Constitutive protein expression of monocyte chemotactic protein-1 (MCP-1) by myelomonocytic cell lines and regulation of the secretion by anti- and proinflammatory stimuli

Leukemia Research 23 (1999) 843 – 849 www.elsevier.com/locate/leukres

Constitutive protein expression of monocyte chemotactic protein-1 (MCP-1) by myelomonocytic cell lines and regulation of the secretion by anti- and proinflammatory stimuli Klaus G. Steube *, Corinna Meyer, Hans G. Drexler Department of Human and Animal Cell Cultures, DSMZ-Deutsche Sammlung 6on Mikroorganismen und Zellkulturen, Mascheroder Weg 1B, D-38124 Braunschweig, Germany Received 18 January 1999; accepted 1 May 1999

Abstract We have investigated the protein expression of the chemokine monocyte chemotactic/chemoattractant protein-1 (MCP-1) in various human myelomonocytic leukemia cell lines. Applying specific ELISA, we demonstrated that this chemokine is produced constitutively by the cell lines HL-60, ML-2, MONO-MAC-6 and MUTZ-3 ranging between 440 and 1400 pg/ml MCP-1 per million cells. In the culture medium of two other unstimulated cell lines, MONO-MAC-1 and THP-1, almost no MCP-1 was detected. Stimulation of HL-60 and MONO-MAC-6 with lipopolysaccharide (LPS), and stimulation of ML-2 and MUTZ-3 with 12-tetradecanoyl phorbol 13-acetate (TPA) dramatically increased the MCP-1 level in the culture medium. The highest amount of MCP-1 ( \80 ng/ml within 24 h) was achieved by TPA stimulation of MUTZ-3 cells. Out of 15 cytokines tested for induction or enhancement of MCP-1 secretion, interleukin-3 (IL-3), IL-6, interferon-g (IFN-g), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) and tumor necrosis factor (TNFa) were able to augment (twofold to 12-fold) the MCP-1 level in the culture medium of MONO-MAC-6 cells. While the antinflammatory cytokines IL-4, IL-10 and IL-13 failed to suppress MCP-1 secretion, the glucocorticoid dexamethasone strongly inhibited the MCP-1 production of unstimulated and stimulated MONO-MAC-6 cells. Thus, several regulatory elements are involved in MCP-1 secretion. Despite the quantitative differences of MCP-1 production among the cell lines analyzed, our results demonstrated a constitutive secretion in differentiation-arrested myelomonocytic leukemia cell lines and emphasize the usefulness of these malignant cell lines as models to study MCP-1 secretion and regulation. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Chemokine; MCP-1; Monocytic leukemia; Cell lines; MONO-MAC-6

1. Introduction Anti-inflammatory responses by circulating leukocytes to infection or tissue injury is a multistep series of adhesive and signaling events and represent an important component of the immune system. Besides adhesion molecules, chemoattracting-signaling proteins are involved in this process of effector cell recruitment during the local immune response. These specialized cytokines, commonly designated as chemokines, do not only activate chemotaxis of leucocytes, but also cytoskeletal organization, regulation of adhesion * Corresponding author. Tel.: +49-531-2616-159; fax: +49-5312616-150. E-mail address: [email protected] (K.G. Steube)

molecules, enzyme release from intracellular vesicles, and oxygen radical formation. Chemokines are small proteins (8–12 kDa) and constitute to date four subfamilies (C, CC, CXC, and CXXXC), distinguished by the spacing of cysteine residues in the amino-terminal part of the polypeptides (for reviews see [1–4]). The monocyte chemotactic protein-1 (MCP-1) was purified 1989 as a monocyte chemotactic and activating factor (MCAF, MCF) from the LPS-stimulated cell line THP-1 [5], as well as from phytohemagglutinin-stimulated mononuclear cells [6]. Cloning and sequencing of the cDNA confirmed it as product of the human JE gene [7,8] and the protein was henceforth termed MCP1 [9]. MCP-1 is a potent chemoattractant for monocytes, activated CD4 and CD8 memory T-lymphocytes and natural killer cells, but not for B-lymphocytes

0145-2126/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 5 - 2 1 2 6 ( 9 9 ) 0 0 1 0 7 - 1

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[10,11]. MCP-1 has been reported to augment the cytostatic activity of monocytes against several tumor cell lines [5] and to enhance the tumor-specific immunity of the host [12]. Additional interest has arisen from the recent discovery of the involvement of chemokine receptors in human immunodeficiency virus (HIV) infection [13,14]. A constitutive production of human MCP-1 has been reported only for several solid tumor cell lines [15–17] and fibroblasts [18] while endothelial cells require certain stimuli to secrete MCP-1 [19–22]. Although hematopoietic cells represent the major target for MCP-1, some studies indicate a production of the chemokine by the hematopoietic cells themselves. Since most of these investigations have used only stimulated blood cells, we analyzed whether differentiationarrested myelocytic and monocytic human cell lines were able to constitutively produce and secrete MCP-1, and furthermore which factors quantitatively affect the protein production.

