Dose-response studies of the suppression of whole blood histamine and basophil counts by prednisone

If some clinical problems (e.g., radiographic contrast media reactions) arise from mediator release by circulating basophils, prednisone's capacity to prevent such is likely to be at least partly related to its suppressive effects on whole blood histamine and basophil levels. To establish an optimal dosage schedule, 15 healthy male volunteers entered a two-phased study to determine (1) the single dose of prednisone required to produce maximal suppression of histamine and basophil levels and (2) the effects of repeated prednisone doses. Parameters monitored were whole blood histamine, quantitative basophil counts, white blood cell (WBC) and differential counts, and plasma prednisone, prednisolone, and cortisol levels. Fifty milligrams prednisone suppressed whole blood histamine levels as much as a larger dose and also showed a marked effect on circulating basophils and other leukocytes. Three 50-mg prednisone doses given at 6-hr intervals had a greater effect on whole blood histamine and circulating leukocytes than fewer doses. Thus, the commonly used empirical prednisone dosage regimen is supported. One implication of the results of this study is that greater suppression of blood basophils and histamine levels might be obtained by administering the last prednisone dose about 6 hr before procedures in which a very rapid release of mediators from basophils is anticipated.     

Spontaneous histamine release and histamine content in normal subjects and subjects with asthma

Spontaneous histamine release (SHR) from basophils by simple incubation at 37 degrees C for 60 minutes and histamine content of basophils were assessed in normal subjects, patients with asthma, and methacholine-sensitive subjects without asthma (NAMS). SHR from basophils of normal subjects did not exceed 10% of the total histamine. A significantly higher SHR was observed in basophils from subjects with asthma than from normal subjects (p less than 0.002). Basophil SHR in patients with asthma not receiving medication was significantly greater than that in patients with asthma receiving medication (p less than 0.05). SHR from basophils in NAMS subjects was similar to that in normal subjects. SHR was highly dependent on temperature and Ca++ and Mg++ ions and appeared to be a slower event than anaphylactic release. The mean histamine content per basophil from normal subjects was 1.48 +/- 0.13 pg (mean +/- SEM). Basophils from subjects with asthma contained significantly less histamine than basophils from normal subjects (p less than 0.002). Histamine content per basophil from NAMS subjects was slightly lower than histamine content per normal basophil. No apparent relationship was found between the magnitude of SHR and the histamine content per basophil in the patients with asthma not receiving medication. Hypersensitivity to food or exercise does not appear to be essential for high SHR. High SHR appears to bear little, if any, relationship to cell damage or cell death. High SHR may be a factor that could serve as a marker for bronchial asthma. Further studies are needed to define the clinical relationship and pathophysiologic mechanism of SHR.     

Histamine suppression of in vivo eosinophil accumulation and histamine release in human allergic reactions

Although it has been shown that histamine inhibits antigen-induced in vitro histamine release from basophils, it is unclear whether histamine inhibits in vivo mediator release in human allergic reactions. We report effects of exogenous histamine on histamine release and inflammatory cell responses in antigen-challenged skin sites in eight ragweed-sensitive individuals. Four heat-suction blisters in each subject were unroofed, and a collection chamber was appended to each blister base. Chamber A contained 1000 PNU/ml ragweed extract; chamber B contained buffered saline (control fluid); chamber C contained 1000 PNU/ml ragweed and 50 ng (5 x 10(-7) M) of histamine; and chamber D contained histamine alone (50 ng). Comparative analyses of chamber histamine levels in individual subjects showed that (1) histamine levels in chamber A were significantly greater than those in chamber B (p less than 0.01) and that histamine levels in chamber C were not significantly different than those in chamber D (p less than 0.5). Likewise, comparison of eosinophils attaching to membrane filters appended to the chamber bases for 2 hr showed that there were significantly more eosinophils in chamber A than in chamber B (p less than 0.01) and that there was no significant difference in eosinophil numbers on filters appended to chamber C vs chamber D. In three of four subjects studied, addition of exogenous histamine (50 ng/ml) to ragweed before intradermal injection inhibited the ultrastructural mast cell alterations seen within 10 min after injection of ragweed alone. In the one subject in which mast cell alterations were not prevented, exogenous histamine also did not inhibit antigen-induced histamine release or subsequent eosinophil accumulation in the skin chambers.     

