From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
Immunohistochemical
Characterization
(LN-4,
LN-5)
Reactive
With
Human
Malignancies
in B5-Fixed,
By Lertlakana
Bhoopat,
Clive
Two
monoclonal
antibodies
macrophages
in B5
sue
sections
cell
extracts
have
of
monoclonal
the
been
of
stain
the
and
human
cytoplasm
hematopoietic
positivity
with
positive
and
Both
mantle
H E
IM
and
using
by
zone
strongly
reactive
cells and related
methods
has been
of
localization
and
tive
LN-5
have
benign
in
liver
was
node
stomach.
described
requirement
for
in the literature,
fresh or frozen
are
fore
not
routinely
suitable
for
processed
cell
of
types.’’2
tissue
enabled
macro-
investigators
of specific
demonstration
poietic
sues.3’
immunoperoxidase
enzymatic
cell types
require
Antisera
that
are
processed
tissue
normal
and
a,-antitrypsin
and
using
Similarly,
reactions
in
the
hemato-
fresh
or
effective
specially
processed
in routinely
fixed
and
can
that
neoplastic
macrophages
These
include
antisera
specificity.
antigen,36
specimens
by the
there-
studies
specimens.
be used
tisand
to identify
suffer
from
a lack of
to lysozyme,32’35
5-100
a,-antichymotrypsin,32’34’37
fac-
tor XIII,38
cathepsin
B,39 and monocytic
the metalophil
method,4”42
which
has
esterase.#{176} Finally,
been
used
to stain
dendritic
cells,
therefore
In the
not widely
last several
generated
of
their
From
these
antibodies
normal
tissue
laboratory
has
to lymphoid
LN-4
processed
the
macrophage-related
that
LN-3,
tiswhich
new
which
and
specimens
diagnosis
including
persistent
new
and
by Grune
& Stratton,
for
benign
and
of
their
their
ability
LN-4
and
diagnosis
histiocytic
of
histiocytosis.
Because
the
posi-
generalized
tissues.
reagents
Hodg-
also
in a variety
sinus
macrophages.
of malignant
1988
present
paraffin-embedded
important
and
to
LN-5
classifi-
lesions.
Inc.
AND
METHODS
A complete
list of the tissues used in these experiments
in Tables
I to 5. For the immunohistochemical
studies,
human
tissues fixed with B5 formalin
were obtained
at biopsies
performed
on patients
for diagnostic
procedures
at the Los Angeles
County-University
of Southern
California
Medical
Center
and
Northwestern
University
Hospital.
Bone marrow
aspirates
from
leukemia
patients were prepared
as clot preparations
and fixed in B5
formalin
before paraffin-embedding
procedures.
All patients used in
this study were advised
of procedures
and attendant
risks, in
accordance
with institutional
guidelines,
and gave informed
consent.
Antigen
preparation.
B5-flxed
paraffin
blocks of Ficoll-Hypaque-separated
normal human peripheral
blood mononuclear
cells
were cut into multiple
30-pm-thick
sections and deparaffinized
with
xylene containing
I % sodium iodide to remove the mercuric
chloride.
The rescued cells were then rehydrated
by washing with decreasing
concentrations
of alcohol followed
by two washes with phosphatebuffered saline. Aliquots of the rescued cells were frozen at - 70#{176}C
for use in the immunization
procedures.
Immunization
protocol.
One-milliliter
aliquots
of the deparaffinized cell preparations
were thawed,
sonicated
to reduce
the
viscosity,
and emulsified
in I .3 mL complete
Freund’s adjuvant
by
using two glass syringes
and a 20-gauge
microemulsifying
needle
(Bolab,
Inc. Lake Havasu
City, AZ). Three 6-week-old
BALB/c
Tissues.
is shown
are
derived
should
and
the
reactive
Submitted
September
in part
Santa
Ana,
of
Angeles,
greatly
of
reprint
requests
USC
payment.
J 988
of
of Pathblogy,
1 987,’ accepted
December
Techniclone
10.
Southern
North1987.
International.
Inc.
to Alan
Medical
L. Epstein,
MD.
Center,
2025
were
defrayed
PhD.
Zonal
DepartAve.
Los
90033.
“advertisement”
()
University
Department
by a grantfrom
Pathology.
The publication
charge
21.
Pathology.
the
CA.
CA
indicate
of
Department
Supported
ment
neoplas-
study
From
(‘alifornia,
Los Angeles,
and
western
University.
Chicago.
Address
monoclonal
diseases.
The availability
of
identify
these cell types in rou-
immunohistological
disorders.
1988:
is
cells, respecAs an extension
of
LN-5,
B5-fixed,
are
histiocytes
disease.
the
were
successfully
subsets
and
two
subsets
pathological
Blood, Vol 71, No4(April),
LN-2,
and
and
paraffin-embedded
on the characterization
generated
macrophage
benign
proliferative
monoclonal
antibodies
that
enhance
our
LN-l,
and
tinely
of specificity
and HLA-DR-positive
malignancies.43’47
we have
designated
antibodies,
tic
a lack
in B5-fixed,
studies,
we reported
investigations,
with
from
antibodies
B cell subsets
and their derived
tively,
these
used.
years,
reactivity
monoclonal
identify
suffers
monoclonal
retain
sues.
also
human
macrophagelike
antibodies
MATERIALS
the precise
however,
are limited
tissue
specimens
and
pathological
stain
in
and
sclerosis
surrounding
lymphadenopathy.
for
macrodisease
of nodular
positive
Gaucher’s
dermatopathic
infiltrating
Hodgkin’s
bands
lesions
lymphadenopathy.
and
cation
of true
and
lymphoid
specificity
to study
the derivation
and antigenic
composition
of histiocytic
disorders.23’3#{176} Currently
available
monoclonal
antibodies
in macrophages
macrophages
permitted
subsets
and
monoclonal
cases
to these
have
specific
dendritic
Both
proliferative
disorders
greatly
aided
by the
antibodies
studies
was
lesions.
lymph
of
LN-4
kin’s
using
LN-5
with
disease.
by
strong
of the
identification
M UNOLOGIC
of monoclonal
anatomic
cells
Hodgkin’s
for
in
In all cases
collagen
showed
of the
chief
lymphomas.
in the
of the
whereas
B lymphocytes
immunoperoxidase
phages
stomach,
except
negative
present
histiocytes
also
but.
entirely
cells
and
cells
LN-4
and
were
dendritic
in situ
availability
These
the
spermatogonia,
antibodies
T
of
and
Kuppfer’s
skin.
