“
“The prevalence of treated patients with end-stage renal disease (ESRD) has been increasing steadily in Japan. High ESRD prevalence could be explained by multiple factors such as better survival on dialysis therapy, luxury acceptance due to insurance system to cover dialysis therapy, and ‘truly’ high incidence and prevalence of chronic kidney disease (CKD). The growing elderly population

may also contribute to this trend. The Japanese Society of Nephrology estimated the prevalence of CKD stage 3 as 10.4%, 7.6% within the range of 50–59 mL/min per 1.73 m2 selleck compound in a screened population. Strong predictors of treated ESRD shown by using community-based screening programs and an ESRD registry in Okinawa are dip-stick-positive proteinuria and hypertension. Low glomerular filtration rate per se, which is often observed in the elderly population, is not

a significant predictor of developing ESRD unless associated with proteinuria. CKD is common in Japan and is expected to increase, particularly in the elderly population. Benefits of proteinuria screening and automatic reporting of estimated glomerular filtration rate on the incidence of ESRD remain to be determined. According to the annual report of the Japanese Society for Dialysis Therapy (JSDT), the prevalence of treated end-stage renal disease (ESRD) patients has been increasing for the past 20 years (Fig. 1).1 In the population aged 75 years and over, the prevalence is more than 0.5%. The incidence of ESRD is also increasing, particularly Selleckchem Dasatinib in those aged 75 years and over (Fig. 2). The main causes of ESRD incidence are diabetes mellitus (DM), chronic glomerulonephritis and nephrosclerosis. The incidence of DM is now more than 300 per million populations in those aged 65 years and over (Fig. 3). The mean age at start of dialysis therapy is over 65 years. There is a north (low) to south (high) gradient in the incidence and prevalence of ESRD without obvious explanation. GBA3 The CKD prevalence seemed to be increasing in Japan. According to a community-based

study in Hisayama, the age-adjusted prevalence of CKD stage 3 and 4 was 4.1% in 1974, 4.8% in 1988 and 8.7% in 2002 in men, and 7.3% in 1974, 11.2% in 1988 and 10.7% in 2002 in women.2 This secular trend may be related to both genetic and environmental factors. Low birthweight, which is associated with lower nephron number, might develop DM and hypertension and therefore increase risk of ESRD.3 However, such data is not available in Japan. Lifestyle-related factors that are often associated with obesity and metabolic syndrome may have a role in the development and progression of CKD.4,5 Japan has a long history of universal screening systems including urine test for proteinuria and haematuria.6,7 It is not mandatory, however, so the fraction of people participating has been low at approximately 20–30%.

The first was the one induced with multiple low doses of streptozotocin (MLD–STZ). STZ is a chemical substance with alkylation properties that interferes with glucose transportation. A single high-dose strategy results in severe toxicity and acute diabetes. Conversely, the multiple low-dose regimen, characterized by minimal β cell toxicity, BMN673 results in autoantigen release and a possible break in self-tolerance [3]. The T cell dependence of this model is a debated topic, and needs

further evaluation. What is well established is that diabetes in this model cannot be transferred reliably to syngenic recipients by transfer of splenocytes [4]. Non-obese diabetic (NOD) mice are an inbred strain derived from Jcl:ICR mice [5], which develop type 1 diabetes spontaneously. The infiltration in the islets starts around 4–5 weeks, when pockets of lymphocytes are first observed juxtaposed to the pancreatic islets of young NOD mice. As the animals grow older, these mononuclear cells migrate into the islets, and by the time hyperglycaemia occurs destructive insulitis is present. This model is very similar to the human disease. Disease onset, for example, is preceded by infiltration of pancreatic islets by mononuclear cells and is controlled by many quantitative trait loci, particularly major histocompatibility

complex (MHC) class II genes. Diabetes in NOD mice is the most extensively studied model of autoimmune

