Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0307158 on September 12, 2007

Published online before print September 12, 2007
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(Journal of Leukocyte Biology. 2007;82:1592-1604.)
© 2007 by Society for Leukocyte Biology

Transcriptional profiling of human monocytes reveals complex changes in the expression pattern of inflammation-related genes in response to the annexin A1-derived peptide Ac1-25

Carsten Lange, Diane J. Starrett, Julia Goetsch, Volker Gerke and Ursula Rescher1

Institute of Medical Biochemistry, Centre for Molecular Biology of Inflammation, and Interdisciplinary Clinical Research Centre, University of Muenster, Muenster, Germany

1Correspondence: Institute of Medical Biochemistry, Centre for Molecular Biology of Inflammation, Von-Esmarch-Str. 56, 48149 Muenster, Germany. E-mail: rescher{at}uni-muenster.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Annexin A1 is a glucocorticoid-regulated, anti-inflammatory protein, which plays an important role as an endogenous regulator of the inflammatory response. Many of these anti-inflammatory properties are retained in the N-terminal annexin A1 peptide Ac1-25, which is released from the full-length protein by a neutrophil elastase. To elucidate whether the anti-inflammatory activity of the bioactive peptide is solely a result of immediate post-translational effects, which include the shedding of L-selectin or also involve transcriptional changes affecting leukocyte function, we recorded global gene expression changes in human monocytes stimulated with exogenously applied Ac1-25. Applying stringent selection criteria, we show that ~100 genes are up-regulated, and ~230 are down-regulated by a factor of at least two in the Ac1-25-treated monocytes. It is important that the profiling reveals that Ac1-25 induces an anti-inflammatory phenotype by down-regulating proinflammatory and up-regulating anti-inflammatory mediators. These effects, elicited by exogenously applied Ac1-25, depend, to different extents, on ERK1/2 and p38 signaling pathways. This identifies the annexin A1 N-terminal peptide as a stimulus, eliciting not only short-term, post-translational effects in human monocytes but also transcriptional changes, defining a more anti-inflammatory profile.

Key Words: gene regulation • anti-inflammation • endogenous mediator • IL-1Ra • CCR2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inflammation, the standard reaction of the organism in response to any disturbances of vascularized tissue, can be initiated by infection or trauma or in autoimmune diseases. The complex inflammatory scenario is characterized by the recruitment of leukocytes of the innate immune system to the site of inflammation. As acute inflammation is accompanied by destruction of the surrounding host tissue, tight spatial and temporal control of the duration and severity of the proinflammatory phase is required to ensure its transient nature and therefore, its host-protective function. Several proinflammatory mediators such as cytokines and chemokines as well as several types of adhesion molecules ensure a tight regulation at the onset of inflammatory responses. In the subsequent resolution phase, infiltrating phagocytic macrophages clear apoptotic and damaged cells, thereby limiting inflammation, and the tissue returns to its functional state [1 ]. Excessive inflammation with impaired resolution, i.e., an imbalance between proinflammatory signals, which initiate and maintain inflammation, and signals, which terminate the process, leads to the development of chronic inflammation [2 ].

One of the first proteins identified to be induced by glucocorticoids, which are immunosuppressive and anti-inflammatory agents widely used for the suppression of inflammation, is annexin A1, formerly known as lipocortin I [3 , 4 ]. Annexin A1, which is expressed abundantly in neutrophils and monocytes, is an endogenous, anti-inflammatory mediator that plays an important role in the down-regulation of the host response, ensuring that the inflammatory reaction is of transient nature. Its pharmacologic property of reducing inflammation has been reported in many models of acute inflammation in animals and in vitro, where it inhibits neutrophil transendothelial migration [5 , 6 ]. The importance of annexin A1 as an endogenous, anti-inflammatory mediator and key regulator of leukocyte extravasation is strongly supported by the observation that annexin A1-deficient mice show an exacerbated, inflammatory response in models of acute and chronic inflammation [7 , 8 ].

The unique N-terminal part of annexin A1, which is probably released from the full-length protein through the proteolytic activity of human leukocyte elastase [9 ], has been shown to exert the anti-inflammatory activity. Moreover, a peptide corresponding to this region, Ac1-25, also known as Ac2-26, when amino acid numbering is based on the cDNA and not the processed protein sequence, acts as mimetic, retaining the full pharmacologic property of the whole protein when exogenously applied in all experimental studies [10 , 11 ]. The recent identification of annexin A1 as a novel, endogenous ligand for all known members of the formyl peptide receptor (FPR) family of chemoattractant receptors [12 13 14 ], together with the enhanced annexin A1 expression following treatment with the glucocorticoid dexamethasone, underscore its important role as an endogenous, anti-inflammatory mediator [15 ].

