Defending the Ocular Surface in

Contact Lens Wear

D1

2016-Sep-14 © The International Association of Contact Lens Educators

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Attributions

Lecture contributors* Fiona Stapleton, Mark Willcox

Lecture editor** William Miller
IACLE executive editor** Etty Bitton
IACLE reviewer** Nilesh Thite, Lewis Williams
Lecture updated 2016-July
Lecture content rechecked 2016-Sep

* Original author of lecture **2016 Editor & reviewers

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 IACLE SPONSORS

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supported through educational grants & in-kind contributions

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 Published by
The International Association of Contact Lens Educators (IACLE)

Revised Edition 2016

© The International Association of Contact Lens Educators 2000-2015

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or
transmitted, in any form or by any means, without the prior permission, in writing, of:
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Micro-organisms: Types

• Bacteria
‒ chlamydiae
• Fungi
• Viruses
• Protozoans

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Abbreviations used in this lecture

• cfu : Colony Forming Unit(s)

• mm : micrometres (m–6)
• nm : nanometres (m–9)
• DNA : DeoxyriboNucleic Acid
• RNA : RiboNucleic Acid
• sp. : species* (singular)
• spp. : species (plural)
• LCP(s) : Lens Care Product(s)
• pg: picograms (g–12)
• ng: nanograms (g–9)
• mg : micrograms (g–6)
• mg: milligrams (g–3) * species is both singular & plural (specie
is a technical term for the physical form of
money, esp. coins)
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Micro-organisms: Chemical Characteristics

• Nucleic acids
‒ DNA
‒ RNA
• Proteins

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Micro-organisms: Physical Characteristics

• Eukaryotic or prokaryotic cell

• Size <0.5 μm (viruses)
1-2 μm (bacteria)
3-5 μm (fungi)
15-30 μm (some protozoans)

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Micro-organism Cell Types: Eukaryotes vs Prokaryotes

Eukaryotic Cells Prokaryotic Cells

Nuclear membrane present No nuclear membrane
Multiple chromosomes Single chromosome
Membrane bound organelles present Membrane bound organelles absent
(mitochondria, lysosomes)
Intracellular digestive vacuoles present Intracellular digestive vacuoles absent
Cells divide by mitosis Cells divide by binary fission

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Micro-organisms: Bacterium/Bacteria (generalized)
Mesosome Cytoplasmic
Cytoplasmic membrane
inclusions
Flagella

Cell Wall
Pili (fimbriae)

Cytoplasm
Capsule Septum
(forming)
Nuclear Material

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Classification of Bacteria

Nucleic acids RNA & DNA

Nuclear membrane No

External cell wall Yes (usually), rigid peptidoglycan

Antibiotic sensitivity Yes

Replication / reproduction Within & outside host cells

by binary fission

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Bacteria: Morphology

• Rods

• Cocci (spheres)

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Bacteria: Cell Division
ivision
fD (c)
Pla ne o

(a)
Diplococci

Sarcinae

Streptococci (d)

(b)

Tetrad Staphylococci

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Bacteria: Rods

(a) (e)
Single bacillus Vibrios

(b) (f)
Diplobacilli
Spirillum
(c)

Streptobacilli (g)
Spirochaete
(d)
Coccobacillus
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Bacteria: Cell Walls
GRAM-NEGATIVE CELL GRAM-POSITIVE CELL
WALL WALL

Outer membrane

Periplasmic space

Peptidoglycan

Cytoplasmic
membrane

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Bacteria: Cell Walls – Gram positive & Gram negative
Staphylococcus aureus Neisseria sp.

Gram negative
Gram positive
Propionibacterium sp. Pseudomonas aeruginosa

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Gram-positive Bacteria: Examples…

• Staphylococcus
• Streptococcus
• Pneumococcus
• Bacillus
• Corynebacterium
• Clostridium

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Gram-negative Bacteria: Examples…

• Pseudomonas
• Haemophilus
• Escherichia
• Neisseria
• Moraxella
• Shigella
• Salmonella

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Classification of Bacteria: Growth Requirements

• Atmospheric: • Temperature:

‒ aerobes ‒ thermophile
‒ anaerobes ‒ mesophile
‒ facultative anaerobes ‒ psychrophile
‒ microaerophiles

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Classification of Bacteria: Growth Requirements

