Human Basophil Releasability VI. Changes in Basophil Releasability in Patients with Allergic Rhinitis or Bronchial Asthma1- 3
VINCENZO CASOLARO, GIUSEPPE SPADARO, and GIANNI MARONE Introduction
Chemical mediators synthesized by human basophils and mast cells playa significant role in the pathogenesis of a variety of allergic disorders, e.g., bronchial asthma, allergic rhinitis, atopic dermatitis, and urticarial syndromes (1-4). The release of chemical mediators from basophils and mast cells is influenced both by the surface density of IgE molecules, and by a largely unknown biochemical process, defined "releasability" (5). Basophil and mast cell releasability implies that biochemical events determine the capacity of these cellsto release mediators in response to IgE- and non-lgEmediated stimuli (5, 6). Recent evidence indicates that basophil releasability and mast cell releasability are independently controlled (7). Basophil releasability appears to be under genetic control (8) and is also linked, in a continuous fashion, to cell donor age (9). Alterations of basophil releasability, both in terms of augmentation or decrease, have been detected in atopic dermatitis (3) and chronic urticaria (4), respectively. Basophil and mast cell releasability may be important also in the pathogenesis of respiratory allergy. However, the few studies on basophil releasability in these patients have yielded contradictory results (7, 10-12). In an attempt to help clarify this aspect, we conducted a study to ascertain whether basophil releasability in adults with bronchial asthma or allergic rhinitis and positive skin tests to Dermatophagoides pteronyssinus major antigen (Der p I) differed from that in control subjects of matched ages. To probe the release mechanisms in detail, we used a variety of releasing agents: two IgE-specific stimuli (Der p I and antiIgE); the formylated tripeptide (f-met peptide), which interacts with a specific cell-surface receptor independent of the IgE receptor (13); and the Ca 2 + ionophore A23187, which bypasses some of the earliest stages of the release process (3). 1108
SUMMARY Weevaluated basophil releasability In two groups of allergic patients with positive skin tests to De,matophagoldes pte,onyss/nus malor allergen (De, p I) (29 adults with bronchial asthma and 17 with allergic rhinitis) and In 31 age-matched normal donors. Both basophil reactivity (maXImal percent histamine release)and basophil sensitivity (the concentration that causes 50% of maximal percent histamine release: HC50) to De, p I In patients with asthma were similar to those In patients with allergic rhinitis. On the contrary, basophil reactivity to antl-lgE was significantly higher In patients with asthma (58.0 ::I:: 3.6%) than In patients with allergic rhinitis (46.3 ::I:: 5.2%; P < 0.05). Both groups of patients showed an Increased ...leasabllity compared to control subJects(27.3 ::I:: 4.6%; P < 0.001),whereas there were no significant dlff.renc.s In basophil sensitivity to antl-lgE among the three groups of donors. Dlff.renc.s were also found with respect to basophil reactivity and sensitivity to f-m.t peptld., wh.reas no dlfferenc.s appeared wh.n basophlls from the three groups of donors were chall.nged with the Ca2 + lonophore A23187. Th.re was a significant correlation betwe.n basophil reactivity and sensitivity to De, p I and to antl-lgE In both asthmatic and all.rglc rhinitis patl.nts. A significant correlation was found betwe.n basophil reactivity and sensitivity to antl-lgE and s.rum IgE level only In patl.nts with bronchial asthma, wh.reas no correlations were found In patients with allergic rhinitis. Th.... was no correlation between In vivo mast cell rel.asability and In vlt,o basophil rel.asablllty In response to De, p I In .Ith.r group of all.rglc patl.nts. Thes. results show that basophil rel.asablllty In response to IgE-m.dlated stimuli Is Inc...as.d In respiratory all.rgy, and that this Increas. Is mo... marked In bronchial asthma. In addition, In vlt,o basophil rel.asablllty does not correlat. with In vivo skin mast c.1I rel.asablllty. AM REV RESPIR DIS 1990; 142:1108-1111
Methods Twenty-nine patients with asthma and 17patients with allergic rhinitis were studied. All 29 asthmatic subjects met the American Thoracic Society's definition for asthma (14). None of the asthma or allergic rhinitis patients used aerosolized or systemic corticosteroids or sodium cromoglycate, all had been free of an upper respiratory tract infection for at least 1 month before the study, and none had received immunotherapy. All patients wereskin test positiveto Der p I. Each patient was prick skin tested with four concentrations of Der pI (0.2, 1.1, 5.6, and 28 ug/ml), a positive control (histamine, 10 mg/ml), and a negative control (vehicle). The reported values represent the mean of duplicate tests performed on the right and left volar surface of the forearms of each patient and are expressed as the area in squared millimeters of wheal. Thirty-one control subjects were studied, all with negative prick skin test responses to common allergens; none had a history of asthma or hay fever, upper respiratory tract infection, or had used any medications for at least 1 month before the study. The procedure for histamine release from human basophils in vitro has been described in detail (3, 7). Cells were challenged with Der
p I, rabbit antihuman IgE (anti-IgE), N-formyl-methionyl-Ieucyl-phenylalanine (f-met peptide), and the Ca:H ionophore A23187. The cell-free supernatant was assayed for histamine with an automated fluorometric technique (15). The serum IgE levels were determined by radioimmunoassay (7), and all analysesweredone on a log-transformed scale (log IgE) when serum IgE level was examined as a continuous variable. The results are expressed as the mean ± SEM. The KruskalWallistest was used for comparisons between the groups. This test reduces to the Mann-
(Received in original form May 21, 1990) 1 From the Division of Clinical Immunology, Department of Medicine, University of Naples Federico II, Second School of Medicine, Naples, Italy. 2 Supported in part by Grant No. 88.00559.04, 89.02672.04, and 89.02967.04 from the C.N.R., by the Ministero della Pubblica Istruzione, and by the Ministero della Sanita-Istituto Superiore di Sanita, AIDS Project 1989 (Rome, Italy). 3 Correspondence and requests for reprints should be addressed to Gianni Marone, M.D., Department of Medicine, University of Naples Federico II, Second School of Medicine, Via S. Pansini 5, 80131 Naples, Italy.
