Michael A. Lett-Brown

Monocyte chemotactic and activating factor is a potent histamine-releasing factor for basophils.

Monocyte chemotactic and activating factor (MCAF) is a recently cloned cytokine that causes chemotaxis of basophils. In our pursuit of cytokines affecting basophil function, we studied the effect of MCAF on histamine secretion from basophils. Leukocytes from 20 donors, 10 allergic and 10 normal subjects, were studied. MCAF caused dose-dependent release of histamine at concentrations of 10(-8) and 10(-7) M, and the mean release was 31.25 +/- 2.9% at the highest concentration. In the same experiments the mean histamine release by anti-IgE and histamine releasing factor (HRF) was 27.05 +/- 4% and 32.70 +/- 2.7%, respectively. All 20 subjects responded to MCAF with significant histamine release. Allergic subjects released significantly more histamine than normals in response to anti-IgE (P less than 0.01) but not to MCAF (P = 0.2) and HRF (P = 0.1). The histamine release was significantly correlated between MCAF and HRF (P less than 0.01), but not between MCAF and anti-IgE (P greater than 0.05). The histamine release by MCAF was complete within the first 3 min. MCAF-induced degranulation was a calcium-dependent process. Leukocytes depleted of monocytes responded equally well to MCAF. Using an anti-MCAF affinity column we determined that greater than 50% of HRF activity of crude PBMC supernatant could be attributed to MCAF. Thus, we established that MCAF is a potent secretagogue for basophils.

Enhancement of basophil chemotaxis in vitro by virus-induced interferon.

It is well established that viral infections may precipitate or worsen attacks of bronchial asthma. Furthermore, in symptomatic atopic subjects, the local accumulation of basophils and the production of a basophil chemotactic factor have been reported. We have investigated the effect of cell-free supernates from viral stimulated cultures of human mononuclear cells on the in vitro migration of human basophils. Our results show the presence of a factor in these culture supernates that enhances the migration of basophils toward two separate chemoattractants, a peptide from C5 and a lymphokine. The enhancing activity, while affecting basophil migration, did not change the response of monocytes. The enhancing activity resembled viral-induced interferon when (a) pH 2 stability, (b) heat resistance, (c) trypsin sensitivity, and (d) species-specificity were compared. Finally, the enhancing activity for basophil chemotaxis and the interferon titer were highly correlated in preparations with a 10(4)-fold difference in interferon specific activity. Our studies show that viral-induced interferon can augment the in vitro chemotactic response of basophils. Because mediators present in basophils may be involved in the pathogenesis of immediate hypersensitivity, the modulation of basophil movement by interferon suggests a possible mechanism for the association between viral infections and atopic disorders.

Histamine-releasing factors and inhibitors: Historical perspectives and possible implications in human illness

The initiation of allergic reactions with the bridging of surface-bound IgE antibodies on mast cells and basophils by allergens is well recognized. However, it is clear that other factors most likely play a role in regulating these cells. A number of cytokines have been identified that modulate the secretory response of mast cells and basophils. Among the well-characterized cytokines, interleukin-3 and connective tissue-activating peptide III (or its degradation product, neutrophil-activating peptide 2) can increase the secretory response, whereas interleukin-8 specifically inhibits the response to cytokines. Additional factors are currently under investigation. Preliminary studies suggest an important role for these histamine-releasing factors in atopic disorders, as well as in other conditions in which an IgE-dependent mechanism is not demonstrable. Furthermore, these cytokines may modulate the response of basophils and mast cells in physiologic conditions, such as tissue repair and host defense.

Histamine-releasing activity. IV. Molecular heterogeneity of the activity from stimulated human thoracic duct lymphocytes

Previous studies with the lymphokine, histamine-releasing activity (HRA), showed that HRA consisted of a heterogeneous group of molecules. The possibility of using thoracic duct lymphocytes (TDL) as a source of large quantities of HRA has been investigated. Antigen-stimulated TDL synthesize and release HRA in quantities similar to an equivalent number of peripheral blood lymphocytes (PBL). Streptokinase (SK) antigen routinely caused TDL to produce HRA approximately 15,000 Da. In contrast, staphylococcus enterotoxin B (SEB) induced the formation of a heterogeneous mixture of HRAs with apparent molecular weights of 50,000 and 15,000. Two peaks of activity (HRA I and II) were recovered when the supernatant from SK-stimulated TDL was subjected to ion-exchange chromatography. Interestingly, basophil chemotactic activity (BCA) was also eluted in these two peaks. Although interferon (IFN) is also released by antigen-stimulated TDL, the nonidentity of IFN and HRA was established by fundamental differences in chromatographic properties and specific antisera to IFN. In contrast, these studies suggest that HRA and BCA may be present on the same molecular entity.

