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(Circulation Research. 1995;76:750-757.)
© 1995 American Heart Association, Inc.

Articles

Induction of Monocyte Chemoattractant Protein-1 Synthesis in Human Monocytes During Transendothelial Migration In Vitro

Masafumi Takahashi, Jun-Ichi Masuyama, Uichi Ikeda, Tadashi Kasahara, Sei-Ichi Kitagawa, Yu-Ichi Takahashi, Kazuyuki Shimada, Shogo Kano

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

Correspondence to Jun-Ichi Masuyama, MD, Department of Clinical Immunology, Jichi Medical School, Minamikawachi-machi, Tochigi 329-04, Japan.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Monocyte chemoattractant protein-1 (MCP-1, or monocyte chemotactic and activating factor) plays important roles in the recruitment of monocytes and thus in the development of atherosclerosis. In this study, we determined whether MCP-1 synthesis was induced by the cellular interaction between monocytes and endothelial cells during the process of transendothelial migration. We found that when human peripheral blood monocytes (2.5x10⁶ cells) and umbilical vein endothelial cells (HUVECs; 5.0x10⁵ cells) were cocultured for 5 hours, 7.9 ng/mL MCP-1 was secreted into the medium, whereas when the two were cultured separately, MCP-1 levels were 1.0 and 0.9 ng/mL, respectively. Furthermore, the use of interleukin-1ß (IL-1ß)–pretreated HUVECs in cocultures induced twice the levels of MCP-1 as in unstimulated HUVEC culture. Conditioned medium had transendothelial chemotactic activity for monocytes, and this activity was completely abolished by addition of anti–MCP-1 antibody. Although MCP-1 mRNA levels were very low or undetectable in HUVECs or monocytes alone, message could be detected after 2 hours of coculture in total mRNA preparations from both monocytes and HUVECs. mRNA levels increased by 4 hours and had declined slightly by 24 hours. The rapid induction of message suggests that cell contact between monocytes and HUVECs induces the de novo synthesis of MCP-1 protein. Anti–interleukin (IL)-1/ß and anti–tumor necrosis factor- antibodies, or anti–lymphocyte function–associated antigen-1 and very late antigen-4 antibodies, had little or no inhibitory effects on MCP-1 secretion by cocultures. Immunohistochemistry revealed that monocytes adherent to or having migrated across unstimulated HUVEC monolayers as well as the HUVECs themselves expressed MCP-1 protein. However, nonadherent monocytes failed to express it. This finding suggests that the monocyte–endothelial cell adhesive interaction results in an MCP-1–inductive signal to each cell type. MCP-1 expression by migrated monocytes may indicate that monocytes are primed to produce MCP-1 during transmigration and can secrete it in normal tissue in which inflammatory cytokines that induce MCP-1 are otherwise absent.

Key Words: atherosclerosis • MCP-1 • monocyte • endothelium • migration

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Monocyte infiltration from the circulation into the extravascular tissue is a characteristic feature of atherosclerosis.^{1 2 3 4 5} Although the mechanisms of monocyte infiltration are not fully understood, it involves the adhesive interaction of blood monocytes with the vascular endothelium and subsequent migration into the subendothelial space. In this regard, it is generally believed that chemoattractants generated in the subendothelial space play an important role in monocyte infiltration.^{6 7 8} Among several cytokines and other factors with chemotactic activity for monocytes, monocyte chemoattractant protein-1 (MCP-1, or monocyte chemotactic and activating factor) may be especially important in the pathogenesis of atherosclerosis.^{9 10 11 12 13} In support of this notion, Ylä-Herttuala et al⁹ demonstrated MCP-1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Takeya et al¹⁰ observed MCP-1 expression in endothelial cells and subendothelial macrophages of human atherosclerotic lesions. Nelken et al¹¹ also reported the expression of MCP-1 mRNA in human atheromatous plaques.

