International Journal of Radiation Biology, November 2014; 90(11): 1095–1103 © 2014 Informa UK, Ltd. ISSN 0955-3002 print / ISSN 1362-3095 online DOI: 10.3109/09553002.2014.943848
Macrophages as key elements of Mixed-oxide [U-Pu(O2)] distribution and pulmonary damage after inhalation? Anne Van der Meeren, Agnes Moureau & Nina M. Griffiths
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Laboratoire de RadioToxicologie, CEA/DSV/iRCM, Bruyères le Châtel, Arpajon, France
consequences of actinide exposure include both stochastic effects, mainly tumors of epithelial origin and determinist effects, such as radiation pneumonitis and interstitial fibrosis (Talbot and Moores 1985, Sanders et al. 1988, Diel et al. 1992, Dudoignon et al. 2003, Newman et al. 2005, Muggenburg et al. 2008, Griffiths et al. 2010, Park et al. 2012). These have been described both in experimental animals and in nuclear workers, such as the cohort from Mayak (Bair et al. 1989, Sanders et al. 1993, Gilbert et al. 2004, Sokolnikov et al. 2008). Shortly after inhalation, the majority of α particles are phagocytized by alveolar macrophages, where they can remain for long periods of time (Sanders 1969, Muller et al. 1989). Inflammatory cells and particularly macrophages are not only central elements in the lung distribution of actinides but also in the development of radiation fibrosis (Johnston et al. 2002). Macrophages are also involved in the early inflammatory response following lung contamination with actinides (Van der Meeren and Gremy 2010, Van der Meeren et al. 2012). It is now recognized that depending on cytokine microenvironment, macrophages can adopt a proinflammatory or a profibrotic phenotype. To parallel macrophage activation with T helper cell polarization, the terms of ‘M1’ and ‘M2’, for macrophages of type 1 or 2, were introduced to describe these functionally distinct subpopulations representing the extremes of a continuum (Mantovani et al. 2004). Excessive scarring and tissue fibrosis may result from an imbalanced action of M1-and M2-polarized macrophages during prolonged lung inflammation (Strieter 2008, Gordon and Martinez 2010). One way to investigate the in vivo function of a particular cell type is to deplete these cells, and to observe the consequences of this depletion. Clodronate entrapped in liposomes has been widely used to selectively deplete macrophages in lungs after intratracheal administration (Berg et al. 1993, Elder et al. 2004, Johnston et al. 2004). Liposomes are used as a ‘Trojan horse’ to deliver the small clodronate molecule inside the macrophages. This drug belonging to bisphosphonate family, induces apoptosis and necrosis in target cells (Van Rooijen and Sanders 1994, Lehenkari et al. 2002).
Abstract Purpose: To investigate the consequences of alveolar macrophage (AM) depletion on Mixed OXide fuel (MOX: U, Pu oxide) distribution and clearance, as well as lung damage following MOX inhalation. Materials and methods: Rats were exposed to MOX by nose only inhalation. AM were depleted with intratracheal administration of liposomal clodronate at 6 weeks. Lung changes, macrophage activation, as well as local and systemic actinide distribution were studied up to 3 months post-inhalation. Results: Clodronate administration modified excretion/retention patterns of a activity. At 3 months post-inhalation lung retention was higher in clodronate-treated rats compared to Phosphate Buffered Saline (PBS)-treated rats, and AM-associated a activity was also increased. Retention in liver was higher in clodronatetreated rats and fecal and urinary excretions were lower. Three months after inhalation, rats exhibited lung fibrotic lesions and alveolitis, with no marked differences between the two groups. Foamy macrophages of M2 subtype [inducible Nitric Oxide Synthase (iNOS) negative but galectin-3 positive] were frequently observed, in correlation with the accumulation of MOX particles. AM from all MOX-exposed rats showed increased chemokine levels as compared to sham controls. Conclusion: Despite the transient reduced AM numbers in clodronate-treated animals no major differences on lung damage were observed as compared to non-treated rats after MOX inhalation. The higher lung activity retention in rats receiving clodronate seems to be part of a general inflammatory response and needs further investigation. Keywords: Actinide inhalation, macrophage depletion, lung damage
Introduction Accidental inhalation of α-emitting particles from Mixed OXide fuel (MOX: U, Pu oxide) poses a potential longterm health risk to nuclear industry workers. Pathological
Correspondence: Dr Anne Van der Meeren, PhD, Laboratoire de RadioToxicologie, CEA/DSV/iRCM, Bruyères le Châtel, 91297 Arpajon, France Tel: ⫹ 33 1 6926 5553. Fax: ⫹ 33 1 6926 7045. E-mail: [email protected] (Received 6 November 2013; revised 23 May 2014; accepted 7 July 2014)
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In the present study we investigated whether the depletion of AM 6 weeks after inhalation of MOX affects retention/ excretion of MOX, α particle distribution and lung damage, and modifies macrophage activation/polarization status.
Materials and methods
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Animals and inhalation procedure Male Sprague Dawley rats, 250 g at the time of exposure, were obtained from Charles River Laboratories (l’Arbresle, France), quarantined at least 7 days before exposure, housed 3 or 4 to a cage and maintained at constant temperature on a 12:12 h light-dark cycle. They received commercial rodent chow and water ad libitum. Housing and experiments were carried out in compliance with the French regulation for animal experimentation (European act 2001-246, June 6, 2001). MOX powder from the rectification step was produced by the MIcronised MASter Blend (MIMAS) procedure at the MELOX installation (Marcoule, France). MOX contained 81% uranium and 7.1% plutonium (by mass). Due to 241Pu decay in 241Am, approximately 23% of total α activity of MOX was accounted for by americium (Am). The specific activity was 123.4 kBq/mg at the time of experimentation, and Activity Median Aerodynamic Diameter was 4.2 μm, σg2.7. Thirty rats restrained in cardboard tubes, were ‘nose-only’ exposed to a MOX aerosol generated as described previously (Andre et al. 1989). Sham-exposed animals were treated in the same manner but were not exposed to the aerosol. The initial lung deposit (ILD) in animals was determined 7 days after inhalation on anesthetized rats (intraperitoneal sodium pentobarbitone, 40 mg/kg, Ceva Santé Animal, Libourne, France) by measurement of thorax-associated radioactivity by γ-ray spectrometry. In the present experiment, ILD was evaluated to be 20.8 ⫾ 4.3 kBq (n ⫽ 27). To determine lung activity excretion, similar counting was done from 2 weeks to 3 months post exposure, for a total of four counting times for all rats. For collection of urine and feces, animals were kept individually in metabolism cages. Lipopolysaccharide (LPS) was used as a positive control to induce M1 differentiation (Sonoki et al. 1997). For this purpose, 2.5 mg LPS (Escherichia Coli 026:B6, Sigma Aldrich, St Quentin-Fallavier, France) was intratracheally administered under light gaseous anesthesia (2.5% isoflurane, Virbac, Carros, France). Rats were euthanized by exsanguination from the dorsal aorta 4 h after administration and lungs harvested for paraffin embedding.
Samples collection and activity measurement Urine and feces were collected daily for 7 days, starting the day of liposome administration. Six weeks and 3 months post exposure, six rats from each group were euthanized. Liver and femurs were taken for measurement of activity. Lungs were collected and divided in two batches for each time-point: One was dedicated to Broncho-Alveolar Lavages (BAL) and determination of total lung activity, and the second one was used for histology. BAL were carried out after euthanasia with four sequential washes to a total of 24 ml of warmed sterile PBS (pH 7.4). Centrifugation of BAL (300 g,
5 min) allowed the separation into a cellular fraction (over 95% AM) and an acellular fraction containing the Epithelial Lining Fluid (ELF). An aliquot of the ELF was taken to determine the total protein content with a BCA assay (Bicinchoninic acid Reagent kit, Fisher Scientific, Illkrich, France). Total α activity was measured in biological samples (femurs, liver, urine, lavaged lungs, BAL, AM and ELF) by liquid scintillation counting using a Packard liquid scintillation counter, after dry ashing and wet ashing with nitric acid (2M) and H2O2 (30%) treatments until a clear solution was obtained. The skeletal retention of actinides was estimated assuming that the two femurs represent 10% of the whole skeleton (International Commission on Radiological Protection [ICRP] 1972). Activity in feces was determined by γ-ray spectrometry.
Cell culture and cytokine determination AM were resuspended in Roswell Park Memorial Institute (RPMI) medium containing 10% Foetal Calf Serum, 200 mM L-Glutamine, 10 IU/ml penicillin and 10 mg/ml streptomycin, all obtained from Sigma Aldrich. Cells were plated into 24-well dishes at a concentration of 105 cells/well. Supernatants were collected 24 h after plating and assessed for cytokine and chemokine content by specific EnzymeLinked Immuno Sorbent Assay (ELISA) as recommended by the manufacturers (R&D Systems Europe, Lille, France) for Tumor Necrosis Factor-α (TNF-α) and Cytokine-Induced neutrophil Chemoattractant-1 (CINC-1) (Biosource, Life technologies, St Aubin, France) for Macrophage Inflammatory Protein-2 (MIP-2) and Amersham Biosciences GE Healthcare Europe (Vélizy-Villacoublay, France) for Monocyte Chemotactic Protein-1 (MCP-1).
