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D S Finbloom

Publications and source records attributed to D S Finbloom.

At least 55 records · Page 3Linked to original sources
PubMed1992

Modulation of interferon signaling in human fibroblasts by phorbol esters.

Phorbol esters activate the expression of a variety of early-response genes through protein kinase C-dependent pathways. In addition, phorbol esters may promote cell growth by the inhibition of expression of cellular gene products regulated by antiproliferative agents such as interferons (IFN)s. In human diploid fibroblasts, phorbol 12-myristate 13-acetate (PMA) selectively inhibits the IFN-alpha-induced cellular gene ISG54. Using transient transfection assays, we have delineated two elements in the promoter of this gene that are necessary for the inhibitory actions of PMA. These elements include (i) the IFN-stimulated response element (ISRE) which is necessary for IFN-alpha-induced cellular gene expression, and (ii) an element located near the site of transcription initiation. IFN-alpha treatment resulted in the rapid induction of ISGF3, a multisubunit transcription factor which binds to the ISRE. PMA caused a substantial reduction in IFN alpha-induced ISGF3 in both nuclear and cytoplasmic extracts, as determined by electrophoretic mobility shift assays with the ISRE as a probe. In vitro reconstitution experiments revealed that IFN-alpha activation of the ISGF3 alpha component of ISGF3 was not affected by PMA. Further experiments were consistent with the possibility that PMA regulated the activity of a cellular factor which competed with ISGF3 gamma for binding of the activated ISGF3 alpha polypeptides. Electrophoretic mobility shift assays using the cap site of ISG54 as a probe demonstrated the formation of a specific complex whose DNA binding activity was not affected by treatment of cells with PMA or IFN-alpha. Competitive inhibition studies were consistent with the DNA-protein complex at the cap site of ISG54 containing proteins with DNA binding sites in common with those which also interact with the ISRE. These data suggest a unique regulatory mechanism by which phorbol esters can modulate IFN signaling.

Base Sequence↗
PubMed1992

Regulation by glutathione of interleukin-4 activity on cytotoxic T cells.

We have previously shown that cellular glutathione (GSH) regulates the T-cell proliferative activity of interleukin-2 (IL-2). Here, we examined whether and how GSH affects the activity of interleukin-4 (IL-4) on murine cytotoxic T cells. CT.4R, a T-cell line that is responsive to both IL-4 and IL-2, was used as a model. Although GSH alone had little effect on the thymidine incorporation of CT.4R cells, it enhanced the response of CT.4R to IL-4 and increased the level of thymidine incorporation up to more than 60-fold in a concentration-dependent manner. GSH affected the binding of IL-4 to cellular receptors. Scatchard plot analysis showed that GSH treatment did not change the dissociation constant significantly; however, it increased the receptor number from 1173 +/- 126 to 2112 +/- 492 molecules per cell. Internalization and degradation studies of IL-4 showed that the amount of IL-4 internalized and degraded in the GSH-treated cells was about twofold higher than those in the cells without GSH treatment. These results suggest that GSH regulates the binding, internalization, degradation and T-cell proliferative activity of IL-4; alteration of cellular GSH levels may thus affect the growth and replication of cytotoxic T cells through growth stimulating cytokines such as IL-2 and IL-4.

Animals↗
PubMed1991

The receptor for interferon-gamma on human peripheral blood monocytes consists of multiple distinct subunits.

The interaction of interferon-gamma (IFN gamma) (a product of activated T lymphocytes) and monocytes is essential for immune responsiveness, host defense, and chronic inflammation. In this report we define the IFN gamma receptor (IFN gamma R) on human monocytes as a receptor complex consisting of at least three subunits. Solubilization and immunoprecipitation of [35S]methionine- and [35S]cysteine-labeled monocytes were optimized by controlling the detergent concentration during solubilization and washing of the immunoprecipitates. This enabled subunits to be coimmunoprecipitated by several different anti-IFN gamma R antibodies raised against the 90-kDa cloned binding protein. Immunoprecipitation under stringent (1% sodium dodecyl sulfate) conditions resulted in the visualization of only the 80-90-kDa binding protein. Under less stringent conditions at least two coimmunoprecipitated subunits (molecular mass of 200 and 38 kDa) were consistently associated with the 80-kDa (90-92 kDa reduced) binding protein. The 38-kDa subunit was shown to be distinct from the 80-kDa subunit by proteolytic fragment analysis. Cross-linking of 125I-rIFN gamma to monocytes yielded receptor-IFN gamma complexes consistent with the existence of multiple subunits.

