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Immune responses of palatine tonsil against bacterial antigens
International Congress Series 1257 (2003) 141 – 144
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Immune responses of palatine tonsil against bacterial antigens Norimitsu Tanaka, Satoshi Fukuyama, Masato Ushikai, Keiichi Miyashita, Yuichi Kurono * Department of Otolaryngology, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan Received 5 August 2003; received in revised form 15 September 2003; accepted 16 September 2003
Abstract. Background: Although various bacterial antigens are frequently found in upper respiratory tract mucosal secretions, fatal responses, such as endotoxin shock, are rare, suggesting the presence of some regulatory system in mucosal as well as systemic tissues. In the present study, we examined the immune responses of tonsillar mononuclear cells against bacterial antigens. Materials and methods: Mononuclear cells were isolated from the palatine tonsils and peripheral blood (tonsil mononuclear cells [TNMC] and peripheral blood mononuclear cells [PBMC]) of patients who underwent palatine tonsillectomy. TNMC and PBMC were stimulated using lipopolysaccharide (LPS) or lipoteichoic acid (LTA) for 24 h, and cytokine production were determined by ELISA. Furthermore, the expression of Toll-like receptor (TLR) 4 and TLR2 on CD14+ and CD11c+ cells were examined by FACS. Results: The production of TNFa, IL-1h, IL-6 and IL-8 from TNMC after stimulation with LPS or LTA were significantly lower than those from PBMC. Although CD11c+ cells were observed in both TNMC and PBMC, the incidence of TLR4 or TLR2 expressing cells was lower in TNMC than in PBMC. Conclusions: Results suggest that the decreased expression of TLR4 and TLR2 in TNMC might be associated with the hyporesponses of palatine tonsils against LPS and LTA. D 2003 Published by Elsevier B.V. Keywords: Tonsil; Toll-like receptor; Lipopolysaccharide; Cytokine; Human
1. Background Mucosal surfaces of upper respiratory tract are the first barrier protecting the host from microorganisms and various bacterial antigens such as lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Palatine tonsils are known to be
* Corresponding author. Tel.: +81-9-9275-5407; fax: +81-9-9264-8296. E-mail address: [email protected] (Y. Kurono). 0531-5131/ D 2003 Published by Elsevier B.V. doi:10.1016/S0531-5131(03)01607-8
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associated with the mucosal immune responses as an inductive as well as an effector site, while the tonsils are harbored by pathogenic bacteria. The facts indicate that the immune responses of palatine tonsils against bacterial flora are suppressed by some mechanisms. For instance, LPS is contained in Gram-negative bacteria and is one of the most potent stimulants inducing inflammation, immune responses, microcirculatory dysfunction, tissue damage and septic shock. However, severe inflammatory or immune responses are usually rare to be observed in tonsils in spite of the presence of Gramnegative bacteria such as Haemophilus influenzae as a parasitic bacteria. Recently, innate immune systems mediated by Toll-like receptors (TLR) have been shown as playing an important role in causing inflammation as well as responding against bacteria and bacterial antigens. TLR4 is a receptor for LPS and initiates LPS signaling cascades [1,2]. MD-2 also has been shown to be involved in LPS signaling of TLR4 in human and murine cells [3,4]. However, it is still controversial if TLR2 and TLR4 are involved in LTA- and PGN-induced signaling [5,6]. Moreover, the role of MyD88 in TLR2- and TLR4-mediated signaling is not yet clarified. In the present study, the immune responses of tonsillar mononuclear cells against LPS, LTA and PGN were examined. Further, the expression of TLR on tonsillar mononuclear cells was investigated in order to reveal the suppressive mechanism in tonsils against bacterial antigens. 2. Materials and methods 2.1. Preparation of mononuclear cells from human palatine tonsil and peripheral blood Patients with recurrent tonsillitis or chronic tonsillitis who underwent tonsillectomy in our clinic were enrolled in the study after having given informed consent. All patients had tonsillectomy during the period when they had no symptom of acute inflammation. Venous blood was obtained at the same time with tonsillectomy. Tonsil mononuclear cells (TNMC) and peripheral blood mononuclear cells (PBMC) were isolated by density-gradient centrifugation. 2.2. Bacterial antigens and stimulants H. influenzae endotoxin (HIE) was prepared from nontypable H. influenzae isolated from the nasopharynx of the patients with otitis media as previously described [7]. LPS of Escherichia coli, LTA and PGN of S. pyogenes were purchased from commercial sources. TNMC and PBMC (1.0 106/ml) were cultured in a 24-well tissue culture plate (Becton Dickinson Labware, USA) with or without a bacterial antigens such as HIE, LPS, LTA or PGN (10 Ag/ml) at 37 jC in 5% CO2 for 24 h. Then, the supernatants were collected and used for enzyme-linked immunosorbent assay (ELISA). 2.3. ELISA The concentrations of cytokines such as TNFa, IL-1h, IL-6 and IL-8 were determined by ELISA using immunoassay kits (BioSource International, CA, USA) according to the manufacturer’s protocol.