Exercise and isocapnic hyperventilation-induced bronchoconstriction in asthma: relevance of circulating basophils to measurements of plasma histamine

The relationship of airway cooling during exercise to changes in airway caliber, plasma histamine levels, and circulating basophils was investigated in eight allergic asthmatic and eight normal subjects. In asthma matched RHE during exercise and ICH produced almost identical bronchoconstriction with maximum falls in SGaw of 61.0 +/- 4.5% and 57.9 +/- 5.2%, respectively. A similar RHE in normal subjects was associated with a 7.9 +/- 3.3% fall in SGaw. The resting plasma-histamine levels were higher in the asthmatic (0.52 +/- 0.06 ng/ml) than in the normal (0.31 +/- 0.07 ng/ml, p less than 0.05) subjects. No significant change in plasma histamine occurred after exercise in either group nor in the asthmatic subjects with ICH. In contrast, exercise but not ICH stimulated an increase in leukocytes, basophils, and total blood histamine in parallel with the airway response that reached a maximum at 2 to 5 min in both normal and asthmatic subjects. There was a positive correlation between basal plasma and total blood-histamine levels (r = 0.67, p less than 0.01) in normal and asthmatic subjects suggesting that basophils contribute significantly to plasma histamine. The spontaneous basophil release of histamine was greater in asthmatic (13.4 +/- 2%) than in normal subjects (6.46 +/- 7%, p less than 0.005), which is consistent with the higher resting plasma-histamine levels in the asthmatic subjects. These findings suggest that plasma-histamine changes with exercise in asthma but not ICH may be related to the associated basophilia and sample handling rather than intrapulmonary mast cell degranulation.     

Basophil histamine release remains unaffected by clinical desensitization to penicillin

A penicillin-allergic patient who required therapy with beta-lactam antibiotics was desensitized with increasing parenteral doses of benzylpenicillin. After desensitization, the patient tolerated, without signs of allergic reaction, intravenous piperacillin. Immunologic studies were undertaken to investigate humoral and cellular changes accompanying desensitization. Serum penicilloyl IgG and IgE antibodies did not change significantly during induction of clinical tolerance to the drug. The patient's previously positive immediate skin reaction to penicilloyl polylysine (PPL) converted to negative. Nonspecific releasability of skin mast cells as tested with polymyxin B skin test was unchanged. In contrast, in vitro blood basophil activation, both by specific antigen (penicilloyl-human albumin) and a nonspecific IgE-dependent stimulus (anti-IgE) remained strong after the desensitization procedure and during weeks of high-dose piperacillin therapy. A monovalent penicilloyl hapten inhibited penicilloyl-albumin-induced basophil histamine release, whereas the patient's own serum taken while the patient was receiving treatment did not. Moreover, the patient's treatment serum, while the patient was receiving piperacillin, was able to trigger histamine release from passively sensitized basophils of a donor not allergic to penicillin, suggesting the presence of a sufficient amount of complete multivalent antigen to initiate release and/or desensitization. The presence of high concentrations of penicilloated serum proteins in the patient's treatment serum was confirmed by immunoassay. Basophil histamine release could, nevertheless, be elicited from the patient's whole blood sample taken while the patient was receiving piperacillin treatment by adding penicilloyl-human albumin in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic urticaria serum induces histamine release, leukotriene production, and basophil CD63 surface expression--inhibitory effects ofanti-inflammatory drugs

BACKGROUND: A role of potential histamine-releasing autoantibodies against the high-affinity IgE receptor on the surface of basophils and mast cells is discussed in the pathogenesis of chronic urticaria. This so-called autoimmune urticaria may be diagnosed by a positive intracutaneous autologous serum skin test, which is found in about 30% of patients with chronic urticaria. OBJECTIVE: Our purpose was, first, to compare the effect of complement-inactivated sera of 20 patients with chronic urticaria and positive autologous serum skin tests, 20 patients with chronic urticaria and negative skin tests, and 20 control subjects without chronic urticaria (10 atopic and 10 nonatopic subjects) and, second, to analyze the effect of anti-inflammatory drugs on the serum activity. METHODS: The following assay systems were used: release of histamine in whole blood samples, surface expression of the activation marker CD63 on basophils, and sulfidoleukotriene de novo production in leukocyte suspensions. Whole blood, basophils, and leukocyte suspensions were obtained from a nonatopic and an atopic donor. RESULTS: Sera of patients with autologous serum skin test positive chronic urticaria resulted not only in significantly increased histamine release compared with skin test-negative chronic urticaria sera but also in a significant higher induction of basophil CD63 surface expression and sulfidoleukotriene de novo production. However, serum activity was neither characteristic for chronic urticaria nor for chronic urticaria with a positive autologous serum skin test. Preincubation with dapsone, chloroquine, and lidocaine dose dependently resulted in a significant reduction of all histamine release, CD63 expression, and sulfidoleukotriene production. In addition, mizolastine was able to inhibit serum-induced sulfidoleukotriene production. CONCLUSION: Further studies investigating the in vivo effect of these drugs will have to clarify their role in the management of the subset of patients with chronic urticaria demonstrating serum-induced inflammatory effects.     

