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Published as doi: 10.1096/fj.07-8473com.
 (The FASEB Journal. 2007;21:3162-3170.)
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Resolvin E1 promotes mucosal surface clearance of neutrophils: a new paradigm for inflammatory resolution

Eric L. Campbell*,{dagger}, Nancy A. Louis{dagger}, Sarah E. Tomassetti*, Geraldine O. Canny{dagger}, Makoto Arita{dagger},1, Charles N. Serhan{dagger} and Sean P. Colgan*,{dagger},2

* Mucosal Inflammation Program, Division of Gastroenterology, University of Colorado Health Sciences Center, Denver, Colorado, USA; and

{dagger} Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA

2Correspondence: Mucosal Inflammation Program, University of Colorado Health Science Center, Biomedical Research Bldg. Rm. 702, 4200 E. 9th Ave, Denver, CO 80262, USA. E-mail: sean.colgan{at}uchsc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Migration of neutrophils (PMN) across epithelia is a pathological hallmark of numerous mucosal diseases. Whereas lesions at mucosal surfaces are generally self-limiting, endogenous mechanisms of resolution are incompletely understood. Previous studies revealed that resolvins directly act on PMN to attenuate transendothelial migration, less is known about the influence of resolvins on PMN-epithelial interactions and whether they act on epithelia. We studied the dynamics of resolvin E1 (RvE1) actions on leukocyte transepithelial migration. PMN exposure to RvE1 or chemerin (peptide agonist of ChemR23) reduced transepithelial migration in a concentration-dependent manner. Conversely, activation of epithelial ChemR23 promoted apical clearance of PMN. A nonbiased screen of known PMN ligands expressed on epithelial cells in response to RvE1 revealed selective induction of CD55, an apically expressed antiadhesive molecule. CD55 promoter analysis demonstrated that both RvE1 and chemerin activate the CD55 promoter. Inhibition of CD55 by neutralizing antibody prevented RvE1-dependent augmentation of apical PMN clearance. Taken together these findings implicate a "two-hit" model of inflammatory resolution, whereby activation of the PMN RvE1 receptor attenuates transepithelial migration and subsequent actions on the epithelium promote CD55-dependent clearance of PMN across the epithelial cell surface promoting active inflammatory resolution.—Campbell, E. L., Louis, N. A., Tomassetti, S. E., Canny, G. O., Arita, M., Serhan, C. N., Colgan, S. P. Resolvin E1 promotes mucosal surface clearance of neutrophils: a new paradigm for inflammatory resolution.


Key Words: transmigration • CD55 • ICAM-1 transmigration • lipid mediator • epithelia


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
NEUTROPHILS (PMN) constitute a first-line immune defense by migrating to sites of injury or infection. Transepithelial migration is instrumental to mucosal defense during inflammation (1 , 2) . Transmigrating PMN undergo a potent respiratory burst and degranulation response to invading pathogens. However, excessive or inappropriate PMN activity can cause tissue damage and contribute to the pathogenesis of numerous inflammatory diseases (3) . Currently, mechanisms of PMN clearance as they relate to inflammatory resolution are unknown.

Before interaction with the epithelium, PMN escape the vasculature into the interstitium via extravasation. Although transendothelial migration is relatively well characterized, transmigration (TM) across the epithelium is less well understood. Evidence suggests that upon interfacing with epithelia, PMN initiate migration by interacting with desmosomal junctional adhesion molecule-C (JAM-C), mediated via PMN ß2-integrin CD11b/18 (4) . Subsequently, the PMN junctional adhesion molecule protein (JAM) associates with epithelial tight junction protein CAR (coxsackie and adenovirus receptor; ref 5 ). Interactions between epithelial CD47 and PMN signal regulatory protein-{alpha} (SIRP{alpha}) facilitate traversing of the paracellular space (6) . Tight junctions serve as a barrier sealing the epithelium; to complete migration, PMN must traverse the tight junction without disrupting barrier function. Occludin, a component of the tight junction, has been implicated in providing passage of PMN across the barrier (7) . Once PMN reach the apical surface of the epithelium, it has been suggested that they bind to intracellular adhesion molecule-1 (ICAM-1) on the apical surface (8) . More recently, studies (9 , 10) have revealed that apically the localized decay accelerating factor (DAF, also termed CD55) functions as an antiadhesive molecule promoting the clearance of epithelial bound PMN.

