foamy cells associated with phagocytosis of glutaraldehyde-treated red blood cells and red cell...
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Acta Pathol. Jpn. 37(4) : 627-637, 1987
FOAMY CELLS ASSOCIATED WITH PHAGOCYTOSIS OF GLUTARALDEHYDE-TREATEDREDBLOODCELLS
AND RED CELL MEMBRANES
Tokuhiro ISHIHARA, Jun-ichi SANO, Sadayoshi YAMANAMI, Yoshimi YAMASHITA, Mutsuo TAKAHASHI, Fumiya UCHINO,
and Noboru MATSUMOTO*
First Department of Pathology, Yamaguchi University School of Medicine, and 'the School of Allied Health Sciences,
Yamaguchi University, Ube
In order to clarify the mechanism for the formation of foamy cells (macro- phages with foamy appearance) associated with increased erythrophagocytosis, we tried to reproduce these cells in mice by subcutaneous injection of intact red blood cells (RBCs), OsO, -treated RBCs (Os-RBCs), glutaraldehyde-treated RBCs (G-RBCs), or isolated red cell membranes, and time-course observation was done by light and electron microscopy. Foamy cells were induced by the latter two methods. Within the macrophages, G-RBCs were fragmented into spherules by newly formed small vacuoles, and with time these spherules lost their hemoglobin content transforming into small vacuoles with translucent matrix. In most of these vacuoles, red cell membrane structure was discern- ible adjacent to the phagocytic vacuole. Such macrophages containing abun- dant small vacuoles appear foamy in light microscopy. Foamy cells induced by injection of red blood cell membranes were positive for lipid stains and contained abundant laminated membrane structures in electron microscopy. These results suggest that the foamy cells related with increased erythro- phagocytosis are heterogeneous with respect to their pathogenesis and cellular inclusions, and proteinaceous constituents resistant to intracellular digestion are also responsible for the occurrence of foamy cells. ACTA PATHOL. JPN. 37 : 627-637, 1987.
Introduction Foamy cells named as Gaucher-like cells are reported in the bone marrow and
spleens from patients with thalassemia1~2~11~12 and le~kemia.~? ' Formation of Gaucher- like cells in thalassemia is presumed to be associated with the accelerated catabolism of erythroblasts and erythrocytes. Recently, we observed foamy cells in the spleen
Accepted for publication August 4, 1986. Mailing address : Tokuhiro ISHIHARA, M.D. (fiR@jt$), First Department of Pathology, Yamaguchi University School of Medicine, Ube 755, JAPAN. The contents of this paper were presented partly a t the IXth International RES Congress (September, 1984).
628 PHACOCYTOSIS OF OLUTARALDEHYDE-TREATED R B C ~ Acta Pathol. Jpn.
from a patient with congenital hemolytic anemia associated with red cell adenosin deaminase overprod~ction.~
It is likely that the accelerated phagocytosis of red blood cells and accumulation of incompletely degraded cellular materials are causely related to the occurrence of foamy cells. To .test this hypothesis, we tried to induce foamy cells in mice by subcutaneous injection of intact red blood cells (RBCs), RBCs treated with glutaralde- hyde (G-RBCs) or with osmium tetroxide (0s-RBCs), and isolated red cell membranes. Time-course observations by light and electron microscopy disclosed typical foamy cells reproduced only in mice which were injected with G-RBCs or isolated red cell membranes.
Matekals and Methods Treatment of murine RBCs and isolation of red cell membranes : The blood obtained in 1%
EDTA-2Na from ICR mice was centrifuged at 3,000 rpm for 15 minutes. The RBCs washed with saline three times were separated into the following three groups. In Group I, intact RBCs were suspended in saline. In Group 11, the RBCs were treated with 0.5% glutaraldehyde for 15 minutes a t 4C. In Group 111, they were treated with 0.1% osmium tetroxide (OsO,) for 15 minutes at 4C. Following glutaraldehyde or osmium treatment, RBCs were washed with saline three times.
To isolate red cell membranes, mice were bled with 1% EDTA-2Na and the whole blood was centrifuged at 3,000rpm for 15minutes. The packed RBCs hemolyzed in 5mM PBS were centrifuged at 10,000 rpm for 20 minutes. The membrane pellet was washed with 0.01 M PBS three times to remove hemoglobin.
Induction of foamy cells in mice : In Groups 1-111, each RBCs suspension (4.5-5.2 x 108/ml) was subcutaneously injected into ICR mice. As a control, 0.05 ml of red cell membrane suspension was injected (Group IV). The subcutaneous tissue materials from Group I and I1 were obtained a t 6, 12, and 24 hours, and a t Days 2, 4, 5, 7, 9, 12, and 15 following injection. The tissue materials from Group I11 and IV were removed at 2, 3, 5, 7, and 15 days after injection.
