Summary B‐lymphocyte stimulator (BLyS) acts as survival factor for B lymphocytes. As Hodgkin and Reed‐Sternberg (HRS) cells express receptors through which BLyS promotes their growth and chemotherapy resistance, we investgated whether this molecule was increased in sera from patients with classical Hodgkin lymphoma (cHL) and whether it correlates with clinical‐pathological features and outcomes. Enzyme‐linked immunosorbent assay was used to measure soluble BLyS (sBLyS) in sera from 87 patients and 33 donors; higher levels were detected in patients (mean ± standard error 4493·9 ± 264·9 pg/ml vs. 2687·0 ± 200·9 pg/ml; P < 0·0001). Levels above the median value (4242·0 pg/ml) were associated with age ≥45 years ( P = 0·042), advanced stages of disease ( P = 0·005), systemic symptoms ( P = 0·014) and extranodal involvement ( P = 0·009). Five‐year failure‐free survival (FFS) of patients with sBLyS below or equal to median levels was 88·6% as compared to 65·1% of those with levels above the median ( P = 0·009). Statistical analyses confirmed the prognostic significance of sBLyS ( P = 0·046). When patients were analysed according to variables associated with high levels, sBLyS showed an independent predictive power in terms of FFS. Our findings support the involvement of BLyS in cHL pathogenesis. The association between high serum levels and an inferior FFS indicates that sBLyS is a possible prognostic predictor with a potential significance as a therapeutic target.

Vascular endothelial cells (EC) participate in the process of bone formation through the production of factors regulating osteoclast differentiation and function. In this study, we report the selective expression in primary human microvascular EC of Osteostat/TNF superfamily 18, a ligand of the TNF superfamily. Osteostat protein is detectable in human microvascular EC and is highly up-regulated by IFN-alpha and IFN-beta. Moreover, an anti-Osteostat antibody strongly binds to the vascular endothelium in human tissues, demonstrating that the protein is present in the EC layers surrounding blood vessels. Functional in vitro assays were used to define Osteostat involvement in osteoclastogenesis. Both recombinant and membrane-bound Osteostat inhibit differentiation of osteoclasts from monocytic precursor cells. Osteostat suppresses the early stage of osteoclastogenesis via inhibition of macrophage colony-stimulating factor-induced receptor activator of NF-kappaB (RANK) expression in the osteoclast precursor cells. This effect appears to be specific for the differentiation pathway of the osteoclast lineage, because Osteostat does not inhibit lipopolysaccharide-induced RANK expression in monocytes and dendritic cells, or activation-induced RANK expression in T cells. These findings demonstrate that Osteostat is a novel regulator of osteoclast generation and substantiate the major role played by the endothelium in bone physiology.

Interferon-kappa (IFN-kappa) is a type I IFN expressed by keratinocytes, monocytes and dendritic cells (DCs). In human keratinocytes, it is produced in response to double-stranded RNA ( dsRNA) and other IFNs and protects from viral infections. In monocytes and DCs, IFN-kappa induces tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) and inhibits lipopolysaccharide (LPS)-induced IL-12. In this study, we evaluated IFN-kappa expression in skin lesions of patients with common immune-mediated inflammatory disorders using immunohistochemical techniques. IFN-kappa was not detectable in healthy skin but was strongly expressed in allergic contact dermatitis and lichen planus-affected skin. IFN-kappa was localized mainly in basal and suprabasal keratinocytes and in some leukocytes infiltrating the dermis. In contrast, IFN-kappa expression in psoriatic or atopic dermatitis ( AD) epidermis was weak and detectable in only 2 of 5 patients examined. Consistently, cultured keratinocytes and monocytes obtained from psoriatic and AD patients expressed null or low levels of IFN-kappa in response to IFN-kappa, which strongly upregulates IFN-kappa in normal keratinocytes. IFN-kappa accumulated in keratinocyte cytoplasm and plasma membrane, and only limited amounts were released extracellularly. Soluble IFN-kappa did not influence keratinocyte proliferation or chemokine and membrane molecule expression, and only its membrane-associated form activated IFN-stimulated response element (ISRE) signaling. Given the difference in IFN-kappa expression levels in the skin disorders examined, IFN-kappa presence or deficiency might have different pathogenetic consequences depending also on other disease-specific intrinsic alterations.