disease [6, 7]. The discovery of regulatory T cells learn more (Tregs) disclosed a new field to be explored in the control of autoimmune pathologies [8]. Heat shock proteins (hsps) are molecules up-regulated in conditions of stress that are highly conserved throughout evolution [9]. Although recent research implicates hsp60 as an autoantigen involved in type 1 diabetes pathogenesis [10], this protein also contributes to protection against autoimmune diseases. It has been described that microbial homologues of mammalian hsps could induce the recruitment of Tregs to inflamed tissues [9]. In this study, we investigated the possible protection against type 1 diabetes through a prime-boost vaccination strategy. This strategy consists in priming the system with the antigen administered in one vector and then boosting it with the same antigen, but through another PAK6 vector [11]. Thus, we made use of two different vaccines containing mycobacterial hsp65: bacille Calmette–Guérin (BCG) and pVAXhsp65, a DNA vaccine. This association could, theoretically, be interesting because both vaccines have been already tested separately against diabetes and other autoimmune diseases and showed positive results [12-15]. We hypothesized that the prime-boost strategy could expand these beneficial effects. Female NOD mice and male C57BL/6 mice were obtained from the animal facility of State University of Campinas (UNICAMP, Campinas, São Paulo, Brazil).

The autocrine role of IL-10 in B cell differentiation was demonstrated further by the inhibitory effect of anti-IL-10 treatment on IgA secretion that was induced selleck chemicals by the dual ligation of CD40 and antigen-receptor without alterations in cell growth [60]. Altogether, our experiments show that IL-10 directly activates the STAT3 pathway so that there is co-operation between the STAT3 pathway and the classical NF-κB pathway that is activated downstream of CD40 ligation (anti-pNF-κB p65 inhibited the STAT3 pathway and vice versa). Because blocking peptides to pNF-kB p50 did not interfere with IgA production, we suggest that p65 homodimers interact with pSTAT3 for enhancing/sustaining AID transcription and IgA production. As p50 does

not possess a DNA binding

motif, this complex would contain another Rel subunit to bind to κB motifs. It seems that complexes formed between p50 homodimers and STAT3 bind to GAS sites, whereas p65/STAT3 complexes bind to κB motifs, as was described previously in another model [18]. In this context, the NF-κB and STAT3 pathways affect each other via an unknown mechanism. It is plausible that after stimulation by IL-1 or IL-6 that STAT3 would form a complex with pNF-κB p65 to facilitate NF-κB binding to DNA [17]. However, we did not focus on IL-1 in this study because we found IL-1 to be unable to phosphorylate STAT3 (unpublished data and [26]). pSTAT3 is able to form a complex with unphosphorylated NF-κB dimers, which bind to κB sites [19]. Summarizing, we suggest that (i) CD40L stimulation induces pNF-κB dimers (interacting or not with unphosphorylated STAT3) to bind to κB sites, (ii) CD40L stimulation promotes IL-10R expression on the B ICG-001 clinical trial cell surface, rendering STAT3 more reactive to IL-10 signalling and many (iii) IL-10 stimulation induces pSTAT3 dimers to bind to GAS sites and pSTAT3 dimers interacting with unphosphorylated NF-κB to bind to κB sites. The fact that IL-10 induces the binding of dimers on both κB and GAS sites can account for the enhanced IgA production. Deciphering the machinery of IgA differentiation is valuable to mucosal immunology and vaccinology, as IgA represents the major protective barrier of mucosal surfaces. Immunological

protection composed of a targeted, specific IgA response provided by either conventional or bioengineering vaccines, especially against invading microbes, may prove to be an achievable goal in the future. The authors gratefully acknowledge Françoise Boussoulade, Patricia Chavarin and Sophie Acquart for their technical help, Philip Lawrence and Samantha Pauls for kindly revising the manuscript and Professors Christian Genin and Frederic Lucht for valuable support. Financial support was provided by grants from the Convention Interregional du Massif Central ‘Réseau switch’ MENRT 01Y0242b and the Regional Blood Bank, EFS Auvergne-Loire, France. Sandrine Lafarge holds a fellowship from the French Ministry for Education, Research and Technology (MENRT).