BASOPHIL RELEASABILITY IN RESPIRATORY ALLERGY
1109
80
Whitney (Wilcoxon) test when there are only two groups. The rank correlations were calculated by the Spearman rank coefficient (rs).
60 40
Results
The age, sex, and serum IgE level of control subjects and of our two groups of patients with respiratory allergy are shown in table 1. Serum IgE levels were significantly higher in both groups with respiratory allergy than in control subjects and were significantly higher in patients with asthma than in patients with allergic rhinitis. Patients with asthma had a reduction of 25.8 ± 3.1% of peak expiratory flow (PEF) compared with predicted values for age-, sex- and heightmatched control subjects, and patients with allergic rhinitis. Skin reactivity to histamine and skin sensitivity to De, p I (i.e., the concentration of De, p I that causes a wheal area equal to that caused by 10 mg/ml histamine in that patient) did not differ between the two groups of patients (data not shown). We examined the basophil response of patients with allergic rhinitis or asthma to a range of concentrations (10-3 to 1 ug/ml) of De, p I optimal for histamine release, and thus obtained an entire doseresponse curve for each donor. Figure lA shows that, although the mean percent histamine release caused by all the concentrations of De, p I antigen used was higher in patients with bronchial asthma, a significant difference (P < 0.05) was observed only with a suboptimal (10-2 ug/ ml) concentration of De, p I. Basophil reactivity (maximal percent histamine release) and basophil sensitivity to De, p I (the concentration of De, p I required for 50070 of maximal percent histamine release: HC 50 ) , in patients with asthma weresimilar to those of patients with allergic rhinitis (68.5 ± 4.5% and 6.4 ± 2.1 x
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F-met peptide (M) Fig. 1. Histamine release induced by Der p I (A), antiIgE (B), and f-met peptide (C) from basophils from 29 patients with bronchial asthma (open triangles), 17 patients with allergic rhinitis (open circles), and 31 normal donors (closed circles). Values are expressed as the mean ± SEM. * Significantly different when compared with the group of controls. 0 Significantly different when compared with the group of rhinitics.
10-2 versus 62.8 ± 6.4% and 9.8 ± 5.9 x 10-2 ug/ml, respectively; not significant). Basophilsobtained from normal donors and patients with respiratory allergy were also exposed to a range of concentrations of anti-IgE (10-2 to 1 ug/ml; figure IB), f-met peptide (10-8 to 10-5 M; figure lC), and A23187(3 x 10-2 to 1 ug/ml) optimal
TABLE 1 AGE, SEX, SERUM IgE LEVEL AND PERCENT REDUCTION OF PEF OF CONTROL SUBJECTS AND PATIENTS WITH RESPIRATORY ALLERGY Sex
Age
Serum IgE (1UIml)
Percent Reduction of PEF
13
301 ± 102 (42-1,390)
2.6 ± 1.5 (0.0-14.5)
7
10
556 ± 151* (56-2,048)
3.2 ± 1.2 (0.0-15.8)
11
18
1,164 ± 352t (38-10,000)
25.8 ± 3.fl: (0.0-58.0)
(yr)
Males
Females
Control subjects
21.7 ± 1.9 (9-40)
18
Allergic rhinitis
19.3 ± 1.9 (11-34)
Bronchial asthma
20.6 ± 1.4 (11-41)
Group
Values are the means :t: SEM. Numbers in parentheses represent the range. Percent reduction of PEF was calculated versus predicted PEF for age-, sex-, and height-matched controls. * p < 0.05 when compared with the group of control subjects. t p < 0.0005 when compared with the group of control subjects. :j: p < 0.0005 when compared with the group of allergic rhinitis patients.
