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Proteome Comparison of Alveolar Macrophages with Monocytes Reveals Distinct Protein Characteristics

Departments of Pathology and Medicine, Dorothy M. Davis Heart and Lung Research Institutes, Ohio State University College of Medicine and Public Health, Columbus, Ohio

Address correspondence to: Haifeng M. Wu, M.D., Assistant Professor and Director of Clinical Coagulation Laboratory, Department of Pathology, Ohio State University College of Medicine and Public Health, 288 Medical Research Facility, 420 West 12th Ave., Columbus, OH 43210. E-mail: wu-6{at}medctr.osu.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Alveolar macrophages (AMs) are a subset of tissue macrophages situated in the alveolar milieu. Compared with their precursor blood monocytes, AMs exhibit distinct physiologic functions unique to their anatomic location. However, the molecular details that control monocyte differentiation into AMs remain unknown. This study employed a proteomic approach to define protein characteristics that distinguish AMs from monocytes. AMs and monocytes were obtained from six nonsmoking, healthy donors. Whole cell lysates from each donor's AMs and monocytes were analyzed by two-dimensional (2D) gel electrophoreses. The protein density for each protein spot in a 2D gel was compared between these two cell types. Proteins that demonstrated consistent level changes of greater than 2.5-fold in all six donors were subjected to tandem mass spectrometry for protein identity. Using this process, we revealed proteome changes in AMs that relate to their physiologic roles in proteolysis, actin reorganization, and cellular adaptation in the unique alveolar milieu. By comparison, blood monocytes displayed higher levels of the proteins involved in transcription, metabolism, inflammation, and in the control of proteolysis. These results provide new insights into the biology of mononuclear phagocytes and set a basis for future causality studies.

Abbreviations: two-dimensional, 2D • alveolar macrophages, AMs • bronchoalveolar lavage, BAL • chronic obstructive pulmonary disease, COPD • interleukin, IL • IL-1ß converting enzyme, ICE • immobilized pH gradient, IPG • mass spectrometry, MS • reactive oxygen species, ROS • selenium-binding proteins, SBP • superoxide dismutase, SOD • tripeptide peptidase I, TPP I

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mononuclear phagocytic system consists of bone marrow monoblasts, promonocytes, blood monocytes, and tissue macrophages. Under normal circumstances, monocytes derived from bone marrow circulate in the blood for less than 48 h and enter into various tissues to differentiate into macrophages. Compared with monocytes, macrophages have a longer life span and display heterogeneity in structure and function (1). Macrophages in different anatomic locations express different biological activities. Moreover, even within tissues, there appears to be subpopulations of macrophages that exhibit unique characteristics (2). These findings suggest that the tissue's microenvironment influences the differentiation of blood monocytes into the appropriate phenotype of macrophages with unique biological functions. However, the cellular mechanisms that control these processes are largely unknown.

Alveolar macrophages (AMs) are a unique subset of macrophage that function primarily in the host defense of the lung against inhaled particulate matter, microorganisms, and environmental toxins. They possess Fc and complement receptors that respond to the Fc portion of IgG and C3 components, respectively. This enables AMs to participate in initial host defense (3). In addition, AMs have other roles in the regulation of adaptive immune response and inflammation, including antigen presentation and production of multiple mediators such as cytokines and metalloproteinases (4–6). Furthermore, AMs have been linked to the pathogenesis of multiple lung diseases such as smoking-related chronic obstructive pulmonary disease (COPD), asthma, and idiopathic pulmonary fibrosis (7–9).

