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Amnion: Implications for Human Pregnancy Anti-Inflammatory Component in the Surfactant Protein-A as an

The mechanism of mouse parturition is thought to involve myometrial inﬁltration by amniotic ﬂuid (AF) macrophages, activated by surfactant protein-A (SP-A). In humans, the concentration of AF SP-A decreases during labor, and no fetal macrophages are found in the myometrium after labor. Therefore, it appears that the mechanisms of labor in mice and humans are different. We investigated a potential role for SP-A in human pregnancy and parturition by examining SP-A expression patterns in AF and amnion. High molecular mass (>250 kDa) oligomeric SP-A was increased in AF with advancing gestation. Interestingly, these oligomers were more abundant in placental amnion before labor at term, while they increased primarily in reﬂected amnion during labor ( p , 0.05). Immunoblotting showed a binding of high molecular mass SP-A in AF to amnion. In C57BL/6 mice, oligomeric SP-Awas also readily detected in AF from E15 onwards, but not in amnion. Macrophage density in mice myometrium did not change with advancing gestational age. Microarray analysis of human amnion explants incubated with SP-A revealed a molecular signature of inhibited cytokine–cytokine receptor interaction with downregulation of IL-1 b , CXCL2, and CXCL5 mRNA expression. The ﬁndings in this study strongly suggest that SP-A signals amniotic anti-inﬂammatory response via AF during pregnancy. We propose that an SP-A interaction among AF, placental amnion, and reﬂected amnion is a unique mechanism for immunoregulation in human pregnancy akin to that established in lung biology. However, AF SP-A and fetal macrophages by themselves do not seem to be exclusive effectors of parturition in humans. The Journal of Immunology , 2010, 184: 6479–6491. To oligomeric molecular SP-A in amniotic of immunoreactive 25 m of amniotic ﬂuid were immunoprecipitated with rabbit polyclonal anti–SP-A Ab (Chemicon International) as previously described. Amniotic ﬂuid immunoprecipitates and puriﬁed SP-Awere resolved in 4–15% gradient SDS- PAGE under nonreducing conditions, the region corresponding to the high molecular mass SP-A immunoreactive band ( . 250 kDa) was cut from the gel, and proteins were extracted as described earlier. Isolated proteins underwent electrophoresis in 4–15% gradient SDS-PAGE under reducing conditions. Both immunoblotting for SP-A and Coomassie blue staining were performed. After destaining, slices corresponding to SP-A bands (32 and 64 kDa) were cut and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gel slices were cut into 1 mm 3 cubes and each was washed with water for 15 min; the liquid was replaced with 50 mM ammonium bicarbonate (AB)/acetonitrile (ACN; 1:1, v/v). Liquid was removed and gel plugs were shrunk with ACN. The gel pieces were rehydrated with AB, and after 5 min an equal volume of ACN was added. After 15 min, the liquid was removed, and ACN was added. After shrinking the gel plugs, the ACN was removed and the gel pieces dried in a vacuum centrifuge. The gel plugs were allowed to swell in 10 mM DTT/ AB and then to reduce for 1 h at 55˚C. After reaching room temperature, the excess liquid was replaced with 55 mM iodoacetamide/AB and incubated for 1 h in the dark. The liquid was removed and the gel plugs were washed twice for 15 min with AB/ACN (1:1).