Inflammation, Vol. 22, No. 1, 1998

A STUDY OF GRANULOCYTERESPIRATORY BURST IN PATIENTS

WITH ALLERGIC BRONCHIAL ASTHMA

Abstract—The respiratory burst of phagocytes plays an important role in the tissuedamage that accompanies the inflammatory response. One of these conditions isallergic bronchial asthma, therefore, to evaluate the activation state of peripheralgranulocytes the generation of reactive oxygen metabolites was evaluated usingLuminol-enhanced chemiluminescence (LCL) and reduction of cytochrome C bysuperoxide. The resting granulocytes of the asthmatic patients under crisis showed ahigher LCL compared to the noncrisis patients and control subjects. The granulocytesstimulated with PMA presented a significant increase in the respiratory burst inboth groups of asthmatics. The granulocytes of noncrisis asthmatics challenged withOps-Zym and with fMLP + Ops-Zym showed a higher metabolic activity, whereas theasthmatics under crisis presented no difference between reactive oxygen generationand that of the control group. The quantitative analysis of superoxide generationby granulocytes of the same patients did not show differences among the groups.Our findings suggest that the granulocytes of crisis and noncrisis asthmatics seemto be in a hyperreactive state and with a higher metabolic response when comparedto the control group. However, the patients present a different behavior dependingon stimulus used to activate cells. This could indicate that in peripheral blood existdifferent granulocyte populations depending on the inflammatory response takingplace in the respiratory tract.

INTRODUCTION

The phagocytic cells, particularly neutrophils, are fundamental in the hostresponse against foreign agents. However, they are involved in the generation

LEONARDO VARGAS,1 PABLO J. PATINO,1FERNANDO MONTOYA,1 ANA CATALINA VANEGAS,2

ALEJANDRO ECHAVARRiA,2 andDIANA GARCIA DE OLARTE1

' Immunology Section, School of MedicineUniversity of Anlioquia, Medellin, Colombia

2Pneumology SectionHospital Universitario San Weenie de Paul

Medellin, Colombia

45

0360-3997/98/0200-0045$13.00/0 ©1998 Plenum Publishing Corporation

of tissue damage during the inflammatory response (1-3) due to the liberationof reactive oxygen intermediates (ROIs) and of multiple lysosomal enzymes inresponse to various chemical mediators (1). Allergic bronchial asthma, as otheratopic diseases, is characterized by different alterations in the immune systemresponse; such as disturbances in the immunoregulation of IgE (4), alteration inthe function of various inflammatory cells (mast cells, neutrophils, eosinophils,lymphocytes, macrophages) (5, 6) and an increase in the production of a widerange of inflammatory mediators (platelet activating factor, leukotrienes, his-tamine, cytokines) in response to diverse antigens (5-8). It has been demon-strated that when neutrophils of atopic patients are stimulated in vitro they showan increase in the respiratory burst activity (9). The activation of these cells inatopic patients has also been established by an increase in the expression ofcomplement receptors, synthesis of leukotriene 04, in the liberation of granularproteins, such as myeloperoxidase (MPO) and by a greater response to chemotactic stimuli (10-13). The oxygen radicals and other toxic metabolites generatedduring the neutrophils respiratory burst can produce damage in the respiratorytract tissues and can increase the symptoms of bronchial hyperreactivity (12, 13

On the other hand, atopic patients present an alteration in the regulationof IgE synthesis due to an imbalance among the subpopulations of helper Tcells with a prevalence of TH2 cells in response to the allergens. Such an imbal-ance causes a larger production of IL-4 by allergen specific T cells (14). Theuncontrolled action of this subpopulation of lymphocytes leads to an excessiveproduction of cytokines or other soluble mediators which can prime neutrophilsto respond in an exaggerated manner against different stimuli (15-18), givingrise to reported abnormalities in the physiology of these cells during allergicprocesses.

The above discussion reveals the importance of understanding the physiol-ogy of the phagocytes in atopic patients. To address these issues we studied theproduction of reactive oxygen metabolites by peripheral granulocytes isolated ofasthmatic patients under crisis or with no evidence of crisis.

MATERIALS AND METHODS

Study Population. We studied forty-two subjects of both sexes. Nine patients with acutebronchial asthma and 12 patients in an intercurrent period of the illness; these patients were between20 and 42 years of age, nonsmokers, and not undergoing systemic treatment with antihistamines,steroids, or theophylline compounds at least 48 h before the blood sample was taken. They alsopresented positive intradermal reaction to a mite extract Dermatophagoides pteronyssinus. A thirdgroup in the study consisted of 21 healthy subjects, nonsmokers, with normal pulmonary function,without respiratory infections, and without any type of allergic signs and negative response to thecutaneous test with D. pteronyssinus.

