Background
The innate immune response to bacteria in the lungs requires the recruitment and activation of neutrophils, mediated by the coordinated expression of diverse genes (see [
1] for review). In rodents, neutrophils recognize at least 2 chemokines (KC and macrophage inflammatory protein-2, MIP-2) that are synthesized
de novo in response to gram-negative bacteria or LPS in the lungs, and each is independently essential to maximal neutrophil recruitment [
2‐
4]. Neutrophils also recognize the adhesion molecule intercellular adhesion molecule-1 (ICAM-1). ICAM-1 is constitutively expressed, but LPS and gram-negative bacteria in the lungs result in increased expression [
5,
6] and ICAM-1 is required for maximal neutrophil emigration [
7,
8]. In addition, the early response cytokines TNF-α and IL-1β are synthesized in response to LPS or gram-negative bacteria in the lungs, and receptors for these cytokines are essential to neutrophil emigration [
9].
The coordinated expression of diverse genes may be mediated in part by common transcription factors. All of the above genes contain κB sites in their 5'-untranslated promoter regions, and mutation of these sites inhibits the inducible expression of reporter genes driven by these promoter regions (see reference [
10] and references therein). Thus, NF-κB proteins may mediate the LPS-induced expression of genes controlling neutrophil emigration in the lungs.
LPS in the lungs induces the nuclear translocation of the NF-κB proteins RelA (also known as p65) and p50 [
11,
12]. RelA contains a transactivation domain which recruits coactivator complexes, increasing gene expression [
13,
14]. The genetic deficiency of RelA inhibits the LPS-induced pulmonary expression of the chemokines KC and MIP-2 and the adhesion molecule ICAM-1, resulting in decreased neutrophil emigration [
15]. Thus, RelA is essential to the gene expression mediating neutrophil emigration induced by LPS in the lungs. p50 is more complex, capable of either increasing or decreasing gene expression
in vitro [
16‐
24]. During
E. coli pneumonia, the deficiency of p50 increases the expression of multiple κB-regulated genes, resulting in increased neutrophil recruitment and excessive inflammatory injury [
25].
In the present manuscript, we report that p50 deficiency had a very different effect on neutrophil recruitment elicited by
E. coli LPS in the lungs. In marked contrast to
E. coli,
E. coli LPS elicited neutrophil accumulation which was significantly decreased by p50 deficiency. p50 deficiency did not diminish neutrophil accumulation elicited by heat-killed
E. coli, indicating that p50 dependency could be overcome by bacterial products other than LPS. Peripheral blood neutrophils remained responsive to the chemokine KC in p50-deficient mice, arguing against a functional desensitization of chemokine receptors. IL-6 was overexpressed in p50-deficient lungs, and IL-6 can decrease neutrophil recruitment to intrapulmonary LPS [
26,
27], but excessive IL-6 was not responsible for the decreased neutrophil recruitment of p50-deficient mice. Although the mechanism remains unclear, these studies demonstrate for the first time that p50 facilitates neutrophil accumulation in response to some stimuli in the lungs, including
E. coli LPS.
Discussion
The deficiency of NF-κB p50 decreased neutrophil recruitment elicited by
E. coli LPS in the lungs to 30% of WT levels, indicating that maximal neutrophil recruitment to this stimulus requires this transcription factor. In contrast, neutrophil recruitment elicited by either living [
25] or heat-killed
E. coli was not decreased in p50-deficient mice compared to WT, but was 130% or 120% of WT levels, respectively. Thus, select pathways for neutrophil emigration depend on NF-κB p50, and these pathways are essential in response to LPS but not more complex gram-negative bacterial stimuli.
The LPS preparations used in these studies likely contained both LPS, which activates TLR4, and bacterial lipoproteins that signal through TLR2 [
33]. Because this semi-purified LPS preparation induced neutrophil recruitment that was decreased by p50 deficiency, TLR2 and TLR4 together induce p50-dependent pathways. Based on an approximation of several ng LPS per million gram-negative bacteria [
34], the instillate of heat-killed
E. coli used here contained 4-5 orders of magnitude less LPS than the instillate of semi-purified LPS used here. Such an amount of semi-purified LPS is insufficient to induce neutrophil recruitment in the lungs (data not shown), indicating that factors other than LPS or in combination with the low levels of LPS are responsible for inducing neutrophil recruitment to heat-killed
E. coli. The present results suggest that these bacterial products, in contrast to LPS, induce neutrophil recruitment that does not require p50. Candidate bacterial products include unmethylated CpG DNA and n-formyl-methionine peptides; both are likely included with killed bacteria, and both are capable of binding host cell receptors and inducing neutrophil recruitment. In addition, killed bacteria may differ from semi-purified LPS in forming complexes with soluble host proteins, such as complement, natural antibody, and surfactant proteins, which may alter host signaling and molecular pathways used for neutrophil emigration.
p50 deficiency increased the expression of κB-regulated genes induced by LPS in the lungs. Similarly, p50 deficiency increases the expression of κB-regulated genes induced by living
E. coli in the lungs [
25]. These data together suggest that p50 usually functions as a gene repressor rather than activator during innate immune responses to bacterial stimuli in the lungs. The data interestingly contrast with the observation that p50 serves as a gene activator during acquired immune responses to antigen in the lungs, elicited by ovalbumin sensitization and challenge [
35,
36]. The molecular mechanisms responsible for determining whether p50 functions as a repressor or an activator remain to be determined, but may involve intracellular signaling pathways which alter the nuclear proteins associating with p50. For example, p50 homodimers usually recruit a repressor complex containing histone deacetylase 3 to the
KAI1 promoter, but MEKK1-mediated phosphorylation of a component of this complex causes it to dissociate from the p50 homodimer and be replaced by a histone acetyl transferase-containing complex which activates gene transcription [
37]. p50 may recruit different complexes to different promoter regions for different purposes during innate and acquired immune responses in the lungs.
