Extracellular proteomes of M-CSF (CSF-1) and GM-CSF ... - Nature

Immunology and Cell Biology (2011) 89, 283–293 & 2011 Australasian Society for Immunology Inc. All rights reserved 0818-9641/11 www.nature.com/icb

ORIGINAL ARTICLE

Extracellular proteomes of M-CSF (CSF-1) and GM-CSF-dependent macrophages Mark J Bailey1,3, Derek C Lacey1, Bernard VA de Kok1, Paul D Veith2, Eric C Reynolds2 and John A Hamilton1 Macrophage colony-stimulating factor (M-CSF) (also known as CSF-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) have distinct effects on macrophage lineage populations, which are likely to be contributing to their functional heterogeneity. A comparative proteomic analysis of proteins released into culture media from such populations after M-CSF and GM-CSF exposure was carried out. Adherent macrophage populations, termed bone marrow-derived macrophage (BMM) and GM-BMM, were generated after treatment of murine bone marrow precursors with M-CSF and GM-CSF, respectively. Proteins in 16-h serum-free conditioned media (CM) were identified by two-dimensional gel electrophoresis and mass spectrometry. Respective protein profiles from BMM and GM-BMM CM were distinct and there was the suggestion of a switch from primarily signal peptide-driven secretion to non-classical secretion pathways from BMM to GM-BMM. Extracellular expression of cathepsins (lysosomal proteases) and their inhibitors seems to be a characteristic difference between these macrophage cell types with higher levels usually observed in BMM-CM. Furthermore, we have identified a number of proteins in BMM-CM and GM-BMM-CM that could be involved in various tissue regeneration and inflammatory (immune) processes, respectively. The uncharacterized protein C19orf10, a protein found at high levels in the synovial fluid of arthritis patients, was also differentially regulated; its extracellular levels were upregulated in the presence of GM-CSF. Immunology and Cell Biology (2011) 89, 283–293; doi:10.1038/icb.2010.92; published online 27 July 2010 Keywords: granulocyte-macrophage colony-stimulating factor (GM-CSF); macrophage colony-stimulating factor (M-CSF); macrophages; proteomics; secretion

Macrophages have numerous roles in tissue homeostasis, innate and acquired immunity, and inflammatory disease progression and resolution.1–4 Apart from their phagocytic and antigen-presenting functions, they secrete a large variety of molecules (including coagulation factors, adhesion molecules, soluble receptors or receptor antagonists, enzymes and enzyme inhibitors, stress proteins and signalling molecules) that can modify their own biology and that of nearby cells. They also show considerable heterogeneity and plasticity depending on their tissue residence and exposure to local and systemic stimuli.1–3 The terms M1 (or ‘classically activated’) and M2 (or ‘alternatively activated’) have been introduced recently to describe polarized subsets of macrophages at opposite ends of a functional spectrum.1,3 These cell populations differ in cell surface expression, immune function and cytokine/chemokine secretion. M1 macrophages are potent effector cells with bactericidal and tumoricidal properties, and they produce relatively large amounts of pro-inflammatory cytokines. M2 macrophages are immunomodulatory, promote angiogenesis, are involved in tissue remodelling and repair, and are resident in tumors in which they have immunosuppressive actions mediated through secreted effector molecules (for example, interleukin (IL)-10).1,3,5,6

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF or CSF-1) were originally defined as hemopoietic growth factors on account of their ability to generate in vitro colonies of myeloid populations from bone marrow precursor cells.7 However, it has become apparent that they have other functions on account of their activities on mature myeloid populations.8–10 As a result of these other functions, GM-CSF and M-CSF have become therapeutic targets with their antagonists being currently in clinical trials in a number of inflammatory/autoimmune conditions.10 The cell lineage on which both GM-CSF and M-CSF act is the macrophage lineage. Studies in which the responses and/or the resultant properties of cells in this lineage, after exposure to each of these CSFs, have been compared indicate that there are similarities but quite significant variation, for example, in cytokine production, surface expression, endocytosis, and so on.9–14 In the mouse, addition of M-CSF to bone marrow cells can give rise, after proliferation and differentiation, to an adherent population, termed bone marrowderived macrophages (BMMs).13–15 However, replacement of M-CSF with GM-CSF can give rise to a population with features of both immature dendritic cells and macrophages;12–18 they have been

1Department of Medicine, CRC for Chronic Inflammatory Diseases, The University of Melbourne, The Royal Melbourne Hospital, Parkville, Victoria, Australia and 2CRC for Oral Health Science, School of Dental Science, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, Australia 3Current address: UQ Centre for Clinical Research, University of Queensland, Queensland, Australia. Correspondence: Dr MJ Bailey, UQ Centre for Clinical Research, Royal Brisbane and Women’s Hospital, Herston, Queensland 4029, Australia. E-mail: [email protected] Received 19 January 2010; revised 16 June 2010; accepted 17 June 2010; published online 27 July 2010

The macrophage secretome MJ Bailey et al 284

termed GM-BMM in some studies.13,14,19 BMM and GM-BMM have recently been termed ‘M2-like’ and ‘M1-like’, respectively, based mainly on their cytokine expression patterns.14 There is also evidence that these populations show some degree of plasticity in that their respective phenotypes can be ‘switched’ to the other by addition of the appropriate CSF.13,14 For example, BMM can adopt some of the features of GM-BMM in the presence of GM-CSF, a process that has been termed ‘priming’.14,20,21 A similar ‘polarization’ by GM-CSF and M-CSF has also been noted in vitro with human peripheral blood monocytes.9,22 Although the influences of GM-CSF and M-CSF on monocytes/ macrophages have been studied individually in a number of reports, very few studies have made direct comparisons. When comparisons have been made, they have usually been limited to specific changes such as in cytokine or surface marker expression.9–14,16–18 We have previously shown that GM-BMM are characterized by their production of high levels of tumor necrosis factor-a, IL-6, IL-12, IL-23 and NO, after stimulation with LPS. In contrast, BMM produce large amounts of IL-10, interferon-b and CCL2 on stimulation.14 GMBMM express CD11c on their surface while BMM do not.13 Recently, gene microarray profiling has been carried out on BMM and GMBMM providing information, at least at the transcriptome level, on their similarities and differences.14,19 However, gene expression does not always parallel that of the corresponding protein product and global protein expression data (proteomics) is needed to complement transcriptome data. A few studies have adopted a proteomic approach to study macrophages23–26 but none for the purpose of comparing the responses to the above CSFs. As mentioned, when appropriately stimulated, macrophages can secrete a plethora of different molecules thereby communicating with their neighboring milieu. Therefore we decided to compare the proteomic profile of conditioned media (CM) from BMM, BMM ‘primed’ with GM-CSF and GM-BMM. For this purpose, we developed novel sample preparation methods for processing large volumes of CM for analysis by two-dimensional gel electrophoresis (2-DE) and

