receptor (CD44) expression and function in human peripheral blood monocytes and alveolar macrophages. Martine. Culty Thomas. E. O'Mara,T. Charles.
Hyaluronan receptor (CD44) expression and function in human peripheral blood monocytes and alveolar macrophages Culty Thomas P Swartz’
Martine
Rodney
Abstract:
*Department
of Cell
Washington,
DC
CD44
Biology
glycoproteins
are
E. O’Mara,T and
tpulmonary
present
on
Charles and
the
B. Underhill
Critical
sur-
Care
ple
Medicine,
Henry Georgetown
proinflammatory
and cytokine release [14, density of CD44 is increased with the degree of synovial
explanted human peripheral hibit a major CD44 band
tous
alveolar ranging began
180
macrophages from 85 to 200 expressing new
kDa.
Newly
(AM) kDa. CD44
(PBMs) exautologous
express multiple isoforms Within 4 h in culture, PBMs isoforms of 120, 150, and
explanted
AM
specifically
bound
[3H]hyaluronan (135 cpm/cg protein), but newly cxplanted PBMs did not. However, in vitro cultured PBM progressively acquired the ability to bind [3H]hyaluronan and exhibited specific binding of hyaluronan similar to that of AMcb (113 cpm/tg protein) after 4 days in culture. In both cases, the binding of [3H]hyaluronan was specifically inhibited by the addition of monoclonal antibody directed against CD44. AM4 readily degraded 3H]hyaluronan and reached a plateau after 4 days in culture (115 cpm/g protein). Newly explanted PBM exhibit no hyaluronan degradation and only a small degradative activity after 4 days in culture (6 to 11 cpm/sg protein). Thus, CD44 expression and function appear to change as PBM mature in vitro resembling more that found in AMcb.J.
Key ronan
Leukoc.
Words: human
Biol. macrophage
56:
605-611; monocyle
1994. CD44
receptor
diseases,
Jr.,’
University
Medical
functions,
faces of many hematopoietic cells and in some cases can bind hyaluronan, a major component of the extracellular matrix. In the present study, we have found that newly blood monocytes of 85 kDa, whereas
Yeager,
CD44
Center,
including
15].
T
cell
In
rheumatoid on synovial cells inflammation [16].
expression
granuloma formation The expression of change with maturation
and
and CD44
is
increased
fibrosis
[4].
has previously granulocyte,
in
activation
arthritis, the and correlates In granulomain the area of been cell,
T
shown to and B cell
lineages [17-19]. The presence of CD44 complexes on monocytes and alveolar macrophages has also been reported [1, 15]. Although monocytes are the immature hematopoietic precursors of macrophages, there has been no comparative study In
of CD44 this
peripheral
different explanted hyaluronan. bind and maturation
expression
in humans. human blood monocytes (PBM5) and AM express isoforms of CD44. We also demonstrate that newly PBMs are incapable of binding and degrading In contrast, newly explanted AM4 efficiently degrade hyaluronan. Moreover, we find that upon in culture, PBMs express different CD44 isopaper
we
and
function
report
in these
that
newly
cells
explanted
forms than are present on newly explanted PBMs. More important, they also develop the ability to bind hyaluronan. However, the ability of cultured PBMs to degrade hyaluronan does not increase to the same extent and stays very low compared with that of mature AM, suggesting that the acquisition of this function may require other regulatory factors.
hyalu-
MATERIALS INTRODUCTION
AND METHODS
Volunteers
Hyaluronan, a major component of the extracellular matrix, has been shown to increase in certain stages of the developing lung [1] and tissues undergoing inflammation and repair [2-4]. Elevated levels of hyaluronan have also been reported in experimental bacille Calmette-Gu#{233}rin (BCG) infections in rodents, and hyaluronan-protein complexes have been referred to as “macrophage agglutinating factors” [5]. In vitro, hyaluronan has been shown to have variable effects on phagocytic cells including human alveolar macrophages (AM4) [6, 7], and has been shown to bind cytokines [8]. The turnover of hyaluronan is in part mediated by its cell surface receptor, which is a member of the CD44 family of glycoproteins. CD44 is expressed on several human cell types, including lymphocytes, alveolar macrophages, fibroblasts, and numerous tumor cells [9]. CD44 mediates the uptake and degradation of hyaluronan by rodent alveolar macrophages and transformed fibroblasts. In addition, CD44 is considered to be a cell adhesion molecule and has been involved in functions such as lymphocyte homing, hyaluronan-dependent cell aggregation, and cell migration [10-13]. CD44 has multi-
After rum
informed consent was obtained, were obtained from 19 healthy,
and senons-
mokers, 18-34 years of age, with no indications of respiratory tract infections and with normal chest roentgenograms and ventilatory tests. The protocol was approved by the Human Research Committee, Georgetown Medical Center, and was carried out according to the principles of the Declaration of Helsinki.
