Membrane CD14 Expression Research Articles

The Effect of Honey Bee Venom on CD14 Protein Expression as Monocyte Marker in D-Alpha-Tocopheryl Succinate (Vitamin E)-Treated HL-60 Cells

Background: The membrane form of a Cluster of Differentiation 14 (CD14) is anchored to the phospholipid bilayer via glycosylphosphatidylinositol anchor. This molecule is expressed on the intrinsic surface of monocytes and neutrophils. This protein is not expressed on the HL60 cell surface. It is believed that the differentiation of HL-60 cells stops in the promyelocytic stage. The differentiation of HL-60 cells with compounds such as vitamin A, D, E results in membrane CD14 expression.
 Objectives: The objective of this study was an evaluation of CD14 expression by Honey Bee Venom (HBV) in HL60 cells treated by D-alpha-Tocopheryl Succinate (D-α-TS). Methods: HL-60 cells were cultured in RPMI Media 1640 medium and treated with different concentrations of D-α-TS and HBV. Cellular differentiation was tested by Nitro Blue Tetrazolium (NBT) staining, immunocytochemistry, and flow cytometry. The studied data were analyzed using one-way analysis of variance and InStat 3 software.
 Methods: HL-60 cells were cultured in RPMI medium and treated with different concentrations of D-α-TS and HBV. Cellular differentiation was tested by NBT staining, immunocytochemistry, and flow cytometry. The studied data were analyzed using one-way analysis of variance and InStat 3 software.
 Results: MTT assay demonstrated that HBV and D-alpha-tocopheryl induce death in HL-60 cell lines in a time and dose-dependent manner. Also, Wright-Giemsa, NBT staining showed morphologically differentiation. Immunocytochemistry and flow cytometry analysis shows that cells treated with 6 µg/mL D-α-TS and 2.5 µg/mL HBV for 5 days significantly increase the expression of CD14 in HL-60 compared to cells treated with D-α-TS.
 Conclusion: HBV can induce cell death and inhibit cell proliferation. Also, increase differentiation potency of D-α-TS via HBV can increase the differentiation by inhibiting NFκB and COX-2 and increasing the expression of P21 that plays an essential role in increasing CD14 protein expression and subsequently induce differentiation in HL-60 cells to monocytes.

Chronic Kidney Disease-Associated Inflammation Increases the Risks of Acute Kidney Injury and Mortality after Cardiac Surgery.

Cardiovascular mortality increases with decreasing renal function although the cause is yet unknown. Here, we have investigated whether low chronic inflammation in chronic kidney diseases (CKD) could contribute to increased risk for coronary artery diseases (CAD). Thus, a prospective case–control study was conducted in patients with CAD and CKD undergoing coronary artery bypass graft surgery with the aim of detecting differences in cardiovascular outcomes, epicardial adipose tissue volume, and inflammatory marker activity associated with renal dysfunction. Expression of membrane CD14 and CD16, inflammatory cytokines and chemokines, mitogen-activated protein (MAP) kinases and hsa-miR-30a-5p were analyzed in peripheral blood mononuclear cells (PBMCs). Epicardial fat volume and tissue inflammation in perivascular adipose tissue and in the aorta were also studied. In the present study, 151 patients were included, 110 with CAD (51 with CKD) and 41 nonCAD controls (15 with CKD). CKD increased the risk of cardiac surgery–associated acute kidney injury (CSA-AKI) as well as the 30-day mortality after cardiac surgery. Higher counts of CD14++CD16+ monocytes were associated with vascular inflammation, with an increased expression of IL1β, and with CKD in CAD patients. Expression of hsa-miR-30a-5p was correlated with hypertension. We conclude that CKD patients show an increased risk of CSA-AKI and mortality after cardiovascular surgery, associated with the expansion of the CD14++CD16+ subset of proinflammatory monocytes and with IL1β expression. We propose that inflammation associated with CKD may contribute to atherosclerosis (ATH) pathogenesis.

