The authors declare no financial or commercial conflict of interest. “
“Systemic lupus erythematosus (SLE) is an

autoimmune disease characterized by the presence of pathogenic IgG antinuclear antibodies. Pathogenic IgG autoantibody production requires B-cell activation, leading to the production of activation-induced deaminase (AID) and class switching of IgM genes to IgG. To understand how and when B cells are activated to produce these IgG autoantibodies, we studied cells from 564Igi, a mouse model of SLE. 564Igi mice develop a disease small molecule library screening profile closely resembling that found in human SLE patients, including the presence of IgG antinucleic acid Abs. We have generated 564Igi mice that conditionally express an activation-induced cytidine deaminase transgene (Aicdatg), either in all B cells or only in mature B cells. Here, we show that class-switched pathogenic IgG autoantibodies were produced only in 564Igi mice in which AID was functional in early-developing B cells, resulting in loss of tolerance. Furthermore, we show that the absence of AID in early-developing B cells also results in increased production of self-reactive IgM, indicating MG-132 manufacturer that AID, through somatic hypermutation, contributes to tolerance. Our results suggest that the pathophysiology of clinical SLE might also be dependent

on AID expression in early-developing B cells. “
“The novel immunosuppressant sotrastaurin is a selective inhibitor of protein kinase C isoforms that are critical in signalling pathways downstream of the T cell receptor. Sotrastaurin inhibits nuclear factor (NF)-κB, which directly promotes the transcription of forkhead box protein 3 (FoxP3), the key regulator for the development and function of regulatory T cells (Tregs). Our center participated in a randomized trial comparing sotrastaurin (n = 14) and the calcineurin inhibitor Neoral (n = 7) in renal transplant recipients. We conducted ex vivo mixed lymphocyte reaction (MLR) and flow cytometry Interleukin-2 receptor studies on these patient samples, as well as in vitro studies on samples

of blood bank volunteers (n = 38). Treg numbers remained stable after transplantation and correlated with higher trough levels of sotrastaurin (r = 0·68, P = 0·03). A dose-dependent effect of sotrastaurin on alloresponsiveness was observed: the half maximal inhibitory concentration (IC50) to inhibit alloactivated T cell proliferation was 45 ng/ml (90 nM). In contrast, Treg function was not affected by sotrastaurin: in the presence of in vitro-added sotrastaurin (50 ng/ml) Tregs suppressed the proliferation of alloactivated T effector cells at a 1:5 ratio by 35 versus 47% in the absence of the drug (P = 0·33). Signal transducer and activator of transcription 5 (STAT)-5 phosphorylation in Tregs remained intact after incubation with sotrastaurin.

The majority of reported Caucasian patients with desminopathy typically presented with lower distal myopathy in early adulthood, which gradually progressed to the upper limbs, trunk,

and bulbar muscles, and ultimately they lost ambulation ability in the later stages of the disease [8,24,25]. However, most of our patients initially had proximal muscle weakness, despite a few patients initially presenting with distal weakness. In addition, our patients were not all wheelchair-bound Pictilisib supplier in the sixth decade of life. Restrictive respiratory insufficiency requiring nocturnal ventilator support was not a common symptom. The clinical picture of desminopathy manifested as highly heterogeneous because of the different mutations in the desmin gene, varying from isolated skeletal myopathy or heart disease to cardiomyopathy AZD0530 chemical structure combined with skeletal myopathy [8,26]. Although cardiac disorders were dominant, cardiac syndromes were not the early or sole manifestations in most of our patients. In contrast to a European report that most patients exhibiting mutations in the tail domain manifested predominantly as cardiomyopathy or cardiomyopathy followed by skeletal myopathy

[23], most affected members in family 4 with the E457V mutation in the tail domain demonstrated skeletal myopathy as the initial symptom followed by conduction block and/or cardiomyopathy. The sporadic second patient with the T445A mutation

in the tail domain presented with skeletal myopathy followed by respiratory insufficiency. Patients with the S13F mutation in the head domain of desmin predominantly showed variable conduction abnormalities at an early age [27]. In another Chinese family with the S13F mutation, cardiomyopathy was the main symptom, and concomitant with asymptomatic skeletal myopathy [22]. However, except for the index case with early onset of dilated cardiomyopathy, most affected individuals with the S12F mutation in the head domain presented with skeletal myopathy followed by cardiomyopathy. Early onset cardiac arrhythmia and conduction block followed by skeletal myopathy have also been described in a series of East European patients with R406W in the helix 2B of the rod domain [25]. Another report suggested that most patients with mutations in helix 2B of the rod domain presented initially with skeletal myopathy, followed by cardiomyopathy [6]. A similar progressive pattern also appeared in the present patients with mutation in the rod domain, including the R355P mutation in helix 2B, a frameshift mutation in helix 1A, as well as L274P and L274R mutations in helix 2A. It is worth stressing that most of the Chinese desminopathy patients suffered from a conduction disorder, which usually occurred after skeletal myopathy.

