Jane A. Warner

Role of human basophils and mast cells in the pathogenesis of allergic diseases

The role of human basophils and mast cells in the pathogenesis of allergic diseases has been analyzed. Purified human basophils and mast cells release several known mediators of allergic reactions, including histamine, sulfidopeptide leukotrienes, kinin-forming enzymes, and, in the case of the mast cell, PGD2. These same mediators are released in vivo after experimental challenge in the upper airways with either allergen or cold, dry air, a stimulus used to simulate exercise-induced bronchospasm. The appearance of mast cell mediators in vivo after such challenges further implicates mast cells in the pathogenesis of allergic diseases of the airways that occur as a result of exposure to allergen or physical stimuli. During the LPR after experimental challenge of the upper airways, the pattern of mediators released (i.e., histamine, leukotrienes, and others, but no PGD2) suggests that basophils may contribute to the LPR. Antiallergic drugs that prevent mediator release in vitro, such as antihistamines, also prevent the appearance of mediators in vivo, strengthening both the validity of the in vitro test as a model of the disease and the hypothesis that mediator release is an essential element of the disease process. A model discussing the pathogenetic mechanism is presented.

Differential release of mediators from human basophils: Differences in arachidonic acid metabolism following activation by unrelated stimuli

We have examined the release of histamine and LTC4 from purified human basophils challenged with several different stimuli, both physiological and nonphysiological. Basophils (n=16) challenged with 0.1 μg/ml anti‐IgE released 38±4% of their available histamine and 39±12 ng LTC4/109 basophils within 15‐30 min. F‐Met peptide (n=8) caused the release of 54±8% histamine and 42±25 ng LTC4/106 basophils within a period of 2‐6 min. C5a caused the release of 22±3% histamine from selected donors but failed to initiate any LTC4 release unless combined with D2O or 5 mM extracellular calcium. The two nonphysiological stimuli A23187 and TPA caused extensive histamine release, 67±8 and 82±11%, respectively, and while A23187 initiated a large and rapid release of leukotriene, TPA failed to release any LTC4 even when combined with D2O or 2‐5 mM extracellular calcium. Increased concentrations of extracellular calcium enhanced anti‐IgE and f‐Met peptide induced release of LTC4 but inhibited the A23187 induced release of leukotriene. A single peak of immunoreactive leukotriene C4 that comigrated with the authentic standard was identified using HPLC followed by radioimmunoassay. No LTD4 or LTE4 could be detected. Purified human basophils incubated with 0.2 μM [3H]AA incorporated 290 pmol/106 cells, or 32±5% of the available label within 60 min. The [3H]AA was taken principally into the phospholipids (73±5%), with 20±3% as neutral lipid, and only 5±2% remaining as the free acid. Three phospholipid subclasses, phosphatidylcholine, PC (24±2%), phosphatidylinositol, PI (22±1%), and phosphatidytethanolamine, PE (15±3%), accounted for the majority of the incorporated [3H]AA while the remainder of the phospholipids accounted for less than 5% of the total cpm. HPLC analysis of the lipid mediators released during stimulation with 0.1 μg/ml anti‐IgE revealed [3H]LTC4 (2.4±1.0%), [3H]5HETE (1.0±0.1%), unmetabollzed [3H]AA (91±2%), and an unidentified peak (3.4±1.4%). The unknown metabolite eluted with the prostaglandins, was inhibited by indomethacin, and appeared to have a relatively high specific activity. It may thus represent an artifact of the labeling procedure rather than a novel basophil‐derived prostaglandin.

Stimulus-dependent leukotriene release from human basophils : a comparative study of C5a and Fmet-leu-phe

Previous studies of human basophil mediator release have noted that the bacterial peptide fmet‐leu‐phe and the anaphylatoxin C5a induce comparable levels of histamine release while only fmet peptide induces leukotriene release. Since 5‐lipoxygenase metabolism of arachidonic acid is calcium dependent, we examined the characteristics of the human basophil [Ca++]i response which follows its activation by either fmet peptide or C5a. While the peak [Ca++]i response was essentially identical for these two stimuli, fmet peptide induced a prolonged increase in [Ca++]i while C5a stimulated only a transient increase in [Ca++]i that was essentially over within 2 minutes of adding the stimulus. Simultaneous addition of EDTA with fmet peptide revealed the two phases of the [Ca++]i response and demonstrated that leukotriene release was dependent on an elevated [Ca++]i level in the 2–5 minutes following challenge. Enhancement of leukotriene release induced by C5a by agents such as staurosporine and interieukin‐3 also produced a [Ca++]i kinetic curve which resembled fmet piptide. Single cell studies of the [Ca++]i response could detect no subpopulations of cells which responded preferentially to fmet peptide or C5a, eliminating the possibility that the ability of fmet peptide to induce leukotriene was a result of its action on a functionally distinct population of basophils.

