Migratory responses of eosinophil and neutrophil granulocytes from patients with asthma Lena Hbkansson, PhD,* Marie Carbon, WC,* Gunnemar StBlenheim, MD,** and Per Venge, MD* Uppsala, Sweden In the present study the migratory function of eosinophil and neutrophil granulocytes from patients with asthma were investigated. Fifty-seven patients with asthmatic disease of varying severity were included. Eosinophil and neutrophil chemotactic responses to 5% pooled normal human serum (NHS), 5% allergen-challenge serum, 2.5% zymosan-activated serum, N-formyl-methionyl-leucyl-phenylalanine (IO nmollL), chemokinetic responses to albumin (2 gmlL) and 5% NHS, and the eosinophil and neutrophil chemotactic and chemokinetic activities of serum were investigated. Eosinophils from patients with asthma demonstrated signtjicantly (p < 0.02) increased chemotactic responses to allergen-challenge serum, zymosan-activated serum, and N-formyl-methionyl-leucyl-phenylalanine, compared with eosinophils from references. The chemokinetic responses to albumin and NHS were increased (p < 0.01) by eosinophils from the patients who had blood eosinophilia (>400 x 106/L). Sera from the patients with asthma demonstrated raised eosinophil chemotactic activity (p < 0.001) and raised eosinophil and neutrophil chemokinetic activity (p < 0.001). The eosinophil chemokinetic activity of serum was correlated to the relative peak expiratory pow rate of the patients (r = -0.43; p < 0.02). The increased migratory responses were specific for the eosinophils, since the migratory responses of their neutrophils were not altered compared with that of the references. These results suggest that the eosinophils from the patients with asthma had been exposed to a priming mechanism in vivo. (J ALLERGY CLIN IMMUNOL 1990;85:743-50.)
Asthma is consideredtoday to be an inflammatory disease.The chronic asthmatic diseasecharacterized by a bronchial hyperreactivity is assumedto be the result of an ongoing inflammatory process.” ’ The mechanismsresponsible for the inflammatory events are not entirely understood, but recent investigations point to a crucial role for the eosinophil granulocyte. Bosinophils have the capacity to induce inflammatory reactionsby secretionof cytotoxic proteins from their granules and production of oxygen radicals and to produce mediators, such as platelet-activating factor
From the *Laboratory for Inflammation Research,Department of Clinical Chemistry, and **Department of Lung Medicine, University Hospital, Uppsala, Sweden. Supportedby grants from the Swedish Medical ResearchCouncil, the MedicaI Faculty of Uppsala University, the SwedishNational Environment Protection Board, the SwedishAssociation against Heart and Lung Diseases,and Bror Hjerpstedts Stiftelse. Received for publication May 8, 1989. Revised Nov. 6, 1989. Accepted for publication Nov. 9, 1989. Reprint requests: Lena Hknsson, PhD, Laboratory for Inflsmmation Research,Department of Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden. l/1/18138
Abbreviations used ACS: Allergen-challenged serum BAL: Bronchoalveolar lavage ECP: Eosinophilcationicprotein PULP: N-formyl-methionyl-leucylphenylalanine HL-ECA: Heat-labile eosinophil chemotactic activity HL-NCA: Heat-labile neutrophil chemotactic activity NHS: Normal human serum PEFR: Peak expiratory flow rate ZAS: Zymosan-activated serum CV: Coefficient of variation
and leukotriene C, that causesmooth muscle contraction3 Asthma is often, but not always, associatedwith blood eosinophilia, and the severity of the asthmatic diseasehasbeendemonstratedto correlateto the blood eosinophil count.4.5 The developmentof a late asthmatic reaction after allergen challengeof patients with asthma has been demonstratedto be associatedwith an influx of eosinophils into the lungs, as demonstrated by BAL.6 Furthermore, studies of a natural 743
744
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H%kansson et al.