Investigating the function of the N-terminal annexin A1 peptide Ac1-25 in the control of inflammation constitutes an approach to better understand the way an organism controls inflammation, which might eventually lead to the development of new compounds mimicking the physiological activity of an endogenous, anti-inflammatory mediator. The present study was undertaken to elucidate if the anti-inflammatory effects elicited by exogenously applied Ac1-25 are solely a result of immediate effects at the post-translational level, such as the inhibition of leukocyte transendothelial migration, or if Ac1-25 treatment also has effects on the transcriptional profile, thereby eliciting longer lasting changes in leukocyte biology. Therefore, we sought to reveal possible anti-inflammatory activities of Ac1-25 on the level of gene expression by using cDNA microarrays, systematically assessing gene regulation in response to Ac1-25 stimulation of human peripheral blood monocytes. Comparison of global gene expression profiles in combination with real-time RT-PCR and flow cytometry revealed that Ac1-25 initiated profound changes in the expression of genes known to play critical roles in the inflammatory process. Also, we were able to show that activation of ERK- and p38-MAPK signaling pathways, which are involved in many physiological processes in mammalian organisms, including most notably, all aspects of immune responses (for an overview, see ref. [16 ]), is involved, to different extents, in mediating the gene expression changes induced by the exogenously applied N-terminal annexin A1 peptide Ac1-25.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Stimulation of monocytes with the annexin A1 peptide
Human peripheral blood monocytes were isolated from buffy coats using Ficoll-Paque gradient centrifugation, essentially as described [17 ]. In brief, density centrifugation was performed using Ficoll separation solution with a density of 1.077 g/ml (PAA Laboratories, Cölbe, Germany) to isolate mononuclear cells. In a second round of centrifugation, monocytes were enriched using Percoll separation solution with a density of 1.139 g/ml (Amersham Biosciences, Freiburg, Germany). After washing, the cells were cultured overnight in McCoy’s 5A medium supplemented with 10% FCS (Biochrom, Berlin, Germany), 100 units/ml penicillin/streptomycin, and 2 mM L-glutamine (BioWhittaker, Verviers, Belgium) in Teflon bags [18 , 19 ] to prevent firm adherence and subsequent activation. Apoptosis levels were consistently low after overnight culturing, as determined by flow cytometrical analysis of annexin V-stained cells. Monocyte purity was routinely >80% when assessed by counting in a Coulter Counter Z2 (Coulter, Krefeld, Germany).

The annexin A1 peptide (Ac1-25, N-acetyl-AMVSEFLKQAWFIENEEQEYVQTVK), which was acetylated during synthesis to mimic the physiological situation, was purchased from Advanced Biotechnology Centre (London, UK). The amino acid numbers 1–25 correspond to the sequence in the actual protein, which is N-terminally processed by removal of the initiating methionine and subsequent N-terminal acetylation, isolated from tissues [20 ]. Thus, this peptide is identical with a peptide, termed Ac2-26, where numbering is based on the amino acid sequence predicted by the nonprocessed cDNA.

The integrity and sequence of the peptide were verified routinely by mass spectrometry. To exclude effects as a result of bacterial LPS contamination, annexin A1 peptide-mediated shedding of L-selectin and activation of ERK1/2 were assayed in the presence of the LPS inhibitor polymyxin B (Sigma-Aldrich, Munich, Germany) with LPS (Sigma-Aldrich) as a positive control. In addition, monocyte experiments were reproduced with Ac1-25 from different independent syntheses to exclude possible effects of potential synthesis variations and impurities.

Monocytes were washed with PBS and then incubated in McCoy’s with 10% FBS (PAA Laboratories), 2 mM glutamine, penicillin and streptomycin (PAA Laboratories) at 37°C, and 5% CO2 for 2 h (real-time PCR and microarray hybridizations, apoptosis assay) or 6 h (ELISA and CCR2 measurements) in the presence or absence of 50 µM Ac1-25. Stimulations were carried out under the above conditions to mimic those used previously, e.g., in studies examining the influence of the peptide on neutrophil adhesion to endothelial cells [21 ] or in chemotaxis assays [12 ].

MAPK activation and inhibition
Monocytes (5x106 cells per 250 µl reaction) were preincubated with medium containing 10 µM ERK1/2 inhibitor UO126 (Promega, Mannheim, Germany), 20 µM p38 inhibitor SB203580 (Merck Biosciences, Darmstadt, Germany), or vehicle control for 30 min at 37°C. Cells were then stimulated with 50 µM Ac1-25 or vehicle control for different periods of time as indicated. To exclude LPS contamination, cells were preincubated with 25 µg/ml LPS inhibitor polymyxin B for 5 min and then stimulated for 15 min with 100 ng/ml LPS or for 5 min with 50 µM Ac1-25. Cells were lysed with hot Laemmli sample buffer containing β-ME. Lysates were heated at 100°C for 10 min and briefly sonicated, and extracted proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Following blocking (5% milk powder/TBST), membranes were incubated overnight at 4°C with primary antibody [mouse monoclonal phospho-p44/42 or mouse monoclonal phospho-p38 (Cell Signaling Technology, Danvers, CT, USA), diluted 1:2000 in 5% milk powder in TBST]. To ensure equal loading, blots were stripped and reprobed for total ERK1/2 (rabbit polyclonal p42/44, Cell Signaling Technologies) or total p38 (mouse p38 mAb, Santa Cruz Biotechnology, Heidelberg, Germany). Immunoreactive bands were visualized according to standard ECL protocols.

