Thymocyte/splenocyte-derived CD4+CD25+Treg stimulated by anti-CD200R2 derived dendritic cells suppress mixed leukocyte cultures and skin graft rejection

BACKGROUND: CD200 delivers immunoregulatory signals following engagement of its receptor, CD200R. A family of CD200Rs (CD200R1-4) has been described. Spleen expresses cell surface CD200R1, while bone marrow shows predominantly expression of cell surface CD200R2/R3. We showed that dendritic cell precursors (DCp) cultured with anti-CD200R2/3 develop the capacity to induce CD4(+)CD25(+) regulatory T cells (Treg) from peripheral lymphocytes. We now characterize DCs involved in induction of antigen-specific Treg from thymocytes or peripheral T cells, and the properties of Treg cells maintained in long-term culture. METHODS: Bone marrow DCp (C3H or BL/6 origin) were cultured for 8 days with GMCSF, IL-4 and anti-CD200R2, or with CD200Fc and a previously described peptide inhibitor of CD200R1 to allow preferential engagement of non-CD200R1 receptors by CD200. Mixed leukocyte cultures (MLCs) were initiated with allogeneic responder lymphocytes/thymocytes (BL/6 or C3H) and mitomycin-c treated DCs to induce Treg. Treg cells were maintained by reculture with DCs derived in the same manner and IL-2, cloned at limiting dilution, and tested for their ability to suppress MLCs and skin graft rejection in vivo. RESULTS: Foxp3(+) CD4(+)CD25(+) Treg were derived from 60-hr thymocyte and splenocyte T cell cultures using both DC populations. Cloned C3H Treg (Foxp3(+)) suppressed both C3H anti-BL/6 reactivity in a fresh MLC and rejection of BL/6 skin allografts in C3H recipients; the converse was true for BL/6 Treg. CONCLUSIONS: We conclude that CD200 triggering of bone-marrow DCs in the absence of CD200R1 engagement induces CD4(+)CD25(+) Treg, and these cloned antigen-specific Treg may have clinical utility.     

烫伤后脂多糖受体CD14和肿瘤坏死因子-α基因表达的变化规律

目的 观察烫伤后机体主要脏器脂多糖受体ＣＤ１４（ＣＤ１４）和肿瘤坏死因子－α（ＴＮＦ－α）ｍＲＮＡ的表达的变化规律及二者的相互关系。方法 采用大鼠３５％Ⅲ度烫伤模型,分别于伤前,伤后１２,２４,４８,７２小时活杀动物,留取组织标本或分离腹腔巨噬细胞,检测组织或巨噬细胞ＣＤ１４,ＴＮＦ－αｍＲＮＡ表达。结果 烫伤后肝,肺,肾,肠等组织ＣＤ１４ｍＲＮＡ水平均有不同程度增高,分别于伤后１２小时和４８小时     

Ontogeny of suppressor cells. II. Suppression of graft-versus-host and mixed leukocyte culture responses by embryonic cells.

Ontogenic development of suppressor cells was studied in mouse embryos. It was found that liver cells of embryos at various stages of gestation were capable of interfering with the mixed leukocyte culture (MLC) reaction. The MLC reaction was also inhibited by embryonic spleen and thymus cells. However, the latter were inhibitory only when originating in 16- and 17-day-old embryos. Embryonic liver cells were also found to interfere with the graft-versus-host (GVH) response induced in neonatal (BALB/c x C57BL)F1 or (C3H X C57BL)F1 mice by adult parental-type C57BL spleen cells. The effect was expressed in the mortality rates but was less pronounced in the splenomegaly reaction measured in vivo or in an in vitro test system. Suppression of the GVH mortality response was manifested predominantly when the liver cells were syngeneic with the effector cells. In contrast, the MLC reaction was inhibited by liver cells syngeneic or allogeneic with the effector cells. The possible developmental patterns of these cells are discussed.     

