Immunology 1976 31 797
Eosinophils and mediators of anaphylaxis HISTAMINE AND IMIDAZOLE ACETIC ACID AS CHEMOTACTIC AGENTS FOR HUMAN EOSINOPHIL LEUCOCYTES
LINDSAY W. TURNBULL & A. B. KAY Departments of Pathology and Respiratory Diseases, University ofEdinburgh, andSouth-East ScotlandRegionalBlood Transfusion Service, Royal Infirmary, Edinburgh
Received 22 March 1976; accepted for publication
Summary. Histamine and one of its major catabolites, imidazole acetic acid (ImAA), were selectively chemotactic for human eosinophils, whereas L-histidine and other histamine catabolites including 1,4-methylhistamine, 1-methyl-4-imidazole acetic acid and N-acetylhistamine were inactive in eosinophilotaxis over a large dose range. The dose response for histamine was dependent on the chemotaxis incubation time and the source of eosinophils, although the latter was not clearly associated with particular disease states. When histamine and ImAA were combined the chemotactic response was similar to that obtained when one agent was assayed alone, no additive or synergistic effects being observed. There was cross-deactivation between histamine and ImAA. These experiments suggest that histamine and ImAA activate the same chemotactic recognition mechanism for eosinophils. Thus ImAA joins histamine and the tetrapeptides (ECF-A) as anaphylaxisassociated selective chemoattractants for human eosinophils.
1
April 1976
cological mediators of anaphylaxis. These include histamine, a slow reacting substance (SRS-A) and an eosinophil chemotactic factor (ECF-A). ECF-A is now recognized as two associated acidic tetrapeptides, Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu (Goetzl & Austen, 1975). An analogue Val-GlyAsp-Glu also selectively attracts human eosinophils from a mixed cell population (Kay, 1976). The role of histamine in the recruitment of eosinophils to the site of allergic tissue reactions has been the subject of some controversy (Parish, 1974). Whereas subcutaneous injections of histamine into the horse resulted in eosinophil accumulation (Archer, 1956) these observations have not been confirmed in the guinea-pig (Kay, 1970a) or man (Felarca & Lowell, 1968). Similarly in vitro experiments failed to show chemoattractant properties for histamine when eosinophils from guinea-pig and man were used as target cells (Kay, Stechschulte & Austen, 1971; Kay & Austen, 1971). However, Clark, Gallin & Kaplan (1975) were able to show human eosinophilotaxis by histamine although the activity was only demonstrable at concentrations of approximately 5 x 10-I mol/l, there being inhibition with higher doses. It now appears that these apparent discrepancies in the chemotactic property of histamine can be explained largely on the type of micropore filters used in the assays. Thus with the more traditional Millipore filter, chemotaxis by
INTRODUCTION Human lung can be passively sensitized by IgE for the antigen-induced release of a number of pharmaCorrespondence: Dr A. B. Kay, South-East Scotland Regional Blood Transfusion Service, Royal Infirmary, Edinburgh EH3 9HB.
797
798
Lindsay W. Turnbull & A. B. Kay
histamine cannot be demonstrated whereas these membranes were suitable for the identification and characterisation of ECF-A (Kay & Austen, 1971). In contrast, cellulose nitrate filters were satisfactory for the assay of both the ECF-A peptides and histamine (Kay, 1976). In the present report we have confirmed and extended the observation on eosinophil chemotaxis by histamine and in addition describe a similar eosinophilotactic property for a major histamine metabolite, imidazole acetic acid (ImAA). The interactions between histamine and ImAA in terms of chemotaxis and chemotactic deactivation are also described.
MATERIALS AND METHODS Materials The materials were obtained as follows. Histamine acid phosphate (BDH Chemicals Ltd, Poole); imidazole acetic acid hydrochloride (ImAA) chromatographically free of histidine, histamine and isopropylester, 98 8 per cent pure; 1-methyl-4-imidazole acetic acid hydrochloride (1,4-MeImAA), L-histidine, free base, 1,4-methylhistamine dihydrochloride (1,4MeHm), N-acetylhistamine (N-AcHm), all chromatographically homogeneous (Calbiochem Ltd, San Diego, California); ovalbumin five times crystallised (Koch-Light Laboratories, Colnbrook, Bucks.); cellulose nitrate filters, 8-0 pm (Sartorius-membrane filters, 34 Gottingen, W. Germany).
