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Circulation. 1996;93:1185-1193

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(Circulation. 1996;93:1185-1193.)
© 1996 American Heart Association, Inc.

Articles

Human Monocyte–Endothelial Cell Interaction Induces Synthesis of Granulocyte-Macrophage Colony-Stimulating Factor

Masafumi Takahashi, MD; Seiichi Kitagawa, MD; Jun-Ichi Masuyama, MD; Uichi Ikeda, MD; Tadashi Kasahara, PhD; Yu-Ichi Takahashi, MD; Yusuke Furukawa, MD; Shogo Kano, MD; Kazuyuki Shimada, MD

From the Department of Cardiology (M.T., U.I., K.S.), Division of Hemopoiesis (S. Kitagawa, Y.F.), and Departments of Clinical Immunology (J.M., S. Kano) and Medical Biology and Parasitology (T.K.), Jichi Medical School, Tochigi, and the Department of Clinical and Laboratory Medicine (Y.T.), Tohoku University School of Medicine, Miyagi, Japan.

Correspondence to Seiichi Kitagawa, MD, Division of Hemopoiesis, Institute of Hematology and Department of Medicine, Jichi Medical School, Minamikawachi-machi, Tochigi 329-04, Japan.


*    Abstract
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 *Abstract
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down arrowResults
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Background Adhesion of monocytes to the endothelium is an initial step in the early stages of atherosclerosis and inflammation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates a range of functional activities of monocytes, including regulation of monocyte adhesion and induction of cytokine production. We investigated in this study whether GM-CSF synthesis was induced by the direct cell-to-cell interaction between human monocytes and human umbilical vein endothelial cells (ECs).

Methods and Results The expressions of GM-CSF mRNA and protein were analyzed by Northern blotting and ELISA, respectively. Coculture of monocytes and ECs induced the high levels of GM-CSF mRNA expression, whereas culture of ECs or monocytes alone or coculture of neutrophils with ECs induced no GM-CSF mRNA expression. A large amount of GM-CSF was secreted into the supernatant upon coculture of monocytes with ECs. The supernatant from the coculture markedly stimulated O2- release in neutrophils, and this effect was significantly inhibited by anti–GM-CSF antibody (Ab). Immunohistochemistry and in situ hybridization revealed that GM-CSF protein and mRNA were clearly detectable in both ECs and monocytes adhered to ECs but not in nonadherent monocytes. The GM-CSF production by the coculture was markedly inhibited by genistein and partially inhibited by Abs against interleukin-1 and tumor necrosis factor-{alpha}.

Conclusions The present results indicate that GM-CSF is produced by direct interaction between monocytes and ECs and suggest that GM-CSF produced locally by monocyte-EC adhesive interaction plays an important role in the pathogenesis of atherosclerosis and inflammation by modulating monocyte/macrophage functions in vivo.


Key Words: endothelium • glycoproteins • leukocytes • atherosclerosis • immunohistochemistry


*    Introduction
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 *Introduction
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down arrowResults
down arrowDiscussion
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The infiltration of monocytes into the vessel wall is an early crucial event in the development of atherosclerosis and inflammation.1 2 3 4 The first step in monocyte infiltration into the subendothelial space is the adhesion of circulating monocytes to the endothelium. Recent studies have shown that adhesion of monocytes to the vascular endothelium is mediated by the interaction of adhesion molecules expressed on the surface of both monocytes and ECs5 6 and may play an important role in several biological functions, such as cytokine and gene induction.7 8 9 For example, adhesion of monocytes to ECs induces the expression of IL-1 and TNF gene transcripts.

GM-CSF is a glycoprotein that regulates the proliferation and differentiation of myeloid progenitor cells.10 11 12 Recent evidence indicates that in addition to its growth-promoting effects, GM-CSF stimulates a range of functional activities of mature neutrophils, monocytes, and eosinophils, including regulation of leukocyte adhesion,13 14 augmentation of surface antigen expression,15 superoxide anion (O2-) generation,16 17 and enhancement or induction of cytokine production.18 19 Thus, it is possible that GM-CSF may contribute to the pathophysiological events involved in atherosclerosis and inflammation. GM-CSF is expressed and produced by a variety of cell types in vitro, including ECs, monocytes, T cells, and fibroblasts, in response to IL-1, TNF, and lipopolysaccharide.10 11 12 20 21 22 23 However, little is known about whether direct cell-to-cell interaction is able to induce the synthesis of GM-CSF. In the present study, we demonstrate that coculture of human monocytes with ECs induces GM-CSF synthesis in both types of cell and that adhesive cell-to-cell interaction may be important for the induction of GM-CSF synthesis.


