rIFN Gamma Research Articles

Recombinant interferon gamma up-regulates in vivo and down-regulates in vitro monocyte CD14 antigen expression in cancer patients.

The expression of the monocyte membrane glycoprotein CD14 was measured and related to the serum interferon gamma (IFN gamma) concentration in thirteen patients with disseminated cancer during treatment with human recombinant interferon gamma (rIFN gamma). The drug was administered by continuous subcutaneous infusion using an escalating dose schedule, starting at 50 micrograms/day or 100 micrograms/day and increasing weekly up to 600 micrograms/day, if tolerated. Treatment was continued at a mean maximal tolerated dose of 200 micrograms/day for a median duration of 43 days. Serum IFN gamma concentration and monocyte CD14 antigen expression (immunofluorescence with the monoclonal antibody LeuM3 and fluorescence-activated cell sorting analysis) were determined weekly. The serum IFN gamma concentration was positively correlated with the rIFN gamma dose (P less than 0.05). Therapy induced a dose-dependent enhancement of CD14 antigen expression. The increase in mean CD14 fluorescence intensity was on average 60% after 3 weeks of treatment at a mean dose of 220 micrograms rIFN gamma/day and was reversed after withdrawal of therapy. Patients with a rapidly rising serum IFN gamma concentration (starting dose 100 micrograms/day) showed a smaller increment in CD14 fluorescence intensity than those with slowly rising serum IFN gamma levels (starting dose 50 micrograms/day). Since rIFN gamma is known to down-regulate CD14 antigen expression in vitro, monocytes from patients off therapy and from healthy volunteers were cultured with this cytokine. A similar decrease of CD14 fluorescence was observed in both groups. In patients several factors, such as IFN gamma concentration, duration of drug effect and type of serum, were evaluated and could not explain the discrepant in vivo and in vitro findings. In conclusion, the monocyte marker CD14 was found to be differentially regulated by rIFN gamma in vivo and in vitro. In vivo, secondary mediators, induced by rIFN gamma and acting on a constantly renewed cell population, may contribute to the enhanced CD14 expression.

Phase I/II study of recombinant interferon alpha and gamma in advanced progressive renal-cell carcinoma.

In vitro studies have documented the synergistic antiviral and antiproliferative activity of recombinant interferon alpha (rIFN alpha) and rIFN gamma. Furthermore, rIFN gamma is a strong immunomodulator with optimal effects at a relative low dose (0.1 mg/m2). On the basis of these observations, we began a phase I/II study with the combination of rIFN gamma at 100 micrograms/m2 (2 x 10(6) IU/m2) and rIFN alpha 2c 6 micrograms/m2 (2 x 10(6) IU/m2), injected twice a week subcutaneously. In cases of stable or progressive disease we increased the dose of rIFN alpha 2c every 2 weeks by 6 micrograms/m2 until the maximum tolerated dose was reached. A total of 32 patients with proven progressive renal-cell carcinoma were included. Of the 31 eligible patients, 21 were male and 10 female, their average age was 57.2 years (range 35-72), 28 had had nephrectomy, their median Karnofsky performance status was 90% (70%-100%), and their tumors were localized predominantly to visceral tissue. In 2, response was complete and in 6 it was partial, for a response rate of 25%. The disease had stabilized in 5 patients and progressed in 16. The median duration of partial response was 14 months (8-16 months); of 2 cases of complete response, 1 persists (23+ months), and the other suffered a relapse after 22 months. The median time to response was 24 weeks (18-24 weeks). The maximum tolerated dose of rIFN alpha was 30 micrograms/m2 (range of 6-36 micrograms/m2). Side-effects included those known to be associated with interferon treatment. One patient developed septicemia during a period with grade 4 leukopenia. Our study permits no conclusion regarding the additional value of rIFN gamma.

Effects of interferon gamma on cultured synovial cells from patients with rheumatoid arthritis: inhibition of cell growth, prostaglandin E2, and collagenase release.

