Effects of bacteria-produced human alpha, beta, and gamma interferons on in vitro immune functions

The effects of bacteria-produced human interferons (HuIFN) alpha, beta, and gamma on in vitro immune functions of human peripheral blood mononuclear cells (PBMC) were studied. Proliferative response to phytohemagglutinin was significantly inhibited by the addition of HuIFN-alpha 2 or HuIFN-beta at 10, 100, or 1000 U/ml. In contrast, HuIFN-gamma showed suppressive activities only when added at 1000 U/ml. HuIFN-alpha 2 or HuIFN-beta caused significant inhibition of human mixed-lymphocyte reaction (MLR) as measured by [3H]thymidine incorporation. Similar inhibition was caused by HuIFN-gamma when it was added only at very low concentrations (1 U/ml); 10, 100, or 1000 U/ml resulted in no or only a modest increase in MLR. All three interferons exhibited dose-related effects on PWM-induced immunoglobulin synthesis in cultures of PBMC. These data demonstrate that purified interferons produced by recombinant DNA technology can significantly alter in vitro immune functions and that HuIFN-gamma has properties which are different from those of HuIFN-alpha 2 or HuIFN-beta.     

Suppression of N-myc over-expression in human neuroblastic cells by human gamma-interferon

In the previous study, it was shown that the treatment of human neuroblastoma cells with human interferon-gamma (HuIFN-gamma) induced the morphological changes. However, the treatment with human interferon-alpha (HuIFN-alpha) or -beta (HuIFN-beta) did not induce them. In the present study, the effect of HuIFNs on the overexpression of N-myc of the human neuroblastoma cells (GOTO strain) is examined. The treatment of GOTO cells with rHuIFN-gamma inhibits the overexpression of N-myc, and its degree is dependent on the duration of the treatment. However, HuIFN-alpha and HuIFN-beta did not inhibit the overexpression of N-myc. This suggests that the oncogene N-myc may have relation to the morphological differentiation of human neuroblastoma cells because only the HuIFN-gamma, which induces the morphological differentiation, inhibits the overexpression of N-myc.     

Effects of hyperthermia on the in vitro antiproliferative activities of HuIFN-alpha, HuIFN-beta, and rHuIFN-gamma employed separately and in combination

The relative enhancing effects of hyperthermia on the three types of interferon were evaluated in cloning studies for three human cell lines: G-361 malignant melanoma cells, WISH ammion cells, and AGS stomach adenocarcinoma cells. Hyperthermia enhanced the antiproliferative activity of rHuIFN-gamma against each of the three cell lines and the levels of enhancement by hyperthermia were seen to increase with increasing concentrations of rHuIFN-gamma. The maximum observed levels of enhancement of rHuIFN-gamma activity by hyperthermia varied from cell line to cell line. However, when the relative sensitivities of the cell lines to rHuIFN-gamma were taken into account, the levels of enhancement of rHuIFN-gamma antiproliferative activity by hyperthermia were seen to be similar for each of the cell lines, indicating that hyperthermia consistently enhanced rHuIFN-gamma antiproliferative activity. Hyperthermia did not consistently enhance the antiproliferative activities of HuIFN-alpha and HuIFN-beta. Further studies indicated that hyperthermia enhanced by approximately 6-fold the antiproliferative effects of combinations of rHuIFN-gamma with HuIFN-alpha and HuIFN-beta. The results support the possibility that a combination treatment protocol of hyperthermia and interferon administration (particularly HuIFN-gamma or combinations of HuIFN-gamma with HuIFN-alpha or HuIFN-beta) may provide an enhanced antitumor effect in man.     

