The Lip Lipoprotein from Neisseria gonorrhoeae Stimulates Cytokine Release and NF-κB Activation in Epithelial Cells in a Toll-like Receptor 2-dependent Manner
2003; Elsevier BV; Volume: 278; Issue: 47 Linguagem: Inglês
10.1074/jbc.m306587200
ISSN1083-351X
AutoresPhilip L. Fisette, Sanjay Ram, Jorunn M. Andersen, Wen Guo, Robin R. Ingalls,
Tópico(s)Streptococcal Infections and Treatments
ResumoThe human pathogen Neisseria gonorrhoeae produces an array of diseases ranging from urethritis to disseminated gonococcal infections. Early events in the establishment of infection involve interactions between N. gonorrhoeae and the mucosal epithelium, which leads to the local release of inflammatory mediators. Because of this, it is important to identify the bacterial virulence factors and host cell components that contribute to inflammation. Using a series of column chromatography steps, we purified a lipoprotein from N. gonorrhoeae strain F62 called Lip. This outer membrane antigen expresses a conserved epitope known as H.8, which is common to all pathogenic Neisseria species. We found the purified preparation of Lip to be a potent inflammatory mediator capable of inducing the release of the chemokine interleukin (IL)-8 and the cytokine IL-6 by immortalized human endocervical epithelial cells and the production of IL-8 and the activation of the transcription factor NF-κB by human embryonic kidney 293 (HEK) cells transfected with toll-like receptor (TLR) 2. Upon removal of Lip by immunoprecipitation, the ability of the H.8/Lip preparation to stimulate NF-κB activation was abolished. In addition to TLR2, the activation of NF-κB by H.8/Lip in HEK cells was enhanced upon coexpression of TLR1 but not TLR6. These observations provide evidence that Lip is capable of inducing the release of inflammatory mediators from epithelial cells in a TLR2-dependent manner. The human pathogen Neisseria gonorrhoeae produces an array of diseases ranging from urethritis to disseminated gonococcal infections. Early events in the establishment of infection involve interactions between N. gonorrhoeae and the mucosal epithelium, which leads to the local release of inflammatory mediators. Because of this, it is important to identify the bacterial virulence factors and host cell components that contribute to inflammation. Using a series of column chromatography steps, we purified a lipoprotein from N. gonorrhoeae strain F62 called Lip. This outer membrane antigen expresses a conserved epitope known as H.8, which is common to all pathogenic Neisseria species. We found the purified preparation of Lip to be a potent inflammatory mediator capable of inducing the release of the chemokine interleukin (IL)-8 and the cytokine IL-6 by immortalized human endocervical epithelial cells and the production of IL-8 and the activation of the transcription factor NF-κB by human embryonic kidney 293 (HEK) cells transfected with toll-like receptor (TLR) 2. Upon removal of Lip by immunoprecipitation, the ability of the H.8/Lip preparation to stimulate NF-κB activation was abolished. In addition to TLR2, the activation of NF-κB by H.8/Lip in HEK cells was enhanced upon coexpression of TLR1 but not TLR6. These observations provide evidence that Lip is capable of inducing the release of inflammatory mediators from epithelial cells in a TLR2-dependent manner. Neisseria gonorrhoeae is a strictly human pathogen and the causative agent of gonorrhea, a sexually transmitted disease of worldwide importance. Infection with gonorrhea usually remains localized to the lower genital tract where it commonly manifests as urethritis in men and cervicitis