Interleukin-tp-Induced Inhibition of Hair Growth
In Vitro Is Mediated by Cyclic AMP
R o lf Hoffmann , Wolfgang Eich elcr, Elke W enzel, and Rudolf H appl e
D e pa rtlll c nt of D c rma to logy. Philipp Uni ve rsity. M a rbuq,;. Ge rman y
Interleukin (1L)-1 has been shown to be a potent
inhibitor of hair growth ;11 .litl'o. We hypothesized
that t his cytokine ITlight be a decisive fa ctor causing
hair loss during the IYITlphocytic attack in alopecia
areata. Neither the intracellular pathways involved in
hair growth inhibition ITlediated by IL-1{3 nor the
signal transduction processes within hair follicles in
general are known. We therefore investigated the
intracellular signals involved in hUluan hair growth i'll
lIitm. Hair follicles were isolated froITl scalp biopsies
by ITlicrodissection, and hair growth was ITleasured
daily by image analysis. We assessed intracellular
signa) trans ducing eleITlents using specific inhibitors
or activators either alone or in combination with
IL-1{3. The calcium ionophore A 23187 induced a
rapid and cOluplete arrest of hair growth, and phor-
boI-12-myristatc-13-acetate (PMA). genistein, or
acto rs t,ha t illAuen ce the g ro wth of the <l na gen fo lli cle o r
F ini t iate the sw itch to a ca ta gen growth pa ttern h ave n o t
bee n di scove red , but the re is in c reasin g ev id e nce that
cy tokin es and g row th f;lc to rs arc in vo lve d in these
processes (Philpott l'f (/1. 1990) . In lin e w it h thi s co nce p t,
in te rl e ukin (l L )-l has bee n sho w n to be a potent inhibi to r of ha il'
fo lli cle g rowth ill "ilro (H arm o n a nd N ev is. 1993). M o reove r.
tran sge nic mi ce tha t ove rcx press in te ri e ukin (I L) - 'I Cl' in basal ke ra-
tin ocytes characte ri stica ll y sh ow patc h y hair loss . 1 Beca use IL-'\ {:3
was fo und to be a he rra n tly expressed in active alo pcc ia <Hea ta
(H o ffill alln ef (/1, 1994 ), w e ha ve h ypo th csi zc d that IL- 1 {:3 migh t Icad
to h air loss durin g t he lymph ocytic a ttac k in alo pccia arc,' ta
(H o ffm ann and H appl c , 199 5), If this h ypothesi s ho ld s tmc , an
effective treatme nt o f alo pecia arcata sh o uld a ntago ni ze les io nal
I L- l J3 o r an tagoni ze I L- J J3- cli cited in trafo lli c ular path w ays leadin g
to hair loss. T he intrafo llic ular sign al transdu c ti o n path ways in -
vo lv cd in I L- I J3-medi ate d arrcs t o f hair g rowth a re unkn o wn, bU l
it is poss ibl e to pharm aco logica ll y mani p ul a te th e sig nal transdu c-
tio n processcs to id e nti fy the pa th ways that a rc : (i) res po nsibl e fo r
IL-1 J3-111 edi ated inhibi tio n o f hair g rowt h , (ii) rcquircd fo r aug-
m e n te d o r pro lo nged h air g row th , and (iii) in vo lve d in chan ges in
M a nu script rece ived D ec e m be r 29. 1995; revised Se ptem bc r 3. 19\)6;
accC).Jted fo r publi Glt io n Se pte mb e r 30. 1996,
R e prin t re qu ests to: D r. Il1 cd, R o lf H o ftillann. Zc n t rulIl flir Haut-
k ..,1I1 k h c ite ll. Phili p ps-U ni vc rsitiit, J) c u tsc hh a usstr" sse I). 35 033 Marburg .
G Crll1 a n y.
Abbrev ia tio n s: d bcA MP . dihut yry l-cA MP ,
I G roves l'l W. Wi lli am s IR . S" rb r S. Naka mu ra 1< . I<up pe r TS: An a lys is
or c pidc rnl :d 11...- 1 f;,1Il1il y In c lnbc rs ;0 lI i l'o us in g t ra nsgeni c 111 0 USC 111 ud d $.
