- Email: [email protected]
MULTIPLE REGULATION OF CONSTITUTIVE AND INDUCED INTERLEUKIN 8 SECRETION IN HUMAN MYELOMONOCYTIC CELL LINES
doi:10.1006/cyto.2000.0702, available online at http://www.idealibrary.com on
SHORT COMMUNICATION
MULTIPLE REGULATION OF CONSTITUTIVE AND INDUCED INTERLEUKIN 8 SECRETION IN HUMAN MYELOMONOCYTIC CELL LINES Klaus G. Steube, Corinna Meyer, Hans G. Drexler Secretion of interleukin 8 (IL-8) and its regulation was investigated in myelomonocytic leukaemia cell lines. Quantification by ELISA revealed a constitutive production in the cell lines HL-60, ML-2, MONO-MAC-6 and MUTZ-3 ranging between 1500 and ca. 5000 pg/ml IL-8 per million cells. No measurable IL-8 was detected in the culture medium of MONO-MAC-1 and THP-1. Stimulation with lipopolysaccharide (LPS) or tetradecanoyl phorbol acetate (TPA) significantly increased the IL-8 level secreted by all cell lines; the best producers were TPA-treated MONO-MAC-6 and MUTZ-3 cultures, generating more than 50 000 pg/ml IL-8. Also the calcium ionophore A-23187, IL-13, macrophage colony-stimulating factor (M-CSF), thapsigargin, an inhibitor of the Ca2+ -ATPase, and tumour necrosis factor- (TNF-) strongly enhanced the IL-8 production in MONO-MAC-6 cells. The glucocorticoid dexamethasone and the protein kinase inhibitor staurosporine distinctively inhibited the IL-8 production of MONO-MAC-6 cells. Thus, our results demonstrate a strong constitutive IL-8 secretion in human myelomonocytic leukaemia cell lines; the variety of different modulators affecting IL-8 production leads to the suggestion of a multiple regulation of IL-8 expression and secretion. 2000 Academic Press
Chemokines (chemotactic cytokines) are well recognized key molecules involved in the recruitment of specific leukocyte subpopulations in several inflammatory disorders and cancer. Chemokines are mostly small proteins (8–12 kDa) and constitute several subfamilies. Interleukin 8 (IL-8) mainly attracts neutrophils and was purified in 1987 by several groups using LPSstimulated peripheral blood mononuclear cells and to date, activated monocytes and neutrophils are believed to be the predominant IL-8-producing cells in the peripheral blood. Besides haematopoietic cells, other cell types also secreted IL-8, especially after stimulation with IL-1, LPS or TNF- (for review see1,2). From the Department of Human and Animal Cell Cultures, DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany Correspondence to: Dr K. Steube, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1B, D-38124 Braunschweig, Germany; E-mail: [email protected] Received 9 August 1999; received in revised form 31 January 2000; accepted for publication 5 April 2000 2000 Academic Press 1043–4666/00/081236+04 $35.00/0 KEY WORDS: chemokine/IL-8/monocytic leukaemia/cell lines/ MONO-MAC-6 1236
Much work on IL-8 secretion was carried out with normal mononuclear cells but less with haematopoietic malignant cells or cell lines. Therefore, we performed studies on IL-8 production in the malignant counterpart of normal myeloid or monocyte precursors and used growth-arrested myelomonocytic leukaemia cell lines. The ability of these cell lines to produce IL-8 and the modulation of its secretion is described.
