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Effects of Cortisol Released Cutaneous Protease Upon the Permeability of the Micro-Circulation*
Vol. 48, No. 4
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1567 by The Williams & Wilkins Co.
EFFECTS OF CORTISOL RELEASED CUTANEOUS PROTEASE UPON THE PERMEABILITY OF THE MICROCIRCULATION* A. W. J. LYKKE, M.D., D. A. WILLOUGHBY, Pu.D. AND J. C. HOUCK, Pn..D.t
It has recently been observed that subse- number of rats which had received only the car-
quent to the injection of cortisol, the ex- rier solvent for the cortisol preparation (Cortril,
C. Pfizer) but none of the steroid. CRPP and the similar preparation made from the non-steroid treated control animals were as-
tracellular, extrafibrillar compartment of rat skin contains a non-lysosomal, neutral pH optimal proteolytic enzyme which can be inhibited by both soybean trypsin inhibitor and salicylates (1, 2). A possibly similar proteolytic activity appears to have been found in
sayed for proteolytic activity upon denatured hemoglobin as described by Houck and Patel (1), and upon the synthetic substrates for chymo-
trypsin (acetyl tyrosine ethyl ester) and trypsin (tosy argine methyl ester) by pH stat technics. necrotic wounds (3, 4). Since some proteolytic Substances were tested for their ability to inenzymes are capable of increasing the per- crease vascular permeability in the rat by the meability of the micro-circulation (5), it was method described by Judah and Willoughby (6). decided to explore the effects of cortisol-re- Animals were given intravenous injections of a 0.1% solution of trypan blue (0.4 ml per 100 gms).
leased protease preparations from rat skin The substances under test were injected intraupon the vascular permeability of the rat.
dermally into the shaved abdominal skin in a
volume of 0.1 ml; 30 minutes later the skin was examined and the blue staining at the injection site was assessed either visually in arbitrary units (0—10) or by extraction and measurement in a colorimeter (6).
MATERIALS AND METHODS
Measurement of vascular permeability Cortisol-released protease preparations (CRPP) were prepared from the shaven and cleaned skin of male Sprague-Dawloy rats weighing 190—210 gm 26 hours after receiving two doses of cortisol.
Leulcocyte emigration.
Action on leukocyte emigration was estimated
by intradermal injection into rats in 0.1 ml
Three separate groups of 12 rats each were injected
amounts. The area was examined at various times
subcutaneously with 3 mg/kg of cortisol, and 24
hours later this injection was repeated intra- commencing 54 minutes after injection and histo-
peritone ally. Two hours after these animals had logical sections were prepared according to the received this second dose of cortisol I.P., they method of Hurley and Spector (7) who discuss were sacrificed and the entire skin was shaven, the reasons for choosing the appropriate time removed and dissected free of adhering fat, fascia intervals. and peniculus coraosum. These cleaned skins from
each group were minced with scissors collected
Antagonism of vascular permeability
the cold in a Serval Omni-mixer for 45 minutes in 10 ml/gm of 0.15 M NaC1. This mixture was allowed to stand overnight at 4°C and after centrifugation the supernatant was dialyzed for 24
permeability factors was carried out by methods described by Spector and Willoughby (8). Indo-
extracts of skin from cortisol treated rats served
The accumulation of colloidal carbon in venules
(CRPP) employed below. Similar preparations were made using a similar
CRPP was assessed by the method described by Majno, Palade and Schoefi (10) as modified for rat skin by Hurley (11). Rats were given an intra-
into three pools of four rats each extracted in
The assessment and antagonism of vascular
methacin was a gift from Merck, Sharpe & Dohme and was used intravenously in doses of 1—10 mg/kg hours with stirring against 1000 volumes of distilled water in the cold. The contents of the dialysis body weight (9). bag was centrifuged and the supernatant lyophi- Carbon leakage lized. These dialyzed, lyophilized isotonic saline
as the cortisol-released protease preparation and capillaries after the intradermal injection of Supported in part by grants from N.I.H. (AM-
venous injection of a colloidal suspension of carbon black (0.1 ml per 100 gms) at varying times after the intradermal injection of CRPP. Hospital, London, England and t Wnehernical The carbon was supplied by Gunther Wagner Research Lab., Children's Hospital, Washington, Pelikan Werke, Hanover, Germany (Batch No D.C. (Address for reprints.) C11/1431a). Thirty minutes after the injection 8168) and O.N.R. (NR-105-325) Received for publication June 1, 1966. * From the Pathology Dept., St. Bartholomew's
318
PROTEASE PERMEABILITY
319
permeable almost immediately after the insite was removed, stretched out on a piece of tradermal injection and thereafter the effect board, fixed in 4% buffered formaldehyde solution for 48 hours, dehydrated in ethanol and placed in rapidly waned. Delaying the injection of dye cedarwood oil to clear. The skin was then examined until 15 minutes after the intradermal injection showed that the onset of increased vascumicroscopically in a perspex chamber (11). All of the above tests were performed on at lar permeability is extremely rapid and that of carbon the animals were killed; the injection
least four animals each.
