Specification of primers used in RT-PCR.

<p>Specification of primers used in RT-PCR.</p

Neutrophil apoptosis and necrosis as a function of the rPVL concentration.

<p>Neutrophils were incubated with PBS, 5 nM/L rPVL and 100 nM/L rPVL for 5 h and then observed by (A) phase contrast microscope, (B) biological microscope, or (C) transmission electron microscopy.</p

Lung histopathology of rabbits treated with PBS or rPVL.

<p>hematoxylin and eosin-stained sections (E–H; magnification 200×) from the lungs of immunocompetent rabbits treated with PBS (A), rPVL (B), or from neutropenic rabbits treated with rPVL (C).</p

Immunohistochemical staining of NF-κB in lung tissues.

<p>NF-κB is mainly expressed in the nucleus of neutrophils. A: Control group negative for NF-κB; B: immunocompetent rabbits inoculated with rPVL; C: neutropenic rabbits inoculated with rPVL.</p

Expression levels of cytokine mRNA in neutrophils.

<p>1, control; 2, treated with 5 nmol/L rPVL for 5 h; 3, treated with 100 nmol/L rPVL for 5 h. Representative agarose-gel photographs showing the expression level of IL-6 (A), IL-8 (B), TNF-α (C) and IL-10 (D) mRNA from neutrophils by RT-PCR analysis. The relative levels of IL-6 (E), IL-8 (F), TNF-α (G) and IL-10 (H) expressed were compared with β-actin levels. * <i>P</i><0.05.</p

Expression of NF-κB protein in neutrophils.

<p>Cells were treated with PBS (1), 5 nmol/L rPVL for 2 h (2) or 100 nmol/L rPVL for 2 h (3) respectively. A: The expression levels of NF-κB proteins in polymorphonuclear cells were analyzed by western blot. B: The relative levels of NF-κB expressed in polymorphonuclear cells. The values indicate the expression indices of the densitometry units relative to the amount of β-actin. Results represent the mean ± SD of three separate measurements. * <i>P</i><0.05.</p

Expression of NF-κB p65 mRNA in neutrophils.

<p>Cells were treated with PBS (1), 5 nmol/L rPVL for 2 h (2) or 100 nmol/L rPVL for 2 h (3), respectively. A: Representative agarose-gel photograph showing the expression levels of NF-κB p65 mRNA in polymorphonuclear cells using RT-PCR. B: Relative levels of NF-κB p65 expressed in polymorphonuclear cells. The values indicate the expression indices of the densitometry units relative to the amount of β-actin. Results represent the mean ± SD of three separate measurements. * <i>P</i><0.05.</p

Expression of cytokine mRNA in neutrophils.

<p>1: Cells were treated with PBS; 2: cells were treated with rPVL; 3: cells pretreated with PDTC for 1 h before treated with rPVL. Representative agarose-gel photographs showing the expression levels of IL-6 (A), IL-8 (B) and TNF-α (C) mRNA in polymorphonuclear cells by RT-PCR analysis. The relative levels of IL-6 (D), IL-8 (E) and TNF-α (F) expressed in polymorphonuclear cells are shown. Results represent the mean ± SD of three separate measurements. * <i>P</i><0.05.</p

Expression of NF-κB protein in neutrophils after NF-κB inhibition.

<p>1: Cells treated with PBS; 2: cells treated with rPVL; 3: cells incubated with PDTC for 1 h before treatment with rPVL. A: The expression levels of NF-κB proteins in polymorphonuclear cells were analyzed by western blot. B: Relative levels of NF-κB expressed in polymorphonuclear cells. The values indicate the expression indices of the densitometry units relative to the amount of β-actin. Results represent the mean ± SD of three separate measurements. * <i>P</i><0.05.</p

Effect of rPVL on cytokine secretion. IL-6 (A), IL-8 (B), TNF-α (C), and IL-10 (D) from lung tissue homogenate was measured by ELISA.

<p>Results shown are representative of two independent experiments. * <i>P</i><0.05.</p