Application of Two-Dimensional Difference Gel Electrophoresis to

Application of Two-Dimensional Difference Gel Electrophoresis to Studying Bone Marrow Macrophages and Their in Vivo Responses to Ionizing Radiation Changwei Chen,*,† Michael T. Boylan,† Caroline A. Evans,‡ Antony D. Whetton,‡ and Eric G. Wright† Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, United Kingdom and Faculty of Medical and Human Sciences, University of Manchester, Christie Hospital, Wilmslow Road, Manchester M20 9BX, United Kingdom Received March 16, 2005

A flow cytometric protocol was developed to isolate primary bone marrow resident macrophages (CD11b(-) Gr-1(-) F4/80(+)) before and 24 h after 0.5 Gy γ-irradiation from mouse strains (C57BL/6 and CBA/Ca) that exhibit significant differences in the response of their hematopoietic tissues to ionizing radiation. The proteins from these populations were analyzed using two-dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry. We identified 36 macrophage proteins from 52 spots in both C57BL/6 and CBA/Ca. Thirty-three spots showed significant difference between genotypes and 16 of them corresponding to 11 proteins were identified. These included G-protein signaling 16, glucoseregulated protein 78, and lactoylglutathione lyase. We detected 16 and 18 spot changes following irradiation in C57BL/6 and CBA/Ca respectively, and in total 16 of them were identified. The identified proteins included calreticulin, lactoylglutathione lyase, regulator of G-protein signaling 16 and peroxiredoxin 5, mitochondrial precursor. The application of DIGE to primary bone marrow resident macrophages has allowed the first description of the proteome of these important components of the hematopoietic microenvironment and an analysis of their in vivo response to ionizing radiation which may shed light on the mechanism underlying the differential radiation-induced leukemogenesis exhibited within these mouse strains. Keywords: bone marrow • flow cytometry • ionizing radiation • macrophage proteome • two-dimensional difference gel electrophoresis

1. Introduction Macrophages make an important contribution to normal development and physiology and the pathogenesis of many diseases.1 In the bone marrow, they are a key component of the stem cell niche and the regulatory stromal microenvironment that controls stem cell biology and blood cell production.2,3 An understanding of macrophage biology is therefore important for normal and abnormal hematopoiesis. Exposure to ionizing radiation often poses significant health risks and the responses of the hematopoietic system are major determinants of outcome. Deleterious effects may be due to stem cell death, with consequent loss of mature functional cells or to stem cell damage that leads to aberrant responses to regulatory mechanisms. Effects on the regulatory microenvironment also result in deleterious effects.4,5 It has been known for many years that macrophages are resistant to * To whom correspondence should be addressed. Tel: +44 (0) 1382 632663. Fax: +44 (0) 1382 633952. E-mail: [email protected]. † Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Hospital and Medical School. ‡ Faculty of Medical and Human Sciences, University of Manchester, Christie Hospital. 10.1021/pr050067r CCC: $30.25

 2005 American Chemical Society

ionizing radiation relative to other eukaryotic cells. Histologically, macrophages in hematopoietic tissues are not damaged by doses that kill very large numbers of the blood-forming cells6 or indeed by potentially lethal myeloablative doses7 and in vivo their activation in irradiated tissues is not a direct effect of radiation damage but a response to effects on more radiosensitive cells.8,9 Until recently, research into the molecular mechanisms of radiation-induced adverse effects mainly focused on the directly irradiated cells with consequences such as death, mutation and malignant transformation being attributed to DNA damage in irradiated cells that had not been correctly restored by metabolic repair processes. In recent years, more attention has been paid to the contribution of the tissue microenvironment with many studies implicating macrophage activation mediating an inflammatory-type response to radiation-induced stress and injury.10-12 These responses have the potential to contribute to longer-term pathological consequences such as radiation-induced genomic instability and leukemia.11,13-16 As with many responses to radiation, the expression of instability phenotype and leukemogenesis are strongly influenced by genetic factors. In mouse model systems, the hematopoietic Journal of Proteome Research 2005, 4, 1371-1380

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Figure 1. Flowchart outlining proteomic analysis of bone marrow macrophages and their in vivo responses to ionizing radiation.

cells of CBA/Ca mice are susceptible to radiation-induced chromosomal instability, whereas those of C57BL/6 mice are relatively resistant, and CBA/Ca but not C57BL/6 mice are susceptible to radiation-induced acute myeloid leukemia.17 We have shown in our previous study, that hematopoietic tissues of C57BL/6 and CBA/Ca exhibited genotype-dependent macrophage activation associated with inflammatory-type responses 24 h post 4 Gy (a leukemogenic dose) γ-irradiation. We suggested that these tissue responses might contribute to the genomic lesions in bystander hematopoietic cells.9 Clearly, understanding macrophage biology in the context of the hematopoietic microenvironment and genetic background is important for understanding both normal and abnormal hematopoiesis. However, it is not possible to investigate many aspects of the relevant macrophage biology using only in situ approaches and macrophage cell lines in vitro may not accurately reflect the in vivo situation. Therefore, we employed a method of isolating primary macrophages from mouse bone marrow to investigate the in vivo pathways involved. DIGE was originally developed by Unlu and colleagues18 to analyze Drosophila embryo and E. coli protein extracts. It was then validated and optimized by Tonge et al.19 in an in vivo mouse toxicology study. The great advantage of this technique as opposed to the conventional 2D gel techniques is the ability to multiplex samples for intra-gel comparison. We applied this technique plus mass spectrometry to characterize the proteome of the bone marrow macrophage from two strains of mice, i.e., C57BL/6 and CBA/Ca that exhibit marked difference in their short- and long-term radiation responses, 9,20,21 (Figure 1). Bone marrow macrophages were isolated from control and irrradiated mice, and macrophage proteins were extracted and labeled with Cy3 and Cy5 or vice versa. Labeled control and irradiated samples were separated on the same gel, and the gel was imaged and analyzed to identify differentially expressed proteins. We have thereby identified significant proteomic differences between the C57BL/6 and CBA/Ca genotypes and between control and irradiated macrophages isolated from the bone marrow of these mice.

