Lecture 8: Chemical Allergy - Toxicological Aspect

What are the characteristics and types of chemical allergens?

• Low molecular weight (<500Da, compared to IgG ~150kDa)

• If reactive, can trigger an allergy

• Two main forms:

  • Skin sensitisation or contact allergy → symptoms appear in skin

  • Respiratory sensitisation → symptoms in respiratory system

What is Immunotoxicity?

• The adverse effects of xenobiotics on the immune system

• This results in:

  • Immunosuppression (or classical immunotoxicity)

  • Immune stimulation (e.g. TGN1412 Ab)

  • Hypersensitivity (or Allergy)

What Can Causes Allergy?

• Chemicals

  • Sensitisation of the respiratory tract/ Rhinitis and asthma

  • Skin sensitisation/ Allergic contact dermatitis

• Proteins

  • Sensitisation of the respiratory tract/ Rhinitis and asthma, e.g. following exposure to pollen or animal dander

  • Sensitisation to food proteins or food allergy

• Drugs

  • Allergic drug reactions

  • Give rise to systemic allergy

What is the Prevelance and Impact of Contact Dermatitis as an Immunotoxicity?

• Most common form of immunotoxicity in humans

• 20% of the UK population has a contact allergy to ≥1 chemical

• Contributes to 1% of GP visits

• Causes severe, non-life-threatening reactions

• Impact is more socio-economic, with occupational implications → individuals require time of work or occupational changes

What is Methyl Isothiazoline?

• A chemical used extensively as a preservative or biocide in cosmetics and household items

• A contact allergen that produced skin reactions and allergy → led to an epidemic in children due to its use in baby wipes, 15 years ago

• Legislation was implemented to ban its use in children’s products

What is Paraphenylene Diamine (PPD)?

• Found Black dye e.g. henna and hair dye

• Concern for intradermal exposure to a potent contact allergen

• Causes allergy

Give Examples of Naturally Occurring Potent Contact Allergens

• Geraniol: Produced by geraniums

  • Causes allergies in gardeners

  • Compound used in fragrances

• Nickel

  • Historically, the most common, but its use has reduced due to EU mandate reducing its use in coins

  • Prevalence due to widespread exposure, e.g. body piercing (introduction of nickel to the inflamed skin)

  • Shift from a weak intrinsic allergen to a strong allergen

  • Acts as a danger signal and stimulates an immune/allergic response

What Happened in the Chinese Sofa-Contact Allergen Case Study?

• Sofas contaminated with mould due to humidity differences

• Treated with dimethyl formamide (fungicide and potent contact allergen)

• Individuals exposed to contact allergens over large body areas → hospitalisations reported

What are Clinical Manifestations of Respiratory Allergy?

• Wheezing

• Hayfever

• Rhinitis

• Asthma

• It can be fatal

  • e.g. Occupational exposure to Toluene Diacetocyanate led to a fatal asthma attack → change in occupation

How Common Are Chemical Respiratory Allergens?

• Few chemicals are known respiratory allergens (~35 identified by Health & Safety Executive)

• “True” respiratory allergens:

  • 7 with compelling evidence

  • 10 with reasonable evidence

• Demonstrates that they are rare but can cause severe consequences

Why are Lymphocytes Important

• They are key features of the adaptive immune response

• They help protect against a wide range of infections, including

  • Virus: Herpes Simplex Virus

  • Fungi: Candida Albicans

  • Bacteria: Mycobacterium Tuberculosis

  • Multicellular parasites: Ascaris Lumbricoides

How Does the Immune System Mediate Proection Against A Wide Range of Infections?

• This is achieved by the immune system shaping the quality of the immune response to the specific challenges

• T-helper cells differentiate and polarise into different T-helper subsets (Th1, Th2, Th17), each with a characteristic profile of cytokines

What are the Thre Main T-helper Subsets Produced in a Polarised Immune Response?

• Th1: Provide protection against viruses and intracellular bacteria

  • Cytokines produced:

    • INF-Y

    • IL-2

• Th2: Provide protection against multicellular parasites

  • Cytokines produced:

    • IL-4

    • IL-5

    • IL-10

    • IL-13

• Th17: Provide protection against fungi

  • Cytokines produced:

    • IL-17

What are the cardianl characteristics of an Adaptive Immune Response?

