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(Journal of Leukocyte Biology. 2000;68:209-215.)
© 2000 by Society for Leukocyte Biology

Human neutrophil cathepsin G down-regulates LPS-mediated monocyte activation through CD14 proteolysis

Karine Le-Barillec, Dominique Pidard, Viviane Balloy and Michel Chignard

Unité de Pharmacologie Cellulaire, Unité Associée IP/INSERM 485, Institut Pasteur, Paris, France

Correspondence: Dr. Michel Chignard, Unité de Pharmacologie Cellulaire, Unité Associée IP/INSERM 485, Institut Pasteur, 25, rue du Docteur Roux, F-75724 Paris Cédex 15, France. E-mail: chignard{at}pasteur.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A major property of monocytes/macrophages is to recognize and to be activated by bacterial wall components such as LPS, through membrane receptors including the key element CD14. We demonstrate that CD14 expression is down-regulated, as judged by flow cytometry analysis, upon incubation of human monocytes with purified cathepsin G (CG), a releasable neutrophil serine proteinase. The progressive decrease of CD14 expression due to increasing concentrations of CG highly correlates (P < 0.0001) with the decreased synthesis of tumor necrosis factor {alpha} (TNF-{alpha}) in response to lipopolysaccharide (LPS). This effect is dependent on the enzymatic activity of CG but is not exerted through an activation of monocytes. Immunoblot analysis reveals that CD14 (Mr = 57,000) is directly cleaved by CG and released into the extracellular medium as a high-Mr species (Mr = 54,000). In this context, incubation of monocytes with activated neutrophils leads to a down-regulation of CD14 expression, a process blocked by a serine proteinase inhibitor. These data suggest a paradoxical anti-inflammatory property for CG.

Key Words: serine proteinase • TNF-{alpha} • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mononuclear phagocytes (monocytes and macrophages) constitute the first line of defense against invading microorganisms and serve as the initial trigger of the innate immune response. Lipopolysaccharides (LPS), which are cell wall components of the outer membrane of gram-negative bacteria, are powerful activators of monocytes/macrophages, inducing the release of a plethora of mediators including cytokines with pro- [such as tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-1, or IL-8] or anti-inflammatory (such as IL-10, IL-1ra) properties [1 , 2 ]. Different types of binding proteins and receptors are known to be involved in the specific interaction with, and activation of monocytes by LPS [3 ]. Among them, the CD14 antigen, a glycosylphosphatidylinositol (GPI)-anchored molecule expressed on cells of the granulomonocytic lineage, functions as a high-affinity LPS binding site. It has recently been shown that, after binding of LPS, a signal is then conveyed across the plasma membrane by Toll-like receptors (TLR), resulting in the activation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) and the regulation of responsive genes [4 , 5 ].

Polymorphonuclear neutrophils (PMN) also take an active part in the natural host defense. Like mononuclear phagocytes, they recognize and eliminate pathogens through phagocytosis. A clue to their functional importance is that their quantitative or qualitative defects both lead to increased susceptibility to infections [6 ]. One paradoxical aspect of PMN activity is that they are also involved in the genesis of various inflammatory conditions. Thus, if PMN activation is excessive or if the signal to turn off the inflammatory process is deficient, tissue damage can ensue [7 ]. The proteolytic enzymes contained in PMN azurophilic granules and released in the extracellular medium are implicated in the injury of host tissues and considered as major mediators of uncontrolled inflammation. Human leukocyte elastase (HLE) and cathepsin G (CG) appear to be the most important of these enzymes [8 ]. Free HLE and CG have been detected in the bronchoalveolar lavage fluids from patients with acute respiratory distress syndrome, in sputum of patients with cystic fibrosis, and in the synovial fluid of patients with arthritis [9 10 11 ]. Numerous studies have demonstrated that various components of the extracellular matrix are degraded by HLE and CG, including collagen [12 ], fibronectin [13 ], elastin [14 ], fibrinogen, and thrombospondin-1 [15 ].