Clodronate administration For AM depletion, Sham- or MOX-exposed rats were administered intratracheally under light gaseous anesthesia with a suspension of 0.25 ml containing either clodronate-encapsulated liposomes (5 mg/ml) or PBS-encapsulated liposomes to serve as a control (Clodronate Foundation, Haarlem, The Netherlands) at 39 days post inhalation, then 2 days later. Rats were euthanized 3 and 49 days after the last administration, equivalent to 6 weeks and 3 months post-inhalation. Table I demonstrates the effects of clodronate administration on macrophage number and MCP-1 production in shamrats, non-exposed to MOX.
Table I. Consequences of clodronate administration in sham-exposed rats: Alveolar macrophage (AM) were obtained from Broncho-Alveolar Lavages (BAL) 3 days and 6 weeks post clodronate treatment, counted and cultured. Monocyte Chemotactic Protein-1 (MCP-1) production was assessed in supernatants from 24 h-cultures of AM. Numbers are mean ⫾ SD (n ⫽ 3). 3 days 6 weeks PBS liposomes Number of macrophages in BAL (⫻ 106) MCP-1 (pg/ml)
8.4 ⫾ 3.5
Clodronate PBS Clodronate liposomes liposomes liposomes 1.67 ⫾ 0.51 10.1 ⫾ 2.8
189.1 ⫾ 56.2 1911 ⫾ 177
PBS, Phosphate Buffered Saline.
37.6 ⫾ 34.9
14.0 ⫾ 4.9 29.0 ⫾ 1.0
Macrophage depletion after MOX inhalation 1097
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Immunohistochemistry
Autoradiography studies Determination of MOX particle distribution was carried out by autoradiography studies on lung sections or macrophages collected on slides by cytocentrifugation. Slides were covered with photographic emulsion (NTB 2K-5, Kodak, Anachem, Luton, UK) and exposed for 7 days. Slides were counterstained with hemalun. The percentage of AM associated with MOX particles was determined by counting approximately 200 cells per slide from three different preparations.
Data analysis Data were analyzed using two-tailed Student’s t-test or the nonparametric Mann-Whitney test. A p value of ⬍ 0.05 was considered statistically significant.
Results Excretion and systemic organ retention An important fraction of the initially deposited activity remained in the lungs over the study period of three months, confirming previous studies obtained with PuO2 or MOX (Matsuoka et al. 1972, Bair 1975, Ramounet et al. 2000, Van der Meeren et al. 2007). In rats receiving PBS-liposomes, 60% of initial activity remained in the lungs 3 months after exposure. However, 77% remained in rats treated with clodronate (Figure 1). Fecal and urinary excretions were significantly decreased (p ⬍ 0.05) in clodronate-treated rats as compared to PBS controls. Activity in urine and feces collected for 10 days following administration represented respectively 0.09 ⫾ 0.01% and 1.62 ⫾ 0.39% in clodronate-treated rats and 0.12 ⫾ 0.04% and 2.49 ⫾ 0.64% of ILD in PBS control rats. At 3 months post inhalation, a slight increase in bone retention was observed in clodronate-treated rats. Liver retention was significantly increased in this group (Table II).
Total lung activity (% ILD)
The left lung was isolated from each exsanguinated rat and expanded to inspiratory volume by intratracheal instillation of 10% neutral buffered formalin (Carlo Erba, Val de Reuil, France). After 48–72 h, lungs were dehydrated and embedded in paraffin. Five μm sections were stained with hematoxylin eosin for microscopic evaluation or processed through autoradiography or immunohistochemistry. Lung sections were deparaffinized. After antigen retrieval using citrate buffer (citrate buffer 0.01 M for 20 min at 98°C), sections were incubated with mouse anti-rat CD68 (ED1), Galectin-3, iNOS (all from Santa Cruz, Heidelberg, Germany) or appropriate IgG control (mouse IgG1 MOPC 21, SigmaAldrich). Sections were then incubated with biotinylated secondary antibody (Vector Labs, Les Ulis, France) and binding was visualized using a peroxidase substrate kit 3-3′ diaminobenzidine (DAB).
110 100 90
*
80 70 60 liposome treatment
50 40
0
20
40
60
80
100
Days after inhalation Figure 1. Consequences of clodronate administration on lung retention after MOX inhalation. Activity is expressed as a percentage of ILD, and represents mean ⫾ SD from 6 animals. *p ⬍ 0.05 between groups as indicated.
time-point, the number of AM in clodronate-treated rats was still 2.8-fold less than PBS controls. This might result from a higher capacity of newly recruited AM to phagocytize MOX particles rather than a higher number of AM able to engulf particles since the percentage of AM-associated activity was only slightly higher (1.3-fold) in clodronate-treated rats. Activity in ELF was also higher in these animals (Table III). The high activity in macrophages in clodronate-treated rats is also illustrated on autoradiographs from isolated AM (Figure 2A, 2B).
Cytokine and chemokine production TNF-α and chemokine production was measured in the supernatant from AM in culture. Immediately after liposome treatment, macrophages from clodronate-treated animals produced more TNF-α and chemokines than those from control rats, however, no difference was observed between sham- or MOX-exposed animals (Figure 3). Three months post-inhalation, chemokine production returned to basal values for both clodronate- and liposome-treated sham animals, although they remained elevated for MOX-exposed rats, whatever liposome treatment. TNF-α returned to basal levels in all groups of rats.
BAL cell analysis No sign of major inflammatory reaction was seen in BAL obtained from MOX-contaminated rats. Cells in BAL were over 98% macrophages and no significant difference Table II. Consequences of clodronate administration on systemic organ retention: Alpha activity was measured by scintillation counting in bone and liver of rats 6 weeks and 3 months post-inhalation. Numbers are % of activity in organs/initial lung deposit (ILD) and are mean ⫾ SD (n ⫽ 6); *p ⬍ 0.05 as compared to PBS-treated rats. 6 weeks 3 months
Lung distribution of a activity Three months after inhalation, activity contained in BAL was higher in clodronate-treated rats, reflecting the results of whole lung counting. At 6 weeks, the proportion of activity recovered in AM compared to activity found in BAL was 1.6 times more for rats receiving clodronate, although at this
PBS clodronate
*
Bone retention (%/ILD) Liver retention (%/ILD)
PBS liposomes
Clodronate liposomes
PBS liposomes
0.48 ⫾ 0.08
0.47 ⫾ 0.05
0.68 ⫾ 0.15 0.82 ⫾ 0.11
0.05 ⫾ 0.02
0.06 ⫾ 0.04
0.04 ⫾ 0.01 0.06 ⫾ 0.02*
PBS, Phosphate Buffered Saline.
Clodronate liposomes
1098 A. Van Der Meeren et al. Table III. Consequences of clodronate administration on lung distribution: Alpha activity was measured by scintillation counting at 6 weeks and 3 months post-inhalation. Numbers are activity in samples expressed as % initial lung deposit (ILD). Percentages of alveolar macrophage (AM) associated with MOX particles were determined after autoradiography of AM. Numbers are mean ⫾ SD (n ⫽ 3). 6 weeks 3 months
Number of macrophages in BAL (⫻ 106) Activity in BAL (kBq) Activity in AM (Bq/106 cells) % of AM associated with MOX particles Activity in ELF (Bq)
PBS liposomes
Clodronate liposomes
PBS liposomes
Clodronate liposomes
8.8 ⫾ 1.38
3.1 ⫾ 0.5
8.1 ⫾ 2.4
11.4 ⫾ 4.0
3.14 ⫾ 0.97 365 ⫾ 125 41.8 ⫾ 8.3
4.02 ⫾ 0.14 1330 ⫾ 221 57.8 ⫾ 6.8
1.45 ⫾ 0.47 178 ⫾ 6.3 11.6 ⫾ 3.8
3.36 ⫾ 0.96 336 ⫾ 182 6.6 ⫾ 3.2
41.3 ⫾ 24.7
370.0 ⫾ 168.2
10.9 ⫾ 6.9
20.7 ⫾ 12.4
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BAL, Broncho-Alveolar Lavages; MOX, Mixed OXide fuel; ELF, Epithelial Lining Fluid; PBS, Phosphate Buffered Saline.
between groups was observed for neutrophils or lymphocytes (Table IV). Three months after inhalation, a higher proportion of binucleated macrophages was observed in MOX rats compared to sham, with respectively in PBS- and clodronate-treated rats 2.5 ⫾ 0.7 and 3.9 ⫾ 1.5% and in sham 1.4 ⫾ 0.7 and 1.2 ⫾ 0.7%. However, the difference was statistically significant only for the PBS-treated rats. Total protein content was higher in MOX-clodronatetreated rats compared to all other groups. Although this seems to result from a combined effect of clodronate and MOX treatments at 6 weeks (significant differences between clodronate-treated rats and PBS-treated rats for both shamor MOX-exposed), the MOX effect seems to be prominent at 3 months (Table IV).
