Proteomics and Leukocytes: An Approach to Understanding Potential Molecular Mechanisms of Inflammatory Responses Xiangdong Wang,*,† Hong Zhao,‡ and Roland Andersson‡ College of Medicine, Zhe-Jiang University, Hang-Zhou, Peoples Republic of China and Department of Surgery, Lund University Hospital, Lund, Sweden Received February 5, 2004
Leukocytes play an important role in the progression of disease and leukocyte-derived proteins are associated with the pathogenesis of the disease. Leukocyte activation causes production of inflammatory mediators, over-expression of cell surface adhesion molecules, and an increase in migration and infiltration, phagocytosis, and degranulation, as well as receptor phosphorylation and signal transduction. An increasing number of studies on the application of leukocyte proteomics have appeared in mapping protein profiles of inflammatory cells, contributing to the understanding of potential mechanisms involved in leukocyte function. Together with improvements in proteomic technology in leukocyte research, leukocyte proteomic analysis becomes more simple, rapid, flexible, sensitive, and specific. This enables proteomic investigation of activated or non-activated leukocytes to be highly focused on defined suborgans or specific signaling pathways. Research in leukocyte proteomics is progressing from fingerprinting to functioning, human cell lines to primary leukocytes, non-activated cells to inflammatory mediator-stimulated cells, in vitro culture to in vivo challenge, and animal models to human disease. A number of newly identified proteins from leukocyte proteomics may offer new mechanism-orientated targets for drug discovery and development. Keywords: leukocytes • proteomics • inflammation • organ injury • proteans profiles • human
Introduction Inflammation was traditionally considered as a local process with a number of local signs and symptoms, including ruber (peripheral vasodilatation with increased blood flow), color (increased skin temperature due to peripheral vasodilatation), dolor (pain), tumor (swelling due to increased capillary permeability to large proteins), and functio laesa (loss of function).1 The inflammatory response could be considered to possess a cyclic pattern of pathophysiological cascades where the local signal passes into the circulation and is then redirected back to the initial site of inflammation. Inflammatory stimuli, bacterial or nonbacterial in nature, can induce generalized inflammatory responses, leading to a systemic inflammatory response and potentially multiple organ dysfunction syndrome.2 Sepsis and a noninfectious systemic inflammatory response syndrome are paradoxically associated with an exacerbated production of inflammatory mediators. Such a systemic inflammation constitutes the leading cause of mortality in intensive care units,3 although the mechanisms responsible for death remain unclear. Leukocyte activation and the production of inflammatory mediators have been suggested to be one of the critical factors that initiate and exacerbate * To whom correspondence should be addressed. Xiangdong Wang MD, PhD, B. Ja¨rnsidas G. 12S-224 77 Lund, Sweden. Tel: 00 46 46 125 931. Fax: 00 46 46 336 624. E-mail:
[email protected]. † College of Medicine, Zhe-Jiang University. ‡ Department of Surgery, Lund University Hospital. 10.1021/pr0499601 CCC: $27.50
2004 American Chemical Society
inflammatory responses (Figure 1), leading to cell and tissue injury and dysfunction.4 Leukocyte activation (hyper-reaction) evokes production and release of inflammatory mediators, overexpression of cell surface adhesion molecules, and an increase in migration and infiltration, phagocytosis, and degranulation, as well as receptor phosphorylation and signal transduction.5-12 The hyperresponsiveness of leukocytes has been noted as a marker of immune reaction and may explain an enhanced sensitivity of patients with inflammation. Leucocytes have been suggested to play an important role in the progression of disease (Figure 2), since leukocyte-derived proteins are associated with the pathogenesis of the disease. For example, the proteomic profile of monocytederived macrophages from HIV-infected patients with cognitive impairment demonstrated that alterations in monocyte function paralleled the development of HIV-1-associated dementia.13 Cytokine productions and the nuclear factor-κ B status within leukocytes have been suggested to represent a useful biomarker of hypo- or hyper-reactivity.4 Furthermore, proteomic approaches have been used to investigate protein profiles undergoing nitration in response to an inflammatory stimuli in disease models.14 Of more than 40 nitrotyrosine-immunopositive proteins, about 30 proteins have been recently discovered, including proteins related to oxidative stress, apoptosis, AT production, and other metabolic functions.14 Although there are still practical issues to be solved, the application of functional proteomics provides a tool to Journal of Proteome Research 2004, 3, 921-929
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Figure 1. During antigen-induced tissue inflammation, a number of cells are activated to produce inflammatory mediators, e.g., tumor necrosis factor-R (TNFR), interleukin 1β (IL-1β), or interferon-γ (IFNγ). These mediators can bind with their specific receptors initiating intracellular signals, activating nuclear factors resulting in overexpression of interleukins (ILs), chemokines (CKs), and/or adhesion molecules (AMs).