Abbreviations: monocyte; BALC, solution; PBS-FT,
AM, alveolar bronchoalveolar phosphate-buffered
0.05%
DOC
Tris,
Tween-20;
pH
Reprint
8.0;
SDS,
requests:
buffer,
of
Leukocyte
macrophage; lavage cell; saline, 0.01%
sodium
dodecyl
Martine
Culty,
Dental Building, Georgetown Road, NW, Washington, DC Received January 26, 1994;
Journal
AM4, PBMs, male and female
Biology
PBM, peripheral blood HBSS, Hanks’ balanced salt 10% fetal bovine serum,
deoxycholate,
sulfate;
0.5
LPS,
Department
University 20007. accepted
Volume
of Cell
Medical June
56,
15,
M
NaCl,
0.02
M
lipopolysaccharide. Center,
Biology,
3900
Medical-
Reservoir
1994.
November
1994
605
Antibodies
temperature.
The hybridoma cells body (rat anti-human Butcher (Department School, Stanford, cites fluid of nude
producing CD44) of
blood
isolation
monocyte
and
culture
purchased
passed MA)
through before
a use.
blood cells and PBMs
produced 90-95% agnostics, 2.0 the
(St.
PBMs
were
performed
Alveolar
macrophage
cell and
counts Nathan
Western
Diof
were determined [21].
by
collection
Bronchoalveolar lavage cells (BALCs) were obtained by fiber-optic bronchoscopy and sterile saline lavage in a bronchoscopy suite [22]. After local anaesthetic, the right lower lobe was lavaged with four 60-ml quantities of sterile, nonpyrogenic, 0.9% sodium chloride, pH 5.5, 25#{176}C.The lavage fluid was removed by gentle suction, pooled, and kept on ice. The pooled lavage fluid was centrifuged (400g 20 mm, 4#{176}C). The pelleted BALCs were washed three times in cold HBSS and resuspended in ExCell-320 medium. BALC viability was 80-85% as determined by trypan blue dye exclusion. For all assays, the BALCs were adjusted to approximately I x I0 viable BALCs/ml of ExCell-320 medium and 0.2-mi aliquots were dispensed into 24-well tissue culture plates. The AM4 were allowed to adhere to the wells for 1 h at 37#{176}C, 5% CO2. The nonadherent cells were removed by vigorous washing with warm HBSS, and 1.0 ml of medium was added to each well. The resulting AM-enriched cultures AM
consisted of 95-97% per well. Total cell
method
of Nakagawara
Serum
collection
and
AM4 counts
at approximately were determined
Nathan
2.0 by
x 10 the
[21].
Fresh autologous human serum was obtained from nonheparinized peripheral whole blood. The blood was allowed to clot in glass tubes at room temperature for 20 mm, placed on ice for 30 mm, then centrifuged at l500g for 1 h at room
606
Journal
of Leukocyte
Biology
Volume
56,
November
cultures were prepared as described above. Immedifollowing the attachment step, the PBMs were washed with HBSS to remove nonadherent cells and medium. PBMs were then cultured in either medium alone or 5 to 100% autologous were harvested for
human Western
serum. Afblot analysis
of CD44.
5 x 106 viable cells/mi. Aliquots (0.2 24-well tissue culture plates (Costar, PBMs were allowed to adhere to the 37#{176}Cin 5% humidified CO2. The removed by vigorous washing of the balanced salt solution (HBSS), and added to the cultures. This protocol
PBMs/well. Total of Nakagawara
studies
medium containing ter 24 h, the PBMs
were suspended in serumBiosciences, Lenaxa, KS). trypan blue dye and ad-
enriched cultures consisting of approximately PBMs as determined by Diff-Quik stain (Dade Aquada, PR), yielding a final average number
x i0 method
PBM ately twice The
[20]. Briefly, peripheral blood was and the mononuclear cells were on Ficoll-Paque solution (PharViability was assessed by trypan
blue dye exclusion, and the cells less medium (ExCell-320; JRH Viable cells were counted using justed to approximately ml) were dispensed into Cambridge, MA). The well bottoms for 1 h at nonadherent cells were wells with warm Hanks’ 1.0 ml of medium was
Sigma
collection of human
as previously described obtained by venipuncture separated by centrifugation macia, Piscataway, NJ).
from
asby
of peripheral
and Bedford,
Serum
was
from IN)
Teflon plate cultures
removed
rat immunoglobulin Louis, MO).
