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A histochemical study of the fibrinolytic activity in dermal tuberculous lesions produced by BCG in rabbits
EXPERIMENTAL
A?il>
~IOLECULAR
A Histochemical Tuberculous ATHANASIOS
SMOKOVITIS,
25, 236-241
PATHOLOGY
Study
(1976)
of the Fibrinolytic
Lesions 2 MINEHARU AND
Produced
by BCG
SUGIMOTO, TACE
Activity
3. 5 ARTHUR
in Dermal
in Rabbits1 M.
DANNENBERG,
JR. 3
ASTRUP 4, B
2 Department of Physiology, lndiana University School of Medicine, NW Center for Medical Education, Gary, Indiana 46408; 3 Departments of Environmental Medicine, Epidemiology and Pathology, School of Hygiene and Public Health, and S&o01 of Medicine, The Johns Hopkins University, Baltimore, MaryZand 21025; 4 The Gaubius Institute, Health Research Organization TNO, Leiden, The Netherlands Receizjcd
February
26, 1976
Dermal tuberculous lesions were produced in rabbits by the injection of BCG, the vaccine strain of the tubercle Bacillus. The lesions showed a remarkable increase of focal fibrinolytic activity throughout a 4- to 53-day period of growth and regression. The fibrinolytic activity was caused by a plasminogen activator. At 11 to 18 days, when hypersensitivity to the BCG vaccine became pronounced and caseous centers developed, fibrinolytic activity was particularly high. It was mainly related to dilated small blood vessels and to new capillaries in the granulation tissue. In the deep dermis near the lesion, dilated larger blood vessels showed increased fibrinolytic activity during the late stages of the healing process. Dilated larger blood vessels in the deep dermis between the lesion area and the normal skin frequently showed increased activity independent of the age of the lesion.
INTRODUCTION While the normal human liver has little or no fibrinolytic activity, the cirrhotic liver contains large amounts of plasminogen activator ( Astrup et al., 1960). The activator is located in the veins of the scar tissue (Todd, 1959). Later, an association between the tissue repair process and the presence of fibrinolytically active vessels was substantiated experimentally in the rat (Kwaan and Astrup, 1964; Peterson et al., 1969; Tympanidis and Astrup, 1972). High fibrinolytic activity was observed in the hyperplastic vessels and capillaries of the richly vascularized repair tissue. During the later stages of the repair process, the fibrinolytic activity disappeared, ultimately leaving inactive fibrous scar tissue. Similar localization of fibrinolytic activity was observed in physiological tissue 1 This study was supported by USPHS Grants HL-05020 and HE-14153 from the National Heart and Lung Institute, and by USPHS Grant AI-08876 from the National Institute of Allergy and Infectious Diseases. Part of the experimental work was performed at the Institute for Medical Research of the James F. Mitchell Foundation, Washington, D.C. 31 5 At Johns Hopkins University on leave of absence from the Department of Medicine, Kumamoto University School of Medicine, Kumamoto, Japan. G To whom all correspondence should be addressed.
Copyright All rights
@ 1976 by Academic Press, Inc. of reproduction in any form reserved.
