Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation

Abstract

Adhesive interactions of leukocytes and endothelial cells initiate leukocyte migration to inflamed tissue and are important for immune surveillance. Acute and chronic inflammatory diseases show a dysregulated immune response and result in a massive efflux of leukocytes that contributes to further tissue damage. Therefore, targeting leukocyte trafficking may provide a potent form of anti-inflammatory therapy. Leukocyte migration is initiated by interactions of the cell adhesion molecules E-, L-, and P-selectin and their corresponding carbohydrate ligands. Compounds that efficiently address these interactions are therefore of high therapeutic interest. Based on this rationale we investigated synthetic dendritic polyglycerol sulfates (dPGS) as macromolecular inhibitors that operate via a multivalent binding mechanism mimicking naturally occurring ligands. dPGS inhibited both leukocytic L-selectin and endothelial P-selectin with high efficacy. Size and degree of sulfation of the polymer core determined selectin binding affinity. Administration of dPGS in a contact dermatitis mouse model dampened leukocyte extravasation as effectively as glucocorticoids did and edema formation was significantly reduced. In addition, dPGS interacted with the complement factors C3 and C5 as was shown in vitro and reduced C5a levels in a mouse model of complement activation. Thus, dPGS represent an innovative class of a fully synthetic polymer therapeutics that may be used for the treatment of inflammatory diseases.

Recruitment of circulating leukocytes to sites of inflammation is a crucial step in the pathogenesis of acute and chronic forms of inflammatory diseases including hypersensitivity reactions, autoimmune diseases, and ischemia-reperfusion injury (1). It occurs through a multistep cascade in which leukocytes interact with the activated endothelium in postcapillary venules followed by transmigration through the vessel wall and further migration into the extravascular tissue. Each step of this process is regulated by cytokines and chemokines and by several families of adhesion molecules including selectins, integrins, and Ig-CAMs (2). Because blockade of the steps of leukocyte recruitment has been predicted to interrupt leukocyte extravasation, pharmacological interference with the function of key molecules in the multistep recruitment cascade represents a promising strategy for therapeutic intervention in inflammatory disorders (3).

The earliest steps of leukocyte recruitment, tethering and rolling on activated endothelium, are mediated by adhesive interactions between selectins and carbohydrate ligand epitopes on mucin-type glycoproteins expressed on both the vascular endothelium and leukocytes (4, 5). Because selectin knock-out mice show a profound defect of neutrophil accumulation at sites of inflammation (6), pharmacological inhibitors of selectin-mediated adhesion may provide a particularly potent treatment for inflammatory diseases. Therefore, specific inhibitors of selectins have been developed including antiselectin antibodies, inhibitors based on sialyl Lewis x (SiaLe^x) and its isomers, derivatives of heparin, and small glycomimetic molecules such as the macrolide efomycine M (3, 7–10). Each of these inhibitors, however, only showed weak anti-inflammatory effects in vivo that most likely reflects the observation that selectin–ligand binding depends on multiple interactions provided by a proper spatial arrangement of binding epitopes (11), a clustered configuration of oligosaccharides on the polypeptide backbone of the glycoprotein ligands (12), and by a multivalent cell surface display of selectins on neutrophils and lymphocytes (13). Overall, the binding strength originates from the large surface area between leukocytes and endothelial cells with numerous individual binding sites.

Recently, we have established a class of compounds based on dendritic polyglycerols (dPG) (14, 15). These unique macromolecules are highly biocompatible and can present multiple functionalities on their surface (16). In contrast to biological macromolecules, a very high number of functional groups can interact with the respective binding region, which results in a significant increase of binding affinity. In the context of designing heparin analogues, we recently developed a one-step synthesis of dendritic polyglycerol sulfates (dPGS) (17). dPGS are well defined polymers with a tree-like, branched architecture presenting anionic sulfate groups (Fig. 1). Given that P- and L-selectin have a region of positive electrostatic potential in the ligand binding pocket that is responsible for the binding of the anionic sulfotyrosine residues of the physiological ligand PSGL-1 (18, 19), the interaction with a high local anionic charge density may lead to a strong binding of dPGS to these proteins.

Fig. 1.

Dendritic polyglycerol sulfate structure. Representative and idealized structure of dPGS. Sulfate endgroups are shown in red. All details of the different dPGS molecules are summarized in Table S1.

