From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
Abnormal
Distribution
of Complex
Patient
By Richard
Previous
ity
to
studies
have
bacterial
T.
identified
infection
With
Parmley,
Lactoferrin
Diana
patients
associated
with
with
Carbohydrates
Y. Tzeng,
Robert
susceptibil-
lactoferrin
defi-
ciency in dysmorphic
neutrophils
containing
abnormal
or
no secondary
granules
and abnormal
nuclear
segmentation. We have investigated
the subcellular
distribution
of
vicinal glycol-containing
complex
carbohydrates
in marrow
and blood myeloid cells of such a patient
using the periodic
acid-thiocarbohydrazide-silver
proteinate
(PA-TCH-SP)
staining
method
and have examined
the response
of these
neutrophils
to the degranulating
agents
N-formylmethionyl-leucyl-phenylalanine
(FMLP)
and phorbol
myristate
acetate
(PMA).
As in normal specimens.
immature
primary
granules
were
strongly
PA-TCH-SP
reactive;
however.
unlike normal specimens.
masking of PA-TCH-SP
reactivity
did not occur
in mature
primary
granules.
Endoplasmic
reticulum
demonstrated
moderately
strong
PA-TCH-SP
staining.
in contrast
to absent staining
of this organelle
in
normal
promyelocytes
and consistent
with abnormal
primary granule
genesis.
Small abnormal
elongated
granules
(0.1
-0.2
zm
in diameter)
S
EVERAL
patients
and
neutrophil
secondary
were
identified
INVESTIGATORS
with susceptibility
dysfunction
or specific
at
the
and
abnormal
morphoin peroxiof these
L. Baehner,
and
ules.
Light
microscopic
studies
demonstrated
absence
or decrease
in alkaline
phosphatase’3
lactoferrin
staining.3
Alkaline
phosphatase
may
rics
the
and
Institute
Pathology,
Birmingham,
the
AL;
Riley
Hospitalfor
cine,
Indianapolis,
Children’s
the
IN;
Hospital,
in
an
and
not be
granules,
since,
in
been localized
in a
HD-l4132,
of
Submitted
Address
© I 983
A.
B.
October
reprint
Alabama
Department
National
to
the
interpretation
killing
ability
appeared
Recently,
Boxer
et al.8
of
an
This
of Medi-
Arbor,
of
work
Established
Whitcomb
Pediatrics,
Ann
Birmingham.
by Grune
University
AL
accepted
to Dr.
Richard
Mott
MI.
Health
was
Investigator
ofAlabama
March
done
during
of
the
in Birmingham,
distribution
are
present
in normal
light
studies.”2
that
not
neutrophils
abnormalities
might
exist.
from such
in complex
Our
numbers,
but
of FMLP
binding
of
are
unrecog-
their
abnormal
can be induced
to
acetate
(PMA),
sites
is altered.
University
The
clinical
previously.’8
a
ultrastruc-
of
MATERIALS
Inc.
and
decreased
in some
have
demonstrated
nizable
morphologically
because
appearance.
Although
these granules
degranulate
with phorbol
myristate
35294.
& Stratton,
biochemical
demonstrate
undescribed
abnormalities
in
of vicinal
glycol-containing
complex
in primary
and
secondary
granules.
the studies
indicate
that secondary
gran-
the recruitment
Institute
of
In this study,
we examined
patient
to determine
whether
ules
Grants
23. 1983.
T. Parmley,
Boxer
only chemotaxis,
but also adherence,
aggregation,
and
the ability
to decrease
cell surface
charge
after
Nformylmethionyl-leucyl-phenylalanine
(FMLP)
stimulation
were diminished
to some extent.
tural studies
the packaging
carbohydrates
Furthermore,
Birmingham,
School
Institutes
AI-20065.
as
in
James
University
ofMichigan.
4, 1982;
requests
0006-4971/83/6203-0004$02.OO/O
538
of
of Pediat-
Association.
Research,
Station.
the
and
L.
Heart
by
Departments
of Pediatrics,
Indiana
and
part
the
American
University
University
DE-02670,
tenure
Research,
Department
Children,
Supported
Dental
of Dental
A.
microscopic
data,
indicating
that lactoferrin
is a component
of secondary
granules.6
It was also reported
that the chemotaxis
of neutrophils
from these patients
seemed
to be impaired.”2
In addition,
the bacterial
carbohydrate
From
Laurence
state of development
and were the predominant
granule
type in late neutrophils.