RNA extraction and DNA microarray hybridization
Total cellular RNA was isolated from monocytes, which were exposed to the annexin A1 peptide Ac1-25 for 2 h, as opposed to medium as control (RNeasy kit, Qiagen, Hilden, Germany). RNA was quantified by UV spectrophotometry and checked for integrity by agarose gel electrophoresis. Samples for microarray hybridization were prepared according to the manufacturer’s instructions, and 6 µg fragmented cRNA was hybridized to Affymetrix Human Genome U133A gene chip arrays (Affymetrix, Santa Clara, CA, USA), which were washed and stained using the Gene Chip Fluidics Station 400 (Affymetrix). Fluorescence signals were detected with the HP G2500A gene array scanner (Affymetrix).

Statistical analysis of microarray data
Data were processed by MicroArray Suite Software 5.0 (Affymetrix). Signals were scaled to a target intensity of 100 and log-transformed. Change P values (statistical significance for change calls) and n-fold changes in expression induced by Ac1-25 treatment were calculated using hybridizations of RNA from medium-treated cells as baseline controls. Average change P values and fold changes were calculated from three independent experiments.

In addition, a more sophisticated statistical analysis of the Affymetrix intensity files was carried out with the Expressionist Pro 2.0 Suite software package (GeneData, Basel, Switzerland). Preprocessing of the Affymetrix arrays used GeneData Refiner software to correct for variations in hybridization intensity because of gradient effects or scratches. GeneData Expressionist Analyst then identified significant gene expression differences, and the n-fold changes were determined and screened for their significance applying a t-test. Only genes showing a P value of less than 0.05 and a fold change of more than 2.0 were retained.

Functionally related genes, which were expressed differentially in Ac1-25-stimulated versus control-treated cells, were also identified using Expressionist Analyst. The genes were classified according to their biological function gene ontology (GO) Level 7 category, and Fisher’s Exact test was performed, revealing statistically over-represented GO categories. Level 7 GO annotation was chosen, as higher GO levels yielded too few genes per GO branch, resulting in seemingly random GO terms found to be over-represented, and classification using Level 6 or lower annotation resulted in reduced statistical significance.

The genes present in the resulting union groups were then assigned to distinct biological processes.

We also used Database for Anotation, Visualization and Integrated Discovery (DAVID 2006 [22 ]) for independent verification of the found GO categorization of genes. Here, we classified the genes according to their Level 3 biological process annotation.

Real-time quantitative PCR
Monocytes were exposed to the annexin A1 peptide Ac1-25 or to medium in four independent experiments. In experiments examining the influence of the MEK/ERK- and p38-signaling pathways on gene expression, prestimulation with the corresponding inhibitors was carried out as stated above. After RNA isolation, RT was performed using 6 µg total RNA and 100 pmol/µl T7-(dT)24 primer (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTT-3'). The reaction was heated to 70°C for 10 min and put on ice prior to the addition of SuperScript II RT, 2 mM dNTPs, 10 mM DTT, and reaction buffer (all reagents: Invitrogen, Karlsruhe, Germany) to a total volume of 20 µl. The reaction was incubated at 42°C for 1 h. cDNA synthesis was terminated by inactivation for 15 min at 70°C, and RNA degraded by RNase H treatment (Invitrogen) for 20 min at 37°C.

The cDNA preparations were then subjected to real-time PCR using an ABI Prism 7900 HT RT-PCR system. Reactions contained 1x Platinum SYBR Green qPCR Supermix-UDG with 6-carboxy-X-rhodamine reference dye (Invitrogen), 15 ng–1 µg cDNA in several serial dilutions, and 340 nM each of the 5' and 3' primers specific for the following genes of interest: CCR2 (sense 5'-GCAGTGAGAGTCATCTTCACCATC-3', antisense 5'-TCTCCCCAACGAAGGCATAG-3'), dominant-negative mutant 3 (DNM3; sense 5'-AGTTGATCCTCAAGGTCTGAGAACC-3', antisense 5'-CCCATTCGGTCAGCGATATG-3'), fatty acid-binding protein 5 (FABP5; sense 5'-GGTGGACAGCAAAGGCTTTG-3', antisense 5'-GCCATCAGCTGTGGTTTCTTC-3'), forkhead box O3A (FOXO3A; sense 5'-AGCACAGAGTTGGATGAAGTCCA-3', antisense 5'-GGAGCGTGATGTTATCCAGCA-3'), gremline1, cysteine knot superfamily, homolog (GREM; sense 5'-CCATCATCAACCGCTTCTGTTAC-3', antisense 5'-TGCAACGACACTGCTTCACAC-3'), homer homolog 1 (HOMER1; sense 5'-TCAGGAATTTAAAGAAGCTGCTCG-3', antisense 5'-GATTGCTGAACTATGTGAAAATGGC-3'), IL-1R antagonist (IL-1Ra; IL1RN; sense 5'-CCAGCTGGAGGCAGTTAACATC-3', antisense 5'-TCGTCCTCCTGGAAGTAGAATTTG-3'), inhibin, β A (INHBA; sense 5'-GCCGTCAAGAAGCACATTTTAAAC-3', antisense 5'-CAAACGTGATGATCTCCGAGGT-3'), plekstrin homology-like domain, family A, member 1 (PHLDA1; sense 5'-CTGGAAGAAAAAGTGTTGCATCC-3', antisense 5'-ATGTACTTGCCCTTGCGCTC-3'), platelet-activating factor receptor (PTAFR; sense 5'-GTTCATCATCTGCTTCGTGCC-3', antisense 5'-CTTTTCGGTGAGGTGCTTGC-3'), ral guanine nucleotide dissociation stimulator-like 1 (RGL1; sense 5'-GATCAGGAATGCAATCGCTTC-3', antisense 5'-GAAGCCCATTGTCAGTTTCCAC-3'), regulator of G-protein signaling 13 (RGS13; sense 5'-AGGCCCCCTTCAAACCTTAC-3', antisense 5'-TTCTTTGCCCTAGAAATTCTGCTC-3'), Ras-induced senescence 1 (RIS1; sense 5'-TCCAATGCTTCAGTCAACGC-3', antisense 5'-TTGGTGGAGAAGAGCAGTAGGTC-3'), TLR4 (sense 5'-TCCATTTCAGCTCTGCCTTCA-3', antisense 5'-ACTGCCAGGTCTGAGCAATCTC-3'), TNF-{alpha}-induced protein 2 (TNFAIP2; sense 5'-CGCGCCTTTAATGAATTTCTG-3', antisense 5'-CTCATGCAGGTTCTGGAGCA-3').