2. Materials and Methods Animals and Irradiation. Mice used in this study were bred in the University of Dundee Medical School Resource Unit in accordance with the guidance issued by the Medical Research Council and Home Office project number PPL 30/1272. Male mice aged 8-10 weeks from each strain were irradiated using a CIS International IBL 637 Cesium irradiator (CIS Bio International, France) with a whole-body dose of 0.5 Gy that would result in a mean reduction of total bone marrow cellularity of 45-50%. 1372

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Preparation of Bone Marrow Cell Suspension. Twenty-four hours post 0.5 Gy γ-irradiation, the mice were killed by cervical dislocation and bone marrow cells were obtained by flushing the femoral shafts with ice-cold Hank’s buffered salt solution (HBSS) (Invitrogen Ltd., Paisely, UK). The bone marrow cells from four to six mice were pooled and suspended in 10 mL of HBSS. Single cell suspensions were prepared by aspirating the clumps through a 21-G needle. Cells were washed twice with ice-cold phosphate buffered saline (PBS), centrifuged at 500 × g for 5 min at 4 °C and re-suspended in 2 mL of PBS containing 1% (w/v) bovine serum albumin (BSA). Control cell suspensions were prepared the same way as described except that the mice were not irradiated. A total nucleated cell count was performed for both irradiated and control bone marrow cells using a haemocytometer. The total mice used for preparing C57BL/6 control, C57BL/6 irradiated, CBA/Ca control and CBA/Ca irradiated cell suspensions were 28, 33, 39, and 66, respectively. Flow Cytometry and Isolation of Macrophages. Flow cytometric analysis and cell sorting were conducted with a presterilized FACSVantage flow cytometer (Becton Dickinson UK Ltd, Oxford). On the basis of the phenotypic characteristics of mouse cells, 22,23 three fluorescence-conjugated rat anti-mouse, lineage-specific monoclonal antibodies (mAbs) were selected to label and isolate bone marrow resident macrophages: fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse CD11b (CD11b-FITC, clone M 1/70, Becton Dickinson UK Ltd., Oxford) (CD11b, the 170 kDa Rm chain of Mac-1 (CD11b/CD18), also known as complement receptor 3 (CR3) is expressed at varying levels on granulocytes, some macrophages, myeloid derived dendritic cells and natural killer cells); allophycocyanin (APC)-conjugated rat anti-mouse Ly-6G (Gr-1) and Ly-6C (Gr1-APC, clone RB6-8C5, Becton Dickinson UK Ltd, Oxford) (Gr1, also known as Ly-6G, is a 21-25 kDa GPI-anchored protein. In the bone marrow, the level of Gr-1 expression is directly correlated with granulocyte differentiation and maturation. It is also expressed transiently on the monocyte linage in the bone marrow, but not on erythroid cells); R-phycoerythrin (PE)conjugated rat anti-mouse F4/80 (F4/80-PE, clone CI: A3-1, Caltag-Medsystems, Ltd, Towcester, UK) (F4/80, also named Ly-71, is a glycoprotein of 160 kDa, and is restricted to many but not all mature macrophages). Bone marrow cells were first incubated with Fc Block (a rat anti-mouse CD16/CD32 [Fcγ III/II receptor] antibody) (Becton Dickinson UK Ltd, Oxford) at 1 µg/106 cells for 10 min at 4°C to reduce background staining.24 The cells were washed with PBS, followed by centrifugation at 500 × g for 5 min at 4°C, and then incubated with the antibodies CD11b-FITC (0.25 µg/106 cells), Gr-1-APC (1 µg/106cells) and F4/80-PE (0.25 µg/106 cells) for 30 min at 4°C. Cells were pelleted at 500 × g, washed in PBS and re-suspended in PBS plus 1% BSA. Control cell staining was carried out at the same time by incubating aliquots of 106 cells with APC-, PE- and FITC-conjugated and isotype-matched rat IgG (Becton Dickinson UK Ltd, Oxford). A schematic representation of the procedures to isolate the CD11b(-) Gr-1(-) F4/ 80(+) (CD11b negative, Gr-1 negative and F4/80 positive) cell population is shown in Figure 2. A sort gate (R1) with a high forward light-scatter and an intermediate side light-scatter was established to isolate monocytic cells. Two additional sort windows, i.e., R2 (CD11b negativity) and R3 (F4/80 positivity and Gr-1 negativity) were established to isolate macrophages. Total sorting criterion was thus (R1 + R2 + R3). Cells were sorted at low pressure (7PSI) to minimize strain on vacuolated cells such as macrophages. All sorting procedures were stand-

Proteomic Analysis of Bone Marrow Macrophages

Figure 2. Schematic representation of the procedures to isolate bone marrow macrophages. Bone marrow cells were washed with ice-cold PBS and stained with CD11b fluorescein isothiocyanate, Gr-1 allophycocyanin and F4/80 phycoerythrin. Cells were washed again with PBS and re-suspended in PBS containing 1% BSA and 5 mM EDTA at a concentration of 107/mL. Cell analysis and isolation were performed on a FACSVantage flow cytometer. (a) A sort gate (R1) with a high forward light-scatter and an intermediate side light-scatter was established to isolate monocytic cells. (b) Monocytic cells that were CD11b negative were selected through gate R2. (c) Cells with the phenotype of CD11b(-)Gr-1(-)F4/80(+), i.e., bone marrow resident macrophages, were selected through gate R3. Dot plot contains events qualifying for selection through gate (R1 + R2) only. Total criterion for sort selection was thus (R1 + R2 + R3).