These serine proteinases are also proteolytically active on different membrane receptors at the surface of immune cells. The present investigation was initiated on the basis of a study by Bazil and Strominger [16 ], demonstrating that exposure of monocytes to PMA induces a loss of the surface expression of CD14. This effect was shown to be associated with a concomitant release of a soluble form of this antigen, a process likely mediated by serine proteinases because specific inhibitors of these enzymes prevented the receptor shedding. Because CG, a chymotrypsin-like proteinase, is released from the azurophilic granules into the extracellular medium upon PMN activation, we wanted to establish whether it could be a candidate for CD14 cleavage. In support of this approach, it was previously noted that chymotrypsin is efficient in digesting soluble CD14, resulting in the generation of several moderate-sized peptides [17 ].

The results of this study demonstrate that monocytes settled in an environment containing CG display scaled-down responses to LPS, an effect related to a restricted proteolytic activity on the CD14 antigen. Such a potential anti-inflammatory activity of CG is susceptible to occur under in vivo conditions, as upon incubation of monocytes with PMN, activation of PMN leads to release of serine proteinases and down-regulation of CD14 expression on monocytes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies
Blood was obtained from the Etablissement de Transfusion Sanguine (Paris, France). LPS from Escherichia coli 055:B5, purchased from Sigma Chemical (St. Louis, MO), was dissolved and sonicated for 1 min in sterile saline solution and kept frozen in aliquots. Eglin C was generously provided by Dr. H. P. Schnebli (Novartis, Basel, Switzerland). The mouse anti-human ß-actin mAb, phorbol 12-myristate 13-acetate (PMA), the HLE inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone (AAPV-CMK) and the serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF), were obtained from Sigma Chemical. Phosphatidylinositol-phospholipase C (PI-PLC) from Bacillus cereus was purchased from Boehringer Mannheim (Mannheim, Germany). Control mouse IgG2b mAb was from Biodesign International (Kennebunk, ME) and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG Ab was from Dako (Copenhagen, Denmark). Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Bio-Rad (Hercules, CA). Nitrocellulose membranes (0.45 µm pores) were from Schleicher and Schuell (Dassel, Germany). Recombinant human CD14 in serum-free culture supernatant of Chinese hamster ovary cell transfectants was purchased from Biometec (Greifswald, Germany). A peroxidase-linked donkey anti-goat IgG Ab was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-human CD14 Abs used in this study included a goat anti-CD14 polyclonal Ab kindly supplied by Dr. J. Pugin (Geneva, Switzerland), and MY4, a murine anti-CD14 mAb, was obtained from Coulter (Miami, FL).

CG was purified as described previously [18 ]. To block its catalytic site, the purified proteinase was incubated for 60 min at 25°C with 1.25 mM PMSF, and the mixture was subsequently dialyzed to remove the free inhibitor. PMSF-treated CG was shown to be proteolytically inactive by testing the lack of hydrolysis of its specific synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma).

Purification of human monocytes
Monocytes were purified from blood collected on heparin and acid citrate dextrose. Blood (2 volumes) was mixed with 3% (w/v) dextran (Sigma) in saline solution (1 volume), and erythrocytes were allowed to sediment for 30 min. The leukocyte-rich supernatant was removed and loaded on Ficoll-Paque gradients (Pharmacia Biotech, Uppsala, Sweden). After centrifugation at 300 g for 45 min at 25°C, peripheral blood mononuclear cells were collected and washed with RPMI 1640 medium containing antibiotics (GIBCO BRL, Paisley, Scotland). After centrifugation at 300 g for 10 min at 25°C, the pellet was resuspended in RPMI 1640 medium with 3% (v/v) fetal calf serum (FCS; Boehringer Mannheim) to a density of 4 x 106 cells/mL. Monocytes were separated from lymphocytes by allowing monocytes to adhere to 12-well plastic dishes (Costar, Cambridge, MA) for 1 h at 37°C. After removal of nonadherent cells, monocytes were washed again with RPMI 1640 medium. When required, the amount of cell proteins in each well was evaluated using the BCA Protein Assay from Pierce (Rockford, IL).

