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Toll-like Receptor 4 Relates to Lipopolysaccharide-induced Mucus Hypersecretion in Rat Airway
Archives of Medical Research 40 (2009) 10e17
ORIGINAL ARTICLE
Toll-like Receptor 4 Relates to Lipopolysaccharide-induced Mucus Hypersecretion in Rat Airway Lei Chen,a Tao Wang,a Jian-Yong Zhang,a Shang-Fu Zhang,b Dai-Shun Liu,a Dan Xu,a Xun Wang,a Ya-Juan Chen,a and Fu-Qiang Wena b
a Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, and Department of Respiratory Medicine, Department of Pathology, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan, China
Received for publication June 13, 2008; accepted September 22, 2008 (ARCMED-D-08-00254).
Background. Toll-like receptor 4 (TLR4) is a transmembrane protein that participates in the recognition of lipopolysaccharide (LPS), a potentially important source of inflammation. To investigate the role of TLR4 in LPS-induced airway mucus hypersecretion (AMH), we used a LPS-induced rat model treated with dexamethasone (DEX). Methods. Rats were randomly divided into four experimental groups: 1) saline (SA)treated with distilled water (DW) (control group); 2) LPS-treated with DW (LPS group); 3) LPS-treated with DEX (LPS plus DEX group); 4) SA-treated with DEX (DEX group). DEX (5 mg/kg) was intraperitoneally injected 1 h before being administered intratracheally with LPS. Expressions of TLR4 and MUC5AC were evaluated with RT-PCR, in situ hybridization, immunohistochemistry and Alcian blue/Periodic acid-schiff (AB/PAS) staining. Results. Increased expressions of TLR4 protein and mRNA were found in rat airway treated with LPS and peaked on day 2 after LPS administration. Following this, LPS increased MUC5AC expression and AB/PAS-stained goblet cells in rat airway. Correlation analysis showed TLR4 correlated well with the expression of MUC5AC (r 5 0.684, p !0.01) and AB/PAS-stained area (r 5 0.781, p !0.01). In addition, DEX pretreatment significantly reduced LPS-induced overexpression of TLR4 ( p !0.05) in rat airway. Conclusions. These results suggest TLR4 relates to LPS-induced AMH and support a role of TLR4 in DEX inhibition of LPS-induced AMH. Ó 2009 IMSS. Published by Elsevier Inc. Key Words: Airway mucus hypersecretion, Dexamethasone, Lipopolysaccharide, Toll-like receptor 4.
Introduction Airway mucus hypersecretion (AMH) is a characteristically pathological change of airway disease. Normally, mucus secretion is an inherent part of airway defense. However, mucus hypersecretion may contribute to the pathophysiology of asthma, chronic bronchitis, and cystic fibrosis (1). In the respiratory tract, mucus consists of water, biomolecules, ions and mucins, including MUC5AC, MUC5B and, on occasion, MUC2. Among these mucins, MUC5AC is considered to be the most important mucin in the pathogenesis of mucus hypersecretion (2). Address reprint requests to: Fu-Qiang Wen, M.D., Ph.D., Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan 610041, P. R. China; E-mail: [email protected]
Lipopolysaccharide (LPS), a major component of the outer membrane of gram-negative bacteria (3), plays an important role in airway inflammation and associated mucus hypersecretion. Consequently, progressive and irreversible limitation of airflow and further impairment of lung function lead to increasing morbidity and mortality. To investigate the mechanisms of AMH is helpful to elucidate the role of mucus hypersecretion in the pathogenesis of airway disease and for clinical therapy. The toll gene and its encoded protein have been documented to be correlated with pathogen recognition (4). Toll-like receptors (TLRs), a family of pattern-recognition receptors, belong to type I transmembrane protein. TLR proteins contain repeated sequences composed of 1831 amino acids with abundant leucines in the ectodomain and also contain |200 amino acids, having high homology
0188-4409/09 $esee front matter. Copyright Ó 2009 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2008.10.005
TLR4 and Airway Mucus Hypersecretion
with interleukin (IL)-1R in the intracellular region. TLRs are thought to be the key accessory receptor of the signal transduction across membranes and through the intracellular IL-1R homogeneous structure, TLR signal transduction evokes the interaction among IL-1R downstream signaling molecules (5). Based on recent studies, TLR4 signaling pathway is probably the essential mode of LPS signal transduction and also the key of pathogenesis of LPS-induced injury (6). However, the role of TLR4 in LPS-stimulated AMH so far remains unclear. Glucocorticoids (GCs) suppress LPS-stimulated AMH (7), which is thought to be involved in their anti-inflammatory properties (8). In the current study, we used a LPSinduced rat model to explore whether TLR4 associates with LPS-induced AMH and whether TLR4 plays a role in dexamethasone (DEX) inhibition of LPS-induced AMH. Materials and Methods Reagents and Animals Male Sprague Dawley rats (200e250 g) were purchased from the Experimental Animal Center of Sichuan University. LPS: E. coli serotype 055:B5 and DEX were purchased from Sigma (St. Louis, MO). Treatment of Animals Animals were specific pathogen free and were kept on a 12 h light/12 h dark cycle at a room temperature of 22 2 C with free access to food and water. Experimental procedures were conducted under aseptic conditions. The protocol was approved by the hospital Animal Care and Use Committee. Forty eight Sprague Dawley rats were divided into four experimental groups, with 12 rats per group as follows: 1) saline (SA)-treated with distilled water (DW) (control group); 2) LPS-treated with DW (LPS group); 3) LPS-treated with DEX (LPS plus DEX group); 4) SAtreated with DEX (DEX group). Rats were anesthetized intraperitoneally with penthiobarbital sodium (50 mg/kg), and LPS (200 mg/rat) in 100 mL of saline was administered by intratracheal instillation with the same volume of intratracheal saline in control animals. DEX (5 mg/kg) treatment was performed by IP injection 1 h before intratracheal instillation of LPS. Rats were sacrificed on days 2, 4 and 7 after LPS or saline administration. Histopathology Lungs of rats in each group were removed and stored at 80 C and frozen immediately. Middle lobes of right lungs were inflated with 1e1.5 mL of 10% neutral formalin and then immersed in this fixative solution and embedded in paraffin. All procedures were performed at standard atmospheric pressure. Four-mm sections were obtained for staining with Alcian blue/Periodic acid-schiff (AB/PAS). To quantitate the positive area of AB/PAS staining, IM-