This occurred when all of the following click here criteria were met: recipient age 18–59 years, deceased donor age less than live donor age, and deceased donor HLA match better than live donor HLA match. The impact of waiting on dialysis was not taken into account in this analysis. The impact of waiting time on the success of transplantation has been examined in several studies. Meier-Kriesche et al. analyzed United States Renal Data System (USRDS) data from 73 103 primary adult renal transplants performed between 1988 and 1997.7 There was a progressive rise in the risk of

death and death-censored graft loss with increasing time on dialysis prior to transplantation. The increases in mortality risk for waiting relative to pre-emptive transplantation were as follows: 6–12 month wait, 21%; 12–24 month wait, 28%; 24–36 month wait, 41%; 36–48 month wait, 53%; and >48 month wait, 72%. In another publication, Meier-Kriesche and Kaplan reported that waiting for a live donor transplant for more than 2 years while on dialysis reduced

graft survival to the same level as that for deceased MI-503 supplier donor transplants performed within 6 months of commencing dialysis.8 Using UNOS Registry data, Gjertson reported that pre-transplant dialysis time accounted for 12–13% of the variation seen in 1-year graft survival rates for both live and deceased donor transplantation.9 Also using UNOS Registry data, Kasiske et al. reported that the relative risk of death or graft failure, was lower in deceased donor and live donor recipients who were transplanted pre-emptively, compared with those transplanted following commencement of dialysis.10 Racial minority groups and those with a lower level of education were less likely to be transplanted pre-emptively. With regards to recipients who are less than 18 years old, a study by Ishitani et al. examined the success of live, related donor transplantation in paediatric recipients using UNOS Registry data.11 When compared with pre-emptive

transplantation, there was a relative risk of graft failure of 1.77 in those transplanted after dialysis had commenced. Kennedy et al. used ANZDATA to examine graft outcomes in transplanted adolescents, and also reported improved outcomes with pre-emptive transplantation.12 Wolfe et al. compared the survival PD184352 (CI-1040) of those on the waiting list with those for individuals receiving a primary deceased donor transplant.13 Standardized mortality ratios were derived from an analysis of 228 552 subjects on dialysis. A total of 46 164 individuals were on the waiting list, of whom 23 275 received a primary deceased donor transplant over a 7-year period of observation. The annual death rate for those on the waiting list was 6.3 per 100 patient-years. By comparison, those transplanted had a long-term annual death rate of 3.8 per 100 patient-years. The improvement in relative risk of mortality was most pronounced for young, white recipients (20–39 years) and for people with diabetes.

The use of mouse models offers a feasible alternative to human observations, when hypothesis-driven studies are needed, but mouse-in-mouse systems do not always reflect the pathology of human diseases. In many aGVHD models, the effector cell is based on infusion of murine splenocytes which may behave differently to human effector cells; furthermore, conventional mice are not well aligned to the study of human cell therapy products. The introduction of the interleukin (IL)-2 receptor gamma mutation onto the non-obese diabetic

(NOD)-severe compromised immunodeficient (SCID) background has allowed for the development Selleck Epigenetics Compound Library of refined mouse models. NOD-SCID IL-2rγnull (NSG) mice are deficient for T, B and NK cell activity and allow engraftment of high levels of human peripheral blood mononuclear cells (PBMC) [29]. The NSG model offers an opportunity to examine human donor cells in combination with clinical cell therapeutics. Using a humanized NSG mouse model of aGVHD, this study sought to examine the effect of human MSC cell therapy, and to investigate the possible therapeutic mechanisms involved. Human MSC cell therapy significantly prolonged the survival of

