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The Journal of Immunology, 2008, 181, 2145 -2154
Copyright © 2008 by The American Association of Immunologists, Inc.
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Points of Control Exerted along the Macrophage-Endothelial Cell-Polymorphonuclear Neutrophil Axis by PECAM-1 in the Innate Immune Response of Acute Colonic Inflammation1

Naohito Sugimoto*,{dagger}, Tao Rui*, Min Yang*, Sulaiman Bharwani*, Osamu Handa*,{ddagger}, Norimasa Yoshida§, Toshikazu Yoshikawa{dagger} and Peter R. Kvietys2,*

* Critical Illness Research, Lawson Health Research Institute, London, Ontario, Canada; {dagger} Inflammation and Immunology, Graduate School of Medical Science, {ddagger} Department of Biomedical Safety Science, and § Molecular Gastroenterology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
PECAM-1 is expressed on endothelial cells and leukocytes. Its extracellular domain has been implicated in leukocyte diapedesis. In this study, we used PECAM-1–/– mice and relevant cells derived from them to assess the role of PECAM-1 in an experimental model of acute colonic inflammation with a predominant innate immune response, i.e., 2,4,6-trinitrobenzine sulfonic acid (TNBS). Using chimeric approaches, we addressed the points of control exerted by PECAM-1 along the macrophage-endothelial cell-polymorphonuclear neutrophil (PMN) axis. In vivo, TNBS-induced colitis was ameliorated in PECAM-1–/– mice, an event attributed to PECAM-1 on hematopoietic cells rather than to PECAM-1 on endothelial cells. The in vivo innate immune response was mimicked in vitro by using a construct of the vascular-interstitial interface, i.e., PMN transendothelial migration was induced by colonic lavage fluid (CLF) from TNBS mice or macrophages (M{Phi}) challenged with CLF. Using the construct, we confirmed that endothelial cell PECAM-1 does not play a role in PMN transendothelial migration. Although M{Phi} activation (NF-{kappa}B nuclear binding) and function (keratinocyte-derived chemokine production) induced by CLF was diminished in PECAM-1–/– M{Phi}, this did not affect their ability to promote PMN transendothelial migration. By contrast, PECAM-1–/– PMN did not adhere to or migrate across endothelial cell monolayers in response to CLF. Further, as compared with PECAM-1+/+ PMN, PECAM-1–/– PMN were less effective in orientating their CXCR2 receptors (polarization) in the direction of a chemotactic gradient. Collectively, our findings indicate that PECAM-1 modulation of PMN function (at a step before diapedesis) most likely contributes to the inflammation in a colitis model with a strong innate immune component.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Platelet endothelial cell adhesion molecule-1, PECAM-1, is a transmembrane glycoprotein localized on both endothelial cells and leukocytes, the most recognized function of which is the facilitation of leukocyte transendothelial migration (1, 2). Its extracellular domain can participate in homotypic adhesive interactions between leukocytes and endothelial cells. Its intracellular domain contains both ITAM and ITIM, which can participate in intracellular signaling (3). These properties have prompted evaluation of the role of PECAM-1 in the pathogenesis of various experimental models of tissue inflammation and associated dysfunction/injury (4, 5, 6, 7, 8). Overall, the results have been inconsistent; i.e., in some models the presence of PECAM-1 is beneficial, while in others it is deleterious.

The lack of consensus regarding the role of PECAM-1 in various models of inflammation can be attributed to a variety of factors including the following. The inflammatory milieu can influence the relative importance of PECAM-1 in leukocyte emigration (9). For example, in the absence of direct activators of leukocytes (e.g., chemokines), ligation of leukocyte PECAM-1 is required for up-regulation/activation of integrins to ensure appropriate adhesive interactions with the endothelium for leukocyte transendothelial migration. Conversely, in the presence of direct activators of leukocytes, leukocyte transendothelial migration can proceed independently of PECAM-1. Another confounding issue is the use of immunoneutralization approaches (Ab directed to PECAM-1) to block leukocyte-endothelial cell adhesive interactions, because ligation of the extracellular domain of PECAM-1 can also result in intracellular signaling mediated by its intracellular domain (3, 10, 11). Even when genetic blockade approaches (PECAM-1 deficient mice) were used, it appears that the requirement for PECAM-1 in the inflammatory response may be dependent on the mouse strain used (12, 13). Finally, the cellular localization of the PECAM-1 deficiency (endothelial cell vs leukocytes) appears to determine whether PECAM-1–/– mice are more or less sensitive to the induced pathology (4, 9).

Most inflammatory disorders are complex, involving both innate and adaptive immune systems (14, 15, 16). Immune cell recognition of nonspecific Ag derived from invading pathogens drives the innate immune response (e.g., LPS engagement of TLR on tissue macrophages). The adaptive immune response involves the processing of specific Ag by resident APC, which subsequently attract and activate circulating lymphocytes (e.g., dendritic cell activation of recruited T cells). Current consensus holds that the innate and adaptive arms of the immune system may not even be discrete components but rather represent a continuum (17), with some immune cells, receptors, and functional properties in common. Nonetheless, perhaps as a matter of convenience, the distinction is still generally maintained. There is some evidence to suggest that PECAM-1 may be proinflammatory in models with a predominant innate immune component (4) and anti-inflammatory in models with a predominant adaptive component (5). Because the innate immune response is more rapid and less complicated as compared with the adaptive immune response and may even be a prerequisite for the launching of the adaptive response (14, 15, 16), we focused on the innate arm of the immune system.