*    Methods
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 up arrowIntroduction
 *Methods
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Reagents
Purified rh-GM-CSF and polyclonal sheep anti–human GM-CSF Ab were provided by Schering-Plough Co, Ltd. Human GM-CSF cDNA was provided by Dr Y. Itoh (Tsukuba Research Institute, Sandoz Pharmaceuticals Ltd). rh-IL-1ß and TNF-{alpha} were gifts from Otsuka Pharmaceutical Co, Ltd. A solution of type I collagen, extracted from porcine skin (Cellmatrix I-A), was purchased from Nitta Gelatin Co. FCS and EC growth supplement were purchased from Cell Culture Laboratories and Collaborative Research, respectively. BSA, HEPES, gelatin, EDTA, collagenase (type I-A), 3,3'-diaminobenzidine tetrahydrochloride, cytochrome c type III, superoxide dismutase, verapamil, actinomycin D, and cycloheximide were obtained from Sigma Chemical Co. Porcine heparin was purchased from Nakarai Chemical Co, and M-199 was obtained from Gibco. Genistein was obtained from Wako Pure Chemical Industries, Ltd. UCN-01 and calphostin C were provided by Kyowa Hakko Kogyo. Biotinylated rabbit anti-sheep IgG and streptavidin reagent (Histofin) were purchased from Dako Japan Co, Ltd and Nichirei Co, respectively. Polyclonal Abs against IL-1{alpha} (R38.3G) and IL-1ß were developed and purified as described previously.24 MAb against TNF-{alpha} (14E3) was a gift from Dainippon Pharmaceutical Co. MAbs against L-selectin (Dreg 56), CD18 (BL5), and ICAM-1 (84H10) were purchased from Immunotech.25 26 27 MAbs against VLA-4 (CD49d; P4G9) and VCAM-1 (4B9) were purchased from Telios and Genzyme, respectively.28 29

Human EC Culture
Primary human umbilical cord–derived ECs were harvested from human umbilical cord veins treated with 0.1% collagenase as described elsewhere30 and grown on 5% gelatin–precoated 60-mm culture dishes (Nunclon) in M-199 containing 20% heat-inactivated FCS, 1% penicillin/streptomycin solution, glutamine (2 mmol/L), HEPES (15 mmol/L), heparin (100 µg/mL), and EC growth supplement (60 µg/mL) (EC medium). Cells between passages 2 and 4 were used.

Isolation of Human Monocytes and Neutrophils
Neutrophils and mononuclear cells were prepared from heparinized venous blood of healthy adult donors as described previously16 17 by use of dextran sedimentation, centrifugation with Ficoll-Conray, and hypotonic lysis of contaminated erythrocytes. Monocytes were further purified from mononuclear cells by centrifugal elution with a Hitachi SRR6Y elution rotor).17 Neutrophil fractions contained more than 95% neutrophils. Mononuclear cell fractions contained 15% to 25% monocytes, 75% to 85% lymphocytes, and <1% neutrophils. Monocyte fractions contained >85% monocytes as determined by Wright-Giemsa staining of cytospun preparations. All the fractions were resuspended in M-199 supplemented with 0.1% BSA, 1% penicillin/streptomycin solution, and 2 mmol/L glutamine (assay medium).

Northern Blot Analysis
Total RNA was prepared by the guanidine isothiocyanate–cesium chloride method. Equal amounts of total RNA (10 to 15 µg) were size-fractionated by electrophoresis on denaturing 1.0% agarose/formaldehyde gels and transferred to nylon membranes (Hybond N+, Amersham). Hybridizations were performed with an excess of [32P]dCTP-labeled human GM-CSF cDNA probe (specific activity, >1x108 cpm/µg DNA) at 65°C for 24 hours. The GM-CSF probe consisted of a 0.4-kb Not I/Pst I restriction fragment.31 At the end of hybridization, the filters were washed twice in 0.2x SSC at 60°C (1x SSC contains 0.15 mol/L NaCl, 0.015 mol/L sodium citrate, pH 7.0), then exposed to Kodak XAR-5 film overnight at -70°C with one intensifying screen.

