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Quantitative studies on langerhans cells in mouse corneal epithelium following infection with herpes simplex virus
E.vp. Eye Re.~. {il~7} 4S~ i27-1~}
Q u a n t i t a t i v e S t u d i e s o n L a n g e r h a n s C e l l s in Mouse Corneal Epithelium Following I n f e c t i o n w i t h H e x e s S i m p l e x Virus S. J. L ~ w ~ o w ~ c z - M o s s * ¢ + C+ S H I ~ L ~ ¢ ,
K. L~I'WOaTH$. T. ,L HILLS,
Department~ ~ ¢ ()phthalmol~!! and +M iertdriol~!/, University ~0~ Bri~stol, Bristol, U.K. (Received 27 Octolmr 1986) l$~endritic ~ n g e r h a n s c~|ls (LCs) w e ~ identified in flat-mount pr~|mratk~t~s o f t n o u ~ t~)rneal epithelium a f a r staining for A ' F l h ~ activity. T h e y w e ~ found pn,~tominant|y in the limbus, buL after inoculating the com~=~ with HSV ! strain S(TI 6 LC~ numbers i n c ~ d both in the limbus and the ¢~ntral euJrnea. N u m ~ r ~ o f l~Cs n:ached a m a x i m u m on d a y 8 and if m v e ~ keratitis was pre.~nt ~ m a i n ~ high a t ~ s t until d a y 22. A small but, significant increase in i.~3s was al~) t~und in the op|~site, anit~oculat(M (~ye in micu2 with revere damage in the imw~ulat(~t eye. After HSV inoculation on the snout. 6 0 % of mi¢~ had cornea| disem*~ in the (~*e oil the i n o c u l a t ~ side; in such micuz corneal Lg.~'sw e ~ a t a tnaximum 18 d a y s after itmcu|atiom The iue~-a~se in LC n u m ~ m w ~ similar whether inoculation was into ttie (u~m~ea or i~l the snout. Aft~-r cornea! inoculation the c.||s w e ~ d i s t r i b u t e | fairly evelily over the (u)nmaI surface, with accumulations |imitM to epithelia| ult~:rs. However~ after inoculation on file snout, numerous clusters w e ~ e~.n over the epithelial surfa(~, often surroumi by epithelium devoid of LCs. Key u~rd.: ]~lngerhans t*|ts; c~ruea; herl~s simplex vita,s: viral keraiitis: dendt4ti~ (a+i|s.
1. I n t r o d u c t i o n
Dendritic cells in skin epithelium were f i ~ t described by Paul Langerhans in 1868. They have since bc~en identified ~ antigen presenting cells of bone marrow origin (Katz, Tamaki and Sachs, 1979; Stingte, Tamaki and Katz, I989)and are now known as Langerhans cells (LC). They can be e~uily distinguished fro~L epithelial cells by their high membrane-associa~d adenosine triphospha~se (ATPa.~z) activity (Mackenzie and Squier, 1975), and this histochemicai proF~rty hmu ~ e n u~
add~
c~r~s~)ndenc~ to U.K. T r a n s p l a n t f~ervi~, S o u t h m ~ d , Bristol B S I 0 5ND, U.K.
~ J 1 4 ~ 8 3 5 / 8 7 / 0 7 0 1 2 7 + 14 $~J.~9/0
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Ine~ ( ~ n d o n ) Limited
128
S.J. L E W K O W I ( 2 Z . M O S S ET AL.
o c u l a r LCs a f t e r a specific viral i n t ~ c t i o n h a v e n o t b e e n r e p o r t e d . W e n o w ~ p o r t s u c h o b s e r v a t i o n s following direct inoculation o f the cornea with herpes simplex virus ( H S V ) a n d following z o s t e r i f o r m s p r e a d ( B l y t h , H a r b o u r a n d Hill~ !984) o f t h e v i r u s t o t h e e y e a f t e r i n o e u l a t i o n o f t h e s k i n o f t h e s n o u t ( S h i m e l d , Easty~ T u l l o , B l y t h a n d Hill, 1985).
2. M a t e ~ a l s a n d M e t h o d s Animals Eight-week-old male outbr(~t Swiss white Bristol/2 mice (Blyth c t a l . , 1 9 ~ ) we~ur u ~ l t h l ~ u g h o u t . T h e ( ~ s w e ~ e x a m i n e d with a slit lamp and a n i m a l s with a b n o r m a l eyes w e ~ excluded. hu~ulation~ Animals were auaesthetised with sodium i~mtabarbitone a n d were inoeulattut by scarification t h o u g h 5 l d o f m ~ l i u m c o n ~ i n i n g I × l()~ p l a q u e - f o r m i n g units (Pfi0 o f HSV! strain S O l 6 (Hill, Field and Btyth, 1975) either on t h e left c~rnea ( T u l l o Shimeld, Blyth, Hill a n d Easty, t983) or on a s h a v e d area o f skin, close to t h e t i p o f tile left s n o u t (Shimeld e t a l , 1985). Control mice ~ t~u~at~ in a n identical way with a mock inoculum prepared in a similar way to t h e virus I)~)l b u t c o n t a i n i n g no virus. Examination of eyes and isolation of vir~a~ fi'om egewashin~ T h e p r o c ~ l u r e s for e x a m i n i n g m o u s e c~yes a n d i ~ l a t i n g virus from eye washings have b-e~n dcscribc~t previously (Tullo et al., 1983) e x c e p t t h a t in this s t u d y no corneal stains were used to assist t h e eye e x a m i n a t i o n s in case t h e y interfered with t h e s t a i n i n g o f LCs, l)emotmtration of Lai~erhans cells in corneal e~pithelium Mice wez-e killed Irv" an i n t r a j ~ r i t o n e a l injection o f sodium !~ntabarbitone~ The c~rneal e p i t h e l i u m from both eyes was r e m o v e d a n d proces~ql for A T P a s e e~tivi~, according to t h e m e t h o d o f J u h l i n and Shelly (1977) b u t using the modification su ~ d by Chaker, T h a r p a n d B e r g s t r e ~ z r (I984), w h e r e b y a d e n o s i n e - 5 - d i p h o ~ ) h a ~ (ADP, s ~ t i u m ~ l t from equine muscle Sigma grade IV) r a t h e r t h a n A T P was used as 8 u b s t r a ~ . F~yJq)ad epithelia w e ~ stained simultaneously with corneal st~zeimens. If the s t a i n i n g o f LCs in the footpad was consistently very pale or t h e b a c k g r o u n d excessively high, t h e n t h e a c e o m i ~ n y i n g corneal shc~vts we~e discarded, After s~aining, the corneal e p i t h e l i u m w ~ plac~d on t o slides basal ¢~11 la)~zr u p p e r m o s t , a n d the epithelium was f l a t t e n s ! by inaking t h ~ radial incisions a r o u n d t h e circumference. A p a t h y ' s m o u n t i n g m e d i u m ( R a y m o n d A. l ~ m b ) was u e ~ l)ar m o u n t i n g the eove~lip, which was t h e n ~ealed with nail v a r n i s h Positively staining d e n d r i t i c cells were c~unt~t in five limbal fields, (total area ~ 0"6! 25 ram2), a n d 10 c~ntral c~rnea| felds,
(to~l area ~ 1"225 mmZ); t h e r e s u l ~ are e x p
~u+ t h e n u m b e r of ~ l l s per m m L
S~t~stic~l analy.Ms N o n - p a r a m e t r i c s ~ t i s t i c a l test~ w e ~ u ~ l tbr analysis o f all t h e d a t a . T h e Wilcoxon ~ n k s u m t e s t were used ~ c o m p a r e g r o u p s o f d a t a , p ~ l ~ over t h e entir~ t i m e c o u p e enabling t h e m¢~ian a n d range of LC ~ u n t ~ for t h e inoculated g r o u ~ to be ~ m l ~ r e d with one a n o t h e r a n d with controls. T h e K r u s k w a l J , Vallis analysis of variance was used ~a ~ s t for h o m o g e n e i t y within a g r o u p a n d therefor~e ~ m p a r ~ c o u n ~ carried o u t at different times after inoculation. T h e level o f significance was ~ t a t P ~ 0"05.
Y
3,4.5,6,7.9
1,2,4,5,6,7,8,9 -1,2,4,5.6.7,8,9
Eye washings {days)
D.K.
9.K.
D.K,
3+ 4, 5, 6, 7+ 9+ D+K.
I~:~.,4, 5, fi, 7, 8, 9, ILK~ D.K. t, 2, 4, O, 6. 7 8, 9, O.K.
D.K.
D.K~
Clinical examinations (days)
s (4) 3 {2) |3 (2) 8 (4} t8 {4)
3 (4}
6 {2) ! s (2) ' I ! {4} ~ {8)
I ! {4) t8 {5)
4 {2) I5 {2} ' :~) {4) 15{4) 30 {t2) 18 (2)
4 {4) J~ {4) 30 {4)
Jt {2) 2~ (2} 13 (4)
t3 (4) 25 {3)
.2 (.1
18(4)
8 (2} I8 (2}
8 {7) is (4} 43' (4 }
LC studi~ on day (n) 2 {4) i J {4} 22 (4) ~5 {3} 2 (2) 1| (2} 22 {2) I1{5) 22 {4) ! ! (2} 3O {2)
C, cvrn~; S, s~out; V, HSVi S.31fi, ! × l0 s pfu; n, ~um~r of mi¢~e;M J . m ~ k inoculum; D.K. day on which mi~ were killed,
S
40
Y
S
M.I.
