- Email: [email protected]
Isolation of Stromal Collagenase in Corneal Inflammation
ISOLATION O F STROMAL COLLAGENASE IN CORNEAL INFLAMMATION STUART I. BROWN, M.D.,
AND CAROL W E L L E R H O O K , P H . D .
New York, New York Although there have been numerous inves tigations of infections of the cornea, few re port the nature of acute and especially chronic corneal inflammation. The present study establishes a model for acute and chronic corneal inflammation, and in addi tion, reports the isolation of a stromal coUag enase. MATERIAL AND METHODS
Albino rabbits weighing from 2 to 4 kg were anesthetized with intravenous pentabarbital and topical tetracaine HC1. Corneal inflammation was produced by injection of 0.05 ml complete Freund's adjuvant in min eral oil (Difco) into the peripheral stroma (2-3 mm from the limbus) with a 25-gauge needle. The corneas were examined daily with loupe and light and were removed at various times before and after onset of in flammation. The excised corneas were cut into quad rants and each inflamed area was bisected. One piece was saved for a collagenase assay and the other was placed in 3 % glutaraldehyde in 0.2M sodium cacodylate buffer for 15 minutes. Following this, it was post-fixed in 2% osmium tetroxide in cacodylate buffer for one hour and dehydrated in alcohols and propylene oxide. Finally, it was embedded in Epon 812 and thick sections (5[<.) were cut and stained with basic fuchsin and alkalinized methylene blue.1 The tissue saved for collagenase assay was dipped into mammalian Tyrode's solution. The epithelium was scraped free of the stroma and both were cultured on a rigid From the Cornea Research Laboratory, Depart ment of Ophthalmology, New York Hospital-Cor nell Medical Center, New York. This study was supported in part by Public Health Service Grant EY-00502 from the National Eye Institute. Reprint requests to Stuart I. Brown, M.D., De partment of Ophthalmology, New York Hospital, 525 East 68th Street, New York, New York 10021.
opalescent gel of 0.2% reconstituted collagen which was incubated at 37° C for up to four days.2 Clearing of the gel surrounding the tissue explant indicated collagen lysis. Additional pieces of corneas were excised seven days after intralamellar injection. The epithelium was scraped free and the stromas were trimmed to approximately 2 X 2 mm. Collagenase was harvested from these tissues by growing them in Dulbecco's medium.3 The effect of the harvested enzyme on the viscosity of a 0.1% collagen solution was tested using an Ostwald viscometer with a water flow rate of 53 seconds at 25° C and at pH 7.4. All measurements were made at 27° C. The effect of the harvested enzyme on the optical rotation of reconstituted collagen was determined using the MSP-3 photoelectric spectrophotometer at 25° C and 589 my.. Three methods were used to determine the cell origin of the stromal enzyme. The first was simply to repeatedly freeze-thaw stro mas which were injected five days previously with Freund's adjuvant. This material was then transferred to gels of reconstituted col lagen and incubated at 37° C for up to four days. The second method was to apply puromy cin 10_3M, which is an inhibitor of protein synthesis (and therefore enzyme synthesis), to the surface of the corneal explant twice a day for four days. For controls, alkali burns were produced in rabbit corneas,2 and when ulcerations were noted, the epithelium which surrounded the ulcers was removed and soaked in puromycin 10"3M and assayed on collagen gels. The third method was to reduce the circu lating granulocytes by the intravenous injec tion of mechlorethamine HC1 (nitrogen mustard), 5 mg three days before, and 3 mg on the day of intralamellar injection of Freund's adjuvant. Three, six, and 10 days later, the corneas were excised and bisected.
AMERICAN JOURNAL OF OPHTHALMOLOGY
1140
DECEMBER, 1971 ?
ML
i*
i
^
i
1?'^
VW
Fig. 1 (Brown and Hook). High concentration of white blood cells, predominantly granulocytes, and a few histiocytes or epitheliod cells (basic fuchsin and alkalinized methylene blue, X400).
