Clinical and Experimental Allergy. 1992. Volume 22. pages 1020-1025
Effects of cytokines on human basophil chemotaxis Y. TANIMOTO, K. TAKAHASHI and I. KIMURA Second Department of Internal Medicine. Okayama University Medical School. Okayanm. Japan Summary Basophil chemotactic activity (BCA) of eight recombinant human (rh) cytokines was examined. Highly purified basophils were obtained by Percoll discontinuous gradients, followed by negative selection using flow cytometry. Then BCA was measured by means of modified Boyden chamber method. Both interleukin (IL)-3 and granulocytemacrophage colony-stimulating factor (GM-CSF) had much more potent BCA than complement C5a, leukotriene B4 and platelet activating factor, well known as granulocyte chemotactic factors. Chemota.xis rather than chemokinesis was shown in chequerboard analysis of basophii migration induced by IL-3 and GM-CSF. Relatively high concentrations of IL-5 also induced basophil migration, although predominantly chemokinetic. IL-8 had apparent BCA. which was not so high as that of C5a. In contrast, IL-2. IL-4. interferon(IFN}--/ and granulocyte colony-stimulating factor (GCSF) had no significant BCA. These findings suggest that IL-3, IL-5. GM-CSF and, perhaps, IL-8 have an effect on basophil migration as well as modulation of basophil mediator release and may provide some insight into the basophil accumulation observed in late-phase allergic responses. Clinical and Experimental Allergy. Vol. 22. pp. 1020-1025. Submitted ! October IQ*?!: revised 30 March 1992; accepted 5 May 1992. Introduction Basophils as well as mast cells play an important role in type I allergy [1,2]. Moreover, basophils infiltrate the skin in delayed type reactions termed cutaneous basophil hypersensitivity (CBH) [3,4], and it is also well known that there is an increase in the number of basophils at sites of late-phase allergic responses such as skin and airways [5-7]. In our previous studies [8,9], it has been suggested that circulating basophils increase in number during the pre-attack stage of bronchial asthma and decrease during attacks with the appearance of basophils in the sputum. However., the mechanisms o( basophii migration to the extravascular sites of allergic responses are not well understood. Recently, it has been reported that several cytokines such as interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) modulate the functions of mature basophils (e.g. enhancement of histamine release upon stimulation with anti-IgF or FMLP) [10] as well as basophil growth and differentiation [II]. We hypothesized that these cytokines, especially derived Correspondence: Dr Y. Tanimoto. Second Department of Internal Medicine. Okayama University Medical School. 2-5-1 Shlkalii-cho. Okayama 700, Japan.
1020
from lymphocytes activated by allergens [12], might also induce basophil migration observed in late-phase allergic responses, and now demonstrate chemotactic effects of several recombinant cytokines on human peripheral blood basophils in vitro. Materials and methods Reagents Hanks" balanced salt solution (HBSS), bovine serum albumin (BSA) and recombinant human complement C5a were purchased from Sigma Chemical Co. {St Louis, Missouri, U.S.A.); Percoll from Pharmacia (Uppsala. Sweden); leukotriene B4 (LTB4) from Cayman Chemical Co. (Ann Arbor, Michigan, U.S.A.); platelet activating factor (PAF) from Bachem Feinchemikalien AG (Bubendorf. Switzerland). Recombinant human {rh) eytokines ami monoclonal antibodies to rh cytokines rhIL-3, rhIL-4, rhGM-CSF, mouse anti-human IL-3 monoclonal antibody and mouse anti-human GM-CSF monoclonal antibody were purchased from Gen/^yme (Boston, Massachussetts, U.S.A.); rhIL-8 was purchased
Effects of cyiokines on human basophil chemotaxis
from Peprotech Inc. (Rocky Hill. New Jersey, U.S.A.). Other recombinant human cytokines were kindly provided; rhIL-2, rhIFN-/ (Shionogi Pharmaceutical Co., Osaka, Japan). rhG-CSF (Kirin Brewery Co.. Tokyo, Japan), and rhTL-5 (The Suntory Institute for Biomedical Research, Osaka, Japan). Specific activities of rhIL-2. rhIL-3,rhIL-4and rhlFN-y were approximately 11 x 10^ U/mg, 10« U/mg, 10^ U/mg and 2 x 10' U/mg. Purificaiion of human basophils Highly purified basophils were used in all experiments. The detail of purification procedures was described previously [13]. Briefly, basophils were separated from peripheral blood by Percoll discontinuous gradients. followed by negative