Rosanna Marsella University of Florida

Rosanna Marsella University of Florida
Update on the Pathogenesis of Canine Atopic Dermatitis: a comparative review Rosanna Marsella University of Florida

R Marsella ACVD Resident Review 2003
Important points Pathogenesis The old theory: Type I hypersensitivity IgE, mast cells, histamine, LT Increased PDE The new theories: T cell imbalances Biphasic responses of T helper cells Cytokines and chemokines Implications for therapy R Marsella ACVD Resident Review 2003

Canine Atopic Dermatitis (cAD) Definition: Relapsing dermatitis with characteristic clinical features familiar predilection mostly associated with increased IgE antibodies against environmental allergens (Task Force, 2001) R Marsella ACVD Resident Review 2003

Canine Human R Marsella ACVD Resident Review 2003

Experimental canine AD R Marsella ACVD Resident Review 2003

Canine Human R Marsella ACVD Resident Review 2003

Spongiotic superficial perivascular mononuclear dermatitis
R Marsella ACVD Resident Review 2003

Human Canine R Marsella ACVD Resident Review 2003

Prevalence and risk factors
Increased prevalence in humans Risk factors Genetics Environmental exposure Nature of the allergen, dose, timing Cytokine profile Foods R Marsella ACVD Resident Review 2003

Co-factors in canine AD Bacteria IgE against Staphylococcus Modulation of T cells (e.g.Protein A) Yeast IgE against Malassezia Zymogen and direct complement activation R Marsella ACVD Resident Review 2003

Epidemiology of AD Canine AD 10% of canine population Increased prevalence More cases diagnosed? Genetic selection? Human AD Children 10-20% Adults 1-3% Increase in prevalence in developed countries Hygiene hypothesis? R Marsella ACVD Resident Review 2003

Genetics and AD Numerous genetic abnormalities reported in humans Little is known in dogs Severity of disease in individual patient may depend on the cumulative effect Genotype Phenotype Severity of AD R Marsella ACVD Resident Review 2003

Genetics Canine AD Familial history Strong breed predilection Increased frequency of haplotype DL-A3 and R15 Only evaluated genetic inheritance of IgE production (Dominant) Human AD Familial history Gene candidates IL-3 IL-4 IL-5 IL-13 IL-4 Ra CD 80 and CD 86 R Marsella ACVD Resident Review 2003

Genetics and AD Response to treatment varies in individual patients depending on the specific abnormality Identification of genotype may be useful in predict clinical response to therapies R Marsella ACVD Resident Review 2003

and type I hypersensitivity in AD
The old theory: Role of IgE, mast cells and type I hypersensitivity in AD R Marsella ACVD Resident Review 2003

IgE Th1 Th2 MC APC Eos IL-4 TNF-a Let’s not put all our “atopic” eggs in one basket…. R Marsella ACVD Resident Review 2003

IgE Destroyed by heating to 56oC for 4 hrs Transferable by ID injection Persistence at the site of injection for > 48 hrs Isotype switching regulated by: IL-4, IL-13 g-IFN and TGF-b R Marsella ACVD Resident Review 2003

Canine IgE Constant region gene isolated and sequenced Heavy chain 75kDa Heterogeneity in canine IgE Different subisotypes Different chromatic properties Different ability to bind antibodies R Marsella ACVD Resident Review 2003

IgE receptors High affinity (FceRI) Tetrameric (abg2) expressed constitutively on mast cells and basophils Trimeric (ag2) only in patients with extrinsic AD (skin and monocytes) Low affinity (FceRII, CD 23) IgE binding lectin galectin-3 R Marsella ACVD Resident Review 2003

FceRI on APC in AD Antigen uptake APC maturation Release of IL-10 R Marsella ACVD Resident Review 2003

Route of allergen exposure Inhalatory Percutaneous R Marsella ACVD Resident Review 2003

Route of allergen exposure - Inhalation
Pathogenesis: Route of allergen exposure - Inhalation Y IgE Allergen IgE are produced systemically Y Y Y Allergen is inhaled and systemically absorbed Y Y Y Y Y IgE migrate to tissue and bind to mast cells Y R Marsella ACVD Resident Review 2003

Route of allergen exposure – Percutaneous absorption
Pathogenesis: Route of allergen exposure – Percutaneous absorption Y IgE Allergen 2) Local production of IgE 1) Allergen is captured by LC in the skin Y Y Y R Marsella ACVD Resident Review 2003

Role of IgE- Facts in support 1) Role in allergen capture
Canine AD Increased expression of surface bound IgE on LC in lesional skin (Olivry et al, 1996) Human AD LC capture allergen via their high affinity IgE receptor (FcRI) Important for LC maturation R Marsella ACVD Resident Review 2003

Role of IgE - Facts in support 2) Role in the effector pathway
Canine AD Most dogs with AD have detectable allergen-specific IgE Human AD Most patients with AD exhibit both elevated total and allergen specific IgE (extrinsic AD, 80% of patients) R Marsella ACVD Resident Review 2003

Role of IgE - Controversies Canine AD IgE do not correlate with severity of disease No difference in serum total IgE between normal and atopic dogs Negative IDST and serology testing in some dogs with clinical AD Human AD People with genetic inability to produce IgE may develop AD Patients treated with g-IFN improve despite raising IgE Intrinsic AD or non-allergic AD (20%) R Marsella ACVD Resident Review 2003

Role of IgE – Controversies
Canine AD Negative IDST and serology testing in some dogs with clinical AD Anergy? Allergens not included in the test? Wrong season? Heterogeneity of IgE R Marsella ACVD Resident Review 2003

Role of IgE- Controversies Canine AD
Positive IDST and serology testing in normal dogs with no signs of AD IgE heterogeneity Positive IDST and serology testing: a secondary criteria for diagnosis R Marsella ACVD Resident Review 2003

Role of IgE- Controversies Canine AD
Past attempts to create a model for cAD in high IgE producing dogs have failed No correlation existed between serum levels of IgE and development of disease R Marsella ACVD Resident Review 2003

IgE in canine AD Evaluation of total and cell bound IgE in normal and atopic dogs (Jackson, 2002) No significant difference in levels of total IgE ** No significant difference in % of B cells expressing IgE Total IgE did not correlate with cell bound IgE in any of the leukocyte populations studied ** **difference between canine and human AD R Marsella ACVD Resident Review 2003