The effects of recombinant interferon gamma (rIFN gamma) on the in vitro growth of adherent synovial fibroblast-like cells from patients with rheumatoid arthritis (RA) and also on the release of prostaglandin E2 and collagenase from these cells stimulated with recombinant interleukin-1 beta (rIL-1 beta) were investigated. The growth of adherent synovial cells from six of nine samples, determined by [3H]thymidine incorporation, was inhibited by rIFN gamma in a manner dependent on dose. The release of prostaglandin E2 and collagenase from adherent synovial cells stimulated with rIL-1 beta was also suppressed by rIFN gamma in all samples tested, though the basal release of these inflammatory mediators was little influenced. No apparent correlation between inhibition of proliferation by rIFN gamma and either inhibition by rIFN gamma of rIL-1 beta stimulated prostaglandin E2 release or the endogenous synthesis of prostaglandins was found.

In vivo biological response to recombinant interferon-gamma during a phase I dose-response trial in patients with metastatic melanoma.

Interferon-gamma (IFN gamma), as produced by recombinant DNA technology, has shown a wide range of immunomodulatory activity in vitro and in vivo. Clinical studies have attempted to establish a dose-response relationship to define optimal dosage ranges for induction of effector cell function and host response in patients with cancer. We conducted a randomized trial to test the in vivo biologic activity of five daily dosages ranging from 3 to 3,000 micrograms/m2, administered by daily 2-hour bolus injection or by continuous infusion for 14 days. We demonstrate comparable immunobiologic effects of recombinant IFN gamma (rIFN gamma; Biogen, Inc, Cambridge, MA) administered by these two schedules at the various dosages tested, and have defined a relationship of dose to biologic response over this 3-log10 dose range. Oligo 2'5' adenylate synthetase (2'5'As) induction, natural-killer (NK) cell activity, and T-cell subset distribution (heightened T helper/suppressor ratio) showed the most consistent treatment-associated changes and the greatest immunobiologic effects at dosages of 300 to 1,000 micrograms/m2. Mononuclear cell DR and DQ antigen expression showed no consistent dose-related treatment effect. The relevance of the phenotypic, functional, and enzymologic effects observed in this trial to any clinical antitumor effects of IFN gamma in cancer therapy must now be established.

Recombinant Gamma-Interferon Has Activity in Chronic Myeloid Leukemia

We demonstrated the clinical effectiveness of recombinant interferon-gamma (rIFN gamma) (Biogen) in 18 patients with Philadelphia-positive chronic myeloid leukemia. Sequential cytogenetic studies and molecular analyses of the breakpoint cluster region and for immunoglobulin and T cell rearrangements were performed every 3-4 months. In 13 patients who received treatment for a minimum of 3 months, the majority were treated with 1.5 mg/m2, t.i.w., i.v. Nonhematologic effects--particularly chills, rigors, myalgia, fatigue, headaches, and nausea--were significant. Complete or partial hematologic responses were observed in six patients, two of whom had approximately 20% normal metaphases after an average of 74 weeks of treatment. However, reversion to 100% Ph+ cells occurred 30 weeks later. In these two patients, in whom normal metaphases were found, no changes were observed in the presence of rearrangements of the breakpoint cluster region. In addition, the marrows remained hypercellular, and the leukocyte alkaline phosphatase score and B12 levels remained abnormal. No immunoglobulin or T cell beta-chain gene rearrangements were found. These data indicate the clinical effectiveness of rIFN gamma in some patients with chronic myeloid leukemia, although the fundamental nature of the disease is unaltered by this form of treatment.

Lysing of fresh human tumor by a cytotoxic factor derived from autologous large granular lymphocytes independently of other known cytokines.

During interaction with autologous tumor cells large granular lymphocytes (LGL) of cancer patients released a soluble cytotoxic factor, termed LGL-derived cytotoxic factor, which mediated lysing of autologous fresh tumor cells. The cytotoxic factor was compared with purified human recombinant cytotoxic cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) alpha, IFN gamma, interleukin-1 alpha (IL-1 alpha) and IL-2. The LGL cytotoxic factor exhibited cytotoxicity against autologous and allogeneic fresh human tumor cells in an 18-h 51Cr-release assay, while these target cells were resistant to lysing by any of the recombinant cytokines. Mixtures of recombinant(r) TNF, rLT, rIFN alpha, rIFN gamma, rIL-1 alpha and rIL-2 were still unable to produce cytotoxic effects on fresh human tumor cells. Treatment with monoclonal and polyclonal antibodies directed against rTNF, rLT, rIFN alpha, rIFN gamma, or rIL-1 alpha did not inhibit the cytotoxic activity of LGL-derived cytotoxic factor against fresh human tumor cells. Even a mixture of all the antibodies was incapable of blocking the cytolytic activity of the factor to fresh human tumor cells. Furthermore, intact LGL-mediated lysing of autologous tumor cells was not inhibited by any of the antibodies. These results may indicate that a cytotoxic factor produced by LGL in response to autologous tumor cells mediates lysing of fresh human tumor cells independently of TNF, LT, IFN, IL-1 and IL-2.

