Aging Enhances Production of Reactive Oxygen Species and Bactericidal Activity in Peritoneal Macrophages by Up-Regulating Classical Activation Pathways

Abstract

Maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection is central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. To identify possible age-related alterations in macrophage function, we have assayed the function of peritoneal macrophages from young (3–4 mo) and aged (14–15 mo) Balb/c mice. In agreement with prior suggestions, we observe age-dependent increases in macrophage recruitment into the peritoneum, as well as ex vivo functional changes involving enhanced nitric oxide production under resting conditions that contribute to a reduction in the time needed for full activation of senescent macrophages following exposure to LPS. Further, we observe enhanced bactericidal activity following Salmonella uptake by macrophages isolated from aged Balb/c mice in comparison with those isolated from young animals. Pathways responsible for observed phenotypic changes were interrogated using tandem mass spectrometry, which identified age-dependent increases in proteins linked to immune cell pathways under both basal conditions and following LPS activation. Immune pathways up-regulated in macrophages isolated from aged mice include proteins critical to formation of the immunoproteasome. Detection of these latter proteins are dramatically enhanced following LPS exposure for macrophages isolated from aged animals; in comparison, the identification of immunoproteasome subunits is insensitive to LPS exposure for macrophages isolated from young animals. Consistent with observed global changes in the proteome, quantitative proteomic measurements indicate that there are age-dependent abundance changes involving specific proteins linked to immune cell function under basal conditions. LPS exposure selectively increases many proteins involved in immune cell function in aged Balb/c mice. Collectively these results indicate that macrophages isolated from old mice are in a pre-activated state that enhances their sensitivities of LPS exposure. The hyper-responsive activation of macrophages in aged animals may act to minimize infection to general bacterial threats that arise due to age-dependent declines in adaptive immunity. However, this hypersensitivity and the associated increase in the formation of reactive oxygen species is likely to contribute to observed age-dependent increases in oxidative damage that underlie many diseases of the elderly.

Within the animal kingdom, the innate immune response is highly conserved. Specific classes of immune cells recognize characteristic pathogen-associated biomolecules (e.g., conserved cell wall components like lipopolysaccharides (LPS¹)) to orchestrate their rapid clearance at localized sites. For example, macrophages rapidly engulf and kill entrapped pathogens through a coordinated oxidative burst, simultaneously releasing inflammatory cytokines (e.g., TNFα) to recruit additional immune cells to the site of infection. In vertebrates, activated macrophages also act as an interface with the adaptive immune system through the presentation of antigenic determinants derived from engulfed pathogens that act to activate and maintain T cell activation following their recruitment to sites of infection. Microbial infections, in turn, reprogram cytotoxic lymphocytes and T helper cell responses as part of the adaptive immune system to induce their proliferation and promote the release of IFNγ to sensitize macrophages at distant sites. IFNγ exposure acts to promote macrophage activation upon bacterial recognition to up-regulate antigen presentation, the release of inflammatory cytokines, and the production of reactive oxygen species (ROS) that act to kill microorganisms. Control of macrophage activation is critical as the oxidative burst, resulting from their activation, inflicts collateral damage to host macromolecules and tissues, which contribute to a range of different age-related diseases (1–2).

Common models of macrophage aging emphasize the accumulation of oxidative DNA damage that results in their functional dysregulation, reported to impair the ability to respond to LPS, generate ROS, and present antigens through class-I and class-II major histocompatibilty complex (i.e., MHC-I and MHC-II) pathways necessary for the activation of cytotoxic lymphocytes and T-helper cells (3). Alternatively, changes in levels of circulating cytokines may induce differentiation to alter macrophage polarity, acting to modify the repertoire and magnitude of available functional responses. Complicating an understanding of age-dependent declines in immune function is the coupling between the innate and adaptive immune systems, which act together to coordinate cellular responses against pathogen exposures. While it is understood that adaptive immunity declines with age (4–5), discrepant results have been reported regarding age-dependent changes in macrophage function and their importance in downgrading immune defenses, which predispose aged animals to infection and contribute to the development of many of the diseases of the elderly (6–8). For example, isolated resident peritoneal macrophages from middle-aged (12 mo) and old (21 mo) mice have been reported to show an enhanced ability to kill herpes simplex virus in comparison to young (2 mo) controls, despite reported decreases in their ability to generate nitric oxide and other reactive oxygen species (ROS) known to mediate bacterial killing (9). Similar increases in pathogen clearance are observed for aged Balb/c mice (18 mo) when animals were challenged with a bacterial infection involving Leishmania major, despite the absence of any significant differences in rates of phagocytosis, bacterial killing, or nitric oxide production following isolation and uniform sensitization of peritoneal macrophages by IFNγ (10). An important clue to these apparently contradictory results comes from the observation that enhanced rates of bacterial killing observed in aged mice depend on their prior exposure to normal circulating pathogens, since housing under clean room conditions abrogate age-dependent increases in bacterial resistance (10). These latter results suggest that age-dependent alterations in macrophage function result from an enhanced sensitivity to environmental exposures, which may occur due to age-dependent reductions in physical barriers that limit pathogen entry and act to sensitize macrophages to promote their rapid activation in response to infection.

As environmentally induced changes in macrophage function should result in characteristic alterations in the proteome that are indicative of shifts in activation pathways, we have examined possible linkages between protein abundance changes and molecular pathways involving immune function, and their relationship to phenotypic changes in macrophage function. Peritoneal macrophages were isolated from young (3–4 mo) and aged (14–15 mo) Balb/c mice, which represents a normal model for use in aging measurements (9–10). The aged mice (14–15 mo) used in our measurements are near the average lifespan of this mouse strain, which is 485 ± 9 days (about 16 months) (11), permitting an investigation of age-dependent cellular changes prior to the onset of late-life pathologies that can complicate mechanistic interpretations. Macrophage functions were assayed using simple in vivo measurements that indicate age-dependent increases in the abundance of macrophages in the peritoneum in response to elicitation by an irritant injected into the peritoneum that is indicative of increased motility and macrophage activation. Mechanistic linkages between observed phenotypic changes in macrophage function and molecular pathways involving immune function were identified using mass spectrometry based measurements of protein abundance changes, which involved a shotgun proteomic analysis in which tandem mass spectrometry was used to identify 1,847 proteins with annotated functions for subsequent quantitation of protein abundance changes using the accurate mass and elution time (AMT) tag proteomic strategy. Consistent with observed functional changes and hypotheses developed using tandem MS data, we observe an age-dependent up-regulation of specific immune cell pathways (e.g., antigen presentation) and abundance changes of 77 proteins linked to macrophage activation. There is no indication of any impairment of normal cellular pathways involving macrophage activation in aged animals; rather, these results are indicative of an age-dependent up-regulation of normal classical activation pathways.

