INTRODUCTION
Atherosclerotic vascular disease remains the most common cause of death and disability in westernized countries. Although atherosclerosis was recognized for many years as a simple lipid storage disease, nowadays, it is widely accepted that both—disturbed lipid metabolism leading to accumulation of low-density lipoproteins (LDLs) in the arteries and chronic inflammation of the vascular wall—are the most prominent features of this illness [
1,
2]. Even though, oxidized forms of low-density lipoproteins (oxLDLs) are major risk factor, recently accumulating evidences have implicated that infectious agents can accelerate atherosclerosis [
3‐
5]. It has been documented that certain chronic infections such as periodontitis and chlamydial infection exacerbate clinical manifestation of atherosclerosis [
6,
7]. The presence of the pathogen-associated molecular patterns (PAMPs), like lipopolysaccharide (LPS), peptidoglican, and bacterial DNA, which are shed and released by all growing and dividing bacteria, was demonstrated in significant proportion of lesions. It is believed that predominantly PAMPs and only exceptionally viable microbes accumulate within atheroma [
8‐
10]. Thus, human atherosclerosis lesions may be directly exposed to bacterial ligands of Toll-like receptors (TLR). TLR receptors is a family of innate immune recognition receptors that detect PAMPs and play a key role in initiating inflammatory responses and, most likely, in the pathogenesis of atherosclerosis [
11‐
14]. TLRs are expressed on monocytes and monocyte-derived macrophages, which are involved in the atherosclerotic lesion development as well as in coordination of innate and acquired immune responses. Cells of monocytic lineage express a variety of receptors, which enable them to detect and integrate complex signals derived from other cells, lipoprotein products, and pathogens (reviewed in [
15]). As immunoregulatory cells, in response to stimuli, monocytes and macrophages produce considerable amounts of pro- and anti-inflammatory cytokines, which in the context of atherosclerosis can be considered as pro- or anti-atherogenic (reviewed in [
16]) and play an important role in the development, progression, and complications of atherosclerosis.
Tumor necrosis factor (TNF) as a potent proinflammatory cytokine is involved in the induction of expression of adhesion molecules and chemokines in the vascular wall, the first step during development of atherosclerotic lesions. Its key role in atherosclerosis was demonstrated in mice model showing that atherosclerotic lesion size was significantly smaller in animals deficient in TNF, which was associated with decreased expression of intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemotactic protein-1 (MCP-1) [
17]. Moreover, it was shown that TNF level correlates strongly with the burden of atherosclerosis in healthy middle-aged men, severity of peripheral arterial disease, and also elevated risk of recurrent myocardial infarction [
18]. In contrast, interleukin-10 (IL-10) is a powerful antiatherogenic cytokine. As a pleiotropic, anti-inflammatory cytokine, it inhibits a broad array of immune functions. The role of IL-10 has been clearly established in mouse model of atherosclerosis. It was demonstrated that IL-10 deficiency promotes early atherosclerotic lesions formation [
19]. Consistent with a protective role of IL-10 in atherosclerosis, overexpression of IL-10 decreases formation of early fatty streak, prevents exaggerated advanced atherosclerosis development, and modulates cellular and collagen plaque composition in mice [
20‐
22]. Furthermore, elevated IL-10 serum levels were associated consistently with significantly improved endothelial function and outcome of patients with acute coronary syndrome [
23,
24]. It is believed that the balance between pro- and anti-inflammatory factors and magnitude of cytokine release at a site of lesion formation may affect further disease fate. In early stages of atherosclerosis, cytokine balance can alter endothelial function favoring or hampering the recruitment, adherence, and migration of leucocytes into the inflamed vessel wall. At a more advanced stage of disease, pro- and anti-inflammatory balance may regulate processes of atherosclerotic plaques destabilization. For example, the imbalance between matrix degradation and synthesis, destroying fibrous cap structure and leading to its rupture was correlated with prevailing proinflammtory cytokine expression over IL-10 and transforming growth factor secretion (reviewed in [
16]). In the context of atherosclerosis, research has focused on the potential of proatherogenic lipids to modulate proinflammatory events, but as atherosclerosis is a chronic inflammatory disease, regulation of anti-inflammatory cytokines may be equally if not more important. As a matter of fact, experimental and epidemiological data support the concept that endothelial function, plaque instability, and patient outcome in atherosclerotic vascular disease depend on the pro- and anti-inflammatory balance at the sites of disease development.
