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Phagocytosis
By
E
by
J.
MARn
OSINOPHILS
over
100
Mesnil
in
eases
CuE,
were
years
remains
semble
other
granulocytic
locomotion,
response
to
fer in the specific
levels
of peroxidase
and
precise
role
general,
cells
chemotactic
activities
of certain
and aryl sulfatase
is available
cent knowledge
of phagocytosis
studies
of Archer
and Hirsch,
peritoneal
exudates.5
with
a 3 year
the
Cancer
Medical
submitted
MARTIN
Associate
Francisco,
Medical
J.
Research
Center,
This
investigation
(GM-01791)
from
San
)
for
and
the
in tissues
Eosinophils
.
normal
also dif-
and
functions
of
the difficulty
of obtaining
elements.
Most
of our re-
has been provided
separated
from
horse
period
in
example,
they
have
high
low levels
of hexokinase
metabolism
we
had
eosinophilia
AND
and
Francisco,
the
of
by the
blood
elegant
or rat
opportunity
the
of
peripheral
ob-
blood.
studies
of the metaboWhenever
possible,
we
that of eosinophils
from
METHODS
March
5, 1968;
M.D.:
a research
Service.
for
Associate
Professor
Research
Institute,
Cancer
California.
accepted
JoN
the
Department
of
Medicine,
either
with
drug
reaction
MT.).
They
University
of
California.
was
supported
by
the U. S. Public Health
CLIME,
Director,
institute
San
Center,
San Francisco,
University
of California
Medical
Requests
for reprints
should
University
of California
Medical
922
re-
of Leukocytes
California
First
dis-
mammals
Leukocytes
were
obtained
from
3 patients
with
eosinophilia,
associated
parasitic
disease
(patient
V.P.),
with
asthmatic
bronchitis
and
possible
(patient
B.C.)
or with
a syndrome
resembling
eosinophilic
leukemia
(patient
From
and
various
subjects.
MATERIALS
Source
in
higher
distribution
infections
These
3 patients
provided
the material
for detailed
lism and phagocytic
functions
of human
eosinophils.
have
tried
to compare
the activity
of these
cells with
healthy
morphology
and
physiologic
behavior,
including
and
particle
ingestion.3
They
on
pronounced
granulocyte
of phagocytosis
the
from
stems
from
other
cellular
by eosinophils
using
cells
During
3 patients
on
man
enzymes;
activity
human
eosinophils.
This
lack no doubt
large
numbers
of these
cells free from
of
capable
literature
their
stimuli
LEH.RER
type
be
in normal
in their
pattern
of
states
( e.g., parasitic
alkaline
phosphatase.4
Only
limited
information
serving
to
eosinophils
in
I.
ROBERT
a distinctive
observed
a voluminous
In
differ
from
neutrophils
and in certain
disease
AND
as
were
their
unclear.
Eosinophils
HANIFIN
recognized
and
Despite
cells,
Human
JON
first
ago,’
1895.2
of these
functions
by
grant
(CA-07723)
publication
July
of Medicine,
University
11,
and
grant
1968.
Department
of
a training
of Medicine,
California
Medical
and
Center,
M.D.:
Fellow
in Medicine,
University
of California
California.
ROBERT
I. LEHRER,
M.D.: Fellow
in Medicine,
Center,
San Francisco,
California.
be addressed
to Dr. M. J. Cline,
Cancer
Research
Institute,
Center,
San Francisco,
Calif. 94122.
HANIFIN,
BLOOD,
VOL.
32,
No.
6
(DECEMBER),
1968
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PHAGOCYTOSIS
were
the
also obtained
peripheral
matory
from
blood
an otherwise
(R.I.L.)
and
normal
from
subject with 8 to 10 per cent
with neutrophilia
associated
7 patients
eosinophils
with
in
inflam-
or polycythemia.
diseases
Preparation
of Leukocytes
Leukocytes
were
isolated
by dextran
sedimentation,
washed
in Hanks’
balanced
salt
solution
containing
1/10 volume
fetal calf serum,
and
suspended
at 2 X 10 or 2 X lOVrnl.
in Eagle’s
minimal
essential
medium
(MEM)
containing
either
3 per cent human
albumin,
10 per cent fresh or fresh frozen
human
serum,
or 20 per cent fetal calf serum.6
In studies
involving
glucose
Leukocyte
per cent
phils
utilization,
populations
contaminating
designated
eosinophils
at or beyond
at least 90 per cent
cells
were
removed
by
hypotonic
lysis.7
at least 80 and usually
95 to 100
state.
Populations
designated
neutrometamyelocytes
and myelocytes.
contained
the metamyelocyte
mature
granulocytes,
contained
Preparation
red
eosinophils
of Particles
Polystyrene
particles,*
1.3 p in diameter,
were suspended
in MEM
at a final concentration
of 0.5 to 1.0 mg./ml.
Candida
albicans
grown
in Sabouraud’s
broth
was washed
with saline
and killed
by heating
at 100 C. for 10 minutes.