ELISA Determination of GM-CSF
GM-CSF concentrations in the conditioned medium were determined by ELISA (R&D Systems) according to the manufacturer's instructions.

Immunohistochemistry
Monocytes (1.5x105 cells) or IL-1ß (25 U/mL) was added to the confluent EC layers on coverslips in eight-well culture plates (Lab-Tek, chamber slide, Nunc). After incubation at 37°C for 8 hours, the ECs were rinsed with PBS and fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature. Before staining, the slides were again fixed for 20 minutes in 0.3% H2O2 in methanol and rinsed in 0.1% Triton X-100/PBS, and nonspecific binding sites were blocked with 10% normal rabbit serum. The slides were rinsed in 0.1% Triton X-100/PBS followed by the addition of anti–GM-CSF Ab (20 ng/mL). After incubation at 4°C for 12 hours, the slides were rinsed again in PBS, overlaid with biotinylated rabbit anti-sheep IgG, incubated for 60 minutes at 37°C, and rinsed in PBS. The slides were treated with streptavidin reagent (Histofin) for 30 minutes at room temperature, rinsed in PBS, overlaid with a solution of 0.05% 3,3'-diaminobenzidine tetrahydrochloride in 0.05 mol/L Tris-HCl buffer (pH 7.6) and 0.01% H2O2 for 5 minutes at room temperature to allow color development, then rinsed with distilled water. Mayer's hematoxylin was used as a counterstain. Nonadherent monocytes were also collected by cytospin and were processed for immunohistochemistry as described above.

In Situ Hybridization Analysis
In situ hybridization was performed as previously described.32 Briefly, monocytes were added to the EC layers on coverslips (Lab-Tek, chamber slide, Nunc). After incubation at 37°C for 4 hours, the cultured cells were rinsed in PBS, fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature, and stored at -80°C until hybridization. Sense and antisense GM-CSF RNA probes were labeled with digoxigenin-UTP by linearization of the cDNA with the appropriate restriction enzyme from pCR-Script SK(+) vector (Stratagene) according to the manufacturer's instructions (Boehringer Mannheim). Cells were hybridized overnight at 50°C in a humidified chamber. After hybridization, slides were rinsed, treated with RNase, and reacted with alkaline phosphatase–conjugated anti-digoxigenin Abs (Boehringer Mannheim). After rinsing, colorimetric reactions were performed.

Determination of O2- Release
O2- release was determined by the end-point assay using polypropylene tubes (Falcon, No. 2063) as described previously.16 33 The reaction mixture contained 110 µmol/L ferricytochrome c, the desired concentration of stimulus, and 6.0x105 neutrophils in 0.4 mL HBSS supplemented with 0.1% BSA, with or without 200 U/mL superoxide dismutase. After incubation for 3 hours at 37°C in a shaking water bath, O2- release was determined as superoxide dismutase–inhibitable reduction of ferricytochrome c.

Statistical Analysis
All values are expressed as mean±SD. In comparisons of two groups, probability values were calculated by Student's t test. In experiments involving comparisons of multiple groups, the probability that differences existed between the means of the groups was determined by ANOVA using the least significant difference for multiple comparisons. Differences at P<.05 were considered to be statistically significant.


*    Results
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 up arrowMethods
*Results
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Induction of GM-CSF mRNA Expression by Coculture of Monocytes With ECs
Neither ECs nor monocytes expressed detectable levels of GM-CSF mRNA transcripts when cultured alone (Fig 1ADown). However, coculture of monocytes (1.0x107 cells) with ECs (2.0x106 cells) for 4 hours induced the expression of high levels of GM-CSF mRNA transcripts. Similarly, good induction of GM-CSF mRNA transcripts occurred in ECs on exposure to IL-1ß (25 U/mL) for 4 hours. In contrast, no induction of GM-CSF mRNA transcripts was detected upon coculture of neutrophils (1.0x107 cells) with ECs. As shown in Fig 1BDown, actinomycin D (5 µg/mL) completely inhibited the expression of GM-CSF mRNA induced by coculture of monocytes with ECs, whereas cycloheximide (10 µg/mL) caused a superinduction of GM-CSF mRNA transcripts.



