neutrophil infiltration and release of il-8 in airway mucosa from subjects with grain dust-induced...

Neutrophil infiltration and release of IL-8 in airway mucosafrom subjects with grain dust-induced occupational asthma

H.-S. PARK, K.-S. JUNG*, S.-C. HWANG†, D.-H. NAHM and H.-E. YIM‡

Departments of Allergy and Clinical Immunology,†Pulmonary and Critical Care Medicine and‡Pathology, Ajou UniversitySchool of Medicine, Suwon and*Department of Internal Medicine, Hallym University School of Medicine, Seoul, Korea

Summary

Background The immuno-pathological mechanism for occupational asthma induced bygrain dust (GD) remains to be clarified. There have been few reports suggesting theinvolvement of neutrophils inducing bronchoconstriction after inhalation of GD.Objective To further understand the role of neutrophil in the pathogenesis of GD-inducedasthma.Materials and methods We studied the phenotype of leucocytes of the bronchial mucosain patients with GD-induced asthma. Bronchial biopsy specimens were obtained byfibreoptic bronchoscopy from six subjects with GD-induced asthma. Six allergic asthmapatients sensitive to house dust mite were enrolled as controls. Bronchial biopsy specimenswere examined by immunohistochemistry with a panel of monoclonal antibodies totryptase-containing mast cell (AA1), activated eosinophil (EG2), pan T-lymphocyte(CD3) and neutrophil elastase (NE). Induced sputum was collected before and after theGD-bronchoprovocation test. The IL-8 level in the sputum was measured using ELISA.Results There was a significant increase in the number of AA1þ and NEþ cells inbronchial mucosa of GD-induced asthma, compared with those of allergic asthma(P¼ 0.01, P¼ 0.01, respectively). No significant differences were observed in thenumber of EG2þ and CD3þ cells (P¼ 0.13, P¼ 0.15, respectively). IL-8 was abundantin the sputum of all GD-induced asthma patients and significantly increased after thebronchial challenges compared with the baseline value (P¼ 0.03).Conclusion These findings support the view that neutrophil recruitment together withmast cells may contribute to the bronchoconstriction induced by GD. A possible involve-ment of IL-8 was suggested.

Keywords: neutrophil, grain dust-induced occupational asthma, IL-8, induced sputum

Clinical and Experimental Allergy, Vol. 28, pp. 724–730. Submitted 6 May 1997; revised11 November 1997; accepted 18 December 1997.

Introduction

Chronic inhalation of grain dust (GD) has been shown tocause acute and chronic airway injury characterized bybronchitis and airflow obstruction [1–4]. Longitudinalstudies have shown accelerated deterioration of pulmonaryfunction in these grain workers [5], the severity of whichappears to be related to the concentration of airborne GD inthe work environment [4,6]. The presence of specific IgE

antibodies in individuals who have clinical lower respira-tory symptoms with exposure to GD, has been demonstratedin up to 40% of symptomatic workers in the same workplace[7].

Airway inflammation is clearly an important character-istic of asthma. Inhalation of GD causes an inflammatoryresponse characterized by neutrophil recruitment to thelower respiratory tract.In vitro studies indicate that extractsof GD are capable of recruiting neutrophils by severalmechanisms; both endotoxin- and non-endotoxin-inducedchemotaxis, activation of complement, and release of alveo-lar macrophagederived neutrophil chemotactic activity [8].

Clinical and Experimental Allergy,1998, Volume 28, pages 724–730

724 q 1998 Blackwell Science Ltd

Correspondence: Dr H.-S. Park, Department of Allergy and ClinicalImmunology, Ajou University School of Medicine, Paldalgu Wonchon-dong San-5, Suwon, Korea.

IL-8 has been recognized as an inflammatory cytokine withstrong potency in neutrophil activation and chemotaxis[9,10].

