SHP-1 inhibits LPS-mediated TNF and iNOS production in murine macrophages

Several lines of evidence have suggested that protein tyrosine phosphatases, including CD45 and SHP-1, regulate macrophage activation. Macrophages from mice lacking SHP-1 (motheaten mice) are hyper-responsive to many stimuli, suggesting that SHP-1 may negatively regulate macrophage activation. Herein we report that the repressible/inducible over-expression of wild-type SHP-1 in a subclone of RAW 264.7 macrophages (RAW-TT10 cells) inhibited both TNF secretion and iNOS protein accumulation in response to stimulation with lipopolysaccharide (LPS) and recombinant murine interferon-gamma and led to diminished LPS-mediated tyrosine phosphorylation of vav1. In contrast, expression of a truncated SHP-1 construct previously shown to interfere with endogenous SHP-1 function modestly augmented LPS-mediated TNF and iNOS production and did not inhibit vav1 tyrosine phosphorylation. Taken together, these data provide the first direct evidence that SHP-1 inhibits macrophage activation by LPS and suggest that this effect may be mediated in part by dephosphorylation of vav1.     

Modulation of allergic inflammation in mice deficient in TNF receptors

Tumor necrosis factor-alpha (TNF) is implicated as an important proinflammatory cytokine in asthma. We evaluated mice deficient in TNF receptor 1 (TNFR1) and TNFR2 [TNFR(-/-) mice] in a murine model of allergic inflammation and found that TNFR(-/-) mice had comparable or accentuated responses compared with wild-type [TNFR(+/+)] mice. The responses were consistent among multiple end points. Airway responsiveness after methacholine challenge and bronchoalveolar lavage (BAL) fluid leukocyte and eosinophil numbers in TNFR(-/-) mice were equivalent or greater than those observed in TNFR(+/+) mice. Likewise, serum and BAL fluid IgE; lung interleukin (IL)-2, IL-4, and IL-5 levels; and lung histological lesion scores were comparable or greater in TNFR(-/-) mice compared with those in TNFR(+/+) mice. TNFR(+/+) mice chronically treated with anti-murine TNF antibody had BAL fluid leukocyte numbers and lung lesion scores comparable to control antibody-treated mice. These results suggest that, by itself, TNF does not have a critical proinflammatory role in the development of allergic inflammation in this mouse model and that the production of other cytokines associated with allergic disease may compensate for the loss of TNF bioactivity in the TNFR(-/-) mouse.     

Prostaglandin E2 inhibits TNF production in murine bone marrow-derived dendritic cells

Exposure to pathogens induces dendritic cells to release inflammatory cytokines and chemokines. The inflammatory response is controlled by endogenous agents such as anti-inflammatory cytokines, glucocorticoids, anti-inflammatory neuropeptides, and lipid mediators. This study is the first report on the inhibition by prostaglandin E2 (PGE2) of TNF release from bone marrow-derived dendritic cells stimulated with lipopolysaccharide (LPS), a TLR4 ligand, or peptidoglycan, a TLR2 ligand. The inhibition of TNF occurs at both mRNA and protein level. The inhibitory effect of PGE2 is mediated by the EP2 and EP4 receptors, and involves both PKA signaling and mediation by DC-derived IL-10. Intraperitoneal administration of PGE2 together with LPS results in a reduction in serum TNF and intracellular TNF in peritoneal exudate cells, compared to LPS alone. In addition, administration of PGE2 in vivo reduces the numbers of CD11c+ DCc that accumulate in the peritoneal cavity in response to LPS. The various implications of the PGE2-induced reduction in TNF are discussed.     

Chromosomal localization and molecular organization of the human genomic fragment containing the TNF/LT locus in transgenic mice

The molecular organization, copy number, and chromosome location of the human TNF/LT transgenes were studied in the genomes of two transgenic mouse strains. One strain proved to carry two transgene copies arranged head-to-tail and detected on chromosome 8 by karyotyping. The other strain had one transgene copy observed on chromosome 5. The strains provide a model for studying the physiological functions of the tumor necrosis factor and lymphotoxin.     