RESULTS AND DISCUSSION Constitutive production and LPS- and TPA-induced secretion of IL-8 To our knowledge, this is the first report showing constitutive IL-8 secretion in haematopoietic cell lines: HL-60, ML-2, MONO-MAC-6, and MUTZ-3 constitutively secreted >1000 pg/ml, whereas MONOMAC-1 and THP-1 only <100 pg/ml soluble IL-8 per 106 cells (Table 1). In some myeloid cell lines low levels of IL-8 mRNA were found,3 but not in the histiocytic lymphoma U-937.4 LPS and phorbol esters are able to modulate gene expression in myelomonocytic leukaemia cells. We found that TPA (10 7 M or 10 8 M) and LPS CYTOKINE, Vol. 12, No. 8 (August), 2000: pp 1236–1239
Chemokine secretion by cell lines / 1237
TABLE 1. of IL-8
(0.1–100 ng/ml) either enhanced or newly induced secretion of IL-8 in all six cell lines, whereby TPA was more efficient than LPS (Table 1). TPA-stimulated cell lines MONO-MAC-6 and MUTZ-3 both elaborated >50 000 pg/ml IL-8. MONO-MAC-1, genetically identical to MONO-MAC-6, secreted much less IL-8 in response to TPA or LPS. Since we did not find intracellular IL-8 in MONO-MAC-1, we assume that IL-8 is indeed induced in these cells. TPA-induction of IL-8, as well as of monocyte chemotactic protein-1, MCP-1,5 indicates that chemokine expression can be activated via protein kinase C. IL-8 secretion was enhanced by LPS, even in those cell lines (HL-60, ML-2, MONO-MAC-1, THP-1) which do not express the membrane-bound LPS receptor, CD14. Thus, an CD14-independent pathway or soluble forms of CD14 may exist.6 Also, low amounts of membrane-bound CD14 may be present on the CD14-negative cell lines which are below the detection limit.
Constitutive, LPS- and TPA-induced production Treatment
Cell line HL-60 ML-2 MONO-MAC-1 MONO-MAC-6 MUTZ-3 THP-1
a
None
LPS
1750400 1530620 <100 51001240 3025680 <100
2150370 2900410 ca. 900 24 5002600 12 3002100 ca. 500
TPAb 62001200 16 9002100 7600900 >50 000 >50 000 <1000
Cell lines are deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). A detailed description of cell lines and culture conditions are available via. The cell line MUTZ-3 is growth factor-dependent and was cultivated in the presence of GM-CSF. Experiments were performed as described in Materials and Methods; the soluble, extracellular IL-8 concentration was determined by specific ELISA and mean values (pg/ml) of three to eight independent experiments were calculated as for 106 cells within 24 h. a LPS (0.1 ng/ml for MONO-MAC-6, 100 ng/ml for MONO-MAC-1, 1 and 10 ng/ml for the other cell lines), or bTPA (10 8 M except for MONO-MAC-1, 10 7 M) were present during the 24 h incubation period.
IL-8 (ng/ml) in the culture supernatant
25
20
15
10
5
0
Figure 1.
None
10 100 Treatment with A-23187
5
500 or
10 20 Thapsigargin (nM)
40
Stimulation of IL-8 secretion by Ca2+ -mobilizing agents.
MONO-MAC-6 cells were incubated with the indicated concentrations of the calcium ionophore A-23187 for 24 h and with the inhibitor of the intracellular Ca2+ -ATPase, thapsigargin, for 6 h, respectively. Soluble, extracellular IL-8 was determined by ELISA. The data shown are mean values of two independent experiments with less then 15% standard deviation and are given as for 106 cells/ml. Concentrations higher than 510 7 M of A-23187 and 510 8 M of thapsigargin turned out to be too toxic within a 24 h cultivation time and results were therefore not included in this study.
1238 / Steube et al.