FIBRINOLYSIS
IN
TUBERCULOUS
LESIONS
237
repair, such as during ,the obliteration of the ductus arteriosus (Glas-Greenwalt et al. 1972). Studying skin wounds in the rabbit ear chamber, Clark and Clark (1953) suggested that regenerating epidermis secretes a “fibrinolytic enzyme” which facilitates the movement of cells beneath the fibrin clot and tissue debris. This report describes the distribution of fibrinolytic activity in dermal tuberculous lesions of rabbits. MATERIALS
AND
METHODS
White New Zealand rabbits of both sexes (2 to 3 kgm) were injected intradermally in the paravertebral area ( in five sites for Series I and in two sites for Series II) with 0.1 ml of double strength BCG (Danish strain No. 1331, supplied by Dr. K. Bunch-Christensen, Statens Seruminstitut, Copenhagen) as described by Courtade et al. (1975). At various times thereafter, the epidermis was removed from the lesion, and a plastic chamber was aflixed over the denuded area by gluing its edges to the surrounding skin (Sheldon et al. 1967). It was filled with 0.2 ml of Medium 199. A corresponding chamber covered a control “lesion” caused by the removal of epidermis from an area of the normal skin on the contralateral side of the same animal. After 2 days, the chamber contents were removed and analyzed for various enzymes ( Sugimoto et uZ., 1975)) and the lesion beneath the chambers was surgically biopsied. Tuberculin tests were made weekly by injecting intradermally a 1:lO dilution of Old Tuberculin (Eli Lilly and Co., Indianapolis, Indiana). In the first series of eight rabbits, two chamber-covered BCG lesions and two chamber-covered control “lesions” were surgically biopsied across their centers at 11, 18, 25, and 27 days. In the second series of 12 rabbits, three lesions of each type were biopsied at 4, 18, 32, and 53 days. Each BCG lesion and control “lesion” on a given day was from a different rabbit. No lesion was biopsied twice, The biopsies were immediately frozen on the outside of a beaker containing acetone and dry ice. They were then stored at a temperature below -20°C until sectioned in a cryostat. One-half of each specimen was used to study fibrinolytic activity; the other half was used to study p-galactosidase activity (Sugimoto et al., 1975). A total of 20 BCG lesions and 20 control “lesions” were evaluated. These were supplemented with further control biopsies of normal, undisturbed skin from untreated rabbits. On an average, 100 serial frozen sections were cut from each BCG lesion and 50 from each control “lesion.” Sections were cut at 9 pm perpendicular to the skin surface, starting from the center of the lesion. The sections were collected on precleaned microscope slides, briefly dried in the air at room temperature, and then covered with a mixture of (a) 60 ~1 of a 0.7% solution of bovine plasminogen-rich fibrinogen (prepared by ammonium sulfate precipitation after Brakman and As&up, 1971) or in some instances, with plasminogen-free fibrinogen and (b) 10 pl of bovine thrombin (20 NIH units/ml in saline, Leo Pharmaceuticals, Copenhagen, Denmark). After stabilization of the fibrin clot for 20 min in a moist chamber at lo-15”C, the slides were incubated at 37°C for 30, 60, 90, 120, and 180 min. They were then fixed
‘3X
SMOKOVITIS
ET AL.
FIG. 1. Demlal BCG lesion 18 days of age. An area of intense lysis is related to the lesion. Active dilated small blood vessels are seen in the mid-dermal area of the lesion. Necrotic material near the area of lysis shows no activity. The absence of activity in dilated blood vessels in the deep dermis is obscured by the Iysis extending from above. (Control “lesions,” made by chambers placed on the normal skin of the same rabbit, showed no activity during the same incubation period.) Fibrin slide technique; incubated 30 min. X 19.
in formaldehyde, rinsed in tap water, stained with Harris’ hematoxylin, and mounted in glycerin jelly. Sites of fibrinolytic activity produced clear foci or areas of lysis in the stained fibrin.
FIBRINOLYSIS
IN TUBERCULOUS
LESIONS
239
RESULTS BCG Lesions
