Surfactant Protein A Inhibits Peptidoglycan-induced Tumor Necrosis Factor-α Secretion in U937 Cells and Alveolar Macrophages by Direct Interaction with Toll-like Receptor 2
2002; Elsevier BV; Volume: 277; Issue: 9 Linguagem: Inglês
10.1074/jbc.m106671200
ISSN1083-351X
AutoresSeiji Murakami, Daisuke Iwaki, Hiroaki Mitsuzawa, Hitomi Sano, Hiroki Takahashi, Dennis R. Voelker, Toyoaki Akino, Yoshio Kuroki,
Tópico(s)Pediatric health and respiratory diseases
ResumoPulmonary surfactant protein A (SP-A) plays an important role in modulation of the innate immune system of the lung. Peptidoglycan (PGN), a cell wall component of Gram-positive bacteria, is known to elicit excessive proinflammatory cytokine production from immune cells. In this study we investigated whether SP-A interacts with PGN and alters PGN-elicited cellular responses. Binding studies demonstrate that PGN is not a ligand for SP-A. However, SP-A significantly reduced PGN-elicited tumor necrosis factor α (TNF-α) secretion by U937 cells and rat alveolar macrophages. The inhibitory effect on TNF-α secretion was dependent upon SP-A concentrations in physiological range. Coincubation of SP-A and PGN with human embryonic kidney 293 cells that had been transiently transfected with the cDNA of Toll-like receptor 2 (TLR2), a cell signaling receptor for PGN, significantly attenuated PGN-induced nuclear factor-κB activity. SP-A directly bound to a soluble form of the recombinant extracellular TLR2 domain (sTLR2). Coincubation of sTLR2 with SP-A significantly reduced the binding of sTLR2 to PGN. These results indicate that the direct interaction of SP-A with TLR2 alters PGN-induced cell signaling. We propose that SP-A modulates inflammatory responses against the bacterial components by interactions with pattern-recognition receptors. Pulmonary surfactant protein A (SP-A) plays an important role in modulation of the innate immune system of the lung. Peptidoglycan (PGN), a cell wall component of Gram-positive bacteria, is known to elicit excessive proinflammatory cytokine production from immune cells. In this study we investigated whether SP-A interacts with PGN and alters PGN-elicited cellular responses. Binding studies demonstrate that PGN is not a ligand for SP-A. However, SP-A significantly reduced PGN-elicited tumor necrosis factor α (TNF-α) secretion by U937 cells and rat alveolar macrophages. The inhibitory effect on TNF-α secretion was dependent upon SP-A concentrations in physiological range. Coincubation of SP-A and PGN with human embryonic kidney 293 cells that had been transiently transfected with the cDNA of Toll-like receptor 2 (TLR2), a cell signaling receptor for PGN, significantly attenuated PGN-induced nuclear factor-κB activity. SP-A directly bound to a soluble form of the recombinant extracellular TLR2 domain (sTLR2). Coincubation of sTLR2 with SP-A significantly reduced the binding of sTLR2 to PGN. These results indicate that the direct interaction of SP-A with TLR2 alters PGN-induced cell signaling. We propose that SP-A modulates inflammatory responses against the bacterial components by interactions with pattern-recognition receptors. Pulmonary surfactant is a mixture of lipids and proteins that functions to keep alveoli from collapsing at expiration (1King R.J. Clements J.A. Am. J. Physiol. 