In this work, we present the function of dPGS for multivalent selectin binding and the inhibition of complement activation in vitro. Furthermore, we show the high anti-inflammatory effect of dPGS in vivo.

Results

L- and P-Selectin-Mediated Adhesion Is Blocked by dPGS.

Dendritic polyglycerol sulfates (Fig. 1) can easily be synthesized in only one synthetic step by sulfation starting from the readily available dendritic polyglycerol (17). For a detailed structure-activity relationship, we varied two parameters: the dPG core size and the degree of sulfation. We obtained seven derivatives with 0–61 sulfate groups per macromolecule (Table S1). In addition, the commercially available triglycerol was also used as a scaffold for sulfation.

To evaluate the selectin binding potential of dPGS in vitro we applied a competitive and highly sensitive surface plasmon resonance (SPR)-based binding assay (Fig. S1) that allows the determination of IC₅₀ values for inhibitory compounds over a large concentration range (pM–mM) (20, 21). Initially, we analyzed the selectin specificity of dPGS. Whereas E-selectin binding was not affected by dPGS₄₆, L- and P-selectin were efficiently inhibited with IC₅₀ values of 30 and 90 nM, respectively (Fig. 2A). This binding pattern was expected. As only L- and P-selectin have positively charged motifs in their ligand binding pocket, they were particularly suited for our studies. The nonbinding E-selectin molecule therefore served as a perfect control.

Fig. 2.

In vitro selectin binding studies of dPGS with different degrees of sulfation. (A) dPGS, here dPGS₄₆, act as competitive selectin binder and addresses L- and P-selectin, but not E-selectin. (B) The number of sulfate groups per molecule and the core size determine selectin inhibition efficacy (shown for L-selectin). (C) Dose-dependent inhibition of leukocyte binding to HUVECs in the flow chamber. Depending on the shear stress and the amount of dPGS applied, leukocyte adhesion decreased. L-selectin transfected NALM-6 cells were preincubated with dPGS₆₁ (0, 1, 10, and 100 nM) and perfused into the system. Adhesion to activated HUVECs was determined by cell counting. As a control, the number of untreated adherent cells was set to 100%. The flow chamber geometry allowed to measure adhesion at defined shear stresses between 0.6 and 2.7 dyn/cm². Mean values ± SEM, n = 4. Student’s t-test was used for statistical comparison. ***P < 0.001, **P < 0.01, *P < 0.05.

Next, the influence of the dPGS core size on L-selectin inhibition was analyzed (Fig. 2B). Three dPGS with nearly the same degree of sulfation (75%–85%, Table S1) but different core sizes were used. For comparison, unfractionated heparin (UHF) was applied as a positive control. UHF is a well-known anti-inflammatory polysaccharide (22) that has an average molecular weight of 15,000 Da and carries approximately 63 sulfate groups (23). Presenting a comparable number of functional groups as dPGS₆₁, but on a linear carbohydrate backbone, the inhibition of L-selectin binding by UFH resulted only in an IC₅₀ value of 12 μM. The backbone architecture and spatial ligand presentation are therefore important for efficient receptor–ligand interaction. Furthermore, the core size and the ligand spacing of the respective dPGS molecules contribute to the binding efficiency. Triglycerol sulfate (TGS₄) with the smallest core size (MW 240 Da, Table S1) showed an IC₅₀ value of only 2 mM. In contrast, the macromolecular compound dPGS₂₉ with a medium core size (MW 2,500 Da, Table S1), but same degree of sulfation, showed a strong inhibition with an IC₅₀ value of 90 nM. A clear dependency of core size and the absolute amount of sulfate groups per molecule could be observed for dPGS₆₁ (MW core 6,000 Da, Table S1), resulting in an IC₅₀ value of 8 nM. In addition, we could demonstrate the sulfate dependency of selectin binding. By increasing the degree of sulfation on the same dPG core (MW 6,000 Da) from 10% (dPGS₈) over 40% (dPGS₃₂) to 75% (dPGS₆₁), binding of L-selectin functionalized Au nanoparticles to immobilized ligands was reduced, which demonstrates the inhibitory capacity of the respective compounds (Fig. S1). In this context it is remarkable that the IC₅₀ of dPGS₃₂ was less than the value of dPGS₄₆, which carries more sulfates (Table S1). Obviously, it is not solely the number of sulfate groups per polymer but ligand spacing and core size that determines the IC₅₀ value. The larger core of dPGS₃₂ (6,000 Da) shields the selectin binding sites presented on the Au particle surface more efficiently than the smaller core of dPGS₄₆ (4,000 Da). In this particular case, steric shielding of L-selectin by the dPGS core renders potential binding sites nonaddressable. In addition, it is noteworthy that a certain number of sulfate groups per molecule (i.e., dPGS₂₉) was required for high affinity binding. A further increase of sulfate groups led to only a minor increase in binding affinity. High functionalization per molecule appears to be a prerequisite for tight binding as well as the architecture of dPGS₂₉ seems to address perfectly the selectin functionalized Au particles. This again is a strong hint for the importance of ligand spacing for high affinity binding.

