IMMUNE MEDIATED BULLOUS LESIONS

OF SKIN

BY EKTA JAJODIA

Blister – fluid filled cavity within or beneath the epidermis

VESICLES( <0.5cm in diam)

BULLAE(>0.5cm in diam)

PATHOLOGICAL EVELUATIONApproach to diagnosis-

1. Anatomical level of split/blister separation plane2. Mech. of blister formation3. In case of subepidermal blisters – presence/absence of

inflammation, pattern and specific cell types involved

ANATOMICAL LEVEL OF SPLIT Blister may form at any one of four diff levels-

1. Subcorneal/intracorneal2. Within spinous or malphigian layers3. Suprabasilar4. Subepidermal

MECHANISM OF BLISTER FORMATION

1. SPONGIOSIS – Accumulation of ECF within epidermis with resultant separation of keratinocytes

Stellate appearance of keratinocytes

2. ACANTHOLYSIS – Loss of keratinocyte cell-cell contact

Keratinocytes are rounded with condensed cytoplasm, large nuclei,peripheral condensation of chromatin and prominent nucleoli

Acantholytic cells are viable

3. RETICULAR DEGENERATION (Ballooning degeneration) – intracellular edema with secondary rupture of keratinocytes and its death

Desmosomal attachments connects strands of ruptured keratinocytic membrane to intact keratinocyte – gives irregular meshwork appearance to epidermis

4. CYTOLYSIS – disruption of keratinocytes- occurs when high levels of friction/heat damages structural matrix(keartin) and desmosomes

5. BASEMENT MEMBRANE ZONE DISRUPTION

AUTOIMMUNE BULLOUS DISEASES -TARGET ANTIGENS

DISEASE TARGET ANTIGENS

Pemphigus foliaceus Desmoglein 1

Herpetiform pemphigus Desmoglein 1

IgA pemphigus(SPD type) desmocollin 1

IgA pemphigus(IEN type) Desmoglein 1 or 3

Pemphigus vulgaris Desmoglein 3

Epidermolysis bullosa acquisita Type VII collagen

DISEASES TARGET ANTIGENS

Paraneoplastic pemphigus Desmoglein1,3Desmoplakin 1,2

Bullous pemphigoid BPAg1(230kDa)BPAg2(180kDa)

Pemphigoid gestationis BPAg2(180kDa)

Dermatitis herpetiformis Tissue transglutaminase

Linear IgA bullous dermatosis LABD97

Ocular cicatricial pemphigoid plectin

Cicatricial pemphigoid BPAg2, epiligrin

Deep lamina lucida pemphigoid 105kDa

Anti-p200 pemphigoid 200kDa

PEMPHIGOUS GROUP

Acantholysis – mech. of bulla formation here

Divided into 5 types-1. P. vulgaris – reactive form is P. vegetans2. P. foliaceus – lupus like variant- P. erythematosus Endemic variant – folo selvagem3. IgA pemphigus4. Drug induced pemphigus5. Paraneoplastic pemphigus

PATHOPHYSIOLOGY

Target Ags of P. are located in desmosomes which are prominent adhesion molecules Desmosome complex contains- a. Transmembrane constituents – desmoglein(DSG) and

desmocollins (DSC)b. Cytoplasmic constituents – plakoglobin(PG), plakophilin(PP)

and desmoplakin(DP) ECD of transmembrane constituent form dimers with adjacent

cell

Cytoplasmic domain of transmemb. constituent bind to PG

This PG links intermediate filaments (keratins) to desmosome via DP

DESMOSOME COMPLEX

ECD of DSG and DSC are targets for Abs

DSG1 – expressed in upper layers of epidermisIf Abs form- skin lesions occurNo mucous membrane lesions

DSG3 – expressed in deep layers of epidermis and mucous membraneIf Abs form- oral lesions with or without skin lesions occur

