Treatment Of Spleen Cells Research Articles

A163 INFECTION WITH HYMENOLEPIS DIMINUTA ELICITS A TH2 IMMUNITY AND ALLEVIATES DNBS-COLITIS IN A MOUSE MODEL WITH MINIMAL INTESTINAL MICROBIOTA

Abstract Background Immunocompetent mice infected with the rat tapeworm Hymenolepis diminuta are protected from dinitrobenzene sulphonic-acid (DNBS)-induced colitis that was accompanied by the increased relative abundance of short-chain fatty acid (SCFA)-producing bacteria and fecal SCFA levels at 8 days post-infection (dpi). These changes were not observed in antibiotic-treated BALB/c mice or germ-free mice. To further characterize the host-parasite-microbiota interaction in the control of colitis, experiments have been conducted in a reductionist system in which the H. diminuta anti-colitic response was tested in mice colonized with 12 commensal bacteria representing the major five phyla of the mouse gut. Purpose To identify key bacterial species and microbe-derived mediators involved in the H. diminuta-induced protection from DNBS-induced colitis. Method OligoMM12 (Oligo-Mouse-Microbiota) mice (n=4-8/group) were infected with 10 viable antibiotic-treated H. diminuta cysticercoids eight days prior to challenge with DNBS (3mg, i.r.). Mice were monitored daily and necropsied 72h post-DNBS. Disease was assessed by a macroscopic disease activity score, colon length and histopathology on H&E sections of mid-colon. Production of IL-4, -5, -10 and -13 by concanavalin-A treated spleen cells by ELISA was used as an index of successful helminth infection. Result(s) OligoMM12 mice treated with DNBS developed acute colitis, with 3/8 mice requiring humane euthanization, while all mice infected with H. diminuta survived the DNBS challenge. Successful infection was confirmed by statistically significant increases in splenic output of the Th2-cytokines in response to mitogenic stimulation and efficient expulsion of the worms burden by 11 dpi. The severity of DNBS-induced colitis was reduced in H. diminuta-infected mice (n=4), although additional experiments are required to confirm the statistical significance of these data. Conclusion(s) Reproducible DNBS-induced colitis in mice with the simplified OligoMM12 consortium of bacteria and reduced disease in H. diminuta-infected mice has created a model by which the participation of exact bacterial groups or species in H. diminuta-induced suppression of colitis can be tested. This is of translational value, since the failure of helminth therapy in IBD could be predicated by dysbiosis in the patient, and therefore potentially overcome by combination therapy of ‘probiotic’ and helminth. Please acknowledge all funding agencies by checking the applicable boxes below CIHR, Other Please indicate your source of funding; NSERC-CGS D, Eyes High doctoral Recruitment Scholarship Disclosure of Interest None Declared

Differential effects of fucoidans with low and high molecular weight on the viability and function of spleen cells

Fucoidan is an edible sulfated polysaccharide purified from brown algae that has multiple biological activities. However, the effects of fucoidans of different molecular weights on immune cells have not been determined. Thus, we treated spleen cells with low- and high-molecular-weight fucoidans (LMF and HMF, respectively). Viability assays demonstrated that HMF enhanced the viability and prevented the death of spleen cells. Furthermore, functional analysis revealed that HMF significantly increased the production of interferon-γ and nitric oxide. In contrast, LMF had low activity and was relatively toxic to spleen cells. Taken together, these results indicate that HMF makes the greatest contribution to the immunostimulatory activity of fucoidan mixtures. Additionally, fucoidans with different molecular weights may have different effects on the viability and function of immune cells. This study increases our understanding of fucoidans, and may broaden their use in the basic research and clinical fields.

