Destruction and loss of bronchial cartilage in cystic fibrosis

Destruction and Loss of Bronchial Cartilage in Cystic Fibrosis GREGORY OGRINC, MD, BAL KAMPALATH, MD, AND JOSEPH F. TOMASHEFSKI, JR, MD We studied by means of serial sections of intact isolated bronchi, the distribution and morphology of bronchial cartilage in lobar and segmental airways of 6 patients with cystic fibrosis (CF). Findings were compared to those of 4 young adults without CF who served as controls. Compared to the controls, cartilage in CF airways extended for a shorter absolute distance along the bronchial tree and disappeared at a more proximal branching level. Loss of cartilage appeared to correlate with the severity of bronchiectasis. In proximal airways chronic inflammation, destruction and fibrous replacement of cartilage preceded its disappearance. Immunohistochemical staining indi-

cated that cells of monocyte/macrophage lineage (CD68, MAC387

Cystic fibrosis (CF) is an autosomal recessive disease in which a genetic mutation results in a dysfunctional chloride transporter protein (cystic fibrosis transmembrane regulator) that results in exocrine gland failure consequent to the obstruction of glandular ducts by viscous, dehydrated secretions, t Mucus impaction of bronchi and chronic bronchocentric bacterial infection lead to bronchiectasis, the hallmark of CF-associated lung disease. 2 The defining pathological features of bronchiectasis in CF and other conditions include airway dilatation, acute and chronic inflammation, and mural destruction. 2,s Classical studies o f bronchiectasis in patients w i t h o u t CF have d o c u m e n t e d simplification o f b r o n chial divisions a n d loss o f b r o n c h i a l cartilage. 4,5 Histologically, in p e r i p h e r a l l u n g tissue o f CF patients, dilated, thin-walled airways are typically devoid o f cartilage, i m p a r t i n g u n c e r t a i n t y a b o u t the b r o n c h i a l o r b r o n c h i o l a r derivation o f the affected airway. Additionally, in a d v a n c e d CF l u n g disease, partially destroyed b r o n c h i a l cartilage plates m a y be infiltrated by inflammat o r y cellsY T h e e x t e n t o f b r o n c h i a l c h o n d r a l destruction a n d elimination in CF has not, however, b e e n systematically studie d . T h e c u r r e n t retrospective autopsy study critically evaluates the abnormalities a n d distribution o f cartilage in the b r o n c h i a l tree o f y o u n g adult CF patients in c o m p a r i s o n with that seen in the n o r m a l lung. We describe the level at which cartilage disappears, the histological evolution o f i n f l a m m a t o r y lesions, the types o f i n f l a m m a t o r y cells associated with the loss o f cartilage, a n d the degenerative c h a n g e s that o c c u r within

From the Department of Pathology, Case Western Reserve University School of Medicine at MetroHealth Medical Center, Cleveland, OH. Accepted for publication April 10, 1997. Supported by a grant from the CysticFibrosis Foundation. Address correspondence and reprint requests to Joseph E Tomashefski,Jr, MD, Department of Pathology, MetroHealth Medical Center, 2500 MetroHealth Dr, Cleveland, OH 44109. Copyright ©1998 by W.B. Saunders Company 0046-8177/98/2901-001258.00/0

65

positive) were most closely associated with chondr01ysis. Dystrophic calcification and ossification were more commonly seen in CF bronchi and dysu'ophic calcification was present even in the lobar branc~hes. Destruction of bronchial cartilage is the result of sustained bronchial infection and chronic inflammation and is an additional contributory factor to bronchiectasis and airway instability in patients with CF. HuM PATHOL29:65-73. Copyright © 1998 by W.B. Saunders Company Key words: cystic fibrosis, bronchiectasis, bronchial cartilage, inflammatory mediators.

Abbreviations: CF, cystic fibrosis; NTM, nontuberculous mycobacteria; IL, interleukin; TNF-~x,tumor necrosis factor-alpha.

the cartilage of the most proximal airways. Our findings confirm the presence of widespread destruction of bronchial cartilage in CF-associated lung disease. The loss of bronchial cartilage contributes to airway instability and the progression of bronchiectasis.