V
V V M.I.
~I.|.
V
ino~'ulum
S
S
~
3
C C C
10
N
C
2;5
2
C
~
1
laoeulation site
Number of mi~,e
gx~fiment mlm~r
Total number of mice w~ed, inoculation site, inoculum aml days on which ~,ariou.~procedures (eye ~z~s/~i~ls,clinical examination, amt Lav£erhans celt wunts) were ~rformed
TABLE l
:c <.
.
>
O
13:0
s.,i. I.E$~, K(}X$ I ( Z - M O S , , Is 3.
AL.
l~esults
G r o u p s o f m i c e w e r e inoeulatx~,d o n t h e cornea, o r t h e s n o u t w i t h v i r u s o r w i t h m o c k i t , o e u l u m ( T a b l e I). A t v a r i o u s it~tervals a f t e r i n o c u l a t i o n , e y e s w e r e e x a m i n e d f o r clinical d i s e a s e , e y e w a s h i n g s w e r e t a k e n f o r i s o l a t i o n o f v i r u s a n d c o r n e a l e p i t h e l i a l s h e e t ~ w e r e t a k e n f o r LC: s t a i n i n g .
SVatisticai ¢~neH!t.~is by the W n r~nk-;~nm te,~t ~0 ~"p~ded lanfferhans cell ( L C ) counts in control~ a n d %¢ter tion o f v i r ~ * on the cornea Nmnb,w ~*f tA's {Irt,up?
Ni,mber t~f ini(~,
hh~iian
Rangt~
Limbus 178
~18-2~
186
147~:t21|
44:1 227
2|11-764 ~q55 124-
Viv~s mm di~-a~.d (N I/I
62 ;t2 :~t :t4
V i r u s contralaiera| We , (i,')E
fi8
| 9:{
(',rot r,d (('} Mt,~.k im~t.uh~m {MY) Vireos dis~.a~, (VD} Virus non diseas~l (N I))
~t2 32
(',,n| ral cor¢~va 9 i)
:t4
~|~
5~94
ViMls v(mtraiatt,ra| v~'e ( E}
fi8
!2
3qt5
5hwk im~t-uhlm (MI)
;~1t4
2~36 4~42
~ta¢ is| |cat ~.~m~parislm
MI vso (' VI} vs. (" N1) vs. M| Nl) w, VI) {'E v:~, U
1'
< < < < <
0"115 O~IiH tHXI5 iHXH O'U!
M l w+ (+
~ +s+
V|) vs+ (+ Ni) vs. MI NI) v s VI) CE vs~ U
< {HMH < o,~2 < tH~*I ~.s.
t Ea ,h grm~p ~pr~,senis the I,C e~m~ts |,~u,l~t ~ver the el/t}~ time v~u~e,
Clinical ~ s e r v a t i o ~ s Corneal inoculation° S u p e r f i c 4 a l d a m a g e w a s ~ e n in ~ l e c o r n e a l e p i t h e l i u m o f all e y e s f r o m t h e f i r s t d a y artier i n o c u l a t i o n w i t h virl!s. T h i s e i t h e r r e s o l v e d w i t h i n 3 - 4 days or more usually p s~d to severe ulcerative keratitis by days 8-I0. Evidence o f s t r o m a l o p a c i f i c a t i o n a n d iris h y p e r a e m i a w a s a l s o ~ e n in m o s t m i c e f r o m t h e f i r s t or second day after inoculation but these signs of disease could be transient, S u p e r f i c i a l d a m a g e t o t h e e p i t h e l i u m w a s s ~ n in t h e m ~ o r i t y o f m i c e s c a r i f i e d through mock inoeulum, and approximately half of thc~ also showed small stromal o p a c i t i e s . H o w e v e r , b y t h e fi~urth d a y all e y e s w e r e n o r m a l . Snout inoculat{on. Zosterifcnnn s p r e a d o f d i s e a s e w i t h l e s i o n s o n o r a r o u n d t h e e y e w a s ~ e n in 9() ~ o f m i c e i n o c u l a t e d w i t h v i r u s o n t h e t i p o f t h e s n o u t . S i g n s o f c o r n e a l d i s e a s e , s u c h a s t h e ' g r o u n d g l a s s ' a p p e a r a n c e w e r e first o b s e r v e d o n d a y 5 a n d r e a c h e d a p ~ a k incidence2 o f 5 0 ~ o n d a y I0. O n d a y 7 s t r o m a l o p a c i t i e s | i o n w a s s e e n in a b o u t h a l f t h e m i c e . A f t e r s c a r i f i c a t i o n o f m o c k i n o c u l u m o n t o t h e s n o u t , n o e y e di was seen. Isolation o f virus f r o m eyewashings Corneal ir~mulation. V i r u s w a s i s o l a t e d f r o m all c y e w a s h i n g s f o r t i m f i r s t 2 d a y s a f t e r i n o c u l a t i o n ; tile i n c i d e n c e o f i s o l a t i o n d e c l i n e d o v e r t h e n e x t 7 d a y s a n d n o v i r u s we~s i s o l a t e d a f a r d a y 9.
|,C8 IN MOUSE
CORNEA
AFTER
HSgl
INFECTION
tal
F r o . I. A n e p i t h e l i a l s h e e t from a n e y e o f a [~)ntrol m o u s e . L a n g e r h a n s tu~ils (IX2s) a~a pre.~mnt m a i n l y in t h e l i m h u s ( r i g h t o f p i c t u r e ) o c c a s i o n a l ceils a ~ p r e s e n t in t h e t ~ n t r a } ~;ornca. l a m g e r h a n s cells welu2 s t a i n ~ l u s i n g a his~gehcmical t e c h n i q u e for d e m o n s t r a t i n g A T P a ~ a c t i v i t y . B a r = ~00 ~till,
60~
0
! J,,
days after ~ u ~
Fm~ 2~ T h e h u m o r o f ~ s o b s e r v e d in t h e l i m b s | c o ~ c a l e p i t h e l i u m a t v a ~ i o u s t i m e s a f t e r a c o r n e a l i n ~ u l a t l o n w i t h ! × l 0 s pfu o f R S V ! s t r a i n SC16~ T h e m e d i a n w i t h r a n ~ is s h o w n a n d t h e n u m b e r o f o b ~ r v a t i o u s is a b o v e e a c h ~ ) i n t , T h e c ~ n t r o ! v a | u ~ at*e s h o w n o a t h e v e r t i c a l axls~ ~ , diseas~ group; - - - . n o n - d i s e a s ~ g r o u p . T h e K r u s k w a l - ~ V a i l i s al~al:;sis o f v a r i a n o e w a s u ~ to e×amine the'~ data. Sigififieant d i f f e r e n t ~ s : d i ~ a ~ d g r o u p , P < ~ O I ; non-dise~e~d g r o u p P < f f O l .
132
S.J. LEWKOWICZ-MOSS
6~
E
ET AL.
4 3
400
Q U
0
20 days Offer ~
a'0 |
£o
.......
5'o
~
Fm~ 3. Tlm number o f ~ n g e r h a n s ~ | l s o b ~ r v e d iu the central c~rneai epithe|ium a t various times after a eort~ea| inoculation with I x 10Spfu H S V I S~2|6. The media~l with ra~*ge is shown a~d tile n u m b e r o f o b ~ r v a t i o n s is abe, r e each point. The ~ n t r o l valu¢~ a ~ shown on the vertical axis~ d i s e ~ d group; - - - ~ a o n . d i ~ d group. T h e Kruskwai-X,Vai|is analysis o f v a r i a n ~ was u ~ d to examine t h e m d a ~ . Significant diffe~3n~s: d I group~ P < 0 ~ ! ; n o ~ d i g r o u p P < ~01.
Fxo. 4. An epithelial s h ~ t from an eye o f a m o u ~ 65 d a y s after a c o r n e l i n s u l a t i o n with i × I0 ~ pfu HSV ! ,c~16. Bar = 2 ~ ~m~
LCs IN MOUSE CORNEA A F T E R HSVI I N F E C T I O N
I~
IIIII ¢0~II |IIIIII|
I~O~t
800 limbus 600
400
| I
~
P ~
0
I|~II
O~O0~
P ~
0.001
700
500"
!l
central cornea 200
100
....
........
l
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
Fro. 5. The number of ~ s observed in ¢o~aeal epithelium after inoculation ~ith 1 × 10s pfu HSV ! SC16 on either ~ e snout or the c,3mea. The histograms m p ~ n t the m ~ i a n number of ~lis o b ~ r v ~ between days 8 and 34 after snout inoculation and betw~n days 8 and ~ a ~ r ~rneal inoculation. The range and number of observations a ~ shown° Differences ~ t w ~ n ~ u ~ were t e s ~ with the Wtleoxon rank-sum t~st~ ~ v e l of significance~ P ~ G~05. +, Comp~son n virus, snout, n o n - d t ~ z ~ and virus, ~ r n e a non-di~a~.l.