We further examined the selectin binding effect of dPGS under more physiological conditions in a flow chamber that mimics the rheological conditions in the blood stream. In these experiments the effect of increasing concentrations of dPGS₆₁ on the attachment of L-selectin expressing NALM-6 cells to TNF-α activated human umbilical vein endothelial cells (HUVECs) was investigated at shear stress conditions between 0.6 and 2.7 dyn/cm². dPGS inhibited leukocyte attachment to endothelial cells dose-dependently from 1 to 100 nM (Fig. 2C). L-selectin ligand binding is flow dependent and requires a threshold shear to support cell adhesion both in vitro and in vivo (24). Flow-enhanced adhesion has been shown to be caused by the formation of catch bonds at the selectin–ligand interface (24, 25). The increase of dPGS inhibitory activity under flow is hence attributed to the availability of multiple sulfate groups of the polymer for rebinding events.

dPGS Reduces Leukocyte Extravasation to Inflamed Tissues in Vivo.

Given that dPGS targets L-selectin on leukocytes and P-selectin on activated endothelia and hence block extravasation, we tested the anti-inflammatory activity of the dPGS in vivo in a skin inflammation mouse model that is accompanied by a massive leukocyte efflux. Acute allergic contact dermatitis was induced with the allergen trimellitic anhydride (TMA), which caused typical symptoms of contact dermatitis (redness, ear swelling, edema, and cellular infiltration). Both the commercial glucocorticoid prednisolone (positive control) and dPGS₆₁ clearly reduced the ear swelling dose-dependently (Fig. 3A). In addition, the benefit of the dPGS₆₁ application could be assigned to the reduction of leukocytic extravasation to inflamed tissue by measuring the neutrophil elastase (NE) activity in mice ear homogenates (Fig. 3B). Administration of the compound dPGS₆₁ resulted in a clear reduction of NE activity already at a concentration of 3 mg/kg and was comparably effective as prednisolone. Blocking leukocyte extravasation and consequential NE activity was beneficial, because as was shown recently, NE contributes to inflammation by inactivating the anti-inflammatory growth factor progranulin (26). Interestingly, ear thickness decreased with dPGS concentration and NE activity indicated that leukocyte infiltration was already maximally inhibited at the lowest dose applied (3 mg/kg). This finding suggests that dPGS also reduces vascular permeability by addressing an additional target.

Fig. 3.

Contact dermatitis. (A) Anti-inflammatory effect of dPGS₆₁ in an acute contact dermatitis mouse model. The administration of dPGS prior to challenge with TMA results in a dose dependent reduction of ear swelling. (B) Reduction of neutrophil extravasation, as measured by specific elastase activity from ear homogenates. The elastase activity was determined by fluorescence of AMC, released by elastase activity from the substrate MeO-Succ-Ala-Ala-Pro-Val-AMC. Data are presented as mean ± SEM. Results are from 7–8 mice per group. Student’s t-test was used for statistical comparison. ***P < 0.001, **P < 0.01, *P < 0.05. TMA, trimellitic anhydride; PRED, prednisolone.

In Vivo Generation of Anaphylatoxins Is Reduced by dPGS.

As C5a is a relevant target in inflammatory processes (27), we investigated the interaction of dPGS binding to the C5a precursor in vitro. The effects of dPGS on the complement system were then examined in vivo in a mouse model of complement activation based on intraperitoneal injection of lipopolysaccharide (LPS). The potent anaphylatoxin C5a was determined 1.5 h and 5.5 h after LPS challenge (Fig. 4). Pretreatment of mice with dPGS₆₁, heparin (10 mg/kg), or dexamethasone (3 mg/kg) 30 min before challenge with a low dose of LPS (0.2 mg/kg) attenuated C5a generation below the control level. This effect could be attributed to a binding of the complement factor C5 to dPGS (Fig. S2), which might block specific proteolysis to generate C5a. As C5a has a chemotactic activity on leukocytes (28) and reduces vascular permeability on endothelial cells (29), C5a reduction could also contribute to alleviate edema formation in the allergic contact dermatitis reaction (Fig. 3).

