Developmental& ComparativeImmunology

ARTICLE IN PRESS

0145-305X/$ - se

doi:10.1016/j.dc

Abbreviations

BlyS, B cell stim

Differential dete

ontology; HSP,

electrospray ion

identification te

electrophoresis;

exchange; SDS,

X-100; VSGP/F�CorrespondiE-mail addre

Developmental and Comparative Immunology 30 (2006) 1070–1083

www.elsevier.com/locate/devcompimm

Differential detergent fractionation for non-electrophoreticbovine peripheral blood monocyte proteomics reveals proteins

involved in professional antigen presentation

S.-R. Lee, G.T. Pharr, A.M. Cooksey, F.M. McCarthy, B.L. Boyd, L.M. Pinchuk�

Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, P. O. Box 6100, Mississippi State,

MS, 39762-6100, USA

Received 26 August 2005; received in revised form 8 February 2006; accepted 9 February 2006

Available online 13 March 2006

Abstract

Professional antigen presenting cells (APC), dendritic cells (DC) and their myeloid progenitors, monocytes/macrophages

are critical controllers of innate and adaptive immunity. Here we show that differential detergent fractionation (DDF)

analysis of bovine monocytes reveals proteins related to antigen pattern recognition, uptake and presentation to

immunocompetent lymphocytes. We identify 53 bovine proteins involved in immune function of professional APC. In

particular, 13 adhesion molecules, three toll-like receptors (TLR1, 6 and 8), three antigen uptake-related proteins

(including mannose receptor [MR] precursor), and eight actin-like proteins involved in active endocytosis were identified.

In addition, MHC class I and II-related proteins, cytokines, active substances and growth factors have been identified. We

conclude that the DDF approach can provide interpretable and meaningful functional information concerning protein

expression profiles associated with monocyte activation, transformation into macrophages and/or immature DC, and

maturation of monocyte-derived DC in the presence of multiple bovine pathogens.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Monocytes; Progenitors; DC; DDF; TLR; Lectin; Actin; MHC; Cytokines; Growth factors

e front matter r 2006 Elsevier Ltd. All rights reserved.

i.2006.02.002

: 1D, one-dimensional; 2D, two- dimensional; APC, Antigen presenting cells; APRIL, A proliferation-inducing ligand;

ulator protein; BVDV, Bovine Viral Diarrhea Virus; CTL, Cytotoxic T cells; DB, Database; DC, Dendritic cells; DDF,

rgent fractionation; DOC, Deoxycholate; GM-CSF, Granulocyte macrophage colony stimulating factor; GO, Gene

Heat shock protein; IFN, Interferon; Ig, Immunoglobulin; IL, Interleukin; LC-ESI/MS2, Liquid chromatography

ization tandem mass spectrometry; LPS, Lipopolysaccharide; MR, Mannose receptor; MudPIT, Multidimensional protein

chnology; NCBI, National Center for Biotechnology Information; NK, Natural killer; PAGE, Polyacrylamide gel

PBMC, Peripheral blood mononuclear cells; RP, Reverse phase; SCO, Subcommissural organ; SCX, Strong cation

Sodium dodecyl sulfate; Th1, T helper cell type 1; TNF-a, Tumor necrosis factor a; TLR, Toll-like receptor; TX, Triton

, Vascular smooth muscle cell growth-promoting; vWF, Von Willebrand factor

ng author. Tel.: +1 662 325 1436; fax: +1 662 325 1031.

ss: pinchuk@cvm.msstate.edu (L.M. Pinchuk).

ARTICLE IN PRESSS.-R. Lee et al. / Developmental and Comparative Immunology 30 (2006) 1070–1083 1071

1. Introduction

Cells of the monocyte/macrophage lineage areimportant elements of the immune defense systembecause these cells can phagocytose foreign materi-al, present antigens to immunocompetent cells andproduce cytokines, including tumor necrosis factora (TNF-a) and interleukins 1 and 6 (IL-1, IL-6).Cytokine expression by monocytes is efficientlytriggered by stimulation with bacterial productssuch as lipopolysaccharide (LPS) acting via theTLR [1–4]. Bovine monocytes, as professional APC,express relatively high levels of MHC class I andclass II molecules, costimulatory molecules inducedupon activation, receptors for endo- and phagocy-tosis, and adhesion molecules [5]. Recent datasuggest that bovine monocytes are capable ofdirectly inducing immunoglobulin (Ig) secretion inactivated bovine peripheral blood B cells [5,6] aswas previously demonstrated in humans [7,8].Before entering the tissues, under the influence ofcertain cytokines, monocytes are capable of differ-entiating into macrophages or DC [9]. DC, highlyspecialized and the most powerful APC, are pivotalin immune responses, and are considered to beterminally differentiated cells derived from hemato-poietic progenitors or monocytes [10,11]. Cells withphenotypic and functional characteristics of DChave been produced from fully differentiated bovinemonocytes using granulocyte macrophage colonystimulating factor (GM-CSF) and IL-4 [12,13].