Stimulatory index
8
7.4
6
4.9
4
3
2
2.4
1.8
1.4
1
3.9
3.7
3.2
2.5
2.5
0.5
N on e D A -2 A- ex 31 23 87 18 + 7 D IF I ex N FN y + y D IL I ex L -1 3 13 + M M De -C - x S F CS + F D e LP L x P S + S D e TN T x F NF + D ex
0
IL-8 (ng/ml) in the culture supernatant
B
41
40
30.1
30 18.7
20
9.6
10
11.1 5.6
2.05 Fo s +
LP
S LP +
TP A
TP A
+
+ S
S
Fo s
S LP
Fo s +
LP
on
TP A
e
(F os
TP A
)
0
25
23.5
C
20.1
20 15
12.3 10
10
10.2
9.5 5.8
5
7.1
3.4
2
4.9
1.7 87 318 + 7 St au LP L S PS + St au M M -C C SF S + F St au TN T N F + F St au TP T A PA + St au
u
St a A
-2
31
A
-2
on
e
0 N
The effect of several cytokines was analyzed in MONO-MAC-6 cells. None of the cytokines tested was inhibitory. A low stimulation index (<2) was observed with IFN- (100 U/ml), IL-1 (1 ng/ml), IL-4 (10 ng/ml), IL-6 (100 ng/ml), and IL-10 (100 ng/ml). A moderate effect (stimulation index 2–3) was induced by GM-CSF (500 U/ml), IL-2 (0.1 ng/ml), IL-3 (100 ng/ml), IL-15 (250 ng/ml); strong enhancers were 50 ng/ml IL-13 (index >3), 20 ng/ml M-CSF (index >4), and 10 ng/ml TNF- (index >8). The strong response to TNF-, also observed in other systems,7,8 may be due to activation of NFB which is involved in transcription of the IL-8 gene.9,10 IL-4 and IL-10 decreased chemokine expression in normal cells,11,12 however, these cytokines exhibit variable functions in different cell systems.5,13 Intracellular free calcium (Ca2+ ) is important for an efficient secretion of IL-8. The calcium ionophore A-23187 and the inhibitor of the intracellular Ca2+ ATPase, thapsigargin,14 dose-dependently elevated the IL-8 secretion of MONO-MAC-6 cells by 4–5-fold (Fig. 1). Thapsigargin further induced cytokine expression in T-lymphocytes15 and normal neutrophils.16 Inhibitors of induced IL-8 expression are shown in Figure 2. Dexamethasone efficiently suppressed IL-8 production by 36–66%. A glucocorticoid response element is present in the 5 region of the IL-8 gene17 and, consequently, inhibition by dexamethasone was reported in other systems.5,18 Thus, glucocorticoids may be important regulators of chemokine gene expression. The immunomodulatory antibiotic fosfomycin inhibited IL-8 production by 27–50% which is in agreement with results published recently.19 Inhibition of secretion by 15–75% by the protein kinase (PK) inhibitor staurosporine, in non-toxic concentrations, demonstrates the involvement of PKs in the signalling pathway controlling IL-8 production and may propose this natural compound as an antiinflammatory agent. In conclusion, we described constitutive and induced IL-8 secretion in human myelomonocytic leukaemia cell lines. The modulation by other cytokines, protein kinases, intracellular free calcium, glucocorticoids and NFB indicates multiple regulation at different cellular levels and suggests the cell lines used as model systems for studies on chemokine expression.
9.3
N
Other modulators of IL-8 production
10 A
IL-8 (ng/ml) in the culture supernatant
The cell line MUTZ-3 is growth factor-dependent and was cultivated with GM-CSF. Thus, the question remains whether its secretion is constitutive or induced. Indeed, IL-8 mRNA expression and protein secretion was inducible by GM-CSF.4
CYTOKINE, Vol. 12, No. 8 (August, 2000: 1236–1239)
Figure 2.
Inhibition of the induced IL-8 production.
(A) MONO-MAC-6 cells were incubated for 24 h without or with dexamethasone (Dex, 10 6 M) and the following inducers: A-23187 (510 7 M), IFN- (200 U/ml), IL-13 (50 ng/ml), LPS (1 ng/ml), M-CSF (20 ng/ml), and TNF- (10 ng/ml). IL-8 in the supernatants was determined by ELISA, mean values of at least three experiments were calculated and data are expressed as stimulatory (inhibitory) index in relation to untreated control cultures. (B) Cells were incubated without or with LPS (1 ng/ml), TPA (10 8 M), LPS plus TPA in the absence or presence of fosfomycin (Fos, 500 g/ml) or (C) with A-23187, LPS, M-CSF, TNF-, or TPA in the absence or presence of staurosporine (Stau, 10 ng/ml) for 24 h. The IL-8 amount secreted was determined by ELISA and the values depicted are representative of at least three experiments.