The ratio of yeast to leukocytes
in phagocytosis experiments
was 1 : 1 or 5:1.
Escherichia
coli or Staphylococcus
aureus
502A,
grown
for
18 hours
in Trypticase
soy
agar
and
washed
in Hanks’
solution,
were added
to leukocyte
suspensions
in ratios
of 8:1
or
20:1.
Blood
0 Rh-positive
group
red cells were
with
a serum
containing
a high
were
washed
and
added
to leukocytes
E. coli endotoxinf
was used.
2
Pliagocytosis
washed
with
buffered
saline
titer (1:2000)
of anti-D
in a final
concentration
hours
and incubated
antibody.
of 0.5
The
per
for
red cells
cent
(v/v).
of Particles
Leukocytes
the presence
were incubated
in 95 per cent 02-5 per cent CO2 at 37 C. for 60 minutes
in
or absence
of metabolic
inhibitors
before
the addition
of the test particles.
Two types
of leukocyte
preparations
were
used:
1) suspensions,
consisting
of 1 ml. of
cells (2 x 107/ml.)
which
had been rotated
at 30 r.p.m. in 5-mi. plastic
tubes in an incubator
or of 2 ml. of cells shaken
in 50-ml.
siliconized
Erlenmeyer
flasks in a Dubnoff
metabolic
shaker
(2
(3 cycles/second),
x 107/ml.)
capacity)
for
warm
60
MEM.
minutes.
was
with
or
Ciemsa
were
counted
per cent.
In studies
in
were
they
added
by
of
duplicate
on pour-plates
and
the
for
added
of
a phase
samples
hypotonic
of
lysis
contrast
of
with
fixed
in
coefficient
white
of cell
of nonadherent
was continued
Four
of
of leukocytes
cells
and
the
methanol
microscope.
mixture
the
1 ml.
in Leighton
along
smears
the
the
free
incubation
were
of
coverslips
washed
preparations;
killing,
minutes
were
examination
with
consisting
to glass
radioisotopes
observation
each
30
variation
and
colony
suspension
tubes
(1 ml.
cells with
for 5 to 120
test particles.
and
stained
hundred
cells
was
bacteria
counts
6
±
were
of
the
of agar.8
Studies
Incorporation
phosphatase
The
direct
to adhere
which
quantitated
of bacterial
microorganism
and
production
except
after
particles
by
preparations,
allowed
experiments,
then
every
2) glass-fixed
been
minutes,
some
Phagocytosis
Metabolic
had
Test
In
removed
and
which
that
of H3-uridine
into
lysozyme
activities
of C1402
Hyamine
flask as CO2-absorber.
*Dow
Chemical
fDifco,
Detroit,
was
from
glucose-i-C14
added
Michigan;
RNA,
production
by previously
of lactate
and acid
described
methods.6’9
was determined
as described
previously,9
to the center
well of a modified
Erlenmeyer
of variation
for radioactive
counts
was ± 7 per cent.
dfrectly
The coefficient
Co., Midlands,
acid-precipitable
were
measured
Michigan.
lot 491-028.
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924
CLINE,
Isolation
of Leukocyte
suspended
granule
LEHBER
Granules
were isolated
from eosinophils
or neutrophils
by the method
in phosphate-buffered
saline
at pH 7.4.
Additions
were
suspension,
and the change
in optical
density
at 450 mt with time
Granules
HANIFIN,
CASE
of Williams’0
and
then
made
to the
was determined.
SUMMARIES
a 47 year old woman
from El Salvador
was admitted
to the University
of California
Medical
Center
because
of progressive
loss of hearing.
She had a history
of previous
infestation
with
Trichuris,
Strongyloides
and Ascaris.
Examination
of the
stools
revealed
the larvae of Strongyloides
stercoralis
and the eggs of Trichuris
trichiura.
A routine
WBC
count
showed
12,000
cells/cu.
mm.,
with 58 to 78 per cent eosinophils.
She was treated
for parasitic
disease,
and the eosinophilia
eventually
disappeared.
B.C.,
a 45 year old white housewife,
entered
the University
of California
Medical
Center
with a complaint
of hay fever and asthma
of 3 years’ duration.
She had been
hospitalized
on several
occasions
previously
because
of pulmonary
and
sinus
infections,
symptoms
of
depression
and suicide
attempts.
She had
been
treated
with
a variety
of medications:
V.P.,
antibiotics,
antihistasnines,
“antidepressants,”
bronchodilators
and
expectorants,
including
iodide.
Two weeks
before
the present
admission
cough
and
sputum
production
increased.
X-ray
examination
showed
infiltration
at the base of both lungs
and the right
middle
lobe. A sputum
culture
grew
E. coli,
and treatment
with ampicillin
was begun.
The
WBC
count
then and at the time of admission
was 42,000
celLs/cu.
mm.,
with 68 to 72
per cent eosinophils.
Physical
examination
was
unremarkable
except
for
evidence
of
respiratory
distress.