Antibodies, Monoclonal↗
PubMed1991

Regulation of interleukin-4 receptors on murine myeloid progenitor cells by interleukin-6.

Interleukin-4 (IL-4) is a T-cell-derived cytokine that regulates induction of proliferation of resting B cells and acts on various other immunocompetent cells, such as monocytes/macrophages and mast cells, as well as hematopoietic progenitor cells. On hematopoietic progenitor cells, cooperation with another cytokine (such as granulocyte-macrophage colony-stimulating factor [GM-CSF], G-CSF, IL-3, or IL-6) is required to render the cells responsive to IL-4. The present study was undertaken to determine if such an interaction entails induction of IL-4 receptor (IL-4R) expression. Using the murine myeloid leukemia M1 cell line and mature, bone marrow (BM)-derived macrophages, we investigated whether IL-4R expression can be induced during differentiation. We detected no high-affinity IL-4R on the surface of either cell, but with exposure to IL-6 a significant induction of IL-4R was measured on both cell types by fluorescence-activated cell sorter analysis. This increase in IL-4R was first noted 6 hours after exposure of the cells to IL-6 and continued to increase up to 48 hours. By RNase protection analysis we found that the expression of IL-4R mRNA also appeared within 6 hours, continuing to increase up to 48 hours. Nuclear run-on assays showed that this increase in steady-state level of IL-4R mRNA results from a transcriptional activation of the IL-4R gene. These data suggest that regulation of IL-4R expression by IL-6 is under transcriptional control.

Animals↗
PubMed1991

Regulation of cell-surface receptors for human interferon-gamma on the human histiocytic lymphoma cell line U937.

Interferon-gamma (IFN gamma) binds to high-affinity receptors on monocytes and is rapidly internalized. This study investigates the ability of the human monocyte-like cell line, U937, to regulate the cell-surface expression of the IFN gamma receptor (IFN gamma R) during endocytosis of ligand. Recombinant IFN gamma was radiolabelled to high specific radioactivity with Bolton-Hunter reagent and used to enumerate IFN gamma R on treated U937 cells. Cells which had internalized IFN gamma for up to 3 h displayed maximal levels of IFN gamma R at all time points tested after all unlabelled IFN gamma had been acid-stripped from the cell at pH 2.78. Therefore there was no evidence of down-modulation of the receptor. After trypsin treatment of the IFN gamma R, the cells were able to synthesize and insert into the cell membrane up to 1000 IFN gamma R molecules/h after a 60 min lag. Since biosynthesis played a minor role during the first 30 min of endocytosis, I examined other possibilities to explain the lack of down-modulation of the receptor. A solubilized-receptor assay revealed the presence of an intracellular pool of receptors equal to about 25% of the number of cell surface receptors. Using trypsin to differentiate between intracellular and surface receptors, I observed that 43% of those receptors that were internalized after a 30 min exposure to IFN gamma (580 molecules) could be recycled back to the plasma membrane. In addition, equal rates of receptor decay (t1/2 = 5 h) were observed in the presence of cycloheximide with or without IFN gamma. All the data taken together suggest that during the first 30 min of endocytosis both the expression of an intracellular source of receptor and recycling of internalized receptors contribute to maintain optimal receptor expression.

Cycloheximide↗
PubMed1991

Binding of recombinant HIV coat protein gp120 to human monocytes.

