This document provides information on eosinophils: - Eosinophils are white blood cells that are characterized by distinctive granules and a bilobed nucleus. They play a role in fighting parasites and allergic responses. - Eosinophils develop from bone marrow stem cells and are regulated by cytokines like IL-5, IL-3 and GM-CSF. Transcription factors like C/EBP, GATA-1 and PU.1 are required for eosinophil development. - Eosinophils traffic to tissues where they can persist for over a week. Their granules contain toxic proteins that are released during degranulation which is regulated by cytokines and chemokines. - Eosin

1. THE HUMAN
EOSINOPHIL

2. •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

3. •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

4. 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

5. IL-5
IL-3
GM-CSF

6. •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

7. •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

8. •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

9. •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

10. TRANSCRIPTION FACTORS
REUIRED FOR EOSINOPHIL
DEVELOPMENT
C/EBP
GATA-1
PU.1
FOG

11. 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)

12. 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

13. 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

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

15. 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

16. • 2 additional trascription factor in eosinophil
development are –
1. IFN consensus sequence binding protein (Icsbp)
2. Id protein –
Exp of Id1 protein – inhibits eosinophil development
Exp of Id2 protein – accelerates final maturation of
eosinophil

17. 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

18. 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

19. 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

20. •They finally enhances exp of antiapoptotic protein
Bcl-x in eosinophils and decreases proapoptotic
factors like Bax
•So increase eosinophil survival

22. EOSINOPHIL HETEROGENEITY
3 different population of eosinophils is
known
1. Normodense eosinophils- constitutes
>90% of blood eosinophils
2.Hypodense eosinophils – raised in
Hypereosinophilia
3.Primed eosinophils

23. 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

24. EOSINOPHIL TISSUE
ACCUMULATION
• Recruitment of eosinophils across vascular
endothelium occurs in 4 steps-
1. TETHERING – of eos to luminal surface of
vascular endothelium
2.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

25. SELECTINS α 4 INTEGRINS
FOR TETHERING AND ROLLING
β 2 INTEGRINSFOR FIRM ADHESION
C-C CHEMOKINES
FOR TRANSMIGRATION

27. EOSINOPHIL MEDIATORS
MEMBRANE DERIVED MEDIATORS
Eos produce a wide variety of lipid derived
mediators-
Membrane phospholipids
Arachidonic acid
Phospholipase A2

28. Leukotriens (LTC4)
Prostaglandins (PGE2)
Thomboxane
Lipoxins(LXA4)
PAF

29. 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

30. •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

31. SMALL GRANULES
•Coreless granules
•Contain acid phosphatase, arylsulfatase ,
catalase and cytb558
LIPID BODIES
•5 LB/mature eosinophils
•Increased in idiopathic hypereosinophilia

32. SECRETORY VESICLES
•Dumbbell shaped in cross section
•Contain albumin
•Aka microgranules/tubulovesicular
structure

35. 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

36. CHARCOT LEYDEN CRYSTAL (CLC)
PROTEIN
•Produced at very high levels
•Designated as galectin-10
•Needle shaped structures that are colorless

37. 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

38. 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

39. Granule contents are
subsequently expelled to
the exterior of cell

40. 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 granules
VAMP-2 – in secretory vesicles
Syntaxin-4
SNAP23
Binds VAMP-2

41. APPROACH TO
EOSINOPHILIA

44. Normal range of peripheral blood eosinophils
is 0-500/microl
Mild eosinophilia 500-1500/microl
Moderate eosinophilia 1500-5000/microl
Severe eosinophilia >5000/microl
Hypereosinophilia (HE) >1500/microl

45. 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 organs
In BM – HE is applied when >20% of all
nucleated cells are eosinophils
•Exact definition for tissue HE is
extramedullary organs is not available

46. 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

47. HYPEREOSINOPHILIC
SYNDROME
Idiopathic HES
PDGFRA associated MPN
Lymphocytic variant HES (L-HES)
Familial hypereosinophilia
Churg strauss syndrome
Eosinophil associated GI disease

48. Causes of
NON-HES

49. MECHANISM RESULTING IN HE
ARE TWO-FOLD
Neoplastic HE
Eosinophils are either a part
(In Myeloid neoplasms
associated with eosinophilia
Or the major
representative(in CEL) of a
malignant clone
Reactive/non
neoplastic HE
Non clonal
eosinophilic
proliferation

50. NEOPLASTIC HE
•c/o myeloid neoplasms associated with HE
1. CML
2.CEL
3.JAK2 mutated MPN
4.Certain variant of AML
5. Some cases of advanced Systemic
mastocytosis