DNA damage and nitric oxide production in mice following infection with L. chagasi

Leishmania chagasi, which causes visceral leishmaniasis in South America, is an obligate intracellular protozoan. Production of nitric oxide by macrophages during the inflammatory response is one of the main microbicidal mechanisms against this parasite. The goal of this study was to evaluate whether L. chagasi infection causes DNA damage in peripheral blood and spleen cells of Balb/c mice and whether such damage may be related to NO production. Balb/c mice were either infected with L. chagasi or maintained as controls. The single-cell gel electrophoresis (comet) assay was used to measure DNA damage in peripheral blood and spleen cells, and the Griess reaction was used to measure NO production in the spleen. L. chagasi infection induced DNA damage in peripheral blood and spleen cells of infected mice. Macrophages from the control group, challenged with L. chagasi, showed significantly ( p < 0.05) greater NO production, compared to non-challenged cells. Treatment of spleen cells with N G-monomethyl- l-arginine (LNMMA) caused a significant reduction of NO production and DNA damage ( p < 0.05). Our results indicate that L. chagasi induces DNA damage in the peripheral blood and spleen cells and that NO not only causes killing of the parasite but also induces DNA damage in adjacent cells.

Staphylococcal enterotoxin A induction of pro‐inflammatory cytokines and lethality in mice is primarily dependent on MyD88

Staphylococcal enterotoxin (SE) -induced toxic shock is triggered by inflammatory cytokine signal amplification after SE binding to major histocompatibility complex class II molecules on antigen-presenting cells and T-cell receptors. Identifying host cellular elements contributing to this pro-inflammatory signal amplification is critical for developing a strategy for therapeutic intervention. Myeloid differentiation primary-response protein 88 (MyD88) is an intracellular signalling adaptor protein primarily known for mediating pro-inflammatory cytokine responses. We investigated the role of MyD88 in staphylococcal enterotoxin A (SEA) -treated cell cultures and mouse models of toxic shock. Our results demonstrated that elevated levels of tumour necrosis factor-alpha, interferon-gamma, interleukin-1alpha/beta (IL-1alpha/beta), IL-2 and IL-6 production correlated with up-regulation of MyD88 after treatment of spleen cells and mice with SEA alone or in combination with lipopolysaccharide (LPS). The SEA-induced lethality was also observed in (LPS-independent) D-galactosamine-sensitized mice. While LPS potentiated SEA-induced cytokine responses, D-galactosamine treatment had no additive effect. Most importantly, our results demonstrated that MyD88(-/-) mice were resistant to SEA-induced toxic shock and had reduced pro-inflammatory cytokine responses. These results suggest that SEA-induced lethality is primarily dependent on MyD88. Our findings offer an important insight on potential therapeutic treatment of SEA-induced toxic shock targeting MyD88.

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)-1beta 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 microg 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-1beta but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.

A Liposomal Formulation of KRN7000 (RGI-2001) Potently Reduces GvHD Lethality through the Expansion of CD4+Foxp3+ Regulatory T Cells in Murine Models