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Classification of Bacteria: Antigenic

Serotyping, antigenic determinants:

• Envelope

• Flagellae

• Capsule

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Bacteria & Disease: Definitions

• Infection

• Colonization

• Asymptomatic carriage

• Virulence or pathogenicity

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Bacteria & Disease: Causation

• Via direct tissue invasion

• Via activation of the host’s immune system

‒ toxin/enzyme production  direct tissue damage

‒ lipopolysaccharide (LPS)

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Bacteria & Disease: Virulence Factors

• Adhesion:

‒ pili

‒ surface adhesins

‒ biofilm

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Bacteria & Disease: Virulence Factors

• Invasion:
‒ actin filament reorganization
‒ pseudopod formation
‒ engulfment of bacteria in a phagocytic vesicle
‒ release from phagocyte
‒ replication in host cytoplasm

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Bacteria & Disease: Virulence Factors

• Survival & acquisition of nutrients:

‒ siderophores

‒ surface proteins which sequester iron (Fe)

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Bacteria & Disease: Virulence Factors

• Evasion of host’s immune system:

‒ capsules

‒ survival of phagocytosis

‒ interaction with host proteins

‒ alteration of surface antigens

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Chlamydiae

• Small intracellular parasite (recognized as a bacterial species)

• When Gram stained - appear as tiny (0.2-1 mm) Gram-negative

bacteria

• Able to synthesize protein, DNA, & RNA

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Chlamydiae: Life Cycle

• Elementary Body (EB) internalized by host cells

• Organism survives in phagosome, & EB becomes a Reticulate
Body (RB)
• RB is an intracellular, non-infectious replicating form
• Phagosome enlarges forming an Inclusion Body (IB)
• Some RBs transform (revert) into the EB form
• Host cell releases EBs on lysis

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Chlamydiae & Ocular Disease

• Trachoma

• Inclusion conjunctivitis

• Diagnosis is difficult

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Fungi: Examples

• Candida albicans

• Aspergillus

• Fusarium

• Histoplasma

• Trichophyton

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Fungi: Forms

• Eukaryotes

• Unicellular - reproduce by budding

• Multicellular - composed of filaments (hyphae)

• Dimorphic - can be uni or multi-cellular

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Fungi: Classification

Based on spore type & mode of spore production

Types include:

• Zygomycetes/phycomycetes

• Ascomycetes

• Basidomycetes

• Deuteromyctes/fungi imperfecta

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Fungi: Classification

Nucleic acids RNA & DNA

Nuclear membrane Yes

External cell wall Yes, rigid chitin

Antibiotic sensitivity No

Replication / reproduction Within & outside host cells by

binary fission & sexually

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Fungi: Dissemination

• Spores released into air/water

• Spores resist harsh environments ← surface components

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Fungi & Disease

• In disease, fungi must penetrate tissue & germinate

• Human disease includes infection of keratinized tissue

• Systemic infections are rare

‒ immuno-compromised

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Viruses: Description

• Small acellular obligate parasites

• A subcellular agent, consisting of a core nucleic acid surrounded

by a protein coat, & sometimes an outer protein & lipid envelope

• Must use the metabolic machinery of a living host to replicate

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Viruses: Classification

Nucleic acids RNA or DNA

Nuclear membrane No

External cell wall No

Antibiotic sensitivity No

Replication / reproduction Within host cells

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Viruses: Life Cycle

• Extracellular form = virion

‒ double/single stranded DNA or RNA molecule

(nucleocapsid) within a protein coat or capsid

• Intracellular form

• Differentiation based on nucleic acid packaging

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Virus Invasion

(a)
x x x

Adsorption of enveloped or Invagination of Lysosomal-vesicle

non-enveloped virus to cell membrane to form fusion causes release
membrane receptors vesicle. Vesicle fuses of nucleocapsid.
with lysosome Enveloped virus has
envelope digested by
lysosomal enzyme

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Virus Invasion

(b) x x x

Adsorption of Fusion of viral Release of

enveloped virus to envelope with nucleocapsid into
cytoplasmic cytoplasmic cytoplasm. Viral
membrane membrane envelope retained in
cytoplasmic
membrane
(c) x

Absorption of Virus penetrates

non-eveloped directly into
virus cytoplasm
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Viruses & Human Disease

