Binding of recombinant-produced interferon beta ser to human lymphoblastoid cells. Evidence for two binding domains

Human interferon beta (IFN beta ser), produced by recombinant DNA technology, was radiolabeled to approximately one atom of iodine-125/molecule of interferon without detectable loss of antiviral activity. At 37 degrees C, binding of 125I IFN beta ser occurred rapidly (t1/2max less than or equal to 15 min) followed by internalization and degradation of bound ligand. Kinetic analysis at 4 degrees C indicated diffusion-limited association kinetics independent of 125I IFN beta ser concentration. Dissociation of bound 125I IFN beta ser from Daudi cells was slow (t1/2 = 1.2 h) of bound radiolabeled ligand was observed in the presence of unlabeled IFN beta ser, naturally produced IFN beta, and IFN alpha 6, but was not observed with unlabeled IFN gamma or nonspecific proteins. Concomitantly, equilibrium analysis indicated heterogeneous binding of 125I IFN beta ser to six cell lines of lymphoid origin consistent with either negative cooperativity or two populations of receptors. Analysis of binding of 125I IFN beta ser to Daudi cell receptors in the presence of unlabeled IFN alpha 6 suggested that one receptor served both ligands. The latter conclusion was supported by results of chemical cross-linking experiments in which an 125I IFN beta ser/receptor complex (Mr 120,000-130,000) was observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This complex was absent when binding occurred in the presence of either excess unlabeled IFN beta ser or IFN alpha 6.     

Two interferons alpha influence each other during their interaction with the extracellular domain of human type interferon receptor subunit 2

The interaction between two human interferons alpha (IFN-alphas) and the extracellular (EC) domain of human type I IFN receptor subunit 2 (IFNAR2) was analyzed. Previous experiments using Daudi cells showed that IFN-alpha21b and some IFN-alpha hybrids (made from IFN-alpha2c and 21b) competed poorly for the IFN-alpha2b binding site. This study examined the causes of the poor competition between these IFN-alphas. IFN-alpha2c and the IFN hybrid CM3 {IFN-alpha21b(1-75)(81-95)/IFN-alpha2c(76-80) (96-166), Y86K} were selected for this study based on their cell binding and biological properties. Competitive binding ELISA, native electrophoresis followed by Western blot, electrospray ionization mass spectrometry (ESI-MS), surface plasmon resonance biosensor (SPR) analysis, as well as neutralization of antiproliferative activities on Daudi cells in the presence of soluble IFNAR2-EC show evidence that each of the described IFN-alpha subtypes affected the binding of the other IFN-alpha to IFNAR2-EC by affecting the stability of the complex, i.e., dissociation of the complex. Moreover, native electrophoresis with different IFNAR2-EC mutants showed that IFN-alpha2c and CM3 utilize different amino acids in the binding domain of IFNAR2-EC. In addition to that, analytical ultracentrifugation (AUC) revealed differences in the oligomeric state of the two studied interferons. Our results demonstrated that two individual IFN-alphas interact differentially with IFNAR2-EC and influence each other during this interaction. This study contributes to the understanding of the mutual interaction between multiple IFN-alpha subtypes during the competition for binding to the receptor.     

Expression of IFN-alpha and IFN-gamma receptors on normal human small resting T lymphocytes and large granular lymphocytes

The expression of interferon (IFN) receptors was studied on freshly isolated human lymphocytes from normal donors. Highly enriched populations of small resting T lymphocytes and large granular lymphocytes (LGL) were found to constitutively express high-affinity receptors for IFN-alpha and IFN-gamma. Both types of lymphocytes also had lower-affinity IFN-alpha binding sites. T lymphocytes had a mean of 230 IFN-alpha and 520 IFN-gamma high-affinity receptors per cell, whereas LGL had 520 IFN-alpha and 760 IFN-gamma receptors. However, because LGL were larger than the T lymphocytes, the IFN receptor density was similar on the two types of lymphocytes. The affinity of binding was similar on the two types of normal lymphocytes and on the cultured lymphoblastoid cell line Daudi. The number of IFN receptors per cell and the affinities of the IFN-receptor interactions varied little among the normal donors. Both the freshly isolated normal lymphocytes and the cultured cell line Daudi had separate receptors for type I (alpha and beta) and type II (gamma) IFN. Taken together, our data indicate that two types of freshly isolated normal lymphocytes constitutively express IFN receptors that are similar to those present on the lymphoblastoid cell line Daudi derived from a patient with Burkitt's lymphoma.     