We hypothesized that AMs have a unique cellular proteome that distinguishes their unique physiologic and structural properties from their precursor blood monocytes. Because proteins determine all biological processes of the cell, a detailed analysis into the AM proteome has the potential to shed light into the biological events controlling the differentiation and function of AMs and to define the role of AMs in disease development. In the present study, AMs and blood monocytes were obtained from six healthy donors and were subjected to two-dimensional (2D) electrophoresis/proteomic analysis. This process identified distinct proteome characteristics that underlie the functional and phenotypic differences between these two cell types. These data provide new insight into the biology of mononuclear phagocytes and form the basis for future investigations.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
All the reagents for cell preparations, RPMI 1640 (containing L-glutamine), fetal bovine serum, and penicillin/streptomycin were obtained from Gibco (Grand Island, NY). For 2D experiments, Sypro-Ruby Fluorescence dye came from Molecular Probes (Eugene, OR). Immobilized pH Gradient (IPG) buffer and IPG strips (pH 3–10) came from Amersham (Uppsala, Sweden). Phosphatase inhibitor cocktail and protease inhibitor cocktail were obtained from Boehringer Mannheim (Mannheim, Germany). DTT came from Invitrogen (Carlsbad, CA). Trypsin was obtained from Promega (Madison, WI). All other reagents and chemicals were obtained from Sigma (St. Louis, MO) unless otherwise noted.

Donor Characteristics
A total of six donors were recruited and their AMs and blood monocytes were used for this study under a research protocol approved by the IRB committee at the Ohio State University. These six donors are healthy, nonsmoking, white males with ages between 26 and 33 yr. Because this initial proteomic study only focused on the fundamental biological differences between AMs and blood monocytes, no additional efforts were made at the moment to recruit females and minority donors.

Isolation of Peripheral Blood Monocytes
Isolation of blood monocytes was performed as described (10). A volume of 120 ml blood, anticoagulated with heparin, was isolated by density gradient centrifugation over Histopaque. The mononuclear layer was pooled, resuspended in RPMI 1640, and the monocytes isolated by negative selection using a monocyte isolation kit (Miltenyi Biotech, Auburn, CA). Monocyte purity in each cell preparation was evaluated by light microscopic examination of Diff-Quik cytospins to ensure at least 90% purity. Contaminated cells were mainly lymphocytes, and the percentage of contaminated cells varied from 1–6% in all six donors used in our studies. Because each monocyte preparation has a different level of lymphocyte contamination, the observed proteome differences consistently present between AMs and monocytes in all six donors were less likely affected by lymphocyte contamination. The number of monocytes obtained from each donation ranged from 15 x 10⁶ to 30 x 10⁶ cells.

Isolation of AMs
Five 20-ml aliquots of sterile 0.9% NaCl were instilled into the right middle lobe or left lingular segment of the lung through a wedged flexible fiber optic bronchoscope and then gently suctioned. The fluid returned from the first aliquot was discarded because it may contain cells and mucous materials/proteins from large airways. The remaining lavage fluid was combined and collected for isolation of AMs. Cells obtained in the bronchoalveolar lavage (BAL) were then washed with cold RPMI. An aliquot was then subjected to cytospin and Wright staining and examined microscopically for purity. Because greater than 95% of the cells in a BAL were AMs, they were used directly for proteomic studies. The numbers of cells obtained ranged from 3–8 x 10⁶ for all AM preparations.

2D . Electrophoresis
Isolated monocytes or AMs were washed with cold Hanks' buffered saline solution buffer and resuspended immediately in 100 µl 2D lysis buffer containing 2 M thiourea, 5 M urea, 0.25% CHAPS, 0.25% Tween 20, 0.25% SB 3–10 (D-4266), 100 mM DTT, 10% isopropanol, 12.5% isobutanol (2-methyl-1-propanol), 5% glycerol, 1 x phosphatase inhibitor cocktail, 1 x protease inhibitor cocktail, and 1 x IPG buffer corresponding to the pH range of IPG strip. The cell lysates were set on a rotator at room temperature for 60 min and then spun at 14,000 rpm at 4°C in a refrigerated Eppendorf microcentrifuge for 20 min to remove cell debris and DNA aggregates. The supernatant was collected and frozen at –80°C for later 2D electrophoresis. For first D electrophoresis, 100 µl of the cell lysates collected above were mixed with 400 µl rehydration buffer (8 M urea, 2% CHAPS, 40 mM DTT, 0.002% bromophenol blue, and 0.5% ampholine buffer matching the IPG strip used for first D electrophoresis). The sample was then spun at 14,000 x g at 4°C for 10 min. Four hundred fifty microliters of the supernatant was subjected to first D electrophoresis at 20°C on an Amersham IPGphor using premade 24 cm IPG strips. Running conditions for first D electrophoresis consisted of rehydration for the first 12 h followed by 500 V for the next hour, 1,000 V for another hour, and finally 8,000 V for the last 11 h. After first D electrophoresis, each strip was equilibrated in a buffer containing 50 mM Tris HCl, pH 8.8, 30% glycerol, 2% SDS, and 6 M urea with 1% DTT for 10 min at room temperature. This is followed by a second equilibration for 10 min using the same buffer, except that DTT was replaced with 2.5% iodoacetamide to prevent thiol reoxidation. Second D electrophoresis was run on a 20 x 26 cm 12% SDS-PAGE on an Amersham Dalt II system.