These granules
were identified
as
secondary
granules
on the basis of their PA-TCH-SP
positivity and were
differentiated
from primary
and tertiary
granules
on the basis of a lack of peroxidase.
acid phosphatase.
and sulfate
staining.
When
the neutrophils
were
exposed
to PMA. cell aggregation
occurred.
and the abnormal granules
degranulated
in a manner
similar
to the
degranulation
observed
with normal
secondary
granules.
Although
PA-TCH-SP
staining
of the plasma
membrane
appeared
normal. a decrease
in FMLP receptors
was demonstrated.
Thus. a defect(s)
is present
in complex
carbohydrate distribution
and staining
that involves
primary
and
secondary
granules
and possibly the plasmalemma
of neutrophils from this patient.
This results in abnormal
packaging of primary granules
and synthesis
of normal numbers
of
secondary
granules
that are qualitatively
and morphologically abnormal.
but can be recruited
to degranulate
with
PMA.
trast
vesicle-like
granappearance
at
Strauss
et al.’
secondary
gran-
a definitive
marker
of secondary
humans,
alkaline
phosphatase
has
Deficiency
vesicle
fraction
distinct
from
secondary
granules.5’6
Similarly,
ultrastructural
immunocytochemical
and
iron
binding
studies
have
localized
lactoferrin
in
mature
primary
granules
in our laboratory,7
in con-
nuclear
lobulation.’
Primary
or azurophilic
granules
logically
appeared
to be normal
or increased
dase-stained
specimens.
The
neutrophils
patients
contained
abundant
small
ules. On the basis of their morphological
the myelocyte
stage
of development,
hypothesized
that they were abnormal
of a
myelocyte
have now reported
to bacterial
infections
associated
with
absent
granules
in Neutrophils
data
regarding
A decrease
Blood,
AND
the patient
in cytoplasmic
METHODS
studied
have been reported
lactoferrin
was documented
Vol. 62, No. 3 (September),
1983:
pp. 538-548
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
LACTOFERRIN
DEFICIENCY
using
an immunoperoxidase
Rare
neutrophils
539
method
from
at the
the patient
light
microscope
demonstrated
normal
level.8
lactoferrin
Mg-free
HBSS
studies
content.
were
Gallin,’7
(mHBSS)
carried
with
minor
To measure
Ultrastructural
Blood
and
syringe
marrow
specimens
by venipuncture
crest,
after
marrow
samples
specimens
and
were
healthy
centrifuged
removed,
the
minced,
buffer,
pH
cacodylate
sucrose
buffer,
method
performed
were
used as controls.
coat
for
phosphate,
in
by
Spicer.”
Specimens
phatase,
and
whereas
specimens
37#{176}C
to a 275
solution
Sigma
Chemical
buffer
(pH
7.0)
All
specimens
previously.’2
and
to remove
propylene
oxide
and
acid
of
phos-
in cacodylate
HID
processed
or
exposed
from
Some
for
porcine
3.2.1.1,
Louis,
for
not osmicated.
were
type
MO)
cytoplasmic
were
to the method
were
EC
of
of specimens
1% 0504
of ct-amylase
St.
was
f3-glycero-
morphology,
in
glycols
Company,
to the
staining
containing
with
(1,4-a-D-Glucan-glucanohydrolase;
according
Anderson.’#{176} Staining
glycols
for vicinal
U/mI
was accomplished
according
stained
in
for routine
by incubation
routine
ofvicinal
coats
0. 1 M
phosphatase
postfixed
specimens
to be stained
alcohols
staining
and
the
rinsing
processed
medium
for
were
The
removing
glutaraldehyde,
were
solution,
processed
staining
Normal
the buffy
solution,
Barka
(HID)
peroxidase
postembedment
phate
3%
was performed
diamine
After
fixative,
Acid
in substrate
as described
patient.
I hr at 4#{176}C-lO#{176}C.
After
Karnovsky.9
glycoconjugates
in a high-iron
mg,
with
Peroxidase
by incubation
buffer,
volunteers
the specimens
and
iliac
the
in a 3,3’-diaminobenzidine
of Graham
of the posterior
from
fixed
7.35,
or cytochemistry.
sulfated
aspiration
3 hr at
pancreas
1-A,
550
in 0.09
glycogen,
M
U/
phos-
as described
routinely
dehydrated
embedded
in Spurr
in
graded
low-viscosity
medium.