During
the patient’s
hospital
stay pronounced
eosinophilia
of the peripheral
blood persisted,
and
repeated
bone
marrow
aspirations
revealed
eosinophiic
hyperplasia.
A marrow
chromosome
preparation
showed
no specific
abnormalities.
A liver biopsy
revealed
normal
potassium
hepatic
architecture
without
for ova and parasites
were
The patient’s
symptoms
was
costeroids
patient
was
instituted.
discharged,
evidence
of
infiltration.
Numerous
examinations
of
the
stool
negative;
and
no evidence
of lymphoma
was found.
the
eosinophilia
continued
until
treatment
with
cortiThereafter,
the WBC
count
decreased
rapidly.
At the
time
the
the WBC
count
was 16,000
cells/cu.
mm.,
with
1 to 7 per
cent
eosinophils.
man, was transferred
to the University
of California
Medical
of fatigability,
anorexia
and persistent
cough
of 11 months’
duration
of 30 pounds,
he had been
admitted
to another
hospital
5
months
and spleen
were
enlarged,
and
the WBC
count
was
26,000
cells/cu.
cent
eosinophils.
The
patient
underwent
splenectomy
and
biopsy
nodes
at that
time.
Histologic
examination
revealed
diffuse
infiltration
the spleen with mature
eosinophils.
Infiltration
by eosinophils
was also seen
in periarteriolar
sheaths
and within
the subendothelium
of trabecular
veins.
The lymph
nodes
showed
reticular
and lymphoid
hyperplasia
and diffuse
infiltration
of the
cortical
and medullaiy
areas by mature
eosinophils.
M.T.,
Center
In
a 25 year
old white
for evaluation.
Because
and a weight
loss
previously.
The liver
mm.,
with
47 per
of mesenteric
lymph
of the red pulp
of
studies
at the
Medical
and by biopsy,
showed
granulocytes.
An attempt
Center,
specimens
of bone
marrow,
obtained
by
aspiration
solidly
cellular
marrow
with
a predominance
of eosinophulic
to prepare
marrow
chromosomes
was unsuccessful.
Numerous
attempts
to demonstrate
parasitic
disease
failed
to reveal
evidence
of infestation.
The
patient
received
60 mg. of prednisone
daily
for 1 week,
but the hematologic
findings
remained
unchanged.
Unfortunately,
he left
the hospital
before
a definitive
diagnosis
could be established.
a
REsULTh
Particle
Ingestion
Eosinophils
polystyrene
in suspension
ingested
spherules,
gram-positive
a variety
of particles
and
gram-negative
in vitro,
bacteria,
including
and living
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925
PHAGOCYTOSIS
Table
1.-Phagocytosis
Ratio
Test
l)lVSt
Human
to
Eosinophils
and
Phago
.
incubation
TVeutrophils
cvtic
.
lndex*
Eosinophils
Time
Leukocvtes
(minutes)
Neutrophils
from
B.G.
V.P.
MT.
R.I.L.
vrene
spherules
(
of
Particles
Particles
by
1 mg.
:1 x l0
30
70
±
9
29
54
33
I :1
60
93
±
3
-
-
-
S :1
1 20
69
±
17
-
-
40
96
±
0.5
-
-
-
(i(lid(l
‘U
(ilblCa7lS
E.seheric/j
coli
‘1(1/)/i
20:1
index
1)olystyrene
tlw phagocytic
the
for
mean
±
of dul)licate
and
dead
particles
one
S.D.
samples
in at
particularly
evident
containing
a large
corresponding
7
0
less
of
in
Table
apparent
nor
glass-fixed
The
In
in detail
Using
ficiency
glass
minutes
50
contrast,
conditions
and
elsewhere.’2
cells isolated
of ingestion
and
in
after
free
the
particles
to
70
per
of
the
from
patient
of heat-killed
suspension.
addition
results
mean
cells
±
ingested
in
(Table
S.D.
was
was
of
was
considerably
at
ingesting
a
was
employed.
eosinophils
example,
these
1 ). This
percentage
but
inability
from
only
less
than
eosinophils
less
than
the
20:1
ratio
of
12 per
the
of
preparations
cent
cent
ingested
eosinophils
to
smears
of
time
ingest
the
of eosinophils
times
large
incubation
phagocytized
a 2 hour
period.
Under
similar
condineutrophils
showed
no erythrophagof human
experiments
monocytes
are
on
monocytes
M.T.,
we studied
C. albicans
by
eosinophils
yeast
had
at various
sampling
from
representative
stained
Phagocytosis
of
The
the
the
For
cells over
of human
results
as
to leukocytes
and
1. The
was
ocytosis.
or
neutrophils
studied
phagocytic
phagocytic,
particles
human
red
preparations
5
more
per cent of neutrophils
were
phagocytic
and
or more
organisms.
Under
similar
conditions,
mixture
(Fig.
1).
Neither
suspensions
antibody-coated
tions
glass-fixed
than
fungi
neutrophils.
20
populations.
the
of particles
of ingested
were
which
efficient
and
ratio
100
±
6 or
cell. For C. albicans,
Results
are expressed
1er
samples
Similar
results
were
obtained
periods
up to 120 minutes.