The following PCR conditions were used: 50°C for 2 min, then 95°C for 2 min, followed by 40 cycles at 95°C/15 s and 60°C/60 s. For each pair of primers, a template concentration was chosen, which yielded a comparative threshold (Ct) value between 25 and 30. All RT-PCR reactions were carried out in duplicate.

The expression level of the genes of interest was normalized to GAPDH controls (sense 5'-CTTCATTGACCTCAACTACATG-3', antisense 5'-TGTCATGGATGACCTTGGCCAG-3') using the 2–{Delta}{Delta}Ct method [23 ]. All primers were designed using the Primer Express software (Applied Biosystems, Darmstadt, Germany) and synthesized by Proligo (Paris, France).

Effects of MAPK inhibition on the annexin A1 peptide-induced changes in gene expression, observed in the four independent experiments, were averaged and categorized according to the following criteria: (o), below 10%; (+), more than 10%; (++), more than 30%; (n.a.), inhibition alone altered the expression level profoundly.

Flow cytometry
Data analysis was based on examination of 10,000 cells per sample on a FACSCalibur cytometer (BD Biosciences, Heidelberg, Germany). Integrated mean fluorescence values were corrected for values with control IgGs. Average mean fluorescence values ± SEM were calculated from at least three independent experiments, each using independently isolated and stimulated monocytes, and statistical significance of differences was evaluated by unpaired t-test.

For determination of cell surface L-selectin [CD62 ligand (CD62L)], 1 x 106 cells per sample were resuspended in 200 µl McCoy’s medium in 96-well plates and stimulated with 100 ng/ml LPS or 50 µM Ac1-25 for 15 min at 37°C, with or without a 5 min pretreatment with the LPS antagonist polymyxin B. Subsequently, cells were washed with ice-cold PBS (supplemented with 1% BSA) and incubated for 1 h with PE-CY5-anti-human CD62L antibody (BD PharMingen, Heidelberg, Germany). Cells were then washed three times, resuspended, and analyzed.

For analysis of the cell surface expression of CCR2, cells were washed, resuspended in PBS containing 3% BSA to minimize unspecific binding, and labeled with 10 µg/ml polyclonal rabbit antibody against CCR2 (Abcam, Cambridge, UK) or 10 µg/ml rabbit Igs (Dianova, Hamburg, Germany) for 45 min. Cells were then washed twice, incubated for 30 min with a goat anti-rabbit, FITC-conjugated, secondary antibody (Dianova) in the dark, washed again, and analyzed for fluorescence.

Quantification of IL-1Ra levels
Protein levels of the IL-1Ra were measured by sandwich ELISA in Ac1-25-stimulated cells and vehicle-treated control cells, according to the manufacturer’s recommendations (BioCat, Heidelberg, Germany). The assays were carried out in triplicate; they did not discriminate between the different isoforms of IL-1Ra. Statistical significance of the results was evaluated by unpaired t-tests.

Chemotaxis assay
Chemotactic response of monocytes toward recombinant human CCL2/MCP-1 was assessed as described previously [12 ] using a microchemotaxis chamber (Neuroprobe, Gaithersburg, MD, USA) with the two compartments separated by a 10-µm pore-size filter. The lower wells were filled with McCoy’s medium alone or 10 nM MCP-1/CCL2 (Calbiochem, La Jolla, CA, USA) in McCoy’s medium. Monocytes (1x106/ml) pretreated for 5 h with medium alone or with the annexin A1 peptide in medium were then placed in the upper wells and allowed to migrate for 90 min at 37°C and 5% CO2. Subsequently, the filters were fixed and stained with DiffQuick (Dade Behring, Marburg, Germany), according to the manufacturer’s protocol, and the migrated cells were counted. The chemotactic index was determined from the number of cells migrating in response to MCP-1/CCL2 compared with the number of cells migrating spontaneously, i.e., in the absence of chemoattractant. Independent experiments using individual preparations of monocytes were performed at least three times with triplicate wells. Statistically significant differences were evaluated by unpaired t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ERK1/2- and p38-signal transduction pathways mediate short-term, anti-inflammatory effects of the bioactive annexin A1 peptide Ac1-25
To examine the role of MAPKs in the transduction of signals elicited following exogenous application of the bioactive annexin A1 peptide Ac1-25, we stimulated human peripheral blood monocytes with 50 µM Ac1-25 for different time periods and probed for p42/44 (ERK1/2) and p38 phosphorylation as an index of activation. Whereas in unstimulated control cells, no elevated phosphorylation levels of ERK1/2 or p38 were found at the time-points indicated (data not shown), ERK1/2 was phosphorylated quickly upon Ac1-25 stimulation, the peak phosphorylation levels were reached after 5 min, and a decreased but detectable phosphorylation level was retained even 3 h after addition of the peptide (Fig. 1A ). p38 phosphorylation levels were also affected but with different kinetics, rising within 3 min after Ac1-25 stimulation, but quickly returning to base levels after reaching a maximum at 10 min poststimulation (Fig. 1B) .


