Martin K. Church

Inhibition of IgE-dependent histamine release from human dispersed lung mast cells by anti-allergic drugs and salbutamol.

1 The ability of the anti‐allergic drugs, sodium cromoglycate (SCG), lodoxamide, traxanox, RU31156 and the β‐adrenoceptor agonist sulbutamol to inhibit IgE‐dependent histamine and prostaglandin D2 (PGD2) release was assessed using human dispersed lung mast cells. 2 The anti‐allergic drugs were weak inhibitors of histamine release, high concentrations (100–1000 μm) producing < 35% inhibition. Salbutamol produced 39% inhibition at 10 μm. 3 The efficacy of both SCG and salbutamol was inversely related to the concentration of anti‐IgE used for challenge and to the degree of histamine release. 4 Rapid tachyphylaxis was observed with all anti‐allergic drugs but not with salbutamol. 5 Cross‐tachyphylaxis was observed between SCG and the other anti‐allergic drugs, suggesting a common mechanism of action. No cross‐tachyphylaxis was observed between SCG and salbutamol. 6 SCG was significantly (P < 0.001) more effective in inhibiting PGD2 than it was histamine release. Preferential inhibition of PGD2 compared with histamine release was less marked (P < 0.05) with salbutamol and not significant with the other anti‐allergic drugs. 7 Mast cells dispersed by enzymatic digestion of human lung released more histamine on immunological challenge than mechanically dispersed cells obtained by fine chopping of tissue. Enzyme treatment of mechanically dispersed cells removed this difference. Enzymatically and mechanically dispersed cells responded similarly to the inhibitory effects of SCG and salbutamol. 8 Our results suggest that salbutamol is a more effective inhibitor of mediator release from human lung mast cells than anti‐allergic drugs. However, with the low levels of mediator release achieved during an allergic reaction in man in vivo, both salbutamol and SCG are likely to be effective inhibitors of both preformed and newly generated mediators.

Human mast cell .heterogeneity: Histamine release from mast cells dispersed from skin, lung, adenoids, tonsils, and colon in response to IgE-dependent and nonimmunologic stimuli

We have compared the ability of anti-IgE, calcium ionophore A23187, substance P, compound 48/80, poly-L-lysine, and morphine to release histamine from mast cells of human skin, lung, adenoids, tonsils, and colon. Use of a single collagenase/hyaluronidase dispersion technique for all tissues has allowed comparisons of reactivity to be made that are free from methodological variations. Mast cells from all tissues examined secreted histamine in response to anti-IgE and calcium ionophore A23187. However, only skin mast cells were responsive to substance P, compound 48/80, poly-L-lysine, and morphine. Activation of human skin mast cells by these nonimmunologic stimuli clearly distinguishes them from the mast cells of human lung, adenoids, tonsils, and colon and is indicative of functional heterogeneity within the human mast cells population. We propose that the presence of functional receptor sites for neuropeptides and basic compounds on skin mast cells that are not present in mast cell populations from mucosal or lymphoid sources reflects a specialized role for these cells in vascular homeostasis.

Characterization of neuropeptide-induced histamine release from human dispersed skin mast cells.

