Airway Host Defenses and Oxidative Stress

types of defense mechanisms that protect airways

physical, chemical, cellular

physical defense mechanisms examples

cough, barrier function, mucociliary clearance

chemical defense mechanisms examples

mucus composition and viscosity, antimicrobial peptides

cellular defense mechanisms examples

phagocytosis by macrophages, immune reponse

clearance of inhaled material is determined by

their size

impaction

largest particles fail to turn at the corners of the respiratory tract

particle hits wet surface and sticks, 95% particles >5 microns filtered by nose

representative site is nasopharynx

sedimentation

gradual settling of particles due to their weight, mechanism dominates in small airways

particles are medium size, 1-5 microns

representative site is small airways

diffusion

random movement of particles as a result of their continuous bombardment by gas molecules

only for the smallest particles (<0.1 microns)

representative site is alveoli

cough

protective reflex that is vital to remove foreign material and secretions from the airways

integral part of host defense mechanisms against inhaled particles (e.g. dust) and noxious substances (e.g. cigarette smoke, ammonia fumes)

especially important when other methods of clearance overwhelmed

can be excessive in obstructive diseases

cough and babies

absent at birth, takes approx one month to develop—newborns at increased risk for respiratory infections

what is cough initiated by?

airway obstruction, mechanical irritation, dust, cigarette smoke, chemical fumes, changes in ion concentration or osmolarity of the airway surface liquid (ASL)

specialized cough receptors

stimuli triggered, located in or under airway epithelium

exact identity unknown

cough step by step mechanism

cough receptor activation

peripheral sensory nerve activation

information relayed to brainstem (exact area unknown)

reflex stimulation of efferent limb results in contraction of skeletal muscle of abdomen and thorax

phases of cough

starts with a deep and rapid respiratory effort

deep inspiration followed immediately by compression brought about by closure of the glottis, activation of the diaphragm and chest wall muscles. acts to increase pleural, alveolar, and abdominal pressures to very high levels

sudden opening of the glottis resulting in high expiratory airflow, velocity approx 200km/hr—collapse of airways can occur

relaxation of muscles and reversal of pressures

bronchial epithelia

ciliated, pseudostratified columnar epithelium

tight junction

provide structural support

inhibit movement of materials via paracellular space (“barrier function”)

separate apical and basolateral domains (“fence function”)

mucus

sources: submucosal glands and goblet cells

5-10 microns thick: gel (more viscous) and sol (less viscous) layers

top gel layer sticky

cilia

5-7 microns length in trachea, 2-3 microns in 7th airways generation

beat in synchronized manner, 1000-1500x/min

tips of cilia interact with gel layer to propel forward

paralyzed by inhalation of certain toxic gases, smoke

together, what do mucus and cilia do?

move mucus blanket at approx 1mm/min in small peripheral airways and 2cm/min in trachea

end point: pharynx

swallowed

ciliated epithelium line

bronchi, bronchioles of lungs plus nasal cavity, paranasal sinuses, eustacian tubes, middle ear, pharynx, trachea

cilia found down to approx

16th bronchial division

structure of cilia

9+2 arrangement

9 interconnected doublet microtubules surrounding and joined by cross-bridges to 2 central microtubules

each double formed by an A- and B- subfiber

subfiber A complete microtubule, subfiber B shares part of A

paired ATPase or dynein arms on subfiber A

radial links or spokes on subfire A, which connect outer doublets to sheath surrounding 2 central microtubules

nexon links connect doublets

microtubule pattern changes at different levels of (this)

recovery stroke of cilia

from rest position, bend sideways and back

takes place near cell surface and accounts for ¾ of cycle time

effective stroke of cilia

move perpendicular to cell surface

this phase is when claws on tip of these engage

with overlying mucus and sweep it in a cephalic direction (towards head)

bases aligned in same direction so this takes place in same direction—beat cooperatively to propel mucus

overlying mucus penetrated by tips in this phase only—optimizes propulsive force

ciliary movement

coordinated by metachronal waves, coupling the beat of each to its neighbour

what is critical for effective mucus movement?

thickness of periciliary liquid layer

ciliary regulation

beat with a frequency between 12-15 Hz in nose and major airways, metachronal wave initially thought to be due to them physically interacting with each other but those separated by a gap of 10 microns are still able to display metachronal waves

maybe intracellular communication involved?

