Macrophage TNF mRNA Expression Is Modulated by Protease Inhibitors

doi:10.1006/jsre.1997.5103

Journal of Surgical Research

Volume 69, Issue 2, May 1997, Pages 408-412

https://doi.org/10.1006/jsre.1997.5103 Get rights and content

Abstract

Background: Nuclear factor κB (NFκB) is an important transcriptional activator protein and is a crucial component of the host's response to infection. The activation of NFκB is correlated with the phosphorylation of inhibitory κB (IκB) and its subsequent degradation. We hypothesized that protease inhibitors which prevented IκB degradation could inhibit the macrophage gene activation and reduce the production of inflammatory cytokines. Methods: Rabbit alveolar macrophages (Mφ) were obtained by bronchoalveolar lavage. Mφ were exposed toEscherichia colilipopolysaccharide (LPS) (10 ng/ml) in the presence of various concentrations of protease inhibitors, eitherN-tosyl-l-phenylalanine chloromethyl ketone (TPCK) orN-benzoyl-l-tyrosine ethyl ester (BTEE). Total RNA was extracted for Northern blot assay of tumor necrosis factor (TNF) mRNA expression using a rabbit genomic DNA probe. Total nuclear extracts were also obtained for the measurement of the NFκB activity with the electrophoretic mobility shift assay. The TNF production in the Mφ supernatant was measured by L929 bioassays. Results: NFκB activity induced by LPS was inhibited by either BTEE or TPCK. Inhibition of NFκB activity by these agents also prevented TNF mRNA expression and TNF production induced by LPS. The cellular mechanism leading to NFκB activation was further studied. TNF mRNA expression and NFκB activation were inhibited by D609, a phospholipase C (PLC) inhibitor, as well as by protein kinase C (PKC) inhibitors. In addition, direct stimulation of PKC led to NFκB activation and TNF mRNA expression. Conclusions: These data suggest that TNF mRNA expression of LPS-stimulated Mφ is mediated through NFκB. NFκB activation is intimately regulated by the PLC signaling pathway.

References (0)

Cited by (24)