1 Human skin mast cells, unlike other human mast cells so far studied, released histamine in a concentration‐related manner in response to substance P, vasoactive intestinal peptide (VIP) and somatostatin (1 μM to 30 μM). In contrast, eledoisin, physalaemin, neurokinin A, neurokinin B, calcitonin gene‐related peptide (CGRP), neurotensin, bradykinin and Lys‐bradykinin induced negligible histamine release. 2 The low histamine releasing activity of physalaemin, eledoisin, neurokinin A and neurokinin B relative to substance P suggests that the human skin mast cell activation site is distinct from the tachykinin NK‐1, NK‐2 or NK‐3 receptors described in smooth muscle. 3 The relative potencies of substance P and its fragments SP2–11, SP3–11, SP4–11 and SP1–4 in releasing histamine from human skin mast cells suggests that both the basic N‐terminal amino acids and the lipophilic C‐terminal portion of substance P are essential for activity. 4 Peptide‐induced histamine release, like that induced by compound 48/80, morphine and poly‐L‐lysine, is rapid, reaching completion in 10–20 s, is largely independent of extracellular calcium but requires intact glycolysis and oxidative phosphorylation. 5 The substance P analogue, [D‐Pro4, D‐Trp7,9,10] SP4–11 (SPA), not only reduced substance P‐induced histamine release in a concentration‐related manner but also inhibited that induced by VIP, somatostatin, compound 48/80, poly‐L‐lysine and morphine but not anti‐IgE. 6 The similar characteristics of histamine release induced by substance P, VIP, somatostatin, compound 48/80, poly‐L‐lysine and morphine suggest that they share a common pathway of activation‐secretion coupling distinct from that of IgE‐dependent activation. Furthermore, the ability of human skin mast cells to respond to basic non‐immunological stimuli including neuropeptides may reflect a specialised function for these cells.

Differential release of histamine and eicosanoids from human skin mast cells activated by IgE‐dependent and non‐immunological stimuli

1 Cells were dispersed from human foreskin using a mixture of collagenase and hyaluronidase and separated into mast cell‐depleted (<1%) or enriched (>75%) preparations by density‐gradient centrifugation. 2 Challenge of gradient fractions with ∍‐chain‐specific anti‐human IgE stimulated the release of histamine, prostaglandin D2 (PGD2) and leukotriene C4 (LTC4). The release of eicosanoids was significantly correlated with that of histamine, suggesting that they are derived from the mast cell population of the dispersate. In highly purified (76.2 ± 4.2%) mast cell preparations, maximum net release of histamine, PGD2 and LTC4 was 3432 ± 725, 84.9 ± 10.8 and 6.6 ± 1.2 pmol/106 nucleated cells. 3 The non‐immunological stimuli substance P, vasoactive intestinal peptide (VIP), somatostatin, compound 48/80, morphine and poly‐L‐lysine released similar amounts of histamine to anti‐IgE, but 12 to 21 fold less PGD2 and LTC4. 4 These studies suggest that IgE‐dependent and non‐immunological stimuli activate human skin mast cells by different secretory mechanisms, a hypothesis supported by our previous findings of differences in Ca2+ requirements and time‐course of histamine release. Activation by the non‐immunological mechanism may be of importance in vivo due to the close anatomical association between skin mast cells and dermal nerve‐terminals containing neuropeptides.

Dendritic cells in normal and asthmatic airways: expression of the α subunit of the high affinity immunoglobulin E receptor (FcεRI‐α)

Background Immunoglobulin E (IgE) plays an important role in asthma, with total serum IgE levels closely related to both clinical expression of the disease and airway hyperresponsiveness. IgE binds to a high affinity cell‐surface receptor (FcεRI) which is present on mast cells and which has also recently been demonstrated on cutaneous dendritic cells. If pulmonary dendritic cells were also able to express this receptor, this would have important implications with regard to their potential role in asthma.

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

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.

Human mast cells express stem cell factor.

Stem cell factor (SCF) is a major cytokine regulator of mast cell growth and function. The present study demonstrates that human mast cells are able to produce SCF. Constitutive synthesis of SCF mRNA was seen in the mast cells isolated from human lung and skin by RT‐PCR. This was confirmed by in situ hybridization in conjunctival mast cells of both tryptase‐only (MCT) and tryptase/chymase (MCTC) subsets. SCF protein product was found in conjunctival MCT and MCTC mast cells by immunohistochemistry. Soluble SCF protein was detected in the culture supernatant of isolated lung mast cells by ELISA, and cross‐linkage of IgE receptor (Fcε–RI) on the lung mast cells in culture did not alter SCF mRNA expression, or the secreted soluble SCF protein. This was consistent with the finding that levels of SCF mRNA expression in conjunctival mast cells were similar between normal subjects and patients with seasonal allergic conjunctivitis (SAC). This study shows that human mast cells themselves are a cellular source of SCF, as well as being target cells for this growth factor. SCF may regulate mast cell growth and function via both paracrine and autocrine mechanisms. The production of SCF by mast cells may be regulated via mechanisms other than IgE receptor‐mediated pathways.