Recent studies have provided important cluesabout the mechanisms of TLR-mediated control ofadaptive immunity orchestrated by DC populations[14]. Professional APC antigen uptake and presen-tation of antigenic peptides on the relevant MHCmolecules involve: phagocytosis, the upregulation ofcostimulatory and MHC molecules, a switch inchemokine receptor expression, and the secretion ofcytokines [14–16]. Furthermore, chemokines areregulated through the recognition of pathogens viareceptors expressed by monocyte-derived DC lead-ing to the generation of effector responses includingT helper cell type 1 (Th1) [15] and cytotoxic T cells(CTL) responses [16]. Activation of monocyte-derived DC through TLR modulated the expressionof a common set of genes in response to differentpathogens [14]. After pathogen recognition, DCdownregulate mRNA levels for mannose receptor(MR) [17] and upregulate mRNA for the pro-inflammatory cytokines TNF-a, IL-1b and chemo-kines IL-8 and MIP-1a [18]. In the later stages of

inflammation, the pathogen-induced differentiationand maturation of DC from monocytes becomesmore apparent with the expression of chemokinereceptors CCR7 and CXCR4, and cell-adhesionproteins integrin, LFA-1 and E-cadherin which arerequired for migration to secondary lymphoidtissues [19,20]. Finally, in the secondary lymphoidtissues, mature DC express genes associated with Tcell activation and differentiation [19,20]. ThemRNA levels for CD80, MHC, as well as compo-nents of the proteosome Imp7 and Tap1 areupregulated at the mature DC stage [20–22].

Given the disconcordance between mRNA andprotein levels [23], we have chosen to identifyproteins in subcellular fractions of bovine mono-cytes using a proteomics-based approach. Once theproteins have been identified and characterized, itwill then be possible to address the modulation inexpression of these proteins during the conversionof monocytes to macrophages or DC by cytokinesor infectious agents.

DDF of whole cells yields four electrophoreticallydistinct fractions enriched in cytosolic, membrane-organelle, nuclear membrane and cytoskeletal-ma-trix markers, respectively [24]. Furthermore, multi-dimensional protein identification technology(MudPIT) couples two-dimensional (2D) chroma-tography of peptides with tandem mass spectro-metry (MS2), allowing for the identification ofproteins from highly complex mixtures [25]. Thecombination of DDF-MudPIT was used to increasethe proteome coverage over that of standard DDFand MudPIT [26]. Here we provide the baselineinformation on expression of protein profiles relatedto immune function, in particular, professionalantigen presentation, in bovine peripheral bloodmonocytes.

2. Materials and methods

2.1. Animals

Two conventionally reared and healthy Jerseycattle from the Mississippi State University DairyFacility (Mississippi State, MS) were used. TheMississippi State University Institutional AnimalCare and Use Committee approved all animal use.

2.2. Cell preparation

Bovine peripheral blood mononuclear cells(PBMC) were separated as described elsewhere

ARTICLE IN PRESSS.-R. Lee et al. / Developmental and Comparative Immunology 30 (2006) 1070–10831072

[12,13,27]. Briefly, PBMC were isolated usingHistopaque gradients (1.077 g/ml, Amersham Bios-ciences) and resuspended in RPMI-1640 supple-mented with 10% FBS, 1% Glutamax-1 (Gibco LifeTechnologies), 5� 10�5M 2-mercaptoethanol and100 IU/ml gentamicin (Gibco Life Technologies).Monocytes were separated from PBMC as de-scribed elsewhere [5]. Briefly, sheep red blood cellswere used to separate E-rosette negative andpositive fractions of PBMC. To isolate monocytes,the E-rosette negative fractions of PBMC wereincubated for 2 h at 37 1C and after removing non-adherent populations (mostly B cells), adherent cellswere incubated with mAbs to CD14 (MM61A,VMRD) followed by the addition of magnetic beadsconjugated with mouse anti-IgG1 (Miltenyi Biotech,Auburn, CA) [12,27]. CD14+ monocytes werepositively selected by using magnetic cell separationtechnique according to the manufacturer’s instruc-tions (Miltenyi Biotech). CD14+ monocytes wereobtained from two animals and stored at –70 1Cseparately. The bleeding procedures for the sameanimals have been repeated until the desirablenumber of 2� 107 cells was achieved. Then all thesamples were thawed and mixed followed byimmediate protein separation procedure.

2.3. Protein extraction, trypsin digestion and 2D-LC

ESI MS2

Proteins were isolated using DDF as described[26]. Briefly, DDF sequentially extracts proteinsfrom cellular compartments using a series ofdetergents. Cytosolic proteins were isolated byrepeated washes in digitonin buffer. After thedigitonin washes, the isolation of membrane,nuclear and cytoskeletal proteins were performedwith triton X-100 (TX), deoxycholate (DOC),Tween 40, and SDS buffers, respectively. Toevaluate the quality of isolated proteins, 1% ofthe protein samples were compared using 10%SDS-PAGE. For each of the detergent fractions,equal amounts of protein were precipitated with25% tricholoroacetic acid to remove salts anddetergents. Protein pellets were solubilized andthen digested with 100 ng of trypsin (50:1 ratioof substrate to enzyme) overnight at 37 1C.Peptides were desalted using a peptide microtrap(Michrom BioResources, Inc.) and eluted by a 0.1%triflouroacetic acid, 95% acetonitrile solution.Desalted peptides were dried and resuspended in0.1% formic acid.

