Adrenomedullin in monocytes and macrophages - Semantic Scholar

Clinical Science (1999) 97, 247–251 (Printed in Great Britain)

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Adrenomedullin in monocytes and macrophages: possible involvement of macrophage-derived adrenomedullin in atherogenesis Masaharu NAKAYAMA, Kazuhiro TAKAHASHI, Osamu MURAKAMI*, Hiroshi MURAKAMI†, Hironobu SASANO†, Kunio SHIRATO‡ and Shigeki SHIBAHARA Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8574, Japan, *Second Department of Internal Medicine, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8574, Japan, †Department of Pathology, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8574, Japan, and ‡First Department of Internal Medicine, Tohoku University School of Medicine, Aoba-ku, Sendai, Miyagi 980-8574, Japan

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Macrophages secrete a variety of growth factors, cytokines and vasoactive peptides, which are related to the progression of atherosclerosis. Adrenomedullin (ADM) is a potent vasodilator peptide and inhibits proliferation and migration of vascular smooth muscle cells. In this study, we investigated the production and secretion of ADM by monocytes and macrophages by Northern blot analysis, RIA and immunocytochemistry. Northern blot analysis showed that ADM mRNA was expressed in human monocytes obtained from peripheral blood and monocyte-derived macrophages. The expression level of ADM mRNA in monocyte-derived macrophages was about five times higher than that in monocytes. Treatment with lipopolysaccharide (100 ng/ml) for 24 h increased ADM mRNA expression levels in both monocytes and monocyte-derived macrophages. The levels of immunoreactive ADM in the media of monocyte-derived macrophages were about three times higher than that of monocytes (0.718p0.046 fmol/24 h/105 cells, n l 8 compared with 0.259p0.018 fmol/24 h/105 cells, n l 8 ; meanpS.E.M., P 0.01). The secretion was also increased by treatment with lipopolysaccharide. Immunocytochemistry showed positive ADM immunostaining in macrophages in atherosclerotic lesions of human aorta obtained at autopsy. ADM secreted from activated macrophages may play an inhibitory role in atherogenesis.

INTRODUCTION Adrenomedullin (ADM) was originally identified as a potent vasodilator peptide from human phaeochromocytoma [1]. ADM consists of 52 amino acids and has about 20 % similarity to calcitonin gene related peptide. Besides its vasodilator effect, ADM has been reported to have various functions, such as suppression of endothelin production from vascular smooth muscle cells (VSMCs), and chemoattractant release from macrophages and

inhibition of the growth and migration of VSMCs [2–5]. Therefore ADM may suppress the progress of atherogenesis. Macrophages play a pivotal role in atherogenesis [6]. In the early phase of atherosclerosis, monocytes adhere to the surface of injured vascular endothelial cells (ECs) and invade the subendothelial space. After entering the vessel wall, monocytes, now called macrophages, internalize the oxidized low-density lipoprotein via the scavenger receptors and become foam cells. Macrophages

Key words : adrenomedullin, atherosclerosis, macrophage, monocyte. Abbreviations : ADM, adrenomedullin ; ECs, endothelial cells ; FBS, fetal bovine serum ; Ir-ADM, immunoreactive adrenomedullin ; LPS, lipopolysaccharide ; VSMCs, vascular smooth muscle cells. Correspondence : Dr K. Takahashi.