0.6× 106 U/mg) were received from R&D Systems (Wiesbaden, Germany). IL-10 (specific activity 5× 105 U/mg) was obtained from Pharma Biotechnologie (Hannover, Germany) and tumor necrosis factor-a (TNFa, specific activity \2×106 U/mg) from Biermann (Bad Nauheim, Germany).

2.3. Treatment of cell lines Experiments were performed in 24-well culture plates (Nunc, Wiesbaden, Germany) with starting cell concentrations ranging between 1 and 5× 105 cells/ml. Stimulation was carried out in fresh culture medium with dexamethasone, GM-CSF, IFN-g, IL-1b, IL-2, IL-3, IL-4, IL-6, IL-10, IL-13, IL-15, LIF, LPS, M-CSF, TNFa and TPA. Culture supernatants were collected 6 and 24 h thereafter, centrifuged (5 min at 10 000×g) and stored frozen at − 20°C.

2.4. Quantitati6e chemokine determination 2. Materials and methods

2.1. Cell lines and culture conditions All cell lines (HL-60, ML-2, MONO-MAC-1, MONO-MAC-6, MUTZ-3, THP-1) are deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). The cultures are mycoplasma-free and were cultivated without antibiotics at 37°C in a humidified atmosphere containing 5% CO2 using appropriate growth media supplemented with 10–20% fetal bovine serum or other nutrients as outlined in the DSMZ Catalogue of Human and Animal Cell Lines [23].

2.2. Chemicals Culture media and supplements were from Life-Technologies (GIBCO-BRL, Karlsruhe, Germany), fetal bovine serum, dexamethasone, the calcium ionophore A-23187, TPA and LPS from Sigma (Deisenhofen, Germany). Interleukin-1b (IL-1b, specific activity 5 × 108 U/mg) were obtained from Amersham (Braunschweig, Germany). Granulocyte-macrophage colony-stimulating factor (GM-CSF, specific activity \ 1 ×107 U/mg), interferon-g (IFN-g, specific activity \ 2× 107 U/mg), IL-2 (specific activity \2 × 106 U/ mg), IL-4 (specific activity \ 5 ×105 U/mg), IL-6 (specific activity \ 1× 108 U/mg) and leukemia inhibitory factor (LIF, specific activity 1×107 U/mg) were purchased from Roche-Diagnostics (Boehringer Mannheim, Germany). IL-3 (specific activity 4× 106 U/mg), IL-13 (specific activity \2 × 105 U/mg), IL-15 (specific activity \ 0.5 ×106 U/mg) and macrophage colony-stimulating factor (M-CSF, specific activity \

The concentrations of the chemokine MCP-1 was determined by a specific double-ligand ELISA assay according to the manufacturer’s protocol (QuantikineR, R&D) with test and control samples. Samples that gave results outside the standard range were assayed again at an appropriate dilution.

3. Results

3.1. Constituti6e production of MCP-1 by myelomonocytic cell lines First, we examined the ability of six human leukemia cell lines to constitutively secrete MCP-1. The cell lines had been established from patients with myeloid or monocytic leukemia defined by the French–American– British classification (FAB M2, M4 and M5). Immunological analysis of the lineage-specific antigens (cluster of differentiation, CD 13, CD14, CD15, CD33, CD34, CD68 and HLA-DR) confirmed the different stages of arrested maturation of the cell lines (Table 1). We determined that four cell lines (HL-60, ML-2, MONOMAC-6, MUTZ-3) secreted large amounts MCP-1 (400–1400 pg/ml per 1 million cells within a 24 h incubation period) into the culture medium; two other cell lines (MONO-MAC-1, THP-1) elaborated MCP-1 below the detection level of the assay (Table 1).

