biology of basophils

Cells and Tissues of the Immune System:BASOPHILS

Alric V. Mondragon, MDSection of Allergy and Immunology

University of the Philippines – Philippine General Hospital

Summary of Concepts

Bone Marrow < 1% In Vivo Processes ?

Mediators and Cytokines

IL-4, IL-13, Cytokines IgE Synthesis

Innate Immunity

Middleton’s Allergy Principles and Practice 8th ed. (2014) by Adkinson et al.

OUTLINE1. Development and Maturity 2. Functional and Phenotypic Markers3. Inflammatory Mediators4. Basophil Activation

a. IgE-dependentb. IgE-independent

5. Basophil Involvement in Disease

DEVELOPMENT AND MATURITY


Property Basophil-specific

Origin Bone Marrow

Lineage Myeloid

Frequency in Circulation <1% of WBCs

Size 5-8 um

Nucleus Lobed

Chromatin appearance Condensed

Metachromatic granules Yes (but less than in mast cells)

Granule glycosaminoglycan

Chondroitin sulfate

Proliferative capacity None

Survival in Circulation ~3-7

Development and Maturity

Janeway’s Immunobiology, 8th Edition (2012). By K. Murphy

Development and Maturity

Exact Factors ?

Survival

Activation of Mature Basophils

IL-3


Development and MaturityAnaphylactic

Degranulation

• Rapid and “explosive” event

• Granule extrusion via exocytosis

• Complete degranulation

Piecemeal Degranulation

• Induced vesicular transport of granular content

• NOT direct granule extrusion


FUNCTIONAL AND PHENOTYPIC MARKERS

Functional and Phenotypic Markers

Selectins

β1- and β2-integrins, along with ICAMs

Basophils could potentially facilitate their own migration into tissue.


Functional and Phenotypic MarkersIL-3

(CD123)

pDC

IFN-α/β

Basophil


-

Functional and Phenotypic Markers• Other cytokine receptors found on basophils:– GM-CSF, IFN-γ, TNF, and the high-affinity receptor

for nerve growth factor (NGF), also known as TrkA.• ST2, an IL-1–like receptor– most recently identified cytokine receptor– Ligand, IL-33, plays an important role in augmenting

basophil secretion in vitroMiddleton’s Allergy Principles and Practice 8th ed. (2014) by Adkinson et al.

Functional and Phenotypic MarkersCCR3 • Most prominently expressed on basophils

• Accounts for the overlapping activity mediated by several of the CC chemokines.• The monocyte chemotactic protein (MCP) subfamily all bind this receptor and are reported

to possess potent chemotactic activity for basophils. • (e.g., CCL2/MCP-1, CCL7/MCP-3), RANTES/CCL5, and eotaxins-1 (CCL11), -2 (CCL24), -3 (CCL26)

CXCR2 • Accounts for the histamine-releasing activity described for IL-8 CXCL8, one of its ligands.

CXCR4 • stromal cell–derived factor (SDF)-1 CXCL12 is an important ligand for chemotaxis and mediator release.

• found on lymphocytes and is a cofactor necessary for infection by HIV.

(CRTH2), or DP2

• Chemoattractant receptor–homologous molecule expressed on Th2 cells• Receptor for PGD2 important for cell trafficking.


Functional and Phenotypic MarkersFcεRIα • The most distinguishing functional characteristic of basophils (and mast cells) is their ability

to bind IgE immunoglobulin with high affinity (α-subunit of FcεRI) • Express between 5000 and 1 million FcεRI sites per cell• FcεRI levels increase with higher serum IgE concentrations • All IgE binding to basophils is mediated through FcεRIα

FcγRII (CD32)

• Binds various subclasses of IgG antibody• To induce intracellular signals that oppose those mediated through FcεRI, thereby

downregulating IgE-mediated responses in basophil and mast cells• FcγRII/FcεRI costimulation attenuates mediator release and cytokine production by basophils

CD40L • can relay signals to develop into immunoglobulin-producing cells• By expressing CD40L and secreting IL-4 and IL-13 provide the two necessary signals for B

cells to produce IgE


Functional and Phenotypic MarkersRECEPTORS ACTION

C5aR • mediates degranulation induced by C5a.

