A Pathogenic Role for IgE in Autoimmunity: Bullous Pemphigoid IgE Reproduces the Early Phase of Lesion Development in Human Skin Grafted to nu/nu Mice

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1 ORIGINAL ARTICLE : Bullous Pemphigoid IgE Reprodues the Erly Phse of Lesion Development in Humn Skin Grfted to nu/nu Mie Jnet A. Firley 1,4, Christopher T. Burnett 1, Chng-Ling Fu 1, Dvid L. Lrson 2, Mtthew G. Fleming 1 nd George J. Giudie 1,3 Bullous pemphigoid (BP) is n utoimmune disese hrterized y suepiderml listering. Bsed on previous work, IgG utontiodies direted ginst BP180 re thought to e the primry pthogeni gent in BP. In ddition to these IgG utontiodies, however, most BP ptients produe IgE lss utontiodies tht lso ret with BP180, nd totl IgE levels re often elevted in this disese. To diretly test whether BP IgE is pthogeni, 6 ng of totl IgE isolted from two BP nd two norml ser were injeted into humn skin grfted onto thymi, nude mie. Twenty-four hours fter injetion, erythemtous, elevted plques were oserved in ll humn skin grfts reeiving BP IgE (n ¼ 11), ut not ontrol IgE (n ¼ 9). Histologi nd ultrstruturl exmintion of the lesions showed engorgement of lood vessels nd derml infiltrte omposed of neutrophils, eosinophils, nd mst ells, mny of whih were degrnulted. At higher dose of BP IgE (47 ng), histologil seprtion of the epidermis from the dermis ws oserved in two of the three grfts. The BP IgE-indued erythemtous plques were reminisent of those linilly seen in BP. This provides erly evidene of diret demonstrtion of pthogeni role for IgE lss utontiodies in humn utoimmune disese. Journl of Investigtive Dermtology (2007) 127, ; doi: /sj.jid ; pulished online 5 July 2007 INTRODUCTION Bullous pemphigoid (BP) is n quired listering skin disese hrterized y humorl utoimmune retion direted ginst omponents of the hemidesmosome, ellulr struture involved in nhoring the epidermis to the dermis. The initil phse of lesion development in BP is hrterized y urtiril plques onsisting of derml edem ompnied y eosinophili infiltrtion of the upper dermis, termed eosinophili spongiosis. As the lesions progress, there is linil development of tense, fluid-filled vesiles, whih orrespond histologilly to seprtion of the skin through the sement memrne zone (BMZ). In ddition, there is superfiil nd deep perivsulr infiltrte of mny eosinophils, lymphoytes, mst ells, nd neutrophils. The seminl work of Beutner nd Jordon (Jordon et l., 1967), in whih immunogloulins nd C3 were shown to e 1 Deprtment of Dermtology, Medil College of Wisonsin, Milwukee, Wisonsin, USA; 2 Deprtment of Plsti Surgery, Medil College of Wisonsin, Milwukee, Wisonsin, USA; 3 Deprtment of Biohemistry, Medil College of Wisonsin, Milwukee, Wisonsin, USA nd 4 Dermtology Setion, Veterns Affirs Medil Center, Milwukee, Wisonsin, USA Correspondene: Dr Jnet A. Firley, Deprtment of Dermtology, University of Iow Hospitls nd Clinis, BT-2090, 200 Hwkins Drive, Iow City, Iow 52246, USA. E-mil: jnet-firley@uiow.edu Arevitions: BP, ullous pemphigoid; BMZ, sement memrne zone Reeived 25 August 2006; revised 10 April 2007; epted 15 April 2007; pulished online 5 July 2007 deposited long the utneous BMZ of BP ptients, pointed to the immunologil sis of this disese. These investigtors were lso the first to identify nti-bmz ntiodies in the ser of BP ptients. The primry ntigeni trgets of BP utontiodies were susequently identified s two hemidesmosoml proteins, BPAG1 (Stnley et l., 1981) nd BP180 (lso termed ollgen XVII nd BPAG2) (Li et l., 1986; Diz et l., 1990; Giudie et l., 1992). BPAG1 is omponent of the intrellulr hemidesmosoml plque (Tnk et l., 1991), nd its role in pthogeniity remins unknown. In ontrst, numerous