Thin
sections
amylase
for
of
digestion,
vicinal
silver
glycols
proteinate
previously
using
unosmicated
were
specimens,
collected
according
to the
(PA-TCH-SP)
for
marrow
TCH-SP,
HID
as described
or
steel
without
grids
a-
and stained
periodate-thiocarbohydrazide-
method
cells.’4
with
on stainless
was similarly
180
ofPhorbol
To examine
were
for
degranulation,
incubated
(PMA)
10 mm
presence
enzyme,
the
lactic
Cells
1 5 mm
nM)
were
or
control).
(55.6
Ci/mM)
San
Carlos,
3H-FMLP
sules
Labs,
mine
nonspecific
bath
for
4.5
washed
twice
dissolved
Measurement
The
ionophore
Robert
were
and
exposure
into
sucrose
as described
oJFMLP
assayed
the
to PMA,
pellets
for
the
cytoplasmic
butTer,
and
and
and
5 and
fixed
in 3%
processed
for
above.
Receptors
polymorphonuclear
were
salt
cells
and
A23l87,
were
incubated
with
lated
and control
precipitates
(HBSS)
incubated
Reversibility
Lilly
cells
were
and
HBSS
were
obtained
were
and
only.
(kindly
Indianapolis,
After
centrifuged
washed
30 mm
stimulus
Co.,
twice
from
washed
resuspended
at 37#{176}C
for
a degranulating
Eli
(PMNs)
patient8’5
solution
then
Hamill,
The
acetate
at 400 g
centrifuged
Binding
buffered
cells
myristate
then
fluids
centrifuged
or a lactoferrin-deficient
Hanks’
phorbol
were
before
were
examination
of3H-FMLP
to
of
both
at 350 g for
10 mm
resuspended
with
by Dr.
Control
the incubation,
and
with
in HBSS
cells
stimuat 4#{176}C.
in Ca-
FMLP,
tosyl-L-phenylalanyl
San Diego,
with
at varying
1 ml
(5
x
Ca)
and
at l0
were
The
shaking.
ice-cold
HBSS,
HBSS
cell
suspension
the
FMLP
then
The
the
The
to deter-
incubated
assay
and
was
cell
in an ice
terminated
by
suspensions
precipitates
and radioactivity
above
(Penni-
simultaneously
were
(5 ml each).
in 0.5 ml of H20,
of
(10-
containing
assayed
En-
concentrations
nonradioactive
cells
M and
New
106 cells)
105M
CA)
final
samples
constant
For
the
reversibility
of
binding)
and
3H-FMLP
binding)
were
added
ice bath,
the cell
start
were
were
was counted
40 p.M
melting
triplicate
bath
binding
excess
of
finally
in 10 ml of
aliquots
of
cells
binding
= total
binding
= total
binding
addition
of excess
specific
= residual
-
To displace
radioactivity
The
binding;
and
the addition
(3)
an
residual
compo-
total
specific
displaceable
radioactivity
FMLP;
the
washed,
different
(2)
surface-
triplicate
the
(I)
in a
points,
from
terminated,
above.
to
containing
determine
was
residual
after
the
intervals,
as follows:
-
nonradioactive
nonspecific
to
nonspecific
-
kept
time
set of aliquots
set of tubes
assay
obtained
binding
various
time
collected
as described
were
were
collected.
certain
In an
added
for 3H-FMLP
mixtures
a paralleled
The
counted
binding
40 nM
At
At
were
were
shaking.
to a separate
FMLP.
and
the
binding
added
radioactivity.
resuspended,
of
were
tubes.
mHBSS)
were
(total
(nonspecific
polypropylene
reaction
p.l) were
the cell,
these
cell-associated
The
3H-FMLP
FMLP
106/ml
concentrations
from
unlabeled
x
constant
(250
tube
50-ml
(5
FMLP.
with
3H-FMLP
nents
final
aliquots
bound
total
The
binding,
nonradioactive
to 2 separate
for cold
ice
3H-FMLP
plus
suspensions
the assay.
and
after
the
nondisplaceable
of nonradioactive
binding.
and
Microscopy
ultrastructural
disorder
has
morphology
been described
of the myeloid
previously.’3
cells
Elon-
gated
abnormal
small granules,
0.1-0.2
tm in diameter, were observed
with increasing
frequency
from the
myelocyte
to the band
neutrophil
stage
of development.