for histamine release (3, 13), and entire dose-response curves were obtained for each donor. As shown in figure IB, IgEmediated releasability was enhanced in patients with respiratory allergy,particularly in those with asthma. The percent histamine releasecaused by anti-1gBin patients with bronchial asthma was significantly higher than in the group with allergic rhinitis, except at the two lowestanti-1gB concentrations. Basophil reactivityto anti-1gB in bronchial asthma was 58.0 ± 3.6070 versus 46.3 ± 5.2% in allergic rhinitis versus 27.3 ± 4.6070 in controls (P < 0.(01). Figure 2 shows the frequency distribution of the maximal percent anti-1gB-induced histamine release from basophils of control subjects, and patients with bronchial asthma or allergic rhinitis. In control subjects there are two peaks: one corresponding to donors whose basophils did not release in response to anti-1gB, and another corresponding to donors whose basophils releasedbetween40 and 80070 of their histamine content. In patients with respiratory allergy, there is a marked reduction of the peak corresponding to nonreleasers and a shift into the peak of releasers. Basophil sensitivity to anti-IgE (HC 40 ) did not vary significantly between patients with asthma (6.3 ± 3.4 x 10-2 ug/ml), with allergic rhinitis (1.1 ± 0.5 x 10-1 ug/ml), and control subjects (1.4 ± 0.5 x 10-1 ug/ml). The f-met peptide-induced releasability in the group of patients with asthma was higher compared to control subjects (p < 0.001 at the three highest concentrations) and to patients with allergic rhinitis (p < 0.05 at the two highest concentrations) (figure lC). Basophil reactivity, but not basophil sensitivity (HC 30 ) , to f-met peptide in bronchial asthma (54.6 ± 3.4%) and in allergic rhinitis (45.0' ± 4.4%) was higher than in control subjects (37.8 ± 3.6070; p < 0.005). . No significant changes in basophil response to A23187 occurred in patients with respiratory allergy compared with control subjects (data not shown). Also, basophil reactivity and sensitivity (HC 50 ) to A23187 did not vary between the three groups of subjects. There was a significant correlation between basophil reactivity to anti-IgE and to De, p I in both patients with allergic rhinitis (r, = 0.66; p < 0.01) and bronchial asthma (r, = 0.84; p < 0.01) (figure 3). In patients with allergic rhinitis, there was a significant correlation (r, = 0.52; p < 0.05) between the maximal percent histamine release induced by f-met peptide and A23187. In patients with asthma, there was a significant correla-
CASOLARO. SPADARO, AND MARONE
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tion between the maximal percent histamine release with anti-IgE and their response to A23187 (r, = 0.37; p < 0.05) and between the maximal percent hista mine release by Der p I and A23187 (r, = 0.46; p < 0.05). Significant correlations were also found between basophil sensitivity to anti-IgE and to Der p I in patients with asthma (r, = 0.53; p < 0.01),between basophil sensitivity to antiIgE and f-met peptide in patients with
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allergic rhinitis (r, = 0.59; p < 0.05), and between serum IgE level and basophil reactivity (r, = 0.44; p < 0.05) and sensitivity (r, = -0.38; p < 0.05) in response to anti-1gB in patients with asthma, but not in those with allergic rhinitis. The range of basophil sensitivity to Der p I was approximately 3 to 4 logs in patients with respiratory allergy. In contrast, the range of Derp I antigen that caused in vivo the same wheal area as 10mg/rnl of histamine (i,e., skin mast cell sensitivity to Der p I) in both groups of patients was rather narrow (approximately I log). There was no correlation between basophil and skin mast cell sensitivity to Der p I in either group of patients, or between basophil reactivity and sensitivity to IgB- and non-IgE-mediated stimuli and skin mast cell sensitivity to Der p I (data not shown).
Acknowledgment The writers thank Drs. Kirk Watson and Ray Harris for critical discussions. Gianni Marone is the recipient of the Angelo Minich Award.
References
20 0
+---.----,r-..........--.----,
Discussion
Basophil releasability in response to di0 0 0 0 ,a:. rect (Der p I) or inverse (anti-IgE) anao '8 phylaxis in patients with asthma or allero ~ o :g 80 :E o 0 .... gic rhinitis was increased when compared o .... o to age-matched control subjects. Both ~ 60 · 0 ." a: types of stimuli might be clinically rele..~ . vant. Antigens are, of course, involved ;N 40 in the pathogenesis of these disorders; o anti-IgE autoantibodies have been found :~ 20 .......... o ... in patients with asthma (16), and recent .:::E O +-- --r- ---,- - ..---- --r- ---, evidence suggests that these autoantibod80 100 40 o 20 60 ies induce mediator release from human basophils and mast cells (17). Although Max imal X Histamine Relea se basophil sensitivity to f-met peptide was !.~ ~~ ~ ~ ~ Py .~ .~ ~.i -!~E similar in the three groups examined, the f-met peptide-induced releasability in the Fig, 3. Correlations between the maximal percent group of patients with asthma and basobasophil histamine release induced byanti·lgE and by phil reactivity to f-met peptide in both Der p I in 17 patients with allergic rhinitis (A) and 29 patients with bronchial asthma (B). Each symbol groups of patients were higher than in control subjects. Basophil releasability represents the mean of duplicate determinations. ."i) 100
®
it
...