Inhibitory effect of interferons and tumor necrosis factor on syncytium formation by bovine leukemia virus

The treatments of the bovine spleen cells BESp with recombinant human leukocyte interferon (HuIFN-alpha A) and human fibroblast interferon (HuIFN-beta) inhibit the syncytium formation by bovine leukemia virus (BLV) and the virus yield, depending on the IFN doses. But HuIFN-gamma did not inhibit the syncytium formation. The tumor-necrosis factor (TNF) also inhibits the syncytium formation and the virus yield, depending on the TNF doses. Furthermore, the combination of the HuIFN-alpha A with the TNF inhibits the syncytium formation more strongly than the HuIFN-alpha A alone or the TNF alone.     

Characterization of receptors for immune interferon in U937 cells with 32P-labeled human recombinant immune interferon

Recombinant human immune interferon (HuIFN-gamma) was labeled with [gamma-32P]ATP and cyclic-AMP-dependent protein kinase from bovine heart to a specific radioactivity of 11,000 Ci/mmol. At least two molecules of phosphate were incorporated per molecule of interferon. The binding of [32P]HuIFN-gamma to human U937 histiocytic lymphoma cells was time dependent, and displaceable by HuIFN-gamma but not by HuIFN-alpha A or HuIFN-beta. The specific binding was saturable with less than 10% nonspecific binding. The dissociation constant of [32P]HuIFN-gamma for U937 interferon receptors was calculated to be 1.5 X 10(-10) M with a total of 1,800 binding sites/cell. Dissociation of bound [32P]IFN-gamma at 24 degrees C exhibited two distinct rates. A fast dissociation with a specific rate constant of 0.141 min-1, and a slow dissociation with a specific rate constant of 0.0027 min-1. The Kd for [32P]HuIFN-gamma was calculated from kinetic constants to be 5.4 X 10(-10) M.     

Analysis of antigenic domains on natural and recombinant human IFN-beta by the inhibition of biologic activities with monoclonal antibodies

Human IFN-beta (HuIFN-beta) is a biologically potent protein with both antiviral and antiproliferative activities. To understand better its mode of action, a number of murine mAb were developed against a recombinant (serine-17) HuIFN-beta (rHuIFN-beta ser) and screened by ELISA and neutralization of antiviral activity. The panel of antibodies, composed of both IgA and IgG immunoglobulins, were specific for HuIFN-beta and did not crossreact with HuIFN-alpha or gamma. Furthermore, three functionally distinct epitopes (designated as sites I, II, and III) were identified based on mAb neutralization of antiviral and antiproliferative activities of recombinant and natural HuIFN-beta. Antibodies directed to sites I and II neutralized the antiviral and antiproliferative activities of rHuIFN-beta ser, though the antiviral neutralization potency of the mAb to site II was approximately 10-fold less than mAb to site I. Antibodies directed to site I neutralized both recombinant and natural HuIFN-beta, although the antiviral neutralization potency was approximately 10-fold higher against rHuIFN-beta ser than the native protein. The mAb directed to site II did not demonstrate any significant neutralization of the antiviral or antiproliferative activity of natural HuIFN-beta but neutralized a recombinant HuIFN-beta containing the native sequence. Antibodies recognizing site III did not neutralize the biologic activity of either recombinant or natural HuIFN-beta. Thus, three epitopes on HuIFN-beta have been identified, two of which are associated with both antiviral and antiproliferative activities.     

Interferon receptor interaction. Internalization of interferon alpha 2 and modulation of its receptor on human cells