3.2. LPS- and TPA-induced secretion of MCP-1 Since LPS and the phorbol ester TPA are potent inducers of differentiation in myelomonocytic cells and modulators of gene expression of cytokine genes, we investigated whether these compounds affected MCP-1

K.G. Steube et al. / Leukemia Research 23 (1999) 843–849

protein expression in our cell lines. As shown in Fig. 1, TPA (10 − 8 M) or LPS (already 1 ng/ml) stimulated secretion of MCP-1 in all cell lines investigated, however to different extents. The panel of cell lines can be divided in two parts, namely into low and high MCP-1 producers. In the first group (Fig. 1A), the level of MCP-1 was increased two to fourfold by TPA (in MONO-MAC-1) and LPS (in HL-60 and THP-1), but did not exceed 1200 pg/ml in 106 HL-60 cells per 24 h. In the second group, consisting of ML-2, MONOMAC-6 and MUTZ-3, the MCP-1 concentration was dramatically raised by LPS in MONO-MAC-6 and by TPA in ML-2 and MUTZ-3 reaching 30 – 90 ng/ml (Fig. 1B). This very strong increase of the MCP-1 content in the culture media could be detected already 4 – 6 h after start of incubation with TPA and LPS, and was followed by a further steady increase of MCP-1 within a 24 h incubation period (exemplified for MONO-MAC-6 in Fig. 2).

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ingly, in the same series of experiments none of these compounds induced any significant MCP-1 secretion in MONO-MAC-1 cells. Analysis of the intracellular MCP-1 concentration in hypotonically lysed unstimulated or stimulated MONO-MAC-1 cells revealed none or only very low amounts of MCP-1.

3.4. Inhibition of the induced MCP-1 secretion of MONO-MAC-6 cells Next, we studied whether inhibitory drugs affected MCP-1 secretion of MONO-MAC-6 cells and incubated unstimulated and stimulated cultures additionally with the glucocorticoid dexamethasone or with IL-4, IL-10, IL-13 or the calcium ionophore A-23187 (Table 2). A very strong inhibitor was dexamethasone which very clearly prevented the MCP-1 secretion induced by IFN-g, IL-3, LPS, M-CSF and TNFa by 50–100%. A-23187 and the cytokines IL-4, IL-10 and IL-13 had no inhibitory effect on the induced MCP-1 secretion of MONO-MAC-6 cells; IL-13 even stimulated MCP-1 secretion approximately fourfold. Also the constitutive expression of MCP-1 in MONO-MAC-6 cells was inhibited only by dexamethasone and not by IL-4, IL-10 or IL-13.

3.3. Incubation of MONO-MAC-6 cells with 6arious cytokines In order to discover any other positive or negative effectors of MCP-1 secretion, we incubated MONOMAC-1 and MONO-MAC-6 cells with a series of proinflammatory or anti-inflammatory agents for 24 h. Both cell lines have been established from the same patient and share many, but not all properties. Fig. 3 shows that GM-CSF, IFN-g, IL-3, IL-6, M-CSF and TNFa were able to significantly (i.e. stimulation index \2) and reproducibly raise the amounts of MCP-1 secreted by MONO-MAC-6 cells. The strongest stimulatory cytokines were IL-3 and TNFa leading to the elaboration of \10 ng/ml MCP-1 per 106 MONOMAC-6 cells, respectively. The TNFa concentration applied was rather high (10 ng/ml), while lower concentrations failed to significantly induce MCP-1 secretion. However, all cytokines tested were less efficient than LPS in mobilizing the secretion of MCP-1. Interest-

4. Discussion Monocytes/macrophages fulfill many important functions in the immunological defense of the host. As a response to various chemotactic stimuli (e.g. chemokines) monocytes/macrophages are present in such diverse physiological and pathophysiological states as inflammation [1], tumor cell growth, joint diseases, allergic responses and development of atherosclerotic lesions [12,24–26]. The chemokine MCP-1 belongs to the b- (or C–C) subfamily of chemokines and is commonly secreted by accessory cells in injured tissues, such as fibroblasts, endothelial or mesothelial cells [18–

Table 1 Constitutive secretion of MCP-1 in myelomonocytic cell linesa Cell Line

HL-60 ML-2 MONO-MAC-1 MONO-MAC-6 MUTZ-3 THP-1

Patient’s disease

FAB FAB FAB FAB FAB FAB

M2 M4 M5 M5 M4 M5

Cell surface marker expression

MCP-1 production (pg/ml)

CD13

CD14

CD15

CD33

CD34

CD68

HLA-DR

+ + + + + +

− − (+) + + (+)

+ + + + + +

+ + − + − +

− − − − − −

n.t. n.t. + + + −

− + + + + +

440 880 B50 700 1400 B50

a Cells were incubated as described in Section 2; the soluble, extracellular MCP-1 concentrations were determined by specific ELISA. Elaborated amounts (pg/ml) are calculated as for 106 cells within 24 h (n.t., not tested). FAB, French-American-British Classification; CD, Cluster of differentiation; HLA-DR, human leukocyte antigen.