Snout inoc~ation. V i r u s w a s first i s o l a ~
4 days after insulation. The ~ak i n c i d e n c e o f i s o l a t i o n w a s 7 5 % on d a y s 5 a n d 6, a n d n o v i r u s w a s i s o l a t e d a f t e r d a y 8.
La~e cell studies Courding error. T o e s t i m a t e t h e c o u n t i n g e r r o r , six c o r n e l e p i t h e l i M sheet,s w e r e c o u n t e d on five s e p a r a ~ o c c a s i o n s : T h e s h e e t s w e r e t a k e n f r o m o n e u n i n o c u l a t e d m o u s e ~ d five o t h e r s a t t h e following t i m e s inoculation with mock inoculum or v i r u s : o n e a t 30 d a y s a f t e r c o r n e a l i n o c u l a t i o n w i t h m o c k i:~oculum, t w o a t 15 d a y s afl~zr c o r n e a l i n o c u l a t i o n w i t h v i r u s (one w i t h n o %ve di a n d o n e w i t h e y e disease), o n e a t ~ dab, s c o r n e a l i n o c u l a t i o n w i t h v i r u s ( w i t h e y e di ), o n e a t 15 d a y s a f t e r s n o u t i n o c u l a t i o n w i t h v i r u s ( w i t h e y e disease). I n t h e l i m b u s , t h e c o u n t i n g e r r o r v a r i e d f r o m 3 - 5 % ~ 2 2 . 3 % , a n d in t h e c e n t r a l c o r n e a f r o m 1 4 - 6 % t o 3 9 % . Controls. T w o g r o u p s o f c o n t r o l s w e r e u s e d : t h e first w a s u n i n o c u l a ~ d e y e s , t h e s e c o n d w a s e y e s scarified t h r o u g h m o c k i n o e u l u r n ( T a b l e I I ) . I n b o t h ~ o u p s , LCs w e r e a b u n d a n t in t h e l i m b u s , l e ~ so in t h e ~ r i p h e r a l a n d almost, a b s e n t f r o m t h e c ~ n t r a l c o r n e a (Fig. 1). N o s i g n i f i c a n t d i f f e r e n c e s w e r e ~ n in t h e LC ~ u n ~ in t h e limbus or the central cornea b e t w ~ n these two groups (Table II).
1,'t 4
H.,I, I , I ~ W K O I ~ ' I C Z - M | } S S
ETA
1,.
SOft
600
400 U
0
days a f t e r inoculation
F I ( I It. q}l(~ nlll|lht~r ~ f I.{Y {~l~.rv{.t ill l b . ~ ' o t n d ¢~rn{.d ~.pilhelinm a t w ~ r b u s t i m e s a l l e r ~ s~t~ut il)Oe~|)alioll tl'ith I × It) ~ I~t}I (~f H S V I s l r a i l t t~l(l. T | l e ln(.Iitlll Wi|l| rllIIge iS 8}l(I.m aI/d t h e IlUlIIIIPI~ (If ~ b ~ ' r v a t i . n . is a l . ~ v e e a c h 1.~il~t+ T h e +~)~tr¢~l valut,s are ~h(~wl~ o n tile v . r t i c a l a x b . ............ d i ~ , a ~ , d g ~ l l p ; - - = , o~nodis~+a~ul g r . l q ) : 'Fh~+ K r u s k w ~ d ~ W a l l b a ~ a l y s i s o f v a r i a l ( ~ w a s osed t o e x a m i n e tlw:~e d a t i t .
F [ o . 7. A n e p i t h e l i a l sht-~t f r o m a ~ e y e o f ~ m o u ~ I 5 d a y s a f a r ~ cg~meal ino~.~utation w i t h 1 × t 0 ~ pfu H S V 1 S C l 6 . A n a c c u m m u b t i o ¢ ~ o f ~ l ) s is ~:~n s u r ~ . } u ~ d i , g a n e p i t h e l i a | , k s : r {13). B a r = ~ ) / t i n .
l,(~s I N M O U S E C O R N E A
AFTER
HSV! INFECTION
Fx(~. 8. h~) epithelial sh~,t IYum a~ eye o f a m(~u~ 18 days after a u~out inocuIatk~n with I × I|l~.iffl~ HSV I SCt(L l~n~erha~s ~ l l s an~ p n ~ n t I ~ t h in t)~e ]|rebus {L) a~(| the ~.ntrat c~r~ea (C) Small
Corneal inoculation (a) LC count:s in mi(ae with or w i t h o u t eye d i s e a ~ Mice sampled between d a y s 8 and 43 were divided into taro groups, those with eye disease (diseased group) and those with no signs of eye d i s e a ~ a t the t i m e the), were killed (non diseased group). W h e n LC counts for d a y s 8 to 43 in the disemued group w e ~ pooled and compared with similarly pooled d a t a for the non d i ~ a s e d group, the LC n u m b e r s in the former group were significantly higher t h a n in t h e l a t t e r (Table If). Both groups had significantly higher counts t h a n controls. Mot~cover, the I;C' counts in the limbus of the eontralaterai (uninoculatcd) eye of mice inoculat~J with virus wen~ significantly tdgher t h a n those o f controls. (b) Changes in LC counts with time L i m b u s : In the diseased group LC n u m l ~ r s reached a m a x i m u m on d a y 8, ~ m a i n e d high until d a y 22 and l~ll to within the normal range b y d a y 30. In the non-d| group t h e rise in LC n u m b e m was small a n d t r a n s i e n t (Fig. 2). Statistical analysis of the d a t a showed t h a t the results in both these groups were not homogeneous indicating difl:erenccs between t h e n u m b e r s of LCs a t d i f f e ~ n t times after inoculation (Fig. 2). Central cornea: N u m b e r s of h ~ s in disea~3d mice ~uzaehed a m a x i m u m on d a y 8 and were still i ~ i ~ d b y d a y 30, b u t had r~turned ~ normal levels b y d a y 43 (Fig. 3). However, two o u t of fc)ur mice s a m p l e ] on d a y 65 ha~J raised LC n u m b e r s in the cex~tral cornea (Fig° 4). StatistAeal analysis o f t h e d a t a for both the di d a n d non d i s e ~ e d groups showc~ t h a t t h e ~zsults w e ~ n o t horn eous i n d i c a t i n g significant d i f f e ~ n e e s between t h e n u m b e r s of LCs ~ d i f f e ~ n t times aster inoculation (Fig. 3).
I36
N. J . L E W K O ~ , ¢ ' I C Z - M O S N
ET AL.
Fro. 9+ A n e p i t h e l i a l s h t + t |Yore a n e y e o f a m o u ~ 25 d ~ v s a f a r a s n o u t i n o c u l a t i o n w i t h 1 × I 0 ~ p f u H SV ! SC l 6~ A n area o f IX~'a c c u m m u l a t i o n is s ~ n ~ w h e r e a s o t h e r a ~ a s o f t h e c ~ n t r a l ~ r ~ m a a p l ~ a r fr¢~ o f IA?s+ B a r = 5 { ~ / i r a ,
Snout inoculation (a) LC counts in mice with or w i t h o u t eye di~¢ea~ Mice sampled between d
LCs IN MOUSE
CORNEA
AFTER
HSVI
INFECTION
t37
Flo~ 10. A high*lrower light m i c r o ~ a p h o f lmh~gerhans t~lls from within the d u s ~ r d e m o n s t r a ~ in Fig. 9~ T h e ¢~ndritic p r o ~ s ~ s are highly ar~orized at~d dendr4tes from different ~ l / a a p ~ a r ~ form connections (arn~ws). l~ar = 50/tin.
