scholarly journals cis-acting sequences required for class II gene regulation by interferon gamma and tumor necrosis factor alpha in a murine macrophage cell line.

1990 ◽  
Vol 171 (4) ◽  
pp. 1283-1299 ◽  
Author(s):  
Y R Freund ◽  
R L Dedrick ◽  
P P Jones
Keyword(s):  
Cell Line ◽  
Class Ii ◽  
Tnf Alpha ◽  
Sequence Element ◽  
Ifn Gamma ◽  
Enhancer Element ◽  
T Box ◽  
Hla Dq ◽  
Mouse Class ◽  
Class Ii Alpha

In this report, we have demonstrated that IFN-gamma and TNF-alpha increase expression of both the I-A and I-E region gene products on the surface of the myelomonocytic cell line WEHI-3, and that they mediate this increase via an increase in A alpha transcription. Constructs containing 5' deletion mutations of the A alpha promoter attached to the bacterial chloramphenicol acetyl transferase gene were used to delineate the minimum 5' flanking sequences required for promoter activity, and for inducibility by IFN-gamma and TNF-alpha. Approximately 115 bp of 5' sequences are required for minimum induction by IFN-gamma or TNF-alpha when the cytokines are present separately. This includes the three conserved promoter elements, the X, Y, and H boxes. Nested linker-scanner mutations demonstrated that additional regions were also critical for optimal induction by IFN-gamma or TNF-alpha. These include the kappa B-like enhancer and a TNF-alpha-specific sequence that we have tentatively called the T box. The T box sequence was also found in the promoter regions of the human HLA-DQ alpha and rat RT1.B alpha genes. Although the entire T box sequence element was not found in the other mouse class II genes, all class II alpha genes contained the SV40 core enhancer element in the regions included by the T box. Mouse class II beta genes appear to contain neither the T box nor the core enhancer element in this region, suggesting differential regulation of class II alpha and beta genes by TNF-alpha.

scholarly journals Cytokines in chronic inflammatory arthritis. IV. Granulocyte/macrophage colony-stimulating factor-mediated induction of class II MHC antigen on human monocytes: a possible role in rheumatoid arthritis.

1989 ◽  
Vol 170 (3) ◽  
pp. 865-875 ◽  
Author(s):  
J M Alvaro-Gracia ◽  
N J Zvaifler ◽  
G S Firestein
Keyword(s):  
Rheumatoid Arthritis ◽  
Class Ii ◽  
Tnf Alpha ◽  
Blood Monocytes ◽  
Ifn Gamma ◽  
Normal Peripheral Blood ◽  
Gm Csf ◽  
Hla Dr ◽  
Class Ii Mhc ◽  
Hla Dq

Granulocyte/macrophage CSF (GM-CSF) has recently been identified in rheumatoid arthritis (RA) synovial effusions. To study a potential role for GM-CSF and other cytokines on the induction of HLA-DR expression on monocytes and synovial macrophages, we analyzed the relative ability of recombinant human cytokines to induce the surface expression of class II MHC antigens on normal peripheral blood monocytes by FACS analysis. GM-CSF (800 U/ml) (mean fluorescence channel 2.54 +/- 0.33 times the control, p less than 0.001) and IFN-gamma (100 U/ml) (5.14 +/- 0.60, p less than 0.001) were the most potent inducers of HLA-DR. TNF-alpha and IL-4 also increased HLA-DR expression, although to a lesser degree [1.31 +/- 0.06 (p less than 0.02) and 1.20 +/- 0.03 (p less than 0.01), respectively]. IL-1 (40 U/ml), IL-2 (10 ng/ml), IL-3 (50 U/ml), IL-6 (100 U/ml), and CSF-1 (1,000 U/ml) did not affect surface HLA-DR density. GM-CSF also increased HLA-DR mRNA expression and surface HLA-DQ expression, but decreased CD14 (a monocyte/macrophage antigen) expression. The effect of GM-CSF on HLA-DR was not mediated by the generation of IFN-gamma in vitro because it was not blocked by anti-IFN-gamma mAb. GM-CSF was additive with IL-4 and low amounts (less than 3 U/ml) of IFN-gamma and synergistic with TNF-alpha. Because we have recently reported that supernatants of cultured RA synovial cells produce a non-IFN-gamma factor that induces HLA-DR on monocytes, we then attempted to neutralize this factor with specific anti-GM-CSF mAb. Four separate synovial tissue supernatants were studied, and the antibody neutralized the HLA-DR-inducing factor in each (p less than 0.01).

scholarly journals Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes.

