pneumophila activity induced by LPS in macrophages from susceptible A/J mice was reversed in vitro by ... to cultures of murine macrophages from A/J mice suscepti- ..... W. E. Bullock, and N. Hanna (ed.) ... Taffet, S. M., and S. W. Russell. 1981 ...
INFECTION AND IMMUNITY, May 1992, p. 1936-1940 0019-9567/92/051936-05$02.00/0 Copyright 3 1992, American Society for Microbiology
Vol. 60, No. 5
Cyclic AMP Inhibition of Lipopolysaccharide-Induced Restriction of Legionella pneumophila Growth in Macrophage Cultures KIYOSHI EGAWA,t THOMAS W. KLEIN, YOSHIMASA YAMAMOTO, CATHERINE A. NEWTON, AND HERMAN FRIEDMAN* Department of Medical Microbiology and Immunology, University of South Florida College of Medicine, Tampa, Florida 33612 Received 24 October 1991/Accepted 19 February 1992
The mechanism of the effects of lipopolysaccharide (LPS) on macrophages in terms of replication of intracellular facultative bacteria is unclear. It was found in the present study that the anti-Legionela pneumophila activity induced by LPS in macrophages from susceptible A/J mice was reversed in vitro by dibubtryl cyclic AMP (DcAMP). A 24-h pretreatment of murine thioglycolate-elicited macrophages with LPS resulted in an enhanced ability of these cells to inhibit the intracellular growth of L. pneumophila. This anti-L. pneumophila activity of macrophages induced by LPS was inhibited when DcAMP (10-3 to 10-5 M) was present during preincubation with LPS. The addition of DcAMP to the cultures was more effective before LPS treatment than after treatment. The effect of DcAMP was dose dependent. The secretion and production of acid phosphatase by LPS-activated macrophages were also inhibited by the addition of DcAMP before LPS treatment. Furthermore, the anti-L. pneumophila activity of macrophages induced by LPS could also be reversed in vitro by treatment with prostaglandin E2, colchicine, isoproterenol, theophylline, or hydrocortisone, all of which are known to increase the intracellular levels of cyclic AMP in various tissues. These observations indicate that the anti-L. pneumophila activity induced by LPS treatment can be modified by mechanisms involving cyclic nucleotide metabolism. Bacterial lipopolysaccharides (LPS) from cell walls of gram-negative bacteria are known to activate numerous functions of macrophages from a variety of species, including mice. LPS-treated macrophages are efficient in inhibiting the growth of intracellular microbial parasites, including bacteria, as compared with non-LPS-treated cells (15, 19, 25). Recently, we reported that bacterial LPS, when added to cultures of murine macrophages from A/J mice susceptible to Legionella pneumophila, an intracellular opportunistic bacterium which is the etiologic cause of pulmonary as well as systemic opportunistic infections in humans, result in a conversion from permissiveness to nonpermissiveness (10). In the present study, we report that this effect is linked to cyclic nucleotide metabolism, since cyclic AMP (cAMP) reverses the LPS-induced inhibition of macrophage growth restriction of L. pneumophila in vitro.
Bacteria. A virulent strain of L. pneumophila serogroup 1 obtained at autopsy from a case of fatal legionellosis at Tampa General Hospital and cultured on buffered charcoalyeast extract medium obtained from GIBCO Laboratories, Madison, Wis., exactly as described previously (11). Macrophage cultures. Thioglycolate-elicited peritoneal macrophages were obtained from test animals as described previously (34). The elicited peritoneal macrophages were suspended in RPMI 1640 medium (GIBCO) supplemented with 15% heat-inactivated fetal calf serum, allowed to adhere to 24-well tissue culture plates (Costar, Cambridge, Mass.) for 2 h in 5% CO2 at 37°C, washed to remove nonphagocytized bacteria, and supplied with medium (RPMI 1640 medium containing 15% fetal calf serum). Infection. The resulting macrophage monolayers (approximately 106 cells per well) were infected with a suspension of L. pneumophila and incubated at 37°C for 30 min. The nonphagocytized bacteria were removed by three consecutive washes with warmed Hanks' balanced salt solution. A total of 1 ml of RPMI 1640 medium containing 15% fetal calf serum was added to each well, and the plates were incubated for an additional 48 h. The number of viable bacteria (CFU) in distilled water lysates of the macrophages was determined by standard plating on buffered charcoal-yeast extract agar (34). Acid phosphatase assay. Acid phosphatase activity was measured as previously described (14). In brief, 20-,ul culture supernatants or macrophage lysates obtained from 0.1% Triton X-100-treated macrophages and 20 RI of 0.2 M acetate buffer (pH 5.0) containing 24 mM p-nitrophenol phosphate solution were added successively to each well of a 96-well Costar plate. After incubation for 30 min, 200 RI of 0.2 M was
MATERIALS AND METHODS Reagents. LPS from Escherichia coli O111:B4, dibutyryl cAMP (DcAMP), colchicine, theophylline, isoproterenol, and hydrocortisone were purchased from Sigma Chemical Co., St. Louis, Mo. Prostaglandin E2 (PGE2) was purchased from The Upjohn Co., Kalamazoo, Mich. Pyrogen-free saline was used to prepare stock solutions of these materials. Concentrated solutions of PGE2 were prepared in absolute ethyl alcohol. Animals. AJJ female mice were obtained from Jackson Laboratory, Bar Harbor, Maine, and used in this study at 8 to 12 weeks of age. They were housed and cared for in the National Institutes of Health-approved animal vivarium at our institution under the direction of a veterinarian.
sodium carbonate solution
added to each well and the an enzyme immunoassay reader (EL 309; Bio-TEK Instrument, Inc.). Enzyme activity was measured as the release of p-nitrophenol was
A405 of p-nitrophenol was measured with
Corresponding author. t Present address: Department of Microbial Chemistry, Showa University School of Pharmaceutical Sciences, Tokyo, Japan. *
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DcAMP INHIBITION OF LPS ACTIVATION
VOL. 60, 1992
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TABLE 1. Inhibition by DcAMP of A/J mouse macrophage anti-L. pneumophila and acid phosphatase activities induced by LPSa
107
DcAMP
(mM)
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