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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Adhesion molecule expression by pulmonary endothelial cells is considered to play an important role in the recruitment of circulating leukocytes to sites of inflammation in the lung. We have used P-selectin- and intercellular adhesion molecule type 1 (ICAM-1)-deficient mice to determine whether these adhesion molecules are important to pulmonary eosinophil recruitment after allergen challenge. There was a significant inhibition of lung tissue eosinophil recruitment in ICAM-1-deficient mice (~ 84% inhibition compared to wild-type mice) and P-selectin-deficient mice (~ 67% inhibition compared to wild-type mice) 3 h after allergen challenge. The number of bronchoalveolar lavage (BAL) eosinophils in P-selectin-deficient and ICAM-1-deficient mice was also significantly reduced compared with wild-type mice. Levels of BAL eosinophil peroxidase (EPO) were significantly lower in ICAM-1-deficient mice (0.21 ± 0.03 EPO units) compared with wild-type mice (3.34 ± 0.65 EPO units). There was no significant difference in the degree of inhibition of eosinophil recruitment in ICAM-1-deficient mice at the three time points (3, 12, and 24 h) of study after allergen challenge. However, in P-selectin-deficient mice there was a decline in the degree of inhibition of eosinophil recruitment from 3 h (67% inhibition) and 12 h (72% inhibition) postchallenge, to 24 h postchallenge (38% inhibition), suggesting that other adhesion molecules may be playing a more prominent role than P-selectin at later time points. These studies suggest an important role for ICAM-1 and P-selectin in eosinophil recruitment to the lung after allergen challenge.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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The inducible expression by pulmonary endothelial cells of adhesion molecules is considered to play an important role in the localization and recruitment of circulating leukocytes to sites of inflammation in the lung (1, 2). The prominent recruitment of eosinophils to sites of allergic inflammation in the airway in patients with asthma (1, 3) has led to studies attempting to define whether either a particular profile of adhesion molecules expressed by endothelium (1, 4), or alternatively eosinophil-selective chemoattractants released at sites of allergic inflammation such as eotaxin (8, 9), could explain this selective eosinophil recruitment. The recruitment of eosinophils to sites of allergic inflammation in vivo is a multistep process characterized by initial eosinophil intravascular rolling and firm adhesion to endothelium, followed by sequential eosinophil diapedesis between endothelial cells and chemotaxis into tissues (10). Although in vivo this paradigm holds true for both eosinophils (11, 12) and neutrophils (13, 14), the endothelial counterreceptors used by these circulating leukocytes differ. For example, studies using flow chambers in vitro or intravital videomicroscopy of the microcirculation in vivo have demonstrated that eosinophils roll on endothelial cell-expressed P-selectin (15), but not on E-selectin (12). In contrast, neutrophils differ from eosinophils and roll on both E-selectin (14) and P-selectin (16). Thus, neutrophils and eosinophils share an ability to roll on some endothelial cell-expressed ligands (i.e., P-selectin), but differ in their ability to roll on other endothelial cell-expressed ligands (i.e., E-selectin). In addition, eosinophils, but not neutrophils, can use very late activation antigen 4 (VLA-4) to roll on endothelial cell-expressed counterreceptors (11). These differences were not previously fully appreciated from in vitro studies of eosinophils using static adhesion assays, in which eosinophils were noted to bind to E-selectin albeit in lower numbers compared to neutrophils (17).
In this study we have focused on determining the relative contribution to eosinophil recruitment of one endothelial cell-expressed rolling receptor (i.e., P-selectin) and one endothelial cell-expressed firm adhesion receptor (i.e., intercellular adhesion molecule type 1 [ICAM-1]). Endothelial cells store P-selectin preformed in Weibel-Palade bodies (18). On in vitro stimulation with histamine or thrombin, Weibel-Palade bodies fuse with the endothelial cell surface membrane and expose P-selectin rapidly (within a few minutes of stimulation) and transiently (peak expression in 20 to 30 min) to the lumenal cell surface (18). In contrast to the transient expression of P-selectin induced by histamine and thrombin, cytokines such as tumor necrosis factor (TNF) induce sustained levels of P-selectin expression by endothelial cells in vitro (19). In in vitro studies of eosinophils and P-selectin, eosinophils have been shown to bind both to purified P-selectin (20) and to P-selectin expressed by nasal polyp endothelium (21). Eosinophils roll on P-selectin in vitro as demonstrated in a flow chamber assay (15). Eosinophils express CD11a/CD18 (LFA-1), the counterreceptor for ICAM-1, and bind to ICAM in vitro (4) and in vivo (6). ICAM-1 expression by endothelial cells (2) is induced by interleukin 1 (IL-1) and TNF, cytokines that have been detected at sites of allergic inflammation (22).