In order to improve understanding of the role of neutro-phil in GD-induced occupational asthma, we performedimmunohistochemical analysis of bronchial mucosa usingmonoclonal antibodies to inflammatory cells includingneutrophil, T lymphocyte, mast cell and eosinophil, andlooked at the changes of IL-8 level in the induced sputumbefore and after the GD inhalation challenges.

Materials and methods

Subjects

All of the 43 subjects participating in this study had beenexposed to grain dust composed of corn, rye, wheat andbarley while working for the Dongbang Animal FeedIndustry in Suwon, Korea. Among them, 15 (34.9%) hadcomplained of work-related respiratory symptoms. Whenbronchoprovocation test with grain dust was performed on15 symptomatic workers, six were confirmed to haveoccupational asthma based upon significant bronchocon-strictions on GD-bronchoprovocation test, clinical historyand work-related symptoms (group I). Three of them hadserum specific IgE antibodies to GD which were detected byELISA and all had specific IgG antibody. Six allergicasthma patients sensitive to house dust mite, never exposedto GD were enrolled as controls (group II). Their clinicalcharacteristics are summarized in Table 1. None of thesubjects had taken any antiasthmatic medication includingcorticosteroid for 8 weeks prior to the study. The subjectsgave their written informed consent as regulated by AjouUniversity Hospital, Suwon, Korea.

Preparation of extracts

GD was obtained from the subjects’ workplace. It wasextracted with phosphate buffered saline [(PBS, pH 7.5),L5w/v] at 408C for 1 h followed by centrifugation at5000 rpm. The supernatants were passed through the syringefilter (MIS, USA:025mm pore-sized) and used for thebronchoprovocation tests at a concentration of 1:10 w/v.Some of the supernatant was dialysed (the cut-off molecularweight was 6000 Da) against 4 L of distilled water at 408Cfor 48 h, and lyophilized at¹ 7008C for the preparation ofantigens and used in ELISA for detection of serum specificIgE antibody.

Protocol

All six subjects who had complained of work-relatedrespiratory symptoms when exposed to GD in the workplace

were admitted to the Allergy Clinic at Ajou UniversityHospital, and before the inhalation challenge, a standardprotocol was followed in all cases. A screening evaluationincluding a complete history and physical examination,pulmonary function tests including methacholine bronchialchallenge test, chest X-ray, and electrocardiogram wereperformed upon admission. A peripheral blood sample wasobtained for total and differential counts of leucocytes.Baseline forced expiratory flow were performed using aMultiSPIRO SX/PC (Multi SPIRO Inc, USA) with standardprotocols.

Bronchoprovocation test with grain dust

Airway responsiveness to methacholine was tested as pre-viously described else where with some modifications [11].Bronchoprovocation tests were performed according tostandard studies of occupational asthma [7,12]. On thefirst day, normal saline was administered from a nebulizer646 connected to a dosimeter (DeVilbiss Co., USA) oper-ated at a pressure of 20 psi. The subject was asked to inhalethe aerosol five times to the vital capacity, and then wasobserved for 7 h. A sputum induction test was done andcollected sputum was used as a baseline sample. On adifferent day, GD extracts were inhaled from 1:1000 to1:10 w/v until FEV1 declined by 20%. In the pulmonaryfunction test, forced vital capacity, forced expiratoryvolume in one second (FEV1), and forced expiratory flowrate (FEF25–75) were measured with MultiSPIRO SX/PC(MultiSPIRO Inc, USA) before and 10 min after eachinhalation. Then, FEV1 and FEF25–75 were measuredevery 10 min during the first hour, and then hourly for 7 hafter the challenge. And then, a second sputum inductiontest was performed and collected sputum was used as apostchallenge sample. A positive reaction was defined as adecrease in FEV1 of more than 20% from the baseline value.

