A nonpeptidyl small molecule SB 247464, capable of activating granulocyte-colony-stimulating factor (G-CSF) signal transduction pathways, was identified in a high-throughput assay in cultured cells. Like G-CSF, SB 247464 induced tyrosine phosphorylation of multiple signaling proteins and stimulated primary murine bone marrow cells to form granulocytic colonies in vitro. It also elevated peripheral blood neutrophil counts in mice. The extracellular domain of the murine G-CSF receptor was required for the activity of SB 247464, suggesting that the compound acts by oligomerizing receptor chains. The results indicate that a small molecule can activate a receptor that normally binds a relatively large protein ligand.

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The stress-activated protein kinase p38 plays a central role in the regulation of cytokine biosynthesis by various cell types in response to a wide range of stimuli. Because the local inflammatory response and the infiltration of neutrophils is thought to contribute to the symptoms and sequelae of rhinovirus infection, we investigated the role of p38 kinase in cytokine and chemokine elaboration in airway epithelial cells infected with human rhinovirus. Rhinovirus-39 infection of BEAS-2B cells resulted in synthesis of cytokines (IL-1, IL-6, G-CSF, and GM-CSF) and CXC chemokines (IL-8, epithelial neutrophil-activating protein-78, and growth-related oncogene-α), evident 24–72 h postinfection. Rhinovirus infection induced a time- and dose-dependent increase in tyrosine phosphorylation of p38 kinase, which peaked 30 min postinfection and remained elevated for 1 h. Treatment of infected cells with SB 239063, a potent pyridinyl imidazole inhibitor of p38 kinase, resulted in up to 100% inhibition of mediator production and partially reduced levels of IL-8 mRNA as determined by quantitative RT-PCR. Treatment with SB 239063 had no effect on virus replication and was not cytotoxic at concentrations ≤ 70 μM. These studies provide the first evidence that early activation of p38 kinase by rhinovirus infection is a key event in regulation of virus-induced cytokine transcription, and may provide a new target for inhibition of symptoms and airway inflammation associated with rhinovirus infection.

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Unmethlylated CpG dinucleotides induce a strong T-helper-1-like inflammatory response, presumably mediated by Toll-like receptor 9 (TLR9). However, the nature and cellular localization of TLR9 in primary human cells remain controversial. Here we demonstrate, using flow cytometry and immunofluorescence microscopy techniques, that TLR9 can be expressed at the cell surface. The primary human cell subsets that were positive for TLR9 expression were distinct depending on the tissues analyzed. Specifically, in human peripheral blood mononuclear cells (PBMC) the majority of cell surface TLR9؉ cells were confined to the major histocompatibility complex (MHC) class II ؉ CD19 ؊ populations that express CD11c and/or CD14, whereas in tonsils the same gated population contained primarily MHC class II ؉ CD19 ؉ cells. Cells positive for surface expression represented a minor fraction of the total cell populations examined, varying between 2 and 10%. In addition, we found that TLR9 expression at the surface of PBMC was up-regulated approximately fourfold following stimulation with the gram-negative bacterial cell wall component lipopolysaccharide, suggesting a potential modulatory role of TLR4 agonists on TLR9 expression. Taken together, these data validate human TLR9 expression at the surface of primary cells, in addition to the previously described intracellular localization. Further, our results suggest that human antigen-presenting cells comprise the major cell populations expressing cell surface TLR9.To discriminate between self and nonself antigens, the immune system has evolved a series of pattern recognition receptors to identify invading pathogens and initiate the host immune response. The newly identified Toll-like family of receptors function in this fashion to activate both the innate and adaptive arms of the immune response (21). Toll was originally identified in Drosophila as a receptor required for the establishment of dorso-ventral polarity (19). However, it was also found to play a critical role in immunity, as flies lacking this protein were highly susceptible to infection with Aspergillus fumigatus (27). The mammalian Toll-like receptors (TLRs) were cloned based on sequence homology to the Drosophila Toll protein (12,13,15,32,35,37). Activation of human Toll results in NF-B activation and up-regulation of B7-1, interleukin-1 (IL-1), IL-8, and IL-6 mRNAs, suggesting a role for TLR in bridging innate and adaptive immunities (32). To date, 10 TLRs in humans and 9 TLRs in mice have been identified. TLR9 has been reported to be the receptor for unmethylated CpG dinucleotides found within bacterial but not human DNA (20,24). Expression profiling revealed TLR9 mRNA or protein in B cells, plasmacytoid dendritic cells (DCs), and cells of the monocyte/macrophage lineage (6,20,22,24,25). Synthetic CpG oligodeoxynucleotides (ODN) were used for TLR9 stimulation or ligation and were found to mediate adjuvant activity (10, 30) resulting in the stimulation of T-helper-like-1 (Th1) cytokine production (23) and maturation of D...