In vivo studies using adhesion molecule-deficient mice have provided important insights into the role of P-selectin and ICAM-1 in neutrophil recruitment (23). In a model of bacterial infection in mice, P-selectin- and ICAM-1- deficient mice with either mutation alone show a 60-70% reduction in acute neutrophil emigration into the peritoneum 2-4 h after Streptococcus pneumoniae-induced peritonitis, and double-mutant mice showed a complete loss of neutrophil emigration into the peritoneal cavity (23). In contrast to studies demonstrating inhibition of neutrophil recruitment into the peritoneum, neutrophil emigration into the alveolar space during acute S. pneumoniae-induced pneumonia is normal in the single- and double-mutant mice (23). These data suggest organ-specific differences in neutrophil adhesion, because neutrophil emigration into the peritoneum requires both adhesion molecules (P-selectin and ICAM-1) whereas neutrophil emigration into the lung appears to be P-selectin- and ICAM-1-independent (23, 24).
In contrast to neutrophils, which are recruited to sites of bacterial infection, eosinophils are recruited to sites of allergic inflammation. The eosinophil, like the neutrophil, expresses counterreceptors for P-selectin (15) and ICAM-1 (4). Using the same P-selectin- and ICAM-1-deficient mice, we have sought to determine the importance of these adhesion molecules to eosinophil recruitment into the lung following allergen challenge.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Adhesion Molecule Knockout Mice
P-selectin-deficient, ICAM-1-deficient, as well as C57BL/6 background control wild-type female mice aged 8 to 10 wk, were purchased from Jackson Laboratories (Bar Harbor, ME). These mice were developed by Dr. Arthur Beaudet (Department of Molecular Genetics, Baylor College of Medicine, Waco, TX) and used previously in studies of neutrophil adhesion in a model of bacterial infection in mice (23).
Mouse Model of Eosinophilic Pulmonary Inflammation
Pulmonary eosinophilia in mice was induced according to the method of Foster and coworkers (25). In brief, wild-type or adhesion molecule-deficient mice were sensitized by intraperitoneal injection with 50 µg ovalbumin/1 mg Alhydrogel (Aldrich Chemical Co., Milwaukee, WI) in 0.9% sterile saline on days 0 and 12. Nonsensitized mice received 1 mg of alhydrogel in 0.9% saline. On day 24, the appropriate groups of mice (n = 3-4 mice/group) were exposed three times (at 1-h intervals) to an aerosol of ovalbumin (10 mg/ml) in 0.9% saline (nonsensitized mice received saline only) for 30 min. The aerosolized ovalbumin protocol was then repeated every second day thereafter for 8 d. The aerosol is generated at 6 liters/min by a nebulizer (Ultra-Neb 99; Devilbiss, Somerset, PA) that produces a mean particle diameter of 3.9 µm into a closed chamber of 800 cm3. Three hours (or in selected experiments 12 h and 24 h) after the last aeroallergen challenge, mice were killed by CO2 asphyxiation.
Bronchoalveolar Lavage Cells
Bronchoalveolar lavage (BAL) cells from wild-type and
adhesion molecule-deficient mice were recovered by lavage with 1 ml of phosphate-buffered saline (PBS) via a tracheal catheter. The resulting BAL cells were immediately
separated from BAL fluid by centrifugation (700 × g for
5 min). BAL fluid was snap frozen and stored at 
70°C
until used in an assay for eosinophil peroxidase (EPO). An
appropriate phosphate-buffered saline dilution of the recovered BAL cells was added to trypan blue, and the viability and total number of BAL white blood cells were determined with a hemocytometer. Differential leukocyte
counts were performed after brief acetone fixation and
staining of the BAL cells with May-Grünwald-Giemsa
stains. The percentages of eosinophils, neutrophils, and
mononuclear cells present on each slide were assessed by
counting a minimum of 300 cells in random high-power
fields using a light microscope (×40 magnification) to display the slide image on a television monitor (Videometric
150 Image Analysis Program; American Innovision, San
Diego, CA).
