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Simultaneous localization of histamine and factor VIII-related antigen in the endothelium of the human umbilical vein.

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THE ANATOMICAL RECORD 232:257-261 (1992)
Simultaneous Localization of Histamine and Factor VIII-Related
Antigen in the Endothelium of the Human Umbilical Vein
HIROSHI UEDA, YOSHIAKI DOI, YOSHITAKA SAKAMOTO,
KUNSHIGE HAMASAKI, AND SUNAO FUJIMOTO
Department of Anatomy, School of Medicine (H.U., Y.D., Y.S., S.F.) and Division of
Maternal Health, School of Nursing (K.H.), University of Occupational and Environmental
Health, Kitakyushu, Japan
ABSTRACT
Simultaneous immunoelectron microscopic localization of histamine and factor VIII-related antigen was examined on the same ultrathin section
of the endothelium of the human umbilical vein from full-term deliveries by means
of the double-immunolabeling technique.
Small gold particles demonstrating antibody reaction with histamine are preferentially located in the cytoplasmic matrix and organelles, especially in mitochondria and on the luminal membrane surface of the endothelial cells. The gold
particles representing histamine immunoreactivity also located on some of WeibelPalade (WP) bodies. In contrast, large gold particles demonstrating factor VIIIrelated antigen are concentrated preferentially on most WP bodies. Single labeling
of either histamine or factor VIII-related antigen shows similar results to those of
the double labeling.
The present study indicates that some W P bodies are involved in storage of both
factor VIII-related antigen and histamine, but others store factor VIII-related antigen only. This difference in contents of WP bodies may be induced during the
development and maturation process of this inclusion. At any rate, it is reasonable
to consider that WP bodies have important roles in both vascular tonus and hemostasis during the vascular obliteration.
Endothelial specific granules, Weibel-Palade (WP)
bodies, were first described by Weibel and Palade
(1964), and since then their biochemical nature and
function have been studied (Jaffe et al., 1973; Sakariassen et al., 1979; Kumar et al., 1980; Fujimoto and
Ueda, 1989).Recent immunocytochemical studies have
indicated that WP bodies react with factor VIII-related
antigen (Wagner et al., 1982; Hormia et al., 1984; Kagawa and Fujimoto, 1987), von Willebrand factor
(Warhol and Sweet, 1984),and its cleaved pro-sequence
(Ewenstein et al., 1987) in the human umbilical vein
and in a variety of human capillaries. In addition, Kita
and Furuya (1989) reported the ultrastructural localization of histamine on WP bodies in the asphyxia1
guinea pig pulmonary vein.
Our previous chromatographic studies also suggested the presence of histamine on WP bodies in the
endothelium of the toad aorta (Fujimoto, 1982; Fujimot0 et al., 1984) and of the umbilical vein of the postnatal rabbit (Fujimoto et al., 1982). In the umbilical
vein of rabbits (Fujimoto et al., 1982) and humans (Kagawa and Fujimoto, 1987),both remarkable increase in
the number of WP bodies and degranulation of WP
bodies by treatment with a histamine releaser (compound 48/80) were observed. So, it has been postulated
that WP bodies might be a reservoir of histamine and
might be involved in the physiological vascular obliteration.
It is well known that factor VIII-related antigen has
an important role in the adhesion of blood platelet to
0 1992 WILEY-LISS, INC.
the endothelium after vascular injuries (Sakariassen et
al., 1979) and histamine does that in the regulation of
vascular tonus (Furchgott, 1983). Therefore, the problem of whether factor VIII-related antigen and histamine are stored in the same W P bodies is of great interest. However, to date, the colocalization of
histamine and factor VIII-related antigen on the same
ultrathin section has not been reported. The purpose of
the present immunoelectron microscopic study was to
obtain direct evidence of simultaneous localization of
histamine and factor VIII-related antigen on the same
ultrathin section by means of the immunoglobulingold, double-staining technique.