EXPERIMENTAL PROCEDURES

Materials

Male Balb/c mice (3–4 mo. and 14–15 mo.) were from Charles River Laboratories International Inc. (Wilmington, MA). Bio-Gel P-100 polyacrylamide beads (45–90 µm) were from Bio-Rad (Richmond, CA). Bacterial LPS from E. coli strain O127:B8 was from Sigma Chemical (St. Louis, MO). Interferon was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Ampicillin, RPMI 1640 medium (#0030078DJ), fetal bovine serum (FBS), penicillin, and streptomycin were from Gibco (Carlsbad, CA). FITC-labeled anti-mouse F4/80 macrophage specific antibodies were from eBioscience, Inc. (San Diego, CA). Calcein acetoxymethyl (AM) ester and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) were from Invitrogen (Carlsbad, CA).

Macrophage Elicitation

When indicated, macrophages were elicited by injection (1mL) of a 2% (v/v) sterile solution of washed and hydrated polyacrylamide beads in phosphate buffered saline (PBS) into the peritoneal cavity five days prior to their isolation, as previously described (12). Our experimental design is in accordance with all prior reports that address age-dependent changes in macrophage function, which commonly isolate peritoneal macrophages following elicitation at a single time point 3–5 days following the introduction of an irritant (in our case Bio-Gel polyacrylamide beads) (8, 13–20). As described by Melnicoff and coworkers (21), homogeneous populations of peritoneal macrophages are isolated within this time window to avoid contamination by neutrophils and resident macrophages that becomes problematic when macrophages are collected at longer times following elicitation.

Macrophage Isolation

Resident or elicited peritoneal macrophages were isolated using standard peritoneal lavage procedures following asphyxiation using CO₂ (12). Briefly, phosphate buffered saline (PBS) (10 mL) was injected into the caudal half of the peritoneal cavity using a 25-gauge needle, whole mice were gently rocked for 10 seconds, and peritoneal cells were slowly withdrawn using a 19-gauge needle and collected into a conical tube on ice. Isolated cells were plated in RPMI 1640 medium supplemented with heat-inactivated fetal bovine serum (10% v/v), penicillin (1% v/v), and ampicillin (1% v/v) at a constant cell density (1 × 10⁶ cells per p100 plate) and incubated for 1 hr at 37 °C. Nonadherent cells were removed by washing plates five times in warm PBS (500 µL), and remaining adherent macrophages were maintained in a humidified atmosphere of 5% CO₂ and 95% air at 37 °C overnight prior to treatment and harvest. Purity and viability of isolated cells were analyzed using an Agilent 2100 Bioanalyzer Microfluidics platform for flow cytometry with 2-color analysis of fluorescently stained cells and standard immunohistochemistry methods using a Nikon Eclipse TE200 epifluorescence microscope equipped with Metamorph imaging software to quantify the correspondence between multiple fluorescence signatures. Macrophage purity was determined to be greater that 95% of isolated cells through a comparison of FITC-labeled anti-mouse F4/80 macrophage specific antibodies to identify cells in comparison to total cells measured using DAPI labeling of double stranded DNA. Viability was measured by comparing fluorescence signals associated with cell esterase activity visible upon cleavage of calcein AM in comparison to FITC-labeled anti-mouse F4/80 macrophage specific antibodies.

Nitric Oxide Measurements

Time-dependent increases in nitric oxide were measured by following the accumulation of the stable nitrite end product using the Griess reagent (Pierce Inc., Rockford, IL). Prior to measurement, all nitrate was enzymatically converted to nitrite using nitrate reductase. Nitrite concentrations in conditioned media were determined on the basis of standard curves calibrated using sodium nitrite as a standard, as we have previously described (22).

Bactericidal Activity

Primary peritoneal or RAW 264.7 macrophages were challenged with S. typhimurium at a multiplicity of infection of 100, essentially as previously described (23–25). Briefly, S. typhimurium 14028 cells were cultured the day before the assay in LB broth at 37 °C overnight, and were harvested by centrifugation (12,000 rpm for 1 minute) before resuspension at a final density of 5 × 10⁵ cells/ml in 24-well cell-culture plates (i.e., 500,000 macrophages/well). Prior to bacterial challenge, macrophages were treated with 100 U/mL of interferon gamma (IFNγ), rinsed, and the prepared S. typhimurium-laden media were directly added to the plate of macrophages (26). The infection proceeded as plates were placed back into the incubator under standard conditions (37 °C in 5% CO₂). Following incubation for 30 minutes to permit macrophages to phagocytize the S. typhimurium, extracellular bacteria were rinsed off with PBS. Fresh media (12.5 µg G418 per mL) replaced the bacteria-laden media to eliminate extracellular bacteria and prevent the extracellular replication and invasion of any S. typhimurium remaining on the plate. Macrophages infected with S. typhimurium were then further incubated, and at indicated times cells were washed and lysed (PBS, 1% triton X-100, and 0.1% sodium dodecyl sulfate [SDS] for 5 minutes at room temperature). Remaining viable bacteria were detected as colonies following the serial dilution of the cellular lysates onto LB agarose plates that were incubated overnight at 37°C.

