Mark Baer

BCL-3 and NF-κB p50 attenuate lipopolysaccharide-induced. Inflammatory responses in macrophages

Lipopolysaccharide (LPS) induces expression of tumor necrosis factor α (TNFα) and other pro-inflammatory cytokines in macrophages. Following its induction, TNFα gene transcription is rapidly attenuated, in part due to the accumulation of NF-κB p50 homodimers that bind to three κB sites in the TNFα promoter. Here we have investigated the inhibitory role of BCL-3, an IκB-like protein that interacts exclusively with p50 and p52 homodimers. BCL-3 was induced by LPS with delayed kinetics and was associated with p50 in the nucleus. Forced expression of BCL-3 suppressed LPS-induced transcription from the TNFα promoter and inhibited two artificial promoters composed of TNFακB sites that preferentially bind p50 dimers. BCL-3-mediated repression was reversed by trichostatin A and was enhanced by overexpression of HDAC-1, indicating that transcriptional attenuation involves recruitment of histone deacetylase. Analysis of macrophages from p50 and BCL-3 knock-out mice revealed that both transcription factors negatively regulate TNFα expression and that BCL-3 inhibits IL-1α and IL-1β. In contrast, induction of the anti-inflammatory cytokine IL-10 was reduced in BCL-3 null macrophages. BCL-3 was not required for the production of p50 homodimers but BCL-3 expression was severely diminished in p50-deficient cells. Together, these findings indicate that p50 and BCL-3 function as anti-inflammatory regulators in macrophages by attenuating transcription of pro-inflammatory cytokines and activating IL-10 expression.

Tumor Necrosis Factor Alpha Transcription in Macrophages Is Attenuated by an Autocrine Factor That Preferentially Induces NF-κB p50

ABSTRACT Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), which are expressed during conditions of inflammation, infection, or injury. We identified an activity secreted by a macrophage tumor cell line that negatively regulates bacterial lipopolysaccharide (LPS)-induced expression of TNF-α. This activity, termed TNF-α-inhibiting factor (TIF), suppressed the induction of TNF-α expression in macrophages, whereas induction of three other proinflammatory cytokines (interleukin-1β [IL-1β], IL-6, and monocyte chemoattractant protein 1) was accelerated or enhanced. A similar or identical inhibitory activity was secreted by IC-21 macrophages following LPS stimulation. Inhibition of TNF-α expression by macrophage conditioned medium was associated with selective induction of the NF-κB p50 subunit. Hyperinduction of p50 occurred with delayed kinetics in LPS-stimulated macrophages but not in fibroblasts. Overexpression of p50 blocked LPS-induced transcription from a TNF-α promoter reporter construct, showing that this transcription factor is an inhibitor of the TNF-α gene. Repression of the TNF-α promoter by TIF required a distal region that includes three NF-κB binding sites with preferential affinity for p50 homodimers. Thus, the selective repression of the TNF-α promoter by TIF may be explained by the specific binding of inhibitory p50 homodimers. We propose that TIF serves as a negative autocrine signal to attenuate TNF-α expression in activated macrophages. TIF is distinct from the known TNF-α-inhibiting factors IL-4, IL-10, and transforming growth factor β and may represent a novel cytokine.

The ability of C/EBP beta but not C/EBP alpha to synergize with an Sp1 protein is specified by the leucine zipper and activation domain.

