INFECrION AND IMMUNITY, OCt. 1992, p. 4402-4406

Vol. 60, No. 10

0019-9567/92V104402-05$02.00/0 Copyright X 1992, American Society for Microbiology

Recombinant Interleukin-6 Protects Mice against Experimental Bacterial Infection ZHANQI LIU,1 RICHARD J. SIMPSON,2 AND CHRISTINA CHEERS'* Department ofMicrobiology, University ofMelbourne,1 and Ludwig Institute for Cancer Research, Royal Melbourne Hospital, 2 Parkville, Victoria 3052, Australia Received 20 April 1992/Accepted 3 August 1992

Because of reports of high levels of interleukin-6 (IL-6) in patients during infection, we studied the role of

IL.6 in experimental infection. Mice infected with the facultative intracellular pathogen Listeria monocytogenes displayed high levels of IL-6 in their sera and tissues, particularly the spleen, 1 to 3 days after infection. At this time, the IL-6 titers correlated with bacterial numbers in individual mice and in groups of mice given graded doses of Listeria organisms. However, the presence of IL-6 in serum declined after 4 days, even when a large initial dose of bacteria meant that bacterial numbers were still increasing at this time. Recombinant mouse IL-6 injected intraperitoneally before infection protected mice in a dose-dependent manner. It was effective when given 4 h before infection but not when administration was delayed for 24 h postinfection. It is therefore believed that IL-6 plays a role in early priming of the immune response to infection. Its exact function in this model is being investigated.

Interleukin-6 (IL-6) was first described as a weakly acting interferon (beta interferon subtype 2), as a B-cell differentiation factor (BSF-2) favoring immunoglobulin A production, and as a B-cell hybridoma growth factor (26). It has since become apparent that IL-6 has a multitude of other roles, including stimulation of T-cell proliferation, differentiation, and activation (26); initiation of the acute-phase response (12); synergistic (with colony-stimulating factors [CSFs]) stimulation of hemopoiesis (14); and under certain conditions, stimulation of macrophage antibacterial activity (6). IL-6 is elevated in the sera of patients suffering from various infections (5, 10) and during experimental infection (8, 11, 13). However, given the multitude of effects of IL-6, it is by no means clear whether IL-6 contributes to the resistance mechanisms or mediates the immunopathology of infection. Listeria monocytogenes has been extensively studied as one of a group of facultative intracellular bacteria which survive within normal macrophages and perhaps within certain tissue cells. During experimental infection of mice, the bacteria are initially phagocytosed by resident tissue macrophages, which kill a proportion, but not all, of the bacteria (23). Natural resistance of the mice is critically dependent on the number of colony-forming cells (hemopoietic precursors) in the bone marrow and spleen, which provide a rapid inflammatory response of highly bactericidal cells in genetically resistant mouse strains (28, 29). Later, T lymphocytes are activated; these secrete lymphokines which increase the bactericidal efficiency of tissue macrophages and attract further inflammatory monocytes to the site. Thus, acquired cell-mediated immunity is dependent on continued hemopoiesis to supply these inflammatory cells (15, 23). The T cells involved include both the CD4+ population and the CD8+ population (15), which in other systems are favored by IL-6 (25). Hence, there are a number of targets for IL-6 to act upon during infection with these bacteria. We described here the presence of IL-6 in serum and tissue during experimental infection of mice with L. mono*

cytogenes and show that, when injected intraperitoneally immediately before infection, recombinant IL-6 (rIL-6) suppresses bacterial growth.

MATERIALS AND METHODS Mice and bacterial infection. C57BL/10 female mice, bred at the Department of Microbiology, University of Melbourne, Melbourne, Australia, were used at 6 to 8 weeks of age. L. monocytogenes (50% lethal dose, 2 x 105 for genetically resistant C57BL/10 mice) was cultured for 24 h at 37°C on horse blood agar. The actively growing organisms were washed from the plates with endotoxin-free saline (STR; Astra Pharmaceuticals Pty). Bacterial concentration was adjusted turbidimetrically, and the dose was confirmed retrospectively by viable counts as previously described (29). Mice were infected intravenously with 104 listeriae per mouse, and at various intervals after infection, groups of five mice were killed by fluothane overdose. For bacterial counts, spleens and livers were removed aseptically and fragments were weighed and homogenized individually in 5 ml of saline by using an Ultra-Turrax homogenizer (Janke & Kunkel, K.S.). Serial 10-fold dilutions were prepared in 96-well microtiter plates. Triplicate 25-,u samples were applied to horse blood agar plates, and colonies were counted after incubation for 1 day at 37°C. Tissue preparation for IL-6 assay. Under fluothane anesthesia, mice were bled from the heart with a syringe to collect the maximum amount of blood aseptically. The blood was allowed to clot for 1 h at room temperature, and the clots were retracted overnight at 4°C to collect the serum. After bleeding, peritoneal washings were collected with 5 ml

of HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered Dulbecco's modified Eagle medium with 5% fetal calf serum and 10 U of heparin per ml. Cells were pelleted by centrifugation at 800 x g for 7 min, and IL-6 was measured in the supernatants. Spleens, livers, kidneys, and lungs were removed, weighed, and suspended in 20 times their weight of HEPES-buffered Dulbecco's medium with 5% fetal calf serum. They were homogenized individually on ice and centrifuged at 800 x g for 30 min at 4°C to remove

Corresponding author. 4402


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Recombinant interleukin-6 protects mice against experimental bacterial infection.

Because of reports of high levels of interleukin-6 (IL-6) in patients during infection, we studied the role of IL-6 in experimental infection. Mice in...
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