Peripheral Blood Lymphocytes Resistant to Epstein-Barr Virus Immortalization Manifest High Natural Killer (NK) Type Activity Against NK-Resistant Target Cells

Epstein-Barr virus (EBV) readily immortalizes human peripheral blood lymphocytes (PBL) in vitro. We found recently that PBL from two EBV-seropositive healthy adults were exceptionally resistant to immortalization by EBV. In contrast to PBL from other EBV-seropositive donors sensitive to immortalization by EBV (S-PBL), the "resistant" PBL (R-PBL) respond to EBV infection with an early interleukin-2 (IL-2) synthesis and high interferon gamma (IFN gamma) production. In order to determine whether these differences in cytokine responses between R-PBL and S-PBL could be associated with a detectable difference in lymphocyte cytotoxicity, we compared the natural killer (NK) activity of R-PBL and S-PBL effectors by using both NK-sensitive (i.e. K562) and NK-resistant (i.e. Raji) targets. We found that, while effectors from EBV-infected R-PBL and S-PBL cultures exhibited comparable NK activity against the K562 targets, they differed remarkably in their cytolytic activity against Raji cells. At days 3 and 5 of culture, effectors from EBV-infected R-PBL showed a significantly higher lytic activity against Raji targets, whereas S-PBL did not. Culture of EBV-infected R-PBL and S-PBL effectors in the presence of recombinant IL-2 (rIL-2) for 5 days resulted in increases of their lytic activity against Raji cells, whereas pretreatment of these effectors with recombinant IFN gamma (rIFN gamma) was found to increase only R-PBL cytotoxicity. These results suggest that the resistance of R-PBL to EBV immortalization could be associated with a lymphokine-mediated early cellular cytotoxic response of the NK/LAK (lymphokine-activated killer cell) type against EBV-infected cells.

Tumour necrosis factors (α, β) induced by HIV-1 in peripheral blood mononuclear cells potentiate virus replication

Cytokines such as tumour necrosis factor (TNF) can induce HIV-1 production in T-cell tumour lines. However, it is not known if the same occurs in freshly isolated mononuclear cells, nor is it known if the virus can itself regulate cellular cytokine production. In this paper we report that HIV-1 induces peripheral blood mononuclear cells (PBMC) and CD4+ T lymphocytes to secrete TNF alpha, TNF beta and interferon-gamma (IFN gamma), three cytokines having multifunctional activities and complex physiological roles. We also show that separate addition of exogenous recombinant (r) TNF alpha or rTNF beta or rIFN gamma increases HIV-1-induced syncytium formation in both PBMC and CD4+ cells by up to 10,000-fold, with TNF alpha being most potent in this regard. Finally, we show that syncytium formation induced by diverse HIV-1 isolates and LAV-2 is inhibited without the addition of exogenous r-cytokines by the respective anti-cytokine antibodies. Our study therefore demonstrates that efficient HIV replication in primary mononuclear cells is associated with the ability of the virus to induce TNF and IFN gamma secretion.

Influence of Tumor Necrosis Factor-α on the Modulation by Interferon-γ of HLA Class II Molecules in Human Thyroid Cells and Its Effect on Interferon-γ Binding*

