Proteoglycans are m acrom olecules com posed of glycosam inoglycan chains covalently linked to a p ro tein core [322]. The glycosam inoglycan chains consist of d isaccharide rep eatin g u n its of uronic acid and hexosam ine
m oieties. These chains are variably sulfated and hence give rise to a net n eg ativ e charge. The m ast cell proteoglycans, h e p a rin an d c h o n d ro itin sulfate, bind histam ine, neutral proteases and acid hydrolases in the granules a n d m ay facilitate the u p tak e and storage of these m ediators [276]. U pon d eg ran u latio n , the n eu tral proteases rem ain associated w ith h ep arin at the cell surface, while histam ine and the acid hydrolases rap id ly diffuse aw ay [304].
The biological effects of heparin include inhibition of blood coagulation [323], regulation of tryptase activation [304], m odulation of grow th factors [324], and inhibition of the com plem ent cascade [325].
R ecent stu d ies have show n th at h ep arin has anti-allergic p ro p e rtie s, as in h aled h e p arin atten u ates antigen-induced bronchoconstriction in sheep [326] and hum ans [327]. In vitro, heparin selectively inhibits anti-IgE-induced histam ine secretion from hu m an uterine and rat peritoneal m ast cells w ith no effect on A23187-induced secretion [327].
H e p a rin also in h ib its the activ ity of a n u m b e r of e o sin o p h il p ro - in fla m m a to ry m ed iato rs th a t h av e been im p licated in a irw a y h y p e r reactivity. These include major basic protein, eosinophil cationic p ro tein and eo sin o p h il m ajor basic p ro tein [328,329]. The late p h a se of a sth m a is ch aracterised by a m arked eosinophilia, w hich m ay resu lt in p a rt from chemotactic action of platelet factor 4 [330]. H eparin has been show n to b in d to this cationic protein [331].
In m arked conflict to established theories, it has been suggested th at the m ast cell m ay have an anti-inflam m atory role in asthm a [332]. M ast cell-derived h e p arin m ay lim it eosinophil recruitm ent and also the actions of cationic p ro tein s in vivo [333]. Furtherm ore, conventional anti-asthm atic trea tm e n t w ith p2 agonists m ay, in fact, exacerbate chronic inflam m atory processes in
the airw ays [333], because these drugs inhibit m ast cell secretion [334,335]. A lthough there is some evidence to sup p o rt this theory [336,337], it does not take into account the potent inflam m atory effects of other m ast cell secretory products.
C h a p te r 1
1.7.1.6 Chemotactic factors
In vitro studies have show n th at m ast cells release eosinophil chem otactic factor of anaphylaxis (ECF-A). Specifically, rat peritoneal m ast cells [338], g u in ea p ig lung [339] and h u m an lung [340] tissues release this p e p tid e follow ing im m unological activation. As late phase reactions in the skin and lu n g are characterised by eosinophil recruitm ent, m ast cell ECF-A secretion m ay contribute to the eosinophilia.
A second chem otactic agent, neu tro p h il chem otactic factor of anaphylaxis (NCF-A) m ay also be m ast cell-d eriv ed . A llerg e n -in d u c ed b ro n c h ia l provocation results in a sustained increase in plasm a NCF-A concentration [341]. In vitro im m unological challenge of guinea-pig lu n g [342] and h u m an lung fragm ents [343] also results in release of NCF-A.
1.7.2 N ew ly generated m ediators
A ctivation of m ast cells can also lead to cellular synthesis of a n u m b er of p o te n t in flam m ato ry agents. M ed iato rs arisin g from arach id o n ic acid m etabolism are rapidly synthesised and secreted, beginning w ith in m inutes of activation and lasting up to 30 min. In contrast, synthesis of m ast cell cytokines occurs hours after activation, although preform ed cytokines m ay be secreted m ore rapidly.
1.7.2.1 A rachidonic acid m etabolites (eicosanoids)
M ast cell eicosanoid synthesis begins w ith the liberation of arachidonic acid (5,8,11,14-eicosatetraenoic acid) from m em brane phospholipids. This reaction is catalysed by the enzym atic action of phospholipase A2 [344] and is the rate
lim iting step in eicosanoid synthesis [345]. In addition, a recent stu d y on rat peritoneal m ast cells has show n that arachidonic acid is also m obilised by the sequential action of phospholipase D, p hosphatidate p h osphohydrolase and diacylglcerol lipase [346]. A rachidonic acid is th en rap id ly m etab o lised p r o d u c in g e ith e r p r o s ta g la n d in s a n d th ro m b o x a n e s th r o u g h th e cyclooxygenase p a th w ay or leu k o trien es via the lipoxygenase p a th w a y (review ed in reference 347), as detailed in Fig. 1.5. Eicosanoids exert their biological effects by acting on specific receptors (reviewed in reference 348).
1.7.2.1.1 PCD;
PG D2 is the m ain cyclooxygenase p roduct released follow ing im m unological
an d calcium ionophore activation of hum an and rat m ast cells [149,150,172- 174,349]. In contrast, substance P selectively stim ulates histam ine secretion from rat peritoneal and hum an skin m ast cells w ith o u t concom itant release of PG D2 [350,351]. C o m p o u n d 4 8 /8 0 stim u lates PG D2 release from rat
peritoneal m ast cells [349] b u t not from hum an skin m ast cells [351].
