Albrecht v. Graefes Arch. klin. exp. Ophthal. 211,221 227 (1979)
Graefes Archiv Ophthalmologie for klinische und experimentelle
9 by Springer-Verlag 1979
Cellular Localization of Prostaglandin E in the Rabbit Iris by Indirect Immunofluorescence Method Hiroshi Sakata and Hideto Yoshida Department of Ophthalmology, Hiroshima University School of Medicine (Head: Prof. Dr. K. Choshi), 1-2-3-Kasumi, Hiroshima 734, Japan
Abstract. To demonstrate the cellular localization of prostaglandin E (PGE) in the normal and irritated rabbit iris, the indirect immunofluorescence method was employed. The amounts of PGE1 and PGF2~ in the aqueous humor were determined with radioimmunoassay. Immunofluorescence of P G E was observed in the cytoplasm of mesenchymal cells of the iris. PGEpositive cells in the normal iris were scattered in the fibrous stroma, and the shape of these cells was oval to spindle-shaped. The numbers of these cells were remarkably increased in the iridectomized and inflamed iris. The amounts of PGE1 in the aqueous humor was 2.0 + 0.53 ng/ml in the normal eyes and 7.92-+2.93 ng/ml in the irritated eyes. The mean level of PGF2~ was 0.4-+ 0.27 ng/ml in the normal eyes and 0.8 + 0.58 ng/ml in the irritated eyes.
Zusammenfassung. Um die cellul/ire Lokalisation von Prostaglandin E in normaler und entzfindeter Iris darzustellen, wurde die Methode der indirekten Immunofluoreszenz angewandt. AuBerdem stellte sich die Aufgabe, die Menge yon P G E 1 und PGF2~ im Kammerwasser radioimmunologisch zu bestimmen. Die Immunofluoreszenz yon PGE liel3 sich im Cytoplasma der mesenchymalen Zellen der Iris beobachten. Die PGE-positiven Zellen lagen im Normalfall in die fibrillgren Fasern des Sromas verstreut und zeigten eine ovale bis spindelartige Form. In der ausgeschnittenen und entztindeten Iris fand sich die Anzahl dieser Zellen merklich erh6ht. - Die Menge der PGE1 im Kammerwasser betrug im normalen Auge 2,0+0,53 ng/ml und im gereizten Auge 7,92_+ 2,93 ng/ml. Im normalen Auge lag der Durchschnittswert von PGF2~ bei 0.4_+0.27 ng/ml und im gereizten Auge bei 0.8 _+0.58 ng/ml.
Introduction It is well recognized that prostaglandin (PG) mediates inflammation (Jampol et al., 1975) and P G itself, when topically applied, causes irritation of eyes
0065-6100/79/0211/0221/$1.40
222
H. Sakata and H. Yoshida
s u c h as m i o s i s ( C a s e y , 1974), b r e a k d o w n o f t h e b l o o d - a q u e o u s b a r r i e r ( N e u f e l d et al., 1973; T a m u r a , 1974) a n d i n c r e a s e d i n t r a o c u l a r p r e s s u r e ( K a s s et al., 1972; G r e e n et al., 1975). P G s y n t h e s i s is k n o w n to b e e n h a n c e d u n d e r t h e m e c h a n i c a l s t i m u l i ( I z a w a et al., 1976). A l t h o u g h P G is g e n e r a l l y s u p p o s e d to be p r o d u c e d in o r v e r y close to t h e p l a c e w h e r e it exerts a c h a r a c t e r i s t i c a c t i o n ( D o r p et al., 1967), t h e e x a c t l o c a l i z a t i o n o f P G - p r o d u c i n g cells in t h e eye has n o t been clearly defined. T h e p u r p o s e o f this p a p e r is to d e m o n s t r a t e t h e c e l l u l a r l o c a l i z a t i o n o f P G E in the n o r m a l a n d i r r i t a t e d iris u s i n g t h e i n d i r e c t i m m u n o f l u o r e s c e n c e m e t h o d . I n a d d i t i o n , to c o n f i r m t h e i n c r e a s e d s e c r e t i o n in the i r i d e c t o m i z e d eyes, t h e a m o u n t s o f P G E 1 a n d PGFz~ in t h e a q u e o u s h u m o r o f t h e n o r m a l a n d i r r i t a t e d eyes w e r e d e t e r m i n e d b y t h e r a d i o i m m u n o a s s a y t e c h n i q u e .
