IgG1 antibody-dependent mediator release after passive systemic sensitization of basophils arriving at cutaneous basophil hypersensitivity reactions

When antigen is injected into a 24-hr cutaneous basophil hypersensitivity (CBH) reaction of an actively sensitized guinea pig, local basophils degranulate and release histamine. This reaction is called cutaneous basophil anaphylaxis and may be antibody mediated. We now report passive sensitization of basophils at CBH sites by systemic transfer of anti-picryl immune serum. Keyhole limpet hemocyanin- (KLH) immunized animals were skin tested with KLH to elicit 24-hr CBH reactions at day 7. Anti-picryl serum was injected i.v. at various times. On day 7, blue dye was injected i.v., and then 24-hr CBH sites vs nearby normal skin were challenged with 0.1 microgram picryl-human serum albumin (Pic-HSA). An immediate increase in vascular permeability (blueing) was noted at normal skin sites due to systemic passive cutaneous anaphylaxis (PCA), and augmented blueing occurred at CBH sites compared with normal skin. Systemic passive sensitization of CBH sites occurred when antiserum was administered as little as 1 hr before challenge of CBH site. However, local administration of anti-picryl serum (as in a local PCA reaction) was not able to sensitize tissue basophils, whether antigen was administered locally or systemically. The serum factor that mediated cutaneous basophil anaphylaxis was heat-stable (56 degrees C X 4 hr) 7S IgG1 antibody. Electron microscopy of Pic-HSA-challenged CBH sites in animals that received IgG1 antibody showed that local basophils undergo anaphylactic degranulation by exocytosis. These studies suggest that basophils arriving at CBH reactions are sensitized for anaphylactic function by antibody that can be acquired in the circulation, but possibly not at the local site.     

Characterization of a localized basophil hypersensitivity lesion in guinea pig conjunctiva

Basophils accumulate in response to antigen challenge in cutaneous basophil hypersensitivity (CBH) reactions. Two ocular diseases, vernal conjunctivitis and contact-lens-associated conjunctivitis, are also characterized by this histopathology. We have refined a model previously developed in guinea pig conjunctiva by precisely defining the site of antigen injection and correlating the site with the clinical and histologic changes. Guinea pigs were primed by an intradermal injection of keyhole limpet hemocyanin (KLH) in the flank and challenged (Day 6) by injection of a small bolus of KLH just under the conjunctival epithelium. Twenty-four hours later histologic examination showed a perivascular infiltrate of inflammatory cells containing large numbers of basophils. Eosinophils, neutrophils, macrophages, and mast cells were also seen. Serial sections of the reaction site showed discrete boundaries. At all sites examined in antigen-challenged tissues, there were significantly more basophils than in control-injected conjunctiva. Insertion of a sterile needle or injection of PBS or KLH into normal conjunctiva induced a significant increase in neutrophils and some macrophages. Injection of graded doses of antigen into the conjunctiva of primed animals, resulted in a dose-dependent increase in basophils up to 50 micrograms KLH (optimal dose).     

Rejection of ticks from guinea pigs by anti-hapten-antibody-mediated degranulation of basophils at cutaneous basophil hypersensitivity sites: role of mediators other than histamine