1972; 223: 707-714Crossref PubMed Scopus (146) Google Scholar). Surfactant protein A (SP-A) 1SP-Asurfactant protein ASP-Dsurfactant protein DMBPmannose-binding proteinPGNpeptidoglycanLPSlipopolysaccharideTLR2Toll-like receptor 2sTLR2a soluble form of recombinant extracellular TLR2 domainHEK293 cellshuman embryonic kidney 293 cellsNF-κBnuclear factor-κBTNFtumor necrosis factorFCSfetal calf serumPBSphosphate-buffered salinePMAphorbol 12-myristate 13-acetateBSAbovine serum albumin 1SP-Asurfactant protein ASP-Dsurfactant protein DMBPmannose-binding proteinPGNpeptidoglycanLPSlipopolysaccharideTLR2Toll-like receptor 2sTLR2a soluble form of recombinant extracellular TLR2 domainHEK293 cellshuman embryonic kidney 293 cellsNF-κBnuclear factor-κBTNFtumor necrosis factorFCSfetal calf serumPBSphosphate-buffered salinePMAphorbol 12-myristate 13-acetateBSAbovine serum albumin is the major protein constituent of the surfactant (2Kuroki Y. Voelker D.R. J. Biol. Chem. 1994; 269: 25943-25946Abstract Full Text PDF PubMed Google Scholar). SP-A belongs to the collectin subgroup of the C-type lectin superfamily along with surfactant protein D (SP-D), mannose-binding protein (MBP), and conglutinin (3Day A.J. Biochem. Soc. Trans. 1994; 22: 83-88Crossref PubMed Scopus (150) Google Scholar). SP-A is now recognized as playing an important role in regulating innate immunity within the lung. This protein enhances the phagocytosis of Staphylococcus aureus (4van Iwaarden F. Welmers B. Verhoef J. Haagsman H.P. van Golde L.M.G. Am. J. Respir. Cell Mol. Biol. 1990; 2: 91-98Crossref PubMed Scopus (311) Google Scholar), herpes simplex virus type I (5van Iwaarden J.F. van Strijp J.A.G. Ebskamp M.J.M. Welmers A.C. Verhoef J. van Golde L.M.G. Am. J. Physiol. 1991; 261: L204-L209PubMed Google Scholar), type A Hemophilus influenzae (6McNeely T.B. Coonrod J.D. Am. J. Respir. Cell Mol. Biol. 1994; 11: 114-122Crossref PubMed Scopus (99) Google Scholar),Mycobacterium tuberculosis (7Gaynor C.D. McCormack F.X. Voelker D.R. McGowan S.E. Schlesinger L.S. J. Immunol. 1995; 155: 5343-5351PubMed Google Scholar), and Klebsiella(8Kabha K. Schmegner J. Keisari Y. Parolis H. Schlepper-Schaefer J. Ofek I. Am. J. Physiol. 1997; 272: L344-L352PubMed Google Scholar) by alveolar macrophages. SP-A can bind with broad specificity to a variety of microorganisms including herpes simplex virus type I (9Van Iwaarden J.F. van Strijp J.A.G. Visser H. Haagsman H.P. Verhoef J. van Golde L.M.G. J. Biol. Chem. 1992; 267: 25039-25043Abstract Full Text PDF PubMed Google Scholar),Pneumocystis carinii (10Zimmerman P.E. Voelker D.R. McCormack F.X. Paulsrud J.R. Martin W.J.I. J. Clin. Invest. 1992; 89: 143-149Crossref PubMed Scopus (170) Google Scholar), and Aspergillus fumigatus (11Allen M.J. Harbeck R. Smith B. Voelker D.R. Mason R.J. Infect. Immun. 1999; 67: 4563-4569Crossref PubMed Google Scholar). The characteristics of transgenic mice with null alleles for SP-A (12LeVine A.M. Bruno M.D. Huelsman K.M. Ross G.F. Whitsett J.A. Korfhagen T.R. J. Immunol. 1997; 158: 4336-4340PubMed Google Scholar, 13LeVine A.M. Kurak K.E. Bruno M.D. Stark J.M. Whitsett J.A. Korfhagen T.M. Am. J. Respir. Cell Mol. Biol. 1998; 19: 700-708Crossref PubMed Scopus (281) Google Scholar, 14LeVine A.M. Kurak K.E. Wright J.R. Watford W.T. Bruno M.D. Ross G.F. Whitsett J.A. Korfhagen T.R. Am. J. Respir. Cell Mol. Biol. 1999; 20: 279-286Crossref PubMed Scopus (170) Google Scholar) provide compelling in vivoevidence that SP-A is an important component of the innate immune system within the lung. Animals lacking SP-A exhibit reduced bacterial clearance and elevated pulmonary inflammation in response to microbial challenge.Gram-positive bacteria including S. aureus cause infections that can be life-threatening. Peptidoglycan (PGN), a major cell wall component of Gram-positive bacteria, is a polymer of alternatingN-acetylglucosaminyl and N-acetylmuramyl glycan whose residues are cross-linked by short peptides (15Schleifer K.H. Kandler O. Bacteriol. Rev. 1972; 36: 407-477Crossref PubMed Google Scholar). PGN, like lipopolysaccharides (LPS) from Gram-negative bacteria, can elicit the excessive release of proinflammatory cytokines from immune cells, which contribute to many of the adverse clinical manifestations of bacterial infections (16Gupta D. Jin Y. Dziarski R. J. Immunol. 