Published ahead of print on October 5, 2006, doi:10.1165/rcmb.2006-0288SM Am. J. Respir. Cell Mol. Biol., Volume 36, Number 2, February 2007, 152-157 A more recent version of this article appeared on February 1, 2007
Submitted on August 9, 2006 Identification of Immunoglobulins that Recognize 3-Nitrotyrosine in Patients with Acute Lung Injury Following Major TraumaLeonor Thomson1,1 Stokes Research Institute and Department of Pediatrics and Pharmacology, Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, PA, USA; Facultad de Ciencias, Universidad de la Republica, Montevideo, Uruguay, 2 Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA; Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA, 3 Stokes Research Institute and Department of Pediatrics and Pharmacology, Children's Hospital of Philadelphia and University of Pennsylvania School of Medicine, Philadelphia, PA, USA, 4 Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA, 5 Departments of Cell Biology and Cardiovascular Medicine, Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Cleveland, OH, USA * To whom correspondence should be addressed. E-mail: ischirop{at}mail.med.upenn.edu.
Tyrosine nitration is a nitric oxide-derived post-translational modification of proteins. Elevated levels of specific plasma proteins modified by tyrosine nitration have been detected during acute and chronic inflammatory conditions including acute lung injury. In the present study we examined if circulating immunoglobulins against nitrated proteins are present in the plasma of subjects with clinically documented acute lung injury. Affinity chromatography using covalently linked 3-nitrotyrosine was employed to identify plasma proteins that bind to this unusual amino acid. Western blotting and liquid chromatography-tandem mass spectrometry of in-gel digested protein bands revealed that the major proteins eluted from the affinity column were IgM and IgG. An enzyme-linked immunoassay based on competition of HRP-derivatized 3-nitrotyrosine binding to plasma with unlabeled 3-nitrotyrosine was developed and validated. Using this ELISA, the levels of immunoglobulins that recognize 3-nitrotyrosine were significantly higher in the plasma of acute lung injury subjects as compared with both normal controls as well as subjects with major trauma who did not developed ALI (0.36 ± 0.14 vs. 0.03 ± 0.05, and 0.25 ± 0.15; p<0.001, and p=0.006 respectively). These data indicate that tyrosine nitrated proteins induce the production of specific immunoglobulins during acute phase response and inflammation.
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