Inflammatory cytokine levels in paw tissues during development of rat collagen-induced arthritis: Effect of FK506, an inhibitor of T cell activation

Objective and design: To characterize rat collagen-induced arthritis (CIA) on the basis of levels of inflammatory cytokines, tumor necrosis factor (TNF)-, interleukin (IL)-1 and IL-6 in paw tissues, and further investigate the effect of FK506 (tacrolimus), a potent inhibitor of T cell activation, on cytokine levels.Methods: CIA was induced in female Lewis rats. The volume of hindpaws was measured before and after collagen immunization. TNF-, IL-1 and IL-6 levels in paw tissue extracts were determined by ELISA. Proteoglycan contents of cartilage in femoral heads was measured as an indication of cartilage destruction. To assess the effect of FK506 on inflammatory cytokine levels, rats were orally treated with 5 mg/kg of FK506 from days 14–21.Results: TNF- a level in paw tissues did not significantly change compared to levels found before collagen immunization, throughout development of CIA. In contrast, IL-1 and IL-6 levels in paw tissues significantly increased between day 14 and day 28 after collagen imuninization, when the arthritis was at a developed stage. Therapeutic treatment with FK506 reduced the elevated level of IL-6, but not IL-1, in paw tissue. FK506 treatment was effective in suppressing paw swelling and also recovering the loss of proteoglycan contents in the cartilage.Conclusions: Levels of IL-1 and IL-6, but not TNF- , in paw tissue were upregulated in association with the development of arthritis in rat CIA. These results suggest that IL-1 and IL-6, rather than TNF- , may play important roles at local inflammatory sites in producing joint destruction in rat CIA. FK506 may improve arthritis in established stages of CIA, by reducing the elevated level of IL-6.Received 4 March 2004; returned for revision 2 April 2004; accepted by M. J. Parnham 9 April 2004     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Genetic characterization of an α-specific gene responsible for sexual agglutinability in Saccharomyces cerevisiae: mapping and gene dose effect

Summary A recessive ag1 mutation leads to specific defect in sexual agglutinability specifically in cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cry^R 1, closely linked to the mating type locus, was used to select / strains which emerged from / strains by mitotic nonreciprocal recombination, to genetically analyse ag1, since ag1 is expressed only in mating type. The ag1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of cells was shown to be dependent on the dose of the AG1 gene, using / isogenic strains carrying AG1/AG1, AG1/ag1 or ag1/ag1. The sst2-1 mutation did not suppress the ag1 mutation. Based on these results, function of the AG1 gene is discussed.Abbreviations cM centimorgan - FDS first division segregation - NPD nonparental ditype - PD parental ditype - SDS second division segregation - TT tetratype     

The differential effects of IL-1 and TNF-α on proinflammatory cytokine and matrix metalloproteinase expression in human chondrosarcoma cells

Objective and design:Interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis (OA). In the present study, using Affymetrix oligonucleotide array technology and real-time quantitative RT-PCR we have investigated the molecular mechanisms underlying the differential effect of IL-1 and TNF- on gene expression in the human chondrosarcoma cell line, SW1353. Materials and methods:SW1353 cells were stimulated singularly with IL-1, TNF-, Phorbol 12-myristate 13-acetate (PMA), or treated with the combination of cytokine and PMA. Total RNA was collected at multiple time points over a 24-h period followed by biotinylated cRNA target preparation and hybridization onto the Affymetrix HG-U95Av2 array. The differential expression patterns of several cytokine and MMP genes were further confirmed by real time quantitative RT-PCR, Western blot, and ELISA. Results:Our microarray experiments have broadly confirmed previously published data on chondrocyte gene expression regulated by IL-1 and TNF-. The expression pattern of proIL-1, MMP-1, and MMP-13 in chondrocytes is differentially regulated when stimulated with proinflammatory cytokines. IL-1, but not TNF-, can induce IL-6, bone morphogenic protein 2 (BMP-2), and cyclooxygenase (COX-2) expression in SW1353 cells. Additionally, our Western blot results provide the first evidence that IL-1 is produced in the proform in IL-1-activated chondrosarcoma cells and that additional signals are required for its posttranslational processing/activation. Conclusions:IL-1 and TNF- each activate a distinct set of genes in chondrosarcoma cells, and gene expression in these cells is regulated by groups of genes related in part by their function. Chondrocyte IL-1 appears to serve an important role in the pathogenesis OA contributing to joint inflammation and cartilage destruction.Received 15 September 2003; returned for revision 16 October 2003; accepted by J. S. Skotnicki 11 March 2004     

