the human eosinophils

THE HUMAN EOSINOPHIL

•Eosinophil was 1st described for its characteristic cytoplasmic granules

•8 microm in diam•Nuclei – usually bilobed•Has 5 different types of granules•Half life in circulation is 18hrs•Normal range in blood is 0-500/microl•Shows diurnal variation – •Lowest in morning (due to high level of corticosteroids•Highest in evening

•Predominantly tissue cells – major target organ is GIT in healthy individuals

•Once they enter target tissue – do not return to circulation

•In tissues persist for 8-12 days

EOSINOPHIL DIFFERENTIATION

•Derived from CD34+ hematopoietic stem cells in bone marrow

•3 key cytokines are required for its production , differentiation, proliferation, trafficking and survival

•These are produced by T-cells in Bone marrow

IL-5

IL-3

GM-CSF

CYTO

KINE

S

•Early development of Eo/B progenitors is driven by IL-3 and GM-CSF, while at later stages IL-5 regulates terminal differentiation of eosinophils

•Potent Eosinophil chemoattractant

•Facilitate degranulation and release of cationic mediators from eosinophils

IL-5

•Several disease process characterised by hypereosinophilia show increased production of IL-5

•So IL-5 has become therapeutic target

•Monoclonal Ab Mepolizumab acts against IL-5

•Has greatest effect on eosinophil survival

•Eosinophils have antigen presenting properties for which GM-CSF is necessary

GM-CSF (granulocyte macrophage – colony stimulating factor

•Activates eosinophils and make them hypodense

IL-3

•In Bone marrow committed eosinophil precursors are recognised by their exp of IL-5 R, CCR3 and CD34

•C-C chemokines are RANTES and eotaxin

•When eotaxin binds to CCR3 receptor on eosinophils – facilitate the efflux of fully matured eosinophils into peripheral circulation

TRANSCRIPTION FACTORS REUIRED FOR EOSINOPHIL

DEVELOPMENT

C/EBP

GATA-1

PU.1

FOG

C/EBP (CCAAT/ enhancer binding protein)

•C/EBP α -If deficient – defects in formation of granulocytes

•C/EBP β - if deficient - immature eosinophils are formed (as it is required for eosinophil maturation)

GATA - 1•Contains 2 zinc finger motifs

•Expressed in hematopoietic system – eosinophils, erythroid cells, megakaryocytes and mast cells

•The C-terminal of GATA-1 is necessary for eosionphil formation

•Level of GATA-1 expression is important – it activates eosinophil specific gene at low concentration and represses it at high concentration

GMP (granulocte-macrophage progenitors

May express C/EBP α

May not express C/EBP α

GATA-2 acts on such GMPs

GATA-2 acts on such GMPs

Exclusively induces eosinophil formation

Basophils/ mast cells are formed

PU.1

•Mechanism by which PU.1 induces eosinophil commitment involves downregulation of GATA-1 expression

FOG

•Acts as a repressor of eosinophil lineage

•Expression of FOG in eosinophil leads to a loss of eosinophil markers and acquisition of multipotent lineage

• 2 additional trascription factor in eosinophil development are –

1. IFN consensus sequence binding protein (Icsbp)2. Id protein –

Exp of Id1 protein – inhibits eosinophil developmentExp of Id2 protein – accelerates final maturation of

eosinophil

EOSINOPHIL PRODUCTION AND SURVIVAL IN

PERIPHERAL TISSUES

•Eosinophil development and maturation can occur in peripheral / extramedullary sites

Eo/B progenitors are released into blood stream directly from bone marrow

goes to specific target organs on response to cytokines and chemokines

• local tissues such as epithelial cells, endothelial cells and fibroblast produce IL-5 and GM-CSF

•Eosinophils themselves produce IL-5 and GM-CSF that leads to autocrine activation

This is responsible for eosinophil maturation and survival in peripheral tissues

ROLE OF IL-5 IN EOSINOPHIL SURVIVAL

•IL-5 consist of 2 subunits - α subunit and βc subunit

•βc subunit is shared by IL-3 R and GM-CSF R

•IL-5 stimulation through βc subunit leads to phosphorylation of tyrosine kinases Jak2 , Lyn and Syk•Jak 2 signals through STAT1 pathway•Lyn and Syk signals through Ras-Raf-MEK-ERK pathway

•They finally enhances exp of antiapoptotic protein Bcl-x in eosinophils and decreases proapoptotic factors like Bax