Data
shown
of
with
conditions.
bacteria
of
neutrophil
of duplicate
neutrophils,
number
anreus
leukocytes.
100
7
containing
experiments.
percentage
of eosinophils
are
five
least
independent
phagocytic
20 or more
bacteria.
during
incubation
at
mean
12
leukocytes
Staph.
per
of
the
a high
to leukocytes,
had
ingested
cent
numbers
as
were
percentage
staphylococci
96 per cent
periods
a series
the
the
of
of fungi
The
efficiency
experimental
when
of
percentage
number
three
and
experiments,
percentage
78 per
least
the
coli or 20 or more
of eosinophils
particles
such
as
either
albicams.
with the
polystyrene
In
(1 1 ) for
expressed
Suspensions
(Iefifle(1
10 or more
E.
is defined
as the
are
C.
varied
60
here
15
parti(les,
ifl(Iex
eosinophils
the
±
ylo(oeeos
(IU1CU$
as
15
i(i
was
to
the
the
complete
cells.
After
phagocytic.
are
reported
course
time
and
ef-
5 and
15
adherent
between
60
minutes,
to
fungi
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926
CLINE,
.
. ‘
LEHRER
.
..
.
S.
\
HANIFIN,
h4.
..
“S
p
.
.
.,
.
.
_(>
*..
..*
,..
1
Fig.
1.-A
smear
of suspension
A central
neutrophil
has
aureus.
rounding
were
eosinophils
found
in
suspended
effects
were
containing
either
serum
cent
or no bacterial
of the
fixed
phagocytosis.
eosinophils
5 to
the
close
30
cells
ingested
when
serum
ratio
in
at
pelleted
37
between
yeast
was
determine
at a variety
when
present,
the
of
Staphylococcus
but the
Giemsa
and
63.5
stain,
per
C.
in
the
medium
suspended
serum
x
cent
sur1000.
of
the
heated
the
presence
and
eosinophils,
17 to 25 per
of pH,
of pH’s
between
serum
in
centrifugation
incubating
effect
or
to
cent
or
were
and
which
at
56
C.
for
g.
of
than
lacked
2);
for
eosinophils
150
absence
less
medium
eosinophils
5.5
on phagoin MEM
required
native
serum
(Table
heated
serum.
The
requirement
experiments,
by
yeast
of the suspending
from
M.T.
were
yeast
or in
additional
were
minutes
contact
autologous
ingestion
in albumin
confirmed
a 1:3
component
Eosinophils
albumin,
Maximal
was
less
was
in
for
MEM
per
of the protein
then
studied.
30 minutes.
phagocytosis
To
91
little
eosinophils
and
of microorganisms,
cells.
The
cytosis
yeast
show
of neutrophils,
ingested
a number
,
,.
...j
and
and
incubated
serum.
1 per
Despite
cent
serum;
of
the
however,
phagocytic.
were
9 and
suspended
then
incubated
in
serum
with
and
heat-
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927
PHAGOCYTOSIS
Table
2.---Effects
of
of
Candida
Suspending
Protein
Serum
albicans
and
by
pH
the
Phagocytosis
(Patient
M.T.)5
Medium
Phagocytic
Content
3% albumin
10% serum
10% serum
20% serum
on
Eosinophils
PH
heated
at 56 C. for 30 mm.
Index
7.4
7.4
7.4
5.5
1.0
60.5
22.0
82.5
20%
serum
6.5
90.5
20%
20%
serum
serum
7.4
8.0
91.0
20%
serum
9.0
70.0
*The
results
killed
yeast.
proached
15
are
the
As
7.4;
minutes,
means
the
of three
incubation
which
experiments.
proceeded,
consequently,
at
independent
the
phagocytosis
time
there
72.0
was
was
no
pH
of
all
recorded
significant
suspensions
only
during
deviation
from
pH.
As shown
in Table
range,
and only
above
2, particle
pH 8 does
ingestion
phagocytosis
takes
place
over
decline
slightly.
sults
polystyrene
as the
particle.
for
Ingestion
were
obtained
Metabolic
As
Requirements
an
approach
ingestion
by
metabolic
to
.
of
these
the
eosinophils
3.-Effect
Particle
study
of
these
cells
before
In all
eosinophils
affected
Table
the
eosinophils,
inhibitors
)
spherules
rations
had
with
were
the
of Metabolic
of
as
to
cause
on
Human
the
test
Phagocytosis
of
the
Conditions
Co ntaini
Potassium
cyanide
73.0
80.0
--
1 x iO
69.
73.2
21
33
1 x 10
Sodium
2 x 10
fluoride
lodoacetate
1 x 10
ng More
Than
indicates
(‘ont.
Exp.
Exp.
54
40
23
24
54
45
72.9
12.4
21
3
54
it;
1d
75.1
25.3
21
0
54
6
M
-
Cont.
-
M
Exp.