1995; 155: 2620-2630PubMed Google Scholar, 17Raetz C.R.H. Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1030) Google Scholar, 18Rosenthal R.S. Dziarski R. Methods Enzymol. 1994; 235: 253-285Crossref PubMed Scopus (134) Google Scholar). CD14 and Toll-like receptors (TLRs) function as pattern-recognition receptors for these bacterial ligands (19Lien E. Sellati T.J. Yoshimura A. Flo T.H. Rawadi G. Finberg R.W. Carroll J.D. Espevik T. Ingalls R.R. Radolf J.D. Golenbock D.T. J. Biol. Chem. 1999; 274: 33419-33425Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar, 20Pugin J. Heumann I.D. Tomasz A. Kravchenko V.V. Akamatsu Y. Nishijima M. Glauser M.P. Tobias P.S. Ulvetch R.J. Immunity. 1994; 6: 509-516Abstract Full Text PDF Scopus (622) Google Scholar). TLRs possess an intracellular domain homologous to that of interleukin-1 receptor (21Medzhitov R. Preston-Hurlburt P. Janeway J.C.A. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4379) Google Scholar) and participate in NF-κB signaling cascades elicited by LPS and PGN. In vivo studies with mice harboring null alleles for TLR2 provide strong evidence that TLR2 is responsible for PGN-induced signaling (22Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2759) Google Scholar, 23Takeuchi O. Hoshino K. Akira S. J. Immunol. 2000; 165: 5392-5396Crossref PubMed Scopus (898) Google Scholar). Recent in vitro studies with overexpression experiments also demonstrate that TLR2 confers cell responsiveness to PGN (24Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1421) Google Scholar, 25Yoshimura A. Lien E. Ingalls R.R. Tuomanen E. Dziarski R. Golenbock D. J. Immunol. 1999; 163: 1-5PubMed Google Scholar). Although CD14 alone appears incapable of signaling because it lacks a transmembrane domain, it is still capable of enhancing PGN-induced NF-κB signaling mediated by TLR2 (24Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1421) Google Scholar).SP-A binds rough serotypes but not smooth serotypes of LPS (26Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar, 27Van Iwaarden J.F. Pikaar J.C. Storm J. Brouwer E. Verhoef J. Oosting R.S. Van Golde L.M.G. Van Strip J.A.G. Biochem. J. 1994; 303: 407-411Crossref PubMed Scopus (144) Google Scholar). The protein inhibits TNF-α secretion induced by smooth LPS (26Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar, 28McIntosh J.C. Mervin-Blake S. Conner E. Wright J.R. Am. J. Physiol. 1996; 271: L310-L319Crossref PubMed Google Scholar) but modestly enhances TNF-α release induced by rough LPS (26Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar) in alveolar macrophages and U937 cells. We have shown that the direct interaction of SP-A with CD14 is the likely mechanism for modulating LPS-elicited cellular responses (26Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar). In addition to SP-A, the collectins, SP-D, and MBP also bind CD14 (29Sano H. Chiba H. Iwaki D. Sohma H. Voelker D.R. Kuroki Y. J. Biol. Chem. 2000; 275: 22442-22451Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 30Chiba H. Sano H. Iwaki D. Murakami S. Mutsuzawa H. Takahashi T. Konishi M. Takahashi H. Kuroki Y. Infect. Immun. 