RESULTS
All three CRPP were found to be equally active against denatured hemoglobin and as active as these preparations previously employed (1) and to be equally capable of hydrolyzing significantly (10%) the synthetic
its duration is short. Site of increased vascular permeability
Injection of colloidal carbon with subsequent
microscopical examination revealed that carbon accumulated in both venules and capillaries although the effect was more marked in the venules (Figs. 3, 4). By delaying the insubstrate for chymotrypsin (acetyl ethyl ester). travenous injection of the carbon for various All of the CRPP prepared as described above times after the intradermal injection of had essentially identical quantitative effects CRPP it was possible to determine the time upon this synthetic substrate. None of these course of the effect of the protease on capilpreparations demonstrated any effects upon the lary and venular accumulation of carbon. It synthetic substrate for trypsin (tosyl arginine can be seen from Fig. 5 that the carbon accumulation persisted for a longer time period methyl ester). Extracts from the skin of three groups of in the venules than the capillaries. However, normal rats failed to demonstrate any pro- the effect on both venules and capillaries was teolytic activity on denatured hemoglobin or negligible in 15 minutes, confirming the short on either synthetic substrate mentioned duration of action determined above by trypan
above; similarly all of these extracts from blue injections. normal skin were inactive in the rat skin
permeability test. However, rats pretreated in The effect of antagonists Table I compares the effects of various such a way as to release protease activity into the skin, i.e. following systemic cortisol treat- treatments and antagonists on the permeabilment, contained significant amounts of the ity enhancing activity of CRPP and a variety protease (CRPP) and all nine pools represent- of other permeability factors. It can be seen
ing three groups of 12 animals each were
that with this screen of antagonists CRPP
found to be equally active in increasing vascular permeability in the skin of rats (Fig. 1). Thus 32 p.g of CRPP gave a maximal dye leakage, and increasing the dosage above this level led to the production of hemorrhagic lesion which obscured dye leakage. Activity was just detectable when 4 pg of CRPP was injected. From these results a dose response
may be readily distinguished from histamine or 5-hydroxyproline, by the use of the specific
antagonists, mepyramine maleate and 2-
acid diethylamide (BOL 148). Differentiation from bradykinin is best effected by the use of local sodium salicylate (2.5 mgs/ml of inoculum) or incubation with soy bean trypsin inhibitor (SBTI 100 pg/ml) curve was plotted (Fig. 2). Quantitatively Both these treatments reduced the effect of bromo-d-lysergie
none of the preparations from normal rats CRPP while not affecting bradykinin. The demonstrated this activity and all of the prep- kinin forming enzyme kallikrein is antago-
arations derived from cortisol treated animals nized by pretreating animals with either were equally active in increasing the vascular diisopropyl fluorophosphonate (DFP 2.5 mgs/ kg) or indomethacin (10 mgs/kg). The treatpermeability of rat skin.
Delaying the injection of the trypsin blue until various times after the intradermal injection of CRPP enabled us to determine the time course of the permeability enhancing effect. It was found that the vessels became
ment had no effect on the permeability increasing activity of CRPP. Globulin PF, one of the permeability factors in serum associated with the a globulins (12), may also be antagonized by treatment of the test animal
320
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
V
1'
B
FIG. 1. Abdominal skin of rat after intravenous trypan blue and intradermal injections 16 CRPP. B 16 g CRPP. C = 16 g CRPP + 250 g salicylate. D 16 jg CRPP pre-incubated with SBTI. (0.1 ml) of CRPP. A =
0 LU Iow_J LU L oi I—
Ui <
4
(n LU
U,
2
4
8
16
J.Jg CRPP INJECTED IN IRADERMALLY FIG. 2. The
effect of various doses of CRPP on vascular permeability in the rat
with DFP. SBTI incubation, which so effectively suppresses CRPP, had no effect on the permeability factor which may be activated in rat plasma by the addition of turpentine, no effect on pleural exudates from rat (8), no effect on the permeability factor associated with the lymph node cells (13), and finally no
effect on RNA (14). It appears that this protease is bringing about an increase in vas-
cular permeability by a mechanism or via mediators hitherto not described.
