Effects of Glucocorticoid Administration on Glucocorticoid Receptor and NF-κB Interaction in Unresolving ARDS

C. R. Yates, G. U. Meduri

University of Tennessee Health Science Center, Memphis, Tennessee, U.S.A.

Excessive systemic inflammation is the pathophysiologic hallmark of pulmonary and extrapulmonary organ dysfunction in patients with acute respiratory distress syndrome (ARDS), a frequent cause of hypoxemic respiratory failure associated with a 40 - 60 % mortality. Failure to down-regulate inflammatory mediators is associated with maladaptive lung repair and inability to improve gas exchange and lung mechanics (clinically defined as unresolving ARDS). In immunohistochemical analyses of lung tissue obtained from patients with unresolving ARDS, we found that histologically severe ARDS was associated with predominant nuclear NF-κB uptake in resident cells, while mild ARDS was associated with predominant nuclear GRα uptake.

NF-κB activation is critical to the experimental development of lung inflammation. NF-κB is found in essentially all cell types and is involved in activation of numerous target genes. Heterodimeric p65:p50 is the most abundant form of NF-κB in most cell types. NF-κB is maintained in an inactive form in the cytoplasm through interaction with inhibitory proteins IκBs (most important being IκBα) (Fig. [1]). In response to various inflammatory signals, the latent NF-κB/IκB complex is activated by phosphorylation and proteolytic degradation of IκB, with exposure of the NF-κB nuclear localization sequence (NSL). The liberated NF-κB then translocates to the nucleus and binds to promoter regions of target genes to initiate the transcription of numerous inflammatory genes including tumor necrosis factor-alpha (TNF-α) and the interleukins (IL) IL-1β, IL-2, IL-6, chemokines (e. g., IL-8), and cell adhesion molecules (e. g., intercellular adhesion molecule-1, E-selectin).

Fig. 1 Cellular events: Activation of inflammation.

As a dimer and/or a monomer, GR-mediated transcriptional interference with NF-κB is achieved by five important mechanisms (Fig. [1]): (i) physically interacting with the p65 subunit and formation of an inactive (GR-NF-κB) complex, (ii) by inducing the transcription of the inhibitory protein IκBα gene (iii) by blocking degradation of IκBα via enhanced synthesis of IL-10, (iv) by impairing TNF-α induced degradation of IκBα, and (v) by competing for limited amounts of GR co-activators such as CREB-binding protein (CBP) and steroid receptor coactivator-1 (SRC-1).

Using an ex vivo model of systemic inflammation we investigated intracellular upstream and downstream events (cytosolic and nuclear) associated with DNA-binding of NF-κB and GRα in PBLs (naïve cells) obtained from a healthy volunteer and incubated for 3 hours with plasma samples obtained longitudinally from patients with unresolving ARDS before and after randomization prolonged methylprednisolone (MP) vs. placebo administration. In the plasma, we measured the concentrations of TNF-α and IL-1β, ACTH, and cortisol. In the observation period prior to randomization, the biologic and physiologic characteristics of the two groups were similar. Patients had persistent elevations in plasma TNF-α, IL-1β, ACTH and cortisol levels, and similar severity of organ dysfunction scores. The intracellular changes observed in exposed normal PBLs included escalating increases in NF-κB-mediated activities (NF-κB DNA-binding, p50 and p65 DNA-binding, and transcription of TNF-α and IL-1β, and modest changes in GRα-mediated activities. The reduction in cytoplasmic IκBα levels observed prior to randomization indicated that NF-κB-mediated IκBα degradation predominated over GRα-mediated IκBα formation.

Following randomization, MP-treated patients had progressive and sustained reductions of TNF-α, IL-1β, ACTH and cortisol levels over time. Normal PBL exposed to plasma exhibited significant progressive increases in all aspects of GR-mediated activity and significant reductions in NF-κB DNA-binding and transcription of TNF-α and IL-1β. Patients treated with MP had rapid, progressive and sustained reductions in plasma TNF-α, IL-1β, IL-6, ACTH and cortisol levels over time, which paralleled improvements in pulmonary and extrapulmonary organ dysfunction scores. Normal PBL exposed to plasma collected during MP vs. placebo treatment exhibited rapid, progressive significant increases in GRα-mediated activities (GRα binding to NF-κB, GRα binding to GRE DNA, stimulation of inhibitory protein IκBα, and stimulation of IL-10 transcription), and significant reductions in NF-κB binding and transcription of TNF-α and IL-1β (Fig. [2]). With MP, the intracellular relations between the NF-κB and GRα signaling pathways changed from an initial NF-κB-driven and GR-resistant state to a GR-driven and GR-sensitive one. The responses observed during MP support the concept of inflammation-dependent acquired glucocorticoid resistance in patients with unresolving ARDS and underscore the central role played by the activated GRα in regulating NF-κB-driven inflammation.

Fig. 2 Inflammatory marker response.