NSG mice with aGVHD, reducing target organ pathology. MSC therapy did not interfere with donor PBMC engraftment or involve the induction of donor T Autophagy Compound Library cell apoptosis, anergy or regulatory cell expansion, but rather the direct inhibition of both donor CD4+ T cell proliferation and tumour necrosis factor (TNF)-α production. All procedures involving animals or human material were carried out by licensed personnel according to approved guidelines. Ethical approval for all work was received from the ethics committee of National University of Ireland (NUI) Maynooth. A humanized mouse model of aGVHD was adapted and optimized from a protocol described by Pearson et al. [29]. NOD.Cg-PrkdcscidIL2tmlWjl/Szj mice (NOD-SCID IL-2rγnull) (NSG) (Jackson Laboratories, Bar Harbour, ME, USA) were exposed to a conditioning dose of 2·4 Gray (Gy) of whole-body gamma irradiation. Human PBMC from healthy volunteer donors were isolated by Ficoll-density

centrifugation and administered intravenously (i.v.) to NSG mice (6·3 × 105 g−1) via the tail vein 4 h following irradiation. Negative control mice received a sham infusion of phosphate-buffered saline (PBS) alone. Signs of aGVHD occurred typically between days 12 and 15 post-PBMC transfusion. Cobimetinib research buy In some mice, conventional human mesenchymal stem cell (MSC) (4·4 × 104 g−1) therapy was administered on day 7 post-PBMC transfusion. In other groups, interferon (IFN)-γ stimulated MSC (4·4 × 104 g−1) were administered concurrent with PBMC on day 0. The level of human cell chimerism was analysed by flow cytometry (days 4, 8 and 12), examining the expression of CD45+ cells and the ratios between human CD4 and CD8 T cells. aGVHD development was determined by examining features daily including body weight, ruffled fur, locomotor activity, posture and diarrhoea.

01% Tween 20/PBS for 30 min. Subsequently, cells were incubated with fluorochrome-conjugated secondary antibodies [Ax488 goat anti-mouse IgG1/2a, Ax546 goat anti-mouse IgG1, Ax546 goat anti-rabbit IgG, Ax546 donkey anti-goat IgG (Invitrogen)] in 2% BSA/0.01% Tween 20/PBS

for 30 min and mounted using DakoCytomation mounting medium. Imaging was performed using a Zeiss EX-527 LSM 510 META confocal microscope equipped with a 63 × /1.4 NA oil-immersion objective and an AxioCam HR (Carl Zeiss, Göttingen, Germany), using laser excitation at 488, 561 and 633 nm. DPC localization was evaluated as the area fraction of fluorescent pixels at the DPC relative to total area of fluorescent pixels for the cell/bead conjugate https://www.selleckchem.com/products/PD-0325901.html using the image analysis software ImageJ developed by Wayne Rasband, National Institute of Health, Bethesda, MD, USA. Graphs were made in SigmaPlot 8.0 (SPSS, Chicago, IL, USA). Statistical analyses were performed using the Mann–Whitney U-test, conducted in spss 16.0 for Windows (Chicago, IL, USA). Upon sustained T cell activation, maintained type II PKA association with the centrosome and the microtubule organizing centre [16] and redistribution of type I PKA (in mouse T cells) [17] have been described. Additionally, type I PKA localization has been observed at the IS and at the DPC of primary human T cells activated by SEB-pulsed Raji B cells [5]. We found type

