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Published ahead of print on April 15, 2004, doi:10.1165/rcmb.2003-0404OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 246-255, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0404OC

Role for Platelet-Endothelial Cell Adhesion Molecule-1 in Macrophage Fc{gamma} Receptor Function

Steven M. Albelda, Kelvin C. Lau, Paul Chien, Zhen-Yu Huang, Eugenia Arguiris, Alyssa Bohen, Jing Sun, Jessica A. Billet, Melpo Christofidou-Solomidou, Zena K. Indik and Alan D. Schreiber

Pulmonary, Allergy, and Critical Care Division, and Hematology Division, Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania

Address correspondence to: Steven M. Albelda, M.D., 421 Curie Blvd., BRBII/III, University of Pennsylvania School of Medicine, Philadelphia, PA 19104. E-mail: albelda{at}mail.med.upenn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Platelet-endothelial cell adhesion molecule-1 (PECAM-1) (CD31), a 130-kD transmembrane glycoprotein that functions in adhesion and signaling, is thought to play a role in some forms of leukocyte transmigration. In the lung, PECAM-1 is highly expressed, yet there have been few studies examining its role in pulmonary pathology. We therefore examined the inflammatory response (measured by bronchoalveolar lavage cell counts and protein content) after several types of lung injury in wild-type and PECAM-1 knockout mice. Consistent with studies in other organs, instillation of an endothelial stimulant (interleukin-1) was PECAM-1–dependent. In contrast, we noted that three other forms of acute lung injury (acid aspiration, adenoviral instillation, and tumor necrosis factor instillation) were completely PECAM-1–independent. Interestingly, in situ immune complex deposition injury, another complex lung disease, was also PECAM-1–dependent. This surprising finding was investigated in more detail and found to be due to a defect in macrophage activation, and not to a blockade of leukocyte transmigration. Experiments in bone marrow chimeric mice as well as ex vivo data demonstrated that Fc{gamma} receptor–dependent phagocytosis and tumor necrosis factor release were significantly reduced in macrophages derived from PECAM-1 knockout mice. Although PECAM-1 may not be required for transmigration of leukocytes into the alveolar space in many forms of complex lung inflammation, it is important in the function of Fc{gamma} receptors on alveolar macrophages.

Abbreviations: bronchoalveolar lavage, BAL • BAL fluid, BALF • bovine serum albumin, BSA • IgG-sensitized erythrocytes, EA • enzyme-linked immunosorbent assay, ELISA • immune complex deposition-induced injury, ICD • interleukin, IL • immunoreceptor tyrosine-based activation motif, ITAM • knockout, KO • lipopolysaccharide, LPS • phosphate-buffered saline, PBS • polymerase chain reaction, PCR • platelet-endothelial cell adhesion molecule-1, PECAM-1 • red blood cells, RBCs • tumor necrosis factor, TNF • white blood cells, WBCs • wild-type, WT


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD transmembrane glycoprotein of the immunoglobulin superfamily expressed on the surface of platelets and most subsets of leukocytes, including macrophages and neutrophils. It is also found in significant concentrations on endothelial cell intercellular junctions (1, 2). Its presence on both leukocytes and endothelial cell junctions, combined with its cell–cell adhesive properties via homophilic and/or heterophilic interactions, suggested a possible role for PECAM-1 in leukocyte transmigration at sites of inflammation (3).

A critical role for PECAM-1 in leukocyte transmigration was first demonstrated by Muller and coworkers (3), using an in vitro transmigration model in which antibodies against PECAM-1 blocked leukocyte transmigration, but not adhesion to the underlying endothelial cell monolayer. These observations were extended in vivo by showing that antibodies against PECAM-1 could block transmigration of neutrophils into (i) the peritoneum of rats after instillation of thioglycollate, (ii) the alveolar space of rats after formation of in situ immune complexes, and (iii) human skin grafts on SCID mice that had been injected with tumor necrosis factor (TNF) (4, 5). Comparable findings were subsequently reported in models of feline and murine peritonitis (6, 7), ischemia-reperfusion injury in the heart (6, 8), and in muscle flaps (9). A soluble PECAM-1 construct (a bivalent chimeric protein consisting of the two extracellular regions of PECAM-1 fused onto an IgG backbone) given exogenously or produced endogenously in transgenic mice has also been shown to block neutrophil migration by 80% in thioglycollate-induced peritonitis (10, 11).

Intravital microscopy of leukocyte adhesion rolling and transmigration in mesenteric vessels in rats showed that polyclonal anti–PECAM-1 blocked leukocyte transmigration, but not rolling or adhesion following interleukin (IL)-1 stimulation (12). In contrast, leukocyte transmigration induced by the bacterial peptide FMLP was not inhibited by an anti–PECAM-1 antibody. Repetition of these studies using monoclonal anti-PECAM antibodies reactive in rats provided almost identical results (13). Similarly, anti-PECAM antibodies blocked L-NAME and hydrogen peroxide-induced migration of leukocytes, but did not prevent extravasation of leukocytes induced by thrombin (14). Neutrophil emigration during acute bacterial pneumonia in mice and rats was unaffected by anti-PECAM-1 antibodies (15). Thus, there appear to be both PECAM-dependent and PECAM-1–independent leukocyte transmigration events.

Studies with PECAM-1 knockout (KO) mice provided quite different results (16). The initial observations suggested that the absence of PECAM-1 was of little physiologic consequence. The influx of neutrophils into the peritoneal cavities of KO mice injected with IL-1 or thioglycolate at 4 and 24 h was identical to that of wild-type (WT) mice. Similarly, leukocyte counts in air pouch models injected with formalin-inactivated bacteria or macrophage inhibitory protein-1{alpha} were the same in KO and WT mice. The only differences noted were subtle trappings of neutrophils at the perivascular basement membrane in the KO animals. However, more detailed intravital microscopic studies of cremasteric venules with inflammation induced by intrascrotal administration of TNF-{alpha} or IL-1ß have implicated PECAM-1 in some types of inflammation (17). In PECAM-1 KO mice, transmigration was inhibited after IL-1 treatment, but not after TNF exposure. Thus, there is genetic support for the antibody inhibition studies that suggest the existence of both PECAM-1–dependent and –independent leukocyte transmigration pathways.