Inflammatory events can either resolve or persist and exacerbate the condition. Understanding inflammatory resolution is currently an area of intense investigation. Increasing evidence suggests that the proresolving lipid mediator resolvin E1 (RvE1: 5S,12R,18R-trihydroxyeicosapentaenoic acid), a derivative of omega-3 fatty acid, contributes to resolution of inflammation and has been demonstrated to stimulate the orphan receptor ChemR23 (11 , 12) . RvE1 is generated at sites of inflammation through transcellular metabolism and has been shown to potently inhibit PMN transendothelial migration (13) , to attenuate colonic mucosal inflammation in vivo (14 , 15) , and to resolve oral inflammation in a rabbit periodontitis model (16) . In the present study, we hypothesized that mucosal epithelial cells actively contribute to the resolution of inflammation via activation of ChemR23. We demonstrate, for the first time, that both RvE1 and chemerin promote the apical clearance of PMN through the transcriptional induction of epithelial CD55. These results provide a new paradigm for inflammatory resolution wherein epithelia actively clear surface PMN.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Materials
RvE1 was prepared by total organic synthesis (synthetic core P50 DE016191) and qualified according to the published physical and biological properties (11 , 17) . Chemerin peptide (linear sequence YHSFFFPGQFAFS; from ref. 12 ) was synthesized by New England Peptide (Garner, MA, USA). Antibodies for CD55 (OE-1) and W6/32 were isolated from hybridomas purchased from the ATCC. ß-Actin antibody (ab8227) was from Abcam. ICAM-1 antibody for immunofluorescence was purchased from Cell Signaling Technology (Danvers, MA, USA), and antibody for Western blotting and immunopreciptation (sc-7891) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TNF-{alpha}, IL-1ß, and TGF-ß were purchased from BioSource (Carlsbad, CA, USA). IL-4, IL-6, and IFN-{gamma} were purchased from R&D Systems (Minneapolis, MN, USA). PGE2 was purchased from Biomol (Plymouth Meeting, PA, USA).

Cell culture
KB-wt and KB-ChemR23 cells were grown on either permeable 0.33-cm2 ring-supported polycarbonate filters (3 µm pore size; Costar, Lowell, MA, USA) or plastic polystyrene tissue culture dishes (Costar), as indicated, using techniques described previously (9) . For migration experiments, cells were plated on the underside of permeable supports to allow physiologically relevant basolateral-to-apical transmigration, as reported previously (9) .

Generation of epithelia expressing ChemR23
KB oral epithelial cells stably expressing human ChemR23 (KB-ChemR23) were established by transfecting pcDNA3-hChemR23 (11) , and positive transformants were selected and maintained with 500 µg/ml G418 (Cellgro, Herndon, VA, USA). Cell surface expression was examined by FACS analysis using mouse monoclonal anti-human ChemR23 antibody (clone 84939; R&D Systems) or control mouse IgG (BD Pharmingen, San Jose, CA, USA), followed by FITC-conjugated goat anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA, USA).

RNA isolation and transcriptional analysis
RNA was isolated and cDNA synthesized as described previously (10) . Potential contaminating genomic DNA was digested using DNA-free (Ambion, Austin, TX, USA). Semiquantitative and real-time PCR were performed using primer sets and conditions reported previously (10) . The following primers and conditions were used to quantify ChemR23 (CMKLR1; NM_004072): forward 5'-ATAGAATGGAGGATGAAGATTACAACACT-3' and reverse 5'-TCCCGAGGAAGCAGACGATG-3'; 35 cycles of PCR with 59°C annealing temperature. Transcript levels and fold change in mRNA were determined as described previously (18) .