A mutation affecting sexual agglutinability in MATα locus of Saccharomyces cerevisiae

Summary A temperature-sensitive non-agglutinative mutant of Saccharomyces cerevisiae was isolated and characterized. The mutation, sag2, affected sexual agglutination, conjugation and production of -mating pheromone at a restrictive temperature, but not the response to -mating pheromone. Genetic analyses showed that the mutation was recessive and in the MAT locus. The sag2 mutation complemented with mat2 but not with mat1 These results suggest that sag2 is in the MAT1 gene and that at a restrictive temperature the mutation, sag2, inactivated the MAT1 product, a positive regulator of -mating functions. The sag2 mutation is like mat1-5 in its retention of response to -mating pheromone. However, at 25 °C, sag2 cells were competent to mate, whereas mat1-5 cells were not. Hence, sag2 is regarded as a new allele in the MAT1 gene, which we designate mat1-11.     

Immunohistochemical localization of αl-antitrypsin and αl-antichymotrypsin in salivary pleomorphic adenomas

Summary Immunohistochemical identification of l-antitrypsin (l-AT) and l-antichymotrypsin (l-ACh) in pleomorphic adenomas of salivary glands is reported in order to compare their distribution profiles with those of lysozyme and lactoferrin, already described elsewhere.Normal salivary glands indicated positive l-AT staining in ductal segments and had no l-ACh in any glandular cell. Pleomorphic adenomas displayed moderate positivity to l-AT staining in duct-like, tubular and glandular epithelia which was particularly intense in luminal cells. The limited number of tumour cells which showed duct-like structures with a single cellular layer arrangement, displayed the highest staining to l-ACh. Strongly l-AT positive tumour cells located on the inner side of luminal cavities were also markedly positive to l-ACh. Spindle shaped tumour cells existed outside tubular and ductal structures and were negative to l-AT and l-ACh.Distribution of l-AT in salivary glands was similar to that of lysozyme as is usual in ductal segments or their transformed cells, and occurrence of l-ACh localization rather resembled that of lactoferrin, with occurrence in acinar compartments and changed epithelia within acini.The biological role of a specific immunohistochemical distribution of l-AT and l-ACh in pleomorphic adenomas may be associated with a self regulating mechanism which inhibits degradation by tissue proteinases.     

Proteinase — Antiproteinase imbalance in meningitis: Determination of α 1 proteinase inhibitor (α 1PI), elastase — α-1PI complex,and elastase inhibition capacity in cerebrospinal fluid

Summary Mortality and long-term neurologic sequelae are still frequent complications of meningitis despite effective antibiotic treatment. This suggests that pathogen-independent inflammatory mechanisms may play an important role in the course of this illness.Neutrophil granulocytes form the primary immune defense in meningitis. Once activated, these cells release elastase into the cerebrospinal fluid (CSF). Elastase may induce tissue damage if local antiproteinase capacity is low as under normal conditions.To define the relevance of this mechanism we studied 22 patients with meningitis. Concentrations of elastase in complex with the main antiproteinase 1-proteinase inhibitor (elastase- 1PI), 1-proteinase inhibitor ( 1PI), and elastase inhibition capacity (EIC) were measured in CSF of 9 patients with bacterial meningitis (BM), aged 1 month-214 years; 13 patients with non-bacterial meningitis (NBM), aged 1 month–15 years; and 20 patients in whom meningitis was excluded after spinal tap (control group), aged 6 months–15 years. The concentration of elastase- 1PI in the BM group (median 552 g/l) was significantly higher than in either the NBM group (median 30 g/l,p<0.01) or the control group (median 30 g/l,p<0.01). Similarly, the 1PI-concentration in the BM group was significantly higher (median 113 mg/l) than either the NBM group (median 13.7 mg/l,p<0.025) or the control group (median 6.3 mg/l,p<0.001). The concentration of elastase- 1PI shows a significant correlation with the duration of the infectious symptoms before admission to the hospital (r=0.51,p<0.02), but not with the number of neutrophil granulocytesr=0.23, p=0.21).Free elastolytic capacity in CSF could be demonstrated in 4 patients: 1 with BM, 2 with NBM, and 1 with pertussis pneumonia and enzephalitis.The measured insufficiency of the proteinase-antiproteinase system may indicate high-risk patients in need of additional anti-inflammatory therapy, e.g., with corticosteroids, during the initial phase of meningitis.Abbreviations 1PI 1-proteinase inhibitor, 1-antitrypsin - elastase- 1PI complex elastase- 1-proteinase inhibitor complex - EIC elastase inhibition capacity - BM group: bacterial meningitis - NBM group: non-bacterial meningitis - CSF cerebrospinal fluid     

