Modulation of Interleukin-8
Receptor Expression by Lipopolysaccharide (LPS) and Phorbol Myristate
Acetate (PMA) in Human Peripheral Monocytes - A Preliminary Study
BISWADEV BISHAYI* AND AJOY K. SAMANTA**
*Department of Physiology,
University of Calcutta,
92, A.P.C. Road,
Kolkata – 700 009
**Division of Immunobiology, Indian Institute of Chemical Biology,
** Present Address: 7220, Cambridge St. # 4 Houston, Texas – 77030
(Received on August 12, 2001)
Interleukin-8, a monocyte derived neutrophil chemotactic agent
is known to play as a key mediator in the pathogenesis of a
large number of neutrophil driven inflammatory diseases. Since
the cytokine activates the target cells through a cell surface
receptor, study of the regulation of IL-8 receptor expression
in monocytes is very important. We found that two very known
modulators, lipopolysaccharide (LPS) in presence of homologous
serum and Phorbol myristate acetate (PMA) resulted in induction
of IL-8 receptor by 100-120% and 75-125% respectively within
1 h in monocytes. Based on the inhibitory effect of cycloheximide,
actinomycin-D we may suggest that PMA and LPS could upregulate
IL-8 receptor in monocytes through denovo protein synthesis.
Prior incubation of polymixin B and anti-CD14 antibody to the
monocytes and subsequent stimulation of the cell with ser.act.LPS
resulted in >90% inhibition of IL-8 binding. Scatchard analysis
showed that estimated receptor number in control cell was 7,500
per cell and it increased to 15,500 per cell in ser.act.LPS
stimulate cell. The receptor number in PMA stimulated cells
was 13,000 per cell. Chemical cross-linking of the IL-8 receptor
with 125I labeled IL-8 in the ser.act.LPS and PMA
stimulated cells-indicated that signals at 59 KD were considerably
increased with respect to control. A correlation between LPS
and Ser. act.LPS induced upregulation of IL-8 receptor expression
has been shown. The study with bacterial product and co-carcinogenic
agent thus provides information about the differential expression
of IL-8 receptor for sustained IL-8 mediated biological response.
words: IL-8 receptor LPS monocytes
Monocytes play a vital role in the defensive mechanism;
immunological response and thus the cells play an important
role in host immunity. In some inflammatory diseases, excessive
accumulation of monocytes is observed along with neutrophils
at the involved sites (1). It is reported that the directional
migration of monocytes from the blood compartment into tissues
is regulated by the chemoattractant obtained from cell-secreted
proteins, bacterial peptides and products of phospholipid metabolism
(2,3). A monocyte chemoattractant protein-1 (MCP-1) has been
reported to be secreted constitutively or after induction with
mitogens, cytokines, and growth factors by a number of cell
types including fibroblast, endothelial cells, mesangial cells,
lymphocyte and various tumor cell lines (4,5). Two other chemoattractants
of monocytes have been purified from cytokine stimulated osteosarcoma
cells (MG-63) designated as MCP-2 and MCP-3 which were homologous
to MCP-1 (62% and 71% respectively) (6). All these chemoattractants
belong to the class of C-C chemokine subfamily where two conserved
cysteine residues remain side by side without presence of intervening
amino acids between the two cysteine residues. These chemoattractant
cytokines belong to the class of chemokine that primarily chemoattract
monocytes and T lymphocytes whereas other class of chemokines
predominantly act on neutrophils (3, 7) although the individual
chemokine has specific effects or the monocytes, during inflammation
more complicated situation may arise due to presence of a cocktail
of several inflammatory cytokine in the inflamed tissues. For
that reason knowledge about the contribution of individual cytokine
on the target cells is needed for detailed understanding of
the pathogenesis of the inflammatory diseases.
(IL-8), a neutrophil activating and chemotactic agent is known
to be released primarily from monocytes in presence of lipopolysaccharide,
LPS (8, 0). IL-8 is known to be implicated in the pathogenesis
of a large number of neutrophil driven inflammatory diseases
like skin diseases, lung diseases and joint diseases. In many
such diseases, monocytes have been observed to migrate together
with neutrophils at the site of inflammation (1). Although the
effect of IL-8 on neutrophils has been studied in detail and
monocytes are known as the predominant source of the cytokine,
knowledge about the direct effect of IL-8 on monocytes is very
limited. Leonard et al. have demonstrated under fluorescent
microscope that a chemical conjugation of NAP-1/IL-8 with FITC
binds to monocytes (10). Chuntharapai et. al detected both
type-A and type-B IL-8 receptor in human monocytes and in other
leukocytes by flow cytometry (11). Recently we have also presented
biochemical evidences for identification of IL-8 receptor in
human monocytes (12). Since IL-8 is an inflammatory cytokine,
and monocytes actively participate in many inflammatory diseases,
the information about the regulation ofIL-8 receptor expression
to normal and diseased state is very important.
