Induction of Tumor Necrosis Factor Alpha (TNFα) and Enhancement of HIV-1 Replication in the J22HL60 Cell Line by Mycoplasma penetrans

Mycoplasma penetrans isolated from clinical specimens of AIDS patients showed potent activity in tumor necrosis factor alpha (TNFα) production in THP-1, U937 and J22HL60 cell lines, and in the enhancement of HIV-1 replication in a dormantly-infected J22HL60 cell line as compared with the activities of other mycoplasmas. Both activities were found in the methanol layer but not in the chloroform layer of the membrane extracted by the Bligh-Dyer method. TNFα production was observed in the peritoneal macrophages from both lipopolysaccharide-responsive and -unresponsive mouse strains, and was not inhibited by polymyxin B. The induction of TNFα production and enhancement of HIV-1 replication were strongly inhibited by Concanavalin A-Sepharose. The inhibitory effect of Concanavalin A-Sepharose was partially prevented by sugars in the order methyl-α-D -mannopyranoside and methyl-α-D -glucopyranoside but not methyl-α-D -galactopyranoside. Anti-human TNFα antibody, however, did not reduce the activity of the methanol layer to enhance HIV-1 replication, suggesting that the methanol layer could enhance HIV-1 replication directly. These results suggest that the carbohydrate derived from M. penetrans might be responsible for the progression of HIV-1 infection.     

Synovial Mononuclear Cells Consist with T Cells Which Produce High Levels of Tumor Necrosis Factor α

To determine whether synovial mononuclear cells include a population of tumor necrosis factor α-produeing T cells, we measured tumor necrosis α levels in culture supernatants of synovial mononuclear cells by ELISA and analyzed tumor necrosis α mRNA-positive cell frequencies. There were no significant differences in the spontaneous levels of TNF α between synovial mononuclear cells and peripheral mononuclear cells. The frequency of tumor necrosis factor α mRNA-positive cells in synovial mononuclear cells was higher than that of peripheral mononuclear cells. When stimulated with a superantigen, mononuclear cells from the synovial fluid of rheumatoid arthritis patients showed higher levels of tumor necrosis factor α production (1,035 ± 817 pg/ml) than did mononuclear cells from their peripheral blood (236 ± 180 pg/ml). In addition, we observed that a few T cell clones were resistant to superantigenic restimulation in vitro. We conclude that when these types of T cells persist in the synovium, they play a role in the development of rheumatoid arthritis via a mechanism involving tumor necrosis factor α production.     

Endogenous Tumor Necrosis Factor (TNF) α Mediates Neutrophil Accumulation at the Mid-Phase of a Murine Model of Pseudomonas aeruginosa Pneumonia

To determine the role of endogenous tumor necrosis factor (TNF) α on neutrophil influx into the lungs in acute Pseudomonas aeruginosa pneumonia, we evaluated TNF α activity, inflammatory cell response and neutrophil chemotactic activity in the bronchoalveolar lavage fluids (BALFs) of P. aeruginosa-infected mice. In the case of fatal pneumonia, the TNF α activity in the BALFs appeared within 3 hr, peaked at 6–12 hr and attenuated within 24 hr after intratracheal challenging, while no TNF α activity was detected in the plasma. The elevation of TNF α activity in the BALFs was closely associated with neutrophil accumulation. Mirroring the TNF α activity response and the influx of neutrophils into the murine airway, the number of neutrophils in the BALFs increased within 3 hr, peaked at 6–12 hr and remained elevated up to 24 hr after challenging. Neutralization of the TNF α activity in the BALFs with anti-murine TNF antiserum decreased the level of neutrophil migration by BALF 45.0–49.7% at 6 hr and 49.3–54.2% at 12 hr, while the neutralizing antiserum had no effect on the level of neutrophil migration by BALFs at 3 and 24 hr. Furthermore, the intravenous administration of anti-murine TNF antiserum 2 hr before challenging significantly inhibited neutrophil migration into the lungs of mice with sublethal pneumonia (P < 0.05; compared with mice receiving pre-immune serum). These data suggest that intra-alveolar TNF α plays an important role in causing lung neutrophil accumulation at the mid-phase of murine P. aeruginosa pneumonia.     