monocytes/macrophages, endothelial cells, eosinophils, and lymphocytes. Neutrophils. The neutrophil is widely accepted as playing a critical role in eliminating endogenous and exogenous stimuli by phagocytosis, respiratory burst, and release of cytotoxic mediators and proteases, as the first line of host defense. Under inflammatory conditions, rolling neutrophils attach onto endothelial cells by reverse binding to transmembrane cell surface glycoproteins.16 Receptor-mediated firm adherence on endothelial cells occurs in response to inflammatory cytokines or an event propagated from activated selectin signaling.17 The activated neutrophil migrates through the endothelial lining, causing cell tissue damage, tissue remodeling and potentially organ dysfunction.18
Figure 2. Activation of leukocytes or other cells can increase production and secretion of proteases which induces the occurrence of inflammatory cascades, leading to tissue/cell damage and dysfunction.
investigate signal transduction networks in inflammation and sepsis, potentially enabling the discovery of disease-associated targets and more effective therapies.15 The current applications of proteomics and the potential molecular mechanisms of leukocyte activation have been reviewed in view of the footprinting of cytoplastic, structural and functional protein profiles in inflammatory cells, including neutrophils, dendritic cells, 922
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Lipopolysaccharides (LPS) are a constituent of the outer membrane of Gram-negative bacterial cells, consisting of a polysaccharide part attached to lipid A and toxin,19 causing the development of the acute respiratory distress syndrome. LPS is also a bioactive element in the cigarette responsible for the formation of chronic inflammation,20 e.g., chronic obstructive pulmonary disease. LPS can activate human neutrophils by binding to a plasma membrane receptor complex involving Toll-like receptors. This is followed by actin assembly, adhesion, activation of nuclear factor-κ B, increased secretary responses and production of inflammatory mediators.21-23 Studies on proteomic changes after exposure of human neutrophils to LPS have identified some proteins involved in the innate immune response.24,25 Fessler et al. have performed a solid in vitro study to compare genomic and proteomic profiles of activated human neutrophils by a 4-hour challenge with LPS.24 Studies on this
Proteomics and Leukocytes
cell type identified that LPS resulted in an up-regulation of inflammatory regulators (annexin III), signaling molecules (Rab-GDP dissociation inhibitor β), several actin fragments, and the proteasome β chain and the down-regulation of various signaling proteins (Rho GTRase activating protein 1) at pHrange 3.0-10.0.24 These results indicate that a 4 hour-challenge with LPS on human neutrophils may induce cytoskeletal remodeling by activation of an actin-cleaving enzyme or calpain.26 Such cytoskeletal reorganization or remodeling may explain LPS-induced priming of neutrophil granule release and migration. The corresponding rate of up-regulated proteins and mRNA transcripts was about 15% (2/13, proteasome β chain and leukocyte elastase inhibitor), and the down-regulated rate was about 60% (3/5, adenylyl cyclase-associated protein1, Rho-GAP 1, and Ficolin 1). In addition to those inflammatory mediators recognized by specific surface receptors, Ionomycin, as the fastest and most potent inducer of neutrophil secretion, can activate neutrophils by binding with high extracellular calcium as a calcium ionophore, resulting in maximum exocytosis of all granule subsets.27 By using such molecules involved in signal transduction to stimulate freshly purified human neutrophils, 19 proteins were identifed, out of which 13 were known to be stored in neutrophil granules, and others were considered as new proteins secreted by activated neutrophils, namely calgranulins A, B, and C, annexin XI, HC gp-39 and chitotriosidase.28 These proteins may explain the number of neutrophil-driven activities, such as neutrophil activation, migration, and accumulation in inflammatory sites, adhesion, antimicrobial capacity, apoptosis, growth inhibition, anticoagulation, tissue remodeling, and antifungal infection. It might be difficult to describe the reproducibility of findings from a particular study, due to lack of information if the proteomic analysis was repeated or a statistical analysis was performed. It would be of benefit if the activating status of these neutrophils was also monitored, e.g., production of cytokines and superoxides, protease activity, and cell