Figure 1
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  		Figure 1. Annexin A1 (Ac1-25)-mediated activation of monocytes. Cells were stimulated with 50 µM Ac1-25 or vehicle control for different periods of time as indicated. The activation kinetics of ERK1/2 (A) and p38 (B) MAPKs after Ac1-25 stimulation was examined by Western blot analysis using phospho-ERK (pERK)- or phospho-p38 (pp38)-specific antibodies. To ensure equal loading, blots were stripped and reprobed using antibodies against ERK and p38. Representative Western blots for at least three separate experiments are shown. (C) To ensure the specificity of annexin A1 activation, ERK1/2 phosphorylation following Ac1-25 treatment was assessed in monocytes pretreated with the LPS antagonist polymyxin B. (D) Cell surface L-selectin (CD62L) levels of monocytes were determined by flow cytometry. Cells were preincubated with polymyxin B or vehicle control for 5 min at 37°C, stimulated with LPS or Ac1-25 for 15 min at 37°C, and stained with PE-CY5-anti-human CD62L antibody. FACS analysis was based on examination of mean fluorescence of 10,000 cells per sample. Mean fluorescence intensity of CD62L on the cell surface was calculated as a percentage of untreated control cells. Bars represent results of four independent experiments ± SEM; *, statistical significance of P < 0.05.

 
To elucidate whether this MAPK activation is specific for Ac1-25 and to rule out that monocytes were activated through contamination of the peptide syntheses with the bacterial endotoxin LPS, we determined ERK1/2 activation following stimulation with Ac1-25 or LPS in the presence or absence of the LPS inhibitor polymyxin B. Signal transduction after LPS stimulation has been shown to include MAPK activation [24 ]. As shown in Figure 1C , polymyxin B did not block annexin A1 peptide-mediated ERK1/2 activation, but LPS-mediated ERK activation was inhibited totally. To further confirm the specificity of monocyte activation through Ac1-25, we investigated the regulation of cell surface L-selectin (CD62L) upon challenge with Ac1-25 or LPS in the presence and absence of polymyxin B (Fig. 1D) . Ac1-25 stimulation led to a decrease in the cell surface L-selectin levels down to 49% of the medium control, corroborating previous data about annexin A1-induced L-selectin shedding [13 , 25 ]. Pretreatment with the LPS antagonist polymyxin B inhibited LPS-induced L-selectin shedding completely, whereas the Ac1-25-induced L-selectin shedding was not affected (Fig. 1D) . Taken together, these results indicate that MAPK signaling is critically involved in mediating rapid, post-translational responses to Ac1-25 challenge in human monocytes.

The annexin A1 peptide Ac1-25 induces gene expression changes in peripheral blood monocytes
To further assess molecular details underlying the anti-inflammatory properties of annexin A1 and its N-terminal peptide Ac1-25, focusing particularly on changes in the transcriptional profile, we analyzed gene expression patterns in peripheral blood monocytes stimulated with the bioactive annexin A1 N-terminal peptide Ac1-25 [6 ]. Cells were exposed to medium control or 50 µM Ac1-25, and their global gene expression profile was assessed by microarray hybridization of RNA prepared from the differently treated cells. A 2-h incubation period was chosen to minimize indirect autocrine or paracrine effects. An average of 10,407 or 46.7% out of 22,283 total probe sets on the Affymetrix U133A microarray was called present, according to Microarray Suite 5.0 software. Including only genes showing at least an average twofold change and change P values of less than 0.05 in each of the experiments, we identified 228 down-regulated and 106 up-regulated genes. The expression changes ranged from 14.3-fold down-regulation (CCR2) to 35.9-fold up-regulation [TNF superfamily 15 (TNFSF15)]. Table 1 lists the genes whose expression is affected by Ac1-25 treatment, sorted by GO categories.


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  		Table 1. Ac1-25-Induced Gene Expression Changes in Peripheral Blood Monocytes

 
GO categorizing of differentially expressed genes
The genes differentially expressed following Ac1-25 stimulation fell into diverse, functional categories. However, further characterization by screening the microarray data for statistically over-represented, biological processes using GO annotation (Level 7) suggested that the common theme of the Ac1-25-induced transcriptional profile was related to inflammation/immunity. Taking into consideration all genes showing at least twofold expression induction or repression and change P values <0.05 as input and applying Fisher’s exact test, we identified 28 GO nodes to be statistically enhanced with a P value of at least 0.05 (data not shown). Although these GO nodes again fell into different categories, eight of the over-represented GO terms were related to apoptosis or inflammation (Table 2 ). We also confirmed the results of this GO categorization by independent analysis with DAVID 2006 [22 ] (data not shown).


