Clin. exp. Immunol. (1991). 86, 322-327 Suppressive effect of hyperbaric oxygenation on immune responses of normal and autoimmune mice K. SAITO, Y. TANAKA, T. OTA, S. ETO & U. YAMASHITA* First Department of Internal Medicine, University of Occupational and Environmental Health, School of Medicine, Kitakyushi and *Department of Immunology and Parasitology, Hiroshima University, School of Medicine, Hiroshima, Japan (Accepted for publication 15 May 1991) SUMMARY We studied the effect of hyperbaric oxygenation (HBO) on immune responses in normal and autoimmune mice. Mice were exposed to HBO in an animal chamber at a pressure of 252-5 kPa for 1 h and once a day for 5 days. The immunization of C3H/He mice with sheep erythrocytes induced marked anti-sheep erythrocyte antibody response on day 7. However, this response was markedly suppressed in HBO-treated mice. The suppression is dependent on the duration of HBO and it works on the early and the late stage of antibody responses. HBO suppresses the development of both sheep erythrocyte-specific B cells and helper T cells after the immunization. Then, we tried to expose autoimmune mice to HBO. Spontaneous immunoglobulin production of NZB and MRL/lpr spleen cells was also significantly suppressed by HBO. Furthermore, long term HBO exposure results in the suppression of the development of autoimmune symptoms such as proteinuria, facial erythema and lymphadenopathy in MRL/lpr mice. All these treatment of autoimmune diseases. results suggest that HBO is applicable for the Keywords HBO lupus mouse autoantibody INTRODUCTION Hyperbaric oxygenation (HBO) therapy is clinically applied for patients with local hypoxia such as skin ulcer, cerebral infarc- tion and sudden deafness. It is widely accepted that the marked increase of dissolved oxygen in the blood and tissues results in the promotion of recovery from such diseases. Some investiga- tors demonstrated that the exposure of experimental animals to HBO induced the suppression of several cell-mediated immune responses such as allograft rejection (Jacob, Thurning & Sacksteder, 1978) and delayed type hypersensitivity responses to tuberculin protein (Warren, Sacksteder & Jacob 1978a). It is also reported that the functions of leukocytes and macrophages are suppressed by in vitro hyperoxidation (Murphy, Hyams & Fisher, 1975; Babior, 1978). However, there has not been a full examination of the mechanism of the immunosuppressive effect of HBO. In a previous report (Saito et al., 1989) we showed that HBO markedly reduced the level of serum IgG, anti-DNA antibody and immune complexes in patients with systemic lupus erythematosus. This evidence suggests that HBO may have some regulatory functions on immune responses. In this paper we studied the effect of HBO on immune responses using sheep erythrocyte-immunized mice as a model system. Furthermore, Correspondence: Dr Uki Yamashita, Department of Immunology and Parasitology, Hiroshima University, School of Medicine, 1-2-3, Kasumi, Minamiku, Hiroshima, 734, Japan. we tried to expose autoimmune mice to HBO in order to evaluate the therapeutic application of HBO to autoimmune diseases. MATERIALS