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. 2005 Mar;73(3):1754-63.
doi: 10.1128/IAI.73.3.1754-1763.2005.

Distinct roles of pattern recognition receptors CD14 and Toll-like receptor 4 in acute lung injury

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

Distinct roles of pattern recognition receptors CD14 and Toll-like receptor 4 in acute lung injury

Samithamby Jeyaseelan et al. Infect Immun. 2005 Mar.

Erratum in

Abstract

Acute lung injury (ALI) induced by lipopolysaccharide (LPS) is a major cause of mortality among humans. ALI is characterized by microvascular protein leakage, neutrophil influx, and expression of proinflammatory mediators, followed by severe lung damage. LPS binding to its receptors is the crucial step in the causation of these multistep events. LPS binding and signaling involves CD14 and Toll-like receptor 4 (TLR4). However, the relative contributions of CD14 and TLR4 in the induction of ALI and their therapeutic potentials are not clear in vivo. Therefore, the aim of the present study was to compare the roles of CD14 and TLR4 in LPS-induced ALI to determine which of these molecules is the more critical target for attenuating ALI in a mouse model. Our results show that CD14 and TLR4 are necessary for low-dose (300-microg/ml) LPS-induced microvascular leakage, NF-kappaB activation, neutrophil influx, cytokine and chemokine (KC, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-6) expression, and subsequent lung damage. On the other hand, when a 10-fold-higher dose of LPS (3 mg/ml) was used, these responses were only partially dependent on CD14 and they were totally dependent on TLR4. The CD14-independent LPS response was dependent on CD11b. A TLR4 blocking antibody abolished microvascular leakage, neutrophil accumulation, cytokine responses, and lung pathology with a low dose of LPS but only attenuated the responses with a high dose of LPS. These data are the first to demonstrate that LPS-induced CD14-dependent and -independent (CD11b-dependent) signaling pathways in the lung are entirely dependent on TLR4 and that blocking TLR4 might be beneficial in lung diseases caused by LPS from gram-negative pathogens.

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Figures

FIG. 1.
FIG. 1.
Effect of E. coli LPS on protein contents, total white blood cell counts, and neutrophil counts in the BALF recovered from LPS- or saline-instilled lungs at 2, 8, and 24 h for CD14−/−, TLR4mt, and wild-type (CD14+/+ and TLR4wt) mice. Each group contained seven animals. The values are means ± standard deviations. Significant differences between LPS- and saline-treated groups are indicated by asterisks (P < 0.05).
FIG. 2.
FIG. 2.
Effect of E. coli LPS on lung histopathology at 24 h after inhalation of LPS for CD14−/− and TLR4mt mice, as determined with hematoxylin and eosin staining. The representative photomicrographs are from one of eight separate experiments which yielded similar results. The brightness, contrast, and magnification are the same for all images. Original magnification, ×100.
FIG. 3.
FIG. 3.
Nuclear translocation of the p65 subunit of NF-κB as detected by p65 ELISA of nuclear extracts of mouse lungs at 2 h after LPS or saline instillation. Note that LPS induced a dramatic increase in nuclear translocation of p65 in CD14+/+ (A) and TLR4wt (B) mice. The values are means ± standard deviations. Values that are significantly different for the LPS- and saline-treated groups are indicated by asterisks (P < 0.05; six mice/group). OD450 nm, optical density at 450 nm.
FIG. 4.
FIG. 4.
Effect of E. coli LPS on KC, MIP-2, TNF-α, and IL-6 protein responses in the BALF at 2, 8, and 24 h after inhalation of LPS or saline for CD14−/− and TLR4mt mice, as determined by ELISA. The values are means ± standard deviations. Significant differences between LPS- and saline-treated groups are indicated by asterisks (P < 0.05; seven mice/group).
FIG. 5.
FIG. 5.
Effect of a high dose of E. coli LPS on the total protein content, the total white blood cell (A and B) and neutrophil (B) counts in BALF, neutrophil sequestration in the lung, and lung histology at 24 h after inhalation of LPS or saline for CD14−/−, TLR4mt, CD14/TLR4 double-knockout (DKO), and wild-type mice. The values are means ± standard deviations. Significant differences between LPS- and saline-treated groups are indicated by asterisks (P < 0.05; eight mice/group).
FIG. 6.
FIG. 6.
Anti-CD11b Ab inhibits CD14-independent signaling in response to a high dose of LPS, as shown for CD14−/− mice by neutrophil counts in BALF (A) and lung MPO activity (B) at 24 h. The values are means ± standard deviations. Values that are significantly different for the anti-CD11b Ab-treated and isotype Ab-treated groups are indicated by asterisks (P < 0.05; nine mice/group). PBS, phosphate-buffered saline.
FIG. 7.
FIG. 7.
Blocking TLR4 abolishes ALI induced by a low dose of LPS, as demonstrated by the neutrophil counts in BALF (A), the MPO activity in the lungs at 8 and 24 h (B), the BALF cytokine profiles at 2 h (C), and the lung histopathology at 24 h after inhalation of LPS (D and E). The values are means ± standard deviations. Values that are significantly different for the anti-TLR4-Ab-treated and isotype control Ab-treated groups prior to LPS treatment are indicated by asterisks (P < 0.05; eight mice/group). The representative photomicrographs are from one of eight separate experiments in which identical results were obtained. Original magnification, ×100. PBS, phosphate-buffered saline.
FIG. 8.
FIG. 8.
Blocking TLR4 attenuates ALI in response to a high dose of LPS, as shown by the neutrophil counts in BALF (A), the lung MPO activity at 8 and 24 h (B), the BALF cytokine profiles at 2 h (C), and the lung histopathology at 24 h after inhalation of saline (D and E) or LPS (F and G). The values are means ± standard deviations. Values that are significantly different for anti-TLR4-Ab-treated and isotype Ab-treated groups prior to the LPS challenge are indicated by asterisks (P < 0.05; eight mice/group). The representative photomicrographs are from one of nine independent experiments in which the same results were obtained. Original magnification, ×100. PBS, phosphate-buffered saline.
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