G (IgG)
was isolated Indianapolis,
was
(Millipore,
filter
chromatography on DEAE Affi-gel blue (Bio-Rad, Richmond, CA). The K-3 monoclonal antibody (mouse antihamster CD44) was isolated from ascites fluid by chromatography on a protein A-Sepharose column [ii]. The BU-52 monocional antibody (mouse anti-human CD44) was purchased from The Binding Site (San Diego, CA). Nonspecific
The
The antibody (Bioproducts,
antiby Dr. Medical
serum
membrane
Mononuclear cells (2.0 x 106) were isolated from blood as described above and were added directly to Teflon culture plates (Scientific Specialties Service, Randallstown, MD) in 5 ml of ExCell-320 medium. PBMs, allowed to attach to plastic for 30 mm, were scraped free, washed in HBSS, and 1.0 x 106 were added to Teflon culture plates in 5 ml of ExCell-320 medium. At 24 h, the cultures were harvested for Western blot analysis of CD44.
Peripheral
CA). mice
Hermes-i monoclonal were kindly donated Pathology, Stanford
The
0.45-sm
blotting
The medium was removed from cultures and the cell layers were washed with HBSS and then dissolved in Laemmli sample buffer [23] lacking $-mercaptoethanol. Each sample (standardized to protein content or cell number) and highmolecular-weight prestained standards (Bio-Rad Laboratories, Melville, NY) were electrophoresed on an 8% sodium dodecyl sulfate (SDS)-polyacrylamide gel (Miniprotean II, Bio-Rad Laboratories) and then transferred to a sheet of nitrocellulose (Immobilon-NC, Millipore, Bedford, MA) at 0.9 amperes for 30 mm using a Trans-Blot Cell (Idea, Corvalis, OR). The nitrocellulose sheet was blocked in 5% nonfat milk for I h and incubated for 1 h with mouse antihuman CD44 monoclonal antibody, BU52, diluted to 1:10,000 in PBS-FT (phosphate-buffered saline, 10% fetal bovine serum, 0.05% Tween-20). The blots were washed twice with PBS-FT and incubated for 1 h with a peroxidaselabeled anti-mouse immunoglobulin (Ig) secondary antibody. The blots were extensively washed with distilled water and incubated in a color reagent solution (0.03% H202, 0.2 mg/ml 3-amino-9-ethylcarbazole in 0.05 M sodium acetate, pH 5.0).
Assay for [3Hlhyaluronan Hyaluronan modified 25]. The M NaCI, quots volume
rated and tubes
binding
activity
binding activity was determined using a version of the method previously described [24, cells were dissolved in 0.1% Na deoxycholate, 0.5 0.02 M Tris, pH 8.0 (DOC buffer) and 200-1d ali-
were
incubated
with
1 tg
of
[3H]hyaluronan
in
a final
250 p1 After shaking for 20 mm, 250 l of satu(NH4)2SO4 was added followed by 25 l of nonfat milk the samples were centrifuged at 9000g for 5 mm. The were rinsed twice with 50% saturated (NH+)2SO4 and of
the pellets were tion counting.
dissolved in water The background
and level
processed of binding
for scintillawas deter-
mined by including an excess of nonlabeled hyaluronan (60 cg) in some samples and was subtracted from each value. The results are expressed as cpm of bound [3Hjhyaluronan normalized to protein, which was measured for each cell extract using the bicinchoninic acid assay (Pierce, Rsckford, IL). To assess the effect of blocking antibodies on the binding of [3H]hyaluronan to CD44, the monoclonal antibodies
1994
Hermes-i or K-3 were incubated with the prior to the addition of the [3H]hyaluronan. IgG
was
Assay
used
as
control
cell
extracts Nonspecific
15
mm rat
antibody.
for [3H]hyaluronan
2O5-.
degradation
The extent of [3H]hyaluronan degradation by AM and PBM cultures was determined as previously described [13]. To each well of AM and PBMs, 2 tg/ml [3H]hyaluronan was added. At various times after the addition, the incubations were stopped by adding 200 jl of pronase E (20 mg/ml solution;
Sigma
Chemical
Company)
to
the
cultures.
