High systemic levels of the cytokine-inducing HMGB1 isoform secreted in severe macrophage activation syndrome

Abstract

Macrophage activation syndrome (MAS) is a potentially fatal complication of systemic inflammation. High mobility group box 1 (HMGB1) is a nuclear protein extensively leaked extracellularly during necrotic cell death or actively secreted by natural killer (NK) cells, macrophages and additional cells during infection or sterile injury. Extracellular HMGB1 orchestrates key events in inflammation as a prototypic alarmin. The redox states of its three cysteines render the molecule mutually exclusive functions: fully reduced "all-thiol HMGB1" exerts chemotactic activity; "disulfide HMGB1" has cytokine-inducing, toll-like receptor 4 (TLR4)-mediated effects—while terminally oxidized "sulfonyl HMGB1" lacks inflammatory activity. This study examines the kinetic pattern of systemic HMGB1 isoform expression during therapy in four children with severe MAS. Three of the four patients with underlying systemic rheumatic diseases were treated with biologics and two suffered from triggering herpes virus infections at the onset of MAS. All patients required intensive care unit therapy due to life-threatening illness. Tandem mass-spectrometric analysis revealed dramatically increased systemic levels of the cytokine-inducing HMGB1 isoform during early MAS. Disease control coincided with supplementary etoposide therapy initiated to boost apoptotic cell death, when systemic HMGB1 levels drastically declined and the molecule emerged mainly in its oxidized, noninflammatory isoform. Systemic interferon (IFN)-γ and ferritin peaked concomitantly with HMGB1, whereas interleukin (IL)-18 and monocyte chemotactic protein (MCP)-1 levels developed differently. In conclusion, this work provides new insights in HMGB1 biology, suggesting that the molecule is not merely a biomarker of inflammation, but most likely also contributes to the pathogenesis of MAS. These observations encourage further studies of disulfide HMGB1 antagonists to improve outcome of MAS.

Figure 1

High systemic HMGB1 levels in MAS patients. Serum concentrations of total HMGB1 levels were markedly increased in plasma during severe MAS as compared with children with uncomplicated JIA and healthy pediatric controls. HMGB1 levels were measured by ELISA and correlation was calculated using the Spearman rank correlation test. The HMGB1 levels in the same cohort of JIA patients and healthy control children have been published previously in (38).

Figure 2

Longitudinal serum analyses before and after introduction of etoposide treatment in patient 1. Serum concentration of total HMGB1 and hyperacetylated HMGB1 during the course of MAS are demonstrated in (A) with the different HMGB1 redox-forms illustrated in (B). High levels of HMGB1 in both fully reduced and disulfide isoforms were documented during severe disease, which rapidly declined after initiation of etoposide treatment concomitantly with serum concentrations of (C) the clinical biomarker ferritin as well as (D) IFN-γ. In the clinical resolution phase, serum HMGB1 appeared predominantly in the terminally oxidized sulfonyl isoform. Serum concentrations of (E) IL-18 and (F) MCP-1 peaked weeks later when the patient was recovering. MP-pulses: methylprednisolone pulses.

Figure 3

Longitudinal serum analyses before and after introduction of etoposide treatment in patient 2. Serum concentration of total HMGB1 and hyperacetylated HMGB1 are illustrated in (A) with the different HMGB1 redox-forms illustrated in (B). The first two serum samples were collected at onset of SoJIA without MAS manifestations when HMGB1 levels appeared mainly in its cytokine-inducing, hyperacetylated isoform. These HMGB1 levels increased at onset of MAS and declined promptly after treatment with etoposide infusions. Increased serum concentrations of (C) ferritin and (D) IFN-γ were documented during MAS followed by a normalization post etoposide treatment. (E) Serum IL-18 levels were distinctly increased during the entire observation period with peak values during MAS. (F) MCP-1 did not mimic the clinical course. MP-pulses: methyl-prednisolone pulses.

Figure 4

Longitudinal serum analyses before and after introduction of etoposide treatment in patient 3. Serum concentration of total HMGB1 and hyperacetylated HMGB1 are demonstrated in (A) with the different HMGB1 redox-forms illustrated in (B). The expression of serum levels of HMGB1 as well as (C) ferritin and (D) IFN-γ corresponded very well to the clinical course of MAS with a rapid decline and clinical improvement after etoposide treatment. IL-18 and MCP-1 levels were increased during critical disease with a prolonged release period (E,F). CsA: cyclosporine A; MP-pulses: methylprednisolone pulses.

Figure 5

Serum analyses in patient 4 before and after treatment with etoposide. Serum concentration of total HMGB1 and hyperacetylated HMGB1 are presented in (A) with the different HMGB1 redox-forms illustrated in (B). Two serum samples were analyzed where normalized levels of (A) HMGB1, (C) ferritin and (D) IFN-γ were documented after intervention with etoposide and subsequent clinical improvement. IL-18 declined but was still elevated (E) while MCP-1 increased (F). MP-pulses: methylprednisolone pulses.