Splenic Adherent Cells Research Articles

In-situ proliferation contributes to the accumulation of myeloid cells in the spleen during progressive experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is characterized by expansion of myeloid cells in the liver and spleen, which leads to a severe splenomegaly associated with higher risk of mortality. This increased cellularity is thought to be a consequence of recruitment of cells to the viscera. We studied whether the local proliferation of splenic myeloid cells contributes to increased splenic cellularity. We found that a monocyte-like population of adherent splenic cells from Leishmania donovani-infected hamsters had enhanced replicative capacity ex vivo and in vivo (BrdU incorporation, p<0.0001). In vitro assays demonstrated that proliferation was more pronounced in the proinflammatory M1 environment and that intracellular infection prevented proliferation. Secondary analysis of the published splenic transcriptome in the hamster model of progressive VL revealed a gene expression signature that included division of tumoral cells (Z = 2.0), cell cycle progression (Z = 2.3), hematopoiesis (Z = 2.8), proliferation of stem cells (Z = 2.5) and overexpression of proto-oncogenes. Regulators of myeloid cell proliferation were predicted in-silico (CSF2, TLR4, IFNG, IL-6, IL-4, RTK signaling, and STAT3). The in-silico prediction was confirmed with chemical inhibitors of PI3K/AKT, MAPK and STAT3 which decreased splenic myeloid cell division ex vivo. Hamsters infected with L. donovani treated with a STAT3 inhibitor had reduced in situ splenic myeloid proliferation (p = 0.03) and parasite burden. We conclude that monocyte-like myeloid cells have increased STAT3-dependent proliferation in the spleen of hamsters with visceral leishmaniasis and that inhibition of STAT3 reduces myeloid cell proliferation and parasite burden.

Identification and characterization of splenic adherent cells forming densely-packed colonies.

It is thought that the spleen contains stem cells that differentiate into somatic cells other than immune cells. We investigated the presence of these hypothetical splenic cells with stem cell characteristics and identified adherent cells forming densely-packed colonies (Splenic Adherent Colony-forming Cell; SACC) in the spleen. Splenic Adherent Colony-forming Cell was positive for alkaline phosphatase staining and stage-specific embryonic antigen (SSEA)-1 antigen. However, the self-renewal properties of SACCs were limited because they stopped cell proliferation once colonies visible to the naked eye were formed. Gene expression analyses by semi-quantitative RT-PCR revealed the significant expression of c-Myc and Klf4, whereas faint or no expression was evident for Nanog, Oct3/4, and Sox2. Global expression analyses by DNA microarray and subsequent gene ontology analyses revealed that the expression levels of genes related to the immune system were significantly lower in SACCs than in control splenic cells. In contrast, genes unrelated to the immune system, such as those involved in cell adhesion and axon guidance, were relatively highly expressed in SACCs compared with control splenic cells. Taken together, we identified a novel cell type residing in the spleen that is different from the hypothetical splenic stem cell, but which bears some, but not all, characteristics that represent an undifferentiated state.

Effect of gamma irradiation doses in the structural and functional properties of mice splenic cells

Purpose: Ionizing radiation is nowadays effectively used in cancer treatments. However, the effect of irradiation in immune-system cells is poorly understood and remains controversial. The aim of this work was to determine the effect of γ-irradiation in the structural and functional properties of mice splenic cells.Materials and methods: Structural traits of irradiated splenic cells were evaluated by Atomic Force Microscopy and Raman spectroscopy. Functional properties were measured by gene and protein expression by RT-qPCR and ELISA, respectively. The induced cytotoxic effect was evaluated by MTT assay and the phagocytic capability by flow cytometry.Results: Membrane roughness and molecular composition of splenic adherent cells are not changed by irradiation doses exposure. An increase in transcription of pro-inflammatory cytokines was observed. While protein expression decreased in IL-2 dose-dependent, relevant differences were identified in the anti-inflammatory marker IL-10 at 27 Gy. An increase of cytotoxicity in irradiated cells at 7 Gy and 27 Gy doses was observed, while phagocytosis was slight increased at 7 Gy dose but not statistically significant.Conclusions: We have demonstrated that γ-irradiation affects the splenic cells and changes the cytokines profile toward a pro-inflammatory phenotype and a tendency to increase the cytotoxicity was found, which implies a stimulation of immune response induced by γ-irradiation.