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AGE-Pro plus 4.5 software (Media Cybernetics Inc., Bethesda, MD) was used to evaluate AB/PAS-stained area in term of the means previously mentioned (9,10). Briefly, AB/PASstained area in rat airway and total bronchial epithelial area were measured, and the value for statistics was expressed as percentage of the area stained by AB/PAS. Image analysis was performed strictly following the blind principle. Immunohistochemistry (IHC) Primary antibodies used for IHC studies included rabbit polyclonal anti-rat TLR4 antibody (Boster, Wuhan, China) and mouse monoclonal anti-rat MUC5AC antibody (Santa Cruz Biotechnology, Santa Cruz, CA). IHC was performed with the conventional avidin-biotin-peroxidase histochemical technique using SP kit (Vector Laboratories Ltd., Burlingame, CA). Briefly, toluene was used to remove the paraffin from sequestered 4-mm-thick paraffin sections, and the sections were rinsed thoroughly with ethanol. Sections were then soaked in 0.3% H2O2 with absolute methanol for 20 min at room temperature to inactivate endogenous peroxidase activity. They were incubated with blocking serum for 30 min and then covered with primary antibodies and incubated for 1 h. After washing in phosphate-buffered saline, sections were processed further using kits according to the instructions provided by the manufacturer and then developed with 3,30 -diaminobenzidine and H2O2, followed by Mayer’s hematoxylin staining method. Integral optical density (IOD) was used to quantitate the immunostained area using IMAGE-Pro plus 4.5 software. In brief, IOD of TLR4- and MUC5AC-stained area in per-unit area of bronchial epithelial was assessed as the value for statistics, according to the methods described above (9,10). RT-PCR For RNA isolation, the whole left lungs were frozen in liquid nitrogen, stored at 80 C and frozen immediately after removal from rats in each group. Total RNA was extracted from frozen lung tissue using Trizol reagent (Gibco-BRL, Gaithersburg, MD) and amplified using Progema PCR single-step kit (Progema, Madison, WI), according to the manufacturer’s instructions. RT-PCR analysis was used (PTC-200 DNA Engine, PCR Cycler, MJ Research, Watertown, MA). Each sample was run in duplicate. The primers, which were designed based on published sequence of these genes and synthesized by Shenneng Bio-tech Co. (Shanghai, China) as follows: MUC5AC, forward (50 -GCTCAT CCTAAG CGAC G TCT-30 ); reverse (50 -GGGGGCATAACTTCTCTTGG-30 ), TLR4, forward (50 -CATCAGTGT ATCGG TG G T CAGT-30 ); reverse (50 -CGAGGTAGGTGTTTCTGCTAAG-30 ), b-actin, forward (50 -CCTCA TGA A GAT C CTGACCG-30 ); reverse (50 -ACCGCTCATTGCCGATAGTG-30 ). b-actin served as the constitutive control. The products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.
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Chen et al./ Archives of Medical Research 40 (2009) 10e17
TLR4 and Airway Mucus Hypersecretion
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Bands were digitized by a Bio-Rad Universal Hood system (Bio-Rad, Hercules, CA). Means of the ratio of MUC5AC, TLR4 band photodensity to b-actin band photodensity in various groups were presented.
( p !0.01), which peaked on day 2, then started to decrease on day 4 and reduced to the control level on day 7 (Figure 3a).
In Situ Hybridization (ISH)
Using semiquantitative RT-PCR, we found that LPS stimulated mRNA expression of TLR4 significantly ( p !0.01), which also peaked on day 2 and decreased to the control level on day 7 (Figure 3b).
Lower lobes of right lungs were prepared for ISH. After removal from rats, lower lobes of right lungs were routinely fixed in 10% buffered formalin, dehydrated in 75% alcohol, and embedded in paraffin. Sections |6-mm thick were then ready for hybridization. All reagents used in this course described above were deactivated of RNase with 0.1% DEPC. The probe of TLR4 mRNA was labeled with digoxigenin as follows: 50 (DIG)-TTgTgCCCTgTgAggTTgAggTTAg-30 . ISH was performed according to the instructions of the ISH kit (Boster).
RT-PCR
ISH ISH (Figure 4) showed positive-stained area of TLR4 in airway epithelial cells and inflammatory cells. Increased positive-stained area of TLR4 was found in LPS group ( p !0.01) and peaked on day 2, which was in accordance with the findings in RT-PCR and IHC studies.
Statistical Analysis
Correlation Analysis
All values were expressed as means SD. One-way analysis of variance followed by Student-Newman-Keuls test was used to compare differences among multiple groups. Correlations of TLR4 expression (IOD) in rat aiway with MUC5AC expression (IOD) and AB/PAS-stained area (%) were performed with Pearson correlation analysis. Significance was defined by a p value of 0.05 (two-tailed). SPSS v.13.0 software package (SPSS Inc., Chicago, IL) was used for statistical analysis.
Correlation analysis (Figure 5) showed TLR4 expression significantly correlated well with AB/PAS-stained area (r 5 0.781, p !0.01) and the expression of MUC5AC (r 5 0.684, p !0.01) in rat airway.
