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Cardiovascular Research 1998 38(2):516-521; doi:10.1016/S0008-6363(98)00014-5
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Release of chemoattractants for human monocytes from endothelial cells by interaction with neutrophils

Peter Schratzberger, Stefan Dunzendorfer, Norbert Reinisch, Christian M Kähler, Manfred Herold and Christian J Wiedermann*

Division of General Internal Medicine, Department of Internal Medicine, Medical Faculty, University of Innsbruck, Anichstraße 35, A-6020 Innsbruck, Austria

* Corresponding author. Tel.: +43 (512) 504-3255; Fax: +43 (512) 504-4201; E-mail: christian.wiedermann@uibk.ac.at

Received 10 June 1997; accepted 22 December 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: The release of monocyte chemoattractant protein-1 (MCP-1) in the vessel wall may lead to accumulation of monocytes in the subendothelial space. The role of neutrophils (PMNL) in the initiation of this process is unknown. We tested whether PMNL are able to induce the production and release of MCP-1 in endothelial cells. Methods: PMNL were allowed to interact with human umbilical vein endothelial cell (HUVEC) monolayers in culture. Culture media were collected and assessed for chemotactic activity on mononuclear leukocytes (MNC) or purified monocytes in a modified Boyden chamber assay. Additionally, MCP-1 levels in supernatants were quantified by ELISA. Results: Media from unstimulated HUVEC culture supernatants induced a slight increase (1.2-fold) of MNC and purified monocyte chemotaxis, which was significantly augmented by addition of PMNL for 1 h (1.4-fold; P<0.05). The increase in chemotaxis was time- and dose-dependent and could be blocked by an anti-MCP-1 monoclonal antibody. Media obtained after coculture of PMNL and HUVEC for 1–5 h contained increased amounts of MCP-1 as measured by ELISA; addition of cycloheximide abolished this response. Conclusions: Interaction of PMNL with endothelium induces the release of functionally active MCP-1 suggesting that in the vascular wall, PMNL may play a role in the recruitment of MNC.

KEYWORDS Monocyte chemoattractant protein-1; Human; Granulocyte; Endothelium; Monocyte


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
All subclasses of granulocytes, i.e. neutrophils (PMNL), eosinophils and basophils, have been reported as being present in arteriosclerotic arterial thickening in several models of experimental arteriosclerosis [1–4], but their role in atherogenesis is less well understood. In a rabbit model of intimal thickening by electrical stimulation of carotid artery, PMNL are among the predominant types of leukocytes that migrate across an intact endothelium into the subendothelial space [5]. In experimental arteriosclerosis, blockade of leukocyte infiltration resulted in a 70% reduction of intimal smooth muscle cell accumulation [6]and invasion of the arterial intima by PMNL in this model was completely prevented by an antibody directed toward the common β2 chain (CD18) of the leukocyte adherence complex CD11/CD18, whereas the influx of monocuclear leukocytes (MNC) was only partially inhibited [6]. Under this condition, smooth muscle cell migration into the intima was not affected, thus excluding PMNL invasion as a prerequisite for the movement of smooth muscle cells into the intimal compartment [7]. Data suggest that MNC, but not PMNL, promote lesion development by stimulating smooth muscle cell migration [7].

Oxidized low-density lipoprotein increases endothelial susceptibility to PMNL-induced endothelial dysfunction in porcine coronary arteries [8]. Additionally, it has been shown that atherosclerosis enhances the endothelial adhesiveness for monocytes and lymphocytes [9, 10], which suggests that the interaction of intact endothelium with PMNL may lead to the release of secretory products which are chemotactic for monocytes and lymphocytes.

The purpose of the present in vitro study was to investigate the role of PMNL for monocyte recruitment in atherosclerosis upon interaction of PMNL with intact endothelium in the absence of additional inflammatory or infectious stimuli. The effects on the release of MNC chemoattractants by the interaction between PMNL and intact endothelial monolayers were tested. Our experiments suggest that the initial contact of PMNL with endothelial cells as it occurs in models of experimental arteriosclerosis, contributes to the recruitment of monocytes and lymphocytes by release of monocyte chemoattractant protein-1 (MCP-1).