(LPS), the major outer membrane constituent of the negative
bacteria is considered a major factor in the pathogenesis of
gram negative bacterial infections (13). Although monocytes
are considered as the predominant source if IL-8 produced upon
interaction with LPS during bacterial infection, no report is
available on the expression of IL-8 receptor on the surface
of monocytes. Phorbol myrisate acetate (PMA), a co-carcinogenic
agent, exerts pleiotropic effects on a number of cell types.
The effects of PMA on the regulation of IL-8 receptor expression
in human monocytes is another relevant point that needs to be
addressed. The aim of this work was to investigate the effects
of LPS and PMA on the expression of cell surface IL-8 receptor
in human monocytes.
human recombinant IL-8 (2x106 U/mg) was obtained
from Prof. K. Matsushima, Kanazawa University, Japan. Bovine
serum albumin, LPS (Serotype 055:B5), PMA cycloheximide, actinomycin
D, polymyxin B sulfate, anti-rabbit IgG (raised in goat), anti-human
CD14 MoAB (UCHM-1), protease inhibitors, Histopaque-1077, dextran
500, were obtained from Sigma chemical Company, St. Louis, USA.
Disuccinimidyl suberate (DSS), CHAPS, were purchased from the
Pierce chemical company, Rockford, IL, USA. Polyclonal human
anti-IL-8 receptor antibody was raised in rabbits against 59kD
receptor protein of human neutrophils and the antibody was characterized
by 60-70% binding inhibition of 125I IL-8 to neutrophils
and western blotting (14, 15). Other reagents and chemicals
were of analytical grade.
Preparation of human peripheral monocytes
venous blood (oxalated) was obtained from healthy donors. Peripheral
blood mononuclear cells (PBMC) were separated from whole blood
by dextran sedimentation followed by Histopaque density gradient
centrifugation method as described earlier (16, 12) PBMC fraction
after washing with DPBS was suspended in RPMI-1640 containing
20mM HEPES (pH 7.2) and 10% FBS. The cells were allowed to adhere
on the glass surface for 2 h at 37°C in a CO2 incubator.
The non-adherent cells were aspirated and the adhered cells
were recovered using a rubber policeman. After washing, the
cells were suspended in binding medium containing 20mM HEPES
buffer (pH 7.2) 0.5% BSA. Monocytes Obtained from this preparation
was devoid of neutrophils and the viability of the cells was
examined by trypan blue dye exclusion test which showed 94-97%
Iodination of IL-8
recombinant IL-8 was labeled with 125I using chloramine-T
according to the method of Grob et. al. (17) with
slight modification as described earlier (18, 19). The specific
activity of the labeled IL-8 as determined by 10% TCA precipitation
method was from 3x107 to 5x107 cpm/µg
vitro treatment of monocytes with immunomodulatory agents
order to investigate the effect of LPS on IL-8 receptor expression,
freshly purified human monocytes (2x106/200
µl) suspended in RPMI-1640 were incubated with different amounts
of LPS at 37°C for different time periods and the effect of
serum activated LPS on the expression of IL-8 receptor, LPS
was preincubated with autologous serum at 37°C, then the cells
were incubated with different amounts of serum mixed LPS.
order to examine the effects of PMA on the modulation of IL-8
receptor, the cells were incubated with different amounts of
PMA for 1 h at 37°C. After incubation the cells were washed,
binding of 125I labeled IL-8 to the cells were carried
out at 4°C for 90 min and the total binding of IL-8 to the cells
were measured by a Gamma Counter.
