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The Journal of Immunology, 2005, 174: 6399-6405.
Copyright © 2005 by The American Association of Immunologists

Neutrophil-Derived Heparin-Binding Protein (HBP/CAP37) Deposited on Endothelium Enhances Monocyte Arrest under Flow Conditions 1

Oliver Soehnlein*, Xun Xie*, Holger Ulbrich*, Ellinor Kenne*, Pierre Rotzius*, Hans Flodgaard{dagger}, Einar E. Eriksson* and Lennart Lindbom2,*

* Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden; and{dagger} Leukotech, Copenhagen, Denmark


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca2+ that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mobilization of circulating leukocytes is an essential part of the inflammatory response following tissue injury or invasion of pathogens. Initially, leukocytes of the innate immune system, predominantly neutrophilic granulocytes, extravasate and contribute to host defense through their cytotoxic and phagocytic activities (1). Subsequently, peripheral blood monocytes are recruited and accumulate in the inflammatory lesion, contributing to the resolution and repair of damaged tissue. Apart from their function as principal phagocytic cells, monocytes constitute an important source of proinflammatory cytokines and serve as APCs in adaptive immunity. Thus, the temporal pattern of leukocyte recruitment to inflammatory loci often reveals a first wave of invasion of cells of the innate immune system preceding a second wave of infiltration of leukocytes that are important in the initiation of adaptive immune responses. Interestingly, data in the literature suggest an important role for initial neutrophil invasion in the control of the succeeding infiltration of monocytes, indicating that these events may not only be temporally linked but also causally related (2, 3, 4, 5). For instance, animals rendered neutropenic show decreased monocyte accumulation at inflammatory sites, and restoring neutrophils to the circulation in these animals re-establishes the normal sequence of events (3). Moreover, case reports from human systems also suggest that secretion products from neutrophils are important regulators of monocyte infiltration into inflamed tissues (6). However, the signals that govern this progression of events have remained largely undefined. Neutrophils are capable of releasing proinflammatory cytokines and chemokines, which may directly or indirectly stimulate monocyte recruitment (7, 8, 9). Moreover, neutrophil activation results in exocytosis of preformed granule proteins (10), several of which have been shown to influence activity of mononuclear cells. In particular, constituents of neutrophil azurophilic granules including the members of the serprocidin family, such as cathepsin G and heparin-binding protein (HBP) 3 also known as cationic antimicrobial protein of molecular mass 37 kDa (CAP37/azurocidin), have attracted attention in this respect, and accordingly, have been suggested to serve as links between initial and delayed phases of cellular immune responses (11). In contrast to cathepsin G, HBP is not only localized in azurophilic granules but also in more readily mobilized compartments close to the plasma membrane (12) and can therefore be released rapidly upon neutrophil activation and adhesion (13). Once secreted, HBP carries out powerful monocyte-activating properties. It is an effective chemoattractant for monocytes in vitro (14) and in vivo (15), it stimulates monocyte survival (16), and potentiates LPS-induced production of proinflammatory cytokines (17, 18).

In this report, we investigate the role for neutrophil-derived HBP in monocyte arrest on endothelial cells (EC). Because HBP is released from activated neutrophils and because it has the capacity to activate mononuclear cells, we hypothesized that neutrophil-derived HBP may assemble on the endothelial surface and influence the recruitment of monocytes to inflamed tissues. Our results show that HBP deposited on endothelium triggers an active response in monocytes and promotes firm arrest of these cells under flow conditions. This mechanism may contribute to the capability of activated neutrophils to stimulate monocyte infiltration at inflammatory loci.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

