Proc. Natl. Acad. Sci. USA Vol. 93, pp. 4885-4890, May 1996 Immunology Elevated interleukin 6 is induced by prostaglandin E2 in a murine model of inflammation: Possible role of cyclooxygenase-2 ROGER M. HINSON*, JoY A. WILLIAMSt, AND EMILY SHACTERt *Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814; and tLaboratory of Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892 Communicated by Michael Potter, National Institutes of Health, Bethesda, MD, January 2, 1996 (received for review July 25, 1995) ABSTRACT Injection of mineral oils such as pristane have used a murine model that involves injection of mineral into the peritoneal cavities of BALB/c mice results in a oils such as pristane (2,6,10,14-tetramethylpentadecane) into chronic peritonitis associated with high tissue levels of inter- the peritoneal cavities of BALB/c mice (14). This treatment leukin 6 (IL-6). Here we show that increased prostaglandin E2 induces a chronic peritonitis that is associated with prolonged (PGE2) synthesis causes induction of IL-6 and that expression high levels of IL-6 (15). A high percentage of the mice go on of an inducible cyclooxygenase, Cox-2, may mediate this to develop IL-6-dependent (16) plasmacytomas (17, 18). The process. Levels of both PGE2 and IL-6 are elevated in inflam- prolonged abundance of macrophages in the peritoneal cavity matory exudates from pristane-treated mice compared with and the constitutive secretion of IL-6 by these cells (15) suggest lavage samples from untreated mice. The Cox-2 gene is that macrophages are the primary source of IL-6 in the induced in the peritoneal macrophage fraction isolated from pristane model. The mechanism by which IL-6 production is the mice. A cause and effect relationship between increased stimulated in vivo has not been determined. macrophage PGE2 and IL-6 production is shown in vitro. When A clue to identifying the pathway that controls IL-6 synthesis peritoneal macrophages are activated with an inflammatory in vivo derives from the observation that chronic administra- stimulus (polymerized albumin), the Cox-2 gene is induced tion of the nonsteroidal anti-inflammatory drug indomethacin and secretion of PGE2 and IL-6 increases, with elevated PGE2 markedly reduces the levels of IL-6 induced by pristane (15). appearing before IL-6. Cotreatment with 1 1,M indomethacin Indomethacin also inhibits pristane-induced plasmacyto- inhibits PGE2 production by the cells and reduces the induc- magenesis (19). Because the main bioactivity of indomethacin tion of IL-6 mRNA but has no effect on Cox-2 mRNA, is the inhibition of prostaglandin synthesis (20), this finding consistent with the fact that the drug inhibits catalytic activity raises the possibility that prostaglandin E2 (PGE2) and/or of the cyclooxygenase but does not affect expression of the other cyclooxygenase products may be involved in stimulating gene. Addition of exogenous PGE2 to macrophages induces IL-6 synthesis in vivo. However, because indomethacin can IL-6 protein and mRNA synthesis, indicating that the eico- have biological activities separate from its effects on prosta- sanoid stimulates IL-6 production at the level of gene expres- glandin metabolism (21-23), the role of prostaglandins needs sion. PGE2-stimulated IL-6 production is unaffected by addi- to be established directly. tion of indomethacin. Taken together with the earlier finding Two gene products, Cox-1 and Cox-2, have been identified that indomethacin diminishes the elevation of IL-6 in as having prostaglandin synthase (EC 1.14.99.1) activity (24, pristane-treated mice, the results show that PGE2 can induce 25). Cox-1 is expressed constitutively in most mammalian IL-6 production in vivo and implicate expression of the Cox-2 tissues (26) and is thought to be responsible for housekeeping gene in the regulation of this cytokine. functions of prostaglandins such as regulation of gastric acid secretion (27). Cox-2 is an inducible enzyme that is thought to Interleukin 6 (IL-6) is a multifunctional cytokine that is give rise to the increased prostaglandin levels produced during produced in a variety of inflammatory conditions in vivo (1, 2). inflammation (28). Cox-2 expression is known to be induced in It and differentiation of B and T macrophages exposed to inflammatory stimuli such as lipo- stimulates activation lym- polysaccharide (LPS) (29-31). This report provides evidence phocytes, induces fever, and regulates acute phase protein that prostaglandins serve as the predominant stimulus for IL-6 synthesis (3-6). Because of its central role in modulating production in the pristane model. Elevated PGE2 probably immunity, IL-6 production is normally tightly regulated. On derives from induction of Cox-2 exposure to an inflammatory stimulus (e.g., infectious agent, expression. endotoxin, wounding), IL-6 levels in vivo rise transiently and then return to background levels on resolution of the insult EXPERIMENTAL PROCEDURES (7-10). Dysregulation of IL-6 production or turnover can lead Mice. BALB/c mice were from a conventional mouse colony to chronic elevations of the cytokine level and may have at Organon Teknika-BRL (National Cancer Institute contract pathologic consequences (5). For example, IL-6 is an impor- NO1CB21075) or were purchased from Charles River Breeding tant growth factor for multiple myeloma (11) and levels are Laboratories and fed Purina laboratory chow and water ad lib. correlated with severity of disease (12). Similarly, synovial Animals were cared for in accordance with the National fluids from patients with rheumatoid arthritis, a chronic Institutes of Health Animal Care Guidelines. Female mice (8 inflammatory disease, contain high levels of IL-6 (13) that may to 16 week old) received a single 0.5-ml i.p. injection of be responsible for some of the systemic abnormalities common pristane. in these patients. In most conditions involving dysregulated Preparation of Peritoneal Macrophages. Peritoneal lavage IL-6, the mechanisms responsible for maintaining the high samples were collected 2-4 months after pristane treatment cytokine levels are not known. (15). The peritoneal macrophage population was isolated as The goal of our work is to identify the pathways responsible described (15). Briefly, neutrophils and macrophages were for inducing IL-6 production during chronic inflammation. We Abbreviations: IL-6, interleukin 6; LPS, lipopolysaccharide; pAlb, The publication costs of this article were defrayed in part by page charge polymerized albumin; PGE2, prostaglandin E2; bicyclo-PGE2, 11- payment. This article must therefore be hereby marked "advertisement" in deoxy-13,14-dihydro-15-keto-11f3,166-cyclo-PGE2; IBMX, 3-isobutyl- accordance with 18 U.S.C. §1734 solely to indicate this fact. 1-methylxanthine. 4885 Downloaded by guest on September 29, 2021 4886 Immunology: Hinson et al. Proc. Natl. Acad. Sci. USA 93 (1996) 25- acute inflammatory stimuli in vivo, similar to LPS (33). pAlb 7 is somewhat more potent and less toxic to the cells than LPS 1 1- and thus was used in the studies described in this report. s 0 However, most of these experiments have been confirmed J5 2 using LPS as stimulant, indicating that the results are not unique to treatment with pAlb. )o Macrophages were cultured in DMEM at 37°C in a humid- 0 ified atmosphere containing 5% CO2. pAlb (10-200 ,ug/ml; ref. 33) or PGE2 (0.001-1 ,uM) was added to washed cells in fresh DMEM. Where indicated, indomethacin was added to S the cell cultures 30 min before adding the stimulus. 3-Isobutyl- * [E1 1-methylxanthine (IBMX; 0.5 mM), a phosphodiesterase in- I-- 6o0- ; hibitor, was added to inhibit breakdown of cAMP in some experiments with exogenously added PGE2. In experiments 41 that contained no added protein, ultrapure BSA (Boehringer Mannheim; ref. 33) was added to each macrophage superna- 2 tant at the time of collection to decrease the adsorption of IL-6 0 to the walls of the collection tube. After various times of incubation, the culture medium was removed from each well Sio and centrifuged. Supernatants were stored frozen until assayed for the presence IL-6 or PGE2. 5 - * EI Purification of RNA and Northern Blot Analysis. Total '~ 60 .siis RNA was purified from in vitro-plated macrophages after 0 various times of incubation using TRIzol (GIBCO/BRL) following the protocol recommended by the manufacturer. For *m examination of in vivo expression of Cox-2, RNA was purified * 2 from freshly isolated peritoneal cells (the macrophage fraction of pristane-elicited cells and the total resident cell population from control mice) without plating. RNA samples (4 or 15 jig each) were run on 1% agarose/0.7% formaldehyde gels con- Pristane Control taining ethidium bromide and transferred to nitrocellulose. FIG. 1. Measurement of IL-6 and prostaglandins in inflammatory 32P-labeled IL-6 and actin cDNA probes were prepared using exudates from pristane-treated mice. Pristane-primed BALB/c mice a random priming system (GIBCO/BRL) and [a-32P]dCTP were lavaged with PBS containing 1 jiM indomethacin (to inhibit (Amersham). Blots were hybridized overnight with probe (1.2 induction of prostaglandin production during the lavage procedure). x 106 cpm/ml, 5 x 108 cpm/,jg) at 65°C in Hybrisol II solution (A) IL-6 in each lavage sample was measured by the B9 bioassay, which (Oncor) and then washed by standard procedures. Autora- was validated by ELISA. (B and C) PGE2 and bicyclo-PGE2 were diography was performed using Kodak XAR film. Films were measured in freshly collected and extracted lavage samples. Values scanned (Microtek Scanmaker) and analyzed using the Macin- represent the concentrations present in each lavage sample and are not corrected for dilution by the lavage process. The levels of PGE2 and tosh densitometry program NIH IMAGE (National Institutes of bicyclo-PGE2 found in control mice are below or near the limits of Health, Bethesda).