Materials and Methods Ten adult white rabbits, weighing about 2.5 kg, were used. 1. Peripheral Iridectomy To produce ocular inflamation, peripheal iridectomy of one eye was done after corneoscleral section with the keratome. A suture thread was placed in the wound. The fellow eyes served as controls. Five animals were used for the indirect immunofluorescence method and the other five were reserved for the radioimmunoassay of PGE~ and PGF2e.
2. Indirect Immunofluorescence Method In the preliminary study, the antiserum was titered for optimal immunofluorescence. The highest dilution giving definite fluorescence was found to be 1:20 and designated as end point. The indirect immunofluorescence method was used modifying Perez' method (1972). The globes were enucleated from the rabbits 12 h after peripheral iridectomy and the anterior segments were immediately excised from the globes. The specimens were then fixed in neutral buffered formalin (10%) for 4 h at 4~ followed by an overnight wash in 40% sucrose solution. A portion of fixed tissue was mounted in OCT compound (Miles Laboratory, Naperville, Illinois, USA) and quick-frozen in isopentane dry ice. Cryostat sections were washed on 0.5 M PBS (pH 7.2) three times for 30 rain and incubated with the normal goat serum to inhibit a non-specific sedimentation to collagen fibers. Sections were then incubated with the specific antiserum for 30 rain, washed in PBS three times for 30 min, incubated with FITC-conjugated goat antirabbit IgG serum (1:20) for 30 min, washed in PBS three times for 30 min and mounted in buffered glycerol. All slides were viewed with Nikon fluorescence microscope. Control experiments including staining tissue with : 1) normal rabbit serum and the FITC-conjugated goat antirabbit IgG serum; 2) only FITC-conjugated goat antirabbit IgG serum; 3) anti-PGE 1 serum absorbed with purified PGE1 and FITC-conjugated goat antirabbit IgG serum to test for inhibition of fluorescence. FITC-conjugated goat antirabbit IgG serum was obtained from Miles Biochemicals, Elkhart, Indiana, USA. This antiserum was absorbed with mouse liver powder prior to use. Normal goat serum was purchased from Granite Diagnostic Inc., Barlington, North Carolina, USA. Anti-PGEI serum was supplied by Ono Pharmaceutical Co., Osaka, Japan. The preparation, potency, and specificity of the anti-PGE1 have been described by Inagawa et al. (1972). Adjacent sections were obtained and stained with H & E and examined with light microscope.
Prostaglandin E in the Iris
223
3. Determination of the Amounts of PGE 1 and PGF2~ in the aqueous Humor The amounts of PGE1 and PGF2~ in the aqueous humor 12 h after the iridectomy were assayed with the radioimmunoassay technique after Inagawa et al. (1972). Crude lipid extracts of sample obtained by the procedure of Folch et al. (1957) were separated from nonlipid contaminants on Sephadex G-25 columns. After evaporation of the eluted solution, the residue was dissolved in carbon tetrachloride and PG was extracted with water. The aqueous phase was acidified with ethyl acetate, the ethyl acetate layer was neutralized with ammonium hydroxide. After evaporation of the solvent, the residue was dissolved in methyl alcohol and PG fraction was obtained by thin-layer chromatography. Each sample was incubated with 3H-PGE1 (or 3H-PGF2~) and PGE~ (or PGF2~)-antiserum for 1 h in a water bath at 37~ C. After incubation, charcoal was added to each sample in a water bath at 4~ and immediately centrifuged at 2,000 rpm, 10 rain at 4~ C. Radioactivity of each vial was counted by a scintillation counter. Anti-PGE1, anti-PGF2~, 3H-PGE1, and 3H-PGF2~ were supplied by Ono Pharmaceutical Co., Osaka, Japan, The specifications of these reagents were reported in the literature cited above (Inagawa et aI., 1972).
Results 1. I m m u n o f l u o r e s c e n c e Findings As the antiserum used may not completely discriminate between PGE 1 and P G E 2 , t h e t e r m P G E will b e u s e d f o r a n y m a t e r i a l in t i s s u e t h a t c a n b e d e m o n strated by said antiserum.
