R

B

ED

a

AA

KMBBTII

1

tasUvTblcppc

2

lscidMc

B

0d

Immunology Letters 138 (2011) 28–31

Contents lists available at ScienceDirect

Immunology Letters

journa l homepage: www.e lsev ier .com/ locate / immlet

eview

asophils and mast cells: Underdog in immune regulation?

dward F. Knol ∗, Maciej Olszewski1

epartment of Dermatology/Allergology, University Medical Center Utrecht, Utrecht, The Netherlands

r t i c l e i n f o

rticle history:vailable online 17 February 2011

a b s t r a c t

Mast cells and basophilic granulocytes have recently been recognized as potent immunomodulatorycells. Whereas these cells originally were described as potent cells that release their pro-inflammatory

eywords:ast cells

asophilic granulocytesasophilsNF

contents (i.e. histamine) unconstrained after activation, nowadays this process is considered to be muchmore subtle. Especially, via the release of pro- and anti-inflammatory cytokines basophils and mast cellsnow can steer an immune response. Recently, it has become evident that at least in murine modelsbasophils are crucial for the initial induction of a Th2 response in immunologically naive mouse.

© 2011 Elsevier B.V. All rights reserved.

gEL-4

. Introduction

Mast cells and basophilic granulocytes are most recognized forheir effector roles in the immune response towards parasites andllergens. Both cells are rather unique in the immune system byharing the expression of the high affinity Fc receptor for IgE (Fc�RI).pon cross linking of IgE on the Fc�RI by allergen these cells are acti-ated and release their inflammatory mediators via degranulation.he most well-known granule product is histamine. Recently, it haseen demonstrated that both cell types are potent immunomodu-

ating cells, not only via the release of stored and novel synthesizedytokines and chemokines, but also via their antigen-presentingroperties. In this review we will discuss in more detail the specificroperties of these separate lineage cell types particularly in theontext of their immunomodulatory functions.

. Mast cells and their granules

Mast cells are tissue-dwelling cells that are predominantlyocated at the interfaces of the organism and the exterior, such askin, gut mucosal membranes and lung. They are evolutionarily oldells that play multiple roles in many modes of immune response,

ncluding innate and antibody-dependent reactions. Mast cellserive their name from the original name given by Paul Ehrlich,astzellen (well-fed cells) reflecting the fact that a mature mast

ell contains large number of cytoplasmic granules. These granules

∗ Corresponding author.E-mail address: e.f.knol@umcutrecht.nl (E.F. Knol).

1 Current address: Department of Cell Biology, NHLBI, National Institute of Health,ethesda, United States.

165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.imlet.2011.02.012

are specialized organelles, found primarily in granulocytes, whichpossess the unique capability of rapid release. Initial studies ofhuman mast cell granule composition by enzyme-affinity labellingand ultrastructural immunocytochemical techniques allowed foridentification of proteases chymase and tryptase a proinflam-matory biogenic amine histamine and the proteoglycan heparin.Subsequently, new techniques allowed for establishing granu-lar localization of many more mediators, among them severalcytokines such as bFGF, SCF, VEGF, IL-4 and TNF [1]. The presence ofcytokines in granules adds a new dimension to a role of mast cellsin cytokine biology. Perhaps the best example of novel mechanismsin cytokine biology is the role of mast cell granule-derived TNF. Thiscytokine plays critical role in host defence against bacterial infec-tions [2] and its lack results in drastically reduced neutrophil influxand significantly increased mortality of experimental animals. Mastcells are considered the only cells storing preformed TNF. Remark-ably the selective granule targeting of TNF appears to be differentlyregulated in mast cells from human and rat origin [3]. The uniquecapability of the fast release of a considerable amount of preformedcytokines might enable mast cells to influence the course of theimmune processes being initiated, directing it towards inflamma-tory or allergic response, depending on the profile of cytokinesreleased.

Until recently it was unclear how mast cells released cytokinescan exert effects in draining lymph nodes. Kunder and cowork-ers demonstrated that mast cell-released TNF is partly packedinto submicrometer heparin-based particles [4]. Upon their release

these stable particles enter the lymphatic vessels and end up inthe draining lymph nodes. This is illustrating that in mast cellsa dedicated physiological drug delivery system facilitating com-munication between peripheral sites of inflammation and remotesecondary lymphoid tissues is active.