The major objective of the present study was to systematically assess the role of PECAM-1 in a model of tissue inflammation and dysfunction that maximizes the role of the innate immune response in its pathogenesis. To this end, we used an experimental model of colonic inflammation induced by a single rectal administration of 2,4,6-trinitrobenzine sulfonic acid (TNBS).3 This model has the following advantages relevant to the innate immune response: 1) mucosal injury with the potential for bacterial contamination (18); 2) a prominent role for macrophages and polymorphonuclear neutrophils (PMN) (19, 20, 21); and 3) development of colitis in the absence of T cells/B cells (22, 23). Further, it provides accessibility to a representative sample of mucosal interstitial fluid (via lavage) to address the points of control exerted by PECAM-1 along the macrophage (M{Phi})-endothelial cell-PMN axis, a hallmark feature of the innate immune response.

Using PECAM-1–/– mice, we show that PECAM-1 deficiency ameliorates the TNBS-induced colitis. Using a chimera approach, we provide evidence indicating that the presence of PECAM-1 on endothelial cells is not a prerequisite for the following: 1) pathogenesis of TNBS colitis in vivo; or 2) PMN transendothelial migration induced by a relevant milieu (colonic lavage fluid (CLF)) in vitro. Further in vitro studies indicate that PECAM-1 plays an important role in the conversion of both M{Phi} and PMN to a proinflammatory phenotype. However, PMN PECAM-1, but not M{Phi} PECAM-1, plays an important role in PMN transendothelial migration.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Male C57BL/6 mice were purchased from Charles River Canada and breeding pairs of PECAM-1–/– mice on a C57BL/6 background (backcrossed >10 generations) were a gift from W. A. Muller (Department of Pathology, Cornell University, New York, NY). For one set of experiments, CXCR2–/– mice were generated as follows. CXCR2+/– mice on a BALB/c background (The Jackson Laboratory) were bred with wild type (CXCR2+/+) C57BL/6 mice. The heterozygous hybrids were then mated to generate CXCR2–/– on a BALB/c-C57BL/6 hybrid background. Homozygous (CXCR2+/+) hybrid littermates were used as controls. All breeding and confirmatory genotyping was performed by the Mouse Core at the Lawson Health Research Institute (London, Ontario, Canada). The mice were housed in open-top shoe box caging in a 12:12 h light/dark cycle with controlled temperature (22–24°C) and humidity (38–42%) in rooms under positive pressure with respect to the external environment to avoid exogenous pathogen contamination. This animal study was reviewed and approved by the University of Western Ontario Committee on Animal Care (London, Ontario, Canada).

TNBS-induced colitis

To induce colitis, 200 mg/kg TNBS (Sigma-Aldrich) dissolved in 30% ethanol was administered intrarectally to lightly anesthetized (ketamine/xylazine) mice via a catheter (20). Control mice received 30% ethanol. Three days after TNBS administration, the colon was excised and the severity of damage was graded according to established criteria (20). Macroscopic grading included the presence of visible damage, serosal adhesions, diarrhea, strictures, and bowel wall thickening. Histological (H&E-stained paraffin sections) grading included the extent of damage (from superficial mucosal to transmural), degree of inflammatory cell infiltrate, and signs of regeneration (assessed blindly by author S.B.). In addition, samples of colon (same regions as used for histology) were used for measurement of myeloperoxidase (MPO) activity.

Chimeras

Donor bone marrow (BM) was infused into irradiated recipient mice, and the BM was allowed to reconstitute over 6 wk (24). Two reciprocal PECAM-1 chimeras were created: PECAM-1 knockout (KO) BM transplanted into wild-type (WT) recipient (KO to WT; PECAM-1 localized to recipient parenchymal cells) and WT BM transplanted into PECAM-1–/– recipient (WT to KO; PECAM-1 localized to donor hematopoietic cells). WT BM transplanted into WT recipient (WT to WT) served as a control. After BM reconstitution, the mice were treated with TNBS.

M{Phi} depletion

Liposomes containing dichloromethylene diphosphonate (Cl2MDP) were prepared as previously described (24). Briefly, phosphatidylcholine and cholesterol were dissolved in chloroform and the solution was evaporated and then dispersed by mixing with 10 ml of PBS containing 2.5 g of Cl2MDP (Sigma-Aldrich). This mixture was sonicated and the resulting liposomes were washed to remove the nonencapsulated Cl2MDP. WT mice were treated with 10 mg of Cl2MDP-liposome suspended in 100 µl of PBS by rectal administration on days 1 and 3. The mice were treated with TNBS on day 7.

Cells from mice

Mesenteric microvessel endothelial cells (MMEC) were isolated using a modification of a previously described method (25). Briefly, the mesentery was minced, digested (300 U/ml collagenase II and 0.6 U/ml dispase II in HBSS) for 35 min at 37°C, and filtered (100-µm nylon mesh). The filtrate was washed and subsequently coincubated with rat anti-mouse PECAM-1 Ab (BD Biosciences) at 4°C for 30 min. For some experiments (in which PECAM-1–/– endothelial cells were required), a rat anti-mouse ICAM-2 Ab was used. The suspension was washed and coincubated with microbeads coated with sheep anti-rat IgG for 20 min, and cells were captured by a Dynal Magnet. The captured MMEC were seeded in fibronectin-coated flasks and cultured in Endothelial Cell Basal Medium-2 (EBM-2; Clonetics) supplemented with EGM-2-MV (Clonetics). The MMEC cultures were >85% pure as assessed by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine (Dil)-acetylated low density lipoprotein uptake and E-selectin expression in response to LPS. An inherent assumption is that the endothelial cells were of blood vessel origin. However, contamination by lymphatic endothelial cells cannot be excluded. Irrespective of that possibility, when the MMEC were activated by IL-1β, TNF-{alpha}, or LPS, PMN migration across MMEC monolayers was increased (Table I) as predicted by studies using endothelial cells obtained from the lining of blood vessels (HUVEC). First and second passage cells were used for experiments.