51. 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

53. REACTIVE /NON-
NEOPLASTIC HE

54. 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-

55. SYSTEMIC MASTOCYTOSIS (SM)
INDOLENT SM
SMOULDERING SM
AGGRESSIVE SM
MAST CELL
LEUKEMIA
EOSINOPHILS
ARE NON-
CLONAL EOSINOPHILS
ARE CLONAL

56. LYMPHOPROLIFERATIVE
DISORDERS
ASSOCIATED WITH
EOSINOPHILIA

57. LPD ASSOCIATED WITH
HYPEREOSINOPHILIA ARE –
1. Hodgkin lymphoma (B-cell neoplasm)
2.Lymphoid neoplasms derived from mature
CD4+ Tcells
3.Lymphoid neoplasms derived from CD8+
Tcells
4.Immature B-cells and T-cells

58. 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

59. NON MALIGNANT T -CELL
LYMPHOMA ASSOCIATED WITH
HYPEREOSINOPHILIA
LYMPHOCYTIC VARIANT HYPEREOSINOPHILIC
SYNDROME (HES)
LYMPHOID DYSCRASIAS OF UNKNOWN
SIGNIFICANCE

60. B- CELL LYMPHOPROLIFERATIVE
DISORDERS
CLASSICAL HODGIN LYMPHOMA
B CELL ACUTE LYMPHOBLASTIC
LEUKEMIA/LYMPHOMA WITH HE

61. 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 tissues
Classified according to their site of involvement
NODAL - Primary in lymph
node
Extranodal sites (spleen
sin GIT)

62. 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

63. 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 syndrome
MYCOSIS 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

64. •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

65. •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

66. ANGIOIMMUNOBLASTIC T CELL
LYMPHOMA (AITL)
•Neoplastic cells are mature CD4+ T cells
•Eosinophilia > 500/microl
•Has no impact on prognosis

67. 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

68. PRIMARY CUTANEOUS CD30+ LPD
This include-
Primary cutaneous ALCL (cALCL)
Lymphomatoid papulosis (LyP)

69. cALCL
•2nd MC CTCL
•Peripheral blood eosinophilia - uncommon
Tumor cells exp IL-4
Induces exp of eotaxin from
surrounding cells
Cause eosinophil chemotaxis

70. 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

71. ENTEROPATHY ASSOCIATED T
CELL LYMPHOMA (ETL)
•Aggressive disorder
•Typically involves small intestine
•Arise from intraepithelial lymphocytes

72. •In T-cell LPD tissue eosinophilia >
peripheral blood eosinophilia
•In B-cell LPD peripheral blood
eosinophilia> tissue eosinophilia

74. EOSINOPHILIC
MYELOID DISORDERS

75. •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)
PDGFRB
(5q)
FGFR1
(8p)

76. •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

77. CLASSIFICATION OF EMD
1. MYELOID AND LYMPHOID
NEOPLASMS WITH EOSINOPHILIA
AND ABNORMALITIES OF PDGFRA,
PDGFRB AND FGFR1
2.CHRONIC EOSINOPHILIC LEUKEMIA ,
NOS (CEL-NOS)

78. MYELOID AND LYMPHOID
NEOPLASMS WITH EOSINOPHILIA
AND ABNORMALITIES OF PDGFRA
In case of PDGFRA related disorders
presentation is usually as
Chronic eosinophilic leukemia (CEL)
AML
Precursor T lymphoblastic lymphoma (T-
LBL)

79. •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

80. •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

81. •Peripheral blood – HALLMARK IS EOSINOPHILIA
•Mostly mature eosinophils with only small
numbers of eosinophil myelocytes or
promyelocytes
•Anemia and thrombocytopenia – frequently seen

82. 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

83. Bone marrow findings
Hypercellular with increased
eosinophils and precursors
Charcot leyden crystals +
Increased CD25+ spindle
shaped atypical mast cells

84. •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

85. •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

86. Diagnostic criteria for FIP1L1-PDGFRA
associated with eosinophilia

87. 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+

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

89. 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

90. Diagnostic criteria for PDGFRB rearrangement
with eosinophilia

91. 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

92. Various fusion genes - associated with FGFR1
are
1. 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 poor
Not sensitive to imatinib

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

95. IDIOPATHIC HYPEREOSINOPHILIC
SYNDROME
1. Eosinophil count >1500/microl persisting for
at least 6 months
2.Reactive eosinophilia is excluded
3.AML, MDS, MPN, MDS/MPN and systemic
mastocytosis is excluded
4.A cytoine producing Immunophenotypically
aberrant T-cell population is excluded
5. Evidence of Tissue damage as a result of HE
is present

96. IDIOPATHIC HYPEREOSINOPHILIA
Definition –
If criteria 1-4 is present but there is
absence of tissue damage

97. THANK YOU