What has hitherto remained unexplained is if oxLDLs influence cytokine production activated by innate recognition of PAMPs, if such regulation favors pro- or anti-inflammatory response, and if other serum factors, which are not considered to interact directly with TLRs, participate in this regulation. Therefore, we set out to challenge these concepts in a simple experimental model employing human peripheral blood monocytes or derived macrophages, a set of well-defined TLR ligands and readily reproducible oxLDLs. We suppose that this model may reflect the pathophysiological milieu during development of atherosclerosis lesions when monocytes are exposed directly to proatherogenic lipids and pathogen-associated molecules.
MATERIALS AND METHODS
Human Peripheral Blood Monocytes and Monocyte-Derived Macrophages
Peripheral blood mononuclear cells (PBMCs) were isolated from citrate-treated blood of healthy donors using standard density gradient centrifugation (Ficoll-Paque PLUS, Amersham Biosciences, Uppsala, Sweden) and plated at 3.5 × 10
6/well in 24-well Cell + plates (Sarstedt, Newton, NC, USA) in RPMI1640 (Gibco Invitrogen Corp., Paisley, UK) supplemented with 2 mM
l-glutamine, 50 μg/ml gentamycin (Sigma), in later parts of text called complete medium, and 10 % fetal calf serum (FCS, Biochrom). After 2 h of incubation at 37°C in humidified atmosphere containing 5 % CO
2, nonadherent cells were removed by washing with complete medium. To obtain elutriation-purified nonadherent monocytes, PBMCs were subjected to counterflow centrifugation as described previously [
25]. The monocytes phenotype was routinely controlled (in the case of adherent cells after nonenzymatic detachment) by immunofluorescence staining with mAb anti-CD14 (clone: TŰK4, DakoCytomation) and subsequent flow cytometry analysis (LSRII, Becton Dickinson). The cultures selected for further experiments were positive in at least 90 % for the CD14. To obtain monocyte-derived macrophages, adherent monocytes were cultured in complete medium supplemented with 10 % pooled heat-inactivated human serum (HS) for at least 7 days. The medium was changed every 2 days. The human monocytes-derived macrophages (hMDMs) phenotype was routinely controlled, after nonenzymatic detachment of cells, by immunofluorescence staining of CD14 (clone: TŰK4, DakoCytomation), CD11b (clone: ICRF44, Becton Dickinson and Co, Franklin Lakes, USA), and CD209 (clone: DCN46, Becton Dickinson) and subsequent flow cytometry analysis. The cultures selected for further experiments were positive in at least 90 % for the first two markers and <1 % for CD209. The adherent cells acquired typical macrophage morphology. Resting (unstimulated) cells did not produce cytokines: IL-1, TNF, IL-6, and IL-10.