•So increase eosinophil survival

EOSINOPHIL HETEROGENEITY

3 different population of eosinophils is known1.Normodense eosinophils-

constitutes >90% of blood eosinophils

2.Hypodense eosinophils – raised in Hypereosinophilia

3.Primed eosinophils

HYPODENSE EOSINOPHILS –

• Morphology – vacuolated , contain more lipid bodies, express less MBP (major basic protein) and possess small size crystalloid granules

• Has greater cell volume• Appears more activated , release more

LTC4 and shows increased cyttoxicity towards helminths

EOSINOPHIL TISSUE ACCUMULATION

• Recruitment of eosinophils across vascular endothelium occurs in 4 steps-1.TETHERING – of eos to luminal surface

of vascular endothelium2.ROLLING – along luminal surface of

activated endothelium in a reversible manner

3.FIRM ADHESION – of eos to endothelium

4.TRANSMIGRATION – through endothelial cells into target tissues

SELECTINS

α 4 INTEGRINS

FOR TETHERING AND ROLLING

β 2 INTEGRINS

FOR FIRM ADHESION

C-C CHEMOKINES

FOR TRANSMIGRATION

EOSINOPHIL MEDIATORS

MEMBRANE DERIVED MEDIATORSEos produce a wide variety of lipid

derived mediators-

Membrane phospholipids

Arachidonic acid

Phospholipase A2

Leukotriens (LTC4)Prostaglandins (PGE2)ThomboxaneLipoxins(LXA4)PAF

GRANULE DERIVED PROTEINS•Eosinophil contain 5 different

population of phospholipid bilayer membrane bound granules-

CRYSTALLOID GRANULES

•Contain crystalline electron dense cores surrounded by electron lucent materx•Take up acidic dyes as they are cationic in nature•Present in mature eosinophils•In immature eosinophils – coreless granules may be seen

•Approx 200 such granules / cell present•CD63 – marker of these granules•The cores contain – MBP•Matrix contain EPO, ECP, EDN

PRIMARY GRANULES•Coreless granules•Enriched with CHARCOT LEYDEN CRYSTAL (CLC) proteins•Present in immature and mature eosinophils•Less abundant than crystalloid granules

SMALL GRANULES

•Coreless granules•Contain acid phosphatase, arylsulfatase , catalase and cytb558

LIPID BODIES

•5 LB/mature eosinophils•Increased in idiopathic hypereosinophilia

SECRETORY VESICLES

•Dumbbell shaped in cross section•Contain albumin•Aka microgranules/tubulovesicular structure

MAJOR BASIC PROTEIN

•Synthesized during promyelocytic stage of eosinophil development as pre pro MBP ------- pro MBP ---------MBP

•Once eos have reached full maturity – MBP is no longer synthesized

•EPO, ECN, EDN continue to be synthesized even in mature Eosinophil

CHARCOT LEYDEN CRYSTAL (CLC) PROTEIN

•Produced at very high levels•Designated as galectin-10•Needle shaped structures that are colorless

DEGRANULATION MECHANISM

•Defined as exocytic fusion of granules with plasma membrane during receptor mediated secretion

Outer leaflet of lipid bilayer membrane surrounding the granule encounters inner leaflet of plasma membrane of the cell

After docking, granule and plasma membrane fuse together and form a reversible structure known as FUSION PORE

Depending on intensity of stimulus, the fusion pore may either

Retreat- leading to separation of granule from plasma membrane

Expand and allow complete integration of granule membrane into plasma membrane as a continous sheet

Granule contents are subsequently expelled to the exterior of cell

FOR DOCKING AND FUSION

SNARES (SNAP RECEPTORS) are required

Granule associated SNAREs (vesicular/V- SNARE)

Plasma membrane associated SNAREs (target / t- SNAREs)

VAMP-7 and VAMP-8 –in crystalloid granulesVAMP-2 – in secretory vesicles

Syntaxin-4SNAP23

Binds VAMP-2

APPROACH TO EOSINOPHILIA

WHAT IS HYPEREOSINOPHILIA?

