Frequency of B cells in normal mice which recognize self proteins

The mechanism whereby the immune system avoids self-aggression is one of the central issues of Immunology. The discovery of natural autoantibodies, mainly of IgM isotype, and of idiotypic interactions between antibodies indicates that elements of the immune system interact with self constituents and with themselves. Results of studies with soluble antibodies have indicated that the pool of circulating IgM represents the end result of a highly selective process of B cells activation and differentiation by self proteins resulting in the formation of a network. The objective of the present work was to determine the frequency of self-reacting B cells in normal mice. We were able to detect B cells that recognize self proteins present in extracts of different organs in normal adult, 2-3-month old, BALB/c and C57BL/6 mice with an ELISA spot assay. About 1% of total IgM-secreting cells among small, LPS-stimulated spleen cells reacted with organ extracts, whereas among large spleen cells the frequency was 5- to 10-fold lower. Immunization induced an increase in the frequency of IgM-secreting cells. The present results provide cellular evidence for the results of studies done at the serological level. The physiological role of these self-recognizing cells, as well as their participation in autoimmune processes, remain to be established

Immunization with PIII, a fraction of Schistosoma mansoni soluble adult worm antigenic preparation, affects nitric oxide production by murine spleen cells

Nitric oxide (NO) is an important effector molecule involved in immune regulation and defense. NO produced by cytokine-activated macrophages was reported to be cytotoxic against the helminth Schistosoma mansoni. Identification and characterization of S. mansoni antigens that can provide protective immunity is crucial for understanding the complex immunoregulatory events that modulate the immune response in schistosomiasis. It is, then, essential to have available defined, purified parasite antigens. Previous work by our laboratory identified a fraction of S. mansoni soluble adult worm antigenic preparation (SWAP), named PIII, able to elicit significant in vitro cell proliferation and at the same time lower in vitro and in vivo granuloma formation when compared either to SEA (soluble egg antigen) or to SWAP. In the present work we report the effect of different in vivo trials with mice on their spleen cells ability to produce NO. We demonstrate that PIII-immunization is able to significantly increase NO production by spleen cells after in vitro stimulation with LPS. These data suggest a possible role for NO on the protective immunity induced by PIII.

Cramoll 1,4 lectin increases ROS production, calcium levels, and cytokine expression in treated spleen cells of rats

This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca(2+) levels, and interleukin (IL)-1 beta expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 mu g ml(-1) single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca(2+), and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca(2+) levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1 beta but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.

11-Oxoaerothionin isolated from the marine sponge Aplysina fistularis shows anti-inflammatory activity in LPS-stimulated macrophages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

11-Oxoaerothionin isolated from the marine sponge Aplysina fistularis shows anti-inflammatory activity in LPS-stimulated macrophages

Marine sponges of the order Verongida are a rich source of biologically active bromotyrosine-derived secondary metabolites. However, none of these compounds are known to display anti-inflammatory activity. In the present investigation, we report the anti-inflammatory effects of 11-oxoaerothionin isolated from the Verongida sponge Aplysina fistularis. When RAW264.7 cells and primary macrophages were preincubated with 11-oxoaerothionin and stimulated with LPS (lipopolysaccharide), a concentration-dependent inhibition of iNOS (inducible nitric oxide synthase) protein and NO2- (Nitrite) production were observed. The same effect was observed when proinflammatory cytokines and PGE(2) (Prostaglandin E2) production was evaluated. In summary, we demonstrated that in the presence of LPS, 11-oxoaerothionin suppresses NO2 and iNOS expression as well as inflammatory cytokines and PGE(2).