Eosinophil chemotaxis Peripheral blood was withdrawn from patients with an eosinophilia of between 10 and 25 percent. Leucocyte rich plasma, obtained by dextran sedimentation of heparinised blood (Kay, 1970b) was applied to a density gradient of 9 per cent Ficoll solution and sodium diatrizoate (density 1 140) in the proportions 2-4: 1 (English & Andersen, 1974). Following centrifugation, at 400 g for 40 min at 200, the eosinophilrich pellet was retained, washed in Tyrode's buffer and resuspended in Tyrode's containing 0-5 per cent ovalbumin. This procedure increased the eosinophil concentration two- to three-fold, the contaminating cells being mostly neutrophils. The cell counts were then adjusted to 2 x 106 cells/ml, with a final eosinophil concentration of 20-30 per cent. Blood with an initial eosinophil concentration in this range was prepared by dextran sedimentation alone. The
chemotactic assay procedure was as previously described (Kay, 1970b) but using 8 0 pm Sartorius micropores. All agents tested for chemotaxis were dissolved in Tyrode's solution and the pH adjusted to 7-24. Statistics Measurements from duplicate filters from three separate experiments were pooled, the mean and standard error determined and observations compared using the Student's t-test. Such pooling was considered to be statistically valid since in the present study the variation between duplicate filters was similar to variations between individual experiments as previously observed (Clark et al., 1975). RESULTS (1) Eosinophil chemotactic activity of histamine and histamine metabolites Histamine, L-histidine, ImAA, 1,4-MeHm, 1,4MeImAA and N-AcHm were assayed for chemo tactic activity at the concentrations shown in Fig. 1. With a 135-min incubation period eosinophil chemotaxis by histamine was demonstrable at concentrations of 10-5 and 10-6 mol/l with apparent inhibition of migration at 10-4 mol/l. In contrast, chemotaxis by ImAA gave a linear-dose response from 10-3 to 10-5 mol/l. No chemotactic activity was found with histidine or the other histamine catabolites. Within the dose-ranges tested in Fig. 1, 18 _ 16-
W
6 4
-X
T-
IF4
10-7 10-5 io-6 Concentration (mol/) Figure 1. Chemotaxis for human eosinophils by histamine (o), ImAA (o), 1,4-MeImAA (A), 1,4-MeHm (A), N-AcHm (P) and L-histidine (o). Incubation period 135 min. The bars for Figs 1-5 represent + s.e.m. The P values refer to the points immediately above or below.
10-3
Eosinophil chemotaxis
799
Table 1. Pattern of chemotactic response to histamine by human eosinophils from various patients No. of
Patient
Associated disease
experiments
Pattern of response
H.W. J.P. G.M. E.S.
Microfilariasis (D. perstans) Microfilariasis (D. perstans) Drug reaction Allergic asthma
4 1 2 1
R.G.
Drug reaction
2
F.F.
Hypereosinophilic syndrome
2
Linear dose-response with maximal density at 10-3 mol/l Linear dose-response with maximal density at 10-3 mol/1 Linear dose-response with maximal density at 10-3 mol/I Peak activity at 5 x 10-6 mol/1 with inhibition at higher dose Peak activity at 5 x 10-6 mol/l with inhibition at higher dose Peak activity at 5 x 10-6 mol/l with inhibition at higher dose
neutrophil migration was not observed with histamine, ImAA or the other compounds.
(2) Time-course studies Using a 180-min incubation time eosinophil chemotactic activity by histamine was found to give a linear-dose response between 10-3 and 10-7 mol/l (Fig. 2). However at 135 min there was an inhibition of chemotaxis with a dose of 10-3 mol/l. Negligible activity was found at all doses with a 90-min incubation time. Therefore by increasing the incubation period the apparent inhibition of migration by higher concentrations of histamine could be abolished. However eosinophils from certain individuals gave a peak of activity at approximately
5 x 10-6 mol/l with inhibition at higher doses even with a 180-min incubation period (Table 1). The two types of chemotactic profile, i.e. maximal activity at 10-3 or at 5 x 10-6 mol/l were not clearly associated with the disease state. In subsequent studies only the eosinophils from patients giving the linear doseresponse pattern of activity shown in Fig. 2 (180 min) were used. Similar experiments were carried out with ImAA. The latter was selectively chemotactic for human eosinophils between 10- 3 and 10' mol/l using either 135- or 180-min incubations and invariably gave a linear-dose response irrespective of the sour'e of eosinophils. As with histamine little activity was seen at 90 min. In all further experiments a 180-min incubation time was used.
