Monocyte CD14 Expression Research Articles

AMPK inactivation in mononuclear cells: a potential intracellular mechanism for exercise-induced immunosuppression

There is much evidence that prolonged intense exercise suppresses the immune system. However, the intracellular biochemical mechanisms linking exercise and immunosuppression remain obscure. The purpose of this study was to investigate the hypothesis that exercise-induced inactivation of 5'AMP-activated protein kinase (AMPK) disrupts individual immune cell function, and thus may be linked to exercise-induced immunosuppression. To confirm AMPK's role in immune cells, AMPK activity was assessed in cultured monocytic Mono Mac 6 (MM6) cells. The effects of single bouts of intense exercise (45 min cycling; 70% VO2 max) on several immune parameters including mononuclear cell AMPK phosphorylation were investigated in 10 male volunteers. In vitro, the mitochondrial ATP synthase inhibitor oligomycin brought about transient decreases in cellular [ATP] (0.41+/-0.04 pmol/cell to 0.31+/-0.02 pmol/cell), and activation of AMPKalpha1 (170.7%+/-31.2% basal) and the glycolytic enzyme inducible phosphofructokinase 2 (iPFK-2) (225.0%+/-46.1% basal), with the latter effects coinciding with recovery from ATP depletion. In contrast, exercise-induced transient (approximately 1 h) decreases in AMPKalpha1 phosphorylation (64.4%+/-17.6% basal). This AMPK inactivation coincided with comparable transient decreases in other immune parameters (salivary IgA levels, serum cytokine levels, monocyte CD36 expression). Although the brief exercise bout employed here is not sufficient to cause full-fledged immunosuppression, exercise-induced transient decreases in mononuclear cell AMPK activation (as seen in this study) may cause energy depletion within individual immune cells, and therefore have an impact upon their ability to carry out their functions. Thus, we suggest that prolonged, repeated, high-intensity exercise that leads to clinically relevant immunosuppression may do so via AMPK inactivation within immune cells.

Quantitative Flow Cytometric Assessment of CD36 (Platelet Glycoprotein IV) Expression on Monocytes and Platelets: Assay Development, Role in Plasmodium falciparum Risk Assessment, and Relationship to Sickle Hemoglobin.

Background: CD36 (platelet glycoprotein IV) is a known binding site for red cells infected with Plasmodium falciparum (iRBC). The pathologic adhesion of iRBC to CD36 accounts for much of the endothelial sequestration and platelet rosetting observed. Deficient expression of CD36 is hypothesized to be another adaptation to P. falciparum malaria, because a 10-fold higher prevalence of deficiency (3–12%) exists in malaria-endemic areas.Purpose: A technique to quantitatively assess CD36 expression was developed for a future study on factors affecting P. falciparum malaria severity. In this pilot, CD36 levels were assessed in African-Americans (AA), non-African-Americans (non-AA), and a cohort of patients screened for sickle hemoglobin (HbS), in order to determine the prevalence and types of CD36 deficiency, and deficiency co-inheritance with HbS, a known malaria-adaptive mutation.Method: EDTA-anticoagulated blood was retrieved from clinical specimens. Antibody-conjugated fluorochromes (CD36-PE, CD14-FITC/− APC, CD61-PerCP, and CD54-FITC) were used. Effects of different assay conditions were investigated on samples from AA (N=57), non-AA (N=36), and those screened for HbS (N=27). FacsLyse lysis was used in the AA and non-AA subjects, but NH4Cl was selected in HbSS samples because of its unique ability to lyse otherwise lysisresistant sickle cells. After washing, samples were analysed by flow cytometry (FACSCalibur). Monocytes and platelets were identified by light-scatter and CD14- and CD61-expression respectively, and analyzed for CD36 at different storage times. Viability was assessed in 2 non-AA controls with 7-AAD. The median fluorescence intensity (MFI) of CD36 expression was log transformed.Results: Monocyte CD36 MFI follows a log-normal distribution:number testedmeanSDAA*572.42.45non-AA362.58.22HbS-pos152.51.35HbS-neg122.73.28*5% of AA were type I CD36-deficient (logMFI <1.5; MFI<32.)Monocyte CD36 MFI at day 4 of room temperature storage was not significantly different from day 1 [N=28; mean MFI 96% of fresh, mean Δ = 65±241, p=NS, paired t-test], despite reduced monocyte viability and CD14 expression after 3 days. Results of platelet CD36 paralleled monocyte CD36. Platelet-monocyte aggregates gave spuriously high CD36, occurring commonly in citrated, heparinized, and ACD anticoagulants, but were rare in EDTA. Sample size was insufficient to detect an association between reduced CD36 and HbS+ (p=0.23, Fisher's exact test).Conclusions: Flow cytometry using EDTA blood can quantify CD36 expression on monocytes and platelets. NH4Cl lysis is preferred for Hb SS specimens. Monocyte CD36 follows a log-normal distribution with 5% of AA deficient in CD36. Monocyte CD36 appears stable during room temperature storage for 4 days. This assay should be useful in studies of malaria and CD36-expression. Further work is required to assess if the reduced expression of CD36 is associated with adaptations to malaria.