• Protective immunity is acheived via:

  • Memory → allows for a rapid and aggressive response to a challenge

  • (Exquisite) specificty

  • Discrimination between self and non-self

What is hypersensitivity, and how does it develop?

• Immune system recognises a non-harmful substance and generates a specific immune response to the innocuous antigen, causing pathology and tissue damage

  • Here, the chemical does not cause pathology, but the specific immune response does.

• Sensitisation to a substance produces memory cells → lifelong sensitivity

• Secondary exposure triggers a rapid, specific immune response even at low levels

How Do Chemicals Cause Allergic Sensitisation?

• Most chemicals are small, ~500Da → too small to be recognised by the immune system

• Chemicals that cause allergens are haptens → form covalent bonds and are associated with macromolecular carriers

• The hapten (macromolecular protein-chemical complex) is recognised by the immune system

• Immune recognition of this complex triggers T-lymphocyte activation → sensitisation depends on an intact adaptive immunity

What are the Two Phases of Allergic Sensitsation/Manifestation?

• Induction:

  • sensitisation phase

  • first exposure to a chemical

  • Become primed

  • Memory cells formed

  • Not symptomatic (not yet primed)

• Elicitation:

  • Secondary response

  • Aggressive

  • Rapid

  • Symptomatic (primed)

What Are Contact Allergens?

• Second commonest occupational health disease,

• Hundreds to thousands of chemicals have been identified

  • ~4000 chemicals identified with the property of contact allergy to date

• Symptoms can be severe/impact on quality of life

  • Impacts are largely socioeconomic

• Cell-mediated (delayed type) hypersensitivity

  • Clinical manifestations of symptoms occur after a ~72-hour delay

• Validated tests available for decades

  • Animal and in vitro tests are available to investigate the potential for a chemical to cause contact allergy

What are Respiratory Allergens?

• Less common

• Few chemicals identified

• Can be life-threatening

• Immediate type hypersensitivity reaction, within seconds to minutes of exposure

  • Delayed response is also possible

• No validated tests available

• Controversy regarding the mechanism

What Happens During The Induction (Sensitisation) Phase of Contact Allergy?

• Chemical allergens are lipophilic → penetrate stratum corneum (outer layer of lipid-dense dead keratinocytes

• Enter viable epidermis → interact with dendritic cells (APCs)

• Allergens act as danger signals, leading to cytokine release and an immune response

  • Allergens are oxidants (electrophilic) and stimulate danger

• Dendritic cells take up allergens and migrate to lymph nodes

• In the lymph node, antigen presentation to T-cells (interaction of complementary TCR-allergen-protein complex) leads to clonal expansion of allergen-specific T-cells

• Sensitised T-cells enter circulation

• Individual becomes sensitised/primed

What happens during the Elicitation phase of contact allergy?

• Secondary exposure to the same allergen

• Allergen-specific T-cells migrate to the exposure site

• Allergen recognition occurs

• Cytokine release

• Recruitment of additional inflammatory cells to site of insult

• Clinical manifestation of disease (contact dermatitis)

When is the Mechanism for Chemical Respiratory Allergy Manifestation?

• There is no consensus on the mechanism

• Due to a problem of definition

How are chemical respiratory allergens classified?

• Health and Safety Executive defines “Asthmagens” as anything causing asthma via three mechanisms:

  • Immunological (respiratory sensitisation)

    • Induction + elicitation phases

  • Non-immunological

    • Irritant-induced airway damage

  • Work-exacerbated asthma

    • Pre-existing asthma worsened at work

• ~35  materials classified currently by HSE as asthmagens

  o    (includes non-immune mediated and families of chemicals)

Why is it difficult to define and identify chemical respiratory allergens?

• Pre-existing asthma complicates assessment as

  • Airway remodelling is already present (lung function already compromised)→ non-specific reactions

  • Respond to non-noxious stimuli (cold air, exercise)

• Symptoms may be worsened at work without a specific immunological trigger

• Highlights the need to differentiate immunological vs non-immunological mechanisms for proper effective intervention

What are the features of immunological (respiratory sensitiser) asthmagens?