ELISA for specific IgE antibodies to grain dust extract

The presence of specific IgE antibody to GD was deter-mined by ELISA according to the previously describedmethod [7,12]. Microtitre plates (Dynatech, USA) werefirst coated with 50mL/well of GD (5mg/well) extract, andleft at 408C overnight. Each well was washed three timeswith 0.05% Tween-phosphate buffered saline (PBST), andthe remaining binding sites were blocked by incubation with350mL of 3% BSA-PBST for 1 h atroom temperature. Thewells were then incubated for 2 h at room temperature with50mL of either the patients’ sera (undiluted) or control serafrom 27 patients who showed negative skin-prick testresponses to common inhaled allergens as well as GD.After washing three times with PBST, 50mL of the 1:1000 v/v biotin-labelled goat antihuman IgE antibody

Neutrophil infiltration and IL-8 in grain dust asthma 725

q 1998 Blackwell Science Ltd,Clinical and Experimental Allergy, 28, 724–730

(Vector Co., USA) was added to the wells and incubated for2 h at room temperature. The wells were then washed threetimes with PBST and incubated with 1:1000 v/v streptavidin-peroxidase (Sigma Co. USA) for 30 min before anotherwashing step which was followed by incubation with100mL 2 N sodium azide. The absorbance was read at 410nm by an automated microplate reader. All assays wereperformed in triplicate. The cut-off absorbance of positiveIgE binding was determined as 0.064, which was derivedfrom the mean and two standard deviations of theabsorbance value of the control subjects.

Induction of sputum methodology

Sputum was induced using a previously described method[13,14]. Immediately before the sputum expectoration, eachsubject was pretreated with 200mg salbutamol, adminis-tered by means of a metered dose inhaler. The subjects theninhaled nebulized sterile 3% saline solution for 20 minthrough an ultrasonic nebulizer (Omron Co., Japan).Sputum sample was collected throughout the procedure.After being instructed to wash the mouth, spit out the salivaand blow the nose, the subjects were asked to cough andexpectorate sputum into a clean plastic container.

Sputum processing

Collected sputum and saliva samples were immediatelyprocessed as previously reported [14]. The volumes of theinduced sputum were determined, and equal volumes ofphosphate buffered saline (PBST) were added. The sampleswere then mixed by vortex mixer for 1 min and centrifugedfor 20 min at 3000 rpm. The supernatants were aspirated andfrozen at¹ 2008C.

ELISA for IL-8 in the induced sputum

To evaluate the role of IL-8, the induced sputum from all sixsubjects with GD asthma were collected before the chal-lenge as baseline value and at 7 h after the GD bronchialchallenge test. The IL-8 level was measured using ELISAkit (R&D system, USA) under the manufacturer’s guidance.The albumin content within the sputum was measured bynephelometry and the IL-8 level was presented asmg/albumin content.

Bronchoscope and bronchial biopsies

Fibreoptic bronchoscopy was performed after the inhalationchallenge with GD as described previously [15]. At leasttwo endobronchial biopsies were taken through thebronchoscope (Olympus BF, type IT 200; Olympus Co.,Tokyo, Japan) with sterile forceps (FB 15C; Olympus Co.)

from the bronchial mucosa of the right intermediatebronchus. The biopsy specimen was fixed in 4% formalde-hyde/l% gluteraldehyde in 0.1 M PBS, then dehydrated andembedded in paraffin for immunohistochemistry.

Immunohistochemistry and quantification

Sections (4mm thick) were obtained from biopsy specimensusing microtome (Shandon, HM340I, Japan). Activationmarkers of inflammatory cells were assessed by immuno-histochemistry on paraffin-embedded sections using a panelof inflammatory cells to monoclonal antibodies: anti-CD3(Dako, Denmark), antibody to secretory form of eosinophilcationic protein (EG2, Pharmacia, Sweden), antineutrophilelastase (NE, Dako, Denmark), and antibody to mast celltryptase (AA1, Southampton University, UK). The mousemonoclonal antibody to mast cell tryptase was kindlyprovided by Andrew Walls from Southampton University,which had been previously characterized for their specificityand reactivity. Biotin-conjugated rabbit antimouse anti-bodies (1:200 in TBS, Sigma, USA) were applied assecondary antibody and detected with the streptavidin-biotin complex using LSAB kit (Dako, CA, USA) andaminoethyl carbazole as chromogen. They were counter-stained with Mayers’ haematoxylin. The numbers of posi-tively stained cells were counted in four fields per one tissueby superimposing a grid of 100 points (intersection ofcrosses covering a surface area of 0.5 mm2) under an eye-piece graticulate at a magnification of x200, and expressedas the mean number of cells per mm2 as reported byFokkenset al. [16]. For negative controls, the mouse IgG,antibodies (1:200 v/v) were incubated with omission ofprimary antibodies. To avoid observer bias, the slideswere coded before analysis and read blind.