Inasmuch as the exact level of CD4 Ag expression on macrophages is controversial and because HIV may interact with macrophages in a manner different from that on T cells, we analyzed the binding of gp120 to freshly isolated and cultured monocytes. rgp120 was iodinated using the lactoperoxidase method to a sp. act. of 600 Ci/mmol. Highly purified monocytes (greater than 90%) were isolated from the leukapheresed blood of normal volunteers by Ficoll-Hypaque sedimentation followed by countercurrent centrifugal elutriation and cultured 7 days in DMEM supplemented with 1000 U/ml macrophage CSF in 10% human serum. Whereas MOLT/4 cells consistently bound freshly prepared 125I-rgp120 at 80% specificity with 5100 +/- 700 mol/cell, MCSF cultured monocytes bound rgp120 at only 0 to 20% specificity and 420 +/- 200 mol/cell. Most of the radioactivity bound by these cells could not be blocked by the addition of unlabeled rgp120. In contrast, the U937 myeloid cell line bound rgp120 with 50% specificity and about 2500 mol/cell. Whereas the antibody OKT4a (anti-CD4) blocked 80% of the binding on MOLT/4 cells and 50% on U937 cells, binding was only inhibited on the average of 6% on cultured monocytes. When soluble rCD4 was used as an inhibitor, binding to MOLT/4 cells was blocked by 80%. In contrast, binding to cultured monocytes was inhibited by 28%. HIV infectivity was blocked by similar concentrations of OKT4a. These observations suggest that although most binding of gp120 to cultured monocytes is not to the CD4 determinant, several hundred molecules do bind to a CD4-like molecule which promotes virus entry and replication.

Binding, Competitive↗
PubMed1990

IL-2. A cofactor for induction of activated macrophage resistance to infection.

Macrophages cultured with IL-2 and IFN-gamma before exposure to microorganisms developed the ability to resist infection with the obligate intracellular parasite, Leishmania major. The induction of this macrophage effector response was maximal by 6 to 8 h after lymphokine addition, and was independent of lymphokine treatment sequence. Activation of macrophages for resistance to infection was the result of the direct action of IL-2 and IFN-gamma on macrophages: the effector reaction was demonstrated in both resident peritoneal macrophages depleted of T cells and bone marrow-derived cells, a homogeneous macrophage population. Radiolabeled murine rIFN-gamma, human rIL-2, and mAb to the IL-2R (7D4), each bound to murine bone marrow-derived macrophages in a specific and saturable manner, which suggested that unstimulated macrophages have receptors for both lymphokines. Treatment of macrophages with IFN-gamma increased the specific binding of IL-2; treatment of cells with IL-2, however, did not up-regulate the IFN-gamma-R. Addition of protein or RNA synthesis inhibitors (cycloheximide, emetine, actinomycin D) during exposure to rIL-2 and rIFN-gamma totally abrogated the ability of macrophages to express this effector reaction; inhibitors of protein kinase C, PG, or calcium redistribution had no effect. Soluble polyclonal anti-TNF-alpha antibodies in culture fluids after activation of macrophages with IL-2 and IFN-gamma totally abrogated the expression of resistance to infection. The T cell growth hormone IL-2 acts as cofactor with IFN-gamma for induction of a macrophage antimicrobial activity, and TNF-alpha may be the effector molecule for resistance to infection regulated by these two lymphokines.

Animals↗
PubMed1990

Induction and regulation of IL-4 receptor expression on murine macrophage cell lines and bone marrow-derived macrophages by IFN-gamma.

IL-4 is a T cell-derived cytokine that regulates the induction of proliferation of resting B cells, and appears to act on various other cells involved in the immune response. The pluripotential effects of IL-4 are dependent on the interaction of IL-4 with its receptor (IL-4R). Although the regulation and metabolism of these receptors have been examined on B and T cells, little is known about the metabolism or regulation of the IL-4R on macrophages. In studying the dynamics of IL-4R expression on the murine macrophage-like cell line J774.16, we detected the presence of low numbers of high affinity IL-4R (234 +/- 38, Kd = 110 +/- 11 pM) on the surface membrane. However, upon exposure to IFN-gamma, a potent macrophage activating cytokine, there was a rapid upregulation (within 45 min) of IL-4R (2750 +/- 178) on the cell surface, with no change in receptor affinity (Kd = 205 +/- 37 pM). Maximum expression occurred at 2 to 4 h with no further increase in IL-4R expression over the next 48 h. Cells pulsed with IFN-gamma for 45 min displayed maximum IL-4R expression by 4 h. The induction of IL-4R by IFN-gamma was dose dependent: as little as 0.5 ng/ml of IFN-gamma was capable of inducing IL-4R expression, with optimal induction at 10 ng/ml. The addition of the metabolic inhibitors actinomycin D and cycloheximide before the addition of IFN-gamma indicated that both RNA transcription and protein translation were required for this upregulation to occur.