Cytokines are important modulators of immunological reactions, but it has been postulated that they might act on other unrelated epithelial cells. We studied the effects of recombinant interferon-gamma (rIFN gamma) and recombinant tumor necrosis factor-alpha (rTNF alpha) on normal human thyroid cells. We found that the combination of these two cytokines enhanced HLA class II molecule expression on these cells compared with the effect of rIFN gamma alone. This was proven by both immunofluorescence as well as a more sensitive and quantitative RIA. rTNF alpha alone had no effect on HLA class II molecule induction on the same thyrocytes, suggesting a synergistic rather than an additive action in combination with rIFN gamma. The addition of 600 U/ml rTNF alpha to low dose rIFN gamma (10 U/mL) enhanced class II expression by 50%, as quantified by RIA. We also demonstrated that normal thyrocytes possess distinct receptors for the two cytokines and that rTNF alpha probably augments IFN gamma binding, since it increased when the cells were first incubated with rTNF alpha. This increased binding provides an explanation for the synergistic action of rTNF alpha in enhancing class II molecule expression by rIFN gamma. We conclude that the presence of receptors for these cytokines on human thyroid cells gives a direct demonstration of their potential biological action on cells normally not involved in the immunological circuit. The phenomenon might also explain their direct or indirect involvement in vivo, such as in influencing inappropriate HLA class II molecule expression in epithelial cells affected by autoimmunity.

Acute and chronic inflammatory responses to local administration of recombinant IL-1α, IL-β, TNFα, IL-2 and Ifnγ in mice

The contention that cytokines are important mediators of inflammation prompted the present studies which were designed to compare acute and chronic pathological effects of locally-administered recombinant (r) IL-1 alpha, IL-1 beta, TNF alpha, IL-2 and Ifn gamma. Acute (6 hr), resolving (48 hr) inflammation was induced by the following, in order of potency: rIL-1 alpha greater than rIL-1 beta greater than rTNF alpha greater than rIfn gamma = BSA (control) following a single sc. injection. However, only rIL-1 beta and rIL-2 initiated and maintained chronic granulomatous reactions when delivered locally from a sc. ethylene vinyl acetate (EVA) slow-release polymer. The predominance of macrophages in EVA-rIL-1 beta lesions contrasted with the proliferative lymphoid granulomata induced by EVA-rIL-2 implants. These "in vivo" observations reinforce the roles of both IL-1 beta and IL-2 as potent mediators of chronic immunoinflammatory disease.

Antimetastatic effect of endogenous tumor necrosis factor induced by the treatment of recombinant interferon gamma followed by an analogue (GLA-60) to synthetic lipid A subunit.

We have investigated the effect of endogenous production of tumor necrosis factor (TNF) induced by the combination of recombinant interferon gamma (rIFN gamma) as a primer followed by GLA-60 as a trigger (rIFN gamma/GLA-60) on murine lung metastases caused by B16-BL6 melanoma. In order to examine the therapeutic effect of endogenous TNF on tumor metastasis, the ability of multiple administrations of rIFN gamma/GLA-60 to induce TNF production was also tested. The multiple administrations of rIFN gamma/GLA-60 at intervals of 2 days were effective for the induction of endogenous TNF in mice but continuous multiple administrations of them for 2-4 days were not. In tumor-bearing mice, the production of endogenous TNF by rIFN gamma/GLA-60 was less than that of normal mice, but treatment 3 days after the surgical excision of primary tumors showed the endogenous TNF production to be similar to that in normal mice. In the experimental lung metastasis model, intravenous administration of rIFN gamma followed by intravenous or intranasal administration of GLA-60 showed potent inhibition of lung metastases of B16-BL6 melanoma, whereas the reverse sequence of administration (GLA-60/rIFN gamma) or administration of a mixture of rIFN gamma and GLA-60, which cannot induce the production of TNF, caused no inhibition of lung metastases. These results indicated that the regression of tumor metastases by rIFN gamma/GLA-60 was mediated by the production of endogenous TNF in addition to the direct effects of both immunostimulants. Furthermore, the administration of rIFN gamma and GLA-60 significantly inhibited the tumor metastases in spontaneous lung metastasis model. These results may provide a promising approach for the treatment of cancer metastasis as a result of its ability to induce endogenous TNF.

Effect of interferon α, γ, and tumor necrosis factor α on the HLA‐A, B, C expression of cell lines derived from human liver

Our study was undertaken to determine whether human recombinant interferon alpha(rIFN alpha), gamma(rIFN gamma), and tumor necrosis factor alpha(rTNF alpha) exert an effect on the HLA-A, B, C expression of human liver cell lines. The HLA-A, B, C expression was assayed by immunoperoxidase staining and enzyme-linked immunosorbent assay. rIFN alpha and gamma enhanced the HLA-A, B, C expression of the three cell lines tested, Chang cells, SK-Hep-1, and PLC/PRF/5. The activity of rIFN gamma proved more than 8000 times more potent than that of rIFN alpha in Chang cells, 30 times in SK-Hep-1, and 20 times in PLC/PRF/5, respectively. rTNF alpha also enhanced the HLA-A, B, C expression of the three cell lines. The enhancement of HLA-A, B, C expression by rIFN alpha and gamma reached a peak on day 3, and that by rTNF alpha on day 5. These findings suggest that IFN alpha, IFN gamma, and TNF alpha may play similar roles in enhancement of HLA-A, B, C expression of hepatocytes in hepatitis and hepatoma cells.