Studies reported earlier [ Joshi et al. (1982) J. Biol. Chem. 257, 13884-13887] have indicated that human interferon-alpha 2 (HuIFN-alpha 2) binds to a specific macromolecular receptor on human cells as identified by cross-linking with bifunctional cross-linking reagents and analysis by polyacrylamide gel electrophoresis. We have carried out experiments to investigate the fate of the interferon-receptor complex on the cell surface under conditions which lead to cellular response. As analyzed by cross-linking and gel electrophoresis, the interferon-receptor complex, formed on incubation with 125I-IFN-alpha 2 at 4 degrees C, persisted at the cell surface for several hours at 4 degrees C; however, if the cells were switched to 37 degrees C, there was a rapid decline in the complex, apparently due to a loss of the interferon receptors from the cell surface. This was associated with an internalization of the 125I-interferon as indicated by the fact that, on incubation at 37 degrees C, an appreciable fraction of the cell-associated interferon (approximately equal to 50%) became resistant to trypsin digestion, or dissociation on incubation in growth medium or low-pH buffer. A large fraction of the trypsin-resistant (internalized) 125I-labeled material migrated as intact interferon in polyacrylamide gels, and it was immunoprecipitated by anti-(HuIFN-alpha)antibodies but not by anti-(HuIFN-beta)antibodies. The bulk of the internalized 125I-interferon was recovered in a particulate fraction and, on cross-linking with disuccinimidyl suberate, a 150000-Mr complex could be detected. The results suggest that interferon may be internalized as a complex with the receptor, which may account for the loss of the interferon-receptors on the cell surface. This modulation of the IFN-alpha/beta receptors was induced by HuIFN-alpha and HuIFN-beta but not by HuIFN-gamma. The recovery of the IFN-alpha/beta receptors, lost upon incubation with HuIFN-alpha, took several hours and required protein synthesis. The significance of the results is discussed.     

Enhancement of cell growth by human interferons

The effects of human interferons (HuIFN) on the human osteosarcoma cells were examined. HuIFN-alpha, -beta and -gamma enhanced dose-dependently the cell growth. There was no difference in the degree of the enhancement of the cell growth among HuIFN-alpha, -beta and -gamma. The higher the cell density was, the lower the degree of the enhancement of the cell growth. When HuIFN-gamma was neutralized with anti-HuIFN-gamma, the enhancement of cell growth was not found.     

Effect of protein kinase C inhibitors on the antiviral activity of human alpha interferon in herpes simplex virus-infected human neuroblastoma cells.

Pretreatment of human neuroblastoma cells with an inhibitor of protein kinase C (PKC), staurosporine or H-7, prior to the addition of human alpha interferon (HuIFN-alpha), recombinant HuIFN-alpha, or recombinant HuIFN-beta blocked the inhibitory effect of these IFNs on the release of infectious herpes simplex virus type 1 from treated cells. In addition, staurosporine blocked the inhibitory effect of HuIFNs on the expressions of herpes simplex type 1 glycoproteins B, C, and D in treated neuroblastoma cells. Furthermore, addition of HuIFNs resulted in an increased expression of PKC in treated neuroblastoma cells. These results suggest that inhibitors of PKC block the expression of HuIFN-induced genes in treated human neuroblastoma cells. Thus, the activation of PKC is an important step in the HuIFN-treated cells of neuronal origin.     

Cutting edge: human eosinophils regulate T cell subset selection through indoleamine 2,3-dioxygenase

Allergy involves eosinophilia and Th2 polarization. Indoleamine 2,3-dioxygenase (IDO)-catalyzed conversion of tryptophan to kynurenines (KYN) regulates T cell function. We show that human eosinophils constitutively express IDO. Eosinophils treated with IFN-gamma showed an 8-fold increase in IDO mRNA within 4 h; IL-3, IL-5, and GM-CSF had no effect on baseline IDO expression. IL-3 pretreatment of eosinophils reduced IFN-gamma-induced IDO mRNA expression below baseline. Conversely, GM-CSF, but not IL-5, resulted in a 2-fold increase in IFN-gamma-induced IDO. Treatment with IL-3, IL-5, GM-CSF, or IFN-gamma alone expressed IDO enzymatic activity (the presence of KYN in supernatants 48 h postculture). CD28 cross-linking resulted in measurable KYN in culture supernatants, inhibitable by a neutralizing anti-IFN-gamma. Coculture of eosinophils with an IFN-gamma-producing T cell line, but not IL-4-producing T cell clone, led to apoptosis and inhibition of CD3 or CD3/CD28-induced proliferation. Eosinophils infiltrating asthmatic lung and associated lymphoid tissue exhibited intracellular IDO immunoreactivity. Eosinophils may, therefore, maintain Th2 bias through IDO.     