Results Changes of MUC5AC Expression and AB/PAS-Stained Area after LPS Stimulation LPS stimulation significantly increased MUC5ac expression in rat airway in IHC (Figure 1a) and RT-PCR (Figure 1b) studies, which peaked on day 4 after LPS treatment ( p !0.01). In addition, similar changes were observed in the evaluation of AB/PAS-stained area (Figure 2). TLR4 Overexpression Correlates Well with LPS-induced AMH IHC Immunoreactive TLR4 expression was detected by IHC in airway epithelial cells and inflammatory cells. Weak expression of TLR4 was found in control and DEX groups. An increased immunostaining of TLR4 was found in LPS group
Effects of DEX on TLR4 Expression and Mucus Hypersecretion in Rat Airway DEX treatment significantly suppressed the peaked expression of TLR4 on day 2 after LPS stimulation in IHC and RT-PCR studies (p !0.01; Figure 3). Compared with the corresponding LPS groups, DEX subsequently reduced AB/PAS-stained area (p !0.01; Figure 2) and expression of MUC5AC protein and mRNA (p !0.01; Figure 1), which peaked on day 4 after LPS instillation. Discussion In the present study, LPS administration markedly increased the expression of TLR4 and subsequently induced overexpression of MUC5AC. Correlation analysis showed TLR4 positively correlated well with AMH. DEX significantly attenuated LPS-induced overexpression of TLR4 and MUC5AC. These findings demonstrate that TLR4 definitely relates to LPS-induced AMH and may play a role in inhibition of LPS-induced AMH by DEX. Bacterial infection, especially gram-negative bacterial infection, plays an important role in the pathogenesis of airway
= Figure 1a. MUC5AC protein expression in rat airway. MUC5AC protein was measured by routine IHC analysis and quantified by IOD measurement (K). MUC5AC positive-stained area was presented with the color of brown in airway epithelium. (A) and (B) were negative control sections from rat spleen and blank for MUC5AC, respectively. Weak positive staining was presented in representative bronchial sections from control (C) and DEX (D) groups. MUC5AC positive-stained area was significantly shown in photomicrographs from LPS group on days 2 (E), 4 (G) and 7 (I) after LPS stimulation and peaked on day 4. In contrast, DEX significantly decreased MUC5AC positive-stained area on days 2 (F), 4 (H) and 7 (J) after LPS treatment, compared with those in the corresponding LPS groups (*p !0.05,**p !0.01 vs. control group; #p !0.05 , ##p !0.01 vs. LPS group). Bar 5 100 mm. Figure 1b. MUC5AC mRNA expression in lung tissue. MUC5AC mRNA levels in the lung tissue samples harvested in every experimental group at each day point are shown in (A) and (B). The relative amounts of MUC5AC mRNA in LPS groups peaked on day 4 after LPS treatment, and this was in accordance with the changes of MUC5AC protein expression in airway epithelium. DEX effectively suppressed LPS-induced overexpression of MUC5AC in transcription level (*p !0.05, **p !0.01 vs. control group; #p !0.05, ##p !0.01 vs. LPS group). Bar 5 100 mm. Color version of the figure available online at www.arcmedres.com.
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Chen et al./ Archives of Medical Research 40 (2009) 10e17
Figure 2. AB/PAS-stained area in rat airway. Shown are representative photomicrographs of AB/PAS stained sections of lung tissue. Positive area of AB/PAS staining (I) was expressed as % stained area of airway epithelium occupied by AB/PAS-stained goblet cells. No obvious AB/PAS positive staining was presented in control (A) and DEX (B) groups. Notable positive-stained goblet cells (blue dots in airway epithelium) were presented in representative bronchial sections from LPS group on days 2 (C), 4 (E) and 7 (G) after LPS treatment and peaked on day 4. Positive-stained goblet cells significantly decreased on days 2 (D), 4 (F) and 7 (H) in LPSþDEX groups, compared with that in the corresponding LPS groups (*p !0.05, **p !0.01 vs. control group; #p !0.05, ##p !0.01 vs. LPS group). Bar 5 100 mm. Color version of the figure available online at www.arcmedres.com.
inflammation. LPS, a major component of gram-negative bacteria, serves as a foremost trigger in this process (10,11). AMH is defined as excessive secretion of mucus with enhancement of viscidity and acidity in airway disorders. Previous studies have reported that several cellular signaling pathways correlate well with mucus secretion as follows: 1) protein kinase C/G (PKC/G); myristoylated alanine-rich C kinase substrate (MARCKS); 2) epidermal growth factor (EGF) signaling pathway; 3) p38 mitogen-activated protein kinase (MAPK) pathway; 4) transforming growth factor (TGF)-Smads signaling pathway; 5) calcium-activated chloride channels (CLCA) (12e17). However, the molecular mechanism of AMH induced by LPS is not fully understood. TLR4 participates in the recognition of LPS through its interaction with CD14 and the accessory molecule, MD-2 (6). Several investigators have confirmed the effects of TLR4 in
LPS-induced inflammation (18,19). Nevertheless, LPS can activate many target intracellular signaling pathways, including not only TLRs and its downstream IL-1R signaling pathway but also G-protein, protein kinase A/C (PKA/C), MAPK pathway, and calcium signalling system (20e23). The novel finding in our study is the expression of TLR4 in rat airway peaks on day 2 after LPS treatment, earlier than MUC5AC expression and AB/PAS-stained area, which peak on day 4 after LPS stimulation. Our data support a definite correlation between TLR4 and LPS-induced AMH and also suggest a potential role of TLR4 in this process, whereas its signal transductions still need further investigations. DEX is a glucocorticosteroid that has a strong immunosuppressive action and anti-inflammatory effect. The mechanisms of DEX suppressing LPS-induced AMH are intricate, probably involved in many links of LPS signal
TLR4 and Airway Mucus Hypersecretion
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Figure 3a. (A) TLR4 protein expression in rat airway. TLR4 protein expression was assessed by routine IHC analysis and quantified by IOD measurement (K). TLR4 positive-stained area was presented with the color of brown in airway epithelium. Representative photomicrographs from each experimental group were shown. (A) and (B) were positive control sections from rat spleen and blank for TLR4, respectively. No obvious TLR4 positive staining was presented in representative bronchial sections from control (C) and DEX (D) groups. TLR4 positive-stained area was obviously presented on days 2 (E) and 4 (G) after LPS treatment, and time-dependently reduced to the control level on day 7 (I). DEX significantly decreased TLR4 positive-stained area on days 2 (F) and 4 (H) after LPS treatment, compared with those in corresponding LPS groups. No significant difference was presented between sections from LPS (I) and LPSþDEX (J) groups on day 7 (**p !0.01 vs. control group; #p !0.05, ##p !0.01 vs. LPS group). Bar 5 100 mm. Figure 3b. TLR4 mRNA expression in lung tissue. TLR4 mRNA levels in the lung tissue samples harvested in every experimental group at each day point were shown in (A) and (B). The relative amounts of TLR4 mRNA expression peaked on day 2 after LPS stimulation, which was in accordance with the changes of TLR4 protein expression in airway epithelium. DEX effectively suppressed LPS-induced overexpression of TLR4 in transcription level (**p !0.01 vs. control group; #p !0.05, ##p !0.01 vs. LPS group). Bar 5 100 mm. Color version of the figure available online at www.arcmedres.com.