LN-4
were
paraffin-
Specificity
that
macrophages
of the
acini
with
spleen.
show
including
cells
nodes
procedure.
tissues
of
organs
Langerhans’
lymph
Both
Paul R. Meyer,
lymphoma
phages.
Antibodies
Derived
L. Epstein
non-Hodgkin’s
cells.
on
Monoclonal
Subsets
and
Tissues
Stathopoulos,
and Alan
histiocytic
tis-
deparaffinized
identified
Efstathios
J. Marder,
to human
mononuclear
staining
on normal
using
initially
hyperplastic
immunoperoxidase
reactive
by
blood
were
R. Turner,
Robert
paraffin-embedded
produced
peripheral
sections
screens
LN-5)
formalin-fixed,
antibodies
embedded
(LN-4.
Roderick
R. Taylor,
of Two New
Macrophage
Paraffin-Embedded
costs
ofthis
article
This
article
must
in
accordance
with
therefore
/8
U.S.C.
be
in part
hereby
§1734
by page
marked
solely
to
this fact.
by Grune
& Stratton,
Inc.
0006-497l/88/7104-0023$3.00/0
pp
1079-1085
1079
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1080
BHOOPAT
Table
1.
Reactivity
Lymphoid
of LN-4
Paraffin-Embe
on Normal
Tissues
dded
Table
Human
center
Mantle
zone
Paracortical
Plasma
B cells
B cells
T cells
cells
Endothelium
Sinus
histiocytes
Tingible
body
Follicular
macrophages
dendritic
Interdigitating
cells
reticulum
Epithelioid
Sections
cells
-
-
-
+ + +
+
Bronchus
-
-
Lung
-
Kidney
+ (tubular
Prostate
-
-
-
+ + (spermatogonia)
+ + +
+ + +
Testes
+ + +
+ + +
Adrenal
gland
-
-
Thyroid
gland
-
-
-
+ + +
-
Parathyroid
-
+
-
-
Liver
-
-
Gallbladder
-
Stomach
+ + + (chief
+ + +
Colon
-
-
+ +
Heart
-
-
Brain
-
-
-
-
-
-
muscle
-
-
-
-
-
-
-
-
-
-
Cartilage
Hassall’s
-
Peripheral
Skin,
Spleen
-t
(mantle
+++
zone)
#{149}Weak
variable
Erythroid
precursors
+
-
-
-
-
Megakaryocytes
-
-
Plasma
-
-
-
+ + +
cells
Macrophages
cells
+
lntensity
tPositive
Positivity
cells
of immunoperoxidase
( + + + I staining
observed
in two
staining
+ +
ranging
for red pulp sinus
of four
samples
from
macrophages
-
elongated
to + +
cells
in adrenal
cortex
and
were
characterization
of
monoclonal
antibody
iso-
Hybridoma
+.
only.
Table
Reactivity
3.
of LN-4
Leukemias,
studied.
and
and
LN-5
Related
With
Diseases
Paraffin-Embedded
Human
female mice were injected subcutaneously
at multiple sites by using
a 22-gauge
needle and glass syringe. Two weeks later, the mice were
reinoculated
as before except that the deparaffinized
cell preparation was prepared
in incomplete
adjuvant.
Ten days later, the mice
received
a third inoculation
of antigen,
this time without adjuvant
and by intravenous
injection.
Four days later, the mice were killed by
cervical
dislocation
and the spleens removed by aseptic techniques.
Cellfusion
and cloning
procedures.
Spleen cells were fused with
8-azaguanine-resistant
mouse myeloma
NS- I cells at a ratio of 5:1,
respectively,
by using 40% polyethylene
glycol,
I ,540 molecular
weight
(Baker
Chemical
Co. Phillipsburg,
NJ) as described
by
Frazekas
de St. Groth and Scheidegger.
Hybridoma
cells from
wells with active cell growth
were expanded
to 24-well plates, at
which time the supernatants
were collected
and tested on B5-fixed,
paraffin-embedded
sections
of hyperplastic
lymph nodes by using
the immunoperoxidase
staining
technique
described
later. Positive
cultures
were cloned
on 0.5% Noble agar (Difco
Laboratories,
Detroit)
containing
RPMI
1640 medium,
20% fetal calfserum,
and
Non-Hodgkin’s
Sections
LN-4
(all categories
of
working
formulation)
sclerosis
and mixed
myelogenous
(M1-M6
cellularity)
of FAB classification)
0/ 1 Ot
0/10
0/27
0/27
0/5
0/5
0/10
0/10
and myelomonocytic
myelogenous
lymphoblastic
(common,
Malignant
leukemia
null cell, and T cell)
fibrous
histiocytoma
Mycosisfungoides
Abbreviation:
FAB,
Data are expressed
of
2/29
leukemia
leukemia
Acute
2/29
disease
(nodular
Chronic
LN-5
lymphomas
Hodgkin’s
Acute
Lymphomas,
in B5-Fixed,
Diagnosis
antibiotics.49
medulla
supernatants
from four-day
cultures
were concentrated
20-fold
in B5 Minicon
concentrators
(Amicon
Corp.
Lexington,
MA) and tested in double-diffusion
Ouchterlony
plates
against rabbit antimouse
immunoglobulin
heavy chain-specific
antisera (Miles Laboratories,
Inc. Elkhart,
IN). The precipitin
bands
were read after two days’ incubation.
+ +
+ +
scattered
Serological
Liver
Kuppfer’s
few
+.
types.
Skin
Langerhans’
staining.
