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The Journal of Immunology, 2004, 172: 4493-4502.
Copyright © 2004 by The American Association of Immunologists

Neutrophil Elastase (NE)-Deficient Mice Demonstrate a Nonredundant Role for NE in Neutrophil Migration, Generation of Proinflammatory Mediators, and Phagocytosis in Response to Zymosan Particles In Vivo1

Rebecca E. Young*, Richard D. Thompson*, Karen Y. Larbi*, Mylinh La{dagger}, Clare E. Roberts*, Steven D. Shapiro{ddagger}, Mauro Perretti{dagger} and Sussan Nourshargh2,*

* Cardiovascular Medicine Unit, Eric Bywaters Centre, Faculty of Medicine, Imperial College, Hammersmith Hospital, and {dagger} William Harvey Research Institute, Bart’s and the London, Queen Mary University of London, London, United Kingdom; and {ddagger} Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neutrophil elastase (NE) remains a controversial player in the process of leukocyte transmigration and much of this controversy stems from conflicting reports on the effects of NE inhibitors. The availability of NE-deficient mice (NE-/-) provides a clean and elegant tool for the study of leukocyte migration in vivo. In this study, NE-/- mice were used to investigate the role of NE in leukocyte migration through cremasteric venules, as observed by intravital microscopy, induced by locally administered cytokines IL-1{beta} and TNF-{alpha} and the particulate stimulus, zymosan. Although no defects in leukocyte responses induced by the cytokines were observed, zymosan-induced leukocyte firm adhesion and transmigration was suppressed in NE-/- mice. These responses were also inhibited in wild-type mice when zymosan was coinjected with a specific NE inhibitor. Quantification of inflammatory mediator levels in homogenates of zymosan-stimulated tissues indicated reductions in levels of IL-1{beta}, KC, and macrophage inflammatory protein-1{alpha} in NE-/- mice. Furthermore, phagocytosis of fluorescent zymosan particles, as observed by intravital microscopy, was diminished in NE-deficient animals. Collectively, the findings of this study indicate a nonredundant role for NE in zymosan-induced leukocyte firm adhesion and transmigration, and that this defect is associated with impaired generation of proinflammatory mediators as well as phagocytosis of zymosan particles in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The migration of leukocytes from the vascular lumen into the surrounding extravascular tissue is a characteristic feature of an inflammatory response and has been associated with the pathogenesis of numerous inflammatory conditions. The final stage of leukocyte migration involves the movement of leukocytes through two distinct barriers namely the endothelium and the perivascular basement membrane (1, 2). With respect to the former, a number of endothelial cell-associated adhesion molecules expressed at high concentrations at endothelial cell junctions, such as platelet endothelial cell adhesion molecule (PECAM-1)3 (CD31), members of the junctional adhesion molecule family, CD99 and ICAM-2 (CD102), have been implicated in leukocyte transendothelial cell migration as supported by convincing in vitro and/or in vivo studies (2, 3, 4). Once through the endothelial cell barrier, leukocytes arrive at the subendothelial cell basement membrane, a tough, thin, and highly distensible matrix composed predominantly of collagen type IV and laminin. The mechanism by which leukocytes penetrate this barrier are unclear though recent findings have demonstrated a role for PECAM-1 and the integrin {alpha}6{beta}1 in leukocyte migration through the perivascular basement membrane in vivo (5, 6, 7, 8). In addition to adhesion molecules, leukocyte proteases, through their ability to disrupt endothelial cell junctional complexes (9) and to degrade key components of the basement membrane (10, 11, 12), have also been implicated in the process of leukocyte migration through the vessel wall. One such protease that has been extensively studied in this context is neutrophil elastase.

Neutrophil elastase (NE) is a serine proteinase, stored in neutrophil azurophilic granules, along with other serine proteinases such as cathepsin G. Due to its capacity to cause damage to endothelial cells (13) and to cleave key endothelial cell-associated adhesion molecules (e.g., ICAM-1 and VCAM-1) (14, 15) as well as its ability to hydrolyze components of the basement membrane and extravascular tissue (16), NE is widely viewed as a mediator of vascular and tissue injury. Indeed, inappropriate release of NE has been associated with the pathology of a number of inflammatory disease states such as ischemia/reperfusion injury (17) and chronic obstructive pulmonary disease (18, 19) and in animal investigations, elastase inhibitors exhibit protective effects against neutrophil-mediated tissue injury in numerous models of inflammation (20, 21, 22, 23). In support of these findings, while there is evidence to suggest that NE inhibitors can suppress leukocyte migration in vivo (24) other studies have found no such effects (25). Similar discrepancies in the role of NE in neutrophil migration through endothelial cells and/or basement membrane-like structures have also been found in vitro (26, 27, 28, 29, 30). As some of these inconsistencies may be related to potential nonspecific, or variable, activities of the inhibitors in different animal species, the availability of NE-deficient mice has provided a valuable tool for further investigations into the functional role of this protease in leukocyte migration in vivo.

NE-deficient (NE-/-) mice have categorically demonstrated a protective role for NE in host immunity in that they exhibit a significant reduction in clearance of Gram-negative bacteria and fungal pathogens (31, 32). However, the limited studies performed with these mice to date have not indicated a generalized role for NE in leukocyte migration in vivo. Specifically, NE-/- mice exhibit normal leukocyte infiltration in response to bacterial infections (31) and to a nonspecific inflammatory stimulus, thioglycolate (32). However, in a model of acute arthritis induced by passive transfer of Abs against collagen, NE-/- mice exhibited reduced leukocyte infiltration into the subsynovial tissue space (33). In the same study, mice deficient in both NE and cathepsin G showed reduced neutrophil migration in response to a reverse passive Arthus reaction and zymosan particles in an air-pouch model (33). In the light of the existing discrepancies in the studies using NE inhibitors and the limited number of in vivo investigations using the NE-/- mice, the aim of the present work was to extend the above studies by using a combined approach of genetic deletion and pharmacological blockade to further investigate the role of this enzyme in leukocyte migration in vivo using primarily the experimental approach of intravital microscopy (IVM). Collectively, while the results do not demonstrate a role for NE in leukocyte migration induced by the cytokines IL-1{beta} and TNF-{alpha}, the findings indicate a nonredundant role for NE in zymosan-induced neutrophil migration, a role that appears to be associated with generation of proinflammatory cytokines and chemokines and phagocytosis of zymosan particles in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

NE-/- mice were generated by targeted gene disruption as previously detailed (31). These mice (~25g), on a mixed C57BL/6/129Sv or C57BL/6 background (F6), were used with appropriate genetic background, age, and weight-matched wild-type (WT) controls with the majority of the studies being conducted using C57BL/6 mice.