Mice with Nlrp3 mutations were developed independently by investigators in two laboratories. One group introduced a R258W mutation in the third exon of the Nlrp3 gene of C57BL/6 mice 9. This corresponds to the R260W mutation frequently found in humans

with the Muckle–Wells syndrome 7. A second group introduced either an A350V or an L351P mutation in exon selleck chemicals llc 3 of Nlrp3 in 129SvJ mice 10. These mutations occur frequently in patients with Muckle–Wells syndrome and familial cold autoinflammatory syndrome, respectively 10. The targeting strategy used to obtain these strains required that the mice co-express Cre-recombinase to delete a neomycin cassette inserted in reverse orientation that when present causes gene silencing. This allowed studies of mice in which the Cre-recombinase was expressed under tissue-specific promoters and thus enabled tissue-specific expression of the mutated gene 10. In studies

to determine if the R258W mice exhibit the basic immunologic abnormality of patients with CAPS, BM-derived macrophages and BM-derived dendritic cells (BMDC) from these mice were stimulated with a TLR ligand (LPS) in the presence and absence of ATP, the latter an essential co-factor in NLRP3 inflammasome activation in WT cells. It was shown that while cells from R258W mice were unable to produce IL-1β and IL-18 in the absence of stimulation, they produced large amounts of these cytokines upon LPS stimulation in the presence or absence of exogenous ATP. These cells therefore differed from WT cells in that the latter only exhibited IL-1β production upon LPS stimulation in the presence of ATP and thus were similar to cells of patients LEE011 order click here with CAPS. Interestingly, both WT and R258W cells produced equivalent amounts of other cytokines upon LPS stimulation. This suggested that the abnormality was limited to the NLRP3 inflammasome and that elevations in non-inflammasome cytokine production occurring during prolonged inflammation was due to secondary stimulation of cells by increased levels of IL-1β

6, 9. In parallel studies of peritoneal macrophages and BMDC from the A350V and L351P knock-in (KI) mice, production of IL-1β in the absence of ATP was also found. In addition, it was shown that BMDC from L351P mice secreted IL-1β when incubated at 32°C, as do CAPS patients with similar mutations. Thus, cold conditions seem to be an inflammasome activator in the presence of this mutation. Finally, cold-challenged dendritic cells from L351P KI mice exhibited spontaneous IL-1β secretion, whereas A350V KI cells were more dependent on LPS priming; this may explain the greater neonatal mortality of the L351P KI mice when compared with A350V KI mice 10. The mechanism of ATP co-activation of the NLRP3 inflammasome was studied in the R258W KI mice. Previous work has shown that this ATP function is an extracellular activity that involves activation of a membrane receptor, P2X7R 11.

vulnificus. Vibrio vulnificus is a halophilic estuarine bacterium that causes septicemia and necrotizing wound infections in susceptible patients with underlying hepatic diseases, heavy alcohol drinking habits and other immunocompromised conditions [1]. PF-6463922 price Primary septicemia has a rapidly progressive and fulminant course, resulting in a mortality rate of over 50%. Several virulence factors reportedly play important roles in the pathogenesis of V. vulnificus septicemia, including hemolysin [2], protease [3], phospholipase A2 [4], siderophores [5] and capsular polysaccharide [6]. We have previously reported that the RtxA1 toxin is the primary acute cytotoxin of V. vulnificus [7-9]. Vibrio vulnificus