Assay for Eosinophil Peroxidase
In addition to enumerating the number of eosinophils in BAL fluid, EPO, an eosinophil cytoplasmic granule protein, was assayed using the substrate solution o-phenylenediamine dihydrochloride (OPD) and a colorimetric assay (26). BAL fluid (100 µl) was added to 2 ml assay buffer (0.1 M phosphate buffer [pH 6.8], 8 mM OPD, 0.01% H2O2). Reaction volumes were incubated in duplicate for 10 min at room temperature, stopped with the addition of 0.4 N sulfuric acid, and read in a spectrophotometer at 492-nm wavelength (UV160U; Shimadza, Tokyo, Japan).
Quantitation of Lung Eosinophils
The number of eosinophils in lung tissue was determined
using a sensitive method dependent on the presence of a
cyanide-resistant eosinophil peroxidase (27). In brief, excised lungs from ovalbumin- or PBS-challenged wild-type
or adhesion molecule-deficient mice were placed in Tissue
Tek O.C.T. compound, snap frozen in liquid nitrogen, and
stored at 70°C. Cryosections (5-µm thickness) were cut
onto microscope slides, and fixed for 5 min in acetone.
Slides were then rehydrated in PBS for 5 min. The lung
sections were incubated at room temperature for 1 min in
the presence of cyanide buffer (10 mM potassium cyanide,
pH 6.0). Slides were then rinsed in PBS and incubated for
10 min with the peroxidase substrate DAB (3,3'-diaminobenzine) (Vector Laboratories, Burlingame, CA). The
tissue sections were subsequently washed in PBS, before
counterstaining with hematoxylin. Cells having dark brown
staining, characteristic of the eosinophil cyanide-resistant
peroxidase (27), were counted in four random high-power
fields (×40 magnification) in each lung section. The area
of the high-power field was quantitated using a calibrated
slide. Results are expressed as the number of eosinophils/ mm2 of lung tissue.
Immediate Hypersensitivity Skin Test
Wild-type and adhesion molecule-deficient mice were sensitized to ovalbumin as described. On day 32, 50 µl of ovalbumin antigen or diluent control was injected into the shaved backs of the different groups of mice. Immediately after antigen administration, 200 µl of 1% Evans blue dye was injected into the tail vein of the mice (28). The size of blueing of the skin (measured as the largest transverse diameter, in millimeters) at the challenged site was assessed 15 min later.
Statistical Analysis
The number of BAL and lung eosinophils, as well as EPO levels in BAL fluid, were compared by multiple comparisons of data by Mann-Whitney test using a statistical software package (InStat, San Diego, CA). P values of < 0.05 were considered to be statistically significant. All results are given as mean ± SEM.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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Mouse Model of Eosinophil Lung Inflammation: Wild-type Mice
As previously described (25), sensitization and ovalbumin allergen challenge of wild-type mice (n = 7 experiments) induce significant BAL eosinophilia (69.3 ± 4.6% BAL eosinophils) compared with mice that were not sensitized or challenged with ovalbumin (0.8 ± 0.2% BAL eosinophils) (P = 0.0003), or compared with mice immunized with ovalbumin and challenged with PBS diluent (0.9 ± 0.4% BAL eosinophils) (P = 0.0001). Ovalbumin sensitization and challenge also induced a significant lung tissue eosinophilia (170.2 ± 19.2 lung eosinophils/mm2) (n = 7 experiments) as compared with mice that were not sensitized or challenged with ovalbumin (6.6 ± 2.0 lung eosinophils/mm2) (P = 0.0001), or compared with mice immunized with ovalbumin and challenged with PBS diluent (4.5 ± 0.5 lung eosinophils/mm2) (P = 0.0001) (Figure 1). Neutrophils composed less than 2% of BAL cells preallergen, postallergen, or postdiluent challenge. Mononuclear cells composed the remainder of the BAL cells.