Potentiation of human basophil histamine release by protamine: a new role for a polycation recognition site

Protamine is an arginine-rich basic polypeptide that stimulates histamine release from rat mast cells but not from human basophils. In this report, we show that protamine causes a non-cytolytic potentiation of IgE-mediated histamine release from human basophils. A direct effect of protamine on basophils was supported by results obtained using cell preparations containing 35-65% basophils. The potentiation occurred at all concentrations of antigen that initiated release and was most pronounced at antigen concentrations that alone stimulated minimal histamine release. The kinetics of potentiated release were parallel to those for IgE-mediated histamine release, and addition of protamine did not overcome the block caused by antigenic desensitization. Staging experiments indicated that enhancement occurred only when protamine and antigen were added together in a single step reaction. Protamine also potentiated release stimulated by eosinophil granule major basic protein or poly-L-lysine, but inhibited release initiated by poly-L-arginine; poly-L-arginine stimulated release was also inhibited by polymyxin B. Arginine-rich histone mimicked the protamine effect, while lysine-rich histone, polymyxin B, and compound 48/80 had minimal or no effect on IgE-mediated release. These results suggest that a polycation recognition site on human basophils similar to that described for rat mast cells may mediate potentiation of basophil secretory events by arginine-rich basic polypeptides.     

Basophil FcepsilonRI histamine release parallels expression of Src-homology 2-containing inositol phosphatases in chronic idiopathic urticaria

BACKGROUND: Basophils are implicated in the pathogenesis of chronic idiopathic urticaria (CIU). Autoantibodies to the IgE receptor (FcepsilonRI) and serum histamine releasing activity have been detected in some subjects with CIU, although their role in vivo is unclear. Basophils of patients with CIU have altered FcepsilonRI-mediated histamine release (HR); however, the mechanism is unknown. In the basophil FcepsilonRI signaling pathway, protein levels of Src-homology 2-containing-5'-inositol phosphatase (SHIP)-1 are inversely correlated with the release of mediators or releasability. A related phosphatase, SHIP-2, is a negative regulator of monocyte IgG receptor (FcgammaR) signaling . We hypothesized that SHIP levels are altered in CIU basophils. METHODS: Blood basophils were isolated from cold urticaria, CIU, or normal donors, and FcepsilonRI-dependent and independent HR were quantified. Protein levels of SHIP-1, SHIP-2, spleen tyrosine kinase, and phosphorylated Akt were determined by Western blotting. Subjects' serum was tested for serum histamine releasing activity and anti-FcepsilonRIalpha antibodies. RESULTS: CIU basophils displayed a bimodal response to anti-IgE activation. One half of CIU subjects' basophils had reductions in anti-IgE-induced HR and were designated nonresponders (CIU NR). CIU NR basophil HR remained diminished at 10-fold to 30-fold higher doses of anti-IgE. CIU anti-IgE responder basophils had HR similar to normal subjects. SHIP-1 and SHIP-2 proteins were increased in CIU NR basophils and were linked to reduced phosphoAkt after anti-IgE stimulation. CIU basophil anti-IgE response was not related to the presence of serologic factors. CONCLUSION: In CIU basophils, the observed changes in FcepsilonRI signaling pathway molecule expression may underlie changes in releasability. CLINICAL IMPLICATIONS: Patients with CIU can be segregated on the basis of basophil functional phenotype.     

Immunoallergological studies on chironomid antigen in bronchial asthma

Immuno-allergological studies on Tokunagayusurika akamusi (TA), a chironomid, were carried out in 217 patients with bronchial asthma. 1. Skin reaction to TA was positive in 72 (33.2%) out of the 217 patients with bronchial asthma. 2. Fifteen (13.8%) of the 109 patients showed a significant amount of histamine release (more than 15%) from basophils stimulated by TA antigen. 3. There was a significant correlation between skin reaction and histamine release by TA antigen. 4. A significant amount of basophil histamine release was observed in young patients, and in those who were under 40 years old of age at the onset of the disease. The serum IgE concentration of these patients was relatively high. 5. There was a significant correlation between basophil histamine release by TA antigen and specific IgE antibody to CTT. 6. There were no significant differences in skin reaction or histamine release by TA antigen between asthmatics in Okayama and Tottori prefectures.     