HlyU protein is reportedly a positive regulator of RtxA1 toxin [10]. We previously reported that the ToxRS system and LuxS quorum-sensing system of V. vulnificus play important roles in coordinating the expression of virulence factors [11, 12]. We identified the essential role of cya, the structural gene for adenylate cyclase, which catalyzes the synthesis of cAMP [13]. The cAMP-CRP system is a well-known global regulator of catabolic repression in enteric MAPK Inhibitor Library price bacteria. In addition to the known roles of this protein in catabolic repression and carbon source utilization,

the cAMP-CRP global regulatory system regulates numerous bacterial cell functions. This system has received attention for its role in modulating virulence gene expression in various pathogenic bacteria [14-19]. There have been reports that V. vulnificus CRP has essential roles in controlling the expression of various genes [20-24]. We have also reported that the V. vulnificus crp mutant extends the time to death in a Caenorhabditis elegans infection model [25]. These reports suggest that CRP may act as a major virulence regulator in V. vulnificus pathogenesis. In the present study, we investigated the regulatory roles of CRP in various virulence traits of V. vulnificus. The following V. vulnificus strains were cultured in 2.5% NaCl HI medium at 37°C: Methamphetamine MO6-24/O, a highly virulent clinical isolate of V. vulnificus [26] and CMM710 (crp −),

a crp deletion mutant strain of MO6-24/O background [25, 27]. To restore the defect, a plasmid pLAFR3::crp was transferred into the CMM710 strain by triparental mating using a conjugative helper plasmid pRK2013 [28], as described previously [7]. CMM770 (rtxA1-) is MO6-24/O with a deletion of the rtxA1 gene [7, 8]. Overnight cultures of bacterial cells were inoculated into 2.5% NaCl HI broth at a concentration of 5 × 106 (CFU/mL and cultured at 37°C with shaking at 200 rpm. At 3 hr intervals, V. vulnificus growth was determined by measuring absorbance at 600 nm using a biophotometer (Eppendorf, Hamburg, Germany). In vivo growth was assayed using the rabbit ileal loop model described by Xu et al. [29] and Haralalka et al. [30].

Efficient responses to the fungus require a complex network of immunological mechanisms. Together with alveolar macrophages and neutrophils, which constitute a primary line of innate cellular defence against A. fumigatus,1,2 the crucial role of the adaptive immunity has been extensively demonstrated.3 Indeed, besides the well-characterized protective role of T helper type 1 (Th1) lymphocytes,4–7 the newly described regulatory T cells and interleukin-17 (IL-17) -producing cells (Th17) represent important mediators of the inflammatory and anti-inflammatory

Ganetespib cost host responses against A. fumigatus.8 However, dendritic cells (DCs) also play a fundamental function in initiating and modulating the specific immune responses upon recognition of A. fumigatus.5,9,10 After internalization of A. fumigatus conidia, DCs mature and acquire the capacity to polarize

naive T cells and, in turn, to promote a protective response.9 In keeping with these findings, in vivo results on the migration of lung DCs into lymphoid organs, where they drive an appropriate T-cell response to fungal antigens,11 have brought DCs centre stage as promising targets for intervention in immunotherapy and fungal vaccine development.12 In addition, it is important this website to consider several studies that have recently pointed to DCs and type I interferons (IFNs) as special players in the immune response tailored to combat tumours and infections.13–15 Indeed, although the anti-microbial properties of these cytokines have not been fully characterized yet, type I IFNs represent important immunomodulators of the innate, as well as the adaptive, arm of the immune system. Type I IFN can promote

the differentiation of human blood monocytes into DCs and contribute to their maturation.16,17 This leads to the generation of DCs able to stimulate a primary human antibody response, a Th1 proliferation,18 and a cross-priming of CD8 T cells against viral antigens.19 In addition, one crucial outcome of type I IFN-induced effects is the ability to directly stimulate IFN-γ production in natural killer and T cells,20–22 which in turn promotes the development of a cell-mediated immune response. Based on these immunoregulatory properties, in this work the expression and the mafosfamide capacity of type I IFN, namely IFN-β, to modulate the T-cell polarizing capacity of A. fumigatus-infected DCs was investigated in an attempt to evaluate the effects induced by this cytokine on anti-fungal immunity. Although the phagocytosis of the fungus was not affected by IFN-β treatment, the maturation induced by A. fumigatus infection was enhanced in IFN-β-primed DCs, as evaluated by analysing the immunophenotype and the release of pro-inflammatory and regulatory cytokines. Accordingly, IFN-β endowed DCs with potent Th1 polarizing capacity because an enhanced IFN-γ production in T cells co-cultured with A. fumigatus-infected DCs was observed in the presence of IFN-β.

Data are presented as mean±SD of independently analysed mice.

