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Ninth Industrial Toxicology and Pathology Short Course
23‐27 July, 2012
Contemporary Concepts in Target Organ Toxicologic Pathology
Respiratory system
Respiratory Response to Toxic Injury (Lung)
Eric Wheeldon
Safety Assessment
GlaxoSmithKline
27 July 2012
Respiratory Response to Toxic Injury (Lung)
Species Differences*Rat
Lung volume: 10.8mlELF volume : ~200µlNumber of alveoli: 19.7 x 106
Alveolar diameter: 70µM
DogLung volume: 1322mlELF volume: ~14.6mlNumber of alveoli: 1040 x 106
Alveolar diameter :126µM
HumanLung volume: 4341mlELF volume: ~24mlNumber of alveoli: 486 x 106
Alveolar diameter: 219µM
Structure* and Function*
architecture, branching
physiology
inhalation
air handling
gas exchange
Histology and response to injury*
cell types and distribution
vulnerability, clearance, inflammation
background changes
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Orders of branching
Length (mm) Volume (ml) Area (cm2)
1 220 80 2.0
14 130 71 13.9
3 5 43 76.1
3 3.6 865 281.0
3‐9 2.6 3000 7x105
Tidal Volume (ml) Minute Ventilation (ml/min)
Rat 2 150
Dog 200 3100
Monkey 20 750
Human 500 7000
Respiratory Response to Toxic Injury (Lung)
Respiratory Response to Toxic Injury (Lung)
Respiratory Response to Toxic Injury (Lung)
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Bronchial Cell typesAirway Type
Lining Cell Secretory Cell
Bronchi Ciliated cell Goblet cell
Terminal bronchiole
Ciliated cell Clara cell
Alveolus Type I Type II
Respiratory Response to Toxic Injury (Lung)
Epithelia of conducting airwaysSpecies Differences
Airway Species Cell types (approx %)$
Basal Ciliated Clara Serous Mucous
Trachea Rat 20 30 0 40 <1
Mouse 10 40 50 0 <1
Bronchi Rat 20 40 0 20 <1
Mouse 2 40 55 0 <1
Bronchiole Rat 0 60 40 0 0
Mouse 0 40 60 0 0
*other cells e.g., neurosecretory, eccrine also present
$ exact figures vary between sources
Respiratory Response to Toxic Injury (Lung)
Mucociliary clearance
• Defense against insoluble particulates
• Ciliated cells ‐ co‐ordinated movement
• Secreting cells ‐mucus, serous
Respiratory Response to Toxic Injury (Lung)
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• 2 layers of fluid
– sol ‐ lower level, watery allows cilia to move
– gel ‐ upper layer of mucus
• Lining fluid contains secretions ‐ IgA, lysozyme, anti‐proteases ‐ SLPI, elafin (elastase‐specific inhibitor), cytokines
Respiratory Response to Toxic Injury (Lung)
http://reni.item.fraunhofer.de/reni/trimming/trimm.php
Respiratory Response to Toxic Injury (Lung)
Particle impaction:
• Airflow changes direction• Particles near airway surface deposits by inertial impaction• Depends on particle stopping distance at the airway velocity
Respiratory Response to Toxic Injury (Lung)
Response to Toxic Injury – Airway epithelium (carina)
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Respiratory Response to Toxic Injury (Lung)
Response to Toxic Injury – Airway epithelium (carina)
Respiratory Response to Toxic Injury (Lung)
Response to Toxic Injury – Airway epithelium (carina)
RESISTANCE THROUGH A TUBE
IS PROPORTIONAL TO1___________
(radius)4
Respiratory Response to Toxic Injury ‐ Airways
Diffuse large airway disease
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Respiratory Response to Toxic Injury ‐ Airways
Airway Type Lining Cell Secretory Cell
Bronchi Ciliated cell Goblet cell
Respiratory/ Terminal bronchiole
Ciliated cell Clara cell
Alveolus Type I Type II
Respiratory Response to Toxic Injury ‐ Airways
Respiratory Response to Toxic Injury ‐ Small Airways
Small Airways as Critical Sites for Damage
• Particle size of most microorganisms and irritant dusts (0.5‐2.0 m) effectively reach the bronchioalveolar junction (BAD) level and deposit