Recombinant IFN-gamma synergizes with lipopolysaccharide to induce macrophage membrane procoagulants.

Fibrin deposition is an important histopathologic feature of inflammation and is mediated, in part, by monocyte/macrophage procoagulants. rIFN gamma acted in synergy with suboptimal levels of bacterial LPS by priming thioglycollate-induced mouse peritoneal exudate cells (TG-PEC) to express high levels of surface procoagulant. TFN-alpha beta, TFN-alpha, IL-1, either alone or in combination with LPS or IFN-gamma, had no effect on macrophage procoagulant activity expression. In contrast to the dramatic increases of macrophage procoagulant activity induced by IFN-gamma/LPS, on exudate macrophages, normal peritoneal macrophages, or peripheral blood monocytes were unresponsive suggesting that the state of activation of the macrophage determines reactivity. IFN-gamma induced a Factor VIIa-like activity detected only after cell disruption. Synergy between LPS and IFN-gamma-induced procoagulants may occur as the result of the assembly of the thromboplastin (induced by LPS), Factor VII/VIIa complex on the macrophage surface. RNA synthesis was required for procoagulant induction. Procoagulant expression may, as for other cytokines involved in inflammatory responses, be regulated by short lived repressor proteins as low dose cycloheximide superinduced procoagulant responses to both LPS and IFN-gamma and caused the extracellular expression of procoagulant in response to IFN-gamma. This study suggests an important role for IFN-gamma in the assembly of components of the extrinsic coagulant cascade on the macrophage surface. The synergy between IFN-gamma and LPS may moderate macrophage-initiated fibrin deposition characteristic of inflammatory responses.

Molecular and biochemical characterization of a novel gamma-interferon-inducible protein.

A cDNA clone has been isolated from mRNA derived from the monocytic cell line U937, which detects an mRNA that is present and inducible by gamma-interferon (IFN gamma) in cells of the hematopoietic lineage and absent but inducible in non-hematopoietic cells. Following recombinant IFN gamma (rIFN gamma) treatment, mRNA accumulation can be detected within 30 min, plateaus at 24 h, and remains elevated for several days. This mRNA accumulation is due, at least in part, to increased transcription and is not inhibited by cycloheximide, although cycloheximide alone induces both mRNA accumulation and transcription of this gene. rIFN gamma is a more potent inducing agent than either rIFN alpha or rIFN beta. Polyclonal monospecific antiserum raised to the longest open reading frame expressed in Escherichia coli immunoprecipitates multiple polypeptides that are inducible by rIFN gamma in human monocytes, fibroblasts, endothelial cells, and keratinocytes. In pulse-chase experiments analyzed under reducing conditions, a 30-kDa polypeptide (referred to as IP-30) is either secreted or converted intracellularly into a 25-kDa protein; when analyzed under nonreducing conditions, the two intracellular forms have apparent molecular masses of 25 and 20 kDa, and the extracellular protein is found in two forms of apparent molecular mass 50 and 25 kDa. These data suggest that the intracellular form contains intrachain disulfide bonds, and the extracellular form is involved in both intrachain and interchain disulfide bonding. Indirect immunofluorescence microscopy reveals a punctate fluorescence pattern in monocytes consistent with a vesicular subcellular location. These data are consistent with IP-30 being a novel IFN gamma-inducible protein which may be lysosomal in location.

Lymphocyte function-associated antigen-1 (LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1) is one of at least three mechanisms for lymphocyte adhesion to cultured endothelial cells.