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  		Figure 1. Induction of GM-CSF mRNA expression by coculture of monocytes and ECs. ECs (2.0x106 cells) were untreated or pretreated with IL-1ß (25 U/mL) for 4 hours, washed, and then cocultured in assay medium with or without monocytes (1.0x107 cells) for 4 hours. A, Lane 1, unstimulated ECs; lane 2, monocytes alone; lane 3, coculture of monocytes with unstimulated ECs; lane 4, IL-1ß–stimulated ECs; lane 5, coculture of monocytes with IL-1ß–prestimulated ECs; lane 6, coculture of neutrophils with unstimulated ECs. B, Lane 1, unstimulated ECs; lane 2, coculture of monocytes with unstimulated ECs; lane 3, coculture in the presence of actinomycin D (5 µg/mL); lane 4, coculture in the presence of cycloheximide (10 µg/mL).

The time course of induction of GM-CSF mRNA transcripts upon coculture of monocytes with ECs is shown in Fig 2Down. GM-CSF mRNA expression as a result of coculture was detectable at 2 hours of incubation, reached a maximal level at 4 hours, and declined by 24 hours. This rapid induction of GM-CSF mRNA transcripts and the complete inhibition of the mRNA induction by actinomycin D suggest that GM-CSF protein is produced by de novo synthesis.



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  		Figure 2. Time course of induction of GM-CSF mRNA expression upon coculture of monocytes with ECs. Unstimulated ECs (2.0x106 cells) were cocultured with monocytes (1.0x107 cells) for the indicated periods (lane 1, 0 hours; lane 2, 2 hours; lane 3, 4 hours; lane 4, 24 hours).

Production of GM-CSF by Coculture of Monocytes With ECs
We next examined the induction of GM-CSF synthesis by coculture of monocytes with ECs at the protein level. Coculture of monocytes (8.0x105 cells) with ECs (1.6x105 cells) induced the secretion of a large amount of GM-CSF into the culture supernatant in a time-dependent manner (Fig 3ADown). In contrast, no secretion of GM-CSF was detected in the culture supernatants from ECs alone or cocultures of neutrophils (8.0x105 cells) with ECs. A lesser amount of GM-CSF was also secreted into the culture supernatant from monocytes adherent to a plastic surface and monocytes treated with IL-1ß (25 U/mL). It has been reported that ECs treated with IL-1 produce GM-CSF.20 21 22 23 In agreement with this, ECs pretreated with IL-1ß (25 U/mL) for 4 hours secreted a significant amount of GM-CSF in our assay system (Fig 3BDown). On the other hand, coculture of monocytes with IL-1ß–pretreated ECs resulted in the marked secretion of GM-CSF, and this secretion was also time dependent.



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  		Figure 3. Production of GM-CSF upon coculture of monocytes (Mo) with ECs. ECs (1.6x105 cells) were untreated (A) or pretreated (B) with IL-1ß (25 U/mL) for 4 hours, washed, and then cocultured with or without monocytes (8.0x105 cells) or neutrophils (8.0x105 cells) for the indicated periods. Values represent mean±SD of four independent experiments, each done in duplicate ({circ}, ECs alone; {square}, monocytes alone; {bullet}, coculture of monocytes with ECs; {triangleup}, coculture of neutrophils with ECs).

The above findings suggest that adhesion between monocytes and ECs induces GM-CSF production. To further determine whether GM-CSF production by the coculture is mediated by direct contact between monocytes and ECs, we performed a separate coculture assay using an inner well system (Cell Culture Insert). As shown in the TableDown, GM-CSF secretion into the wells induced by coculture of monocytes with ECs was substantially greater than that induced by separate coculture of monocytes with ECs, also supporting the assumption that direct adhesion between monocytes and ECs is necessary for GM-CSF production.


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  		Table 1. GM-CSF Production Upon Separate-Culture and Coculture Systems

Fig 4Down shows GM-CSF secretion upon coculture of monocytes with ECs, in which different numbers of monocytes were added to a constant number of ECs. Significant secretion of GM-CSF was detected at a monocyte-to-EC numerical ratio of 0.5. The secretion of GM-CSF was further increased by increasing the number of added monocytes up to a ratio of 5.0.



