Biology of basophils & basophil activation test

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Biology of Basophils &
Basophil Activation Test
Suda Sibunruang, M.D.

Outline
• Development and morphology
• Functional and Phenotypic Markers
• Mediators and effector functions
• Role in health and diseases
• Basophil activation test

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http://intranet.tdmu.edu.ua,
access November 2015
Paul Ehrlich (1854-1915)
1879
Identified by staining with
basic dyes

Access from www.commons.wikimedia.org, November 2015

Schroeder JT. Immunological Reviews 2011;242:144–60
Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014
IL-3 affects essentially every aspect of basophil biology including
development and maturation, survival, acute or late effects on
mediator release, and direct stimulation of IL-13 production

Siracusa MC. et. al. J Allergy Clin Immunol 2013;132:789-801

Picture from http://static.wikidoc.org &
Siracusa MC. et. al. Immunology and Cell Biology 2010;88:275–84
CD34+ myeloid
hematopoietic
progenitors
IL - 3
IL – 5
IL-3, GM-CSF
SCF

Stone KD., et. al. J Allergy Clin Immunol 2010;125:S73-80
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Storage granule contents
• Major preformed mediator is histamine
• Histamine complexes with proteoglycans,
most notably chondroitin sulfate,
as opposed to heparin sulfate in the mast cell,
and dissociates after exocytosis by
ion exchange and changes in pH
• Contain less heparin than mast cell
• Tryptase in basophil are thought to be much
lower than in mast cells;
however, there can be variability
Clearly caution in using tryptase as the sole
specific marker for identifying the
presence of mast cells

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Morphology

Schroeder JT. Immunological Reviews 2011;242:144–60

Kawakami T. and Galli SJ. Nature Reviews Immunology 2002;2:773-86
Differ more than alike
Basophils
• Segmented nucleus
• Larger & fewer granules
Mast cell
Basophil

Functional and Phenotypic Markers

• Immunoglobulin Fc receptors
FcεRI, FcγRIIb
• Cytokine receptors
IL-3R,IL-5R, GM-CSFR
• Chemokine receptors
CCR2, CCR3
• Complement receptors
CD11b, CD11c, CD35, CD88
• PG receptors
CRTH2
• TLRs

Basophil Activation
• Immunoglobulin E dependent pathway
• Immunoglobulin E independent pathway

Karasuyama H. et. al. Nature Reviews Immunology 2009;9:9-13
Human basophils may express between
5,000 and 1 million FcεRI sites per cell
Estimated 200 IgE-receptor cross-links
needed to initiate mediator release from basophils
Immunoglobulin E dependent pathway

Moon TC. et. al. Front Immunol 2014;5:1-18
Spencer LA. et. al. Front Immunol 2014;5:496
IgE dependent pathway
Mast cell
Piecemeal degranulation: vesicular transport of small packets of materials from secretory granules to cell surface

Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408
Hausmann OV. et. al. Immunol Allergy Clin N Am 2009;29:555–66
Basophil activation markers: CD63 & CD203c
glycoprotein expressed
in membranes of
cytoplasmic granules
CD203c is a glycosylated transmembrane molecule
Basophils express low level of CD203c, which is increased
with basophil activation and may correspond to piecemeal degranulation

Mediators
- Preformed mediators
- Newly syn. Leukotrienes &
prostaglandins
- Cytokines & chemokines
Significant source of IL-4
and IL-13 vital to
pathogenesis of allergic
disease

Basophils play a dual role in allergic inflammation, depending on
antigen concentrations:
At low concentrations of antigen, basophils may modulate immune
responses of B cells, T cells, and eosinophils through production
of IL-4 and IL-13
As they infiltrate lesion site, and concentration of antigen increases,
basophils may take on more of an effector role by secreting
two substances (i.e., histamine and LTC4)

IgE dependent & independent pathway

Marone G. et. al. Trends Immunol 2001;22:229-32
Shed- or virus-bound-gp120 can interact with variable,
heavy chain 3 (VH3)+ IgE bound to FcεRI+ cells
of individuals at an early stage of infection
Induces IL-4 and IL-13 release and
favours a shift towards a
Th2 phenotype
HIV-1 transactivation (Tat) protein
secreted by infected cells interacts
with CCR3 receptor and is a potent
chemoattractant
Extracellular Tat can also induce
IL-4 and IL-13 release
Human FcεRI+ cells express CCR3 and CXCR4,
which are co-receptors for several strains of HIV-1,
suggesting that these cells could be
infected by certain strains of HIV-1
High IgE levels often observed
in some HIV-infected patients

Schroeder JT. Adv Immunol 2009;101:123-61
Activation of TLR 2 and 4 can lead to production of BAFF and IL-13,
indicating that microbiota might modulate immunity by
interacting with basophils

