Single-particle tracking methods allow detailed analysis of protein movement in cells, but existing tracking algorithms have substantial limitations, particularly at high particle densities. A new software tool overcomes some of these limitations and is used to track CD36 receptors and assay the lifetime of clathrin-coated pits. Also in this issue, Sergé et al. describe an alternative software tool for high-density single-particle tracking. Single-particle tracking (SPT) is often the rate-limiting step in live-cell imaging studies of subcellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose solution identifies the overall most likely set of particle trajectories throughout a movie. Using this approach, we show that the GTPase dynamin differentially affects the kinetics of long- and short-lived endocytic structures and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.

Toll-like receptors (TLRs) recognize microbial components and trigger the inflammatory and immune responses against pathogens. IκBζ (also known as MAIL and INAP) is an ankyrin-repeat-containing nuclear protein that is highly homologous to the IκB family member Bcl-3 (refs 1–6). Transcription of IκBζ is rapidly induced by stimulation with TLR ligands and interleukin-1 (IL-1). Here we show that IκBζ is indispensable for the expression of a subset of genes activated in TLR/IL-1R signalling pathways. IκBζ-deficient cells show severe impairment of IL-6 production in response to a variety of TLR ligands as well as IL-1, but not in response to tumour-necrosis factor-α. Endogenous IκBζ specifically associates with the p50 subunit of NF-κB, and is recruited to the NF-κB binding site of the IL-6 promoter on stimulation. Moreover, NF-κB1/p50-deficient mice show responses to TLR/IL-1R ligands similar to those of IκBζ-deficient mice. Endotoxin-induced expression of other genes such as Il12b and Csf2 is also abrogated in IκBζ-deficient macrophages. Given that the lipopolysaccharide-induced transcription of IκBζ occurs earlier than transcription of these genes, some TLR/IL-1R-mediated responses may be regulated in a gene expression process of at least two steps that requires inducible IκBζ.

Interleukin-10 (IL-10) plays an important role in prevention of chronic inflammation in vivo. However, the molecular mechanism by which IL-10 exerts its anti-inflammatory response is poorly understood. Here, we performed a microarray analysis and identified Bcl-3 as an IL-10-inducible gene in macrophages. Lentiviral vector-mediated expression of Bcl-3 inhibited lipopolysaccharide (LPS)-induced production of tumor necrosis factor α (TNF-α), but not IL-6, in macrophages. In Bcl-3-transduced and IL-10-pretreated macrophages, LPS-induced nuclear translocation of nuclear factor κB (NF-κB) p65 was not impaired. However, DNA binding by NF-κB p50/p65 was profoundly inhibited. Nuclear localization of Bcl-3 was associated with inhibition of LPS-induced TNF-α production. Overexpression of Bcl-3 suppressed activation of the TNF-α promoter, but not the IL-6 promoter. Bcl-3 interacted with NF-κB p50 and was recruited to the TNF-α promoter, but not the IL-6 promoter, indicating that Bcl-3 facilitates p50-mediated inhibition of TNF-α expression. Furthermore, Bcl-3-deficient macrophages showed defective IL-10-mediated suppression of LPS induction of TNF-α, but not IL-6. These findings suggest that IL-10-induced Bcl-3 is required for suppression of TNF-α production in macrophages. (Blood. 2003; 102:4123-4129)

Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPMφ). CLPMφ of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine production and costimulatory molecule expression. We compared CLPMφ gene expression profiles of wild-type mice with IL-10-deficient mice, and identified genes that are selectively expressed in wild-type CLPMφ. These genes included nuclear IκB proteins such as Bcl-3 and IκBNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-α production, we analyzed the role of IκBNS in macrophages. Lentiviral introduction of IκBNS resulted in impaired LPS-induced IL-6 production, but not TNF-α production in the murine macrophage cell line RAW264.7. IκBNS expression led to constitutive and intense DNA binding of NF-κB p50/p50 homodimers. IκBNS was recruited to the IL-6 promoter, but not to the TNF-α promoter, together with p50. Furthermore, small interference RNA-mediated reduction in IκBNS expression in RAW264.7 cells resulted in increased LPS-induced production of IL-6, but not TNF-α. Thus, IκBNS selectively suppresses LPS-induced IL-6 production in macrophages. This study established that nuclear IκB proteins differentially regulate LPS-induced inflammatory cytokine production in macrophages.