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2.4. Flow cytometry analysis The cell populations were analyzed with a flow cytometer (COULTERR EPICSR XL flow cytometer, Coulter, USA). TNMC and PBMC were stained with fluorescein (FITC)conjugate anti-human CD14 (eBioscience, San Diego, CA, USA), FITC-conjugate antihuman CD11c (Southern Biotechnology Associates, USA), phycoerythrin (PE)-conjugate anti-human TLR2 (eBioscience) and PE-conjugate anti-human TLR4 (eBioscience). 2.5. Reverse transcription – polymerase chain reaction analysis (RT-PCR) The total RNA was extracted from the cells using high pure RNA kit (Roche, Germany) and reverse-transcribed using Superscript II (GibcoBRL). Then, PCR was performed with Taq polymerase together with specific primer sets of TLR2, TLR4 and MyD88. 3. Results 3.1. Production of cytokines from TNMC stimulated with bacterial antigens The production of TNFa, IL-1h, IL-6 and IL-8 from TNMC after the stimulation with HIE, LPS, LTA and PGN was significantly less than that from PBMC. The results of the TNFa production after the stimulation with the several bacterial antigens are shown on Fig. 1.
Fig. 1. The production of TNFa in TNMC and PBMC detected by ELISA. *p < 0.05, **p < 0.01 compared with PBMC.
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3.2. Expression of TLR2, TLR4 on CD14+ or CD11c+ cells in TNMC Only a few CD14+ cells were observed in TNMC and the incidence of TLR4expressing CD14+ cells in TNMC was lower than that in PBMC. In contrast, TLR4 was expressed on most of CD14+ cells in PBMC. CD11c+ cells were found in 4% of TNMC. However, TLR4 and TLR2 were expressed on few CD11c+ cells in TNMC. 3.3. Expression of gene targets of TLR2, TLR4 and MyD88 in TNMC RT-PCR showed that TLR2 but not TLR4 mRNA was expressed in TNMC. On the other hand, the expression of both TLR2 and TLR4 mRNA was found in PBMC. Interestingly, the expression of MyD88 mRNA was observed in both TNMC and PBMC. 4. Conclusions The production of cytokines in TNMC in response to LPS, LTA or PGN was significantly lower than that in PBMC. The results might explain the hyporesponses of palatine tonsils against bacterial antigens and indicate the presence of some mechanisms suppressing the immune responses in palatine tonsils. Since the incidence of CD14+ cells and the numbers of TLR4 or TLR2 expressing cells were lower in TNMC than in PBMC, those factors might be associated with the hyporesponsiveness of palatine tonsils against bacterial antigens. References [1] K. Hoshino, O. Takeuchi, T. Kawai, et al., Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the LPS gene product, J. Immunol. 162 (1999) 3749 – 3752. [2] N.C. Arbour, E. Lorenz, B.C. Schutte, et al., TLR4 mutations are associated with endotoxin hyporesponsiveness in humans, Nat. Genet. 25 (2000) 187 – 191. [3] R. Shimazu, S. Akashi, H. Ogata, et al., MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4, J. Exp. Med. 189 (1999) 1777 – 1782. [4] S. Akashi, R. Shimazu, H. Ogata, et al., Cutting edge: cell surface expression and lipopolysaccharede signaling via the Toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages, J. Immunol. 164 (2000) 3471 – 3475. [5] R. Schwandner, R. Dziarski, H. Wesche, et al., Peptidoglycan-and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2, J. Biol. Chem. 274 (1999) 17406 – 17409. [6] O. Takeuchi, K. Hoshino, T. Kawai, et al., Differential roles of TLR2 and TLR4 in recognition of gramnegative and gram-positive bacterial cell wall components, Immunity 11 (1999) 443 – 451. [7] T. Miyanohara, M. Ushikai, S. Matsune, et al., Effects of clarithromycin on cultured human nasal epithelial cells and fibroblasts, Laryngoscope 110 (2000) 126 – 131.
