Tumor necrosis factor alpha transcription in macrophages is attenuated by an autocrine factor that preferentially induces NF-kappaB p50 - PubMed (original) (raw)

Tumor necrosis factor alpha transcription in macrophages is attenuated by an autocrine factor that preferentially induces NF-kappaB p50

M Baer et al. Mol Cell Biol. 1998 Oct.

Abstract

Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), which are expressed during conditions of inflammation, infection, or injury. We identified an activity secreted by a macrophage tumor cell line that negatively regulates bacterial lipopolysaccharide (LPS)-induced expression of TNF-alpha. This activity, termed TNF-alpha-inhibiting factor (TIF), suppressed the induction of TNF-alpha expression in macrophages, whereas induction of three other proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-6, and monocyte chemoattractant protein 1) was accelerated or enhanced. A similar or identical inhibitory activity was secreted by IC-21 macrophages following LPS stimulation. Inhibition of TNF-alpha expression by macrophage conditioned medium was associated with selective induction of the NF-kappaB p50 subunit. Hyperinduction of p50 occurred with delayed kinetics in LPS-stimulated macrophages but not in fibroblasts. Overexpression of p50 blocked LPS-induced transcription from a TNF-alpha promoter reporter construct, showing that this transcription factor is an inhibitor of the TNF-alpha gene. Repression of the TNF-alpha promoter by TIF required a distal region that includes three NF-kappaB binding sites with preferential affinity for p50 homodimers. Thus, the selective repression of the TNF-alpha promoter by TIF may be explained by the specific binding of inhibitory p50 homodimers. We propose that TIF serves as a negative autocrine signal to attenuate TNF-alpha expression in activated macrophages. TIF is distinct from the known TNF-alpha-inhibiting factors IL-4, IL-10, and transforming growth factor beta and may represent a novel cytokine.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

Identification of TNF-α-inhibitory activity in CM from P388D1(IL1) macrophages. (A) Analysis of TNF-α, IL-6, MCP-1, and IL-1β RNA expression in IC-21 macrophages. IC-21 cells were pretreated with P388D1(IL1) CM (concentrated by ultrafiltration) or unconditioned medium (UCM) for 16 h and induced with LPS (10 μg/ml), and RNA was harvested over an 8-h time course. One microgram of total RNA from each time point was blotted onto a nylon membrane (slot blot), and duplicate blots were hybridized with the indicated cytokine probes. Cytokine RNA expression was quantitated with a PhosphorImager. Cytokine inductions were normalized to actin mRNA and are expressed as percent maximal induction in control (UCM-treated) cells. (B) Effect of CM on TNF-α expression in murine bone marrow (BM) and peritoneal macrophages. Primary macrophages were cultured for 3 to 4 days and treated for 16 h with CM or UCM. The cells were then stimulated with LPS, and RNA was prepared at 0, 3, and 6 h as described for panel A. TNF-α expression was analyzed by slot blotting and quantitated (normalized to actin) with a radioanalytical scanner.

FIG. 2

FIG. 2

A TNF-α-inhibiting activity is secreted by LPS-stimulated IC-21 macrophages. (A) CM was prepared from control and LPS-treated (10 μg/ml, 16 h) IC-21 cells and concentrated by ultrafiltration. The CM preparations were then added to naive IC-21 cells together with 20 μg of LPS per ml, and RNA was harvested over an 8-h time course. Parallel inductions were performed on cells treated with LPS alone, pretreated with P388D1(IL1) CM for 16 h, or treated simultaneously with P388D1(IL1) CM and LPS (see key at right). One microgram of total RNA from each time point was analyzed by slot blot hybridization using the indicated cytokine probes. Hybridization signals were quantitated by scanning (normalized to actin) and are expressed as a percentage of the maximal induction observed in untreated cells (no CM pretreatment). TNF-α expression was determined in two independent experiments (top panels). (B) Cytokine expression in IC-21 macrophages treated with LPS− CM. The culture medium was removed from naive IC-21 macrophages and replaced with LPS− CM or control CM (see Materials and Methods). After 16 h, the cells were treated with LPS and cytokine mRNA levels were analyzed as described for Fig. 1A.

FIG. 3

FIG. 3

Effects of IL-6 and TNF-α pretreatment on cytokine mRNA expression in IC-21 cells. IC-21 cells were grown for 16 to 20 h in fresh medium (control) or fresh medium supplemented with recombinant IL-6 or TNF-α (10 ng/ml). The cells were then stimulated with LPS, and RNA was harvested over an 8-h time course. Northern blots were prepared (10 μg of RNA per lane) and hybridized with the indicated cDNA probes.

FIG. 4

FIG. 4

CM suppresses LPS-induced transcription from the TNF-α promoter in transfected macrophages. (A) ANA-1 macrophages were transfected with the TNF-Luc reporter plasmid (1 μg/2 × 106 cells), treated with LPS− CM or control CM for 16 h, and then stimulated with LPS over a 12-h time course. Relative luciferase expression was calculated by normalizing to luciferase activity in control cells at 0 h. The data represent the average of three independent experiments. (B) The same experiment was performed with a control reporter plasmid, pRSV-βgal. Relative β-galactosidase (β-gal) expression was calculated as described above for luciferase activity.