Statistical analysis

The Mann–WhitneyU and Wilcoxon-signed rank testswere applied using the SPSS version 7.0 (Chicago, USA)to evaluate the statistical differences between the data: aP-value of less than 0.05 was regarded as significant.

Results

Bronchoprovocation test

Table 2 summarized the changes of FEV1 during theasthmatic response. Five subjects of group I showed earlyasthmatic responses. One showed a dual asthmatic response.The inhaled dose of GD antigen to induce 20% fall of FEV,was described in Table 2.

726 H.-S. Parket al.


Inflammatory cell infiltrate in bronchial mucosa

Figure 1 shows AA1þ (1a) and NEþ cell (1b) in thebronchial mucosa from the subjects with GD-inducedasthma. Figure 2 demonstrates the comparison of individualinflammatory cell counts expressing AA1, CD3, EG2 andNE cell markers between GD-induced asthma and allergicasthma patients. All subjects had both AA1þ and EG2þ

cells. The numbers of AA1þ and NEþ cells were signifi-cantly higher in GD-induced asthma than those of allergicasthma (P¼ 0.01, P¼ 0.01, respectively). No significantdifferences were observed in the numbers of EG2þ andCD3þ cells (P¼ 0.13,P¼ 0.15, respectively).

IL-8 level in the induced sputum

Table 2 shows changes of IL-8/albumin level in the induced

sputum before and after GD- bronchoprovocation test. IL-8was abundantly present in the baseline value of inducedsputum from all subjects with GD-induced asthma and itsignificantly increased after the challenge (P¼ 0.03), incontrast to no significant changes in those working in thesame workplace, but without occupational asthma (P> 0.05,data not shown).

Discussion

Several mechanisms may be involved in pathogenesis ofGD-induced occupational asthma. Our previous study [7]demonstrated the serum specific IgE antibodies and IgE-binding components within GD in six subjects with GD-induced asthma, and suggested that the IgE-mediatedreaction is likely to be responsible for their asthmaticsymptoms. The failure to detect specific IgE antibodies toGD in the sera of three workers with positive challengesmight indicate the involvement of other immunological ornon-immunological mechanism in the induction of theirasthmatic symptoms. Although there has been speculationthat IgG may have a role in the pathogenesis of asthma [17],our previous study on the effect of anti-IgG4 antibody onbasophil histamine release from six challenge-proven GD-asthma patients, suggested that the possibility of specificIgG4 as a sensitizing antibody seemed to be very low [12].

Recently, neutrophil involvement in the pathogenesis ofoccupational asthma has been suggested. Neutrophilia hadbeen noted in bronchoalveolar lavage fluid in subjects withlate asthmatic reaction induced by isocyanate challenge test[18]. Inhalation of grain sorghum dust in normal volunteerscould induce an increased peripheral neutrophil count andchemotactic response to GD [19].In vitro study revealed therelease of substances presenting neutrophil chemotacticactivity by bronchial epithelial cell in response to GD[20]. So far, the mechanism of neutrophil chemotaxis andthe function of the neutrophils in GD-induced asthma arenot well documented. In this study, the numbers of NEþ

cells were significantly higher in the bronchial mucosa ofGD-induced asthma than in those of allergic asthma. Ourprevious study dealing with neutrophil chemotactic activityof GD-asthma [15] revealed that serum neutrophil chemo-tactic activity significantly increased at 30 min after GDinhalation challenge relative to the baseline value, and thensignificantly decreased at 240 min, whereas no significantchanges of neutrophil chemotactic activity occurred inasthmatic patients with negative result on grain dust-bronchoprovocation test. These findings suggest possibleinvolvement of neutrophils as an effector cell in the devel-opment of GD-induced bronchoconstriction in exposedworkers.