in women. In some cases it can ascend to the upper genital tract, which in women can lead to endometritis, pelvic inflammatory disease, and fallopian tube scarring. In addition, certain strains are capable of dissemination, resulting in bacteremia, septic arthritis, and pustular skin lesions. During the early stages of infection, the initial interaction of N. gonorrhoeae with mucosal epithelial cells triggers the release of inflammatory cytokines and chemokines including interleukin (IL)-6 1The abbreviations used are: ILinterleukinTLRtoll-like receptorNF-κBnuclear factor κBLPSlipopolysaccharideLOSlipooligosaccharideTNFαtumor necrosis factor αMALP-2mycoplasmal macrophage-activating lipopeptide 2mAbmonoclonal antibodyPBSphosphate-buffered salineGLCgas/liquid chromatographyHEKhuman embryonic kidneyDEAE2-diethylamino-ethylIPimmunoprecipitation. and IL-8 (1Fichorova R.N. Desai P.J. Gibson F.C.R. Genco C.A. Infect. Immun. 2001; 69: 5840-5848Crossref PubMed Scopus (131) Google Scholar, 2Harvey H. Post D. Apicella M. Infect. Immun. 2002; 70: 5808-5815Crossref PubMed Scopus (52) Google Scholar, 3Naumann M. Weβler S. Bartsch C. Wieland B. Meyer T.F. J. Exp. 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Immunol. 2001; 13: 933-940Crossref PubMed Scopus (1009) Google Scholar). Moreover, macrophages derived from mice lacking TLR1 are hindered in the release of TNFα when treated with triacylated lipopeptides but respond normally to MALP-2, peptidoglycan, and LPS (17Takeuchi O. Sato S. Horiuchi T. Hoshino K. Takeda K. Dong Z. Modlin R.L. Akira S. J. Immunol. 2002; 169: 10-14Crossref PubMed Scopus (1080) Google Scholar). interleukin toll-like receptor nuclear factor κB lipopolysaccharide lipooligosaccharide tumor necrosis factor α mycoplasmal macrophage-activating lipopeptide 2 monoclonal antibody phosphate-buffered saline gas/liquid chromatography human embryonic kidney 2-diethylamino-ethyl immunoprecipitation. In light of reports that an LPS-deficient strain of N. meningitidis primarily activates macrophages through TLR2 (20Ingalls R.R. Lien E. Golenbock D.T. Infect. Immun. 2001; 69: 2230-2236Crossref PubMed Scopus (67) Google Scholar, 21Pridmore A.C. Wyllie D.H. Abdillahi F. Steeghs L. van der Ley P. Dower S.K. Read R.C. J. Infect. Dis. 2001; 183: 89-96Crossref PubMed Scopus (131) Google Scholar) and that purified preparations of the Neisseria outer membrane porin protein signal through TLR2 (22Massari P. Henneke P. Ho Y. Latz E. Golenbock D. Wetzler L. J. Immunol. 2002; 168: 1533-1537Crossref PubMed Scopus (260) Google Scholar), we examined other potential TLR2 ligands from the outer membrane of Neisseria. Many of the surface proteins of Neisseria are known to undergo significant antigenic variability; comparatively fewer are conserved within the pathogenic Neisseria species. One such conserved antigen is the Lip (or H.8 antigen) lipoprotein. The H.8 antigen is named after a mouse hybridoma antibody that recognizes a surface epitope that is common to pathogenic Neisseria species including N. gonorrhoeae and Neisseria meningitidis but absent from other commensal Neisseria species (23Cannon J.G. Black W.J. Nachamkin I. Stewart P.W. Infect. Immun. 1984; 43: 994-999Crossref PubMed Google Scholar). Although the size and amino acid sequence of Lip can differ between Neisseria species and strains (24Trees D.L. Schultz A.J. Knapp J.S. J. Clin. Microbiol. 2000; 38: 2914-2916Crossref PubMed Google Scholar), it primarily consists of 11–20 pentameric AAEAP amino acid repeats and a lipid-modified N-terminal cysteine residue and lacks any aromatic residues (25Wagner D.A. Young V.R. Tannenbaum S.R. Proc. Nat. Acad. Sci. U. S. A. 1993; 80: 4518-4521Crossref Scopus (193) Google Scholar, 26Woods J.P. Spinola S.M. Strobel S.M. Cannon J.G. Mol. Microbiol. 