一直以來，我們的肺臟含有某些物質，在自然性免疫系統中提供了第一道防線的屏障來對抗外來的感染物。第一道防線中，其中一個最重要的細胞就是吞噬細胞，它是由樹枝狀細胞而來的，而它的功能就像是自然性免疫系統和獲得性免疫系統的連接線，藉由辨識外來的病原體，經由直接和T細胞接觸並活化T細胞，釋放出一些發炎反應的物質、細胞激素以及氮化物…..等等。近年來，越來越多的文獻顯示肺泡介面活性蛋白質中的A和D，它們在寄主肺臟的防禦上扮演一個很重要的角色。肺泡介面活性蛋白質A和D皆是屬於collectin家族中的一個成員，它們可以和許多種的微生物和過敏原結合，並且可以辨識微生物表面的醣類分子，幫助吞噬細胞進行吞噬作用，或者調控一些免疫細胞，例如：T細胞和吞噬細胞，直接毒殺外來的病原體。我的研究目的主要是想看在自然性免疫中，肺泡介面活性蛋白質D所扮演的角色，肺泡介面活性蛋白質D（SP-D）是否能調節肺泡吞噬細胞受到脂多醣（LPS）或是抑制主要過敏原中的家塵蹣（Der-p）活化所產生的一氧化氮。在這個實驗中，我們將蘭嶼迷你豬的肺泡沖洗液，通過maltose-sepharose的管柱層析，純化出我們要的肺泡介面活性蛋白質D。我們去測MH-S（BALB/c品系）及AMJ2-C11 (C57BL/6 品系)細胞株一氧化氮（NO）的含量，結果發現LPS和Der-p在不同細胞株會引起不同NO產生的反應。我們看到Der-p的刺激在MH-S細胞中，NO濃度會隨著Der-p濃度增加而上升，可是在AMJ2-C11細胞中卻看不到這個情形，然而LPS引發NO的產生，在兩株細胞中都可以看到，我們也發現SP-D只有在MH-S中才會引起NO。同時，我們也偵測到iNOS蛋白質的表現。在之前的文獻中，我們清楚的知道LPS活化肺泡吞噬細胞是經由和TLR4及CD14的結合才發生作用。於是我們假設LPS和Der-p是參與不同細胞表面分子活化的機制。結果發現MH-S和AMJ2-C11受到LPS和Der-p刺激後所產生的NO會伴隨著細胞表面CD14和TLR4分子表現量的上升。並且在兩株細胞中LPS和Der-p都會活化NF-κB的活性，經由這個轉譯因子，將訊息往下傳遞。在AMJ2-C11細胞株中，我們看到Der-p與LPS都可能刺激產生TNF-α，但只有LPS刺激才能有NO和TGF-β的製造，而Der-p刺激皆無NO及TGF-β的製造，因此，在Der-p刺激下，AMJ2-C11其 NO和TNF-α的產生是不同類型的，而NO 和TGF-β則是相同的。在競爭性結合的實驗中可以看到在這兩株細胞中，預先與SP-D作用下，再以培養液清洗，可以抑制LPS和Der-p刺激所產生的NO，若是同時刺激SP-D與LPS或Der-p，則無此抑制作用。我們也去看細胞型態的改變，有趣的是我們發現AMJ2-C11在受到LPS刺激時會有型態上的改變，但對於Der-p和SP-D刺激下，並不會有；而MH-S在3種刺激物下都不會有型態上的變化。因此，我們認為在不同細胞中，LPS和Der-p參與著不同的機制來促使NO的產生，並且伴隨著細胞表面CD14和TLR4的上升，SP-D可以降低LPS和Der-p刺激所產生的NO。SP-D的確在過敏氣喘的自然性免疫中扮演一個非常重要的角色。

