Rat Splenocytes Research Articles

Su.84. A Time Course Analysis of the Protection of Pulmonary Arterial Hypertension by Splenocytes in Athymic Rats

Su.84. A Time Course Analysis of the Protection of Pulmonary Arterial Hypertension by Splenocytes in Athymic Rats

Effect of a New Immunosuppressant Histon Deacetylase (HDAC) Inhibitor FR276457 in a Rat Cardiac Transplant Model

Histone deacetylase (HDAC) is a known modulator of gene transcription, and the immunosuppressive activity of HDAC inhibitors has been demonstrated in recent several reports. In this study, the HDAC inhibitor FR276457, a hydroxamic derivative, was found to have a similar inhibitory effect on all mammalian HDACs tested, but no isozyme selectivity. Both FR276457 and tacrolimus exerted an immunosuppressive effect on in vitro rat splenocyte proliferation stimulated with Concanavalin A. Next, the effect of FR276457 on allograft rejection when administered either as a monotherapy or in combination with tacrolimus was investigated in a rat heterotopic cardiac transplant model. Orally administered FR276457 prolonged the median survival times (MST) of the transplanted grafts in the vehicle group from 6 d to 17 or 21 d at doses of 20 or 40 mg/kg, respectively. Histopathological analysis showed the structures of the myocardium were not affected, but interstitial cellular infiltration could not be suppressed completely. Tacrolimus (0.032 mg/kg) prolonged allograft MST to 16 d. FR276457, when combined with tacrolimus, prevented allograft rejection at a dose lower than that of the monotherapy. The combination dose prolonged the MST in the groups treated with 10 and 20 mg/kg to >28 d, and cellular infiltration was suppressed completely. In conclusion, this study demonstrated that the oral administration of HDAC inhibitor FR276457 can prevent allograft rejection as a monotherapy, and has additive or synergistic effects when combined with tacrolimus.

Reduced methylation-induced mutagenesis in rat splenocytes in vivo by sub-chronic low dose exposure to N-metyl-N-nitrosourea

Estimates of genotoxic effects of mutagens at low and protracted doses are often based on linear extrapolation of data obtained at relatively high doses. To test the validity of such an approach, a comparison was made between the mutagenicity of N-methyl-N-nitrosourea (MNU) in T-lymphocytes of the rat following two treatment protocols, i.e. sub-chronic exposure to a low dose (15–45 repeated exposures to 1mg/kg of MNU) or acute exposure to a single high dose (15, 30 or 45mg/kg of MNU). Mutation induction appeared dramatically lower following sub-chronic treatment compared to treatment with a single high exposure. Furthermore, DNA sequence analysis of the coding region of the hprt gene in MNU-induced mutants showed that acute high dose treatment causes mainly GC→AT base pair changes, whereas sub-chronic treatment results in a significant contribution of AT base pair changes to mutation induction. We hypothesize that O6-methylguanine-DNA methyltransferase is saturated after acute treatments, while after sub-chronic treatment most O6-methylguanine is efficiently repaired. These data suggest (i) that risk estimations at low and protracted doses of MNU on the basis of linear extrapolation of effects measured at high dose are too high and (ii) that the protective effects of DNA repair processes are relatively strong at low sub-chronic exposure.

Preparing T Cell Growth Factor from Rat Splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization (3). Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro (3). Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later and excess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20 degrees C.

Preparing T Cell Growth Factor from Rat Splenocytes

Preparing T Cell Growth Factor from Rat Splenocytes

Regulation of nociceptin/orphaninFQ secretion by immune cells and functional modulation of interleukin-2

Previous research has demonstrated that numerous populations of immune cell, including lymphocytes, synthesize nociceptin (N/OFQ) precursor mRNA although little is known regarding the immunological role of N/OFQ. In the present study we have demonstrated significant effects of mitogens, pro-inflammatory cytokines, cyclic AMP analogues, glucocorticoids and CRF on N/OFQ secretion by rat splenocytes in vitro. N/OFQ (10 −14 to 10 −10 M) was also shown to inhibit proliferation of Con A-activated splenocytes and production of IL-2 in vitro. In summary we have shown how a variety of stimuli relevant to inflammation can regulate endogenous N/OFQ secretion by splenocytes in vitro. We also suggest that N/OFQ may promote anti-inflammatory actions via suppression of IL-2 in vivo.