Statistical significance was calculated using the paired Student’s t-test. A value of p<0.05 was considered significant (*p<0.01; **p<0.001; ***p<0.0001). This work was supported by the FWF project P-19017, P-22419, W-1213 the OeNB grant 11710 and the DocForte fellowship 22174 of the OeAW. Work at SIAF was supported by the Swiss National Science Foundation grants no. 320030_127618/1 and 316030_128813/1 and by the Christine Kühne-Center for Allergy Research and Education, Davos (CK-CARE). Conflict of interest: The authors declare no financial or www.selleckchem.com/products/dabrafenib-gsk2118436.html commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“This chapter contains sections titled: Introduction to the innate immune system Innate immune receptors and cells TLRs and pattern recognition TLR signalling in response to LPS Peptidoglycan and Nods

Nod-like receptors recognize PAMPs and DAMPs Damage associated molecular patterns (DAMPs) Complement proteins perform several innate immune functions The classical complement pathway The lectin and alternative complement pathways Biological properties of complement cleavage products Opsonization by complement proteins Phagocytosis Fc receptors induce phagocytosis Palbociclib nmr Neutrophil function and the respiratory burst ADCC NK cells recognize missing self Activating adaptive immunity Dendritic cells link innate and adaptive immunity Summary “
“Inflammatory bowel disease (IBD) is associated with neutrophil

infiltration into the mucosa and crypt abscesses. The chemokine interleukin (IL)-8 [murine homologues (KC) and macrophage inflammatory protein (MIP)-2] and its receptor CXCR2 are required for neutrophil recruitment; thus, blocking this engagement is a potential therapeutic strategy. In the present study, we developed ADAMTS5 a preclinical model of neutrophil migration suitable for investigating the biology of and testing new drugs that target neutrophil trafficking. Peritoneal exudate neutrophils from transgenic β-actin-luciferase mice were isolated 12 h after intraperitoneal injection with thioglycollate, and were assessed phenotypically and functionally. Exudate cells were injected intravenously into recipients with dextran sodium sulphate (DSS)-induced colitis followed by bioluminescence imaging of whole-body and ex vivo organs at 2, 4 and 16–22 h post-transfer. Anti-KC antibody or an isotype control were administered at 20 µg/mouse 1 h before transfer, followed by whole-body and organ imaging 4 h post-transfer. The peritoneal exudate consisted of 80% neutrophils, 39% of which were CXCR2+. In vitro migration towards KC was inhibited by anti-KC.

6D and

E). In contrast, Daporinad solubility dmso the addition of only viable DC, necrotic DC, viable DC and necrotic DC, or apoptotic DC alone or viable DC and apoptotic splenocytes, even with a very high ratio of apoptotic splenocytes to the upper well of the transwell only resulted in approximately 5–6% of naïve CD4+CD25– T cells differentiating into Foxp3+ Treg. Overall, these findings indicate that only upon uptake of apoptotic DC, viable DC acquire the ability to induce Foxp3+ Treg, which is mediated by soluble factors released by viable DC upon apoptotic DC uptake. Additionally, as tolerance is a balance between effector and suppressor T cells, we looked at the effect of apoptotic/necrotic DC on the ability of viable DC

to induce Th17. Our findings demonstrated that it is only upon apoptotic DC uptake that viable DC had a diminished ability to induce Th17 (Fig. 6F). As TGF-β is a known inducer of Foxp3, we looked at the induction of TGF-β1 and TGF-β2 at the mRNA level in viable DC that had taken up apoptotic DC in the presence/absence of LPS. Our findings indicate that at basal levels without any stimulation, there is some expression of TGF-β1 in DC which is suppressed in response to LPS. This suppression is also observed in viable DC incubated with necrotic DC followed by LPS exposure (Fig. 7A). However, no suppression Cabozantinib in vitro of TGF-β1 expression is observed in viable DC incubated with apoptotic DC prior to LPS exposure. At the same time, no induction of TGF-β1 is observed in this group. In contrast to TGF-β1, TGF-β2 levels were upregulated approximately 12–13-fold in viable DC incubated with apoptotic DC followed by LPS exposure compared with viable immature DC without any treatment (Fig. 7B). As cytokines are also regulated at translational level, we also looked at the protein levels of total as well as active TGF-β1 by ELISA. Results show that upon uptake of apoptotic DC, there was a significant increase in the secretion

of total as well active TGF-β1 by viable DC (Fig. 7C and D). However, this was not observed upon uptake of necrotic DC or apoptotic splenocytes by viable DC. In addition, viable immature DC upon incubation with apoptotic DC followed by LPS exposure also find more secreted significantly higher levels of both total and active TGF-β1 compared with viable immature DC treated with LPS or viable immature DC incubated with necrotic DC and then treated with LPS. Collectively, these findings clearly show that only upon uptake of apoptotic DC, viable DC secrete increased levels of TGF-β1, which is regulated at the protein level. In order to confirm that it was specifically the release of TGF-β upon uptake of apoptotic DC by live DC, which was mediating induction of Foxp3+ Treg, we repeated Treg differentiation experiments in the presence of TGF-β neutralizing Ab (Fig. 7E).