It has been recognized that the lung contains resident components of the innate immune system that provide a first-line defense against infectious challenge. Alveolar macrophages (AMs), derived from dentritic cells, function as a unique link between the innate and adaptive immune system by detecting pathogens and triggering T cell activation through direct contact and release inflammatory cytokines and nitric oxide (NO). Recent studies have shown that two surfactant-associated proteins, surfactant protein (SP)-A and SP-D, play important roles in pulmonary host defense. Both collectins bind to a variety of microoganisms, allergens included, by recognizing the patterns of surface carbohydrate on pathgens and target them for phagocytosis and killing through macrophages and effector T cells. The objectives of my study is to determine the role of SP-D, a molecule of innate immunity, in the modulation of NO production from alveolar macrophage activated by lipopolysaccharide (LPS) and major allergen of house dust mite, Dermatophagoides pteronyassinus (Der-p). In this study, porcine SP-D was purifed from broncholaveolar lavage (BAL) fluids of Lan-Yu minipigs through maltose-sepharose column. NO production from different AMs cell lines, MH-S (BALB/c strains), AMJ2-C11 (C57BL/6 strains) was determined by nitrite levels. We found that LPS and Der-p caused different responses of NO production in different cell lines. Der-p stimulation caused NO production in a dose-dependent manner in MH-S, but not in AMJ2-C11, while LPS induced NO production in both cell lines. We also found that SP-D caused NO production in MH-S but not in AMJ2-C11. Inducible NOS (iNOS) protein expression were also measured. Previously, it was known that LPS-induced AMs activation was via TLR4/CD14 complex. We hypothesized that LPS and Der-p may involve different mechanism of cellular receptor activation, and we found that the production of NO from MH-S and AMJ2-C11 after stimulation were accompanied by up-regulation of surface CD14 and TLR4 receptors. LPS and Der-p activated NF-κB of both MH-S and AMJ2-C11, and there is different pattern of NO and TNF-α production and is same pattern of NO and TGF-β production for Der-p-activated and SP-D-activated AMJ2-C11 cell line. Binding competition experiments further showed that SP-D can inhibit LPS-induced and DerP-induced NO production in MH-S and AMJ2-C11 when cell first incubate with SP-D（pre-incubation）, but not in co-incubation. AMJ2-C11 can change its morphology when stimulating with LPS. Therefore, we suggest that LPS and Der-p may involve in different pathway of NO production accompanied by up-regulation of surface CD14 and TLR4 receptors in MH-S and AMJ2-C11, and SP-D can reduce LPS-induced and DerP-induced NO production in MH-S and AMJ2-C11, respectively. SP-D may plays an important role in modulation of NO production in the innate immunity of lung.

Augusto LA, Synguelakis M, Johansson J, Pedron T, Girard R, Chaby R. 2003. Interaction of pulmonary surfactant protein C with CD14 and lipopolysaccharide. Infect Immun. 71(1):61-7

Ann Marie LeVine. 2002. News about the Pulmonary Collectins and Infection. Mod. Asp. Immunobiol. 2(5):228-231

Beutler, B. 2002. LPS in microbial pathogenesis: promise and fulfilment. J Endotoxin Res. 8:329-335.

Calhoun, W. J., H. E. Reed, D. R. Moest, and C. A. Stevens. 1992. Enhanced superoxide production by alveolar macrophages and air-space cells, airway inflammation, and alveolar macrophage density changes after segmental antigen bronchoprovocation in allergic subjects. Am. Rev. Respir. Dis. 145:317.

Comhair, S. A., P. R. Bhathena, R. A. Dweik, M. Kavuru, and S. C. Erzurum. 2000. Rapid loss of superoxide dismutase activity during antigen-induced asthmatic response. Lancet 355:624.

Crapo, J. D., and B. J. Day. 1999. Modulation of nitric oxide responses in asthma by extracellular antioxidants. J. Allergy Clin. Immunol. 104:743.
Keane-Myers, A. M., W. C. Gause, F. D. Finkelman, X. D. Xhou, and M. Wills-Karp. 1998. Development of murine allergic asthma is dependent upon B7-2 costimulation. J. Immunol. 160:1036.

Capote, K.R., F.X. McCormack, and F. Possmayer. 2003. Pulmonary surfactant protein-A (SP-A) restores the surface properties of surfactant after oxidation by a mechanism that requires the Cys6 interchain disulfide bond and the phospholipid binding domain. J Biol Chem. 278:20461-74.