• Latency period: individuals must be priming and develop an immune response

• Respond to the same chemical → Chemical-specific response

• Triggered by low concentrations (hypersensitivity)

• Life-long sensitisation: memory cells present

• Other exposure routes may cause sensitisation

• Mechanisms sometimes uncertain

What are the features of non-immunological asthmagens?

• No latency period

• Triggered by a single, high-intensity exposure (“irritant asthma)

• <10% of chemicals are non-immune mediated

What is the Mechanism of Protein Respiratory Allergy (e.g. Pollen and Dander)?

• Priming event:

  • Production of IgE → binds to the surface of specialised receptors on mast cells and basophils (primed and sensitised)

• Secondary exposure:

  • Allergy causes cross-linking of IgE molecules on mast cells

  • Results in the degranulation of mast cells

  • Release of leukotrienes and vasoactive amines → facilitates immediate response, bronchocontriction

• Delayed response:

  • T-cell and eosinophil influc to site of damage(lung

  • Leads to chronic inflammation and remodelling

What is the Guinea Pig Test Used for Respiratory Sensitisation?

• Mechanism behind chemical allergens is uncertain

• Test mimics asthma-like symptoms (respiratory distress) to identify potential chemical respiratory allergens

• The guinea pig lung acts as a “shock organ”, where respiratory responses are observed

• Limitations:

  • Only a few chemicals tested - not all were respiratory allergen positive

  • Expensive test

  • Ethical concerns: endpoint is respiratory distress

Is IgE Always Involved in Chemical Respiratory Allergies?

• IgE is not always present in chemical respiratory allergy

  • B-cells require T-cell help to produce immunoglobulins, e.g. IgE

• Symptoms and downstream events are the same regardless

• This suggests common pathways in allergic responses, even if IgE-independent

What is the Role of IgE in Chemical Respiratory Allergy?

• IgE is highly diagnostic if present and is strongly associated with some chemicals, e.g. acid anhydrides, platinum salts

• For other chemicals (e.g. diisocyanates), IgE is detected in <5% of asthmatic individuals.

Why is it difficult to detect IgE?

• This may be due to the detection methods used:

  • Need for proper protein-hapten conjugate for assays → no single best conjugate identified as the process occurs in vivo

    • Hapten does not bind to the ELISA plate

  • Timing of measurement affects detection → may be absent/eliminated at time of assessment

  • Anatomical location: IgE half-life longer on mast cells than in serum; lung biopsy preferred but not feasible for routine detection/testing

• Clinical features are similar/indistinguishable regardless of detectable IgE, suggesting a common underlying mechanism

  • Insight required into the mechanism

What is the Polar Reponse Observed with the Two Refernce Chemicals Tremolitic Anhydride and DNCB in Mouse Model Studies?

• Tremolitic anhydride: reference respiratory allergen

  • Causes asthma in humans and experimental animals’ models

  • Protein reactive

  • Immunogenic

    • Lymphocyte proliferation

    • IgG and IgE production

• DNCB: reference contact allergen

  • Causes asthma in humans and experimental animal models

  • DCNB was previously used as an algicide in the ventilation shaft

  • Causes no respiratory allergy

  • Protein reactive

  • Immunogenic

    • Lymphocyte proliferation

    • IgG production

How is the Immune System Shaped to Specific Threats?

• The immune response generated is governed by the T-helper subset involved

  • Th2 cells and associated cytokines required for IgE-mediated immunity

  • Th1 and Th17 cells and associated cytokines required for cell-mediated (delayed-type) immunity

• Chemical allergens

  • Contact allergens (e.g. DNCB) are misrecognised by the immune system as viruses/intracellular bacteria/ fungi → induce Th1 and Th17 responses

  • Respiratory allergens (e.g. TMA) are misrecognised by the immune system as multicellular parasites → induce a Th2 response

What Cytokines are Involved in the Th2 Respiratory Allergy

• These cytokines are crucial for IgE antibody production and have other functions that lead to respiratory responses → explains respiratory allergic response in the absence of IgE

  • IL-4/IL13: IgE antibody production

  • IL-13:  hypersecretion of mucin; airway remodelling and muscle cell proliferation

  • IL-5/IL-13: activation and recruitment of eosinophils

  • IL-4/IL-5/IL-10: differentiation and activation of mast cells

How Do Contact and Respiratory Allergens Induce Differential Cytokine Expression and Responses in BALB/c Mice?

