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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 575-582, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0015OC

Functions of I{kappa}B Proteins in Inflammatory Responses to Escherichia coli LPS in Mouse Lungs

Joseph P. Mizgerd, Martin L. Scott, Matt R. Spieker and Claire M. Doerschuk

Physiology Program, Harvard School of Public Health, Boston; and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts

Address correspondence to: Joseph P. Mizgerd, S.D., Physiology Program, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. E-mail: jmizgerd{at}hsph.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Acute inflammation induced by intrapulmonary LPS requires nuclear factor (NF)-{kappa}B RelA. This study elucidates the effects of intrapulmonary LPS on I{kappa}B proteins, endogenous inhibitors of RelA, and the effects of deficiency of I{kappa}B-ß. I{kappa}B-{alpha}, I{kappa}B-ß, and I{kappa}B-{epsilon} each complexed with RelA in uninfected murine lungs. Intratracheal instillation of LPS induced the degradation of I{kappa}B-{alpha} and I{kappa}B-ß, as measured by the loss of immunoreactive proteins in non-nuclear fractions. Degradation was apparent by 2 h and sustained through 6 h. In contrast, net I{kappa}B-{epsilon} content increased over this period. The small amounts of I{kappa}B-{alpha} and I{kappa}B-ß that were detected in nuclear fractions from the lungs also decreased over this time frame, whereas intranuclear NF-{kappa}B content (including both RelA and p50) increased. The hypophosphorylated form of I{kappa}B-ß, which facilitates transcription induced by NF-{kappa}B, was not detected. Neutrophil recruitment and edema accumulation did not differ between wild type mice and gene-targeted mice deficient in I{kappa}B-ß, suggesting that I{kappa}B-ß is not specifically required for these responses. Altogether, these data suggest that RelA is liberated during LPS-induced pulmonary inflammation by the regulated degradation of both I{kappa}B-{alpha} and I{kappa}B-ß. In the absence of I{kappa}B-ß, I{kappa}B-{alpha} or other inhibitory proteins can regulate NF-{kappa}B functions essential to acute neutrophil emigration in the lungs.

Abbreviations: antibody, Ab • electrophoretic mobility shift assay, EMSA • nuclear factor, NF • red blood cell, RBC • wild type, WT


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lower respiratory infections are the leading cause of lost disability-adjusted life years worldwide (1). Gram-negative bacteria are common causes of both community- and hospital-acquired pneumonias (2, 3). LPS from Gram-negative bacteria is recognized in the lungs by pattern recognition receptors (4) that initiate local innate immune responses, including neutrophil emigration and edema accumulation. Neutrophil emigration elicited by LPS or Gram-negative bacteria in the lungs requires the regulated expression of a variety of genes, including chemokines (5–7), adhesion molecules (8, 9), and early response cytokines (10). The transcription of many of these genes (including KC, macrophage inflammatory protein-2, intercellular adhesion molecule-1, and tumor necrosis factor-{alpha}) is mediated at least in part by the nuclear factor (NF)-{kappa}B family of transcription factors (reviewed in (11)).

The intrapulmonary deposition of bacteria or LPS induces the nuclear translocation of NF-{kappa}B complexes, including RelA and p50 subunits (12, 13). The RelA subunit is essential for effectively responding to bacterial stimuli in the lungs. The gene-targeted deletion of RelA renders mice susceptible to spontaneous pulmonary infections (14). Neutrophil emigration and the pulmonary expression of KC, macrophage inflammatory protein-2, and intercellular adhesion molecule-1 induced by intranasal insufflation of Escherichia coli LPS are inhibited by the absence of RelA (14). These data suggest that nuclear translocation of NF-{kappa}B RelA is critical to inducing the gene expression mediating neutrophil emigration elicited by LPS in the lungs.

NF-{kappa}B transcription factors are controlled by the I{kappa}B family of inhibitor proteins (15). I{kappa}B proteins bind to the Rel-homology domains of NF-{kappa}B proteins, masking the nuclear localization and DNA-binding sequences, and preventing NF-{kappa}B–mediated gene transcription. The stimulus-induced phosphorylation, ubiquitination, and proteasomal degradation of I{kappa}B proteins liberates the NF-{kappa}B complexes, which then translocate to the nucleus, bind specific DNA sequences, and induce the transcription of downstream genes.

Three I{kappa}B family members, I{kappa}B-{alpha}, -ß, and -{epsilon}, are encoded by distinct genes and are capable of inhibiting NF-{kappa}B–mediated transcription. The extent to which these proteins have unique or overlapping functions remains unclear. They possess subtle differences in the quality of their biochemical interactions with individual NF-{kappa}B complexes (16–18), but no particular NF-{kappa}B complexes or NF-{kappa}B activities have been demonstrated to require distinct I{kappa}B proteins. Each of the I{kappa}B proteins is capable of inhibiting NF-{kappa}B translocation, DNA binding, and transcriptional activation when overexpressed (16–18). Degradation of each of the I{kappa}B proteins is associated with NF-{kappa}B translocation and expression of {kappa}B-associated genes. However, the kinetics of activation-induced degradation and resynthesis appears to differ for the three I{kappa}B proteins, with I{kappa}B-{alpha} being degraded and resynthesized most quickly and I{kappa}B-ß most slowly (18–21). The I{kappa}B proteins are inducibly phosphorylated by multiple kinases (22–25); to our knowledge, no specific kinases have been selectively associated with distinct I{kappa}B proteins. The biochemical mechanisms responsible for the independent regulation of these I{kappa}B proteins remain to be elucidated.