Interaction of Neuropeptides with Human Mast Cells

Mast cells of human skin, but not lung, adenoids, tonsils, or intestine, release histamine in response to substance P, vasoactive intestinal polypeptide, and somatostatin. The substance P receptor of skin mast cells is not of the NK-1, NK-2 or NK-3 subtypes of smooth muscle. Time course and calcium dependency of release by peptides differed from anti-IgE. With anti-IgE, the molar ratios of histamine:PGD2:LTC4 generated by skin mast cells was 1,000:25:2, whereas with substance P these ratios were 1,000:1:0.1. Similar results were obtained with the other neuropeptides. The ability of peptides to stimulate skin mast cell histamine release suggests a mechanism whereby their release from dermal nerve endings is coupled to changes in microvasculature.

Allergic eye disease mechanisms

Allergic eye disease is a common ocular problem. It is one part of the whole spectrum of allergic diseases which share a common initiating mechanism and a characteristic pattern of inflammation. The association between exposure to airborne plant pollens and the development of ocular and nasal symptoms in itself was noted by Charles Blackley in 1873.1 The classification of allergic eye diseases is based entirely on clinical features, which in the past has resulted in the use of inappropriate or unsatisfactory treatments, owing to our lack of knowledge of the underlying mechanisms. Recent work has defined such underlying pathogenic mechanisms of allergic eye disease, extending our knowledge of the cellular and mediator mechanisms involved, and enabling pathophysiological correlates to be made between the clinical patterns of disease and responses to treatment. This is now resulting in a clearer understanding of the disease processes which, in the long term, holds promise for the development of more effective treatments. As the prevalence of allergic disease is increasing, probably through environmental factors, it is appropriate at this stage to review our current knowledge of the mechanisms of allergic eye disease. The morbidity and mortality of asthma have increased in the past few years2-5 with no corresponding rise in the underlying rate of atopy itself.6 Environmental causes have been cited as responsible for this, as the rising trends have occurred too quickly for genetic causes to have exerted their effects.6The increase in asthma has coincided with an increase in house dust mite levels, and is greatest in communities exposed to high allergen levels.2 However, there are no firm data available on which to base any change in the incidence of allergic eye disease. The reported incidence rates for the different forms of allergic eye disease have been varied. …

Inhibition profiles of sodium cromoglycate and nedocromil sodium on mediator release from mast cells of human skin, lung, tonsil, adenoid and intestine

We have studied an aspect of the functional heterogeneity of human mast cells, namely responsiveness to the inhibitory effects of sodium cromoglycate and nedocromil sodium. The effects of these drugs were examined on the release of histamine and PGD2 from mast cells of human skin, lung, tonsils, adenoids and intestine. A high concentration, 1000 μM, of sodium cromoglycate was required to significantly inhibit histamine release from lung and tonsillar mast cells. Nedocromil sodium, 1000 μM, was more effective than sodium cromoglycate against histamine release from lung, tonsillar and adenoidal cells. Both compounds showed tachyphylaxis in lung and tonsillar mast cells but not in adenoidal and intestinal mast cells. In contrast, in intestinal mast cells, the effect of nedocromil sodium was weaker and more variable than sodium cromoglycate. Skin mast cells differed from mast cells of the other anatomical sites in being unresponsive to sodium cromoglycate and nedocromil sodium. Our results confirm that high concentrations of sodium cromoglycate and nedocromil sodium are required to achieve even modest inhibition of mediator release from human mast cells under in vitro conditions. Notwithstanding this, the results also indicate that differences exist among skin, lung, tonsillar, adenoidal and intestinal mast cells with respect to their sensitivity to sodium cromoglycate and nedocromil sodium, thus extending our knowledge of functional heterogeneity within the human mast cell populations.