• No protective mucus layer (due to gas exchange function)
• Absence of ciliary apparatus
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Respiratory Response to Toxic Injury ‐ Small Airways
Bronchiolar‐ alveolar duct junction (BADJ) common site for pathology
Respiratory Response to Toxic Injury ‐ Small Airways
Respiratory Response to Toxic Injury ‐ Small Airways
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Respiratory Response to Toxic Injury ‐ Alveoli
Terminal Airflow in the airspaces
red blood cell
type I pneumocyte
alveolar macrophage movingthrough alveolar pore of Kohn
fused basement membranesof capillary and type I pneumocyte
at areas of contactalveolar capillaries
interalveolar septumcontaining elastic and
collagen fibers
Respiratory Response to Toxic Injury ‐ Alveoli
Alveolar Cell Types
Airway Type Lining Cell Secretory Cell
Bronchi Ciliated cell Goblet cell
Terminal bronchiole
Ciliated cell Clara cell
Alveolus Type I Type II
Respiratory Response to Toxic Injury ‐ Alveoli
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Respiratory Response to Toxic Injury ‐ Alveoli
Alveolar epithelium
• Type I cells ‐
– squamous, large surface area ‐ susceptible to damage
– maintain barrier to fluid loss, allow diffusion of gases,
– cannot divide
• Type II cells ‐
– cuboidal, lamellar bodies (surfactant), corners of alveoli
– divide to replace type I and II cells
– synthesizes surfactant, type IV collagen, PAF,
– aa metabolites
– modulate macrophage function
Respiratory Response to Toxic Injury – Alveoli
Retention and Clearance of Deposited Particles
Alveolar Region:
– No protective mucus layer due to gas exchange function
– Soluble particles
• Pass through membrane
into blood
– Insoluble particles
• Engulfed by alveolar macrophages
– Lymph nodes
– Mucociliary escalator
Respiratory Response to Toxic Injury – Alveoli
Alveolar macrophages
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Respiratory Response to Toxic Injury – Airspaces (Alveoli)
Retention and Clearance of Deposited ParticlesMacrophage accumulation
Respiratory Response to Toxic Injury – Alveoli
Alveolar inflammation
Respiratory Response to Toxic Injury – Alveoli
Alveolar edema
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Respiratory Response to Toxic Injury – Alveoli
Diffuse alveolar damage
Respiratory Response to Toxic Injury – Alveoli
Diffuse alveolar damage
Respiratory Response to Toxic Injury – Alveoli
Diffuse alveolar damage ‐ sequel
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Respiratory Response to Toxic Injury – Alveoli
Alveolar destruction ‐ emphysema
Response to Foreign Material
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Respiratory Response to Toxic Injury – Alveoli
Inhaled inert material
Respiratory Response to Toxic Injury – Alveoli
Alveolar fibrosis (Silica – 30 days)
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Respiratory Response to Toxic Injury
Collection / Inflation Procedures for Lung
I. Syringe technique
II. Constant pressure technique
Respiratory Response to Toxic Injury – Alveoli
Importance of Fixation
Respiratory Response to Toxic Injury
Syringe technique
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Respiratory Response to Toxic Injury
Constant pressure technique
– Advantages
• Reasonably inexpensive
• Excellent control over pressure
• Least amount of artifact
– Disadvantages
• Requires more time
• Availability of apparatus
Airway Macrophages
Air inflation/immersion fixationMigrating macrophages in situ
Intratracheal fixationMacrophages flushed from airway
Respiratory Response to Toxic Injury (Lung)
Case Study
Macrophage aggregations in the lung
Adverse? Non‐adverse? Incidental?
