Immunology Letters, 28 (1991) 121-126 Elsevier IMLET 01568
Interleukin-3 enhances cytokine production by LPS-stimulated macrophages L u c h i n o C o h e n , B e r n a r d D a v i d a n d J e a n - M a r c Cavaillon Unitd d'Immuno-Allergie, Institut Pasteur, Paris, France (Received 9 November 1990; revision received 10 January 1991; accepted 17 January 1991)
1. Summary
In addition to its hematopoietic activities, interleukin-3 (IL-3) can modulate macrophage functions. We have studied the production of interleukin1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor (TNF) by mouse peritoneal macrophages triggered by lipopolysaccharide (LPS) in the presence or absence of IL-3. Interleukin-3 at the concentration used (i.e., 100 U/ml) did not induce the production of any cytokines, whereas it enhanced significantly the secretion of IL-1, IL-6 and TNF by LPSstimulated macrophages. The synergistic activity of IL-3 was observed over a wide range of Escherichia coli or Salmonella enteritidis LPS concentrations. No additive effect was noticed between IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF), another factor able to enhance LPSinduced IL-1 production. Thus, IL-3 can potentiate the inflammatory response induced by endotoxin from Gram-negative bacteria through a potentiation of cytokine production. 2. Introduction
Interleukin-3 (IL-3) is one of the hematopoietic growth factors produced by activated T cells. It was initially defined as a factor which could induce the expression of the enzyme 20-a-hydroxysteroid dehydrogenase by splenic lymphocytes from athymic mice [1]. IL-3 has been found to support the prolifer-
ation and differentiation of early hematopoietic stem cells, which differentiate into macrophages, granulocytes, eosinophiles, megakaryocytes or mast cells [2]. Other cytokines such as IL-1 [3], IL-6 [4, 5] and granulocyte-macrophage colony-stimulating factor (GM-CSF) [6] are able to enhance the IL-3induced hematopoiesis. Recent studies have described the ability of IL-3 to regulate the proliferation of blood monocytes and peritoneal exudate macrophages [7], the killing of Candida albicans by murine macrophages [8], the expression of tumor necrosis factor (TNFc0 mRNA by human monocytes [9] and the adhesion of human monocytes to endothelial cells [10]. Another study suggests that IL-3 enhances the antigen-presenting function of macrophages [11]. Taken together, these data imply that monocytes and macrophages are receptive to IL-3. Weiss et al. [12] have previously reported that IL-3 can enhance the IL-I release induced by lipopolysaccharide (LPS) in serum-free cultures of human monocytes. In the present study, we have investigated whether LPS-induced IL-1 secretion by more mature cells like mouse peritoneal macrophages, could also be regulated by IL-3. In addition, because of the evolutionary linkage between IL-3 and GM-CSF, we compared the action of the two cytokines. We also studied the regulatory effect of IL-3 on IL-6 and TNF release by these cells. 3. Materials and Methods
Key words: lnterleukin-3; Cytokine production; Macrophage; Lipopolysaccharide
3.1. Mice
Correspondence to: J.-M. Cavaillon, Unit6 d'Immuno-Allergie, Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France
Eight to twelve-week-old C57BI/6 and BALB/c mice were purchased from the Pasteur Institute
0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V.
121
breeding center and were maintained in our animal facility.
rophages. The lysates were then collected and stored as described for the supernatants, and tested for the cell-associated IL-1 activity.