Gabapentin and (S)-pregabalin decrease intracellular d-serine concentrations in PC-12 cells
2013, Neuroscience Letters
Citation Excerpt :
Modulation of SR activity, determined by the efflux of d-Ser, has been achieved through the reduction of intracellular Ca2+ through the addition of chelating agents to the incubation media [26] or increased using the calcium ionophore, A23187 [6,9]. We have also recently shown that the treatment of PC-12 cells with Escherichia coli lipopolysaccharide (LPS) increased intracellular d-Ser [20], which is consistent with LPS induced Ca2+ influx, see for example [10,26]. Thus, this approach would involve the reduction of NMDAR activity via alterations in intracellular Ca2+ produced by effects at receptors and transporters involved in Ca2+ flux.
The effects of gabapentin (GBP) and (S)-pregabalin (PGB) on the intracellular concentrations of d-serine and the expression of serine racemase (SR) in PC-12 cells were determined. Intracellular d-serine concentrations were determined using an enantioselective capillary electrophoresis assay with laser-induced fluorescence detection. Increasing concentrations of GBP, 0.1–20 μM, produced a significant decrease in d-serine concentration relative to control, 22.9 ± 6.7% at 20 μM (*p < 0.05), with an IC₅₀ value of 3.40 ± 0.29 μM. Increasing concentrations of PGB, 0.1–10 μM, produced a significant decrease in d-serine concentration relative to control, 25.3 ± 7.6% at 10 μM (*p < 0.05), with an IC₅₀ value of 3.38 ± 0.21 μM. The compounds had no effect on the expression of monomeric-SR or dimeric-SR as determined by Western blotting. The results suggest that incubation of PC-12 cells with GBP and PGB reduced the basal activity of SR, which is most likely a result of the decreased Ca²⁺ flux produced via interaction of the drugs with the α₂-δ subunit of voltage-gated calcium channels. d-Serine is a co-agonist of the N-methyl d-aspartate receptor (NMDAR) and reduced d-serine concentrations have been associated with reduced NMDAR activity. Thus, GBP and PGB may act as indirect antagonists of NMDAR, a mechanism that may contribute to the clinical effects of the drugs in neuropathic pain.
Effect of synthetic protease inhibitor gabexate mesilate on the attenuation of ischemia/reperfusion injury in canine kidney autotransplantation
2005, Surgery
Kidneys from non–heart-beating donors are associated with delayed graft function and a high rejection rate due to the long period of warm ischemia. Gabexate mesilate (GM), a synthetic serine protease inhibitor, has been shown to improve organ function by suppressing cytokine activity and neutrophil function after ischemia/reperfusion. In this study, we evaluated the effect of GM on renal function after warm ischemia in a canine kidney autotransplantation model.
After 60 minutes of warm ischemia, the left kidney was transplanted into the iliac fossa, and the right kidney was removed. The control group (n = 7) and GM group (n = 7) were evaluated for serum creatinine and blood urea nitrogen (BUN) concentrations, renal tissue blood flow, resistive index, pulsatility index, interleukin (IL)-1β and tumor necrosis factor (TNF)-α mRNA expression levels in peripheral blood mononuclear cells, apoptotic index, CD10 immunolabeling as an indicator of brush border injury, and standard histopathology.
Compared with controls, administration of GM resulted in lower serum creatinine concentrations (11.3 ± 2.4 vs 5.2 ± 3.3 mg/dL at 72 hours; P = .04) and BUN concentrations (188 ± 26 mg/dL vs 98 ± 41 mg/dL at 72 hours; P = .04), as well as better tissue blood flow, improvement of brush border injury and apoptotic index (each P < .05). The expression of IL-1β and TNF-α mRNA did not change after administration of GM.
The present study shows that GM protected renal function after warm ischemia/reperfusion by inhibition of serine proteases, maintenance of tissue blood flow, and amelioration of tubular apoptosis.
Prostaglandin Levels in Stimulated Macrophages Are Controlled by Phospholipase A<inf>2</inf>-activating Protein and by Activation of Phospholipase C and D
2001, Journal of Biological Chemistry
Prostaglandins (PG), which are responsible for a large array of biological functions in eukaryotic cells, are produced from arachidonic acid by phospholipases and cyclooxygenase enzymes COX-1 and COX-2. We demonstrated that PG levels in cells were partly controlled by a regulatory protein, phospholipase A₂(PLA₂)-activating protein (PLAA). Treatment of murine macrophages with lipopolysaccharide, interleukin-1β, and tumor necrosis factor-α increased PLAA levels at early time points (2–30 min), which correlated with an up-regulation in cytosolic PLA₂ and PGE₂ levels. Both COX-2 and secretory PLA₂ were also increased in lipopolysaccharide-stimulated macrophages, however, at later time points of 4–24 h. The role of PLAA in eicosanoid formation in macrophages was confirmed by the use of an antisense plaa oligonucleotide. Within amino acid residues 503–538, PLAA exhibited homology with melittin, and increased PGE₂ production was noted in macrophages stimulated with melittin. In addition to PLA₂, we demonstrated that activation of phospholipase C and D significantly controlled PGE₂ production. Finally, increased antigen levels of PLAA, COX-2, and phospholipases were demonstrated in biopsy specimens from patients with varying amounts of intestinal mucosal inflammation, which corresponded to increased levels of phospholipase activity. These results could provide a basis for the development of new therapeutic tools to control inflammation.