PMN isolation and transmigration assays
PMN were isolated from whole venous blood by venipuncture from healthy human donors as described previously (10) . PMN (>97% pure as determined by microscopic evaluation) were resuspended to a final concentration of 5 x 107 in HBSS (with 10 mM HEPES, pH 7.4, and without Ca2+ or Mg2+; Sigma-Aldrich, St. Louis, MO, USA). PMN were used within 2 h of isolation.

Migration assays were performed as described previously (9 , 10) . Briefly, 106 PMN were added to the upper chambers of transwell inverts in which KB-ChemR23 cell monolayers were plated on the opposing side. A chemotactic gradient was established by adding 100 nM n-formyl-methionyl-leucyl-phenylalanine (fMLP) to the lower chambers. PMN TM was performed at 37°C for 20 min, after which filters were transferred to a new plate with fresh 100 nM fMLP. Filters were transferred every 20 min for a total of 80 min (9) . For neutralizing antibody experiments, monolayers were preincubated with mouse monoclonal antibody (clone OE-1) (9) or bind control directed against MHC class I (clone W6/32).

CD55 promoter analysis
The CD55 promoter, truncated promoter, and mutation constructs were generated previously (10) , transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) with 1 µg construct DNA, and cotransfected with 0.05 µg Renilla luciferase per well of a 24-well plate as directed by the manufacturer. Dual-luciferase assays were performed as described previously (10) .

Sample preparation and immunodetection
Cell lysates for Western blotting and coimmunoprecipiation were prepared with the following lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% TX-100, 1 mM Na3VO4, and 1 mM PMSF). Crude lipid raft enrichment was performed as described previously (19) , with the exception that the TX-100 soluble fraction was termed "MCF" for membrane cytosolic fraction, and the ß-octylglucoside soluble fraction was termed "LRF" for lipid raft fraction. Western blotting, coimmunoprecipitation, and immunofluorescence were all performed as described previously (20) . CD55 was detected by Western blotting and immunofluorescence using OE-1 at 1 µg/ml. ICAM-1 was detected by immunofluorescence at 1:500 or by Western blotting at 1:750. ß-actin protein was detected using antibody at 1:10,000 in 5% nonfat milk/TBST.

Coimmunoprecipitations were performed on cleared total lysates from vehicle- or chemerin-treated cells. Lysates were incubated end over end with 1 µg antibody or isotype-matched control per 500 µg protein, for 3 h at 4°C and subsequently incubated with µMACS protein-A or -G microbeads (Miltenyi Biotech, Auburn, CA, USA) for 1 h. Antibody-bound proteins were isolated by magnetic separation and subjected to SDS-PAGE.

For immunofluorescence cells were grown directly on acid-washed glass coverslips (Fisher Scientific, Pittsburgh, PA, USA), fixed with 4% paraformaldehyde/PBS, and permeabilized with 0.1% TX-100/PBS. After appropriate 1° antibody incubation at 37°C for 1 h, AlexaFluor-488 and AlexaFluor-555 conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA). Images were visualized and captured using an AxioCam MRc5 attached to an AxioImager A1 microscope (Zeiss, Oberkochen, Germany). Colocalization analysis was performed using Image J software on 8-bit images taken from six separate frames of each treatment, from three separate experiments. The degree of colocalization was expressed as a percentage of the region of interest (ROI).

