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EXPRESSION OF IL-8 GENE IN HUMAN MONOCYTES AND LYMPHOCYTES: DIFFERENTIAL REGULATION BY TNF AND IL-1
doi:10.1006/cyto.1999.0664, available online at http://www.idealibrary.com on
EXPRESSION OF IL-8 GENE IN HUMAN MONOCYTES AND LYMPHOCYTES: DIFFERENTIAL REGULATION BY TNF AND IL-1 Yuri V. Chaly,1,2 Rathinam S. Selvan,3 Konstantin V. Fegeding,4 Tatyana S. Kolesnikova,2 Nikolai N. Voitenok1,5 TNF- and IL-1 were reported to be the most powerful inducers of IL-8 in a multitude of cells, including leukocytes. In this study, we investigated TNF-- and IL-1-mediated regulation of IL-8 gene expression in non-fractionated PBMC, and purified monocyte (MO) and lymphocyte (LY) fractions. Our analysis revealed that purified human MO did not respond to exogenous TNF- with the induction of IL-8 mRNA or protein, nor require endogenous TNF- for IL-8 expression. In contrast, in the presence of exogenous IL-1 and IL-1 a substantial enhancement of IL-8 mRNA and protein expression in MO was observed. Nevertheless, antibodies to IL-1 and IL-1 were unable to downregulate the expression of IL-8 in resting adherent or Staphylococcus aureus Cowan 1 (SAC)-stimulated MO. In contrast with MO, purified LY and non-fractionated PBMC expressed IL-8 in response to exogenous TNF-, similar to exogenous IL-1 and IL-1. As was seen with MO, antibodies to TNF-, IL-1 and IL-1 did not inhibit the expression of IL-8 in purified LY and non-fractionated PBMC stimulated with SAC and LPS. Taken together, our data demonstrate major differences in responsiveness of MO and LY to exogenous TNF- and IL-1, and suggest relative autonomy of IL-8 gene expression in these cells that does not require accessory cytokines but can be induced directly by exogenous stimuli. 2000 Academic Press
Interleukin (IL)-8, a member of the CXC chemokine family, is a key chemoattractant and activator of neutrophils.1,2 The most prominent producer of IL-8 in vitro is the monocyte (MO) population of peripheral blood mononuclear cells (PBMC).3 There are numerous non-specific bacterial products, cytokines, ligands and metabolic stimulants that induce IL-8 gene expression in MO and PBMC.1–3 The observation that IL-8 expression may be induced in virtually all types of cells was very important for the understanding of the role of IL-8 in inflammation.1 Tumour necrosis factor (TNF-) and IL-1 are the most powerful inducers of IL-8 in a multitude of From the 1Foundation for Fundamental Researches of Republic of Belarus, 2Institute of Hematology and Blood Transfusion, Minsk, Belarus; 3Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA; 4Hematology Research Center, Moscow, Russia; 5Fund for Molecular Hematology and Immunology, Moscow, Russia. Correspondence to: Nikolai N. Voitenok, Foundation for Fundamental Research of Republic of Belarus, F. Skaryna Av. 66, room 414, Minsk, 220072, Belarus; E-mail: [email protected] Received 2 September 1999; accepted for publication 6 December 1999 2000 Academic Press 1043–4666/00/060636+08 $35.00/0 KEY WORDS: chemokines/leukocytes/induction/endogenous cytokines 636
cell lines and primary cultured cells of non-leukocyte lineage, including endothelium, hepatocytes, fibroblasts and others1,2 It is generally believed that IL-8 expression in monocytes and other cells is also TNF-dependent.2,3,5 However, recent studies do not see a role for TNF- in the induction of IL-8 gene expression in MO by nitric oxide and reactive oxygen intermediates,6 some cytokines and immunomodulators,7,8,9 and in intercellular interactions.10 Previous studies concerning the role of TNF- in MO IL-8 induction have been performed mostly using unfractionated PBMC.4,5 Given the important contribution of MO and lymphocytes (LY) to inflammatory reactions, the present study was conducted to analyse TNF-- and IL-1-mediated regulation of expression of IL-8 in non-fractionated PBMC and purified MO and LY subsets. We show here that IL-8 gene expression in purified human MO is TNF--independent, whereas exogenous TNF- may induce the expression of IL-8 transcripts and secreted IL-8 protein in LY and nonpurified PBMC. In contrast to TNF, exogenous IL-1 and IL-1 enhanced the expression of IL-8 mRNA and protein in MO. Nevertheless, antibodies to TNF, IL-1 and IL-1 were unable to downregulate the expression of IL-8 in resting and activated MO and LY. These CYTOKINE, Vol. 12, No. 6 (June), 2000: pp 636–643
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results demonstrate differences in the responsiveness of MO and LY to exogenous TNF and IL-1, and suggest relative autonomy of their IL-8 gene expression that does not require endogenous TNF and IL-1.
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To probe whether exogenous TNF-, IL-1 and IL-1 could alone induce the expression of IL-8 mRNA and IL-8 protein, purified MO were incubated with recombinant cytokines. Similarly to PBMC, freshly isolated MO contained no IL-8 mRNA, while MO incubated adherent on plastic surface for 4 h or 20 h expressed substantial levels of IL-8 mRNA and secreted IL-8 protein (Fig. 2). As shown in Figure 2, constitutive production of IL-8 protein and IL-8 mRNA accumulation induced by adherence was not enhanced by exogenous TNF- at any given time. The inability of exogenous TNF- to upregulate the expression of IL-8 was confirmed in eight separate experiments using MO from different donors. In contrast to exogenous TNF-, expression of IL-8 mRNA and IL-8 protein was significantly upregulated in adherent MO exposed to exogenous IL-1 or IL-1 (Fig. 2). These data reveal the difference in responsiveness of IL-8 gene to exogenous TNF- in PBMC and purified MO.
