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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Paul Ehrlich (1854-1915)

1879

Identified by staining with basic dyes

http://www.nobelprize.org/

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

Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014

Stone KD., et. al. J Allergy Clin Immunol 2010;125:S73-80 Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014

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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

Stone KD., et. al. J Allergy Clin Immunol 2010;125:S73-80 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

Siracusa MC. et. al. Immunology and Cell Biology 2010;88:275–84

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 Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014

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)

Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408

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 Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408

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 Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014

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

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 Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408

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 Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014

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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

Karasuyama H. et. al. Nature Reviews Immunology 2009;9:9-13 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


Role in health and diseases

Karasuyama H. et. al. Nature Reviews Immunology 2009;9:9-13

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 Schroeder JT. Middleton’s Allergy Principle and Practice. 8th edition, 2014

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


• Infections

• Autoimmunity

• Malignancy


Charles N and Rivera J. Curr Allergy Asthma Rep 2011;11:378–87 Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408

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 Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408

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

Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408 Stone KD., et. al. J Allergy Clin Immunol 2010;125:S73-80

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


• Infections

• Autoimmunity

• Malignancy


Wimazal F. et. al. Cancer 2010;116:2372-81 Cromheecke JL. et. al. Curr Allergy Asthma Rep 2014;14:408

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 Karasuyama H. et. al. Nature Reviews Immunology 2009;9:9-13

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

Hoffmann HJ. et. al. Allergy 2015;70:1393-405


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


• 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



• 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.


Thank you for your attention

Health & Medicine

Biology of basophils & basophil activation test