IMMUNOCHEMICAL CHARACTERIZATION OF THE FUNCTIONAL CONSTITUENTS OF TRIPTERYGIUM WILFORDII CONTRIBUTING TO ITS ANTI‐INFLAMMATORY PROPERTY

1. Tripterygium wilfordii (TW) contains bioactive compounds that possess immunosuppressive properties. These compounds are considered to be potential drugs in the treatment of acute graft rejections. However, their structure-activity relationships remain unknown. 2. The aim of the present study was to delineate the molecular moieties of triptolide that could account for its ability to inhibit inflammatory responses. In this context, purified TW active compounds (triptolide and triptonide) and synthetic triptolide derivatives were prepared to investigate the structure-activity relationships of triptolide. To this end, rat splenocytes were treated with increasing concentrations of the compounds and then allogenically stimulated using a mixed lymphocyte reaction to determine their antiproliferative activities. From the results, the IC50 value of each compound was calculated. 3. Modification of the beta-hydroxyl group at the C-14 position of the triptolide molecule significantly affected the immunosuppressive activity of T59, as demonstrated by a sevenfold increase of the IC50. Conversely, reduction of the gamma-butyrolactone group in T60 and T61 completely abrogated the antiproliferative effect. Alterations in the C-14 beta-hydroxyl and gamma-butyrolactone groups also resulted in reduced cytotoxicity. 4. The present findings demonstrate that the C-14 beta-hydroxyl and gamma-butyrolactone moieties of the triptolide molecule are crucial for its anti-inflammatory properties and cytotoxicity and are responsible for the compound's antiproliferative activity.

Urotensin II is a New Chemotactic Factor for UT Receptor-Expressing Monocytes

Urotensin II (U-II), a vasoactive cyclic neuropeptide which activates the G protein-coupled receptor UT receptor, exerts various cardiovascular effects and may play a role in the pathophysiology of atherosclerosis. In this study, we report that the UT receptor is expressed and functional on human PBMC and rat splenocytes. PBMC surface expression of the UT receptor was mainly found in monocytes and NK cells, also in a minority of B cells, but not in T cells. Stimulation of monocytes with LPS increased UT receptor mRNA and protein expression. Cloning and functional characterization of the human UT receptor gene promoter revealed the presence of NF-kappaB-binding sites involved in the stimulation of UT receptor gene expression by LPS. Activation of the UT receptor by U-II induced chemotaxis with maximal activity at 10 and 100 nM. This U-II effect was restricted to monocytes. Analysis of the signaling pathway involved indicated that U-II-mediated chemotaxis was related to RhoA and Rho kinase activation and actin cytoskeleton reorganization. The present results thus identify U-II as a chemoattractant for UT receptor-expressing monocytes and indicate a pivotal role of the RhoA-Rho kinase signaling cascade in the chemotaxis induced by U-II.

Periodontal Bacterial DNA Suppresses the Immune Response to Mutans Streptococcal Glucosyltransferase

Certain CpG motifs found in bacterial DNA enhance immune responses through Toll-like receptor 9 (TLR-9) and may also demonstrate adjuvant properties. Our objective was to determine if DNA from bacteria associated with periodontal disease could affect the immune response to other bacterial antigens in the oral cavity. Streptococcus sobrinus glucosyltransferase (GTF), an enzyme involved in dental caries pathogenesis, was used as a test antigen. Rowett rats were injected with aluminum hydroxide (alum) with buffer, alum-GTF, or alum-GTF together with either Escherichia coli DNA, Fusobacterium nucleatum DNA, or Porphyromonas gingivalis DNA. Contrary to expectation, animals receiving alum-GTF plus bacterial DNA (P. gingivalis in particular) demonstrated significantly reduced serum immunoglobulin G (IgG) antibody, salivary IgA antibody, and T-cell proliferation to GTF compared to animals immunized with alum-GTF alone. A diminished antibody response was also observed after administration of alum-GTF with the P. gingivalis DNA either together or separately, indicating that physical complexing of antigen and DNA was not responsible for the reduction in antibody. Since TLR triggering by DNA induces synthesis of prospective suppressive factors (e.g., suppressor of cytokine signaling [SOCS]), the effects of P. gingivalis DNA and GTF exposure on rat splenocyte production of SOCS family molecules and inflammatory cytokines were investigated in vitro. P. gingivalis DNA significantly up-regulated SOCS1 and SOCS5 expression and down-regulated interleukin-10 expression by cultured splenocytes. These results suggested that DNA from periodontal disease-associated bacteria did not enhance, but in fact suppressed, the immune response to a protein antigen from cariogenic streptococci, potentially through suppressive SOCS components triggered by innate mechanisms.

