NF-κB and TNF-α: A Positive Autocrine Loop in Human Lung Mast Cells?

William R. Coward, Yoshimichi Okayama, Hironori Sagara, Susan J. Wilson, Stephen T. Holgate and Martin K. Church
J Immunol November 1, 2002, 169 (9) 5287-5293; DOI: https://doi.org/10.4049/jimmunol.169.9.5287

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.

Materials and Methods

Purification and culture of human lung mast cells

Human lung cells were dispersed from macroscopically normal human lung by an enzymatic procedure described previously (11). Mast cell numbers were counted in a Neubauer hemocytometer after metachromatic staining with Kimura stain (12). Cell dispersates initially contained 0.4 ± 0.1 × 106 mast cells/g lung tissue in a purity of ∼5%. Erythrocytes were removed by centrifugation through a 65% continuous Percoll gradient (1.084 g/ml) and T lymphocytes by serial affinity selection using anti-CD2-coated magnetic beads (Dynabeads M-450 Pan-T; Dynal Biotech, Oslo, Norway), a technique which removes >99% of T lymphocytes. Nucleated cells were then incubated with the anti-c-kit mAb YB5.B8 (13, 14) (donated by Dr. L. K. Ashman, Institute of Medical and Veterinary Science, Adelaide, South Australia), washed, and subjected to positive magnetic affinity using goat anti-mouse IgG-coated Dynabeads (15). This procedure yielded mast cells, identified by the mAb AA1 against tryptase (16), in purities of >95%. Following purification, to allow the cells to recover the rigors of purification and to sensitize them, mast cells were incubated for 16 h with human myeloma IgE (3 μg/ml; Calbiochem-Novabiochem, San Diego, CA). Following sensitization mast cells were cultured at 1 × 105 cells/well in a 96-well tissue culture plate in humidified 95% air with 5% CO2 at 37°C in DMEM supplemented with 2 mM l-glutamine, 200 U penicillin/200 μg/ml streptomycin, and 1% FCS.

Activation of NF-κB with immunological mediators

To investigate the kinetic activation of NF-κB, degradation of IκB and the immunoreactivity for TNF-α, 1 × 105 human lung mast cells following purification and passive sensitization were incubated in medium alone and compared with mast cells activated with recombinant human stem cell factor (SCF5; 10/50 ng/ml, donated by Cytomed, Cambridge, MA) in the presence or absence of either anti-IgE (1 μg/ml; Serotec, Oxford, U.K.) or TNF-α (5 ng/ml) for various time intervals (0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h) up to 48 h. Following experimentation, the cells were spun in a cytocentrifuge (20 × g) for 7 min onto poly-l-lysine-coated glass slides and stained with specific mAbs: for activated NF-κBa, 2C7 (17) (Pharmacia Upjohn, Kalamazoo, MI), IκBα (Santa Cruz Biotechnology, Santa Cruz, CA) or TNF-α (18) (Celltech, Slough, U.K.), GM-CSF (Genzyme, Cambridge, U.K.), and IL-8 (Dr. I. Lindley, Novartis, Vienna, Austria). Slides were developed using an indirect peroxidase detection system with the substrate 3-amino-9-ethylcarbazole. Two hundred to 300 cells were counted, depending upon the quality of the cytospin, and the percentage of positive cells was calculated.

Colocalization of activated NF-κB to the nuclei of human lung mast cells

Two-channel confocal microscopy was used to produce a combined image of cytospin preparations in which activated NF-κB was stained with mAb 2C7 using a biotin and fluorescein detection system (2C7-biotin-FITC), and the cell nuclei were counterstained with propidium iodide. Colocalization analysis was performed and a multicolor look-up table was used to show the extent of colocalization of activated NF-κB and the nucleus.

EMSA

Nuclear proteins were isolated from human purified lung mast cells and incubated with a radiolabeled NF-κB consensus DNA sequence. The reaction mixture was then subjected to gel electrophoresis using a nondenaturing polyacrylamide gel. The gel was then subjected to autoradiography and the heavier DNA-NF-κB complexes were identified by their slower electrophoretic migration. Supershift assays were performed by preincubating the nuclear extracts with Abs to subunits, Rel-A, Rel-B, c-Rel, p50, p52, and p65 of NF-κB (Santa Cruz Biotechnology).