DISEASES TARGET ANTIGENS

P. vulgaris- 1.mucosal mainly 2. mucocutaneous

DSG3DSG1 and 3

P. foliaceus DSG1

PNP DSG1,3 and DP

IgA pemphigus- 1. SPD type 2. IEN type

DSC1DSG1 and 3

PEMPHIGUS FOLIACEUS Recurrent crops of flaccid bullae that easily rupture – so shallow erosions and erythematous plaques Initially in face and trunk – then spreads to involve large areas of body Mucous membrane involvement is rare Target Ag – DSG1Ab – IgG4 subclass 2 types- a. lupus like – P. erythematosus b. endemic variant - fogo selvagem(wild fire) Develops in those who lives near rivers- its said that a black fly(simulium) may be a cause

Pemphigus : foliaceus erythematous : vulgaris : vegetans

SUPERFICIAL

DEEP

HISTOPATHOLOGY Earliest change – formation of vacuoles in intercellular spaces in upper layers of epidermis – these expand leading to cleft formation

Late lesions – epidermis- hyperplastic with parakeratosis and orthokeratosis

Superficial bulla with split high in granular layer or just beneath stratum corneum

Dyskeratotic cells with hyperchromatic nuclei – distinctive feature of granular layer

Bulla contains fibrin, neutrophils and scattered acantholytic keratinocytes (no bacteria present)

Superficial dermis is edematous with mixed inflammation with both eosinophils and neutrophils

Direct immunofluorescence – intercellular staining for IgG and C3 in both affected and normal skin

Indirect immunofluorescence – demonstrates circulating antibodies in nearly 90% of non endemic cases

2. Subcorneal blister c/o neutrophils and acantholytic cells3.Dyskeratotic hyperchromatic granular cells are diagnostic4. IgG deposited in upper layers of epidermis

PEMPHIGUS VULGARIS Target antigen DSG3 in only mucous lesion In case of mucocutaneous DSG3 and 1 both Abs are predominantly IgG4 subclass Initial lesion is oral blister with ulcers and erosions – weeks to months later cutaneous lesions In addition to oral lesions , other mucosal lesions may develop - conjunctiva, larynx and rarely esophagus, urethra, vulva, vagina

HISTOPATHOLOGY - earliest changes- edema and disappearance of intercellular bridges of keratinocytes in lower epidermis

Established lesions – suprabasal bullae with acantholysis Basal cells lose their intercellular bridges but remain attached to dermis giving a tombstone appearance Blister cavity contains acantholytic cells with some eosinophils and neutrophils Dermal changes are usually mild, superficial mixed inflammatory cell infiltrate No histological difference between drug induced P. vulgaris from idiopathic cases Immunostaining with monoclonal Ab 32-2B – detects DSG 1 and 3 – patchy pattern of staining seen in idiopathic cases and not in drug induced type DIF – demonstrates IgG in intercellular regions of epidermis

The earliest changes consist of intercellular edema with eosinophilic spongiosis leading to loss of intercellular bridges in the lower epidermis

SUPRABASAL BLISTER

The suprabasal blister contains few acantholytic cells, neutrophils and eosinophils. Note the dermal papillae lined by a single layer of basal keratinocytes, so-called villi

Lace like intercellular space deposition of IgG

PV – suprabasilarPF – subcorneal/within granular layer

IgA PEMPHIGUS

Intraepidermal vesicobullous eruption

Most common sites are axilla and groin. Can involve trunk and proximal extremities Mucous membranes are usually spared

2 types-1. Subcorneal pustular dermatosis (SPD) type2. Intraepidermal neutrophilic(IEN) type – lesions with central

crusts and peripheral vesiculation- so called sunflower lesions target Ags – in SPD type – DSC 1 in IEN type – DSG 1and 3

HISTOPATHOLOGY

In SPD type – sub corneal pustules with mild acantholysis In IEN type – intraepidermal pustules Underlying dermis shows mild inflammatory cell infiltrate

DIF – Intercellular deposition of IgA in epidermis In SPD type – increased intensity of staining in upper epidermis In IEN type – IgA staining is throughout the entire epidermis