Mr

Q61207

kDa

P63017

matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We have so far identified 94 different proteins in CM from BMM and GM-BMM, many of which were restricted to one macrophage phenotype. Extracellular expression of cathepsins (lysosomal proteases) and their inhibitors seems to be a characteristic difference between these macrophage cell types. Furthermore, we have identified a number of proteins in BMM-CM and GMBMM-CM that could be involved in various tissue regeneration and inflammatory (immune) processes, respectively. We also identified the novel, uncharacterized protein, C19orf10 that seemed to be differentially regulated by GM-CSF. To the best of our knowledge, this is the first report comparing the extracellular protein profiles of macrophage phenotypes using a proteomic approach. RESULTS Proteins in the CM from CSF-1- and GM-CSF-treated macrophage populations Preliminary experiments determined that cell viability in 70–80% confluent cultures after 16 h in serum-free medium was 497% by Trypan blue exclusion. CM was therefore collected from such cultures, using reproducible preparation protocols optimized for large volumes of sample, before subjecting them to analysis by 2-DE. Owing to the very low protein concentrations in these CM, each gel represents pooled protein from up to 12 replicate tissue culture experiments. CM from three separate macrophage populations were tested: BMM (CSF1-dependent bone marrow-derived macrophages), GM-primed BMM (BMM treated for the 16 h with GM-CSF) and GM-BMM (GM-CSFdependent bone marrow-derived macrophages).14 MALDI-MS identified only four bovine proteins (from fetal bovine serum) and they were generally in low abundance, showing the effectiveness of our washing protocol. Initially, protein spots were excised from BMM-CM gels to generate a 2-DE reference map (Figure 1). Subsequently, this map was used to prioritize protein spots in GM-primed BMM and GM-BMM gels for analysis. Prioritization was based on their presence or absence in these other conditions and not on statistical fold change.

P28798

P20029 P63038 Q02819

P27773

P10605

50

P52480

P17182

P18242

P13020

P09411 O70370 Q8CDN6

P18242 P24452

P60710 Q9D8Y0 Q9WUU7

P21107

P06797 P28798

P16110

P57759 P25785

O70370

20

Q9JII6

Q8VE43

P17918

Q9DBJ1

Q99PT1

P20152

Q923D2

Q61171 P63028

Q9WVA4

Q61599 P35700

Q9Z0J0

P17742

P60710

15

P17751

P08228 P54227

P16045

Q9CPT4 Q62426

Q91VW3

pI 5

P01887

Q9Z0J0 P18760

P08905 P35175

P01887

8

Figure 1 2-DE reference map for BMM-CM (pI 5-8, 12% acrylamide, MOPS running buffer). In total, 238 spots were detected and 131 were excised and analyzed. In all, 106 of these were identified (81% success) as 51 distinct murine proteins. Annotated with Swiss-Prot accession numbers. Immunology and Cell Biology


GM-primed BMM

BMM Mr kDa 50

B

B

20 C

C

15

pI 5

8

pI 5

8 GM-primed BMM

BMM

BMM

GM-primed BMM Stefin-3

C19orf10

CTSL Figure 2 Representative 2-DE separation of BMM-CM and GM-primed BMM-CM. Sixteen hour serum-free CM from BMM and GM-primed BMM were harvested, concentrated and 300 mg (in 2D sample buffer, 9.5 M urea, 2% CHAPS (w/v), 1% dithiothreitol (DTT) (w/v), 0.4% pharmalytes, bromophenol blue, 40 mM Tris base) subjected to 2-DE analysis (pI 5-8, 12% acrylamide, MOPS running buffer). Using PDQuest software, a total of 238 spots were detected, 131 spots analyzed and 106 spots identified as 51 distinct proteins. (a) The few differentially secreted proteins are circled. (b) Detail of gels showing downregulation of cathepsin L (CTSL) by GM-CSF priming. (c) Detail of gels showing upregulation of stefin-3 and C19orf10 by GM-CSF priming.

We excised and analyzed 131 gel spots from BMM and GM-primed BMM gels (total detected spots¼238) and 201 from GM-BMM gels (total¼336). In BMM and GM-primed BMM, we identified 106/131 spots (81% success) comprising 51 distinct murine proteins. Of the 201 spots from GM-BMM gels, we identified 159 (79% success) comprising 77 distinct proteins. There were only a few differences in the protein secretion profiles of BMM and GM-primed BMM (Figure 2a). In contrast, the 2-DE patterns for BMM and GM-BMM were quite different (Figure 3a). 34 proteins (unshaded in Tables 1 and 2) were identified in the CM from both BMM and GM-BMM (by MS or by correlation with co-migration on 2D gels and/or partial sequence data). The shaded entries in Tables 1 and 2 are those proteins found to be restricted to only one or other of these CM by these same approaches (that is, no correlating MS or co-migration evidence). On the basis of these data there are no evidence in GM-BMM CM for 12 of the proteins found in BMM CM. Reciprocally, there are 23 proteins identified that are restricted to GM-BMM CM. Cathepsin L, a protein of high abundance in the BMM-CM, was present at much lower levels after GM-CSF priming (Figure 2b) while conversely C19orf10 and stefin-3 (two spots) were upregulated by