Normal range of peripheral blood eosinophils is 0-500/microl

Mild eosinophilia 500-1500/microlModerate eosinophilia 1500-5000/microlSevere eosinophilia >5000/microlHypereosinophilia (HE) >1500/microl

TISSUE HYPEREOSINOPHILIA •In bone marrow and GIT normally

increased numbers of eosinophils may be seen

•Other than BM and GIT - eosinophils are undetectable in other organsIn BM – HE is applied when >20% of all nucleated cells are eosinophils

•Exact definition for tissue HE is extramedullary organs is not available

HYPEREOSINOPHILIA

HYPEREOSINOPHILIC SYNDROME(HES)

NON -HYPEREOSINOPHILIC SYNDROME(NON-HES)

Heterogenous group of uncommon disorders characterised by marked eosinophilia in peripheral blood, tissues or both often without an identifiable cause

HYPEREOSINOPHILIC SYNDROME

Idiopathic HES

PDGFRA associated MPNLymphocytic variant HES (L-HES)Familial hypereosinophilia

Churg strauss syndromeEosinophil associated GI disease

Causes of NON-HES

MECHANISM RESULTING IN HE ARE TWO-FOLD

Neoplastic HE

Eosinophils are either a part (In Myeloid neoplasms associated with eosinophiliaOr the major representative(in CEL) of a malignant clone

Reactive/non neoplastic HE

Non clonal eosinophilic proliferation

NEOPLASTIC HE

•c/o myeloid neoplasms associated with HE1.CML2.CEL3.JAK2 mutated MPN4.Certain variant of AML5.Some cases of advanced Systemic

mastocytosis

1.Lymphoid neoplasms associated with eosinophilia

2.Solid tumors associted with eosinophilia

3.Reactive conditions associated with eosinophilia

4.Some cases of mastocytosis

REACTIVE /NON NEOPLASTIC HE

it includes

REACTIVE /NON-NEOPLASTIC HE

1.Lymphoid neoplasms associated with eosinophilia

2.Solid tumors associated with eosinophilia

3.Reactive conditions associated with eosinophilia

4.Some cases of mastocytosis

•Here eosinophils are non neoplastic cells

•It includes-

SYSTEMIC MASTOCYTOSIS (SM)

INDOLENT SMSMOULDERING SMAGGRESSIVE SMMAST CELL LEUKEMIAEOSINOPHILS

ARE NON-CLONAL EOSINOPHILS

ARE CLONAL

LYMPHOPROLIFERATIVE DISORDERS

ASSOCIATED WITH EOSINOPHILIA

LPD ASSOCIATED WITH HYPEREOSINOPHILIA ARE –

1.Hodgkin lymphoma (B-cell neoplasm)2.Lymphoid neoplasms derived from

mature CD4+ Tcells3.Lymphoid neoplasms derived from

CD8+ Tcells4.Immature B-cells and T-cells

PERIPHERAL T CELL LYMPHOMAS (PTCL) ASSOCIATED WITH HYPEREOSINOPHILIA

MYCOSIS FUNGOIDES AND SEZARY SYNDROME

ANGIOIMMUNOBLASTIC T CELL LYMPHOMA

ADULT T CELL LEUKEMIA/LYMPHOMA

SYSTEMIC ANAPLASTIC LARGE CELL LYMPHOMA

PRIMARY CUTANEOUS CD30+ LPD

ENTEROPATHY ASSOCIATED T CELL LYMPHOMA

PTCL,NOS

NON MALIGNANT T -CELL LYMPHOMA ASSOCIATED WITH HYPEREOSINOPHILIA

LYMPHOCYTIC VARIANT HYPEREOSINOPHILIC SYNDROME (HES)

LYMPHOID DYSCRASIAS OF UNKNOWN SIGNIFICANCE

B- CELL LYMPHOPROLIFERATIVE DISORDERS

CLASSICAL HODGIN LYMPHOMA

B CELL ACUTE LYMPHOBLASTIC LEUKEMIA/LYMPHOMA WITH HE

PERIPHERAL T CELL LYMPHOMA (PTCL)•Most T cell malignancy associated with HE derive from mature CD4+ T cells – that can produce eosinophilopoietic factors

•PTCL are malignancies of mature T cells typically involving peripheral tissuesClassified according to their site of involvementNODAL - Primary in lymph node

Extranodal sites (spleen sin GIT)