studies using in vivo nd in vitro models of BP strongly suggest tht ntiodies direted ginst BP180 re of primry pthogeni importne (reviewed in Liu nd Diz 2001). Injetion of IgG ntiodies ginst BP180 into neontl mouse skin initites n inflmmtory retion resulting in skin frgility t the level of the BMZ; however, urtiril plque formtion, reruitment of eosinophils nd gross linil listering re not seen. Likewise, the injetion of humn BP IgG into humn skin trnsplnted onto SCID mie leds to histologil suepiderml seprtion, ut no linil disese tht mimis BP or ny eosinophili infiltrtion (Zillikens et l., 2001). The mjority of the experimentl work on BP hs foused on IgG lss utontiodies, even though it hs een long known tht there is lso n IgE response ssoited with this disese (Provost nd Tomsi, 1974). The presene of IgE utontiodies in BP is prtiulrly interesting in light of the ft tht BP ptients typilly hve n erly urtiril phse of & 2007 The Soiety for Investigtive Dermtology

2 the eruption, nd the role of IgE-medited degrnultion of mst ells in llergi forms of urtiri is well estlished (Friedmnn 1999). During this erly phse of BP, ptients hve lrge urtiril plques ut my mnifest few, if ny, listers. Sequentil histologil exmintion of the skin of BP ptients during the development of individul lesions hs shown tht mst ell degrnultion is the erliest event tht n e identified histologilly, followed y lymphoyti infiltrtion nd lter eosinophil nd neutrophil migrtion into the lesion (Wintrou et l., 1978; Dvork et l., 1982). Further evidene for mst ell degrnultion is the presene of eotxin (Shrikhnde et l., 2000) nd tryptse (D Auri et l., 2000) in the lister fluid of BP lesions. Also of note, mst ell tryptse hs een shown experimentlly to e ple of using epiderml derml seprtion (Kminsk et l., 1999). Totl irulting IgE levels re elevted in 70% of untreted BP ptients (Dimson et l., 2003, Aresmn et l., 1974), nd IgE lss utontiodies direted ginst BP180 n e deteted in 90% of untreted BP ptients (Dimson et l., 2003). Moreover, sophils isolted from the peripherl lood of untreted BP ptients were shown to degrnulte when exposed to reominnt BP180 (Dimson et l., 2003). The importnt implition of this finding is tht IgE utontiodies speifi for BP180 re present t the surfe of sophils, ound to the IgE reeptor. We hve hypothesized tht n nlogous proess is ourring in mst ells in the skin of BP ptients, nd tht utontigen-indued mst ell degrnultion is key trigger for the urtiril phse of BP. To test the pthogeni potentil of IgE lss utontiodies in BP, we isolted IgE from BP ptients ser nd injeted it into humn skin trnsplnted to thymi nude (nu/nu) mie. RESULTS Levels of totl IgE re elevted in BP ser Totl serum IgE levels were determined y stndrd pture ELISA. The IgE onentrtions in ser BP-W7714 nd BP- G5554 were 18 nd 4 mg/ml, respetively. Compred to the upper limit of the norml rnge for serum IgE (240 ng/ml), the IgE levels for BP-W7714 nd BP-G5554 re 75 nd 17-fold higher, respetively. Western lot nlysis reveled tht oth BP ser ontined IgE tht rets with the NC16A domin of type XVII ollgen (see elow). Purified BP IgE ontinues to ind to NC16A, the BMZ, nd mst ells IgE ws purified from ser BP-W7714 nd BP-G5554 using omintion of negtive nd positive seletion (see Mterils nd Methods). This purifition sheme yielded pproximtely 10,000-fold purifition of IgE reltive to IgG. The IgE nd IgG onentrtions in oth of the purified BP IgE preprtions were nd ng/ml, respetively. The IgE ontinued to ind to the BMZ of humn skin nd utneous mst ells, s determined y immunofluoresene using speies-speifi FITC-onjugted nti-ige (Chemion, Temeul, CA) (Figure 1). The purified IgE lso retined its ility to ind to