Bovine aorta endothelial cells (BAEC) were isolated as previously described (19). Harvested cells were cultured in medium containing RPMI 1640 and M199 (1:1), penicillin (100 U/ml) and streptomycin (100 µg/ml) in 20% FBS. Primary cultures of HUVEC were obtained from fresh umbilical cords. The umbilical vein was incubated with 0.1% dispase for 30 min. After washing with PBS, HUVEC were cultured in M199 containing 20% human serum. Human mononuclear leukocytes were isolated from buffy coat by density gradient centrifugation. Buffy coats were diluted 3-fold with PBS and layered on Ficoll-Paque PLUS (Amersham Pharmacia Biotech), centrifuged at 400 x g for 40 min at room temperature, and mononuclear cells were collected. The cells were then washed twice with PBS and seeded in culture flasks. After 1 h incubation, lymphocytes in the supernatant were centrifuged and resuspended in RPMI 1640 supplemented with 1% FBS and subsequently used in flow chamber experiments. Cells adherent to the flask bottom after four washes were considered as representing the monocyte fraction. These cells were detached with 1 mM EDTA, washed four times, and resuspended in RPMI 1640 containing 1% FBS and then used. Monocyte purity assessed by FACS analysis always exceeded 80%. To determine activation of monocytes by the isolation procedure, the surface expression of L-selectin (anti-human CD62L FITC; BD Pharmingen) and CD11b (anti-human CD11b PE; BD Pharmingen) was analyzed by FACS before and after the adherence. Expression of both molecules was only marginally affected indicating a low activation of the monocytes. In contrast, treatment with MCP-1 (10 ng/ml, 60 min) resulted in a distinct induction of CD11b on the monocyte cell surface and profound shedding of L-selectin. Human polymorphonuclear leukocytes (PMN) were isolated from whole blood by single-step density centrifugation over Polymorphprep (Nycomed), washed twice, and resuspended in RPMI 1640 containing 1% FBS. Mono Mac 6 (MM6) cells, a monocytic cell line, were maintained as previously described (20). The cells were washed before use and resuspended in RPMI 1640 containing 1% FBS. Cell culture media, PBS, dispase, collagenase, and antibiotics were obtained from Invitrogen Life Technologies, and supplements for MM6 medium were purchased from Sigma-Aldrich.

Preparation of neutrophil secretion

PMN activation and secretion was induced through Ab cross-linking of integrin {beta}2-chain CD18 as previously described (21). In brief, isolated PMN were incubated with mAb IB4 against CD18 (3 µg of IB4 per 106 neutrophils), washed and subjected to CD18 receptor cross-linking through addition of goat anti-mouse F(ab')2 (diluted 1/20; Jackson ImmunoResearch Laboratories). PMN were sedimented by centrifugation and the cell-free supernatant containing neutrophil secretion was collected and stored at –70°C until use.

HBP binding assays

Binding of HBP to EC was assessed in a fluorescence plate reader or by FACS analysis. In fluorescence plate reader experiments BAEC and HUVEC were grown to confluence in 96-well plates. Recombinant HBP (17) at different concentrations, CD18 cross-linking-induced PMN secretion, or culture media alone serving as control, was added for 15 min. In some wells BAEC were treated with heparinase II (Sigma-Aldrich) at 37°C for 60 min, chondroitinaseABC (avidin-biotin peroxidase complex; Sigma-Aldrich) at 37°C for 60 min, or combinations of both and intensively washed before addition of HBP. Additionally, some EC monolayers were pretreated with TNF-{alpha} (50 ng/ml, 24 h; R&D Systems) or LPS (from Salmonella Minnesota, 1 µg/ml for 4 h; Sigma-Aldrich). The EC monolayers were then washed twice with PBS, fixed with 4% paraformaldehyde (room temperature, 30 min) or methanol (–20°C, 5 min), and incubated with 1% BSA in PBS for 20 min at room temperature. EC were subsequently incubated with rabbit anti-HBP polyclonal Ab (5 µg/ml) (22) or purified rabbit IgG (5 µg/ml; Sigma-Aldrich) in 1% BSA (room temperature, 30 min). After washing, the cells were incubated with FITC-labeled goat anti-rabbit F(ab')2 (room temperature, 30 min, 1/100; Jackson ImmunoResearch Laboratories), washed again and analyzed in a fluorescence plate reader (Fluoroscan II; Labsystems). To adjust for autofluorescence in the experiments, mean fluorescence intensity (MFI) obtained in wells treated with the isotype control Ab was subtracted from MFI in wells that were treated with rabbit anti-HBP. Statistical comparisons of the subtracted MFI values were performed between control wells (wells treated with culture medium only) and wells that were exposed to PMN secretion or recombinant HBP (compared wells were thus all subjected to an adequate staining procedure). For FACS analysis BAEC or HUVEC were detached from the culture plate and incubated with recombinant HBP for 15 min. Cells were centrifuged, washed twice with PBS, and then fixed with paraformaldehyde. Staining for HBP on EC surface was performed as described.