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Enhancement of adherence-induced IL-8 expression in peripheral blood mononuclear cells (PBMC) by exogenous TNF-, IL-1 and IL-1 The capacity of exogenous TNF- and IL-1 to induce the expression of IL-8 was directly tested in non-fractionated PBMC that were incubated in the presence of recombinant human TNF-, IL-1 and IL-1. In agreement with previously published data,5 freshly isolated PBMC did not contain IL-8 mRNA, while PBMC cultured adherent on a plastic surface for 4 or 20 h expressed substantial levels of IL-8 mRNA and secreted IL-8 protein (Fig. 1). As shown in Figure 1, adherence-induced expression of IL-8 mRNA and protein was significantly enhanced in the presence of recombinant TNF- (2.5 ng/ml), IL-1 (2 ng/ml) and IL-1(2 ng/ml) both at 4 and 20 hours of incubation. Addition of anti-TNF- antibody (Fig. 1), anti-IL-1 and anti-IL-1 (data not shown) abolished the effects of corresponding exogenous cytokines. These results indicate that exogenous TNF-, IL-1 and IL-1 appear to be stimulants of IL-8 expression in resting non-fractionated PBMC.
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Figure 1. Effects of IL-1, IL-1, TNF- and anti-TNF- on IL-8 protein production in human PBMC as detected by ELISA and IL-8 mRNA induction as measured by quantitative RT-PCR. Cells were incubated for 4 h (open bars) or 20 h (shaded bars) alone or in the presence of IL-1 (2 ng/ml), IL-1 (2 ng/ml), TNF- (2.5 ng/ml) or SAC. Anti-TNF- antibody was added from the beginning of incubation. Each bar represents the meanSE of triplicate cultures. Similar data obtained in five independent experiments.
IL-8 expression in purified lymphocytes (LY) is up-regulated by exogenous TNF- Since TNF- was unable to stimulate IL-8 expression in MO, we reasoned that the induction of IL-8 expression observed in non-fractionated PBMC may be due to effects of TNF- stimulating other subset of PBMC, i.e. lymphocytes. To test this possibility, purified lymphocytes were exposed to recombinant human TNF- as well as IL-1 or IL-1. Freshly purified LY contained no IL-8 mRNA and when cultured alone these cells synthesized a little IL-8 mRNA and protein (Fig. 3). In contrast to MO, exposure of these cells to exogenous TNF- induced
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Figure 2. Effects of TNF-, IL-1 and IL-1 on IL-8 protein production in purified MO as detected by ELISA and IL-8 mRNA induction as measured by quantitative RT-PCR. Percoll-isolated MO were incubated for 4 h (open bars) or 20 h (shaded bars) alone or in the presence of TNF- (2.5 ng/ml), IL-1 (2 ng/ml), IL-1 (2 ng/ml) or SAC. Each bar represents the meanSE of triplicate cultures. Similar data obtained in five independent experiments.
substantial expression of IL-8 mRNA and protein as was seen with PBMC. The effect was abolished by anti-TNF- antibody. As was seen with PBMC and MO, high levels of IL-8 mRNA and IL-8 protein were induced in the presence of exogenous IL-1 or IL-1 (Fig. 3). These results provide evidence that LY but not MO in PBMC respond directly to TNF- with the induction of IL-8 expression.
IL-8 expression in PBMC, MO and LY requires no endogenous TNF-, IL-1 or IL-1 Since it was shown that endogenous cytokines may be required for induction of chemokine expression in monocytes,17 we then investigated endogenous
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TNF-, IL-1 and IL-1 for a possible role in the induction of IL-8 gene expression in PBMC, purified MO and LY stimulated with SAC. As shown in Figure 4, SAC substantially enhanced the expression of IL-8 mRNA and protein in PBMC, purified MO and LY as compared to adherence-induced levels. Addition of anti-TNF-, anti-IL-1 or anti-IL-1 antibodies had no effect on expression of IL-8 mRNA and protein in PBMC and their MO and LY subsets stimulated with SAC. Similar results were observed when resting purified MO and LY were incubated in the presence of anti-TNF-, anti-IL-1 or anti-IL-1 antibodies. Figure 5 shows that substantial production of IL-8 protein induced in MO by adherence was not affected by antibodies to TNF-, IL-1 and IL-1. Similarly, background production of IL-8 in resting purified LY
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Figure 4. Role of endogenous TNF-, IL-1 and IL-1 in the expression of IL-8 protein and mRNA in (A) human PBMC, (B) MO and (C) LY stimulated by SAC. SAC and corresponding antibodies were added from the beginning of cell incubation: polyclonal rabbit anti-human IL-1 and anti-human IL-1 at 30 g/ml, monoclonal anti-human TNF- at 30 g/ml. Each bar represents the meanSE of triplicate cultures. Similar data obtained in three independent experiments.
was also retained in the presence of anti-TNF-, anti-IL-1 and anti-IL-1. We have showed before that adherence itself may induce high levels of nuclear transcription of IL-8 and IL-1 genes in MO.16 Since it was shown here that IL-1 is a potent inducer of IL-8 expression in MO, we tested purified resting adherent MO for spontaneous induction of IL-1, IL-8 and TNF- expression. As shown in Figure 6, MO freshly isolated from PBMC using a Percoll gradient contained no IL-8, IL-1 or TNF- transcripts. Incubation (adhesion) of MO in a plastic dish for 4 h caused accumulation of IL-8 mRNA and IL-1 mRNA. However, adherence per se did not induce TNF- mRNA in these cells. Adherent MO secreted substantial level of IL-8 protein during 4 h of incubation (Fig. 6), whereas no IL-1 or TNF- protein production was detected by corresponding ELISAs in the same cultures. Dissociation between induction of IL-1 mRNA and protein in adherence stimulated MO has been previously shown.24 This results further support our demonstration that endogenous TNF- and IL-1 play no role in adherence-induced IL-8 gene expression.