In band
and segmented
neutrophils
(Fig.
1),
these
granules
outnumbered
dense
primary
granules
by a ratio
of approximately
4: 1 . In morphological
preparations,
smaller
and
granules
in
these abnormal
granules
less dense
when
compared
normal
neutrophils.
The
contained
moderately
dense content,
sicular
structures,
which
were also
and normal
neutrophils.
in HBSS.
provided
IN).
healthy
twice
the
Duplicate
binding.
ml
and
Aquasol-2.
The
in this
of 5 x 106 cells/mI
lactoferrin,
0. 1 M cacodylate
morphological
Purified
tubes
protein,
immediately
glutaraldehyde,
donors
20 ng/ml
The
and binding
of Fletcher
RESULTS
dehydrogenase.’6
exposure
added
1 hr with
adding
MA)
enhanced
on Granules
consisting
the supernatant
granule
obtained
after
aliquots
15 mm.’5
at 4#{176}C,
and
of
Acetate
at 37#{176}C
with
for 5 and
Boston,
previously.’4
Myristate
for
Calbiochem,
(stimulated
Electron
Effect
Receptor
(formylmethionyl-leucyl-3H-phenylalanine;
Nuclear,
FMLP
of Thi#{233}ry,’3 as described
staining
gland
receptors
(TPCK,
3H-FMLP
in a heparinized
consent
buffy
and
cacodylate
by incubation
obtained
at 1,500 g for 3 mm.
overlaying
morphology
needle
informed
from
were
plasma
and
obtaining
were
assays.
to the procedure
modifications.
PMN
chloromethane
Cytochemistry
for binding
out according
was
Peroxidase,
performed
acid phosphatase,
to determine
the
appeared
much
to secondary
small
granules
unlike
present
and sulfate
relationship
tubulovein patient
staining
of the
abnormal
granules
to primary
and tertiary
granules.
Primary
granules
demonstrated
normal
peroxidase
(Fig.
2), acid
phosphatase,
and
sulfate
staining.
In
most primary
granules
of late neutrophils
or PMNs,
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
LACTOFERRIN
staining
masked
DEFICIENCY
541
of acid
phosphatase
and
(Fig. 3). From 1 to 4 tertiary
sulfate
granules
Primary
granules
stage of development
appeared
measur-
ing 0.1-0.3
tm in diameter
were stained
in profiles
of
late neutrophils
and appeared
normal
(Fig. 3). Positive
identification
of tertiary
granules
was possible
on the
basis
of their
acid phosphatase
and sulfate
staining
(Fig. 3) and ofGolgi
segmented
neutrophils
ate
not
staining
ticulum
in
and
contrast
specimens.
moderately
matrix;
patient
staining
The
granule
treated
with
aof glycogen
was
Golgi
to
ticulum,
reported
which
observed
despite
nuclear
stained
lamellae
normal
endoplasmic
in promyelocytes
promyelocyte
lacked
staining
Staining,
(Fig.
with this
however,
in the nuclear
envelope
with
the observed
peroxidase
reactivity
envelope
and the endoplasmic
promyelocytes.
The
matrix
and
ture
primary
granules
stained
The abnormal
small granules
myelocyte
stage of development
as
not
of imma-
tivity
in
structures
patient
were
phic,
were
and
abnormal
tacted
and
primary
7). Several
demonstrated
canaliculi
and
PA-TCH-SP
granules.
appeared
granules
and
to moderate
secondary
larger
than
normal
observed
fusing
vacuoles
or
cells were
membranes
abnormal
small grantime of exposure
to
the abnormal
small
with the
indentations
of
to
and appeared
to be a collection
(Fig. I 1 In specimens
exposed
of
for
These
vesicles
frequently
conto contact
or fuse with immature
1 5 mm, the small
were outnumbered
granules
were markedly
by the larger,
more
granules
12).
(Fig.
of promyelocytes
and
7) than
myelocytes.
plasmalemma
(Fig.
10).
observed
with an accumulation
at one pole that were adherent
the plasmalemma
extruded
granules
stained
weak
Specimens
granules
were
and cytoplasmic
Several
vesicular
of the patient
PA-TCH-SP
was masked
in
neutrophils
structures
demonstrated
A progressive
decrease
in the
ules was observed
with increasing
PMA.