2D LC ESI MS2 was done as described elsewhere[26]. Briefly, LC analysis was accomplished bystrong cation exchange (SCX) followed by reversephase (RP) liquid chromatography (LC) coupleddirectly in line with electrospray (ESI) ion trap MS.Samples were loaded into a LC gradient ionexchange system including a Thermo SeparationsP4000 quaternary gradient pump (ThermoElectronCorporation) coupled with a 0.32� 100mm BioBa-sic SCX column. A flow rate of 3 mL/min was usedfor both SCX and RP columns. A salt gradient wasapplied in steps of 0, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 57, 64, 71, 79, 90, 110, 300, and 700mMammonium acetate in 5% acetonitrile, 0.1% formicacid and the resultant peptides were loaded directlyinto the sample loop of a 0.18� 100mm BioBasicC18 RP LC column of a Proteome X workstation(ThermoElectron). The RP gradient used 0.1%formic acid in acetonitrile and increased theacetonitrile concentration in a linear gradient from5% to 30% in 30min and then 30% to 65% in 9minfollowed by 95% for 5min and 5% for 15min.TheLC peak ion chromatograms of four bovinemonocyte DDF are presented as Supplementarydata online (see Appendix A).

The spectrum collection time was 59min for everySCX step. The LCQ Deca ion trap mass spectro-meter was configured to optimize the duty cyclelength with the quality of data acquired byalternating between a single full MS scan followedby three tandem MS scans on the three most intenseprecursor masses from full scan. The collisionenergy was normalized to 35%. Dynamic massexclusion windows were set at 2min, and all of thespectra were measured with an overall mass/charge(m/z) ration range of 200–2000. Shown in Fig. 1 isone example of a MS chromatogram (Fig. 1A) andMS2 spectrum (Fig. 1B) for MHC class I heavychain peptide, GI# 1864812.

2.4. Protein identification and analysis

Proteins were identified and analyzed as pre-viously described [26]. The non-redundant proteindatabase (DB) downloaded from the NationalCenter for Biotechnology Information (NCBI;3/29/05) by TurboSEQUEST (Bioworks Browser3.1; ThermoElectron) was used to create a bovinesubset (bovine DB; search terms: bos taurus and bos

indicus). Trypsin digestion was applied in silico tobovine DB and mass changes due to cysteinecarbamidomethylation and methionine oxidation

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RT :0.00 - 37.05

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 360

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100 26.00

19.24

12.247.57

8.78

16.66 21.75 24.424.72 14.6331.8729.2623.49

3.47 33.6935.710.96

NL:4.11E8Base PeakF: MSMyco26_15

Rel

ativ

e A

bund

ance

Time (min)

#430-430 RT:10.50-10.50 NL:3.01E5

0 100 200 300 400 500 600 700 800 900 1000 1100 12000

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y5+1

617.3

y9+1

1046.4y4+1

502.2b2+1

276.9y8+1

917.3

y6+1

745.4b3+1

406.0

b7+1

821.2

y3+1

402.0b6+1

705.3

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577.3

y2+1

287.9b10

+1

1147.9

b9+1

1035.1

m/z

Rel

ativ

e A

bund

ance

(A)

(B)

Fig. 1. MS chromatogram (A) and MS2 spectrum (B) for the MHC class I heavy chain peptide sequence ‘‘YLENGKDTLLR’’. The MS

chromatogram was obtained from a salt step gradient of 15mM ammonium acetate in acetonitrile, 0.1% formic acid in the TX fraction.

The scan number and RT of the MS2 spectrum were 430 and 10.5min, respectively. The arrow on the bottom of panel A indicates RT.

S.-R. Lee et al. / Developmental and Comparative Immunology 30 (2006) 1070–1083 1073

ARTICLE IN PRESS

DOC 9

SDS2

Digitonin1

1

4

30

0 0

0

0

Triton X-10033

Fig. 2. Distribution of immune-related proteins identified from

bovine monocyte DDF. The MS2 data from each of the DDF

samples were analyzed using the bovine DB subset and the

resulting proteins were classified with the respect to their

subcellular location.

Table 1

Proteins related to major cellular functions identified in bovine

monocytes by DDF-MudPIT

Biological functions Number of

protein

DDF

Proteins related to immune

functions

53 D/T/TD/S

Sodium ion transport 2 T

Protein transport 1 T

Thyroid hormone

generation; transport

1 T

ATP synthesis coupled

proton transport

14 T/TD/S

Lipid transport 5 T

Oxygen transport 7 T

Electron transport 27 T/TD/S

Protein folding 9 T/TD

Lactose biosynthesis 14 D/T/TD

Protein biosynthesis 12 T

Cytochrome-c oxidase

activity

6 T/TD/S

Nucleosome assembly 5 T/TD/S

Signal transduction 10 D/T/TD

Proteolysis and peptidolysis 2 T/TD

Gene regulation 7 T/TD

Blood coagulation 3 T/S

Protein amino acid

dephosphorylation

4 D/T/TD/S

Regulation of apoptosis 1 T

Muscle contraction 3 T/TD/S

Metabolism 7 T

Unannotated 91 D/T/TD/S

Total 284

Key: DDF, Differential detergent fraction; D, Digitonin; T,

Triton X-100; TD, Tween 40-deoxycholate; S, Sodium dodecyl

sulfate.