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Reagents
HBSS was purchased from Gibco (Vienna, Austria), Lymphoprep was from Nycomed Pharma AS (Oslo, Norway), and Dulbecco's phosphate-buffered saline (PBS) and RPMI-1640 were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). Bovine serum albumin (BSA) was purchased from Behring Werke AG (Marburg, Germany), and complete endothelial cell (EC) growth medium (ECGM) with low serum contents was from Promo Cell (Heidelberg, Germany). Fibronectin, purified from human plasma, was obtained from Biomedica (Vienna, Austria), and collagenase, type CLS, was from Seromed (Berlin, Germany). Dextran (MW 500 000), formyl-methionine–leucine–phenylalanine (fMLP) and cycloheximide were purchased from Sigma Chemical (St. Louis, MO, USA). Human recombinant tumor necrosis factor-{alpha} (TNF-{alpha}) was obtained from Boehringer Mannheim Biochemica (Mannheim, Germany) and anti-monocyte chemoattractant protein-1 (MCP-1, or monocyte chemotactic and activating factor) monoclonal antibody (mAb) was purchased from Serotec (Vienna, Austria).

2.2 Preparation of leukocytes
MNC were isolated from EDTA blood of healthy volunteers by Lymphoprep density gradient centrifugation and washed twice in HBSS as described previously [11]. The MNC preparations (>98% viability by Trypan dye exclusion) were counted and adjusted to 1.0x106 cells/ml in RPMI-1640 containing 0.5% BSA for use in chemotaxis assays.

Positive selection of monocytes (CD14+ cells) was performed according to the manufacturer's recommendations as described previously [12]. Purity of sorted CD14+ cells was >98%, as determined by FACS analysis. Monocytes were washed and resuspended in RPMI-1640/0.5% BSA.

PMNL were obtained from EDTA blood of healthy volunteers by Lymphoprep density gradient centrifugation and removal of contaminating erythrocytes by dextran sedimentation and subsequent lysis using sodium chloride [11]. Cell preparations yielded >95% PMNL (by morphology in Giemsa stains) and >99% viability (by Trypan dye exclusion).

2.3 EC culture
Human umbilical vein endothelial cells (HUVEC) from fresh placental cords were isolated by previously described methods [13]and grown to confluence at 37°C in 5% CO2 and humidified atmosphere in ECGM. Tissue culture flasks (Falcon, Lincoln Park, NJ, USA) were coated with 1 µg/cm2 human fibronectin prior to seeding with HUVEC. Cells were detached by treatment with collagenase and used for experiments from passage 2 to 5 [12].

2.4 Coculture of HUVEC with PMNL or MNC
HUVEC were grown to confluence in 24-well tissue culture plates or in Transwell filter insets before 600 or 100 µl of PMNL suspensions (1.0x106 cells/ml ECGM) were added to the monolayers and allowed to interact for various periods of time (0.5–5 h) or monolayers remained untreated. In another set of experiments, HUVEC were preincubated for 30 min with cycloheximide (5 µg/ml) prior to washing with PBS and addition of PMNL with or without TNF-{alpha} (10 ng/ml) preincubation. Supernatants were collected, purified by centrifugation and subjected either to the Boyden microchemotaxis chamber assays or always to ELISA detection of MCP-1 contents. At least, HUVEC monolayers were examined microscopically and appeared to be unharmed.