Enhanced Levels of Oxidized Low-Density Lipoprotein Prime Monocytes to Cytokine Overproduction via Upregulation of CD14 and Toll-Like Receptor 4 in Unstable Angina

The purpose of this study was to establish whether oxidized low-density lipoprotein (oxLDL) contributes to cytokine overproduction via upregulation of CD14 and toll-like receptor-4 (TLR-4) expression on circulating monocytes of unstable angina (UA) patients. Expression of CD14 and TLR-4 on circulating monocytes, and the concentration of plasma oxLDL, (interleukin [IL])-6, IL-1 beta, IL-8, tumor necrosis factor (TNF)-alpha, monocyte chemoattractant protein-1 (MCP-1) were measured in 27 control (C) subjects, 29 patients with stable angina (SA), and 27 with UA. CD14 and TLR-4 expression on monocytes and circulating IL-6, IL-1 beta, and oxLDL were higher in UA than in SA and C subjects (P<0.001). In in vitro experiments, oxLDL increased CD14 and TLR-4 expression (P<0.001) in control monocytes as well as IL-6, IL-1 beta, and at a lower extent TNF-alpha and MCP-1 levels in the supernatant (P from <0.05 to <0.001). The preincubation of sera derived from UA patients but with control monocytes also induced a significant increase of CD14 and TLR-4 expression (P<0.001) and of IL-6 and IL-1 beta production (P<0.001) in the supernatant. In UA patients oxLDL may contribute to monocyte overproduction of some cytokines by upregulating CD14 and TLR-4 expression.

IL‐13 induces expression of CD36 in human monocytes through PPARγ activation

The class B scavenger receptor CD36 is a component of the pattern recognition receptors on monocytes that recognizes a variety of molecules. CD36 expression in monocytes depends on exposure to soluble mediators. We demonstrate here that CD36 expression is induced in human monocytes following exposure to IL-13, a Th2 cytokine, via the peroxisome proliferator-activated receptor (PPAR)gamma pathway. Induction of CD36 protein was paralleled by an increase in CD36 mRNA. The PPARgamma pathway was demonstrated using transfection of a PPARgamma expression plasmid into the murine macrophage cell line RAW264.7, expressing very low levels of PPARgamma, and in peritoneal macrophages from PPARgamma-conditional null mice. We also show that CD36 induction by IL-13 via PPARgamma is dependent on phospholipase A2 activation and that IL-13 induces the production of endogenous 15-deoxy-Delta12,14-prostaglandin J2, an endogenous PPARgamma ligand, and its nuclear localization in human monocytes. Finally, we demonstrate that CD36 and PPARgamma are involved in IL-13-mediated phagocytosis of Plasmodium falciparum-parasitized erythrocytes. These results reveal a novel role for PPARgamma in the alternative activation of monocytes by IL-13, suggesting that endogenous PPARgamma ligands, produced by phospholipase A2 activation, could contribute to the biochemical and cellular functions of CD36.

Corticosteroid enhances heme oxygenase-1 production by circulating monocytes by up-regulating hemoglobin scavenger receptor and amplifying the receptor-mediated uptake of hemoglobin–haptoglobin complex

This study examined the relationship between steroid treatment and CD163-mediated downstream pathways linked to inflammatory resolution. Twelve patients referred for congenital heart disease surgery were divided into two groups based on the severity of intravascular hemolysis during cardiopulmonary bypass surgery. Patients with severe intravascular hemolysis were administered haptoglobin during the procedure. Flow cytometry indicated a peak in monocyte CD163 expression on post-operative day 1 in both groups. Enhanced and prolonged heme oxygenase-1 (HO-1) mRNA expression levels were observed in patients who received haptoglobin. Binding of hemoglobin–haptoglobin complex (Hb/Hp) to CD163 resulted in significant induction of HO-1 by peripheral blood mononuclear cells after exposure to dexamethasone prior to culture. This effect was significantly inhibited by anti-CD163 antibody. Our results demonstrated up-regulation of CD163 expression on the monocyte surface by steroid treatment. Steroid treatment was suggested to facilitate CD163-mediated endocytosis of hemoglobin to monocytes/macrophages and thereby induce acceleration of HO-1 synthesis.