Fig. 4.

Inhibition of anaphylatoxin generation. dPGS₆₁ inhibits the generation of C5a in LPS challenged mice. LPS (0.2 mg/kg) was injected intraperitoneally at time point zero. Thirty minutes before test start, the compounds DEX [dexamethasone (3 mg/kg)], UFH [unfractionated heparin (10 mg/kg)], and dPGS₆₁ (10 mg/kg) were administered subcutaneously in the nuchal fold (n = 4). 1.5 h and 5.5 h after LPS shock, C5a was quantified by ELISA from plasma samples. Both, UFH and dPGS₆₁ showed C5a levels below the untreated control. Data are presented as mean ± SEM; LPS, lipopolysaccharide.

Discussion

The selectins are cell adhesion molecules that act as molecular brakes to slow down leukocyte velocity in blood. Tethering and rolling of leukocytes mediated by binding to their respective carbohydrate ligands on endothelial cells prime leukocytes for firm adhesion and subsequent extravasation. In a recent study, we showed that galactose functionalized polyglycerol dendrimers were able to bind the three selectins, but with low affinity (21). In contrast sulfation of the carbohydrate tuned the selectivity of the polymer toward L- and P-selectin and IC₅₀ values decreased by two orders of magnitude. Sulfation of L- and P-selectin ligands is obviously an essential modification and contributes to the binding affinity. Recently, it has been shown that the proteoglycan heparan sulfate is a major L-selectin ligand on the inflamed endothelium (30). Sulfate deficiency of this ligand did not decelerate neutrophil velocity with the result that L-selectin-mediated cell trafficking was reduced.

Although the precise selectin binding site of dPGS is unknown, the competitive binding to the well-known ligands sialyl Lewis x (SiaLe^x) and tyrosine sulfate () used as the immobilized biligand structure in our SPR measurements is a significant hint. Both epitopes are naturally located on the physiological ligand P-selectin glycoprotein ligand-1 (PSGL-1) (31, 32). The crystal structure of P-selectin in complex with its natural high-affinity ligand PSGL-1 shows that the binding interface of the P-selectin surface contacts the tyrosine sulfate residues of PSGL-1 by certain arginine and histidine residues (18). A homologous binding mode is proposed for L-selectin. This region may also support the binding of inhibitory anionic compounds including heparan sulfate, heparin, fucoidan, sulfatides (4), and the polyanion dPGS. Because the basic patch is missing in E-selectin (18, 19), anionic compounds do not bind, which is consistent with our findings. dPGS addresses proteins through electrostatic interactions. High anionic surface charge is the basis for efficient interaction. A multivalent display of addressable ligands generally leads to an enhancement of binding strength (33). Inside one dPGS molecule multiple sulfate groups also enable rebinding events that probably contribute to the binding strength and the low nanomolar IC₅₀ values (Fig. 2 and Table S1). In this context, the ideal spatial presentation of sulfate ligands is of great importance.

A proper presentation of ligands also applies to heparin’s anti-inflammatory effect (34). Efficient binding to L- and P-selectin critically depends on glucosamine 6-O-sulfation, whereas sulfate groups at position C3 of the glucosamine residues or at C2 of the uronic acid residues are less important. In vivo investigations in a contact dermatitis and peritonitis model confirmed the need for site-specific sulfation. Furthermore, studies of selectin deficient mice indicate that heparin’s interaction with L-and P-selectin completely accounts for its anti-inflammatory effect in vivo. In comparison to heparin, highly functionalized dPGS binds to L-and P-selectin with increased affinity (Fig. 2 and Table S1). Therefore, it is tempting to speculate that dPGS is the more potent anti-inflammatory compound.