# 1999 The Biochemical Society and the Medical Research Society

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produce and secrete a variety of bioactive substances, including growth factors, cytokines and lytic enzymes. Growth factors and cytokines enhance migration and proliferation of VSMCs, which also produce growth factors and inflammatory mediators. These factors act in a paracrine and autocrine fashion to amplify the signals leading to cellular proliferation and connective tissue formation, and cause the vascular damage with lytic enzymes. ADM is produced and secreted not only by phaeochromocytoma but also by various cell types, such as ECs [7], VSMCs [8] and many tumour cell lines [9]. We have previously reported the synthesis of ADM in cultured choroid plexus carcinoma cells [10], T98G glioblastoma cells [11] and DLD-1 colorectal adenocarcinoma cells [12]. Martinez et al. [13] showed the expression of ADM mRNA in alveolar macrophages by in situ reverse transcriptase PCR. The secretion of ADM by THP-1 acute monocytic leukaemia cells and monocytes has recently been reported [14]. On the other hand, ADM mRNA expression has not been studied in either peripheral monocytes or monocyte-derived macrophages. It has not been clarified whether macrophages in atherosclerotic lesions express ADM. In this study, we therefore examined expression of ADM mRNA and protein in monocytes prepared from peripheral blood and monocyte-derived macrophages. In addition, we have studied the expression of ADM by immunocytochemistry in macrophages in atherosclerotic lesions of human aorta obtained at autopsy.

MATERIALS AND METHODS Materials Lipopolysaccharide (LPS) (Escherichia coli O55 : B5) and AB human serum were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). RPMI 1640 medium was purchased from Life Technologies Inc. (Grand Island, NY, U.S.A.), Limulus Amebocyte Lysate assay, Pyrogent, was from BioWhittaker Inc. (Walkersville, MD, U.S.A.) and macrophage-colony stimulating factor was from Peprotech (London, U.K). Monoclonal antibodies against human α-smooth muscle actin and CD68 antigen were purchased from NeoMarkers (Union City, CA, U.S.A.)

Cell culture Peripheral blood mononuclear cells were collected from blood obtained from healthy volunteers with FicollPaque (Eurobio, Paris, France). The mononuclear cells were suspended in fetal bovine serum (FBS)-precoated plastic flasks (Becton Dickinson, New Jersey, USA) in RPMI 1640 medium with 10 % (v\v) FBS and cultured for 3 h in 5 % CO \95 % air. Human monocytes were

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selected out by repeated washing with FBS to remove non-adherent cells [15]. Human monocytes were cultivated in RPMI 1640 medium with 10 % (v\v) FBS at 37 mC in 5 % CO \95 % air and were treated with or # without LPS (100 ng\ml) for 12 and 24 h. All the reagents used for monocyte isolation and culture were endotoxinfree as evaluated by the Limulus Amebocyte Lysate assay, Pyrogent. Monocyte-derived macrophages were generated by culturing monocytes in RPMI 1640 medium with 10 % (v\v) pooled AB human serum and 100 ng\ml macrophage-colony stimulating factor for 5 days [16]. After the medium was replaced, they were cultivated with or without LPS (100 ng\ml) for 12 and 24 h.

Northern blot analysis Total RNA was extracted from cultured cells by the guanidium thiocyanate–caesium chloride method, and subjected to Northern blot analysis, as previously reported [10]. The probe for ADM mRNA was the HindIII–EcoRI fragment of pBS-hADM2 [10]. The probe for β-actin mRNA was the SmaI–ScaI fragment (nucleotide positions 124\1050) derived from a fulllength human β-actin cDNA [17]. Radioactive signals were detected by exposing the filters to X-ray films (XAR5, Kodak, Rochester, NY, U.S.A.) or with a Bioimage analyser (BAS 1000 ; Fuji Film, Tokyo, Japan). The intensity of hybridization signals was quantified with a Bioimage analyser. The intensity representing ADM mRNA was normalized with the intensity of the β-actin band, and the normalized values were compared.

RIA Peptides in the medium were extracted with a Sep-Pak C cartridge (Waters, Milford, MA, U.S.A.). Immuno") reactive adrenomedullin (Ir-ADM) in the extract was measured by RIA using the antiserum against human ADM as previously reported [10,18]. Chromatographic characterization of the extract of the culture medium was performed by reverse-phase HPLC using a µBondapak C column (3.9 mmi300 mm ; ") Waters.), as previously reported [10,18].