Differential local and systemic tumor necrosis factor-alpha responses to a second hit of lipopolysaccharide after hemorrhagic shock

BACKGROUND: The immune response to subsequent stressors after traumatic hemorrhage and resuscitation (HR) may be dependent on timing and counterinflammatory cytokine expression. Our hypothesis was that the timing of the second hit would influence the immune response, and we investigated whether an early second stimulus after HR would result in worse acute lung injury. METHODS: One hour after HR or sham shock (Sham), mice were given intraperitoneal (IP) injections of lipopolysaccharide (LPS) or saline (Sal). Mortality, pulmonary function (PF), bronchoalveolar lavage neutrophil infiltration, and bronchoalveolar lavage (BAL), in addition to serum interleukin (IL)-10, IL-6, and tumor necrosis factor-alpha (TNF-alpha), were assessed. RESULTS: HR blunted serum TNF-alpha expression to LPS (HR+LPS, 424.8 pg/mL; Sham+LPS, 2,248.8 pg/mL; p < 0.05), but primed for increased bronchoalveolar lavage TNF-alpha (HR+LPS, 259.5 pg/mL; Sham+LPS, 23.5 pg/mL; p < 0.05). Elevated serum TNF-alpha corresponded with greater bronchoalveolar lavage neutrophil infiltration (HR+LPS, 0.93%; Sham+LPS, 17.5%; p < 0.05). IL-10 expression was similar in HR and Sham. There were no significant differences in mortality or PF between HR+LPS and Sham+LPS. CONCLUSION: Priming and blunting of the LPS-induced TNF-alpha response occurred concomitantly in two-hit mice, corresponding to an altered pattern of pulmonary inflammation, but no change in PF.     

Impact of interleukin-1 receptor antagonist and tumor necrosis factor-alpha gene polymorphism on IgA nephropathy

BACKGROUND: It is evident that cytokines play an important role in the pathogenesis as well as disease progression in IgA nephropathy (IgAN). The level of cytokine production is influenced by different genotypes that reflect gene polymorphism of the pertinent cytokine. Interleukin-1 receptor antagonist (IL-1ra) and tumor necrosis factor-alpha (TNF-alpha) gene polymorphism have been found to affect disease susceptibility and activity in several inflammatory diseases. However, the impact of these polymorphisms in IgAN patients has not previously been thoroughly studied. METHODS: We investigated 111 cases of biopsy-proven IgAN and 100 healthy, normal controls for their IL-1ra and TNF-alpha gene polymorphism. IL-1ra gene polymorphism was characterized as a variable number of tandem repeats of a 86 bp sequence within intron 2. Five alleles were identified and were designated as IL1RN*1, IL1RN*2, IL1RN*3, IL1RN*4, and IL1RN*5, corresponding to 4, 2, 5, 3, 6 repeats, respectively. A polymorphism in the promoter region of the TNF-alpha gene was also studied. This polymorphism involved a guanidine to adenosine transition at position -308 and was designated as TNF1 (-308G) and TNF2 (-308A). RESULTS: There were 54 male and 57 female patients with a mean age of 30.3 +/- 12.5 years and a disease duration of 66. 8 +/- 47.2 months. The mean duration of the follow-up period was 47. 3 +/- 32.6 months. In the patient group, the allele frequencies of IL1RN*1, IL1RN*2, IL1RN*3, IL1RN*4, and IL1RN*5 were 89.6%, 9.9%, 0%, 0.5%, and 0%, respectively, whereas the corresponding carriage rates were 100%, 19.8%, 0%, 0.9%, and 0%, respectively. An excessive carriage of IL2RN*2 was found in the patients when compared with normal controls (allele frequency, 9.9 vs. 2.5%, P < 0.0001). The allele frequencies of TNF1 and TNF2 were 94.1 and 5.9%, respectively, and the carriage rates were 99.1 and 10.8%, respectively, in the patients, which was not significantly different from those of normal controls. When the patients were stratified into mild and severe groups according to their initial presentation, none of the studied alleles correlated with the severity. However, patients with gross hematuria were associated with a higher carriage rate of TNF2 when compared with patients without gross hematuria (allele frequency, 15. 4 vs. 4.6%, P = 0.0552; carriage rate, 30.8% vs. 8.2%, P = 0.0272). Renal survival analysis revealed that the TNF2 carrier had a renal survival comparable with TNF2 (-) patients. However, the carriage of the IL1RN*2 allele was associated with a significantly poorer long-term outcome with a median survival time of 72 months, as compared with those without IL1RN*2 (134 months, P < 0.01). CONCLUSION: IL-1ra and TNF-alpha gene polymorphism may affect disease susceptibility as well as disease activity and long-term outcome in human IgAN. Treatment with an IL-1ra or IL-1 blocking agent may be relevant in those carrying the IL1RN*2 allele.     