Previous studies have established that recruitment of basophils to sites of tick feeding in guinea pigs is required to effect immune resistance. In the current study, actively sensitized guinea pigs treated three times daily with H-1 (mepyramine) and H-2 (cimetidine) histamine receptor antagonists, during the challenge tick infestation period, expressed normal resistance to Amblyomma americanum larvae. Similarly, naive guinea pigs treated with anti-histamines four times daily, beginning 7 days before transfer of immune serum and tick challenge and continuing through the tick infestation period, also expressed normal antibody-mediated resistance to A. americanum. These results indicated that histamine was not an important basophil mediator of the resistance response. Ticks allowed to feed on tissue rich in basophils that were induced by sensitization and subsequent local challenge with non-tick protein antigen, keyhole limpet hemocyanin (KLH), expressed normal yield. Ticks that fed on similar tissue rich in basophils induced by sensitization and challenge with KLH, in which the basophils expressed anti-picryl specificity due to systemic passive transfer of anti-picryl antibodies, were rejected when basophils were induced to degranulate by i.v. challenge with picryl antigen at 6 hr (29% rejection), 12 hr (18% rejection), 24 hr (22% rejection), and 48 hr (37% rejection) post-tick attachment. However, basophil degranulation at 18, 72 and 96 hr post-tick attachment had no adverse effect on tick feeding. These hosts were protected from systemic anaphylaxis by treatment with the anti-histamine mepyramine. Release of histamine occurred at tick feeding sites, but vasoactive effects were blocked by mepyramine treatment as evidenced by a lack of increased vascular permeability (bluing) at these sites compared with non-tick-infested tissues, or to cutaneous basophil hypersensitivity (CBH) sites of animals not protected with mepyramine. These results indicate that local recruitment and subsequent degranulation of basophils via immune mechanisms dependent on non-tick antigens can lead to tick rejection, and that basophil-derived mediators other than histamine are involved in this immune resistance response to A. americanum ticks. The identity of the crucial basophil mediator(s) is not known. The significant susceptibility of ticks to basophil-mediator release at 6 to 12 hr and 24 to 48 hr post-attachment coincides with the tick attaching and fast-feeding phases, respectively, suggesting that these phases of tick parasitism are particularly susceptible to the effect of basophil mediators other than histamine.     

Mast cell degranulation and its inhibition by an anti-allergic agent tranilast. An electron microscopic study

Degranulation of IgE-sensitized rat mast cells by antigen was studied quantitatively in vitro and in vivo by electron microscopy. The inhibition of this degranulation by an anti-allergic drug, N-(3,4-dimethoxycinnamoyl)anthranilic acid (Tranilast), was also examined both in vitro and in vivo. In the in vitro study using peritoneal mast cells, alteration of the granules, cavity formation by fusion of the perigranular membrane and granule discharge due to fusion of the cavity membrane with the cell membrane were observed and were accompanied by histamine release. Scanning electron microscopy disclosed the extrusion of smooth, round bodies from pores formed on the cell surface. In the in vivo study of passive cutaneous anaphylaxis (PCA), the characteristic features of mast cell degranulation were obvious 5 min after the injection of antigen; leakage of dye increased progressively from 5 to 30 min but was not found at 6 h. From quantitative analysis of the substructure of mast cells, it was demonstrated that degranulation of IgE-sensitized mast cell induced by antigen was achieved by sequential exocytosis both in vitro and in vivo. Tranilast inhibited these changes to a remarkable extent and it was concluded that the inhibition of mast cell degranulation by this drug might play an important role in anti-allergic treatment.     

Involvement of host Fc receptors in antibody-mediated cutaneous basophil hypersensitivity reactions.

Cutaneous basophil hypersensitivity (CBH) reactions are heterogeneous delayed time course basophil-rich responses that can be mediated by either T cells, B cells, or serum antibodies. The current study examined the mechanism by which antibodies mediate CBH in guinea pigs. Fc competition experiments were constructed by passively transferring mixtures of anti-KLH serum and normal heterologous gamma-globulins. It was found that rabbit IgG and its isolated and purified Fc fragment [but not the (Fab')2 fragment] inhibited the ability of guinea pig immune serum to transfer CBH. Concurrent inhibition of transferred KLH-specific CBH and systemic passive cutaneous anaphylaxis (PCA) reactions by rabbit IgG or its Fc fragment, and not by sheep or bovine gamma-globulins, indicated that Fc receptors on cutaneous mast cells were probably involved in both CBH and PCA. It was also found that the basophil aspect of delayed cutaneous responses elicited by PHA was inhibited by Fc competition maneuvers. This could mean that some forms of apparently T cell-mediated CBH may be T cell dependent, but via secretion of molecules that bind to Fc receptors, as seems required in antibody-mediated CBH.     