The rat CYP2D5 P-450 gene is activated in the liver during postnatal development. We previously showed that liver-specific transcription of the CYP2D5 gene is dictated by a proximal promoter element, termed 2D5, that is composed of a binding site for Sp1 or a related factor, and an adjacent cryptic C/EBP (CCAAT/enhancer-binding protein) site. Despite the fact that both C/EBP alpha and C/EBP beta are expressed abundantly in liver, only C/EBP beta is capable of stimulating the 2D5 promoter in HepG2 hepatocarcinoma cells. In addition, activation of the 2D5 promoter by C/EBP beta is completely dependent on the presence of the Sp1 site. Domain switch experiments reveal that C/EBP beta proteins containing either the leucine zipper or the activation domain of C/EBP alpha are unable to stimulate the 2D5 promoter yet are fully capable of transactivating an artificial promoter bearing a high-affinity C/EBP site. Thus, the leucine zipper and the activation domain of C/EBP beta are absolutely required to support transactivation of the 2D5 promoter. Using Drosophila cells that lack endogenous Sp1 activity, we show that the serine/threonine- and glutamine-rich activation domains A and B of Sp1 are required for efficient cooperatively with C/EBP beta. Furthermore, analysis of c/ebp beta-deficient mice shows that mutant animals are defective in expression of a murine CYP2D5 homolog in hepatic cells, confirming the selective ability of C/EBP beta to activate this liver-specific P-450 gene in vivo. Our findings illustrate that two members of a transcription factor family can achieve distinct target gene specificities through differential interactions with a cooperating Sp1 protein.

The C/EBP bZIP Domain Can Mediate Lipopolysaccharide Induction of the Proinflammatory Cytokines Interleukin-6 and Monocyte Chemoattractant Protein-1

C/EBPα, β, and δ are all expressed by bone marrow-derived macrophages. Ectopic expression of any of these transcription factors is sufficient to confer lipopolysaccharide (LPS)-inducible expression of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) to a B lymphoblast cell line, which normally lacks C/EBP factors and does not display LPS induction of proinflammatory cytokines. Thus, the activities of C/EBPα, β, and δ are redundant in regard to expression of IL-6 and MCP-1. Surprisingly, the bZIP region of C/EBPβ, which lacks any previously described activation domains, can also confer LPS-inducible expression of IL-6 and MCP-1 in stable transfectants. Transient transfections reveal that the bZIP regions of C/EBPβ, C/EBPδ, and, to a lesser extent, C/EBPα can activate the IL-6 promoter and augment its induction by LPS. Furthermore, the transdominant inhibitor, LIP, can activate expression from the IL-6 promoter. The ability of the C/EBPβ bZIP region to activate the IL-6 promoter in transient transfections is completely dependent upon an intact NF-κB-binding site, supporting a model where the bZIP protein primarily functions to augment the activity of NF-κB. Replacement of the leucine zipper of C/EBPβ with that of GCN4 yields a chimeric protein that can dimerize and specifically bind to a C/EBP consensus sequence, but shows a markedly reduced ability to activate IL-6 and MCP-1 expression. These results implicate the leucine zipper domain in some function other than dimerization with known C/EBP family members, and suggest that C/EBP redundancy in regulating cytokine expression may result from their highly related bZIP regions.

GM-CSF–dependent pSTAT5 sensitivity is a feature with therapeutic potential in chronic myelomonocytic leukemia

Granulocyte-macrophage-colony-stimulating factor (GM-CSF) hypersensitivity is a hallmark of juvenile myelomonocytic leukemia (JMML) but has not been systematically shown in the related human disease chronic myelomonocytic leukemia (CMML). We find that primary CMML samples demonstrate GM-CSF-dependent hypersensitivity by hematopoietic colony formation assays and phospho-STAT5 (pSTAT5) flow cytometry compared with healthy donors. Among CMML patients, the pSTAT5 hypersensitive response positively correlated with high-risk disease, peripheral leukocytes, monocytes, and signaling-associated mutations. When compared with IL-3 and G-CSF, GM-CSF hypersensitivity was cytokine specific and thus a possible target for intervention in CMML. To explore this possibility, we treated primary CMML cells with KB003, a novel monoclonal anti-GM-CSF antibody, and JAK2 inhibitors. We found that an elevated proportion of immature GM-CSF receptor-α(R) subunit-expressing cells were present in the bone marrow myeloid compartment of CMML. In survival assays, we found that myeloid and monocytic progenitors were sensitive to GM-CSF signal inhibition. Our data indicate that a committed myeloid precursor expressing CD38 may represent the progenitor population with enhanced GM-CSF dependence in CMML, consistent with results in JMML. These preclinical data indicate that GM-CSF signaling inhibitors merit further investigation in CMML and that GM-CSFR expression on myeloid progenitors may be a biomarker for this therapy.