Effects of OPC-6535 on lipopolysaccharide-induced acute liver injury in the rat: involvement of superoxide and tumor necrosis factor-alpha from hepatic macrophages

BACKGROUND: The objective of this study was to investigate the effects of OPC-6535 on Propionibacterium acnes-primed and lipopolysaccharide-induced liver injury in the rat. METHODS: P. acnes was administered intravenously to the rat at 16 mg/kg 7 days before the experiments. In liver perfusion experiments, lipopolysaccharide was mixed in perfusion buffer at 2.5 microg/mL. The chemiluminescence method and histochemical reduction of nitro blue tetrazolium were used for detecting superoxide. Release of cytokines into the perfusate was examined. In in vivo experiments, lipopolysaccharide was administered intravenously to the rat at 200 microg/kg. Concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and cytokines were determined in the plasma, and myeloperoxidase activity was measured in the liver tissue. OPC-6535 was given intravenously at 1 mg/kg 30 minutes before lipopolysaccharide challenge, and was then, in perfusion experiments, added to the buffer at 10 micromol/L. RESULTS: In perfusion experiments, P. acnes and lipopolysaccharide caused dramatic production of superoxide, tumor necrosis factor-alpha (TNF-alpha) and growth-related oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1). Superoxide was mainly from hepatic macrophages. Treatment with OPC-6535 suppressed superoxide and TNF-alpha but did not affect GRO/CINC-1. In in vivo experiments, P. acnes and lipopolysaccharide increased the level of TNF-alpha, GRO/CINC-1, AST and ALT in the plasma, and myeloperoxidase activity in the liver. OPC-6535 reduced TNF-alpha, AST, and ALT, but did not affect GRO/CINC-1 or myeloperoxidase. CONCLUSION: Attenuation of liver injury by OPC-6535 is believed to be due to its inhibitory effects on superoxide and TNF-alpha production by hepatic macrophages in P. acnes- and lipopolysaccharide-treated rats.     

Differential effects of tumor necrosis factor-alpha and interleukin-1beta on cell death in human articular chondrocytes