Statistical analysis
Data was compared by Student’s t test or ANOVA where appropriate. Values are mean ± SE from at least three separate experiments.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Influence of RvE1 on PMN transepithelial migration
Transepithelial migration of PMN represents an important component of the innate immune response and is a pathological hallmark of active mucosal inflammation (1) . Studies with anti-inflammatory lipids, the resolvins RvE1 and RvD1, have demonstrated that exposure to PMN attenuates migration across endothelial monolayers (13 , 21) . Here we defined whether RvE1 similarly influences PMN migration across epithelial monolayers. For these purposes, PMN were pre-exposed to either RvE1 or the inactive metabolite 13-hydroxy-docosahexaenoic acid (13-HDHA) for 15 min and applied to "inverts" (KB cell monolayers grown on the underside of a Transwell filter and inverted, such that PMN migrate in a physiologically relevant basolateral-to-apical direction) and allowed to migrate for 60 min. As shown in Fig. 1 A, PMN exposure to RvE1 potently (EC50=~25 nM) attenuated transepithelial migration in a concentration-dependent manner (ANOVA, P<0.01). 13-HDHA did not significantly influence PMN transepithelial migration. Such findings are similar to previous work with endothelial cells, wherein PMN exposure to RvE1 resulted in a 40–60% decrease in PMN transendothelial migration (13 , 17) .


Figure 1
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  		Figure 1. Influence of RvE1 on PMN TM and characterization of ChemR23 in KB oral epithelial cells. A) PMN were exposed to RvE1 (1–1000 nM) or vehicle for 15 min and applied to basolateral surface of inverted KB epithelial monolayers and allowed to migrate for 90 min. Migrated cells were subsequently lysed and quantified by MPO assay. Data were analyzed by ANOVA and are presented as mean ± SE (n=6). P < 0.01 compared to 13-HDHA. B) KB oral epithelial cells were transfected with empty vector (KB-wt) or human ChemR23 (KB-ChemR23) and selected with G418 (500 µg/ml). Semiquantitative PCR was used to detect expression of hChemR23 in wild-type and stably transfected KB cells. Reactions were stopped at 20, 25, 30, and 35 cycles and visualized by UV-transillumination. C) FACS was used to define cell surface expression using anti-human ChemR23 antibody (light gray) or control mouse IgG (dark gray) and FITC-labeled secondary antibody. D) Inverted KB-ChemR23 monolayers were treated with RvE1 (100 nM) or chemerin (1 µM) for 12 h before PMN exposure. RvE1 and chemerin kinetic migration of PMN across epithelial monolayers was examined by MPO at 20, 40, 60, and 80 min postexposure. Data were analyzed by ANOVA and are presented as mean ± SE (n=6). P < 0.01 compared to vehicle control. E) Results expressed as rate of transmigration of PMN across RvE1- and chemerin-exposed monolayers.

Expression of ChemR23 in epithelial cells after exposure to cytokines
Lipid mediators with anti-inflammatory actions, such as the lipoxins, exert their influences through both leukocyte- and parenchymal-expressed receptors (2) . Furthermore, cytokines induce lipoxin A4 receptor (ALX) expression in intestinal epithelia (22) . Given the beneficial redundancy between lipoxin A4 and RvE1 activity and that ChemR23 and ALX receptors share 36.4% sequence homology, we examined whether cytokines also drive epithelial expression of ChemR23 and whether epithelial receptor activation contributes to the inflammatory resolution actions of RvE1. Initial mRNA-based screens revealed that KB cells express low levels of endogenous ChemR23. As shown in Table 1 , a real-time PCR screen of inflammatory mediators on epithelial ChemR23 expression revealed that TNF-{alpha} significantly suppressed ChemR23 mRNA (P<0.05) and more notably that TGF-ß1 induced ChemR23 expression by 4 ± 0.5-fold (P<0.01). Important in this regard, TGF-ß has been implicated as an end point target for anti-inflammatory lipids (e.g., lipoxins, resolvins, and protectins; ref 23 24 25 ), and thus, these findings of TGF-ß-mediated induction of ChemR23 support the sustained induction of epithelial ChemR23 receptor as a feed-forward mechanism. Moreover, mouse ChemR23 was recently shown to be induced by both TGF-ß1 and TGF-ß2 in murine macrophages (26) . These findings provide for epithelial ChemR23 expression in the inflammatory milieu.