Recently, cytokines with chemotactic activity have been termed chemokines and included in a superfamily based on the homology of their cDNA sequences.^{14 15} The chemokine family is divided into - and ß-subfamilies by the difference of repeat amino acid motifs, the former containing Cys-X-Cys and the latter Cys-Cys sequences. MCP-1 is a relatively specific chemoattractant for monocytes classified in the ß(C-C) subfamily. This chemokine can be produced by a wide variety of cell types, such as monocytes,^{16 17} vascular endothelial cells,¹⁸ smooth muscle cells,¹⁹ fibroblasts,²⁰ cardiac myocytes,²¹ hepatic cells,²² synovial cells,²³ and certain tumor cell lines.²⁴ Stimuli for MCP-1 production include minimally modified low-density lipoprotein (mmLDL)²⁵ and cytokines such as interleukin-1 (IL-1), tumor necrosis factor (TNF), interferon gamma,¹⁸ and macrophage–colony stimulating factor.²⁶ Recently, it has been reported that unstimulated human monocytes express MCP-1 mRNA in relation to their cellular density but not to their adherence to a substrate.²⁷

Direct cell-cell interaction may induce MCP-1 synthesis. For example, it has been reported that endothelial cells act as a costimulator to augment IL-2 synthesis by phytohemagglutinin-activated CD4⁺ T cells.²⁸ The costimulatory activities are dependent on contact between endothelial cells and the responding T cells, partially mediated by the specific interaction of lymphocyte function–associated antigen-3 (LFA-3) on endothelial cells with CD2 on the T cells. The adhesive interaction of blood monocytes with vascular endothelial cells occurs continually during monocyte recruitment. It is thus possible that both are stimulated to secrete MCP-1 during transendothelial migration of monocytes. If MCP-1 is produced by monocytes that have migrated into normal vascular tissue in which inflammatory cytokines are absent, it would promote further extravasation of monocytes into the tissue and thus play an important role in early atherogenesis. In this study, we determined whether the cellular interaction of monocytes with endothelial cells induces both MCP-1 protein and mRNA in these cells and examined the expression of MCP-1 protein by migrated monocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
MCP-1 Preparation
Purified recombinant human MCP-1 (rhMCP-1) was produced in X63-Ag8-653 myeloma cells transfected with BCMGSneo (kindly provided by H. Karasuyama, Basel Institute) harboring a 400-bp Pst I fragment of human MCP-1 cDNA.²⁹ MCP-1 was purified to homogeneity from the serum-free culture supernatants by concentration on an Amicon YM-5 membrane (Grace Japan), passage through a heparin-Sepharose column, and carboxymethyl high-performance liquid chromatography (HPLC) and reversed-phase HPLC as described elsewhere.³⁰ Purified MCP-1 showed a major band with a molecular weight of 8000 kD on tricinesodium dodecyl sulfate–polyacrylamide gel electrophoresis and on the Western blot (immunoblot) analysis using rabbit anti–human MCP-1 antibody (Ab).³¹ The MCP-1 preparation had monocyte-chemotactic activity at 1 ng/mL and induced Ca²⁺ influx at 1 to 10 ng/mL on human peripheral blood monocytes. The endotoxin content of the MCP-1 preparation was <0.2 ng/mL of protein as determined by a Toxicolor kit (Seikagaku Kougyou).

Reagents
Purified recombinant human IL-8 was prepared as described previously.³¹ Recombinant human IL-1ß was obtained from Genzyme Corp. Type I collagen, extracted from porcine skin, in solution (Cellmatrix I-A) was purchased from Nitta Gelatin Co. Fetal calf serum and endothelial cell growth supplement (ECGS) were purchased from Cell Culture Laboratories and Collaborative Research, respectively. Bovine serum albumin, HEPES, gelatin, EDTA, collagenase (type I-A), and 3,3'-diaminobenzidine tetrahydrochloride were obtained from Sigma Chemical Co. Porcine heparin was purchased from Nakarai Chemical Co, and medium 199 (M-199) was obtained from Gibco. Biotinylated rabbit anti-mouse Ig and streptavidin reagent (Histofin) were purchased from Nichirei Co. Polyclonal Abs against MCP-1, IL-8, IL-1 (R38.3G), and IL-1ß were developed and purified as described previously.^{31 32 33} Monoclonal Abs (mAbs) against TNF (14E3) and HLA-ABC were gifts from Dainippon Pharmaceutical Co and Dr C. Morimoto (Dana Farber Cancer Institute), respectively. mAbs against CD18 (BL5) and intercellular adhesion molecule-1 (ICAM-1; 84H10) were purchased from Immunotech.^{34 35} mAbs against very late antigen-4 (VLA-4; CD49d, P4G9) and vascular cell adhesion molecule-1 (VCAM-1; 4B9) were purchased from Telios and Genzyme, respectively.^{36 37}

Human Endothelial Cell Cultures
Primary human umbilical cord–derived endothelial cells (HUVECs) were harvested from human umbilical cord veins treated with 0.1% collagenase as described elsewhere³⁸ and grown on 5% gelatin–precoated 60-mm culture dishes (Nunclon) in M-199 containing 20% heat-inactivated fetal calf serum, 1% penicillin/streptomycin solution, glutamine (2 mmol/L), HEPES (15 mmol/L), heparin (100 µg/mL), and ECGS (60 µg/mL) (EC medium). Cells between passages 2 and 4 were used.