Binding of HBP released from human PMN adhering to EC under flow was determined by perfusing PMN (106/ml) over TNF-{alpha}-activated (50 ng/ml, 24 h) EC for 1 h in a flow chamber model (see Flow chamber below). To block PMN adhesion, PMN were treated with IB4 (3 µg/106 PMN) for 30 min, washed twice, and then used in flow chamber assays. Staining of HBP on EC was performed as described earlier. Fluorescence intensity was quantified by fluorescence microscopy (Nikon TE300), digital camera recording (Nikon DN100), and ImageJ 1.32j software (<rsb.info.nih.gov/ij>).

Flow chamber

Laminar flow chamber assays (Glycotech) were performed on confluent BAEC or HUVEC monolayers or on plates coated with recombinant cell adhesion molecules (CAM). Confluent EC were activated with LPS at a concentration of 1 µg/ml for 4 h. Preperfusion of EC with PMN was achieved by perfusing freshly isolated PMN over LPS-activated BAEC for 1 h, followed by perfusion with medium for 5 min to wash away nonadherent cells. Thereafter MM6 cells were infused for 4 min. The number of adherent MM6 cells was quantified after nuclear staining to allow distinction between PMN and MM6. Preincubation of EC with HBP was achieved by adding 10 µg/ml recombinant HBP to the wells 15 min before the experiment. PMN secretion was similarly added to EC 15 min before the experiment when indicated. Plates coated with CAM (P-selectin, E-selectin, VCAM-1, ICAM-1) and recombinant HBP were produced by adding 50 µl of recombinant human CAM (1 µg/ml; R&D Systems) to the center of a well in a six-well plate. Recombinant HBP (1 µg/ml) was added together with CAM when indicated. Plates were then incubated overnight at 4°C. The coated plates and the plates with EC were washed with PBS before the experiment to remove excessive reagents. Flow chamber experiments were performed with MM6 cells and with isolated human monocytes, lymphocytes, and neutrophils (106 cells/ml) resuspended in medium containing 1% FBS and infused with a syringe pump (Univentor 864; Zejtun) at 1 dyne/cm2 for 4 min. The experiments were televised and recorded on video for offline analysis. Leukocyte adhesion was quantified by counting the number of arrested cells in four separate randomly chosen fields in each well.

Intracellular Ca2+ mobilization

MM6 cells were incubated (37°C, 30 min) with the Ca2+ sensitive fluorophore fluo-4-acetoxymethyl ester (fluo-4-AM; Molecular Probes) according to manufacturer’s instructions and washed twice with PBS before use. A total of 500 µl of MM6 cells (106 cells/ml) were subjected to stimulation with recombinant HBP 500 ng/ml. Changes in intracellular free Ca2+ were analyzed by flow cytometry (FACSort; BD Biosciences) immediately and every 30 s after stimulation with HBP for up to 3 min. Additional experiments were performed by fluorescence plate readings. A total of 100 µl of MM6 cells (106 cells/ml) loaded with fluo-4-AM were added to wells of a 96-well plate, which had been coated with CAM (1 µg/ml) and recombinant HBP as described earlier (see Flow chamber). Fluorescence intensity was measured after cells were added to the well. Fluorescence intensity of MM6 cells added to BSA-coated wells was considered as background fluorescence and subtracted from fluorescence intensity values of CAM-coated wells. Similar experiments were performed on 24-well plates coated as described. Fluorescence of fluo-4-AM-labeled MM6 was measured by fluorescence microscopy for individual cells 30, 60, and 90 s after cell injection.

In separate experiments, increase in cytosolic free Ca2+ in MM6 cells was prevented by treatment with 5 µM of the cell-permeable Ca2+ chelator BAPTA-AM (37°C, 30 min; Molecular Probes). Cells were washed twice and used in flow chamber experiments as described (see Flow chamber).