METHODS Five right lungs and one left lung were obtained at autopsy from six patients with CE The fight lung ~vas also selected for study from autopsy specimens of each o f four young adults who died without CF or significant chronic lung disease. All lungs were expanded and fixed with 10% neutral buffered formalin perfused through the main bronchus. The fixed lungs were sagittally sliced, beginning at the lateral edge, at 1-cm intervals to the midsagittal plane. The lateral lung slices were then used for routine histotogica! sampling after they were macroscopically p0int-counted for four anatomic compartments: parenchyma, nonparenchyma, bronchi, and blood vessels, as previously described. 6 The volume proportion of each anatomic compartment was then calculated. The medial (unsliced) p0rtion of the lung was used for bronchial dissections. Selected segmental bronchi, including the upper lobe apical segment (all patients and controls) and the middle lobe medial segment (three CF patients and three controls), were carefully dissected, along with their subsegmental branches, as far as possible to the lung periphery and were removed intact from the surrounding lung tissue. The linear distance from the origin of the segmental bronchus to the terminus of selected, prominent, subsegmental branches ("axial paths") was either directly measured or calculated (using the average thickness of serial sectiofls obtained for histological examination). The number of subsegmental bronchial divisions along each of the axial paths was counted, and each division was consecutively numbered. For the purposes of localization, the bronchial "level" refers to the interval between divisions and is numbered the same as the division immediately proximal to it. The lobar, segmental, and subsegmental bronchi were then serially cross-sectioned into slices averaging 3.5 mm in thickness. Each cross-section was localized on a diagram of the dissected bronchus and processed for histological evaluation. All bronchial cross-sections were stained with hematoxylin-

HUMAN PATHOLOGY Volume29, No. 1 (January 1998) eosin. Selected sections were stained with alizarin red, von Kossa, Masson trichrome, or Movat pentachrome stain. Each histological section was examined by light microscopy for the presence or absence of cartilage and tor pathological changes such as inflammation and destruction, fibrous replacement, calcification, and ossification involving cartilage. After histological review, the furthest subsegmental level at which cartilage was found ("cartilage level") and the linear distance to which cartilage extended along each subsegmental axial path ("cartilage extension") were determined. For each dissected bronchus, the ratio of the mean cartilage extension to the mean length of the axial paths was calculated and expressed as a percentage ("% cartilage extension"). To standardize and compare the distribution of cartilage in CF and control bronchi, the number of subsegmental bronchial branches, with or without cartilage, at a specified subsegmental level were also respectively counted. For the upper lobe apical segmental bronchi, the proportion of level 4 subsegmental airways that contained cartilage was recorded. For the middle lobe bronchi, the proportion of cartilage-bearing level 5 airways was noted. A similar calculation of the percentage of axial pathways with cartilage at or exceeding a set distance along the axial path (40 mm for apical bronchi, 60 mm for medial bronchi) was also obtained. To further evaluate the cellular infiltration and fibrous replacement of bronchial cartilage, immunohistochemical stains were performed on deparaffinized sections from five CF lung specimens using antibodies to factor VIII-related antigen (DAKO Corporation, Carpinteria, CA; polyclonal; 1:1,000 dilution); muscle-specific actin (Enzo Diagnostics, Farmingdale, NY; monoclonal; 1:4,000), Vimentin (Biogenex Laboratories, San Ramon, CA; monoclonal; 1:200); CD20 (L26) (DAKO Corporation; monoclonal; 1:200); CD45RO (UCHL-1) (DAKO Corporation; monoclonal; 1:200); CD3 (DAKO Corporation; polyclonal; 1:200); CD68 (KP1) (DAKO Corporation; monoclonal; 1:125); and myeloid histiocyte antigen (MAC387) (DAKO Corporation; monoclonal; 1:600). All slides were processed using the avidin-biotin complex method. To enhance antigen retrieval for some markers (CD20, vimentin, CD3, CD68, MAC387) sections were placed in citrate buffer (10 mmol/L, pH 6.0) and heated in a 700-plus Watt microwave oven on high power for 6 minutes X 3 (total, 18 minutes). Slides were cooled for 20 minutes, after which the routine staining procedure was applied. 7 Positive control slides consisted of appropriate human tissues processed in parallel. Negative controls substituted phosphate-buffered saline for the primary antiserum.