Refractory cholinergic urticaria successfully treated with ketotifen

Four patients with cholinergic urticaria associated with additional cardiorespiratory manifestations are described. Two patients reported cold, in addition to heat and exercise, as a factor inducing symptoms. Prospective exercise challenge documented a rise in in plasma histamine sixfold to 20-fold above baseline values that accompanied onset of symptoms. All four subjects had proved refractory to conventional antihistamine therapy. Institution of ketotifen at doses ranging from 3 to 8 mg per day resulted in symptomatic improvement, and in all four subjects a repeat exercise challenge confirmed clinical improvement. In three subjects exercise challenge with ketotifen demonstrated blockade of mast cell-mediator release. Plasma histamine levels remained at baseline. In the fourth patient, histamine rose to about half the peak observed before ketotifen therapy. These findings confirm the observation that ketotifen is both an H1 histamine-receptor antagonist as well as a stabilizer of mast cell-mediator release. We speculate that ketotifen may prove more effective than conventional antihistamines in the management of severe urticaria.

Identification of a histamine release inhibitory factor produced by human mononuclear cells in vitro.

Human mononuclear cells (MNC) secrete histamine-releasing factor(s) (HRF) when cultured in vitro. HRF induces the release of histamine and other mediators from basophils and mast cells. We have shown that MNC also produced a histamine release inhibitory factor (HRIF), and that the synthesis is augmented by culture with physiologic concentrations of histamine (10(-10) to 10(-6) M) and by the mitogen concanavalin A (Con A). HRIF does not affect release initiated by other secretagogues such as allergen, anti-IgE, C5a, Con A, and phorbol myristate acetate. HRIF requires a preincubation with the cells for 5-10 min for maximal inhibition, and this effect is not abolished by washing the cells after the preincubation. The biological activity of HRIF is protease-sensitive, neuraminidase-resistant, and relatively heat-stable. HRIF can be distinguished from HRF by a lower apparent molecular mass (8,000-10,000 D).

Middle ear fluid histamine and leukotriene B4 in acute otitis media: effect of antihistamine or corticosteroid treatment.

OBJECTIVE Two potent mediators of acute inflammation, histamine and leukotriene B4 (LTB4), have been shown to play important roles in the pathogenesis and clinical course of acute otitis media (AOM) in children. The purpose of this study was to evaluate the ability of adjuvant drugs, antihistamine and corticosteroid, in reduction of the levels of histamine and LTB4 in the middle ear and their ability to improve outcomes of AOM. METHODS Eighty children with AOM (aged 3 months to 6 years) were enrolled in a prospective, randomized, double-blind, placebo controlled study. All children received one dose of intramuscular ceftriaxone and were randomly assigned to receive either chlorpheniramine maleate (0.35 mg/kg per day) and/or prednisolone (2 mg/kg per day) or placebos three times a day for 5 days. Tympanocentesis was performed at enrollment and after 5 days of adjuvant drug treatment. MEFs were collected for bacterial and viral studies and histamine and LTB4 levels. The subjects were followed for the duration of middle ear effusion or up to 3 months. RESULTS Histamine or LTB4 levels in the MEF after 5 days of treatment were not significantly reduced by adjuvant drug treatment. However, subjects receiving corticosteroid had a lower rate of treatment failure during the first 2 weeks and shorter duration of middle ear effusion. CONCLUSIONS Five day of antihistamine or corticosteroid treatment does not reduce the levels of histamine or leukotriene B4 in the MEF of children with AOM. Positive clinical outcomes of AOM cases associated with corticosteroid treatment needs to be confirmed in a larger clinical trial of children with intact tympanic membranes, who do not receive tympanocentesis.