AND METHODS Animals C3H/He, NZB and MRL/lpr mice were purchased from Seiwa Experimental Animal Ltd. (Yoshitomi, Japan) and bred in our laboratory. Six-month old female C3H/He, 4-month old female NZB and 2-month old female MRL/lpr mice were used for experiments. Exposure to HBO Mice caged in groups of 10 were exposed to HBO in an animal chamber at the pressure of 252-5 kPa for 1 h once a day. After at least 5 min of flushing, the exhaust valve was closed and the flow from the commercial oxygen tank was adjusted to the rate of compression of 252 5 kPa. At the end of the treatment, the tank was decompressed slowly (50 5 kPa/min). Immunization of mice C3H/He mice were immunized with an i.p. injection of 5 x 108 sheep erythrocytes. Anti-sheep erythrocyte antibody response was detected by a direct haemolytic plaque-forming cell (PFC) assay using spleen cells. 322
HBOT and Autoimmune Disease
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C l i n . e x p . I m m u n o l . ( 1 9 9 1 ) . 8 6 , 3 2 2 - 3 2 7S u p p r e s s i v e e f f e c t o f h y p e r b a r i c o x y g e n a t i o n o n i m m u n e r e s p o n s e so f n o r m a l a n da u t o i m m u n e m i c eK . S AI TO, Y . TANAK A, T. OTA, S . ETO&U. Y AMAS HI TA*F i r s t D e p a r t m e n t o f I n t e r n a l Me di c i n e , Un i v e r s i t y o fOc c u p a t i o n a l a n d En v i r o n m e n t a l He a l t h , S c h o o l o f Me di c i n e , K i t a k y u s h i a n d *D e p a r t m e n t o f I m m u n o l o g y a n d P a r a s i t o l o g y ,Hi r o s h i m a Un i v e r s i t y , S c h o o l o f Me di c i n e , Hi r o s h i m a , J a p a n( Ac c e p t e d f o r p u b l i c a t i o n 1 5 Ma y 1 9 9 1 )S UMMARYWe s t u di e d t h e e f f e c t o f h y p e r b a r i c o x y g e n a t i o n ( HBO) o n i m m u n e r e s p o n s e s i n n o r m a l a n da u t o i m m u n e m i c e . Mi c e we r e e x p o s e dt o HBOi n a n a n i m a l c h a m b e r a t a p r e s s u r e o f 2 5 2 - 5 k P a f o r 1 ha n d o n c e a da y f o r 5 da y s . Th e i m m u n i z a t i o n o f C 3 H/He m i c e wi t h s h e e p e r y t h r o c y t e s i n du c e dm a r k e d a n t i - s h e e p e r y t h r o c y t e a n t i b o dy r e s p o n s e o n da y 7 . Ho we v e r , t h i s r e s p o n s e wa s m a r k e dl ys u p p r e s s e d i n HBO- t r e a t e dm i c e . Th e s u p p r e s s i o n i s de p e n de n t o n t h e du r a t i o n o f HBOa n d i t wo r k so n t h e e a r l y a n dt h e l a t e s t a g e o f a n t i b o dy r e s p o n s e s . HBOs u p p r e s s e s t h e de v e l o p m e n t o f b o t h s h e e pe r y t h r o c y t e - s p e c i f i c B c e l l s a n d h e l p e r T c e l l s a f t e r t h e i m m u n i z a t i o n . Th e n , we t r i e d t o e x p o s ea u t o i m m u n e m i c e t o HBO. S p o n t a n e o u s i m m u n o g l o b u l i n p r o du c t i o n o f NZBa n d MRL/l p r s p l e e nc e l l s wa s a l s o s i g n i f i c a n t l y s u p p r e s s e d b y HBO. F u r t h e r m o r e , l o n g t e r m HBOe x p o s u r e r e s u l t s i n t h es u p p r e s s i o n o f t h e de v e l o p m e n t o f a u t o i m m u n e s y m p t o m s s u c h a s p r o t e i n u r