1991 ◽  
Vol 174 (5) ◽  
pp. 1209-1220 ◽  
Author(s):  
R de Waal Malefyt ◽  
J Abrams ◽  
B Bennett ◽  
C G Figdor ◽  
J E de Vries
Keyword(s):  
Class Ii ◽  
Interleukin 10 ◽  
Tnf Alpha ◽  
Human Monocytes ◽  
Colony Stimulating Factor ◽  
Ifn Gamma ◽  
Gm Csf ◽  
Cytokine Synthesis ◽  
Activated Monocytes ◽  
Stimulating Factor

In the present study we demonstrate that human monocytes activated by lipopolysaccharides (LPS) were able to produce high levels of interleukin 10 (IL-10), previously designated cytokine synthesis inhibitory factor (CSIF), in a dose dependent fashion. IL-10 was detectable 7 h after activation of the monocytes and maximal levels of IL-10 production were observed after 24-48 h. These kinetics indicated that the production of IL-10 by human monocytes was relatively late as compared to the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, tumor necrosis factor alpha (TNF alpha), and granulocyte colony-stimulating factor (G-CSF), which were all secreted at high levels 4-8 h after activation. The production of IL-10 by LPS activated monocytes was, similar to that of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF, inhibited by IL-4. Furthermore we demonstrate here that IL-10, added to monocytes, activated by interferon gamma (IFN-gamma), LPS, or combinations of LPS and IFN-gamma at the onset of the cultures, strongly inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF at the transcriptional level. Viral-IL-10, which has similar biological activities on human cells, also inhibited the production of TNF alpha and GM-CSF by monocytes following LPS activation. Activation of monocytes by LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies resulted in the production of higher amounts of cytokines relative to LPS treatment alone, indicating that endogenously produced IL-10 inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF. In addition, IL-10 had autoregulatory effects since it strongly inhibited IL-10 mRNA synthesis in LPS activated monocytes. Furthermore, endogenously produced IL-10 was found to be responsible for the reduction in class II major histocompatibility complex (MHC) expression following activation of monocytes with LPS. Taken together our results indicate that IL-10 has important regulatory effects on immunological and inflammatory responses because of its capacity to downregulate class II MHC expression and to inhibit the production of proinflammatory cytokines by monocytes.

scholarly journals Supernatants of human leukocytes contain mediator, different from interferon gamma, which induces expression of MHC class II antigens.

1986 ◽  
Vol 164 (1) ◽  
pp. 131-143 ◽  
Author(s):  
G Groenewegen ◽  
M de Ley ◽  
G M Jeunhomme ◽  
W A Buurman
Keyword(s):  
Mhc Class Ii ◽  
Antigen Expression ◽  
Class Ii ◽  
Ifn Gamma ◽  
Human Leukocytes ◽  
Hla Dr ◽  
Hla Dq ◽  
Mhc Class Ii Antigens ◽  
Class Ii Antigen ◽  
Mhc Class Ii Antigen

In this report, data are presented on the regulation of MHC class II antigen expression by a mediator present in supernatants of human mixed leukocyte cultures (MLC-SN), and which is different from IFN-gamma. The capacity of supernatants to induce antigen expression did not correspond to titers of IFN-gamma. Removal of IFN-gamma using either dialysis against pH 2 or neutralizing mAb against human IFN-gamma did not abrogate the MHC class II antigen expression-inducing capacity of MLC-SN when tested on adenocarcinoma cell lines, kidney epithelial cells, and fibroblasts in vitro in an indirect immunofluorescence assay. Therefore, supernatants of human leukocytes contain a mediator, different from IFN-gamma, which induces expression of MHC class II antigens. Dose-response studies revealed that the mediator is produced after allogeneic and lectin stimulation of human leukocytes, and by unstimulated leukocytes. Activation of leukocytes resulted in increased titers of the mediator. The mediator markedly enhances expression of both HLA-DR and HLA-DQ antigens, whereas IFN-gamma had a similar effect on HLA-DR antigens, and only a minor effect on HLA-DQ antigens. Interaction of the mediator and IFN-gamma resulted in a potentiating effect of these two factors on MHC class II antigen expression. Biochemical analysis revealed a mediator, distinguishable by FPLC from IL-1, IL-2, and human IFN-gamma, and which has a molecular mass of 32 kD.