Macrophage Lysis

Following removal of media, cells were rinsed once with chilled D-PBS (Invitrogen, Carlsbad, CA) and incubated in chilled 20 mM Tris (pH 8.0), 1% Nonidet P-40, 0.15 M NaCl, 1 mM Na₂PO₄, and 1 mM EGTA. Cell lifters were used to manually scrape and transfer cells into a chilled glass homogenizer, and following cell disruption (a ten-stroke homogenization), lysates were immediately centrifuged for 30 minutes at 9,300 × g at 4°C. Supernatant was removed, and stored at −80°C. Cellular disruption in the presence of detergent permits quantitative measurements of total lysates to be evaluated as membrane disruption and protein solubilization enhances overall proteomic coverage. In the absence of protein solubilization, membrane associated proteins are lost from the sample during sample processing. As many of the key proteins associated with macrophage activity are linked to membrane processes, it is critical to include a solubilization step for accurate assessments of proteomic changes. Lysates were subjected to low-speed centrifugation (9,300 × g on a table top centrifuge) prior to analysis to prevent any large particles from clogging the LC equipment, and there was no detectable loss of protein following this procedure.

Cysteine Alkylation and Trypsin Digestion

Lysates were dialyzed against ammonium bicarbonate prior to addition of 8 M urea to denature proteins. Cysteine residues in lysates were reduced using tris(2-carboxyethyl)phosphine (TCEP) (Bond Breaker TCEP solution) (5 mM) (Thermo Scientific, Rockford IL), alkylated using iodoacetamide (25 mM), and sonicated in a UTR200 sonoractor (Hischler, Teltow,Germany) at 50% intensity for 3 min, as described before (27). Following a 10-fold dilution of samples into freshly prepared 50 mM ammonium bicarbonate solution (pH 7.8) and 1 mM CaCl₂, samples were digested using trypsin (1:50, wt/wt ratio of trypsin to sample protein)for 1 min in a Barocycler™ NEP-3229 instrument, as previously described (28). Each digest was desalted using Supelco (St. Louis, MO) Supelclean C-18 tubes, as described elsewhere (29), and concentrated (1 mg/mL) using vacuum centrifugation.

LC-MS Analysis and Peptide Identification

A quantitative analysis of changes in the cellular proteome involves of two-step process. First, in stage 1 tandem MS (MS/MS) measurements are made following proteolytic digestion and strong cation exchange (SCX) fractionation of proteins from peritoneal macrophage lysates obtained from young and aged animals into 30 liquid chromatography (LC) fractions for analysis using a linear trap quadrupole (LTQ) iontrap mass spectrometer (ThermoElectron Corp., San Jose, CA), as described previously (30). The MS/MS spectra were analyzed using the SEQUEST Bioworks 3 release version (31) against an IPI Mus musculus database downloaded from NCBI (i.e., M_Musculus_2006-07-25_IPI which contains 94,146 entries). The mass tolerance for the precursor and fragment ions were respectively 2.5 Da and 0.8 Da. Database search parameters included dynamic modification of +16 Da for Met oxidation and fixed +57 for Cys carbamylation. Only peptides with +2 and +3 charges were analyzed for fragmentation and SEQUEST analysis. Confident peptide identification with a false discovery rate of 1% (i.e., q < 0.01) involved fitting the data to a sum of Gaussian distributions following correction of X_corr values to take into account peptide length, where corrected X_corr = ln(X_corr)/ln(peptide length) and corrected ΔC_n = (ΔC_n)^½ (27, 32). Sequences, charge states, masses, and peptide identification scores obtained from the SEQUEST algorithm for all identified peptides are included as an excel file in Supporting Online Information. No upper limit was prescribed for the number of missed or non-specific peptide cleavages. However, as tabulated in Supporting Online Material, the vast majority (>92%) of identified peptides contain two tryptic cleavage sites – providing enhanced support that the SEQUEST algorithm combined with the mass resolution of our instrumentation provides accurate peptide assignments. We find that 6% of the identified peptides contain a single tryptic cleavage site. Peptides identified from these tandem MS measurements were used to build an accurate mass and elution time (AMT) database that matches the masses of individual peptide sequences and normalized elution times to permit unique peptide identifications, as previously described (33). The AMT database is necessary for all quantitative changes in the abundances of individual proteins.

Quantitative Measurements of Protein Abundance Changes

Using the AMT database, in stage 2 we use a high-resolution reversed phase capillary chromatography coupled to a high-resolution LTQ-Orbitrap XL MS to quantify changes in the abundance of individual peptides from a consideration of the ratio of ion currents for individual AMT tags, as previously described (34–35). LC-MS spectra are first processed to detect charge and isotopic masses in individual mass spectra using the program Decon2LS (http://ncrr.pnl.gov/software/) and identified spectra are further processed using the VIPER program to calibrate elution times, refine the mass calculation, and match the LC-MS features to AMT tags in the reference database (36). In these latter measurements, the grouped features for each identified peptide are represented by the median value obtained across three LC-MS runs. These data were loaded into the software tool DAnTE for quantitative data analysis, which allows a direct comparison of identified peptides across data sets to analyze and visualize abundance differences (37). Peptide abundances were first log base 2 transformed, and an outlier check was applied by observing the Pearson correlations between datasets. Any prominent outlier dataset that had weak correlations (< 0.7) is excluded from further analysis, as previously described (38). A linear regression based normalization method (available in the program DAnTE) was applied next, within each replicate category. The central tendency adjusted peptide abundances were used to infer the corresponding protein abundances via the ‘Rrollup’ algorithm in DAnTE (37). This tool permitted a determination of protein abundance changes, where the most abundant peptide across all data sets is used as a reference to calculate the ratios of protein abundances. During the Rrollup step the Grubbs outlier test was applied with a p-value cutoff of 0.05 to further remove any outlying peptides. Finally, a t-test identified significant abundance differences, using a p-value< 0.05. Protein abundances are the median of the resulting peptide abundances, where statistical significance is calculated from the ratio of the protein abundances using a cut-off at 5% FDR. IPI protein identifiers obtained from the individual proteins were used for data mining and retrieval using internal cross identifier mappings (i.e., mapping form protein to gene identifiers) and further pathway and functional enrichment data retrieval in conjunction with the Bioinformatics Resource Manager (BRM) software (39). Estimates of total proteome coverage utilized David Bioinformatics Resources (http://david.abcc.ncifcrf.gov/content.jsp?file=citation.htm) to analyze 28 major cellular pathways expected to be present in macrophages (40–41).