3.2. Reagents 3.5. IL-1 bioassay Murine rIL-3 and rGM-CSF were purchased from Genzyme Corp. (Boston, MA), human rIL-1t3 was obtained from Rh6ne-Poulenc (France), murine rIL6 was a kind gift of Dr. J. van Snick and rTNFc~ was generously provided by Rh6ne-Poulenc (France). Escherichia coli (Olll:B4) LPS was obtained from Sigma (U.S.A.), Salmonella enteritidis LPS was from Difco (U.S.A.) and concanavalin A (Con A) was purchased from Miles (U.S.A.). 3.3. Isolation of peritoneal exudate macrophages
(PEM) PEM were harvested from the animals by peritoneal lavages with RPMI-1640 (Gibco, U.K.) supplemented with heparin (Roche, France), 3 or 4 days after the intraperitoneal injection of 1.5 ml of thioglycolate medium (Diagnostic Pasteur, France), and were washed with RPMI-1640 supplemented with 100 U/ml penicillin and 100 m g / m l streptomycin (Eurobio, France). The cell number, viability and percentage of macrophages were determined under a microscope. Cells were then plated into 24-well flat-bottomed culture trays (Falcon, Becton Dickinson Labware, U.K.) at a density of 5 x 105 macrophages/well. After 1 h, non-adherent cells were removed by vigorous washing with RPMI-1640. 3.4. Induction of IL-1 expression PEM were cultured in the absence or presence of LPS and cytokines for 24 h, in 0.5 ml of a culture medium composed of RPMI-1640 supplemented with 2 mM L-glutamine (Eurobia, France), 1 ~g/ml of indomethacin (Sigma, U.S.A.) and antibiotics. Preliminary experiments have established that 100 U/ml of rIL-3 or 60 U/ml of rGM-CSF led to optimal results. The supernatants were collected at the end of the induction period, centrifuged at 2 0 0 × g and stored frozen at - 3 0 ° C before determining their secreted cytokine activities. A volume of 0.5 ml/well of culture medium was added after the collection of the supernatants, the culture trays were then frozen and thawed 3 times to lyse the mac122
IL-1 activity in the PEM supernatants (l:10) was determined in the murine thymocyte proliferation assay: the supernatants were tested for their ability to enhance C 3 H / H e J routine thymocyte incorporation of [3H]TdR in the presence of a suboptimal concentration of Concanavalin A (0.9 ~g/ml) as previously described [13]. We ensured that residual amounts of IL-3 (i.e., < 10 U/ml) neither influence the IL-1 thymocyte proliferation as reported for high concentrations [14], nor modify the other biological assays. 3.6. TNF bioassay The T N F activity was determined by testing the cytotoxicity of serial dilutions of the supernatants in the presence of L929 fibroblasts, as previously described [15]. One unit of T N F activity was defined as the amount required to lyse 50% of L929 target cells. 3.7. IL-6 bioassay IL-6 activity was determined by using the IL-6dependent 7TD1 cell line [16]. Cells were cultured at a density of 1300 cells/well, in 100 ~1 of RPMI-1640 medium supplemented with antibiotics, 2mercaptoethanol (5 × 10 5 M) and 10% FCS, in the presence of serial dilutions of supernatants from stimulated PEM. After 4 days of culture at 37 °C, the proliferation was determined by a dye method [171:125 txg of tetrazolium salts (MTT) were added to each well and after 1 or 2 h of incubation at 37 ° C, the test was stopped with 100 ~l/well of an extraction buffer (20% SDS, 50% DMF in H20, 2.5% HC1 1 N, 2.5% of an 80% acetic acid solution, pH 7.4). After an overnight incubation at 37 °C, the absorbance at 540 nm was measured using an automated microELISA autoreader. One unit of IL-6 corresponds to half-maximal growth of the hybridoma cells.
***
4. Results 100
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4.1. Enhancement of IL-1 production by IL-3 ¢:
o
We have previously observed that IL-3 could enhance the secretion of IL-I by LPS-activated human monocytes [12]. We now report IL-1 activity assessed in the supernatants of murine peritoneal macrophages, stimulated with increasing concentrations of Salmonella enteritidis LPS (LPS Se), in the absence or presence of 100 U/ml IL-3. As shown in Fig. 1, an enhancing effect of IL-3 on the secretion of IL-1 activity was observed at various concentrations of LPS ranging from 0.2 ng/ml to 2/zg/ml. IL3 by itself was unable to induce the secretion of IL-1 in the absence of LPS. We then investigated whether the enhancing effect due to IL-3 correlates with an increase in cell-associated IL-1. Using Escherichia coli LPS (LPS Ec), we found that within the range of LPS concentrations used (i.e., 2 ng/ml to 20/zg/ml), the synergistic action of IL-3 was mainly observed when measuring IL-1 in the cell supernatants (Fig. 2). Titration curves of cell lysates led to similar conclusions (data not shown).