unolo

3

ccmgrBmaWTtvaaTrmoustheagbsM

4

mssmdmmdtsobtcbmc

5

5

awaathc

E.F. Knol, M. Olszewski / Imm

. Basophilic granulocytes and their granules

Basophilic granulocytes (basophils) reside, in contrast to mastells, in blood. It was again Paul Ehrlich that first described theseells in 1879 as cells in blood that contained granules and stainedetachromatically when exposed to basic dyes, naming this set of

ranulocytes basophilic granulocytes. Basophilic granulocytes rep-esent only about 0.5% of the leukocyte population in human blood.asophils are spheric cells with a diameter of about 10 �m and aultilobe nucleus. The most evident characteristics of basophils

re the round cytoplasmic granules with diameters up to 1.2 �m.ithin the granules histamine is stored, at about 1–2 pg per cell.

he characteristic metachromatic staining of basophils is due tohe presence of proteoglycans in the granule. Basophils release aariety of pre-stored and newly sensitized pro-inflammatory medi-tors (e.g. Histamine and LTC4) and cytokines (e.g. IL-3, IL-4, IL-13nd VEGF) which are involved in the pathogenesis of allergies.he basophil population in blood represent a population that canapidly migrate in tissues. At tissue sites of allergic inflammation aarked influx of basophils can be found. The influx of basophils is

ften accompanied by a simultaneous influx of eosinophilic gran-locytes and Th2 lymphocytes. Basophils enter tissue sites withineveral hours after exposure to allergens. However, it is conceivablehat by the time basophils enter these tissues the allergens may wellave been cleared. This evidently leads to the question as to whatlse, other than allergen-mediated stimulation, can drive basophilctivation following extravasation into tissue sites affected by aller-ic inflammation. Recently, it was demonstrated that basophils cane activated by IL-18 and IL-33 to release large amounts of cytokinesuch as IL-4, IL-13, IL-6, IL-9, RANTES, GM-CSF, MIP-1a, MIP1b andCP-1, but not IL-17, IL-5 and IFN-� [5].

. Relation between mast cells and basophils

There is much confusion on the relation between basophils andast cells. It is not uncommon that basophils are mistakenly con-

idered to be the blood-derived progenitor of tissue mast cells,uch as the link between monocytes and macrophages. Althoughast cells and basophils share several unique properties, they are

erived from distinct progenitors. Basophils differentiate in bonearrow and are released in the blood as mature basophils, whereasast cells progenitors are found in bone marrow and blood, but

ifferentiation of these cells does not occur before entering theissue. For the development of basophils IL-3 is crucial, whereastem cell factor (c-kit ligand) is important for the differentiationf mast cells. The mast cell development in tissue is fine-tunedy additional cytokines, such as TGF-�, IL-4, IL-9 and matrix pro-eins, resulting in different phenotypes of mucosal tissue mast cellsompared to connective tissue mast cells. In general it seems thatasophils are much more related to eosinophils and mast cells areuch related to monocytes/macrophages. In Table 1, we provide a

omprehensive list of differences between mast cells and basophils.

. Mast cells and basophils in immune responses

.1. General remarks

Mast cells and basophils are important elements of both innatend acquired immunity. They express numerous receptors that,hen stimulated, may induce production of a plethora of medi-

tors. These receptors include IgE and IgG, complement, IL-1, TNFnd several Toll-like receptors, to name just a few most impor-ant. Upon stimulation they can degranulate, release and synthesizeighly bioactive, proinflammatory, vasodilative, chemotactic, andytotoxic substances. These cells are crucial for the function of sev-

gy Letters 138 (2011) 28–31 29

eral biologically and clinically important mechanisms of immuneresponse such as allergy, inflammation and, as shown recently, alsoimmune tolerance [6]. Some of the more important mediators pre-stored and synthesized by mast cells and basophils and their majorpathophysiological effects are summarized in Table 2.

5.2. Innate immunity

Looking from a broader perspective of host defence especiallymast cells play several roles in innate and acquired immunity.Although there is some evidence of mast cells exhibiting directlygermicidal activity by phagocytosis or bactericidal peptide release,several lines of evidence suggest that the most important way bywhich mast cells contribute to innate immune response is initia-tion and regulation of the magnitude of leukocyte infiltration intothe site of inflammation. It has been demonstrated using mast-celldeficient mice that at least TNF and leukotrienes are important fac-tors in neutrophil recruitment towards sites of bacterial infectionand mast cells deficiency correlates with much worse prognosisin experimentally infected mice. Experiments in other model sys-tems have shown that secretion of TNF and leukotrienes in the acutephase of inflammatory processes may also promote influx of leuko-cytes other than neutrophils, such as T cells or macrophages whichare typical for chronic inflammatory state. Such leukocytes can theninitiate and maintain features characteristic of the chronic inflam-matory state. Another function mast cell play in innate responsesis limiting the toxicity of certain substances generated by the hostwhich have adverse effects when present in high concentrations.An example of such activity is degradation of endothelin-1, a pep-tide that is involved in sepsis, by the proteases released from mastcell granules. Mast cells are also capable of releasing mediatorsinfluencing (positively or negatively) the transition from innate toacquired immunity. It has been reported that mast-cell derived TNFplays a role in draining lymph node hypertrophy and T cell recruit-ment to these nodes in a model of E. coli infection in mouse. Thisindicates that while there is a mast cell-dependent component inthe development of adaptive immune response, the mechanismsare likely to be more redundant as compared to innate response.