IgG is present in small number of cases – known as IgA/IgG pemphigus

SPD – subcorneal pustule

Neutrophils and fibrin in blister cavity

PARANEOPLASTIC PEMPHIGUS Characterized by polymorphous skin lesions with features of both

erythema multiforme and pemphigus vulgaris in association with internal neoplasms, esp. NHL Other ass. are Hodgkin's disease, thymoma, hepatocellular carcinoma, MFH, castleman’s disease, etc PNP can precede diagnosis of malignancy, particularly in patients who develop castleman’s disease, or it may develop many years after development of tumor

7 clinical variants are recognized -1. Erythema multiforme-like (vacuolar interface dermatitis)2. pemphigoid-like (subepidermal blister)3. Pemphigus-like (suprabasilar blister)

4. GVHD-like5. Lichen planus pemphigoid/ lichen planus like (lichenoid inflammation)6. Cicatricial pemphigoid-like7. Linear IgA dermatosis-like

Poor prognosis

3 or more Abs are present in same patient-1. DP 1 ,22. Envoplakin3. DSC 2 ,34. DSG 1 , 35. Periplakin6. Plectin

Abs against DSG 3 are IgG 1 or 2 ( in P.vulgaris against DSG3 is IgG4) Autoantibodies may get deposited in skin, lungs, and other organs and may produce damage to these organs. Known as “paraneoplastic autoimmune multiorgan syndrome”(PAMS) Treatment is aimed at underlying malignancy to control the autoantibody production

HISTOPATHOLOGY - Lichenoid tissue reaction/ interface dermatitis with extension of infiltrate into reticular dermis Mostly lymphocytes but occasional eosinophils and neutrophils Suprabasal acantholysis and clefting may be seen

DIF – Intercellular and BM staining with C3 and IgG

Lichen planus like (lichenoid tissue reaction) and P. vulgaris like (suprabasal blister)

Both intercellular and BM staining with IgG

SUBEPIDERMAL BLISTERS To understand this , it is essential to have some knowledge of epidermal basement membrane zone(BMZ) From epidermis to dermis – 4 distinct components of BM1. Basal keratinocyte plasma membrane2. Lamina lucida3. Lamina densa4. Sublamina densa zone – including anchoring fibrils

BASAL KERATINOCYTE PLASMA MEMBRANE Plasma membrane incorporates hemidesmosomes Components of hemidesmosomes- 1. Intracellular component – Major bullous pemphigoid

antigen(BPAg1) / 230 kDa and plectin

2. Transmembrane component – Minor bullous pemphigoid antigen (BPAg2)/180kDa and α6β4 integrin

Abs to BPAg1 – in bullous pemphigoid

Abs to BPAg2 – in bullous pemphigoid pemphigoid gestationis cicatricial pemphigoid linear IgA bullous dermatoses

Abs to β4 of α6β4 - ocular cicatricial pemphigoid

Mutation of β4 - junctional epidermolysis bullosa

LAMINA LUCIDA

Weakest link in dermal-epidermal junction (DEJ) – so easily severed

M/L Ags ass with lamina lucida – laminin 5, 6, 1 uncein nidogen

defect in this Ags are common in Epidermolysis bullosa

LAMINA DENSA

main component is type IV collagen

Consist of 6 different chains alpha 1-6

Other components are- laminin1, nidogen, precelan

SUBLAMINA DENSA ZONE

Main component is anchoring fibrils(AF) which tether epidermis to papillary dermis

AF are composed of type VII collagen and laminin 332

Abs to type VII collagen seen in – EBA Bullous SLE

SALT-SPLIT SKIN IMMUNOFLUORESCENCE

Variant of indirect immunofluorescence Normal skin from a normal person is taken and soaked in 5ml NaCl

After 24hrs , the salt solution induces a subepidermal blister in the normal skin at the level of lamina lucida