Excessive gene expression due to p50 deficiency may be responsible for the decreased neutrophil recruitment during LPS-induced pulmonary inflammation. Excess levels of chemokines can desensitize neutrophils
in vitro [
30,
31], and can inhibit neutrophil recruitment
in vivo [
32]. Although KC and MIP-2 concentrations were increased in the BALF and KC concentrations were increased in the blood of p50-deficient mice, circulating neutrophils in p50-deficient mice remained responsive to the chemokine KC. Desensitization of chemokine receptors therefore seems unlikely to mediate the decreased neutrophil recruitment. However, excessive chemokine elaboration could still contribute to the decreased neutrophil recruitment. The dose-response curves for neutrophil chemoattractants is typically bell-shaped, with
in vitro chemotaxis peaking at an optimum concentration and then decreasing at higher concentrations [
38]. Net chemokine concentrations in p50-deficient lungs may have been beyond the optimal concentrations. Furthermore, increasing chemokine concentrations in the blood may collapse gradients of soluble and/or matrix-bound chemokine, preventing the directed migration of neutrophils [
39]. The increased KC concentration in the blood of p50-deficient mice could have contributed to such a collapse of chemokine gradients.
Another strong candidate for decreasing neutrophil recruitment when overexpressed is IL-6. The effect of IL-6 on neutrophil emigration in the lungs is complex and stimulus-specific [
40,
41]. During LPS-induced pulmonary inflammation, adding IL-6 decreases neutrophil emigration [
26] and interrupting IL-6 increases neutrophil emigration [
27], suggesting that this cytokine limits LPS-induced neutrophil recruitment in the lungs. LPS-induced IL-6 was increased in the BALF of p50-deficient mice compared to WT. Therefore, we hypothesized that excessive IL-6 expression decreased LPS-induced neutrophil emigration in p50-deficient mice. We tested this hypothesis by generating double mutant mice deficient in both p50 and IL-6, and comparing neutrophil recruitment in double mutants and mice deficient in IL-6 alone. p50 deficiency decreased neutrophil recruitment in the absence of IL-6. These data demonstrate conclusively that p50 facilitates neutrophil recruitment independent of any requirement for IL-6.
The precise mechanism by which p50 facilitates neutrophil recruitment to
E. coli LPS in the lungs remains to be elucidated. We have measured over 30 genes at the RNA or protein level (including the present data and data not shown), and all of them have been either increased or unchanged by p50 deficiency during LPS-induced pulmonary inflammation. We have observed no significant decreases in gene expression during innate immune responses in p50-deficient mice. Since p50 clearly limits the expression of many κB-regulated genes during innate immune responses to bacterial stimuli in the lungs, we favor the interpretation that p50 facilitates neutrophil recruitment in the lungs by decreasing gene expression which brakes neutrophil recruitment to LPS but not
E. coli in the lungs. However, even within an extremely simplified system, such as a single cell type (fibroblasts) treated
in vitro with a single stimulus (TNF-α), each NF-κB subunit has distinct roles that differ among different genes [
42]. Although not yet observed, p50 may increase the expression of some genes during LPS-induced pulmonary inflammation. Thus, an alternative mechanistic hypothesis is that NF-κB p50 is essential for the production of genes yet to be identified which are essential for maximal neutrophil recruitment to LPS in the lungs.
Conclusions
In response to E. coli or E. coli LPS, p50 deficiency increases the expression of multiple κB-regulated genes, suggesting that p50 usually limits the expression of these genes. LPS induces a p50-dependent pathway for neutrophil recruitment in the lungs, which can be overcome or bypassed with bacterial products other than LPS. The mechanism by which p50 facilitates neutrophil accumulation during LPS-induced pulmonary inflammation remains to be determined, but we hypothesize that p50 may limit gene expression that interferes with neutrophil emigration. Deficiency of p50 increases concentrations of chemokines and of IL-6, each of which can suppress neutrophil emigration. However, p50-deficient neutrophils remain responsive to chemokines, and p50 deficiency compromises neutrophil recruitment even when IL-6 is abrogated by gene targeting. Identifying the genes regulated by p50 to facilitate neutrophil recruitment to LPS in the lungs is an important goal for future studies.
Authors' contributions
JM conceived of the study, participated in its design and coordination, performed the statistical analyses, and drafted the manuscript. ML carried out in vivo studies of neutrophil function, and participated in their design and interpretation. MS carried out molecular studies including immunoassays, RNA analyses, and genotyping, and participated in their design and interpretation. All authors read and approved the final manuscript.