The regulated expression of these three different proteins is likely critical to their independent functions. I{kappa}B-{alpha} deficiency results in neonatal lethality secondary to dysregulated NF-{kappa}B activity and spontaneous inflammatory disease (26), suggesting that I{kappa}B-{alpha} is essential to the basal inhibition of NF-{kappa}B. However, replacing the I{kappa}B-{alpha} gene with an I{kappa}B-ß gene abrogates these effects, demonstrating that I{kappa}B-ß can perform similar basal inhibitory functions as I{kappa}B-{alpha}, but only when it is controlled by the cis regulatory elements of I{kappa}B-{alpha} (27). In contrast to I{kappa}B-{alpha} deficiency, gene-targeted mutation of I{kappa}B-ß does not affect survival or result in spontaneous inflammatory disease (M.L. Scott and D. Baltimore, unpublished observations), suggesting that I{kappa}B-ß is not essential to the basal inhibition of NF-{kappa}B.

Although all three of these I{kappa}B proteins concentrate NF-{kappa}B proteins in the cytoplasm, both I{kappa}B-{alpha} and I{kappa}B-ß appear in the nucleus as well. The regulation and function of these I{kappa}B proteins within this organelle may differ. I{kappa}B-{alpha} does not completely mask the nuclear localization signals of RelA, and hence, I{kappa}B-{alpha} is regularly imported into nuclei along with NF-{kappa}B proteins (28–30). However, a nuclear export signal on I{kappa}B-{alpha} efficiently returns the complexes to the cytoplasm via the Crm1 export receptor (28–30). Thus, I{kappa}B-{alpha} regularly shuttles through the nucleus, and export of NF-{kappa}B complexes displaced from the DNA by I{kappa}B-{alpha} may be critical to turning off gene expression induced by NF-{kappa}B. I{kappa}B-ß can also be found in the nucleus, but the mechanisms of its import and export are different from I{kappa}B-{alpha} and remain to elucidated (28, 29, 31–33). I{kappa}B-ß in particular may increase in the nucleus subsequent to cell stimulation (31, 33). The intranuclear form of I{kappa}B-ß appears to be hypophosphorylated, and incapable of inhibiting NF-{kappa}B–mediated gene expression (31). Since it prevents binding of NF-{kappa}B by other I{kappa}B proteins (31), hypophosphorylated I{kappa}B-ß may enhance rather than inhibit gene expression mediated by NF-{kappa}B.

We hypothesized that the different I{kappa}B proteins perform distinct functions in regulating inflammatory responses to LPS in the lungs. The present study characterizes I{kappa}B-{alpha}, -ß, and -{epsilon} in the lungs during pulmonary inflammation induced by E. coli LPS. The functional requirements for I{kappa}B-ß in regulating pulmonary inflammation were specifically examined using mice with targeted deletion of the I{kappa}B-ß gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
I{kappa}B-ß–deficient mice were generated by standard procedures using homologous recombination in embryonic stem cells. The targeting of mouse Nfkbib deleted the genomic DNA encoding nucleotides 253 to 987 of the I{kappa}B-ß cDNA (GenBank Accession U19799, National Center for Biotechnology Information, Bethesda, MD). This targeted deletion eliminated > 60% of the coding region, including all ankyrin repeats. I{kappa}B-ß–deficient mice and wild type (WT) mice of similar random hybrid genetic background (C57BL/6 x 129/Sv) were maintained under specific pathogen-free conditions in a full barrier facility. C57BL/6 mice were purchased from Taconic (Germantown, NY). Mice were 6 to 10 wk of age at the time of experiments. All experimental protocols were approved by the Harvard Medical Area Standing Committee on Animals.

NF-{kappa}B and I{kappa}B Proteins in the Lungs
NF-{kappa}B translocation in murine lungs was measured as previously described (10, 13). Mice were anesthetized by intramuscular injection of ketamine hydrochloride (100 mg/kg) and acepromazine maleate (5 mg/kg). The trachea was surgically exposed, and an angiocatheter was inserted via the trachea into the left bronchus. Sterile saline containing 100 µg E. coli LPS serotype O55:B5 (Sigma, St. Louis, MO) and 5% colloidal carbon, to mark the site of instillation, was instilled in a volume of 50 µl per mouse. Mice were killed by inhalation of a halothane overdose at the indicated times. Colloidal carbon-containing lung lobes from mice instilled with LPS, as well as left lung lobes from mice that did not receive LPS instillation, were excised, snap-frozen in liquid nitrogen, and stored at -80°C until protein extraction. Nuclear and non-nuclear proteins were collected from the frozen lung samples (10, 13), and total protein concentrations were measured using a bicinchonic acid assay with bovine serum albumin as the standard.