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  		Figure 4. Effects of increasing number of monocytes on GM-CSF production upon coculture of monocytes with ECs. Different numbers of monocytes (0.016x105 to 1.6x105 cells) were added to a constant number of ECs (1.6x105 cells); the numerical ratios of monocytes to ECs were 0, 0.05, 0.5, 1.0, 2.5, and 5.0. After coculture for 5 hours, GM-CSF concentration in the supernatants was measured by an ELISA method. Values represent mean±SD of four independent experiments, each done in duplicate. *P<.001.

Effects of Supernatants From Cocultures of Monocytes and ECs on O2- Release by Neutrophils
We previously reported that GM-CSF directly stimulates the release of O2- from suspended human neutrophils.16 To test whether the coculture supernatants have GM-CSF biological activity, we examined the effects of the supernatants on O2- release by neutrophils. As shown in Fig 5Down, the supernatant from the coculture of monocytes with ECs for 5 hours markedly stimulated O2- release by neutrophils, and this effect was partially but significantly inhibited by neutralizing Ab against GM-CSF (P<.01). rh-GM-CSF (5 ng/mL) and TNF-{alpha} (100 U/mL) also stimulated the O2- release. The release of O2- stimulated by rh-GM-CSF (5 ng/mL) was almost completely inhibited by anti–GM-CSF Ab, whereas that stimulated by TNF-{alpha} (100 U/mL) was not affected, indicating the specific neutralizing action of anti–GM-CSF Ab. It is likely that GM-CSF in the supernatant is almost completely neutralized by anti–GM-CSF Ab at the concentration used, because the GM-CSF concentration in the supernatant from the coculture incubated for 5 hours was 200 to 300 pg/mL (Fig 3Up). Since IL-1 and TNF-{alpha} are known to stimulate O2- release from neutrophils, it is suggested that the supernatants may contain other stimulants, such as IL-1 and TNF-{alpha}. IL-8 produced by activated ECs and monocytes may also contribute to O2- generation by neutrophils.34 35 36



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  		Figure 5. Effects of supernatants from coculture of monocytes with ECs, rh-GM-CSF, and TNF-{alpha} on O2- release by neutrophils. Neutrophils (6x105 cells/0.4 mL) were incubated with the supernatants (75%), rh-GM-CSF (5 ng/mL), or TNF-{alpha} (100 U/mL) in the presence (open bar) or absence (solid bar) of anti–GM-CSF Ab (550 µg/mL). After 3 hours of incubation, the O2- release was measured as described in "Methods." Values represent mean±SD of three independent experiments, each done in triplicate. *P<.01.

Immunohistochemical Staining and In Situ Hybridization of GM-CSF–Producing Cells
To verify the cells producing GM-CSF in the coculture system, we performed immunohistochemical staining. As shown in Fig 6Down, GM-CSF protein was not immunolocalized in unstimulated ECs, whereas it was clearly detected in ECs prestimulated with IL-1ß (25 U/mL) for 8 hours (Fig 6ADown and 6BDown). Both ECs and monocytes adhering to the ECs for 8 hours of the coculture were strongly stained with anti–GM-CSF Ab (Fig 6CDown) but not with control sheep IgG (Fig 6EDown). Interestingly, nonadherent monocytes that did not attach to the ECs and were collected by cytospin showed no staining (Fig 6DDown).



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  		Figure 6. Immunohistochemical staining of monocytes and ECs. After ECs had been cultured for 8 hours with or without IL-1ß (25 U/mL) or monocytes, immunohistochemistry was performed with anti–GM-CSF Ab or control IgG. A, Unstimulated ECs. B, IL-1ß–stimulated ECs. C, Unstimulated ECs and adherent monocytes. D, Nonadherent monocytes. E, Unstimulated ECs and adherent monocytes (control IgG). Magnification x100.

Furthermore, as shown in Fig 7Down, in situ hybridization also clearly showed the localization of GM-CSF mRNA in both monocytes and ECs by their coculture for 4 hours. These findings indicate that both monocytes and ECs are responsible for GM-CSF synthesis and that the adhesive interaction between monocytes and ECs is required for induction of GM-CSF expression.




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  		Figure 7. In situ hybridization of monocytes and ECs. After ECs were cultured for 4 hours with monocytes, in situ hybridization was performed using sense and antisense GM-CSF RNA probes, as described in "Methods." A, Antisense probe. B, Sense probe.

