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. JANO~F~'~ S. SCHAEFER, J. SCHERER, AND M. A. BEAN
tions or known mastocytolytic agents such as compound 48/80 (Burroughs Wellcome and
Co., Inc., Tuckahoe, New York). Thirty minutes of incubation at room temperature was
routinely employed in all tests; and, incubation in Ringer-Locke alone was always included
as a control for spontaneous mast cell rupture. Following the period of incubation, the tissues
were fixed in 10 per cent formalin containing 0.1 per cent toluidine blue, dehydrated in ace-
tone, cleared in xylene, mounted in permount (Fisher Scientific Co., Fairlawn, New Jersey),
and examined microscopically for the enumeration of intact vs. disrupted mast cells.
Details of methodology which were varied according to the needs of each experiment are
described in the appropriate sections of the Results.
Time Course of Vascular Permeability Change Produced by PMNL Lysosomes in Rats
protein (20 per cent
Reaction/No. of rats in test
30 rain.* 60 mln.*/120 rain.*
+ (skin), fiat, pale-blue, uneven reaction, 5 mm or less in diameter.
++ (cremaster), postcapillary collecting veunles show carbon deposits over area 10 to
15 mm in diameter.
0 (no reaction), no blue dye grossly visible (skin) or carbon deposits microscopically visible
* Elapsed time between intracutaneous (skin) or subcutaneous (cremaster) injection of
test agent and intravenous injection of Evan's blue (skin) or carbon suspension (eremaster).
(skin), raised, intensely blue wheal, 12 to 15 mm diameter.
Time Course of the Permeability Change Produced in Skin and Cremaster
Muscle of Rats by PMNL Lysosomes.--Table I shows that the permeability
changes induced in skin and cremaster muscle of rats by single injections of
PMNL lysosomes (both extracts of frozen-thawed granules and cationic pro-
tein fractions) are acute, transient changes which disappear rapidly.
When animals were injected intravenously with Evan's blue either immedi-
ately before or after intracutaneous application of lysosomal materials, an
intense exudation of plasma-bound dye developed, rapidly reaching a maxi-
mum diameter of 12 to 15 mm in 15 or 20 minutes. However, when dye was
IN'FL.A MMA.TORY MECHANISM
injected 30 or 60 minutes after the lysosomal fractions, only trace reactions
occurred (pale, incomplete staining); and, at 2 and 3 hours after injection of
materials into the skin, no discernible exudation of dye was observed except
at the finely circumscribed point of passage of the needle into the skin (see
Table I). The absence of dye exudation at later intervals was not exclusively
the result of mechanical interference with local blood flow following engorge-
ment of tissue spaces with fluid during the early phase of the reaction. This was
shown by the fact that injection of 2/zg of histamine into the same skin sites
previously treated with lysosome fractions produced typical histamine blueing
reactions at these sites in the later time-groups (2 and 3 hours).
When animals received an intravenous injection of carbon suspension im-
mediately after subcutaneous application of lysosomal materials over the
cremaster muscle (frozen-thawed granules or cationic protein), a widespread
blackening of venules developed in the muscle, in a pattern identical with that
produced in this tissue by similar injections of histamine. However, if carbon
suspension was administered 2 hours after injection of lysosomal materials
over the muscle, no labelling of vessels could be discerned (see Table I). Again,
it could be shown that potential vascular reaction was still present in the
cremaster at this time, since reinjection of the scrotal sac with the standard
dose of histamine just before the administration of carbon produced a normal
venular-labelling pattern in animals that had been treated with lysosomal
materials 2 hours earlier.
Although the doses of lysed granule protein and cationic protein shown in
Table I were the ones most frequently employed in permeability tests, other
experiments have shown that strong permeability changes (2+ and 3+ re-
actions, see Table I) can be produced by as little as 2 #g of protein in the skin
and 10 #g in cremaster muscle.
Mastocytolytic Activity of PMNL Lysosomes in Vitro.--In view of the acute,
transient nature of the vascular permeability change produced in rat tissues by
PMNL lysosomal fractions, it seemed reasonable to suppose that the reaction
might be mediated by vasotropic agents released from tissue mast cells which
had been disrupted by contact with the injected lysosomal material. It was
therefore decided to investigate the effect of PMNL lysosomes upon rat mast
cells in vitro, in order to determine whether the lysosomal fractions employed
in our permeability tests were, in fact, capable of rupturing these cells.
Table II shows the results of these experiments. Both lysosomal cationic
protein (20 per cent ethanol fraction) and extracts of frozen-thawed lysosomes
were compared with a known mast cell-rupturing agent, compound 48/80
(Burroughs Wellcome and Co., Inc., Batch No. NS280). It can be seen that
both of the lysosomal preparations produced a marked mastocytolyfic effect
in these tests, although the activity of the lysosome fractions was less than
one-tenth that of compound 48/80 when these agents were compared on a
A. JANOFF, S. SCHAEFER, J. SCHERER, AND M. A. BEAN
simple weight per weight basis. However, if the lysosomal cationic protein
was compared with compound 48/80 on a molar weight basis (see Discussion),
then the two agents appeared to be more nearly equal in mastocytolytic po-
Effects of PMNL Lysosomes on Rat Mast Cells In Vitro
Percentage disruption of mast cells*
Test material Concentration (/~g) $ EDio§
".P. (lysosomal cationic protein, 20
per cent ethanol fraction)
~.P. in presence of trasylol[[
~.T. (extract of frozen-thawed lyso-
~.T. in presence of trasytolll
C.P. = cationic protein fraction (20 per cent ethanol); F.T. ffi extract of frozen-thawed
* Percentage disruption is based on the number of mast cells showing granule extrusion
out of 1500 cells examined (X640).
~g axe per ml in the case of compound 48/80 and per 0.1 ml in the case of all other
§ Concentration of test agent calculated to produce 50 per cent disruption of mast cells
(estimated from log dose/response plot).
II Trasylol -~ zymofren (Societe Paxisienne d'Expansion Chimique---S.A., Lot No. ES
1402) present in a concentration of 10 kallikrein-inhibiting units per ml.
Preliminary evidence was also obtained which suggested that the action of
PMNL lysosomal cationic protein upon mast cells was not simply due to
the basic groups on these protein molecules, but rather may have been asso-
ciated with some as yet unidentified, specific component of the granules, present
only in the 20 per cent ethanol-precipitated fraction of acid extracts of the
lysosomes (see Discussion).