Purification of human PMN
After the centrifugation of the leukocyte-rich plasma on Ficoll-Paque gradients, the PMN pellet was collected and resuspended in a red blood cell lytic solution (155 mM NH4Cl; 2.96 mM KHCO3; 3.72 mM EDTA-Na2). The cell suspension was gently inverted for 5 min in order to lyse the remaining erythrocytes, then centrifuged at 350 g for 10 min and after two washes, PMN were resuspended in HBSS (GIBCO BRL) at 107 cells/mL. Conditioned medium of PMN (PMN-cm) was obtained by incubating PMN with 0.5 µM fMLP, 10 µM cytochalasin B, 1 mM CaCl2, and 1 mM MgCl2 for 5 min at 37°C with gentle agitation, followed by sedimentation of the cells at 300 g and 25°C for 10 min.

TNF-{alpha} and CD14 quantitation
TNF-{alpha} and CD14 concentrations in the supernatants of treated monocytes were determined as previously described [19 ], and using a commercial sandwich enzyme immunoassay according to the manufacturer’s protocol (ICN, Costa Mesa, CA), respectively.

Flow cytometry analysis
At the end of the incubation periods of monocytes under various conditions (see Results and figure legends), reactions were stopped by addition of 20 µM eglin C, an inhibitor of CG [20 ]. Cells were then washed twice with Hanks’ balanced salt solution (HBSS) and incubated with 1.5 mL of cold HBSS containing 0.1% (w/v) bovine serum albumin (BSA). Plates were then let rest on ice for 15 min to facilitate cell detachment. Then, cells were resuspended by vigorous pipetting, centrifuged at 300 g for 10 min at 4°C, and fixed with 0.33% (w/v) formaldehyde for 30 min at room temperature. After fixation, cells were added to conical-bottomed 96-well plastic plates (Nunc, Roskilde, Denmark) at 1.5 x 105 cells/well. Next, plates were centrifuged at 80 g for 5 min at 4°C and monocytes were subsequently incubated with MY4 or with matched IgG2b control mAb, 1 µg/mL each, for 30 min at 4°C. Cells were centrifuged again at 80 g for 5 min at 4°C and washed twice with HBSS-BSA before incubation for 30 min at 4°C with the FITC-labeled anti-IgG Ab (5 µg/mL). Finally, monocytes were centrifuged and washed as above and resuspended in the same buffer.

Samples were analyzed with a FACScan cytometer (Becton Dickinson Immunocytometry System, Mountain View, CA). Binding of the MY4 mAb to its specific epitope on CD14 at the surface of monocytes was expressed as the percentage of the median fluorescence intensity (MFI) over control values measured on untreated cells after subtraction of the background binding measured with the control isotype Ab.

SDS-PAGE and immunoblot analysis
At the end of the exposure of monocytes to CG, eglin C was added to block the enzymatic activity. Extracellular milieus and the monocyte cell fractions were separately solubilized as previously described [21 ] and protein disulfide bonds were reduced by adding 5% (v/v) 2-mercaptoethanol (2-ME). SDS-PAGE was subsequently performed according to the procedure of Laemmli [22 ] using 10 µg of total monocyte proteins loaded per well on 10% acrylamide gels. Proteins were electrotransferred to nitrocellulose membranes, probed with the polyclonal anti-CD14 antiserum diluted 1/3,000, and bound Ab revealed with an anti-goat IgG, horseradish peroxidase-linked donkey Ab diluted 1/1,000 and reacted with ECL detection reagents (Amersham, Little Chalfont, UK) before exposure to Kodak X-Omat AR films (Kodak-Pathe, Paris, France). For Mr determinations, polyacrylamide gels were calibrated using standard proteins (Bio-Rad) with Mr within the range 14,400–200,000.