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Figure 4. TLR4 mRNA expression in situ. TLR4 mRNA was assessed by in situ hybridization and quantified by IOD measurement (I). TLR4 positive-stained area was presented with the color of fawn in airway epithelium. Representative photomicrographs from each experimental group are shown. (A) and (B) were positive control section from rat spleen and blank for TLR4, respectively. No obvious TLR4 positive staining was presented in representative bronchial sections from control (C) and DEX (D) groups. TLR4-positive stained area was obviously presented in the photomicrograph from LPS group on day 2 (E) after LPS treatment and significantly decreased on day 4 (G). DEX markedly decreased TLR4-positive stained area on days 2 (F) and 4 (H) after LPS treatment, compared with that in the corresponding LPS groups (*p !0.05, **p !0.01 vs. control group; #p !0.05, ##p !0.01 vs. LPS group). Bar 5 100 mm. Color version of the figure available online at www.arcmedres.com.
Figure 5. Correlation analysis. The two scatter graphs showed correlations between TLR4 expression (IOD) and AB/PAS-stained area (%) (A), and MUC5AC expression (IOD) (B) in rat airways after LPS treatment, respectively.
TLR4 and Airway Mucus Hypersecretion
transduction. Although DEX can directly inhibit MUC5AC production by goblet cell and mucin gene expression (24,25), in general, DEX exerts its effect indirectly by suppressing activation of inflammatory cells, production and release of cytokines and other inflammatory mediators and even attenuating expression of inflammatory factor genes (26,27). As TLR4 plays a key role in pathogenesis of LPS, our results suggest DEX probably suppresses LPS-induced AMH through inhibition of TLR4 expression in CD14-positive inflammatory cells and TLR4associated inflammatory mediators, which stimulates mucus hypersecretion. Taken together, the current findings demonstrate that TLR4 is associated with LPS-stimulated AMH and probably plays a key role in DEX inhibition of LPS-induced AMH, which contributes to furtherunderstanding of GCs suppression of AMH stimulated by LPS. Further studies will be required to investigate whether TLR4 serves as a mediator in the process of LPS-induced AMH and the signaling pathway in which DEX inhibits TLR4 expression. Acknowledgments This study was supported by grants #30425007, 30370627, 30670921 from the National Natural Science Foundation of China (Beijing, China) and #00-722 and #06-834 from China Medical Board of New York (Cambridge, MA) to F.Q.W.
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11. Snelgrove R, Williams A, Thorpe C, Hussel T. Manipulation of immunity to and pathology of respiratory infections. Expert Rev Anti Infect Ther 2004;2:413e426. 12. Chen Y, Zhao YH, Wu R. Differential regulation of airway mucin gene expression and mucin secretion by extracellular nucleotide triphosphates. Am J Respir Cell Mol Biol 2001;25:409e417. 13. Singer M, Martin LD, Vargaftig BB, Park J, Gruber AD, Li Y, et al. A MARCKS-related peptide blocks mucus hypersecretion in a mouse model of asthma. Nat Med 2004;10:193e196. 14. Takeyama K, Dabbagh K, Lee HM, Agustı´ C, Lausier JA, Ueki IF, et al. Epidermal growth factor system regulates mucin production in airways. Proc Natl Acad Sci USA 1999;96:3081e3086. 15. Wang B, Lim DJ, Han J, Kim YS, Basbaum CB, Li JD. Novel cytoplasmic proteins of nontypeable Haemophilus influenzae upregulate human MUC5AC mucin transcription via a positive p38 mitogenactivated protein kinase pathway and a negative phosphoinositide 3kinase-Akt pathway. J Biol Chem 2002;277:949e957. 16. Jono H, Xu H, Kai H, Lim DJ, Kim YS, Feng XH, et al. Transforming growth factor-beta-Smad signaling pathway negatively regulates nontypeable Haemophilus influenzae-induced MUC5AC mucin transcription via mitogen-activated protein kinase (MAPK) phosphatase1-dependent inhibition of p38 MAPK. J Biol Chem 2003;278: 27811e27219. 17. Toda M, Tulic MK, Levitt RC, Hamid Q. A calcium-activated chloride channel (HCLCA1) is strongly related to IL-9 expression and mucus production in bronchial epithelium of patients with asthma. J Allergy Clin Immuol 2002;109:246e250. 