In ,nice with eye disease after snout, inoculation, LCs initially spread from t h e limbus over t h e e n t i ~ surfac~ of t h e cornea, b u t in certain a r e ~ t h e y w e ~ c o n c e n t r a t e d in small c l u s ~ r s (Fig. 8). B y day 25 when t h e LC n u m b e r s in the central cornea veerc d r o p p i n g a n d a ~ o f epithelium were ~ c o m i n g devoid o f thee~ cells, a variable n u m b e r of dense foe| of L t ~ remained (Fig. 9). T h e LCs within thcJse foe| were generally highly dendritic (Fig. 10). 4. D i s c u s s i o n
In c o m m o n with previous ~ p o r t s on I ~ distribution in t h e cornea of various animal s ~ c i ~ (Brown e t a ] . , I968; K | og, Forsum, ~ e r n l u n d , and Petez~on, !979; Rodrigues e t ai., 1981 ; G i | l e t ~ e t a|o, I982) we have found t h a t in n o r m a l mouse eyes, corneal LCs wez~ a b u n d a n t in t h e limbus, b u t sparse or totally a b s e n t from the central ~ r n e a . i ~ the high c o u n t i n g error, which could accounted for by low hUm bel~ o f LCs in some specimens a n d n o n uniform distributions in othem, t h e r e was clearly a highly significant i n c ~ e ~ in LCs following H S V infection. HSV antigens can be d e t e c t e d in t h e cornea one day, after diz~ect corneal inoculation with virus, whereas after inoculation on t h e snout, from w h e ~ virus spreads ~ the eye via t h e n e r v o u s system (Shimeld e t aL, 1987) a n t i g e n s can be d e l e t e d in t h e cornea from day" 3 (Shimeld e t a l . 1986). I n t h e p ~ s e n t s t u d y we h a v e d e m o n s d increased LCs in corneal e p i t h d i u m 3 days a f a r cornoal inoculation and 6 days an inoculation on t h e snout. In each ~ e this indicates a lag of 3 ~ d a y s b~tween t h e arrival of t h e virus in t h e cornea and t h e initial ~se in LC numbers. A similar delay hms been reported following chemical injuT3, (Brown e t al., 1968L
138 s..I.I.EWKOWICZ*MOSS ET AI.. The f a c t o ~ contro|ling LC migration into the com~ca art~ not fldIy understood. Passive infiltration after wound healing m a y play a part (Giaconmtti, 1969)~ However. this ~ e m s an unlikely explanation of our Tvsults, as no increase in LC n u m b e r s was seen after scarification of the e~)rnea t h r o u g h mock inoculum. However, it is now rccognised t h a t LCs act ~ a n t i g e n - p r e ~ n t i n g cells l)oth in delayed hypersensitivity reactions (Silerberg-Sinakin and Thorheeke, 1980) a n d in Mlograft I<~jection (Rowden, 1980). Moreover, these cells have b~en shown ~ present HSV antigens to T i y m p h o c y t e s (Braathen, Berle, Mobech-Hanssen and T h o ~ b y , 1980). Therefore the iuc~zase in I~5:s ah~er herpetic infections m a y be a response to an antigenic stimulus. in delayc~J hypersensitivity reactions, the allergen is believed to induce t h e release of lymphokines, which in turn h a v e a ehemotactic effect on LCs (Roussel, Osato and Withebnus, ! 983). The plaque-like distribution of HSV antigens in corneal epithelium after an inoculation on the s n o u t (Shimeld et a i , 1986) and t h e similar clustering of LCs when HSV is i n o e u l a ~ d by this route, suggests t h a t the~e cells m a y be d ~ w n to the ~ e i of viral antigens. With either r o u ~ of inoculation, LC numbet~ ~zmained high long after d a y 10, particularly in mice with obvious clinical c~mmal d i ~ a ~ . By this time infectious virus and viral antigens would have been clea~
LCs IN MOUSE CORNEA AFT E R HSVI INFECTION
139
REFERENCES Blyth, W. A., Harbour, D. A. and Hill, T. J. (1984). Pathogenesis of zost~riform s p e n d of h e r ~ s simplex virus in the mouse~ J . Gen. ViroL 65, 1 4 7 7 - ~ Braathen, L. R , l~rle, E , Mobech-Han~en, N. and Thorsby, E. (19~). Studies on human epidermal Langerhans c~lls: 11 activation of human T lymphocytes to herl>eS simplex virus. Acts Dervnatovener. (Stockholm)6~0, 381~7. Brown, J~, ~ d e m t r o m , C. W~ and Winkelmann, R. K, (1968). ~ n g e r h a n 8 c~lls in guinea-pig cor~ea. Pceswan~ ~ chemical injury. Invest. OptdhalmoL 7, 668-71. Chaker, M. B., Tharp, M. D, and B e r g s t ~ r , P. R. (1984). Rodent epidermal l~ngerhans c~ells demonstrate g ~ a t e r histochemieal sl~cificity for A D P than fi)r ATP and AMP. J. Iuve~t. DermatoL 82, 4 ~ - 5 ~ 9 . Cole. S., L~wkowicu, 8. ,L and Townsend, K, M. 8. (1984). Imngerhan8 c~l| number and morpholo~¢ in mouse footpad epidermis after X irradiation. . Res. I ~ , 594606. Giaeometti, L. (1969). The healing of skin wounds in primates. III behaviour of the c~l!s of Langerhans. J. Invest. Dermat~. 53, t 5 1 4 , Gillette, T. E., Chandler, ,L W. and Greiner, J . V . (1982L Imngerhans ce}ls of the ocular surfa(~. Olddk~lmoloyy, 89, 7 ~ - 1 0 . Gschnait, F. and Br~enner, W. C. (1979). Kineti¢~ ofepididermal Langerhans ceils, d. l)ermat~. 73, 566-9, Hazlett~ L~ 1), Grevengood, C. and Berk, g. 8. (1984L Respon~ of routine ocular Langerhans' cells ~ P, Auruginosa corneal infection . ~ d d h a h n ~ . Vi~. Sci. ~ , 21.
Hill, T. J., Field, H. J. and Blyth, VV. A. (1975). A c u ~ and r e e u r ~ n t infection with herpes simplex virus in the mouse: a model for studying latency and ~ c u r r e n t d i ~ a ~ , d. Gen. ViroL 28~ 341-53. Juhlin, L. and Shelley, W. B. (!977). New s~ining techniques for the I~ngerhans cells. Acta r~r. (~t~kholrn) 57, 289-96. Katz, S~ I., Tamaki, K. and Sachs~ D. H. (1979)~ Epidermal Langerhans ~lls are derived ~om ~ l l s originating in bone marrx~w. Nature ( L o ~ o n ) 282, 324-6. Klar~skog, L., Forsum, U., Tjernlund, U.M., R ~ k , L. and Pe~r:~on, P . A . (1979L Expression of Ia antigen-like moleeul~s on cells in the corneal epithelium lmoL 18, 310-3. Lang, R. M., Fri~laender, M. H. and Sehoenrock, B. d. (1981). ~ n g e r h a n s ~!1 induction and migration in guinea pig cornea, lnvesL @hthalmoL Vis. 8eL 20 (Suppl.L 1. Mackenzie, I.C. and Squier, C. A. (1975L Cytoehemical identification of ATl~se-wositive cells in EDTA s e p a ~ t e d sheets of m o u ~ epidermis~ Br. J, DernaatoL 92, 523-33. Pep(me, J. S., Gardner, K. M., N e s ~ r , M. S., Foos, R. Y. and Pettit, T. H. (1985), Detection of HLA class I and II antigens in ~jected humau corneal all . 92, Rodrigu~, M. M., Rowden, G., Hackett, J . and Bakos, I. (1981). Langerhans cells in normal ~ n j u n e t i v a and Feripheral ~ r n e a of ~ l e c ~ d settles. Invest. O p ~ . Vi~. Sci~ 21, 759~65. Roussel, T. J., Osato, M. 8. and Vgi!helmu8, K. R. (1983). C~rneal Langerhans cell migration following ocular e o n ~ e t hy~mensitivityo C o ~ e a 2, 27-30. Rowden, G. (1980). Exp~ssion of Ia antigens on Langerhans c~lls in mice, guinea pigs and man. J, I ~ t . De . 75, 22-31. Rubsamen, P. E., MeCulley, J.~ Bergstresser, P. R. and St~ilein, J. W. (!984). On the la immunogenicity of mouse c~rneal allografts infil with Langerhans ¢~ils. . Op I. Vis. Sci. 25, 513-8. Shimeld, Co, Easty, D. L . Tullo, A. B,. Biyth, W. Ao and Hill~ T. J. (1985), Spread of h e r ~ s simplex virus ~ the eye following cutane~Jus inoculation in the skin of the snout of the mouse. I n D ~ Op 44, t ~ ~ye . (Eds Maudgal, D, C. and Missotten, L.). Dr W. J u n k : D o r d ~ c h t / B ~ t o n / L a n c a s t e r . Shirneld, C., ~ w k o w i ~ - M o g s , S. J., Lipworth, K., Hi!l, T. J., Blyth, W. A. and Easty, D. L. (1986). Antigens of herpes simplex virus in whole corneal epithelial shee~ from mice. Arch. OphlhalmoL 104, 1 8 3 0 ~
140
8. J. LEWKOWI(:Z.MOSS ET AL.
Shimeld, C., Dyson, H., Lewkowiez-Moss, S,, Hill, T. ,I., Blyth~ W. A. and Ea~ ty. t). 1,, (1987}. Spread of HSV-I to tile mou~ ~¢e after inoculation in the skin of the snout ~quires an intmet nerve supply to ~he inoeu|ation site. Curr. I'(tle Re.~, 6. 9~-12. Sil~rberg-Sinakin, 1. and Thorb~ke, G. J. (|980), Contact hypersensitivity and Langerhans ~lls. J. Invest. De~atoL 75, 61-7. Sting, G., Tamaki, K. and Katz, S. L (1980). Origin and function of epidermal I~angerhans ~lls, [mmunol. Ret,. 53, 149~74. Streilein, J. W., Toews, O. B. and Be r, P. R+ (1980}, Langerhans (~lls: flmetional ~imct~ m;~aled by in vhv ~ a ~ i n g studies. J. Invest. Dern~tol, 75, 17~21. Toews, G . B . B e r g s t ~ r , P.R., St~ilein, J. W, and Sullivan~ S. (i9~). Epiderma| ~ngerhans ~ll density de~rmin~ whether contact hyi~r~nsitivity or unto.~* sponsivene~ follows skin painting with DNFB. J. lmmunoL 124, 445-53. Tu|to, A. B., Shimeld, C., Blyth, W, A., Hill, T. J, and Easty D. L. (I983). Ocular infection with herk~s simplex virus in non immune and immune mic~. Arch. Ol~thalmol, 101. ~1-4.