in response to A23187 was essentially unchanged in the three groups of donors. The range of basophil sensitivity to Der p I is several logs higher than the range of skin mast cell sensitivity to Der p I in both groups of patients. This suggests that the in vitro study of basophil sensitivity to Der p I is a more sensitive way to analyze the immunologic sensitization of these patients. In this study, skin mast cell sensitivity to Der p I was not correlated with IgE- or non-lgE-mediated basophil releasability, which reinforces the concept of the functional heterogeneity between human basophils and mast cells in allergic disorders (7). Basophil sensitivity to anti-IgE and to Der p I were correlated only in patients with asthma. Similarly, a positive correlation between serum IgE levelsand both basophil reactivity and sensitivity to antiIgE was found only in patients with asthma. The lack of correlation between serum IgE levels and both basophil reactivity and sensitivity to anti-1gB in patients with allergic rhinitis agrees with a report that the prevalence of asthma is closely related to serum IgE levels, whereas allergic rhinitis appears independent of this parameter (18). In conclusion, we demonstrate that patients with an allergic involvement of the upper or lower respiratory tract possess a variable degree of alteration of IgE- and non-Igli-dependent basophil releasability.
Kimura I, Tanizaki Y, Saito K, Takahashi K, Veda N, Sato S. Appearance of basophils in the sputum of patients with bronchialasthma. Clin Allergy 1975; 5:95-102. 2. Neijens HJ, Raatgeep RE, Degenhart HJ. DuivermanEJ. Kerrebijn KF. Altered leukocyteresponse in relation to the basic abnormality in children with asthma and bronchial hyperresponsiveness. Am Rev Respir Dis 1984; 130:744-7. 3. Marone G, Giugliano R, Lembo G. Ayala F. Human basophil releasability. II. Changes in basophil releasability in patients with atopic dermatitis. J Invest Dermatol 1986; 87:19-23 . 4. Kern F, Lichtenstein LM. Defective histamine release in chronic urticaria. J Clin Invest 1976; I.
57:1369-77.
5. ConroyMC, Adkinson NF Jr, Lichtenstein LM. Measurement of IgE on human basophils: relation to serum IgE and anti-IgE-induced histamine -release. J Immunol 1977; 118:1317-21. 6. MacOlashan DW Jr, Warner JA, Nguyen K. Biochemical mechanisms underlying human basophil and mast cell responsiveness. In: Melillo G, Norman PS, Marone G, eds, Clinical immunology.Vol. 2. Respiratoryallergy. Toronto: B.C. Decker, Inc., 1990; 79-84.
BASOPHIL RELEASABILITY IN RESPIRATORY ALLERGY
7. Casolaro V, Galeone D, Giacummo A, Sanduzzi A, Melillo G, Marone G. Human basophil/mast cell releasability. V. Functional comparisons of cells obtained from peripheral blood, lung parenchyma and from bronchoalveolar lavage in asthmatics. Am Rev Respir Dis 1989; 139:1375-82. 8. Marone G, Poto S, Celestino D, Bonini S. Human basophil releasability. III. Genetic control of human basophil releasability. J Immunol 1986; 137:3588-92. 9. Marone G, Poto S, di Martino L, Condorelli M. Human basophil releasability. I. Age-related changes in basophil releasability. J Allergy Clin Immunol 1986; 77:377-83. 10. Findlay SR, Lichtenstein LM. Basophil "releasability" in patients with asthma. Am Rev Respir
1111 Dis 1980; 122:53-9. 11. Gaddy IN, Busse WW. Enhanced IgEdependent basophil histamine release and airway reactivity in asthma. Am Rev Respir Dis 1986; 134:969-74. 12. Busse WW, Swenson CA, Sharpe G, Koschat M. Enhanced basophil histamine release to concanavalin A in allergic rhinitis. J Allergy Clin Immunol 1986; 78:90-7. 13. Marone G, Columbo M, Soppelsa L, Condorelli M. The mechanism of basophil histamine release induced by pepstatin A. J Immunol 1984; 133:1542-6. 14. American Thoracic Society. Definitions and classifications of chronic bronchitis, asthma, and pulmonary emphysema. Am Rev Respir Dis 1962;
85:762-8. 15. Siraganian RP. An automated continuous-flow system for the extraction and fluorometric analysis of histamine. Anal Biochem 1974; 57:383-94. 16. Nawata Y, Koike T, Yanagisawa T, et al. AntiIgE autoantibody in patients with bronchial asthma. Clin Exp Immunol 1984; 58:348-56. 17. Marone G, Casolaro V, Paganelli R, Quinti 1. IgG anti-lgE from atopic dermatitis induces mediator release from basophils and mast cells. J Invest Dermatol 1989; 93:246-52. 18. Burrows B, Martinez FD, Halonen M, Barbee RA, Cline MG. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med 1989; 320:271-7.