1989; 3: 43-48Crossref PubMed Scopus (27) Google Scholar). This latter property makes it virtually undetectable by most protein assay and staining methods and thus makes it an elusive potential contaminant of preparations purified from outer membranes derived from Neisseria. In addition to recognizing Lip, the H.8-specific monoclonal antibody is weakly cross-reactive to a second conserved protein from Neisseria known as Laz. This lipid-modified azurin-like protein contains imperfect AAEAP motifs at the N-terminal domain of the mature protein and a second domain with homology to the azurin protein of Pseudomonas aeruginosa (27Gotschlich E. Seiff M. FEMS Microbiol. Lett. 1987; 43: 253-255Crossref Scopus (28) Google Scholar, 28Kawula T. Spinola S. Klapper D. Cannon J. Mol. Microbiol. 1987; 1: 179-185Crossref PubMed Scopus (29) Google Scholar, 29Woods J. Dempsey J. Kawula T. Barritt D. Cannon J. Mol. Microbiol. 1989; 3: 583-591Crossref PubMed Scopus (20) Google Scholar). In this report, we describe the proinflammatory activity of the Lip lipoprotein in terms of its ability to stimulate chemokine and cytokine production in human endocervical epithelial cells. In addition, we provide evidence using transfected human embryonic kidney (HEK) 293 cells suggesting that TLR2 and TLR1 are required for recognition of the Lip lipoprotein. Finally, preliminary biochemical analysis confirms the presence of lipid modification in the Lip lipoprotein and suggests that it may contain both short (C8–C10) and medium (C16) chain fatty acids. Reagents—The anti-H.8 murine monoclonal antibody (mAb 2C3) was obtained from Drs. Sunita Gulati and Peter Rice (Boston Medical Center, Boston, MA; Ref. 30Apicella M. Mandrell R. Shero M. Wilson M. Griffiss J. Brooks G. Lammel C. Breen J. Rice P. J. Infect. Dis. 1990; 162: 506-512Crossref PubMed Scopus (105) Google Scholar). LPS from Escherichia coli strain K235 was purchased from List Pharmaceuticals (Woburn, MA) and repurified by phenol re-extraction to remove potentially contaminating proteins, as described previously (31Manthey C.L. Perera P.Y. Henricson B.E. Hamilton T.A. Qureshi N. Vogel S.N. J. Immunol. 1994; 153: 2653-2663PubMed Google Scholar, 32Hirschfeld M. Ma Y. Weis J. Vogel S. Weis J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (971) Google Scholar). Lipooligosaccharide (LOS) was purified from N. gonorrhoeae strain F62 using a modification (33Gnehm H. Pelton S. Gulati S. Rice P. J. Clin. Invest. 1985; 75: 1645-1648Crossref PubMed Scopus (47) Google Scholar, 34Densen P. Gulati S. Rice P. J. Clin. Invest. 1987; 80: 78-87Crossref PubMed Scopus (27) Google Scholar) of the hot phenol water extraction method (35Westphal O. Luderitz O. Bister F. Z. Naturforsch. 1952; B7: 148-155Crossref Scopus (1043) Google Scholar). Recombinant human IL-1β and TNFα were obtained from Endogen (Woburn, MA). The lysate from Borrelia burgdorferi strain cN40 was a gift from Dr. Timothy Sellati (Albany Medical College, Albany, NY). Recombinant human soluble CD14 was obtained from Dr. Henri Lichenstein (Amgen, Thousand Oaks, CA). The synthetic lipopeptide Pam3Cys-Lip, the nonlipidated Lip peptide, the Pam3Cys-SK4 lipopeptide, and the mycoplasmal lipopeptide MALP-2 were purchased from EMC Microcollections (Tuebingen, Germany). The Pam3Cys-Lip, which contains a tripalmitoyl-S-glyceryl cysteine at the N terminus, was based on the N-terminal sequence of the N. gonorrhoeae F62 Lip protein (sequence CGGEKAAEAPAAEAS). The MALP-2 lipopeptide was derived from the MALP-2 membrane lipoprotein from Mycoplasma fermentans (sequence CGNNDESNISFKEK) and possesses a dipalmitoyl-S-glyceryl cysteine at the N terminus. Protein G-agarose was from Sigma. Bacterial Strains—The N. gonorrhoeae laboratory strain F62 (nonpiliated, nonopaque; pil–/opa–), which was originally isolated from an individual with pelvic inflammatory disease, was used for these studies (36Schneider H. Griffiss J.M. Williams G.D. Pier G.B. J. Gen. Microbiol. 