IgE - Controversies Additional factors besides IgE are necessary to cause disease T cells abnormalities Abnormality in lipid composition in the skin Altered reactivity in the skin β adrenergic hyporesponsiveness Increased PDE, decreased cAMP R Marsella ACVD Resident Review 2003

Intrinsic AD or non allergic atopiform dermatitis
10-30% of humans with AD do not have increased IgE levels Negative IDST Negative APT Negative serology R Marsella ACVD Resident Review 2003

Human AD Extrinsic AD  IgE and FceRI + APT + serology + IDST  IL-13  IL-5  IL-4 Intrinsic AD Normal IgE, low FceRI - APT - serology - IDST  IL-13  IL-5  IL-4 but  IL-4Ra (receptor for both IL-4 and IL-13) R Marsella ACVD Resident Review 2003

Intrinsic AD IL-13 and IL-4 important for IgE production, but non sufficient Additional co-stimulatory factors missing in patients with intrinsic AD? IL-13 may cause disease directly bypassing production of IgE R Marsella ACVD Resident Review 2003

IgE as epiphenomena in human AD?
Increased LC in lesions and increased expression of FceRI may derive from excessive transcutaneous allergen exposure in traumatized skin R Marsella ACVD Resident Review 2003

ROLE OF MAST CELLS AND HISTAMINE IN AD R Marsella ACVD Resident Review 2003

Role of histamine in AD Circulating levels
Human AD Elevated during exacerbation of disease (plasma) and return to normal during clinical remission Correlation between histamine release and IgE levels is controversial Canine AD Serum concentrations in dogs with AD are same or lower than in controls No correlation with IgE R Marsella ACVD Resident Review 2003

Role of histamine in AD Skin
Human AD Increased concentrations in AD patients Good correlation between cutaneous and circulating levels of histamine Canine AD Levels are greater in dogs with AD than normal dogs No correlation between cutaneous and plasma histamine concentrations R Marsella ACVD Resident Review 2003

IDST reactivity to histamine in AD
Human AD Decreased response in people with AD Down regulation of target structures? Canine AD Decreased response in dogs with AD Down regulation of target structures? R Marsella ACVD Resident Review 2003

Role of histamine in AD Peripheral leukocytes
Human AD Increased histamine releasability after stimulation High and low histamine responders Pre-medication with PDE inhibitors normalized histamine”releasability” Canine AD Greater tendency to release histamine than normal dogs R Marsella ACVD Resident Review 2003

Role of Mast cells in AD Traditionally seen as the effector cell for Type I hypersensitivity Insufficient evidence to support an important role in canine AD R Marsella ACVD Resident Review 2003

Allergen IgE MAST CELL Y Y FceRI Codein Substance P Y Anti-IgE Y C5a Stem cell factor Y Anti-FceRI R Marsella ACVD Resident Review 2003

MAST CELL DEGRANULATION Newly-formed mediators PGD2 LTC4 PAF Pre-formed mediators Proteases (e.g. tryptase) Heparin Histamine Cytokines (e.g. IL-1, 2, 3, 4, 5, 6, 8, 9, 13, GM-CSF, TNF-a, IFN-g, MIP-1a and MIP-1b) R Marsella ACVD Resident Review 2003

TYPE I HYPERSENSIVITY Histamine FceRI Y Allergen IgE LT IL-1, 2, 3, 4, 5, 13 IL-6, TNF- a Blood vessel R Marsella ACVD Resident Review 2003

Canine Mast cells Heterogeneity, not tissue specific Highest number on pinnae and feet R Marsella ACVD Resident Review 2003

Mast cells in canine AD Total histamine content, per skin mast cell, is higher in dogs with AD than controls (Nimmo Wilkie, 1990) Increased histamine/cell? Increased reactivity to non-immunological and immunological stimuli (Demora, 1996) R Marsella ACVD Resident Review 2003

Mast cells in canine AD Number of mast cells in atopic skin is controversial Increased, decreased, normal (Scott et al, 1981) Same as normal dogs (Olivry, 1997 and Welle 1999) Increased but not in all sites (Nimmo Wilkie, 1990) R Marsella ACVD Resident Review 2003

Leukotrienes in AD Peripheral leukocytes
Human AD Enhanced release of LTB4 and LTC4 in patients with AD when compared to controls Increased LTA4 hydrolase Canine AD No differences in s-LT synthesis between normal and dogs with AD after challenge R Marsella ACVD Resident Review 2003

Leukotrienes in AD Skin
Human AD Increased LTB4 in lesional skin of atopic patients Increased production of LT after allergen challenge Canine AD No differences in s-LT between controls and non-lesional skin of AD dogs Within AD,no differences in s-LT between lesional and non-lesional skin R Marsella ACVD Resident Review 2003

Role of T cells, cytokines, chemokines
The new theories: Role of T cells, cytokines, chemokines and type IV hypersensitivity in AD R Marsella ACVD Resident Review 2003

Additional players T cells Dendritic cells Cytokines Function Kinetics APC Chemokines Neuropeptides R Marsella ACVD Resident Review 2003

T cell sub-populations - Human AD Cutaneous lesions CD4+, CLA+ Imbalance in the T helper cells (BIPHASIC response) Acute phase Th2 cytokines (IL-3, 4, 5, and 13) Chronic phase Th1 cytokines (IL-2, 12 and g-IFN) R Marsella ACVD Resident Review 2003

T helper 2 T helper 1 IL-4 IL-5 IL-2 -IFN Atopic Dermatitis (acute lesions) R Marsella ACVD Resident Review 2003

T helper 2 T helper 1 IL-4 IL-5 IL-2 -IFN Atopic Dermatitis (chronic lesions) R Marsella ACVD Resident Review 2003

Epidermal dendritic cells in AD
Langerhans cells (LC) Resident cells Polarization toward Th2 response (IL-4, IL-13) Stimulation by TLR (early phase) Stimulation of FceRI (later phase) Inflammatory dendritic epidermal cells (IDEC) Recruited ex novo from dermis Upon stimulation of FceRI they release IL-12 and IL18  switch of Th response into Th1 R Marsella ACVD Resident Review 2003

Dendritic cells in AD Receptors FceRI, FceRII, TLR2 and 4 Important for antigen presentation Important for cytokine release and modulation of T cell population Increased number in chronic lesions of AD R Marsella ACVD Resident Review 2003