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  		Table I. Effects of cytokines and LPS on the transendothelial migration of WT and PECAM-1–/– (KO) PMNa

 
PMN were isolated from the BM of adult mice as previously described (25). The long bones of the hind legs were removed and flushed with ice-cold Ca2+- and Mg2+- free HBSS. The obtained marrow was centrifuged, resuspended, and subjected to a Percoll (Sigma-Aldrich) step gradient. Cells were removed from the neutrophil-enriched fraction. This procedure yielded 4–5 million white blood cells, 95% of which were adult PMN.

Nonelicited M{Phi} were isolated from the peritoneal cavities of mice by lavage with PBS containing 5 IU/ml sodium heparin. Cells were washed and resuspended in DMEM containing 10% FCS, 100 U/ml penicillin, and streptomycin. Cells were seeded in 24-well plates and allowed to adhere for 1 h at 37°C. Nonadherent cells were removed by washing with DMEM. The remaining adherent cells consisted of M{Phi} (>95% purity) and were used in experiments after 24 h in culture.

Construct of the vascular-interstitial interface

MMEC were grown to confluence on fibronectin (25 mg/ml)-coated inserts (3-µm pore diameter). A chemotactic gradient was established in the basal compartment and 51Cr-labeled PMN were added to the apical aspect of the endothelial cell monolayers. PMN transendothelial cell migration was assessed 1 h later and quantitated as the percentage of added PMN that entered the basal compartment (25). To mimic the colonic mucosal vascular-interstitial interface, the chemotactic gradient was established using CLF or CLF-challenged M{Phi}.

CLF was obtained using a modification of a previously described approach used in inflammatory bowel disease (IBD) patients to gain insight into the colonic mucosal inflammatory status (26). Briefly, 2 days after the induction of colitis with TNBS, the colonic lumen was washed with MEM until particulate matter was removed. Subsequently, 1 ml of MEM was instilled into the rectum for 3 min to allow for equilibration with the mucosal interstitium. The fluid was removed and filtered (0.2 µm). This approach to obtaining a sample representative of the colonic mucosal milieu is based on the premise that the mucosal barrier is breached in colitis, allowing for equilibration of the contents of the mucosal interstitium with the instilled fluid. In this respect it provides a sample of the colonic interstitium akin to that in draining lymphatics. As a control, CLF was obtained from ethanol-treated mice.

Assays

MPO activity in the colon tissue (index of PMN infiltration) was measured as previously described (27). Briefly, colonic tissue was homogenized and sonicated in detergent buffer. The prepared samples were used in reactions for MPO activity determined spectrophotometrically (650 nm) by measuring hydrogen peroxide-dependent oxidation of 3,3',5,5'-tetramethylbenzidine.

EMSA was used to measure the nuclear binding of NF-{kappa}B as previously described (28). Nuclear protein was extracted from M{Phi} and 3 µg of the extract was incubated with 1 picomole of double-stranded [{gamma}-32P]ATP end-labeled oligonucleotide containing a consensus (sense strand: 5'-AGGGACTTTCCGCTGGGGACTTTCC-3') binding sequence for NF-{kappa}B. After 30 min of coincubation, the samples were electrophoresed (4% nondenaturing polyacrylamide gel), dried, and exposed to x-ray film.

ELISA was used to quantitate the concentration of the chemokines KC (keratinocyte-derived chemokine) and LIX (LPS-induced CXC chemokine) in colonic tissue, CLF, and supernatants from M{Phi} as described previously (29). Samples (tissue homogenates and supernatants) were incubated with rat anti-mouse mAb directed to KC (R&D Systems), LIX (R&D Systems), and TNF-{alpha} (BioLegend) and Ab binding intensity measured (ExtrAvidin peroxidase staining kit; Sigma-Aldrich).

PMN elastase activity was assessed as previously described (30). In brief, suspensions of PMN were reacted with a fluorogenic elastase substrate (N-succinyl-(L-alanine)3-p-nitroanilide) for 1 h. Absorbance was read at 415 nm and elastase activity was determined from a standard curve using purified human PMN elastase.

PMN adhesion to endothelium was assessed by adding 51Cr-labeled PMN to confluent MMEC monolayers (31). After 30 min. of coincubation, the percentage of added PMN remaining adherent after a wash procedure was quantitated.

PMN CXCR2 polarization was assessed as previously described (32). Suspensions of PMN were added to glass coverslips and the PMN were allowed to adhere for 10 min at 37°C. The coverslips were inverted and the cell-covered portion was centered above the bridge of Zigmond chambers. IL-8 (20 nM) in Hank’s buffer (containing 1% BSA) was added to one groove along the bridge and the vehicle was added to the other groove. After a 10-min incubation at 37°C, the PMN were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were blocked with 2% BSA for 30 min and a rabbit anti-mouse polyclonal Ab to CXCR2 (Abcam) was added. After an overnight incubation at 4°C, the cells were washed and a FITC-conjugated goat anti-rabbit IgG Ab (Bethyl Laboratories) was added. The coverslips were treated with a drop of anti-fade reagent and examined using fluorescence microscopy.

Statistics

All values are expressed as mean ± SEM. Histological scores were analyzed by the Mann-Whitney U test. All other data were analyzed by ANOVA and differences were analyzed by Fischer’s protected least significant difference multiple-comparison test. Differences between the groups were considered significant if the p < 0.05. All analyses were performed using the Stat View 5.0-J program (Abacus Concepts).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TNBS-induced colitis is mitigated in PECAM-1–/– mice

By 3 days after a single intrarectal administration of TNBS (200 mg/kg) in 30% ethanol (EtOH), the mice had lost a significantly greater amount of weight (18.5 ± 2.6%) than control animals receiving only EtOH (2.7 ± 2.1%). The TNBS-induced weight loss was significantly minimized in PECAM-1–/– mice (12.4 ± 3.5%).