116-.’80-.’
The
cells were digested with protease overnight. The digests were centrifuged in Centricon-30 microconcentrators (Amicon, Danvers, MA). The material passing through the membrane was collected and processed for scintillation counting. This procedure has been shown to give similar results to molecular-sieve chromatography for determining the degradation of [3H]hyaluronan [13]. Background levels of degradation were determined by incubating [3H]hyaluronan in a cell-free medium for identical time periods and were subtracted from each value. The data are expressed in terms of cpm of hyaluronan fragments produced per tg of protein in the cell layer. This latter value was determined by extracting representative wells with DOC buffer and assaying them for protein as described above.
0.5
-205 -116
1. Western
peripheral
blood
blot
analysis monocytes
of CD44
(PBMs)
expression
and
on
alveolar
newly
explanted
macrophages
human
(AM).
Lanes I and 3 are the PBMs and AM& respectively, from donor A. Lanes 2 and 4 are the PBMs and AM, respectively, from donor B. Cells (2 x l05/well) were solubilized in 60 sl of Laemmli buffer without 3mercaptoethanol and 20 jslwas electrophoresed in 8% SDS-polyacrylamide gel. Presence of CD44 on nitrocellulose was detected using a monoclonal antibody (BU52) and a peroxidase-coupled secondary monodonal antibody. Arrows on the left indicate the location of CD44 bands. Arrows on the right
indicate
the positions
of various
molecular
weight
standards.
6
tured
Western
analysis
PBMs
ExCell-320
were
medium.
Laemmli
in
blot
PBMs. buffer
8%
of CD44
cultured
Cells
without
(2
antibody
expression plastic
x l05/well)
gel.
and
culturing
the the
of AM
at
and
tissue
times culture
solubilized 20
isoforms a
various
24-well
were
CD44
(BU52)
monoclonal antibody. Arrows on bands. Arrows on the left indicate standards.
vitro
(hr)
fi-mercaptoethanol
SDS-polyacrylamide
monoclonal
on
24
tl
was
were
in
60
detected
in
sl
of
using
a
secondary
right indicate the positions of various
7 days
cul-
electrophoresed
peroxidase-coupled
for
in plates
location molecular
resulted
of CD44 weight
in
little
change in the CD44 profIle. However, under the same conditions, CD44 expression by PBMs changed within the first 24 h in culture (Fig. 2). While the 85-kDa CD44 band remained unchanged, there was an increased expression of the 120-, 150-, and 180-kDa isoforms. Newly explanted AM4 were able to bind [3H]hyaluronan (135 cpm/&g protein) (Fig. 3). In contrast, newly explanted PBMs However,
exhibited the
no PBMs
significant progressively
binding
of acquired
[3H]hyaluronan. the ability
to
bind [3H]hyaluronan and exhibited a specific binding of [3Hjhyaluronan of 113 cpm/ig protein after 4 days in culture, remaining at similar binding levels for the following days. When monoclonal antibodies that have been shown to block CD44 function (Hermes-I; K-3) [25] were added to the 2- and 24-h cell extracts 15 mm prior to the addition of [3H]hyaluronan, the binding of [3H]hyaluronan was significantly reduced, whereas the addition of nonspecific rat IgG had little effect (Table 1). AM4 exhibited the ability to degrade [3H]hyaluronan in vitro, with the level of hyaluronan fragments formed plateauing by 4 days (Fig. 4). In contrast, PBMs exhibited no ability to degrade hyaluronan in the first days of incubation and reached very low levels of hyaluronan fragment production after 4 days in culture (ranging from 6 to 11 cpmJtg protein in different experiments). To determine what mechanisms may be altering the expression of CD44 in cultured PBMs, we examined the effects of altering the culture conditions of the PBMs (Fig 5). Since lipopolysaccharide (LPS) has been shown to alter PBM ac-
-80 Fig.
2.
Fig.
In
band at 85 kDa. Of the 19 individuals tested, PBMs from 13 showed only the 85-kDa form of CD44 and PBMs from 6 individuals exhibited three additional faint bands at higher molecular masses of 120, 150, and 180 kDa.