RESULTS Clinical Data The six patients with CF included five males and one female who ranged from 16 to 34 years o f age. T h e diagnosis of CF had b e e n confirmed by an elevated sweat chloride concentration along with compatible symptoms or a family history of CE All patients had severe respiratory complications and died of respiratory failure. Microbiological studies indicated that all patients bad respiratory tract infection with Pseudomonas aeruginosa and two with Burkholderia cepacia. O t h e r respiratory pathogens included yeast (four patients), Aspergillus species (two patients), and nontuberculous mycobacteria (NTM) (three patients). The control patients consisted of two m e n and two women who ranged from 26 to 37 years of age. Two

persons died suddenly of head trauma, one died of cerebellar herniation due to p s e u d o t u m o r cerebri, and one died o f alcohol-induced liver disease complicated by r u p t u r e d esophageal varices and empyema. Autopsy and clinical records did not disclose evidence of chronic lung disease or CF.

Cardiopulmonary Pathology T h e six patients with CF exhibited evidence of severe lung disease, including chronic bronchitis, bronchiectasis, obliterative bronchiolitis, and paracicatricial emphysema. Focal acute b r o n c h o p n e u m o n i a or mild interstitial p n e u m o n i a was present in four patients each, whereas regional alveolar damage or focal organizing p n e u m o n i a was seen in one person. T h e r e was n o histological evidence of mycobacterial infection in the three patients with positive a n t e m o r t e m sputum cultures for NTM. The volume p r o p o r t i o n of bronchi in the lateral lung slices of CF patients 1 through 6 was, respectively: 4.3%, 14.8%, 8.2%, 10.1%, 6.3%, and 40.5%. Four patients had right ventricular cardiac hypertrophy. A m o n g the controls, one patient had acute bronc h o p n e u m o n i a with p u l m o n a r y e d e m a and regional alveolar damage, and one had organizing empyema. O t h e r m i n o r histological features included mild centriacinar emphysema (two patients), increased alveolar macrophages (two patients), and small airways disease (one patient). No patient exhibited bronchiectasis. T h e volume proportion of bronchi in the lateral lung slices o f patients ! through 4, respectively, was: 1.8%, 1,9%, 2%, and 0.8%. T h e r e was n o significant cardiac pathology in any of the controls.

Bronchial Dissections

Controls. In all cases, bronchi tapered rapidly and could not be macroscopically dissected beyond the fifth subsegmental level o f the u p p e r lobe apical bronchi or the seventh subsegmental level o f the middle lobe (Fig 1). T h e data derived from the control bronchial dissections are presented in Tables 1 and 2. In each patient, cartilage was present along the entire length o f the bronchus to the limit of the dissection. Cartilage was present in all bronchi beyond the fourth apical bronchial or fifth medial bronchial divisions. Rarely, short skip areas devoid of cartilage were observed near the periphery o f the dissections (Fig 1). Histologically, there were few degenerative changes seen in bronchial cartilage of the control patients. T h e r e was focal basophilic, stippled calcification of cartilage in the main and lobar bronchi of one patient. In the segmental and subsegmental bronchi, stippled calcification (two patients), coarse calcifications (one patient), or metaplastic ossification (one patient) were minimal and focal. CF Patients. In all patients, bronchi were dilated in a fusiform fashion to form thin-walled, blind-ended pouches that could be dissected to the subpleural region (Figs 2 through 4). The degree of bronchiectasis varied from mild (patient 5) to severe (patient 6) (Fig

BRONCHIAL CARTILAGE DESTRUCTION IN CF (Ogrinc et al)

B

5 4

4 ~2

2

1 [

Normal cartilage I Cartilage absent

FIGURE 1. Control, (A) Dissected upper lobe apical bronchus. Bronchial branches taper peripherally. (Scale equals 0.83 cm). (B) Schematic of A, showing distribution of cartilage. Numerals represent level of bronchial branching, (C) Subsegmental level 5 bronchus. Two cartilagenous plates are indicated by arrowheads. (H&E, original magnification 31 x .)

4). Within an individual segment, the degree ofbronchiectasis occasionally varied among axial paths (Fig 2). The data derived from the bronchial dissections of the CF lungs are seen in Tables 3 and 4. Compared with the control bronchi, the length of the axial paths and the number of subsegmental bronchial divisions tended to be greater in the CF patients, with the exception of patient 6 (Fig 4), in whom there was near complete effacement of subsegmental divisions. The mean number of divisions per centimeter of axial path was similar in CF and control patients for the apical bronchi (0.99 v 0.85) but was less than control values for the CF middle lobe medial bronchi (0.71 v 0.97). The distribution of cartilage in the CF bronchi differed from that seen in the control patients. The linear extent of cartilage along the axial paths and the proportion of the axial path that contained cartilage was less in the CF lungs than in the controls. Cartilage began to disappear from the CF bronchi about the third or fourth subsegmental division. In the apical bronchi, cartilage was not present beyond the fifth subsegmental division in four patients; for the middle lobe bronchi, cartilage disappeared at the fourth subsegrnental division in two of three bronchi dissected. All axial pathways