Lung damage and macrophage activation Histological analyses were carried out on lungs from MOXcontaminated rats. Six weeks post-inhalation, no overt damage in the lungs of MOX-exposed animals was observed, and MOX particles were homogeneously distributed at the tissue level. At 3 months, lung autoradiograph sections showed retention of activity in regions of alveolitis (areas with numerous enlarged and vacuolized AM in alveoli, as well as proteinaceous material) as well as in fibrotic lesions (areas with accumulation of dense connective tissue) (Figure 4A, 4B). No differences were observed between PBS and clodronateliposome-treated rats. In these animals, foci of inflammatory lesions were observed, such as congestion and occlusion
of alveoli with exudates, as well as macrophagic alveolitis, with the presence of a high number of foamy macrophages. Fibrotic lesions were also evidenced by Sirius red staining of collagen (Figure 4C, 4D). No major differences were seen between clodronate- or PBS-treated rats. Nonetheless, the number of lesions on each slice of lung seemed lower in clodronate-treated rats than in PBS controls. Unfortunately, the small number of animals dedicated to histological analysis in each group was not sufficient to powerfully compare the two groups. CD68 immunolabelling showed increased numbers of activated macrophages 3 months post-inhalation, with no differences observed between PBS- and clodronate-treated animals (Figure 4E, 4F). CD68 was also expressed by macrophages in LPS-treated rats used as positive controls (Figure 4G). Specific markers for M1 or M2 phenotypes were used to determine which macrophage subtypes were present in macrophagic alveolitis. iNOS, a marker of classicallyactivated macrophages (M1) was expressed in macrophages from LPS-treated rats as expected (Figure 4J). However, iNOS was barely detectable in lungs of MOX-contaminated rats whatever the time after inhalation. The increased labeling observed in PBS-treated rats 3 months post inhalation seemed to be associated with labeling of Pneumocyte II cells and/or interstitial macrophages (Figure 4H). Galectin-3 was used as a marker of the M2 phenotype. As shown in Figure 4K, and 4L, foamy macrophages were strongly labeled with Galectin-3 antibody and Galectin-3 positive
Figure 2. Pu retention in alveolar macrophages. Autoradiographs from cells isolated from Broncho-Alveolar Lavages (BAL) 6 weeks after MOX inhalation in PBS controls (A) or clodronate-treated rats (B), original magnification ⫻ 400.
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Sham PBS Sham clodronate MOX PBS MOX clodronate
180 160
TNF (pg/ml)
140 120 100 80 60
(B) 600 500
CINC-1 (pg/ml)
(A) 200
400 300 200
40
100
20 0
0
6 weeks
3 months
6 weeks
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(C)
(D)
8000
2500 2000
MCP-1 (pg/ml)
MIP-2 (pg/ml)
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3 months
Time after inhalation
4000
2000
1500 1000 500
0
0
6 weeks
3 months
6 weeks
Time after inhalation
3 months
Time after inhalation
Figure 3. Cytokine and chemokine production by alveolar macrophages. Alveolar macrophages were recovered by lavage 6 weeks and 3 months after MOX inhalation. Concentrations of Tumor Necrosis Factor-α (TNF-α) (panel A), Cytokine-Induced neutrophil Chemoattractant-1 (CINC-1) (B), Macrophage Inflammatory Protein-2 (MIP-2) (C) and Monocyte Chemotactic Protein-1 (MCP-1) (D) were measured with ELISA in culture supernatants collected 24 h after plating. Results are expressed as means ⫾ SD from three animals.
cells accumulated in fibrotic and inflammatory lesions. Galectin-3 labelling followed by autoradiography showed that M2 macrophages do not necessarily contain MOX particles (Figure 4N and 4O). No Galectin 3 positive cells were observed in lungs from LPS-treated rats (Figure 4M).
Discussion In addition to their role in local distribution of activity and early inflammatory response of the lungs, macrophages may also play a role in fibrosis development after actinide oxide inhalation. To further investigate this hypothesis, a depletion of AM was induced 6 weeks post MOX inhalation prior the appearance of alveolitis or fibrotic lesions in the lungs. Depletion of macrophages followed by functional studies provides a generally accepted approach to establish their role in any particular event. At 6 weeks, most of the MOX
particles were expected to be retained in AM. Thus, following clodronate treatment, the majority of AM exposed to α particle irradiation consecutive to MOX particle engulfment were eliminated and replaced by newly recruited cells. Our experiment was designed to evaluate the consequence of this elimination of potentially activated macrophages. Administration of liposome-clodronate effectively depleted AM in MOX-exposed rats. This induced a redistribution of MOX particles in different lung compartments (from macrophages to ELF). As a consequence of AM apoptosis, an important fraction of activity may be released. During the time AM numbers are below normal levels, the activity released by macrophages might remain trapped at other retention sites within lungs such as interstitial macrophages or some components of the epithelial lining fluid. This is illustrated by a 10-fold increase in activity recovered in ELF in clodronate-treated rats as compared
Table IV. Consequences of clodronate administration on Broncho-Alveolar Lavages (BAL) differential and protein content: BAL was collected at 6 weeks and 3 months post-inhalation. Percentages of alveolar macrophage (AM), lymphocytes (lympho) and neutrophils (PMN) were based on morphological analysis of 100–400 cells. Protein content was measured in the acellular fraction of BAL. Numbers are the mean ⫾ SD (n ⫽ 3). 6 weeks 3 months Sham PBS % AM % lympho % PMN Protein (μg/ml)
Clodronate
99.2 ⫾ 0.2 99.0 ⫾ 0.9 0 0 0 0 151 ⫾ 66 392 ⫾ 25*
MOX PBS
Clodronate
Sham PBS
98.8 ⫾ 0.7 98.5 ⫾ 0.8 98.6 ⫾ 0.8 0 0 0.2 ⫾ 0.2 0 0 0.1 ⫾ 0.2 265 ⫾ 68# 640 ⫾ 212* 91 ⫾ 22
PBS, Phosphate Buffered Saline; *p ⬍ 0.05 clodronate vs. PBS; #p ⬍ 0.05 MOX vs. Sham.
Clodronate 98.6 ⫾ 0.5 0.1 ⫾ 0.2 0 104 ⫾ 50#
MOX PBS
Clodronate
99.2 ⫾ 0.2 99.0 ⫾ 0.9 0 0.3 ⫾ 0.5 0.3 ⫾ 0.5 0.3 ⫾ 0.3 239 ⫾ 86 334 ⫾ 116#
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Figure 4. Lung damage and macrophage activation after MOX inhalation. Lungs were harvested for paraffin inclusion 3 months post-MOX inhalation, or 4 h after LPS challenge. Panels A and B show autoradiographs of rat lung sections followed by hemalun staining in rats treated with PBS-liposomes (A) or clodronate-liposomes (B). Sirius red staining shows collagen accumulation after MOX inhalation in rats treated with PBS-liposomes (C) or clodronate-liposomes (D). Bars represent 100 μm. Panels E–M are rat lung sections stained with anti-CD68 (E–G), anti-iNOS (H–J) or anti-galectin 3 (K–M) antibodies in rats treated with PBS-liposomes (E, H, K) or clodronate-liposomes (F, I, L) or in positive controls treated with LPS (G, J, M). Panels N and O are galectin-3 immunostaining followed by autoradiographs in rats treated with PBS-liposomes (N) or clodronate-liposomes (O). Bars represent 50 μm (A–M) or 25 μm (N–O). This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
to controls. ELF represents an acellular lung compartment that transiently retains dissolved actinides prior to their transfer to the bloodstream and their subsequent systemic
deposition (Gremy et al. 2010, Van der Meeren and Gremy 2010). Another consequence of AM depletion is the increase in surfactant pool sizes (Forbes et al. 2007), which could
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Macrophage depletion after MOX inhalation 1101 mathematically increase fraction of actinide retained in ELF in the absence of AM. A few weeks after AM depletion, a return to a distribution of activity similar to rats receiving PBS-liposomes was observed, although the α activity level remained higher in the clodronate-treated rats. In addition to the local redistribution of activity, we also report changes in the excretion/retention pattern of MOX. The percentage of activity transferred from lungs to blood is determined by urinary excretion and bone and liver retention measurements. The decrease in fecal excretion in clodronatetreated rats is likely to result from a smaller number of AM excreted into the gastro-intestinal tract through muco-ciliary escalator. MOX particles retained in macrophages during the first 6 weeks post MOX inhalation may undergo partial solubilization in phagolysosomes (Muller et al. 1989, Henge-Napoli et al. 1996). After clodronate-induced AM apoptosis, a fraction of solubilized elements from MOX particles can transfer to the systemic compartment and be retained by bone and liver. The slight decrease observed in urinary excretion is more difficult to understand. A possible explanation is the incomplete solubilization of Pu leading to the release of ultrafine particles, able to cross the alveolo-capillary barrier and recognized by cells from reticulo-endothelial system such as liver, where