Effects of Echinaforce® treatment on ex vivo-stimulated blood cells

The herb Echinacea purpurea, also called purple coneflower, is regarded as an immune modulator. This study examined changes in cytokine production in blood samples from 30 volunteers before and during 8-day oral administration with an ethanolic extract of fresh Echinacea purpurea (Echinaforce(®)). Daily blood samples were ex vivo stimulated by LPS/SEB or Zymosan and analysed for a series of cytokines and haematological and metabolic parameters. Treatment reduced the proinflammatory mediators TNF-α and IL-1β by up to 24% (p<0.05) and increased anti-inflammatory IL-10 levels by 13% (p<0.05) in comparison to baseline. This demonstrated a substantial overall anti-inflammatory effect of Echinaforce(®) for the whole group (n=28). Chemokines MCP-1 and IL-8 were upregulated by 15% in samples from subjects treated with Echinaforce(®) (p<0.05). An analysis of a subgroup of volunteers who showed low pre-treatment levels of the cytokines MCP-1, IL-8, IL-10 or IFN-γ (n=8) showed significant stimulation of these factors upon Echinaforce(®) treatment (30-49% increases; p<0.05), whereas the levels in subjects with higher pre-treatment levels remained unaffected. We chose the term "adapted immune-modulation" to describe this observation. Volunteers who reported high stress levels (n=7) and more than 2 colds per year experienced a significant transient increase in IFN-γ upon Echinaforce(®) treatment (>50%). Subjects with low cortisol levels (n=11) showed significant down-regulation of the acute-phase proteins IL1-β, IL-6, IL-12 and TNF-α by Echinaforce(®) (range, 13-25%), while subjects with higher cortisol levels showed no such down-regulation. This is the first ex vivo study to demonstrate adapted immune-modulation by an Echinacea preparation. While Echinaforce(®) did not affect leukocyte counts, we speculate that the underlying therapeutic mechanism is based on differential multi-level modulation of the responses of the different types of leukocytes. Echinaforce(®) thus regulates the production of chemokines and cytokines according to current immune status, such as responsiveness to exogenous stimuli, susceptibility to viral infection and exposure to stress.

Gene expression, synthesis, and secretion of interleukin 18 and interleukin 1β are differentially regulated in human blood mononuclear cells and mouse spleen cells

Interleukin (IL)-18, formerly called interferon γ (IFN-γ)-inducing factor, is biologically and structurally related to IL-1β. A comparison of gene expression, synthesis, and processing of IL-18 with that of IL-1β was made in human peripheral blood mononuclear cells (PBMCs) and in human whole blood. Similar to IL-1β, the precursor for IL-18 requires processing by caspase 1. In PBMCs, mature but not precursor IL-18 induces IFN-γ; in whole human blood stimulated with endotoxin, inhibition of caspase 1 reduces IFN-γ production by an IL-1β-independent mechanism. Unlike the precursor for IL-1β, precursor for IL-18 was expressed constitutively in PBMCs and in fresh whole blood from healthy human donors. Western blotting of endotoxin-stimulated PBMCs revealed processed IL-1β in the supernatants via an caspase 1-dependent pathway. However, in the same supernatants, only unprocessed precursor IL-18 was found. Unexpectedly, precursor IL-18 was found in freshly obtained PBMCs and constitutive IL-18 gene expression was present in whole blood of healthy donors, whereas constitutive IL-1β gene expression is absent. Similar to human PBMCs, mouse spleen cells also constitutively contained the preformed precursor for IL-18 and expressed steady-state IL-18 mRNA, but there was no IL-1β protein and no spontaneous gene expression for IL-1β in these same preparations. We conclude that although IL-18 and IL-1β are likely members of the same family, constitutive gene expression, synthesis, and processing are different for the two cytokines.

Contribution of TIR domain-containing adapter inducing IFN-beta-mediated IL-18 release to LPS-induced liver injury in mice.