Equitoxic doses of 5-(3-3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) and aryl-triazene derivatives (compounds all capable of inducing a marked increase in murine tumor cell immunogenicity) were studied for their effects on the host immune system. At different times after drug exposure the animals were tested for allograft responses, competence in producing lymphocytes active in lethal graft-versus-host disease, delayed-type hypersensitivity, humoral antibody production, and mitogen responsiveness. While some of the aryl-triazenes tested (DM-COOK DM-NO2) showed a pattern of immunodepression similar to that of DTIC, others were less (MIC, MM-COOK, MM-Cl) or far less (DM-Cl, MM-NO2) active than DTIC in impairing host immunocompetence, although all retained or even augmented their ability to induce chemical xenogenization.

ABSTRACT Tumor necrosis factor (TNF) receptor 6/decoy receptor 3 (TR6/DcR3) is an antiapoptosis soluble receptor of the TNF family produced by tumor cells. In this study, TR6 expression in human immune cells was investigated. TR6 mRNA and protein were detectable in selected antigen-presenting cells. Monocytes and myeloid-derived dendritic cells (MDC) released the protein exclusively following stimulation of Toll-like receptor 2 (TLR2) and TLR4 by gram-positive and gram-negative bacterial antigens. Plasmacytoid dendritic cells, activated by bacterial antigens via TLR9 or by viral infection, did not produce the protein. Similarly, activated T cells did not release TR6. The release of TR6 by MDC was dependent on the activation of p42/p44 mitogen-activated protein kinases, Src-like protein tyrosine kinases, and phosphatidylinositol 3-kinase, signaling pathways important for MDC maturation and survival. In agreement with the in vitro data, TR6 levels in serum were significantly elevated in patients with bacterial infections. Overall, these data suggest a novel role for TR6 in immune responses to bacteria.

We have recently shown that granulocyte-colony-stimulating factor (G-CSF)- and interferon-gamma (IFN-gamma)-activated human neutrophils accumulate and release remarkable amounts of soluble B-lymphocyte stimulator (BLyS) in vitro. In this study, we provide evidence that neutrophils migrating into skin window exudates (SWEs) developed in healthy volunteers and in patients with rheumatoid arthritis (RA), synthesized, and released BLyS in response to locally produced G-CSF. Accordingly, the concentrations of soluble BLyS in SWEs were significantly more elevated than in serum. Because the levels of SWE BLyS, but not SWE G-CSF, were higher in patients with RA than in healthy subjects, we examined the effect of CXCL8/IL-8, C5a, and other proinflammatory mediators that dramatically accumulate in RA SWEs and in inflamed synovial fluids. We show that CXCL1/GROalpha, CXCL8/IL-8, C5a, immune complexes, tumor necrosis factor-alpha (TNF-alpha), leukotriene B4, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and lipopolysaccharide (LPS), which by themselves do not induce BLyS de novo synthesis, act as potent secretagogues for BLyS, which is mainly stored in Golgi-related compartments within G-CSF-treated neutrophils, as determined by immunogold electron microscopy. This action is pivotal in greatly amplifying neutrophil-dependent BLyS release in SWEs of patients with RA compared with healthy subjects. Collectively, our data uncover a novel mechanism that might dramatically exacerbate the release of BLyS by neutrophils during pathologic inflammatory responses.