Production of Tumor Necrosis Factor-α in Granulocytopenic Mice with Pulmonary Candidiasis and Its Modification with Granulocyte Colony-Stimulating Factor

In the present study, a lethal model of pulmonary candidiasis was established using granulocytopenic mice with cyclophosphamide. These mice started to die 1 day after infection and had all died within the next 48 hr. The counts of live C. albicans in the lung gradually increased with time, while the organisms were quickly eliminated in the normal mice. From the histology and measurements on bronchoalveolar lavage fluid (BALF), polymorphonuclear cells (PMN) response was almost zero up to 24 hr, and then a weak but significant response was observed at 48 hr, while a marked accumulation of PMN was detected from as early as 6 hr in normal mice. In contrast, macrophages had accumulated in BALF by 48 hr in granulocytopenic mice, but not in normal mice. Both in serum and BALF, a considerable level of tumor necrosis factor-α (TNF-α) was detected from 6 hr, peaking at 24 to 48 hr, while in normal mice the quantity was under the detection limit in serum and very low in BALF. The effects of administering granulocyte colony-stimulating factor (G-CSF) on these parameters were next examined. G-CSF significantly prolonged the survival time of granulocytopenic mice, promoted the clearance of organisms through increasing the counts of PMN in the lung, and strongly inhibited the production of TNF-α both in BALF and serum. These results suggest that this cytokine does not protect them, but plays some role in their death due to candidial infection in granulocytopenic mice.     

PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

Sympathetic Regulation of Glucose Uptake by the α1‐Adrenoceptor in Human Obesity

Objective: To investigate the involvement of α₁‐adrenoceptors in the sympathetic regulation of glucose uptake in human adipocytes. Research Methods and Procedures: Twenty‐four severely obese subjects participated in this study. The microdialysis technique was used to determine interstitial glucose concentration after stimulation of abdominal subcutaneous adipose tissue with the α₁‐agonist norfenefrine, the α_{1, 2}β‐agonist norepinephrine, and both agents in combination with the α₁‐antagonist urapidil. The effect of β‐adrenoceptor stimulation was assessed by orciprenaline. Changes in local blood flow were determined using the ethanol escape technique. Results: Both norfenefrine and norepinephrine induced a concentration‐dependent decrease of interstitial glucose concentration, with a greater decrease observed with norepinephrine. Preperfusion of adipose tissue with urapidil inhibited glucose decrease. The inhibition was overcome with high concentrations of norfenefrine and norepinephrine, respectively. Both adrenergic agents induced tachyphylaxia. Urapidil enhanced extracellular glucose level at high concentration. Blood flow decreased in the presence of norfenefrine and norepinephrine but increased in response to urapidil. The accelerated blood flow due to urapidil was counteracted by norepinephrine and norfenefrine. Orciprenaline decreased interstitial glucose concentration and increased nutritive blood flow. The observed changes in blood flow induced by adrenergic agents were not related to glucose uptake. Discussion: The stimulatory effect of the sympathetic nerves on glucose uptake in subcutaneous adipose tissue appears to be mediated by the α₁‐adrenoceptor. Norepinephrine enhances glucose entry into adipocytes independently of insulin action. In obese subjects with insulin resistance, the α₁‐adrenergic receptor may provide an important alternative pathway for glucose uptake.     

Effect of lactoferrin on the production of tumor necrosis factor‐α and nitric oxide

One of the biological functions of lactoferrin is the modulation of the host defense systems, including cytokine production and immune responses. We have tested the effect of lactoferrin on the productions of tumor necrosis factor‐α and nitric oxide in some cells. Lactoferrin itself did not induce either tumor necrosis factor‐α production or nitric oxide production, but lipopolysaccharide‐stimulated tumor necrosis factor‐α production of macrophages and monocytes were inhibited by lactoferrin treatment combined with stimulant. The induction of nitric oxide synthesis in stimulated macrophages and vascular smooth muscle cells was not affected by the lactoferrin. J. Cell. Biochem. 76:30–36, 1999. © 1999 Wiley‐Liss, Inc.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