Reagents

ONO-5046 was a gift from ONO Pharmaceuticals (Osaka, Japan). Soluble TNFR (p55) IgG-fusion protein and its control IgG-fusion protein, cSF25, reactive to human pan adenocarcinoma Ag, were a gift from Centocor (Malvern, PA). The following rat anti-mouse mAbs were purchased from BD Biosciences (Cowley, U.K.) unless otherwise stated: HM{alpha}2 (anti-{alpha}2); PS/2 (anti-{alpha}4; Serotec, Oxford, U.K.); 5H10-27 (anti-{alpha}5); GoH3 (anti-{alpha}6); 9EG7 (anti-{beta}1); GAME-46 (anti-{beta}2); MEL-14 (anti-L-selectin); MEC 13 · 3 (anti-PECAM-1); M1/70 (anti-CD11b); 2A11 (anti-dectin 1; a gift from Dr. G. Brown (University of Oxford, Oxford, U.K.)). All other general reagents were purchased from Sigma-Aldrich (Poole, U.K.), unless indicated in the text.

Peritoneal inflammation

Leukocyte migration into the peritoneal cavity of mice was induced by i.p. administration of 1 ml of recombinant murine IL-1{beta} (10 ng) or TNF-{alpha} (100 ng) (Serotec). Control mice received saline. After 4 h, mice were sacrificed by CO2 asphyxiation and the peritoneal cavity was lavaged using PBS supplemented with 2 mM EDTA and 0.25% BSA. Total cell counts were determined following staining of exudate samples with Kimura stain and differential cell analysis was determined in exudate smears prepared in a cytocentrifuge and stained with May-Grünwald/Giemsa stains.

IVM of murine cremasteric venules

IVM was used to observe leukocyte responses within mouse cremasteric venules following intrascrotal (i.s.) administration of IL-1{beta} (30 ng), TNF-{alpha} (300 ng), or zymosan particles (30–300 µg). Control mice received i.s. saline (400 µl). In selected animals, stimuli were coinjected i.s. with other reagents such as a soluble TNFR (p55) IgG fusion protein or the specific NE inhibitor, ONO-5046.

The cremaster muscle of anesthetized mice was surgically exteriorized for observation by IVM as previously described (6, 7, 34). Leukocyte rolling flux, rolling velocity, firm adhesion, and transmigration in postcapillary venules were quantified as previously detailed (35). To visualize leukocyte subtypes, cremaster muscles were stained with topical application of the nuclear dye, acridine orange (1 mg/ml), and were observed using a Zeiss LSM 5 Pascal laser-scanning confocal microscope (Göttingen, Germany) attached to a Zeiss Axioscop 2 FS using a 488 nm Argon laser line as the excitation source. In selected experiments, microvascular centerline erythrocyte velocity and peripheral leukocyte counts were determined as previously detailed (5, 7).

Quantification of phagocytosis in vivo

Fluorescent zymosan particles (Alexa Fluor 488-labeled; 30 µg/mouse) or Escherichia coli particles (FITC-labeled; 1 x 107 particles/mouse) (Molecular Probes, Eugene, OR), were injected i.s. and 6 h later the cremaster muscle was prepared for IVM. Fluorescent particles were visualized using a silicon-intensified low-level light camera (Hamamatsu Photonics, Enfield, U.K.). Phagocytosis was quantified as the number of transmigrated leukocytes containing fluorescent particles per field of view (>12/tissue were quantified). In some experiments, the phagocytosis response was normalized for the number of infiltrating leukocytes and hence the data is presented as index of phagocytosis (%).

Quantification of neutrophil phagocytosis and elastase cell surface expression/release in vitro

Neutrophils were isolated from the bone marrow of WT and NE-/- mice. Briefly, dissected femurs were flushed with sterile HBSS and neutrophils were purified using a discontinuous Percoll gradient (purity typically >80%). Neutrophils were added to BSA-coated (1 µg/ml) 96-well plates (5 x 105 neutrophils/well) and allowed to adhere for 15 min at 37°C before addition of stimuli. Opsonized (serum-treated) zymosan particles (0.003–3 mg/ml) or modified PBS (0.25% BSA/5 mM glucose/1 mM Ca2+/Mg2+) was added to neutrophils and plates were incubated at 37°C for a further 30 min. Plates were then centrifuged to allow for removal of supernatants for subsequent analysis of released elastase. Pelleted cells were then fixed using 3% paraformaldehyde/0.5% glutaraldehyde and finally resuspended in Tris buffer (0.2M Tris Base, 0.15M NaCl, 0.02M CaCl2 at pH 8.5) for assay of cell surface NE as previously described (36).

Enzyme activity was assayed using a fluorogenic substrate specific for elastase, MeOSuc-Ala-Ala-Pro-Val-AFC (Enzyme Systems Products, Livermore, CA) (36). Briefly, fixed cells, supernatants, or purified human NE (50 µl; Merck Biosciences, Nottingham, U.K.) were incubated with 400 µM substrate in Tris buffer (50 µl). After incubation at 37°C for 25 min, the level of liberated 7-amino-4-trifluoromethyl coumarin was quantified in a fluorescent plate reader (Millipore, Watford, U.K.) using excitation 409 nm and emission 530 nm. A standard curve was established using commercially obtained purified human NE and was used to represent the results in terms of murine neutrophil enzyme activity equivalent to the activity detected from nanograms per milliliter of purified human NE. Finally, assay supernatants were removed from the plates and the cells were washed and stained with May-Grünwald and Giemsa stains to enable quantification of phagocytosis of zymosan particles by microscopy.

Chemokine and cytokine assays

Cremaster tissues were dissected and snap-frozen in liquid nitrogen. Tissues were then homogenized in ice-cold PBS containing a commercially available mixture of protease inhibitors, centrifuged, and supernatants were collected for assays. Levels of IL-1{beta}, TNF-{alpha}, KC, and macrophage inflammatory protein (MIP)-1{alpha}, were quantified using commercially available ELISA kits (R&D Systems, Abingdon, U.K.). Levels were normalized according to the weight of tissue samples and presented as picograms per milligram of tissue.

Flow cytometry analysis

Whole blood taken by cardiac puncture from WT and NE-/- animals was collected in EDTA. Indirect immunostaining and flow cytometry was used to quantify cell surface expressions of selected molecules as previously described (6). The ratio of fluorescence intensities associated with the binding of primary mAbs and isotype-matched control mAbs was used to express specific binding of test mAbs in terms of relative fluorescence intensity (RFI).

Statistical analysis

Results are given as mean ± SEM and analyzed using either an unpaired t test or one-way ANOVA and Newman-Keuls post test with p < 0.05 taken as significant (Prism; GraphPad Software, San Diego, CA). Calculation of unknown values in the assay of NE and ELISAs was achieved using a GraphPad Prism linear regression standard curve algorithm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leukocyte transmigration induced by IL-1{beta} and TNF-{alpha} is normal in NE-deficient mice

The role of NE in IL-1{beta}- and TNF-{alpha}-induced leukocyte migration was investigated using two models of leukocyte infiltration, migration into the peritoneal cavity, and through cremasteric venules as observed by IVM. In the peritonitis model, in both WT and NE-/- mice, local injections of IL-1{beta} (10 ng) and TNF-{alpha} (100 ng) induced comparable and significant degrees of neutrophil infiltration within 4 h, as compared with saline-injected mice (Fig. 1, a and b). However, no significant differences were observed between responses in the two strains of animals.



