Failure of interferon gamma to induce the anti-inflammatory interleukin 18 binding protein in familial hemophagocytosis

Abstract

Background: Familial hemophagocytosis (FHL) is a rare disease associated with defects in proteins involved in CD8+ T-cell cytotoxicity. Hyperactivation of immune cells results in a perilous, Th1-driven cytokine storm. We set out to explore the regulation of cytokines in an FHL patient who was clinically stable on low-dose immunosuppressive therapy after bone marrow transplantation over a six-month period. During this period, chimerism analyses showed that the fraction of host cells was between 1 and 10%. Both parents of the patient as well as healthy volunteers were studied for comparison.

Methods/principal findings: Using ELISA, quantitative real-time PCR, and clinical laboratory methods, we investigated constitutive and inducible cytokines, polymorphisms, and clinical parameters in whole blood and whole blood cultures. Although routine laboratory tests were within the normal range, the chemokines IP-10 and IL-8 as well as the cytokine IL-27p28 were increased up to 10-fold under constitutive and stimulated conditions compared to healthy controls. Moreover, high levels of IFNgamma and TNFalpha were produced upon stimulation. Unexpectedly, IFNgamma induction of IL-18 binding protein (IL-18BP) was markedly reduced (1.6-fold vs 5-fold in controls). The patient's mother featured intermediately increased cytokine levels, whereas levels in the father were similar to those in the controls.

Conclusions/significance: Since IL-18 plays a major role in perpetuating hemophagocytosis, the failure of IFNgamma to induce IL-18BP may constitute a fundamental pathogenetic mechanism. Furthermore, increased production of IL-8 and IL-27 appears to be associated with this disease. Such dysregulation of cytokines was also found in the heterozygous parents, providing a novel insight into genotype-phenotype correlation of FHL which may encourage future research of this rare disease.

Figure 1. Excessive cytokine production in WB cultures from the FHL patient.

WB culture were incubated for 20 h; thereafter, ELISA was performed on lysates (IL-8) or supernatants (others). Data are shown as cytokine protein in pg normalized to 1 million white blood cells ± SEM, n = 4 time points for IP-10 and IL-8 and n = 3 for IFNγ and TNFα spanning 6 months for all subjects; the same three healthy donors were tested on each time point. *, p<0.05 and **, p<0.01 for healthy controls vs patient, mother, and father; #, p<0.05 and ##, p<0.01 for patient vs mother and father.

Figure 2. Cytokine levels in WB cultures stimulated with LPS.

WB cultures were incubated in the presence of 100 ng/ml LPS for 20 h. In panel D, IL-12 (20 ng/ml) was also added. IL-8 was measured in cell lysates, IP-10, TNFα, and IFNγ were determined in the supernatants. Data are depicted as cytokine protein in pg normalized to 1 million white blood cells ± SEM, n = 4 time points for IP-10 and IL-8 and n = 3 for IFNγ and TNFα over a period of 6 months for all subjects; the identical healthy volunteers were tested on each time point. *, p<0.05 and **, p<0.01 for healthy controls vs patient, mother, and father; #, p<0.05 and ##, p<0.01 for patient vs mother and father.

Figure 3. Regulation of IL-18BP and IL-27p28.

RNA was isolated from WB culture after a 20 h incubation with or without 50 ng/ml IFNγ. A, Data are shown as absolute copy numbers of IL-18BP normalized to GAPDH×10³± SEM, n = 4 time points for all subjects; three control individuals were assayed on each time point. B, IFNγ-induced fold-changes in normalized IL-18BP mRNA copy numbers are shown. A and B, *, p<0.05 and **, p<0.01 for healthy donors vs patient, mother, and father; #, p<0.05 for patient vs mother and father. C, IL-27p28 PCR from the same samples. Differences in GAPDH copy numbers were negligible. One representative of 3 different gels showing similar results is depicted. Healthy donor C exhibited no expression of IL-27p28 (not shown).