Statistical analyses

Data, expressed as the mean ± SEM, were tested for normality using the Shapiro-Wilk test. In normally distributed data, the difference between two means was tested for significance using Student’s t test for paired data. In skewed nonparametric data, the difference between two means was tested for significance using the Mann-Whitney U test. The difference between two means was said to be significant when p < 0.05 and greatly significant when p < 0.001. To determine whether or not the immunoreactivity of NF-κBa correlated with the immunoreactivity for TNF-α, Pearson’s correlation was used.

Results

Activation of NF-κB

To determine the optimal concentrations of SCF, anti-IgE and TNF-α required to activate NF-κB to its active form (NF-κBa), five preliminary experiments were performed in which mast cells were incubated with the stimulants for 4 h and the percentage of cells staining positive for immunoreactive NF-κBa was determined. SCF (1–100 ng/ml) caused a concentration-related increase in NF-κBa+ mast cells from a baseline of 10 ± 5% (medium control) to a maximum of 52 ± 12% (p < 0.005) at 100 ng/ml. A concentration of 10 ng/ml SCF, which induced NF-κB activation in 26 ± 4% cells (p < 0.02), was used in additional experiments. Anti-IgE (0.1–10 μg/ml) also induced a concentration-related increase in the number of NF-κBa+ mast cells from 10 ± 5% to a maximum of 44 ± 6% (p < 0.001) at 5 μg/ml. A concentration of 1 μg/ml anti-IgE, which caused 35 ± 6% (p < 0.005) activation, was used in additional experiments. TNF-α (0.01–50 ng/ml) again caused a concentration-related increase in the number of NF-κBa+ mast cells from 15 ± 5% to a maximum of 70 ± 4% (p < 0.001) at 50 ng/ml. A concentration of 5 ng/ml TNF-α, which caused 59 ± 2% (p < 0.001) activation, was used in additional experiments.

Kinetics of NF-κB activation

Fig. 1⇓a shows that when mast cells were incubated with 10 ng/ml SCF for up to 48 h, a maximum percentage of NF-κBa+ mast cells of 34 ± 9% (p < 0.05, n = 5) was seen at 4 h, after which numbers waned. In the same experiments, 1 μg/ml anti-IgE in the presence of 10 ng/ml SCF significantly (p < 0.05) enhanced NF-κB activation at 30 min, 1 h, and 2 h, a maximum of 38 ± 5% being observed at 2 h.

FIGURE 1.
FIGURE 1.

Kinetics of NF-κB activation. A, Effects of anti-IgE and SCF. The symbols represent mast cells incubated with 1 μg/ml anti-IgE in the presence of 10 ng/ml SCF (▪), 10 ng/ml SCF alone (▴), or tissue culture medium (▿, unstimulated control). At the end of the incubation, cytospin preparations were made and stained immunocytochemically for NF-κBa. All stimulated values were significantly (p < 0.001) different from those of unstimulated controls, and ∗ indicates the times when anti-IgE plus SCF was significantly (p < 0.05) different from SCF alone. B, Effect of TNF-α. In the first series of experiments in which the earliest measurements were made at 15 min, the symbols represent mast cells incubated with: 5 ng/ml TNF-α in the presence of 10 ng/ml SCF, 5 ng/ml TNF-α alone, or tissue culture medium (▿, unstimulated control). At the end of the incubation, cytospin preparations were made and stained immunocytochemically for NF-κBa. All stimulated values were significantly (p < 0.001) different from those of unstimulated controls and from each other. In the second series of experiments to examine the first 15 min after stimulation, the symbols represent mast cells incubated with 5 ng/ml TNF-α alone (♦) or tissue culture medium (▿, unstimulated control). Again, all stimulated values were significantly (p < 0.001) different from those of unstimulated controls. All results are expressed as the mean ± SEM for five experiments.