A remarkable increase in focal fibrinolytic activity was found in BCG lesions of all ages (4, 11, 18, 25, 27, 32, and 53 days). Areas of intense lysis were observed after 60 min of incubation of the pIasminogen-rich fibrin slides. Specimens obtained at 11 and 18 days showed particularly intense lysis, appearing in most of the sections after 30 min of incubation (Fig. 1). After 60 min of incubation the areas of lysis produced in these specimens were larger than those produced in lesions of other ages. At 11 and 18 days hypersensitivity to tuberculin had become pronounced and caseous centers had developed. When the sections were incubated for short periods of time, or in specimens which showed less extensive lysis, the activity could be localized to dilated small blood vessels and to capillaries in the granulation tissue. Dilated larger blood vessels, mainly in the deep dermis, were seen in all BCG lesions. Usually these vessels showed little or no activity in the early stages of repair (lesions biopsied from 4 to 32 days) after 60 min of incubation of the fibrin slides (Fig. 2). However, in the biopsies taken at 53 days, the dilated vessels in deep dermis were usually quite active after 60 min of incubation. Interestingly, independent of the age of the lesion, the dilated larger blood vessels in the deep dermis between the lesion area and the normal skin frequently showed 1ysi.s after 60 or 90 min of incubation, while the fibrinolytic activity of the vessels in the mid and upper dermis usually was negligible after the same period of incubation. A slight cellular infiltration was observed in all dermal layers. Sections of BCG lesions of all ages placed on plasminogen-free fibrin slides showed only weak activity after prolonged incubation ( 180 min). Thus, the observed marked activity on plasminogen-rich fibrin slides was due to a plasminogen activator and not due to proteases directly hydrolyzing fibrin, Assays of fibrinolytic activity in samples of the chamber fluid were negative, probably because of the small volumes of fluid collected, their dilution (10x with Medium 199), and the presence of inhibitors. When incubated for 17 hr at 37”C, none of them showed fibrinolytic activity on plasminogen-rich fibrin plates (Brakman and Astrup, 1971). ContruE Specimens
Most of the biopsies of control “lesions” showed a slight cellular infiltration with some dilation of blood vessels in the dermis. Foci of fibrinolysis usually appeared in the dermis after 120 min of incubation of the slides, and in two specimens after 60 min of incubation. Lysis was most frequent around the dilated blood vessels in the deep dermis and sometimes occurred around blood vessels in the mid and upper dermis. The lytic activity was always much weaker than that seen in the corresponding BCG lesions. Four control “lesions” showed undilated blood vessels and negligible fibrinolytic activity even after 120 min of incubation. The control specimens from the skin of rabbits bearing no chambers nor BCG lesions showed focal fibrinolytic activity only after 150-180 min of incubation, and this activity was mainly related to blood vessels in the deep dermis.
240
FIG. 2. Dermal BCG lesion large blood vessels are mostly
SMOKOVITIS
4 days inactive.
ET AL.
of age. An area of intense lysis Fibrin slide technique; incubated
is visible. 60 min.
The
dilated
X22.
DISCUSSION Dermal tuberculous lesions produced in rabbits by injection of BCG showed a remarkable increase in focal fibrinolytic activity (due to a plasminogen activator) from 4 days to 53 days of age (the time of the last biopsy). The increased activity was mainly related to dilated small blood vesselsand capillaries in the granulation tissue, and to dilated larger vesselsin the deep dermis during the period of healing. The period during which the fibrinolytic activity was particularly marked (11 to
FIBRINOLYSIS
IN
TUBERCULOUS
LESIONS
241
18 days) corresponded to the period of increased capillary density reported elsewhere (Courtade et al., 1975). This was also the period in which delayed hypersensitivity to tuberculin became pronounced, caseousnecrosis was increased, and the lesions reached their maximal size. At this time the host was sensitive to the products of the tubercle bacillus, and large amounts of these products were probably present in the lesion. The enhancement of the fibrinolytic activity following the injection of BCG was easy to observe, because the fibrinolytic activity of normal rabbit skin is low, being related mainly to vessels in the deep dermis and requiring 150 to 180 min of incubation. A similar pattern of focaIIy increased fibrinolytic activity was observed in rats after the intradermal injection of aluminum hydroxide or complete Freund’s adjuvant (Smokovitis, et al., 1975, 1976), as well as in rabbits or guinea