BP180 y immunolotting (Figure 1). BP serum Injetion of BP IgE produes urtiril plques nd mst ell degrnultion in humn skin grfts The model system tht ws used to test the pthogeniity of BP IgE involved grfting humn skin onto the ks of nude mie. Cryosetions of 2-week grfts exhiited norml leling ptterns with ntiodies to humn BP180, lminin 5, nd ollgen VII, thus showing the integrity of the BMZ (dt not shown). Retention of humn mst ells in the grfts ws verified y stining with Giems stin nd nti-humn mst ell tryptse ntiodies (Chemion, Temeul, CA; dt not shown). IgE purified from BP-W7714 nd BP-G5554 ws injeted intrdermlly into the 2-week humn skin grfts. Injetion volumes were kept onstnt t 100 ml. Within 15 minutes fter injetion of 6 ng of BP IgE, edem ws noted within the grfted skin. At 24 hours, n edemtous, erythemtous plque developed in ll of the grfts tht reeived IgE from either of the BP ser (n ¼ 11, Figure 2 nd ). When the higher mount of BP IgE (47 ng, n ¼ 3) ws injeted into the grft, erythem developed round the injetion site within 5 minutes, eoming muh more intense t the 15-minute time point. No swelling or erythem ws noted in ny of the ontrol grfts tht reeived 6 ng of IgE isolted from norml humn serum (n ¼ 9, Figure 2 nd d) or in the surrounding murine skin. To test whether the residul IgG remining in the BP IgE preprtion ws responsile for the oserved hnges, we injeted 1.25 ng of purified BP-W7714 IgG (pproximtely 4 to 5 times the mount of IgG present in the 6 ng dose of BP IgE) into the humn skin grft. The purified BP IgG produed no linil or histologil evidene of disese (dt not shown). These results re summrized in Tle 1. Hemtoxylin nd eosin stining of the lesionl grfts showed ongestion of the lood vessels in the upper dermis BP IgE N G N G Figure 1. Purified BP IgE inds to sl epiderml kertinoytes nd derml mst ells y IF, nd to the NC16A region of BP180 y immunolotting. Following purifition, IgE from serum BP-W7714 ws tested y indiret immunofluoresene for its ility to ind to omponents of humn skin. () A ryosetion of norml humn skin ws leled with the purified BP IgE followed y FITC-nti-humn IgE. Stining n e seen t the BMZ (rrowheds), s well s on some derml ells (rrows). () This is n immunolot of GST-NC16A fusion protein nd GST (designted N nd G, respetively) leled with either the unfrtionted BP-W7714 serum (left two lnes) or IgE purified from BP-W7714 (right two lnes). The seondry ntiody used for ll four lnes ws horserdish peroxidse-ntihumn IgE. The two rrowheds to the left of the lot indite the positions of GST-NC16A (upper) nd GST (lower). IgE retivity with the GST-NC16A fusion protein is seen in the first nd third lnes. Identil results were seen with BP-G5554. Br ¼ 100 mm Journl of Investigtive Dermtology (2007), Volume 127

3 d d Figure 2. Humn skin grfts injeted with BP or ontrol IgE. ( nd ) Grfts injeted with 6 ng of BP IgE show well-developed urtiril plque (11/11). ( nd d) No elevtion or erythem ws seen, the ontrol grfts injeted with 6 ng of norml IgE. e f Tle 1. Summry of pssive trnsfer experiments Injetion No. of mie Inflmmtory plque BP IgE, 6 ng 11 11/11 BP IgE, 47 ng 3 3/3 1 BP IgG, 1.25 ng 4 0/4 Control IgE, 6 ng 9 0/9 Control IgE, 47 ng 3 0/3 BMZ, sement memrne zone; BP, ullous pemphigoid. 