Isolation and properties of genetic engineered human leukocyte interferons alphaI1 and alphaN

Using controlled pore glass chromatography and immunoaffinity chromatography on monoclonal antibodies NK-2 immobilized on Sepharose 4B, the electrophoretically homogeneous interferons alpha N and alpha I1 were isolated from the biomass of gene-engineered Pseudomonas sp. strains. In terms of specific activity on human fibroblast diploid cells, interferon alpha I1 does not differ from interferon alpha A, whereas the specific antiviral activity of interferon alpha N is as low as 2.10(7) JU/mg. The procedures for immunometric assay of interferons alpha N and alpha I1 have been elaborated. Various monoclonal antibodies to interferon alpha A and to natural leucocyte interferon were analyzed; among those the antibodies specifically interacting with interferons alpha N and alpha I1 (but not with interferon alpha A) were identified.     

The interferon-inducible 15-kDa ubiquitin homolog conjugates to intracellular proteins.

We have previously identified a 15-kDa interferon-induced protein that is recognized by affinity-purified rabbit polyclonal antibodies against ubiquitin (Haas, A. L., Ahrens, P., Bright, P. M., and Ankel, H. (1987) J. Biol. Chem. 262, 11315-11323). This ubiquitin cross-reactive protein (UCRP) possesses significant homology to a tandem diubiquitin sequence. Since the biological effects of ubiquitin arise from its covalent ligation to intracellular target proteins, we hypothesized that the multiple cellular responses to inteferon are mediated in part by an analogous conjugation pathway for UCRP. Rabbit polyclonal antibodies specific for UCRP were prepared against homogeneous recombinant protein. Affinity-purified anti-UCRP antibodies detected the induction of UCRP in interferon-beta-treated A549 cells and recognized a group of high molecular weight UCRP conjugates on immunoblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved cell extracts. Both free and conjugated UCRP are constitutively present at low levels in untreated cells, suggesting a role for UCRP ligation in normal cellular regulation, and significantly accumulate following interferon treatment. The temporal induction of free UCRP following interferon treatment preceded a delayed increase in UCRP conjugates. Treatment of A549 cells with type I interferons (alpha and beta) strongly induced the expression of free and conjugated UCRP, whereas the response to type II interferon (gamma) was significantly less. A survey of selected cultured cell lines showed differential induction of free versus conjugated UCRP pools in response to interferon. Interferon-beta treatment of A549, MG63, and U937 cells induced high levels of both free and conjugated UCRP, whereas only free UCRP levels increased in Daudi, Namalwa, and K562 cells. These results confirm that UCRP represents a functional ubiquitin homolog participating in a parallel pathway of post-translational ligation and provides a novel mechanism for the response of susceptible cells to the effects of interferon exposure.     

Conjugates of ubiquitin cross-reactive protein distribute in a cytoskeletal pattern.

Ubiquitin cross-reactive protein (UCRP), a 15-kDa interferon-induced protein, is a sequence homolog of ubiquitin that is covalently ligated to intracellular proteins in a parallel enzymatic reaction and is found at low levels within cultured cell lines and human tissues not exposed to interferon. Ubiquitin and UCRP ligation reactions apparently target distinct subsets of intracellular proteins, as judged from differences in the distributions of the respective adducts revealed on immunoblots. In this study, successive passages of the human lung carcinoma line A549 in the presence of neutralizing antibodies against alpha and beta interferons had no effect on the levels of either free or conjugated UCRP, indicating that these UCRP pools are constitutively present within uninduced cells and are thus not a consequence of autoinduction by low levels of secreted alpha/beta interferon. In an effort to identify potential targets for UCRP conjugation, the immunocytochemical distribution of UCRP was examined by using affinity-purified polyclonal antibodies against recombinant polypeptide. UCRP distributes in a punctate cytoskeletal pattern that is resistant to extraction by nonionic detergents (e.g., Triton X-100) in both uninduced and interferon-treated A549 cells. The cytoskeletal pattern colocalizes with the intermediate filament network of epithelial and mesothelial cell lines. Immunoblots of parallel Triton X-100-insoluble cell extracts suggest that the cytoskeletal association largely results from the noncovalent association of UCRP conjugates with the intermediate filaments rather than direct ligation of the polypeptide to structural components of the filaments. A significant increase in the sequestration of UCRP adducts on intermediate filaments accompanies interferon induction. These results suggest that UCRP may serve as a trans-acting binding factor directing the association of ligated target proteins to intermediate filaments.     