Methods for histology and electron microscopy : For histologic examination, the subcutaneous tissue fragments were fixed in 10% formalin. Sections from paraffin blocks were stained with hematoxylin and eosin (H.E.), periodic acid-Schiff (PAS), van Gieson, PTAH and Prussian blue. The tissues were also fixed in formalin-calcium. Frozen sections were stained with Sudan 111, Sudan black B, Smith-Dietrich, acid phosphatase, and beta-glucuronidase.
For transmission electron microscopy, the subcutaneous tissues were fixed in 2.1% glutaraldehyde at 4C and postfixed in 1% osmium tetroxide. They were dehydrated in gradient ethanol and embedded in Epon 812. Semithin sections stained with alkaline toluidine blue were examined with a light microscope for selection of the appropriate areas. The ultrathin sections cut with LKB ultramicrotome were stained with uranyl acetate and lead citrate. They were examined in a Hitachi H-800 electron microscope.
Resu 1 ts Group I (Subcutaneous injection of intact RBCs).
At 6 hrs. after injection, there was a subcutaneous mass composed of RBCs with
Fig. 1. Intact RBCs phagocytized by macrophages reveal a variety of intracellular degradation a t 24 hrs. after injection.
Fig. 2. At the advanced stage of intracellular digestion (Day 5), non-treated RBCs are degraded into dense granular deposits, laminated membrane structures, and marble-like struc- tures. x 9,000.
37(4) : 1987 T. ISHIHARA el al. 629
630 PHAGOCYTOSIS OF GLUTARALDEHYDE-TREATED R B C ~ Acta Pathol. Jpn.
variable shape. At 12 hrs., a few macrophages appeared around and within the RBC mass. At 24 hrs., occasional macrophages with erythrophagocytic activity appeared around the mass. Ultra- structurally, such macrophages contained various kinds of degraded structures of RBCs (Fig. 1). Recently phagocytized and still intact appearing RBCs were sur- rounded by a single membrane and myelin-like materials were occasionally noted within the phagocytized RBCs. After 2 and 4 days, a large number of macrophages revealed phagocytosis of several RBCs. At Day 7, the subcutaneous mass almost disappeared, and a few remaining macrophages revealed erythrophagocytosis. These macrophages contained a large amount of positive materials for acid phosphatase. In these macrophages, the matrix of the phagocytized RBCs was markedly degraded and was condensed into coarsely granular or mottled materials with increased electron density (Fig. 2). These macrophages were stained blue with Prussian blue. At 15 days after injection, a large number of hemosiderin granules were demonstrated in the cytoplasm of scattered macrophages. During the observation period, foamy cells were not induced in this group.
A few neutrophils infiltrated around the RBC mass.
Group 11 (Subcutaneous injection of G -RBCs). Erythrophagocytosis by macrophages was conspicuous early a t 6 to 24 hrs. after
injection. Although some phagocytized G-RBCs retained their discoid shape, small spherules with an increased electron density were noted within the cytoplasm of macrophages (Fig. 3). At Day 4, the enlarged macrophages phagocytosed many G-RBCs and contained reddish brown granular materials. Macrophages showing foamy appearance (foamy cells) appeared around the mass of G-RBCs. Thereafter, these foamy cells were gradually increased in number. Most of the foamy cells had a variable number of pale eosinophilic granules. At Day 7, a mantle of foamy cells surrounded the G-RBC mass with an accompanied proliferation of capillaries. A t this stage the foamy cells contained an eccentric nucleus and clear cytoplasm with a few reddish brown granules (Fig. 4). These foamy cells were not stained with PAS and PTAH, but were stained pale yellow with van Gieson. In Prussian blue staining, several iron containing materials were demonstrated and Sudan black B staining disclosed a few positive materials in the cytoplasm of foamy cells. Moderate activity of acid phosphatase and beta-glucuronidase was demonstrated in the macrophages without foamy appearance which were located in the peripheral area of the nodule. On the contrary, these enzyme activities were diminished and hardly demonstrable in the foamy cells. Ultrastructurally, a large number of G-RBCs were engulfed by the macrophages. A few small vacuoles and membrane structures were formed in most of
Fig. 3. Macrophages contain many G-RBCs a t 24hrs. after injection. Though most of phagocytized G-RBCs retain their discoid shape, fragmented spherules are noted. x 7,200.
Fig. 4. Insert : The foamy cells have an eccentric nucleus and clear cytoplasm with a few reddish brown granules. Day 7. H.E.
A mantle of foamy cells surrounding the mass of G-RBCs at Day 7.
37(4) : 1987 T. ISHIHARA el al 631
632 PHACOCYTOSIS OF CLUTARALDEHYDE-TREATED R B C ~ Actu Puthol. Jpn.
37(4) : 1987 T. ISHIHARA et al. 633
the engulfed G-RBCs. These small vacuoles initially appeared at the peripheral area of the engulfed G-RBCs, and moved into the central area of the engulfed G-RBCs. By these vacuoles most of the engulfed G-RBCs were divided into several smaller spherules as shown in Figs.