CD163-expressing monocytes constitute an endotoxin-sensitive Hb clearance compartment within the vascular system

Hemoglobin (Hb) is released into the circulation during intravascular hemolysis and exerts toxic effects through oxidative damage and NO scavenging. According to the traditional concept of Hb clearance, free Hb is bound to the plasma protein haptoglobin (Hp), and the Hb-Hp complexes are cleared by liver and spleen macrophages via the Hb scavenger receptor CD163. Using a novel whole blood assay, we demonstrate that clearance of Hb-Hp is also mediated by CD14(high)/CD64(high) peripheral blood monocytes, which express CD163. Hb-Hp uptake by these cells is Ca(2+)-dependent and is abrogated by the addition of CD163-blocking antibodies. Accordingly, LPS treatment reduces monocyte surface CD163 and impairs Hb-Hp uptake. Monocytes likely mediate Hp-Hb uptake in vivo, as a high expression of the heme breakdown enzyme heme oxygenase-1 was observed in CD163(+) monocytes but not in other leukocyte populations obtained from healthy blood donors. We propose that CD163-mediated Hb-Hp uptake by peripheral blood monocytes constitutes an Hb-Hp clearance pathway, which acts at the site of intravascular hemolysis to reduce Hb-Hp circulation time and toxicity. Disruption of monocyte Hb-Hp clearance may increase Hb-Hp toxicity and contribute to the pathogenesis of systemic inflammatory diseases associated with reduced monocyte CD163 expression.

Tumor necrosis factor alpha and adalimumab differentially regulate CD36 expression in human monocytes

In chronic inflammatory diseases, such as rheumatoid arthritis, inflammation acts as an independent cardiovascular risk factor and the use of anti-inflammatory drugs, such as anti-tumor necrosis factor alpha (anti-TNFα), may decrease this risk. The phagocytosis of oxidized low density lipoproteins (LDLs) accumulated in the subendothelium by mononuclear cells influences atherosclerosis and depends on CD36 expression. We investigated the role of TNFα and adalimumab, a human anti-TNFα monoclonal antibody widely used in human pathology, in CD36 expression in human monocytes. Human monocytes were prepared by adherence from whole-blood buffy-coat fractions from healthy donors. CD36 expression was assessed by RT-PCR and flow cytometry, with various TNFα or adalimumab concentrations. Implication of peroxisome proliferator-activated receptor (PPAR)γ in the regulation of CD36 expression was assessed using specific inhibitor or gel shift assays. The impact of redox signaling was investigated using quantification of reactive oxygen species, antioxidant and a NADPH oxidase inhibitor. The F(ab')2 fragment of adalimumab was isolated and its effect was analyzed. TNFα inhibits both CD36 membrane expression and mRNA expression. This inhibition involves a reduction in PPARγ activation. In contrast, adalimumab increases both CD36 membrane expression and mRNA expression. This induction is independent of the Fc portion of adalimumab and involves redox signaling via NADPH oxidase activation. CD36 expression on human monocytes is inhibited by TNFα and independently increased by adalimumab. These data highlight that pro-inflammatory cytokines and their specific neutralization influence the expression of cellular receptors implicated in atherosclerosis. Further studies are needed to investigate the clinical implications of these results in accelerated atherosclerosis observed in rheumatoid arthritis.