Intercellular adhesion molecule-1 (ICAM-1) on the surface of cultured umbilical vein and saphenous vein endothelial cells was upregulated between 2.5- and 40-fold by rIL-1, rTNF, LPS and rIFN gamma corresponding to up to 5 X 10(6) sites/cell. Endothelial cell ICAM-1 was a single band of 90 kD in SDS-PAGE. Purified endothelial cell ICAM-1 reconstituted into liposomes and bound to plastic was an excellent substrate for both JY B lymphoblastoid cell and T lymphoblast adhesion. Adhesion to endothelial cell ICAM-1 in planar membranes was blocked completely by monoclonal antibodies to lymphocyte function associated antigen-1 (LFA-1) or ICAM-1. Adhesion to artificial membranes was most sensitive to ICAM-1 density within the physiological range found on resting and stimulated endothelial cells. Adhesion of JY B lymphoblastoid cells, normal and genetically LFA-1 deficient T lymphoblasts and resting peripheral blood lymphocytes to endothelial cell monolayers was also assayed. In summary, LFA-1 dependent (60-90% of total adhesion) and LFA-1-independent basal adhesion was observed and the use of both adhesion pathways by different interacting cell pairs was increased by monokine or lipopolysaccharide stimulation of endothelial cells. The LFA-1-dependent adhesion could be further subdivided into an LFA-1/ICAM-1-dependent component which was increased by cytokines and a basal LFA-1-dependent, ICAM-1-independent component which did not appear to be affected by cytokines. We conclude that ICAM-1 is a regulated ligand for lymphocyte-endothelial cell adhesion, but at least two other major adhesion pathways exist.

Tumor necrosis factor induces tissue factor-like activity in human leukemia cell line U937 and peripheral blood monocytes

The induction of procoagulant activity (PCA) by human recombinant tumor necrosis factor (rTNF) was studied in human monoblastic leukemia cell line U937 and human peripheral blood monocytes. Using a one-step recalcificating clotting assay, PCA in cell lysates or whole cell preparations was measured by comparison to a rabbit brain thromboplastin standard. There was a dose- and time-dependent increase in PCA when U937 cells were cultured with rTNF. The effect of rTNF was not enhanced by recombinant human interferon-gamma (rIFN gamma). Cycloheximide inhibited the expression of PCA by U937 cells, showing that protein synthesis was necessary to mediate the effects of rTNF. Whole cell preparations demonstrated that greater than 80% of the PCA was expressed on the surface of the cells. The PCA functioned as a tissue factor-like substance, since it required coagulation factor VII and factor X. rTNF also increased PCA in human monocytes in a dose- and time-dependent manner. This effect was abrogated by boiling the rTNF for ten minutes, and was not inhibited by adding polymyxin-B to the cultures, making it unlikely that endotoxin accounted for the observed effects. These results suggest that TNF-induced expression of tissue factor by mononuclear phagocytes may modulate immunologic, inflammatory, and hemostatic processes.

Tumor Necrosis Factor Induces Tissue Factor-Like Activity in Human Leukemia Cell Line U937 and Peripheral Blood Monocytes

The induction of procoagulant activity (PCA) by human recombinant tumor necrosis factor (rTNF) was studied in human monoblastic leukemia cell line U937 and human peripheral blood monocytes. Using a one-step recalcificating clotting assay, PCA in cell lysates or whole cell preparations was measured by comparison to a rabbit brain thromboplastin standard. There was a dose- and time-dependent increase in PCA when U937 cells were cultured with rTNF. The effect of rTNF was not enhanced by recombinant human interferon-gamma (rIFN gamma). Cycloheximide inhibited the expression of PCA by U937 cells, showing that protein synthesis was necessary to mediate the effects of rTNF. Whole cell preparations demonstrated that greater than 80% of the PCA was expressed on the surface of the cells. The PCA functioned as a tissue factor-like substance, since it required coagulation factor VII and factor X. rTNF also increased PCA in human monocytes in a dose- and time-dependent manner. This effect was abrogated by boiling the rTNF for ten minutes, and was not inhibited by adding polymyxin-B to the cultures, making it unlikely that endotoxin accounted for the observed effects. These results suggest that TNF-induced expression of tissue factor by mononuclear phagocytes may modulate immunologic, inflammatory, and hemostatic processes.

Activation of monocytes by interferon-gamma has no effect on the level or affinity of the nicotinamide adenine dinucleotide-phosphate oxidase and on agonist-dependent superoxide formation.

