Interferons (IFNs) are secretory proteins produced by virus-infected cells. The name interferon refers to their ability to interfere with viral replication. However, IFNs induce several pleiotropic responses, including antiviral, antitumor, immunomodulatory, antiparasitic, and antiproliferative activities. In addition to viral infection, double-stranded RNA (dsRNA), fungal cell wall products, and other cytokines also induce the production of IFNs. dsRNA may be a physiologically relevant regulator of IFN synthesis because it is thought to be an intermediate product of viral infection. It not only induces the expression of IFN and IFN-inducible genes but also serves as a cofactor for some IFN-induced enzymes.
I. INTERFERON GENES
Distinct cellular genes encode for various IFNs. The current IFN nomenclature is based on gene sequences. The major types IFN-α and -ω, IFN as IFN-β, and IFN-γ are produced by leukocytes, fibroblasts, and lymphocytes, respectively. There are 18 IFN-α nonallelic genes in humans among which four are pseudogenes. IFN-ω is represented as six nonallelic genes of which five are pseudogenes. Single genes encode for IFN-β and -γ. IFN-α, -ω, and -β genes are clustered on the short arm of human chromosome 9. An IFN-γ gene with three introns is located on human chromosome 12. All these genes are thought to have arisen from a single ancestral gene. A secretory signal sequence is present in all IFNs, which is cleaved off prior to their secretion. The mature forms of IFNs contain 165-172 amino acids.
Expression of IFNs is primarily regulated at the transcriptional level, although the rate of mRNA decay may also contribute. Among IFNs, regulation of the IFN-β gene is relatively well understood. Virus or dsRNA activated transcription factors, IFNgene regulatory factor-1 (IRF-1) and NF-κB (nuclear factor-κB), bind to specific elements in the IFN-β gene promoter and stimulate transcription. A negative factor IRF-2, related to but distinct from IRF-1, inhibits gene expression. IFN-α genes are regulated by distinct promoter elements and the corresponding cognate factors. Although viruses induce both IFN-α and IFN-β, there are distinguishable differences between the patterns of induction of these genes. The virus-regulated element (VRE) of IFN-α contains IRF-binding elements but not NF-κB-binding sites. The exact sequence of IRF-1- like elements present in the IFN-α gene determines its inducibility by various agents such as virus infection or IRF-1. IRF3 and IRF-7, the recently discovered members of the IRF-family, also induce IFN- α gene expression.
The expression of IFN-γ gene is induced in T cells activated by antigens, mitogen anti-CD3, anti-CD28 antibodies, IL-2, IL-12, or IL-18. Unlike IFN-α/β, expression of the IFN-γ gene is regulated by elements both upstream of the transcription start site and those within the introns. Pregnant domestic ruminants express a novel type of IFN-ω called IFN-τ. These proteins are produced in the absence of any stimuli by the trophoectoderm of the concepti and appear to signal via specific receptors in the endometrium to maintain an appropriate milieu for the embryo.
II. INTERFERON RECEPTORS
IFN-α and IFN-β bind to a similar receptor(s), whereas IFN- γ binds to a different one. IFN-α and IFN-β receptors have high affinity for their ligand (10-10M) with an abundance of 0.5-5 × 103/cell. After generating the necessary response, the ligand- receptor complexes are internalized and degraded. The exact composition of the IFN-α/β receptor is still controversial. The minimal receptor consists of two polypeptides, IFNAR1 and IFNAR2. Human IFN- α/β receptor genes are located on chromosome 21. Mice lacking IFNAR1 gene are highly susceptible to infectious agents.
The IFN-γ receptor is composed of two distinct polypeptides: the ligand-binding α chain and the signal transducing β chain. α chain genes are present on chromosomes 6 and 10 of human and murine species. Genes for the β chain have been localized on chromosomes 21 and 16 in humans and mice, respectively. Functional IFN-γ is a dimer. Therefore, two α and two β chains form the active receptor in a ligand dependent manner. Mice lacking the IFN-γ receptor or patients with a mutant receptor are highly susceptible to mycobacterial infections.