1982; 128: 13-22PubMed Google Scholar). Strain FA1090 and the H.8 deletion mutant FA1090ΔH.8 and the plasmid pHSS6 (ΔH.8) containing the FA1090 H.8 gene insertionally inactivated with a chloramphenicol resistance gene were gifts from Dr. Janne Cannon (University of North Carolina, Chapel Hill, NC) (26Woods J.P. Spinola S.M. Strobel S.M. Cannon J.G. Mol. Microbiol. 1989; 3: 43-48Crossref PubMed Scopus (27) Google Scholar). Piliated N. gonorrhoeae strain F62 was transformed with purified plasmid pHSS6 as previously described (37Biswas G.D. Sox T. Blackman E. Sparling P.F. J. Bacteriol. 1977; 129: 983-992Crossref PubMed Google Scholar, 38Carbonetti N. Simnad V. Elkins C. Sparling P.F. Mol. Microbiol. 1990; 4: 1009-1018Crossref PubMed Scopus (40) Google Scholar). Chloramphenicol-resistant N. gonorrhoeae F62 colonies were selected on standard gonococcal culture medium supplemented with chloramphenicol at a concentration of 1 μg/ml. Transformants were confirmed by immunoblot analysis showing the absence of the 2C3 epitope as described below. Nucleotide Sequencing of Lip from N. gonorrhoeae—N. gonorrhoeae strain F62 was grown overnight on chocolate agar. Crude DNA was isolated by resuspending approximately five colonies in 100 μl of sterile water, as described by Hobbs et al. (39Hobbs M.M. Alcorn T.M. Davis R.H. Fischer W. Thomas J.C. Martin I. Ison C. Sparling P.F. Cohen M.S. J. Infect. Dis. 1999; 179: 371-381Crossref PubMed Scopus (67) Google Scholar). The Lip gene was amplified by polymerase chain reaction using Taq DNA polymerase (Promega) and chromosomal DNA from N. gonorrhoeae strain F62. The primers, derived from Trees et al. (24Trees D.L. Schultz A.J. Knapp J.S. J. Clin. Microbiol. 2000; 38: 2914-2916Crossref PubMed Google Scholar) that were used to amplify the Lip gene were CAAATTCAGCGATGAATTTCCAACCC-3′ (forward primer) and 5′-TATGAAGGTCAGGCATGTTTGTCGG-3′ (reverse primer). The PCR conditions consisted of 25 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and elongation at 68 °C for 2 min 30 s. The amplified PCR product of ∼500 base pairs was gel-purified and sequenced by the Boston University Molecular Genetics Core Facility using the ABI Prism Big DYE Terminator Cycle Sequencing Ready reaction kit (Applied Biosystems) performed on the ABI Prism 377-96 DNA sequencer. Outer Membrane Preparations—N. gonorrhoeae strain F62 was grown overnight on chocolate agar, and the outer membranes were prepared as described previously (33Gnehm H. Pelton S. Gulati S. Rice P. J. Clin. Invest. 1985; 75: 1645-1648Crossref PubMed Scopus (47) Google Scholar). Briefly, bacteria were harvested into shearing buffer (PBS containing 10 mm EDTA, pH 7.4). The suspension was incubated at 60 °C for 30 min, and the outer membranes were sheared by passage through a 25-gauge needle, followed by homogenization for 10 s in a Waring blender. After two rounds of centrifugation at 12,000 × g for 20 min to remove unlysed bacteria and large cellular debris, the resulting supernatant was centrifuged at 80,000 × g for 2 h. The pellet from this ultracentrifugation step was resuspended in deoxycholate buffer (50 mm glycine, 