After
1 mm exposure,
tubuloreac-
which
gran-
staining.
the
intensely
were
demonmatrix
granules
(Fig.
9),
the abnormal
small
neutrophils
PA-TCH-SP
PMA-Treated
and
normal
myeloid
cells.
These
generally
more
elongated,
pleomorless
normal
much
in patient
moderately.
were first noted at the
and stained
intensely
with
the PA-TCH-SP
method
(Fig.
6). Often,
the
granule
membrane
staining
was more intense
than the
matrix
staining
and was similar
to the intensity
of the
matrix
and membrane
staining
seen in normal
secondary granules
(Fig.
vesicular
structures
was
normal
of maturation
P-I granules
of flocculent
ules of the patient.
Primary
granules
continued
to demonstrate
moderate
staining
(Fig. 8), whereas
this staining
primary
granules
of normal
segmented
(Fig. 9). Some pleomorphic
tubulovesicular
this method,
in both the
reticulum
of
membrane
stages
myelocytes:
staining
of
and P-3 granules
lacked
matrix
staining.
In the
myelocytes,
granules
with the P-3 pattern
of
were markedly
decreased
or absent.
abnormal
small granules
were the predominant
type seen in segmented
neutrophils
and gener-
stained
appeared
5),
re-
method
was
three
in normal
PA-TCH-SP
masking
granules
ally outnumbered
primary
granules
by a ratio of 4:1
(Fig. 8). The staining
of the abnormal
granules
was
similar
to that
seen
in the patient’s
myelocytes.
In
normal
segmented
neutrophils,
PA-TCH-SP
strongly
re-
endoplasmic
least
primary
myelocyte
moder-
material
and were often
contacted
by PA-TCH-SPreactive
vesicles
that presumably
transport
glycoprotein to the forming
granule;
P-2 granules
demonstrated
moderated
PA-TCH-SP
staining
throughout
the
observed
in myeloid
cells.
This
staining
was most
abundant
in segmented
neutrophils,
and it obscured
the staining
of cytoplasmic
organelles
(Fig. 4). Consequently,
staining
of organelles
was evaluated
in aamylase-treated
PA-TCH-SP
In contrast,
mature
At
identified
strated
reactivity
for these components
in
as described
for normal
human
specimens
particulate
staining.
in more
myelocytes.
neutrophils.’8
In contrast,
positively
stained
profiles
similar
to the abnormal
granules
were
not observed
with
the peroxidase,
acid
phosphatase,
and sulfate
methods.
In unosmicated
amylase,
strong
PA-TCH-SP
observed
of the patient
at the
continued
to demonstrate
).
(Fig.
In
addition,
depleted
and
dense
primary
the
neutrophils
Fig. 1 .
This segmented
neutrophil
contains
several
dense primary
granules
(P) and numerous
abnormal
small granules
(arrows)
that
appear less dense than primary granules (enlarged in inset). In longitudinal
section.
these granules
appear
to be disc- or cup-shaped.
whereas in transverse
section.
they appear
more round. The morphology
does overlap with short tubulovesicular
structures;
however.
the
latter structures
are more pleomorphic
and lack matrix
density
( x22.000;
inset. x 55.000).
Fig. 2.
Diaminobenzidine
stains
peroxidase
in primary
granules
in this segmented
neutrophil
from
the patient.
Few
or no
normal-appearing
mitochondria.
5The
thin
preparations
were
not.
secondary
granules
are
( x22.000;
inset, x 55.000.)
sections
for Figs.
and were
The
specimens
I and
10-1 2 were stained
not counterstained.
for Figs.
present.
The
1-9 are from
specimens
marrow,
Staining
with
uranyl
for Figs.
and those
is not
seen
acetate
in small
and lead citrate,
I , 2, and
10-1 2 were
for
10-12
Figs.
granule
whereas
postfixed
are from
profiles
blood.
(arrows.
the other
in 0504,
enlarged
figures
whereas
are from
those
in
inset).
M.
cytochemical
for the other
figures
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
542
PARMLEY
ET AL.
1Prn’
-
I/4
*0
C
(
.
.
.
:
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p4_
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Fig.
3.
granules.
Fig. 4.
HID-TCH-SP
stains
sulfate
Profiles of the stained granules
Without
a-amylase
digestion.
in presumed
tertiary
do not resemble
PA-TCH-SP
granules
(arrows)
the frequency
intensely
stains
from
or morphology
cytoplasmic
the
patient.