Cell-membrane phospholipase C is involved in inducing the antiviral effect of interferon

A monospecific inhibitory antibody directed to phospholipase C (phosphoinositidase C) blocked the antiviral effect of human interferons alpha and beta when tested on human quiescent fibroblasts challenged with the vesicular stomatitis virus. This action was due to specific inhibition of polyphosphoinositide hydrolysis because (a) the F(ab)₂ fragment of the antibody molecule was also inhibitory; (b) excess antibodies directed to phospholipase A₂ and to a phosphatidylcholine-preferring phospholipase C did not have any inhibitory effect, and (c) the combination of 12-O-tetradecanoylphorbol-acetate and calcium ionophore A23187 had an interferon-like antiviral effect which was not influenced by the inhibitory anti-phospholipase C antibodies. To avoid an interferon-like effect due to induction of interferon by second messengers, Vero cells, which lack interferon biosynthesis, were also used. Liposomes containing inositol 1,4,5-triphosphate and 1-oleoyl-2-acetyl-rac-glycerol protected Vero cells against the infection with the vesicular stomatitis virus. These results taken together show that phosphoinositide-derived second messengers are involved in triggering the antiviral effect of interferons alpha and beta.     

Modification of DNA repair by human interferons

We have found a new biological function of interferons, namely, their capacity to protect human cells from the action of some physical and chemical mutagens. To evaluate the protective effect of interferons the following criteria were applied: formation of sister chromatid exchanges (SCE) and chromosomal aberrations (CA), as well as viability of cells and intensity of DNA repair synthesis. Pretreatment of cells with natural interferon decreased the number of sister chromatid exchanges and chromosomal aberrations, induced by different mutagens, and increased the intensity of DNA repair synthesis. This is attributed to the ability of interferon to enhance certain phases of DNA repair. In the case of photomutagenic action of 8-methoxypsoralen (8-MOP) on the lymphocytes, when monoadducts (MA) only, or both monoadducts and interstrand cross-links (ICL) are formed, the antimutagenic effect of interferon is exhibited only with respect to ICL. Unlike the natural interferon, the recombinant alpha 2-interferon failed to have any effect on the lymphocytes of clinically healthy donors exposed to gamma-radiation. In the repair- deficient cells (Marfan's syndrome) the protection of natural interferon against the action of 4-nitroquinoline-1'-oxide and gamma- radiation was found to be reduced significantly and that of alpha 2-interferon was not manifested at all. Thus, the capacity of interferons to alter the DNA repair, conceivably, depends on the type of interferon and on the cell genotype.     

Microinjection of anti-interferon antibodies into cells does not inhibit the induction of an antiviral state by interferon.

Affinity-purified polyclonal antibodies directed against human lymphoblastoid interferon (IFN), Escherichia coli-derived human IFN-alpha 2, or two synthetic fragments of human IFN-alpha 1 all neutralized the antiviral activity of human alpha IFNs when added to the culture medium of MDBK cells together with IFNs. However, when these antibodies were microinjected into the cytoplasm or the nucleus of cells, subsequent treatment of the cells with IFNs induced full protection against vesicular stomatitis virus. This suggests that IFNs themselves need not act in the cytoplasmic compartment or the nucleus to induce an antiviral state.     

Interferons in the management of neuroendocrine tumors and their possible mechanism of action.