S.-R. Lee et al. / Developmental and Comparative Immunology 30 (2006) 1070–10831074

were included. The bovine DB was used to searchtandem MS using peptide (MS precursor ion) masstolerance of 1.5Da and a fragment ion (MS2) masstolerance of 1.0Da. Peptide matches were consid-ered genuine if they were X6 amino acids with a X

correlation values of 1.5, 2.0 and 2.5 (+1, +2, and+3 ions, respectively) and DCn (X0.1) values [28].Each protein was classified with respect to itsbiological process using gene ontology (GO) anno-tation [29]. GO: biological process was determinedfor each protein by searching the UniProt database(http://www.pir.uniprot.org/).

3. Results and discussion

3.1. Subcellular fractionation of bovine CD14+

peripheral blood monocytes

A widely used 2D separation technique inproteomics has been 2D gel electrophoresis. How-ever, this technique is limited by its inability todetect and identify low abundance proteins, pro-teins with extremes in isoelectric point and mole-cular weight and very hydrophobic proteins [30]. Toovercome these obstacles, several comprehensivetechniques have been developed [31], includingMudPIT. The combination of DDF and MudPITallows obtaining a comprehensive cellular proteomeas well as information about a protein’s subcellularlocalization [26]. We identified a total of 284proteins from monocytes using this non-electro-phoretic technique. In comparison, only 250 pro-teins were identified from bovine neutrophils usingone-dimensional (1D) electrophoresis coupled withMudPIT [32].

In our study proteins from four different sub-cellular fractions were obtained using differentdetergent buffers. Different proteins are isolatedfrom the different detergent fractions, and aftereight washes with DDF buffer 1, most of thecytosolic proteins had been extracted (Fig. 2).

In this study tandem mass spectra were analyzedusing a bovine DB and the identified proteins wereclassified according to their GO biological process(Table 1). We found a total of 284 proteins of which91 (32.0%) were not annotated and not related tothe immune function based on the availableliterature. Of the remaining proteins, 53 wererelated to immune function (27.5%), including 13unannotated bovine proteins that have been as-signed to this group based on the available literaturein humans, mice and cattle (Table 2). Proteins

ARTICLE IN PRESSS.-R. Lee et al. / Developmental and Comparative Immunology 30 (2006) 1070–1083 1075

annotated to immune response were examined inmore detail to determine the specific biologicalprocess such as antigen uptake, processing andpresentation (Tables 2 and 3).

Information on the immunologically importantproteins is summarized in Tables 2 and 3. Weidentified eight categories of immune responseproteins: antigen uptake; innate antigen recognition;immune response; cell proliferation; adhesion,integrin-mediated signal pathway; actin cytoskele-ton organization and biogenesis; antigen presenta-tion, immune response; response to unfoldedprotein (Tables 2 and 3). Most of the proteinsinvolved in immune function were found in the TXfraction (62.3%) (Fig. 2).

Overall, 13 bovine proteins related to cell adhe-sion, three TLR proteins (bovine TLR1, 6, 8)involved in innate antigen pattern recognition, threeproteins related to antigen uptake including the MRprecursor, and eight actin-related proteins involvedin active endocytosis in monocytes/macrophages andimmature DC were identified (Table 2). In addition,a total of nine MHC class I- and II-related proteinswere identified in bovine monocytes (Table 3).Finally, cytokines, including active substances andgrowth factor proteins were also found in bovineCD14+ monocytes. Namely, interferon- g (IFN- g),transforming growth factor beta 1 precursor, vascu-lar endothelial growth factor C and heat shockprotein (HSP) have been identified by DDF-MudPIT(Table 2).

3.2. Proteins involved in antigen uptake, processing

and presentation identified by DDF

Different mechanisms of antigen uptake are usedby different APC types and determine their relativeefficiency in priming MHC restricted T cells [27,33].Capture of antigens by surface receptors, such asIgR, FcR or MR allows efficient delivery of antigento the processing compartment via receptor-mediated endocytosis [34]. Recently, we haveidentified MR as the important receptor involvedin endocytosis in bovine monocytes [27]. We havealso demonstrated that antigen uptake throughMR-mediated endocytosis, an important APCfunction, is affected in monocytes in the early stageof Bovine viral diarrhea virus (BVDV) infectionduring the first 24 h, with both BVDV biotypes [27].Endocytosis can occur by several mechanisms, inparticular, clathrin-, non-coated pit-, caveolar-,membrane ruffles-dependent and others [34]. In

our research, we identified three proteins related toantigen uptake: the galactose binding lectin, MyD-1, and MR precursor (Table 2). In addition, weidentified eight proteins related to cytoskeleton suchas actin, beta actin and gamma actin (Table 2).Previous data in humans suggest that lectins, inparticular galectins (galactose binding lectins) andtheir ligands, regulate immune cell homeostasiswhereas some members of this family, such asgalectin-3, behave as amplifiers of the inflammatorycascade [35]. MyD-1, found in bovine monocytes,macrophages, granulocytes, and a subset of afferentlymph veiled cells, plays a role in regulation of theinnate immunity in humans by pathogen productssuch as LPS, purified protein derivative andzymosan [36,37]. MR, a C-type lectin containingcarbohydrate recognition domains with broadspecificity for sugars, mediates phagocytosis ofmannose-coated particles, such as yeast and en-docytosis of mannosylated glycoproteins in macro-phages and immature DC [38–41]. MR can beviewed as a pattern recognition molecule that canprovide APC with some capacity of self non-selfdiscrimination at the level of antigen uptake on thebasis of glycosylation and perhaps hydrophobicity[27,38]. Antigens that fail to bind to cell surfacereceptors can still be taken up by micro- andmacropinocytosis [42]. While micropinocytosis oc-curs constitutively in all cells, macropinocytosis islimited to few cell types, such as macrophages andendothelial cells stimulated by growth factors[43–45]. In this study, we found two growth factorsthat could function in antigen uptake: transforminggrowth factor beta 1 precursor and vascularendothelial growth factor C (Table 2).