Fig. 1. Normal iris stained with anti-PGE1 serum and FITC-conjugated antirabbit serum. Fluorescence was found in oval to spindlelike cells in the stroma. They were often located around the capillary (C). x 600
224
H. Sakata and H. Yoshida
Fig. 2. Inflamed iris stained with anti PGE1 serum and FITC-conjugated antirabbit serum. Positive cells were clustered around the capillaries (C). x 600
Fig. 3. Control iris stained with only FITC-conjugated arftirabbit serum, x 600
225
Prostaglandin E in the Iris Table 1. The amounts of PGE 1 and PGF2~ in the aqueous humor 12 h after peripheral iridectomy
PGE 1 (mean + SD ng/ml) PGF2~ (mean+SD ng/ml)
Normal eyes (N:4)
Operated eyes (N:5)
2.0 _+0.53 0.4_+0.27
7.92 _+2.93a (P < 0.005) 0.8+0.58 b
N=number a Differencestatistically significant u Not significant
A PGE-positive cell was one which demonstrated intense fluorescence in the cytoplasm. A negative cell was one in which no fluorescence was localized, but diffuse non-specific background fluorescence of variable degrees was also present. A c o m m o n characterisitc of positive cells was a spindle Or oval shape of the cellular cytoplasm with a slightly eccentric round nucleus (Fig. 1). Only a few PGE-positive cells in one tissue slice were detected in the normal eyes. They were often located around the capillaries. Numerous cells were observed in the inflamed iris with varying fluorescent intensity (Fig. 2). Blood plasma in the capillary lumen exhibited non-specific fluorescence of weaker intensity. Polymorphonuclear leucocytes did not show fluorescence, although it was reported that leucocytes m a y release P G during phagocytosis (Higgs et al., 1975). N o fluorescence was seen when normal rabbit serum was used instead of specific anti-PGE1 or when FITC-conjugated goat antirabbit I g G serum was used alone (Fig. 3). Absorption of anti-PGE~ with PGE1 decreased markedly the fluorescence of PGE-positive cells. Light microscopic examination of H & E stained specimens of adjacent sections showed polymorphonuclear leucocytes and oval to spindle-shaped mesenchymal cells around the dilated capillaries. These mesenchymal cells were thought to be young fibroblasts or macrophages.
2. The Amounts of PGE a and PGF2~ in the Aqueous Humor The mean PGE1 level in the aqueous h u m o r of the five inflamed eyes, as shown in Table 1, was 7.92_+2.93 ng/ml, and it was 2 . 0 + 0 . 5 3 n g / m l in the four control animals. The mean PGF2~ was 0.8+0.58 ng/ml in the inflamed eyes and 0.4+0.27 ng/ml in the normal eyes. The rise of PGE1 level in the inflamed eyes was statistically significant (p
Graefes Archiv Ophthalmologie for klinische und experimentelle
9 by Springer-Verlag 1979
Cellular Localization of Prostaglandin E in the Rabbit Iris by Indirect Immunofluorescence Method Hiroshi Sakata and Hideto Yoshida Department of Ophthalmology, Hiroshima University School of Medicine (Head: Prof. Dr. K. Choshi), 1-2-3-Kasumi, Hiroshima 734, Japan
Abstract. To demonstrate the cellular localization of prostaglandin E (PGE) in the normal and irritated rabbit iris, the indirect immunofluorescence method was employed. The amounts of PGE1 and PGF2~ in the aqueous humor were determined with radioimmunoassay. Immunofluorescence of P G E was observed in the cytoplasm of mesenchymal cells of the iris. PGEpositive cells in the normal iris were scattered in the fibrous stroma, and the shape of these cells was oval to spindle-shaped. The numbers of these cells were remarkably increased in the iridectomized and inflamed iris. The amounts of PGE1 in the aqueous humor was 2.0 + 0.53 ng/ml in the normal eyes and 7.92-+2.93 ng/ml in the irritated eyes. The mean level of PGF2~ was 0.4-+ 0.27 ng/ml in the normal eyes and 0.8 + 0.58 ng/ml in the irritated eyes.
Zusammenfassung. Um die cellul/ire Lokalisation von Prostaglandin E in normaler und entzfindeter Iris darzustellen, wurde die Methode der indirekten Immunofluoreszenz angewandt. AuBerdem stellte sich die Aufgabe, die Menge yon P G E 1 und PGF2~ im Kammerwasser radioimmunologisch zu bestimmen. Die Immunofluoreszenz yon PGE liel3 sich im Cytoplasma der mesenchymalen Zellen der Iris beobachten. Die PGE-positiven Zellen lagen im Normalfall in die fibrillgren Fasern des Sromas verstreut und zeigten eine ovale bis spindelartige Form. In der ausgeschnittenen und entztindeten Iris fand sich die Anzahl dieser Zellen merklich erh6ht. - Die Menge der PGE1 im Kammerwasser betrug im normalen Auge 2,0+0,53 ng/ml und im gereizten Auge 7,92_+ 2,93 ng/ml. Im normalen Auge lag der Durchschnittswert von PGF2~ bei 0.4_+0.27 ng/ml und im gereizten Auge bei 0.8 _+0.58 ng/ml.