Splenic Micro Environmental Cells from Patients with Myelofibrosis Elaborate a Cascade of Cytokines and Serve As a Niche for Malignant Hematopoiesis

Although the clinical course of patients with myelofibrosis (MF) is punctuated by the development of extramedullary hematopoiesis leading to symptomatic splenomegaly, the spleen is not a hematopoietic organ in normal adults. Spleens from patients with MF are characterized by increased numbers of hematopoietic cells and increased micro vessel density within the red pulp. Previously, we reported that the siderophore, lipocalin-2 (LCN2), was elaborated by MF myeloid cells which leads to increased MF marrow fibrosis, osteoblastogenesis,and depletion of the pool of normal hematopoietic cells but promotion of the proliferation of MF cells (Lu M, Blood, 2015). Using nanostring nCounter technology, we have now demonstrated that LCN2 transcript levels were significantly higher in MF spleen (4 fold) than in normal spleen samples (p=0.008) and that LCN2 transcript levels were only modestly increased in MF marrow (p=0.48). The data identified the spleen as a major source of LCN2 in MF patients. These findings led us to further examine the effects of LCN2 on MF spleen CD34+ cells and micro environmental cells. Normal spleen adherent cells (AC) were treated with LCN2 and conditioned media (CM) were collected. CD34+ cells isolated from normal BM or MF spleen were cultured with this CM for an additional 5 days. LCN2 treated AC CM increased MF hematopoietic cell numbers by 2 fold and increased MF CFU-GM and BFU-E numbers by 40% (p<0.05) and 90% (p<0.01), respectively, but decreased the proliferation of normal HPC. These effects were far greater than that observed with the addition of LCN2 alone, suggesting that cytokines beyond LCN2 are elaborated by splenic AC that are capable of affecting MF hematopoiesis. We next demonstrated that protein levels of IL-8 and CXCL1, but not CXCL2 were dramatically elevated in AC CM following treatment with LCN2 (p=0.037 and p=0.046). By contrast, LCN2 treatment of marrow AC led to the generation of CM with increased levels of CXCL2 but not CXCL1, demonstrating that a differential responses of splenic and marrow AC to LCN2. Furthermore, LCN2 increased VEGF, IL-8 and CXCL1 mRNA levels in splenic stromal cells. Splenic stromal cell IL-8 transcript levels were knocked down using siRNA resulting in a reduction in VEGF mRNA levels, which could be overcome by addition of greater amounts of IL-8. Furthermore, the effect of the additional IL-8 was blocked by adding an anti-IL8 antibody, thereby confirming the regulation of VEGF by IL-8. These results indicate that LCN2 promotes the elaboration of a cascade of cytokines by splenic AC which are capable of affecting splenic endothelial cell proliferation as well as malignant hematopoiesis.We, then, cultured MF splenic and normal BM CD34+ cells in media alone and in a co-culture system with MF splenic endothelial cells in the absence of exogenous cytokines. The total MF and normal BM cell number was dramatically increased when they were co-cultured with EC as compared to the CD34+ cells cultured alone. The absolute MF CD34+ cell number was increased more than 50 fold (p<0.001), while the numbers of normal BM CD34+ cells were increased to a lesser degree (18 fold). LCN2 treatment of the endothelial cells layers did not further increase their ability to promote the proliferation of MF CD34+ cells. These data indicate that splenic endothelial cells serve as a niche which supports hematopoiesis within the spleens of MF patients and that splenic fibroblasts elaborate a number of cytokines which affect splenic micro vessel density. We attempted to target this MF hematopoietic / endothelial cell interaction by adding a CXCR1/2 receptor antagonist, reparixin, to the endothelial cell based cultures which decreased MF CD34+ cell numbers by 35 % (p=0.04). These data suggest that CXCR1/2 receptor antagonists might be used to disrupt the endothelial cell niche within the MF spleen. In summary, our data indicate that LCN2 promotes the elaboration of cytokines such as CXCL1, IL-8 and VEGF within the splenic microenvironment which leads to an increase in splenic endothelial cell proliferation which is supportive of MF CD34+ cell proliferation. Furthermore this cascade of events can be interrupted by potential treatments which reduce LCN2 levels as well as reparixin which disrupts the hematopoietic cell niche supporting activity of splenic endothelial cells, thereby providing a means of correcting the dysregulated tumor microenvironment present within the MF spleen. DisclosuresNo relevant conflicts of interest to declare.