Chemokine secretion by cell lines / 1239
MATERIALS AND METHODS Reagents Culture media and supplements were from LifeTechnologies (Karlsruhe, Germany); fetal bovine serum, dexamethasone, the calcium ionophore A-23187, LPS, staurosporine, thapsigargin and TPA from Sigma (Deisenhofen, Germany). Fosfomycin was from ICN, Aurora, Ohio, USA. Interleukin-1 (IL-1, specific activity 5108 U/mg) was obtained from Amersham (Braunschweig, Germany). Granulocyte-macrophage colony-stimulating factor (GM-CSF, specific activity >1107 U/mg), interferon- (IFN-, specific activity >2107 U/mg), IL-2 (specific activity >2106 U/mg), IL-4 (specific activity >5105 U/mg), IL-6 (specific activity >1108 U/mg), and leukaemia inhibitory factor (LIF, specific activity 1107 U/mg) were purchased from Roche-Diagnostics (Mannheim, Germany). IL-3 (specific activity 4106 U/mg), IL-13 (specific activity >2105 U/mg), IL-15 (specific activity >0.5106 U/mg), and macrophage colony-stimulating factor (M-CSF, specific activity >0.6106 U/mg) were received from R & D Systems (Wiesbaden, Germany). IL-10 (specific activity 5105 U/mg) was obtained from Pharma Biotechnologie (Hannover, Germany) and tumor necrosis factor- (TNF-, specific activity >2106 U/mg) from Biermann (Bad Nauheim, Germany).
Stimulation of cells and quantitative determination of IL-8 Experiments were carried out in 24-well culture plates (Nunc, Wiesbaden, Germany) with starting cell concentrations of 5105 cells/ml. Culture supernatants were routinely collected 6 and 24 h after start of the cultivation, centrifuged (5 min at 10 000g) and stored frozen. The concentrations of IL-8 were determined by a double-ligand ELISA assay according to the manufacturer’s protocol (R & D). Samples that gave results outside the standard range were assayed again at an appropriate dilution.
REFERENCES 1. Bagglioni M, Dewald B, Moser B (1997) Human chemokines: an update. Annu Rev Immunol 15:675–705. 2. Rollins BJ (1997) Chemokines. Blood 90:909–928. 3. Meier RW, Niklaus G, Dewald B, Fey MF, Tobler A (1995) Inhibition of the arachidonic acid pathway prevents induction of IL-8 mRNA by phorbol ester and changes the release of IL-8 from HL-60 cells: differential inhibition of induced expression of IL-8, TNF-, IL-1 and IL-1. J Cell Physiol 165:62–70. 4. Takahashi GW, Andrews DF, Lilly MB, Singer JW, Alderson MR (1993) Effect of GM-CSF and IL-3 on IL-8
production by human neutrophils and monocytes. Blood 81: 357–364. 5. Steube KG, Meyer C, Drexler HG (1999) Constitutive protein expression of MCP-1 by myelomonocytic cell lines and regulation of the secretion by anti- and proinflammatory stimuli. Leuk Res 23:843–849. 6. Shapira L, Takashiba S, Champagne C, Amar S, Van Dyke TE (1994) Involvement of PKC and PTK in LPS-induced TNF- and IL-1 production by human monocytes. J Immunol 153: 1818–1824. 7. Strieter RM, Kunkel SL, Showell HJ, Remick DG, Phan SH, Ward PS, Marks RM (1989) Endothelial cell gene expression of a neutrophil chemotactic factor by TNF and IL-1. Science 243:1467–1469. 8. Schu¨ rer-Maly CC, Eckmann L, Kagnoff MF, Falco MT, Maly FE (1994) Colonic epithelial cell lines as a source of IL-8: stimulation by inflammatory cytokines and bacterial LPS. Immunology 81:85–91. 9. Yasumoto K, Okamoto S, Mukaida N, Murakami S, Mai M, Matsushima K (1992) TNF and IFN synergistically induce IL-8 production in a human gastric cancer cell line through acting concurrently on AP-1 and NF-B-like binding sites of the IL-8 gene. J Biol Chem 267:22506–22511. 