It is of interest that the antagonists, while
often causing a dramatic reduction in the leakage of protein-bound trypan blue, failed
321
PROTEASE PERMEABILITY
I
if
St
•
¼ FIG. 3. Carbon accumulation in the venules of rat skin following intradermal injection of CRPP (16 pg). X 25. FIG. 4. Carbon accumulation in the capillaries of rat skin following intradermal injection of CRPP (16 pg). X 100.
322
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Histology of the lesions produced by CRPP
I
jO
at various times after administration of the extract
0<0 o
1. 45 minutes. A very prominent vascular reaction occurs at this time. Small blood vessels of the capillary and venular size are con-
o
C
I-0
U-
WI-.
8.
0< 00
IES
Fia. 5. Time course of the accumulation of
carbon in both venules and capillaries following intradermal injection of CRPP (16 pg).
to exert such a marked supprestlon in the
<
0
o 2 U) U)
Li
Z
(I) — U)
accumulation of carbon in the capillaries. It
5
can be seen from Fig. 6 that sodium salicylate,
10
15
20
TIME OF INJECTION OF CARBON (mm)
an effective antagonist of the permeability effect of CRPP as judged by leakage of pro-
AFTER IN TRADERMAL CRPP
course of the accumulation of tein-bound dye, caused a suppression of the ac- carbon6.inTime both venules and capillaries after cumulation of carbon in the venules but had intradermal injection of CRPP (16 /Lg) + local virtually no effect on carbon accumulation in salicylate. Compared to Fig. 5 there is a marked suppression in accumulation of carbon in the the capillaries. Fia.
venules with only a modest effect in the capillaries.
TABLE
I
Differentiation of protease from various vascular permeability factors by the antagonistic effect of various drugs Drugs
Mepyramine
Protease Histamine 5-HT
Bradykinin
P.F. dil Plasma inoculated c mitochondria Globulin P.F. Kallikrein: Padutin Saliva Substance P RNA
Turpentine activated plasma Pleural exudate 6 hrs LNPF
0
+ 0 0 0 0 0 0 0 0 0 0 0 0
BOL 148 Salicylate
0 0
+
+ 0
SBTI
Guinea pig plasma
DFP
Indomethacin
+
+
0
0
0
0
0 0
0
0
+
+ 0 0 0
0
0
+
0
0 0
0 0
+
0
0
0 0
0
0
+
0
0 0
0
0
0
0 0 0
+
+
+
NT
+
0 0
+ +
+ +
0 0 0 0
0 0
0
0
+ + + +
0 0 0 0
0 0
0 0
+
323
.
Fio. 7. Section of rat skin removed 45 mins. after intradermal CRPP (16 g) showing
leakage of red blood cells. H & E. X 312.
Fic. 8. Section of rat skin removed 6 hr. after intradermal CRPP (16 g) showing emigra-
tion of polymorphonuclear leukocytes. H & E. X 254.
Fic. 9. Section of rat skin removed 18 hr. after intradermal CRPP showing infiltration
with mononuclear cells. H & E. X 3120.
.e
..e_
a
a-
S —
a. .4 I
•
e
4
a C
a.
S.
-t
.5
S
,- "- v_see • I •tt
SI
S
I.S.
S
:"' tb' tLt
a
4S a
S
p
.
S
a
a
a
V
I
S
a
a
S
I
I
p
I
'S
"S
a
a
PROTEASE PERMEABilITY
324
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
gested and there is evidence of acute exuda- unique properties unlike the polypeptides tion which is distending the tissue spaces tested during this investigation. It has been (Fig. 7). There is a leakage of red blood cells reported that the injection of eortisol into from small vessels into the interstitial space. rats led to the release of RNA into the cxThis effect increased to frank hemorrhage traeellular compartment of the skin (1). We when the concentration of the protease was were unable to obtain any histological eviincreased. Fig. 4 shows a series of capillary dence of such a release, possibly because of loops labelled with carbon and apparently in the relative insensitivity of our staining a condition of complete stasis, with packed methods. It was also possible to exclude RNA red cells and with escape of cells and carbon as a mediator of CRPP induced increased through the walls.
vascular permeability as SBTI failed to reduce
2: 2—C hours. In this period there is a the effect of RNA. This was the only point massive emigration of neutrophil polymor- of difference between the permeability-enphonuelear leukoeytes from the small blood hancing activity of RNA and that due to vessels into the interstitial tissue (fig. 8). CRPP. It is of course possible that SBTI,
Numbers of polymorphs are found marginating
while preventing the protease activity of
in the vascular endothelium. In this period CRPP, prevented the release of tissue RNA there is no evidence of the prominent dia- which would normally have brought about the pedesis of erythroeytes as seen in section increase in vascular permeability and leukotaken at earlier times. Very few mononuclear cells are seen in the inflammatory cell migration at. this time.