such priming (Figure 2c). For the CM from BMM and GM-BMM, while a number of proteins remained relatively unchanged in abundance, many clearly different spots were present in each condition (Figure 3a). One easily observable difference was the almost complete absence of cathepsins in GM-BMM-CM (Figure 3b). These proteins (cathepsins B, D, L, S and Z) made up a significant proportion of the total number of identified spots (that is, 29 out of 131) in BMM-CM while they accounted for only two such spots (both cathepsin S) in GM-BMM-CM (Figure 3b). C19orf10 western blotting One of the few secreted proteins detected for which there seemed to be modulation by GM-CSF priming of BMM was C19orf10 (Figure 2c). To further examine the relative C19orf10 abundance in the CM, we performed western blots of the CM from the three macrophage populations. This analysis confirmed that this protein was upregulated in CM from GM-primed BMM and GM-BMM compared with BMMCM (Figure 4). As the expression of many pro-inflammatory cytokines follows this pattern,14 we treated the three populations with the anti-inflammatory glucocorticoid, dexamethasone. Interestingly, the Immunology and Cell Biology


BMM

GM-BMM

Mr

kDa

50

20

15

CTSD 50 CTSB

CTSL

CTSZ 20

CTSS

CTSS

C19orf10

15 pI 5

8

5

8

Figure 3 Representative 2-DE separation of BMM-CM and GM-BMM-CM. Sixteen hour serum-free CM from BMM and GM-BMM were harvested, concentrated and 300 mg (in 2D sample buffer, 9.5 M urea, 2% CHAPS (w/v), 1% dithiothreitol (DTT) (w/v), 0.4% pharmalytes, bromophenol blue, 40 mM Tris base) subjected to 2-DE analysis (pI 5-8, 12% acrylamide, MOPS running buffer). Using PDQuest software, a total of 336 spots were detected, 206 spots analyzed and 159 spots identified as 77 distinct proteins. (a) Although these macrophage cell types have quite different spot patterns, there are a number of common proteins (some of these are highlighted by squares). (b) Among the most obvious differences in protein abundance are the changes in cathepsin enzyme abundance. The position of C19orf10 is also indicated.

Table 1 Proteins identified in BMM-CM Protein

Accession #

No. of spots

Coverage (%)

Mass values

Mascot

matched

expect value

Extracellulara 60 kDa chaperonin b-2-Microglobulin precursor

(Swiss-Prot: P63038) (Swiss-Prot: P01887)

1 4

31 57

14 6

4.8E-04 3.7E-06

BiP Cathepsin B


1 10

41 33

27 9

4.4E-22 4.0E-22b

Cathepsin D Cathepsin L


12 4

14 35

5 16

4.0E-07b 1.1E-09b

Cathepsin S Cathepsin Z

(Swiss-Prot: O70370) (Swiss-Prot: Q9WUU7)

2 1

56 34

14 9

1.3E-35b 3.2E-09b

Cu/Zn superoxide dismutase Cystatin C


1 1

36 39

4 7

1.1E-04b 8.0E-15b

Endoplasmic reticulum protein 29 Epididymal secretory protein E1 precursor

(Swiss-Prot: P57759) (Swiss-Prot: Q9Z0J0)

1 3

46 38

23 6

6.9E-17 3.80E-06

Galectin-1 Galectin-3 (Mac-2 antigen)


2 2

54 23

7 7

5.0E-17b 1.3E-13b

Gelsolin—C terminal Granulin


1 2

3 39

1 18

1.5E-06c 3.5E-09b

Lysozyme

(Swiss-Prot: P08905)

5

67

7

2.0E-27b

Immunology and Cell Biology


Table 1 Continued Protein

Accession #

No. of spots

Coverage (%)

Mass values

Mascot

matched

expect value

Macrophage-capping protein


2

49

12

3.5E-08

Meteorin, glial cell differentiation regulator-like Nucleobindin 1

(Swiss-Prot: Q8VE43) (Swiss-Prot: Q02819)

1 2

29 22

9 7

3.5E-04b 1.8E-05b

Prosaposin Protein disulfide-isomerase A3

(Swiss-Prot: Q61207) (Swiss-Prot: P27773)

4 1

46 58

27 29

6.9E-18 6.9E-22

Tissue inhibitor of metalloproteinases 2 Uncharacterized protein C19orf10

(Swiss-Prot: P25785) (Swiss-Prot: Q9CPT4)

1 1

20 30

5 6

1.3E-02 6.3E-08

b-Actin Biliverdin reductase B (flavin reductase (NADPH))

(Swiss-Prot: P60710) (Swiss-Prot: Q923D2)

3 2

50 39

16 5

8.0E-10b 1.5E-03

Cofilin 1 Cystatin B

(Swiss-Prot: P18760) (Swiss-Prot: Q62426)

1 1

55 63

7 6

7.8E-04b 6.3E-09b

Heat shock protein 8 Translationally controlled tumor protein


1 1

44 41

24 10

5.5E-19 1.4E-05

Peroxiredoxin 1 Peroxiredoxin 2


1 1

59 37

13 7

4.0E-17b 7.9E-05

Proliferating cell nuclear antigen Ras-related protein Rab-6A


1 1

29 40

6 6

3.2E-06 1.1E-03

SH3 domain-binding glutamic acid-rich protein-like 3 Stathmin

(Swiss-Prot: Q91VW3) (Swiss-Prot: P54227)

1 2

43 39

5 6

1.3E-10b 6.8E-03

Thioredoxin-like 1 Transgelin 2

(Swiss-Prot: Q8CDN6) (Swiss-Prot: Q9WVA4)

1 1

53 36

13 8

6.9E-11 1.4E-02

Tropomyosin a-3 chain Vimentin


1 3

26 30

11 19

2.9E-04 5.0E-10


3 1

64 37

25 14

2.8E-23 2.2E-09b

Non-classical secretiona

Predicted transmembrane helix(s)a a-Enolase Phosphoglycerate kinase 1 No predicted sequencesa Alcohol dehydrogenase (NADP+)