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  		FIGURE 1. Cytokine-induced peritoneal and cremasteric inflammation in WT and NE-/- mice. Mice were treated with i.p. (a) IL-1{beta} (10 ng) or (b) TNF-{alpha} (100 ng) in 1 ml of sterile saline, with control mice receiving saline only. Four hours later, the peritoneal cavities were lavaged and total and differential leukocyte infiltration was quantified as detailed in Materials and Methods. Number of neutrophils in each cavity are presented as mean ± SEM for n = 4-5 mice per group. In a parallel series of experiments, mice were treated i.s. for 4 h with IL-1{beta} (30 ng) or TNF-{alpha} (300 ng), both in 400 µl of sterile saline with control mice receiving saline only. Leukocyte firm adhesion (c) and transmigration (d) were quantified by IVM as described in Materials and Methods. Results are expressed as mean ± SEM for n = 5 mice per group. Statistically significant difference between control and cytokine-treated groups are represented by asterisks: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
IVM was used to further investigate the role of NE in the different stages of leukocyte responses. For this purpose, IL-1{beta} (30 ng) or TNF-{alpha} (300 ng) were injected i.s. and responses in cremasteric venules were quantified 4 h later. In agreement with the findings in the peritonitis model, no defects in leukocyte responses of firm adhesion or transmigration were identified in the NE-/- mice, as compared with WTs (Fig. 1, c and d). The two strains of mice also exhibited similar levels of leukocyte rolling flux (results not shown).

Characterization of zymosan-induced leukocyte responses within murine cremasteric venules

To extend the above studies, leukocyte responses elicited by the particulate inflammatory stimulus, zymosan, was next investigated. Using a 4-h in vivo test period, i.s. zymosan (30–300 µg) induced significant, and largely dose-dependent, leukocyte firm adhesion and transmigration responses within mouse cremasteric venules (Fig. 2a). The submaximal dose of 30 µg was chosen for all subsequent studies including investigations into the time course of responses induced by zymosan. Although no significant increases in leukocyte rolling flux were observed in response to zymosan particles (results not shown), leukocyte firm adhesion and transmigration increased in a time-dependent manner during the 4-h observation period (Fig. 2b). Within this time period, the primary infiltrating leukocyte subtype in zymosan-stimulated cremasters was the neutrophil, as identified based on its characteristic size and nuclear morphology in acridine orange-stained tissues (Fig. 2, c and d).



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  		FIGURE 2. Characterization of zymosan-induced leukocyte responses within the mouse cremaster muscle. The results shown are all from WT mice. a, To establish the zymosan-induced dose-response relationship, mice were treated i.s. for 4 h with 30–300 µg of zymosan particles (suspended in 400 µl of sterile saline) with control mice receiving saline. Leukocyte firm adhesion and transmigration were quantified by IVM as described in Materials and Methods. b, To establish the time-course profile of zymosan-induced leukocyte responses, mice were treated with saline or 30 µg of zymosan for 30 min to 4 h before leukocyte firm adhesion and transmigration were quantified. Results are expressed as mean ± SEM for n = 3-5 mice per group. Statistically significant difference between control and zymosan-treated groups are represented by asterisks: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Finally, the photomicrographs illustrate the neutrophilic profile of zymosan-induced leukocyte migration in this model. Saline-injected (c) or zymosan-injected (d) tissues were stained with topical acridine orange (1.0 mg/ml) and images obtained using a Zeiss LSM 5 Pascal confocal microscope.

 
Zymosan-induced leukocyte responses within cremasteric venules is suppressed in NE-/- mice and in WT tissues treated with a specific NE inhibitor

Leukocyte firm adhesion and transmigration in zymosan (30 µg)-stimulated cremaster muscles was significantly suppressed in NE-/- mice (inhibitions of 65 and 66%, respectively; p < 0.001) (Fig. 3, a and b). In contrast, no statistically significant differences were observed in leukocyte rolling flux or rolling velocity in WT and NE-/- mice (results not shown). Furthermore, quantification of peripheral blood counts, mean arterial pressure, and erythrocyte velocity showed no significant differences between the two strains (results not shown).



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  		FIGURE 3. Zymosan-induced leukocyte responses within cremasteric venules of NE-/- mice and WT mice treated with an NE inhibitor. Mice were treated i.s. for 4 h with zymosan (30 µg) in 400 µl of sterile saline with control mice receiving saline. Leukocyte firm adhesion (a) and transmigration (b) were quantified by IVM. In additional experiments, WT mice received zymosan and the NE-specific inhibitor, ONO-5046 (30 or 100 µM concentration within the injection fluid of 400 µl of sterile saline). Panels show leukocyte firm adhesion (c) and leukocyte transmigration (d). Results are expressed as mean ± SEM for n = 3-5 mice per group. Statistically significant differences between WT and NE-/- mice or control and ONO-5046-treated mice are represented by asterisks: *, p < 0.05; ***, p < 0.001.

 
In agreement with the above findings, local administration of a specific NE inhibitor, ONO-5046 (37), significantly inhibited zymosan-induced leukocyte firm adhesion and transmigration in cremasteric venules (Fig. 3, c and d). For these experiments, zymosan particles were coinjected with ONO-5046 (30 or 100 µM solutions). Although 30 µM ONO-5046 had no inhibitory effects, the higher dose of 100 µM exhibited a significant inhibition of both leukocyte firm adhesion and transmigration (inhibitions of 74 and 61%, respectively; p < 0.05), inhibitory effects that were comparable with those observed in NE-/- mice.

As collectively, the above series of in vivo experiments indicated the functional importance of local NE enzyme activity in regulation of leukocyte responses elicited by zymosan, we next examined whether this stimulus could directly induce the cell surface expression and/or release of active NE from neutrophils in vitro. For this purpose, bone-marrow mouse neutrophils were stimulated with zymosan particles (0.003–3 mg/ml) for 30 min before NE was assayed. Zymosan induced a dose-dependent increase in both cell-associated (Fig. 4a) and released (Fig. 4b) NE activity above PBS-treated cells. Neutrophils isolated from NE-/- mice were included in the experiments to demonstrate the specificity of the enzyme assay for NE. Of relevance, bone marrow-derived neutrophils exhibited a similar profile of responsiveness to that obtained from murine blood neutrophils with respect to NE release/cell surface expression (data not shown).



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  		FIGURE 4. Zymosan-stimulated murine neutrophils exhibit both cell surface expression and released NE. Wild-type and NE-/- neutrophils (5 x 105/well) were purified from bone marrow and stimulated with a range of zymosan particle concentrations (0.003–3 mg/ml, final concentrations in wells) for 30 min at 37°C. Plates were spun and supernatants were collected for assay of NE activity before the pelleted cells were fixed. Cells or supernatants were then incubated for 25 min at 37°C with 400 µM of the NE-specific fluorogenic substrate. NE activity per 5 x 105 neutrophils was detected using a fluorescent plate reader and quantified as compared with a standard curve created using purified human NE, as detailed in Materials and Methods. Results are expressed as mean ± SEM of triplicates for n = 3-4 separate experiments. Statistically significant difference between control and zymosan-treated groups are represented by asterisks: ***, p < 0.001.