ardized throughout the experimental time course. Macrophages isolated by flow cytometric sorting have been shown by others to retain their physiological functions.25 Macrophages were directly sorted into a 15 mL Falcon centrifuge tube on ice. Single sorting procedures were restricted to 2 h at which time the tubes were removed from the cytometer and cells pelleted

research articles at 500 × g for 5 min at 4 °C. Each 2-hour sorting resulted in the isolation of 1.6-3.4 × 106 cells depending on the strain of mice and radiation dose received. Cell viability was assessed with a trypan blue assay and exceeded 95%. At the end of each sorting, cells were stored at -80 °C. As macrophages are a minority population in bone marrow and the protein yield from primary cells is much lower than that from cultured cells, macrophages from 7 to 11 enrichment procedures for each experimental mouse group were pooled to obtain sufficient proteins for analysis. Cytological Analysis. For a comparison of whole bone marrow cell profiles between control and irradiated mice, cell smears were prepared using cell suspensions in HBSS (106 cells/ mL) and air-dried. For characterizing different cell populations, cells with phenotypes of CD11b(+) Gr-1(-) F4/80(+) (CD11b positive, Gr-1 negative and F4/80 positive) and CD11b(-) Gr1(-) F4/80(+) (CD11b negative, Gr-1 negative and F4/80 positive), respectively, were directly sorted onto glass slides coated with 0.1% BSA in PBS (50 000 cells /slide) and air-dried. Cells were fixed in 100% methanol for 10 min at room temperature and stained with Giemsa’s stain. Cells with the phenotype of CD11b(-) Gr-1(-) F4/80(+) were subjected to morphological evaluation, then further characterized by cytochemistry. Cell monolayers were prepared as described above and stained for peroxidase (Px),26 acid phosphatase (AP) and nonspecific esterase 1 (NSE-1),27 respectively. Light microscopy was performed using a Leitz Wetzlar Dialux 20 microscope. Preparation of Protein Samples. Purified macrophages from each group were suspended in 100 µL of lysis buffer (10 mM Tris, pH 8.8, 4% (w/v) CHAPS, 8 M urea, 5 mM magnesium acetate) and lysed on ice for 20 min with occasional vortexing. Cell suspensions were centrifuged at 14 000 rpm for 20 min at 10 °C and the protein contents in the supernatants were measured with a DC protein assay (Bio-Rad laboratories UK Ltd., Hemel Hempstead, Herts.) using BSA as a standard, according to the manufacturer’s instructions. Two-Dimensional Difference Gel Electrophoresis. In the standard 2D DIGE approach, the experimental/system variation can be reduced by including an internal standard which is a pooled sample containing equal amounts of all samples in the experiment.28,29 In our study, due to limited proteins available for analysis, we used a strategy of labeling each sample with two different dyes and running all gels simultaneously to reduce the experimental variation. Aliquots of 100 µg of proteins from each sample were labeled with N-hydroxy succinimidyl esterderivatives of the cyanine dyes, Cy3 and Cy5 (Amersham Biosciences), according to Tonge et al.19 For one set of analyses, control and irradiated macrophage (CM and IM) proteins labeled with Cy3 and Cy5, respectively, were mixed and diluted with rehydration buffer (final concentration: 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2% (v/v) IPG buffer, 10 mg/mL DTT) to 450 µL. For a second set of analyses, the dyes were used in reverse ordersCy5 was used to label CM proteins and Cy3 was used to label IM proteins. The mixed 200 µg of proteins were loaded on a 24 cm IPG strip (pH 3-10, NL) (Amersham Biosciences) by rehydration overnight, according to the manufacturer’s instructions. The first dimension, isoelectric focusing (IEF), was performed using a Multiphor II electrophoresis unit (Amersham Biosciences) for a total of 115 kVh at 20 °C. The IPG strips were incubated for 15 min in 50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 1% (w/v) SDS containing 1% (w/v) dithiothreitol (DTT), and then for 15 min in the same buffer containing 2.5% (w/v) iodoacetamide (IAA). The second Journal of Proteome Research • Vol. 4, No. 4, 2005 1373