Cytokines in AD Pro-inflammatory cytokines IL-1, IL-6 and TNF-a Th2 cytokines IL3, 4, 5, 13 Th1 cytokines IL-2, g-IFN, IL-12 IL-18 “Suppressive cytokines” TGF-b and IL-10 R Marsella ACVD Resident Review 2003

TNF-a Produced by: Mononuclear cells Neutrophils Activated T cells NK cells Mast cells Keratinocytes R Marsella ACVD Resident Review 2003

TNF-a Inducers Trauma LPS Toll-like receptors (TLR2 and TLR4) R Marsella ACVD Resident Review 2003

Role of TNF-α in AD Induces adhesion molecules expression ICAM-1, ELAM-1, VCAM-1 No direct effect on lymphocyte proliferation TNF-a increases: IL-1 (activation and recruiting of inflammatory cells, expression of adhesion molecules) IL-3 (mast cell growth factor) IL-4 (IgE synthesis) R Marsella ACVD Resident Review 2003

Role of TNF-a in AD Increases: IL-5 (maturation and activation of eosinophils) IL-6 (important for IL-4 induced IgE synthesis) IL-8 (chemotactic for neutrophils) GM-CSF (activation and prolonged survival of eosinophils) R Marsella ACVD Resident Review 2003

TNF-a and AD Expression of TNF-a up-regulated 2-4 hours after allergen challenge in atopic people (Gosset et al, 1992) Expression of TNF-a correlates with severity of late phase cutaneous reactions after allergen challenge in atopic people (Gosset et al, 1992) R Marsella ACVD Resident Review 2003

TNF-a and AD TNF-a produced by mast cells after IgE mediated activation (Gordon et al, 1990) Anti-TNF-a antibodies abrogate LPR in mice (Wershill et al, 1991) R Marsella ACVD Resident Review 2003

IL-1 Family of peptides IL-1a, IL-1b, IL-1ra, IL-18 Two receptors Type I - Active Type II – Inactive, anti-inflammatory functions R Marsella ACVD Resident Review 2003

IL-1 Produced by: Mononuclear cells Endothelial cells Keratinocytes R Marsella ACVD Resident Review 2003

IL-1 Activation of T cells Increased IL-2 and IL-2 receptors Expression of adhesion molecules ICAM-1, VCAM-1, E-selectin R Marsella ACVD Resident Review 2003

IL-1ra (receptor antagonist)
Important for down-regulation of inflammatory process Up-regulated by: IL-4, IL-13, IL-6 R Marsella ACVD Resident Review 2003

IL-6 Produced by: Mononuclear cells T cells B cells Fibroblasts Endothelial cells Keratinocytes R Marsella ACVD Resident Review 2003

IL-6 Pro-inflammatory properties T cell activation and differentiation B cell differentiation and Ig synthesis Anti-inflammatory properties Inhibits IL-1 and TNF-a Increases IL-1ra R Marsella ACVD Resident Review 2003

Cytokines in human AD IL-6 Role in IL-4 induced IgE synthesis Released after allergen challenge in humans with AD Correlates with the size of cutaneous LPR R Marsella ACVD Resident Review 2003

IL-6 and AD IL-6 released after allergen challenge in atopic people (Lee et al, 1992) IL-6 production correlates with severity of LPR (Yamada et al, 1995) Obligatory role of IL-6 in IL-4 induced IgE synthesis in humans (Vercelli et al, 1989) IL-6 produced by mast cells after IgE mediated activation (Gordon et al, 1990) R Marsella ACVD Resident Review 2003

Cytokines in AD Pro-inflammatory cytokines IL-1, IL-6 and TNF-a Th2 cytokines IL 4, 5, 13 Th1 cytokines IL-2, g-IFN, IL-12 IL-18 “Suppressive cytokines” TGF-b and IL-10 R Marsella ACVD Resident Review 2003

IL-4 Produced by: T cells Eosinophils Mast cells Basophils R Marsella ACVD Resident Review 2003

IL-4 IL-4 and 13 share Common receptor subunit (IL-4Ra) – 8 allelic variants reported, some associated with AD 25% homology Overlap in functions Stimulates antigen presentation MHCII, B7, CD40 Increases expression of CD23 (FceRII) On B cells, Macrophages, APC Isotype switching (IgE) Increases LTC4 synthetase R Marsella ACVD Resident Review 2003

IL-4 and AD Increases expression of adhesion molecules Differentiation of Th0 into Th2 (function not shared with IL-13) Prevents apoptosis of T cells Increases resistance to glucocorticoids R Marsella ACVD Resident Review 2003

Actions of IL-4 Anti-inflammatory properties Decreases IL-1, IL-6, TNF-a Increases IL-1ra R Marsella ACVD Resident Review 2003

IL-13 and AD Receptor IL-4Ra chain of the receptor IL13Ra chain (not expressed on mast cells and T cells) No ability to induce Th2 differentiation or mast cells activation Shares many actions with IL-4 IgE isotype switching Expression of VCAM-1 R Marsella ACVD Resident Review 2003

IL-5 Produced by: Th2 cells Mast cells Eosinophils? Actions on eosinophils Stimulation of eosinophil production Activation of mature eosinophils Increased survival (blockade of apoptosis) Up-regulation of responses to chemokines (increases expression of CCR3) R Marsella ACVD Resident Review 2003

IL-5 and AD Positive correlation between IL-5 release and size of LPR after allergen challenge (Okada et al, 2002) Prevention of blockade of eosinophil apoptosis by oxatomide as a potential treatment for atopic disease (Domae et al, 2003) R Marsella ACVD Resident Review 2003

Eosinophils and s-LT Number of Eosinophils Recruitment Survival + + + + S-LT IL-4, IL-5, IL-13 R Marsella ACVD Resident Review 2003

Cytokines in AD Pro-inflammatory cytokines IL-1, IL-6 and TNF-a Th2 cytokines IL3, 4, 5, 13 Th1 cytokines IL-2, g-IFN, IL-12 IL-18 “Suppressive cytokines” TGF-b and IL-10 R Marsella ACVD Resident Review 2003

IL-2 Produced by T cells (Th1) after antigen stimulation Clonal T cell proliferation and differentiation Activation of NK cells Activation of cytotoxic T cells Activation of macrophages R Marsella ACVD Resident Review 2003