(Swiss-Prot: Q9JII6)

1

37

8

2.8E-08

Peptidylprolyl isomerase A (cyclophilin A) Phosphoglycerate mutase

(Swiss-Prot: P17742) (Swiss-Prot: Q9DBJ1)

3 1

60 46

7 9

4.0E-09 3.5E-06

Pyruvate kinase isozymes M1/M2 Rho GDP-dissociation inhibitor 1

(Swiss-Prot: P52480) (Swiss-Prot: Q99PT1)

1 2

37 31

15 5

2.1E-06 2.4E-04

Rho GDP-dissociation inhibitor 2 Stefin-3


2 2

35 33

5 4

8.0E-10 3.2E-02

Swiprosin 1 Triosephosphate isomerase 1 (Mus musculus)

(Swiss-Prot: Q9D8Y0) (Swiss-Prot: P17751)

2 1

29 43

9 8

4.3E-05b 6.2E-05b

Abbreviations: BMM, bone marrow-derived macrophage; CM, conditioned media; GM, granulocyte-macrophage; MS, mass spectrometry; NADPH, nicotinamide adenine dinucleotide phosphate; PMF, peptide mass fingerprinting. Shaded entries have not been identified in GM-BMM (by MS or co-migration on gels). aSubcellular localization sequences: extracellular—annotated as extracellular in Swiss-Prot database or contain predicted secretory signal peptides; non-classical secretion—containing predicted non-classical protein secretory sequences or propeptide cleavage sites; predicted transmembrane helix(s); no predicted sequences—all other proteins. bMascot expect value generated by PMF unless annotated: combined cMascot expect value generated by PMF unless annotated: MS/MS.

steroid decreased extracellular C19orf10 in BMM-CM and GMBMM-CM but increased it in GM-primed BMM-CM. The identity of this protein is discussed in more detail below. In silico cellular localization prediction In silico cellular localization programs are valuable tools for the analysis of proteins identified in the extracellular space.27,28 We therefore used online sequence analysis programs to predict the cellular localization of our identified extracellular proteins. For BMM-CM (and GM-primed BMM-CM), of the 51 proteins identified, 24 (47%) were annotated as extracellular in Swiss-Prot and/or

contained predicted signal peptides (see Table 1 and also Figure 5 for a diagrammatic representation). Of the 27 remaining proteins, 16 (31%) were predicted to undergo non-classical secretion and/or contain proprotein cleavage sites. Two of the remaining proteins (4%) contained predicted transmembrane domains and nine (18%) no predicted sequences. The predicted localization pattern for the extracellular proteins from GM-BMM seems to show some differences from that for BMM—of the 77 proteins identified in GM-BMM CM, only 16 (21%) were extracellular, 35 (45%) were predicted as nonclassically secreted, 4 (5%) had transmembrane domains and 22 (29%) had no predicted sequences (see Table 2 and also Figure 5). Immunology and Cell Biology


Table 2 Proteins identified in GM-BMM-CM Protein

Accession #

No. of spots

Coverage (%)

Mass values

Mascot expect

matched

value

Extracellulara b-2-Microglobulin precursor Cathepsin S

(Swiss-Prot: P01887) (Swiss-Prot: O70370)

4 2

39 37

5 11

2.3E-02 9.3E-07

Chitinase-3-like protein 3 precursor Cu/Zn superoxide dismutase

(Swiss-Prot: O35744) (Swiss-Prot: P08228)

2 1

46 38

16 6

1.2E-12 8.9E-05

Cystatin C Endoplasmic reticulum protein 29


7 1

60 33

14 10

7.4E-13 3.7E-06

Galectin-1 Galectin-3


2 12

60 41

9 12

3.7E-08 1.5E-08

Ganglioside GM2 activator precursor Gelsolin—C-terminal fragment


3 3

41 9

8 9

1.3E-04 2.3E-06

High mobility group protein B1 Lysozyme


1 10

54 53

13 8

9.3E-12 6.6E-06

Macrophage metalloelastase precursor—MMP12 Macrophage-capping protein


1D band 3

19 28

11 11

1.1E-04 1.0E-05

Protein disulfide-isomerase A3 precursor Uncharacterized protein C19orf10

(Swiss-Prot: P27773) (Swiss-Prot: Q9CPT4)

1 1

23 28

15 5

1.5E-08 1.2E-05b

Actin, a cardiac muscle 1 -N-term fragment 16 kDa b-Actin


1 7

20 29

8 11

1.5E-06 3.7E-08

Actin-related protein 2/3 complex subunit 5 Acyl-CoA-binding protein

(Swiss-Prot: Q9CPW4) (Swiss-Prot: P31786)

1 1

37 52

5 8

7.6E-03 6.8E-04

Biliverdin reductase B (flavin reductase (NADPH)) Coactosin-like protein

(Swiss-Prot: Q923D2) (Swiss-Prot: Q9CQI6)

2 2

68 47

12 8

1.2E-11 8.5E-06

Cofilin-1 Coronin-1A

(Swiss-Prot: P18760) (Swiss-Prot: O89053)

3 1

77 18

11 13

1.5E-09b 1.5E-05

Cystatin B Cysteine-rich protein 1


2 1

63 48

7 4

1.9E-07 6.2E-05b

Fumarate hydratase, mitochondrial precursor Glia maturation factor gamma

(Swiss-Prot: P97807) (Swiss-Prot: Q9ERL7)

1 1

19 38

10 9

1.9E-05 2.9E-08

Heterogeneous nuclear ribonucleoproteins A2/B1 Lamin-B1


3 1

43 19

14 13

3.7E-07 4.2E-03

Osteoclast-stimulating factor 1 Peroxiredoxin-1


2 2

50 63

8 19

1.2E-05 1.2E-17

Peroxiredoxin-2 Peroxiredoxin-5, mitochondrial precursor


1 1

56 51

11 13

1.2E-14 3.0E-06

Non-classical secretiona

Phosphatidylethanolamine-binding protein 1


1

48

6

3.5E-05

Phosphomannomutase 2 Profilin-1

(Swiss-Prot: Q9Z2M7) (Swiss-Prot: P62962)

