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Explant cultures of mouse duodenum were used to show that interleukin-1 (IL-1) causes release of mucus from epithelial goblet cells. Our experiments made use of a newly described enzyme-linked lectin assay (ELLA) which employs enzyme-conjugated soybean agglutinin to detect mucus glycoproteins secreted from explant cultures of mouse duodenum. Supernatants from cultures of lipopolysaccharide-stimulated peritoneal macrophages as well as partially purified rabbit alveolar macrophage-derived IL-1 and human rIL-1β all induced mucus release in a rapid and dose-dependent fashion. This observation may be important for investigating a link between the immune response and mucus hypersecretion from inflamed intestinal mucosa.

We have used the bradykinin analog, [ d Arg0-Hyp3- d Phe7]-bradykinin, as a model stimulus with which to examine peptide-induced mediator release from human skin mast cells (SMC) and to compare it with IgE-mediated release from the same cells. The bradykinin analog was an effective histamine secretagogue, inducing a comparable maximal level of release to that observed for anti-IgE. By contrast to anti-IgE, however, [ d Arg0-Hyp3- d Phe7]-bradykinin did not stimulate marked release of prostaglandin D2 (PGD2) from these cells. In experiments where cells were exposed to both stimuli simultaneously, histamine release was additive, while PGD2 release was the same as that observed for anti-IgE alone. The kinetics of [ d Arg0-Hyp3- d Phe7]-bradykinin-stimulated histamine release were rapid, with 50% of maximal release being achieved within 30sec, compared to 2–3 min for anti-IgE. Interestingly, when both stimuli were applied simultaneously, the kinetics of release were intermediate between those of either stimulus alone. Studies of the signal transduction pathways that may be involved in [ d Arg0-Hyp3- d Phe7]-bradykinin-induced histamine release revealed striking differences to results obtained with anti-IgE. While agents that increase intracellular cyclic AMP have a pronounced inhibitory effect on IgE-mediated release, forskolin, isobutylmethylxanthine and isoproterenol were all totally ineffective at inhibiting histamine release induced by the bradykinin analog. Similarly, staurosporine, a relatively selective inhibitor of protein kinase C, and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) an activator of this enzyme, both have pronounced effects on IgE-mediated histamine release from SMC but were completely inactive with regard to [ d Arg0-Hyp3- d Phe7]-bradykinin-stimulated release. SMC stimulated with this peptide showed characteristic changes in intracellular free calcium levels, as assessed by digital video microscopy. This response differs from that induced by anti-IgE in that it had a more rapid onset, achieved a lower peak, and decayed much more rapidly. Analysis at the single cell level showed that cells that responded in this fashion upon exposure to the bradykinin analog were capable of showing an additional response upon subsequent exposure to anti-IgE. We conclude that histamine release from SMC in response to [ d Arg0-Hyp3- d Phe7]-bradykinin occurs via a completely different mechanism from that in response to IgE-mediated stimuli. Peptide-induced release is rapid and is not susceptible to pharmacologie manipulation of intracellular cyclic AMP or protein kinase C but utilizes a rapid transient shift in intracellular calcium concentrations as part of its signal transduction pathway.

The effects of a putatively specific 5-lipoxygenase inhibitor, 2(12-hydroxydodeca-5,10-dinyl)-3,5,6-trimethyl-1,4-benzoquin one (AA-861), and its major metabolite, M-I, were assessed using anti-IgE activated human basophils, lung mast cells and skin mast cells. In basophils and lung mast cells, no effects on histamine release were observed, whereas leukotriene C4 (LTC4) production was inhibited (IC50 values less than 1 microM). In addition, prostaglandin D2 (PGD2) production was inhibited in lung mast cells (IC50 congruent to 5 microM). In contrast, in skin mast cells both histamine release and PGD2 production were reduced by AA-861 and M-I, with IC50 values of congruent to 5 and 0.4 microM for histamine and PGD2, respectively. These data reveal biochemical heterogeneity among human histamine-containing cells and underscore the necessity of assessing a pharmacologic agonist in relevant cell systems.

Kinins are potent proinflammatory peptides that induce histamine release from rodent mast cells. We examined the ability of bradykinin, lysylbradykinin and a series of kinin analogs to cause histamine release from human basophils, human lung mast cells and human skin mast cells. At concentrations ranging from 0.1 microM to 1 mM, bradykinin failed to cause histamine release from any of the human histamine-containing cells studied. Lysylbradykinin was also without effect on basophils and lung mast cells, but was a weak secretagogue for human skin mast cells, inducing 5.5 +/- 3% (mean +/- SD) of total cellular histamine release at a concentration of 10(-5) M. Similarly, when sixteen recently developed bradykinin antagonists were examined, these compounds had no effect on basophils or lung mast cells but all sixteen induced dose-dependent histamine release from skin mast cells. The release process was temperature dependent and, at a concentration of 10(-5) M, the antagonists induced 8-27% histamine release. Although preincubation of cells with 10(-3) M bradykinin or des(Arg9) bradykinin significantly inhibited antagonist-induced histamine release, the requirement for such high concentrations of these peptides to cause inhibition suggested that histamine release is not mediated by either B1 or B2 kinin receptors. To understand further the mechanism of histamine release, we examined a series of bradykinin analogs with single amino acid substitutions in the bradykinin sequence. Replacement of proline7 in the bradykinin sequence with D-phenylalanine is the essential change used to convert kinin analogs into antagonists, and 10(-5) M [DPhe7]-bradykinin induced 8-10% histamine release. Other analogs, devoid of antagonist activity, however, such as [DPhe6]-bradykinin and [LPhe7]-bradykinin were able to induce equivalent levels of histamine release. The ability to induce histamine release appears to be related, at least in part, to aromaticity, since [DTrp6]-bradykinin and [DTrp7]-bradykinin induced greater amounts of histamine release than equivalent [DPhe]-analogs, causing approximately 20% histamine release at 10(-5) M. By contrast, [DAla7]-bradykinin was an ineffective stimulus. In summary, a single amino acid substitution can convert bradykinin into a secretagogue for human skin mast cells. The ability of kinin analogs to induce histamine release from skin mast cells, but not lung mast cells or basophils, emphasizes the heterogeneity of human histamine-containing cells.

The known abilities of kinins to increase vascular permeability, to cause vasodilatation and pain, and to stimulate arachidonic acid metabolism have led several investigators to suggest that these potent peptides may be important mediators of allergic and inflammatory diseases in humans. In recent years, we have provided the first direct evidence to support this concept by demonstrating that bradykinin and lysylbradykinin are generated in nasal secretions during both the immediate and late responses to allergen challenge (1,2). Kinins also appear to be generated in allergic events in the lower airways, since immunoreactive kinin levels in bronchoalveolar lavage fluids from symptomatic asthmatics, or from asthmatics responding to allergen challenge are significantly elevated compared to levels in lavages from normals (3). In addition, recent studies by Atkins and coworkers (4) suggest that activation of plasma kallikrein, presumably accompanied by kinin generation, occurs during ongoing allergic reactions in the skin.