migration. One of the potential mechanisms responsible for these proinflammatory stimuli in the activation of neutrophils may be p38 mitogen-activated protein kinase (MAPK). A number of studies have demonstrated that p38 MAPK signal transduction is a central pathway in regulating neutrophil function including chemotaxis, chemokine expression, respiratory burst activity, exocytosis, and priming, thereby contributing to the severity of inflammatory diseases.29-32 A genomic and proteomic analysis of human neutrophils pretreated with a p38 MAPK inhibitor and challenged by LPS showed that the p38 MAPK inhibitor could attenuate approximately 20% of LPS-induced genes and proteins, but had no effects on corresponding changes of upor down-regulated protein and transcript levels.24 This indicates that p38 may have a selective effect on LPS-induced inflammation. MAPK-activated protein kinase2 (MAPKAPK2), a serinetheronine kinase, in neutrophils is thought to be involved in the neutrophil respiratory burst activity, exocytosis, chemotaxis, and migration.33,34 The p38 MAPK pathway regulates multiple neutrophil functions through activation of MAPKAPK2. Singh et al. have applied a proteomic approach to identify substrates of MAPKAPK2 in human neutrophil lysates after a combination of in vitro phosphorylation with exogenous active recombinant MAPKAPK2.35 Studies on this cell type identified about 30 proteins phosphorylated by MAPKAPK2, of which 6 proteins were considered as candidate MAPKAPK2 substrates, including leukocyte-specific protein-1, myosin regulatory light chain and 16-kDa subunit of the Arp23 complex.35 This particular study
reviews explored a new approach to identify phosphorylated proteins and to investigate the functional consequences of p16-Arc phosphorylation by MAPKKAPK2 and the role of differences in phosphorylation between p16-A and p16-B isoforms. It is possible that MAPKAPK2 is involved in the actin cytoskeletondependent regulation of neutrophil infiltration and secretion by phosphorylating the actin-binding proteins,36 heat shock protein 2737 or the 16-kDa subunit of the Arp23 complex. Neutrophil skeletons, cytoplasmic, membrane, and/or cortical cytoskeletons, play an important role in the maintenance of chemotactic signaling, cell polarity, movement, adhesion, and phagocytosis.38,39 Intracellular signal transductions occur at cytoskeleton-stabilized membrane domains and lipid rafts,40 which contain resident integral membrane proteins. To investigate the proteomic profile of the endogenous raft lipids and proteins in neutrophils, a subset of plasma membrane skeleton proteins was isolated with cholesterol-rich, detergent-resistant membrane fragments from bovine neutrophils.41 Although the difference between bovine and human neutrophils remains unclear, studies on this cell type have identified about 19 detergent-resistant membrane proteins, including fodrin, myosins, R-actinins, vimentin, the F-actin-binding protein, lipid raft-associated integral membrane proteins, and intracellulardually acylated signal proteins. The proteomic characteristics of surface-exposed membrane proteins on normal rat neutrophils have been mapped and about 50 spots (belonging to the most visible spots when stained with colloidal Coomassie) were identified.42 To understand the molecular mechanisms of neutrophil differentiation a mouse promyelocytic cell line derived from murine bone marrow cells, arrested developmentally by a dominant-negative retinoic acid receptor, was morphologically differentiated into mature neutrophils during incubation with retinoic acid.43,44 In differentiated murine neutrophils, more than 500 protein spots were detected and 50 proteins were identified on the basis of the range of molecular weights, isoelectric points and density, of which 28 proteins were sequenced.43 During murine neutrophil differentiation, protein expression patterns exhibited 18% cytoskeletal proteins, 15% metabolism-related molecules, 10% signaling pathway-related proteins, 7% possible transcription factors, 4% cytokines and others (e.g., kinases and cromatin remodeling factors).44 The importance of these studies on animal neutrophil proteomics and the variation of proteomic profiles between animal and human neutrophils and between normal and pathological conditions should be further clarified, to provide more powerful tools for the further understanding of the pathophysiology of inflammatory diseases in humans. Dendritic Cells. Dendritic cells, present in most tissues in a relatively immature state, are highly specialized as antigenpresenting cells with an essential role in the