Isolation of Human Monocytes
Mononuclear cells were prepared from heparinized venous blood of healthy adult donors by Ficoll-Conray density gradient centrifugation, and monocytes were purified from these cells by centrifugal elutriation with a Hitachi SRR6Y elutriation rotor (Hitachi Ltd) as described previously.³⁹ The monocyte fractions contained 85% to 95% monocytes but only 5% to 15% lymphocytes as determined by Giemsa staining on cytospun preparations. All the fractions were resuspended in M-199 supplemented with 0.1% bovine serum albumin, 1% penicillin/streptomycin solution, and 2 mmol/L glutamine (assay medium).

Radioimmunoassay for MCP-1
Competitive radioimmunoassay (RIA) for the quantification of MCP-1 was developed by use of ¹²⁵I-labeled rhMCP-1 and polyclonal rabbit anti-human MCP-1 Ab.³¹ Briefly, rhMCP-1 was radioiodinated with Bolton-Hunter reagent (Amersham-Japan). Each 50 µL of ¹²⁵I-MCP-1 (2x10⁴ cpm), sample, and antiserum (1:3200) were mixed and incubated at 4°C overnight. Then magnetic goat anti-rabbit IgG (Advanced Magnetics Inc) was added; the mixture was further incubated for 2 hours at 4°C and spun down, and the radioactivity in the pellet was counted. The intra-assay variation (coefficient of variation) of this MCP-1 RIA was <5%. This MCP-1 RIA demonstrated a linear detection of specific MCP-1 at concentrations >0.1 ng/mL and did not cross-react with MCP-2, RANTES (regulated on activation, normal T expressed and secreted), IL-8, macrophage inflammatory protein-1 (MIP-1), GRO-, IL-1, IL-1ß, IL-6, TNF, or platelet-activating factor.

Northern Blot Analysis
Total RNA was prepared by the guanidine isothiocyanate–cesium chloride (GITC-CsCl) 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 at 65°C for 24 hours with an excess of dCTP-labeled human MCP-1 cDNA probe (specific activity, >1x10⁸ cpm/µg DNA) at 60°C for 24 hours. The MCP-1 probe consisted of a 0.4-kb Pst I restriction fragment. At the end of hybridization, the filters were washed twice in 0.2xstandard saline citrate (SSC) at 60°C (1xSSC contains 0.15 mol/L NaCl, 0.015 mol/L sodium citrate, pH 7.0). Filters were exposed to Kodak XAR-5 film overnight at -70°C with one intensifying screen.

Monocyte Transendothelial Migration Assay
Transendothelial chemotactic activity of samples for monocytes were examined with inner wells (Cell Culture Insert, catalogue No. 3095, Falcon) to divide each well of 24-well multiwell plates (Falcon) into two compartments. For microscopic evaluation of the monocyte transendothelial migration, a thin layer of collagen gel (100 µL per well), prepared as described previously,^{40 41} was placed on the filters of inner wells. The wells were seeded with HUVECs at 6x10⁴ cells per inner well in 300 µL of EC medium and placed in wells of 24-well multiwell plates containing 500 µL of EC medium without ECGS. The HUVEC monolayers were grown to confluence and further cultured for at least 48 hours at 37°C in a humidified incubator under 95% air/5% CO₂. The inner wells were washed and then transferred to new 24-well multiwell plates. Monocyte suspensions containing 2x10⁵ cells in 300 µL assay medium were added to HUVEC monolayers in the inner wells. rhMCP-1 or the supernatants from cocultures of monocytes and HUVECs were placed in the upper or lower compartment of inner wells. After 1 hour of incubation at 37°C, unbound monocytes were removed from the inner wells by washing with warmed assay medium. To assess monocytes that had migrated into collagen gels across the HUVEC monolayers, the inner wells were further incubated for 60 minutes at 37°C with 0.4% EDTA in PBS, and adherent monocytes and HUVECs were removed by washing from the surface of the collagen gels. The collagen gels containing migrated cells were fixed with 1% paraformaldehyde. The numbers of migrated cells were counted in at least eight fields under a phase-contrast microscope (Nikon) at x100 magnification. All experiments were done in duplicate.