(3) The effect of combining histamine and ImAA When histamine was combined with ImAA (10-3 mol/l) there was virtually no difference in the eosinophil chemotactic response when compared to hista-
20 18 16 0
cE
12
=
10
a0
8
0. 0
)Histomine+T3M ImAA Histamine
24 22 20 0 18 1~2anE 16 .s-
w
6
14
P
Eosinophils and mediators of anaphylaxis HISTAMINE AND IMIDAZOLE ACETIC ACID AS CHEMOTACTIC AGENTS FOR HUMAN EOSINOPHIL LEUCOCYTES
LINDSAY W. TURNBULL & A. B. KAY Departments of Pathology and Respiratory Diseases, University ofEdinburgh, andSouth-East ScotlandRegionalBlood Transfusion Service, Royal Infirmary, Edinburgh
Received 22 March 1976; accepted for publication
Summary. Histamine and one of its major catabolites, imidazole acetic acid (ImAA), were selectively chemotactic for human eosinophils, whereas L-histidine and other histamine catabolites including 1,4-methylhistamine, 1-methyl-4-imidazole acetic acid and N-acetylhistamine were inactive in eosinophilotaxis over a large dose range. The dose response for histamine was dependent on the chemotaxis incubation time and the source of eosinophils, although the latter was not clearly associated with particular disease states. When histamine and ImAA were combined the chemotactic response was similar to that obtained when one agent was assayed alone, no additive or synergistic effects being observed. There was cross-deactivation between histamine and ImAA. These experiments suggest that histamine and ImAA activate the same chemotactic recognition mechanism for eosinophils. Thus ImAA joins histamine and the tetrapeptides (ECF-A) as anaphylaxisassociated selective chemoattractants for human eosinophils.
1
April 1976
cological mediators of anaphylaxis. These include histamine, a slow reacting substance (SRS-A) and an eosinophil chemotactic factor (ECF-A). ECF-A is now recognized as two associated acidic tetrapeptides, Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu (Goetzl & Austen, 1975). An analogue Val-GlyAsp-Glu also selectively attracts human eosinophils from a mixed cell population (Kay, 1976). The role of histamine in the recruitment of eosinophils to the site of allergic tissue reactions has been the subject of some controversy (Parish, 1974). Whereas subcutaneous injections of histamine into the horse resulted in eosinophil accumulation (Archer, 1956) these observations have not been confirmed in the guinea-pig (Kay, 1970a) or man (Felarca & Lowell, 1968). Similarly in vitro experiments failed to show chemoattractant properties for histamine when eosinophils from guinea-pig and man were used as target cells (Kay, Stechschulte & Austen, 1971; Kay & Austen, 1971). However, Clark, Gallin & Kaplan (1975) were able to show human eosinophilotaxis by histamine although the activity was only demonstrable at concentrations of approximately 5 x 10-I mol/l, there being inhibition with higher doses. It now appears that these apparent discrepancies in the chemotactic property of histamine can be explained largely on the type of micropore filters used in the assays. Thus with the more traditional Millipore filter, chemotaxis by
INTRODUCTION Human lung can be passively sensitized by IgE for the antigen-induced release of a number of pharmaCorrespondence: Dr A. B. Kay, South-East Scotland Regional Blood Transfusion Service, Royal Infirmary, Edinburgh EH3 9HB.
797
798
Lindsay W. Turnbull & A. B. Kay
histamine cannot be demonstrated whereas these membranes were suitable for the identification and characterisation of ECF-A (Kay & Austen, 1971). In contrast, cellulose nitrate filters were satisfactory for the assay of both the ECF-A peptides and histamine (Kay, 1976). In the present report we have confirmed and extended the observation on eosinophil chemotaxis by histamine and in addition describe a similar eosinophilotactic property for a major histamine metabolite, imidazole acetic acid (ImAA). The interactions between histamine and ImAA in terms of chemotaxis and chemotactic deactivation are also described.
MATERIALS AND METHODS Materials The materials were obtained as follows. Histamine acid phosphate (BDH Chemicals Ltd, Poole); imidazole acetic acid hydrochloride (ImAA) chromatographically free of histidine, histamine and isopropylester, 98 8 per cent pure; 1-methyl-4-imidazole acetic acid hydrochloride (1,4-MeImAA), L-histidine, free base, 1,4-methylhistamine dihydrochloride (1,4MeHm), N-acetylhistamine (N-AcHm), all chromatographically homogeneous (Calbiochem Ltd, San Diego, California); ovalbumin five times crystallised (Koch-Light Laboratories, Colnbrook, Bucks.); cellulose nitrate filters, 8-0 pm (Sartorius-membrane filters, 34 Gottingen, W. Germany).