18. Togbe D, Schnyder-Candrian S, Schnyder B, Couillin I, Maillet I, Bihl F, et al. TLR4 gene dosage contributes to endotoxininduced acute respiratory inflammation. J Leukocyte Biol 2006;80: 451e457. 19. Xu Z, Huang CX, Li Y, Wang PZ, Ren GL, Chen CS, et al. Toll-like receptor 4 siRNA attenuates LPS-induced secretion of inflammatory cytokines and chemokines by macrophages. J Infect 2007;55:e1ee9. 20. Ishii I, Fukushima N, Ye X, Chun J. Lysophospholipid receptors: signaling and biology. Annu Rev Biochem 2004;73:321e354. 21. Jefferies CA, O’Neill LA. Bruton’s tyrosine kinase (Btk)—the critical tyrosine kinase in LPS signalling? Immunol Lett 2004;92:15e22. 22. Diks SH, Van Deventer SJ, Peppelenbosch MP. Lipopolysaccharide recognition, internalisation, signalling and other cellular effects. J Endotoxin Res 2001;7:335e348. 23. Diks SH, Richel DJ, Peppelenbosch MP. LPS signal transduction: the picture is becoming more complex. Curr Top Med Chem 2004;4: 1115e1126. 24. Lu W, Lillehoj EP, Kim KC. Effects of dexamethasone on Muc5ac mucin production by primary airway goblet cells. Am J Physiol Lung Cell Mol Physiol 2005;288:L52eL60. 25. Ishinaga H, Takeuchi K, Kishioka C, Yagawa M, Majima Y. Effects of dexamethasone on mucin gene expression in cultured human nasal epithelial cells. Laryngoscope 2002;112(8 Pt 1):1436e1440. 26. Kiku Y, Matsuzawa H, Ohtsuka H, Terasaki N, Fukuda S, Kon-Nai S, et al. Effects of chlorpromazine, pentoxifylline and dexamethasone on mRNA expression of lipopolysaccharide-induced inflammatory cytokines in bovine peripheral blood mononuclear cells. J Vet Med Sci 2002;64:723e726. 27. Ma W, Gee K, Lim W, Chambers K, Angel JB, Kozlowski M, et al. Dexamethasone inhibits IL-12p40 production in lipopolysaccharidestimulated human monocytic cells by down-regulating the activity of c-Jun N-terminal kinase, the activation protein-1, and NF-kB transcription factors. J Immunol 2004;172:318e330.
ORIGINAL ARTICLE
Toll-like Receptor 4 Relates to Lipopolysaccharide-induced Mucus Hypersecretion in Rat Airway Lei Chen,a Tao Wang,a Jian-Yong Zhang,a Shang-Fu Zhang,b Dai-Shun Liu,a Dan Xu,a Xun Wang,a Ya-Juan Chen,a and Fu-Qiang Wena b
a Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, and Department of Respiratory Medicine, Department of Pathology, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan, China
Received for publication June 13, 2008; accepted September 22, 2008 (ARCMED-D-08-00254).
Background. Toll-like receptor 4 (TLR4) is a transmembrane protein that participates in the recognition of lipopolysaccharide (LPS), a potentially important source of inflammation. To investigate the role of TLR4 in LPS-induced airway mucus hypersecretion (AMH), we used a LPS-induced rat model treated with dexamethasone (DEX). Methods. Rats were randomly divided into four experimental groups: 1) saline (SA)treated with distilled water (DW) (control group); 2) LPS-treated with DW (LPS group); 3) LPS-treated with DEX (LPS plus DEX group); 4) SA-treated with DEX (DEX group). DEX (5 mg/kg) was intraperitoneally injected 1 h before being administered intratracheally with LPS. Expressions of TLR4 and MUC5AC were evaluated with RT-PCR, in situ hybridization, immunohistochemistry and Alcian blue/Periodic acid-schiff (AB/PAS) staining. Results. Increased expressions of TLR4 protein and mRNA were found in rat airway treated with LPS and peaked on day 2 after LPS administration. Following this, LPS increased MUC5AC expression and AB/PAS-stained goblet cells in rat airway. Correlation analysis showed TLR4 correlated well with the expression of MUC5AC (r 5 0.684, p !0.01) and AB/PAS-stained area (r 5 0.781, p !0.01). In addition, DEX pretreatment significantly reduced LPS-induced overexpression of TLR4 ( p !0.05) in rat airway. Conclusions. These results suggest TLR4 relates to LPS-induced AMH and support a role of TLR4 in DEX inhibition of LPS-induced AMH. Ó 2009 IMSS. Published by Elsevier Inc. Key Words: Airway mucus hypersecretion, Dexamethasone, Lipopolysaccharide, Toll-like receptor 4.