Protein Quantification and Profiling
A total of twelve 2D gels were performed to analyze all AM and monocyte proteomes from all six donors. After 2D electrophoresis, the gels were fixed and stained with SyproRuby fluorescence dye according to manufacturer's protocol. Gel images were captured on a Typhoon 9,200 laser scanner (Amersham) that offers high resolution and quantification of protein spots. Protein quantification on all twelve 2D gels was performed using ImageMaster 2D software (Nonlinear Dynamics, Durham, NC). The monocyte 2D gel from Donor 1 was selected as the reference gel for this 2D experiment because it has 1,752 spots, the highest number of observed protein spot of all twelve gels. The remaining eleven 2D gels were matched to the reference gel for spot identification. On average, > 80% of all protein spots on each 2D gel was successfully matched to their respective protein spots on the reference gel. All de novo protein spots on nonreference gels were added to the reference gel image during the matching process, giving the reference gel a total of 2,021 spots that encompass all the protein spots in these twelve 2D gels. For each protein spot in the reference gel, a molecular weight (mol wt) was assigned using mol wt standard markers (Invitrogen) and pI values were estimated based on manufacturer's instruction (Amersham). The density of each protein spot on a 2D gel was calculated as a percentage of total protein volume of the 2D gel to minimize variations caused by staining and destaining procedures between all 2D gels. For each protein spot, six density ratios were calculated that correspond with the six donors in the study. Depending on whether or not the AM density was higher or lower than the monocyte density, each ratio was expressed either as AM/monocyte or as monocyte/AM. This kept all protein ratios at 1.0. A mean density ratio for each spot was calculated and target proteins were selected if their mean density ratio was > 2.5. Differences in protein densities between these two cell types were then visually reconfirmed with ImageMaster 2D software that provided a simultaneous and enlarged display of the protein spot as it appeared within all twelve 2D gels. Target spots were then cut by a Bio-Rad robotic 2D spotcutter platform and subjected to mass spectrometry (MS) analysis.

Statistical Determination of Proteome Differences between AMs and Blood Monocytes
Statistics was performed using Stata version 8 to analyze the data derived from the twelve 2D gels of all six donors. A total of 1,415 protein spots were analyzed in this study. Each spot had twelve density values, of which six were from the AM 2D gels and six were from the monocyte 2D gels. All values were then subjected to a logarithmic transformation to obtain an approximate normal distribution of the six values for each protein spot in either AMs or monocytes. For each spot, the transformed values between the two cell types were compared by a two-tailed t test to determine if they are statistically different using a significance level of 5% (P values 0.05).

Protein Identification by Tandem MS
Protein identification was performed using MALDI TOF/TOF (Applied Biosystems, Foster City, CA). Briefly, the spots cut from each 2D gel were washed two times with water and then washed three times with 25 mM ammonium bicarbonate followed by acetonitrile. Once dehydrated with acetonitrile, the gels were then treated with trypsin at 5 ng/µl in 25 mM ammonium bicarbonate for 10 min at room temperature. Following this, the samples were digested for 6 h at 37°C and treated with 20 µl of 0.3% formic acid bound to millipore zip tips. Any bound peptides were eluted onto a MALDI plate set in an 8 mg/ml solution of -cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% trifluoroacetic acid. Samples were then placed in the 4,700 Proteomics Analyzer. The resulting mass spectra were interpreted using Mascot software from Matrix Sciences (www.matrixsciences.com).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Proteomic Analysis into Common Proteome Differences between AMs and Blood Monocytes
We hypothesized that AMs and monocytes would display distinct proteome expression patterns due to great anatomic and functional differences between the two cell types. To test this hypothesis, we analyzed the AMs and monocytes obtained from six donors. 2D gel electrophoresis was performed using pH 3–10 IPG strip on the first D and 12% SDS-PAGE on the second D. Despite our efforts in applying a similar amount of proteins ( 2 mg) onto each 2D gel, the number of observed protein spots from all six monocyte 2D gels (1,404–1,752) was consistently higher than that in each of the six AM 2D gels (1,292–1,594). It is intriguing that compared with AMs, monocytes displayed a larger number of small cationic proteins. However, the low abundance of these proteins makes it difficult to obtain protein identification during this study, leaving the significance of these proteins uncertain. All twelve 2D gels (six AMs and six blood monocytes) were matched to the monocyte reference 2D map and the relative densities of all protein spots were quantified using ImageMaster software.