Statistics

Data are presented as mean ± SD. Statistical significances were calculated using the Mann-Whitney U test for independent samples. Statistical significant difference was set at a value p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Neutrophil-derived HBP binds to EC

Surface deposition of recombinant HBP and HBP derived from activated PMN was demonstrated by FACS analysis and fluorescence plate reader experiments. Cultured EC monolayers were exposed to either recombinant HBP or to secretion derived from PMN activated by Ab cross-linking of CD18. EC were then stained and analyzed for surface-bound and intracellular HBP. As illustrated for BAEC and HUVEC in Fig. 1A, specific immunofluorescence, reflecting binding of HBP to the EC surface, was demonstrated after incubation with recombinant HBP or PMN secretion but not on EC exposed to culture medium alone, indicating a lack of expression of HBP on EC under resting conditions. Fluorescence intensity in wells treated with neutrophil secretion was in the range of the intensities obtained in wells treated with recombinant HBP at concentrations 10 ng/ml and 10 µg/ml. HBP in this concentration range has previously been described as capable of evoking responses in monocytes (14, 17, 18). No difference was found in the binding of recombinant HBP to resting EC compared with monolayers treated with TNF-{alpha} (50 ng/ml, 24 h), indicating that binding of HBP to endothelium is independent of endothelial activation (data not shown). Treatment of EC with TNF-{alpha} (50 ng/ml, 24 h) or LPS (1 µg/ml, 4 h) alone did not result in positive staining on the EC surface (Fig. 1A). However, staining after fixation with methanol, which permeabilizes the cells, showed enhanced immunoreactivity for HBP in EC stimulated with TNF-{alpha} or LPS (data not shown), confirming previous observations by Lee et al. (23). Treatment of BAEC with heparinase II before addition of recombinant HBP (10 µg/ml) or PMN secretion significantly reduced HBP binding, suggesting that heparan sulfate proteoglycans on EC serve as primary binding sites for HBP (Fig. 1B). Cotreatment of BAEC with heparinase II and chondroitinaseABC before injection of recombinant HBP further reduced binding of HBP.



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  		FIGURE 1. Binding of HBP to EC monolayers. A, BAEC and HUVEC were treated with culture medium serving as negative control (ctrl), recombinant HBP (10 ng/ml or 10 µg/ml, 15 min, room temperature), PMN secretion (15 min, room temperature), TNF-{alpha} (50 ng/ml, 24 h), or LPS (1 µg/ml, 4 h). Cells were fixed with 4% paraformaldehyde, stained with an anti-HBP Ab and a FITC-conjugated secondary Ab, and analyzed in a fluorescence plate reader. Bars indicate a difference between sample MFI and MFI for isotype control. Values are given as mean ± SD (n = 6–9 EC monolayers). B, BAEC were treated as indicated. Heparinase II (Hep, 2 U/ml, 1 h, 37°C) and chondroitinaseABC (CABC, 150 mU/ml, 1 h, 37°C) were added before addition of 10 µg of HBP/ml where indicated. Cells were stained and analyzed as described in A (n = 6). *, Significant difference (p < 0.05). C, BAEC were detached from culture flasks, resuspended in culture medium and treated with recombinant HBP (10 µg/ml, 15 min, room temperature) or culture medium. Cells were fixed with 4% paraformaldehyde, stained as described in A, and analyzed by FACS. C, Histogram overlay represents MFI of BAEC treated with either HBP (thick line) or medium alone (thin line) and stained with an anti-HBP Ab. Dashed line represents isotype control. Overlay is representative of five independent experiments.

 
Moreover, BAEC in suspension were incubated with recombinant HBP, stained as described earlier, and analyzed by FACS (Fig. 1C). We found no significant difference between isotype control and negative control indicating a lack of HBP on resting EC. Addition of recombinant HBP (10 µg/ml) caused a significant increase in MFI (p < 0.01). Similar data were obtained for HUVEC treated and stained in the identical manner (data not shown).

In a more physiologic assay we perfused freshly isolated human PMN over TNF-{alpha}-activated BAEC for 1 h (Fig. 2, A and B). After perfusion we found a clear accumulation of HBP on the EC surface, whereas resting cells and activated cells in the absence of PMN stained negative. Treatment of PMN with anti-CD18 mAb IB4 (3 µg/ml, 30 min) before injection, which prevented PMN arrest on EC while rolling remained intact, markedly reduced HBP deposition. This observation emphasizes the importance of firm PMN adhesion to EC and signaling via {beta}2 integrin in the release of HBP from PMN storage compartments (13).