Histamine-releasing activity (HRA): III. HRA induces human basophil histamine release by provoking noncytotoxic granule exocytosis

Histamine-releasing activity (HRA) is an approximately 10,000-15,000 dalton, protease-sensitive factor that induces the rapid liberation of histamine from human basophils. Production of HRA by human peripheral blood mononuclear cells in vitro is augmented by concanavalin A or antigen, suggesting a mechanism whereby lymphocytes may regulate basophil mediator release in vivo. In order to determine whether HRA provokes conventional exocytosis of basophil granules or, alternatively, results in mediator release by some other mechanism such as vesicular transport or cytotoxicity, we investigated the ultrastructural features of human blood basophils purified over Percoll and exposed to HRA in vitro. HRA preparations induced a noncytotoxic pattern of basophil degranulation very similar to that previously observed in basophils triggered to release histamine in response to specific antigen, C5a, or mannitol. Thus, cytoplasmic granules were extruded singly through multiple separate points of fusion between perigranular membranes and the plasma membrane. Degranulating basophils exhibited plasma membrane activation but lacked a polarized configuration. By contrast, those basophils exposed to HRA that did not exhibit evidence of degranulation displayed a single elongated cellular process. The development of this polarized cellular configuration, similar in some respects to that of uropod-bearing motile guinea pig basophils, may have reflected chemokinetic or chemotactic effects of preparations containing HRA activity.

Histamine-releasing activity. V. Characterization and purification using high-performance liquid chromatography.

The production of large quantities of the lymphokine(s) histamine-releasing activity (HRA) and its partial purification by Sephadex G-75 and ion-exchange chromatography on carboxymethyl (CM) Sepharose 6B have been detailed (M. A. Lett-Brown, D. O. Thueson, D. E. Plank, M. P. Langford, and J. A. Grant, Cell. Immunol. 87, 434-444, 1984). Two peaks of activity (HRA I and II) were recovered. Preparations of HRA have now been analyzed by high-performance liquid chromatography (HPLC). Thoracic duct lymphocytes stimulated with 200 U/ml streptokinase were used as a source of HRA. Gel-filtration HPLC on a TSK 3000 column separated HRA into two peaks of activity (10,000-20,000 and 1300 Da). Reverse-phase high-performance liquid chromatography using a Nucleosil C-8 column showed that HRA II (the activity eluted at a conductivity of 18-20 mmho on the CM-Sepharose column) eluted as a single sharp peak, the main protein contaminant being cytochrome c, the carrier protein added to enhance the yield of HRA. High-performance liquid chromatography was found to be a useful analytical tool and may be suitable for the large-scale purification of HRA.

Measurement of histamine-releasing factor activity in individual nasal washings: Relationship with atopy, basdphil response, and membrane-bound IgE

We collected individual pools of nasal washings (NWs) from 15 allergic and 15 nonallergic subjects to determine histamine-releasing factor (HRF) activity and to ascertain the relationship of these cytokines with atopic status, basophil releasability, and cell membrane-bound IgE. NWs were concentrated, dialyzed, and assayed with basophils from a single donor. Samples from 12 of 15 allergic subjects and from all the nonallergic subjects revealed greater than or equal to 15% histamine release (HR), 33.5% +/- 21.3% (mean +/- SD) and 38.6% +/- 19.6%, respectively (p greater than 0.05). When we assayed the same samples with autologous basophils, the allergic group demonstrated higher HR than the nonallergic group (31.9% +/- 19.7% versus 4.8% +/- 4.3%; p less than 0.001). A standard lot of mononuclear cell-derived HRFs was also screened with basophils from both groups. Means for HR from basophils of allergic and nonallergic subjects were 51.9% +/- 16.7% versus 26.3% +/- 8.2%, respectively (p less than 0.001). Pretreatment of basophils with lactic acid led to abrogation of sensitivity to HRF. Acid-stripped cells incubated with sera from patients with asthma regained their capacity to release histamine. We found that HRF activity can be detected in NWs of most donors, and there is no difference among allergic and nonallergic subjects. Our results suggest that the capacity of these cytokines to induce HR depends on several factors: atopic status, basophil releasability, and membrane-bound IgE.