Treatment of metabolic inhibitors on the SA-LPS and PMA stimulated
monocytes (2 x 106/200 µl) suspended in binding medium
were incubated at 37°C separately with cycloheximide (10 µg/ml),
actinomycin D (I µM), colchicines (5 µg/ml), tunicanycin (10
µg/ml) and polymyxin-B (50 µg/ml) for 15 min. Then serum activated
LPS (10 µg/ml) and PMA was added separately and incubated at
37°C for 1 h as before. After incubation cells were washed,
cooled and binding of IL-8 was examined using 5 mg 125I
labeled IL-8/tube. To determine the effects of salicylate and
hydrocortisome on the SA-LPS and PMA induced activation of monocytes
we incubated the cells with 100 µg/ml salicylate and 100 µM
hydrocortisome for 15 min 37°C then SA-LPS (10 ng/ml) and PMA
(10 ng/ml) were added and the incubation was continued as before.
After washing, binding ability of 125I IL-8 to the
cells was examined.
of anti-CD14 and anti-IL-8 receptor antibody
order to investigate the involvement of CD14 in SA-LPS mediated
IL-8 receptor expression, cells were first incubated with anti-CD14
MoAB followed by stimulation with SA-LPS as before. A control
set without addition of anti-CD14 Ab was run in parallel. Then
the cells were washed and the binding of IL-8 to the cells were
examined with 125I labeled IL-8.
another experiment, after stimulation with SA-LPS or PMA the
cells were incubated with anti-IL-8 receptor antibody for 45
min at 37°C. After washing away the excess first antibody, the
cells were incubated with 125I IgG for 1 h. The binding
of 125I labelled anti-rabbit IgG was measured in
(2 x 106/200 µl) suspended in binding medium were
incubated with SA-LPS (10 ng/ml) and PMA (100 ng/ml) for 1 h
at 37°C as before. After washing, cells were incubated with
various amounts of 125I IL-8 for 90 min at 4°C. A
control set, without addition of SA-LPS and PMA were also run
in parallel. To determine specific binding, the non-specific
binding (obtained from experiments done in presence of 100 fold
excess cold IL-8) was subtracted.
IL-8 induced migration of monocytes was carried out in a Boyden
chemotactic chamber using IL-8 (500 ng/ml) as a chemoattractant
in the lower chamber. In the upper chamber, control and treated
monocytes (50 x 303 cell/well) were put in each well.
In between the two chambers a PVP free polycarbonate membrane
(pore size 5 µm) was placed for specific migration. The migrated
cells were stained with Giemsa and counted under a phase contrast
microscope as stated earlier (20).
crosslinking and autoradiography
chemical crosslinking was carried out following the method of
Dower et al. (21). Human monocytes (1 x 107/ml) were
suspended in RPMI-1640 containing 0.5% BSA and incubated at
37°c with the stimulus for 1 h as indicated in the legends of
respective figures. After incubation, cells were washed with
Dulbeccos Phosphate buffered saline (DPBS), cooled and then
allowed to bind 125I labeled IL-8 at 4°C for 90 mins.
After washing with cold DPBS, the cells were finally suspended
in 100µl DPBS. Then DSS (1 mg/ml) was added with gentle stirring
and incubated at 4°C for 1 h. After washing the cells, 100 µl
CHAPS (9mM in DPMS) and a cocktail of protease inhibitors containing
PMSF (200 µM), leupeptin (1 µM), EDTA (100 µM) and pepstatin
(1 µM) were added and kept for 10 min in ice. After mixing thoroughly,
the cells were centrifuged at 10,000 x g for 15 min at 4°C.
The cell free extract was analyzed in SDS-PAGE (10%) under reducing
condition following the method of Laemmli et al. (22). The gel
was dried and using KODAK X-OMAT film at –70°C.
prepared monocytes (4 x 106 cells/tube) were treated
with 10 ng/ml serum activated LPS and was incubated at 37°C
for 1 h as before. A parallel set without addition of serum
activated LPS was run. After incubation, the cells were extracted
with 9mM CHAPS in DPBS containing a cocktail of protease inhibitors
and the extract was resolved in a 10% SDS-PAGE under reducing
condition (12). The proteins were electrotransfered onto a nitrocellulose
paper for 2 h at room temperature following the method described
earlier (12). The nitrocellulose paper was blocked overnight
by 1% BSA, immersed in anti-IL-8 receptor antibody solution
(1:50) and finally immersed in 125I labeled anti-rabbit
IgG. After washing, the paper was dried and autoradiography
was carried out using Kodak X-Omat film at –70°C.
