Immunolocalization of inducible nitric oxide syn-thase in human amnionTo the Editors: 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 localiza-tion of inducible nitric oxide synthase on human fetalamnion in intra-amniotic infection. Am J Obstet Gynecol1998;179:1271-4), which concludes that inducible nitricoxide synthase is not normally present in human amnionepithelial cells but is present in these cells after intra-amniotic infection.

These findings are in contrast to our data, which showinducible nitric oxide synthase expression in human amni-otic epithelial cells in tissues collected before and after theonset of spontaneous labor at term in the absence of in-trauterine infection.1 Concern has been expressed regard-ing the specificity of antibodies used in immunolocaliza-tion studies of inducible nitric oxide synthase in humantissues. Therefore we used 3 different inducible nitricoxide synthase antibodies and investigated both cryosec-tions and paraffin-embedded tissue. In addition, we testedliquid-phase absorption controls using the appropriate im-munogen peptides to ensure specificity of each of the anti-bodies. Inducible nitric oxide synthase was consistentlyidentified in the amniotic epithelial cells with each of thedifferent antibodies and techniques. In support of ourfindings, Dennes et al2 identified inducible nitric oxidesynthase messenger ribonucleic acid in human amnioncollected at term with the polymerase chain reaction.

We were surprised that Hsu et al identified induciblenitric oxide synthase expression within infiltrating neu-trophils in patients with intra-amniotic infection. Theauthors stated that this finding was not unexpected,since neutrophils can be stimulated during infection toexpress inducible nitric oxide synthase. However, the 5references provided to support this statement related towork performed with rodent macrophages. In contrastto rodent data, Miles et al,3 in studies on human neu-trophils with Western and Northern blotting, measure-ments of nitric oxide synthase enzyme activity (radiola-beled arginine to citrulline conversion assay), and theGriess reaction (which measures the byproducts of nitricoxide production), concluded that neither circulatingnor extravasated human neutrophils contain protein ormessage for inducible nitric oxide synthase nor do theypossess inducible nitric oxide synthase enzyme activity.

We suggest that studies investigating changes in nitricoxide synthase expression in human tissues should notrely on immunohistochemical data alone. The findingof inducible nitric oxide synthase expression within neu-trophils raises doubts about the specificity of the anti-body used by Hsu et al, whereas the absence of adequatecontrols is also of concern. The authors indicated thatthey are currently analyzing patients in labor withoutchorioamnionitis; we encourage them to use techniquesin addition to immunohistochemistry in these studies.

Andrew Thomson, MD, Ian Greer, MD, and Jane Norman,MD

University Department of Obstetrics and Gynaecology, Glasgow RoyalInfirmary, 10 Alexandra Parade, Glasgow, United Kingdom G31 2ER

REFERENCES

1. Thomson AJ, Telfer JF, Kohnen G, et al. Nitric oxide synthaseactivity and localization do not change in uterus and pla-centa during human parturition. Hum Reprod 1997;12:2546-52.

2. Dennes WJB, Slater DM, Bennett PR. Nitric oxide synthasemRNA expression in human fetal membranes: a possible rolein parturition. Biochem Biophys Res Commun 1997;233:276-8.

3. Miles AM, Owens MW, Milligan S. Nitric oxide synthase in circu-lating vs. extravasated polymorphonuclear leukocytes. J LeukocBiol 1995;58:616-22.

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ReplyTo the Editors: We thank Thomson et al for their thought-ful letter and questions regarding our study and will elab-orate on their comments. Generally, the specificity of dif-ferent antibodies used in immunolocalization studies canbe varied. Indeed, Eis et al1 agreed with our findings thatno inducible nitric oxide synthase immunostaining wasidentified in human amnion epithelium but was found infibroblasts of human amnion. Although Dennes et al(see reference 2 of Thomson et al) identified induciblenitric oxide synthase messenger ribonucleic acid inhuman amnion at term by polymerase chain reaction,the expression was at least 30-fold lower than in chorion-decidua. We suspect that a weak expression of induciblenitric oxide synthase in human amnion in that study maybe caused by fibroblasts but not epithelium of humanamnion.

We agree that we should provide the inducible nitricoxide synthase expression on human neutrophils2, 3 in-stead of rodent macrophages. However, we disagree withthe comments by Thomson et al regarding no expres-sion of inducible nitric oxide synthase in human neu-trophils. Evidence that human neutrophils express in-ducible nitric oxide synthase or its products has beeninconclusive and controversial. Recently, Wheeler et al2and Tsukahara et al3 have clearly demonstrated that in-ducible nitric oxide synthase can be induced in humanneutrophils.

Chaur-Dong Hsu, MD, MPH, and Vinita Parkash, MDDivision of Maternal-Fetal Medicine, Department of Obstetrics andGynecology and Department of Pathology, Yale University School ofMedicine, 333 Cedar St, New Haven, CT 06520

REFERENCES

1. Eis ALW, Brockman DE, Myatt L. Immunolocalization of the in-ducible nitric oxide synthase isoform in human fetal mem-branes. Am J Reprod Immunol 1997;38:289-94.

2. Wheeler MA, Smith SD, García-Cardeña G, Nathan CF, WeissRM, Sessa WC. Bacteria infection induces nitric oxide synthasein human neutrophils. J Clin Invest 1997;99:110-6.

3. Tsukahara Y, Morisaki T, Horita Y, Torisu M, Tanaka M.Expression of inducible nitric oxide synthase in circulating neu-trophils of the systemic inflammatory response syndrome andseptic patients. World J Surg 1998;22:771-7.

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770 Letters September 1999Am J Obstet Gynecol