Q u a n t i t a t i v e S t u d i e s o n L a n g e r h a n s C e l l s in Mouse Corneal Epithelium Following I n f e c t i o n w i t h H e x e s S i m p l e x Virus S. J. L ~ w ~ o w ~ c z - M o s s * ¢ + C+ S H I ~ L ~ ¢ ,
K. L~I'WOaTH$. T. ,L HILLS,
Department~ ~ ¢ ()phthalmol~!! and +M iertdriol~!/, University ~0~ Bri~stol, Bristol, U.K. (Received 27 Octolmr 1986) l$~endritic ~ n g e r h a n s c~|ls (LCs) w e ~ identified in flat-mount pr~|mratk~t~s o f t n o u ~ t~)rneal epithelium a f a r staining for A ' F l h ~ activity. T h e y w e ~ found pn,~tominant|y in the limbus, buL after inoculating the com~=~ with HSV ! strain S(TI 6 LC~ numbers i n c ~ d both in the limbus and the ¢~ntral euJrnea. N u m ~ r ~ o f l~Cs n:ached a m a x i m u m on d a y 8 and if m v e ~ keratitis was pre.~nt ~ m a i n ~ high a t ~ s t until d a y 22. A small but, significant increase in i.~3s was al~) t~und in the op|~site, anit~oculat(M (~ye in micu2 with revere damage in the imw~ulat(~t eye. After HSV inoculation on the snout. 6 0 % of mi¢~ had cornea| disem*~ in the (~*e oil the i n o c u l a t ~ side; in such micuz corneal Lg.~'sw e ~ a t a tnaximum 18 d a y s after itmcu|atiom The iue~-a~se in LC n u m ~ m w ~ similar whether inoculation was into ttie (u~m~ea or i~l the snout. Aft~-r cornea! inoculation the c.||s w e ~ d i s t r i b u t e | fairly evelily over the (u)nmaI surface, with accumulations |imitM to epithelia| ult~:rs. However~ after inoculation on file snout, numerous clusters w e ~ e~.n over the epithelial surfa(~, often surroumi by epithelium devoid of LCs. Key u~rd.: ]~lngerhans t*|ts; c~ruea; herl~s simplex vita,s: viral keraiitis: dendt4ti~ (a+i|s.
1. I n t r o d u c t i o n
Dendritic cells in skin epithelium were f i ~ t described by Paul Langerhans in 1868. They have since bc~en identified ~ antigen presenting cells of bone marrow origin (Katz, Tamaki and Sachs, 1979; Stingte, Tamaki and Katz, I989)and are now known as Langerhans cells (LC). They can be e~uily distinguished fro~L epithelial cells by their high membrane-associa~d adenosine triphospha~se (ATPa.~z) activity (Mackenzie and Squier, 1975), and this histochemicai proF~rty hmu ~ e n u~
add~
c~r~s~)ndenc~ to U.K. T r a n s p l a n t f~ervi~, S o u t h m ~ d , Bristol B S I 0 5ND, U.K.
~ J 1 4 ~ 8 3 5 / 8 7 / 0 7 0 1 2 7 + 14 $~J.~9/0
© 1 ~ 7 Academic P r ~
Ine~ ( ~ n d o n ) Limited
128
S.J. L E W K O W I ( 2 Z . M O S S ET AL.
o c u l a r LCs a f t e r a specific viral i n t ~ c t i o n h a v e n o t b e e n r e p o r t e d . W e n o w ~ p o r t s u c h o b s e r v a t i o n s following direct inoculation o f the cornea with herpes simplex virus ( H S V ) a n d following z o s t e r i f o r m s p r e a d ( B l y t h , H a r b o u r a n d Hill~ !984) o f t h e v i r u s t o t h e e y e a f t e r i n o e u l a t i o n o f t h e s k i n o f t h e s n o u t ( S h i m e l d , Easty~ T u l l o , B l y t h a n d Hill, 1985).
2. M a t e ~ a l s a n d M e t h o d s Animals Eight-week-old male outbr(~t Swiss white Bristol/2 mice (Blyth c t a l . , 1 9 ~ ) we~ur u ~ l t h l ~ u g h o u t . T h e ( ~ s w e ~ e x a m i n e d with a slit lamp and a n i m a l s with a b n o r m a l eyes w e ~ excluded. hu~ulation~ Animals were auaesthetised with sodium i~mtabarbitone a n d were inoeulattut by scarification t h o u g h 5 l d o f m ~ l i u m c o n ~ i n i n g I × l()~ p l a q u e - f o r m i n g units (Pfi0 o f HSV! strain S O l 6 (Hill, Field and Btyth, 1975) either on t h e left c~rnea ( T u l l o Shimeld, Blyth, Hill a n d Easty, t983) or on a s h a v e d area o f skin, close to t h e t i p o f tile left s n o u t (Shimeld e t a l , 1985). Control mice ~ t~u~at~ in a n identical way with a mock inoculum prepared in a similar way to t h e virus I)~)l b u t c o n t a i n i n g no virus. Examination of eyes and isolation of vir~a~ fi'om egewashin~ T h e p r o c ~ l u r e s for e x a m i n i n g m o u s e c~yes a n d i ~ l a t i n g virus from eye washings have b-e~n dcscribc~t previously (Tullo et al., 1983) e x c e p t t h a t in this s t u d y no corneal stains were used to assist t h e eye e x a m i n a t i o n s in case t h e y interfered with t h e s t a i n i n g o f LCs, l)emotmtration of Lai~erhans cells in corneal e~pithelium Mice wez-e killed Irv" an i n t r a j ~ r i t o n e a l injection o f sodium !~ntabarbitone~ The c~rneal e p i t h e l i u m from both eyes was r e m o v e d a n d proces~ql for A T P a s e e~tivi~, according to t h e m e t h o d o f J u h l i n and Shelly (1977) b u t using the modification su ~ d by Chaker, T h a r p a n d B e r g s t r e ~ z r (I984), w h e r e b y a d e n o s i n e - 5 - d i p h o ~ ) h a ~ (ADP, s ~ t i u m ~ l t from equine muscle Sigma grade IV) r a t h e r t h a n A T P was used as 8 u b s t r a ~ . F~yJq)ad epithelia w e ~ stained simultaneously with corneal st~zeimens. If the s t a i n i n g o f LCs in the footpad was consistently very pale or t h e b a c k g r o u n d excessively high, t h e n t h e a c e o m i ~ n y i n g corneal shc~vts we~e discarded, After s~aining, the corneal e p i t h e l i u m w ~ plac~d on t o slides basal ¢~11 la)~zr u p p e r m o s t , a n d the epithelium was f l a t t e n s ! by inaking t h ~ radial incisions a r o u n d t h e circumference. A p a t h y ' s m o u n t i n g m e d i u m ( R a y m o n d A. l ~ m b ) was u e ~ l)ar m o u n t i n g the eove~lip, which was t h e n ~ealed with nail v a r n i s h Positively staining d e n d r i t i c cells were c~unt~t in five limbal fields, (total area ~ 0"6! 25 ram2), a n d 10 c~ntral c~rnea| felds,
(to~l area ~ 1"225 mmZ); t h e r e s u l ~ are e x p
~u+ t h e n u m b e r of ~ l l s per m m L
S~t~stic~l analy.Ms N o n - p a r a m e t r i c s ~ t i s t i c a l test~ w e ~ u ~ l tbr analysis o f all t h e d a t a . T h e Wilcoxon ~ n k s u m t e s t were used ~ c o m p a r e g r o u p s o f d a t a , p ~ l ~ over t h e entir~ t i m e c o u p e enabling t h e m¢~ian a n d range of LC ~ u n t ~ for t h e inoculated g r o u ~ to be ~ m l ~ r e d with one a n o t h e r a n d with controls. T h e K r u s k w a l J , Vallis analysis of variance was used ~a ~ s t for h o m o g e n e i t y within a g r o u p a n d therefor~e ~ m p a r ~ c o u n ~ carried o u t at different times after inoculation. T h e level o f significance was ~ t a t P ~ 0"05.
Y
3,4.5,6,7.9
1,2,4,5,6,7,8,9 -1,2,4,5.6.7,8,9
Eye washings {days)
D.K.
9.K.
D.K,
3+ 4, 5, 6, 7+ 9+ D+K.
I~:~.,4, 5, fi, 7, 8, 9, ILK~ D.K. t, 2, 4, O, 6. 7 8, 9, O.K.
D.K.
D.K~
Clinical examinations (days)
s (4) 3 {2) |3 (2) 8 (4} t8 {4)
3 (4}
6 {2) ! s (2) ' I ! {4} ~ {8)
I ! {4) t8 {5)
4 {2) I5 {2} ' :~) {4) 15{4) 30 {t2) 18 (2)
4 {4) J~ {4) 30 {4)
Jt {2) 2~ (2} 13 (4)
t3 (4) 25 {3)
.2 (.1
18(4)
8 (2} I8 (2}
8 {7) is (4} 43' (4 }
LC studi~ on day (n) 2 {4) i J {4} 22 (4) ~5 {3} 2 (2) 1| (2} 22 {2) I1{5) 22 {4) ! ! (2} 3O {2)
C, cvrn~; S, s~out; V, HSVi S.31fi, ! × l0 s pfu; n, ~um~r of mi¢~e;M J . m ~ k inoculum; D.K. day on which mi~ were killed,
S
40
Y
S
M.I.