Inhibition of Inducible Nitric Oxide Synthase Enhances Anti‐tumour Immune Responses in Rats Immunized with IFN‐γ‐Secreting Glioma Cells

Interferon gamma (IFN-gamma) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN-gamma has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN-gamma (N32-IFN-gamma), could enhance the anti-tumour immune response. Initially, both a selective iNOS, l-N(6)-(1-Iminoethyl)-l-lysine (l-NIL), and non-selective, N-nitro-l-arginine methyl ester (l-NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both l-NIL and l-NAME enhanced proliferation and production of IFN-gamma from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, l-NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32-IFN-gamma, and administered NOS inhibitors by intraperitoneal (i.p.) mini-osmotic pumps, only splenocytes of rats treated with l-NIL, but not l-NAME, displayed an enhanced proliferation and production of IFN-gamma when re-stimulated with N32 tumour cells. Based on these findings, l-NIL was administered concurrently with N32-IFN-gamma cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN-gamma-based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects.

Th1/Th2 xenogenic antibody responses are associated with recipient dendritic cells

We characterized dendritic cells (DC) phenotypically and functionally between C57BL/6 (Th1-prone) and BALB/c (Th2-prone) mouse recipients in an in vivo sensitization model. Two strains of mice were presensitized with Lewis rat splenocytes as xenogeneic antigens. We found that BALB/c recipients mounted a significantly higher total IgG response to the xeno-antigens when compared with C57BL/6 recipients, 10 days after rat splenocyte infusion. A Th2-mediated antibody response with high ratio of IgG1/IgG2a was seen in the BALB/c recipients, while a Th1 antibody response with low ratio of IgG1/IgG2a was detected in C57BL/6 recipients. CD11c(+)DC isolated from C57BL/6 recipients possessed increased expression of CD8alpha(+) (DC-1 type). The administration of bone marrow derived-DC from IL-12 knockout mice into C57BL/6 recipients induced a shift of Th-mediated anti-xenogeneic antibody responses from Th1 to Th2 domain. Our findings suggest that DC could play an important role to regulate the balance of Th1/Th2 cytokine profiles and rejection patterns in xenotransplantation.

Enhanced transduction of antigen-stimulated T lymphocytes with recombinant retroviruses concentrated by centrifugal filtration

Retroviral gene transduction of antigen-specific T cells and reintroduction of the gene-modified T cells into animals or human subjects is attractive for experimental disease-modeling applications and gene therapy approaches for autoimmune or allergic diseases. However, retrovirus titers are often a limiting factor for the efficient gene transfer of mature T cells, which have proven to be relatively refractory to gene transduction. Retrovirus-containing supernatants with titers sufficient for effective transduction of immortalized T cell lines may fail to transduce peripheral T cells. The use of high-titer retroviruses pseudotyped with vesicular stomatitis virus G protein and concentrated by ultracentrifugation is limited by the loss of specific tropism, lower lymphocyte transduction efficiency on infectious particle basis and pseudotransduction. Herein, we present a simple method to concentrate retroviruses by centrifugal filtration at low g force. We compared the ability of unconcentrated and concentrated retroviruses to transduce immortalized fibroblasts as well as primary rat splenocytes activated with antigen and we evaluated transduction efficiency and mean fluorescence intensity of transgene expression in transduced cells. Our data demonstrate that, with this technique, retrovirus titers were increased nearly 10-fold without significant loss of infectious particles. Compared to unconcentrated retroviral preparations, the concentrated retrovirus supernatants more effectively transduced antigen-stimulated, primary rat T cells. This simple method of concentrating retroviruses may be exploited to generate gene-modified T cells for gene therapy applications in animal models of human autoimmune or allergic disease and may also be applicable for T lymphocyte-based gene therapy approaches in humans.