RESULTS

Macrophage Isolation and Characterization

Homogenous populations of viable macrophages were isolated from Balb/c mice. Macrophage purity was determined to be greater that 95% of isolated cells through a comparison of FITC-labeled anti-mouse F4/80 macrophage specific antibodies to identify macrophages in comparison to total cells measured using DAPI labeling of double stranded DNA (Figure S1 in Supporting Information). Viability was measured by comparing fluorescence signals associated with cell esterase activity visible upon cleavage of calcein AM in comparison to FITC-labeled anti-mouse F4/80 macrophage specific antibodies, and represents greater than 90% of isolated macrophages following correction for signals associated with uncomplexed FITC-labeled antibody visible even in the absence of added cells.

In vivo Functional Measurements of Macrophage Recruitment

To assess age-dependent alterations in the inflammatory response of macrophages, in vivo macrophage recruitment was measured following injection of an irritant (i.e., sterile Bio-Gel P-100 polyacrylamide beads) into the peritoneal cavity of Balb/c mice. The large 45–90 micron size of the Bio-Gel P-100 polyacrylamide beads avoids possible artifacts associated with phagocytosis and results in the isolation of a population of macrophages that remain largely quiescent in comparison with other elicitation protocols using, for example, thioglycollate (TG) (42–43). The number of macrophages isolated from young (3–4 mo) mice were compared with those isolated from older animals near the average mouse lifespan (14–15 mo) (44). Prior to elicitation, we respectively isolate an average of 1.3 ± 0.2 × 10⁶ and 0.90 ± 0.02 × 10⁶ resident macrophages from each young and old mouse (Figure 1A). The observation that young mice yield more resident macrophages than aged mice is consistent with earlier observations (13). Following elicitation, there are substantial increases in the number of isolated macrophages, equaling an average of 9 ± 2 × 10⁶ and 12.6 ± 0.8 × 10⁶ macrophages respectively isolated from each young and old mouse (Figure 1B); overall yields are not statistically different, which is in agreement with prior reports that indicate age-dependent differences in the yield of resident macrophages disappear following elicitation (13). These results indicate an age-dependent enhancement in the recruitment of macrophages into the peritoneal cavity of aged animals (14 ± 1 fold) in comparison to young control (7 ± 2 fold) to yield equivalent numbers of macrophages. Age-dependent increases in macrophage recruitment may be relevant to understanding previous measurements that have identified age-dependent increases in cellular inflammatory responses and an enhanced resistance of infected mice to introduced bacterial pathogens (10).

Figure 1. Yields and Age-Dependent Functional Changes for Peritoneal Macrophages.

Average yields and variance of isolated peritoneal resident macrophages from young (3–4 mo) and aged (14–15 mo) Balb/c mice before (A) and following (B) challenge with Bio-Gel polyacrylamide beads (elicited response) for three independent experiments involving more than 6 mice in each experiment, where * indicates statistically significant differences based on Student’s t-test (p = 0.05). (C) Nitric oxide production determined by measuring release of the stable nitrite end product using Griess reagent and monitoring absorbance changes at 560 nm based on standard curves calibrated using sodium nitrite as a standard, as previously described (22). (D) Surviving bacterial colonies measured 4 hours after exposure and internalization of S. typhimurium 14028 cells for lysates prepared from 500,000 isolated peritoneal macrophages or RAW 264.7 macrophage cell line (n = 9), where the number of S. typhimurium colonies apparent for lysates prepared from 500,000 macrophages following the 30 minute incubation associated with infection were: 0.9 ± 0.6 × 10⁶ (young), 1.4 ± 0.5 × 10⁶ (old), and 2.4 ± 0.1 × 10⁶ (RAW 264.7). (E) Time-dependence of surviving bacterial colonies measured following lysis of peritoneal macrophages (n = 9) infected with S. typhimurium 14028 cells. Peritoneal macrophages were isolated from young (3–4 mo) (n = 15) and aged (14–15 mo) BALB/c mice (n = 11).

Ex vivo ROS Generation and Bactericidal Activity and

To assess possible age-dependent alterations in macrophage function, we measured nitric oxide production, measured as total nitrite (Figure 1C). We observe increased levels of nitric oxide production during the first two hours following exposure to bacterial endotoxin LPS. These latter results are in agreement with suggestions that increases in the generation of reactive oxygen species by macrophages contribute to age-dependent increases in the oxidative damage to cellular proteins (45). At longer times, comparable levels of nitric oxide are detected irrespective of the age of the donor animals. These observations indicate that the age-dependent increase in nitric oxide production does not represent an intrinsic limitation in the capacity of macrophages isolated from the younger animal to produce nitric oxide. Rather, macrophages isolated from aged animals are highly responsive to activation upon exposure to the bacterial endotoxin LPS.

Additional clarification of functional differences involved measurements of bacterial killing following their phagocytosis, which involves increases in nitric oxide production as part the oxidative burst. These measurements involved assays of the bactericidal response of isolated macrophages following phagocytosis of live bacteria (i.e. Salmonella typhimurium). Macrophages were plated to the same density in all cases (i.e., 500,000 macrophages/well). At various times following phagocytosis macrophages were lysed and colonies that arise from surviving intracellular bacteria were measured using a plate assay, as previously described (25). In comparison to a commonly used macrophage-like cell line derived from Abelson leukemic virus-induced tumors in Balb/c mice (i.e., RAW 264.7 cells), all isolated peritoneal macrophages have a substantially increased bactericidal activity that is indicative of highly coordinated metabolic pathways that are retained in aged animals (Figure 1D). However, macrophages isolated from old mice effectively kill 99.9% of phagocytosed bacteria during the first two hours following infection (Figure 1E). In comparison, macrophages isolated from young control animals kill only 90% of the phagocytosed bacteria. Increased bacterial killing is consistent with observations that there is an enhanced basal level of nitric oxide production and a more rapid response following exposure to LPS. These results, in total, suggest that measurements of protein abundance changes following LPS exposure provide a realistic indication of age-dependent changes in macrophage response pathways.