For electrophoretic mobility shift assay (EMSA), nuclear proteins (0.5 mg/ml) incubated with 3.5 nM {gamma}[32P]ATP-labeled NF-{kappa}B consensus oligonucleotide (Promega, Madison, WI). In supershift assays, nuclear proteins (0.3 mg/ml) were incubated with 3.5 nM {gamma}[32P]ATP-labeled NF-{kappa}B consensus oligonucleotide and an antibody (Ab) against one subunit of NF-{kappa}B (0.2 mg/ml) for 30 min prior to electrophoresis. All polyclonal Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), including the following: sc-7151 against RelA, sc-1192 against p50, sc-71 against c-Rel, sc-226 against RelB, and sc-298 against p52. Protein-oligonucleotide complexes were separated from protein-free oligonucleotides by polyacrylamide gel electrophoresis and detected by autoradiography. Independent experiments with proteins collected from the lungs of different mice yielded consistent results.

For most Western analyses, nuclear and non-nuclear proteins (30 µg/well) were separated on 4–12% gradient gels by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P PVDF membranes. Membranes were probed with the following polyclonal Ab (Santa Cruz Biotechnology): sc-371 against I{kappa}B-{alpha}, sc-945 against I{kappa}B-ß, and sc-7156 against I{kappa}B-{epsilon}. After washing, primary Ab associated with the membranes were detected on autoradiographic film by horseradish peroxidase-conjugated secondary Ab and the ECL+Plus chemiluminescent system (Amersham Pharmacia Biotech; Piscataway, NJ). Densitometric data were collected and analyzed using Scion ImagePC software (Scion; Frederick, MD).

For Western analyses designed to differentiate the electrophoretic mobilities of basally phosphorylated and hypophosphorylated I{kappa}B-ß, gel electrophoresis conditions which separated these isoforms were empirically determined. Nuclear and non-nuclear proteins (30 µg/well) were separated by SDS-PAGE over 20 cm in 10% polyacrylamide at a constant current of 24 mA. Proteins were transferred to Immobilon-P PVDF membranes, and Western analyses were performed as above. To render I{kappa}B-ß hypophosphorylated, protein samples were incubated with calf intestinal phosphatase (Promega) at 0.5 U/µl for 30 min at 37°C prior to electrophoresis.

For immunoprecipitations, left lung lobes were homogenized in the lysis buffer containing antiproteases designed by Shenkar and colleagues for immunoprecipitation of mouse lung proteins (34), and cleared by centrifugation. Aliquots from each extract were saved for Western analyses. Protein concentrations were determined using the bicinchonic acid assay (Sigma), and aliquots containing 8 mg of lung protein were rotated end-over-end with primary Ab at a final concentration of 5 µg/ml, overnight at 4°C. Ab-binding complexes were precipitated with Protein A-sepharose (Sigma); protein samples which did not precipitate with protein A-sepharose (supernatant fractions) were collected for comparison. Precipitates were washed three times with lysis buffer containing antiproteases, and suspended in loading dye. Equal volumes of protein samples were separated by SDS-PAGE, and the presence of specific proteins in a given preparation (extract, supernatant, or precipitate) was assessed by Western analyses as described above. Immunoprecipitations using proteins collected from the lungs of different mice yielded reproducible, consistent results.

Neutrophil Emigration and Edema Accumulation
Mice received intratracheal instillations of LPS as described above. After 6 h, mice were killed by inhalation of a halothane overdose. Control WT mice received no intratracheal instillations. The hearts were tied off to maintain pulmonary blood contents, and peripheral blood samples were collected from the inferior vena cava. Excised lungs were fixed by intratracheal instillation of 6% glutaraldehyde at a pressure of 23 cm H2O. Emigrated and sequestered neutrophils were quantified by morphometric analyses of histologic lung sections (10, 35). Investigators were blinded to the genotype of the mice during counting procedures.

Circulating neutrophils were quantified in peripheral blood samples. After red blood cell (RBC) lysis, leukocytes were counted using a hemacytometer, and differential distributions were assessed in blood smears stained with LeukoStat (Fisher Scientific, Pittsburgh, PA).

Pulmonary edema, as measured by the extravascular accumulation of 125I-albumin, was quantified as previously described (10, 35). Specific activities of 125I-albumin and 51Cr-RBC were measured in blood and plasma samples and in excised, fixed lungs from each mouse. Hematocrits were calculated from 125I-albumin activities in the blood and plasma samples. Pulmonary blood volume was derived from the 51Cr-RBC activity in the lung and blood samples. Volumes of total plasma equivalents in the lungs were calculated from the 125I-albumin activity in the lung and the plasma samples, and volumes of intravascular plasma were calculated using the hematocrits and the pulmonary blood volumes. Volumes of extravascular plasma equivalents in the lungs were derived from the difference between the volumes of total plasma equivalents and the volumes of intravascular plasma. Edema fluid accumulation was expressed as µl per lung of extravascular plasma equivalents.