research articles dimension, SDS-PAGE (polyacrylamide gel electrophoresis), was carried out using an ISO-DALT vertical system which can accommodate 10 gels (Amersham Biosciences). Equilibrated IPG strips were transferred onto 10% gels between low fluorescence glass plates and overlaid with 0.5% (w/v) low melting point agarose in running buffer. Four gels were run simultaneously overnight at 30 mA/gel at 10 °C. Gel Imaging and Data Analysis. 2D gels were scanned directly between glass plates at 532 nm (Cy3) and 633 nm (Cy5) using a Typhoon 8600 fluorescent scanner (Amersham Biosciences, Bucks UK). Electronic gel images (.gel format) were recorded with ImageQuant software (Amersham Biosciences) and converted to tagged-image format (TIF). After scanning, gels were removed from plates and fixed in 50% (v/v) methanol plus 5% (v/v) acetic acid for 20 min, followed by three washes (5 min each) with Milli-Q water. Gels were then stored at 4 °C for spot picking. Gel images were analyzed with Progenesis software. Briefly, Cy3 and Cy5 images were loaded into the program and grouped. Spot detection was carried out automatically, followed by the manual editing of each image to remove artifacts such as streaks. Each spot was checked and edited, if required, by filtering, drawing, erasing and splitting, etc. Background subtraction and spot volume normalization were then carried out for each gel image, and the experiment was saved at this stage. Paired images in each group were warped and the spots matched using the Cy3- or Cy5-labeled control gel image as a reference. Spot differences were identified through running ‘Difference Maps’ and ‘Comparison’ functions of Progenesis. Various thresholds such as 1.5-, 2-, and 5-fold were applied to identify the differences between samples. To validate results and exclude the possibility that observed spot differences were due to preferential dye labeling, differences between macrophage samples were only considered real if they appeared on reciprocally labeled samples. Mass Spectrometry. Spots of interest were excised as 1.5 mm diameter gel plugs. This was performed manually using an One Touch 2D Gel Spot Picker (Web Scientific Ltd, Crewe, Cheshire, England) from gels that had been aligned to full-sized printouts of their corresponding Cy5 images, followed by destaining, reduction, alkylation and digestion with modified porcine trypsin (Promega, Southampton, UK) as described previously.30 To prepare for mass spectrometry, dried peptides were resuspended in 1 µL of 50% (v/v) ACN plus 0.1% TFA (v/v) in water, mixed with 1 µL of saturated R-cyano-4-hydroxycinnamic acid (Sigma-Aldrich Company Ltd., Poole, Dorset, UK) (10 mg/mL in 50% ACN plus 0.1% TFA in water) and immediately spotted onto a target plate (Waters, Manchester, UK). The peptide mass fingerprint data were obtained with a reflectron MALDI-TOF (matrix-assisted laser desorption/ ionization-time-of-flight) mass spectrometer (Waters, Manchester, UK). All mass spectra were internally calibrated with trypsin autolysis peaks (m/z 842.51 or 2211.10). Protein identification was performed with Mascot software (Matrix Science Ltd., London, UK) against Swiss-Prot and NCBInr databases with the following parameters: taxonomy (Rodential); enzyme (trypsin); maximum missed cleavages by trypsin (up to 1); peptide tolerance (( 0.2 Da); fixed modification (carbomidomethylation); variable modification (oxidation). Protein identity was based on matched peptides (at least 5), sequence coverage and the probability of score (p e 0.05). Candidates from peptide matching analysis were further evaluated by comparison with their calculated masses and pIs using the experimental values obtained from 2D DIGE. 1374


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3. Results Isolation and Characterization of Two Marrow Cell Populations Expressing F4/80. Mononuclear phagocytic cells in both control and irradiated bone marrow were identified by flow cytometric analysis and isolated by labeling with fluorescenceconjugated mAbs against 3 lineage-specific markers. Two cell populations expressing F4/80 were isolated and characterized. Cells with the phenotype of CD11b(+)Gr-1(-)F4/80(+) were heterogeneous in size and staining characteristics. Under highpower magnification (400×), many cells displayed monocytic characteristics with a curved nucleus and no obvious granules. As these cells were positive for F4/80, they were likely to be monocytes and their precursors. Cells with the phenotype of CD11b(-)Gr-1(-)F4/80(+) displayed typical macrophage morphology. They were large cells with a small nucleus-tocytoplasm ratio. Under high-power magnification (400×), they had a small round nucleus with prominent nucleoli. The cytoplasm contained multiple large granules and vacuoles. All the cells had fine-branched processes. To further characterize the cells in this population, we analyzed them for cytochemical markers normally associated with macrophages. These cells were positively stained for AP, NSE-1 but negatively stained for Px. These staining patterns were consistent with the cytochemical characteristics of macrophages.23, 31 Purification of Macrophages and Preparations of Protein Samples. As the cells with the phenotype of CD11b(-)Gr-1(-)F4/ 80(+) were characterized as marrow macrophages, we then purified these cells by cell sorting from control and irradiated bone marrow of both C57BL/6 and CBA/Ca mice. Purified macrophages were lysed and the proteins extracted. The total numbers of purified macrophages obtained from control and irradiated (CM and IM) C57BL/6 mice and CBA/Ca mice were 20.1 × 106, 13.9 × 106, 20.3 × 106, and 17.6 × 106, respectively. The corresponding proteins extracted were 255 µg, 225 µg, 255 µg, and 275 µg. The protein yield calculated per 106 cells from each group of macrophages varied from 12.7 to 16.2 µg, with slightly higher yields from IM than from CM. This might reflect the increased phagocytosis of apoptotic cells by macrophages post irradiation. Identification of Macrophage Proteins. To better understand the proteome of bone marrow resident macrophages and also for purposes of in-gel calibration for pI and molecular mass, we analyzed relatively abundant protein spots along with those differentially expressed spots in both C57BL/6 and CBA/ Ca with mass spectrometry. As labeled in Figure 3 and summarized in Table 1, we identified in total 52 protein spots. Using proteins pooled from two gels, we achieved an identification efficiency of ca. 75% and 22% for those relatively abundant (normalized spot volume > 0.3) and those less abundant spots (normalized spot volume < 0.3), respectively. A number of proteins gave rise to several spots with different pI and/or mass values, which indicates the presence of protein isoforms that may arise by various post-translational modifications. The identified proteins have functions in the regulation of a number of cell processes including cell structure, signaling, molecular chaperoning of proteins, cellular defense and calcium metabolism. Detection and Identification of Proteins Differentially Expressed in C57BL/6 and CBA/Ca Macrophages. An important question we asked was whether there is genotypic difference in macrophage protein profile between C57BL/6 and CBA/ Ca. To answer this question, we performed differential analysis of C57BL/6 and CBA/Ca control unirradiated macrophage

