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Migratory responses of eosinophil and neutrophil granulocytes from patients with asthma
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
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5% NHS
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A (10nmolll)
FIG. 1. Chemotactic responses of eosinophils from patients with asthma (A/ (N = 57) and references (R) (N = 36). The respective means and +I SD interval of the patient and reference groups are listed. The significant differences between the two groups as determined by means of Student’s t test are indicated.
challenge situation, the birch-pollen season,demonstrated an increased amount of eosinophils in BAL from birch pollen-allergic patients with asthmaduring season,comparedwith BAL obtained before season.’ One mechanismresponsible for the usually selective accumulation of eosinophils in the lungs of patients with asthmais believed to be the production of chemotacticfactors that attract the eosinophils from the blood stream. IncreasedECA has also been demonstratedin BAL during allergen challenge.‘, ’ However, chemotactic factors are probably not the only explanation for the accumulation of eosinophils. IncreasedNCA was also found in BAL during allergen challenge despitethe fact that no increaseof the number of neutrophils was found.’ An increasedresponsivenessto chemotactic factors of eosinophils compared with other cells may theoretically result in a selective accumulation of eosinophils to the lungs. We hypothesized that one reason for the selective accumulationof eosinophils in the lungs of individuals with asthma, in spite of the nonselective production of both eosinophil and neutrophil chemotacticfactors, was an increasedresponsivenessof the blood eosinophils; that is, the eosinophils were selectively primed. To test this hypothesis we have investigated the chemotacticand chemokinetic responsesof eosinophil and neutrophil granulocytes from patients with asthmaas well as the chemotactic and chemokinetic activities of their sera. Fifty-seven patients with an asthmatic diseaseof varying severity were included in the study.
MATERIAL Patients
AND METHODS
The patientgroupconsistedof 57 patientswith asthma (accordingto the definitionof the AmericanThoracicSociety of 1962),33 womenand24 men,with a meanageof 48 years(range,20 to 91 years).Nine patientsweresmokers, and of the 48 nonsmokers, 16 were former smokers. Thirteen of the patients were consideredatopic as a result of positive skin prick tests to more than one common allergen. PEFRsat the time of examination had a meanvalue of 87% (range, 19% to 130%) of expected. To evaluate further the severity of the disease,the amount of treatment needed to control symptoms was registered. The patients were divided into four groups: (1) using only inhaled &agonists, five patients; (2) using inhaled steroidsor oral pzagonistsin addition to inhaled P,-agonists, 16 patients; (3) using a combination of inhaled P,-agonists,inhaled steroids, and oral &-agonists or oral theophylline, 31 patients; and (4) using oral corticosteroids, five patients. It thus appears that the group of patients generally neededquite large treatments, but, according to the current PEFR value, had a well-controlled diseaseat the time of the study. The patients had had asthmafor 1 to 60 years (median, 10 years). References The reference group consisted of 36 apparently healthy blood donors with no allergic symptoms and had serum concentrationsof total IgE within the referenceinterval. PEFRs. PEFRswere measured with a Wright peak flow meter. The highest value of three attemptswas recorded. Blood samples. Blood samplesfrom patients and referenceswere drawn in the morning within 90 minutes. Samples from the patients and referenceswere tested simultaneously.