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1
-
i
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p 0.05). Migratory responses of eosinophils and neutrophils from atopic and nonatopic patients The presentgroup of patientswith asthmaconsisted of 44 nonatopic and 13 atopic patients. The eosinophil
and neutrophil chemotactic and chemokinetic responses,as well as chemotactic activities of serum, did not in general demonstrate any differences between the two groups (p > 0.05). The only exception wasthe significantly (p < 0.006, Mann-Whitney test) decreasedchemokinetic responseto albumin of neutrophils from atopic patients compared with that of nonatopicpatients. The eosinophil and neutrophil chemokinetic activities were significantly (p < 0.03, Mann-Whitney test) higher in sera from nonatopic patients than in sera from atopic patients. However, the mean relative PEFR of the atopic patients tended to be higher than the relative PEFR of the nonatopic subjects, 94% comparedwith 86%. Eosinophil chemotactic and chemokinetic responses of patients with asthma in relation to blood eosinophil count and serum concentration of ECP About half the patients had a normal blood eosinophil count, that is, 400 (N = 29)
p400 x 106/L). The raised migratory responsesof the eosinophils were not related to the severity of the asthmatic diseaseof the patients, when estimated as PEFRsand were specific for the eosinophils, since the
I 100 Serum-Eosinophil
I 120
I 110
chemokinetic
I 160 activity
I (%)
FIG. 3. Correlation between eosinophil chemokinetic activity of serum and relative PEF of patients with asthma (n = 49). The coefficient of correlation calculated by linear regression analysis is presented.
responsesof the neutrophils werenot alteredcompared with neutrophils of the references.Although no direct correlations between the eosinophil chemotactic and chemokinetic responsesand blood eosinophil count were found, the chemotacticresponsesto ZAS and the chemokinetic response to NHS were significantly higher in the group of patients with eosinophilia compared with patients with a normal blood eosinophil count. Furthermore, patients with eosinophilia demonstrated raised chemokinetic responses compared with references, whereas chemokinetic responsesof the patientswith a normal blood eosinophil count were not different from responsesof the references.These findings suggest a parallel stimulation of eosinopoesis and the migratory responsivenessof eosinophils. The resultsare in accordancewith anotherstudy of patients with severe labile asthma and blood eosinophilia, who were subjectedto 5 weeks of treatmentintended to stabilize their clinical condition, and that demonstrated increased chemotactic and chemokinetic responsesof their eosinophils both at the beginning and end of the treatmentperiod.*’ A previous investigation has demonstratedan increased fraction of light-density eosinophils in the blood of patient with asthma.*’ Light-density eosinophils have been found in the blood of patients with eosinophilia of different causesand have been suggested to represent activated eosinophils.**-*’ How-
748
HBkansson
et al.
J. ALLERGY
200-
.
.
.
CLIN. IMMUNOL. APRIL 1990
r=a509 pco.02
. z
loo.
Yi?
Eosinophil
chemokinetic
response
(pm/h)
FIG. 4. Correlation between chemokinetic response to 5% NHS of eosinophils from patients with asthma (n = 24) and the serum concentration of ECP. The coefficient of correlation as calculated by linear regression analysis is illustrated in the figure.
ever, all investigations comparing the activity of lightdensity eosinophils with that of eosinophils with normal density are not in accordancewith this suggestion. A reducedchemotacticresponseto leukotriene B, and reduced PMA-induced chemiluminescenceof lightdensity eosinophils compared with normodense eosinophils have also been reported.26,*’ Furthermore, a study on normal volunteers who developed eosinophilia after hookworm infection demonstratedan increased chemotactic response to Escherichiu coli endotoxin-activated serum as well as an increasedsuperoxide production by their normodense eosinophils.28 Besidesthe increasedmigratory activity of the eosinophils, raised ECA and eosinophil and neutrophil chemokinetic activities were demonstratedin serum from the patients with asthma. The method used in the presentinvestigation to assaychemotacticactivity of serumis designedto measurethe HL-NCA and HLECA. I43“129,3oHL-NCA and HL-ECA have previously been described in serum from patients with asthma after allergen challenge, as well as in serum of birch pollen-allergic patients during birch-pollen season.“, 3’ The finding of ECA in serum of the patients in the present investigation probably reflects the continuous challenge that these patients are exposedto. It is, however,noteworthy that the increasedECA was found in sera from both the atopic and nonatopic patients. HL-NCA has previously been demonstratedto be related to the developmentof late asthmaticreaction after allergen challenge.30Furthermore, in the investigation of birch pollen-allergic patients, the ECA in BAL was demonstratedto correlate with HL-ECA in serum,’ which indicatesthat HL-ECA in serumreflects the production of chemoattractantsin the lungs.