Half of each specimen was assayed for collagenase activity and the other half was ex amined histologically. RESULTS
Grossly, the limbal blood vessels became dilated within 12 hours after injection of Freund's adjuvant. At 36 hours, the corneas were semitranslucent and slightly edematous. Neovascularization began at about four days postinjection. At this time, the stromas were grossly edematous and opaque. Histologic examination of the corneas at 24 hours showed granulocytes and an occasional mononuclear cell. The concentration of white blood cells increased markedly over the next 48 hours, especially in the area of the for eign protein. At four days, there were many granulocytes, and fewer monocytes and his tiocytes (Fig. 1). The cellular infiltrate increased in concen tration until the third week, when the num ber and types of cells in the infiltrate became constant. Six weeks after injection of
Freund's adjuvant, all cell types, but espe cially the granulocytes, began to decrease in number, and the stromal edema was grossly reduced. During postinjection weeks nine to 12 there were only a few histiocytes or epitheloid cells and granulocytes. The epithelium from the inflamed stromas did not produce detectable collagen lysis in any specimen. On the other hand, in every instance culturing of the inflamed stromas produced collagen lysis at least 10 mm in di ameter, beginning at 36 hours and continu ing through nine weeks after intralamellar injection of Freund's adjuvant (Table 1). The specific viscosity of the mixture of harvested stromal enzyme and reconstituted collagen at pH 7.4 gradually decreased over a one-hour period to about 60 percent of the original value (Fig. 2). The optical rota tion measurement of reconstituted collagen (—442) was not changed by the addition of the harvested enzyme (—450). All specimens that had been repeatedly f reeze-thawed produced at least 10 mm in di-
VOL. 72. NO. 6
CORNEAL INFLAMMATION TABLE 1
PUROMYCIN (10~ 3 M) AND COLLAGEN LYSIS*
Diameter of Lysis Alkali-burned epithelium plus Tyrode's solution plus puromycin Granuloma (only stroma) puromycin
> 10 mm 0 > 10 mm
* Five assays for each category.
ameter of collagen lysis. Puromycin com pletely inhibited collagen lysis by the epithe lium from alkali-burned corneas (controls), but allowed lysis by the inflamed stromas. The nitrogen mustard treatment schedule resulted in a circulating white blood count between 1200 to 1800 on the day of intralamellar injection. The granulocytes in the pe ripheral blood were mainly blast cells and were about 30% of the white blood cells. Specimens of the corneas of the nitrogen mustard-treated animals did not lyse the col lagen gels at four, six, and 10 days after in jection of Freund's adjuvant. The inflamma tory reaction that resulted from injection of Freund's adjuvant was markedly reduced in comparison with those animals that had not received nitrogen mustard (i.e., there was less edema and neovascularization was de layed to the end of the second week). Histologically, there were many less white blood cells in the stromal infiltrate and these were heavily granulated. COMMENTS
Inflammation was consistently produced in the rabbit corneas by the intralamellar in jection of Freund's adjuvant. This was first noted in 24 hours, reached its peak in inten sity by the end of the third week and began to quiet after two months. The stromal cellu lar infiltrate surrounding the foreign protein was mainly granulocytic in the first few days, but later took on the characteristics of a granuloma with many large histiocytes or epitheliod cells in addition to the granulo cytes. Tissue culture assays showed that the
1141
stromas of these corneas caused collagen ly sis that was similar in quantity to that caused by the ulcerated alkali-burned cornea.2 Har vest of the enzyme showed it to be a true collagenase because of its characteristic effect on the viscosity and optical rotations of recon stituted collagen.4 The molecular weight of the enzyme and the dynamics of its inhibition will be reported. Gross and Lapiere 6 demonstrated that ani mal collagenase is produced by living cells (epithelium and possibly fibroblasts) and that destruction of the cells by freezing stops collagenase production. An exception is the collagenase from the granulocytes which originates in the granules and can be de tected only after cell disruption.6 Another unusual feature of the granulocyte collage nase is that it is only weakly active against collagen fibrils. Since no other blood cell ele ments contain collagenase,0 the only cells that could have produced the collagenase found in the inflamed corneal stromas were either the fibroblasts, the histiocytes, or the granulocytes. Collagenase was detected after freeze-thawing, which indicated that the en zyme originated from the granulocytes. A less likely possibility was that the enzyme was produced and stored in the tissues. Since stored collagenase has never been demon-
s
0>
.2
3
£
*-. o ^
1
100 ( \ \ 80 - \ 60 40
t \
\
1V ~
J * ^
■5
£
£
20 ,
L
1
1
m
'
hours
Fig. 2 (Brown and Hock). Graph showing a re duction in specific viscosity of reconstituted colla gen after exposure to the stromal collagenase.