CBF rate

basal one can increase in response to insult

neuropeptide Y can inhibit this, via reducing intracellular Ca2+

rises in Cai2+ increase this

epithelium has mechanoreceptors that increase this in response to mechanical stimulation

factors affecting mucociliary host defense in healthy individuals

age, sex, sleep, exercise, gravity

how does age affect mucociliary clearance?

CBF is higher in neonates and teens, lower in adults

how does sex affect mucociliary clearance?

clearance slower in males than females

how does sleep affect mucociliary clearance?

clearance decreased during sleep

how does exercise affect mucociliary clearance?

strenuous exercise increases MCC (no effect of gentle exercise)

how does gravity affect mucociliary clearance?

no effect on healthy individuals, but postural drainage may assist in CF

primary ciliary dyskinesia (immotile cilia syndrome)

impaired ciliary activity, so incidence of respiratory infection high

usually caused by absence of dynein arms normally found in 9 peripheral microtubular doublets

missing radial spokes also observed

ciliary motion grossly abnormal and not coordinated into metachronal waves

common clinical findings with primary ciliary dyskinesia

bronchitis, rhinitis, sinusitis, otitis media (middle ear infection), obstructive lung disease

males usually sterile due to sperm immotility

respiratory tract is site of repeated infection

bronchiectasis

abnormal widening of bronchi

local damage to respiratory tract epithelium due to infection is thought to result in defective MCC

cystic fibrosis

defective ion permeability results in defective MCC, resulting in repeated bacterial infection

how is inhaled material cleared?

mucociliary system and alveolar macrophages

alveolar macrophages

no mucus or cilia in this area, instead particles engulfed by these

phagocytose foreign particles: migrate to small airways to load in mucociliary escalator or leave via lymphatics

contain lysozyme so can directly kill bacteria

this area remains sterile, although it may take time for dead organisms to be removed from the lung

what impairs alveolar macrophage activity?

cigarette smoke, alcohol, alveolar hypoxia, ozone, radiation

composition of respiratory mucus

water with high molecule mass cross-linked glycoproteins + serum and cellular proteins, e.g. albumin, enzymes, immunoglobulins

DNA present in pathological conditions (increases viscosity)

glycoproteins and water →viscoelastic gel properties

mucins

gigantic biopolymers of glycoproteins characterized by presence of one or more large region rich in serine and threonine

amino acids covalently attached via linkage sugar N-acetylgalactosamine

polyanionic

usually 70%+ carbohydrate

provide structural framework of defense barrier, prevent barrier dehydration, present carbohydrate sites which pathogens attach to

human respiratory tract mucus

mixture of MUC5AC and MUC5B (some MUC2 also)

antibacterial, antiviral, antifungal agents

airway mucus is a mixture of products from

alveolar liquid, secretory products from cells lining the surface epithelium, submucosal gland products, serum transudate

nose, trachea, larger bronchi contain

goblet cells and SMGs (both serous and mucous cells)

volume of SMGs is approx 40 times that of goblet cells

goblet cells and SMGs decrease peripherally

club cells may transform into

goblet cells in disease

how are mucins stored?

condensed and folded within cell

secretion of respiratory mucus

when secretory granule docks with plasma membrane, secretory pore forms

pore has high ionic conductance and Ca2+ inside the granule is exchanged for extracellular Na+

this Na+/Ca2+ exchange triggers a polymer-gel phase transition, when the mucin polymer matrix undergoes extensive swelling and switched from condensed to hydrated state

physical characteristics of mucus

complex, non-Newtonian properties

viscosity and elasticity classify it

viscosity of mucus

loss modulus G, the extent to which the gel resists the tendency to flow

elasticity of mucus

storage modulus G, measures the tendency for gel to recover its original shape following stress-induced deformation

physiologically, changes in physical properties/rheology of mucus

may greatly affect its ability to function as a lubricant, selective barrier, and the airways first line of defense against inhaled particles

what happens when mucus is too thick?