Predictively, intrarectal administration of TNBS induced colitis, i.e., colonic inflammation and injury. There was evidence of both macroscopic and histologic injury (Fig. 1, A–C). Colonic PMN infiltration was evidenced by an increase in MPO activity (Fig. 1D). Histologic assessment of the infiltrates (Fig. 1B) indicated that PMN comprised ~60% while monocytes/macrophages comprised 40% of the infiltrated cells. Colonic edema was also present as indicated by an increase in the colonic wet/dry weight ratio (data not shown) and histologic examination (Fig. 1B). All of the above salient features of colitis were significantly reduced when TNBS was administered to PECAM-1–/– mice (Fig. 1). These observations indicate that PECAM-1 facilitates the development of TNBS-induced colitis, a response consistent with the proposed paradigm predicting a proinflammatory role for PECAM-1 in the innate immune response.


Figure 1
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  		FIGURE 1. PECAM-1 plays a role in TNBS-induced colitis. TNBS (200 mg/kg) in 30% EtOH was given intrarectally to WT and PECAM-1–/– (KO) mice. As a control, 30% EtOH was given intrarectally to WT mice. Indices of colonic mucosal inflammation and injury were evaluated 3 days later. A, Macroscopic injury. B, Histologic injury. C, Quantitation of macroscopic and histologic injury. D, Colonic MPO activity (neutrophil infiltration). n = 10; *, p < 0.05 compared with EtOH-treated WT mice; #, p < 0.05 compared with TNBS treated WT mice.

 
Hematopoietic cell PECAM-1, but not endothelial cell PECAM-1, is involved in TNBS-induced colitis

PECAM-1 is ideally localized to facilitate leukocyte transendothelial migration, i.e., on hematopoietic cells (monocytes, PMN, and some subsets of lymphocytes) and endothelial cell junctions, the major sites of leukocyte penetration of the endothelial lining of microvessels (1, 2). To determine, the relative contribution of endothelial and hematopoietic cell PECAM-1 to the TNBS-induced colitis, we generated reciprocal chimeras deficient in endothelial cell or hematopoietic cell PECAM-1. As expected, the WT to WT chimeras (control; all cells PECAM-1+/+) developed colitis as evidenced by an increase in PMN infiltration (increased MPO activity) and histologically demonstrable injury (Fig. 2A). The WT to KO chimeras (PECAM-1–/– endothelial cells and PECAM-1+/+ hematopoietic cells) also developed colitis. By contrast, the KO to WT chimeras (PECAM-1+/+ endothelial cells and PECAM-1–/– hematopoietic cells) exhibited a significantly reduced colitis (Fig. 2A). These findings indicate that endothelial cell PECAM-1 does not play a role in the pathogenesis of TNBS colitis. Rather, hematopoietic cell PECAM-1 is a prerequisite for the development of TNBS-induced colitis.


Figure 2
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  		FIGURE 2. A, PECAM-1 on hematopoietic cells, but not endothelial cells, is involved in TNBS-induced colitis. Reciprocal PECAM-1–/– chimeras were created by BM reconstitution of BM-depleted (irradiated) mice. Two reciprocal chimeras were generated: PECAM- 1–/– BM transplanted into WT recipient (KO to WT) and WT BM transplanted into PECAM-1–/– recipient (WT to KO). WT BM transplanted into WT recipient (WT to WT) served as a control. Six weeks after reconstitution, TNBS colitis was induced according to the protocol described in Fig. 1. n = 7; *, p < 0.05 compared with EtOH-treated WT mice; #, p < 0.05 compared with TNBS-treated WT to KO chimeras or WT to WT mice. B, To deplete colonic mucosal macrophages, mice were given liposomes containing dichloromethylene diphosphonate (CL2MDP liposome; 10 mg/mouse) intrarectally on days 1 and 4. PBS containing liposomes was used as a control. The mice were treated with TNBS on day 7. Three days after the induction of colitis by TNBS the colons were harvested for assessment of MPO activity and histologic evaluation. n = 5; *, p < 0.05 compared with EtOH within the relevant liposome group; #, p < 0.05 compared with TNBS in PBS liposome-treated mice.

 
PMN infiltration of tissue is facilitated by the generation of a chemotactic gradient by resident immune cells. Colonic M{Phi} have been implicated in other models of colonic inflammation (33) and, of particular relevance to the present study, the Fc{gamma}R function of peritoneal M{Phi} can be modulated by PECAM-1 (4). Thus, we verified whether M{Phi} play a role in our model of TNBS colitis by using Cl2MDP liposomes to deplete colonic M{Phi}. As shown in Fig. 2B, in control mice (given PBS liposomes), there were the expected TNBS-induced colonic inflammation and injury. In mice treated with Cl2MDP liposomes, the severity of the inflammation and injury noted in the TNBS model was significantly diminished.

An in vitro construct of the vascular-interstitial interface mimics the innate immune response of TNBS-induced colitis

To address the potential mechanism(s) by which PECAM-1 modulates TNBS-induced colitis, we used an in vitro approach designed to mimic the situation in vivo. To this end, we developed an in vitro construct of the colonic vascular-interstitial interface incorporating the following: 1) cell types relevant to the innate immune response (M{Phi}, endothelial cells, and PMN); and 2) a fluid phase milieu (CLF) relevant to the TNBS model (CLF cytokine/chemokine levels reflect colonic levels) (Fig. 3, A–D).


Figure 3
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  		FIGURE 3. A comparison of the concentration of the chemokines KC and LIX and the cytokine TNF-{alpha} in CLF) and homogenates of the colon. A–C, KC (A), LIX (B), and TNF-{alpha} (C) were measured by ELISA in samples of CLF (undiluted) or homogenates of the colon 2 days after TNBS treatment of WT or PECAM-1–/– (KO) mice. *, p < 0.05 compared with WT; #, p < 0.05 compared with TNBS treated WT. D, Regression analysis of the TNF-{alpha} data presented in C. Similar correlations were noted with the chemokine data presented in A and B (r2 values of 0.981 and 0.856, respectively).