PERIPHERAL T CELL LYMPHOMAS (PTCL) ASSOCIATED WITH HYPEREOSINOPHILIA

MYCOSIS FUNGOIDES AND SEZARY SYNDROME

ANGIOIMMUNOBLASTIC T CELL LYMPHOMA

ADULT T CELL LEUKEMIA/LYMPHOMA

SYSTEMIC ANAPLASTIC LARGE CELL LYMPHOMA

PRIMARY CUTANEOUS CD30+ LPD

ENTEROPATHY ASSOCIATED T CELL LYMPHOMA

PTCL,NOS

CUTANEOUS T- CELL LYMPHOMA (CTCL)•CTCL are the most common PTCL associated with HE •Most common CTCL associated with HE are – mycosis fungoides and sezary syndromeMYCOSIS FUNGOIDES (MF) AND SEZARY SYNDROME (SS)

•MF – presence of epidermotropic clonal T cells showing convoluted cerebriform nuclei•These T cells are mature CD4+ memory T cells

•SS – aggressive leukemic variant of CTCL•Epidermotropism –ve•Sezary cells in circulation +ve (derived from TH2 cells)•Disseminated LN involvement +ve•Can arise de-novo or as a progression of preexisting MF

•SS is more commonly ass with eosinophilia than MF•Blood eosinophilia is >700/microl•Peripheral blood eosinophilia in MF and SS – poor prognosis

•Eosinophil mediated end organ damage is uncommon•Mechanism of eosinophilia –

Sezary cells produce IL-4 this induces exp of eotaxin by surrounding cells chemoattractant for CCR3+ eosinophils

ANGIOIMMUNOBLASTIC T CELL LYMPHOMA (AITL)

•Neoplastic cells are mature CD4+ T cells

•Eosinophilia > 500/microl

•Has no impact on prognosis

ADULT T- CELL LEUKEMIA/LYMPHOMA (ATLL)

•Associated with HTLV1•Neoplastic cells are mature CD4+ T cells – multilobated nucleus- aka FLOWER CELLS•Peripheral blood eosinophilia significantly associated with skin and spleen involvement•HTLV-1 encoded transactivator – tax may contribute to eosinophilopoietic cytokine production by the ATLL cells•Ass with poor prognosis

PRIMARY CUTANEOUS CD30+ LPDThis include-

Primary cutaneous ALCL (cALCL)

Lymphomatoid papulosis (LyP)

cALCL•2nd MC CTCL•Peripheral blood eosinophilia - uncommon Tumor cells exp IL-4

Induces exp of eotaxin from surrounding cells

Cause eosinophil chemotaxis

LYMPHOMATOID PAPULOSIS (LyP)

•Clinically benign disorder

•Recurrent and self healing skin lesions

•May be associated with malignant lymphomas – MF and HL

•Peripheral blood eosinophilia - uncommon

ENTEROPATHY ASSOCIATED T CELL LYMPHOMA (ETL)

•Aggressive disorder

•Typically involves small intestine

•Arise from intraepithelial lymphocytes

•In T-cell LPD tissue eosinophilia > peripheral blood eosinophilia

•In B-cell LPD peripheral blood eosinophilia> tissue eosinophilia

EOSINOPHILIC MYELOID DISORDERS

•Imatinib sensitive fusion genes in a subset of patients with eosinophilia has changed the way we approach the diagnosis and treatment of this group of patients

•Associated with 28 fusion genes encoding an aberrant tyrosine kinase and involving either-

PDGFRA (4q)

PDGFR

B (5q)

FGFR1(8p)

•Patients with fusion genes involving PDGFRA/B are responsive to imatinib with a very low rates of primary and secondary resistance

•In contrast , fusion genes involving FGFR1 are imatinib resistant and associated with aggressive clinical course

•All 3 disorders can present as chronic MPN but frequency of manifestation as lymphoid neoplasm varies

CLASSIFICATION OF EMD

1.MYELOID AND LYMPHOID NEOPLASMS WITH EOSINOPHILIA AND ABNORMALITIES OF PDGFRA, PDGFRB AND FGFR1

2.CHRONIC EOSINOPHILIC LEUKEMIA , NOS (CEL-NOS)

MYELOID AND LYMPHOID NEOPLASMS WITH EOSINOPHILIA AND ABNORMALITIES OF PDGFRAIn case of PDGFRA related disorders presentation is usually as

Chronic eosinophilic leukemia (CEL)

AML

Precursor T lymphoblastic lymphoma (T-LBL)

•Most common MPN associated with PDGFRA rearrangement is associated with –•FIP1L1-PDGFRA•This is formed by a cryptic deletion of CHIC2 at 4q12 in exon 12•The breakpoint in FIP1L1 is variable