pigs after the intradermal injection of aluminum hydroxide or following a skin crush injury (Smokovitis and Astrup, in preparation). Products from necrotic tissue in the BCG lesions may have stimulated capillary proliferation. Such proliferating capillaries are rich in plasminogen activator ( Kwaan and Astrup, 1964). Fibrin present in the region immediately surrounding new capillaries in repair tissue was reported to undergo resolution at the same time as blood begins to circulate in them (Clark and Clark, 1953). Fibrin lysis in the dermal lesions appears to depend upon the ceIIular release ‘of a pIasminogen activator rather than upon the release of a fibrinolytic enzyme. REFERENCES ASTRUP, T., RAshfussEN,
J,, AMERY, A., and POULSEN, H. E. (1960). Fibrinolytic activity of cirrhotic liver. Nature (Lond.) 185, 619-620. method for assay of fibrinolytic BRAKMAN, P., and ASTRUP, T. (1971). The fib rin plate agents. In “Thrombosis and Bleeding Disorders,” (N. U. Bang, F. K. Beller, E. Deutsch, and E. F. Mammen, eds.), pp. 332-336. Academic Press, New York. CLARK, E. R., and CLARK, E. L. ( 1953). Growth and behavior of epidermis as observed microscopically in observation chambers inserted in the ears of rabbits. Amer. J. Anat. 93, 171219. COURTADE, E. T., TSUDA, T., THOMAS, C. R., and DANNENBERG, A. M., JR. ( 1975). Capillary density in developing and healing tuberculous lesions produced by BCG in rabbits. Amer. 1. Pathol. 78, 243-256. GLAS-GREENWALT, P., STRAND, C., and ASTRUP, T. ( 1972). Fibrinolytic activity in the closed ductus arteriosus. Experientia 28, 448. KWAAN, H. C., and ASTRUP, T. ( 1964). Fibrinolytic activity of reparative connective tissue. J. Puthol. Bacterial. 87, 409-414. PETERSON, H. I., PETERSON, A., and ZEDERFELDT, B. (1969). Fibrinolytic activity in healing wound. Acta Chir. &and. 135, 649-652. SHELDON, W. H., MILDVAN, D., and ALLEN, J. C. (1967). Some serum protein and cellular constituents of inflammatory lesions. Collection of exudates in a chamber adhered over skin wounds of rabbits. Johns Hopkins Med. J. 121, 113-133. SMOKOVITIS, A., KOK, P., and ASTRUP, T. ( 1975). Tissue repair in rats in presence of locally applied tissue plasminogen activator. Exp. Mol. Puthol. 22, 109-117. SMOKOVITIS, A., KOK, P., and ASTRUP, T. ( 1976). Presence of fibrin prolongs the retainment of locally injected tissue plasminogen activator. Exp. Mol. Patho’l. 24, 48-58. SUGIMOTO, M., DANNENBERG, A. M., JR., WAHL, L. M., DANIELS, D. C., ETTINGER, W. H., JR., and THOMAS, C. R. ( 1975). Extracellular hydrolytic enzymes collected in skin chambers placed on rabbit BCG lesions. (Abstract). J. Reticuloendothel. Sot. 18, 29b. TODD, A. S. ( 1959). The histological loca!ization of fibrinolysin activator. T. Putlzol. BocterioE. 78, 281-283. TYMPANIDIS, K., and ASTRUP, T. ( 1972). Fibrinolytic activity in injured rat skin. Erp. Mol. Puthol. 16, 101-108.
A?il>
~IOLECULAR
A Histochemical Tuberculous ATHANASIOS
SMOKOVITIS,
25, 236-241
PATHOLOGY
Study
(1976)
of the Fibrinolytic
Lesions 2 MINEHARU AND
Produced
by BCG
SUGIMOTO, TACE
Activity
3. 5 ARTHUR
in Dermal
in Rabbits1 M.
DANNENBERG,
JR. 3
ASTRUP 4, B
2 Department of Physiology, lndiana University School of Medicine, NW Center for Medical Education, Gary, Indiana 46408; 3 Departments of Environmental Medicine, Epidemiology and Pathology, School of Hygiene and Public Health, and S&o01 of Medicine, The Johns Hopkins University, Baltimore, MaryZand 21025; 4 The Gaubius Institute, Health Research Organization TNO, Leiden, The Netherlands Receizjcd
February
26, 1976
Dermal tuberculous lesions were produced in rabbits by the injection of BCG, the vaccine strain of the tubercle Bacillus. The lesions showed a remarkable increase of focal fibrinolytic activity throughout a 4- to 53-day period of growth and regression. The fibrinolytic activity was caused by a plasminogen activator. At 11 to 18 days, when hypersensitivity to the BCG vaccine became pronounced and caseous centers developed, fibrinolytic activity was particularly high. It was mainly related to dilated small blood vessels and to new capillaries in the granulation tissue. In the deep dermis near the lesion, dilated larger blood vessels showed increased fibrinolytic activity during the late stages of the healing process. Dilated larger blood vessels in the deep dermis between the lesion area and the normal skin frequently showed increased activity independent of the age of the lesion.
