Tissue- and time-dependent upregulation of cytokine mRNA in a murine model for the multiple organ dysfunction syndrome

Abstract

Objective: We sought to quantitate the course of specific cytokine mRNA expression in tissues that exhibit increasing histopathological changes in time in an animal model for the multiple organ dysfunction syndrome (MODS).

Summary background data: The development of treatment protocols for MODS requires elucidation of the mechanisms and mediators involved. To devise logical interventions, it is necessary to collect data on cytokine expression at tissue level during the development of MODS.

Methods: Ninety-four C57BL/6 mice were given an intraperitoneal injection of 40 microg of lipopolysaccharide (LPS), followed by zymosan at a dose of 0.8 mg/g body weight 6 days later (day 0). Six additional animals did not receive zymosan and acted as controls. At several time points after zymosan injection, 6 randomly assigned, zymosan-treated animals were killed, and their livers, lungs, spleens, and kidneys were collected. mRNA expression of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, macrophage migration inhibiting factor, IL-12, interferon-gamma, and IL-10 was measured using a real-time reverse transcription-polymerase chain reaction assay.

Results: The injection of zymosan induced an acute peritonitis, followed by an apparent recovery. From approximately day 6 onwards, animals started to display MODS-like symptoms. During the peritonitis phase, up-regulation of cytokine mRNA was limited. During the period of apparent recovery, cytokine mRNA expression strongly increased, mostly reaching its maximum at day 9 when deterioration of the clinical condition had already set in. The up-regulation of tumor necrosis factor-alpha mRNA was most pronounced, especially in the lungs and liver.

Conclusions: Interventions should preferentially be targeted against multiple cytokines and, at least in this model, there may be a treatment window well after the initial challenge.

FIGURE 1. Clinical course after zymosan administration. A group of 10 animals (group 3) was followed to monitor survival (A), body temperature (B), and body weight (C). The latter are depicted as average + SD.

FIGURE 2. Survival and development of lung damage after zymosan administration. A, Kaplan-Meier survival curve in group 1 (n = 84 at day 0), which was used for cytokine mRNA quantitation; B, Lung score for mice in groups 1 and 2 (controls). Bars represent average values (+ SD, n = 6 for controls and days 2–15 and n = 11 for day 17).

FIGURE 3. Up-regulation of TNF-α (A), IL-1β (B), and IL-6 (C) mRNA. The degree of up-regulation was calculated for each animal as the mRNA expression at any time point in relation to the median mRNA expression in control mice. Data are represented as medians. +, liver; ▪, lungs; Δ, spleen; ×, kidneys, n = 6 per organ per time point.

FIGURE 4. Cytokine mRNA expression in the liver. Individual values of mRNA for TNF-α (•) and IL-1β (○), quantitated by a real-time RT-PCR, in livers from untreated control mice and zymosan-treated animals.

FIGURE 5. Up-regulation of MIF (A), IL-12 (B), IFN-γ (C), and IL-10 (D) mRNA. The degree of up-regulation was calculated for each animal as the mRNA expression at any time point in relation to the median mRNA expression in control mice. Data are represented as medians. +, liver; ▪, lungs; Δ, spleen; ×, kidneys, n = 6 per organ per time point.