CXCR4 signalling via second messenger was found distinctly regulated between DRL and DV. In this context, it has been demonstrated that migration of human T cells to pancreatic islets was controlled by the beta cell–produced SDF-1 and its receptor CXCR4 [39]. Our group has previously reported findings related to differences in the production of RANTES, MCP and other chemokines in T1D [40, 41]. Moreover, our recent study detected the presence of activated eosinophils in patients with T1D, suggesting that these cells could be involved in an intricate cellular network underlying T1D development (manuscript

submitted). When DRL group was compared to controls, the top-scored immune response–related pathway was the delta-type opioid receptor signalling in T cells. Nguyen and Miller [42] provided evidence that CD28 costimulation-induced delta opioid receptor Ku-0059436 manufacturer expression plays a role in antibody-mediated CD3 activation of T cells in mice. Indeed, our analysis revealed Navitoclax ic50 that CD28 signalling was the third top-scored pathway in this pair comparison. However, among the top-scored pathways, CD40 signalling ranked highest in the term of literature sources linking this molecule to T1D. CD40 was differentially expressed in both DRL and DRLN versus

DV comparisons. Interestingly, in a mouse http://www.selleck.co.jp/products/ch5424802.html model of T1D, CD40 marks a unique pathogenic T cell population in which CD40 ligation induces rapid activation of NFKB [43]. The molecule CD137, also known as TNFRSF9 (tumour necrosis factor receptor superfamily, member 9), influences T cell reactivity and modulates CD28-mediated costimulation to promote Th1 cell responses [33]. It has been demonstrated that anti-CD137 treatment protects NOD mice from diabetes, probably via increasing the

number of regulatory CD4+CD25+ T cells [44]. Finally, it is necessary to emphasize that we were not able to find any information concerning the possible link between some of differentially activated immunorelevant genes and autoimmune diabetes. For example, TGF-βRAP1– transforming growth factor-beta receptor-associated protein 1, CD79β, HELLS– lymphoid-specific helicase, CIAPIN1– cytokine-induced apoptosis inhibitor 1 and ILF3 – interleukin enhancer–binding factor 3, to mention just a few. However, we have already reported a correlation between the expression of TGF-β and a prediabetic stage of this disease [11, 40, 41]. It cannot be overlooked that the signalling element on which many of the above-described pathways converge and proceed via its activation is NF-KB. A few years ago, Pieper and colleagues [32] suggested that NF-KB together with the inducible nitric oxide synthase could play an important role in diabetogenesis.

“
“Malnutrition is highly prevalent in haemodialysis (HD) patients, and it contributes to morbidity and mortality. Fibroblast growth factor-23 (FGF-23) and Klotho contribute to chronic Selleck Y 27632 kidney disease-mineral and bone disorder (CKD-MBD) in HD patients, but

the role that these molecules play in determining nutritional status is currently unknown. A cross-sectional study examining 77 HD patients was performed. The plasma concentrations of FGF-23 and soluble Klotho (s-Klotho) were studied to evaluate their association with muscle mass, which was investigated by abdominal muscle areas measured using computed tomography and by creatinine (Cr) production estimated using the Cr kinetic model. Plasma FGF-23 concentrations were significantly and positively correlated with abdominal muscle areas and Cr production