initiation and control of the cytotoxic T cell response. During the inflammatory reaction, dendrite cells rapidly take up foreign antigens and undergo maturation into potent antigen-presenting cells. These migrate to lymphoid organs and initiate an immune response. Phenotypic and functional characteristics of dendritic cells are highly dependent upon the stage of their maturation.45,46 Additional proteomic and genomic profiles were analyzed during dendritic cell differentiation and maturation derived from human circulating CD14+ monocytes treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 posttreated with tumor necrosis factor-R.47,48 Of the 900 matching protein spots quantified from cultured cells, these studied on this cell type identified 37 regulated Journal of Proteome Research • Vol. 3, No. 5, 2004 923
reviews proteins (25 up- and 12 down-regulated) during the differentiation of CD14+ monocytes into immature dendritic cells. The additional alterations were unchanged during the differentiation of immature into mature cells. During differentiation into immature dendritic cells, the expression of the fatty-acid binding protein (FABP) family (FABP4, FABP5, and acyl-CoAbinding protein), heat shock proteins (hsp73 and hsp27), vimentin, macrophage capping protein, guanylate kinase, and ferritin light chain protein were substantially increased, whereas the cognate chaperone protein (carlreticulin) and the myeloidrelated proteins (MRP8 and 14) were down regulated. Although statistical analysis was not indicated in these studies, such outstanding experimental designs and clear investigations definitely contribute to the further understanding of novel genes and proteins in dendritic cell function, differentiation and/or maturation. It is possible that increased hsps may bind to the surface of monocytes and dendritic cells to participate in antigen processing and presentation, activate T lymphocytes and promote resistance of monocytes from apoptosis.49-51 Inflammatory mediators have been suggested to be critical in the differentiation and maturation of dendritic cells.52 Although the expression of some genes encoding pro-inflammatory cytokines or chemokines was down-regulated during the differentiation of dendritic cells, these inflammatory proteins displayed an unchanged proteomic profile.47 Sensitivity of human dendritic cells to isolation or stimulation makes it difficult to investigate proteomic profiles of human primary dendritic cells, which is even highly expected in future studies. Monocytes/Macrophages. Reactive oxygen species such as superoxide anions and hydrogen peroxide are generated at sites of inflammation and injury. At low levels, these oxygen free radicals can function as signaling molecules in regulation of fundamental cell activities such as cell growth and adaptation responses,53 whereas at higher concentrations they can result in cellular injury and death.54 Reactive oxygen species have the cytotoxic effects by initiating lipid peroxidation, inhibiting mitochondrial respiratory chain enzymes and membrane Na+/ K+ ATP-ase activity, inactivating gyceraldehyde-3-phosphate dehydrogenase and membrane sodium channels, modifying other oxidative proteins, as well as triggering DNA strand breakage and severe energy depletion of the cells. Oxidant stress also increases vascular endothelial permeability and promotes leukocyte adhesion.55 Such oxidant stress has been suggested to influence the proteomic profile of monocytes.56 In this particular study, U937 cells representative of monocytes were exposed to 50 µM H2O2 for 24 h and then analyzed by two-dimensional polyacrylamide gel electrophoresis. The study on this cell type identified an oxidant stress-induced increase in the staining intensity of 34 spots, of which 28 proteins were identified and involved in energy metabolism (8 spots), translation and RNA processing (6 spots), chaperones or mediations of protein folding (5 spots), cellular signaling (3 spots), redox regulations (2 spots), mitochondrial channel (1 spot), actinbundling (1 spot) and others (2 spots).56 However, no conclusions could be drawn on down-regulated proteomic alterations. There is still a need to clarify the difference between oxidant stress-induced changes in the proteomic profile and the pattern of monocyte differentiation processes, since U937 cells can be induced to differentiate into phagocytic macrophages under stimulation.57 Monocyte differentiation to macrophages is an important process of local inflammation after that circulating monocytes undergoes chemotaxis and migrates to the local tissues. When 924