Statistics
Results are expressed as means ± SEM for the indicated number of experiments performed independently. Statistical significance between the different values was analyzed by Student’s t-test for unpaired data with a threshold of P <= 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition by CG of TNF-{alpha} synthesis by LPS-activated human monocytes
As shown in Figure 1 , pretreatment of monocytes for 30 min with CG at concentrations ranging from 0.25 to 3 µM increasingly inhibited the synthesis of TNF-{alpha} induced by 1 ng/mL LPS added for 3 h, from 9.4 ± 1.3 pg/µg protein for untreated monocytes to 0.8 ± 0.4 pg/µg protein when they were pretreated with 3 µM CG (n = 3, P < 0.05).



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  		Figure 1. Inhibition by CG of TNF-{alpha} synthesis by LPS-activated human monocytes. Monocytes were pretreated with CG concentrations ranging from 0.25 to 3 µM, or with the MY4 mAb (10 µg/mL) for 30 min at 37°C. Reactions were stopped with 20 µM eglin C, cells were washed twice and then incubated for 3 h at 37°C with LPS at 1 ng/mL (filled circles) or 10 µg/mL (open circles). Supernatants were analyzed for TNF-{alpha} content by ELISA. Data are means ± SEM of three experiments with cells from different donors.

 
It is well-established that when monocytes are exposed to a low concentration of LPS such as 1 ng/mL, only one activating pathway is likely to be involved, namely the CD14 pathway [23 ]. To ensure that under our experimental conditions monocytes were activated as such, they were pretreated, before LPS stimulation, with an anti-CD14 mAb (MY4) known to specifically inhibit the binding of LPS to the CD14 molecule [24 ]. Incubation of monocytes for 30 min with 10 µg/mL MY4 resulted in a marked decrease of TNF-{alpha} production triggered by 1 ng/mL LPS, with 2.6 ± 0.6 pg/µg protein released in the presence of MY4 compared with 8.9 ± 2.1 pg/µg protein in the presence of an isotype-matched IgG2b Ab (n = 3, P < 0.05), indicating the major involvement of CD14 for monocyte activation under these conditions. By contrast and as expected, when cells were stimulated with a high LPS concentration, namely 10 µg/mL, the MY4 Ab did not blunt the response (Fig. 1) . It is interesting that pretreatment of the cells with 3 µM CG also did not produce an inhibitory effect on the production of TNF-{alpha} induced by this concentration of LPS (10.3 ± 1.7 pg/µg protein compared with 12.6 ± 2.0 pg/µg protein for control LPS-activated cells; n = 3, P > 0.05). This was an indication that CG affected the CD14 pathway.

Disappearance of the CD14 molecule from the surface of human monocytes treated with CG
Simultaneously with the inhibition of TNF-{alpha} synthesis, the disappearance of CD14 from the surface of monocytes was assessed by flow cytometry using the anti-CD14 mAb MY4. As shown in Figure 2A , a progressive decrease of CD14 expression was observed upon exposure of cells to increasing concentrations of CG, down to 9.3 ± 3.1% (n = 3) of the control values for 3 µM CG. The kinetics of disappearance of the surface expression of CD14 showed that after a 5-min exposure of the cells to this concentration of CG, values already dropped to {approx}55% of the initial value, and were and remained <10% by 30 min. No decrease in the level of CD14 expression was observed after a 30-min exposure to 3 µM PMSF-inactivated CG, indicating an absolute requirement for the enzymatic activity of the proteinase in reducing the antigen recognition. Figure 2B indicates that the disappearance of the CD14 antigen from the surface of monocytes strongly correlates with the decrease of TNF-{alpha} synthesis (r2 = 0.984, P < 0.0001).



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  		Figure 2. Disappearance of the CD14 molecule from the surface of human monocytes treated with CG and relationship with the inhibition of the TNF-{alpha} synthesis. (A) Monocytes were treated with CG as described in the legend to Figure 1 . Binding of the MY4 mAb was then analyzed by flow cytometry; (B) the same data are plotted vs. the concentrations of TNF-{alpha} produced by LPS-activated monocytes pretreated with the same range of CG concentrations (data from Fig. 1 ). Results are means ± SEM of three experiments with cells from different donors.