Interferon-alpha down-regulates insulin receptors in lymphoblastoid (Daudi) cells. Relationship to inhibition of cell proliferation

The Daudi line of human lymphoblastoid cells requires insulin and transferrin for growth in serum-free medium and is highly sensitive to the inhibitory effect of human leukocyte interferon (IFN-alpha) on cell proliferation. A variant subline of Daudi cells, which is resistant to the antiproliferative action of IFN-alpha, also has been grown in serum-free medium containing insulin and transferrin. The proliferation of IFN-sensitive and -resistant Daudi cells is dependent on the occupancy of insulin receptors, with optimal cell proliferation observed at high receptor occupancy (nearly 100%). No evidence was found for receptors for insulin-like growth factor I on Daudi cells. IFN treatment of IFN-sensitive cells decreased the capacity of the cells to bind 125I-insulin. The altered binding capacity was due to diminished specific, lower affinity insulin binding, as detected at high 125I-insulin concentrations. Higher affinity insulin binding was not altered by IFN. Insulin binding was also reduced in detergent-solubilized extracts from IFN-treated sensitive Daudi cells and the magnitude of the effect was comparable to that observed in intact cells. This indicates that the total number of insulin binding sites (surface + internal) is decreased in IFN-treated sensitive cells. Insulin binding to IFN-sensitive cells decreased linearly with time between 6 and 48 h from the addition of IFN. The effect on lower affinity insulin binding developed more rapidly than the inhibitory effect of IFN on cell proliferation. The insulin-binding capacity of Daudi cells resistant to the antiproliferative effect of IFN was unaffected by IFN, despite the fact that these cells contain as many cell surface IFN receptors as sensitive cells. These observations raise the possibility that lower affinity insulin binding is important in the growth-promoting actions of insulin.     

Formation of a uniquely stable type I interferon receptor complex by interferon beta is dependent upon particular interactions between interferon beta and its receptor and independent of tyrosine phosphorylation

Human type I interferons (IFN) require two receptor chains, IFNAR1 and IFNAR2c for high affinity (pM) binding and biological activity. Our previous studies have shown that the ligand dependent assembly of the type I IFN receptor chains is not identical for all type I IFNs. IFNbeta appears unique in its ability to assemble a stable complex of receptor chains, as demonstrated by the observation that IFNAR2c co-immunoprecipitates with IFNAR1 when cells are stimulated with IFNbeta but not with IFNalpha. The characteristics of such a receptor complex are not well defined nor is it understood if differential signaling events can be mediated by variations in receptor assembly. To further characterize the factors required for formation of such a stable receptor complex we demonstrate using IFN stimulated Daudi cells that (1) IFNAR2c co-immunoprecipitates with IFNAR1 even when tyrosine phosphorylation of receptor chains is blocked with staurosporine, and (2) IFNbeta1b but not IFNalpha2, is present in the immunoprecipitated receptor complex. These results demonstrate that the unique IFNbeta induced assembly of type I IFN receptor chains is independent of receptor tyrosine phosphorylation and the recruitment of additional proteins to the receptor by such events. Furthermore, the presence of IFNbeta1b in the immunoprecipitated IFN receptor complex suggests that IFNbeta interacts and binds differently to the receptor than IFNalpha2. These results suggest that the specific assembly of type I IFN receptor chains is ligand dependent and may represent an early event which leads to the differential biological responses observed among type I IFNs.     

Insulin receptor expression in the Burkitt lymphoma cells Daudi and Raji

The specific binding of insulin to either intact or Triton-solubilized Daudi cells (a Burkitt lymphoma cell line) was reduced by over 95% compared to that to control IM-9 lymphocytes due to a decrease in receptor number without a change in affinity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that 125I-labeled Daudi cells had reduced amounts (approximately 1/20th) of immunoprecipitable binding (alpha) subunit [mol wt (Mr), 130,000] of the receptor and a relatively abundant 210,000 Mr form not seen in IM-9 cells. The transmembranous (beta) subunit (Mr, 90,000) of the receptor, although not detected by 125I surface labeling, could be phosphorylated and, together with the 210,000 Mr form, exhibited the same 2-fold stimulation of phosphorylation by insulin as that in IM-9 cells. Northern blot hybridization revealed a decrease in Daudi cells of all four major species of insulin receptor mRNA. The Raji cell, another Burkitt lymphoma cell line, also exhibited reduced protein and genetic expression of the insulin receptor, indicating that reduced insulin receptor expression may be representative of other Burkitt lymphoma cell lines.     