Involvement of Cytokines and Adhesion Molecules in GM-CSF Production Upon Coculture of Monocytes With ECs
GM-CSF is produced in ECs exposed to proinflammatory cytokines such as IL-1{alpha}, IL-1ß, and TNF-{alpha}.20 21 22 23 To investigate the mechanisms of GM-CSF production by coculture of monocytes with ECs, we carried out Ab-blocking studies using anti–IL-1{alpha}, anti–IL-1ß, and anti–TNF-{alpha} neutralizing Abs, either separately or in combination. As shown in Fig 8Down, in comparison with the use of control Ab (anti-KLH), GM-CSF production upon coculture was inhibited by 34%, 69%, and 59% in the presence of anti–IL-1{alpha}, anti–IL-1ß, and anti–TNF-{alpha} Abs, respectively (P=NS, P<.001, and P<.01). Furthermore, when the Abs were used in combination (anti–IL-1{alpha}, anti–IL-1ß, and anti–TNF-{alpha} Abs together), GM-CSF production was significantly inhibited, by 76% (P<.001).



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  		Figure 8. Effects of Abs against proinflammatory cytokines on GM-CSF production upon coculture of monocytes with ECs. After monocytes (2.5x106 cells) and ECs (5.0x105 cells) were cocultured for 5 hours in the presence of anti-KLH peptide Ab (1:200, control Ab), anti–IL-1{alpha} Ab (50 µg/mL), anti–IL-1ß Ab (1:400), anti–TNF-{alpha} Ab (1:100), or their combination (anti–IL-1{alpha} plus anti–IL-1ß plus anti–TNF-{alpha} Abs), GM-CSF concentration in the supernatants was measured by an ELISA method. The values obtained with anti-KLH peptide Ab were taken as the 100% control, which represents 742.3±78.0 pg/mL (n=5). Values represent mean±SD of five independent experiments, each done in duplicate. *P<.01; **P<.001.

Recent findings have indicated that monocyte–EC adhesive interaction is directly mediated by adhesion molecules expressed on the surfaces of both cells.5 6 Our previous study also demonstrated that adhesion of monocytes to ECs is partially mediated by both the CD18–ICAM-1 and VLA-4–VCAM-1 pathways.37 To investigate the involvement of these pathways in GM-CSF production, we carried out Ab-blocking studies using anti–L-selectin (10 µg/mL), anti-CD18 (10 µg/mL), anti–VLA-4 (10 µg/mL), anti–ICAM-1 (10 µg/mL), and anti–VCAM-1 (10 µg/mL) MAbs, either separately or in combination. However, no significant inhibition of GM-CSF production by these MAbs was observed (Fig 9Down).



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  		Figure 9. Effects of MAbs against adhesion molecules on GM-CSF production upon coculture of monocytes with ECs. After monocytes (2.5x106 cells) and ECs (5.0x105 cells) were cocultured for 5 hours in the presence or absence (control) of mouse IgG (10 µg/mL), anti–L-selectin (10 µg/mL) MAb, anti-CD18 MAb (10 µg/mL), anti–VLA-4 MAb (10 µg/mL), anti–ICAM-1 (10 µg/mL), anti–VCAM-1 (10 µg/mL), or their combination (anti–L-selectin plus anti-CD18 plus anti–VLA-4 plus anti–ICAM-1 plus anti–VCAM-1 MAbs), GM-CSF concentration in the supernatants was measured by an ELISA method. The control values were taken as the 100% control, which represents 342.6±82.5 pg/mL (n=5). Values represent mean±SD of five independent experiments, each done in duplicate.

Effects of Various Inhibitors on GM-CSF Production Upon Coculture of Monocytes With ECs
We then investigated the effects of various inhibitors on GM-CSF production upon coculture of monocytes with ECs. As shown in Fig 10Down, genistein (10 µg/mL), actinomycin D (5 µg/mL), and cycloheximide (10 µg/mL) showed significant inhibition of GM-CSF production upon coculture (P<.001), whereas neither verapamil (10-6 mol/L), UCN-01 (20 ng/mL), nor calphostin C (10-7 mol/L) exerted any effects on the GM-CSF production. No cytotoxic effect of genistein, calphostin C, and UCN-01 on monocytes and ECs was observed after 6 hours of incubation (data not shown).