+++
tA
precursors
cells)
squamous
Placenta
Bone marrow
Myeloid
nerve
epithelium
-t
+ + +
+ (chief
muscle
+ +
-
-
cells)
Smooth
-
-
gland
t
Pancreas
-
Macrophages
Macrophages
epithelium)
+ (glands)
Skeletal
pulp
+ (tubular
-
+ + +
White
-
epithelium)
-
-
Redpulp
epithelium)
Endometrium
-
corpuscles
(ductal
Ovary
+ +
cells
LN-5
-
-
Medulla
Dendritic
LN-4
Breast
-
-
Lymphocytes
Nonlymphoid
Tissues
-
-
Macrophages
Normal
bedded
-
Cortex
cells
With
Paraffin-Em
-
Thymus
Dendritic
and LN-5
-
+ + +
Lymphocytes
of LN-4
i n B5-Fixed,
Tissue
LN-5
-
histiocytes
Reactivity
Tissues
node and tonsil
Germinal
2.
in B5-Fixed,
LN-4
Organ
Lymph
and LN-5
and Hematopoietic
ET AL
0/2
2/2
0/4
0/4
French-American-British.
as a
percentage
of positive
cases
per
t otal
number
cases.
The
enzymatic
tStrong
positive
cases
were
and immunophenotyping
positivity
in sclerotic
true
histiocytic
in origin
as determined
studies.
bands
in nodular
sclerosis
type.
by
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MONOCLONAL
Table
ANTIBODIES
4.
Reactivity
Lesions
of LN-4
in B5-Fixed,
Diagnosis
Reactive
IN LN-4
and LN-5
Lymphoid
sinus
and
body
phages
histiocytes
Sinus
histiocytes,
Sinus
histiocytes
+ + ,
thy
and
sinus
+ + +,
Sinus
5
macro-
and
histiocytes
+ + ,
lymphadenopa-
the immunoreactivity
of the tissue sections.
To identify
of nonspecific
binding
or the presence
of endogenous
peroxidase
activity,
monoclonal
antibody
43H3, which is nonreactive
in paraffin-embedded
sections, was used with each run.
Fixed and live cell indirect immunofluorescence.
To determine
the indirect
immunofluorescence
staining
patterns
of LN-4 and
LN-5
monoclonal
antibodies,
live cell and paraformaldehydeacetone-fixed
cell preparations
were used. For these studies,
a
variety of normal
and malignant
cell lines and strains were used. A
detailed
description
of these indirect
immunofluorescence
methods
has been published
previously.5’
+ +,
Tingible
mac-
histiocytes
5
determine
the level
Sections
LN-5
body
rophages
Dermatopathic
Benign
+ ,
Tingible
histiocytosis
With
LN-4
9
sia
Sinus
1081
LN-5
Paraffin-Embedded
n
hyperpla-
AND
+ +,
Histiocytes
+ , interdigi-
tating
RESULTS
reticulum
Generation
cells
Persistent
general-
10
+ + + ,
ized lymphade-
Tingible
nopathy
body
mac-
rophages,
and
perisinus
4
Sarcoidosis
body
phages
sinus
histiocytes.
Tuberculosis
Tingible
fusion
sinus
cytes
and
cells
+
Epithelioid
histio-
cytes
and Langer-
haus’
cells
3
macro-
macrophages
+ + + ,
+ +
Epithelioid
Epithelioid
+.
disease
2
.
Epithelioid
cells
staining.
Frozen and paraffin sections from
human
tissues were stained
with monoclonal
antibodies
LN-4
and LN-5 by using goat antimouse
1gM, avidinbiotin complex,
immunoperoxidase-staining
reagents
(Vector Laboratories,
Inc. Burlingame,
CA) as described
by Hsu et al.#{176}
For these
a 1:2 or
was used.
LN- I and LN-2
Table
5.
Reactivity
Tumors
1:4 dilution
of LN-4
were run in parallel
of LN-4
in B5-Fixed,
and LN-5
of ovary
Adenocarcinoma
of colon
With
and rectum
-
-
3
-
-
Medullary
Tubular
of lung
carcinoma
carcinoma
of breast
Seminoma
Endometrial
Papillary
carcinoma
carcinoma
Anaplastic
carcinoma
Parathyroid
of thyroid
of
thyroid
adenoma
Renal cell carcinoma
Transitional
cell carcinoma
of kidney
Melanoma
Colonic
polyps
Granulocytic
Glioblastoma
Variable
both
cases.
staining
in one
case,
glandular
of
of the
in
the
-
-
-
-
shown
2
-
2
-
1
-
1
-
1
-
-
1
-
-
2
-
-
3
-
thymic
cells,
present
-
testinal
tract
-
shown).
B5-fixed,
normal
peripheral
but gave
interpret.
Reactivity
tissues.
1
-
-
human
tissues
-
-
biopsy
specimens
-
-
identify
-
-
tern.
in
In general,
stomach
Slight
where
positivity
LN-4
+ cells
cells
+.
distinctive
staining
were
marrow
LN-5+.
were
also
LN-4-
and
LN-5-positive
buffy
stained
+
As
.
liver
Macro-
.
and gastroin(data
coat
with
+
and
LN-5
as the lung
staining
therefore
LN-5
cell
such
were
Macro-
also
Langerhans’
were
reticulum
macrophages
not
pellets
from
and
LN-5
LN-4
and were difficult
excluded
from Table
to
I.
of LN-4
and LN-5
with normal
nonlymphoid
shown
in Table
2, a large
panel
of normal
-
positive
to contain
in Fig 2. In
the only
organs
blood
-
their
medulla
bone
5, skin
also
in reactive
but Langerhans’
liver were LN-4
a very
gave
seen
center,
interdigitating
Dendritic
cells and
background
data were
-
As
of
were
of the germihistiocytes.
In
were
paraffin-embedded
high
These
Because
specimens
was found
as shown
were
respectively,
were
on nor-
nodes and tonsils.
Strong
reactivity
zone B cells,
tingible
body
macro-
and
in other
1
strongly
results.
were LN-4-.
cells of the
and
4 and
-
secretions
LN-5
tissues.
Initial
tissues
fixed with
biopsy
cells
3, LN-5
cortex
phages
to + +
and
inconsistent
macrophages
spleen
Figs
Kuppfer’s
-
deparafanalysis
B5 formalin.
in
the spleen
macrophages
germinal
histiocytes.