MATERIALS AND METHODS
Tissue Preparation
Human umbilical veins obtained from full-term normal deliveries were fixed for 18 hr at 4°C with 2%
paraformaldehyde- 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. Specimens were washed for 2 hr
in 0.1 M phosphate buffer containing 10% sucrose, de-
Received December 14, 1990; accepted August 6, 1991.
Address reprint requests to Dr. Sunao Fujimoto, Dept. of Anatomy,
University of Occupational and Environmental Health, School of
Medicine, 1-1Iseigaoka, Yahatanishi-ku, Kitakyushu 807, Japan.
Dr. H. Ueda is now at Toya Lake Station for Environmental Biology, Faculty of Fisheries, Hokkaido University, 122 Tsukiura, Abutacho, Abuta-gun, Hokkaido 049-57, Japan.
H. UEDA ET AL.
258
Fig. 1. Electron micrograph of a n endothelial cell (EC) of the human
umbilical vein from full-term delivery in osmium-postfixed and Eponembedded specimens. Abundant round or rod-shape Weibel-Palade
bodies with tubular structures (WP) exist in the endothelial cell
(A).Immature WP bodies of which tubular structures are obscure and
vesicular components are observed near the Golgi apparatus (GA) (B).
A: X 40,000; B: X 20,000.
hydrated through graded concentrations of ethanol,
embedded in Lowicryl K4M (Polaron Equipment, Watford, England), and polymerized at 4°C with a n ultraviolet polymerizer (Dosaka EM, Kyoto, Japan). Some
specimens were postfixed for 2 h r at 4°C with 1% osmium tetroxide in 0.1 M phosphate buffer, dehydrated
through graded concentrations of acetone, and embedded in Epon 812 (Taab Laboratories, Reading, UK). For
ordinary electron microscopy, ultrathin sections of
Epon-embedded specimens were stained with 5% uranyl acetate for 5 min and lead nitrate for 1 min.
(E.Y Labs) for antifactor VIII-related antigen serum
diluted both at 1:lOO in 0.1% EA-PBS. The grids were
washed in PBS and then in distilled water.
For the double labeling of histamine and factor VIIIrelated antigen in the same ultrathin section, Bendayan’s two-face method (Bendayan, 1982) was used. The
first face (face A) of the grid was stained by the same
procedure as for the single labeling of histamine. After
complete drying with air, the grids were turned over to
the second face (face B), which was treated with the
same procedure as for the single labeling of factor VIIIrelated antigen.
The sections were finally stained with 5%uranyl acetate for 5 min and examined on a JEM 100 CX electron microscope. The specificity of the present double
immunolabeling was confirmed by the following controls: (1)antihistamine serum and antifactor VIII-re-
lmmunoelectron Microscopy
All procedures were done a t room temperature. U1trathin sections of Lowicryl-embedded specimens indicating gold interference color were collected on uncoated 150-mesh nickel grids and placed for 10 min on
drops of 0.1% egg albumin (EA) in phosphate-buffered
saline (PBS). For the single labeling of either histamine or factor VIII-related antigen, sections were incubated for 2 hr with rabbit antihistamine serum
Fig. 2. Single immunoelectron microscopic localization of histamine
(Chemicon International, Los Angeles) diluted a t 1:100
and factor VIII-related antigen (B) in the endothelial cells in
in 0.1% EA-PBS, or with goat antihuman factor VIII- (A)
Lowicryl-embedded specimens. Five nm gold particles indicating hisrelated antigen serum (Nordic Immunological Labs, tamine immunoreaction are localized on the cytoplasmic matrix and
Tilburg, Netherlands) diluted a t 1:lOOO in 0.1% EA- the luminal membrane surface (arrowheads) of the endothelial cell
PBS. After rinsing in PBS, the grids were reacted for 1 (A). Some Weibel-Palade bodies (WP) also show specific histamine
Asterisks in A represent WP bodies that retain feah r with goat antirabbit IgG-coated 5 nm colloidal gold immunoreaction.
tures of tubular structure. In B, factor VIII-related antigen-reacted 20
(E.Y Labs, San Mateo, CA) for antihistamine serum, or nm gold particles are located on most WP bodies (WP). EL, elastic
with swine antigoat IgG-coated 20 nm colloidal gold lamina. A: x 50,000; B: x 50,000.