DISCUSSION Recent data demonstrated that several stimuli may induce the expression of IL-8 gene in MO without activating TNF- and IL-1 genes,6–10 implying autonomy of IL-8 gene regulation. The present study was undertaken to clarify the role of TNF-, IL-1 and IL-1 in the induction of IL-8 gene in the leukocyte
fractions of human PBMC. Our analysis reveals that purified human MO do not respond to exogenous TNF- with the induction of IL-8 mRNA and protein, while exogenous IL-1 and IL-1 induced the expression of IL-8 in purified MO culture. In contrast, purified LY and non-fractionated PBMC express IL-8 mRNA and IL-8 protein in response to TNF-. Similar to MO, IL-8 expression in LY and PBMC was upregulated by exogenous IL-1 and IL-1. Described herein, the inability of TNF- to induce IL-8 expressaon in purified monocytes seems to be in conflict with previous reports demonstrating enhancement of IL-8 expression in human blood MO in the presence of TNF-.5 This discrepancy may be due to the use of non-fractionated PBMC, not purified MO, in previous studies to demonstrate the effect of TNF- on IL-8 expression.4,5 Thus, according to our results, LY can respond to TNF- with upregulation of IL-8 expression in non-fractionated PBMC. Adherence per se is a potent stimulus to MO population of PBMC.5,22,23 Using nuclear run-on analysis, we demonstrated previously that adherence alone was sufficient to induce transcription of IL-8 and IL-1 genes, but not the TNF- gene.16 In agreement with previous studies, adherence of PBMC and purified MO induces IL-8 mRNA expression and IL-8 protein secretion. The substantial enhancement of IL-8 mRNA and protein expression in purified MO by exogenous IL-1 suggests that endogenous IL-1 may contribute to the expression of IL-8 production in MO. However, our data demonstrating inability of antibodies to IL-1 and IL-1 to downregulate the expression of IL-8 mRNA in purified MO argue against the role of
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Figure 5. Role of endogenous TNF-, IL-1 and IL-1 in the expression of IL-8 protein and mRNA in (A) purified resting MO and (B) LY. TNF- (2.5 ng/ml), IL-1 (2 ng/ml), IL-1 (2 ng/ml) and corresponding antibodies at 30 g/ml were added from the beginning of cell incubation. Incubation was 4 h (open bars) or 20 h (shaded bars).
endogenous IL-1 in adherence-induced MO IL-8 expression. In agreement with that observation, it was further demonstrated that in the cultures of purified MO, adherence alone induced no secretion of immunoreactive IL-1, while IL-1 mRNA was induced. Our observation is in agreement with a previous study describing the dissociation of adherence induced IL-1 mRNA and protein secretion in human MO in vitro.24,25 Taken together, our data further confirm the suggestion that adherence directly activates IL-8 gene expression in MO, and endogenous TNF- and IL-1 play no role in this process. Similarly to adherence, expression of IL-8 in MO stimulated with SAC (or LPS, not shown) was not affected by anti-TNF-, anti-IL-1 or anti-IL-1 antibodies. This may be due to the sustained primary response of IL-8 gene transcription to SAC. There are few studies that report the requirement of endogenous TNF or IL-1 for induction of chemokine genes in MO. In spite of the inability of TNF- to upregulate IL-8 expression in purified MO, it was demonstrated
that exogenous TNF- enhances expression of the CC chemokine RANTES and endogenous TNF- plays a crucial role in the expression of RANTES gene in MO.26 Similarly, the induction of I-309 mRNA in MO was mediated by endogenous IL-1, since antiserum to IL-1 abolished I-309 expression induced by LPS or immunoglobulin plus LPS.17 The regulation of IL-8 expression in MO was thus revealed to be different from the regulation of these CC chemokines in terms of dependence on TNF- and IL-1. Differential expression of IL-8 and CC chemokines by TNF- and IL-1 is in agreement with recent data concerning 5 -upstream regulatory regions of IL-8 and chemokine genes.27–31 These data suggest a complex regulation of chemokine genes at multiple levels by several members of the NF--B family that are able to differentially activate IL-8 gene, compared to RANTES,29 ENA-7830 and some other CC-chemokine genes.31 Similarly to MO, the expression of IL-8 in PBMC and their LY subset activated with SAC was not inhibited by antibodies to TNF- and IL-1. Nevertheless, it is noteworthy that we observed reduction of SAC-induced IL-8 production in part of PBMC cultures (six experiments of 11, mean inhibition 165%) as well as in their purified LY cultures (227% inhibition) in the presence of anti-TNF- antibody. This may be due to the presence of minor fraction of LY that is dependent on endogenous TNF- to express IL-8. Taken together, the findings suggest a relative autonomy of IL-8 gene expression in MO that does not require autocrine or paracrine accessory cytokines but can be induced directly by exogenous stimuli. Therefore, it could be reasoned that this mechanism operates to produce IL-8 and to attract neutrophils in the site of MO activation in an inflammatory reaction more rapidly. Described herein, the differences in responsiveness of MO and LY to exogenous TNF- suggest that LY but not MO will produce potent neutrophil chemoattractant IL-8 in the sites of TNF- accumulation, while IL-1 will induce IL-8 expression in both compartments. Further, our observation suggests that the selective expression and regulation of chemokine genes in MO and LY in inflammatory responses may depend upon surrounding cells and accessory exogenous cytokines.
MATERIALS AND METHODS Reagents Staphylococcus aureus Cowan 1 (SAC) was prepared as described11 and stored at 4C. RPMI 1640 and fetal calf serum (FCS) were purchased from Gibco, Paisley, UK. Percoll was obtained from Pharmacia, Uppsala, Sweden. Standards of recombinant human TNF-, IL-8 and IL-1
Expression of IL-8 gene in human leukocytes / 641
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Comparison of production of IL-8, IL-1 and TNF- and their mRNA expression in resting adherent human MO.
MO were purified by Percoll fractionation and incubated for 4 h alone or in the presence of SAC. (A) Production of IL-1 (grey bars), TNF- (black bars) and IL-8 (open bars): each bar represents the meanSE of triplicate cultures. (B) Northern blot of total RNA samples was sequentially hybridized with 32P-labelled probes as indicated. Comparable RNA loading was confirmed by ethidium bromide staining of gel. Similar data obtained in 3 independent experiments. *TNF- was below 30 pg/ml in adherent MO cultured alone, IL-1 protein production was below 100 pg/ml.
were kindly provided by Dr A. Meager (NIBSC, South Mimms, UK). Recombinant IL-8 and polyclonal antibodies against human IL-8 were kindly donated by Prof. K. Matsushima, Cancer Research Institute, Kanazawa University, Japan. High neutralizing monoclonal antibody against human TNF- 5N and monoclonal antibody to human IL-8 WS-4 were produced in our laboratory.12,13 Neutralizing antibodies against human IL-1 and IL-1 were purchased from Endogen, MA, USA; Code: P-420A and 420B. Samples of recombinant human IL-1 and IL-1 were also kindly provided by Dr S. Ketlinsky (Institute of Ultrapure Biochemicals, St. Petersburg, Russia). Plasmid pQA-114 was kindly provided by Dr A. Shakhov (Institute of Toxicology, Schwerzenbach, Switzerland). Oligonucleotides for reverse transcriptase–polymerase chain reaction (RT-PCR) were synthesized by MWG-BIOTECH, Ebersberg, Germany.