i a , f a c i a l e r y t h e m a a n dl y m p h a de n o p a t h y i n MRL/l p r m i c e . Al l t h e s et r e a t m e n t o f a u t o i m m u n e di s e a s e s .r e s u l t s s u g g e s t t h a t HBO i s a p p l i c a b l e f o r t h eK e y wo r ds HBO l u p u s m o u s e a u t o a n t i b o dyI NTROD UC TI ONHy p e r b a r i c o x y g e n a t i o n ( HBO) t h e r a p y i s c l i n i c a l l y a p p l i e d f o rp a t i e n t s wi t h l o c a l h y p o x i a s u c h a s s k i n u l c e r , c e r e b r a l i n f a r c -t i o n a n d s u dde n de a f n e s s . I t i s wi de l y a c c e p t e d t h a t t h e m a r k e di n c r e a s e o f di s s o l v e d o x y g e n i n t h e b l o o d a n d t i s s u e s r e s u l t s i nt h e p r o m o t i o n o f r e c o v e r y f r o m s u c h di s e a s e s . S o m e i n v e s t i g a -t o r s de m o n s t r a t e d t h a t t h e e x p o s u r e o f e x p e r i m e n t a l a n i m a l s t oHBOi n du c e d t h e s u p p r e s s i o n o f s e v e r a l c e l l - m e di a t e d i m m u n er e s p o n s e s s u c h a s a l l o g r a f t r e j e c t i o n ( J a c o b , Th u r n i n g &S a c k s t e de r , 1 9 7 8 ) a n dde l a y e dt y p e h y p e r s e n s i t i v i t y r e s p o n s e s t ot u b e r c u l i n p r o t e i n ( Wa r r e n , S a c k s t e de r &J a c o b 1 9 7 8 a ) . I t i sa l s o r e p o r t e d t h a t t h e f u n c t i o n s o f l e u k o c y t e s a n dm a c r o p h a g e sa r e s u p p r e s s e d b y i n v i t r o h y p e r o x i da t i o n ( Mu r p h y , Hy a m s &F i s h e r , 1 9 7 5 ; Ba b i o r , 1 9 7 8 ) . Ho we v e r , t h e r e h a s n o t b e e n a f u l le x a m i n a t i o n o f t h e m e c h a n i s m o f t h e i m m u n o s u p p r e s s i v e e f f e c to f HBO. I n a p r e v i o u s r e p o r t ( S a i t o e t a l . , 1 9 8 9 ) we s h o we d t h a tHBO m a r k e dl y r e du c e d t h e l e v e l o f s e r u m I g G , a n t i - D NAa n t i b o dy a n di m m u n e c o m p l e x e s i n p a t i e n t s wi t h s y s t e m i c l u p u se r y t h e m a t o s u s . Th i s e v i de n c e s u g g e s t s t h a t HBO m a y h a v es o m e r e g u l a t o r y f u n c t i o n s o n i m m u n e r e s p o n s e s . I n t h i s p a p e rwe s t u di e d t h e e f f e c t o f HBOo n i m m u n e r e s p o n s e s u s i n g s h e e pe r y t h r o c y t e - i m m u n i z e d m i c e a s a m o de l s y s t e m . F u r t h e r m o r e ,C o r r e s p o n de n c e : D r Uk i Y a m a s h i t a , D e p a r t m e n t o f I m m u n o l o g ya n d P a r a s i t o l o g y , Hi r o s h i m a Un i v e r s i t y , S c h o o l o f Me di c i n e , 1 - 2 - 3 ,K a s u m i , Mi n a m i k u , Hi r o s h i m a , 7 3 4 , J a p a n .we t r i e d t o e x p o s e a u t o i m m u n e m i c e t o HBO i n o r de r t oe v a l u a t e t h e t h e r a p e u t i c a p p l i c a t i o n o f HBO t o a u t o i m m u n edi s e a s e s .MATERI ALS AND METHOD SAn i m a l sC 3 H/He , NZBa n d MRL/l p r m i c e we r e p u r c h a s e d f r o m S e i waEx p e r i m e n t a l An i m a l Lt d. ( Y o s h i t o m i , J a p a n ) a n d b r e d i n o u rl a b o r a t o r y . S i x - m o n t h o l d f e m a l