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  		Table 2. Analysis of Over-Represented GO Categories

 
Underscoring the anti-inflammatory activities of the annexin A1 N-terminal peptide Ac1-25, the inflammation-related genes in these categories generally showed an up-regulation in the case of anti-inflammatory gene products and a down-regulation for proinflammatory factors. We recorded, e.g., a more than ninefold up-regulation of IL-1Ra, which has been shown previously to negatively regulate the proinflammatory cytokine IL-1 [26 ], and a 14-fold down-regulation of the chemokine receptor CCR2, whose activity in the inflammatory response of monocytes has been well-documented (see, for example, ref. [27 ] and references therein), together with a twofold down-regulation of the cognate ligand, MCP-1, also known as CCL2 (Table 1 ).

For validation of the microarray data, we chose 12 examples from the up-regulated (FABP5, GREM1, HOMER1, IL1RN, INHBA, PHLDA1, RIS1) and down-regulated genes (CCR2, TLR4, PTAFR, RGL1, TNFAIP2) and analyzed their expression by RT-PCR in independent experiments. This selection placed an emphasis on inflammation-related genes but also considered genes involved in other physiological processes. The average regulations found in the microarray hybridizations were only considered to be verified successfully if at least three out of four independent RT-PCR experiments showed the same result. Applying these strict inclusion criteria, the regulation of 11 out of the 12 chosen genes could be validated by RT-PCR (Table 3 ). Only the expression of RIS1 appeared inconclusive and was not rigorously verified by real-time RT-PCR experiments, possibly because of the low, absolute expression of RIS1 in monocytes. Overall, the real-time RT-PCR results were consistent with the microarray data, confirming the validity of our approach.


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  		Table 3. Involvement of MAPK Signaling in Ac1-25-Induced Gene Expression Changes

 
ERK1/2- and p38-signaling pathways affect gene expression changes induced by Ac1-25
To reveal whether ERK1/2- and p38-mediated signal transduction pathways are not only involved in mediating the annexin A1-induced rapid and short-term anti-inflammatory effects, such as regulation of L-selectin shedding, but also in mediating the annexin A1-induced gene expression changes observed, we conducted an independent set of three real-time RT-PCR experiments, focusing on the 12 genes confirmed in PCR analysis (Table 3 ). Human peripheral blood monocytes were pretreated with the UO126 and SB203580 MAPK inhibitors for 20 min and then challenged with Ac1-25 for 2 h. Pretreatment of the monocytes with the ERK1/2 inhibitor UO126 resulted in a significant repression (>30%, ++) of the Ac1-25 effect on CCR2, HOMER1, IL1RN, INHBA, PHLDA, and RGL1 expression, and pretreatment with the p38 inhibitor SB203580 only affected IL1RN, INHBA, and less distinctive PHLDA1 expression. In the case of GREM1, PTAFR, and TNFAIP2, the influence of the pretreatment with the MAPK inhibitor on Ac1-25-induced gene expression could not be determined (n.a.), as neither inhibitor alone had a severe impact on the gene expression.

To exemplify the role of p38 and ERK1/2 in the regulation of the annexin A1-induced gene expression changes, effects of the inhibitors on one down-regulated (CCR2) and one up-regulated gene (IL-1Ra) are shown in Figure 2 . Whereas Ac1-25 stimulation alone induced an average 27-fold repression of CCR2 transcript levels, this reduction in expression was only 11-fold when ERK1/2 was inhibited in the monocytes. The p38 inhibitor SB203580 had no statistically significant effect on CCR2 expression levels (Fig. 2A) . In contrast, both inhibitors strongly affected the annexin A1-induced IL-1Ra expression changes. Ac1-25-stimulated monocytes, pretreated with SB203580 or UO126, only showed a 6.5-fold or ninefold change in IL-1Ra expression, as opposed to the 18-fold induction of expression elicited by Ac1-25 alone (Fig. 2B) . Taken together, both MAPK signaling pathways investigated contributed to the expression changes induced by Ac1-25 stimulation of human peripheral blood monocytes but are probably not equally involved in the mediation of all gene expression changes observed.


Figure 2
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  		Figure 2. p38 and ERK1/2 signaling differentially affects Ac1-25-induced gene expression changes in human monocytes. Graphs show fold changes over unstimulated control cells of (A) CCR2 and (B) Il-1Ra expression. Monocytes were pretreated for 20 min with medium alone or medium containing 10 µM UO126 or 20 µM SB203580, respectively, and then stimulated with 50 µM Ac1-25 for 2 h. Data are expressed as means ± SEM from three independent experiments. *, Statistical significance of P < 0.05.