• Herpes group • Adenovirus

• Rubella • Papilloma

• Influenza • Polio
• AIDS • Rhinovirus
• Measles • Rabies
• Mumps • Rotavirus
• Smallpox • Hepatitis
• Zika
• Ebola

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Viruses & Disease: Possible Mechanisms

• Inhibiting cell metabolism & synthesis

• Compromising host defences allowing infection by other

opportunistic organisms

• Inducing tumour formation (oncogenic viruses)

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Oncogenic Viruses & Disease: Cell Transformations

• Viral nucleic acid becomes associated with cell genome

‒ then cell does not lyse
• Transformed cells show altered:
‒ morphology
‒ metabolism
‒ growth patterns
‒ chromosomal abnormalities
• Cells  tumours in animal models

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Protozoans

• Unicellular

• Parasitic or free living

• Often dual life cycle

• Few cause human disease

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Protozoans: Classification

• Magistophora

• Sarcodina/Rhizopodia

• Sporozoa

• Ciliata

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Protozoa: Classification

Nucleic acids RNA & DNA

Nuclear membrane Yes

External cell wall No

Antibiotic sensitivity Some

Within & outside host cells by
Replication / reproduction binary fission & sexual

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Micro-organisms: Identification

• Presence of micro-organisms in disease

• Assists in choice of therapeutic agent

• Collection techniques

• Type of specimen

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Specimens for Culture

• Swabs

• Fluids
‒ exudate
‒ excreta

• Tissue

• Volume

• Transport to lab

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Specimens for Culture

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Microbiological Testing

• Macroscopic observations

• Microscopic examination

• Cultures

• Antigen detection or gene sequence

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Microbiological Findings

• Require interpretation

‒ type of organism?

‒ patient's condition?

‒ therapy optimized

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Examination of Cultures

• Rapid tests

• Incubation

‒ short-term

‒ prolonged

• Antibiotic susceptibility tests

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Culture of Micro-organisms

• Solid nutrient media

‒ blood agar

‒ chocolate agar

• Liquid media

‒ thioglycollate broth

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Culture of Micro-organisms

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Bacteria Identification: Based on Biochemical Properties

• Ability to  enzymes
• Ability to metabolize sugars
‒ oxidatively
‒ fermentatively
• Ability to utilize growth substrates
‒ glucose
‒ lactose
‒ sucrose

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External Ocular Biota

• Sparse colonization of ocular surface

‒ lids 60% of cases
‒ conjunctiva 40% of cases
• Mainly gram-positive species
‒ coagulase-negative staphylococci
‒ Corynebacterium spp.
‒ others: Propionibacterium spp., S.Aureus, Micrococcus
spp., Bacillus spp.
• Gram-negative organisms isolated infrequently (<5% of total)

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External Ocular Biota

• Physiological variations:
‒ transient/permanent biota
‒ geographical regional variations
‒ ∆s with sleep:
- ↑ in Gram-positive organisms
- no ∆ in Gram-negative organisms

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Functions of Mucosal Microbiota

• Mediate mucosal defences

• Prevent colonization by pathogens by:
‒ biocompetition
‒ production of antimicrobial agents
‒ production of toxins

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Functions of Ocular Microbiota

• S.epidermidis & Corynebacterium sp. may act

synergistically to inhibit S.aureus in the nasal mucosa

• Propionibacterium sp. may ↑ specific & local immune

defence

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Effect of DW CL Wear on the Ocular Biota
1.6
Mean level of colonization (grade)
(Stapleton et al., 1995)
1.4

1.2

0.8

0.6

0.4
Conjunctiva
0.2
Lid (p<0.0001)
0
0 2 4 6 8 10 12

Duration of wear (months)

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Neophyte EW CL Users
Mean level of colonization (grade)
1.6
(Stapleton et al., 1995) (n=26)
1.4

1.2

0.8

0.6

0.4

0.2
Conjunctiva (p=ns)
Lid (p=ns)
0
0 2 4 6 8 10 12

Duration of wear (months)

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Effect of DW CL Wear on the Ocular Biota

• In vitro studies reveal that bacterial adhesion is > in unworn

silicone hydrogel (SiHy) CLs versus HEMA-based SCLs
• CL wear can affect microbial adhesion (affected by type of CL &
microbial species studied)
• Microbial adhesion in SiHy CLs occurs & is associated with
corneal infiltrative events (CIEs) during CL wear

Willcox M, 2013. Microbial adhesion to silicone hydrogel lenses.