III. INTERFERON-REGULATED PROMOTER ELEMENTS
IFN actions are mediated by IFN-stimulated genes (ISG). Some ISGs are exclusively induced by IFN-α/β or IFN-γ. Other ISGs are induced by both IFNs. Specific response elements present in the promoters of the IFN-stimulated genes (ISGs) sense IFN-stimulated signals. Induction of ISGs by IFN-α/β is a rapid but transient process, which often lasts 3-4 h. The temporal control of IFN-γ- stimulated genes is variable. After the initial induction, ISG transcription declines and returns to basal level.
A conserved 15-bp element, the IFN-stimulated response element (ISRE), is essential for inducing ISGs by IFN-α/β. The consensus sequence for ISRE is AGGTTTCNNTTTCCT. In contrast, a variety of elements respond to IFN-γ. These include ISRE, the X and Y box elements of HLA class II genes, and the IFN-γ activation sequence (GAS). GAS-like sequences are present in the promoters of several cytokine or growth factor-inducible genes. The consensus sequence for GAS is TTNNNNNAA.
IV. INTERFERON-STIMULATED TRANSCRIPTION FACTORS
IFN-stimulated gene factors (ISGFs) bind to ISRE and regulate gene expression. Two such factors, ISGF1 and ISGF2, are constitutive, although ISGF2 is further induced by IFN treatment. ISGF1 and ISGF2 are not the primary regulators of several ISGs. ISGF2 is identical to IRF-1. ISGF3 is rapidly activated in the cytoplasm by IFN-α/β, which then translocates to the nucleus and stimulates ISG transcription. ISGF3 is composed of four proteins: p48 (ISGF3γ or IRF9), STAT1α (p91), STAT1β (p84), and STAT2 (p113). The acronym STAT (signal transducing activator of transcription) defines the bifunctional nature of these proteins. IFNs activate the STAT proteins using Janus tyrosine kinases (JAK). STAT1α and STAT1β are generated from the same gene. STAT1α and STAT1β are identical except that the former has an additional 38 amino acid long sequence at the C terminus. STAT1α alone can fully restore IFN responses in STAT1-/- cells. STAT2 is encoded by a distinct gene. p48 (IRF-9 or ISGF3γ) is a member of the IRF-myb family of DNA-binding proteins.
V. STAT PROTEINS
These proteins not only transduce the signals but also act as transcription factors. The first two members of the STAT family, STAT1 and STAT2, specifically participate in the IFN-initiated JAK-STAT pathway. Five other STATs, discovered later, have been shown to participate in various cytokine-induced signaling pathways. Most STATs have the following characteristics: (1) interact with cytoplasmic tails of cytokine or growth factor receptors, (2) are tyrosine phosphorylated by JAKs, (3) contain SH2 and SH3 (SH src homology) domains, (4) homo- or heterodimerize with other members of their family, (5) translocate to the nucleus after activation and dimerization, (6) bind to DNA in a sequence-specific and activation dependent manner, and (7) induce gene transcription.
SH2 domains are critical for homo- or heterodimerization in response to ligand-induced tyrosine phosphorylation and maintenance of the signal specificity. A conserved tyrosine residue at six to eight amino acids downstream of the SH2 domain is crucial for STAT dimerization. A transactivation domain (TAD) is present at the C terminus. Phosphorylation of a specific serine residue in the TAD is also crucial for deriving optimal transcription.