 
Neutrophils from NE-deficient mice exhibit normal levels of key adhesion molecules

Having observed a defect in leukocyte responses induced by zymosan particles in NE-/- mice, we next aimed to address the mechanism by which this occurred. For this purpose, initial studies investigated the expression profile of key adhesion and zymosan-binding sites on the cell surface of neutrophils from WT and NE-/- mice, as quantified by flow cytometry. Neutrophils from WT and NE-/- mice exhibited comparable levels of expression of the adhesion molecules {alpha}2, {alpha}4, {alpha}5, {alpha}6, {beta}1, and {beta}2 integrins, as well as L-selectin and PECAM-1 (Table I). Furthermore, NE-/- neutrophils responded normally to an exogenous stimulus, PMA (100 ng/ml for 20 min), with respect to increased expression of {beta}2 integrins. Finally, no significant difference between expressions of zymosan binding molecules CD11b and dectin-1 was found on the cell surface of neutrophils obtained from the two strains of animals.


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  		Table I. Profile of selected receptors and adhesion molecules on the cell surface of WT and NE-/- mouse neutrophilsa

 
Zymosan-induced generation of endogenous inflammatory mediators is suppressed in NE-deficient mice

As zymosan-induced leukocyte migration is associated with the generation of endogenous inflammatory mediators, we next examined the role of NE in this response. For this purpose, control or zymosan-stimulated cremaster muscles, obtained from both WT and NE-/- mice, were homogenized and assayed by ELISA for the cytokines IL-1{beta} and TNF-{alpha}, and the chemokines, KC and MIP-1{alpha}.

As compared with saline-treated tissues, zymosan-stimulated cremasters exhibited a significantly elevated level of immunoreactive IL-1{beta} in WT mice (Fig. 5a), a response that was significantly suppressed in NE-/- animals (61% inhibition, p < 0.01). In contrast, no significant levels of TNF-{alpha} could be detected in zymosan-stimulated tissues from either WT or NE-/- mice (Fig. 5a). These negative detection results were in agreement with functional observations where a soluble TNF-{alpha}-receptor (p55) fusion protein (10 µg), when coinjected with zymosan particles, exerted no inhibitory effects (Fig. 5, b and c). In parallel studies, this reagent did however inhibit TNF-{alpha}-induced leukocyte migration in the cremaster muscle (data not shown), in agreement with its effects on TNF-{alpha}-elicited responses in other models (38).



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  		FIGURE 5. Analysis of cytokine generation in homogenates of zymosan-stimulated cremaster muscles from WT and NE-/- mice. a, Cremaster muscles from mice treated with saline or zymosan (30 µg, 4-h test period) were removed, homogenized and levels of IL-1{beta} and TNF-{alpha} were measured by ELISA. The functional role of TNF-{alpha} in leukocyte firm adhesion (b) and transmigration (c) induced by zymosan was further analyzed by IVM using a soluble TNFR (p55) fusion protein. For these studies, zymosan was coinjected i.s. with either the p55 fusion protein, or a control fusion protein (CSF25), both at the dose of 10 µg. Results are expressed as mean ± SEM for n = 3-5 mice per group. Statistically significant differences between saline and zymosan-treated groups or WT and NE-/- mice are represented by asterisks: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
Zymosan-stimulated tissues also contained significantly elevated levels of the chemokines KC and MIP-1{alpha} (Fig. 6, a and b), responses that were again significantly reduced in tissues from NE-/- animals (inhibitions of 70 and 57% for KC and MIP-1{alpha}, respectively, p < 0.05). To investigate the possibility that the reduced level of KC in zymosan-stimulated tissues of NE-/- mice was as result of reduced leukocyte infiltration, NE-/- mice were injected with a combination of zymosan particles and IL-1{beta} (30 ng), the latter being used as a mediator that induces normal leukocyte transmigration in NE-/- animals (Fig. 1). Although coadministration of zymosan and IL-1{beta} in NE-/- mice elicited a leukocyte transmigration response comparable to that induced by zymosan alone in WT animals (Fig. 6c), the levels of KC in the NE-/- mice were still significantly reduced (Fig. 6d). These results indicate that the low KC level in NE-/- mice was not attributable to the reduced infiltration of leukocytes in the tissues analyzed but was as a result of the animal’s NE deficiency.



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  		FIGURE 6. Chemokine levels in zymosan-stimulated cremaster muscles of WT and NE-/- mice. Cremaster muscles were injected with saline or zymosan (30 µg) and 4 h later the tissues were removed, homogenized and levels of KC (a) and MIP-1{alpha} (b) were measured by ELISA. In a second series of experiments, leukocyte transmigration (c) and levels of KC (d) were quantified in tissues when zymosan (30 µg) was injected alone or coinjected with IL-1{beta} (30 ng) in the NE-deficient mice. Both graphs show responses induced by zymosan alone in WT animals for comparison. Results are expressed as mean ± SEM for n = 3-5 mice per group. Statistically significant differences between WT and NE-/- mice or other indicated comparisons are represented by asterisks: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
NE-deficient mice exhibit impaired phagocytosis of zymosan particles in vivo

As phagocytosis of particulate stimuli is closely associated with generation of inflammatory mediators, in a final series of experiments, we investigated the ability of NE-/- neutrophils to phagocytose zymosan particles in vitro and in vivo. In vitro, bone-marrow derived neutrophils were stimulated with serum-opsonized zymosan particles (0.3–3 mg/ml) and the number of phagocytosing neutrophils was quantified by microscopy. All concentrations of zymosan tested led to a phagocytic response from both WT and NE-/- neutrophils with no significant difference between the two cell types being detected (results not shown).

As in vitro assays of phagocytosis are highly simplistic, we also investigated neutrophil phagocytosis using a novel in vivo experimental approach. Briefly, the above cremaster muscle IVM model was extended so that we could simultaneously quantify established parameters of leukocyte responses as well as leukocyte phagocytosis. For this purpose, responses to fluorescent zymosan particles (30 µg i.s.) were quantified using an in vivo test period of 6 h, an observation period during which we observed readily quantifiable, reproducible, and significant phagocytosis responses in WTs. Of relevance, zymosan-induced leukocyte transmigration, quantified in this set of studies as per field of view, was suppressed in NE-/- mice (81%, p < 0.01, Fig. 7a), in agreement with previous observations using a 4-h reaction time (Fig. 3b).



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  		FIGURE 7. Phagocytosis of fluorescent zymosan particles in WT and NE-/- mice. WT and NE-/- mice were treated i.s. for 6 h with Alexa Fluor 488-conjugated zymosan (30 µg) in 400 µl of sterile saline. Leukocyte transmigration (a) and phagocytosis (b) were quantified by IVM as described in Materials and Methods. c, The same data when it has been normalized for the number of infiltrating leukocytes and is described as index of phagocytosis (%). For comparison, d shows phagocytosis of E. coli particles in WT and NE-/- mice. For these experiments, mice were injected i.s. with FITC-labeled E. coli (1 x 107 particles) for 6 h before transmigration and phagocytosis were quantified as above and results are presented as index of phagocytosis (%). Results are mean ± SEM for n = 5 mice per group. Statistically significant differences between WT and NE-/- groups are indicated by asterisks: * p < 0.05; ** p < 0.01; *** p < 0.001.