Scavenger receptor for lipoteichoic acid is involved in the potent ability of Lactobacillus plantarum strain L-137 to stimulate production of interleukin-12p40.

Heat-killed Lactobacillus plantarum strain L-137 (HK L-137) is a more potent inducer of interleukin (IL)-12 than other heat-killed Lactobacillus strains. To elucidate the mechanism involved in this IL-12p40 induction, we compared HK L-137 with heat-killed L. plantarum strain JCM1149 (HK JCM1149) by nuclear magnetic resonance and mass spectrometry. Results showed that HK L-137 contained lipoteichoic acid (LTA) with a chemical structure similar to that of JCM1149, except for a lower degree of glucosyl substitution in the poly(glycerol phosphate) backbone. Lysozyme sensitivity and electrophoretic moiety analysis revealed that HK L-137 exposed more LTA on its cell surface than HK JCM1149. Phagocytosis of HK L-137 by splenic adherent cells was significantly greater than that of HK JCM1149. Anti-LTA antibody and anti-scavenger receptor-A (SR-A) antibody selectively inhibited phagocytosis of HK L-137, as well as IL-12p40 production, by splenic adherent cells. Thus, a higher efficiency of phagocytosis of HK L-137 via SR-A for LTA is responsible for the potent IL-12p40 induction.

Splenic Niche Cells from Young Heterochronic Parabionts Have Decreased Capability to Amplify T-cell Proliferation in Vitro

Immune system dysfunction during aging is well documented fact. Changes in the adaptive immune system are the most marked, especially in the T-cell compartment. In multiple preliminary studies it has been shown that parabiosis between 2 animals of different age can induce age-related changes in adaptive immune response and T-cell subpopulation composition of younger animal. In present study we evaluated the age-related changes in functions of rapidly renewing (macrophages) and slowly renewing cells (CD11c+ DCs) cells in spleens of young and old parabionts. We observed impaired capacity of splenic adherent cells from younger heterochronic parabionts to co-stimulate proliferation of autologous T-cells in vitro up to the level of old animals. Also we observed negative effect of this splenic cell population on intracellular signaling mechanisms that regulate PHA-activated proliferation of T-cells from young animals: statistically significant decrease of NFκB p65 expression and increased expression of IκBα during early activation events. This fact suggests that splenic macrophages may be involved in the induction of age-related changes of the immune system and they can be prospective target for further investigation.

A Brucella Virulence Factor Targets Macrophages to Trigger B-cell Proliferation

Brucella spp. and Trypanosoma cruzi are two intracellular pathogens that have no evolutionary common origins but share a similar lifestyle as they establish chronic infections for which they have to circumvent the host immune response. Both pathogens have a virulence factor (prpA in Brucella and tcPrac in T. cruzi) that induces B-cell proliferation and promotes the establishment of the chronic phase of the infectious process. We show here that, even though PrpA promotes B-cell proliferation, it targets macrophages in vitro and is translocated to the cytoplasm during the intracellular replication phase. We observed that PrpA-treated macrophages induce the secretion of a soluble factor responsible for B-cell proliferation and identified nonmuscular myosin IIA (NMM-IIA) as a receptor required for binding and function of this virulence factor. Finally, we show that the Trypanosoma cruzi homologue of PrpA also targets macrophages to induce B-cell proliferation through the same receptor, indicating that this virulence strategy is conserved between a bacterial and a protozoan pathogen.

β&lt;sub&gt;2&lt;/sub&gt;-Agonist Clenbuterol Suppresses Bacterial Phagocytosis of Splenic Macrophages Expressing High Levels of Macrophage Receptor with Collagenous Structure

Splenic marginal zone macrophages expressing macrophage receptor with collagenous structure (MARCO) contribute to the clearance of blood-borne pathogens. We determined a splenic adherent cell fraction abundantly containing cells expressing a higher level of MARCO by flow cytometry, and examined the effects of daily administration of an anabolic dose of β2-agonist clenbuterol on the phagocytic capacity of the cells in mice. After 6 weeks of clenbuterol (1.0 mg/kg body weight/d) or vehicle administration to the mice, splenic adherent cells were isolated. These cells were separated into three cell-size subpopulations. Among them, the small-cell subpopulation contained abundantly the cells with markedly higher levels of MARCO and exhibited more intense phagocytic capacity against Escherichia coli, as compared with the other subpopulations. The phagocytic capacity of the small cells was significantly reduced after clenbuterol administration. These results suggest that the utilization of clenbuterol as doping drug impairs bacterial clearance in the spleen.

