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A model system in haematology and immunology: The human monocytic cell line MONO-MAC-1
Leukemia Research Vol. 21, No. 4, pp. 327-335, 1997. Copyright 0 1997 Elsevier Science Ltd. All rights reserved Printed m Great Britain 0145-2126197 $17.W + 0.00
Pergamon PII: SO1452126(96)00129-4
A MODEL
SYSTEM IN HAEMATOLOGY AND IMMUNOLOGY: HUMAN MONOCYTIC CELL LINE MONO-MAC-l
THE
Klaus G. Steube, Dorthe Teepe, Corinna Meyer, Margarete Zaborski and Hans G. Drexler DSMZ -
German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig, Germany
Abstract-MONO-MAC-l is a human cell line with properties of blood monocytes, which can be used as a model system to study monocytic functions in vitro. In the present study, we prepared a karyotype of MONO-MAC-l, analysed the growth behaviour, determined the presence of differentiation-associated antigens and studied the expression and secretion of several cytokines upon stimulation with 12-0-tetradecanoyl phorbol 13-acetate (TPA) and lipopolysaccharide (LPS). The MONO-MAC-l cells have a near diploid karyotype and contain several recurrent chromosomal rearrangements, in particular the translocation (9;ll) commonly found in AML-M5. Stimulation with TPA or LPS induced changes in morphology and gene expression, especially an increase in the level of the differentiation marker CD14 and the production of monocyte-related cytokines. Both biomodulators alone were sufficient to promote TNFcl release; however, the combination of TPA and LPS resulted in a synergistic increase of TNFu secretion. Northern blot analysis indicated that upregulated production of TNFa was due to induced synthesis of mRNA. The mRNA accumulation peaked approximately 2 h after stimulation and maximum levels of TNFcl were found in the supernatants after 4-8 h of culture. The MONO-MAC-l cells could not be restimulated with the same inducer to release TNFcc when a 48 h pre-treatment was carried out with LPS or TPA. LPS induced the release of granulocyte colony-stimulating factor (G-CSF), while TPA failed to do so. Vice versa, secretion of macrophage CSF (M-CSF) could be induced by TPA, but not by LPS. However, LPS enhanced the TPA-induced M-CSF production. Similarly, incubation of MONO-MAC-l, simultaneously with TPA and LPS, led to granulocyte macrophage CSF (GM-CSF) and interleukin-l/I (ILIp)secretion, while both stimulators alone had almost no (TPA) or only a weak (LPS) effect on the secretion of GM-CSF and IL-l/?. Our results demonstrate that MONO-MAC-l is a unique cell line with distinct monocytic features; certain monocytic properties can be upregulated by activation of intracellular signalling pathway(s). We suggest that, besides the LPS receptor CD14, activation of PKC participates in these process, especially in the production and secretion of cytokines by MONO-MAC-l cells. Ic 1997 Elsevier Science Ltd. All rights reserved. Key words: Phorbol ester, protein monocytic cell line MONO-MAC-l.
kinase C, differentiation,
Introduction
LPS, TNFc(, cytokine
secretion,
tions, there in fact exist only few haematopoietic cell lines with pronounced characteristics of the-monocytic lineage [l]. The leukaemia cell lines U-937, THP-1, ML-2 or HL-60, frequently used as in vitro model systems for monocytic cells, either are clearly myeloid (HL-60) or appear to be arrested at early stages of monocvtic differentiation, manifested bv the absence. weak or incomplete expression of certain monocyteassociated features, e.g. typical morphology, phagocytosis, adherence, NaF sensitive esterase or the cell surface antigen CD14. In 1985, the cell line MONO-MAC-6, originated from a patient with monoblastic leukaemia, was described to exhibit morphological, phenotypical and functional characteristics of mature blood monocytes [2]; prior to MONO-MAC-6, another subclone termed MONO-
Established tumour cell lines provide excellent and reproducible tools for the investigation of many biochemical, immunological or genetic functions of cells and tissues. However, to study monocytic funcAbbreviations: G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage CSF; IL-lfi, interleukin-l/?; LPS, lipopolysaccharide; M-CSF, macrophage CSF; mAb. monoclonal antibodv: PKC, orotein kinase C: TNFc(. turn&r necrosis factor?; Tki, 12-G-ietradecanoyl phorbol 13: acetate; SCF, stem cell factor. Correspondence to: Dr K. Steube, Ph.D., DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg lB, D-38124 Braunschweig, Germany (Tel: +49 531 2616.159; Fax: +49 531 2616.150; E-mail: kst.gbfbraunschweig.de). 327
K. G. Steube et al.
328
MAC-l was also established. The second subclone, MONO-MAC-6, was studied in more detail and thereafter widely used. However, the first subclone, MONO MAC-l, originally described as CD14 negative, remained less characterized. In the following, we present a thorough description of this cell line, MONO-MAC-l, with respect to growth characteristics, cytogenetic aberrations, cytokine and cell surface marker expression, both constitutively and following treatment with differentiation inducing agents. In particular, the induced expression of TNFcr at the mRNA and protein level was investigated in more detail.
Materials and Methods Chemicals Lipopolysaccharide (LPS) from Salmonella typhimurium, 12-0-tetradecanoyl phorbol 13-acetate (TPA), [3(4,5-dimethylthiazol-2-yl)2,.5 diphenyltetrazoliumbromide] (MTT), 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazin (H7) and actinomycin D were purchased from Sigma (Deisenhofen, Germany). The TPA and H7 were dissolved in dimethylsulfoxide (DMSO), stored frozen and diluted prior to the experiments in culture medium. At the final dilution used, the DMSO concentration was 0.1% or less. A polyclonal antibody against human TNFc( was purchased from Endogen (Cambridge, MA, U.S.A.) and the monoclonal antibody (mAb) MY4 (Clone 322, from ascites), directed against human CD14, was from Coulter (Krefeld, Germany). Kits for the quantitative determination of secreted interleukin-lg (IL-l@, IL-2, IL-3, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) (Quantikine Immunoassays) were from R&D Systems (Wiesbaden, Germany). The [a-32P]-deoxycytosine-triphosphate (specific activity 110 TBq/mmol) was obtained from Amersham-Life Science (Braunschweig, Germany). Culture media and supplements were from Gibco-BRL (Eggenstein, Germany). Cell line MONO-MAC-l The parental cell line MONO-MAC was established in 1985 from a peripheral blood sample obtained from a 60-year-old male diagnosed with acute monoblastic leukaemia [2]. The first series of sequential cloning gave rise to MONO-MAC-l found as CD14 negative; a second series resulted in CD14 positive cells, designated MONO-MAC-6. According to morphological, cytochemical and further immunological criteria, both cell lines were assigned to the monocytic lineage. Both cell lines expressed the NaF-sensitive, non-specific esterase, produced reactive oxygen; MONO-MAC-6 was described to carry out phagocytosis [2].