VINCENZO CASOLARO, GIUSEPPE SPADARO, and GIANNI MARONE Introduction
Chemical mediators synthesized by human basophils and mast cells playa significant role in the pathogenesis of a variety of allergic disorders, e.g., bronchial asthma, allergic rhinitis, atopic dermatitis, and urticarial syndromes (1-4). The release of chemical mediators from basophils and mast cells is influenced both by the surface density of IgE molecules, and by a largely unknown biochemical process, defined "releasability" (5). Basophil and mast cell releasability implies that biochemical events determine the capacity of these cellsto release mediators in response to IgE- and non-lgEmediated stimuli (5, 6). Recent evidence indicates that basophil releasability and mast cell releasability are independently controlled (7). Basophil releasability appears to be under genetic control (8) and is also linked, in a continuous fashion, to cell donor age (9). Alterations of basophil releasability, both in terms of augmentation or decrease, have been detected in atopic dermatitis (3) and chronic urticaria (4), respectively. Basophil and mast cell releasability may be important also in the pathogenesis of respiratory allergy. However, the few studies on basophil releasability in these patients have yielded contradictory results (7, 10-12). In an attempt to help clarify this aspect, we conducted a study to ascertain whether basophil releasability in adults with bronchial asthma or allergic rhinitis and positive skin tests to Dermatophagoides pteronyssinus major antigen (Der p I) differed from that in control subjects of matched ages. To probe the release mechanisms in detail, we used a variety of releasing agents: two IgE-specific stimuli (Der p I and antiIgE); the formylated tripeptide (f-met peptide), which interacts with a specific cell-surface receptor independent of the IgE receptor (13); and the Ca 2 + ionophore A23187, which bypasses some of the earliest stages of the release process (3). 1108
SUMMARY Weevaluated basophil releasability In two groups of allergic patients with positive skin tests to De,matophagoldes pte,onyss/nus malor allergen (De, p I) (29 adults with bronchial asthma and 17 with allergic rhinitis) and In 31 age-matched normal donors. Both basophil reactivity (maXImal percent histamine release)and basophil sensitivity (the concentration that causes 50% of maximal percent histamine release: HC50) to De, p I In patients with asthma were similar to those In patients with allergic rhinitis. On the contrary, basophil reactivity to antl-lgE was significantly higher In patients with asthma (58.0 ::I:: 3.6%) than In patients with allergic rhinitis (46.3 ::I:: 5.2%; P < 0.05). Both groups of patients showed an Increased ...leasabllity compared to control subJects(27.3 ::I:: 4.6%; P < 0.001),whereas there were no significant dlff.renc.s In basophil sensitivity to antl-lgE among the three groups of donors. Dlff.renc.s were also found with respect to basophil reactivity and sensitivity to f-m.t peptld., wh.reas no dlfferenc.s appeared wh.n basophlls from the three groups of donors were chall.nged with the Ca2 + lonophore A23187. Th.re was a significant correlation betwe.n basophil reactivity and sensitivity to De, p I and to antl-lgE In both asthmatic and all.rglc rhinitis patl.nts. A significant correlation was found betwe.n basophil reactivity and sensitivity to antl-lgE and s.rum IgE level only In patl.nts with bronchial asthma, wh.reas no correlations were found In patients with allergic rhinitis. Th.... was no correlation between In vivo mast cell rel.asability and In vlt,o basophil rel.asablllty In response to De, p I In .Ith.r group of all.rglc patl.nts. Thes. results show that basophil rel.asablllty In response to IgE-m.dlated stimuli Is Inc...as.d In respiratory all.rgy, and that this Increas. Is mo... marked In bronchial asthma. In addition, In vlt,o basophil rel.asablllty does not correlat. with In vivo skin mast c.1I rel.asablllty. AM REV RESPIR DIS 1990; 142:1108-1111
Methods Twenty-nine patients with asthma and 17patients with allergic rhinitis were studied. All 29 asthmatic subjects met the American Thoracic Society's definition for asthma (14). None of the asthma or allergic rhinitis patients used aerosolized or systemic corticosteroids or sodium cromoglycate, all had been free of an upper respiratory tract infection for at least 1 month before the study, and none had received immunotherapy. All patients wereskin test positiveto Der p I. Each patient was prick skin tested with four concentrations of Der pI (0.2, 1.1, 5.6, and 28 ug/ml), a positive control (histamine, 10 mg/ml), and a negative control (vehicle). The reported values represent the mean of duplicate tests performed on the right and left volar surface of the forearms of each patient and are expressed as the area in squared millimeters of wheal. Thirty-one control subjects were studied, all with negative prick skin test responses to common allergens; none had a history of asthma or hay fever, upper respiratory tract infection, or had used any medications for at least 1 month before the study. The procedure for histamine release from human basophils in vitro has been described in detail (3, 7). Cells were challenged with Der
p I, rabbit antihuman IgE (anti-IgE), N-formyl-methionyl-Ieucyl-phenylalanine (f-met peptide), and the Ca:H ionophore A23187. The cell-free supernatant was assayed for histamine with an automated fluorometric technique (15). The serum IgE levels were determined by radioimmunoassay (7), and all analysesweredone on a log-transformed scale (log IgE) when serum IgE level was examined as a continuous variable. The results are expressed as the mean ± SEM. The KruskalWallistest was used for comparisons between the groups. This test reduces to the Mann-
(Received in original form May 21, 1990) 1 From the Division of Clinical Immunology, Department of Medicine, University of Naples Federico II, Second School of Medicine, Naples, Italy. 2 Supported in part by Grant No. 88.00559.04, 89.02672.04, and 89.02967.04 from the C.N.R., by the Ministero della Pubblica Istruzione, and by the Ministero della Sanita-Istituto Superiore di Sanita, AIDS Project 1989 (Rome, Italy). 3 Correspondence and requests for reprints should be addressed to Gianni Marone, M.D., Department of Medicine, University of Naples Federico II, Second School of Medicine, Via S. Pansini 5, 80131 Naples, Italy.