B lymphocyte stimulator (BLyS) is a novel member of the TNF ligand superfamily that is important in B cell maturation and survival. We demonstrate that human neutrophils, after incubation with G-CSF or, less efficiently, IFN gamma, express high levels of BLyS mRNA and release elevated amounts of biologically active BLyS. In contrast, surface expression of the membrane-bound BLyS was not detected in activated neutrophils. Indeed, in neutrophils, uniquely among other myeloid cells, soluble BLyS is processed intracellularly by a furin-type convertase. Worthy of note, the absolute capacity of G-CSF-stimulated neutrophils to release BLyS was similar to that of activated monocytes or dendritic cells, suggesting that neutrophils might represent an important source of BLyS. In this regard, we show that BLyS serum levels as well as neutrophil-associated BLyS are significantly enhanced after in vivo administration of G-CSF in patients. In addition, serum obtained from two of these patients induced a remarkable accumulation of neutrophil-associated BLyS in vitro. This effect was neutralized by anti-G-CSF antibodies, indicating that G-CSF, present in the serum, stimulated neutrophils to produce BLyS. Collectively, our findings suggest that neutrophils, through the production of BLyS, might play an unsuspected role in the regulation of B cell homeostasis.

The long half-life and stability of human serum albumin (HSA) make it an attractive candidate for fusion to short-lived therapeutic proteins. Albuferon (Human Genome Sciences [HGS], Inc., Rockville, MD) beta is a novel recombinant protein derived from a gene fusion of interferon-beta (IFN-beta ) and HSA. In vitro, Albuferon beta displays antiviral and antiproliferative activities and triggers the IFN-stimulated response element (ISRE) signal transduction pathway. Array analysis of 5694 independent genes in Daudi-treated cells revealed that Albuferon beta and IFN-beta induce the expression of an identical set of 30 genes, including 9 previously not identified. In rhesus monkeys administered a dose of 50 microg/kg intravenously (i.v.) or subcutaneously (s.c.) or 300 microg/kg s.c., Albuferon beta demonstrated favorable pharmacokinetic properties. Subcutaneous bioavailability was 87%, plasma clearance at 4.7-5.7 ml/h/kg was approximately 140-fold lower than that of IFN-beta, and the terminal half-life was 36-40 h compared with 8 h for IFN-beta. Importantly, Albuferon beta induced sustained increases in serum neopterin levels and 2',5' mRNA expression. At a molar dose equivalent to one-half the dose of IFN-beta, Albuferon beta elicited comparable neopterin responses and significantly higher 2',5'-OAS mRNA levels in rhesus monkeys. The enhanced in vivo pharmacologic properties of IFN-beta when fused to serum albumin suggest a clinical opportunity for improved IFN-beta therapy.

BLyS and APRIL are two members of the TNF superfamily that are secreted by activated myeloid cells and have costimulatory activity on B cells. BLyS and APRIL share two receptors, TACI and BCMA, whereas a third receptor, BAFF-R, specifically binds BLyS. Both BLyS and APRIL have been described as homotrimeric molecules, a feature common to members of the TNF superfamily. In this study, we show that APRIL and BLyS can form active heterotrimeric molecules when coexpressed and that circulating heterotrimers are present in serum samples from patients with systemic immune-based rheumatic diseases. These findings raise the possibility that active BLyS/APRIL heterotrimers may play a role in rheumatic and other autoimmune diseases and that other members of the TNF ligand superfamily may also form active soluble heterotrimers.