 
Such an alteration of the CD14 expression could be due to an indirect effect of CG via the activation of monocytes. Indeed, CD14 shedding has been observed upon activation of monocytes with PMA [16 ], and CG is able to activate intracellular signaling pathways, as shown in platelets [18 ]. To evaluate such a possibility, monocytes were pretreated with formaldehyde in order to block any possible activation. To prove that such fixed cells indeed could not be activated, PMA was used to induce the shedding of CD14. Surface expression of CD14, as evaluated by flow cytometry, was diminished upon PMA treatment of unfixed control cells, but not when fixed cells were used, with 47.8 ± 3.4 vs. 106.4 ± 7.3% of the basal expression measured on resting unfixed and fixed cells, respectively (n = 3, P < 0.05). By contrast, the effect of CG (3 µM, 30 min) on the CD14 expression was observed with both unfixed and fixed cells, with values lowered to 6.6 ± 0.3 and 25.2 ± 6.8% (n = 3), respectively, compared with control untreated cells. This result indicated that CG was not acting indirectly on CD14 through a signal transduction pathway, but most probably directly modified the receptor.

Immunoblot analysis of CD14 expression by human monocytes
The structure and expression of the membrane CD14 receptor on monocytes exposed to 3 µM CG for 30 min were compared to those resulting from exposure of the cells to PI-PLC. PI-PLC was used because this enzyme specifically detaches membrane GPI-anchored molecules by hydrolyzing the GPI moiety, releasing in the medium an intact, uncleaved soluble form of the protein [25 ]. Lysates and extracellular milieus from monocyte suspensions were thus subjected to gel electrophoresis and immunoblotting (Fig. 3 ). Immunoblot analysis of the cell lysate indicated that the bulk of the intact CD14 that is detected in control, untreated monocytes with Mr {approx}57,000 was indeed extensively removed from the cell surface after treatment with PI-PLC. Similarly, exposure to CG resulted in a nearly complete disappearance of CD14, with no appearance of membrane-bound fragments. Immunoblot analysis of the extracellular milieus indicated that, on PI-PLC treatment, the intact CD14 antigen was released (Mr {approx}57,000), whereas incubation with CG produced a soluble form of CD14 with a slightly lower Mr value ({approx}54,000). This suggested that CG may cleave CD14 within its carboxy-terminal domain, a few amino acids above the linkage site between the protein moiety and the GPI anchor. Control, untreated monocytes showed no detectable shedding of CD14.



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  		Figure 3. Immunoblot analysis of CD14 expression by human monocytes. Monocytes were either untreated (C) or treated with 3 µM CG or 0.6 U/mL PI-PLC for 30 min at 37°C. Reactions were stopped with 20 µM eglin C; cell fractions and extracellular fluids were immediately separated and extracted with 1% (v/v) Triton X-100 in the presence of a cocktail of proteinase inhibitor [21 ] then solubilized with 2% SDS and reduced with 5% (v/v) 2-ME. After SDS-PAGE on 10% polyacrylamide gels and transfer to nitrocellulose membranes, protein samples were probed with a polyclonal anti-CD14 Ab. In addition, the same membranes were probed with an anti-ß-actin Ab to ensure an equal protein loading between the different wells. Mr were calculated with respect to calibration standard proteins included in the gel.

 
Surface expression of CD14 upon incubation of monocytes with PMN stimulated by fMLP
We next performed coincubations of monocytes and PMN activated with the chemotactic peptide agonist fMLP in order to induce the release of the content of the neutrophil azurophilic granules, and to test its proteolytic activity on CD14.

The surface expression of CD14 was first analyzed by flow cytometry on both cell types taken separately. In agreement with previous data [26 ], the binding of the specific anti-CD14 mAb MY4 to PMN was found to be very low, being in the range of that found for the matched control IgG isotype (data not shown), whereas monocytes were strongly labeled. Whether PMN or monocytes were treated with fMLP, CD14 expression was not affected and remained comparable to that of resting cells (data not shown). This indicated that challenging cells with fMLP did not modify the expression of CD14 or the ability of the anti-CD14 mAb MY4 to recognize the surface receptor.