For each spot on the 2D gel, the mean density ratio between AMs and monocytes was obtained along with a standard deviation (n = 6). Protein spots showing a ratio of > 2.5-fold were selected, and differences in protein quantity were reconfirmed through visual examination of a simultaneous display of the same gel area within all twelve 2D gels. Figure 1 illustrates the comparison of protein density between AMs and blood monocytes by depicting the same area within all twelve 2D gels of the six donors in the study. Red arrows highlight two proteins that demonstrated a significant increase in protein level in AMs. These candidate proteins were identified as Cathepsin H by tandem MS. Black arrows illustrate a few protein spots present in both AM and monocyte 2D gels, indicating that the same gel areas were compared between AM and monocytes. Meanwhile, Figure 1 demonstrates the excellent reproducibility of our proteomic analysis. Within each cell type, there was magnificent reproduction in protein pattern and protein density between all six donors. Moreover, between these two different cell types, proteome differences were highly consistent among all six donors. Such reproducible studies allow for an accurate examination of cellular proteome changes between AMs and monocytes.

View larger version (99K): [in this window] [in a new window]   Figure 1. Demonstration of how the protein spots initially selected by statistical analysis were visually examined using the montage window of a specific gel area in the proteomes of AMs and blood monocytes of all six donors. (A) AM 2D gel from Donor 1. (B) Monocyte 2D gel from Donor 1. (C) Magnified image of the same gel area for both AM and monocyte 2D gels of all six donors in the study.

Figure 2 shows one of six AM 2D gels in this experiment. The arrow and spot number illustrate protein spots with a significant increase in protein levels in AMs. Each of these spots has had their identity reconfirmed at least once by repeated MS/MS. Protein spot numbering in Table 1 corresponds to the spot number of each protein in Figure 2. Also listed in Table 1 are the protein characteristics of these proteins such as pI, M_r, and the extent of quantity increase defined by the protein's mean density ratio (AM/monocyte). Some of the proteins in this table, such as various cathepsins (B or D) and Cap G, were known to be expressed in high levels in AMs (11–13). However, most proteins that had higher expression in AMs are described here for the first time. Analogous to Figure 2, the proteins identified in Figure 3 have also been reconfirmed by Tandem MS. However, Figure 3 illustrates proteins that were expressed in greater levels in blood monocytes compared with AMs. Table 2 lists the detailed identities and properties of all proteins shown in Figure 3. The level of each protein quantity changes is illustrated by the protein's density ratio of monocyte/AMs. As noted for AMs, many of the proteins found to be greater in monocytes than AMs were not previously described.

View larger version (103K): [in this window] [in a new window]   Figure 2. A 2D gel of alveolar macrophages. Arrows point to proteins spots that when compared with the proteome of blood monocytes, displayed a 2.5-fold or greater increase in protein levels. Protein density changes appear as a ratio of alveolar macrophage to monocyte and are listed in Table 1.