1142
AMERICAN JOURNAL OF OPHTHALMOLOGY
strated in tissues before, it is unlikely that the collagenolytic activity after freeze-thawing was from enzyme produced and stored in the tissues by the fibroblasts and the histiocytes before their destruction. Soaking the inflamed stromas with puromycin, which is a potent inhibitor of protein metabolism, allowed collagenolytic activity. This indicates that the stromal enzyme was not a product of cell metabolism, which again points to the granulocytes as the collagenase carrier. Finally, when the number of the circulating granulocytes was reduced by approximately 80% by intravenous nitro gen mustard, collagenase production was not detected after intralamellar injection. Except for the findings that the harvested enzyme was active against heat-gelled collagen fibrils, the results of the present study indi cate that at least part of the collagenase of the stromal granuloma originates from the granulocytes. The obvious reduction in stromal edema and neovascularization in the animals pretreated with nitrogen mustard suggests that the granulocyte may also play a role in medi ating various aspects of the inflammatory re sponse. Studies are in progress to isolate this effect of the granulocyte. Collagenolytic lysozomal enzymes have been isolated from inflamed tissues in other parts of the body. However, these were effective only when pH acidity fell between 3 and 5.5. There are no studies showing that the pH of inflamed tissue is in the acid range. Consequently, there is doubt that these acid hydrolases could destroy collagen in vivo. The present study demonstrates a neutral collagenase in inflammation of the corneal stroma, indicating the destructive po tential of the inflammatory process. Though there was no gross loss of tissue in these in flamed corneas, preliminary study of the
DECEMBER, 1971
electron micrographs of the inflamed areas showed various types of collagen fibril alter ations.7 Recently, we found that the collagenases of the alkali-burned cornea and the inflamed stroma are each inhibited by dilute concen trations of fresh serum. It may be that the minimal corneal destruction relative to the large amount of collagenase produced by the inflamed stroma is due to enzyme inhibition by elements in the serum. This inhibition would probably be related to new vessel in growth into the inflamed area. This will be expanded in a later report. SUMMARY
Inflammation of the corneal stroma was produced in rabbits by the intralamellar in jection of Freund's adjuvant. The inflamma tion progressed from a typically acute to a chronic granulomatous type of inflammation. A collagenase was isolated from the inflamed stromas and partially characterized. The en zyme appeared early in the course of the in flammation, thus indicating that it originated in the granulocytes. REFERENCES
1. Huber, J. D., Parker, F., and Odland, G. F.: A basic fuschin and alkalinized methylene blue rapid stain for epoxy-embedded tissue. Stain Tech. 43:83,1968. 2. Brown, S. I., Weller, C. A., and Wassermann, H. E.: Collagenolytic activity of alkali-burned cor neas. Arch. Ophth. 81:370, 1969. 3. Brown, S. I., Weller, C. A., and Akiya, S.: The pathogenesis of ulcers of alkali-burned cor neas. Arch. Ophth. 83:205, 1970. 4. Dische, Z.: A new specific color reaction of hexuronic acids. J. Biol. Chem. 167:189, 1947. 5. Gross, G., and Lapiere, C. M.: Activity in am phibian tissues: A tissue culture assay. Proc. Nat. Acad. Sci. 48:1014,1962. 6. Lazarus, G. S., Brown, R. S., Daniels, J. R., and Fullmer, H. M.: Human granulocyte collagen ase. Science 159:1483, 1968. 7. Nakanishi, I., and Brown, S. I.: Unpublished data, 1970.