Monocyte CD14 expression was then analyzed under conditions in which the two cell populations were coincubated for 2 h. First of all, it was observed that the labeling of the cells with the control matched IgG isotype gave one peak corresponding to low levels of cell fluorescence (Fig. 4A ). Upon labeling with the specific anti-CD14 mAb, two peaks of fluorescence were displayed (Fig. 4B) . With reference to data obtained with each cell population taken separately, the left peak corresponded to PMN (MFI <= 20) and the right peak to monocytes (MFI >= 800). Treatment of the mixed cell population with fMLP induced a marked drop of CD14 expression within the monocyte population, with a decrease of MFI values within the range observed for the control matched IgG isotype (Fig. 4C) . Our hypothesis that serine proteinases released from activated PMN were responsible for the down-regulation of CD14 was ascertained by the addition of eglin C, a potent serine proteinase inhibitor, before activation of PMN with fMLP. Under these conditions, the disappearance of CD14 from the monocyte surface was prevented (Fig. 4D) .



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  		Figure 4. Surface expression of CD14 upon coincubation of monocytes with PMN stimulated by fMLP. Monocytes (5 x 105/mL) and PMN (5 x 106/mL) were coincubated in suspension and stimulated (C, D) or not (A, B) with 0.5 µM fMLP for 2 h at 37°C. One sample was stimulated in the presence of 20 µM eglin C (D). Binding of the MY4 mAb (B–D) or a matched IgG isotype (A) was then analyzed by flow cytometry. Fluorescence histograms are representative of three distinct experiments.

 
Cleavage of recombinant CD14 in the presence of the conditioned medium of fMLP-activated PMN
The decrease of CD14 surface expression on monocytes when the two cell populations were coincubated and activated with fMLP was not accompanied by the recovery of CD14 in the extracellular milieu when checked by immunoblot analysis, as one would expect from an effect of CG on this receptor like that illustrated in Figure 3 (data not shown). We hypothesized that this result could be explained by the release of HLE along with CG in the milieu. Indeed, HLE is known to proteolyze CD14 into numerous small fragments undetectable by our immunoblotting assay [27 ]. Furthermore, quantitation of each proteinase activity, as measured with specific synthetic substrates [28 ] in the extracellular milieu of fMLP-activated PMN suspensions, indicated concentrations of 215 and 440 nM for CG and HLE, respectively. These concentrations were indeed within the range of those inducing decreases of CD14 expression on monocytes (see Fig. 2A [27 ]).

To verify the hypothesis of a participation of HLE in monocyte CD14 proteolysis, recombinant CD14 was reacted with the conditioned medium of fMLP-activated PMN for different periods of time up to 2 h, and analyzed by immunoblot. A progressive, time-dependent disappearance of the intact recombinant CD14 was observed (Fig. 5A ). However, when the reaction was performed in the presence of a specific synthetic HLE inhibitor, AAPV-CMK, the degradation of the CD14 antigen was prevented. This result pointed out the active role of HLE and confirmed its extensive proteolytic activity on CD14. By contrast, treatment of recombinant CD14 with 0.5 or 3 µM of purified CG for 2 or 0.5 h, respectively, did not detectably change the Mr of the protein and its recovery (Fig. 5B) . The observation that the electrophoretic mobility of the recombinant molecule is not visibly affected by CG confirms our previous assumption that CG cleaves the CD14 antigen from the cell surface of monocytes at an amino acid(s) located near the GPI anchor.