-
-
:34
69
:i
0
M
73(1
67.3
21
11
54
23
:34
77.0
72.0
21
7
54
26
-
25
M
7 x I0
M
77.0
73.
54
45
34
28
control
and
exp.
indicates
experimental
-
cell
-
suspensions.
cubated
in 5 per cent C02-95 per cent O with saline as the only addition.
Results
independent
experiments
performed
at different
times on different
populations
population
MT.
Coat.
4 x 10
(‘ortisol
*Cont
i
Exp.
5 Particles
from
2.5 x 10
Puromcin
Colchicine
i
to
Particles
V.P.
(‘ont.
1)initrophenol
prepainhibitors
population
Po1ystrene
B.C.
Neutrophils
Concentration
Anaerobic
of
Leukocytes
Eosinophils
Experimental
particle
(polystyrene
glass-fixed
If the
of
pH
re-
a variety
particles
than
reason.
initial
for
with
a fraction
(7 of Le ukocytes
Inhibitor
the
first
a broad
Similar
requirements
preincubated
suspensions
rather
for the following
Inhibitors
by
metabolic
were
addition
studies,
used
so
the
test
apthe
of eosinophils.
Control
cell
suspensions
are the means
of neutrophils
-
were
in-
of at least three
or on the same
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
928
cu,
Table
4.-Effect
of
Metabolic
RTVA
Inhibitors
Human
by
H1-Uridi,ie
on
Leukocytes
HANIFIN,
Incorporation
Conditions
I nhihitor
N(Lit
98
±
5
95
1 12
i 03
±
S
74
1 00
I x H)
\l
96
±
10
I 06
57
2 x 10
\I
6
±
4
5
\l
7 1 ±
12
71
\l
51
±
16
85
±
13
1)initrophenol
IO(loac(tat(
1 x 10
2.5
(‘olchicine
*l.t.siil1
s
ts
leave
(Xl)I(’SS((1
tie
I li(’
ire
the
reflected
hibition
surface,
staining
exceed
8 per
itors
of
on
and
the
The
results
an
that
intact
of
these
neither
poration
nitrogen
and
eosinophils,
terfered
with
particle
uptake
of
uridine
incorporation,
H3-uridine
vital
experiments
are
nor
32
79
85
.
rol
(OUt
unIv
1
leukocyte
s.
1(l(lit
.
ion
would
not
have
To differentiate
between
cells,
all suspensions
as
nonviable
into
RNA
given
in
of the
judged
cells
control,
not
effect
was
inwere
experiments.
by supravital
did
the
affect
of
In
the
the
inhib-
in
control
measured
glycolytic
In
particle
ingestion
these
studies
was
fluoride
iodoacetate
and
or
(fluoride
there
3
or
included
a
both
neuin-
effect
on
inhibited
slightly
ap-
incor-
iodoacetate)
a significant
only
is
uridine
tested
With
and
It
conditions
for
profoundly
was
4.
aerobic
metabolism
dinitrophenol.
inhibitors
before
whereas
Tables
require
of oxidative
cyanide
and
_____
60
111
t he
t exl)eIini(llt
eosinophils
for
ingestion
dyes.
-
preparations.
The inhibitors
potassium
trophils
In
of
of
100
as
rol
2
eosinophils
of
additional
pathway
into RNA.
atmosphere,
(liffel(’n
in
at the conclusion
nonviable
cells
fraction
an
neutrophils
mitochondrial
us
adherent
blue
of
As
leukocyte
ti
total
population.
simple
killing
This
data.
incorporation
experimental
parent
cent.
the
inrurl)orat
h salimu’
( ) it
of (cait
152
((‘nt
remaining
the
and
(
I 1-mimidiiu
e1
let erniiii;i
2 per cent
trypan
did the percentage
interpretation
of
i
( 2-5
I limee
of
ag(
ni
(
the
the behavior
of phagocytosis
stained
with
no instance
mir
((‘lit
if :i t least
mmo:iiis
glass
5 1)(1
ifl
sI
x 11)
t lie
LS
111(111 )tt ((l
5iiS1)flSli)fl5
I(’ill
x I 0-
1
liromiiyein
:i
rol )
#{188}1
‘1’.
\I
I x 10
fluoride
\‘.P.
(‘ont
(Vttfli(l(
SO(liLIIII
from
SiIR)j)hils
B.(.
of
nu)sphere
it
Potassiuni
rophils
rat lou
(‘omuent
Nitrogen
into
:5
Eo
I’x%)eriml1(’ntal
LEHRER
the
H3-
inhibitory.
Phag-
‘I
0
_-*-_._.
ln_J
40-
00
I”,.
0.0
5
‘S.’
5-
viz:5
5%’
o
5-.-.-
b-
20-
.-..
-----#{149}
.--
SW
Neutrophils
m
Eosinoph0s
5-55
I
0
I
20
I
I
40
I
I
60
I
I
20
1
I
40
I
and
phagocytic
TIME, MINUTES
Fig.