46 Vargas et al.

Collection and Preparation of Granulocytes. A modified version of the Yanai et al. techniquewas used (19). Twenty milliliters of heparinized venous blood (10 U of heparin/ml) were mixed with6 ml of 6% Dextran 70 (Pharmacia, Uppsala, Sweden) and left to sediment for one h. The supernatantwas placed on a Ficoll-hypaque (Histopaque, 1077, Sigma Chemical Co., St. Louis, Missouri) densitygradient, and centrifuged at 400 xg/25 min/4°C. Hypotonic lysis of red blood cells in the pellet wascarried out in deionized water, then granulocytes were centrifuged at 250 xg/10 min/4°C. The cellswere resuspended in a Hank's solution (Gibco-BRL Laboratories, New York), supplemented with0.1% gelatin (Difco Laboratories, Detroit, Michigan). Afterwards, the number, purity and viabilityof the granulocytes were determined with gentian violet and trypan blue; these last two parameterswere always greater than 95%.

Luminol-Enhanced Chemiluminescence. In order to measure oxygen intermediates metabo-lites and free oxygen radicals produced by granulocytes we used a modification of Luminol-enhancedChemiluminescence technique described by Dechatelet et al. (20). The reaction was carried out, bytriplicate, in scintillation counter vials, using PBS as solvent, with a total volume of 2 ml. Luminol(Sigma) at a final concentration of 2.2 x 10~6 M was used to increase the photonic reaction. Thephagocytes respiratory burst activating agents were Zymosan (Sigma) previously opsonized (Ops-Zym) with a mixture of normal human serum, PMA (12-0-tetradecanoyl-phorbol-13-acetate, Sigma)and fMLP (Sigma), at final concentrations of 1.66 mg/ml, 0.1 jig/ml and 10~5 M, respectively. Insome experiments fMLP was used as a priming agent for a later stimulus with Ops-Zym, The cells,2.5 x 104 cells/ml final concentration, were added to the vials immediately before taking the read-ings. The test was also carried out in the absence of stimuli, which corresponds to granulocytes ina resting state. The light production in each vial was measured every 10 min during one hour on aB-scintillation counter (Beckman LS 3801, Columbia, Maryland). From the readings of each trip-licate, expressed in counts per min (cpm), the median of triplicates were obtained and from eachtreatment the given value of the median of the nonspecific triplicate was subtracted in order to obtainthe net cpm.

Measurement of the Production of the Superoxide Anion by Means of the Reduction ofCytochrome C. The release of superoxide anion (O^) by granulocytes was measured by reduc-tion of cytochrome C and its specificity was determined by the addition of superoxide dismutase(SOD) (21). Briefly, reactions were prepared by duplicate in 12 x 75-mm polyestyrene tubes with afinal volume of 500 n\. To adjust the volume of each tube, PBS supplemented with 0.9 mM of CaCh,0.5 mM of MgCh and 7.5 mM of glucose pH 7.4 was used. Two resting tubes contained 0.213 mMof cytochrome C (cytochrome C type VI, Sigma) and two test tubes contained cytochrome C plus0.1 fig/ml of PMA. Each reaction was compared to an identical control mixture that contained, inaddition to the above, 0.2 mg/ml of SOD (Sigma) to determine the specificity of the test. The blankwas a mixture of supplemented PBS, cylochrome C and cells which were always kept at 4°C. 150 /tlof each of the reaction supernatants were placed into 96 well plates and absorbance was measured at550 nm in an ELISA minireader (Dynatech, Minireader II, Alexandria, Virginia). Superoxide aniongeneration was calculated using the extinction coefficient of reduced cytochrome C (2.1 x 104 M~'cm"1) and was expressed in nmols of reduced cytochrome C/l x 106 granulocytes/15 min.

Statistical Analysis. A one-way analysis of variance was used to compare the superoxideanion production, taking the treatment group (resting cells and PMA) as the dependent variable andthe study group (noncrisis asthmatics, asthmatics under crisis, and healthy controls) as the inde-pendent variable. A one-way variance analysis was carried out on the LCL data, taking time as thecontrol variable, type of treatment as the dependent variable (resting, PMA, Ops-Zym, and Ops-Zym+ fMLP), and the study groups as the independent variable. In all cases the Levine test was usedto determine homogeneity of variance. When the difference between the averages was statisticallysignificant with respect to the 5% critical level, the lowest square difference test (LSD) was appliedto decide which groups were different. The program used for the statistical analysis was Statisticaversion 1.0 for Windows.