Eye & CL. 39: 61 – 66

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Effect of CL Wear on the Ocular Biota: Staphylococci

• In EW, most numerous isolate = S. epidermidis

• In DW most numerous isolate = S. capitis / S. warneri

• In DW/EW greatest isolation frequency = S. capitis / S. warneri

• S. haemolyticus, S. lugdenesis, S. hyicus, S. schleferi,

S. intermedius, & S. aureus isolated infrequently

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CL Biota: In Asymptomatic CL Wear

• CL contamination is infrequent
• Commonest organisms = coagulase-negative staphylococci (CNS)
• Occasional isolation of S. aureus, Streptococcus spp., &
Propionibacterium spp.
• Gram-negative bacteria isolated rarely
• No ∆ in frequency of isolation for DW & EW

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CL Biota: In Asymptomatic CL Wear

• Contamination occurs during CL handling

• Higher rates of contamination follow CL storage
• Main sources:
‒ skin, air, pets
• Gram-negative bacteria from water/low levels of
environmental contamination

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LCP Contamination: In Asymptomatic CL Wear

• Case contamination in >80% of CL wearers

• CNS, environmental, & enteric Gram-negative bacteria
commonly associated
• Ps. aeruginosa rare in asymptomatic wear
• Acanthamoeba sp. in 7%
• Fungi in 3 - 5%
• Lack of association between compliance & LCP contamination

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CL Biota: CL-induced Acute Red Eye (CLARE)

• High numbers of Gram-negative bacteria recovered from the CLs

• H. influenza, Ps. aeruginosa, Serr. marcescens, &

Stenotrophomonas spp.

• No corneal colonization by organisms

• Rôle of endotoxin in pathogenesis

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CL Biota: ‘Sterile’ Infiltrates/CL-induced Peripheral Ulcer (CLPU)

• CLPUs associated with ocular carriage of Gram-positive

bacteria especially S. aureus

• Toxin related?

• So-called ‘sterile’ infiltrates associated with bacterial

contamination of the CL storage case

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Corneal Infiltrative Events (CIEs): 12-mth UManchester Study

12-month UManchester Keratitis Study

Infiltrates (CIEs) tend to occur in the:
• Superior cornea in EW of SiHy CLs
• Central cornea in DW hydrogel CLs & DD CLs
• Peripheral cornea in DW hydrogel CLs but NOT DD CLs
Generally:
• CIEs near the limbus are ↓ severe
• CIEs ↑ with overnight wear

Efron N, Morgan P, 2006. Rethinking contact lens-associated

keratitis. Clin Exp Optom. 89(5): 280 – 298

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Risk of a CIE

• Risk of developing a severe CIE is ↑ 5X with conventional

hydrogel EW versus SiHy EW

• ↑ risk of CIE in male gender, smoking, a healthy eye & body, & in

the late winter months

• CL-induced Acute Red Eye (CLARE) with a CIE should prompt

discontinuation of CL wear & appropriate antimicrobial therapy

Efron N, Morgan P, 2006. Rethinking contact lens-
associated keratitis. Clin Exp Optom. 89(5): 280 – 298
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CL Biota: CL-Associated Corneal Infection - Aetiologies

• Ps. aeruginosa predominates (60-70% of culture-proved cases)

• Others: Serratia spp., Proteus spp., Moraxella spp., other

Pseudomonads

• Acanthamoeba spp. strongly associated with CL-related infections

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CL Biota: Corneal Infections - LCPs

• Causative organism isolated from CL care system or CLs

• CL & storage contamination can occur despite good compliance

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CL Biota: Rôle of Bacterial Biofilm

• Explains unexpected persistence of organisms in CL storage cases

• Can play a part in pathogenesis of infection

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CL Biota: Corneal Infection - Biofilm

• Biofilm defined as:

‒ a functional consortia of micro-organisms, organized at

interfaces, within an exopolymer matrix

• Organisms in biofilms resist antimicrobials by:

‒ physical exclusion

‒ phenotypic alterations

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CL Biota: Corneal Infection - CL Cases

• Biofilm formation on CLs & storage cases in bacterial & amoebic

infections:
‒ 17/20 storage cases vs 11/20 CLs
‒ greater density of biofilm in storage cases
• CL case hygiene can be ↑ by effective communication of
instructions.