e C 3 H/He , 4 - m o n t h o l d f e m a l eNZB a n d 2 - m o n t h o l d f e m a l e MRL/l p r m i c e we r e u s e d f o re x p e r i m e n t s .Ex p o s u r e t o HBOMi c e c a g e d i n g r o u p s o f 1 0 we r e e x p o s e d t o HBOi n a n a n i m a lc h a m b e r a t t h e p r e s s u r e o f 2 5 2 - 5 k P a f o r 1 h o n c e a da y . Af t e r a tl e a s t 5 m i n o f f l u s h i n g , t h e e x h a u s t v a l v e wa s c l o s e da n dt h e f l o wf r o m t h e c o m m e r c i a l o x y g e n t a n k wa s a dj u s t e d t o t h e r a t e o fc o m p r e s s i o n o f 2 5 2 5 k P a . At t h e e n d o f t h e t r e a t m e n t , t h e t a n kwa s de c o m p r e s s e d s l o wl y ( 5 0 5 k P a /m i n ) .I m m u n i z a t i o n o f m i c eC 3 H/He m i c e we r e i m m u n i z e d wi t h a n i . p . i n j e c t i o n o f 5 x 1 0 8s h e e p e r y t h r o c y t e s . An t i - s h e e p e r y t h r o c y t e a n t i b o dy r e s p o n s ewa s de t e c t e d b y a di r e c t h a e m o l y t i c p l a q u e - f o r m i n g c e l l ( P F C )a s s a y u s i n g s p l e e n c e l l s .3 2 2S u p p r e s s i v e e f f e c t o f HBOo n i m m u n e r e s p o n s eP r e p a r a t i o n o fTc e l l s a n dB c e l l sS p l e e n c e l l s u s p e n s i o n s we r e p r e p a r e d f r o m m i c e b y g e n t l ydi s p e r s i n g t h e t i s s u e i n HBS S ( Ni s s u i S e i y a k u C o . , To k y o ,J a p a n ) a n d e r y t h r o c y t e s we r e l y s e d b y t r e a t m e n t wi t h Tr i s -NH4 C l . Th e s p l e e n c e l l s s u s p e n de d i n RP MI 1 6 4 0 m e di u m( Ni s s u i S e i y a k u C o . ) c o n t a i n i n g 5 % f e t a l c a l f s e r u m ( F C S ,G r a n d I s l a n d Bi o l o g i c a l C o . , G r a n d I s l a n d, NY ) we r e a p p l i e do n n y l o n wo o l c o l u m n ( F e n wa l La b o r a t o r i e s , Mo r t o n G r o v e ,I L) . Af t e r i n c u b a t i o n o f c e l l s o n t h e c o l u m n f o r I h a t 3 7 C , n o n -a dh e r e n t c e l l s we r e e l u t e d dr o p wi s e . Th e c e l l r e c o v e r y f r o m t h ec o l u m n wa s a b o u t 1 0 % o f t h e o r i g i n a l c e l l s a p p l i e d. Th er e c o v e r e d c e l l s we r e c o m p o s e d o f m o r e t h a n 9 5 %Th y l + c e l l sa n d di d n o t r e s p o n d t o B c e l l m i t o g e n s u c h a s l i p o p o l y s a c c h a r -i de ( LP S ) f r o m Es c h e r i c h i a c o l i a n dwe r e u s e da s p u r i f i e d Tc e l l s .S p l e e n c e l l s we r e a l s o t r e a t e d wi t h a n t i - Th y l - 2 m o n o c l o n a la n t i b o dy ( C e da r l a n e La b o r a t o r i e s , Ot t a wa , C a n a da ) a n dr a b b i tc o m p l e m e n t ( C e da r l a n e La b o r a t o r i e s ) a t 3 7 C f o r 1 h . Th e c e l lr e c o v e r y wa s a b o u t 4 0 % o f t h e o r i g i n a l c e l l n u m b e r s t r e a t e d.Th e r e c o v e r e d c e l l s we r e c o m p o s e d o f m o r e t h a n 9 0 %i m m u n o -g l o b u l i n - p o s i t i v e c e l l s a n d di d n o t r e s p o n d t o T c e l l m i t o g e ns u c h a s c o n c a n a v a l i n A a n d we r e u s e d a s p u r i f i e d B c e l l s( Y a m a s h i t a &Ha m