Sources of alveolar soluble TNF receptors during acute lung injury of different etiologies

Elevated soluble tumor necrosis factor-α receptor (sTNFR) levels in bronchoalveolar lavage fluid (BALF) are associated with poor patient outcome in acute lung injury (ALI). The mechanisms underlying these increases are unknown, but it is possible that pulmonary inflammation and increased alveolar epithelial permeability may individually contribute. We investigated mechanisms of elevated BALF sTNFRs in two in vivo mouse models of ALI. Anesthetized mice were challenged with intratracheal lipopolysaccharide or subjected to injurious mechanical ventilation. Lipopolysaccharide instillation produced acute intra-alveolar inflammation, but minimal alveolar epithelial permeability changes, with increased BALF sTNFR p75, but not p55. Increased p75 levels were markedly attenuated by alveolar macrophage depletion. In contrast, injurious ventilation induced substantial alveolar epithelial permeability, with increased BALF p75 and p55, which strongly correlated with total protein. BALF sTNFRs were not increased in isolated buffer-perfused lungs (devoid of circulating sTNFRs) subjected to injurious ventilation. These results suggest that lipopolysaccharide-induced intra-alveolar inflammation upregulates alveolar macrophage-mediated production of sTNFR p75, whereas enhanced alveolar epithelial permeability following mechanical ventilation leads to increased BALF p75 and p55 via plasma leakage. These data provide new insights into differential regulation of intra-alveolar sTNFR levels during ALI and may suggest sTNFRs as potential markers for evaluating the pathophysiology of ALI.     

Tissue specific distribution, clearance and vascular effects of endothelin in the pig

Endothelin-like immunoreactivity (-LI) was detected in the kidney, spleen, skeletal muscle, lung and in plasma of the pig. The highest tissue levels were found in the lung (1.5 +/- 0.07 pmol/g) and the lowest in the muscle (0.08 +/- 0.02 pmol/g). I.v. infusion of endothelin (20 pmol/kg/min for 20 min) increased plasma endothelin-LI from 18 +/- 3 to 628 +/- 109 pM and renal, splenic and femoral vascular resistance by 570, 430 and 50%, respectively. There was a close correlation between the increase in plasma endothelin-LI and the vasoconstrictor effects. During the infusion 92, 82 and 76% of arterial endothelin-LI was removed by the kidney, spleen and hindlimb, respectively, whereas there was no clearance over the lung or degradation in plasma. After the infusion, arterial endothelin-LI decreased with a half life of 77 s, while the renal and splenic vasoconstriction persisted for 60 min. Characterization by reversed phase HPLC revealed that endothelin-LI in plasma and tissue co-eluted with synthetic endothelin. It is concluded that endothelin has tissue specific distribution, clearance and vascular effects in the pig.     

广东汉族人TNF多态性

采用PCR－RFLP技术对103例广东汉族人TNF多态性进行了检测,结果是：TNFβ^*1纯合子表型频率为0．2524,TNFβ^*2纯合子表型频率为0．2913,TNFβ^*1／TNFβ^*2杂合子表型频率为0．4563,TNFβ^*1基因频率0．4806,TNFβ^*1基因频率0．5194。本结果与其他人群的结果进行了比较。     

鲢（Hypophthalmichthys molitrix）肿瘤坏死因子基因的克隆与表达分析

肿瘤坏死因子(TNF)是一种炎症细胞因子,在非特异性免疫系统中发挥着重要作用.TNF由单核细胞或巨噬细胞产生,能直接造成肿瘤细胞的死亡,并参与机体炎症和免疫应答的调节.用RACE(rapid amplification of cDNAends)-PCR方法,从鲢总RNA反转录产物中获得了1 254 bpTNF cDNA全序列.该序列包含394 bp的5'端非编码区,398 bp的3'端非编码区和462 bp的开放阅读框.鲢TNF的开放阅读框编码239个氨基酸,其中包含构成一对二硫键的2个保守半胱氨酸.同时利用RT-PCR技术,对该基因在鲢鱼体内不同组织之间的表达差异进行了分析研究,结果表明鲢TNF mRNA主要在脑、鳃和头肾中表达,肠和脾中有少量表达,而在肝中几乎没有表达.     