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Measurements of the migration of eosinophil and neutrophil granulocytes The migmtion of eosinophils and neutrophils was assayed with a modification of the Boyden chamber technique as previously describedin detail.g,‘OGranulocyteswere isolated from heparinized blood by dextran sedimentation.” The granulocyte mixture obtained by this procedurehad a purity of 78 2 9?&(SD) (references)and 80 2 9% (SD) (patients with asthma), respectively. The relative amount of eosinophils was, on average, 4% (range, 0% to 10%) and 9% (range, 1% to 29%), respectively, in the granulocyte populations obtained from referencesand patients with asthma. The final concentration of granulocytes was 1.5 X 109/L in all assays.The filter pore size was 5 km (Millipore Corp., Bedford, Mlass.). Incubation was performed for 1 hour at 37” C. The jilters were stainedwith Mayer’s Hlmalunlosung (Merck, Darmstadt, W. Germany) and Chromotrope 2R (Sigma Chemical Co., St. Louis, MO.) according to a procedure describedelsewhere, to enable the detection of both neutrophils and eosinophils.“’ Migration of neutrophils and eosinophils was measuredin the same filter according to the leading..fronttechnique.‘* A chemotacticresponseis defined as the increasedmigration causedby the attraction of the cells by a gradient of a chemotactic stimulus formed through the filter.“, I4 A chemokinetic responseis defined as the enh,ancementof random migration caused by the presenceof a chemokineticstimulus in the cell suspension.13, Is Random migration, cells in buffer migrating toward the same buffer, was 17 4 4 (SD) and 21 +- 3 (SD) pm/hr for eosinophils and neutrophils, respectively. The chemotacticresponsesof eosinophils and neutrophils were assayedwith four different stimuli in the following way: (1) granulocytes in Gey’s solution and 5% (vol/vol) of fresh-frozen pooled NHS or 5% pooled ACS below the filter (ACS was obtained from pooled serumof 10 patients with asthma25 minutes to 2 hours after allergen inhalation challenge.) and (2) granulocytes in Gey’s solution with 2 gm of albumin per liter and 2.5% (vol/vol) of ZASI” or 10 nmol of FMLP per liter below the filter. The intra-assay variation of neutrophils from referencesand patients with asthmawas 6.8% (CV) and 8.0% (CV), respectively, and of eosinophils, 15.4% (CV) and 14.1% (CV), respectively. The interassay (interday) variation of neutrophils and eosinophils from referenceswas 13.O%(CV) and 15.8%(CV), respectively. The chemokinetic responsesof eosinophils and neutrophils were assayedwith two different stimuli in the following way: (1) granulocytesin the presenceof 2 gm of albumin (AB Kabi. Stockholm, Sweden) per literlb and Gey’s solution and (2) granulocytesin the presenceof 5% (vol/vol) of pooled fresh-frozen NHS and Gey’s solution below the filter in both cases.The intra-assayvariation of neutrophils from referencesand patients with asthmawas 8.3% (CV) and 8.8% (CV), respectively, and of eosinophils, 13.2% (CV) and 14.9% (CV), respectively. The interassay(interday) variation of neutrophils and eosinophils from referenceswas 12.1% (CV) and 15.7% (CV), respectively.
migration
and asthma
I. Chemotactic and chemokinetic responses of neutrophils from patients asthma and references
745
TABLE
(pm/hr,
Variable
Response Chemotactic NHS (5%) ACS (5%) ZAS (2.5%) FMLP (10 nmol/L) Chemokinetic Albumin (2 gm/L) NHS (5%)
with
Migration mean f SD)
References (N = 36)
Patients with asthma (N = 57)
17 16 10 17
90 k 20 95 2 22 115 If- 11 103 I? 19
82 k 12 76 e 13
79 -+ 13 82 c 13
93 k 90 _’ 116 101 +
The ECA and NCA of serum were measuredas the migration of granulocytesfrom healthy blood donors in Gey’s solution toward 5% (vol/vol) of fresh-frozen patient serum below the filter. Pooled NHS served as reference, and the chemotactic activity was expressedin relation to the migration toward normal serum (lOO%).” The eosinophil and neutrophil chemokinetic activity of serum was assayedas the migration of granulocytes from healthy blood donors in the presenceof 5% (vol/vol) of fresh-frozen patient serum with Gey’s solution below the filter. PooledNHS servedasreference,asdescribed above.15 Blood eosinophil COUI?I. The number of eosinophil granulocytes in blood was assayedaccording to the method of Forshamet al.lB Serum concentration of ECP. The serum concentration of ECP was assayedby meansof a radioimmunoassay. Statistical evaluation. Student’s t test, Mann-Whitney test, andlinear regressionanalysiswere usedin the statistical evaluation of the results.
RESULTS Chemotactic and chemokinetic eosinophils and neutrophils
responses of
Eosinophils from the patients with asthma demonstrated significantly increased chemotactic re-
sponsesto ACS, ZAS, and FMLP comparedwith the referencegroup (Fig. 1). The chemokinetic responses of eosinophils were not altered compared with the responsesof eosinophils from the reference group. The average chemokinetic response to albumin (2 gml L) of eosinophils from patientsandreferenceswas 59 t 12 (SD) ~m/hr and 53 or 12 pm&r, respectively, and the average response to NHS (5%) was
73 + 12 (SD) and 67 + 10 (SD), respectively. Neu-
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p4001
lC0
B
4 R A neutrophil Serum-Chemotactic
R A eosinophil activity
R A neutrophil Serum-Chemokinetic
R A eosinophil activity.