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  		Table 1. Influence of cytokines on ChemR23 expression in KB cells

Generation and characterization of KB-ChemR23
We next endeavored to define the influence of epithelial ChemR23 activation on the kinetics of PMN TM. To circumvent the use of cytokines, which could independently influence our epithelial phenotype, we utilized a plasmid-based approach to generate an epithelial cell line (KB-ChemR23) stably expressing ChemR23. Expression of ChemR23 mRNA in KB-wt and KB-ChemR23 was readily detected by semiquantitative PCR. Low basal expression of ChemR23 was detected in KB-wt cells between 30–35 cycles; however, ChemR23 expression was detected after just 25 cycles in the stable cell line (Fig. 1B ). Similarly, FACS analysis indicated background levels of ChemR23 surface protein in KB-wt cells and substantial ChemR23 surface expression in KB-ChemR23 (Fig. 1C ).

RvE1 and chemerin increase the rate of PMN clearance
To define the role of the epithelial ChemR23 in PMN TM, we utilized a kinetic model (27) to examine the dynamics of PMN TM over an 80 min time period. As shown in Fig. 1D , KB-ChemR23 epithelial pre-exposure to RvE1 (100 nM, 12 h) and the ChemR23 peptide ligand chemerin (1 µM, 12 h) resulted in a significant shift in the kinetics of fMLP-stimulated PMN TM (both P<0.01 by ANOVA). These studies indicated that at as early as 40 min, significantly more PMN had migrated across ChemR23-stimulated epithelial monolayers (P<0.01). Such enhanced TM was evident at each point over the 80 min time period (Fig. 1D ). Calculated rates of PMN TM (Fig. 1E ) revealed a >2-fold increase in PMN TM across ChemR23-stimulated epithelia (rates of 4.1±.0.49, 9.9±2.66, and 11.4±3.9x103/min/cm2 for vehicle-, chemerin-, and RvE1-exposed epithelia, respectively, P<0.01).

Influence of RvE1 on known epithelial PMN ligands
Based on these findings of increased PMN transepithelial migration kinetics in response to epithelial exposure to RvE1, we profiled the influence of RvE1 on epithelial mRNA expression of a panel of molecules previously shown to regulate PMN TM. Included in this screen were occludin (7) , CD47 (28) , intercellular adhesion molecule-1 (ICAM-1; ref. 8 ), and members of the junctional adhesion molecule (JAM) family of desmosomal proteins (5) . As shown in Table 2 , we used real-time PCR to profile mRNA derived from KB-wt and KB-ChemR23 cells exposed RvE1 (100 nM, 8 h). Expression patterns of these ligands in response to RvE1 were surprisingly stable. No differences in mRNA expression were observed for occludin, JAM-A, JAM-B, or JAM-C after RvE1 stimulation. A small but significant decrease in ICAM-1 (30±4% compared to KB-Wt cells, P<0.05) was observed in response to RvE1. More notable were changes in CD55 expression (Table 1) , wherein CD55 mRNA was significantly induced by RvE1 in KB-ChemR23 but not KB-wt cells (maximal 2.3±0.1-fold increase over KB-wt, P<0.025). These studies suggest that ChemR23 activation represses the proadhesive PMN ligand ICAM-1 and significantly induces antiadhesive GPI-linked molecule CD55 in epithelia expressing ChemR23. Of interest in this regard, it was recently demonstrated that CD55 represents an apically expressed PMN ligand that functions in a novel fashion to promote the detachment of PMN from the luminal surface (9) . These studies implicated CD55 as a PMN clearance mechanism controlled by the total amount of accessible CD55 on the apical membrane surface. For instance, heterologous over-expression of CD55 promotes PMN clearance, and synthetic peptides directed against the PMN-binding site on CD55 effectively block this response (9 , 10) .