5.3. IgE-associated adaptive responses

Another aspect of mast cells and basophils contribution toimmune response is their involvement in adaptive immunity. Orig-inally, these activities were connected to antigen-specific IgE that,when bound to Fc�RI and crosslinked by an antigen, activate mul-tiple pathways in these cells. Recent findings demonstrate, that,in mast cells, IgE at high concentrations has more than just a pas-sively sensitizing activity. Some antibodies are able to elicit fullresponses in the absence of antigen while other only upregulateFc�RI and enhance mast cells survival. This survival enhancementis mediated by autocrine IL-3 stimulation and activation of Bcl-xL/Bcl-2. The extent of mast cell activation in absence of antigendepends on a particular antibody, although the molecular determi-nants of this anti-apoptotic activities are not defined. Additionally,the increased survival after Fc�RI stimulation differs between mastcell subpopulations [7].

It has been widely accepted that mast cells contribute sig-nificantly to acute inflammatory reactions to antigens/allergensagainst which the host bears antibodies of the IgE class. Mastcells are responsible for virtually all of the increased vascular per-meability and tissue swelling early in the IgE-dependent passive

cutaneous anaphylactic response. If the stimulation is of morepersistent or more severe nature, acute response may undergo tran-sition into late-phase reaction (LPR) which, except for the time scaleranging from few to several hours from initial antigen challenge, ischaracterized by recruitment of leukocytes to the site of inflamma-

30 E.F. Knol, M. Olszewski / Immunology Letters 138 (2011) 28–31

Table 1Distinct properties of basophils and mast cells.

Properties Basophils Mast cellsa

Size 7–10 �m 14–20 �mNucleus Multi-lobed Single-lobedGranules Fewer and larger than those of mast cells Smaller, more numerous than those of basophilsLocation Blood, can migrate into tissue TissueLife span Days Weeks to monthsDevelopment

Maturation site Bone marrow Tissue, progenitors move from bone marrow via blood in tissueDifferentiation factors IL-3 Stem cell factor (fine tuning with other local cytokines and tissue matrix

ContentsHistamine 1–2 pg/cell 1–15 pg/cellMajor proteases – TryptaseArachidonic acid metabolites LTC4 LTC4, PGD2, thromboxanesGranule contents Major basic protein, Charcot Leyden crystals No major basic protein or Charcot Leyden crystals

ActivationfMLP Activation No effectPMA/A23187 Activation by each separate agent Only activation when added togetherCompound 48/80 No effect ActivationSubstance P No effect ActivationMorphine No effect Activation

Pharmacological inhibitionH2 agonists Inhibition No effect (also no H2 receptor)

Thereft

titmh

rio

TM

Steroids InhibitionIndomethacin Enhances

a Within the mast cell population different cell types have been demonstrated.ypes.

ion. In this chronic response basophils can play an important rolen the sustained nature of inflammation upon entering the affectedissue, not only due to the release of stored and newly synthesized

ediators, such as histamine and LTC4, but also via the release ofigh amounts of the cytokines IL-4 and IL-13 [8].

In this view, a wide range of innate and IgE-associated immuneesponses appear to represent a situation in which mast cell activ-ty, depending on particular circumstances, may be either beneficialr detrimental to the host.

able 2ast cell and basophil mediators.