FIG. 2. ECA and NCA and eosinophil and neutrophil chemokinetic activites of serum from patients with asthma and references. The respective means and ?l SD interval of the patient and reference groups are listed. The significant differences between the two groups as determined by Student’s t test are indicated. -
trophils from the patients with asthma and the references demonstratedsimilar chemotactic and chemokinetic responses(Table I). Eosinophil and neutrophil chemotactic and chemokinetic activities of serum Sera from the patients with asthma demonstrated significantly increasedECA and increasedeosinophil and neutrophil chemokinetic activity compared with serafrom the referencegroup (Fig. 2). The ECA and NCA of serumfrom referencesand from patients with asthma were significantly correlated to each other (r = 0.70 and p < 0.0003; r = 0.59 and p < 0.0001, respectively) and also the eosinophil and neutrophil chemokinetic activities (r = 0.82 and p < 0.0001; r = 0.87 and p < 0.0001, respectively). However, the respective chemotactic and chemokinetic activities were not related to each other (p > 0.05). Relationship between eosinophil and neutrophil migratory responses and lung function of the patients with asthma The eosinophil chemokinetic activity of serumdemonstrateda significant correlation to the lung function of the patients with asthmameasuredasrelative PEFR (Fig. 3). The neutrophil chemokinetic activity of serum was also correlated to relative PEFR of the patients (r = 0.273; p < 0.05). However, none of the chemotactic or chemokinetic responsesof the cells were related to the lung function of the patients (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 x 106/L, whereas the other half of the patientshad eosinophilia. Eosinophils from the patients with blood eosinophilia demonstrated a significantly higher chemotacticresponseto ZAS and chemokinetic responseto NHS than eosinophils from patients with a normal blood eosinophil count (Table II). Furthermore, eosinophils from patients with eosinophilia also demonstrated significantly increased chemotactic responsesto ACS and FMLP and chemokinetic responseto albumin compared with references. The corresponding responses of eosinophils from patients with a normal blood eosinophil count were not altered comparedwith that of the references. The chemokinetic responseto NHS by eosinophils from patients with asthma demonstrated a significant correlation to the serum concentration of ECP (Fig. 4).
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TABLE II. Chemotactic and chemokinetic responses of eosinophils from patients with asthma in relation to the blood eosinophil count Migration
Variable Blood eosinophil count (x 1O-B/L)
(pm/hr,
mean +
.
>400 (N = 29)
p
5
References (N = 57)
I2 OI
z 5 !Y
Response Chemotactic
NHS (5%) ACS(5%) ZAS (2.5%)
75 k 25
FML.P
70 2 15 76 -+ 20t
64 + 15
Albumin (2 gm/L)
56 e 12 62 + 12*
53 +- 12
NHS (5%)
67 k 11 78 + ll*t
67 ? 10
82 e 22*
747
7
.?
81 + 21 82 2 20
.
and asthma
SD)
Patients with asthma 400 (N = 25)
migration
75 + 19 65 r 18
60. LO-
. .
.
91 r 17 99 ? 13*t 86 + 19
.
.
(10 nmol/L) Chemokinetic
The statistical significancesof the differencesbetween the groups were calculated by Mann-Whitney test. *p
Relationship between the migratory responses of eosinophils and neutrophils and the medical treatment of the patients Neither the migratory responsesof eosinophils and neutrophils nor the ECA and NCA of serum demonstratedany differencesin relation to the medical treatment of the patients (p > 0.05). The only exception was demonstratedby the eosinophil chemokinetic activity of serum. Sera from group 4 demonstrateda significantly higher eosinophil chemokinetic activity than sera from groups 1 and 2 (p < 0.03, MannWhitney test). DISCUSSION The present investigation of granulocytes from patients with asthmahasrevealedan increaseof the chemotactic responsesof the eosinophils. Furthermore, the eosinophil chemokinetic responseswere increased in the group of patients who had blood eosinophilia (>400 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-
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200-
.
.
.