Interval shifts in basophil measures correlate with disease activity in chronic spontaneous urticaria

Chronic spontaneous urticaria (CSU) significantly impacts the quality of life of those affected through symptoms of pruritus and recurrent skin lesions. In active CSU disease, reduced IgE‐mediated basophil histamine release (HR) and basopenia are observed. We sought to examine the relationship between interval changes in basophil measures and shifts in patient‐reported disease impairment. Simultaneous symptom and basophil evaluations were completed at two sequential study visits, and interval changes in measures were compared between visits for each subject (n = 38). These measures included Skindex‐29, current itch and hives scores, total leukocyte histamine content (an indirect measure of blood basophil presence), and basophil HR in response to anti‐IgE and formyl‐methionine‐leucine‐phenylalanine. Overall, interval improvements in disease measures in CSU subjects were associated with increased basophil numbers (total leukocyte histamine content) and IgE‐mediated HR. This suggests these measures are potential biomarkers for CSU disease improvement and further implicates a role for basophils in CSU.     

Cytokine modulation of basophil histamine release in wasp-venom allergy

We report the effect of interleukin-3 (IL-3) and of other cytokines on antigen-induced basophil histamine release in wasp-venom-allergic subjects. Leukocytes from 12 patients with documented anaphylactic sensitivity to wasp venom were preincubated in the presence or absence of IL-3, granulocyte/macrophage-colony stimulating factor (GM-CSF), IL-5, IL-8, or stem cell factor (SCF). Washed cells were then exposed to venom and to other secretagogues, and histamine release in the supernatant was measured fluorometrically. Preincubation of leukocytes with IL-3, GM-CSF, or IL-5 (0.02–2 ng/ml), but not with IL-8 and SCF, caused a dose-dependent enhancement of antigen-induced basophilic histamine release in all subjects tested. Mean maximum increase was about 100% for IL-3, IL-5, and GM-CSF. The priming effect of IL-3 was rapid, persisted up to 12 h, and was not accompanied by a change in cellular histamine. IL-3 had a comparable enhancing effect when basophils were triggered with anti-IgE or N-formylmethionylphenylalanine (FMP). By contrast, IL-3 had no effect on substance-P-induced histamine release. The significant enhancement of basophil releasability to antigen in wasp-venom allergy by very low concentrations of IL-3, GM-CSF, and IL-5 suggests that cytokines in the basophil (mast-cell?) microenvironment could be critical factors in determining the variability of sting reactions in Hymenoptera-venom-allergic subjects.     

Basophil histamine release, IgE, eosinophil counts, ECP, and EPX are related to the severity of symptoms in seasonal allergic rhinitis

BACKGROUND: Serum specific IgE, basophil histamine release, and blood eosinophil parameters are associated with allergic rhinitis, but investigations of the relationship to the severity of allergic symptoms are few and conflicting. Our study aimed to investigate the seasonal changes in the following laboratory tests: specific IgE, basophil histamine release, eosinophil counts, and serum and plasma eosinophil cationic protein (ECP) and eosinophil protein X (EPX), and to analyze, in detail, the relationship of each individual test to the severity of symptoms in rhinitis patients allergic to both birch and grass pollen. METHODS: The above tests were performed on blood samples obtained from 49 allergic rhinitis patients during the birch-pollen season, during the grass-pollen season, and after the seasons. Symptom-medication diaries were filled in during both pollen seasons. We used partial least square (PLS) analysis to establish an optimal statistical link between the symptom score and medication and the laboratory tests, in an investigator-independent way. RESULTS: Increases in specific IgE, basophil histamine release, eosinophil counts, serum ECP and EPX, and plasma EPX were observed from the birch-pollen season to the grass-pollen season, followed by a decrease from the grass-pollen season to after the pollen seasons, except for the specific IgE. No seasonal changes in plasma ECP and total IgE were seen. The PLS analysis found a relationship between symptom score and medication and the aggregate laboratory tests (F-test value 40.2, correlation 0.34 for the cumulative relation). However, the variation in laboratory tests could explain only half of the total variation in symptoms and less than a quarter of the total variation in medication. The symptom score and, to a minor degree, medication were especially correlated with the basophil histamine-release results, with a decreasing relevance of specific IgE, eosinophil counts, total IgE, serum and plasma EPX, and serum ECP. Plasma ECP was not related to the symptom score and medication. CONCLUSIONS: A significant relationship between the severity of allergic rhinitis and various allergic inflammatory markers was found but could account for only a minor part of the variation in the patients' evaluation of their disease.     