IFN-kappa is a recently identified type I IFN that exhibits both structural and functional homology with the other type I IFN subclasses. In this study, we have investigated the effect of IFN-kappa on cells of the innate immune system by comparing cytokine release following treatment of human cells with either IFN-kappa or two recombinant IFN subtypes, IFN-beta and IFN-alpha2a. Although IFN-alpha2a failed to stimulate monocyte cytokine secretion, IFN-kappa, like IFN-beta, induced the release of several cytokines from both monocytes and dendritic cells, without the requirement of a costimulatory signal. IFN-kappa was particularly effective in inhibiting inducible IL-12 release from monocytes. Unlike IFN-beta, IFN-kappa did not induce release of IFN-gamma by PBL. Expression of the IFN-kappa mRNA was observed in resting dendritic cells and monocytes, and it was up-regulated by IFN-gamma stimulation in monocytes, while IFN-beta mRNA was minimally detectable under the same conditions. Monocyte and dendritic cell expression of IFN-kappa was also confirmed in vivo in chronic lesions of psoriasis vulgaris and atopic dermatitis. Finally, biosensor-based binding kinetic analysis revealed that IFN-kappa, like IFN-beta, binds strongly to heparin (K-d: 2.1 nM), suggesting that the cytokine can be retained close to the local site of production. The pattern of cytokines induced by IFN-kappa in monocytes, coupled with the unique induction of IFN-kappa mRNA by IFN-gamma, indicates a potential role for IFN-kappa in the regulation of immune cell functions.

B Lymphocyte Stimulator (BLyS™3; protein) is a member of the tumor necrosis factor (TNF) family of ligands and functions as an essential in vivo regulator of B lymphocyte homeostasis. As such, changes in systemic BlyS protein expression caused by disruption of the gene encoding BLyS or administration of neutralizing soluble receptors have resulted in profound losses in mature B cell numbers and impaired humoral immunity. A similar phenotype has been observed in A/WySnJ mice that express a truncated BLyS receptor and are thus defective in BLyS signal transduction. In contrast, overexpression of BLyS protein in BLyS-transgenic mice results in B cell hyperplasia, hypergammaglobulinemia, and development of autoimmune-like disease. The ability of BLyS to regulate both the size and repertoire of the peripheral B cell compartment raises the possibility that BLyS and antagonists thereof may form the basis of a therapeutic trichotomy. As an agonist, BLyS protein may enhance humoral immunity in congenital or acquired immunodeficiencies such as those resulting from viral infection or cancer therapy. BLyS-specific antagonists (antibodies or soluble receptors) that inhibit the biological activity of BLyS may be effective therapies for those autoimmune diseases characterized by polyclonal hypergammaglobulinemia and elevated autoantibody titers. Finally, the specificity of BLyS for B-lineage cells raises the possibility that BLyS may be used as a targeting vehicle for delivery of a cytotoxic or cytolytic signal to neoplastic B-lineage cells expressing one or more of the three known BLyS receptors. This review discusses the therapeutic potential of BLyS in the context of BLyS structure, function and receptor specificity.

Interferon-alpha (IFN-alpha) is indicated for the treatment of certain viral infections including hepatitis B and C, and cancers such as melanoma. The short circulating half-life of unmodified IFN-alpha makes frequent dosing (daily or three times weekly) over an extended period (6-12 months or more) necessary. To improve the pharmacokinetics of IFN-alpha and decrease dosing frequency, IFN-alpha was fused to human serum albumin producing a new protein, Albuferon. In vitro comparisons of Albuferon and IFN-alpha showed similar antiviral and antiproliferative activities, although Albuferon was less potent on a molar basis than IFN-alpha. Pharmacokinetic and pharmacodynamic properties of the fusion protein were enhanced in monkeys. After a single intravenous injection (30 microg/kg,) clearance was 0.9 ml/h/kg, and the terminal half-life was 68 h. After 30 microg/kg subcutaneous injection, apparent clearance (clearance divided by bioavailability) was 1.4 ml/h/kg, the terminal half-life was 93 h, and bioavailability was 64%. The rate of clearance of Albuferon was approximately 140-fold slower, and the half-life 18-fold longer, than for IFN-alpha given by the subcutaneous route in other monkey studies. Sera from Albuferon-treated monkeys demonstrated dose-related antiviral activity for > or =8 days based on an in vitro bioassay, whereas antiviral activity from IFN-alpha-treated animals was only slightly elevated relative to vehicle on day 0. Significant increases in 2',5'-oligoadenylate synthetase mRNA relative to IFN-alpha- or vehicle-treated animals were maintained for > or =10 days after subcutaneous dosing. The improved pharmacokinetics of Albuferon are accompanied by an improved pharmacodynamic response suggesting that Albuferon may offer the benefits of less frequent dosing and a potentially improved efficacy profile compared with IFN-alpha.