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  		Figure 10. Effects of various inhibitors on GM-CSF production upon coculture of monocytes with ECs. After monocytes (2.5x106 cells) and ECs (5.0x105 cells) were cocultured for 5 hours in the presence or absence (control) of verapamil (10-6 mol/L), UCN-01 (25 ng/mL), calphostin C (10-7 mol/L), genistein (10 µg/mL), actinomycin D (5 µg/mL), or cycloheximide (10 µg/mL), GM-CSF concentration in the supernatants was measured by an ELISA method. Values represent mean±SD of five independent experiments, each done in duplicate. *P<.001.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
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*Discussion
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Adhesion of monocytes to the endothelium and their subsequent migration from the circulation into the subendothelium are the prominent features of atherosclerosis and inflammation.1 2 3 4 The adhesion of monocytes to the endothelium of large vessels during the early stages of experimentally induced atherosclerosis has been observed in several animal model systems.38 39 In addition, immunohistochemical analyses of human necropsy and carotid endarterectomy specimens have demonstrated similar involvement of monocytes in the early stages of atherosclerosis,40 41 indicating that adhesion of monocytes to the endothelium is an initial step in the early stage of atherogenesis. Furthermore, after their migration into the subendothelium, monocytes are exposed to a milieu of cytokines, chemoattractants, growth factors, and modified lipoproteins, all of which can trigger their activation and differentiation into macrophages. The present study demonstrated that interaction between monocytes and ECs can induce marked GM-CSF synthesis in both types of cells, as evidenced by the expression of both mRNA and protein of GM-CSF and its biological activity. Since GM-CSF has biological activities not only on myelopoietic progenitor cells but also on mature monocytes,10 11 12 we speculate that GM-CSF synthesis resulting from monocyte-EC interaction may contribute to the recruitment of monocytes/macrophages and their proliferation and differentiation in the atherosclerotic lesions.

Known cellular sources of GM-CSF are monocytes, T lymphocytes, ECs, mast cells, and fibroblasts in response to cytokines, such as IL-1 and TNF-{alpha}, and other inflammatory stimuli.20 21 22 23 However, GM-CSF production through direct cell-to-cell interaction has not been reported. We demonstrate here that GM-CSF mRNA and protein were expressed in both monocytes and ECs as a result of their adhesive interaction (Figs 1Up and 3Up). Adhesive interaction is an important regulatory signal in monocytes for several biological functions, including cytokine production and gene induction. Haskill et al7 described that adhesion of monocytes to Petri dishes induces the expression of IL-1 gene transcripts and proto-oncogenes such as c-fos and c-fms. Sporn et al9 and Eierman et al8 demonstrated that adhesive interaction between monocytes and ECs results in the induction of several cytokines, such as TNF-{alpha} and colony-stimulating factor-1 (CSF-1/M-CSF). The present study showed that the expression of GM-CSF mRNA resulting from monocyte–EC adhesive interaction increased and peaked at 2 to 4 hours and had declined by 24 hours (Fig 2Up). This rapid and transient expression of GM-CSF mRNA and the inhibition of GM-CSF mRNA expression by actinomycin D (Fig 1BUp) suggest, when taken together, that the protein is produced by de novo synthesis and that the direct cell-to-cell interaction may be an important signal for GM-CSF synthesis. Indeed, we confirmed immunohistochemically that GM-CSF protein was expressed predominantly in both ECs and monocytes that adhered to them, whereas no significant expression of the protein was detected in monocytes that failed to adhere to ECs (Fig 6Up). Furthermore, in situ hybridization clearly revealed the localization of GM-CSF mRNA in both cell types (Fig 7Up), indicating that monocytes and ECs actually synthesize GM-CSF. These findings also support the idea that adhesive interaction between monocytes and ECs is an important event for induction of GM-CSF synthesis.

