Jackie C. Timans

Novel p19 Protein Engages IL-12p40 to Form a Cytokine, IL-23, with Biological Activities Similar as Well as Distinct from IL-12

A novel sequence discovered in a computational screen appears distantly related to the p35 subunit of IL-12. This factor, which we term p19, shows no biological activity by itself; instead, it combines with the p40 subunit of IL-12 to form a novel, biologically active, composite cytokine, which we term IL-23. Activated dendritic cells secrete detectable levels of this complex. IL-23 binds to IL-12R beta 1 but fails to engage IL-12R beta 2; nonetheless, IL-23 activates Stat4 in PHA blast T cells. IL-23 induces strong proliferation of mouse memory (CD4(+)CD45Rb(low)) T cells, a unique activity of IL-23 as IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-gamma production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.

IL-27, a Heterodimeric Cytokine Composed of EBI3 and p28 Protein, Induces Proliferation of Naive CD4+ T Cells

An efficient Th1-driven adaptive immune response requires activation of the T cell receptor and secretion of the T cell stimulatory cytokine IL-12 by activated antigen-presenting cells. IL-12 triggers Th1 polarization of naive CD4(+) T cells and secretion of IFN-gamma. We describe a new heterodimeric cytokine termed IL-27 that consists of EBI3, an IL-12p40-related protein, and p28, a newly discovered IL-12p35-related polypeptide. IL-27 is an early product of activated antigen-presenting cells and drives rapid clonal expansion of naive but not memory CD4(+) T cells. It also strongly synergizes with IL-12 to trigger IFN-gamma production of naive CD4(+) T cells. IL-27 mediates its biologic effects through the orphan cytokine receptor WSX-1/TCCR.

Two Novel IL-1 Family Members, IL-1δ and IL-1ε, Function as an Antagonist and Agonist of NF-κB Activation Through the Orphan IL-1 Receptor-Related Protein 2

IL-1 is of utmost importance in the host response to immunological challenges. We identified and functionally characterized two novel IL-1 ligands termed IL-1δ and IL-1ε. Northern blot analyses show that these IL-1s are highly abundant in embryonic tissue and tissues containing epithelial cells (i.e., skin, lung, and stomach). In extension, quantitative real-time PCR revealed that of human skin-derived cells, only keratinocytes but not fibroblasts, endothelial cells, or melanocytes express IL-1δ and ε. Levels of keratinocyte IL-1δ are ∼10-fold higher than those of IL-1ε. In vitro stimulation of keratinocytes with IL-1β/TNF-α significantly up-regulates the expression of IL-1ε mRNA, and to a lesser extent of IL-1δ mRNA. In NF-κB-luciferase reporter assays, we demonstrated that IL-1δ and ε proteins do not initiate a functional response via classical IL-1R pairs, which confer responsiveness to IL-1α and β or IL-18. However, IL-1ε activates NF-κB through the orphan IL-1R-related protein 2 (IL-1Rrp2), whereas IL-1δ, which shows striking homology to IL-1 receptor antagonist, specifically and potently inhibits this IL-1ε response. In lesional psoriasis skin, characterized by chronic cutaneous inflammation, the mRNA expression of both IL-1 ligands as well as IL-1Rrp2 are increased relative to normal healthy skin. In total, IL-1δ and ε and IL-1Rrp2 may constitute an independent signaling system, analogous to IL-1αβ/receptor agonist and IL-1R1, that is present in epithelial barriers of our body and takes part in local inflammatory responses.