Discovery of Immature Thymocyte Proliferation Factor

An athymic mouse-derived immature T-cell clone, N-9F, was not maintained by interleukin-2 alone but required another soluble factor, contained in concanavalin A-stimulated rat splenocyte culture supernatant, namely T cell growth factor (TCGF), for its proliferation. An N-9F-proliferation factor (NPF) was isolated in a pure form from TCGF. N-9F cells and immature thymocytes proliferated in the presence of N-9F at 10(-12)-10(-9)M in a dose-dependent manner, but adult thymocytes were not stimulated by NPF. NPF increased DNA synthesis of N-9F. NPF increased CD4 and CD8 double negative, single positive and double positive thymocytes in fetal thymus organ culture. A hamster anti-NPF antiserum possessing the capacity to neutralize N-9F proliferation activity of NPF neutralized the increasing effect of NPF on immature thymocytes. All effects of NPF was inhibited by mAb QR6.6 to recognize a 100 kDa surface molecule of N-9F. The amino-terminal 20 amino acid sequence of NPF was identified and identical to that of rat saposin A. The apparent molecular weight of NPF, 16000, was comparable to that of saposin A. A Hitrap-mouse recombinant His-tag-saposin A antibody column bound NPF, pulled down the NPF activity in TCGF, and the antibody recognized a 16kDa molecule in western-blotting of TCGF. Thus, NPF in TCGF was a saposin A-like protein possessing the capacity for growth and differentiation of immature thymocytes. The physiological significance of NPF in the growth and differentiation of immature thymocytes was discussed in view of the characteristic distributions of NPF and the molecule recognized by its mAb QR6.6 in fetal thymi.

Adenovirus-mediated CTLA4Ig and OX40Ig gene transfer induces long-term survival of cardiac allografts in rats

To investigate the potential role of CTLA4Ig gene and OX40Ig protein in inducing transplantation tolerance and the mechanisms thereof. Thirty Lewis rats underwent transplantation of the hearts of DA rats and then randomly divided into five equal groups: control group, blank virus AdEGFP treated group (adenovirus containing EGFP at the dose of 1-5 x 10(9) pfu/ml was infused via portal vein immediately after the operation), AdCTLA4Ig treated group, AdOX40Ig treated group, and AdCTLA4Ig-IRES-OX40Ig treated group. The cardiac allograft survival was monitored by daily palpation. The total cessation of beating was defined as rejection and was confirmed by histology. Peripheral venous blood samples were collected 0, 3, 7, 10, 14, 21 and 28 days after the administration of adenovirus. ELISA was used to detect the expression of CTLA4Ig and OX40Ig. Twenty days after the heart transplantation single splenocyte suspension was prepared from surviving Lewis rats to be used as responder. The spleens of the normal donor-DA rats and the third strain DA rats to prepare single cell suspension of the same density to perform mixed lymphocyte reaction (MLR). Then recombinant IL-2 was added into the mixed MLR system to observe t\if the MLR could be reversed. Twenty days after the heart transplantation the splenocytes of the tolerating Lewis rats were injected into the lingual vein of the normal Lewis rats to observe the delayed type hypersensitivity (DTH) of the transferred Lewis rat to normal rat splenocytes. RT-PCR was used to detect the mRNA expression of IL-2, interferon-gamma, IL-4, and IL-10. The survival time of the AdCTLA4Ig-IRES-OX40Ig treated group was 151.5 d +/- 42.6 d, significantly longer than those of the AdOX40Ig treated group (60.2 d +/- 11.4 d (P = 0.003), AdCTLA4Ig (43.2 d +/- 11.1 d, P = 0.0026), control group (5.7 d +/- 0.5 d, P = 0.000 43), and AdEGFP treated group (5.2 d +/- 0.4 d, P = 0.000 43). CTLA4Ig and/or OX40Ig proteins were expressed at a high level in the adenoviral treated rats. Compared with the control group the splenocytes of the AdCTLA4Ig-IRES-OX40Ig, AdCTLA4Ig, and AdOX40Ig treated groups displayed donor-specific hyporesponsiveness (P = 0.0016, 0.0026 and 0.001), which could be partly reversed by the addition of exogenous IL-2. Moreover, the hyporesponsiveness could be transferred to the same strain rats through adoptive transfer. In comparison with the normal controls, the expression of Th1 type cytokines, such as IL-2 and IFN-gamma, was significantly decreased in the tolerating rats and significantly increased in the rats with rejection; however the expression of the Th2 type cytokines, such as IL03 and IL-10, was significantly increased in the tolerating rats and significantly decreased in the rats with rejection, showing a deviation of Th1/Th2 type cytokines. AdCTLA4Ig-IRES-OX40Ig-mediated genes transfer renders prolonged expression of CTLA4Ig and OX40Ig in Lewis recipient rats, leading to a long-term survival of cardiac allografts. The induced tolerance is donor-specific, and the mechanisms may be associated with T cell anergy, deviation of Th1/Th2, and the regulatory T cells.