Nonadherent and migrated monocytes prepared as described previously^{40 41} were collected by cytospin preparation for subsequent immunohistochemistry. Briefly, monocyte suspensions (5.0x10⁶ cells) were added to HUVECs cultured on collagen gels in 60-mm dishes and incubated at 37°C for 12 hours, after which the nonadherent cells were collected. After the adherent monocytes and HUVECs had been removed from the collagen gel surfaces, the collagen gels containing migrated cells were digested twice with 0.05% collagenase, and the migrated cells were collected.

Immunohistochemistry
Monocytes (1.5x10⁵ cells) or IL-1ß (25 U/mL) was added to the confluent HUVEC layers on coverslips in eight-well culture plates (Lab-Tek, chamber slide, Nunc). After incubation at 37°C for 12 hours, the HUVECs 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–MCP-1 Ab (4 µg/mL). After incubation at 4°C for 12 hours, the slides were rinsed again in PBS, overlaid with biotinylated rabbit anti-mouse Ig, incubated for 60 minutes, and rinsed in PBS. The slides were treated with streptavidin reagent (Histofin) for 30 minutes at room temperature, 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, and rinsed with distilled water. Mayer's hematoxylin was used as a counterstain. Furthermore, nonadherent and migrated monocytes were collected by cytospin, as described above, for subsequent immunohistochemistry using anti–MCP-1 Ab.

Statistical Analysis
All values are expressed as mean±SEM. In comparing two groups, P 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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Production of MCP-1 Protein in the Conditioned Medium of Cocultures of Monocytes and HUVECs
First, to determine whether the interaction of monocytes and unstimulated HUVECs stimulates the production of MCP-1 by these cells, we measured MCP-1 protein by RIA using ¹²⁵I-labeled MCP-1 in the culture medium of either HUVECs (5.0x10⁵ cells) or monocytes (2.5x10⁶ cells) alone and in cocultures. As shown in Fig 1A, unstimulated HUVECs or monocytes alone produced a minimal amount of MCP-1, 0.9±0.7 and 1.0±0.3 ng/mL, respectively, after 5 hours of incubation. However, cocultures of these cells increased MCP-1 production up to 7.9±1.4 ng/mL at 5 hours, although levels were as low as 1.1±0.2 ng/mL at 3 hours. The amount of MCP-1 in cocultures was approximately eightfold greater than that of either HUVECs or monocytes alone, suggesting that the effect of cocultures on MCP-1 secretion was synergistic.

View larger version (10K): [in this window] [in a new window]   Figure 1. Graphs showing monocyte chemoattractant protein-1 (MCP-1) levels in the conditioned media of cocultures of monocytes and human umbilical vein endothelial cells (HUVECs). After pretreatment with or without interleukin (IL)-1ß (25 U/mL) for 4 hours, HUVECs were coincubated in assay medium with () or without () monocytes for the indicated time. Monocytes alone () were also incubated for 5 hours. MCP-1 concentration in the conditioned medium was measured by radioimmunoassay. A, Unstimulated HUVECs. B, IL-1ß (25 U/mL)–pretreated HUVECs (4 hours). Values represent mean±SEM for duplicate determinations in four independent experiments.

IL-1ß stimulates MCP-1 production from various cell types.^{12 18 22} In our cultures, we confirmed that HUVECs produced 10.5±2.2 ng/mL MCP-1 during 4 hours of culture with IL-1ß at a concentration of 25 U/mL. As the gradual accumulation of MCP-1 in the conditioned medium shows (Fig 1B), the ability to produce MCP-1 was retained even after IL-1ß had been removed from the HUVEC culture. However, the addition of monocytes to the HUVEC monolayer that had been stimulated with IL-1ß roughly doubled the production of MCP-1 compared with unstimulated HUVECs at 5 hours. This enhancement appears to be an additive effect.