Based on the finding of the increased eosinophil and neutrophil chemokinetic activity of serum and its negative correlation to relative PEFR of the patients, it could be speculatedthat there is a mechanismthat activates the eosinophils, which thereby acquire an increased responsiveness to chemoattractants produced in the lungs. Accumulation of activated eosinophils in the lungs might maintain an inflammatory process,which eventually results in a decreaseof the PEFR. An alternative hypothesis would be that the increasedchemokinetic activity in serumis formed as a consequenceof the inflammation in the lungs. The highly significant correlation between the eosinophil and neutrophil chemokinetic activities of serum suggeststhat the chemokinetic responseof the two types of cells is governed by essentially the same serum components.However, the lack of relation to the chemotactic activity of serum indicates that the chemokinetic and chemotacticcomponentsin serum are different from each other. The fact that the chemokinetic responseto NHS of the eosinophils from patients with asthmawas related to the serum concentration of ECP may suggestthat the increased migratory responsesof eosinophils is also related to the secretory activity of the cells. The current finding is supported by the demonstration of a correlation betweenthe eosinophil chemokinetic response to autologous serum and serum ECP in the abovementionedinvestigation of patients with severe labile asthma.” Furthermore, a recent study of the secretory function of eosinophils demonstrated a raised secretion of ECP by cells from patients with asthmacomparedwith references.32 In conclusion, the present investigation of granulocytes from patients with asthma has demonstrated
VOLUME NUMBER
85 4
increased eosinophil migratory responses, partly related to an increased amount of eosinophils in the blood and raised activity of migratory stimulating substances in serum. These results lead to the hypothesis that asthma is characterized by a mechanism that primes the eosinophils to an increased responsiveness to migratory stimuli in vitro and in vivo, induces the production of serum components that stimulate migration, and, in addition, affects the eosinopoesis, resulting in blood eosinophilia. Which components that might be responsible for such a mechanism is a matter of speculation, but in vitro studies of interleukin-5 and granulocyte macrophage colonystimulating factor have demonstrated that they are able to stimulate both eosinopoesis and the activity of mature eosinophils.33-37 We appreciatethe skillful technical assistanceof Kerstin Lindblad and Lena Moberg. REFERENCES I. Laitinen LA, Heino M, Laitinen A, Kava T, Haahtela T. Damage of the airways epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 198.5;131:599. 2. Cockcroft DW, Ruffin RE, Dolovitch J, Hargreave FE. Allergen-induced increase in nonallergic bronchial reactivity. Clin Allergy 1977;7:503. 3. Spry CJF. Eosinophils: a comprehensive review and guide to the scientific and medical literature. Oxford: Oxford University Press, 1988. 4. Horn BR, Robin ED, Theodore J, van Kessel A. Total eosinophil counts in the management of bronchial asthma. N Engl 3 Med ‘1975;292:1152. 5. Taylor KJ, Luksza AR. Peripheral blood eosinophil counts and bronchial responsiveness. Thorax 1987;42:35. 6. de Morchy JGR, Kauffman HF, Venge P, et al. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reaction Am Rev Respir Dis 1985;131:373. 7. Rak S, Bjomson A, H&ansson L, Sorenson S, Venge P. Immunotherapy prevents eosinophil accumulation and production of eosinophil chemotactic activity in the lung of asthmatics during iallergen exposure [Dissertation]. In: Rak S, ed. Bronchial h:y-perresponsiveness and cellular and humoral factors involved in allergic inflammation during pollen season. Gothenburg, Sweden: Medical Faculty, University of Gothenburg, 1988:161. 8. de Monchy JGR, Postma DS, Kauffman HF. Venge P, Weeke B, Vries K. Pretreatment with inhaled corticosteroids reduces eosinophil chemotaxis of BAL fluid following house dust mite challenge. Montreux, Switzerland: Proceedings of the International Congress of Allergology and Clinical Immunology, 1988:249. 9. Venge P, Arthursson G. Locomotion of neutrophil granulocytes from patients with thermal injury: identification of serumderived inhibitors. Burns 1981;8:6. 10. H&ansson L, Westerlund D, Venge P. New method for the measurement of eosinophil migration. J Leukocyte Biol 1987;42:689. 11. H&ansson L, Venge P. The influence of serum on random migratil,n and chemomxis of polymorphonuclear leukocytes:
Eosinophil
migration
and asthma
749
methodological evaluation using sera from infection-prone patients and normals. Stand J Immunol 1980; 11:27 1. 12. Zigmond SH, Hirsch JG. Leukocyte locomotion and chemotaxis: new methods for evaluation, and demonstration of cellderived chemotactic factors. J Exp Med 1973;137:387. 13. Keller HU, Wilkinson PC, Abercombie M, et al. A proposal for definition of terms related to locomotion of leukocytes and other cells. Clin Exp Immunol 1977;27:377. 14. Hakansson L, Venge P. The chemotactic activity of normal and complement-activated serum as measured by the leadingfront method using a Boyden chamber. Stand J Immunol 1984;19:63. 15. H&ansson L, Venge P. Partial characterization and identification of chemokinetic factors in serum. Stand J Immunol 1983;18:531. 16. H&ansson L, Venge P. The chemotactic response of granulocytes to the low molecular weight chemoattractants f-MLP, CSf, and LTB,, is dependent on chemokinetic factors. J Leukocyte Biol 1985;38:521. 17. H&kansson L, Rak S, Dahl R, Venge P. The formation of eosinophil and neutrophil chemtoactic activity during a pollen season and after allergen challenge. J ALLERGYCLIN IMMUNOL 1989;83:933. 18. Forsham PH, Thorn GW, Prunty FTG, Hills AG. Clinical studies with pituitary adrenocorticotropin. J Endocrinol 1948; 8:15. 19. Venge P, Roxin L-E, Olsson I. Radioimmunoassay of human eosinophil cationic protein. Br J Haematol 1977;37:331. 20. Griffin E, H&+nsson L, Formgren H, Jorgensen K, Venge P. Increased chemokinetic and chemotactic responses of eosinophils in asthmatic patients [submitted]. 21. Fukuda T, Dunnette SL, Reed CE, Ackerman SJ, Peters MS, Gleich GJ. Increased numbers of hypodense eosinophils in the blood of patients with bronchial asthma. Am Rev Respir Dis 1985;132:981. 22. Bass DA, Grover WH, Lewis JC, Szejda P, Dechatelet LR, McCall CE. Comparison of human eosinophils from normals and patients with eosinophilia. J Clin Invest 1980;66: 1265. 23. Winquist I, Olofsson T, Olsson I, Persson AM, Hallberg T. Altered density, metabolism, and surface receptors of eosinophils in eosinophilia. Immunology 1982;47:531. 24. De Simone C, Doneli G, Meli D, Rosati F, Sorice F. Human eosinophils and parasitic diseases. II. Characterization of two cell fractions isolated at different densities. Clin Exp Immunol 1982;48:249. 25. Prin L, Capron M, Tonne1 AB, Bletry 0, Capron A. Heterogeneity of human peripheral blood eosinophils: variability in cell density and cytotoxic ability in relation to the level and origin of hypereosinophilia. Int Arch Allergy Appl Immunol 1983;72:336. 26. Gosset P, Prin L, Capron M, Auriault C, Tonne1 AB, Capron A. Presence of factors chemotactic for granulocytes in hypcreosinophilic syndrome sera: relation with alterations in eosinophil migration. Clin Exp Immunol 1986;65:654. 27. Prin L, Charon J, Gosset P, Taelman H, Tonne1 AB, Capron A. Heterogeneity of human eosinophils. II. Variability of respiratory burst activity related to cell density. Clin Exp Immunol 1984;57:735. 28. White CJ, Maxwell CJ, Gallin JI. Changes in the structural and functional properties of human eosinophilia during experimental hookworm infection. J Infect Dis 1986;154:778. 29. Venge P, Dal-11R, H&ansson L, Peterson C. Generation of heat-labile chemotactic activity in blood after allergen challenge and its relationship to neutrophil and monocyte/macrophage turnover and activity. Allergy 1982;37:55.
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et al.