Bovine plasmacytoid dendritic cells are the major source of type I interferon in response to foot-and-mouth disease virus in vitro and in vivo

Type I interferons (alpha/beta interferons [IFN-α/β]) are the main innate cytokines that are able to induce a cellular antiviral state, thereby limiting viral replication and disease pathology. Plasmacytoid dendritic cells (pDCs) play a crucial role in the control of viral infections, especially in response to viruses that have evolved mechanisms to block the type I IFN signal transduction pathway. Using density gradient separation and cell sorting, we have highly enriched a population of bovine cells capable of producing high levels of biologically active type I IFN. These cells represented less than 0.1% of the total lymphocyte population in blood, pseudoafferent lymph, and lymph nodes. Phenotypic analysis identified these cells as bovine pDCs (CD3(-) CD14(-) CD21(-) CD11c(-) NK(-) TCRδ(-) CD4(+) MHC II(+) CD45RB(+) CD172a(+) CD32(+)). High levels of type I IFN were generated by these cells in vitro in response to Toll-like receptor 9 (TLR-9) agonist CpG and foot-and-mouth disease virus (FMDV) immune complexes. In contrast, immune complexes formed with UV-inactivated FMDV or FMDV empty capsids failed to elicit a type I IFN response. Depletion of CD4 cells in vivo resulted in levels of type I IFN in serum early during FMDV infection that were significantly lower than those for control animals. In conclusion, pDCs interacting with immune-complexed virus are the major source of type I interferon production during acute FMDV infection in cattle.     

A glycosylation-deficient endothelial cell mutant with modified responses to transforming growth factor-beta and other growth inhibitory cytokines: evidence for multiple growth inhibitory signal transduction pathways.

An endothelial cell line (M40) resistant to growth inhibition by transforming growth factor-beta type 1 (TGF beta 1) was isolated by chemical mutagenesis and growth in the presence of TGF beta 1. Like normal endothelial cells, this mutant is characterized by high expression of type II TGF beta receptor and low expression of type I TGF beta receptor. However, the mutant cells display a type II TGF beta receptor of reduced molecular weight as a result of a general defect in N-glycosylation of proteins. The alteration does not impair TGF beta 1 binding to cell surface receptors or the ability of TGF beta 1 to induce fibronectin or plasminogen activator inhibitor-type I production. M40 cells were also resistant to growth inhibition by tumor necrosis factor alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) but were inhibited by interferon-gamma (IFN gamma) and heparin. These results imply that TGF beta 1, TNF alpha, and IL-1 alpha act through signal transducing pathways that are separate from pathways for IFN gamma and heparin. Basic fibroblast growth factor was still mitogenic for M40, further suggesting that TGF beta 1, TNF alpha, and IL-1 alpha act by direct inhibition of cell growth rather than by interfering with growth stimulatory pathways.     

Effect of in vitro interferon treatment on the rapid clearance of murine leukemia cells in vivo.

Previous work showed that interferon (IFN) can protect target cells from NK mediated lysis in vitro. In the present study we investigate the effect of IFN alpha/beta or IFN gamma treatment of three different murine leukemia cell lines. For this purpose FLC-745 (susceptible to the antiproliferative activity of IFN alpha/beta and gamma), FLC-3C18 (IFN alpha/beta -resistant and IFN gamma - susceptible) of DBA/2 origin and EL-4 (IFN alpha/beta - susceptible and IFN gamma - resistant) leukemia of C57B1/6 origin were treated with IFN alpha/beta or gamma in vitro and assayed for their susceptibility to natural resistance measured in vivo as organ rapid clearance 4 hr after iv injection into syngeneic mice. Using young or Poly I:C stimulated hosts, but not mice with low levels of natural resistance (i.e. older animals or mice treated with cyclophosphamide), slower elimination of treated cells was observed with: (a) FLC-745 cells treated with IFN alpha/beta and IFN gamma and (b) FLC 3C18 treated with IFN gamma. Such a delayed clearance was not observed with: (a) FLC-3C18 cells treated with IFN alpha/beta and (b) EL-4 leukemia cells preincubated with IFN alpha/beta or IFN gamma. These results suggest that under selected conditions IFNs can protect leukemic cells from in vivo natural reactivity.     