KRN7000 is a potent synthetic derivative of alpha-galactosylceramide and ligand for CD1d molecules expressed on antigen-presenting cells. Subsequent presentation of KRN7000 by CD1d to invariant natural killer T (iNKT) cells, a subset of natural killer T cells expressing invariant T cell receptor alpha, results in the rapid release of Th1, Th2, or immune regulatory cytokines and initiation of multiple downstream cellular events such as T cell polarization and expansion of dendritic cell subsets. Previously we have demonstrated that liposomal formulation of KRN7000 containing a model antigen induces antigen specific immune tolerance by way of regulatory dendritic cells and induction of Foxp3+ regulatory T (Treg) cells. In this study, we examined the ability of KRN7000 embedded in a liposomal bilayer (RGI-2001) to prevent acute graft-versus-host disease (aGvHD), as Tregs have been shown to have a pivotal role in regulating immune responses to alloantigens. RGI-2001 demonstrated potent activity in reducing GvHD lethality in mice that received fully mismatched bone marrow cells (BMC) and whole spleen cells (WSC). A single intravenous administration of RGI-2001 given on day 0 reproducibly demonstrated efficacy in prolonging the survival of mice relative to untreated mice in a total of 16 independent experiments using lethally-irradiated (9 Gys) Balb/c (H-2d) recipients transplanted with 5×10e6 C57BL/6 (H-2b) T cell depleted BMC with 5×10e6 or 2.5×10e6 WSC. Statistically significant prolongation by Log-rank test was observed at the doses > 1 ng/kg in mice that received 2.5×10e6 WSC, and at the doses > 10 ng/kg in those that received 5×10e6 WSC. Investigation of mice that survived greater than 100 days demonstrated multi-lineage hematopoietic reconstitution by donor derived cells. Next, we investigated the kinetics of Treg expansion following BMT using the model receiving 2.5×10e6 WSC. The results revealed significantly accelerated expansion of donor-derived Tregs in RGI-2001 treated animals as compared with untreated animals. In a representative experiment, 3–4 fold higher percentages of Foxp3+ cells were found in H-2b+CD4+ cells in RGI-2001 treated animals on day 15 post BMT as follows: untreated (n=5) vs RGI-2001 (n=5), 4.5±0.7 % vs 20.7±6.0% (spleen), 2.4±1.3% vs 10.6±5.0% (mesenteric lymph nodes) and 5.4±2.8% vs 15.7±6.0% (peripheral lymph nodes). Furthermore, allo-responses of RGI-2001 treated spleen cells were suppressed in an antigen-specific manner. Proliferation of RGI-2001 treated spleen cells was significantly reduced when stimulated with host (Balb/c) antigen presenting cells (APCs) in vitro while responsiveness to a third party (C3H) APCs remained. Based on these findings, we investigated if the second injection of RGI-2001 on day 15 could further support the expansion/maintenance of Tregs post BMT. Results demonstrated further improvement in the survival as well as clinical score of mice that received RGI-2001 treatment on both day 0 and day 15 as compared to those received the treatment only on day 0. Taken together, these results strongly suggest that RGI-2001 promotes the expansion of donor-derived Tregs which specifically suppress T-cell responses against host alloantigens, thereby reducing the GvHD lethality.

Influence of chronic exposure to low doses of γ-radiation and 90Sr on the level of DNA breaks and cell sensitivity to hydrogen peroxide in the mouse spleen

Using comet assay, a statistically significant increase (p < 0.05) in the level of DNA breaks in spleen cells was revealed in male CBA/lac mice exposed to gamma-radiation (1.7 cGy/day) or 90Sr (150-250 Bq/day) for 210 days. The level of DNA breaks also increased under combined exposure to both gamma-radiation and 90Sr (p < 0.05), but to a lesser degree than under exposure to each of these factors alone. Upon additional in vitro treatment of spleen cells with hydrogen peroxide, the relative increase in the level of DNA breaks was smaller in cells of irradiated mice than in the control. The ratio of the level of DNA breaks after hydrogen peroxide treatment to that before this treatment in control mice was 4.2 +/- 0.9, compared to 1.4 +/- 0.6 in gamma-irradiated mice, 1.9 +/- 0.8 in 90Sr-irradiated mice, and 2.3 +/- 0.8 in mice exposed to both gamma- and 90Sr-irradiation.

C-Reactive Protein Stimulated Spleen Cells Transfer Suppression of Experimental Immune Thrombocytopenia (53.9)

Abstract C-reactive protein (CRP) is an innate recognition molecule, which regulates immune responses by interaction with FcγR. CRP suppresses inflammation and autoimmunity in diverse experimental models. Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease in which platelets are cleared by splenic macrophages through FcγR. IVIG is used to treat human ITP. IVIG induces dendritic cells to transfer suppression of ITP (Siragam et al., 2006, Nat Med). In this study, splenocytes were treated with 200 μg/ml CRP or BSA or 1.8 mg/ml IVIG for 30 min, washed and transferred to naïve recipients. After 24 h, ITP was induced by injection of anti-CD41 mAb. Platelets were counted 24 h later. Anti-CD41 decreased platelet counts from 945±29 to 312±61 x 103/mm3. Platelet counts in recipients of 106 treated spleen cells were: BSA, 225±35; CRP, 672±25; and IVIG 590±35. Spleen cells from FcR γ-chain−/− or FcγRI−/− mice treated with CRP or IVIG were not protective and no protection was observed in FcγRIIb−/− mice. Macrophage depletion in vivo by Clodronate liposome treatment of the donor eliminated protection by CRP (363±9, p&amp;lt;0.001 vs CRP), but not IVIG (560±38). Thus, CRP treatment of spleen cells in vitro generates a cell that transfers suppression of ITP. Generation of this cell by CRP is dependent on FcγRI and macrophages. The findings allow for the characterization of the cells involved in the immunoregulatory properties of CRP.