Purification of MO and LY Mononuclear leukocytes were isolated from fresh heparinized blood of healthy donors using standard Ficoll/ Hypaque protocol.15,16 Mononuclear leukocytes from Ficoll/ Hypaque were then fractionated on continuous Percoll gradient at 4C.15,16 LY fraction from Percoll gradient was
additionally purified by plastic adherence for 1 h to eliminate few contaminating MO. Isolated MO were 95–98% naphthyl acetate esterase-positive (NAE kit from Sigma), purified LY contained 0–2% of NAE + cells. Purified MO and LY were incubated in 24-well plastic plates (Corning, New York, USA) or 50-mm polystyrene plastic dishes (Corning) in 1% FCS–RPMI 1640 at 1.5–2106 cells/ml. Part of the MO and LY cultures was activated by SAC at a final concentration of 0.001% (V/V).15,16 Recombinant human cytokines IL-1, IL-1 and TNF-, and neutralizing antibodies against human IL-1, IL-1 and TNF- were added at the beginning of MO and LY incubation as indicated in Results.
Transcript analysis Total RNA from cells was extracted by acidic guanidinium thiocyanate.18 Northern blotting of IL-8, IL-1 and TNF- mRNA has been carried out as described.16,17,19 Reverse transcription (RT) was carried out in 5 g of total cellular RNA with M-MLV reverse transcriptase (GIBCO-BRL) using the recommended conditions in a total reaction volume of 20 l containing 1 g oligo (dT)12–18 (Pharmacia, Dubendorf, Switzerland). The fixed amount of cDNA was co-amplified by PCR in the presence of serial dilutions of control plasmids pQA-1 containing sequences
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complementary to the primers specific for human IL-8 and 2-microglobulin cDNA.14 Amplification was achieved over 30 cycles on a Perkin-Elmer Thermal Cycler 9600 (UK) (denaturation for 20 s at 94C, annealing for 20 s at 55C—two-step PCR) framed by initiating incubation for 2 min at 94C and a final incubation for 5 min at 72C. The PCR products were analysed by agarose gel electrophoresis (2% agarose) in the presence of ethidium bromide. Quantitation of fluorescence of both cellular and competitive template product bands was performed on Gelprint System with Onedscan Software. To correct for any variation in RNA content and cDNA synthesis in the different preparations, each sample was normalized on the basis of its 2-microglobulin content. Results were expressed as the calculated ratio of cytokine mRNAs to 2-microglobulin mRNA as external control.20
Protein analysis Supernatants from cultured cells were harvested, centrifuged for 5 min at 13 000 rpm in Eppendorf tubes at 4C, and stored at 80C. Cytokine concentration was quantified by ELISAs for IL-8,12,13 TNF-13 and IL-1.21
Acknowledgements The work was supported in part by the Department of Surgery, Duke University Medical Center, Durham, NC, USA; by Grant 94-1634 from INTAS, Brussels, Grant MWO 300 from International Science Foundation, NY, USA and B24-343 from Foundation for Fundamental Researches of the Republic of Belarus. We would like to thank Dr A. Shakhov (Institute of Toxicology, Schwerzenbach, Switzerland) for providing plasmid pQA-1 and support with quantitative RT–PCR analysis. The authors are grateful to Dr A. Meager (NIBSC, South Mimms, UK) for providing cytokine standards, Prof. K. Matsushima (Cancer Research Institute, Kanazawa University, Japan) for the gift of anti-IL-8 polyclonal antibody and recombinant IL-8 and Dr S. Ketlinsky (Institute of Ultrapure Biochemicals, St. Petersburg, Russia) for providing recombinant IL-1 and IL-1.
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CYTOKINE, Vol. 12, No. 6 (June, 2000: 636–643) 5. Kasahara K, Strieter RM, Standiford TJ, Kunkel SL (1993) Adherence in combination with lipopolysaccharide, tumor necrosis factor or interleukin-1 potentiates the induction of monocyte-derived interleukin-8. Pathobiology 61:57–66. 6. Remick DG, Villarete L (1996) Regulation of cytokine gene expression by reactive oxygen and reactive nitrogen intermediates. J Leukoc Biol 59:471–475. 7. Muzzo T, Badolato R, Longo D, Gusella GL, Varesio L (1995) Leukemia inhibitory factor induces interleukin-8 and monocyte chemotactic and activating factor in human monocytes: Differential regulation by interferon-. Blood 86:1961–1967. 8. Yoshimura T, Usami E, Kurita C, Watanabe S, Nakao T, Kobayashi J, Yamazaki F, Nagai H (1995) Effect of theophylline on the production of interleukin-1 beta, tumor necrosis-alpha, and interleukin-8 by human peripheral blood mononuclear cells. Biol Pharm Bull 18:1405–1408. 9. Meloni F, Ballabio P, Bianchi L, Grassi FA, Gialdroni-Grassi GG (1995) Cefozidime modulates in vitro tumor necrosis factor-alpha, interleukin-6 and interleukin-8 release from human peripheral monocytes. Chemotherapy 41:289–295. 10. Lukacs NW, Strieter RM, Elner V, Evanoff HL, Burdick MD, Kunkel SL (1995) Production of chemokines, interleukin-8 and monocyte chemoattractant protein-1, during monocyte: endothelial cell interactions. Blood 86:2767–2773. 11. Kessler SW (1975) Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol 115:1617–1624. 12. Ko Y, Mukaida N, Panyutich A, Voitenok N, Matsushima K, Kawai T, Kasahara T (1992) A sensitive enzyme-linked immunosorbent assay for human interleukin-8. J Immunol Meth 149: 227–235. 13. Petyovka NV, Lyakh LA, Voitenok NN (1995) Homologous ELISA for detection of oligomeric TNF: properties of the assay. J Immunol Meth 186:161–170. 14. Bouaboula M, Legoux P, Pessegue B, Delpech B, Dumont X, Piechaczyk M, Casellas P, Shire D (1992) Standardization of mRNA titration using a polimerase chain reaction method involving co-amplification with multispecific internal control. J Biol Chem 267:21830–21838. 15. Misuno NI, Osipovich OA, Sudarikov AB, Idelson GL, Kolesnikova TS, Panyutich AV, Voitenok NN (1990) TNF- induction in human monocytes. Cytokine 2:464–469. 16. Osipovich O, Fegeding K, Misuno N, Kolesnikova T, Savostin I, Sudarikov A, Voitenok N (1993) Differential action of cycloheximide and activation stimuli on transcription of tumor necrosis factor-, IL-1, IL-8, and P53 genes in human monocytes. J Immunol 150:4958–4965. 17. Selvan RS, Zhou L-J, Krangel MS (1997) Regulation of I-309 gene expression in human monocytes by endogenous interleukin-1. Eur J Immunol 27:687–694. 18. Chomczynski P, Sacchi N (1987) Single step method of RNA isolation by acid guanidinum-thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159. 19. Selvan RS, Butterfield JH, Krangel MS (1994) Expression of multiple chemokine genes in a human mast cell leukemia. J Biol Chem 269:13893–13898. 20. Shakhov AN, Woerly G, Car BD, Ryffel B (1996) Interferon- enchances tumor necrosis factor- production by inhibiting early phase interleukin-10 transcription. Eur Cytokine Netw 7:741–750. 21. Simbirtsev A, Prokopieva E, Ivanova E, Konusova V, Perumov N, Ketlinsky SA (1992) Interleukin-1 and tumor necrosis factor production by human monocytoid cells: study on a single cell level. Eur Cytokine Netw 3:421–428. 22. Kasahara K, Strieter R, Chensue S, Standiford T, Kunkel S (1991) Mononuclear cell adherence induces neutrophil chemotactic factor/interleukin-8 gene expression. J Leukoc Biol 50:287–295. 23. Sporn SA, Eierman DF, Johnson CE, Morris J, Martin G, Ladner M, Haskill S (1990) Monocyte adherence results in selective induction of novel genes sharing homology with mediators of inflammation and tissue repair. J Immunol 144:4434–4441.