Induction of histamine release from human skin mast cells by bradykinin analogs.

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.

A rapid percoll technique for the purification of human basophils

We have refined a rapid single-step Percoll technique to obtain a basophil-enriched mononuclear cell fraction suitable for further purification. Greater than 75% of the total blood basophils were recovered from a blood-Percoll interface at a purity of between 5 and 23%. The contaminating cells were principally lymphocytes with a smaller (20-25%) percentage of monocytes. Further purification using the penicillin affinity column led to the recovery of between 28 and 64% of the total blood basophils at purities ranging from 52 to 92%, a substantial improvement over our earlier protocol. There was also a marked increase in the recovery of basophils from the affinity column over our previous technique, which lead to a moderate (5-10%) increase in overall recovery.

Mediator release during nasal provocation: A model to investigate the pathophysiology of rhinitis

The pathogenesis of rhinitis was investigated using a model of nasal provocation with different types of stimuli. Allergic subjects had an immediate response to antigenic challenge with symptoms of rhinitis highly correlated with increments in the concentrations of histamine, prostaglandin D2, kinins and kininogens, leukotrienes, and toluene sulfonyl arginine methyl ester esterase activity in their nasal secretions. This reaction was abated by a tricyclic antihistamine also capable of inhibiting mediator release from human mast cells in vitro and, in some subjects, by disodium cromoglycate. In a number of patients, symptoms reappeared three to 12 hours after nasal provocation. This late reaction also involves release of all of the aforementioned mediators except for prostaglandin D2, and preliminary data suggest that it can be inhibited by oral or topical steroids. Cold, dry air can induce rhinitis with mast cell mediator release from selected subjects. The pathogenesis of this reaction is unclear, but there are indications that osmolarity changes are responsible for mast cell activation. Thus, mast cells can be induced to release mediators and cause nasal symptoms by both immunologic and physical mechanisms, which may account for the pathophysiology of several types of rhinitis.

Graded changes in the response of individual human basophils to stimulation: distributional behavior of early activation events.

These studies examine the distribution of single‐cell responses in basophil preparations in the context of four events that may be associated with early activation by anti‐immunoglobulin E (IgE) antibody and the bacterial peptide fMet‐Leu‐Phe (fMLP). In general, we measured the single‐cell response distributions after challenge with a concentration of stimulus that resulted in an optimal response and compared this with the distribution that occurred after challenge with suboptimal concentrations of the same stimulus. The elevation in cytosolic calcium, as detected in Fura‐2‐labeled basophils, after challenge with anti‐IgE or fMLP showed graded characteristics in that the distributions were unimodal under conditions of optimal or suboptimal challenge with little skewing from a normal distribution. Similarly, the up‐regulation of the cell surface adhesion molecule CD11b, as determined by flow cytometry, showed graded unimodal increases after challenge with anti‐IgE antibody at optimal and suboptimal concentrations. In addition, stimulation of basophils led to increased F‐actin polymerization. After challenge with an optimal concentration of anti‐IgE antibody, the F‐actin content of basophils increased to a maximum between 10 and 15 min and returned to near prechallenge levels by 60 min. There was a close correlation between the maximum increase in F‐actin content and histamine release regardless of the stimulus; anti‐IgE antibody, fMLP, and phorbol ester (PMA) responses lay on the same regression line. The single‐cell F‐actin polymerization distributions were also unimodal and graded according to the magnitude of the histamine release response. During measurements of the calcium response under the microscope we noted that basophils underwent significant changes in morphology after challenge with any stimulus. These changes were related to both degranulation and nondegranulation events and could be quantitated by a series of imageprocessing algorithms, which are presented. The kinetics of the morphological change, measured as a change in cell perimeter, paralleled degranulation. Single‐cell distributions of the morphologic changes were also unimodal under conditions of both optimal and suboptimal stimulation. Therefore, no evidence of all‐or‐nothing responses could be observed in the context of these four early activation events. In general, the response distributions resembled normal distributions at both optimal and suboptimal levels of stimulation, which indicated that single basophils responded in a graded manner. J. Leukoc. Biol. 55: 13–23; 1994.