they remained trapped. We next evaluated whether the higher MOX retention in the lungs of rats receiving clodronate would lead to increased lung damage after MOX inhalation. The consequences of MOX inhalation were similar to those described after PuO2 by us and others, i.e., at 3 months, alveolitis and fibrosis. The presence of abnormal macrophages was also reported (foamy, activated, binucleated) despite the high recognized radioresistance of these cells (Talbot et al. 1989). The type of lesions in lung parenchyma observed in both groups of MOX-contaminated rats, were similar, with a tendency for lower number of lesions observed in clodronate-treated rats. Similarly, a lower number of binucleated cells was found in clodronate-treated rats as compare to PBS-treated animals. Because diminution in lung excretion might result from activation of macrophages after clodronate instillation, and since we have previously shown that Pu lung exposure induced early activation of macrophages (Van der Meeren and Gremy 2010, Van der Meeren et al. 2012) we evaluated AM activation using different parameters. First, we measured the production of TNF-α and chemokines by AM. The production and release of cytokines from macrophages are critical for inflammatory responses. The type of cytokines and chemokines significantly influences the quality, duration, and magnitude of most inflammatory reactions. Depending on the cytokine microenvironment, macrophages can adopt a proinflammatory or a profibrotic phenotype. Amongst others, the production TNF-α has been associated to the M1 phenotype. MCP-1 belongs to the C-C chemokine family and promotes both the recruitment of monocytes and their maturation to macrophages (Maus et al. 2002). MCP-1 is produced by AM of M1 phenotype, but also by M2a (alternative type II inflammation) (Lech and Anders 2013). MIP-2 and CINC-1 are both members of the CXC chemokine family. Both have been shown to participate in inflammatory responses involving neutrophils, and MIP-2 to participate in fibrogenesis. To
our knowledge, their related expression with polarization has not been described following internal contamination with actinides. Our results show an increase in inflammatory mediator production at 6 weeks, but more likely as a consequence of clodronate treatment, which can mask the effect of MOX. At 3 months, TNF-α returned to basal levels although chemokines remained elevated in both groups of MOX-contaminated rats. In another approach, we performed immunostaining in lung slices, using various markers of macrophage activation/polarization. Similarly to what we described after Pu contamination (Van der Meeren et al. 2008, 2012), CD68 positive cells were increased after MOX. Because CD68 is expressed on all macrophages (Murray and Wynn 2011), we used specific markers for M1 and M2 phenotypes. The role of macrophages in fibrosis is now recognized and has been identified in all stages of fibrogenesis. Particularly, M2 macrophages are more supportive of a fibro-proliferative microenvironment (Strieter 2008, Boorsma et al. 2013). Markers found on M2 macrophages have also be found to be increased in pulmonary fibrosis, such as Galectin 3 (Nishi et al. 2007), supporting the use of Galectin-3 as a good marker for M2 macrophages. iNOS is the most common marker used for M1 macrophages. It has been proposed that classical activation of macrophages (M1) is an important player in inducing radiation pneumonitis while the alternative activation (M2) is associated with radiation fibrosis (Zhang et al. 2011). Because of the high level of expression of galectin-3 and the lack of expression of iNOS in the areas of lungs showing accumulation of foamy macrophages 3 months post-MOX inhalation, we can conclude that the M2 phenotype is associated with fibrotic areas. However, Galectin-3 expression is not necessarily linked to the presence of particles. M2 macrophages are observed whatever the liposome treatment indicating that, at this time of treatment, clodronate does not greatly affect MOX-induced fibrotic development. This confirms previous studies where depletion of tissue-resident macrophages during the inflammatory phase did not affect the degree of fibrosis that develops after this inflammatory phase, although depletion during the fibrotic phase of lung fibrosis reduced the extra-cellular matrix deposition in this organ (Gibbons et al. 2011). The phenotype of macrophages, regardless of prior macrophage depletion, were similar, underlying the importance of local microenvironment, including the presence of particles, on macrophage polarization status following actinide inhalation. It has been described by others (Hong et al. 2003) that following external irradiation, differential roles for interstitial and AM, interstitial macrophages being involved in late pneumonitis, and alveolar in the early inflammatory response. However, we demonstrate in our study that AM are highly activated three months post actinide inhalation and express markers of M2 phenotype, raising the hypothesis of the role of AM in late pneumonitis. Our data indicate that the transient AM depletion induces a delay in MOX excretion as well as a transient modification of lung activity distribution. However, no major changes on long-term (3 months) macrophage activation status and lung damage were observed.
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Acknowledgements We acknowledge financial support from AREVA. The assistance of Quang Chau, Daniel Renault, Françoise Tourdes and Marie-Claire Abram is highly appreciated.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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References Andre S, Charuau J, Rateau G, Vavasseur C, Metivier H. 1989. Design of a new inhalation device for rodents and primates. J Aerosol Sci 20:647–656. Bair WJ. 1975. Recent animal studies on the deposition, retention and translocation of plutonium and other transuranic compounds. In: Proceedings of Seminar by the International Atomic Energy Agency and the World Health Organization. Vienna, December 8–12, 1975. Diagnosis and treatment of incorporated radionuclides. pp. 51–83. Bair WJ, Park GE, Dagle GE, James AC. 1989. Overview of biological consequences of exposure to plutonium and higher actinides. Radiat Protect Dosimetry 26:125–135. Berg JT, Lee ST, Thepen T, Lee CY, Tsan MF. 1993. Depletion of alveolar macrophages by liposome-encapsulated dichloromethylene diphosphonate. J Appl Physiol 74:2812–2819. Boorsma CE, Draijer C, Melgert BN. 2013. Macrophage heterogeneity in respiratory diseases. Mediators of Inflamm 2013:769214. Diel JH, Guilmette RA , Muggenburg BA , Hahn FF, Chang IY. 1992. Influence of dose rate on survival time for 239PuO2-induced radiation pneumonitis or pulmonary fibrosis in dogs. Radiat Res 129: 53–60. Dudoignon N, Guillet K , Fritsch P. 2003. Evaluation of risk factors for lung tumour induction in rats exposed to either NpO(2) or PuO(2) aerosols. Int J Radiat Biol 79:169–174. Elder AC, Gelein R, Oberdorster G, Finkelstein J, Notter R, Wang Z. 2004. Efficient depletion of alveolar macrophages using intratracheally inhaled aerosols of liposome-encapsulated clodronate. Experim Lung Res 30:105–120. Forbes A , Pickell M, Foroughian M, Yao LJ, Lewis J, Veldhuizen R. 2007. Alveolar macrophage depletion is associated with increased surfactant pool sizes in adult rats. J Appl Physiol 103:637–645. Gibbons MA , MacKinnon AC, Ramachandran P, Dhaliwal K , Duffin R, Phythian-Adams AT, van Rooijen N, Haslett C, Howie SE, Simpson AJ, et al. 2011. Ly6Chi monocytes direct alternatively activated profibrotic macrophage regulation of lung fibrosis. Am J Respirat Crit Care Med 184:569–581. Gilbert ES, Koshurnikova NA , Sokolnikov ME, Shilnikova NS, Preston DL, Ron E, Okatenko PV, Khokhryakov VF, Vasilenko EK, Miller S, et al. 2004. Lung cancer in Mayak workers. Radiat Res 162:505–516. Gordon S, Martinez FO. 2010. Alternative activation of macrophages: Mechanism and functions. Immunity 32:593–604. Gremy O, Tsapis N, Chau Q, Renault D, Abram MC, Van der Meeren A . 2010. Preferential decorporation of americium by pulmonary administration of DTPA dry powder after inhalation of aged PuO(2) containing americium in rats. Radiat Res 174:637–644. Griffiths NM, Van der Meeren A , Fritsch P, Abram MC, Bernaudin JF, Poncy JL. 2010. Late-occurring pulmonary pathologies following inhalation of mixed oxide (uranium ⫹ plutonium oxide) aerosol in the rat. Health Phys 99:347–356. Henge-Napoli MH, Ansoborlo E, Claraz M, Berry JP, Cheynet MC. 1996. Role of alveolar macrophages in the dissolution of two different industrial uranium oxides. Cell Molec Biol (Noisy-le-grand) 42:413–420. Hong JH, Jung SM, Tsao TC, Wu CJ, Lee CY, Chen FH, Hsu CH, McBride WH, Chiang CS. 2003. Bronchoalveolar lavage and interstitial cells have differnet roles in radiation-induced lung injury. Int J Radiat Biol 79:159–167. International Commission on Radiological Protection (ICRP). 1972. The metabolism of compounds of plutonium and other actinides. ICRP publication 19.