Expression of IL-8 gene in human leukocytes / 643 24. Schindler R, Clark BD, Dinarello CA (1990) Dissociation between interleukin-1 mRNA and protein synthesis in human peripheral blood mononuclear cells. J Biol Chem 265:10232– 10237. 25. Eierman DF, Johnson CE, Haskill JS (1989) Human monocyte inflammatory mediator gene expression is selectively regulated by adherence substrates. J Immunol 142:1970–1976. 26. Selvan RS, Krangel MS, Winter S (1997) Expression of RANTES gene in human macrophages stimulated with LPS and/or IFN: A role for endogenous TNF-. J Leuk Biol (Suppl.) 12. 27. Kunsch C, Rosen CA (1993) NF-kappa B subunit-specific regulation of the interleukin-8 promoter. Mol Cell Biol 13: 6137–6146.
28. Stein B, Baldwin AS (1993) Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF- B. Mol Cell Biol 13:7191–7198. 29. Ray P, Yang L, Zhang DH, Ghosh SK, Ray A (1997) Selective up-regulation of cytokine-induced RANTES gene expression in lung epithelial cells by overexpression of IBR. J Biol Chem 272:20191–20197. 30. Chang MS, McNinch J, Basu R, Simonet S (1994) Cloning and characterization of the human neutrophil-activating peptide (ENA-78) gene. J Biol Chem 269:25277–25282. 31. Rovin BH, Dickerson JA, Tan LC, Hebert CA (1995) Activation of nuclear factor-kappa B correlates with MCP-1 expression by human mesangial cells. Kidney Int 48:1263–1271.
EXPRESSION OF IL-8 GENE IN HUMAN MONOCYTES AND LYMPHOCYTES: DIFFERENTIAL REGULATION BY TNF AND IL-1 Yuri V. Chaly,1,2 Rathinam S. Selvan,3 Konstantin V. Fegeding,4 Tatyana S. Kolesnikova,2 Nikolai N. Voitenok1,5 TNF- and IL-1 were reported to be the most powerful inducers of IL-8 in a multitude of cells, including leukocytes. In this study, we investigated TNF-- and IL-1-mediated regulation of IL-8 gene expression in non-fractionated PBMC, and purified monocyte (MO) and lymphocyte (LY) fractions. Our analysis revealed that purified human MO did not respond to exogenous TNF- with the induction of IL-8 mRNA or protein, nor require endogenous TNF- for IL-8 expression. In contrast, in the presence of exogenous IL-1 and IL-1 a substantial enhancement of IL-8 mRNA and protein expression in MO was observed. Nevertheless, antibodies to IL-1 and IL-1 were unable to downregulate the expression of IL-8 in resting adherent or Staphylococcus aureus Cowan 1 (SAC)-stimulated MO. In contrast with MO, purified LY and non-fractionated PBMC expressed IL-8 in response to exogenous TNF-, similar to exogenous IL-1 and IL-1. As was seen with MO, antibodies to TNF-, IL-1 and IL-1 did not inhibit the expression of IL-8 in purified LY and non-fractionated PBMC stimulated with SAC and LPS. Taken together, our data demonstrate major differences in responsiveness of MO and LY to exogenous TNF- and IL-1, and suggest relative autonomy of IL-8 gene expression in these cells that does not require accessory cytokines but can be induced directly by exogenous stimuli. 2000 Academic Press
Interleukin (IL)-8, a member of the CXC chemokine family, is a key chemoattractant and activator of neutrophils.1,2 The most prominent producer of IL-8 in vitro is the monocyte (MO) population of peripheral blood mononuclear cells (PBMC).3 There are numerous non-specific bacterial products, cytokines, ligands and metabolic stimulants that induce IL-8 gene expression in MO and PBMC.1–3 The observation that IL-8 expression may be induced in virtually all types of cells was very important for the understanding of the role of IL-8 in inflammation.1 Tumour necrosis factor (TNF-) and IL-1 are the most powerful inducers of IL-8 in a multitude of From the 1Foundation for Fundamental Researches of Republic of Belarus, 2Institute of Hematology and Blood Transfusion, Minsk, Belarus; 3Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA; 4Hematology Research Center, Moscow, Russia; 5Fund for Molecular Hematology and Immunology, Moscow, Russia. Correspondence to: Nikolai N. Voitenok, Foundation for Fundamental Research of Republic of Belarus, F. Skaryna Av. 66, room 414, Minsk, 220072, Belarus; E-mail: [email protected] Received 2 September 1999; accepted for publication 6 December 1999 2000 Academic Press 1043–4666/00/060636+08 $35.00/0 KEY WORDS: chemokines/leukocytes/induction/endogenous cytokines 636
cell lines and primary cultured cells of non-leukocyte lineage, including endothelium, hepatocytes, fibroblasts and others1,2 It is generally believed that IL-8 expression in monocytes and other cells is also TNF-dependent.2,3,5 However, recent studies do not see a role for TNF- in the induction of IL-8 gene expression in MO by nitric oxide and reactive oxygen intermediates,6 some cytokines and immunomodulators,7,8,9 and in intercellular interactions.10 Previous studies concerning the role of TNF- in MO IL-8 induction have been performed mostly using unfractionated PBMC.4,5 Given the important contribution of MO and lymphocytes (LY) to inflammatory reactions, the present study was conducted to analyse TNF-- and IL-1-mediated regulation of expression of IL-8 in non-fractionated PBMC and purified MO and LY subsets. We show here that IL-8 gene expression in purified human MO is TNF--independent, whereas exogenous TNF- may induce the expression of IL-8 transcripts and secreted IL-8 protein in LY and nonpurified PBMC. In contrast to TNF, exogenous IL-1 and IL-1 enhanced the expression of IL-8 mRNA and protein in MO. Nevertheless, antibodies to TNF, IL-1 and IL-1 were unable to downregulate the expression of IL-8 in resting and activated MO and LY. These CYTOKINE, Vol. 12, No. 6 (June), 2000: pp 636–643
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results demonstrate differences in the responsiveness of MO and LY to exogenous TNF and IL-1, and suggest relative autonomy of their IL-8 gene expression that does not require endogenous TNF and IL-1.