One of the most important functions in profes-sional APC is an innate form of antigen recognition[46]. TLR recognize specific molecular patterns thatare present in microbial components, and triggerDC maturation. Mature human DC induce costi-mulatory molecule expression and increase antigenpresenting capacity [46]. Therefore, TLR help todirect adaptive immune responses to antigensderived from microbial pathogens [46]. Stimulationof different TLR induces distinct patterns of geneexpression which both lead to the activation ofinnate immunity and instruct the development ofantigen specific acquired immunity in humans [47].In this study, three bovine TLR were identified:TLR 1, 6 and 8 (Table 2). In addition, cathelicidin[48] and fetuin [49], which are involved in innateantigen recognition, were also identified (Table 2).

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Table 2

Proteins related to professional antigen presentation identified in bovine monocytes by DDF-MudPIT

GI# Protein DDF # Peptides

identified

Function

28189272* Similar to galactose-binding lectin T 1 Antigen uptake

2842388* MyD-1 antigen T/TD 4 Antigen uptake

163336 Mannose-6-phosphate receptor

precursor

T 3 Antigen uptake

48995741* Toll-like receptor 1 T 1 Innate antigen recognition

44804291* Toll-like receptor 6 T 1 Innate antigen recognition

61875304* Similar to Toll-like receptor 8, partial T/TD/S 4 Innate antigen recognition

463 Cathelicidin T 2 Innate antigen recognition

344 Fetuin T 1 Innate antigen recognition

508 Clathrin light chain A T 1 Innate antigen recognition

514 Clathrin light chain B TD 1 Innate antigen recognition

28195120 Gamma interferon T 1 Immune response

61864535 Predicted: Similar to CD80 antigen T 1 Immune response

163748 Transforming growth factor-beta-1

precursor

S 1 Cell proliferation

4850314 Vascular endothelial growth factor C TD 1 Cell proliferation

520521 Beta-1 integrin subunit T 3 Adhesion, integrin-mediated signaling pathway

53680685 Integrin beta-2 T 3 Adhesion, integrin-mediated signaling pathway

162819 Antigen CD18 T 18 Adhesion, integrin-mediated signaling pathway

4996449 CD34 T 1 Adhesion, integrin-mediated signaling pathway

187 Bovine CD44 T 2 Adhesion, integrin-mediated signaling pathway

33086939* Brain microvascular membrane

cofactor protein CD46

T 2 Adhesion, integrin-mediated signaling pathway

37811992* CD46 splice variant T 1 Adhesion, integrin-mediated signaling pathway

1044811 Metalloproteinase D 1 Adhesion, integrin-mediated signaling pathway

11320818 VSGP/F-spondin TD 1 Adhesion, integrin-mediated signaling pathway

20145484 SCO-spondin TD 1 Adhesion, integrin-mediated signaling pathway

2230771 Von Willebrand factor TD 1 Adhesion, integrin-mediated signaling pathway

20336613 Plakoglobin TD 1 Adhesion, integrin-mediated signaling pathway

6707084* Low-affinity IgE receptor (CD23) TD 1 Adhesion, integrin-mediated signaling pathway

162606 Actin T/TD/S 11 Actin cytoskeleton organization and biogenesis

2239063 Actin-binding protein CP3 TD 1 Actin cytoskeleton organization and biogenesis

22655316* Beta-actin T/TD 14 Actin cytoskeleton organization and biogenesis

28189611* Similar to beta actin S 1 Actin cytoskeleton organization and biogenesis

409694 Alpha skeletal actin precursor T/S 9 Actin cytoskeleton organization and biogenesis

59858375* Actin, gamma 2 T/TD/S 10 Actin cytoskeleton organization and biogenesis

2852385 14-3-3 protein gamma TD 1 Actin cytoskeleton organization and biogenesis

1838956 Capping protein, beta 3 isoform T/TD/S 10 Actin cytoskeleton organization and biogenesis

116 Beta 2 microglobulin precursor T/TD 6 Antigen presentation, immune response

28189424* Similar to beta 2-microglobulin T 3 Antigen presentation, immune response

32306980* Beta-2-microglobulin T 3 Antigen presentation, immune response

163161 70 kDa heat shock protein T 1 Response to unfolded protein

414975 70 kDa heat shock protein-2 T 1 Response to unfolded protein

28189915 Similar to 70 kDa heat shock cognate

protein

T 1 Response to unfolded protein

32364477 Heat-shock 70-kilodalton protein 1A T 1 Response to unfolded protein

32364478 Heat-shock 70-kilodalton protein 1B T 1 Response to unfolded protein

34392343 90-kDa heat shock protein alpha T 1 Response to unfolded protein

Key: GI#, Genbank accession number; DDF, Differential detergent fractionation; D, Digitonin; T, Triton X-100; TD, Tween 40-

deoxycholate; S, Sodium dodecyl sulfate; *, Proteins not annotated with GO biological process.