 
Ac1-25 regulates CCR2 and IL1-Ra protein levels in human monocytes
To elucidate whether the observed mRNA regulations reflect themselves in the respective protein abundance, we chose the above-mentioned examples, IL-1Ra and CCR2, for a more detailed analysis of protein levels, as both proteins have been shown to play central roles in inflammatory processes [28 , 29 ]. Intracellular IL-1Ra and CCR2 cell surface levels following Ac1-25 challenge of monocytes were assessed using ELISA and flow cytometry, respectively, in three independent experiments each. To allow for de novo synthesis of proteins, we stimulated the monocytes with the annexin A1 peptide for 6 h as compared with the 2-h stimulation in the microarray and real-time RT-PCR experiments. IL-1Ra ELISAs showed an approximate 4.5-fold up-regulation of intracellular protein levels (Fig. 3A ), in line with the significant Ac1-25-induced up-regulation of the IL-1Ra mRNA. In contrast, CCR2 levels were found to be decreased by ~35% as compared with control levels, and the decrease is reproducible and significant (Fig. 3B) and rather modest compared with the marked reduction in the mRNA levels, probably reflecting a relatively long half-life of the receptor. To confirm the functional relevance of annexin A1-induced CCR2 down-regulation, we performed chemotaxis assays with the CCR2 ligand MCP-1/CCL2 using annexin A1 peptide-pretreated monocytes. We first established a dose-response curve for MCP-1/CCL2, which showed maximal chemotactic activity at ~10 nM concentration (not shown). Using this agonist concentration, we evaluated the migratory activity of monocytes, which were pretreated for 5 h with the Ac1-25 peptide, in comparison with monocytes, which were kept in assay medium for the same period of time. In the absence of chemoattractant, the annexin-pretreated monocytes showed a slightly increased migratory ability, although this was not statistically significant. In contrast to that, directed migration in response to MCP-1/CCL2 was greatly reduced in the monocytes, which were exposed to the annexin peptide (Fig. 3C) , indicating that the inhibitory effect is functionally related to the observed CCR2 down-regulation after Ac1-25 treatment.


Figure 3
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  		Figure 3. Annexin A1 (Ac1-25) stimulation of monocytes alters CCR2 cell surface receptor and IL-1Ra total protein levels. Monocytes were stimulated with 50 µM of the Ac1-25 peptide for 6 h and compared with nonstimulated, control cells. (A) Cells were lysed, and IL-1Ra protein levels were measured by sandwich ELISA. Data are means ± SEM of three independent experiments (*, P<0.05); control levels equal 2.4 ng per 106 cells. (B) Cells were stained with polyclonal rabbit antibodies against CCR2, washed, and stained with secondary, FITC-conjugated goat anti-rabbit antibodies. FACS analysis was based on examination of 10,000 cells per sample and shows mean fluorescence. Data are means ± SD of three independent experiments (*, P<0.05). (C) Pretreatment of monocytes with the Ac1-25 peptide reduces MCP-1/CCL2-induced chemotaxis. Cells were treated for 5 h with assay medium alone (open bars) or medium containing 50 µM Ac1-25 (shaded bars). Subsequently, cells were placed in the upper compartment of the chemotaxis chamber and allowed to migrate in response to 10 nM MCP-1/CCL2 in the lower compartment. The average chemotaxis index (±SEM) was calculated from three independent experiments with three parallel samples each (*, significant inhibition of MCP-1/CCL2-dependent chemotaxis; P<0.001).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the anti-inflammatory activity of exogenously applied annexin A1 has been established in numerous animal and in vitro models [4 , 30 ], a global analysis of elicited effects in human leukocyte populations has never been carried out. Most of the biological activity of annexin A1 is retained in the N-terminal portion of the molecule, namely the peptide Ac1-25 [31 ], and annexin A1 N-terminal peptides, most likely generated at sites of inflammation by the proteolytic activity of human leukocyte elastase [9 ], have been shown to act as mimetics of the entire protein, retaining the full pharmacologic activity, i.e., to act as a potent anti-inflammatory agent, limiting the extravasation of leukocytes [6 ]. Therefore, we here used microarray technology to fully analyze the global gene expression changes elicited in human peripheral blood monocytes by the N-terminal annexin A1 peptide Ac1-25, possibly explaining anti-inflammatory effects other than the established immediate effects, such as L-selectin shedding [13 , 25 ]. Although it had been proposed before that annexin A1 N-terminal peptides modulate the expression of certain genes [7 , 32 ], our approach for the first time yielded a full expression profile of Ac1-25-regulated genes in human monocytes.

The statistical relevance and validity of the findings were guaranteed by several features in our experimental setup and data evaluation [33 ]. First, only genes showing up in all of the three independent hybridization experiments were considered as being regulated by stimulation with the annexin A1 peptide. Second, we pooled leukocytes from several donors in each of the experiments to overcome possible pharmacogenetic effects, e.g., receptor polymorphisms responsible for atypical responses to stimuli or anomalies in the underlying regulatory pathways in single donors [34 , 35 ]. Third, verification by real-time RT-PCR revealed that the vast majority of genes identified in the global microarray screen (11 out of 12 genes chosen) shows the same quality of regulation when the individual mRNA is highlighted. The fold changes were mostly higher in the real-time RT-PCR experiments, which is in accordance with generally accepted knowledge about correlation between these two methods [36 ].

Apart from obtaining an overview of the global gene expression changes occurring after stimulation of monocytes with the N-terminal annexin A1 peptide, we also identified functional groups, which were statistically overrepresented among the differentially expressed genes. Our GO analysis took into account all known annotations concerning biological processes in which the genes are involved. Although previous studies documented immediate anti-inflammatory activities of exogenously applied Ac1-25 in acute models of inflammation in vivo [37 38 39 ] as well as in vitro [13 , 40 , 41 ], our GO analysis establishes for the first time that the N-terminal annexin A1 peptide induces a stable anti-inflammatory phenotype in monocytes by regulating the expression of molecules playing important roles in inflammatory processes.

