The Journal Copyright
of Histochemistry and Cytochemistry © 1990 by The Histochemical Society,
Vol. 38, No. 8, pp. 1179-1186, Inc.
Heterogeneity Peroxidase-positive HATICE
G of Human
1, 1989 and
in a variety information trophils
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
tri.x components, including Elastase is also active against
proteoglycans elastin and types
and fibronectin (3). II, ifi, and IV collagen.
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
50 N. Medical
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.
and Respiratory Missouri
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.
for mononuclear more difficult
the capacAll of these
contained elastase and CG that were elastase and CG of PMN by a variety
We also reported
In the present
dence that antigenically
of human serine
phagocytes than for PMN, to obtain in pure populations
ocyte-like cell line, U937, distinguishable from the at low levels
ity to bind and internalize proteinases factors have impeded experimentation.
concerning the nature of the activity, its subcelluand its relationship to the degree of differentia-
per cell are lower the cells are often high
formation exists lar localization, tion
of pathological processes. A considerable amount of has been accumulated about the proteinases of neu-
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,
a very small
human peripheral blood similar to HLE and CG,
monocytes contain enzymes which could not be detected
histochemically in one type ofmature mononuclear alveolar macrophage. In marked contrast to PMN,
the are 1179
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.
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