Respiratory Response to Toxic Injury (Lung)
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Respiratory Response to Toxic Injury – Alveoli
Variable terminology for macrophages in the lung
• alveolar foamy macrophages• alveolar histiocytosis• prominent alveolar macrophages• intra‐alveolar macrophages• foamy alveolar macrophages• increased macrophage aggregates• alveolar macrophage aggregation• macrophage aggregation, alveolar
Respiratory Response to Toxic Injury – Alveoli
Alveolar macrophages
Female High Dose
x5 x40
Respiratory Response to Toxic Injury – Alveoli
Rat 4 Week Tox Study
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Respiratory Response to Toxic Injury – Alveoli
Rat 4 Week Tox Study
Female Control
x5 x40
Respiratory Response to Toxic Injury – Alveoli
Foamy Macrophages (2)
• Rat 26‐week study (3, 30 and 300mg/kg/day), alveolar foamy macrophages in control rats (7/12 control males & 5/12 control females) in rats at the end of treatment period
• In males, foamy macrophages similar to concurrent controls, but at slight grade in a small proportion of animals with no clear dose response
• In females, an increased alveolar foamy macrophages in all treated groups. Some rats at slight grade & single rat at 300 mg/kg/day at moderate grade
• Recovery group: increased incidence in males & an increase in severity in females at 300 mg/kg/day at end of recovery phase
Male Female Dose (mg/kg/day) 0 3 30 300 0 3 30 300 Number Examined 12 12 10 11 12 12 12 12 Alveolar Foamy Macrophage/s
Minimal 7 6 5 6 5 6 5 7 Slight 0 2 3 2 0 3 3 2
Moderate 0 0 0 0 0 0 0 1 Total 7 8 8 8 5 9 8 10
End of dosing phase:
End of recovery phase:
Male Female Dose (mg/kg/day) 0 300 0 300 Number Examined 6 6 6 6 Alveolar Foamy Macrophage/s
Minimal 1 4 3 0 Slight 1 1 0 3 Total 2 5 3 3
Respiratory Response to Toxic Injury – Alveoli
Rat Foamy Macrophages –Historical Data
Incidence and Severity of Foamy Alveolar Macrophages in Lung from aCarcinogenicity Study in Rats
MALE FEMALE
Group No. 1 2 3 4 5 1 2 3 4 5
Group Designation C L LI HI H C L LI HI H
No. of Animals/Group 60 60 60 60 60 60 60 60 60 60
Lung
Foamy Alveolar Macrophages
Grade 1
Grade 2
Grade 3
Grade 4
Total
38
3
3
0
44
36
2
0
1
39
31
8
0
0
39
30
3
1
1
35
30
12
4
1
47
27
4
0
0
31
21
9
2
0
32
36
8
1
0
45
23
10
4
0
37
23
9
4
1
37
C = Control. L = Low. LI = Low intermediate. HI = High intermediate. H = High.
Grade 1 = Minimal.Grade 2 = Slight.Grade 3 = Moderate.Grade 4 = Moderately severe.
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Respiratory Response to Toxic Injury – Alveoli
Alveolar macrophage response
What is clinical & regulatory significance of foamy macrophages in lungs from oral tox studies in rat (4 and 26 wk) ?
“Prior to long term clinical trials, provide adequate justification that the finding is within spontaneous background and/or is not an adverse finding, or identify a NOAEL dose in the rat by conducting an additional toxicity study”.
Respiratory Response to Toxic Injury – Alveoli
Unique aspects of Respiratory System
• Tremendous surface area
• Highly elastic tissue
• Low tissue density
• Delicate fluid air interface
• Dual blood supply
• Multiple types of air conducting units
• Complexities of cell types and locations
• Species variations
•Back ups
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Differences ‐ rodent and human
Anatomical features Human Rodent
Pleura Thick Thin
Airway Size Small calibre Large calibre
Submucosal glands Abundant Rare
Respiratory bronchioles Present Absent
Clara cell Low numbers High numbers
Mucus cell Abundant Rare (serous)
Cardiac muscle in pulmonary veins
Absent Present
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Inflation Fixation
• Syringe technique
– Advantages
• Quick, Inexpensive, Practical, Convenient
– Disadvantages
• Risk of variation in pressure
• Risk of overinflation, alveolar rupture