Toxoplasma gondii Inhibits Apoptosis in Infected Cells by Caspase Inactivation and NF-κB Activation

Our experiments aimed to clarify the mechanism by which host cell apoptosis is inhibited by infection with the intracellular protozoan parasite, Toxoplasma gondii (T. gondii). Mouse spleen cells were cultured in 6-well plates with RPMI 1640/10% FBS at 37℃, in a 5% CO2 atmosphere. Apoptosis of spleen cells was induced by actinomycin-D (AD) treatment for 1 h prior to infection with T. gondii. A variety of assays were used to assess the progression of apoptosis: DNA size analysis on agarose gel electrophoresis, flow cytometry with annexin V/PI staining, and analysis of expression levels of Bcl-2 family and NF-κB mRNA and proteins by RT-PCR, Western blotting, and EMSA. Additionally, transmission electron microscopy (TEM) was performed to observe changes in cell morphology. Fragmentation of DNA was inhibited in spleen cells treated with AD and T. gondii 5 h and 18 h post infection, respectively, and flow cytometry studies showed a decreased apoptotic rates in AD and T. gondii treated spleen cells. We observed decreased expression of Bax mRNA and protein, while levels of Bcl-2 mRNA remained constant in spleen cells treated with AD and T. gondii. Caspase 3 and PARP were inactivated in cells treated with AD and T. gondii, and increased levels of cleaved caspase 8 were also observed. Analysis of EMSA and Western blot data suggests that activation of transcription factor NF-κB may be involved in the blockade of apoptosis by T. gondii. TEM analysis showed nuclear fragmentation and chromatin condensation occurring in spleen cells treated with AD; however, such apoptosis-associated morphological changes were not observed in cells treated with both AD and T. gondii tachyzoites. Together, these data show that T. gondii infection inhibits AD induced apoptosis via caspase inactivation and NF-κB activation in mouse spleen cells.

Experimental hypersensitivity pneumonitis: cellular requirements.

We previously demonstrated that Thy1.2+, CD4+, Ia-T cells are responsible for transfer of murine adoptive experimental hypersensitivity pneumonitis (adoptive EHP). To characterize the culture conditions necessary for development of these cells, we depleted cell cultures of Thy1.2+, CD4+, CD8+, or Ia+ cells using MoAbs and complement or magnetic beads, prior to culture of sensitized C3H/HeJ murine spleen cells (SC) with Micropolyspora faeni. After culture, cells were transferred to recipients which were later challenged intratracheally with M. faeni. The extent of pulmonary inflammatory changes in these animals was determined 4 days after intratracheal (i.t.) challenge with M. faeni. Cultured M. faeni-sensitized SC which had been treated before culture with media, complement only, anti-CD8 plus complement or magnetic beads alone could transfer EHP to naive animals. SC treated with anti-Thy1.2 or anti-CD4 plus complement could not transfer EHP. Treatment of SC with anti-Iak plus magnetic beads diminished the ability of cultured cells to transfer EHP. We conclude that the ability to produce cells able to adoptively transfer EPH is dependent on the presence of Thy1.2+, CD4+, and Ia+ cells, but not CD8+ cells, at the onset of culture.

Production of nitrite by dengue virus-induced cytotoxic factor.

