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Immunohistochemical analysis of the distribution of vimentin in human peripheral lymphoid tissues.

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Immunohistochemical Analysis of the Distribution of
Vimentin in Human Peripheral Lymphoid Tissues
Division of Clinical Immunology, Department of Medicine, University of Colorado Health
Sciences Center, Denver, CO 80262
The distribution of the intermediate filament protein vimentin in
peripheral lymphoid tissues was determined using a monoclonal antibody. Frozen
sections of tissue were stained using a n avidin-biotin immunoperoxidase
method. The antibody stained endothelial cells in spleen, lymph node, and tonsil.
Unusual rod-like structures were revealed in the sinusoid-lining cells of the spleen.
A variety of reticulum cells was detected, including fibroblastic reticulum cells,
histiocytic reticulum cells (tingible body macrophages), and splenic marginal zone
macrophages. Very few lymphocytes were immunoreactive. Only weak cytoplasmic immunoreactivity was observed in lymphocytes of the periarteriolar lymphocyte
sheath of the spleen. The monoclonal antibody employed appears to be of limited
usefulness in detecting normal lymphocytes, but is strongly reactive with endothelial structures and some types of reticulum cells.
The use of monoclonal antibodies to intermediate filament (IF)proteins is finding widespread application in
diagnostic histopatholorn (Osborn and Weber, 1983).Intermediate filaments are filamentous structures (7-11
nm diameter) that are intermediate in size between
smaller microfilaments and larger microtubules (Osborn
et al., 1977; Hynes and Destree, 1978). These structural
proteins represent families of related, though not identical, polypeptides whose expression in various cells
and tissues appears to depend on processes of cellular
Epithelial cells contain I%s related to epidermal tonofilaments which are classified as cytokeratins (Franke
et al., 1978).Mesenchymal cells contain only one type of
IF protein, vimentin (Franke et al., 1979). Other IF Proteins appear to be rather restricted in their distribution,
such as desmin in muscle cells (Gard et al., 19791, m u rofilament in neurons (Liem et al., 1978), and dial fibrillary acidic protein in aStrOCyteS (Goldman et al.,
These findings suggest that IF proteins could Serve as
differentiation markers and aid in classifying the histogenesis of cells of unknown origin, including those occurring in malignant tumors. Indeed, antibodies to the IF
protein vimentin have been used to classify tumors as
being Of mesenchYmal origin,
sarcomas, 'ymphomas, and melanomas (Gabbiani et al., 1981).
Nevertheless, the distribution of IF proteins in normal
tissues has often not been well documented. It is particularly important to define the IF protein distribution in
normal tissues if one is to apply antibody reagents for
diagnostic purposes, since cross reactions With various
cell types may be misleading.
The purpose of this report is to describe the distribution of vimentin in normal human peripheral lymphoid
tissues using immunohistochemical techniques.
0 1985 ALAN R. LISS, INC.
~i~~~~specimens were obtained at
blocks of fresh tissue were embedded in a cryoprotectant
(ocT, Ames) in an
foil vessel and rapidly
frozen by immersion in a dry ice.isopentane bath. F ~ ~ .
blocks were stored at - 7 0 0 ~until ready for section.
ing. Cryostat sections were cut at 6 pm, air h i e d a t
room temperature 1-2 hr, fixed in reagent grade acetone
for 20 min at
temperature, and air dried again.
Sections were rehydrated briefly in modified phosphatebuffered saline (PBS; Wood and Warnke, 1981) and incubated for 20 min at
temperaturein the follow.
ing avidin.biotin immunoperoxi~ase staining protoco~
(Warnke and Levy, 1980; Giorno, 1984a): 1) unlabeled
murine monoclonal antibody to vimentin (obtained from
Lab Systemsand diluted
in PBS supplemented with
0.2% bovine albumin [ ~ i 1gg4bl);
~ ~ 2) ~
~ ,
antibody (obtained from Biomeda) to primary antibody;
and 3) avidin-peroxidase (obtained from Biomeda). Set.
tions were washed with PBS between incubations. After
the last incubation and PBS wash, sections were incubated with 3,31-diaminobemidine (DAB, 0.2%) in 0.01%
in PBS for 5 min and washed. The DAB staining
was intensified by incubation for 5 min in 0.5% cuso4
in o.9% NaCl (Hanker et al., 1979). After washing in
water, sections were dehydrated through alcohol, cleared
with xylene, and mounted with Permount.
proper staining patterns, similar sections
were stained with a pan-leukocyte monoc~ona~
HLe-l (pizzolo et al., 1980; obtained from Becton-Dickinson and used a t 1:50) for a positive control or with a n
irrelevant monoclonal antibody to keratin (obtained from
Lab Systems and used at 1 : l O ) as a negative control.
Received May 14,1984;accepted July 31,1984.
Fig. 1 . Cryostat section of spleen immunostained for vimentin. Central arteriolar (open arrow) and sinusoidal (closed arrows) endothelium
is immunoreactive. W, white pulp; R, red pulp.
Fig. 2. Cryostat section of spleen immunostained for vimentin. Marginal zone macrophages are immunoreactive (arrows). W, white pulp;
R, red pulp.