selection using flow cytometry (i.e. sortingCD2-CDM-CD16-CD19"^ cells from basophilrich mononuclear cell layers). Purified basophils were resuspended in HBSS containing 0 rvi, BSA before chemotactic assay, and basophil counts were performed by the method of Kimura et al. [8]. Thus basophils, of which viability exceeded 90%, were enriched to a purity of >80%. The contaminating cells of purified basoph\l suspensions were mostly lymphocytes, neither neutrophils nor eosinophils. Chemotactic assay Basophil chemotactic activity (BCA) was measured by modified Boyden chamber method. The lower chamber (Neuro Probe Inc., Cabin John, Maryland, U.S.A.). Lo which 50 /il of chemoattractant was added, was separated from the upper one by a 10 /nn ihick polycarbonate membrane filter with 5 ftm diameter pores (Nuclepore, Pleasanton, California, U.S.A.). A volume of 100 //I of basophil suspension (2 x lO'/ml) was added to the upper chamber. Chambers were incubated for 60 min at 37 C in a humidified atmosphere of 5'>n CO: in air. Then filters were removed and slatned by the method described previously [Sj. Migrated basophils were counted in 20 randomly selected fields at a magnification of x 400. Degranulation, even though it occurred during migration, did not alTect the ability to lind basophils. Measurements were made in duplicate chambers; results were expressed as mean + s.c.m of duplicate determinations from three separate experiments, and statistically analysed by means of Student's Mest.
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compared with recombinant human cytokines. Figure I showed that BCA of C5a was significantly high at 10^ " M (7±1 basophils 20 h.p.f. vs control values; l ± 0 basophils/20 h.p.f; /'
Effects of cytokines on human basophil chemotaxis Y. TANIMOTO, K. TAKAHASHI and I. KIMURA Second Department of Internal Medicine. Okayama University Medical School. Okayanm. Japan Summary Basophil chemotactic activity (BCA) of eight recombinant human (rh) cytokines was examined. Highly purified basophils were obtained by Percoll discontinuous gradients, followed by negative selection using flow cytometry. Then BCA was measured by means of modified Boyden chamber method. Both interleukin (IL)-3 and granulocytemacrophage colony-stimulating factor (GM-CSF) had much more potent BCA than complement C5a, leukotriene B4 and platelet activating factor, well known as granulocyte chemotactic factors. Chemota.xis rather than chemokinesis was shown in chequerboard analysis of basophii migration induced by IL-3 and GM-CSF. Relatively high concentrations of IL-5 also induced basophil migration, although predominantly chemokinetic. IL-8 had apparent BCA. which was not so high as that of C5a. In contrast, IL-2. IL-4. interferon(IFN}--/ and granulocyte colony-stimulating factor (GCSF) had no significant BCA. These findings suggest that IL-3, IL-5. GM-CSF and, perhaps, IL-8 have an effect on basophil migration as well as modulation of basophil mediator release and may provide some insight into the basophil accumulation observed in late-phase allergic responses. Clinical and Experimental Allergy. Vol. 22. pp. 1020-1025. Submitted ! October IQ*?!: revised 30 March 1992; accepted 5 May 1992. Introduction Basophils as well as mast cells play an important role in type I allergy [1,2]. Moreover, basophils infiltrate the skin in delayed type reactions termed cutaneous basophil hypersensitivity (CBH) [3,4], and it is also well known that there is an increase in the number of basophils at sites of late-phase allergic responses such as skin and airways [5-7]. In our previous studies [8,9], it has been suggested that circulating basophils increase in number during the pre-attack stage of bronchial asthma and decrease during attacks with the appearance of basophils in the sputum. However., the mechanisms o( basophii migration to the extravascular sites of allergic responses are not well understood. Recently, it has been reported that several cytokines such as interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) modulate the functions of mature basophils (e.g. enhancement of histamine release upon stimulation with anti-IgF or FMLP) [10] as well as basophil growth and differentiation [II]. We hypothesized that these cytokines, especially derived Correspondence: Dr Y. Tanimoto. Second Department of Internal Medicine. Okayama University Medical School. 2-5-1 Shlkalii-cho. Okayama 700, Japan.