Moraxella catarrhalis activates murine macrophages through multiple toll like receptors and has reduced clearance in lungs from TLR4 mutant mice

Moraxella catarrhalis is a gram negative bacterium and a leading causative agent of otitis media (OM) in children. Several recent reports have provided strong evidence for an association between toll like receptors and OM. It has been found that both Streptococcus pneumoniae and nontypeable Haemophilus influenzae activate host protective immune responses through toll like receptors (TLRs), however, the precise mechanism by which Moraxella catarrhalis initiates the host immune response is currently unknown. In this report, using murine macrophages generated from a series of knock-out mice, we have demonstrated that M. catarrhalis lipooligosaccharide (LOS) and either heat killed or live bacteria are recognized by one or more TLRs. LOS activates the host immune response through a membrane bound CD14-TLR4 complex, while both heat killed and live M.cat require recognition by multiple toll like receptors such as TLR2, TLR4 and TLR9 without the requirement of CD14. We have also shown that M.cat stimuli are capable of triggering the host innate immune response by both MyD88- and TRIF- dependent signaling pathways. We further showed that M.cat induced activation of mitogen activated protein kinase (MAPK) is essential in order to achieve optimal secretion of pro-inflammatory cytokine TNF-α. We finally showed that TLR4 mutant C3H/HeJ mice produce significantly lower levels of pro-inflammatory cytokines TNF-α and IL-6 in vivo, An increased bacterial loads at 12 and 24 hours (P<0.001) in their lungs upon challenge with live M.cat in an aerosol chamber compared to wild-type (WT) control mice. These data suggest that TLRs are crucial for an effective innate immune response induced by M.cat. The results of these studies contribute to an increased understanding of molecular mechanism and possible novel treatment strategies for diseases caused by M.cat by specifically targeting TLRs and their signaling pathways.     

Tissue distribution and half-life of 125I-endothelin in the rat: importance of pulmonary clearance

Plasma half-life and organ uptake of endothelin 1 (ET-1) was studied in anaesthetized Wistar rats administering a bolus of 125I-labeled ET-1 into jugular vein, with and without a preceding dose of 1-80 ng of unlabeled ET-1, via the same route. Plasma half-life of intact 125I-ET-1 was 40 sec and that of total radioactivity 68 sec. Immunoreactivity of circulating 125I-ET was rapidly reduced, by 23% at 30 sec and by 43% at 360 sec, respectively. High power liquid chromatography of plasma extracts revealed a corresponding decrease of intact 125I-ET-1 peak with time. There was a predominant uptake of 125I-ET-1 in the lungs, 82%, and lesser by kidneys, 10%, heart 3.6%, liver, 2.7%, and spleen, 1.5%, respectively. Pretreatment with unlabeled ET-1 caused a dose-dependent shift of uptake from lungs to kidneys. We conclude that circulating ET-1 is rapidly cleared, primarily by the lungs, which may play an important role in protecting the systemic circulation from the vasoconstricting actions of ET-1.     

Structure-Function Relationship of Tumor Necrosis Factor (TNF) and Its Receptor Interaction Based on 3D Structural Analysis of a Fully Active TNFR1-Selective TNF Mutant

Tumor necrosis factor (TNF) is an important cytokine that suppresses carcinogenesis and excludes infectious pathogens to maintain homeostasis. TNF activates its two receptors [TNF receptor (TNFR) 1 and TNFR2], but the contribution of each receptor to various host defense functions and immunologic surveillance is not yet clear. Here, we used phage display techniques to generate receptor-selective TNF mutants that activate only one TNFR. These TNF mutants will be useful in the functional analysis of TNFR.Six amino acids in the receptor binding interface (near TNF residues 30, 80, and 140) were randomly mutated by polymerase chain reaction. Two phage libraries comprising over 5 million TNF mutants were constructed. By selecting the mutants without affinity for TNFR1 or TNFR2, we successfully isolated 4 TNFR2-selective candidates and 16 TNFR1-selective candidates, respectively. The TNFR1-selective candidates were highly mutated near residue 30, whereas TNFR2-selective candidates were highly mutated near residue 140, although both had conserved sequences near residues 140 and 30, respectively. This finding suggested that the phage display technique was suitable for identifying important regions for the TNF interaction with TNFR1 and TNFR2. Purified clone R1-6, a TNFR1-selective candidate, remained fully bioactive and had full affinity for TNFR1 without activating TNFR2, indicating the usefulness of the R1-6 TNF mutant in analyzing TNFR1 receptor function.To further elucidate the receptor selectivity of R1-6, we examined the structure of R1-6 by X-ray crystallography. The results suggested that R31A and R32G mutations strongly influenced electrostatic interaction with TNFR2, and that L29K mutation contributed to the binding of R1-6 to TNFR1. This phage display technique can be used to efficiently construct functional mutants for analysis of the TNF structure-function relationship, which might facilitate in silico drug design based on receptor selectivity.     