Inhibition of the release of soluble tumor necrosis factor receptors in experimental endotoxemia by an anti-tumor necrosis factor-α antibody

The role of tumor necrosis factor- in the shedding of soluble tumor necrosis factor receptors in endotoxemia was investigated. The appearance of the soluble tumor necrosis factor receptors was assessed in four healthy volunteers following an intravenous injection of tumor necrosis factor- and in eight chimpanzees after intravenous administration of endotoxin in the absence or presence of concurrent treatment with a neutralizing anti-tumor necrosis factor- monoclonal antibody. Injection of tumor necrosis factor- in humans elicited a significant, instantaneous (after 15 min) increase in the plasma concentrations of both types of soluble tumor necrosis factor receptors. In chimpanzees, treatment with the anti-tumor necrosis factor- antibody completely neutralized endotoxin-induced tumor necrosis factor- activity. The release of soluble tumor necrosis factor receptors was strongly (80–90%) inhibited in the presence of the neutralizing antibody. Our results indicate that tumor necrosis factor- is a prime mediator of endotoxin-induced release of its own soluble receptors.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Antitumor effects of liposomal IL1α and TNFα against the pulmonary metastases of the B16F10 murine melanoma in syngeneic mice

Interleukin 1 alpha (IL1) and tumor necrosis factor alpha (TNF) have been successfully incorporated into specific phosphatidylcholine (PC) and phosphatidylserine (PS) multilamellar vesicle (MLV) liposomes by modifying the concentration of calcium ion and pH of the encapsulation buffer. Under these conditions, some of the cytokines may attach to the exterior surface of the MLV and therefore be readily accessible to target cells for receptor binding and signal transduction. These cytokine-associated liposomes are stable for up to 2 weeks in serum-free buffer, and leakage of cytokines into medium containing 10% fetal bovine serum was about 50% at the end of a 3-day incubation period at 37°C. The biological activities mediated by liposomal IL1 and TNF were specific: the stimulation of thymidine uptake in T-helper D10 lymphocytes and the cytolysis of TNF-sensitive L929 target cells could be blocked by specific neutralizing antibodies in a dose-dependent fashion. When administered intravenously into C57BL/6 mice bearing the syngeneic B16F10 murine melanoma cells, dual entrapment of liposomal IL1 and TNF significantly reduced the number of metastatic tumor nodules in the lungs and prolonged the life span of the animals. Thus, liposomal IL1 and TNF displayed significant in vivo antitumor activity against the IL1- and TNF-resistant B16F10 metastatic murine melanoma.     

Immunohistochemical colocalization of growth hormone (GH) and α subunit in human GH secreting pituitary adenomas

Summary This immunohistochemical study disclosed that 9 of 15 GH secreting pituitary adenomas contained subunit positive cells. These cases also contained PRL positive adenoma cells, but LH was negative. Of these 9 cases, 4 cases showed occasional FSH containing cells, 2 of these also contained a few TSH positive cells. By mirror section technique, variable numbers of adenoma cells were found to contain both GH and subunit. Immunoelectron microscopically, both GH and subunit were localized in secretory granules which suggested their co-release from the tumour cells. The presence of GH and subunit in rough endoplasmic reticulum indicated their active production in the tumour. In the normal adult anterior pituitary gland, about 10% of GH cells contain FSH , and LH subunits and had appearances suggesting the co-production of GH and FSH as well as LH. The colocalization of GH and FSH is considered to be associated with the neoplastic transformation GH cells which possess the intrinsic potentiality of differentiation toward subunit. However, the mechanism for the lack or deficiency of subunits in the neoplastic condition remains to be further investigated.     

Expression ofβ1 integrins in non-neoplastic mammary epithelium,fibroadenoma and carcinoma of the breast