AND CAROL W E L L E R H O O K , P H . D .
New York, New York Although there have been numerous inves tigations of infections of the cornea, few re port the nature of acute and especially chronic corneal inflammation. The present study establishes a model for acute and chronic corneal inflammation, and in addi tion, reports the isolation of a stromal coUag enase. MATERIAL AND METHODS
Albino rabbits weighing from 2 to 4 kg were anesthetized with intravenous pentabarbital and topical tetracaine HC1. Corneal inflammation was produced by injection of 0.05 ml complete Freund's adjuvant in min eral oil (Difco) into the peripheral stroma (2-3 mm from the limbus) with a 25-gauge needle. The corneas were examined daily with loupe and light and were removed at various times before and after onset of in flammation. The excised corneas were cut into quad rants and each inflamed area was bisected. One piece was saved for a collagenase assay and the other was placed in 3 % glutaraldehyde in 0.2M sodium cacodylate buffer for 15 minutes. Following this, it was post-fixed in 2% osmium tetroxide in cacodylate buffer for one hour and dehydrated in alcohols and propylene oxide. Finally, it was embedded in Epon 812 and thick sections (5[<.) were cut and stained with basic fuchsin and alkalinized methylene blue.1 The tissue saved for collagenase assay was dipped into mammalian Tyrode's solution. The epithelium was scraped free of the stroma and both were cultured on a rigid From the Cornea Research Laboratory, Depart ment of Ophthalmology, New York Hospital-Cor nell Medical Center, New York. This study was supported in part by Public Health Service Grant EY-00502 from the National Eye Institute. Reprint requests to Stuart I. Brown, M.D., De partment of Ophthalmology, New York Hospital, 525 East 68th Street, New York, New York 10021.
opalescent gel of 0.2% reconstituted collagen which was incubated at 37° C for up to four days.2 Clearing of the gel surrounding the tissue explant indicated collagen lysis. Additional pieces of corneas were excised seven days after intralamellar injection. The epithelium was scraped free and the stromas were trimmed to approximately 2 X 2 mm. Collagenase was harvested from these tissues by growing them in Dulbecco's medium.3 The effect of the harvested enzyme on the viscosity of a 0.1% collagen solution was tested using an Ostwald viscometer with a water flow rate of 53 seconds at 25° C and at pH 7.4. All measurements were made at 27° C. The effect of the harvested enzyme on the optical rotation of reconstituted collagen was determined using the MSP-3 photoelectric spectrophotometer at 25° C and 589 my.. Three methods were used to determine the cell origin of the stromal enzyme. The first was simply to repeatedly freeze-thaw stro mas which were injected five days previously with Freund's adjuvant. This material was then transferred to gels of reconstituted col lagen and incubated at 37° C for up to four days. The second method was to apply puromy cin 10_3M, which is an inhibitor of protein synthesis (and therefore enzyme synthesis), to the surface of the corneal explant twice a day for four days. For controls, alkali burns were produced in rabbit corneas,2 and when ulcerations were noted, the epithelium which surrounded the ulcers was removed and soaked in puromycin 10"3M and assayed on collagen gels. The third method was to reduce the circu lating granulocytes by the intravenous injec tion of mechlorethamine HC1 (nitrogen mustard), 5 mg three days before, and 3 mg on the day of intralamellar injection of Freund's adjuvant. Three, six, and 10 days later, the corneas were excised and bisected.
AMERICAN JOURNAL OF OPHTHALMOLOGY
1140
DECEMBER, 1971 ?
ML
i*
i
^
i
1?'^
VW
Fig. 1 (Brown and Hook). High concentration of white blood cells, predominantly granulocytes, and a few histiocytes or epitheliod cells (basic fuchsin and alkalinized methylene blue, X400).