Human monocytes purified by elutriation were cultured for 3 d in Teflon bags with or without human recombinant interferon-gamma (rIFN gamma). The cells were then collected and used in suspension to determine the rate of stimulus-dependent superoxide or hydrogen peroxide formation as a measure of the NADPH-oxidase. The treatment with IFN gamma increased this rate two- to threefold when phorbol myristate acetate (PMA) was used as the stimulus. By contrast, no IFN gamma-dependent increase in superoxide production was observed when the cells were stimulated with different concentrations of the receptor agonist N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) alone or in combination with another receptor agonist, platelet-activating factor (PAF). At optimum concentrations, f-Met-Leu-Phe elicited rates of superoxide formation that could not be exceeded under other stimulatory conditions including PMA after treatment with IFN gamma. It thus appears that f-Met-Leu-Phe can lead to maximum activation of the NADPH-oxidase, and that this response is not influenced by IFN gamma. Treatment with IFN gamma also failed to affect the affinity of PMA- or f-Met-Leu-Phe-stimulated oxidase for NADPH, the Km values being 30 to 40 microM under all conditions. IFN gamma did not alter the cellular levels of cytochrome b558, as measured by low-temperature spectroscopy, and protein kinase C, as measured by [3H]phorbol dibutyrate binding, and did not appreciably influence the stimulus-dependent increase of cytosolic free calcium. These results indicate that activation of human mononuclear phagocytes by IFN gamma does not affect the level and the kinetic properties of NADPH-oxidase or its activation by receptor agonists. They confirm, however, that IFN gamma enhances the respiratory burst response to PMA.

Regulation by interferon gamma of function in the acute monocytic leukemia cell line, THP-1.

THP-1 is an acute monocytic leukemia cell line which acquires phenotypic and functional monocytoid-like features following incubation with mezerein. The current study concerned the modulation of these features by rIFN gamma. rIFN gamma induces the time-dependent enhancement of HLA-DR expression in the presence or absence of mezerein but has no effect on the expression of Leu-M1, Leu-M2, or Leu-M3 antigens. CSF-1 production following mezerein activation was reduced by incubation in the presence of 10(3) and 10(4) units/ml rIFN gamma. This was confirmed through both biological assays with mouse bone marrow cells and an indirect ELISA. In contrast, the concentration of growth inhibitory activity in conditioned medium was increased by rIFN gamma. A small but significant increase in IL-1 beta concentration in conditioned medium was detected using a sensitive double-antibody ELISA and a radioimmunoassay. The results infer that the functional characteristics of this leukemia cell line are modulated by rIFN gamma in a manner qualitatively similar to that reported for IFN gamma treated normal monocytes.

The release of tumor necrosis factor from endotoxin-stimulated rat Kupffer cells is regulated by prostaglandin E 2 and dexamethasone

Evidence is presented that upon stimulation with endotoxin (lipopolysaccharide, LPS), Kupffer cells, the body's largest pool of sessile macrophages, synthesize and liberate a factor whose immunological, cytotoxic and chemical properties are those described for tumor necrosis factor (TNF)-alpha. Hepatocytes and sinusoidal endothelial cells do not produce detectable amounts of this protein. Ten nanograms of LPS per ml medium are sufficient to stimulate a substantial release of this mediator. Recombinant interferon-gamma (rIFN gamma) per se is a poor inducer of TNF release. Costimulation with endotoxin and rIFN gamma shows only a slight increment in the release of this cytotoxic factor, relative to LPS alone. Exposure of Kupffer cells to the Ca2+ ionophore A23187 or to elicitors of the oxidative burst and superoxide production, e.g. zymosan or phorbol 12-myristate 13-acetate, stimulates only a fraction (20%) of the TNF release seen after endotoxin challenge. Prostaglandin E2, the synthesis of which is strongly enhanced after challenge of rat Kupffer cells with LPS, suppresses the release of TNF by these cells. This autoregulatory mechanism may explain the kinetics of TNF production by stimulated Kupffer cells. Dexamethasone is another important mediator capable of reducing the LPS-elicited TNF formation. An effect of the glucocorticoid hormone can still be provoked if it is added simultaneously with or shortly after LPS. This rapid action requires a mechanism that is different from the time-consuming one leading to the inhibition of prostaglandin synthesis in Kupffer cells.