IL-2 and AD IL-2 levels have significant inverse correlation with serum IgE levels (Yoshizawa et al, 2002) IL-2 may inhibit IgE synthesis in patients with AD Prevents apoptosis of T cells in skin of patients with AD R Marsella ACVD Resident Review 2003

g-IFN Produced by: Th1 cells Cytotoxic T cells NK cells Increases expression of MHCI and II Stimulation of cytokines release by monocytes IL-12, IL-18 and IL-23 Activation of macrophages Improves killing by NK cells Inhibits IL-4 and regulates IgE synthesis R Marsella ACVD Resident Review 2003

g-IFN and AD Reduced production by peripheral mononuclear cells in AD Important in chronic lesions of AD Recruitment of mononuclear cells Up-regulation of Fas on keratinocytes which increases susceptibility to apoptosis and leads to spongiosis R Marsella ACVD Resident Review 2003

IL-12 Produced by: Monocytes Macrophages B cells Dendritic cells (IDEC) Induces Th1 response Proliferation of cytotoxic cells R Marsella ACVD Resident Review 2003

IL-12 and AD IL-12 p40 polymorphism is associated with development of AD (Tsunemi et al, 2002) IL-12 serum levels correlate with IL-10 (Yoshizawa et al, 2002) R Marsella ACVD Resident Review 2003

Cytokines in AD Pro-inflammatory cytokines IL-1, IL-6 and TNF-α Th2 cytokines IL3, 4, 5, 13 Th1 cytokines IL-2, -IFN, IL-12 IL-18 “Suppressive cytokines” TGF-β and IL-10 R Marsella ACVD Resident Review 2003

IL-18 Produced by: Macrophages IDEC Keratinocytes Pleiotropic properties Inducer of Th-1 response Stimulates production of g-IFN Co-stimulant for IL-12 Increases expression of ICAM-1 R Marsella ACVD Resident Review 2003

IL-18 Inducer of Th-2 response In presence of IL-3, stimulates basophil degranulation (in absence of IgE) Stimulates IL-4 and IL-13 synthesis  IgE R Marsella ACVD Resident Review 2003

IL-18 in AD Elevated serum levels in human patients with AD (Shida et al 2002, El-Mezzein et al, 2001) Marked expression in skin biopsies from lesional skin in AD patients (Higashi et al, 2002) R Marsella ACVD Resident Review 2003

Cytokines in AD Pro-inflammatory cytokines IL-1, IL-6 and TNF-α Th2 cytokines IL3, 4, 5, 13 Th1 cytokines IL-2, -IFN, IL-12 IL-18 “Suppressive cytokines” TGF-β and IL-10 R Marsella ACVD Resident Review 2003

TGF-b Produced by Th3 or T repressors Inhibits B cells and T helper and cytotoxic Inhibits IgE and mast cell proliferation R Marsella ACVD Resident Review 2003

TGF-b and AD Patients with AD have decreased ability to produce TGF-b TGF-b suppresses AD lesions in NC/Nga mice through down-regulation of IFN-g R Marsella ACVD Resident Review 2003

IL-10 Pleiotropic properties according to circumstances Generally considered as a Th-2 cytokine Suppression of Th-1 response Enhancement of Th-2 response Suppression of pro-inflammatory cytokines Used as a marker of response to IT R Marsella ACVD Resident Review 2003

Redundancy and divergence in the cytokine system Redundancy IL 4 and IL13 IL-1 and TNF-a Cross-reaction of pathways IL Th2 IL Th1 Divergence IL-18 IL-10 R Marsella ACVD Resident Review 2003

Chemokines (Chemotactic cytokines)
Group of small molecules (8-14kD) Released at site of infection or trauma 4 families, classified based on the position of cysteine residues CXC – target neutrophils and lymphocytes CC - target eosinophils, basophils, dendritic cells, T cells, and monocytes C CX3C R Marsella ACVD Resident Review 2003

Chemokines Link with innate immune system Triggered by: Bacterial products Pro-inflammatory cytokines Released in a short time (within 1-2 hours) Binding to seven specific trans-membrane G protein coupled surface receptors (GPCR) R Marsella ACVD Resident Review 2003

Chemokines Induce chemotaxis By up-regulating selectins Affect T cell differentiation Altering cytokine secretion Altering APC trafficking R Marsella ACVD Resident Review 2003

Chemokines and Th cytokines
g-IFN induced chemokines 10kd IFN Inducible Protein (IP-10) Monokine Induced by g-IFN (MIG) IFN-Inducible T cell a chemoattractant (I-TAC) IL-4 and IL-13 induced chemokines Macrophage Chemoattractant Protein (MCP) Eotaxin R Marsella ACVD Resident Review 2003

Chemokine receptors and Th cells
CXCR3 - binds MIG and IP-10 CCR5 - binds RANTES Th2 CCR3 - binds MCP 4 CCR4 - binds TARC, MDC CCR8 R Marsella ACVD Resident Review 2003

Chemokines and eosinophils
CCR3 (high expression) CCR1 (low expression) Chemotactic stimuli Macrophage Inflammatory Protein (MIP) 1a RANTES Eotaxin-1, 2, 3 MCP-2, 3, 4 R Marsella ACVD Resident Review 2003

Antagonism between Chemokines
MIG inhibits CCR3 mediated responses IV administration before allergen challenge inhibits recruitment of eosinophils R Marsella ACVD Resident Review 2003

Chemokines and AD Macrophage Chemoattractant Proteins (MCP) - MCP 2, 3, 4 Bind to CCR3 - Expressed on eosinophils and Th2 Bind to CCR2- Expressed on monocytes (constitutively) and memory T cells (after IL-2 stimulation) R Marsella ACVD Resident Review 2003

Chemokines and AD Macrophage Derived Chemokine (MDC, CCL22) Produced by DC and macrophages Binds to CCR4 - Expressed on Th2 cells Increased expression in patients with AD (both serum and lesional skin) R Marsella ACVD Resident Review 2003

Chemokines and AD Eotaxin (CCL11) Attracts eosinophils Binds to CCR3 - Expressed on eosinophils and Th2 cells RANTES (Regulated on Activation Normal T-cell-Expressed and Secreted or CCL5) Binds to CCR1, CCR3 and CCR5 R Marsella ACVD Resident Review 2003

CC Chemokines and AD Spontaneous production of RANTES, MCP-1 and eotaxin by peripheral mononuclear cells in patients with AD R Marsella ACVD Resident Review 2003