The anti-inflammatory effects of vitamin D in mammary epithelial cells through sCD14 (P1329)

Abstract Many studies have shown vitamin D to regulate immune cell responses, yet few have investigated whether vitamin D alters the immune responses of epithelial cells in barrier tissues, such as the mammary gland, to alter immune signaling within tissue microenvironments. Microarray screening and qPCR of human mammary epithelial cells treated with 1α,25-dihydroxyvitamin D (the active form of vitamin D) identified CD14 as a 1,25D induced gene. To follow up these findings, we examined whether 1,25D induced the expression of membrane CD14 or soluble CD14 and found that 1,25D increased the accumulation of sCD14 in a dose- and time- dependent manner but did not alter mCD14. To determine the effects of 1,25D-induced sCD14 from HME cells on Toll-like receptor 4 activation and cytokine secretion from monocyte-derived macrophages, we incubated HEK-Blue TLR4 reporter cells and differentiated THP1 macrophages in conditioned media from 1,25D-treated HME cells ± purified E.coli lipopolysaccharide. We found that conditioned media from 1,25D-treated cells had no significant effects on TLR4 activation in HEK Blue cells yet did promote the secretion of IL-10 from differentiated THP-1 macrophages in the presence of E.coli lipopolysaccharide. These results are the first to demonstrate vitamin D’s effects on the expression and secretion of CD14 in mammary epithelial cells and support the hypothesis that vitamin D promotes an anti-inflammatory environment via secretion of sCD14 in mammary tissue.

Modulation of lipopolysaccharide receptor expression by lactate dehydrogenase-elevating virus

Lactate dehydrogenase-elevating virus (LDV) exacerbates mouse susceptibility to endotoxin shock through enhanced tumour necrosis factor (TNF) production by macrophages exposed to lipopolysaccharide (LPS). However, the in vivo enhancement of TNF production in response to LPS induced by the virus largely exceeds that found in vitro with cells derived from infected animals. Infection was followed by a moderate increase of Toll-like receptor (TLR)-4/MD2, but not of membrane CD14 expression on peritoneal macrophages. Peritoneal macrophages from LDV-infected mice unresponsive to type I interferons (IFNs) did not show enhanced expression of TLR-4/MD2 nor of CD14, and did not produce more TNF in response to LPS than cells from infected normal counterparts, although the in vivo response of these animals to LPS was strongly enhanced. In contrast, the virus triggered a sharp increase of soluble CD14 and of LPS-binding protein serum levels in normal mice. However, production of these LPS soluble receptors was similar in LDV-infected type I IFN-receptor deficient mice and in their normal counterparts. Moreover, serum of LDV-infected mice that contained these soluble receptors had little effect if any on cell response to LPS. These results suggest that enhanced response of LDV-infected mice to LPS results mostly from mechanisms independent of LPS receptor expression.

Expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases

BackgroundInflammatory lung diseases are a major morbidity factor in children. Therefore, novel strategies for early detection of inflammatory lung diseases are of high interest. Bacterial lipopolysaccharide (LPS) is recognized via Toll-like receptors and CD14. CD14 exists as a soluble (sCD14) and membrane-associated (mCD14) protein, present on the surface of leukocytes. Previous studies suggest sCD14 as potential marker for inflammatory diseases, but their potential role in pediatric lung diseases remained elusive. Therefore, we examined the expression, regulation and significance of sCD14 and mCD14 in pediatric lung diseases.MethodssCD14 levels were quantified in serum and bronchoalveolar lavage fluid (BALF) of children with infective (pneumonia, cystic fibrosis, CF) and non-infective (asthma) inflammatory lung diseases and healthy control subjects by ELISA. Membrane CD14 expression levels on monocytes in peripheral blood and on alveolar macrophages in BALF were quantified by flow cytometry. In vitro studies were performed to investigate which factors regulate sCD14 release and mCD14 expression.ResultssCD14 serum levels were specifically increased in serum of children with pneumonia compared to CF, asthma and control subjects. In vitro, CpG induced the release of sCD14 levels in a protease-independent manner, whereas LPS-mediated mCD14 shedding was prevented by serine protease inhibition.ConclusionsThis study demonstrates for the first time the expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases and suggests sCD14 as potential marker for pneumonia in children.