J /II llrs/ Dc'flII (/lo / 1()2:556 . 1994 (a bstL),
0022- 202X/97/S I O,SO • Cop y ri g ht © 1997 b y T h e Soc iety fo r Ill ves ti gati\'e De rm ato logy.
40
IL-1{3 decreased hair growth by approximately 60%-
80 IX,. IL-1{3-elicited hair growth arrest was not antago-
nized by calphostin C, a specific inhibitor of protein
kinase C. In contrast, coincubation of IL-1{3 with
pertussis toxin or H 1004 neutralized the effect o f
IL-1{3, and dibutyryl-cAMP and cholera toxin, an
activator of adenylate cyclase, inhibited hair growth.
These data suggest that cAMP acts as a second m.es-
senger for IL-1{3-induced inhibition of hair growth.
Moreover, our data indicate that;II lIitm hair growth is
dependent on intracellular Ca 2 + levels and activation
of tyrosine kinase as well as protein kinase C. We
were unable to detect a signal transducing eleluent
responsible for enhanced hair growth ill lIitro. Ke},
lVo vds: ""ilia II/alopecia al'eata/cytok;IIes/adellyiate cyclase .
] IIIlIest DeI'lIIatoi 108:40-42, 1997
m o rph o logy o f hair fo lli c k s ill lIilro , With thi s approac h w c w antc d
to elu cid ate sign al transdu cti o n pa th w ays ill sin gle human hair
fo lli clcs afte r in c ub ation with I L- I J3 and trall sdu c ti o n c le m e nts t hat
arc ge ne rall y impo rtan t fo r hair g ro w t h ill lIil 1'0 ,
M AT EI'l IA l S AN I) M ET H O D S
C h c nli c al s L-G lutalllin l! . il1 : < ulin. rra nsferrill. so diuI11 scl e lli tl.! . F Ull g i-
zo ne . pe ni c illin , strepto lll ycin . ami ph o rb o l es ter (PM A) were frolll Sig m a
(Dc isenh o fe n . Ge rm a n y) , Itec o l1lbi na n t hUlllan Il- I i3 was b o ng ht 1;'0 111
Labosc r v (G icSSCJl , Gc nn a n y) . Will ia nls E Ill Cdiul1l \v a:-; purc ha :-;c d fr o n l
G I13 CO (H e ide lbe rg. Ge rman y), Ca lph os tin C. Ill e litt in , U 73 122 . p rn p 'III O-
10 1. perruss is tox in . c h o lera toxi n . dibuty ryl-cA MP (dbc AMP) . lll e th ), le n e
bill e . d ibu ty r yl-cG MP. cth yle neglycol- bi s(i3-a mill o eth yl e th e r)-
N .N .N ' .N'-tetraaceti c acid (EGTA) , calcium io no ph o r e A 23 11:17 . I<.T-
5823 , C2-ce ralllide. sphin g ios in e- I-ph ospha te, ,nld ge ni ste in we re o btain e d
fro l11 131 0 M O l (PI Yl11 o u th M eeti ng. PAl,
Isolation, C ultivation, and M a intenance of Single Hlunan Hair
F ollicles Afte r info rm e d co n se n t h ad b ee n o bwin ed . excisio nal scal p
bi o psies we re take n unde r loca l an csth es i" li'o lll 20 h ca lt h ), vo lun tcers,
Ill tact. v iable a ll agc ll h ~lir fo llicl es were iso la te d b y Ill ic ro disscctio ll as
p rev io usly d escribe d ( Philpott ('I ,, [ . 191:\9. 1,)1)2), I" b ri" f. sCll p sp "c im c n s
were p lace d in su p pl e lllen ted \ V illialll s E l11 ediulll in petri d ish cs, U nd er a
stc n ..:o- cii sscctill g mi c rosco pe, ;l sca lpel blad e w as lI scd to rClll Ovc ril e
c pi dc nni s and uppe r parts o f th e co riulil . T h e inta c t h air fo lli cles We re
iso la te d fro nt t he SUbClI tilll CO LI S f:H w it h tI w atc hlliak c r 's fo rceps h y ge ntl y