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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
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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:
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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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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
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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
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1
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i
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p
p
r g IOO-
It
.-s z 80.-&l I: 60-
-
pco.02
60
t
b
403
40
1
R
A
5% NHS
R
A
5% ACS
R
A
2.5% ZAS
R f-MLP
A (10nmolll)
FIG. 1. Chemotactic responses of eosinophils from patients with asthma (A/ (N = 57) and references (R) (N = 36). The respective means and +I SD interval of the patient and reference groups are listed. The significant differences between the two groups as determined by means of Student’s t test are indicated.
challenge situation, the birch-pollen season,demonstrated an increased amount of eosinophils in BAL from birch pollen-allergic patients with asthmaduring season,comparedwith BAL obtained before season.’ One mechanismresponsible for the usually selective accumulation of eosinophils in the lungs of patients with asthmais believed to be the production of chemotacticfactors that attract the eosinophils from the blood stream. IncreasedECA has also been demonstratedin BAL during allergen challenge.‘, ’ However, chemotactic factors are probably not the only explanation for the accumulation of eosinophils. IncreasedNCA was also found in BAL during allergen challenge despitethe fact that no increaseof the number of neutrophils was found.’ An increasedresponsivenessto chemotactic factors of eosinophils compared with other cells may theoretically result in a selective accumulation of eosinophils to the lungs. We hypothesized that one reason for the selective accumulationof eosinophils in the lungs of individuals with asthma, in spite of the nonselective production of both eosinophil and neutrophil chemotacticfactors, was an increasedresponsivenessof the blood eosinophils; that is, the eosinophils were selectively primed. To test this hypothesis we have investigated the chemotacticand chemokinetic responsesof eosinophil and neutrophil granulocytes from patients with asthmaas well as the chemotactic and chemokinetic activities of their sera. Fifty-seven patients with an asthmatic diseaseof varying severity were included in the study.
MATERIAL Patients
AND METHODS
The patientgroupconsistedof 57 patientswith asthma (accordingto the definitionof the AmericanThoracicSociety of 1962),33 womenand24 men,with a meanageof 48 years(range,20 to 91 years).Nine patientsweresmokers, and of the 48 nonsmokers, 16 were former smokers. Thirteen of the patients were consideredatopic as a result of positive skin prick tests to more than one common allergen. PEFRsat the time of examination had a meanvalue of 87% (range, 19% to 130%) of expected. To evaluate further the severity of the disease,the amount of treatment needed to control symptoms was registered. The patients were divided into four groups: (1) using only inhaled &agonists, five patients; (2) using inhaled steroidsor oral pzagonistsin addition to inhaled P,-agonists, 16 patients; (3) using a combination of inhaled P,-agonists,inhaled steroids, and oral &-agonists or oral theophylline, 31 patients; and (4) using oral corticosteroids, five patients. It thus appears that the group of patients generally neededquite large treatments, but, according to the current PEFR value, had a well-controlled diseaseat the time of the study. The patients had had asthmafor 1 to 60 years (median, 10 years). References The reference group consisted of 36 apparently healthy blood donors with no allergic symptoms and had serum concentrationsof total IgE within the referenceinterval. PEFRs. PEFRswere measured with a Wright peak flow meter. The highest value of three attemptswas recorded. Blood samples. Blood samplesfrom patients and referenceswere drawn in the morning within 90 minutes. Samples from the patients and referenceswere tested simultaneously.