Statistical Analysis
For comparing NF-{kappa}B and I{kappa}B levels at multiple time points, all groups were compared by ANOVA, and intergroup comparisons were performed using post hoc Scheffé's tests. For emigration and edema studies, WT and I{kappa}B-ß–deficient mice were compared using Student's t test. All pooled data were presented as mean and SE values. Differences were considered significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NF-{kappa}B Translocation Elicited by LPS in the Lungs
E. coli LPS induced the nuclear translocation of NF-{kappa}B complexes in murine lungs. By 2 h after the intratracheal instillation of E. coli LPS, a significant increase in NF-{kappa}B binding activity was detectable in the nuclear fractions (Figure 1A) . The NF-{kappa}B activity in the nuclear fractions was further increased after 6 h (Figure 1A). The electrophoretic mobilities of the NF-{kappa}B complexes differed in the nuclear fractions from lungs collected 2 h compared with that collected 6 h after the instillation of LPS (Figure 1A). In particular, faster moving complexes became apparent after 6 h, while the slowest moving complexes became less prominent.



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  		Figure 1. Nuclear translocation of NF-{kappa}B proteins induced by LPS in the lungs. (A) Increased levels of NF-{kappa}B proteins in nuclear fractions from the lungs of C57BL/6 mice after the intratracheal instillation of E. coli LPS. NF-{kappa}B proteins were identified by their ability to bind a consensus oligonucleotide probe using EMSA. (B) Complexes induced to translocate in the lungs include both RelA and p50 at each time point examined after LPS instillation. Components of NF-{kappa}B Complexes I, II, and III were identified by the ability of polyclonal Ab to supershift complexes (please see RESULTS). All EMSA and supershift experiments using protein from the lungs of different mice were repeated with consistent results.

 
The identities of NF-{kappa}B proteins translocating to the nucleus in response to E. coli LPS were investigated using supershift analyses. Ab against c-Rel, RelB, and p52 did not supershift bands at either time point (Figure 1B). Bands were supershifted by Ab against RelA or p50 at both time points (Figure 1B), indicating that these proteins translocated to the nucleus in response to the pulmonary deposition of LPS. The band labeled Complex II in Figure 1B was shifted only by Ab against p50, suggesting that this band contains p50/p50 homodimers. Ab against RelA decreased the intensity of a distinct band at either time point (Complex I, present after 2 h, and Complex III, present after 6 h; Figure 1B), but the electrophoretic mobility of the supershifted band differed at the two time points. The intensities of Complexes I and III in Figure 1B were also decreased by Ab against p50. These data suggest that Complexes I and III both contain RelA/p50 heterodimers. The greater electrophoretic mobility of Complex III compared with either Complexes I or II suggests that RelA may be partially degraded in nuclear fractions from lungs collected 6 h after LPS instillation, perhaps as a result of neutrophil proteases, as has been previously described (12, 36, and unpublished observations).

Selective Degradation of I{kappa}B Proteins Elicited by LPS in the Lungs
The content of I{kappa}B proteins was assessed in the non-nuclear fractions from the same lungs as that of the NF-{kappa}B analyses. I{kappa}B-{alpha} was readily detected in the lungs prior to instillation of LPS. By 2 h after instillation, LPS in the air spaces induced a significant loss of I{kappa}B-{alpha}, to 39 ± 4% of the basal content (Figure 2A) . I{kappa}B-{alpha} content remained low, with only 30 ± 5% of the basal content detected 6 h after LPS instillation (Figure 2A). Thus, LPS in the lungs induced a rapid, substantial, and sustained loss of I{kappa}B-{alpha}.



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  		Figure 2. Altered I{kappa}B levels in response to LPS in the lungs. I{kappa}B levels in the non-nuclear fractions from the lungs of C57BL/6 mice were detected by Western analyses 0, 2, and 6 h after LPS instillation, and quantitated using densitometry. Graphs depict the mean and SE from six mice, with insets demonstrating representative immunoblots with three mice at each time point. Asterisks (*) indicate significant differences (P < 0.05) compared with hour 0. (A) I{kappa}B-{alpha} levels decreased within 2 h and remained low at 6 h after LPS instillation. (B) I{kappa}B-ß levels decreased within 2 h and remained low at 6 h after LPS instillation. (C) I{kappa}B-{epsilon} levels did not decrease but increased 2 and 6 h after LPS instillation. (D) Phosphorylated I{kappa}B-ß in murine lungs. I{kappa}B-ß was detected by Western analyses after incubation of non-nuclear fractions from lungs of two different 0 h C57BL/6 mice with calf intestinal phosphatase, as indicated (+). Adjacent (-) lanes show the same proteins not treated with phosphatase. Increased electrophoretic mobility induced by phosphatase was interpreted as evidence of I{kappa}B-ß phosphorylation.