2.-Cellular
(#{149} #{149})
leukocytes.
acid
phosphatase
activity
control
(0
0)
I
60
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
929
PHACOCYTOSLS
Table
5.-Effect
of
Phagocytosis
on
Incorporation
Metabolisni
of
of
Production
113-Uridine
Cell
Control
from
Phagocvtic
Type
Neutrophils
696
124
±
Lactate
(‘ontrol
Production
Phagocytic
1342
194
±
163
5
±
2534
124
65
±
4
±
148
12
±
from:
435
676
2505
--
.-
291
:3782
572
1284
MT.
172
*The
methods
of measuring
trophils
differed
from
the counting
rate,
408
H3-uridine
used
employed
mycin
a slight
60
x
effect
less
the
80
per
cent
studies
ingestion,
transferred
particles
titate
fraction
behaved
determine
however,
often
intracellular
and
than
the magnitude
and
V.P.)
of
are from
for neutrophils
(13G.
or three
in-
into
the
assessed
the increase
gives
in Figure
particle
other
precise
suspending
free
by
their
lost
at
contrast
37 C.
nonphagocytic
dense
an
of
were
loss
granule
by
residual
of
of
activity
data
eosinophils
were
the
in
on
the
acid
and
measured
cellular
fraction
granparticle
and
difficult
in
residual
total
the medium,
of
and
to quanthe
granule
is most
cellular
although
enzyme
neither
degranulation.
phosphatase
activity
As
occurred
neutrophils.
in
are
of polystyrene
enzymes
into the
degranulation
extent
The
size
granule
After
enzyme
Therefore,
the
occasional
It is technically
of
in cellular
events
microscopy
Suspensions
of
performed.
the
cytoplasm.
measurement
in enzyme
30
to col5M ) had
neighbors.
particles
that
from
medium.
in the
quantitative
ingestion
phase
period
observations
measurements
2, a decrease
metabolic
by
vacuoles.
When
large
numbers
there
is some
leakage
of granule
by
within
PhagocytosLi
activity
are
synthesis
eosinophils.
suggesting
phosphatase
enzymes
inhibition
of protein
The
dose
of puro-
protein
by
by
granules,
acid
or of enzyme
shown
Three
of
degranulation
method
following
of neu-
similarly.
The
fate
of the
individual
because
of the motion
of the cells
and
the phagocytic
eosinophils
clearly
had
surrounded
into
phagocytic
are phagocytized,
conveniently
activity
or
affected
The data
studies
Accompanying
granules
were
lysosomal
cytoplasm
-
tested
were
sensitive
of cortisol
(7 x 10
uptake
be extruded
intact
from the cell.
order
to support
the morphologic
ulation,
-
by populations
differences
single
of
yeast
were
observed
over
a 15- to 30-minute
prominent
cells
of
production
values.
are from
of eosinophils
concentrations
Changes
ingesting
granules
phagocytic
to
on particle
Morphologic
and
glass-fixed
preparations
granules
was
difficult
to
their
cytoplasmic
contents;
shape
these
94
--
appears
to be more
sensitive
to
than
phagocytosis
by neutrophils.
inhibitory
Eosinophils
lose their
and
C’402
to control
for eosinophils
populations
10M.
High
two
and
fewer
and
Although
of phagocytic
Those
inhibited
The
at 2.5
Metabolic
I{NA
populations.
the ratios
populations.
into
83
(MT.).
by eosinophils
by puromycin
minutes.
chicine
incorporation
for eosinophil
did not affect
different
studies
ocytosis
synthesis
those
they
of seven
dependent
could
In
Phagocytic
(Mg.)
220
to
Control
(c.p.m.)
B.G.
only
Eosinophils
of C’O
V.P.
studies
and
Glucose-I-C1tm
c.p.m.
Eosinophils
Neutrophils
phagocytic
eosinophils:
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
CUNE,
930
Table
6.-Effect
of
Endotoxin
Eosinophils
on
the
Metabolis,mi
in Autologous
of
Seruv,a
Incorporation
(2
Human
x 1O
No. of
Neutrophils
of
Neutrophils
the
from
MT.
of
(a
shunt),
in Table
All
three
was
metabolic
The
more
eosinophils.
tion
the
of the
higher
Fig.
dotoxin,
±
200
3532
rise
into
pronounced
This
events
study,
the
was
were
Endotoxin
after
than
does
the
not
incubation
matter
of
in both
types
ingestion
of
of the
ingested
30 minutes
a vacuole
866
±
C1402
from
hexose-monophos-
of lactic
in neutrophils,
for
within
of
the
acid.
eosinophils
necessarily
showing
6434
6524
production
in two
shunt
162
±
3424
by
stimulated
of neutrophils
3.-An
eosinophil
after
demonstrating
particulate
3280
production
exhibited
production
hexose-monophosphate
percentage
RNA,
in neutrophils
observation
388
measurement
pattern
in C1402
±
748
H-uridine
in a single
same
(‘ontrol
(c.p.m./hr.)
817
semiquantitative
and,
5, the
kocytes.