View larger version (107K): [in this window] [in a new window]   Figure 3. A 2D gel of blood monocytes. Arrows point to protein spots that when compared with the proteome of alveolar macrophages, displayed a 2.5-fold or greater increase in protein levels. Protein density changes appear as a ratio of blood monocytes to alveolar macrophages and are listed in Table 2.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Candidate proteins with distinct quantity changes between AMs and monocytes are apportioned into categories according to function. The first of these categories consists of proteins related to lysosomal proteolysis. Our study confirms many previous observations that AMs contain higher protein levels of lysosomal thiol proteases such as cathepsins B, D, H, and X, as well as aspartic protease napsin A (11, 12, 14, 15). In addition, our study demonstrates for the first time that AMs showed an increased level of tripeptide peptidase I (TPP I), a lysosomal serine peptidase that removes tripeptides from the free N-terminal of small polypeptides. Furthermore, AMs' serine protease inhibitor, leukocyte elastase inhibitor, was on the contrary significantly decreased when compared with monocytes. Because AMs are exposed to the external environment where they encounter microbes and particulate materials, a shifted balance of AMs toward proteolysis likely represents an adaptation of AMs to their unique physiologic condition.

Moreover, higher protein resolution power within 2D gel electrophoresis enabled the detection of various isoforms of these lysosomal proteases, all exhibiting higher protein levels in AMs compared with blood monocytes (Table 1). For example, pronapsin A is a precursor protein that undergoes post-translational cleavage to the active enzyme napsin A with an M_r of 34–36 kD (16). Seven different spots of napsin A were uncovered via 2D gel electrophoresis, and their levels were all elevated in AMs. These isoforms were characterized with molecular weights ranging from 32.43–37.54 kD and pI values from 4.96–5.47. Isoforms were also detected for TPP I. Details about the physiologic substrates of TPP I remain unclear at the present time. However, an inherited deficiency of TPP I activity causes fatal lysosomal storage disorder, a classic late infantile neuronal ceroid lipofuscinosis (17). Previous studies estimated the molecular weight of TPP I to range from 47–49 kD (18). Our report details four major isoforms of TPP I that have the M_r ranges as reported and pI values between 5.61 and 5.72. Differences in their electrophoretic properties suggest that these TPP I isoforms differ either in post-translational modification or in the de novo protein structures. In contrast to previous predictions that native TPP I, with a molecular weight of 280 kD, was composed of six identical subunits (18, 19), our data suggest that native TPP I likely consists of the subunits that differ either in post-translational modification or in the primary protein structure. The discovery of the many isoforms of both TPP I and napsin A necessitates further studies to elucidate the biochemical basis of these isoforms and to understand their respective physiologic properties in AMs.

A second category consists of proteins involved in the regulation of cytoskeleton function. Phagocytes exhibit many cellular activities such as chemotaxis, phagocytosis, shape changes, and degranulation that all involve cytoskeletal reorganization. AMs and blood monocytes are expected to display different actin dynamics due to their unique biological activities. As predicted, distinctive differences in the actin regulatory components were detected for each cell type. One observation consisted of two different isoforms of Heat Shock Protein 27 (HSP 27) that exhibited significant level increase in AMs. HSP 27 belongs to the family of small chaperone molecules that modulate cytoskeleton through its actin-capping activity (20). HSP 27 has several forms of post-translational modification that includes phosphorylation occurring at ser 15 or ser 78. Future studies are needed to detail HSP 27's phosphorylation states and their respective roles in AMs. Of note, HSP27 is also involved in cellular survival, which also correlates to an enhanced life span in AMs versus blood monocytes (21). Second, our results support previous observations that Cap G is abundant in macrophages (13). Importantly, we demonstrate for the first time that Cap G has four isoforms with analogous molecular weights but different pI values. Cap G belongs to the gelsolin/villin family of actin regulatory proteins that controls the actin assembly process by interacting with the barbed end of actin filaments. Cap G null mice have impaired macrophage ruffling and phagocytosis, suggesting an essential role of CapG in the regulation of host immunity (22). Further studies are required to elucidate the biochemical basis of these four different isoforms of Cap G and their roles in controlling AMs' functions.