OBJECTIVE: The death of chondrocytes by apoptosis is characteristic of degenerative joint diseases, such as osteoarthritis (OA). Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) have been shown to play an important role in the development of OA. In this study we analyzed the effects of TNF-alpha and IL-1beta on cell death in normal human chondrocytes. METHODS: Normal human chondrocytes were isolated from knee cartilage obtained at autopsy from 30 adult cadaveric donors. The cells were stimulated with TNF-alpha (10 ng/ml) or IL-1beta (5 ng/ml) in the presence or absence of Ro 31-8220 (Ro: a structurally related analog of bisindolylmaleimide that inhibits mitogen-activated protein kinase phosphatase 1 [MKP-1]) (Ro; 10 microM), an MKP-1 inhibitor, which induces apoptosis in chondrocytes. Apoptosis was evaluated by flow cytometry (propidium iodide) and nuclear morphology was evaluated with 4',6'-dianidino-2-phenylindole dihydrochloride. The expressions of caspase-8, -7 and -3 and Bcl-2 were analyzed by Western blot and the activation of caspase-3 and -8 was measured by flow cytometry. Prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay. RESULTS: At 24 h the percentage of apoptotic (hypodiploid) nuclei induced by TNF-alpha+Ro was higher than the level induced by Ro alone. The combination of IL-1beta (5 ng/ml) with Ro did not show a synergistic effect. A morphological analysis demonstrated that treatment with TNF-alpha+Ro resulted in a large number of cells with condensed nuclei and DNA fragmentation. Western blot studies indicated that IL-1beta+Ro did not induce the time-dependent activation of caspase-8, -7 and -3 as seen with TNF-alpha+Ro. As quantified by flow cytometry, TNF-alpha+Ro induced a higher level of caspase-3 and -8 activation than that seen with IL-1beta+Ro. Pre-incubation for 2h with caspase inhibitors for caspase-3, -7, -8 and pan-caspase significantly decreased the hypodiploid DNA peak induced by treatment with TNF-alpha+Ro at 24 h. Indomethacin increased the cell death induced by IL-1beta+Ro; however, apoptosis induced by TNF-alpha+Ro was not modified by indomethacin. CONCLUSIONS: These results confirm that TNF-alpha and IL-1beta regulate apoptosis differently in this human chondrocyte model and that the differing effects of these cytokines are PGE2-independent. Indomethacin potentiates the effect of IL-1 on cell death and this may explain the reported effect of indomethacin on the progression of joint destruction.     

Neutralization of tumor necrosis factor-alpha action delays but does not prevent lung injury induced by alloreactive T helper 1 cells

BACKGROUND: Lung injury occurs frequently after allogeneic bone marrow transplantation in association with graft-versus-host disease, an immune response that involves both cellular and cytokine components. In a murine model, we recently showed that cloned alloreactive T helper (Th)1 cells can cause lung injury associated with increased production of tumor necrosis factor (TNF)-alpha by alveolar macrophages (J Immunol 1998; 161: 1913). METHODS: To evaluate the role of TNF-alpha in this model, we injected in vitro-activated Th1 cells into the following: (1) recipients deficient in receptors for TNF; (2) C57BL/6 control mice; (3) C57BL/6 mice, pretreated with soluble TNFRIIFc (a dimorphic high-affinity TNF antagonist); (4) mice expressing TNFRIIFc transgene under control of the surfactant apoprotein C promoter (SPCTNFRIIFc); and (5) wild-type littermate controls (C57BL/6) (n=3-6 mice/group). RESULTS: At 1 and 3 days after i.v. Th1 cell transfer, recipients were killed for analysis of lung histology, bronchoalveolar lavage (BAL) protein, and BAL cell counts. Control mice (wild type) at day 1 after injection had a mild to moderate mononuclear perivasculitis and increased interstitial cellularity. At day 3, lesions were more severe and perivasculitis also involved larger veins. TNFR-deficient mice had normal lung or minimal lung inflammation at day 1. At day 3, perivasculitis of medium-sized vessels was present, but there was no apparent involvement of larger veins. Results in mice treated with soluble TNFRIIFc and transgenic mice (SPCsTNFRIIFc) were similar to controls. BAL protein and BAL cell counts did not differ between any of the experimental groups. CONCLUSIONS: We conclude that lung inflammation induced by Th1 cells may be only delayed when TNF-alpha action is blocked. The persistence of abnormalities indicates that other proinflammatory pathways are involved in injury caused by these cells.     