2006
I
incorporation
glucose-i-C’4
phate
Endotoxin
of Ci402
Glucose-1-C
(c.p.m.’hr.)
7
F:osinophils
and
Production
from
(‘ontrol
Studies
LEHRER
cells/mi.)
I13-Uridine
(‘eli Type
HANIFIN,
of
As
phagocytic
polystyrene
three
reflect
leuparticles
populations
a greater
but
shown
neutrophils.
arid
of
stimula-
is consistent
with
particles.
with
Escherichia
(arrow).
coil
en-
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
931
PHAGOCYTOSIS
40
-
20
-
0
,
Granules
Neutrophil
8
Eosinophil
310
210
Granules
510
40
TIME, MINUTES
Fig. 4.-The
ules suspended
change
in optical
in buffered
saline
absorpancy
at pH 7.4
-s).
S)orO.1NHCi(
(S
Effects
at 450
m of isolated
leukocyte
gran0), 0.8 per cent deoxycholate
(0
of Endotoxin
We
have
changes
in
shown
previously
that
E.
coli
endotoxin
produces
human
neutrophils
resembling
those
associated
sis.13
The
changes
include
increased
production
of C’402
and of lactate,
increased
incorporation
of H3-uridine
into
tide
and
RNA,
panied
ides.
and
by
the
These
which
particles
contaminate
the
than
of
the
present
studies
the
almost
certainly
cells
effect
0.i
of
endotoxin
indicate
of
the
on
same
those
walls
eosinophils
seen
105,000
was
produces
RNA
by
effect
examinaat
on
endotoxin-treated
to
part-
cell
microscopic
preparations
in diameter.
accom-
small
bacterial
that
endotoxin
H3-uridine
into
Occasional
similar
of
Electron
such
to i
lysosomal
are
containing
fragments
of
certain
events
of vacuoles
approximately
particles
of
metabolic
preparations.
the
has
activity
These
.
centrifugation
approximately
glucose-i-C’4.
containing
cellular
)
within
(Table
6)
incorporation
but
from
vacuoles
by
the
lysozyme
endotoxin
results
in the
neutrophils,
C1402
are
obtained
measuring
similarly.
The
matic
changes
in
and
appearance
tion of pellets
reveals
particles
In
a decrease
( a-glucosidase
enzymes
metabolic
with
phagocytofrom
glucose-i-C14
acid-soluble
nucleo-
the
tested
less draeosinophils
production
eosinophils
in
g
had
( Fig.
neutrophils
3).
Degranulation
Is
the
integrity
influenced
be
by
obtained
In two
tration
and
that
granule
factors?
of
separate
the
the
the
experimental
of
Only
on the
absorbancy
granules
at 450
of eosinophils
conditions.
the
eosinophils
a limited
difficulty
experiments,
of deoxycholate
by measuring
these
the
same
because
phils.
strate
of
the
in
the
mp.
acquiring
effects
integrity
The
and
and
answer
large
of a high
of eosinophil
results,
neutrophils
of
the
to this
shown
neutrophils
question
numbers
of
could
eosino-
hydrogen
ion
concen-
granules
was
studied
in Figure
behave
similarly
4, demonunder
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
932
CLINE,
HANIFIN,
LEHRER
DiscussioN
The
that
the
precise
cells
function
of the
are phagocytic,
teria
and
bacterial
toxins,
that
are
in contact
with
gest
a role
in
host
and
Certainly,
other
phages,
are
abundant
The
present
are
phils.
and
The
The
role
as yet
sequestration
and
the
processing
in that
hydrogen
ion
as efficient
serum
Watson
in
is
than
of
that
occur
eosinophils
in
these
are
efforts
are
of
subsequent
antibody
as
the
free
late
suspension.15
in an infectious
studies
resemble
ingestion
Their
oxidative
metabolism,
slightly
reduced
study
on
in
and
by
In
limited
),
and
enzymatic
studies,
sensitive
into
the
glass
range
are
at
suspension,
suggested
least
provided
that
surface
phago-
antibodies,
whereas
phagothe high
levels
of antibodies
from
are
occurs
the
granules
consequences
be
unaffected
reduced
protein
by
of
by
particle
inhibitors
of
and
production
and
phagocytosis
both
ingestion
and
particle
of glycolysis
Lactate
shunt
eosinophils
deoxycholate.
known
inhibitors
observed
eosino-
for
of phagocytosis.
by
synthesis.
all stimulated
following
with
metabolic
Degranulation,
tests,
patients
requirements3’#{176}”6
to
of
RNA
and
to
free
hexose-monophosphate
neutrophils.
to acid
resemble
a broad
metabolic
is strikingly
inhibitors
eosinophils
of H3-uridine
eosinophils
by
of the
appears
but
the
neutrophils,
Wood,
Smith
and
may be a more
efficient
process
eosinophils
their
some
capacity
The
their
physioeosinophil
or
over
adherent
They
that
in
in at least
and
in
Eosinophils
ingestion
cells
possible
phagocytosis
process.
indicate
neutrophils
phagocytic
by
production.
phagocytes.
same
allergic
by antigenprocesses.14
Among
may
be important
are
unique
to
of particle
fungi
various
is unknown.