In contrast, we observed that two other actin regulatory components, cofilin and F-actin–capping protein, were expressed in significantly higher levels in blood monocytes. Cofilin negatively regulates actin polymerization. However, when phosphorylated by LIM kinase at ser 3, cofilin appears to lose its inhibitory activity (23). We discovered that two different isoforms of cofilin both displayed significant level increases in the monocyte proteome. Studies are under way to fully examine the changes of all possible cofilin isoforms and their phosphorylation states between AMs and blood monocytes. F-actin–capping protein is a heterodimeric protein composed of and ß subunits that is present in a wide variety of tissues and organisms. This protein binds to the barbed ends of actin filaments and thus controls actin polymerization (24). We predict that F-actin–capping protein in blood monocytes confines actin filament growth and maintains a steady-state level of actin monomers necessary for actin polymerization to occur upon the activation of cells. Future studies are required to test this hypothesis.

A third category of proteins relates to the adaptation of phagocytes to the unique alveolar milieu. AMs are the mononuclear phagocytic cells situated within the alveoli. Their location within the alveolar walls exposes them to the external atmosphere that allows direct modification by environmental hazards. Within this anatomic environment, AMs also encounter a much higher partial oxygen pressure. Accordingly, we observed high abundances of aldehyde dehydrogenase, a key component of the respiratory oxidation chain in the AM proteome. In contrast, glycolysis appears to be downregulated in AMs, because protein levels of four key enzymes in the glycolysis pathways, glyceraldehydes-3-phosphate dehydrogenase, pyruvate kinase, aldolase, and phosphoglycerate mutase, were all significantly decreased in AMs. We also observed that the level of selenium binding protein 1 was significantly increased in AMs. Selenium-binding proteins (SBP) have been identified in liver cells (25). Our study for the first time demonstrates that SBP is present in AMs with an M_r of 24.8 kD. Because AMs are exposed to selenium compounds in the external environment (26), this interesting discovery prompts us to predict that higher level of SBP in AMs may contribute to the regulation of selenium homeostasis. Future studies are needed to test this hypothesis.

Further findings revealed elevated levels of two antioxidant enzymes, superoxide dismutase (SOD) and peroxiredoxin in AMs. SOD effectively eliminates one of the most potent reactive oxygen species (ROS), superoxide anions. Peroxiredoxin efficiently reduces H₂O₂ and alkyl hydroperoxide to water and alcohol, respectively (27). The high partial oxygen pressure within the alveolar space likely increases AMs' production of ROS such as superoxide, hydroxyl radicals, and hydrogen peroxide. Increased levels of antioxidants in AMs reduce excessive ROS levels in AMs that may otherwise causes cell injury and perhaps cell death.

The next category of proteins consists of proteins contributing to host inflammatory responses. AMs are tailored to function in the alveolar environment, whereas blood monocytes are more metabolically active with the potential to differentiate and engage in a variety of inflammatory processes. Accordingly, we observed a significant elevation of three different Heterogeneous Nuclear Ribonucleoproteins (hnRNPs) in blood monocytes. hnRNPs are a family of more than 20 proteins designated with letters A to U and are known to be involved in regulating transcription, translation, nuclear transport, and signal transduction (28). hnRNP A1, hnRNP H, and hnRNP K were significantly elevated in blood monocytes, implying active processes in transcription and protein synthesis. In addition, we discovered two isoforms of interleukin (IL)-1ß–converting enzyme (ICE), each displaying increased expression levels in blood monocytes. ICE is a member of the cysteine–aspartic acid protease (caspase) family that cleaves IL-1ß precursor intracellularly to generate an active 17-kD cytokine. IL-1ß is one of the major cytokines regulating host inflammatory responses. Furthermore, we show that two calgranulins, calgranulin A and calgranulin B, displayed significantly increased levels in blood monocytes. Calgranulins, also named as myeloid-related proteins (MRP), with MRP 8 for calgranulin A and MRP 14 for calgranulin B, belong to the S-100 protein family that associates to form heterodimers/heterotetramers in the presence of calcium (29). Calgranulins have been used as markers for activation of inflammatory phagocytes, although their exact roles remain undefined. Our observation supports previous discovery that calgranulin expression were downregulated during differentiation of monocytes into tissue macrophages (30). Four different isoforms of the calgranulin B and one isoform of calgranulin A all exhibited higher levels in blood monocytes. Finally, we confirm that ficolin-1, a subtype of the ficolin protein family involved in recognizing pathogens, is significantly higher in blood monocytes (31). Ficolin is a group of pattern recognition proteins that bind to oligosaccharide structures on the surface of microorganisms and initiate the complement activation system that kills the bound microbes (32). This report reveals two isoforms of ficolin-1 in monocytes that have similar M_r but different pI values. Future studies are needed to better understand the structure and roles of these various isoforms of ficolins. In summary, our findings lend support to monocytes playing a more active role in host proinflammatory responses.

