© 2005 Oxford University Press
BRIEF COMMUNICATION |
Toll-Like Receptor 4 in Butylated Hydroxytoluene–Induced Mouse Pulmonary Inflammation and Tumorigenesis
Affiliations of authors: Laboratories of Respiratory Biology (AKB, LMD, H-YC, CRW, SRK) and Experimental Pathology (DD), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC; Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO (AMM)
Correspondence to: Alison K. Bauer, PhD, Laboratory of Respiratory Biology, National Institute of Environmental Health Sciences, National Institutes of Health, 111 T.W. Alexander Drive, Building 101, Rm. E214, Research Triangle Park, NC 27709 (e-mail: bauer1{at}niehs.nih.gov).
 |
ABSTRACT |
|---|
 TopNotes Abstract References |
|---|
Because chronic pulmonary diseases predispose to lung neoplasia, the identification of the molecular mechanisms involved could provide novel preventive, diagnostic, and therapeutic strategies. Toll-like receptors (TLRs) transduce exogenous and endogenous signals into the production of inflammatory cytokines to coordinate adaptive immune responses. To determine the role of Tlr4 in chronic lung inflammation, we compared lung permeability, leukocyte infiltration, and nuclear factor kappa B (NF
B) and activator protein 1 (AP-1) DNA binding in butylated hydroxytoluene (BHT)-treated (four weekly injections of 125–200 mg/kg each) inbred mouse strains with functional Tlr4 (OuJ and BALB) and mutated Tlr4 (HeJ and BALBLps–d). We also measured primary tumor formation in these mice after single-carcinogen injection (3-methylcholanthrene; 10 µg/kg), followed by BHT treatment (six weekly injections of 125–200 mg/kg each). Mice with functional Tlr4 had reduced lung permeability, leukocyte inflammation, and primary tumor formation (BALBLps–d, mean = 22.3 tumors/mouse, versus BALB, mean = 13.9 tumors/mouse, difference = 8.4 tumors/mouse, 95% confidence interval = 4.6 to 12.1 tumors/mouse; P = .025) compared with mice with mutated Tlr4. NFB DNA binding activity was higher in OuJ than in HeJ mice; however, AP-1 activity was elevated in HeJ mice. To our knowledge, this is the first model to demonstrate a modulatory role for Tlr4 in chronic lung inflammation and tumorigenesis.
Lung cancer has a mortality rate greater than that of breast, colorectal, prostate, and pancreatic cancers combined, largely due to the inability to diagnose early-stage disease coupled with therapeutic intransigence after metastatic spread (1). Pulmonary diseases characterized by chronic inflammation, such as bronchitis, emphysema, and asthma, enhance lung cancer risk (2–4). As a preclinical model system, primary mouse lung neoplasms resemble human lung adenocarcinoma in their anatomy, histogenesis, and molecular features (5). The absence of functional Toll-like 125N receptor 4 (Tlr4), an innate immunity gene, enhances susceptibility to lung injury caused by bacterial infections (6,7). A single-nucleotide polymorphism in TLR4 has been associated with prostate cancer (8), and Tlr4 mediates anticancer efficacy by the streptococcal agent OK-432 (9). We hypothesized that functional Tlr4 would reduce lung neoplasia in a two-stage chemical carcinogenesis mouse model in which inflammation mediates the promotion phase.
Butylated hydroxytoluene (BHT) is not carcinogenic (10) and is metabolized in the lungs of mice to oxidative species that cause reversible lung injury and inflammation and promote lung tumorigenesis following protooncogene activation (11–13). Promotion is dependent on inflammation elicited by BHT metabolites (14). C3H/HeJ (HeJ) mice have a dominant negative proline-to-histidine substitution (15) in Tlr4, whereas the coisogenic C3H/HeOuJ (OuJ) strain has functional Tlr4 (15). C.C3H-TlrLps–d (BALBLps–d ) mice are congenic for a 10 centimorgan region of HeJ chromosome 4 that contains Tlr4 and thus lack Tlr4 function compared with wild-type BALB/cJ (BALB) mice (16).