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  		FIGURE 2. EC surface binding of HBP released from adherent PMN. BAEC were grown to confluence in six-well plates and activated with TNF-{alpha} (50 ng/ml, 24 h). Freshly isolated PMN were perfused over the EC monolayer for 1 h. Cells were fixed with 4% paraformaldehyde and stained for HBP. To prevent PMN adhesion neutrophils were pretreated with IB4 (3 µg/ml, 30 min, 37°C) and washed twice before perfusion. A, Fluorescent light images show staining for HBP on resting (ctrl) and TNF-{alpha}-activated BAEC as well as BAEC perfused with PMN with or without IB4 treatment. Bars indicate 20 µm. Images are representative of four independent experiments. B, Fluorescence intensity of images captured in the perfusion assay was analyzed by ImageJ 1.32j software. Ordinate minimum was adjusted to MFI of control images. Values are presented as mean ± SD (n = 4 fields). *, Significant difference (p < 0.05).

 
HBP stimulates monocyte arrest under flow

Having confirmed that HBP released from activated neutrophils binds to endothelial surface, we investigated the role for HBP in leukocyte arrest on BAEC and HUVEC under flow conditions. Leukocytes were perfused over cultured EC monolayers in a parallel plate flow chamber for 4 min at a shear stress of 1 dyne/cm2 and the number of leukocytes that arrested were subsequently assessed based on counting in four separate fields in each experiment. Activation of EC with LPS (1 µg/ml, 4 h) caused a significant increase in the accumulation of all leukocyte subclasses compared with nonstimulated EC (p < 0.01). The addition of recombinant HBP (10 µg/ml) to LPS-stimulated EC for 15 min before the experiment significantly increased the number of monocytes (p < 0.01) and MM6 cells (p < 0.01) adhering to BAEC (Fig. 3A) or HUVEC (Fig. 3B) as compared with monolayers treated with LPS alone. However, recombinant HBP did not influence the number of adherent lymphocytes or neutrophils. Similar results were obtained when TNF-{alpha} was used to activate EC (data not shown). In additional experiments, PMN were perfused over activated BAEC for 1 h and MM6 cells were then infused. PMN preperfusion in this way clearly enhanced MM6 arrest to activated EC. Likewise, deposition of PMN secretion on activated BAEC stimulated arrest of MM6 cells in a similar manner (Fig. 3C).



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  		FIGURE 3. Adhesion of leukocytes to BAEC and HUVEC under flow conditions. A and B, Leukocytes were perfused over BAEC (A) or HUVEC (B) monolayers for 4 min and the number of adherent leukocytes was subsequently counted. Basal and stimulated adhesion varied between leukocyte subtypes. To allow comparisons between groups, the number of cells of respective leukocyte subclass that arrested on LPS-stimulated (1 µg/ml, 4 h) EC was used as reference and set to 100 (dashed line). Bars represent leukocyte adhesion to untreated EC (ctrl) or to EC treated with LPS (1 µg/ml, 4 h) and HBP (10 µg/ml, 15 min) (LPS+HBP) expressed as a percentage of adhesion to LPS-treated EC for respective leukocyte subclass. C, Freshly isolated PMN were perfused over a LPS-activated (1 µg/ml, 4 h) BAEC monolayer for 1 h. MM6 cells were then infused for 4 min and the number of adherent MM6 cells were subsequently counted. In separate experiments, PMN secretion was added to activated BAEC for 15 min followed by infusion of MM6 cells. Bars represent adhesion of MM6 cells to untreated EC (ctrl) and to LPS-activated EC combined with PMN preperfusion ({blacksquare}) or incubation with PMN secretion ({square}) expressed as a percentage of adhesion to EC treated with LPS alone (dashed line).

 
Because increased accumulation of monocytes on BAEC and HUVEC could be due to responses in monocytes, EC, or both, we investigated the possibility of an effect of HBP specifically on monocytes by excluding EC from the assay. In this situation, monocytes were perfused over surfaces coated with recombinant CAM. Recombinant CAM (ICAM-1, VCAM-1, E-selectin, and P-selectin) increased arrest of monocytes as compared with BSA. The addition of recombinant HBP to the CAM-coated plates triggered a 2-fold increase in shear-resistant monocyte arrest on plates coated with E-selectin (p < 0.01), P-selectin (p < 0.01), and VCAM-1 (p < 0.001), but not on plates coated with ICAM-1 or BSA (Fig. 4A). Similar data were found for MM6 cells (Fig. 4B), whereas no effect of HBP on the arrest of lymphocytes or neutrophils was detected (data not shown). Taken together, these data indicate that HBP, released from neutrophils upon adhesion and activation, specifically influences monocyte arrest on EC.