I PKA [regulatory subunit (R)Iα] to mainly localize with filamentous

(F)-actin close to the cell membrane in resting primary human T cells (Fig. 1B, upper panel). Upon activation with CD3/CD28-coated beads, F-actin accumulated at the cell/bead contact zone, a known hallmark of productive TCR engagement alongside reorientation of the microtubule organizing centre identified here by β-tubulin staining (Fig. 1A, [3]). The accumulation intensified and persisted for at least 20 min (Fig. 1B, left column, Interleukin-3 receptor and A) and was used as a marker for activated conjugates. About 1 min after activation, RIα was recruited to the IS, then distributed back in the membrane at 5 min before translocating to the distal pole (DP) of the cell (20 min) (Fig. 1B, middle column). After 20 min, RIα was localized at the DP in 69 ± 4% of activated T cells (mean ± SEM, n = 100 T cells from each of three donors). Thus, CD3/CD28-coated beads robustly and reproducibly generated a high percentage of activated T cells, in which RIα was consistently found to migrate via the IS to the DP. To align cross-ligation with CD3/CD28-coated beads with a more physiological mode of activation, we stimulated primary human T cells for 30 min with SE-primed Raji B cells (Fig. 1C). In successfully activated T cells (31 ± 10% of the conjugates, mean ± SEM, n = 100 T cells from each of two donors), CD3 accumulated at the IS at the T cell/Raji B cell interface (Fig. 1C, left column).

7D). Thus, in vivo infusion with DC-FcγRIIb could protect MRL/lpr mice from obvious nephritis injuries. Finally, in vivo administration of DC-FcγRIIb, before (4-wk-old) or after (10-wk-old) the onset of clinic lupus, was found to be able to significantly prolong the survival of MRL/lpr mice, whereas MRL/lpr mice receiving DC-GFP or DCs all died by 40 wk (Fig. 7E). Thus, in vivo administration of DC-FcγRIIb CHIR-99021 mw could protect MRL/lpr mice from lupus progression, both preventively and therapeutically. SLE is a progressive systemic autoimmune disease, for which current therapy relies largely on long-term suppression of the immune system. We

here provide a short-term treatment regimen to attenuate lupus progression. Single infusion of DC-FcγRIIb, either before or after the onset of clinic lupus, into lupus-prone mice exerts a significant protection from lupus progression. The presence of large amounts of circulating IC in SLE may be potent stimulator for DCs. However, selleck chemicals llc for DC-FcγRIIb, these IC might become potent inhibitor of DC maturation through binding to the preferentially expressed FcγRIIb. FcγRIIb-mediated negative signal contributes to the maintenance of immature/tolerogenic property of DCs. The consequence of this event results in suppression of antigen-specific

T-cell responses and thereby inhibition of B-cell responses, furthermore reducing the generation of autoreactive T cells and autoantibodies. It has been previously reported that decreased FcγRIIb expression is associated with the progression of lupus;

it would therefore make sense that artificial enhancement of the inhibitory FcγRIIb expression on some cell types could possibly provide an efficient approach for the treatment of lupus. In addition to the maintenance of DC tolerogenecity, IC also induce massive PGE2 production from DCs and more PGE2 from DC-FcγRIIb. PGE2 might play a protective role in autoimmune responses via directly inhibiting both CD4+ and CD8+ T-cell responses, inducing Foxp3+ Treg differentiation, suppressing B-cell activation and Ig production 28–32. Moreover, PGE2 Dipeptidyl peptidase may be also responsible for the inhibition of TLR-induced DC maturation because PGE2-triggered signal is involved in the downregulation of TLR4 expression 27. It is worth investigating whether PGE2 also contributes to inhibition of TLR7 and TLR9 expression, because natural activators of TLR9 and TLR7 can be found in the blood of lupus patients. FcγRIIb seems to be a redundant receptor to mediate PGE2 production, because FcγRIIb−/− DCs can also produce certain amount of PGE2 although much less than that produced by WT DCs in response to stimuli. We found that DCs express more FcγRIIa than FcγRIIb (Supporting Information Fig. 5), suggesting that other activating FcγRs might contribute to the production of PGE2 by IC. Once pretreated with IC and then triggered with TLR-ligands, FcγRIIb−/− DCs could secrete certain level of PGE2.