1 mm EDTA, and 1.5% deoxycholate, pH 9.5). Uniform solubilization of the membranes was achieved by increasing the pH of the suspension to 13.5 with 1 m NaOH, followed by immediate readjustment back to pH 9.5 with 0.5 n HCl. Purification of the H.8/Lip Lipoprotein from N. gonorrhoeae—The procedure for purifying H.8/Lip from Neisseria was described by Zollinger et al. (40Zollinger W. Ray J. Moran E. Seid R. Schoolnik G. The Pathogenic Neisseriae: Proceedings of the Fourth International Symposium. American Society for Microbiology, Asilomar, CA1984: 579-584Google Scholar). Briefly, the solubilized outer membrane preparation was loaded onto a Sephadex G-100 column (2.5 cm × 60 cm) equilibrated with deoxycholate buffer. Fractions (5 ml) were collected at a flow rate of 0.25 ml/min. The presence of H.8 was confirmed by dot-blot analysis using the anti-H.8 mouse monoclonal IgG antibody 2C3, and the presence of other proteins and LOS was monitored by silver staining of SDS-PAGE gels and/or BCA protein assay reagent A (Pierce). The fractions that were positive for H.8 and contained a low level of other proteins were pooled and dialyzed for 18 h against PBS (NaPO4, pH 7.4, and 50 mm NaCl). This fraction was further clarified by ion exchange chromatography on a DEAE-Sephacel CL-4B (Amersham Biosciences) column (1.5 × 12 cm; 20 ml) that was equilibrated with PBS. The bound material was eluted with a 50–400 mm NaCl gradient in PBS at a flow rate of 0.5 ml/min, and the fractions were collected every 4 min. Fatty Acid Analysis—The purified H.8/Lip fraction, the synthetic Pam3Cys-Lip lipopeptide, and gonococcal LOS were hydrolyzed in 6 m HCl for 20 h at 95 °C. After extraction of the hydrolyzed products with isopropanol/hexane and 1 n H2SO4 (40:10:1), the hexane phase containing total lipid was concentrated by evaporation of the organic solvent under nitrogen gas, and the resulting product was redissolved in 100 μl of hexane and separated by TLC (hexane/ethyl ether/acetic acid, 70:30: 1). The fraction that contained free fatty acids was scraped off and eluted with hexane. After drying down under N2 gas, methylation was performed by incubation in BF3-methanol solution (14%, v/v, BF3 in methanol) at 60 °C for 30 min. The fatty acid methyl ester was extracted into a hexane solution. The hexane solution was dried with anhydrous sodium sulfate and used directly for gas/liquid chromatography (GLC) analysis without further condensation. GLC analysis was performed on a Shimuzu 14A gas chromatograph with a Supelco SP™-2380 capillary column, with an initial oven temperature of 150 °C, a final temperature of 240 °C, a heating rate of 4 °C/min, an injector temperature of 220 °C, and a detector temperature of 240 °C. The carrier gas (helium) was at 50 kPa, make-up carrier gas (helium) was at 100 kPa, hydrogen gas was at 55 kPa, and compressed air was at 50 kPa. The sample was injected in 1–1.5 μl with splitting rate of 1:25. The peak assignment was done using Nu-Chek GLC reference for free fatty acids (063A) and bacterial acid methyl ester reference standards (catalog number 47080-U) from Supelco (Bellefonte, PA). The results are presented as the percentages of each fatty acyl chain in the sample. Cell Culture—The HEK 293 cell line (ATCC, Manassas, VA) was cultured in Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (HyClone, Logan UT) and 10 μg/ml ciprofloxacin (Bayer Corp., West Haven, CT). HEK 293 cells stably transfected with TLR2 (HEK-TLR2) were provided by Dr. Douglas Golenbock (University of Massachusetts Medical School, Worcester, MA). The human papillomavirus 16/E6E7 immortalized endocervical