INTRODUCTION While the normal human liver has little or no fibrinolytic activity, the cirrhotic liver contains large amounts of plasminogen activator ( Astrup et al., 1960). The activator is located in the veins of the scar tissue (Todd, 1959). Later, an association between the tissue repair process and the presence of fibrinolytically active vessels was substantiated experimentally in the rat (Kwaan and Astrup, 1964; Peterson et al., 1969; Tympanidis and Astrup, 1972). High fibrinolytic activity was observed in the hyperplastic vessels and capillaries of the richly vascularized repair tissue. During the later stages of the repair process, the fibrinolytic activity disappeared, ultimately leaving inactive fibrous scar tissue. Similar localization of fibrinolytic activity was observed in physiological tissue 1 This study was supported by USPHS Grants HL-05020 and HE-14153 from the National Heart and Lung Institute, and by USPHS Grant AI-08876 from the National Institute of Allergy and Infectious Diseases. Part of the experimental work was performed at the Institute for Medical Research of the James F. Mitchell Foundation, Washington, D.C. 31 5 At Johns Hopkins University on leave of absence from the Department of Medicine, Kumamoto University School of Medicine, Kumamoto, Japan. G To whom all correspondence should be addressed.
Copyright All rights
@ 1976 by Academic Press, Inc. of reproduction in any form reserved.
FIBRINOLYSIS
IN
TUBERCULOUS
LESIONS
237
repair, such as during ,the obliteration of the ductus arteriosus (Glas-Greenwalt et al. 1972). Studying skin wounds in the rabbit ear chamber, Clark and Clark (1953) suggested that regenerating epidermis secretes a “fibrinolytic enzyme” which facilitates the movement of cells beneath the fibrin clot and tissue debris. This report describes the distribution of fibrinolytic activity in dermal tuberculous lesions of rabbits. MATERIALS
AND
METHODS
White New Zealand rabbits of both sexes (2 to 3 kgm) were injected intradermally in the paravertebral area ( in five sites for Series I and in two sites for Series II) with 0.1 ml of double strength BCG (Danish strain No. 1331, supplied by Dr. K. Bunch-Christensen, Statens Seruminstitut, Copenhagen) as described by Courtade et al. (1975). At various times thereafter, the epidermis was removed from the lesion, and a plastic chamber was aflixed over the denuded area by gluing its edges to the surrounding skin (Sheldon et al. 1967). It was filled with 0.2 ml of Medium 199. A corresponding chamber covered a control “lesion” caused by the removal of epidermis from an area of the normal skin on the contralateral side of the same animal. After 2 days, the chamber contents were removed and analyzed for various enzymes ( Sugimoto et uZ., 1975)) and the lesion beneath the chambers was surgically biopsied. Tuberculin tests were made weekly by injecting intradermally a 1:lO dilution of Old Tuberculin (Eli Lilly and Co., Indianapolis, Indiana). In the first series of eight rabbits, two chamber-covered BCG lesions and two chamber-covered control “lesions” were surgically biopsied across their centers at 11, 18, 25, and 27 days. In the second series of 12 rabbits, three lesions of each type were biopsied at 4, 18, 32, and 53 days. Each BCG lesion and control “lesion” on a given day was from a different rabbit. No lesion was biopsied twice, The biopsies were immediately frozen on the outside of a beaker containing acetone and dry ice. They were then stored at a temperature below -20°C until sectioned in a cryostat. One-half of each specimen was used to study fibrinolytic activity; the other half was used to study p-galactosidase activity (Sugimoto et al., 1975). A total of 20 BCG lesions and 20 control “lesions” were evaluated. These were supplemented with further control biopsies of normal, undisturbed skin from untreated rabbits. On an average, 100 serial frozen sections were cut from each BCG lesion and 50 from each control “lesion.” Sections were cut at 9 pm perpendicular to the skin surface, starting from the center of the lesion. The sections were collected on precleaned microscope slides, briefly dried in the air at room temperature, and then covered with a mixture of (a) 60 ~1 of a 0.7% solution of bovine plasminogen-rich fibrinogen (prepared by ammonium sulfate precipitation after Brakman and As&up, 1971) or in some instances, with plasminogen-free fibrinogen and (b) 10 pl of bovine thrombin (20 NIH units/ml in saline, Leo Pharmaceuticals, Copenhagen, Denmark). After stabilization of the fibrin clot for 20 min in a moist chamber at lo-15”C, the slides were incubated at 37°C for 30, 60, 90, 120, and 180 min. They were then fixed
‘3X
SMOKOVITIS
ET AL.