Immunocytochemistry Human aortic tissues with atherosclerotic lesions were collected from six patients (five male and one female, 56–84 years old) at autopsy within 4 h post-mortem. Pathological diagnosis of these patients was pulmonary emboli, hepatocellular carcinoma and squamous cell carcinoma of the urinary bladder, sepsis, oesophageal squamous cell carcinoma, malignant melanoma, and gastric ulcer, respectively. This study has been approved by the Ethics Committee on human study of Tohoku University School of Medicine. Aortic tissues were fixed in 4 % paraformaldehyde, adjusted to pH 7.4, for 18 h at room temperature and embedded in paraffin.

Adrenomedullin in monocytes and macrophages

Immunocytochemistry of ADM was performed using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA, U.S.A.), as previously reported [19]. The antiserum against human ADM(1–52) (No. 102) [19] was used at a dilution of 1 : 1000. The preabsorption of the antiserum with human ADM(1–52) (5.5 nmol peptide\ml of the diluted antiserum) abolished positive immunostaining. Immunopositive cells were characterized by immunostaining the serial sections for α-smooth muscle actin (smooth muscle cell marker), ADM and CD68 (macrophage marker). Monoclonal antibodies against human α-smooth muscle actin and CD68 antigen were used at dilutions of 1 : 400 and 1 : 100 respectively.

Statistics

Figure 2 Reverse-phase HPLC of the medium extract of monocytes

Statistical analysis was performed by Student’s t-test or Welch’s t-test. Results are given as meanspS.E.M.

The Ir-ADM level in each fraction was measured by RIA. The arrow indicates the elution position of human ADM. The dotted line indicates a gradient of acetonitrile (ACN).

RESULTS

macrophages without LPS treatment was about five times higher than that in monocytes without LPS treatment (Figure 1, top). Treatment with LPS (100 ng\ml) for 24 h increased ADM mRNA expression levels 5-fold and 2fold in monocytes and monocyte-derived macrophages respectively. The Ir-ADM levels in the medium in which monocyte-derived macrophages were cultured for 24 h without LPS treatment were about three times higher than those of monocytes without LPS treatment (0.718p0.046 fmol\24 h\10& cells, n l 8 compared with 0.259p0.018 fmol\24 h\10& cells, n l 8 ; P 0.01) (Figure 1, bottom). The treatment with LPS significantly increased the levels of Ir-ADM in the culture media both of monocytes and monocyte-derived macrophages at 12 h and 24 h. Reverse-phase HPLC of the extract of the culture medium of peripheral monocytes showed one major peak which was eluted in the position of the human ADM standard. Two other minor peaks were eluted earlier than the ADM standard (Figure 2). ADM with an oxidized methionine, which was generated by incubating ADM with 3 % (v\v) H O for 1 h at room temperature, was # # eluted in the same position as ADM, indicating that these two minor peaks do not represent ADM with an oxidized methionine. Material eluting in these two peaks may be ADM with some other types of modifications. Figure 3(A) shows a representive atherosclerotic lesion stained with haematoxylin-eosin. An atheromatous plaque of the specimen immunostained for CD68 and ADM was magnified (Figures 3B and 3C). Immunocytochemistry of CD68 showed that cells surrounding the atheromatous plaque were positive for CD68 (Figure 3B) but negative for α-smooth muscle actin, indicating that these cells were macrophages. Immunocytochemistry of ADM showed that some of the CD68positive cells were also positive for ADM (Figure 3C). In addition to macrophages, positive immunostaining of

Northern blot analysis showed that ADM mRNA was expressed in the human monocytes obtained from peripheral blood and monocyte-derived macrophages. The expression level of ADM mRNA in monocyte-derived

Figure 1 ADM expression in human peripheral monocytes and monocyte-derived macrophages

Human monocytes (mono) and monocyte-derived macrophages (MDM) were cultivated with or without LPS (100 ng/ml) for 24 h. Top : Northern blot analysis of ADM mRNA. Each lane contains 15 µg of total RNA. Lower panel shows β-actin mRNA as an internal control. Bottom : Ir-ADM concentrations in the culture media of human monocytes and monocyte-derived macrophages (meanspS.E.M. ; n l 8). Monocytes without LPS (#) ; monocytes with LPS ($) ; monocytederived macrophages without LPS ( ) ; monocytes-derived macrophages with LPS ( ). *P 0.05 compared with untreated ; **P 0.01 compared with untreated.