Treatment of rats with heat killed cells (HKC) of Cryptococcus neoformans var. grubii induces cellular activation in spleen and lymphatic nodes

Our previous studies showed that the subcutaneous pretreatment of rats with heat killed cells of Cryptococcus neoformans (HKC) emulsified in complete Freund adjuvant (CFA) promotes protection against an intraperitoneal challenge with viable C. neoformans. In this model, an appropriate activation of adherent peritoneal cells after antigenic treatment is very important for the control of the infection. Here, we investigated the immune response developed in spleen and lymphatic nodes as a result of treatment with HKC-CFA, which might also contribute in the protective phenomenon of this treatment against cryptococcal infection. The results show that, compared with adjuvant alone, rats which received treatment with HKC-CFA presented a greater activation of adherent splenic cells, with up-regulation of major histocompatibility complex class II (MHC II) and CD86 expression and secretion of anticryptococcal metabolites. Furthermore, this treatment also induced an increase in the blastogenic response and the secretion of Th1 and Th2 cytokines by spleen cells in comparison with cells from CFA-phosphate-buffered saline (PBS) treated rats. On the other hand, lymph node cells from animals treated with HKC-CFA presented a rise in the expression of MHCII but not of CD86 with respect to control cells from rats treated with CFA-PBS. These cells also showed a high proliferative response and secretion of Th1-related cytokines, interleukin (IL)-12 and tumor necrosis factor (TNF). These results show that treatment of rats with HKC-CFA is able to induce an early immune response in secondary lymphoid organs, which may contribute to the protective effect induced by this treatment.

Abstract: P626 LACTOBACILLUS PLANTARUM LIPOTEICHOIC ACID DOWNREGULATES LPS-INDUCED INFLAMMATORY RESPONSES AND SUPPRESSES ATHEROSCLEROTIC PLAQUE INFLAMMATION

Heat-killed Lactobacillus plantarum strain L-137 (HK L-137) is a more potent inducer of interleukin (IL)-12 than other heat-killed Lactobacillus strains. To elucidate the mechanism involved in this IL-12p40 induction, we compared HK L-137 with heat-killed L. plantarum strain JCM1149 (HK JCM1149) by nuclear magnetic resonance and mass spectrometry. Results showed that HK L-137 contained lipoteichoic acid (LTA) with a chemical structure similar to that of JCM1149, except for a lower degree of glucosyl substitution in the poly(glycerol phosphate) backbone. Lysozyme sensitivity and electrophoretic moiety analysis revealed that HK L-137 exposed more LTA on its cell surface than HK JCM1149. Phagocytosis of HK L-137 by splenic adherent cells was significantly greater than that of HK JCM1149. Anti-LTA antibody and anti-scavenger receptor-A (SR-A) antibody selectively inhibited phagocytosis of HK L-137, as well as IL-12p40 production, by splenic adherent cells. Thus, a higher efficiency of phagocytosis of HK L-137 via SR-A for LTA is responsible for the potent IL-12p40 induction.

STAT6 signalling is important in CD8+ T‐cell activation and defence against Toxoplasma gondii infection in the brain

Signal transducer and activator of transcription (STAT) 6 is a molecule involved in interleukin (IL)-4 and -13 signalling. We investigated the role of STAT6 signalling in Toxoplasma gondii-infected mice using STAT6-deficient (STAT6(-/-)) and wild-type (WT) mice. A significantly larger number of cysts were recovered from the brain in STAT6(-/-) than in WT mice on days 28 and 56 post-infection. CD8(+) T cells in cerebrospinal fluid and spleen stimulated with T. gondii antigen produced higher levels of interferon (IFN)-gamma in WT than in STAT6(-/-) mice. CD8(+) T-cell function, estimated by expression of CD25 and cytotoxic activity, was lower in STAT6(-/-) than in WT mice. Transfer of CD8(+) but not CD4(+) T cells, purified from infected WT mice, into STAT6(-/-) mice successfully prevented formation of cysts in the brain. However, transfer of naïve CD8(+) T cells from WT into STAT6(-/-) mice did not show either activation of CD8(+) T cells or a decrease in the number of cysts in the brain. Transfer of splenic adherent cells from WT into STAT6(-/-) mice induced activation of CD8(+) T cells and decreased the number of cysts in the brain. Expression of CD86 on splenic dendritic cells and IL-12 p40 production were weaker in STAT6(-/-) than in WT mice after T. gondii infection. These results indicate that STAT6 signalling is important in CD8(+) T-cell activation, possibly through regulation of antigen-presenting cells, which could suppress T. gondii infection in the brain.