4
time
RESULTS Western blot analysis of newly explanted AM/ demonstrated a diffuse pattern indicative of CD44 isoforms ranging in molecular size from approximately 85 to 200 kDa (Fig. 1, lanes 3 and 4). When less protein was loaded, the same diffuse pattern was observed (data not shown). In contrast, newly explanted PBMs (lanes 1 and 2) exhibited a major
2
tivity myxin negate
[26, 27], we supplemented the medium with polyB, an antibiotic that binds to and inactivates LPS, to the effect of any contaminating LPS. Western blot analysis (Fig. 5A) showed no difference in CD44 expression between PBMs cultured with polymyxin B (lane 2) and control cultures (lane 1). We assessed the effect of the presence of lymphocytes (Fig. 5B) on the expression of CD44 at 0.5 and 24 h by comparing cultures of nonfractionated buffy coat cells (lymphocytes and
Cu!ty
et a!.
CD44
expression
and
function
in monocytes
607
monocytes = B), adherent PBMs (P), and nonadherent cells (predominantly lymphocytes = L). Both lymphocytes and PBMs expressed the 85-kDa CD44. The presence of lymphocytes in the PBM cultures did not decrease the appearance of the higher-molecular-weight CD44 isoforms but rather the combination of both cell populations showed in-
250
200
creased expression of all CD44 bands cultured in the absence of lymphocytes. Adherence of PBMs has been shown [28-30]. Therefore we assessed whether
J
150
.AM#{216} 100
5/1
PBM
.01234567
Incubation Fig.
3.
Binding
of
3Hjhyaluronan.
blood monocytes were cultured ExCell-320 medium supplemented
time
periods
quots room
were
cpm
for
of two
to
were
solubilized
with
1 sg
scintillation Each
nine
and
of
[3H]hyaluronan
counting.
to
point
independent
results
protein
(mean
and
are as
range)
±
activity may alter
culture of CD44 nonadherent
plates (T). The appeared in PBMs cells. However,
higher-molecular-weight cultured as both the magnitude
adherof the
150
in
mm
20
sulfate,
The
normalized
time
plates
At various buffer. Mifor
ammonium
their
peripheral
culture
serum. in deoxycholate
with
bound,
Methods.
days
macrophages
24-well plastic tissue with 5% autologous
precipitated
[3Hhyaluronan
and
average
incubated
then
processed
of
Materials
on
the cells
of 250 sl were temperature,
pellets as
in culture,
time,
Alveolar
to alter adherence
PBMs
expression was greater in the PBMs cultured in Teflon plates. Buffy coat cells (PBM + lymphocytes), which had always been cultured in Teflon and thus never adherent (NA), expressed very low amounts of the higher-molecular-weight isoforms. We tested whether the presence of autologous serum may alter the CD44 profile observed after 24 h culture of PBMs. Adherent cultures of PBMs were incubated for 24 h in ExCell-320 medium, alone or supplemented with 5, 10, 25, 50, 75, or 100% autologous serum. Western blot analysis indicates that the expression of CD44 is enhanced by increasing the concentration of autologous serum up to 50%.
7
rnO
0
with
CD44 expression (Fig. SC). After the initial purification by adherence for 30 mi PBMs were incubated for 24 h as adherent cultures on plastic wells (A) or as suspension cultures
I
in Teflon isoforms ent and
50
compared
at
the
C
in
0
expressed
described represents
the
experiments.
C)
100
E 0 TABLE
1.
Effect
of Specific
Antibodies
AM4
and
on
[3HjHyaluronan
Binding
C
to
PBM?
0 Cell
Culture (h)
type
added
2
PBM
24
0
234
±
10
212
±
7
K-3
30
Alveolar
bodies:
monoclonal The
of [‘Hjhyaluronan 8
sg for
Mean
608
and
range
Journal
4
0
28±4
5
6
and
of duplicate
normalized
to
24
h AM;
CD44 and
2
-
±
3
79
monocytes with
8
Biology
both
anti-
blocking
monodonal
content.
sg for
are
(PBM5)
CD44
antibody. Results
Protein 2 and
#{176} cpm
content:
24 h PBMs.
given.
Volume
56,
November
0 7
0123456
Aliquots
the various
Methods.
protein
determinations
of Leukocyte
53
±
anti-hamster
anti-human Materials
4
24 h in culture.