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Cells of the monocytic lineage are one major target of the chemokines and are also known to produce a large series of signaling and immunomodulating peptide factors. Therefore, we have investigated whether maturation-arrested and non-terminally differentiated myelomonocytic cells were themselves capable to produce and secrete MCP-1; we further analyzed which factors may interfere with that protein production. We have chosen established human cell lines instead of freshly prepared blood cells in order to exclude the involvement of accessory cells, either as direct producers of MCP-1 or as producers of ‘second messengers’ which hence may affect the MCP-1 producing cells. To our knowledge, this report is the first which demonstrates a strong constitutive secretion of MCP-1 in myelomonocytic leukemia cell lines. Normal leukocytes did not constitutively produce MCP-1, and only in fibroblastic and glioma cell lines a constitutive production has been reported [16,18]. Thus, established myelomonocytic cell lines represent an attractive source for MCP-1 production and are useful tools for studies on its secretion. Also the commonly used modulators TPA and LPS, well-known to affect several myelomonocytic functions, turned out to be very strong enhancers of MCP-1 secretion in our systems. Originally, THP-1 cells, stimulated with 1 mg/ml LPS, were

Fig. 1. LPS- and TPA-induced secretion of MCP-1. Myelomonocytic cell lines were seeded out in 24-well culture plates and incubated without or with LPS (1 ng/ml) or TPA (10 − 8 M) as outlined in Section 2. Supernatants were collected and analyzed by specific ELISA. Data given are mean values of at least three independent experiments. For the exact values of the untreated cell lines see Table 1. Please note that the MCP-1 concentration of part A (upper) is pg/ml, and that of part B (lower) is ng/ml, each referring to 106 cells/ml.

21,27]. MCP-1 represents a specific chemoattractant for monocytes and T-lymphocytes, but not for neutrophils [1,11].

Fig. 2. Time course of MCP-1 secretion by MONO-MAC-6 cells. MONO-MAC-6 cells were seeded out in a 24-well culture plate and incubated without or with 1 ng/ml LPS or 10 − 8 M TPA for up to 24 h. At various time intervals the MCP-1 amounts were determined in the culture medium. For each time point one mini-culture was used and the starting cell concentration was 2 ×105 cells/ml. Data shown is one representative out of three independent experiments.

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Fig. 3. Induced secretion of MCP-1 of MONO-MAC-6 cells. MONOMAC-6 cells were seeded out in 24-well culture plates (2 × 105 cells/ml) and incubated without or with the following inducers: GM-CSF (500 U, equalize 50 ng/ml), IFN-g (1000 U, equalize 50 ng/ml), IL-1b (10 ng/ml), IL-2 (0.1 ng/ml), IL-3 (100 ng/ml), IL-4 (10 ng/ml), IL-6 (100 ng/ml), IL-10 (100 ng/ml), IL-15 (500 ng/ml), LIF (50 ng/ml), M-CSF (20 ng/ml) and TNFa (10 ng/ml). After 24 h, cell free supernatants were collected and MCP-1 concentrations were determined by specific ELISA. Since several stimulations were carried out repeatedly at various days, these representative data are given as stimulation index relative to the untreated cultures (set as 1.0). The control cultures elaborated ca. 640 pg/ml MCP-1 by 106 cells.

used for isolation and purification of MCP-1 [5], but the cell lines MONO-MAC-6, ML-2, HL-60 and MUTZ-3 turned out to be much more effective MCP-1 producers. MONO-MAC-6, even when stimulated with only 0.1 ng/ml LPS, elaborated 100-fold more MCP-1 than stimulated THP-1. TPA alone was effective in the same order of magnitude, with the strongest response seen in MUTZ-3 cells, elaborating more than 90 ng/ml MCP-1 within 24 h. To date, TPA stimulation had been used only in combination with high levels of LPS and retinoic acids [8]. Table 2 Inhibition of induced MCP-1 secretion of MONO-MAC-6 cells Stimulusa

Stimulation index

Number of experiments

Nil Dexa IFN-g IFN-g+Dexa IL-3 IL-3+Dexa LPS LPS+Dexa M-CSF M-CSF+Dexa TNFa TNFa+Dexa

1.0 0.5 4.2 9 0.8 0.7 6.9 92.8 0.8 31 912 13 93 8.6 94 0.9 12.9 9 3 3.7

15 4 3 3 6 3 8 3 4 3 2 1

a

Dexa: dexamethasone (10−6 M and 10−7 M were equally effective), LPS, 1 ng/ml; IFN-g, 100 U/ml; IL-3 100 ng/ml; M-CSF, 20 ng/ml; TNFa, 10ng/ml.