Antiallergic/antiasthmatic effect of novel antiallergic hexapeptide-95/220 in various experimental models

The effects of newly synthesized antiallergic hexapeptide 95/220 was investigated on various allergic and asthmatic test models. This newly developed peptide was found to be more potent than clinically used drug disodium cromoglycate (DSCG). Hexapeptide 95/220 inhibited immediate hypersensitivity reactions such as passive cutaneous anaphylaxis (PCA) and mast cell degranulation in rats, antigen-induced bronchoconstriction in actively sensitized guinea pigs in dose dependent manner like DSCG. Antigen-induced contraction of guinea pig ileum was also markedly inhibited by this newly developed hexapeptide in the same fashion as ketotifen and DSCG did but at comparatively lower dose. Egg albumin-induced histamine release was also blocked by this hexapeptide from chopped lung tissues of sensitized guinea pigs. These results suggest that hexapeptide' 95/220 has potent inhibitory effect on immediate hypersensitivity reactions thereby inhibiting mediator release from mast cell. Moreover, this newly synthesized peptide is orally active and effective at lower doses as compared to standard drugs.     

Effect of disodium cromoglycate on mast cell-mediated immediate-type allergic reactions

We investigated the effect of disodium cromoglycate (DSCG) on mast cell-mediated immediate-type hypersensitivity. DSCG inhibited systemic allergic reaction induced by compound 48/80 dose-dependently. Passive cutaneous anaphylaxis was inhibited by 71.6% by oral administration of DSCG (1 g/kg). When DSCG was pretreated at concentration rang from 0.01-1000 g/kg, the serum histamine levels were reduced in a dose dependent manner. DSCG also significantly inhibited histamine release from rat peritoneal mast cell (RPMC) by compound 48/80. We confirmed that DSCG inhibited compound 48/80-induced degranulation of RPMC by alcian blue/nuclear fast red staining. In addition, DSCG showed a significant inhibitory effect on anti-dinitrophenyl IgE-mediated tumor necrosis factor-alpha production. These results indicate that DSCG inhibits mast cell-mediated immediate-type allergic reaction.     

Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigen-induced secretion and involves SHIP-Grb2-Dok complexes

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.     

Tumor-basophil interactions in vitro--a scanning and transmission electron microscopic study.

Purified guinea pig basophils, or basophils either specifically degranulated with antigen or nonspecifically degranulated with lectin, were cultured with guinea pig line 1 hepatoma cells for 1 to 24 hr and studied ultrastructurally. As early as 1 hr of culture, degranulated or nongranulated basophils and tumor cells formed close contacts by mutually intertwined elongated cell processes and also in cultures containing degranulated basophils, extruded membrane-free basophil cytoplasmic granules became firmly attached to tumor cells. At later intervals, some tumor cells cultured with basophils exhibited cytostatic and cytopathic changes, including dense mitochondria, centralization of organelles, dilated perinuclear and rough endoplasmic cisternae, cell swelling and cytoplasmic lucency, disrupted cytoplasmic organelle and plasma membranes, nuclear pyknosis and fragmentation. Some tumor cell specialized surface attachments were either disrupted or damaged at points of basophil or basophil granule adhesion. Tumor damage was most extensive in cultures containing degranulated basophils, although only a minority of tumor cells (less than 10%) was affected. Tumor injury was seen much less frequently in the presence of nondegranulated basophils, and was absent in control cultures of tumor alone. The occasional viable tumor cells that phagocytosed basophil granules were apparently unharmed, suggesting that internalization of basophil granules by tumor cells is not cytotoxic.     

