PROSTAGLANDINS
LET'IERTO THE EDITOR PROSTAGLANDINSIN HUMAN MIDDLE EAR EFFUSIONS Dear Sir: Otitis media with effJsions is a common disease, especially in children. Supposedly, the effusions are caused when the Eustachian tube becomes inflammed and closes, shutting off the only portal to the middle ear (1). A portion of the air of the middle ear is absorbed, causing a small, negative pressure in the middle ear space. Usually, this is followed by the appearance of a middle ear effusion (MEE) that can often be seen behind the intact tympanic membrane. The fluid impairs hearing and is routinely removed prior to the insertion of a ventilation tube in the tympanic membrane. Because this fluid is pathological, i.e., an inflammatory exudate, it seemed reasonable to seek chemical mediators of inflammation in the fluid (2). Samples of MEE from clinic patients were suctioned into a Senturia suction trap and frozen at O" C until testing. The volume of fluid from an individual varied from 0.1 to 0.5 ml. Aliquots of the individual samples were tested on the gerbil colon using the technique of Eakins et al. (3). The smooth muscle stimulating activity of MEE was compared to the activity of authentic samples of bradykinin, prostaglandin El (PGE~), histamine and acetylcholine. An attempt was made to discover if the smooth muscle stimulating activity of MEE could be antagonized by polyphloretin phosphate (PPP), a PG inhibitor; pyrilamine, an antihistamine; or atropine sulfate, an acetylcholine inhibitor. Concurrently, three pooled samples of MEE from many patients were analyzed for the presence of PGs using a modified radioimmunoassay (RIA) procedure (4). (Commercial RIA kits from Clinical Assays, Inc., Cambridge, Massachusetts were used.) The first pooled sample was analyzed for PGEl, PGFl and PGF2, only. In the second and third pooled samples, the Technique was modified to include an assay for PGE2 (5). Gerbil colon - To date, every individual sample (14 samples) of MEE tested on the gerbil colon has shown some degree of smooth muscle stimulating activity. We directed our efforts toward
AUGUST
1975 VOL. 10 NO. 2
365
PROSTAGLANDINS
identifyingthe active agent in MEE. In our experience,the gerbil colon is relatively insensitive to acetylcholineand histamine and it seemed unlikely that the large contractionsproduced by 0.01 to 0.1 ml of MEE could be due to their action. Fig. 1 demonstratesthat MEE does not contain significantamounts of acetylcholine. Similar experiments were performed using 20 ug/ml of pyrilamine in the bathing solution in an attempt to block any histamine response induced by MEE. Again, an antihistamineblock had no effect on the colon response to MEE. Fig. 2 is an example of the simultaneousblock of PGEl and MEE with PPP. This kind of result was obtained in three consecutive experiments. In most samples of MEE, we were able to obtain an estimate of the relative activity in relation to authentic samples of PGEl. If all of the smooth muscle stimulatingactivity of MEE the concentrationwould vary between 40 and were due to PGE 800 nanograms/m 1' . The estimatesof PG concentrationin 7 MEE samples are: 40, 120, 250, 300, 400, 750 and 800 ng/ml. Radioimmunoassay- The first pooled sample was 3 ml of MEE of all types. The second and third samples each contained 2 ml. The second sample was of the serous type. At collection,it was clear to pale yellow and had a low viscosity. The third sample was of the mucoid type. It was quite viscous and cloudy. The PG values obtained are shown in Table I. TABLE I RADIOIMMUNOASSAYOF MIDDLE EAR FLUID #
1 2
Mixture (Serous E Mucus) Serous
3 MUCUS 5kNot analyzed
366
ng/ml ear fluid
Sample
El + Al + Bl
E2
Total
F1o
F2cz
1.2.