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30. Venge P, Dahl R, H&ansson L. Heat-labile neutrophil chemotactic activity in subjects with asthma after allergen inhalation: relation to the late asthmatic reaction and effects of asthma medication. J ALLERGY CLIN IMMUNOL 1987;80:679. 3 I. Rak S, H&msson L, Venge P. Immunotherapy abrogates the generation of eosinophil and neutrophil chemotactic activity during pollen season [submitted]. 32. Carlson M, H&ansson L, St&lenheim G, Venge P. Increased degranulation of eosinophils and neutrophils from asthmatic patients. Eur J Clin Invest 1989;19:A59. 33. Campbell HD, Tucker WQ, Hort Y, et al. Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinphil differentiation factor (interleukin-5). Proc Nat1 Acad Sci USA 1987;84:6629. 34. Lopez AF, Sanderson CJ, Gamble JR, Campbell HD, Young
Relationship theophylline
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IG, Vadas MA. Recombinant human interleukin-5 is a selective activator of human eosinophil function. J Exp Med 1988; 167:219. 35. Metcalf D, Begley CG, Johnson GR, et al. Biologic properties in vitro of recombinant human granulocyte-macrophage colony-stimulating factor. Blood 1986;67:37. 36. Tomonoga M, Golde DW, Gasson JC. Biosynthetic (recombinant) human granulocyte-macrophage colony-stimulating factor: effect on normal bone marrow and leukemia cell lines. Blood 1986;67:31. 37. Silberstein DS, Owen WF, Gasson JC, et al. Enhancement of human eosinophil cytotoxicity and leukotriene synthesis by biosynthetic (recombinant) granulocyte-macrophage colonystimulating factor. J Immunol 1986;137:3290.
of different serum levels of on methacholine sensitivity
David G. Tinkelman, MD, Bikramjit S. Garcha, PhD, and Cheryl N. Lutz, MT Atlanta, Ga. With an increased awareness of the importance of bronchial hyperreactivity on the clinical state of asthma, efforts at establishing effective treatment for asthma have been aimed at trying to reduce the level of bronchial hyperreactivity. This study investigated whether or not theophylline at different serum concentrations had any effect on bronchial hyperreactivity, as measured by methacholine challenges. Twelve adult subjects with mild asthma participated. All subjects wre atopic and were nonsmokers. Subjects were studied on three separate occasions according to a double-blind, randomized, crossover study design with one of the following medications: two placebo capsules in the morning and evening, a placebo and 250 mg of sustained-release theophylline in the morning and evening, or two 250 mg sustained-release theophylline capsules each morning and evening. Subjects were administered their medication between 5 and 15 days before performing methacholine challenges in the morning at the presumed trough level, and in the afternoon at the presumed peak serum theophylline level. Despite statistically significant difSerences in the pulmonary function and theophylline levels between the two active dosages (p < 0.05 for both), there was no difSerence in methucholine sensivity at the time of ingestion of the two dosage forms. There was also no correlation between a particular theophylline level and degree of methacholine sensitivity. It was concluded that, although theophylline has sign$cant bronchodilator capability, it has little, if any, effect on bronchial hyperreactivity, as measured by methacholine challanges. Thus, other forms of long-term therapy should be used in reducing bronchial hyperreactivity. (J ALLERGY CLINIMMUNOL1990;85:750-5.)