 
When used in the construct, CLF obtained from EtOH-treated mice had no effect on PMN transendothelial migration relative to MEM (compare Fig. 4, A and B). CLF from TNBS-treated mice promoted PMN migration in a dose-dependent manner (Fig. 4A). Although CLF contained ~10 ng/ml LPS, this concentration of LPS did not directly promote PMN transendothelial migration (Fig. 4B). Based on the submaximal effect of 20% CLF on PMN transendothelial migration, this concentration of CLF was used for further studies.


Figure 4
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  		FIGURE 4. The CLF-induced PMN transendothelial migration is not due to LPS. A, CLF was obtained from TNBS- or EtOH-treated mice and added to the basal aspect of the endothelial monolayers. PMN were added to the apical aspect. After 1 h of coincubation, PMN transendothelial migration was assessed. The CLF-induced PMN transendothelial migration was dependent on the concentration of CLF. *, p < 0.05 compared with EtOH. #, p < 0.05 compared with 50% CLF. B, LPS at concentrations measured in CLF (10 ng/ml; inset) did not induce PMN transendothelial migration.

 
M{Phi} challenged with 20% CLF could promote PMN migration. The PMN transendothelial migration induced by CLF-challenged M{Phi} was blunted by an Ab directed to the LPS receptor, TLR4 (Fig. 5). However, the addition of polymyxin B (10–30 µg/ml) to CLF to neutralize LPS did not affect the PMN migration induced by CLF-challenged M{Phi} (data not shown). This finding suggests that other constituents of CLF, other than LPS, are interacting with TLR4 to activate the M{Phi}. Likely candidates include breakdown products of interstitial matrix (e.g., fibronectin) (34) or cells (e.g., HMGB1) (35) that have entered the CLF from the inflamed/injured colonic mucosa. Irrespective of this, the ability of CLF to activate M{Phi} (with respect to promoting PMN migration) is dependent to a large extent on the engagement of M{Phi} TLR4 receptors.


Figure 5
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  		FIGURE 5. CLF can stimulate peritoneal macrophages (M{Phi}) to promote PMN transendothelial migration, an effect mediated by TLR4. M{Phi} adherent to the bottom of the basal compartment of the insert were challenged with CLF (20%) for 2 h, washed, incubated with endothelial monolayers for 4 h, and PMN transendothelial migration was assessed. Unstimulated M{Phi} did not promote migration whereas CLF-challenged M{Phi} increased PMN migration. Pretreatment of the M{Phi} with an Ab directed to TLR4 (10 ng/ml) for 30 min reduced the PMN migration induced by CLF-challenged M{Phi}. n = 3; *, p < 0.05 compared with M{Phi} treated with MEM. #, p < 0.05 compared with M{Phi} treated with CLF.

 
Using both immunoneutralization (Ab to KC and LIX) and a genetic blockade (CXCR2–/– PMN), it appears that the PMN transendothelial migration induced by CLF-challenged M{Phi} could be attributed to the engagement of CXCR2 on PMN by the M{Phi}-derived chemokines KC and LIX (Fig. 6).


Figure 6
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  		FIGURE 6. Chemokines play a role in the PMN transendothelial migration induced by CLF or CLF-challenged M{Phi}. A, PMN transendothelial migration induced by 20% CLF was assessed (see Fig. 4A for details) in the absence or presence of an Ab to LIX or KC (1 µg/ml) in the basal compartment. The Ab reduced the CLF-induced PMN migration. n = 3; *, p < 0.05 compared with MEM control; #, p < 0.05 compared with CLF in the absence of Ab. B, PMN transendothelial migration induced by macrophages challenged with 20% CLF was assessed (see Fig. 4C for details) in the absence or presence of an Ab to LIX or KC (1 µg/ml) in the basal compartment. The Ab reduced the PMN migration induced by CLF-challenged M{Phi}. *, p < 0.05 compared with MEM control; #, p < 0.05 compared with CLF-treated M{Phi} in the absence of Ab. C, Same experimental protocol as in B, except that PMN isolated from CXCR2–/– mice (BALB/c-C57BL/6 hybrid) or WT mice (WT; BALB/c-C57BL/6 hybrid) were used. The CLF-induced PMN migration was abrogated when CXCR2–/– PMN were used. n = 3; *, p < 0.05 compared with MEM-M{Phi} within relevant group; #, p < 0.05 compared with CLF-M{Phi} using CXCR2–/– PMN.

 
Collectively, these findings indicate that this in vitro construct allows for a systematic assessment of the points of control exerted by PECAM-1 along a M{Phi}-endothelial cell-PMN axis relevant to the role of the innate immune response in our TNBS model.

PECAM-1 on M{Phi} is not a prerequisite for M{Phi}-induced PMN transendothelial migration

PECAM-1 can modulate M{Phi} Fc{gamma}R-mediated phagocytosis and cytokine production (4). Our results indicate that PECAM-1 can also modulate M{Phi} Fc{gamma}R-independent activation and function. WT M{Phi} had increased binding of NF-{kappa}B to nuclear protein by 4 h after exposure to CLF, whereas CLF-challenged PECAM-1–/– M{Phi} did not (Fig. 7A). In addition, WT M{Phi} challenged with CLF generated the chemokine KC into the surrounding milieu 4 h after challenge, a response blunted in PECAM-1–/– M{Phi} (Fig. 7B). However, when PMN transendothelial migration induced by CLF-challenged M{Phi} was assessed 4 h after challenge, the migration was similar irrespective of whether PECAM-1-competent or PECAM-1-deficient M{Phi} were used (Fig. 7C). To determine whether M{Phi} PECAM-1 could modulate PMN transendothelial migration before translation/transcription events, CLF-challenged M{Phi} were immediately used in the migration assay. Transendothelial migration of WT PECAM-1 was significantly increased (1.7 ± 0.2-fold) by M{Phi} challenged with CLF from TNBS-treated mice as compared with M{Phi} challenged with CLF from EtOH-treated mice. The results were similar when PECAM-1–/– M{Phi} were used (1.9 ± 0.4-fold increase). Collectively, these findings indicate that while PECAM-1 facilitates CLF-induced M{Phi} activation (with respect to nuclear translocation of NF-{kappa}B and KC production), this effect does not translate into an effect on PMN transendothelial migration.