Exposure to particles stimulates superoxide production by human THP-1 macrophages and avian HD-11EM osteoclasts activated by tumor necrosis factor-alpha and PMA

Wear of orthopaedic implants generates particles capable of inducing bone resorption and aseptic loosening of the implant. The present study shows the combined effect of particles and cell activation on macrophage (THP-1) and osteoclast (HD-11EM) release of reactive oxygen and nitrogen species, providing insight into mechanisms that can lead to osteolysis. In the absence of cell activation, exposure of either cell type to submicron zirconia or latex particles did not elicit an increase in reactive oxygen and nitrogen species production. Suboptimal stimulation with 4 beta-phorbol-12-myristate-13-acetate (PMA) plus particles resulted in a synergistic release of superoxide (O2-), however, and a low-level production of nitric oxide small middle dot by THP-1 macrophages. Similarly, particle stimulation of tumor necrosis factor-alpha-activated THP-1 cells increased O2- release. Our findings show the synergistic effect of cell activation and wear particles on O2- production by activated macrophages and osteoclasts, suggesting O2- involvement in mediating osteolysis.     

The role of tumor necrosis factor-alpha and interleukin-1beta in ischemia-reperfusion injury of the rat small intestine.

BACKGROUND: To determine the role of tumor necrosis factor (TNF) and interleukin (IL)-1 in small-intestinal ischemia-reperfusion (I-R) injury, we investigated the effect of FR 167653, a specific IL-1 and TNF inhibitor, on warm I-R injury of the rat small intestine. MATERIALS AND METHODS: Male rats treated with either saline (NS group) or FR 167653 (FR group) underwent 150 min of warm small-intestinal ischemia by applying a vascular clip at the origin of the superior mesenteric artery. In addition to the survival analyses, we investigated plasma TNF-alpha and endotoxin levels, intestinal tissue TNF-alpha and IL-1beta levels, hematocrit values and the amount of exudates in the intestinal lumen, glutamic aspartate aminotransferase (AST), and histological findings up to 120 min after reperfusion. RESULTS: TNF-alpha and IL-1beta levels in the intestinal tissue, and plasma TNF-alpha and endotoxin levels, were significantly (P < 0.05) reduced in the FR group. Severe mucosal damage on histological findings (120 min after reperfusion) and a large amount of intraluminal exudates (60 min after reperfusion) were shown in the NS group, but these findings were significantly (P < 0.05) ameliorated in the FR group. Serum AST levels in the NS group increased 120 min after reperfusion, but this change was significantly (P<0.05) reduced in the FR group. The 30-day survival rate was 80% in the FR group and 30% in the NS group (P<0.05). CONCLUSIONS: Dual inhibition of TNF and IL-1 effectively alleviated intestinal I-R injury, suggesting the key role of TNF and IL-1 in this pathophysiology.     

Stimulation of lipolysis by tumor necrosis factor-alpha in 3T3-L1 adipocytes is glucose dependent: implications for long-term regulation of lipolysis

Tumor necrosis factor-alpha (TNF-alpha) and hyperglycemia both impair insulin sensitivity in vivo. This may be secondary to stimulation of adipose tissue lipolysis and consequent increased circulating free fatty acids (FFAs). Here we report that neither TNF-alpha nor glucose alone has a pronounced effect on lipolysis in 3T3-L1 adipocytes. However, the combination of TNF-alpha plus glucose markedly stimulates lipolysis. Glucose does not affect the ability of isoproterenol to stimulate lipolysis. Alternative substrates such as acetate, pyruvate, and lactate do not allow the TNF-alpha effect. Mannose was almost as effective as glucose; fructose was marginally effective, but galactose was ineffective. The effectiveness of the sugars corresponded with production of lactate, i.e., the cells readily produced lactate from glucose or mannose, slightly from fructose, and not at all from galactose. The ability of TNF-alpha to phosphorylate extracellular signal-regulated kinase 1 (ERK1) and ERK2 and to downregulate perilipin (which has been implicated in the lipolytic effect of TNF-alpha) was not affected by glucose. We conclude that the lipolytic action of TNF-alpha is influenced by glucose in 3T3-L1 adipocytes. The findings suggest that glucose metabolism is required for the lipolytic response to TNF-alpha but not for early signaling events. These findings suggest novel mechanisms by which TNF-alpha and hyperglycemia raise FFA levels and induce insulin resistance.     