Dengue type 2 virus (DV) infection induces production of a cytokine, the cytotoxic factor (CF) in the spleen of mice. The present study was undertaken to investigate the production of nitrite (NO2-) by the spleen cells of mice in vitro and in vivo following inoculation of DV or CF. Maximum NO2- production occurred at 45 min after inoculation of 5 microg CF, both in vitro and in vivo. The NO2- was produced by macrophages and T cells and not by B cells. Pretreatment of CF with anti-CF antisera inhibited production of NO2-. DV-stimulated spleen cell culture supernatants showed peak production of CF and NO2- at 72h. In DV-infected mouse spleen, maximum NO2- production occurred at 8-11 days post-infection, which correlated with peak cytotoxic activity in the spleen. Pretreatment of spleen cells with N(G)-monomethyl L-arginine (NMMA) inhibited NO2- production. NO2- production was abrogated in a dose-dependent manner by treatment of spleen cells with Ca2+ channel blocking drug, Nifedipine. The findings demonstrate that DV-induced CF induces production of NO2- in spleen cells, probably in a Ca2+-dependent manner, and may be a mechanism of target cell killing.

An immunostimulatory oligodeoxynucleotide containing a cytidine-guanosine motif protects senescence-accelerated mice from lethal influenza virus by augmenting the T helper type 1 response.

The SAM-P1 strain of senescence-accelerated model mice shows an impaired T helper type 1 (Th1) immune response upon infection with influenza virus, which results in high susceptibility to the virus. Treatment of spleen cells from SAM-P1 mice with an immunostimulatory oligodeoxynucleotide containing a cytidine-guanosine motif (CpG ODN) in vitro increased the ratio of the titre of IFN-gamma to that of IL-4. Administration of CpG ODN to SAM-P1 mice generated satisfactory virus-specific cytotoxic T-lymphocyte responses and natural killer cell activation and the virus-specific immunoglobulin (Ig) isotype switched from IgG1 to IgG2a. Virus growth in the lungs of CpG ODN-treated SAM-P1 mice was cleared quickly and mice survived the lethal influenza virus infection. It could be inferred that a possible mechanism of CpG ODN for normalization of senescence-associated dysregulation of the Th1/Th2 balance involves the upregulated expression of CD154 and CD40 molecules on immune-competent cells. These results suggest that CpG ODN could contribute to the development of a protective strategy against infectious diseases, especially among immunocompromised elderly persons, by stimulating Th1 immune responses.

Allogeneic cell stimulation enhances cytomegalovirus replication in the early period of primary infection in an experimental rat model

Background: Cytomegalovirus (CMV) diseases commonly occur in allograft recipients in the early post-transplant period. However, factors responsible for the high incidence of CMV diseases during this period are not yet fully defined. Methods: Wistar–Furth (WF; RT- 1 u ) rats were inoculated with 10 4 plaque-forming units (PFU) of rat CMV (RCMV) intraperitoneally, and then transplanted with allogeneic lungs from Dark Agouti (DA; RT-1avl) rats or stimulated with 10 7 mitomycin C–treated spleen cells from DA rats by daily sub-cutaneous injections for 2 weeks. No immunosuppressive agent was used. Naive WF rats and WF rats grafted with syngeneic lungs or cells were used as controls. The level of RCMV replication in rats was assessed by infectious virus titers in tissues. Results: The virus titers in salivary glands of allogeneic and syngeneic lung graft recipients were significantly higher than in naive WF rats. The level of RCMV replication in rats stimulated with allogeneic spleen cells was significantly higher than in the syngeneic recipient rats: virus titers in the salivary gland of allogeneic and syngeneic recipients reached 4.61 ± 0.33 and 4.00 ± 0.37 log 10 PFU/g tissue, respectively, at 14 days post-infection ( p = 0.015). The augmented viral replication in allogeneic recipients was confirmed by an increase in the number of RCMV antigen–positive macrophages present in tissue sections of the salivary gland. Conclusions: Acute lung allograft rejection and allogeneic spleen cell stimulation enhance CMV replication in the salivary gland of rats. Various responses to allogeneic antigens occurring in the process of acute allograft rejection could be risk factors for post-transplant CMV replication and infection.

Mitogenic activity of gelatin to murine spleen cells.