TARC and AD Thymus and activation-regulated chemokine (TARC, CCL17) Produced by keratinocytes, peripheral mononuclear cells, dendritic cells, macrophages, endothelial cells Production is stimulated by: IL-4 and IL-13 Pro-inflammatory cytokines such as -IFN and TNF-α LPS R Marsella ACVD Resident Review 2003

TARC and AD Binds to CCR4 - Expressed on Th2 cells Recruits CLA+, CCR4+ T cells (Th2) into lesional skin Increases integrin-dependent adhesion of T cells to endothelial ICAM-1 Increased expression in patients with AD R Marsella ACVD Resident Review 2003

TARC and AD Plasma TARC levels Significantly increased in patients with AD and correlate with severity of AD TARC expression in the skin Not expressed in normal skin or psoriatic skin Highly expressed in lesional atopic skin Higher expression of TARC in acute lesions than chronic Keratinocytes in the basal layer showed the strongest staining for TARC R Marsella ACVD Resident Review 2003

CCR4 and AD Circulating CCR4+ cells (CD4+ T cells) Significantly higher in AD patients than that in healthy controls CCR4+ cells in the skin Present only in the lesional skin of AD patients, but not in the non-lesional skin R Marsella ACVD Resident Review 2003

CCR4 and AD Pre-administration of CCR4 neutralizing antibody before challenge prevents recruitment of eosinophils and T cells after allergen challenge (Kawasaki et al, 2003) R Marsella ACVD Resident Review 2003

Cytokines in Canine AD (Nuttal et al, 2002)
RNA isolated from atopic and normal skin  IL-4 in atopic skin  TGF-b  IFN-g, TNF-a and IL-2 in lesional vs. non lesional (infections?) R Marsella ACVD Resident Review 2003

Cytokines in Canine AD (Maeda et al, 2002)
mRNA isolated from atopic and normal skin TARC mRNA selectively expressed in lesional skin of dogs with AD  IL-1b, g-IFN and TNF-a in lesional skin compared to non-lesional skin of dogs with AD CCR4 mRNA preferentially expressed in lesional skin of dogs with AD R Marsella ACVD Resident Review 2003

T cell sub-populations in canine AD (Hayahiya et al, 2002)
Expression of cytokine mRNA from peripheral blood mononuclear cells  g-IFN and  IL-5 in dogs with AD No difference in IL-4 and IL-10 between normal and atopic dogs R Marsella ACVD Resident Review 2003

T cell populations in canine AD (Olivry et al, 1999)
Increase in CD4+ and CD8+ T-cells in lesional skin of dogs with AD Predominance of CD4+ T-cells in the epidermis IL-4 and IL-5 cytokine-gene transcripts are detected more commonly in atopic skin biopsies ¼ of atopic samples has type-2 cytokine profiles R Marsella ACVD Resident Review 2003

The Hygiene Hypothesis A protective role for endotoxin?

Bacterial infections and AD
Protective effect (hygiene hypothesis) Toll-like receptors (TLR) and DC Contributing factor in clinical relapses Serving as antigen Stimulating TLR on mast cells and LC Activating keratinocytes Recruiting macrophages R Marsella ACVD Resident Review 2003

Protective effects of innate immunity
Toll-like receptors Expressed on variety of cells (DC, Mast cells, B and T cells) Recognize invariant pathogen associated molecular patterns 10 different types recognized in humans and 11 in mice Type of antigen, dose and receptor determine the outcome R Marsella ACVD Resident Review 2003

Toll-like receptors Stimulation of receptor TLR4 on APC by high doses of LPS Release of antibacterial peptides Release of cytokines important for Th-1 (IL-12, IL-18) Maturation of APC (expression of co-stimulatory molecules, CD 80) R Marsella ACVD Resident Review 2003

-IFN ACQUIRED IMMUNITY IL-12 IL-10 IL-18 Th1 cell NK cell Memory Th2 cell IL-4, 5, 13 ATOPY X INNATE IMMUNITY Dendritic cell Macrophage Monocyte TLR4 Endotoxin (high doses) R Marsella ACVD Resident Review 2003

ACQUIRED IMMUNITY IL-4 Th2 cell IL-5 IL- 13 Memory ATOPY INNATE IMMUNITY Dendritic cell Macrophage Monocyte TLR4 Endotoxin (low doses) R Marsella ACVD Resident Review 2003

Deleterious effects of bacteria TLR on mast cells (TLR 2 and TLR 4) Mast cell degranulation in absence of IgE cross-linking Release of s-LT Release of IL-1, TNF-a, IL-4, IL-5, IL-13 R Marsella ACVD Resident Review 2003

Different actions of bacteria in AD patients Staphilococcus MC Y LC IL-1, IL-6, TNF-a TARC IL-4 Th2 IL-4, 5, 13 IL-1, IL-6 TNF-a, LT, Histamine Th2 IL-4, 5, 13 R Marsella ACVD Resident Review 2003

Trauma of the skin in AD Trauma leads to release of IL-10 (stimulation of Th2) C3 TARC (chemotactic for Th2) Eotaxin (chemotactic for eosinophils) IL-1, TNF-α (increased expression of adhesion molecules leading to leukocytes extravasation) R Marsella ACVD Resident Review 2003

Immunologic phases of AD Trauma Allergens LC Th2 IL-4, 5, 13 Y Staph IDEC YY IL-1, IL-6, TNF-a, TARC, Eotaxin IL-12, 18 Eos Th0 IL-2, g-IFN Th1 MC Y IL-1, IL-6 TNF-a, LT, Histamine Th2 CLA+ Circulation R Marsella ACVD Resident Review 2003

Additional theories on AD
Abnormalities in cyclic nucleotide regulation Excessive activity of PDE  Decreased cAMP  Hyperreactivity and excessive releasability of mediators Role of neuropeptides R Marsella ACVD Resident Review 2003

PDE and AD Increased PDE Dogs with AD (Chan et al, 1985) People with AD (Hanifin et al, 1992) Atopic PDE in man has higher sensitivity to a variety of enzyme inhibitors than healthy controls (Crocker et al, 1998) Deficient cAMP response Dogs with AD (Emala et al, 1995) People with AD (Butler et al, 1983) R Marsella ACVD Resident Review 2003

PDE Various PDE isoenzymes exist PDE-4 important for mediator release Mast cells Macrophages T-lymphocytes Eosinophils Keratinocytes R Marsella ACVD Resident Review 2003