S.-R. Lee et al. / Developmental and Comparative Immunology 30 (2006) 1070–10831076

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Table

3

Proteinsrelatedto

MHC

class

IandII

alleles

identified

inbovinemonocytesbyDDF-M

udPIT

GI#

Protein

DDF

#Peptides

identified

Function

Peptidesequence

2864742

MHC

class

Iheavychain

T2

Antigen

Presentation,im

muneresponse

–.Y

FYTAVSRPGLGEPR.–

–.LLSGFTQFGYDGR.–

2864812

MHC

class

Iheavychain

T4

Antigen

Presentation,im

muneresponse

–.Y

LENGKDTLLR.–

–.Y

FYTAVSRPGLGEPR.–

–.Y

TC*HVQHEGLQEPLIL

R.–

–.W

AALVVPSGEEQR.–

3688211

MHC

class

Iantigen

T2

Antigen

Presentation,im

muneresponse

–.W

AALVVPSGEEQR.–

–.LLSGFTQFGYDGR.–

42563807

MHC

class

IIantigen

T1

Antigen

Presentation,im

muneresponse

–.H

NYQVEAPFTWQR.–

163359

MHC

lymphocyte

antigen

(DQA)

T4

Antigen

Presentation,im

muneresponse

–.VEHWGLDKPLLK.–

–.VEHWGLDKPLLK.–

–.ISYLTFLPSDDDVYDC*K.–

–.ETVWNLPLFSK.–

493485

MHC

class

IIDQ-betachain

T1

Antigen

Presentation,im

muneresponse

–.H

NYQLDAPFTWQR.–

1246367

MHC

class

IIDM

beta-chain

T1

Antigen

Presentation,im

muneresponse

–.TRPPTVQVAK.–

1256156

MHC

class

IIDM

alpha-chain

T1

Antigen

Presentation,im

muneresponse

–.LPEFADWAHQHGDTSDIM

FDK.–

163371

MHC

BoLA-D

R-alphachain

(AA

at265)

T1

Antigen

Presentation,im

muneresponse

–.FSPPVISVTWLR.–

Key:GI#,Genbankaccessionnumber;DDF,Differentialdetergentfractionation;D,Digitonin;T,TritonX-100;TD,Tween40-deoxycholate;S,Sodium

dodecylsulfate.

S.-R. Lee et al. / Developmental and Comparative Immunology 30 (2006) 1070–1083 1077

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Gi# 2864812 ---------------LTETRAGSHSLRYFYTAVSRPGLGEPRFISVGYVD 35 Gi# 3688211 MGPRALFMLLLGALVLIETRAGSHFLRYFHTAVSRPGLREPLFITVGYVD 50

* ******* ****:******** ** **:*****

DTQFVRFDSDAPDPRMEPRAPWIEKEGPEYWDRETRISKENTLVYRGSLN 85 DTQFVRFDSDARDPRKEPRQPWMEKEGPEYWDRETQISKENALWYREALN 100 *********** *** *** **:************:*****:* ** :**

NLRGYYNQSEAGSHTLQLMYGCDVGPDGRLLRGYRQDAYDGRDYLALNED 135 NLRGYYNQSEAGSHTYQRMYGCDVGPDGRLLSGFTQFGYDGRDYIALNED 150 *************** * ************* *: * .******:*****

LRSWTAADTAAQITKRKWEAEGYAESLRNYLEGRCVEGLRRYLENGKDTL 185 LRSWTAADTAAQITKRKWEAAGEAERFRNYVEGRCVEGLRRYLENGKDAL 200 ******************** * ** :***:*****************:*

LRADPPKAHVTHHSISDREVTLRCWALGFYPEEISLTWQHNGEDQTQDME 235 LRADPPKAHVTHHPISEHEVTLRCWALGFYPEEISLTWQRNGEDQTQDME 250 *************.**::*********************:**********

LVETRPSGDGTFQKWAALVVPSGEEQRYTCHVQHEGLQEPLILRWEPPQT 285 LVETRPSGDGNFQKWAALVVPSGEEQRYTCRVQHEGLQEPLTLRWEPPQT 300 **********.*******************:********** ********

SFLIMGIIVGLVLLVVAVVAGAVIWRKKRSGEKGRIYTQAASSDSTQGSD 335 SFLTMGIIVGLVLLVVAVVAGAVIWMKKHSGEKRRTYTQAASNDSAQGSD 350 *** ********************* **:**** * ******.**:****

MSLTVPKV 343 VSLTVPKV 358 :*******

Fig. 3. The identification of different MHC class I protein alleles with peptide sequences detected in MHC class I a 1 domain. Two MHC

class I heavy chain alleles (GI# 2864812 and GI# 3688211) were aligned using ClustalW (http://www.ebi.ac.uk/clustalw/) to reveal the

allelic differences between the proteins. MHC class I peptide sequences identified in bovine monocytes by DDF-MudPIT are marked with

gray background (GI# 2864812—dark gray, GI# 3688211—light gray). *, identical residues; conserved substitutions; semi-conserved

substitutions; gaps were introduced to maximize homology.