Cell culture conditions All cell lines, MONO-MAC-l, MONO-MAC-6 [2], ML-2 [3], L-929 [4] and U-937 [5], are deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany [6]). The cultures are mycoplasma-free and were cultivated without antibiotics at 37°C in a humidified atmosphere containing 5% CO2 using RPM1 1640 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS, Sigma). In the case of MONO-MAC-l and MONO-MAC-6, 1 mM sodium pyruvic acid, 2 mM Lglutamine and 1 x MEM non-essential amino acids were added, as well as 10 pg./ml bovine insulin to MONOMAC-6 cultures. For the experiments, exponentially growing cells (viability of >80% by trypan blue exclusion) were washed and resuspended in a final concentration ranging from 0.5-2 x lo6 cells/ml in 24well culture plates (Nunc, Wiesbaden, Germany) for 1 h. Then, medium or different dilutions of activators were added from 100-fold stock solutions to the cultures. After the indicated incubation periods, culture supernatants were removed, centrifuged and either stored frozen or analysed immediately for the presence of the cytokines. Immunophenotyping Expression of cell surface antigens was determined by immunofluorescence analysis with mAbs against CD3 (Becton Dickinson, Heidelberg, Germany), CD13 (Coulter, Krefeld, Germany), CD14 (Prof. Knapp, Vienna, Austria), CD15 (Becton Dickinson), CD19 (Prof. Janossy, London, U.K.), CD33 (Coulter) and CD68 (Dako). Non-reactive antibodies were applied as controls and binding was visualized by fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin heavy chain-specific antisera (Dunn, Asbach, Germany). Distribution of antigens was analysed by flow cytometry (FACSscan; Becton Dickinson). Addition of 10 ug/ml propidium iodide to the cells prior to analysis allowed the gating out of dead cells. Cytokine assays Biologically active TNFcl was assessed by its ability to induce lysis of the TNF-sensitive murine cell line L929. To that end, 2 x lo4 cells per well were plated out in 96-well flat-bottom culture plates (Nunc) and incubated at 37°C. After 16 h, the medium was replaced by 50 ul fresh medium containing 10 pg/ml actinomycin D and 50 ul of the samples to be tested [7]. After further incubation for 20 h, the degree of cytotoxicity was determined by the MTT-assay [8]. Recombinant human TNFc( (rhTNFa, Boehringer Mannheim, Germany) was used as a standard for the quantitative determination of TNFc( released from MONO-MAC-l cells. All samples were tested in triplicate. Polyclonal anti-TNFa antibody
Cell line
329
MONO-MAC-l
Table 1. Cell surface marker analysis of unstimulated and stimulated monocytic cell lines
Growth Curves of MONO-MAC Cell Lines 16-
Cell line/treatment
CD33
CD14
MONO-MAC-l None
o%*
4% 8% 38%
TPA
0%
LPS
0%
MONO-MAC-6 None TPA 0’
l&50% 14%
LPS
0%
37% 53% 60%
ML-2 None TPA LPS
0% NW ND
0% 4% 48%
u-937 None TPA LPS
0% ND ND
0% 0% 0%
I 0123456789
Days Fig. 1. Growth curves of MONO-MAC-l and MONO-MAC-6. The MONO-MAC-l (2 x lo6 cells) and MONO-MAC-6
(3 x lo6 cells) were seededout in 10 ml RPM1 1640 medium containing 10% FBS, 2 mM L-glutamine, 1 x MEM nonessential amino acids and 1 mM Na pyruvate with or without 10 pg/ml bovine insulin and cultivated for several days without replacement or addition of fresh medium. At the time points indicated, small aliquots were removed and cell numbers and
*Percentage of cells positive by flow cytometry. tND, not determined.
viabilities were determined. Data given as total viable cell number.
Results and Discussion (Endogen) was used at a final concentration of 10-100 &ml. The other cytokines were determined by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (QuantikineR, R&D Systems).
Cytogenetic
analysis
The G-banded metaphase chromosomal preparations were obtained from MONO-MAC-l and MONO-MAC6 using standard cytogenetic protocols. Several rearrangements were confirmed by fluorescence in situ hybridization (FISH) analysis which was carried out as described previously in detail [6].
RNA isolation
and Northern
blot analysis
Total cellular RNA was isolated by the guanidinium isothiocyanate/cesium chloride method. Ten micrograms per lane were separated in a 1% agarose/2.2 M formaldehyde gel, transferred to a nylon membrane (Schleicher & Schilll, Dassel, Germany) and crosslinked with UV light (UV Stratalinker 1800, Stratagene, Heidelberg). Filters were hybridized overnight at 62°C with [32P]-labelled probes (USB Random Primed Labelling Kit, USB, Bad Homburg, Germany) specific for TNFc( (1.1 kb PstI fragment) and p-actin (1.3 kb PstI fragment [9]) and exposed at -80°C to X-ray films with intensifying screens for autoradiography.
Growth
characteristics
of MONO-MAC-1
and MONO-
MAC-6 The cell line MONO-MAC-l is growth factorindependent, continuously cultivated in RPM1 1640 medium supplemented with 5-10% FBS. Addition of glutamine, pyruvate and a cocktail of non-essential amino acids was beneficial for growth, but was not absolutely required. Figure 1 demonstrates cell expansion over a period of several days without addition or replacement of medium. Bovine insulin, which seemed to be necessary for the growth of MONO-MAC-6, was not essential for cultivation of MONO-MAC-l. Also, MONO-MAC-l cells usually were easier to handle than MONO-MAC-6 and subcultures could be initiated easily from the single cells. Starting with l-2 x lo5 cells/ml, MONO-MAC-l cultures reached a cell concentration higher than 2 x lo6 cells/ml, whereas the saturation density of MONO-MAC-6 was 1 x lo6 cells/ml, and the optimal concentration lay below 0.6 x lo6 cells/ml. Changes in morphology during differentiation
and surface antigen expression
Cultivation of MONO-MAC-l cells in the presence of LPS or TPA resulted in morphological changes usually associated with induction of differentiation, e.g. cell clustering, adherence and/or formation of pseudopodia. Cell clustering and adherence appeared rapidly within 6 h after addition of the inducers and were most prominent in TPA + LPS double-stimulated cultures. As evidenced by flow cytometry, the B- and T-cell
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K. G. Steube et al.
Table 2. Time course of TNFc( secretion by MONO-MAC-l cells stimulated with different LPS concentrations* Time (h)
LPS Concentration (@ml) used 0.1 1.0 10
2 4 8 16 24 48
It 5.5 11
70 60 11 16 21 15
NDS 300 280 110 90 53
*The MONO-MAC-l cells (0.5 x 106/ml) were precultivated overnight in 24-well plates. Then medium, 0.1, 1 or 10 @ml LPS, respectively, were added. After the indicated time periods, supernatants of the cultures were analysed for TNFc( bioactivity. Cell-free medium with or without 10 @ml LPS served as controls and did not result in measurable cell lysis of L-929 cells. The rhTNFcc, in concentrations from 1 to 1000 pg/ml, was used in a parallel set-up for the quantitation of the induced TNFc( production. tTNFcc in pgiml. $ND, not determined.
marker CD3 and CD19 were clearly absent on MONOMAC-l and MONO-MAC-6 cells, whereas antigens characteristically found on cells committed to myeloid/ monocytic lineages, namely CD13, CD14, CD1.5, CD68 and HLA-DR, were present [6]. Table 1 shows a comparison of CD33 and CD14 expression after LPS or TPA treatment of MONO-MAC-l, and -6, and the two other monocytic cell lines U-937 and ML-2. The MONO-MAC-6 had variable levels of CD33 in several determinations ranging between 10 and 50%, while the other cell lines were negative or very weakly positive. Stimulation of the cell lines with TPA or LPS had no significant effect on the CD33 surface expression of any of the cell lines. On the other hand, the level of the CD14 molecules clearly increased on the surface of LPS- or TPA-treated MONO-MAC-l and MONOMAC-6 cells. Unstimulated ML-2 and U-937 were negative for CD14. Lipopolysaccharide, but not TPA, could induce CD14 expression on ML-2 cells. However, these markers remained unaffected after treatment of U937 with either of these drugs. Recently, the levels of CD14 and CD33 have been described as varying depending on the mAb used, the drug concentration and the mode of stimulation [lo, 111. Cytogenetic analyses The karyotype of MONO-MAC-l is near-diploid and has the following characteristics: XY, +8, +13, +21, t(10;13)(q23.3;q13) t(wl)(pl3;q23), adW)(W), t(12;17)(q21;qll), t(21;21)(922.l;qll/22), -17, +18, -20. Most rearrangements also occur in MONOMAC-6, implying their common origin in a precursor
