitre plate (Dynatech Laboratories. Life Technologies.9 mMNaHCO3. 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. The cells were washed twice in PBS and nucleated cells lysed on ice for 10 min in 1 ml 0. Paisley. Peterborough. pre-absorbed with mouse immunoglobulins (Vector Laboratories. 100 mM NaC1. Two hundred l of either 4 mg/ml ortho-nitrophenyl.1% Triton X-100 in water.-Dgalactopyranoside (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. The F4/80 macrophagespecific rat monoclonal antibody (AMS Biotechnology. 800 l of lysate was added to 200 l of 5 assay buffer (40 mM citric acid/sodium phosphate. UK) was diluted 1/10 in PBS containing 5% normal rabbit serum and 5% normal mouse serum (Harlan Sera-Lab Ltd. UK) and 10 l were added to 1106cells in PBS plus rabbit and mouse sera.

NY. Immunoreactive sites were revealed with hydrogen peroxide in the presence of diaminobenzidine and cell nuclei were counterstained with haematoxylin. 30% chloroform. N Kernohan. 10% glacial acetic acid). USA) diluted to 1 g/ml in PBS containing 3% BSA. Results are presented as the mean number per high power fieldthe standard error of the mean. Following washes in PBS. condensed chromatin margination at the nuclear periphery. 2000). antigens were detected using biotinylated rabbit anti-rat or swine anti-rabbit immunoglobulins (Dako Ltd. 1980). Apoptosis was identified by the characteristic morphological features of nuclear condensation. Neutrophils were identified in haematoxylin/eosin stained paraffin sections by their characteristic nuclear morphology and by myeloperoxidase immunocytochemistry. Quantitation of apoptosis and neutrophil infiltration Apoptotic cells were quantitated in 4 m sections of formalin fixed. 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. Acknowledgements The authors thank R Mitchell.. Neutrophils were identified using a polyclonal rabbit serum to myeloperoxidase (Dako. 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 nonspecific binding of the fluorochrome. processed to paraffin wax and 4 m sections cut for immunocytochemistry. paraffin processed tissues stained with haematoxylin and eosin. cytoplasmic blebbing and nuclear fragmentation (Wyllie et al. Lake Placid. S Macfarlane. UK) diluted 1/2000 in 5% normal swine serum. bone marrow plugs or bisected spleens were fixed in neutral buffered 10% formalin or in Methacarn solution (60% methanol. J Gibbs. 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. Nitrotyrosine was identified using an immunoaffinity purified rabbit polyclonal antibody (Upstate Biotechnology.FACSVantage SE (Becton Dickinson. followed by a pre-formed complex of avidin-biotin-peroxidase (Dako) each for 30 min at room temperature. UK) diluted 1/500 in PBS containing 3% BSA. UK). For immunohistochemistry. 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. M Boylan and R ..

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United Kingdom 1 Address for correspondence: Jacqui Wood Centre. we show that the ability of macrophages to interact with apoptotic cells and their responses to interaction varies significantly according to macrophage phenotype. Eric G. able to contribute to a damaging microenvironment. Ninewells Hospital and Medical School. superoxide and NO.Interactions of Apoptotic Cells with Macrophages in Radiation-Induced Bystander Signaling Shubhra Rastogi 1. Modulation of macrophage phenotypes may represent a novel approach for reducing the nontargeted effects of radiation. Macrophages from CBA/Ca exhibit an M1 (proinflammatory) phenotype compared to the M2 (anti-inflammatory) phenotype of C57BL/6 macrophages. and these cells are a source of bystander signaling involving cytokines and reactive oxygen and nitrogen species. Medical Research Institute. but not C57BL/6. . Macrophages stimulated with both inflammatory and anti-inflammatory agents prior to exposure to apoptotic cells induce a mixed response. University of Dundee. In vivo. Ninewells Hospital and Medical School. Wright and Philip J. exhibiting sensitivity to induced genomic instability. Using the murine RAW264. email: s.rastogi@dundee. DD1 9SY. UK. but express the proinflammatory markers NOS2. M1 macrophages do not induce anti-inflammatory responses. radiation exposure is associated with genotypedependent macrophage activation. In contrast. The results indicate a complex crosstalk 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. Michael Boylan .7 macrophage-like cell line. The mechanisms responsible for macrophage activation and production of damaging bystander signals after irradiation are unclear. Nontargeted effects that result in ongoing cellular and tissue damage show genotypedependency in murine models with CBA/Ca. TNFα. DD1 Coates Medical Research Institute. Nonstimulated and M2 macrophages induce anti-inflammatory markers arginase and TGFβ after engulfment of apoptotic cells. University of Dundee.

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