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Figure 3. Representative 2D gel image of bone marrow macrophage proteins showing identified protein spots by MALDI-TOF mass spectrometry. The gel image was obtained from Cy3-labeled C57BL/6 macrophage proteins. Spot numbers were assigned by Progenesis and protein identities are shown in Table 1.

proteins. The data from paired Cy3 or Cy5 2D gel images revealed that although the global protein maps of C57BL/6 and CBA/Ca macrophages were quite similar in overall expression profiles there were significant differences between samples (Figure 4). Thirty-three spots were found to be different between C57BL/6 and CBA/Ca. Of these spots, 2 were unique to C57BL/6 (Figure 4a, circled). Eleven spots (arrow-labeled) were more abundantly expressed in C57BL/6 macrophages, whereas 20 (line-labeled) were expressed at higher levels in CBA/Ca macrophages. These spots were selected for MALDITOF mass spectrometry, and 16 of them were identified (Table 2). Two of the identified proteins, i.e., regulator of G-protein signaling 16 (RGS16) (spot 991) and actin isoforms (spots 702, 707, 837, 1290) were substantially more expressed in C57BL/6. However, several metabolic enzymes were less abundant in C57BL/6 macrophages. These enzymes included delta-aminolevulinic acid dehydratase (spots 799, 1257, and 1258), cholinephosphate cytidylyltransferase A (spot 1254), lactoylglutathione lyase (spot 1167) and seryl-tRNA synthetase (spot 569). CBA/ Ca macrophages were found to express more DNA replication licensing factor MCM7 (spot 457), 78 kDa glucose-regulated protein precursor (spot 1058) and complement component 1, Q subunit-binding protein, mitochondrial precursor (spot 847). Identification of Proteins Altered by Irradiation. Figure 5 shows 2D gel images of C57BL/6 CM (a), C57BL/6 IM (b), CBA/ Ca CM (c), and CBA/Ca IM (d) proteins, with CM and IM proteins labeled with Cy3 and Cy5, respectively. In C57BL/6, sixteen spots were found to have changed at least 1.5-fold as a result of irradiation (Figure 5a,b). Of these spots, 9 (arrow labeled) were increased and 7 (dashed arrow-labeled) decreased. Two spots (spots 886 and 1441) changed 2-fold or more. In CBA/Ca, 18 spot changes were detected between samples (Figure 5c,d). Seven spots (arrow-labeled) were increased and 11 spots (dashed arrow-labeled) decreased. Nine spots (707, 767, 837, 848, 842, 982, 991, 1013, and 1152) changed 2-fold or more, and spot 991 changed more than 5-fold. One spot (spot 916) change was observed in both C57BL/6 and CBA/ Ca. All the other spot changes observed were unique to an individual strain.

We then picked these spots for MALDI-TOF mass spectrometry. The resulting monoisotopic mass values from each spectrum were used to search protein databases with Mascot software. Among the 16 altered spots in C57BL/6, 7 were identified (Table 3) and they were from 5 proteins. These include actin, cytoplasmic 1, calreticulin, proliferating cell nuclear antigen (PCNA), and two metabolic enzymes, i.e., alcohol dehydrogenase and delta-aminolevulinic acid dehydratase. In CBA/Ca, we have identifications on 9 of the 18 spots (Table 3). Four of the identified proteins were increased including 40S ribosomal protein SA, actin cytoplasmic 1, aspartyl-tRNA synthetase and regulator of G-protein signaling 16, and 3 were decreased including carbonic anhydrase I, lactoylglutathione lyase and peroxiredoxin 5, mitochondrial precursor. Apart from actin, cytoplasmic 1 (spots 886 and 1165 in C57BL/6 and spots 707, 837, and 842 in CBA/Ca), all other identified proteins were specific to an individual strain. It is interesting that two proteins, i.e., actin, cytoplasmic 1 and alcohol dehydrogenase were identified from multiple spots. This might be due to either posttranslational modification of these proteins or the fragmentation of these proteins caused by in vivo biological processing.

4. Discussion In this study, we used a flow cytometric protocol to obtain highly pure macrophages for characterizing the proteome of the bone marrow resident macrophage and the genotypedependent macrophage response to γ-irradiation. We expected that the cells with CD11b(+)Gr-1(-)F4/80(+) would be macrophages based on the characteristics of mouse cell surface antigens22 but morphological evaluation revealed that these cells were quite heterogeneous and generally small. However, the CD11b(-)Gr-1(-)F4/80(+) population had typical macrophage morphology and was shown to express AP and NSE-1 but not Px. As these cells were unexpectedly CD11b(-), they were obviously different from other tissue macrophages such as peritoneal macrophages that are CD11b(+).22 The macrophages isolated by our protocol were similar to the resident macrophages obtained by the method of Crocker and Gordon,23 in Journal of Proteome Research • Vol. 4, No. 4, 2005 1375

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Table 1. List of C57BL/6 and CBA/Ca Macrophage Protein Spots Identified by MALDI-TOF Mass Spectrometry spot no. a