of appropriate length were evaluated for the presence or absence of cartilage at a specified distance (40 mm for apical, 60 mm for medial) (Table 5). All control bronchi contained cartilage beyond the specified distance, whereas less than 50% of CF bronchi contained cartilage. Degenerative and inflammatory changes anteceded the disappearance of cartilage in all CF bronchi dissected. The main and lobar bronchi, studied in five patients, showed coarse, basophilic, platelike calcification in three patients (Fig 5), basophilic stippled calcification in one, and peripheral fibrous replacement in patient 6. Discrete calcification centered on the chondroyte lacunae was focally prominent (Fig 5). In subsegmental bronchi fine, stippled calcification and coarse platelike calcification were seen in two cases. Metaplastic bone replacement was prominent in three cases (patients 1, 2, and 3) and minimally present in the other three. Typically, bone with either cellular or fatty marrow was formed at the narrowed edge of the cartilage plate (Fig 6). Inflammation and replacement of cartilage by cellular fibrous tissue was usually present by the second or third subsegmental division and became progressively more prevalent distally (Fig 7). Inflamma-

67

HUMAN PATHOLOGY

Volume 29, No. 1 (January 1998)

Bronchial Measurements, Controls, Apical Segment-Upper Lobe

TABLE 1.

Patients Axial Paths (n)* Length (mm)t Mean Range Divisions + (range) Divisions/cm§ Cartilage levelll Mean Maximal Cartilage extension (ram)¶ Mean Maximal % Cartilage extension# Extent of cartilage/ maximal division** % Cartilaget# Subseg 4

1

2

3

4

1

2

2

4

40 40 4 1

47,5 45,6-49.4 ~5 0,84

49,4 38-60.8 4 0.86

57,2 26-82 3-5 0.77

4 4

4 5

4 4

4 5

40 40 100

47.5 49.4 100

47,5 57 96.0

57,25 82 100

4/4 100

5/5 100

4/4 100

5/5 100

*Number of axial pathways measured. tLinear distance of axial pathway from origin of segmental bronchus to end point of'dissection. SMaximal number ofsubsegmental divisions along axial pathway. §Mean number of subsegmental divisions per cm length of axial pathway, IISubsegrnental level to which cartilage extends, ~Linear distance to which cartilage extends along axial pathway. #Ratio of mean cartilage extension to mean length of axial pathway x 100. **Cartilage level versus maximal subsegmental bronchial division, ttPercentage of subsegmental level 4 branches with cartilage.

tion was typically mild and consisted of mononuclear cells consistent with macrophages, and fewer lymphocytes, p l a s m a cells, a n d n e u t r o p h i l s . T h e i n f l a m m a t o r y tissue t h a t r e p l a c e d t h e c a r t i l a g e also c o n t a i n e d s p i n d l e s h a p e d f i b r o b l a s t s a n d t h i n - w a l l e d ectafic c a p i l l a r i e s (Fig 7). T h e last r e m a i n i n g c a r t i l a g e i n a s u b s e g m e n t a l axial p a t h u s u a l l y was f o u n d at t h e b r o n c h i a l b i f u r c a t i o n . T h e r e w e r e n o d i s c r e t e scars i n t h e b r o n c h i a l wall t h a t c o u l d b e c o n s t r u e d as p r i o r sites o f c a r t i l a g e plates. I n g e n e r a l , t h e level at w h i c h c a r t i l a g e d i s a p p e a r e d c o r r e l a t e d w i t h t h e m a c r o s c o p i c severity o f b r o n c h i e c t a -

TABLE 2.

- - - -o............... ,~¢edcarlitage [ ] Cartilage absent

BronchialMeasurements, Controls, Medial Segment-Middle Lobe

Patients Axial paths (n)* Length (mm) * Mean Range Division* (range) Divisions/cm* Cartilage level* Mean Maximal Cartilage extension (mm)* Mean Maximal % Cartilage extension* Extent of cartilage/ maximal division* % Cartilage subseg 5t

1 1

2 2

3 2

70 70 6 0.86

60.8 57-64.6 6-7 1.1

60.8 53.2-68.4 5-7 0,98

6 6

6,5 7

70 70 100

60.8 64,6 100

6/6 100

7/7 100

6 7 58.9 68,4 96,9

FIGURE 2. CF, Patient 3. (A) Dissected upper lobe apical bronchus. Note variable severity of bronchiectasis. (Scale equals 1.3 cm), (B) Schematic showing greater degree of cartilage loss in more severely ectatic airways (right). (C) Subsegmental level 5 bronchus with thin fibrous wall devoid of cartilage. Compare with figure 1C. (H&E, original magnification 31 × .)