Regulation of CCAAT/Enhancer-binding Protein (C/EBP) Activator Proteins by Heterodimerization with C/EBPγ (Ig/EBP)

The CCAAT/enhancer-binding proteins (C/EBPs) are basic leucine zipper transcription factors that play important roles in regulating cell growth and differentiation. C/EBP proteins form leucine zipper-mediated homodimers but are also capable of heterodimerizing with other C/EBPs in vitro. Here we show that C/EBPβ occurs predominantly as a heterodimer that displays rapid mobility in gel shift assays. Biochemical fractionation and antibody supershift assays demonstrate that the C/EBPβ heterodimeric partner is C/EBPγ (Ig/EBP), a C/EBP protein that has been implicated as an inhibitor of other family members. Although most cell types express C/EBPβ·C/EBPγ heterodimers, macrophages contain a C/EBPβ partner that is serologically distinct from C/EBPγ. We found that C/EBPγ blocked the ability of C/EBPβ and C/EBPγ to activate a reporter gene in L cell fibroblasts but did not inhibit a chimeric C/EBPβ protein containing the GCN4 leucine zipper. Repression by C/EBPγ occurs at the level of transactivation and requires heterodimerization with the C/EBP partner. C/EBPγ was an ineffective repressor in HepG2 hepatoma cells despite forming C/EBP heterodimers, and C/EBPα was not effectively inhibited in either L or HepG2 cells. Our findings demonstrate that C/EBPγ modulates C/EBP activity in a cell- and isoform-specific manner.

PcrV antibody–antibiotic combination improves survival in Pseudomonas aeruginosa -infected mice

The type III secretion system (TTSS) of Pseudomonas aeruginosa, associated with acute infection, facilitates the direct injection of cytotoxins into the host cell cytoplasm. Mab166, a murine monoclonal antibody against PcrV, a protein located at the tip of the injectisome, has demonstrated efficacy against P. aeruginosa infection, resulting in reduced lung injury and increased survival in murine models of infection. We hypothesised that the administration of Mab166 in combination with an antibiotic would further improve the survival of P. aeruginosa-infected mice. A murine model of P. aeruginosa acute infection, three clinically relevant antibiotics (ciprofloxacin, tobramycin and ceftazidime) and the Mab166 antibody were used for this study. Consistently, compared to other treatment groups (antibiotic or antibody administered in isolation), the combination of Mab166 and antibiotic significantly improved the survival of mice infected with three times the lethal dose (LD90) of the highly cytotoxic ExoU-secreting strain, PA103. This synergistic effect was primarily due to enhanced bactericidal effect and protection against lung injury, which prevented bacterial dissemination to other organs. Hence, the combination of Mab166 with antibiotic administration provides a new, more effective strategy against P. aeruginosa airway infection, especially when large numbers of highly virulent strains are present.

A high affinity recombinant antibody to the human EphA3 receptor with enhanced ADCC activity.

Abstract EphA3is expressed in solid tumors and leukemias and is an attractive target for the therapy. We have generated a panel of Humaneered® antibodies to the ligand-binding domain using a Fab epitope-focused library that has the same specificity as monoclonal antibody mIIIA4. A high-affinity antibody was selected that competes with the mIIIA4 antibody for binding to EphA3 and has an improved affinity of ∼1 nM. In order to generate an antibody with potent cell-killing activity the variable regions were assembled with human IgG1k constant regions and expressed in a Chinese hamster ovary (CHO) cell line deficient in fucosyl transferase. Non-fucosylated antibodies have been reported to have enhanced binding affinity for the IgG receptor CD16a (FcγRIIIa). The affinity of the antibody for recombinant CD16a was enhanced approximately 10-fold. This resulted in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity against EphA3-expressing leukemic cells, providing a potent antibody for the evaluation as a therapeutic agent.