Chabot, S., K. Koumanov, G. Lambeau, M.H. Gelb, V. Balloy, M. Chignard, J.A. Whitsett, and L. Touqui. 2003. Inhibitory effects of surfactant protein a on surfactant phospholipid hydrolysis by secreted phospholipases A2. J Immunol. 171:995-1000.

Chabot, S., L. Salez, F.X. McCormack, L. Touqui, and M. Chignard. 2003. Surfactant protein A inhibits lipopolysaccharide-induced in vivo production of interleukin-10 by mononuclear phagocytes during lung inflammation. Am J Respir Cell Mol Biol. 28:347-53.

Chiba, H., S. Pattanajitvilai, H. Mitsuzawa, Y. Kuroki, A. Evans, and D.R. Voelker. 2003. Pulmonary surfactant proteins A and D recognize lipid ligands on Mycoplasma pneumoniae and markedly augment the innate immune response to the organism. Chest. 123:426S.

Fan, J., and A.B. Malik. 2003. Toll-like receptor-4 (TLR4) signaling augments chemokine-induced neutrophil migration by modulating cell surface expression of chemokine receptors. Nat Med. 9:315-21.

Feng, J., Y. Liu, J. Shi, and R. Zhang. 2002. [The role of LPS in the CD14 expression and the activation of Kupffer cells]. Zhonghua Shao Shang Za Zhi. 18:107-11.

Flak, T. A., and W. E. Goldman. 1996. Autotoxicity of nitric oxide in airway disease. Am. J. Respir. Crit. Care Med. 154:S202.

Gant, V., M. Cluzel, I. Shakoor, P. J. Rees, T. H. Lee, and A. S. Hamblin. 1992. Alveolar macrophage accessory cell function in bronchial asthma. Am. Rev. Respir. Dis. 146:900.

Gosset, P., A. Tsicopoulos, B. Wallaert, C. Vannimenus, M. Joseph, A.-B. Tonnel, and A. Capron. 1991. Increased secretion of tumor necrosis factor –αand interleukin-6 by alveolar macrophages consecutive to the development of the late asthmatic reaction. J. Allergy Clin. Immunol. 88:561

Guo, F. H., K. Uetani, S. J. Haque, B. R. G. Williams, R. A. Dweik, F. B. J. M. Thunnissen, W. Calhoun, and S. C. Erzurum. 1997. Interferon-γand interleukin-4 stimulate prolonged expression of inducible nitric oxide synthase in human airway epithelium through synthesis of soluble mediators. J. Clin. Invest.100:829.

Hart, M.L., D.A. Mosier, and S.K. Chapes. 2003. Toll-like receptor 4-positive macrophages protect mice from Pasteurella pneumotropica-induced pneumonia. Infect Immun. 71:663-70.

Hussain, S., J.R. Wright, and W.J. Martin, 2nd. 2003. Surfactant protein A decreases nitric oxide production by macrophages in a tumor necrosis factor-alpha-dependent mechanism. Am J Respir Cell Mol Biol. 28:520-7.

Herbert, C. A., C. M. King, P. C. Ring, S. T. Holgate, G. A. Stewart, P. J. Thompson, and C. Robinson. 1995. Augmentation of permeability in the bronchial epithelium by the house dust mite allergen Der p1. Am. J. Respir. Cell Mol. Biol. 12:369

Jiang, Y., and R.J. Ulevitch. 1999. [The signal transduction of cell activation by LPS: the studies from CD14 to p38 MAPK]. Sheng Li Ke Xue Jin Zhan. 30:29-34.

Kamath, A.B., J. Alt, H. Debbabi, and S.M. Behar. 2003. Toll-like receptor 4-defective C3H/HeJ mice are not more susceptible than other C3H substrains to infection with Mycobacterium tuberculosis. Infect Immun. 71:4112-8.

Kiechl, S., C.J. Wiedermann, and J. Willeit. 2003. Toll-like receptor 4 and atherogenesis. Ann Med. 35:164-71.