α1-Antitrypsin regulates CD14 expression and soluble CD14 levels in human monocytes in vitro

The recognition of bacterial lipopolysaccharide (LPS) is principally mediated by either membrane-bound or soluble form of the glycoprotein CD14 and CD14-associated signal transducer, toll-like receptor 4 (TLR4). Recent findings indicate that the serine protease inhibitor, α1-antitrypsin (AAT), may not only afford protection against proteolytic injury, but may also neutralize microbial activities and affect regulation of innate immunity. We postulated that AAT affects monocyte responses to LPS by regulating CD14 expression and soluble CD14 release. Here we show that a short-term (up to 2 h) monocyte exposure to AAT alone or in combination with LPS leads to a remarkable induction of CD14 levels. In parallel, a short-term (2 h) cell exposure to AAT/LPS significantly enhances LPS-induced NFκB (p50 and p65) activation in conjunction with increased TNFα, IL-1β and IL-8 release. In contrast, longer term incubation (18 h) of monocytes with combined AAT/LPS results in a significant reduction in expression of both CD14 and TLR4, inhibition of LPS-induced TNFα, IL-1β and IL-8 mRNA and protein expression. These findings provide evidence that AAT is an important regulator of CD14 expression and release in monocytes and suggest that AAT may be involved in LPS neutralization and prevention of over-activation of monocytes in vivo.

Differential expression of monocyte CD163 in single – and dual‐asthmatic responders during allergen‐induced bronchoconstriction

Mononuclear phagocytes play an important role in modulating inflammatory reactions in response to antigen challenge. To investigate the regulation of CD163, a marker of anti-inflammatory macrophages, during Dermatophagoides pteronyssinus (Dp)-induced bronchoconstriction. Among 110 Dp-sensitive patients who underwent bronchial challenge with Dp, there were 51 dual responders (DR), 32 single responders (SR) and 27 non-responders (NR). Monocyte expression of CD14 and CD163 was evaluated by flow cytometry. Exhaled NO (eNO) concentration was determined on-line using a chemiluminescence analyser. In 21 DR, nine SR and 13 NR-soluble CD163 in plasma was measured by ELISA before, 1, 8 and 24 h after the challenge. In DR, the baseline expression of monocyte CD163 and eNO were significantly greater than in SR and NR. A pattern of (1) decreased monocyte CD163 expression, (2) unchanged sCD163 and (3) increased eNO in DR was contrasted by a pattern of (1) increased CD163 expression, (2) increased sCD163 and (3) unchanged eNO in SR. During allergen challenge, the changes in monocyte CD163 expression and sCD163 inversely correlated with the changes in eNO. Both pro-inflammatory and anti-inflammatory responses to allergen challenge are uniquely expressed among SR and DR.

Monocyte CD163 and CD36 Expression in Human Whole Blood and Isolated Mononuclear Cell Samples: Influence of Different Anticoagulants

We investigated whether the choice of anticoagulant or the application of density gradient mononuclear cell isolation may account for conflicting published data regarding the levels of the scavenger receptors' expression in healthy individuals. We demonstrate that the detection of CD163, but not CD36, differs dramatically among the methods.

Su.58. Donor-Specific Differences in the Regulation of Human Monocyte Cd163 Expression and Cytokine Production Upon Toll-Like Receptor Activation

Su.58. Donor-Specific Differences in the Regulation of Human Monocyte Cd163 Expression and Cytokine Production Upon Toll-Like Receptor Activation

Anti-proteinase 3 antibodies (c-ANCA) prime CD14-dependent leukocyte activation

In Wegener's granulomatosis (WG), a pathogenetic role has been proposed for circulating anti-neutrophil-cytoplasmic antibodies (ANCA) targeting proteinase 3 (PR3). Disease activation in WG appears to be triggered by bacterial infections. In the present study, we characterized the effect of anti-PR3 antibodies on in vitro activation of isolated monocytes and neutrophils by the bacterial cell-wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Although sole incubation of monocytes and neutrophils with monoclonal anti-PR3 antibodies induced the release of minor quantities of the chemokine interleukin-8 (IL-8), preincubation with anti-PR3 antibodies, but not with isotype-matched control immunogloblin G (IgG), resulted in a markedly enhanced IL-8 liberation upon LPS challenge. The priming response was evident after 2 h of preincubation with anti-PR3 and peaked after 6 h. The anti-PR3-related priming was also observed for tumor necrosis factor alpha (TNF-alpha) and IL-6 synthesis. Comparable priming occurred when leukocytes were preincubated with ANCA-IgG derived from WG serum but not with normal IgG. The priming effect of the anti-PR3 antibody pretreatment was reproduced for LTA challenge of monocytes and neutrophils but not for leukocyte stimulation with TNF-alpha. Flow cytometric analysis revealed an increase in monocyte and neutrophil membrane CD14 expression during the anti-PR3 priming. We conclude that cytoplasmic ANCA specifically prime CD14-dependent monocytes and neutrophils for activation. The resulting enhanced responsiveness to bacterial pathogens may contribute to the development and maintenance of inflammatory lesions during active WG.