Alpha interferons at doses of 3-9 MU subcutaneously, three to seven times/week, have been administered to 32 patients with malignant endocrine pancreatic tumors. The objective biochemical response rate was 63 percent with a median duration of 20.5 months. Significant reduction of tumor size was only noticed in 20 percent of the patients. Alpha interferon administered to 111 patients with malignant carcinoid tumors showed objective biochemical responses in 42 percent of the patients with a median duration of 32 months. Another 39 percent of the patients showed stabilization of disease without any further tumor growth. Subjective improvement was noticed in 70 percent of the patients. When survival data are analyzed in patients with malignant carcinoid tumors, the median survival from start of treatment was 80+ months in the group of patients treated with alpha interferon, which should be compared with only eight months in a historical group treated with chemotherapy (streptozotocin plus 5-fluorouracil). The adverse reactions to alpha-interferon treatment are dose-dependent and include, mainly, flu-like symptoms, fatigue, and low-grade weight loss. Autoimmune reactions are noted in about 20 percent of the patients. Patients treated with recombinant alpha interferons might develop neutralizing interferon antibodies (6-27 percent), which abrogate the anti-tumor response. The anti-tumor effect in neuroendocrine tumors includes anti-proliferation, apoptosis, differentiations, and cytotoxic/cytostatic effects. Furthermore, immunomodulation is obtained by increased expression of class I antigens on tumor cells. Four patients also developed antibodies directed against carcinoid tumor cells. Alpha interferons induce several nuclear enzymes such as 2'-5'-A synthetase, p-68 kinase, and Mx-A proteins, which are involved in a downregulation of expression of growth factors, oncogenes, and peptide hormones, leading to anti-proliferation and/or apoptosis. The response to alpha-interferon treatment might be predicted by analysis of the induction of 2'-5'-A synthetase in samples from neuroendocrine tumors. Stimulatory tests of hormone secretion, such as meal stimulation of pancreatic polypeptide secretion or secretin test, clearly demonstrate a normalization during alpha-interferon treatment, which might depend on reduced peptide production and/or secretion but also on eradication of malignant cell clones. In summary, alpha interferons have demonstrated significant anti-tumor effects in patients with malignant neuroendocrine gut and pancreatic tumors. The adverse reactions are dose-dependent and manageable. The anti-tumor effects of alpha interferons are pleiotropic and include several direct effects on tumor cells but also immunomodulation.     

Antiviral and protein-inducing activities of recombinant human leukocyte interferons and their hybrids.

The antiviral activities of recombinant human leukocyte interferons IFN-alpha A and IFN-alpha D as well as five hybrids of these interferons against retroviruses, vesicular stomatitis virus, and encephalomyocarditis virus were studied in feline, human, and murine cells. Although these interferon species had widely different potencies, their activities against these viruses were, in general, proportional. The IFN-alpha A/D (Bgl) hybrid was the most potent species, and the IFN-alpha D/A (Bgl) hybrid was the least potent. However, the latter species did not interfere with the action of the former species. Like natural human leukocyte interferon, each of the seven species of recombinant interferons induced the synthesis of at least five proteins in human fibroblasts, whereas induction of only one such protein was readily detected in a feline fibroblast line in which these interferon species inhibited the replication of all three viruses.     

Differential effects of pure human alpha and gamma interferons on fibroblast cell growth and the cell cycle

Pure human alpha and recombinant gamma interferons had differential effects on two strains of fetal lung fibroblasts in vitro. Alpha interferon had little effect on long-term cell growth, whereas gamma interferons, both glycosylated and non-glycosylated, were cytotoxic. However, when synchronized cells were studied, alpha interferon prolonged both G1 and S + G2 phases of the cell cycle, whereas gamma interferon only affected the G1 phase.     

Polyclonal antibodies to chlorosome proteins as probes for green sulfur bacteria.

We found that polyclonal antibodies raised against chlorosome polypeptides from green sulfur bacteria reacted to Chlorobium tepidum, Chlorobium limicola, and Chlorobium phaeobacteroides but not to Chloroflexus aurantiacus. These antibodies successfully labeled only green sulfur species in marine microbial mat samples. Our results suggest that these antibodies may be useful as immunohistochemical probes.     