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Mouse Model of Eosinophilic Lung Inflammation: P-Selectin-deficient Mice
In contrast to wild-type mice, P-selectin-deficient mice (n = 4 experiments) immunized and challenged with ovalbumin developed significantly less BAL eosinophilia (P-selectin-deficient mice, 31.2 ± 8.2% BAL eosinophils versus wild-type mice, 58.0 ± 9.1% BAL eosinophils) (P = 0.04) (Figure 2A). Neither P-selectin-deficient mice that were not sensitized and challenged with ovalbumin (1.3 ± 0.2% BAL eosinophils) nor P-selectin-deficient mice immunized with ovalbumin and challenged with PBS diluent (1.5 ± 0.7% BAL eosinophils) developed BAL eosinophilia. There was a greater increase in BAL total leukocytes in wild-type mice after allergen challenge (wild-type mice, 0.57 [± 0.13] × 105 BAL leukocytes preallergen versus 14.2 [± 4.73] × 105 BAL leukocytes postallergen) (P = 0.0007) compared with the increase in total BAL leukocytes in P-selectin-deficient mice after allergen challenge (P-selectin-deficient mice, 0.54 [± 0.08] × 105 BAL leukocytes preallergen versus 3.21 [± 0.85] × 105 BAL leukocytes postallergen) (P = 0.001).
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Analysis of lung sections of P-selectin-deficient mice immunized and challenged with ovalbumin also demonstrated a significant reduction in lung eosinophils (P-selectin-deficient mice, 67.6 ± 12.3 lung eosinophils/mm2 versus wild-type mice, 177.5 ± 31.0 lung eosinophils/mm2) (n = 4 experiments) (P = 0.03) (Figure 2B).
Mouse Model of Eosinophil Lung Inflammation: ICAM-1-deficient Mice
In contrast to wild-type mice, ICAM-1-deficient mice (n = 3 experiments) immunized and challenged with ovalbumin developed significantly less BAL eosinophilia (ICAM-1-deficient mice, 2.6 ± 1.0% BAL eosinophils versus wild-type mice, 75.5 ± 4.5% BAL eosinophils) (P = 0.0001) (Figure 3A). Neither ICAM-1-deficient mice that were not sensitized and challenged with ovalbumin (0.3 ± 0.2% BAL eosinophils) nor ICAM-1-deficient mice immunized with ovalbumin and challenged with PBS diluent (1.5 ± 0.5% BAL eosinophils) developed BAL eosinophilia. There was a greater increase in BAL total leukocytes in wild-type mice after allergen challenge (wild-type mice, 0.57 [± 0.13] × 105 BAL leukocytes preallergen versus 14.2 [± 4.73] × 105 BAL leukocytes postallergen) (P = 0.0007) compared with the increase in total BAL leukocytes in ICAM-1-deficient mice after allergen challenge (ICAM-1-deficient mice, 0.46 [± 0.21] × 105 BAL leukocytes preallergen versus 3.59 [± 0.49] × 105 BAL leukocytes postallergen) (P = 0.02).
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Analysis of lung sections of ICAM-1-deficient mice immunized and challenged with ovalbumin also demonstrated a significant reduction in lung eosinophils (ICAM-1-deficient mice, 23.1 ± 10.9 lung eosinophils/mm2 versus wild-type mice, 160.4 ± 19.0 lung eosinophils/mm2) (n = 3 experiments) (P = 0.0001) (Figure 3B).
BAL Eosinophil Peroxidase Levels
Control wild-type mice (n = 3 experiments) when immunized and challenged with ovalbumin allergen developed a significant increase in BAL EPO levels (3.34 ± 0.65 EPO units) compared with wild-type mice that were not challenged with ovalbumin (0.35 ± 0.08 EPO units) (P = 0.01), or compared with wild-type mice that were immunized with ovalbumin and challenged with PBS diluent (0.63 ± 0.29 EPO units) (P = 0.01). In contrast, ICAM-1-deficient mice immunized with ovalbumin developed lower levels of BAL EPO compared with wild-type mice when challenged with ovalbumin allergen (ICAM-1-deficient mice, 0.21 ± 0.04 BAL EPO units, ~ 94% inhibition compared with ovalbumin-challenged wild-type mice, 3.34 ± 0.65 BAL EPO units, P = 0.0002; Figure 4). There was a trend (~ 30% inhibition) for P-selectin-deficient mice to have lower EPO levels than wild-type mice, but this did not reach statistical significance (P-selectin-deficient mice, 1.59 ± 0.32 BAL EPO units, compared with ovalbumin-challenged wild-type mice, 2.27 ± 0.83 BAL EPO units, P = NS).
