0022-1554/90/$3.30
The Journal Copyright
of Histochemistry and Cytochemistry © 1990 by The Histochemical Society,
Vol. 38, No. 8, pp. 1179-1186, Inc.
Printed
Original
Elastase
and
Cathepsin
Heterogeneity Peroxidase-positive HATICE
AYDANUR
Department
ofPatbology
Critical
Division,
Care
Received
and
KARGI,
Jewish
for publication
G of Human
1, 1989 and
Localization
Washington
at Washington
form
Medical
Center
St. Louis,
October
6, 1989
in a variety information trophils
contain kocyte
phagocytes
from
been
have
their degranulation
subcellular localization, and their release dur(25,26,47,54). Azurophil granules of PMN
major neutral elastase; HLE) and
serine proteinases, elastase (human leucathepsin G (CG), which can hydrolyze
two
extracellular
substrates
including
components,
fibrinogen,
vasoactive
tri.x components, including Elastase is also active against
I Supported
immunoglobulins,
by USPHS
peptides,
proteoglycans elastin and types
training
grant
complement
and extracellular
ma-
and fibronectin (3). II, ifi, and IV collagen.
ES 07066
(HAK),
grants
research
HL 29594 and HL 30341, and by the Council forTobacco Research, USA, Inc. 2 Correspondence to: Dr. EdwardJ. Campbell, Division of Respiratory,
Critical cine, Lake
Care, and Occupational
University City, UT 3
Present
ofUtah 84132.
address:
Health
Pulmonary Sciences
Medicine, Center,
Department
50 N. Medical
of McdiDrive,
Salt
of Respiratory,
Critical
Care, and Occupa-
tional Pulmonary Medicine, Department ofMedicine, University of Utah, Salt Lake City, UT 4 Present address: Department of Pathology, Memorial Hospital, Pawtucket, RI 02860.
KUHN
and Respiratory Missouri
16,
1990;
iii
and
63110. accepted
March
24,
1990
(9A1707).
combined with histochemistry, to cytoplasmic granules which had peroxidase activity. Alveolar macrophages were unstained. Therefore, a subpopulation of peripheral blood monocytes contains leukocyte elastase and cathepsin G in a cell compartment from which these enzymes may potentially be released into the extracellular space. The occurrence of peroxidase and neutral proteinases in the same granules in monocytes could permit the H202-myeloperoxidase-halide system and the neutral proteinases to act in concert in such functions as microbe killing and extracellular proteolysis. (J Hiscochem Cytochem 38:1179-1186, 1990) KEY WORDS: Monocyte; Elastase; Cathepsin G; Neutrophil; Immunogold; Immunoperoxidase.
less
is known
and/or
of the
yield.
serine
activity
in human
phagocyte.
for mononuclear more difficult
Furthermore,
bution
studies
(46,48).
Levels
(21,31,42,45) definitive
of enzymes
in-
activity
that
have
PMN(5,9).
a neoplastic
and and
the capacAll of these
human
mon-
contained elastase and CG that were elastase and CG of PMN by a variety
might from
of enzymatic
phagocytes from
we showed
We also reported
which
In the present
dence that antigenically
little
of human serine
phagocytes than for PMN, to obtain in pure populations
mononuclear
ocyte-like cell line, U937, distinguishable from the at low levels
described
monocytes but
ity to bind and internalize proteinases factors have impeded experimentation.
criteria
have
concerning the nature of the activity, its subcelluand its relationship to the degree of differentia-
mononuclear
In previous
proteinases
authors
(12,19,23,27,34,43),
per cell are lower the cells are often high
the
Several
or elastolytic
macrophages
formation exists lar localization, tion
about
phagocytes.
elastase-like
PMN. Division
March
mononuclear
implicated
of pathological processes. A considerable amount of has been accumulated about the proteinases of neu-
(PMN),
ing PMN
and
Much released
CHARLES
ofMedicine,
Introduction enzymes
and
School
We used antibodies to human leukocyte (“neutrophil”) elastase and cathepsin G to localize the corresponding antigem in human neutrophils, monocytes, and alveolar macrophages by immunohistochemistry. Furthermore, we cornbined immunogold localization with enzyme histOChernistry to localize proteinase antigens and endogenous peroxidase activity in the same sections. As expected, all ncutrophils contained both elastase and cathepsin G, and the proteinases localized to granules with peroxidase activity. In contrast, marked heterogeneity in monocyte staining for elastase, cathepsin G, and endogenous peroxidase was found. Sixty percent or more were unstained, while the remainder varied greatly in staining intensity. The elastase and cathepsin G in monocytes were localized by immunoelectron microscopy,
Proteolytic
to
University
University
in revised
Monocytes:
3. CAMPBELL,2’3
EDWARD Medicine,
Hospital
June
Artide
Subcellular Granules’
and Internal
1990
in U,S.A.
lead
such
a very small
report
activity
to concern
we present
human peripheral blood similar to HLE and CG,
in monocytes,
about
proportion
a possible
inof but
contri-
of contaminating
immunohistochemical
evi-
monocytes contain enzymes which could not be detected
histochemically in one type ofmature mononuclear alveolar macrophage. In marked contrast to PMN,
phagocyte, monocytes
the are 1179
1180
MONOCYTE
strikingly
heterogeneous
demonstrate
that
cytoplasmic
in their
these
granules
Materials
and
antigens in
of these
content
are present
enzymes.
We also
in peroxidase-positive
monocytes.
Methods
Ultrastructural
Separation of PMN and Monocytes. PMN and monocytes were separated from the blood of 10 normal volunteers by Ficoll-Hypaque centrifugation and centrifugal elutriation, as has been described in greater detail (8). Alveolar Macrophages. Alveolar macrophages were obtained from two normal volunteers, by bronchoalveolar lavage as described previously (9, 49). These subjects also donated blood for preparation ofPMN and monocytes. To ensure adequate yield of macrophages, both subjects were cigarette smokers. Antibodies.
Antibodies
to human
leukocyte
elastase
and
cathepsin
G
were generously provided as IgG fractions by Drs. D. Burnett and R. A. Stockley(University ofBirmingham, UK)(15,16). Their specificity was verifled by competitive binding enzyme-linked immunosorbent assays as described in detail elsewhere (8). The antibodies each had