g rippin g th e o ute r rOO t she at h o f rh e hair fo llicl e w ith s ubsequ e nt gentl e
t ra crio n , The iso la te d sin g le h a ir fo llicl es did n ot show v isible d amage a nd
we re Ill ailltaill c d in SU O J-LI of W illi a m s E Ilw diulll co n ta inin g 2 111M
I.-glutamin e, in sulin (10 }J.g pc r ml ), tran sfe rrin (1 0 }J.g p e r 1111). sodium
sel e ni te (10 n g pe r 1111) . Fun g izon e (2,5 p..g p er 1111). an d pe ni c illi n /
stre pto m yci n ( 100 U p CI' 1111 : 1no }J.g p" r ml ), Fo llicl es we re in c u b atcd in a
hUlllidifi e d a tlll osph e re of S'y" CO , /9S% air an d we re ke p t li p to 6 d ill
III C
VOL. I nK. NO. I JAN UA ltY 1 ~~7
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COlll ro l A 23 187 EGTA PMA IL-I fl Gcni slein IL-fl plus
Genistein
F ig ure 1. Inhibition of human h a ir growth ill "iII'O. Six sill gle hair
fo llicles wcre ill cubated with a givell sllhsta ll ce for 6 d. Th e Iellgth or th e
hair fo ll icles w as 111c a s lln.:d b y digital illl agc anal ),sis. and rh e g rowth ra te
was co mpared 10 cO ll tro l hair lo lli ck s ( IOII'X,). i1l1m ed iate in hibi rion of hair
growth was l! li cired by the ca lciulI l iOll oph orc A 23 1H7 (2 /.LM). Ph orbo l
l!stcr (I'MA. I /.LM). ge llisteill (50 flM). or IL-I /3 ( 1110 IIg per ml) inhibited
hair g ro\tvt h ain l O S[ to dw ~;l111 C d egrce . b y app rox-inliltd y UOIXI. afte r () d in
c ul t ure . I L-I {3-c li citcd a rres t of h air WilS tl ug lllc llred by co in Clibatlo ll wit h
gl! nistcin. EGTA ( I flM) had 11 0 "fl'l!cr O il hai r growth. EX l'erinll!lI ts were
perfon ll ed ar Ieasl ill trip li cate. Vailles are mea ns ::+: SD (htln).
c ul ture. At th e begillnill g of till! ex pl! ril11 ellts all hair fo lli cl es we re nl!a rl v
eq ua l ill leng th . T h e I..: lIl t un.! l1l ed iu l11 was c h an ged every 3 d . a ll d v ia hili r;,
of hair fo llicl es w as assessed by lig hL III ic rosco p y and da il y 1l1 C aS lIrC lll C il t o r
hair growt h.
A nal ysis of Sig n a l Transd u ction Pathways in Sin gle Huma n Hair
Fo ll icles I-lair folli cl es wc re kl!p t ill 2·1-ln lll tiwe ll plates at Oll e tn ll iclc per
w,, 11 ill SOO I.d or supple lll ellted "Villi ;lllls E mediulll . Every expe rilll ent lo r
a g ivcll sub.'\tan ce includ e d six sill g le hair fo llicl es ti'O lll o ll e dO ll o r an d \vas
pcrfonned at least ill t ri pli ca te. D ail y IIl L'a SlIrCI11 Cllts of hair growth w e rc
pcrfo nll cd by t h e li se of :111 illv c rt ed Illicroscopc conn ected to a v ide o
ca mera all d a digita l il1 1agin g process in g llni t (L lI cia M. Nik oll. Diisscl dorf.
Germany). O ur l!x l' eril11 ell t:l 1 obj ecrive was (i) to amagollize or (ii) [0
silllu late the IL- I/3 efl;'cr by the lise of either inhibi to rs or :1crivato rs of
sig ll a l -tra n s dll c il1 ~
e lClll CJl ts wit h in hair fo ll icl es. In h ih ito rs were lIsed
toget h er wit h I L- I (3: ;H.: ti v;,tto rs wcre ll sed al o ne. O IlC ho ur afrcr isolatio n
rh e h:1i r fo lli cles. the slIhstall ce, we re added to rh e cul ture dishes. T he
cxpcri nl c n tS we re sl'o lJPcd a ft c r 6 d .