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae

Schistosoma mansoni: the cutaneous response to cercarial challenge in naive guinea pigs and guinea pigs vaccinated with highly irradiated cercariae. International Journal for Parasitology16: 491–510. Naive guinea pigs and guinea pigs vaccinated 4 weeks previously with highly irradiated cercariae were challenged percutaneously with normal cercariae. Skin samples from the challenge site were then harvested at varying times to provide histological, quantitative and ultrastructural data on the respective cellular responses to cercarial invasion. The primary cutaneous reaction was characterised by neutrophils; these cells reached peak numbers (16% of total cells) by 18 h. Eosinophils and basophils made only a small contribution to the infiltrate (2.9 and 5.7% respectively). Some basophils showed evidence of anaphylactic degranulation, others seemed to be damaged, but most appeared normal. Mononuclear cells of varied morphology were present at all times, but activated fibroblasts were prominent, and collagen deposition increased with time. Degranulating mast cells were recognised at 24 and 48 h. Dead schistosomula were never seen in naive-challenged skin, although one or two of the observed larvae showed minor tegumental abnormalities. In vaccinated guinea pigs, the cutaneous cellular response to challenge was significantly enhanced, with basophils dominating the reaction (33% of total cells at 24 h). Many basophils were already degranulating by the anaphylactic mechanism at 3 h post challenge, and free basophil granules were seen frequently. Both intact cells and free granules congregated in close proximity to invading larvae. Eosinophils were also present in greater numbers at secondary reaction sites, but they never exceeded 6% of the total infiltrate. Mononuclear cells believed to be immature eosinophils were prominent from 3 h. For the first time, the mechanism of eosinophil attachment and degranulation onto a multicellular target, described previously only from in vitro investigations, was recognized in vivo. Neutrophil numbers matched those recorded in naive-challenged skin at 3 and 6 h, but declined thereafter, while mast cells were seen degranulating at these early times. Mononuclear cells again presented a variety of morphological appearances; of particular note were large cells that had phagocytosed debris and were presumed to be macrophages, and small rounded cells with scant cytoplasm and few organelles, that may have been lymphocytes. By 12 h, large areas of the dermis had become severely disorganised and numerous, free basophil granules were distributed amongst the other cellular constituents. Dead schistosomula, denuded of tegument, were clearly recognised at 6 h, and such individuals invariably had neutrophils attached to their exposed muscle layers. Since dead schistosomula were not identified in naive-challenged guinea pig skin, it is concluded that a percentage of the challenge larvae, however small, is preferentially killed in the skin of the vaccinated animals.     

A role for vesicles in human basophil secretion

The evidence for vesicular transport as a mechanism for secretion by human basophils is reviewed. Initially, direct electron-microscopic inspection of experimentally produced and sequentially biopsied contact allergy skin lesions revealed a unique form of secretion termed piecemeal degranulation, characterized by the slow emptying of secretory granule contents (with retention of empty containers) in the absence of extrusion of entire granules. Budding of small vesicles to/from secretory granules was observed, and cytoplasmic vesicles were abundant. A generalized degranulation model was proposed to unify classical regulated secretion and this new form of secretion. Investigation of the mechanism(s) of secretion from human basophils required the development of numerous tools and resources. Chief among these were: (a) isolation and purification of circulating basophils; (b) identification of specific growth factors to increase the supply of this rare granulocyte; (c) understanding of secretogogue mechanisms and reliable analyses of secreted basophil products; and (d) development of ultrastructural preparations allowing imaging of small vesicles and quantifiable small electron-dense tags for granule materials in small vesicles. Applications of these tools to well-defined models of basophil secretion have established a role for vesicles as a mechanism for effecting secretion of histamine and the Charcot-Leyden crystal protein from activated human basophils.     

IgE antibody-mediated cutaneous basophil hypersensitivity reactions in guinea pigs