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Fig. 2. Effect of IL-3 on the release of IL-1 by cells stimulated with LPS (A) and on intracellular IL-1 activity (B) of BALB/c mouse PEM. Adherent cells were cultured as described in Fig. 1, in the presence ofE. coli LPS. (A) Secreted IL-1 activity (supernatants 1:10), in the absence ( o ) or presence ( • ) of 100 U / m l of rlL-3. (B) Intracellular IL-1 activity (as assessed by testing IL-1 activity in cell lysates diluted 1:10), in the absence ( o ) or the presence ( • ) of 100 U / m l of rlL-3. Responses that differ significantly from those of ceils not exposed to 1L-3 are denoted by asterisks (*p
Interleukin-3 enhances cytokine production by LPS-stimulated macrophages L u c h i n o C o h e n , B e r n a r d D a v i d a n d J e a n - M a r c Cavaillon Unitd d'Immuno-Allergie, Institut Pasteur, Paris, France (Received 9 November 1990; revision received 10 January 1991; accepted 17 January 1991)
1. Summary
In addition to its hematopoietic activities, interleukin-3 (IL-3) can modulate macrophage functions. We have studied the production of interleukin1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor (TNF) by mouse peritoneal macrophages triggered by lipopolysaccharide (LPS) in the presence or absence of IL-3. Interleukin-3 at the concentration used (i.e., 100 U/ml) did not induce the production of any cytokines, whereas it enhanced significantly the secretion of IL-1, IL-6 and TNF by LPSstimulated macrophages. The synergistic activity of IL-3 was observed over a wide range of Escherichia coli or Salmonella enteritidis LPS concentrations. No additive effect was noticed between IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF), another factor able to enhance LPSinduced IL-1 production. Thus, IL-3 can potentiate the inflammatory response induced by endotoxin from Gram-negative bacteria through a potentiation of cytokine production. 2. Introduction
Interleukin-3 (IL-3) is one of the hematopoietic growth factors produced by activated T cells. It was initially defined as a factor which could induce the expression of the enzyme 20-a-hydroxysteroid dehydrogenase by splenic lymphocytes from athymic mice [1]. IL-3 has been found to support the prolifer-
ation and differentiation of early hematopoietic stem cells, which differentiate into macrophages, granulocytes, eosinophiles, megakaryocytes or mast cells [2]. Other cytokines such as IL-1 [3], IL-6 [4, 5] and granulocyte-macrophage colony-stimulating factor (GM-CSF) [6] are able to enhance the IL-3induced hematopoiesis. Recent studies have described the ability of IL-3 to regulate the proliferation of blood monocytes and peritoneal exudate macrophages [7], the killing of Candida albicans by murine macrophages [8], the expression of tumor necrosis factor (TNFc0 mRNA by human monocytes [9] and the adhesion of human monocytes to endothelial cells [10]. Another study suggests that IL-3 enhances the antigen-presenting function of macrophages [11]. Taken together, these data imply that monocytes and macrophages are receptive to IL-3. Weiss et al. [12] have previously reported that IL-3 can enhance the IL-I release induced by lipopolysaccharide (LPS) in serum-free cultures of human monocytes. In the present study, we have investigated whether LPS-induced IL-1 secretion by more mature cells like mouse peritoneal macrophages, could also be regulated by IL-3. In addition, because of the evolutionary linkage between IL-3 and GM-CSF, we compared the action of the two cytokines. We also studied the regulatory effect of IL-3 on IL-6 and TNF release by these cells. 3. Materials and Methods
Key words: lnterleukin-3; Cytokine production; Macrophage; Lipopolysaccharide
3.1. Mice
Correspondence to: J.-M. Cavaillon, Unit6 d'Immuno-Allergie, Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France
Eight to twelve-week-old C57BI/6 and BALB/c mice were purchased from the Pasteur Institute
0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V.
121
breeding center and were maintained in our animal facility.
rophages. The lysates were then collected and stored as described for the supernatants, and tested for the cell-associated IL-1 activity.