BASOPHIL RELEASABILITY IN RESPIRATORY ALLERGY
1109
80
Whitney (Wilcoxon) test when there are only two groups. The rank correlations were calculated by the Spearman rank coefficient (rs).
60 40
Results
The age, sex, and serum IgE level of control subjects and of our two groups of patients with respiratory allergy are shown in table 1. Serum IgE levels were significantly higher in both groups with respiratory allergy than in control subjects and were significantly higher in patients with asthma than in patients with allergic rhinitis. Patients with asthma had a reduction of 25.8 ± 3.1% of peak expiratory flow (PEF) compared with predicted values for age-, sex- and heightmatched control subjects, and patients with allergic rhinitis. Skin reactivity to histamine and skin sensitivity to De, p I (i.e., the concentration of De, p I that causes a wheal area equal to that caused by 10 mg/ml histamine in that patient) did not differ between the two groups of patients (data not shown). We examined the basophil response of patients with allergic rhinitis or asthma to a range of concentrations (10-3 to 1 ug/ml) of De, p I optimal for histamine release, and thus obtained an entire doseresponse curve for each donor. Figure lA shows that, although the mean percent histamine release caused by all the concentrations of De, p I antigen used was higher in patients with bronchial asthma, a significant difference (P < 0.05) was observed only with a suboptimal (10-2 ug/ ml) concentration of De, p I. Basophil reactivity (maximal percent histamine release) and basophil sensitivity to De, p I (the concentration of De, p I required for 50070 of maximal percent histamine release: HC 50 ) , in patients with asthma weresimilar to those of patients with allergic rhinitis (68.5 ± 4.5% and 6.4 ± 2.1 x
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F-met peptide (M) Fig. 1. Histamine release induced by Der p I (A), antiIgE (B), and f-met peptide (C) from basophils from 29 patients with bronchial asthma (open triangles), 17 patients with allergic rhinitis (open circles), and 31 normal donors (closed circles). Values are expressed as the mean ± SEM. * Significantly different when compared with the group of controls. 0 Significantly different when compared with the group of rhinitics.
10-2 versus 62.8 ± 6.4% and 9.8 ± 5.9 x 10-2 ug/ml, respectively; not significant). Basophilsobtained from normal donors and patients with respiratory allergy were also exposed to a range of concentrations of anti-IgE (10-2 to 1 ug/ml; figure IB), f-met peptide (10-8 to 10-5 M; figure lC), and A23187(3 x 10-2 to 1 ug/ml) optimal
TABLE 1 AGE, SEX, SERUM IgE LEVEL AND PERCENT REDUCTION OF PEF OF CONTROL SUBJECTS AND PATIENTS WITH RESPIRATORY ALLERGY Sex
Age
Serum IgE (1UIml)
Percent Reduction of PEF
13
301 ± 102 (42-1,390)
2.6 ± 1.5 (0.0-14.5)
7
10
556 ± 151* (56-2,048)
3.2 ± 1.2 (0.0-15.8)
11
18
1,164 ± 352t (38-10,000)
25.8 ± 3.fl: (0.0-58.0)
(yr)
Males
Females
Control subjects
21.7 ± 1.9 (9-40)
18
Allergic rhinitis
19.3 ± 1.9 (11-34)
Bronchial asthma
20.6 ± 1.4 (11-41)
Group
Values are the means :t: SEM. Numbers in parentheses represent the range. Percent reduction of PEF was calculated versus predicted PEF for age-, sex-, and height-matched controls. * p < 0.05 when compared with the group of control subjects. t p < 0.0005 when compared with the group of control subjects. :j: p < 0.0005 when compared with the group of allergic rhinitis patients.