Eosinophil chemotaxis Peripheral blood was withdrawn from patients with an eosinophilia of between 10 and 25 percent. Leucocyte rich plasma, obtained by dextran sedimentation of heparinised blood (Kay, 1970b) was applied to a density gradient of 9 per cent Ficoll solution and sodium diatrizoate (density 1 140) in the proportions 2-4: 1 (English & Andersen, 1974). Following centrifugation, at 400 g for 40 min at 200, the eosinophilrich pellet was retained, washed in Tyrode's buffer and resuspended in Tyrode's containing 0-5 per cent ovalbumin. This procedure increased the eosinophil concentration two- to three-fold, the contaminating cells being mostly neutrophils. The cell counts were then adjusted to 2 x 106 cells/ml, with a final eosinophil concentration of 20-30 per cent. Blood with an initial eosinophil concentration in this range was prepared by dextran sedimentation alone. The
chemotactic assay procedure was as previously described (Kay, 1970b) but using 8 0 pm Sartorius micropores. All agents tested for chemotaxis were dissolved in Tyrode's solution and the pH adjusted to 7-24. Statistics Measurements from duplicate filters from three separate experiments were pooled, the mean and standard error determined and observations compared using the Student's t-test. Such pooling was considered to be statistically valid since in the present study the variation between duplicate filters was similar to variations between individual experiments as previously observed (Clark et al., 1975). RESULTS (1) Eosinophil chemotactic activity of histamine and histamine metabolites Histamine, L-histidine, ImAA, 1,4-MeHm, 1,4MeImAA and N-AcHm were assayed for chemo tactic activity at the concentrations shown in Fig. 1. With a 135-min incubation period eosinophil chemotaxis by histamine was demonstrable at concentrations of 10-5 and 10-6 mol/l with apparent inhibition of migration at 10-4 mol/l. In contrast, chemotaxis by ImAA gave a linear-dose response from 10-3 to 10-5 mol/l. No chemotactic activity was found with histidine or the other histamine catabolites. Within the dose-ranges tested in Fig. 1, 18 _ 16-
W
6 4
-X
T-
IF4
10-7 10-5 io-6 Concentration (mol/) Figure 1. Chemotaxis for human eosinophils by histamine (o), ImAA (o), 1,4-MeImAA (A), 1,4-MeHm (A), N-AcHm (P) and L-histidine (o). Incubation period 135 min. The bars for Figs 1-5 represent + s.e.m. The P values refer to the points immediately above or below.
10-3
Eosinophil chemotaxis
799
Table 1. Pattern of chemotactic response to histamine by human eosinophils from various patients No. of
Patient
Associated disease
experiments
Pattern of response
H.W. J.P. G.M. E.S.
Microfilariasis (D. perstans) Microfilariasis (D. perstans) Drug reaction Allergic asthma
4 1 2 1
R.G.
Drug reaction
2
F.F.
Hypereosinophilic syndrome
2
Linear dose-response with maximal density at 10-3 mol/l Linear dose-response with maximal density at 10-3 mol/1 Linear dose-response with maximal density at 10-3 mol/I Peak activity at 5 x 10-6 mol/1 with inhibition at higher dose Peak activity at 5 x 10-6 mol/l with inhibition at higher dose Peak activity at 5 x 10-6 mol/l with inhibition at higher dose
neutrophil migration was not observed with histamine, ImAA or the other compounds.
(2) Time-course studies Using a 180-min incubation time eosinophil chemotactic activity by histamine was found to give a linear-dose response between 10-3 and 10-7 mol/l (Fig. 2). However at 135 min there was an inhibition of chemotaxis with a dose of 10-3 mol/l. Negligible activity was found at all doses with a 90-min incubation time. Therefore by increasing the incubation period the apparent inhibition of migration by higher concentrations of histamine could be abolished. However eosinophils from certain individuals gave a peak of activity at approximately
5 x 10-6 mol/l with inhibition at higher doses even with a 180-min incubation period (Table 1). The two types of chemotactic profile, i.e. maximal activity at 10-3 or at 5 x 10-6 mol/l were not clearly associated with the disease state. In subsequent studies only the eosinophils from patients giving the linear doseresponse pattern of activity shown in Fig. 2 (180 min) were used. Similar experiments were carried out with ImAA. The latter was selectively chemotactic for human eosinophils between 10- 3 and 10' mol/l using either 135- or 180-min incubations and invariably gave a linear-dose response irrespective of the sour'e of eosinophils. As with histamine little activity was seen at 90 min. In all further experiments a 180-min incubation time was used.
(3) The effect of combining histamine and ImAA When histamine was combined with ImAA (10-3 mol/l) there was virtually no difference in the eosinophil chemotactic response when compared to hista-
20 18 16 0
cE
12
=
10
a0
8
0. 0
)Histomine+T3M ImAA Histamine
24 22 20 0 18 1~2anE 16 .s-
w
6
14
P