IL-8 was recognized as a key chemokine to induceneutrophil recruitment and activation. Early evidence for



Fig. 1. Localization of tryptase-containing mast cell (a) andelastase-containing neutrophils (b) in the bronchial biopsy fromsubjects with grain dust-induced occupational asthma. Magnifica-tion in a & b ×200.

the involvement of this chemokine in allergic inflammationwas obtained from a study which demonstrated increasedexpression of IL-8 immunoreactivity in bronchial biopsiesof asthmatic patients when compared with biopsies fromnormal subjects [21]. Release of IL-8 had also been reportedto be increased in the bronchoalveolar large (BAL) fluid ofasthmatic patients [22] and in the nasal secretions of atopicpatients in response to nasal allergen challenge [23]. How-ever, neutrophils have not only been known as merelyterminal effector cells; they have also been known toaffect other cells by releasing various cytokines [9, 24–26]. Recently, it has been reported that mast cells are able torelease IL-4, TNFa, IL-8, leukotrienes and platelet activat-ing factors which could induce neutrophil chemotaxis[27,28]. In the present study, the numbers of tryptase-secreting mast cells and neutrophils were significantlyhigher in GD-induced asthma than in allergic asthma. The

IL-8 level in the induced sputum significantly increasedafter the bronchial challenge test compared with the base-line level in contrary to no significant change in asthmaticsubjects showing a negative result on GD-bronchoprovoca-tion test. These results suggest that activated neutrophilsassociated with mast cell may participate in the develop-ment of GD-induced bronchoconstriction in exposedworkers, and IL-8 released after exposure to GD mightcon-tribute to neutrophil infiltration into the airway mucosa.Further investigations will be needed to elucidate the exactmechanism of IL-8 released after exposure to GD.

Induction of sputum production by inhalation of hyper-tonic saline was widely used to investigate the bronchialconstituents of airway secretions in asthmatic patients. Theresults obtained from them are consistent and could yieldmore concentrated secretions than those obtained frombronchoscopy [29–32]. Biochemical analysis of the fluid



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Fig. 2. Individual cell count expressing AA1 (mast cells), EG2 (activated eosinophils), CD3 (pan T cells) and NE (neutrophils) in bronchialmucosa of grain dust-induced occupational asthma and allergic asthma as controls, expressed as the number of positive cells per mm2.Median values are represented by horizontal bars.

phase of the induced sputum samples should take intoconsideration the variable dilutions by hypertonic salineand saliva [13]. In this study, we measured albumin in bothsaliva and sputum from the same subjects. The albuminlevel in saliva was much lower than those in induced samplein the same subjects, which suggested that sputum IL-8 andalbumin level presented in this study was derived from thesubglottic airway. Therefore, in order to correct the dilutionfactor and IL-8 level in the induced sputum was presented asratio to albumin (IL-8/albumin).

In conclusion, these findings support the view that bothneutrophils and mast cells are involved in the developmentof GD-induced asthma and that IL-8 may contribute toneutrophil recruitment.

Acknowledgements

The authors are grateful to Ms Sun-Hye Hong and Jinn Yoofor their help in preparing the manuscript. This study wasfunded by Ajou Graduate School of Medicine (1996).

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Fig. 3. Release of IL-8 in the induced sputum before and after thebronchial challenge in six grain dust-asthma patients showing apositive result on the bronchial challenge test. A significantincrease of IL-8 relative to the baseline value was noted after thebronchial challenge testð p ¼ 0:03Þ.

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