Isolation and Oxidation of Lipoproteins
Low density lipoproteins (LDLs) were isolated from the fresh EDTA-treated plasma of healthy donors by the method of sequential ultracentrifugation through a discontinuous KBr gradient according to Havel
et al. [
26]. Plasma density was adjusted to 1.019 g/ml with solid KBr, and plasma was centrifuged at 180,000×
g for 24 h at 4°C (Beckman L7-65 ultracentrifuge with Ti 60 rotor, Beckman, USA). The top fraction containing very low-density lipoproteins and intermediate density lipoproteins was removed and density of the remaining solution was raised to 1.063 g/ml by addition of solid KBr followed by repeated centrifugation. LDL fraction from the top layer was collected and dialysed overnight at 4°C against phosphate-buffered saline (PBS) with 0.05 % EDTA pH 7.4 for native LDLs (nLDLs) or against PBS pH 7.4 for LDLs prepared to be oxidized. Oxidation of LDLs was performed by incubation with Cu
2+ for 20 h at 37°C (final Cu
2+ concentration, 5 μM) after adjustment of protein concentration to 0.12 mg/ml. Obtained oxLDLs were dialyzed overnight against PBS pH 7.4 at 4°C. nLDLs and oxLDLs preparations were concentrated by ultrafiltration (Amicon Ultra Centifugal Filters,100 K NMWL, Millipore, USA) at 3,500×
g at 4°C, and sterilized by filtration through a 0.22-μm syringe filter (Millex-GV, Millipore, USA). Minimally modified LDLs were obtained by storage of nLDLs at 4°C for 6 months. All materials used during isolation procedures were endotoxin free, and we have not observed LDL-induced stimulation of cytokine production. Protein concentration was quantified by the Lowry method using Total Protein Kit (MicroLowry, Peterson’s Modification, Sigma, USA). The purity of LDLs preparations was routinely controlled by polyacrylamide gel electrophoresis and subsequent gels staining with Red Oil O for lipids detection (Sigma, USA) and with Coomasie Briliant Blue R for protein detection (Sigma, USA).
Treatment with LDLs and Stimulation
In most experiments elutriation-purified, adherent monocytes or macrophages were placed in complete medium supplemented with 10% FCS, treated for 30 min with native, minimally modified, or oxidized low-density lipoproteins (0–50 μg/ml) and then stimulated with PAMPs: Escherichia coli 0127:B8 LPS (stLPS, Sigma), ultrapure E. coli 011:B4 LPS (upLPS, Invivogen), synthetic lipopeptides: diacylated Pam2CysSerLys4 (Pam2, Invivogen), and triacylated Pam3CysSerLys4 (Pam3, Invivogen) at a final concentration of 10 ng/ml, ultrapure Porphyromonas gingivalis LPS (pgLPS, Invivogen) at a final concentration of 1 μg/ml. In some cases, cells were pretreated with oxidized LDLs as described above or in complete medium supplemented with 0.15 % bovine serum albumin (BSA, Sigma). After pretreatment, oxLDLs were washed out, and cells were stimulated in complete medium supplemented with 10 % FCS. For the experiments concerning the influence of soluble serum factors on cytokine production, elutriation-purified monocytes were placed in culture plates or in polypropylene culture tubes (BD Falcon) in complete medium supplemented with 0.15 % BSA, 1 %, 10 %, 30 % FCS, or 10 % HS and stimulated as described above.
Treatment with LDLs and Immunofluorescence Staining
To analyze the effect of oxLDLs on expression of surface receptors on monocytes, cells were placed in complete medium supplemented with 10 % FCS and treated for 30 min or 3 h with oxidized low-density lipoproteins (15 μg/ml). After nonenzymatic detachment monocytes were suspended in complete medium supplemented with 5% FCS (5 × 105/sample/100 μl) and incubated with PE-conjugated antihuman TLR2 (clone TL2.1, eBiosciences), TLR4 (clone HTA125, eBiosciences), CD36 (clone CB38, BD Pharmingen), CD11b (clone ICRF44, BD Pharmingen) or Fluorescein isothiocyanate (FITC)-conjugated antihuman CD14 (clone RM052, Immunotech) mAbs, or appropriate isotype controls (BD Pharmingen) for 30 min at 4°C. After washing with cold medium, the cells were resuspended and analyzed by flow cytometry using an LSRII cytometer (Becton Dickinson). The analysis was performed using the FACSDiva program to determine the percentage and mean fluorescence intensity (MFI) of positive cells.
Blocking of TLR Receptors
Adherent monocytes were placed in complete medium supplemented with 10% FCS and incubated with 10 μg/ml of blocking monoclonal antibodies against human TLR2 (clone: TL2.1, IgG2a, eBiosciences) or TLR4 (clone: HTA125, IgG2a, eBiosciences) or appropriate isotype controls (eBiosciences) for 30 min at room temperature (RT). Then, the cells were stimulated with LPS from P. gingivalis at final concentration 1 μg/ml for 20 h, and supernatants were collected.