In the lung, PECAM is intensely expressed in large and small vessel endothelium, but not by epithelial cells. However, other than the aforementioned immune complex deposition (4) and bacterial pneumonia studies (15), there have been few studies of the role of PECAM-1 in the lung. Basic questions, such as which lung diseases are PECAM-dependent and how PECAM-1 actually functions in regulating transmigration remain unanswered. To study these issues, we have examined a variety of forms of lung injury in WT and PECAM-1 KO mice. We noted that most forms of complex lung injury (i.e., acid aspiration, adenoviral instillation, hyperoxia, and TNF instillation) were completely PECAM-1–independent. However, after intratracheal instillation of IL-1, PECAM-1 KO mice suffered significantly less alveolar inflammation than did WT mice, indicating that the effect of IL-1 instillation into the lung was PECAM-1–dependent.

In our previous antibody study, we demonstrated that in situ immune complex deposition is also almost completely PECAM-1–dependent (4). This result was surprising, given that this is a complex form of lung injury that is largely TNF-mediated (18–19). In extension of these studies, we examined TNF release (presumably secondary to macrophage activation) in the bronchoalveolar fluid of WT and PECAM-1 KO mice. In contrast to WT mice, PECAM-1 KO mice lack high levels of TNF in the bronchoalveolar fluid after immune complex deposition, suggesting that immune complex induced lung inflammation involves a defect in macrophage Fc{gamma} receptor function rather than a problem in neutrophil transmigration. This dependence of an Fc{gamma} receptor function on PECAM-1 expression was confirmed in ex vivo assays of macrophage function. To our knowledge, this is the first evidence to link the presence of PECAM-1 to normal function of Fc{gamma} receptors in macrophages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Except where noted, male and female C57BL/6 mice of ages 6–10 wk were used throughout this study. Fc{gamma}R KO mice were purchased from Jackson Laboratory (Bar Harbor, ME). A breeding pair of PECAM-1 null mice originally created by Dr. Tak Mak (16) was kindly provided by Dr. Joseph Madri (Yale University). These mice were backcrossed for over 10 generations into the C57BL/B6 background. All protocols were performed in accordance with National Institutes of Health guidelines and with the approval the University of Pennsylvania Animal Use Committee.

Generation of Bone Marrow Chimeric Animals
Bone marrow chimeric mice were generated as described in detail by Mahooti and colleagues (20). Three groups of mice were produced: (i) WT mice with PECAM-1 KO bone marrow (KO->WT); (ii) KO mice with reconstituted WT bone marrow (WT->KO); and (iii) WT mice with reconstituted WT bone marrow (WT->WT). The latter served as a control. All experiments were conducted 4–6 wk post-transplantation. Flow-cytometric analysis (FACS) of white blood cells (WBCs) using an anti-mouse CD31 antibody (BD Biosciences, San Jose, CA) verified the phenotypes of the chimeric mice for each group (see Figure 4).



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  		Figure 4. Flow cytometry from blood of chimeric animals. Blood was isolated from chimeric animals 6 wk after bone marrow transplantation. RBCs were lysed, and the leukocytes subjected to flow cytometry with a control antibody (A, C, and E) or with antibody against mouse PECAM-1 (B, D, and F). Leukocytes from WT->WT controls showed high levels of PECAM-1 expression (B). Leukocytes from WT->KO animals showed high levels of PECAM-1 expression (D), and leukocytes from KO->WT mice (F) showed very low levels of PECAM-1. These results are consistent with efficient engraftment of the donor leukocytes.

 
Mouse Models of Lung Injury
Five of the six lung injury models tested required the intratracheal administration of the proinflammatory agents. After anesthesia, a 0.5-cm midline cervical incision was made, the trachea exposed, and inflammatory challenge was performed with a bent 28.5-gauge insulin syringe as follows.

Immune complex deposition-induced injury. Immediately after injection of 500 µg/100 µl endotoxin-free bovine serum albumin (BSA; Roche, Indianapolis, IN) dissolved in saline through the tail vein, 60 µl of 1 mg/ml anti-BSA antibody (ICN, Aurora, OH) dissolved in sterile saline was administered intratracheally to the anesthesized animals.

Acid aspiration–induced injury. A volume of 40 µl of 0.1 M HCl was administered per mouse as adapted from Goldman and coworkers (21).

TNF-{alpha}–induced injury. Inflammation was elicited in a manner similar to that described in Harrod (22). A volume of 100 µl (5 µg/ ml) murine TNF-{alpha} (Roche) was injected.

IL-1{alpha}–induced injury. Mouse recombinant Il-1{alpha} (obtained from Sigma, St. Louis, MO) was reconstituted in 0.9% NaCl containing 0.1% BSA. The stock solution was further diluted in saline and filtered sterilized through a 0.2 µm syringe filter. A working dosage of 4.2 ng in 100 µl, as adapted from Leff (23), was used for the injections.

Lipopolysacchride-induced lung injury. Lipopolysaccharide (LPS)-induced lung injury was induced via intratracheal injection of 7.5 µg of LPS (serotype 0111:B4; Sigma) in 100 µl of saline.

Adenovirus-induced lung injury. Adenovirus-induced lung injury was induced through the intranasal administration of adenovirus. After anesthesia, animals were positioned vertically, and 50 µL of adenovirus (Ad.LacZ, 20 particles/ pfu, 2.0 x 109 pfu) was gradually pipetted into the nose of each mouse.