bacterial LPS is known to activate monocytes and induce release
of IL-8 for the recruitment of neutrophils and other cells,
the question arises whether LPS itself has any role in alteration
of IL-8 receptor level on the surface of monocytes concomitant
with the release of IL-8 from the same source. The data presented
in Fig. 1 show that an optimum dose of 10 ng/ml serum activated
LPS can increase binding of IL-8 of monocytes by 100-110% with
respect to control (P<0.001). Under identical condition,
either 10 ng/ml LPS or 20 µl serum only (which was transferred
with LPS during serum activated LPS) had no significant effect
for expression on IL-8 receptor (Fig. 1). Time kinetic study
shows that the serum activated LPS mediated expression of IL-8
receptor was maximum at 60 min. After that period IL-8 binding
was observed to be reduced very slowly About 20% decrease in
IL-8 binding was obtained after 2 h incubation of monocytes
with serum activated LPS (Data not presented)
click for full view
Effect of PMA
The effect of PMA, a co-carcinogenic immunomodulating
agent, has been extensively studied in a number of cells. Whether
such compound has any effect on modulation of the function of
IL-8 was a question. The result presented in Fig. 1 shows that
addition of 100ng/ml PMA to the incubation mixture resulted
in an increase of 70-125% binding in IL-8 with respect to control
(P<0.01) (Fig. 1).
results show that cycloheximide and actinomycin D treated cells
showed 85-90% inhibition of IL-8 binding with respect to the
control (Fig. 2). Preincubation of monocytes with tunicamycin
had no effect on ser.act.LPS stimulated cells. For PMA stimulated
cells, preincubation of the cells with colchicine, actinomycin-D
before showed that addition of colchicine to the incubation
mixture did not affect IL-8 binding increase in any way. On
the other hand presence of cycloheximide, actinomycin-D strongly
inhibited the PMA mediated increase in binding of IL-8 to the
click for full view
I: Effect of polymixin and anti-cd-14 antibody on SA-LPS induced
upregulation of IL-8 receptor expression.
+ Ser.act.LPS (10 ng/ml)
+ polymyxin-B + SA-LPS (10 ng/ml)
+ Anti-CD-14 AB
+ Anti-CD-14 AB + SA-LPS (10 ng/ml)
of salicylate and hydrocortisone on SA-LPS and PMA treated cells
and hydrocortisone are know to inhibit NFkB mediated
gene activation, therefore, de novo protein synthesis
is strongly inhibited (23, 24). Whether these agents have any
effect on the SA-LPS and PMA induced activation of monocytes
was a question to us. The data presented in Table II and Table
III with respect to control, salicylate and hydrocortisone treated
cells strongly inhibited the LPS and PMA induced increased binding
of IL-8 to the cells. In a separate experiment we have examined
that none of these agents have any significant effect on the
II: Effect of salicylate and hydrocortisone on the expression
of IL-8 receptor in human monocytes.
of 125I IL-8
+ Hydrocortisone (100 µM)
µM) + PMA (100 ng/ml)
ng/ml) + PMA (100 ng/ml)
III: Effect of hydrocortisone and salicylate on ser. activated
LPS induced IL-8 receptor expression in human monocyte.
of 125 IL-8 (in cpm±SD)
for 1h incubation
binding for 2h
+ serum activated LPS (10 ng/ml)
+ Salicylate (100 µg/ml) + serum activated LPS (10 ng/ml)
+ Hydrocortisone (100 µg/ml)
+ Hydrocortisone (100 µg/ml) + serum activated LPS (10
Polymyxin B and anti-CD14 antibody
B is known to prevent the binding of LPS to its carrier protein
lipopolysaccharide binding protein (LBP) present in the serum.
Whether SA-LPS acts on the target cells through LPS binding
protein was a relevant question. To examine this, polymyxin
B was added during incubation of LPS with serum at 37°C. Incubation
of the cells with polymixin B and LPS mixed serum caused no
alteration of IL-8 binding on the surface of monocytes.
LPS acts on the target cell through a cell surface receptor,
CD-14 therefore we wanted to address the question that, whether
ser.act.LPS involves CD-14. Our results showed that IL-8 binding
to the cells reduced with respect to control (Table I).
anti-IL-8 receptor antibody
increase in binding of IL-8 in ser.act. LPS and PMA stimulated
monocytes was due to total increase in induction of IL-8 receptor
protein on the surface of the cell or due to increased binding
of the receptor without altering protein content of the receptor
can be clarified by titrating the antigenic epitopes of the
IL-8 receptor protein on the surface. The results showed that
binding of 125I labeled IgG to ser.act. LPS treated
monocytes was increased 2 fold with respect to that of that
of unstimulated cells (Fig. 3).
click for full view
Scatchard analysis of the binding data showed that receptor
number in unstimulated cell in about 7,500 per cell, in ser.act.LPS
stimulated monocytes it was about 15,550 receptor/monocyte (P<0.05)
and the Kd values for these receptors of control and SA-LPS
stimulated cells are 12.78 nM and 11.97 nM respectively (Fig.