The biological actions of PGD2 include bronchoconstriction [352], perip h eral
vasodilatation [353], inhibition of platelet aggregation [354], p ro d u ctio n of a w heal and flare response in hum an skin [355] and augm enting LTB^-induced accum ulation of neutrophils in hum an skin [356].
1.7.2.1.2 Leukotrienes
The p ro d u cts of the lipoxygenase-directed m etabolism of arachidonic acid include LTB4, LTC4 and LTD4. Im m unologically-stim ulated rat m ucosal m ast
cells p ro d u c e LTB4 an d LTC4 [150]. P u rified h u m a n lu n g m a st cells
p red o m in an tly release LTC4 u pon im m unological challenge [174] as well as
sm all am ounts of LTB4 [357].
In vitro, LTB4 is a p o ten t chemotactic agent for h u m an an d rat n eutrophils
[358]. This leukotriene also enhances the expression and release of CD23 from B lym phocytes, and potentiates IL-4-m ediated B lym phocyte pro liferatio n [359]. In vivo, cutaneous injection of LTB4 causes a transient w heal and flare,
followed by a delayed infiltration of neutrophils [360].
The biological responses to LTC4 and LTD4 arise from the contractile potency
of these leukotrienes. Specifically, their biological effects include constriction of arterial, arteriolar, intestinal and bronchial sm ooth m uscle, increased v a so p erm e ab ility d u e to contraction of e n d o th elial cells an d en h an ced bronchial m ucus secretion (review ed in reference 361). LTC4 and LTD4 are
over 1 0 0 0 tim es m ore potent constrictors of hu m an bronchial sm ooth m uscle
C h a p te r 1
1.7.2.2 Platelet activating factor
In addition to the eicosanoids, cellular activation can result in the generation of the ether-linked phosholipid, platelet activating factor (PAF) (l-O -alkyl-2- acetyl-sn-glyceryl-phosphorylcholine). The biosynthesis of PAF occurs in a concerted tw o-step process, phospholipase A2 hydrolyses p h o sp h o lip id s to
p ro d u c e 1 -O -a lk y l-sn -g ly ce ry l-p h o sp h o ry lch o lin e (lyso-PA F), th e n an acetyltransferase enzyme acetylates lyso-PAF generating PAF [364].
PAF is released from m ouse BMCMC follow ing antigen activation [365,366]. A ctivated hu m an lung m ast cells generate PAF, although this lipid-derived m ediator is not secreted [367]. PAF is released from a variety of other activated inflam m atory cells, including rabbit basophils [368], hum an neutrophils [379], hum an alveolar m acrophages [370], hum an eosinophils [371] and h u m an and rabbit platelets [372].
The biological actions of PAF in vitro include th e activ atio n of h u m an platelets [373] and h u m an eosinophils [374]. PAF also induces histam ine release from h u m an basophils [375] and ra t p erito n eal m ast cells [376], although m ast cells isolated from hum an lung, skin or u teru s fail to respond to PAF [375,377]. PAF exhibits chemotactic activity for eosinophils, and it has therefore b een suggested th at this lip id -d eriv ed m ed iato r m ay have an im p o rtan t role in the d ev elo p m en t of the characteristic eo sin o p h ilia in allergic asthm a [378]. In addition, PAF m ay have a role in the cutaneous response follow ing antigen-m ediated m ast cell activation [379,380].
1.7.2.3 Cytokines
Cytokines are m ultifunctional proteins produced by m any activated cell types. They alter the behaviour of target cells through their action on specific cell surface receptors [381]. Essentially, cytokines influence m any physiological an d p a th o p h y sio lo g ica l processes in clu d in g c ellu lar p ro life ra tio n an d d iffe re n tia tio n , cellu lar p h e n o ty p e ex p ressio n , re g u la tio n of im m u n e responses, control of host defences against viral and parasitic infections, tissue rem odelling and bone form ation (review ed in references 382 and 383). The m ain biological activities of the cytokines discussed in this section are listed in Table 1.2.
The first direct dem onstration of a m ast cell p opulation releasing a cytokine w as reported in 1986 [384]. The Abelson m urine leukaem ia virus-transform ed tum origenic m ouse m ast cell line constitutively p ro d u ced GM-CSF m RNA a n d rele ased GM-CSF. S u b seq u en tly , c u ltu re d m ast cells stim u la te d im m unologically or w ith the calcium ionophore A23187 have been show n to secrete IL-1 [385], IL-3 [386], IL-4 [385,386] and IL- 6 [385,386].
Studies on cytokine release from freshly isolated m am m alian m ast cells are essentially still in their infancy. M ouse peritoneal m ast cells show increased tran scrip tio n a n d /o r secretion of TN F-a [387-389] and transform ing g ro w th factor (TGF-P) [389,390].
H u m an skin m ast cells contain preform ed TNF-a w hich is released follow ing cellu lar activation [198]. Im m unologically-activated skin m ast cells also ap p ear to p roduce IL- 8 [391]. Purified h u m an lu n g m ast cells secrete IL-4
follow ing im m unological challenge [197] and contain preform ed TN F-a [392]. A recent im m unohistochem ical stu d y com paring bronchial biopsies of atopic asthm atics and non-asthm atics confirm ed th at lung m ast cells contain both IL-4 and TNF-a, as well as IL-5 and IL- 6 [393]. Furtherm ore, asthm atic subjects
h a d increased nu m b ers of m ast cells th at stain ed for IL-4 an d T N F-a com pared to norm al subjects.
1.8 SIGNAL TRA NSD UC TIO N PATHWAYS CON TRO LLING MAST