• CL cases should NOT be rinsed with tap H2O because of the ↑ risk
of Gram-negative bacterial contamination.
(McLaughlin-Borlace et al., 1998 &
Tilia et al., 2014)
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Summary: Microbiology & CL Wear

• Gram-positive organisms are present in low numbers on the

normal ocular surface
• Some evidence exists that the ocular biota is ↕ by hydrogel CLs in
DW/EW, but the implications of this are unclear
• In asymptomatic wearers, CL contamination is infrequent
• Instances of contamination usually reflect the normal biota

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Summary: Microbiology & CL Wear

• CL-related inflammation may be due to CL colonization by Gram-

negative species (CLARE) or ocular carriage of Gram-positive

species (CLPU)

• CL-related microbial keratitis (MK) is predominantly bacterial, with

Ps. aeruginosa the commonest corneal isolate

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OCULAR HOST DEFENCE SYSTEMS
&
CL WEAR

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Ocular Non-Specific Host Defences

• Lids & lashes

• Blink mechanism
• Tear flow
• Epithelial shedding
• Epithelial integrity
• Tear proteins

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Major Tear Proteins

• Lysozyme

• Lactoferrin

• b lysin

• Lipocalin

• Mucin

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Bacterial Cell Wall
GRAM-NEGATIVE CELL GRAM-POSITIVE CELL
WALL WALL

Outer membrane

Periplasmic space

Peptidoglycan

Cytoplasmic
membrane

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Lysozyme

• Is an enzyme, muramidase

• Catalyses the hydrolysis of N-acetyl

muramic acid & N-acetyl glucosamine

• More active against Gram-positive

bacteria than Gram-negative bacteria

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Lysozyme: Action on Bacteria

LYSOZYME

NAG NAM NAG NAM NAG

PEPTIDE PEPTIDE
BRIDGE BRIDGE

NAG NAM NAG NAM

LYSOZYME

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Lactoferrin

• Chelates Fe3+ ions:

‒ essential for bacterial growth

• Disrupts outer membrane of gram-negative bacteria:

‒ chelates Mg2+/Ca2+ or binds to lipopolysaccharide

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b Lysin & Lipocalin

• Reported to be antibacterial

• Action unknown

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Mucin

• Bacteria bind via lectin/carbohydrate interactions

• Prevents bacterial adhesion by acting as a non-specific blocker

• Aids in removal by tears &/or blink mechanism

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Effect of Sleep on Non-Specific Tear Components
Sack et al., 1992

Concentration % Total tear

Protein Tear type [mg/mL] protein
(Mean ± SD)
Total Reflex 6 ± 0.8
Closed Eye 18 ± 6.2

Lactoferrin Reflex 1.8 30 ± 10

Closed Eye 1.8 10 ±
10

Lysozyme Reflex 1.6 26 ± 4

Closed Eye 1.8 10 ± 4
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Minor Tear Proteins Involved in Host Defence

• Enzymes & enzyme inhibitors

• Complement

• Cytokines/chemokines

• Arachidonic acid metabolites

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Minor Proteins: Antimicrobial Enzymes & Enzyme Inhibitors

• Secretory Phospholipase A2

• Specific Leukocyte Protease Inhibitor (SLPI)

• Elafin

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Alternative Pathway

BACTERIUM
C3a
C3

C3b Factor B
C3 convertase Factor D
C3bBb
Ba
C3
C5 convertase C3bBb3b

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Classical Pathway

Antibody

BACTERIUM
C1 C1
C4b C4
C2
C3 convertase C4b2b

C3

C5 convertase C4b2b3b

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Terminal Pathway

C3bBb3b
C5 convertase or
C4b2b3b

C5a

BACTERIUM
C5
C5b
C6
C7
C8
C9
C5b-9 (MAC)

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Key Molecules of the Complement Pathway

C3b Opsonizes micro-organisms

C3a Anaphylatoxin - vasoactive -
activates mast cells
C5a Anaphylatoxin – recruits polymorphonuclear
leukocytes
MAC Bores holes in cell membranes

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Complement Activation Regulators: Tear Soluble Factors