•This fusion results in an activated tyrosine kinase which is inhibited by imatinib•Mutation can be detected by FISH or PCR•Some are negative for fusion gene – instead can overexpress PDGFRA – also candidates for imatinib

•M>F•MC in 4th decade•Manifestation are due to eosinophilic tissue infiltration and release of proinflammatory mediators and toxic granule products

•Most feared complication - ENDOMYOCARDIAL FIBROSIS WITH ENSUING RESTRICTIVE CARDIOMYOPATHY

•So prior to initiating imatinib serum troponin and echocardiogram must be done

•Peripheral blood – HALLMARK IS EOSINOPHILIA

•Mostly mature eosinophils with only small numbers of eosinophil myelocytes or promyelocytes

•Anemia and thrombocytopenia – frequently seen

Increased eosinophil size

Sparse granules with clear areas of cytoplasm

Cytoplasmic vacuolation

Smaller size granules

Immature purplish granules in romanowsky stain

Nuclear hypo/hyper segmentation

Eosin

ophi

l m

orph

olog

y

Bone marrow findings

Hypercellular with increased eosinophils and precursors

Charcot leyden crystals +

Increased CD25+ spindle shaped atypical mast cells

•Serum tryptase and Vit B12 levels increased

•FIP1L1-PDGFRA related disease is more sensitive to imatinib than is BCR-ABL

•Effective dose is lower in these cases compared to BCR-ABL

•Rarely imatinib resistance develops – due to hypermethylation of PTEN gene

•Eosinophilia can develop in patients with systemic mastocytosis (SM-Eo) and increased mast cells are seen in FIP1L1-PDGFRA related eosinophilia

•So confusion can arise between the two diseases

•Diagnostic criteria for SM-Eo is –• Persistent eosinophilia of at least 6

months duration• Absolute eosinophil count

>1500/microl• WHO diagnostic criteria for SM

Diagnostic criteria for FIP1L1-PDGFRA associated with eosinophilia

FIP1L1-PDGFRA vs SM-Eo•Mast cell aggregates contain <15 mast cells•Serum tryptase <50ng/ml•PDGFRA rearrangement +•Endomyocardial fibrosis found exclusively in this•Absolute eosinophil count >10000/microl•Mast cells are CD2 -ve

•Mast cell aggregates contain >15 mast cells•Serum tryptase >100ng/ml•KIT D816V mutation +•Urticaria pigmentosa exclusive in this•Absolute eosinophil count > 1500/microl•Mast cells are CD2+

PROGNOSIS – Favourable if cardiac damage has not occurred and imatinib treatment is available

MYELOID NEOPLASM WITH EOSINOPHILA AND ABNORMALITIES OF

PDGFRB•20 PDGFRB fusion partners have been reported•Most common – ETV6-PDGFRB – t (5;12)•Result in activated tyrosine inase – sensitive to imatinib•Commonly manifested as CMML with eosinophilia

Diagnostic criteria for PDGFRB rearrangement with eosinophilia

MYELOID AND LYMPHOID NEOPLASM WITH

EOSINOPHILA AND ABNORMALITIES OF FGFR1

•In FGFR1 related diseases the manifestations are most commonly lymphomatous with most common being T-LBL•Lymphadenopathy is common•LN biopsy – Lymphoblatic lymphoma – most often of T-cell or mixed myeloid/T-cell lineage

Various fusion genes - associated with FGFR1 are1.t(8;13) – MC translocation -

associated with T-LBL and lymphadenopathy

2.t(8;22) – (BCR;FGFR1) – associated with leucocytosis and basophilia

3.t(8;9) – tonsillar involvement and monocytosis

4.T(8;6) – associated with polycythemia

Prognosis is poorNot sensitive to imatinib

CHRONIC EOSINOPHILIC LEUKEMIA , NOS (CEL-NOS)

IDIOPATHIC HYPEREOSINOPHILIC SYNDROME

1.Eosinophil count >1500/microl persisting for at least 6 months

2.Reactive eosinophilia is excluded3.AML, MDS, MPN, MDS/MPN and

systemic mastocytosis is excluded4.A cytoine producing

Immunophenotypically aberrant T-cell population is excluded

5.Evidence of Tissue damage as a result of HE is present

IDIOPATHIC HYPEREOSINOPHILIA

Definition –If criteria 1-4 is present but there is absence of tissue damage

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