Bronchial Epithelial Inflammation in Children with Chronic Cough after Early Lower Respiratory Tract lllness'?

M. HEINO, K. JUNTUNEN-BACKMAN, M. LEIJALA, J. RAPOLA, and L. A. LAITINEN

Introduction

Lower respiratory tract illness (LRI) in children is a poorly defined entity with a nonuniform terminology. LRI illness may be related to later respiratory symptomatology, possibly on the basis of epithelial damage and airway hyperresponsiveness (1). In bronchopulmonary dysplasia (BPD), the presence and initial severity of airway hyperresponsiveness correlated to the severity and duration of respiratory failure regardless of family history of atopia or asthma (2). At the ultrastructural level, the severity of BPD paralleled the degree of epithelial damage (3). Epithelial damage and inflammation are closely related to hyperreactive airways (4-8). In adults with chronic bronchitis and productive cough, there is inflammation of the bronchial epithelium (9). To further clarify the ultrastructural nature of epithelial damage in respiratory symptoms, we report inflammatory changes in the epithelium of biopsies from the main carina of children with chronic cough who previously had LRI. To our knowledge, there are no previous similar studies. We also introduce a new wayof assessing the relationship between epithelial inflammation and the number of ciliated cells. Methods Subjects Seven school-aged children, three boys and four girls, werereferred to the Children's Hospital, University of Helsinki, because of persistent productive cough that had lasted more than 3 months. They had already been treated with antibiotics and symptomatically with oral bronchodilators (salbutamol, theophylline) without response. One child (Subject 4) used inhaled steroids (beclomethasone dipropionate 100ug x 2) in addition in an attempt to suppress epithelial inflammation. All had been operated on for chronic ear and nose problems or hypertrophied adenoids. None of them had experienced dyspnea or wheezing. On auscultation, no signs of bronchial obstruction were evident at the time of study or in history records. Patient data and family 428

SUMMARY Westudied the ultrastructural findings in biopsies from the main carina of seven schoolaged children who had had chronic cough for at least 3 months and who all had a history of early lower respiratory illness (LRI). They had their first LRI between birth and 7 yr of age (range, 5 to 11 yr). The cross-sectional area of the epithelium was quantified by point counting for the percentage area of intercellular spaces (ICS) denoting edema, and the numbers of both inflammatory cells (leukocytes, including eosinophils, and mast cells) and ciliated cells. The children (excluding the one using inhaled steroids) demonstrated nearly 17-and more than sevenfold increases in the mean area of ICS and number of inflammatory cells per epithelial area, respectively, and a nearly threefold decrease in the mean number of ciliated cells per epithelial area compared with the biopsy specimens from the orifice of the right upper lobe bronchus of two healthy adults. In the children, the increase in inflammatory cells (greater than 91% were lymphocytes) was more prominent in the children with two LRI before the age of 1 yr. Our findings imply a close association of early LRI and later epithelial inflammation during chronic cough. Allergic mechanisms in the epithelial inflammation cannot be ruled out as six of the patients had, either alone or in combination, signs of atopia, positive family history of allergic rhinitis or asthma, and eosinophils or mast cells in the epithelium. All eight subjects (six children, two adults) showed a very high and significant inverse correlation between the number of ciliated cells and the percentages of both ICS (edema index: r = -0.8n, p < 0.004) and inflammatory cells (inflammatory cell index: r = -0.826, P < 0.011) in the upper half of the epithelium. There was an upward spreading of epithelial inflammation in the patients with concomitant reduction in the number of ciliated cells; with less inflammation, the number of ciliated cells approached that of the adult reference subjects. Using multiple correlation analysis, the joint impact of both indexes on the number of ciliated cells was even higher (R - 0.960, P < 0.002), and omission of the adult reference subjects did not alter this correlation, which remained very high and significant (R = - 0.988, P < 0.003). The fit of this model, by which both indexes together account for the number of ciliated cells, is extremely good.

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AM REV RESPIR DIS 1990; 141:428-432

history of atopia or asthma and the first LRI of the children are shown in the table (table 1).This study wasperformed, with signed consent of the parents, as part of a routine examination involving bronchoscopy. It is ethically doubtful to include healthy nonhospitalized children as control subjects and subject them to general anesthesia and bronchoscopy without reason. If hospitalized children had been included as control subjects, there certainly would have been some underlying respiratory cause for bronchoscopy; hence, their epithelium would hardly have been normal. Tosomewhat overcome the problem of control subjects, we bronchoscoped two healthy nonsmoking women, 34 and 44 yr of age, who suffered from symptoms of primary hyperventilation. We call them reference subjects in this report. Their bronchial biopsies from the orifice of the right upper lobe bronchus, handled identically with biopsies from the main carina of the children, served as control biopsies for any artifacts in the preparation of specimens and also for guidance to interpretation of normality. Our findings are discussed also in terms of nor-

mal ultrastructure as defined in recent literature (10-14).