VI. JANUS TYROSINE KINASES
These enzymes are essential for cytokine signaling. Most JAKs have been isolated by polymerase chain reaction based on conserved oligonucleotides of protein tyrosine kinase domains. Because their physiologic functions were uncertain at the time of discovery, they were designated as just another kinases (JAK). JAKs are 120-130 kDa in size and contain seven conserved JAK homology (JH) domains. The functional tyrosine kinase activity is located in the JH1 domain. The JH2 domain, also known as the pseudokinase domain, is highly homologous to JH1 but does not have kinase activity (Fig. 1B).Therefore, the acronym JAK also refers to their structural analogy to Janus, the Roman god of gateways who possessed two heads. JAKs do not have SH2 or SH3 domains. JAKs do not have substrate specificity toward STATs. Most JAKs require another JAK to be functional because the intermolecular tyrosine phosphorylation of the JH1 domain stimulates their action. To date, four JAKs--JAK1, JAK2, JAK3, and Tyk2--have been identified.
FIGURE 1 Signal transduction by interferons. (A) IFNs-α/β, employing tyk-2 and JAK1, induce the tyrosine phosphorylation of STAT1 and STAT2 on binding to their receptor. STATs are ligand bond receptors recruited to the receptor, phosphorylated. Following this, a STAT1:STAT2 dimer is formed, which migrates to the nucleus, associates with the p48 subunit, and stimulates ISRE-dependent gene expression. Similarly, FN-γ induces the tyrosine phosphorylation of STAT1 using JAK1 and JAK2, which binds to GAS and activates gene expression. (B) Structure of a typical STAT molecule. DBD, DNA-binding domain; SH, Src homology domain; Y, critical tyrosine; S, serine; TAD, transcription activating domain; N, amino terminus; C, carboxyl terminus. (C) Structure of a typical JAK. JH, JAK homology domain; PKD, pseudokinase domain; KD, kinase domain.
VII. SIGNAL TRANSDUCTION BY INTERFERONS
A. IFN-γ/α Signaling
The IFN-α/β receptor is composed of two subunits, IFNAR1 and IFNAR2. The cytoplasmic tail of IFNAR1 is preassociated with Tyk2. JAK1 is associated to the intracellular domain of IFNAR2. Binding of IFN-α/β to the receptor causes mutual tyrosine phosphorylation of JAK1 and Tyk2. Activated Tyk2 then phosphorylates the tyrosine (at 466) of IFNAR1. This phosphotyrosine serves as a docking site for STAT2. Tyrosyl phosphorylation of STAT2 at position 690 creates a binding site for STAT1. STAT1 is then phosphorylated by JAK1 at tyrosine 701. The STAT2-STAT1 dimer dissociates from its receptor, migrates to the nucleus, and associates with p48 (ISGF3γ) to form ISGF3. STAT2, unlike STAT1, is not phosphorylated at serine. STAT2 contains a critical transcription-activating domain at the C terminus.
B. IFN-ω Signaling
The dimeric IFN-γ first interacts with two molecules of the ligand-binding IFNGR-α chain, associated with JAK1. Subsequently, two molecules of the signal- transducing IFNGR-β chain, preassociated with JAK2, are recruited into the complex. Neither JAK1 nor JAK2 is phosphorylated in the absence of the other. Thus, the JAK-STAT pathway does not involve classical kinase cascades as in the case of Ras-Raf-Map kinase pathways. Aggregation of receptor polypeptides brings the JAKs closer, leading to mutual phosphorylation and the phosphorylation of IFNGR-α at tyrosine 440 by JAK1. Tyrosinephosphorylated IFNGR-α serves as a docking site for the SH2 domain of unphosphorylated STAT1α.
JAK2 then phosphorylates STAT1α at tyrosine 701. Phosphorylated STAT1α serves as an anchor for the second STAT1α molecule, leading to its tyrosine phosphorylation. The STAT1 dimer then migrates to the nucleus, binds to GAS, and induces transcription. Mutant STAT1α (lacking tyrosine 701) is not phosphorylated and therefore cannot translocate to the nucleus. As mentioned earlier, serine phosphorylation at 727 is essential for strong transcriptional stimulation by STAT1α. The kinase responsible for this activity is unknown. A nuclear GTP-binding protein, Ran, facilitates the nuclear import of STAT1. Finally, the transcriptional activity of STAT1 is terminated by unknown tyrosine phosphatases. Thus, JAK1 and STAT1 are shared components of IFN-α/β and IFN-γ signaling pathways.