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U937 cells were stimulated and differentiated into macrophagelike cells by the incubation with phorbol 12-myristate 13acetate (PMA) for 24 h, these cells had limited proteolytic cleavage and maturation of a discrete number of lysosomal hydrolases.57,58 Journet et al. demonstrated that the soluble lysosomal proteomic profile correlated to the differentiation process of U937 cells.58 Studies on this cell type identified the following proteins: acid ceramidases, actin R-L-iduronidase, R-mannosidases, β-galactosidase, β-glucuronidase, β-hexosaminidases, cathepsin A, D, S, and Z, cellular repressor of E1A-stimulated genes, cystain F, cytokeratin 9, dipeptidyl peptidase II, DNase II and lysyl hydroxylase 1.58 Maturation of secreted hydrolases may be associated with cell invasion, tissue destruction or the early secretory process. Proteomic analysis of macrophage differentiation was performed in the murine hemopoietic FD/WT cells which with Y706 mutation can be differentiated into macrophages by induction of GM-CSF and FD/807 cells with a defect in differentiation.59 Results from proteomic analysis demonstrated that these two cells had common and distinct profiles of tyrosine phosphorylated proteins by the GM-CSF,80 of which three comigrated with p46/ 52Shc. Transfection with a nontyrosine-phosphorylatable form of p46/52Shc prevented GM-CSF -mediated macrophage differentiation. Activity of extracellular signal-regulated kinase as a key-regulating factor correlated with the degree of differentiation induced by GM-CSF in both cell lines. By using the differentiated mouse macrophage cell line BAC1.2F5 for studying CSF-induced signal transduction, investigations on this cell type identified tyrosine-phosphorylated proteins and nonphosphotyrosine proteins in the anti-phosphotyrosine reactive fraction from CSF-1-stimulated macrophages.60 In addition, the number of cytoskeletal and contractile proteins were identified in CSF-1-stimulated macrophages, indicating that cellular movement and reshape as well as cytoskeleton reorganization and remodeling are important in the differentiation process of monocytes to macrophages. Proteomic analysis of early events in CSF-1 signal transduction demonstrated that the first wave of CSF-1-induced CSF-1R tyrosine phosphorylation was the active signaling phase for cell proliferation and activation of the Ras-mitogen-activated protein kinase pathway. The second wave increased serine phosphorylation following ligand-receptor extracellular domain disulfide bonding followed by the third wave, this being the internalization of the ligand-receptor complex.60 In a study on proteomic fingerprinting of the differentiation of human acute myelogenous leukemia HL-60 cells into macrophages induced by 12-O-tetradecanoyl-phorbol-13-acetate, Juan et al.61 found that about 20% of totally detected protein spots significantly differed. The seven main groups classified by subcellular locations and functions include secreted proteins, nuclear proteins, proteins corresponding with signal transduction/growth control, metabolism/enzymes, cytoskeleton related proteins, RNA or calcium binding proteins and unclassified proteins. LPS induced major changes in the two-dimensional protein patterns using cyanine-3 and -5 prelabeling of proteins compared to unstimulated U937 cells.62 In this study, incubation of U937 cells with LPS for 30 min caused the appearance of phosphorylated species of hsp 27 which could be inhibited by a p38 kinase inhibitor.62 Another important proteomic study carried out in monocytes harvested from the normal human and incubated with LPS for 48 h, demonstrated that 16 proteins of the approximate 500 detected were altered after LPS challenge.63 Of these LPS-challenged human monocytes, the pro-
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tective enzymes (superoxide dismutase and catalase), calgranulins, pre-B cell-enhancing factor, and serine/threonine protein kinase-10 were significantly increased, whereas integrin R-IIB, protein disulfide isomerase and platelet-activating factor acetylhydrolase decreased compared to unchallenged cells. An abundance of detected spots on the two-dimensional gels were attributed to calgranulin B (∼45 000) and A ( ∼30 000) from LPS-challenged monocytes.63 It is possible that phosphorylation of calgranulin B increases calcium binding and translocation of the calgranulin A-B heterodimer to the membrane. Subsequently, calgranulin A may be oxidized to generate intrachain disulfide bonds, protect oxygen free radical-induced tissue injury, and inactivate protease activities as induced