From the Atlanta Allergy and Immunology Research Foundation, Atlanta, Ga. Supported by a grant from Riker Laboratories Inc., St. Paul, Minn. Received for publication March 29, 1989. Revised Nov. 6, 1989. Accepted for publication Nov. 9, 1989. Reprint requests: David G. Tinkelman, MD, Atlanta Allergy Clinic, 6667 Vernon Woods Drive, Atlanta, GA 30328. l/1/18139
Abbreviatons used
BHR: SRT: CBU: ANOVA:
Bronchial hyperreactivity Sustained-releasetheophylline Cumulative breath unit Analysis of variance
Asthma is consideredtoday to be an inflammatory disease.The chronic asthmatic diseasecharacterized by a bronchial hyperreactivity is assumedto be the result of an ongoing inflammatory process.” ’ The mechanismsresponsible for the inflammatory events are not entirely understood, but recent investigations point to a crucial role for the eosinophil granulocyte. Bosinophils have the capacity to induce inflammatory reactionsby secretionof cytotoxic proteins from their granules and production of oxygen radicals and to produce mediators, such as platelet-activating factor
From the *Laboratory for Inflammation Research,Department of Clinical Chemistry, and **Department of Lung Medicine, University Hospital, Uppsala, Sweden. Supportedby grants from the Swedish Medical ResearchCouncil, the MedicaI Faculty of Uppsala University, the SwedishNational Environment Protection Board, the SwedishAssociation against Heart and Lung Diseases,and Bror Hjerpstedts Stiftelse. Received for publication May 8, 1989. Revised Nov. 6, 1989. Accepted for publication Nov. 9, 1989. Reprint requests: Lena Hknsson, PhD, Laboratory for Inflsmmation Research,Department of Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden. l/1/18138
Abbreviations used ACS: Allergen-challenged serum BAL: Bronchoalveolar lavage ECP: Eosinophilcationicprotein PULP: N-formyl-methionyl-leucylphenylalanine HL-ECA: Heat-labile eosinophil chemotactic activity HL-NCA: Heat-labile neutrophil chemotactic activity NHS: Normal human serum PEFR: Peak expiratory flow rate ZAS: Zymosan-activated serum CV: Coefficient of variation
and leukotriene C, that causesmooth muscle contraction3 Asthma is often, but not always, associatedwith blood eosinophilia, and the severity of the asthmatic diseasehasbeendemonstratedto correlateto the blood eosinophil count.4.5 The developmentof a late asthmatic reaction after allergen challengeof patients with asthma has been demonstratedto be associatedwith an influx of eosinophils into the lungs, as demonstrated by BAL.6 Furthermore, studies of a natural 743
744
J. ALLERGY
H%kansson et al.
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1
-
i
1990
p 0.05). Migratory responses of eosinophils and neutrophils from atopic and nonatopic patients The presentgroup of patientswith asthmaconsisted of 44 nonatopic and 13 atopic patients. The eosinophil
and neutrophil chemotactic and chemokinetic responses,as well as chemotactic activities of serum, did not in general demonstrate any differences between the two groups (p > 0.05). The only exception wasthe significantly (p < 0.006, Mann-Whitney test) decreasedchemokinetic responseto albumin of neutrophils from atopic patients compared with that of nonatopicpatients. The eosinophil and neutrophil chemokinetic activities were significantly (p < 0.03, Mann-Whitney test) higher in sera from nonatopic patients than in sera from atopic patients. However, the mean relative PEFR of the atopic patients tended to be higher than the relative PEFR of the nonatopic subjects, 94% comparedwith 86%. Eosinophil chemotactic and chemokinetic responses of patients with asthma in relation to blood eosinophil count and serum concentration of ECP About half the patients had a normal blood eosinophil count, that is, 400 (N = 29)
p400 x 106/L). The raised migratory responsesof the eosinophils were not related to the severity of the asthmatic diseaseof the patients, when estimated as PEFRsand were specific for the eosinophils, since the
I 100 Serum-Eosinophil
I 120
I 110
chemokinetic
I 160 activity
I (%)
FIG. 3. Correlation between eosinophil chemokinetic activity of serum and relative PEF of patients with asthma (n = 49). The coefficient of correlation calculated by linear regression analysis is presented.
responsesof the neutrophils werenot alteredcompared with neutrophils of the references.Although no direct correlations between the eosinophil chemotactic and chemokinetic responsesand blood eosinophil count were found, the chemotacticresponsesto ZAS and the chemokinetic response to NHS were significantly higher in the group of patients with eosinophilia compared with patients with a normal blood eosinophil count. Furthermore, patients with eosinophilia demonstrated raised chemokinetic responses compared with references, whereas chemokinetic responsesof the patientswith a normal blood eosinophil count were not different from responsesof the references.These findings suggest a parallel stimulation of eosinopoesis and the migratory responsivenessof eosinophils. The resultsare in accordancewith anotherstudy of patients with severe labile asthma and blood eosinophilia, who were subjectedto 5 weeks of treatmentintended to stabilize their clinical condition, and that demonstrated increased chemotactic and chemokinetic responsesof their eosinophils both at the beginning and end of the treatmentperiod.*’ A previous investigation has demonstratedan increased fraction of light-density eosinophils in the blood of patient with asthma.*’ Light-density eosinophils have been found in the blood of patients with eosinophilia of different causesand have been suggested to represent activated eosinophils.**-*’ How-
748
HBkansson
et al.
J. ALLERGY
200-
.
.
.