a o k a , 1 9 7 9 ) .S e c o n da r y a n t i b o dy p r o du c t i o n a g a i n s t s h e e p e r y t h r o c y t e sP u r i f i e d B c e l l s ( 2 - 5 x 1 0 5 ) a n d T c e l l s ( 2 - 5 x 1 0 5 ) f r o m HBO-t r e a t e d o r u n t r e a t e d C 3 H/He m i c e p r i m e d wi t h s h e e p e r y t h r o -c y t e s we r e m i x e d i n s e v e r a l c o m b i n a t i o n s a n d c u l t u r e d wi t hI x 1 0 6 s h e e p e r y t h r o c y t e s i n RP MI 1 6 4 0 m e di u m ( Ni s s u iS e i y a k u C o . ) c o n t a i n i n g 1 0 % F C S u s i n g m i c r o t i t r e c u l t u r ep l a t e s ( F a l c o n No . 3 0 7 2 , F a l c o n P l a s t i c s , Ox n a r d, C A) a t 3 7 Cf o r 5 da y s i n 5 % C O2 a n d 9 5 % a i r . An t i - s h e e p e r y t h r o c y t ea n t i b o dy r e s p o n s e wa s de t e c t e d b y P F C a s s a y o n da y 5 .As s a y o f a n t i b o dy p r o du c t i o n b y s p l e e n c e l l sAn t i b o dy - p r o du c i n g c e l l s we r e de t e c t e d b y a di r e c t h a e m o l y t i cP F C a s s a y o n s l i de c h a m b e r s wi t h s h e e p e r y t h r o c y t e s . I nm e a s u r i n g t o t a l i m m u n o g l o b u l i n p r o du c t i o n o f s p l e e n c e l l sf r o m NZBa n dMRL/l p r m i c e , i m m u n o g l o b u l i n - p r o du c i n g c e l l swe r e de t e c t e d b y a r e v e r s e h a e m o l y t i c P F C a s s a y o n s l i dec h a m b e r s wi t h p r o t e i n A( P h a r m a c i a F i n e C h e m i c a l s , Up p s a l a ,S we de n ) c o a t e d s h e e p e r y t h r o c y t e s ( P i e r c e e t a l . 1 9 7 1 ; G r o n o w-i c z , C o u t i n h o &Me l c h e r s , 1 9 7 6 ) . I n di r e c t P F C , f a c i l i t a t e d b yr a b b i t a n t i - m o u s e I g G s e r u m , we r e de t e c t e d. Th e r e s u l t s we r ee x p r e s s e d a s t h e m e a n n u m b e r s a n ds . e . m . o fP F C /5 x 1 0 5 s p l e e nc e l l s i n t r i p l i c a t e c u l t u r e s .Ev a l u a t i o n o f c l i n i c a l e f f e c t o f l o n g t e r m HBOe x p o s u r eTwo - m o n t h - o l d f e m a l e MRL/l p r m i c e we r e e x p o s e d t o HBOo n c e ada y f o r 2 m o n t h s i n t h e c o n di t i o n s de s c r i b e da b o v e . Th e nt h e s u r v i v a l o f m i c e , b o dy we i g h t a n d we i g h t o f b i l a t e r a li n g u i n a l l y m p h n o de we r e m e a s u r e d. Ur i n e p r o t e i n wa s m e a -s u r e d s e m i q u a n t i t a t i v e l y b y m e a n s o f C o m b i s t i c s ( S a n k y o C o . ,To k y o , J a p a n ) . Ag r a di n g o f 0 - 4 wa s m a de o n t h e b a s i s o f t h ep r o t e i n c o n c e n t r a t i o n i n t h e u r i n e , t h e f i v e c a t e g o r i e s b e i n g0 - 3 0 , 3 0 - 1 0 0 , 1 0 0 - 3 0 0 , 3 0 0 - 1 0 0 0 a n d 1 0 0 0 m g /dl , r e s p e c t i v e l y .Ur i n e o f e a c h m o u s e wa s m e a s u r e d f o r 3 da y s a n dm e a n s c o r e swe r e a v e r a g e d i n b o t h c o n t r o l a n d HBOg r o u p . An t i - n u c l e a ra n t i b o di e s i n t h e s e r a we r e de t e c t e d b y ELI S A. Th e r e s u l t s we r ee x p r e s s e d a s t h e o p t i c a l de n s i t y u n i t s a t 4 0 5 n m o