3: 18—24 hours. In this period the lesion is composed of a dense infiltration of inflammatory cells composed of mononuclear cells and some polymorphs. The mononuclear cell population now outnumbers the polymorphs by approximately 10 to 1. There is not evidence of a deposition of fibrinoid such as is seen when
eyte emigration.
It does not seem surprising that a potent permeability factor be associated with systemic treatment of rats with steroids. This could well explain the apparent lack of effect of steroids on actute inflammation consisting
mainly of increased vascular permeability eq. thermal injury (15), turpentine induced pleurisy (16), whereas it is effective against the more chronic type of inflammatory le-
LNPF is introduced to the skin (3). By 24 sion, e.g. cotton wool pellet granuloma (17). hours the mononuclear cells begin to change
It is difficult to attempt to explain the effect
into plump elongated cells rather than the of this protease on emigration of cells since rounded migrating forms seen in lesions of emigration of white cells seems difficult to some hours later (fig. 9).
reconcile with the anti-inflammatory effects of
4: Progression of the lesion beyond 1 doy. cortisol which releases this protease. It is of From 24 hours onward there is evidence of interest, however, that indomethaein injected resolution of the inflammatory process. By 5 locally into rats skin will cause emigration of days almost all signs of the lesion have dis- leukocytes (9). Indomethaein administration appeared. to rats will apparently also result in the appearance of cutaneous protease similar to that DISCUSSION
The cortisol-released protease preparation
(CRPP) has been shown to be a possible
produced by eortisol (2). SUMMARY
mediator of increased vascular permeability.
Extracts from the skin of rats treated with It is of great interest that this protease ap- eortisol have been found to contain a propears to exert its vascular permeability-en- tease. Extracts of the skinS of normal unhancing effect by a mechanism that would treated rats do not contain this protease. not seem to rely on the release or activation of Injections of low concentrations of the eortisolmany of the well recognized mediators. Thus released protease preparation (CRPP) gave it is possible to exclude from its mode of increased vascular permeability and emigraaction the release of histamine, 5-HT or form- tion of leukoeytes. It has been found that the ation of vasoaetive kinins unless these have action on vascular permeability is different
325
PROTEASE PERMEABILITY
from that invoked by many of the known
methacin, a new anti-inflammatory drug: Path. Bact., 90: 641, 1965.
its potential use as a laboratory tool. J.
permeability factors. The significance of this factor is discussed.
10. Maino, C., Palade, C. E. and Schoefi, C,: Studies
on inflammation. II. The site of
histamine and serotonin along the vascular
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1. buck, J. C. and Patel, Y. M.: Proposed
mode of action of eorticosteroids on the connective tissue. Nature 206: 158, 1965. 2. buck, J. C. aod Pate!, Y. M.: Hormone and
tree: a topographic study. J. Biophys. Biochem., Cytol., 11:
607, 1961.
An electron microscopic study of leucocytie emigration and vascular per-
11. burley, J. V.:
meability in rat skin. Aust. J. Exp. Biol.
drug released dermal protease. Fed. Proc., Med. Sci., 41: 177, 1963. 12. Miles, A. A. and Wilhelm, D. L.: Enzyme-like 25: 762 #3241, 1966. globulins from serum reproducing the vas3. Houck, J. C. and Jacob, R. A.: The effects cular phenomena of inflammation. Brit. J. of some non-steroidal drugs upon the chemistry of inflammation. Proceedings of InterExp. Path., 36: 71, 1966. nat. Sympos. A Non-Steroidal Anti-Inflam- 13. Willoughby, D. A., Boughton, B. and Schild, H.: A factor capable of increasing vascular matory Drugs, Excerpta Medica Internat. permeability present in lymph node cells. Cong. Series No. 82, pg. 44, 1965.
4. buck, J. C. and Jacob, H. A.: The chemistry of necrosis. Lab. Invest. 15: 181, 1966. 5. Spector, W. C.: Substances which affect capillary permeability. Pharmacol. Rev., 10:
Immunology, 6: 484, 1963.