Bacterial peptide (fMLP) • mediates basophil degranulation

Toll-like receptors (e.g., TLR1, -2, -4, -6, -9)

• bind a variety of microbial products

Leukocyte immunoglobulin-like receptors (e.g., LIR3, -7)

• mediate either inhibitory or stimulatory activity


Functional and Phenotypic MarkersOTHER RECEPTORS/SPECIFIC MARKERS• Receptors for:

1. Prostacyclin (i.e., prostaglandin I2) 2. Platelet-activating factor (PAF) 3. Adenosine, 4. Histamine


Functional and Phenotypic Markers• At least 3 monoclonal antibodies have been described that

immunologically detect proteins unique to basophils or their progenitors


mAb2D7 • Recognizes a 72-kD protein, released upon degranulation

BB1 • Specific for a large complex (basogranulin)CD203c • Detect an ectonucleotide pyrophosphatase/ phosphodiesterase

• Also used to identify basophils (mature and immature)

INFLAMMATORY MEDIATORS

Inflammatory MediatorsHISTAMINE• Store 1 pg of histamine per cell• Synthesized by the actions of histidine decarboxylase• Storage in basophils is mediated with the highly charged

proteoglycan chondroitin sulfate, (mast cell = heparin sulfate) • Histamine acts as a spasmogen

– Causes smooth muscle contraction– Causes vascular leakage by dilating terminal arterioles.


Inflammatory MediatorsOTHER PREFORMED MEDIATORS• Small quantities stored in Basophil

1. Tryptase2. Major Basic Protein (MBP)

• Significance = unclear at present– Caution in the use of tryptase as the sole specific marker

for identifying the presence of mast cells in tissueMiddleton’s Allergy Principles and Practice 8th ed. (2014) by Adkinson et al.

Inflammatory MediatorsLEUKOTRIENE C4• Arachidonic acid (AA) through the lipoxygenase pathway

LTC4


LTC4 compared to HistamineStorage NOT stored in the cytoplasmic granulesRate of Secretion Slightly slower than that of histamineAmount Generated Far less than the amounts of histamine stored in these cells

Potency 6000 times more potent than histamine in contracting airway smooth muscle

Inflammatory MediatorsCYTOKINES• Basophils produce only Th2-like cytokines. There are no reports of

Th1-like cytokines secretion.• Basophils are a significant source of IL-4 and IL-13• Basophils express CD40L and when engaged by CD40 on the surface of

B cells Synthesis of IgE• IL-4: promotes the Th2 phenotype

– amplify Th2 responses. – primary source of IL-4. – Mice studies: basophil may provide initial source of IL-4.


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Inflammatory MediatorsCYTOKINES• Rapidly produce IL-3• IL-17–associated cytokines and growth factors (e.g.,

VEGF, GM-CSF, leptin)• Several chemokines are secreted

– may modulate cell trafficking into lesion sites.• Synthesize and release granzyme B, suggesting perhaps

yet another class of mediator produced by these cells.Middleton’s Allergy Principles and Practice 8th ed. (2014) by Adkinson et al.

BASOPHIL ACTIVATION

Basophil Activation1. IgE-dependent Pathway2. IgE-Independent Pathway


IgE-dependent Pathway• Allergen + specific IgE occupying FcεRI receptors

on the surface of basophils secretion of mediators and cytokines. – Similarities in sensitivity of release– Different parameters for release

• Basophil release of mediators: typically bell-shaped dose-response curves


IgE-dependent PathwayBasophil release of mediators: • Typically bell-shaped dose-response curves• Released on IgE-mediated stimulation


MEDIATORS RATE of SECRETION

Histamine(preformed)

nearly complete within 20 minutes

LTC4 synthesis and release also are nearly complete within minuteCytokines dependent on signals

IgE-dependent PathwayCYTOKINES RATE of SECRETIONIL-3 and IL-4 Secreted within 1 hour

Half-maximal: 2 hoursPeak: 4-6 hours

mRNA for IL-3, IL-4 Detected within 30minutes of activationPeak at 2 hours

IL-13 Minutes to hours AFTER IL-4 secretionPeak: 18-20 hours later


IgE-dependent Pathway• Dissociation in cytokine secretion and mediator release

– Dual role in allergic inflammation– Different concentration gradient: Optimal cytokine levels produced

when stimulus is 10x LESS than those required for release of histamine and LTC4


ANTIGENCONCENTRATION

ROLE RELEASE

LOW Modulate the immune responses of B cells, T cells, and eosinophils

Produce IL-4, IL-13

HIGH Effector Role Histamine and LTC4

MCP Family • Cause degranulation: interact with several CCR receptor

CC Chemokines • RANTES/CCL5, MIP-1α/CCL3, and the eotaxins (types 1/CCL11 and 2/CCL24), • Limited potential for inducing histamine release• Interact with the CCR3 receptor• Greater role in the selective recruitment of eosinophils, basophils, and

lymphocytes into allergic lesions.

SDF-1/CXCL12 • Potent chemoattractant for basophils• Capable of inducing histamine release from these cells.

IgE-Independent Pathway• Basophils more reactive than mast cells to a greater number

of substances that induce histamine release– Amplifying allergic reactions as they infiltrate allergic lesions during

late phase responses.