Interferon-gamma induces persistent depletion of internal Ca2+ stores in a human salivary gland cell line

1996 ◽  
Vol 270 (2) ◽  
pp. C514-C521 ◽  
Author(s):  
A. J. Wu ◽  
Z. J. Chen ◽  
B. J. Baum ◽  
I. S. Ambudkar
Keyword(s):  
Cell Proliferation ◽  
Salivary Gland ◽  
Cell Line ◽  
Interferon Gamma ◽  
Prolonged Treatment ◽  
Tnf Alpha ◽  
Salivary Gland Cell ◽  
Ifn Gamma ◽  
Human Salivary Gland ◽  
In Cells

Interferon-gamma (IFN-gamma), in the presence of tumor necrosis factor-alpha (TNF-alpha), decreases proliferation of a human salivary gland ductal cell line, HSG (Wu, A., R. Kurrasch, J. Katz, P. Fox, B. Baum, and J. Atkinson. J. Cell. Physiol. 161:217-226, 1994). We examined the possible effects of these cytokines (1,000 U/ml IFN-gamma +/- 20 U/ml TNF-alpha for 7 days) on Ca2+ mobilization in HSG cells. In HSG cells, fetal bovine serum (10%) or carbachol (100 microM) stimulated rapid increases in cytosolic Ca2+ concentration ([Ca2+]i), apparently mobilized from different thapsigargin-sensitive intracellular Ca2+ stores. Serum induced a proliferative effect on HSG cells, which was suppressed (> 90%) by treatment with IFN-gamma +/- TNF-alpha, but not with TNF-alpha alone. Serum-, carbachol-, and thapsigargin-stimulated [Ca2+]i elevations were reduced by 90, 60, and > 65%, respectively, in cells treated with IFN-gamma +/- TNF-alpha and 30, 45, and 45%, respectively, in cells treated with TNF-alpha. Removal of the cytokines from the growth medium induced recovery of both cell proliferation and Ca2+ mobilization responses within 7 days. Treatment of HSG cells with thapsigargin (0.02-2 nM) induced a dose-dependent decrease in cell proliferation. Additionally, acute treatment (< 10 min) of cells with IFN-gamma did not affect [Ca2+]i or alter carbachol-, thapsigargin-, or serum-induced changes in [Ca2+]i. These data demonstrate that prolonged treatment of HSG cells with IFN-gamma +/- TNF-alpha leads to a persistent depletion of intracellular Ca2+ stores. We suggest that this may have a role in cell growth.

scholarly journals Modulation of iron metabolism in monocyte cell line U937 by inflammatory cytokines: changes in transferrin uptake, iron handling and ferritin mRNA

1993 ◽  
Vol 296 (1) ◽  
pp. 175-181 ◽  
Author(s):  
M Fahmy ◽  
S P Young
Keyword(s):  
Cell Line ◽  
Inflammatory Cytokines ◽  
Iron Metabolism ◽  
Transferrin Receptor ◽  
Iron Uptake ◽  
Interleukin 1 ◽  
Receptor Expression ◽  
Tnf Alpha ◽  
Ifn Gamma ◽  
Transferrin Receptor Expression

We have investigated the effects of the pro-inflammatory cytokines interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on the iron metabolism of the human monocytic cell line U937. Cells were treated with each cytokine for up to 24 h, and then iron uptake from diferric transferrin was determined. The intracellular distribution of this iron, the expression of the transferrin receptor and levels of mRNA for the two ferritin subunits were also studied. IL-1 beta, TNF alpha and IFN gamma all decreased transferrin-iron uptake into cells, and all three cytokines had effects on the proportion of iron associated with ferritin. With TNF alpha there was a marked enhancement of the fraction incorporated into ferritin. Transferrin-receptor expression was diminished by TNF alpha and IL-1 beta, but not IFN gamma, suggesting different effector mechanisms. Both TNF alpha and IFN gamma increased the amount of cellular mRNA for ferritin H-chain, but not the L-chain; IL-1 beta affected mRNA for neither ferritin. These data demonstrate that cytokines, which can be present at high concentrations in inflammation, have the capacity to affect macrophage iron uptake, transferrin receptor expression, intracellular iron handling and the relative abundance of ferritin-subunit mRNA, and may therefore be important mediators in the observed perturbations of iron metabolism in inflammatory diseases.