e.g. cystic fibrosis, where sputum viscosity can be 100,000x that of water = mucociliary clearance impaired with bacterial overgrowth

phlegm

purulent secretion that is a product of airway inflammation, containing breakdown products of inflammatory and epithelial cells; including DNA and actin fragments, bacteria, cell debris, mucins

what is phlegm called when it is expectorated?

sputum

sol layer or periciliary liquid (PCL)

low viscosity medium to facilitate ciliary beating

approx 7 microns depth

volume of ~1 microliter per cm² mucosal surface

contains many antibacterial factors e.g. lysozymes, lactoferrin, human beta-defensins, cathelecidin LL-37

has broad antibacterial spectrum, e.g. against S Aureus and P aeruginosa

hydration of mucus and depth of sol layer controlled by

transepithelial movement of ions and water

regulation of periciliary liquid layers- absorption

driven by active Na+ absorption via apical ENaC membrane and Na+/K+-ATPase in basolateral membrane creating an electrochemical driving force for paracellular passive Cl- transport

water then follows either through aquaporins or the paracellular pathway

regulation of periciliary liquid layers- secretion

driven by Cl- secretion through CFTR and other Cl- channels in the apical membrane

NKCC1 and the coupled action of an anion exchanger and NBC in the basolateral membrane accumulate Cl- in the cell

active Cl- secretion creates the driving force for Na+ movement across the epithelium through the paracellular pathway and water transport occurs paracellularly and/or transcellularly

antimicrobial peptides (AMP)

expressed by epithelial cells lining airways as part of innate host defense mechanism

generally cationic, allowing them to bind to negatively charged prokaryotic cell membranes

after binding to microbial surface, they lead to cell wall disruption

sequester nutrients and iron

exhibit broad-spectrum antimicrobial activity

activity of many inhibited by high levels of salt or serum proteins

secreted from surface epithelial cells and submucosal glands

may also have some activity against viruses and fungi

common bacterial pathogens in lung infections

P aeruginosa, H influenzae, K pneumoniae, B cepacia, S aureus—AMPs active against these

types of small AMPs

defensins, cathelicidins

defensins

human Beta (these) 1-4 are all expressed in airways epithelial cells, and so the PCL

expressed in neutrophils

36-42 amino acid residues

characteristic 6-cysteine motif, which results in a 3-disulphide-bonded secondary structure

human beta (these) 1 constitutively secreted into the PCL

human beta (these) 2, 3, 4 induced in response to pathogens, inflammatory cytokines

broad-spectrum activity against gram-positive and gram-negative bacteria, mycobacteria, and fungi

cathelicidins

only human example is LL-37, 37 amino acids

similar broad-spectrum activity to defensins, both gram-positive and gram-negative bacteria, plus Candida albicans

induced by pathogens such as P aeruginosa

over-expression in mice results in increased protection against bacterial challenge

chemotactic for neutrophils, monocytes, mast cells, and T cells

large AMP types

lysozyme, lactoferrin

lysozymes

secreted from epithelial cells and SMGs

induce lysis of gram-positive bacteria

lactoferrin

activated by inflammatory and infectious stimuli

sequesters iron from microbes

how do small and large AMPs work together?

synergistically, e.g. antibacterial activity of hBD-2 improved in presence of lysozyme and lactoferrin

sources of exogenous (inhaled) and endogenous ROS and RNS include

1. environmental factors such as ozone, air pollution (particulates such as those from diesel fuel combustion), particulates containing metals and cigarette smoke