Animals↗
PubMed1990

Differential regulation of IL-1 production in human monocytes by IFN-gamma and IL-4.

We have demonstrated that IL-4 markedly inhibits IL-1 production by highly purified normal human monocytes. When added to monocyte cultures, IL-4 suppressed LPS-induced IL-1 production in a time- and dose-dependent manner. Concentrations of IL-4 as low as 100 pg/ml reduced IL-1 production by approximately 50%, and doses of 1 ng/ml or higher suppressed IL-1 production by more than 90%. Maximal inhibition required that IL-4 be added before or simultaneous with LPS. Northern dot blot analyses revealed that IL-4 not only dramatically reduced the steady-state IL-1 beta mRNA levels in LPS-stimulated monocytes, but also those of TNF-alpha and IL-6. The inhibitory effect was not stimulus-specific because IL-4 suppressed IL-1 production induced by a variety of monocyte activation stimuli, including LPS, PMA, and Staphylococcus aureus Cowan strain. Monocytes expressed a relatively small number of high affinity IL-4R (approximately 150/cell; Ka = 3.15 +/- 1.13 x 10(10) M-1) indicating that relatively few receptors are necessary to generate the inhibitory effect. IL-4 enhanced monocyte MHC class II Ag (HLA-DR) expression in a manner similar to that of IFN-gamma. However, although both IFN-gamma and IL-4 up-regulated HLA-DR expression, they exhibited opposite effects on IL-1 production: IFN-gamma significantly enhanced monocyte IL-1 production induced by submaximal concentrations of LPS; whereas, IL-4 suppressed IL-1 production. Moreover, IL-4 largely neutralized the potentiating effect of IFN-gamma suggesting that IL-4 may be an effective antagonist of certain IFN-gamma-induced effects. Together these findings demonstrate that the relative levels of IFN-gamma and IL-4 may profoundly influence the state of monocyte activation by differentially regulating the expression of IL-1.

Cells, Cultured↗
PubMed1990

Specific prolongation of MHC class II disparate skin allografts by in vivo administration of anti-IFN-gamma monoclonal antibody.

In vivo rejection of MHC class II disparate skin allografts has been thought to involve IFN-gamma-induced expression of MHC class II alloantigens because less than 3% of skin epidermal cells express MHC class II alloantigens constitutively. In our study we directly tested this hypothesis by examining the effect of in vivo administered anti-IFN-gamma mAb on rejection of MHC class II disparate skin allografts, and comparing its effect on rejection of MHC class I disparate skin allografts placed on the same individual mice. We found that anti-IFN-gamma mAb blocked the rejection of MHC class II disparate skin allografts, but had no effect on the rejection of MHC class I disparate skin allografts. These results demonstrate that endogenously produced IFN-gamma is critical for rejection of MHC class II disparate skin allografts, but not for rejection of MHC class I disparate skin allografts. Thus, this study strongly supports the concept that MHC class II rejection responses require IFN-gamma induced MHC class II expression on keratinocytes of the allograft.

Animals↗
PubMed1990

Inhibition of phospholipase activity in human monocytes by IFN-gamma blocks endogenous prostaglandin E2-dependent collagenase production.