• Contact allergen (e.g. DNCB induces Th1/Th17 response):

  • Cytokines: IFN-γ, IL-12, IL-17

• Respiratory allergen (e.g. TMA induces Th2 response):

  • Cytokines: IL-4, IL-5, IL-10, IL-13

• Both allergens are protein-reactive, immunogenic, and T-cell activating, but they stimulate different T-helper subsets and associated cytokine production.

  • This can be used to help identify if a chemical is allergenic and the allergen type (contact vs respiratory).

Why is there a push for the development of in vitro tests in toxicology?

• Regulatory & economic pressures:

  • REACH legislation requires 30,000 chemicals to have new safety data → expensive with in vivo tests

  • Government aim to phase out animal testing and research by 2030.

    • in vivo are only used if no alternative (in vitro) method exists

• Societal/ethical pressures: growing public support (~70%) for reduced animal use (ethics)

• Technological advances:

  • Microfluidics and organ-on-a-chip replicate aspects of in vivo physiology

What are the key properties of a contact allergen?

• It must:

  • Penetrate the skin - Epidermal bioavailability

  • Produce local trauma to activate the immune system (danger signal)

    • proinflammatory cytokine production

    • dendritic cell activation

    • Keap1-Nfr2-ARE activation –antioxidant response

  • Show protein reactivity (otherwise too small for immune detection)

  • Immunogenicity (must be recognised as foreign and stimulate an immune response

How Has In Vitro Assay Development Changed Over the Last 15-20 Years?

• Shift to assess epidermal bioavailability, danger signals and DC activation, protein reactivity and immunogenicity in in vitro development

• Some success has been seen

How is epidermal bioavailability assessed in in-vitro contact allergy testing?

• Human skin used (e.g., tummy tuck or breast reduction samples) to assess chemical absorption in vitro

• (Q)SAR dermal absorption analysis compares chemical structure to known allergens and families of chemicals

• Epidermal bioavailability is not a major driver of contact allergy

How are danger signals assessed in in-vitro contact allergy testing?

• Focus on dendritic cell activation

• Measured by:

  • Cytokine production (DCs/epithelial cells)

  • DC activation/maturation

  • DC migration/mobilisation

  • Keap1-Nrf2-ARE pathway activation

How is protein reactivity assessed in in-vitro contact allergy testing?

• (Q)SAR modelling

• Peptide reactivity tests

• Identifies chemicals that can bind proteins (hapten formation) and trigger sensitisation

Why is immunogenicity difficult to assess in-vitro, and how is it tested?

• Difficult because:

  • Limited T-cells obtained from blood samples

  • Low chance of finding allergen-specific TCR

  • Individuals are immunologically naïve to test chemicals → unlikely to find specific receptor

• Method used:

  • Primary T-lymphocyte activation assays

What is the Direct Peptide Activation Test?

• Measures chemical–protein interaction and reactivity using HPLC (high-pressure liquid chromatograph)

• Hapten (electrophile) reacts with protein (nucleophile)

  • Free cysteine/lysine residues enable covalent binding between hapten and protein

• Procedure:

  • Peptide produced with free lysine/cysteine group

  • Peptide run on HPLC → Baseline peptide peak measured

  • Test chemical added to the peptide protein

  • Assess peptide peak depletion → indicates protein reactivity

• Greater peptide depletion = higher allergenic potential

What is the KEAP1-Nrf2-ARE Pathway and How is It Activated?

• Detects oxidative stress caused by chemical allergens (danger signal)

• Normal conditions:

  • Keap1-Nrf2 complex (held together by disulfide bonds via Cys residues) in the cytoplasm

• Oxidative stress generated by hapten:

  • Reduces disulfide bonds

  • Nrf2 dissociates from Keap1

  • Nrf2 translocates to the nucleus

  • Nrf2 binds transcription factor MAF

  • Nrf2-MAF bind to ARE (antioxidant response element) → drives gene expression (e.g., IL-8)

How is the KEAP1-Nrf2-ARE Pathway Used In In-Vitro Sensitisation Assays?