 
The number of transmigrated leukocytes that had engulfed fluorescent zymosan particles was quantified and the results are presented both in terms of absolute numbers of leukocytes (Fig. 7b) or, to correct for the reduced leukocyte transmigration response in the NE-/- mice, as a percentage of infiltrating leukocytes (Fig. 7c). Both modes of data presentation indicated a significant suppression of phagocytosis of fluorescent zymosan particles in NE-/- animals as compared with WTs (78% inhibition, p < 0.05). These findings did not appear to reflect a generalized defect in phagocytosis in the NE-/- mice, as phagocytosis of E. coli particles (FITC-conjugated/1 x 107 particles) was not affected in mice lacking NE (Fig. 7d).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recent advancements in the field of leukocyte transmigration have led to the identification of several new molecular interactions that may mediate and/or regulate this complex cellular response critical to both innate and adaptive immunity (2). Despite such developments, certain controversies related to the functional role of specific molecules implicated in the process of leukocyte migration remain unresolved. The role of NE falls within this category in that despite many reports into the effects of pharmacological inhibitors of NE on leukocyte migration in vitro and in vivo, there is evidence to both support and refute the potential role of NE in this response (24, 25, 27, 29, 30, 39, 40). The availability of genetically modified mice deficient in NE has provided an important experimental tool for further investigations into the role of this serine protease in leukocyte migration at sites of inflammation. These mice have demonstrated the importance of NE in host defense but the studies performed to date have not shown a critical role for this enzyme in leukocyte migration in vivo (31, 32). The aim of the present study was to use NE-/- mice to investigate the functional role of NE in leukocyte migration as investigated by the technique of IVM, an experimental approach that enabled us to address the potential role of NE in the different stages of leukocyte migration in vivo. Collectively, while leukocyte responses induced by the cytokines IL-1{beta} and TNF-{alpha} were found to be unaltered in mice deficient in NE, zymosan-induced leukocyte firm adhesion and transmigration was significantly suppressed. This inhibition appeared to be associated with a defect in zymosan-induced generation of proinflammatory cytokines and chemokines and phagocytosis of zymosan particles in vivo, demonstrating a role for NE in a broad-spectrum of inflammatory responses induced by this particulate stimulus.

NE-/- mice were used to investigate the functional role of NE in leukocyte migration induced by the cytokines IL-1{beta} and TNF-{alpha} using two inflammatory models, namely leukocyte infiltration into the peritoneal cavity and migration through cremasteric venules, as observed by IVM. In both models, locally administered cytokines elicited significant leukocyte transmigration in WT and NE-/- mice with no significant differences being observed between the responses in the two strains of animals. Although NE inhibitors have previously been indicated to suppress IL-1{beta}- and/or TNF-{alpha}-induced neutrophil adhesion to cultured endothelial cells in vitro and cytokine-induced neutrophil migration in vivo (20, 41), this is the first reported study on the profile of leukocyte responses elicited by these cytokines in NE-/- mice. The normal neutrophil infiltration responses observed in the present models are in agreement with the reported normal infiltration of neutrophils induced by i.p. injection of Gram-negative bacteria or the nonspecific inflammatory stimulus, thioglycolate (31, 32). In addition, in vitro, NE-/- bone-marrow-derived neutrophils exhibited normal transmigration through TNF-{alpha}-stimulated cultured murine endothelial cells under conditions of flow (42). Taken together, these findings indicate that NE does not have an essential role in mediating neutrophil migration elicited by IL-1{beta} or TNF-{alpha}.

To extend the study to investigations into the role of NE in a broader range of inflammatory responses associated with leukocyte migration, e.g., endogenous mediator generation and phagocytosis, responses elicited by the particulate stimulus, zymosan, were studied. Local administration of zymosan elicited a dose- and time-dependent increase in leukocyte firm adhesion and transmigration in mouse cremasteric venules (within 4–6 h), with the predominant infiltrating leukocytes being neutrophils. In this model, in contrast to the responses elicited by preformed cytokines, zymosan-induced leukocyte firm adhesion and transmigration was significantly inhibited in NE-/- animals (65 and 66%, respectively). As the ratio of firmly adherent leukocytes to transmigrated leukocytes in the two mouse strains was identical (2.5 for both WT and NE-/- mice), the findings indicate that the resultant suppressed leukocyte transmigration response was directly associated with defects in leukocyte firm adhesion.

In agreement with the findings in NE-/- mice, coinjection of the specific elastase inhibitor, ONO-5046 (37), with zymosan particles significantly inhibited leukocyte firm adhesion and transmigration in cremasteric venules of WT mice. The levels of inhibition detected under conditions of pharmacological blockade or genetic deletion of NE were highly comparable. As the NE inhibitor was administered i.s. together with zymosan particles, its efficacy indicates the importance of locally generated NE, and its enzyme activity, in the process of leukocyte migration. These results provide the first direct evidence for a nonredundant role for locally generated NE in regulation of leukocyte firm adhesion to venular endothelium in vivo. Although in vitro studies indicated that opsonized zymosan particles could directly stimulate murine neutrophils for increased cell surface and released NE activity, the mechanism by which this response was associated with leukocyte migration was unclear and required further investigation.

Quantification of expression of key adhesion molecules on the cell surface of control or PMA-stimulated neutrophils from WT and NE-/- mice did not show any significant differences. In addition, no defects in expression of key zymosan-binding molecules, Mac-1 and dectin-1 as opsonic and nonopsonic receptors for zymosan, respectively (43, 44), were detected on the surface of NE-/- neutrophils. Thus, potential defects in expression profiles of key leukocyte adhesion molecules (or receptors) does not appear to account for the defect in leukocyte firm adhesion observed in zymosan-treated NE-/- mice. The normal leukocyte infiltration detected in the NE-/- mice in response to IL-1{beta} and TNF-{alpha} also strongly argues against any potential defects in expression of key endothelial cell-associated adhesion molecules such as ICAM-1 or E-selectin.

