Departments of 1Pediatrics, 2Internal Medicine, 3Microbiology, 5Biochemistry, and 4The Inflammation Program, Carver College of Medicine, University of Iowa and Iowa City Veterans Administration, Iowa City, Iowa
Submitted 3 November 2003 ; accepted in final form 25 April 2004
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ABSTRACT |
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cell surface molecules; mucosa; lung; inflammation
Inducible antimicrobial peptides, including the cationic -defensins, represent an important component of the innate immune system (15, 47, 58). In humans, the expression of four
-defensins has been reported in pulmonary epithelia (5, 16, 23, 29, 36, 46), and a recent genome-wide search uncovered evidence of a much larger family of
-defensin genes encoded in five chromosomal clusters (48). Recent data from several groups suggest that the regulation of inducible
-defensin expression in epithelia may parallel many aspects of antimicrobial peptide expression in Drosophila, including sensing of the extracellular environment by the Toll-like receptor (TLR) family of pattern recognition receptors (7, 31, 50, 56) and intracellular signaling through NF-
B and other pathways (20, 52).
Human -defensin-2 (HBD-2) is an inducible cationic antimicrobial peptide expressed at many muscosal surfaces, including the skin, cornea, gut, gingiva, and airway epithelium (5, 22, 33, 35, 38, 43). In the airways, HBD-2 mRNA is expressed at low levels in resting cells but is markedly induced by proinflammatory stimuli, including IL-1
, TNF-
, and Pseudomonas aeruginosa (24, 50). Furthermore, the 5'-flanking sequence of the HBD-2 gene contains several cis-acting elements that may mediate transcription in response to inflammatory stimuli, including NF-
B, IFN-
, AP-1, and NF-IL-6 response elements (24, 33, 53). These properties suggest that endotoxins derived from airborne gram-negative bacteria could also induce HBD-2 gene expression.
Sensitive cellular responses to many endotoxins require the concerted action of at least four host extracellular and cellular proteins: LPS binding protein (LBP), CD14, MD-2, and TLR-4 (49). We now show that in well-differentiated primary cultures of human airway epithelia, TLR-4 but little or no MD-2 is expressed, and these cells are relatively unresponsive to added endotoxin, even in the presence of LBP and CD14. However, the responsiveness of these cells to endotoxin is markedly amplified by either the endogenous expression or the exogenous addition of MD-2, indicating that the constitutively low levels of MD-2 expression in these cells at "rest" are important in maintaining their hyporesponsiveness to endotoxin. Changes in MD-2 expression in the airway epithelium and/or neighboring cells can be achieved by exposure of these cells to specific bacterial and host products and may thereby regulate airway responsiveness to endotoxin.
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MATERIALS AND METHODS |
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Phorbol myristate acetate (PMA), human recombinant IL-1, TNF-
, and IFN-
were obtained from Sigma (St. Louis, MO). Soluble CD14 (sCD14) and LBP were provided by Xoma (Berkeley, CA). Lipooligosaccharide (LOS) from nontypeable Haemophilus influenzae was isolated by a mini-phenol-water extraction procedure, as previously described (26). LOS was also isolated from Neisseria meningitidis and used as purified aggregates and as monomeric LOS:sCD14 complexes as previously described (17, 19). P6 from nontypeable H. influenzae was a generous gift from Dr. Timothy F. Murphy (SUNY Buffalo, Buffalo, NY). Nontypeable Haemophilus influenzae (NTHi) strain 12 (14) was a kind gift of Dr. Dwight Look.
Cell Culture
Primary cultures of human airway epithelia were prepared from tracheal and bronchial tissues obtained from donated lungs not used for transplantation. Epithelia were isolated by enzymatic dispersion using well-established methods (30). Briefly, epithelial cells were dissociated and seeded onto collagen-coated, semipermeable membranes with a 0.4-µm pore size (Millicell-HA; surface area 0.6 cm2; Millipore, Bedford, MA). Human airway epithelial cultures were maintained in Ultroser G media at 37°C, 5% CO2. Millicell inserts were placed into 24-well plastic cell culture plates (Costar, Cambridge, MA). Twenty-four hours after seeding, the mucosal medium was removed, and the cells were allowed to grow at the air-liquid interface as reported previously (30). Only unpassaged, well-differentiated cultures (>2 wk old) were used in these studies. The presence of tight junctions was confirmed by transepithelial resistance measurements using a voltohmeter (World Precision Instruments, Sarasota, FL; resistance >500 ·cm2). For each experimental protocol, the interventions were performed on the indicated number of primary culture specimens from different donors. For each individual experiment, a matched, unstimulated sample from the same specimen served as the control. This study was approved by the Institutional Review Board of the University of Iowa.