for histamine release (3, 13), and entire dose-response curves were obtained for each donor. As shown in figure IB, IgEmediated releasability was enhanced in patients with respiratory allergy,particularly in those with asthma. The percent histamine releasecaused by anti-1gBin patients with bronchial asthma was significantly higher than in the group with allergic rhinitis, except at the two lowestanti-1gB concentrations. Basophil reactivityto anti-1gB in bronchial asthma was 58.0 ± 3.6070 versus 46.3 ± 5.2% in allergic rhinitis versus 27.3 ± 4.6070 in controls (P < 0.(01). Figure 2 shows the frequency distribution of the maximal percent anti-1gB-induced histamine release from basophils of control subjects, and patients with bronchial asthma or allergic rhinitis. In control subjects there are two peaks: one corresponding to donors whose basophils did not release in response to anti-1gB, and another corresponding to donors whose basophils releasedbetween40 and 80070 of their histamine content. In patients with respiratory allergy, there is a marked reduction of the peak corresponding to nonreleasers and a shift into the peak of releasers. Basophil sensitivity to anti-IgE (HC 40 ) did not vary significantly between patients with asthma (6.3 ± 3.4 x 10-2 ug/ml), with allergic rhinitis (1.1 ± 0.5 x 10-1 ug/ml), and control subjects (1.4 ± 0.5 x 10-1 ug/ml). The f-met peptide-induced releasability in the group of patients with asthma was higher compared to control subjects (p < 0.001 at the three highest concentrations) and to patients with allergic rhinitis (p < 0.05 at the two highest concentrations) (figure lC). Basophil reactivity, but not basophil sensitivity (HC 30 ) , to f-met peptide in bronchial asthma (54.6 ± 3.4%) and in allergic rhinitis (45.0' ± 4.4%) was higher than in control subjects (37.8 ± 3.6070; p < 0.005). . No significant changes in basophil response to A23187 occurred in patients with respiratory allergy compared with control subjects (data not shown). Also, basophil reactivity and sensitivity (HC 50 ) to A23187 did not vary between the three groups of subjects. There was a significant correlation between basophil reactivity to anti-IgE and to De, p I in both patients with allergic rhinitis (r, = 0.66; p < 0.01) and bronchial asthma (r, = 0.84; p < 0.01) (figure 3). In patients with allergic rhinitis, there was a significant correlation (r, = 0.52; p < 0.05) between the maximal percent histamine release induced by f-met peptide and A23187. In patients with asthma, there was a significant correla-
CASOLARO. SPADARO, AND MARONE
1110
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Fig. 2. Frequency distribution of the maximal percent anti-lgE-induced histaminesecretionfrom basophils obtained from 31 normal donors(open bars). from 29 patients with bronchial asthma (closed bars) and from 17 patientswith allargic rhinitis (hatched bars).
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Max imal % Histam ine Release Induced by Ant i-igE
tion between the maximal percent histamine release with anti-IgE and their response to A23187 (r, = 0.37; p < 0.05) and between the maximal percent hista mine release by Der p I and A23187 (r, = 0.46; p < 0.05). Significant correlations were also found between basophil sensitivity to anti-IgE and to Der p I in patients with asthma (r, = 0.53; p < 0.01),between basophil sensitivity to antiIgE and f-met peptide in patients with
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allergic rhinitis (r, = 0.59; p < 0.05), and between serum IgE level and basophil reactivity (r, = 0.44; p < 0.05) and sensitivity (r, = -0.38; p < 0.05) in response to anti-1gB in patients with asthma, but not in those with allergic rhinitis. The range of basophil sensitivity to Der p I was approximately 3 to 4 logs in patients with respiratory allergy. In contrast, the range of Derp I antigen that caused in vivo the same wheal area as 10mg/rnl of histamine (i,e., skin mast cell sensitivity to Der p I) in both groups of patients was rather narrow (approximately I log). There was no correlation between basophil and skin mast cell sensitivity to Der p I in either group of patients, or between basophil reactivity and sensitivity to IgB- and non-IgE-mediated stimuli and skin mast cell sensitivity to Der p I (data not shown).
Acknowledgment The writers thank Drs. Kirk Watson and Ray Harris for critical discussions. Gianni Marone is the recipient of the Angelo Minich Award.
References
20 0
+---.----,r-..........--.----,
Discussion
Basophil releasability in response to di0 0 0 0 ,a:. rect (Der p I) or inverse (anti-IgE) anao '8 phylaxis in patients with asthma or allero ~ o :g 80 :E o 0 .... gic rhinitis was increased when compared o .... o to age-matched control subjects. Both ~ 60 · 0 ." a: types of stimuli might be clinically rele..~ . vant. Antigens are, of course, involved ;N 40 in the pathogenesis of these disorders; o anti-IgE autoantibodies have been found :~ 20 .......... o ... in patients with asthma (16), and recent .:::E O +-- --r- ---,- - ..---- --r- ---, evidence suggests that these autoantibod80 100 40 o 20 60 ies induce mediator release from human basophils and mast cells (17). Although Max imal X Histamine Relea se basophil sensitivity to f-met peptide was !.~ ~~ ~ ~ ~ Py .~ .~ ~.i -!~E similar in the three groups examined, the f-met peptide-induced releasability in the Fig, 3. Correlations between the maximal percent group of patients with asthma and basobasophil histamine release induced byanti·lgE and by phil reactivity to f-met peptide in both Der p I in 17 patients with allergic rhinitis (A) and 29 patients with bronchial asthma (B). Each symbol groups of patients were higher than in control subjects. Basophil releasability represents the mean of duplicate determinations. ."i) 100
®
it
...