14. Willoughby, D. A. and Walters, M:: The effect of RNA on vascular permeability and
475,
its
1958.
6. Judah, J. D. and Willoughby, D. A.: A quan-
titative method for the study of capillary
15.
possible relationship to LIVPF. J. Path.
Baet., 90: 193, 1965. Spector, W. G., Walters, M. and Willoughby, D. A.: Venular and capillary permeability
thermal injury. J. Path. Bact., 90: 635, 16. Spector, W. C. and Willoughby, D. A.: The 567, 1962. demonstration of the role of mediators in 7. burley, J. V. and Spector, W. C.: Endogenous turpentine pleurisy in rats by experimental factors responsible for leucocytic emigration suppression of the inflammatory changes. J. in vivo. J. Path. Baet., 82: 403, 1961. Path. Bact., 77: 1, 1959. 8. Spector, W. C. and Willoughby, D. A.: The antagonism of substances that increase vas- 17. Spector, W. C. and Willoughby, D. A.: in permeability; extraction and determination of trypan blue in tissues. J. Path. Bact., 83:
in
cular permeability in the rat. J. Path. Bact.,
Evaluation
86:
487, 1963. 9. Walters, M. and Willoughby, D. A.: Indo-
1965.
of Drug Activities: Pharmacometrics, pg. 815. Ed. Lawrence, D. H. and Bachrack, A. L., London, Academic Press.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Copyright
Printed in U.S.A.
1567 by The Williams & Wilkins Co.
EFFECTS OF CORTISOL RELEASED CUTANEOUS PROTEASE UPON THE PERMEABILITY OF THE MICROCIRCULATION* A. W. J. LYKKE, M.D., D. A. WILLOUGHBY, Pu.D. AND J. C. HOUCK, Pn..D.t
It has recently been observed that subse- number of rats which had received only the car-
quent to the injection of cortisol, the ex- rier solvent for the cortisol preparation (Cortril,
C. Pfizer) but none of the steroid. CRPP and the similar preparation made from the non-steroid treated control animals were as-
tracellular, extrafibrillar compartment of rat skin contains a non-lysosomal, neutral pH optimal proteolytic enzyme which can be inhibited by both soybean trypsin inhibitor and salicylates (1, 2). A possibly similar proteolytic activity appears to have been found in
sayed for proteolytic activity upon denatured hemoglobin as described by Houck and Patel (1), and upon the synthetic substrates for chymo-
trypsin (acetyl tyrosine ethyl ester) and trypsin (tosy argine methyl ester) by pH stat technics. necrotic wounds (3, 4). Since some proteolytic Substances were tested for their ability to inenzymes are capable of increasing the per- crease vascular permeability in the rat by the meability of the micro-circulation (5), it was method described by Judah and Willoughby (6). decided to explore the effects of cortisol-re- Animals were given intravenous injections of a 0.1% solution of trypan blue (0.4 ml per 100 gms).
leased protease preparations from rat skin The substances under test were injected intraupon the vascular permeability of the rat.
dermally into the shaved abdominal skin in a
volume of 0.1 ml; 30 minutes later the skin was examined and the blue staining at the injection site was assessed either visually in arbitrary units (0—10) or by extraction and measurement in a colorimeter (6).
MATERIALS AND METHODS
Measurement of vascular permeability Cortisol-released protease preparations (CRPP) were prepared from the shaven and cleaned skin of male Sprague-Dawloy rats weighing 190—210 gm 26 hours after receiving two doses of cortisol.
Leulcocyte emigration.