Discovery of the IFN-stimulated JAK-STAT pathway served a paradigm for most cytokine signal transduction pathways. Following this, five other STATs and four JAKs that govern the cellular responses of various cytokines and other growth factors were discovered.
VIII. INHIBITORS OF JAK-STAT SIGNALING
Studies have identified several cellular inhibitors of the JAK-STAT pathway. A family of proteins, SOCS (suppressor of cytokine signaling) or SSI (STATinduced STAT inhibitor), inhibit the activation of STATs. These proteins contain a SH2 domain, bind to JAKs, and suppress their tyrosine kinase activity. They are induced by the same ligands that activate STATs. For example, SOCS-1 is inducible by GM-CSF, IL-3, IL-13, and IFN-γ. Overexpression of SOCS-1 blocks the antiproliferative and antiviral actions of IFN-α. Certain ubiquitously expressed protein inhibitors of activated STATs (PIAS) also suppress STAT functions. PIAS1 inhibits STAT1. It appears that physical levels of PIAS and STAT molecules determine the outcome of cellular response to a given cytokine.
IX. BIOLOGICAL EFFECTS OF JAK-STAT GENE DISRUPTION
Targeted disruption of STATs and JAK genes in mice has been reported (Table I). STAT1-/- mice are normal, except for a loss of innate immunity against viral infections and high susceptibility to cancers. Disruption of STAT2 causes embryonic lethality, indicating its crucial role in the development. Disruption of the JAK1 gene causes postnatal lethality. JAK1-/- pups fail to nurse and die. Neurons from these mice undergo rapid apoptosis compared to those from wild-type mice. Thus, neuronal development seems to be incomplete in these mice. Disruption of JAK2 causes embryonic lethality due to defective embryonic hematopoiesis. Tyk2-/- mice are normal in their appearance but their physiological defects are unclear at this stage.
TABLE I Defective IFN System and PathogenesisGene product Defects detected in knockout/human patients IFN-γ receptor Susceptibility to bacterial infections in mice and humans and carcinogens in mice STAT1 Loss of innate immunity and susceptibility to carcinogens in mice STAT2 Embryonic lethality in mice JAK1 Postnatal lethality, neuronal development defects in mice JAK2 Lack of hematopoiesis, leukemia in humans IFNAR1 Loss of antiviral responses in mice IRF1 Immunodeficiency, loss of tumor suppressor functions in mice and humans IRF2 Loss of antiviral effects and B-cell proliferation ICSBP Immunodeficiency and chronic myelogenous leukemia in mice p48 Loss of antiviral responses and immune system defects in mice PKR Loss of stress responses, antiviral functions, and signaling defects in mice RNAseL Loss of antiviral responses, defective thymic apoptosis in mice CIITA Loss of antigen presentation, bare lymphocyte syndrome DAPK Loss of antimetastatic function, loss of expression in human lymphomas
X. INTERFERON-STIMULATED GENES
A number of ISGs induced by IFNs-α/β and IFN-γ regulate the cellular responses (Table II). Nearly 250 genes are induced and 10 others are repressed by IFNs. This section discusses a few of the well-studied gene products that have influences on viral replication and neoplastic cell proliferation. For example, protein kinase R (PKR), a dsRNA-activated enzyme, phosphorylates several cellular proteins, including the eukaryotic protein synthesis initiation factor-2α (eIF-2α). Phosphorylation of eIF-2α results in the cessation of polypeptide chain initiation, thus leading to cellular and viral growth inhibition. PKR also activates transcription factor NF-κB by phosphorylating its inhibitor IκB. PKR is essential for certain cellular stress responses.