1 1

28 72

9 16

3.7E-05 3.7E-11

Putative RNA-binding protein 3 Ras-related protein Rab-6A


2 2

58 38

9 6

2.5E-06 4.2E-03

Ras-related protein Rab-6B SH3 domain-binding glutamic acid-rich-like protein

(Swiss-Prot: P61294) (Swiss-Prot: Q9JJU8)

2 1

41 34

6 4

3.2E-02 5.2E-03

SH3 domain-binding glutamic acid-rich-like protein 3 Sorcin

(Swiss-Prot: Q91VW3) (Swiss-Prot: Q6P069)

1 1

39 39

5 9

2.4E-04 2.3E-06

Stathmin Thioredoxin


1 1

30 75

4 8

9.3E-03 4.7E-08

Transgelin-2 Translationally controlled tumor protein

(Swiss-Prot: Q9WVA4) (Swiss-Prot: P63028)

4 1

72 40

15 7

1.9E-13 2.9E-05

Tropomyosin a-3 chain Ubiquitin-conjugating enzyme E2 L3


1 1

10 57

5 5

1.0E-02 2.9E-04

Ubiquitin-conjugating enzyme E2 N Vimentin


1 4

54 38

8 28

1.9E-07 2.9E-19


3 2

52 43

21 9

1.2E-19 1.4E-04

Predicted transmembrane helix(s)a a-Enolase Core-binding factor subunit b



Table 2 Continued Protein

Accession #

No. of spots

Coverage (%)

Mass values

Mascot expect

matched

value

Heat shock protein HSP 90-b


1D band

30

24

2.3E-11

Phosphoglycerate kinase 1


2

47

16

3.7E-14

No predicted sequencesa 14-3-3 Protein zeta/delta


1D band

44

14

1.4E-02

Alcohol dehydrogenase (NADP+) Eukaryotic translation initiation factor 1A, X-chromosomal

(Swiss-Prot: Q9JII6) (Swiss-Prot: Q8BMJ3)

1 1

45 33

13 5

2.9E-17 5.0E-02

Eukaryotic translation initiation factor 5A-1 F-actin-capping protein subunit a-2


2 1

42 18

6 7

8.1E-03 4.8E-05

Fatty acid-binding protein, epidermal FK506-binding protein 1A


2 1

73 41

17 6

7.4E-17 1.1E-04b

Fructose-bisphosphate aldolase A Growth factor receptor-bound protein 2


1 1

64 50

16 13

2.3E-16 2.9E-11

Histidine triad nucleotide-binding protein 1 Nucleoside diphosphate kinase A


1 1

51 79

5 11

1.3E-03 9.3E-08

Nucleoside diphosphate kinase B Peptidylisomerase A (cyclophilin A)


1 7

79 57

14 16

5.9E-13 4.7E-12

Peroxiredoxin-6 Polymerase delta-interacting protein 3

(Swiss-Prot: O08709) (Swiss-Prot: Q8BG81)

1 1D band

65 30

13 11

2.3E-19 1.7E-03

Protein S100-A9 Rho GDP-dissociation inhibitor 1

(Swiss-Prot: P31725) (Swiss-Prot: Q99PT1)

5 4

52 32

11 10

3.1E-06 9.3E-09

Rho GDP-dissociation inhibitor 2 Stefin-3


3 2

41 51

7 7

6.5E-05 4.1E-04

Stress-induced-phosphoprotein 1 Swiprosin 1

(Swiss-Prot: Q60864) (Swiss-Prot: Q9D8Y0)

1 1

16 37

10 11

3.8E-02 2.3E-12

Triosephosphate isomerase


3

81

16

2.3E-19

Abbreviations: BMM, bone marrow-derived macrophage; CM, conditioned media; GM, granulocyte-macrophage; MS, mass spectrometry; NADPH, nicotinamide adenine dinucleotide phosphate; PMF, peptide mass fingerprinting. Shaded entries have not been identified in BMM (or GM-primed BMM) (by MS or co-migration on gels). aSubcellular localization sequences as for Table 1. bMascot expect value generated by PMF unless annotated: combined.

+

GM-BMM

+

GM-p BMM

BMM

17kDa 14kDa

+

Figure 4 C19orf10 western blot. BMM, GM-primed BMM and GM-BMM were cultured with (+) or without () dexamethasone (107 M) for 16 h, and serum-free CM subjected to western blot analysis.

It should be noted that most of the proteins that had no predicted sequences have also been reported in the extracellular compartment in other studies (see Discussion). DISCUSSION We developed methods for enrichment of proteins from CM and compared the protein composition of CM from three different macrophage populations using proteomics techniques. Cells were extensively washed in serum-free medium to reduce as much as possible contamination from serum proteins. Some bovine proteins were detected but only in a few spots at low abundance, relative to other published studies,23,29–32 suggesting effective cell washing.