Action on leukocyte emigration was estimated
by intradermal injection into rats in 0.1 ml
Three separate groups of 12 rats each were injected
amounts. The area was examined at various times
subcutaneously with 3 mg/kg of cortisol, and 24
hours later this injection was repeated intra- commencing 54 minutes after injection and histo-
peritone ally. Two hours after these animals had logical sections were prepared according to the received this second dose of cortisol I.P., they method of Hurley and Spector (7) who discuss were sacrificed and the entire skin was shaven, the reasons for choosing the appropriate time removed and dissected free of adhering fat, fascia intervals. and peniculus coraosum. These cleaned skins from
each group were minced with scissors collected
Antagonism of vascular permeability
the cold in a Serval Omni-mixer for 45 minutes in 10 ml/gm of 0.15 M NaC1. This mixture was allowed to stand overnight at 4°C and after centrifugation the supernatant was dialyzed for 24
permeability factors was carried out by methods described by Spector and Willoughby (8). Indo-
extracts of skin from cortisol treated rats served
The accumulation of colloidal carbon in venules
(CRPP) employed below. Similar preparations were made using a similar
CRPP was assessed by the method described by Majno, Palade and Schoefi (10) as modified for rat skin by Hurley (11). Rats were given an intra-
into three pools of four rats each extracted in
The assessment and antagonism of vascular
methacin was a gift from Merck, Sharpe & Dohme and was used intravenously in doses of 1—10 mg/kg hours with stirring against 1000 volumes of distilled water in the cold. The contents of the dialysis body weight (9). bag was centrifuged and the supernatant lyophi- Carbon leakage lized. These dialyzed, lyophilized isotonic saline
as the cortisol-released protease preparation and capillaries after the intradermal injection of Supported in part by grants from N.I.H. (AM-
venous injection of a colloidal suspension of carbon black (0.1 ml per 100 gms) at varying times after the intradermal injection of CRPP. Hospital, London, England and t Wnehernical The carbon was supplied by Gunther Wagner Research Lab., Children's Hospital, Washington, Pelikan Werke, Hanover, Germany (Batch No D.C. (Address for reprints.) C11/1431a). Thirty minutes after the injection 8168) and O.N.R. (NR-105-325) Received for publication June 1, 1966. * From the Pathology Dept., St. Bartholomew's
318
PROTEASE PERMEABILITY
319
permeable almost immediately after the insite was removed, stretched out on a piece of tradermal injection and thereafter the effect board, fixed in 4% buffered formaldehyde solution for 48 hours, dehydrated in ethanol and placed in rapidly waned. Delaying the injection of dye cedarwood oil to clear. The skin was then examined until 15 minutes after the intradermal injection showed that the onset of increased vascumicroscopically in a perspex chamber (11). All of the above tests were performed on at lar permeability is extremely rapid and that of carbon the animals were killed; the injection
least four animals each.
RESULTS
All three CRPP were found to be equally active against denatured hemoglobin and as active as these preparations previously employed (1) and to be equally capable of hydrolyzing significantly (10%) the synthetic
its duration is short. Site of increased vascular permeability
Injection of colloidal carbon with subsequent
microscopical examination revealed that carbon accumulated in both venules and capillaries although the effect was more marked in the venules (Figs. 3, 4). By delaying the insubstrate for chymotrypsin (acetyl ethyl ester). travenous injection of the carbon for various All of the CRPP prepared as described above times after the intradermal injection of had essentially identical quantitative effects CRPP it was possible to determine the time upon this synthetic substrate. None of these course of the effect of the protease on capilpreparations demonstrated any effects upon the lary and venular accumulation of carbon. It synthetic substrate for trypsin (tosyl arginine can be seen from Fig. 5 that the carbon accumulation persisted for a longer time period methyl ester). Extracts from the skin of three groups of in the venules than the capillaries. However, normal rats failed to demonstrate any pro- the effect on both venules and capillaries was teolytic activity on denatured hemoglobin or negligible in 15 minutes, confirming the short on either synthetic substrate mentioned duration of action determined above by trypan
above; similarly all of these extracts from blue injections. normal skin were inactive in the rat skin
permeability test. However, rats pretreated in The effect of antagonists Table I compares the effects of various such a way as to release protease activity into the skin, i.e. following systemic cortisol treat- treatments and antagonists on the permeabilment, contained significant amounts of the ity enhancing activity of CRPP and a variety protease (CRPP) and all nine pools represent- of other permeability factors. It can be seen
ing three groups of 12 animals each were
that with this screen of antagonists CRPP
found to be equally active in increasing vascular permeability in the skin of rats (Fig. 1). Thus 32 p.g of CRPP gave a maximal dye leakage, and increasing the dosage above this level led to the production of hemorrhagic lesion which obscured dye leakage. Activity was just detectable when 4 pg of CRPP was injected. From these results a dose response
may be readily distinguished from histamine or 5-hydroxyproline, by the use of the specific
antagonists, mepyramine maleate and 2-
acid diethylamide (BOL 148). Differentiation from bradykinin is best effected by the use of local sodium salicylate (2.5 mgs/ml of inoculum) or incubation with soy bean trypsin inhibitor (SBTI 100 pg/ml) curve was plotted (Fig. 2). Quantitatively Both these treatments reduced the effect of bromo-d-lysergie
none of the preparations from normal rats CRPP while not affecting bradykinin. The demonstrated this activity and all of the prep- kinin forming enzyme kallikrein is antago-
arations derived from cortisol treated animals nized by pretreating animals with either were equally active in increasing the vascular diisopropyl fluorophosphonate (DFP 2.5 mgs/ kg) or indomethacin (10 mgs/kg). The treatpermeability of rat skin.