Regulation of αM, αvβ3, and αvβ5 Integrins
Adherent monocytes were placed in complete medium supplemented with 1% FCS and incubated with ligands for integrins: recombinant human ICAM-1 (10 μg/ml, R&D), human plasma fibrinogen (20–200 μg/ml, Millipore), fibronectin (0.1–1 μg/ml, Millipore), or vitronectin (3–30 μg/ml, Millipore) for 30 min at 37°C in humidified atmosphere containing 5 % CO2. Alternatively, cells were placed in complete medium supplemented with 10 % FCS and incubated with 150 mM N-acetyl-d-glucosamine (Sigma), 10 μg/ml of blocking monoclonal antibodies against human CD11b (αM integrin, clone Vim12, IgG1, Santa Cruz Biotechnology, Inc.) or agonistic monoclonal antibodies against human β3 (clone RUU-PL 7 F12, IgG1, BD Pharmingen), αvβ3 (clone LM609, IgG1, Chemicon), and αvβ5 (clone P1F6, IgG1, Chemicon) integrins for 30 min at RT. For applied antibodies corresponding isotype controls (BD Pharmingen) were used, all at concentration 10 μg/ml. Then, the cells were stimulated with LPS from P. gingivalis at final concentration of 1 μg/ml for 20 h, and supernatants were collected.
Membrane Disruption
Membrane-disrupting agent methyl-β-cyclodextrin (MβCD, Sigma) was added to monocytes at 0.5 and 1 mM. Then, the cells were stimulated with LPS from E. coli or P. gingivalis at final concentration 10 ng/ml or 1 μg/ml for 20 h. Then, supernatants were collected, and cell viability was monitored by propidium iodide exclusion test.
Viability Assay
Cells were evaluated for viability and apoptosis after LDLs treatment by morphology using bright-field microscopy or by the binding of FITC-labeled annexin V and exclusion of propidium iodide (PI) according to the manufacturer’s recommendations (Annexin V-FITC kit, Bender MedSystem, Vienna, Austria) followed by analysis with LSR II flow cytometer (Becton Dickinson).
Measurement of IL-10 and TNF Production
For the cytokine measurements, supernatants were collected at different time points after stimulation as indicated in the figures. All supernatants were centrifuged at 500×
g for 5 min to remove particulate debris and stored at −20°C. The concentrations of IL-10 and TNF in culture supernatants were determined by enzyme-linked immunosorbent assays (ELISAs) using the OptEIA Sets (BD Pharmingen) according to the instructions provided with each set of antibodies. The assay was sensitive down to concentration of 7 pg/ml. Selected data (Fig.
2c, d) were presented as IL-10/TNF biological activity ratio obtained from recalculation of ELISA results to international units of biological activity per ml (based on data of ELISA standards against NIBSC/WHO international standards).
Statistical Analyses
All experiments were performed at least in triplicate. The data are presented as means ± SD. All statistics were calculated using Origin 8.1 (OriginLab Corporation, Northampton, MA, USA). Statistical significance was asset at 0.05 and calculated using one-way ANOVA test.
DISCUSSION
In this paper, we describe an unexpected aspect of pathogen-accelerated atherosclerosis resulting from altered recognition of selected PAMPs by monocytes exposed to proatherogenic oxidized low-density lipoproteins. The dysfunctional recognition was apparently shifting the physiological balance between pro- and anti-inflammatory cytokines, represented in our study by the most biologically active members of both groups, TNF and IL-10, respectively. Strong inhibition of IL-10 induced with TLR2 and TLR4 ligands was opposed by only moderate inhibition of TNF. Thus, it appears that monocytes recognizing PAMPs in the presence of established risk factor of atherosclerosis—oxLDLs—create a cytokine milieu promoting chronic inflammation. The inhibitory effect of low-density lipoproteins depended significantly on their oxidation state, and it was not attributable to oxLDLs toxicity. A potent antiatherogenic activity of IL-10 has already been shown by many groups [
20‐
24].