8.57
trace
trace
1.09
0.99
1.24
3.32
0.94
5.74
2.53
7.44
16.65
AUGUST
1975
*
9.78
VOL. 10 NO. 2
PROSTAGLANDINS
The gerbil colon assay of samples of human MEE indicates the presence of an agent that appears to be a prostaglandin. The consistent results with PPP not only implicate a prostaglandin but seem to remove bradykinin from consideration. The results from the radioimmunoassay confirm the presence of several PGs in MEE. PGF2o and PGE2 are in relatively high concentrations. These PGs have been reported to produce inflammation (2). There is an obvious discrepancy between the estimates of PG concentration using the gerbil colon assay and the radioimmunoassay. Several possibilities exist as an explanation. MEE could contain a mediator that was not tested (e.g., SRS-A). The MEE was compared to authentic PGEIY Perhaps some other PG or a combination would be more appropriate. Also, the known and unknown mediators in MEE could be acting synergistically on the colon. This information poses several problems. First, does the type of MEE determine its prostaglandin concentration? Classifications of this fluid vary, but one system lists four kinds: serous, mucoid, mucopurulent and purulent. The type is determined by the cytological appearance of the fluid (6). As yet, we have made no serious attempt to relate the type of MEE to the mediator concentration. Our crude division of fluid by physical properties (Table I) shows some potential. Second, we will attempt to resolve the discrepancy between the values obtained for PG concentrations in MEE. Third, what is the source of the PG? A portion of MEE could enter the middle ear space as a serum transudate (1). The other candidate for the source is the middle ear mucosa. It is known that mucus glands proliferate in the middle ear mucosa in the course of otitis media (7). These glands or some other component of the mucosa could respond to middle ear inflammation by secreting PGs. Fourth, does the secretion of PG into the middle ear space produce the effusion or act to enhance or prolong the inflammation? Meyers et al. (8) have shown that intraocular injection of PG severely altered the fine structure of the epithelium of ciliary process tissue (e.g., a disappearance of apical tight junctions). These alterations coincide with the breakdown of
AUGUST
1975
VOL. 10 NO. 2
367
PROSTAGLANDINS
the blood-aqueousbarrier. Such a breakdown could account for proteinaceous(1) fluid in the middle ear space. We have shown previously that 50 ng of PGE is easily capable of inducing a vasodilationin the mucosa o$ the Eustachiantube if applied topically (9) or injected into the carotid artery of a dog (10). This implies that, once the inflammatorystate occurs, an inflammatorymediator appears in the MEE that enhances or prolongs the inflammation. Further, work with intestine,stomach and gall bladder has shown that PGEl not only inhibitswater, glucose and electrolytenet absorptionbut reverses the movements to net secretion (11). Acknowledgements: This work was supportedby NIH grants NS 09570 and EY00243. The authors would like to thank Drs. J. Per Lee, D. Rooker and J. Turner, Jr. for their cooperationin the collectionof middle ear effusions. We would also like to thank Dr. John E. Pike of the Upjohn Company, Kalamazoo,Michigan for the samples of PGEl and Dr. B. Hogberg of the A. B. Leo Company, Helsinborg,Sweden for the sample of polyphloretinphosphate. The excellent technical assistanceof Miss Mary Louise Law and Mr. Michael Barron is acknowledgedwith appreciation.
R. T. Jackson, Ph.D., M. B. Waitzman, Ph.D.*, L. Pickford,M.D. and S. E. Nathanson,M.D. From the Division of Otolaryngologyand the Laboratoryfor OphthalmicResearch of the Departmentof Ophthalmology*, Emory University School of Medicine, Atlanta, Georgia 30322 REFERENCES 1. Sade, J.:Ann. Otol. Rhinol. Laryngol.,-83 (Suppl. 11):59-70, 1974. 2. Ferreira, S. H., and Vane, J. R.:Ann. Rev. Pharmacol.,-14: 57-73, 1974.
368
AUGUST
1975
VOL. 10 NO. 2
PROSTAGLANDINS
3.
Eakins, K. E,, Karim, S. M. M. and Miller, J. D.:Brit J. Pharmacol., -39:556-563, 1970.
4.
Waitzman, M. B.:Submitted for publication.
5.
Levine, L., Hinkle, P. M., Voelkel, E. F. and Tashjian, A. H.: Biochem. Biophys. Res. Comm., -47:888-896, 1972.
6.