In preliminary studies, the 24-h time point was chosen for experiments since inflammation was maximal at this time.

Evaluation of Lung Injury
The mice were killed at various time points after the induction of lung injury. Bronchoalveolar lavage (BAL) was then performed using intratracheal instillation of 1 ml phosphate-buffered saline (PBS) containing an anti-protease cocktail (Sigma) and 5 mM EDTA through a 20-gauge angiocatheter (BD Biosciences), given in 0.5-ml increments. Total leukocyte cell counts were determined and the remaining lavage fluid was centrifuged at 1,200 rpm for 10 min, and the cell-free supernatant was frozen at –80°C.

Neutrophils. The concentration of neutrophils in BAL fluid was determined from staining of cytospins remaining after removal of the cell-free supernatant.

Protein assay. The amount of protein in the BAL fluid was assayed using the BCA Protein Assay Kit (Pierce, Rockford, IL) in accordance with manufacturer's instructions.

TNF analysis by enzyme-linked immunosorbent assay. Enzyme-linked immunosorbent assays (ELISAs) for BAL fluid TNF were conducted using a PharMingen OptEIA Kit (San Diego, CA) following the manufacturer's instructions.

Collection of Peritoneal Macrophages
Five days after intraperitoneal injection with 2 ml of thioglycollate broth (Sigma), resident macrophages were washed from the peritoneal cavity of mice using 10 ml of PBS. After centrifugation and hypotonic lysis to remove red blood cells (RBCs), the cells were resuspended in Dulbecco's modified Eagle's medium with 1% fetal calf serum or PBS and adhered to tissue culture wells. Cytostaining indicated that > 95% of the adhered cells were macrophages.

Ex Vivo TNF Secretion Assay
To stimulate Fc{gamma} receptor–mediated TNF secretion, cells were exposed to IgG-opsonized particles (24). Latex beads, 3 µm in diameter (Polysciences, Warrington, PA), were opsonized by incubation in a solution of 100 µg/ml endotoxin-free human IgG (ICN) for 1 h at 37°C. Elicited macrophages were plated into 96-well plates at a concentration of 2 x 105 cells per well. After 24 h, nonadherent cells were washed off and each well exposed to 100 µl of: (i) media (Dulbecco's modified Eagle's medium) alone; (ii) human IgG at 100 µg/ml; (iii) uncoated latex beads (10 beads per cell); (iv) IgG-coated beads (10 beads/cell); or (v) media containing LPS at 10 ng/ml as a positive control. Assays were performed in at least triplicate for each condition. After 6 h incubation, the solutions were removed, centrifuged, and the supernatants frozen at –80°C. TNF concentration of each solution was measured by ELISA as described above.

Ex Vivo Phagocytosis Assay
Sterile sheep RBCs (109/ml) in calcium and magnesium-free PBS were sensitized by incubation with an equal volume of a subagglutinating concentration of rabbit anti-sheep RBC antibody (Cappel Laboratories, Cochranville, PA). The IgG-sensitized erythrocytes (EA) were washed twice with PBS and resuspended to a concentration of 109 cells/ml. Peritoneal macrophages in 35-mm wells were overlaid with 1 ml PBS and incubated with EA at a ratio of 100 to 1 (EA to macrophages) for 30 min at 37°C. After removal of unbound EA by washing with PBS, macrophages were subjected to a brief period of hypotonic shock (35 s) to remove surface bound EA. The cells were stained with Wright's-Giemsa and phagocytosis (ingested EA) was determined by light microscopy. Results are expressed as the phagocytic index (PI, the number of RBCs internalized/100 macrophages) and as the percentage of phagocytic cells.

Fc{gamma}R Real-Time Polymerase Chain Reaction
Analysis of mRNA expression was performed to compare expression levels of the various Fc{gamma}R's. The polymerase chain reaction (PCR) primers were as follows: Fc{gamma}RI (sense 5'-CAGTTCCACACAATGGTTTA-3' and antisense 5'-AGGAGAAGTGAAGCTGTAAG-3'); Fc{gamma}RIIb Primer set 1 (sense 5'-GAGGCTGAGAATACGATCAC-3' and antisense 5'-GTTGCTGCAGTCTCTTAACG-3') and Primer set 2 (sense 5'-GCTACACGTTTAAGGCCACA-3' and antisense 5'-TGGTTCTGGTAATCATGCTC-3'); Fc{gamma}RIII (sense 5'-AGGTGCTCAAGGAAGACATG-3' and antisense 5'-TCTGATTGACAGGGACTTCC-3'); and the common Fc{gamma} {gamma} chain (sense 5'-AGCCGTGATCTTGTTCTTGC-3' and antisense 5'-AGGTCTCTGGCAGCTTTATT-3').

Two micrograms of total RNA were reverse transcribed to cDNA using Oligo(dT)15 primer (Promega, Madison, WI) and powerscript reverse transcriptase (BD Biosciences). Synthesized cDNA was then subjected to real-time PCR using the SmartCycler System (Cepheid, Sunnydale, CA). The amounts of cDNA were normalized using ß-actin primers. Equivalent amounts of cDNA were then analyzed. A minimum of three samples from each group was analyzed and the relative expression level based on cycle number was compared among groups.

Fc{gamma}R Surface Analysis
Binding studies were performed as previously described (25) using mAb 2.4G2 IgG antibody (BD Biosciences) directed against mouse Fc{gamma}RII and Fc{gamma}RIII (26), mouse IgG2a (BD Biosciences) for binding to mouse Fc{gamma}RI (27), Ly17.2 antibody (Monoclonal Antibody Core of the Memorial Sloan-Kettering Cancer Center, New York, NY) directed against mouse Fc{gamma}RIIb (28) and mouse IgG1 (BD Biosciences) as nonbinding control. The data were analyzed by Scatchard plot, and the number of IgG binding sites on WT and PECAM-1 KO macrophages were estimated by linear regression using the least squares method.