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  		Figure 5. Cleavage of recombinant CD14 by the conditioned medium of fMLP-activated PMN. (A) Recombinant CD14 (0.25 µM) was incubated for various periods of time at 37°C with PMN-cm. A mixture of 2 mM AAPV-CMK and PMN-cm was incubated with recombinant CD14 for 2 h under the same conditions; (B) recombinant CD14 (0.25 µM) was incubated without or with 0.5 or 3 µM CG for 120 or 30 min, respectively. In all cases, reactions were stopped with 20 µM eglin C and 2-ME-reduced samples processed for CD14 immunoblot analysis as described in the legend to Figure 3 . Mr were calculated with respect to calibration standard proteins included in the gel.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CD14 is a 55-kDa glycoprotein found in two forms in all mammalian species: a membrane protein (mCD14) expressed in cells of the granulomonocytic lineage, in which it is anchored to the outer leaflet of the plasma membrane via a GPI moiety, and a soluble plasma protein (sCD14) lacking the GPI tail. mCD14 is important for the activation of monocytes/macrophages by LPS, whereas sCD14 participates in LPS activation of nonmyeloid cell types such as endothelial and epithelial cells [29 ]. Support for an essential role of CD14 in LPS-induced physiopathological manifestations comes from demonstration that mice with targeted deletion of the gene encoding CD14 are resistant to the lethal effects of LPS [30 ], whereas anti-CD14 mAb treatment provides therapeutic benefit after exposure to LPS [31 ]. The most important target cells for LPS are components of the innate immune system, specifically monocytes and macrophages, which, in response to this agonist, produce a large array of bioactive proteins that include TNF-{alpha} and IL-1, two cytokines considered as the main proximal mediators of the inflammatory process [32 ].

Our present data demonstrate that CG inhibits the response of monocytes to LPS under experimental conditions for which the CD14 pathway is the major operative one. At this stage, however, two different targets could be contemplated, i.e. CD14 itself and Toll proteins. Indeed, it is only recently that the elusive proteins working in concert with the GPI-anchored CD14 and responsible for the intracellular signaling have been formally considered to include members of the TLR family. It has been shown that human TLR2 confers LPS responsiveness to cells transfected with this receptor [4 , 5 ], while it turns out that TLR4-deficient mice are hyporesponsive to LPS [33 , 34 ]. In fact, this recent information only partially explains the complexity of cellular responses to LPS. Thus, it is conceivable that different combinations of TLRs with CD14 make up various LPS receptors [35 , 36 ]. It is obvious that a great deal of work is still needed to fully apprehend the contribution of the TLRs in the cellular responses to LPS. Regardless, one can speculate that TLRs can be most probably ruled out as potential targets for CG. Indeed, TLRs apparently bind LPS and signal even in the absence of CD14 as far as high concentrations of LPS are used [5 ]. This is reminiscent of what happens under our experimental conditions, in that CG did not suppress monocyte activation when the high concentration of 10 µg/mL LPS was used, indicating that CG has no major functional impact on the receptors involved, i.e. the TLR(s). Consequently, only an effect of CG on the CD14 molecule was taken into consideration as the mechanism accountable for the inhibition of TNF-{alpha} synthesis at low LPS concentrations. As a confirmation, cell function and antigen analysis performed in parallel indicated a strong correlation between CG-induced inhibition of TNF-{alpha} synthesis and the decrease in expression of CD14.

The underlying mechanism of CG-dependent down-expression of CD14 appears to be a direct proteolytic cleavage. Internalization of this antigen can be excluded as (1) free CD14 is detectable in the extracellular milieu after cell exposure to CG, to levels comparable to those observed after hydrolysis by PI-PLC (see Fig. 3 ), and (2) CD14 is releasable from the surface of fixed monocytes. The latter feature also rules out a potential indirect proteolytic process depending on monocyte activation, as it occurs with PMA [16 ]. The presence of intact soluble CD14 in the conditioned medium of CG-treated monocytes contrasts with data obtained with the other neutrophil-derived proteinase HLE. Indeed, HLE cleaves CD14 in multiple small fragments [27 ], whereas CG generates a single large molecular species with a Mr value slightly lower than that of the molecule released upon PI-PLC treatment, which contains the whole protein and part of the GPI anchor. The difference between the two Mr corresponds to about 3 kDa, which allows us to hypothesize that CG cleaves CD14 at less than 30 amino acids from the carboxy terminus of the polypeptide chain, considering that part of the GPI moiety participates for an undefined proportion to this Mr difference.