We then determined whether the protein in the supernatants indeed exhibited the same biological activity as rhMCP-1 on transendothelial migration of monocytes. In these experiments, we used a double-chamber transendothelial migration assay as described in "Materials and Methods." As shown in Fig 2, when compared with control conditions, rhMCP-1 (100 ng/mL) present in the lower compartment induced a 4.0-fold increase in monocyte transmigration across HUVEC monolayers. Like rhMCP-1, the supernatants from the 24-hour cocultures increased monocyte transmigration 2.6-fold, and this increase was completely abolished by treatment with anti–MCP-1 Ab (25 µg/mL). On the other hand, rhIL-8 (50 ng/mL), which is also secreted from activated endothelial cells but does not act on monocytes, did not affect the monocyte transmigration. Moreover, anti–IL-8 Ab (25 µg/mL) did not change the increased transmigration induced by the supernatants. Thus, this finding suggests that MCP-1 protein in the supernatants from the cocultures had functionally the same activity for monocytes as rhMCP-1.

View larger version (13K): [in this window] [in a new window]   Figure 2. Bar graphs showing transendothelial chemotactic activity for monocytes in supernatants (Sup) from cocultures of monocytes and human umbilical vein endothelial cells (HUVECs). Recombinant human monocyte chemoattractant protein-1 (rhMCP-1) was added to the supernatants on the upper or lower sides of HUVECs, and the migrated cells were counted as described in "Materials and Methods." Left, Induction of monocyte transendothelial chemotactic activity by the supernatants. rhMCP-1, 100 ng/mL; recombinant human interleukin-8 (rhIL-8), 50 ng/mL. Right, Inhibition of monocyte transendothelial chemotactic activity by anti()–MCP-1 antibody (Ab). Anti–MCP-1 Ab, 25 µg/mL; anti–IL-8 Ab, 25 µg/mL. Data for monocyte migration without MCP-1 or supernatants were taken as controls (Nil, 100% value for migration index). The data were expressed as the index values in comparison with the control. Values represent mean±SEM for duplicate determinations in six independent experiments. **P<.01 compared with the controls.

Induction of MCP-1 mRNA Expression by Cocultures of Monocytes and HUVECs
Next, we examined the induction of MCP-1 by cocultures of monocytes and HUVECs at the mRNA level. Northern blot analysis revealed that although MCP-1 mRNA was very slight or undetectable in cultures of either HUVECs or monocytes alone, it could be detected at 2 hours of coculture in the total RNA extracted from both monocytes and HUVECs (Fig 3, left). The level of mRNA increased by 4 hours and then declined slightly by 24 hours. The magnitude of expression corresponded to those of IL-1–stimulated HUVECs (Fig 3, right), suggesting that the MCP-1–inductive signal in cocultures is equal to that in IL-1–stimulated HUVECs. The rapid induction of MCP-1 mRNA indicates that MCP-1 protein is produced by de novo synthesis and that cell contact between monocytes and HUVECs is necessary for it.

View larger version (49K): [in this window] [in a new window]   Figure 3. Induction of monocyte chemoattractant protein-1 (MCP-1) mRNA expression by coculture of monocytes and human umbilical vein endothelial cells (HUVECs) analyzed by Northern blotting. Left, Cocultures of monocytes and unstimulated HUVECs (lane 1) or interleukin (IL)-1ß–pretreated HUVECs for 4 hours (lane 2) were incubated for 4 hours. HUVECs were incubated for 4 hours in the absence (lane 4) or presence (lane 5) of IL-1ß (25 U/mL). Monocytes alone were incubated for 4 hours (lane 3). Right, Cocultures of monocytes and HUVECs were incubated for 0 (lane 1), 2 (lane 2), 4 (lane 3), and 24 hours (lane 4), respectively.

Involvement of Cytokines and Adhesion Molecules in MCP-1 Production Induced by Cocultures of Monocytes and HUVECs
We analyzed the mechanism of MCP-1 induction by the cocultures of monocytes and HUVECs. Cocultures of monocytes and HUVECs induce secretion of IL-1 and TNF from these cells.^{42 43} Thus, it is possible that the MCP-1 induction is mediated by IL-1 and/or TNF produced by coculture. To test this possibility, we examined the effect of Abs against IL-1, IL-1ß, and TNF on MCP-1 production. As shown in Fig 4, when Abs were used in combination (anti–IL-1+anti–IL-1ß+anti-TNF Abs), MCP-1 production was inhibited by only 20%, but this decrease was significant (P<.05). The doses of antibodies used in this experiment were sufficient to neutralize 10 to 20 ng of IL-1, IL-1ß, and TNF, respectively.³³ This suggests that the involvement of IL-1/ß and TNF was only partial in our system.