(rho = 0.301, P < 0.01 and rho = 0.345, P < 0.01, respectively). In contrast, s-Klotho was not significantly correlated with these muscle mass indices and plasma FGF-23 concentrations. Multiple regression analyses showed that FGF-23 was a significant independent predictor of both muscle mass indices (P < 0.01 and P < 0.05, respectively). Plasma FGF-23 concentrations were associated with muscle mass indices in HD patients. Our findings suggest that FGF-23 and nutritional status are linked and this link is most likely independent of s-Klotho. "
“Aortic Dissection (AD) is the most common life-threatening disease involving Raf inhibition the aorta. It is rarely associated with systemic disorders

such as Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic syndrome characterized by cystic degeneration of kidneys, possible presence of cysts in other organs and extra-renal manifestations, including cardiovascular disorders. We performed a systematic literature search focused on the occurrence of AD associated with ADPKD (25 cases identified), and reported 2 cases from our experience. Acyl CoA dehydrogenase We selected data on sex, age, family history of ADPKD and/or AD, habitus, hypertension, renal function, presence of hepatic/pancreatic/splenic cysts, clinical presentation of AD, AD type according to the Stanford classification, treatment and outcome. Furthermore we compared this dataset with the data of the overall population with AD from International Registry of Acute Aortic Dissection (IRAD). Stanford A type AD was documented in 62% of patients. As expected, the initial manifestation of AD was most commonly chest and back pain (80%). The mean age of AD occurrence appears significantly reduced in ADPKD patients compared to the general population with AD (49 ± 12 vs 62 ± 14, p < 0,001). Of note, our analysis shows a remarkably higher frequency of hypertension (90%) compared to the overall AD population (75%), although not significantly (p = 0,133).

Allantoic fluid was collected PF-6463922 order and stored at −80 °C as a stock solution of the virus. Virus titers in the stock solution were determined to be 1.2 × 107 plaque-forming unit (pfu) mL−1 by the plaque assay described below. The following antimouse antibodies (Abs) were used in the neutralization studies: anti-IL-1β monoclonal Ab (mAb), 30311; anti-IL-15 polyclonal Ab, AF447; anti-IL-21 polyclonal Ab, AF594; IgG1 isotype control mAb, 43413; IgG2a isotype control mAb, 54447 (R&D Systems, Minneapolis, MN); anti-IL-12 mAb, C17.8 (BD Pharmingen, San Diego, CA); and anti-IL-18 mAb, 93-10C (Medical

& Biological Laboratories, Woburn, MA). The following antimouse mAbs conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), and PE-Cy5 were used in flow cytometric analysis: FITC-anti-CD69 mAb, H1.2F3; FITC-anti-CD49b mAb, DX5; PE-anti-IFN-γ mAb, XMG1.2; PE-Cy5-anti-CD3e mAb, 145-2C11 (eBioscience, San Diego, CA); FITC-anti-CD4 mAb, RM4-5; FITC-anti-CD8a mAb, 53-6.7 (BD Pharmingen); and PE-anti-CD49b mAb, DX5 (Biolegend, San Diego, CA). Splenocytes were obtained from mice euthanized by cervical dislocation and treated with Tris-buffered NH4Cl solution to MAPK Inhibitor Library deplete erythrocytes. Splenocytes were cultured in RPMI 1640 containing 10% FBS, 100 U mL−1 penicillin,

100 μg mL−1 streptomycin, 50 μM 2-mercaptoethanol, and 0.03% l-glutamine for an indicated period. Unless otherwise indicated, cells were cultured at a dilution of 2.0 × 106 cells mL−1 in a 96-well culture plate (0.2-mL per well) at 37 °C in 5% CO2. The culture supernatants were collected and kept frozen until use. CD90.2− cells, B220− cells, CD11b− cells, CD11c− cells, DX5− cells, and Ly-6G− cells were prepared using MACS system (Miltenyi Biotech, Bergisch Gladbach, Germany), according to the manufacturer’s protocols. The purities as determined

by flow cytometry were > 90% for B220− cells and > 95% for the others. CD11b+ cells and DX5+ cells were positively selected using CD11b and Methamphetamine DX5 microbeads (Miltenyi Biotech), respectively. The purity of these fractions as determined by flow cytometry was > 80% and > 70%, respectively. In the neutralization study, cells were cultured in the presence of 5 μg mL−1 of neutralizing antibodies. When the neutralizing antibody was a monoclonal antibody, an isotype-matched control antibody was used in control experiments. When the neutralizing antibody was a polyclonal antibody, cells in control experiments were cultured without any antibodies. Mouse IL-12p70 and mouse IFN-γ in the culture supernatants were quantified using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) in accordance with the manufacturer’s instructions. Mouse IL-18 was quantified using ELISA kits manufactured by Medical & Biological Laboratories. Cells for flow cytometric analysis were preincubated with anti-CD16/CD32 Ab (2.4G2; BD Pharmingen) to block nonspecific Fc receptor binding.