OB-BP1/Siglec-6 A LEPTIN- AND SIALIC ACID-BINDING PROTEIN OF THE IMMUNOGLOBULIN SUPERFAMILY

We report the expression cloning of a novel leptin-binding protein of the immunoglobulin superfamily (OB-BP1) and a cross-hybridizing clone (OB-BP2) that is identical to a recently described sialic acid-binding I-type lectin called Siglec-5. Comparisons to other known Siglec family members (CD22, CD33, myelin-associated glycoprotein, and sialoadhesin) show that OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 constitute a unique related subgroup with a high level of overall amino acid identity: OB-BP1versus Siglec-5 (59%), OB-BP1 versus CD33 (63%), and OB-BP2/Siglec-5 versus CD33 (56%). The cytoplasmic domains are not as highly conserved, but display novel motifs which are putative sites of tyrosine phosphorylation, including an immunoreceptor tyrosine kinase inhibitory motif and a motif found in SLAM and SLAM-like proteins. Human tissues showed high levels of OB-BP1 mRNA in placenta and moderate expression in spleen, peripheral blood leukocytes, and small intestine. OB-BP2/Siglec-5 mRNA was detected in peripheral blood leukocytes, lung, spleen, and placenta. A monoclonal antibody specific for OB-BP1 confirmed high expression in the cyto- and syncytiotrophoblasts of the placenta. Using this antibody on peripheral blood leukocytes showed an almost exclusive expression pattern on B cells. Recombinant forms of the extracellular domains of OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 were assayed for specific binding of leptin. While OB-BP1 exhibited tight binding (K d 91 nm), the other two showed weak binding with K d values in the 1–2 μmrange. Studies with sialylated ligands indicated that OB-BP1 selectively bound Neu5Acα2–6GalNAcα (sialyl-Tn) allowing its formal designation as Siglec-6. The identification of OB-BP1/Siglec-6 as a Siglec family member, coupled with its restricted expression pattern, suggests that it may mediate cell-cell recognition events by interacting with sialylated glycoprotein ligands expressed on specific cell populations. We also propose a role for OB-BP1 in leptin physiology, as a molecular sink to regulate leptin serum levels.

IL-18 receptors, their role in ligand binding and function: anti-IL-1RAcPL antibody, a potent antagonist of IL-18.

IL-18 is critical in eliciting IFN-γ production from Th1 cells both in vitro and in vivo. Th1 cells have been implicated in the pathogenesis of autoimmune disorders, making antagonists of IL-18 promising therapeutics. However, specificity and binding characteristics of IL-18R components have only been superficially explored. In this study, we show that IL-1R related protein 1 (IL-1Rrp1) and IL-1R accessory protein-like (IL-1RAcPL) confer responsiveness to IL-18 in a highly specific (no response to other IL-1 ligands) and unique manner (no functional pairing with other IL-1Rs and IL-1R-like molecules). Cotransfection with both receptor components resulted in expression of both low and high affinity binding sites for IL-18 (Kd of 11 and 0.4 nM, respectively). We prepared anti-IL-1RAcPL mAb TC30-28E3, which, in contrast to soluble R proteins, effectively inhibited the IL-18-induced activation of NF-κB. Quantitative PCR showed that Th1 but not Th2 cells are unique in that they coexpress IL-1Rrp1 and IL-1RAcPL. mAb TC30-28E3 inhibited IL-18-induced production of IFN-γ by Th1 cells, being at least 10-fold more potent than anti-IL-18 ligand mAb. This study shows that IL-1RAcPL is highly specific to IL-18, is required for high affinity binding of IL-18, and that the anti-IL-1RAcPL mAb TC30-28E3 potently antagonizes IL-18 responses in vitro, providing a rationale for the use of anti-IL-1RAcPL Abs to inhibit Th1-mediated inflammatory pathologies.

Functional Replacement of Cytokine Receptor Extracellular Domains by Leucine Zippers

Granulocyte-macrophage colony-stimulating factor receptor signals by a complex which includes the ligand and two different receptor subunits: a low affinity α receptor binding chain (granulocyte-macrophage colony-stimulating factor receptor α subunit (GM-Rα)) and a signal-transducing β chain (GM-Rβ). To investigate two unresolved issues in the initiation of signaling, the role of receptor extracellular domains and receptor stoichiometry, we replaced the mouse GM-Rα and GM-Rβ extracellular domains with the leucine zipper domain of either the Fos or Jun molecule. We co-transfected combinations of chimeric receptors into Ba/F3 cells and found that both simple heterodimers of the GM-Rα and GM-Rβ intracellular domains and homodimers of the GM-Rβ intracellular domain signaled for proliferation. Surprisingly, homodimers of the GM-Rα intracellular domain also signaled for prevention of apoptosis in transfected cells. We similarly engineered dimers of the intracellular domain of the human interferon γ receptor β subunit and found that homodimers of the intracellular domain signaled for proliferation. When Fos peptide was added to Ba/F3 cells expressing the human interferon γ receptor β subunit construct, thereby preventing homodimer formation, the cells no longer proliferated in the absence of mouse interleukin 3.