Protein Identification and Pathway Interrogation

To access mechanisms associated with increased recruitment and pathogen killing in macrophages isolated from aged mice, we have identified expressed proteins using tandem mass spectrometry. To achieve in-depth proteome coverage and to build an AMT database, proteolytically digested macrophage lysates were fractionated using strong anion exchange (SCX) chromatography, permitting the facile identification of 19,055 peptides using a 1% false-discovery threshold (q < 0.01) (Table 1; see Figure S2A in Supporting Information)(27, 32). The 19,055 identified peptides correspond to 6,578 unique peptides that were used to build an accurate mass and elution time (AMT) database useful for quantitative measurements of protein abundance changes (see below), that matches the masses of individual peptide sequences and normalized elution times to permit unique peptide identifications (33). Identified proteins demonstrate, as expected, that pathways involving macrophage function are substantially enriched (Table S1 in Supporting Information).

Table 1.

Tandem MS Identification of Peptides and Proteins^a

MS/MS Identifications	Young		Old
	NT	+ LPS	NT	+ LPS	All Datasets
Total Identified Peptides	3,133	3,992	5,601	6,329	19,055
Uniquely Identified Peptides	2,062	2,379	3,781	4,296	6,578
Uniquely Identified Proteins	901	1,096	1,334	1,459	2,006b
Uniquely Identified Proteins Linked to Immune Responsesc	110 (12.2%)	136 (12.4%)	257 (19.3%)	282 (19.3%)	361c

^aNumber of total and uniquely identified peptides and corresponding proteins from tandem mass spectra from 30 SCX fractions from tryptic digests of lysates from peritoneal macrophages isolated from young or old mice prior to (NT) and following LPS activation, obtained using the 2006 international protein index (IPI) protein database (http://www.ebi.ac.uk.IPI/), as described in Experimental Procedures.

^bIncludes 159 hypothetical proteins not included in listing summarizing 1,847 known proteins that are found to be expressed in macrophages (Table S6).

^cComplete listing of proteins identified as part of macrophage specific pathways are documented in Supplementary Table S5, and involved searching complete listing of identified proteins using the following search terms involving cytoskeleton/motility (search terms: transendothelia migration, cell motion, cell migration and chemotaxis), phagocytosis/signaling (search terms: phagocytosis, ruffle, phagocytic cup, cell projection, and filopodium), antigen presentation/differentiation (search terms: antigen processing and presentation, antigen presentation, cytokine production, macrophage differentiation, complement, classical pathway, alternative pathway, and immunological synapse), and activation/stress response (search terms: immune cell activation, stress response, immune response, MAPKK, activation, macrophage, mast cell, platelet, B cell, T cell, tumor necrosis factor, interleukin, acute inflammatory response, immunologic).

Using gene ontology software to link individual proteins to cellular function, we have identified 1,847 macrophage proteins (see Table S5 in Supporting Materials). Irrespective of aging or macrophage activation, the distribution of proteins within gene ontology categories are very similar (Figure 2A,B). Nevertheless, there are substantial gaps in the proteome coverage, as we identify only 36% of the proteins in highly conserved central metabolic pathways (see Figure S3 in Supporting Information). These results indicate that MS/MS proteomic measurements selectively identify the most abundant proteins, and that increases in the identification of proteins from specific pathways are indicative of increases in the proteins within these pathways. As a result, the substantial (greater than 2-fold) increase in the number of identified proteins linked to immune response pathways for macrophages isolated from aged Balb/c mice, in comparison to young controls, suggests that there is an age-dependent up-regulation of inflammatory pathways in macrophages that is broadly consistent with observed age-dependent increases in inflammatory damage reported for a range of animal models (Figure 2C,D).

Figure 2. Age-Dependent Increases in Protein Identifications for Immune Response Pathways.

Fractional contributions for protein classes within different gene ontology categories (http://amigo.geneontology.org/cgi-bin/amigo/go.cgi) for all 1,847 identified proteins with annotated functions (see Table S6 in Supporting Information) in peritoneal macrophages isolated from young (3–4 mo.) (n = 15) (open) and old (14–15 mos.)(n = 11) (gray) BALB/c mice following cell lysis and mass spectrometric protein identification for macrophages under resting conditions (no treatment) (A) or 2 hours following exposure to LPS (B). Number of proteins and fractional contributions (in brackets) for macrophage response pathways under resting conditions (C, E, G) or following LPS activation (D, F, H). Groupings were binned using gene ontology terms linked to all (total) immune response pathways (C, D) or specific terms relating to either the progressive activation of macrophage specific pathways (E, F) or the classical or alternative pathways of macrophage activation (G, H) (summarized in Table S4 in Supporting Information). Proteins were counted more than once if annotations map onto multiple macrophage response pathways (E, F). Specific response pathways were identified by searching individual protein annotations using the following terms: cytoskeleton/motility (search terms: transendothelia migration, cell motion, cell migration and chemotaxis), phagocytosis/signaling (search terms: phagocytosis, ruffle, phagocytic cup, cell projection, and filopodium), antigen presentation/differentiation (search terms: antigen processing and presentation, antigen presentation, cytokine production, macrophage differentiation, complement, classical pathway, alternative pathway, and immunological synapse), and activation/stress response (search terms: immune cell activation, stress response, immune response, MAPKK, activation, macrophage, mast cell, platelet, B cell, T cell, tumor necrosis factor, interleukin, acute inflammatory response, immunologic).

