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Cardiovascular Research 1998 37(3):820-825; doi:10.1016/S0008-6363(97)00265-4
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Monocyte–vascular smooth muscle cell interaction enhances nitric oxide production

Uichi Ikedaa,*, Yoshikazu Maedaa, Hiroshi Funayamaa, Yukihiro Hojoa, Michiyo Ikedab, Seiji Minotab, Shogo Kanob and Kazuyuki Shimadaa

aDepartment of Cardiology, Jichi Medical School, Tochigi, Japan
bDepartment of Clinical Immunology, Jichi Medical School, Tochigi, Japan

* Corresponding author. Department of Cardiology, Jichi Medical School, Minamikawachi-machi, Tochigi 329-04, Japan. Tel. (+81-285) 44-2111, ext. 3556; Fax (+81-285) 44-5317; E-mail: uikeda@jichi.ac.jp

Received 10 June 1997; accepted 18 October 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The adhesive interaction of monocytes and vascular smooth muscle cells (VSMCs) has been suggested to be a regulatory signal in the cellular activation that is involved in the pathogenesis of atherosclerosis. We investigated the effects of monocyte–VSMC interaction on inducible nitric oxide (NO) synthase expression. Methods: NO production by the cultured cells was determined by measuring the nitrite content of the culture media using the Griess reagent. The expression of inducible NO synthase protein was assayed by Western blotting. Results: Interleukin-1β (IL-1β) induced nitrite production by VSMCs in a time-dependent manner. The addition of the mouse monocyte cell line J774 to IL-1β-stimulated VSMCs further increased nitrite production in a monocyte number-dependent manner. Enhanced nitrite production by coculture was accompanied by increased inducible NO synthase protein accumulation. Addition of tumor necrosis factor-{alpha} (TNF-{alpha}) also enhanced IL-1β-induced nitrite production by VSMCs, but TNF-{alpha} showed no effect in the presence of monocytes. Coculture of monocytes and VSMCs in the presence of IL-1β secreted substantial amounts of TNF-{alpha}. The production of nitrite by coculture was markedly inhibited by an anti-TNF-{alpha} antibody. Conclusions: The present study revealed that direct cell-to-cell interaction between monocytes and VSMCs enhances NO production, suggesting an important role for their interaction in the pathogenesis of atherosclerosis.

KEYWORDS Monocyte; Vascular smooth muscle; Nitrite; Atherosclerosis


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Nitric oxide (NO), the extensively characterized endothelium-derived relaxing factor, is a short-lived free radical. NO is synthesized from L-arginine by three isoenzymes expressed either constitutively (neuronal, type I cNOS; endothelial, type III cNOS) or following stimulation by cytokines and endotoxins (inducible, type II iNOS) [1, 2]. Inducible NO synthase has been identified in monocytes/macrophages, hepatocytes, endothelial cells and myocardium [1, 3]. NO synthase activity is also induced in aortic rings and cultured vascular smooth muscle cells (VSMCs) by cytokines and endotoxins [4, 5]. Since exogenous NO-containing compounds inhibit DNA synthesis and proliferation of VSMCs, it has been suggested that endogenously produced NO may also control VSMC proliferation in vivo [6–8]. Indeed, L-arginine, the substrate of NO synthesis, exhibits anti-atherogenic effects in cholesterol-fed rabbits, whereas inhibition of NO production potentiates intimal lesion formation in the same model [9]. It has been demonstrated that inducible NO synthase is expressed in the balloon-inured rat carotid artery [10]or human atherosclerotic lesions [11]. Therefore, inducible NO synthase induction in the vascular tissue may play a role in vaso-occlusive disorders such as atherosclerosis, and restenosis after angioplasty and bypass surgery.