1
1
2
Astrocytoma
the
sinus
-
7
2
sarcoma
of
-
Trace
In contrast,
of LN-4+
in Fig
-
to + + +
with
Isotypic
of LN-4
B5-fixed
marrow
cells
and Kuppfer’s
-
Trace
fixed
few antigen-positive
cortical
and
the
spleno-
,
in reactive
lymph
seen in the mantle
cells,
phages
1
of breast
very
phages
1
of breast
pattern
LN-5.
by
As shown
in Table
I and Fig I the
reactivity
of LN-4
in human
lymph
staining
the thymus,
was
2
BALB/c
hematopoietic
indicated
that
and
only
experiments.
nodes.
numbers
lymph
seen. Bone
of the skin
Solid
variable
As shown
1
of lung
carcinoma
to
and
node and tonsil included
tingible
macrophages
nal center,
sinus histiocytes,
and epithelioid
Sections
LN-5
of stomach
cell carcinoma
Human
LN-4
Adenocarcinoma
Ductal
Selected
controls
nodes
and
LN-5
findings,
used in these
predominant
large
supernatants
n
Adenocarcinoma
Small
LN-5
as positive
Paraffin-Embedded
Diagnosis
Adenocarcinoma
and
lymph
lymphoid
LN-4 and
early
general,
and
produced
hyperimmunized
blood
cells.
reactivity
gave
these
formalin-fixed
experiments,
of reactive
formalin
+ + +,
Gaucher’s
cells
cells
LN-4
were
both clones
are of the 1gM heavy-chain
submonoclonal
antibodies
were identified
by immuscreening
procedures
using paraffin-embedded
ma! human
studies
with
histiocytes
LN-5
NS-l
from a mouse
peripheral
Immunoperoxidase
Immunoperixodase
B5
that
sections
+ ,
Gaucher’s
revealed
and
myeloma
obtained
human
antibodies
LN-4
mouse
Both
noperoxidase
cytes
Gaucher’s
of
class.
histiocytes
monoclonal
clones
finized
+.
histio-
of
Hybridoma
+ +,
obtained
from
stained
were
staining
LN-4
reactivity
had
one
the
acini
were
by
LN-4
was
B5-fixed,
paraffin-embedded
with
LN-4
outside
major
site
the
and
LN-5
lymphoid
of reactivity
to
sys-
in the
strongly
positive
(Fig
6).
also found
in the tubular
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1082
BHOOPAT
ET AL
Figs 1 -1 5.
(1 1 Immunoperoxidase
staining
reactivity
of monoclonal
antibodies
LN-4 and LN-5 on B5-fixed,
paraffin-embedded
tissue
sections
(refers
to Figs. 1 to 1 5. all of which
were
counterstained
with hematoxylin).
LN-4 staining
of germinal
center.
tingible
body
macrophages
in hyperplastic
lymph node (original
magnification
x 221 ). (2) LN-4-stain
macrophages
in spleen
(original
magnification
x
514). (3) LN-5 staining of hyperplastic
lymph node showing
specific
reactivity
for tingible
macrophages
of the germinal
center.
mantle
zone B lymphocytes.
and interdigitating
reticulum
cells in between
follicles
(original
magnification
x 41 ). (4) LN-5-stained
Langerhans’
cell of the skin (original
magnification
x 1 .31 8). (5) LN-5-stained
Kuppfer’s
cells
in the liver
(original
magnification
x 658).
(6) LN-4-stained
acini of stomach
(original
magnification
x 130).
(7) LN-4-stained
malignant
cells of true histiocytic
lymphoma
(original
magnification
x 800).
(8) LN-4-stained
histiocytes
along collagen
bands in nodular
sclerosis
Hodgkin’s
disease
(original
magnification
x
45).
(9) Higher magnification
of LN-4-stained
histiocytes
in collagen
bands. Note that the Hodgkin’s
disease
cells at the left are unstained
(original
magnification
x 265).
(10) LN-5 staining
of ‘starry
sky” macrophages
in Burkitt’s
lymphoma
(original
magnification
x 814).
(1 1 ) LN-4-stained
macrophages
in reactive
hyperplastic
lymph node from patient
with persistent
generalized
lymphadenopathy
who was
infected
with the human immunodeficiency
virus (HIV) (original
magnification
x 708).
(12) LN-5-stained
histiocytes
in lymph
node
with
sinus histiocytosis
(original
magnification
x 640).
(1 3) LN-4-stained
sinus histiocytes
in lymph
node with dermatopathic
lymphadenopathy (original
magnification
x 129).
(14) IN-S-stained
Gaucher’s
cells and histiocytes
in the red pulp of the spleen
(original
magnification
x
446).
(1 5) LN-4 staining
of the cytoplasm
of epithelioid
histiocytes
and Langhans’
giant cells in a lymph node with tuberculous
granuloma
(original
magnification
x 280).
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
MONOCLONAL
ANTIBODIES
epithelium
the
of
kidney.
of breast
endometrial
hum,
Reactivity
leukemias,
LN-4
and
of LN-4
and
sections
from
diseases
are
negative
in
related
LN-5
with
myelogenous
that
ously
were
diagnosed
described
reactivity
of
cytic
lymphoma
also
found
areas.
in Fig
in
Burkitt’s
showed
no positivity
was positive
of
of
reactivity
lymph
tingible
are
body
indicating
and
LN-5
and
staining
benign
with
benign
monoclonal
reactive
in 30 to
lesions
15,
of
the
lesions
of
antibodies
histiocytes,
in
varying
with
most
the
staining
nomas
ofLN-4
and
and
the
stained
and
Table
5 along
tumor
type.
LN-5
was
brain
tumors.
with
LN-4
with
the
types
thus
of macro-
selected
human
data
did not show
any positivity
positivity
carcinoma
surface
and
of the breast
of the
secretions
in the
and
cases.
renal
cell
Fixed
and
live
ity
studied
cell
by using
rations
because
located
predominantly
used
with
These
an
internal
cell
cellular
reactiv-
and tonsils,
respect
thus
to cell
indicating
lymphoma
gives
results
that
are in contradiction
LN-4
could
with B5reactivities
find-
of this
when
paper;
designing
present
that
react
sections
antigens.
strongly
have
sinus
In general,
in germinal
and
pro-
LN-4
center
epithelioid
liver
are
has
hisnodes
tingible
staining
body
where
histiocytes
cytoplasmic
in
been
macrophages
and epithelioid
tissues.