Johnston CJ, Williams JP, Okunieff P, Finkelstein JN. 2002. Radiationinduced pulmonary fibrosis: Examination of chemokine and chemokine receptor families. Radiat Res 157:256–265. Johnston CJ, Williams JP, Elder A , Hernady E, Finkelstein JN. 2004. Inflammatory cell recruitment following thoracic irradiation. Experim Lung Res 30:369–382. Lech M, Anders HJ. 2013. Macrophages and fibrosis: How resident and infiltrating mononuclear phagocytes orchestrate all phases of tissue injury and repair. Biochim Biophys Acta 1832:989–997. Lehenkari PP, Kellinsalmi M, Näpakangas JP, Ylitalo KV, Mönkkönen J, Rogers MJ, Azhayev A , Väänänen HK , Hassinen IE. 2002. Further insight into mechanism of action of clodronate: Inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite. Molec Pharmacol 61: 1255–1262. Mantovani A , Sica A , Sozzani S, Allavena P, Vecchi A , Locati M. 2004. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25:677–686. Matsuoka O, Kashima M, Joshima H, Noda Y. 1972. Whole-body autoradiographic studies on plutonium metabolism as affected by its physico-chemical state and route of administration. Health Phys 22:713–722. Maus U, von Grote K, Kuziel WA , Mack M, Miller EJ, Cihak J, Stangassinger M, Maus R, Schlondorff D, Seeger W, et al. 2002. The role of CC chemokine receptor 2 in alveolar monocyte and neutrophil immigration in intact mice. Am J Respirat Crit Care Med 166:268–273. Muggenburg BA , Guilmette RA , Hahn FF, Diel JH, Mauderly JL, Seilkop SK, Boecker BB. 2008. Radiotoxicity of inhaled 239PuO2 in dogs. Radiat Res 170:736–757. Muller HL, Drosselmeyer E, Hotz G, Seidel A , Thiele H, Pickering S. 1989. Behaviour of spherical and irregular (U,Pu)O2 particles after inhalation or intratracheal instillation in rat lung and during in vitro culture with bovine alveolar macrophages. Int J Radiat Biol 55:829–842. Murray PJ, Wynn TA . 2011. Protective and pathogenic functions of macrophage subsets. Nature Rev Immunol 11:723–737. Newman LS, Mroz MM, Ruttenber AJ. 2005. Lung fibrosis in plutonium workers. Radiat Res 164:123–131. Nishi Y, Sano H, Kawashima T, Okada T, Kuroda T, Kikkawa K , Kawashima S, Tanabe M, Goto T, Matsuzawa Y, et al. 2007. Role of galectin-3 in human pulmonary fibrosis. Allergol Int 56: 57–65. Park JF, Watson CR, Buschbom RL, Dagle GE, Strom DJ, Weller RE. 2012. Biological effects of inhaled 239PuO2 in beagles. Radiat Res 178:447–467. Ramounet B, Matton S, Guezingar-Liebard F, Abram MC, Rateau G, Grillon G, Fritsch P. 2000. Comparative biokinetics of plutonium and americium after inhalation of PuO2 and mixed oxides (U, Pu)O2 in rat. Int J Radiat Biol 76:215–222. Sanders CL. 1969. The distribution of inhaled plutonium-239 dioxide particles within pulmonary macrophages. Arch Environ Health 18:904–912. Sanders CL, McDonald KE, Lauhala KE. 1988. Promotion of pulmonary carcinogenesis by plutonium particle aggregation following inhalation of 239PuO2. Radiat Res 116:393–405. Sanders CL, Lauhala KE, McDonald KE. 1993. Lifespan studies in rats exposed to 239PuO2 aerosol. III. Survival and lung tumours. Arch Environ Health 64:417–430. Sokolnikov ME, Gilbert ES, Preston DL, Ron E, Shilnikova NS, Khokhryakov VV, Vasilenko EK , Koshurnikova NA . 2008. Lung, liver and bone cancer mortality in Mayak workers. Int J Cancer 123: 905–911. Sonoki T, Nagasaki A , Gotoh T, Takiguchi M, Takeya M, Matsuzaki H, Mori M. 1997. Coinduction of nitric-oxide synthase and arginase I in cultured rat peritoneal macrophages and rat tissues in vivoby lipopolysaccharide. J Biologic Chem 272:3689–3693. Strieter RM. 2008. What differentiates normal lung repair and fibrosis? Inflammation, resolution of repair, and fibrosis. Proc Am Thoracic Soc 5:305–310. Talbot RJ, Moores SR. 1985. The development and interlobar distribution of plutonium-induced pulmonary fibrosis in mice. Radiat Res 103:135–148. Talbot RJ, Nicholls L, Morgan A , Moores SR. 1989. Effect of inhaled alpha-emitting nuclides on mouse alveolar macrophages. Radiat Res 119:271–285.
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Van der Meeren A , Gremy O. 2010. Isotopic and elemental composition of plutonium/americium oxides influence pulmonary and extra-pulmonary distribution after inhalation in rats. Health Phys 99:380–387. Van der Meeren A , Grillon G, Tourdes F, Rateau S, Le Gall B, Griffiths N. 2007. Influence of initial lung deposit on pulmonary clearance after plutonium oxide inhalation in rat. Radiat Protect Dosimetry 127:50–54. Van der Meeren A , Tourdes F, Gremy O, Grillon G, Abram MC, Poncy JL, Griffiths N. 2008. Activation of alveolar macrophages after plutonium oxide inhalation in rats: Involvement in the early inflammatory response. Radiat Res 17:591–603.
Van der Meeren A , Gremy O, Renault D, Miroux A , Bruel S, Griffiths N, Tourdes F. 2012. Plutonium behavior after pulmonary administration according to solubility properties, and consequences on alveolar macrophage activation. J Radiat Res 53:184–194. Van Rooijen N, Sanders A . 1994. Liposome mediated depletion of macrophages: Mechanism of action, preparation of liposomes and applications. J Immunol Meth 174:83–93. Zhang H, Han G, Liu H, Chen J, Ji X, Zhou F, Zhou Y, Xie C . 2011. The development of classically and alternatively activated macrophages has different effects on the varied stages of radiation-induced pulmonary injury in mice. J Radiat Res 52: 717–726.
Macrophages as key elements of Mixed-oxide [U-Pu(O2)] distribution and pulmonary damage after inhalation? Anne Van der Meeren, Agnes Moureau & Nina M. Griffiths
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Laboratoire de RadioToxicologie, CEA/DSV/iRCM, Bruyères le Châtel, Arpajon, France
consequences of actinide exposure include both stochastic effects, mainly tumors of epithelial origin and determinist effects, such as radiation pneumonitis and interstitial fibrosis (Talbot and Moores 1985, Sanders et al. 1988, Diel et al. 1992, Dudoignon et al. 2003, Newman et al. 2005, Muggenburg et al. 2008, Griffiths et al. 2010, Park et al. 2012). These have been described both in experimental animals and in nuclear workers, such as the cohort from Mayak (Bair et al. 1989, Sanders et al. 1993, Gilbert et al. 2004, Sokolnikov et al. 2008). Shortly after inhalation, the majority of α particles are phagocytized by alveolar macrophages, where they can remain for long periods of time (Sanders 1969, Muller et al. 1989). Inflammatory cells and particularly macrophages are not only central elements in the lung distribution of actinides but also in the development of radiation fibrosis (Johnston et al. 2002). Macrophages are also involved in the early inflammatory response following lung contamination with actinides (Van der Meeren and Gremy 2010, Van der Meeren et al. 2012). It is now recognized that depending on cytokine microenvironment, macrophages can adopt a proinflammatory or a profibrotic phenotype. To parallel macrophage activation with T helper cell polarization, the terms of ‘M1’ and ‘M2’, for macrophages of type 1 or 2, were introduced to describe these functionally distinct subpopulations representing the extremes of a continuum (Mantovani et al. 2004). Excessive scarring and tissue fibrosis may result from an imbalanced action of M1-and M2-polarized macrophages during prolonged lung inflammation (Strieter 2008, Gordon and Martinez 2010). One way to investigate the in vivo function of a particular cell type is to deplete these cells, and to observe the consequences of this depletion. Clodronate entrapped in liposomes has been widely used to selectively deplete macrophages in lungs after intratracheal administration (Berg et al. 1993, Elder et al. 2004, Johnston et al. 2004). Liposomes are used as a ‘Trojan horse’ to deliver the small clodronate molecule inside the macrophages. This drug belonging to bisphosphonate family, induces apoptosis and necrosis in target cells (Van Rooijen and Sanders 1994, Lehenkari et al. 2002).
Abstract Purpose: To investigate the consequences of alveolar macrophage (AM) depletion on Mixed OXide fuel (MOX: U, Pu oxide) distribution and clearance, as well as lung damage following MOX inhalation. Materials and methods: Rats were exposed to MOX by nose only inhalation. AM were depleted with intratracheal administration of liposomal clodronate at 6 weeks. Lung changes, macrophage activation, as well as local and systemic actinide distribution were studied up to 3 months post-inhalation. Results: Clodronate administration modified excretion/retention patterns of a activity. At 3 months post-inhalation lung retention was higher in clodronate-treated rats compared to Phosphate Buffered Saline (PBS)-treated rats, and AM-associated a activity was also increased. Retention in liver was higher in clodronatetreated rats and fecal and urinary excretions were lower. Three months after inhalation, rats exhibited lung fibrotic lesions and alveolitis, with no marked differences between the two groups. Foamy macrophages of M2 subtype [inducible Nitric Oxide Synthase (iNOS) negative but galectin-3 positive] were frequently observed, in correlation with the accumulation of MOX particles. AM from all MOX-exposed rats showed increased chemokine levels as compared to sham controls. Conclusion: Despite the transient reduced AM numbers in clodronate-treated animals no major differences on lung damage were observed as compared to non-treated rats after MOX inhalation. The higher lung activity retention in rats receiving clodronate seems to be part of a general inflammatory response and needs further investigation. Keywords: Actinide inhalation, macrophage depletion, lung damage
Introduction Accidental inhalation of α-emitting particles from Mixed OXide fuel (MOX: U, Pu oxide) poses a potential longterm health risk to nuclear industry workers. Pathological
Correspondence: Dr Anne Van der Meeren, PhD, Laboratoire de RadioToxicologie, CEA/DSV/iRCM, Bruyères le Châtel, 91297 Arpajon, France Tel: ⫹ 33 1 6926 5553. Fax: ⫹ 33 1 6926 7045. E-mail: [email protected] (Received 6 November 2013; revised 23 May 2014; accepted 7 July 2014)
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In the present study we investigated whether the depletion of AM 6 weeks after inhalation of MOX affects retention/ excretion of MOX, α particle distribution and lung damage, and modifies macrophage activation/polarization status.