Staining
of the abnormal
glycogen
is masked
granules
of this segmented
in larger
primary
( x 28.500).
neutrophil
from
the patient.
The reactive
glycogen
particles
obscure
the staining
of cytoplasmic
granules
( x 11.250).
Fig. 5. PA-TCH-SP stains vicinal glycol-containing
glycoconjugates
in this a-amylase-digested
specimen.
This early promyelocyte
from
the patient contains several dilated segments
of endoplasmic
reticulum
(ER). with moderate
PA-TCH-SP
reactivity
(enlarged
in far right
inset) not seen in normal specimens. Only a few primary granules (P) are present. The Golgi Iamellae (G) are intensely PA-TCH-SP reactive.
with
increasing
staining
on the mature
face
(enlarged
in inset).
( x 13.120;
inset.
x40.000;
far right
inset,
x 50.000.)
Fig. 6.
The patient’s
myelocyte
from an a-amylase-digested
PA-TCH-SP-stained
specimen
contains
several
reactive
primary
granules.
a Golgi zone (G). and intensely
reactive
abnormal
small granules
(enlarged
in inset. arrows).
Primary
granules
(P) stain intensely.
and some
weak staining
persists
in endoplasmic
reticulum
(ER). These abnormal
primary
granules
are not as readily appreciated
in morphological
specimens
(of.. Fig. 1). (x22.000;
inset. x55.000.)
Fig. 7.
This normal
marrow
myelocyte
(after
cr-amylase
digestion)
contains
several
intensely
reactive
intense
staining
is present
in immature
primary
granules
(P-2). and staining
is completely
masked
in mature
immature
primary
granules
(P-i ) or condensing
vacuoles
are contacted
by PA-TCH-SP-reactive
tubulovesicular
presumably transport glycoprotein matrix material to the forming primary granule (enlarged in B. arrows).
secondary
granules
(5). Less
primary
granules
(P-3). Very
structures
(arrows).
which
(A) x22.000;
(B) x55.000.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
ER
1
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p
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#{149}:;:.
:
Fig. 8.
This segmented
neutrophil
from
the patient
(after
a-amylase
digestion)
demonstrates
numerous
intensely
PA-TCHSP-reactive
abnormal
small granules
(enlarged
in upper left inset).
Primary
granules
(P) are also PA-TCH-SP
reactive
(enlarged
in upper
right inset) and do not demonstrate
the masking
of staining
observed
in normal
primary
granules
(as is shown in Figs. 7 and 9). Specimen
digested
with
a-amylase ( x 22.000;
insets.
x 62.000).
Fig. 9.
This normal segmented
n.utrophil
(after a-amylase
digestion)
contains
abundant
PA-TCH-SP-reactive
secondary
granules
(5).
Staining
is present
in the membrane
and matrix
of the normal secondary
granule.
Vicinal glycols in mature
primary
granules
(P) are masked
to staining
(enlarged
in B). (A) x22.000;
(B) x 55.000.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
LACTOFERRIN
DEFICIENCY
545
appeared
exposed
in large
specimens.
aggregates
Binding
of Chemoattractant
in
the
and
5-
1 5-mm
Table i
Comparison
.
Between
Normal
of the
and the
3H-FMLP
Patient
Number
plot
A23
Figure
for
1 3 indicates
the effects
FMLP
the representative
the degranulating
of
1 87 on 3H-FMLP
Stimulus
binding
of PMN
donor
and the lactoferrin-deficient
was derived
from
the method
Scatchard
stimulus
from
a normal
patient.
of linear
analysis
to obtain
the best fitted
lines. Comparison
of
the calculated
available
FMLP
binding
sites per PMN
(Table
1, without
A23187
exposure)
shows
that the
lactoferrin-deficient
patient
had
only
36%
of that
found in normal
cells
PMN).
Under
the
3H-FMLP
to normal
exposure
to 10 nM
an exposure
caused
sites to 72% of the
deficient
PMN.
binding
of
degranulating
the normal
(10,245
3H-FMLP
binding
sites/
present
conditions,
the binding
of
PMN
was enhanced
by 46% by
of A23187.