TNF-α induced a more rapid activation of NF-κB (Fig. 1⇑b), 19 ± 1% of mast cells expressing NF-κBa within 1 min and rising to a maximum of 63 ± 2% at 15 min (both p < 0.001, n = 5). In the presence of SCF, this was significantly (p < 0.001) enhanced to 77 ± 2% at 15 min. NF-κB activation remained significantly (p < 0.05) elevated throughout the 48-h observation period.

IκB immunoreactivity

The activation of NF-κB within mast cells should be accompanied by a fall in its inhibitory protein, IκB. This was demonstrated in five experiments in which the percentage of mast cells showing immunoreactivity for IκB at baseline was 46 ± 2%. Incubation of these cells with 10 ng/ml SCF, 1 μg/ml anti-IgE, and 5 ng/ml TNF-α (the last two both in the presence of 10 ng/ml SCF) all caused an inhibition of IκB immunoreactivity which paralleled the kinetics of the increase in NF-κB immunoreactivity (Fig. 2⇓).

FIGURE 2.
FIGURE 2.

Kinetics of IκB degradation. The symbols represent mast cells incubated with 1 μg/ml anti-IgE in the presence of 10 ng/ml SCF (▪), 5 ng/ml TNF-α in the presence of 10 ng/ml SCF, or tissue culture medium (▿, unstimulated control). At the end of the incubation, cytospin preparations were made and stained immunocytochemically for IκB. All stimulated values were significantly (p < 0.001) different from those of unstimulated controls. Results are expressed as the mean ± SEM for five experiments.

Gel shift assay

To verify NF-κB activation, a gel shift assay was used in which the electrophoretic migration of the heavier DNA-NF-κB complexes is slower than inactive NF-κB (Fig. 3⇓). Incubation of mast cells for 4 h in medium alone contained very little activated NF-κB bound to the radiolabeled NF-κB-binding motif. Activation with 1 μg/ml anti-IgE for 4 h and 5 ng/ml TNF-α for 15 min induced the activation of NF-κB, the latter appearing to be more dense on the gel. Preincubation of mast cells for 1 h with 10 μM of the NF-κB inhibitor, calpain inhibitor 1, prevented activation of NF-κB by TNF-α.

FIGURE 3.
FIGURE 3.

Gel shift assay of NF-κB. Mast cells were activated with 1 μg/ml anti-IgE for 4 h, 5 ng/ml TNF-α for 15 min, or 10 μM calpain inhibitor for 1 h before activation with 5 ng/ml TNF-α for 15 min or in medium alone for 15 min (unstimulated control). Following incubation, experimentation nuclear proteins were purified and incubated with a radiolabeled NF-κB oligonucleotide-binding motif and run on a 5% acrylamide gel.

Supershift assay

To gain further insight into the composition of activated NF-κB, a gel supershift assay was used. Nuclear protein purified from mast cells that had been activated with 5 ng/ml TNF-α for 15 min was incubated with mAbs for Rel-A, Rel-B, c-Rel, p50, p52, and p65. Fig. 4⇓ shows the presence of large amounts of p50 and smaller amounts of p65. No interaction with the other Abs was seen.

FIGURE 4.
FIGURE 4.

Supershift assay of NF-κB. Mast cells were activated with 5 ng/ml TNF-α for 15 min and the nuclear proteins were purified. These proteins were then incubated with mAbs against p65, c-Rel, Rel-B, p50, p52, and then a radiolabeled NF-κB oligonucleotide-binding motif. Running the protein mAb complex on a 5% acrylamide gel allowed the components of NF-κB to be determined.

Colocalization of NF-κB to the nucleus

To visualize NF-κBa within the nucleus, mast cells were incubated for 4 h in medium alone or activated with anti-IgE (1 μg/ml). Two-channel confocal microscopy was used to produce a combined image of cytospin preparations stained with 2C7-biotin-FITC in conjunction with the nuclear stain propidium iodide (Fig. 5⇓). Colocalization analysis using multicolor look-up tables demonstrated enhanced levels of NF-κBa, clearly enhanced within the nuclei of mast cells activated with anti-IgE compared with controls.

FIGURE 5.
FIGURE 5.