457 473 568 569 585 616 660 702 707 772 799 837 842 847 857 861 864 869 886 925 928 934 945 960 970 991 1058 1064 1120 1140 1165 1167 1186 1192 1254 1257 1258 1277 1282 1290 1313 1315 1317 1329 1330 1344 1345 1362 1376 1405 1421 1422 a

protein name

predicated Mr/pI

peptide matched

sequence coverage %

accession no.b

DNA replication licensing factor MCM7 Lamin A Protein disulfide isomerase A3 [precursor ] Seryl-tRNA synthetase Aspartyl-tRNA synthetase T-complex protein 1, beta subunit Alpha enolase Actin, cytoplasmic 1 Actin, cytoplasmic 1 Transaldolase Delta-aminolevulinic acid dehydratase Actin, cytoplasmic 1 Actin, cytoplasmic 1 Complement component 1, Q subcomponent binding protein, mitochondrial [precursor] Purine nucleoside phosphorylase Guanine nucleotide-binding protein beta subunit 2-like 1 Chloride intracellular channel protein 1 Alcohol dehydrogenase [NADP+] Actin, cytoplasmic 1 Peroxiredoxin 6 Carbonic anhydrase I Ras-related protein Rab-33B Peroxiredoxin 5, mitochondrial [precursor] Glutathione S-transferase Mu 1 Peroxiredoxin 2 Regulator of G-protein signaling 16 78 kDa glucose-regulated protein precursor Proliferating cell nuclear antigen Heat shock cognate 71 kDa protein 60 kDa heat shock protein, mitochondrial [precursor] Actin, cytoplasmic 1 Lactoylglutathione lyase Actin, cytoplasmic 1 Actin, cytoplasmic 1 Choline-phosphate cytidylyltransferase A Delta-aminolevulinic acid dehydratase Delta-aminolevulinic acid dehydratase Proteasome activator complex subunit 1 Malate dehydrogenase, cytoplasmic Actin, cytoplasmic 1 Carbonic anhydrase II Carbonic anhydrase I Alcohol dehydrogenase [NADP+] Actin, cytoplasmic 1 Actin, cytoplasmic 1 Transketolase Transketolase Heterogeneous nuclear ribonucleoprotein A3 L-lactate dehydrogenase A chain Carbonic anhydrase I 14-3-3 protein tau Calreticulin [precursor]

81,787/5.98 74,450/6.54 57,042/5.98 58,734/5.95 57,537/6.07 57,652/5.98 47,322/6.36 42,052/5.29 42,052/5.29 37,534/6.57 36,456/6.32 42,052/5.29 42,052/5.29 31,336/4.82

34 29 12 10 8 19 15 15 15 9 12 7 6 6

47 43 26 19 21 50 47 45 47 28 48 24 20 22

Q61881 P48678 P27773 P26638 Q922B2 P80314 P17182 P60710 P60710 Q93092 P10518 P60710 P60710 O35658

32,538/5.93 35,511/7.60

9 13

42 43

P23492 P68040

27,207/5.09 36,661/6.87 42,052/5.29 24,794/6.01 28,229/6.47 26,206/7.62 22,491/8.94 25,936/8.13 21,936/5.20 22,905/7.05 72,492/5.07 29,108/4.66 71,055/5.37 61,088/5.91 42,052/5.29 20,836/5.25 42,052/5.29 42,052/5.29 42,040/6.58 36,456/6.32 36,456/6.32 28,826/5.73 36,494/6.16 42,052/5.29 29,056/6.52 28,229/6.47 36,661/6.87 42,052/5.29 42,052/5.29 68,272/7.23 68,272/7.23 37,291/8.46 36,686/7.77 28,229/6.47 28,046/4.69 48,136/4.33

7 8 8 9 8 7 6 10 7 8 13 9 20 11 6 9 6 12 8 8 9 10 12 7 7 9 8 11 12 13 13 8 13 7 11 15

36 29 33 49 44 31 29 37 37 33 26 35 37 29 24 32 19 39 22 27 30 46 39 26 34 50 27 38 44 25 25 31 37 32 49 27

Q9Z1Q5 Q9JII6 P60710 O08709 P13634 O35963 Q9R063 P10649 Q61171 P97428 P20029 P17918 P63017 P63038 P60710 Q9CPU0 P60710 P60710 P49586 P10518 P10518 P97371 P14152 P60710 P00920 P13634 Q9JII6 P60710 P60710 P40142 P40142 Q8BG05 P06151 P13634 P68254 P14211

Spot numbers were assigned by Progenesis software and labeled in Figure 3. b Accession through Swiss-Prot database.

cell surface antigens and cytochemical characteristics. It has been shown by various investigators that macrophages isolated by these approaches retain many of their functional properties.23,25 The advantage of our protocol is that a much greater purity (>95%) can be obtained enabling us to investigate the in vivo characteristics and activities of the macrophage population that is an important component of the hematopoietic microenvironment. Using protein samples prepared from highly pure macrophages and 2D DIGE technique, we were able to detect the genotype-dependent differences between C57BL/6 and CBA/ Ca and the differences between control and irradiated macrophages. We detected 33 protein spots that displayed differ1376


ence between C57BL/6 and CBA/Ca (Figure 4). Sixteen of these spots, corresponding to 11 distinct proteins, have so far been identified (Table 2). Two proteins, i.e., regulator of G-protein signaling 16 (RGS16) and actin, were substantially more expressed in C57BL/6 than in CBA/Ca. Actin cytoplasmic 1 form was detected as multiple spots on 2D gels, indicating either the post-translational modifications of this protein or the fragmentation of this protein. Similar observations have been reported in 2D gel analyses of the protein from rat polymorphonuclear leucocytes, human neutrophils and HeLa cells.32-34 As actin is closely associated with cell motility, phagocytosis and signaling,35-37 and RGS16 is a member of the RGS protein family, which is involved in the G-protein-