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  		FIGURE 4. Adhesion of monocytes and MM6 cells to recombinant adhesion molecules. Leukocytes were perfused over plates coated with recombinant CAM ({blacksquare}) or CAM plus recombinant HBP ({square}) for 4 min and the number of adherent cells was subsequently counted. Addition of recombinant HBP caused significantly increased binding of monocytes (A) (n = 3 experiments) and MM6 cells (B) (n = 4 experiments) to VCAM-1, P-selectin, and E-selectin but not to ICAM-1 and BSA. Values are presented as mean ± SD. *, Significant difference (p < 0.01).

 
HBP activates monocytes through mobilization of intracellular free Ca2+

To investigate whether HBP induces an active response in monocytes we analyzed the effect of HBP on calcium mobilization in MM6 cells by flow cytometry. Cells were labeled with the calcium sensitive fluorophore fluo-4-AM. The addition of recombinant HBP (500 ng/ml, which was found to be the dose optimum) to cells in suspension caused an immediate and significant increase of MFI compared with control. Fluorescence intensity was maximal after 30 s (p < 0.001) and then gradually declined, indicating a rapid and transient response (Fig. 5A). As data from the flow chamber assay indicated an activation of monocytes by HBP during the early contact between CAM and monocytes, we analyzed calcium responses in MM6 cells interacting with combinations of immobilized HBP and CAM in a fluorescence plate reader (Fig. 5B) and by fluorescence microscopy (Fig. 5C). In experiments in the fluorescence plate reader, both P-selectin and E-selectin could alone evoke an increase in cytosolic free Ca2+ in the monocytic cells. However, this response was significantly augmented when the cells were exposed to combinations of the respective selectin and recombinant HBP (P-selectin, p < 0.01; E-selectin, p < 0.05; Fig. 5B). Furthermore, although VCAM-1 did not alone evoke a Ca2+ response in MM6 cells, the combination of VCAM-1 and recombinant HBP produced a clear-cut increase in intracellular free calcium as compared with VCAM-1 alone (p < 0.05). Comparable data were obtained in experiments performed in the fluorescence microscope under similar conditions as described earlier (Fig. 5C). Although hardly any fluorescence was detectable in MM6 cells injected into BSA coated wells, injection into E-selectin-coated wells resulted in enhancement of MM6 fluorescence (p < 0.01). Coating with E-selectin plus recombinant HBP further increased calcium mobilization compared with E-selectin coating alone (p < 0.01). Fluorescence of cells increased between 30 and 60 s after injection indicating a rise in cytosolic free Ca2+ after the primary contact was established (Fig. 5C). Similar data were obtained for combinations of P-selectin and HBP or VCAM-1 and HBP (data not shown). Collectively, these data demonstrate that HBP triggers a rapid active response in monocytes.



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  		FIGURE 5. Intracellular Ca2+ mobilization in MM6 cells after stimulation with recombinant HBP. A, Dynamic change in fluorescence intensity of MM6 cells loaded with fluo-4-AM after stimulation with recombinant HBP 500 ng/ml ({circ}) or sham treatment (•). Measurements were made by flow cytometry in MM6 cell suspensions before and immediately after stimulation, and then at 30-s intervals. Values are expressed as a percentage of MFI before treatment. *, Significant difference (p < 0.05) from sham treatment value (n = 5). Values are presented as mean ± SD. B, Fluorescence intensity of fluo-4-loaded MM6 cells added to wells coated with individual CAM ({blacksquare}) or combinations of CAM and recombinant HBP ({square}). Measurements were made in a fluorescence plate reader. Fluorescence intensity of MM6 cells in BSA-coated wells was considered as background fluorescence and subtracted from MFI of CAM-coated wells. *, Significant difference (p < 0.05) from MFI in the absence of recombinant HBP (n = 6). Values represent mean ± SD. C, Dynamic changes in fluorescence intensity of individual MM6 cells loaded with fluo-4-AM after contact with the coated surfaces had been established. Fluorescent images were captured at 30, 60, and 90s after injection. Images are representative of three independent experiments with three wells in each experiment. Bars indicate 20 µm.