Another important point is that dPGS interacts with proteins of the complement system. The site-specific binding of dPGS to basic amino acid sequences of the complement factors C3 and C5 (Fig. S2) might block proteolysis and inhibit the release of the bioactive anaphylatoxins. Although the respective calculated K_D values were only in the micromolar range, we clearly demonstrated that administration of dPGS affects the C5a level in vivo (Fig. 4). In addition to the selectin binding function, this second mode of action of dPGS further contributes to its anti-inflammatory activity. By diminishing the level of anaphylatoxins, vascular permeability did not increase and C5a directed chemotaxis of leukocytes was limited. Although the inhibition of C5a generation has also been shown for heparin (35), other sulfated compounds like oversulfated chondroitin sulfate (OSCS) detected recently in batches of UFH have the adverse effect. By activating the kinin-kallikrein pathway, OSCS also induced the generation of the anaphylatoxins C3a and C5a (36). This clinical observation emphasizes the high demand for defined synthetic macromolecules for in vivo application.

In summary, the decrease of the inflammatory response is a result of different interactions of polyglycerol sulfate (Fig. 5). Binding of dPGS to L-selectin on leukocytes and P-selectin on inflamed vascular endothelium reduces leukocyte extravasation by shielding the adhesion molecule. Furthermore, the inhibition of C5a generation inhibits leukocyte chemotaxis.

Fig. 5.

Pleiotropic anti-inflammatory effect of dPGS diminishes the inflammatory response and reduces leukocyte extravasation. Molecular targets of dPGS are the adhesion molecules L- and P-selectin. dPGS prevents leukocyte extravasation by shielding the selectins. Binding to complement factors C3 and C5 inhibits the formation of the proinflammatory anaphylatoxins. Reduction of the C5a level decreases vascular permeability and further leukocyte extravasation. By addressing these inflammatory targets simultaneously, dPGS promote the resolution of inflammation.

Due to their high local sulfate concentration, dPGS macromolecules establish high multivalent binding affinities by targeting positively charged protein motifs such as those of L- and P-selectin and of the complement factors C3 and C5.

In conclusion, we found that highly sulfated dendritic polyglycerols as fully synthetic macromolecules exert high-affinity binding to mediators of inflammation and strong therapeutic effects in vivo. Investigations on the structure-activity relationship established a rationale for polyanionic drugs that target positively charged motifs that are present in several mediators of inflammation (37). Thus, dPGS may represent an alternative therapeutic to ameliorate inflammatory processes.

Materials and Methods

Synthesis of dPGS.

dPGS of different molecular weights and degree of sulfation were synthesized by anionic polymerization of glycidol (14, 15) and subsequent sulfation using SO₃/Pyridinum complex (Fluka) according to Turk et al. in 2004 (17). Elemental analysis was used to determine the degrees of sulfation. Conjugation of dPGS to biotin will be described elsewhere. Briefly, the polyglycerol intermediate was reacted with an aliphatic linker chain followed by sulfation. Biotin was conjugated to this linker followed by HPLC purification, which yielded dPGS-biotin with a mean biotin-to-polymer ratio of 1.

Surface Plasmon Resonance Measurements.

Surface plasmon resonance experiments were carried out on a BIAcore X instrument (GE Healthcare) at 25 °C. The competitive selectin–ligand binding assay has been described in detail previously (20). In brief, selectin-IgG chimeras (R&D Systems) were immobilized on protein A coated Au nanoparticles (Biotrend) and passed over a standard ligand (20 mol% SiaLe^x and 5 mol% conjugated to polyacrylamide, Lectinity) immobilized on a sensor chip. The resulting binding signal was set to 100% and served as a control (Fig. S1A). To evaluate selectin binding of potential inhibitors, a defined preincubation step with the selectin nanoparticles was performed before its passage over the sensor chip. Reduction of the binding signal with respect to the inhibitor concentration was recorded and calculated as X% binding of the control. The inhibitor concentration that caused 50% reduction of binding was referred to as IC₅₀ value. Each concentration was applied at least in triplicate. Heparin was purchased from Sigma.

Direct binding assays were applied to detect the interaction of dPGS with the human complement proteins C3 and C5 that were purchased from Sigma (Fig. S2). Therefore, dPGS₆₁-biotin was immobilized on a streptavidin precoated sensor chip (sensor chip SA, GE Healthcare) with a ligand density of approximately 700 resonance units (RU). Direct binding was recorded in running buffer (15 mM NaHPO₄, 150 mM NaCl, pH 7.2) at a flow rate of 30 μL/ min with an association phase of 200 sec and a dissociation phase of 400 sec. Regeneration of the surface was done by injection of 4 M MgCl₂ at 100 μL/ min for 60 sec. Proteins were used at five concentrations (10, 20, 30, 40, and 50 nM for C3 and 1, 1.56, 3.12, 6.25, and 10 nM for C5). Rate constants were calculated by using BIAevaluation software 4.1 assuming a 1∶1 mode of binding.