View larger version (15K): [in this window] [in a new window]   Figure 4. Bar graph showing effect of anti()–interleukin (IL)-1 and anti–tumor necrosis factor (TNF) antibodies (Abs) on monocyte chemoattractant protein-1 (MCP-1) production induced by cocultures of monocytes and human umbilical vein endothelial cells (HUVECs). Monocytes (6.0x105 cells) and HUVECs (1.2x106 cells) were coincubated for 5 hours with or without (Nil) control IgG (anti–keyhole limpet hemocyanin [KLH] peptide; 1:200) or anti–IL-1 (50 µg/mL), anti-IL-1ß (1:400), anti-TNF (1:100) Abs, or combinations (anti–IL-1+anti–IL-1ß+anti-TNF Abs), and MCP-1 concentration in the conditioned medium was measured by radioimmunoassay. Values represent mean±SEM for duplicate determinations in six independent experiments. *P<.05 compared with Nil; MCP-1 concentration, 6.2 ng/mL.

We and other investigators have previously reported that monocyte–endothelial cell adhesion is mediated by the lymphocyte function–associated antigen-1 (LFA-1; CD11a/CD18) or Mac-1 (CD11b/CD18)/ICAM-1 pathway as well as the VLA-4/VCAM-1 pathway.^{40 44} To determine whether these integrin adhesion pathways initiate MCP-1 protein synthesis in cocultures of monocytes and unstimulated HUVECs, we carried out inhibition experiments using mAbs against CD18 (common ß chain of LFA-1, Mac-1, and p150/95), VLA-4, ICAM-1, and VCAM-1, separately and in combination. Anti–VLA-4 mAb was used because the VCAM-1 counterligand might be induced on unstimulated HUVECs by production of IL-1 or TNF in cocultures.⁴⁰ The presence of these mAbs used separately and in combination in coculture did not significantly inhibit MCP-1 secretion (Fig 5). These results suggest that coculture-induced MCP-1 expression is mediated by undefined adhesion pathways but not by soluble factors such as IL-1 and TNF.

View larger version (20K): [in this window] [in a new window]   Figure 5. Bar graph showing effect of anti()-CD18, anti–very late antigen (VLA)-4, anti–intercellular adhesion molecule (ICAM)-1, and anti–vascular cell adhesion molecule (VCAM)-1 monoclonal antibodies (mAbs) on monocyte chemoattractant protein-1 (MCP-1) production induced by cocultures of monocytes and human umbilical vein endothelial cells (HUVECs). Monocytes (6.0x105 cells) and HUVECs (1.2x106 cells) were coincubated for 5 hours with or without (Nil) control IgG (anti–HLA-ABC, 1:100), anti-CD18 (10 µg/mL), anti–VLA-4 (10 µg/mL), anti–ICAM-1 (10 µg/mL), or anti–VCAM-1 (10 µg/mL) mAbs, separately and in combination. MCP-1 concentrations in the conditioned media were measured by radioimmunoassay. Values represent mean±SEM for duplicate determinations in six independent experiments. Nil indicates MCP-1 concentration of 4.3 ng/mL.

Immunohistochemical Staining of MCP-1–Producing Cells
Both monocytes and endothelial cells can produce MCP-1.^{16 17 18} We next defined which cell type, the monocyte or the endothelial cell, is responsible for MCP-1 induction by cocultures. To examine MCP-1 protein on HUVECs and nonadherent and adherent monocytes, we used HUVEC monolayers cultured on dishes for 5 or 12 hours with monocytes. We isolated monocytes that had migrated through HUVEC monolayers into collagen gels as described in "Materials and Methods." Then the cells were immunohistochemically identified with anti–MCP-1 Ab. Although MCP-1 protein was not detected on any cells at 5 hours of coculture (not shown), it was induced by coculture for 12 hours on both HUVECs and the adherent monocytes at a similar level (Fig 6). The staining of anti–MCP-1 Ab on migrated monocytes was comparable to that on adherent monocytes (Fig 7B), whereas nonadherent monocytes did not react with anti–MCP-1 Ab (Fig 7A). These results indicate that an adhesive interaction between monocytes and endothelial cells is necessary to induce MCP-1 in both cell types and that an MCP-1–inductive signal is transduced to each cell type by the interaction.