Although we detected actin as minor spots (full length and fragments) on our gels, it has been reported as being secreted by a number of cell types (including macrophages) in several papers,23,33–36 as well as being on the cell surface.37 Furthermore, the majority of proteins identified have been reported to be secreted (see below). We compared BMM with (a) BMM treated acutely (16 h) with GMCSF (GM-primed BMM), and with (b) GM-CSF-differentiated macrophages (derived by culturing bone marrow precursors with GM-CSF) (GM-BMM). Although there were few differences between BMM and GM-primed BMM CM, the 2-DE spot patterns for GM-BMM-CM and BMM-CM were quite different. GM-BMM and BMM are phenotypically diverse cells, with different morphology and cytokine secretion profiles11–14,17,19,38 and their contrasting spot patterns reflect this (see Figure 3). On the basis of protein identification and co-migration data, 12 proteins identified in BMM CM seem to be restricted to this phenotype and 23 proteins in GM-BMM CM were specific to these cells (see shaded entries in Tables 1 and 2). Identified proteins were divided into four groups depending on their Swiss-Prot annotation and possession of sequences found in extracellular or membrane proteins. For BMM- and GM-BMM-CM, 82 and 71% of the proteins, respectively, had sequences predicted to place them in a membrane or potentially outside the cell (Figure 5). A majority of the proteins that we identified as having no predicted sequences have been reported in the extracellular compartment in other studies.23,30,32,33,39 Thus, the large majority of the proteins detected in the CM of these cell types are known or predicted to be extracellular. Given the difference in the distribution of proteins in the Immunology and Cell Biology

The macrophage secretome MJ Bailey et al 290 BMM

predicted transmembrane helix(s) 2 (4%)

GM-BMM

no predicted sequences 9 (18%)

no predcted sequences 22 (29%)

extracellular 16 (21%)

extracellular 24 (47%) non-classical secretion 16 (31%)

predicted transmembrane helix(s) 4 (5%)

non-classical secretion 35 (45%)

Figure 5 Pie charts showing cellular localizations of BMM and GM-BMM extracellular proteins as predicted by online sequence analysis algorithms as in Tables 1 and 2. Both the number of proteins and their percentage (in parentheses) in the various localizations are provided.

extracellular and non-classical secretion classifications for BMM vs GM-BMM (Tables 1 and 2, Figure 5), a possible switch from primarily signal peptide-driven secretion to non-classical secretion pathways from BMM to GM-BMM, respectively, is suggested. Of the 12 proteins restricted to BMM-CM, 11 were predicted to be in the extracellular compartment, while of the 23 proteins restricted to GM-BMM-CM, only three were similarly predicted. These figures also suggest a possible relative preference for different secretory mechanisms for these two cell phenotypes. However, a larger data set and/or mechanistic studies would be needed before such a conclusion could be drawn. The most striking of the differences between BMM and GM-BMM is the relative abundance of the lysosomal cathepsin enzymes in the CM of the former. These enzymes are thiol (cathepsins B, L, S and Z) or aspartyl (cathepsin D) proteases, contain a signal peptide and are translated as inactive zymogens (proenzymes) that need cleavage and/ or disulfide bonding of heavy and light chains for activity. All the identified cathepsins were at molecular masses consistent with the inactive zymogens, except cathepsin S, which was present at both its zymogen mass (38 kDa) and that of its active enzyme (24 kDa). The latter cathepsin S form was the only cathepsin detected in GM-BMM (Figure 2b). Cathepsins have important roles in physiologic processes including antigen presentation, bone remodelling and wound healing, and are implicated in pathologies such as rheumatoid arthritis and osteoarthritis.40 Trombetta et al.12 have shown that, while macrophages (in fact, BMM) contained high levels of lysosomal proteases and rapidly degraded internalized proteins, GM-CSF-derived dendritic cells (in fact, derived in a similar way to GM-BMM) were protease poor and retained antigen for extended periods. This maintenance of an antigen pool was conducive to antigen presentation. Our cathepsin data would seem to be consistent with these literature findings. Interestingly, one difference between BMM-CM and GM-primed BMM-CM was a sharp drop in the level of cathepsin L in the latter (Figure 2b). Reilly et al.41 have shown uptake of cathepsin L from CM of human alveolar macrophages and THP-1 cells (a human monocyte/ macrophage line) after addition of human serum. These results, in conjunction with ours, suggest that GM-CSF might provide a stimulus that drives active uptake of cathepsin L by macrophages from the extracellular environment. In addition to these thiol proteases, thiol protease inhibitors (stefin-3, cystatin C and cystatin B) were identified. One of the few differences between BMM-CM and GM-primed BMM-CM was a marked increase in stefin-3 in the CM of the latter. Cystatin C was also much more prevalent in GM-BMM-CM (seven spots) compared with BMM-CM (one spot). One protein that is present in the CM of both GM-primed BMM and GM-BMM, but at very low levels in BMM-CM, is the Immunology and Cell Biology

uncharacterized protein, C19orf10. High levels of this molecule have been found levels in the synovial fluid of patients presenting with different arthropathies (7–184 mg ml1).42 These researchers also found it in human fibroblast-like synoviocytes and the synovium. There is considerable confusion in the literature,42–44 in protein databases and with reagent companies regarding this protein. The protein we have identified is the gene product of D17Wsu104e (the human gene is called C19orf10) and has the synonyms, stromal cellderived growth factor SF20 and IL-25 and IL-27W. Indeed, literature searches for either of these genes (C19orf10 and D17Wsu104e) give results overwhelmingly for other proteins. Another protein with homology to the IL-17 family has also been designated as IL-25 and has the synonym IL-17E. These two proteins share no homology but both continue to be labelled as IL-25 in Swiss-Prot and NCBI databases. Commercial antibody datasheets labelled IL-25 may contain antibodies to either of these proteins. Furthermore, research attributing growth factor activity to the molecule we have identified could not be repeated and was later withdrawn.45,46 With this in mind and the fact that no function has been established for this molecule, we have followed the lead of Wilkins et al.42 and referred to it as C19orf10. We found C19orf10, which contains a secretory signal peptide, in GMprimed BMM-CM and GM-BMM-CM (Figures 2c and 3b); western blotting confirmed its relative upregulation in CM from these cells (Figure 4). Furthermore, dexamethasone treatment decreased extracellular C19orf10 in BMM-CM and GM-BMM-CM but increased it in GM-primed BMM-CM. This protein has been named in a patent for possible diagnostic and therapeutic applications (http:// www.freshpatents.com/Characterization-of-c19orf10-a-novel-synovialprotein-dt20080103ptan20080004232.php). We intend to further study the regulation of the expression and the secretion of this molecule in macrophages with different phenotypes and in response to various stimuli. BMM and CSF-1-differentiated human monocytes have been suggested as having a ‘M2-like’ phenotype, that is, one that is less inflammatory than GM-BMM and GM-CSF-differentiated monocytes (that have a ‘M1-like’ phenotype). This type of macrophage is also thought to have a role in homeostasis.10,14,22,47–49 A number of proteins found in the CM of BMM but not GM-BMM could perhaps fit with the M2 phenotype, with putative roles in tissue growth, repair or protection from damage. These include meteorin,50,51 granulin,52,53 Timp-2,54–56 chaperonin 60,57–59 prosaposin60 and nucleobindin 1.61,62 In contrast, there were a number of proteins identified in GM-BMM CM but not BMM CM, which have been implicated in inflammatory processes, namely chitinase-3-like protein 3 (YM1, eosinophil chemotactic cytokine),63 MMP12,64,65 peroxiredoxin-5,66,67 14-3-3 protein zeta/delta,68 epidermal fatty acid-binding