BACKGROUND/AIMS: After treatment with heat-killed Propionibacterium acnes mice show dense hepatic granuloma formation. Such mice develop liver injury in an interleukin (IL)-18-dependent manner after challenge with a sublethal dose LPS. As previously shown, LPS-stimulated Kupffer cells secrete IL-18 depending on caspase-1 and Toll-like receptor (TLR)-4 but independently of its signal adaptor myeloid differentiation factor 88 (MyD88), suggesting importance of another signal adaptor TIR domain-containing adapter inducing IFN-beta (TRIF). Nalp3 inflammasome reportedly controls caspase-1 activation. Here we investigated the roles of MyD88 and TRIF in P. acnes-induced hepatic granuloma formation and LPS-induced caspase-1 activation for IL-18 release. METHODS: Mice were sequentially treated with P. acnes and LPS, and their serum IL-18 levels and liver injuries were determined by ELISA and ALT/AST measurement, respectively. Active caspase-1 in LPS-stimulated Kupffer cells was determined by Western blotting. RESULTS: Macrophage-ablated mice lacked P. acnes-induced hepatic granuloma formation and LPS-induced serum IL-18 elevation and liver injury. Myd88(-/-) Kupffer cells, but not Trif(-/-) cells, exhibited normal caspase-1 activation upon TLR4 engagement in vitro. Myd88(-/-) mice failed to develop hepatic granulomas after P. acnes treatment and liver injury induced by LPS challenge. In contrast, Trif(-/-) mice normally formed the hepatic granulomas, but could not release IL-18 or develop the liver injury. Nalp3(-/-) mice showed the same phenotypes of Trif(-/-) mice. CONCLUSIONS: Propionibacterium acnes treatment MyD88-dependently induced hepatic granuloma formation. Subsequent LPS TRIF-dependently activated caspase-1 via Nalp3 inflammasome and induced IL-18 release, eventually leading to the liver injury.

Conditioned medium from activated spleen cells supports the survival of rat retinal cells in vitro

Cytokines are a heterogeneous group of molecules that have been associated with several functions in the nervous system, such as survival and differentiation of neuronal and glial cells. In the present study, we demonstrated that conditioned medium from spleen cells activated with concanavalin A increased neuritogenesis and survival of retinal cells, as measured by biochemical and morphological criteria. Our data showed that conditioned medium induced a five-fold increase in the amount of protein after 120 h in vitro. This effect was not inhibited by the blockade of voltage-dependent L-type calcium channels with 5.0 µM nifedipine. However, the use of an intracellular calcium chelator (15.0 µM BAPTA-AM) inhibited this effect. Our results support the idea that factors secreted by activated lymphocytes, such as cytokines, can modulate the maintenance and the differentiation of rat retinal cells in vitro, indicating a possible role of these molecules in the development of retinal cells, as well as in its protection against pathological conditions

Stimulated mast cells promote maturation of myocardial microvascular endothelial cell neovessels by modulating the angiopoietin-Tie-2 signaling pathway

Angiopoietin (Ang)-1 and Ang-2 interact in angiogenesis to activate the Tie-2 receptor, which may be involved in new vessel maturation and regression. Mast cells (MCs) are also involved in formation of new blood vessels and angiogenesis. The present study was designed to test whether MCs can mediate angiogenesis in myocardial microvascular endothelial cells (MMVECs). Using a rat MMVEC and MC co-culture system, we observed that Ang-1 protein levels were very low even though its mRNA levels were increased by MCs. Interestingly, MCs were able to enhance migration, proliferation, and capillary-like tube formation, which were associated with suppressed Ang-2 protein expression, but not Tie-2 expression levels. These MCs induced effects that could be reversed by either tryptase inhibitor [N-tosyl-L-lysine chloromethyl ketone (TLCK)] or chymase inhibitor (N-tosyl-L-phenylalanyl chloromethyl ketone), with TLCK showing greater effects. In conclusion, our data indicated that MCs can interrupt neovessel maturation via suppression of the Ang-2/Tie-2 signaling pathway.

Donor bone marrow cells play a role in the prevention of accelerated graft rejection induced by semi-allogeneic spleen cells in transplantation

Spleen or spleen plus bone marrow cells from (BALB/c x C57Bl/6)F1 donors were transferred into BALB/c recipients 21 days before skin or cardiac transplantation. Prolonged graft survival was observed on recipients treated with the mixture of donor-derived cells as compared to those treated with spleen cells alone. We evaluated the expression of CD45RB and CD44 by splenic CD4(+) and CD8(+) T cells 7 and 21 days after donor cell transfer. The populations of CD8(+)CD45RB(low) and CD8(+)CD44(high) cells were significantly decreased in mice pre-treated with donor spleen and bone marrow cells as compared to animals treated with spleen cells only, although these cells expanded in both groups when compared to an earlier time-point. No differences were observed regarding CD4+ T cell population when recipients of donor-derived cells were compared. An enhanced production of IL-10 was observed seven days after transplantation in the supernatants of spleen cell cultures of mice treated with spleen and bone marrow cells. Taken together these data suggest that donor-derived bone marrow cells modulate the sensitization of the recipient by semi-allogeneic spleen cells in part by delaying the generation of activated/memory CD8(+) T cells leading to enhanced graft survival. (c) 2007 Elsevier B.V. All rights reserved.