2.5 MNC and monocyte chemotaxis assay
Chemotaxis through cellulose nitrate to gradients of soluble attractants was measured as described [12, 15]using a 48-well microchemotaxis chamber (Neuroprobe, Bethesda, MA, USA) in which a 5-µm pore-sized cellulose nitrate filter (Sartorius, Göttingen, Germany) separates the upper and lower chambers, allowing only the actively migrating cells to get through the pores. MNC or purified monocytes (5x104 cells/well in RPMI-1640/0.5% BSA) were placed in the upper chamber and soluble attractants in the lower chamber. Attractants were either 10 nmol/l of fMLP as positive control or supernatants derived from treated or untreated HUVEC cultures. In some experiments, a purified anti-MCP-1 mAb (1.0 µg/ml) was added to the supernatants in the lower compartment. After 90 min at 37°C, the nitrocellulose filters were dehydrated, fixed, and stained with hematoxylin–eosin as described [11]. Migration along the filter was quantified by microscopy, measuring the distance from the surface of the filter to the leading front of cells [12]. The data are expressed as chemotaxis index (CI), which is the ratio between the distance of migration towards the test attractants and the distance of migration towards control medium. The migration depth of the leading front in the medium controls was 50–75 µm.

2.6 ELISA for MCP-1
For direct measurement of MCP-1 contents in supernatants from cocultures of HUVEC and PMNL, a commercially available sandwich ELISA kit (R&D Systems Quantikine human MCP-1, Minneapolis, MN, USA) was performed in accordance with the manufacturer's instructions. This assay is stated to recognize both natural and rMCP-1, but exhibits no cross-reactivity with any other molecules tested, including IL-8, RANTES, IL-1β, serum amyloid protein, and epidermal growth factor.

2.7 Statistical analyses
All values are expressed as mean±s.e.m. Prior to two-group comparisons using the Mann–Whitney U-test, all data sets were analyzed for multigroup comparison by the Kruskal–Wallis analysis of variance. Probability values below 0.05 were considered to be statistically significant. For all statistical analyses, the StatView software package (Abacus Concepts, Berkeley, CA, USA) was used.

The investigation conforms to the principles outlined in the Declaration of Helsinki.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Involvement of MCP-1 in MNC chemotactic activity of supernatants from cocultures of HUVEC and PMNL
MNC migration toward supernatant from cultures of HUVEC without PMNL remained almost unaffected, whereas addition of PMNL to HUVEC for 60 min produced supernatants which induced significant MNC migration. Supernatants harvested from HUVEC that were primed with TNF-{alpha} for 4 h and subsequently interacted with PMNL or medium for 60 min, stimulated MNC migration to an equal extent (Fig. 1).


Figure 1
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  		Fig. 1 Effect of an anti-monocyte chemoattractant protein-1 (MCP-1) monoclonal antibody (mAb; at 1 µg/ml) on mononuclear leukocyte (MNC) chemotaxis into cellulose nitrate micropore filters induced by supernatants that were obtained from endothelial cell cultures. HUVEC were preincubated with plain medium (left) or with human recombinant tumor necrosis factor-{alpha} (TNF; 10 ng/ml) (right) for 4 h prior to addition of PMNL. Chemotactic migration of MNC toward supernatants is shown with and without addition of anti-MCP-1 mAb. Chemotaxis Index (CI) represents the ratio between the distance of MNC migration toward chemoattractant and the distance of migration toward control medium. Values represent mean±s.e.m. for quadruplicate determinations in six independent experiments (n=6). n.s., not significant; *P<0.05; **P<0.01.

 
In a following set of experiments, we tried to clarify a substrate for the chemotactic activity of the supernatants from PMNL/HUVEC-cocultures. Chemotactic activities of each supernatant could be significantly decreased by an anti-MCP-1 mAb (1 µg/ml). Addition of the specific antibody to the supernatants from PMNL-incubated HUVEC suppressed the chemotactic effect to random migration levels (Fig. 1, left), the CI of supernatants from TNF-{alpha}-primed HUVEC without and TNF-{alpha}-primed HUVEC with PMNL incubation then amounting to 1.09±0.04 and 1.13±0.03, respectively (Fig. 1, right). fMLP (10 nmol/l) served as positive control for migration of MNC. The anti-MCP-1 mAb itself affected neither random migration nor fMLP-stimulated migration of MNC (data not shown).