Intracellular Monocyte Cytokine Production and CD14 Expression Are Up-Regulated in Severe vs Mild Chronic Heart Failure

The role of circulating monocytes in the process of low-grade inflammation, characteristic of chronic heart failure (CHF), has recently been questioned. Lipopolysaccharide (LPS) desensitization has been proposed to mediate reduced monocyte cytokine elaboration in patients with severe CHF. Intracellular monocyte production of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha, and monocyte CD 14 expression were measured flow-cytometrically without and after 8-hour LPS stimulation in 46 patients with CHF and in a healthy control group. Basal cytokine concentrations were similar for the control and the mild CHF groups (New York Heart Association [NYHA] Class I or II). After LPS stimulation, IL-6 (p=0.002) and TNF-alpha levels (p=0.001) were lower in the latter group, whereas IL-1 beta production was comparable. For the moderate-severe CHF patients, unstimulated IL-1 beta (p=0.04) was higher, whereas IL-6 (p=0.2) and TNF-alpha (p=0.1) levels were not different from the controls. Measurement of LPS-stimulated cytokine production showed no differences between the control group and patients with moderate-severe CHF (all p= 0.5). Upon comparing mild vs moderate-severe CHF patients, higher levels of unstimulated cytokine production (IL-1 beta, p=0.002; IL-6, p=0.01; TNF-alpha, p=0.003), stimulated IL-1 beta (p=0.002) and IL-6 (p=0.008) were found in the latter patients. CD 14 expression in the moderate-severe CHF group was higher than in the mild-CHF group (p = 0.03) and was strongly related to stimulated IL-1 beta (r=0.62, p<0.0001), IL-6 (r=0.56, p=0.0002) and TNF-alpha (r=0.41, p=0.006) production. CD 14 expression and monocyte cytokine production, both unstimulated and after LPS stimulation, are increased in moderate-severe CHF when compared with mild CHF. These data suggest that circulating monocytes, possibly via increased CD 14 expression, may play a significant role in the immunologic dysbalance observed in advanced CHF.

Human monocyte CD163 expression inversely correlates with soluble CD163 plasma levels

CD163 is a monocyte/macrophage-restricted receptor involved in the clearance of hemoglobin-haptoglobin complexes and regulation of inflammatory processes. CD163 is shed from the cell surface and exists as a soluble form in plasma (sCD163). Monocyte CD163 and sCD163 are potential diagnostic tools in variety of disease states. We determined the relation between plasma sCD163 levels by enzyme-linked immunosorbent assay, membrane expressions of CD163, CD64, and CD14 on blood monocytes by flow cytometry, and monocyte counts in 129 random blood samples. A strong inverse correlation was found between membrane CD163 expression and sCD163 levels (r = -0.65, P < 0.001). Monocyte CD163 expression and SCD163 levels did not correlate with the monocyte absolute count. The inverse relation between monocyte surface CD163 expression and sCD163 levels in human blood suggests that plasma sCD163 is derived from circulating monocytes, in addition to an unknown component from tissue macrophages. The lack of correlation with the absolute monocyte number suggests that such a balance is driven by the functional state of monocytes, rather than simply by numerical changes in circulating cells. We propose that further clinical evaluations of CD163 as a diagnostic parameter should include simultaneous measurements of soluble and cell-bound forms of this antigen.

Long-distance running modulates the expression of leucocyte and endothelial adhesion molecules.

There still exist many unanswered questions whether physical exercise is beneficial or harmful to the immune system. The 'open-window' post-exercise hypothesis states that athletes are more susceptible to infections after exercise, but there is a need for further elucidation. The aim of the present study was to investigate the effect of long-distance running on leucocyte expression of selected adhesion molecules as well as the plasma levels of soluble leucocyte- and endothelium-derived adhesion molecules. Twenty-seven men participating in Oslo marathon together with 16 entrants (eight men and eight women) in the Oslo half-marathon were recruited to this study. Venous blood was collected before and immediately after the races for analysing the leucocyte expression of CD62L, CD11b and CD14 with the help of flow cytometry, and plasma concentrations of soluble (s) sE-selectin, sL-selectin, sP-selectin, sVCAM-1, sICAM-1 and sCD14 were assessed by means of enzyme-linked immunosorbent assays. A significant increase of leucocyte CD11b expression was observed following both races, compared to the pre-race situation. Monocyte CD14 expression increased only after the marathon race. After both races, CD62L expression was significantly lowered on all leucocyte subsets, whereas the plasma levels of sE-selectin, sP-selectin, sL-selectin, sVCAM-1, sICAM-1 and sCD14 were all increased. Altogether, these changes negatively influence the ability of leucocytes to adhere to and actively transmigrate the endothelium to reach the tissues. Our study thus supports the 'open-window' hypothesis, indicating a reduced capacity to combat infectious agents during the immediate post-exercise period.