This is apparent from a consideration of identified proteins linked to antigen presentation as part of the MHC-1 pathway (see Figure S4 in Supporting Information), where substantially more of the proteins linked to the formation of the immunoproteasome necessary for peptide degradation and antigen presentation are detected in macrophages isolated from aged animals (see Table S2 in Supporting Information). Further, there are substantial increases in the number of detected proteasomal subunits following LPS exposure for macrophages isolated from aged mice; in comparison, LPS exposure has essentially no effect with respect to the number of identified proteosomal proteins in macrophages isolated from young animals. As antigen presentation is highly sensitive to macrophage activation (46), these latter results are consistent with enhanced levels of macrophage activation in aged animals. Likewise, there are age-dependent increases in the number of identified proteins associated with immune response pathways within a range of macrophage response pathways, including those linked to cytoskeleton/motility, phagocytosis/signaling, antigen presentation/differentiation, and activation/stress response (Figure 2E,F). Within the list of identified proteins, there are also substantial age-dependent increases in the number of proteins assigned to play a role in classical activation pathway (Figure 2G,H). However, for the majority of detected proteins LPS exposure results in only modest (24–28%) increases in the fraction of identified proteins linked to these immune response pathways that is very similar for macrophages isolated from either young and aged mice, suggesting the involvement of a subset of pathways (e.g., proteasome function) in age-dependent alterations in macrophage function. A similar insensitivity to LPS activation is apparent from a consideration of a range of proteins linked to antigen presentation pathways not involving the proteasome, which appear to be constitutively present irrespective of activation (Table S3). These latter results are consistent with known regulatory control mechanisms in which pathway control typically involves the first committed step of a pathway (in this case antigen presentation as part of the MHC-1 immune response).

Identification of Protein Abundance Changes

Age-dependent differences in the abundance of specific proteins identified in both young and aged animals were resolved using quantitative proteomics using an accurate mass and time tag (AMT) database to compare ion currents of individual peptides (33). In all reported proteins, abundance differences represent averages and standard deviations for more than two unique peptides in each protein, as previously described. For example, in the case of arginase 1, abundance changes during aging involve pair-wise comparisons of ion current for seven different peptides, as fully described in Experimental Procedures (see section entitled Quantitative Measurements of Protein Abundance Changes)(Figure 3). Observed decreases in the peptide abundances for the seven resolved peptides vary from a 99.5% decrease (i.e., REGLYITEEIYK) to a maximal decrease in abundance of > 99.9% (i.e., LKETEYDVRDHGDL). Collectively, the mean decrease in arginase I abundance is 99.7 ± 0.1%.

Figure 3. Age-Dependent Decrease in the Abundance of Arginase I.

Abundance changes of seven individual peptides in arginase from lysates obtained from macrophages isolated from young and aged Balb/c mice (no LPS treatment) were obtained from a consideration of the ratio of their ion currents using AMT reference database (36), where averages and standard deviations are obtained for four technical replicate measurements, as fully described in Experimental Procedures.

Following LPS exposure, there are significant changes in the abundance of 54 proteins, where 90% of affected proteins increase in their abundance in macrophages isolated from aged mice (Figure 4; see Table S4 in Supporting Information). Of these proteins, we observe substantial increases in the abundance of diagnostic proteins linked to nitric oxide generation (e.g., annexin I, aldehyde dehydrogenase 2, and cystatin B) as well as co-regulated antioxidant proteins that are part of the classical activation pathway of macrophage activation (47–51). Further, of the 54 sensitive proteins, 26 have previously been co-localized within intracellular vacuoles associated with the compartmentalization and killing of bacterial pathogens (52). The other 28 proteins that undergo age-dependent changes in abundance are all linked to known pathways associated with the macrophage immune response involving cytoskeletal motility, phagocytosis and signaling, antigen presentation and macrophage differentiation, and activation pathways involving reactive oxygen species and stress responses (see Table S4 in Supporting Information). Thus, age-dependent alterations in macrophage function involving enhanced mobility, increases in the formation of nitric oxide, and higher rates of bacterial killing are all the result of a coordinated up-regulation of normal pathways involving immune cell activation. There is no indication of any dysregulation of normal cellular pathways linked to macrophage activation.

Figure 4. Selective Age-Dependent Increases of Macrophage Proteins Linked to Antimicrobial Responses.

Heat map depictions of protein abundance changes (rows) for resting macrophages (No Treatment) (A) and two hours following LPS exposure (LPS Treatment) (B) for macrophages isolated from aged (14–15 mo) Balb/c mice (right panels) in comparison to macrophages isolated from young (3–4 mo) Balb/c mice (left), comparing 5–6 independent mass spectrometric measurements of protein abundance changes (columns). Color code shown above each heat map represents a logarithmic (base 2) Z-scale commonly used due to the ease of identification of 2-fold changes in abundance (i.e., Z = 1). The dynamic range of the Z-scale varies between 0.0002 (i.e., Z = −12) and 4,096 (i.e., Z = 12). Relationships between identified proteins that undergo age-dependent abundance changes to pathways linked to macrophage activation and antimicrobial responses are shown to the right of each heat map (A, B) and are summarized (C, D) to indicate total numbers of proteins in each pathway that are up-regulated (gray) or down-regulated (black) in resting macrophages (C) or following LPS exposure (D). Proteins in heat maps are listed according to the magnitude of the abundance change within each identified pathway and are in the same order as in Table 5, which indicate average abundance changes and describe protein functions. Age-dependent protein abundance changes were mapped onto antimicrobial response pathways, depicting decreases (green) or increases (red) in protein abundances for resting macrophages (E, no LPS treatment) or two hours following exposure to bacterial endotoxin (F, LPS treatment) according to different cellular pathways known to be sequentially up-regulated as part of the antimicrobial response. Indicated antimicrobial pathways involve (left-to-right) abundance changes of cytoskeletal proteins linked to motility, increases in phagocytosis and signaling linked to bacterial sequestration, antigen presentation for sustained activation of T-helper cells, and final activation involving cytokine production, oxidative stress responses, and apoptosis. Proteins previously identified in Salmonella containing vesicles (SCV) linked to bacterial killing are indicated (* or **), where (**) indicates that proteins were observed only following infection (52). (G) Cartoon summarizing age-induced phenotypic shift from alternative activation pathways (high arginase activity) to classical activation pathways (high nitric oxide levels). Cartoon is consistent with tandem MS identification of key enzymes involving arginine uptake, utilization, or recycling detected only in macrophages isolated from aged animals, including the cationic amino acid transporter associated with arginine uptake (entrez 11987), argininosuccinate lyase (As1; entrez 109900), arginine-glycine amidinotransferase (AGAT; entrez 11987), and arginosuccinate synthetase (Ass1; entrez 11898) (only following LPS activation). Enhanced S-adenosyl methionine levels resulting from AGAT activity protect macrophages against oxidative stress to decrease rates of apoptosis. Quantitative AMT data identifies a 99% decrease in arginase (entrez 11846 and 11847) abundance. Ornithine is metabolized to citrulline by ornithine carbamoyltransferase (within the urea cycle), not detected by tandem MS.