Two protocols were used in this study based on a previous study that demonstrated a correlation, but not a causal relationship, between BHT-induced inflammation and promotion (14). To study the effects of Tlr4 on inflammation (protocol 1), HeJ, OuJ, BALBLps–d, and BALB mice (6–8 weeks of age; Jackson Laboratories, Bar Harbor, ME) were injected intraperitoneally with BHT (Sigma, St. Louis, MO) once a week for 4 weeks (125–150 mg/kg for the first dose, followed by 200 mg/kg for the next three doses). Mice were killed 1 day after the last injection to assess vascular leakage of proteins into bronchoalveolar lavage fluid and 3 days after BHT injection to assess leukocyte infiltration. The right lung was lavaged, total protein concentration was determined as an indicator of hyperpermeability, and leukocyte infiltration was estimated as described previously (17). To assess the role of Tlr4 in BHT-induced lung tumor promotion (protocol 2), mice were injected intraperitoneally with 10 µg 3-methylcholanthrene (MCA; Sigma)/gm of body weight or with corn oil vehicle. At this dose, MCA did not cause lung inflammation (data not shown). One week later, 150 mg of BHT/kg was administered intraperitoneally, followed by five weekly injections of 200 mg/kg each. The two additional BHT injections were given (compared with four in protocol 1) to maximize promotional efficacy; the number of tumors that arise is a linear function of the number of BHT injections (18). Mice were killed 20 weeks after the MCA injection, lungs were removed en bloc and fixed in Tellyesniczky's fixative (19) for 48 hours, and tumors were counted using a dissecting microscope. The National Institute of Environmental Health Sciences Animal Care and Use Committee approved all protocols. Differences between groups were tested by two-way analysis of variance and Tukey a posteriori comparisons of means (SigmaStat, Jandel Scientific Software, San Rafael, CA). All statistical tests were two-sided; P<.05 was considered statistically significant.
To identify the potential mechanisms by which functional Tlr4 inibits BHT-induced injury, we examined signaling elements myeloid differentiation factor 88 (MyD88), nuclear factor kappa B (NFB), and activator protein 1 (AP-1), all of which act downstream of TLR4 in HeJ and OuJ mice. Immunoblotting was performed using 100 µg of protein obtained from total lung homogenates, and blots were quantified (BioRad Quantity Image One Software; BioRad, Hercules, CA) as previously described (12). TLR4 (goat polyclonal; L14) and MyD88 (goat polyclonal; N-19) antibodies were obtained (Santa Cruz Biotechnology, Santa Cruz, CA), and nuclear protein was prepared according to the manufacturer's instructions (Nuclear Extraction Kit; Active Motif, Carlsbad, CA). To determine transcription factor DNA binding activity, 7 µg of nuclear protein was incubated with consensus sequences for either NFB or AP-1, and specific activity was determined with p65 NFB (rabbit polyclonal), phosphorylated (p)-c-jun, or c-fos (both rabbit polyclonal) antibodies by colorimetric analysis (TransAm NFB and AP-1 kits; Active Motif).
Functional Tlr4 inhibited BHT-induced weight loss, inflammation, and MCA/BHT-induced tumor formation. Statistically significantly greater weight loss was observed in Tlr4-mutated HeJ and BALBLps–d mice compared with their respective Tlr4 normal controls (HeJ versus OuJ, difference = –6.7%, 95% confidence interval [CI] = –10.2% to –3.1%, P = .006; BALBLps–d versus BALB, difference = –7.9%, 95% CI = 11.2% to –4.6%, P<.002) (Fig. 1, A.) Survival rates were 100% for OuJ, BALB, and BALBLps–d mice and 62% for HeJ mice (95% CI = 39.0% to 85.0%; P = .032). In addition, bronchoalveolar lavage fluid from HeJ and BALBLps–d mice contained more protein, alveolar macrophages, and lymphocytes than that from OuJ and BALB mice, respectively (Fig. 1, B), suggesting a protective role for TLR4 in lung inflammation and permeability. Lung tumors did not develop in MCA/BHT-treated OuJ or HeJ mice because these strains contain a tumor resistance allele at the Kras locus (20). Tumor-sensitive BALB mice contain one copy of a 37-bp intronic sequence in Kras (21), whereas two copies are found in resistant strains, such as OuJ and HeJ (22). The results described herein confirm earlier findings in C3H mice (23). Tumor multiplicity increased 60% in BALBLps–d mice compared with that in BALB mice (means = 22.3 and 13.9 tumors/mouse, respectively; difference = 8.4 tumors/mouse, 95% CI = 4.6 to 12.1 tumors/mouse; P = .025) (Fig. 1, C), but no differences in overall tumor size or morphology were noted. Interestingly, increases in numbers of pulmonary lymphocytic aggregates were found in Tlr4-mutated mice compared with wild-type mice following MCA or MCA/BHT treatment, although the importance of these cellular aggregates remains unclear.