Substance P in human AD Substance P has numerous modulatory effects on inflammation Chemotactic for neutrophils, monocytes, T cells, and eosinophils Induces the expression of cell adhesion molecules Induces proliferation of T helper lymphocytes R Marsella ACVD Resident Review 2003

Substance P in human AD SP receptors identified on mast cells Stimulation of SP receptors triggers degranulation and histamine release R Marsella ACVD Resident Review 2003

Substance P and AD Human AD Decreased reactivity of ID injections of SP SP release is triggered by allergen challenge and by histamine Canine AD Decreased reactivity to ID injections of SP in dogs with AD Altered sensitivity? Altered sensitivity to histamine (triggered by SP)? R Marsella ACVD Resident Review 2003

Substance P and AD Human AD Patients with AD have increased SP immunoreactivity compared to controls Canine AD No difference in SP concentration between normal and AD dogs R Marsella ACVD Resident Review 2003

Treatments for AD Targeted treatments Antihistamines PDE inhibitors (e.g. pentoxifylline) Anti-IgE, Anti-LT, Anti-TNF-a Non-specific treatments Glucocorticoids Calcineurin inhibitors (e.g. Cyclosporine, FK-506) PG analogues R Marsella ACVD Resident Review 2003

Considerations Advantage of targeted treatments Mediator targeted is produced in large quantities Receptors are expressed Other pathways are not affected Disadvantages Alternative circuits may compensate for blockade of mediators Insignificant clinical improvement R Marsella ACVD Resident Review 2003

Considerations Advantages of non-specific treatments Broad spectrum effects Disadvantages Unwanted effects Steroids Decrease IL-10 Increase LTB4 receptor expression Poor inhibitors of LT R Marsella ACVD Resident Review 2003

Alternative treatments for AD The present
Misoprostol PDE inhibitors Pentoxifylline Leukotriene inhibitors Zileuton Calcineurin inhibitors Cyclosporine Tacrolimus Neuropeptide modulators R Marsella ACVD Resident Review 2003

Misoprostol PGE1 analog Rationale for use in cAD: PGE1 increases cAMP  stabilizes cells and blocks secretion of pro-inflammatory cytokines Strong anti-allergic properties in people R Marsella ACVD Resident Review 2003

Misoprostol (Cytotec®)
Open, uncontrolled study (Olivry) 20 dogs with AD received 3-6mcg/kg of misoprostol PO 3x/day for 30 days Pruritus decreased by 50% in 56% of dogs Skin lesions decreased by 50% in 61% of dogs R Marsella ACVD Resident Review 2003

Misoprostol (Cytotec®)
Blinded, placebo controlled, study (Olivry) 20 dogs were either given 5mcg/kg of misoprostol TID for 3 weeks or placebo At the end of the trial, in the misoprostol group Pruritus decreased by 30% Skin lesions decreased by 30% No change was noted in placebo group R Marsella ACVD Resident Review 2003

PDE inhibitors Individuals with AD have high levels of PDE and low cAMP PDE inhibitors Decrease production of IL-10 Decrease production of PGE2 by monocytes and of IL-4 by T cells PDE inhibitors might reduce inflammation in AD R Marsella ACVD Resident Review 2003

Pentoxifylline Methyl-xanthine derivative Strong PDE inhibitor Rationale for use in cAD: It inhibits PDE  increases cAMP  stabilizes cells It suppresses synthesis of pro-inflammatory cytokines (IL-1, IL-6 and TNF-α) R Marsella ACVD Resident Review 2003

Pentoxifylline (Trental®) Double blinded, placebo controlled, cross over clinical trial (Marsella et al, 2000) 10 dogs with AD were randomly divided into 2 groups (A and B) Group A received PTX at 10mg/kg BID for 4 weeks Groups B received placebo for 4 weeks R Marsella ACVD Resident Review 2003

Pentoxifylline (Trental®) After 4 weeks, a 2 week wash-out period was observed and then the treatments were switched R Marsella ACVD Resident Review 2003

Pentoxifylline (Trental®)
At the end of the trial Significant decrease in pruritus and erythema was observed in PTX treated dogs when compared to placebo Residual pruritus was observed in all dogs R Marsella ACVD Resident Review 2003

Pentoxifylline (Trental®) Conclusions PTX is a moderately effective adjunctive treatment for canine AD Well tolerated in dogs R Marsella ACVD Resident Review 2003

Pentoxifylline (Trental®) Efficacy seems to be dose dependent Half life is short thus 3x/day is better than 2x/day Dogs with grass allergy respond better than dogs allergic to HDM and molds Older dogs respond better than younger dogs R Marsella ACVD Resident Review 2003

Arofylline (Almirall®)
Oral PDE-4 inhibitor Controlled study in dogs with AD (Ferrer et al, 1999) Arophylline (1 mg/kg BID) for 4 weeks Prednisone (0.5 mg/kg BID for the first week, SID for the second week and EOD for two additional weeks) Pruritus and skin lesions were evaluated and graded (scale from 0 to 3) weekly R Marsella ACVD Resident Review 2003

Arofylline (Almirall®)
No significant difference was noted among treatments Frequent adverse effects in the arofylline group Vomiting, diarrhea and anorexia R Marsella ACVD Resident Review 2003

Other PDE inhibitors In human medicine Topical PDE inhibitors (e.g. Ro ) R Marsella ACVD Resident Review 2003

Capsaicin Capsaicin is a derivative of chili pepper Decreases pain and itch Mechanism of action unknown Depletion of Substance P (SP) from sensory nerve endings? Desensitization of C fibers? R Marsella ACVD Resident Review 2003

Capsaicin Rationale for use in AD In humans, there is evidence that SP plays a role in AD SP release is triggered by allergen challenge and by histamine SP receptors have been identified on mast cells R Marsella ACVD Resident Review 2003

Capsaicin clinical trial Double blinded, placebo-controlled clinical trial (Marsella et al, 2002) 12 dogs with AD were selected and randomly divided into 2 groups Group A received 0.025% capsaicin lotion 2x/day for 6 weeks Group B received placebo lotion R Marsella ACVD Resident Review 2003

Capsaicin clinical trial After 6 weeks, there was a 4 week washout period and groups were crossed over Investigator scored pruritus before and after each treatment Owner scored pruritus on a weekly basis On week 0 and 6 skin biopsies were taken (to extract and measure SP (via ELISA) R Marsella ACVD Resident Review 2003