In reactive
lymph
is positive
and
Langerhans’
cells,
macrophages
pattern.
it
Skin
Kuppfer’s
cells,
and
spleen
and
also positive,
as are macrophages
in nonlymphoid
In malignant
organs.
lesions,
LN-4
true
histiocytic
lymphomas
and collagen
banded
regions
in nodular
sclerosis
Hodgkin’s
disease.
Malignant
lymphomas
and
Hodgkin’s
disease
Reed-Sternberg
and
benign
cells
are
lymphoid
negative
lesions,
in all cases
LN-4
shows
of the
persistent
staining,
tubular
positive.
LN-4
epithelium
showed
of
the
some
LN-5
positivity
demonstrated
In
reactive
and
sinus
body
histiocytes,
In addition
weak
and
bone
of the
epithelioid
stained
mantle
than
epithelium,
and
stomach
zone
marrow;
normal
staining
skin
cells
in the
B cells
LN-4.
ductal
gla nds,
were
all
positive
reacstained
center,
mantle
reticulum
cells, sinus
In other
lymphoid
and macrophages
of the
spleen,
and macrophages
Langerhans’
Breast
of
carcinomas.
germinal
histiocytes.
dendritic
endometrial
acini
macro-
with
with a wide selection
although
one might
cells
pfer’s cells were also LN-5-positive
like
topoietic
tissues,
LN-5
had a somewhat
staining
to tissue
strong
stomach
interdigitating
and
lymphade-
reactivity
and
certain
macrophages
LN-5
histiocytes
a wider range of hematopoietic
lymph
nodes and tonsils,
LN-5
B lymphocytes,
zone
in
cells show
epithelioid
in dermatopathic
kidney
with
Among
staining
Gaucher’s
cells and
stomach
acini. No reactivity
was found
of human
solid
tumor
malignancies,
of the
be
cells
strongly
are
phage
antigen
subtypes
by this
to the immunope-
reticulum
studied.
prominent
germinal
center
in lymph
generalized
lymphade-
lesions
in tuberculosis
interdigitating
thymus,
not
using
and
secondary
information
tissue
a predominantly
prepa-
of most cell lines tested.
types was identified
by this
or leukemia
this
macrophage
LN-4
macrophages
tissues,
in the nucleus
to distinguish
method.
LN-4
primary
antibodies
for phagocytic
in hematopoietic
tingible
studies.
methods.
method
LN-4
reagents.
monoclonal
to human
tiocytes
carci-
LN-4 and LN-5 on
leukemia
cell lines
paraformaldehyde-fixed
it recognized
of analysis,
method
immunofluorescence
by immunofluorescence
was assessed
No specificity
indirect
of monoclonal
antibodies
human
lymphoma
and
these
paraffin-embedded
tivity.
The reactivities
40 established
are
the
to know
with
novel
expect
luminal
noma.
were
Two
B5-fixed,
nopathy
in
each
but did show
specimens
of
(particularly
glands)
paraformaldehydeare quite different
focus
not
of
DISCUSSION
presented
in these
negative
in the majority
of cases
with variable
intensity
in B5-fixed
tubular
studies
histiocytes
for
after
results
nopathy
in HIV-positive
individuals.
weak
binding,
but Langerhans’
giant
of LN-4
studied
the cell surface
cell lines but failed
to
cell lines. LN-5
showed
immunofluorescence
it is useful
future
lesions, these
carcinomas,
are
of cases
with
are
tingible
body macrophages
nodes
from patients
with
card-
the specificity
These
number
as such,
mononuclear
epithelioid
degrees,
and malignant
histiocytic
to stain a variety
of human
and LN-5
in benign
reagents
were used
sarcomas,
LN-5
To determine
sarcomas.
by indirect
and,
ings
one
stains
subsets.
Reactivity
shown
specificity
but not
histio-
cells
these
it appears
antigens,
fixation
and another
paraffin-embedding
and
to react
with
these
Like LN-4,
thymic
I I to
cells
are
LN-5
fibrous
found
data,
were initially
discovered
by their reactivity
paraffin-embedded
tissues,
these
new
LN-5
fixed,
duced
the “starry
samples
but
was
these
different
obtained
by the immunoperoxidase
paraffin-embedded
tissues.
Because
however,
in the
lymph
seen
pattern
Figs
sinus
Gaucher’s
they
of
as
tumor.
with
LN-5
Both
macrophages,
that
stained
staining
the
4 and
shown.
and
histiocytes,
phage
LN-4
LN-4
node
positive
lymphoma
throughout
In Table
node.
10, LN-5
a speckled
scattered
Reactivity
lymph
The
two
were previ-
From
distinctly
lymphoma
and leukemia
the large cell lymphoma
from those
B5-fixed,
chronic
with true histiobut not LN-5
was
with the lymphoma
cells.
in both cases of malignant
it gave
where
of cells
with
no reactivity
acetone
fixation.
were
earlier.
two
by paraformaldehyde
by B5 fixation
LN-5
react
were negative
for LN-4
despite
histiocytes
in the surrounding
As shown
macrophages
50%
cells
of the
is preserved
is revealed
and related
on the basis
findings
et al.52 The
that
that
B cell
and
enzymatic
detailed
recognizes
most
non-Hodgkin’s
lymphomas
and
studies
LN-4
procedures.