Fig. 3.Cryostat section of spleen immunostained for vimentin. Central arteriolar endothelium is strongly immunoreactive (closed arrow).
Lymphocytes in surrounding sheath exhibit patchly weak immunoreactivity (open arrow).
Fig. 4. Cryostat section of spleen immunostained for vimentin. Unusual rod-like structures are associated with sinusoidal endothelium
Fig. 5. CryoaLaL section of spleen immunostained for vimentin. A
longitudinal section of a sinusoid wall shows strongly immunoreactive
rod-like structures running parallel to the long axis (arrow).
Fig. 7. Cryostat section of lymph node irnmunostained for vimentin.
Capillary endothelium is strongly immunoreactive. Surrounding lymphocytes are negative.
Fig. 6. Cryostat section of lymph node immunostained for vimentin.
Capillary endothelium (open arrow) and mantle zone (closed arrow) are
Fig. 8. Cryostat section of tonsil immunostained for vimentin. The
mantle zone (M) appears uniformly immunoreactive. Scattered cells in
the germinal center (GC)are positive.
Fig. 9 Cryostat section of tonsil immunostained for vimentin. The
mantle zone contains parallel fibers that are strongly immunoreactive
(open arrows). Lymphocytes are not stained. Capillary endothelium
(closed arrow) is also strongly immunoreactive.
Fig. 10. Cryostat section of tonsil immunostained for vimentin. Scattered branching cells in the germinal center are strongly immunoreactive (arrows).
Photomicrographs were made using a n interference
filter combination which enhances the contrast of the
DAB reaction product (Moller et al., 1984).
cytes was not appreciated in any regions of the lymph
node. The antibody also revealed intense staining of
branching cells in the germinal centers of the secondary
follicles, which appear to represent histiocytic reticulum
cells (see below). The antibody also revealed intense
staining of endothelial cells in capillaries and high endothelial venules (Fig. 7).
Within the spleen, vimentin was localized in vascular
endothelium of central arterioles of the white pulp and
the sinusoids of the red pulp (Fig. 1).Vimentin was also
present, but less intensely stained, in marginal zone
macrophages and reticulum fibers of the mantle zone of
secondary lymphoid follicles (Fig. 2). There was very
weak reactivity of lymphocytes in the periarteriolar
lymphocyte sheath (Fig. 3). The lymphocyte staining
could not be localized to the cell membrane and appeared to be cytoplasmic in nature. Staining of sinusoidal endothelium revealed unusual rod-like structures
(Figs. 4, 5) which probably correspond to filamentous
structures which maintain the rigidity of endothelial
slits (Chen and Weiss, 1973; Giorno, 1984~).
Palatine Tonsil
The distribution of immunoreactivity in the tonsil was
similar to that seen in lymph nodes (Fig. 8). The reticulum fibers in the mantle zone (Fig. 9) ran between lymphocytes. The branching cells in the germinal center
(Fig. 10) were quite large, irregularly shaped, and intensely immunoreactive. Their distribution was similar
to that of tingible body macrophages noted in routine
histologic sections. Definitive staining of lymphocytes
was not appreciated.
I have described the distribution in peripheral lymLymph Node
phoid tissues of material immunoreactive with a monoImmunostaining in the lymph node revealed what ap- clonal antibody to the IF protein vimentin. Rather unpeared to be relatively specific staining in the mantle expectedly, lymphocytes were not immunoreactive. Only
zone of the secondary follicle (Fig. 6). On higher power, in the periarteriolar lymphocyte sheath of the spleen
the staining was localized to filamentous reticulum fi- was some apparent immunoreactivity noted. This imbers between lymphocytes. Definite staining of lympho- munoreactivity was not membranous in nature, as seen
with most monoclonal antibodies to lymphocyte surface
markers, but rather appeared to be cytoplasmic. The
periarteriolar lymphocyte sheath is a T-cell zone without any unique properties, as it is presently understood
(Giorno, 1984d).
Not all types of reticulum cells (Muller-Hermelink and
Kaiserling, 1980)exhibited immunoreactivity with the
monoclonal antibody to vimentin. Histiocytic reticulum
cells (tingible body macrophages) in germinal centers of
secondary follicles were strongly immunoreactive. Macrophages in the marginal zone of the spleen were also
immunoreactive. The antibody failed to detect dendritic
reticulum cells in germinal centers or interdigitating
reticulum cells in T-cell zones (lymph node paracortex).
The antibody appears to detect a t least some portions of
fibroblastic reticulum cells, i.e., the fibrous reticulum
structures in the mantle zone of the secondary follicle
which coursed between lymphocytes.
These findings suggest either that not all normal cells
of mesenchymal origin contain vimentin or that the
monoclonal antibody used only recognizes a n epitope of
the vimentin polypeptide in certain cell types, but not
in others. Further consideration of these possibilities is
important in the understanding of the histogenesis of
normal lymphoid tissues as well as in attempts to classify malignant tumors based on the presence or absence
of vimentin.
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periphery, distributions, immunohistochemical, vimentin, lymphoid, analysis, tissue, human
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