RESULTS
Inhibition of Human Hair Growth ill V i tI'/) Hair g rowth was
imll1ed ia te ly inhihi ted b y ca lc ium io n op ho re A 23 187 (2 J.LM)
w h ere"s EGTA ( I J.LM ) h ad n o ellect o n hair growth . PM A ( I J.LM)
il n d IL-1 {3 ( I J) n g pe r ml) in hibi te d h a ir g ro wth n ot immediatel y.
but slow ly and n ea rl y eq u ,dl y. by approx im ate ly GO'X,. after 6 d in
c u lture. Ge ni ste in (5 () J.LM) inh ibi ted h a ir g ro wth b y approx ima te ly
80% and a ug m e n ted th e I L- I /3- e1i c ited h air g rowth inhibi tio n . (Fig
1). T reatm e n t of h air fo lli cl es w ith propan o lo l ( I J.LM) , KT 5823
(0.5 /.L M) , dibuty ryl- cG MP (II)() J.LM). C2- ce rami de (0.5 J.LM).
sp hingios in e- l-ph osp ha te ( I J.LM). m eth ylen e blu e ( I J.LM), U
73122 (1 J.LM). o r ll1 e li tt in CI J.Lg pe r ml) alo n e or in co mbin atio n
w ith I L- I /3 h ad n o e ffect o n ha ir g rowth ;11 /' ;11"0 (data n ot shown) .
T h e m o rph o logy of th e hair fo lli cles w as n ot altered b y an y
treatlllent (data n ot sho wn) .
IL-1J3-Elicited Hair Growth Inhibition Is Mediated b y
cAMP T h e I L- I /3- e1 icite d h air g rowth arrest was n ea rl y -0 111-
plete ly antagon ize d by additio n of pe rtu ssis toxi n (,lO t) n g pe r 1111) o r
H 1004 (2 J.LM) into th e c ulture d ish es but was n ot influ e n ced b y
ad diti o n of ca lp h ostin C (0.1 J.LM). W h e n use d alone, the c AMI'
SIGNAL TRANSDUCT ION AN I) HA li t C l tOWTH I," I Tmo 41
120
, 0
e
a.!::: 100
8
'0
80
"i;l0-
0
u
60
'>
'" 40
ED
20
.iii
~
Control IL-Ill IL- Ifl + IL- lfl + dbcAMP CTX IL-Ifl +
PTX Calpho. H 1004
Figure 2. IL-lf3-elicited hair growth inhibition is mediated by
cAMP. In cuh;lti on of hair f" lIicles with IL-I !3 (100 ng per ml) led to
t
consistellt inhi b it io ll of hair growth , by apprOX il11:lrcl y 60 X" cO Jllparcd to
llnstinlld ared hair fo llicles. T hi s growt h inhi hition was nea rl y co mpletely
anrago ni zed by addition ot pertussis tox in ( 100 ng per ml. PTX) or H I tJO~
(2 flM). \Xfhcn lls"d al one. th e cA MP ana log dhcA MP (501) flM) or th"
adcnyia te cyclasl! activator cholera tox in ( I flf!. per 1111. CTX) in hib ited hair
growth ;/1 ";/,.,, to rh e same degree as IL- I/:l. Co in cllbntion at IL- I /3 with
ca lph ostin C (0. 1 flM , C alph o.) did not in fluen ce inhibition of lwir gro wth.
ExperilllCllts w crc pc rfo l'Tll cd at le ast in rr i p li L~Hc . VJ lu cs arc In C<lnS :!: SD
(bars).
an a log, db cA M P (500 J.LM) o r th e aden ylate cyclase ac ti vato r
c h o le ra toxin ( I J.Lg p e r m l) inhibi ted hair growth as d id IL- I {3 (Fig
2).
D ISCUSS IO N
Th e e xa c t m ec h a ni sm o f I L- l sig n a lin g in h a ir fo ll icles is
unkn ow n . l3 eca u se of its p le iotr op ic efl'Ccts, I L-I ut ili zes difle r-
e nt sig n alin g pathwa ys (Foxwe ll cr al. '1992) and h as been sh ow n
to act iva te. e.g., p h osp h o lip ase: A2 (C h a n g er ai, 1 986). p rote in
kina se C (PKC) -. cA MP-a ctivated p rote in kin ase: -, an d cGM p-
ac ti v ate d pro te in kina se- d e pendent sig n a l tra n sd u c ti o n pat h ways
in va ri o u s ce ll s ( R.osofF I'r al. 1 988: l3 eas ley 1'1 al. 199 1).
or (L eszcy n ski 1'1 al. 1 994). Fu rth ermore, ce ra mi de a n d sp h ingomy-
elin. w hi c h arc link ed to ph os p ho li pid h yd ro lys is. h ave b ee n
id e n titi ed a s m e diat o rs o f so l11 e of th e effects of IL-1 {3 (l3a ll ou CI
ai, 1992; Mathia s ('( al. 1993).