Isolation of Macrophages from Bronchoalveolar Lavage Fluid
Bronchoalveolar lavage fluid (BALF) was obtained from normal volunteers as previously reported (37). BALF was filtered through two layers of gauze and spun down at 3,500 rpm at 4°C for 5 min. The cell pellets were resuspended in 10 ml of sterile saline and quantified using a Coulter counter.
Adenoviral Vector Constructs
A commercially available NF-B-Luc plasmid (Clontech Laboratories, Palo Alto, CA) was used as a template to generate a recombinant adenovirus vector (Ad-NF-
B-Luc). The fragment containing the firefly luciferase gene driven by four tandem copies of the NF-
B consensus sequence fused to a TATA-like promoter from herpes simplex virus thymidine kinase gene was released by KpnI and XbaI double digestion. The fragment was inserted into a promoterless adenoviral shuttle plasmid (pAd5mcspA), and Ad-NF-
B-Luc virus was generated by homologous recombination, as previously described, and stored in 10 mM Tris with 20% glycerol at 80°C (3). The particle titer of adenoviral stock was determined by A260 reading. The functional titer of the adenoviral stock was determined by plaque titering on HEK-293 cells and expression assays for the encoded protein.
An adenoviral vector expressing human MD-2 was generated. Total RNA was isolated from human alveolar macrophages using TRI reagent (Molecular Research Center, Cincinnati, OH). RNA was reverse transcribed to generate cDNA using SuperScript II reverse transcriptase (Life Technologies, Rockville, MD) following the manufacturer's protocol. PCR was employed to amplify a cDNA comprising the open reading frame of human MD-2 (GenBank AB018549). The primers consisted of forward: 5'-CTTGTCGACATTTGTAAAGCTTTGGAGATATTGAA-3' and reverse: 5'-ATTGAATTCTAATTTGAATTAGGTTGGTGTAGGA-3' (49).The reaction was performed at 95°C for 5 min, followed by 35 cycles at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min. The chain reaction was elongated at 72°C for 10 min. The fidelity of the PCR product was confirmed by DNA sequencing. The MD-2 cDNA was digested by SalI at the 5' end and EcoRI at the 3' end and then inserted into an adenoviral shuttle plasmid (pAd5cmcpA) containing the cytomegalovirus promoter, and Ad-MD-2 virus was generated by homologous recombination. The titering and storage of the Ad-MD-2 virus were identical to those described above for Ad-NF-B-Luc. An adenoviral vector expressing the human TLR-4 cDNA was also used in these studies. The methods for the construction of this vector were published previously (4).
Messenger RNA Analysis
Semiquantitative RT-PCR.
RT-PCR was used to detect expression of TLR-4, CD14, and MD-2 mRNA in human airway epithelia and alveolar macrophages. One microgram of total RNA from each sample was reverse transcribed using random hexamer primers with SuperScript (GIBCO). First-strand cDNA was amplified by PCR. The primer set for TLR-4 consisted of forward: 5'-TGAGCAGTCGTGCTGGTATC-3'; reverse: 5'-CAGGGCTTTTCTGAGTCGTC-3' and amplified a product of 166 bp. The primer set for CD14 consisted of forward: 5'-CTGCAACTTCTCCGAACCTC-3' and reverse: 5'-CCAGTAGCTGAGCAGGAACC-3' and produced a cDNA fragment of 215 bp. The primer set for MD-2 comprised forward: 5'-TGTAAAGCTTTGGAGATATTGAA-3' and reverse: 5'-TTTGAATTAGGTTGGTGTAGGA-3' and amplified a product of 508 bp. As a control for amplification, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified in each reaction using the following primers: forward: 5'-GTCAGTGGTGGACCTGACC-3'; reverse: 5'-AGGGGTCTACATGGCAACTG-3'. Each reaction contained 1.25 pM of the primers, 3 mM Mg2+. After an initial denaturing step (95°C for 3 min), 35 cycles of denaturing (94°C for 30 s), annealing (60°C for 30 s), and extension (72°C for 30 s), followed by 5 min at 72°C for elongation, were conducted. PCR products were electrophoresed on a 2% agarose gel and visualized using ethidium bromide.
