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Proteinase activity in effusion from children with otitis media with effusion
te~ationa~ Journal of sevkr
atric
182
If
not inactivated, the proteolytic enzymes could attack healthy as well as diseased tissues. Protection against the action of the granulocyte pr offered by systemic as well as by locally produced protease plasma protease inhibitors have been demonstrated in elastase and neutral pro-
The subjects for this study were c who underwent m than 3 months.
status, effusions were obtained from 58 ears.
culturing. The material was transported to the labor in a few hours. Each specimen was cultivated aerobically and anaerobically. aerobic plates were read after 24 and 48 h of incubation and the anaerobic plates after 5 days. The identification of isolated bacteria were made by conventional technique. 1:lOwithO.l (w/v) Triton homogeuized cooled using a ground glass hornogenkr. After 5 min, tire material was centrifuged at 25 000 X g 5 min. The supixnatant was either direct without further preparation or sto at - 20 OC for further use. ghss test tubes containing 6
oalbumin (Sigma), 500 ~10.1 and 40 fivll0.5 m
These were (a) nor
teincontents0f al. [I91 using b0viw se by the Student’s E-test.
1 1.5 r
1 t:
Z
‘ii
1.0
L
f
K
72
0.5
x a
IO
20
30
40
50
68
70
temp. ( ‘C’s
Fi. 1. Optimumincubation temperatureof MEESproteinax Incubation was performedat temperatures fokwed by determination as described under Materials and Metho&. Op indicated for 3Q incubation temperaturewas 37 * C.
incubation temperature was found to be 37OC. inactivated above 45OC (Fig. 1). and 37OC were compared to the @zxxWWd when all of 811series of experiment
5
10
15
28
2,c
time (mid Fig. 2.Kinetics of MEES proteinaseexamined at pH 7.5, To separate soh&ionsOr1mg Tryph (a) md
0.3 mg
protein (Iv)was addt9. At times indicated, ihe optical absorption ws tested.
BBS
Pncubations were perform average of 4-5 reactions
e results iere the
__--sl_.---&_I Control CaCl 2
Diisopropyl fluorophosphate l,lO-phenanthroKne
V
*-.P
3 % 50 10 1 10 10 30 1 1 5
68 60 80 :oo 65 68 66 58 87 31
A
.__I
186 TABLE II &-oreW activity in MEES in patients tympanic *Wennbrone status
with
otitis media with effrcsio?&correlated
Nwmal tympanic
Mucoid eftusion XfS.D. n Serouseffusion XfSD. n
to Qpe
of
efhion and
Thickenedtyqanic
membrane
Atty@$ed
tvmpMic _iCA. mei&&& -
Tt?e?!c
? 282f 0.28 o-o.915 19
0.412f 0.22 0.128-0.863 21
0.346f 0.14 0.029-0.537 17
0.275.&0.12 0.195-0.389 6
0.162*0.11 o-o.291 6
0.126f0.10 O-O.272 5
e proteinase was rapidly inactivated and after 15 ti
_.. ----
virtually no activity
e nature of the effects of various compo ts reduced the e
activity to tyxnpanic TABLE III Broteinaseactivg in MEES from patients with OME correlatedto type of bacteria Streptococ~
Diphteroides Haemophilus a-Streptococcus Stap&coccus
pneumoniae Rf
S.D. 0.387
n
1
injhenzae
0.103
0.317
0.516
1
P
1
Culture-
epidhmieiis
neggth7.e
0.203 f0.127 O.O29-0.405 5
0.251 f 0.187 0 -0.537 *)
TABLE IV Proteinasraci&qr in M.iTS porn patients with OME correlatedto number of
granulocyte~ in efbim
Group N, nope or just a few grenUcytes in 10 high power microscopic fields. Group G, more than 3 granuIocyttsin 10 high power E-AL. Statistical significancewas tested by the Studarit’st-test.
X&SD.