1 Integrins were examined immunohisto-chemically in normal and mastopathic mammary glands, 12 benign tumours and 90 carcinomas of the breast using monoclonal antibodies against1 and1 to6 subunits. When compared with epithelial cells of non-neoplastic mammary glands and of benign tumours, carcinoma cells showed considerable quantitative changes in the pattern of2,3 and6 subunit expression. In contrast, the distribution pattern of1,1,4 and5 antigens corresponded to the situation observed in non-neoplastic mammary gland epithelium in most instances. An abnormal expression of2 was found in 71.0% of the carcinomas ranging from a remarkably low number of2-positive tumour cells in 27.5% of the cases to a complete absence of the2 molecule in 43.5% of the carcinomas. Of the carcinomas 39.9% exhibited quantitative changes in3 expression with an abnormally low content of3-positive neoplastic cells in 15.4% and a complete absence of this molecule in 24.5% of the cases. Expression of6 was abnormal in 73.2% of the carcinomas, consisting in a greater number of6-negative tumour cells in 31.9% and in a complete absence of6 in 41.3% of the tumours. The abnormally low expression/absence of2 and3 subunits correlated with oestrogen receptor negativity (P<0.033 andP<0.04, respectively). In addition, abnormally low expression/absence of2 correlated with poor differentiation of the tumours (P< 0.014). The quantitative changes in the expression pattern of1-associated subunits in breast carcinomas may cause a disturbed cell-cell and/or cell-matrix interaction that increases the invasive and migratory property of the tumour cells.     

Expression of transforming growth factor-alpha and its receptor during human liver development and maturation

We investigated the expression of transforming growth factor-alpha (TGF-) and its receptor during human liver development and maturation, using immunohistochemistry. In the fetal liver, strong immunoreactivity for TGF- and its receptor was noted in intrahepatic bile duct cells of various developmental stages; moderate immunoreactivity for TGF- and mild immunoreactivity for TGF- receptor were found in immature hepatocytes. In the postnatal liver, reactivity for TGF- in hepatocytes decreased gradually and was negative or only weakly positive in the adult liver, while reactivity for TGF- receptor in hepatocytes increased gradually and was strongly positive in the adult liver. In contrast, immunoreactivity of TGF- and its receptor in intrahepatic bile duct cells persisted in the postnatal liver and was positive in the adult liver. These data suggest that the system of TGF- and its receptor has an important role in the proliferation and differentiation of intrahepatic biliary cells and hepatocytes in the fetal liver. The decreasing expression of TGF- in hepatocytes in the postnatal liver may indicate that proliferative activity of hepatocytes gradually decreases with liver maturation. The presence of TGF- and its receptor in intrahepatic bile ducts in the postnatal liver suggests that the system of TGF- and TGF- receptor is operative postnatally.     

Temporal and mechanistic effects of heat shock on LPS-mediated degradation of IkappaBalpha in macrophages

Previous studies demonstrated important interactions between the heat shock response and the IB/NF-B pathway when these two pathways are induced sequentially. One such interaction involves the ability of heat shock to inhibit subsequent degradation of IB in response to a proinflammatory signal. Herein we investigated the temporal relationship between recovery from heat shock and inhibition of IB degradation, and the proximal mechanisms by which heat shock inhibits degradation of IB in macrophages. In RAW 264.7 murine macrophages, prior heat shock inhibited LPS-mediated IB degradation up to 4 h after recovery from heat shock, and this effect correlated with inhibition of LPS-mediated activation of NF-B. Beyond these recovery periods, heat shock did not inhibit IB degradation. IB kinase (IKK) assays demonstrated that heat shock inhibited LPS-mediated activation of IKK up to 1 h after recovery from heat shock. Heat shock also increased intracellular phosphatase activity, and inhibition of intracellular phosphatase activity partially reversed the ability of heat shock to inhibit both LPS-mediated degradation of IB and LPS-mediated activation of IKK. These data demonstrate that the ability of heat shock to inhibit degradation of IB is dependent on the recovery period between the heat shock stimulus and the proinflammatory stimulus. The mechanism by which heat shock inhibits degradation of IB involves dual modulation of IKK and intracellular phosphatase activity.     

Differential effect of Fc gamma receptor ligation on LPS-stimulated TNF-alpha secretion by hepatic,splenic, and peritoneal macrophages

Lipopolysaccharide (LPS) increases serum TNF- levels due to TNF- secretion by macrophages. The serum TNF- response to LPS was augmented 10× when FcR ligation was induced by the intravenous injection of Gig-coated erythrocytes (IgG) prior to the administration of LPS. The macrophage population responsible for the augmented TNF- secretion was determined by isolating Kupffer cells, splenic macrophages and peritoneal macrophages from mice that had been given ElgG prior to LPS and determining TNF- secretion ex vivo. The intravenous injection of ElgG augmented LPS-stimulate TNF- secretion by Kupffer cells and splenic macrophages. In contrast, LPS-stimulated TNF- secretion by peritoneal macrophages was not altered by either the intravenous or intraperitoneal injection of ElgG. In vitro phagocytosis of ElgG by isolated peritoneal macrophages also did not augment LPS-stimulated TNF- secretion. These results show that FcR ligation augments LPS-stimulated TNF- secretion by Kupffer cells and splenic macrophages but not by peritoneal macrophages. Thus, the ability of FcR ligation to influence TNF- secretion may be specific to the tissue source of the macrophages.     