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Inhibition of Eosinophil Recruitment in Adhesion Molecule-deficient Mice
The percentage inhibition of lung eosinophil recruitment 3 h postallergen in adhesion molecule-deficient compared with wild-type mice was greater in ICAM-1-deficient compared with P-selectin-deficient mice, in all three indices measured including total lung eosinophils (ICAM-1-deficient mice, 84 ± 14% inhibition versus P-selectin-deficient mice, 67 ± 14% inhibition), BAL eosinophils (ICAM-1-deficient mice, 97 ± 2% inhibition versus P-selectin-deficient mice, 51 ± 18% inhibition), and BAL EPO levels (ICAM-1-deficient mice, 94 ± 2% inhibition versus P-selectin-deficient mice, 30 ± 10% inhibition).
To determine whether differences in peripheral blood eosinophil levels or sensitization to antigen could account for the differences in eosinophil recruitment between wild-type and adhesion molecule-deficient mice, we determined both the percentage of peripheral blood eosinophils as well as immediate hypersensitivity skin reactions in the different groups of mice. There was no significant difference in the percentage of peripheral blood eosinophils in wild-type and adhesion molecule-deficient mice (ICAM-1-deficient mice, 2.2%; P-selectin-deficient mice, 2.0%; wild-type mice, 2.1%). Wild-type and adhesion molecule-deficient mice sensitized with ovalbumin and challenged intradermally with ovalbumin developed equivalent-sized immediate hypersensitivity skin "blueing" reactions when Evans blue dye was injected into the tail vein of these mice (wild-type, 13 ± 2 mm; ICAM-1, 10 ± 3 mm; P-selectin, 11 ± 1 mm). Therefore, neither a lack of eosinophils nor an absence of response to ovalbumin sensitization is likely to explain the differences noted in eosinophil recruitment in wild-type and adhesion molecule-deficient mice.
Kinetic Studies of Eosinophil Recruitment
There was no significant difference in the degree of inhibition of BAL eosinophilia in ICAM-1-deficient compared with wild-type mice at the three time points studied (3, 12, and 24 h) after allergen challenge (Figure 5A). Analysis of lung sections of ICAM-1-deficient mice immunized and challenged with ovalbumin also demonstrated a similar significant reduction in lung eosinophils at all time points studied (Figure 5B).
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In contrast, inhibition of BAL eosinophil recruitment was maximal at 3 h after allergen challenge in P-selectin-deficient compared to wild-type mice (51% inhibition), with a trend to lesser degrees of inhibition noted at 12 h (40%) and 24 h (36%) after allergen challenge (Figure 6A). Similar results were noted in the analysis of the number of eosinophils in lung sections of P-selectin-deficient compared with wild-type mice at 3 and 12 h compared with 24 h (65% inhibition at 3 h, 72% inhibition at 12 h, and 26% inhibition at 24 h) (Figure 6B). These results suggest that eosinophil recruitment is inhibited maximally in P-selectin-deficient mice between 3 and 12 h after allergen challenge in this model.
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Discussion |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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In this study, we have demonstrated the importance of ICAM-1 (~ 84% inhibition of lung eosinophils) and P-selectin (~ 65% inhibition of lung eosinophils) in eosinophil recruitment to the lung after allergen challenge. P-selectin-deficient mice exhibited a lesser reduction in lung eosinophils (~ 65% inhibition) compared with ICAM-1-deficient mice (~ 84% inhibition), suggesting that alternate endothelial cell-expressed rolling receptors vascular cell adhesion molecule (VCAM) (29), in addition to P-selectin, were important to eosinophil recruitment into the lung. Our studies of the kinetics of eosinophil recruitment also suggest a more important role for P-selectin at 3-12 h after allergen challenge than at 24 h after allergen challenge. In contrast, ICAM-1 was important to eosinophil recruitment at all time points studied.