clone. Both the reciprocal translocation between specific bands of chromosomes 9 and 11 (associated with AMLM.5) and the rearrangement of the long arm of chromosome 3 (associated with disordered platelet production) are known as primary changes in acute myeloid leukaemia [ 121. The remaining changes may be merely random ‘noise’ or, instead, actively promote tumour development. Recurrence of such chromosomal alterations in other reports of AML would tend to favour the latter explanation. The MONO-MAC-6 has acquired additional cytogenetic changes, including chromosome doubling (S = 94), which appears to have arisen in vitro. The MONO-MAC-l appears to have preserved the karyotypic features of the original primary leukaemia cells - one of only a minority of AML cell lines to have done so. Induction of TNFa secretion in MONO-MAC-l cells Tumour necrosis factorct is one of several cytokines produced by monocyteslmacrophages upon appropriate stimulation [13, 141. It activates a large number of cellular genes, i.e. other cytokines, is involved in the process of differentiation of haematopoietic cells and contributes to severe systemic reactions in mammals; thus, it plays a crucial role in inflammation and the immune response. The polyfunctional role of TNFcl in haematopoiesis, both as growth supporter and suppressor of progenitor cells, has led to its attribution as a ‘ying-yang’ molecule [15]. The TNFa is expressed initially as a 26 kDa membrane-anchored precursor protein which is processed proteolytically by a metalloproteinase to yield the mature 17 kDa polypeptide [16, 171. The biologically active form of TNFcr is a homotrimer with an apparent molecular weight of 52 kDa [18, 191. Since LPS is a potent inducer of TNFc( synthesis and secretion in several cellular systems [20221, we examined modes of TNFc! production by MONO-MAC-l by stimulating the cells for different periods and with various concentrations of LPS. In order to assess only the bioactive TNFc(, we measured its cytotoxic activity against L-929 cells. Culture medium of unstimulated MONO-MAC-l cells did not contain measurable TNFa activity. Soluble TNFc( activity was detected as early as 2 h after LPS addition and had a maximum between 4 and 8 h (Table 2). Thereafter, measurable TNFc( activity decreased in those MONOMAC-l cultures treated with low doses of LPS (0.1 ng/ ml or less) while, in cultures stimulated with high LPS doses (10 rig/ml), TNFc( activity could still be detected after 24 h. This result is in agreement with those of previous reports on LPS-induced TNFc( secretion in MONO MAC-6 cells [7,23-261. In other cellular systems, the combination of phorbol esters with LPS was used to induce TNFa secretion [17,27-291. We observed that TPA alone clearly
331
Cell line MONO-MAC-l [pg/mtl
TNF
TPA-pretreated
2
4
61624
h
2
4
61624
MAC-l
cells. Cell-free
from MONO-
culture medium containing
TPA
was unable to destroy the L-929 target cells. The released activity was not due to TNFa released from dead MONO-MAC-l cells because the viability of TPAtreated MONO MAC-l cultures were about 90% and remained unchanged during this 48 h incubation period. The time course and amount
of secreted
TNFa
was
similar to that seen after LPS stimulation (Fig. 2, lefthand side). When MONO-MAC-l cells TPA and LPS simultaneously,
LPS-pretreated
cells
h
Fig. 2. Synergistic effect of TPA and LPS on TNFcr secretion by MONO-MAC-l cells. The MONO-MAC-l cells (1.0 x 106/ ml) were seeded out in 24-well plates. After 1 h, medium, 100 nM TPA (left) or 0.1 rig/ml LPS + 100 nM TPA (right) were added. At the indicated time points, supernatants of the cultures were analysed for TNFc( bioactivity. Cell-free medium without or with 100 nM TPA, 1 @ml LPS or both served as controls in the TNFMTT-assay and did not result in measurable lysis of L-929 cells. For quantitation of the secreted TNFcl from the MONO-MAC-l cells, rhTNFcc, in concentrations from 1 to 1000 pg/ml, was added to L-929 cells in a parallel MlT-assay.
induced secretion of a TNFcr activity
cells
were stimulated with the concentration of
bioactive TNFa increased several-fold compared with those cultures stimulated with LPS or TPA alone (Fig. 2, right-hand side). This synergistic effect of TPA and LPS was even visible when only 0.05 or 0.1 rig/ml LPS (plus TPA) was used. To ensure that the toxic effects on L929 cells really were caused by TNFa, we added polyclonal anti-TNFa antibodies to the supernatants of TPA- or TPA+LPS-stimulated MONO-MAC-l cells and were able to inhibit completely the lysis of L-929 cells (data not shown). Since several binding sites for different transcription factors have been detected within the promoter region of the TNFc( gene ([23,30] and references therein), it is conceivable that the simultaneous stimulation by LPS and TPA results in a super-induction of TNFc( gene expression. The glycosyl-phosphatidylinositol anchored
Fig. 3. Effect of LPS or TPA pre-treatment on the stimulated TNFcl release. The MONO-MAC-l cells (1.0 x 106/ml) were seeded out in 24-well plates and cultivated in the presence of 100 nM TPA (left part) or 1 @ml LPS (right part) for 48 h. Then, 700 pl of medium was replaced by fresh medium containing TPA (final concentration 100 nM), LPS (final concentration 1 rig/ml) or TPA + LPS. A fourth miniculture remained untreated and served as control. After 5 h, the media were removed and assayed for TNFc( bioactivity. Results are expressed in percentage of dead L-929 cells (lysis) related to 0% dead (=lOO% viable cells) of the unstimulated control cultures. No measurable TNFcl activity was detected in the cultures treated for 48 h.
membrane protein CD14 is the receptor for LPS [31-331 and protein kinase C (PKC) is the molecular target of phorbol esters [34,35]. Incubation of MONO-MAC-l cells with either TPA and the PKC inhibitor H7, or with LPS and a mAb against CD14, decreased the TNFc( level in the culture medium by 50% for both settings (not shown). Conversely, H7 did not inhibit the response to LPS and anti-CD14 mAb did not prevent TPA-induced release
of
TNFc(.
These
results
suggest
that
both
receptors PKC and CD14 are involved individually in the signal transduction leading to TNFc( secretion of MONO-MAC-l cells. Pre-incubation of MONO-MAC-l with LPS or TPA modulates TNFcr secretion Since induction of differentiation is accompanied by changes in gene expression, we examined whether MONO-MAC-l cells, which have been cultivated for 48 h in the presence of TPA or LPS, were still able to release TNFr after a second challenge with LPS, TPA or both agents. Figure 3 (left-hand side) demonstrates that pre-incubation of MONO-MAC-l with 100 nM TPA decreased the amount of TNFx secreted after a second stimulus with TPA. Interestingly, when the second stimulus was carried out with LPS or LPS+TPA, the TNFz secretion was not inhibited. Vice versa, LPS-pre-
treated cells did not secrete TNFc( after a second LPS
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K. G. Steube et al.
TNF
1.6 kb
actin
2.2 kb I
I
12345678910 Fig. 4. Induction of TNFc( mRNA in MONO-MAC-l cells. MONO-MAC-l cells were incubated without (lanes 1 and 6) or with 1.0 rig/ml LPS (lanes 2-5), or 100 nM TPA (lanes 7-10). After 2 (lanes 2 and 7) and 24 h (lanes 3 and 8), cells either were harvested directly or subsequently restimulated for 2 h with LPS (lanes 4 and 10) or TPA (lane 5 and 9) and then harvested. Total RNA isolated and Northern blot analysis was carried out with 10 ug RNA per lane and the blot was hybridized first to a 32P-labelled TNFa DNA probe and 6 weeks thereafter to a /I-actin DNA probe.
challenge. However, they were still able to release TNFc( when the second stimulation was carried out with TPA. Recently, it has been reported for MONO-MAC-6, but not for MONO-MAC-l, that LPS pre-treatment mediates tolerance to further stimuli with LPS [lo, 23, 251 and that this process was dependent on the LPS concentration used [lo]. Our experiments further demonstrate that TPA pre-treatment reprogrammed the cellular response of MONO-MAC-l to a subsequent stimulation, such that TNFa release was markedly inhibited, an observation which until now has been reported only for murine macrophages [36]. The LPS- and TPA-mediated regulation of TNFu mRNA The LPS stimulated transiently the de novo synthesis of TNFa mRNA in MONO-MAC-6 cells [23,28,37]. In other human cells, activation of PKC induced or promoted TNFc( mRNA expression with or without subsequent protein secretion [38-43]. Performing Northern blot analysis, we found that both LPS and TPA strongly increased the TNFa mRNA level in MONOMAC-l cells within 2 h; after 24 or 48 h, the amount of mRNA
declined back to that of unstimulated
cells (Fig.