Indirect immunofluorescence done- patient’s serum directed against the salt-split skin

IgG Abs go above the split (epidermal side) in BP IgG Abs go below the split (dermal side) in EBA

B- Bullous pemphigoid; C - EBA

SUBEPIDERMAL BLISTERS

INFLAMMATORY CELLS DISEASES

With litle inflammation EB

With lymphocytes PNP

With eosinophils BPPemphigoid gestationis

With neutrophils DHLinear IgA bullous dermatosis

EPIDERMOLYSIS BULLOSA ACQUISITA Non inflammatory subepidermal bullae develop in areas subjected to minor trauma such as extensor surface of limbs Target antigen – EBA antigen which is a 290 kDa type VII collagen – a major component of anchoring fibrils

HISTOPATHOLOGY- classic form – non inflammatory subepidermal blisters bullous pemphigoid like – inflammatory blisters – mostly lymphocytes and neutrophils PAS stain- BM is split and most of the PAS positive material are in blister roof

DIF – linear deposition of IgG / C3/ C5 along BM zone

A useful clue to the presence of Abs targeting type VII collagen is presence of u-serrated pattern of linear IgG deposition Routine DIF cannot distinguish between EBA and bullous pemphigoid

SALT-SPLIT SKIN TECHNIQUE – Abs bind to dermal floor, deposited below lamina densa

Fluorescence overlay antigen mapping (FOAM) technique – can be used to determine site of deposits

Non inflammatory subepidermal bulla

Serrated deposition of IgG in blister base

BULLOUS PEMPHIGOID

Most common subepidermal blister Occurs primarily in elderly M/L tense bullae develop on normal or erythematous skin Oral lesions seen in 10-40% , but involvement of other mucosal areas is rare May occur in childhood – two types1. Infantile BP – in 1st yr of life – in acral areas2. Localised vulval BP- confined to vulva only Many clinical variants – 1. vesicular pemphigoid2. Pemphigoid vegetans3. Polymorphic pemphigoid 4. Pemphigoid excoriee

5. Drug induced pemphigoid6. Pemphigoid nodularis

Target antigens – BPAg1/230 kDa and BPAg2/180kDa Abs are – IgG4 and IgG1 Abs to BPAg2 is more associated with oral lesions, less responsive to steroids and poor prognosis

HISTOPATHOLOGY- Unilocular subepidermal blisters cell rich type – blister develop on erythematous skin - eosinophils predominant cell in blister cavity and in dermis – more than in arthropod bite reaction cell poor type – blister develop on normal skin – scant perivascular lymphocytic infiltrate with few eosinophils

In lesions of several days duration - blister may appear intraepidermal at its periphery as a result of regeneration

‘Caterpillar bodies’ – segmented , eosinophilic, PAS positive globules arranged in linear array in roof of blisters – feature of porphyria cutanea tarda – sometimes seem in BP

DIF – linear , homogenous deposition of IgG/C3 along BM

Salt-split skin technique- deposition found on the epidermal side of the blister

FIGURE 9-16. Bullous pemphigoid. (A) Prebullous phase, eosinophils are present at the dermoepidermal junction and in the dermis. (B) Cell-rich variant, subepidermal blister formation and an inflammatory infiltrate composed predominantly of eosinophils and a few neutrophils in the dermis and bullous cavity. (C) Cell-poor variant, subepidermal blister with few inflammatory cells.

Vesicular pemphigoid. There is a small subepidermal blister

subepidermal blister, the lumenof which contains eosinophils

Bullous pemphigoid. The basement membrane zone shows a linearpattern of staining for C3.