1 Two of the three grfts injeted with 47 ng of BP IgE developed histologil seprtion t the BMZ. Figure 3. Histology of skin grfts injeted with BP IgE. () The low-power view of humn skin grft injeted with 6 ng of BP IgE. Mild ongestion of the vessels is seen (rrow) nd grnuloyti infiltrte. () Are of dense infiltrtion showing the grnuloyti hrter of the infiltrte. () Two of the three humn skin grfts reeiving 47 ng of BP IgE split long the sement memrne zone. (d) Control grfts tht reeived norml IgE show no normlities. Immunofluoresent mpping with ntiodies to (e) BPAG1 nd (f) type VII ollgen indited the split ws predominntly through the lmin luid/lmin dens region. Br ¼ 100 mm. nd grnuloyti infiltrte, whih vried from sprse to more dense ner the mrgins of the grfts (Figure 3 nd ). The denser infiltrte ws generlly seen ner the mrgins of the grfts, where we hd previously noted n inresed numer of mst ells on Giems stin. At the higher dose of IgE, mirosopi seprtion t the BMZ ws seen in two of the three grfts (Figure 3). The histologil level of the lister ws mpped y indiret immunofluoresene using BPAG1- nd ollgen VII-speifi ntiodies. BPAG1 nd ollgen VII leling were deteted in the eiling nd floor of the IgEindued vesile, respetively (Figure 3 e nd f). This indited tht the level of the split ws in the lmin luid, the sme site s lesionl BP. Diret immunofluoresent stining showed wek fol deposition of humn IgE t the BMZ of the grft nd stronger stining on mst ells (Figure 4 nd ). No stining ws seen with nti-humn IgG or nti-murine C3 (Figure 4 nd d). Eletron mirosopy identified mst ells within the lesionl region of the grfts (Figure 5). Mny of the mst ells ontined empty grnules, inditing reent degrnultion. In ddition, eosinophils, neutrophils, nd lymphoytes were lso identified in the infiltrte (Figure 5 nd ). DISCUSSION Historilly, the primry role for IgE lss ntiodies ws in the immune response in llergi nd prsiti diseses. Reently, role for IgE nd mst ells hs een proposed in the regultion of immune responses nd in the development of utoimmune disese (Frossi et l., 2004). This hypothesis is

4 d Figure 4. IgE, IgG, nd C3 leling in grfts injeted with BP IgE. Following hrvest of the grfts 24 hours fter injetion with 6 ng of BP IgE, ryosetions were leled with FITC-onjugted nti-humn IgE, IgG or nti-murine C3. () Anti-humn IgE reveled wek stining t the BMZ. () Mst ells were lso leled y nti-ige s indited y doule leling with nti-humn mst ell tryptse. ( nd d) No BMZ stining ws seen with nti-humn IgG or nti-murine C3. Br ¼ 50 mm. supported y the reent identifition of IgE lss utontiodies in numer of utoimmune diseses, inluding Grve s disese nd Hshimoto s thyroiditis (Guo et l., 1997, Sto et l., 1999), 3); however, the pthogeni relevne of these IgE s remins unknown. Elevted levels of IgE hve lso een demonstrted in nti-ssa-positive women with lupus who experiene fetl loss, ut gin the speifiity of the IgE nd ny pthogeni role it plys hve not een determined (Sekigw et l., 2004). In ontrst, there is growing ody of dt regrding the speifiity nd potentil pthogeni role of IgE lss utontiodies in the orgnspeifi utoimmune disese, BP (Provost nd Tomsi, 1974; Delporte et l., 1996; Christophoridis et l., 2000; Dopp et l., 2000, Chen et l., 2002, Dimson et l., 2003). In this report, we showed tht injetion of BP IgE into humn skin grfts resulted in lesions tht repitulted the linil fetures of the urtiril lesions of BP, inluding the presene of degrnulted mst ells nd eosinophils. In erly BP, the ptients my hve lrge urtiril plques ut mnifest few, if ny, listers. Sequentil histologil exmintion of the skin of BP ptients during the development of individul lesions hs shown tht mst ell degrnultion is the erliest event followed y migrtion of lymphoytes nd lter eosinophils nd neutrophils into the lesion (Wintrou Figure 5. Eletron mirogrphs of the infiltrte indued y BP IgE. The infiltrte ws omposed of () mst ells, mny of whih hve empty grnules (rrows), inditing reent degrnultion. () Eosinophils, reognized y their hrteristi grnules nd iloed nuleus, were lso present, s were () neutrophils nd lymphoytes (dt not shown). Br ¼ 1 mm. et l., 1978; Dvork et l., 1982). Further evidene for mst ell degrnultion is the presene of eotxin in the lister fluid of BP lesions (Shrikhnde et l., 2000). The level of IgE used in these studies (60 ng/ml) pproximtes the level of IgE present in norml humn ser. The higher level (470 ng/ml) is slightly ove the norml rnge of IgE, ut pproximtes tht seen in lrger group of our BP ptients (Dimson et l., 2003). Thus, the level of IgE tht we re testing in these experiments is within the physiologi rnge. The IgE isoltion proedure used in the present investigtion yielded greter thn 10,000-fold purifition of IgE reltive to IgG. The level of totl IgG remining in the BP IgE purified preprtions ws in the rnge of 2 3 ng/ml. This mens tht eh 6 ng dose of BP IgE ontined roughly 0.3 ng of totl IgG. To determine whether this level of IgG ould e responsile for the oserved effets indued y our IgE injetions, we performed the sme type of pssive trnsfer experiments using 1.25 ng of IgG purified from serum BP- W7714. This key ontrol demonstrted tht BP IgG t this onentrtion, whih orresponds to pproximtely 4 5 times the mount of IgG present in the 6 ng dose of BP IgE, did not trigger ny detetle hnges in the skin grfts. This finding is supported y previous report y Zillikens nd oworkers (Zillikens et l., 2001), who injeted s muh s 400 mg of purified BP IgG (over 800,000 times greter thn the mount of IgG present in our BP IgE preprtion) into 2608 Journl of Investigtive Dermtology (2007), Volume 127

5 humn skin grfted onto SCID mie nd deteted no linil or histologil lesions. Another importnt ontrol tht we implemented ws to purify IgE from norml ontrol ser nd injet it into the humn skin grfts. This norml IgE ws purified using the sme protool tht ws used to purify BP IgE nd ws used t the sme dose s tht used for the BP IgE. The ontrol IgE filed to indue ny speifi hnges in the injeted skin grfts. This ontrol rules out the possiility tht the oserved hnges in the skin grfts indued y our BP IgE injetions were non-speifi effets of IgE or elements resulting from the purifition proedure. Bsed on the results of these ontrols, it is ler tht the hnges tht we oserved in the humn skin grfts n e ttriuted to the IgE from the BP serum. Although not diretly tested in this study, it is proly tht the IgE ntiodies responsile for the oserved pthologi response re direted ginst the kertinoyte surfe protein, BP180, or frgments of it formed y metoli degrdtion. It is well estlished tht high perentge of BP ptients produe nti-bp180 utontiodies of oth IgG nd IgE isotypes. Our reserh group hs lso reently shown tht these IgE utontiodies lrgely trget NC16A, the nonollgenous linker region of this ntigen s etodomin (Firley et l., 2005). Furthermore, ntigen-speifi degrnultion of irulting sophils from BP ptients n e eliited y tretment with the NC16A peptide (Dimson et l., 2003). We therefore hypothesize tht the mehnism for the pthogeni retion oserved in our present humn skin grft model involves the inding of the injeted BP IgE to the IgE reeptor on the surfe of humn derml mst ells, nd degrnultion of these ells fter inding of the surfeound IgE to its ntigeni trget, thus relesing histmine nd other inflmmtory meditors. The reltively wek stining for IgE t the BMZ in the BP IgE-injeted grfts when ompred to the stining seen on mst ells would support this hypothesis. The stronger stining seen on the mst ells my e due to higher ffinity of the IgE for its reeptor on mst ells thn for the BMZ. It is known tht lrge stle frgment of the BP180 etodomin is shed from the kertinoyte surfe (Shke et l., 1998; Shumnn et l., 2000). This 120 kd shed form ould well serve s the ntigeni tivtor in this pthogeni