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Immune responses of palatine tonsil against bacterial antigens Norimitsu Tanaka, Satoshi Fukuyama, Masato Ushikai, Keiichi Miyashita, Yuichi Kurono * Department of Otolaryngology, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan Received 5 August 2003; received in revised form 15 September 2003; accepted 16 September 2003
Abstract. Background: Although various bacterial antigens are frequently found in upper respiratory tract mucosal secretions, fatal responses, such as endotoxin shock, are rare, suggesting the presence of some regulatory system in mucosal as well as systemic tissues. In the present study, we examined the immune responses of tonsillar mononuclear cells against bacterial antigens. Materials and methods: Mononuclear cells were isolated from the palatine tonsils and peripheral blood (tonsil mononuclear cells [TNMC] and peripheral blood mononuclear cells [PBMC]) of patients who underwent palatine tonsillectomy. TNMC and PBMC were stimulated using lipopolysaccharide (LPS) or lipoteichoic acid (LTA) for 24 h, and cytokine production were determined by ELISA. Furthermore, the expression of Toll-like receptor (TLR) 4 and TLR2 on CD14+ and CD11c+ cells were examined by FACS. Results: The production of TNFa, IL-1h, IL-6 and IL-8 from TNMC after stimulation with LPS or LTA were significantly lower than those from PBMC. Although CD11c+ cells were observed in both TNMC and PBMC, the incidence of TLR4 or TLR2 expressing cells was lower in TNMC than in PBMC. Conclusions: Results suggest that the decreased expression of TLR4 and TLR2 in TNMC might be associated with the hyporesponses of palatine tonsils against LPS and LTA. D 2003 Published by Elsevier B.V. Keywords: Tonsil; Toll-like receptor; Lipopolysaccharide; Cytokine; Human
1. Background Mucosal surfaces of upper respiratory tract are the first barrier protecting the host from microorganisms and various bacterial antigens such as lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Palatine tonsils are known to be
* Corresponding author. Tel.: +81-9-9275-5407; fax: +81-9-9264-8296. E-mail address: [email protected] (Y. Kurono). 0531-5131/ D 2003 Published by Elsevier B.V. doi:10.1016/S0531-5131(03)01607-8
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associated with the mucosal immune responses as an inductive as well as an effector site, while the tonsils are harbored by pathogenic bacteria. The facts indicate that the immune responses of palatine tonsils against bacterial flora are suppressed by some mechanisms. For instance, LPS is contained in Gram-negative bacteria and is one of the most potent stimulants inducing inflammation, immune responses, microcirculatory dysfunction, tissue damage and septic shock. However, severe inflammatory or immune responses are usually rare to be observed in tonsils in spite of the presence of Gramnegative bacteria such as Haemophilus influenzae as a parasitic bacteria. Recently, innate immune systems mediated by Toll-like receptors (TLR) have been shown as playing an important role in causing inflammation as well as responding against bacteria and bacterial antigens. TLR4 is a receptor for LPS and initiates LPS signaling cascades [1,2]. MD-2 also has been shown to be involved in LPS signaling of TLR4 in human and murine cells [3,4]. However, it is still controversial if TLR2 and TLR4 are involved in LTA- and PGN-induced signaling [5,6]. Moreover, the role of MyD88 in TLR2- and TLR4-mediated signaling is not yet clarified. In the present study, the immune responses of tonsillar mononuclear cells against LPS, LTA and PGN were examined. Further, the expression of TLR on tonsillar mononuclear cells was investigated in order to reveal the suppressive mechanism in tonsils against bacterial antigens. 2. Materials and methods 2.1. Preparation of mononuclear cells from human palatine tonsil and peripheral blood Patients with recurrent tonsillitis or chronic tonsillitis who underwent tonsillectomy in our clinic were enrolled in the study after having given informed consent. All patients had tonsillectomy during the period when they had no symptom of acute inflammation. Venous blood was obtained at the same time with tonsillectomy. Tonsil mononuclear cells (TNMC) and peripheral blood mononuclear cells (PBMC) were isolated by density-gradient centrifugation. 2.2. Bacterial antigens and stimulants H. influenzae endotoxin (HIE) was prepared from nontypable H. influenzae isolated from the nasopharynx of the patients with otitis media as previously described [7]. LPS of Escherichia coli, LTA and PGN of S. pyogenes were purchased from commercial sources. TNMC and PBMC (1.0 106/ml) were cultured in a 24-well tissue culture plate (Becton Dickinson Labware, USA) with or without a bacterial antigens such as HIE, LPS, LTA or PGN (10 Ag/ml) at 37 jC in 5% CO2 for 24 h. Then, the supernatants were collected and used for enzyme-linked immunosorbent assay (ELISA). 2.3. ELISA The concentrations of cytokines such as TNFa, IL-1h, IL-6 and IL-8 were determined by ELISA using immunoassay kits (BioSource International, CA, USA) according to the manufacturer’s protocol.