Real-time quantitative PCR for detecting HBD-2 and MD-2. Real-time PCR was employed to detect HBD-2 and MD-2 and to quantify changes in expression. Real-time quantitative PCR was performed using a sequence detector (ABI PRISM 7700; Applied Biosystems, Foster City, CA) and Taqman technology (Roche Molecular Diagnostic Systems) following the manufacturer's protocols (9). The primers and probes were designed using the Primer Express program (Applied Biosystems). For MD-2, the primers consisted of forward: 5'-CAACAATATCATTCTCCTTCAAGGG-3', reverse: 5'-GCATTTCTTCTGGGCTCCC-3', and the probe 5'-AAAATTTTCTAAGGGAAAATACAAATGTGTTGTTGAAGC-3'. For HBD-2, forward: 5'-CCTGTTACCTGCCTTAAGAGTGGA-3', reverse: 5'-ACCACAGGTGCCAATTTGTTTA-3', and the probe was 5'-CCATATGTCATCCAGTCTTTTGCCCTAGAAGG-3'. Both probes contained a fluorescent reporter [6-carboxyfluorescein (FAM)] at the 5' end and a fluorescent quencher [6-carboxytetramethylrhodamine (TAMRA)] at the 3' end. As an internal control for normalization, human GAPDH real-time quantitative PCR was conducted in every reaction. The primers and probes were purchased from Roche Molecular Diagnostic Systems. The PCR fragments were amplified for 40 cycles (15 s at 95°C and 1 min at 60°C). The comparative threshold cycle method of quantitation was employed following methods described in the ABI PRISM 7700 manual. For each experimental probe set, a preliminary validation experiment was performed to demonstrate that the efficiencies of target (HBD-2 or MD-2) and reference (GAPDH) amplification were approximately equal (data not shown).
Transduction of Human Airway Epithelia With Adenoviral Expression Vectors
Primary cultures of well-differentiated human airway epithelia were prepared as described above. In vector transduction experiments, the cells were transduced either with Ad-NF-B-Luc alone or with both Ad-NF-
B-Luc and Ad-MD-2 2 days before performing experimental interventions. The vectors were formulated in 5 mM EGTA and applied to the apical surface [50 multiplicities of infection (MOI)] of the cells for 2 h in a 100-µl volume, as described previously, to access receptors (55). The vector solution was removed, and the cells were transferred to another multiwell culture dish with fresh culture medium. On the experimental day, LOS ± specific endotoxin-binding proteins (as indicated in the individual figure legends) were applied in a volume of 50 µl to the apical or basolateral side of the cells as noted. Control groups received PBS (negative control) or IL-1
(100 ng/ml, applied apically and basolaterally as positive control) (50). Eighteen to twenty-four hours later, the cells were disrupted in the 1x lysis buffer provided with the luciferase assay kit (Promega) to measure luciferase activity. In addition, some samples were prepared to isolate total RNA.
Human Embryonic Kidney Cell Activation Assay
Human embryonic kidney (HEK) cells ± TLR-4 were obtained from Dr. Jesse Chow (Eisai Research Institute, Andover, MA) and were cultured as described (18, 57). For cell activation assays, cells were grown to confluency in 48-well plates. Epithelia were washed with warm PBS 2x and incubated overnight at 37°C, 5% CO2, and 95% humidity in Hanks' balanced salt solution and 0.1% human serum albumin with the supplements indicated (see Fig. 3). Activation of HEK cells was assessed by measuring accumulation of extracellular IL-8 by ELISA as previously described (10).