B lymphocyte stimulator (BLyS) is a novel member of the TNF family of proteins expressed by myeloid cells as membrane-bound and soluble forms. BLyS was shown to act specifically on B cells, inducing proliferation and immunoglobulin production both in vitro and in vivo. The present study was undertaken to characterize binding of radiolabeled BLyS to its cognate receptor on human B lymphocytes and examine intracellular events initiated by BLyS binding. Similar to other TNF family members, BLyS is present in solution as a homotrimer as determined by gel filtration chromatography and light scattering analysis. BLyS binding to B cells is specific as other TNF family members tested did not compete for I-125-BLyS binding. Analysis of equilibrium binding of I-125-labeled BLyS to purified human tonsillar B cells demonstrated saturable binding. Scatchard analysis of the binding data revealed a single class of high-affinity binding on human B cells with approximately 2600 binding sites per cell and an apparent dissociation constant (K-D,) of about 0.1 nM. In addition we report that BLyS binding to B cells results in the activation of NF-kappaB and the Ets family transcription factor, ELF-1, and in the induction of mRNA for Polo-like kinase (PLK). (C) 2001 Academic Press.

Myeloid progenitor inhibitory factor (MPIF)-2 is a beta-chemokine with select and potent activities on eosinophils and myeloid progenitors. In the beta-chemokine family, biological activity is modulated by differential processing of the amino-terminus. Here, for MPIF-2, we describe the biological activities of NH(2)-terminal deletion mutants and compare regions necessary for eosinophil and myeloid progenitor activities. Five MPIF-2 proteins with deletions at the amino-terminus were produced in Escherichia coli and assayed for calcium mobilization, chemotaxis and receptor binding activities on eosinophils, and for their ability to inhibit colony formation of human myeloid bone marrow progenitors. For eosinophils, deletion of the first two amino acids did not markedly alter activity, while subsequent truncations result in a complete loss of activity. One of the MPIF-2 mutants, MPIF-2 (P30-R99) was converted from an agonist to an antagonist of eotaxin, MPIF-2 and MCP-4 functional responses in eosinophil calcium flux and chemotaxis assays. Surprisingly, while displaying a complete loss of agonist activity toward eosinophils, MPIF-2 (P30-R99) retains ability to inhibit human bone marrow myeloid progenitor cell colony formation. In addition, processing at the amino terminus of MPIF-2 in vivo, may result in a chemokine with altered biological activities.

High throughput cDNA sequencing has led to the identification of interferon-kappa, a novel subclass of type I interferon that displays similar to 30% homology to other family members. Interferon-kappa consists of 207 amino acids, including a 27-amino acid signal peptide and a series of cysteines conserved in type I interferons. The gene encoding interferon-kappa is located on the short arm of chromosome 9 adjacent to the type I interferon gene cluster and is selectively expressed in epidermal keratinocytes. Expression of interferon-kappa is significantly enhanced in keratinocytes upon viral infection, upon exposure to double-stranded RNA, or upon treatment with either interferon-gamma or interferon-beta. Administration of interferon-kappa recombinant protein imparts cellular protection against viral infection in a species-specific manner. Interferon-kappa activates the interferon-stimulated response element signaling pathway and a panel of genes similar to those regulated by other type I interferons including anti-viral mediators and transcriptional regulators. An antibody that neutralizes the type I interferon receptor completely blocks interferon-kappa signaling, demonstrating that interferon-kappa utilizes the same receptor as other type I interferons. Interferon-kappa therefore defines a novel subclass of type I interferon that is expressed in keratinocytes and expands the repertoire of known proteins mediating host defense.