First, data from the OT1 system using recombinant TCR, where no triggering is present, shows that 2D off-rate for agonist is even faster than that of native TCR on the cell surface (Liu, B. et al., our unpublished data). Second, all the TCRs in the current study showed fast kinetics, reaching adhesion plateau from the shortest contact time of 0.1 s. The fact that the adhesion did not increase further in longer contact times suggests that factors contributing to the adhesion did not change significantly over the time scale of our 2D measurement. Thus, should signaling have occurred, it was within 0.1 s, which, to the best of

GSI-IX order our knowledge, is faster than any documented T-cell

signaling events. Third, in our 2D assays, off-rate measurement was performed at zero-force condition. As we elaborated previously [27, 37], in the adhesion frequency assay, the stretch at the end of each contact is merely a means of detecting whether a bond was present at the very end of the contact; the binary readout (bond or no bond) but not bond duration are analyzed PLX3397 in vitro with a mathematical model to derive the off-rate. In the thermal fluctuation assay, more direct evidence is available for the zero-force condition because we quantitatively monitor the force (by tracking the position of the biomembrane force probe (BFP) probe bead). If any cellular processes impose forces significantly deviate from zero on individual TCR–pMHC bonds, we should have observed them (the BFP has a ∼1 pN force resolution). Fourth, the surface density of pMHC http://www.selleck.co.jp/products/CHIR-99021.html is carefully controlled such that, at any moment of contact, the majority of adhesion events are mediated by a single bond [37]. Therefore, although we cannot rule out a possible

role of T-cell signaling, these factors would favor the proposition that 2D TCR–pMHC off-rate most likely reflects an intrinsic property of the native TCR in the cell membrane. One intriguing property of 2D off-rate (or bond lifetime) for the gp100 system is that higher potency corresponds to a faster off-rate (thus shorter bond lifetime), which was also observed in the OT1 [27], 42F3 [33], and 2B4 and 5C.C7 [28] TCR systems. However, higher potency interactions have much higher on-rates. Evaluation based on both on-rates and off-rates is actually consistent with the serial engagement model [27] and the total confinement time model [42]. Take 19LF6 TCR as an example. The measured on-rate (Ackon) is 0.072 μm4s−1 and off-rate (koff) is 11.4/s. For typical surface densities of 15 TCR/μm2 (mTCR) and 6 pMHC/μm2 (mpMHC) on a T cell and an RBC, respectively, it takes on average 0.15 s (1/(Ackon×mTCR×mpMHC)) for a new TCR–pMHC bond to form and 0.088 s (1/koff) to dissociate.

[69] Both in vitro and in vivo stimulation of microglial expression

of inflammatory molecules by MIF was associated with up-regulated expression of CCAAT/enhancer binding protein-β (C/EBP-β) that participates in the regulation of inflammatory cytokines,[70] suggesting a role for MIF in promoting microglia activation through induction of C/EBP-β, possibly through binding to CD74,[71] a marker of activated microglia.[72] Together these studies confirm a role for microglia in the pathogenesis and progression of EAE, with a beneficial effect on disease progression of inhibitors of microglial activation. However, microglia do not only contribute to the disease in an adverse manner, and the impact of microglial activation on disease outcome depends on the form and timing of activation. Indeed, evidence has accumulated indicating that microglia can also exert a neuroprotective Vorinostat cell line role in EAE/MS. One of the most important beneficial roles of microglia in EAE is the phagocytic removal of apoptotic cells and myelin debris, without the induction of inflammation, which is crucial for the maintenance of a microenvironment that supports tissue regeneration. Indeed, myelin debris has an inhibitory effect on maturation of oligodendrocyte progenitor cells[30] and www.selleckchem.com/products/MK-2206.html on axonal regeneration.[73] In this context, the role of TREM-2 in the control of

excessive inflammation was recently demonstrated in EAE. TREM-2, which stimulates phagocytosis and down-regulates inflammatory signals in microglia via the signalling adaptor molecule DAP12,[22] is up-regulated on microglia and macrophages, mainly in the spinal cord, during EAE[27, 29] and its blockade during the effector phase of EAE leads to disease exacerbation with