Role of IL-27-producing dentritic cells in Th1-immunity polarization in Lewis rats

Lewis and Brown Norway rats are entirely different with respect to the polarization of their immune responses (Th1 and Th2, respectively). We found that naive Lewis rat splenocytes treated in vitro with heat-killed Mycobacterium tuberculosis (Mtb) upregulate the expression of both subunits of IL-27 (IL-27p28 and EBI3). Mtb treatment caused naive Lewis rat splenocytes to express 4.6-fold more IL-27p28 than Mtb-treated Brown Norway rat splenocytes 6 h after the treatment. Although WSX-1, the IL-27 receptor, was not induced by Mtb treatment in splenocytes from either rat strain, Lewis rats expressed significantly higher levels of the IL-27 signal transducers T-bet and IL-12Rbeta2 than Brown Norway rats. Flow cytometric analysis of dendritic cells from bone marrow cells revealed Lewis rats had more IL-27p28-positive cells. Thus, early in the immune response, Lewis rats appear to produce higher levels of IL-27 than Brown Norway rats, resulting in polarization towards Th1-immunity.

T-kininogen, a cystatin-like molecule, inhibits ERK-dependent lymphocyte proliferation

Plasma levels of kininogens increase with age in both rats and humans. Kininogens are inhibitors of cysteine proteinases, and filarial cysteine proteinase inhibitors (cystatins) reduce the proliferation of T cells. We evaluated whether T-kininogen (T-KG) might mimic this effect, and here we present data indicating that exposure of either rat splenocytes or Jurkat cells to purified T-KG results in inhibition of both ERK activation and [ 3H]-thymidine incorporation, both basal and in response to ConA or PHA. Interestingly, T-KG did not impair [ 3H]-thymidine incorporation in response to IL-2, which requires primarily the activation of the JNK and Jak/STAT pathways. These effects were neither the consequence of increased cell death, nor required the activity of kinin receptors. Furthermore, when T cell receptor proximal events were bypassed by the use of PMA plus Calcium ionophore, T-KG no longer inhibited ERK activation, suggesting that inhibition occurs upstream of these events, possibly at the level of membrane associated signal transduction molecules. We conclude that, like filarial cystatins, T-KG inhibits ERK-dependent T cell proliferation, and these observations suggest a possible role for T-KG in immunosenescence.

Tolerance to Rat Heart Grafts Induced by Intrathymic Immunomodulation Is Mediated by Indirect Recognition Primed CD4+CD25+ Treg Cells

In a rat model (PVG.R8-to-PVG.1U) disparate for one class I antigen, RT.1Aa, we previously demonstrated that intrathymic immunomodulation with donor antigens resulted in prolonged survival of cardiac allografts that underwent chronic rejection. However, long-term survivors developed a regulatory cell population that prevented both acute and chronic rejection when adoptively transferred into secondary graft recipients. The purpose of this study was to characterize these regulatory cells with particular emphasis on CD4+CD25+ Treg cells. Spleens, lymph nodes, and peripheral blood lymphocytes of secondary tolerant recipients were characterized using antibodies to various T cell markers in flow cytometry. In vitro MLR and in vivo adoptive transfer experiments were conducted to investigate the involvement of CD4+CD25+ T cells in the observed tolerance. The presence of various cytokines in the sera of graft recipients and MLR culture supernatants was tested using ELISA. Tolerant recipients compared with naive rats had substantially higher percentages of CD4+CD25+ T cells in the spleen (28+/-3% vs. 11+/-5%) and blood (23+/-6% vs. 9+/-4%). Tolerant animals also had higher levels of serum IL-10 than naive and rejecting animals. CD4+CD25+ T cells from secondary long-term graft survivors inhibited donor-specific proliferative responses in vitro that was associated with high IL-10 production. Importantly, depletion of CD4+CD25+ T cells from splenocytes of tolerant rats abrogated their ability to transfer tolerance to tertiary graft recipients. Our data demonstrate that cardiac allograft tolerance in this model is mediated by CD4+CD25+ Treg cells primed by indirect recognition and is associated with high levels of IL-10.