Evaluation system for an experimental study of low-pathogenic avian influenza virus (H9N2) infection in specific pathogen free chickens using lactic acid bacteria, Lactobacillus plantarum KFCC11389P

In low-pathogenic avian influenza (LPAI) virus, the effects of prophylactic oral administration of Lactobacillus plantarum KFCC11389P on immunity, viral loads and levels of nitric oxide and interferon-γ in splenocytes from specific pathogen free (SPF) chickens were investigated. For in vitro screening of the antiviral effect, haemagglutination activities of four different lactic acid bacterial strains by direct contact with virus were measured. L. plantarum KFCC11389P only neutralized the virus on the SPF eggs. For in vivo studies, four groups of chickens were fed with various L. plantarum KFCC11389P bacterial components and were then challenged with LPAI virus at 28 days. Additionally, two control groups were used as negative (virus-unchallenged) control and positive (virus-challenged) control. We show that some parameters such as nitric oxide production in serum and splenic adherent cells and interferon-γ levels in splenic cells from SPF chickens could be used for the evaluation of LPAI virus infection. This is the first report demonstrating the interferon-γ production in cultured splenic mononuclear cells and nitric oxide production in serum as well as virus shedding in trachea and cloacae in LPAI virus-infected SPF chickens fed specific lactic acid bacterial components.

Facilitation of hematopoietic recovery by bone grafts with intra-bone marrow–bone marrow transplantation

We have previously shown that T cells can acquire donor-type major histocompatibility complex (MHC) restriction and can interact with both donor-type antigen-presenting cells (APCs) and B cells, when adult donor bones are co-grafted with intravenous (IV) injection of bone marrow cells (BMCs) in order to supply donor bone marrow (BM) stromal cells. We have also found that the direct injection of donor BMCs into recipient BM (intra-bone marrow–bone marrow transplantation: IBM–BMT) produces more rapid reconstitution (including T-cell functions) and higher survival rates than IV injection (IV-BMT) even in chimerism-resistant combinations. In the present study, we show that the co-administration of bones from suckling (2–3 days old) donor mice is also effective in the IBM–BMT system. Even when a relatively low number of BMCs were injected into adult (more than 15 weeks old) mice, complete reconstitution was achieved in the mice that had received IBM–BMT+bone grafts, but not in the mice that had received IBM–BMT alone. Most BM and splenic adherent cells obtained from the recipients that had received IBM–BMT+bone grafts were reconstituted by donor-type cells. Both T-cell proliferation and plaque-forming cell assays indicated that the T cells of such mice showed donor-type MHC restriction. Moreover, the analyses of thymic sections using confocal microscopy revealed that donor BM stromal cells had migrated into the thymus. Thus, the co-administration of donor bones has great advantages for allogeneic BMT in adult mice.

Human splenic macrophages as a model for in vitro infection with Mycobacterium tuberculosis

Macrophages play an important role during Mycobacterium tuberculosis (MTB) infection. In humans most of the studies on MTB-macrophage interactions have been performed using circulating monocytes and monocyte-derived macrophages. However, little research has been performed on this interaction using tissue macrophages. Herein, we used human splenic macrophages to characterize particular responses to MTB infection. Based on morphological, biochemical, and immunological markers, splenic adherent cells exhibit characteristics of tissue macrophages. They were able to efficiently phagocytose both live and heat-killed (h-k) MTB H37Rv. Upon infection with live, but not h-k MTB, an increase in secreted TNF-alpha was elicited. Splenic macrophages produced high basal levels of IL-10; however, infection with live or h-k MTB resulted in decrease IL-10 secretion. Both IL-12p40 and IL-12p70 basal levels were also decreased upon infection with live or h-k MTB; however, while the reduction for IL-12p40 levels was observed at earlier time points (4h) for both live and h-k MTB, infection with live MTB, but not h-k MTB, resulted in a time-dependent secretion of IL-12p40 at 24 and 48h after infection. IL-12p70 levels were completely reduced upon infection by either live or h-k MTB. These results support that human splenic macrophages may represent a potential useful model to study MTB-macrophage interactions in vitro.

