David O. Thueson

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.

Enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G antibodies against insect venoms

IgG blocking antibodies were measured in patients receiving insect venom immunotherapy. The enzyme-linked immunosorbent assay (ELISA) described herein was found to be sensitive and reproducible. Results with ELISA correlated well with values obtained with a radioimmunoassay and with inhibition of the release of histamine from sensitive basophils. Also, specific antibody titers against phospholipase A and whole bee venom were correlated. Serial determinations of venom-specific IgG antibodies were made in 17 patients receiving Polistes wasp or bee venom immunotherapy. The majority of patients showed a rise in IgG antibodies, which peaked after administration of approximately 500 micrograms of venom. Only one out of 13 of these venom-treated patients had allergic symptoms after an insect sting while on maintenance therapy.

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.

Complement-mediated release of histamine from human basophils: III. Possible regulatory role of microtubules and microfilaments

The release of histamine by normal human leukocytes (basophils) following in vitro challenge with activated complement (zymosan-treated serum) was previously reported. In this study, the effects of various pharmacologic agents on this release mechanism were compared with allergen-induced release of histamine. Colchicine and vinblastine antagonize the polymerization of tubulin to form microtubules, and both agents inhibited complement-and allergen-triggered release of histamine from basophils. Finally, treatment with cytochalasin B, a fungal product known to interfere with microfilament formatin, resulted in enhanced release of histamine from complement-treated basophils but no significant change in the percentage of histamine released from allergen-treated basophils. These findings suggest that microtubules and/or microfilaments are involved in complement-induced secretion of histamine by human basophils.

Selective regulation of human neutrophil functions by the cell activation inhibitor CI-959.

The cell activation inhibitor CI‐959 [5‐methoxy‐3‐(1‐methylethoxy)‐N‐1H‐tetrazol‐5‐ylbenzo[b]thiophene‐2‐carboxamide, monosodium salt] was evaluated for its effects on human neutrophil functions. CI‐959 inhibited spontaneous migration and chemotaxis toward N‐formyl–methionyl‐l‐leucyl‐l‐phenylalanine (fMLP) with 50% inhibition (IC50) values of 3.6 and 3.1 μM, respectively. CI‐959 also inhibited superoxide anion generation in response to C5a, fMLP, serum‐opsonized zymosan (SOZ), concanavalin A (Con A), and calcium ionophore A23187 with IC50 values of 2.5, 4.7, 14.5, 5.4, and 14.8 μM, respectively. In comparison, CI‐959 inhibited myeloperoxidase release in response to C5a, fMLP, SOZ, and Con A with IC50 values of 11.6, 16.1, 7.5, and <1.0 μM, respectively, while inhibiting the response to A23187 by only 5.5% at 100 μM. At concentrations up to 100 μM, CI‐959 had no effect on the respiratory burst or degranulation in response to L‐α‐1,2‐dioctanoylglycerol (DiC8) or phorbol 12‐myristate 13‐acetate (PMA). In addition, the compound inhibited leukotriene B4 release stimulated by fMLP and SOZ (IC50 values 4.0 and 2.5 μM, respectively), while having less activity against the A23187‐stimulated response (IC50>100 μM). These results demonstrate that CI‐959 inhibits cellular responses to stimuli that mobilize intracellular calcium. For cellular responses to ionophore‐mediated calcium influx, only oxygen radical production was inhibited by CI‐959. CI‐959 was further evaluated for its effects on neutrophil stimulus‐response coupling. At 100 μM, CI‐959 had no effect on human neutrophil phospholipase C or protein kinase C. CI‐959 inhibited fMLP‐stimulated intracellular calcium mobilization and calcium influx with IC50 values of 16.7 and 3.1 μM, respectively, and exhibited less potent calmodulin antagonist activity (IC50 = 90.5 μM). These results indicate that CI‐959 may exert its stimulus‐ and response‐specific inhibitory effects on neutrophil functions, in part, through inhibition of calcium‐regulated signalling mechanisms. J. Leukoc. Biol. 55: 443–451; 1994.

Regular articleHistamine-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.

Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-949.