f wa v e l e n g t h .S t a t i s t i c a l a n a l y s i sS t a t i s t i c a l a n a l y s i s o f t h e di f f e r e n c e b e t we e n t h e c o n t r o l a n dt h ee x p e r i m e n t a l g r o u p wa s p e r f o r m e d u s i n g n o n - p a r a m e t r i cMa n n - Wh i t n e y Ut e s t .RES ULTSEf f e c t o f HBOo n a n t i - s h e e p e r y t h r o c y t e a n t i b o dy r e s p o n s eC 3 H/He m i c e we r e i m m u n i z e dwi t h s h e e p e r y t h r o c y t e , e x p o s e dt o HBO a t 2 5 2 5 k P a f o r I h o n c e a da y a n d a n t i - s h e e pe r y t h r o c y t e a n t i b o dy r e s p o n s e wa s de t e c t e d b y a di r e c t h a e m o -l y t i c a s s a y u s i n g s p l e e n c e l l s a t s e v e r a l i n t e r v a l s a f t e r i m m u n i z a -t i o n . As s h o wn i n F i g . 1 , a n t i - s h e e p e r y t h r o c y t e P F C r e s p o n s e so f c o n t r o l m i c e we r e i n c r e a s e d t o t h e l e v e l o f 4 0 0 /1 x 1 0 6 s p l e e nc e l l s o n da y 7 a n d t h e n g r a du a l l y de c r e a s e d. Ho we v e r , a n t i -s h e e p e r y t h r o c y t e P F C r e s p o n s e s o f HBO- e x p o s e d m i c e we r em a r k e dl y r e du c e d t o 1 5 0 /1 x 1 0 6 s p l e e n c e l l s , a l t h o u g h t h e t i m ec o u r s e o f a n t i b o dy r e s p o n s e o f HBO- t r e a t e dm i c e wa s s i m i l a r t ot h a t o f HBO- u n t r e a t e d m i c e . Th e n u m b e r o f s p l e e n c e l l sr e c o v e r e d a n d t h e c e l l v i a b i l i t y de t e r m i n e d b y a t r y p a n b l u e dy ee x c l u s i o n t e s t we r e n o t di f f e r e n t b e t we e n b o t hHBOa n dc o n t r o lg r o u p s . Th i s r e s u l t s u g g e s t s t h a t HBOh a s s u p p r e s s i v e a c t i v i t yo n a n t i g e n - i n du c e d i m m u n e r e s p o n s e s .K i n e t i c s t u dy o f t h e s u p p r e s s i v e e f f e c t o f HBOo n t h e a n t i b o dyr e s p o n s eNe x t , we s t u di e d t h e t i m i n g o fHBOe x p o s u r e o n t h e a n t i - s h e e pe r y t h r o c y t e P F C r e s p o n s e . We e x p o s e d s h e e p - e r y t h r o c y t e -i m m u n i z e dm i c e t o HBOf r o m da y 0 t o i n di c a t e d da y a n d s h e e p4 0 0a )4 )0xQLLI L3 0 02 0 01 0 000 ' 5 7 1 0D a y a f t e r i m m u n i z a t i o nF i g . 1 . Ef f e c t o f HBO t r e a t m e n t o n a n t i - s h e e p e r y t h r o c y t e P F Cr e s p o n s e . C 3 H/He m i c e we r e i m m u n i z e dwi t h 5 x I o 8 s h e e p e r y t h r o c y t e si n t r a p e r i t o n e a l l y o n da y 0 . Th e n , t h e m i c e we r e e x p o s e dt o HBOa t 2 5 2 5k P a f o r 1 h e v e r y da y u n t i l k i l l e d. An t i - s h e e p e r y t h r o c y t e P F C r e s p o n s ewa s de t e c t e d b y a di r e c t h a e m o l y t i c p l a q u e a s s a y u s i n g s p l e e n c e l l s o ni n di c a t e d da y . Th e r e s u l t s we r e e x p r e s s e d a s t h e m e a n P F C /1 x 1 0 6s p l e e n c e l l s + s . e . m . o f f i v e m i c e i n e a c h g r o u p .*S i g n i f i c a n t l y di f f e r e n tf r o m t h e c o n t r o l m i c e ( P
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