The lack of effect of tumor necrosis factor-alpha, interleukin-1-alpha, and interferon-gamma on human sperm motility in vitro.

Whether cytokines present in human peritoneal fluid reduce sperm motility, and thus contribute to infertility, is investigated. The human recombinant cytokines, tumor necrosis factor-alpha, interleukin-1-alpha, and interferon-gamma, were incubated with motile human sperm obtained from fertile men and separated by the swim-up technique. These cytokines, alone or in combination, in higher doses than those observed in vivo (greater than or equal to 25,000 U/ml), did not alter the percentage of motile sperm after 90 minutes, 24 hours, and 48 hours under standard culture conditions. Similarly, penetration of a column of bovine cervical mucus was unchanged after preincubation of the sperm with individual cytokines or combinations of several cytokines for 24 hours. In contrast to those given in previous reports, these dta do not support a direct effect of tumor necrosis factor-alpha, interleukin-1-alpha, or interferon-gamma on sperm motility, and suggest that other soluble factors are responsible for the observed effects of peritoneal fluid on sperm motility in vitro.     

Temporal correlation of tumor necrosis factor-alpha release, upregulation of pulmonary ICAM-1 and VCAM-1, neutrophil sequestration, and lung injury in diet-induced pancreatitis

Lung injury is a major cause of patient morbidity in acute pancreatitis. The purpose of this study was to examine the mechanism of pulmonary infiltration and lung injury in acute pancreatitis. Mice were fed a choline-deficient/ethionine-supplemented (CDE) diet for 144 hours to induce severe acute pancreatitis. Serum samples were collected for measurement of biochemical markers of disease and for the detection of tumor necrosis factor-alpha (TNF-α). Cell surface adhesion molecule expression was quantified by the sensitive radiolabeled dual monoclonal antibody technique. Neutrophil sequestration in lung tissue was measured by the myeloperoxidase assay. Lung injury was determined histologically and lung edema was assessed by wet/dry ratios. Pancreatic injury was demonstrated to occur in all CDE-fed mice, which developed significant hyperamylasemia and hypoglycemia by 48 hours (P <0.0001). Serum TNF-a levels increased significantly by 48 hours over baseline values (P < 0.02). E x p ression of intracellular adhesion molecule (ICAM-1) in pulmonary endothelia was significantly increased above baseline by 30% at 48 hours (P < 0.02) and peaked at 120 hours by 100% (P < 0.000l). Vascular cellular adhesion molecule (XXVI-l) was constitutively expressed at baseline and was upregulated threefold by 48 hours (P < 0.000l). Neutrophil infiltration increased gradually 24 hours after ICAM- and VCAM-1 were upregulated with significant elevation of myeloperoxidase activity over baseline at 72 hours (7.2 ±1.2 vs. 18.1 ±2.2 activity units/gram tissue; P < 0.05). Neutrophil infiltration peaked at 144 hours (26.24 ±10.49 activity units/gram tissue P <0.000l), and its kinetics correlated with the onset and progression of morphologic injury as well as increased lung edema. These results show that acute pancreatitis is associated with a systemic release of inflammatory cytokines, followed by increased expression of pulmonary ICAM- and VCAM-1, neutrophil infiltration, and histologic lung injury. The adhesion molecule axis may be a potential target for practical intervention to ameliorate lung injury and morbidity in acute pancreatitis. Supported by the Transplant Surgical Immunology Laboratory at the University of Tennessee at Memphis and by grant PO I DK-43 785 from the National Institutes of Health (Dr. Granger). Presented at the SSAT/Ross Resident Research Symposium at Howey-in-the-Hills, Fla., (oral presentation), and the Fortieth Annual Meeting of The Society for Surgery of the Alimentary Tract, Orlando, Fla., May 16–19, 1999.