 
LPS similarly affected I{kappa}B-ß content in the lungs. Like I{kappa}B-{alpha}, I{kappa}B-ß levels were decreased to 40 ± 11% and 31 ± 14% of basal content, 2 and 6 h after LPS instillation, respectively (Figure 2B).

In contrast, LPS instillation did not decrease I{kappa}B-{epsilon} levels in the non-nuclear fractions recovered from whole lungs. Ab against I{kappa}B-{epsilon} detected multiple bands in the expected 45 kD size range in protein fractions from mouse lungs with or without LPS instillation (Figure 2C). Multiple isoforms of I{kappa}B-{epsilon} have been detected in mouse cell lines, more prominently than in human cell lines, and the slower migrating bands have been demonstrated to be hyperphosphorylated forms of I{kappa}B-{epsilon} (18). The biological significance of this I{kappa}B-{epsilon} phosphorylation is unknown. In the present study, all isoforms, but perhaps especially the slower migrating hyperphosphorylated isoforms, of I{kappa}B-{epsilon} proteins detected by Western blotting increased in the lungs 2 and 6 h after the instillation of E. coli LPS (Figure 2C). Thus, in contrast to the other I{kappa}B proteins, E. coli LPS resulted in a net gain, rather than loss, of I{kappa}B-{epsilon} in the lungs.

Nuclear I{kappa}B Proteins
Although both are concentrated in the cytoplasm, I{kappa}B-{alpha} and I{kappa}B-ß each appear in the nuclear compartment as well (28, 29, 31–33). Furthermore, I{kappa}B-ß content in the nucleus increases subsequent to stimulation in some settings (31, 33). The levels of I{kappa}B proteins in the nuclear fractions collected from mouse lungs before and after LPS instillation were examined by Western analyses. Although the majority of I{kappa}B-{alpha} and I{kappa}B-ß in the lungs was cytoplasmic, both proteins were detected in the nuclear fractions prior to LPS instillation. In contrast, I{kappa}B-{epsilon} in the nuclear compartments was present, if at all, at levels too low to reliably visualize or quantitate. These results are consistent with the transit of I{kappa}B-{alpha} and I{kappa}B-ß through the nuclear compartment in resting lungs. Contents of both I{kappa}B-{alpha} and I{kappa}B-ß decreased in the nuclear fraction after LPS instillation (Table 1). I{kappa}B-{epsilon} remained at levels too low to reliably quantify. Thus, none of the I{kappa}B proteins, including I{kappa}B-ß, accumulated in the nuclei after LPS instillation to the lungs.


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  		TABLE 1 Loss of nuclear I{kappa}B-{alpha} and I{kappa}B-ß after LPS instillation to the lungs

 
Basal Phosphorylation of I{kappa}B-ß in the Lungs
A hypophosphorylated form of I{kappa}B-ß may emerge subsequent to stimulation of NF-{kappa}B translocation (31, 32). Electrophoretic mobility was used to differentiate hypophosphorylated forms of I{kappa}B-ß from basally phosphorylated forms. Incubation with phosphatase prior to electrophoresis increased the electrophoretic mobility of I{kappa}B-ß collected from the non-nuclear fractions of mouse lungs (Figure 2D). These data demonstrate that hypophosphorylated I{kappa}B-ß could be differentiated from basally phosphorylated I{kappa}B-ß using this assay. Furthermore, the results indicate that the majority of I{kappa}B-ß in resting lungs was basally phosphorylated. No differences in electrophoretic mobility were detected between I{kappa}B-ß proteins in nuclear compartments and those in non-nuclear compartments, before or 6 h after LPS instillation (data not shown). Thus, these studies did not demonstrate an accumulation of hypophosphorylated I{kappa}B-ß induced by LPS in the lungs.

Complexes Between I{kappa}B Proteins and RelA in the Lungs
LPS induced the degradation of both I{kappa}B-{alpha} and I{kappa}B-ß in the lungs (Figure 2) and the nuclear translocation of p50/p50 and p50/RelA (Figure 1). RelA is essential for LPS-induced neutrophil emigration in the lungs (14). To determine whether either or both of these I{kappa}B proteins physically interacted with RelA in the lungs, reciprocal co-immunoprecipitations were performed. Immunoprecipitation of either I{kappa}B-{alpha} or I{kappa}B-ß coprecipitated RelA, but did not coprecipitate the other I{kappa}B protein (Figures 3A and 3B) . Immunoprecipitation of RelA coprecipitated both I{kappa}B proteins (Figure 3C). Thus, in resting lungs, RelA forms complexes with I{kappa}B-{alpha} and with I{kappa}B-ß. These data suggest that the LPS-induced degradation of either I{kappa}B-{alpha} or I{kappa}B-ß liberates RelA to translocate to the nucleus in the lungs.