2',5'-Oligoadenylate synthetases (2'5' AS) are a family of unique enzymes induced by IFNs. Isoforms of 2'5' AS, whose molecular masses range from 20 to 100 kDa, are either derived from a single gene due to alternate splicing or from different genes. Their subcellular location is also different. Using dsRNA as a cofactor, they polymerize ATP into 2'-5'-linked oligoadenylates [2'-5' (A)n]. These products activate an endoribonuclease, RNase L, which cleaves cellular or viral RNAs.
IFN-α and IFN-β also induce a number of GTPbinding proteins, including the Mx family, which inhibits the replication of several viruses. The IGTP protein is critical for antitaxoplasma responses in vivo. Inducible nitric oxide synthase is critical for macrophage-dependent antimicrobial and antitumor responses. MHC class I and II antigens and proteasome components are critical for antigen presentation and development of immune responses.
Cell Growth Regulators
In addition to regulating IFN and ISGs, IRF (IFN gene regulatory factor) proteins control cell growth and immune responses. IRFs are a family of proteins that include IRF1, p48 (ISGF3γ), IRF2, IRF3, IRF4 (Pip), IRF7, ICSBP, and v-IRF of Kaposi's sarcomaassociated herpes virus. All these proteins have a structurally similar DNA-binding domain at their N termini and nonconserved C termini. However, except for this similarity, these proteins are quite diverse in their functions. Both IFN-dependent and IFNindependent actions of IRFs are known.
IRF1-/- cells fail to undergo apoptosis and are transformed readily by activated oncogenes, indicating a tumor suppressor role for this protein. The IRF1 gene is deleted in myelodysplasia and myelocytic leukemia. IRF1-/- mice are immunodeficient due to defects in T cell, NK cell maturation. Mice lacking IRF1 are also defective in antigen presentation. p48 is critical for antiviral responses and antigen presentation.
Cells lacking the p48 gene die rapidly upon exposure to cytotoxic drugs compared to wild-type cells. These results suggest that p48 plays other roles in cell growth control in an IFN-independent manner. In contrast, IRF2 induces cell proliferation and acts as an oncogene. ICSBP, lymphoid transcription factor, is also a tumor suppressor. ICSBP-/- mice develop a chronic myelogenous leukemia (CML)-like disease and immunodeficiency. ICSBP gene expression is suppressed in cells derived from patients with myeloid leukemia. ICSBP suppresses the expression of the BCR-ABL oncogene. IRF3 and IRF7 appear to be essential for IFN gene expression.
TABLE II Mammalian Interferon-Regulated GenesGene Function Inducer β2-Microglobulin MHC class I light chain α,β,γ CIITA Transcription factor γ C56,561,PIF-2 Unknown α,β>γ,dsRNA CRG-2 Monokine α,β,γ C/EBP-E Transcription factor γ>α,β Cytochrome b Mitochondrial gene product α inhibits Cytochrome c oxidase, subunit I Mitochondrial gene product α inhibits 2-5 (A)synthetase Antiviral enzyme α,β>γ,dsRNA Inducible nitric oxide synthase Macrophage effector LPS, >γ ISG 54,PIF-2 Unknown α,β>γ,dsRNA FcγRI Binds IgG-Fc γ GBP,γ67 GTP binding γ>α,β IGTP Antitaxoplasma activity γ Indoleamine-2,3-dioxygenase Protozoan inhibition γ>α,β ISG-15 Ubiquitin like α,β>γ,dsRNA ISG-20 PML nuclear body α,β ISG-43 Deubiquitinating enzyme β α,>γ,dsRNA IFP-35 Leucine zipper protein α,β,γ Invariant chain MHC class II assembly γ IP-10 Platelet factor 4 related γ>α,β IP-30 Unknown γ>α,β IRF-1 Transcription factor, tumor suppressor α, β, γ,dsRNA IRF-2 Transcription factor, growth promoter α, β,dsRNA ICSBP Transcription factor, tumor suppressor γ; α/β inhibits Leucine amino peptidase Exopeptidase γ Mn-leucine amino peptidase Mitochondrial superoxide scavenger γ Lysyl oxidase Anti-ras activity α,β Tryptophanyl-tRNA synthetase, γ56 Trp-tRNA synthetase γ>α,β MxA Antiinfluenza virus and VSV α,β>γ,dsRNA MxB Unknown α,β>γ,dsRNA MHC class I Antigen presentation α,β,γ MHC class II Antigen presentation γ Phagocyte gp91-phox NADPH oxidase cytochrome b subunit γ, α inhibits PKR Inhibits protein synthesis, signaling α,β>γ RING 12 Proteasome complex γ RING 4 Putative peptide transporter γ γ.1 Unknown γ>>α,β 202 Cell cycle regulator α,β 204 Growth regulator α,β 1-8/9-27 Unknown α,β,γ 6-16 Unknown α,β>γ,dsRNA RNase L Viral RNA degradation α,β