2001; 69: 1587-1592Crossref PubMed Scopus (28) Google Scholar), suggesting this may be an important property of this protein family. The interaction of SP-D with CD14 may be also accompanied by modulation of the cellular response to ligands such as LPS. SP-A-deficient mice exhibit significant increases in the production of TNF-α and nitric oxide after intratracheal instillation of smooth LPS when compared with wild type mice (31Borron P. McIntosh J.C. Korfhagen T.R. Whitsett J.A. Taylor J. Wright J.R. Am. J. Physiol. 2000; 278: L840-L847Crossref PubMed Google Scholar). Intratracheal administration of SP-A to SP-A-deficient mice diminished the production of the proinflammatory cytokines. Taken together with the in vitro observations of the inhibitory function of SP-A on smooth LPS-elicited TNF-α secretion (26Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar, 28McIntosh J.C. Mervin-Blake S. Conner E. Wright J.R. Am. J. Physiol. 1996; 271: L310-L319Crossref PubMed Google Scholar), there is growing evidence that SP-A promotes an anti-inflammatory response to some bacterial ligands.The role of SP-A in modulating innate immunity may be a key element to understanding the dual requirement of the lung to remain relatively quiescent in its inflammatory response to routine daily burdens of inspired LPS and easily dispatched microorganisms while remaining competent to mount a potent and vigorous response to specialized pulmonary pathogens. Many inhaled pathogens that reach the alveolus are thought to interact immediately with lung collectins because these proteins are highly enriched at this biological interface.PGN elicits many of the clinical manifestations of Gram-positive organisms, and we focused on the interactions of SP-A with PGN and the consequences of the interaction upon TNF-α secretion by alveolar macrophages and U937 cells. The specific objectives of this study were to determine 1) the interaction of SP-A with PGN, 2) the role of SP-A in modulating leukocyte cytokine responses to PGN, 3) the interaction between SP-A and TLR2, and 4) the role of SP-A in altering PGN interaction with TLR2. Our findings demonstrate that PGN is not a ligand for SP-A and that SP-A reduces the PGN-elicited proinflammatory cytokine release by reducing TLR2-PGN interactions.DISCUSSIONThis study demonstrates that SP-A inhibits PGN-induced TNF-α secretion by U937 cells and alveolar macrophages. In addition, SP-A attenuates PGN-elicited NF-κB activation in TLR2-transfected HEK293 cells. Direct binding studies also provide clear evidence that SP-A and PGN bind the extracellular TLR2 domain. Binding competition analysis demonstrates that SP-A can alter the interaction of TLR2 with PGN. Direct interaction of SP-A with TLR2 and down-regulation of TLR signaling by SP-A constitute the likely mechanisms by which SP-A inhibits PGN-induced cellular responses. This study supports an antiinflammatory role for SP-A in controlling the host response to PGN from S. aureus.Initial experiments performed in this study focused on interactions between SP-A and PGN. Solid phase binding did not reveal any interaction between SP-A and PGN. However, we were not able to quantify the amount of PGN bound to the wells. We therefore conducted binding assays in solution with sedimentable PGN. Consistent with the solid phase binding results, SP-A failed to bind to PGN in solution. Taken together, we conclude that PGN derived from S. aureus is not a ligand for SP-A.Previous studies from this (26Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar) and other (28McIntosh J.C. Mervin-Blake S. Conner E. Wright J.R. Am. J. Physiol. 1996; 271: L310-L319Crossref PubMed Google Scholar) laboratories demonstrate that SP-A inhibits smooth LPS-elicited TNF-α secretion by alveolar macrophages and U937 cells. SP-A has also been shown to down-regulate proinflammatory cytokine production elicited by Candida albicans (44Rosseau S. Hammerl P. Maus U. Günther A. Seeger W. Grimminger F. Lohmeyer J. J. Immunol. 