endogenously produced cytokines in the development of LPS tolerance, we administered IL-6, TNF, or IL-lc, using the same treatment schedule as that for LPS.
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INFECrION AND IMMUNITY, Oct. 1993, p. 4356-4359 0019-9567/93/104356-04$02.00/0 Copyright C 1993, American Society for Microbiology
Differential Regulation of Cytokine Production in Lipopolysaccharide Tolerance in Mice ANNALAURA ERROI,' GIAMILA FANTUZZI,1 MANUELA MENGOZZI,' MARINA SIRONI,' SCOTT F. ORENCOLE,2 BURTON D. CLARK 2 CHARLES A. DINARELLO 2 ANNA ISETTA,3 PAOLA GNOCCHI,3 MIRELLA GIOVARELLI,4 AND PIETRO GHEZZIl* Mario Negri Institute for Pharnacological Research, Via Eritrea 62, 20157, Milan, 1 Department of Immunology, Farmitalia Carlo Erba R&D, Nerviano)3 and Institute of Microbiology, University of Turin, Turin,4 Italy, and Division of Geographic Medicine and Infectious Diseases, New England Medical Center, Boston, Massachusetts2 Received 16 February 1993/Returned for modification 16 April 1993/Accepted 16 July 1993
We investigated the pattern of down-regulation of cytokine production in endotoxin (lipopolysaccharide
[LPS]) tolerance. A 4-day treatment with LPS (35 ,ug per mouse) was followed by a challenge on day 6 with one more injection of LPS. Circulating tumor necrosis factor (TNF) and interleukin-6 (IL-6) could not be induced (>99%o inhibition) by LPS in LPS-tolerant mice; colony-stimulating factor (CSF) was also down-regulated by than 95%, whereas interferon (IFN) and IL-1 syntheses were only partially inhibited. To study the mechanism of cytokine down-regulation in tolerance, we attempted to reverse the tolerant state by pretreatment with phorbol 12-myristate 13-acetate (PMA) (4 ,ug per mouse) 10 min before the LPS challenge. PMA completely restored IL-6 production and partially that of CSF. PMA had no effect on IFN production and inhibited the induction of IL-1. TNF production was also not restored by PMA. To investigate the role of endogenously produced cytokines in the development of LPS tolerance, we administered IL-6, TNF, or IL-lc, using the same treatment schedule as that for LPS. Whereas IL-6 had no effect, IL-lae or TNF induced partial tolerance to LPS in terms of inhibition of LPS-stimulated TNF and IL-6 production. However, a full LPS-tolerant state could not be induced by administration of recombinant cytokines, suggesting the existence of additional mechanisms, such as a loss of LPS receptors or changes in release of soluble binding proteins. more
Repeated administration of endotoxin (lipopolysaccharide [LPS]) leads to the rapid development of a state of tolerance in experimental animals. Tolerance develops to the lethal (2), metabolic (3), and pyrogenic (7) effects of LPS, and treatment with LPS induces, among other mediators, circulating levels of tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6, interferon (IFN), and colony-stimulating factor (CSF). Mice treated chronically with LPS fail to respond to a subsequent LPS challenge in terms of induction of TNF, IL-6, IFN and CSF (8, 12-14). Furthermore, peritoneal macrophages obtained from LPS-tolerant mice do not respond to LPS stimulation in terms of production of TNF and IL-1 (4, 10, 17), suggesting that LPS tolerance involves a down-regulation of the LPS response directly in the producing cells. However, the role of down-regulation of cytokine production in LPS tolerance has not yet been fully clarified. In particular, it is not known whether different cytokines are down-regulated to similar extents in the same model of LPS tolerance (i.e., whether some cytokines are down-regulated more than others). We studied the effect of LPS tolerance
loss of responsiveness to LPS, which is common for each of its effects and due, for instance, to down-regulation of LPS receptors or associated signalling mechanisms. To investigate the second aspect, we used phorbol 12-myristate 13acetate (PMA), a classical macrophage activator and, at the molecular level, a protein kinase C activator. Previous work by some of the present investigators had shown that coadministration of PMA could restore IL-6 induction by LPS in LPS-tolerant mice (12). TNF production, on the other hand, was not only not restored by PMA, but it was inhibited. In the present work we tested the effect of PMA on the production of different cytokines in response to LPS in normal and LPS-tolerant mice, using an experimental schedule previously described (12). We also investigated the possibility that cytokines induced by LPS might be at least partly responsible for the down-regulation of their own production in LPS tolerance. For instance, crosstolerance between TNF and LPS was previously reported (3), leading to the idea that part of the LPS tolerance might be due to TNF produced after the earlier treatments with LPS. The same might hold true for other cytokines such as IL-1 since the IL-1 receptor antagonist was shown to partially reverse the induction of early tolerance to LPS in mice
induced in mice by a 4-day treatment with LPS on the subsequent production of TNF, IL-lao, IL-11, IL-6, IFN, and CSF. A second question is the mechanism involved in the establishment of LPS tolerance. Inhibition of cytokine production in LPS tolerance could result either from a downregulation of specific biochemical mechanisms for LPS triggering of cytokine production or from a merely nonspecific *
(9). MATERIALS AND METHODS Reagents. LPS (phenol-extracted preparation from Escherichia coli 055:B5; protein content less than 3%) was from Sigma Chemical Co., St. Louis, Mo. PMA was obtained from Sigma, dissolved in dimethyl sulfoxide at a concentration of 1 mg/ml, and diluted in saline at the time of treatment.
Corresponding author. 4356
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TABLE 1. Down-regulation of cytokine production in LPS-tolerant micea Treatment
IL-6 (U/ml)
TNF (U/ml)
CSF (CFU/ml)
IFN (U/ml)
IL-113 (ng/g)
IL-la (ng/g)
Saline/saline Saline/LPS LPS/saline LPS/LPS