Korsgren, M., C. G. Persson, F. Sundler, T. Bjerke, T. Hansson, B. J. Chambers, S. Hong, L. Van Kaer, H. G. Ljunggren, and O. Korsgren. 1999. Natural killer cells determine development of allergen-induced eosinophilic airway inflammation in mice. J. Exp. Med. 189:553.

Kishore U, Wang JY, Hoppe HJ, Reid KB. 1996. The alpha-helical neck region of human lung surfactant protein D is essential for the binding of the carbohydrate recognition domains to lipopolysaccharides and phospholipids. Biochem J. 318 ( Pt 2):505-11.

Lakhani, S.A., and C.W. Bogue. 2003. Toll-like receptor signaling in sepsis. Curr Opin Pediatr. 15:278-82.

Leth-Larsen, R., C. Nordenbaek, I. Tornoe, V. Moeller, A. Schlosser, C. Koch, B. Teisner, P. Junker, and U. Holmskov. 2003. Surfactant protein D (SP-D) serum levels in patients with community-acquired pneumonia small star, filled. Clin Immunol. 108:29-37.

Li, X., J.C. Tupper, D.D. Bannerman, R.K. Winn, C.J. Rhodes, and J.M. Harlan. 2003. Phosphoinositide 3 Kinase Mediates Toll-Like Receptor 4-Induced Activation of NF-kappaB in Endothelial Cells. Infect Immun. 71:4414-4420.

Lim BL, Wang JY, Holmskov U, Hoppe HJ, Reid KB. 1994. Expression of the carbohydrate recognition domain of lung surfactant protein D and demonstration of its binding to lipopolysaccharides of gram-negative bacteria. Biochem Biophys Res Commun. 202(3):1674-80.

Matsuguchi, T. 2002. [Toll-like receptor signals and innate immunity]. Seikagaku. 74:1463-8.

McNamara, K.M., S.E. Hall, R.S. Wilder, H.P. Lawrence, and S. Offenbacher. 1999. Periodontitis and cytokine expression in CD14 deficient patients. J Int Acad Periodontol. 1:95-100.

Madan T, Kishore U, Shah A, Eggleton P, Strong P, Wang JY, Aggrawal SS, Sarma PU, Reid KB. 1997. Lung surfactant proteins A and D can inhibit specific IgE binding to the allergens of Aspergillus fumigatus and block allergen-induced histamine release from human basophils. Clin Exp Immunol. 110(2):241-9.

Medvedev, A.E., and S.N. Vogel. 2003. Overexpression of CD14, TLR4, and MD-2 in HEK 293T cells does not prevent induction of in vitro endotoxin tolerance. J Endotoxin Res. 9:60-4.

Meier, A., C.J. Kirschning, T. Nikolaus, H. Wagner, J. Heesemann, and F. Ebel. 2003. Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of murine macrophages. Cell Microbiol. 5:561-70.

Mizel, S.B., A.N. Honko, M.A. Moors, P.S. Smith, and A.P. West. 2003. Induction of macrophage nitric oxide production by gram-negative flagellin involves signaling via heteromeric toll-like receptor 5/toll-like receptor 4 complexes. J Immunol. 170:6217-23.

Moonsom, S., and W. Kasinrerk. 2000. Production of anti-CD14 monoclonal antibodies using CD14 expressing COS cells as immunizing antigen. Asian Pac J Allergy Immunol. 18:53-61.

Maruo, K., T. Akaike, T. Ono, T. Okamoto, and H. Maeda. 1997. Generation of anaphylatoxins through proteolytic processing of C3 and C5 by house dust mite protease. J. Allergy Clin. Immunol. 100:253.

Michel, O., J. Kips, J. Duchateau, F. Vertongen, L. Robert, H. Collet, R. Pauwels, and R. Sergysels. 1996. Severity of asthma is related to endotoxin in house dust. Am. J. Respir. Crit. Care Med. 154:1641.