Senturia, B. H., Gessert, C. F., Carr, C. D. et al.:Ann. Otol. Rhinol. Laryngol., -67:440-468, 1958.
7.
Tos, M. and Bak-Pedersen, K.:Arch. Otolaryngol., -101:123-128, 1975.
8.
Meyers, R. L., Shabo, A. L. and Maxwell, D. S.: glandins, 2:167-173, 1975.
9.
Davis, L. J., Sheffield, P. A. and Jackson, R. T.:Arch. Otolaryngol., -92:325-328, 1970.
Prosta-
10.
Jackson, R. T.:Ann. Otol. Rhinol. Laryngol., -80:313-318, 1971.
11.
Leyssac, P. P., Bukhave, K. and Frederiksen, O.:Acta Physiol. Stand., -92:496-507, 1974.
AUGUST
1975
VOL. 10 NO. 2
369
PROSTAGLANDINS
25 ng
I 0 0 0 ng
0.1 ml
BK
ACh
MEE- 7
AFTER 15rain of IOug/ml ATROPINE
Fig. i.
370
Responses of gerbil colon to 25 nanograms of bradykinin, 1000 nanograms of acetylcholine and a 0.1 ml aliquot of MEE before and after bathing the colon for 15 minutes in i0 ~g/ml of atropine sulfate. Bath volume is i0 ml.
AUGUST 1975
VOL. 10 NO. 2
PROSTAGLANDINS
50
100
5
50
ng
43
ng
w
ng
-f~c/‘-r”“I
PGE,
MEE-I5
5
50ng
I
50
AFTER
80
NO
PPP
MEE-
min
15
J
BK
-
PGE,
5
0.1 ml MEE-I5
5Ona
ng
.m.--
0. I ml
P I
in PPP
min
50
MEE-
15
ng
Fig. 2.
AUGUST
Responses of the gerbil colon to bradykinin, PGEl and aliquots of a sample of MEE before, during and after bathing the colon in 20 pg/ml of polyphloretin phosphate (PPP), a prostaglandin inhibitor.
1975
VOL. 10 NO. 2
371
LET'IERTO THE EDITOR PROSTAGLANDINSIN HUMAN MIDDLE EAR EFFUSIONS Dear Sir: Otitis media with effJsions is a common disease, especially in children. Supposedly, the effusions are caused when the Eustachian tube becomes inflammed and closes, shutting off the only portal to the middle ear (1). A portion of the air of the middle ear is absorbed, causing a small, negative pressure in the middle ear space. Usually, this is followed by the appearance of a middle ear effusion (MEE) that can often be seen behind the intact tympanic membrane. The fluid impairs hearing and is routinely removed prior to the insertion of a ventilation tube in the tympanic membrane. Because this fluid is pathological, i.e., an inflammatory exudate, it seemed reasonable to seek chemical mediators of inflammation in the fluid (2). Samples of MEE from clinic patients were suctioned into a Senturia suction trap and frozen at O" C until testing. The volume of fluid from an individual varied from 0.1 to 0.5 ml. Aliquots of the individual samples were tested on the gerbil colon using the technique of Eakins et al. (3). The smooth muscle stimulating activity of MEE was compared to the activity of authentic samples of bradykinin, prostaglandin El (PGE~), histamine and acetylcholine. An attempt was made to discover if the smooth muscle stimulating activity of MEE could be antagonized by polyphloretin phosphate (PPP), a PG inhibitor; pyrilamine, an antihistamine; or atropine sulfate, an acetylcholine inhibitor. Concurrently, three pooled samples of MEE from many patients were analyzed for the presence of PGs using a modified radioimmunoassay (RIA) procedure (4). (Commercial RIA kits from Clinical Assays, Inc., Cambridge, Massachusetts were used.) The first pooled sample was analyzed for PGEl, PGFl and PGF2, only. In the second and third pooled samples, the Technique was modified to include an assay for PGE2 (5). Gerbil colon - To date, every individual sample (14 samples) of MEE tested on the gerbil colon has shown some degree of smooth muscle stimulating activity. We directed our efforts toward
AUGUST
1975 VOL. 10 NO. 2
365
PROSTAGLANDINS
identifyingthe active agent in MEE. In our experience,the gerbil colon is relatively insensitive to acetylcholineand histamine and it seemed unlikely that the large contractionsproduced by 0.01 to 0.1 ml of MEE could be due to their action. Fig. 1 demonstratesthat MEE does not contain significantamounts of acetylcholine. Similar experiments were performed using 20 ug/ml of pyrilamine in the bathing solution in an attempt to block any histamine response induced by MEE. Again, an antihistamineblock had no effect on the colon response to MEE. Fig. 2 is an example of the simultaneousblock of PGEl and MEE with PPP. This kind of result was obtained in three consecutive experiments. In most samples of MEE, we were able to obtain an estimate of the relative activity in relation to authentic samples of PGEl. If all of the smooth muscle stimulatingactivity of MEE the concentrationwould vary between 40 and were due to PGE 800 nanograms/m 1' . The estimatesof PG concentrationin 7 MEE samples are: 40, 120, 250, 300, 400, 750 and 800 ng/ml. Radioimmunoassay- The first pooled sample was 3 ml of MEE of all types. The second and third samples each contained 2 ml. The second sample was of the serous type. At collection,it was clear to pale yellow and had a low viscosity. The third sample was of the mucoid type. It was quite viscous and cloudy. The PG values obtained are shown in Table I. TABLE I RADIOIMMUNOASSAYOF MIDDLE EAR FLUID #