1999; 163: 4495-4502PubMed Google Scholar). In contrast to these studies, another study (45Kremlev S.G. Phelps D.S. Am. J. Physiol. 1994; 267: L712-L719PubMed Google Scholar) reports that SP-A stimulates the production of cytokines including TNF-α, interleukin-1, and interleukin-6. However, in vivostudies (31Borron P. McIntosh J.C. Korfhagen T.R. Whitsett J.A. Taylor J. Wright J.R. Am. J. Physiol. 2000; 278: L840-L847Crossref PubMed Google Scholar) using SP-A (−/−) mice give results consistent with an inhibitory role of SP-A on inflammatory cytokine production. SP-A (−/−) mice produced significantly increased TNF-α and nitric oxide in the lung compared with SP-A (+/+) mice after intratracheal administration of smooth LPS. Instillation of SP-A to SP-A (−/−) mice restored regulation of proinflammatory cytokine production. Another study (43LeVine A.M. Whitsett J.A. Gwozdz J.A. Richradson T.R. Fisher J.H. Burhans M.S. Korfhagen T.R. J. Immunol. 2000; 165: 3934-3940Crossref PubMed Scopus (315) Google Scholar) with SP-A (−/−) mice has shown that infection with group B streptococcus and H. influenzae increased the proinflammatory cytokines in the lung. In this report we sought to determine whether the antiinflammatory role for SP-A was also applicable to a PGN stimulus. This study demonstrates in vitro that SP-A attenuates TNF-α release induced by PGN derived from S. aureus. The SP-A effect on PGN responsiveness occurs with the U937 cell line as well as primary alveolar macrophages. The results obtained from this and the previous in vivo (31Borron P. McIntosh J.C. Korfhagen T.R. Whitsett J.A. Taylor J. Wright J.R. Am. J. Physiol. 2000; 278: L840-L847Crossref PubMed Google Scholar, 43LeVine A.M. Whitsett J.A. Gwozdz J.A. Richradson T.R. Fisher J.H. Burhans M.S. Korfhagen T.R. J. Immunol. 2000; 165: 3934-3940Crossref PubMed Scopus (315) Google Scholar) and in vitro (26Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar, 28McIntosh J.C. Mervin-Blake S. Conner E. Wright J.R. Am. J. Physiol. 1996; 271: L310-L319Crossref PubMed Google Scholar, 44Rosseau S. Hammerl P. Maus U. Günther A. Seeger W. Grimminger F. Lohmeyer J. J. Immunol. 1999; 163: 4495-4502PubMed Google Scholar) studies are consistent with the idea that SP-A plays a role in modulating cytokine production and inflammatory responses during bacterial infection within the lung.We also sought to elucidate the mechanism by which SP-A inhibits PGN-elicited TNF-α secretion. Because this study has shown that SP-A does not bind to PGN, the mechanism of the inhibitory effect must be different from that by which MBP inhibits cellular responses caused by streptococcal cell wall components (46Soell M. Lett E. Holveck F. Scholler M. Wachsmann D. Klein J.-P. J. Immunol. 1995; 154: 851-860PubMed Google Scholar). In the latter case, the direct interaction of MBP with streptococcal rhamnose glucose polymer (RGP) inhibits RGP-induced TNF-α secretion. A previous study (26Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar) from this laboratory suggests that one of the possible mechanisms by which SP-A modulates LPS-induced cytokine expression is likely to be due to the interaction of SP-A with the LPS receptor, CD14. CD14 and TLR2 function as pattern recognition receptors for PGN (19Lien E. Sellati T.J. Yoshimura A. Flo T.H. Rawadi G. Finberg R.W. Carroll J.D. Espevik T. Ingalls R.R. Radolf J.D. Golenbock D.T. J. Biol. Chem. 1999; 274: 33419-33425Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar, 20Pugin J. Heumann I.D. Tomasz A. Kravchenko V.V. Akamatsu Y. Nishijima M. Glauser M.P. Tobias P.S. Ulvetch R.J. Immunity. 