Immunolocalization of inducible nitric oxide synthase in human amnion

Abstract

We read with interest the article by Hsu et al (Hsu C-D, Meaddough E, Lu L-C, Chelouche A, Liang R-I, Copel JA, Parkash V. Immunohistochemical localization of inducible nitric oxide synthase on human fetal amnion in intra-amniotic infection. Am J Obstet Gynecol 1998;179:1271-4), which concludes that inducible nitric oxide synthase is not normally present in human amnion epithelial cells but is present in these cells after intraamniotic infection. These findings are in contrast to our data, which show inducible nitric oxide synthase expression in human amniotic epithelial cells in tissues collected before and after the onset of spontaneous labor at term in the absence of intrauterine infection.1Thomson Aj Telfer Jf Kohnen G et al.Nitric Oxide Synthase Activity And Localization Do Not Change In Uterus And Placenta During Human Parturition.Hum Reprod. 1997; 12: 2546-2552Crossref PubMed Scopus (70) Google Scholar Concern has been expressed regarding the specificity of antibodies used in immunolocalization studies of inducible nitric oxide synthase in human tissues. Therefore we used 3 different inducible nitric oxide synthase antibodies and investigated both cryosections and paraffin-embedded tissue. In addition, we tested liquid-phase absorption controls using the appropriate immunogen peptides to ensure specificity of each of the antibodies. Inducible nitric oxide synthase was consistently identified in the amniotic epithelial cells with each of the different antibodies and techniques. In support of our findings, Dennes et al2Dennes Wjb Slater Dm Bennett Pr Nitric Oxide Synthase Mrna Expression In Human Fetal Membranes: A Possible Role In Parturition.Biochem Biophys Res Commun. 1997; 233: 276-278Crossref PubMed Scopus (23) Google Scholar identified inducible nitric oxide synthase messenger ribonucleic acid in human amnion collected at term with the polymerase chain reaction. We were surprised that Hsu et al identified inducible nitric oxide synthase expression within infiltrating neutrophils in patients with intra-amniotic infection. The authors stated that this finding was not unexpected, since neutrophils can be stimulated during infection to express inducible nitric oxide synthase. However, the 5 references provided to support this statement related to work performed with rodent macrophages. In contrast to rodent data, Miles et al,3Miles Am Owens Mw Milligan S. Nitric Oxide Synthase In Circulating Vs. Extravasated Polymorphonuclear Leukocytes.J Leukoc Biol. 1995; 58: 616-622PubMed Google Scholar in studies on human neutrophils with Western and Northern blotting, measurements of nitric oxide synthase enzyme activity (radiolabeled arginine to citrulline conversion assay), and the Griess reaction (which measures the byproducts of nitric oxide production), concluded that neither circulating nor extravasated human neutrophils contain protein or message for inducible nitric oxide synthase nor do they possess inducible nitric oxide synthase enzyme activity. We suggest that studies investigating changes in nitric oxide synthase expression in human tissues should not rely on immunohistochemical data alone. The finding of inducible nitric oxide synthase expression within neutrophils raises doubts about the specificity of the antibody used by Hsu et al, whereas the absence of adequate controls is also of concern. The authors indicated that they are currently analyzing patients in labor without chorioamnionitis; we encourage them to use techniques in addition to immunohistochemistry in these studies.