10. Tanaka C, Kamata H, Takeshita H, Yagisawa H, Hirata H (1997) Redox regulation of LPS-induced IL-8 gene expression mediated by NF-B and AP-1 in human astrocytoma U-373 cells. Biochem Biophys Res Commun 232:568–573. 11. Casatella MA, Meda L, Bonora S, Ceska M, Constantin G (1993) Interleukin 10 inhibits the release of proinflammatory cytokines from human polymorphonuclear leukocytes. J Exp Med 178:2207–2211. 12. De Waal-Malefyt R, Abrams J, Bennett B, Figdor CG, Vries JE (1991) IL-10 inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J Exp Med 174:1209–1220. 13. Seitz M, Loetscher P, Dewald B, Towbin H, Gallati H, Baggiolini M (1995) IL-10 differentially regulates cytokine inhibition and chemokine release from blood mononuclear cells and fibroblasts. Eur J Immunol 25:1129–1132. 14. Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson EJ (1990) Thapsigargin, a tumor promotor, discharges intracellular Ca + + stores by specific inhibition of the endoplasmic reticulum Ca + + ATPase. Proc Natl Acad Sci USA 87:2466–2470. 15. Negulescu PA, Shastri N, Cahalan MD (1994) Intracellular calcium dependence of gene expression in single T lymphocytes. Proc Natl Acad Sci USA 91:2873–2877. 16. Kuhns DB, Young HA, Gallin EK, Gallin JI (1998) Ca + + dependent production and release of IL-8 in human neutrophils. J Immunol 161:4332–4339. 17. Mukaida N, Gusella GL, Kasahara T, Ko Y, Zachariae COC, Kawai T, Matsushima K (1992) Molecular analysis of the inhibition of IL-8 production by dexamethasone in a human fibrosarcoma cell line. Immunology 75:674–679. 18. Levine SJ, Larivee P, Logun C, Angus CW, Shelhamer JH (1993) Corticosteroids differentially regulate secretion of IL-6, IL-8, and G-CSF by a human bronchial epithelial cell line. Am J Physiol 265:L360–L368. 19. Honda J, Okubo Y, Kusaba M, Kumagi M, Saruwatari N, Oizumi K (1998) Fosfomycin suppresses the production of IL-8 from monocytes via the suppression of neutrophil function. Immunopharmacology 39:149–155.
SHORT COMMUNICATION
MULTIPLE REGULATION OF CONSTITUTIVE AND INDUCED INTERLEUKIN 8 SECRETION IN HUMAN MYELOMONOCYTIC CELL LINES Klaus G. Steube, Corinna Meyer, Hans G. Drexler Secretion of interleukin 8 (IL-8) and its regulation was investigated in myelomonocytic leukaemia cell lines. Quantification by ELISA revealed a constitutive production in the cell lines HL-60, ML-2, MONO-MAC-6 and MUTZ-3 ranging between 1500 and ca. 5000 pg/ml IL-8 per million cells. No measurable IL-8 was detected in the culture medium of MONO-MAC-1 and THP-1. Stimulation with lipopolysaccharide (LPS) or tetradecanoyl phorbol acetate (TPA) significantly increased the IL-8 level secreted by all cell lines; the best producers were TPA-treated MONO-MAC-6 and MUTZ-3 cultures, generating more than 50 000 pg/ml IL-8. Also the calcium ionophore A-23187, IL-13, macrophage colony-stimulating factor (M-CSF), thapsigargin, an inhibitor of the Ca2+ -ATPase, and tumour necrosis factor- (TNF-) strongly enhanced the IL-8 production in MONO-MAC-6 cells. The glucocorticoid dexamethasone and the protein kinase inhibitor staurosporine distinctively inhibited the IL-8 production of MONO-MAC-6 cells. Thus, our results demonstrate a strong constitutive IL-8 secretion in human myelomonocytic leukaemia cell lines; the variety of different modulators affecting IL-8 production leads to the suggestion of a multiple regulation of IL-8 expression and secretion. 2000 Academic Press