LN-5
acute
roxidase
that
reactivities
tissue
stain
a macrophage-like
cell found
of nodular
sclerosis
Hodgkin’s
disease
in Fig 8. Under
the higher
magnification
to
sky”
cytoma
histiocytic
by Thomas
in Fig 9, the Hodgkin’s
the strong
positivity
LN-4
disease,
with one of the patients
is shown
in Fig 7. LN-4
LN-4
bands
as shown
sclerotic
and
mycosis
fungoides.
and LN-5-positive
cytochemical,
previously
LN-4
exceptions,
and
as true
leukemia,
Hodgkin’s
LN-4-
the
weak
lymphomas,
The staining
paraffin-embedded
3. Both
notable
both
morphological,
nodes
of
two
stain
to have
human
lymphoma,
leukemias,
cases
with
B5-fixed,
in Table
cases
all
LN-5
with
shown
lymphomas
collagen
with
to
and
kidney
tubular
epithecells of the stomach.
diseases.
patients
found
testes
epithelium,
and chief
and
1083
LN-5
was
of the
ductal
glands,
of
AND
LN-5
of spermatogonia
cytoplasm
staining
IN LN-4
and
LN-4.
wider
epithelium,
testicular
liver
Kup-
In nonhemaspectrum
of
renal
tubular
spermatogonia,
in varying
degrees.
In
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1084
BHOOPAT
malignant
diseases
strongly
histiocytic
The
of LN-5
diseases
binding
that
LN-4.
tumors,
was
variable
Unlike
-3,
showed
similar
selection
types
of
renal
different
or
paraffin
sections.
nancies,
LN-4
staining
pattern
tissues,43’
cell
With
a broad
gave
preparations.
a
in
patterns
or
histiocytic
believed
carcinoma
methods,
LN-5
showed
that
showed
LN-5
LN-5
with
Because
related
made
antigens
by standard
immunobiochemical
more difficult
and may depend
on the
and their
of
this,
identification
of
the
to
be
bodies.
Finally,
phage
infiltrates
ing those
prognostic
cytoplasmic
cell
no reactivity.
other
lesions
from
diseases.
histiocytosis
the
are
reactivity
as specific
material
histopathologist.
were
LN-4disorder
may
histiocytic
such as
derived
the
warranted
of these
reagents
for
in inflammation
and
serniquantitative
antibody
markers
Further
X,
B
in
macrophage-
image
to
tissue
paraffin-embedded
analysis
systems
cell,28
identifying
these studies.
Along
with
further
delineate
antigenic
and help
processes
is
and
further
antimacro-
malignancies,
LN-4
cells in
includ-
et a154 have studied
the
in tumors
by using
that
sections
may
studies
which
and will
monoclonal
The availability
macrophages
techniques.
Of
and
LN-5be antigeni-
Langerhans’
of Milas et a153 and Luebber
significance
of macrophages
reactivity
with
and certain
B lymphomas
on the cell surface.
and LN-5
antigens
in B5-fixed,
paraffin-embedappear
to be different
in these other cell prepara-
tions.
from
pathological
for
and LN-5 may be used to investigate
the role of these
infection
and tumor
immunity.
Several
investigations
malig-
reacted
processed
reagents
malignant
histiocytosis25’27
help to characterize
the
LN-1,
in B5-
of human
routinely
true histiocytic
lymphoand benign
lesions
of the
Hodgkin’s
disease
cells
which
indicates
that this
distinct
formaldehyde-acetone-fixed
however,
valuable
them
with
and
occasional
in
makes
cally
compared
spectrum
macrophage
LN-5
in paraformal-
preparations
nuclear
para
By these
studies,
staining
unfixed
LN-4
to identify
positively
as other
malignant
of solid
carcinomas,
cell
Their
ability
mas as well
interest,
negative,
lesions
with
studies
with monoclonal
antibodies
retained
their
immunoreactivity
distinctly
lymphocytes
The LN-4
ded tissues
and
lymphoid
a wide
two
had
reactive
of the breast
and
was observed.
which
dehyde-fixed
cell
benign
in
paraffin-embedded
fixed,
LN-5
more
among
histiocytorna.
staining
previous
and
in
were
true
lymphornas
elsewhere.
positive
carcinoma
where
Live
Finally,
LN-5
tubular
cells
zone
was
LN-5
namely,
fibrous
mantle
LN-4
to
system,
of tumors,
malignant
with
Gaucher’s
with
types
will be reported
reactivities
except
-2,
and
lyrnphoma
related
hematopoietic
two
in only
reactivity
than
the
of
positive
ET AL
greatly
of monoclonal
retain
their
and
enhance
the
the
use
of
value
of
LN-1,
-2, and -3, LN-4
and LN-5
subsets
in the lymphoid
system
to identify
the cell of origin
in these tissues.
of benign
and malignant
methods
is
source
of cells
ACKNOWLEDMENT
fixation.
For the immunodiagnosis
LN-5
are unique
markers
of lymphoid
lesions,
of the macrophage
and
LN-4
and
histiocyte.
The authors wish to thank Helen
their expert technical
assistance.
Salwen
and Lillian
Flanagan
for
REFERENCES
1. Radzun
HJ,
Parwaresch
Hansmann
ML:
Ki-M 1 recognizes interdigitating
reticulum
cells. Am J Pathol I 17:441, 1984
2. Wright
SD, Rao PE, Van Voorhis WC, Craigmyle
LS, lida K,
Talle MA, Westberg
EF, Goldstein
C, Silverstein
SC: Identification
of the C3bi receptor of human monocytes
and macrophages
by using
monoclonal
antibodies.
Proc NatI Acad Sci USA 80:5699,
1983
3. Ugolini
V, Nunez G, Smith
RG, Stastny
P. Capra JD: Initial
characterization
of monoclonal
antibodies
against
human
monocytes. Proc NatI Acad Sci USA 77:6764,
1980
4. Adreesen
R, Bross KJ, Osterholz
J, Emmrich
F: Human
macrophage
maturation
and heterogeneity:
Analysis
with a newly
generated
set of monoclonal
antibodies
to differentiation
antigens.
Blood 67:1257,
1986
5. Zwadlo
G, Brocker EB, von Bassewitz
DB, Feige U, Sorg C: A
monoclonal
antibody
to a differentiation
antigen
present on mature
human
macrophages
and absent
from monocytes.
J Immunol
Monocyte/macrophage-specific
134:1487,
MR.
Feller
monoclonal
AC,
antibody
1985
6. Naiem
M, Gerdes
J, Abdulaziz
Z, Stein H, Mason
DY:
Production
of a monoclonal
antibody
reactive with human dendritic
reticulum
cells and its use in the immunohistological
analysis
of
lymphoid
tissue. J Clin Pathol 36:167,
1983
7.