Lastly, we report that Galectin 3 level is significantly increased in AMs. Galectin-3 belongs to the lectin family of the carbohydrate-binding proteins present in various epithelial and inflammatory cells including peritoneal macrophages and AMs. Galectin-3 is localized within the cells and is also secreted into extracellular space. Extracellular galectin-3 interacts with ß-galactoside residues of several extracellular matrices and regulates the cell adhesion process. Intracellularly, galectin-3 may regulate diverse cellular processes such as RNA processing, cell replication, and apoptosis. A recent study demonstrates that galectin 3 plays a critical role during phagocytosis (33). Further studies are required, however, to define the biological properties of galactin-3 in AMs.

In this study, we focused on a global comparison of cellular proteomes between AMs and blood monocytes. A large format 2D gel (20 x 24 cm) in combination with SyproRuby fluorescence staining enabled detection of over 1,000 protein spots on each 2D gel. By using 2.5-fold changes as the cutoff point, we were able to detect 124 protein spots that demonstrated reproducible and apparent quantity differences between AMs and monocytes among all six donors. These candidate protein spots were then repeatedly subjected to tandem MS for protein identity. Eventually, 61 out of 124 protein spots were successfully identified. Failure to obtain other protein's identity determination was due either to low protein abundance or due to insufficient spot resolution on a 2D gel. To ameliorate protein resolution and to enrich protein abundance on 2D gels, a subproteomic approach focusing on a particular pH/or protein size ranges and subcellular proteomic analysis are necessary. Both methods can significantly increase protein resolution/detection power by focusing only on subsets of a total proteome.

Tandem MS such as MALDI-TOF-TOF is becoming the gold standard for determining protein identity. Tryptic peptides of target proteins were subjected to tandem MS for sequencing and the resulting sequences matched against a protein database to obtain a protein ID. This method of matching amino acid sequences enables protein identification to be obtained with a higher confidence score. Yet, limitations exist in the fact that a portion of the tryptic peptides is sequenced rather than all tryptic peptides of the protein. As a result, a partial determination of the protein sequences is made without the ability to distinguish the identified protein as an active protein or the protein's precursors, post-translational modifications, or proteolytic fragments. In this report, we reported a target protein's ID as its mature protein unless the sequenced amino acids cover either a pre- or pro-peptide of the protein.

Post-translational modification of protein by phosphorylation plays a central role in transducing signals responsible for a variety of biological processes. In macrophages for example, the expression of cellular activities involves numerous protein kinases and their downstream phosphorylated substrates (34). Conceivably, large-scale phosphorylation proteomics using Pro Q phosphoprotein staining (35) or tandem mass spectrometry in combination with multidimensional chromatographies will add significant more knowledge into understanding AMs' biology.

In this study, we did not directly address the functional role of each protein in human monocytes and AMs. Instead, the proteomics analysis provided in this article offers the protein characteristics that are fundamental to biology and disease processes. In addition, proteomic analysis allows for a rational selection of target proteins for further corroborative studies using specific assays or model systems. In this light, our study provided unprecedented information on potential target proteins that underlie the phagocyte functions, thus creating a basis for future causality studies. At the present time, our laboratories are engaging in specific studies based on these proteomic discoveries and we welcome other laboratories to join us in this endeavor.

	Acknowledgments

 
This work is supported in part by grants from National Institutes of Health K08HL03279 (to H.W.), HL63800 (to C.M.), and HL67176 (to C.M.), and by the grant support from Ohio Biomedical Research and Technology Transfer Commission.

	Footnotes

 
Conflict of Interest Statement: M.J. has no declared conflicts of interest; J.M.O. has no declared conflicts of interest; C.B.M. has no declared conflicts of interest; H.M.W. has no declared conflicts of interest.

Received in original form March 1, 2004

Received in final form April 25, 2004

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