Phosphate restriction using a processed clay mineral reduces vascular pathologies and microalbuminuria in rats with chronic renal failure.

BACKGROUND: The progression of chronic kidney disease (CKD) is associated with an increasing risk of cardiovascular morbidity and mortality due to elevated serum phosphate levels. Besides low phosphate diets and hemodialysis, oral phosphate binders are prescribed to treat hyperphosphatemia in CKD patients. This study reports on a processed clay mineral as a novel and efficient phosphate sorbent with comparable efficacy of a clinically approved phosphate binder. METHODS: 5/6 nephrectomized rats, which develop chronic renal failure (CRF), received a high phosphate and calcium diet supplemented with either a processed Montmorillonite-Illite clay mineral (pClM) or lanthanum carbonate (LaC) for 12 weeks. Levels of plasma uremic toxins, glomerular filtration rates and microalbuminuria were determined and the histomorphology of blood vessels and smooth muscle cells was analyzed. RESULTS: 5/6 nephrectomy induced an increase in plasma uremic toxins levels and progressive proteinuria. Treatment of CRF rats with pClM decreased observed vascular pathologies such as vascular fibrosis, especially in coronary vessels. The transition of vascular smooth muscle cells from a contractile to a secretory phenotype was delayed. Moreover, pClM administration resulted in decreased blood creatinine and urea levels, and increased glomerular filtration rates, reduced microalbuminuria and eventually the mortality rate in CRF rats. CONCLUSION: Our study reveals pClM as a potent phosphate binding agent with beneficial impacts on pathophysiological processes in an animal model of CKD. pClM effectively attenuates the progression of vascular damage and loss of renal function which are the most severe consequences of chronic renal failure.

Smectite as a Preventive Oral Treatment to Reduce Clinical Symptoms of DSS Induced Colitis in Balb/c Mice.

Natural smectites have demonstrated efficacy in the treatment of diarrhea. The present study evaluated the prophylactic effect of a diosmectite (FI5pp) on the clinical course, colon damage, expression of tight junction (TJ) proteins and the composition of the gut microbiota in dextran sulfate sodium (DSS) colitis. Diosmectite was administered daily to Balb/c mice from day 1 to 7 by oral gavage, followed by induction of acute DSS-colitis from day 8 to 14 ("Control", n = 6; "DSS", n = 10; "FI5pp + DSS", n = 11). Mice were sacrificed on day 21. Clinical symptoms (body weight, stool consistency and occult blood) were checked daily after colitis induction. Colon tissue was collected for histological damage scoring and quantification of tight junction protein expression. Stool samples were collected for microbiome analysis. Our study revealed prophylactic diosmectite treatment attenuated the severity of DSS colitis, which was apparent by significantly reduced weight loss (p = 0.022 vs. DSS), disease activity index (p = 0.0025 vs. DSS) and histological damage score (p = 0.023 vs. DSS). No significant effects were obtained for the expression of TJ proteins (claudin-2 and claudin-3) after diosmectite treatment. Characterization of the microbial composition by 16S amplicon NGS showed that diosmectite treatment modified the DSS-associated dysbiosis. Thus, diosmectites are promising candidates for therapeutic approaches to target intestinal inflammation and to identify possible underlying mechanisms of diosmectites in further studies.

Targeting of NADPH oxidase in vitro and in vivo suppresses fibroblast activation and experimental skin fibrosis.

Although there is increasing evidence that oxidative stress is involved in collagen synthesis and myofibroblast activation, the NADPH oxidase (Nox) system is incompletely investigated in the context of human dermal fibroblasts (HDFs) and skin fibrosis. Using the pan-Nox inhibitor diphenyleneiodonium (DPI) as an initial tool, we show that gene expression of collagen type I, α-smooth muscle actin (α-SMA) and fibronectin 1 is suppressed in HDFs. Detailed expression analysis of all Nox isoforms and adaptors revealed expression of RNA and protein expression of Nox4, p22phox and Poldip2 but neither Nox1 nor Nox2. Nox4 could be immunolocalized to the endoplasmic reticulum. Importantly, TGF-ß1 had a dose- and time-dependent upregulating effect on NADH activity and Nox4 gene expression in HDFs. Genetic silencing of Nox4 as demonstrated by siRNA in HDFs as well as in murine fibroblasts established from Nox4 knockout mice confirmed that TGF-ß1 -mediated collagen type I gene, α-SMA and fibronectin 1 gene expressions were Nox4-dependent. This TGF-ß1 effect was mediated by Smad3 as shown by in silico promoter analysis, pharmacological inhibition and gene silencing of Smad3. The relevance of these findings is highlighted in the bleomycin-induced scleroderma mouse model. DPI treatment attenuated skin fibrosis and myofibroblast activation. Moreover, Nox4 knockdown by siRNA reduced skin collagen synthesis, α-SMA and fibronectin 1 expression in vivo. Finally, analyses of HDFs from patients with systemic sclerosis confirmed the expression of Nox4 and its adaptors, whereas Nox1 and Nox2 were not detectable. Our findings indicate that Nox4 targeting is a promising future treatment for fibrotic skin diseases.

Is Nox4 a key regulator of the activated state of fibroblasts in systemic sclerosis?

The family of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases consists of phagocytic gp91(phox) and six-related isoforms. Recent evidence indicates that the NADPH oxidase isoform Nox4 controls vascular, renal and pulmonary injury. We propose that Nox4 is an intrinsic regulator of the activated state of dermal fibroblasts in systemic sclerosis (SSc). Profibrotic cytokines on the one hand and antifibrogenic factors such as α-melanocyte-stimulating hormone on the other hand may target Nox4 as an intracellular nodal point. Via increased or decreased generation of reactive oxygen species and/or hydrogen peroxide, Nox4 could orchestrate collagen synthesis, differentiation of dermal fibroblasts into a profibrotic myofibroblast phenotype and thus dermal fibrosis. Confirmation of this hypothesis will have important consequences in our understanding of the activated state of dermal fibroblasts in SSc. Based on the availability of clinically useful Nox4 inhibitors, novel antifibrotic therapies of SSc can be envisioned.

Alteration of DSS-mediated immune cell redistribution in murine colitis by oral colostral immunoglobulin.

BACKGROUND: Oral bovine colostrum prophylaxis accelerates the recovery of dextran sulfate sodium (DSS)-induced colitis. In the present study the beneficial effects on acute intestinal inflammation of two major colostral components, secretory immunoglobulin A and lactoferrin, were investigated. Outbred NMRI mice received whole bovine colostrum (BC, 20 mg/kg body weight), colostral bovine lactoferrin (bLf, 150 mg/kg), or secretory immunoglobulin A (sIgA, 1-2 mg/kg body weight) daily by oral gavage, either two weeks before induction of colitis (prophylaxis) or after disease establishment (therapy). Bovine serum albumin (BSA, 150 mg/kg body weight) and immunoglobulin G (IgG, 1 and 2 mg/kg body weight) served as protein controls. Colitis was induced by providing 5% DSS solution ad libitum for seven days. RESULTS: Compared to BSA, BC therapy improved occult blood, stool consistency, and clinical recovery from colitis but did not prevent initial weight loss. In contrast, administration of bLf did not influence the course of colitis in either the prophylactic or the therapeutic setting. Therapeutic application of sIgA promoted weight gain in the recovery phase of colitis but failed to improve other clinical parameters. Prophylactically-fed sIgA influenced immune cell redistribution, normalized peripheral blood CD11c⁺CD83⁺ mature dendritic cells, modulated colonic immune cell infiltration, and altered the numbers of both DSS-induced regulatory γδ TCR⁺ T cells and CD11b⁺Gr-1⁺ myeloid suppressor cells in the lymph nodes and spleens of mice. CONCLUSIONS: These data demonstrated the potential of colostrum in disease recovery and epithelial homeostasis following intestinal injury. Colostral sIgA failed to improve acute disease activity but promoted weight gain and modulated immune cell responses that are involved in the genesis of colitis.

S100A8 and S100A9 in cardiovascular biology and disease.

There is recent and widespread interest in the damage-associated molecular pattern molecules S100A8 and S100A9 in cardiovascular science. These proteins have a number of interesting features and functions. For example, S100A8 and S100A9 (S100A8/A9) have both intracellular and extracellular actions, they are abundantly expressed in inflammatory and autoimmune states, primarily by myeloid cells but also by other vascular cells, and they modulate inflammatory processes, in part through Toll-like receptor 4 and the receptor for advanced glycation end products. S100A8/A9 also have anti-inflammatory and immune regulatory actions. Furthermore, increased plasma levels of S100A8/A9 predict cardiovascular events in humans, and deletion of these proteins partly protects Apoe(-)(/)(-) mice from atherosclerosis. Understanding the roles of S100A8 and S100A9 in vascular cell types and the mechanisms whereby these proteins mediate their biological effects may offer new therapeutic strategies to prevent, treat, and predict cardiovascular diseases.

A novel intronic cAMP response element modulator (CREM) promoter is regulated by activator protein-1 (AP-1) and accounts for altered activation-induced CREM expression in T cells from patients with systemic lupus erythematosus.

The transcriptional repressor cAMP response element modulator (CREM) α has important roles in normal T cell physiology and contributes to aberrant T cell function in patients with systemic lupus erythematosus (SLE). Recently, we characterized a specificity protein-1-dependent promoter located upstream of the CREM gene that accounts for increased basal CREM expression in SLE T cells and reflects disease activity. Here, we identify a novel intronic CREM promoter (denoted P2) in front of the second exon of the CREM gene that harbors putative binding sites for TATA-binding proteins and the transcriptional activator AP-1. DNA binding studies, chromatin immunoprecipitation, and reporter assays confirmed the functional relevance of these sites, and T cell activation through CD3/CD28 stimulation or phorbol 12-myristate 13-acetate/ionomycin treatment enhances P2 promoter activity. Although the basal CREM levels are increased in T cells from SLE patients compared with healthy controls, there are remarkable differences in the regulation of CREM expression in response to T cell activation. Whereas T cells from healthy individuals display increased CREM expression after T cell activation, most likely through AP-1-dependent up-regulation of the P2 promoter, SLE T cells fail to further increase their basal CREM levels upon T cell activation due to a decreased content of the AP-1 family member c-Fos. Because CREM trans-represses c-fos transcription in SLE T cells, we propose an autoregulatory feedback mechanism between CREM and AP-1. Our findings extend the understanding of CREM gene regulation in the context of T cell activation and disclose another difference in the transcriptional machinery in SLE T cells.

S100A9 differentially modifies phenotypic states of neutrophils, macrophages, and dendritic cells: implications for atherosclerosis and adipose tissue inflammation.

BACKGROUND: S100A9 is constitutively expressed in neutrophils, dendritic cells, and monocytes; is associated with acute and chronic inflammatory conditions; and is implicated in obesity and cardiovascular disease in humans. Most of the constitutively secreted S100A9 is derived from myeloid cells. A recent report demonstrated that mice deficient in S100A9 exhibit reduced atherosclerosis compared with controls and suggested that this effect was due in large part to loss of S100A9 in bone marrow-derived cells. METHODS AND RESULTS: To directly investigate the role of bone marrow-derived S100A9 in atherosclerosis and insulin resistance in mice, low-density lipoprotein receptor-deficient, S100A9-deficient bone marrow chimeras were generated. Neither atherosclerosis nor insulin resistance was reduced in S100A9-deficient chimeras fed a diet rich in fat and carbohydrates. To investigate the reason for this lack of effect, myeloid cells were isolated from the peritoneal cavity or bone marrow. S100A9-deficient neutrophils exhibited a reduced secretion of cytokines in response to toll-like receptor-4 stimulation. In striking contrast, S100A9-deficient dendritic cells showed an exacerbated release of cytokines after toll-like receptor stimulation. Macrophages rapidly lost S100A9 expression during maturation; hence, S100A9 deficiency did not affect the inflammatory status of macrophages. CONCLUSIONS: S100A9 differentially modifies phenotypic states of neutrophils, macrophages, and dendritic cells. The effect of S100A9 deficiency on atherosclerosis and other inflammatory diseases is therefore predicted to depend on the relative contribution of these cell types at different stages of disease progression. Furthermore, S100A9 expression in nonmyeloid cells is likely to contribute to atherosclerosis.

Novel insights into the role of S100A8/A9 in skin biology.

S100A8 and S100A9 belong to the damage-associated molecular pattern molecules. They are upregulated in a number of inflammatory skin disorders. Owing to their abundance in myeloid cells, the main function of S100A8/A9 has been attributed to their role in inflammatory cells. However, it is becoming increasingly clear that they also exert important roles in epithelial cells. In this review, we discuss the context-dependent function of S100A8/A9 in epithelial cells and their impact on wound healing, psoriasis and other skin diseases.

S100A2 induces metastasis in non-small cell lung cancer.

PURPOSE: S100 proteins are implicated in metastasis development in several cancers. In this study, we analyzed the prognostic role of mRNA levels of all S100 proteins in early stage non-small cell lung cancer (NSCLC) patients as well as the pathogenetic of S100A2 in the development of metastasis in NSCLC. EXPERIMENTAL DESIGN: Microarray data from a large NSCLC patient cohort was analyzed for the prognostic role of S100 proteins for survival in surgically resected NSCLC. Metastatic potential of the S100A2 gene was analyzed in vitro and in a lung cancer mouse model in vivo. Overexpression and RNAi approaches were used for analysis of the biological functions of S100A2. RESULTS: High mRNA expression levels of several S100 proteins and especially S100A2 were associated with poor survival in surgically resected NSCLC patients. Upon stable transfection into NSCLC cell lines, S100A2 did not alter proliferation. However, S100A2 enhanced transwell migration as well as transendothelial migration in vitro. NOD/SCID mice injected s.c. with NSCLC cells overexpressing S100A2 developed significantly more distant metastasis (64%) than mice with control vector transfected tumor cells (17%; P < 0.05). When mice with S100A2 expressing tumors were treated i.v. with shRNA against S100A2, these mice developed significantly fewer lung metastasis than mice treated with control shRNA (P = 0.021). CONCLUSIONS: These findings identify S100A2 as a strong metastasis inducer in vivo. S100A2 might be a potential biomarker as well as a novel therapeutic target in NSCLC metastasis.

S100A8/9 induces cell death via a novel, RAGE-independent pathway that involves selective release of Smac/DIABLO and Omi/HtrA2.

A complex of two S100 EF-hand calcium-binding proteins S100A8/A9 induces apoptosis in various cells, especially tumor cells. Using several cell lines, we have shown that S100A8/A9-induced cell death is not mediated by the receptor for advanced glycation endproducts (RAGE), a receptor previously demonstrated to engage S100 proteins. Investigation of cell lines either deficient in, or over-expressing components of the death signaling machinery provided insight into the S100A8/A9-mediated cell death pathway. Treatment of cells with S100A8/A9 caused a rapid decrease in the mitochondrial membrane potential (DeltaPsi(m)) and activated Bak, but did not cause release of apoptosis-inducing factor (AIF), endonuclease G (Endo G) or cytochrome c. However, both Smac/DIABLO and Omi/HtrA2 were selectively released into the cytoplasm concomitantly with a decrease in Drp1 expression, which inhibits mitochondrial fission machinery. S100A8/A9 treatment also resulted in decreased expression of the anti-apoptotic proteins Bcl2 and Bcl-X(L), whereas expression of the pro-apoptotic proteins Bax, Bad and BNIP3 was not altered. Over-expression of Bcl2 partially reversed the cytotoxicity of S100A8/A9. Together, these data indicate that S100A8/A9-induced cell death involves Bak, selective release of Smac/DIABLO and Omi/HtrA2 from mitochondria, and modulation of the balance between pro- and anti-apoptotic proteins.

S100A8/A9 at low concentration promotes tumor cell growth via RAGE ligation and MAP kinase-dependent pathway.

The complex formed by two members of the S100 calcium-binding protein family, S100A8/A9, exerts apoptosis-inducing activity against various cells, especially tumor cells. Here, we present evidence that S100A8/A9 also has cell growth-promoting activity at low concentrations. Receptor of advanced glycation end product (RAGE) gene silencing and cotreatment with a RAGE-specific blocking antibody revealed that this activity was mediated via RAGE ligation. To investigate the signaling pathways, MAPK phosphorylation and NF-kappaB activation were characterized in S100A8/A9-treated cells. S100A8/A9 caused a significant increase in p38 MAPK and p44/42 kinase phosphorylation, and the status of stress-activated protein kinase/JNK phosphorylation remained unchanged. Treatment of cells with S100A8/A9 also enhanced NF-kappaB activation. RAGE small interfering RNA pretreatment abrogated the S100A8/A9-induced NF-kappaB activation. Our data indicate that S100A8/A9-promoted cell growth occurs through RAGE signaling and activation of NF-kappaB.

The calcium binding protein S100A9 is essential for pancreatic leukocyte infiltration and induces disruption of cell-cell contacts.

Leukocyte infiltration is an early and critical event in the development of acute pancreatitis. However, the mechanism of leukocyte transmigration into the pancreas and the function of leukocytes in initiating acute pancreatitis are still poorly understood. Here, we studied the role of S100A9 (MRP14), a calcium binding protein specifically released by polymorph nuclear leukocytes (PMN), in the course of acute experimental pancreatitis. Acute pancreatitis was induced by repeated supramaximal caerulein injections in S100A9 deficient or S100A9 wild-type mice. We then determined S100A9 expression, trypsinogen activation peptide (TAP) levels, serum amylase and lipase activities, and tissue myeloperoxidase (MPO) activity. Cell-cell contact dissociation was analyzed in vitro with biovolume measurements of isolated acini after incubation with purified S100A8/A9 heterodimers, and in vivo as measurement of Evans Blue extravasation after intravenous application of S100A8/A9. Pancreatitis induced increased levels of S100A9 in the pancreas. However, infiltration of leukocytes and MPO activity in the lungs and pancreas during acute pancreatitis was decreased in S100A9-deficient mice and associated with significantly lower serum amylase and lipase activities as well as reduced intrapancreatic TAP-levels. Incubation of isolated pancreatic acini with purified S100A8/A9-heterodimers resulted in a rapid dissociation of acinar cell-cell contacts which was highly calcium-dependent. Consistent with these findings, in vivo application of S100A8/A9 in mice was in itself sufficient to induce pancreatic cell-cell contract dissociation as indicated by Evans Blue extravasation. These data show that the degree of intrapancreatic trypsinogen activation is influenced by the extent of leukocyte infiltration into the pancreas which, in turn, depends on the presence of S100A9 that is secreted from PMN. S100A9 directly affects leukocyte tissue invasion and mediates cell contact dissociation via its calcium binding properties.

The hetero-oligomeric complex of the S100A8/S100A9 protein is extremely protease resistant.

S100A8, S100A9 and S100A12 proteins are associated with inflammation and tissue remodelling, both processes known to be associated with high protease activity. Here, we report that homo-oligomeric forms of S100A8 and S100A9 are readily degraded by proteases, but that the preferred hetero-oligomeric S100A8/A9 complex displays a high resistance even against proteinase K degradation. S100A12 is not as protease resistant as the S100A8/A9 complex. Since specific functions have been assigned to the homo- and heterooligomeric forms of the S100A8 and A9 proteins, this finding may point to a post-translational level of regulation of the various functions of these proteins in inflammation and tissue remodelling.

Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression.

BACKGROUND: S100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca2+-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879-41887). RESULTS: In the present study, we investigated transcription factors that bind to MRE. Using the MRE motif for a pull-down assay, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80 were identified by mass spectrometry and confirmed by chromatin immunoprecipitation. Furthermore, TPA-induced S100A9 gene expression in HaCaT keratinocytes was blocked after the pharmacologic inhibition of PARP-1 with 1,5-isoquinolinediol (DiQ). CONCLUSION: The candidates, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80, are known to participate in inflammatory disorders as well as tumorgenesis. The latter may indicate a possible link between S100 and inflammation-associated cancer.

The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

S100A9 deficiency alters adenosine-5'-triphosphate induced calcium signalling but does not generally interfere with calcium and zinc homeostasis in murine neutrophils.

The two calcium- and zinc-binding proteins, S100A9 and S100 A8, abundant in myeloid cells are considered to play important roles in both calcium signalling and zinc homeostasis. Polymorphonuclear neutrophils from S100A9 ko mice are also devoid of S100A8. Therefore, S100A9-deficient neutrophils were used as a model to study the role of the two S100 proteins in the neutrophils's calcium and zinc metabolism. Analysis of the intracellular zinc level upon pyrithione and (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide (NOR-1) treatment revealed no differences between S100A9-deficient and wildtype neutrophils. Similar, the calcium signals were not distinguishable from S100A9-deficient and wildtype neutrophils upon stimulation with platelet activating factor (PAF), thapsigargin or macrophage inflammatory protein 1 alpha (MIP-1 alpha), indicating despite their massive expression S100A8/A9 do neither serve as calcium nor as zinc buffering proteins in granulocytes. In contrast, stimulation with adenosine-5'-triphosphate (ATP) induces a significant stronger increase of the intracellular free calcium level in S100A9-deficient cells compared to wildtype cells. Moreover, the ATP-induced calcium signal was still different when the cells were incubated in calcium free buffer suggesting that pirinergic receptors of the P(2Y) class could be involved in this signalling pathway.

Mechanism of apoptosis induced by S100A8/A9 in colon cancer cell lines: the role of ROS and the effect of metal ions.

The protein complex S100A8/A9, abundant in the cytosol of neutrophils, is secreted from the cells upon cellular activation and induces apoptosis in tumor cell lines and normal fibroblasts in a zinc-reversible manner. In the present study, we present evidence that the S100A8/A9 also exerts its apoptotic effect by a zinc-independent mechanism. Treatment of the colon carcinoma cells with different concentrations of human S100A8/A9 or the metal ion chelator diethylenetriaminepentacetic acid (DTPA) resulted in a significant increase of cell death. Annexin V/phosphatidylinositol and Hoechst 33258 staining revealed that cell death was mainly of the apoptotic type. A significant increase in the activity of caspase-3 and -9 was observed in both cell lines after treatment. Caspase-8 activation was negligible in both cell lines. The cytotoxicity/apoptotic effect of human S100A8/A9 and DTPA was inhibited significantly (P<0.05) by Zn(+2) and Cu(+2), more effectively than by Ca(2+) and Mg(2+). The antioxidant N-acetyl-L-cysteine inhibited the cytotoxicity/apoptotic effect of S100A8/A9 and DTPA. However, as a result of the different time-courses of both agents and that the S100A8/A9-induced apoptosis was not completely reversed, we conclude that S100A8/A9 exerts its apoptotic effect on two colon carcinoma cell lines through a dual mechanism: one via zinc exclusion from the target cells and the other through a yet-undefined mechanism, probably relaying on the cell-surface receptor(s).

L-FABP is exclusively expressed in alveolar macrophages within the myeloid lineage: evidence for a PPARalpha-independent expression.

Peroxisome proliferator-activated receptors (PPARs) play a role in inflammation and, in particular, PPARgamma is involved in monocyte/macrophage differentiation. Members of the fatty acid-binding protein (FABP) family have been reported to function as transactivators for PPARs. Therefore, the expression of PPARs and FABPs in the myeloid lineage was investigated by real-time PCR and immunofluorescence analysis. We found adipocyte-, epidermal-, and heart-type FABP to be ubiquitously expressed within the myeloid lineage. In contrast, liver-type FABP was exclusively detected in murine alveolar macrophages (AM), confirmed on protein level by double fluorescence analysis. The PPAR subtypes also showed a temporally and spatially regulated expression pattern in myeloid cells: the beta-subtype was expressed in bone marrow, peritoneal, and alveolar macrophages, whereas it was not detected in dendritic cells (DCs). The gamma1-isoform was present in all cells, however, at different levels, whereas the gamma2-isoform was expressed in alveolar macrophages and dendritic cells. A low level PPARalpha mRNA could be detected in peritoneal macrophages and immature dendritic cells but not in mature dendritic cells and bone marrow macrophages. Interestingly, PPARalpha mRNA was also absent in the alveolar macrophages although liver-type FABP was expressed, indicating that gene expression of liver-type FABP was independent of PPARalpha. Since liver-type FABP is known as transactivator of PPARgamma the simultaneous expression of both proteins may have general implications for the activation of PPARgamma in alveolar macrophages.

The myeloid expressed EF-hand proteins display a diverse pattern of lipid raft association.

EF-hand proteins are known to translocate to membranes, suggesting that they are involved in signaling events located in the cell membrane. Many proteins involved in signaling events associate cholesterol rich membrane domains, so called lipid rafts, which serve as platforms for controlled protein-protein interaction. Here, we demonstrate that the myeloid expressed EF-hand proteins can be distinguished into three classes with respect to their membrane association. Grancalcin, a myeloid expressed penta EF-hand protein, is constitutively located in lipid rafts. S100A9 (MRP14) and S100A8 (MRP8) are translocated into detergent resistant lipid structures only after calcium activation of the neutrophils. However, the S100A9/A8 membrane association is cholesterol and sphingolipid independent. On the other hand, the association of S100A12 (EN-RAGE) and S100A6 (calcyclin) with membranes is detergent sensitive. These diverse affinities to lipid structures of the myeloid expressed EF-hand proteins most likely reflect their different functions in neutrophils.