IL-3

Histamine • at relatively high concentrations, IL-3 can act as a complete stimulus

LTC4 • NO ability to induce synthesis

IL-4 • Poor activator

• Released to a greater extent after IgE-mediated activation of cells primed in IL-3.

• Late priming effects on LTC4 secretion

IgE-Independent Pathway• Several cytokines play a role in enhancing basophil responses:

– Enhanced histamine release in response to IgE-dependent activation.


IgE-Independent Pathway

IL-3

Activate basophils for IL-13 secretion directly- IL-3 is more potent than antigen or anti-IgE- Relatively slow to start, begin 4 hours after incubation- But it remains ongoing beyond 20 hours

Basophils secrete IL-3 - By direct contact with allergen- Priming phenomenon is autocrine in natureAffects essentially every aspect of basophil biology - including development and maturation, survival, - acute or late effects on mediator release, - direct stimulation of IL-13 production.


IgE-Independent Pathway• Only type I interferons (IFN-α and IFN-β) have been shown to negatively regulate

basophil function. • Anaphylatoxins C5a and C3a and the bacteria-derived peptide fMLP are some of

the most potent basophil secretagogues described.– Not necessarily linked to allergic inflammation– fMLP: active and rapid inducer of basophil histamine release and mediates degranulation

• Intracellular pathway for degranulation different from those using FcεRI-mediated• Neither fMLP nor the chemokines that activate basophils for histamine release, when used alone,

typically induce IL-4 and IL-13 secretion.• Cytokine generation (particularly IL-4) is primarily a FcεRI-mediated response

– C5a: does not normally activate basophils for cytokine secretion. – Both stimuli: capable of promoting LTC4 synthesis


TLR2 ligands • Ex. peptidoglygan (PGN)—major constituent of G(+) bacteria cell wall• Directly induce IL-4 and IL-13 from human basophils• PGN and the synthetic TLR2 agonist Pam3Cys when combined with other stimuli, both IgE-

dependent and IgE-independent:- augmented mediator release or cytokine secretion, or both

TLR4 agonists • Ex. lipopolysaccharide (LPS), a constituent of G(-) bacteria• Unresponsiveness to LPS is from basophils without CD14

LIR • Similar differential effects on basophil function- LIR3 mediated inhibitory activity on basophil secretion- LIR7 caused mediator release and cytokine secretion.

IgE-Independent Pathway• Basophils

– express various receptors associated with innate immunity– responsive to specific agonists known to bind these receptors.


Pharmacologic Modulation of Secretion

Drug

CytokineHistamine


- - - -

Pharmacologic Modulation of Secretion

cAMP

β2-agonists, PGE2, histamine Theophylline

Histamine-release


-

Via phosphodiesterase inhibitionVia activation of adenylate cyclase

Pharmacologic Modulation of Secretionβ2-agonist Salmeterol

• 2 to 9x more potent at inhibiting IL-4 and IL-13 secretion from basophils than blocking histamine release

Theophylline • Nearly 10 times more active at inhibiting IL-4 than either histamine or IL-13

Antihistamines • Inhibit IgE-mediated histamine release by basophils in vitro. • Desloratadine – 7 times more potent at inhibiting IL-4 than inhibiting either

histamine or LTC4.• Terfenadine – inhibit mediator release and cytokine secretion, but with

similar potencies.• Cetirizine – slight enhancing effect on cytokine secretion


Pharmacologic Modulation of SecretionCorticosteroids • Inhibit in vitro histamine release

• Far more effective in blocking basophil cytokine secretion; • immediate inhibition• concentrations much lower than those required for the inhibition of

mediator release.

Tacrolimus and Cyclosporine

• Some of the most potent inhibitors of IgE-mediated cytokine secretion• NFAT family of transcription factors are involved in the transcription of IL-4

and IL-13 initiated with FcεRI activation.

Inhibitor of Bruton’s tyrosine kinase

• Inhibit IgE-dependent secretion of mediators and cytokines from basophils


BASOPHIL INVOLVEMENT IN DISEASE

CORRELATES of ALLERGIC DISEASE• Clinical correlates have linked the basophil response to disease

severity – Seen in urticaria, asthma, and food allergy– Histamine released by basophils predict the severity of the respiratory

symptoms• Most food-allergic children and many asthmatic subjects in

general:– have basophils that spontaneously secrete histamine in vitro.– Basophils from allergic asthmatics also are reported to possess an overall

increased releasability to various stimuliMiddleton’s Allergy Principles and Practice 8th ed. (2014) by Adkinson et al.