NOD2 Transgenic Mice Exhibit Enhanced MDP-Mediated Down-Regulation of TLR2 Responses and Resistance to Colitis Induction

Mutations in the CARD15 gene encoding NOD2 are susceptibility factors in Crohn's disease. We explored the mechanism of this susceptibility using mice that over express NOD2. Cellular and molecular responses of mice bearing an NOD2 transgene or administered plasmids that express wild-type and mutated NOD2 constructs were examined. In initial studies, we showed that splenocytes from NOD2 transgenic mice as compared with littermate controls exhibit decreased interleukin (IL)-12p70 responses to peptidoglycan (PGN), a TLR2 ligand that contains muramyl dipeptide, but not other TLR ligands; in contrast, IL-12 responses to PAM(3)CSK(4), a TLR2 ligand that does not contain muramyl dipeptide, were normal. Similarly, transgenic mice as compared with controls exhibited greatly decreased IL-12p40 responses to intraperitoneal administration of PGN but not to lipopolysaccharide. In further studies, we showed using electrophoretic mobility shift assay that PGN-stimulated cells from transgenic mice exhibited decreased activation of nuclear factor kappaB. Finally, in a series of studies on the effect of the NOD2 on susceptibility to induced colitis, we found that (1) transgenic mice were highly resistant to induction of PGN colitis and partially resistant to induction of trinitrobenzene sulfonic acid (TNBS) colitis and (2) mice administered a plasmid expressing a wild-type NOD2 gene were completely resistant to TNBS colitis whereas mice administered a plasmid expressing an NOD2 gene with the Crohn's disease frameshift mutation were only slightly resistant to TNBS colitis. These data offer new evidence that NOD2 mutations contribute to inflammatory bowel disease by causing excessive TLR2 cytokine responses.

The splenic microenvironment is a source of proangiogenesis/inflammatory mediators accelerating the expansion of murine erythroleukemic cells

AbstractThe stromal compartments of hematopoietic organs (eg, spleen) are known to influence the viability and growth of diseased hematopoietic progenitors. Here we have used Friend murine leukemia virus (F-MuLV)–induced erythroleukemia to investigate factors of the splenic microenvironment that may make it fertile for the expansion and survival of malignant erythroblasts. We found that splenectomized, erythroleukemic mice exhibited extended survival compared with age-matched sham controls. In vitro, the proliferation of primary erythroleukemic cells cocultured with leukemic-derived splenic adherent cells or their conditioned media was found to be significantly higher than that observed in cocultures with healthy-derived adherent splenic cells. Cytokine protein arrays revealed that F-MuLV–infected splenocytes secreted elevated levels of interleukin-6 (IL-6), vascular endothelial growth factor-A (VEGF-A), macrophage chemoattractant protein-5 (MCP-5), soluble tumor necrosis factor receptor-1 (sTNFR1), IL-12p70, tumor necrosis factor-α (TNF-α), and IL-2 over normal splenocytes. Medium supplemented with both VEGF-A and MCP-5 could sustain proliferation of primary erythroleukemic cells in vitro, and significant proliferative suppression was observed upon addition of neutralizing antibodies to either of these factors. Furthermore, in vivo administration of a neutralizing antibody to VEGF-A extended survival times of erythroleukemic mice in comparison with controls. These findings suggest that VEGF-A and MCP-5 are potentially pivotal paracrine mediators occurring within the diseased splenic microenvironment capable of promoting disease acceleration and expansion of erythroleukemic blasts.