Delaying the injection of the trypsin blue until various times after the intradermal injection of CRPP enabled us to determine the time course of the permeability enhancing effect. It was found that the vessels became
ment had no effect on the permeability increasing activity of CRPP. Globulin PF, one of the permeability factors in serum associated with the a globulins (12), may also be antagonized by treatment of the test animal
320
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
V
1'
B
FIG. 1. Abdominal skin of rat after intravenous trypan blue and intradermal injections 16 CRPP. B 16 g CRPP. C = 16 g CRPP + 250 g salicylate. D 16 jg CRPP pre-incubated with SBTI. (0.1 ml) of CRPP. A =
0 LU Iow_J LU L oi I—
Ui <
4
(n LU
U,
2
4
8
16
J.Jg CRPP INJECTED IN IRADERMALLY FIG. 2. The
effect of various doses of CRPP on vascular permeability in the rat
with DFP. SBTI incubation, which so effectively suppresses CRPP, had no effect on the permeability factor which may be activated in rat plasma by the addition of turpentine, no effect on pleural exudates from rat (8), no effect on the permeability factor associated with the lymph node cells (13), and finally no
effect on RNA (14). It appears that this protease is bringing about an increase in vas-
cular permeability by a mechanism or via mediators hitherto not described.
It is of interest that the antagonists, while
often causing a dramatic reduction in the leakage of protein-bound trypan blue, failed
321
PROTEASE PERMEABILITY
I
if
St
•
¼ FIG. 3. Carbon accumulation in the venules of rat skin following intradermal injection of CRPP (16 pg). X 25. FIG. 4. Carbon accumulation in the capillaries of rat skin following intradermal injection of CRPP (16 pg). X 100.
322
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Histology of the lesions produced by CRPP
I
jO
at various times after administration of the extract
0<0 o
1. 45 minutes. A very prominent vascular reaction occurs at this time. Small blood vessels of the capillary and venular size are con-
o
C
I-0
U-
WI-.
8.
0< 00
IES
Fia. 5. Time course of the accumulation of
carbon in both venules and capillaries following intradermal injection of CRPP (16 pg).
to exert such a marked supprestlon in the
<
0
o 2 U) U)
Li
Z
(I) — U)
accumulation of carbon in the capillaries. It
5
can be seen from Fig. 6 that sodium salicylate,
10
15
20
TIME OF INJECTION OF CARBON (mm)
an effective antagonist of the permeability effect of CRPP as judged by leakage of pro-
AFTER IN TRADERMAL CRPP
course of the accumulation of tein-bound dye, caused a suppression of the ac- carbon6.inTime both venules and capillaries after cumulation of carbon in the venules but had intradermal injection of CRPP (16 /Lg) + local virtually no effect on carbon accumulation in salicylate. Compared to Fig. 5 there is a marked suppression in accumulation of carbon in the the capillaries. Fia.
venules with only a modest effect in the capillaries.
TABLE
I
Differentiation of protease from various vascular permeability factors by the antagonistic effect of various drugs Drugs
Mepyramine
Protease Histamine 5-HT
Bradykinin
P.F. dil Plasma inoculated c mitochondria Globulin P.F. Kallikrein: Padutin Saliva Substance P RNA
Turpentine activated plasma Pleural exudate 6 hrs LNPF
0
+ 0 0 0 0 0 0 0 0 0 0 0 0
BOL 148 Salicylate
0 0
+
+ 0
SBTI
Guinea pig plasma
DFP
Indomethacin
+
+
0
0
0
0
0 0
0
0
+
+ 0 0 0
0
0
+
0
0 0
0 0
+
0
0
0 0
0
0
+
0
0 0
0
0
0
0 0 0
+
+
+
NT
+
0 0
+ +
+ +
0 0 0 0
0 0
0
0
+ + + +
0 0 0 0
0 0
0 0
+
323
.
Fio. 7. Section of rat skin removed 45 mins. after intradermal CRPP (16 g) showing
leakage of red blood cells. H & E. X 312.
Fic. 8. Section of rat skin removed 6 hr. after intradermal CRPP (16 g) showing emigra-
tion of polymorphonuclear leukocytes. H & E. X 254.