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Recombinant MD-2 protein (rMD-2) was produced in baculovirus for application to airway epithelia. The human MD-2 cDNA was first subcloned into pGEM-T easy vector for transformation of Escherichia coli JM109 and amplification. The DNA was then isolated, linearized, and inserted into pBAC11 (using NcoI- and XhoI-sensitive restriction sites) for transfection into insect cells. A vector encoding a six-histidine ("poly-HIS") extension of the COOH terminus was used. The DNA encoding MD-2 was sequenced in both directions to confirm the fidelity of the product. Sf9 cells were used for production and multiplication of virus containing pBAC11 plasmids. To maximize recombinant protein production, High Five (Invitrogen) cells were inoculated with recombinant virus in serum-free medium, incubated for 2448 h, and culture medium was then collected for analysis. The presence of rMD-2-(HIS)6 was determined by SDS-PAGE and immunoblots of the culture medium using an anti-His4 MAb (Qiagen). The culture medium was dialyzed against sterile Hanks' balanced salt solution buffered with 10 mM HEPES, pH 7.4, and supplemented with 0.1% human serum albumin before use in bioassays.
Stimulation of Airway Epithelia With Proinflammatory Products
Several agents were applied to human airway epithelia to investigate the regulation of MD-2 expression. PMA, TNF-, and IFN-
were applied to the apical and basolateral surfaces at 100 ng/ml. Bacterial products, including the NTHi membrane protein P6 (5 µg/ml) and heat-killed NTHi (strain 12,
100 bacteria per epithelial cell, Ref. 14), were applied to the apical surface. After an 18- to 24-h stimulation, the cells were lysed and total RNA was extracted. Real-time PCR was conducted as described above.
Statistical Analysis
Unless otherwise noted, all numerical data are presented as means ± SE. Statistical analysis was performed using a two-tailed, unpaired Student's t-test using Microsoft Excel software.
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RESULTS |
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NTHi is a common commensal, and sometimes a pathogen, of the respiratory tract (6, 32, 51). We asked whether endotoxin (LOS) from NTHi increased HBD-2 expression after application to the apical surface of human airway epithelia. As shown in Fig. 1, A and B, after the apical application of LOS in the presence of sCD14 and LBP, there was little or no change in the HBD-2 mRNA abundance. In contrast, IL-1 stimulated a large increase in HBD-2 expression (>10,000-fold), consistent with our previous studies (50).
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MD-2 Expression in Human Airway Epithelia Enhances Endotoxin Responsiveness
Pattern recognition receptors from the family of TLRs play a central role in the recognition of bacterial products, including endotoxin from gram-negative bacteria (39). TLR-4 is a key receptor for recognition and signaling in response to endotoxin (40), and optimal responses require presentation of the bacterial product in the presence of LBP, CD14, and MD-2 (2, 18, 49). We screened human airway epithelia for the expression of TLR-4, CD14, and MD-2 mRNAs using RT-PCR. Alveolar macrophages served as a positive control. Figure 2 demonstrates that airway epithelia express the mRNAs for TLR-4 and CD14. However, although macrophages demonstrated an MD-2 signal, no significant MD-2 transcripts were detected in airway epithelia following 35 cycles of PCR.
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Transduction of the human airway epithelial cells with Ad-MD-2, but not with the control Ad-vector, markedly increased the cellular levels of MD-2 mRNA (data not shown). Importantly, the Ad-MD-2-complemented epithelia exhibited markedly enhanced endotoxin responsiveness following apical application of either purified LOS (17) + LBP and sCD14 (Fig. 4A) or purified LOS:sCD14 aggregates (19) (Fig. 4C).
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Extracellular Complementation With rMD-2 Protein Enhances Endotoxin Signaling in Human Airway Epithelia
Genetically manipulated cells, such as HEK/TLR-4+ cells, have been used to show that endogenous (co)expression of MD-2 or addition of secreted MD-2 to TLR-4+/MD-2 cells confers increased endotoxin responsiveness. The primary cultures of human airway epithelia provide a more natural setting to test the effect of exogenous addition of rMD-2 on cellular responsiveness to endotoxin (i.e., LOS:sCD14). As shown in Fig. 4D, addition of conditioned insect cell culture medium containing rMD-2 increased the response of the human airway epithelial cultures to LOS:sCD14 by 100-fold. Control-conditioned medium, by contrast, had no effect (data not shown).