A novel class of IFN-induced growth regulatory proteins is encoded by the gene 200 cluster of mouse chromosome 1. Murine p202, p203, p204, D3, and human MNDA and IFI16 genes belong to this family. These proteins share a characteristic 200 residue segment at their C termini. p202, a well-characterized member of this family, is a nuclear protein that inhibits cell proliferation. By binding to the pRb-E2F complex, p202 inhibits the action of transcription factor E2F, which regulates the expression of a number of genes involved in DNA synthesis and cell division.
p202 also inhibits the action of a number of growth-promoting transcription factors, such as NF- κ B and AP1. A role for the 200 family of genes in growth control is suggested by the translocation of the MNDA gene in human myeloid leukemias. The role of other members of this family in cell growth control needs to be defined.
IFN-γ activates the expression of p21/WAF/Cip-1, a cyclin-dependent kinase inhibitor, to inhibit cell growth. IFN-β induces lysyl oxidase, a specific regressor of Ha-ras-induced transformation. IFN-α downregulates c-myc gene expression and increases the levels or dephosphorylation of pRb to cause growth arrest.
A number of novel apoptosis-regulating genes are controlled by IFN-γ. These include thioredoxin, death associated protein-1(DAP-1), death associate protein kinase (DAPK), cathepsin D, and DAP-5, a homologue of eukaryotic translation initiation factor 4G. Among these DAPK is a potent tumor suppressor. DAPK, a calmodulin-dependent serine-threonine protein kinase, disrupts cytoskeleton during cell death. The DAPK gene is deleted in several carcinomas and lymphomas. It suppresses tumor metastasis. A number of DAPK-related genes have been identified. These enzymes also induce apoptosis.
XI. DYSFUNCTION OF INTERFERONS AND PATHOGENESIS
A number of studies have identified various aspects of IFN resistance and their roles in pathogenesis (see Table III). The following section discusses a few of these observations.
TABLE III Viral Resistance to IFNsVirus Component Target Mechanism Adeno E1A ISG induction Blocks signaling VAI RNA PKR Blocks activation Hepatitis B TP ISG induction Blocks signaling ORF-C IFN-β gene Inhibits gene expression Epstein-Barr EBNA-2 ISG induction Inhibits anticellular action EBER PKR Blocks activation BCRF-1 IFN-γ gene Inhibits IFN-γ synthesis Herpes simplex 2-5A analog RNase L Blocks activation HIV-1 Tar RNA PKR Blocks activation Tat protein PKR Inhibits PKR Influenza Cellular p58 PKR Inhibits PKR Polio Unknown PKR Degrades PKR Reo σ3 PKR Sequesters dsRNA Vaccinia SKI PKR Sequesters dsRNA K3L PKR Analog of eIF-2α E3L PKR Inhibits PKR Myxoma MT2 IFN-γ receptor analog Neutralizes IFN-γ Polyoma virus T antigen ISG induction Blocks JAK1 Papilloma virus E6 IFN-D-induced genes BLocks Tyk2
A. Inactivation of ISG Products
PKR regulates cellular or viral growth by inhibiting protein synthesis. A number of viral gene products inhibit PKR. These include the virus-associated I (VAI) RNA product of adenoviruses, Epstein-Barr virus (EBV)-encoded small molecular weight RNAs (EBERs), and HIV-TAR RNA, HIV-tat protein, reovirus σ3 protein. The influenza virus inhibits PKR using a cellular protein, p58. The vaccinia virus encodes several inhibitors, which either serve as pseudosubstrates or sequester dsRNA to prevent PKR activation. The polio virus targets PKR for degradation.