3.2 Dose dependency of chemotactic migration of purified human monocytes toward supernatants from cocultures of HUVEC and PMNL
To investigate whether chemotactic effects of the supernatants are dose-dependent, we performed a series of chemotaxis experiments in which we exposed purified monocytes (CD14+) to various dilutions (1:1, 1:10, 1:100, 1:1000) of supernatants from HUVEC–PMNL cocultures. Results show that the more the supernatants were diluted, the less was the chemotactic response. Addition of an anti-MCP-1 mAb (1 µg/ml) to the various dilutions of supernatants was able to diminish the chemotactic response to baseline (Table 1).


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  		Table 1 Chemotactic response of magnetically separated human monocytes (CD14+) to various dilutions of supernatants obtained from human umbilical vein endothelial cell (HUVEC) neutrophil (PMNL) cocultures

 
3.3 Time-dependent release of MCP-1 from HUVEC into supernatants from HUVEC–PMNL cocultures and effects of cycloheximide
In order to investigate a possible time dependency of MCP-1 release from HUVEC–PMNL cocultures, time periods allowed for PMNL to interact with HUVEC monolayers were varied from 0.5 to 5 h. After these periods of coincubation, supernatants were assayed for immunoreactive MCP-1 as well as monocyte chemotactic activity. Fig. 2 shows that exposure of HUVEC to PMNL gives rise to rapid increase in secreted MCP-1, being significant after 60 min and increasing for up to 5 h, the longest time period tested. To examine whether de novo protein synthesis is involved in the observed MCP-1 release, we preincubated HUVEC monolayers with cycloheximide (5 µg/ml) for 30 min prior to interaction with PMNL. Pretreatment of HUVEC with cycloheximide led to a suppression in MCP-1 release over the whole period of time.


Figure 2
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  		Fig. 2 Monocyte chemoattractant protein-1 (MCP-1) levels and monocyte chemotactic activity of conditioned media of cocultures of neutrophils (PMNL) and human umbilical vein endothelial cells (HUVEC). After pretreatment with or without cycloheximide (5 µg/ml), confluent HUVEC monolayers were coincubated with PMNL for the indicated time periods and MCP-1 concentration was measured by ELISA or chemotactic activity was determined by the Boyden chamber micropore filter assay. Migration of monocytes toward f–Met–Leu–Phe (10 nmol/l) revealed a chemotaxis index (CI) of 1.66±0.085. CI represents the ratio between the distance of monocyte migration toward chemoattractant and the distance of migration toward control medium. Values represent mean±s.e.m. for quadruplicate determinations in six independent experiments (n=6).

 
When supernatants were tested for chemotactic activity for purified human monocytes in the Boyden chamber assay, the migratory response was significantly increased at 60 min to 5 h. Pretreatment of HUVEC with cycloheximide completely blunted the chemotactic action of the supernatants (Fig. 2).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In the pathophysiology of cardiovascular diseases, in which there is an accumulation of monocytes in the subendothelial space, an interaction between PMNL and EC is known to occur. Therefore, the question arises whether such interaction induces production of MNC chemoattractants. We attempted to answer this question by investigating in vitro the interaction between PMNL and HUVEC. Our experiments show that cocultures of resting PMNL and unstimulated or TNF-{alpha}-primed HUVEC monolayers induced an increase in the amounts of soluble MCP-1 secreted from EC. MCP-1 secreted into supernatants exhibited chemotactic activity for MNC and this effect was abolished in the presence of anti-MCP-1 mAb. Quantification of MCP-1 protein in medium indicates that augmented production of MCP-1 was induced by PMNL in HUVEC to levels comparable to MCP-1 released by TNF-{alpha} or direct cell-to-cell contact with monocytes [16]. These results indicate that during the process of adhesion, PMNL transmit signals to EC inducing MCP-1 synthesis and release.

Previous results suggested that the monocyte–EC adhesive interaction induces an MCP-1-inductive signal to each cell type [16]. Since PMNL fail to produce MCP-1 [17], the MCP-1 activity obtained in media of PMNL-exposed HUVEC can be assumed to originate exclusively from the HUVEC.