Immunoprecipitation and Immunoblotting
Peritoneal macrophages from WT mice and PECAM-1 KO mice were stimulated by crosslinking Fc{gamma}R with EA or with mAb 2.4G2 (anti-mouse Fc{gamma}RII/III) at 10 µg/ml plus goat anti-rat IgG F(ab')2 (25 µg/ml). Cells were lysed with Triton X100 lysis buffer in the presence of protease and phosphatase inhibitors. Immunoprecipitated proteins were resolved on 7.5% SDS polyacrylamide. After electrophoretic transfer to nitrocellulose, proteins were immunoblotted with 1 µg/ml anti-phosphotyrosine mAb 4G10 (UBI, Lake Placid, NY) or 0.2 µg/ml anti-Syk antibody (Santa Cruz Biotechnology Inc, Santa Cruz., CA). Immunoblots were developed with horseradish peroxidase–conjugated goat anti-mouse IgG (Bio-Rad Inc., Richmond, CA) and specific bands detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ).

Statistics
Statistical significance in experiments involving two groups was determined using Student's t test. For phagocytosis experiments, paired t tests were used to compare values for macrophages from WT and PECAM-1 KO mice within a given experiment. In experiments with more than two groups, comparisons were made using one-factor ANOVA, with appropriate post hoc testing for more than two samples. A P value of < 0.05 was deemed significant. Values were expressed as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Acid Aspiration–, TNF-{alpha}–, and Adenovirus-Induced Pulmonary Inflammation Are Independent of PECAM-1
To measure the inflammatory response generated by the intratracheal instillation of TNF-{alpha}, adenovirus, and acid, we obtained BAL fluid (BALF) and measured WBC counts, neutrophil counts, and protein concentrations 24 h after injury. At baseline, the total WBC (Figure 1A) and neutrophil (Figure 1B) counts were the same in both WT and KO mice. There was, however, a small, but statistically significant increase (P < 0.05) in BALF protein levels in the KO mice compared with WT animals (Figure 1C). Figure 1 shows a dramatic increase in these parameters in both WT and PECAM KO mice 24 h after treatment compared with untreated controls in all three models. Importantly, PECAM-1–null mice showed responses similar to WT mice in all three parameters. These data show that PECAM-1 deficiency does not affect the various pathways necessary for neutrophil transmigration and inflammation in TNF-{alpha}–, adenovirus-, and acid-induced injuries.



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  		Figure 1. Intracheal instillation of acid, adenovirus, and TNF-{alpha} induces similar degrees of lung injury in PECAM-1 KO and WT mice. Lung injury was induced by injecting WT (solid bars) or PECAM-1 KO (hatched bars) mice intratracheally with acid (AA), adenovirus (AdV), and TNF-{alpha}. 24 h later, BAL was performed. (A) Total number of WBCs in BALF. (B) Number of neutrophils in BALF. (C) Protein concentrations (mg/ml) in BALFs. For each treatment, there were no significant differences in WBC and neutrophil counts and in protein concentrations in KO mice versus WT mice. Each group has six or more animals. Error bars represent SEM.

 
IL-1{alpha} and Immune Complex Deposition Induce PECAM-1–Dependent Pulmonary Injury
Like the above models of lung injury, intratracheal instillation of IL-1{alpha} and in situ IgG immune complex deposition (ICD) resulted in striking increase in numbers of WBCs and neutrophils in BALF as well as increases in protein concentration in WT mice compared with untreated control mice (Figure 2). However, PECAM-1–null mice exhibited a significantly smaller increase in total WBC counts (Figure 2A) and neutrophil counts (Figure 2B) when challenged by IL-1{alpha} and ICD (P < 0.05). After ICD, the number of transmigrated neutrophils into the pulmonary space was ~ 8-fold lower (164 x 103 cells/ml versus 23.6 x 103 cells/ml; P < 0.05) in KO versus WT mice. The protein levels were significantly lower after ICD in the PECAM KO mice, as well (Figure 2C; P < 0.05). These data demonstrate an important role for PECAM-1 in IL-1{alpha} and ICD-triggered pulmonary inflammation.



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  		Figure 2. Lung injury after IL-1 instillation and immune complex deposition is blunted in PECAM-1 KO mice. Lung ICD treatment was induced by injecting WT (solid bars) or PECAM-1 KO (hatched bars) mice intratracheally with IL-1 or anti-BSA antibodies and intravenously with BSA. Twenty-four hours later, BAL was performed. (A) Total number of WBCs in BALF. (B) Number of neutrophils in BALF. (C) Protein concentrations (mg/ml) in BALFs. Note significantly smaller increases in WBC and neutrophil counts and in protein concentrations in KO mice versus WT mice after IL-1 and ICD treatment. *P < 0.05 compared with WT control. Each group had at least 10 animals. Error bars represent SEM.

 
KO Mice Fail to Release TNF after ICD
The data indicate that ICD disease, thought to be due to activation of alveolar macrophages via Fc{gamma} receptors with subsequent release of TNF and other cytokines (18), is PECAM-1–dependent, whereas direct TNF instillation and adenovirus instillation, thought to be mediated by macrophage activation through non–Fc{gamma} receptor–mediated pathways (29) are both PECAM-1–independent.