Prior to macrophage activation by bacterial endotoxin LPS a smaller number of proteins change their abundance in macrophages isolated from aged mice, involving the down-regulation of 11 proteins and the up-regulation of 12 proteins (Figure 4). Of particular interest is the large decrease in the abundance of arginase I in macrophages isolated from aged animals (Figure 3), whose expression is under the control of cytosolic interleukins (such as IL-4) that act to induce a phenotypic switch that favors an alternative activation pathway (3). This latter result is consistent with earlier observations that have demonstrated age-dependent declines in the ability of T helper (CD4) cells to mount antigen-specific Th2 responses involving the release of IL-4 and other cytokines that promote this alternative pathway of macrophage activation (10). Rather, T helper cells in aged animals primarily exhibit a Th1 phenotype involving the release of inflammatory cytokines IFNγ, IL-2, and TNFα that promote classical pathways of macrophage activation that enhance generalized antimicrobial responses involving the up-regulation of iNOS. These latter results are consistent with the functional data demonstrating higher levels of nitric oxide production in the resting state of aged macrophages as well as a hypersensitivity of macrophages isolated form aged animals to activation by LPS (Figure 1C).

DISCUSSION

We report an age-dependent up-regulation of coordinated pathways normally associated with macrophage activation, which provides a strong indication that increases in macrophage sensitivity to activation is fundamental to observed age-dependent changes involving innate immunity. These results do not support models involving genetically programmed cellular changes during aging that might be causal in the induction of a pathological state that involves a loss of coordination between normal pathways. Rather, our results support prior suggestions that “many of the aberrant responses seem to be dependent on the host environment, the milieu in which the cells reside, and might not be entirely dependent on the innate immune cells themselves” (53). Consistent with this model, our data suggests that environmental effects associated with immune cell exposure act to sensitize macrophage to induce a chronic inflammatory response that is consistent with the vast majority of data concerning age-dependent phenomena. Apparent contradictory results in the literature can be understood in terms of differences in environmental exposures.

The data presented here represents the first global assessment of protein abundance changes of the aging process in macrophages, demonstrating an up-regulation of immune pathways in macrophages isolated from aged mice. Prior to the identification of age-dependent changes in the macrophage proteome, we first confirmed prior observations of in vivo and ex vivo functional differences between peritoneal macrophages isolated from young (3–4 mo) and aged (14–15 mo) Balb/c mice (Figure 1). Observed age-dependent functional changes involving increases in: i) macrophage recruitment following elicitation, ii) basal rates of nitric oxide generation that result in a reduction in the time needed for full activation of senescent macrophages following LPS exposure, and iii) bactericidal activity following phagocytosis of Salmonella are all in agreement with prior observations, as well as suggestions that age-dependent increases in the generation of reactive oxygen species by macrophages contribute to age-dependent increases in oxidative damage to a range of biomolecules (9–10, 13, 45). Using these characteristic macrophage samples, it is therefore possible to employ proteomic measurements that identify specific proteins and related pathways to investigate fundamental mechanisms responsible for age-dependent changes in macrophage function.

To quantitatively address possible changes in immune specific pathways, protein abundance changes were measured using the quantitative AMT-tag approach. We observe age-dependent abundance changes of 77 proteins known to play central roles in promoting macrophage activation (Figure 4; Table S4 in Supporting Information). Virtually all identified proteins that undergo age-dependent abundance changes map onto normal processes associated with macrophage activation in terms of processes (Figure 4) or cellular locations (i.e., 26 of the 54 proteins whose abundance is altered during aging following LPS exposure co-localize with intracellular vacuoles associated with bacterial killing, a process linked to macrophage activation) (52). As reflected in the heat maps in Figure 4, observed abundance differences are highly reproducible (Figure 3; see tabulated errors in Table S4). Of particular interest is the large 99% decrease in the abundance of arginase I in macrophages isolated from aged animals, whose expression is under the control of cytosolic interleukins (such as IL-4) that act to induce a phenotypic switch that favors an alternative activation (Th2-like) pathway. These latter results are consistent with substantial increases in the abundance of diagnostic proteins linked to nitric oxide generation (e.g., annexin I, aldehyde dehydrogenase 2, and cystatin B) as well as co-regulated antioxidant proteins that are part of the classical (Th1-like) macrophage activation pathway. The vast majority of the other proteins that undergo age-dependent changes in abundance are linked to known pathways associated with the macrophage immune response involving cytoskeletal motility, phagocytosis and signaling, antigen presentation and macrophage differentiation, and activation pathways involving reactive oxygen species and stress responses (Figure 4) that are consistent with observed age-dependent changes in macrophage function (Figure 1).