Augmentation and Suppression of TNF Release from Macrophages by Inflammatory Polymorphonuclear Leukocytes

It is known that polymorphonuclear leukocytes (PMNs) emerge first in local inflammatory sites, and then they are followed and scavenged by macrophages. We focused on the effect of PMN on tumor necrosis factor (TNF) release activity of macrophages, which is viewed as a possible indicator of the status of macrophage activation. One day after macrophages were cultured with fresh, intact murine PMNs which were induced with sodium casein, the release of TNF triggered by lipopolysaccharide (LPS) was augmented by low concentrations of PMNs, but suppressed by their high concentrations. When the PMN samples were fractionated into soluble and insoluble fractions, the augmenting and suppressing activity was partitioned; the relatively high concentrations of soluble fraction showed the suppressive effect whereas the insoluble fraction in lower concentrations showed augmentation. The suppressive activity was stable at 100 C, but the filtrates of the soluble fraction with membranes having cut-offs of 5,000 or 10,000 were not suppressive at all, suggesting the suppression is not due to low molecular compounds. It was also suggested that the suppressive effect for TNF release was not due to contaminating LPS or transforming growth factor-β. Inflammatory processes may thus be positively and negatively controlled by a quantitative factor of initial PMN populations by regulating the TNF release activity of the subsequent macrophages.     

Tissue distribution of the death ligand TRAIL and its receptors.

Recombinant human (rh) TNF-related apoptosis-inducing ligand (TRAIL) harbors potential as an anticancer agent. RhTRAIL induces apoptosis via the TRAIL receptors TRAIL-R1 and TRAIL-R2 in tumors and is non-toxic to nonhuman primates. Because limited data are available about TRAIL receptor distribution, we performed an immunohistochemical (IHC) analysis of the expression of TRAIL-R1, TRAIL-R2, the anti-apoptotic TRAIL receptor TRAIL-R3, and TRAIL in normal human and chimpanzee tissues. In humans, hepatocytes stained positive for TRAIL and TRAIL receptors and bile duct epithelium for TRAIL, TRAIL-R1, and TRAIL-R3. In brains, neurons expressed TRAIL-R1, TRAIL-R2, TRAIL-R3 but no TRAIL. In kidneys, TRAIL-R3 was negative, tubuli contorti expressed TRAIL-R1, TRAIL-R2, and TRAIL, and cells in Henle's loop expressed only TRAIL-R2. Heart myocytes showed positivity for all proteins studied. In colon, TRAIL-R1, TRAIL-R2, and TRAIL were present. Germ and Leydig cells were positive for all proteins studied. Endothelium in liver, heart, kidney, and testis lacked TRAIL-R1 and TRAIL-R2. In alveolar septa and bronchial epithelium TRAIL-R2 was expressed, brain vascular endothelium expressed TRAIL-R2 and TRAIL-R3, and in heart vascular endothelium only TRAIL-R3 was present. Only a few differences were observed between human and chimpanzee liver, brain, and kidney. In contrast to human, chimpanzee bile duct epithelium lacked TRAIL, TRAIL-R1, and TRAIL-R3, lung and colon showed no TRAIL or its receptors, TRAIL-R3 was absent in germ and Leydig cells, and vascular endothelium showed only TRAIL-R2 expression in the brain. In conclusion, comparable expression of TRAIL and TRAIL receptors was observed in human and chimpanzee tissues. Lack of liver toxicity in chimpanzees after rhTRAIL administration despite TRAIL-R1 and TRAIL-R2 expression is reassuring for rhTRAIL application in humans.     

Tissue distribution of insulin-like growth factor receptors in the turkey.