As reported t'O r oth er ce ll s (Sh i"i bwa 1'1 al. 1988; O n ozak i CI ai,
1992), o ur data suggest that I L- I f3-e li cite d h air grow th inhibi tio n is
m e dia te d b y cA MP . T hi s co nclu sio n is ba sed o n th e fo ll owin g
pie ces of ev idence: (i) IL-I- e licited h air g rowrh inhibiti o n is simil ar
to h a ir growt h inhibiti o n evoke d by th e cAMP an alo g db cA MP, (ii)
n o n toxic co n ce n trati o ns of th e ad eny latc cyc lase inhibi to r. pe rtussis
tox in. inhibi ted the IL- I f3- in d ll ce d arreSt of h air g rowth . (iii)
n ontox ic co n ce n trarions of the cAM P-depende nt prote in kinase A
inhibito r 1-11004 an tago ni ze d th e IL-l /3-ind ll ce d arrest of h ai r
g1'Owth. (iv) " cA M P-c1 evatin g agent (ch ole ra toxi n ) co uld substi-
tute for I L- 1 in inhibitin g h air growth . Due to th e lim ited numb ers
of h air fo lli cles prese nt in sm all scalp bi ops ies o btain e d from h ea lth y
vo lun teers, we we re u nable to m eas ure cA MP leve ls o r adelly late
cyc la se ac tivity in iso la ted h air fo ll icles.
It h as bee n h yp o thesi ze d that IL-l mi g h t be a cru c ia lm cd iator in
th e pathop h ys io logy of va ri o u s di seases acco mpani ed b y hair loss
and a regul ator of the cyc li caJ h a ir g rowth pattern ( I-lann on and
N ev is. 1993). IL- ln and IL-I{3 share cO lllm o n rccepto rs. an d th e
biol ogic fun ctio n s arc indistin g ui shable . T h e m ec h anisms wi thin
th e: h ai r fo lli c le th at arc evoke d b y I L- I and th e I L-I-res p o n sive
42 HOFFMANN ET AL
hair fo ll ic le ce ll s are no t knovvn , but an inhibitio n o f m;ltrix cell
d ifferen tiatio n and pro life rati o n has been ass umed (Harmo n and
Ne vis, " 994). W e performed ex perim en ts w ith hair fo llicles that
we re iso late d by a techniqu e similar to that d esc/'ibed by p/'ev ious
aut ho rs (Westgate ('I (/ /, 1993) . whi ch repo rted a co ntinu o us m ;ltl·ix
cell divi sio n and format io n of ke ratini ze d hair shafts, It has bee n
shown chat IL-l is able to inhibi t hair loss induced by cytos ine
arabin osid e Oimen ez ct (//. 1991). T hi s cy tostatic d ru g interferes
w ith n orm al DNA synth es is and is th ere fo re selec tively tox ic to
hi g hl y pro li feratin g cells. In t hi s situ atio n I L-l may inhjbit l11 atrix
ce ll differe ntiation and thi s, in turn , protects the hair follicl e frOI1l
the toxic effects mediated by th e che m otherap e uti c agent.