Okamoto, M., T. Oshikawa, T. Tano, G. Ohe, S. Furuichi, H. Nishikawa, S.U. Ahmed, S. Akashi, K. Miyake, O. Takeuchi, S. Akira, Y. Moriya, S. Matsubara, Y. Ryoma, M. Saito, and M. Sato. 2003. Involvement of Toll-like receptor 4 signaling in interferon-gamma production and antitumor effect by streptococcal agent OK-432. J Natl Cancer Inst. 95:316-26.

O’Neill, L. A., and C. A. Dinarello. 2000. The IL-1 receptor/Toll-like receptor superfamily: crucial receptors for inflammation and host defense. Immunol. Today 21:206.

Paik, Y.H., R.F. Schwabe, R. Bataller, M.P. Russo, C. Jobin, and D.A. Brenner. 2003. Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells. Hepatology. 37:1043-55.

Palaniyar N, Nadesalingam J, Reid KB. 2002. Pulmonary innate immune proteins and receptors that interact with gram-positivebacterial ligands. Immunobiology 205(4-5):575-94

Rodriguez, D., A.C. Keller, E.L. Faquim-Mauro, M.S. de Macedo, F.Q. Cunha, J. Lefort, B.B. Vargaftig, and M. Russo. 2003. Bacterial lipopolysaccharide signaling through toll-like receptor 4 suppresses asthma-like responses via nitric oxide synthase 2 activity. J Immunol. 171:1001-8.

Raychaudhuri, B., R. Dweik, M. J. Connors, L. Buhrow, A. Malur, J. Drazba, A. C. Arroliga, S. C. Erzurum, M. S. Kavuru, and M. J. Thomassen. 1999. Nitric oxide blocks nuclear factor-_B activation in alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 21:311.

Rinco´n, M., J. Anguita, T. Nakamura, E. Fikrig, and R. A. Flavell. 1997. Interleukin (IL)-6 directs the differentiation of IL-4-producing CD4_ T cells. J. Exp. Med. 185:461.

Roger, T., C. Froidevaux, C. Martin, and T. Calandra. 2003. Macrophage migration inhibitory factor (MIF) regulates host responses to endotoxin through modulation of Toll-like receptor 4 (TLR4). J Endotoxin Res. 9:119-23.

Stamme C, Muller M, Hamann L, Gutsmann T, Seydel U. 2002. Surfactant protein a inhibits lipopolysaccharide-induced immune cell activationby preventing the interaction of lipopolysaccharide with lipopolysaccharide-binding protein. Am J Respir Cell Mol Biol 27(3):353-60

Sano H, Chiba H, Iwaki D, Sohma H, Voelker DR, Kuroki Y. Surfactant proteins A and D bind CD14 by different mechanisms. 2000 J Biol Chem. 275(29):22442-51

Sano H, Sohma H, Muta T, Nomura S, Voelker DR, Kuroki Y. 1999. Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by interaction with CD14. J Immunol. 163(1):387-95

Stewart, G. A., S. M. Boyd, C. H. Bird, K. D. Krska, M. R. Kollinger, and P. J. Thompson. 1994. Immunobiology of the serine protease allergens from house dust mites. Am. J. Ind. Med. 25:105.

Schulz, O., H. F. Sewell, and F. Shakib. 1998. Proteolytic cleavage of CD25, the subunit of the human T cell interleukin 2 receptor, by Der p 1, a major mite allergen with cysteine protease activity. J. Exp. Med. 187:271.

Shakib, F., O. Schulz, and H. Sewell. 1998. A mite subversive: cleavage of CD23 and CD25 by Der p I enhances allergenicity. Immunol. Today 19:313.

Simultaneous Blocking of Human Toll-Like Receptors 2 and 4 Suppresses Myeloid Dendritic Cell Activation Induced by Mycobacterium bovis Bacillus Calmette-Guerin Peptidoglycan. Infect Immun. 71:4238-4249.

Takeshige, K., S. Yamazaki, A. Eto, K. Kataoka, and T. Muta. 2002. [Mechanism of macrophage activation by LPS]. Masui. 51 Suppl:S50-62.