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  		Figure 3. Biochemical interactions of RelA with I{kappa}B proteins. Whole lung lobes were homogenized, and protein complexes containing RelA, I{kappa}B-{alpha}, I{kappa}B-ß, or I{kappa}B-{epsilon} were immunoprecipitated using Ab specific for the proteins indicated under IP. An additional lung homogenate was precipitated with nonspecific (NS) IgG. Proteins present in the whole lung extract (E), proteins which were not precipitated but remained in the supernatant (S), and proteins which were precipitated (P) were separated by SDS-PAGE. Separated proteins were transferred to membranes, and the presence of I{kappa}B-{alpha}, I{kappa}B-ß, or RelA in the protein samples was assessed by immunoblot (as indicated under IB). All immunoprecipitations were repeated with proteins from the lungs of multiple mice with consistent results. (A) I{kappa}B-{alpha} was precipitated by IP of RelA or I{kappa}B-{alpha}, but not I{kappa}B-ß. (B) I{kappa}B-ß was precipitated by IP of RelA or I{kappa}B-ß, but not I{kappa}B-{alpha}. Because Ab used to precipitate I{kappa}B-{alpha} and -ß (but not RelA) were from same species as I{kappa}B-ß–specific Ab used in IB, the Ig used for IP heavy chains were revealed by secondary Ab staining, apparent in the precipitate lanes running slightly higher than the I{kappa}B-ß band. (C) RelA was precipitated by IP of I{kappa}B-{alpha} or I{kappa}B-ß, but not by non-specific IgG. (D) RelA, but not I{kappa}B-{alpha} or I{kappa}B-ß, was precipitated by IP of I{kappa}B-{epsilon}.

 
Although I{kappa}B-{epsilon} levels were not decreased during this inflammatory response, we immunoprecipitated I{kappa}B-{epsilon} to determine whether I{kappa}B-{epsilon} formed complexes with RelA in mouse lungs. The immunoprecipitation of I{kappa}B-{epsilon} coprecipitated RelA, but not I{kappa}B-{alpha} or -ß (Figure 3D). Thus, conditions in which I{kappa}B-{epsilon} is degraded in the lungs may also result in the nuclear translocation of RelA.

Responses to Intrapulmonary LPS in the Absence of I{kappa}B-ß
The above data implicate the regulated degradation of I{kappa}B-{alpha} and/or I{kappa}B-ß as potential regulatory steps determining NF-{kappa}B function and acute inflammatory responses elicited by bacterial LPS in the lungs. To determine whether I{kappa}B-ß has unique essential roles in regulating pulmonary responses to LPS, biochemical and functional responses to intratracheally instilled E. coli LPS were compared in WT mice and mice deficient in I{kappa}B-ß due to gene targeting.

Gene targeting resulted in the complete loss of immunoreactive I{kappa}B-ß (Figure 4A) . I{kappa}B-{alpha} and I{kappa}B-{epsilon} were detected in non-nuclear fractions from the lungs of I{kappa}B-ß–deficient mice (Figure 4A), at levels similar to or greater than that in WT mice. I{kappa}B-ß remained undetectable after LPS instillation to I{kappa}B-ß–deficient mice (Figure 4B). As in WT mice, LPS instillation to I{kappa}B-ß–deficient mice diminished levels of I{kappa}B-{alpha} but not I{kappa}B-{epsilon} (Figure 4B). The intratracheal instillation of LPS resulted in NF-{kappa}B translocation in I{kappa}B-ß–deficient mice, which persisted at least 6 h, as was the case in WT mice (Figure 4B). Thus, over this time frame, I{kappa}B-ß does not appear essential to the nuclear accumulation of NF-{kappa}B proteins induced by LPS in the lungs.



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  		Figure 4. I{kappa}B and NF-{kappa}B in I{kappa}B-ß–deficient mice. (A) I{kappa}B expression detected by Western analyses of non-nuclear fractions from the lungs of mice prior to LPS instillation. Membranes were immunoblotted with polyclonal Ab against the indicated I{kappa}B protein, or were stained for total protein using BLOT-FastStain (Genotech; St. Louis, MO). (B) Effect of LPS instillation on I{kappa}B protein levels and NF-{kappa}B nuclear translocation in the lungs of WT and I{kappa}B-ß–deficient mice. I{kappa}B protein levels were detected in the non-nuclear fractions by Western analyses using polyclonal Ab against the indicated I{kappa}B proteins. NF-{kappa}B levels in the nuclear fractions were detected by EMSA.

 
Potential roles of I{kappa}B-ß in regulating pulmonary inflammation were examined by comparing neutrophil emigration and edema accumulation induced by E. coli LPS in the lungs of WT and I{kappa}B-ß–deficient mice. Neutrophil emigration was induced by LPS instillation (Figure 5A) , as measured by morphometric quantitation of neutrophils in the alveolar air spaces. The quantities of emigrated neutrophils did not differ between WT and I{kappa}B-ß–deficient mice (Figure 5A), suggesting that I{kappa}B-ß was not essential for this process. Circulating neutrophil numbers also did not differ between WT and I{kappa}B-ß–deficient mice with LPS-induced pulmonary inflammation (1.5 ± 0.3 x 106 and 1.7 ± 0.3 x 106 per ml blood in WT and mutant mice, respectively). Similarly, pulmonary edema, as measured by the accumulation of extravascular albumin in the lungs, was induced by the intratracheal instillation of LPS (Figure 5B). Again, the amount of extravasated albumin did not differ between WT and I{kappa}B-ß–deficient mice (Figure 5B), suggesting that I{kappa}B-ß was not essential for this process. Therefore, I{kappa}B-ß did not perform unique and required roles in regulating these acute inflammatory responses to LPS in the lungs when studied using gene-targeted mutant mice.