Oxidized Phospholipid Inhibition of Toll-like Receptor (TLR) Signaling Is Restricted to TLR2 and TLR4: ROLES FOR CD14, LPS-BINDING PROTEIN, AND MD2 AS TARGETS FOR SPECIFICITY OF INHIBITION

The generation of reactive oxygen species is a central feature of inflammation that results in the oxidation of host phospholipids. Oxidized phospholipids, such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC), have been shown to inhibit signaling induced by bacterial lipopeptide or lipopolysaccharide (LPS), yet the mechanisms responsible for the inhibition of Toll-like receptor (TLR) signaling by OxPAPC remain incompletely understood. Here, we examined the mechanisms by which OxPAPC inhibits TLR signaling induced by diverse ligands in macrophages, smooth muscle cells, and epithelial cells. OxPAPC inhibited tumor necrosis factor-alpha production, IkappaBalpha degradation, p38 MAPK phosphorylation, and NF-kappaB-dependent reporter activation induced by stimulants of TLR2 and TLR4 (Pam3CSK4 and LPS) but not by stimulants of other TLRs (poly(I.C), flagellin, loxoribine, single-stranded RNA, or CpG DNA) in macrophages and HEK-293 cells transfected with respective TLRs and significantly reduced inflammatory responses in mice injected subcutaneously or intraperitoneally with Pam3CSK4. Serum proteins, including CD14 and LPS-binding protein, were identified as key targets for the specificity of TLR inhibition as supplementation with excess serum or recombinant CD14 or LBP reversed TLR2 inhibition by OxPAPC, whereas serum accessory proteins or expression of membrane CD14 potentiated signaling via TLR2 and TLR4 but not other TLRs. Binding experiments and functional assays identified MD2 as a novel additional target of OxPAPC inhibition of LPS signaling. Synthetic phospholipid oxidation products 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine inhibited TLR2 signaling from approximately 30 microm. Taken together, these results suggest that oxidized phospholipid-mediated inhibition of TLR signaling occurs mainly by competitive interaction with accessory proteins that interact directly with bacterial lipids to promote signaling via TLR2 or TLR4.

Critical Investigation of the CD14 Promoter Polymorphism: Lack of a Role for In Vitro Cytokine Response and Membrane CD14 Expression

Blood of volunteers, genotyped for the CD14 C(-159)-->T polymorphism, showed no difference in cytokine release when stimulated with nine CD14-dependent immune stimuli. An analysis of the published data on the proposed association of CD14 genotype with membrane CD14 density revealed no significant correlation, questioning a functional impact of the CD14 polymorphism.

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.

Gamma-interferon enhances expression of CD14/MyD88 and subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts.