epithelial cell line (End/E6E7) was a gift from Dr. Raina Fichorova (Brigham and Women's Hospital, Harvard Medical School, Boston, MA). The End/E6E7 cells were grown in keratinocyte serum-free medium (Invitrogen) supplemented with the provided 50 μg/ml bovine pituitary extract, 0.1 ng/ml recombinant epidermal growth factor, and 0.4 mm CaCl2 (keratinocyte serum-free growth medium). The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Assays for IL-6 and IL-8 Secretion—End/E6E7, HEK 293, and HEK-TLR2 cell were grown in 96-well tissue culture dishes at a density of 5 × 104/well and allowed to grow for 24 h. The cells were treated with various concentrations of purified DEAE H.8/Lip fraction in a reaction volume of 100 μl. Control treatments were PBS or DEAE column elution buffer, IL-1β (5 ng/ml), TNFα (5 ng/ml), and LPS (100 ng/ml). Treatments of the End/E6E7 cells were in keratinocyte serum-free growth medium. Soluble CD14, at a concentration of 500 ng/ml, was added to the medium when indicated (41Fichorova R.N. Cronin A.O. Lien E. Anderson D.J. Ingalls R.R. J. Immunol. 2002; 168: 2424-2432Crossref PubMed Scopus (206) Google Scholar). Treatments for the HEK 293 cells were in medium supplemented with 10% fetal bovine serum. After incubation for 20 h at 37 °C, the culture supernatants were removed, diluted to the appropriate concentrations in keratinocyte serum-free medium or Dulbecco's modified Eagle's medium, and assayed for IL-6 or IL-8 using Eli-pair enzyme-linked immunosorbent assay kits from Cell Sciences (Norwood, MA). All of the cytokines were assayed in duplicate or triplicate, and the data are reported as the means ± the standard error or standard deviation, respectively. Expression Plasmids—The ELAM-1-luciferase reporter plasmid (pELAM-luc) was generated as described (5Chow J.C. Young D.W. Golenbock D.T. Christ W.J. Gusovsky F. J. Biol. Chem. 1999; 274: 10689-10692Abstract Full Text Full Text PDF PubMed Scopus (1617) Google Scholar) by cloning a fragment (–241 to –54 base pairs) of the human E-selectin promoter into the pGL3 reporter plasmid (Promega). This expression plasmid reports luciferase in an NF-κB-dependent manner. Expression plasmids for human TLR1 (in the pFLAG-CMV vector), TLR2 (in the pFLAG-CMV vector), and TLR6 (in the pEF-BOS vector) were a gift of Douglas Golenbock (University of Massachusetts Medical School, Worcester, MA). All of the plasmid DNA was isolated with Endo-Free™ Maxi-prep columns from Qiagen. Transient Transfection and NF-κB Reporter Luciferase Assay—HEK 293 or HEK-TLR2 cells were plated in 6-well tissue culture plates (4 × 105 cells/well) and maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum for 18 h. Using the SuperFect™ Transfection Protocol (Qiagen), the cells were transfected with 250 ng of pELAM-luc reporter construct and one of the following expression vectors (described above): 100 ng of human TLR2, 500 ng of human TLR1, or 10 ng of human TLR6. The empty vector pcDNA3 (Invitrogen) was used as a control and to normalize the DNA concentration for all of the transfection reactions. The transfected cells were then allowed to recover for a period of 20 h. After triplicate treatments for 5 h, the cells were harvested in lysis buffer (Promega), snap frozen on dry ice, and assayed for luciferase activity by the use of a commercial luciferase assay kit (Promega) according to the manufacturer's protocol. The amount of luciferase activity in each sample was quantified by a Monolight™ 3010 Luminometer (PharMingen, San Diego, CA). The data are reported as the means ± the standard deviation. All of the transfection experiments were