1020
from lymphocytes activated by allergens [12], might also induce basophil migration observed in late-phase allergic responses, and now demonstrate chemotactic effects of several recombinant cytokines on human peripheral blood basophils in vitro. Materials and methods Reagents Hanks" balanced salt solution (HBSS), bovine serum albumin (BSA) and recombinant human complement C5a were purchased from Sigma Chemical Co. {St Louis, Missouri, U.S.A.); Percoll from Pharmacia (Uppsala. Sweden); leukotriene B4 (LTB4) from Cayman Chemical Co. (Ann Arbor, Michigan, U.S.A.); platelet activating factor (PAF) from Bachem Feinchemikalien AG (Bubendorf. Switzerland). Recombinant human {rh) eytokines ami monoclonal antibodies to rh cytokines rhIL-3, rhIL-4, rhGM-CSF, mouse anti-human IL-3 monoclonal antibody and mouse anti-human GM-CSF monoclonal antibody were purchased from Gen/^yme (Boston, Massachussetts, U.S.A.); rhIL-8 was purchased
Effects of cyiokines on human basophil chemotaxis
from Peprotech Inc. (Rocky Hill. New Jersey, U.S.A.). Other recombinant human cytokines were kindly provided; rhIL-2, rhIFN-/ (Shionogi Pharmaceutical Co., Osaka, Japan). rhG-CSF (Kirin Brewery Co.. Tokyo, Japan), and rhTL-5 (The Suntory Institute for Biomedical Research, Osaka, Japan). Specific activities of rhIL-2. rhIL-3,rhIL-4and rhlFN-y were approximately 11 x 10^ U/mg, 10« U/mg, 10^ U/mg and 2 x 10' U/mg. Purificaiion of human basophils Highly purified basophils were used in all experiments. The detail of purification procedures was described previously [13]. Briefly, basophils were separated from peripheral blood by Percoll discontinuous gradients. followed by negative selection using flow cytometry (i.e. sortingCD2-CDM-CD16-CD19"^ cells from basophilrich mononuclear cell layers). Purified basophils were resuspended in HBSS containing 0 rvi, BSA before chemotactic assay, and basophil counts were performed by the method of Kimura et al. [8]. Thus basophils, of which viability exceeded 90%, were enriched to a purity of >80%. The contaminating cells of purified basoph\l suspensions were mostly lymphocytes, neither neutrophils nor eosinophils. Chemotactic assay Basophil chemotactic activity (BCA) was measured by modified Boyden chamber method. The lower chamber (Neuro Probe Inc., Cabin John, Maryland, U.S.A.). Lo which 50 /il of chemoattractant was added, was separated from the upper one by a 10 /nn ihick polycarbonate membrane filter with 5 ftm diameter pores (Nuclepore, Pleasanton, California, U.S.A.). A volume of 100 //I of basophil suspension (2 x lO'/ml) was added to the upper chamber. Chambers were incubated for 60 min at 37 C in a humidified atmosphere of 5'>n CO: in air. Then filters were removed and slatned by the method described previously [Sj. Migrated basophils were counted in 20 randomly selected fields at a magnification of x 400. Degranulation, even though it occurred during migration, did not alTect the ability to lind basophils. Measurements were made in duplicate chambers; results were expressed as mean + s.c.m of duplicate determinations from three separate experiments, and statistically analysed by means of Student's Mest.
1021
compared with recombinant human cytokines. Figure I showed that BCA of C5a was significantly high at 10^ " M (7±1 basophils 20 h.p.f. vs control values; l ± 0 basophils/20 h.p.f; /'