KaIue
N
G
0.066*0.041 o-o.115 6
0.378 i 0.076 0.231-0.537 10 0.001
-.m
188
a amplex of many proteins [II], the specific activity of the in&tidud proteirum~s) is likely to be much higher. me tests to study the amount of substrate, enzyme, incubation temperature and incubation properties. iippearity with time were performed to obtain as been established [5] ever, the presence of proteinase inhibitors in j&&ng that the enzyme activity recorded hzzGz+- 0nIy a min pr&mse activity present. Precautions to distinguish the proteinase the protease enzymes were nut taken here as we ,were may interested in the in vivc,situation, namely whether proteinase activity might be present in such amounts as to destroy the tympanic membranes. A number of chelating agents tested caused a deerease in dye release from gzoeolhtgen indicating the MEES proteinase is a metalloprotease. A sensitivity to 0 indicated by the deelute in compounds reacting with suMhydry1 groups was othreitol or plchlorornercurispecific activity after the addition of either ES proteinase was not affected by diisopropyl fluorophosphate, benzoate. the group of serine proteases which are known to be sensitive to ehhatin this substance [S]. Comparing the properties of the EES proteinase with proteinases from other rial proteinases are sourceq *he most likely source is granulocytes. secretions. Of the c produc~I by other species than those determined . samples 75% wete negative for bacteria but h t!?: proteinase activity. The number of granulocytes o with the amount of proteinase present. Wide ranges of proteiiase activity in EES were found within each category studied s is a feature in cxhrnon with previ r lysosomal enzymes [U$33,34]. As ’ 151and inflammation fhrctuates, and the wide ranges can be reg inflammatory con&eon in BME. The difference in be attributed to a difference in the proteinaseprotease mhibitor balance in the mid&e ear as first suggest4 by Carlss~n [S]. As to the function of neutral proteinascs, these enzymes may play an de not only in phagocytosis but alss in other reactions of the organkm. Neutral p in the phagolysome either directly [ Ehstase, Catlx+ps~ G and other n proKferation of c-ohsin general f35]. latory effect. Rrrthermore, these enzymes are able to activate the complement system [Id] and to modify the ‘self’ proteins of the organism, which may resuh in the formation of autsantibodks 9351. After th c&s t.hcy might participate furt&er in pathological pheuomena, m estruction of tipoue [2,3,9,i6,?2], In a pretious study, WC sttdkd the effect on temporal fascia - use reCarastmctionsfog tympanic membranes ..- after incubatiori with MEE. It found that after 10 days of incubation, a significant reduction in t,h,e9’rLicknessof the WaS Seen as compared to incubation in FM.2 1131.T& presmx iif EE thus indicates one possible mechanism of tissue
on collagenous
tissue. In J. Sade (
., Lactate de~yaro~e~~e
activity
ami
e ear mucosa:
21
Murphy, G., Reynolds, J., Bretz, II. and Bagglioni, hf., CoIla8enase is a component of specific gmm& of human neutrophil leukocytes, B&hem. J., 162 (1977) 195-197. 22 &leberS, II. and Ohson, I., Microbicida mechanisms of human 8mmtIoeytes. Synergistic effects of granulocytes elastase and myeloperoxidase of chymotrypsin-like cationic protein, Infect. Lmnun., 14 (1976) 1276-1283. 23 ohlsson, K.., Coliagenase and eIastase released during peritonitis am complexed by plasma protease inhibitors, Surgexy, 79 (1976) 652-657. 24 Oldson, K. and Olsson, I., The neutral protease of human gramtlocytes. Isolation and partial character&ion of two grauuiocyte coIIagenases, Eur. J. Biochem., 36 (1973) 473-481. 