Chen K. and Cerutti A. Current Opinion in Immunology 2011;23:345–52
IgD immune complexes
Augment mucosal immune responses by promoting B and T cell activation,
leukocyte recruitment and direct microbial killing

Basophil degranulation is restricted to signals induced
by FcεRI crosslinking, C5a, and IL-3

Granzyme B, a novel mediator of allergic inflammation:
its induction and release in blood basophils and human asthma
Tschopp CM, et al. Blood 2006;108:2290-9
Basophils synthesize and
release granzyme B in
response to IL-3 stimulation
The significance of this finding
remains undetermined

Schroeder JT. Adv Immunol 2009;101:123-61
Only cytokines reported to negatively regulate basophils are
type 1 interferons which limit IL-3-induced cytokine production

Abbas AK, et al. Cellular and Molecular immunology 2014 Eighth Edition

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Naclerio RM, et al. N Engl J Med 1985;313:65-70
Inflammatory mediators in late antigen-induced rhinitis
Obj: To investigate mechanisms responsible for
late-phase response in patients with allergies
Method: We measured four biochemical mediators
(histamine, tosyl-L-arginine methyl ester [TAME]-esterase,
kinin, and prostaglandin D2) in nasal secretions
after nasal challenge with pollen antigen in
12 patients with allergy and 3 persons for control

Kepley CL, et. al. Am J Resp Crit Care Med 2011;164:1053-8
Contribution of basophils to LPR
is based on their presence in
tissues within hours following
experimental allergen challenge
Late response is largely mediated
by basophils
Immunohistochemical Detection of Human Basophils in
Postmortem Cases of Fatal Asthma

Dvorak HF, et al. J Exp Med 1970;132:558-82
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Delayed-type hypersensitivity
When given rhus toxoid (the active agent
in poison ivy) or dinitrochlorobenzene
in a patch test,
sensitized subjects developed skin reactions
that were characterized by a cellular
infiltrate into dermis consisting of up to
approximately 16% basophils by
3 - 6 days after application of antigen

Basophils in immune regulation
Interact with T or B cell

Sokol CL, Medzhitov R. Mucosal Immunol 2010;3:129-37
Basophils secrete IL-4 & TSLP, which induce differentiation of
naive T cells into TH2 cells in cooperation with dendritic cells
TSLP is capable of inhibiting IL-12
production by DCs, thereby
further promoting Th2 cell
differentiation

Voehringer D. Trends Parasitol 2009;25:551-6

Gibbs BF. Clin Exp Med 2005;5:43–9

Siracusa MC. et. al. J Allergy Clin Immunol 2013;132:789-801
Role in health and diseases

Roles of basophils in allergy
1) In sensitized animals, specific IgE are
produced and captured by circulating
basophils through FcεrI
2) When the same antigens enter skin,
IgE-bearing basophils are recruited to lesion,
possibly by chemokines released by
phagocytic cells that have captured antigen,
and are activated when Ag binds to
IgE–FcεrI complex
3) Activated basophils secrete cytokines
and other factors that in turn act on
tissue-resident cells, such as fibroblasts,
4) Induce them to produce more chemokines
5) Inflammatory cells, such as eosinophils and
neutrophils, are then recruited to skin lesion
in response to chemokines that are released
by tissue-resident cells , resulting in
chronic allergic inflammation

Role of basophils in allergic diseases
Important in many if not all allergic diseases:
• Anaphylaxis
• Allergic rhinitis
• Asthma
• Urticaria
• Food allergies

Role of basophils in other clinical conditions
• Infections
• Autoimmunity
• Malignancy

http://www.dailymail.co.uk/sciencetech/article-2957265/When-white-blood-cells-ATTACK-Amazing-video-captures-moment-immune-takes-parasitic-worm.html
Immune system takes down a worm
Access from http://i.dailymail.co.uk, November 2015
Caenorhabditis elegans

Voehringer D. Trends Parasitol 2009;25:551-6
Long-held belief that basophils
(and eosinophils) are involved in natural
immunity to parasitic infections
IL-4–secreting basophils are critical
for expulsion of these worms

Charles N and Rivera J. Curr Allergy Asthma Rep 2011;11:378–87
Correlations between elevated serum IgE autoantibodies,
and activated basophils with severity of lupus nephritis
IgE anti-dsDNA/dsDNA immune complexes
bind to basophils FcεRI, causing basophil
activation with homing to lymphoid organs
Activated basophils produce IL-4 and promote
Th2 adaptive immune response that enhances
autoantibody production

Schuerwegh JM, et al. PNAS. 2010;107:2586–91
Rheumatoid arthritis
Citrullinated proteins can activate basophils
due to IgE anti-citrullinated protein
Basophil activation may contribute
to pathogenesis of autoimmune
diseases as diverse as:
• Antiglomerular basement
membrane disease
• Membranous nephropathy
• ANCA associated vasculitis