Regulation of hepcidin and ferroportin expression by lipopolysaccharide in splenic macrophages

Acute and chronic inflammatory states are associated with many changes in intracellular iron metabolism including sequestration of iron in the mononuclear-phagocyte system (MPS) and a decline in serum iron. Previous work in rodent models of acute inflammation has demonstrated inflammation-induced downregulation of intestinal and MPS iron exporter, ferroportin 1, mRNA and protein. In addition, these models have also demonstrated hepatic induction of mRNA of the small 25 amino acid peptide hepcidin. Hepcidin has been hypothesized to be the mediator of iron- and inflammation-induced changes in iron metabolism. The molecular details of the connection between iron metabolism, hepcidin and inflammation have become clearer with the recent finding of hepcidin-induced internalization and degradation of FPN1. The work presented here demonstrates that the lipopolysaccharide-induced splenic macrophage FPN1 mRNA downregulation is not dependent upon the action of a single cytokine such as IL-6, IL-1 or TNF-α because mice deficient in these pathways downregulate FPN1 normally. Furthermore, hepcidin is also synthesized in the spleen of normal mice and induced by lipopolysaccharide. Additionally, in vitro, splenic adherent cells produce hepcidin in response to lipopolysaccharide in an IL-6-dependent manner. There appear to be both probable transcriptional and post-transcriptional control of FPN1 expression by lipopolysaccharide-induced inflammation. The former effect is on mRNA expression and is independent of hepcidin, whereas the latter is IL-6- and hepcidin-dependent.

IgM production of lymphocytes from C57BL/6N mice was stimulated by estrogen treated splenic adherent cells

Estrogens have diverse effects on cell growth, differentiation and homeostatic functions, and have been shown to play an important role in regulating immune system. In this study, we examined the effect of 17β-estradiol (E2) on antibody production by splenocytes isolated from C57BL/6N mice. Our results suggest that the activation of immunoglobulin (Ig) M production by E2 requires direct cell–cell interaction between adherent and non-adherent cells in mouse splenocyte population, and the primary target of E2 is adherent cell population. In addition, we indicated that ER antagonist ICI 182780 suppressed this enhancing effect of E2. Both ERα agonist and ERα agonist enhanced IgM production of mouse splenocytes. ERs are expressed on plasma membrane as well as in nucleus. However, a plasma membrane-associated ER specific ligand has no stimulation effect on IgM production. In conclusion, our results indicate that adherent cells stimulated by E2 up-regulate IgM production of lymphocytes through the direct cell–cell interactions, and the enhancing effect of E2 is arouse through ERα and ERβ on these cells.

Induction of antigen-dependent interleukin-12 production by negatively charged liposomes encapsulating antigens

The aim of this study was to clarify the mechanism of adjuvant action induced by negatively charged liposomes composed of phosphatidylserine (PS-liposomes). Coculture of mouse splenic adherent cells with ovalbumin (OVA)-specific Th1 clone (42-6A) cells in the presence of PS-liposomes encapsulating OVA resulted in high levels of IL-12 and IFN-γ productions compared with those of positively charged or neutral liposomes. IL-12 and IFN-γ productions were dose-dependent on antigens in PS-liposomes. The expression of CD40L on 42-6A cells was enhanced by treatment with PS-liposomes encapsulating OVA. IL-12 production, but not IFN-γ, was completely inhibited by the addition of anti-CD40L mAb. These results indicate that PS-liposomes encapsulating antigens could enhance antigen-dependent IL-12 production through the interaction between CD40 on macrophage/dendritic cells and CD40L on 42-6A cells.

Immuno-potentiating effects of the antler-shaped fruiting body of Ganoderma lucidum (Rokkaku-Reishi).

The immuno-potentiating effects of the antler-shaped fruiting body of Ganoderma lucidum (Rokkaku-Reishi, RR), which has been used as a traditional supplement for human health, were investigated in mice. BALB/c mice were administered orally with RR for 3 days at a dose of 50 mg/kg or 500 mg/kg, and interferon-gamma (IFN-gamma) production by splenocytes in response to lipopolysaccharide (LPS) was examined on day 4. The oral administration of 500 mg/kg of RR resulted in a significant increase (p<0.05) in IFN-gamma production. Stimulation of splenic adherent cells from these mice with LPS also resulted in a significant increase (p<0.05) in interleukin-12 (IL-12) production compared with that from the control mice, suggesting that splenic macrophages were activated by RR administration. Furthermore, 500 mg/kg of RR administered for 14 days resulted in a significant increase (p<0.05) in IFN-gamma production by splenocytes in response to both LPS and concanavalin A (Con A). These results suggest that not only splenic macrophages but also T cells were activated by the long-term treatment with RR in vivo. On the other hand, the production of interleukin-4 (IL-4), which is known as an allergic disease-related cytokine, was not affected by the long-term treatment with RR. Our results suggest that the oral administration of RR resulted in Th1-associated immuno-potentiating activities in vivo.