The findings presented above indicate that MD-2 expression is the principal limiting factor for responsiveness of human airway epithelia to endotoxin. The need for MD-2 could reflect its role either in TLR-4 trafficking, posttranslational modifications and surface expression (42), and/or in endotoxin recognition and delivery to TLR-4 (18). To test the latter possibility more directly, we examined the responsiveness of airway epithelia to purified LOS:MD-2 complex. Figure 5 shows that the apical application of 2 ng/ml (400 pM) of LOS:MD-2 produced significant activation of resting epithelia, but not of cells induced by adenoviral transduction, to express MD-2 along with TLR-4. This contrasts with effects of LOS:sCD14 that potently activated cells transduced with Ad-MD-2, but not resting cells (Fig. 4C). Because cell activation by LOS:MD-2 requires expression by cells of TLR-4 without MD-2 (18), these findings confirm that the well-differentiated primary cultures of airway epithelia express and produce TLR-4 without MD-2.
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The demonstration that expression (or addition) of MD-2 could markedly increase the responsiveness of the airway epithelia to endotoxin prompted a search for more natural conditions in which endogenous MD-2 expression might be upregulated. We therefore examined the effects of specific host or bacterial products on cellular MD-2 levels. As shown in Fig. 6A, real-time PCR demonstrated marked increases in steady-state MD-2 mRNA levels after exposure of the apical surface of the cultured airway epithelium to heat-killed NTHi, the NTHi outer membrane protein P6, or the combination of TNF- and IFN-
. The levels of MD-2 mRNA attained under these conditions were similar to that induced by PMA but were still significantly less than MD-2 mRNA levels in human alveolar macrophages. These findings demonstrate that, in response to specific stimuli, levels of MD-2 transcript can be upregulated in human airway epithelia.
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DISCUSSION |
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Hyporesponsiveness to endotoxin is a common characteristic of epithelial cells lining mucosal surfaces that are repeatedly exposed to gram-negative bacteria or cell-free (sterile) forms of endotoxin. The molecular basis of endotoxin hyporesponsiveness is generally unknown. Because sensitive responses to endotoxin generally depend on TLR-4-dependent signaling (49), hyporesponsiveness likely represents the functional deficiency of one or more elements of pathway(s) leading to and resulting from TLR-4 activation. TLR-4 is a membrane protein containing repeats of a leucine-rich motif in the extracellular portion of the protein and a cytoplasmic domain homologous to the intracellular domain of the human IL-1 receptor (40). The IL-1 responsiveness of airway epithelia indicates that the overlapping intracellular signaling pathways for activated TLR and IL-1 receptors (8, 25, 28) are present and functionally intact in human airway epithelia, including those important in NF-B-regulated HBD-2 expression. Even though we and others (4, 7) have demonstrated that airway epithelia express TLR-4, the cells responded poorly to LOS:sCD14. Therefore, it is likely that there is a defect in TLR-4-dependent recognition and/or response to endotoxin.
MD-2, also termed lymphocyte antigen 96, was first identified by Shimazu and colleagues (49, also see Ref. 57) as a molecule that conferred TLR-4-dependent responses to minute amounts of endotoxin. Cells that are TLR-4+/MD-2 are virtually unresponsive to endotoxin (42, 49). In the current study, we observed that human airway epithelia exhibit endotoxin responses consistent with a TLR-4+/MD-2 phenotype. The complementation of this deficiency of MD-2 via endogenous expression or extracellular addition was sufficient to convert human airway epithelia from endotoxin hyporesponsive to highly endotoxin responsive without changing basal NF-B-regulated HBD-2 expression of these cells or those stimulated by treatment with an unrelated agonist (e.g., IL-1
). These data support the notion that the hyporesponsiveness of resting airway epithelia is due specifically to the absence of MD-2 expression. Our findings confirm and extend earlier observations of Becker and coworkers (7), who detected expression of TLRs 16 and CD14 in differentiated human airway epithelia. Although they reported that P. aeruginosa LPS activated CD14-dependent NF-
B signaling, the increase in HBD-2 mRNA they observed in response to P. aeruginosa LPS was no greater (
3-fold) than what we found with added LOS in the absence of MD-2 complementation; i.e., very modest when contrasted to the induction of HBD-2 expression caused by IL-1
or by endotoxin in the presence of MD-2. In contrast to our findings in primary cultures, Guillot and colleagues (21) recently reported that human alveolar and airway epithelial cell lines express detectable levels of MD-2 as well as TLR-4 mRNA by RT-PCR. However, even in those cell lines, levels of MD-2 (protein) may be limiting, since little or no TLR-4 is detected on the cell surface. Our prejudice is that the well-differentiated primary cultures of human airway epithelia better mimic the phenotype of these cells in vivo and therefore provide an important model for studies of the regulation of MD-2 expression in future studies. Additional methods of investigating MD-2 expression, such as in situ hybridization, will be required in future studies using more complex cell culture and animal models to test this prediction more rigorously.
