Staphylococcal Panton-Valentine leukocidin induces pro-inflammatory cytokine production and nuclear factor-kappa B activation in neutrophils

Abstract

Panton-Valentine leukocidin (PVL) is a cytotoxin secreted by Staphylococcus aureus and associated with severe necrotizing infections. PVL targets polymorphonuclear leukocytes, especially neutrophils, which are the first line of defense against infections. Although PVL can induce neutrophil death by necrosis or apoptosis, the specific inflammatory responses of neutrophils to this toxin are unclear. In this study, both in vivo and in vitro studies demonstrated that recombinant PVL has an important cytotoxic role in human neutrophils, leading to apoptosis at low concentrations and necrosis at high concentrations. Recombinant PVL also increased the levels of pro-inflammatory cytokine secretion from neutrophils. The up-regulation of pro-inflammatory cytokines was due to nuclear factor-kappa B (NF-κB) activation induced by PVL. Moreover, blocking NF-κB inhibited the production of inflammatory cytokines. To test the role of neutrophil immune responses during the pathogenesis of PVL-induced acute lung injury, we used immunocompetent or neutropenic rabbits to develop a model of necrotizing pneumonia. Immunocompetent rabbits challenged with PVL demonstrated increased inflammation containing neutrophilic infiltrates. In addition, there were elevated levels of inflammatory cytokines (IL-6, IL-8, TNF-α and IL-10) and NF-κB in the lung homogenate. In contrast, the lung tissues from neutropenic rabbits contained mild or moderate inflammation, and the levels of inflammatory cytokines and NF-κB increased only slightly. Data from the current study support growing evidence that neutrophils play an important role in the pathogenesis of PVL-induced tissue injury and inflammation. PVL can stimulate neutrophils to release pro-inflammatory mediators, thereby causing an acute inflammatory response. The ability of PVL to induce inflammatory cytokine release may be associated with the activation of NF-κB or its pore-forming properties.

Figure 1. Neutrophil apoptosis and necrosis as a function of the rPVL concentration.

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.

Figure 2. Effect of rPVL on cytokine secretion.

1, control; 2, treated with 5 nmol/L rPVL for 5 h; 3, treated with 100 nmol/L rPVL for 5 h. The supernatants of neutrophils were collected and IL-6 (A), IL-8 (B), TNF-α (C), IL-10 (D) secretion was measured by ELISA. Results represent the mean ± SD of three separate measurements. * P<0.05.

Figure 3. Expression levels of cytokine mRNA in neutrophils.

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. * P<0.05.

Figure 4. Expression of NF-κB p65 mRNA in neutrophils.

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. * P<0.05.

Figure 5. Expression of NF-κB protein in neutrophils.

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. * P<0.05.

Figure 6. Expression of NF-κB protein in neutrophils after NF-κB inhibition.

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. * P<0.05.

Figure 7. Effect of rPVL on cytokine secretion in neutrophils.

1: Cells were treated with PBS; 2: cells were treated with rPVL; 3: cells pretreated with PDTC for 1 h before treatment with rPVL. The supernatants of polymorphonuclear cells were collected and IL-6 (A), IL-8 (B), TNF-α (C) levels were measured by ELISA. Results shown are representative of three independent experiments. * P<0.05.

Figure 8. Expression of cytokine mRNA in neutrophils.

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. * P<0.05.

Figure 9. Lung histopathology of rabbits treated with PBS or rPVL.

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).

Figure 10. 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.

Results shown are representative of two independent experiments. * P<0.05.

Figure 11. Immunohistochemical staining of NF-κB in lung tissues.

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.