4, lanes l-3 and 68). When LPS-precultured MONO-
MAC-l cells were stimulated a second time with the same concentration LPS or with TPA, only a weak increase of TNFcl mRNA was detectable (lanes 4 and 5), which is in agreement with results obtained with MONO-MAC-6 [23,25]. Even so, when TPA-pretreated cells were stimulated the second time with TPA or LPS (lanes 9 and lo), only LPS was able to reinduce the TNFa mRNA suggesting a regulatory modulation of the signal transduction via PKC after long-term treatment with phorbol esters. The stimulation of CD14 and PKC involves a cascade of protein phosphorylations on members of the Raf, Src and MAP kinase family [44-46]. Also, NFkB and IkB has been shown to be involved in regulation of TNFa transcription [23,47,48], and PKC is one of the kinases that phosphorylate IkB directly and also mediates the activation of NFkB [49,50]. Taken together, our results and these studies imply multiple and complex regulations of the TNFa gene expression in the monocytic system. Production of other cytokines Besides TNFr, the production of seven additional cytokines (SCF, IL-lp, IL-2, IL-3, M-CSF, G-CSF and
Cell line MONO-MAC-l
M-CSF (pglml)
I.200
333
G-CSF (pg/ml)
1
l.wor------
”
CO
LPS
TPA
TPA
LPS+TPA
GM-CSF (pg/ml)
IL-1 I3 (pg/ml)
Ifi
0
CO
LPS
TPA
LPS+TPA
0
CO
LPS
TPA
LPS+TPA
Fig. 5. Stimulation-dependent cytokine production of MONO-MAC-l cells. MONO-MAC-l cells (2 x 106/ml) were cultivated in 24-well plates in the absence or presence of 1 @ml LPS, 100 nM TPA or a combination of both. After 6 and 24 h, the supernatants were collected, centrifuged and stored frozen. Determination of M-CSF, G-CSF, GM-CSF, and IL-lp were carried out by ELISA. For the quantification of the secreted cytokines, recombinant standardized material was used.
GM-CSF) was studied. Unstimulated MONO-MAC-l cells did not secrete any of these cytokines but, 24 h after LPS treatment, 750 pg/ml G-CSF, 14 pg/ml IL-lb and 20 pg/ml GM-CSF was measured (Fig. 5). Other investigators had reported the LPS-induced production of IL-6 and IL-l by MONO-MAC-6 cells. The low amount of IL-lb we measured in the culture medium of MONO-MAC-l probably was due to either the intracellular storage of IL-l/I [25], or the low dose of LPS (1 rig/ml) we used. Interestingly, release of M-CSF could be induced by TPA but not by LPS. The TPA, which was inefficient in inducing release of the other three cytokines, turned out to exhibit a strong co-enhancing effect on the production of all four cytokines when used in combination with LPS (most dramatically for M-CSF and GM-CSF, with approximately 1 r&ml). Our results indicate that both signalling pathways, via PKC and via CD14, are involved in the release of M-CSF, GM-CSF, G-CSF and IL-l/I from MONO-MAC-l cells and that TPA and LPS act synergistically to increase further the amount of cytokines secreted into the medium.
Synopsis The present data show that the human cell line MONO-MAC-l represents a model system that appears to be very useful for the analysis of various aspects of monocytic (patho)biology, including processes involved in signal transduction pathways and the regulatory mechanisms of cytokine production. References 1. Drexler, H. G., Gignac, S. M. & Minowada J. Hematopoietic cell lines. In Atlas of Human Tumor Cell Lines, ed. R. J. Hay, J. G. Park and A. Gazdar. Academic Press, San Diego, CA, 1994, pp. 213-250. 2. Ziegler-Heitbrock, H. W. L., Thiel, E., Ftitterer, A., Herzog, V., Wirtz, A. & Riethmtiller, G. Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes. International Journal of Cancer, 1988, 41, 456461. 3. Herrmann, R., Han, T., Barcos, M. P., Lok, M. S. & Henderson, E. S. Malignant lymphoma of pre-T-cell terminating in acute myelocytic leukemia. Cancer, 1980, 46, 1383-1388. 4. Sanford, K. K., Earle, W. R. & Likely, G. D. The growth in
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vitro of single isolated tissue cells. Journal of the National Cancer Institute, 1948, 9, 229-246. 5. SundstrGm, C. & Nilsson, K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). International Journal of Cancer, 1976, 17, 565-577. 6. Drexler, H. G., Dirks, W., Ma&cod, R. A. F., Quentmeier, H. & Steube, K. G. DSM Catalogue of Human and Animal Cell Lines, 5th edn. DSMZ, Braunschweig, 1995. 7. Steube, K. G. & Drexler, H. G. The protein kinase C activator Bryostatin-1 induces the rapid release of TNFc( from MONO-MAC-6 cells. Biochemical and Biophysical Research Communication, 1995, 214, 1197-1203. 8. Steube, K. G., Grunicke, G., Pietsch, T., Gignac, S. M., P&tit, G. R. & Drexler, H. G. Dolastatin 10 and Dolastatin 15: Effect of two natural peptides on growth and differentiation of leukemia cells. Leukemia, 1992, 6, 1048-1053. 9. Dodemont, H. J., Soriano, P., Quax, W. J., Ramaeker, F., Lenstra, J. A., Groenen, M. A. M., Bernardi, G. & Bloemendahl, H. The genes coding for the cytoskeletal protein actin and vimentin in warm-blood vertebrates. EMBO Journal, 1992, 1, 167-171. 10. Labeta, M. O., Durieux, .I. J., Spagnoli, G., Fernandez, N., Wijdenes, J. & Herrmann, R. CD14 and tolerance to lipopolysaccharide: biochemical and functional analysis. Immunology, 1993, 80, 415423. 11. Ziegler-Heitbrock, H. W. L., Schraut, W., Wendelgass, P., StrGebel, M., Sternsdorf, T., Weber, C., Aepfelbacher, M., Ehlers, M., Schiitt, C. & Haas, J. G. Distinct pattern of differentiation induced in the monocytic cell line MONOMAC-6. Journal of Leukocyte Biology, 1995, 55, 73-80. 12. MacLeod, R. A. F., Voges, M. & Drexler, H. G. MONOMAC-6: a mature monoblastic leukemia cell line with t(9;11)(p21;q23). Blood, 1993, 82, 3221-3222. 13. Beutler B. (ed.) Tumor Necrosis Factor: The Molecules and Their Emerging Role in Medicine. Raven Press, NY, 1992. 14. Beutler, B. & Cerami, A. The biology of cachectin/TNF: A primary mediator of the host response. Annual Review of Immunology, 1989, 7, 625-6.56. 15. Jacobsen, S. E. W., Jacobsen, F. W., Fahlman, C. F. & Rusten, L. S. TNFc(, the great imitator: role of p55 and p75 TNF receptors in hematopoiesis. In: Polyfunctionality of hemopoietic regulators: The Metcalf forum. Stem Cells, (Suppl. l), 1994, 12, 111-128. 16. Gearing, A. J. H., Beckett, P., Christodouiou, M., Churchill, M., Clements, J., Davidson, A. H., Drummond, A. H., Galloway, W. A., Gilbert, R., Gordon, J. L., Leber, T. M., Mangan, M., Miller, K., Nayee, P., Owen, K., Pate& S., Thomas, W., Wells, G., Wood, L. M. & Woolley, K. Processing of tumour necrosis factor-cc precursor by metallo-proteinases. Nature, 1994, 370, 555-557. 17. McGeehan, G. M., Becherer, J. D., Bast, R. C., Boyer, C. M., Champion, B., Conolly, K. M., Conway, J. G., Furdon, P., Karp, S., Kidao, S., McElroy, A. B., Nichols, J., Pryzwanski, K. M., Schoenen, F., Sekut, L., Truesdale, A., Verghese, M., Warner, J. & Ways, J. P. Regulation of tumour necrosis factor-cc processing by a metalloproteinase inhibitor. Nature, 1994, 370, 558-561. 18. Fiers, W. Tumor necrosis factor: characterization at the molecular, cellular and in vivo level. FEBS Letters, 1992, 285, 199-212. 19. Wingfield, P., Pain, R. H. & Craig, S. Tumor necrosis factor is a compact trimer. FEBS Letters, 1987, 211, 179184.