|
, P = .016 compared with oil vehicle; +, P<.001 compared with OuJ and BALB controls; 
, P = .002 compared with BALB control. C) Tumor multiplicity (the total number of tumors per total number of mice) in OuJ, BALB, HeJ, and BALBLps–d mice 20 weeks after oil, MCA, or MCA/BHT treatment. Tumor incidence (the total number of mice with tumors) was 100% in BALB and BALBLps–d mice in response to MCA/BHT treatment and <70% in the control treatment groups, as shown previously in this promotion model (Chronic BHT treatment increased the protein levels of TLR4 and the MyD88 adaptor protein responsible for TLR4-mediated signal transduction (24) 3 days after the final BHT injection in OuJ mice (Fig. 2, A). Although TLR4 protein levels increased in the HeJ mice, the levels remained statistically significantly lower than those in OuJ mice, and no changes in MyD88 concentration were found in HeJ mice (Fig. 2, A). BHT treatment led to elevated p65 NF
B transcriptional activity in both strains at 3 days (Fig. 2, B), but binding was statistically significantly greater in OuJ mice compared with HeJ mice (Fig. 2, B). Compared with HeJ mice, a larger increase in p50 and p65 NFB DNA binding was found in OuJ mice after 3 days (electromobility shift assay) (data not shown). NFB activation can be proapoptotic (25); hence, it can inhibit tumor formation by altering the balance between apoptosis and proliferation. In contrast with the increase in NFB activation observed in the TLR4 wild-type mice, AP-1 transcriptional activity was elevated in HeJ but not OuJ mice 1 day following BHT treatment, as evidenced by increased p-c-jun and c-fos binding to the AP-1 response element (Fig. 2, C). Therefore, differential transcriptional activities following BHT treatment of mice depend on the presence or absence of functional Tlr4. The functional importance of the different transcription factor kinetics observed is not clear.
|
These novel findings, in a chronic pulmonary inflammation model, demonstrate the importance of Tlr4 in chronic disease states. Our results suggest that Tlr4 inhibits lung carcinogenesis by inhibiting tumor promotion and that it may be an effective target for alternate preventive, diagnostic, and therapeutic strategies.
|
NOTES |
|---|
|
TopNotesAbstractReferences |
|---|
Supported by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health (grants CA33497 [A. M. Malkinson] and CA96133 [A. M. Malkinson]).
|
REFERENCES |
|---|
|
TopNotesAbstractReferences |
|---|
(1) Jemal A, Murray T, Samuels A, Ghafoor A, Ward E, Thun MJ. Cancer statistics, 2003. CA Cancer J Clin 2003;53:5–26.
(2) Mayne ST, Buenconsejo J, Janerich DT. Previous lung disease and risk of lung cancer among men and women nonsmokers. Am J Epidemiol 1999;149:13–20.
(3) Vesterinen E, Pukkala E, Timonen T, Aromaa A. Cancer incidence among 78 000 asthmatic patients. Int J Epidemiol 1993;22:976–82.
(4) Cohen BH, Diamond EL, Graves CG, Kreiss P, Levy DA, Menkes HA, et al. A common familial component in lung cancer and chronic obstructive pulmonary disease. Lancet 1977;2:523–6.[ISI][Medline]
(5) Malkinson AM. Molecular comparison of human and mouse pulmonary adenocarcinomas. Exp Lung Res 1998;24:541–55.[ISI][Medline]
(6) Faure K, Sawa T, Ajayi T, Fujimoto J, Moriyama K, Shime N, et al. TLR4 signaling is essential for survival in acute lung injury induced by virulent Pseudomonas aeruginosa secreting type III secretory toxins. Respir Res 2004;5:1.[CrossRef][ISI][Medline]
(7) Branger J, Knapp S, Weijer S, Leemans JC, Pater JM, Speelman P, et al. Role of Toll-like receptor 4 in gram-positive and gram-negative pneumonia in mice. Infect Immun 2004;72:788–94.
(8) Zheng SL, Augustsson-Balter K, Chang B, Hedelin M, Li L, Adami HO, et al. Sequence variants of toll-like receptor 4 are associated with prostate cancer risk: results from the Cancer Prostate in Sweden Study. Cancer Res 2004;64:2918–22.
(9) Okamoto M, Sato M. Toll-like receptor signaling in anti-cancer immunity. J Med Invest 2003;50:9–24.[Medline]
(10) Witschi H, Williamson D, Lock S. Enhancement of urethane tumorigenesis in mouse lung by butylated hydroxytoluene. J Natl Cancer Inst 1977;58:301–5.[ISI][Medline]
(11) Miller AC, Dwyer LD, Auerbach CE, Miley FB, Dinsdale D, Malkinson AM. Strain-related differences in the pneumotoxic effects of chronically administered butylated hydroxytoluene on protein kinase C and calpain. Toxicology 1994;90:141–59.[CrossRef][ISI][Medline]
(12) Bauer AK, Dwyer-Nield LD, Hankin JA, Murphy RC, Malkinson AM. The lung tumor promoter, butylated hydroxytoluene (BHT), causes chronic inflammation in promotion-sensitive BALB/cByJ mice but not in promotion-resistant CXB4 mice. Toxicology 2001;169:1–15.[CrossRef][ISI][Medline]
(13) Malkinson AM, Koski KM, Evans WA, Festing MF. Butylated hydroxytoluene exposure is necessary to induce lung tumors in BALB mice treated with 3-methylcholanthrene. Cancer Res 1997;57:2832–4.