As zymosan is a potent inducer of endogenously generated inflammatory mediators (45, 46, 47, 48, 49, 50, 51), we investigated the potential role of NE in generation of mediators in zymosan-stimulated cremaster muscles. For this purpose, levels of the cytokines, IL-1{beta} and TNF-{alpha}, and the chemokines, KC and MIP-1{alpha}, in homogenates of zymosan-stimulated cremaster muscles were quantified by ELISA. In WT mice, zymosan-stimulated cremaster muscles exhibited a marked increase in levels of IL-1{beta}, KC, and MIP-1{alpha}, but not TNF-{alpha}, as compared with saline-injected tissues, responses that were all significantly suppressed in tissues obtained from NE-/- animals. These findings are in agreement with the observations of Adkison et al. (33) where, using mice deficient in both NE and cathepsin G, reduced levels of cytokines were detected in response to zymosan within an air pouch model. The present findings could clearly provide an explanation for the reduced leukocyte firm adhesion response detected in zymosan-stimulated cremasteric venules of NE-/- mice. However, because neutrophils are themselves a rich source of inflammatory mediators (e.g., chemokines) (52, 53, 54), it is possible that the reduced level of mediators in tissues from NE-deficient mice was at least partly due to the reduced neutrophil infiltration response. To address this possibility, the ability of IL-1{beta} to induce normal neutrophil migration in NE-/- mice was exploited. Coinjection of IL-1{beta} and zymosan into NE-/- mice induced a neutrophil migration response similar to that induced by zymosan alone in WT animals. Despite this comparable leukocyte infiltration response, tissue levels of KC remained significantly low in the NE-/- mice demonstrating that the reduced levels of KC quantified were attributable to the absence of NE and not impaired neutrophil transmigration. The mechanism by which NE enhances mediator levels in zymosan-stimulated tissues is unclear but may involve direct effects of released NE on resident inflammatory cells and/or infiltrating leukocytes. In support of this possibility, neutrophil-derived NE has been shown to directly stimulate the release of proinflammatory mediators from macrophages (55). Furthermore, in recent years a number of proteases have been associated with the cleavage and/or processing of cytokines/chemokines and/or their receptors and as a result there is much interest in the role of proteases as regulators of mediator bioactivity and availability (56). In line with these findings, the results of the present study, together with other published data (33, 57), strongly support such a role for NE. However, while it is conceivably possible that NE may be directly involved in regulation of mediator generation and/or action in response to zymosan, it is also possible that NE may have an indirect role in this process. In this context, as phagocytosis is a key cellular response leading to inflammatory mediator generation (46, 53, 54, 58), in a final series of experiments the potential role of NE in phagocytosis of zymosan particles was investigated both in vitro and in vivo.

Initial studies compared the ability of neutrophils from WT and NE-/- mice to phagocytose opsonized zymosan particles in vitro. In this assay, no significant difference in phagocytosis was observed between WT and NE-/- neutrophils. To investigate phagocytosis in vivo, fluorescently conjugated zymosan particles were injected i.s. in WT and NE-/- mice and responses were observed by IVM as a novel extension of the cremaster muscle model. The findings indicated that NE-/- mice had an impaired ability to phagocytose zymosan particles. This defect appeared to be stimulus specific as phagocytosis of fluorescently labeled E. coli particles was normal in the NE-/- mice, in agreement with the work of Belaaouaj et al. (31). Furthermore, Tkalcevic et al. (32) found no defects in the ability of peritoneal neutrophils derived from NE-/- mice in phagocytosis of Candida albicans. The reason for the contrasting findings of our in vitro and in vivo assays of phagocytosis are unknown but may clearly be due to the complexities of this response within the in vivo scenario, as opposed to the simple conditions used in vitro. For example, while in vitro, zymosan particles were opsonized using serum, in vivo, other mechanisms, such as cell-derived complement products, (59, 60) may be used which may lead to phagocytosis via more heterogeneous mechanisms, some involving NE. Furthermore, the observed in vivo defect in phagocytosis of zymosan particles may be associated with a potential role for NE in regulation of expression of uncharacterized zymosan-binding molecules, physical breakdown of the particles (thus aiding their uptake), and/or priming of neutrophils for the phagocytic response through regulated availability and activity of inflammatory mediators or adhesive mechanisms.

Collectively, we have identified a significant nonredundant role for neutrophil elastase in zymosan-induced leukocyte firm adhesion and transmigration in vivo. Although the precise mechanism by which NE mediates this response is unclear, the results indicate that it is associated with regulation of phagocytosis of zymosan particles and subsequent generation and/or release of proinflammatory mediators. However, because zymosan-induced neutrophil migration was not totally blocked in the NE-deficient mice, in the light of the present findings, and previous observations reported by others (46, 47, 49, 58), one can hypothesize that zymosan-induced neutrophil infiltration may have two broad phases, an NE-independent and an NE-dependent phase. The initial NE-independent phase may be mediated by early generation of potent chemoattractants through activation of the complement cascade leading to the generation of C5a and/or zymosan-induced generation of lipid mediators such as leukotriene B4 and platelet-activating factor from resident inflammatory cells. Once in the extravascular tissue, neutrophils within this first phase of infiltrate may then phagocytose opsonized zymosan particles, a response that can lead to further release, generation, and/or processing of inflammatory mediators, initiating the second NE-dependent phase of leukocyte migration. Although details of the mechanisms by which NE mediates these latter cellular and molecular events are at present unknown, the findings provide further evidence for a significant role for neutrophil elastase in the host immune reaction in response to infiltrating pathogens.


   Footnotes
 
1 This work was supported by the British Heart Foundation and the Wellcome Trust. R.E.Y. was funded by a British Heart Foundation PhD studentship. Back

2 Address correspondence and reprint requests to Dr. Sussan Nourshargh, Reader in Microvascular Biology, Cardiovascular Medicine Unit, Eric Bywaters Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 ONN, U.K. E-mail address: s.nourshargh{at}imperial.ac.uk Back

3 Abbreviations used in this paper: PECAM-1, platelet endothelial cell adhesion molecule-1; NE, neutrophil elastase; WT, wild type; IVM, intravital microscopy; i.s., intrascrotal; MIP, macrophage inflammatory protein; RFI, relative fluorescence intensity. Back