proess. The rpidity with whih we sw retion (less thn 5 minutes in the high-dose grfts) oupled with the eletron mirosopy findings strongly support mst ell degrnultion s the mehnism for the erly-phse lesion development in the grfts. In ddition to mplifying the inflmmtory retion, the tivted mst ells lso relese proteses tht ould well ontriute diretly to derml epiderml seprtion. Another possile omponent of the BP IgE-medited response my involve the inding of IgE to BP180 on the surfe of sl kertinoytes. This inding ould impir BP180 funtion diretly, or, more likely, initite proinflmmtory signling sde. One onern with this senrio is tht IgG would out ompete for the ville inding sites on BP180, diminishing the importne of IgE utontiodies. Another is tht the stining for IgE seen in our model ws wek ompred to tht on mst ells. Our reserh group hs reently initited study direted t the quntifition nd omprison of BP180-speifi IgE nd IgG levels in BP. In the mteril utilized in this study, the onentrtions of irulting BP180-speifi IgE nd IgG were similr (6 9 nd 24 mg/ml, respetively; unpulished oservtions), lthough the reltive vidities were not mesured. More work is needed to determine the plusiility of this type of IgE-medited effet on kertinoytes in the pthogenesis of BP. Complement fixtion t the BMZ, hrteristi immunopthologi finding in BP, ws one feture sent in our BP IgE-injeted grfts. Sine IgE does not tivte omplement, this ws not unexpeted. Clerly, IgE lone is not ple of repliting ll of the pthologi fetures of BP. Previous work from our lortory nd others point to IgG lss utontiodies direted ginst BP180 s the trigger for omplement fixtion t the BMZ nd for the susequent reruitment of inflmmtory ells involved in derml epiderml seprtion (Liu et l., 1993, 1995; Chen et l., 2002; Sitru et l., 2002). Just s in the se of IgE, it is lso ler tht IgG lone nnot ount for ll of the pthologi fetures of BP. Conspiuously sent from the IgG pssive trnsfer model re the key hnges ssoited with the urtiril stge of BP. It is highly likely tht the linil nd immunohistohemil mnifesttions we see in ptients represent the effets of oth IgE nd IgG lss utontiodies. In previous report, we demonstrted tht the fine speifiities exhiited y the IgG nd IgE lss utontiodies re quite similr, nd thus re likely to hve risen vi lonl seletion (Firley et l., 2005). After n initil humorl immune response, whih is typilly IgM, ytokine-medited DNA reomintion events n our leding to swith in the isotype, while lrgely mintining the ntigen-inding properties. Sutle hnges in the inding ffinity to the trget ntigen n our during isotype swithing vi somti hypermuttion of the omplementrity determining region (Chudhuri nd Alt, 2004). Owing to the hromosoml positions of the g nd e genes, lss swith from IgG to IgE is possile, while the reverse is not. Prodution of IgE requires the ytokines IL-4 nd -13 s well s ligtion of CD40 to CD40L on B ells (reviewed in Prussin nd Metlfe, 2003). The findings of this study represent erly evidene pthogeniity of IgE utontiodies. We showed tht injetion of BP IgE into humn skin grfts resulted in lesions tht produed urtiril plques hrterized y the presene of degrnulted mst ells. The degrnultion of mst ells repitulted the erliest events of lesion development reported y previous investigtors (Wintrou et l., 1978; Dvork et l., 1982). The presene of pthogeni IgE utontiodies my e inditive of the type of initil trigger whih leds to this disese or the mnner in whih the ntigen is presented to the immune system. BP provides unique opportunity to study further the role of IgE in utoimmunity. These findings my lso open up new res for potentil therpeuti intervention in BP or other humn utoimmune diseses