Introduction It is well recognized that prostaglandin (PG) mediates inflammation (Jampol et al., 1975) and P G itself, when topically applied, causes irritation of eyes
0065-6100/79/0211/0221/$1.40
222
H. Sakata and H. Yoshida
s u c h as m i o s i s ( C a s e y , 1974), b r e a k d o w n o f t h e b l o o d - a q u e o u s b a r r i e r ( N e u f e l d et al., 1973; T a m u r a , 1974) a n d i n c r e a s e d i n t r a o c u l a r p r e s s u r e ( K a s s et al., 1972; G r e e n et al., 1975). P G s y n t h e s i s is k n o w n to b e e n h a n c e d u n d e r t h e m e c h a n i c a l s t i m u l i ( I z a w a et al., 1976). A l t h o u g h P G is g e n e r a l l y s u p p o s e d to be p r o d u c e d in o r v e r y close to t h e p l a c e w h e r e it exerts a c h a r a c t e r i s t i c a c t i o n ( D o r p et al., 1967), t h e e x a c t l o c a l i z a t i o n o f P G - p r o d u c i n g cells in t h e eye has n o t been clearly defined. T h e p u r p o s e o f this p a p e r is to d e m o n s t r a t e t h e c e l l u l a r l o c a l i z a t i o n o f P G E in the n o r m a l a n d i r r i t a t e d iris u s i n g t h e i n d i r e c t i m m u n o f l u o r e s c e n c e m e t h o d . I n a d d i t i o n , to c o n f i r m t h e i n c r e a s e d s e c r e t i o n in the i r i d e c t o m i z e d eyes, t h e a m o u n t s o f P G E 1 a n d PGFz~ in t h e a q u e o u s h u m o r o f t h e n o r m a l a n d i r r i t a t e d eyes w e r e d e t e r m i n e d b y t h e r a d i o i m m u n o a s s a y t e c h n i q u e .
Materials and Methods Ten adult white rabbits, weighing about 2.5 kg, were used. 1. Peripheral Iridectomy To produce ocular inflamation, peripheal iridectomy of one eye was done after corneoscleral section with the keratome. A suture thread was placed in the wound. The fellow eyes served as controls. Five animals were used for the indirect immunofluorescence method and the other five were reserved for the radioimmunoassay of PGE~ and PGF2e.
2. Indirect Immunofluorescence Method In the preliminary study, the antiserum was titered for optimal immunofluorescence. The highest dilution giving definite fluorescence was found to be 1:20 and designated as end point. The indirect immunofluorescence method was used modifying Perez' method (1972). The globes were enucleated from the rabbits 12 h after peripheral iridectomy and the anterior segments were immediately excised from the globes. The specimens were then fixed in neutral buffered formalin (10%) for 4 h at 4~ followed by an overnight wash in 40% sucrose solution. A portion of fixed tissue was mounted in OCT compound (Miles Laboratory, Naperville, Illinois, USA) and quick-frozen in isopentane dry ice. Cryostat sections were washed on 0.5 M PBS (pH 7.2) three times for 30 rain and incubated with the normal goat serum to inhibit a non-specific sedimentation to collagen fibers. Sections were then incubated with the specific antiserum for 30 rain, washed in PBS three times for 30 min, incubated with FITC-conjugated goat antirabbit IgG serum (1:20) for 30 min, washed in PBS three times for 30 min and mounted in buffered glycerol. All slides were viewed with Nikon fluorescence microscope. Control experiments including staining tissue with : 1) normal rabbit serum and the FITC-conjugated goat antirabbit IgG serum; 2) only FITC-conjugated goat antirabbit IgG serum; 3) anti-PGE 1 serum absorbed with purified PGE1 and FITC-conjugated goat antirabbit IgG serum to test for inhibition of fluorescence. FITC-conjugated goat antirabbit IgG serum was obtained from Miles Biochemicals, Elkhart, Indiana, USA. This antiserum was absorbed with mouse liver powder prior to use. Normal goat serum was purchased from Granite Diagnostic Inc., Barlington, North Carolina, USA. Anti-PGEI serum was supplied by Ono Pharmaceutical Co., Osaka, Japan. The preparation, potency, and specificity of the anti-PGE1 have been described by Inagawa et al. (1972). Adjacent sections were obtained and stained with H & E and examined with light microscope.