Interferon action: nucleolar and nucleoplasmic localization of the interferon-inducible 72-kD protein that is encoded by the Ifi 204 gene from the gene 200 cluster

The interferons are cytokines with antiviral, cell growth regulatory, and immunomodulatory activities. These activities are mediated by the proteins induced by the interferons. Earlier we described a gene cluster (the 200 cluster) consisting of at least six adjacent, interferon-activatable genes located next to the erythroid alpha-spectrin locus on murine chromosome 1. The genes of the cluster arose by repeated gene duplication and they specify proteins with pronounced sequence similarity. We have now raised polyclonal antibodies against a segment from one of these proteins (the 204 protein of 72 kD). Using these, we established that the 204 protein is a phosphoprotein whose level in cells from various murine lines can be increased up to 75-fold upon treatment with alpha interferon. Experiments involving fractionation of cell lysates and indirect immunofluorescence microscopy of control and interferon-treated cells revealed that the 204 protein is nucleolar and nucleoplasmic. This conclusion was confirmed by co-localization with B23, a known nucleolar protein. The 204 protein is the first interferon-induced protein found to be located in the nucleoli, the subcellular organelles of ribosomal RNA production and ribosome assembly. It remains to be seen whether the 204 protein affects any of these processes. Studies on 204 protein function should be facilitated by the availability of complete cDNA clones and the finding of cell lines in which the expression of this protein is impaired.     

Heterogeneity of tubulin epitopes in mouse fetal tissues

A panel of six monoclonal antibodies against alpha (TU-01, TU-03, TU-04, TU-05, TU-09) or beta (TU-13) subunits of tubulin was used to study expression of tubulin epitopes in 14-day-old mouse embryos. Specificity of antibodies was confirmed by immunoblotting experiments. Monoclonal antibodies TU-01, TU-09 and TU-13, like the polyclonal antibody reacted essentially with all tissues, whereas other antibodies displayed differential reactivity. Most notably, TU-03 reacted very strongly with simple epithelia and basal layer of stratified epithelial layers. TU-04 recognized maturation related changes in spinal cord. Reactivity of TU-05 was restricted to central nervous system and peripheral nerves. Present results document immunohistochemical heterogeneity of tubulin in fetal tissues and suggest the existence of maturation and tissue specific epitopes of tubulin in developing organs.     

Effect of human interferons on cAMP phosphodiesterase activity in a culture of human ovarian carcinoma cells (CaOv)

Crude and purified human interferons of alpha type exerted 2 step inhibition of cAMP phosphodiesterase activity in CaOv cells: in 4 and 24 hours after cells treatment with interferon. The maximal inhibition was obtained in response to interferon doses 1200-2000 IU/ml. In contrast to natural interferons the human alpha 2 recombinant interferon (20-25000 IU/ml) did not inhibit the cAMP phosphodiesterase activity in CaOv cells.     

Analysis of mammalian dynein using antibodies against a polypeptides of sea urchin sperm flagellar dynein

Two different affinity-purified polyclonal antibodies were prepared against A polypeptides of dynein 1 extracted from sea urchin sperm. These antibodies, named AD1 and AD2, reacted exclusively with the alpha and beta heavy chains of dynein 1. Using these antibodies, we analyzed their cross-reactivity with dynein of mammalian cells. Immunohistochemically, both AD1 and AD2 stained dynein-related structures such as cilia of rabbit tracheal epithelia and flagella of rat spermatozoa. Immunoblots of the proteins extracted from mammalian cilia and flagella revealed the presence of A polypeptide-like proteins which cross-reacted with AD1 and AD2. Immunoblot analysis showed that the cross-reactive proteins were localized to the 370-kDa band of rabbit cilia and the 390- and 350-kDa bands of rat sperms. The reaction patterns showed that there were some differences between the two antibodies. On ciliary protein immunoblots, AD1 recognized about half of the broad band region which reacted with AD2, and AD1 also recognized only the 350-kDa band of the flagella extract, suggesting that the antibody reveals only a beta-like polypeptide. Immunoprecipitation studies using the ciliary proteins and AD2 confirmed that the immunoreactive protein had ATPase activity. Given these results, we have characterized mammalian dyneins previously reported by other laboratories.     