Parwaresch
MR.
Radzun
HJ,
Hansmann
ML,
Peters
KP:
Ki-M4
specifically
recognizes
human
dendritic
reticulum
cells (follicular
dendritic
cells) and their possible
precursor
in blood. Blood 62:585, 1983
8. Van Voorhis
WC, Steinman
RM, Hair LS, Luban J, Witmer
MD,
Koide 5, Cohn ZA: Specific
antimononuclear
phagocyte
monoclonal
antibodies.
Application
to the purification
of dendritic
cells and the tissue localization
of macrophages.
J Exp Med 158:126,
Monoclonal
1983
antibody
9. Knowles DM, Tolidjian
B, Marboe C, D’Agati
V, Grimes
M,
Chess L: Monoclonal
anti-human
monocyte
antibodies
0KM 1 and
OKM5
possess
distinctive
tissue distributions
including
differential
reactivity
with vascular
endothelium.
J Immunol
I 32:21 70, 1984
10. Koestler
TP, Rieman
D, Muirhead
K, Grieg RG, Poste G:
Identification
and characterization
of a monoclonal
antibody
to an
antigen
expressed
on activated
macrophages.
Proc NatI Acad Sci
USA
81:4505,
I 1. Weiss
6-expressing
1984
Beckstead
JH, Warnke
RA, Wood
GS: Leuin lymph nodes: Dendritic
cells phenotypically
similar
to interdigitating
cells. Hum Pathol 17:179, 1986
12. Hancock
WW, Rickles FR, Ewan VA, Atkins RC: Immunohistochemical
studies with A I -3, a monoclonal
antibody
to activated
human
LM,
cells
monocytes
and macrophages.
J Immunol
136:2416,
1986
GS, Turner
RR, Shiruba
RA, Eng L, Warnke
RA:
Human dendritic
cells and macrophages:
In situ immunophenotypic
definition
of subsets that exhibit specific morphologic
and microenvironmental
characteristics.
Am J Pathol I 19:73, 1985
14. Franklin
WA, Mason
DY, Pulford
K, Falini B, Bliss E,
Gatter
KC, Stein H, Clarke LC, McGee JOD: Immunohistochemical analysis of human mononuclear
phagocytes
and dendritic
cells by
using monoclonal
antibodies.
Lab Invest 54:322, 1986
I 5. Hofman
FM, Lopez D, Husmann
L, Mayer PR, Taylor CR:
Heterogeneity
of macrophage
populations
in human lymphoid
tissue
and peripheral
blood. Cell Immunol
88:61, 1984
16. Guettier
C, Gatter
KC, Heryet
A, Mason
DY: Dendritic
reticulum
cells in reactive
lymph nodes and tonsils: An immunohis13.
tological
Wood
study.
Histopathology
10:1 5, 1986
Van Voorhis WC, Witmer
MD,
type of dendritic
cells and macrophages.
18. Van der Valk P. Van der Loo
17.
Steinman
RM: The phenoFed Proc 42:3 1 14, 1983
EM,
Jansen
J, Daha MR.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
MONOCLONAL
ANTIBODIES
IN
LN-4
AND
LN-5
1085
CJLM:
Analysis
of lymphoid
and dendritic
cells in human
node, tonsil, and spleen. Virchows Arch [Cell Pathol] 45:169,
Meijer
lymph
I984
19. Flotte TJ, Springer
TA, Thorbecke
GJ: Dendritic
cell and
macrophage staining by monoclonal antibodies in tissue sections and
epithelial
sheets. Am J Pathol
1 1 1:1 12, 1983
20. Todd
RF III, Schlossman
SF: Analysis
of antigenic
determinants
on human
monocytes
and
macrophages.
Blood
59:775,
1982
21 . Freemont
AJ, Matthews
5, Stoddart
RW, Jones CJP: The
distribution
of cells of the monocytic-lineage
in reactive lymph nodes
and non-Hodgkin’s
lymphomata.
Characterization
using protein
histochemistry,
lectin binding,
and monoclonal
antibodies.
J Pathol
146:139, 1985
22. Hsu SM: Phenotypic
expression
of cells of stationary
dcments in human lymphoid
tissues. A histochemical
and immunohistochemical
study. Hematol
Pathol I :45, 1987
23. Turner RR, Wood
GS, Beckstead
JH, Colby TV, Horning SJ,
Warnke RA: Histiocytic
malignancies:
Morphologic,
immunologic,
and enzymatic
heterogeneity.
Am J Surg Pathol 8:485, 1984
24. Gatter KC, Morris
HB, Roach B, Mortimer
P, Fleming KA,
Mason DA: Langerhans’
cells and T cells in human skin tumors: An
immunohistochemical
study. Histopathology
8:229, 1984
25. Meister
P. Huhn D, Nathrath
W: Malignant
histiocytosis:
Immunohistochemical
characterization
on paraffin
embedded
tis-
sue. Virchows Arch [Al 385:233,
1980
26. Isaacson P, Jones DB, Sworn MJ, Wright
DH: Malignant
histiocytosis
of the intestine:
Report of three cases with immunological and cytochemical
analysis. J Clin Pathol 35:510, 1982
27. Ducatman
BS, Wick MR. Morgan
TW, Banks PM, Pierre
RV: Malignant
histiocytosis:
A clinical, histologic, and immunohistochemical study of 20 cases. Hum Pathol 15:368, 1984
28. Beckstead
JH, Wood GS, Turner
RR: Histiocytosis-X
cells
and Langerhans’
cells: Enzyme
histochemical
and immunologic
similarities.
Hum Pathol 15:826,
1984
29. Daum GS, Liepman
M, Woda BA: Dendritic
cell phenotype
in localized
malignant
histiocytosis
of the small
intestine.
Arch
Pathol Lab
30.
Unnik
Med
109:647,
1985
Van Heerde PV, Feltkamp
J, Vroom
TM: Malignant
CA, Hart
histiocytosis
clinicopathologic
study
and ultrastructural
3 1 . Beckstead
parameters.