High concentrations of CG were actually tested in this study, but it is known that under certain acute inflammatory conditions, the concentration of PMN may increase by 14-fold in the peripheral circulation and even by 100-fold at inflammatory foci [37 , 38 ]. This means that in a confined injured tissue environment, much higher concentrations of CG may even be reached, up to >= 250 µM [39 ]. Although our in vitro studies using the coincubation of monocytes and PMN clearly demonstrate a proteolysis of CD14, they also obviously bring evidence that the presence of antiproteinases impairs the phenomenon when the cells are present at physiological blood concentrations. Nonetheless, at inflammatory sites, and in addition to the local accumulation of cells and enzymes, close contacts between monocytes and PMN may create a microenvironment in which proteinases are preserved from their inhibitors. More importantly, it has been shown that activated PMN express at their surface an active form of CG, which has an increased resistance to inhibition by proteinase inhibitors [40 , 41 ]. Therefore, bound CG could potentially interact with CD14 at the surface of monocytes. Data recently reported provide a strong argument in support of a possible biological activity of CG in vivo. It was shown that CG is a monocyte chemoattractant that, when given subcutaneously to rats, induces an influx of inflammatory cells, including monocytes, into the site of injection [42 ]. Thus, despite high concentrations of inhibitors in plasma, monocytes can encounter free CG, which may thus proteolyze CD14.

The present observation may provide an as yet unsuspected way to generate soluble CD14, through CG proteolysis. Soluble CD14 levels are known to be increased in various biological fluids of patients presenting severe, often fatal acute inflammatory syndromes [43 44 45 46 ]. The formation of LPS-sCD14 complexes activates endothelial, epithelial, and smooth muscle cells via an unknown receptor [47 ]. By contrast, sCD14 acts on myeloid cells by competing with mCD14 for LPS binding and reduces LPS-induced cytokine production in whole blood [48 ]. This in vitro LPS-neutralizing capacity of sCD14 makes it a putative therapeutic molecule in endotoxin-induced shock [49 ]. In this context, the capacity of CG to proteolyze CD14 may be a physiological advantage by both removing mCD14 and generating sCD14. However, it could be put forward that the in vivo generation of sCD14 by CG is doubtful, despite the fact that we clearly showed that PMN granular exocytosis effectively produces a down-regulation of CD14 expression at the surface of nearby monocytes, an effect related to serine proteinase release. Indeed, under these conditions, CD14 is extensively proteolyzed due to the concomitant presence of HLE in the milieu, a neutrophil proteinase for which we previously demonstrated the ability to cleave CD14 in a large number of small peptides [27 ]. However, we believe that CG may play a role of its own because HLE is more prone to inhibition by plasma antiproteinases than CG is. It has thus been observed that CG and HLE enzymatic activities present in the extracellular medium of activated PMN were progressively inhibited by the addition of increasing amounts of plasma. Nonetheless, within a given range, plasma inhibited HLE >= 80%, whereas CG was only reduced by <=10% [50 ]. These data plead for the possibility that CG can still be active at inflammatory sites where HLE is blocked, particularly in the context of a microenvironment in which the concentrations of inhibitors are restricted. Moreover, it can be speculated that during the early phase of an inflammatory process, when PMN are already activated and have degranulated, but before that vascular permeability has reached its acme, the presence of a reduced amount of exudated plasma favored the participation of CG in CD14 cleavage as opposed to HLE.

Finally, whether CG is bound to cells or free in the extracellular medium, the ultimate result is that monocytes may become refractory to LPS, thus conferring to this neutrophil proteinase a potential unusual anti-inflammatory property.


   ACKNOWLEDGEMENTS
 
Karine Le-Barillec was supported by the Délégation Générale pour l’Armement, and Dominique Pidard by the Centre National de la Recherche Scientifique (CNRS), Paris, France. The authors wish to thank Dr. Mustapha Si-Tahar (Epithelial Pathobiology Unit, Emory University, Atlanta, GA) for critically reading the manuscript.

Received February 1, 2000; revised April 11, 2000; accepted April 21, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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