Markedly Elevated Levels of Interferon (IFN)-γ, IFN-α, Interleukin (IL)-2, IL-10, and Tumor Necrosis Factor-α Associated with Fatal Ebola Virus Infection
- Francois Villinger,
- Pierre E. Rollin,
- Sukhdev S. Brar,
- Nathaniel F. Chikkala,
- Jorn Winter,
- J. Bruce Sundstrom,
- Sherif R. Zaki,
- Robert Swanepoel,
- Aftab A. Ansari and
- Clarence J. Peters
- Department of Pathology and Laboratory Medicine, School of Medicine, Emory University, and Special Pathogens Branch, Molecular Pathology and Ultrastructure Activity, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; National Institute of Virology, Sandringham, South Africa
- Reprints or correspondence: Dr. F. Villinger, Winship Cancer Center, Emory University, 1327 Clifton Rd., Atlanta GA 30322 (fvillin{at}emory.edu).
Abstract
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)-γ levels (>100 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)-α, and IFN-α 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-α and IFN-α 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.
Since its first recognition in 1976, Ebola virus or closely related strains have caused intermittent outbreaks of human disease mostly in or associated with infected animals originating from the African continent [1]. The mechanisms of pathogenesis responsible for the Ebola hemorrhagic fever (EHF) have yet to be fully defined [2]. The viruses replicate in endothelial cells [3], and pathologic examination of fatal EHF cases reveals extensive infection of vessel endothelium and the reticuloendothelial system, including spleen and Kupffer's cells [4]. Invading microorganisms usually trigger antigen-specific and nonspecific immune responses by various effector cells of the immune system. The orchestration of the responses involves signaling via cell surface molecules and soluble cytokines. The presence and pattern of cytokine profiles associated with a particular syndrome provide valuable information regarding the type and magnitude of protective or potentially detrimental responses that occur.
Herein we present the first analysis of cytokine responses in EHF patients. This information may help elucidate the mechanisms of EHF pathogenesis and provide a basis for future novel immune therapeutic strategies.
Materials and Methods
Patient samples
Sequential blood samples were collected from 11 patients in the Kikwit General Hospital, Bandundu region, Democratic Republic of the Congo, between 17 May and 7 June, 1995 [5]. Of the 11 patients, 9 (7 fatal cases and 2 survivors) had confirmed EHF (Ebola antigen or IgM antibody positive by ELISA). The 2 remaining patients, who were in the same ward, died of unconfirmed infectious diseases; however, they did not have EHF (Ebola antigen and antibody negative by ELISA and virus-isolation negative).
Serum samples and blood clots were transported in dry ice to Atlanta and stored at −70°C. Twenty-eight serum samples were analyzed by EIA, and 33 blood clots were analyzed by mRNA quantitation.
Cytokine EIAs
For safety reasons, aliquots of serum samples were inactivated using gamma radiation (5.106 rads). The irradiation was shown not to interfere with the antigenic detection or the biologic activity of the cytokines tested (unpublished data). Commercial EIAs for the detection of serum levels of human interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-α were purchased from BioSource International (Camarillo, CA). The assays were done according to the manufacturer's instructions. The EIA for the quantitation of IFN-γ was done as described previously [6].
RNA isolation
Total RNA was extracted from blood clots in a biosafety level 4 laboratory using 4M guanidium isothiocyanate, and the suspensions were transferred to a biosafety level 2 laboratory for chloroform—isoamyl alcohol purification and precipitation with isopropanol.
Reverse transcription (RT)
Air-dried RNA pellets were resuspended in 50 µL of RT cocktail (50 mM Tris, pH 8.3, 6 mM MgCl2, 40 mM KCl, 10 mM dithiothreitol, 0.01% Nonidet P-40, 50 µM random hexamers, 25 µM 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.
Polymerase chain reaction amplification
Polymerase chain reaction amplification for cytokine message was performed according to a previously described protocol [6–8] with primers specific for human CD3, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12α and β, IL-15, IFN-γ, TNF-α, and IFN-α. Thirty to 35 cycles of denaturation (95°C), annealing (60°C), and elongation (72°C) were done in a thermocycler (Perkin-Elmer; Norwalk, CT). Amplification products were separated by electrophoresis through 1.6% agarose gels and visualized with ethidium bromide staining. The identity of the amplified fragments was verified by Southern blot and hybridization to an internal digoxigenin-labeled oligoprobe. Semiquantitation of the amplified products was achieved by amplification of defined copy numbers of each corresponding cytokine gene in parallel. The relative densities of these amplification products were used to derive a standard curve for each product. The amplification products of the respective samples were calculated by using this standard curve and expressed in copy equivalent numbers [6, 8].
Statistical analyses
The data were analyzed using the two-tailed Student's t test.