FIG. 1. Demlal BCG lesion 18 days of age. An area of intense lysis is related to the lesion. Active dilated small blood vessels are seen in the mid-dermal area of the lesion. Necrotic material near the area of lysis shows no activity. The absence of activity in dilated blood vessels in the deep dermis is obscured by the Iysis extending from above. (Control “lesions,” made by chambers placed on the normal skin of the same rabbit, showed no activity during the same incubation period.) Fibrin slide technique; incubated 30 min. X 19.
in formaldehyde, rinsed in tap water, stained with Harris’ hematoxylin, and mounted in glycerin jelly. Sites of fibrinolytic activity produced clear foci or areas of lysis in the stained fibrin.
FIBRINOLYSIS
IN TUBERCULOUS
LESIONS
239
RESULTS BCG Lesions
A remarkable increase in focal fibrinolytic activity was found in BCG lesions of all ages (4, 11, 18, 25, 27, 32, and 53 days). Areas of intense lysis were observed after 60 min of incubation of the pIasminogen-rich fibrin slides. Specimens obtained at 11 and 18 days showed particularly intense lysis, appearing in most of the sections after 30 min of incubation (Fig. 1). After 60 min of incubation the areas of lysis produced in these specimens were larger than those produced in lesions of other ages. At 11 and 18 days hypersensitivity to tuberculin had become pronounced and caseous centers had developed. When the sections were incubated for short periods of time, or in specimens which showed less extensive lysis, the activity could be localized to dilated small blood vessels and to capillaries in the granulation tissue. Dilated larger blood vessels, mainly in the deep dermis, were seen in all BCG lesions. Usually these vessels showed little or no activity in the early stages of repair (lesions biopsied from 4 to 32 days) after 60 min of incubation of the fibrin slides (Fig. 2). However, in the biopsies taken at 53 days, the dilated vessels in deep dermis were usually quite active after 60 min of incubation. Interestingly, independent of the age of the lesion, the dilated larger blood vessels in the deep dermis between the lesion area and the normal skin frequently showed 1ysi.s after 60 or 90 min of incubation, while the fibrinolytic activity of the vessels in the mid and upper dermis usually was negligible after the same period of incubation. A slight cellular infiltration was observed in all dermal layers. Sections of BCG lesions of all ages placed on plasminogen-free fibrin slides showed only weak activity after prolonged incubation ( 180 min). Thus, the observed marked activity on plasminogen-rich fibrin slides was due to a plasminogen activator and not due to proteases directly hydrolyzing fibrin, Assays of fibrinolytic activity in samples of the chamber fluid were negative, probably because of the small volumes of fluid collected, their dilution (10x with Medium 199), and the presence of inhibitors. When incubated for 17 hr at 37”C, none of them showed fibrinolytic activity on plasminogen-rich fibrin plates (Brakman and Astrup, 1971). ContruE Specimens
Most of the biopsies of control “lesions” showed a slight cellular infiltration with some dilation of blood vessels in the dermis. Foci of fibrinolysis usually appeared in the dermis after 120 min of incubation of the slides, and in two specimens after 60 min of incubation. Lysis was most frequent around the dilated blood vessels in the deep dermis and sometimes occurred around blood vessels in the mid and upper dermis. The lytic activity was always much weaker than that seen in the corresponding BCG lesions. Four control “lesions” showed undilated blood vessels and negligible fibrinolytic activity even after 120 min of incubation. The control specimens from the skin of rabbits bearing no chambers nor BCG lesions showed focal fibrinolytic activity only after 150-180 min of incubation, and this activity was mainly related to blood vessels in the deep dermis.
240
FIG. 2. Dermal BCG lesion large blood vessels are mostly
SMOKOVITIS
4 days inactive.
ET AL.
of age. An area of intense lysis Fibrin slide technique; incubated
is visible. 60 min.
The
dilated
X22.