Figure 7
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  		FIGURE 7. M{Phi} PECAM-1 modulates CLF-induced M{Phi} activation, but not M{Phi}-induced PMN transendothelial migration. A, WT or PECAM-1–/– (KO) M{Phi} were coincubated with CLF for 4 h. Subsequently, nuclear proteins were extracted and the binding of NF-{kappa}B was assessed by EMSA. The CLF-induced increase in M{Phi} nuclear NF-{kappa}B was abolished in KO M{Phi}. Representative EMSA is shown and the experiments were repeated twice with similar results. B, WT or PECAM-1–/– (KO) M{Phi} were coincubated with CLF for 2 h, washed, and incubated with MEM for an additional 4 h. Subsequently, the supernatants were collected and assayed for KC using ELISA. The CLF-induced increase in M{Phi} generation of KC was mitigated in KO M{Phi}. C, PMN transendothelial migration induced by WT and PECAM-1–/– (KO) M{Phi} challenged with CLF was assessed as described in Fig. 4C. n = 3; *, p < 0.05 compared with MEM-treated WT; #, p < 0.05 compared with CLF-treated WT.

 
PECAM-1 on PMN facilitates PMN-endothelial cell adhesive interactions

It is generally accepted that homotypic PECAM-1 adhesive interactions play an important role in leukocyte transendothelial migration (1, 2). Thus, the roles of endothelial cell and PMN PECAM-1 were evaluated in the in vitro construct of the vascular-interstitial interface. As shown in Fig. 8A, the PMN migration induced by CLF was not affected when PECAM-1–/– endothelial cells were used. By contrast, when PECAM-1–/– PMN were used in the construct, the CLF-induced PMN migration was blunted (Fig. 8B). These observations are in general agreement with our in vivo studies using chimeras (Fig. 2A) and indicate that PECAM-1 may be modulating PMN function rather than simply acting as an adhesion molecule facilitating PMN transendothelial migration.


Figure 8
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  		FIGURE 8. PMN PECAM-1 modulates PMN transendothelial migration. PMN transendothelial migration induced by 20% CLF was assessed as described in Fig. 4A. A, WT or PECAM-1-deficient (KO) MMEC were used in the migration assay; the PMN were from WT mice. The CLF-induced PMN migration was increased to the same extent whether WT or KO MMEC were used. n = 3. B, WT or PECAM-1–/– (KO) PMN were used in the migration assay; the MMEC were from WT mice. The CLF-induced PMN migration was mitigated when KO-PMN were used. n = 3; *, p < 0.05 compared with WT PMN treated with CLF.

 
PMN transendothelial migration is facilitated by PMN-derived elastase (1, 2, 30). As shown in Fig. 9A, PMN elastase activity was increased by CLF regardless of whether WT or PECAM-1–/– PMN were assayed. These observations indicate that PECAM-1 deficiency impairs an important step upstream of the migration process itself. Thus, we assessed the role of PMN PECAM-1 in the PMN adhesion to endothelium in response to CLF. As shown in Fig. 9B, CLF increased the adhesion of WT PMN to MMEC, an effect not noted when PECAM-1–/– PMN were used in the assay.


Figure 9
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  		FIGURE 9. PMN PECAM-1 modulates PMN function at a step upstream to diapedesis. A, PMN were challenged with 20% CLF from TNBS-treated mice (TNBS) or EtOH-treated mice (EtOH). A, PMN elastase activity was increased to the same extant by CLF from TNBS mice whether WT or KO (PECAM-1–/–) PMN were used in the assay. n = 3; *, p < 0.05 compared with EtOH in the relevant group. B, The increase in PMN adhesion to endothelial cell monolayers induced by CLF from TNBS treated mice was reduced when KO PMN were used. n = 3; * p < 0.05 compared with EtOH in relevant group. C, CXCR2 immunostaining of WT and PECAM-1–/– (KO) PMN in an IL-8 gradient (arrow). Representative images from 2–3 experiments are shown. The percentages of PMN exhibiting CXCR2 polarization are shown below relevant image. The percentage values were obtained by counting at least 100 cells for each experimental condition.

 
Because PECAM-1-deficient megakaryocytes have a defect in the polarization of their chemokine receptor (CXCR4) in response to a gradient of stromal cell-derived factor 1 (32) and because PMN CXCR2 is critical for the PMN transendothelial migration in our model (Fig. 6), we assessed whether PMN PECAM-1 modulates PMN CXCR2 polarization in response to a chemotactic gradient of IL-8. IL-8 was chosen as the chemotactic stimulus based on previous studies indicating that IL-8 was much more potent then KC in promoting PMN migration on coverslips in Zigmond chambers (36). In this assay, PMN migrated in the direction of the IL-8 gradient (our unpublished observations), confirming the results of the latter report. As shown in Fig. 9C, the distribution CXCR2 staining was uniform in the cell membranes of WT PMN not exposed to a chemotactic gradient. The establishment of an IL-8 gradient resulted in a much greater degree of CXCR2 staining in the portion of the membrane exposed to the highest concentration of IL-8 (CXCR2 polarization in the direction of the IL-8 gradient). Quantitative analyses indicated that 50% of the WT PMN exhibited CXCR2 polarization in the direction of the IL-8 gradient, whereas only 12% of the PECAM-1–/– PMN responded to the gradient with CXCR2 polarization.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we assessed the role of PECAM-1 in a model of tissue inflammation and dysfunction that maximizes the role of the innate immune response in its pathogenesis, i.e., TNBS-induced acute colitis. Herein, we show that PECAM-1 deficiency ameliorates the inflammation and dysfunction in TNBS-induced colitis (Fig. 1). Further, a systematic in vivo and in vitro analysis of the points of control exerted by PECAM-1 along the macrophage-endothelial cell-PMN axis, a key anatomical feature of the innate immune system, provided the following novel insights. First, a chimeric approach indicated that PECAM-1 on endothelial cells does not play an important role in PMN emigration in vivo and PMN transendothelial migration in vitro. Second, PECAM-1 appears to serve as a facilitator/enhancer of both macrophage function (chemokine production) and PMN function (CXCR2 polarization and adhesivity for endothelium) in this milieu. Third, PMN PECAM-1, rather than M{Phi} PECAM-1, is the primary determinant of PMN transendothelial migration.

