Detection of cytokine protein expression in mouse lung homogenates using suspension bead array

The chemically purified endotoxin, lipopolysaccharide (LPS) has no or only traces amounts of cell wall proteins [1]. Alveolar macrophages have a primary role in mediating the effects of LPS entering airways and lungs. CD-1 mice are relatively sensitive to LPS treatment [2, 3]. Previous reports suggested that similar to that seen with systemic inflammatory response in CD-1 mice, pro-inflammatory cytokines, interleukin-6 (IL-6) and macrophage inflammatory protein-2 (MIP-2) protein levels in mouse lung tissue homogenates are increased following LPS treatment as determined by ELISA [2]. Increased neutrophil infiltration is also detectable in lung sections in parallel with increased IL-6 and MIP-2 expression in lung tissue homogenates from LPS-treated mice. Additional studies are needed to further elucidate diagnostic markers indicative of the LPS-evoked lung inflammatory injury response.

While, traditional immunohistochemical staining methods for detection of cytokine protein expression in tissue sections is semi-quantitative, current and emerging quantitative methods and their applications including the use of tissue homogenates have been reviewed [3]. Suspension bead array affords multiplexing of microspheres labeled with fluorescent dyes and contain surface carboxyl groups for covalent attachment analytes (i.e., cytokine proteins) to conform a solid phase sandwich immunoassay for measuring multiple analytes simultaneously. We determined in our laboratory that suspension bead array is a useful tool for measuring mouse plasma levels of cytokine protein expression following LPS treatment [4]. In LPS-treated female CD-1 mice, increased plasma levels of IL-6, interleukin-10 (IL-10), interferonγ (IFNγ) and tumor necrosis factorα (TNFα) were detectable at both 2-hours and 24-hours post treatment; while increased plasma protein levels of interleukin-1β (IL-1β), interleukin-5 (IL-5), interleukin-12 (IL-12) and granulocyte/macrophage colony-stimulating factor (GM-CSF) were detectable at 24-hours post-treatment only. Whether or not suspension bead array can be used to measure mouse lung tissue homogenate levels of cytokine protein expression has not been reported in the literature. Procedures for simultaneous measurement of multiple cytokines in rat serum and brain tissue by suspension bead array have been optimized [5, 6]. Methods for antibody array analysis of peripheral and blood cytokine levels in rats after masseter inflammation induced by injection of complete Freund's adjuvant have also been described [7].

The objective for this present study was to determine whether or not suspension bead array is a feasible method to detect changes in cytokine protein expression in mouse lung tissue homogenates.

The feasibility of simultaneous measurement of cytokine levels may lend to the elucidation of potential diagnostic markers of compound-induced inflammatory responses in tissues.

CD-1 mice are relatively sensitive to endotoxin and the relative effects of endotoxin (specifically LPS) on lung inflammation in CD-1 mice have been previously described [2]. The purpose of the present study was to investigate the feasibility of using suspension bead array for detection of LPS (i.p. administration of 0.25 mg/ml)-evoked changes in levels of cytokine protein expression (at 2- and 24-hours post treatment) in mouse lung tissue homogenates. Simultaneous detection of changes in levels of lung cytokine proteins could prove useful during the preclinical safety evaluation phase of compound development.

LPS treatment was well tolerated in all animals; and there were no adverse clinical observations in this study. LPS-related histopathology changes were identified in the left lung lobe in female CD-1 mice after treatment with LPS (0.25 mg/ml) for both 2- and 24-hours. The inflammatory changes were characterized by perivascular edema and the accumulation of mixed cell infiltrates within blood and lymphatic vessels, as well as in the adjacent interstitium at both 2-hours and 24-hours following LPS treatment. A minimal increase in the number of alveolar macrophages was also observed in the 24-hour LPS-treated mice. No histopathology findings were observed in the lungs of 2-hour PBS-treated control mice.

In the present study, among the 24-hour LPS-treated animals, increased IL-6 (574-fold) and GM-CSF (8-fold) and decreased IL-2 (1.3-fold) protein concentrations in lung homogenates were detected using suspension bead array. Previously, we reported observations of statistically significant increases in CD-1 mouse plasma IL-6 protein expression at both 2- and 24-hours post LPS treatment and GM-CSF protein expression at 2-hours post LPS treatment [4]. Suspension bead array-detectable changes in lung homogenate levels of IL-2, -6 and GM-CSF protein expression 24-hours following LPS treatment correlated with qualitative protein expression profile data (i.e., as low as 10 pg/ml of IL-2) detected using antibody array. In this study, antibody arrays findings suggested significantly increased expression of IL-6 and -13 and L-selectin proteins and significantly decreased IL-2 protein in lung homogenates from 24-hour LPS-treated mice. Similar to the protein expression levels detected using suspension bead array, significantly increased IL-6 protein expression was detected in lung homogenates from 24-hour LPS-treated mice using ELISA.

LPS exerts adjuvant effects on macrophages, resulting in an inflammatory cascade defined by early production of TNFα followed by subsequent production of IL-1, IL-6 [8] and MIP-2 [2]. The production of TNFα by macrophages and the recruitment of inflammatory cells are regulated by T cells. High dose (1000 μg/ml) LPS exposure causes activation of both T cells and macrophages, while low dose (100 μg/ml) treatment evokes activation of macrophages only [8]. Perivascular edema and accumulation of mixed cell infiltrates in the lung is typical of lung inflammatory responses [2]. In the present study, the LPS dose of 250 μg/ml evoked lung perivascular edema, accumulation of mixed cell infiltrates within blood and lymphatic vessels and in the adjacent interstitium. These changes parallel the significantly increased levels of IL-6 and GM-CSF in lung tissue homogenate from LPS-treated mice versus controls. Only a minimal increase in the number of alveolar macrophages was observed 24-hours post LPS treatment, which also corroborates with the "minimally" elevated levels of macrophage-origin cytokines (IL-6 and GM-CSF) in lung tissue homogenates. Taken together, the enhanced cytokine levels detected in lung tissue homogenates suggest an increased local cytokine production in the lungs by LPS-activated macrophages. Alternatively, histopathology changes observed in the LPS-treated mice may reflect local effects due to the highly elevated circulating cytokines (TNFα, IFNγ, GM-CSF, IL-1β, IL-5, IL-6, IL-10, IL-12), which were reported previously for these animals [4]. Circulating cytokine levels were log-folds higher than those observed for the lung tissue homogenates, which may indicate a tissue dilution effect and/or a tissue sampling phenomenon, since the histopathology changes were multi-focal and not diffusely distributed. Similarly, the decrease in IL-2 expression in lung tissue homogenates following LPS treatment could possibly be due to tissue dilution effect and/or a tissue sampling phenomenon, but requires further investigation.

In summary, we found that a single i.p. administration of LPS at 0.25 mg/ml yielded significantly increased IL-6 and GM-CSF proteins and decreased IL-2 protein expression in lung tissue homogenates using suspension bead array. These findings correlated with cytokine protein expression profiles detected using antibody array and were confirmed using conventional ELISA (IL-6 protein concentrations).

Overall, the present study shows that suspension bead array is a feasible method for detecting changes in cytokine protein expression in mouse lung tissue homogenates.