1 2
Mixture (Serous E Mucus) Serous
3 MUCUS 5kNot analyzed
366
ng/ml ear fluid
Sample
El + Al + Bl
E2
Total
F1o
F2cz
1.2.
8.57
trace
trace
1.09
0.99
1.24
3.32
0.94
5.74
2.53
7.44
16.65
AUGUST
1975
*
9.78
VOL. 10 NO. 2
PROSTAGLANDINS
The gerbil colon assay of samples of human MEE indicates the presence of an agent that appears to be a prostaglandin. The consistent results with PPP not only implicate a prostaglandin but seem to remove bradykinin from consideration. The results from the radioimmunoassay confirm the presence of several PGs in MEE. PGF2o and PGE2 are in relatively high concentrations. These PGs have been reported to produce inflammation (2). There is an obvious discrepancy between the estimates of PG concentration using the gerbil colon assay and the radioimmunoassay. Several possibilities exist as an explanation. MEE could contain a mediator that was not tested (e.g., SRS-A). The MEE was compared to authentic PGEIY Perhaps some other PG or a combination would be more appropriate. Also, the known and unknown mediators in MEE could be acting synergistically on the colon. This information poses several problems. First, does the type of MEE determine its prostaglandin concentration? Classifications of this fluid vary, but one system lists four kinds: serous, mucoid, mucopurulent and purulent. The type is determined by the cytological appearance of the fluid (6). As yet, we have made no serious attempt to relate the type of MEE to the mediator concentration. Our crude division of fluid by physical properties (Table I) shows some potential. Second, we will attempt to resolve the discrepancy between the values obtained for PG concentrations in MEE. Third, what is the source of the PG? A portion of MEE could enter the middle ear space as a serum transudate (1). The other candidate for the source is the middle ear mucosa. It is known that mucus glands proliferate in the middle ear mucosa in the course of otitis media (7). These glands or some other component of the mucosa could respond to middle ear inflammation by secreting PGs. Fourth, does the secretion of PG into the middle ear space produce the effusion or act to enhance or prolong the inflammation? Meyers et al. (8) have shown that intraocular injection of PG severely altered the fine structure of the epithelium of ciliary process tissue (e.g., a disappearance of apical tight junctions). These alterations coincide with the breakdown of
AUGUST
1975
VOL. 10 NO. 2
367
PROSTAGLANDINS
the blood-aqueousbarrier. Such a breakdown could account for proteinaceous(1) fluid in the middle ear space. We have shown previously that 50 ng of PGE is easily capable of inducing a vasodilationin the mucosa o$ the Eustachiantube if applied topically (9) or injected into the carotid artery of a dog (10). This implies that, once the inflammatorystate occurs, an inflammatorymediator appears in the MEE that enhances or prolongs the inflammation. Further, work with intestine,stomach and gall bladder has shown that PGEl not only inhibitswater, glucose and electrolytenet absorptionbut reverses the movements to net secretion (11). Acknowledgements: This work was supportedby NIH grants NS 09570 and EY00243. The authors would like to thank Drs. J. Per Lee, D. Rooker and J. Turner, Jr. for their cooperationin the collectionof middle ear effusions. We would also like to thank Dr. John E. Pike of the Upjohn Company, Kalamazoo,Michigan for the samples of PGEl and Dr. B. Hogberg of the A. B. Leo Company, Helsinborg,Sweden for the sample of polyphloretinphosphate. The excellent technical assistanceof Miss Mary Louise Law and Mr. Michael Barron is acknowledgedwith appreciation.