4 panel A). The number of PMA treated cells was about 13,000
receptor/cell (P<0.05) and the Kd values of control and PMA
treated cells are 10.69 nM and 9.27nM respectively (Fig. 4 panel
click for full view
crosslinking and autoradiography
the autoradiography picture it is observed that with respect
to control the intensity of the signal at 59kD is considerably
increased in ser.act.LPS and PMA treated cells (Fig. 5, panel-1
click for full view
To understand that ser.act.LPS
stimulated monocytes specifically increased the binding of IL-8
to the cell surface, the immunoblotting was carried out in control
and ser.act.LPS stimulated cells. The autoradiographic signals
show that the band at 59 kD (molecular mass) has been considerably
increased in ser.act.LPS treated monocytes with respect to control
click for full view
we have demonstrated that monocytes migrate towards IL-8 in
a Boyden chemotactic chamber (12). The data presented in Fig.
7 shows that the chemotactic index of migrating cells in control
was 10.66 which was reduced to 5.22 in anti-IL-8 receptor antibody
treated cells. Stimulation of monocytes with ser.act. LPS could
enhance the chemotactic index to 20.88 which was reduced by
the antibody to 11.5 as indicated in the Fig. 7. In PMA stimulated
cells the chemotactic index was 21.25 with respect to 12.25
in control cells (p<0.05).
of LPS and ser.act.LPS mediated stimulated of IL-8 receptor
LPS elicited poor response and ser.act.LPS showed considerably
upregulation of IL-8 receptor in monocytes, our question was,
whether there is any correlation between LPS and ser.act.LPS
for activation of monocytes. The result showed that there was
no alteration of IL-8 binding (Table IV). Since it is reported
that LPS can induce the expression of a protein- bactericidal/permeability-increasing
protein (BPI) released from the azurophilic granules and cytochalasin
B can remove the protein from the cell surface of monocytes
(25), we wanted to explore the possibility of the involvement
of this type of protein. Therefore, in LPS stimulated cells,
we treated cytochalasin-B (5 µg/ml) for 5 min, then after washing
the cells, serum activated LPS (10 ng/ml) was added. Finally,
IL-8 binding ability of the cells was examined. It was found
that after cytochalasin treatment, stimulation of ser.act.LPS
was almost equal to stimulation of cells with ser.act.LPS alone
IV: Interaction of different doses of activated LPS on IL-8
receptor expression on human monocytes.
37°C For 1h
+ LPS (10 µg/ml)
+ LPS (10 µg/ml)
+ serum (20µl)
+ serum activate
+ LPS (10 µg/ml)
+ LPS (10 µg/ml)
cytochalasin-B (5 µg/ml)
+ LPS (10 µg/ml)
cytochalasin-B (5 µg/ml)
of human monocytes with high doses of bacterial LPS causes synthesis
and release of IL-8 which is essential for the recruitment of
neutrophils and other cells during inflammation in healthy and
diseased state. We found that LPS not only induced the production
of the cytokines, but also increased synthesis of IL-8 receptor
protein which caused increase in binding of IL-8 on the surface
of human monocytes. Since very low number of receptors are expressed
in presence of LPS, it may not be very potent for the synthesis
if IL-8 receptor on the surface of monocytes. LPS may induce
the degranulation process as a result of which presynthesized
receptor derived from degranulation may be the possible source
for IL-8 receptor. Colchicine, a microtubule disrupting agent,
frequently used for blocking degranulation of neutrophils (14,
15), can strongly inhibit LPS induced increased binding of IL-8
on the surface of monocyte. Recently it is reported that presynthesized
IL-8 receptors remained stored in the package of secretary vesicles
in the cytosol which upon stimulation with LPS, can mobilize
towards the cell surface, fuse with the membrane and IL-8 receptor
s are expressed on the cell surface (15). In presence of serum,
the situation for the mode of LPS action seems to be different.