(14) Bauer AK, Dwyer-Nield LD, Keil K, Koski K, Malkinson AM. Butylated hydroxytoluene (BHT) induction of pulmonary inflammation: a role in tumor promotion. Exp Lung Res 2001;27:197–216.[ISI][Medline]
(15) Poltorak A, He X, Smirnova I, Liu MY, Huffel CV, Du X, et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998;282:2085–8.
(16) Vogel SN, Wax JS, Perera PY, Padlan C, Potter M, Mock BA. Construction of a BALB/c congenic mouse, C.C3H-Lpsd, that expresses the Lpsd allele: analysis of chromosome 4 markers surrounding the Lps gene. Infect Immun 1994;62:4454–9.
(17) Cho HY, Zhang LY, Kleeberger SR. Ozone-induced lung inflammation and hyperreactivity are mediated via tumor necrosis factor-alpha receptors. Am J Physiol Lung Cell Mol Physiol 2001;280:L537–46.
(18) Brown LM, Malkinson AM, Rannels DE, Rannels SR. Compensatory lung growth after partial pneumonectomy enhances lung tumorigenesis induced by 3-methylcholanthrene. Cancer Res 1999;59:5089–92.
(19) Obermueller-Jevic UC, Espiritu I, Corbacho AM, Cross CE, Witschi H. Lung tumor development in mice exposed to tobacco smoke and fed beta-carotene diets. Toxicol Sci 2002;69:23–9.
(20) You M, Wang Y, Stoner G, You L, Maronpot R, Reynolds SH, et al. Parental bias of Ki-ras oncogenes detected in lung tumors from mouse hybrids. Proc Natl Acad Sci U S A 1992;89:5804–8.
(21) Malkinson AM, You M. The intronic structure of cancer-related genes regulates susceptibility to cancer. Mol Carcinog 1994;10:61–5.[ISI][Medline]
(22) Lin L, Festing MF, Devereux TR, Crist KA, Christiansen SC, Wang Y, et al. Additional evidence that the K-ras protooncogene is a candidate for the major mouse pulmonary adenoma susceptibility (Pas-1) gene. Exp Lung Res 1998;24:481–97.[ISI][Medline]
(23) Malkinson AM. The genetic basis of susceptibility to lung tumors in mice. Toxicology 1989;54:241–71.[CrossRef][ISI][Medline]
(24) Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R. Toll-like receptors control activation of adaptive immune responses. Nat Immunol 2001;2:947–50.[CrossRef][ISI][Medline]
(25) Kimura M, Haisa M, Uetsuka H, Takaoka M, Ohkawa T, Kawashima R, et al. TNF combined with IFN-alpha accelerates NF-kappaB-mediated apoptosis through enhancement of Fas expression in colon cancer cells. Cell Death Differ 2003;10:718–28.[CrossRef][ISI][Medline]
Manuscript received April 5, 2005; revised September 14, 2005; accepted September 29, 2005.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. S. Roberts, R. S. Thomas, and D. C. Dorman Gene Expression Changes Following Acute Hydrogen Sulfide (H2S)-induced Nasal Respiratory Epithelial Injury Toxicol Pathol, June 1, 2008; 36(4): 560 - 567. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Andreani, G. Gatti, L. Simonella, V. Rivero, and M. Maccioni Activation of Toll-like Receptor 4 on Tumor Cells In vitro Inhibits Subsequent Tumor Growth In vivo Cancer Res., November 1, 2007; 67(21): 10519 - 10527. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Astrakianakis, N. S. Seixas, R. Ray, J. E. Camp, D. L. Gao, Z. Feng, W. Li, K. J. Wernli, E. D. Fitzgibbons, D. B. Thomas, et al. Lung Cancer Risk Among Female Textile Workers Exposed to Endotoxin J Natl Cancer Inst, March 7, 2007; 99(5): 357 - 364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. P Singh and R. Agarwal Mechanisms of action of novel agents for prostate cancer chemoprevention. Endocr. Relat. Cancer, September 1, 2006; 13(3): 751 - 778. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||