Introduction Airway mucus hypersecretion (AMH) is a characteristically pathological change of airway disease. Normally, mucus secretion is an inherent part of airway defense. However, mucus hypersecretion may contribute to the pathophysiology of asthma, chronic bronchitis, and cystic fibrosis (1). In the respiratory tract, mucus consists of water, biomolecules, ions and mucins, including MUC5AC, MUC5B and, on occasion, MUC2. Among these mucins, MUC5AC is considered to be the most important mucin in the pathogenesis of mucus hypersecretion (2). Address reprint requests to: Fu-Qiang Wen, M.D., Ph.D., Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan 610041, P. R. China; E-mail: [email protected]
Lipopolysaccharide (LPS), a major component of the outer membrane of gram-negative bacteria (3), plays an important role in airway inflammation and associated mucus hypersecretion. Consequently, progressive and irreversible limitation of airflow and further impairment of lung function lead to increasing morbidity and mortality. To investigate the mechanisms of AMH is helpful to elucidate the role of mucus hypersecretion in the pathogenesis of airway disease and for clinical therapy. The toll gene and its encoded protein have been documented to be correlated with pathogen recognition (4). Toll-like receptors (TLRs), a family of pattern-recognition receptors, belong to type I transmembrane protein. TLR proteins contain repeated sequences composed of 1831 amino acids with abundant leucines in the ectodomain and also contain |200 amino acids, having high homology
0188-4409/09 $esee front matter. Copyright Ó 2009 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2008.10.005
TLR4 and Airway Mucus Hypersecretion
with interleukin (IL)-1R in the intracellular region. TLRs are thought to be the key accessory receptor of the signal transduction across membranes and through the intracellular IL-1R homogeneous structure, TLR signal transduction evokes the interaction among IL-1R downstream signaling molecules (5). Based on recent studies, TLR4 signaling pathway is probably the essential mode of LPS signal transduction and also the key of pathogenesis of LPS-induced injury (6). However, the role of TLR4 in LPS-stimulated AMH so far remains unclear. Glucocorticoids (GCs) suppress LPS-stimulated AMH (7), which is thought to be involved in their anti-inflammatory properties (8). In the current study, we used a LPSinduced rat model to explore whether TLR4 associates with LPS-induced AMH and whether TLR4 plays a role in dexamethasone (DEX) inhibition of LPS-induced AMH. Materials and Methods Reagents and Animals Male Sprague Dawley rats (200e250 g) were purchased from the Experimental Animal Center of Sichuan University. LPS: E. coli serotype 055:B5 and DEX were purchased from Sigma (St. Louis, MO). Treatment of Animals Animals were specific pathogen free and were kept on a 12 h light/12 h dark cycle at a room temperature of 22 2 C with free access to food and water. Experimental procedures were conducted under aseptic conditions. The protocol was approved by the hospital Animal Care and Use Committee. Forty eight Sprague Dawley rats were divided into four experimental groups, with 12 rats per group as follows: 1) saline (SA)-treated with distilled water (DW) (control group); 2) LPS-treated with DW (LPS group); 3) LPS-treated with DEX (LPS plus DEX group); 4) SAtreated with DEX (DEX group). Rats were anesthetized intraperitoneally with penthiobarbital sodium (50 mg/kg), and LPS (200 mg/rat) in 100 mL of saline was administered by intratracheal instillation with the same volume of intratracheal saline in control animals. DEX (5 mg/kg) treatment was performed by IP injection 1 h before intratracheal instillation of LPS. Rats were sacrificed on days 2, 4 and 7 after LPS or saline administration. Histopathology Lungs of rats in each group were removed and stored at 80 C and frozen immediately. Middle lobes of right lungs were inflated with 1e1.5 mL of 10% neutral formalin and then immersed in this fixative solution and embedded in paraffin. All procedures were performed at standard atmospheric pressure. Four-mm sections were obtained for staining with Alcian blue/Periodic acid-schiff (AB/PAS). To quantitate the positive area of AB/PAS staining, IM-
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AGE-Pro plus 4.5 software (Media Cybernetics Inc., Bethesda, MD) was used to evaluate AB/PAS-stained area in term of the means previously mentioned (9,10). Briefly, AB/PASstained area in rat airway and total bronchial epithelial area were measured, and the value for statistics was expressed as percentage of the area stained by AB/PAS. Image analysis was performed strictly following the blind principle. Immunohistochemistry (IHC) Primary antibodies used for IHC studies included rabbit polyclonal anti-rat TLR4 antibody (Boster, Wuhan, China) and mouse monoclonal anti-rat MUC5AC antibody (Santa Cruz Biotechnology, Santa Cruz, CA). IHC was performed with the conventional avidin-biotin-peroxidase histochemical technique using SP kit (Vector Laboratories Ltd., Burlingame, CA). Briefly, toluene was used to remove the paraffin from sequestered 4-mm-thick paraffin sections, and the sections were rinsed thoroughly with ethanol. Sections were then soaked in 0.3% H2O2 with absolute methanol for 20 min at room temperature to inactivate endogenous peroxidase activity. They were incubated with blocking serum for 30 min and then covered with primary antibodies and incubated for 1 h. After washing in phosphate-buffered saline, sections were processed further using kits according to the instructions provided by the manufacturer and then developed with 3,30 -diaminobenzidine and H2O2, followed by Mayer’s hematoxylin staining method. Integral optical density (IOD) was used to quantitate the immunostained area using IMAGE-Pro plus 4.5 software. In brief, IOD of TLR4- and MUC5AC-stained area in per-unit area of bronchial epithelial was assessed as the value for statistics, according to the methods described above (9,10). RT-PCR For RNA isolation, the whole left lungs were frozen in liquid nitrogen, stored at 80 C and frozen immediately after removal from rats in each group. Total RNA was extracted from frozen lung tissue using Trizol reagent (Gibco-BRL, Gaithersburg, MD) and amplified using Progema PCR single-step kit (Progema, Madison, WI), according to the manufacturer’s instructions. RT-PCR analysis was used (PTC-200 DNA Engine, PCR Cycler, MJ Research, Watertown, MA). Each sample was run in duplicate. The primers, which were designed based on published sequence of these genes and synthesized by Shenneng Bio-tech Co. (Shanghai, China) as follows: MUC5AC, forward (50 -GCTCAT CCTAAG CGAC G TCT-30 ); reverse (50 -GGGGGCATAACTTCTCTTGG-30 ), TLR4, forward (50 -CATCAGTGT ATCGG TG G T CAGT-30 ); reverse (50 -CGAGGTAGGTGTTTCTGCTAAG-30 ), b-actin, forward (50 -CCTCA TGA A GAT C CTGACCG-30 ); reverse (50 -ACCGCTCATTGCCGATAGTG-30 ). b-actin served as the constitutive control. The products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.
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Chen et al./ Archives of Medical Research 40 (2009) 10e17
TLR4 and Airway Mucus Hypersecretion
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Bands were digitized by a Bio-Rad Universal Hood system (Bio-Rad, Hercules, CA). Means of the ratio of MUC5AC, TLR4 band photodensity to b-actin band photodensity in various groups were presented.