We hypothesized that one potential explanation for this puzzling observation may be a failure of the macrophages in the PECAM-1 KO mice to be normally activated by immune complexes, resulting in a decreased inflammatory response. We therefore conducted experiments to assess macrophage activation in vivo using TNF release as a marker. In mice and rats, immune complex–triggered alveolitis has been shown to induce the secretion of TNF from macrophages. The secretion of TNF likely attracts neutrophils to the alveolar space, thus promoting lung injury (18, 19). Measurement of TNF levels in BALF from WT mice verified this observation in our mice, showing a progressive increase in TNF-{alpha} concentrations that peaked at around 4 h (~ 680 pg/ml) before resolving by 24 h in immune complex–challenged WT mice (Figure 3A). Interestingly, TNF-{alpha} concentrations were significantly lower (< 50 pg/ml) at 4 h in KO animals (P < 0.001). This study was repeated with an additional time point at 8 h with similar results, ruling out a delayed response in the KO animals (data not shown).



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  		Figure 3. PECAM-1 KO mice fail to secrete TNF after immune complex deposition but not after instillation of LPS. (A) BAL was performed at various time points after immune complex deposition in WT (solid bars) or PECAM-1 KO (hatched bars) mice. TNF concentrations in BALF (pg/ml) were measured by ELISA. Four hours after ICD, a marked increase in TNF was observed in the BALF in WT but not in KO mice (*P < 0.001 compared with WT control). Each group had at least six animals. Error bars represent SEM. (B) BAL was performed at various time points after intratracheal instillation of bacterial LPS in WT (solid bars) or PECAM-1 KO (hatched bars) mice. TNF concentrations in BALF (pg/ml) were measured by ELISA. Marked, but equivalent increases in TNF levels were observed at 4 and 8 h after LPS treatment. Each group had five animals.

 
To confirm that the KO macrophages were fully capable of releasing TNF, a control experiment was performed in which bacterial endotoxin (a strong stimulator of alveolar macrophage TNF release) was instilled intratracheally into WT and PECAM KO mice. As shown in Figure 3B, TNF levels were markedly increased at 4 and 8 h after endotoxin instillation; however, there were no statistically significant differences between the WT and KO animals.

These studies demonstrate that the lack of PECAM-1 inhibits immune complex–induced release of TNF-{alpha} into alveolar fluid, but not release of TNF-{alpha} after LPS instillation. Because macrophages are the primary source of TNF-{alpha} in BAL, these data suggest that PECAM-1–deficient animals may have a defect in macrophage/IgG interaction (a function mediated by Fc{gamma} receptors).

Immune Complex–Mediated Pulmonary Inflammation Is Dependent on Leukocyte Rather than on Endothelial Cell PECAM-1
Because PECAM-1–mediated transmigration requires homophilic interactions of PECAM-1 on both leukocytes and endothelial cells (30), we generated chimeric mice lacking endothelial cell PECAM-1. We investigated the ability of PECAM-1–positive leukocytes from chimeric mice lacking endothelial cell PECAM-1 to support ICD inflammation, postulating that lack of PECAM-1 on leukocytes, but not on endothelial cells, would diminish response to ICD. To generate chimeric mice, the bone marrow of irradiated WT mice was reconstituted with bone marrow cells from either PECAM-1 KO mice (KO->WT) or PECAM-1 WT mice as controls (WT->WT). Conversely, the bone marrow of irradiated PECAM-1 KO mice was reconstituted with bone marrow cells from WT mice (WT->KO).

Analysis by flow cytometry using anti–PECAM-1 antibody was performed 4–6 wk after bone marrow transplantation to confirm that transplantation was successful for predicted chimeric phenotypes (Figure 4). Total WBCs (Figure 5A) and neutrophil concentration (Figure 5B) in BALF were examined in chimeric mice subjected to intrapulmonary ICD. We observed a statistically significant decrease (P < 0.05) in total WBC counts and in neutrophil concentrations in BALF from WT mice reconstituted with KO marrow (KO->WT) compared with BALF derived from control mice (WT->WT) and from KO mice reconstituted with WT leukocytes (WT->KO). These data indicate that the presence of PECAM-1 on only leukocytes is sufficient to support the response to ICD, and that the absence of leukocyte PECAM-1 results in the inhibition of neutrophil migration into the alveolar space.



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  		Figure 5. Immune complex-mediated pulmonary inflammation is dependent on leukocyte rather than endothelial cell PECAM-1. Lung immune complex deposition (ICD treatment) was induced by injecting bone marrow chimeric mice intratracheally with anti-BSA antibodies and intravenously with BSA. BAL was performed 24 h later. Bone marrow chimeric mice tested include: WT bone marrow into irradiated WT mice (WT->WT), WT bone marrow into irradiated PECAM-1 KO mice (WT->KO), or PECAM-1 KO bone marrow into irradiated WT mice (KO->WT). Each group had five animals. (A) Number of WBCs in BALF. (B) Total number of neutrophils in BALF. Only animals with PECAM-1–deficient leukocytes showed inhibition of white cell infiltration into BALF. *P < 0.05 compared with WT control. Error bars represent SEM.

 
Macrophages Harvested from PECAM-1 KO Mice Show Decreased Ability to Secrete TNF-{alpha} after Exposure to IgG-Coated Beads
To test directly the hypothesis that macrophages from PECAM-1 KO animals have impaired Fc{gamma} receptor function, we examined the ability of peritoneal macrophages (thioglycollate-elicited) to release TNF-{alpha} 4 h after immune complex stimulation ex vivo. Fc{gamma} receptors were stimulated using IgG-coated beads (24). As negative controls, macrophages were exposed to purified IgG (100 µg/ml) or uncoated beads at the same 10:1 ratio. As positive controls, macrophages were exposed to 10 ng of LPS.

Macrophages exposed to media alone, purified IgG, or uncoated beads did not release detectable levels of TNF (Figure 6A). In contrast, both WT and PECAM-1 KO macrophages secreted high levels of TNF at virtually identical levels after exposure to LPS (Figure 6B). However, only WT macrophages exposed to IgG-coated beads secreted TNF (428 pg/ml). No TNF secretion was observed in PECAM-1 KO macrophages treated with IgG-coated beads (P < 0.001 WT versus KO). These data, demonstrating marked inhibition of TNF release after engagement of Fc{gamma} receptors, are consistent with our studies of TNF release in vivo (Figure 3).