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  		Table 2. Influence of RvE1 on expression of PMN ligands

RvE1 and chemerin induce CD55
Further experiments were conducted to elucidate the increase in CD55 expression. Real-time PCR analysis (relative to ß-actin) revealed that RvE1 selectively induced KB-ChemR23 CD55 mRNA in a concentration-dependent manner (Fig. 2 A). Significant induction of CD55 mRNA was observed at concentrations of RvE1 as low as 10 nM (P<0.05). No changes in KB-Wt CD55 were observed at any concentration tested (P=not significant for all), thereby suggesting a selective induction of CD55 in epithelia expressing ChemR23 receptor. In parallel, we examined CD55 protein expression in KB-ChemR23. CD55 is a heavily glycosylated 70 kDa, GPI-linked protein expressed on the apical surface of epithelia (29) . As shown in Fig. 2B , Western blot analysis of CD55 protein in KB-ChemR23 exposed to RvE1 (100 nM, 12 h) revealed a nearly 4-fold increase (relative to ß-actin) in CD55 over that of vehicle-exposed cells, indicating that changes in mRNA are also reflected at the protein level.


Figure 2
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  		Figure 2. Influence of RvE1 CD55 expression and promoter activity. A) Real-time PCR analysis of CD55 mRNA levels in KB-wt and KB-ChemR23 following treatment with RvE1 (100 nM, 8 h). RvE1 treatment results in a dose-dependent increase in CD55 expression only in KB-ChemR23 cells. Data are presented as fold change ± SE (n=3). P < 0.05 compared to wild type. B) Representative Western blot of CD55 from cells exposed to RvE1 (100 nM, 24 h) or vehicle control. C) KB-ChemR23 were transfected with full-length CD-55 promoter (DAF-733) or deletion constructs (DAF-352/157/106/60). Cells were subsequently exposed chemerin (1µM). Results expressed as fold over background (PGL3 vector) ± SE (n=3). *P < 0.05 compared to vehicle treated. D) Results expressed as fold change with chemerin over vehicle control ± SE (n=3). *P < 0.05 compared to vehicle treated. **P < 0.01 compared to vehicle control.

To examine whether the induction of CD55 by ChemR23 stimulation occurs through transcriptional pathways, CD55 luciferase promoter constructs were utilized. As shown in Fig. 2C, D , KB-ChemR23 cells were transiently transfected with CD55 promoter constructs (constructs –733, –352, –157, –106, and –60, all representing 5' truncations relative to the transcription start site; ref 10 ) and examined for changes after exposure to the ChemR23 peptide ligand chemerin. Consistent with previous work (10) , successive truncations each maintained activity well above the empty PGL3 vector, although increasing truncation at the 5' end resulted in a progressive decrease in baseline activity (e.g., –60 construct contained <15% activity relative to the wild-type –733 construct). Subsequent examination of these CD55 promoter truncations in response to ChemR23 stimulation revealed significant chemerin-inducibility in all but the –60 construct (P<0.05 for all). Chemerin-inducibility was particularly prominent in the region spanning positions +88 to –106 (2.2±0.3-fold increase over vehicle control, P<0.01). This region of the promoter contains binding sites for multiple transcription factors, including CRE, AP1, AP2, SP1, and USF. Further studies will be necessary to define the mechanisms underlying this response.

Influence of CD55 inhibition on PMN migration
Our screen of known epithelial ligands responsive to ChemR23 stimulation indicate that adhesion molecules, per se, are less likely to be the limiting factor for PMN trafficking under such conditions. Rather, these studies implicate the antiadhesive properties of CD55 as the critical control point for the resolution of PMN TM. Such events at the apical surface reflect the terminal steps of TM (9) and, as such, provide a mechanism for the clearance of PMN from the apical membrane surface. To directly test the specificity of CD55 in ChemR23-dependent clearance, we utilized a monoclonal antibody directed against CD55 that functionally inhibits the release of PMN from the epithelial membrane surface (clone OE-1; ref. 9 ). KB-ChemR23 inverted monolayers were stimulated with ChemR23 agonists RvE1 (100 nM, 12 h) or chemerin (1 µM, 12 h), and the rate of fMLP-stimulated PMN TM was examined the in the presence of mAb OE-1 (10 µg/ml) or binding control W6/32 directed against MHC class I (10 µg/ml). As shown in Fig. 3 A, B, this analysis revealed that mAb OE-1 significantly attenuated the clearance of PMN from the epithelial surface after exposure to RvE1 and chemerin (P<0.001 by ANOVA). As shown in Fig. 3C , calculated rates revealed that mAb OE-1 decreased PMN clearance by >4-fold in RvE1 (rates of 11.4±.3.4 and 2.6±0.4x103 /min/cm2 in the presence of W6/32 and OE-1, respectively, P<0.01)- and chemerin-exposed monolayers (10.0±2.9 and 2.5±0.3x103/min/cm2 n the presence of W6/32 and OE-1, respectively, P<0.01). Taken together, these findings indicate that the initial increase in TM observed with RvE1/chemerin represents accelerated clearance from the apical surface due to elevated expression of CD55.