in response to A23187 was essentially unchanged in the three groups of donors. The range of basophil sensitivity to Der p I is several logs higher than the range of skin mast cell sensitivity to Der p I in both groups of patients. This suggests that the in vitro study of basophil sensitivity to Der p I is a more sensitive way to analyze the immunologic sensitization of these patients. In this study, skin mast cell sensitivity to Der p I was not correlated with IgE- or non-lgE-mediated basophil releasability, which reinforces the concept of the functional heterogeneity between human basophils and mast cells in allergic disorders (7). Basophil sensitivity to anti-IgE and to Der p I were correlated only in patients with asthma. Similarly, a positive correlation between serum IgE levelsand both basophil reactivity and sensitivity to antiIgE was found only in patients with asthma. The lack of correlation between serum IgE levels and both basophil reactivity and sensitivity to anti-1gB in patients with allergic rhinitis agrees with a report that the prevalence of asthma is closely related to serum IgE levels, whereas allergic rhinitis appears independent of this parameter (18). In conclusion, we demonstrate that patients with an allergic involvement of the upper or lower respiratory tract possess a variable degree of alteration of IgE- and non-Igli-dependent basophil releasability.
Kimura I, Tanizaki Y, Saito K, Takahashi K, Veda N, Sato S. Appearance of basophils in the sputum of patients with bronchialasthma. Clin Allergy 1975; 5:95-102. 2. Neijens HJ, Raatgeep RE, Degenhart HJ. DuivermanEJ. Kerrebijn KF. Altered leukocyteresponse in relation to the basic abnormality in children with asthma and bronchial hyperresponsiveness. Am Rev Respir Dis 1984; 130:744-7. 3. Marone G, Giugliano R, Lembo G. Ayala F. Human basophil releasability. II. Changes in basophil releasability in patients with atopic dermatitis. J Invest Dermatol 1986; 87:19-23 . 4. Kern F, Lichtenstein LM. Defective histamine release in chronic urticaria. J Clin Invest 1976; I.
57:1369-77.
5. ConroyMC, Adkinson NF Jr, Lichtenstein LM. Measurement of IgE on human basophils: relation to serum IgE and anti-IgE-induced histamine -release. J Immunol 1977; 118:1317-21. 6. MacOlashan DW Jr, Warner JA, Nguyen K. Biochemical mechanisms underlying human basophil and mast cell responsiveness. In: Melillo G, Norman PS, Marone G, eds, Clinical immunology.Vol. 2. Respiratoryallergy. Toronto: B.C. Decker, Inc., 1990; 79-84.
BASOPHIL RELEASABILITY IN RESPIRATORY ALLERGY
7. Casolaro V, Galeone D, Giacummo A, Sanduzzi A, Melillo G, Marone G. Human basophil/mast cell releasability. V. Functional comparisons of cells obtained from peripheral blood, lung parenchyma and from bronchoalveolar lavage in asthmatics. Am Rev Respir Dis 1989; 139:1375-82. 8. Marone G, Poto S, Celestino D, Bonini S. Human basophil releasability. III. Genetic control of human basophil releasability. J Immunol 1986; 137:3588-92. 9. Marone G, Poto S, di Martino L, Condorelli M. Human basophil releasability. I. Age-related changes in basophil releasability. J Allergy Clin Immunol 1986; 77:377-83. 10. Findlay SR, Lichtenstein LM. Basophil "releasability" in patients with asthma. Am Rev Respir
1111 Dis 1980; 122:53-9. 11. Gaddy IN, Busse WW. Enhanced IgEdependent basophil histamine release and airway reactivity in asthma. Am Rev Respir Dis 1986; 134:969-74. 12. Busse WW, Swenson CA, Sharpe G, Koschat M. Enhanced basophil histamine release to concanavalin A in allergic rhinitis. J Allergy Clin Immunol 1986; 78:90-7. 13. Marone G, Columbo M, Soppelsa L, Condorelli M. The mechanism of basophil histamine release induced by pepstatin A. J Immunol 1984; 133:1542-6. 14. American Thoracic Society. Definitions and classifications of chronic bronchitis, asthma, and pulmonary emphysema. Am Rev Respir Dis 1962;
85:762-8. 15. Siraganian RP. An automated continuous-flow system for the extraction and fluorometric analysis of histamine. Anal Biochem 1974; 57:383-94. 16. Nawata Y, Koike T, Yanagisawa T, et al. AntiIgE autoantibody in patients with bronchial asthma. Clin Exp Immunol 1984; 58:348-56. 17. Marone G, Casolaro V, Paganelli R, Quinti 1. IgG anti-lgE from atopic dermatitis induces mediator release from basophils and mast cells. J Invest Dermatol 1989; 93:246-52. 18. Burrows B, Martinez FD, Halonen M, Barbee RA, Cline MG. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med 1989; 320:271-7.