Half of each specimen was assayed for collagenase activity and the other half was ex amined histologically. RESULTS
Grossly, the limbal blood vessels became dilated within 12 hours after injection of Freund's adjuvant. At 36 hours, the corneas were semitranslucent and slightly edematous. Neovascularization began at about four days postinjection. At this time, the stromas were grossly edematous and opaque. Histologic examination of the corneas at 24 hours showed granulocytes and an occasional mononuclear cell. The concentration of white blood cells increased markedly over the next 48 hours, especially in the area of the for eign protein. At four days, there were many granulocytes, and fewer monocytes and his tiocytes (Fig. 1). The cellular infiltrate increased in concen tration until the third week, when the num ber and types of cells in the infiltrate became constant. Six weeks after injection of
Freund's adjuvant, all cell types, but espe cially the granulocytes, began to decrease in number, and the stromal edema was grossly reduced. During postinjection weeks nine to 12 there were only a few histiocytes or epitheloid cells and granulocytes. The epithelium from the inflamed stromas did not produce detectable collagen lysis in any specimen. On the other hand, in every instance culturing of the inflamed stromas produced collagen lysis at least 10 mm in di ameter, beginning at 36 hours and continu ing through nine weeks after intralamellar injection of Freund's adjuvant (Table 1). The specific viscosity of the mixture of harvested stromal enzyme and reconstituted collagen at pH 7.4 gradually decreased over a one-hour period to about 60 percent of the original value (Fig. 2). The optical rota tion measurement of reconstituted collagen (—442) was not changed by the addition of the harvested enzyme (—450). All specimens that had been repeatedly f reeze-thawed produced at least 10 mm in di-
VOL. 72. NO. 6
CORNEAL INFLAMMATION TABLE 1
PUROMYCIN (10~ 3 M) AND COLLAGEN LYSIS*
Diameter of Lysis Alkali-burned epithelium plus Tyrode's solution plus puromycin Granuloma (only stroma) puromycin
> 10 mm 0 > 10 mm
* Five assays for each category.
ameter of collagen lysis. Puromycin com pletely inhibited collagen lysis by the epithe lium from alkali-burned corneas (controls), but allowed lysis by the inflamed stromas. The nitrogen mustard treatment schedule resulted in a circulating white blood count between 1200 to 1800 on the day of intralamellar injection. The granulocytes in the pe ripheral blood were mainly blast cells and were about 30% of the white blood cells. Specimens of the corneas of the nitrogen mustard-treated animals did not lyse the col lagen gels at four, six, and 10 days after in jection of Freund's adjuvant. The inflamma tory reaction that resulted from injection of Freund's adjuvant was markedly reduced in comparison with those animals that had not received nitrogen mustard (i.e., there was less edema and neovascularization was de layed to the end of the second week). Histologically, there were many less white blood cells in the stromal infiltrate and these were heavily granulated. COMMENTS
Inflammation was consistently produced in the rabbit corneas by the intralamellar in jection of Freund's adjuvant. This was first noted in 24 hours, reached its peak in inten sity by the end of the third week and began to quiet after two months. The stromal cellu lar infiltrate surrounding the foreign protein was mainly granulocytic in the first few days, but later took on the characteristics of a granuloma with many large histiocytes or epitheliod cells in addition to the granulo cytes. Tissue culture assays showed that the
1141
stromas of these corneas caused collagen ly sis that was similar in quantity to that caused by the ulcerated alkali-burned cornea.2 Har vest of the enzyme showed it to be a true collagenase because of its characteristic effect on the viscosity and optical rotations of recon stituted collagen.4 The molecular weight of the enzyme and the dynamics of its inhibition will be reported. Gross and Lapiere 6 demonstrated that ani mal collagenase is produced by living cells (epithelium and possibly fibroblasts) and that destruction of the cells by freezing stops collagenase production. An exception is the collagenase from the granulocytes which originates in the granules and can be de tected only after cell disruption.6 Another unusual feature of the granulocyte collage nase is that it is only weakly active against collagen fibrils. Since no other blood cell ele ments contain collagenase,0 the only cells that could have produced the collagenase found in the inflamed corneal stromas were either the fibroblasts, the histiocytes, or the granulocytes. Collagenase was detected after freeze-thawing, which indicated that the en zyme originated from the granulocytes. A less likely possibility was that the enzyme was produced and stored in the tissues. Since stored collagenase has never been demon-
s
0>
.2
3
£
*-. o ^
1
100 ( \ \ 80 - \ 60 40
t \
\
1V ~
J * ^
■5
£
£
20 ,
L
1
1
m
'
hours
Fig. 2 (Brown and Hock). Graph showing a re duction in specific viscosity of reconstituted colla gen after exposure to the stromal collagenase.