CD14, toll-like receptor 4 (TLR4) and MyD88 have been shown to mediate responsiveness in host cells to lipopolysaccharide. We investigated here the regulatory effects of inflammatory cytokines on the expression of membrane CD14 (mCD14), TLR4 and MyD88, and on subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts. Following treatment with either interleukin-1beta, tumor necrosis factor-alpha (TNF-alpha) or gamma-interferon (IFN-gamma), expression of mCD14/TLR4 and MyD88 was determined by flow cytometry and western blotting, respectively. After pretreatment with IFN-gamma, cells were pre-incubated with either anti-CD14 antibody MY4 or anti-TLR4 antibody HTA125 and subsequently treated with A. actinomycetemcomitans lipopolysaccharide. Then, phosphorylation of mitogen-activated protein (MAP) kinases and IkappaBalpha was examined by western blotting, and production of interleukin-6 and interleukin-8 was measured by their respective enzyme-linked immunosorbent assay (ELISA) kits. IFN-gamma stimulated expression of mCD14, whereas -1beta and TNF-alpha did not. Expression of MyD88 but not TLR4 was also enhanced by IFN-gamma. The lipopolysaccharide activated MAP kinases, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, and IkappaBalpha and stimulated production of interleukin-6 and interleukin-8. The lipopolysaccharide-stimulated interleukin-6 and interleukin-8 production was markedly inhibited by MY4 or HTA125. Pretreatment with IFN-gamma augmented the following activation of MAP kinases and IkappaBalpha and production of interleukin-6 and interleukin-8 in response to the lipopolysaccharide. These results suggest that the augmentation by IFN-gamma of the responsiveness to A. actinomycetemcomitans lipopolysaccharide, such as activation of MAP kinases and IkappaBalpha and terminal cytokine production in human gingival fibroblasts, may be partially mediated by up-regulation of CD14 and MyD88 expression.

A CD14 promoter polymorphism is associated with CD14 expression and Chlamydia-stimulated TNF alpha production.

CD14, a pattern recognition receptor on monocyte and macrophage, plays a central role in innate immunity through recognition of bacterial lipopolysaccharide and initiation of inflammatory response. Recently, CD14/-260C>T promoter gene polymorphism has been found to be related to a risk of inflammatory diseases. Our results showed that the C allele frequency among Chinese in Taiwan was lower than those in Western countries. The membrane CD14 expression was significantly higher in TT as compared with CT and CC genotypes (P=0.034, 0.044, respectively). There was a higher level of soluble CD14 in TT and CT genotypes than in CC genotypes. In addition, TNFalpha production in whole blood was significantly higher in TT genotype than in CC genotype after stimulation by Chlamydiae. In conclusion, the single base pair polymorphism of CD14 promoter gene is associated with CD14 expression and Chlamydia-stimulated TNFalpha production, and may thus play some role in the chlamydia-induced inflammatory response.

Differential regulation of membrane CD14 expression and endotoxin-tolerance in alveolar macrophages.

CD14 is important in the clearance of bacterial pathogens from lungs. However, the mechanisms that regulate the expression of membrane CD14 (mCD14) on alveolar macrophages (AM) have not been studied in detail. This study examines the regulation of mCD14 on AM exposed to Escherichia coli in vivo and in vitro, and explores the consequences of changes in mCD14 expression. The expression of mCD14 was decreased on AM exposed to E. coli in vivo and AM incubated with lipopolysaccharide (LPS) or E. coli in vitro. Polymyxin B abolished LPS effects, but only partially blocked the effects of E. coli. Blockade of extracellular signal-regulated kinase pathways attenuated LPS and E. coli-induced decrease in mCD14 expression. Inhibition of proteases abrogated the LPS-induced decrease in mCD14 expression on AM and the release of sCD14 into the supernatants, but did not affect the response to E. coli. The production of tumor necrosis factor-alpha in response to a second challenge with Staphylococcus aureus or zymosan was decreased in AM after incubation with E. coli but not LPS. These studies show that distinct mechanisms regulate the expression of mCD14 and the induction of endotoxin tolerance in AM, and suggest that AM function is impaired at sites of bacterial infection.

Batimastat reduces Mycobacterium tuberculosis-induced apoptosis in macrophages

In this study, we report evidences that Mycobacterium tuberculosis (MTB)-induced apoptosis in macrophages is reduced by a broad-spectrum hydroxamic acid-based matrix metalloproteinase (MMP) inhibitor, Batimastat (BB-94). In particular, we show that BB-94 administration to MTB-infected macrophages inhibits apoptosis and the downmodulation of membrane CD14 expression. Moreover, the addition of broad spectrum matrix metalloproteinase inhibitor to cell culture, during MTB infection, decreases the release of soluble TNF-α and leads to a simultaneous increase of membrane TNF-α. These results show that MTB-induced apoptosis in macrophages is reduced by a MMP inhibitor and most probably is related to TNF-α release. This identifies BB-94 as a simultaneous anti-apoptotic and anti-inflammatory molecule during MTB infection.