LBP is present constitutively in human serum as a 60 kD glycoprotein
which is synthesized as a single polypeptide chain (50 kD) in
the hepatocytes (26. 27). LBP has a high affinity binding site
for lipid a part of LPS. Preincubation of LPS with serum perhaps
forms an active LBP-LPS complex which can efficiently induce
the synthesis of IL-8 receptor. However, LPS forms a high affinity
stoichiometric complex with LBP and serves as a carrier protein
that brings LPS to the surface antigen CD-14, a 55kD cell surface
glycoprotein (glycosyl phosphatidyl inositol anchored membrane
protein), which is strongly expressed on mononuclear phagocytes
is known to be an opsonic receptor for LBP, when it is in complex
form with LPS. Since CD-14 is abundantly present on the surface
of monocytes, interaction of LPS-LBP with CD-14 leads to formation
of a complex, which in cooperation facilitates interaction of
LPS with cells and induced potent activating signals for the
upregulation of IL-8 receptor in monocytes. Thus, we can postulate
the role of LBP and CD-14 for the recognition of LPS on the
surface of monocytes.
actinomycin-D and cycloheximide, the inhibitors of protein synthesis,
could inhibit the expression of IL-8 receptor. It can be concluded
that the de novo protein synthesis and not the degranulation
process is the principal reason for SA-LPS stimulated induction
of IL-8 receptor synthesis in monocytes.
myristate acetate (PMA) exerts pleiotropic effects on a number
of cell types. PMA can bind and activate protein kinase-C and
phosphorylate a numbers of amino acids, like serine and theronine
residues of cytoplasmic proteins, resulting in activation of
the biochemical machinery of IL-8 receptor synthesis. Although
the mode of action of PMA in inducing IL-8 receptor is not well
defined, but at this stage it can be suggested that activation
of some cytoplasmic protein may collectively stimulate the uperegulation
of IL-8 receptor in monocytes through de novo protein
is reported that NF/kB/Rel belongs to the family of transcriptional
factors that participate in activation of a diverse range of
genes involved in inflammatory and immunological responses (29).
NF/kB/Rel is a cytosolic protein which remains in latent from
as a complex consisting of a dimmer of a DNA binding subunits
bound to an inhibitor IkB. It is reported that the modulators
like LPS and PMA activate the Rel proteins and as a result,
translation of the proteins from cytosol to nucleus occur (30).
A large number of genes have binding sites for NF/kB in their
promoters and the transcription of the genes is modulated to
various extents upon binding of activated NF/kB. The transcriptional
factors are known to rapidly modulate gene expression needed
for host defence, since it is reported that lipopolysacharide
and phorbol esters induce the degradation of cytosolic IkBα,
as a result NFkB is released and translocated to nucleus for
activation of the genes involved in the inflammatory response
(29, 30). Recently it is demonstrated that a number of cytokine
genes are repressed by glucocorticoids and aspirin or salicylate
inhibit NFkB activity and IkB degradation (24). As hydrocortisone
and salicylate can also strongly block the SA-LPS and PMA induced
IL-8 receptor synthesis both the agents seem to be involved
in modulating the gene for IL-8 receptor synthesis.
in protein level of IL-8 receptor was supported by the measurement
of IL-8 receptor level as detected by anti-IL-8 receptor antibody
measured by radiolabeled anti-rabbit IgG. Anti-IL-8 receptor
antibody could recognize the antigenic epitopes of IL-8 receptor
efficiently in SA-LPS and PMA treated cells, it suggests that
the modulators can not induce the expression of Fc receptor
in monocytes. It clearly demonstrates that the modulation of
IL-8 receptor in SA-LPS and PMA treated cells increased the
total level of IL-8 receptor.
considerable increase in IL-8 receptor number in SA-LPS and
PMA stimulated cells binding affinity of the ligands remained
almost same. It excludes the possibility of the conversion of
low affinity form of the receptor (control) to high affinity
form of the receptor that is responsible for increase binding
of IL-8 in SA-LPS and PMA stimulated cells. Autoradiographic
signals of chemically coupled radioactive IL-8 (molecular mass
8 kDa) with its receptor (molecular mass, 59kDa) appeared at
67 kDa. Since SA-LPS increased the 59kD signal intensely and
the other band (molecular mass 67kD) was not altered markedly,
it suggests that the C-X-C-R-1 type of receptor was induced
by serum activated LPS. This is further supported by immunoblotting
experiment were the level of type-1 receptor 59kD is considerably
increased in serum activated LPS with respect to unstimulated
cells. The autoradiographic signals for PMA mediated IL-8 receptor
expression was also triggered at 59kD indicating again that
C-X-C-R-I type of receptor is induced for expression.