View larger version (0K): [in this window] [in a new window]   Figure 6. Immunohistochemistry of human umbilical vein endothelial cells (HUVECs) and monocytes adherent to HUVECs. Unstimulated HUVECs, interleukin (IL)-1ß (25 U/mL)–stimulated HUVECs, and cocultures of monocytes and HUVECs were incubated for 12 hours. After cells were washed three times, immunohistochemistry was performed with anti–monocyte chemoattractant protein-1 (MCP-1) antibodies. A, Unstimulated HUVECs. B, IL-1ß–stimulated HUVECs. C, Cocultures of monocytes and HUVECs (magnification x100).

View larger version (0K): [in this window] [in a new window]   Figure 7. Immunohistochemistry of monocytes that failed to adhere to and those that migrated across human umbilical vein endothelial cells (HUVECs). Monocytes were coincubated with HUVECs for 12 hours, and then nonadherent and transendothelially migrated cells were collected and stained as described in "Materials and Methods." A, Nonadherent monocytes. B, Migrated monocytes (magnification, x100).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Direct cell-to-cell contact regulates a variety of cellular functions, including cell activation and cytokine production.^{42 43 45} We attempted to determine whether the interaction between monocytes and endothelial cells induces MCP-1 production from these cell types, because this interaction constantly occurs in recruitment of monocytes and because production of MCP-1 would affect subsequent monocyte transendothelial migration. Our results indicate that migrated monocytes express MCP-1 protein during the process of transmigration. Cocultures of resting monocytes and unstimulated or IL-1–prestimulated endothelial cell monolayers induced an increase in the amounts of soluble MCP-1 secreted into the medium. MCP-1 secreted in supernatants exhibited chemotactic and transendothelial activities for monocytes, and its biological effect was confirmed by the disappearance of the activity in the presence of anti–MCP-1 Ab. Quantification of mRNA levels indicates that augmentation was mediated at the level of MCP-1 synthesis. Both IL-1 and TNF stimulate MCP-1 production,¹⁸ and both are produced by endothelial cells.⁴² Anti–IL-1/ß and anti-TNF Abs used in combination inhibited MCP-1 production by only 20%, but this inhibition was statistically significant. However, monoclonal antibodies directed against CD18, VLA-4, ICAM-1, and VCAM-1, which can partially inhibit monocyte–endothelial cell adhesion,⁴⁰ had no effect. Immunohistochemical studies showed that adherent and transmigrated monocytes as well as endothelial cells expressed MCP-1 protein, but nonadherent monocytes did not express MCP-1 (Figs 6 and 7). These results indicate that monocytes and endothelial cells transmit signals to each other to induce MCP-1 synthesis during the process of cell contact of transmigration.

Although the kinetics of monocyte transendothelial migration in vivo are unknown, we have previously shown in an in vitro system that monocytes in the subendothelial space can be seen as early as 30 minutes after monocytes are added to endothelial cell monolayers, and their numbers reach a plateau by 3 to 5 hours of incubation (approximately 40% of total added monocytes).⁴⁰ When monocytes and endothelial cells were cocultured for 5 hours, MCP-1 secretion was rapid, appearing within 3 hours, and was markedly elevated by 5 hours (Fig 1). The induction of MCP-1 mRNA in cocultured cells occurred early, at 2 and 4 hours, and declined slightly at 24 hours, suggesting that de novo synthesis of MCP-1 commenced early and was sustained at 24 hours (Fig 3). Thus, the rapid induction of MCP-1 mRNA by coculture indicates that direct cell contact between monocytes and endothelial cells, rather than the effect of soluble factors such as IL-1, is required in the early phase of MCP-1 induction. The fact that inhibition of MCP-1 secretion by anti–IL-1 and anti-TNF antibodies was incomplete also supports this.

What processes are involved in the cell contact that result in a signal to induce both MCP-1 mRNA and protein? It has been reported that during T-cell activation, ICAM-1, VCAM-1, and LFA-3 are capable of providing costimulatory functions.^{28 46 47 48} For example, the CD2/LFA-3 pathway transduces a costimulatory signal to induce IL-2 mRNA in T-cell–endothelial cell interaction.²⁸ Since MCP-1 could be visualized immunohistologically in adherent and migrated monocytes but not in nonadherent monocytes, we suspected that LFA-1, Mac-1, and VLA-4 on monocytes mediated MCP-1 secretion. However, mAbs directed against CD18 (common ß chain of LFA-1 and Mac-1) and VLA-4 failed to inhibit secretion. Since the contribution of these adhesion molecules to monocyte–endothelial cell adhesion is only partial,⁴⁰ undefined adhesion pathways other than LFA-1, Mac-1, and VLA-4 may be responsible. Indeed, a novel endothelial molecule promoted by mmLDL has been reported that induces adhesion of monocytes.⁴⁸ Alternatively, since platelet-activating factor is expressed on activated endothelial cells and enhances neutrophil–endothelial cell adhesion in cooperation with P-selectin,^{49 50} unknown accessory factors other than adhesion molecules might be involved in MCP-1 induction in cocultures. At present, the precise mechanism of the MCP-1 induction is unclear, and further study is required.

