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. 2004 Dec;72(12):7247-56.
doi: 10.1128/IAI.72.12.7247-7256.2004.

Transcriptional profiling of lipopolysaccharide-induced acute lung injury

Samithamby Jeyaseelan  1 , Hong Wei Chu, Scott K Young, G Scott Worthen
Affiliations

Transcriptional profiling of lipopolysaccharide-induced acute lung injury

Samithamby Jeyaseelan et al. Infect Immun. 2004 Dec.

Abstract

Mortality associated with acute lung injury (ALI) induced by lipopolysaccharide (LPS) remains high in humans, warranting improved treatment and prevention strategies. ALI is characterized by the expression of proinflammatory mediators and extensive neutrophil influx into the lung, followed by severe lung damage. Understanding the pathogenesis of LPS-induced ALI is a prerequisite for designing better therapeutic strategies. In the present study, we used microarrays to gain a global view of the transcriptional responses of the lung to LPS in a mouse model of ALI that mimics ALI in humans. A total of 71 inflammation-associated genes were up-regulated in LPS-treated lungs, including a chemokine, LPS-induced CXC chemokine (LIX), whose role in the induction of ALI is unknown. Most of the inflammatory genes peaked at 2 h post-LPS treatment. Real-time reverse transcription-PCR confirmed the LPS-induced up-regulation of selected genes identified by microarray analysis, including LIX. The up-regulation of LIX, tumor necrosis factor alpha, and macrophage inflammatory protein 2 was confirmed at the protein level by enzyme-linked immunosorbent assays. To determine the role of LIX in the induction of ALI, we used both exogenous LIX and a LIX blocking antibody. Exogenous LIX alone elicited a neutrophil influx in the lungs, and the anti-LIX antibody attenuated the LPS-induced neutrophil accumulation in the lungs. Taken together, the results of our study demonstrate for the first time the temporal expression of inflammatory genes during LPS-induced ALI and suggest that early therapeutic intervention is crucial to attenuate lung damage. Moreover, we identified a role for LIX in the induction of ALI, and therefore LIX may serve as a novel therapeutic target for the minimization of ALI.

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Figures

FIG. 1.
FIG. 1.
Hematoxylin and eosin staining of lung tissues at 2 h (A), 8 h (B), and 24 h (C) after aerosolization with LPS or at 24 h (D) after aerosolization with 0.9% saline. Note that infiltration of neutrophils into the air spaces and interstitial edema were evident in LPS-treated lungs in a temporal manner. The arrow indicates infiltrating inflammatory cells. Cytospin preparations of BALF cells stained with Diff-Quick at 2 h (E), 8 h (F), and 24 h (G) after aerosolization with LPS or at 24 h (H) after aerosolization with 0.9% saline are also shown. Photomicrographs are representative of five independent experiments; the brightness, contrast, and magnification of the pictures are similar. Five mice were used for each group. Original magnification, ×400. The graphs show the quantitation of total WBC (I) and neutrophil (J) counts in BALF. Five mice were tested for each group, and significance was calculated by a one-way analysis of variance (ANOVA). Values that were significantly different between the LPS- and saline-treated groups (P < 0.05) are indicated by asterisks.
FIG. 2.
FIG. 2.
Validation of LPS-induced up-regulation of selected genes in lungs by real-time quantitative RT-PCR. The LPS-induced changes were calculated against saline controls as described in Materials and Methods. There was a strong correlation between the microarray analysis and real-time RT-PCR data.
FIG. 3.
FIG. 3.
Confirmation of LPS-induced up-regulation of LIX, TNF-α, and MIP-2 proteins by ELISAs. LPS- or 0.9% saline-treated BALF (A to C) and lung tissue homogenates (D to F) were used to measure the levels of LIX, TNF-α, and MIP-2 by ELISAs. Data are expressed in picograms per milliliter for BALF and in picograms per milligram of total protein for lung tissue. Groups of five mice each were used. A one-way ANOVA was used to determine the significance between the LPS- and saline-treated groups. Values that were significantly different between the LPS- and saline-treated groups (P < 0.05) are indicated by asterisks.
FIG. 4.
FIG. 4.
Exogenous LIX induces neutrophil influx in lungs. Three micrograms of exogenous LIX, antibody-treated LIX (with anti-LIX antibody or an isotype-matched control), or PBS was administrated intratracheally to mice, and BALF and lungs were collected at 2, 8, and 24 h posttreatment. Total WBC (A) and neutrophil (B) counts in BALF and MPO activities in the lungs (C) were determined as described in Materials and Methods. A one-way ANOVA was used to determine the significance between the LIX- and saline-treated groups (n = 8 animals/group). Values that were significantly different between LIX- and saline-treated mice (P < 0.05) are indicated by asterisks.
FIG. 5.
FIG. 5.
Neutralization of LIX attenuates neutrophil recruitment in lungs during LPS-induced ALI. Thirty micrograms of anti-LIX antibody, an isotype-matched control antibody, or PBS was administrated intratracheally to mice 2 h prior to LPS treatment. BALF and lungs were collected at 2, 8, and 24 h post-LPS treatment, and total WBC (A and D) and neutrophil (B and E) counts in BALF and MPO activities in lungs (C and F) were determined as described in Materials and Methods. A one-way ANOVA was used to determine the significance between the LPS- and saline-treated groups (n = 8 mice/group). Values that were significantly different between anti-LIX antibody-treated mice and isotype-matched control antibody-treated mice (P < 0.05) are indicated by asterisks.
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