Flow Chamber Assay.

The assay was performed as previously described (20, 38). In brief, HUVECs on cover slips were activated with TNF-α (human, 100 U/mL, 5 h exposure, purchased from Alexis), and exposed to nearly physiological conditions in a parallel-plate flow chamber. At a constant flow rate of 116 μL/ min, the flow chamber geometry permitted the examination of wall shear stress between 0.6 and 2.9 dyn/cm². Activation with TNF-α induced the expression of L-selectin-specific ligands on HUVECs. L-selectin transfected NALM-6 cells were added to the perfusion medium (Medium 199 with Hanks’s salts (PAA Laboratories) supplemented with 5% (v/v) fetal calf serum (Biochrom) at the entrance to the flow channel. The volume of the added cell suspension was 100 μL (10⁶ cells per mL). Cell adhesion was allowed to occur during a 5 min run. The number of firmly attached cells counted after 5 min was used for curve creation. Each point represents the mean value (± SEM) of four measurements. Binding of L-selectin-expressing NALM-6 cells to TNF-α-activated HUVECs was set at 100% and used as control. Cells were preincubated with dPGS₆₁ (1, 10, and 100 nM concentration) for 10 min and then added to the perfusion medium. Endothelial cells were preincubated with anti-hCD62E (2.4 μg/mL, R&D Systems) and anti-hCD62P (1.2 μg/mL, R&D Systems) 1 h prior to a run. The ability of dPGS to block L-selectin-dependent adhesion in the flow chamber emerged in the reduction of firmly attached cells. The adhesion rates were compared to the positive control set at 100%.

Animal Studies.

All animals were obtained from Charles River Laboratories. Specific pathogen-free female Naval Medical Research Institute outbred mice were used during the experiments of the contact dermatitis model and the LPS shock model. Animals had access to drinking water ad libitum and received standard diet (Altromin). All animal experiments were performed in accordance with institutional, state, and federal guidelines.

Complement Activation Assay.

Inhibition of C5a generation in mice after low dose LPS shock by anti-inflammatory compounds was analyzed. LPS from Escherichia coli serotype O111∶B4 (Sigma) was injected intraperitoneally (0.2 mg/kg) at time point zero. Thirty min before test compounds dexamethasone (3 mg/kg, Sigma), UFH (10 mg/kg, Sigma) or dPGS₆₁ (10 mg/kg) were administered subcutaneously in the nuchal fold; n = 4. 1.5 h and 5.5 h after LPS injection, blood samples were collected by S-monovettes (Sarstedt) from anaesthetized mice. After centrifugation C5a was quantified by ELISA (BD Biosciences) from plasma samples.

Contact Dermatitis Model.

In the sensitization phase mice received on day 0 a single application of 50 μL TMA solution (3% w/v TMA in acetone/isopropylmyristate (80∶20) onto a shaven area of 2 cm² on the right flank. Before challenge the individual ear thickness was determined on day 5. Test compounds were injected subcutaneously into the mouse nuchal fold 1 h prior to challenge. dPGS₆₁ and heparin were dissolved in PBS as vehicle, prednisolone in a mixture of 5% DMSO, 3% ethanol, and 92% peanut oil. The contact hypersensitivity reaction was then induced by challenging the animals with a single application of 10 μL 3% w/v TMA in acetone/isopropylmyristate (80∶20) onto the dorsal sides of both ears (area per ear approximately 2 cm²). Quantification of the challenge response was performed 24 h later. Ear thickness was determined and animals were euthanized with CO₂. Ears (area ∼ 1 cm²) were homogenized in 2 mL buffer containing 0.5% hexadecyltrimethylammonium bromide, 10 mM 3-(N-morpholino)propanesulfonic acid pH 7.0 under constant cooling for 20 s in a Kinematica Polytron(R) PT 3000 homogenizer set at maximum speed (30,000 rpm). Samples were centrifuged at 24,000  × g for 20 min at 10 °C. The supernatants were then used to determine elastase activity as an indicator for infiltrating neutrophils. Elastase activity was determined by fluorescence of 7-amino-4-methyl-coumarin (AMC) that is released by elastase activity from the substrate MeO-Succ-Ala-Ala-Pro-Val-AMC (Bachem). A standard curve was recorded with AMC. Measurements were done in a SpectraMax Gemini (Molecular Device) at 380 nm.