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  		Figure 6. Macrophages from PECAM-1 KO mice show decreased ability to secrete TNF. Thioglycollate-elicited peritoneal macro-phages from WT (solid bars) or PECAM-1 KO mice (hatched bars) were isolated and exposed to various reagents. Culture supernatant was collected after 6 h and TNF-{alpha} levels measured by ELISA. (A) Cells were exposed to soluble IgG, to uncoated latex beads, or to IgG-coated latex beads. WT, but not KO, macrophages secreted TNF after exposure to the IgG-coated beads (*P < 0.001 compared with WT control). (B) Both WT and KO macrophages secreted large amounts of TNF after exposure to 10 ng/ml of bacterial lipopolysaccharide. The experiment was repeated twice with similar results.

 
Macrophages Harvested from PECAM-1 KO Mice Have Decreased Ability to Phagocytose IgG-Coated Erythrocytes
To determine whether the abnormalities we observed in PECAM-1–deficient animals were present in another well-established function of macrophage Fc{gamma}Rs, phagocytosis, we examined functional characteristics of macrophages isolated from WT and PECAM-1 KO animals. To obtain sufficient numbers of macrophages for these in vitro experiments, thioglycollate-elicted peritoneal macrophages were primarily used. The phagocytosis of IgG-coated RBCs was examined in peritoneal macrophages harvested from WT and PECAM-1 KO animals. Phagocytosis by PECAM-1 KO macrophages (as measured by the % phagocytic cells and by phagocytic index) was ~ 50% lower than in WT macrophages (P < 0.05) (Figures 7A and 7B). Similar differences in phagocytosis were observed when WT and PECAM-1 KO alveolar macrophages (30% phagocytosis for KO alveolar macrophages versus 87% phagocytosis for WT alveolar macrophages) or when macrophages from non-thioglycollate treated animals were examined (data not shown).



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  		Figure 7. Macrophages from PECAM-1 KO mice have decreased ability to phagocytose IgG-coated erythrocytes. Phagocytosis of IgG-coated erythrocytes by thioglycollate-elicited peritoneal macrophages from WT or PECAM-1 KO mice was determined (A and B). (A) Percentage of phagocytic cells. (B) The phagocytic index (number of erythrocytes internalized per 100 macrophages). The graphs represent data from four independent experiments. Phagocytosis was significantly lower (as indicated by t tests, *P < 0.01) in the macrophages derived from KO mice.

 
Surface Expression of Fc{gamma} Receptors Is Not Diminished on PECAM-1 KO Macrophages
To address whether the diminishment in Fc{gamma} receptor function in macrophages lacking PECAM-1 is due to a decrease in receptor expression, we evaluated the expression levels of the different macrophage Fc{gamma} receptor mRNAs in WT and PECAM-1 KO macrophages using real-time PCR. As shown in Table 1, after normalization of mRNA using ß-actin, there were no significant differences in the expression levels of Fc{gamma}RI, Fc{gamma}RIIb (data from two different sets of primers), Fc{gamma}RIII, or the common Fc{gamma} {gamma} chain. In addition, we performed formal binding studies using Scatchard analysis in two independent experiments to determine the numbers of Fc{gamma} receptors on the cell surface (Table 2). The well-characterized mAb 2.4G2 (26) was used to quantify surface expression of mouse Fc{gamma}RII and Fc{gamma}RIII. Monomeric mouse immunoglobulin of the IgG2a class was used for analysis of mouse Fc{gamma}RI because antibody specific for mouse Fc{gamma}RI was not available and IgG2a binds specifically to Fc{gamma}RI (27). The Ly17.2 antibody was used to quantify the expression of the Fc{gamma}IIb receptor. This antibody does not crossreact with other Fc{gamma}Rs, including Fc{gamma}RIII (28). As a control, we included binding of a monomeric form of an irrelevant monoclonal antibody of the IgG1 subclass.


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  		TABLE 1. Ratio of mRNA expression of macrophages (KO/WT)

 

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  		TABLE 2. Scatchard analysis: number of binding sites per cell

 
As shown in Table 2, the number of Fc{gamma} receptors on macrophages isolated from WT mice and from PECAM-1 KO mice was similar. There was no specific binding of the labeled IgG1 antibody on either cell type. These data indicate that surface expression of Fc{gamma} receptors on PECAM-1 KO macrophages is not downregulated. Thus, the impairment of Fc{gamma} receptor function in PECAM-1 KO mice is likely due to the absence of a functional interaction between PECAM-1 and Fc{gamma} receptors.

Lack of PECAM-1 Does Not Inhibit Syk Phosphorylation Induced by Fc{gamma}R Crosslinking
It is well established that one of the first steps in Fc{gamma}R signaling involves phosphorylation of the tyrosine kinase Syk (31). To determine if the deficits in macrophage function in the PECAM-1 KO macrophages are due to an inhibition of this initial signal, Fc{gamma}Rs on the surface of thioglycollate-elicited macrophages from WT or KO animals were crosslinked by exposure to IgG-coated erythrocytes or by treatment with mAb 2.4G2 followed by anti-mouse F(ab)2' antibody. Cell lysates were then immunoprecipitated with anti-Syk antibody and immunoblotted with either anti-phosphotyrosine or anti-Syk antibodies. As shown in Figure 8, the degree of Syk phosphorylation after Fc{gamma}R crosslinking was equivalent in macrophages from WT and KO mice.