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ADM was observed in endothelial cells and smooth muscle cells of the aorta (results not shown).

DISCUSSION

Figure 3 An atherosclerotic lesion of human aorta stained with haematoxylin-eosin (A) and a magnified field of the serial sections immunostained with CD68 (macrophage marker) (B) and ADM (C)

ADM immunoreactivity was observed in cells surrounding the atheromatous plaque. Two photomicrographs are combined in (A). An asterisk (*) indicates the atheromatous plaque. M indicates medium. Magnification i23 (A) and i165 (B and C). Scale bar l 200 µm. # 1999 The Biochemical Society and the Medical Research Society

In the present study, we have shown the production and secretion of ADM in monocytes prepared from peripheral blood and monocyte-derived macrophages. In addition, we have demonstrated for the first time that macrophages in atherosclerotic lesions of human aorta, as well as ECs and smooth muscle cells, were immunostained for ADM. ADM mRNA expression and secretion of ADM by monocyte-derived macrophages were greater than those by monocytes, suggesting that the differentiation from monocytes to macrophages caused an increased expression of ADM. The differentiation caused a 5-fold increase in ADM mRNA levels, whereas it caused a 3fold increase in Ir-ADM levels in the medium at 24 h. This discrepancy between ADM mRNA levels and IrADM levels may be explained by increased consumption of ADM by monocyte-derived macrophages themselves. The levels of ADM mRNA and Ir-ADM were augmented by LPS treatment. LPS is known to be a potent stimulator of monocytes and macrophages to secrete cytokines such as tumour necrosis factor-α and interleukin 6 [20]. Since monocytes and macrophages are activated by LPS, these findings suggest that activation of monocytes and macrophages may be accompanied by increased production of ADM. Recent studies revealed that human monocytes, monocyte-derived macrophages and murine peritoneal macrophages secrete ADM [14,21,22]. Contrary to our observation, Kubo et al. [14] showed that augmentation by LPS was detected only in monocyte-derived macrophages but not in monocytes. The difference between the results of Kubo et al. [14] and ours might be due to the different method of monocyte isolation from mononuclear cells or the different cultivating time. We have found that some of the CD68-positive cells surrounding the atheromatous plaques are immunostained for ADM, indicating that some macrophages in atheromatous lesions produce ADM. Macrophages are activated in atheroscleroic lesions and have a key role in atherogenesis. It has been reported that VSMCs and ECs secrete ADM [7,8,23] and express ADM receptors [24,25]. ADM secreted from VSMCs and ECs is thought to act as a vasodilator in an autocrine and\or paracrine fashion. The target cells of ADM secreted by macrophages may include VSMCs and ECs. ADM is known to inhibit endothelin production from VSMCs and growth and migration of VSMCs [2,4,5]. ADM has also been found to suppress apoptosis of rat ECs via a cAMPindependent mechanism [25]. Another possible target of ADM secreted from macrophages may be macrophages

Adrenomedullin in monocytes and macrophages

themselves. It has been reported that ADM inhibits chemoattractant release from macrophages [3]. ADM secreted from macrophages in the atherosclerotic lesion may act against the progression of atherosclerosis through these effects together with ADM secreted from VSMCs and ECs. In conclusion, we have shown that ADM is expressed in human monocytes obtained from peripheral blood and monocyte-derived macrophages. The secretion was augmented by treatment with LPS. In addition, we have shown the expression of ADM by immunocytochemistry in macrophages in atherosclerotic lesions of human aorta obtained at autopsy.

ACKNOWLEDGMENTS We thank Dr T. Yamamoto for β-actin cDNA. This work was supported in part by the Gonryou Medical Foundation, a Grant-in-aid for Brain Science Research from the Ministry of Health and Welfare of Japan, and Grants-in-aid for Scientific Research (B) and (C) and for Exploratory Research from the Ministry of Education, Science, Sports and Culture of Japan.

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