Previous studies from our laboratory have demonstrated that exposure of human monocytes to a stimulant, such as Con A, results in the production of the enzyme collagenase through PGE2-dependent pathway. Inasmuch as rIFN-gamma has been shown to modulate monocyte/macrophage PG synthesis, we examined the effect of rIFN-gamma on the activation sequence leading to collagenase production. The addition of rIFN-gamma (10 to 1000 U/ml) to Con A-stimulated monocytes resulted in a dose-dependent inhibition of PGE2 and collagenase synthesis. The suppression of collagenase production by rIFN-gamma was related to its ability to reduce PGE2 levels as demonstrated by the restoration of collagenase activity by the addition of PGE2. HPLC analysis of the arachidonic acid (AA) metabolites released by monocytes showed that rIFN-gamma caused a reduction in the release of AA and products of the cyclooxygenase and lipoxygenase pathways. These data indicated that rIFN-gamma decreased eicosanoid production by inhibiting the release of AA from phospholipids. This conclusion was supported by the reduction in membrane bound phospholipase activity in rIFN-gamma-treated monocytes. Moreover, the inhibition by rIFN-gamma of PGE2 and collagenase was reversed by the addition of phospholipase A2. Our findings demonstrate that rIFN-gamma inhibits phospholipase activity in activated monocytes and as a result blocks PGE2-dependent collagenase synthesis.

Cells, Cultured↗
PubMed1990

Characterization of the interaction between recombinant human interferon-gamma and its receptor on human polymorphonuclear leukocytes.

The interaction of human recombinant interferon-gamma (rIFN-gamma) with human polymorphonuclear cells (PMN) was investigated. Bolton-Hunter radioiodinated rIFN-gamma bound to PMN in a specific and saturable manner. Eleven hundred binding sites were observed with a Ka of 0.56 x 10(10) M-1. Binding to PMN was rapid with a K1 of 9 x 10(5) M-1 sec-1 at 4 degrees C. At 37 degrees C binding was complete within 6 min. About 50% of bound ligand was internalized within 30 min at 37 degrees C. The receptor demonstrated moderate lability at 37 degrees C in culture. After 1 h at 37 degrees C, PMN lost 80% of their 125I-rIFN-gamma binding sites. This loss was reversed in part by the presence of interleukin-1 in the culture, but not tumor necrosis factor. These studies provide a framework for further investigation into the signalling process of rIFN-gamma on PMN.

Humans↗
PubMed1990

Expression of high-affinity IL-4 receptors on murine tumour infiltrating lymphocytes and their up-regulation by IL-2.

Since interleukin-2 (IL-2) and IL-4 act in concert to support the development of cytotoxic T lymphocytes (CTL) and the generation of antigen-specific tumour infiltrating lymphocytes (TIL), we investigated the interaction of these cytokines with an established TIL line. TIL proliferated in an additive fashion in response to suboptimal concentrations of IL-2 and various concentrations of IL-4. TIL possessed high-affinity IL-4 receptors whether cultured in recombinant IL-2 (rIL-2) or rIL-4, but cells cultured in rIL-2 had higher numbers of IL-4 receptors than cells cultured in rIL-4. When TIL were cultured in increasing concentrations of rIL-2, a dose-dependent enhancement in IL-4 receptor number was observed. The maximum induction of IL-4 receptor expression was achieved by 4 hr of incubation with rIL-2 and was completely blocked by cycloheximide. Other cytokines, such as rIL-1, recombinant tumour necrosis factor (rTNF), recombinant interferon-alpha (rIFN-alpha) and rIFN-gamma, had no effect on IL-4 receptor number. rIL-2 also up-regulated IL-4 receptors on CTLL-2, a murine CTL line. These data indicate that high-affinity IL-4 receptors exist on murine TIL and they can be up-regulated by IL-2. Our observation that IL-2 up-regulates IL-4 receptor may help explain the additive effects of these lymphokines on the proliferation of TIL and other cell lines. It may also help explain their co-operative effects on the generation of antigen-specific TIL and the differentiation of CTL.

Animals↗
PubMed1989

Administration of monoclonal anti-IFN-gamma antibodies in vivo abrogates natural resistance of C3H/HeN mice to infection with Leishmania major.