CLIN. IMMUNOL. APRIL 1990
r=a509 pco.02
. z
loo.
Yi?
Eosinophil
chemokinetic
response
(pm/h)
FIG. 4. Correlation between chemokinetic response to 5% NHS of eosinophils from patients with asthma (n = 24) and the serum concentration of ECP. The coefficient of correlation as calculated by linear regression analysis is illustrated in the figure.
ever, all investigations comparing the activity of lightdensity eosinophils with that of eosinophils with normal density are not in accordancewith this suggestion. A reducedchemotacticresponseto leukotriene B, and reduced PMA-induced chemiluminescenceof lightdensity eosinophils compared with normodense eosinophils have also been reported.26,*’ Furthermore, a study on normal volunteers who developed eosinophilia after hookworm infection demonstratedan increased chemotactic response to Escherichiu coli endotoxin-activated serum as well as an increasedsuperoxide production by their normodense eosinophils.28 Besidesthe increasedmigratory activity of the eosinophils, raised ECA and eosinophil and neutrophil chemokinetic activities were demonstratedin serum from the patients with asthma. The method used in the presentinvestigation to assaychemotacticactivity of serumis designedto measurethe HL-NCA and HLECA. I43“129,3oHL-NCA and HL-ECA have previously been described in serum from patients with asthma after allergen challenge, as well as in serum of birch pollen-allergic patients during birch-pollen season.“, 3’ The finding of ECA in serum of the patients in the present investigation probably reflects the continuous challenge that these patients are exposedto. It is, however,noteworthy that the increasedECA was found in sera from both the atopic and nonatopic patients. HL-NCA has previously been demonstratedto be related to the developmentof late asthmaticreaction after allergen challenge.30Furthermore, in the investigation of birch pollen-allergic patients, the ECA in BAL was demonstratedto correlate with HL-ECA in serum,’ which indicatesthat HL-ECA in serumreflects the production of chemoattractantsin the lungs.
Based on the finding of the increased eosinophil and neutrophil chemokinetic activity of serum and its negative correlation to relative PEFR of the patients, it could be speculatedthat there is a mechanismthat activates the eosinophils, which thereby acquire an increased responsiveness to chemoattractants produced in the lungs. Accumulation of activated eosinophils in the lungs might maintain an inflammatory process,which eventually results in a decreaseof the PEFR. An alternative hypothesis would be that the increasedchemokinetic activity in serumis formed as a consequenceof the inflammation in the lungs. The highly significant correlation between the eosinophil and neutrophil chemokinetic activities of serum suggeststhat the chemokinetic responseof the two types of cells is governed by essentially the same serum components.However, the lack of relation to the chemotactic activity of serum indicates that the chemokinetic and chemotacticcomponentsin serum are different from each other. The fact that the chemokinetic responseto NHS of the eosinophils from patients with asthmawas related to the serum concentration of ECP may suggestthat the increased migratory responsesof eosinophils is also related to the secretory activity of the cells. The current finding is supported by the demonstration of a correlation betweenthe eosinophil chemokinetic response to autologous serum and serum ECP in the abovementionedinvestigation of patients with severe labile asthma.” Furthermore, a recent study of the secretory function of eosinophils demonstrated a raised secretion of ECP by cells from patients with asthmacomparedwith references.32 In conclusion, the present investigation of granulocytes from patients with asthma has demonstrated
VOLUME NUMBER
85 4
increased eosinophil migratory responses, partly related to an increased amount of eosinophils in the blood and raised activity of migratory stimulating substances in serum. These results lead to the hypothesis that asthma is characterized by a mechanism that primes the eosinophils to an increased responsiveness to migratory stimuli in vitro and in vivo, induces the production of serum components that stimulate migration, and, in addition, affects the eosinopoesis, resulting in blood eosinophilia. Which components that might be responsible for such a mechanism is a matter of speculation, but in vitro studies of interleukin-5 and granulocyte macrophage colonystimulating factor have demonstrated that they are able to stimulate both eosinopoesis and the activity of mature eosinophils.33-37 We appreciatethe skillful technical assistanceof Kerstin Lindblad and Lena Moberg. REFERENCES I. Laitinen LA, Heino M, Laitinen A, Kava T, Haahtela T. Damage of the airways epithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 198.5;131:599. 