20. Sung, S. S. J., Bjomdahl, J. M., Wang, C. Y., Kao, H. T. & Fu, S. M. Production of tumor necrosis factor/cachectin by human T-cell lines and peripheral blood T lymphocytes stimulated by phorbol myristate acetate and anti-CD3 antibody. Journal of Experimental Medicine, 1988, 167, 937-944. 21. Cuturi, M. C., Murphy, M., Costa-Giomo, M. P., Weinmann, R., Perussia, P. & Trinchieri, G. Independent regulation of tumor necrosis factor and lymphotoxin production by human peripheral blood lymphocytes. Journal of Experimental Medicine, 1987, 165, 1582-1590. 22. Chen, C. C. & Manning, A. M. TGF-/3, IL-10 and IL-4 differentially modulate the cytokine-induced expression of IL-6 and IL-8 in human endothelial cells. Cytokine, 1996, 8, 58-65. 23. Haas, J. G., Baeuerle, P. A., Riethmiiller, G. & ZieglerHeitbrock, H. W. L. Molecular mechanisms in down regulation of tumor necrosis factor expression. Proceedings of the National Academy of Science, U.S.A., 1990, 87, 9563-9567. 24. Neustock, P., Brand, J. M., Kruse, A. & Kirchner, H. Cytokine production of the human monocytic cell line MONO-MAC-6 in comparison to mature monocytes in peripheral blood mononuclear cells. Immunobiology, 1993, 188, 293-302. 25. Ziegler-Heitbrock, H. W. L., Blumenstein, M., KBferlein, E., Kieper, D., Petersmann, I., Endres, S., Flegel, W. A., Northoff, H., Riethmiiller, G. & Haas, J. G. In vitro desensitization to lipopolysaccharide suppresses tumour necrosis factor, interleukin-1 and interleukin-6 gene expression in a similar fashion. Immunology, 1992, 75, 264-268. 26. Eisenhut, T., Sinha, B., Groettrup-Wolfers, E., Semmler, J., Siess, W. & Endres, S. Prostacyclin analogs suppress the synthesis of tumor necrosis factor-cc in LPS-stimulated human peripheral blood mononuclear cells. Immunopharmacology, 1993, 26, 259-264. 27. Golenbook, D. T., Hampton, R. Y., Qureshi, N., Takayama, K. & Raetz, C. R. H. Lipid A-like molecules that antagonize the effects of endotoxins on human monocytes. Journal of Biological Chemistry, 1991, 266, 19490-19498. 28. Pradines-Figueres, A. & Raetz, C. R. H. Processing and secretion of tumor necrosis factor tc in endotoxin treated MONO-MAC-6 cells are dependent on phorbol myristate acetate. Journal of Biological Chemistry, 1992, 267, 23261-23268. 29. Daniel-Issakani, S., Spiegel, A. & Strulovici, B. Lipopolysaccharide response is linked to the GTP binding protein, Gi2, in the promonocytic cell line U937. Journal of Biological Chemistry, 1989, 264, 2024@20247. 30. Krlmer, B., Wiegmann, K. & Kr6nke, M. Regulation of the human TNF promoter by the transcription factor Ets. Journal of Biological Chemistry, 1995, 270, 65774583. 31. Schumann, R. R., Leong, S. R., Flaggs, G. W., Gray, P. W., Wright, S. D., Mathison, J. C., Tobias, P. S. & Ulevitch, R. J. Structure and function of lipopolysaccharide binding protein. Science, 1990, 249, 1429-1431. 32. Wright, S. D., Ramos, R. A., Tobias, P. S., Ulevitch, R. J. & Mathison, J. C. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science, 1990, 249, 1431-1433. 33. Dentener, M. A., Bazil, V. B., von Asmuth, E. J. U., Ceska, M. & Buurman, W. A. Involvement of CD14 in lipopolysaccharide-induced tumor necrosis factor?, IL-6 and IL-8 release by human monocytes and alveolar
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35. 36.
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macrophages. Journal of Immunology, 1993, 150, 2885% 2891. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, V. & Nishizuka, Y. Direct activation of calcium activated, phospholipid-dependent, protein kinase C by tumor esters. Journal of Biological promoting phorbol Chemistry, 1982, 257, 7847-7851. Blumberg, P. M. Protein kinase C as the receptor for the phorbol ester tumor promoters: Sixth Rhoads’ Memorial Award lecture. Cancer Research, 1988, 48, 1-8. Seatter, S. C., Clair, L., Bennet, T., Bubrick, M. & West, M. A. Independent signal transduction pathways for IL-1 and TNF in LPS-tolerant macrophages. Journal of Surgical Research, 1995, 58, 651-658. Quentmeier, H., Kolsdorf, K., Zaborski, M. & Drexler, H. G. IL-4 inhibits the LPS-induced expression of CD14 and monocyte-specific esterase mRNA in MONO-MAC-6 cells. Leukemia, 1994, 8, 1551-1556. Goldfeld, A. E. & Maniatis, T. Coordinate viral induction of tumor necrosis factor c( and interferon fi in human Bcells and monocytes. Proceedings of the National Academy of Sciences, U.S.A., 1989, 86, 1490-1494. Sung, J. S. S., Walters, J. A., Hudson, J. & Gimble, J. M. Tumor necrosis factor !X mRNA accumulation in human myelomonocytic cell lines. Journal of Immunology, 1991, 147, 2047-2054.
40. Horiguchi,
J., Spriggs, D., Imamura, K., Stone, R., Luebbers, R. & Kufe, D., Role of arachidonic acid metabolism in the transcriptional induction of tumor necrosis factor x gene expression by phorbol ester. Molecular Cellular Biology, 1989, 9, 252-258. 41. Martin, C. A. & Dorf, M. E. Interleukin-6 production by murine macrophage cell lines P388Dl and J774A.l: stimulation requirements and kinetics. Cellular Immunology, 1990, 128, 555-568. 42. Sariban, E., Imamura, K., Luebbers, R. & Kufe, D. Transcriptional and posttranscriptional regulation of tumor
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Lohmann-Matthes, M. L. & Baccarini, M. Lipopolysaccharide induces activation of the Raf-l/MAP kinase pathway. Journal of Immunology, 1994, 153, 574G5748. 45. Weinstein, S. L., Gold, M. R. & DeFranco, A. L. Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages. Proceedings of the National Academy of Sciences, U.S.A., 1991, 88, 41484152. 46. Stefanova, I., Corcoran, M. L., Horak, E. M., Wahl, L. M., Bolen, J. B. & Horak, I. D. Lipopolysaccharide induces activation of CD14-associated protein tyrosine kinase ~531 561y”. Journal of Biological Chemistry, 1993, 268, 2072520728. 47. Takasuka, N., Matsuura, K., Yamamoto, S. & Akagawa, K.
S. Suppression of TNFcr mRNA expression in LPS-primed macrophages occurs at the level of nuclear factor kappaB activation, but not at the level of protein kinase C or CD14 expression. Journal of Immunology, 1995, 154, 48034812. 48. Shakhov, A. N., Collart, M. A., Vassalli, P., Nedospasov,
S. A. & Jongeneel, C. V. KB-type enhancers are involved in LPS-mediated transcriptional activation of the TNFc( gene in primary macrophages. Experimental Medicine, 1990, 171,35-49. 49. Shirakawa, F. & Mizel, S. B. In vitro activation and nuclear translocation of NFkB catalyzed by CAMPdependent protein kinase and protein kinase. Molecular and Cellular Biology, 1989, 9, 2424-2430. 50. Gosh, S. & Baltimore, D. Activation in vitro of NFkB by phosphorylation of its inhibitor IkB. Nature, 1990, 344, 678-682.
Pergamon PII: SO1452126(96)00129-4
A MODEL
SYSTEM IN HAEMATOLOGY AND IMMUNOLOGY: HUMAN MONOCYTIC CELL LINE MONO-MAC-l
THE
Klaus G. Steube, Dorthe Teepe, Corinna Meyer, Margarete Zaborski and Hans G. Drexler DSMZ -
German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures, Braunschweig, Germany
Abstract-MONO-MAC-l is a human cell line with properties of blood monocytes, which can be used as a model system to study monocytic functions in vitro. In the present study, we prepared a karyotype of MONO-MAC-l, analysed the growth behaviour, determined the presence of differentiation-associated antigens and studied the expression and secretion of several cytokines upon stimulation with 12-0-tetradecanoyl phorbol 13-acetate (TPA) and lipopolysaccharide (LPS). The MONO-MAC-l cells have a near diploid karyotype and contain several recurrent chromosomal rearrangements, in particular the translocation (9;ll) commonly found in AML-M5. Stimulation with TPA or LPS induced changes in morphology and gene expression, especially an increase in the level of the differentiation marker CD14 and the production of monocyte-related cytokines. Both biomodulators alone were sufficient to promote TNFcl release; however, the combination of TPA and LPS resulted in a synergistic increase of TNFu secretion. Northern blot analysis indicated that upregulated production of TNFa was due to induced synthesis of mRNA. The mRNA accumulation peaked approximately 2 h after stimulation and maximum levels of TNFcl were found in the supernatants after 4-8 h of culture. The MONO-MAC-l cells could not be restimulated with the same inducer to release TNFcc when a 48 h pre-treatment was carried out with LPS or TPA. LPS induced the release of granulocyte colony-stimulating factor (G-CSF), while TPA failed to do so. Vice versa, secretion of macrophage CSF (M-CSF) could be induced by TPA, but not by LPS. However, LPS enhanced the TPA-induced M-CSF production. Similarly, incubation of MONO-MAC-l, simultaneously with TPA and LPS, led to granulocyte macrophage CSF (GM-CSF) and interleukin-l/I (ILIp)secretion, while both stimulators alone had almost no (TPA) or only a weak (LPS) effect on the secretion of GM-CSF and IL-l/?. Our results demonstrate that MONO-MAC-l is a unique cell line with distinct monocytic features; certain monocytic properties can be upregulated by activation of intracellular signalling pathway(s). We suggest that, besides the LPS receptor CD14, activation of PKC participates in these process, especially in the production and secretion of cytokines by MONO-MAC-l cells. Ic 1997 Elsevier Science Ltd. All rights reserved. Key words: Phorbol ester, protein monocytic cell line MONO-MAC-l.