Interferon-gamma synergizes with tumor necrosis factor and with interleukin 1 and requires the presence of both monokines to induce antitumor cytotoxic activity in macrophages

Small concentrations of recombinant murine interferon-gamma (MuIFN-gamma), recombinant human interleukin 1 (HuIL-1), and recombinant murine tumor necrosis factor (MuTNF), added separately to cultures of thioglycolate-elicited peritoneal macrophages, produced no cytotoxic activity against L5178Y cells, a tumor cell line which is resistant to the direct toxic effects of these cytokines, either alone or in combination. However, small concentrations of MuIFN-gamma when combined with small concentrations of either HuIL-1 or MuTNF activated these macrophages to produce cytotoxic effects against L5178Y cells; small concentrations of HuIL-1 and MuTNF in combination had no macrophage activating activity. Specific antibody to MuTNF blocked the macrophage-activating synergistic effects of MuIFN-gamma + HuIL-1, and specific antibody to HuIL-1 blocked the macrophage-activating activity of MuIFN-gamma + MuTNF, indicating that MuTNF was induced in macrophage cultures treated with MuIFN-gamma + HuIL-1, and that murine IL-1 was induced in macrophage cultures treated with MuIFN-gamma + MuTNF. These results indicate that all three cytokines are required for induction of antitumor cytotoxic activation of macrophages. Experiments with a concentration of MuIFN-gamma which alone could activate macrophages revealed that both MuTNF and murine IL-1 were required for this activation. The demonstration that small concentrations of these three cytokines can act synergistically, but not separately, to activate macrophages indicates the importance of cytokine combinations in immunoregulation and in anti-tumor cell-mediated immune responses.     

Effects of recombinant human interferon alpha on human megakaryocyte and fibroblast colony formation

Effects of recombinant human interferon alpha (HuIFN-alpha) on human megakaryocyte (CFU-MK) and fibroblast (CFU-F) colony-forming cell growth were studied. Concentration-dependent inhibition of both CFU-MK and CFU-F by HuIFN-alpha was demonstrated. Statistically significant suppression of both CFU-MK and CFU-F was seen at a HuIFN-alpha concentration of 1000 U/ml or greater. No significant difference was found between HuIFN-alpha treated cultures and controls for the distribution of CFU-MK types and for the size and cell morphology of CFU-F. When a concentration of 1000 u/ml HuIFN-alpha was added at varying time points during the marrow cultures, decreased numbers of megakaryocyte and fibroblast colonies only appeared at the early days of cultures. When bone marrow cells were incubated with HuIFN-alpha for different periods of time prior to initiation of cultures, a reduction of megakaryocyte colony formation also occurred. These studies demonstrate a suppressive effect of HuIFN-alpha on human CFU-MK and CFU-F growth. This effect seems to occur at the initial stages of CFU-MK and CFU-F development.     

Role of endogenously produced interleukin-6 as a second signal in murine thymocyte proliferation induced by multiple cytokines: regulatory effects of transforming growth factor-beta

Previous studies have demonstrated that murine thymocytes proliferate in the presence of submitogenic concentrations of phytohemagglutinin-P (PHA-P) and various cytokines such as interleukin-1 (IL-1), interleukin-4 (IL-4), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6). We report that C3H/HeJ thymocytes stimulated with PHA-P and IL-1, IL-4, or TNF-alpha secrete significant levels of IL-6 as determined on B9 hybridoma cells. The possibility that thymocyte proliferation induced by these cytokines was mediated through IL-6 was investigated utilizing a neutralizing monoclonal antibody against murine IL-6, MP5 20F3.1. The results demonstrate that MP5 20F3.1 inhibited the proliferative response of thymocytes and B9 hybridoma cells to recombinant MuIL-6 (but not HuIL-6) and neutralized the endogenous IL-6 produced in the thymocyte cultures, but did not have any measurable effects on the proliferative responses induced by IL-1, IL-4, or TNF-alpha. Although the level of endogeneously produced IL-6 did not play a measurable role in the proliferative response induced by TNF-alpha, the addition of higher concentrations of IL-6 augmented the proliferation of murine thymocytes induced by rMu TNF-alpha. In addition, recombinant human transforming growth factor-beta 1 (rHu TGF-beta 1) significantly inhibited thymocyte proliferation induced by HuIL-1, rMuIL-4, rMuIL-6, and rMuTNF-alpha. The studies suggest that IL-1, IL-4, or TNF-alpha mediate a proliferative signal on murine thymocytes independent of IL-6 and that the proliferative signals provided by these cytokines as well as IL-6 are inhibitable by rHu TGF-beta 1.     