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  		Figure 8. Syk tyrosine phosphorylation after Fc{gamma} receptor engagement is equivalent on macrophages from WT and PECAM-deficient mice. Fc{gamma}Rs on the surface of thioglycollate-elicited macrophages from WT or KO animals were crosslinked by exposure to IgG-coated erythrocytes (lanes 3 and 4) or by treatment with mAb 2.4G2 followed by anti-mouse F(ab)2' antibody (lanes 5 and 6). Cell lysates were then immunoprecipitated with anti-Syk antibody and immunoblotted with either anti-phosphotyrosine (A) or anti-Syk antibodies (B). To control for unequal loading of protein, the intensity of each band was determined by densitometry (expressed in arbitrary units) and the ratio of the densities of phospho-Syk to total Syk determined (C, table). The degree of Syk phosphorylation after Fc{gamma}R crosslinking was equivalent in macrophages from WT and KO mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the mechanisms by which PECAM-1 affects leukocyte transmigration are not yet known, data accumulating from in vitro and in vivo experiments suggest that its most important role may be its ability to activate leukocyte integrins. Thus, in settings where endothelial cell (versus leukocyte) activation is predominant, engagement of the PECAM-1 on tethered neutrophils by the PECAM-1 expressed on the surface of endothelial cells is needed to activate ß2 (CD18) integrins and allow transmigration to proceed (32). This appears to be the situation after IL-1 stimulation, but not after TFN-{alpha} stimulation. An explanation for this difference may be that although both TNF and IL-1 are strong endothelial cell stimulants, TNF is a much more potent stimulator of neutrophil activation than is IL-1 (33). However, in settings where direct activation of neutrophil ß2 integrins occurs through G protein–mediated binding of chemotactic substances (such as IL-8, FMLP, or LTB4), PECAM-1-activation of neutrophil integrins may be redundant and inhibition of PECAM-1 function may have little effect on transmigration. We have direct support for this paradigm using in vitro transmigration models where IL-1–mediated neutrophil transmigration is PECAM-1–dependent, whereas IL-8–mediated neutrophil transmigration is PECAM-1–independent (32). This hypothesis explains the in vivo observations of Norshaugh and coworkers, who showed in both WT and KO mice that IL-1–mediated neutrophil transmigration is PECAM-1–dependent, whereas fMLP-induced transmigration is PECAM-1–independent (13, 17). This theory also predicts that the leukocyte activation seen in most "naturally occurring" types of inflammation (which involves both cytokine and chemokine release) would not require PECAM-1.

With this paradigm in mind, we were not surprised to see similar degrees of neutrophil migration and protein leaking into the alveolar space in the complex models of lung injury induced by intratracheal instillation of acid, adenovirus, or TNF (Figure 1). Using anti–PECAM-1 antibodies, we have also recently shown that neutrophil migration into the alveoli in bacterial pneumonia is PECAM-1–independent (15). Our finding that intratracheal instillation of IL-1 was PECAM-1–dependent was also consistent with previous data and the aforementioned paradigm.

However, even though our previous study using antibodies had shown blockade of ICD-induced lung inflammation (4), the finding that ICD (a complex lung injury [18, 19]) was markedly PECAM-1–dependent (Figure 2) did not fit the paradigm described above. We therefore explored the idea that the failure of neutrophil infiltration after ICD was not due to a block in transmigration, but rather to a defect in the ability of the PECAM-1 KO mice to initiate an inflammatory response. This hypothesis was supported by two observations. First, after ICD, PECAM-1 KO mice had much lower levels of TNF-{alpha} in their BAL fluids (Figure 3). Second, the inhibition of ICD-dependent inflammation was seen in bone marrow chimeric mice who had PECAM-null leukocytes, whereas mice with PECAM-null endothelial cells had normal responses to ICD. Because PECAM-1–mediated transmigration would require homophilic neutrophil–endothelial cell interactions (30), seeing strong PECAM-dependent effects in the mice missing PECAM only on their WBCs suggested another explanation (i.e., a leukocyte defect). One caveat to these experiments is that although the blood leukocytes in the chimeric animals showed complete engraftment of donor cells (Figure 4), alveolar macrophages may take longer to repopulate than blood cells, and it is possible that some of the alveolar cells remained from the donor. If this occurred, it would tend to lessen the differences among the groups. The fact that our results were significantly different (even with possible donor contamination) supports the validity of our findings.

Our data also suggest that there is a specific deficit in the Fc{gamma} receptor function of PECAM-null macrophages. ICD is caused by Fc{gamma} receptor–dependent stimulation of macrophage secretion of a mixture of cytokines (TNF, being prominent) and chemokines (18, 19). Although well established in the literature (34), we confirmed this finding in our own pilot studies. Two mice lacking the Fc{gamma} receptor {gamma} chain ({gamma} KO) necessary for Fc{gamma}R signaling were challenged with intratracheal immune complexes. BAL revealed marked blunting of the inflammatory responses in the {gamma} KO mice. After ICD, the average total WBC count in {gamma} KO mice was 80,367/ml, ~ 40% of the increase seen in WT mice. The average number of neutrophils in the {gamma} KO mice was 8,586/ml, only ~ 5% of the increase seen in WT mice. Supporting specificity of the macrophage dysfunction were the observations that LPS instillation, another stimulus for macrophage release of TNF (Figure 3B), and intratracheal adenovirus instillation, an injury that is macrophage-dependent but Fc{gamma} receptor–independent (Figure 1) (29), were not affected. To confirm this hypothesis, we explored Fc{gamma} receptor function in macrophages isolated from WT and PECAM KO animals. Our in vitro experiments demonstrated that both TNF release induced by opsonized beads and phagocytosis of IgG-coated RBCs were diminished in macrophages isolated from PECAM-1 KO animals (Figures 6 and 7). Furthermore, real-time PCR studies (Table 1) and Scatchard analyses (Table 2) showed that our observed differences in activity were not due to differences in the number of Fc{gamma} receptors on the macrophages. Specifically, we saw no differences in the inhibitory Fc{gamma}RIIb receptor that could explain these differerences. Although we did not specifically test the function of the Fc{gamma}RIIb receptor in the PECAM KO macrophages, we are unaware of any reports of functional defects in this receptor. Taken together, our data suggest a functional interaction between PECAM-1 and Fc{gamma} receptors on macrophages.