25 Oldsson, K. and O&on, I., The neutraI peoliease of human granulocytes. Isolation and partial characterization of granukyte elastase, Eur. J. Biochem., 42 (1974) 519-527. 26 Ohlsson, K and Olsson, I., Neutral proteases of human granulocytes. IV. Interaction between human gmmdocyte coliagenase and pIasma protease inhibitors, J. Lab. Chn. Med., 89 (1977) 269-277. 27 OIsson, I. and Venge, P., Cationic proteins of human granuktcytes. II. Separation of the cationic proteins of the grant&s of leucaemic myeloid ‘&Is, Blood, 44 (1974) 235-246. 28 PaIva, T., Wolopainen, E. an P., Protein and cehuhtr pattern of glue ear secretions, Ann. -109. OtoL Bhinol., LaryngoI., 85 ( 29 Schiessier, II., IIochstrasser, K. and Phisson, K., Acid-stable inhibitors of gmnuiocyte neutral proteases iu human mucous secretions. Biochemistry an n K. Havemann and A. Janoff (Bds.), Neutral Proteam of Human Urban and Schwamenberg Baltimore, 1978, pp. 195-87. 30 Theme, K.J.I., Oliver, RC. and Barrett, A.J., Lysis ano hiBin of bacteria by lysosomai proknases, Infect. Immun., 14 (1976) 555-563. 31 Thomson, J., Bretlau, P. and Biistensen, II., Bone resq%ion in chronic o&is microscopicaI and histochemicai investigation of acid phosphatase activity, Acta (1975) 400-408. 32 Weissman, G., Zurier, RB. and IIoffstein, S., Leukocytic proteases aud the immunologic release of lysosomai enzymes, Am. J. Pathol., 68 (1972) 539-560. 33 Vehri, RW. and Sprinlde, P.M., Secretory otitis media. An immune complex disease, Ann. Otoll. 34
Virtanen, S. and en, E., Lysozyme activity and ogiob s in middle ear effusion fhtid in acute puruknt otitis media and in otitis media with effusion, Scand. J. Infect. Dis., 11 (1979) 63-67. 35 Viseher, T.L. and Bertrand, L., Stimulating effect of neutral proteinases on cells in vitro, Agents Actions, 6 (1976) 180-182. 36 Wright, D.G. and Maiawista, SE., The mobilization and extraceiluiar release of gramdar engmes from human leukooytes during phagoeytosis, J. Celi Biol., 53 (1972) 788-7.57.
atric
182
If
not inactivated, the proteolytic enzymes could attack healthy as well as diseased tissues. Protection against the action of the granulocyte pr offered by systemic as well as by locally produced protease plasma protease inhibitors have been demonstrated in elastase and neutral pro-
The subjects for this study were c who underwent m than 3 months.
status, effusions were obtained from 58 ears.
culturing. The material was transported to the labor in a few hours. Each specimen was cultivated aerobically and anaerobically. aerobic plates were read after 24 and 48 h of incubation and the anaerobic plates after 5 days. The identification of isolated bacteria were made by conventional technique. 1:lOwithO.l (w/v) Triton homogeuized cooled using a ground glass hornogenkr. After 5 min, tire material was centrifuged at 25 000 X g 5 min. The supixnatant was either direct without further preparation or sto at - 20 OC for further use. ghss test tubes containing 6
oalbumin (Sigma), 500 ~10.1 and 40 fivll0.5 m
These were (a) nor
teincontents0f al. [I91 using b0viw se by the Student’s E-test.
1 1.5 r
1 t:
Z
‘ii
1.0
L
f
K
72
0.5
x a
IO
20
30
40
50
68
70
temp. ( ‘C’s
Fi. 1. Optimumincubation temperatureof MEESproteinax Incubation was performedat temperatures fokwed by determination as described under Materials and Metho&. Op indicated for 3Q incubation temperaturewas 37 * C.
incubation temperature was found to be 37OC. inactivated above 45OC (Fig. 1). and 37OC were compared to the @zxxWWd when all of 811series of experiment
5
10
15
28
2,c
time (mid Fig. 2.Kinetics of MEES proteinaseexamined at pH 7.5, To separate soh&ionsOr1mg Tryph (a) md
0.3 mg
protein (Iv)was addt9. At times indicated, ihe optical absorption ws tested.