Upregulation of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cultures by lipopolysaccharide, muramyl tripeptide, and interleukin-1

The tryptophan decyclizing enzyme indoleamine 2,3-dioxygenase (IDO) was induced in human monocyte-derived macrophages (MDM) treated with human recombinant interferon-β (IFN-β) or interferon-γ (IFN-γ). Treated cells exhibited dose-dependent increases in IDO when assayed 48 hr after treatment. Cells exposed to IFN-γ were observed to exhibit consistently higher peak levels of IDO when compared with cells incubated in the presence of IFN-β. When IFN-β-treated cells were incubated in the presence of specified amounts of bacterial lipopolysaccharide (LPS) or liposome-encapsulated muramyl tripeptide (MTP), peak IDO activity increased such that enzyme activity was comparable to maximal activity observed with IFN-γ-treated cells. LPS and MTP also upregulated IFN-γ-mediated IDO activity when suboptimal amounts of IFN-γ were used. When macrophages were costimulated with various concentrations of human recombinant interleukin 1α (IL-1α), along with either maximum-stimulating amounts of IFN-β or suboptimal amounts of IFN-γ, IDO activity was upregulated in a manner similar to results obtained using the microbial products as stimuli. While neither IL-1α or IL-1β was detected in culture supernatants from macrophages treated with either LPS or MTP (alone or in combination with IFN), IL-1α was detected in cell lysates of macrophages treated with these upregulators. Although neutralizing antibody to IL-1α abolished the upregulatory effect of exogenous IL-1α, it had no effect on upregulation by LPS or MTP. This suggests that although LPS and MTP may induce production of cell-associated IL-1α, upregulation of IDO activity by these agents is independent of IL-1α production and may be mediated through distinct pathways.     

IFN-gamma is the inducer of indoleamine 2,3-dioxygenase in allografted tumor cells undergoing rejection

The depletion of an essential amino acid, tryptophan, caused by induction of indoleamine 2,3-dioxygenase (IDO), has been shown to be a mechanism involving self-defense against inhaled microorganisms and tumor growth. We recently reported that the IDO is dramatically (approximately 50-fold) induced in allografted tumor (3-methylcholanthrene-induced ascites type tumor cells) cells undergoing rejection, and that the enzyme is induced by factor(s) released through the interaction of allografted tumor cells with infiltrating leukocytes. The culture supernatant of infiltrating leukocytes, which were harvested on day 7 after tumor transplantation, induced the highest IDO activity in the tumor cells. The inducer activity was completely neutralized by the addition of antibody to IFN-gamma but not by antibody to IFN-alpha/beta. Approximately 6 U/ml of IFN-gamma was detected by an ELISA assay in the 12-h culture supernatant with 2 x 10(6) leukocytes/ml, and rIFN-gamma at 6 U/ml induced IDO in 3-methylcholanthrene-induced ascites type tumor cells to the same extent as IFN-gamma in the culture supernatant. Moreover, i.p. administration of antibody to IFN-gamma almost completely inhibited the induction of IDO in the allografted tumor cells. These observations indicate that the factor responsible for IDO induction in the allografted tumor cells is IFN-gamma.     