We reported previously that gelatin stimulates the growth of spleen cells in vitro. Tritium thymidine (3H-TdR) uptake into phytohemagglutinin (PHA)-stimulated spleen cells as well as intact spleen cells was augmented by gelatin. These findings suggest that gelatin serves as a mitogen for lymphoid cells. In this study, the target of action of gelatin was investigated. Tritium thymidine uptake into T cell-rich fraction was enhanced by incubation with 7.5 mg/ml of gelatin for 48 hours. The level of 3H-TdR uptake into B cell-rich fraction was not definitely increased by gelatin. Flow cytometric analysis confirmed these findings. Namely, it showed that treatment of spleen cells with 7.5 mg/ml gelatin increased a ratio of CD3-positive cells and decreased that of CD19-positive cells. Tritium thymidine uptake into natural killer cell-rich fraction was augmented by gelatin in a similar fashion to T cell-rich fraction. Tritium thymidine uptake into macrophages was very low and not affected by gelatin. Tritium thymidine uptake into macrophage-precursors was very low but was enhanced by gelatin. These findings suggest that gelatin could be used as an agent of cancer biotherapy.

Characterization of a surface membrane molecule expressed by natural killer cells in most inbred mouse strains: monoclonal antibody C9.1 identifies an allelic form of the 2B4 antigen.

A newly generated monoclonal antibody (mAb C9.1) described in this study identifies a surface membrane molecule that is involved in the lytic programme of activated natural killer (NK) cells. This conclusion is based on the facts that, first, this antigen was expressed on the vast majority of surface immunoglobulin (sIg)- CD3- CD4- CD8- spleen lymphocytes, albeit it was also present on minor subsets of sIg+ B (approximately 7%) and CD3+ T (approximately 2%) lymphocytes; second, that all splenic NK activity was contained within the C9.1+ cell population, and was almost totally abolished by treatment of spleen cells with mAb C9.1 and complement; third, that mAb C9.1 was capable of increasing interleukin-2-cultured and in vivo polyinosinic:polycytidylic acid-activated, NK cell-mediated, antibody-redirected lysis, but not freshly isolated NK cell-mediated killing. Furthermore, the strain distribution of the C9.1 antigen was shown to be antithetical to that of the 2B4 antigen already described as a molecule associated with major histocompatibility complex-unrestricted killing mediated by activated NK cells. The gene encoding C9.1 antigen was linked to the Akp1 isozyme locus on chromosome 1 close to the 2B4 gene. Although C9.1 and 2B4 were monomeric glycoproteins of 78 000 MW and 66 000 MW, respectively, removal of N-linked sugars from both antigens by endoglycosidase F yielded identical protein backbones of 38 000 MW. Thus, all of these results suggest that mAb C9.1 recognizes an allelic form of the 2B4 antigen. However, the detection of mAb C9.1-reactive antigen on a minor subset of B cells may suggest a possible reactivity of mAb C9. 1 with some product of other members of the 2B4 family genes.

Immunomodulating activity of Chinese medicinal herbs and Oldenlandia diffusa in particular

The effect of eight different Chinese medicinal herbs (CMHs) on lymphocytes was studied in vitro using murine spleen cells. Among the studied eight CMHs, Astragalus membranaceus and Oldenlandia diffusa markedly stimulated murine spleen cells to proliferate. The responder cells for CMHs were B cells, because the response was depleted by the treatment of spleen cells with anti-immunoglobulin (Ig) antibody and complement and after purification by nylon wool column. This response was not due to contamination by lipopolysaccharide (LPS), because CMHs could stimulate C3H/HeJ spleen cells which are low responders to LPS. CMHs enhanced the production of Ig. CMHs also enhanced the induction of allo-antigen specific cytotoxic T lymphocytes. However, CMHs had no effect on natural killer cells. Furthermore, CMHs stimulated macrophages to produce interleukin-6 and tumor necrosis factor. The electroelution of the proteins from SDS-PAGE gel showed that the active components of Oldenlandia diffusa had an apparent molecular weight of 9-200 kD and were sensitive to pronase E and NaIO 4 treatment, suggesting glycoproteins in nature. These results suggest that CMHs have immunomodulating activity in vitro and this activity could be used clinically for the modulation of immune responses.