Chemokines (chemotactic cytokines) are well recognized key molecules involved in the recruitment of specific leukocyte subpopulations in several inflammatory disorders and cancer. Chemokines are mostly small proteins (8–12 kDa) and constitute several subfamilies. Interleukin 8 (IL-8) mainly attracts neutrophils and was purified in 1987 by several groups using LPSstimulated peripheral blood mononuclear cells and to date, activated monocytes and neutrophils are believed to be the predominant IL-8-producing cells in the peripheral blood. Besides haematopoietic cells, other cell types also secreted IL-8, especially after stimulation with IL-1, LPS or TNF- (for review see1,2). From the Department of Human and Animal Cell Cultures, DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany Correspondence to: Dr K. Steube, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1B, D-38124 Braunschweig, Germany; E-mail: [email protected] Received 9 August 1999; received in revised form 31 January 2000; accepted for publication 5 April 2000 2000 Academic Press 1043–4666/00/081236+04 $35.00/0 KEY WORDS: chemokine/IL-8/monocytic leukaemia/cell lines/ MONO-MAC-6 1236
Much work on IL-8 secretion was carried out with normal mononuclear cells but less with haematopoietic malignant cells or cell lines. Therefore, we performed studies on IL-8 production in the malignant counterpart of normal myeloid or monocyte precursors and used growth-arrested myelomonocytic leukaemia cell lines. The ability of these cell lines to produce IL-8 and the modulation of its secretion is described.
RESULTS AND DISCUSSION Constitutive production and LPS- and TPA-induced secretion of IL-8 To our knowledge, this is the first report showing constitutive IL-8 secretion in haematopoietic cell lines: HL-60, ML-2, MONO-MAC-6, and MUTZ-3 constitutively secreted >1000 pg/ml, whereas MONOMAC-1 and THP-1 only <100 pg/ml soluble IL-8 per 106 cells (Table 1). In some myeloid cell lines low levels of IL-8 mRNA were found,3 but not in the histiocytic lymphoma U-937.4 LPS and phorbol esters are able to modulate gene expression in myelomonocytic leukaemia cells. We found that TPA (10 7 M or 10 8 M) and LPS CYTOKINE, Vol. 12, No. 8 (August), 2000: pp 1236–1239
Chemokine secretion by cell lines / 1237
TABLE 1. of IL-8
(0.1–100 ng/ml) either enhanced or newly induced secretion of IL-8 in all six cell lines, whereby TPA was more efficient than LPS (Table 1). TPA-stimulated cell lines MONO-MAC-6 and MUTZ-3 both elaborated >50 000 pg/ml IL-8. MONO-MAC-1, genetically identical to MONO-MAC-6, secreted much less IL-8 in response to TPA or LPS. Since we did not find intracellular IL-8 in MONO-MAC-1, we assume that IL-8 is indeed induced in these cells. TPA-induction of IL-8, as well as of monocyte chemotactic protein-1, MCP-1,5 indicates that chemokine expression can be activated via protein kinase C. IL-8 secretion was enhanced by LPS, even in those cell lines (HL-60, ML-2, MONO-MAC-1, THP-1) which do not express the membrane-bound LPS receptor, CD14. Thus, an CD14-independent pathway or soluble forms of CD14 may exist.6 Also, low amounts of membrane-bound CD14 may be present on the CD14-negative cell lines which are below the detection limit.