Results – Owner scores of pruritus A week 6 in the capsaicin group Scores were significantly lower scores than the placebo group (p=0.005) Scores were significantly lower than the beginning of the study (p=0.0006) Scores worsened after first week of treatment (not significant) R Marsella ACVD Resident Review 2003

Correlation between pruritus and SP No correlation was found between pruritus and SP concentrations in the skin R Marsella ACVD Resident Review 2003

Capsaicin Conclusions Capsaicin at 0.025% was well tolerated Alternative topical treatment for cAD Initial worsening may be observed R Marsella ACVD Resident Review 2003

Leukotrienes inhibitors in hAD
Leukotriene receptor antagonists and inhibitors have been used successfully in the management of atopic disease in man R Marsella ACVD Resident Review 2003

Zileuton in man Zileuton inhibits LT production (5-LO inhibitor) Used successfully in the management of atopic disease in man R Marsella ACVD Resident Review 2003

Zileuton in dogs Zileuton is rapidly absorbed in the dog after oral administration Peak levels are achieved within 1 hour Half-life (t1/2) is approximately 7.5 hours R Marsella ACVD Resident Review 2003

Clinical trial with Zileuton
Controlled study (Crow and Marsella, 2001) 9 dogs with AD were selected and randomly divided into two groups Group A received zileuton (Zyflo) orally at 2 mg/kg TID for 30 days Group B received placebo carrier suspension TID for 30 days R Marsella ACVD Resident Review 2003

Zileuton for canine AD Double-blinded, placebo controlled, cross-over pilot study Dogs were assigned to either Zileuton or placebo for 30 days After a wash out period of 5 days, the treatments were switched R Marsella ACVD Resident Review 2003

Zileuton for canine AD Clinical signs (erythema and pruritus) were evaluated and scored by the investigator on day 0 and 30 of each treatment R Marsella ACVD Resident Review 2003

Results – Investigator scores of erythema
On Day 30 in the zileuton group Erythema scores were lower than the placebo treated dogs (p=0.02) Erythema was significantly decreased (p=0.02) compared to day 0 On Day 30 in the placebo group No significant change compared to day 0 R Marsella ACVD Resident Review 2003

Results – Investigator scores of pruritus
On day 0 and 30 There were no differences between zileuton or placebo treated dogs Scores did not change over time between treatment groups or within each group R Marsella ACVD Resident Review 2003

Results – Owners scores of Pruritus
No significant changes over time in either group R Marsella ACVD Resident Review 2003

Conclusions Pilot study using Zileuton
Zileuton was well tolerated 4 weeks of treatment significantly decreased erythema, but had no effect on pruritus R Marsella ACVD Resident Review 2003

Cyclosporine A (Sandimmune®, Neoral®) Fungal metabolite with immuno-modulatory properties Rationale for use in cAD: It suppresses T cell proliferation and cytokine production (IL-1, IL-2, IL4, IL-6, and TNF-α) It suppresses histamine release from mast cells R Marsella ACVD Resident Review 2003

Cyclosporine: Mode of Action
CaN P- NFAT TCR CM calmodulin Ca2+ CycloP NFAT AP1 calcineurin + cyclophilin R Marsella ACVD Resident Review 2003

X Cyclosporine in Atopic Dermatitis IL-4 ACUTE CHRONIC Y Y Y LC Th2
MC Y IL-1, IL-6 TNF-a, LT, Histamine Th1 IL-2 Circulation R Marsella ACVD Resident Review 2003

Cyclosporine A Pharmacokinetics Absorption Metabolism Drug interactions Different formulations Adverse effects R Marsella ACVD Resident Review 2003

Cyclosporine A Very effective in human AD after oral administration Not effective after topical application R Marsella ACVD Resident Review 2003

Cyclosporine A (Neoral®) Open study (Fontaine et al, 2001) 14 dogs with AD received 5mg/kg once daily for 2 weeks At the end of the trial: Pruritus decreased by 100% Skin lesions decreased by 60% R Marsella ACVD Resident Review 2003

Cyclosporine A (Neoral®) Controlled study (Olivry et al, 2002) 30 dogs with AD were randomly allocated to receive either: Oral solution of Neoral® once daily (5 mg/kg) or Prednisolone (0.5 mg/kg) for 6 weeks R Marsella ACVD Resident Review 2003

Cyclosporine A (Neoral®) Controlled study (Olivry et al, 2002) % reduction of skin lesions at 6 weeks: 68-70% in the prednisolone group 58% in the cyclosporine group % reduction in pruritus at 6 weeks: 81% in the prednisolone group 78% in the cyclosporine group R Marsella ACVD Resident Review 2003

Cyclosporine A (Neoral®)
Controlled study (Steffan et al, 2003) Multicentre, parallel, blinded, randomized, controlled, long-term study Comparison between cyclosporine (117 dogs) and methylprednisolone (59 dogs) for cAD for 4 months Cs (induction): 5mg/kg Tapered according to clinical response R Marsella ACVD Resident Review 2003

Cyclosporine A (Neoral®)
Controlled study (Steffan et al, 2003) % reduction of lesion scores 52% in Cs 45% in MP % reduction in pruritus score 36% in Cs 33% in MP No significant changes in CBC and chemistry Cs dogs had more GI problems MP dogs tended to have more skin infections R Marsella ACVD Resident Review 2003

Cyclosporine A Conclusions Effective treatment for refractory cases of cAD Same efficacy of prednisone It may work in cases where steroids are not effective or stop working No increased incidence of skin infections and demodicosis was noted R Marsella ACVD Resident Review 2003

Cyclosporine A Conclusions Efficacy may be noted within the first week of therapy Side effects: Vomiting, diarrhea, bone marrow suppression, nephropathy, papillomatous dermatitis, gingival hyperplasia R Marsella ACVD Resident Review 2003

Tacrolimus Tacrolimus (FK-506) is a macrolide lactone produced by the fungus Streptomyces tsukubaensis Mechanism of action similar to Cyclosporine A R Marsella ACVD Resident Review 2003

Tacrolimus and DC IDEC are reduced after only one week Decreased inflammation LC are not affected No effect on risk for secondary infections R Marsella ACVD Resident Review 2003

Tacrolimus and cytokines
Suppression of synthesis of: IL-2 IL-4 IL-5 g-IFN R Marsella ACVD Resident Review 2003