Relevance of basophil histamine release changes during venom immunotherapy

We investigated the effect of rush and long-term venom immunotherapy on histamine release parameters in bee venom allergic patients. Ten patients received rush venom immunotherapy, and histamine release data were obtained immediately before and after treatment. 17 patients were assessed by histamine release 24 to 63 months after termination of long-term venom immunotherapy. A control group of 10 non-allergic subjects was included in this study. Histamine released from whole blood was determined in a sensitive radio-enzymatic assay using a single isotope technique. Bee venom phospholipase A-induced histamine release from whole blood proved to be a test procedure of high specificity and sensitivity. Eight of 10 untreated patients and no control subject showed significant antigen-induced histamine release. Results obtained from patients immediately after successful rush venom immunotherapy showed an important decrease (mean 45.9%) of total histamine content of basophil leukocytes in all patients. Antigen-induced maximum histamine release was found to be increased in one, decreased in two and unchanged in seven patients. In patients who received long-term immunotherapy cell sensitivity to phospholipase A was significantly lower than in a group of untreated patients (P less than or equal to 0.002). These results suggest that even years after discontinuation of immunotherapy, histamine release parameters reflect patients' protection from systemic sting reactions as assessed by sting challenges. Histamine depletion of basophils induced by rush immunotherapy may play an important role in patients' protection immediately after termination of the rush regimen.     

Influence of bronchial allergen challenge on histamine release by human basophils

BACKGROUND: Basophils can be primed by cytokines such as interleukin (IL) -3, IL-5 or granulocyte macrophage-colony stimulating factor (GM-CSF). It has been described that the concentrations of these cytokines are enhanced at sites of allergic inflammation as well as systemic in allergic asthma. OBJECTIVE: To investigate the priming status of basophils as detected by thapsigargin-induced histamine release during bronchial allergen challenge. METHODS: Ten subjects allergic to house dust mite were challenged via an aerosol delivery system. Spontaneous leucocyte histamine release as well as histamine release induced by various stimuli was measured in vitro at several time points. In addition, lung function parameters, serum IL-5 and blood eosinophil counts were evaluated. RESULTS: We found no effect of bronchial allergen challenge upon spontaneous leucocyte histamine release, nor upon histamine release induced by anti-immunoglobulin (Ig) E, house dust mite extract, C5a, fMLP, IL-3, PMA+ thapsigargin or IL-3+ thapsigargin. However, the priming status of basophils as measured by thapsigargin-induced histamine release was enhanced at 24 h after bronchial allergen challenge. Analysis of the individual data showed a heterogeneous initial response (30 min, 6 h) followed by a predominant increase at 24 h after allergen challenge. This increase in the thapsigargin-induced histamine release correlated with the increase in serum IL-5 levels at 24 h after allergen challenge. CONCLUSION: The priming status of human basophils as measured by thapsigargin-induced histamine release is enhanced 24 h after allergen challenge.     

Azelastine inhibits IgE-mediated human basophil histamine release

To examine the mechanism potentially contributing to therapeutic efficacy of azelastine in allergic rhinitis and asthma, we studied the effect of azelastine on stimulated histamine release from basophils prepared as a mixed leukocyte suspension from human blood. Azelastine was found to significantly inhibit anti-IgE-stimulated basophil histamine release. Time-course experiments indicated that the inhibitory effect of azelastine was immediate and that preincubation of basophils in azelastine was not necessary. In dose-response experiments with azelastine, 1 to 100 mumol/L, significant inhibition of histamine release was consistently observed in azelastine concentrations greater than or equal to 10 mumol/L. This inhibition was dose dependent (r = 0.96; p less than 0.001) with maximal mean inhibition of 91 +/- 8% at 100 mumol/L of azelastine.     