Allogeneic apoptotic thymocyte-stimulated dendritic cells expand functional regulatory T cells

P>Dendritic cells (DCs) play an important role in the clearance of apoptotic cells. The removal of apoptotic cells leads to peripheral tolerance, although their role is still not clear. We show that the uptake of apoptotic thymocytes by DCs converts these cells into tolerogenic DCs resistant to maturation by lipopolysaccharide, modulating the production of interleukin-12 and up-regulating the expression of transforming growth factor-beta(1) latency associated peptide. We also observed that DCs pulsed with apoptotic cells in the allogeneic context were more efficient in the expansion of regulatory T cells (Tregs), and that this expansion requires contact between DCs and the T cell. The Tregs sorted from in vitro culture suppressed the proliferation of splenocytes in vitro in a specific and non-specific manner. In the in vivo model, the transfer of CD4+ CD25- cells to Nude mice induced autoimmunity, with cell infiltrate found in the stomach, colon, liver and kidneys. The co-transfer of CD4+ CD25- and CD4+ CD25+ prevented the presence of cell infiltrates in several organs and increased the total cell count in lymph nodes. Our data indicate that apoptotic cells have an important role in peripheral tolerance via induction of tolerogenic DCs and CD4+ CD25+ Foxp3+ cells that present regulatory functions.

Cytotoxic effects of permethrin on mouse liver and spleen cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Propolis and its constituent caffeic acid suppress LPS-stimulated pro-inflammatory response by blocking NF-κB and MAPK activation in macrophages

Ethnopharmacological relevance Propolis is a bee product with numerous biological and pharmacological properties, such as immunomodulatory and anti-inflammatory activities. It has been used in folk medicine as a healthy drink and in food to improve health and prevent inflammatory diseases. However, little is known about its mechanism of action. Thus, the goal of this study was to verify the antioxidant activity and to explore the anti-inflammatory properties of propolis by addressing its intracellular mechanism of action. Caffeic acid was investigated as a possible compound responsible for propolis action. Materials and methods The antioxidant properties of propolis and caffeic acid were evaluated by using the 2,2-Diphenyl-1-picrylhydrazyl free radical (DPPH) scavenging method. To analyze the anti-inflammatory activity, Raw 264.7 macrophages were treated with different concentrations of propolis or caffeic acid, and nitric oxide (NO) production, a strong pro-inflammatory mediator, was evaluated by the Griess reaction. The concentrations of propolis and caffeic acid that inhibited NO production were evaluated on intracellular signaling pathways triggered during inflammation, namely p38 mitogen-activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK1/2), the transcription nuclear factor (NF)-κB and extracellular signal-regulated kinase (ERK1/2), through Western blot using specific antibodies. A possible effect of propolis on the cytotoxicity of hepatocytes was also evaluated, since this product can be used in human diets. Results Caffeic acid showed a higher antioxidant activity than propolis extract. Propolis and caffeic acid inhibited NO production in macrophages, at concentrations without cytotoxicity. Furthermore, both propolis and caffeic acid suppressed LPS-induced signaling pathways, namely p38 MAPK, JNK1/2 and NF-κB. ERK1/2 was not affected by propolis extract and caffeic acid. In addition, propolis and caffeic acid did not induce hepatotoxicity at concentrations with strong anti-inflammatory potential. Conclusions Propolis exerted an antioxidant and anti-inflammatory action and caffeic acid may be involved in its inhibitory effects on NO production and intracellular signaling cascades, suggesting its use as a natural source of safe anti-inflammatory drugs. © 2013 Elsevier B.V.

Biomechanical Loading Modulates Proinflammatory and Bone Resorptive Mediators in Bacterial-Stimulated PDL Cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)