more diffuse CNS inflammatory infiltrates and demyelination in the brain parenchyma.[29] Intravenous treatment of EAE-affected mice at disease peak with TREM-2-transduced myeloid precursor cells, which migrated to the perivascular inflammatory lesions, led to increased SB-3CT phagocytosis of debris in these mice, together with a decrease in expression of inflammatory cytokines in the spinal cord, some diminution of the inflammatory infiltrate, and a clear reduction of axonal damage and demyelination. These effects were associated with a marked amelioration of the clinical course in mice treated at disease peak, with early and almost complete recovery from clinical symptoms.[27] More recently, microRNA-124 (miR-124) was identified through EAE studies as a key regulator of microglia quiescence. In healthy mice, CNS-resident microglia, but not peripheral macrophages, were found to express high levels of miR-124, and EAE studies with chimeric mice showed that miR-124 expression by microglia decreased by ~ 70% during the course of the disease.

The Gas6 mRNA level was markedly decreased in macrophages treated with 1 ng/ml LPS for 16 hr, and was abolished by 10 ng/ml LPS (Fig. 5a). A striking down-regulation of Gas6 mRNA was initially observed at 4 hr after treatment with 10 ng/ml LPS, and was abolished at 16 hr (Fig. 5b). An enzyme-linked immunosorbent assay (ELISA) showed that the Gas6 concentration

in the medium was significantly decreased at 8 hr after LPS treatment, and declined to a very low level by 16 hr (Fig. 5c). Given that Gas6 specifically promotes phagocytosis of apoptotic cells by macrophages,20 we speculated that LPS inhibition of phagocytosis might be also attributable Cobimetinib solubility dmso to the down-regulation of Gas6. We found that neutralizing Gas6 activity with 5 ng/ml anti-Gas6 CP-673451 concentration antibodies, following the manufacturer’s instructions, significantly inhibited macrophage phagocytosis (Fig. 5d), suggesting that Gas6 positively regulated macrophage phagocytosis in an autocrine manner. Exogenous Gas6 increased macrophage phagocytosis in a dose-dependent manner

(Fig. 5e). Moreover, exogenous Gas6 significantly reduced the LPS inhibition of phagocytosis (Fig. 5f). In particular, when Gas6 and anti-TNF-α were given to the macrophages simultaneously, they restored LPS-inhibited phagocytosis to a normal level (Fig. 5f). Whether TLR4 signalling is necessary for LPS-inhibited Gas6 expression, since it is by activating TLR4 that LPS induces TNF-α production. To address this question, we analysed the effects of LPS on TLR4-deficient (TLR4−/−) macrophages.

Gas6 expression in TLR4−/− macrophages was also abolished by LPS, and displayed a similar pattern to that observed in wild-type (WT) macrophages (Fig. 6a). In contrast, LPS-induced TNF-α expression was blocked in TLR4−/− macrophages (Fig. 6b). The concentrations of Gas6 and TNF-α in the medium corresponded to MG-132 nmr their mRNA levels (Fig. 6c). Next, we analysed the phagocytosis of apoptotic cells by TLR4−/− macrophages. In the absence of LPS, the phagocytic ability of TLR4−/− macrophages was similar to that of WT controls (Fig. 6d). Although LPS significantly inhibited phagocytosis of apoptotic cells by TLR4−/− macrophages, there was a latency in this inhibitory effect compared with WT macrophages. The LPS inhibition of phagocytosis by TLR4−/− macrophages was initially observed at 12 hr after treatment, and the inhibition became more evident at 16 and 24 hr (Fig. 6d). Moreover, the LPS-inhibited phagocytosis by TLR4−/− macrophages was significantly reduced compared with that by WT controls (Fig. 6d). Anti-TNF-α did not affect LPS inhibition of phagocytosis by TLR4−/− macrophages (Fig. 6e). In contrast, exogenous Gas6 reversed LPS-inhibited phagocytosis by TLR4−/− macrophages to the control level. These observations suggest that down-regulation of Gas6 production is entirely responsible for LPS inhibition of phagocytosis by TLR4−/− macrophages.