On the contrary,
such
a decrease
of 3H-FMLP
binding
unstimulated
cells in lactoferrin-
Therefore,
it
is apparent
that
3H-FMLP
to PMN
in response
agent
A23 1 87 was different
and
lactoferrin-deficient
cells.
the
to the
between
Further-
more,
the total
specific
saturable
binding
of the 3HFMLP
to the patient’s
PMN
was only
16% of that
observed
from the normal
cells (Table
2). This drastic
decrease
could
be attributed
to a decrease
in both
displaceable
and
nondisplaceable
binding
sites.
DISCUSSION
This
study
demonstrates
a variety
of
neutrophils
related
to complex
carbohydrate
tion and staining
in a patient
with lactoferrin
cy.
Specifically,
complex
carbohydrates
defects
in
distribudeficien-
appear
to
be
abnormally
packaged
in both the primary
and secondary granules
of this patient.
In contrast
to previous
speculation,
the number
of secondary
granules
is not
truly
decreased.
Using
the a-amylase
method,
the abnormal
small granules
PA-TCH-SP
appear
intensely
reactive,
secondary
as is seen
ules’4”9
and
glycoproteins
for larger
normal
is consistent
with
the high
in these granules.2#{176} As noted
et al.,’ these
abnormal
granules
myelocyte
stage
of development,
with normal
secondary
ent study
does indicate
granule
that the
gran-
content
of
by Strauss
first appear
at the
which
is consistent
genesis.21’22
The
abnormal
small
10,245
A23187(1OnM)
21,830(146)
presgran-
in parentheses
in the control
observed
comparable
Abnormal
observed.
granules
degranulation
to normal
packaging
This
was
is supported
by their relawith
PMA,
which
is
secondary
granules.23
of primary
granules
evident
in the
failure
endoplasmic
TCH-SP-reactive
also
Patient
(100)’
represent
(100)
2,700(72)
the
or unstimulated
3,738
percentages
of
the
values
cells.
reticulum
of
material
promyelocytes.
in endoplasmic
The
PAreticulum
endoplasmic
reticulum
or increased
synthesis
of glycoprotein
by this organelle.
Other
studies
have demonstrated
increased
amounts
or reactivity
of the primary
granule
substances,
peroxidase,3’t
and lysosome,8
consistent
with
increased
synthesis
of primary
granule
components.
The masking
of vicinal
glycol
groups
in
normal
primary
granules
may reflect
interaction
with
other added
granule
substances.’4”9’24
Thus, the lack of
masking
in this
patient
could
represent
either
an
abnormal
responsible
glycoprotein
for masking
The
deficiency
patients
may
dary
granule
of
represent
defect.
or lack of a granule
of vicinal
glycols.
lactoferrin
substance
observed
in
a primary
rather
than
Recent
ultrastructural
these
a seconstudies7
have localized
iron-binding
protein(s),
including
lactoferrin,
in mature
primary
granules
and not in secondary granules.
The lactoferrin-positive
primary
granules are secreted
with PMA
and function
differently
than immature
primary
granules.7
Thus,
the decrease
in lactoferrin
observed
in this patient
appears
to be
consistent
with the primary
granule
maturation
defect
in complex
TCH-SP
in the
carbohydrates
method.
The
neutrophils
demonstrated
with the PAproduction
of some lactoferrin
of this
Table 2. Reversibility
Lactoferrin-Deficient
patient,
of 3H-FMLP
Total
Origm
Normal
for total
Granulocytes
(fmole
Bound/5
Specific
in Materials
in
10
PMN)#{176}
20(100)
5.2(26)
bindings
were
obtained
as
and Methods.
parentheses
in normal
and displaceable
x
Displaceable
21.7(16)
specific
by
Binding to Normal and
131.7(100)t
Lactoferrin-deficient
tEntries
observed
as demonstrated
Polymorphonuclear
3H-FMLP
Cell
described
was
of FMLP Receptors/PMN
and
immature
primary
granules
presumably
represents
glycoprotein
enzymes,
which
may include
acid
hydrolases.25
The endoplasmic
reticulum
staining
mdicates
increased
amounts
of glycoprotein,
which
could
result
from delayed
transit
of glycoprotein
from
the
Values
of vicinal
of PMN
Deficiency
glycol-reactive
groups
to become
masked
with granule
maturation,
as is seen in normal
primary
granules’4”9
and
eosinophil-specific
granules.24
Similarly,
PATCH-SP
moderately
stained
the normally
unreactive
ules lack the peroxidase,
acid phosphatase,
and sulfate
seen
in primary
granules
and/or
tertiary
granules.