3.2. Reagents 3.5. IL-1 bioassay Murine rIL-3 and rGM-CSF were purchased from Genzyme Corp. (Boston, MA), human rIL-1t3 was obtained from Rh6ne-Poulenc (France), murine rIL6 was a kind gift of Dr. J. van Snick and rTNFc~ was generously provided by Rh6ne-Poulenc (France). Escherichia coli (Olll:B4) LPS was obtained from Sigma (U.S.A.), Salmonella enteritidis LPS was from Difco (U.S.A.) and concanavalin A (Con A) was purchased from Miles (U.S.A.). 3.3. Isolation of peritoneal exudate macrophages
(PEM) PEM were harvested from the animals by peritoneal lavages with RPMI-1640 (Gibco, U.K.) supplemented with heparin (Roche, France), 3 or 4 days after the intraperitoneal injection of 1.5 ml of thioglycolate medium (Diagnostic Pasteur, France), and were washed with RPMI-1640 supplemented with 100 U/ml penicillin and 100 m g / m l streptomycin (Eurobio, France). The cell number, viability and percentage of macrophages were determined under a microscope. Cells were then plated into 24-well flat-bottomed culture trays (Falcon, Becton Dickinson Labware, U.K.) at a density of 5 x 105 macrophages/well. After 1 h, non-adherent cells were removed by vigorous washing with RPMI-1640. 3.4. Induction of IL-1 expression PEM were cultured in the absence or presence of LPS and cytokines for 24 h, in 0.5 ml of a culture medium composed of RPMI-1640 supplemented with 2 mM L-glutamine (Eurobia, France), 1 ~g/ml of indomethacin (Sigma, U.S.A.) and antibiotics. Preliminary experiments have established that 100 U/ml of rIL-3 or 60 U/ml of rGM-CSF led to optimal results. The supernatants were collected at the end of the induction period, centrifuged at 2 0 0 × g and stored frozen at - 3 0 ° C before determining their secreted cytokine activities. A volume of 0.5 ml/well of culture medium was added after the collection of the supernatants, the culture trays were then frozen and thawed 3 times to lyse the mac122
IL-1 activity in the PEM supernatants (l:10) was determined in the murine thymocyte proliferation assay: the supernatants were tested for their ability to enhance C 3 H / H e J routine thymocyte incorporation of [3H]TdR in the presence of a suboptimal concentration of Concanavalin A (0.9 ~g/ml) as previously described [13]. We ensured that residual amounts of IL-3 (i.e., < 10 U/ml) neither influence the IL-1 thymocyte proliferation as reported for high concentrations [14], nor modify the other biological assays. 3.6. TNF bioassay The T N F activity was determined by testing the cytotoxicity of serial dilutions of the supernatants in the presence of L929 fibroblasts, as previously described [15]. One unit of T N F activity was defined as the amount required to lyse 50% of L929 target cells. 3.7. IL-6 bioassay IL-6 activity was determined by using the IL-6dependent 7TD1 cell line [16]. Cells were cultured at a density of 1300 cells/well, in 100 ~1 of RPMI-1640 medium supplemented with antibiotics, 2mercaptoethanol (5 × 10 5 M) and 10% FCS, in the presence of serial dilutions of supernatants from stimulated PEM. After 4 days of culture at 37 °C, the proliferation was determined by a dye method [171:125 txg of tetrazolium salts (MTT) were added to each well and after 1 or 2 h of incubation at 37 ° C, the test was stopped with 100 ~l/well of an extraction buffer (20% SDS, 50% DMF in H20, 2.5% HC1 1 N, 2.5% of an 80% acetic acid solution, pH 7.4). After an overnight incubation at 37 °C, the absorbance at 540 nm was measured using an automated microELISA autoreader. One unit of IL-6 corresponds to half-maximal growth of the hybridoma cells.
***
4. Results 100
$$
$$$
#~
[~
•
4.1. Enhancement of IL-1 production by IL-3 ¢:
o
We have previously observed that IL-3 could enhance the secretion of IL-I by LPS-activated human monocytes [12]. We now report IL-1 activity assessed in the supernatants of murine peritoneal macrophages, stimulated with increasing concentrations of Salmonella enteritidis LPS (LPS Se), in the absence or presence of 100 U/ml IL-3. As shown in Fig. 1, an enhancing effect of IL-3 on the secretion of IL-1 activity was observed at various concentrations of LPS ranging from 0.2 ng/ml to 2/zg/ml. IL3 by itself was unable to induce the secretion of IL-1 in the absence of LPS. We then investigated whether the enhancing effect due to IL-3 correlates with an increase in cell-associated IL-1. Using Escherichia coli LPS (LPS Ec), we found that within the range of LPS concentrations used (i.e., 2 ng/ml to 20/zg/ml), the synergistic action of IL-3 was mainly observed when measuring IL-1 in the cell supernatants (Fig. 2). Titration curves of cell lysates led to similar conclusions (data not shown).
m ¢p
80 60
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40' 20'
LPS Se + IL-3
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1 02
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102
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100 -
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80 60 mE m
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1 01 LPS
60'
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102
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(ng/well)
Fig. 2. Effect of IL-3 on the release of IL-1 by cells stimulated with LPS (A) and on intracellular IL-1 activity (B) of BALB/c mouse PEM. Adherent cells were cultured as described in Fig. 1, in the presence ofE. coli LPS. (A) Secreted IL-1 activity (supernatants 1:10), in the absence ( o ) or presence ( • ) of 100 U / m l of rlL-3. (B) Intracellular IL-1 activity (as assessed by testing IL-1 activity in cell lysates diluted 1:10), in the absence ( o ) or the presence ( • ) of 100 U / m l of rlL-3. Responses that differ significantly from those of ceils not exposed to 1L-3 are denoted by asterisks (*p