Materials and methods
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Animals and inhalation procedure Male Sprague Dawley rats, 250 g at the time of exposure, were obtained from Charles River Laboratories (l’Arbresle, France), quarantined at least 7 days before exposure, housed 3 or 4 to a cage and maintained at constant temperature on a 12:12 h light-dark cycle. They received commercial rodent chow and water ad libitum. Housing and experiments were carried out in compliance with the French regulation for animal experimentation (European act 2001-246, June 6, 2001). MOX powder from the rectification step was produced by the MIcronised MASter Blend (MIMAS) procedure at the MELOX installation (Marcoule, France). MOX contained 81% uranium and 7.1% plutonium (by mass). Due to 241Pu decay in 241Am, approximately 23% of total α activity of MOX was accounted for by americium (Am). The specific activity was 123.4 kBq/mg at the time of experimentation, and Activity Median Aerodynamic Diameter was 4.2 μm, σg2.7. Thirty rats restrained in cardboard tubes, were ‘nose-only’ exposed to a MOX aerosol generated as described previously (Andre et al. 1989). Sham-exposed animals were treated in the same manner but were not exposed to the aerosol. The initial lung deposit (ILD) in animals was determined 7 days after inhalation on anesthetized rats (intraperitoneal sodium pentobarbitone, 40 mg/kg, Ceva Santé Animal, Libourne, France) by measurement of thorax-associated radioactivity by γ-ray spectrometry. In the present experiment, ILD was evaluated to be 20.8 ⫾ 4.3 kBq (n ⫽ 27). To determine lung activity excretion, similar counting was done from 2 weeks to 3 months post exposure, for a total of four counting times for all rats. For collection of urine and feces, animals were kept individually in metabolism cages. Lipopolysaccharide (LPS) was used as a positive control to induce M1 differentiation (Sonoki et al. 1997). For this purpose, 2.5 mg LPS (Escherichia Coli 026:B6, Sigma Aldrich, St Quentin-Fallavier, France) was intratracheally administered under light gaseous anesthesia (2.5% isoflurane, Virbac, Carros, France). Rats were euthanized by exsanguination from the dorsal aorta 4 h after administration and lungs harvested for paraffin embedding.
Samples collection and activity measurement Urine and feces were collected daily for 7 days, starting the day of liposome administration. Six weeks and 3 months post exposure, six rats from each group were euthanized. Liver and femurs were taken for measurement of activity. Lungs were collected and divided in two batches for each time-point: One was dedicated to Broncho-Alveolar Lavages (BAL) and determination of total lung activity, and the second one was used for histology. BAL were carried out after euthanasia with four sequential washes to a total of 24 ml of warmed sterile PBS (pH 7.4). Centrifugation of BAL (300 g,
5 min) allowed the separation into a cellular fraction (over 95% AM) and an acellular fraction containing the Epithelial Lining Fluid (ELF). An aliquot of the ELF was taken to determine the total protein content with a BCA assay (Bicinchoninic acid Reagent kit, Fisher Scientific, Illkrich, France). Total α activity was measured in biological samples (femurs, liver, urine, lavaged lungs, BAL, AM and ELF) by liquid scintillation counting using a Packard liquid scintillation counter, after dry ashing and wet ashing with nitric acid (2M) and H2O2 (30%) treatments until a clear solution was obtained. The skeletal retention of actinides was estimated assuming that the two femurs represent 10% of the whole skeleton (International Commission on Radiological Protection [ICRP] 1972). Activity in feces was determined by γ-ray spectrometry.
Cell culture and cytokine determination AM were resuspended in Roswell Park Memorial Institute (RPMI) medium containing 10% Foetal Calf Serum, 200 mM L-Glutamine, 10 IU/ml penicillin and 10 mg/ml streptomycin, all obtained from Sigma Aldrich. Cells were plated into 24-well dishes at a concentration of 105 cells/well. Supernatants were collected 24 h after plating and assessed for cytokine and chemokine content by specific EnzymeLinked Immuno Sorbent Assay (ELISA) as recommended by the manufacturers (R&D Systems Europe, Lille, France) for Tumor Necrosis Factor-α (TNF-α) and Cytokine-Induced neutrophil Chemoattractant-1 (CINC-1) (Biosource, Life technologies, St Aubin, France) for Macrophage Inflammatory Protein-2 (MIP-2) and Amersham Biosciences GE Healthcare Europe (Vélizy-Villacoublay, France) for Monocyte Chemotactic Protein-1 (MCP-1).
Clodronate administration For AM depletion, Sham- or MOX-exposed rats were administered intratracheally under light gaseous anesthesia with a suspension of 0.25 ml containing either clodronate-encapsulated liposomes (5 mg/ml) or PBS-encapsulated liposomes to serve as a control (Clodronate Foundation, Haarlem, The Netherlands) at 39 days post inhalation, then 2 days later. Rats were euthanized 3 and 49 days after the last administration, equivalent to 6 weeks and 3 months post-inhalation. Table I demonstrates the effects of clodronate administration on macrophage number and MCP-1 production in shamrats, non-exposed to MOX.
Table I. Consequences of clodronate administration in sham-exposed rats: Alveolar macrophage (AM) were obtained from Broncho-Alveolar Lavages (BAL) 3 days and 6 weeks post clodronate treatment, counted and cultured. Monocyte Chemotactic Protein-1 (MCP-1) production was assessed in supernatants from 24 h-cultures of AM. Numbers are mean ⫾ SD (n ⫽ 3). 3 days 6 weeks PBS liposomes Number of macrophages in BAL (⫻ 106) MCP-1 (pg/ml)
8.4 ⫾ 3.5
Clodronate PBS Clodronate liposomes liposomes liposomes 1.67 ⫾ 0.51 10.1 ⫾ 2.8
189.1 ⫾ 56.2 1911 ⫾ 177
PBS, Phosphate Buffered Saline.
37.6 ⫾ 34.9
14.0 ⫾ 4.9 29.0 ⫾ 1.0
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Immunohistochemistry
Autoradiography studies Determination of MOX particle distribution was carried out by autoradiography studies on lung sections or macrophages collected on slides by cytocentrifugation. Slides were covered with photographic emulsion (NTB 2K-5, Kodak, Anachem, Luton, UK) and exposed for 7 days. Slides were counterstained with hemalun. The percentage of AM associated with MOX particles was determined by counting approximately 200 cells per slide from three different preparations.
Data analysis Data were analyzed using two-tailed Student’s t-test or the nonparametric Mann-Whitney test. A p value of ⬍ 0.05 was considered statistically significant.
Results Excretion and systemic organ retention An important fraction of the initially deposited activity remained in the lungs over the study period of three months, confirming previous studies obtained with PuO2 or MOX (Matsuoka et al. 1972, Bair 1975, Ramounet et al. 2000, Van der Meeren et al. 2007). In rats receiving PBS-liposomes, 60% of initial activity remained in the lungs 3 months after exposure. However, 77% remained in rats treated with clodronate (Figure 1). Fecal and urinary excretions were significantly decreased (p ⬍ 0.05) in clodronate-treated rats as compared to PBS controls. Activity in urine and feces collected for 10 days following administration represented respectively 0.09 ⫾ 0.01% and 1.62 ⫾ 0.39% in clodronate-treated rats and 0.12 ⫾ 0.04% and 2.49 ⫾ 0.64% of ILD in PBS control rats. At 3 months post inhalation, a slight increase in bone retention was observed in clodronate-treated rats. Liver retention was significantly increased in this group (Table II).
Total lung activity (% ILD)
The left lung was isolated from each exsanguinated rat and expanded to inspiratory volume by intratracheal instillation of 10% neutral buffered formalin (Carlo Erba, Val de Reuil, France). After 48–72 h, lungs were dehydrated and embedded in paraffin. Five μm sections were stained with hematoxylin eosin for microscopic evaluation or processed through autoradiography or immunohistochemistry. Lung sections were deparaffinized. After antigen retrieval using citrate buffer (citrate buffer 0.01 M for 20 min at 98°C), sections were incubated with mouse anti-rat CD68 (ED1), Galectin-3, iNOS (all from Santa Cruz, Heidelberg, Germany) or appropriate IgG control (mouse IgG1 MOPC 21, SigmaAldrich). Sections were then incubated with biotinylated secondary antibody (Vector Labs, Les Ulis, France) and binding was visualized using a peroxidase substrate kit 3-3′ diaminobenzidine (DAB).