Constitutive, LPS- and TPA-induced production Treatment
Cell line HL-60 ML-2 MONO-MAC-1 MONO-MAC-6 MUTZ-3 THP-1
a
None
LPS
1750400 1530620 <100 51001240 3025680 <100
2150370 2900410 ca. 900 24 5002600 12 3002100 ca. 500
TPAb 62001200 16 9002100 7600900 >50 000 >50 000 <1000
Cell lines are deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). A detailed description of cell lines and culture conditions are available via
IL-8 (ng/ml) in the culture supernatant
25
20
15
10
5
0
Figure 1.
None
10 100 Treatment with A-23187
5
500 or
10 20 Thapsigargin (nM)
40
Stimulation of IL-8 secretion by Ca2+ -mobilizing agents.
MONO-MAC-6 cells were incubated with the indicated concentrations of the calcium ionophore A-23187 for 24 h and with the inhibitor of the intracellular Ca2+ -ATPase, thapsigargin, for 6 h, respectively. Soluble, extracellular IL-8 was determined by ELISA. The data shown are mean values of two independent experiments with less then 15% standard deviation and are given as for 106 cells/ml. Concentrations higher than 510 7 M of A-23187 and 510 8 M of thapsigargin turned out to be too toxic within a 24 h cultivation time and results were therefore not included in this study.
1238 / Steube et al.
Stimulatory index
8
7.4
6
4.9
4
3
2
2.4
1.8
1.4
1
3.9
3.7
3.2
2.5
2.5
0.5
N on e D A -2 A- ex 31 23 87 18 + 7 D IF I ex N FN y + y D IL I ex L -1 3 13 + M M De -C - x S F CS + F D e LP L x P S + S D e TN T x F NF + D ex
0
IL-8 (ng/ml) in the culture supernatant
B
41
40
30.1
30 18.7
20
9.6
10
11.1 5.6
2.05 Fo s +
LP
S LP +
TP A
TP A
+
+ S
S
Fo s
S LP
Fo s +
LP
on
TP A
e
(F os
TP A
)
0
25
23.5
C
20.1
20 15
12.3 10
10
10.2
9.5 5.8
5
7.1
3.4
2
4.9
1.7 87 318 + 7 St au LP L S PS + St au M M -C C SF S + F St au TN T N F + F St au TP T A PA + St au
u
St a A
-2
31
A
-2
on
e
0 N
The effect of several cytokines was analyzed in MONO-MAC-6 cells. None of the cytokines tested was inhibitory. A low stimulation index (<2) was observed with IFN- (100 U/ml), IL-1 (1 ng/ml), IL-4 (10 ng/ml), IL-6 (100 ng/ml), and IL-10 (100 ng/ml). A moderate effect (stimulation index 2–3) was induced by GM-CSF (500 U/ml), IL-2 (0.1 ng/ml), IL-3 (100 ng/ml), IL-15 (250 ng/ml); strong enhancers were 50 ng/ml IL-13 (index >3), 20 ng/ml M-CSF (index >4), and 10 ng/ml TNF- (index >8). The strong response to TNF-, also observed in other systems,7,8 may be due to activation of NFB which is involved in transcription of the IL-8 gene.9,10 IL-4 and IL-10 decreased chemokine expression in normal cells,11,12 however, these cytokines exhibit variable functions in different cell systems.5,13 Intracellular free calcium (Ca2+ ) is important for an efficient secretion of IL-8. The calcium ionophore A-23187 and the inhibitor of the intracellular Ca2+ ATPase, thapsigargin,14 dose-dependently elevated the IL-8 secretion of MONO-MAC-6 cells by 4–5-fold (Fig. 1). Thapsigargin further induced cytokine expression in T-lymphocytes15 and normal neutrophils.16 Inhibitors of induced IL-8 expression are shown in Figure 2. Dexamethasone efficiently suppressed IL-8 production by 36–66%. A glucocorticoid response element is present in the 5 region of the IL-8 gene17 and, consequently, inhibition by dexamethasone was reported in other systems.5,18 Thus, glucocorticoids may be important regulators of chemokine gene expression. The immunomodulatory antibiotic fosfomycin inhibited IL-8 production by 27–50% which is in agreement with results published recently.19 Inhibition of secretion by 15–75% by the protein kinase (PK) inhibitor staurosporine, in non-toxic concentrations, demonstrates the involvement of PKs in the signalling pathway controlling IL-8 production and may propose this natural compound as an antiinflammatory agent. In conclusion, we described constitutive and induced IL-8 secretion in human myelomonocytic leukaemia cell lines. The modulation by other cytokines, protein kinases, intracellular free calcium, glucocorticoids and NFB indicates multiple regulation at different cellular levels and suggests the cell lines used as model systems for studies on chemokine expression.