V
V V M.I.
~I.|.
V
ino~'ulum
S
S
~
3
C C C
10
N
C
2;5
2
C
~
1
laoeulation site
Number of mi~,e
gx~fiment mlm~r
Total number of mice w~ed, inoculation site, inoculum aml days on which ~,ariou.~procedures (eye ~z~s/~i~ls,clinical examination, amt Lav£erhans celt wunts) were ~rformed
TABLE l
:c <.
.
>
O
13:0
s.,i. I.E$~, K(}X$ I ( Z - M O S , , Is 3.
AL.
l~esults
G r o u p s o f m i c e w e r e inoeulatx~,d o n t h e cornea, o r t h e s n o u t w i t h v i r u s o r w i t h m o c k i t , o e u l u m ( T a b l e I). A t v a r i o u s it~tervals a f t e r i n o c u l a t i o n , e y e s w e r e e x a m i n e d f o r clinical d i s e a s e , e y e w a s h i n g s w e r e t a k e n f o r i s o l a t i o n o f v i r u s a n d c o r n e a l e p i t h e l i a l s h e e t ~ w e r e t a k e n f o r LC: s t a i n i n g .
SVatisticai ¢~neH!t.~is by the W n r~nk-;~nm te,~t ~0 ~"p~ded lanfferhans cell ( L C ) counts in control~ a n d %¢ter tion o f v i r ~ * on the cornea Nmnb,w ~*f tA's {Irt,up?
Ni,mber t~f ini(~,
hh~iian
Rangt~
Limbus 178
~18-2~
186
147~:t21|
44:1 227
2|11-764 ~q55 124-
Viv~s mm di~-a~.d (N I/I
62 ;t2 :~t :t4
V i r u s contralaiera| We , (i,')E
fi8
| 9:{
(',rot r,d (('} Mt,~.k im~t.uh~m {MY) Vireos dis~.a~, (VD} Virus non diseas~l (N I))
~t2 32
(',,n| ral cor¢~va 9 i)
:t4
~|~
5~94
ViMls v(mtraiatt,ra| v~'e ( E}
fi8
!2
3qt5
5hwk im~t-uhlm (MI)
;~1t4
2~36 4~42
~ta¢ is| |cat ~.~m~parislm
MI vso (' VI} vs. (" N1) vs. M| Nl) w, VI) {'E v:~, U
1'
< < < < <
0"115 O~IiH tHXI5 iHXH O'U!
M l w+ (+
~ +s+
V|) vs+ (+ Ni) vs. MI NI) v s VI) CE vs~ U
< {HMH < o,~2 < tH~*I ~.s.
t Ea ,h grm~p ~pr~,senis the I,C e~m~ts |,~u,l~t ~ver the el/t}~ time v~u~e,
Clinical ~ s e r v a t i o ~ s Corneal inoculation° S u p e r f i c 4 a l d a m a g e w a s ~ e n in ~ l e c o r n e a l e p i t h e l i u m o f all e y e s f r o m t h e f i r s t d a y artier i n o c u l a t i o n w i t h virl!s. T h i s e i t h e r r e s o l v e d w i t h i n 3 - 4 days or more usually p s~d to severe ulcerative keratitis by days 8-I0. Evidence o f s t r o m a l o p a c i f i c a t i o n a n d iris h y p e r a e m i a w a s a l s o ~ e n in m o s t m i c e f r o m t h e f i r s t or second day after inoculation but these signs of disease could be transient, S u p e r f i c i a l d a m a g e t o t h e e p i t h e l i u m w a s s ~ n in t h e m ~ o r i t y o f m i c e s c a r i f i e d through mock inoeulum, and approximately half of thc~ also showed small stromal o p a c i t i e s . H o w e v e r , b y t h e fi~urth d a y all e y e s w e r e n o r m a l . Snout inoculat{on. Zosterifcnnn s p r e a d o f d i s e a s e w i t h l e s i o n s o n o r a r o u n d t h e e y e w a s ~ e n in 9() ~ o f m i c e i n o c u l a t e d w i t h v i r u s o n t h e t i p o f t h e s n o u t . S i g n s o f c o r n e a l d i s e a s e , s u c h a s t h e ' g r o u n d g l a s s ' a p p e a r a n c e w e r e first o b s e r v e d o n d a y 5 a n d r e a c h e d a p ~ a k incidence2 o f 5 0 ~ o n d a y I0. O n d a y 7 s t r o m a l o p a c i t i e s | i o n w a s s e e n in a b o u t h a l f t h e m i c e . A f t e r s c a r i f i c a t i o n o f m o c k i n o c u l u m o n t o t h e s n o u t , n o e y e di was seen. Isolation o f virus f r o m eyewashings Corneal ir~mulation. V i r u s w a s i s o l a t e d f r o m all c y e w a s h i n g s f o r t i m f i r s t 2 d a y s a f t e r i n o c u l a t i o n ; tile i n c i d e n c e o f i s o l a t i o n d e c l i n e d o v e r t h e n e x t 7 d a y s a n d n o v i r u s we~s i s o l a t e d a f a r d a y 9.
|,C8 IN MOUSE
CORNEA
AFTER
HSgl
INFECTION
tal
F r o . I. A n e p i t h e l i a l s h e e t from a n e y e o f a [~)ntrol m o u s e . L a n g e r h a n s tu~ils (IX2s) a~a pre.~mnt m a i n l y in t h e l i m h u s ( r i g h t o f p i c t u r e ) o c c a s i o n a l ceils a ~ p r e s e n t in t h e t ~ n t r a } ~;ornca. l a m g e r h a n s cells welu2 s t a i n ~ l u s i n g a his~gehcmical t e c h n i q u e for d e m o n s t r a t i n g A T P a ~ a c t i v i t y . B a r = ~00 ~till,
60~
0
! J,,
days after ~ u ~
Fm~ 2~ T h e h u m o r o f ~ s o b s e r v e d in t h e l i m b s | c o ~ c a l e p i t h e l i u m a t v a ~ i o u s t i m e s a f t e r a c o r n e a l i n ~ u l a t l o n w i t h ! × l 0 s pfu o f R S V ! s t r a i n SC16~ T h e m e d i a n w i t h r a n ~ is s h o w n a n d t h e n u m b e r o f o b ~ r v a t i o u s is a b o v e e a c h ~ ) i n t , T h e c ~ n t r o ! v a | u ~ at*e s h o w n o a t h e v e r t i c a l axls~ ~ , diseas~ group; - - - . n o n - d i s e a s ~ g r o u p . T h e K r u s k w a l - ~ V a i l i s al~al:;sis o f v a r i a n o e w a s u ~ to e×amine the'~ data. Sigififieant d i f f e r e n t ~ s : d i ~ a ~ d g r o u p , P < ~ O I ; non-dise~e~d g r o u p P < f f O l .
132
S.J. LEWKOWICZ-MOSS
6~
E
ET AL.
4 3
400
Q U
0
20 days Offer ~
a'0 |
£o
.......
5'o
~
Fm~ 3. Tlm number o f ~ n g e r h a n s ~ | l s o b ~ r v e d iu the central c~rneai epithe|ium a t various times after a eort~ea| inoculation with I x 10Spfu H S V I S~2|6. The media~l with ra~*ge is shown a~d tile n u m b e r o f o b ~ r v a t i o n s is abe, r e each point. The ~ n t r o l valu¢~ a ~ shown on the vertical axis~ d i s e ~ d group; - - - ~ a o n . d i ~ d group. T h e Kruskwai-X,Vai|is analysis o f v a r i a n ~ was u ~ d to examine t h e m d a ~ . Significant diffe~3n~s: d I group~ P < 0 ~ ! ; n o ~ d i g r o u p P < ~01.
Fxo. 4. An epithelial s h ~ t from an eye o f a m o u ~ 65 d a y s after a c o r n e l i n s u l a t i o n with i × I0 ~ pfu HSV ! ,c~16. Bar = 2 ~ ~m~
LCs IN MOUSE CORNEA A F T E R HSVI I N F E C T I O N
I~
IIIII ¢0~II |IIIIII|
I~O~t
800 limbus 600
400
| I
~
P ~
0
I|~II
O~O0~
P ~
0.001
700
500"
!l
central cornea 200
100
....
........
l
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Fro. 5. The number of ~ s observed in ¢o~aeal epithelium after inoculation ~ith 1 × 10s pfu HSV ! SC16 on either ~ e snout or the c,3mea. The histograms m p ~ n t the m ~ i a n number of ~lis o b ~ r v ~ between days 8 and 34 after snout inoculation and betw~n days 8 and ~ a ~ r ~rneal inoculation. The range and number of observations a ~ shown° Differences ~ t w ~ n ~ u ~ were t e s ~ with the Wtleoxon rank-sum t~st~ ~ v e l of significance~ P ~ G~05. +, Comp~son n virus, snout, n o n - d t ~ z ~ and virus, ~ r n e a non-di~a~.l.