by other stimuli.64 Superoxide dismutase and annexin II corresponded to high-density protein spots on the gel. This indicates that LPS-induced annexin II may initiate the recruitment and activation of plasmin and cathepsin B at the monocyte surface, resulting in matrix invasion and degradation by activated monocytes during inflammation. However, activities of protective enzymes were increased in parallel to protect against LPSinduced production of oxygen free radicals.63 Such protective responses in macrophages may be a common innate immune reaction, as demonstrated by the proteomic analysis of human macrophages (THP1 cells) challenged by live Mycobacterium tuberculosis, bacille Calmette-Guerin, or heat-killed Mycobacterium tuberculosis.65 Phagosomes, pivotal organelles in macrophages, are involved in the management of apoptotic cells, tissue remodeling and limitations of intracellular pathogen spreading.66 Formation of the phagosome results from binding of the particle to cell surface receptors and reorganization of the plasma membrane and cytoskeletal elements. Garin et al.59 used the proteomic approach for identifying more than 140 proteins associated with latex bead-containing phagosomes to determine the functions of phagosomes and propose new concepts on its biogenesis. Such an important approach can be used for charactering phagosome proteins in human cells and investigating phagosome function during cell differentiation or response to challenges. This particular study demonstrated that phagosome proteins isolated and identified from the J774 mouse macrophage-like cells may be involved in membrane fusion with subsets of endosomes, phagosome maturation, the interaction between ER elements and phagosomes, lipid rafts, and apoptosis.59 On the other hand, intracellular microorganisms can influence or manipulate the nature of the phagosomal microenvironment and induce sophisticated mechanisms to detect and respond to environmental conditions by phenotypic alterations that ensure pathogen adaptation, survival, and proliferation within the cell.67 Endothelial Cells. The endothelial barrier plays a critical and central role in the development of systemic inflammatory response syndrome, by interacting with leukocytes, producing inflammatory mediators, expressing cell surface adhesion molecules and increasing surface permeability resulting in plasma exudation, tissue edema, and organ dysfunction.68 Alterations in hemodynamic conditions may also cause dysfunction of the endothelial barrier, initiating several pathological processes.69 A proteomic analysis on endothelial cells exposed in vitro to plaque-free and plaque-prone flows demonstrated an increased expression of macrophage-capping protein, a member of the gelsolin protein superfamily, in cells exposed to a splaque-free flow.70 After proteomic analysis, this outstanding study continued to explore expression and func-
reviews tioning of macrophage-capping protein. It is one of a few studies on proteomic profiles and functions to use statistical analysis through all measurements. However, a few spots showed differences between the two flow patterns, indicating that hemodynamic alterations may have limited effects on the induction of endothelial barrier dysfunction during systemic inflammatory responses. Oxygen free radicals have been proposed as interorgan signaling agents responsible for communication from the origin dysfunctional organ to the distant secondary compromised organ.71 Pretreatment with antioxidants may prevent the development of endothelial barrier dysfunction in multiple organs after the induction of acute pancreatitis.72 Proteomic profiles of human endothelial cells have been investigated in precultured human umbilical vein endothelial cells.73 Studies on this normal cell type identified 53 proteins, as shown in Table 1. Furthermore, when human endothelial cells isolated from fresh umbilical cords were exposed to low concentrations of hydrogen peroxide, one spot on the two-dimensional gel was consistently altered and corresponded with molecular masses of 39 and 41 kDa and a pI of -5.0.74 Altered protein spots from endothelial cells 20 min post-challenge with hydrogen peroxide were identified as heterogeneous nuclear ribonucleoprotein C1/ C2 (hnRNP-C1/C2), a nuclear restricted RNA-binding protein.74 It was suggested that hydrogen peroxide might directly cause the increased phosphorylation of hnRNP-C1/C2 leading to the mobilization or removal of a population of hnRNP-C1/C2 from the nuclear mRNA pool to regulate the post-transcriptional response to the radicals. Protein shedding is another