1994; 6: 509-516Abstract Full Text PDF Scopus (622) Google Scholar). Because TLR2-deficient mice were hyporesponsive to PGN (22Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2759) Google Scholar, 23Takeuchi O. Hoshino K. Akira S. J. Immunol. 2000; 165: 5392-5396Crossref PubMed Scopus (898) Google Scholar) and transfection with a TLR2 cDNA conferred cell responsiveness to PGN on HEK293 cells (24Schwandner R. Dziarski R. Wesche H. Rothe M. Kirschning C.J. J. Biol. Chem. 1999; 274: 17406-17409Abstract Full Text Full Text PDF PubMed Scopus (1421) Google Scholar), TLR2 is concluded to be responsible for PGN-induced cellular responses. Although CD14 enhances PGN-induced cell signaling mediated through TLR2, TLR2 alone can induce significant NF-κB activation in response to PGN. In addition, because PGN- or LPS-elicited TNF-α expression is coupled with TLR-mediated NF-κB signaling, we examined the effect of SP-A on PGN-induced NF-κB activation in TLR2-transfected HEK293 cells. SP-A significantly reduced the measured NF-κB activity, indicating that SP-A can alter TLR2-mediated NF-κB signaling.Next, we constructed a soluble form of recombinant extracellular TLR2 domain (sTLR2) and isolated sTLR2 protein expressed by the baculovirus-insect cell expression system. The direct binding of PGN to the extracellular TLR2 domain has now been demonstrated. Additional details about this binding reaction will be described elsewhere.2 The preincubation of SP-A with sTLR2 significantly reduced the binding of sTLR2 to PGN. These results obtained from the cell-free system are essentially consistent with those obtained using U937 cells and alveolar macrophages, although the magnitudes of the SP-A inhibition are different. The expression of TLR2 has been demonstrated in U937 cells (38Kirschning C.J. Wesche H. Ayres T.M. Rothe M. J. Exp. Med. 1998; 188: 2091-2097Crossref PubMed Scopus (654) Google Scholar), leukocytes (47Chaudhary P.M. Ferguson C. Nguyen V. Nguyen O. Massa H.F. Eby M. Jasmin A. Trask B.J. Hood L. Nelson P., S. Blood. 1998; 91: 4020-4027Crossref PubMed Google Scholar), and lung (48Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1438) Google Scholar). Taken together, these data support the idea that the direct interaction of SP-A with the extracellular TLR2 domain interfere with PGN binding to TLR2, resulting in decreased TLR2-mediated NF-κB signaling and reduced TNF-α secretion from immune cells. From the present and previous (26Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. J. Immunol. 1999; 163: 387-395PubMed Google Scholar) studies we now propose that SP-A modulates cellular responses induced by bacterial ligands through direct interactions with pattern recognition receptors, CD14, and/or TLR.SP-A almost completely abrogated PGN-induced TNF-α secretion from U937 cells. However, this protein did not completely inhibit PGN-induced cytokine release in alveolar macrophages. The difference between SP-A's inhibitory effects on U937 cells and alveolar macrophages may be due to the different capacity of these cells to secrete TNF-α. Ten μg/ml PGN induced secretion of more than 150 ng/ml TNF-α in alveolar macrophages, whereas the same concentration of PGN applied to U937 cells produced 7.5 ng/ml TNF-α. SP-A exhibited a greater inhibitory effect at lower concentrations of PGN than at higher concentrations in alveolar macrophages, with 80% inhibition at 1 μg/ml versus 33% inhibition at 10 μg/ml. SP-A did significantly attenuate, but did not completely abrogate, PGN-induced NF-κB activation in TLR2-transfected cells or sTLR2 binding to solid phase PGN. The absence of a one to one correlation of the SP-A effect between TNF-α secretion and NF-κB activity or sTLR2 binding to PGN may be a consequence of TLR2 overexpression or altered affinity for sTLR2. The naturally occurring soluble form of mouse TLR4, which is expressed by alternatively spliced mouse TLR4 mRNA, has been shown to only partially block LPS-elicited NF-κB activation (49Iwami K. Matsuguchi T. Masuda A. Kikuchi T. Musikacharoen T. Yoshikai Y. J. Immunol. 