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To probe whether exogenous TNF-, IL-1 and IL-1 could alone induce the expression of IL-8 mRNA and IL-8 protein, purified MO were incubated with recombinant cytokines. Similarly to PBMC, freshly isolated MO contained no IL-8 mRNA, while MO incubated adherent on plastic surface for 4 h or 20 h expressed substantial levels of IL-8 mRNA and secreted IL-8 protein (Fig. 2). As shown in Figure 2, constitutive production of IL-8 protein and IL-8 mRNA accumulation induced by adherence was not enhanced by exogenous TNF- at any given time. The inability of exogenous TNF- to upregulate the expression of IL-8 was confirmed in eight separate experiments using MO from different donors. In contrast to exogenous TNF-, expression of IL-8 mRNA and IL-8 protein was significantly upregulated in adherent MO exposed to exogenous IL-1 or IL-1 (Fig. 2). These data reveal the difference in responsiveness of IL-8 gene to exogenous TNF- in PBMC and purified MO.
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Enhancement of adherence-induced IL-8 expression in peripheral blood mononuclear cells (PBMC) by exogenous TNF-, IL-1 and IL-1 The capacity of exogenous TNF- and IL-1 to induce the expression of IL-8 was directly tested in non-fractionated PBMC that were incubated in the presence of recombinant human TNF-, IL-1 and IL-1. In agreement with previously published data,5 freshly isolated PBMC did not contain IL-8 mRNA, while PBMC cultured adherent on a plastic surface for 4 or 20 h expressed substantial levels of IL-8 mRNA and secreted IL-8 protein (Fig. 1). As shown in Figure 1, adherence-induced expression of IL-8 mRNA and protein was significantly enhanced in the presence of recombinant TNF- (2.5 ng/ml), IL-1 (2 ng/ml) and IL-1(2 ng/ml) both at 4 and 20 hours of incubation. Addition of anti-TNF- antibody (Fig. 1), anti-IL-1 and anti-IL-1 (data not shown) abolished the effects of corresponding exogenous cytokines. These results indicate that exogenous TNF-, IL-1 and IL-1 appear to be stimulants of IL-8 expression in resting non-fractionated PBMC.
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Figure 1. Effects of IL-1, IL-1, TNF- and anti-TNF- on IL-8 protein production in human PBMC as detected by ELISA and IL-8 mRNA induction as measured by quantitative RT-PCR. Cells were incubated for 4 h (open bars) or 20 h (shaded bars) alone or in the presence of IL-1 (2 ng/ml), IL-1 (2 ng/ml), TNF- (2.5 ng/ml) or SAC. Anti-TNF- antibody was added from the beginning of incubation. Each bar represents the meanSE of triplicate cultures. Similar data obtained in five independent experiments.
IL-8 expression in purified lymphocytes (LY) is up-regulated by exogenous TNF- Since TNF- was unable to stimulate IL-8 expression in MO, we reasoned that the induction of IL-8 expression observed in non-fractionated PBMC may be due to effects of TNF- stimulating other subset of PBMC, i.e. lymphocytes. To test this possibility, purified lymphocytes were exposed to recombinant human TNF- as well as IL-1 or IL-1. Freshly purified LY contained no IL-8 mRNA and when cultured alone these cells synthesized a little IL-8 mRNA and protein (Fig. 3). In contrast to MO, exposure of these cells to exogenous TNF- induced
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substantial expression of IL-8 mRNA and protein as was seen with PBMC. The effect was abolished by anti-TNF- antibody. As was seen with PBMC and MO, high levels of IL-8 mRNA and IL-8 protein were induced in the presence of exogenous IL-1 or IL-1 (Fig. 3). These results provide evidence that LY but not MO in PBMC respond directly to TNF- with the induction of IL-8 expression.
IL-8 expression in PBMC, MO and LY requires no endogenous TNF-, IL-1 or IL-1 Since it was shown that endogenous cytokines may be required for induction of chemokine expression in monocytes,17 we then investigated endogenous
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TNF-, IL-1 and IL-1 for a possible role in the induction of IL-8 gene expression in PBMC, purified MO and LY stimulated with SAC. As shown in Figure 4, SAC substantially enhanced the expression of IL-8 mRNA and protein in PBMC, purified MO and LY as compared to adherence-induced levels. Addition of anti-TNF-, anti-IL-1 or anti-IL-1 antibodies had no effect on expression of IL-8 mRNA and protein in PBMC and their MO and LY subsets stimulated with SAC. Similar results were observed when resting purified MO and LY were incubated in the presence of anti-TNF-, anti-IL-1 or anti-IL-1 antibodies. Figure 5 shows that substantial production of IL-8 protein induced in MO by adherence was not affected by antibodies to TNF-, IL-1 and IL-1. Similarly, background production of IL-8 in resting purified LY
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Figure 4. Role of endogenous TNF-, IL-1 and IL-1 in the expression of IL-8 protein and mRNA in (A) human PBMC, (B) MO and (C) LY stimulated by SAC. SAC and corresponding antibodies were added from the beginning of cell incubation: polyclonal rabbit anti-human IL-1 and anti-human IL-1 at 30 g/ml, monoclonal anti-human TNF- at 30 g/ml. Each bar represents the meanSE of triplicate cultures. Similar data obtained in three independent experiments.