protein,69 HMGB1,70–72 heterogeneous nuclear ribonucleoproteins A2/B1,73–76 glia maturation factor gamma,77,78 HSP9079–82 and S100A9.83–86 To the best of our knowledge this is the first report comparing the extracellular protein profiles of macrophage phenotypes using a proteomic approach. We have shown that BMM-CM and GMBMM-CM have rather different protein profiles with the latter cell type seeming to favor non-classical secretion pathways. Extracellular expression of cathepsins (lysosomal proteases) and their inhibitors seems to be a characteristic difference between these macrophage cell types with higher levels usually observed in BMM-CM. Furthermore, we have identified a number of proteins in BMM-CM and GM-BMMCM that could be involved in various tissue regeneration and inflammatory (immune) processes, respectively. We have also identified the novel, uncharacterized protein, C19orf10, in the CM of macrophages and whose extracellular levels are upregulated by GM-CSF and differentially regulated by dexamethasone. METHODS All animal experiments were preformed in accordance with Australian law and under the approval of the ethics committee of The University of Melbourne.

Preparation of BMM and GM-BMM The preparation of adherent BMM grown in M-CSF or GM-CSF (GM-BMM) has been described.13 Briefly, bone marrow cells were flushed from long bones of C57Bl6 mice (Central Animal Services, Monash University, Victoria, Australia) in RPMI 1640 medium (Invitrogen, Grand Island, NY, USA), centrifuged, the cell pellet resuspended (106cells ml–1) in RPMI containing 10% heat-inactivated fetal bovine serum (CSL Biosciences, Parkville, Australia), penicillin (100 U ml–1), streptomycin (100 mg ml–1) and L-glutamine (2 mM), and either M-CSF (CSF-1) (5000 U ml–1, a gift from Chiron, Emeryville, CA, USA) for BMM or GM-CSF (1000 U ml–1, PeproTech, Rocky Hill, NJ, USA) for GM-BMM. After 3 days, non-adherent cells were harvested and 3106 cells were seeded in 10 ml of the same medium in non-treated, 100 mm dishes (Iwaki, Tokyo, Japan). After 4 days, the adherent cells were harvested as BMM or GM-BMM.

Preparation of secreted protein fractions Three separate CM were prepared from BMM, ‘GM-primed’ BMM and GMBMM. To do this, BMM and GM-BMM were plated in 150 mm Falcon dishes at a density of 1107 cells per dish (50 ml total volume) in the respective growth media and cultured at 37 1C for 48 h or until 70–80% confluent. The CM was then removed and the dish washed with phenol red-free RPMI. This washing was repeated five times to remove bovine proteins. Phenol red-freeRPMI (40 ml) containing M-CSF, M-CSF+GM-CSF (for the GM-primed BMM) or GM-CSF was added. Plates were cultured overnight (16 h) at 37 1C before the CM was removed and centrifuged at 1500 r.p.m. for 5 min at 4 1C. Complete protease inhibitor cocktail (Roche, Basel, Switzerland) was added to the CM before filtering (0.45 mm). CM was acidified by addition of trifluoroacetic acid (TFA, 0.1% v/v) and stored at 20 1C before processing. The protein content in the CM was very low (o1 mg ml–1) requiring the concentration of the large volumes before electrophoresis. This was achieved by immobilizing acidified CM on reverse phase cartridges (tC2 SepPak Light, Waters, Milford, MA, USA) before desalting and eluting in small volumes of organic solvent. Protein binding was optimized and no protein breakthrough was observed with up to 500 mg protein. Elution of protein from the cartridges was also optimized using increasing 5% steps of acetonitrile (ACN) in 0.1% TFA—all protein eluted between 30 and 80% ACN (cf.87) and the solution was vacuum dried and stored at 20 1C.

Two-dimensional gel electrophoresis A minimum of three 2D gels was run for each experimental condition. The dried CM protein was resuspended in first dimension IEF buffer (9.5 M urea, 2% CHAPS (w/v), 1% dithiothreitol (w/v), 0.4% pharmalytes, bromophenol

blue, 40 mM Tris base) over 90 min at room temperature with occasional vortexing. Samples were clarified by centrifugation and protein concentration determined using the two-dimensional Quant Kit (GE Healthcare Bio-Sciences, Uppsala, Sweden). In all, 300 mg of protein in 185 ml was loaded onto 11 cm pI 5-8 or 3-10NL IPG strips (Bio-Rad, Hercules, CA, USA) and after 12-h rehydration subjected to first dimension isoelectric focusing with rapid voltage ramping to a total of 30 000 V h–1 in the Bio-Rad Protean IEF cell. Strips were washed briefly in deionized water and stored at 20 1C or immediately subjected to reduction/alkylation and equilibration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer before electrophoresis on Bio-Rad Criterion Bis-Tris precast gels (10 or 12%) using the MOPS running buffer. Gels were stained with PageBlue colloidal Coomassie stain (Fermentas, Burlington, Ontario, Canada) and imaged on a Bio-Rad GS710 scanner. The images obtained were analyzed by PDQuest 2-DE analysis software (Bio-Rad). Parameters for spot detection were defined using the spot detection wizard. After filtering to remove speckles and clarify spots, three-dimensional Gaussian spots were created. Gel images were aligned and matchsets were produced for comparison. The software identified matched and unmatched spots, which we inspected manually for errors. Protein spots of interest were excised for in-gel digestion with blunt-ended 16 gauge needles and deposited in microcentrifuge tubes. Gel plugs were destained by consecutive 15 min washes with vortexing in 150 ml of 25 mM ammonium bicarbonate, 25 mM ammonium bicarbonate in 50% ACN and 100% ACN. This treatment was repeated if needed. Gel plugs were either processed immediately or stored in ACN. We found that gel plugs could be stored in this final ACN wash for a number of weeks at 20 1C before digestion without affecting mass spectra.