 
HBP-mediated rise in cytosolic free Ca2+ is required for HBP-induced increase in monocyte arrest

Knowing that HBP triggers a transient increase in cytosolic free calcium in monocytes, we investigated the role for this calcium response in monocyte arrest under flow conditions. In these experiments, the rise in cytosolic calcium triggered by HBP was prevented by pretreatment of MM6 cells with the cell-permeable calcium-chelating agent BAPTA-AM. As in previous experiments, HBP augmented arrest of MM6 cells on plates coated with VCAM-1, P-selectin, and E-selectin as well as on LPS-activated BAEC. However, the HBP-evoked increase in monocyte arrest on CAM-coated surfaces or BAEC monolayers was abolished in experiments in which MM6 cells were pretreated with BAPTA-AM: VCAM, p < 0.001; P-selectin, p < 0.01; E-selectin, p < 0.001; and BAEC monolayers, p < 0.001 (Fig. 6).



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  		FIGURE 6. Effect of intracellular calcium chelator BAPTA-AM on arrest of MM6 cells on recombinant adhesion molecules or on LPS-stimulated BAEC. MM6 cells were perfused over CAM-coated plates (A) or BAEC monolayers (B) for 4 min in the absence or presence of recombinant HBP and arrested cells were subsequently counted. Pretreatment of MM6 cells with BAPTA-AM consistently prevented the increase in adhesion evoked by recombinant HBP on all substrates. Values are presented as mean ± SD. *, Significant difference (p < 0.01). ns, Not significant.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In inflammation, infiltration of neutrophils often precedes a second wave of monocyte invasion, and several reports indicate that neutrophils may directly stimulate the mobilization of monocytes (2, 3, 5). Although not well characterized, mechanisms of neutrophil-evoked activation of mononuclear cells have been suggested to involve neutrophil degranulation and signaling via granule-derived proteins (6, 11, 15). In this study, we demonstrate that the neutrophil granule protein HBP/CAP37 is deposited on the EC surface and evokes an active response in monocytes that significantly enhances monocyte firm arrest on endothelium.

HBP/CAP37 has important functions in inflammatory actions of neutrophils. Besides its localization in azurophilic granules (10), it is also found in secretory vesicles (12) from which it can be rapidly released (13). Consequently, HBP has the capacity to exert not only functions typical of proteins of the azurophilic granules (24), which primarily come about when the neutrophil has extravasated, but also to rapidly affect EC at the luminal side of the vessel wall. We have previously shown that HBP is released from PMN upon {beta}2 integrin engagement (13). Our current data suggest that HBP released from activated neutrophils interacting with EC accumulates on the surface of the EC and that the protein in this position is capable of activating and promoting firm arrest of rolling monocytes. We show that liberation of HBP and its consequent binding to the EC surface depends on integrin-mediated tight adhesion of PMN to EC, whereas the transient rolling interaction is insufficient in causing substantial HBP release. The accumulation of HBP on the endothelium is reduced by treatment with heparinase, indicating that the cationic nature of the protein favors binding to negatively charged proteoglycans in the endothelial glycocalyx (25). In response to treatment with TNF-{alpha} or LPS we found intracellular immunoreactivity for HBP, which is in agreement with findings by Lee et al. (23). However, HBP was detected on the EC surface only after being extracellularly deposited, thus indicating that HBP-induced monocyte arrest requires the presence of HBP seeded by PMN. Of interest, we have preliminary data to indicate an in vivo correlate to the capacity of HBP to bind to inflamed endothelium. Clear positive staining for HBP is revealed on EC of blood vessels in inflamed appendicitis specimens obtained after clinically indicated appendectomy (our unpublished observations).