Assuming the proportion between IL-10 and TNF produced in the absence of oxLDLs as physiological, we observed that oxLDLs strongly disturbed the balance between the pro- and anti-inflammatory cytokines produced by monocytes upon TLR stimulation (Fig.
2a ,c, d, and supplementary data Table
1 and Figure
1). Irrespective of TLR-ligand used to challenge the monocytes, we observed dramatic inhibition of IL-10 and a moderate change in secretion of TNF, varying from subtle stimulation to slight inhibition. In kinetics experiments, oxLDLs inhibited the anti-inflammatory response following less or more intensely pronounced pro-inflammatory reaction (Fig.
2c, d and supplementary data Figure
1). From the same experiments, we also concluded that modulation of cytokine production by oxLDLs did not result from altered kinetics of cytokine release (supplementary data Figure
1). A spectacular example of the differential regulation of TNF and IL-10 by oxLDLs was seen upon stimulation of monocytes with LPS from
P. gingivalis (PG). In spite of strong IL-10 suppresion, oxLDLs did not inhibit TNF production, which was even slightly increased (Fig.
2a and supplementary data Table
1). PG is a primary etiological agent of human periodontal disease, but epidemiological and experimental studies support the hypothesis that chronic infection with this bacterium may be associated with pathogen-accelerated atherosclerosis [
7,
34‐
37]. Recently, it has been demonstrated that patients with periodontal disease are at greater risk of developing vascular dysfunction [
38] as well as the association between periodontal pathogens, and subclinical atherosclerosis has been revealed [
39]. Some
in vitro studies have demonstrated that endothelial cells and monocytes respond to
P. gingivalis, pgLPS, or FimA secreting various cytokines and chemokines [
40‐
42], but to the best of our knowledge, there are no reports concerning the effect of oxLDLs on recognition of LPS from
P. gingivalis. We demonstrated for the first time that oxLDLs strongly suppress production of IL-10 by monocytes challenged with PG. PAMP hypothesis of atherosclerosis postulates that the occasional presence of PAMPs in the blood may promote the activation of endothelial cells and the recruitment of monocytes into the vessel wall. Based on our results, we propose that also the anti-inflammatory response of monocytes is inhibited, thus leading to chronic inflammation. In this context, the altered recognition of PAMPs by monocytes in the presence of oxLDLs may be proposed as a new, general mechanism governing pathogen-accelerated atherosclerosis. Interestingly, we have demonstrated that the differential regulation of IL-10 and TNF is specific for monocytes. In hMDMs, both cytokines were proportionally inhibited (Fig.
2b and supplementary data: Table
1). Combined with the finding by Fuhrman
et al. [
43] that oxLDLs accelerate monocyte to macrophage differentiation, our observation suggests a coherent picture of pathogenic mechanism in which a modified, proinflammatory microenvironment can be created before monocytes differentiate into macrophages—at early stages of fatty streak development or at later stages by currently recruited cells.
In this work, we used copper oxidized LDLs, which do not occur
in vivo and are not a physiologic molecule. Like variety of minimally modified LDLs, extensively oxidized LDLs contain a myriad of bioactive compounds including oxidized phospholipids, lysophospholipids, oxysterols, oxidized fatty acids, and variably modified ApoB (reviewed in [
44]). However, it is not clear which of these components is predominant
in vivo and has the most significant pathophysiological effect. Moreover, some of the biological effects of oxLDLs are dependent on individual components, while others are due to the complex signal provided by whole oxLDL particle. For these reasons, there is no accepted “gold standard” for preparing oxidized LDLs
ex vivo. Nevertheless, copper oxidized LDLs resemble naturally occurring oxidized LDLs as antibodies raised against Cu
2+LDLs recognize epitopes present
in vivo and are successfully used to detect oxLDLs in patients [
45]. We observed that both copper oxidized as well as minimally modified LDL obtained by storage of LDLs at 4°C for 6 months inhibited IL-10 production by monocytes stimulated with TLR ligands, but detailed analysis of oxidized LDL compounds was beyond of the scope of this study.