Specific Pancreatic Enzymes Activate Macrophages to Produce Tumor Necrosis Factor-Alpha: Role of Nuclear Factor Kappa B and Inhibitory Kappa B Proteins
2000, Journal of Gastrointestinal Surgery
The triggering events by which mononuclear cells throughout the body are induced to produce large amounts of cytokines during acute pancreatitis are unclear. However, recent work in our laboratory demonstrated that three specific pancreatic enzymes (elastase, carboxypeptidase A, and lipase) induced dramatic tumor necrosis factor-alpha (TNF-α) protein production from macrophages, whereas all others could not. This series of experiments was designed to examine the second messenger system by which this occurs. The rat macrophage cell line NR8383 was incubated for 3 hours with elastase, carboxypeptidase A, lipase, trypsin, or lipopolysaccharide (positive control). Activation of nuclear factor kappa B (NF-κB) was demonstrated by electrophoretic mobility shift assay, presence of inhibitory kappa B alpha and beta (IκB-α and IκB-β) by Western blot analysis, and TNF-α protein production by enzyme-linked immunosorbent assay. Elastase, carboxypeptidase A, and lipase induced degradation of IκB-β (but not IκB-α), activation of NF-κB, and production of TNF-α protein, whereas inhibition of IκB with pyrrolidine dithiocarbamate attenuated this response. Trypsin was unable to elicit any of these responses. Macrophages can be induced by specific activated pancreatic enzymes—elastase, carboxypeptidase A, and lipase—to produce TNF-α. This process is dependent on IκB-β degradation and NF-κB activation, suggesting that these enzymes trigger this second messenger system through specific membrane-bound receptors.
Elastase mimics pancreatitis-induced hepatic injury via inflammatory mediators
2000, Journal of Surgical Research
Background. Recent evidence suggests that pancreatitis-associated hepatic injury is regulated by inflammatory mediator production. Our laboratory demonstrated in vitro that pancreatic elastase is a pancreatic enzyme that can induce inflammatory cell cytokine production. Therefore we now explore the in vivo effects of elastase on the liver.
Materials and methods. Elastase (1.5 U) ± CNI-1493, which attenuates mediator production through p38 MAP kinase inhibition, was administered intraperitoneally to mice while control animals received saline. Acute pancreatitis (AP) was induced with a choline-deficient, ethionine-supplemented (CDE) diet. Serum hepatic enzymes and hepatic neutrophil infiltration by myeloperoxidase (MPO) activity were measured as indicators of hepatic insult. Serum tumor necrosis factor (TNF) protein (ELISA), hepatic TNF mRNA (reverse transcription polymerase chain reaction), and hepatic activation of the transcription factor nuclear factor kappa B (electrophoretic mobility shift assay) were also determined.
Results. A significant increase in hepatic enzymes and MPO activity was induced by AP and mirrored by intraperitoneal elastase. Both types of liver injury resulted in near identical elevations in serum TNF protein and hepatic TNF mRNA. Elastase-treated animals with mediator production inhibited (CNI-1493) had attenuated hepatic enzymes, MPO activity, TNF protein, and TNF mRNA. Activation of nuclear factor kappa B occurred 30 min after elastase administration.
Conclusion. Exposure of the liver to pancreatic elastase results in hepatic inflammation and injury which appears identical to that seen during severe AP. Prevention of inflammatory mediator production by intrahepatic leukocytes attenuates injury and supports recent adult respiratory distress syndrome and in vitro data suggesting that elastase is the principal factor that propagates pancreatic inflammation into a systemic illness through direct activation of systemic inflammatory cells.
Fish oil decreases macrophage tumor necrosis factor gene transcription by altering the NFκB activity
1999, Journal of Surgical Research
Background.Fish oil-supplemented diets have anti-inflammatory and immunomodulating effects, though the exact mechanism(s) are unknown. This study investigated the effects of eicosapentanenoic acid (EPA), a major component of fish oil, on transcriptional regulation of tumor necrosis factor (TNF) gene in lipopolysaccharide (LPS)-stimulated macrophages (MØ).
Methods.RAW 264.7 cells, a mouse MØ cell line, were grown in EPA-rich media for 24–48 h. MØ were washed and exposed toEscherichia coliLPS (1 μg/ml) for 2 h. TNF mRNA expression was measured by Northern blot assays. Total nuclear extracts were harvested for the measurement of NFκB with electrophoretic mobility shift assays. Supershift assays were performed with anti-P50 or anti-P65 antibodies to show components of NFκB dimers. TNF production was determined by L929 bioassays.
Results.LPS stimulated RAW cell TNF mRNA expression and NFκB activity. In contrast, RAW cells grown in EPA-rich media had less TNF mRNA expression and an altered composition of the NFκB subunits (P65/P50 dimers) in the presence of LPS. TNF production by LPS-stimulated MØ was reduced by EPA.
Conclusions.The inhibitory effect of EPA on LPS-stimulated MØ TNF gene transcription and protein elaboration is, in part, mediated through altering NFκB activation by reducing the P65/P50 dimers.

View all citing articles on Scopus

^☆: Supported by the American Association for the Surgery of Trauma/John H. Davis, M.D., Research Scholarship and a grant from the Sence Foundation, Los Angeles, California.

View full text

Regular ArticleMacrophage TNF mRNA Expression Is Modulated by Protease Inhibitors☆

Abstract

Regular Article
Macrophage TNF mRNA Expression Is Modulated by Protease Inhibitors☆