Heterogeneity of tubulin epitopes in mouse fetal tissues

Summary A panel of six monoclonal antibodies against alpha (TU-01, TU-03, TU-04, TU-05, TU-09) or beta (TU-13) subunits of tubulin was used to study expression of tubulin epitopes in 14-day-old mouse embryos. Specificity of antibodies was confirmed by immunoblotting experiments. Monoclonal antibodies TU-01, TU-09 and TU-13, like the polyclonal antibody reacted essentially with all tissues, whereas other antibodies displayed differential reactivity. Most notably, TU-03 reacted very strongly with simple epithelia and basal layer of stratified epithelial layers. TU-04 recognized maturation related changes in spinal cord. Reactivity of TU-05 was restricted to central nervous system and peripheral nerves.Present results document immunohistochemical heterogeneity of tubulin in fetal tissues and suggest the existence of maturation and tissue specific epitopes of tubulin in developing organs.     

The integrin beta 1 subunit associates with the vitronectin receptor alpha v subunit to form a novel vitronectin receptor in a human embryonic kidney cell line.

We describe a novel integrin heterodimer on the surface of the human embryonic kidney cell line 293. This receptor is comprised of alpha v and beta 1 subunits, each of which has been previously found in association with other integrin subunits. This alpha v.beta 1 complex was identified as the predominant vitronectin receptor (VnR) on the surface of 293 cells by immunoprecipitation with antibodies raised against the alpha v subunit. Polymerase chain reaction analysis detected mRNAs for alpha v and beta 1 subunits while no evidence was obtained for beta 2, beta 3, or alpha IIb integrin subunit mRNA. Immunoprecipitation of surface-iodinated proteins with antibodies to alpha v gave bands of 150 and 120 kDa. The 120-kDa band reacted with antibodies to beta 1 in immunoblotting experiments. 293 cells adhere to vitronectin, fibronectin, laminin, and collagen IV, while von Willebrand factor and fibrinogen, known ligands of the VnR (alpha v.beta 3), did not support adhesion. A polyclonal antibody directed against both subunits of the VnR (alpha v, beta 3) inhibits attachment of 293 cells to vitronectin but not to other adhesive proteins. A beta 1-specific monoclonal inhibited attachment to fibronectin, laminin, and collagen IV, known ligands of beta 1 integrins, as well as vitronectin. This novel (alpha v. beta 1) VnR thus appears to mediate cell adhesion exclusively to vitronectin, in contrast to previously described VnRs which have multiple ligands.     

Interferon levels in human pulmonary tumors are lower than plasma levels

It is uncertain whether interferon levels in the interstitial fluid of tumors are equivalent to interferon plasma levels and we have investigated this problem in human pulmonary tumors by infusing human recombinant interferon alpha A and natural interferon Beta for about three hours before surgery. By determining the hematocrit and hemoglobin content it was possible to calculate interferon values (International Units/g wet tissue) present in the interstitial fluid of tumor and lung samples, simultaneously. In 14 patients (epidermoids, n = 9 and adenocarcinomas, n = 5) interferon levels in tumor and "normal" lung expressed as percentages of interferon plasma levels were: 9.5 +/- 3.9 and 29.8 +/- 6.9 for recombinant interferon alpha A and 3.1 +/- 0.4 and 10.1 +/- 2.4 for natural interferon Beta, respectively. Differences for both interferons are statistically significant (p less than 0.05). To our knowledge these are the first data indicating that interferon levels in pulmonary tumor interstitial fluid are markedly lower than those in normal lung although they do not clarify the main factor responsible for the decrease, they explain at least in part the negligible therapeutic activity of interferons in these tumors and emphasize the need for new approaches for improving the therapeutic index of interferons.     