Factor H Accelerates C3  iC3b

Lactoferrin Inhibits C3 convertase

Vitronectin Inhibits MAC formation

Sack et el., 1993; Tipireddy et al., 1997

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Complement Activation Regulators: Cell-Bound Factors

DAF Stimulates C3 convertase

decay

C8bp Prevents C8/C9 binding to MAC

CD-59 Prevents MAC formation

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Effect of Sleep on Complement
Willcox et al., 1997
Concentration
% Plasma
Protein Tear type mg/mL
concentration
[Mean ± SD]
Reflex 4.0 ± 5.6 0.6

C3 Closed Eye 107 ± 84 15

Reflex 0.1 ± 0.1 0.1

Factor B Closed Eye 21 ± 8 20

Reflex 0 0
Closed Eye 1.0 ± 0.4 1.9
C5

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Rôles of Complement in Tears & Anterior Eye

• In tears:

‒ recruit PMNs

‒ opsonizes entrapped bacteria

• In the tissues:

‒ tissue rejection after corneal grafting

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Cytokines Found in Tears

• Interleukins: IL - 1 & IL - 6

• Colony stimulating factors: GM-CSF

• Growth factors: TGFb (TGF beta) & HGF

• Interferons: IFNg (InterFeroN gamma)

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Interleukins: IL - 1 & IL - 6

• Potent inflammatory cytokines

• Activate PMNs

• Stimulate release of other ILs

• Stimulate proliferation of epithelial cells

• Enhance dendritic cell/T-cell interactions

• Stimulate B cells to synthesize antibodies

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Colony Stimulating Factors: GM-CSF - Main Ocular Functions

• Stimulate dendritic (antigen presenting) cells in the cornea

• Stimulate PMNs for ↑ IgA-mediated phagocytosis

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Growth Factors: TGF b (An Anti-Inflammatory Cytokine)

• Inhibits certain types of antibody production

• Promotes B cells to synthesize IgA

• Promotes wound healing by stimulating fibroblasts

• Down-regulates inflammatory functions of IL – 1 &/or IL - 6

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Interferons: IFN g (gamma) – as an Ocular Cytokine

• Influences antigen presentation to T cells

• Activates phagocytes

• Inhibits IgE production

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Chemokines Found in Tears: IL - 8

• A specific attractant for PMNs

• Produced by epithelial cells

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Arachidonic Acid Metabolites

Phospholipid

PLA2

AA PAF

Cyclo-oxygenase Lipo-oxygenase

Prostaglandins Leukotrienes

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Tear Arachidonic Acid Metabolites: Possible Functions

• Prostaglandins ↑ vasodilation / ↑ vascular permeability

• Leukotrienes chemo-attractant for PMNs & macrophages,

stimulate PMNs

• PAF activation of PMNs,

vascular permeability

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Effect of Sleep on Cytokines & Arachidonic Acid Metabolites

Concentration
Cytokines/AAs Tear type [pg/mL]

IL - 6 Reflex 0
Closed Eye 150 ± 110

IL - 8 Reflex 2000 ± 2000

Closed Eye 150 x 103 ± 100 x 103

LTB4 Reflex 232 ± 35

Closed Eye
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1005 ± 205
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WHITE BLOOD CELL TYPES INVOLVED
IN NON-SPECIFIC DEFENCES OF THE
ANTERIOR EYE

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Macrophages

• Resident white blood cells

• Phagocytose micro-organisms

• Kill micro-organisms

• Signal for recruitment of other cells

• Found in all tissues - sparse in the cornea

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Phagocyte Secretory Products

• Enzymes - lysozyme

- protease

• Complement components

• Cytokines & arachidonic acid metabolites

• Reactive O2 radicals - H2O2 (hydrogen peroxide)

- O 2– (superoxide)

- OH– (hydroxide)
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RECRUITED WHITE BLOOD CELLS
INVOLVED IN
NON-SPECIFIC HOST DEFENCE

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The PolyMorphoNuclear (PMN) Leukocyte

• Phagocytose micro-organisms

• Kills micro-organisms

• Signal for further PMN recruitment

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PMN Secretory Products: Microbicidal Proteins

Azurophillic granules Specific granules

• Defensins • Lysozyme

• Myeloperoxidase • NADPH oxidase

• Lysozyme • Lactoferrin

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PMN Recruitment During Sleep