post-translational event in regulating protein function, independent of the expression level of mRNA. Proteolysis of cell membrane-bound proteins can control the production of soluble cytokines, receptors, adhesion molecules and growth factors through the process termed ectodomain shedding.75 When human adult dermal microvascular endothelial cells were treated with a mixture of inflammatory cytokines (e.g., CD40L, IL-1β, TNFR, interferon-γ, fibroblast growth factor, TWEAK, and vascular endothelial growth factor) for 4 h followed by addition of PMA for an hour to induce shedding, two endothelial cell-derived proteins, Jagged 1 and endothelial cell protein C receptorwere identified from a proteomic analysis for cell surface shedding.76 Both of these transmembrane proteins may be released by shedding, a process dependent on metalloproteases.76,77 Alterations of such metalloprotease-shed proteins in endothelial cells were different from those in monocytes stimulated with LPS and shed by PMA. These include amyloid A4 protein, AXL receptor tyrosine kinase, interleukin 1 receptor type II, interleukin 6 receptor-R, low-density lipoprotein receptor, L-selectin, GM-CSF 1 receptor, hepatocyte growth factor receptor, SHP substrate 1 protein, saposin, and tubulin.76 Eosinophils. Activation and infiltration of eosinophils play an important role during allergic inflammatory responses (Figure 3). Proteomic analysis was performed to understand the potential mechanisms involved in human peripheral blood eosinophil survival and activation after incubation with human mast cells, TNFR, and GM-CSF.78 The proteomic pattern of labeled polypeptides was measured after challenge with these three stimuli in a qualitative and quantitative manner. GM-CSF induced the strongest signal and the highest rate of protein synthesis with 1018 spots, whereas TNFR induction and mast cell sonicate produced 747 spots and 611 spots, respectively. This contributory study on human eosinophils compared Journal of Proteome Research • Vol. 3, No. 5, 2004 925
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Table 1. Summary of Identified Up-Regulated or Down-Regulated Proteins in Human Primary Leukocytes Challenged with Inflammatory Factors cells
CD14+ monocytes
challenges
GMCSF +IL4-7days; GMCSF +IL4+TNFa-7days
monocytes
LPS-48 h
neutrophils
ionomycin or N-formyl-methionyl-leucylphenylalanine 1µm-15 min
neutrophils
LPS-4h
HUVECs
hydrogen peroxide-60 min
up-regulated
Hsp73 vimentin macrophage capping protein guanylate kinase Hep27 ferritin light chain FABP4,5 ACBP S100c calgranulins superoxide dismutase annexins macrophage capping proetin pre-B cell enhancing factor catalase transketolase pyruvate kinase serine threonine proteins kinase 10 myeloperoxidase gelatinase B lactoferrin serum albumin annexin XI collagenase chitotriosidase HC gp-39 cathepsine G elastase proteinase 3 azurocidin NGAL FALL-39 lysozyme C cagranulins defensins proteasome b chain leukocyte elastase inhibitor Rab-GDI b grancalcin transaldolase moesin nonmuscle myosin heavy chain glutathione S-transferase P pre-B cell enhancing factor isocitrate dehydrogenase PO4-stathmin protein phosphatase 1, β catalytic subunit annexin III nuclear restricted, RNA-binding protein-C1/C2
categories of up- or down-regulated spots in various treatment groups and correlated the expressed polypeptides with various treatments, although these proteins were not identified. These results support the findings that GM-CSF plays an important role in eosinophilopoiesis and eosinophil activation.79 Lymphocytes. Lymphocytes and their cytokines, together with other leukocytes (e.g., eosinophils) play major roles in the initiation and perpetuation of the inflammatory response. In antigen-induced inflammation, lymphocyte hyperreactivity was considered to play a major role in the development of the disease,81 whereas circulating lymphocytes became hyporeactive as revealed by a diminished capacity of ex vivo mediator production in response to inflammatory stimuli in septic patients.82 Recent studies have applied quantitative proteomic analysis for identifying and quantifying chromatin-associated proteins, e.g., transcription and replication factors, in human B lymphocytes (P493-6 cells) expressing c-Myc in a tetracycline926
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calreticulin RNCC protein MRP14 MRP8
47
integrin R-IIB chain 1 protein disulfide isomerase platelet-activating factor acetylhydrolase
63
28
adenylyl cyclaseassociated bprotein 1 Rho-GAP 1 ficolin 1 adenosylhomocysteinase calponin H2
24
74