Fig. 2
260
H. UEDA ET AL.
Fig. 3. Double imrnunoelectron microscopic labeling of histamine (5
nm gold particles) and factor VIII-related antigen (20 nm gold particles) in an endothelial cell (EC) in Lowicryl-embedded specimen. The
small gold particles are distributed in the cytoplasmic matrix and
organelles especially in mitochondria (MT),and on the luminal
plasma membrane (arrows) of the endothelial cell. Some W P bodies
(WP,-WP,) are immunoreacted with both histamine and factor VIIIrelated antigen; others (WP,) are labeled with the large gold particles
only. x 50,000.
lated antigen serum were absorbed with histamine and
factor VIII-related antigen, respectively, (2) normal
rabbit and goat sera were substituted for the specific
antisera, (3) both antisera were reacted on the same
face (face A) followed by the reaction with immunogold solutions.
ticles is observed preferentially on WP bodies, although some of them are fused to each other (Fig. 2B).
Figure 3 shows simultaneous localization of histamine labeled by small gold particles (5 nm) and factor
VIII-related antigen by large gold particles (20 nm).
The large particles are concentrated on most of the WP
bodies as shown in this figure. Some nonspecific binding to the cytoplasm is also observed. In contrast, the
small particles are localized in the cytoplasmic matrix
and organelles, especially in mitochondria and on the
luminal plasma membrane of the endothelial cell.
Some WP bodies were reacted with both factor VIIIrelated antigen and histamine, whereas others were
labeled with factor VIII-related antigen only.
In control sections reacted with specific antisera that
were absorbed with their antigens or reacted with normal sera, no specific gold particles were observed. Double labelings performed on the same face showed artifactual codistribution of large and small gold particles.
In these control sections, both particles are always associated with each other. On the contrary, the different
localization of small and large gold particles on similar
WP bodies in the double-reacted sections labeled on
opposite surfaces has confirmed the specificity of the
present double labeling (Fig. 3).
RESULTS
Endothelial cells of the human umbilical vein from
full-term deliveries are charcterized by the presence of
abundant electron-dense WP bodies with tubular structures in osmium-postfixed and Epon-embedded specimens (Fig. 1A). In the Golgi apparatus, WP bodies are
actively segregated, but their tubular structures are
obscure, and many vesicular components exist near
WP bodies (Fig. 1B). At first, we tried to immunoreact
with histamine and factor VIII-related antigen after
de-osmification with saturated sodium metaperiodate.
In these specimens, the antigenicity to factor VIII-related antigen is poorly preserved, but that to histamine
is completely destroyed. In Lowicryl-embedded ones,
WP bodies considerably reduce their electron densities,
but their tubular structures were sometimes detectable
(asterisk in Fig. 2A). Histamine antigenic sites are
clearly demonstrated by these samples. The gold-labeled (5 nm) histamine immunoreactivity is located
mainly on the cytoplasmic matrix and the luminal
membrane surface of the endothelial cell (Fig. 2A).
