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,
Immunology Laboratory,
University of Calcutta,
92, A.P.C. Road,
Kolkata – 700 009
*Corresponding Author
**Division of Immunobiology, Indian Institute of Chemical Biology, Kolkata–700052
** Present Address: 7220, Cambridge St. # 4 Houston, Texas – 77030 (USA)
(Received on August 12, 2001)

 

Abstract: 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.

Key words:     IL-8 receptor               LPS     monocytes        PMA

 

Introduction
Methods
Results
Discussion
References



 

INTRODUCTION

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.

Interleukin-8 (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.

Lipopolysaccharide (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. 
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Purified 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

Peripheral 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% viable cells.

Iodination of IL-8

Human 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 protein.

In vitro treatment of monocytes with immunomodulatory agents

In 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.

In 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 cells

Human 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.

Involvement of anti-CD14 and anti-IL-8 receptor antibody

In 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.     

In 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 Gamma Counter. 

Scatchard analysis 

Monocytes (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.

Chemotaxis asay

The 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).

Chemical crosslinking and autoradiography

The 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.

Immunoblotting

Freshly 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.
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RESULTS

Effects of LPS

Since 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)

Fig.1 click for full view

Source of IL-8 receptor     

The 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 cells (Fig.2).

Fig.2 click for full view

 

TABLE I: Effect of polymixin and anti-cd-14 antibody on SA-LPS induced upregulation of IL-8 receptor expression.

	Incubation condition	125I IL-8 bound (cpm±SD)
Monocytes (Control)	37°C for 1h	2043±82
Monocytes + Ser.act.LPS (10 ng/ml)		4068±1
Monocytes + polymyxin-B		2257±260
Monocytes + polymyxin-B + SA-LPS (10 ng/ml)		2336±232
Monocytes + Anti-CD-14 AB		2125±169
Monocyte + Anti-CD-14 AB + SA-LPS (10 ng/ml)		2199±144

Effect of salicylate and hydrocortisone on SA-LPS and PMA treated cells      

Salicylate 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 control cells.

TABLE II: Effect of salicylate and hydrocortisone on the expression of IL-8 receptor in human monocytes.

	Condition of Incubation	Binding of 125I IL-8 (Mean cpm±SD)
Monocytes	37°C for 1h	2071±45
Monocytes + PMA (100 ng/ml)		3866±89
Monocytes + Hydrocortisone (100 µM)		1525±116
Monocytes + Hydrocortisone (100 µM) + PMA (100 ng/ml)		1924±125
Monocytes + Salicylate (100 ng/ml)		1822±95
Monocytes + Salicylate (100 ng/ml) + PMA (100 ng/ml)		1739±168

TABLE III: Effect of hydrocortisone and salicylate on ser. activated LPS induced IL-8 receptor expression in human monocyte.

Incubation condition			Binding of 125 IL-8 (in cpm±SD)
Monocytes	37°C for 1h incubation	4°C binding for 2h	1947±207
Monocytes + serum activated LPS (10 ng/ml)			4235±37
Monocytes + Salicylate (100 µg/ml) + serum activated LPS (10 ng/ml)			1784±224
Monocytes + Hydrocortisone (100 µg/ml)			1832±275
Monocytes + Hydrocortisone (100 µg/ml) + serum activated LPS (10 ng/ml)			1924±196

Effect of Polymyxin B and anti-CD14 antibody 

Polymyxin 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. 

Since 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). 

Effect of anti-IL-8 receptor antibody 

Whether 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).  

Fig.3 click for full view

Scatchard analysis 

The 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 B). 

Fig.4 click for full view

Chemical crosslinking and autoradiography

From 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 and 2).

Fig.5 click for full view

Immunoblotting

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 (Fig. 6).

Fig.6 click for full view

IL-8 induced migration

Recently 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).

Correlation of LPS and ser.act.LPS mediated stimulated of IL-8 receptor

Since 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 (Table IV).

TABLE IV: Interaction of different doses of activated LPS on IL-8 receptor expression on human monocytes.       

	Incubation Condition	Binding of 125 IL-8 (2h) to the cells (in cpm±SD)
Monocytes	At 37°C For 1h	3513±51
Monocytes + LPS (10 µg/ml)   Monocytes + LPS (10 µg/ml)		3472±273   4128±101
Monocytes + serum (20µl)		3563±189
Monocytes + serum activate LPS (10ng/ml)		7396±44
Monocytes + LPS (10 µg/ml) +serum activated LPS (10 µg/ml)		3644±273
Monocytes + LPS (10 µg/ml) + cytochalasin-B (5 µg/ml)		3683±132
Monocytes + LPS (10 µg/ml) + cytochalasin-B (5 µg/ml)
+serum activated  LPS (10 µg/ml		7259±243

DISCUSSION

Interaction 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.

As 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.

Phorbol 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 synthesis.

It 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.

Increase 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.

Despite 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.

Since 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 functionally inactive.

Direct 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.

During 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.

 It 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.

The 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.  

Although 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.

Thus, 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.

ACKNOWLEDGEMENTS

We 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 Pharmaceutical company, Japan for supplying recombinant human IL-8 for our experiments.
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