R. T. Jackson, Ph.D., M. B. Waitzman, Ph.D.*, L. Pickford,M.D. and S. E. Nathanson,M.D. From the Division of Otolaryngologyand the Laboratoryfor OphthalmicResearch of the Departmentof Ophthalmology*, Emory University School of Medicine, Atlanta, Georgia 30322 REFERENCES 1. Sade, J.:Ann. Otol. Rhinol. Laryngol.,-83 (Suppl. 11):59-70, 1974. 2. Ferreira, S. H., and Vane, J. R.:Ann. Rev. Pharmacol.,-14: 57-73, 1974.
368
AUGUST
1975
VOL. 10 NO. 2
PROSTAGLANDINS
3.
Eakins, K. E,, Karim, S. M. M. and Miller, J. D.:Brit J. Pharmacol., -39:556-563, 1970.
4.
Waitzman, M. B.:Submitted for publication.
5.
Levine, L., Hinkle, P. M., Voelkel, E. F. and Tashjian, A. H.: Biochem. Biophys. Res. Comm., -47:888-896, 1972.
6.
Senturia, B. H., Gessert, C. F., Carr, C. D. et al.:Ann. Otol. Rhinol. Laryngol., -67:440-468, 1958.
7.
Tos, M. and Bak-Pedersen, K.:Arch. Otolaryngol., -101:123-128, 1975.
8.
Meyers, R. L., Shabo, A. L. and Maxwell, D. S.: glandins, 2:167-173, 1975.
9.
Davis, L. J., Sheffield, P. A. and Jackson, R. T.:Arch. Otolaryngol., -92:325-328, 1970.
Prosta-
10.
Jackson, R. T.:Ann. Otol. Rhinol. Laryngol., -80:313-318, 1971.
11.
Leyssac, P. P., Bukhave, K. and Frederiksen, O.:Acta Physiol. Stand., -92:496-507, 1974.
AUGUST
1975
VOL. 10 NO. 2
369
PROSTAGLANDINS
25 ng
I 0 0 0 ng
0.1 ml
BK
ACh
MEE- 7
AFTER 15rain of IOug/ml ATROPINE
Fig. i.
370
Responses of gerbil colon to 25 nanograms of bradykinin, 1000 nanograms of acetylcholine and a 0.1 ml aliquot of MEE before and after bathing the colon for 15 minutes in i0 ~g/ml of atropine sulfate. Bath volume is i0 ml.
AUGUST 1975
VOL. 10 NO. 2
PROSTAGLANDINS
50
100
5
50
ng
43
ng
w
ng
-f~c/‘-r”“I
PGE,
MEE-I5
5
50ng
I
50
AFTER
80
NO
PPP
MEE-
min
15
J
BK
-
PGE,
5
0.1 ml MEE-I5
5Ona
ng
.m.--
0. I ml
P I
in PPP
min
50
MEE-
15
ng
Fig. 2.
AUGUST
Responses of the gerbil colon to bradykinin, PGEl and aliquots of a sample of MEE before, during and after bathing the colon in 20 pg/ml of polyphloretin phosphate (PPP), a prostaglandin inhibitor.
1975
VOL. 10 NO. 2
371