Fic. 9. Section of rat skin removed 18 hr. after intradermal CRPP showing infiltration
with mononuclear cells. H & E. X 3120.
.e
..e_
a
a-
S —
a. .4 I
•
e
4
a C
a.
S.
-t
.5
S
,- "- v_see • I •tt
SI
S
I.S.
S
:"' tb' tLt
a
4S a
S
p
.
S
a
a
a
V
I
S
a
a
S
I
I
p
I
'S
"S
a
a
PROTEASE PERMEABilITY
324
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
gested and there is evidence of acute exuda- unique properties unlike the polypeptides tion which is distending the tissue spaces tested during this investigation. It has been (Fig. 7). There is a leakage of red blood cells reported that the injection of eortisol into from small vessels into the interstitial space. rats led to the release of RNA into the cxThis effect increased to frank hemorrhage traeellular compartment of the skin (1). We when the concentration of the protease was were unable to obtain any histological eviincreased. Fig. 4 shows a series of capillary dence of such a release, possibly because of loops labelled with carbon and apparently in the relative insensitivity of our staining a condition of complete stasis, with packed methods. It was also possible to exclude RNA red cells and with escape of cells and carbon as a mediator of CRPP induced increased through the walls.
vascular permeability as SBTI failed to reduce
2: 2—C hours. In this period there is a the effect of RNA. This was the only point massive emigration of neutrophil polymor- of difference between the permeability-enphonuelear leukoeytes from the small blood hancing activity of RNA and that due to vessels into the interstitial tissue (fig. 8). CRPP. It is of course possible that SBTI,
Numbers of polymorphs are found marginating
while preventing the protease activity of
in the vascular endothelium. In this period CRPP, prevented the release of tissue RNA there is no evidence of the prominent dia- which would normally have brought about the pedesis of erythroeytes as seen in section increase in vascular permeability and leukotaken at earlier times. Very few mononuclear cells are seen in the inflammatory cell migration at. this time.
3: 18—24 hours. In this period the lesion is composed of a dense infiltration of inflammatory cells composed of mononuclear cells and some polymorphs. The mononuclear cell population now outnumbers the polymorphs by approximately 10 to 1. There is not evidence of a deposition of fibrinoid such as is seen when
eyte emigration.
It does not seem surprising that a potent permeability factor be associated with systemic treatment of rats with steroids. This could well explain the apparent lack of effect of steroids on actute inflammation consisting
mainly of increased vascular permeability eq. thermal injury (15), turpentine induced pleurisy (16), whereas it is effective against the more chronic type of inflammatory le-
LNPF is introduced to the skin (3). By 24 sion, e.g. cotton wool pellet granuloma (17). hours the mononuclear cells begin to change
It is difficult to attempt to explain the effect
into plump elongated cells rather than the of this protease on emigration of cells since rounded migrating forms seen in lesions of emigration of white cells seems difficult to some hours later (fig. 9).
reconcile with the anti-inflammatory effects of
4: Progression of the lesion beyond 1 doy. cortisol which releases this protease. It is of From 24 hours onward there is evidence of interest, however, that indomethaein injected resolution of the inflammatory process. By 5 locally into rats skin will cause emigration of days almost all signs of the lesion have dis- leukocytes (9). Indomethaein administration appeared. to rats will apparently also result in the appearance of cutaneous protease similar to that DISCUSSION
The cortisol-released protease preparation
(CRPP) has been shown to be a possible
produced by eortisol (2). SUMMARY
mediator of increased vascular permeability.
Extracts from the skin of rats treated with It is of great interest that this protease ap- eortisol have been found to contain a propears to exert its vascular permeability-en- tease. Extracts of the skinS of normal unhancing effect by a mechanism that would treated rats do not contain this protease. not seem to rely on the release or activation of Injections of low concentrations of the eortisolmany of the well recognized mediators. Thus released protease preparation (CRPP) gave it is possible to exclude from its mode of increased vascular permeability and emigraaction the release of histamine, 5-HT or form- tion of leukoeytes. It has been found that the ation of vasoaetive kinins unless these have action on vascular permeability is different
325
PROTEASE PERMEABILITY
from that invoked by many of the known
methacin, a new anti-inflammatory drug: Path. Bact., 90: 641, 1965.
its potential use as a laboratory tool. J.
permeability factors. The significance of this factor is discussed.
10. Maino, C., Palade, C. E. and Schoefi, C,: Studies
on inflammation. II. The site of
histamine and serotonin along the vascular
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