Colocalization of NF-κB to the nucleus of human lung mast cells. Following purification and passive sensitization, human lung mast cells were incubated in medium alone (A) or activated with anti-IgE (1 μg ml) for 4 h (B). Two-channel confocal microscopy was used to produce a combined image of cytospin preparations stained with the mAb 2C7 in conjunction with fluorescein and the cell nuclei counterstained with propidium iodide (original magnification, ×400). Colocalization analysis was then performed; a multicolor look-up table was used to show the extent of colocalization between activated NF-κB and the nucleus. Enhanced levels of activated NF-κB are clearly evident within the nuclei of mast cells activated with anti-IgE compared with controls.

Production TNF-α and other cytokines

Because the measurement of cytokines by ELISA requires large numbers of cells, initial studies on TNF-α production were performed by counting in cytospin preparations the number of mast cells that stained positively for NF-κBa and TNF-α. These studies confirmed the presence at 4 h and 15 min, respectively, of NF-κBa when mast cells were activated with 1 μg/ml anti-IgE and 5 ng/ml TNF-α, both in the presence of 10 ng/ml SCF. These stimuli also led to a time-related increase in TNF-α-positive cells up to 12 h (Fig. 6⇓a). Preincubation of the cells with 10 μM calpain inhibitor 1 for 1 h before stimulation significantly reduced the number of cells staining for both NF-κBa and TNF-α. Other inhibitors of NF-κB activation, pentoxifylline (0.5 mM), pyrrolidine dithiocarbamate (10 μM), and gliotoxin (1 pg/ml), reduced TNF-α-stimulated NF-κB activation 89, 91, and 63%, respectively (all P < 0.001).

FIGURE 6.
FIGURE 6.

Inhibition of NF-κB and cytokine production by calpain inhibitor 1. A, Mast cells, preincubated for 1 h in the presence or absence of 10 μM calpain inhibitor 1, were activated with 5 ng/ml TNF-α plus 10 ng/ml SCF for 15 min for estimation of NF-κB activation (▪) and 6 h for estimation of TNF-α positivity (▩). Cells incubated in tissue culture medium for 6 h served as unstimulated controls. At the end of the incubation, cytospin preparations were made and stained immunocytochemically for NF-κBa and TNF-α. Inhibition by calpain inhibitor 1 was statistically significant (p < 0.001) for both NF-κBa and TNFα. B, The above experiments were repeated with two differences. The cells were stimulated with 5 ng/ml TNF-α alone and cells staining positive for GM-CSF (▥) and IL-8 (▤) were also counted. In this study, inhibition by calpain inhibitor 1 was statistically significant (p < 0.05) for all cytokines. All results are the mean ± SEM for five experiments.

In parallel studies, activation with 5 ng/ml TNF-α alone significantly increased the number of mast cells staining positive for NF-κBa, TNF-α, GM-CSF, and IL-8. Again, the degree of activation was significantly (p < 0.05) reduced by calpain (Fig. 6⇑b).

The production of TNF-α by mast cells was confirmed in six experiments where mast cells were stimulated with 1 μg/ml anti-IgE and 10 ng/ml SCF for 6, 12, or 24 h, and the supernatant and cell lysate were assayed for TNF-α using ELISA. Baseline TNF-α levels were close to the level of detection, being 0.37 ± 0.06 and 1.06 ± 0.2 pg/1 × 106 mast cells for supernatant and lysate. In unstimulated cells, these levels did not change significantly at any point throughout the 24-h incubation period. Following stimulation, the concentration of TNF-α in the supernatant rose to a maximum of 6.56 ± 0.26 pg/1 × 106 mast cells (p < 0.001) at the first observation point at 6 h and remained significantly elevated thereafter. Only small amounts of TNF-α were found in the lysate, but the concentration of 1.46 ± 0.20 pg/1 × 106 mast cells found at 12 h was significantly (p < 0.005) above that of control.

In an additional 35 experiments, mast cells were stimulated for 24 h with 1 μg/ml anti-IgE in the presence of 50 ng/ml SCF. The TNF-α levels in the supernatant and pellet were 50.4 ± 7.7 and 16.3 ± 1.8 pg/1 × 106 mast cells (both p < 0.001 compared with unstimulated control). Fig. 7⇓ shows that the TNF-α levels within the pellets were correlated significantly (p < 0.005) with the concentrations in the supernatant, the amount stored being ∼30% of that secreted.