The allergic mediator release inhibitor CI‐949 [5‐methoxy‐3‐(1‐methylethoxy)‐1‐phenyl‐N‐1H‐tetwol‐5‐yl‐1H‐indole‐2‐carboxamide, L‐arginine salt] was evaluated for its effects on human neutrophil functions. CI‐949 (100 μM) inhibited spontaneous migration and chemotaxis toward f‐met‐leu‐phe (FMLP) by 49.1% and 45.8%, respectively. At the same concentration, CI‐949 inhibited the phagocytosis of serum‐opsonized zymosan (SOZ) by 39.0%. CI‐949 inhibited leukotriene B4 and thromboxane B2 release in response to SOZ with IC50S of 2.0 μM and 3.3 μM, while inhibiting the response to FMLP with IC50s of 1.7 and 2.0 μM. CI‐949 also inhibited myeloperoxidase release from primary lysosomal granules in response to the following stimuli with the respective IC50s (μM): C5a (40.3); FMLP (34.4): SOZ (21.4); concanavalin A (Con A) (3.9); and calcium ionophore A23187 (91.2). In contrast, CI‐949 inhibited lysozyme release from secondary granules in response to SOZ and Con A with IC50S of 99.3 and 56.1 μM, while inhibiting the response to C5a, FMLP, and A23187 by 41.2%, 52.4%, and 10.0%, respectively, at 100 μM. CI‐949 (100 μM) had no inhibitory effect against lysozyme release in response to L‐α‐1,2 dioctanoylglycerol (DiC8), or phorbol 12‐myristate 13‐acetate (PMA). CI‐949 inhibited superoxide anion generation stimulated by FMLP and Con A with IC50S of 33.9 and 25.8 μM, while inhibiting the response to C5a, SOZ, and A23187 by 36.6%, 24.8%, and 14.1% and having no effect on the response to DIC8 or PMA at 100 μM. These results demonstrate preferential inhibition of arachidonic acid metabolism and degranulation of primary lysosomal granules by CI‐949 with selectivity for stimuli which promote intracellular calcium mobilization or calcium influx.

Inhibition of histamine, leukotriene C4/D4, and thromboxane B2 release from human leukocytes and human chopped lung mast cells by the allergic mediator release inhibitor, CI-949

The novel antiallergy compound, 5-methoxy-3-(1-methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H- indole-2- carboxamide, L-arginine salt (CI-949), inhibited mediator release from human basophilic leukocytes and from human chopped lung mast cells challenged with anti-IgE. In leukocytes, CI-949 was a more potent inhibitor of leukotriene C4/D4 and thromboxane B2 release (concentration of drug that inhibits mediator release by 50% [IC50] 0.5 and 0.1 mumol/L, respectively) than of histamine (IC50, 11.4 mumol/L) when anti-IgE was the challenging stimulus. In human lung, inhibition of release of all three mediators occurred at approximately equal concentrations (IC50s for histamine, 16.6 mumol/L; for leukotriene C4/D4, 7.1 mumol/L; and for thromboxane B2, 6.2 mumol/L). The inhibition of histamine release from basophils by CI-949 was further characterized using a variety of stimuli. Challenge with anti-IgE, histamine-releasing factor derived from lymphocytes, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, and concanavalin A revealed potent inhibition (IC50, 10 to 15 mumol/L). CI-949 was less potent versus calcium ionophore A23187, phorbol myristate acetate (12-o-tetradecanoylphorbol-13-acetate), and C5a (IC50s, 30, 54, and 60 mumol/L, respectively). These results suggest that diverse pathways of cell activation-excitation coupling exist for different stimuli in basophils. Furthermore, the activity and potency of CI-949 in inhibiting release of histamine, leukotrienes, and thromboxane from both human basophils and mast cells suggest that the compound will be effective clinically for indications in which these mediators are implicated, including asthma and allergic rhinitis.

Modulation of human basophil migration in vitro by a soluble factor from virus-stimulated leukocytes

Abstract Although it is clear that respiratory virus infections frequently provoke attacks of asthma, the mechanism of the provocation is unknown. The accumulation of basophils in the sputum and the presence of basophil chemotactic factors in the serum of allergic patients suggests that the basophil may play a causative role in asthmatic attacks. We have investigated the effect of viruses on the in vitro migration of human basophils. When peripheral blood leukocytes from normal subjects were preincubated for 24 hr with uv-inactivated Sendai virus, the chemotactic response of basophils was augmented. This effect was also seen with the cell-free supernatant of mononuclear cells cultured with virus. Our results suggest that the soluble product mediating the enhancement is interferon. While the soluble factor enhanced basophil migration toward two chemoattractants (C5 peptide and a synthetic formyl-tripeptide), it has no effect on the migration of monocytes.