Th1 and Th2 lymphocytes express a different repertoire of chemokine receptors (CCRs). CXCR3, the receptor for I-TAC (interferon-inducible T cell ␣-chemoattractant), Mig (monokine induced by ␥-interferon), and IP10 (interferon-inducible protein 10), is expressed preferentially on Th1 cells, whereas CCR3, the receptor for eotaxin and several other CC chemokines, is characteristic of Th2 cells. While studying responses that are mediated by these two receptors, we found that the agonists for CXCR3 act as antagonists for CCR3. I-TAC, Mig, and IP10 compete for the binding of eotaxin to CCR3-bearing cells and inhibit migration and Ca 2؉ changes induced in such cells by stimulation with eotaxin, eotaxin-2, MCP-2 (monocyte chemottractant protein-2), MCP-3, MCP-4, and RANTES (regulated on activation normal T cell expressed and secreted). A hybrid chemokine generated by substituting the first eight NH 2 -terminal residues of eotaxin with those of I-TAC bound CCR3 with higher affinity than eotaxin or I-TAC (3-and 10-fold, respectively). The hybrid was 5-fold more potent than I-TAC as an inhibitor of eotaxin activity and was effective at concentrations as low as 5 nM. None of the antagonists described induced the internalization of CCR3, indicating that they lack agonistic effects and thus qualify as pure antagonists. These results suggest that chemokines that attract Th1 cells via CXCR3 can concomitantly block the migration of Th2 cells in response to CCR3 ligands, thus enhancing the polarization of T cell recruitment.

Full transcriptional activity of the nuclear, DNA-bound form of NF-κB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans-activating NF-κB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IκBα and IκB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Cθ, and NF-κB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKβ, but not by IKKα. Phosphorylation occurs within the cytoplasmic and intact NF-κB/IκBα complex and requires prior phosphorylation of IκBα at serines 32 and 36. Reconstitution of p65−/− cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IκBα localization and the kinetics of p65 nuclear import.

NF-kappaB is a generic name for an evolutionarily conserved transcription-factor system that contributes to the mounting of an effective immune response but is also involved in the regulation of cell proliferation, development, and apoptosis. The implication of NF-kappaB in central biological processes and its extraordinary connectivity to other signaling pathways raise a need for highly controlled regulation of NF-kappaB activity at several levels. While all NF-kappaB activation pathways share a central and critical proteasome-mediated step that leads to the degradation of inhibitory proteins and the release of DNA-binding subunits, there is evidence for a downstream level of NF-kappaB regulation that employs several mechanisms. These include promoter-specific exchange of dimers and modification of the transactivating p65 subunit by phosphorylation, acetylation, ubiquitination, or prolyl isomerization. The signaling pathways and enzymes controlling this second level of regulation and their potential use as therapeutic targets for the treatment of NF-kappaB-associated pathologies are discussed here.

I-TAC, IP10, and Mig are interferon-␥ inducible CXC chemokines that share the same G-protein-coupled receptor CXCR3, which is preferentially expressed on Th1 lymphocytes. We have explored the structure-function relationship of the CXCR3 ligands, in particular of I-TAC, which has highest affinity for CXCR3 and is the most potent agonist. A potent antagonist for CXCR3 was obtained by NH 2 -terminal truncation of I-TAC. I-TAC (4 -73), which lacks the first three residues, has no agonistic activity but competes for the binding of I-TAC to CXCR3-bearing cells and inhibits migration and Ca 2؉ changes in such cells in response to stimulation with I-TAC, IP10, and Mig. It does also not induce internalization of CXCR3, which is in support of the lack of agonistic effects. Hybrid chemokines between I-TAC and IP10 were used to identify regions responsible for the higher activity of I-TAC. I-TAC-like IP10 analogs are obtained by substituting the NH 2 terminus (residues 1-8) or N-loop region (residues 12-17) of IP10 with those of I-TAC, suggesting that the differences in function of the CXCR3 ligands can be assigned to distinct regions and that these regions are interchangeable. Structureactivity studies with Mig showed that the extended basic COOH-terminal region, which is not present in I-TAC and IP10, is important for binding and activity.

Here we identify IKK⑀ as a novel NF-B p65 kinase that mediates inducible phosphorylation of Ser 468 and Ser 536 in response to T cell costimulation. In addition, the kinase activity of IKK⑀ contributes to the control of p65 nuclear uptake. Serines 468 and 536 are evolutionarily conserved, and the surrounding amino acids display sequence homology. Down-regulation of IKK⑀ levels by small interfering RNA does not affect inducible phosphorylation of Ser 536 but largely prevents Ser 468 phosphorylation induced by T cell costimulation. Ser 536 -phosphorylated p65 is found predominantly in the cytosol. In contrast, the Ser 468 phosphorylated form of this transcription factor occurs mainly in the nucleus, suggesting a function for transactivation. Reconstitution of p65 ؊/؊ cells with either wild type p65 or point-mutated p65 variants showed that inducible phosphorylation of Ser 468 serves to enhance p65-dependent transactivation. These results also provide a mechanistic link that helps to explain the relevance of IKK⑀ for the expression of a subset of NF-B target genes without affecting cytosolic IB␣ degradation.The NF-B transcription factor system serves to control the expression of an extraordinarily wide array of genes in response to infections, inflammation, and other harmful situations (1, 2). NF-B target genes (such as immunoreceptors, cytokines, and chemokines) contribute to the innate immune response but also serve to control cell survival and proliferation (3). NF-B is a collective name for homo-or heterodimers composed of five different DNA-binding subunits, with the most frequently detected form being a heterodimer of p50 and p65 (RelA). The p65 subunit contains two strong, acidic transactivation domains called TAD1 3 and TAD2 in its C-terminal portion (4). A large variety of different inducers leads to NF-B activation by activation of numerous cellular and membrane receptors, including toll-like receptors and the T cell receptor. Thus far, three major pathways mediating NF-B activation have been identified, the so-called canonical and noncanonical pathways and the DNA damage-induced NF-B pathway. All NF-B activating events have in common that they lead to the proteasome-dependent generation of DNA-binding dimers (5). NF-B signals activating the canonical pathway funnel into the IKK complex, which is composed of the enzymatically active subunits IKK␣ and IKK␤ and the regulatory subunits IKK␥/NEMO (6, 7) and ELKS (8). IKK␤-mediated I␤ phosphorylation allows subsequent ubiquitination and proteolytic destruction of this inhibitory protein. This leads to an unmasking of the p65 nuclear localization sequence and results in NF-B nuclear immigration, DNA binding, and gene expression.Once activated, inducible post-translational modifications, including phosphorylation, acetylation, ubiquitination, or prolyl isomerization, allow the regulation of NF-B transcriptional activity (9, 10). Thus far, eight different phosphorylation sites have been mapped for the strongly activating NF-B p65 subunit. Three sites are contain...