was also retained in the presence of anti-TNF-, anti-IL-1 and anti-IL-1. We have showed before that adherence itself may induce high levels of nuclear transcription of IL-8 and IL-1 genes in MO.16 Since it was shown here that IL-1 is a potent inducer of IL-8 expression in MO, we tested purified resting adherent MO for spontaneous induction of IL-1, IL-8 and TNF- expression. As shown in Figure 6, MO freshly isolated from PBMC using a Percoll gradient contained no IL-8, IL-1 or TNF- transcripts. Incubation (adhesion) of MO in a plastic dish for 4 h caused accumulation of IL-8 mRNA and IL-1 mRNA. However, adherence per se did not induce TNF- mRNA in these cells. Adherent MO secreted substantial level of IL-8 protein during 4 h of incubation (Fig. 6), whereas no IL-1 or TNF- protein production was detected by corresponding ELISAs in the same cultures. Dissociation between induction of IL-1 mRNA and protein in adherence stimulated MO has been previously shown.24 This results further support our demonstration that endogenous TNF- and IL-1 play no role in adherence-induced IL-8 gene expression.
DISCUSSION Recent data demonstrated that several stimuli may induce the expression of IL-8 gene in MO without activating TNF- and IL-1 genes,6–10 implying autonomy of IL-8 gene regulation. The present study was undertaken to clarify the role of TNF-, IL-1 and IL-1 in the induction of IL-8 gene in the leukocyte
fractions of human PBMC. Our analysis reveals that purified human MO do not respond to exogenous TNF- with the induction of IL-8 mRNA and protein, while exogenous IL-1 and IL-1 induced the expression of IL-8 in purified MO culture. In contrast, purified LY and non-fractionated PBMC express IL-8 mRNA and IL-8 protein in response to TNF-. Similar to MO, IL-8 expression in LY and PBMC was upregulated by exogenous IL-1 and IL-1. Described herein, the inability of TNF- to induce IL-8 expressaon in purified monocytes seems to be in conflict with previous reports demonstrating enhancement of IL-8 expression in human blood MO in the presence of TNF-.5 This discrepancy may be due to the use of non-fractionated PBMC, not purified MO, in previous studies to demonstrate the effect of TNF- on IL-8 expression.4,5 Thus, according to our results, LY can respond to TNF- with upregulation of IL-8 expression in non-fractionated PBMC. Adherence per se is a potent stimulus to MO population of PBMC.5,22,23 Using nuclear run-on analysis, we demonstrated previously that adherence alone was sufficient to induce transcription of IL-8 and IL-1 genes, but not the TNF- gene.16 In agreement with previous studies, adherence of PBMC and purified MO induces IL-8 mRNA expression and IL-8 protein secretion. The substantial enhancement of IL-8 mRNA and protein expression in purified MO by exogenous IL-1 suggests that endogenous IL-1 may contribute to the expression of IL-8 production in MO. However, our data demonstrating inability of antibodies to IL-1 and IL-1 to downregulate the expression of IL-8 mRNA in purified MO argue against the role of
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Figure 5. Role of endogenous TNF-, IL-1 and IL-1 in the expression of IL-8 protein and mRNA in (A) purified resting MO and (B) LY. TNF- (2.5 ng/ml), IL-1 (2 ng/ml), IL-1 (2 ng/ml) and corresponding antibodies at 30 g/ml were added from the beginning of cell incubation. Incubation was 4 h (open bars) or 20 h (shaded bars).
endogenous IL-1 in adherence-induced MO IL-8 expression. In agreement with that observation, it was further demonstrated that in the cultures of purified MO, adherence alone induced no secretion of immunoreactive IL-1, while IL-1 mRNA was induced. Our observation is in agreement with a previous study describing the dissociation of adherence induced IL-1 mRNA and protein secretion in human MO in vitro.24,25 Taken together, our data further confirm the suggestion that adherence directly activates IL-8 gene expression in MO, and endogenous TNF- and IL-1 play no role in this process. Similarly to adherence, expression of IL-8 in MO stimulated with SAC (or LPS, not shown) was not affected by anti-TNF-, anti-IL-1 or anti-IL-1 antibodies. This may be due to the sustained primary response of IL-8 gene transcription to SAC. There are few studies that report the requirement of endogenous TNF or IL-1 for induction of chemokine genes in MO. In spite of the inability of TNF- to upregulate IL-8 expression in purified MO, it was demonstrated
that exogenous TNF- enhances expression of the CC chemokine RANTES and endogenous TNF- plays a crucial role in the expression of RANTES gene in MO.26 Similarly, the induction of I-309 mRNA in MO was mediated by endogenous IL-1, since antiserum to IL-1 abolished I-309 expression induced by LPS or immunoglobulin plus LPS.17 The regulation of IL-8 expression in MO was thus revealed to be different from the regulation of these CC chemokines in terms of dependence on TNF- and IL-1. Differential expression of IL-8 and CC chemokines by TNF- and IL-1 is in agreement with recent data concerning 5 -upstream regulatory regions of IL-8 and chemokine genes.27–31 These data suggest a complex regulation of chemokine genes at multiple levels by several members of the NF--B family that are able to differentially activate IL-8 gene, compared to RANTES,29 ENA-7830 and some other CC-chemokine genes.31 Similarly to MO, the expression of IL-8 in PBMC and their LY subset activated with SAC was not inhibited by antibodies to TNF- and IL-1. Nevertheless, it is noteworthy that we observed reduction of SAC-induced IL-8 production in part of PBMC cultures (six experiments of 11, mean inhibition 165%) as well as in their purified LY cultures (227% inhibition) in the presence of anti-TNF- antibody. This may be due to the presence of minor fraction of LY that is dependent on endogenous TNF- to express IL-8. Taken together, the findings suggest a relative autonomy of IL-8 gene expression in MO that does not require autocrine or paracrine accessory cytokines but can be induced directly by exogenous stimuli. Therefore, it could be reasoned that this mechanism operates to produce IL-8 and to attract neutrophils in the site of MO activation in an inflammatory reaction more rapidly. Described herein, the differences in responsiveness of MO and LY to exogenous TNF- suggest that LY but not MO will produce potent neutrophil chemoattractant IL-8 in the sites of TNF- accumulation, while IL-1 will induce IL-8 expression in both compartments. Further, our observation suggests that the selective expression and regulation of chemokine genes in MO and LY in inflammatory responses may depend upon surrounding cells and accessory exogenous cytokines.