repeated at least twice. Immunoprecipitation of H.8 —Immunoprecipitations were carried out for 2 h at 4 °C in 70-μl reaction volumes containing 5 μl of purified H.8/Lip, 15 μg of protein G-agarose, 4 μg of bovine serum albumin, and the indicated concentrations of either anti-H.8 2C3 antibody or control IgG (0–10 μg/ml). After centrifugation, the IP supernatants were decanted, and the resulting IP pellets were washed three times with PBS. The proteins from both the pellet and the supernatant were separated by SDS-PAGE and transferred onto an Immobilon-P polyvinylidene difluoride membrane (Millipore, Bedford, MA). H.8 antigen was detected using mAb 2C3 and visualized by alkaline phosphatase-conjugated goat-anti-mouse IgG (Sigma) and substrate as described previously (42Blake M. Johnston K. Russell-Jones G. Gotschlich E. Anal. Biochem. 1984; 136: 175-179Crossref PubMed Scopus (1630) Google Scholar). Gel Purification of Lip—H.8/Lip derived from the DEAE-Sephacel column was further purified by elution after electrophoresis on a 4–12% Bis-Tris gel under denaturing conditions in the presence of lithium dodecyl sulfate sample buffer (Invitrogen). Using the prestained molecular mass samples as a guide, the gel fragment that corresponded to the predicted migration of Lip was excised and incubated with 1 ml of endotoxin-free H2O for 18 h at 37 °C. The eluted material was analyzed by silver staining to assess for contaminants, and the presence of Lip was confirmed by immunoblotting with mAb 2C3 as described above. As a negative control, a gel fragment spanning the 60–70-kDa regions was also excised and treated similarly. Both samples were tested for contaminating endotoxin using the Limulus Amebocyte Lysate Pyrochrome kit from Associates of Cape Cod (Falmouth, MA). The limit of detection for the Pyrochrome kit is 0.005 endotoxin units/ml (12 endotoxin units = ∼1 ng of endotoxin). Endotoxin contamination in the concentrated H.8/Lip and gel control preparations was 6 and 5 endotoxin units/ml, respectively; preparations were used in the biological assays at dilutions of 1:50 to 1:1000. Purification of the H.8 Lipoprotein Lip from N. gonorrhoeae F62 Outer Membranes—It has been documented that small amounts of contaminating lipoproteins in LPS preparations can lead to TLR2-dependent cell activation (32Hirschfeld M. Ma Y. Weis J. Vogel S. Weis J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (971) Google Scholar). We tested an LOS preparation from N. gonorrhoeae for activation of NF-κB in human endocervical epithelial cells. This cell line had previously been shown by our laboratory to be deficient in TLR4 and MD-2, the receptors required for responses to most species of endotoxin (41Fichorova R.N. Cronin A.O. Lien E. Anderson D.J. Ingalls R.R. J. Immunol. 2002; 168: 2424-2432Crossref PubMed Scopus (206) Google Scholar). Rather unexpectedly, we found dose-dependent cell activation with LOS concentrations greater than 1 μg/ml (data not shown). However, upon repurification of the crude LOS preparation by phenol re-extraction (32Hirschfeld M. Ma Y. Weis J. Vogel S. Weis J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (971) Google Scholar), the LOS-dependent activation disappeared. This suggested that cell activation was being driven by a phenol-soluble lipoprotein. Immunoblot analysis using mAb 2C3 revealed that the gonococcal outer membrane protein Lip (or H.8 antigen) was present in the original LOS preparation but not after re-extraction (data not shown). This observation prompted us to further examine the potential of the Lip lipopro
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