kinase C, differentiation,
Introduction
LPS, TNFc(, cytokine
secretion,
tions, there in fact exist only few haematopoietic cell lines with pronounced characteristics of the-monocytic lineage [l]. The leukaemia cell lines U-937, THP-1, ML-2 or HL-60, frequently used as in vitro model systems for monocytic cells, either are clearly myeloid (HL-60) or appear to be arrested at early stages of monocvtic differentiation, manifested bv the absence. weak or incomplete expression of certain monocyteassociated features, e.g. typical morphology, phagocytosis, adherence, NaF sensitive esterase or the cell surface antigen CD14. In 1985, the cell line MONO-MAC-6, originated from a patient with monoblastic leukaemia, was described to exhibit morphological, phenotypical and functional characteristics of mature blood monocytes [2]; prior to MONO-MAC-6, another subclone termed MONO-
Established tumour cell lines provide excellent and reproducible tools for the investigation of many biochemical, immunological or genetic functions of cells and tissues. However, to study monocytic funcAbbreviations: G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage CSF; IL-lfi, interleukin-l/?; LPS, lipopolysaccharide; M-CSF, macrophage CSF; mAb. monoclonal antibodv: PKC, orotein kinase C: TNFc(. turn&r necrosis factor?; Tki, 12-G-ietradecanoyl phorbol 13: acetate; SCF, stem cell factor. Correspondence to: Dr K. Steube, Ph.D., DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg lB, D-38124 Braunschweig, Germany (Tel: +49 531 2616.159; Fax: +49 531 2616.150; E-mail: kst.gbfbraunschweig.de). 327
K. G. Steube et al.
328
MAC-l was also established. The second subclone, MONO-MAC-6, was studied in more detail and thereafter widely used. However, the first subclone, MONO MAC-l, originally described as CD14 negative, remained less characterized. In the following, we present a thorough description of this cell line, MONO-MAC-l, with respect to growth characteristics, cytogenetic aberrations, cytokine and cell surface marker expression, both constitutively and following treatment with differentiation inducing agents. In particular, the induced expression of TNFcr at the mRNA and protein level was investigated in more detail.
Materials and Methods Chemicals Lipopolysaccharide (LPS) from Salmonella typhimurium, 12-0-tetradecanoyl phorbol 13-acetate (TPA), [3(4,5-dimethylthiazol-2-yl)2,.5 diphenyltetrazoliumbromide] (MTT), 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazin (H7) and actinomycin D were purchased from Sigma (Deisenhofen, Germany). The TPA and H7 were dissolved in dimethylsulfoxide (DMSO), stored frozen and diluted prior to the experiments in culture medium. At the final dilution used, the DMSO concentration was 0.1% or less. A polyclonal antibody against human TNFc( was purchased from Endogen (Cambridge, MA, U.S.A.) and the monoclonal antibody (mAb) MY4 (Clone 322, from ascites), directed against human CD14, was from Coulter (Krefeld, Germany). Kits for the quantitative determination of secreted interleukin-lg (IL-l@, IL-2, IL-3, stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) (Quantikine Immunoassays) were from R&D Systems (Wiesbaden, Germany). The [a-32P]-deoxycytosine-triphosphate (specific activity 110 TBq/mmol) was obtained from Amersham-Life Science (Braunschweig, Germany). Culture media and supplements were from Gibco-BRL (Eggenstein, Germany). Cell line MONO-MAC-l The parental cell line MONO-MAC was established in 1985 from a peripheral blood sample obtained from a 60-year-old male diagnosed with acute monoblastic leukaemia [2]. The first series of sequential cloning gave rise to MONO-MAC-l found as CD14 negative; a second series resulted in CD14 positive cells, designated MONO-MAC-6. According to morphological, cytochemical and further immunological criteria, both cell lines were assigned to the monocytic lineage. Both cell lines expressed the NaF-sensitive, non-specific esterase, produced reactive oxygen; MONO-MAC-6 was described to carry out phagocytosis [2].
Cell culture conditions All cell lines, MONO-MAC-l, MONO-MAC-6 [2], ML-2 [3], L-929 [4] and U-937 [5], are deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany [6]). The cultures are mycoplasma-free and were cultivated without antibiotics at 37°C in a humidified atmosphere containing 5% CO2 using RPM1 1640 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS, Sigma). In the case of MONO-MAC-l and MONO-MAC-6, 1 mM sodium pyruvic acid, 2 mM Lglutamine and 1 x MEM non-essential amino acids were added, as well as 10 pg./ml bovine insulin to MONOMAC-6 cultures. For the experiments, exponentially growing cells (viability of >80% by trypan blue exclusion) were washed and resuspended in a final concentration ranging from 0.5-2 x lo6 cells/ml in 24well culture plates (Nunc, Wiesbaden, Germany) for 1 h. Then, medium or different dilutions of activators were added from 100-fold stock solutions to the cultures. After the indicated incubation periods, culture supernatants were removed, centrifuged and either stored frozen or analysed immediately for the presence of the cytokines. Immunophenotyping Expression of cell surface antigens was determined by immunofluorescence analysis with mAbs against CD3 (Becton Dickinson, Heidelberg, Germany), CD13 (Coulter, Krefeld, Germany), CD14 (Prof. Knapp, Vienna, Austria), CD15 (Becton Dickinson), CD19 (Prof. Janossy, London, U.K.), CD33 (Coulter) and CD68 (Dako). Non-reactive antibodies were applied as controls and binding was visualized by fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin heavy chain-specific antisera (Dunn, Asbach, Germany). Distribution of antigens was analysed by flow cytometry (FACSscan; Becton Dickinson). Addition of 10 ug/ml propidium iodide to the cells prior to analysis allowed the gating out of dead cells. Cytokine assays Biologically active TNFcl was assessed by its ability to induce lysis of the TNF-sensitive murine cell line L929. To that end, 2 x lo4 cells per well were plated out in 96-well flat-bottom culture plates (Nunc) and incubated at 37°C. After 16 h, the medium was replaced by 50 ul fresh medium containing 10 pg/ml actinomycin D and 50 ul of the samples to be tested [7]. After further incubation for 20 h, the degree of cytotoxicity was determined by the MTT-assay [8]. Recombinant human TNFc( (rhTNFa, Boehringer Mannheim, Germany) was used as a standard for the quantitative determination of TNFc( released from MONO-MAC-l cells. All samples were tested in triplicate. Polyclonal anti-TNFa antibody
Cell line
329
MONO-MAC-l
Table 1. Cell surface marker analysis of unstimulated and stimulated monocytic cell lines
Growth Curves of MONO-MAC Cell Lines 16-
Cell line/treatment
CD33
CD14
MONO-MAC-l None
o%*
4% 8% 38%
TPA
0%
LPS
0%
MONO-MAC-6 None TPA 0’
l&50% 14%
LPS
0%
37% 53% 60%
ML-2 None TPA LPS
0% NW ND
0% 4% 48%
u-937 None TPA LPS
0% ND ND
0% 0% 0%
I 0123456789
Days Fig. 1. Growth curves of MONO-MAC-l and MONO-MAC-6. The MONO-MAC-l (2 x lo6 cells) and MONO-MAC-6
(3 x lo6 cells) were seededout in 10 ml RPM1 1640 medium containing 10% FBS, 2 mM L-glutamine, 1 x MEM nonessential amino acids and 1 mM Na pyruvate with or without 10 pg/ml bovine insulin and cultivated for several days without replacement or addition of fresh medium. At the time points indicated, small aliquots were removed and cell numbers and
*Percentage of cells positive by flow cytometry. tND, not determined.
viabilities were determined. Data given as total viable cell number.