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Figure 4. Protein spots differentially expressed in C57BL/6 and CBA/Ca macrophages. (a) & (b): 2D gel images of Cy3-labeled C57BL/6 and CBA/Ca macrophage proteins, respectively. Two spots labeled by circles were unique to C57BL/6. Spots labeled by arrows were more abundantly expressed in C57BL/6 macrophages, whereas spots labeled by lines were more abundantly expressed in CBA/Ca macrophages. Table 2. Identified Protein Spots Differentially Expressed in C57BL/6 and CBA/Ca Bone Marrow Macrophages normalized volume C57BL/6 spot no.a

991 837 702 707 857 1290 1167 1058 1254 847 799 1257 569 1258 925 457 a

protein name

Cy3

Cy5

ratio in mean normalized volume

CBA/Ca mean b

Cy3

Proteins more abundantly expressed in C57BL/6 Regulator of G-protein signaling 16 0.602 0.507 0.555 0.029 Actin, cytoplasmic 1 1.902 1.178 1.550 0.217 Actin, cytoplasmic 1 0.447 0.277 0.362 0.066 Actin, cytoplasmic 1 1.390 0.999 1.195 0.345 Purine nucleoside phosphorylase 0.388 0.294 0.341 0.140 Actin, cytoplasmic 1 0.296 0.216 0.256 0.111 Proteins more abundantly expressed in CBA/Ca Lactoylglutathione lyase 0.056 0.069 0.063 0.272 78 kDa glucose-regulated protein precursor 0.168 0.118 0.143 0.480 Cholinephosphate cytidylyltransferase A 0.094 0.142 0.118 0.460 Complement component 1, Q subcomponent 0.258 0.045 0.152 0.756 binding protein, mitochondrial precursor Delta-aminolevulinic acid dehydratase 0.168 0.229 0.199 0.685 Delta-aminolevulinic acid dehydratase 0.064 0.084 0.074 0.215 Seryl-tRNA synthetase 0.012 0.013 0.013 0.047 Delta-aminolevulinic acid dehydratase 0.298 0.269 0.284 0.630 Peroxiredoxin 6 0.160 0.139 0.150 0.374 DNA replication licensing factor MCM 7 0.067 0.095 0.081 0.148

Cy5

mean

C57BL/6 vs CBA/Ca

0.042 0.146 0.061 0.259 0.112 0.084

0.036 0.182 0.064 0.302 0.126 0.098

15.4 8.5 5.7 4.0 2.7 2.6

0.268 0.376 0.285 0.219

0.270 0.428 0.373 0.488

0.2 0.3 0.3 0.3

0.650 0.274 0.051 0.667 0.468 0.199

0.668 0.245 0.049 0.649 0.421 0.174

0.3 0.3 0.3 0.4 0.4 0.5

Spot numbers were assigned by Progenesis software and labeled in Figure 2. b Mean value of Cy3- and Cy5-labeled samples.

mediated signaling pathways,38 the differences in basal expression levels of these two proteins may contribute in part to the genotypic difference in macrophage phagocytic activity and activation following ionizing radiation.9 Glucose-regulated protein (GRP) 78 is an endoplasmic reticulum (ER) chaperone protein whose expression is induced during oxidative stress.39 It has been shown that GRP78 could protect cells from apoptosis by blocking caspase activation.40,41 Lactoylglutathione lyase, also named glyoxalase I, is a component of the glyoxalase system believed to have evolved to detoxify reactive 2-oxoaldehydes, mainly methylglyoxal, formed endogenously as a byproduct of the triosephosphate isomerase reaction in glycolysis.42 As the activities of both glyoxalase I and II are linked to states of glutathione (GSH) in the cells, the different expression levels of glyoxalase I in the macrophages of C57BL/6 and CBA/Ca mice point to processes that may result in genotype-dependent signaling mediated by GSH.43 Apart from genotype-dependent spot differences, protein changes were also detected in irradiated macrophage samples. Actin cytoplasmic 1 form was markedly increased in both C57BL/6 (Figure 5a,b, spots 886, 1165) and CBA/Ca (Figure 5c,

d, spots 707, 837, 842). One possible mechanism for this regulation is that exposure of macrophages to radiation activates an actin-cleaving enzyme, as proposed by Fessler et al.44 in their study investigating the activation of neutrophils by lipopolysaccharide (LPS). This enzyme may play a role in remodeling the cytoskeleton. In a separate study of human monocytes exposed to LPS, it was demonstrated that an actin isoform changed in its phosphorylation state, indicating that actin may play a role in the activation of monocytes.45 On the basis of these observations and our result, we suggest that actin may play important roles in modulating macrophage activities such as cell motility and phagocytosis of apoptotic cells following irradiation. An alterative explanation for this change is that the increased actin fragments were derived from either the apoptotic cells phagocytosed by macrophages or the macrophages themselves undergoing apoptosis.46 In this study, we identified mouse strain-specific protein changes that reflect genotype-dependent difference in radiation response between two mouse strains. In C57BL/6 macrophages, calreticulin was increased. Calreticulin is a Ca2+-binding protein with chaperone activity in the endoplasmic reticulum. It is Journal of Proteome Research • Vol. 4, No. 4, 2005 1377

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Chen et al.