Cutaneous basophil hypersensitivity (CBH) reactions are a heterogeneous group of delayed time course basophil-rich immune responses that can be mediated in the guinea pig by T cells, B cells, or IgG1 antibody. This study examined whether guinea pig IgE antibody could also mediate CBH reactions. IgE antibody to picryl or oxazolone determinants was induced by immunizing Hartley strain guinea pigs pretreated with cyclophosphamide. Hyperimmune serum from these animals was passed through a heavy chain-specific anti-IgG1 affinity column. The presence of IgE anti-hapten antibody in the filtrate fraction was verified by passive cutaneous anaphylaxis (PCA) testing with a 7-day period of local passive sensitization and by the heat lability (56 degrees C, 4 hr) of PCA activity. This IgE-rich fraction and the IgG1 fraction eluted from the column with base (0.2 M Na2CO3, pH 11.3) were transferred i.v. to separate groups of normal guinea pigs. Both fractions mediated delayed time course reactions that contained basophils. Macroscopic and microscopic reactions mediated by the IgE-rich fraction were abolished with heat (56 degrees C, 4 hr). Thus, two antigen-specific factors in guinea pig serum can mediate delayed time course basophil-containing reactions: IgG1 and IgE antibodies. IgE-mediated CBH reactions are similar to the late-phase reaction that follows IgE-dependent wheal-and-flare reactions in humans. The finding that guinea pig IgE can mediate a late reaction that contains basophils makes this a possible model for the human late-phase response, and suggests that some forms of CBH may play a role in human allergic disease.     

A Novel Model of IgE-Mediated Passive Pulmonary Anaphylaxis in Rats

Mast cells are central effector cells in allergic asthma and are augmented in the airways of asthma patients. Attenuating mast cell degranulation and with it the early asthmatic response is an important intervention point to inhibit bronchoconstriction, plasma exudation and tissue oedema formation. To validate the efficacy of novel pharmacological interventions, appropriate and practicable in vivo models reflecting mast cell-dependent mechanisms in the lung, are missing. Thus, we developed a novel model of passive pulmonary anaphylaxis in rats. Rats were passively sensitized by concurrent intratracheal and intradermal (ear) application of an anti-DNP IgE antibody. Intravenous application of the antigen, DNP-BSA in combination with Evans blue dye, led to mast cell degranulation in both tissues. Quantification of mast cell degranulation in the lung was determined by (1) mediator release into bronchoalveolar lavage, (2) extravasation of Evans blue dye into tracheal and bronchial lung tissue and (3) invasive measurement of antigen-induced bronchoconstriction. Quantification of mast cell degranulation in the ear was determined by extravasation of Evans blue dye into ear tissue. We pharmacologically validated our model using the SYK inhibitor Fostamatinib, the H₁-receptor antagonist Desloratadine, the mast cell stabilizer disodium cromoglycate (DSCG) and the β₂-adrenergic receptor agonist Formoterol. Fostamatinib was equally efficacious in lung and ear. Desloratadine effectively inhibited bronchoconstriction and ear vascular leakage, but was less effective against pulmonary vascular leakage, perhaps reflecting the differing roles for histamine receptor sub-types. DSCG attenuated both vascular leakage in the lung and bronchoconstriction, but with a very short duration of action. As an inhaled approach, Formoterol was more effective in the lung than in the ear. This model of passive pulmonary anaphylaxis provides a tissue relevant readout of early mast cell activity and pharmacological benchmarking broadly reflects responses observed in patients with asthma.     

A newly described activity of guinea pig IgG1 antibodies: transfer of cutaneous basophil reactions.

Hapten-specific delayed time course skin reactions containing predominant accumulations of basophils and eosinophils were elicited in newborn guinea pigs after i.v. transfer of small amounts of oxazolone immune serum. The immune serum was fractionated by column chromatography procedures, and the fractions were examined for their ability in transferring this form of cutaneous basophil hypersensitivity (CBH). Only the 7S IgG-containing peak from Sephadex G-200 columns, and only the IgG1-containing fractions from DEAE columns, transferred CBH. An affinity column of bound oxazolone removed the activity from immune serum, and it could be recovered from the column by eluting with soluble oxazolone. About 35 microgram of purified IgG1 anti-oxazolone antibody could systemically transfer CBH reactivity. An immunoadsorbant column of anti-IgG1 removed this activity, but a column of anti-IgG2 did not. None of the procedures were able to separate activity in transferring CBH from passive cutaneous anaphylactic (PCA) activity classically associated with guinea pig IgG1 antibody. IgG1 from 8-day immune and 31-day hyperimmune donors were both effective. The average association constant of 8-day antibody was 8 X 10(-4) M-1. Transfer of cutaneous basophil reactions can be mediated by low affinity serum 7S IgG1 antibody.     