Non-IgG1 nature of cutaneous basophil hypersensitivity factor in contact sensitivity. III. Cutaneous basophil hypersensitivity factor promotes histamine release from guinea pig bone marrow basophils after antigenic stimulation

Cutaneous basophil hypersensitivity factor (CBH-F) from the sera of 1-fluoro-2,4-dinitrobenzene contact-sensitized guinea pigs promoted histamine release from bone marrow basophils in the presence of the antigen, dinitrophenylated epidermal microsomes as well as in vivo activity to induce CBH reaction in naive recipients and in vitro activity to mediate antigen-dependent basophil chemotaxis. The amount of histamine released was around 20% of the total release when compared to that of concanavalin A-induced basophil degranulation (50-60%). Divalent cations were required in this reaction because addition of EDTA completely suppressed histamine release from passively sensitized basophils with CBH-F. Though it is not known at present whether the same molecule is involved in basophil chemotaxis and histamine release, this histamine-releasing activity was recovered in the same preparations with basophil chemotactic activity after several purification steps.     

Separation of human basophils into two fractions with different density and histamine content

We designed experiments in this study to test the hypothesis suggested by recent purification data that blood basophils comprise two populations of different density, which circulate in numbers characteristic for each human subject. Basophils were separated into two density bands by single step centrifugation on a discontinuous Percoll gradient. Band 1 cells were at the interface between plasma and Percoll of density 1.070 gm/ml. Band 2 cells were at the Percoll 1.070 to 1.080 interface. When the number of band 1 basophils was expressed as a percentage of the total in bands 1 and 2, this relative amount generally remained in a narrow range for blood obtained from the same donor on 3 successive days but differed markedly in different individuals. In a series of leukapheresis experiments, we demonstrated that the percentage of band 1 basophils in postleukapheresis venous blood was strikingly similar to the preleukapheresis value. If basophils that repopulated the leukapheresis-depleted circulation came from the bone marrow, we can conclude that blood levels of basophils in bands 1 and 2 are under physiologic control and that the two types of basophils are released in amounts characteristic for each human subject. Additional evidence for two distinct blood basophil populations was provided by histamine measurements. The histamine content per basophil was consistently higher in cells from band 1 than from band 2, the mean difference between pairs of values for 30 subjects being 0.3 +/- 0.04 pg or about 27% of the band 1 basophil histamine content of 1.1 pg.     

Cytomorphological variations and blood histamine in bronchial asthma.

The absolute counts on basophil and eosinophil leucocytes, percentage of vacuolated eosinophil cells and whole blood histamine were determined in 30 controls and 34 patients with bronchial asthma. The basophil counts rose from the quiescent to pre-attack stage and then fell in the acute stage of bronchial asthma. The eosinophil counts, the percentage of vacuolated eosinophils and the blood histamine content increased significantly during an asthmatic attack. An attack of bronchial asthma was precipitated if the blood histamine rose to or above 200 ng/ml or a 50% rise occurred from its quiescent level. The inter-relationship between cytomorphological variations and blood histamine content in various stages of bronchial asthma is discussed.     

Diagnostic tests in ragweed-allergic asthma. A comparison of direct skin tests, leukocyte histamine release, and quantitative bronchial challenge

Thirty-six patients with a history of seasonal ragweed asthma were tested. Direct intradermal skin tests, leukocyte histamine release, and quantitative inhalation bronchial challenge correlated significantly. Data reported suggest that quantitative skin tests are as diagnostically valuable as quantitative inhalation bronchial challenge or leukocyte histamine release. Data also suggest that lung and skin mast cells and circulating basophils respond as a single population of cells to ragweed antigens.     

Antigen-induced histamine release from guinea pig basophils

Guinea pig blood was found to contain an average of 106 +/- 21 ng/ml of histamine. Of this total, approximately 85-90% was of platelet origin and the rest from basophils. Basophils contain about 0.72 pg of histamine/cell. Concanavalin A (1-5 microgram/ml) induced the release of approximately 65% of the basophilic histamine. When basophils were isolated from animals sensitized to ovalbumin or keyhole limpet hemocyanin, addition of the appropriate antigen induced histamine release at concentrations of 0.01 microgram/ml or lower. Individual animals were studied over time by repetitive bleeding. The circulating basophils remained sensitized for at least 17 weeks postsensitization. However, release did not occur if animals had been sensitized less than 7 days earlier. This assay facilitates the investigation of basophil sensitization since animals can be studied on several occasions following immunization. The mechanisms, timing and role of basophil sensitization in various types of immune and hypersensitivity reactions can now be evaluated.