HBP is known as a potent chemoattractant for monocytes (15, 26, 27), but may also stimulate chemotaxis of T cells and neutrophils (28, 29). However, the effect on leukocyte arrest was specific for monocytes, suggesting that this molecule represents a pathway by which monocytes can be recruited selectively. Because the effect on monocyte arrest was evident also in experiments in which we used isolated CAM coated on the surface of the flow chamber, the HBP-evoked response is primarily due to an effect in monocytes with no major active support from EC. Indeed, the monocytic response to HBP, both in solution and immobilized on adhesive surfaces, involved a rapid and transient rise in cytosolic free calcium indicating a specific signaling mechanism. Experiments with the calcium chelator BAPTA-AM further support this notion, as the enhanced monocyte adhesion triggered by HBP was totally dependent on the evoked calcium response. In contrast, basal adhesion to cytokine-activated EC (or CAM-coated surfaces) was not affected by BAPTA-AM treatment, indicating that endothelial expression of CAM may determine monocyte arrest in this situation. In addition, the potentiating effect of HBP on monocyte arrest was only seen in the interaction of monocytes with CAM known to support leukocyte rolling, whereas binding to ICAM-1 was not influenced by the presence of HBP. This may indicate that the HBP-induced activation primes the monocyte with regard to rolling efficacy or that it may be important in the transition from rolling to firm arrest. Interestingly, although our data revealed no active contribution of the endothelium in the HBP-evoked monocyte arrest, it is worth noting that prolonged exposure of EC to HBP/CAP37 may result in endothelial CAM expression leading to enhanced leukocyte adhesion (30).

Our observations regarding the effects of HBP on monocyte recruitment reveal a function of the protein that in many respects resembles the function of classical chemokines in inflammation with thr capacity to stimulate the critical steps in the mobilization of a particular leukocyte subclass, i.e., activation and arrest on endothelium, and subsequent chemotaxis. However, HBP does not share structural homology with chemokines (27) and is not known to activate any of the classical chemokine receptors. In fact, the binding site for HBP on immune cells remains elusive (18) and further research is required to uncover the signaling pathways implicated in monocyte activation. The requirement of calcium-dependent signaling in this respect is at variance with chemokine-triggered monocyte arrest on E-selectin-expressing EC monolayers, which was found not to correlate with induction of intracellular calcium fluxes (31).

An interesting feature with regard to the HBP-evoked effect on monocyte arrest is that the activating agent is not derived from the inflammatory lesion itself, but rather seeded on the endothelium by a third party, the emigrating neutrophil. A similar mechanism has been observed previously in relation to leukocyte recruitment during atherogenesis, where activated platelets may spread the proinflammatory chemokine RANTES (32) on the endothelial surface, which in turn triggers increased arrest of monocytes. Thus, evidence is accumulating in support of the notion that agents that stimulate mobilization of leukocytes to inflammatory sites may be derived not only from cells stationary in the inflamed tissue but also from cells in the circulation, and accordingly, may serve a function in bridging acute and delayed host defense reactions.

In conclusion, we demonstrate that HBP derived from activated neutrophils accumulates on the surface of EC. In this location the protein, although by a so far unknown pathway, is capable of triggering a calcium-dependent activation response in monocytes that ultimately results in increased firm arrest on the endothelium. This mechanism may contribute to monocyte trafficking in inflammation and may be of pathophysiologic significance in a number of inflammatory disorders in which recruitment of mononuclear cells at inflammatory foci is preceded by activation of the neutrophil population.


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


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

1 This study was supported by grants from the Swedish Research Council, the Swedish Heart-Lung Foundation, the AFA Health Fund, the Vardal Foundation, the Swedish Society for Medical Research, the Tore Nilsson Foundation, and the Lars Hierta Memorial Fund. H.U. is recipient of a Postdoctoral Fellowship from Deutsche Forschungsgemeinschaft UL-199/1. Back

2 Address correspondence and reprint requests to Dr. Lennart Lindbom, Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden. E-mail address: lennart.lindbom{at}fyfa.ki.se Back

3 Abbreviations used in this paper: HBP, heparin-binding protein; EC, endothelial cell; CAP37, cationic antimicrobial protein of molecular mass 37 kDa; BAEC, bovine aorta endothelial cell; fluo-4-AM, fluo-4-acetoxymethyl ester; MM6, Mono Mac 6; PMN, polymorphonuclear leukocyte; CAM, cell adhesion molecule; MFI, mean fluorescence intensity. Back

Received for publication October 13, 2004. Accepted for publication March 3, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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