Electrostatic interactions in the cellular dynamics of the interferon-receptor complex

Using membrane preparations of the interferon receptor, prepared from cells of the Burkitt line, Daudi, we have examined the binding of three human recombinant alpha-interferons. 1. We discovered a binding titration of the interferons IFN-alpha A and IFN-alpha D in the pH range 6-9. Receptor binding, negligible at pH 6, rises to a maximum close to pH 9. We have shown that binding of IFN-alpha A at basic pH is to the same receptors as at neutrality and that IFN-receptor complexes extracted with digitonin are more stable at basic pH than they are at neutrality. 2. The recombinant interferon, IFN-alpha B, shows little change of binding in the pH range 6-9. At its basic optimum the binding of IFN-alpha A approaches that of IFN-alpha B, while at neutral pH the binding of IFN-alpha A is 3-4 times less. This difference at neutral pH is seen on intact cells as well as on membrane preparations. The specific activity of IFN-alpha B is close to that of IFN-alpha A, both of which are 10-20 times more active than IFN-alpha D; and the binding titration is, therefore, independent of the initial binding affinities. 3. Using hybrid IFNs constructed from the DNA sequences of alpha D and alpha B, we have isolated the sequence responsible for the binding titration to the segment comprising amino acids 61-92. Examination of these sequences reveals that Lys-84 is present in all the IFN-alpha except IFN-alpha B where it is replaced by Glu; and Tyr-90, present in most of the common IFN-alpha including alpha A and alpha D, is replaced by Asp in IFN-alpha B. Lys and Tyr would normally titrate in the pH range 6-9. We conclude that the binding titration is due to an electrostatic interaction and we propose that the interaction is between IFN-receptor complexes. The role of the interaction in the binding losses that accompany the antiproliferative effects of IFN is discussed.     

Interleukin 1 receptors on rabbit articular chondrocytes: relationship between biological activity and receptor binding kinetics

This study gives an account of the biologic and kinetic binding properties of interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta), and Glu-4 (an NH2-terminal mutant of IL 1 beta) to interleukin 1 (IL 1) receptors in rabbit articular chondrocytes. All three IL 1's demonstrated full agonist properties in their ability to stimulate prostaglandin E2 (PGE2) synthesis. IL 1 alpha was 23-fold more biologically active than IL 1 beta, which was around 110-fold more active than Glu-4 based on the concentration of IL 1 required for half-maximal stimulation of PGE2. The binding of all three ligands was concentration-dependent and saturable at 4 degrees C. Scatchard analysis of receptor binding data showed that the dissociation constant (KD) of IL 1 alpha was 46 +/- 12 pM, and the receptor density was 3120 sites/cell. The association of IL 1 alpha at 4 degrees C did not attain equilibrium until after 10 h at 100 pM of 125I-labeled IL 1 alpha. The dissociation of bound IL 1 alpha was very slow, t1/2 of 21 h, although only one class of high-affinity receptors was detected. The KD of IL 1 beta binding was 72 +/- 3 pM with a receptor density of 800 +/- 40 sites/cell. Dissociation of bound 125I-labeled IL 1 beta at 4 degrees C appeared to indicate the presence of two receptor subsets, a fast and a slower component with a t1/2 of 2 min and 5 h, respectively. The receptor binding affinity of Glu-4 was 324 +/- 3 pM, in line with its reduced biologic activity. Both IL 1 alpha and IL 1 beta are rapidly internalized in chondrocytes in a time- and temperature-dependent manner.     

Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response

The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-alpha) receptor. Treatment of cells with recombinant universal IFN-alpha A/D or IFN-beta revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-alpha/beta mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.     

Early plasma membrane depolarization by alpha interferon: biologic correlation with antiproliferative signal

Daudi lymphoma cells, of a line sensitive to growth inhibition by alpha interferon, showed dose-dependent plasma membrane depolarization within 10 min after exposure to natural or recombinant alpha interferons (10 to 1000 IU/ml). This biophysical change was detected flow cytometrically by measuring the intensity of fluorescent emission from cells stained with dye indicators of membrane potential. Subclones of Daudi lymphoma cells, resistant to growth inhibition by alpha interferon, showed no membrane depolarization. Parallel results were obtained in initial tests of an isologous pair of T cell and B cell lines which differ in sensitivity to growth inhibition. Thus, decreased membrane potential may herald an interferon signal for antiproliferative action.     