• The enterprise group genetically engineered a cell line with an antioxidant response element with a luciferase gene (ARE-luciferase reporter gene)

• When a chemical/substrate activates Nrf2 pathway:

  • Luciferase expressed

  • Fluorescent signal

  • Fluorescence = a chemical has sensitisation potential

What are the OECD Guidelines for In Vitro Skin Sensitisation Assays?

• Danger signal / Dendritic cell activation

  • OECD TG 442D:

    • KeratinoSens (Feb 2015)

    • LuSens (Jun 2018)

    • Based on KEAP1-Nrf2-ARE pathway

  • OECD TG 442E:

    • h-CLAT

    • U-SENS

    • IL-8 Luc assay (Oct 2017) → assess BC activation biomarkers

    • GARDskin (2022)

• Protein Reactivity

  • OECD TG 442C:

    • DPRA (Direct Peptide Reactivity Assay)

• Defined approaches for Skin Sensitisations

  • OECD Guideline 497

    • Combines in vitro approaches to identify potential skin sensitisation hazard (contact allergy)

How is In Vitro Data Used for Hazard Identification in Skin Sensitisation?

• In vitro tests assess isolated aspects of the pathway (not holistic)

• “2 of 3” approach suggested → confirmatory tests required to confirm or deny a result

• Tests conducted sequentially and include:

  • Direct peptide reactivity assay (protein reactivity)

  • Keratinosense assay (Danger signals)

  • h-CLAT (DC activation assay)

• 2/3 agreement for classification as a sensitiser or a non-sensitiser

• If disagreement, a third test must be conducted

How is In Vitro Data Used for (Hazard Identification &) Potency Assessments for Skin Sensitisation?

• Potency classification of 1A (strong) or 1B (weak) assigned via:

• Integrated Testing Strategy (ITS-v1)

  • DPRA +h-CLAT + DEREK (QSAR)

• Integrated Testing Strategy (ITS-v2)

  • DPRA +h-CLAT + Toolbox (QSAR)

• Show limited sensitivity for potency classification

What are the Key Properties of Chemical Contact Allergens?

• Well characterised with validated identification methods

• Must be epidermally bioavailable

• Cause local danger signals (trauma) via:

  • Pro-inflammatory cytokines

  • Dendritic cell activation

  • Keap1-Nfr2-ARE activation

• Show Protein reactivity

• Immunogenic → induce Type 1 immune responses

• Classified as Type 1 inducers

What are the Key Properties of Chemical Respiratory Allergens?

• Fewer validated methods due to uncertainty in the mechanisms involved

• Must be epithelially bioavailable

• Cause local danger signals (trauma) via:

  • Proinflammatory cytokine production

  • Dendritic cell activation

  • Keap1-Nrf2-ARE pathway (uncertain but suspected)

• Show protein reactivity

• Immunogenic → induce Type 2 immune response

• Classified as Type 2 inducers

How Does the GARD Assay (Genomic Allegergen Rapid Detection Model) Detect Chmical Allergens?

• An in vitro assay assessing immune response induction

• Uses DC-like cell line, exposed to respiratory sensitisers, contact allergens and non-sensitisers

• After 24 hours, the whole genome (~23,000 genes) was analysed to assess gene expression patterns (up- and down-regulation)

• Respiratory allergens show similar gene expression patterns

• Non-sensitisers show distinct patterns

• Suggests possible biomarker signature for respiratory allergens and contact allergens, related to the differential induction of Th1 vs Th2 cells.

What are GARDskin and GARDair and how are they used?

• Developed by Sensagen using DC-based gene expression profiling

• GARDskin (skin sensitisation):

  • DC-based assay with ~200 gene markers

  • ~90% accuracy claimed

  • Used for skin sensitisation hazard prediction

  • Included in OECD TG 442E → validated test for in vitro contact allergen testing

• GARDair (respiratory sensitisation):

  • DC-based assay assessing a 28-gene signature

  • ~95% accuracy claimed

  • Used for respiratory sensitiser screening and prediction

  • Under EU validation (2020), still under peer review

What are Limitations of the GARD Assays?

• Considered a “black box” approach

• Limited insight into mechanisms involved

• Focus only on Dendritic cell responses and gene signatures

• Does not fully explain underlying immunological pathways

What is the alternative in vitro respiratory sensitisation (RS) model to GARDair?