Purified human basophils do not generate LTB4

We investigated the release of the 5-lipoxygenase derivatives of arachidonic acid (AA) in purified human basophils and compared them with similar results obtained in the human lung mast cell. We have shown that purified basophils (average purity = 51 +/- 6%) challenged with 0.1 microgram/ml anti-IgE released histamine (35 +/- 9%), and LTC4 (32 +/- 10 ng/10(6) cells) but failed to release measurable quantities of immunoreactive LTB4. In contrast, the non-specific stimulus, A23187, caused the release of histamine and both LTC4 (279 +/- 95 ng/10(6) cells) and LTB4 (148 +/- 41 ng/10(6) cells). Closer analysis of the data revealed an inverse relationship between the levels of LTB4 released and the purity of the basophils, strongly suggesting that the contaminating monocytes were responsible for LTB4 synthesis. Purified human lung mast cells have been shown to release 6 ng of immunoreactive LTB4/10(6) cells, indicating that basophils release significantly less LTB4 following an IgE-mediated challenge. In a series of experiments using highly purified basophils prelabeled with [3H]AA, we demonstrated that exposure to 0.1 microgram/ml anti-IgE led to the release of [3H]LTC4, with no detectable [3H]LTB4, whereas exposure to 1.0 micrograms/ml A23187 caused the release of [3H]LTC4 and smaller quantities of [3H]LTB4, [3H]LTD4, and [3H]LTE4. We failed to detect any [3H]LTB4 in the cell pellet following challenge with either anti-IgE or A23187, indicating that LTB4 was not synthesized and retained within the cell pellet. Finally, we found that exogenously added [3H]LTB4 was not metabolized, either by basophils alone or by basophils stimulated with anti-IgE (0.1 microgram/ml).

Mechanisms of mediator release from human skin mast cells upon stimulation by the bradykinin analog, [dArg0-Hyp3-dPhe7]bradykinin☆

We have used the bradykinin analog, [DArg0-Hyp3-DPhe7]-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, [DArg0-Hyp3-DPhe7]-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 [DArg0-Hyp3-DPhe7]-bradykinin-stimulated histamine release were rapid, with 50% of maximal release being achieved within 30 sec, 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 [DArg0-Hyp3-DPhe7]-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 [DArg0-Hyp3-DPhe7]-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 rapd 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 [DArg0-Hyp3-DPhe7]-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 pharmacologic 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.

Vesicular Transport of Charcot-Leyden Crystal Protein in f-Met Peptide-Stimulated Human Basophils

The ultrastructural localization of Charcot-Leyden crystal (CLC) protein during f-Met-peptide-induced degranulation of human basophils was analyzed at multiple times after stimulation. In this secretion model, piecemeal and anaphylactic degranulation occurred sequentially in stimulated cells and were followed by reconstitution of granule contents. This analysis showed that granule number and alteration and location of gold-labeled, formed CLCs changed over time. CLCs were extruded from granules and remained attached to plasma membranes early after stimulation. At later times, similar structures reappeared in granules in quantity. Smooth-membrane-bound vesicles, analyzed by number, by visible particle contents (or lack of contents) and by gold labeling for CLC protein, showed that empty vesicles increased at the earliest time sampled (0 time) and plunged thereafter in actively extruding and completely degranulated cells. Vesicles containing granule particles were elevated initially at 10 s and at later times. Gold-labeled CLC-protein-containing vesicles were of either empty or particle-filled varieties, and both types were involved with CLC protein transport out of cells at early times and into cells at later times as basophils recovered. Thus, vesicle transport of CLC protein is a mechanism for producing piecemeal degranulation and endocytotic recovery of released CLC protein from human basophils. This vesicular shuttle may be an effector mechanism for widespread piecemeal losses from granules in basophils in inflammatory sites in vivo in human disease.