S188 Markedly Elevated Levels of Interferon (IFN)-g, IFN-a, Interleukin (IL)-2, IL- 10, and Tumor Necrosis Factor-a Associated with Fatal Ebola Virus Infection Francois Villinger, Pierre E. Rollin, Sukhdev S. Brar, Department of Pathology and Laboratory Medicine, School of Medicine, Emory University, and Special Pathogens Branch, Molecular Pathology Nathaniel F. Chikkala, Jorn Winter, J. Bruce Sundstrom, and Ultrastructure Activity, Division of Viral and Rickettsial Diseases, Sherif R. Zaki, Robert Swanepoel, Aftab A. Ansari, Centers for Disease Control and Prevention, Atlanta, Georgia; National and Clarence J. Peters Institute of Virology, Sandringham, South Africa The role of immune mechanisms in the pathogenesis of Ebola hemorrhagic fever (EHF) remains to be elucidated. In this report, the serum cytokine levels of patients who died of EHF were compared with those of patients who recovered and those of control patients. A marked elevation of interferon (IFN)-g levels (ú100 pg/mL) was observed in sequential serum samples from all fatal EHF cases compared with patients who recovered or controls. Markedly elevated serum levels of interleukin (IL)-2, IL-10, tumor necrosis factor (TNF)-a, and IFN-a were also noted in fatal EHF cases; however, they had a greater degree of variability. No differences were noted in serum levels of IL-4 and IL-6. mRNA quantitation from blood clots of the same patients showed relatively elevated levels of TNF-a and IFN-a in samples from EHF patients. Taken together, these results suggest that a high degree of immune activation accompanies and potentially contributes to a fatal outcome in EHF patients. 1995 [5]. Of the 11 patients, 9 (7 fatal cases and 2 survivors) had Since its first recognition in 1976, Ebola virus or closely confirmed EHF (Ebola antigen or IgM antibody positive by related strains have caused intermittent outbreaks of human ELISA). The 2 remaining patients, who were in the same ward, disease mostly in or associated with infected animals originat- died of unconfirmed infectious diseases; however, they did not ing from the African continent [1]. The mechanisms of patho- have EHF (Ebola antigen and antibody negative by ELISA and genesis responsible for the Ebola hemorrhagic fever (EHF) virus-isolation negative). have yet to be fully defined [2]. The viruses replicate in endo- Serum samples and blood clots were transported in dry ice to thelial cells [3], and pathologic examination of fatal EHF cases Atlanta and stored at 070°C. Twenty-eight serum samples were reveals extensive infection of vessel endothelium and the retic- analyzed by EIA, and 33 blood clots were analyzed by mRNA uloendothelial system, including spleen and Kupffer’s cells [4]. quantitation. Invading microorganisms usually trigger antigen-specific and Cytokine EIAs. For safety reasons, aliquots of serum samples nonspecific immune responses by various effector cells of the were inactivated using gamma radiation (5.10 6 rads). The irradia- immune system. The orchestration of the responses involves tion was shown not to interfere with the antigenic detection or the biologic activity of the cytokines tested (unpublished data). signaling via cell surface molecules and soluble cytokines. The Commercial EIAs for the detection of serum levels of human presence and pattern of cytokine profiles associated with a interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor particular syndrome provide valuable information regarding the (TNF)-a, and interferon (IFN)-a were purchased from BioSource type and magnitude of protective or potentially detrimental International (Camarillo, CA). The assays were done according to responses that occur. the manufacturer’s instructions. The EIA for the quantitation of Herein we present the first analysis of cytokine responses in IFN-g was done as described previously [6]. EHF patients. This information may help elucidate the mecha- RNA isolation. Total RNA was extracted from blood clots in nisms of EHF pathogenesis and provide a basis for future novel a biosafety level 4 laboratory using 4M guanidium isothiocyanate, immune therapeutic strategies. and the suspensions were transferred to a biosafety level 2 labora- tory for chloroform – isoamyl alcohol purification and precipitation with isopropanol. Materials and Methods Reverse transcription (RT). Air-dried RNA pellets were resus- Patient samples. Sequential blood samples were collected pended in 50 mL of RT cocktail (50 mM Tris, pH 8.3, 6 mM from 11 patients in the Kikwit General Hospital, Bandundu region, MgCl 2 , 40 mM KCl, 10 mM dithiothreitol, 0.01% Nonidet P-40, Democratic Republic of the Congo, between 17 May and 7 June, 50 mM random hexamers, 25 mM dNTP, 3 U RNasin, and 30 U murine leukemia virus reverse transcriptase [Promega, Madison, WI]). RT was allowed to proceed for 10 min at room temperature and 1 h at 42°C and then was heat-inactivated at 95°C for 5 min. Reprints or correspondence: Dr. F. Villinger, Winship Cancer Center, Emory Polymerase chain reaction amplification. Polymerase chain University, 1327 Clifton Rd., Atlanta GA 30322 (fvillin@emory.edu). reaction amplification for cytokine message was performed ac- The Journal of Infectious Diseases 1999; 179(Suppl 1):S188 – 91 cording to a previously described protocol [6 – 8] with primers  1999 by the Infectious Diseases Society of America. All rights reserved. 0022–1899/99/79S1 – 0029$02.00 specific for human CD3, IL-1b, IL-2, IL-4, IL-6, IL-10, IL-12a / 9d49$$se06 12-27-98 12:38:14 jinfa UC: J Infect Downloaded from https://academic.oup.com/jid/article/179/Supplement_1/S188/881048 by guest on 04 December 2021