We were unable to onhem previo us studies showin g that
in creased PKC activity a ugm ents th e I L-l (3-i ndu ced arrest of hair
growth (H arm o n and N evis, 1993). In o ur hands, l L-l {3-c1i cited
inhibi tion of h air growth was ind e penden t of PKC activity, sin ce
add itio n of a se lective inhibi to r of PKC , ca lph ostin C, to lL-l {3-
stimu late d hair fo llicles wa s w ith o ut eHect o n hair growth . N ever-
th eless , o ur data sugges t th at PKC ac tiv ity is impo rtan t to m aintain
ha ir g rowth ;11 ,,;tro, b eca usc PMA stimu lation led to a significa n t
g r owth inhibiti o n . PMA is known to activate PKC transiently in
seve ral cells, but prolo n ge d ex posure of cell s to PMA dow nregll-
lates th e activity of PKC . H e nce, o ur data su ggest th at decreased
PKC ~cti v ity is res po nsib le for in hi bition of hair g rowth . rath er th en
PMA ~e l ic i ted PKC activatio n as previously sugges ted (Harmon and
N evis, 1993). M o reover, o ur data indicate that hair gro wth ill lI it l"('
2
is d e pende n t o n intrace ll u lar C a /- leve ls and tyro sine kina se
beca u se A 2 3187 treatm en t res ulted in an immcd iate and comp lete
arrest of hait· g /·owth . wh ereas ge niste in inhibited hair growt h to a
lesse r d egree. In Ollr system, the inhibition of otber possibl e
sig nal-transd uc in g ele m e n ts, such as phospholipase A2, g uan ylate
cyclase. tyr osin c k inasc , o r pb ospholipa se C/ D, did nor antagoni ze
the I L- J /3--m ediated <lrrcst o f hair g rowth .
O ur results pro v ide evide nce t hat con titu eivc acti vatio n of
seve ra l intracellular sig na l- tr ansducing cle m e nts m aintain s the
growth of sin gle human h air fo lli cles ill vitro and that inhi bition of
hair growth by lL-l {3 is m ediated by cAMP. M o dulation of these
Hlctors ill vil'o m ay be a future too l for the manipulatio n o f hair
g rowth .
THE J OU R NA L or INVES TI G AT IVE I)E lt M ATOLOC;Y
--..,
TO
,,;s IIIo ri.! II'i1S slIpported /J}' ,(!rn IIlS );'(111/ 'h t' ! )(,IIfSfh t' Porscllllllgs.<!t'lIu·;llsr!/(!}t ( H o
/ 598/ / -2), /301111 , C n /ll " " )" '/11".1i·"/ll '''1" Krlll/,I,,·,< S' ij;III1.~ , ,\J " rbll/:~ . Cem"III)'.
IlEFE RE N CES
Ba ll ou LR. C hao C Po 1-l o ll\ \!~s MA. IhrkeI' SC, R ag h nw Ie IIl LCrlcllkil1-1 - lIlcdi;l tcd
PGE2 prod uclioll ;111 d spliill gOlll yclill1l1 ctaho li sll1. j !Jill! CI/C/II 2h7: 2110·14 -2l11\..l 9.
11)1) 2
B ca~' c)' D. Scll'.vartz JH . BrCIIIH.: t HM . Inl c rl c uki ll - I illtiu (;cs p ro lo ll ged L- argi nin e ...
dc pCllde lll" c ~'di c g:((a ll o5ittl! m Ollo phosph;lt\! and "in-ite pro du ct iu ll in r;lt V:1Sr llhu'
SlIl 00 lh IIlll sd c cells. J C'/ill III P('s r H7: ()02-60 H. t IJ I) 1
C lwlI g J. G ilm an SC. Lewis AJ . Il1lCdc ukin - 1 ;lC li v al c~ ph ospho lipase A2 ill rahhit
dlOlldrocytc:-: A possihlc Sig ll:ll ~~lr lL- l actiOll .j /111111 /(1101 13 6: 128.\- 12H7. 1986
Foxwe ll 13MJ. !3;llTc ll K, Fcld l11 ~11111 tV1: CylOki nc rccc pt(lt"s: srn lt"t \1 rc :md sil!l1 n\
transdu c ti o n. e lill I.::xp IlIInllll/oJ 90: I (, I - I (,9. I c)c)2 '
H armon CS , Nev is Tn: IL- l (f inhihits lU1ll1illl hair fo lli cle growth 11 1l1l hair lihrc
productio ll in w ho lc-orv;all (' lIif lircs. L)'Hll'lwk ill" Cy/n/.: ;IIf Res 12: 11)7-203. 1<)1)3
1-larm o l1 CS. Nev is TO : H air fibre produ c ti o ll hy hlll1 1;J 1l hair ro lliclcloo in whn lc \wgn ll
e ultllre Ilr./ [)IT"In,oI1 30"II S- ,123. I IN ~
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