Tacrolimus Advantage of being effective after topical application Successfully used to decrease signs of AD in people Clinical improvement is marked and rapid R Marsella ACVD Resident Review 2003

Tacrolimus in human AD 0.1% ointment (controlled studies) 83% improvement Significant difference with placebo was evident as early as day 8 0.3% ointment (controlled studies) On day 3, significant improvement noted in 81% of adults and 88% of children On day 8, improvement was evident in 94% of adults and 100% of children R Marsella ACVD Resident Review 2003

Tacrolimus in human AD Minimally absorbed and well tolerated 0.3% ointment applied to up to 30% of total body surface Highest blood level of tacrolimus was 1.6ng/ml (Toxic levels are > 20ng/ml) No accumulation was found with repetitive applications R Marsella ACVD Resident Review 2003

Clinical trial in dogs with AD Marsella et al, 2002 Randomized, double blind, placebo controlled, cross-over study to evaluate the efficacy and safety of 0.3% tacrolimus lotion in dogs with AD Marsella et al, 2003 (unpublished) Randomized, double blind, placebo controlled, cross-over study to evaluate the efficacy and safety of 0.1% tacrolimus ointment in dogs with AD R Marsella ACVD Resident Review 2003

TAC (0.3% lotion) clinical trial Six dogs with AD were selected Selected dogs were randomly assigned to either group A (0.3% tacrolimus lotion at 0.1ml/kg/day) or group B (vehicle) for 4 weeks After 2-week wash out, treatments were switched R Marsella ACVD Resident Review 2003

Evaluation of efficacy and safety Owners evaluated pruritus weekly Investigator evaluated pruritus and erythema at at week 0 and 4 (beginning and end of each treatment) Blood was collected for CBC and chemistry panel and to monitor drug absorption (ELISA for TAC) on week 0 and 4 of each treatment R Marsella ACVD Resident Review 2003

Results CBC and Chemistry No significant changes were detected Tacrolimus values On day 28 tacrolimus levels were significantly higher at 2 and 4 hours compared to day 0 Peak concentrations noted at 2 hours All mean values were below toxicity levels R Marsella ACVD Resident Review 2003

Results – Owner score of pruritus No difference was found for both groups overtime R Marsella ACVD Resident Review 2003

Results – Investigator score of pruritus Pruritus decreased significantly within the TAC group (p=0.03) R Marsella ACVD Resident Review 2003

Results – Investigator score of erythema Erythema decreased significantly in the TAC group (p=0.015) R Marsella ACVD Resident Review 2003

Conclusions – Pilot study using 0.3% tacrolimus lotion Tacrolimus was well tolerated No adverse effects were noted Tacrolimus was effective in decreasing scores according to the investigator but not the perception of pruritus by owners R Marsella ACVD Resident Review 2003

New trial using TAC 0.1% ointment (Protopic®)
Stability of lotion could not be guaranteed Need to repeat a study using the ointment, once commercially available Efficacy Safety 2 formulations available in the US 0.1% 0.03% R Marsella ACVD Resident Review 2003

Experimental Design Randomized, double-blinded, placebo-controlled cross-over study Selected dogs were allocated to either tacrolimus or placebo for 4 weeks Wash out period of 2 weeks Treatments were switched (4 additional weeks) R Marsella ACVD Resident Review 2003

Materials and Methods All dogs were evaluated on week 0 and 4 of each treatment PE Blood samples CBC Chemistry panels Tacrolimus levels Cytology Scoring of clinical signs (CADESI) R Marsella ACVD Resident Review 2003

Modified CADESI used by Investigator
Body divided into 40 sites Evaluated for: Erythema Lichenification Excoriations Papules Pruritus On a scale of (3 = severe) R Marsella ACVD Resident Review 2003

Materials and Methods Owners evaluation of pruritus Required to fill out a scoring sheet once a week Body divided into 22 sites Evaluated for pruritus once weekly On a scale of 0 - 3 R Marsella ACVD Resident Review 2003

Results – Owners evaluation of pruritus
Within the TAC group Significant decrease of scores at week 3 and 4, compared to week 0 (p< 0.01 and p=0.055, respectively) Within the placebo group No differences R Marsella ACVD Resident Review 2003

Results – Owners evaluation of pruritus

Results – Investigator scores
Within the TAC group Scores at week 4 were significantly lower than week 0 (p=0.0019) Within the placebo group No differences R Marsella ACVD Resident Review 2003

Results – Investigator scores R Marsella ACVD Resident Review 2003

Results – TAC blood concentrations
Week 0 Week 4 R Marsella ACVD Resident Review 2003

Results – CBC and Chemistry
No changes in CBC and Chemistry parameters were detected between groups or within groups No adverse effects was noted during the trial in either group R Marsella ACVD Resident Review 2003

Results Dogs with localized disease responded better on TAC than dogs with generalized Investigator scores Generalized disease: 24% decrease Localized disease: 61% decrease Owners scores Generalized disease: 19% decrease Localized disease: 58% decrease R Marsella ACVD Resident Review 2003

Conclusions Clinical trial using 0.1% ointment
TAC is well tolerated and minimally absorbed in dogs with AD Protopic® is a useful topical treatment for canine AD Protopic® is best used in dogs with localized disease due to the difficulty in application on extensive parts of the body R Marsella ACVD Resident Review 2003

Additional calcineurin inhibitors
SDZ ASM 981 or Pimecrolimus (Elidel®, 1%) Sirolimus (Rapamycin) Efomycin Everolimus (SDZ RAP) R Marsella ACVD Resident Review 2003

So, what do we know about AD? Conclusions Cause of AD is a complex interaction Genetic susceptibility Environmental factors Abnormal immune responses Likely that not one single mediators is responsible for clinical signs R Marsella ACVD Resident Review 2003

Rejection of old dogma AD may represent a type IV hypersensitivity more than originally thought AD encompasses a spectrum of abnormalities, different for each patient AD is a dynamic process R Marsella ACVD Resident Review 2003

Directions for the future
Identification of mediators of disease in order to develop targeted treatments Anti-IL-5, anti-TNF-α? anti- CCR4? Anti-IgE? Evaluation of genotypes to better predict clinical response of individual patients R Marsella ACVD Resident Review 2003

Questions? R Marsella ACVD Resident Review 2003

Rosanna Marsella University of Florida

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