Eosinophils
yeast
eosinophils
certain
in
aggregates
eosin-
as neutro-
are
attracted
in immunologic
of immune
other
capable
with
normal
abundant
they
processes
for
patients
that
macrodefensive
of bacteria
shown
or in
is un-
and
in phagocytizing
From
studies
of rabbit
that
surface
phagocytosis
in
sug-
Whether
efficient
from
strains
have
disarming
the
ingesting
available.
concluded
present
philia
equally
eosinophils
may require
only low levels
of opsonizing
by freely
suspended
cells
may
require
The
tion
more
unreported,
concentrations.
phagocytosis
cytosis
cytosis
neutrophils
instances
the eosinophil
these
functions
they
flora
microorganisms.
including
of certain
antigens
of
duplications
neutrophils
cell
of
membranes
bacterial
some
evidence
that
their
involvement
and
phagocytic
capacities
logic
functions-whether
its
cells,
that
that
of the
are
of
indicate
observation
roles
merely
in
phagocytes
and
experimental
aggregates
suggest
specific
these
less effective
than
neutrophils
ingested
microorganisms.
the
clinical
conditions
antibody
and
studies,
in mucous
and
against
phagocytic
as efficient
Additional
considerably
in killing
are
abundant
environment
The observations
inactivating
bac-
cells
are present
in the circulating
blood
to be critical
in such
defense
reactions
studies
not
is unknown.
capable
of
particularly
reactions
known.
more
eosinophil
enzymes
external
defense
numbers
of these
active
inflammation
ophiia
are
the
sufficient
sites
of
units.
human
possess
neutrophils
the
(one
incorpora-
in both
human
morphologically
in both
cell
appear
types.
to
be
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
933
PHAGOCYTOSIS
In
in
conclusion,
the
human
range
appear
but
to be
not
eosinophils
in
similar
the
in the
of phagocytosis.
We
of physiologic
functions
appear
efficiency
of
requirements
may
thus
for
add
that
to
particle
these
these
resemble
human
ingestion.
The
and
the
aspects
two
cell types
consequences
metabolic
of
of
types
neutrophils
two
phagocytosis
human
to
white
cells
the
list
have
in
common.
SUMMARY
Eosinophils
tested
were
phagocytic
for
Escherichia
coli,
so less
red
cells.
Metabolic
over
lysosomal
had
of
RNA,
enzymes
similar
a broad
on
glucose
and
in
glycolysis,
extent.
and
neutrophils
oxidation
presence
and
incorporation
degranulation
and
by
the
did
puromycin
enhanced
oxidation
in
but
antibody-coated
range
of
including
similar
albicans,
and
eosinophils.
eosinophils
En-
and
neutro-
in vitro.
SUMMARIO
Eosinophulos
testate
esseva
pro
styreno,
br
minus
coli,
del
Concomitantes
in
enzymas
del
le
stimulation
lysosomal-esseva
effectos
super
le
in un
phagocytose-incluse
del
oxydation
oxydation
de
in
e Candida
area
esseva
de
sed
erythrocytos
in
poly-
isto
ilos
revestite
de pH
glycolyse,
illos
incite
albicans,
de valores
de
e
particulas
phagocytava
large
le
de
glucosa,
neutrophilos
glucosa
eosinophiia,
de
e in ie presentia
puromycina
e colchicina,
grados.
del
simile
non
Inhibitores
a vane
con
ingereva
520A,
Illos
oxydative.
particulas
patientes
eosinophilos
aureus
occurreva
metabolismo
INTERLINGUA
de
Le
neutrophilos.
metabolic
ARN,
IN
sanguine
Staphylococcus
que
le ingestion
le
phagocytic.
efficace
inhibitores
ad
ab
Le phagocytose
anticorpore.
inhibiva
isolate
capacitate
Escherichia
esseva
de
pH
Inhibitors
to a variable
of glucose
were
effects
did
phagocytosis,
stimulation
with
eosinophilia
and
polystyrene
particles,
and Candida
not phagocytize
502A
They
occurred
concomitants
into
dotoxin
aureus
neutrophils.
of oxidative
metabolism.
inhibited
particle
ingestion
of uridine
phils
than
Phagocytosis
of
from
the blood
of patients
Eosinophils
ingested
inert
Staphylococcus
efficiently
of inhibitors
colchicine
loss
isolated
ability.
per
promovite
e le
e eosinophilos.
eosinophilos
de uridina
incorporation
disgranulation
e
le
Endotoxina
e neutrophilos
perdita
habeva
in
studios
de
simile
effectuate
vitro.
in
ACKNOWLEDGMENT
The
electron
were
micrographs
Medical
California
Center,
kindly
prepared
by
Dr.
William
Davis,
University
of
San Francisco.
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1968 32: 922-934
Phagocytosis by Human Eosinophils
MARTIN J. CLINE, JON HANIFIN and ROBERT I. LEHRER
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