( p !0.01), which peaked on day 2, then started to decrease on day 4 and reduced to the control level on day 7 (Figure 3a).
In Situ Hybridization (ISH)
Using semiquantitative RT-PCR, we found that LPS stimulated mRNA expression of TLR4 significantly ( p !0.01), which also peaked on day 2 and decreased to the control level on day 7 (Figure 3b).
Lower lobes of right lungs were prepared for ISH. After removal from rats, lower lobes of right lungs were routinely fixed in 10% buffered formalin, dehydrated in 75% alcohol, and embedded in paraffin. Sections |6-mm thick were then ready for hybridization. All reagents used in this course described above were deactivated of RNase with 0.1% DEPC. The probe of TLR4 mRNA was labeled with digoxigenin as follows: 50 (DIG)-TTgTgCCCTgTgAggTTgAggTTAg-30 . ISH was performed according to the instructions of the ISH kit (Boster).
RT-PCR
ISH ISH (Figure 4) showed positive-stained area of TLR4 in airway epithelial cells and inflammatory cells. Increased positive-stained area of TLR4 was found in LPS group ( p !0.01) and peaked on day 2, which was in accordance with the findings in RT-PCR and IHC studies.
Statistical Analysis
Correlation Analysis
All values were expressed as means SD. One-way analysis of variance followed by Student-Newman-Keuls test was used to compare differences among multiple groups. Correlations of TLR4 expression (IOD) in rat aiway with MUC5AC expression (IOD) and AB/PAS-stained area (%) were performed with Pearson correlation analysis. Significance was defined by a p value of 0.05 (two-tailed). SPSS v.13.0 software package (SPSS Inc., Chicago, IL) was used for statistical analysis.
Correlation analysis (Figure 5) showed TLR4 expression significantly correlated well with AB/PAS-stained area (r 5 0.781, p !0.01) and the expression of MUC5AC (r 5 0.684, p !0.01) in rat airway.
Results Changes of MUC5AC Expression and AB/PAS-Stained Area after LPS Stimulation LPS stimulation significantly increased MUC5ac expression in rat airway in IHC (Figure 1a) and RT-PCR (Figure 1b) studies, which peaked on day 4 after LPS treatment ( p !0.01). In addition, similar changes were observed in the evaluation of AB/PAS-stained area (Figure 2). TLR4 Overexpression Correlates Well with LPS-induced AMH IHC Immunoreactive TLR4 expression was detected by IHC in airway epithelial cells and inflammatory cells. Weak expression of TLR4 was found in control and DEX groups. An increased immunostaining of TLR4 was found in LPS group
Effects of DEX on TLR4 Expression and Mucus Hypersecretion in Rat Airway DEX treatment significantly suppressed the peaked expression of TLR4 on day 2 after LPS stimulation in IHC and RT-PCR studies (p !0.01; Figure 3). Compared with the corresponding LPS groups, DEX subsequently reduced AB/PAS-stained area (p !0.01; Figure 2) and expression of MUC5AC protein and mRNA (p !0.01; Figure 1), which peaked on day 4 after LPS instillation. Discussion In the present study, LPS administration markedly increased the expression of TLR4 and subsequently induced overexpression of MUC5AC. Correlation analysis showed TLR4 positively correlated well with AMH. DEX significantly attenuated LPS-induced overexpression of TLR4 and MUC5AC. These findings demonstrate that TLR4 definitely relates to LPS-induced AMH and may play a role in inhibition of LPS-induced AMH by DEX. Bacterial infection, especially gram-negative bacterial infection, plays an important role in the pathogenesis of airway
= Figure 1a. MUC5AC protein expression in rat airway. MUC5AC protein was measured by routine IHC analysis and quantified by IOD measurement (K). MUC5AC positive-stained area was presented with the color of brown in airway epithelium. (A) and (B) were negative control sections from rat spleen and blank for MUC5AC, respectively. Weak positive staining was presented in representative bronchial sections from control (C) and DEX (D) groups. MUC5AC positive-stained area was significantly shown in photomicrographs from LPS group on days 2 (E), 4 (G) and 7 (I) after LPS stimulation and peaked on day 4. In contrast, DEX significantly decreased MUC5AC positive-stained area on days 2 (F), 4 (H) and 7 (J) after LPS treatment, compared with those in the corresponding LPS groups (*p !0.05,**p !0.01 vs. control group; #p !0.05 , ##p !0.01 vs. LPS group). Bar 5 100 mm. Figure 1b. MUC5AC mRNA expression in lung tissue. MUC5AC mRNA levels in the lung tissue samples harvested in every experimental group at each day point are shown in (A) and (B). The relative amounts of MUC5AC mRNA in LPS groups peaked on day 4 after LPS treatment, and this was in accordance with the changes of MUC5AC protein expression in airway epithelium. DEX effectively suppressed LPS-induced overexpression of MUC5AC in transcription level (*p !0.05, **p !0.01 vs. control group; #p !0.05, ##p !0.01 vs. LPS group). Bar 5 100 mm. Color version of the figure available online at www.arcmedres.com.