Temple, S.E., K.Y. Cheong, C.M. Almeida, P. Price, and G.W. Waterer. 2003. Polymorphisms in lymphotoxin alpha and CD14 genes influence TNFalpha production induced by Gram-positive and Gram-negative bacteria. Genes Immun. 4:283-8.
Takahashi, K., T. Aoki, S. Kohmoto, H. Nishimura, Y. Kodera, A. Matsushima, and Y. Inada. 1990. Activation of kallikrien system in human plasma with purified serine protease from Dermatophagoides farinae. Int. Arch. Allergy Appl. Immunol. 91:80.

Tsitoura, D. C., S. Kim, K. Dabbagh, G. Berry, D. B. Lewis, and D. T. Umetsu. 2000. Respiratory infection with influenza A virus interferes with the induction of tolerance to aeroallergens. J. Immunol. 165:3484.

Vives-Pi, M., N. Somoza, J. Fernandez-Alvarez, F. Vargas, P. Caro, A. Alba, R. Gomis, M.O. Labeta, and R. Pujol-Borrell. 2003. Evidence of expression of endotoxin receptors CD14, toll-like receptors TLR4 and TLR2 and associated molecule MD-2 and of sensitivity to endotoxin (LPS) in islet beta cells. Clin Exp Immunol. 133:208-218.

Viksman, M. Y., M. C. Liu, C. A. Bickel, R. P. Schleimer, and B. S. Bochner.1997. Phenotypic analysis of alveolar macrophages and monocytes in allergic airway inflammation. Am. J. Respir. Crit. Care Med. 155:858.

Van der Veen, R. C., T. A. Dietlin, F. M. Hofman, L. Pen, B. H. Segal, and S. M. Holland. 2000. Superoxide prevents nitric oxide-mediated suppression of helper T lymphocytes: decreased autoimmune encephalomyelitis in nicotinamide adenine dinucleotide phosphate oxidase knockout mice. J. Immunol. 164:5177.

Wu, H., A. Kuzmenko, S. Wan, L. Schaffer, A. Weiss, J.H. Fisher, K.S. Kim, and F.X. McCormack. 2003. Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability. J Clin Invest. 111:1589-602.

Wang, J. Y., U. Kishore, B. L. Lim, P. Strong, and K. B. Reid. 1996. Interaction of human lung surfactant proteins A and D with mite (Dermatophagoides pteronyssinus) allergens. Clin. Exp. Immunol. 106:367.

Wang JY, Shieh CC, You PF, Lei HY, Reid KB. 1998. Inhibitory effect of pulmonary surfactant proteins A and D on allergen-induced lymphocyte proliferation and histamine release in children with asthma. Am J Respir Crit Care Med. 158(2):510-8.

Wang JY, Shieh CC, Yu CK, Lei HY. 2001. Allergen-induced bronchial inflammation is associated with decreased levels of surfactant proteins A and D in a murine model of asthma. Clin Exp Allergy. 31(4):652-62.

Whitekus, M. J., N. Li, M. Zhang, M. Y. Wang, M. A. Horwitz, S. K. Nelson, L. D. Horwitz, N. Brechun, D. Diaz-Sanchez, and A. E. Nel. 2002. Thiol antioxidants inhibit the adjuvant effects of aerosolized diesel exhaust particles in a murine model of ovalbumin sensitization. J. Immunol. 168:2560.

Yang, L., L. Cohn, D. H. Zhang, R. Homer, A. Ray, and P. Ray. 1998. Essential role of nuclear factor _B in the induction of eosinophilia in allergic airway inflammation. J. Exp. Med. 188:1739.

Zhang, Y., and J.B. Bliska. 2003. Role of Toll-like receptor signaling in the apoptotic response of macrophages to Yersinia infection. Infect Immun. 71:1513-9.

Zuany-Amorim, C., C. Ruffie, S. Haile, B. B. Vargaftig, P. Pereira, and M. Pretolani. 1998. Requirement for T cells in allergic airway inflammation. Science 280:1265.

Thierry Roger, John David, Michel P. Glauser, Thierry Calandra. 2001. MIF regulates innate immune responses through modulation of Toll-like receptor 4. Nature 414:920 - 924.