Mediator Major path

PrestoredBiogenic amines

Histamine Vasodilatio5-HT Leukocyte

Chemokines (IL-8, MCP-1, MCP-3, MCP-4, RANTES) ChemoattrEnzymes

Chymase Tissue damTryptase ActivationKinogenases Synthesis oNitric oxide synthase NO producCarboxypeptidase A Degrades e

PolypeptidesCRH InflammatEndothelin SepsisKinins InflammatSomatostatin (SRIF) Anti-inflamVEGF Neovascula

ProteoglycansChondroitin sulfate ConnectiveHeparin Angiogene

De novo synthesizedCytokines

IL-1, -3, -4, -5, -6, -9, -10, -13, -16, IFN-�, MIF, TNF Multiple roGrowth factors

SCF, GM-CSF, GnRH-I �-FGF, NGF, VEGF Growth ofPhospholipid metabolites

LTB4 LeukocyteLTC4 VasoconstrPAF Platelet actPGD2 BronchocoNO Vasodilatio

No effectsNo effects

ore some of the characteristics given in this table do not account for all mast cell

5.4. IgE-independent responses

Apart from IgE-dependent responses, mast cells and basophilshave been implicated in pathogenesis of several autoimmune dis-eases, including multiple sclerosis and rheumatoid arthritis in

humans and experimental autoimmune encephalomyelitis (EAE)and IgG1 antibody-dependent autoimmune arthritis in mice.Moreover, under some experimental conditions mast cells are nec-essary for complete elicitation of inflammation associated with

ophysiologic effect

n, angiogenesis, mitogenesis, suppressor T-cell activationregulation, vasoconstriction, painaction and tissue infiltration of leukocytes

age, pain, angiotensin II synthesisof PAR, inflammation, pain, tissue damage, degradation of antigens and peptidesf kinins, paintionnzymes

ion, vasodilation, mast cell VEGF release

ion, pain, vasodilation, mast cell triggermatory (?), mast cell triggerrization, vasodilation

tissue component, anti-inflammatory, mast cell inhibitorsis, NGF stabilization, mast cell inhibitor

les

a variety of cells, mast cell proliferation

chemotaxisiction, painivation, vasodilation, inflammationnstriction, painn, neuromodulation

unolo

hi

6

psmarUwsassuaantcrmrMcmEiltt

airt

7ap

tditiTriwtTfttcrnbmb

[8] Falcone FH, Haas H, Gibbs BF. The human basophil: a new appreciation of itsrole in immune responses. Blood 2000;96(13):4028–38.

E.F. Knol, M. Olszewski / Imm

apten-induced contact hypersensitivity (CHS) or asthma andnflammatory bowel disease.

. Mast cells do also limit inflammation

Although there is a solid body of evidence that mast cells exertredominantly proinflammatory activities, there are a few reportstating otherwise. Examination of biological activity of mast cellediators, indicates that some of them, including TGF-�, IL-4, IL-10

nd histamine have potentially anti-inflammatory activity. The firsteport of such activity of mast cells in knockin mouse concernedV-induced suppression of contact hypersensitivity to DNFB thatas, at least partly, mediated by IL-10. It has also been demon-

trated, that mice that were bitten by a mosquito display loweredntigen-specific T cell responses in the model of delayed hypersen-itivity to OVA and that this phenomenon requires mast cells at theite of the bite. The mechanism of this regulatory activity remainsnknown. The results of yet another study show that mast cellsre necessary for peripheral tolerance to skin allografts. In toler-nt mice considerable increase of mast cell-specific transcripts andumber of mast cells was observed. This increase correlates withhe influx to the graft of IL-9-producing CD4+Foxp3+ T cells. Mastell-deficient mice cannot be tolerized and experience rapid graftejection, which can be prevented by local skin reconstitution withast cells. IL-9 released by Tregs is the major mediator of mast cells

ecruitment and activation in the dermis of these tolerant grafts.ast cells may then act by limiting the influx of inflammatory T

ells or cooperating with dermal Tregs. Unexpected as it sounds,ast cells do contain TGF-� that is a major Tregs inducing factor.

ven in the response to cobra venom and bee venom it was foundn mice that the presence of mast cells was an important factor inimiting the pathologic effects. Most likely this limitation was dueo release of carboxypeptidase A and possibly other proteases byhe mast cells.

In conclusion, mast cell activators may yield pro- ornti-inflammatory responses [9]; most probably are the pro-nflammatory effect most pronounced in the earlier phase of theesponse and is the anti-inflammatory effects more pronounced athe later phase of the response to limit ongoing inflammation.