Cellular interactions are critical events in vascular biology. Previously, Marczin et al. [12]reported that cell-to-cell interaction between human monocytes and aortic endothelial cells down-regulates steady-state levels of endothelial constitutive NO synthase. We investigated whether monocyte–VSMC interaction modulated inducible NO synthase expression.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Reagents
Recombinant human interleukin-1β (IL-1β) was kindly provided by Otsuka Pharmaceutical Co. (Tokushima, Japan). Recombinant human tumor necrosis factor-{alpha} (TNF-{alpha}) was purchased from Genzyme (Cambridge, MA). The monoclonal antibody against mouse TNF-{alpha} was purchased from Endogen (Woburn, MA). A monoclonal anti-mouse inducible NO synthase antibody, which cross-reacts against rat inducible NO synthase, was obtained from Transduction Lab. (Lexington, KY). All other chemicals used were of the highest grade commercially available.

2.2 Culture of cells
Primary cultures of VSMCs were obtained from the media of thoracic aortas of Sprague–Dawley rats (200–250 g), as described previously [13]. The cells were grown in Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells grown to confluence were detached by treatment with 0.125% trypsin and 0.02% EDTA and reseeded in secondary cultures. The cells exhibited typical ‘hill and valley’ growth morphology of VSMCs, and almost all cells reacted with the anti-{alpha}-actin antibody, which selectively recognizes muscle forms of actin but does not react with endothelial cells or fibroblasts [13]. Typically, cultured cells were used at passage 5–10. Cells (3x104/ml) were plated in 24-well or 100-mm culture dishes in DMEM supplemented as described above, and allowed to grow to subconfluence for 48–72 h. Subsequently, they were preincubated in DMEM containing 0.5% fetal bovine serum and supplemented with insulin (5 µg/ml) and transferrin (5 µg/ml) for 24 h, and used for the experiments described below.

Mouse monocyte cell line J774 was cultured in 20% fetal bovine serum containing RPMI 1640 medium, and used for the experiments in the presence of 0.5% fetal bovine serum.

The investigation was performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.3 Measurement of nitrite contents
VSMCs plated in 24-well dishes were incubated in DMEM containing 0.5% fetal bovine serum at 37°C. The nitrite contents of culture media were determined by mixing 500 µl of medium with an equal volume of Griess reagent (1 part 0.1% naphthylethylenediamine dihydrochloride to 1 part 1% sulfanilamide in 5% phosphoric acid) [14]. The absorbance at 550 nm was measured and the nitrite concentration was determined by interpolation of a calibration curve of standard sodium nitrite concentrations against absorbance. After washing, cells were dissolved in 0.2 ml of 1% sodium dodecyl sulfate (SDS) and used for protein assay (Bio-Rad assay kit, Hercules, CA) with bovine serum albumin as a standard. Nitrite levels were corrected by protein measurement and data are shown as nmol per mg protein.

2.4 Assay for inducible NO synthase protein
The expression of inducible NO synthase protein was analyzed by immunoblotting with an anti-inducible NO synthase antibody [15]. Briefly, VSMCs were lysed in a buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 µM leupeptin, 1 µM pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride and 1 M dithiothreitol, and sonicated. The homogenates were then centrifuged at 100 000xg for 20 min, and the supernatants (60 µg protein) were subjected to 10% SDS-polyacrylamide gel electrophoresis. The separated proteins were electrophoretically transferred onto nitrocellulose membranes, and the resultant blots were incubated with the anti-inducible NO synthase antibody for 2 h followed by peroxidase-labeled anti-mouse IgG for 1 h. Peroxidase-labeled proteins were detected using the ECL detection system (Amersham Int., Bucks, UK) on X-ray film; the results were quantified by densitometric scanning.

2.5 Measurement of TNF-{alpha} levels
Concentrations of TNF-{alpha} were measured with commercially available ELISA kits (Amersham Int.) according to the manufacturer's protocol. The assay was performed with an antibody against mouse TNF-{alpha}, which had been coated onto the wells of a 96-well microplate. After adding 50 µl of standard or sample per well, 50 µl of biotinylated antibody reagent was added to the wells and plates were incubated for 2 h at room temperature. One hundred µl of streptavidin-horseradish peroxidase was then added for 30 min, followed by addition of 100 µl of tetramethylbenzidine substrate solution for 30 min at room temperature. The reaction was stopped by adding stop solution to each well, and the absorbance at 450 nm was measured with an ELISA reader. TNF-{alpha} concentrations were calculated from standard curves. The limit of detection for TNF-{alpha} was 10 pg/ml. All samples were run in duplicate and the average values of the two measurements are reported.