S.-R. Lee et al. / Developmental and Comparative Immunology 30 (2006) 1070–10831078

Several reports that characterize different TLR areavailable. In particular, it is known that TLR 1recognizes triacyl lipopeptides of bacteria andmycobacteria, and soluble factors of Neisseria

meningitis [47]. Another report suggests that diacyllipopeptides, lipoteichoic acid and zymosan, whichare originally released from Mycoplasma, grampositive bacteria and fungi, respectively, are recog-nized by TLR 6 [47]. Finally, TLR 8 recognizesimidazoquinoline, synthetic compounds, and single-stranded RNA of viruses [47]. At present, 11 TLRhave been reported in mammals [47]. Complete andpartial sequences of 10 bovine TLR are available inNCBI database. TLR 2 and TLR 6 mRNAtranscripts have been detected in bovine macro-phages and monocyte-derived DC [4]. The bovinegene, encoding the key pathogen recognitionreceptor, TLR 9 was characterized and mapped tobos taurus chromosome A22 and used to establishreal-time PCR quantification assays to measure themRNA abundances of TLR 9 in bovine mammarygland [50]. In this study, we identified for the first

time TLR 1, 6 and 8 proteins in bovine monocytes(Table 2), thus expanding the knowledge aboutinnate immune responses in cattle.

MHC genes encode class I and II molecules.Recent data indicate that MHC class I moleculesinteract with a range of natural killer (NK) cellreceptors, in addition to CD8+T cells [51]. Wefound nine proteins, including MHC class I, MHCclass II DQ alpha and beta, DM and DR alpha,related to antigen presentation (Table 3). Each ofthe MHC genes exists in an extremely large numberof allelic variations and the exact number of allelesis not known [52]. For example, GI] 2864812 andGI] 3688211 are both MHC class I heavy chainproteins, but they arose from two different MHCclass I alleles (Fig. 3). Both proteins shared the samepeptide sequence WAALVVPSGEEQR (Fig. 3).However, both MHC class I proteins had uniquepeptide sequences to confirm that peptides identifiedby DDF-MudPIT experiments arose from twodifferent MHC class I alleles (Fig. 3). The mostessential features of MHC gene regulation is that

ARTICLE IN PRESSS.-R. Lee et al. / Developmental and Comparative Immunology 30 (2006) 1070–1083 1079

the expression of these molecules is controlled bythe level of their transcription, and is responsive to anumber of cytokines [52]. We found the cytokineIFN -g protein, which enhances the expression ofboth MHC class I and II molecules [52].

A previous study demonstrated that humanmonocytes and DC induce CD40 independent Igclass switching through the B cell stimulator protein(BLyS) and a proliferation-inducing ligand(APRIL) in humans [8]. Furthermore, monocytesand DC upregulate BLyS and APRIL uponexposure to IFN-a, IFN-g or CD40 ligand [8]. Inthis study, we were not able to identify themolecules involved in direct monocyte-B cell inter-actions in cattle. Although recent data suggest thatbovine monocytes and monocyte-derived DC di-rectly regulate Ig secretion in peripheral blood Bcells [5,6,13], the possible bovine counterparts forAPRIL and BLyS have yet to be identified.

CD80 but not CD86 proteins have been detectedin the TX fraction of bovine monocytes (Table 2).Both CD80 and CD86 proteins are crucial forprofessional APC to provide costimulatory signalsto naıve T cells [53]. At present there is no clearmodel for understanding which ligands promote orinhibit specific immune responses [54]. Surfaceexpression of bovine CD80 and CD86moleculeshave been characterized in cattle by flow cytometrywith marker specific antibodies in monocytes andDC [5,13,55]. Low abundant molecules such asCD86 are difficult to detect at the protein levelbecause the expression of the genes encodingcostimulatory molecules is tightly regulated: theyare often expressed at low levels and for the shortperiods of time [55]. Overall, DDF-MudPIT wasvery useful to detect the low abundant cytosolicproteins in our study, suggesting high sensitivity ofthis approach.

Some of the anti-inflammatory proteins known tobe important for signaling in monocytes have notbeen identified in our study. Cytokines such asTNF-a, IL-1, IL-6, and IFN-a and b are difficult todetect on the protein level because they are oftenexpressed at low amounts and their production isefficiently triggered with bacterial products [9]. Inour study, CD14+ monocytes were obtained fromhealthy animals that most likely had not beenchallenged with bacterial infection at the time ofblood drawing. Therefore, we suggest that thesesoluble signaling proteins were downregulatedbelow the detectable level under our experimentalconditions.

3.3. Proteins related to cell adhesion

DC and their myeloid progenitors, monocytesand macrophages, serve as a bridge between theinnate and adaptive immune systems and are criticalin host protection against infectious agents. Theseprofessional APC can contribute to local sites ofinflammation where, after being activated bypathogenic molecules, they uptake antigens and/ormigrate to secondary lymphoid organs [52]. Adhe-sion molecules play an important role in themigration of monocytes from blood to the tissuesand from tissues to the lymph nodes [56]. Thismigration is mediated by adhesive interactionsbetween leukocytes and vascular or lymphaticendothelium [56]. We have identified 12 proteinsrelated to adhesion: b integrins, CD18, 34, 44 and46, metalloproteinase, vascular smooth muscle cellgrowth-promoting (VSGP/F)-spondin, subcommis-sural organ (SCO)-spondin, von Willebrand factor(vWF), plakoglobin, and the low-affinity IgEreceptor (CD23) (Table 2).