1. The distribution and relative insulin-like growth factor (IGF) binding capacities of membranes derived from 14 tissues of the turkey were examined. 2. Affinity cross-linking analyses using [125I]IGF-I and [125I]IGF-II with membranes derived from the liver, pectoralis major muscle, gizzard, heart and brain indicated that both IGFs interact with only type-I IGF receptors on these tissues. 3. There was no evidence for the existence of a type-II IGF receptor in any tissue. 4. Although considerable variation was detected in the molecular weights of the IGF receptor alpha subunits between tissues (112.2-132.9 kDa), these differences did not appear to influence receptor-ligand affinities.     

The significance and occurrence of TNF receptor polymorphisms in the Saudi population

Background and objective: On the basis that the inflammatory effects of TNF (tumour necrosis factor) are predominantly mediated through interaction with the TNF receptor-1 (TNFRSF1A), the current study was designed to establish the prevalence of the mutations, R92Q and P46L TNFRSF1A polymorphisms both in the general healthy Saudi population, and in Saudi patients carrying inflammatory diseases such as atherosclerosis or rheumatoid arthritis. We felt it important to report the frequency of the mutations, R92Q and P46L TNFRSF1A polymorphisms in healthy Saudi individuals, and those with inflammatory conditions, as well as to describe the pattern of immunological factors in individuals expressing R92Q or P46L TNFRSF1A. Patients and methods: We collected in PAX gene blood RNA tubes (for RT-PCR and sequencing) 500 blood samples from normal healthy individuals from the West and Center of Saudi Arabia, as well as 100 from patients with atherosclerosis, and 100 patients diagnosed with rheumatoid arthritis. All were screened for the levels of soluble TNF, C-reactive protein (CRP), interleukin6 (IL-6) and sTNFR1. In addition, they were screened for R92Q and P46L TNFRSF1A by RT-PCR. Moreover, phenotype and expression of peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry (FACS). Results: Across 500 normal individuals, 8 (1.6%) expressed both R92Q and P46L mutations. By contrast, of the 100 patients in our study with atherosclerosis, 34% expressed both the R92Q and P46L mutations, whilst 42% of patients with rheumatoid arthritis expressed both mutations R92Q and P46L. No significant differences were observed between cell markers of normal individuals (CD3, 4, 8, 16, 56, 19, 25, ICAM-1, VLA-4 & l-selectin) and patients with atherosclerosis. There were significantly high values of cell markers in patients with rheumatoid arthritis compared with normal individuals both in terms of percentage and absolute counts (p < 0.05). Soluble IL-6 and sTNFR1 showed significant decreases in atherosclerosis and rheumatoid arthritis when compared with controls (p < 0.05). In addition, CRP and sTNF showed significant increases in the atherosclerosis and rheumatoid arthritis groups when compared to controls (p < 0.05). Conclusion: Our findings reasonably anticipate the presence of TRAPS disease (low penetrance mutations) amongst the Saudi population although further studies are needed to confirm these results.     

Regulation of free and bound leptin and soluble leptin receptors during inflammation in mice

Leptin, an appetite-regulating hormone/cytokine, circulates both free and bound to soluble leptin receptors (s-leptin R). An electrochemiluminescence (ECL) assay for the quantitative measurement of murine s-leptin R was developed. The absence of s-leptin R immunoreactivity in the serum of db(pas)/db(pas)mice demonstrated the specificity of the assay, which detected s-leptin R both in the free form and complexed with leptin. The distribution of free vs bound leptin and the regulation of s-leptin R were evaluated in mice following administration of the pro-inflammatory stimuli endotoxin (100 microg/mouse) and turpentine (100 microl/mouse). Both endotoxin and turpentine significantly increased serum leptin and s-leptin R levels compared to control mice. The distribution of free vs bound leptin was not altered by administration of endotoxin or turpentine. In fact, approximately 50% of total leptin was present in the free form in either control, endotoxin- or turpentine-injected mice. On the contrary, during the hyperleptinemia of pregnancy, only 10% of total leptin was present in the free form. We conclude that inflammation leads to the increase of both bound and free leptin. Therefore, the total amount of bioactive leptin is increased during acute inflammation, suggesting that leptin participates in the host response to inflammation.