2.6 Statistical analysis
Data are expressed as means±S.E.M. of four samples, which represented at least three separate experiments. Differences were analyzed by one-way ANOVA combined with Scheffé's test, and P values less than 0.05 were considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effects of coculture on NO production
NO production by the cultured cells was determined by measuring the nitrite contents of the culture media. Accumulation of nitrite in the medium represents the summation of NO synthase activity during the time period studied, since NO secreted by cells is rapidly decomposed to the more stable products nitrite and nitrate. As shown in Fig. 1, nitrite was produced at a very low level in unstimulated VSMCs. When VSMCs were stimulated with IL-1β (10 ng/ml), nitrite production increased in a time-dependent manner. Culture of IL-1β-stimulated VSMCs with monocytes further increased nitrite production within 9 h of the beginning of the incubation, while culture of unstimulated VSMCs with monocytes showed no significant effect on nitrite accumulation.


Figure 1
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  		Fig. 1 Line graph showing the kinetics of nitrite production by coculture of VSMCs and monocytes. VSMCs (3x104 cells) were stimulated with IL-1β (10 ng/ml) in the presence (closed circles) or absence (open circles) of monocytes (3x104 cells) in 24-multiwell plates for the indicated periods. VSMCs (open squares) or VSMCs plus monocytes (closed squares) were also cultured in the absence of IL-1β. The production of nitrite was determined using the Griess reagent. Values represent means±S.E.M. for four samples. *P<0.05 compared with IL-1β-stimulated VSMCs.

 
Fig. 2 shows nitrite production induced by coculture of monocytes and VSMCs at various ratios. Significant production of nitrite was observed at a monocyte/VSMC ratio of 0.5, and the production increased in a monocyte number-dependent manner. Thus, in the following experiments, we examined the production of nitrite after 24 h coculture with a monocyte/VSMC ratio of 1.0.


Figure 2
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  		Fig. 2 Bar graph showing the effects of different numbers of monocytes on nitrite production. VSMCs (3x104 cells) were cocultured with different numbers of monocytes (monocyte/VSMC ratio=0, 0.1, 0.5, 1.0, 5.0) for 24 h in the presence of IL-1β (10 ng/ml), and the production of nitrite was determined using the Griess reagent. Values represent means±S.E.M. for four samples. *P<0.05 compared with control samples without monocytes.

 
We then investigated the effects of monocyte–VSMC interaction at various concentrations of IL-1β (Fig. 3). IL-1β dose-dependently increased nitrite production by VSMCs at 24 h. When VSMCs were cultured with monocytes, the production of nitrite was also increased by IL-1β in a dose-dependent manner. In contrast, when monocytes alone were incubated with various concentrations of IL-1β, no change in nitrite accumulation was observed.


Figure 3
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  		Fig. 3 Line graph showing the effects of different concentrations of IL-1β on nitrite production in cocultures. VSMCs alone (open circles), monocytes alone (open squares), or VSMCs plus monocytes (closed circles) were cultured in the presence of various concentrations of IL-1β, and the production of nitrite was determined using the Griess reagent. Values represent means±S.E.M. for four samples. *P<0.05 compared with VSMCs alone.

 
The above findings suggest that adhesion between monocytes and VSMCs enhances NO production. To further determine whether NO production by coculture is mediated by direct contact between monocytes and VSMCs, we performed a separate coculture assay using an inner well system (Cell Culture Insert). As shown in Fig. 4, nitrite accumulation induced by coculture of monocytes with VSMCs was substantially greater than that induced by separate coculture of monocytes with VSMCs, also supporting the assumption that direct adhesion between monocytes and VSMCs is necessary for maximal increased NO production.