Flow Cytometric Analysis of In Vitro Proinflammatory Cytokine Secretion in Peripheral Blood from Multiple Sclerosis Patients

The cytokines, interferon- (IFN-), tumor necrosis factor- (TNF-rpar;, and interleukin-2 (IL-2) are important endogenous proinflammatory proteins and have been linked to disease activity in multiple sclerosis. In this study, we use flow cytometric methodology to compare the secretion of IFN-, IL-2, and TNF- from peripheral blood-derived T cells of multiple sclerosis patients to the secretion in healthy controls. The percentages of IFN-, IL-2, and TNF- secreting cells are not significantly different between multiple sclerosis patients and controls. However, the TNF- secreting CDS cell percentage is correlated with the IFN- and IL-2 secreting CD3 cell percentages in multiple sclerosis patients. In the controls, only the TNF- secreting CD3 cell percentage is correlated with IFN-. These findings show that correlated secretion of cytokines occurs in multiple sclerosis and suggest that concerted intercytokine interactions may play an important role in the disease.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Low In Vitro Production of Interferon-γ and Tumor Necrosis Factor-α in HIV-Seronegative Patients with Pulmonary Disease Caused by Nontuberculous Mycobacteria

We studied 32 HIV-seronegative patients with pulmonary disease caused by nontuberculous mycobacteria (NTM). Immunologic studies included lymphocyte subset analysis by flow cytometry, measurement of interferon- (IFN-) and tumor necrosis factor- (TNF-) production followingin vitro stimulation of diluted whole blood (DWB) and peripheral blood mononuclear cells (PBMC) by phytohemagglutinin (PHA), anti-CD3 as well as purified protein derivative of tuberculin (PPD), and in four cases with different amounts of the very mycobacterium, which caused disease in these patients. Data were compared to those of 30 HIV-seronegative patients with disease byMycobacterium tuberculosis (MTb). Following -CD3-stimulation of PBMC, NTM patients showed lower IFN-(P < 0.00005) and lower TNF-(P < 0.02). For a subgroup of tuberculin skin test-positive NTM patients we found significantly lower PPD-induced IFN- releases in cultured DWB(P < 0.0002) and PBMC(P < 0.0004) compared to MTb patients. Data for PPD-induced TNF- release for this subgroup were also significant(P < 0.001 andP < 0.05, respectively). The four NTM patients with poor PPD-induced IFN- response hardly showed increased cytokine production on stimulation with their specific mycobacterium. The lower production capacity of IFN- and TNF- of NTM patients compared to the MTb patients points to an immunologic imbalance forming the basis for their increased susceptibility to pulmonary infections by nontuberculous mycobacteria.     

A murine model of NKT cell-mediated liver injury induced by alpha-galactosylceramide/d-galactosamine

Natural killer-T (NKT) cells are rich in the liver. However, their involvement in liver injury is not fully understood. We developed here a new murine model of NKT-cell-activation-associated liver injury, and investigated a role of tumor necrosis factor alpha (TNF-) and Fas in pathogenesis. We injected intraperitoneally alpha-galactosylceramide (-GalCer), an NKT-cell stimulant, into d-galactosamine (GalN)-sensitized mice. Survival rate, pathological changes of the liver, and plasma concentrations of cytokines were studied. Alpha-GalCer/GalN administration gave a lethal effect within 7 h, making pathological changes such as massive parenchymal hemorrhage, hepatocyte apoptosis, sinusoidal endothelial cell injury, and close apposition of lymphocytes to apoptotic hepatocytes. Anti-NK1.1 mAb-pretreated mice and V14NKT knock out (KO) mice did not develop liver injury. Tumor necrosis factor-alpha (TNF-) and interferon-gamma (IFN-) were elevated at 4 h in the plasma. These cytokines were produced by hepatic lymphocytes as demonstrated by in vitro stimulation with -GalCer. The lethal effect was suppressed in TNF- KO mice, TNF receptor-1 KO mice, and lpr/lpr (Fas deficient) mice, whereas it was not in IFN- KO mice. These results indicate that the present liver injury is characterized by parenchymal hemorrhage and hepatocyte apoptosis, and mediated by TNF- secretion and direct cytotoxicity of -GalCer-activated NKT cells.