Regulation of ICAM-1 by dexamethasone in a human vascular endothelial cell line EAhy926

1996 ◽  
Vol 270 (2) ◽  
pp. C552-C561 ◽  
Author(s):  
A. Burke-Gaffney ◽  
P. G. Hellewell
Keyword(s):  
Endothelial Cell ◽  
Cell Line ◽  
Short Exposure ◽  
Tnf Alpha ◽  
Human Neutrophils ◽  
High Dose ◽  
Dependent Manner ◽  
Ifn Gamma ◽  
Human Vascular Endothelial Cell ◽  
Endothelial Cell Line

Regulation by dexamethasone of intercellular adhesion molecule-1 (ICAM-1) in cultured monolayers of the human umbilical vein endothelial cell line EAhy926 was investigated. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in combination or lipopolysaccharide (LPS) alone gave time- and dose-dependent increases in ICAM-1. Sustained expression of ICAM-1 was observed after short exposure (30 min) to TNF-alpha + IFN-gamma, but not to LPS. LPS-induced ICAM-1 expression was not inhibited by interleukin-1 (IL-1) receptor antagonist (0.01-100 micrograms/ml). Dexamethasone (1,000 nM) did not inhibit TNF-alpha + IFN-gamma-induced ICAM-1 expression or mRNA induction. In contrast, dexamethasone dose dependently (0.1-1,000 nM) inhibited LPS-induced ICAM-1 expression; however, its effect on mRNA was not established, because ICAM-1 mRNA induced by LPS was not detected at the time points investigated in this study (3 and 20 h). Adhesion of unstimulated human neutrophils to EAhy926 monolayers activated with TNF-alpha + IFN-gamma or LPS was increased in the presence of dexamethasone at low doses, whereas neutrophil adhesion to LPS- but not cytokine-stimulated endothelial cells was significantly reduced (P < 0.05) in the presence of a high dose of dexamethasone (1,000 nM). In conclusion, dexamethasone was demonstrated to regulate the expression and function of ICAM-1 in a stimulus-dependent manner.

scholarly journals Efficient killing of chronic B-lymphocytic leukemia cells by superantigen-directed T cells

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1230-1238 ◽  
Author(s):  
A Wallgren ◽  
R Festin ◽  
C Gidlof ◽  
M Dohlsten ◽  
T Kalland ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Class Ii ◽  
Cell Killing ◽  
Lak Cells ◽  
Lymphocytic Leukemia ◽  
Tnf Alpha ◽  
Target Cells

Abstract In vitro studies have indicated that chronic lymphocytic leukemia of B- cell origin (B-CLL) is resistant to cytotoxic effector lymphocytes such as natural killer and lymphokine activated killer (LAK) cells. We show here that B-cell cells are sensitive to Staphylococcal enterotoxin (SE) A-directed T-cell killing. Activation of the target cells by phorbol ester (tetradecanoyl phorbol acetate, [TPA]) greatly enhances their sensitivity to lysis. In SE-dependent cellular cytotoxicity (SDCC), members of the SE superantigen family form a bridge between T cells and target cells expressing major histocompatability complex class II molecules. Binding of SEA to the T-cell-receptor V beta region induces a strong cytotoxic capacity and cytokine production. Cells from 9 B-CLL patients were cultured in the presence or absence of TPA and used as targets in a 4-hour SDCC assay using an allogeneic T-cell line as effector. At an effector:target cell ratio 30:1, 70% to 80% of TPA- induced B-CLL cells were killed. Even at the effector:target ratio of 3:1, 47% +/- 6% of TPA-activated B-cell cells were lysed compared with 13% +/- 2% of resting cells (P < .001). A T-cell line established from a B-CLL patient killed autologous tumor cells as efficiently as allogeneic effectors. SEA-directed T cells were far more lytic to B-CLL cells compared with LAK cells or lectin (phytohemagglutinin-directed T cells. Mechanisms of SDCC lysis were investigated. Effector plus target cell supernatants contained high levels of tumor necrosis factor (TNF)- alpha and interferon-gamma, but these supernatants were not directly toxic to B-CLL cells in short term culture. High concentrations of recombinant TNF-alpha or TNF-beta had no lytic effect. Addition of neutralizing anti-TNF-alpha and anti-TNF-beta antibodies into the SDCC assay did not inhibit SEA-directed T-cell killing. TPA-activated B-CLL cells showed a 1.2- to 13-fold increased expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen (LFA)-1, and LFA-3, whereas expression of HLA class II molecules increased up to 5 times. The expression of CD72, CD40, and BB-1/B7 increased 1.8 to 4.5 times. The role of these surface molecules in SDCC was analyzed in blocking experiments with monoclonal antibodies. Antibodies to ICAM-1, CD18, and HLA-DR abolished the cytotoxicity, and a substantial reduction was seen with antibody to CD72.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Efficient killing of chronic B-lymphocytic leukemia cells by superantigen-directed T cells