Induction of keratinocyte growth factor 1 Expression by lipopolysaccharide is regulated by CD-14 and toll-like receptors 2 and 4.

Periodontal disease is a chronic inflammatory condition that is associated with increased concentrations of gram-negative pathogenic bacteria and epithelial cell proliferation. Regulation of this proliferation is poorly understood but is most likely controlled by locally expressed growth factors. Keratinocyte growth factor 1, an epithelium-specific growth factor, is expressed by gingival fibroblasts, and its expression is regulated in a concentration-dependent manner by lipopolysaccharide. In this study, induction of keratinocyte growth factor 1 protein expression was dependent on gingival fibroblast expression of membrane CD14 (mCD14) and Toll-like receptors 2 and 4. Lipopolysaccharides from Escherichia coli and Porphyromonas gingivalis induced membrane expression of CD14 at 1, 3, and 24 h. Specifically, lipopolysaccharide induced low mCD14 expression gingival fibroblasts to express mCD14 at a level consistent with that of high mCD14 expression cells. Functional studies with specific blocking antibodies for CD14 and Toll-like receptors 2 and 4 implicated all of these molecules in signal transduction. The rapid decrease in cell membrane expression of Toll-like receptors 2 and 4 after treatment with lipopolysaccharide was consistent with receptor internalization, and blocking of either of these receptors completely inhibited keratinocyte growth factor 1 protein expression. The transcription factors AP-1 and NF-kappaB were involved in lipopolysaccharide induction of keratinocyte growth factor 1 mRNA and protein expression. These results suggest that lipopolysaccharide may induce proliferation of periodontal epithelial cells by upregulating keratinocyte growth factor 1 expression via the CD14 and Toll-like receptor signaling pathway.

Soluble CD14 enriched in colostrum and milk induces B cell growth and differentiation.

Induction of resting B cell growth and differentiation requires a complex series of temporally coordinated signals that are initiated on contact with activated helper T cells. These signals complement one another, each rendering the B cell susceptible to factors supporting progressive activation. Here, we demonstrate that soluble CD14 (sCD14) bypasses the physiological sequelae of events that limit B cell activation. B cell growth and differentiation in vitro is induced by both native and recombinant forms of sCD14 at nanomolar concentrations. sCD14-mediated cellular activation does not require membrane CD14 expression, depends on a region of CD14 that is not involved in lipopolysaccharide binding, and requires functional Toll-like receptor 4. Consistent with biological activity of sCD14 in vitro, its administration to neonatal mice enhances Ig secretion. The results presented establish sCD14 as a naturally occurring soluble B cell mitogen of mammalian origin.

Down-modulation through protein kinase C-α of lipopolysaccharide-induced expression of membrane CD14 in mouse bone marrow granulocytes

We have previously shown that stimulation of mouse bone marrow granulocytes (BMC) by lipopolysaccharide (LPS) induces the expression of CD14. We found here that phorbol 12-myristate 13-acetate (PMA) blocks this LPS effect. The aim of this study was to investigate the mechanism by which PMA can block the LPS signaling pathway in BMC. The unmodified binding of a radiolabeled LPS in PMA-treated cells indicated that the PMA effect was not the consequence of a shedding or an internalization of the LPS receptor, but was rather due to a biochemical event that follows the interaction of LPS with its receptor. The observations that a selective activator of protein kinase C (PKC)-α (sapintoxin D) mimics the PMA effect, whereas a selective PKC-α inhibitor (Ro-320432) antagonizes this effect, suggest a regulatory role of PKC-α in the LPS signaling pathway in mouse BMC.

Mycobacterium tuberculosis-induced apoptosis in monocytes/macrophages: early membrane modifications and intracellular mycobacterial viability.