Growth factors induce monocyte binding to vascular smooth muscle cells: implications for monocyte retention in atherosclerosis

Adhesive interactions between monocytes and vascular smooth muscle cells (VSMC) may contribute to subendothelial monocyte-macrophage retention in atherosclerosis. We investigated the effects of angiotensin II (ANG II) and platelet-derived growth factor (PDGF)-BB on VSMC-monocyte interactions. Treatment of human aortic VSMC (HVSMC) with ANG II or PDGF-BB significantly increased binding to human monocytic THP-1 cells and to peripheral blood monocytes. This was inhibited by antibodies to monocyte beta(1)- and beta(2)-integrins. The binding was also attenuated by blocking VSMC arachidonic acid (AA) metabolism by inhibitors of 12/15-lipoxygenase (12/15-LO) or cyclooxygenase-2 (COX-2). Conversely, binding was enhanced by overexpression of 12/15-LO or COX-2. Direct treatment of HVSMC with AA or its metabolites also increased binding. Furthermore, VSMC derived from 12/15-LO knockout mice displayed reduced binding to mouse monocytic cells relative to genetic control mice. Using specific signal transduction inhibitors, we demonstrated the involvement of Src, phosphoinositide 3-kinase, and MAPKs in ANG II- or PDGF-BB-induced binding. Interestingly, after coculture with HVSMC, THP-1 cell surface expression of the scavenger receptor CD36 was increased. These results show for the first time that growth factors may play additional roles in atherosclerosis by increasing monocyte binding to VSMC via AA metabolism and key signaling pathways. This can lead to monocyte subendothelial retention, CD36 expression, and foam cell formation.

Multiple sclerosis: glatiramer acetate inhibits monocyte reactivity in vitro and in vivo.

It is widely assumed that glatiramer acetate (GA), an approved agent for the immunomodulatory treatment of multiple sclerosis, acts primarily as an antigen for T lymphocytes. Recent studies, however, indicated that in vitro, GA directly inhibits dendritic cells, a rare but potent type of professional antigen-presenting cell (APC). To investigate whether these in vitro observations are relevant to the actions of GA in vivo, we studied the effects of GA on monocytes, the major type of circulating APC. In a first series of experiments, we investigated the effects of GA on monocyte reactivity in vitro. Monocytes were stimulated with ligands for Toll-like receptor (TLR)-2 (peptidoglycan and lipoteichoic acid), TLR-4 [lipopolysaccharide (LPS)] and TLR-5 (flagellin), as well as two proinflammatory cytokines (interferon-gamma and granulocyte-monocyte colony-stimulating factor). Monocyte activation was measured by induction of the surface markers signalling lymphocytic activation molecule (SLAM), CD25 and CD69 (detected by cytofluorometry), and by production of monocyte-derived tumour necrosis factor (TNF)-alpha (detected by enzyme-linked immunospot assay). GA had a broad inhibitory effect on all measures of monocyte reactivity, regardless of which stimulator was used. It is unlikely that this reflects a simple toxic effect, because monocyte viability and CD14 expression were unaffected. In a second series of experiments, we investigated the properties of monocytes cultured ex vivo from eight GA-treated multiple sclerosis patients, eight untreated multiple sclerosis patients and eight healthy subjects. We found that LPS-induced SLAM expression and TNF-alpha production were significantly reduced in monocytes from GA-treated patients compared with controls. These results demonstrate for the first time that GA inhibits monocyte reactivity in vitro and in vivo, significantly extending the current concept of the mechanism of action of GA.