FIGURE 7.
FIGURE 7.

Relationship between intracellular and secreted TNF-α. Mast cells were incubated with 1 μg/ml anti-IgE plus 50 ng/ml SCF (1 μg/ml) for 24 h and centrifuged, and the supernatant was removed and the pellets were lysed. TNF-α was assayed simultaneously in both. Each point represents the results from lung mast cells prepared from a separate donor’s tissue (n = 35). TNF-α contents of the pellets were significantly correlated (r2 = 0.213, p = 0.003) with the TNF-α concentration in the supernatant.

Does mast cell-derived TNF-α contribute to NF-κB activation?

To confirm the blocking activity of the anti-TNF-α Ab, mast cells were incubated for 1 h with 1–100 ng/ml of the Ab before stimulation with 5 ng/ml TNF-α for 15 min in five experiments. The results show a concentration-related inhibition of the percentage of cells staining for activated NF-κB (Fig. 8⇓). A concentration of 100 ng/ml blocking Ab was used in additional experiments.

FIGURE 8.
FIGURE 8.

Effect of anti-TNF-α on NF-κB activation by TNF-α. Mast cells preincubated for 1 h with 0–100 ng/ml of a monoclonal blocking Ab for TNF-α before activation with 5 ng/ml TNF-α for 15 min. At the end of the incubation, cytospin preparations were made and stained immunocytochemically for NF-κBa. Results are expressed as the mean ± SEM for five experiments.

The possible participation of TNF-α generated by mast cells in NF-κB activation by anti-IgE in the presence of SCF was investigated in five experiments using a blocking Ab for TNF-α. The results (Fig. 9⇓) show that the percentage of cells containing NF-κBa following incubation in medium alone for 4 h was 9 ± 1%. Activation of these cells with 1 μg/ml anti-IgE in the presence of 10 ng/ml SCF increased the percentage of immunoreactive cells to 38 ± 6%. Preincubation of the cells for 1 h with 100 ng/ml of the blocking mAb significantly reduced the percentage of immunoreactive cells to 18 ± 1% (p < 0.05).

FIGURE 9.
FIGURE 9.

Effect of anti-TNF-α on NF-κB activation by SCF and anti-IgE. Mast cells preincubated for 1 h with 100 ng/ml of a monoclonal blocking Ab for TNF-α before activation with 1 μg/ml anti-IgE in the presence of 10 ng/ml SCF for 4 h. At the end of the incubation, cytospin preparations were made and stained immunocytochemically for NF-κBa. Results are expressed as the mean ± SEM for five experiments.

Discussion

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. The kinetics of NF-κB activation and its involvement in the production of TNF-α have been investigated following activation of purified mast cells with anti-IgE, SCF, and TNF-α itself. To explore the relationship between NF-κB activation and TNF-α production, a range of NF-κB inhibitors has been used.

Activation of mast cells with SCF, anti-IgE, and TNF-α induced the activation of NF-κB in a concentration-dependent manner with a corresponding decline in IκBα immunoreactivity. Activation of mast cells with SCF and anti-IgE induced maximal activation of NF-κB at 4 and 2 h, respectively. In contrast, TNF-α induced a much more rapid activation of NF-κB, maximal activation occurring within 15 min. That this response was initiated by a TNF-α receptor-dependent mechanism was demonstrated by its inhibition by a blocking mAb for TNF-α. That this immunoreactivity was associated with the activation of authentic NF-κB was confirmed in gel shift assays. Furthermore, supershift assays showed p50 to be the major NF-κB subunit in human lung mast cells, with smaller amounts of p65 also being present.