Antibody to staphylococcal enterotoxin A-induced human immune interferon (IFN gamma)

Antiserum to human gamma interferon (IFN gamma) was produced in rabbits immunized with partially purified (10(4.8) to 10(6.2) antiviral U/mg protein) staphylococcal enterotoxin A-induced IFN gamma. Staphylococcal enterotoxins, phytohemagglutinin M, concanavalin A, and pokeweed mitogen-induced antiviral activity in human leukocyte cultures was neutralized to undetectable levels by the antiserum. However, human leukocyte interferon (IFN alpha), human fibroblast interferon (IFN beta), and mouse interferons were not neutralized by the antiserum. After determining the antiserum was specific for IFN gamma and did not neutralize other known types of interferon, it was used with antibody to human IFN alpha to demonstrate the type(s) of interferon stimulated by some new inducers and antigens. Galactose oxidase- and calcium ionophore-induced interferons were neutralized to undetectable levels by the antiserum to IFN gamma. Interferon produced in leukocyte cultures from tuberculin-negative individuals stimulated with tuberculin-purified protein derivative or old tuberculin was IFN alpha, whereas interferon from tuberculin-positive individuals was a combination of alpha and gamma IFN. In addition, the antiserum neutralized the anticellular and natural killer cell enhancement activities of IFN gamma preparations. The specificity of this antiserum for IFN gamma indicates that it is an additional, powerful tool for identifying and classifying known and new interferons produced in vitro or in vivo and for investigating the role(s) of IFN gamma during the course of infectious, neoplastic, and autoimmune diseases.     

Immunity to type XI collagen in mice. Evidence that the alpha 3(XI) chain of type XI collagen and the alpha 1(II) chain of type II collagen share arthritogenic determinants and induce arthritis in DBA/1 mice.

To determine whether native bovine type XI collagen (BXI) is arthritogenic, five strains of inbred mice were immunized with BXI/CFA. Arthritis was not observed in any of these strains, though it was prevalent in DBA/1 and B10.RIII controls immunized with bovine type II collagen (BII). Antisera from BXI-immunized mice reacted with mouse type XI collagen (MsXI), weakly with the alpha-chains of BXI, and minimally with mouse type II collagen (MsII). However, antisera to BII reacted with MsII and MsXI, indicating antibodies to conformation-independent epitopes shared by alpha 1(II) and alpha 3(XI). Mice immunized with BXI containing a small amount of BII developed arthritis much like those immunized with BII; sera from these mice reacted with MsXI and MsII. Delayed-type hypersensitivity responses differed from IgG responses, i.e., BXI elicited responses to alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II); BII, to alpha 3(XI) and alpha 1(II) exclusively. To determine whether alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II) are arthritogenic, DBA/1J mice were immunized with each alpha-chain. Arthritis was seen in mice injected with alpha 3(XI) or alpha 1(II). Sera to both alpha-chains reacted similarly with MsII and peptide fragment alpha 1(II)-CB11. Epitope mapping using polyclonal and mAb to type II collagen revealed that all polyclonal and 11 of 14 mAb reacted with alpha 3(XI) and alpha 1(II), whereas three mAb reacted only with alpha 1(II). In conclusion, BXI is immunogenic but not arthritogenic in five strains of mice, whereas alpha 3(XI) and alpha 1(II) are arthritogenic and immunogenic in DBA/1 mice and share greater than or equal to 11 epitopes recognized by autoantibody.     

Additive and synergistic effects of a novel antiestrogen, toremifene (Fc-1157a), and human interferons on estrogen responsive MCF-7 cells in vitro

The effect of human interferons alpha and gamma alone and in combination with a novel antiestrogen toremifene were studied in vitro using MCF-7 cell line, an estrogen receptor positive and antiestrogen sensitive cell line. The effects were evaluated by a simple bioluminescence method with which the number of living cells was obtained as cellular adenosine triphosphate (ATP) content. The growth of MCF-7 cells was inhibited both by interferon alpha and interferon gamma. At least additive effect was evident when the cells were exposed to combination of interferons and toremifene: the combination was additive with interferon gamma + toremifene and synergistic with interferon alpha + toremifene. The combination of toremifene and interferons may have clinical importance.