Results and Discussion (Endogen) was used at a final concentration of 10-100 &ml. The other cytokines were determined by an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (QuantikineR, R&D Systems).
Cytogenetic
analysis
The G-banded metaphase chromosomal preparations were obtained from MONO-MAC-l and MONO-MAC6 using standard cytogenetic protocols. Several rearrangements were confirmed by fluorescence in situ hybridization (FISH) analysis which was carried out as described previously in detail [6].
RNA isolation
and Northern
blot analysis
Total cellular RNA was isolated by the guanidinium isothiocyanate/cesium chloride method. Ten micrograms per lane were separated in a 1% agarose/2.2 M formaldehyde gel, transferred to a nylon membrane (Schleicher & Schilll, Dassel, Germany) and crosslinked with UV light (UV Stratalinker 1800, Stratagene, Heidelberg). Filters were hybridized overnight at 62°C with [32P]-labelled probes (USB Random Primed Labelling Kit, USB, Bad Homburg, Germany) specific for TNFc( (1.1 kb PstI fragment) and p-actin (1.3 kb PstI fragment [9]) and exposed at -80°C to X-ray films with intensifying screens for autoradiography.
Growth
characteristics
of MONO-MAC-1
and MONO-
MAC-6 The cell line MONO-MAC-l is growth factorindependent, continuously cultivated in RPM1 1640 medium supplemented with 5-10% FBS. Addition of glutamine, pyruvate and a cocktail of non-essential amino acids was beneficial for growth, but was not absolutely required. Figure 1 demonstrates cell expansion over a period of several days without addition or replacement of medium. Bovine insulin, which seemed to be necessary for the growth of MONO-MAC-6, was not essential for cultivation of MONO-MAC-l. Also, MONO-MAC-l cells usually were easier to handle than MONO-MAC-6 and subcultures could be initiated easily from the single cells. Starting with l-2 x lo5 cells/ml, MONO-MAC-l cultures reached a cell concentration higher than 2 x lo6 cells/ml, whereas the saturation density of MONO-MAC-6 was 1 x lo6 cells/ml, and the optimal concentration lay below 0.6 x lo6 cells/ml. Changes in morphology during differentiation
and surface antigen expression
Cultivation of MONO-MAC-l cells in the presence of LPS or TPA resulted in morphological changes usually associated with induction of differentiation, e.g. cell clustering, adherence and/or formation of pseudopodia. Cell clustering and adherence appeared rapidly within 6 h after addition of the inducers and were most prominent in TPA + LPS double-stimulated cultures. As evidenced by flow cytometry, the B- and T-cell
330
K. G. Steube et al.
Table 2. Time course of TNFc( secretion by MONO-MAC-l cells stimulated with different LPS concentrations* Time (h)
LPS Concentration (@ml) used 0.1 1.0 10
2 4 8 16 24 48
It 5.5 11
70 60 11 16 21 15
NDS 300 280 110 90 53
*The MONO-MAC-l cells (0.5 x 106/ml) were precultivated overnight in 24-well plates. Then medium, 0.1, 1 or 10 @ml LPS, respectively, were added. After the indicated time periods, supernatants of the cultures were analysed for TNFc( bioactivity. Cell-free medium with or without 10 @ml LPS served as controls and did not result in measurable cell lysis of L-929 cells. The rhTNFcc, in concentrations from 1 to 1000 pg/ml, was used in a parallel set-up for the quantitation of the induced TNFc( production. tTNFcc in pgiml. $ND, not determined.
marker CD3 and CD19 were clearly absent on MONOMAC-l and MONO-MAC-6 cells, whereas antigens characteristically found on cells committed to myeloid/ monocytic lineages, namely CD13, CD14, CD1.5, CD68 and HLA-DR, were present [6]. Table 1 shows a comparison of CD33 and CD14 expression after LPS or TPA treatment of MONO-MAC-l, and -6, and the two other monocytic cell lines U-937 and ML-2. The MONO-MAC-6 had variable levels of CD33 in several determinations ranging between 10 and 50%, while the other cell lines were negative or very weakly positive. Stimulation of the cell lines with TPA or LPS had no significant effect on the CD33 surface expression of any of the cell lines. On the other hand, the level of the CD14 molecules clearly increased on the surface of LPS- or TPA-treated MONO-MAC-l and MONOMAC-6 cells. Unstimulated ML-2 and U-937 were negative for CD14. Lipopolysaccharide, but not TPA, could induce CD14 expression on ML-2 cells. However, these markers remained unaffected after treatment of U937 with either of these drugs. Recently, the levels of CD14 and CD33 have been described as varying depending on the mAb used, the drug concentration and the mode of stimulation [lo, 111. Cytogenetic analyses The karyotype of MONO-MAC-l is near-diploid and has the following characteristics: XY, +8, +13, +21, t(10;13)(q23.3;q13) t(wl)(pl3;q23), adW)(W), t(12;17)(q21;qll), t(21;21)(922.l;qll/22), -17, +18, -20. Most rearrangements also occur in MONOMAC-6, implying their common origin in a precursor
clone. Both the reciprocal translocation between specific bands of chromosomes 9 and 11 (associated with AMLM.5) and the rearrangement of the long arm of chromosome 3 (associated with disordered platelet production) are known as primary changes in acute myeloid leukaemia [ 121. The remaining changes may be merely random ‘noise’ or, instead, actively promote tumour development. Recurrence of such chromosomal alterations in other reports of AML would tend to favour the latter explanation. The MONO-MAC-6 has acquired additional cytogenetic changes, including chromosome doubling (S = 94), which appears to have arisen in vitro. The MONO-MAC-l appears to have preserved the karyotypic features of the original primary leukaemia cells - one of only a minority of AML cell lines to have done so. Induction of TNFa secretion in MONO-MAC-l cells Tumour necrosis factorct is one of several cytokines produced by monocyteslmacrophages upon appropriate stimulation [13, 141. It activates a large number of cellular genes, i.e. other cytokines, is involved in the process of differentiation of haematopoietic cells and contributes to severe systemic reactions in mammals; thus, it plays a crucial role in inflammation and the immune response. The polyfunctional role of TNFcl in haematopoiesis, both as growth supporter and suppressor of progenitor cells, has led to its attribution as a ‘ying-yang’ molecule [15]. The TNFa is expressed initially as a 26 kDa membrane-anchored precursor protein which is processed proteolytically by a metalloproteinase to yield the mature 17 kDa polypeptide [16, 171. The biologically active form of TNFcr is a homotrimer with an apparent molecular weight of 52 kDa [18, 191. Since LPS is a potent inducer of TNFc( synthesis and secretion in several cellular systems [20221, we examined modes of TNFc! production by MONO-MAC-l by stimulating the cells for different periods and with various concentrations of LPS. In order to assess only the bioactive TNFc(, we measured its cytotoxic activity against L-929 cells. Culture medium of unstimulated MONO-MAC-l cells did not contain measurable TNFa activity. Soluble TNFc( activity was detected as early as 2 h after LPS addition and had a maximum between 4 and 8 h (Table 2). Thereafter, measurable TNFc( activity decreased in those MONOMAC-l cultures treated with low doses of LPS (0.1 ng/ ml or less) while, in cultures stimulated with high LPS doses (10 rig/ml), TNFc( activity could still be detected after 24 h. This result is in agreement with those of previous reports on LPS-induced TNFc( secretion in MONO MAC-6 cells [7,23-261. In other cellular systems, the combination of phorbol esters with LPS was used to induce TNFa secretion [17,27-291. We observed that TPA alone clearly
331
Cell line MONO-MAC-l [pg/mtl
TNF
TPA-pretreated
2
4
61624
h
2
4
61624
MAC-l
cells. Cell-free
from MONO-
culture medium containing
TPA
was unable to destroy the L-929 target cells. The released activity was not due to TNFa released from dead MONO-MAC-l cells because the viability of TPAtreated MONO MAC-l cultures were about 90% and remained unchanged during this 48 h incubation period. The time course and amount
of secreted
TNFa
was
similar to that seen after LPS stimulation (Fig. 2, lefthand side). When MONO-MAC-l cells TPA and LPS simultaneously,
LPS-pretreated
cells
h
Fig. 2. Synergistic effect of TPA and LPS on TNFcr secretion by MONO-MAC-l cells. The MONO-MAC-l cells (1.0 x 106/ ml) were seeded out in 24-well plates. After 1 h, medium, 100 nM TPA (left) or 0.1 rig/ml LPS + 100 nM TPA (right) were added. At the indicated time points, supernatants of the cultures were analysed for TNFc( bioactivity. Cell-free medium without or with 100 nM TPA, 1 @ml LPS or both served as controls in the TNFMTT-assay and did not result in measurable lysis of L-929 cells. For quantitation of the secreted TNFcl from the MONO-MAC-l cells, rhTNFcc, in concentrations from 1 to 1000 pg/ml, was added to L-929 cells in a parallel MlT-assay.