Received for publication November 24, 2003. Accepted for publication January 28, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yadav, R., K. Y. Larbi, R. E. Young, S. Nourshargh. 2003. Migration of leukocytes through the vessel wall and beyond. Thromb. Haemostasis 90:598.[Medline]
  2. Muller, W. A.. 2003. Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trends Immunol. 24:327.[Medline]
  3. Martin-Padura, I., S. Lostaglio, M. Schneemann, L. Williams, M. Romano, P. Fruscella. 1998. Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J. Cell Biol. 142:117.[Abstract/Free Full Text]
  4. Issekutz, A. C., D. Rowter, T. A. Springer. 1999. Role of ICAM-1 and ICAM-2 and alternate CD11/CD18 ligands in neutrophil transendothelial migration. J. Leukocyte Biol. 65:117.[Abstract]
  5. Dangerfield, J. P., K. Y. Larbi, M. T. Huang, A. Dewar, S. Nourshargh. 2002. PECAM-1 (CD31) homophilic interaction up-regulates {alpha}6{beta}1 on transmigrated neutrophils in vivo and plays a functional role in the ability of {alpha}6 integrins to mediate leukocyte migration through the perivascular basement membrane. J. Exp. Med. 196:1201.[Abstract/Free Full Text]
  6. Duncan, G. S., D. P. Andrew, H. Takimoto, S. A. Kaufman, H. Yoshida, J. Spellberg, J. Luis de la Pompa, A. Elia, A. Wakeham, B. Karan-Tamir, et al 1999. Genetic evidence for functional redundancy of platelet-endothelial cell adhesion molecule-1 (PECAM-1): CD31-deficient mice reveal PECAM-1 dependent and PECAM-1-independent functions. J. Immunol. 162:3022.[Abstract/Free Full Text]
  7. Thompson, R. D., K. E. Noble, K. Y. Larbi, A. Dewar, G. S. Duncan, T. W. Mak, S. Nourshargh. 2001. Platelet-endothelial cell adhesion molecule-1 (PECAM-1)-deficient mice demonstrate a transient and cytokine-specific role for PECAM-1 in leukocyte migration through the perivascular basement membrane. Blood 97:1854.[Abstract/Free Full Text]
  8. Wakelin, M. W., M. J. Sanz, A. Dewar, S. M. Albelda, S. W. Larkin, N. Boughton-Smoth, T. J. Williams, S. Nourshargh. 1996. An anti-platelet-endothelial cell adhesion molecule-1 antibody inhibits leukocyte extravasation from mesenteric microvessels in vivo by blocking the passage through the basement membrane. J. Exp. Med. 184:229.[Abstract/Free Full Text]
  9. Moll, T., E. Dejana, D. Vestweber. 1998. In vitro degradation of endothelial catenins by a neutrophil protease. J. Cell Biol. 140:403.[Abstract/Free Full Text]
  10. Bonnefoy, A., C. Legrand. 2000. Proteolysis of subendothelial adhesive glycoproteins (fibronectin, thrombospondin, and von Willebrand factor) by plasmin, leukocyte cathepsin G, and elastase. Thromb. Res. 98:323.[Medline]
  11. Watanabe, H., S. Hattori, S. Katsuda, I. Nakanishi, Y. Nagai. 1990. Human neutrophil elastase: degradation of basement membrane components and immunolocalization in the tissue. J. Biochem. 108:753.[Abstract/Free Full Text]
  12. Zhu, Y. K., X. D. Liu, C. M. Skold, T. Umino, H. J. Wang, J. R. Spurzem, T. Kohyama, R. F. Ertl, S. I. Rennard. 2001. Synergistic neutrophil elastase-cytokine interaction degrades collagen in three-dimensional culture. Am. J. Physiol. 281:L868.
  13. Smedly, L. A., M. G. Tonnesen, R. A. Sandhaus, C. Haslett, L. A. Guthrie, R. B. Johnston, P. M. Henson, G. S. Worthen. 1986. Neutrophil-mediated injury to endothelial cells: enhancement by endotoxin and essential role of neutrophil elastase. J. Clin. Invest. 77:1233.
  14. Champagne, B., P. Tremblay, A. Cantin, Y. St Pierre. 1998. Proteolytic cleavage of ICAM-1 by human neutrophil elastase. J. Immunol. 161:6398.[Abstract/Free Full Text]
  15. Levesque, J. P., Y. Takamatsu, S. K. Nilsson, D. N. Haylock, P. J. Simmons. 2001. Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor. Blood 98:1289.[Abstract/Free Full Text]
  16. Owen, C. A., E. J. Campbell. 1999. The cell biology of leukocyte-mediated proteolysis. J. Leukocyte Biol. 65:137.[Abstract]
  17. Zimmerman, B. J., D. N. Granger. 1990. Reperfusion-induced leukocyte infiltration: role of elastase. Am. J. Physiol. 259:H390.
  18. Shapiro, S. D.. 2002. Proteinases in chronic obstructive pulmonary disease. Biochem. Soc. Trans. 30:98.[Medline]
  19. Stockley, R. A.. 2002. Neutrophils and the pathogenesis of COPD. Chest 121:151S.[Free Full Text]
  20. Ishii, K., Y. Ito, H. Katagiri, Y. Matsumoto, A. Kakita, M. Majima. 2002. Neutrophil elastase inhibitor attenuates lipopolysaccharide-induced hepatic microvascular dysfunction in mice. Shock 18:163.[Medline]
  21. Ishikawa, N., M. Oda, M. Kawaguchi, Y. Tsunezuka, G. Watanabe. 2003. The effects of a specific neutrophil elastase inhibitor (ONO-5046) in pulmonary ischemia-reperfusion injury. Transpl. Int. 16:341.[Medline]
  22. Kakimoto, K., A. Matsukawa, M. Yoshinaga, H. Nakamura. 1995. Suppressive effect of a neutrophil elastase inhibitor on the development of collagen-induced arthritis. Cell. Immunol. 165:26.[Medline]
  23. Kawabata, K., T. Hagio, S. Matsumoto, S. Nakao, S. Orita, Y. Aze, H. Ohno. 2000. Delayed neutrophil elastase inhibition prevents subsequent progression of acute lung injury induced by endotoxin inhalation in hamsters. Am. J. Respir. Crit. Care Med. 161:2013.[Abstract/Free Full Text]
  24. Woodman, R. C., P. H. Reinhardt, S. Kanwar, F. L. Johnston, P. Kubes. 1993. Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels. Blood 82:2188.[Abstract/Free Full Text]
  25. Rosengren, S., K. E. Arfors. 1990. Neutrophil-mediated vascular leakage is not suppressed by leukocyte elastase inhibitors. Am. J. Physiol. 259:H1288.
  26. Delclaux, C., C. Delacourt, M. P. D’Ortho, V. Boyer, C. Lafuma, A. Harf. 1996. Role of gelatinase B and elastase in human polymorphonuclear neutrophil migration across basement membrane. Am. J. Respir. Cell Mol. Biol. 14:288.[Abstract]
  27. Furie, M. B., B. L. Naprstek, S. C. Silverstein. 1987. Migration of neutrophils across monolayers of cultured microvascular endothelial cells. An in vitro model of leucocyte extravasation. J. Cell Sci. 88:161.[Abstract/Free Full Text]
  28. Heck, L. W., W. D. Blackburn, M. H. Irwin, D. R. Abrahamson. 1990. Degradation of basement membrane laminin by human neutrophil elastase and cathepsin G. Am. J. Pathol. 136:1267.[Abstract]
  29. Huber, A. R., S. J. Weiss. 1989. Disruption of the subendothelial basement membrane during neutrophil diapedesis in an in vitro construct of a blood vessel wall. J. Clin. Invest. 83:1122.
  30. Mackarel, A. J., D. C. Cottell, K. J. Russell, M. X. FitzGerald, C. M. O’Connor. 1999. Migration of neutrophils across human pulmonary endothelial cells is not blocked by matrix metalloproteinase or serine protease inhibitors. Am. J. Respir. Cell Mol. Biol. 20:1209.[Abstract/Free Full Text]
  31. Belaaouaj, A., R. McCarthy, M. Baumann, Z. Gao, T. J. Ley, S. N. Abraham, S. D. Shapiro. 1998. Mice lacking neutrophil elastase reveal impaired host defense against Gram negative bacterial sepsis. Nat. Med. 4:615.[Medline]