Similar to the findings in airway epithelia, cell lines derived from intestinal epithelia respond robustly to IL-1 stimulation but not to purified, protein-free LPS with NF-
B signaling (2). However, published data on intestinal epithelia indicate both diminished TLR-4 expression as well as absent MD-2 (2). Accordingly, complementation of both TLR-4 and MD-2 in intestinal epithelia is needed for significant activation of NF-
B and IL-8 reporter genes in response to endotoxin. These data suggest that airway and intestinal epithelia, both derivatives of the endodermal foregut, have evolved pattern recognition receptor profiles that minimize stimulation by endotoxin that is frequently encountered at these mucosal surfaces. Whether or not the apparent differences in the molecular basis of endotoxin hyporesponsiveness observed in these studies vs. our findings reflect differences between airway and intestinal epithelial cells or differences between well-differentiated primary cultures and cell lines requires further study. The ability of the airway to mobilize protective TLR-4-dependent responses without the need for MD-2 in response to specific products of gram-positive bacterial airway pathogens (e.g., pneumococci) (34) is consistent with the apparently greater retention of TLR-4 expression by airway vs. intestinal epithelia.
How might expression of MD-2 in the airway increase epithelial responsiveness to endotoxin? On the basis of current understanding of MD-2 actions, mobilization of MD-2 in the airway could have at least two roles: 1) combining, when endogenously expressed, with endogenously expressed TLR-4 to increase surface expression of TLR-4 (MD-2) (43), and 2) participating directly in endotoxin recognition by extracting endotoxin from monomeric endotoxin:CD14 complexes either before or after interaction of MD-2 with TLR-4 (18) (Fig. 7). TLR-4 alone has no apparent ability to engage the endotoxin:(s)CD14 complex (18). The ability of soluble, extracellular MD-2 plus endotoxin:sCD14 (Fig. 4D) and preformed endotoxin:MD-2 complex (Fig. 5) to activate resting human airway epithelia implies that sufficient TLR-4 is produced by these cells to respond sensitively to endotoxin when endotoxin is presented as a complex with MD-2. No mechanism for TLR-independent interactions of endotoxin:MD-2 complex has been described (18). Therefore, we believe that the ability of picomolar amounts of LOS:MD-2 to activate resting human airway epithelia suggests that there is sufficient apical surface expression of TLR-4 (even without endogenous MD-2) to interact directly with extracellular endotoxin:MD-2 and produce potent cell activation. This could be biologically important because it would permit TLR-4 from one cell to engage MD-2 or endotoxin:MD-2 derived from MD-2 secreted by a neighboring cell. This potency of endotoxin:MD-2-TLR-4 interactions may explain how small amounts of surface TLR-4 may be sufficient for robust cell activation yet insufficient for recognition by fluorescent or neutralizing antibodies (21).
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One advantage of such a hierarchy of responses is that it may help to minimize the frequency of epithelial-induced inflammatory signals from endotoxin. Ambient air contains bacteria and endotoxin (41), and the aerosolized concentrations of endotoxin can increase dramatically in some agricultural and industrial environments from 10 to >1,000 endotoxin units/m3 (12). Under normal conditions, the low expression of MD-2 in epithelia may serve to dampen endotoxin responsiveness to common environmental exposures and thereby avoid unwanted states of chronic inflammation in the face of frequent encounters with environmental endotoxin and other bacterial cell wall components. Conversely, upregulation of MD-2 expression may be important to enhance host defense responses to invading gram-negative bacteria. However, in certain disease states, such as cystic fibrosis or asthma, enhanced expression of MD-2 could lead to exaggerated endotoxin responsiveness with pathological consequences. Future studies will be designed to better define the cells in the airway responsible for MD-2 production and the conditions that regulate its expression.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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