Although PMNL are capable of destroying tissue by secreting proteolytic enzymes [18], they are rare in atherosclerotic lesions [19, 20]. PMNL may occasionally be found in disrupted plaques beneath coronary thrombi, probably entering these plaques shortly after disruption [19]and may also migrate into the arterial wall shortly after reperfusion of occluded arteries in response to ischemia/reperfusion [21, 22]. The relatively low number of PMNL identified in the subendothelial space, however, may lead one to underestimate the actual pathophysiological importance of the cells, since transendothelial migration conveys an apoptotic signal to PMNL, activating the endogenous cell death program [23].

Since MNC comprise a mixture of monocytes and lymphocytes, monocytes were identified as a chemotactically responsive cell population by experiments using purified CD14+ cells. Time course experiments in adhesion assays confirm that the response occurs rapidly and induces chemokine release as early as 60 min and steadily increases for up to 5 h (Fig. 2). Similar kinetics have been previously reported, in particular when experiments were performed in combination with cytokines [16]. As the time course data were similar for MCP-1 protein and functional response of monocytes in the chemotaxis assay and both abrogated by pretreatment of HUVEC with cycloheximide, de novo synthesis of MCP-1 may be primarily responsible for the monocyte chemotactic activity. The time required for de novo synthesis of chemokines, lymphokines and cytokines in vitro, may be between 2 and 6 h; in some experimental systems, initial mediator release, however, may occur after 1 h [16, 24, 25]. Since levels of MCP-1 released from HUVEC by PMNL reached plateau after 4 h, whereas effects on monocyte chemotaxis increased continually over time, MCP-1 may not be the only monocyte-chemotactic factor released upon PMNL stimulation of HUVEC (Fig. 2).

The in vivo kinetics of PMNL transendothelial migration are unknown. We and others have previously shown in an in vitro system that PMNL in the subendothelial space can be seen as early as 30 min after PMNL are added to EC monolayers [14, 22]. A previous study on MCP-1 induction by coculture of monocytes and EC suggested that the induction of MCP-1 mRNA occurs relatively early, 2–4 h after coculture [16]. This rapidity may indicate that direct cell–cell contact rather than the presence of soluble factors such as IL-1 is required in the early phase of MCP-1 induction. We directly compared induction of MCP-1 synthesis by PMNL–HUVEC interaction with that by MNC–HUVEC interaction and observed comparable early responses, also suggesting that the endothelial MCP-1 production is directly induced by interaction with PMNL (unpublished data).

The precise role of endothelial–PMNL interactions in atherogenesis and the development of vascular events is unknown. Interaction of PMNL with EC has previously been shown to result in the release of endothelium-derived growth factors [25]. PMNL-EC interactions may contribute to endothelial vasodilator dysfunction in atherosclerotic arteries [8]with inappropriate vasoconstriction [26]. The present study shows that PMNL induce EC to synthesize MCP-1 which may lead to monocyte accumulation in the vascular wall. Since MCP-1 may be especially important in the pathogenesis of atherosclerosis [27], our results suggest a new pathogenetic link between PMNL and endothelial activation. MCP-1 has been demonstrated in macrophage-rich areas of atherosclerotic lesions [28]. Included in the β(C–C) subfamily of chemokines, MCP-1 is a relatively specific chemoattractant for monocytes and is produced by a variety of cell types [29]. Stimuli for MCP-1 production include modified oxidized LDL [30]and cytokines [29]. Cytokine production and cellular activation have been induced by direct cell-to-cell contact in a variety of cell types [31, 32].

In conclusion, our data indicate that EC are primed to produce MCP-1 through direct cellular contact with PMNL during adhesion even in the absence of additional stimulation by proinflammatory cytokines such as TNF-{alpha}. PMNL-induced production of MCP-1 by endothelium may be an important mechanism in the recruitment of blood monocytes to the vessel wall and thus in the pathogenesis of atherosclerosis.

Time for primary review 21 days.


   Acknowledgements
 
The study was supported by the Austrian Science Funds Grant 09977-Med to C.J.W.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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