Specific examples of the mechanistic relationship between the quantitative AMT data and macrophage function include the identification of increases in the abundance of central proteins linked to normal pathways involving macrophage activation (see Table S4 in Supporting Information). Consistent with age-dependent increases in macrophage elicitation and trafficking, we observe abundance increases in major structural proteins necessary for cytoskeletal motility that include actin, specific myosin isoforms linked to motility, tubulin, and macrophage capping protein (known to be critical to resist infection). Likewise, age-dependent increases in bactericidal activity are consistent with quantitative increases in the abundance of key proteins that have previously been co-localized within intracellular vacuoles associated with bacterial killing (52). These proteins include moesin (linked to TNF production), coronin (actin binding protein linked to phagosome formation), and transgelin (actin binding protein that suppresses MMP-9 and whose expression is triggered by TNF). Key proteins indicative of the up-regulation of activation pathways associated with antigen presentation and macrophage polarization resulting from classical activation pathways include filamin alpha (initiates actin polymerization to reorganize cytoskeletal complexes that promotes MAPK-dependent signaling and ERK phosphorylation), cathepsin D (endopeptidase in lysosomes that promotes apoptosis through activation of caspase 8), histocompatibility-2 (surface glycoprotein linked to antigen processing and presentation via MHC-1 immune response), and heat shock protein 8 (involved in protein transport to endoplasmic reticulum critical for antigen presentation). Observed age-dependent increases in nitric oxide generation are consistent with abundance changes for proteins linked to the respiratory burst and adaptive macrophage functions involving chaperones and antioxidants that are critical to macrophage activation, which include arginase I (decreases in abundance are diagnostic of classical activation pathway, as arginase depletes arginine to inhibit nitric oxide production by NOS), cathepsin B (increases in cysteine protease linked to antigen degradation and inflammatory processes associated with trafficking TNF containing vesicles to membrane), arachidonate-5-lipoxygenase activating protein (downstream of Toll-receptors this protein plays a key in forming leukotrienes linked to inflammatory responses), annexin A2 (promotes tyrosine kinase activation), lymphocyte cytosolic protein (actin binding protein critical to adhesion-dependent respiratory burst), and aldehyde dehydrogenase (antioxidant protein linked to nitric oxide production). Collectively, these results indicate a coordinated increase in normal pathways involving macrophage activation, and do not support models that emphasize a dysregulation of normal cellular pathways linked to macrophage activation (3).

Observed age-dependent alterations in macrophage function are consistent with known age-dependent changes in the adaptive immune system, which involve shifts in T-helper cellular responses that favor the production of the inflammatory cytokines IFNγ, IL-2, and TNFα (i.e., Th1 response) (10). Such soluble factors induce the activation of resting macrophages to enhance nitric oxide production, antigen presentation, enhanced cytokine biosynthesis, and phagocytosis (54). Thus age-dependent increases in the resistance of macrophages to pathogens following their phagocytosis (Figure 1D,E)(9), as well as the increased resistance of old Balb/c mice themselves to introduced infections (10), are consistent with well-documented age-dependent changes involving population shifts in T-lymphocytes to favor Th1 inflammatory responses. Apparent contradictory results in the literature, which detect both increases and decreases in macrophage function (9, 14–15, 55–57), can be understood in terms of environmental factors and assay conditions that uncover age-dependent differences in macrophage sensitization to activating signals (e.g., LPS). For example, increases in nitric oxide levels, routinely observed upon challenge of elicited peritoneal macrophages by bacterial endotoxin (14) (Figure 1C), are abolished if macrophages are first uniformly sensitized by IFNγ exposure prior to endotoxin challenge (8). Likewise, increases in the bacterial resistance of aged mice are dependent on environmental exposures to normal pathogens, and are lost when mice are housed in sterile (clean room) conditions (10). These observations are consistent with the considerable phenotypic heterogeneity present within macrophage populations, which is modulated in response to environmental conditions, which include responses to the generation of inflammatory cytokines (e.g., INF-γ, IL-2, and TNFα) and other soluble factors from T-lymphocytes that act to induce changes in the macrophage proteome and differentiation (58–59).

What is currently unclear in the etiology of immune dysregulation during aging is the role that macrophages may play in promoting long-term shifts in T-lymphocyte population heterogeneity. Understanding the causal relationships that lead to age-related changes in the immune system requires an understanding of the functional coupling between the innate and adaptive immune systems. Specifically, antigen presenting cells (i.e., macrophages, dendritic cells, and B cells) inform and amplify adaptive immune system responses and have the potential to induce shifts in innate and adaptive immunity characteristic of that seen in aged animals. For example, maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection is central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. In aged animals macrophages exhibit a hyper-responsive and coordinated initiation of classical macrophage activation pathways, which enhance nitric oxide production and bactericidal activity to minimize infection from general microbial threats (9–10), which are likely to be exacerbated by age-dependent decreases in the efficacy of physical barriers that limit pathogen entry to enhance environmental exposures. Upon pathogen recognition, macrophages release cytokines (e.g., IL-12) to induce T-helper cell differentiation to favor a Th1 response and release of inflammatory cytokines by T-helper cells that is associated with macrophage activation and T-helper cell proliferation in response to pathogen entry (Figure 5). Cross-talk with T-helper cells thereby amplify inflammatory signals that promote macrophage activation in response to age-dependent decreases in physical barriers to pathogen entry that enhance exposures and further modify adaptive immunity through responses that originate in the innate system. These results suggest that therapies aimed to alleviate immune system dysregulation should include strategies aimed at neutralizing these amplification cascades between macrophage and T-lymphocytes, involving key targets of macrophage function that include not only inflammatory cytokines but also key signaling pathways involving macrophage activation, and pathways associated with antigen presentation.

Figure 5. Aging Induces Persistent Macrophage Activation and Reprogramming of Adaptive Cellular Response.

Age-dependent disruptions in physical barriers increase rates of pathogen entry, which upon recognition by macrophages (e.g., via Toll-like receptors) induce cytokine release (e.g., IL-12) to induce T-helper cell differentiation and Th1-responses involving the release of inflammatory cytokines (IFNγ and IL-2). IFNγ exposure enhances macrophage activation in the presence of bacterial antigens (e.g., LPS) and down-regulates Th2 cellular responses by T-helper cells. IL-2 release by T-helper cells induces their proliferation favoring Th1 cellular responses and activation of cytotoxic T-cells (CTL), thereby amplifying innate immune response and killing of infected (antigen presenting) macrophages.