B cell stimulating activity of seaweed extracts

The activity of seaweed extracts on murine and human lymphocytes was studied in vitro. The extracts of some kinds of seaweed, such as Hizikia fusiformis and Meristotheca papulosa, stimulated normal mouse spleen cells to proliferate. The responder cells are B cells, because the response was depleted by the treatment of spleen cells with anti-immunoglobulin (Ig) antibody and complement and being passed through a nylon wool column. This response is not due to lipopolysaccharide (LPS) contamination, because seaweed extracts can stimulate spleen cells of C3H/HeJ mice which are LPS low responders. Seaweed extracts also enhanced Ig production by B cells and tumor necrosis factor (TNF) production by macrophages. Furthermore, seaweed extracts stimulated human lymphocytes to proliferate. All these B cell stimulating activities of seaweed extracts associated with glycoproteins whose molecular weights resided in 100 kD. These results suggest that seaweed extracts have stimulating activity on B cells and macrophages and this ability could be used clinically for the modulation of immune responses.

Donor-specific transfusion using mitomycin-C treated spleen cells induces significant prolongation of cardiac allograft in rats

Donor-specific transfusion using mitomycin-C treated spleen cells induces significant prolongation of cardiac allograft in rats

Costimulation of CD28- T lymphocytes by 4-1BB ligand.

The interaction of the T cell surface protein CD28 with its ligand, B7-1 or B7-2, provides a critical costimulatory signal for T cell activation. T cells from CD28- mice are deficient in a variety of responses, including those to lectins and allogeneic spleen cells. However, some immune responses do occur in CD28- mice, suggesting the existence of alternate costimulatory pathways. In this work, we show that T cells purified from CD28- mice respond to B lymphomas expressing 4-1BB ligand (4-1BBL), a member of the TNF gene family. This response is inhibited by a soluble form of 4-1BB, the T cell surface receptor for 4-1BBL. Thus, 4-1BBL/4-1BB interaction provides costimulatory signals to T cells independent of signaling through the CD28 receptor. We find that 4-1BBL is inducible on splenic B cells by CD40 ligand/CD40 interaction or by culturing of splenic dendritic cells, treatments that also induce B7 family molecules. CD28- T cells fail to respond in an MLR to resting allogeneic spleen cells. However, treatment of spleen cells with CD40 ligand renders them competent in activation of CD28- T cells. In contrast to results using B lymphomas as APC, soluble 4-1BB fails to inhibit the T cell response to activated spleen cells. This failure of soluble 4-1BB to block an MLR between CD28+ or CD28- T cells and allogeneic spleen cells is in contrast to a previous report with CD28+ cells.

Anti-interleukin-4 modulation of the Th2 polarized response to the parasitic nematode Brugia pahangi

Lymphatic filariasis is a chronic disease characterized by a pronounced Th2 bias in the immune response and impaired antigen (Ag)-specific Th1 responses. We have used a mouse model of filariasis to investigate the role of the infective form (the third-stage larvae [L3]) in modulating the immune response. Subcutaneous infection of BALB/c mice with L3 of Brugia pahangi has a profound effect on Th cell function. By day 12 post-infection, spleen cells from these mice exhibited a dramatic reduction in concanavalin A-driven proliferation and interleukin-2 (IL-2) and gamma interferon (IFN-gamma) secretion in comparison with uninfected controls. However, exposure to L3 did not render the mice completely unresponsive; these animals mounted a strong Th2 response to the parasite, characterized by elevated levels of IL-4, IL-5, and IL-10 and parasite-specific serum immunoglobulin G (IgG), IgG1, and IgE. Treatment of spleen cells from L3-infected mice with neutralizing anti-IL-4 or recombinant IL-2 resulted in a dramatic increase in concanavalin A-induced proliferation and IL-2 and IFN-gamma production. Despite their defective polyclonal Th1 response, cells from L3 infected mice proliferated when stimulated with Ag, and this response was blocked by anti-IL-4. However, anti-IL-4 treatment failed to induce Ag-specific IL-2 or IFN-gamma production, indicating that B.pahangi-primed Th1 cells do not appear to be present or are still unable to respond even in the absence of IL-4.