Sensitization of circulating basophils in guinea pig recipients of passive transfer of cutaneous basophil hypersensitivity (CBH) with immune serum: antigen-specific histamine release in vitro

An in vitro histamine release assay was used to test the hypothesis that passive sensitization of circulating basophils is associated with the activity of immune serum that transfer the ability to elicit cutaneous basophil hypersensitivity (CBH) reactions. Systemic i.v. transfer of several types of immune sera that mediate CBH also led to passive sensitization of circulating basophils for antigen-specific release of histamine in vitro. In addition, we found that immune serum passively sensitizes basophils in vitro. Thus immune sera had three activities that are probably interconnected: sera will 1) passively transfer CBH in vivo, 2) passively sensitize basophils in vivo, and 3) passively sensitize basophils in vitro. These results suggest that passive sensitization of circulating basophils by immune serum contributes to the mechanism by which antibodies transfer the ability to elicit CBH reactions.     

Cutaneous basophil hypersensitivity uncovered in the cell transfer of classical tuberculin hypersensitivity.

Cellular transfer of cutaneous basophil hypersensitivity (CBH) was studied. Guinea pigs immunized for CBH with incomplete Freund's adjuvant (IFA) provided cells which could transfer delayed and basophil-rich reactions in skin tests of recipients. Guinea pigs immunized with complete classical tuberculin-type delayed hypersensitivity reactions (DH), which are characteristically devoid of basophils. However, recipients of cells from donors with DH, surprisingly, were found to have delayed skin reactions containing large basophil infiltrates which were lacking in the donors. Thus, recipients of classical cell transfers of tuberculin-type DH had delayed reactions which resembled CBH. Control experiments verified that the cell transfer of CBH from donors with DH was due to passive transfer with live cells and not transfer of contaminating humoral factors or active sensitization of recipients. It was concluded that cutaneous basophil responses were suppressed in CFA-immunized donors and expressed in cell transfer recipients. Cells from donors immunized with CFA were enriched for nonadherent and nonimmunoglobulin-bearing lymphocytes by passage through nylon wool columns, and these cells transferred conjugate specific CBH reactions. It was concluded that cells mediating these transfers were probably T cells. The finding of basophils in cell transfers of DH and a variety of other findings suggesting complex regulation of basophil numbers in tissue lead to the conclusion that the term CBH be used to simply describe a basophil-containing skin reaction.     

Naturally abundant basophils in the snapping turtle, Chelydra serpentina, possess cytophilic surface antibody with reaginic function

Basophils constitute 50 to 63% of the blood leukocytes in Chelydra serpentina, the snapping turtle. Immunoglobulin (Ig) on the surface of the turtle basophil was detected by indirect immunofluorescence by using an IgG fraction from rabbit anti-turtle Ig serum (RATIg) and a fluoresceinated goat anti-rabbit antibody incubated at 4 degrees C. However, when the cells were incubated with RATIg at 22 degrees C, the basophil number, as determined by Wright's stain and neutral red counts, decreased dramatically. This morphologic evidence of degranulation was directly proportional to the antiserum concentration. Degranulation also correlated with cell histamine release (r = 0.73). In other experiments, turtle basophils were found to express antigen-specific surface Ig after immunization with sheep red blood cells (SRBC). Washed basophils from immunized turtles formed basophil-SRBC rosettes in vitro. Basophils from control turtles did not. Basophil-SRBC rosettes could also be induced by in vitro passive sensitization by preincubation of normal turtle basophils in the SRBC immune turtle sera. This study shows clearly that the turtle basophil has an immune capacity analogous to the mammalian basophil/mast cell. This study also contains the first direct evidence for the existence of reaginic antibody (or antibodies) in an ectothermic vertebrate. Finally, C. serpentina is proposed as a unique animal model for the study of basophil function.     