7/7 100

*For definitions of variables, see footnotes of Table 1. tPercentage of subsegmental 5 branches with cartilage.

68

BRONCHIAL CARTILAGE DESTRUCTION IN CF (Ogrinc et al)

Immunohistochemica! Stains. Within the destroyed cartilage, mononuclear cells ~ith vesicular, folded nuclei were prominently stained with CD68 and MAC387 and were aligned adjacent to the scalloped edges of destroyed cartilage fragments (Fig 8). Small round lymphocytes were positive for CD3. No staining for UCHL-1 and CD20 was observed, probably because of interference by prolonged fixation. Endothelial cells of capillary ingrowths were positive for factor VIII-related antigen (Fig 8). Vimentin was positive in connective tissue fibers, chondrocytes, and inflammatory cells, thus indicating preservation ofimmunogenicity in this formalin-fixed tissue. Stain for muscle-specific actin was negative.

B

ilage ~l/destroyedcartilage ~sent

FIGURE 3. CE Patient 1. (A) Dissected middle lobe bronchi with saccuiar bronchiectasis. Lobar bronchus is at bottom; medial (M) and lateral (L) segmental bronchi are indicated. (Scale equals 1.4 cm). (B) Schematic of medial segmental bronchus showing diffuse loss of bronchial cartilage beyond level 4 divisions. Normal cartilage

sis. In patient 5, with relatively mild bronchiectasis, cartilage could be found in the tenth subsegrnental level, although fibrous replacement of cartilage was present in more proximal branches. In patient 6, with the most severely dilated bronchi, nearly all cartilage was absent by the second subsegrnental level, and fibrous replacement of cartilage was ongoing in the level 1 bronchus.

Degenerated/destroyed cartilage Cartilage absent FIGURE 4. CF, Patient 6. (A) Apical segmental bronchus showing massive bronchiectasis, (Scale equals 1.5 cm), (B) Schematic showing complete effacement of bronchial branching pattern and diffuse loss of cartilage. Destruction of cartilage extends into the segmental bronchus.

69

HUMAN PATHOLOGY TABLE 3.

Volume 29, No. 1 (January 1998)

TABLE 5.

Bronchial Measurements, CF Patients, Apical Segment-Upper Lobe

Patients Axial paths (n)* Length (mm)* Mean Range Divisions* (range) Divisions/cm* Cartilage level* Mean Maximal Cartilage extension (ram)* Mean Maximal % Cartilage extension* Extent of cartilage*/ maximal division % Cartilage subseg 4*

1

2

3

4

5

6

6

5

7

3

2

3

Controls

63.2 72.1 44.7 56 59 64.3 51-76 56-80.5 38-51 38.5-66.5 4%71 61-70 4-7 5-9 4-7 4-7 6-10 3 0.94 0.9 1.2 0.9 1.3 0.5 4

4

5

3.7

8

2

5

5

7

5

10

2

39.3 52

41.3 49

39.1 46

30.3 42

54.5 62

19 20

62.2

57.3

87.5

54.1

92.4

29.5

5/7 83

4/9 75

7/7 88

5/7 25

Bronchopulmonary Segment Distance* Nt Apical, upper lobe Medial, middle lobe

CF

Bronchi With Bronchi With Cartilage:~ CartilageS (%) Nt (%)

40 mm

9

9 (100)

24

11 (46)

60 mm

5

5 (100)

14

5 (36)

*Set distance along axial pathway at which cartilage was evaluated histologically. tN, number of axial pathways studied. :~Number (%) of axial pathways with cartilage at the set distance. e x t e n d s f o r at least 10 g e n e r a t i o n s a l o n g t h e axial p a t h o f s e g m e n t a l airways. I n b r o n c h i e c t a t i c airways, however, b r o n c h i a l subdivisions w e r e r e d u c e d , a n d b r o n c h i ectatic saccules were n e a r l y d e v o i d o f c a r t i l a g e A ~ I n t h e c u r r e n t study, t h e m e a n n u m b e r o f b r o n c h i a l subdivisions p e r c e n t i m e t e r was s i m i l a r i n C F a n d c o n t r o l a p i c a l airways a n d slightly r e d u c e d in C F m e d i a l air-

10/10 2/3+ 100 0

*For definitions of variables, see footnotes of Table 1.