Apoptosis has been observed in monocytes/macrophages in the course of in vivo and in vitro Mycobacterium tuberculosis (MTB) infection. In order to define the early events of MTB-induced apoptosis, membrane CD14 expression and the exposure of Annexin V-binding sites in MTB-infected monocytes/macrophages have been monitored. Moreover, the role of MTB-induced apoptosis was further analyzed in vitro in terms of mycobacterial viability. Results show that monocyte/macrophage apoptosis is a very early event that is strictly dependent on the MTB amount, and this apoptosis is associated with a selective down-regulation of surface CD14 expression. Furthermore, no statistically significant decrease in mycobacterial viability was observed, which indicates that the apoptotic pathway triggered by high doses of MTB is associated with parasite survival rather than with killing of the parasite.

Function of CD14 as a peptidoglycan receptor: differences and similarities with LPS

Peptidoglycan (PGN) is a macrophage activator from Gram-positive bacteria. PGN activates cells of hemopoietic origin through CD14 since: (i) PGN-unresponsive CD14-negative cells become PGNresponsive after transfection with CD14 and expression of membrane CD14; (ii) PGN binds to CD14 with high affinity; and (iii) anti-CD14 mAbs inhibit both binding of PGN to CD14 and activation of CD14-positive cells by PGN. However, there are several differences in the function of CD14 as PGN and LPS receptor: (i) the kinetics of binding are different; (ii) the affinity of binding in the absence of LPS-binding protein (LBP) is higher for PGN than LPS; (iii) LBP does not increase the affinity of binding of PGN to CD14 and does not enhance cell activation by PGN (in contrast to LPS); (iv) the regions of CD14 needed for binding and activation are partially similar and partially different for PGN and LPS; (v) sCD14:PGN complexes, in contrast to sCD14:LPS complexes, do not activate CD14-negative cells; (vi) PGN, in contrast to LPS, does not activate CHO cells expressing mCD14; and (vii) PGN and LPS induce differential activation of MAP kinases, but activate similar transcription factors (NF-κB, ATF1/CREB, and AP-1).

Endotoxin binding and elimination by monocytes: secretion of soluble CD14 represents an inducible mechanism counteracting reduced expression of membrane CD14 in patients with sepsis and in a patient with paroxysmal nocturnal hemoglobinuria.

Little is known about the role of peripheral blood mononuclear cells (PBMCs) in lipopolysaccharide (LPS) elimination. We studied the endotoxin elimination capacities (EEC) of PBMCs of 15 healthy volunteers, 13 patients with sepsis, and 1 patient suffering from paroxysmal nocturnal hemoglobinuria (PNH). Although expression of CD14, the best-characterized receptor for LPS to date, was reduced from 93.6% +/- 0.8% in healthy subjects to 50.5% +/- 6.5% in patients with sepsis and was 0.3% in a patient with septic PNH, EEC were found to be unchanged. There was no difference in the amount of tumor necrosis factor alpha (TNF-alpha) released by PBMCs of healthy donors and patients with sepsis. Anti-CD14 antibodies (MEM-18) completely suppressed EEC, binding of fluorescein isothiocyanate-labeled LPS to monocytes as determined by FACScan analysis, and TNF-alpha release in all three groups studied. The concentrations of soluble CD14 (sCD14) secreted by endotoxin-stimulated PBMCs from healthy donors and patients with sepsis amounted to 4.5 +/- 0.4 and 20.1 +/- 1.8 ng/ml, respectively. Based on our results, we suggest that PBMCs eliminate LPS by at least two different mechanisms; in healthy subjects, the membrane CD14 (mCD14) receptor is the most important factor for LPS elimination, while in patients with sepsis (including the septic state of PNH), increased sCD14 participates in LPS elimination. Secretion of sCD14 is strongly enhanced under conditions of low expression of mCD14 in order to counteract the reduction of mCD14 and maintain the function of monocytes. This sCD14 may substitute the role of mCD14 in LPS elimination during sepsis. The elimination of LPS by PBMCs correlates with the binding reaction and the secretion of TNF-alpha.

Impaired phagocyte responses to lipopolysaccharide in paroxysmal nocturnal hemoglobinuria.

Bone marrow-derived cells from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH) show a defect in the expression of phosphatidylinositol-anchored membrane proteins, including the CD14 molecule. Blocking experiments with anti-CD14 monoclonal antibodies have shown that lipopolysaccharide (LPS)-induced tumor necrosis factor alpha production by monocytes depends on the interaction between CD14 and a complex formed by LPS and LPS-binding protein. We used a whole-blood model to examine the LPS-induced production of tumor necrosis factor alpha and interleukin-6 in PNH patients and healthy volunteers. At low endotoxin concentrations (1 ng/ml), PNH patients displayed a marked defect in the production of both cytokines, whereas at high LPS concentrations (100 ng/ml), cytokine production was similar to that in healthy volunteers. Using flow cytometry, we also studied the expression of the adhesion molecules Mac-1 (CD11b/CD18) and ICAM-1 (CD54) by monocytes and granulocytes after LPS stimulation. Compared with phagocytes from healthy volunteers, CD14-deficient cells showed poor Mac-1 and ICAM-1 upregulation when whole blood was stimulated with LPS (1 ng/ml), whereas their response to higher LPS doses (100 and 1,000 ng/ml) was essentially normal. The importance of the CD14 molecule in the activation of phagocytes by low LPS concentrations was confirmed by the inhibitory effect of an anti-CD14 antibody both in healthy volunteers and in PNH patients. Since these patients produce the soluble form of the CD14 molecule, these data suggest that soluble CD14 could play a role in phagocyte responses to LPS. We conclude that, in whole blood, phagocytes from PNH patients show impaired responsiveness to LPS and this phenomenon is most probably related to their defect in expression of membrane CD14.

Transforming growth factor-beta potentiates vitamin D3-induced terminal monocytic differentiation of human leukemic cell lines.

We have investigated the effects of 1,25-dihydroxyvitamin D3 (D3) and/or transforming growth factor (TGF)-beta on one monocytic (U-937) and two human promyelocytic (HL-60 and AML-193) leukemic cell lines. D3 addition induces a partial monocytic maturation of the cell lines, whereas TGF-beta treatment is largely ineffective. Combined treatment with TGF-beta and D3 causes terminal monocytic maturation, as evaluated both by assessment of a large spectrum of membrane Ag and by functional assays. Furthermore, sequential addition of the two inducers showed that pretreatment with TGF-beta 1 followed by incubation with D3, but not vice versa, induces monocytic maturation as effectively as simultaneous treatment with both agents. In liquid culture the proliferative activity of these cell lines is slightly decreased by D3 and virtually unaffected by TGF-beta, whereas combined treatment with D3 and TGF-beta induces a markedly potentiated inhibitory effect. Furthermore, TGF-beta/D3 treatment (but not D3 alone) elicits the expression of membrane CD14, FcRI, FcRII, CD11a, CD11b, CD11c, ICAM-1, and PECAM-1 Ag at a level comparable to that observed on normal human monocytes. It is noteworthy that several of these Ag play an important role in monocyte physiology (e.g., CD14 Ag mediates the binding of bacterial LPS to monocytes). Treatment with both TGF-beta and D3 (but not D3 alone) induces superoxide anions and H2O2 production similar to that of circulating monocytes. In semisolid culture, D3 and TGF-beta alone cause, respectively, a marked and slight loss of cloning efficiency of the cell lines, whereas their combined addition synergistically results in a complete loss of the cloning capacity. These findings suggest a physiologic role for TGF-beta in monocyte maturation. Furthermore, they may pave the way to the design of clinical protocols combining D3 and TGF-beta in the differentiation therapy of acute promyelocytic/myelomonocytic leukemia.