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Measurements of the migration of eosinophil and neutrophil granulocytes The migmtion of eosinophils and neutrophils was assayed with a modification of the Boyden chamber technique as previously describedin detail.g,‘OGranulocyteswere isolated from heparinized blood by dextran sedimentation.” The granulocyte mixture obtained by this procedurehad a purity of 78 2 9?&(SD) (references)and 80 2 9% (SD) (patients with asthma), respectively. The relative amount of eosinophils was, on average, 4% (range, 0% to 10%) and 9% (range, 1% to 29%), respectively, in the granulocyte populations obtained from referencesand patients with asthma. The final concentration of granulocytes was 1.5 X 109/L in all assays.The filter pore size was 5 km (Millipore Corp., Bedford, Mlass.). Incubation was performed for 1 hour at 37” C. The jilters were stainedwith Mayer’s Hlmalunlosung (Merck, Darmstadt, W. Germany) and Chromotrope 2R (Sigma Chemical Co., St. Louis, MO.) according to a procedure describedelsewhere, to enable the detection of both neutrophils and eosinophils.“’ Migration of neutrophils and eosinophils was measuredin the same filter according to the leading..fronttechnique.‘* A chemotacticresponseis defined as the increasedmigration causedby the attraction of the cells by a gradient of a chemotactic stimulus formed through the filter.“, I4 A chemokinetic responseis defined as the enh,ancementof random migration caused by the presenceof a chemokineticstimulus in the cell suspension.13, Is Random migration, cells in buffer migrating toward the same buffer, was 17 4 4 (SD) and 21 +- 3 (SD) pm/hr for eosinophils and neutrophils, respectively. The chemotacticresponsesof eosinophils and neutrophils were assayedwith four different stimuli in the following way: (1) granulocytes in Gey’s solution and 5% (vol/vol) of fresh-frozen pooled NHS or 5% pooled ACS below the filter (ACS was obtained from pooled serumof 10 patients with asthma25 minutes to 2 hours after allergen inhalation challenge.) and (2) granulocytes in Gey’s solution with 2 gm of albumin per liter and 2.5% (vol/vol) of ZASI” or 10 nmol of FMLP per liter below the filter. The intra-assay variation of neutrophils from referencesand patients with asthmawas 6.8% (CV) and 8.0% (CV), respectively, and of eosinophils, 15.4% (CV) and 14.1% (CV), respectively. The interassay (interday) variation of neutrophils and eosinophils from referenceswas 13.O%(CV) and 15.8%(CV), respectively. The chemokinetic responsesof eosinophils and neutrophils were assayedwith two different stimuli in the following way: (1) granulocytesin the presenceof 2 gm of albumin (AB Kabi. Stockholm, Sweden) per literlb and Gey’s solution and (2) granulocytesin the presenceof 5% (vol/vol) of pooled fresh-frozen NHS and Gey’s solution below the filter in both cases.The intra-assayvariation of neutrophils from referencesand patients with asthmawas 8.3% (CV) and 8.8% (CV), respectively, and of eosinophils, 13.2% (CV) and 14.9% (CV), respectively. The interassay(interday) variation of neutrophils and eosinophils from referenceswas 12.1% (CV) and 15.7% (CV), respectively.
migration
and asthma
I. Chemotactic and chemokinetic responses of neutrophils from patients asthma and references
745
TABLE
(pm/hr,
Variable
Response Chemotactic NHS (5%) ACS (5%) ZAS (2.5%) FMLP (10 nmol/L) Chemokinetic Albumin (2 gm/L) NHS (5%)
with
Migration mean f SD)
References (N = 36)
Patients with asthma (N = 57)
17 16 10 17
90 k 20 95 2 22 115 If- 11 103 I? 19
82 k 12 76 e 13
79 -+ 13 82 c 13
93 k 90 _’ 116 101 +
The ECA and NCA of serum were measuredas the migration of granulocytesfrom healthy blood donors in Gey’s solution toward 5% (vol/vol) of fresh-frozen patient serum below the filter. Pooled NHS served as reference, and the chemotactic activity was expressedin relation to the migration toward normal serum (lOO%).” The eosinophil and neutrophil chemokinetic activity of serum was assayedas the migration of granulocytes from healthy blood donors in the presenceof 5% (vol/vol) of fresh-frozen patient serum with Gey’s solution below the filter. PooledNHS servedasreference,asdescribed above.15 Blood eosinophil COUI?I. The number of eosinophil granulocytes in blood was assayedaccording to the method of Forshamet al.lB Serum concentration of ECP. The serum concentration of ECP was assayedby meansof a radioimmunoassay. Statistical evaluation. Student’s t test, Mann-Whitney test, andlinear regressionanalysiswere usedin the statistical evaluation of the results.
RESULTS Chemotactic and chemokinetic eosinophils and neutrophils
responses of
Eosinophils from the patients with asthma demonstrated significantly increased chemotactic re-
sponsesto ACS, ZAS, and FMLP comparedwith the referencegroup (Fig. 1). The chemokinetic responses of eosinophils were not altered compared with the responsesof eosinophils from the reference group. The average chemokinetic response to albumin (2 gml L) of eosinophils from patientsandreferenceswas 59 t 12 (SD) ~m/hr and 53 or 12 pm&r, respectively, and the average response to NHS (5%) was
73 + 12 (SD) and 67 + 10 (SD), respectively. Neu-
746
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p4001
lC0
B
4 R A neutrophil Serum-Chemotactic
R A eosinophil activity
R A neutrophil Serum-Chemokinetic
R A eosinophil activity.