Figure 4
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  		Fig. 4 Production of nitrite upon separate culture and coculture of monocytes with VSMCs. VSMCs cultured on the lower compartment of the inner well system were coincubated for 24 h with monocytes added to the upper or lower compartment (separate culture, VSMCs in the lower compartment and monocytes in the upper compartment; coculture, VSMCs and monocytes in the lower compartment) in the presence of IL-1β (10 ng/ml). Nitrite accumulation in the lower compartment was determined using the Griess reagent. Values represent means±S.E.M. for four samples. *P<0.05 compared with separate culture.

 
3.2 Effects of coculture on inducible NO synthase protein
We next examined whether monocyte–VSMC interaction increased the production of nitrite at the inducible NO synthase level (Fig. 5). On Western blots, inducible NO synthase was undetectable in unstimulated VSMCs. However, inducible NO synthase was clearly detected in VSMCs stimulated with IL-1β for 24 h, and its expression was further increased in the presence of monocytes.


Figure 5
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  		Fig. 5 Expression of inducible NO synthase protein by coculture of VSMCs and monocytes. (A) Western blot depicting inducible NO synthase (iNOS) protein accumulation. VSMCs were cultured for 24 h with or without monocytes in the presence of IL-1β (10 ng/ml). Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblot analysis. The inducible NO synthase protein was detected as a molecular mass of about 125 kDa. Lane 1: unstimulated VSMCs; lane 2: VSMCs in the presence of IL-1β; lane 3: VSMCs plus monocytes in the presence of IL-1β. (B) Densitometry values (in arbitrary densitometry units) of inducible NO synthase protein in Western blot. Data represent means of two separate experiments.

 
3.3 Involvement of TNF-{alpha} in NO production by coculture
We then analyzed the possible mechanism underlying the coculture-induced NO production. As shown in Fig. 6, addition of TNF-{alpha} showed no effect on the basal level of nitrite by VSMCs, but the production of nitrite by IL-1β-stimulated VSMCs was significantly up-regulated by TNF-{alpha}. Addition of monocytes to IL-1β-stimulated VSMCs enhanced nitrite accumulation, but TNF-{alpha} showed no effect in the presence of monocytes. Coculture of monocytes and VSMCs in the presence of IL-1β secreted substantial amounts of TNF-{alpha} in a time-dependent manner, while IL-1β-stimulated VSMCs did not secrete detectable amounts of TNF-{alpha} (Fig. 7).


Figure 6
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  		Fig. 6 Effects of TNF-{alpha} on nitrite accumulation. VSMCs were incubated for 24 h with IL-1β (10 ng/ml) and/or TNF-{alpha} (10 ng/ml) in the presence (solid bars) or absence (hatched bars) of monocytes. Nitrite accumulation in the culture medium was measured, and values were normalized to the protein content per well. Data represent means±S.E.M. for four samples. *P<0.05.

 

Figure 7
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  		Fig. 7 TNF-{alpha} production by the coculture of VSMCs and monocytes. VSMCs were stimulated with IL-1β (10 ng/ml) in the presence (closed circles) or absence (open circles) of monocytes in 24-multiwell plates for the indicated periods. TNF-{alpha} levels in the culture media were determined by ELISA. Values represent means±S.E.M. for four samples. *P<0.05 compared with VSMCs in the absence of monocytes.

 
These results suggest that the production of nitrite by coculture of monocytes and VSMCs is mediated at least partially by TNF-{alpha}. Thus, we examined the effects of a neutralizing antibody (mouse IgG1) against TNF-{alpha} on nitrite accumulation induced by coculture. As shown in Fig. 8, the use of this antibody dose-dependently (0.1~10 µg/ml) inhibited nitrite production induced by coculture. On the other hand, addition of non-specific mouse IgG1 (0.1~10 µg/ml) showed no effect on the nitrite accumulation (data not shown).