The finding that the absence of PECAM-1 on macrophages decreased the activity of Fc{gamma} receptor on the macrophages was unexpected. Several lines of evidence have suggested that PECAM-1 may function as an inhibitory receptor in functions requiring the participation of immunoreceptor tyrosine-based activation motif (ITAM) containing signaling molecules (2). Functional data supporting the classification of PECAM-1 as an inhibitory receptor have been provided in several cellular and animal models. For example, collagen-induced platelet aggregation by activation of the GPVI/Fc{gamma} receptor chain receptor is augmented in platelets from mice lacking PECAM-1 compared with WT mice (35, 36); B-cell proliferation induced by IgM crosslinking is enhanced in B cells from mice lacking PECAM-1 compared with WT mice (37); and mast cell activation induced by IgE immune complexes is enhanced in cells from mice lacking PECAM-1 (38). In addition, in several cell systems, co-ligation of PECAM-1 with an ITAM-containing receptor leads to inhibition of receptor function. For example, calcium mobilization is inhibited in T-lymphocytes when CD3 crosslinking is combined with PECAM-1 co-ligation (39) and crosslinking PECAM-1 followed by stimulation of platelets through the GPVI/Fc{gamma} receptor chain by collagen leads to inhibition of platelet aggregation (40, 41). Experimental autoimmune encephalomyelitis occurs earlier in PECAM-1–deficient mice (42). Based on these data, it might be anticipated that engagement of lung macrophage Fc{gamma} receptors in PECAM-1 KO mice would augment inflammation following lung immune complex deposition. Such a response, i.e., increased inflammation, was observed when mice lacking the ITAM family member, Fc{gamma}RIIB, were challenged in a similar mouse intratracheal immune complex disease model (34). Surprisingly, we observed instead that responses to immune complex deposition in the lung were decreased in PECAM-1 KO mice. This unexpected observation suggests that PECAM-1 enhances, rather than inhibits, Fc{gamma} receptor–mediated signaling in macrophages

The mechanism by which PECAM-1 affects Fc{gamma}R function is unclear and under active investigation in our laboratory. It appears that PECAM-1 affects both Fc{gamma}R-mediated phagocytosis and cytokine release. Signal transduction propagated by Fc{gamma} receptor engagement is complex; however, a number of key components of the pathway have been identified (31, 43). Signaling begins by phosphorylation of the receptor ITAM by enzymes of the Src tyrosine kinase family (31, 44). Phosphorylated ITAMs then become docking sites for the SH-2 domains of the tyrosine kinase Syk. Because recruitment and activation of Syk kinase are required for Fc{gamma} receptor–mediated phagocytosis (45, 46) and TNF release (47), a distinct possibility was that Syk provides a potential avenue for the PECAM-1/Fc{gamma}R interaction. However, our data indicate no differences in the phosphorylation of Syk after engagement of Fc{gamma} receptors in macrophages obtained from WT and PECAM-1–deficient macrophages (Figure 8). We are thus currently investigating signaling pathways downstream of Fc{gamma} receptor and Syk phosphorylation, i.e., protein kinase C, phospholipase A2, phosphatidyl-inositol 3-kinase, mitogen-activated protein kinases, and GTPases of the Rho family (48). Clearly, many other questions remain from the perspectives of both Fc{gamma} receptor and PECAM-1 biology. We do not yet know which of the Fc{gamma} receptors on macrophages are involved. The actual mechanism of the PECAM-1 interaction with Fc{gamma} receptor signaling also remains unknown. In vitro models using transfection of WT and mutant PECAM-1 into macrophages and macrophage cell lines are currently underway. Future studies will also address the ability of PECAM-1 to affect other Fc{gamma} receptor–mediated disease models such as immune complex–induced renal disease.

In summary, these studies suggest that PECAM-1 may have a limited role in regulating neutrophil influx in most acute lung injuries. Our data suggest that most complex injuries in which both leukocyte and endothelial activators are released will be PECAM-1–independent. It is possible, however, that other functions of PECAM-1, such as its role in angiogenesis (49) and as an endothelial survival factor (50) will be important in other aspects (i.e., healing) of complex lung injuries. In contrast, immune complex–induced lung inflammation appears to require macrophage PECAM-1 to regulate the activity of Fc{gamma} receptors, which themselves mediate the release of proinflammatory agents. Thus, our investigation of the physiologic role of PECAM-1 and its interaction with Fc{gamma} receptors could be important for the development of effective therapeutic agents aimed at alleviating inflammation and injury wherever macrophage Fc{gamma} receptors are activated.


   Acknowledgments
 
The authors thank Drs. Juan-Carlos Murciano and Thomas Dziubla for assistance in iodinating proteins. This work was supported by NIH Grants HL705728 (S.M.A.) and HL69498 (A.D.S.).

Received in original form November 11, 2003

Received in final form March 31, 2004


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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P. Kinniry, Y. Amrani, A. Vachani, C. C. Solomides, E. Arguiri, A. Workman, J. Carter, and M. Christofidou-Solomidou
Dietary Flaxseed Supplementation Ameliorates Inflammation and Oxidative Tissue Damage in Experimental Models of Acute Lung Injury in Mice
J. Nutr., June 1, 2006; 136(6): 1545 - 1551.
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H. M. DeLisser, B. P. Helmke, G. Cao, P. M. Egan, D. Taichman, M. Fehrenbach, A. Zaman, Z. Cui, G. S. Mohan, H. S. Baldwin, et al.
Loss of PECAM-1 Function Impairs Alveolarization
J. Biol. Chem., March 31, 2006; 281(13): 8724 - 8731.
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