2001; 165: 6682-6686Crossref Scopus (224) Google Scholar), indicating that the extracellular domain alone may not explain all the activity of the receptor-ligand interaction. Clearly, more detailed biochemical data regarding SP-A-TLR2 and PGN-TLR2 interactions and the mechanism of signal transduction and the terminal inhibitory event are required.We have previously shown that SP-A and SP-D bind CD14 by different mechanisms (29Sano H. Chiba H. Iwaki D. Sohma H. Voelker D.R. Kuroki Y. J. Biol. Chem. 2000; 275: 22442-22451Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). The SP-A neck domain and SP-D lectin domain participate in CD14 binding. SP-A and SP-D recognize a peptide portion and a carbohydrate moiety, respectively, of CD14. MBP also binds CD14 in a manner similar to that of SP-A (30Chiba H. Sano H. Iwaki D. Murakami S. Mutsuzawa H. Takahashi T. Konishi M. Takahashi H. Kuroki Y. Infect. Immun. 2001; 69: 1587-1592Crossref PubMed Scopus (28) Google Scholar). CD14 and TLRs possess homologous structures consisting of leucine-rich repeats characteristic of a short β-sheet and α-helix (50Kobe B. Deisenhofer J. Curr. Opin. Struct. Biol. 1995; 5: 409-416Crossref PubMed Scopus (322) Google Scholar). CD14 and TLR2 contain 10 and 19 leucine-rich repeats, respectively. Because SP-A binds to the CD14 region containing leucine-rich repeats and also binds to deglycosylated sTLR2, 3D. Iwaki and Y. Kuroki, unpublished data. we infer that SP-A may interact with the leucine-rich repeat region of TLR2. The binding of rat SP-A to sCD14 was blocked by a monoclonal antibody that binds to the SP-A neck domain but was not attenuated in the presence of EDTA (29Sano H. Chiba H. Iwaki D. Sohma H. Voelker D.R. Kuroki Y. J. Biol. Chem. 2000; 275: 22442-22451Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), indicating that the SP-A neck domain participates in CD14 binding. In this study the binding of human SP-A to sTLR2 was abolished by a monoclonal antibody whose epitope is located at a region contiguous to the human SP-A region Thr184–Gly194 (42Chiba H. Sano H. Saitoh M. Sohma H. Voelker D.R. Akino T. Kuroki Y. Biochemistry. 1999; 38: 7321-7331Crossref PubMed Scopus (24) Google Scholar). In addition, it was blocked by the presence of EDTA, indicating that the SP-A binding to sTLR2 is Ca2+-dependent and that the carbohydrate recognition domain is involved in sTLR2 binding. These studies reveal the different mechanisms of the SP-A binding to the pattern recognition receptors. The molecular and mechanistic details by which SP-A and other collectins interact with TLRs are now under investigation.It is relatively difficult to determine the actual SP-A concentrationin vivo since the epithelial lining fluid of the alveolus (alveolar hypophase) cannot be directly measured. Nevertheless, the SP-A concentrations can be estimated based on the recovery of the protein in the bronchoalveolar lavage fluids and the extrapolated hypophase volume (100–1000 μl/lung) (51Rennard S.I. Basset G. Lecossier D. O'Donnell K.M. Pinkston P. Martin P.G. Crystal R.G. J. Appl. Physiol. 1986; 60: 532-538Crossref PubMed Scopus (886) Google Scholar, 52Bastacky J. Lee C.Y.C. Goerke J. Koushafar H. Yager D. Kenaga L. Speed T.P. Chen Y. Clements J.A. J. Appl. Physiol. 