110 100 90
*
80 70 60 liposome treatment
50 40
0
20
40
60
80
100
Days after inhalation Figure 1. Consequences of clodronate administration on lung retention after MOX inhalation. Activity is expressed as a percentage of ILD, and represents mean ⫾ SD from 6 animals. *p ⬍ 0.05 between groups as indicated.
time-point, the number of AM in clodronate-treated rats was still 2.8-fold less than PBS controls. This might result from a higher capacity of newly recruited AM to phagocytize MOX particles rather than a higher number of AM able to engulf particles since the percentage of AM-associated activity was only slightly higher (1.3-fold) in clodronate-treated rats. Activity in ELF was also higher in these animals (Table III). The high activity in macrophages in clodronate-treated rats is also illustrated on autoradiographs from isolated AM (Figure 2A, 2B).
Cytokine and chemokine production TNF-α and chemokine production was measured in the supernatant from AM in culture. Immediately after liposome treatment, macrophages from clodronate-treated animals produced more TNF-α and chemokines than those from control rats, however, no difference was observed between sham- or MOX-exposed animals (Figure 3). Three months post-inhalation, chemokine production returned to basal values for both clodronate- and liposome-treated sham animals, although they remained elevated for MOX-exposed rats, whatever liposome treatment. TNF-α returned to basal levels in all groups of rats.
BAL cell analysis No sign of major inflammatory reaction was seen in BAL obtained from MOX-contaminated rats. Cells in BAL were over 98% macrophages and no significant difference Table II. Consequences of clodronate administration on systemic organ retention: Alpha activity was measured by scintillation counting in bone and liver of rats 6 weeks and 3 months post-inhalation. Numbers are % of activity in organs/initial lung deposit (ILD) and are mean ⫾ SD (n ⫽ 6); *p ⬍ 0.05 as compared to PBS-treated rats. 6 weeks 3 months
Lung distribution of a activity Three months after inhalation, activity contained in BAL was higher in clodronate-treated rats, reflecting the results of whole lung counting. At 6 weeks, the proportion of activity recovered in AM compared to activity found in BAL was 1.6 times more for rats receiving clodronate, although at this
PBS clodronate
*
Bone retention (%/ILD) Liver retention (%/ILD)
PBS liposomes
Clodronate liposomes
PBS liposomes
0.48 ⫾ 0.08
0.47 ⫾ 0.05
0.68 ⫾ 0.15 0.82 ⫾ 0.11
0.05 ⫾ 0.02
0.06 ⫾ 0.04
0.04 ⫾ 0.01 0.06 ⫾ 0.02*
PBS, Phosphate Buffered Saline.
Clodronate liposomes
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Number of macrophages in BAL (⫻ 106) Activity in BAL (kBq) Activity in AM (Bq/106 cells) % of AM associated with MOX particles Activity in ELF (Bq)
PBS liposomes
Clodronate liposomes
PBS liposomes
Clodronate liposomes
8.8 ⫾ 1.38
3.1 ⫾ 0.5
8.1 ⫾ 2.4
11.4 ⫾ 4.0
3.14 ⫾ 0.97 365 ⫾ 125 41.8 ⫾ 8.3
4.02 ⫾ 0.14 1330 ⫾ 221 57.8 ⫾ 6.8
1.45 ⫾ 0.47 178 ⫾ 6.3 11.6 ⫾ 3.8
3.36 ⫾ 0.96 336 ⫾ 182 6.6 ⫾ 3.2
41.3 ⫾ 24.7
370.0 ⫾ 168.2
10.9 ⫾ 6.9
20.7 ⫾ 12.4
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BAL, Broncho-Alveolar Lavages; MOX, Mixed OXide fuel; ELF, Epithelial Lining Fluid; PBS, Phosphate Buffered Saline.
between groups was observed for neutrophils or lymphocytes (Table IV). Three months after inhalation, a higher proportion of binucleated macrophages was observed in MOX rats compared to sham, with respectively in PBS- and clodronate-treated rats 2.5 ⫾ 0.7 and 3.9 ⫾ 1.5% and in sham 1.4 ⫾ 0.7 and 1.2 ⫾ 0.7%. However, the difference was statistically significant only for the PBS-treated rats. Total protein content was higher in MOX-clodronatetreated rats compared to all other groups. Although this seems to result from a combined effect of clodronate and MOX treatments at 6 weeks (significant differences between clodronate-treated rats and PBS-treated rats for both shamor MOX-exposed), the MOX effect seems to be prominent at 3 months (Table IV).
Lung damage and macrophage activation Histological analyses were carried out on lungs from MOXcontaminated rats. Six weeks post-inhalation, no overt damage in the lungs of MOX-exposed animals was observed, and MOX particles were homogeneously distributed at the tissue level. At 3 months, lung autoradiograph sections showed retention of activity in regions of alveolitis (areas with numerous enlarged and vacuolized AM in alveoli, as well as proteinaceous material) as well as in fibrotic lesions (areas with accumulation of dense connective tissue) (Figure 4A, 4B). No differences were observed between PBS and clodronateliposome-treated rats. In these animals, foci of inflammatory lesions were observed, such as congestion and occlusion
of alveoli with exudates, as well as macrophagic alveolitis, with the presence of a high number of foamy macrophages. Fibrotic lesions were also evidenced by Sirius red staining of collagen (Figure 4C, 4D). No major differences were seen between clodronate- or PBS-treated rats. Nonetheless, the number of lesions on each slice of lung seemed lower in clodronate-treated rats than in PBS controls. Unfortunately, the small number of animals dedicated to histological analysis in each group was not sufficient to powerfully compare the two groups. CD68 immunolabelling showed increased numbers of activated macrophages 3 months post-inhalation, with no differences observed between PBS- and clodronate-treated animals (Figure 4E, 4F). CD68 was also expressed by macrophages in LPS-treated rats used as positive controls (Figure 4G). Specific markers for M1 or M2 phenotypes were used to determine which macrophage subtypes were present in macrophagic alveolitis. iNOS, a marker of classicallyactivated macrophages (M1) was expressed in macrophages from LPS-treated rats as expected (Figure 4J). However, iNOS was barely detectable in lungs of MOX-contaminated rats whatever the time after inhalation. The increased labeling observed in PBS-treated rats 3 months post inhalation seemed to be associated with labeling of Pneumocyte II cells and/or interstitial macrophages (Figure 4H). Galectin-3 was used as a marker of the M2 phenotype. As shown in Figure 4K, and 4L, foamy macrophages were strongly labeled with Galectin-3 antibody and Galectin-3 positive
Figure 2. Pu retention in alveolar macrophages. Autoradiographs from cells isolated from Broncho-Alveolar Lavages (BAL) 6 weeks after MOX inhalation in PBS controls (A) or clodronate-treated rats (B), original magnification ⫻ 400.
Macrophage depletion after MOX inhalation 1099
Sham PBS Sham clodronate MOX PBS MOX clodronate
180 160
TNF (pg/ml)
140 120 100 80 60
(B) 600 500
CINC-1 (pg/ml)
(A) 200
400 300 200
40
100
20 0
0
6 weeks
3 months
6 weeks
Time after inhalation
(C)
(D)
8000
2500 2000
MCP-1 (pg/ml)
MIP-2 (pg/ml)
6000
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3 months
Time after inhalation
4000
2000
1500 1000 500
0
0
6 weeks
3 months
6 weeks
Time after inhalation
3 months
Time after inhalation
Figure 3. Cytokine and chemokine production by alveolar macrophages. Alveolar macrophages were recovered by lavage 6 weeks and 3 months after MOX inhalation. Concentrations of Tumor Necrosis Factor-α (TNF-α) (panel A), Cytokine-Induced neutrophil Chemoattractant-1 (CINC-1) (B), Macrophage Inflammatory Protein-2 (MIP-2) (C) and Monocyte Chemotactic Protein-1 (MCP-1) (D) were measured with ELISA in culture supernatants collected 24 h after plating. Results are expressed as means ⫾ SD from three animals.
cells accumulated in fibrotic and inflammatory lesions. Galectin-3 labelling followed by autoradiography showed that M2 macrophages do not necessarily contain MOX particles (Figure 4N and 4O). No Galectin 3 positive cells were observed in lungs from LPS-treated rats (Figure 4M).
