NF-kappaB p50 facilitates neutrophil accumulation during LPS-induced pulmonary inflammation

Abstract

Background: Transcription factors have distinct functions in regulating immune responses. During Escherichia coli pneumonia, deficiency of NF-kappaB p50 increases gene expression and neutrophil recruitment, suggesting that p50 normally limits these innate immune responses. p50-deficient mice were used to determine how p50 regulates responses to a simpler, non-viable bacterial stimulus in the lungs, E. coli lipopolysaccharide (LPS).

Results: In contrast to previous results with living E. coli, neutrophil accumulation elicited by E. coli LPS in the lungs was decreased by p50 deficiency, to approximately 30% of wild type levels. Heat-killed E. coli induced neutrophil accumulation which was not decreased by p50 deficiency, demonstrating that bacterial growth and metabolism were not responsible for the different responses to bacteria and LPS. p50 deficiency increased the LPS-induced expression of kappaB-regulated genes essential to neutrophil recruitment, including KC, MIP-2, ICAM-1, and TNF-alpha suggesting that p50 normally limited this gene expression and that decreased neutrophil recruitment did not result from insufficient expression of these genes. Neutrophils were responsive to the chemokine KC in the peripheral blood of p50-deficient mice with or without LPS-induced pulmonary inflammation. Interleukin-6 (IL-6), previously demonstrated to decrease LPS-induced neutrophil recruitment in the lungs, was increased by p50 deficiency, but LPS-induced neutrophil recruitment was decreased by p50 deficiency even in IL-6 deficient mice.

Conclusion: p50 makes essential contributions to neutrophil accumulation elicited by LPS in the lungs. This p50-dependent pathway for neutrophil accumulation can be overcome by bacterial products other than LPS and does not require IL-6.

Figure 1

Effect of p50 deficiency on neutrophil recruitment elicited by LPS in the lungs. E. coli LPS was instilled intratracheally to WT and p50-deficient mice, and lungs and blood were collected after 24 hours. Representative histologic images from lungs of WT and p50-deficient mice are shown (A and B, respectively). Emigrated neutrophils (C) in the alveolar air spaces, and sequestered neutrophils (D) in the alveolar septae, were quantified using morphometric analyses of histologic sections. Circulating neutrophils (E) were quantified in blood samples collected from the inferior vena cava. Asterisks (*) denote significant differences from WT mice.

Figure 2

Effect of p50 deficiency on neutrophil recruitment elicited by heat-killed E. coli in the lungs. E. coli that had been killed by autoclaving (as indicated by an inability to grow in culture) were instilled intratracheally to WT and p50-deficient mice, and lungs were collected after 24 hours. Emigrated neutrophils in the alveolar air spaces were quantified using morphometric analyses of histologic sections. There were no significant effects of p50 deficiency.

Figure 3

Effect of p50 deficiency on ICAM-1 expression in the lungs. cDNA was prepared from RNA in lungs collected from WT and p50-deficient mice. ICAM-1 expression was measured by semi-quantitative multiplex RT-PCR. The autoradiograph (A) shows representative samples, with each lane containing amplicons of ICAM-1 message and 18S rRNA from cDNA reverse transcribed from lung RNA of an independent mouse. The graph (B) depicts ICAM-1 message for each sample expressed relative to the densitometric value of 18S rRNA from the same PCR reaction tube. Bars depict mean and SEM from 4–7 mice per group, and asterisk (*) denotes significant difference from WT mice.

Figure 4

Effect of p50 deficiency on cytokine concentrations in the air spaces of the lungs during LPS-induced pulmonary inflammation. Bronchoalveolar lavage fluids were collected from WT and p50-deficient mice after E. coli LPS was instilled intratracheally. Concentrations of MIP-2 (A), KC (B), TNF-α (C), and IL-1β (D) in the lavage fluids were quantified by ELISA. Asterisks (*) denote significant differences from WT mice.

Figure 5

Effect of p50 deficiency on chemokine responsiveness of blood neutrophils. (A) Effect of p50 deficiency on concentrations of neutrophil chemokines in the blood. E. coli LPS was instilled intratracheally to WT and p50-deficient mice, blood was collected after 6 hours, and the concentrations of MIP-2 and KC were quantified by ELISA. Asterisks (*) denote significant differences from WT mice. (B) Effect of p50 deficiency on the ability of blood neutrophils to respond to the chemokine KC. Since chemoattractants induce cell stiffening which results in neutropenia, circulating neutrophils (PMN) were counted before and after the intravenous injection of recombinant murine KC. Circulating neutrophils were quantified by differential counting of blood samples collected from the inferior vena cava. There were no significant effects of p50 deficiency. (C) Effect of p50 deficiency on the ability of blood neutrophils to respond to the chemokine KC during LPS-induced pulmonary inflammation. E. coli LPS was instilled intratracheally to WT and p50-deficient mice, and 6 hours later neutrophil responsiveness to recombinant murine KC was assessed as described above. Asterisks (*) denote significant differences from WT mice.

Figure 6

Role of IL-6 in the decreased neutrophil recruitment of p50-deficient mice during LPS-induced pulmonary inflammation. (A) Increased IL-6 in the lungs of p50-deficient mice. E. coli LPS was instilled intratracheally to WT and p50-deficient mice, bronchoalveolar lavage fluids were collected, and IL-6 concentrations were measured by ELISA. Asterisk (*) denotes significant difference from WT mice. (B) Effect of p50 deficiency on LPS-induced neutrophil recruitment in the absence of IL-6. IL-6-deficient and p50-deficient mice were crossed to generate double mutant mice deficient in both IL-6 and p50. E. coli LPS was instilled intratracheally to IL-6-deficient mice and double mutant mice deficient in both IL-6 and p50, and lungs were collected after 24 hours. Emigrated neutrophils in the alveolar air spaces were quantified using morphometric analyses of histologic sections. Asterisk (*) denotes significant differences from IL-6 deficient mice without mutation in the gene for p50.