Hematol Oncol
JH: The evaluation
of human
and enzyme histochemistry.
plastic
sections
80:131,
1983
of 42 cases using
AAM, Somers R, Van
and related tumors. A
cytological,
histochemical,
2:13, 1984
lymph nodes using
Am J Clin Pathol
Mason DY, Taylor
CR: The distribution
of muramidase
(lysozyme)
in human
tissues. J Clin Pathol 28:124, 1975
33. Motoi M, Stein H, Lennert
K: Demonstration
of lysozyme,
alpha- I -antichymotrypsin,
alpha-I
-antitrypsin,
albumin,
and transferrin
with the immunoperoxidase
method in lymph node cells.
Virchows Arch [Cell Patholl 35:73, 1980
34. Krugliak
L, Meyer
PR, Taylor
CR: The distribution
of
lysozyme,
alpha-1-antitrypsin,
and alpha-1-antichymotrypsin
in
normal hematopoietic
cells and in myeloid leukemias: An immunoperoxidase
study on cytocentrifuge
preparations,
smears, and paraffin sections. Am J Hematol 21:99, 1986
35. Mendelsohn
G, Eggleston
JC, Mann RB: Relationship
of
32.
lysozyme
(muramidase)
to histiocytic
differentiation
Cancer 45:273, 1980
36. Kahn HJ, Marks A, Thom H, Baumal
5100 protein
in diagnostic
pathology.
Am
1983
in malignant
histiocytosis.
R: Role of antibody
to
J Clin Pathol 79:341,
Demonstration
of alpha-1-antitrypsin
and
in fibrous histiocytomas
using the immunoperoxidase
technique.
Am J Surg Pathol 6:559, 1982
38. Nemes Z, Thomazy
V, Adany R, Muszbek
L: Identification
of histiocytic
reticulum
cells by the immunohistochemical
demonstration
of factor XIII (F-XIIIa)
in human lymph nodes. J Pathol
149:121, 1986
39. Crocker J, Burnett
D, Jones EL: Immunohistochemical
demonstration
of cathepsin
B in the macrophages
of benign and malignant lymphoid tissues. J Pathol 142:87, 1984
40. Lam KW, Li CY, Siemens M, Yam LY: Immunohistochemical detection
of monocytes
by the antiserum
specific to monocytic
esterase. J Histochem
Cytochem
33:379, 1985
41. Van Unnik JAM: The application
of the metalophil
method
for the demonstration
of histiocytes.
J Pathol 144:233,
1984
42. Van Unnik
JAM, Dc Weger RA: Metalophil
histiocytes
in
non-Hodgkin’s
lymphomas
particularly
in malignant
proliferations
of B-lymphocytes.
J Pathol 144:241
I 984
43. Epstein
AL, Marder
RJ, Winter
JN, Fox RI: Two new
monoclonal antibodies (LN-l,
LN-2)
reactive in B5 formalin-fixed,
37.
du
Boulay
CEH:
alpha-1-antichymotrypsin
,
paraffin-embedded
tissues
with
follicular
center
and
mantle
zone
human B lymphocytes
and derived
tumors.
J Immunol
133:1028,
1984
44. Marder
RJ, Variakojis
D, Silver J, Epstein
AL: Immunohistochemical
analysis
of
human
lymphomas
with
monoclonal
anti-
to B cell and Ia antigens
reactive
in paraffin sections.
Lab
Invest 52:497, 1985
45. Okon
E, Felder
B, Epstein
AL, Lukes
RJ, Taylor
CR:
Monoclonal
antibodies
reactive with B-lymphocytes
and histiocytes
in paraffin
sections. Cancer 56:95, 1985
46. Sherrod
AE, Felder B, Levy N, Epstein
AL, Marder
RJ,
Lukes RJ, Taylor CR: Immunohistologic
identification
of phenotypic antigens associated
with Hodgkin and Reed-Sternberg
cells: A
paraffin study. Cancer 57:2135, 1986
47. Samoszuk
MK, Epstein
AL, Said J, Lukes RJ, Nathwani
BN: Sensitivity
and specificity
of immunostaining
in the diagnosis
of
mantle zone lymphoma.
Am J Clin Pathol 85:557, 1986
48. Frazekas
de St Groth
5, Scheidegger
D: Production
of
monoclonal
antibodies.
Strategy
and tactics.
J Immunol
Methods
bodies
35:1, 1980
AL, Kaplan
HS: Feeder
layer and nutritional
for the establishment
and cloning of human malignant
lymphoma
cell lines. Cancer Res 39:1748,
1979
50. Hsu SM, Raine L, Fanger
H: The use of avidin-biotinperoxidase
complex
(ABC)
in immunoperoxidase
techniques:
A
comparison
between ABC and unlabeled
antibody
(PAP)
procedures. J Histochem Cytochem
29:577, 1981
51. Winter JN, Variakojis
D, Epstein AL: Phenotypic
analysis of
established
diffuse histiocytic
lymphoma
cell lines utilizing
monoclonal antibodies
and cytochemical
techniques. Blood 63:140,
1984
52. Thomas
P. Said JW, Rosenfelt
FP, Heifetz U: True histiocytic
lymphoma:
An immunohistochemical
and ultrastructural
study of two cases. Am J Clin Pathol 81:243, 1984
53. Milas L, Wike J, Hunter
N, Volpe J, Basic I: Macrophage
content
of murine
sarcomas
and carcinomas:
Associations
with
tumor
growth
parameters
and tumor radiocurability.
Cancer
Res
47:1069, 1987
54. Luebbers
EL, Pretlow TP, Emancipator
SN, Boohaker
EA,
Pitts AM, Macfadyen
AJ, Bradley EL Jr. Pretlow TG II: Heterogeneity
and prognostic
significance
of macrophages
in human
colonic carcinomas.
Cancer
Res 45:5196,
1985
49.
Epstein
requirements
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1988 71: 1079-1085
Immunohistochemical characterization of two new monoclonal antibodies
(LN-4, LN-5) reactive with human macrophage subsets and derived
malignancies in B5-fixed, paraffin-embedded tissues
L Bhoopat, RR Turner, E Stathopoulos, PR Meyer, CR Taylor, RJ Marder and AL Epstein
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