Laboratory Investigations The lung function tests wereperformed using Vitalograph ™ Spirotrac II (Vitalograph LTD, Buckingham, UK). The skin prick tests were made using 10 HEP (histamine equivalent prick) biologically standardized solutions to

(Received in original form December 8, 1988 and in revised form July 5, 1989) 1 From the Department of Pulmonary Medicine, Helsinki University Central Hospital, the Department of Electron Microscopy, and the Children's Hospital, University of Helsinki, Finland, and the Department of Lung Medicine, University of Lund, and the Department of Exploratory Clinical Research, AB Draco, Lund, Sweden. 2 Supported by the Sigrid Juselius Foundation and the Anti-Tuberculosis Association in Finland. 3 Correspondence and requests for reprints should be addressed to Dr. M. Heino, Department of Pulmonary Medicine, Helsinki University Central Hospital, 00290 Helsinki, Finland.

429

BRONCHIAL EPITHELIAL INFLAMMATION IN CHILDREN WITH CHRONIC COUGH

TABLE 1

identified if two or more typical granules were present.

CLINICAL DATA ON CHILDREN WITH CHRONIC COUGH Skin Prick Testt

Eos NIB

Family History of Atopia or Asthma

FEV, (% pred)

FVC (% pred)

97

101

Allergic rhinitis (brother)

Pneumonia twice before 1 yr of age

76

102

No history

M

RDS at birth, pneumonia at 2.5 months of age

62

75

9

F

Pneumonia (Mycoplasma) before 1 yr of age

87

104

5

6

F

Relapsing bronchitis starting at 3 yr of age (streptococcal sepsis at birth)

73

97

6

6

F

Pneumonia at 4 yr of age

ND

ND

Subject No.

Age (yr)

First Lower Respiratory Tract Illness and Age*

Sex

8

F

Aspiration at birth, pneumonia and pertussis at 2 months of age

2

9

M

3

5

4

+

Asthma (sister) No information No history

Calculations Apple Statworks was used in a microcomputer to calculate the correlation coefficient (r), the multiple correlation coefficient (R), and their two-tailed levelsof significance for the values in the table (table 2). Values of p < 0.05 were considered significant. Partial correlation was employed when appropriate. The patient treated with inhaled steroids (Subject 4) was omitted from the calculations because of possible effects of steroids on the ciliated cells and inflammation, but her results were included in the table (table 2) for comparison. Results

+

Allergic rhinitis (father)