Salt-split skin IF – deposits on the blister roof (epidermal side)

PEMPHIGOID GESTATIONIS aka herpes gestationis rare, pruritic, vesicobullous dermatosis of pregnancy and puerperium occasionally seen in association with hyadatidiform mole or choriocarcinoma onset is in 2nd or 3rd trimester Subside within several weeks of delivery diagnosis can be made by commercially available BP180 ELISA Rarely neonate develops vesicular lesions due to transfer of maternal BP180 autoantibodies IgG1 class Ab (pemphigoid gestationis factor) is formed against a placental antigen – this cross reacts with BP180 antigen of BM

HISTOPATHOLOGY Early lesion – marked edema of papillary dermis Superficial and mid dermal infiltrate by lymphocytes, eosinophils and histiocytes Infiltrate is predominantly perivascular in location Established blister – is subepidermal , contains similar inflammatory infiltrate within the cavity Eosinophilic microabscesses may be formed in dermal papillae

DIF – Linear pattern of IgG or C3 in BM zoneSalt split skin – deposits in epidermal side

Pemphigoid gestationis. There is subepidermal clefting with both neutrophils and eosinophils in the dermal infiltrate

LINEAR IgA BULLOUS DERMATOSES Subepidermal blistering disorder 2 clinical variants – 1. Chronic bullous dermatosis of childhood2. Adult IgA bullous dermatosis Target Ag – 97 kDa Ag ( degradation products of

180kDa/BPAg2 Chronic bullous dermatosis of childhood – in 1st decade of life

/ large tense bullae/ perioral or genital regions/ polycyclic grouping known as ‘cluster of jewels’ sign

Circulating IgA Abs are present in 70% of childhood disease and in 20% of adult cases

HISTOPATHOLGY – Subepidermal blisters with neutrophil as predominant cell Indistinguishable from DH by presence of papillary microabscesses and neutrophils in light microscope DIF – homogenous linear pattern of IgA deposition along BM zone of non-lesional area

Subepidermal blister with neutrophils in the lumen and in the base

DERMATITIS HERPETIFORMIS Subepidermal blistering disorder – intensely pruritic papules and vesicles – bullae are uncommon Ass. with high incidence of gluten sensitive enteropathy – in 90% cases and also with internal cancers, esp intestinal lymphoma Target Ag – tissue transglutaminase (tTG) sites- elbows, knees, shoulders, nape of neck onset – early adult life Gluten free diet – reversal of villous atrophy and skin lesions / also protective effect against development of lymphoma IgA Abs formed in gut binds with skin transglutaminase(TG) In skin 6 TG isoenzymes are present

Deposits mainly seen are IgA/TG3 aggregates in small blood vessels in papillary dermis This complex activates complement – chemotaxis of neutrophils in papillary dermis Also serum levels of IL-8 increases - increases the expression of CD11b on neutrophil cell surface – increase neutrophil function – enzymes released by these neutrophils destroy two BM components (laminin and type IV collagen) – formation of blisters

HISTOPATHOLOGY – Early lesions – collection of neutrophils and occasional eosinophils at tips of dermal papillae – papillary microabscesses Fibrin is also present at the tips of dermal papillae – imparting a necrotic appearance

In lesions of 36-48 hrs – neutrophil fragmentation occurs Very rarely intraepidermal neutrophils occur – confusion with IgA pemphigus

In older lesions – subepidermal vesiculation occurs Initially multilocular blisters due to interpapillary ridges – but after few days these attachments break down – formation of unilocular blister

Distinction between DH and linear IgA bullous dermatosis is impossible histologically Older lesions may resemble BP – eosinophils more predominant in the latter condition

DIF – Granular / fibrillary/ thready deposits of IgA in dermal papillae of perilesional and uninvolved skin

Deposition is greatest in normal skin adjacent to an active lesion

If DIF testing is negative – repeat test – negative results of DIF testing of 2 appropriately selected biopsy sites are strong indication that patient does not have DH

A- linear deposition of IgA along BM in linear IgA dermatosisB- granular deposits of IgA in the dermal papillae in DH

Dermatitis herpetiformis. A- There is a subepidermal blister , B/C- A microabscess is present in a dermal papilla

DERMATITIS HERPETIFORMIS

PEMPHIGUS FOLIACEUS

THANK

YOU