Our chimera studies indicated that PECAM-1 on BM-derived hematopoietic cells plays a role in TNBS-induced inflammation and injury (Fig. 2A). Macrophages have been implicated in several experimental models of colitis (19, 33), and the results of the present study confirm an important role for M{Phi} in our TNBS model (Fig. 2B). In addition, we show that M{Phi} challenged by CLF can promote PMN transendothelial migration, an effect mediated via TLR4 receptors on M{Phi} and CXCR2 receptors on PMN (Figs. 5 and 6) and a hallmark feature of an innate immune response. Previous studies have shown that M{Phi} generate the cytokine TNF when Fc{gamma}Rs are engaged (IgG-coated beads), a response substantially diminished in PECAM-1-deficient M{Phi} (4). Herein, we show that CLF can induce nuclear translocation of NF-{kappa}B and KC production in WT M{Phi}, effects markedly diminished in PECAM-1-deficient M{Phi} (Fig. 7, A and B). Thus, these latter observations indicate that PECAM-1 can modulate M{Phi} innate immune function independently of Fc{gamma}R ligation.

The question arises as to whether PECAM-1 modulation of M{Phi} activation/function translates into an effect on M{Phi}-induced PMN transendothelial migration. In a previous study in which PECAM-1 was shown to modulate M{Phi} Fc{gamma}R-dependent function, PMN transendothelial migration was not assessed (4). In the present study, the PMN transendothelial migration induced by CLF-activated M{Phi} was similar whether WT or PECAM-1–/– M{Phi} were used in the assay (Fig. 7C). These observations indicate that the ability of PECAM-1 to modulate M{Phi} activation does not necessarily translate into an effect on M{Phi}-induced PMN transendothelial migration. Thus, the impact of PECAM-1–/– M{Phi} dysfunction in our in vivo model is not entirely clear and warrants further attention.

Homotypic adhesive interactions between PECAM-1 on leukocytes and endothelial cells are considered to be important in leukocyte transendothelial migration, particularly in a milieu where endothelial cells are activated without direct activation of leukocytes (e.g., cytokine challenge of endothelium) (1, 2, 9). In this situation, ligation of leukocyte PECAM-1 by endothelial cell PECAM-1 serves to up-regulate/activate leukocyte integrins, thereby facilitating leukocyte adhesion to endothelium and subsequent emigration. In agreement with this contention, we noted that PECAM-1–/– PMN transendothelial migration was defective when the endothelium was activated with cytokines or LPS (Table I). Conversely, in a milieu containing inflammatory mediators that can directly activate leukocytes (e.g., chemokines), endothelial cell PECAM-1 appears to be redundant and not required for the migration process. In the TNBS model of colitis, both endothelial cell activators (e.g., TNF-{alpha}) and leukocyte activators (KC and LIX) were detected in colonic tissue (Fig. 3). Similarly, in our in vitro construct CLF also contained these same inflammatory mediators (Fig. 3). Our in vivo (Fig. 2A) and in vitro (Fig. 8) chimera approaches indicated that endothelial cell PECAM-1 does not play an important role in PMN emigration. Collectively, these findings appear to be consistent with the contention that the role of PECAM-1 homotypic adhesive interactions between leukocytes and endothelium should not be important in the colonic inflammation when the interstitial milieu contains direct activators of leukocytes.

Interestingly, we also found that the CLF-induced PMN transendothelial migration was dependent on PMN PECAM-1 (Fig. 8B) despite the fact that CLF contains KC and LIX (Fig. 3). These findings are consistent with a previous study indicating that PECAM-1–/– PMN transendothelial migration in response to IL-8 is retarded as compared with WT PMN (36). Thus, collectively these findings indicate that PMN PECAM-1 plays an important role in PMN chemotaxis and transendothelial migration even in a milieu containing direct activators of PMN.

PMN transendothelial migration is dependent on PMN elastase activity (30). The role of PECAM-1 in modulating PMN elastase activity is equivocal (37, 38). In the present study, PMN membrane elastase activity was increased by CLF to the same extant in PECAM-1-competent and PECAM-1-null PMN (Fig. 9A). Interestingly, in the study in which PECAM-1 was shown to modulate elastase activity, a PECAM-1 Ab was used (37). This observation may reflect the ability of Ab directed to PECAM-1 to induce PECAM-1-mediated intracellular signaling (3, 10, 11). Irrespectively, in the studies in which PECAM-1–/– PMN are used (Ref. 38 and this study), PECAM-1 has no effect on PMN elastase activity. Taken together, these observations would suggest that the defect in migratory ability of PMN deficient in PECAM-1 may lie proximal to the diapedesis step.