Figure 3
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  		Figure 3. Influence of CD55 blockade on ChemR23-dependent clearance of PMN. A) Inverted KB-ChemR23 monolayers were exposed to RvE1(100 nM for 12 h followed) by a 10 min incubation with OE-1 (anti-CD55) antibody on the apical surface. PMN were then added to the basolateral aspect of cells and stimulated to migrate by the addition of fMLP (100 nM). Kinetic migration was quantified by MPO assay. B) Inverted KB-ChemR23 monolayers were exposed to 1 µM chemerin for 12 h, followed by incubation with OE-1 and migration of PMN was assayed by MPO. C) Rate of migration of PMN across RvE1- and chemerin-exposed monolayers in the presence and absence of OE-1 antibody. Data are mean ± SE (n=6). *P < 0.05 compared to W6/32 antibody control.

CD55 interacts with ICAM-1
A close extracellular spatial interaction has been hypothesized to exist between CD55 and ICAM-1 via the functional short consensus repeat-3 (SCR-3) domain of CD55 (30) . Such an association may limit PMN binding to the proadhesive ICAM-1. Thus, the observed induction of CD55 and repression of ICAM-1 at the apical surface may alter the dynamics of PMN adhesion, through a shift in the balance of the pro- and antiadhesive properties of ICAM-1 and CD55, respectively. Under our defined conditions of resolvin/chemerin exposure, the predominant phenotype is one of antiadhesion (i.e., expression of CD55>>ICAM-1), resulting in a net shift toward PMN clearance. Moreover, in addition to its antiadhesive properties, CD55 may also function as a competitive inhibitor of ICAM-PMN binding through direct binding to ICAM-1. To elucidate if an association exists between these molecules that contributes to an antiadhesive phenotype, we examined CD55 and ICAM-1 colocalization by immunofluorescent microscopy (Fig. 4 A). Colocalization of CD55 and ICAM-1 was apparent in both untreated and in cells exposed to chemerin/RvE1. Analysis of the degree of colocalization (Fig. 4B ) revealed a significant increase in CD55-ICAM colocalization in both chemerin- and RvE1-treated cells. To confirm this spatial colocalization of ICAM-1 and CD55 biochemically, coimmunoprecipitation studies were performed. As shown in Fig. 4C , immunoprecipitation of ICAM-1 from vehicle-treated or chemerin-exposed cells and subsequent Western blotting for CD55 increased association of CD55 and ICAM-1 in chemerin-exposed cells. No change in binding was observed on immunoprecipitation of CD55 and blotting for ICAM-1 (Fig. 4C ), suggesting that increased levels of CD55 expression in chemerin-exposed cells result in increased CD55 bound to ICAM-1 in treated cells.


Figure 4
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  		Figure 4. Interactions of CD55 and ICAM-1. A) KB-ChemR23 were grown on coverslips and exposed to either 100 nM RvE1, 1 µM chemerin or vehicle for 12 h. CD55 and ICAM-1 were imaged by immunofluorescent staining. Images represent 5–6 samples per group. B) Colocalization of CD55 and ICAM-1 was quantified using ImageJ software from 6 frames of 3 separate experiments. Data are mean ± SE. P < 0.05 compared to vehicle treated. C) KB-ChemR23 were grown on 10 cm2 dishes and exposed to either 1 µM chemerin or vehicle for 12 h. Shown are representative images of coimmunoprecipitations of ICAM-1 followed by Western blotting for CD55 (top row), similarly IP of CD55 was followed by blotting for ICAM-1 (bottom row). D) Subcellular fractionation was performed on KB-ChemR23 grown on 10 cm2 dishes and exposed to either 1 µM chemerin or vehicle for 12 h. MCF and LRF fractions were assayed for CD55 and ICAM-1 by Western blotting.