1142
AMERICAN JOURNAL OF OPHTHALMOLOGY
strated in tissues before, it is unlikely that the collagenolytic activity after freeze-thawing was from enzyme produced and stored in the tissues by the fibroblasts and the histiocytes before their destruction. Soaking the inflamed stromas with puromycin, which is a potent inhibitor of protein metabolism, allowed collagenolytic activity. This indicates that the stromal enzyme was not a product of cell metabolism, which again points to the granulocytes as the collagenase carrier. Finally, when the number of the circulating granulocytes was reduced by approximately 80% by intravenous nitro gen mustard, collagenase production was not detected after intralamellar injection. Except for the findings that the harvested enzyme was active against heat-gelled collagen fibrils, the results of the present study indi cate that at least part of the collagenase of the stromal granuloma originates from the granulocytes. The obvious reduction in stromal edema and neovascularization in the animals pretreated with nitrogen mustard suggests that the granulocyte may also play a role in medi ating various aspects of the inflammatory re sponse. Studies are in progress to isolate this effect of the granulocyte. Collagenolytic lysozomal enzymes have been isolated from inflamed tissues in other parts of the body. However, these were effective only when pH acidity fell between 3 and 5.5. There are no studies showing that the pH of inflamed tissue is in the acid range. Consequently, there is doubt that these acid hydrolases could destroy collagen in vivo. The present study demonstrates a neutral collagenase in inflammation of the corneal stroma, indicating the destructive po tential of the inflammatory process. Though there was no gross loss of tissue in these in flamed corneas, preliminary study of the
DECEMBER, 1971
electron micrographs of the inflamed areas showed various types of collagen fibril alter ations.7 Recently, we found that the collagenases of the alkali-burned cornea and the inflamed stroma are each inhibited by dilute concen trations of fresh serum. It may be that the minimal corneal destruction relative to the large amount of collagenase produced by the inflamed stroma is due to enzyme inhibition by elements in the serum. This inhibition would probably be related to new vessel in growth into the inflamed area. This will be expanded in a later report. SUMMARY
Inflammation of the corneal stroma was produced in rabbits by the intralamellar in jection of Freund's adjuvant. The inflamma tion progressed from a typically acute to a chronic granulomatous type of inflammation. A collagenase was isolated from the inflamed stromas and partially characterized. The en zyme appeared early in the course of the in flammation, thus indicating that it originated in the granulocytes. REFERENCES
1. Huber, J. D., Parker, F., and Odland, G. F.: A basic fuschin and alkalinized methylene blue rapid stain for epoxy-embedded tissue. Stain Tech. 43:83,1968. 2. Brown, S. I., Weller, C. A., and Wassermann, H. E.: Collagenolytic activity of alkali-burned cor neas. Arch. Ophth. 81:370, 1969. 3. Brown, S. I., Weller, C. A., and Akiya, S.: The pathogenesis of ulcers of alkali-burned cor neas. Arch. Ophth. 83:205, 1970. 4. Dische, Z.: A new specific color reaction of hexuronic acids. J. Biol. Chem. 167:189, 1947. 5. Gross, G., and Lapiere, C. M.: Activity in am phibian tissues: A tissue culture assay. Proc. Nat. Acad. Sci. 48:1014,1962. 6. Lazarus, G. S., Brown, R. S., Daniels, J. R., and Fullmer, H. M.: Human granulocyte collagen ase. Science 159:1483, 1968. 7. Nakanishi, I., and Brown, S. I.: Unpublished data, 1970.