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