The mechanism of
P. gingivalis LPS recognition is still controversial. It activates host cells through both TLR2 and TLR4, and this may result from heterogeneity of these molecules, containing various forms of lipid A [
29]. A lipoprotein from pgLPS was suggested to be a principal component for TLR2- and highly purified lipid A for TLR4-mediated cell activation [
30,
31]. On the other hand, it has been shown that in human vascular endothelial cells
P. gingivalis LPS-induced cell activation is mediated through TLR2, and it is lipid raft-dependent as well as requires the formation of receptor complex comprising of TLR2/TLR1, CD36, and CD11b/CD18 [
32]. These results support hypothesis that innate immune signaling following PG challenge is cell specific [
46]. We observed that TNF secretion was dependent on TLR4 and TLR2, but IL-10 production was exclusively TLR2 dependent (Fig.
6), which is supported by strong association of TLR2 expression on monocytes with IL-10 inducibility [
47].
There are conflicting data on the possibility of TLR stimulation by LDLs alone. Some authors suggested that minimally modified LDLs and their components—oxidized phospholipids—may stimulate TLR-dependent signaling [
33,
48‐
50]. It has been reported recently that copper oxidized mmLDLs induce both proinflammatory cytokines IL-1β and IL-6 as well as anti-inflammatory cytokine IL-10, in human monocytes and U937-derived macrophages, and this effect can be assigned to CD14, TLR2, and TLR4 activation [
51]. On the contrary, the evidence has been presented that neither oxPAPC nor extensively oxidized LDLs are capable of stimulating TLR2 and TLR4-dependent signaling [
52,
53]. The apparent contradiction may be in part explained by a suggestion that pro- or anti-inflammatory activities of oxidized LDLs may depend on their concentration [
54] experimental designs and/or variable LDLs preparations [
44]. Nevertheless, in our hands, spontaneously oxidized mmLDLs and oxLDLs alone did not stimulate detectable TNF or IL-10 secretion (data not shown).
It is clear that the ability of both TLR4 and TLR2 receptors to activate cells relies on “accessory proteins,” soluble serum or membrane bound factors such as CD14 or LBP [
55,
56] The serum factors were also identified as key targets for oxidized phospholipids, which specifically inhibit TLR2 and TLR4 by competitive interaction [
27,
28]. Consequently, we were interested if IL-10 and TNF production by monocytes upon stimulation via TLR2 and TLR4 was dependent on soluble serum molecules. As shown in Fig.
4, IL-10 and TNF production responded differently to serum concentration only following TLR2 activation. Precisely, IL-10 production was relatively low in serum-free media and at 1 % FCS and was strongly augmented in 10 % FCS, but for maximal TNF production, supplementation with 1 % FCS was sufficient and further raising the FCS concentration suppressed TNF secretion. It is noteworthy that TNF production induced with LPS from
P. gingivalis appeared completely independent on serum (Fig.
4). These results correlate with data showing that serum-soluble CD14 effectively transferred
P. gingivalis LPS to TLR2 plus TLR1, but poorly to TLR4 [
29], and our observation of TLR2-dependent production of IL-10 by monocytes stimulated with pgLPS. Assuming that sCD14 and LBP are main targets for oxPL, the described dependence of cytokine production on serum factors correlated well with the inhibition pattern shown in Fig.
2a. To expand this observation, we treated cells with oxLDLs and then washed them out before stimulation. From this experiment, we concluded that inhibition of IL-10 production occurred mainly by competitive interaction of oxLDLs with accessory proteins as removing the preincubation medium before the stimulation abolished the inhibitory effect of lipoproteins. The “unblocking” effect of medium change was most pronounced for Pam3CSK4 and pgLPS. The apparent IL-10-promoting effect of serum attracted our attention to a possibility that some still unrecognised serum “accessory molecules” may participate the pgLPS-TLR2-IL-10 pathway. As the effect of serum was apparent only in freshly adhered monocytes but neither in hMDMs (where adherence is established) nor in elutriated monocytes incubated in polypropylene tubes (where adherence is prevented), we assumed that the serum “accessory protein” is one of its factors of adherence. Consequently, we tried to substitute FCS using purified fibronectin, fibrinogen, or vitronectin. Among the tested proteins, only vitronectin demonstrated a concentration-dependent effect on IL-10 production (Fig.