1995; 79: 1615-1628Crossref PubMed Scopus (238) Google Scholar). The calculated SP-A concentrations in the alveolar hypophase range from 180 μg/ml to 1.8 mg/ml (53Tino M.J. Wright J.R. Am. J. Physiol. 1996; 270: L677-L688PubMed Google Scholar, 54van de Graaf E.D. Jansen H.M. Lutter R. Alberts C. Kobesen J., De Varies I.J. Out T.A. J. Lab. Clin. Med. 1992; 120: 252-263PubMed Google Scholar, 55Wright J.R. Physiol. Rev. 1997; 77: 931-962Crossref PubMed Scopus (500) Google Scholar). The levels of SP-A appear to vary in diseased states, indicating complex responses under conditions of physiological stress. In the rat model, the levels of SP-A mRNA and protein are elevated in response to intratracheal administration of LPS (56McIntosh J.C. Swyers A.H. Fisher J.H. Wright J.R. Am. J. Respir. Cell Mol. Biol. 1996; 15: 509-519Crossref PubMed Scopus (122) Google Scholar). SP-A recovered in the lavage fluid also increases in AIDS-related pneumonia (57Phelps D.S. Rose R.M. Am. Rev. Respir. Dis. 1991; 143: 1072-1075Crossref PubMed Google Scholar). However, in some situations (58Baughman R.P. Sternberg R.I. Hull W. Buchsbaum J.A. Whitsett J. Am. Rev. Respir. Dis. 1993; 147: 653-657Crossref PubMed Scopus (127) Google Scholar), the SP-A concentration in lavage fluid decreases in patients with bacterial pneumonia. Although one cannot yet determine the exact concentrations of SP-A in the hypophase of healthy and diseased human lungs, the SP-A concentrations used in these studies are within the best estimates of the physiological ranges.The respiratory system continually faces exposure to airborne LPS, PGN, and microbes. SP-A may play an important role in the elimination of these microbes by enhancing phagocytosis by alveolar macrophages. Although SP-A enhances microbial clearance, it also appears to be an important element in dampening the inflammatory response to some organisms and their derivative cell surface components. Alveolar macrophages and neutrophils produce proinflammatory cytokines through the CD14/TLR pathway in response to microbial components including PGN, LPS, and lipoteichoic acid. TNF-α is a pivotal mediator of the host responses to infections and triggers inflammatory responses. Because overproduction of TNF-α can cause chronic pathological states especially in the lung, the inhibitory function of SP-A on TNF-α release may be crucial regulatory component for controlling pulmonary inflammation.In conclusion, this study demonstrates that SP-A inhibits TNF-α secretion induced by PGN. The results also reveal that SP-A directly binds TLR2, alters the interaction of TLR2 with PGN, and attenuates downstream signaling events. These findings provide one mechanistic framework by which SP-A can regulate inflammatory responses in the alveolar compartment. Pulmonary surfactant is a mixture of lipids and proteins that functions to keep alveoli from collapsing at expiration (1King R.J. Clements J.A. Am. J. Physiol. 1972; 223: 707-714Crossref PubMed Scopus (146) Google Scholar). Surfactant protein A (SP-A) 1SP-Asurfactant protein ASP-Dsurfactant protein DMBPmannose-binding proteinPGNpeptidoglycanLPSlipopolysaccharideTLR2Toll-like receptor 2sTLR2a soluble form of recombinant extracellular TLR2 domainHEK293 cellshuman embryonic kidney 293 cellsNF-κBnuclear factor-κBTNFtumor necrosis factorFCSfetal calf serumPBSphosphate-buffered salinePMAphorbol 12-myristate 13-acetateBSAbovine serum albumin 1SP-Asurfactant protein ASP-Dsurfactant protein DMBPmannose-binding proteinPGNpeptidoglycanLPSlipopolysaccharideTLR2Toll-like receptor 2sTLR2a soluble form of recombinant extracellular TLR2 domainHEK293 cells
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