Tan et al., 1993

Tear type Number of PMNs

Reflex / open 0

3 h sleep 41 ± 63

8 h sleep 6583 ± 2354

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PMN Recruitment During Sleep

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Non-Specific Host Defences: Summary

• Based on physical properties of the eye, proteins, & white blood

cells in tears
• Lysozyme, lactoferrin, mucin, & complement are main 'removers'
of micro-organisms
• Complement, cytokines, & arachidonic acid metabolites are main
signalers for white blood cells
• PMNs & macrophages are main cellular 'removers' of micro-
organisms

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Specific Host Defences

• Based on antibody production & T cells

• Humoral immune response based on antibodies (immunoglobulins)

• Cell-mediated response based on T cells

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Function of Antibodies

• Bind to surface of micro-organisms to prevent adhesion to

host surfaces

• Neutralize toxins

• Aid in phagocytosis

• Activate complement

• Activate specific white cells

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The Immunoglobulins Found in Tears

• IgA

• IgG

• IgM

• IgE

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Immunoglobulin A (Ig A)

• Most abundant immunoglobulin in tears

• Two isoforms, IgA & IgA2

• Both forms at equal concentrations

• Both forms have similar functions

• IgA2 is resistant to bacterial proteases

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IgA

Monometer
J - Chain
Heavy Chain

s-s s-s Light Chain

Secretory Component
Fab Fc

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Secretion of IgA
Acinar epithelium

s
B Cell Sig A To Tear Film
s
s

s s

s
s = J chain

= Secretory Component

A lacrimal gland acinus = dimeric IgA

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Function of Secretory IgA

• Binds to micro-organisms preventing:

‒ adhesion to surfaces (epithelia, CLs)

‒ motility & growth

• PMNs have IgA receptors triggering  phagocytosis

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Levels of Tear Secretory IgA
Sack et al., 1992

% Total tear
Concentration
Protein Tear type protein
[mg/mL]
(Mean ± SD)
Total Reflex 6.0
Closed Eye 18.0

sIgA Reflex 0.23 4.9 ± 1.1

Closed Eye 8.4 50.3 ±
13.4

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Immunoglobulin G

Fab s-s s-s

Di-Sulphide Bridge

s-s
Fc s-s

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Immunoglobulin M

s-s

s-s

s-s
s- - s
s s

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Immunoglobulin E

s-s s-s
Fab'

s-s

Fc''
s-s
Fc

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IgE & Ocular Hypersensitivity

Once IgE encounters an allergen/antigen:

• A specific IgE is produced
• That specific IgE binds to conjunctival mast cells
Following a second encounter with the same allergen:
• Allergen binds to mast cell-associated IgE
• Mast cells activate rapidly
• Potent inflammatory mediators are released:
‒ histamine
‒ AA metabolites
‒ cytokines

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Cell Components of Anterior Eye’s Specific Immune System

• B cells
• T cells
• Dendritic cells
• Mast cells
• Basophills
• Macrophages
• PMNs

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B Cells

• Produce & secrete immunoglobulins

• Present in the lacrimal gland

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T Cells

• T helper cells (CD8+)

‒ recognize antigens presented by dendritic cells or
macrophages
‒ stimulate B cell differentiation
• Cytotoxic T cells (CD4+)
‒ destroy infected cells
• All are present in lacrimal gland & conjunctiva?

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Dendritic Cells

• Present antigen to T cells

• Present in cornea, conjunctiva, & lacrimal gland

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Macrophages

• Phagocytic
• Present antigens to T cells
• Present in conjunctiva, lacrimal gland, & cornea (?)

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Mast Cells & Basophils

• Involved in allergic reactions

‒ stimulated by IgE
BASOPHIL
• Present in conjunctiva - mast cells

MAST

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PolyMorphoNuclear (PMN) Leukocytes

• Recognize & destroy:

‒ immunoglobulin-coated particles

‒ micro-organisms

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Specific Host Defences: Summary

• Based on production of immunoglobulins

• sIgA is the main antibody in tears

• IgE is the main antibody in allergic reactions

• Prevent micro-organisms colonizing the eye & aid in

recognition of micro-organisms by cellular components

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Specific Host Defences: Summary

• Dendritic cells signal T cells that micro-organisms are present

• T cells signal B cells to  specific antibody

• PMNs & macrophages ingest & kill micro-organisms coated

with antibody &/or complement

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Effect of CL Wear on Anterior Ocular Host Defences