Some of WP bodies also showed specific immunoreaction with histamine (Fig. 2A). As reported previously
(Kagawa and Fujimoto, 1987), the immunoreactivity of
factor VIII-related antigen revealed by 20 nm gold par-
DISCUSSION
The present immunoelectron microscopic labeling of
factor VIII-related antigen confirms our previous data
that WP bodies are a storage site of this antigen (Kagawa and Fujimoto, 1987). Similarly, i t has been reported that von Willebrand factor and its cleaved pro-
DOUBLE LABELING OF HISTAMINE AND FACTOR VIII
261
sequence can be shown to be localized in W P bodies by preparation. This work was supported in part by a Rethe protein A-gold technique (Ewenstein et al., 1987). search Grant for Cardiovascular Diseases (63C-1)from
We have also suggested that WP bodies might be a the Ministry of Health and Welfare.
reservoir site of histamine because the degranulation
LITERATURE CITED
of these bodies is induced by treatment with a histamine liberator, compound 48/80 (Fujimoto, 1982; Fuji- Antohe, F., C. Heltianu, and N. Simionescu 1986 Further evidence for
the distribution and nature of histamine receptors on microvasmot0 et al., 1982; 1984; Kagawa and Fujimoto, 1987).
cular endothelium. Microcirc. Endothelium Lymphatics., 3:163The present study provides the direct evidence by
185.
means of specific histamine immunoreaction that some Bendayan, M. 1982 Double immunocytochemical labeling applying
the protein A-gold technique. J. Histochem. Cytochem., 3023of the W P bodies are a reservoir of histamine. Hista85.
mine is also observed to be located in the cytoplasmic
B.M., M.J. Warhol, R.I. Handin, and J.S. Pober 1987 Commatrix and organelles including mitochondria, and on Ewenstein,
position of the von Willebrand factor storage organelle (Weibelthe luminal membrane surface of the endothelium. UsPalade body) isolated from cultured human umbilical vein endothelial cells. J. Cell Biol., 104t1423-1433.
ing histamine-ferritin conjugate, Simionescu and his
coworkers (Heltianu et al., 1982; Antohe et al., 1986) Fujimoto, S. 1982 Degranulation of endothelial specific granules of
the toad aorta after treatment with compound 48/80. Anat. Rec.,
revealed that the venular endothelium contained
203t197-204.
mainly H, receptor and that the conjugate is bound on Fujimoto, S., and H. Ueda 1989 Weibel-Palade body. Electron Microsc., 24:33-39.
luminal endothelial cell plasmalemma. They did not
make any reference to WP bodies. Recently, Kita and Fujimoto, S., K. Yamamoto, H. Ueda, K. Hamasaki, and T. Nomiyama
1984 Histamine disposition in endothelial specific granules of the
Furuya (1989) found histamine reactive WP bodies
toad aorta. Acta Anat., 118r38-41.
with the immunoperoxidase technique in asphyxia1 Fujimoto, S., K. Yamamoto, K. Arashidani, I. Hayabuchi, M. Yoshizuka, and T. Nomiyama 1982 Endothelial specific granules in the
guinea pig pulmonary veins but not in exsanguinated
umbilical veins of the postnatal rabbit. Cell Tissue Res., 227:
controls.
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Using simultaneous labeling, some W P bodies react Furchgott,
R.F. 1983 Role of endothelium in responses of vascular
with both factor VIII-related antigen and histamine
smooth muscle. Circ. Res., 53557-573.
but others with factor VIII-related antigen only. Sengel Hattori, R., K.K. Hamilton, R.D. Fugate, R.P. McEver, and P.J. Sims
1989 Stimulated secretion of endothelial van Willebrand factor is
and Stoebner (1967), Takeshige and Fujimoto (1977),
accompanied by rapid redistribution to the cell surface of the
and Fujimoto et al. (1982) have indicated that WP bodintracellular granule membrane protein GMP-140. J . Biol.
ies are segregated from the Golgi apparatus and inChem., 264:7768-7771.
crease their electron density by successive fusion with Heltianu, C., M. Simionescu, and N. Simionescu 1982 Histamine receptors of the microvascular endothelium revealed in situ with a
other vesicular components. In the present study, the
histamine-ferritin conjugate: Characteristic high-affinity bindmorphology of Golgi apparatus indicates active syntheing sites in venules. J . Cell Biol., 93:357-364.
sis of WP bodies, in which tubular structure are rarely Hormia, M., V.P. Lehto, and I. Vitanen 1984 Intracellular localization
of factor VIII-related antigen and fibronectin in cultured human
observed and many vesicular components are present
endothelial cells: Evidence for divergent routes of intracellular
near immature WP bodies. It is reasonable to consider
Eur. J . Cell Biol., 33:217-228.
that factor VIII-related antigen may develop during Jaffe,translocation.