  32. Tkalcevic, J., M. Novelli, M. Phylactides, J. P. Iredale, A. W. Segal, J. Roes. 2000. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12:201.[Medline]
  33. Adkison, A. M., S. Z. Raptis, D. G. Kelley, C. T. Pham. 2002. Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis. J. Clin. Invest. 109:363.[Medline]
  34. Ley, K., D. C. Bullard, M. L. Arbones, R. Bosse, D. Vestweber, T. F. Tedder, A. L. Beaudet. 1995. Sequential contribution of L- and P-selectin to leukocyte rolling in vivo. J. Exp. Med. 181:669.[Abstract/Free Full Text]
  35. Nourshargh, S., S. W. Larkin, A. Das, T. J. Williams. 1995. Interleukin-1-induced leukocyte extravasation across rat mesenteric microvessels is mediated by platelet-activating factor. Blood 85:2553.[Abstract/Free Full Text]
  36. Owen, C. A., M. A. Campbell, P. L. Sannes, S. S. Boukedes, E. J. Campbell. 1995. Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J. Cell Biol. 131:775.[Abstract/Free Full Text]
  37. Kawabata, K., M. Suzuki, M. Sugitani, K. Imaki, M. Toda, T. Miyamoto. 1991. ONO-5046, a novel inhibitor of human neutrophil elastase. Biochem. Biophys. Res. Commun. 177:814.[Medline]
  38. Sanz, M. J., A. Hartnell, P. Chisholm, C. Williams, D. Davies, V. B. Weg, M. Feldmann, M. A. Bolanowski, R. R. Lobb, S. Nourshargh. 1997. Tumor necrosis factor {alpha}-induced eosinophil accumulation in rat skin is dependent on {alpha}4 integrin/vascular cell adhesion molecule-1 adhesion pathways. Blood 90:4144.[Abstract/Free Full Text]
  39. Steadman, R., P. L. St John, R. A. Evans, G. J. Thomas, M. Davies, L. W. Heck, D. R. Abrahamson. 1997. Human neutrophils do not degrade major basement membrane components during chemotactic migration. Int. J. Biochem. Cell Biol. 29:993.[Medline]
  40. Cepinskas, G., M. Sandig, P. R. Kvietys. 1999. PAF-induced elastase-dependent neutrophil transendothelial migration is associated with the mobilization of elastase to the neutrophil surface and localization to the migrating front. J. Cell Sci. 112:1937.[Abstract]
  41. Nozawa, F., M. Hirota, A. Okabe, M. Shibata, T. Iwamura, Y. Haga, M. Ogawa. 2000. Elastase activity enhances the adhesion of neutrophil and cancer cells to vascular endothelial cells. J. Surg. Res. 94:153.[Medline]
  42. Allport, J. R., Y. C. Lim, J. M. Shipley, R. M. Senior, S. D. Shapiro, N. Matsuyoshi, D. Vestweber, F. W. Luscinskas. 2002. Neutrophils from MMP-9- or neutrophil elastase-deficient mice show no defect in transendothelial migration under flow in vitro. J. Leukocyte Biol. 71:821.[Abstract/Free Full Text]
  43. Brown, G. D., P. R. Taylor, D. M. Reid, J. A. Willment, D. L. Williams, L. Martinez-Pomares, S. Y. Wong, S. Gordon. 2002. Dectin-1 is a major {beta}-glucan receptor on macrophages. J. Exp. Med. 196:407.[Abstract/Free Full Text]
  44. Ehlers, M. R.. 2000. CR3: a general purpose adhesion-recognition receptor essential for innate immunity. Microbes Infect. 2:289.[Medline]
  45. Ajuebor, M. N., R. J. Flower, R. Hannon, M. Christie, K. Bowers, A. Verity, M. Perretti. 1998. Endogenous monocyte chemoattractant protein-1 recruits monocytes in the zymosan peritonitis model. J. Leukocyte Biol. 63:108.[Abstract]
  46. Fantuzzi, G., S. Sacco, P. Ghezzi, C. A. Dinarello. 1997. Physiological and cytokine responses in IL-1{beta}-deficient mice after zymosan-induced inflammation. Am. J. Physiol. 273:R400.
  47. Forrest, M. J., P. J. Jose, T. J. Williams. 1986. Kinetics of the generation and action of chemical mediators in zymosan-induced inflammation of the rabbit peritoneal cavity. Br. J. Pharmacol. 89:719.[Medline]
  48. Jose, P. J., M. J. Forrest, T. J. Williams. 1985. Generation of C5a and prostacyclin (PGI2) in an inflammatory response to zymosan and a reversed passive Arthus-type reaction in the peritoneal cavity of the rabbit. Agents Actions 16:39.[Medline]
  49. Perretti, M., E. Solito, L. Parente. 1992. Evidence that endogenous interleukin-1 is involved in leukocyte migration in acute experimental inflammation in rats and mice. Agents Actions 35:71.[Medline]
  50. Sanchez-Crespo, M., F. Alonso, J. Egido. 1980. Platelet-activating factor in anaphylaxis and phagocytosis. I. Release from human peripheral polymorphonuclears and monocytes during the stimulation by ionophore A23187 and phagocytosis but not from degranulating basophils. Immunology 40:645.[Medline]
  51. von Asmuth, E. J., J. G. Maessen, C. J. van der Linden, W. A. Buurman. 1990. Tumour necrosis factor {alpha} (TNF{alpha}) and interleukin 6 in a zymosan-induced shock model. Scand. J. Immunol. 32:313.[Medline]
  52. Cassatella, M. A., F. Bazzoni, M. Ceska, I. Ferro, M. Baggiolini, G. Berton. 1992. IL-8 production by human polymorphonuclear leukocytes: the chemoattractant formyl-methionyl-leucyl-phenylalanine induces the gene expression and release of IL-8 through a pertussis toxin-sensitive pathway. J. Immunol. 148:3216.[Abstract]
  53. Bazzoni, F., M. A. Cassatella, F. Rossi, M. Ceska, B. Dewald, M. Baggiolini. 1991. Phagocytosing neutrophils produce and release high amounts of the neutrophil-activating peptide 1/interleukin 8. J. Exp. Med. 173:771.[Abstract/Free Full Text]
  54. Au, B. T., T. J. Williams, P. D. Collins. 1994. Zymosan-induced IL-8 release from human neutrophils involves activation via the CD11b/CD18 receptor and endogenous platelet-activating factor as an autocrine modulator. J. Immunol. 152:5411.[Abstract]
  55. Fadok, V. A., D. L. Bratton, L. Guthrie, P. M. Henson. 2001. Differential effects of apoptotic versus lysed cells on macrophage production of cytokines: role of proteases. J. Immunol. 166:6847.[Abstract/Free Full Text]
  56. Bank, U., S. Ansorge. 2001. More than destructive: neutrophil-derived serine proteases in cytokine bioactivity control. J. Leukocyte Biol. 69:197.[Abstract/Free Full Text]
  57. Haga, Y., M. Ogawa. 1997. Neutrophil elastase inhibitor (ONO-5046.Na) decreases production of proinflammatory cytokines by lipopolysaccharide-stimulated human monocytes. Res. Commun. Mol. Pathol. Pharmacol. 98:243.[Medline]
  58. Rao, T. S., J. L. Currie, A. F. Shaffer, P. C. Isakson. 1994. In vivo characterization of zymosan-induced mouse peritoneal inflammation. J. Pharmacol. Exp. Ther. 269:917.[Abstract/Free Full Text]
  59. Ezekowitz, R. A., R. B. Sim, G. G. MacPherson, S. Gordon. 1985. Interaction of human monocytes, macrophages, and polymorphonuclear leukocytes with zymosan in vitro: role of type 3 complement receptors and macrophage-derived complement. J. Clin. Invest. 76:2368.
  60. Ezekowitz, R. A., R. B. Sim, M. Hill, S. Gordon. 1984. Local opsonization by secreted macrophage complement components: role of receptors for complement in uptake of zymosan. J. Exp. Med. 159:244.[Abstract/Free Full Text]



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