Cell contact during transmigration may deliver an early priming signal to induce MCP-1 mRNA in migrating monocytes. The primed monocytes would be able to quickly secrete MCP-1 in response to additional stimuli such as IL-1 and TNF in inflammatory lesions. In our cultures, however, collagen gels on which unstimulated endothelial cell layers were grown did not contain IL-1 and TNF. Indeed, MCP-1 protein was immunohistochemically undetectable on migrated monocytes that were isolated from collagen gels at 5 hours of coculture. Because it was detectable at 12 hours, the amount of MCP-1 protein at 5 hours may be below the sensitivity of our detection system. What promotes the secretion of MCP-1 by migrated monocytes that are primed during transmigration? Recently, it has been reported that the ß₁-integrin–mediated interaction with extracellular matrix proteins contributes to persistent cytokine gene expression in synovial fluid T cells from patients with rheumatoid arthritis.⁵¹ Collagen gels from which migrated monocytes have been isolated are a potential source for a factor to stimulate MCP-1 secretion by migrated monocytes. The interaction between collagen and the corresponding receptors on migrated monocytes may maintain and increase the level of MCP-1 mRNA and subsequent protein synthesis after transmigration. Thus, MCP-1 expressed by migrated monocytes will form a chemotactic gradient between blood and extravascular tissue and promote further infiltration of monocytes. In normal vascular tissue, in which inflammatory cytokines are at very low concentrations, MCP-1 may play an important role in the continual immunological surveillance by monocytes.

We also found that MCP-1 secretion from IL-1–prestimulated endothelial cells was increased by the addition of monocytes. As shown in Fig 1, the enhancement appears to be an additive but not synergistic effect, suggesting that the IL-1–derived signal to stimulate MCP-1 secretion from endothelial cells is different from the monocyte-derived signal. The in vivo role of endothelium-derived MCP-1 in transmigration of monocytes is still uncertain. Our preliminary in vitro experiments indicate that approximately 90% of MCP-1 produced by endothelial cell layers is secreted at the apical side. Since this would be washed away in vivo by the bloodstream, secreted MCP-1 probably does not affect the transendothelial gradient of MCP-1. However, the addition of rhMCP-1 (100 ng/mL) to endothelial cell monolayers completely inhibits migration of monocytes adhering to the layers and even induces detachment of the adherent monocytes, although the mechanism is not fully understood (M. Takahashi, submitted for publication). This finding indicates that the subsequent transmigration of adherent monocytes in vivo is suppressed by continuous exposure to endothelium-secreted MCP-1. A role for MCP-1 may therefore be to prevent excessive infiltration of monocytes into peripheral tissues and into inflammatory lesions. More recently, Carr et al⁵² demonstrated that MCP-1 is a chemoattractant for T cells. Thus, it is possible that MCP-1 secreted by the monocyte–endothelial cell interaction also affects the recruitment of T cells as well as monocytes.

In conclusion, our data suggest that monocytes are primed to produce MCP-1 through direct cellular contact with endothelial cells during transendothelial migration into subintimal space even in the absence of stimulation by proinflammatory cytokines such as IL-1¹⁷ and blood components such as mmLDL.²⁵ MCP-1 production by subintimal monocytes/macrophages might be one of the important factors in recruitment of blood monocytes and thus in the pathogenesis of atherosclerosis.

	Acknowledgments

 
This work was supported by grants from the Ministry of Education, Science, and Culture (grant 5671632) and the Molecular Cardiology Study Group (Japan). We are grateful to Drs Naofumi Mukaida and Kouji Matsushima for providing the rabbit anti–MCP-1 antibody and monocyte chemotactic and activating factor/MCP-1 cDNA and to Dr Chikao Morimoto for providing the anti–HLA-ABC antibody. We thank Toshiko Kambe, Mamiko Semba, and Atsuko Okamoto for technical assistance.

Received September 12, 1994; accepted January 23, 1995.

	References

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