Integrins were named for their ability to link theextracellular and intracellular skeletons [57]. In ourstudy two integrins, b1 and b2 have been identifiedin the detergent fractions (Table 2). In bovinesystem, b1 integrin was characterized in bovinetrophoblasts by mRNA PCR and immunohisto-chemistry with anti-b1 integrin specific mAbs [58].In addition, specific mAbs were used to detectbovine b2 integrin expressed in neutrophils [59].Recent work has revealed that the integrin-actincytoskeleton connection is highly dynamic and is asubject to many regulatory processes [60]. Forexample, loss of b1-integrin on keratinocytes leadsto impaired as well as non-directed migrationresulting in severely delayed re-epithelializtion [60].Importantly, the CD18 antigens and b2-integrinscomprise a family of three closely related cell surfaceglycoproteins with CD11 a- and CD18 b-chains [61].Four additional CD molecules related to celladhesion were identified in this study (Table 2).One of them, CD34, a surface expressed glyco-protein [62], was detected in membrane fraction(Table 2). Bovine CD34 was characterized in bonemarrow cells based on the CD34 mRNA level [63].In this study, CD34 protein was detected andcharacterized in bovine monocytes for the first time.In humans CD34 plays a role in adhesion to thestromal microenvironment and may be involved incell transformation [62]. Other members of theadhesion protein family identified in this study were

ARTICLE IN PRESSS.-R. Lee et al. / Developmental and Comparative Immunology 30 (2006) 1070–10831080

CD44, CD46, and CD23 (Table 2). Previously,CD44 cDNA was detected in bovine [64]. CD44 is asurface receptor that has been implicated in celladhesion to a variety of matrix components andmay be associated with cytoskeletal proteins andintracellular signal pathways [65]. CD46 is acomplement regulatory protein that also functionsas the cellular receptor in humans [66]. Recentstudies indicate that CD46 may posses signalingfunctions that modulate cellular response [66]. Inaddition, cross-linking of CD46 with antibody orwith the complement activation product C3binhibited IL-12, which is critical for the generationof cell mediated and humoral immunity, by provid-ing a regulatory link between the complementsystem and cellular immune responses [66]. Impor-tantly, CD46 may act as a cellular receptor for theBVDV strains, in particular, the cytopathic strainNADL in cattle [67]. Finally, CD23 (FceRII), thelow-affinity receptor for IgE is expressed on thesurface of B cells prior to the isotype switching aswell as on monocytes, eosinophils and platelets [67].In cattle, CD23 plays a role in IgE associatedimmune responses, including allergies [68]. How-ever, it may also be important in endocytosis andadhesion because of its relation to the lectin family,which is involved in endocytosis and antigenpresentation [69]. Interestingly, metalloproteinasewas the one adhesion protein found in digitoninfraction (Table 2). Metalloproteinase is critical inpathological processes that depend on dysregulatedcell-cell and cell-matrix interactions in monocytes[69]. Metalloproteinase was also assessed by-Northern analysis and Western blotting in bovinebrain [70]. Our study confirmed that bovine mono-cytes also expressed the metalloproteinase protein.Furthermore, two spondins in bovine system,involved in protein-protein interactions, SCO-spon-din [71] and VSGP/F-spondin [72], were identifiedin our study (Table 2). VSGP/F-spondin is a majorfactor for vascular SMC proliferation promotingfactor, and vWF is a multimeric adhesive glycopro-tein with cystein-rich domains [72,73]. Finally,we found plakoglobin (g-catenin), a close homologof b-catenin, in the membrane protein fraction(Table 2). Plakoglobin shares some of the samefunctions of b-catenin [74]. Plakophilin, the memberof plakoglobin family, was examined in varioustissues and cell cultures including bovine withspecific antibodies and cDNA probes [75].

In conclusion, the data presented in this manu-script are important because the identification of the

proteins present in professional APC is instrumentalto understanding the mechanisms involved in thefunction of monocytes/macrophages and monocyte-derived DC as controllers and stimulators of T andB cell specific immune responses. In particular, thetransformation of monocytes into immature DCand their further maturation to become the mostpowerful APC is a very critical bridge betweeninnate and adaptive immune responses. In futurestudies, comparative protein profiling could be usedto elucidate the mechanisms involved in pathogeninterference of DC maturation to avoid effectiverecognition and elimination by innate and adaptiveimmune responses which are controlled and regu-lated by the most powerful professional APC, DC.Finally, the unannotated proteins will be annotatedin the future with database and literature searches toidentify homologs that are publicly released throughAgBase [76].

Acknowledments

This work was supported by the MississippiAgricultural and Forestry Experiment Station(MAFES) under project ] MIRS-13788 and theCollege of Veterinary Medicine, Mississippi StateUniversity. This paper is MAFES ] J-10791. Theauthors wish to acknowledge Dr. T. Pechan andMSU LSBI facility for the protein analysis ofbovine monocyte DDF. The authors also wish tothank Drs. C. Panuska, M. Lawrence, and L.Hanson for the helpful critique and suggestion onthe manuscript.

Appendix A. Supplementary data

Supplementary data associated with this articlecan be found in the online version at doi:10.1016/j.dci.2006.02.002.

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