9.3
N
Other modulators of IL-8 production
10 A
IL-8 (ng/ml) in the culture supernatant
The cell line MUTZ-3 is growth factor-dependent and was cultivated with GM-CSF. Thus, the question remains whether its secretion is constitutive or induced. Indeed, IL-8 mRNA expression and protein secretion was inducible by GM-CSF.4
CYTOKINE, Vol. 12, No. 8 (August, 2000: 1236–1239)
Figure 2.
Inhibition of the induced IL-8 production.
(A) MONO-MAC-6 cells were incubated for 24 h without or with dexamethasone (Dex, 10 6 M) and the following inducers: A-23187 (510 7 M), IFN- (200 U/ml), IL-13 (50 ng/ml), LPS (1 ng/ml), M-CSF (20 ng/ml), and TNF- (10 ng/ml). IL-8 in the supernatants was determined by ELISA, mean values of at least three experiments were calculated and data are expressed as stimulatory (inhibitory) index in relation to untreated control cultures. (B) Cells were incubated without or with LPS (1 ng/ml), TPA (10 8 M), LPS plus TPA in the absence or presence of fosfomycin (Fos, 500 g/ml) or (C) with A-23187, LPS, M-CSF, TNF-, or TPA in the absence or presence of staurosporine (Stau, 10 ng/ml) for 24 h. The IL-8 amount secreted was determined by ELISA and the values depicted are representative of at least three experiments.
Chemokine secretion by cell lines / 1239
MATERIALS AND METHODS Reagents Culture media and supplements were from LifeTechnologies (Karlsruhe, Germany); fetal bovine serum, dexamethasone, the calcium ionophore A-23187, LPS, staurosporine, thapsigargin and TPA from Sigma (Deisenhofen, Germany). Fosfomycin was from ICN, Aurora, Ohio, USA. Interleukin-1 (IL-1, specific activity 5108 U/mg) was obtained from Amersham (Braunschweig, Germany). Granulocyte-macrophage colony-stimulating factor (GM-CSF, specific activity >1107 U/mg), interferon- (IFN-, specific activity >2107 U/mg), IL-2 (specific activity >2106 U/mg), IL-4 (specific activity >5105 U/mg), IL-6 (specific activity >1108 U/mg), and leukaemia inhibitory factor (LIF, specific activity 1107 U/mg) were purchased from Roche-Diagnostics (Mannheim, Germany). IL-3 (specific activity 4106 U/mg), IL-13 (specific activity >2105 U/mg), IL-15 (specific activity >0.5106 U/mg), and macrophage colony-stimulating factor (M-CSF, specific activity >0.6106 U/mg) were received from R & D Systems (Wiesbaden, Germany). IL-10 (specific activity 5105 U/mg) was obtained from Pharma Biotechnologie (Hannover, Germany) and tumor necrosis factor- (TNF-, specific activity >2106 U/mg) from Biermann (Bad Nauheim, Germany).
Stimulation of cells and quantitative determination of IL-8 Experiments were carried out in 24-well culture plates (Nunc, Wiesbaden, Germany) with starting cell concentrations of 5105 cells/ml. Culture supernatants were routinely collected 6 and 24 h after start of the cultivation, centrifuged (5 min at 10 000g) and stored frozen. The concentrations of IL-8 were determined by a double-ligand ELISA assay according to the manufacturer’s protocol (R & D). Samples that gave results outside the standard range were assayed again at an appropriate dilution.
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