Snout inoc~ation. V i r u s w a s first i s o l a ~
4 days after insulation. The ~ak i n c i d e n c e o f i s o l a t i o n w a s 7 5 % on d a y s 5 a n d 6, a n d n o v i r u s w a s i s o l a t e d a f t e r d a y 8.
La~e cell studies Courding error. T o e s t i m a t e t h e c o u n t i n g e r r o r , six c o r n e l e p i t h e l i M sheet,s w e r e c o u n t e d on five s e p a r a ~ o c c a s i o n s : T h e s h e e t s w e r e t a k e n f r o m o n e u n i n o c u l a t e d m o u s e ~ d five o t h e r s a t t h e following t i m e s inoculation with mock inoculum or v i r u s : o n e a t 30 d a y s a f t e r c o r n e a l i n o c u l a t i o n w i t h m o c k i:~oculum, t w o a t 15 d a y s afl~zr c o r n e a l i n o c u l a t i o n w i t h v i r u s (one w i t h n o %ve di a n d o n e w i t h e y e disease), o n e a t ~ dab, s c o r n e a l i n o c u l a t i o n w i t h v i r u s ( w i t h e y e di ), o n e a t 15 d a y s a f t e r s n o u t i n o c u l a t i o n w i t h v i r u s ( w i t h e y e disease). I n t h e l i m b u s , t h e c o u n t i n g e r r o r v a r i e d f r o m 3 - 5 % ~ 2 2 . 3 % , a n d in t h e c e n t r a l c o r n e a f r o m 1 4 - 6 % t o 3 9 % . Controls. T w o g r o u p s o f c o n t r o l s w e r e u s e d : t h e first w a s u n i n o c u l a ~ d e y e s , t h e s e c o n d w a s e y e s scarified t h r o u g h m o c k i n o e u l u r n ( T a b l e I I ) . I n b o t h ~ o u p s , LCs w e r e a b u n d a n t in t h e l i m b u s , l e ~ so in t h e ~ r i p h e r a l a n d almost, a b s e n t f r o m t h e c ~ n t r a l c o r n e a (Fig. 1). N o s i g n i f i c a n t d i f f e r e n c e s w e r e ~ n in t h e LC ~ u n ~ in t h e limbus or the central cornea b e t w ~ n these two groups (Table II).
1,'t 4
H.,I, I , I ~ W K O I ~ ' I C Z - M | } S S
ETA
1,.
SOft
600
400 U
0
days a f t e r inoculation
F I ( I It. q}l(~ nlll|lht~r ~ f I.{Y {~l~.rv{.t ill l b . ~ ' o t n d ¢~rn{.d ~.pilhelinm a t w ~ r b u s t i m e s a l l e r ~ s~t~ut il)Oe~|)alioll tl'ith I × It) ~ I~t}I (~f H S V I s l r a i l t t~l(l. T | l e ln(.Iitlll Wi|l| rllIIge iS 8}l(I.m aI/d t h e IlUlIIIIPI~ (If ~ b ~ ' r v a t i . n . is a l . ~ v e e a c h 1.~il~t+ T h e +~)~tr¢~l valut,s are ~h(~wl~ o n tile v . r t i c a l a x b . ............ d i ~ , a ~ , d g ~ l l p ; - - = , o~nodis~+a~ul g r . l q ) : 'Fh~+ K r u s k w ~ d ~ W a l l b a ~ a l y s i s o f v a r i a l ( ~ w a s osed t o e x a m i n e tlw:~e d a t i t .
F [ o . 7. A n e p i t h e l i a l sht-~t f r o m a ~ e y e o f ~ m o u ~ I 5 d a y s a f a r ~ cg~meal ino~.~utation w i t h 1 × t 0 ~ pfu H S V 1 S C l 6 . A n a c c u m m u b t i o ¢ ~ o f ~ l ) s is ~:~n s u r ~ . } u ~ d i , g a n e p i t h e l i a | , k s : r {13). B a r = ~ ) / t i n .
l,(~s I N M O U S E C O R N E A
AFTER
HSV! INFECTION
Fx(~. 8. h~) epithelial sh~,t IYum a~ eye o f a m(~u~ 18 days after a u~out inocuIatk~n with I × I|l~.iffl~ HSV I SCt(L l~n~erha~s ~ l l s an~ p n ~ n t I ~ t h in t)~e ]|rebus {L) a~(| the ~.ntrat c~r~ea (C) Small
Corneal inoculation (a) LC count:s in mi(ae with or w i t h o u t eye d i s e a ~ Mice sampled between d a y s 8 and 43 were divided into taro groups, those with eye disease (diseased group) and those with no signs of eye d i s e a ~ a t the t i m e the), were killed (non diseased group). W h e n LC counts for d a y s 8 to 43 in the disemued group w e ~ pooled and compared with similarly pooled d a t a for the non d i ~ a s e d group, the LC n u m b e r s in the former group were significantly higher t h a n in t h e l a t t e r (Table If). Both groups had significantly higher counts t h a n controls. Mot~cover, the I;C' counts in the limbus of the eontralaterai (uninoculatcd) eye of mice inoculat~J with virus wen~ significantly tdgher t h a n those o f controls. (b) Changes in LC counts with time L i m b u s : In the diseased group LC n u m l ~ r s reached a m a x i m u m on d a y 8, ~ m a i n e d high until d a y 22 and l~ll to within the normal range b y d a y 30. In the non-d| group t h e rise in LC n u m b e m was small a n d t r a n s i e n t (Fig. 2). Statistical analysis of the d a t a showed t h a t the results in both these groups were not homogeneous indicating difl:erenccs between t h e n u m b e r s of LCs a t d i f f e ~ n t times after inoculation (Fig. 2). Central cornea: N u m b e r s of h ~ s in disea~3d mice ~uzaehed a m a x i m u m on d a y 8 and were still i ~ i ~ d b y d a y 30, b u t had r~turned ~ normal levels b y d a y 43 (Fig. 3). However, two o u t of fc)ur mice s a m p l e ] on d a y 65 ha~J raised LC n u m b e r s in the cex~tral cornea (Fig° 4). StatistAeal analysis o f t h e d a t a for both the di d a n d non d i s e ~ e d groups showc~ t h a t t h e ~zsults w e ~ n o t horn eous i n d i c a t i n g significant d i f f e ~ n e e s between t h e n u m b e r s of LCs ~ d i f f e ~ n t times aster inoculation (Fig. 3).
I36
N. J . L E W K O ~ , ¢ ' I C Z - M O S N
ET AL.
Fro. 9+ A n e p i t h e l i a l s h t + t |Yore a n e y e o f a m o u ~ 25 d ~ v s a f a r a s n o u t i n o c u l a t i o n w i t h 1 × I 0 ~ p f u H SV ! SC l 6~ A n area o f IX~'a c c u m m u l a t i o n is s ~ n ~ w h e r e a s o t h e r a ~ a s o f t h e c ~ n t r a l ~ r ~ m a a p l ~ a r fr¢~ o f IA?s+ B a r = 5 { ~ / i r a ,
Snout inoculation (a) LC counts in mice with or w i t h o u t eye di~¢ea~ Mice sampled between d
LCs IN MOUSE
CORNEA
AFTER
HSVI
INFECTION
t37
Flo~ 10. A high*lrower light m i c r o ~ a p h o f lmh~gerhans t~lls from within the d u s ~ r d e m o n s t r a ~ in Fig. 9~ T h e ¢~ndritic p r o ~ s ~ s are highly ar~orized at~d dendr4tes from different ~ l / a a p ~ a r ~ form connections (arn~ws). l~ar = 50/tin.