Figure 8
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  		Fig. 8 Effects of TNF-{alpha} antibody on nitrite accumulation. VSMCs were incubated for 24 h with (solid bars) or without (hatched bars) of monocytes in the presence of IL-1β (10 ng/ml) and an antibody against TNF-{alpha} (0.1~10 µg/ml). Nitrite accumulation in the culture medium was measured, and values were normalized to the protein content per well. Data represent means±S.E.M. for four samples. *P<0.05 compared with control cells without the antibody.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The adhesive interaction of monocytes and VSMCs may be an important regulatory signal for cell activation. In the present study, we demonstrated that coculture of monocytes and VSMCs in the presence of IL-1β enhanced NO production. We also performed a separate coculture assay and confirmed that direct adhesion between monocytes and VSMCs was more effective for NO production. Furthermore, the increase in NO production was associated with increased inducible NO synthase protein accumulation.

This raises the question of which types of cells produce nitrite under these coculture conditions. Mouse monocytes alone do not produce nitrite in response to IL-1β (Fig. 3) or TNF-{alpha} [16]. Thus, although we did not perform immunohistochemical analysis, we speculate that VSMCs were the major source of nitrite production in these cocultures. The next question to be addressed is how monocytes enhance NO production by IL-1β-stimulated VSMCs. It is well known that monocytes are potentially large sources of proinflammatory cytokines such as TNF-{alpha}. Addition of TNF-{alpha} to IL-1β-stimulated VSMCs enhanced nitrite accumulation, but TNF-{alpha} showed no effect in the presence of monocytes (Fig. 6). Coculture of monocytes and VSMCs in the presence of IL-1β induced substantial amounts of TNF-{alpha} secretion (Fig. 7), although it remains obscure which cell type is the major source of TNF-{alpha}. We therefore speculated that the production of nitrite induced by the coculture was mediated at least partially by TNF-{alpha}, and indeed an antibody against TNF-{alpha} markedly inhibited the coculture-induced nitrite production (Fig. 8).

Previously, we and others showed that monocyte–endothelial cell interaction induces the expression of cytokines [17–19], adhesion molecules [20]or factors for coagulation and fibrinolysis [21–23]. On the other hand, the cell-to-cell interaction between monocytes and VSMCs has not been extensively investigated, although Lee et al. [24]recently reported that monocytes induced matrix metalloproteinase secretion by VSMCs through an IL-1-dependent mechanism. In this study, we showed that cell-to-cell interaction between monocytes and VSMCs induced TNF-{alpha} production followed by enhanced NO generation. Such in vitro phenomena could also occur in vivo, and indeed in atherosclerotic lesions in humans, TNF-{alpha} has been shown to be present in monocytes/macrophages associated with the progression of atherosclerosis [25].

Inducible NO synthase activity is induced in blood vessel walls and cultured VSMCs by cytokines [5]. Joly et al. [26]demonstrated that balloon injury induced NO synthase activity in rat carotid arteries, even in the absence of endothelium. Hansson et al. [6]detected inducible NO synthase gene expression in neointimal but not medial smooth muscle cells 1 to 14 days after balloon-induced rat carotid artery injury. Recently, Buttery et al. [11]also reported that inducible NO synthase mRNA and protein were present within human arteriosclerotic lesions. NO production by VSMCs may in part compensate for the absence of endothelial NO synthesis and prevent the development of atherosclerotic lesions by inhibiting VSMC proliferation, leukocyte adhesion, and thrombus formation [27–29]. This hypothesis is supported by the observation that, in animals, L-arginine attenuates neointimal formation after balloon injury [30].

In conclusion, the present study revealed that cell-to-cell interaction between monocytes and VSMCs modulates NO production, suggesting an important role for their interaction in the pathogenesis of atherosclerosis.

Time for primary review 34 days.


   Acknowledgements
 
This work was supported by grants from the Ministry of Education, Science, Sports and Culture and the Mitsukoshi Grant-in-Aid 1996 in Japan. We thank Toshiko Kanbe for her technical assistance.


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

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