2. Cockcroft DW, Ruffin RE, Dolovitch J, Hargreave FE. Allergen-induced increase in nonallergic bronchial reactivity. Clin Allergy 1977;7:503. 3. Spry CJF. Eosinophils: a comprehensive review and guide to the scientific and medical literature. Oxford: Oxford University Press, 1988. 4. Horn BR, Robin ED, Theodore J, van Kessel A. Total eosinophil counts in the management of bronchial asthma. N Engl 3 Med ‘1975;292:1152. 5. Taylor KJ, Luksza AR. Peripheral blood eosinophil counts and bronchial responsiveness. Thorax 1987;42:35. 6. de Morchy JGR, Kauffman HF, Venge P, et al. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reaction Am Rev Respir Dis 1985;131:373. 7. Rak S, Bjomson A, H&ansson L, Sorenson S, Venge P. Immunotherapy prevents eosinophil accumulation and production of eosinophil chemotactic activity in the lung of asthmatics during iallergen exposure [Dissertation]. In: Rak S, ed. Bronchial h:y-perresponsiveness and cellular and humoral factors involved in allergic inflammation during pollen season. Gothenburg, Sweden: Medical Faculty, University of Gothenburg, 1988:161. 8. de Monchy JGR, Postma DS, Kauffman HF. Venge P, Weeke B, Vries K. Pretreatment with inhaled corticosteroids reduces eosinophil chemotaxis of BAL fluid following house dust mite challenge. Montreux, Switzerland: Proceedings of the International Congress of Allergology and Clinical Immunology, 1988:249. 9. Venge P, Arthursson G. Locomotion of neutrophil granulocytes from patients with thermal injury: identification of serumderived inhibitors. Burns 1981;8:6. 10. H&ansson L, Westerlund D, Venge P. New method for the measurement of eosinophil migration. J Leukocyte Biol 1987;42:689. 11. H&ansson L, Venge P. The influence of serum on random migratil,n and chemomxis of polymorphonuclear leukocytes:
Eosinophil
migration
and asthma
749
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of different serum levels of on methacholine sensitivity
David G. Tinkelman, MD, Bikramjit S. Garcha, PhD, and Cheryl N. Lutz, MT Atlanta, Ga. With an increased awareness of the importance of bronchial hyperreactivity on the clinical state of asthma, efforts at establishing effective treatment for asthma have been aimed at trying to reduce the level of bronchial hyperreactivity. This study investigated whether or not theophylline at different serum concentrations had any effect on bronchial hyperreactivity, as measured by methacholine challenges. Twelve adult subjects with mild asthma participated. All subjects wre atopic and were nonsmokers. Subjects were studied on three separate occasions according to a double-blind, randomized, crossover study design with one of the following medications: two placebo capsules in the morning and evening, a placebo and 250 mg of sustained-release theophylline in the morning and evening, or two 250 mg sustained-release theophylline capsules each morning and evening. Subjects were administered their medication between 5 and 15 days before performing methacholine challenges in the morning at the presumed trough level, and in the afternoon at the presumed peak serum theophylline level. Despite statistically significant difSerences in the pulmonary function and theophylline levels between the two active dosages (p < 0.05 for both), there was no difSerence in methucholine sensivity at the time of ingestion of the two dosage forms. There was also no correlation between a particular theophylline level and degree of methacholine sensitivity. It was concluded that, although theophylline has sign$cant bronchodilator capability, it has little, if any, effect on bronchial hyperreactivity, as measured by methacholine challanges. Thus, other forms of long-term therapy should be used in reducing bronchial hyperreactivity. (J ALLERGY CLINIMMUNOL1990;85:750-5.)
From the Atlanta Allergy and Immunology Research Foundation, Atlanta, Ga. Supported by a grant from Riker Laboratories Inc., St. Paul, Minn. Received for publication March 29, 1989. Revised Nov. 6, 1989. Accepted for publication Nov. 9, 1989. Reprint requests: David G. Tinkelman, MD, Atlanta Allergy Clinic, 6667 Vernon Woods Drive, Atlanta, GA 30328. l/1/18139
Abbreviatons used
BHR: SRT: CBU: ANOVA:
Bronchial hyperreactivity Sustained-releasetheophylline Cumulative breath unit Analysis of variance