• Tests 10 respiratory sensitisers + 10 controls

• Uses a more physiologically relevant air–liquid interface model

  • Respiratory epithelial cell line grown on a filter (as an air-liquid interface)

  • Addition of DC-like THP-1 cells, which are activated into macrophage-like DCs

• Chemical applied at the air interface (better mimics lung exposure, especially for lipophilic chemicals)

• Cells primed and activated with a danger signal (poly IC), then exposed to a chemical

• Endpoint: Measures cytokines: IL-6, CCL-2 (upregulated in in vivo respiratory allergy)

• The model is more physiologically representative and mechanistic

How well does the RS assay perform, and how is it used in an integrated testing strategy?

• RS assay shows:

  • 80% sensitivity (some false negatives)

  • 100% specificity (no false positives)

  • 90% accuracy

    • … to respiratory sensitisation assessment

• Integrated Testing Strategy (ITS):E*

  • Combines RS + DPRA + in silico models (Combo of direct peptide reactivity and computational approaches)

  • Improved performance:

    • 90% sensitivity

    • 100% specificity

    • 95% accuracy

    • Reduces false positives and improves prediction reliability

What is the 3D Multi-Cell Culture System Model Alternative to GARD Air for Respiratory Sensitisation?

• A 3D co-culture system composed of:

  • Epithelial cells (A549)

  • Macrophages (differentiated U937)

  • Dendritic cells (differentiated ThP-1)

• Test with 2 respiratory sensitisers and 2 controls

• Transcriptomics (whole genome) analysis

  • Gene expression (Up/downregulation) assessed

  • Cross-referenced against 2 databases (Geo and KegSystem) on biological processes and disease pathways

  • Identifies molecules and signatures thought to be implicated

• Aims to develop a respiratory sensitiser gene profile (Still early days)

• Currently no validated in vitro methods for respiratory sensitisers hazard identification, unlikeW contact allergens

What are the Relevant Routes of Sensitisation in Respiratory Allergy?

• Although symptoms occur in the respiratory tract (lungs), sensitisation is not limited to it

• The skin is a key route for priming respiratory allergy

• Many animal models use:

  • Skin = priming site/dose route

  • Lung = challenge site (lung not good for initial sensitisation and priming)

• Evidence: Experimental models show skin contributes to respiratory sensitisation via priming

  • Occupational exposure data support skin involvement

• → must be caution with route of exposure (skin well validated route for respiratory allergy priming)

What Clinical/Occupational Evidence Shows the Importance of Skin Exposure in Respiratory Sensitisation?

• Occupational evidence suggests skin exposure is a significant route of sensitisation

• Regulations focus on protecting the respiratory tract (inhalation exposure)

• No equivalent legislation for skin exposure to respiratory sensitisers

• Therefore individuals may still be exposed through the skin at high levels, contributing to sensitisation risk

Why is the Skin a Suitable Priming Site?

• It has

  • Increased immunogenicity due to exposure through skin

  • Presence of Professional DCs

  • Easy Absorption

  • Many precidents for its immunogenicity → used as an immunisiation route

Why is the Respiratory Tract an Unsuitable Priming Site?

• * It has

  • Decreased immunogenicity

  • Presence of professional DCs and alveolar macrophages → downregulate immune response

  • Easy absorption

  • Fewer presidents

  • Exposure here is tolerising rather than immunogenicity

How Does Skin Exposure Contribute to Peanut (Protein Allergy)?

• Increase in peanut allergy observed over ~20 years

• “First exposure reactions” explained by prior occult sensitisation (no allergic response upon initial exposure to allergen)

• Not primed through food, in utero exposure or breast milk

• Evidence shows environmental/household exposure to peanuts, e.g. via skin, is important → caused allergy

• Skin exposure can prime the immune system → allergy development

How Do Skin and Gut Exposure Routes Differ in Immune Response to Allergens e..g Proteins?

• Skin exposure → immunogenic (sensitising) response

• Gut exposure → tolerogenic(immune-tolerant) response

• Protein allergies e.g. peanut can sensitise via skin contact before ingestion

• Explains why allergic reactions can occur on first known ingestion (individuals already sensitised)