Impact of Different Immunosuppressive Regimens on Antigen-Presenting Blood Cells in Kidney Transplant Patients

Background: Alloantigen-specific and unspecific immune processes contribute to chronic renal graft dysfunction. Despite ‘optimized immunosuppressive therapy’ (IS), the role of chronic cell activation still remains open. Methods: 69 kidney transplant recipients (NTX) were assessed for monocyte surface antigens CD14 (LPS receptor) and CD16 (Fc-γ-III receptor) by flow cytometry including the percentage amount of the proinflammatory CD14+CD16+ subset. 14 non-dialysis patients with chronic renal failure (CRF) and 24 healthy persons served as controls. Results: All 14 patients suffering from CRF revealed higher CRP serum levels compared to healthy controls (p = 0.01). NTX patients had a (not significant) tendency to higher CRP concentrations (p > 0.05). The mean expression of CD14 on monocytes (mCD14) was lower in patients with CRF and in NTX patients (p = 0.024 – p = 0.026). NTX patients revealed low expression of monocytic CD14 with no difference between the single IS therapy groups. The proinflammatory monocyte subpopulation positive for CD14 and CD16 was elevated both in uremic and NTX patients (p < 0.002), despite long-lasting IS therapy. Conclusions: Continuing IS therapy, even under ‘optimized’ drug-monitoring conditions, does not sufficiently prevent or suppress a microinflammatory (and potential fibrotic growth-promoting) status in NTX patients.

Enzymatically modified low-density lipoprotein upregulates CD36 in low-differentiated monocytic cells in a peroxisome proliferator-activated receptor-γ-dependent way

Peroxisome proliferator-activated receptor-γ (PPARγ) has been suggested to upregulate CD36. Since free oxidized polyunsaturated fatty acids are PPARγ ligands, we studied the effects of LDL modified by the simultaneous action of sPLA2 and 15-lipoxygenase (15LO) on CD36 expression and PPARγ activation in monocytic cells. Exposure of MM6 cells, which do not express CD36 or other scavenger receptors, to such enzymatically modified LDL (enzLDL) resulted in upregulation of CD36 surface protein and mRNA expression. Similar effects were observed with free 13-hydroperoxyoctadecadienoic acid but not its esterified counterpart. Less pronounced effects were observed with LDL modified by 15LO alone. Upregulation of CD36 was inversely correlated to the state of cell differentiation, as showed by lower response to enzLDL of the scavenger receptor-expressing MM6-sr and THP1 cells. Importantly, LDL modified by sPLA2 and 15LO did not efficiently induce upregulation CD36 in PPARγ-deficient macrophage-differentiated embryonic stem cells confirming a role of PPARγ in CD36 expression in cells stimulated with enzLDL. Our data show that LDL modified with physiologically relevant enzymes stimulates CD36 expression in non-differentiated monocytes and that this process involves PPARγ activation. These effects of enzLDL can be considered pro-atherogenic in the context of early atherosclerosis.

Statins upregulate CD36 expression in human monocytes, an effect strengthened when combined with PPAR-γ ligands Putative contribution of Rho GTPases in statin-induced CD36 expression

Scavenger receptor CD36 plays important roles in atherosclerosis, inflammation, thrombosis, and angiogenesis. Statins besides lowering serum cholesterol levels, exhibit a variety of effects on inflammation, coagulation and atherosclerosis lesion stability. PPAR-γ ligands influence macrophage responses to many inflammatory stimuli. Herein, we investigated in human monocytes the effect of statins alone, and in combination with PPAR-γ ligands on CD36 expression, as well as the molecular mechanisms underlying the regulatory action of statins. Our results demonstrate that statins upregulate both CD36 surface protein and mRNA by potentiating the transcription of the CD36 gene. Furthermore, the combination of statins and PPAR-γ ligands has an additive effect on CD36 expression. Effects of statins on CD36 expression were prevented by mevalonate and geranylgeraniol, indicating the requirement of geranylgeranylated proteins for CD36 regulation. Rho GTPases inhibitor C3 exoenzyme reproduced the effect of statins, while Rho activator lysophosphatidic acid downregulated CD36. Transient expression of dominant-negative mutants of RhoA and RhoB induced a significant increased in CD36 promoter activity. Finally, the actin cytoskeleton disrupter cytochalasin D upregulated CD36. These data indicate that Rho proteins are important modulators of CD36 expression, and strongly suggest that statins increased CD36 expression by disrupting cytoskeleton organization by inactivating Rho GTPases. These features prompt to investigate the roles of Rho GTPases and actin cytoskeleton modulators on monocytic functions affected by statins.