CL wear may:
• Disrupt the tear film
• Affect epithelial integrity/shedding
• Alter the balance of tear proteins
• Affect PMN recruitment during sleep
• Affect levels of immunoglobulins
• Provide a niche for bacterial colonization & thus predispose
to infection & inflammation

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Effect of CL Wear on Epithelial Integrity & Shedding Rates

• Low Dk/t CLs ↑ levels of LDH in tears

• Low Dk/t CLs ↑ corneal swelling &  ↑ 'space' between cells

• SCLs tend to trap sloughed cells

• Low levels of mechanical abrasion can disrupt epithelium

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CL Wear Can ↕ Balance of Tear’s Non-Specific Defence Proteins

• CLs adsorb & absorb proteins from tears

• Lysozyme/lactoferrin/mucin levels do not ↕
• Absorbed protein :
‒ substratum for bacterial adhesion
‒ potential for immunological reactions as protein can be 'seen' as
foreign

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Effect of CL on Cytokines & Arachidonic Acid Metabolites

Subject IL-8 IL-6 GM-CSF LTB4

No CLW 150 X 103 150 66 1005

EW CLW 230 X 103 218 59 1150

CLs have no statistically significant effect on these important inflammatory mediators

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Complement Activation on CLs

C3 C5

C3a C5a
MAC

C3b C3bBb

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CL Wear ↓ PMN Recruitment into the Tears

Stapleton et al., 1997

Sampling Non-CL wear CL wear

occasion median median

Open Eye 0 0

8 h Sleep 2777 181

N=6

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Effect of CL Wear on Levels of Tear sIgA

sIgA: % of Total
protein
Tear type No CL DW EW

Closed Eye 54 - 51

Open Eye 22 13 10

N=6
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Effect of ↓ Tear sIgA

• Micro-organisms would be more likely to adhere to surfaces in

the eye & grow

• Toxins would be less readily neutralized

• PMNs would be less able to phagocytose

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Effect of CL Wear on Host Defences

• ↕ normal functions of the epithelium (Dk/t dependent)

• ↓ PMN numbers (CL type dependent?)

• ↓ sIgA

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Effect of CL Wear on Host Defences

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Inflammatory Components in CLARE

Inflammatory Reflex tears CLARE tears

component Mean/mL Mean/mL

C3 4.0 mg 4.3 mg

Factor B 0.1 mg 0.3 mg

IL-6 75 pg 116 pg

IL-8 0.5 ng 2.7 ng*

LTB4 250 pg 636 pg*

N=6
*p < 0.05
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Effect of CLARE on Levels of Tear PMNs

Holden et al., 1996

CLARE NORMAL
median median

PMN numbers 4 4

Epithelial cell numbers 28 15

N = 12

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Inflammatory Components in CLPU

Inflammatory Reflex tears CLPU tears

component Mean/mL Mean/mL
C3 4.0 mg 3.7 mg
Factor B 0.1 mg 0.1 mg
IL-6 75 pg 62 pg
IL-8 0.5 ng 0.8 ng
LTB4 250 pg 1271 pg*

N=8
*p < 0.05
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CLPU

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GPC/CLPC

• Aetiology still not fully resolved

• Occurs more frequently in non-ionic hydrogel CLs*

• Frequent replacement of CLs ↓ incidence

*Hart et al., 1989

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GPC/CLPC

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Tear Immunoglobulin Levels in CLPC

Immunoglobulin GPC:Normal % Plasma protein

IgA 1
IgG 2
IgM >5
IgE 3

Donshik et al., 1983; Barishak et al., 1984

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White Blood Cells Seen in Conjunctiva During GPC

• Increased:

‒ granulocytes

‒ mast cells

‒ eosinophils

‒ basophils

Allansmith et al., 1977

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∆s During CL Wear

• Decreased:

‒ PMNs

‒ sIgA

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∆s During Various CL-Induced Adverse Responses

• Increased:

‒ IL-8 (CLARE)

‒ LTB4 (CLARE, CLPU)

‒ PMNs (CLARE, CLPU, CLPC)

‒ IgE, IgG, IgM (CLPC)

‒ Mast cells, eosinophils, basophils (CLPC)

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Symbols

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Abbreviations

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Acronyms

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Acronyms

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THANK YOU
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