B. Disruption of JAK-STAT Pathway
Certain viruses inhibit ISG transcription by preventing ISGF3 formation. Adenoviral E1A and hepatitis B virus terminal protein (TP) mediate such effects. Consistent with this, biopsy samples from patients with HBV infection show a dramatic reduction in HLA class I expression. These data, in part, may explain the IFN-α resistance of a substantial subgroup of chronic HBV-infected patients. EBV nuclear antigen-2, an oncogene that immortalizes B cells, also inhibits the antiproliferative action of IFNs by repressing ISGs. Mouse polyoma viral T antigen, human papilloma viral E6 oncogene inhibit JAK1- and Tyk2-induced gene expression, respectively.
Similarly, deletion of JAKs and STATs leads to deregulation of cell growth. For example, tumor cells expressing a dominant-negative mutant of the IFN-γ receptor are not rejected upon transplantation. The chemical carcinogen 3-methylcolanthrene induces tumors at higher frequency in mice lacking STAT1 or IFN-γ receptor compared to normal ones. A constitutive activation of STAT1 by mutant fibroblast growth factor receptor-3 results in abnormal induction of WAF/Cip-1, an inhibitor of cell cycle, and growth stunting. This appears to be a mechanism in type II thanatophoric chondroplasia, a form of human dwarfism.
Hyperactivated JAK2 appears to play a role in acute lymphoblastic leukemia (ALL). Chromosomal translocation t(9;12)(p24;p13) in childhood ALL causes production of a chimeric protein consisting of TEL (a member of ETS family of transcription factors) and JAK2 genes. The resultant chimeric protein, TEL-JAK2, is a constitutively active oncogene.
C. Virokines and Viroceptors
Pox viruses evade the immune system by producing a number of secreted virulence-associated factors. These factors include homologues of cytokines or their receptors. These so called "virokines" include EGF-like growth factors, complement-binding protein, and serine protease inhibitors. Among the viroceptors, the homologues of cytokine receptors are secreted IFN receptors. These proteins bind to IFNs and inhibit their biological actions.
XII. THERAPEUTIC ACTIVITY IN HUMANS
Several clinical studies indicated the potential use of IFNs in therapy of human diseases. Leukocyte IFNs derived from blood donor units were introduced into clinical trials in the mid-1970s. Subsequently, recombinant IFNs have entered clinical trials. These studies have led to licensure in more than 50 countries for the treatment of neoplastic disease, viral diseases, multiple sclerosis, and certain microbial infections. IFN-α controls the disease in ~40% of patients chronically infected with hepatitis B and C viruses. Approximately half of these patients will remain virus free.
In more than a dozen malignancies, IFNs cause regression or control disease progression. In chronic myelogenous leukemia, more than 75% of patients experience complete regression of the disease with approximately one-third of these experiencing complete cytogenetic remission. IFNs, in combination with other systemic treatments, have been used successfully in hairy cell leukemia, B- and T-cell lymphomas, CML, and multiple myeloma. For some solid tumors, IFN treatment resulted in palliation equal to the best chemotherapeutic regimens. Metastatic melanoma and renal cell carcinoma are two refractory malignancies in which IFN-α is more effective than any available chemotherapeutic agent.