Results
The kinetics of cytokine levels in serum as determined by EIA are presented in figure 1. While there were no detectable levels of IL-4 in serum (data not shown), IL-6 serum levels unexpectedly did not appear to be markedly elevated in patients who died of acute EHF. Sera from only 1 of the 2 convalescent-phase patients showed values of >1 ng/mL after the acute stage of the disease (figure 1, IL-6). In contrast, serum samples from the non-EHF control patients had significant IL-6 levels. Markedly elevated levels of serum IL-2, IL-10, IFN-γ, TNFα, and IFN-α were noted in most samples from fatal EHF patients (figure 1, IL-2, patients C–F; table 1) compared with levels in samples from patients with nonfatal EHF and from controls. While the levels of IL-2 and IL-10 appeared to decline over time, no clear trend could be recognized for IFN-γ, TNF-α, or IFN-α. The TNF-α levels, however, seemed to be elevated in early samples from a nonfatal EHF patient and some non-EHF controls.
Figure 1
Kinetic analyses of serum cytokine levels in Ebola hemorrhagic fever (EHF) patients who survived (lanes A, B), EHF patients who died (lanes, C–I), and non-EHF control patients who died of causes other than EHF (lanes, J, K). * Denotes samples that were not tested.
Table 1
Serum cytokine levels (pg/mL) and mRNA copy equivalents (CE) (mean ± SD) in Ebola hemorrhagic fever (EHF) patients and controls.
The levels of cytokine and CD3 mRNA detected in blood clots varied widely among the patients. The levels of IFN-γ and IL-2 were higher in EHF patients (nonsignificant, F = 0.059 and F = 0.40, one-way analysis of variance). Furthermore, the mRNA values obtained for IL-2 and IFN-γ did not correlate with the cytokine levels detected in serum. This variation may have been partly due to the lymphopenia and coagulopathy that is characteristic of the disease [9, 10], resulting in uneven trapping of peripheral blood mononuclear cells (PBMC) in blood clots. Despite the variations, the levels of TNF-α and IFN-α mRNA expression were higher in EHF patients than in controls and higher in patients with fatal infections than with nonfatal infections (table 1).
Tests for detection of mRNA for IL-4, IL-6, IL-12α, and IL-15 were negative (data not shown). A slight elevation in IL-12β message levels was noted in samples from fatal EHF cases, suggesting a potential participation by this cytokine in the up-regulation of IFN-γ expression. However, the elevated IL-12β values were not correlated with an equivalent increase in IL-12α message levels in the same samples, suggesting that the elevated IL-12β may represent a physiologic response that did not result in significant increases in the secretion of biologically active IL-12.
Discussion
The data presented here suggest that massive immune activation occurs in acute and terminal EHF. Of interest was the finding that despite an apparent lack of histologic inflammatory response [4, 9, 10], the lymphoid cells contribute to the overall cytokine cascade, as evidenced by the up-regulation of IL-2 secretion. However, the massive IFN up-regulation observed most likely originates from monocytes/macrophages and parenchymal cells that are known targets for Ebola virus replication [4]. These in vivo observations confirm the data derived by Feldmann et al. [11] in vitro, suggesting that infection of monocytes/macrophages may contribute to a large extent to TNF up-regulation and capillary leakage.
The dissociation observed between the circulating IL-2 and IFN levels and the corresponding RNA message levels in the blood clot has been described in patients with septic syndrome [12] and is probably due to non-PBMC cytokine production (e.g., splenic and Kupffer's cells), decreased RNA half-life in the clinical samples, or extravasation of activated cytokine secreting PBMC in EHF patients. The absence of detectable IL-6 in patients with acute-phase disease was unexpected in the context of an overall proinflammatory cytokine background, with the exception of the IL-10 up-regulation. This finding may suggest that endothelial cells, a likely source of IL-6 and a target of viral replication, do not appear to respond by cytokine production in fatal EHF. The massive presence of IFNs probably contributes to the capillary leak syndrome and hemorrhagic rash classically associated with EHF, and this hypothesis correlates with the clinical observation in Kikwit that most of the fatal cases died in a state of shock accompanied by fever, polypnea, renal failure, and central nervous system alterations.
The evidence for extensive cytokine up-regulation in EHF adds to the information suggesting that cytokines are important in animal models and human diseases caused by other hemorrhagic fever viruses, such as arenaviruses or dengue hemorrhagic fever viruses [2, 13]. While the reported administration of IFN or IFN inducers may have beneficial effects during the incubation phase [14], our data suggest that treatment with antiinflammatory agents during the acute stage of the disease could be considered. It will be interesting to use the available nonhuman primate model of EHF to confirm this clinical data and to test such a therapeutic strategy.
Acknowledgments
We acknowledge the help of John O'Connor for the editorial review of this manuscript.
- © 1999 by the Infectious Diseases Society of America
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