BBS
Pncubations were perform average of 4-5 reactions
e results iere the
__--sl_.---&_I Control CaCl 2
Diisopropyl fluorophosphate l,lO-phenanthroKne
V
*-.P
3 % 50 10 1 10 10 30 1 1 5
68 60 80 :oo 65 68 66 58 87 31
A
.__I
186 TABLE II &-oreW activity in MEES in patients tympanic *Wennbrone status
with
otitis media with effrcsio?&correlated
Nwmal tympanic
Mucoid eftusion XfS.D. n Serouseffusion XfSD. n
to Qpe
of
efhion and
Thickenedtyqanic
membrane
Atty@$ed
tvmpMic _iCA. mei&&& -
Tt?e?!c
? 282f 0.28 o-o.915 19
0.412f 0.22 0.128-0.863 21
0.346f 0.14 0.029-0.537 17
0.275.&0.12 0.195-0.389 6
0.162*0.11 o-o.291 6
0.126f0.10 O-O.272 5
e proteinase was rapidly inactivated and after 15 ti
_.. ----
virtually no activity
e nature of the effects of various compo ts reduced the e
activity to tyxnpanic TABLE III Broteinaseactivg in MEES from patients with OME correlatedto type of bacteria Streptococ~
Diphteroides Haemophilus a-Streptococcus Stap&coccus
pneumoniae Rf
S.D. 0.387
n
1
injhenzae
0.103
0.317
0.516
1
P
1
Culture-
epidhmieiis
neggth7.e
0.203 f0.127 O.O29-0.405 5
0.251 f 0.187 0 -0.537 *)
TABLE IV Proteinasraci&qr in M.iTS porn patients with OME correlatedto number of
granulocyte~ in efbim
Group N, nope or just a few grenUcytes in 10 high power microscopic fields. Group G, more than 3 granuIocyttsin 10 high power E-AL. Statistical significancewas tested by the Studarit’st-test.
X&SD.
KaIue
N
G
0.066*0.041 o-o.115 6
0.378 i 0.076 0.231-0.537 10 0.001
-.m
188
a amplex of many proteins [II], the specific activity of the in&tidud proteirum~s) is likely to be much higher. me tests to study the amount of substrate, enzyme, incubation temperature and incubation properties. iippearity with time were performed to obtain as been established [5] ever, the presence of proteinase inhibitors in j&&ng that the enzyme activity recorded hzzGz+- 0nIy a min pr&mse activity present. Precautions to distinguish the proteinase the protease enzymes were nut taken here as we ,were may interested in the in vivc,situation, namely whether proteinase activity might be present in such amounts as to destroy the tympanic membranes. A number of chelating agents tested caused a deerease in dye release from gzoeolhtgen indicating the MEES proteinase is a metalloprotease. A sensitivity to 0 indicated by the deelute in compounds reacting with suMhydry1 groups was othreitol or plchlorornercurispecific activity after the addition of either ES proteinase was not affected by diisopropyl fluorophosphate, benzoate. the group of serine proteases which are known to be sensitive to ehhatin this substance [S]. Comparing the properties of the EES proteinase with proteinases from other rial proteinases are sourceq *he most likely source is granulocytes. secretions. Of the c produc~I by other species than those determined . samples 75% wete negative for bacteria but h t!?: proteinase activity. The number of granulocytes o with the amount of proteinase present. Wide ranges of proteiiase activity in EES were found within each category studied s is a feature in cxhrnon with previ r lysosomal enzymes [U$33,34]. As ’ 151and inflammation fhrctuates, and the wide ranges can be reg inflammatory con&eon in BME. The difference in be attributed to a difference in the proteinaseprotease mhibitor balance in the mid&e ear as first suggest4 by Carlss~n [S]. As to the function of neutral proteinascs, these enzymes may play an de not only in phagocytosis but alss in other reactions of the organkm. Neutral p in the phagolysome either directly [ Ehstase, Catlx+ps~ G and other n proKferation of c-ohsin general f35]. latory effect. Rrrthermore, these enzymes are able to activate the complement system [Id] and to modify the ‘self’ proteins of the organism, which may resuh in the formation of autsantibodks 9351. After th c&s t.hcy might participate furt&er in pathological pheuomena, m estruction of tipoue [2,3,9,i6,?2], In a pretious study, WC sttdkd the effect on temporal fascia - use reCarastmctionsfog tympanic membranes ..