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  		Figure 5. Pulmonary inflammation induced by LPS in the lungs of WT and I{kappa}B-ß–deficient mice. LPS was instilled intratracheally, and the lungs were collected after 6 h. Baseline values were collected from WT mice. Data depict mean ± SE from four to five mice per group. (A) Neutrophil emigration was not affected by I{kappa}B-ß deficiency. Emigrated neutrophils were quantified as neutrophils within alveolar air spaces, expressed as a percent of the total volume of distal lung, using morphometric analyses of histologic sections. (B) Edema accumulation was not affected by I{kappa}B-ß deficiency. Edema accumulation was quantified as the volume of extravascular plasma equivalents, expressed as µl/lung, using radiotracer analyses.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that LPS in the lungs resulted in decreased levels of I{kappa}B-{alpha} and I{kappa}B-ß, consistent with the hyperphosphorylation, ubiquitination, and proteasomal degradation of each of these proteins. The LPS-induced nuclear translocation of NF-{kappa}B could therefore be regulated by either or both of these proteins. Coimmunoprecipitations demonstrated that NF-{kappa}B RelA, which is essential for neutrophil emigration and antibacterial host defenses (14), complexed with both I{kappa}B-{alpha} and I{kappa}B-ß proteins. In contrast to RelA, neither I{kappa}B-{alpha} nor I{kappa}B-ß accumulated in the nuclear fractions after LPS instillation. These data suggest that the LPS-induced nuclear translocation of RelA in the lungs results from the degradation of both I{kappa}B-{alpha} and I{kappa}B-ß.

In contrast to I{kappa}B-{alpha} and -ß, the levels of I{kappa}B-{epsilon} did not decrease in response to LPS in the lungs. These data suggest that I{kappa}B-{epsilon} is regulated differently than I{kappa}B-{alpha} and -ß in this setting, and that net I{kappa}B-{epsilon} degradation is not responsible for NF-{kappa}B translocation in the lungs up to 6 h after LPS instillation. Interestingly, I{kappa}B-{epsilon} and RelA did form complexes in mouse lungs, suggesting that the degradation of pulmonary I{kappa}B-{epsilon} in settings other than LPS-induced inflammation may contribute to the nuclear translocation of RelA. I{kappa}B-{epsilon} degradation can be induced in vitro (18), although the degradation of I{kappa}B-{epsilon} in the lungs has not been demonstrated to our knowledge.

The nuclear accumulation of NF-{kappa}B over several hours may specifically require signaling to I{kappa}B-ß proteins (19, 20, 31). This postulate is based in part on evidence that, after stimulation by LPS or cytokines, I{kappa}B-{alpha} re-attains prestimulation values rapidly (typically within 1–2 h), whereas I{kappa}B-ß remains at decreased levels for longer times (typically >= 4 h). Such distinctive kinetics have been observed after LPS stimulation both in vitro, with the 70Z/3 cell line and with cultured murine peritoneal macrophages (19, 21), and in vivo, in the liver and in the lungs after intraperitoneal injection (21, 37). However, in the present study, the intratracheal instillation of LPS decreased the pulmonary levels of both I{kappa}B-{alpha} and I{kappa}B-ß for at least 6 h. After LPS instillation to the lung, I{kappa}B degradation was likely initiated by receptors recognizing LPS, but it may have been prolonged by sequential activation of additional receptors (such as adhesion molecules and cytokine receptors) during neutrophil emigration from the pulmonary capillaries into the alveolar air spaces.

Pulmonary inflammation induced by intra-alveolar IgG immune complexes in rats is associated with a different pattern of I{kappa}B regulation than that observed in the present study with pulmonary inflammation induced by intratracheal LPS. Intrapulmonary formation of IgG immune complexes induces a slower degradation of I{kappa}B-{alpha}, with I{kappa}B-{alpha} content in the lungs decreasing progressively until it eventually reaches a nadir 4 h after the intratracheal instillation of IgG (38), and no decrease in I{kappa}B-ß protein (39). In contrast, the present study demonstrates that LPS induced a rapid drop in the levels of I{kappa}B-{alpha}, apparent less than 1 h after LPS instillation (data not shown) and reaching a nadir by 2 h. Furthermore, I{kappa}B-ß content decreased in parallel with I{kappa}B-{alpha} content after LPS instillation. Thus, I{kappa}B proteins display distinct patterns of regulation and may serve distinct functions in pulmonary inflammations induced by IgG immune complexes and E. coli LPS.