IFN-α can also induce regressions in Kaposi's sarcoma, endocrine-pancreatic tumors, and metastatic colorectal, ovarian, and bladder carcinomas. In malignant melanoma, IFN-α delays recurrence following resection of primary disease. In relapsing multiple sclerosis, IFN-β reduces the frequency of relapse and inhibits demyelination of the central nervous system. IFN-γ is effective in chronic granulomatous disease and has been used in combination with chemotherapy for leprosy. IFN-γ may also be useful against chronic pulmonary fibrosis and antibiotic-resistant mycobacterial infections.
Preclinical and clinical findings form the basis for new therapeutic directions in chronic myelogenous leukemia, lymphomas, myelomas, melanoma, urologic malignancies, primary brain tumors, ovarian carcinoma, multiple sclerosis, chronic viral hepatitis, and papillomatous diseases. Major research challenges for full understanding and clinical application of the IFN system are (i) the diversity of the IFN family, (ii) the role of induction, (iii) molecular mechanism of action, (iv) cellular modulatory effects, (v) advantages of combinations, and (vi) identification of new therapeutic indications.
Clinical Effectiveness of Second-Generation IFNs
Quantitative differences exist between various IFNs in terms of their in vitro and in vivo effects. Pegylated IFN-α has a covalently linked polyethylene glycol (PEG) moiety. PEG reduces immunogenicity, sensitivity to proteolysis, and extends the serum half-life. It has been utilized advantageously for therapeutic proteins such as erythropoietin, thrombopoietin, and adenosine deaminase. Pharmacokinetic studies have demonstrated a dose-related increase in serum concentrations and delayed clearance of PEG-IFN-α2 compared to IFN-β. IFN-β has also been modified with various molecules, including PEG and the soluble portion of its receptor to enhance its stability in vivo. Although IFN-α binds to the same receptor on the cell surface as IFN-β, IFN-β does so with higher affinity. IFN-β has distinct effects on cell differentiation and proliferation in contrast to those of IFN-α2. IFN-β is a more potent of growth inhibition and apoptosis than IFN-α2. These advances suggest that the empirical combination of various IFNs with other modalities will have an increasingly broad impact on clinical therapy.
IFNs have come a long way since their discovery. The IFN-stimulated JAK-STAT signal transduction pathway serves as a paradigm for most cytokines. These signal-transducing molecules have central roles in cell growth, differentiation, and tumorigenesis. Over the past decade, IFNs have been established as effective therapeutic molecules for malignant and viral diseases. Gene knockout studies in mice have established new animal models for studying pathogenic mechanisms. It is likely that the therapeutic spectrum of IFNs has just begun to be exploited. Knowledge of signal transduction mechanisms and the ISG actions may help in the development of novel combination therapeutic strategies in the near future.
The authors thank Daniel Lindner and Robert Freund for a critical reading of the manuscript. DVK is supported by research grants from the National Cancer Institute.
Dhananjaya V. Kalvakolanu
Greenebaum Cancer Center, Baltimore, Maryland
Ernest C. Borden
Taussig Cancer Center, Cleveland, Ohio
ACUTE LYMPHOBLASTIC LEUKEMIA ; CHRONIC MYELOGENOUS LEUKEMIA ; CYTOKINE GENE THERAPY ; EPSTEIN-BARR VIRUS ; JAK/STAT PATHWAY
GAS IFN-γ activated site.
ICSBP IFN consensus sequence binding protein.
ISG IFN-stimulated gene.
ISRE IFN-stimulated response element.
IRF IFN-gene regulatory factor.
ISGF IFN-stimulated gene factor.
JAK Janus kinase (just another kinase).
JH JAK homology domain.
SH src homology.
STAT signal transducing activator of transcription.
tyk2 tyrosine kinase 2.
PKR Protein kinase R.
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