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Figure 2. AB/PAS-stained area in rat airway. Shown are representative photomicrographs of AB/PAS stained sections of lung tissue. Positive area of AB/PAS staining (I) was expressed as % stained area of airway epithelium occupied by AB/PAS-stained goblet cells. No obvious AB/PAS positive staining was presented in control (A) and DEX (B) groups. Notable positive-stained goblet cells (blue dots in airway epithelium) were presented in representative bronchial sections from LPS group on days 2 (C), 4 (E) and 7 (G) after LPS treatment and peaked on day 4. Positive-stained goblet cells significantly decreased on days 2 (D), 4 (F) and 7 (H) in LPSþDEX groups, compared with that in the corresponding LPS groups (*p !0.05, **p !0.01 vs. control group; #p !0.05, ##p !0.01 vs. LPS group). Bar 5 100 mm. Color version of the figure available online at www.arcmedres.com.
inflammation. LPS, a major component of gram-negative bacteria, serves as a foremost trigger in this process (10,11). AMH is defined as excessive secretion of mucus with enhancement of viscidity and acidity in airway disorders. Previous studies have reported that several cellular signaling pathways correlate well with mucus secretion as follows: 1) protein kinase C/G (PKC/G); myristoylated alanine-rich C kinase substrate (MARCKS); 2) epidermal growth factor (EGF) signaling pathway; 3) p38 mitogen-activated protein kinase (MAPK) pathway; 4) transforming growth factor (TGF)-Smads signaling pathway; 5) calcium-activated chloride channels (CLCA) (12e17). However, the molecular mechanism of AMH induced by LPS is not fully understood. TLR4 participates in the recognition of LPS through its interaction with CD14 and the accessory molecule, MD-2 (6). Several investigators have confirmed the effects of TLR4 in
LPS-induced inflammation (18,19). Nevertheless, LPS can activate many target intracellular signaling pathways, including not only TLRs and its downstream IL-1R signaling pathway but also G-protein, protein kinase A/C (PKA/C), MAPK pathway, and calcium signalling system (20e23). The novel finding in our study is the expression of TLR4 in rat airway peaks on day 2 after LPS treatment, earlier than MUC5AC expression and AB/PAS-stained area, which peak on day 4 after LPS stimulation. Our data support a definite correlation between TLR4 and LPS-induced AMH and also suggest a potential role of TLR4 in this process, whereas its signal transductions still need further investigations. DEX is a glucocorticosteroid that has a strong immunosuppressive action and anti-inflammatory effect. The mechanisms of DEX suppressing LPS-induced AMH are intricate, probably involved in many links of LPS signal
TLR4 and Airway Mucus Hypersecretion
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Figure 3a. (A) TLR4 protein expression in rat airway. TLR4 protein expression was assessed by routine IHC analysis and quantified by IOD measurement (K). TLR4 positive-stained area was presented with the color of brown in airway epithelium. Representative photomicrographs from each experimental group were shown. (A) and (B) were positive control sections from rat spleen and blank for TLR4, respectively. No obvious TLR4 positive staining was presented in representative bronchial sections from control (C) and DEX (D) groups. TLR4 positive-stained area was obviously presented on days 2 (E) and 4 (G) after LPS treatment, and time-dependently reduced to the control level on day 7 (I). DEX significantly decreased TLR4 positive-stained area on days 2 (F) and 4 (H) after LPS treatment, compared with those in corresponding LPS groups. No significant difference was presented between sections from LPS (I) and LPSþDEX (J) groups on day 7 (**p !0.01 vs. control group; #p !0.05, ##p !0.01 vs. LPS group). Bar 5 100 mm. Figure 3b. TLR4 mRNA expression in lung tissue. TLR4 mRNA levels in the lung tissue samples harvested in every experimental group at each day point were shown in (A) and (B). The relative amounts of TLR4 mRNA expression peaked on day 2 after LPS stimulation, which was in accordance with the changes of TLR4 protein expression in airway epithelium. DEX effectively suppressed LPS-induced overexpression of TLR4 in transcription level (**p !0.01 vs. control group; #p !0.05, ##p !0.01 vs. LPS group). Bar 5 100 mm. Color version of the figure available online at www.arcmedres.com.
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Figure 4. TLR4 mRNA expression in situ. TLR4 mRNA was assessed by in situ hybridization and quantified by IOD measurement (I). TLR4 positive-stained area was presented with the color of fawn in airway epithelium. Representative photomicrographs from each experimental group are shown. (A) and (B) were positive control section from rat spleen and blank for TLR4, respectively. No obvious TLR4 positive staining was presented in representative bronchial sections from control (C) and DEX (D) groups. TLR4-positive stained area was obviously presented in the photomicrograph from LPS group on day 2 (E) after LPS treatment and significantly decreased on day 4 (G). DEX markedly decreased TLR4-positive stained area on days 2 (F) and 4 (H) after LPS treatment, compared with that in the corresponding LPS groups (*p !0.05, **p !0.01 vs. control group; #p !0.05, ##p !0.01 vs. LPS group). Bar 5 100 mm. Color version of the figure available online at www.arcmedres.com.
Figure 5. Correlation analysis. The two scatter graphs showed correlations between TLR4 expression (IOD) and AB/PAS-stained area (%) (A), and MUC5AC expression (IOD) (B) in rat airways after LPS treatment, respectively.
TLR4 and Airway Mucus Hypersecretion
transduction. Although DEX can directly inhibit MUC5AC production by goblet cell and mucin gene expression (24,25), in general, DEX exerts its effect indirectly by suppressing activation of inflammatory cells, production and release of cytokines and other inflammatory mediators and even attenuating expression of inflammatory factor genes (26,27). As TLR4 plays a key role in pathogenesis of LPS, our results suggest DEX probably suppresses LPS-induced AMH through inhibition of TLR4 expression in CD14-positive inflammatory cells and TLR4associated inflammatory mediators, which stimulates mucus hypersecretion. Taken together, the current findings demonstrate that TLR4 is associated with LPS-stimulated AMH and probably plays a key role in DEX inhibition of LPS-induced AMH, which contributes to furtherunderstanding of GCs suppression of AMH stimulated by LPS. Further studies will be required to investigate whether TLR4 serves as a mediator in the process of LPS-induced AMH and the signaling pathway in which DEX inhibits TLR4 expression. Acknowledgments This study was supported by grants #30425007, 30370627, 30670921 from the National Natural Science Foundation of China (Beijing, China) and #00-722 and #06-834 from China Medical Board of New York (Cambridge, MA) to F.Q.W.
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