C3H/HeN mice that are naturally resistant to cutaneous disease and systemic infections with the protozoan parasite, Leishmania major, were treated at the time of infection, and weekly thereafter, with mouse anti-rat IFN-gamma mAb or an irrelevant antibody of similar isotype. Anti-IFN-gamma-treated mice developed cutaneous lesions; parasites spread to the regional lymph nodes and then metastasized to spleens and livers. The course of disease in these animals was similar to that of genetically susceptible BALB/c mice. Two exceptions in the pathology of L. major infections were noted between BALB/c and anti-IFN-gamma-treated C3H/HeN mice: 1) BALB/c mice died of systemic complications, whereas C3H/HeN mice did not, and 2) multinucleated giant cells were observed in lymph nodes and spleens of infected BALB/c mice, whereas these cells were not observed in infected C3H/HeN mice. Control mice, those treated with either saline or irrelevant antibody of the same isotype as the anti-IFN-gamma monoclonal, showed no evidence of cutaneous disease (development of footpad lesions) or systemic infection (by histopathology). Total abrogation of the natural resistance of C3H/HeN mice could be achieved by treatment with as little as 0.5 mg/mouse/wk of anti-IFN-gamma antibody, or by a single dose of 1 mg/mouse anti-IFN-gamma antibody administered at the time of parasite inoculation. If antibody treatment was delayed for as little as 1 wk after parasite inoculation, the infections in treated animals resembled that of untreated or control antibody-treated mice: no cutaneous lesions (by footpad swelling or viable counts of leishmania in footpad tissue) or systemic disease (by microscopic analysis of touch preparations of internal organs, and histopathology of same). The production of IFN-gamma during the initial interaction of the parasite and host cells appears to be a major component of genetic control of natural resistance to infection with L. major in C3H/HeN mice.

Animals↗
PubMed1989

Sporozoite vaccine induces genetically restricted T cell elimination of malaria from hepatocytes.

The target of the CD8+ T cell-dependent immunity that protects mice immunized with irradiation-attenuated malaria sporozoites has not been established. Immune BALB/c mice were shown to develop malaria-specific, CD8+ T cell-dependent inflammatory infiltrates in their livers after challenge with Plasmodium berghei sporozoites. Spleen cells from immune BALB/c and C57BL/6 mice eliminated hepatocytes infected with the liver stage of P. berghei in vitro. The activity against infected hepatocytes is not inhibited by antibodies to interferon-gamma and is not present in culture supernatants. It is genetically restricted, an indication that malaria antigens on the hepatocyte surface are recognized by immune T effector cells. Subunit vaccine development will require identification of the antigens recognized by these T cells and a method of immunization that induces such immunity.

Animals↗
PubMed1989

Characterization of a novel low affinity receptor for IFN-gamma on adherent human monocytes by radioligand binding studies and chemical cross-linking.

Freshly isolated monocytes in suspension express 2000 to 4000 high affinity receptors for IFN-gamma. Because monocytes change phenotypically as they migrate out of the circulation and adhere to extracellular matrix, modulation of the expression of IFN-gamma receptors may occur. In order to determine if adherence alone modulates the receptor for IFN-gamma, we have studied receptor expression in adherent human peripheral blood monocytes. Elutriation-purified monocytes were allowed to adhere to polystyrene overnight at 37 degrees C. These cells now expressed 1 to 2 x 10(5) low affinity (Ka = 10(8) liters/M) receptors for [125I]rIFN-gamma. Binding to this receptor was specific and saturable. The expression of these receptors occurred rapidly (within 3 h) after adherence and was not inhibited by cycloheximide treatment. Binding to the receptor was abrogated by treating cells with trypsin, but was enhanced after treatment with alkaline protease or proteinase K. mAb against the high affinity receptor did not block binding to the low affinity receptor on adherent cells. The low affinity receptor transduced a signal to the cell as measured by the IFN-gamma-induced enhancement in FcR for human IgG1. The structure of the receptor on adherent cells was investigated by chemical cross-linking techniques. A receptor-[125I]rIFN-gamma complex was observed by SDS-PAGE to have a Mr of 180,000 to 200,000. Reduction of this complex with 2-ME resulted in the loss of the high Mr complex and the appearance of a doublet of lower Mr of 68,000 and 82,000. In contrast, cross-linking of monocytes in suspension yielded a complex of 110,000 to 120,000 Mr, which was unchanged upon reduction. Upon adherence, human monocytes express large numbers of a novel receptor for rIFN-gamma which is capable of stimulating the cell. This receptor appears to be composed of at least two components which are disulfide linked and structurally differs from the high affinity receptor on nonadherent monocytes.

Antibodies, Monoclonal↗