2. endogenous species are produced as byproducts of mitochondrial respiration; inflammatory cells produce high levels in response to allergens and microbial infections

multiplicity and abundance of antioxidant systems available in lung result in

redox status being reducing not oxidizing

vast excess of reduced substances over oxidized ones maintained by

a rich array of antioxidant enzymatic and nonenzymatic effectors on the surface of, and within, epithelial cells in the airways

what happens when body is oxidatively “balanced”?

sufficient antioxidants in body to counter production of small amount of ROS

what happens when body is in oxidative stress?

if either the levels of antioxidants are diminished or the production of ROS is increased, balance of ROS to antioxidants is out of whack

what is a free radical?

any species that contains one or more unpaired electrons

e.g. superoxide anion O2*0-, hydroxyl radical *OH

non-radical reactive species

also important, include nitric oxide NO and hydrogen peroxide H2O2

antioxidants

neutralize oxidants, can either be enzymatic or non-enzymatic

non-enzymatic antioxidants

include vitamins C and E, uric acid, glutathione

enzymatic antioxidants

includes SODs, catalases, glutathione peroxidase

how is the superoxide anion broken down?

by SODs and H2O2 by various enzymatic antioxidants

glutathione

tripeptide composed of glutamic acid, cysteine, glycine

main function element is cysteine residue, which contains reactive thiol group

H2O2/hydroxyperoxidase

reduced by glutathione peroxidase (GPx) to H2O using GSh as the cosubstrate

organic ones reduced by corresponding alcohols in reaction catalyzed by GPx, or glutathione S-transferases

resulting GSSG rapidly reduced by glutathione reductase

what is an important marker of redox status?

ratio of GDH:GSSG

GSH

extremely efficient buffer of oxidative stress

most important antioxidant in airways, lower levels in ECL of CF patients

oxidant stress in CF

GSH levels significantly reduced in epithelial lining fluid

characterized by neutrophil-dominated airway inflammation = increased oxidant production

poor nutritional status associated with this disease, so lack of dietary antioxidants

CFTR channel able to transport GSH, therefore defective CFTR results in less of this antioxidant in ELF

consequences of oxidant stress in CF

CFTR-mediated GSH transport may serve to reduce mucus viscosity by disrupting disulphide bond formation in mucin proteins

decrease in antioxidant plus increase in ROS production = lot of oxidative stress, potential role for antioxidant therapies in this disease?

asthma

chronic inflammatory disease of lower airways, characterized by reversible airway obstruction and hyperresponsiveness

inflammation results in epithelial cell desquamation, mucus production, airway remodeling

inflammatory cells include mast cells, eosinophils, lymphocytes and activated monocytes, macrophages, neutrophils

ROS/RNS increased

oxidant stress in asthma

levels of eosinophil peroxidaze and myeloperoxidase increased in peripheral blood and in induced sputum and BAL

have higher levels of NO in their exhaled breath condensate

peroxynitrite

peroxynitrite

detected in asthmatic tissues and results in apoptosis and necrosis in asthmatics

exogenously applied, stimulates airway hyperresponsiveness

appears to contribute to airway remodeling that occurs in asthma

nitrotyrosine levels

increased in bronchoalveolar lavage (BAL) fluid of patients with severe asthma

ROS and asthma

spontaneously generated at higher levels in BAL cells isolated from patients with mild to moderate this disease compared to normal

presence of this in BAL cells inversely correlated to FEV1 in patients with this disease

what appears related to asthma disease severity?

levels of indirect markers of oxidant stress, e.g. isoprostanes

pathophysiology of oxidant stress in asthma

more inflammatory cells than controls, these cells produce more ROS when compared to controls

airway antigen challenge and (this disease) attacks are associated with immediate formation of O2*-

ROS production by neutrophils correlates with severity of airway hyperresponsiveness

increased biomarkers of eosinophil activation

increased biomarkers of eosinophil activation include

release of granule proteins such as EPO (eosinophil peroxidase) and major basic protein (MBP)