Respiratory Burst of Granulocytes in Bronchial Asthma 47

48 Vargas et al.

RESULTS

The ROIs generation in resting cells to evaluate the activation state ofphagocytes in different moments of the asthmatic process was studied. In Figure1 we show that the nonstimulated granulocytes in asthmatic patients under crisispresented a statistically significant increase of the LCL (P - 0.003) with respectto the other two groups (noncrisis asthmatics and controls). The noncrisis asth-matic patients did not show significant changes during these time periods withrespect to the healthy controls.

The generation of ROIs by granulocytes of asthmatics under crisis or with-out crisis was analyzed after stimulating the cells by different agents. We usedPMA as a direct activator of respiratory burst, Zym-Ops to evaluate the phagocy-tosis mediated activation of the NADPH oxidase, and preincubation with fMLPbefore Zym-Ops activation to evaluate the priming state of phagocytes. The LCLresponse of the granulocytes in both groups of asthmatics stimulated with PMAshowed statistically significant differences due to an increase in the counts perminute in the noncrisis asthmatics at 10, 20, and 30 min and crisis asthmatic

Fig. 1. Luminol-enhanced chemiluminiscence (LCL) of resting granulocytes in asthmatic patientsunder crisis (O), noncrisis asthmatic (O), and healthy controls (D). The nonstimulated granulocytesin asthmatic patients under crisis showed a statistically significant increase in LCL (*/> = 0.003).See Materials and Methods for further experimental details.

Fig. 2. LCL of stimulated granulocytcs with PMA (0.1 ng/ml) in asthmatic patients under crisis(O), noncrisis asthmatic (O), and healthy controls (D). There was a significant difference at 10, 20,and 30 min poststimulation between noncrisis asthmatics and controls (*P = 0.0008, P = 0.009, andP = 0.03, respectively). At 20, 30, and 40 min there were significant differences between under crisisasthmatics and controls ("P = 0.002, P = 0.0002 and P = 0.0002, respectively).

patients at 20, 30, and 40 min (Figure 2). When the granulocytes were stimu-lated with Ops-Zym, a statistically significant increase was observed in the ROIsgeneration by noncrisis asthmatic granulocytes with respect to control group at60 min (P - 0.001) (Figure 3); however, a tendency to a greater response inthese patients was observed from minute 20 poststimulation. In the granulocytespreincubated with fMLP and then stimulated with Ops-Zym, the LCL showed astatistically significant increase in noncrisis patients only at 60 min (P = 0.009);however, as in the case of Ops-Zym the increase in the cpm levels was observedfrom 20 min after activation (Figure 4).

We analyzed the specific production of superoxide anion by restingor PMA-activated cells using a SOD-inhibitable reduction of cytochrome Cmethod. The levels of the superoxide anion produced by the granulocytes inthe three groups analyzed, however, did not reveal any statistically significantdifference in the production of superoxide anion by resting and PMA-stimulatedcells at end point of 15 min (Data not shown).

Respiratory Burst of Granulocytes in Bronchial Asthma 49

so Vargas et al.

Fig. 3. LCL of granulocytes stimulated with Ops-Zym (1.66 mg/tnl) in asthmatic patients undercrisis (O), noncrisis asthmatics (O), and healthy controls (D). The figure shows the increment in themetabolic activity of the cells after 60 min stimulation in noncrisis asthmatics with respect to thecontrol group (*P < 0.001).

DISCUSSION

Allergic bronchial asthma is an inflammatory condition whose clinical pre-sentation depends on the activity of different mediators, reactive oxygen inter-mediates and lysosomal content released by phagocytes, being such mediators.In this study we analyzed the respiratory burst of peripheral granulocytes in orderto determine its metabolic state and possible involvement in pathogenesis of air-way inflammation.

Resting granulocytes of asthmatic patients under crisis showed a greaterROIs production measured by LCL. This could reflect a state of in vivo activa-tion of such cells as the result of their exposition to multiple chemical mediatorsduring the inflammatory response triggered by contact with high concentrationsof the allergen. These data correlate with those reported by Styrt et al. Theyfound that the neutrophils of atopic patients were preactivated and presented agreater oxygen intermediary metabolites generation even when the cells werenot stimulated (9).

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Fig. 4. LCL of preactivated granulocytes with fMLP (10 6 M) and posterior stimulation with Ops-Zym (1.66 mg/ml) in asthmatic patients under crisis (O), noncrisis asthmatics (O), and healthycontrols (D). The figure shows a statistically significant difference between noncrisis asthmatics andthe control group after 60 min stimulation (*/• < 0.01).