CD55 is a well-documented lipid-raft component (31) , and ICAM-1 has been demonstrated to be recruited to caveolae (32) . We next determined if increased CD55-ICAM-1 binding observed in chemerin-treated cells resulted in ICAM-1 internalization into a caveolar or lipid raft microdomain on ChemR23 activation As shown in Fig. 4D , the majority of CD55 increase associated with chemerin exposure occupied the MCF, although readily observable amounts of CD55 remained in the LRF. Notable was the observation that chemerin decreased membrane ICAM-1 and, concomitantly, increased lipid raft-associated ICAM-1 (Fig. 4D ). Such observations suggest that a close physical association exists between CD55 and ICAM-1. The antiadhesive phenotype of chemerin/RvE1-exposed epithelia likely results from both induction of CD55 and the sequestration of free apical ICAM-1 into a lipid raft microdomain, thereby preventing PMN adhesion through increased CD55 and decreased ICAM-1.

Based on these findings, it is likely that ChemR23-induced CD55 represents a proresolving mechanism as a terminal step in mucosal inflammation. In the past, CD55 has been viewed as a regulator of innate immunity through its regulation of the complement pathway (33) . CD55 is expressed in most tissues exposed to serum components, and CD55 expression has been shown, in human endothelial systems, to be augmented in response to numerous inflammatory stimuli, including cytokines (34 , 35) . Moreover, PMN express CD55 (36) , although less is known about the potential contribution of PMN CD55. In this regard, CD55 likely contributes at multiple steps along the inflammatory cascade.

Rodent models have provided significant insight into the role of CD55 in mucosal inflammation and in inflammatory resolution in vivo. Lin et al. (37) revealed that the course of colitis is significantly worsened in cd55–/– mice, reflected by a large increase in PMN accumulation and profound epithelial destruction. These investigations did not address whether ICAM-1 expression was different in cd55–/– animals. Although these observations could reflect aberrant complement activation related to the loss of CD55, it is also possible that loss of CD55-dependent PMN clearance could result in enhanced intestinal epithelial damage with concomitant loss of barrier. Consistent with our finding of resolvin-dependent CD55 induction in the present work, it was recently shown that resolvins are protective during experimental colitis (15) . Moreover, transgenic mice that endogenously synthesize omega-3 fatty acids generate large amounts of resolvins and are protected from colitic disease (14) . On this accord, chemerin represents an endogenous ChemR23 ligand that could be active even in the absence of omega-3 fatty acids. Indeed, chemerin is made as a precursor protein molecule with low biological activity and on proteolytic cleavage of the COOH-terminal domain is converted into a highly specific agonist of ChemR23 (12) . In our investigations, we found no apparent differences between chemerin or RvE1 in their ability to promote clearance of PMN.

Taken together, these studies identify a new paradigm of inflammatory resolution wherein resolvins actively promote mucosal epithelial clearance of PMN. Such a mechanism provides key insight into our understanding of balanced innate immune responses involving the interaction of barrier cell types with potentially autologously toxic PMN.


   ACKNOWLEDGMENTS
 
RvE1 was synthesized in Core C of P50-DE016191 by Dr. N. Petasis (University of Southern California) and validated in Core D of P50-DE016191 by R. Yang (Brigham and Women’s Hospital). We also acknowledge the technical assistance of K. Hamilton, D. Daniels, and M. Scully. This work was supported by National Institutes of Health Grants DK-50189, HL-60569, and DE-13499.


   FOOTNOTES
 
1 Current address: PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan.

Received for publication March 4, 2007. Accepted for publication April 12, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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