Discussion In addition to their role in local distribution of activity and early inflammatory response of the lungs, macrophages may also play a role in fibrosis development after actinide oxide inhalation. To further investigate this hypothesis, a depletion of AM was induced 6 weeks post MOX inhalation prior the appearance of alveolitis or fibrotic lesions in the lungs. Depletion of macrophages followed by functional studies provides a generally accepted approach to establish their role in any particular event. At 6 weeks, most of the MOX
particles were expected to be retained in AM. Thus, following clodronate treatment, the majority of AM exposed to α particle irradiation consecutive to MOX particle engulfment were eliminated and replaced by newly recruited cells. Our experiment was designed to evaluate the consequence of this elimination of potentially activated macrophages. Administration of liposome-clodronate effectively depleted AM in MOX-exposed rats. This induced a redistribution of MOX particles in different lung compartments (from macrophages to ELF). As a consequence of AM apoptosis, an important fraction of activity may be released. During the time AM numbers are below normal levels, the activity released by macrophages might remain trapped at other retention sites within lungs such as interstitial macrophages or some components of the epithelial lining fluid. This is illustrated by a 10-fold increase in activity recovered in ELF in clodronate-treated rats as compared
Table IV. Consequences of clodronate administration on Broncho-Alveolar Lavages (BAL) differential and protein content: BAL was collected at 6 weeks and 3 months post-inhalation. Percentages of alveolar macrophage (AM), lymphocytes (lympho) and neutrophils (PMN) were based on morphological analysis of 100–400 cells. Protein content was measured in the acellular fraction of BAL. Numbers are the mean ⫾ SD (n ⫽ 3). 6 weeks 3 months Sham PBS % AM % lympho % PMN Protein (μg/ml)
Clodronate
99.2 ⫾ 0.2 99.0 ⫾ 0.9 0 0 0 0 151 ⫾ 66 392 ⫾ 25*
MOX PBS
Clodronate
Sham PBS
98.8 ⫾ 0.7 98.5 ⫾ 0.8 98.6 ⫾ 0.8 0 0 0.2 ⫾ 0.2 0 0 0.1 ⫾ 0.2 265 ⫾ 68# 640 ⫾ 212* 91 ⫾ 22
PBS, Phosphate Buffered Saline; *p ⬍ 0.05 clodronate vs. PBS; #p ⬍ 0.05 MOX vs. Sham.
Clodronate 98.6 ⫾ 0.5 0.1 ⫾ 0.2 0 104 ⫾ 50#
MOX PBS
Clodronate
99.2 ⫾ 0.2 99.0 ⫾ 0.9 0 0.3 ⫾ 0.5 0.3 ⫾ 0.5 0.3 ⫾ 0.3 239 ⫾ 86 334 ⫾ 116#
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Figure 4. Lung damage and macrophage activation after MOX inhalation. Lungs were harvested for paraffin inclusion 3 months post-MOX inhalation, or 4 h after LPS challenge. Panels A and B show autoradiographs of rat lung sections followed by hemalun staining in rats treated with PBS-liposomes (A) or clodronate-liposomes (B). Sirius red staining shows collagen accumulation after MOX inhalation in rats treated with PBS-liposomes (C) or clodronate-liposomes (D). Bars represent 100 μm. Panels E–M are rat lung sections stained with anti-CD68 (E–G), anti-iNOS (H–J) or anti-galectin 3 (K–M) antibodies in rats treated with PBS-liposomes (E, H, K) or clodronate-liposomes (F, I, L) or in positive controls treated with LPS (G, J, M). Panels N and O are galectin-3 immunostaining followed by autoradiographs in rats treated with PBS-liposomes (N) or clodronate-liposomes (O). Bars represent 50 μm (A–M) or 25 μm (N–O). This Figure is reproduced in color in the online version of International Journal of Radiation Biology.
to controls. ELF represents an acellular lung compartment that transiently retains dissolved actinides prior to their transfer to the bloodstream and their subsequent systemic
deposition (Gremy et al. 2010, Van der Meeren and Gremy 2010). Another consequence of AM depletion is the increase in surfactant pool sizes (Forbes et al. 2007), which could
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Macrophage depletion after MOX inhalation 1101 mathematically increase fraction of actinide retained in ELF in the absence of AM. A few weeks after AM depletion, a return to a distribution of activity similar to rats receiving PBS-liposomes was observed, although the α activity level remained higher in the clodronate-treated rats. In addition to the local redistribution of activity, we also report changes in the excretion/retention pattern of MOX. The percentage of activity transferred from lungs to blood is determined by urinary excretion and bone and liver retention measurements. The decrease in fecal excretion in clodronatetreated rats is likely to result from a smaller number of AM excreted into the gastro-intestinal tract through muco-ciliary escalator. MOX particles retained in macrophages during the first 6 weeks post MOX inhalation may undergo partial solubilization in phagolysosomes (Muller et al. 1989, Henge-Napoli et al. 1996). After clodronate-induced AM apoptosis, a fraction of solubilized elements from MOX particles can transfer to the systemic compartment and be retained by bone and liver. The slight decrease observed in urinary excretion is more difficult to understand. A possible explanation is the incomplete solubilization of Pu leading to the release of ultrafine particles, able to cross the alveolo-capillary barrier and recognized by cells from reticulo-endothelial system such as liver, where they remained trapped. We next evaluated whether the higher MOX retention in the lungs of rats receiving clodronate would lead to increased lung damage after MOX inhalation. The consequences of MOX inhalation were similar to those described after PuO2 by us and others, i.e., at 3 months, alveolitis and fibrosis. The presence of abnormal macrophages was also reported (foamy, activated, binucleated) despite the high recognized radioresistance of these cells (Talbot et al. 1989). The type of lesions in lung parenchyma observed in both groups of MOX-contaminated rats, were similar, with a tendency for lower number of lesions observed in clodronate-treated rats. Similarly, a lower number of binucleated cells was found in clodronate-treated rats as compare to PBS-treated animals. Because diminution in lung excretion might result from activation of macrophages after clodronate instillation, and since we have previously shown that Pu lung exposure induced early activation of macrophages (Van der Meeren and Gremy 2010, Van der Meeren et al. 2012) we evaluated AM activation using different parameters. First, we measured the production of TNF-α and chemokines by AM. The production and release of cytokines from macrophages are critical for inflammatory responses. The type of cytokines and chemokines significantly influences the quality, duration, and magnitude of most inflammatory reactions. Depending on the cytokine microenvironment, macrophages can adopt a proinflammatory or a profibrotic phenotype. Amongst others, the production TNF-α has been associated to the M1 phenotype. MCP-1 belongs to the C-C chemokine family and promotes both the recruitment of monocytes and their maturation to macrophages (Maus et al. 2002). MCP-1 is produced by AM of M1 phenotype, but also by M2a (alternative type II inflammation) (Lech and Anders 2013). MIP-2 and CINC-1 are both members of the CXC chemokine family. Both have been shown to participate in inflammatory responses involving neutrophils, and MIP-2 to participate in fibrogenesis. To
our knowledge, their related expression with polarization has not been described following internal contamination with actinides. Our results show an increase in inflammatory mediator production at 6 weeks, but more likely as a consequence of clodronate treatment, which can mask the effect of MOX. At 3 months, TNF-α returned to basal levels although chemokines remained elevated in both groups of MOX-contaminated rats. In another approach, we performed immunostaining in lung slices, using various markers of macrophage activation/polarization. Similarly to what we described after Pu contamination (Van der Meeren et al. 2008, 2012), CD68 positive cells were increased after MOX. Because CD68 is expressed on all macrophages (Murray and Wynn 2011), we used specific markers for M1 and M2 phenotypes. The role of macrophages in fibrosis is now recognized and has been identified in all stages of fibrogenesis. Particularly, M2 macrophages are more supportive of a fibro-proliferative microenvironment (Strieter 2008, Boorsma et al. 2013). Markers found on M2 macrophages have also be found to be increased in pulmonary fibrosis, such as Galectin 3 (Nishi et al. 2007), supporting the use of Galectin-3 as a good marker for M2 macrophages. iNOS is the most common marker used for M1 macrophages. It has been proposed that classical activation of macrophages (M1) is an important player in inducing radiation pneumonitis while the alternative activation (M2) is associated with radiation fibrosis (Zhang et al. 2011). Because of the high level of expression of galectin-3 and the lack of expression of iNOS in the areas of lungs showing accumulation of foamy macrophages 3 months post-MOX inhalation, we can conclude that the M2 phenotype is associated with fibrotic areas. However, Galectin-3 expression is not necessarily linked to the presence of particles. M2 macrophages are observed whatever the liposome treatment indicating that, at this time of treatment, clodronate does not greatly affect MOX-induced fibrotic development. This confirms previous studies where depletion of tissue-resident macrophages during the inflammatory phase did not affect the degree of fibrosis that develops after this inflammatory phase, although depletion during the fibrotic phase of lung fibrosis reduced the extra-cellular matrix deposition in this organ (Gibbons et al. 2011). The phenotype of macrophages, regardless of prior macrophage depletion, were similar, underlying the importance of local microenvironment, including the presence of particles, on macrophage polarization status following actinide inhalation. It has been described by others (Hong et al. 2003) that following external irradiation, differential roles for interstitial and AM, interstitial macrophages being involved in late pneumonitis, and alveolar in the early inflammatory response. However, we demonstrate in our study that AM are highly activated three months post actinide inhalation and express markers of M2 phenotype, raising the hypothesis of the role of AM in late pneumonitis. Our data indicate that the transient AM depletion induces a delay in MOX excretion as well as a transient modification of lung activity distribution. However, no major changes on long-term (3 months) macrophage activation status and lung damage were observed.
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Acknowledgements We acknowledge financial support from AREVA. The assistance of Quang Chau, Daniel Renault, Françoise Tourdes and Marie-Claire Abram is highly appreciated.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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