SECOND (COl]NELL) MEDICAL DIVISION
LIBRARY BELLEVUE HOSPITAL
MEDIATORS OF INFLAMMATION IN LEUKOCYTE LYSOSOMES
II. M~CHANIS~ OF AdiOS OF LYSOSO~:AL CATmmc PROTEIN
UPON VASCULAR PERMEABILITY IN THE RAT*
B~¢ AARON JANOFF,:~ PH.D., SONJA SCHAEFER,§ JOAN SCHERER, H A~m
MICHAEL A. BEAN¶
(From the Department of Pathology, New York University Sckool of Medicine,
(Received for publication, June ll, 1965)
Considerable emphasis has been placed on the role of acid-proteases within lyso-
somes of polymorphonuclear leukocytes (PMNL) in the pathogenesis of allergic and
other forms of tissue injury (1, 2). We reported previously (3) that a cationic protein-
containing fraction of PMNL lysosomes, extracted by weak mineral acid and precipi-
tated by ethanol at a 20 per cent (v/v) concentration, produced inflammatory changes
in the microcirculation of rat and rabbit mesentery. However, such cationic protein
fractions of leukocyte granules are reportedly free of acid-proteases (4) and other
hydrolases (3) normally associated with intact leukocyte lysosomes. This apparent
discrepancy led us to suggest alternative mechanisms of action of lysosomal cationic
protein fractions in local tissue injury reactions and to propose that a part, at least,
of the inflammatory changes induced by whole granule preparations might be medi-
ated independently of their protease content.
In the course of these earlier investigations it was found that the cationic
protein fraction, in addition to causing adhesion and emigration of leukocytes,
also produced a marked increase in vascular permeability to circulating Evan's
blue dye when the protein was applied topically onto rat mesentery or when it
was injected intracutaneously into rat skin. However, in vitro tests using iso-
lated smooth muscle preparations failed to reveal any kinin-like, serotonin, or
histamine activity in this extract (3). Further studies have now been carried
out on the mechanism of action of crude extracts of PMNL lysosomes, and
* This work was supported by a grant from the United States Public Health Service
J~ Career Development Award Fellow of the United States Public Health Service (GM-
§ Candidate for the degree of Master of Science. Part of the work reported here has been
submitted in partial fulfillment of the requirements for this degree at the New York Uni-
versity Graduate School of Arts and Science.
 Research trainee of the United States Public Health Service (HF_,-5501).
¶ Research trainee of the United States Public Health Service. Present address: Colorado
University School of Medicine, Denver.
also of the cationic protein isolated from these granules, upon vascular perme-
ability in skin and cremasteric muscle of the rat. The present report will present
evidence which suggests that the early increase in vascular permeability pro-
duced in rat tissues by PMNL lysosomes is caused by vasotropic agents dis-
charged from tissue mast cells and not by protease activity of the granules.
This effect is due to a mastocytolyfic agent present in the cationic protein
fraction of PMNL lysosomes.
Materials and Methods
Preparation of Leukocyte Lysosome Fractions.--Rabbit peritoneal exudate polymorpho-
nuclear leukocytes (PMNL) were collected and their granules isolated by the technique of
Cohn and Hirsch (5). Superuates of frozen-thawed granules and cationic protein-containing
extracts of the granules were prepared by previously described techniques (3) except that in
the present studies dialysis of the fractions was carried out against sodium bicarbonate-
buffered, mammalian-Ringer's balanced salt solution at pH 7.2. Protein content of the various
fractions was determined by the method of Lowry (6), with crystalline bovine serum albumin
as a standard.
General Method of Permeability Tests in Rat Skin.--The method employed to evaluate
permeability effects in rat skin was essentially similar to that described by Miles and Wilhelm
(7). Materials to be tested were injected in a volume of 0.05 ml, intracutaneously, into the
shaved abdominal skin of rats (males, Sprague-Dawley strain, 250 to 350 gm body weight)
anesthetized with sodium pentobarbital (nembutal, Abbott Laboratories, North Chicago,
Ill.). In each of the test animals at least two separate sites were prepared at each dose of
test agent. Either immediately or at varying times after the intracutaneous administration of
test substances, Evan's blue dye was injected intravenously as a 1 per cent solution in iso-
tonic saline and at a dose of 0.4 ml per 100 gm of body weight. Animals were regularly sacri-
ficed 20 minutes after dye injection and the abdominal skin was reflected for examination
of the undersurface. The grade of edema in the injected skin site was estimated according
to the diameter and color intensity (visual observation) of the dye accumulated in the tissues.
General Method of Permeability Tests in Cremaster Muscle of the Rat.--Permeability effects
in rat cremasteric muscle were studied using the carbon-labelling technique described by
Majno (8). Aliquots of 0.05 ml of the materials to be tested were injected subcutaneously
into the scrotal sac just over the mid-ventral surface of the testis. Either immediately or at
varying times after the injection of test substances, carbon suspension (pelikan Cl1-1431a,
Gunther Wagner, Germany) was injected intravenously (0.1 mi per 100 gm body weight).
Animals were sacrificed 1 hour after injecting the carbon suspension, and the testes were
placed briefly in 10 per cent formalin. The muscle was then dissected free of other structures,
stretched, and pinned on dental wax, fixed in formalin for an additional 24 hours, cleared in
xylene, and mounted in glycerine-jelly for low power light microscopic observation. In this
test, vessels whose permeability has been abnormally increased by the subcutaneous injection
of test material appear blackened by carbon particles which have passed between endothelial
cells. Furthermore, the characteristic topography of the microcirculation in rat cremasteric
muscle (8) readily permits identification of the type of vessel (venule, capillary, or arteriole)
most affected by the test agent.
Assay of Mastocytolytic Activity in Vitro.--The effect of leukocyte granule fractions upon
rat mast cells in ~itro was assayed according to the technique described by Norton (9). Briefly,
this involved incubation of pieces of rat mesentery in Ringer-Locke glucose solution buffered
with bicarbonate to pH 7.45 and containing varying concentrations of either leukocyte frac-
A. JAN0~'F, S. SCHAEI~ER, J. SCHERER, AND M. A. BEAN
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