DISCUSSION T h e results o f t h e c u r r e n t s t u d y e m p h a s i z e t h a t c a r t i l a g e is c o n s i s t e n t l y lost in t h e b r o n c h i e c t a t i c airways o f p a t i e n t s with a d ~ a n c e d CF-associated l u n g disease. I n t h e u p p e r l o b e a p i c a l a n d m i d d l e l o b e m e d i a l s e g m e n t s , c a r t i l a g e d r o p o u t o c c u r r e d as early as subsegm e n t a l level 2, a n d c a r t i l a g e was c o n s p i c u o u s l y a b s e n t in b r o n c h i a l levels 4 a n d 5. I n c o n t r a s t , in t h e l u n g s o f c o n t r o l p a t i e n t s , c a r t i l a g e was u n i f o r m a l l y p r e s e n t at t h e s e levels. I n g e n e r a l , t h e m a c r o s c o p i c d e g r e e o f b r o n c h i e c t a s i s d i r e c t l y c o r r e s p o n d e d to t h e e x t e n t o f c a r t i l a g e loss. Destruction and elimination of bronchial cartilage h a s b e e n p r e v i o u s l y d e s c r i b e d in b r o n c h i e c t a s i s d u e to c a u s e s u n r e l a t e d to CF. 3,4 W h i t w e l l 3 o b s e r v e d t h a t cartil a g e was d e s t r o y e d a l o n g with o t h e r m u r a l s t r u c t u r a l c o m p o n e n t s in f o l l i c u l a r a n d s a c c u l a r b r o n c h i e c t a s i s . H a y w a r d a n d R e i d 4 f o u n d t h a t in n o r m a l l u n g s c a r t i l a g e TABLE4.

Bronchial Cartilage at Set Lengths Along Axial Pathways

Bronchial Measurements, CF Patients, Medial Segment-Middle Lobe

Patients

1

2

3

Axial paths (n)* Length (ram)* Mean Range Divisions* (range) Divisions/cm* Cartilage level* Mean Maximal Cartilage extension (mm)* Mean Maximal % Cartilage extension* Extent of cartilage/ maximal division % Cartilaget subseg 5

4

6

6

111.5 9%120 5-6 0.63 4.75 5

86.9 73.5-94.5 5-8 0.74

55.3 42-69 4-6 0.82

3.3 4

4 6

74 76

50.2 59.5

45.7 62

66.4

57.8

82.6

4/6 0

4/8 0

5/6 25

FIGURE 5. Dystrophic calcification of cartilage of central bronchus. A dense band of calcium rims the outer edge of the cartilage plate. Calcification centered on chondrocyte lacunae is concentrated at inner third of plate. (von Kossa, original magnification x53.) Inset: Stippled calcification centered on chondrocytes. (von-Kossa, original magnification x212.)

*For definitions of variables, see footnotes of Table 1. tPercent ofsubsegmental 5 branches with cartilage. 70

BRONCHIAL CARTILAGE DESTRUCTIONIN CF (Ogrinc et ai)

chronic inflammation and subsequent fibrous replacement, which were noted in all cases proximate to the point at which cartilage ultimately disappeared from the bronchial wall. Immunohistochemical stains of the chondrocentric inflammatory cell populations show mainly macrophages (CD68, MAC387 positive) with fewer numbers o f T lymphoctyes (CD3 positive), plasma cells, and neutrophils. 12,1~ The active inflammatory destruction of bronchial cartilage in CF differs from the reported loss of bronchial cartilage due to atrophy in emphysema. 14,15In emphysema, there is little histological evidence of chronic inflammation.14 Reduction in bronchial cartilage, as assessed by toluidine-bluestained whole mounts, has also been reported in patients with chronic bronchitis, in which sustained chronic bronchial inflammation may also play a role. 16 Inflammation in CF-associated lung disease occurs early and is sustained and severe. 17Khan et alls showed that infants with CF at 4 weeks postpartum had increased neutrophils, elevated levels of interleukin-8 (IL-8) (a neutrophil chemotacfic factor), elevated neutrophil elastase, and elastase/alpha-1 protease inhibitor complexes in bronchoalveolar lavage fluid and increased IL-8 mRNA in the cytoplasm of macrophages, is Birrer et aP9 documented significant pulmonary protease-