When the LCL of granulocytes using PMA as the stimulus was evalu-ated, a statistically significant increase in the bioluminescent response of thetwo asthmatic patient groups with respect to the controls was found. These find-ings are interesting since they correlate with those reported by other authors(12, 22, 23), who demonstrated that when the neutrophils of atopic patientswere stimulated with PMA, they showed an increase in the respiratory burst.The observed increase in the levels of LCL after stimulation with PMA in thetwo groups of asthmatics could be the result of various mechanisms. During theasthmatic inflammatory response, a number of chemical mediators are producedand released. Such mediators include leukotrienes, prostaglandins, platelet acti-vating factor, C5a, histamine, mast cell derived chemotactic factors, and var-ious cytokines like TNF-a, IL-1, IL-4, IL-8, IL-10, GM-CSF (8, 22, 24-26)among others, which promote the activation of different inflammatory cells.These molecules could explain the increase in the metabolic activity of restingphagocytes of asthmatic patients as well as those stimulated with PMA.

When the LCL of the granulocytes stimulated with Ops-Zym were ana-

Respiratory Burst of Granulocytes in Bronchial Asthma

lyzed, we observed that the non-crisis asthmatic patients showed a greater lumi-nescence than the asthmatic patients under crisis and the control group from 20min poststimulation, even though there was only a statistically significant differ-ence at 60 minutes. These findings are different from those reported by Fukudaet al. (27), who did not find any differences among the three groups analyzedwhen quantifying the LCL after neutrophils stimulation of asthmatic patientsduring an acute state, a stable period, and healthy controls, with Ops-Zym. Onthe other hand, when they stimulated the neutrophils with fMLP, the LCL ofpatients in an acute state was less than that in the controls, This suggests thatthe peripheral neutrophil activity in the respiratory burst of asthmatics under anacute attack was reduced when the stimulus was fMLP.

Since Ops-Zym is a particle covered with different serum proteins, ourresults can be explained by analyzing the function and expression of adhesionproteins on the phagocytes membranes; among which the complement C3bifragment receptor CDllb/CD18 (CR3), and CDllc/CD18 (CR4) molecules arefound. These are important for the adhesion, chemotaxis, and phagocytosis activ-ity of these cells (24). Carroll et al. (12) reported an increase in the expression ofthe complement receptors in neutrophils of asthmatic patients, suggesting thatthese circulating cells have been exposed to some stimulus. According to theabove, it might be said that the number of adhesion molecules in the neutrophilsmembranes of non-crisis asthmatics is overexpressed, which would explain theincrease in LCL levels due to a particulate stimulus, such as Ops-Zym. Otherwisein patients under crisis, the absence of an increase in the LCL can be explainedby a greater recruitment of granulocytes at the lungs due to intense chemotacticactivity and adherence in the vascular endothelial tissue of this organ.

Since LCL detects the production of various types of oxygen free radicals,without distinguishing between them, we specifically quantified the productionof the superoxide anion. No differences were found among the different studygroups. These findings correlate with those reported by Polla et al. who quan-tified the production of the superoxide anion in the neutrophils of patients withatopic dermatitis, and they did not find any difference between patients and theircontrols (28). On the other hand, Meltzer et al. found a significant increase inthe production of the superoxide by the neutrophils of asthmatic patients at 5minutes poststimulation in vitro with PMA or fMLP (23). From our findingswe can at least deduce that the production of O^ is not increased during thebronchial hyperreactivity that develops in asthmatic patients. It is possible thatother intermediary oxygen metabolites play an important role in tissue damageduring the inflammatory response.

Even though knowledge concerning bronchial asthma is increasing, thedynamics of the process still remains unclear. Furthermore, it is important to takeinto account that it is unlikely that only one type of cell or inflammatory medi-ator can totally explain the pathological findings of asthma. Hence, the results

52 Vargas et al.

obtained from this type of study need to be analyzed in a general way, in orderto reach a better understanding of the clinical manifestations of asthma. There-fore, asthma must be understood to be a complex interactive process in whichthe inflammation of the respiratory tract becomes the focus of the pathogenesisand the treatment. Phagocytes are formidable inflammatory cells that, besidesproducing different intermediary oxygen molecules, liberates different lysoso-mal enzymes capable of destroying many tissues (1, 2). Although its specificrole in atopic asthma is still unclear, our findings show an involvement of gran-ulocytes in the pathogenesis allergic bronchial asthma. Our data also providesevidence, once again, of the role in the general tissue damage associated withsome atopic illnesses.

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