Detection of the deletion of interferon-alpha and beta genes in lymphoblastoid cells by PCR]

The HuIFN-alpha and beta genes were examined by the PCR method in the 11 human lymphoblastoid cell lines. The results showed that the homozygous deletion of HuIFN-alpha and beta genes was detected in 5 of 11 cell lines and in 5 of 11 cell lines, respectively. The deletions of both the HuIFN-alpha and beta genes were observed in 4 of 11 cell lines. One T cell leukemia cell line deleted only HuIFN-alpha gene, while the other T cell leukemia line deleted only HuIFN-beta gene. This suggests that the deletions of HuIFN-alpha and beta genes may be related the development of leukemia or lymphoma.     

High expression of IMPACT protein promotes resistance to indoleamine 2,3‐dioxygenase‐induced cell death

Indoleamine 2,3‐dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO‐induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non‐derepressible‐2(GCN2) kinase stress‐responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO‐induced low tryptophan environment. IDO‐expressing human fibroblasts were co‐cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO‐expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO‐induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co‐cultured with IDO‐expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT‐expressing vector, was rescued significantly in tryptophan‐deficient but not IDO‐expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO‐mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO‐generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non‐immune cells such as skin cells acts as a protective mechanism against IDO‐induced GCN2 activation, therefore, makes them resistant to the amino acid‐deprived environment caused by IDO. J. Cell. Physiol. 225: 196–205, 2010. © 2010 Wiley‐Liss, Inc.     

The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: synergism of tumor necrosis factor and interferon-gamma

The influences of human tumor necrosis factor (TNF) (LuKII), recombinant human TNF-alpha, natural human interferon-gamma (HuIFN-gamma), recombinant HuIFN-gamma, and natural HuIFN-alpha were evaluated alone or in combination for their effects in vitro on colony formation by human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells incubated at 5% CO2 in lowered (5%) O2 tension. TNF (LuKII) and recombinant TNF-alpha caused a similar dose-dependent inhibition of colony formation from CFU-GM, BFU-E, and CFU-GEMM. Day 7 CFU-GM colonies were more sensitive than both day 14 CFU-GM colonies and day 7 CFU-GM clusters to inhibition by TNF. BFU-E colonies and CFU-GEMM colonies were least sensitive to inhibition with TNF. The suppressive effects of TNF (LuKII) and recombinant TNF-alpha were inactivated respectively with hetero-anti-human TNF (LuKII) and monoclonal anti-recombinant human TNF-alpha. The hetero-anti-TNF (LuKII) did not inactivate the suppressive effects of TNF-alpha and the monoclonal anti-recombinant TNF-alpha did not inactivate TNF (LuKII). The suppressive effects of TNF did not appear to be mediated via endogenous T lymphocytes and/or monocytes in the bone marrow preparation, and a pulse exposure of marrow cells with TNF for 60 min resulted in maximal or near maximal inhibition when compared with cells left with TNF for the full culture incubation period. A degree of species specificity was noted in that human TNF were more active against human marrow CFU-GM colonies than against mouse marrow CFU-GM colonies. Samples of bone marrow from patients with non-remission myeloid leukemia were set up in the CFU-GM assay and formed the characteristic abnormal growth pattern of large numbers of small sized clusters. These cluster-forming cells were more sensitive to inhibition by TNF than were the CFU-GM colonies and clusters grown from the bone marrow of normal donors. The sensitivity to TNF of colony formation by CFU-GM of patients with acute myelogenous leukemia in partial or complete remission was comparable with that of normal donors. When combinations of TNF and HuIFN were evaluated together, it was noted that TNF (LuKII) or recombinant TNF synergized with natural or recombinant HuIFN-gamma, but not with HuIFN-alpha, to suppress colony formation of CFU-GM, BFU-E, and CFU-GEMM from bone marrow of normal donors at concentrations that had no suppressive effects when molecules were used alone.(ABSTRACT TRUNCATED AT 400 WORDS)