It has been shown that proinflammatory cytokines such as IL-1 and TNF-{alpha} stimulate GM-CSF production20 21 22 23 and that IL-1 and TNF-{alpha} are produced by adhesive interaction of monocytes.7 8 9 We also found that substantial amounts of IL-1{alpha}, IL-1ß, and TNF-{alpha} were produced upon coculture of monocytes with ECs (unpublished results). Our blocking studies using neutralizing antibodies against IL-1ß and TNF-{alpha} revealed a significant inhibition of GM-CSF production, indicating that these cytokines also play an important role in GM-CSF production. However, it should be noted that GM-CSF protein was not detectable in nonadherent monocytes immunohistochemically, indicating that these cytokines, although present in the culture supernatant, are insufficient to induce the expression of GM-CSF protein in nonadherent monocytes in the present experimental system. In considering that cell adhesion by itself could activate or prime the cells, it seems likely that these cytokines in concert with adhesive interaction may augment the production of GM-CSF in adherent monocytes and ECs. Massive production of GM-CSF by coculture of monocytes with IL-1ß–pretreated ECs may be partly explained by augmented adhesion of monocytes to IL-1ß–pretreated ECs compared with unstimulated ECs.37

Although the molecular mechanisms underlying adhesive interaction between monocytes and ECs are unclear, recent studies have shown that the direct adhesive interaction of cells would be regulated by various adhesion molecules on surfaces of both interacting cells. We previously demonstrated that adhesive interaction between monocytes and ECs is partially mediated by both the CD18 (common ß-chain of lymphocyte function–associated antigen-1, Mac-1, and p150, 95)–ICAM-1 and VLA-4–VCAM-1 pathways.37 Actually, when Abs were used in the combination of anti–L-selectin, anti-CD18, anti–VLA-4, anti–ICAM-1, and anti–VCAM-1 MAbs, {approx}30% inhibition of monocyte adhesion was observed (data not shown). However, MAbs against these molecules failed to inhibit the GM-CSF production resulting from monocyte-EC interaction (Fig 9Up). Although the reason for this discrepancy is unknown, partial inhibition of monocyte adhesion might not be enough to affect GM-CSF production. Another possibility is that intact MAbs used here might interact with Fc receptors of IgG on monocytes and stimulate GM-CSF gene induction, which could counteract the inhibitory effect of MAbs against adhesion molecules on GM-CSF production. We also performed a separate coculture assay and confirmed that direct adhesion between monocytes and ECs is necessary for GM-CSF production (TableUp). Although neutrophils can also interact with ECs through adhesion molecules such as CD18 and ICAM-1,5 6 neutrophil-EC interaction induced no production of GM-CSF, suggesting that the GM-CSF production resulting from monocyte-EC adhesive interaction might be mediated by other, as yet unknown, adhesion molecules.4 42 In this regard, a novel monocyte-EC adhesion molecule recognized by MAb IG9 was recently reported by Calderon et al.43 This molecule is expressed on the cell surface of ECs treated with IL-1, TNF-{alpha}, and minimally modified LDL.

Studies using inhibitors have shown that GM-CSF production was markedly inhibited by genistein, a potent inhibitor of tyrosine kinase,44 but was not affected by UCN-01 and calphostin C, potent inhibitors of protein kinase C45 46 (Fig 10Up). These findings suggest that tyrosine kinase but not protein kinase C is involved in the induction of GM-CSF synthesis by monocyte-EC interaction. Further investigations are required to elucidate the adhesion molecules involved in the induction of GM-CSF synthesis through monocyte-EC interaction and the signaling pathways involving these molecules.

Adhesion of monocytes to the endothelium is an initial step in the early stages of atherosclerosis and inflammation. The present results indicate that GM-CSF is produced by direct interaction between monocytes and ECs and suggest that GM-CSF produced locally by monocyte-EC adhesive interaction plays an important role in the pathogenesis of atherosclerosis and inflammation by modulating monocyte/macrophage functions in vivo.


*    Selected Abbreviations and Acronyms
 
Ab = antibody
EC = endothelial cell
GM-CSF = granulocyte-macrophage colony-stimulating factor
ICAM-1 = intercellular adhesion molecule-1
IL-1 = interleukin-1
KLH = keyhole limpet hemocyanin
M-199 = medium 199
MAb = monoclonal Ab
rh = recombinant human
TNF = tumor necrosis factor
UCN-01 = 7-hydroxystaurosporine
VCAM-1 = vascular cell adhesion molecule-1
VLA-4 = very late antigen-4


*    Acknowledgments
 
This work was supported by grants-in-aid 5671632 and 6671106 from the Ministry of Education, Science, and Culture, Japan, and the Ichiro Kanehara Foundation. We thank Toshiko Kambe, Mamiko Semba, and Taeko Inageta for their technical assistance.

Received June 9, 1995; revision received October 16, 1995; accepted October 18, 1995.


*    References
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*References
 

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