At the time of bronchoscopy, all the children were free of any noticeable infec7 11 M Pneumonia (hemophilus) 91 91 No history + tion (normal ESR and CRP) and had at 7 yr of age normal values for sweat test chloride, Definition of abbreviations: Eos NIB = eosinophils in nasalsmearor blood; ADS = respiratory distress syndrome; NO = not alpha-l-antitrypsin, and immunoglobudone. lins. Normal lung function was seen in • Infectious agent specified if known. t Wheal equal or greaterthan that caused by histamine shown by plus sign. only two patients (table 1). By electron microscopy the child patients and reference subjects showed firm cell junctions spaces (ICS) was assessed as well as the num- near the lumen. The specimens in both common allergens (ALK, Copenhagen, Denber of inflammatory cells (eosinophils, mast mark). The reaction was regarded as positive groups showed equal preservation of celcells, and other leukocytes) and ciliated cells if the wheal caused by one or more allergens (including preciliated cells) per mm' of cross- lular fine structures. No microorganisms was equal to, or larger than, that caused by sectional epithelial area. Ciliated cells,or parts were seen. Differences were seen only in histamine. Nasal smear eosinophilia was posiof ciliated cells, were identified by basal bod- the proportions of ICS and inflammative if more than three eosinophils per field ies below the luminal cell surface whether or tory and ciliated cells (table 2). were seen. Blood eosinophilia was positive if The reference subjects were practicalnot the nucleus was included in the plane of there were equal to, or more than, 400 x 106 section. Preciliated cells wereimmature ciliat- ly free of ICS compared witha nearly eosinophils per liter. Serum IgE was measured ed cells with fibrogranular areas (11). with Prist (Pharmacia, Uppsala, Sweden) and 17-fold increase shown in the patients Weused the following rules for point count- (0.59 versus 10.02% of total epithelial serum immunoglobulins (IgA, IgO, IgM) and ing. Points hitting the areal boundaries were area). Only 8.34% of the ICS in the refserum alpha-l-antitrypsin with laser nepheincluded in the epithelial area. In counting lometry. Blood erythrocyte sedimentation rate erence subjects was found in the luminal the area of ICS, points touching the cell (ESR) and serum C-reactive protein (CRP) boundaries of the ICS were not included. On zone of the epithelium. In the patients, were measured routinely as was the sweat test the other hand, points hitting tiny solitary cy- the ICS appeared more widespread, using chloride electrode. toplasmic processes in the ICS were included 42.88010 being in the luminal zone (table Bronchoscopy and Electron Microscopy in the area of ICS. Inflammatory cellsoccupy- 2). The ICS contained inflammatory cells ing ICS were not included in the area of ICS. and protein-rich exudate, which was Bronchial biopsies were taken under general Wedivided the distribution of both the ICS stained dark. Often there was seen a anesthesia using rigid tube bronchoscopes and the inflammatory cells in the epithelium more-or-Iess double layer of basal cells (Storz, Tuttlingen, FRO) from the main cariinto two zones of equal size, viz., the luminal abovethe basement membrane. The edemna in the children and from the orifice of the right upper lobe bronchus in the adults. The and basal zones. The distance of each point atous epithelium, caused by the ICS, representing part of the ICS was measured specimens were processed in a routine manshowed cells separated from each other to the nearest point representing the luminal ner for electron microscopy (4) and examined as if the epithelium was about to disinteblindlywith a Jeol-JEMTernscan 1200 transmis- border and the basal lamina of the basement membrane. The distance of the inflammato- grate (figure 1). sion electron microscope operated at 60 kv, The inflammatory cells were for the ry cells was measured similarly from the grid Electron micrographs were taken at an point closest to the midpoint of an addition- most part confined to the patients (table original magnification x 800 from a one-slot alline running along the long axis of the cell. 2) and more than 91% of all inflammagrid to ensure as much visible epithelium as Inflammatory cells and points representing tory cells were lymphocytes. There was possible and enlarged 2.6 times to produce ICS were included in the basal zone if their a greater than sevenfold increase in the a montage of the entire epithelium (15).Higher magnifications were used to identify in- distances to the luminal border and basal lam- total number of inflammatory cellsin the ina were the same. Wedefined the percentage patients compared with that in the refflammatory cells. A transparent grid with of ICS in the luminal zone as the edema incross sections 7 mm apart was used for point erence subjects (1710.33 versus 234.02 dex. The inflammatory cell index was defined counting. Best-preserved central areas of the cells/rum'). There were no mast cells or similarly. Both indexes werecalled the inflamepithelium were analyzed to exclude possible eosinophils in the reference subjects, but artificial damage at the margins of the biopmatory indexes. The presence of a nucleus in the plane of a section was compulsory on- a few were seen in the patients (zero versy. On the average, more than 1,700 points ly in counting leukocytes other than eo- sus 34.76 mast cells/mm" and zero verwere counted on the epithelium in each subsinophils. Eosinophils and mast cells were sus 62.13 eosinophils/rnm" respectively). ject. The percentage area of the intercellular

430

HEINO, JUNTUNEN-BACKMAN, LEIJALA, RAPOLA, AND LAITINEN

TABLE 2 ULTRASTRUCTURAL DATA ON BRONCHIAL EPITHELIUM

Subject No.