DISCUSSION Dermal tuberculous lesions produced in rabbits by injection of BCG showed a remarkable increase in focal fibrinolytic activity (due to a plasminogen activator) from 4 days to 53 days of age (the time of the last biopsy). The increased activity was mainly related to dilated small blood vesselsand capillaries in the granulation tissue, and to dilated larger vesselsin the deep dermis during the period of healing. The period during which the fibrinolytic activity was particularly marked (11 to
FIBRINOLYSIS
IN
TUBERCULOUS
LESIONS
241
18 days) corresponded to the period of increased capillary density reported elsewhere (Courtade et al., 1975). This was also the period in which delayed hypersensitivity to tuberculin became pronounced, caseousnecrosis was increased, and the lesions reached their maximal size. At this time the host was sensitive to the products of the tubercle bacillus, and large amounts of these products were probably present in the lesion. The enhancement of the fibrinolytic activity following the injection of BCG was easy to observe, because the fibrinolytic activity of normal rabbit skin is low, being related mainly to vessels in the deep dermis and requiring 150 to 180 min of incubation. A similar pattern of focaIIy increased fibrinolytic activity was observed in rats after the intradermal injection of aluminum hydroxide or complete Freund’s adjuvant (Smokovitis, et al., 1975, 1976), as well as in rabbits or guinea pigs after the intradermal injection of aluminum hydroxide or following a skin crush injury (Smokovitis and Astrup, in preparation). Products from necrotic tissue in the BCG lesions may have stimulated capillary proliferation. Such proliferating capillaries are rich in plasminogen activator ( Kwaan and Astrup, 1964). Fibrin present in the region immediately surrounding new capillaries in repair tissue was reported to undergo resolution at the same time as blood begins to circulate in them (Clark and Clark, 1953). Fibrin lysis in the dermal lesions appears to depend upon the ceIIular release ‘of a pIasminogen activator rather than upon the release of a fibrinolytic enzyme. REFERENCES ASTRUP, T., RAshfussEN,
J,, AMERY, A., and POULSEN, H. E. (1960). Fibrinolytic activity of cirrhotic liver. Nature (Lond.) 185, 619-620. method for assay of fibrinolytic BRAKMAN, P., and ASTRUP, T. (1971). The fib rin plate agents. In “Thrombosis and Bleeding Disorders,” (N. U. Bang, F. K. Beller, E. Deutsch, and E. F. Mammen, eds.), pp. 332-336. Academic Press, New York. CLARK, E. R., and CLARK, E. L. ( 1953). Growth and behavior of epidermis as observed microscopically in observation chambers inserted in the ears of rabbits. Amer. J. Anat. 93, 171219. COURTADE, E. T., TSUDA, T., THOMAS, C. R., and DANNENBERG, A. M., JR. ( 1975). Capillary density in developing and healing tuberculous lesions produced by BCG in rabbits. Amer. 1. Pathol. 78, 243-256. GLAS-GREENWALT, P., STRAND, C., and ASTRUP, T. ( 1972). Fibrinolytic activity in the closed ductus arteriosus. Experientia 28, 448. KWAAN, H. C., and ASTRUP, T. ( 1964). Fibrinolytic activity of reparative connective tissue. J. Puthol. Bacterial. 87, 409-414. PETERSON, H. I., PETERSON, A., and ZEDERFELDT, B. (1969). Fibrinolytic activity in healing wound. Acta Chir. &and. 135, 649-652. SHELDON, W. H., MILDVAN, D., and ALLEN, J. C. (1967). Some serum protein and cellular constituents of inflammatory lesions. Collection of exudates in a chamber adhered over skin wounds of rabbits. Johns Hopkins Med. J. 121, 113-133. SMOKOVITIS, A., KOK, P., and ASTRUP, T. ( 1975). Tissue repair in rats in presence of locally applied tissue plasminogen activator. Exp. Mol. Puthol. 22, 109-117. SMOKOVITIS, A., KOK, P., and ASTRUP, T. ( 1976). Presence of fibrin prolongs the retainment of locally injected tissue plasminogen activator. Exp. Mol. Patho’l. 24, 48-58. SUGIMOTO, M., DANNENBERG, A. M., JR., WAHL, L. M., DANIELS, D. C., ETTINGER, W. H., JR., and THOMAS, C. R. ( 1975). Extracellular hydrolytic enzymes collected in skin chambers placed on rabbit BCG lesions. (Abstract). J. Reticuloendothel. Sot. 18, 29b. TODD, A. S. ( 1959). The histological loca!ization of fibrinolysin activator. T. Putlzol. BocterioE. 78, 281-283. TYMPANIDIS, K., and ASTRUP, T. ( 1972). Fibrinolytic activity in injured rat skin. Erp. Mol. Puthol. 16, 101-108.