As a general rule, we observe the Ac1-25-induced up-regulation of anti-inflammatory genes and the concomitant down-regulation of proinflammatory genes. Two prominent examples whose Ac1-25-dependent regulation was also verified at the protein level are CCR2 and IL-1Ra. CCR2, the cognate receptor for MCPs (MCP-1–5), and its ligands are key players responsible for the directed migration of macrophages and the subsequent infiltration of inflamed tissues. As a consequence, the CCR2/MCP system seems to be of importance in the pathogenesis of atherosclerosis as well as in the host response toward infections with intracellular pathogens [29 ]. In spite of the robustness of the cytokine signaling system, limiting the influence of its single components, it seems that CCR2 acts as a critical node in cytokine signaling, whose attenuation is likely to destabilize the immune response as compared with other, more redundant chemokine receptors [42 ]. It remains to be seen, though, whether the reduced expression of CCR2, which we observed on the mRNA as well as the protein level after stimulation with the Ac1-25 peptide, is an early sign of monocyte differentiation toward the tissue macrophage type as has been proposed recently [43 ] or a differentiation-independent effect, possibly related to the regulation of directed monocyte migration. Clearly, annexin peptide pretreatment diminished monocyte migration significantly in response to MCP-1/CCL2.

IL-1Ra is a member of the IL-1 family, which in its secreted form, binds to IL-1Rs but is also present in an intracellular isoform [44 ]. Studies in IL-1Ra knockout mice have revealed a spontaneous development of arthritis, suggesting that an imbalance between IL-1 and IL-1Ra may predispose to local inflammatory disease [45 ]. Recently, IL-1Ra has been approved to treat rheumatoid arthritis [46 , 47 ] and has also been shown to counteract the IL-1-associated pathogenesis of inflammation in other diseases [48 , 49 ]. The strong up-regulation of IL-1Ra following Ac1-25 stimulation of monocytes could provide a direct link between glucocorticoids and IL-1Ra to limit inflammatory responses.

Along with the strong differential expression of CCR2 and IL-1Ra, we also identified an Ac1-25 regulation of other important inflammation-related genes. This includes the down-regulation of PTAFR, which is, in case of inappropriate activation, associated with proinflammatory responses and subsequent disorders, including allergy and atherosclerosis [50 , 51 ], and TLR4, a part of the bacterial LPS receptor [52 ], which is also implicated in the initiation of septic shock [53 ] and atherosclerotic disease [54 ], as well as other inflammatory and infectious disorders.

As MAPK signaling is involved in all aspects of immune responses, we went on to analyze whether the MAPK signaling pathways are activated upon Ac1-25 stimulation. We observed that the annexin A1 peptide activated p38 and ERK1/2 in a time-dependent manner and that activation of these signaling modules is involved in the gene expression changes occurring with the influence of these two MAPK pathways differing among the examined genes. Whereas Ac1-25-triggered repression of CCR2 expression was sensitive to ERK1/2 inhibition only, Ac1-25 stimulation of IL-1Ra expression was reduced markedly by inhibition of both pathways. Only a few publications have addressed the impact of these signaling pathways on the expression of the candidate genes selected here, but in the case of IL-1Ra, an influence of both MAPKs on IL-1Ra expression in monocytes stimulated with LPS had been observed [55 ]. Altogether, our results show that both MAPK pathways are required for the mediation of certain Ac1-25-induced gene expression changes.

Most likely, the effects of Ac1-25 on MAPK signaling and the gene-expression changes documented here are mediated through members of the FPR family, which have been shown to act as receptors for the mimetic annexin A1 peptide [12 13 14 ]. It is interesting that Ac1-25 serves as a ligand for all three FPRs expressed in monocytes, FPR, FPRL1, and FPRL2. Future studies, which would require the development of specific inhibitors for each of the FPRs, should reveal which FPR family member contributes to what extent to the Ac1-25-induced gene expression changes.

Taken together, our data show that the annexin A1 peptide Ac1-25 not only elicits rapid, anti-inflammatory effects by post-translational modifications, inhibiting monocyte extravasation by, e.g., inducing L-selectin shedding, but also triggers the establishment of an anti-inflammatory phenotype in human monocytes. We provide a comprehensive and statistically validated overview of the global gene expression changes induced in human monocytes by the biologically active annexin A1 peptide Ac1-25. This further supports the role of the N-terminal annexin A1 peptide as a key anti-inflammatory molecule, acting on monocytes by activating them to develop an anti-inflammatory phenotype and triggering apoptosis, both of which are important steps to ensure a proper resolution of the inflammatory response. We also provide evidence that the MAPKs ERK1/2 and p38 are involved in the mediation of the anti-inflammatory effects of the exogenously administered Ac1-25 peptide.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the German Research Society (SFB 293, RE26/11) and the Interdisciplinary Clinical Research Centre of the University of Muenster (Re2/033/04). We thank Frauke Brinkmann and Viola Geissler for help with the experiments and Dr. Arseni Markoff for stimulating discussions.

Received March 14, 2007; revised August 16, 2007; accepted August 23, 2007.


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 DISCUSSION
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