6 Addendum: While this pper ws under review, Nishie et l. (2007) hve pulished n dditionl murine model of BP. This model utilizes humn BP IgG in humnized mouse tht expresses the humn BP180 protein. Dethment of the epidermis ws seen in nimls injeted with BP IgG; however, like other models, the urtiril plques nd eosinophili hrteristi of BP were sent. MATERIALS AND METHODS Chrteriztion of IgE nd IgG in BP nd norml ser This study ws pproved y the Humn Reserh Review Committee of the Medil College of Wisonsin Affilited Hospitls (no ) nd the VA Medil Center nd ws performed in dherene to the Delrtion of Helsinki Priniples. Consent ws otined from ll ptients. Levels of totl IgE nd IgG were mesured y pture ELISA (Bethyl Ls, Montgomery, TX). Purifition of BP IgE nd BP IgG IgE ws purified from BP ser BP-W7714 nd BP-G5554 using multistep proess modified from Kleine-Tee et l. (1995). Serum ws diluted 1:1 in phosphte-uffered sline nd the IgG ws removed y multiple psses over protein G ffinity olumn (Piere, Rokford, IL). By indiret immunofluoresene, the titers of IgG direted ginst the BMZ went from 1:2,560 nd 1:1,280, respetively, in the strting ser to undetetle following the protein G olumn. The resulting IgG-depleted frtion ws inuted with n e-hin-speifi monolonl nti-humn IgE oupled to Affi-Gel HZ or Affigel-10 (Bio-Rd, Herules, CA) nd eluted with 25 mm triethylmine ph 11.2 with 1% BSA, nd immeditely neutrlized. The IgE-ontining elute ws extensively dilyzed ginst sterile phosphte-uffered sline nd onentrted. BP IgG ws purified y ffinity hromtogrphy utilizing protein G ffinity olumn (Piere). The IgG ws eluted with 0.1 M glyine ph 2 3 nd immeditely neutrlized with 1 M Tris ph 8.0. The IgG ws extensively dilyzed ginst sterile phosphte-uffered sline (ph 7.4). The IgG nd IgE onentrtions were determined y ELISA (Bethyl Ls). Injetion of BP IgE into humn skin grfts These experiments were pproved y Animl Studies Suommittee (VA P). A 2 2 m re of skin ws removed from the k of 6-week-old nu/nu mie. A similr sized piee of humn skin otined from redution mmmoplsties ws grfted onto the k of the mie. After 14 dys, the expression of BMZ proteins in the grft ws tested y indiret immunofluoresene utilizing ntiodies direted ginst ollgen VII (gift of Drs Mei Chen nd Dvid Woodley, University of Southern Cliforni), lminin 5 (gift of Kim Yney, Medil College of Wisonsin) or BP180 (Liu et l., 1995). The presene of mst ells in the grft ws determined with Giems stin, nd their humn origin ws estlished y diret IF utilizing nti-humn mst ell tryptse (Chemion, Temeul, CA) nd tehniques s desried previously (Dimson et l., 2003). One hundred miroliters of BP IgE (60 ng/ml, n ¼ 11 or 470 ng/ ml, n ¼ 3) ws injeted into 14-dy humn skin grfts on nu/nu mie. Controls inluded norml humn IgE purified y our lortory in the sme mnner s BP IgE (n ¼ 9). Non-speifi humn IgE ( Oslo, Norwy) ws lso tested t the high onentrtion, 47 mg per grft (n ¼ 3). The ommeril IgE ws used for the more onentrted injetion, sine onentrtion of the IgE we isolted from norml ser would lso hve inresed the onentrtion of ny ontminnts from the purifition sheme tht ould ffet our injetions. The grfts were hrvested 24 hours fter injetion nd fixed for routine histology, diret immunofluoresene, nd eletron mirosopy. Eletron mirosopy ws performed in the Eletron Mirosopy Fility t the Medil College of Wisonsin. CONFLICT OF INTEREST The uthors stte no onflit of interest. ACKNOWLEDGMENTS This work ws supported in prt y Merit Review Awrd from the Veterns Administrtion (2655-2, to Dr Firley), nd y grnt from the Ntionl Institutes of Helth (AR040410, to Dr Giudie). 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