E.A., L.W. Hoyer, and R.L. Nachman 1973 Synthesis of antithe formation of WP bodies in the Golgi apparatus and
hemophilic factor antigen by cultured human endothelial cells. J .
Clin. Invest., 52:2757-2764.
that histamine uptake may take place during the maturation process of WP bodies after segregation from the Kagawa, H., and S. Fujimoto 1987 Electron-microscopic and immunocytochemical analyses of Weibel-Palade bodies in the human
Golgi apparatus. Recently, Hattori et al. (1989) reumbilical vein during pregnancy. Cell Tissue Res., 2 4 9 5 5 7 4 6 3 ,
ported the colocalization of von Willebrand factor and Kita, T., and Y. Furuya 1989 Histamine effects on pulmonary blood
vessels in strangulation. Z. Rechtmed., 103:85-91.
intracellular granule membrane protein GMP-140 on
Kumar, P., S. Kumar, H.B. Marsden, P.G. Lynch, and E. Earnshaw
W P bodies by immunofluorescent microscopy.
1980 Weibel-Palade bodies in endothelial cells as a maker of anThe present work describes simultaneous localizagiogenesis in brain tumors. Cancer Res., 40:2010-2019.
tion of factor VIII-related antigen and histamine on the Sakariassen, K.S., P.A. Bolhuis, and J . J . Sixma 1979 Human blood
platelet adhesion to artery subendothelium is mediated by factor
same ultrathin section. Special attention should be
VIII-von Willebrand factor bound to the subendothelium. Nature,
paid to the colocalization of factor VIII-related antigen
279:636-638.
with histamine in the same WP bodies. Severe endo- Sengel,
A,, and P. Stoebner 1967 Golgi origin of tubular inclusions in
thelial cell damage was observed during vascular conendothelial cells. J. Cell Biol., 44t223-226.
tractions induced by histamine in the in vitro swine Takeshige, Y., and S. Fujimoto 1977 Ultrastructural modifications of
endothelial cells by changes of the vascular function. In: Biliocoronary artery (Ueda et al., 1991). Therefore, it is postheca Anatromica, No. 16. D.H. Lewis, ed. Karger, Basel, pp.
sible to consider that some WP bodies participate in the
364-369.
regulation of both vascular tonus and hemostasis in Ueda, H., Y. Doi, M. Yoshizuka, Y. Sakamoto, N. Mori, H. Araki, and
S. Fujimoto 1991 Electron microscopy of histamine-induced concase of vascular injury and obliteration. More detailed
traction of the in vitro swine coronary artery. Acta Anat., (in
investigations to clarify the functional significance and
press).
the exact biochemical nature of WP bodies are now in Wagner,
D.D., J.B. Olmsted, and V.J. Marder 1982 Immunolocalizaprogress in our laboratory.
tion of von Willebrand protein in Weibel-Palade bodies of human
ACKNOWLEDGMENTS
We thank Ms. Tomoko Nishino for technical assistance, Ms. Toyono Nobukuni for typing the manuscript, and Mr. Mitsuru Yokoyama for photographic
endothelial cells. J. Cell Biol., 95:355-360.
Warhol, M.J., and J.M. Sweet 1984 The ultrastructural localization of
von Willebrand factor in endothelial cells. Am. J . Pathol., 117:
310-315.
Weibel, E.R., and G.E. Palade 1964 New cytoplasmic components in
arterial endothelia. J. Cell Biol., 23:lOl-112.
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