Kawakami T. and Galli SJ. Nature Reviews Immunology 2002;2:773-86
Indirect therapeutic modulation of basophil activation, such as omalizumab, will be extended to
direct therapeutic targeting of basophils to inhibit activation and promotion of a pathogenic Th2
microenvironment

Wimazal F. et. al. Cancer 2010;116:2372-81
Retrospective study of prognostic significance of eosinophils and basophils
in 1008 patients with de novo MDS in Austria & German
Increased no. of basophils are common features of AML
and accelerated phase of CML
Basophils, greater than 250/ul, was associated
with reduced survival

Nievergall E. et. al. Blood 2014;123:1218-28

Hoffmann HJ. et. al. Allergy 2015;70:1393-405
measures basophil response to allergen cross-linking IgE
Basophil activation test (BAT)
Compared with determination of sIgE in serum,
BAT reflects a functional response as basophil activation
can be induced by cross-linking of FceRI, which
requires more than binding of sIgE to allergen

Diagnostic evaluation
Determine clinically relevant allergen in addition
to clinical history, skin prick test, and specific IgE
• Food
• Insect venom
• Drug allergy
• Chronic urticaria:
Alternative for autologous serum skin test (ASST)
Basophils from healthy donors are challenged with patients’ serum

Sensitivity & specificity
• Drug allergy: Beta-lactams, Neuromuscular blocking
agent (NMBA), quinolones , radio contrast media, and
pyrazolones
sensitivity 50%, specificity up to 93%
• Food allergy:
sensitivity 77-98%, specificity 75 – 100 %
• Hymenoptera venom allergy:
sensitivity 85-100%, specificity 83 – 100 %

Monitor patients
• Allergen immunotherapy
• Anti-IgE treatment
• Natural resolution of allergy

Basophil reactivity is the dose (range) at which maximal response occurs
Basophil sensitivity is the dose at which half of the maximal response occurs
Concentration at which 50% of basophils respond = EC50
‘CD-sens’ = 1/EC50 x 100

Basophil reactivity reflected severity
Basophil sensitivity reflected threshold of response to allergen

Advantages
• Fewer resources
• More reproducible than challenge test
• Less stressful for the patient
• Avoids severe allergic reactions

Nonresponders
• 6–17% of population
• Nonresponders can experience allergic
symptoms and have positive SPT with relevant
allergens
• Remain unresponsive to stimulation through
FceRI under standard BAT conditions

Kepley CL, et. al. J Allergy Clin Immunol 1999;104:279-84
Differences in intracellular signaling pathway

Considerations when taking a blood
sample for BAT (1)
• Remember to…
Check absolute basophil counts of above
50 cells/µl within 24 hours prior to
performing BAT
Personal communication, Jettanong Klaewsongklam, M.D., 2015

sample for BAT (2)
• Antihistamines do not influence BAT but
systemic steroids and cyclosporin A should be
avoided
• Blood samples should preferentially be taken
within 1 yr of the most recent exposure to
allergen
• It is possible to use blood samples within 24 h
to document sensitization, but may lose
reactivity

sample for BAT (3)
• Basophil function is mostly assessed in
heparin-stabilized blood

Allergen concentration range
Benzylpenicillin 2 &0.4 mg/ml
Amoxicillin 1.25 & 0.25 mg/ml
PPL 0.25 & 0.05 mg/ml
MDM 0.25 & 0.05 mg/ml
Cephalosporins 1.25 & 0.25 mg/ml
ASA 1 & 0.2 mg/ml
Diclofenac 0.3 & 0.06 mg/ml
Naproxen 1 & 0.2 mg/ml
Paracetamol 1 & 0.2 mg/ml
Metamizol 2.5 & 0.25 mg/ml
Atracurium 0.5 &0.05 mg/ml
Vecuronium 2-0.5 mg/ml
Negative test with a parent drug does thus not
rule out that reacts to a metabolite of drug

Summary (1)
• Basophils have long been seen as a surrogate
with which to study the more elusive mast
cell. This view, however, is no longer valid,
because substantial developmental and
physiologic evidence now shows that these
two cell types have more differences than
similarities

Summary (2)
• Once thought to do little other than to secrete
histamine and LTC4, basophils are now well
recognized to be the predominant cellular
source of IL-4 and IL-13—perhaps the two
most important cytokines with a role in the
pathogenesis of allergic disease

Summary (3)
• This information, along with evidence that
these cells infiltrate allergic lesions and are
capable of responding to a wide variety of
stimuli, has sparked a renewed interest in
basophils and in their role in allergic
inflammation and disease.

Biology of basophils & basophil activation test

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