MATERIALS AND METHODS Reagents Staphylococcus aureus Cowan 1 (SAC) was prepared as described11 and stored at 4C. RPMI 1640 and fetal calf serum (FCS) were purchased from Gibco, Paisley, UK. Percoll was obtained from Pharmacia, Uppsala, Sweden. Standards of recombinant human TNF-, IL-8 and IL-1
Expression of IL-8 gene in human leukocytes / 641
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Comparison of production of IL-8, IL-1 and TNF- and their mRNA expression in resting adherent human MO.
MO were purified by Percoll fractionation and incubated for 4 h alone or in the presence of SAC. (A) Production of IL-1 (grey bars), TNF- (black bars) and IL-8 (open bars): each bar represents the meanSE of triplicate cultures. (B) Northern blot of total RNA samples was sequentially hybridized with 32P-labelled probes as indicated. Comparable RNA loading was confirmed by ethidium bromide staining of gel. Similar data obtained in 3 independent experiments. *TNF- was below 30 pg/ml in adherent MO cultured alone, IL-1 protein production was below 100 pg/ml.
were kindly provided by Dr A. Meager (NIBSC, South Mimms, UK). Recombinant IL-8 and polyclonal antibodies against human IL-8 were kindly donated by Prof. K. Matsushima, Cancer Research Institute, Kanazawa University, Japan. High neutralizing monoclonal antibody against human TNF- 5N and monoclonal antibody to human IL-8 WS-4 were produced in our laboratory.12,13 Neutralizing antibodies against human IL-1 and IL-1 were purchased from Endogen, MA, USA; Code: P-420A and 420B. Samples of recombinant human IL-1 and IL-1 were also kindly provided by Dr S. Ketlinsky (Institute of Ultrapure Biochemicals, St. Petersburg, Russia). Plasmid pQA-114 was kindly provided by Dr A. Shakhov (Institute of Toxicology, Schwerzenbach, Switzerland). Oligonucleotides for reverse transcriptase–polymerase chain reaction (RT-PCR) were synthesized by MWG-BIOTECH, Ebersberg, Germany.
Purification of MO and LY Mononuclear leukocytes were isolated from fresh heparinized blood of healthy donors using standard Ficoll/ Hypaque protocol.15,16 Mononuclear leukocytes from Ficoll/ Hypaque were then fractionated on continuous Percoll gradient at 4C.15,16 LY fraction from Percoll gradient was
additionally purified by plastic adherence for 1 h to eliminate few contaminating MO. Isolated MO were 95–98% naphthyl acetate esterase-positive (NAE kit from Sigma), purified LY contained 0–2% of NAE + cells. Purified MO and LY were incubated in 24-well plastic plates (Corning, New York, USA) or 50-mm polystyrene plastic dishes (Corning) in 1% FCS–RPMI 1640 at 1.5–2106 cells/ml. Part of the MO and LY cultures was activated by SAC at a final concentration of 0.001% (V/V).15,16 Recombinant human cytokines IL-1, IL-1 and TNF-, and neutralizing antibodies against human IL-1, IL-1 and TNF- were added at the beginning of MO and LY incubation as indicated in Results.
Transcript analysis Total RNA from cells was extracted by acidic guanidinium thiocyanate.18 Northern blotting of IL-8, IL-1 and TNF- mRNA has been carried out as described.16,17,19 Reverse transcription (RT) was carried out in 5 g of total cellular RNA with M-MLV reverse transcriptase (GIBCO-BRL) using the recommended conditions in a total reaction volume of 20 l containing 1 g oligo (dT)12–18 (Pharmacia, Dubendorf, Switzerland). The fixed amount of cDNA was co-amplified by PCR in the presence of serial dilutions of control plasmids pQA-1 containing sequences
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complementary to the primers specific for human IL-8 and 2-microglobulin cDNA.14 Amplification was achieved over 30 cycles on a Perkin-Elmer Thermal Cycler 9600 (UK) (denaturation for 20 s at 94C, annealing for 20 s at 55C—two-step PCR) framed by initiating incubation for 2 min at 94C and a final incubation for 5 min at 72C. The PCR products were analysed by agarose gel electrophoresis (2% agarose) in the presence of ethidium bromide. Quantitation of fluorescence of both cellular and competitive template product bands was performed on Gelprint System with Onedscan Software. To correct for any variation in RNA content and cDNA synthesis in the different preparations, each sample was normalized on the basis of its 2-microglobulin content. Results were expressed as the calculated ratio of cytokine mRNAs to 2-microglobulin mRNA as external control.20
Protein analysis Supernatants from cultured cells were harvested, centrifuged for 5 min at 13 000 rpm in Eppendorf tubes at 4C, and stored at 80C. Cytokine concentration was quantified by ELISAs for IL-8,12,13 TNF-13 and IL-1.21
Acknowledgements The work was supported in part by the Department of Surgery, Duke University Medical Center, Durham, NC, USA; by Grant 94-1634 from INTAS, Brussels, Grant MWO 300 from International Science Foundation, NY, USA and B24-343 from Foundation for Fundamental Researches of the Republic of Belarus. We would like to thank Dr A. Shakhov (Institute of Toxicology, Schwerzenbach, Switzerland) for providing plasmid pQA-1 and support with quantitative RT–PCR analysis. The authors are grateful to Dr A. Meager (NIBSC, South Mimms, UK) for providing cytokine standards, Prof. K. Matsushima (Cancer Research Institute, Kanazawa University, Japan) for the gift of anti-IL-8 polyclonal antibody and recombinant IL-8 and Dr S. Ketlinsky (Institute of Ultrapure Biochemicals, St. Petersburg, Russia) for providing recombinant IL-1 and IL-1.
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