PMN diapedesis requires that PMN first adhere to the endothelium via PMN β2 integrin adhesive interactions with the endothelial cell ICAM-1. Ligation of PECAM-1 on monocytes and PMN can induce up-regulation/activation of their β2 integrins (10). Whereas the rate of adhesion of monocytes to endothelial cells is increased after the ligation of monocyte PECAM-1 with Ab, no comparable assessment has been made with PMN. In this study we show for the first time that CLF-induced PMN adhesion to endothelial cells is substantially reduced when PECAM-1-null PMN are used (Fig. 9B). This observation was rather surprising because CLF contains a sufficient amount of chemokines to activate PMN and promote their transendothelial migration (Fig. 6), and previous studies using PECAM-1 Ab indicated that immunoneutralization of PECAM-1 would not affect or increase leukocyte adhesion to endothelium (2, 10). The immunoneutralization approach may have been complicated by PMN activation via outside-in signaling upon the ligation of PMN PECAM-1, and this may in part account for the discrepancy between results obtained using PECAM-1–/– PMN vs PECAM-1 Ab. However, the discrepancy between our observations and those of a previous study using PECAM-1–/– PMN is more difficult to reconcile. The previous study showed that IL-8-induced adhesion of PMN to fibronectin, fibrinogen, or BSA is independent of PMN PECAM-1 (36). Although an explanation for these discordant observations is not readily available, the most obvious difference is that PMN adhesion to protein substrates was addressed in the previous study whereas in the present study PMN adhesion to endothelium was assessed.

Chemokine receptors (CCR2 and CCR5) on lymphocytes, but not a chemotactic receptor (C5aR) on PMN, polarize to the migrating front in response to a chemotactic gradient (39). In the present study, we provide evidence that the CXCR2 on PMN does, in fact, polarize to the leading front in a chemotactic gradient (Fig. 9C). Because PECAM-1–/– PMN exhibit a loss of motility and directionality on coverslips in response to a CXCR2 gradient (36), we assessed whether PECAM-1 plays a role in the chemokine-induced PMN CXCR2 polarization. As shown in Fig. 8C, PECAM-1–/– PMN do not polarize their CXCR2 in response to a chemotactic gradient. These observations provide a possible explanation for the migratory defect in PECAM-1–/– PMN on coverslips (36) and across endothelial cell monolayers (Fig. 8). Further studies are warranted to address the mechanistic linkages between PECAM-1, CXCR2 polarization, and PMN function.

Our findings in an acute model of colonic inflammation, as well as those in a previous study using another model characterized by a predominantly innate immune response (immune complex deposition in the lung) (4), indicate that endothelial cell PECAM-1 does not play a role in the resultant tissue inflammation and dysfunction. However, endothelial cell PECAM-1 does appear to play a role in experimental autoimmune encephalitis; a model with a predominant adaptive immune response involving T cell infiltration (5). In this model, endothelial cell PECAM-1 was not directly involved in promoting T cell transendothelial migration but rather was required for the maintenance of endothelial barrier function (5). Interestingly, in endotoxin shock models (no evidence of an inflammatory response in organ systems), endothelial cell PECAM-1 was also deemed important, again primarily for the maintenance of endothelial barrier integrity (40, 41). There is no readily apparent explanation for these rather discordant findings. However, it is of interest to note that in those models in which PECAM-1 deficiency exacerbated tissue injury, endothelial PECAM-1 was deemed responsible (5, 41), whereas in those models in which PECAM-1 deficiency was protective, PECAM-1 on bone marrow-derived cells (e.g., leukocytes) was deemed responsible (Ref. 4 and this study).

In summary, our results indicate that endothelial cell PECAM-1 does not play a role in leukocyte emigration in a model of inflammation characterized by a strong innate immune component, i.e., TNBS-induced colitis. Furthermore, it appears that PECAM-1 can modulate the function of two cells of the innate immune system involved in the TNBS colitis, i.e., M{Phi} and PMN. PMN PECAM-1 appears to be a prerequisite for PMN polarization of CXCR2 receptors, adherence to, and migration across endothelium. However, while PECAM-1 facilitates M{Phi} activation (nuclear translocation of NF-{kappa}B) and function (KC production), it does not appear to be required for M{Phi}-induced PMN transendothelial migration. Thus, with regard to immune cells, these findings indicate the PECAM-1 modulation of PMN function, rather than M{Phi} function, is the means by which PECAM-1 deficiency affords protection against TNBS colitis. It would be tempting to speculate that anti-PECAM-1 therapy could provide some relief from the colonic inflammation and dysfunction in IBD patients, particularly because anti-PECAM-1 therapy as been shown to be effective in alleviating established colitis in a dextran sodium sulfate (DSS) model (another model with a strong innate immune component) (42). However, because it is generally believed that IBD has a strong adaptive immune component in its pathogenesis and progression (18), caution should be used in considering PECAM-1-directed therapeutic approaches in IBD patients.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Canadian Institutes of Health Research Grants MOP13668, MOP 81303, and MGC 12816. Back

2 Address correspondence and reprint requests to Dr. Peter R. Kvietys, Lawson Health Research Institute, 800 Commissioners Road East, Sixth Floor, Victoria Research Laboratories, London, Ontario, Canada N6A 4G5. E-mail address: peter.kvietys{at}lhsc.on.ca Back

3 Abbreviations used in this paper: TNBS, 2,4,6,-trinitrobenzine sulfonic acid; BM, bone marrow; CLF, colonic lavage fluid; Cl2MDP, dichloromethylene diphosphonate; EtOH, ethanol; IBD, inflammatory bowel disease; KC, keratinocyte-derived chemokine; KO, knockout; LIX, LPS-induced CXC chemokine; M{Phi}, macrophage; MMEC, mesenteric microvessel endothelial cell; MPO, myeloperoxidase; PMN, polymorphonuclear neutrophil; WT, wild type. Back

Received for publication September 20, 2007. Accepted for publication May 8, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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