NF-κB and TNF-α: A positive autocrine loop in human lung mast cells?

Abstract

The generation of cytokines, particularly TNF-α, by mast cells is crucial for the initiation of the allergic response. A key transcription factor involved in the synthesis of TNF-α is NF-κB. Using a mAb specific for the activated form of NF-κB, immunocytochemistry, confocal microscopy, and gel shift assays have been used in conjunction to localize this transcription factor to human lung mast cells and to study its activation. Activation of mast cells with stem cell factor (10 ng/ml) and anti-IgE (1 μg/ml) induced maximal activation of NF-κB at 4 and 2 h, respectively. In contrast, with TNF-α (5 ng/ml) maximal activation occurred within 15 min. Parallel falls in IκB were demonstrated. Confocal microscopy demonstrated the localization of the activated form of NF-κB to the nuclei of activated mast cells. NF-κB activation was verified using a gel shift assay. A supershift assay showed mast cell NF-κB to be composed primarily of p50 with smaller amounts of p65. No interaction with Abs for Rel-A, c-Rel, Rel-B, and p52 was seen. Immunocytochemistry and ELISAs showed TNF-α to be stored within mast cells and released into the extracellular environment following activation. The possible participation of TNF-α generated by mast cells in NF-κB activation by anti-IgE was investigated using a blocking Ab for TNF-α. The blocking Ab reduced NF-κB activation by anti-IgE by >50%, suggesting that the release of preformed mast cell-associated TNF-α acts as a positive autocrine feedback signal to augment NF-κB activation and production of further cytokine, including GM-CSF and IL-8.

The mast cell has long been considered a pivotal effector cell in allergic disease by virtue of its capacity to respond rapidly to provoking stimuli and its ability to release a wide range of preformed and newly generated proinflammatory mediators. The generation by mast cells of cytokines likely to be crucial for the initiation and maintenance of the allergic responses is now well established (reviewed in Ref. 1). One of the major mast cell-derived cytokines is TNF-α, a pleotropic cytokine encoded on chromosome 6, which exists in its biologically active form as a homotrimer of a 17-kDa subunit cleaved proteolytically from its 26-kDa cell surface-associated form (2, 3). It has a broad range of biological activities associated with inflammatory diseases, many of which are pertinent to allergy (reviewed in Ref. 4). Preformed immunoreactive TNF-α has been observed within the granules of mast cells from human skin (5) and lung (6), suggesting its rapid availability following mast cell activation. That this preformed TNF-α may be biologically relevant has been shown by its release within 2 min of allergen challenge in parallel with tryptase in patients with allergic rhinitis (7).

A key transcription factor involved in the synthesis of TNF-α is NF-κB. NF-κB, which also regulates the transcription of a number of proinflammatory molecules, including GM-CSF, IL-8, IL-2, IL-6, E-selectin, ICAM-1, and VCAM-1, may be of variable composition but is classically presented in a wide range of cells as a heterodimer comprising of p50 and p65, each of which contains the 300-aa NF-κB/rel/dorsal domain (8, 9). In resting cells NF-κB is present in the cytoplasm in an inactive form reversibly bound to proteins of the IκB family (10). On cell stimulation by a range of stimuli including TNF-α, IL-1, IL-2, leukotriene B4, viruses, and free radicals (10), IκB undergoes proteolysis and the nuclear location site of NF-κB becomes revealed. This activation of NF-κB is necessary for its translocation across the nuclear membrane and binding to its target gene promoter regions. In addition to increasing the transcription of cytokines and adhesion proteins, NF-κB also increases the transcription of IκB, thus leading to its own inactivation and subsequent termination of the response.

In human lung mast cells, we demonstrate the presence and activation of NF-κB and the generation of TNF-α. We then examined the hypothesis that when mast cells are stimulated immunologically, the release of preformed TNF-α acts as a positive autocrine feedback signal to augment NF-κB activation.

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