Prostaglandin E in the Iris
223
3. Determination of the Amounts of PGE 1 and PGF2~ in the aqueous Humor The amounts of PGE1 and PGF2~ in the aqueous humor 12 h after the iridectomy were assayed with the radioimmunoassay technique after Inagawa et al. (1972). Crude lipid extracts of sample obtained by the procedure of Folch et al. (1957) were separated from nonlipid contaminants on Sephadex G-25 columns. After evaporation of the eluted solution, the residue was dissolved in carbon tetrachloride and PG was extracted with water. The aqueous phase was acidified with ethyl acetate, the ethyl acetate layer was neutralized with ammonium hydroxide. After evaporation of the solvent, the residue was dissolved in methyl alcohol and PG fraction was obtained by thin-layer chromatography. Each sample was incubated with 3H-PGE1 (or 3H-PGF2~) and PGE~ (or PGF2~)-antiserum for 1 h in a water bath at 37~ C. After incubation, charcoal was added to each sample in a water bath at 4~ and immediately centrifuged at 2,000 rpm, 10 rain at 4~ C. Radioactivity of each vial was counted by a scintillation counter. Anti-PGE1, anti-PGF2~, 3H-PGE1, and 3H-PGF2~ were supplied by Ono Pharmaceutical Co., Osaka, Japan, The specifications of these reagents were reported in the literature cited above (Inagawa et aI., 1972).
Results 1. I m m u n o f l u o r e s c e n c e Findings As the antiserum used may not completely discriminate between PGE 1 and P G E 2 , t h e t e r m P G E will b e u s e d f o r a n y m a t e r i a l in t i s s u e t h a t c a n b e d e m o n strated by said antiserum.
Fig. 1. Normal iris stained with anti-PGE1 serum and FITC-conjugated antirabbit serum. Fluorescence was found in oval to spindlelike cells in the stroma. They were often located around the capillary (C). x 600
224
H. Sakata and H. Yoshida
Fig. 2. Inflamed iris stained with anti PGE1 serum and FITC-conjugated antirabbit serum. Positive cells were clustered around the capillaries (C). x 600
Fig. 3. Control iris stained with only FITC-conjugated arftirabbit serum, x 600
225
Prostaglandin E in the Iris Table 1. The amounts of PGE 1 and PGF2~ in the aqueous humor 12 h after peripheral iridectomy
PGE 1 (mean + SD ng/ml) PGF2~ (mean+SD ng/ml)
Normal eyes (N:4)
Operated eyes (N:5)
2.0 _+0.53 0.4_+0.27
7.92 _+2.93a (P < 0.005) 0.8+0.58 b
N=number a Differencestatistically significant u Not significant
A PGE-positive cell was one which demonstrated intense fluorescence in the cytoplasm. A negative cell was one in which no fluorescence was localized, but diffuse non-specific background fluorescence of variable degrees was also present. A c o m m o n characterisitc of positive cells was a spindle Or oval shape of the cellular cytoplasm with a slightly eccentric round nucleus (Fig. 1). Only a few PGE-positive cells in one tissue slice were detected in the normal eyes. They were often located around the capillaries. Numerous cells were observed in the inflamed iris with varying fluorescent intensity (Fig. 2). Blood plasma in the capillary lumen exhibited non-specific fluorescence of weaker intensity. Polymorphonuclear leucocytes did not show fluorescence, although it was reported that leucocytes m a y release P G during phagocytosis (Higgs et al., 1975). N o fluorescence was seen when normal rabbit serum was used instead of specific anti-PGE1 or when FITC-conjugated goat antirabbit I g G serum was used alone (Fig. 3). Absorption of anti-PGE~ with PGE1 decreased markedly the fluorescence of PGE-positive cells. Light microscopic examination of H & E stained specimens of adjacent sections showed polymorphonuclear leucocytes and oval to spindle-shaped mesenchymal cells around the dilated capillaries. These mesenchymal cells were thought to be young fibroblasts or macrophages.
2. The Amounts of PGE a and PGF2~ in the Aqueous Humor The mean PGE1 level in the aqueous h u m o r of the five inflamed eyes, as shown in Table 1, was 7.92_+2.93 ng/ml, and it was 2 . 0 + 0 . 5 3 n g / m l in the four control animals. The mean PGF2~ was 0.8+0.58 ng/ml in the inflamed eyes and 0.4+0.27 ng/ml in the normal eyes. The rise of PGE1 level in the inflamed eyes was statistically significant (p