In-gel tryptic digestion of gel spots Gels plugs were dried in a vacuum centrifuge and 20 ml of 20 mg ml–1 proteomics grade trypsin (Sigma, St Louis, MO, USA) in 25 mM ammonium bicarbonate was added to the tubes and the gel allowed to rehydrate for 60 min on ice. Excess trypsin solution was then removed, 20 ml of 25 mM ammonium bicarbonate added and digestion was allowed to proceed overnight at 37 1C. Digestion was quenched by addition of 1 ml of 10% TFA. The digest supernatant was either used directly for analysis or the gel plugs further extracted with increasing ACN concentrations before analysis as follows. Direct Anchor Chip analysis of digest supernatant even without extraction gave good results and we tended to favor this method to minimize processing steps. For extracted gel plugs, pooled extracts were dried by vacuum centrifuging and resuspended in 5–10 ml of 0.1% TFA for analysis.

MALDI-MS and protein identification In total, 3 ml of digest supernatant or pooled extract was applied to a Bruker Biosciences Anchorchip MALDI target, prepared with CHCA (see the thin layer affinity method for CHCA in the Bruker Anchorchip manual). After 3 min, this solution was removed and the spot washed with 5 ml of 0.1% TFA. MALDI-MS was performed on a Bruker Ultraflex MALDI-TOF/TOF mass spectrometer or a Bruker Microflex MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). Selected intense peaks were analyzed using MS/MS (LIFT, Ultraflex) or PSD (FAST, Microflex) functions of the mass spectrometers. Calibration was performed using Peptide Calibration mix II (Bruker). Peaklists were generated using Flexanalysis (Bruker). Biotools software (Bruker) and the Mascot search engine were used to interrogate the Swiss-Prot database (release: 14.0/56.0, 22 July 2008) and proteins were identified by peptide mass fingerprinting by sequencing of peptide fragments (MS/MS or PSD) or by using the combined search option in Biotools. Initial search parameters for peptide mass fingerprinting were: Taxonomy: mammalia (to identify possible human and bovine contaminants as well as murine proteins); MS tolerance: 200 p.p.m., missing cleavages: p1; Enzyme: trypsin; Fixed modifications: carbamidomethylation; Variable modifications: oxidation (M). Initial search parameters for MS/MS and PSD were: MS tolerance: 200 p.p.m.; MS/MS tolerance: 0.8 Da; Enzyme: trypsin. Identifications with Mascot expect probability values o0.05 were then manually verified by examination of spectra and/or resubmission of peak lists to Mascot. We took a conservative approach to protein identification and based acceptance on a number of criteria other than these scores. These included theoretical and experimental Mr and pI being Immunology and Cell Biology

The macrophage secretome MJ Bailey et al 292 in accordance, experimental peptide mass accuracy variation across the mass range and repeatability of identification across different gels. If multiple members of a protein family were identified those with the highest ranked hit were selected.

In silico cellular localization prediction Amino acid sequences of identified proteins were submitted to online programs for the prediction of cellular localization sequences. The presence of secretory signal peptides was predicted using SignalP88 (http://www.cbs.dtu.dk/services/ SignalP/). Those proteins that were not annotated in the Swiss-Prot database as extracellular and were not predicted to contain signal peptides were further analyzed by SecretomeP89 (http://www.cbs.dtu.dk/services/SecretomeP/) and ProP 1.090 (http://www.cbs.dtu.dk/services/ProP/) for predicting non-classical protein secretion and proprotein cleavage sites, respectively. Those proteins that were not detected as potentially extracellular with the above software were analyzed for transmembrane domains using http://www.ch.embnet.org/ software/TMPRED_form.html website.91

Western blotting The presence of C19orf10 was analyzed by western blot. Culture conditions for CM preparation were identical to the above with the exception that cells were cultured in the presence and absence of dexamethasone (100 nM). CM from three to four separate experiments were pooled and concentrated in Microcon YM-10 10 kDa spin filters (Millipore, Billerica, MA, USA). The equivalent of 0.75 ml CM was loaded onto 12% Bio-Rad Criterion Bis-Tris precast gels and subjected to electrophoresis with the MES buffer system (which allows separation at lower Mr). Proteins were transferred onto PVDF membranes (Millipore), blocked with 3% bovine serum albumin and 1% fetal bovine serum in phosphate-buffered saline, incubated with rabbit polyclonal antiC19orf10 (1/5000, Abcam, Cambridge, UK), followed by 1/10 000 horseradish peroxidase labelled anti-rabbit IgG (Dako, Glostrup, Denmark). Specific protein bands were visualized using immobilon horseradish peroxidase substrate (Millipore).

CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS This work was funded in part by the Cooperative Research Centre (CRC) for Chronic Inflammatory Diseases, and grants from the National Health and Medical Research Council (NHMRC) to JAH and from the Helen Macpherson Smith Trust (JAH/MJB). JAH is also supported by a NHMRC Senior Principal Research Fellowship. DCL was also supported by AFA-ARA Heald Fellowship from the Arthritis Foundation of Australia. Proteomic data analysis described in this work was supported by the use of the Australian Proteomics Computational Facility funded by the Australian NH&MRC under grant No. 381413.

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