15 mm
for
in
for
86 ±
blood
and
2
K-3,
IgG;
Hermes-i,
sg
after
incubated
rat
is described 18
peripheral
x
24
80
buffer were
72
24±9
None
bound,
2 h AMt’;
±
9
(AM)
9
130±3
0
nonspecific assay
171
None
antibody;
binding
0
5
in deoxycholate
IgG,
6
30
jsl) of the cell extracts
(250
±
Hermes-I
macrophages
solubilized
66
K-3
C) 0
80
5 25
50
-
47±3
IgG
Hermes-l
were
protein)
25
None
Inhibition (%)
bound
IgG
Hermes-I 24
[‘I-l]Hyaluronan (cpm/sg
(pg)
None
2
AMI
Amount
Antibody
Incubation Fig. 4. peripheral plates the
in presence
Degradation of blood monocytes ExCell-320 of 2 sg/ml
medium
time,
13H]hyaluronan. were cultured
Alveolar on
supplemented
[3H]hyaluronan.
days
with At
macrophages
plastic
various
24-well
5%
tissue
autologous
times,
the
and culture serum
cultures
in were
digested with Pronase-E and applied to Centricon 30 microconcentrators. The material passing through the membrane was collected and processed for scintillation counting. The results are expressed as cpm of [3H]hyaluronan fragments formed normalized to protein. Data are mean ± range of duplicate determinations from one representative experiment.
1994
205-*116 80-’-
Si
2
PLB
B -4
P
Ohr 5.
Fig.
Effect
of various
culture
conditions
on
CD44
LPA
TNA
‘
expression
24hr
in PBMs.
PBMs
were
24hr
Ohr
cultured
on
plastic
24-well
tissue
culture
plates
in ExCell-320
medium
except where noted. Arrows on the right indicate the location of CD44 bands. Arrows on the left indicate the positions of various molecular weight standards. Total protein per lane 6 sg. (A) Effect of LPS. S, Standard molecular weight markers; I, PBMs cultured for 24 h in medium alone; 2, PBMs cultured in medium supplemented with 2 g/ml polymyxin B. (B) Effect of lymphocytes. B, PBMs cultured with lymphocytes; P, PBMs cultured alone; L, lymphocytes cultured alone. Cells were harvested after 30 mm (time 0) and 24 h in culture. (C) Effect of adherence on CD44 expression. P, Freshly isolated cells (PBMs + lymphocytes); A, PBMs cultured for 24 h as adherent cells on plastic; T, nonadherent PBMs in Teflon plates; NA, PBMs + lymphocytes never adherent
However, weight
and
cultured
at CD44
totally
50%, isoforms
in
the
Teflon
plates
for
24
h.
expression of the higher-molecularappear to be retarded in the first
day
of culture (Fig. 6). Longer exposures to high amounts of serum led to enhanced expression of all CD44 forms by PBMs (data not shown). However, it is not clear whether the longterm effects are specific or due to a general improvement of
the
cell
by
presence
In
this
tially
study,
form
805
1025
5075100
%serum ‘ig. 6. Effect of autologous serum on CD44 expression of adherent PBMs. BMs (2 x l05/well) were cultured for 24 h on plastic 24-well tissue culture lates in ExCell-320 medium with increasing concentrations of serum. S, tandard molecular weight markers; C, PBMs at 0.5 h; 0-100, PBMs at 24 cultured in medium supplemented with serum. Western blot analysis of D44 was performed on all samples as described in the legend of Figure 1. krrows on the right indicate the location of CD44 bands. Arrows on the left ndicate
the
positions
of
various
molecular
weight
we
have
that
explanted
of
However,
0
their of
morphology
serum
standards.
after
extended
was
a few
our
85
kDa,
of human
whereas
mature
days
improved in
culture.
previous
observations
AM.
PBMs
present
AM4
after
a few
hours
in
a major
express
molecules ranging from 85 to phages represent a heterogeneous phagocytes that have been shown functions and surface markers ogeneity found in the expression the diversity of functions found
$11111.’ I1IIL sc
levels
the hyaluronan receptor (CD44) to the human system. We present evidence that the expression changes as PBM mature in vitro, such that it par-
resembles
Newly
116-
because
of high
DISCUSSION
concerning macrophage of CD44
205-
functions,
a variety
CD44 isoof CD44
200
kDa. Alveolar macropopulation of mature to exhibit a wide variety of [31-33]. Thus, the heterof CD44 may correspond to in alveolar macrophages.
culture,
PBMs
begin
to
express
isoforms of CD44 of 120, 150, and 180 kDa, which are not present or barely detectable in circulating monocytes of most individuals. The expression of fewer CD44 isoforms by PBMs (immature phagocytes) may reflect their more limited repertoire of activities. At present, the mechanism(s) underlying the appearance of these new forms of CD44 remains unknown. This could result either from the formation of splice variants or posttranslational modifications, as both types of alterations have been implicated in the formation of various CD44 molecules [9]. In addition, it is still unclear why the newly explanted PBMs from 6 out of 19 individuals tested presented high-molecular-weight CD44 bands.