Figure 5. Protein spots altered in irradiated C57BL/6 and CBA/Ca macrophage samples. Control and irradiated macrophage proteins from C57BL/6 (C57BL/6 CM and C57BL/6 IM) or CBA/Ca (CBA/Ca CM and CBA/Ca IM) mice, were labeled with Cy3 and Cy5 or Cy5 and Cy3 respectively, and mixed. The protein mixture was first separated by IEF on a 24 cm IPG gel and then by SDS-PAGE on a 10% polyacrylamide gel. Gels were imaged and analyzed with Progenesis. (a) & (b): C57BL/6 CM and C57BL/6 IM, respectively. (c) & (d): CBA/Ca CM and CBA/Ca IM, respectively. Spots labeled by arrows were increased, whereas those labeled by dashed arrows were decreased at least 1.5-fold in irradiated samples compared to controls. Table 3. Identified C57BL/6 and CBA/Ca Protein Spots that Changed in Irradiated Macrophages (IM) Compared to the Control Macrophages (CM) ratio in mean normalized volume (IM/CM)

mean normalized volume b C57BL/6 protein name

Regulator of G-protein signaling 16 40S ribosomal protein SA Actin, cytoplasmic 1 Actin, cytoplasmic 1 Actin, cytoplasmic 1 Actin, cytoplasmic 1 Actin, cytoplasmic 1 Aspartyl-tRNA synthetase Calreticulin Carbonic anhydrase I Lactoylglutathione lyase Alcohol dehydrogenase Delta-aminolevulinic acid dehydratase Peroxiredoxin 5, mitochondrial precursor Alcohol dehydrogenase Proliferating cell nuclear antigen

spot no.a

991 848 707 842 886 837 1165 585 1422

CM

CBA/Ca IM

CM

C57BL/6 IM

Proteins up-regulated in IM 0.036

0.225

6.3

0.098 0.302 0.163

0.353 0.943 0.425

3.6 3.1 2.6

0.182

0.380

0.031 0.269

0.082

2.6

0.481 0.842

928 1167 869 1258 945

0.078

0.043

0.308 0.771

0.204 0.502

2.1 1.8

0.504 0.402 0.610 Proteins down-regulated in IM 0.174 0.270 0.145 0.094 0.235 0.130

1317 1064

CBA/Ca

1.7 1.5

0.090 0.140

0.5 0.5 0.6 0.6 0.6 0.7 0.7

a Spot numbers were assigned by Progenesis software and labeled in Figure 5. b Mean normalized volume was the mean value of Cy3- and Cy5-labeled samples.

involved in various cellular signaling pathways and is important in modulating functions such as cell adhesion, gene expression, apoptosis and phagocytosis.47,48 The increase of calreticulin was consistent with the macrophage activities such as phagocytosis 1378


of apoptotic cells in irradiated bone marrow being greater in C57BL/6 than in CBA/Ca irradiated mice.9 In CBA/Ca macrophages, one protein that markedly increased following irradiation was RGS16. It has been reported that RGS16 mRNA


could be induced by protein kinase C (PKC) and that the induction was mediated via tumor necrosis factor-R (TNF-R) in a calcium-sensitive manner.49 More recently, RGS16 was shown to have a regulatory role in inflammation-induced T lymphocyte migration and activation.50 Given that TNF-R gene expression is mediated by PKC following activation by ionizing radiation,51 it is likely that the increase of RGS16 expression in irradiated macrophages was due to the activation of PKC. Several proteins that were decreased following exposure to radiation in CBA/Ca have been identified. One of them was lactoylglutathione lyase, also named glyoxalase I, which is discussed above. Another decreased protein was peroxiredoxin 5, mitochondrial precursor. Peroxiredoxin 5 belongs to the family of peroxiredoxin that has important functions in antioxidant defense and redox signaling.52 Mouse peroxiredoxin 5 has been shown to inhibit p53-induced apoptosis.53 Taken together, the data indicate that several signaling pathways involving PKC, Ca2+, and reactive species may be operating in bone marrow macrophages in their responses to tissue changes following in vivo exposure to γ-radiation. In summary, we utilized a flow cytometric protocol for isolating marrow macrophages and, for the first time, studied the marrow resident macrophages and their in vivo responses to ionizing irradiation with 2D DIGE technique and MALDITOF mass spectrometry. Proteins that are involved in phagocytosis, apoptosis and cell signaling showed different basal expression levels in C57BL/6 and CBA/Ca macrophages. These protein differences would contribute to the genotype-dependent macrophage response to ionizing radiation. The comparative analyses have revealed that a number of macrophage proteins changed at 24 h post 0.5 Gy γ-irradiation. The data suggest that several signaling pathways involving PKC, Ca2+, and reactive species were activated in macrophages. The proteomic approach makes an important contribution to investigating the response of bone marrow tissue to irradiation, assists in identifying genotype-dependent responses and provides support for the importance of microenvironmental factors contributing to the overall tissue response. The results derived permit hypothesis driven research to model genotypic differences that contribute to response to irradiation, and the quasiinflammatory process governing post-irradiation responses, via macrophages, in the bone marrow. Abbreviations: 2D DIGE, two-dimensional difference gel electrophoresis; HBSS, Hank’s buffered salt solution; PBS, phosphate buffered saline; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; APC, allophycocyanin; PE, phycoerythrin; Px, peroxidase; AP, acid phosphatase; NSE-1, nonspecific esterase 1; CM, control macrophage; IM, irradiated macrophage; RGS16, regulator of G-protein signaling 16; GRP78, glucose-regulated protein 78; GSH, glutathione; LPS, lipopolysaccharide; PKC, protein kinase C; TNF-R, tumor necrosis factor-R

Acknowledgment. This work was supported in part by the Medical Research Council (UK) (Grant No. G9824583) and the Leukaemia Research Fund (UK) (Grant Nos. 02.14 and 00.44). We wish to think Sally A. Lorimore for irradiating mice, Lynda Capper for assistance in mass spectrometry, and Dr. Philip J. Coates for helpful discussions. References (1) van Furth, R. Mononuclear Phagocytes: Biology of Monocytes and Macrophages; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1992.

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PR050067R

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