Glycosylation of Ceramide Potentiates Cellular Resistance to Tumor Necrosis Factor-α-Induced Apoptosis

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor necrosis factor-α (TNF-α)-induced apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. By introduction of the GCS gene, cytotoxic resistance to TNF-α has been conferred in human breast cancer cells. MCF-7/GCS-transfected cells expressed 4.1-fold higher levels of GCS activity and exhibited a 15-fold (P < 0.0005) greater EC₅₀ for TNF-α, compared with the parental MCF-7 cell line. DNA fragmentation and DNA synthesis studies showed that TNF-α had little influence on the induction of apoptosis or on growth arrest in MCF-7/GCS cells, compared to MCF-7 cells. These studies reveal that TNF-α resistance in MCF-7/GCS cells is closely related to ceramide hyperglycosylation, a hallmark of this transfected cell line, and resistance was not aligned with changes in TNF receptor 1 expression. This work demonstrates that GCS, which catalyzes ceramide glycosylation, potentiates cytotoxic resistance to TNF-α.     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

TNF‐α has both stimulatory and inhibitory effects on mouse monocyte‐derived osteoclastogenesis

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31^hiLy‐6C^?), myeloid blasts (CD31⁺Ly‐6C⁺), and monocytes (CD31^?Ly‐6C^hi) were sorted from mouse bone marrow using flow cytometry and cultured with M‐CSF and RANKL, with or without TNF‐α. Surprisingly, TNF‐α prevented the differentiation of TRAcP⁺ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes’ osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes’ osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone.

     

IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis

Inflammatory cytokines are closely related to pigmentary changes. In this study, the effects of IFN‐γ on melanogenesis were investigated. IFN‐γ inhibits basal and α‐MSH‐induced melanogenesis in B16 melanoma cells and normal human melanocytes. MITF mRNA and protein expressions were significantly inhibited in response to IFN‐γ. IFN‐γ inhibited CREB binding to the MITF promoter but did not affect CREB phosphorylation. Instead, IFN‐γ inhibited the association of CBP and CREB through the increased association between CREB binding protein (CBP) and STAT1. These findings suggest that IFN‐γ inhibits both basal and α‐MSH‐induced melanogenesis by inhibiting MITF expression. The inhibitory action of IFN‐γ in α‐MSH‐induced melanogenesis is likely to be associated with the sequestration of CBP via the association between CBP and STAT1. These data suggest that IFN‐γ plays a role in controlling inflammation‐ or UV‐induced pigmentary changes.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Granulocyte-Macrophage Colony-Stimulating Factor and Tumor Necrosis Factor Alpha in Patients with Human Immunodeficiency Virus (HIV) Type 1 Infection

Variations in cytokine production in patients with human immunodeficiency virus (HIV) infection could be involved in the physiopathology and in the progression of the disease. Therefore we studied the level of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor α (TNFα) produced in patients with HIV infection at stage II (asymptomatic seropositives) and stage IV (AIDS) of the CDC classification, by using an enzyme amplified sensitivity immunoassay. We measured the level of GM-CSF and TNFα in supernatant of phytohemagglutinin-activated peripheral blood mononuclear cells from patients and healthy individuals. In one out of 10 stage II patients and 4 out of 14 stage IV patients, we obtained higher levels of GM-CSF than the mean + 2 S.D. of controls, but in 3 stage IV patients with very low CD4+ T lymphocyte counts (< 50/mm^–3) compared to other patients, the GM-CSF values were very low. High levels of TNFα were detected in 3 out of 10 stage II and 6 out of 11 stage IV patients. The high values of TNFα were associated with high values of GM-CSF in stage II and in most of AIDS patients except those with very low CD4⁺ T cell counts, who produced low levels of GM-CSF. Plasma levels of cytokines were evaluated in 10 stage II, 22 stage IV patients and 20 controls. Increased levels of GM-CSF (more than 9 pg/ml) were observed in the plasma from 8 out of 10 stage II patients and 17 out of 22 stage IV patients. The tendency that increased levels of GM-CSF were associated with increased levels of TNFα was observed in plasma from stage IV patients. We report a disarray of GM-CSF production in patients with HIV infection that could be involved in clinical manifestations and progression of the disease.