. Basophils are important cells in the induction of anllergic immune response via their Th2-skewing, antigenresenting functional capacity

In the summer of 2009 in Nature Immunology a rather uniquehree-fold back-to-back set of papers has been published, allescribing a breakthrough in the important role of basophils in the

nduction of allergic immune responses. The take home message ofhese papers is that basophilic granulocytes not only have the abil-ty to function as antigen presenting cells, but they also promoteh2-responses [10]. Sokol and colleagues studied the strong Th2esponse induced by papain in mice. In their setup papain mim-cked the protease activity of allergens and parasites. Remarkably

as that removal of classic antigen presenting cells did not affecthe Th2 response but diminished the induction of both Th1 andh17 responses. The presence of basophils turned out to be crucialor the induction of the Th2 responses. The authors demonstratedhat basophils not only expressed MHC-II molecules, they also con-ained the machinery to take up, process and present antigens to Tells on MHC-II molecules. In addition, Perrigoue et al. studied Th2

esponses that were induced via infection with the gastrointesti-al nematode parasite Trichuris muris. They also demonstrated thatasophils present the antigenic peptides of the parasite on MHC-IIolecules to naïve T cells. Via the simultaneous release of IL-4 the

asophils skewed the T cells towards the Th2 phenotype. Basophils

[

gy Letters 138 (2011) 28–31 31

were crucial for the Th2-response because depletion of peripheralblood basophils with Mar-1 antibodies blocked the developmentof Th2 cells. Finally, further evidence for the important role ofbasophil-derived IL-4 in the induction of Th2-responses was pro-vided by research from the group of Yoshimoto. This group studiedthe role of basophils in the induction of Th2 responses by infec-tion with the nematode Strongyloides venezuelensis. In addition, thisgroup demonstrated that activation of basophils did not depend onthe presence of IgE antibodies on their membrane. This can be con-sidered an important finding because it indicates that basophilsmight be responsible for the initial induction of IgE.

Since the time that IL-4 has been demonstrated as mast cell andbasophil products, now almost 20 years ago, it has been hypothe-sized that these cells are important in the early induction of Th2cells and the development of allergy. However, convincing stud-ies were not yet published. In the three papers described aboveclear indications are provided that basophilic granulocytes induceTH2 responses via the peptide presentation on MHC-II and simul-taneous release of IL-4. Extrapolation of these results obtained inmouse models towards human is not clear yet. Expression of MHC-IIon human basophils has not been convincingly demonstrated andthe mouse basophils appear to be morphologically distinct fromthe human basophils. It will probably be shortly before follow-uppapers will address the potency of human basophils.

8. Final conclusion

Mast cells and basophils have long been considered to be smallbags filled with potent inflammatory mediators that were releasedafter a specific stimulation. Especially in the context of allergicinflammation these cells are thought to be crucial in the initi-ation and maintenance of the inflammatory reactions. Recently,much more subtle roles of these cells have been demonstrated, notonly via the large numbers of cytokines and chemokines that thesecells can produce, but also via their pro- and anti-inflammatoryfunctions. In addition, most pronounced for the basophils, there isa potent antigen-presenting cell activity described that togetherwith the release of IL-4 might be important for the very earlyTh2 skewing of the immune response towards allergens and para-sites.

References

[1] Galli SJ, Kalesnikoff J, Grimbaldeston MA, Piliponsky AM, WilliamsCM, Tsai M. Mast cells as “tunable” effector and immunoregu-latory cells: recent advances. Annu Rev Immunol 2005;23:749–86.

[2] Malaviya R, Ikeda T, Ross E, Abraham SN. Mast cell modulation of neutrophilinflux and bacterial clearance at sites of infection through TNF-alpha. Nature1996;381:77–80.

[3] Olszewski MB, Groot AJ, Dastych J, Knol EF. TNF trafficking to humanmast cell granules: mature chain-dependent endocytosis. J Immunol2007;178(9):5701–9.

[4] Kunder CA, St John AL, Li G, Leong KW, Berwin B, Staats HF, et al. Mast cell-derived particles deliver peripheral signals to remote lymph nodes. J Exp Med2009;206(11):2455–67.

[5] Knol EF, Gibbs BF. Basophil survival and immunomodulatory function areuniquely regulated by a novel MyD88-dependent pathway. J Leukoc Biol2009;86(4):753–5.

[6] Lu LF, Lind EF, Gondek DC, Bennett KA, Gleeson MW, Pino-Lagos K, et al.Mast cells are essential intermediaries in regulatory T-cell tolerance. Nature2006;442(7106):997–1002.

[7] Kawakami T, Kitaura J. Mast cell survival and activation by IgE in the absence ofantigen: a consideration of the biologic mechanisms and relevance. J Immunol2005;175(7):4167–73.

[9] Galli SJ, Grimbaldeston M, Tsai M. Immunomodulatory mast cells: negative, aswell as positive, regulators of immunity. Nat Rev Immunol 2008;8(6):478–86.

10] Wynn TA. Basophils trump dendritic cells as APCs for T(H)2 responses. NatImmunol 2009;10(7):679–81.