FIGURE 6, Metaplastic ossification with fatty marrow replaces lateral tapered portion of cartilage plate. (H&E, original magnification x 108.)

ways. The average length of dissected axial paths was longer in the CF patients than in controls because of the technical difficulty of dissecting bronchi in the normal lung. As normal bronchi taper peripherally, it becomes increasingly difficult to remove them intact from their parenchymal investment. Terminal bronchiectatic airways in CF, however, are sufficientlysclerotic and dilated to enable more peripheral dissection. Our measurements of dissected bronchi indicate that, compared with controls, cartilage in the CF airway disappears not only at a more proximal division, but also at a shorter absolute distance along the segmental axial pathway. The premature disappearance of bronchial cartilage persists when the bronchi are standardized for differences in dissected length. Although chondritis and apparent loss of bronchial cartilage has been observed histologically in CE there has been no comprehensive study of airway cartilage in CF-associated lung disease. 2 Previous morphometric studies of central airways show no apparent difference in the percentage of cartilage in CF patients versus normals, s'l° These studies, however, evaluate only single transections of lobar bronchi and are, therefore, inadequate for assessing cartilage in the peripheral airways, where destruction and loss of cartilage is most likely to occur. In an autopsy study of CF patients, Griscom et aP 1 observed that tracheal cartilage was pale with irregular outlines and empty lacunae, suggesting chondrocyte necrosis. Osseous metaplasia was found in 1 of 15 tracheas studied. These authors suggested that chondromalacia may contribute to abnormal tracheal flaccidity in CE n The major degenerative changes of cartilage in the central bronchi in the current study were punctate or coarse calcification in three patients and fibrous replacement of cartilage in one. Osseous metaplasia was frequently encountered in the segmental or subsegmental branches but not in the main or lobar bronchi. Tracheal cartilage was not studied in our patients. Based on the current study, we conclude that the elimination of bronchial cartilage in the peripheral airways of CF patients is the direct result of sustained

FIGURE 7. Erosion and destruction of bronchial cartilage by chronic inflammatory cells and fibrous tissue. Neovascularization by dilated capillaries is also present at left edge of cartilage, (H&E, original magnification x435.)

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HUMAN PATHOLOGY

Volume 29, No. 1 (January 1998)

FIGURE 8. (A) Mononuclear cells staining positively for CD68 (arrowheads) are notably aligned along destroyed cartilage fragments. (B) A similar appearance of mononuclear cells positive for MAC387 (arrowheads). (C) Endothelial cells of dilated capillary vessels (v) are highlighted by stain for factor VIII related antigen. A fragment of cartilage is seen centrally. (A, B, C, original magnification ×294.)

The role of IL-1 in articular cartilage destruction has been well studied. It is released mainly from the monocyte/macrophage system and is considered the "prototypic inducer of catabolic responses in chondrocytes. ''26 IL-1 also affects the calcification process of cartilage in vitro by inhibiting the onset of calcification in ossifying cartilage. 27 This is discrepant with our findings of increased calcification and ossification of bronchial cartilage plates in CF lungs, but the in vivo actions of IL-1 and its actions on bronchial cartilage have yet to be described. The cause of enhanced calcification of proximal bronchial cartilage in CF patients is unknown, but it is likely to also be secondary to chronic bronchopulmonary infection. In elderly persons without CF, radiographic evidence of calcification of tracheobronchial cartilage represents a common, asymptomatic manifestation of aging. 2a None of our patients received longterm warfarin therapy, which has been associated with premature calcification of tracheobronchial cartilage. 29 Of theoretical concern is destruction of cartilage due to fluoroquinolone antibiotics used to treat bronchopulmonary Pseudomonas infection in CE 3° Although fluoroquinolones cause destruction of articular cartilage in immature, susceptible animal species, there is no evidence of destructive arthropathy (or injury to bronchial cartilage) due to fluoroquinolones in humans. ~ Amelioration of CF-related lung disease with antiinflammatory ibuprofen therapy indicates that modulation of the inflammatory response can be clinically