We have previously demonstrated that eosinophil recruitment into the peritoneal cavity is significantly inhibited in P-selectin-deficient mice and ICAM-1-deficient mice compared with wild-type mice challenged with allergen (Broide, D. H., D. Humber, S. Sullivan, and P. Sriramarao. 1996. Inhibition of Eosinophil Rolling and Recruitment in P-Selectin- and ICAM-deficient Mice. [Submitted]). The ability to visualize fluorescently labeled eosinophil trafficking in the peritoneal microcirculation using intravital microscopy allowed us also to demonstrate reduced eosinophil rolling in P-selectin-deficient mice and reduced firm adhesion in ICAM-1-deficient mice, underscoring the importance of endothelial cell-expressed P-selectin as an eosinophil rolling receptor, and ICAM-1 as a firm adhesion receptor. Interestingly, there was also a significant inhibition of eosinophil firm adhesion to endothelium in P-selectin-deficient mice. The reduced firm adhesion of eosinophils to mesenteric endothelium in P-selectin-deficient mice challenged with allergen suggests that leukocyte rolling and firm adhesion are sequential at sites of allergen challenge. A model of sequential eosinophil adhesion would require initial eosinophil rolling prior to firm adhesion. Thus, the absence of significant eosinophil rolling in the mesenteric venules of P-selectin-deficient mice would not allow for the subsequent firm adhesion of eosinophils to non-P-selectin firm adhesion receptors such as ICAM-1. Similarly, only firm adhesion and not rolling is inhibited in the peritoneal microcirculation of ICAM-1-deficient mice.
In contrast to our studies of eosinophil trafficking in P-selectin-deficient mice, studies of neutrophil trafficking in E-selectin/P-selectin double-mutant mice have demonstrated that streptococcal pneumonia induces an E- and P-selectin-independent increase in leukocyte interactions with noncapillary endothelium, and that migration of neutrophils to alveoli can occur despite a deficiency of E- and P-selectin (30). Whereas these results with E- and P-selectin mutant mice suggest that selectins are not important to neutrophil trafficking into the lung, studies with chimeric selectin antibodies have demonstrated that neutrophil recruitment in a rat model of lung inflammation induced by cobra venom factor is P-selectin dependent (and E-selectin independent) (31). Conversely, neutrophil recruitment in another rat model of lung inflammation induced by immune complex injury is E-selectin-dependent (and P-selectin-independent) (31). These results suggest that there may be differences in requirements for selectins in neutrophil trafficking dependent on the animal species studied, the inflammatory stimulus used, or the method used to neutralize adhesion molecules (adhesion molecule-deficient mice, neutralizing antibodies). Results of studies in adhesion molecule deficient mice may also be complicated by upregulation of alternative adhesion pathways, whereas results of studies using adhesion molecule-blocking antibodies may be confounded by antibodies inhibiting leukocyte function by mechanisms independent of adhesion. Studies assessing the role of P-selectin and ICAM-1 in inducing neutrophil-mediated lung injury using blocking antibodies or mutant mice underscore the different results that may be obtained with two different methods of adhesion molecule blockade (32). When acute lung injury was assessed using blocking antibodies, both P-selectin and ICAM-1 were required for neutrophil sequestration and lung injury, whereas neither adhesion molecule played a role when studied in adhesion molecule-deficient mice (32). The results of our demonstration that eosinophil recruitment to the lung is inhibited in ICAM-1-deficient mice are similar to results obtained with neutralizing antibodies to ICAM in a primate model of asthma (33).
Overall, this study demonstrates an important role for P-selectin and ICAM-1 in eosinophil recruitment to the lung after allergen challenge. An improved understanding of the mechanisms accounting for the difference in the requirements for P-selectin and ICAM-1 by eosinophils and neutrophils awaits the development of methods to visualize these cells in the pulmonary microcirculation in mice.
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Footnotes |
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Address correspondence to: Dr. David H. Broide, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0635. E-mail: dbroide{at}ucsd.edu
(Received in original form October 31, 1996 and in revised form June 10, 1997).
Acknowledgments: The authors wish to thank Lauri Doval for expert secretarial support during the preparation of the manuscript. This study was supported by NIH Grants AI 33977 and AI 38425 (to David H. Broide) and AI 35796 (to P. Sriramarao).
Abbreviations BAL, bronchoalveolar lavage; EPO, eosinophil peroxidase; ICAM-1, intercellular adhesion molecule type 1; OPD, o-phenylenediamine dihydrochloride; PBS, phosphate-buffered saline; TNF, tumor necrosis factor.
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Materials & Methods
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Discussion
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