induced secretion of a TNFcr activity
cells
were stimulated with the concentration of
bioactive TNFa increased several-fold compared with those cultures stimulated with LPS or TPA alone (Fig. 2, right-hand side). This synergistic effect of TPA and LPS was even visible when only 0.05 or 0.1 rig/ml LPS (plus TPA) was used. To ensure that the toxic effects on L929 cells really were caused by TNFa, we added polyclonal anti-TNFa antibodies to the supernatants of TPA- or TPA+LPS-stimulated MONO-MAC-l cells and were able to inhibit completely the lysis of L-929 cells (data not shown). Since several binding sites for different transcription factors have been detected within the promoter region of the TNFc( gene ([23,30] and references therein), it is conceivable that the simultaneous stimulation by LPS and TPA results in a super-induction of TNFc( gene expression. The glycosyl-phosphatidylinositol anchored
Fig. 3. Effect of LPS or TPA pre-treatment on the stimulated TNFcl release. The MONO-MAC-l cells (1.0 x 106/ml) were seeded out in 24-well plates and cultivated in the presence of 100 nM TPA (left part) or 1 @ml LPS (right part) for 48 h. Then, 700 pl of medium was replaced by fresh medium containing TPA (final concentration 100 nM), LPS (final concentration 1 rig/ml) or TPA + LPS. A fourth miniculture remained untreated and served as control. After 5 h, the media were removed and assayed for TNFc( bioactivity. Results are expressed in percentage of dead L-929 cells (lysis) related to 0% dead (=lOO% viable cells) of the unstimulated control cultures. No measurable TNFcl activity was detected in the cultures treated for 48 h.
membrane protein CD14 is the receptor for LPS [31-331 and protein kinase C (PKC) is the molecular target of phorbol esters [34,35]. Incubation of MONO-MAC-l cells with either TPA and the PKC inhibitor H7, or with LPS and a mAb against CD14, decreased the TNFc( level in the culture medium by 50% for both settings (not shown). Conversely, H7 did not inhibit the response to LPS and anti-CD14 mAb did not prevent TPA-induced release
of
TNFc(.
These
results
suggest
that
both
receptors PKC and CD14 are involved individually in the signal transduction leading to TNFc( secretion of MONO-MAC-l cells. Pre-incubation of MONO-MAC-l with LPS or TPA modulates TNFcr secretion Since induction of differentiation is accompanied by changes in gene expression, we examined whether MONO-MAC-l cells, which have been cultivated for 48 h in the presence of TPA or LPS, were still able to release TNFr after a second challenge with LPS, TPA or both agents. Figure 3 (left-hand side) demonstrates that pre-incubation of MONO-MAC-l with 100 nM TPA decreased the amount of TNFx secreted after a second stimulus with TPA. Interestingly, when the second stimulus was carried out with LPS or LPS+TPA, the TNFz secretion was not inhibited. Vice versa, LPS-pre-
treated cells did not secrete TNFc( after a second LPS
332
K. G. Steube et al.
TNF
1.6 kb
actin
2.2 kb I
I
12345678910 Fig. 4. Induction of TNFc( mRNA in MONO-MAC-l cells. MONO-MAC-l cells were incubated without (lanes 1 and 6) or with 1.0 rig/ml LPS (lanes 2-5), or 100 nM TPA (lanes 7-10). After 2 (lanes 2 and 7) and 24 h (lanes 3 and 8), cells either were harvested directly or subsequently restimulated for 2 h with LPS (lanes 4 and 10) or TPA (lane 5 and 9) and then harvested. Total RNA isolated and Northern blot analysis was carried out with 10 ug RNA per lane and the blot was hybridized first to a 32P-labelled TNFa DNA probe and 6 weeks thereafter to a /I-actin DNA probe.
challenge. However, they were still able to release TNFc( when the second stimulation was carried out with TPA. Recently, it has been reported for MONO-MAC-6, but not for MONO-MAC-l, that LPS pre-treatment mediates tolerance to further stimuli with LPS [lo, 23, 251 and that this process was dependent on the LPS concentration used [lo]. Our experiments further demonstrate that TPA pre-treatment reprogrammed the cellular response of MONO-MAC-l to a subsequent stimulation, such that TNFa release was markedly inhibited, an observation which until now has been reported only for murine macrophages [36]. The LPS- and TPA-mediated regulation of TNFu mRNA The LPS stimulated transiently the de novo synthesis of TNFa mRNA in MONO-MAC-6 cells [23,28,37]. In other human cells, activation of PKC induced or promoted TNFc( mRNA expression with or without subsequent protein secretion [38-43]. Performing Northern blot analysis, we found that both LPS and TPA strongly increased the TNFa mRNA level in MONOMAC-l cells within 2 h; after 24 or 48 h, the amount of mRNA
declined back to that of unstimulated
cells (Fig.
4, lanes l-3 and 68). When LPS-precultured MONO-
MAC-l cells were stimulated a second time with the same concentration LPS or with TPA, only a weak increase of TNFcl mRNA was detectable (lanes 4 and 5), which is in agreement with results obtained with MONO-MAC-6 [23,25]. Even so, when TPA-pretreated cells were stimulated the second time with TPA or LPS (lanes 9 and lo), only LPS was able to reinduce the TNFa mRNA suggesting a regulatory modulation of the signal transduction via PKC after long-term treatment with phorbol esters. The stimulation of CD14 and PKC involves a cascade of protein phosphorylations on members of the Raf, Src and MAP kinase family [44-46]. Also, NFkB and IkB has been shown to be involved in regulation of TNFa transcription [23,47,48], and PKC is one of the kinases that phosphorylate IkB directly and also mediates the activation of NFkB [49,50]. Taken together, our results and these studies imply multiple and complex regulations of the TNFa gene expression in the monocytic system. Production of other cytokines Besides TNFr, the production of seven additional cytokines (SCF, IL-lp, IL-2, IL-3, M-CSF, G-CSF and
Cell line MONO-MAC-l
M-CSF (pglml)
I.200
333
G-CSF (pg/ml)
1
l.wor------
”
CO
LPS
TPA
TPA
LPS+TPA
GM-CSF (pg/ml)
IL-1 I3 (pg/ml)
Ifi
0
CO
LPS
TPA
LPS+TPA
0
CO
LPS
TPA
LPS+TPA
Fig. 5. Stimulation-dependent cytokine production of MONO-MAC-l cells. MONO-MAC-l cells (2 x 106/ml) were cultivated in 24-well plates in the absence or presence of 1 @ml LPS, 100 nM TPA or a combination of both. After 6 and 24 h, the supernatants were collected, centrifuged and stored frozen. Determination of M-CSF, G-CSF, GM-CSF, and IL-lp were carried out by ELISA. For the quantification of the secreted cytokines, recombinant standardized material was used.
GM-CSF) was studied. Unstimulated MONO-MAC-l cells did not secrete any of these cytokines but, 24 h after LPS treatment, 750 pg/ml G-CSF, 14 pg/ml IL-lb and 20 pg/ml GM-CSF was measured (Fig. 5). Other investigators had reported the LPS-induced production of IL-6 and IL-l by MONO-MAC-6 cells. The low amount of IL-lb we measured in the culture medium of MONO-MAC-l probably was due to either the intracellular storage of IL-l/I [25], or the low dose of LPS (1 rig/ml) we used. Interestingly, release of M-CSF could be induced by TPA but not by LPS. The TPA, which was inefficient in inducing release of the other three cytokines, turned out to exhibit a strong co-enhancing effect on the production of all four cytokines when used in combination with LPS (most dramatically for M-CSF and GM-CSF, with approximately 1 r&ml). Our results indicate that both signalling pathways, via PKC and via CD14, are involved in the release of M-CSF, GM-CSF, G-CSF and IL-l/I from MONO-MAC-l cells and that TPA and LPS act synergistically to increase further the amount of cytokines secreted into the medium.
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