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Thus, our data indicate that activation of both, protein kinase C and the LPS receptor CD14, induce and enhance MCP-1 protein expression. At the mRNA level, Colotta et al. [28] have shown MCP-1 expression in LPS-stimulated peripheral blood mononuclear cells (PBMC); Ueda et al. [29] demonstrated mRNA induction 2 h after LPS treatment and 8 h after TPA-treatment of THP-1 cells. They further demonstrated binding of different NF-kB-dimers to the A1- and A2-NF-kB binding sites in the distal part of the MCP-1 gene, elevating its transcription. Besides NF-kB, another transcription factor, AP-1, has been described to be involved in regulation of the MCP-1 gene expression [30]. Several protein factors influence MCP-1 production in various systems. By use of the monocytic cell line MONO-MAC-6, we demonstrated that IFN-g, IL-3, M-CSF and TNFa were able to strongly augment the MCP-1 production. A moderate or weak, but still significant induction was achieved by GM-CSF and IL-6. In endothelial cells, IL-1 was found to exert a stimulatory effect on MCP-1 production [19,20,31] as well as in non-hematopoietic tumor cells [32,33]. IL-6 has been described to induce MCP-1 in PBMC and in the histiocytic cell line U-937 [34]. Musso et al. recently introduced LIF as an inducer of IL-8 and MCP-1 expression in blood monocytes while INF-g differentially regulated the expression of both chemokines [35]. IL-4, IL-10 and IL-13 have been described as antiinflammatory cytokines affecting blood monocytes [36– 38] and as inhibitors of MCP-1 secretion in intestinal cells [39]. In mononuclear cells, however, IL-10 was reported to even stimulate MCP-1 release [40]. We found that IL-4, IL-10 or IL-13 did not inhibit MCP-1 secretion, neither in unstimulated nor in stimulated MONO-MAC-6 cells. Thus, differences seem to exist between leukemia cell lines and freshly prepared normal blood monocytes with respect to general MCP-1 secretion and its modulation by cytokines. However, even similar myelomonocytic leukemia cell lines may display different reactions as our results with MONO-MAC-1 and MONO-MAC6 have demonstrated. This is in accordance with experiments comparing the regulation of CD14 and IL-1b expression, in which IL-4 exerted different effects on MONO-MAC-6 and MUTZ-3 cells [41]. Even monocytes explanted from one patient (but from variable tissues) expressed a differential response pattern to IL-4 treatment [42]. Our experiments further demonstrated a very strong inhibition of MCP-1 secretion by dexamethasone, both in unstimulated and stimulated cells. These observations were independent of the inducer chosen (IFN-g, IL-3, M-CSF or TNFa) and confirmed the suggestion that steroids negatively influence transcriptional regulation of the MCP-1 gene [32,43,44].

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Besides its function as chemoattractant, MCP-1 was reported to induce the synthesis of other cytokines such as IL-1 or IL-6 or cellular adhesion factors in fresh monocytes [45]. Preliminary experiments in our laboratory could not demonstrate an induction of IL-1b or IL-6 in MONO-MAC-6 cells incubated for 24 h with up to 100 ng/ml MCP-1. MCP-1 transduces its effect through seventransmembrane-domaine receptors and the activation of the mitogen-activated protein kinase (MAPK) elevating the intracellular Ca + + concentration [46,47]. Naturally occurring posttranslational glycosylation or N-terminal truncation of the 76 amino acid long polypeptide strongly attenuates the bioactivity of this chemokine [48]. The receptor specific for MCP-1 has been cloned recently from MONO-MAC-6 cells [49] and has been found to be distinct from other known b-chemokine receptors. Very recently, b-chemokine receptors have been found to be involved in HIV-infections (for review see [10,50]) and receptor antagonists like the modified chemokines may represent an interesting approach for antiviral applications [51,52] further underlining the important role of this new class of interesting ‘specialized cytokines’.

Acknowledgements Drs Steube and Drexler contributed to all aspects of this manuscript, including concept, design, analysis of data and critical revision of the manuscript.

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