IL-8 directed, migration was significantly increased in serum
activated LPS with respect to control, it can be suggested that
the stimulated cells were functionally active compared to control
cells, therefore the cells migrated faster than that of normal
cells. It also supports that the upregulated receptor is not
interaction of LPS to the peripheral monocytes induce expression
of the protein BPI which can neutralize the effects of LPS on
the cells (25). As result, no significant upregulation of IL-8
receptor was obtained. For the same reason activated LPS failed
to show any alteration of the level cell surface receptor. Cytochalasin
B could remove BPI from the surface of the cells in LPS treated
cells (25). Possibly it makes the cells very sensitive to serum
activated LPS again like normal monocytes since almost equal
extent of IL-8 receptor induction was obtained. Thus a significant
correlation between the effects of LPS and serum activated LPS
on the monocytes is obtained. Phorbol esters are known to modulate
cellular functions and metabolism in many cell types by triggering
calcium and phospholipid-dependent protein kinase-C in cells
of specific kinase substrates leading to the phospshorylation
of the proteins. Therefore, the phosphorylated proteins may
elicit necessary signals for on set of the de novo protein
synthesis for IL-8 receptor. In the present study, however,
no effort as been given to identify the phosphorylated complex
responsible for the signaling of IL-8 receptor synthesis.
bacterial infection, the microorganisms release a number of
products which interact with the host cells, activate the host
defense mechanism against the Gram negative bacteria. Monocytes
are the phagocytic cells engaged in the host defence mechanism.
This study shows that considerable upregulation of the receptors
of inflammatory cytokines like IL-8 is also a simultaneous cellular
event occurring on the surface of monocytes. It suggests that
a single cell stimulant may activate more than one signaling
system for the inflammatory response. Whether a common pathway
is involved in the cell signaling of two different immunomodulators
for monocytes or two different pathways of action on the target
cells are involved is not clear.
can be suggested the cell has a unique regulatory mechanism
through which is emergency, receptors are rapidly expressed
on the cell surface facilitating the interaction of IL-8 with
its receptor the cytokine activated cell may migrate faster
towards the source of the cytokine, recognize the foreign pathogenic
microorganism, release several oxygen free radicals and proteases
and thereby help to eradicate the microorganism from the host.
Thus an emergency service automatically becomes operative in
the host system to combat the adverse situation. We can conclude
that perhaps the two agents have their own independent signaling
pathways in the cells. However. In the current study, no effort
has been made to establish LPS/ser.act.LPS mediated signaling
pathways for stimulating monocytes.
significance of ser.act.LPS stimulation is that, it equips the
cells with additional membrane constituents for interaction
of more cytokine ligands with the cell surface receptor, so
that cells can destroy the bacteria or other pathogenic microorganism
efficiently. LBP, present in plasma, is considered to be an
acute phase protein and the function of LPS-LPB complex is amplified
upon interaction with the membrane bound glycoprotein CD14,
a receptor for LPS. There for, the bacterial product modulate
the receptor mediated functions in human monocytes by increasing
as well as facilitating the interacting sites of IL-8. Thus,
recognition of LPS and PMA by monocytes and the upregulation
of IL-8 receptor may be considered to have a protective role
in post defences.
the significance of PMA induced stimulation of IL-8 receptor
expression in monocyte is nor clearly understood, at least we
can propose that PMA can increase the duration and amplitude
of IL-8 action on the target cells. Since IL-8 is an inflammatory
cytokine and PMA is a tumor promoter and co-carcinogenic agent,
interaction of PMA with monocytes rapidly stimulate the cells
to facilitate the interaction of IL-8 with the cells and activate
the innate immune system of the host for considerable period.
in the present study, we have demonstrated that human monocytes
express functionally active IL-8 receptor, which are upregulated
by LPS and PMA. Detailed study of the LPS and PMA mediated regulatory
mechanisms in monocytes will be helpful for understanding the
bactericidal, tumoricidal and inflammatory functions of phagocytic
cells like monocytes.
thankfully acknowledgment a Research Fellowship provided to
Mr. Biswandey Bishayi during 1993-1998 by Council of Scientific
and Industrial Research, New Delhi, India We are deeply indebted
to Prof. Kouji Matsushima, Kanazawa University, Japan and Dainippon
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