CORRELATES of ALLERGIC DISEASE• Allergen Challenge:

– Increase in basophil secreted IL-13 levels (in both nose and lung) (Allergic > non-allergic)– Phenotypic and Functional changes in circulating basophils– Increase in number of basophils and progenitors

• Systemic activation or “priming” of basophils in patients with clinical inflammatory disease. – Increased responsiveness as a manifestation of the overall inflammatory response– Self- priming by basophils augmented by autocrine-produced IL-3 on encountering allergen.

• CD63 and CD203c: – phenotypic markers increased on basophils after in vitro activation. – CD203c expression higher on basophils from asthmatic subjects who had recent exacerbations


Basophil Involvement in Disease• Use of basophil-specific antibodies confirm

basophil involvement – Chronic Idiopathic Urticaria and AD


MAST CELL BASOPHIL

TIMING Immediate Late phasePGD2 With PGD2 Without PGD2

Histamine ✓ ✓

LATE PHASE RESPONSES• Basophils in Allergic Inflammatory Reactions

– Best evidence with the late phase response following experimental allergen challenge.

– Glucocorticoids given before allergen challenge: NO effect on early response, but ablated late phase response• Block in vitro mediator release from basophils but not mast cells


BASOPHIL IN MOUSE MODELS• Critical role for basophils in the initiation of the Th2 responses • Prior exposure to IL-4 is not necessary for their IL-4–producing

ability, such is the case for development of IL-4–producing Th2 lymphocytes.

• Serine proteases can directly activate basophils to produce IL-4 as well as other pro-Th2 cytokines (e.g., TSLP)– Papain, cause a rapid influx of basophils into lymph nodes at a time

preceding lymphocytes. • Basophils can act as professional antigen-presenting cells (APCs)

critical for the induction of Th2 responses.Middleton’s Allergy Principles and Practice 8th ed. (2014) by Adkinson et al.

DELAYED-TYPE HYPERSENSITIVITY• Rhus toxoid (poison ivy) or dinitrochlorobenzene given by patch test:

Sensitized subjects developed skin reactions showed cellular infiltrate into the dermis – Up to approximately 16% basophils by 3 to 6 days after application of the

antigen– Basophils were the only granulocytes found in these lesions. – Mononuclear cells accounted for most of the cells infiltrating the lesions– Selective recruitment of basophils resulted from the secretion of some

factor(s) released by T cells. • Changes in the dermal microvasculature with these reactions suggested:

– Release of inflammatory mediatorsMiddleton’s Allergy Principles and Practice 8th ed. (2014) by Adkinson et al.

DELAYED-TYPE HYPERSENSITIVITY• Basophils migrating to these reaction sites --- Secrete Cytokines?

– May produce IL-13, because the synthesis of this cytokine (unlike IL-4) seems less dependent on signals generated through FcεRI.

• Degranulation in basophils unlike that seen in immediate hypersensitivity reactions (piecemeal degranulation)

• Increased number of basophils seen in many conditions involving a cellular immune component– Skin allograft and Tumor rejection, Viral hypersensitivity, and Crohn

disease.


DELAYED-TYPE HYPERSENSITIVITY• Basophil involvement in natural immunity to parasites

– IL-4–secreting basophils are critical for the expulsion of these worms, ectoparasites (ticks)

– Yet to be translated to human model

• Human basophils may play a role in impaired immunity to parasitic infections– completely opposite from the work emerging from the animal studies

• Similarity between Immediate Hypersensitivity and Parasitic Infections– increased IgE, eosinophils, basophils, and mast cells– Parasite antigens seldom induce the clinical manifestations typically of immediate

hypersensitivity reactions


DELAYED-TYPE HYPERSENSITIVITY• Basophils from human patients with helminth infections secrete IL-4 and IL-

13 in response to antigens derived from specific life stages of parasites.– hypothesized to be “driving” the Th2 response seen in helminthic infections– favoring of Th2- like responses is associated with impaired immunity to the

parasite– production of IL-4 and IL-13 by basophils may create conditions favorable for the

organism• Translationally controlled tumor protein (TCTP) of Plasmodium falciparum,

the organism responsible for malaria, is homologous to the HRF that causes histamine release and IL-4 secretion described earlier. – “Immune Mimicry” (also seen in certain viruses)


Summary of Concepts

Bone Marrow < 1% In Vivo Processes ?

Mediators and Cytokines

IL-4, IL-13, Cytokines IgE Synthesis

Innate Immunity


THANK YOU

Reference• Middleton’s Allergy

Principles and Practice 8th ed. (2014) by Adkinson et al. – Chapter 15: Biology of

Basophils

• Janeway’s Immunobiology 8th ed.(2012) by Murphy

biology of basophils

Health & Medicine

antigen basophils

number of basophils

circulating basophils

basophils alric

rapid influx of basophils

basophil activation

cytokines il

development of il