In ,nice with eye disease after snout, inoculation, LCs initially spread from t h e limbus over t h e e n t i ~ surfac~ of t h e cornea, b u t in certain a r e ~ t h e y w e ~ c o n c e n t r a t e d in small c l u s ~ r s (Fig. 8). B y day 25 when t h e LC n u m b e r s in the central cornea veerc d r o p p i n g a n d a ~ o f epithelium were ~ c o m i n g devoid o f thee~ cells, a variable n u m b e r of dense foe| of L t ~ remained (Fig. 9). T h e LCs within thcJse foe| were generally highly dendritic (Fig. 10). 4. D i s c u s s i o n
In c o m m o n with previous ~ p o r t s on I ~ distribution in t h e cornea of various animal s ~ c i ~ (Brown e t a ] . , I968; K | og, Forsum, ~ e r n l u n d , and Petez~on, !979; Rodrigues e t ai., 1981 ; G i | l e t ~ e t a|o, I982) we have found t h a t in n o r m a l mouse eyes, corneal LCs wez~ a b u n d a n t in t h e limbus, b u t sparse or totally a b s e n t from the central ~ r n e a . i ~ the high c o u n t i n g error, which could accounted for by low hUm bel~ o f LCs in some specimens a n d n o n uniform distributions in othem, t h e r e was clearly a highly significant i n c ~ e ~ in LCs following H S V infection. HSV antigens can be d e t e c t e d in t h e cornea one day, after diz~ect corneal inoculation with virus, whereas after inoculation on t h e snout, from w h e ~ virus spreads ~ the eye via t h e n e r v o u s system (Shimeld e t aL, 1987) a n t i g e n s can be d e l e t e d in t h e cornea from day" 3 (Shimeld e t a l . 1986). I n t h e p ~ s e n t s t u d y we h a v e d e m o n s d increased LCs in corneal e p i t h d i u m 3 days a f a r cornoal inoculation and 6 days an inoculation on t h e snout. In each ~ e this indicates a lag of 3 ~ d a y s b~tween t h e arrival of t h e virus in t h e cornea and t h e initial ~se in LC numbers. A similar delay hms been reported following chemical injuT3, (Brown e t al., 1968L
138 s..I.I.EWKOWICZ*MOSS ET AI.. The f a c t o ~ contro|ling LC migration into the com~ca art~ not fldIy understood. Passive infiltration after wound healing m a y play a part (Giaconmtti, 1969)~ However. this ~ e m s an unlikely explanation of our Tvsults, as no increase in LC n u m b e r s was seen after scarification of the e~)rnea t h r o u g h mock inoculum. However, it is now rccognised t h a t LCs act ~ a n t i g e n - p r e ~ n t i n g cells l)oth in delayed hypersensitivity reactions (Silerberg-Sinakin and Thorheeke, 1980) a n d in Mlograft I<~jection (Rowden, 1980). Moreover, these cells have b~en shown ~ present HSV antigens to T i y m p h o c y t e s (Braathen, Berle, Mobech-Hanssen and T h o ~ b y , 1980). Therefore the iuc~zase in I~5:s ah~er herpetic infections m a y be a response to an antigenic stimulus. in delayc~J hypersensitivity reactions, the allergen is believed to induce t h e release of lymphokines, which in turn h a v e a ehemotactic effect on LCs (Roussel, Osato and Withebnus, ! 983). The plaque-like distribution of HSV antigens in corneal epithelium after an inoculation on the s n o u t (Shimeld et a i , 1986) and t h e similar clustering of LCs when HSV is i n o e u l a ~ d by this route, suggests t h a t the~e cells m a y be d ~ w n to the ~ e i of viral antigens. With either r o u ~ of inoculation, LC numbet~ ~zmained high long after d a y 10, particularly in mice with obvious clinical c~mmal d i ~ a ~ . By this time infectious virus and viral antigens would have been clea~
LCs IN MOUSE CORNEA AFT E R HSVI INFECTION
139
REFERENCES Blyth, W. A., Harbour, D. A. and Hill, T. J. (1984). Pathogenesis of zost~riform s p e n d of h e r ~ s simplex virus in the mouse~ J . Gen. ViroL 65, 1 4 7 7 - ~ Braathen, L. R , l~rle, E , Mobech-Han~en, N. and Thorsby, E. (19~). Studies on human epidermal Langerhans c~lls: 11 activation of human T lymphocytes to herl>eS simplex virus. Acts Dervnatovener. (Stockholm)6~0, 381~7. Brown, J~, ~ d e m t r o m , C. W~ and Winkelmann, R. K, (1968). ~ n g e r h a n 8 c~lls in guinea-pig cor~ea. Pceswan~ ~ chemical injury. Invest. OptdhalmoL 7, 668-71. Chaker, M. B., Tharp, M. D, and B e r g s t ~ r , P. R. (1984). Rodent epidermal l~ngerhans c~ells demonstrate g ~ a t e r histochemieal sl~cificity for A D P than fi)r ATP and AMP. J. Iuve~t. DermatoL 82, 4 ~ - 5 ~ 9 . Cole. S., L~wkowicu, 8. ,L and Townsend, K, M. 8. (1984). Imngerhan8 c~l| number and morpholo~¢ in mouse footpad epidermis after X irradiation. . Res. I ~ , 594606. Giaeometti, L. (1969). The healing of skin wounds in primates. III behaviour of the c~l!s of Langerhans. J. Invest. Dermat~. 53, t 5 1 4 , Gillette, T. E., Chandler, ,L W. and Greiner, J . V . (1982L Imngerhans ce}ls of the ocular surfa(~. Olddk~lmoloyy, 89, 7 ~ - 1 0 . Gschnait, F. and Br~enner, W. C. (1979). Kineti¢~ ofepididermal Langerhans ceils, d. l)ermat~. 73, 566-9, Hazlett~ L~ 1), Grevengood, C. and Berk, g. 8. (1984L Respon~ of routine ocular Langerhans' cells ~ P, Auruginosa corneal infection . ~ d d h a h n ~ . Vi~. Sci. ~ , 21.
Hill, T. J., Field, H. J. and Blyth, VV. A. (1975). A c u ~ and r e e u r ~ n t infection with herpes simplex virus in the mouse: a model for studying latency and ~ c u r r e n t d i ~ a ~ , d. Gen. ViroL 28~ 341-53. Juhlin, L. and Shelley, W. B. (!977). New s~ining techniques for the I~ngerhans cells. Acta r~r. (~t~kholrn) 57, 289-96. Katz, S~ I., Tamaki, K. and Sachs~ D. H. (1979)~ Epidermal Langerhans ~lls are derived ~om ~ l l s originating in bone marrx~w. Nature ( L o ~ o n ) 282, 324-6. Klar~skog, L., Forsum, U., Tjernlund, U.M., R ~ k , L. and Pe~r:~on, P . A . (1979L Expression of Ia antigen-like moleeul~s on cells in the corneal epithelium lmoL 18, 310-3. Lang, R. M., Fri~laender, M. H. and Sehoenrock, B. d. (1981). ~ n g e r h a n s ~!1 induction and migration in guinea pig cornea, lnvesL @hthalmoL Vis. 8eL 20 (Suppl.L 1. Mackenzie, I.C. and Squier, C. A. (1975L Cytoehemical identification of ATl~se-wositive cells in EDTA s e p a ~ t e d sheets of m o u ~ epidermis~ Br. J, DernaatoL 92, 523-33. Pep(me, J. S., Gardner, K. M., N e s ~ r , M. S., Foos, R. Y. and Pettit, T. H. (1985), Detection of HLA class I and II antigens in ~jected humau corneal all . 92, Rodrigu~, M. M., Rowden, G., Hackett, J . and Bakos, I. (1981). Langerhans cells in normal ~ n j u n e t i v a and Feripheral ~ r n e a of ~ l e c ~ d settles. Invest. O p ~ . Vi~. Sci~ 21, 759~65. Roussel, T. J., Osato, M. 8. and Vgi!helmu8, K. R. (1983). C~rneal Langerhans cell migration following ocular e o n ~ e t hy~mensitivityo C o ~ e a 2, 27-30. Rowden, G. (1980). Exp~ssion of Ia antigens on Langerhans c~lls in mice, guinea pigs and man. J, I ~ t . De . 75, 22-31. Rubsamen, P. E., MeCulley, J.~ Bergstresser, P. R. and St~ilein, J. W. (!984). On the la immunogenicity of mouse c~rneal allografts infil with Langerhans ¢~ils. . Op I. Vis. Sci. 25, 513-8. Shimeld, Co, Easty, D. L . Tullo, A. B,. Biyth, W. Ao and Hill~ T. J. (1985), Spread of h e r ~ s simplex virus ~ the eye following cutane~Jus inoculation in the skin of the snout of the mouse. I n D ~ Op 44, t ~ ~ye . (Eds Maudgal, D, C. and Missotten, L.). Dr W. J u n k : D o r d ~ c h t / B ~ t o n / L a n c a s t e r . Shirneld, C., ~ w k o w i ~ - M o g s , S. J., Lipworth, K., Hi!l, T. J., Blyth, W. A. and Easty, D. L. (1986). Antigens of herpes simplex virus in whole corneal epithelial shee~ from mice. Arch. OphlhalmoL 104, 1 8 3 0 ~
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8. J. LEWKOWI(:Z.MOSS ET AL.
Shimeld, C., Dyson, H., Lewkowiez-Moss, S,, Hill, T. ,I., Blyth~ W. A. and Ea~ ty. t). 1,, (1987}. Spread of HSV-I to tile mou~ ~¢e after inoculation in the skin of the snout ~quires an intmet nerve supply to ~he inoeu|ation site. Curr. I'(tle Re.~, 6. 9~-12. Sil~rberg-Sinakin, 1. and Thorb~ke, G. J. (|980), Contact hypersensitivity and Langerhans ~lls. J. Invest. De~atoL 75, 61-7. Sting, G., Tamaki, K. and Katz, S. L (1980). Origin and function of epidermal I~angerhans ~lls, [mmunol. Ret,. 53, 149~74. Streilein, J. W., Toews, O. B. and Be r, P. R+ (1980}, Langerhans (~lls: flmetional ~imct~ m;~aled by in vhv ~ a ~ i n g studies. J. Invest. Dern~tol, 75, 17~21. Toews, G . B . B e r g s t ~ r , P.R., St~ilein, J. W, and Sullivan~ S. (i9~). Epiderma| ~ngerhans ~ll density de~rmin~ whether contact hyi~r~nsitivity or unto.~* sponsivene~ follows skin painting with DNFB. J. lmmunoL 124, 445-53. Tu|to, A. B., Shimeld, C., Blyth, W, A., Hill, T. J, and Easty D. L. (I983). Ocular infection with herk~s simplex virus in non immune and immune mic~. Arch. Ol~thalmol, 101. ~1-4.