Interferon alpha induces rapid tyrosine phosphorylation of the alpha subunit of its receptor.

The mechanisms of generation of second messengers after binding of interferon alpha (IFN alpha) to its receptor remain unknown. We have studied the phosphorylation of the alpha subunit of the IFN alpha receptor, which is recognized by the monoclonal antibody IFNa receptor 3. Immunoblotting experiments showed that IFN alpha induced rapid tyrosine phosphorylation of the alpha subunit in the IFN alpha-sensitive H-929, U-266, and Daudi cell lines. Immunoprecipitation experiments performed with 32P-labeled cells showed that the alpha subunit is phosphorylated before IFN alpha treatment and that the level of phosphorylation increases after IFN alpha stimulation. Phosphoamino acid analysis confirmed the IFN alpha-induced tyrosine phosphorylation and demonstrated that the base-line phosphorylation corresponded to serine phosphorylation that increased 50% upon IFN alpha treatment. Tyrosine phosphorylation of the alpha subunit was time- and dose-dependent, further demonstrating the specificity of the process. Phosphorylation of the alpha subunit of the receptor occurred rapidly after IFN alpha binding, both at 37 and 4 degrees C. Exposure of the cells to the tyrosine kinase inhibitor genistein blocked the IFN alpha-induced tyrosine phosphorylation of this subunit of the IFN alpha receptor. In contrast H7, a specific protein kinase C inhibitor, and acute and chronic exposure to phorbol esters had no effect on tyrosine phosphorylation, suggesting that protein kinase C does not regulate the tyrosine phosphorylation of the alpha subunit of the IFN alpha receptor. No IFN alpha-induced tyrosine phosphorylation was observed in the IFN alpha-resistant U-937 cell line that expresses a variant IFN alpha receptor. Altogether these data suggest that tyrosine phosphorylation of the alpha subunit may play a role in the signal transduction pathway of IFN alpha.     

Receptor dynamics of closely related ligands: "fast'' and "slow'' interferons.

Two related human alpha interferons with 83% homology in their primary sequences show a similar specific activity on nonhuman cells, but a striking difference on human cells, on which alpha-1 shows 1-5% of the specific molar activity displayed by alpha-2. Both interferons were labelled with 125I, and their binding kinetics followed on growing cultures of the human Burkitt line Daudi. Binding of alpha-1 showed slower rates of association and faster rates of dissociation implying that differences in apparent binding affinity were responsible for the differences in specific molar activity. However, binding was shown to reach steady-state rather than an equilibrium, so differences in the dynamics of the ligand-receptor complexes may represent amplification of differences in the initial binding constant. alpha-2, but not alpha-1, induces a marked loss of binding sites leading to a high affinity steady-state binding. Inhibition of cell multiplication by both interferons depends on a continued stimulation by free ligands at steady-state. It is proposed that the differences in specific molar activity are, in the main, kinetic and cause alpha-1 and alpha-2 to behave respectively as "slow' and "fast' interferons.     

Detection of functional interferon alpha receptors in human neuroendocrine tumor cell lines using a new monoclonal antibody.

While first described as antiviral agents, interferons (IFNs) exhibit significant antiproliferative and antitumor effects as well. IFN alpha has been successfully used in clinical trials to treat several malignancies, including leukemias and certain solid tumors. While many cell types have been studied for IFN alpha receptor expression, very little is known about receptor expression on human neuroendocrine cells. Using a novel anti-IFN alpha receptor monoclonal antibody, we examined IFN alpha receptor expression in 10 human cell lines derived from tumors of neuroendocrine origin, including neuroblastoma, neuroepithelioma and small cell lung carcinoma. All cell lines studied displayed a similar pattern of IFN alpha receptor expression and 5 of 8 cell lines demonstrated reduced thymidine incorporation following IFN alpha treatment. Addition of exogenous IFN alpha caused a decrease in IFN alpha receptor expression, while differentiating agents, such as phorbol esters and retinoic acid, induced an increase in receptor number without altering receptor affinity.