repressible manner.83 Chromatin-associated regulatory factors play central roles in the regulation of cell proliferation, differentiation, senescence, and death. It was emphasized that the proteomic technology was necessary for directly analyzing chromatin-enriched fractions in the detection of chromatinassociated factors,83 and reticulation of the gel could interfere by preventing the uptake of enzyme during the in-gel digestion.86 By using high-resolution two-dimensional polyacrylamide gel electrophoresis or Melanie 3.0, no significant difference in the number of spots and the global protein pattern were evident in CD19-, CD8-, or CD4-positive human lymphocytes, while three spots corresponding to cytoskeletal proteins were identified in CD4- and CD8-positive cells.87 To investigate protein profiles in lymphocytes challenged with immunosuppressors and activators, proteomic analysis was performed in concanavalin A-stimulated mouse spleen cells cultured with or without cyclosporine A and re-stimulated
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on leukocyte activation and translation between genes and proteins. Protein tyrosine phosphorylation is an important signaling event critical for regulation of gene transcription, cell proliferation, differentiation, cytoskeletal organization and survival. Such a process of signal transduction in lymphocytes may be involved in the development of tissue remodeling in chronic inflammation.90 By using a battery of Src homology 2 domain probes, distinct profiles of tyrosine phosphorylation were investigated in different cell lines. Two lymphocyte cell-lines (Daudi and BJAB) showed similar patterns of tyrosine phosphorylation for the Fyn and PI3-kinase Src homology 2 but not in Crk Src homology 2.89 The fingerprinting of signal transduction pathways in response to IL-2 or IFNγ was investigated in primary human lymphocytes using the combination of antiphosphotyrosine immunoprecipitations and proteomic analysis.91 Results from this important study demonstrated that IFNγ induced phosphorylation of the transcription factor STAT2 within minutes, while IL-2 activated PI3K phosphoprotein in human lymphocytes. Transcription factor Jak-1 was phosphorylated after treatment with both IFNγ and IL-2.91 To conclude, an increasing number of studies on the application of leukocyte proteomics have appeared in mapping protein profiles of inflammatory cells, contributing to the understanding of potential mechanisms involved in leukocyte function. Together with improvements in proteomic technology in leukocyte research, leukocyte proteomic analysis becomes more simple, rapid, flexible, sensitive and specific. This enables proteomic investigation of activated or nonactivated leukocytes to be highly focused on defined suborgans or specific signaling pathways. Research in leukocyte proteomics is progressing from fingerprinting to functioning, human cell lines to primary leukocytes, nonactivated cells to inflammatory mediatorstimulated cells (Table 1), in vitro culture to in vivo challenge, and animal models to human disease. A number of newly identified proteins from leukocyte proteomics may offer new mechanism-orientated targets for drug discovery and development. Figure 3. Histography of the nasal mucosa from transmission electronic microscopy (×3000). Eosinophils activated by the antigen exposure have migrated from the capillary to the nasal tissue (see arrows). Granules secreted from activated eosinophils distributed between the epithelial cells and the conjunction between epithelial cells was damaged and enlarged.
in the presence or absence of concanavalin A.84 Results demonstrated that withdrawal of the immunosuppressor and activator induced de novo protein synthesis, and transcription and translation of genes without further reactivation. Cyclosporine A-induced down-regulation of immune responses by inhibiting the calcineurin-dependent dephosphorylation of nuclear factor in activated T cells can cause synthesis of several new and specific synthesized polypeptides, without affecting CD44 and CD69 and early protein tyrosine phosphorylation events.85 When human T cells (E6-1 cell line) were cultured with rapamycin, a potent immunosuppressant, the integrated intensity of about 111 protein spots were changed; 70 increased and 41 decreased.88 Of these identified proteins, Grolleau et al. found that rapamycin induced increased intensities of annexin V and Ro/SSA antigen and a large fall in R enolase and LAMR1, in a good consistence with the microarray data. This is an example of application of proteomic and genomic approaches to investigate the influence of drugs
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