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2.4. Flow cytometry analysis The cell populations were analyzed with a flow cytometer (COULTERR EPICSR XL flow cytometer, Coulter, USA). TNMC and PBMC were stained with fluorescein (FITC)conjugate anti-human CD14 (eBioscience, San Diego, CA, USA), FITC-conjugate antihuman CD11c (Southern Biotechnology Associates, USA), phycoerythrin (PE)-conjugate anti-human TLR2 (eBioscience) and PE-conjugate anti-human TLR4 (eBioscience). 2.5. Reverse transcription – polymerase chain reaction analysis (RT-PCR) The total RNA was extracted from the cells using high pure RNA kit (Roche, Germany) and reverse-transcribed using Superscript II (GibcoBRL). Then, PCR was performed with Taq polymerase together with specific primer sets of TLR2, TLR4 and MyD88. 3. Results 3.1. Production of cytokines from TNMC stimulated with bacterial antigens The production of TNFa, IL-1h, IL-6 and IL-8 from TNMC after the stimulation with HIE, LPS, LTA and PGN was significantly less than that from PBMC. The results of the TNFa production after the stimulation with the several bacterial antigens are shown on Fig. 1.
Fig. 1. The production of TNFa in TNMC and PBMC detected by ELISA. *p < 0.05, **p < 0.01 compared with PBMC.
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3.2. Expression of TLR2, TLR4 on CD14+ or CD11c+ cells in TNMC Only a few CD14+ cells were observed in TNMC and the incidence of TLR4expressing CD14+ cells in TNMC was lower than that in PBMC. In contrast, TLR4 was expressed on most of CD14+ cells in PBMC. CD11c+ cells were found in 4% of TNMC. However, TLR4 and TLR2 were expressed on few CD11c+ cells in TNMC. 3.3. Expression of gene targets of TLR2, TLR4 and MyD88 in TNMC RT-PCR showed that TLR2 but not TLR4 mRNA was expressed in TNMC. On the other hand, the expression of both TLR2 and TLR4 mRNA was found in PBMC. Interestingly, the expression of MyD88 mRNA was observed in both TNMC and PBMC. 4. Conclusions The production of cytokines in TNMC in response to LPS, LTA or PGN was significantly lower than that in PBMC. The results might explain the hyporesponses of palatine tonsils against bacterial antigens and indicate the presence of some mechanisms suppressing the immune responses in palatine tonsils. Since the incidence of CD14+ cells and the numbers of TLR4 or TLR2 expressing cells were lower in TNMC than in PBMC, those factors might be associated with the hyporesponsiveness of palatine tonsils against bacterial antigens. References [1] K. Hoshino, O. Takeuchi, T. Kawai, et al., Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the LPS gene product, J. Immunol. 162 (1999) 3749 – 3752. [2] N.C. Arbour, E. Lorenz, B.C. Schutte, et al., TLR4 mutations are associated with endotoxin hyporesponsiveness in humans, Nat. Genet. 25 (2000) 187 – 191. [3] R. Shimazu, S. Akashi, H. Ogata, et al., MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4, J. Exp. Med. 189 (1999) 1777 – 1782. [4] S. Akashi, R. Shimazu, H. Ogata, et al., Cutting edge: cell surface expression and lipopolysaccharede signaling via the Toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages, J. Immunol. 164 (2000) 3471 – 3475. [5] R. Schwandner, R. Dziarski, H. Wesche, et al., Peptidoglycan-and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2, J. Biol. Chem. 274 (1999) 17406 – 17409. [6] O. Takeuchi, K. Hoshino, T. Kawai, et al., Differential roles of TLR2 and TLR4 in recognition of gramnegative and gram-positive bacterial cell wall components, Immunity 11 (1999) 443 – 451. [7] T. Miyanohara, M. Ushikai, S. Matsune, et al., Effects of clarithromycin on cultured human nasal epithelial cells and fibroblasts, Laryngoscope 110 (2000) 126 – 131.