Cult,
ci al.
CD44
expression
and
function
in
monocytes
609
To assess relevance, hyaluronan the main
whether these changes could have any functional we then examined the interactions of CD44 and in these cells, because this glycosaminoglycan is ligand for CD44. A comparative study of the hyaluronan binding activity of PBMs and AM revealed that AM4 are very efficient in binding hyaluronan whereas newly explanted PBMs do not bind hyaluronan. However, after 4 days in culture, PBMs acquire hyaluronan binding activity similar to that of AM4. Monoclonal antibodies that have previously been shown to block CD44 function (K-3; Hermes-i) [24] effectively reduced the hyaluronan binding capability of both AM and PBMs, indicating that the majority of the binding of hyaluronan is through CD44. Because we have previously shown that CD44 mediates hyaluronan degradation in rodent macrophages [13], we examined whether human PBMs and AMq bility. Our results indicate that, initially, the ability to degrade hyaluronan, but
also have this capaPBMs do not have they slowly acquire
this function when cultured for a few days. However, the observed level of hyaluronan degradation remained very low compared with that observed in AM4, suggesting that an additional signal(s) is most likely necessary to stimulate the hyaluronan degradation in PBMs to levels observed with the mature AM4,. We examined the possible involvement of several parameters in producing the alterations in CD44 expression by PBMs. One possibility is that the cultures may have contained a small amount of contaminating LPS. The effects of LPS are in part mediated through interleukin-1 and tumor necrosis factor, both of which have been shown to influence CD44 expression in human lung fibroblasts [34]. The change in CD44 expression in our studies did not appear to be driven by LPS, as the addition of polymyxin B to negate the LPS had no effect on CD44 expression in PBMs. It is also possible that lymphocytes (and their products) present in the PBM cultures had an effect on CD44 expression. To test this possibility, monocytes were cocultured with lymphocytes. The changes in CD44 expression were enhanced, indicating that the presence of lymphocytes may potentiate this process. Such an effect may be due to the production of specific cytokines from one or both cell populations. This point is now under investigation. Adherence to plastic or glass has been shown to alter some macrophage activities [32]. Our data comparing PBMs adherent to plastic versus PBMs cultured in Teflon plates indi-
At concentrations CD44 isoforms lying this effect It is probably press
610
Journal
of Leukocyte
Biology
Volume
56,
November
be
the
bind
and
blood
serum exact
appears
under-
elucidated. for the immature
degrade
stream
to retard
mechanism(s) PBMs
hyaluronan
to
their
to
until
normal
target
exthey
tissue
or a site of inflammation, where they will encounter hyaluronan, a major component of the extracellular matrix. Therefore, the changes observed in the expression and function of CD44 must be controlled by specific signals. The determination of these factors clearly requires further investigation. Among the probable candidates are cytokines secreted by the monocytes
in the
as
serum
they
or
maturate
at
the
in
surface
Under normal conditions, in regulating hyaluronan
vivo
and
of the mature
levels
present endothelium.
a factor(s)
vascular AMcb
in
the
may be involved as suggested by
lung,
our results showing that they bind and degrade hyaluronan efficiently. The acquisition by PBMs of these activities may reflect (1) their normal maturation into specific macrophage populations and/or (2) their activation under pathological situations. Along these lines, hyaluronan levels have been shown
to
repair reported
[2,
increase
in
tissues
3]. Elevated in experimental
undergoing
could be important properly in both
inflammation
levels of hyaluronan BCG infections
hyaluronan-protein complexes rophage agglutinating factors ring in CD44 expression and
have been [5]. Thus, its interaction
and
have also in rodents,
been and
referred to as macthe changes occurwith hyaluronan
for the maturing macrophage to function normal and pathological situations.
ACKNOWLEDGMENTS The authors thank preparation of the part by U.S. Health
M. Shizari manuscript. Services
for technical support in This work was supported grant HL41565.
the in
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