Inflammatory Cells, (n/mm 2 )

(%)

Edema Index

Eos

Mast

8.92 2.97 20.52 21.02 8.95 9.12 9.62 10.02 2.33

65.45 17.65 44.72 34.37 44.22 51.92 33.33 42.88 6.64

0 85.92 286 .83 0 0 0 0 62.13 47.08

39.82 0 0 0 52.53 0 116.20 34.76 18.80

0.15 1.03 0.59 0.44

0 16.67 8.34 8.34

0 0 0

0 0 0

ICS

Ciliated Cells (n/mm 2 )

Preciliated Cells'

Total

Inflammatory Cell Index

2,110.64 2,362.72 3,155 .11 288.92 682.89 904 .77 1,045 .83 1,710.33 400.81

22.64 24.53 50.00 18./8 7.69 42.86 11.11 26 .47 6.90

0 1,503 .55 204 .88 1,613.92 1,129.40 0 1,611 .61 741 .57 309 .62

0 0 0 40.00 2.33 0 7.14 1.58 1.18

2,001.07 2,366.61 2,183.84 182 .77

2.56 0 1.28 1.28

(%)

Children

1 2 3 4t 5 6 7

Mean SE Reference subjects

8 9 Mean SE

258.02 210.02 234.02 24.00

0 0 0

Definitionof abbreviations: ICS = intercellular spaces (percentage of epithelial area); Edema Index = percentage of rcs in the upper half of the epithelium or the luminal zone; Inflammatory Cell Index = percentage of inflammatory cells in the upper half of the epithelium or the luminal zone; Inflammatory Cells = eosinophils (Eos), mast cells , and all other leukocytes (more than 91% of leukocytes were lymphocytes); Ciliated Cells = ciliated cells and preciliated cells. • Shown as percentage of ciliated cells. t Excluded from calculations because of steroids.

The patients showed a nearly threefold decrease in the number of ciliated cells compared with that in the reference subjects (741.57 versus 2183.84 cells/rum') but equal signs of preciliated cells (1.58

.

versus 1.28% of ciliated cells) (table 2). Deuterosomes and free basal bodies were rare and seen only with cells having fibrogranular areas. No mucociliary cells were seen. The fibrogranular areas in

LU

JO..

~.

,,)

'.

*

Fig. 1. Electron micrograph of patient (SUbject 3) showing wide intracellular spaces (asterisks), some of which stain darker for fixed proteinaceous fluid, and several leukocytes with prominent nuclear chromatin (arrCNVheads) . Apical cell junctions bordering the lumen (LU) are intact. The dark basal cells form a more or less double layer above the basement membrane (BM), which is seen as a ribbon beneath the epithelium. (2.50AJ glutaraldehyde and 1% osmiumtetroxide fixation; uranyl acetate and lead citrate staining. Bar = 10 11m.)

preciliated cells appeared solitary in the immature ciliated cells of the reference subjects. but there were sometimes two such areas in some cells of the patients. In the patients, a reduction in epithelial inflammation, as reflected by the inflammatory indexes, resulted in an increase in the number of ciliated cells, which was found to approach that of the reference subjects with a normal epithelium. There was a high and significant inverse correlation between the number of ciliated cells and the edema index (r = -0.877, p < 0.004) and the number of ciliated cells and the inflammatory cell index (r = -0.826, p < 0.011) (figure 2). Partial correlation was used to check that both indexes had a significant influence on the ciliated cell number that was not accounted by the other index. Using multiple correlation analysis, the joint impact of both indexes on the number of ciliated cells resulted in an even higher inverse correlation (R = - 0.960, p < 0.002), which was not altered even if the reference subjects were omitted (R = -0.988, P < 0.003). This indicated an extremely good fit of this model whereby both inflammatory indexes jointly accounted for the number of ciliated cells. When both indexes were taken separately, the omission of the reference subjects did not greatly affect the correlation between the number of ciliated cells and the edema index (r = -0.834, p < 0.039); the respective correlation between the number of ciliated cells and the inflammatory cell index (r = - 0.687, p < 0.129) did not reach the level of significance as the total number of subjects fell. This emphasizes the importance of the edema index and especially the joint impact of both indexes on the number of ciliated cells. The highest total number of inflammatory cells were seen in Patients 1, 2, and 3 who had had a LRI twice before the age of 1 yr (tables 1 and 2). This may imply a connection between early LRI and later chronic cough and epithelial inflammation. Also, allergic mechanisms may have been related to the more widespread epithelial inflammation since Patients 1, 3, and 6, who had the lowest numbers of ciliated cells with high inflammatory indexes, had close relatives with allergic rhinitis or asthma. Two of these patients also had signs of atopia themselves. Discussion

The main findings in the bronchial epithelium of the children with chronic

BRONCHIAL EPITHELIAL INFLAMMATION IN CHILDREN WITH CHRONIC COUGH

Bronchial epithelial inflammation in children with chronic cough after early lower respiratory tract illness.

We studied the ultrastructural findings in biopsies from the main carina of seven school-aged children who had had chronic cough for at least 3 months...
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