FIG. 2. ECA and NCA and eosinophil and neutrophil chemokinetic activites of serum from patients with asthma and references. The respective means and ?l SD interval of the patient and reference groups are listed. The significant differences between the two groups as determined by Student’s t test are indicated. -
trophils from the patients with asthma and the references demonstratedsimilar chemotactic and chemokinetic responses(Table I). Eosinophil and neutrophil chemotactic and chemokinetic activities of serum Sera from the patients with asthma demonstrated significantly increasedECA and increasedeosinophil and neutrophil chemokinetic activity compared with serafrom the referencegroup (Fig. 2). The ECA and NCA of serumfrom referencesand from patients with asthma were significantly correlated to each other (r = 0.70 and p < 0.0003; r = 0.59 and p < 0.0001, respectively) and also the eosinophil and neutrophil chemokinetic activities (r = 0.82 and p < 0.0001; r = 0.87 and p < 0.0001, respectively). However, the respective chemotactic and chemokinetic activities were not related to each other (p > 0.05). Relationship between eosinophil and neutrophil migratory responses and lung function of the patients with asthma The eosinophil chemokinetic activity of serumdemonstrateda significant correlation to the lung function of the patients with asthmameasuredasrelative PEFR (Fig. 3). The neutrophil chemokinetic activity of serum was also correlated to relative PEFR of the patients (r = 0.273; p < 0.05). However, none of the chemotactic or chemokinetic responsesof the cells were related to the lung function of the patients (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 x 106/L, whereas the other half of the patientshad eosinophilia. Eosinophils from the patients with blood eosinophilia demonstrated a significantly higher chemotacticresponseto ZAS and chemokinetic responseto NHS than eosinophils from patients with a normal blood eosinophil count (Table II). Furthermore, eosinophils from patients with eosinophilia also demonstrated significantly increased chemotactic responsesto ACS and FMLP and chemokinetic responseto albumin compared with references. The corresponding responses of eosinophils from patients with a normal blood eosinophil count were not altered comparedwith that of the references. The chemokinetic responseto NHS by eosinophils from patients with asthma demonstrated a significant correlation to the serum concentration of ECP (Fig. 4).
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TABLE II. Chemotactic and chemokinetic responses of eosinophils from patients with asthma in relation to the blood eosinophil count Migration
Variable Blood eosinophil count (x 1O-B/L)
(pm/hr,
mean +
.
>400 (N = 29)
p
5
References (N = 57)
I2 OI
z 5 !Y
Response Chemotactic
NHS (5%) ACS(5%) ZAS (2.5%)
75 k 25
FML.P
70 2 15 76 -+ 20t
64 + 15
Albumin (2 gm/L)
56 e 12 62 + 12*
53 +- 12
NHS (5%)
67 k 11 78 + ll*t
67 ? 10
82 e 22*
747
7
.?
81 + 21 82 2 20
.
and asthma
SD)
Patients with asthma 400 (N = 25)
migration
75 + 19 65 r 18
60. LO-
. .
.
91 r 17 99 ? 13*t 86 + 19
.
.
(10 nmol/L) Chemokinetic
The statistical significancesof the differencesbetween the groups were calculated by Mann-Whitney test. *p
Relationship between the migratory responses of eosinophils and neutrophils and the medical treatment of the patients Neither the migratory responsesof eosinophils and neutrophils nor the ECA and NCA of serum demonstratedany differencesin relation to the medical treatment of the patients (p > 0.05). The only exception was demonstratedby the eosinophil chemokinetic activity of serum. Sera from group 4 demonstrateda significantly higher eosinophil chemokinetic activity than sera from groups 1 and 2 (p < 0.03, MannWhitney test). DISCUSSION The present investigation of granulocytes from patients with asthmahasrevealedan increaseof the chemotactic responsesof the eosinophils. Furthermore, the eosinophil chemokinetic responseswere increased in the group of patients who had blood eosinophilia (>400 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
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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:
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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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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
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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