FIG. 5

FIG. 5

CM preferentially activates nuclear NF-κB p50 expression. IC-21 cells were treated with control CM (A), LPS− CM (B), or a combination of IL-6 (10 ng/ml) and TNF-α (20 ng/ml) (C), and nuclear extracts were prepared over a 24-h time course. Four micrograms of each protein extract was assayed by Western blotting. The blots were probed simultaneously with polyclonal antibodies specific for p50 and p65.

FIG. 6

FIG. 6

Nuclear NF-κB p50 is selectively induced in LPS-stimulated macrophages. IC-21 macrophages (A) or L fibroblasts (B) were treated with LPS (1 μg/ml), and nuclear extracts were prepared over a 24-h time course. Samples were analyzed for p50 and p65 expression by Western blotting as described in the legend to Fig. 5.

FIG. 7

FIG. 7

Overexpression of NF-κB p50 inhibits LPS induction of the TNF-α promoter. ANA-1 macrophages were transfected with 1 μg of TNF-Luc reporter DNA and 2 μg of either the Rc/CMV control vector, Rc/CMV-p50, Rc/CMV-p65, or Rc/CMV-p50 plus Rc/CMV-p65 (1 μg of each). Forty hours later, the cells were stimulated with LPS (1 μg/ml), and lysates were prepared at 0, 3, and 6 h. Relative luciferase expression was normalized to the luciferase activity of Rc/CMV-transfected cells prior to LPS treatment (0 h). Expression of a reporter gene driven by the simian virus 40 early promoter, pGL2-Luc, was compared as a control (lower panel). The data are the means ± standard errors of the means from three independent experiments.

FIG. 8

FIG. 8

Localization of TNF-α promoter sequences required for inhibition by CM. (A) Diagram of 5′ deletions and point mutations and a summary of their repression by LPS− CM. The locations of known κB elements are depicted. The name of each construct indicates the position of the deletion endpoint relative to the TNF-α transcription startsite. (B) LPS− CM inhibition assays. Each plasmid construct (1 μg/2 × 106 cells) was transfected into ANA-1 macrophages, and 24 h later the cells were treated with control CM (□) or LPS− CM (•) for 16 h. The cells were then stimulated with LPS, and lysates were prepared over a 6-h time course. Relative luciferase expression (fold induction) for each reporter construct was normalized to luciferase activity from cells treated with control CM before LPS stimulation (0 h). The data represent the average means ± standard errors of the means from three independent experiments. Significance was calculated by Student’s t test (∗, P ≤ 0.02; ∗∗, P ≤ 0.14 versus control CM).

FIG. 9

FIG. 9

Three NF-κB sites in the TNF-α promoter preferentially bind NF-κB p50 homodimers. (A) EMSA analysis of nuclear extracts from LPS-stimulated IC-21 cells. The cells were induced with LPS, and nuclear extracts were prepared over a 24-h time course. Double-stranded oligonucleotide probes corresponding to the one of the distal NF-κB sites of the TNF-α promoter (TNF-κB3) or the Ig-κ light-chain NF-κB site (Ig-κ) were incubated with the nuclear extracts, and protein-DNA complexes were separated from free probe by electrophoresis. (B) Supershift analysis of complexes bound to κB sites from the TNF-α promoter or the Ig-κ element. Nuclear extract harvested 8 h after LPS treatment (A) was incubated in the presence or absence of p50- or p65-specific antibodies (Ab), and the indicated oligonucleotide probes were added prior to gel electrophoresis.

FIG. 10

FIG. 10

The distal TNF-α κB sites are poorly activated by p65. Reporter plasmids (1 μg) containing four tandem copies of the indicated κB sites upstream of a minimal promoter reporter gene (TK-Luc) were cotransfected into ANA-1 cells with various ratios of p50 and p65 expression vectors (1 μg in total) and an internal standard, pRSV-βgal (0.5 μg). After 2 days, the cells were induced with LPS for 4 h and harvested, and the lysates were assayed for luciferase and β-galactosidase activities. Luciferase activity was normalized to β-galactosidase activity for each sample. The data are from a representative experiment; similar results were obtained in two independent experiments.

Similar articles

Cited by

References

    1. Akira S, Isshiki H, Sugita T, Tanabe O, Kinoshita S, Nishio Y, Nakajima T, Hirano T, Kishimoto T. A nuclear factor for IL-6 expression (NF-IL6) is a member of a C/EBP family. EMBO J. 1990;9:1897–1906. - PMC - PubMed
    1. Akira S, Taga T, Kishimoto T. Interleukin-6 in biology and medicine. Adv Immunol. 1993;54:1–78. - PubMed
    1. Baer, M., S. C. Williams, R. C. Schwartz, A. J. Dillner, and P. F. Johnson. Autocrine signals control C/EBPβ expression, localization, and activity in macrophages. Blood, in press. - PubMed
    1. Beyaert R, Fiers W. Molecular mechanisms of tumor necrosis factor-induced cytotoxicity. What we do understand and what we do not. FEBS Lett. 1994;340:9–16. - PubMed
    1. Bogdan C, Paik J, Vodovotz Y, Nathan C. Contrasting mechanisms for suppression of macrophage cytokine release by transforming growth factor-β and interleukin-10. J Biol Chem. 1992;267:23301–23308. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources