In agreement, the number of GM-CSF-producing CD4+ T cells, i.e. the essential EAE-inducing CD4+ T-cell subset [7], was significantly increased in the spinal cord of GFAP-Cre FasLfl/fl mice at day 22 p.i. The increased number of infiltrating activated autoreactive CD4+ T cells in GFAP-Cre FasLfl/fl mice was

associated Selleckchem FK228 with an enhanced production of proinflammatory cytokines. At day 15 p.i., IFN-γ, TNF, GM-CSF, IL-27, and iNOS but not IL17 mRNA was increased in GFAP-Cre FasLfl/fl mice as compared with that in FasLfl/fl mice. IFN-γ, TNF, and GM-CSF have been reported to contribute to disease progression and demyelination in EAE [7, 28]. GM-CSF and IFN-γ are mainly produced by encephalitogenic T cells. GM-CSF sustains neuroinflammation via myeloid Proteasome inhibitor cells that infiltrate the spinal cord. In addition to its proinflammatory function, IFN-γ is also a potent stimulator of IL-27 production by astrocytes [29], which might explain the increased production of this immunosuppressive and protective cytokine in the spinal cord of GFAP-Cre

FasLfl/fl mice at day 15 p.i. IL-27 can suppress IL-17 production of primed Th17 cells [29], which might explain that GFAP-Cre FasLfl/fl mice did not show elevated IL-17 mRNA expression in the spinal cord as compared to FasLfl/fl mice. IL-17 is an important cytokine contributing to demyelination and progression of EAE [30]. Comparable levels of IL-17 mRNA transcription in GFAP-Cre FasLfl/fl and FasLfl/fl Amylase mice at day 15 p.i. might, therefore, explain similar clinical scores in the two mouse strains at this stage of disease. However, at day 22 p.i., when numbers of activated CD25+ and GM-CSF-producing CD4+ T cells were significantly increased and numbers of Foxp3+ regulatory CD4+ T cells decreased in GFAP-Cre FasLfl/fl mice, IL-17 mRNA was very prominently increased in addition to IFN-γ, TNF, GM-CSF, and iNOS mRNA. Thus, aggravation of clinical symptoms in GFAP-Cre FasLfl/fl mice correlated with an increased

IL-17 mRNA transcription, indicating that this cytokine was decisive for the more severe and persisting EAE in these mice. IL-23, which drives IL-17 polarization of CD4+ T cells was not increased in the CNS of GFAP-Cre FasLfl/fl mice, which fits to the important role of IL-23 for Th17 polarization in lymphatic organs [31]. Astrocytes play both positive and negative roles in the pathogenesis and development of EAE [32]. As part of the blood-brain barrier, early chemokine release of astrocytes contributes to the recruitment of autoimmune CD4+ T cells to the CNS [33]. At later stages of EAE, astrogliosis develops, which may restrict further invasion of leukocytes into the CNS parenchyma [34]. In fact, genetically induced ablation of reactive astrocytes during EAE led to widespread inflammation and more severe clinical symptoms [35].

The unique receptor repertoire of dNK cells further includes the expression of several Ly49 receptors, the expression of activation markers such as CD69 and KLRG1 (which is considered as a marker for active NK cell proliferation37) and the expression of CD117 (the c-kit receptor). Another study, by Mallidi et al.17 described the phenotype of NK1.1+ dNK cells as DX5+ NKp46+ CD27+ CD11b+ CD11c+ CD69+. Interestingly, the NK1.1+ dNK cells expressed more B220 and CD69 than NK1.1+ eNK cells and also expressed ICOS (which is expressed on activated NK cells38), whereas eNK cells did not express ICOS at all. In the fetal-maternal interface, the maternal uterine tissue is

in close contact with the fetal-derived trophoblast cells. This interface contains immune cells, which constitute click here 40% of the cells in the human decidua.39 Analysis of this immune population has revealed that, unlike any other tissues or mucosal surfaces, 50–70% of the human decidual lymphocytes are NK cells, while the remainder are CD14+ macrophages, dendritic cells,

CD4+ T cells, a few CD8+ T cells, γδ T cells, and NKT cells.35 dNK cell numbers are the highest in the first trimester of pregnancy and their numbers decline during the second trimester. As in mice, only few dNK cells are present in the human decidua at term.36 The majority of dNK cells are CD56bright CD16− (as opposed to mouse dNK cells which express high levels of CD1618). Indeed, dNK cells Selleck Erlotinib resemble peripheral blood CD56bright CD16− NK cells also in the high expression levels of CD94/NKG2.40 However, similar to eNK cells, dNK cells resemble CD56dim CD16+ NK cells in the expression of KIRs41 and in their granules cell content. In fact, dNK cells differ from peripheral Chorioepithelioma blood NK cells both in phenotype and in function. Comparison analysis of the gene expression in dNK cells versus peripheral blood NK cells showed that dNK cells

should be considered as a unique NK subset.27 dNK cells over-expressed several genes, compared with the two peripheral blood NK subsets and several genes were exclusively expressed in dNK cells. For example, granzyme A was significantly over-expressed in dNK cells, as were the C-type lectin-like receptors NKG2C and NKG2E. dNK cells have been shown to express several activating receptors, including NKp46, NKp30, NKp44 (in contrast to human eNK cells which lack NKp30 and NKp44 expression, as discussed above), NKG2D, and 2B4.42–44 The expression of NKp44 (which is not expressed on non-activated peripheral blood NK cells) and the expression of the activation marker CD6945 (which is also expressed on mouse dNK cells) suggest that dNK cells might already be activated in the local environment of the decidua.

However, it must be noted that TD and TI responses are not rigidly compartmentalized within the B-2 and MZ/B-1 cell subsets. check details For instance,

MZ B cells also participate in TD antibody production owing to their ability to shuttle to the follicle and present antigen to T cells [[40, 41]]. Conversely, B-2 cells can initiate TI antibody responses in the intestine [[42]]. Here, we discuss recent advances in our understanding of the mechanisms by which adaptive and innate immune cells provide help to B cells. Protein antigens initiate protective antibody responses in the follicles of secondary lymphoid organs, a microenvironment that favors the interaction of B and T cells with each other as well as with antigen presenting DCs and

antigen exposing follicular dendritic cells (FDCs) (reviewed in [[7]]). After interacting with antigen through the B-cell receptor (BCR), which includes IgM and IgD (Fig. 1), naive B cells migrate Selleck FDA approved Drug Library to the boundary between the follicle and the outer T-cell zone [[43]]. At this location, B cells form dynamic conjugates with TFH cells, which deliver cognate B-cell help through a mechanism involving the tumor necrosis factor (TNF) family member CD40L and cytokines such as interferon-γ (IFN-γ, a cytokine also expressed by TH1 cells) and interleukin-4 (IL-4, a cytokine also expressed by TH2 cells) [[13, 14, 43, 44]]. B cells thereafter differentiate

along one of the two pathways. The follicular pathway generates Bcl6-positive germinal center B cells that further differentiate into long-lived memory B cells and plasma cells producing high-affinity antibodies, whereas the extrafollicular pathway generates Bcl6-negative blasts that further differentiate into short-lived plasma cells secreting low-affinity antibodies [[14, 45]]. After receiving activating signals from TFH cells at the border of the follicle with the T-cell zone, B cells upregulate the expression of the DNA-editing enzyme activation-induced cytidine deaminase (AID) and initiate somatic hypermutation (SHM) and class switch recombination (CSR), two Ig gene diversifying processes highly dependent on AID [[46-49]]. SHM introduces point mutations within V(D)J genes, thereby providing the structural Selleck Gefitinib correlate for selection of high-affinity Ig mutants by antigen (reviewed in [[50]]). By replacing constant (C) μ, and Cδ genes, which encode IgM and IgD, respectively, with Cγ, C, or C genes, which encode IgG, IgA, or IgE, respectively, CSR provides antibodies with novel effector functions without changing antigen specificity (reviewed in [[51]]). In humans, a noncanonical form of CSR from Cμ to Cδ has also been documented in lymphoid structures associated with the upper respiratory tract and generates B cells specialized in IgD production [[52]].

5°C above baseline. Thereafter, they were immersed in a different water tank maintained at 12°C water temperature until their rectal temperature was decreased by 0.5°C below

baseline. This procedure was conducted twice. Auto-Regressive Integrated Moving Average analysis showed that fluctuations in finger blood flow were associated with changes in mean body temperature (Ljung-Box statistic >0.05; R2 = 0.67) and body heat storage (Ljung-Box statistic >0.05; R2 = 0.70), but not with rectal (Ljung-Box statistic ATM/ATR cancer <0.05; R2 = 0.54) or tympanic (Ljung-Box statistic <0.05; R2 = 0.49) temperatures. It is concluded that reflex alterations in finger blood flow during repetitive hot and cold water immersions are associated with selleck chemicals llc mean body temperature and the rate of body heat storage, but not with rectal and tympanic temperatures. “
“Please cite this paper as: Henriksson, Diczfalusy and Freyschuss (2012). Microvascular Reactivity in Response to Smoking and Oral Antioxidants in Humans. Microcirculation 19(1), 86–93. Objective:  To investigate whether a daily intake of a moderate dose

of antioxidants modifies the microcirculatory response to smoking, assuming a major influence of oxidative stress on microcirculation. Methods:  The microvascular response to smoking was assessed in individual capillaries by capillaroscopy before and after two weeks of treatment with oral antioxidants. Results:  Smoking prolonged time to peak (TtP) capillary blood flow velocity in all subjects. When the subjects were pre-treated with ascorbate, TtP was comparable to baseline values of untreated subjects. No significant effect of vitamin E was observed either before or after smoking. Capillary blood flow velocity increased after treatment with ascorbate as well as after vitamin E. However, significant reductions in velocity were still observed Astemizole in response to smoking even after subjects consumed

ascorbate and vitamin E (p < 0.0004 and p < 0.000008 respectively). Conclusions:  This study focused on individual capillaries, and confirms that smoking has a very pronounced, direct and reproducible microvascular effect possible to demonstrate in vivo in human capillaries. Moderate intake of the antioxidant ascorbate clearly mitigated the effects induced by smoking. TtP after smoking in subjects treated with ascorbate was similar to that observed in untreated subjects before smoking a cigarette. Thus, oxidative stress could be assumed to play a role in the effects of smoking on microcirculation. Effects of antioxidants in vivo continue to bewilder science, with contradictory results from different studies. A large body of research has indicated an important role of oxidative processes for vascular function and in the development of atherosclerosis [7,58,67].

Another is to determine what DC learn from

their close interaction with the so-called fibroblastic reticular cells in the stroma of lymphoid tissues. Stromal cells are likely to be distinct in different regions of the lymph node where B cells, T cells and macrophages are enriched. A third challenge, also emphasized in Germain’s laboratory, is how DC orchestrate the interaction of two rare cells, the antigen-specific helper CD4+ T cells and killer CD8+ T cells. The medical impact of the last mentioned interaction of antigen-specific CD4+ and CD8+ T cells is notable. “Helped” CD8+ T cells mobilize better to infection challenge sites CP-690550 datasheet 52, and are a goal for more effective T-cell-based vaccines in the future 53. An obstacle in vivo is to be able to do more imaging of DC in large

animals and humans, e.g. appropriately labeled, DC-targeting antibodies might be visualized by positron emission tomography (PET scanning). The tolerance field has been energized by exceptional progress with Foxp3+Treg as suppressors of immune responses. Rescigno’s Viewpoint54 addresses the valuable DC part of GW-572016 research buy the equation. DC exert significant controls on Treg and, reciprocally, will likely be necessary in understanding how Treg work. During homeostasis, DC regulate the numbers of Treg 21, and when DC present specific antigens, they can expand antigen-specific Treg 55–58. Control of Treg seems to be carried out best by particular DC subsets such as the CD103+ DC (also marked by DEC-205/CD205, Langerin/CD207, occasionally CD8) 59–61. A challenge in going forward will be to learn how to control Treg in an antigen-specific manner. Until now, most research on Treg has involved approaches to totally remove them and then observe the rapid development

of various forms of autoimmunity and chronic inflammatory bowel disease 62. These valuable approaches document the essential role of Treg in suppressing autoinflammatory diseases and have revealed critical mechanisms. A major gap remains: to determine whether one can expand antigen-specific Treg and selectively selleck chemical suppress unwanted immune responses. Early papers on antigen-specific Treg have involved TCR transgenic T cells. DC either expand transgenic natural Treg in the presence of IL-2 or induce adaptive Treg along with TGF-β 63–65. When DC generate natural and induced Treg specific for a single pancreatic islet autoantigen, the Treg suppress autoimmune diabetes, which involves multiple autoantigens 63–65. A clinically relevant goal now is to find out whether antigen-capturing DC expand specific Treg from the polyclonal repertoire. If we could learn to expand antigen-specific Treg, as Rescigno 54 emphasizes in her Viewpoint, we could achieve an entirely new approach to suppress allergy, autoimmunity and transplant rejection.

IL-21 has been implicated in the pathogenesis of type 1 diabetes on the basis of the knowledge of the immune pathophysiology of a non-obese diabetic (NOD) mouse

strain [13, 14]. IL-21 stimulates the proliferation of both T and B cells and terminal differentiation of natural killer (NK) cells, enhances the cytotoxic activity of CD8+ T cells [15-17], counteracts the suppressive effects of regulatory T cells [18] and stimulates non-immune cells to generate inflammatory mediators [19]. Recently, the importance of IL-21 [20] and its related T helper type 17 (Th17) cells [21, 22] has emerged in the pathogenesis of type 1 diabetes as well in other autoimmune diseases [23, 24] in humans. The Th-cell-subset-specific selleck screening library expression of the IL-21 proximal promoter is controlled via the action of several transcription factors, including

nuclear factor-activated T cells, cytoplasmic 2 (NFATc2), T-bet and leucine-zipper transcription factor Maf (c-MAF) [25, 26]. Due to the pleiotropic effects of IL-21 on immune regulation, it is important to elucidate the genetically driven changes in its function and regulation that INCB024360 price might affect the autoimmune process and cause beta cell destruction. The presence of autoantibodies against islet-cell antigens is the first indication of diabetes development and is a well-established fact. Currently, four autoantibodies are used to predict the development of T1AD: antibodies against glutamic acid decarboxylase (GAD65), tyrosine phosphatase-like protein (ICA512, also termed IA-2), insulin and the recently discovered zinc T8 transporter (ZnT8) [1, 2, 27]. T1AD is also associated frequently with other immune-mediated disorders [27, 28] such as autoimmune thyroiditis [29, 30], Addison’s disease [31], pernicious anaemia [32, 33] and coeliac disease [30, 34]. During the past few years, extensive research has been conducted to predict the occurrences of autoimmune diseases through the detection of organ-specific antibodies in T1D patients [27, 35]. Early detection of antibodies and latent organ-specific

dysfunction is important to alert physicians to take appropriate next measures to prevent the progression to full-blown disease. Several autoimmune diseases are related to T1AD and elevated IL-21 expression in both human and animal models, as well as to a high frequency of the PTPN22 C1858T polymorphism. The Brazilian population is one of the most heterogeneous in the world, composed mainly of European (Caucasian descent, 0·771), African (0·143) and Amerindian (Native South American, 0·085) ancestry [36]. We hypothesized that the variants of these genes that regulate immune function would influence not only diabetes risk, but also the expression of other tissue-specific autoantibodies among patients with T1D in a Brazilian population.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by production of autoantibodies against nuclear autoantigens. Almost all the organs can be affected in patients with SLE. A wide range of molecules are involved

in SLE; therefore, the pathogenesis of the disease is complex and still unclear. The receptor for advanced glycation end products (RAGE) is a multi-ligand member belonging to the immunoglobulin superfamily. RAGE is expressed by many types of immune cells, including macrophages, neutrophils and T cells and interacts with a diverse class of ligands [1, 2]. Up to now CHIR-99021 datasheet identified RAGE ligands include high mobility group box-1 (HMGB1) protein, advanced glycation end products (AGEs), members of the S100/calgranulin family. AGEs is a class of compounds resulting from glycation of proteins, lipids or nucleic acids under conditions of oxidative stress and hyperglycaemia [3]. The

stimulation of RAGE through Bortezomib solubility dmso AGEs may contribute to certain disease state such as diabetes and Alzheimer’s disease, in which the accumulation of AGE has been demonstrated [4, 5]. In addition, as a family of over 20 related calcium-binding proteins that exclusively expressed in vertebrates, S100s modulate an array of intracellular functions [6, 7]. S100s released from different cell types during inflammation serve as useful markers of disease activity [8, 9]. It has been demonstrated that increased serum levels of S100A8/A9 correlated to disease activity index in SLE, indicating S100A8/A9 as a more relevant marker of infection in patients with SLE [10]. Besides that, HMGB1 is a ubiquitously expressed

evolutionary conserved chromosomal protein. Intracellular HMGB1 participates in transcriptional regulation [11]. Extracellular HMGB1 binds to cell surface receptors including RAGE, toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4). Studies indicate that interaction between HMGB1 and RAGE results in the production of type I interferon, which plays key role in the pathogenesis of SLE [12–14]. In addition, TNF-α and IL-6, which are implicated in association acetylcholine with disease activity or involvement of some organs in SLE [15, 16], can be induced by extracellular HMGB1 [17]. It has been documented that RAGE seemed to involve in all responses that depend on HMGB1 [18]. Notably, previous studies showed that increased serum level of HMGB1 was associated with lupus disease activity [19, 20]. All these results imply that HMGB1-RAGE pathway may participate in the pathogenesis of SLE. The RAGE protein consists of an N-terminal signal peptide, a V-type immunoglobulin-like domain, two tandem C-type immunoglobulin-like domains, a single transmembrane domain and a short C-terminal intracellular cytoplasmic tail [21].

Taken together, IC pretreatment can significantly inhibit LPS or CpG ODN-induced maturation of DCs in a FcγRIIb-dependent manner. Mature DC-induced Th1 and Th17 responses are involved in the pathogenesis of some autoimmune Akt inhibitor diseases, whereas immature DCs contribute to tolerance induction by downregulation of T-cell response and subsequently attenuate the pathogenesis of some autoimmune diseases. Next we investigated whether IC pretreatment could enhance tolerogenecity of immature DCs. OVA-pulsed immature DCs, which were pretreated with IC/Ig and then stimulated with LPS or CpG ODN, were incubated with OVA323–339-specific CD4+ T cells in vitro. We found that IC pretreatment reduced the

ability of LPS or CpG ODN-stimulated DCs to induce the proliferation and IL-17, IFN-γ secretion of antigen-specific CD4+ T cells (Fig. 1C and D). In contrast, IC/Ig pretreatment could not reduce the ability of FcγRIIb−/− DCs to induce proliferation and IL-17 secretion of antigen-specific CD4+ T cells. Altogether, the data suggest that IC pretreatment could enhance tolerogenecity of immature DCs in FcγRIIb-dependent manner. We previously showed that IC can induce massive amount of PGE2 from macrophages, which is responsible for the inhibition of TLR4-triggered inflammatory response. Similar

to macrophages, immature EX 527 solubility dmso DCs produced large amount of PGE2 once stimulated with IC. LPS or CpG ODN could not further promote IC-induced PGE2 production of immature DCs (Fig. 2A). Also, immature FcγRIIb−/− DCs released some PGE2 in response to IC stimulation, but less than the PGE2 secreted by WT DCs in response to IC stimulation (Fig. 2B). To investigate whether PGE2 was responsible

for the hyporesponsiveness of T cells induced by DCs pretreated with IC, we first observed the direct effect of PGE2 on the proliferation of CD4+ T Paclitaxel cells by anti-CD3/CD28. As expected, PGE2 inhibited the proliferation of T cells in a dose-dependent manner (Supporting Information Fig. 2). Next, OVA323–339-pulsed DCs were incubated with celecoxib, an inhibitor of COX2, 30 min prior to treatment with IC and TLR ligands. The hyporesponsiveness of OVA323–339-specific T cells disappeared when PGE2 secretion was inhibited, and addition of exogenous PGE2 could restore the inhibitory effect on T-cell proliferation in this system (Fig. 2C). Altogether, these data confirmed that IC-induced PGE2 from DCs was responsible for the downregulation of T-cell response by immature DCs that were pretreated with IC and then stimulated with TLR ligands. The data in the previous sections indicated that IC could downregulate DC-initiated T-cell response by inducing PGE2 production from DCs via FcγRIIb. To investigate whether IC could also inhibit in vivo T-cell response triggered by TLR agonists, we i.v. injected mice with OVA323–339-specific CD4+ T cells 24 h and OVA together with IC before i.p. administration of LPS or CpG ODN.

Conclusions: RPGN if diagnosed early and treated aggressively

is salvageable. Early Renal biopsy is useful Doxorubicin cost in deciding treatment plan and prognostication. YAMANARI TOSHIO1, SUGIYAMA HITOSHI1, MORINAGA HIROSHI1, KITAGAWA MASASHI1, ONISHI AKIFUMI1, OGAWA AYU1, KIKUMOTO YOKO1, KITAMURA SHINJI1, MAESHIMA YOHEI1, OGAWA DAISUKE1,2, SHIKATA KENICHI1,3, OHMOTO YASUKAZU4, MAKINO HIROHUMI1 1Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences; 2Department of Diabetic Nephropathy, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences; 3Center for Innovative Clinical Medicine,

Okayama University Hospital; 4Otsuka Pharmaceutical Co., Ltd. Introduction: TFF3 plays essential roles in mucosal surface maintenance and reconstitution. A decrease in the urinary levels of TFF3 is associated with acute kidney injury in animal models. Circulating serum TFF3 is significantly increased GPCR Compound high throughput screening in patients with chronic kidney disease (CKD) in a recent report. However, whether the urinary levels of TFF3 are associated with renal dysfunction in patients with CKD is unclear. Methods: We analyzed the urinary TFF (uTFF) levels in spot urine samples from 216 patients with CKD, and assessed the relationships among the uTFF, proteinuria and kidney function. Patients were prospectively followed for three years for doubling of the baseline serum creatinine concentration Nutlin-3 in vitro and the initiation of renal replacement therapy. Results: The excretion of uTFF3 significantly increased with the extent of albuminuria (P < 0.0001), urinary α1 microglobulin (P < 0.0001) and urinary β2 microglobulin (P < 0.0001) and the decline in the eGFR (P < 0.0001). A multivariate logistic regression analysis

showed that the patients with higher levels of uTFF3 were more likely to have CKD stage ≥G3b (P < 0.01). A longitudinal analysis demonstrated that patients with a higher uTFF3, in combination with macroalbuminuria, had a significantly worse renal prognosis (Log rank, P < 0.0001). The levels of urinary TFF3 in the renal end-point group were significantly higher than those in the renal survival group (P < 0.01). The AUC of uTFF3 for predicting the progression of CKD (0.879) was significantly higher than that of albuminuria (0.692) (P < 0.0001). The levels of uTFF1 and uTFF2 did not correlate with albuminuria. Conclusions: The excretion of uTFF3 is therefore significantly associated with albuminuria and a decline in the renal function. Moreover, the uTFF3 level can be used as a novel biomarker to predict the renal outcomes in CKD patients.

Megalin is expressed on proximal tubule cells in the kidney and also on the

cell surface of macrophages and T cells. However, the functional characterization of the Lcn2/megalin interaction is still elusive [10, 19, 20]. The second receptor, 24p3R, is a membrane-associated protein with 12 predicted transmembrane helices [17]. Overexpression of 24p3R in HeLa cells induces binding and uptake of Lcn2. Depending on the iron content of the ligand, Lcn2 is able to modulate iron status of cells overexpressing 24p3R, thereby influencing the expression of the proapoptotic protein Bim [17]. Via this modulatory effect on cellular apoptosis, Lcn2 has been implicated to play a role in tumor growth and proliferation [10, 21]. Interestingly, Lcn2 has been shown to increase tumor cell mobility [13]. Because Lcn2 is secreted by PMNs as part of their immune response to invading bacteria [3] and because Lcn2 is stored in the same endosomal vesicles as the this website chemotaxis-inducing Selleck PD98059 factors lactoferrin, S100A8 and S100A9, we questioned whether Lcn2 may also affect the migration and chemotaxis of

immune cells, such as neutrophils or macrophages. In the present study, we describe and characterize a new function of Lcn2 as a potent inducer of chemotaxis and migration of PMNs. To study a potential chemotactic effect of Lcn2, we first stimulated primary human PMNs either with recombinant human (rh)IL-8, one of the most powerful chemoattractants, or rhLcn2. The migration of PMNs was analyzed in Boyden chambers using nitrocellulose micropore filters. We found that rhLcn2 already at a concentration of 10 nM significantly induced PMN chemotaxis (p < 0.001; Fig. 1A). There was no further stimulatory effect when using a higher dose of rhLcn2 (50 nM, Fig. 1A). The stimulation of PMNs with rhLcn2 did not result in detectable IL-8 levels in cell culture supernatants after 6 h of treatment (details not

shown). To ensure that the effect observed was due to gradient-dependent chemotaxis, checkerboard analysis was performed (Fig. 1B). Therefore, primary human PMNs were resuspended in medium RPMI containing various concentrations of Lcn2 just before they were transferred to the upper wells of the Boyden chamber. The same concentrations of Lcn2 were put in the lower wells beneath the filter selleck antibody to the Boyden chamber, thus creating distinct concentration gradients. These experiments clearly demonstrated a specific and concentration-dependent chemotactic effect of rhLcn2 toward human PMNs (Fig. 1B). Because some of the biological activities of Lcn2 are dependent on the presence of the specific Lcn2 receptors, 24p3R or megalin, on target cells we studied their expression on human PMNs. As shown in Fig. 1C, 24p3R protein expression could be visualized in human PMNs while megalin was not detected (data not shown). In a next step, we investigated the signaling pathways under-lying Lcn2-dependent PMNs chemotaxis.