Further
identification
of these
abnormal
small
granules as secondary
tively
selective
Sites
Lactoferrin
Normal
Control
‘Entries
This plot
regression
Binding
With
cells.
represent
the
percentages
of
the
values
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
546
PARMLEY
ET AL.
:..
.,
,...
.
.
..
.
1
,
.
-
.
-
2#{149}
.
#{149}..
.
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‘#{149}
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4.
#{149},%7
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.
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.
-
_-“,;
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,
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#{149}
S
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-‘:‘“
,
IS.
‘..
#{149}
;<
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t\#{188}#{149}
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6,
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,
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r
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r
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
LACTOFERRIN
DEFICIENCY
547
EFFECT
ON
OF DEGRANLJLATINC
RECRUITING
PMN
STIMULI
RECEPTORS
O-o
.
.
k
A
NORMAL
PMN;
WI0
NORMAL
PMN;
A23187
TREATMENT
PATIENT’S
PMN;
W/O
PATIENT’S
PMN;
A23157
TREATED
TREATMENT
TREATED
.
4
00
00
00
-S
I-
0
I-
immunoassay
methods
that
a complete
synthesis
has
deletion
of
not
occurred.
observed
some
granules
of these
described
histochemical
deficiency
of
A
in previous
iron-binding
cells
(3H1
studies,8
activity
lactoferrin
we have
in mature
(unpublished)
using
methods.7
alkaline
phosphatase
primary
x io6
BOUND/u.s
ciency.4
indicates
a gene
for
Similarly,
FMLP
Our
data
( p moles
NEUTROPHILS
would
suggest
that
branes,
since
has
and can
evidenced
be induced
in our
these
treated
cells.
The abnormal
formation,
and
membranes
appear
to fuse with
morphological
the
present
PMA-
scribed
nuclear
envelope,
which
shares
protein
tions
with the endoplasmic
reticulum.
receptors
and
in their
characterized
Fig. i 0.
recruitment
in another
After
i -mm
patient
exposure
has
with
FMLP
been
previously
lactoferrin
of the patient’s
neutrophils
to PMA.
this
nuclear
nuclear
previously’3
organelles
defi-
observed
to be
the pathophysiology
The
observed
disorder.
in plasmalemma
the
plasmalemma,
studies
of
described
in these patients.
In humans,
alkaline
phosphatase
may
be localized
in vesicles
distinct
from
secondary
granules.5’6
Thus,
membrane-limited
organelles,
other
than lysosomes,
appear
to be involved
in
of this
decrease
)-
plasma
membrane
defect
in this disorder
may then be a
consequence
of abnormal
secondary
granule
membrane
structure,
rather
than absence
of granule
mem-
previously
been
0.10
0.0$
0.0$
Fig. 1 3.
This graph depicts
the decrease
in recruitment
of FMLP
receptors
in the
patient’s
neutrophils
compared
to controls.
appeared
patient
a small
abnormal
on the
granule
segmentation,
membrane
could
be related
to be
basis
functioning
of PA-TCH-SP
profile.
indicative
nuclear
duplication
to a defect
as
pocket
dein the
synthetic
funcBoth of these
abnormally
staining
of degranulation
in
and
(large
arrow; enlarged in inset). can be seen. although several abnormal small granules remain (small arrows). ( x34.000; inset. x80.000.)
Fig. 1 1 .
Vesiculated structures (arrows) are attached to the plasmalemma
and presumably represent degranulated
abnormal small
granules after PMA exposure ( x25.000).
Fig. 1 2.
Fifteen
minutes after exposure to PMA. virtually all abnormal small granules have degranulated;
some primary granules
remain.
A rare
abnormal
small
granule
is intact
(arrow;
x 22.000).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
548
PARMLEY
nuclear
membrane
segmentation
observed
duplication.
and
other
in
genesis.26
Thus,
cal and functional
Defects
nuclear
disorders
in
nuclear
organelles
membranes
have
been
with
abnormal
granule
the observation
abnormalities
in the
neutrophils
of this
patient
more
generalized
defect
affecting
neutrophil
membrane
genesis
and/or
of several
morphologiof membrane-limited
ET AL.
suggests
many
aspects
glycoprotein
a
of
syn-
thesis.
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1983 62: 538-548
Abnormal distribution of complex carbohydrates in neutrophils of a patient
with lactoferrin deficiency
RT Parmley, DY Tzeng, RL Baehner and LA Boxer
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