SCF is a key factor influencing the survival, maturation, migration, and function of mast cells (15, 19, 20). In this study, SCF was shown to both initiate NF-κB activation and enhance activation induced by TNF-α and anti-IgE. The c-kit receptor has intrinsic tyrosine kinase activity (21) and upon agonist binding undergoes autophosphorylation (22). Even though FcεRI lacks intrinsic tyrosine kinase activity (23) and relies upon closely associated tyrosine kinases (24), the activation of mast cells via SCF or anti-IgE leads to a virtually indistinguishable calcium signaling pattern (25). This has been shown in previous studies to lead to a similar pattern of early response genes including c-fos, c-jun, and c-junβ, and similar patterns of phosphorylation and activation of mitogen-activated protein kinase (26). SCF has been demonstrated to both induce the secretion of mast cell mediators directly (27) and to regulate the extent of mediator release in mast cells activated by anti-IgE-dependent mechanisms (15, 28). In particular, anti-IgE and SCF stimulate the transcription and secretion of TNF-α when used independently and enhance the effects of each other when used together. This was confirmed in the current study in which the activation of mast cells with SCF alone or in combination with anti-IgE led to NF-κB activation and the production of TNF-α.

TNF-α induces a variety of cellular responses via binding to its two receptors (29). The ability of these receptors to transduce signals is dependent upon the recruitment of TNFR-associated factors (30) which initiate a number of downstream events, including the activation of NF-κB-inducing kinases (31, 32). TNF-α has been reported to induce histamine release from human skin mast cells (33, 34) and to enhance IgE-dependent histamine release from sensitized lung mast cells (35). Furthermore, the sustained release of preformed and newly synthesized TNF-α following appropriate stimulation represents a mechanism by which mast cell-derived TNF-α can exert its actions on leukocyte migration and activation and the initiation of late-phase allergic inflammation. With the use of KO mice and adoptive transfer experiments, mast cell-derived TNF-α has been shown to contribute significantly to leukocyte infiltration (36). Also, unlike macrophages and lymphocytes, which contain little or no preformed TNF-α, this cytokine is stored in human mast cells in many tissue sites (5, 6, 37). But is this TNF-α available for rapid release following mast cell activation? Two clinical studies suggest that it is, TNF-α having been detected within 2 min in the nasal lavage fluid following allergen challenge in hay fever (7) and in the venous blood following challenge of the skin in cold urticaria (38). However, it was not possible to confirm this directly by ELISA measurement of TNF-α in our in vitro studies because the levels of “total cell-associated” cytokine were close to the levels of detection. However, we did obtain indirect evidence of TNF-α release by use of a blocking Ab that reduced NF-κB activation by anti-IgE and SCF by >50%. Beside providing evidence for TNF-α release by human mast cells, this observation is highly suggestive of a local autocrine feedback of TNF-α, possibly even membrane-associated TNF-α, onto surface receptors on the cell from which it is released. This would be consistent with the high affinity of TNF-α for its receptors (39).

This work has demonstrated important roles for TNF-α and NF-κB in the activation of human mast cells. The observations that mast cells both release and respond to TNF-α indicates that there is a positive autocrine loop which leads to augmentation of mast cell activation. Our previous studies have shown that activation of bronchial explants and nasal polyps stimulated ex vivo, with recombinant TNF-α, lead to the activation of NF-κB in complex tissue systems in parallel with the up-regulation of proinflammatory cytokines and inflammatory markers (17). It is, therefore, suggested that the secretion of TNF-α from mast cells augments NF-κB activation not only of mast cells but also of surrounding inflammatory cells and thus has a considerable proinflammatory effect on its local microenvironment.

Footnotes

  • ↵1 Current address: Department of Microbiology and Immunology, Faculty of Medicine and Biological Sciences, Maurice Shock Building, Institute for Lung Health, University of Leicester, P.O. Box 138, Leicester, LE1 9HN, U.K.

  • ↵2 Current address: Laboratory for Allergy Transcriptome, The Institute of Physical and Chemical Research Center for Allergy and Immunology, National Research Institute for Child Health and Development, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8567, Japan.

  • ↵3 Current address: Department of Pulmonary Medicine and Clinical Immunology, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, Japan.

  • ↵4 Address correspondence and reprint requests to Dr. Martin K. Church, Dermato-pharmacology Unit, South Block 825, Southampton General Hospital, Southampton SO16 6YD, U.K. E-mail address: mkc{at}soton.ac.uk

  • ↵5 Abbreviation used in this paper: SCF, stem cell factor.

  • Received December 19, 2001.
  • Accepted August 9, 2001.

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