Blood ◽  
1993 ◽  
Vol 82 (4) ◽  
pp. 1230-1238
Author(s):  
A Wallgren ◽  
R Festin ◽  
C Gidlof ◽  
M Dohlsten ◽  
T Kalland ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Line ◽  
Class Ii ◽  
Cell Killing ◽  
Lak Cells ◽  
Lymphocytic Leukemia ◽  
Tnf Alpha ◽  
Target Cells

In vitro studies have indicated that chronic lymphocytic leukemia of B- cell origin (B-CLL) is resistant to cytotoxic effector lymphocytes such as natural killer and lymphokine activated killer (LAK) cells. We show here that B-cell cells are sensitive to Staphylococcal enterotoxin (SE) A-directed T-cell killing. Activation of the target cells by phorbol ester (tetradecanoyl phorbol acetate, [TPA]) greatly enhances their sensitivity to lysis. In SE-dependent cellular cytotoxicity (SDCC), members of the SE superantigen family form a bridge between T cells and target cells expressing major histocompatability complex class II molecules. Binding of SEA to the T-cell-receptor V beta region induces a strong cytotoxic capacity and cytokine production. Cells from 9 B-CLL patients were cultured in the presence or absence of TPA and used as targets in a 4-hour SDCC assay using an allogeneic T-cell line as effector. At an effector:target cell ratio 30:1, 70% to 80% of TPA- induced B-CLL cells were killed. Even at the effector:target ratio of 3:1, 47% +/- 6% of TPA-activated B-cell cells were lysed compared with 13% +/- 2% of resting cells (P < .001). A T-cell line established from a B-CLL patient killed autologous tumor cells as efficiently as allogeneic effectors. SEA-directed T cells were far more lytic to B-CLL cells compared with LAK cells or lectin (phytohemagglutinin-directed T cells. Mechanisms of SDCC lysis were investigated. Effector plus target cell supernatants contained high levels of tumor necrosis factor (TNF)- alpha and interferon-gamma, but these supernatants were not directly toxic to B-CLL cells in short term culture. High concentrations of recombinant TNF-alpha or TNF-beta had no lytic effect. Addition of neutralizing anti-TNF-alpha and anti-TNF-beta antibodies into the SDCC assay did not inhibit SEA-directed T-cell killing. TPA-activated B-CLL cells showed a 1.2- to 13-fold increased expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen (LFA)-1, and LFA-3, whereas expression of HLA class II molecules increased up to 5 times. The expression of CD72, CD40, and BB-1/B7 increased 1.8 to 4.5 times. The role of these surface molecules in SDCC was analyzed in blocking experiments with monoclonal antibodies. Antibodies to ICAM-1, CD18, and HLA-DR abolished the cytotoxicity, and a substantial reduction was seen with antibody to CD72.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals An isotype-specific trans-acting factor is defective in a mutant B cell line that expresses HLA-DQ, but not -DR or -DP.

1991 ◽  
Vol 173 (3) ◽  
pp. 629-637 ◽  
Author(s):  
S J Ono ◽  
V Bazil ◽  
M Sugawara ◽  
J L Strominger
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Mutant Cell ◽  
B Cell Lymphoma ◽  
Surface Expression ◽  
Class Ii ◽  
Parental Cell Line ◽  
Transcription Analysis ◽  
Hla Dq

The B lymphoblastoid cell line clone 13 (a subclone of the mutant cell line P3JHR-1) has been found to express high levels of HLA-DQ; by contrast, HLA-DR and -DP antigens are not expressed and cannot be induced by interferon gamma. Northern blot analysis using gene-specific probes indicated that the lack of surface expression of the DR and DP antigens is due to a marked decrease in the levels of steady-state RNA for both the alpha and beta chains. Southern blots demonstrated that none of the transcriptionally repressed genes are grossly deleted. Preparations of interspecific transient heterokaryons between clone 13 and the class II antigen-positive murine B cell lymphoma, A20, resulted in reactivation of the DRA gene and surface expression of both the DR and DP molecules. The efficiency of the DRA promoter relative to the DQB promoter is markedly and specifically diminished in clone 13 (P3JHR-1) as compared with the parental cell line, Jijoye, as assayed both by transient expression of appropriate chloramphenicol acetyltransferase gene (CAT) constructs and by in vitro transcription analysis. These data clearly demonstrate the existence of an isotype-specific trans-acting factor, and provide direct evidence that the highly homologous class II genes have distinct regulatory mechanisms.

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