ELECTRON MICROSCOPE OBSERVATIONS ON COMPOUND 48/80-INDUCED DEGRANULATION IN RAT MAST CELLS : Evidence for Sequential Exocytosis of Storage Granules

In vitro degranulation of rat mast cells was studied at different intervals ranging from 10 to 60 sec after adding the histamine liberator, compound 48/80 (0.4 µg/ml, 17°C). The ultrastructural changes were followed by electron microscopy, and parallel assays were made to determine the histamine released. In addition, the extracellular tracers lanthanum and hemoglobin (demonstrated by its peroxidative activity) were applied to mast cells to follow communication of the extracellular space with the cavities formed during degranulation. After a lag period of 10 sec, degranulation started in the most peripherally located granules. The perigranular membrane fused with the plasma membrane, resulting in a pore bridged by a thin diaphragm. This was followed by rupture of the diaphragm and extrusion of the granule matrix (exocytosis). The process advanced towards the cell interior by fusion and opening of the deeper situated granules to the formerly opened granule cavities. At the end of the process, the cell was filled by a system of complicated cavities containing a number of altered granules. Extracellular tracers have shown that these intracellular cavities were in unbroken communication with the extracellular space from the very beginning of their formation. Both lanthanum and hemoglobin were found to be adsorbed to the limiting membrane of the cavities and bound to altered mast cell granules. In contrast, no tracer substance was present in nondegranulating mast cells. Degranulation of mast cells by compound 48/80 is regarded as a sequential exocytosis, a process similar to that described for some exocrine gland cells. All the "intracellular" cavities, formed by degranulation, were shown to communicate with the extracellular space; consequently, granules lying in these cavities must be considered as biologically extracellular. The present findings support the view that histamine is released from the granule matrix by the extracellular ionic milieu.     

Human monocytes generate basophil histamine-releasing activities

Human peripheral blood monocytes generated activities during 24-h culture that were capable of triggering histamine release from 17 of 18 human basophil donors. Monocytes and their in vitro transformed macrophages continued to elaborate these basophil histamine-releasing activities for at least 3 wk in culture. In the 18 basophil donors tested, maximum histamine release induced by monocyte supernatants was 33.8 +/- 5.9% (mean +/- SEM) of total basophil histamine content; optimum anti-IgE-induced release was 38.8 +/- 6.2%. Basophil histamine release in response to monocyte activities was optimal at 37 degrees C and at calcium concentrations of 2 to 5 mM. Release was greater than 90% complete 1 min after challenge and was inhibited by anti-allergic drugs. The mechanism of release appeared to be independent of IgE binding. Gel filtration of supernatants derived from both day 1 (monocyte stage) and day 14 (macrophage stage) cultures demonstrated activity peaks with approximate m.w. of 12,000 and 30,000. In contrast to the marked responsiveness of basophils, only 2 of 10 human lung mast cell preparations responded; release in those preparations was low: 3% and 13% histamine release, respectively. Thus, monocytes produce potent histamine-releasing activities with differential actions on basophils and mast cells.     

Basic characteristics of human lung mast cell desensitization

Human lung parenchymal mast cells displayed both specific and nonspecific desensitization. The kinetics of both release and desensitization were approximately equal to 3 times faster than human basophils, but a similar relationship between release and desensitization suggests similar biochemistries in basophils and mast cells. Arachidonic acid metabolite (PGD2 and LTC4) release was slower to desensitize (t1/2 of 8 min) than histamine release (t1/2 of 3 min), the ratio of which is similar to the ratio observed in basophils. Ionophore A23187-induced release was unaffected by desensitization to anti-IgE antibody, and calcium-45 uptake was inhibited by desensitization, suggesting that desensitization inhibits the early post-cross-linking "influx" of calcium that is necessary for mediator release in mast cells. In contrast to the above similarities in basophil and mast cell desensitization, mast cell desensitization, unlike that of basophils was not inhibited by diisopropylfluorophosphate.