B-lymphocyte stimulator (BLyS) is a recently identified novel member of the tumor necrosis factor ligand superfamily shown to exist in a membrane-bound and soluble form. BLyS was found to be specifically expressed on cells of myeloid lineage and to selectively stimulate B-lymphocyte proliferation and immunoglobulin production. The expression of a cytokine involved in potentiation of humoral immune responses, such as BLyS, is expected to be strictly controlled. The goal of the present study was to examine regulation of BLyS levels in monocytic cells in response to cytokines and during their differentiation to macrophages and dendritic cells. The presence of BLyS on the cell surface and in the culture medium of both normal blood monocytes and on tumor cells of myelomonocytic origin was demonstrated. BLyS gene expression and levels of membrane-associated and soluble BLyS were found to be regulated by cytokines, in particular interferon (IFN)-gamma and to a lesser extent interleukin-10 (IL-10). The expression of BLyS on monocyte membranes was retained following differentiation into macrophages, but detection on the surface of monocyte-derived dendritic cells required stimulation with IFN-gamma. Both IFN-gamma and IL-10 enhanced the release of soluble BLyS that was active in B-cell proliferation assays. Cells transfected with BLyS complementary DNA mutated in a predicted cleavage site failed to release BLyS into the culture medium, thereby suggesting that soluble BLyS was derived from the membrane form. These results provide further support for an important role for BLyS expressed in myeloid cells in B-cell expansion and antibody responses.

Allergic reactions are characterized by the infiltration of tissues by activated eosinophils, Th2 lymphocytes, and basophils. The beta-chemokine receptor CCR3, which recognizes the ligands eotaxin, eotaxin-2, monocyte chemotactic protein (MCP) 3, MCP4, and RANTES, plays a central role in this process, and antagonists to this receptor could have potential therapeutic use in the treatment of allergy. We describe here a potent and specific CCR3 antagonist, called Met-chemokine beta 7 (Ckbeta7), that prevents signaling through this receptor and, at concentrations as low as 1 nM, can block eosinophil chemotaxis induced by the most potent CCR3 ligands. Met-Ckbeta7 is a more potent CCR3 antagonist than Met- and aminooxypentane (AOP)-RANTES and, unlike these proteins, exhibits no partial agonist activity and is highly specific for CCR3. Thus, this antagonist may be of use in ameliorating leukocyte infiltration associated with allergic inflammation. Met-Ckbeta7 is a modified form of the beta-chemokine macrophage inflammatory protein (MIP) 4 (alternatively called pulmonary and activation-regulated chemokine (PARC), alternative macrophage activation-associated C-C chemokine (AMAC) 1, or dendritic cell-derived C-C chemokine (DCCK) 1). Surprisingly, the unmodified MIP4 protein, which is known to act as a T cell chemoattractant, also exhibits this CCR3 antagonistic activity, although to a lesser extent than Met-Ckbeta7, but to a level that may be of physiological relevance. MIP4 may therefore use chemokine receptor agonism and antagonism to control leukocyte movement in vivo. The enhanced activity of Met-Ckbeta7 is due to the alteration of the extreme N-terminal residue from an alanine to a methionine.

An expression cloning approach was employed to identify the receptor for B-lymphocyte stimulator (BLyS) and identified the tumor necrosis factor receptor superfamily member TACI as a BLyS-binding protein. Expression of TACI in HEK293T cells confers on the cells the ability to bind BLyS with subnanomolar affinity. Furthermore, a TACI-Fc fusion protein recognizes both the cleaved, soluble form of BLyS as well as the membrane BLyS present on the cell surface of a recombinant cell line. TACI mRNA is found predominantly in B-cells and correlates with BLyS binding in a panel of B-cell lines. We: also demonstrate that TACI interacts with nanomolar affinity with the BLyS-related tumor necrosis factor homologue APRIL for which no clear in vivo role has been described. BLyS and APRIL are capable of signaling through TACI to mediate NF-KB responses in HEK293 cells. We conclude that TACI is a receptor for BLS S and APRIL and discuss the implications for B-cell biology.