- after incubatiori with MEE. It found that after 10 days of incubation, a significant reduction in t,h,e9’rLicknessof the WaS Seen as compared to incubation in FM.2 1131.T& presmx iif EE thus indicates one possible mechanism of tissue
on collagenous
tissue. In J. Sade (
., Lactate de~yaro~e~~e
activity
ami
e ear mucosa:
21
Murphy, G., Reynolds, J., Bretz, II. and Bagglioni, hf., CoIla8enase is a component of specific gmm& of human neutrophil leukocytes, B&hem. J., 162 (1977) 195-197. 22 &leberS, II. and Ohson, I., Microbicida mechanisms of human 8mmtIoeytes. Synergistic effects of granulocytes elastase and myeloperoxidase of chymotrypsin-like cationic protein, Infect. Lmnun., 14 (1976) 1276-1283. 23 ohlsson, K.., Coliagenase and eIastase released during peritonitis am complexed by plasma protease inhibitors, Surgexy, 79 (1976) 652-657. 24 Oldson, K. and Olsson, I., The neutral protease of human gramtlocytes. Isolation and partial character&ion of two grauuiocyte coIIagenases, Eur. J. Biochem., 36 (1973) 473-481. 25 Oldsson, K. and O&on, I., The neutraI peoliease of human granulocytes. Isolation and partial characterization of granukyte elastase, Eur. J. Biochem., 42 (1974) 519-527. 26 Ohlsson, K and Olsson, I., Neutral proteases of human granulocytes. IV. Interaction between human gmmdocyte coliagenase and pIasma protease inhibitors, J. Lab. Chn. Med., 89 (1977) 269-277. 27 OIsson, I. and Venge, P., Cationic proteins of human granuktcytes. II. Separation of the cationic proteins of the grant&s of leucaemic myeloid ‘&Is, Blood, 44 (1974) 235-246. 28 PaIva, T., Wolopainen, E. an P., Protein and cehuhtr pattern of glue ear secretions, Ann. -109. OtoL Bhinol., LaryngoI., 85 ( 29 Schiessier, II., IIochstrasser, K. and Phisson, K., Acid-stable inhibitors of gmnuiocyte neutral proteases iu human mucous secretions. Biochemistry an n K. Havemann and A. Janoff (Bds.), Neutral Proteam of Human Urban and Schwamenberg Baltimore, 1978, pp. 195-87. 30 Theme, K.J.I., Oliver, RC. and Barrett, A.J., Lysis ano hiBin of bacteria by lysosomai proknases, Infect. Immun., 14 (1976) 555-563. 31 Thomson, J., Bretlau, P. and Biistensen, II., Bone resq%ion in chronic o&is microscopicaI and histochemicai investigation of acid phosphatase activity, Acta (1975) 400-408. 32 Weissman, G., Zurier, RB. and IIoffstein, S., Leukocytic proteases aud the immunologic release of lysosomai enzymes, Am. J. Pathol., 68 (1972) 539-560. 33 Vehri, RW. and Sprinlde, P.M., Secretory otitis media. An immune complex disease, Ann. Otoll. 34
Virtanen, S. and en, E., Lysozyme activity and ogiob s in middle ear effusion fhtid in acute puruknt otitis media and in otitis media with effusion, Scand. J. Infect. Dis., 11 (1979) 63-67. 35 Viseher, T.L. and Bertrand, L., Stimulating effect of neutral proteinases on cells in vitro, Agents Actions, 6 (1976) 180-182. 36 Wright, D.G. and Maiawista, SE., The mobilization and extraceiluiar release of gramdar engmes from human leukooytes during phagoeytosis, J. Celi Biol., 53 (1972) 788-7.57.