High levels of endogenous nitric oxide produced after burn injury in rats arrest activated T lymphocytes in the first G1 phase of the cell cycle and then induce their apoptosis

Major physical traumas provoke a systemic inflammatory response and immune dysfunction. In a model of thermal injury in rats, we previously showed that an overproduction of nitric oxide (NO) was responsible for the collapse of lymphoproliferative responses. In the present work, we performed a time-course analysis of cell proliferation and cell death parameters in order to establish the sequence of events triggered by the high NO output in Wistar/Han rat splenocytes activated with Con A, 10 days after burn injury. We demonstrate that activated T cells from burned rats never divided whereas normal T cells underwent four division cycles. However, T cells from both burned and normal rat entered the G1 phase as shown by increase of cell size, mitochondria hyperpolarization, and expression of cyclin D1. Burned rat T cells progressed to the late G1 phase as shown by expression of the nuclear Ki-67 antigen, but they never entered the S phase. They underwent apoptosis as shown by morphological parameters, disruption of transmembrane mitochondrial potential, and DNA fragmentation. Persistent accumulation of the p53 protein accompanied these phenomena. NO synthase inhibitors antagonize alterations of cell proliferation and cell death parameters in burned rat T cells and accelerated p53 turnover.

Pharmacological profile of FR260330, a novel orally active inducible nitric oxide synthase inhibitor

In this study, we examined effects of a newly synthesized chemical compound, FR260330, (2 E)-3-(4-chlorophenyl)- N-[(1 S)-2-oxo-2-{[2-oxo-2-(4-{[6-(trifluoromethyl)-4-pyrimidinyl]oxy}-1-piperidinyl)ethyl]amino}-1-(2-pyridinylmethyl)ethyl]acrylamide on nitric oxide (NO) production in rat splenocytes and human colon cancer cell line, DLD-1 cells. FR260330 inhibited NO x production dose dependently in both cells. In lipopolysaccharide (LPS) and interferon-γ (IFN-γ) treated murine macrophage cell line, RAW264.7, Western blot analysis with gel filtration chromatography revealed FR260330 might prevent dimerization of inducible nitric oxide synthase (iNOS), but had no effect on the expression of iNOS protein. Furthermore, oral administration of FR260330 reduced NO x production dose dependently in plasma from rats exposed to LPS (IC 50=1.6 mg/kg). Meanwhile, higher dose (100 mg/kg) of oral administration of FR260330 did not change mean arterial blood pressure in rats. These results suggest that FR260330 might be a useful therapeutical approach to various inflammatory diseases, in which superoxide or peroxynitrite formed from iNOS-derived NO are involved.

Effects of arginine supplementation on splenocyte cytokine mRNA expression in rats with gut-derived sepsis

To investigate the effects of arginine (Arg)-enriched diets before sepsis and/or Arg-containing total parenteral nutrition (TPN) after sepsis or both on cytokine mRNA expression levels in splenocytes of rats with gut-derived sepsis. Rats were assigned to four experimental groups. Groups 1 and 2 were fed with a semipurified diet, while groups 3 and 4 had part of the casein replaced by Arg which provided 2% of the total calories. After the rats were fed with these diets for 10 d, sepsis was induced by cecal ligation and puncture (CLP), at the same time an internal jugular vein was cannulated. All rats were maintained on TPN for 3 d. Groups 1 and 3 were infused with conventional TPN, while groups 2 and 4 were supplemented with Arg which provided 2% of the total calories in the TPN solution. All rats were killed 3 d after CLP to examine their splenocyte subpopulation distribution and cytokine expression levels. Plasma interleukin (IL)-2, IL-4, tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN-gamma) were not detectable 3 d after CLP. There were no differences in the distributions of CD45Ra(+), CD3(+), CD4(+), and CD8(+) cells in whole blood and splenocytes among the four groups. The splenocyte IL-2 mRNA expression in the Arg-supplemented groups was significantly higher than that in group 1. IL-4 mRNA expression in groups 3 and 4 was significantly higher than that in groups 1 and 2. The mRNA expression of IL-10 and IFN-gamma was significantly higher in group 4 than in the other three groups. There was no difference in TNF-alpha mRNA expression among the four groups. The influence of Arg on the whole blood and splenic lymphocyte subpopulation distribution is not obvious. However, Arg administration, especially before and after CLP, significantly enhances the mRNA expression levels of Th1 and Th2 cytokines in the spleen of rats with gut-derived sepsis.