In addition to stimulating NF-{kappa}B translocation subsequent to its degradation, a hypophosphorylated form of I{kappa}B-ß can actively promote NF-{kappa}B translocation and NF-{kappa}B–mediated gene expression. Hypophosphorylated I{kappa}B-ß proteins competitively inhibit the interaction of NF-{kappa}B proteins with other I{kappa}B proteins, but they do not prevent the nuclear translocation or transcriptional activity of NF-{kappa}B (31). The hypophosphorylated form of I{kappa}B-ß may contribute to the excessive inflammation in the airways of patients with cystic fibrosis, as mutation in the CFTR gene in transformed bronchial epithelial cell lines confers an increase in hypophosphorylated I{kappa}B-ß and in IL-8 expression induced by tumor necrosis factor-{alpha} in vitro (40). In the present study, phosphatase treatment increased the electrophoretic mobility of I{kappa}B-ß from the lungs, demonstrating that the protein was phosphorylated. The I{kappa}B-ß detected after LPS instillation possessed the same electrophoretic mobility as I{kappa}B-ß proteins from uninflamed lungs. These data do not suggest the emergence of a prominent hypophosphorylated (and faster migrating) form of I{kappa}B-ß induced by LPS in the lungs. Further, the genetic deficiency of I{kappa}B-ß did not prevent the nuclear accumulation of NF-{kappa}B proteins through 6 h after LPS instillation, and neither neutrophil emigration nor edema accumulation were compromised by I{kappa}B-ß deficiency over this period. Thus, hypophosphorylated forms of I{kappa}B-ß are not essential to promoting NF-{kappa}B functions critical to the acute inflammatory responses elicited by LPS in the lungs.

As with any work based on targeted mutation as a means to study the function of a gene, other genes may potentially be regulated in ways that are not typical of animals without the mutation, appropriating functions normally mediated by the targeted gene. The genetic deficiency of I{kappa}B-{alpha} results in increased expression of I{kappa}B-{epsilon}, as detected by Western blots of the non-nuclear fractions from embryonic fibroblasts (18), although the overexpressed I{kappa}B-{epsilon} is incapable of performing I{kappa}B-{alpha} functions that are essential to homeostasis ex utero (26). In the present study, two of three I{kappa}B-ß–deficient mice demonstrated increased levels of both I{kappa}B-{alpha} and I{kappa}B-{epsilon} compared with WT mice, as detected by Western blots of the non-nuclear fractions from uninstilled lungs. Thus, I{kappa}B-{alpha} and/or I{kappa}B-{epsilon} may have been altered in the lungs of the I{kappa}B-ß–deficient mice prior to LPS instillation. It is possible that alterations in I{kappa}B-{alpha}, I{kappa}B-{epsilon}, or other genes, resulting indirectly from targeted mutation of the I{kappa}B-ß gene, may have prevented the identification of I{kappa}B-ß functions that are critical to regulation of acute inflammatory responses in the lungs of normal mice. The present results indicate that I{kappa}B-ß does not possess unique attributes, unavailable to other gene products, that are essential to mediating neutrophil emigration and edema accumulation elicited by intrapulmonary LPS.

Altogether, the present results and previous studies suggest that I{kappa}B proteins play diverse roles in different settings of pulmonary inflammation. The intratracheal instillation of LPS elicits signaling events (and downstream cellular and physiological changes) that are directly relevant to infection of the distal lung with Gram-negative bacteria. The present data are, to our knowledge, the first to report the effects of intrapulmonary LPS on I{kappa}B proteins. These findings indicate that the nuclear translocation of RelA and p50 induced by LPS in the lungs is associated with the degradation of both I{kappa}B-{alpha} and I{kappa}B-ß, but not I{kappa}B-{epsilon}. Because both I{kappa}B-{alpha} and I{kappa}B-ß complex with RelA, the translocation of this NF-{kappa}B subunit induced by LPS in the lungs requires the degradation of both of these I{kappa}B proteins. Studies with I{kappa}B-ß–deficient mice demonstrate that I{kappa}B-ß proteins do not possess unique properties which are essential to NF-{kappa}B translocation or to acute inflammation induced by E. coli LPS in the lungs. Thus, I{kappa}B-{alpha} or other proteins, in the absence of I{kappa}B-ß, can regulate the NF-{kappa}B functions induced by intrapulmonary LPS that are required for acute neutrophil emigration.


   Acknowledgments
 
The authors thank Dr. David Baltimore (California Institute of Technology) for generously providing the I{kappa}B-ß–deficient and WT mice, and Drs. Zhimin Yuan and Eric S. Silverman (Harvard School of Public Health) for expert advice related to the biochemical characterization of I{kappa}B proteins. These studies were supported by U.S. Public Health Service grant HL 52466. J.P.M. is a Parker B. Francis Fellow in Pulmonary Research.

Received in original form February 5, 2002

Received in final form May 9, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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