antiprotease imbalance by 1 year of age in children with CE 19 Even clinically stable CF patients with mild lung disease have ongoing evidence of lung inflammation .2o Increased degradation products of elastin and collagen found in the urine of patients with CF have been linked to morphological evidence of destruction of bronchial connective tissue. 2a In the current study, we observed numerous neutrophils in bronchial lumens but relatively few neutrophils in proximity to destroyed cartilage. Although it is possible that bronchial cartilage is destroyed secondarily by proteases that diffuse from bacteria and neutrophils residing in the bronchial lumen, we found little histological evidence of direct injury to cartilage by neutrophils. Other inflammatory mediators such as IL-1 and tumor necrosis factor alpha (TNF-e0 are increased in the serum and bronchial microenvironment of CF patients and likely play a role in mediating cartilage destruction. 22-24 These macrophage-derived mediators have been linked to the destruction of articular cartilage in rheumatoid arthritis and osteoarthritis, in which they act directly on chondrocytes to increase matrix degradation while decreasing matrix synthesis25 Articular hyaline matrix is replaced by fibrous tissue that is not capable of resisting compressive load. z5 An analogous situation may occur in CF, in which macrophages release inflammatory mediators that damage chondrocytes and cartilage matrix, thereby furthering the development of bronchiectasis because the airways are unable to resist increasing expansile pressures.

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BRONCHIAL CARTILAGE DESTRUCTIONIN CF (Ogrinc et al)

beneficial.32Other attempts at decreasing airway inflammation have included systemic corticosteroid therapy or specifc aerosolized protease inhibitors. 3~,~4 A decrease ofgranuloma-mediated articular cartilage destruction in mice has been accomplished with combined cortisone/heparin therapy, which inhibits mononuclear cell influx and angiogenesis into the cartilage. 35 It is unknown to what extent antiinflammatory therapy can alter the development and progression of bronchiectasis in CE In summary, our study clearly indicates extensive loss of cartilage with increasing bronchiectasis attributable to sustained bronchocentric inflammation in the CF lung. Cartilage destruction appears to be linked to the invasion of the chondroid plates by macrophages, which are known to produce cytokines (IL-1 and TNF-a) catabolic to cartilage. The loss of cartilage is obviously not the sole explanation for the development ofbronchiectasis, but it is a reasonably important contributing factor.

Acknowledgment. T h e authors thank Denise Hatala Lewis for technical support, Debra Moscalink for secretarial assistance, Vince Messina for the photographs, and Kathleen Digney for artwork. REFERENCES 1. Ramsey BW: Management of pulmonary disease in patients with cystic fibrosis. N EnglJ Med 335:179-188, 1996 2. TomashefskiJFJr, Dahms BB, Abramowsky CA: The pathology of cystic fibrosis, in Davis PB (ed): Cystic Fibrosis. New York, NY, Marcel Dekker, 1994, pp 435-489 3. Whitwell F: A study of the pathology and pathogenesis of bronchiectasis. Thorax 7:213-239, 1952 4. Hayward J, Reid LM: The cartilage of the intrapulmonary bronchi in normal lungs, in bronchiectasis, and in massive collapse. Thorax 7:98-110, 1952 5. Reid LM: Reduction in bronchial subdivisions in bronchiectasis. Thorax 5:233-247, 1950 6. Tomashefski JF Jr, Bruce M, Goldberg HI, et al: Regional distribution of macroscopic lung disease in cystic fibrosis. Am Rev Respir Dis 133:535-540, 1986 7. Brown RW, Chirala R: Utility of microwave-citrate antigen retrieval in diagnostic immunohistochemistry. Mod Pathol 8:515-520, 1995 8. Matsuba K, Thurlbeck Vv~l: A morphometric study of bronchial and bronchiolar walls in children. Am Rev Respir Dis 105:908913, 1972 9. Sobonya RE, Taussig LM: Quantitative aspects of lung pathology in cystic fibrosis. Am Rev Respir Dis 134:290-295, 1986 10. Tomashefski JFJr, Morgan J, Bruce MC: The central bronchial glands in cystic fibrosis, a morphometric, clinicopathologic study. Am Rev Respir Dis 135:A464, 1987 (suppl) 11. Griscom NT, Vawter GF, Stigol LC: Radiologic and pathologic abnormalities in the trachea of older patients with cystic fibrosis. AmJ Radiol 148:691-693, 1987 12. Flavell DJ, Jones DB, Wright DH: Identification of tissue bistiocytes on paraffin sections by a new monoclonal antibody. J Histochem Cytochem 35:1217-1226, 1987 13. Pulford KAF, Rigney EM, Micklem KJ, et al: A new monoclonal antibody that detects a monocyte/macrophage associated antigen in routinely processed tissue sections.J Clin Patho142:414-421, 1989

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