7). Previous studies concerning vitronectin receptors expression on human monocytes have produced conflicting results. Some groups suggested that, although freshly isolated monocytes lack surface expression of β3 and β5 integrins, it appeared gradually during monocyte adhesion and maturation [
57]. In our hands, expression of αvβ3 on monocytes 5 h post-isolation was evident while αvβ5 was barely detactable. Still, pretreatment with vitronectin receptors agonistic mAbs (clones: RUU-PL 7F12, P1F6, or LM609), which alone did not stimulate cytokine production, resulted in dramatic increase in pgLPS-induced production of IL-10 (Fig.
7b). The agonistic effect was also observed in serum-free media (data not shown) suggesting that costimulation through TLR2 and β3 and β5 integrins leads to increased IL-10 production.
As CD11b is naturally involved in adherence of monocytes to solid substratum, we have demonstrated that some ligands or antagonists of CD11b influenced the IL-10 production induced by TLR2 ligands (Fig.
7b). Significantly, we have not seen any effect of ICAM-1 or fibrinogen, what may suggest that CD11b is involved through lateral interaction with other receptor(s) rather than by direct binding of vitronectin or other serum “accessory molecules.” As TLR2 and 4 form a functional surface complex with integrin CD11b/CD18 [
32], our results may suggest that vitronectin receptor is a novel partner in this interaction. An interaction between CD11b and vitronectin has been described in neutrophils [
58]. The most likely sites for such interactions are cholesterol-rich membrane platforms containing arrays of PRRs and other surface proteins, which are now proposed to play a critical role in interaction of PAMPs with immune cells [
59]. Indeed, disruption of lipid rafts with methyl-β-cyclodextrin inhibited the effect of pgLPS on IL-10 production, while TNF-production was not afflicted. TLR-containing lipid rafts harbor not only other PRRs (e.g., Dectin-1 [
60]) but also receptors and proteins, which have not been directly connected to pattern recognition (e.g., HSP90 [
61]). We have previously reported functional interactions of CD11b with CD16, which stabilized other surface molecules (phosphatidylserine, annexin I) not previously known to interact with the integrin complex [
62‐
64]. In this paper, we suggest that also vitronectin receptor may function as a component of TLR signaling platforms.
Vitronectin has become an important mediator in the pathogenesis of coronary atherosclerosis because of its ability to bind platelet glycoproteins and mediate platelet adhesion and aggregation at sites of vascular injury (extensively reviewed in [
65,
66]). The involvement of vitronectin and CD11b in the described mechanism is also supported by interesting clinical findings: Firstly, elevated levels of vitronectin are connected with coronary atherosclerosis and are postulated to be the result of a compensatory mechanism [
67]; on the other hand, inhibition of vitronectin receptor enhances the uptake of oxLDL and differentiation of monocytes/macrophages into foam cells [
68]; secondly, transmigrating monocytes from patients with coronary artery disease have lower expression of CD11b [
69]; thirdly, in children with hypercholesterolemia, cell surface expression of CD11b and CD18 on PBMC was significantly decreased [
70]; finally, Toll-like receptor 2 and 4 stimulation produced an enhanced inflammatory response in human obese patients with atherosclerosis [
71].
Summarizing, it seems justified to assume that proatherogenic oxidized LDLs disturbed the balance between pro- and anti-inflammtory cytokines produced by monocytes upon TLR2 and TLR4 stimulation. IL-10 release was strongly suppressed by oxLDLs with no respect to the TLR ligand used for activation. In contrast, TNF production was reduced to a lesser extent than IL-10, unaffected or even slightly increased, when stimulated with LPS from P. gingivalis, one of infectious agent of atherosclerosis. Although the precise mechanisms of observed cytokine modulation remain to be determined, our results highlight the differential dependence of IL-10 and TNF production on serum accessory molecules. Significantly, vitronectin or/and its receptor appear to be the main target of oxidized phospholipids inhibitory activity on TLR-induced activation.